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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160201
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 109-115
Potential distribution of Monotropa uniflora as a surrogate for range of
Monotropoideae (Ericaceae) in South Asia
PRAKASH PRADHAN
West Bengal Biodiversity Board, Department of Environment, Government of West Bengal, Salt Lake, Sector-III, FD415A, Poura Bhawan, 4th Floor,
Kolkata, West Bengal, India, 700 106. Tel.+91 8013126863, email: shresthambj@gmail.com.
Manuscript received: 12 February 2015. Revision accepted: 13 May 2015.
Abstract. Pradhan P. 2015. Potential distribution of Monotropa uniflora as a surrogate for range of Monotropoideae (Ericaceae) in
South Asia. Biodiversitas 16: 109-115. Monotropoideae is a mycoheterotrophic subfamily of Ericaceae. Its members are highly specific
to a particular fungal family, which has attributed to the rarity and limited distribution of Monotropoideae. In the past two decades, there
are considerable developments in understanding their biology and biogeography, among which, the distribution of Monotropa uniflora
L. and M. hypopitys L. has been extensively studied. In this contribution, Ecological Niche Modeling of M. uniflora has been conducted
to test its earlier proposed distribution in South Asia, to test the spatial scale of the said proposal, to test its potential distribution as a
surrogate for range of Monotropoideae in South Asia and to prioritize conservation areas for M. uniflora in the region. The model was
built with five occurrence details of the rare plant M. uniflora in Western and Eastern Himalaya, in relation to 19 bioclimatic
explanatory variables, performed in MaxEnt. The results show the good performance of the model with the training AUC of 0.994.
1,50,316 square Km. of suitable areas have been predicted for the growth of M. uniflora (IHS ≥0.5) in South Asia, many areas of which
is in line with earlier distributional reports. The bioclimatic variables are able to predict and suitably justify the spatial distribution of M.
uniflora. The predicted range of the species could be established for potential distribution of other Asian Monotropoids like
Monotropastrum and Cheilotheca.
Key words: Ecological Niche Modeling, Himalaya, MaxEnt, Mycoheterotrophy, Russulaceae
Abbreviations: IHS = Index of Habitat Suitability; Bio = Bioclimatic variable
INTRODUCTION
Mycoheterotrophy includes an obligatory reliance of
achlorophyllous and non-photosynthetic plants upon
specialized mycorrhizal associates for carbon influx
(Klooster and Culley 2009). Monotropoideae is a
mycoheterotrophic subfamily of Ericaceae, consisting of 10
genera and 15 species (Wallace 1975). Leake (1994) has
reported 260 species of Mycoheterotrophic plants as
endemic to Palaeotropics, extending from India in the West
to Papua-New Guinea and Japan in the East. Endemic taxa
of Monotropoideae have been reported from Western North
America (seven species) and Asia (two Monotropastrum
species and two Cheilotheca species and a variety)
(Tsukaya et al. 2008), preferring to grow mostly in shady
old growth forests (Min et al. 2012). Their endemism and
narrow geographic range have been linked to limited
distribution of their taxa specific mycorrhizal fungal
partners (Kruckeberg and Rabinowitz 1985; Bidartondo
and Bruns 2001), and such trophic structure has left the
subfamily with very isolated and rare taxa (Klooster and
Culley 2009). Monotropa uniflora L. and M. hypopitys L.
though distantly related but are the only species within
Monotropoideae which are distributed across Neotropics
and Palaeotropics (Leake 1994). Although Leake (1994)
has indicated broad range of genus Monotropa in Indian
subcontinent, M. uniflora is considered regionally rare in
Indian states of West Bengal (WBBB 2012; FRLHT 2015)
and Meghalaya (Mir et al. 2014).
Suitability of climate and presence of dense and shaded
forest habitat has been attributed for the evolution of
heterotrophy in paleotropics and neotropics (Leake 1994).
Similarly, Ecological Niche/climate modeling has been
proposed to act as a successful marker to infer the
phylogeographic
and
demographic
histories
of
mycoheterotrophic plants by Taylor et al. (2013). In this
regard, paleo-distribution modeling of M. hypopitys in
North America has been successfully conducted in MaxEnt
using bioclimatic variables (Beatty and Provan 2011),
however no study has been focused on Monotropoideae as
a whole or any species therein in South Asia regarding their
distribution in bioclimatic envelope. For planning suitable
conservation action for Monotropoideae in this region,
prime necessity is to prioritize areas for conservation
having high suitability of the ecological niche of the taxa.
In this contribution, Ecological Niche Modeling of M.
uniflora has been conducted to test earlier proposed
distribution of the species in South Asia, to test the spatial
scale of the said proposal, to test potential distribution of
the species as a surrogate for range of Monotropoideae in
South Asia and to prioritize conservation areas for M.
uniflora in the region.
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B I O D I V E R S IT A S 16 (2): 109-115, October 2015
MATERIALS AND METHODS
Occurrence records and site characteristics
Occurrence records were obtained with the help of
Garmin Etrex GPS machine for the locations of
Jorepokhari (Figure 1); data from Rachela were obtained
from Divisional Forest Office (Research Division),
Darjeeling; occurrences in Phedkhal, Jakholi and Mandal
of Uttarakhand were derived from Semwal et al. (2014) and
in communication with Dr. Semwal (Figure 2). All the
occurrence sites have montane topography. Vegetation
wise, Rachela and Phedkhal have Oak dominated
vegetation, Jakholi has mixed woodlands, Mandal has
mixed forest with Oak, Birch and Cedrus, while
Jorepokhari has mixed woodlands of Cryptomeria
japonica, Oak and Bamboos. The geographical features of
the occurrence sites are shown in Table 1.
Figure 2. Monotropa uniflora plants observed in Jorepokhari,
Darjeeling District, West Bengal, India
Figure 1. Occurrence records utilized for modelling potential distribution of Monotropa uniflora
PRADHAN – Potential distribution of Monotropa uniflora in South Asia
111
Table 1. Geographical and climatic features of the occurrence sites; Tmin=mean monthly minimum temperature, Tmax=mean monthly
maximum temperature, PPT=annual precipitation.
Site
Longitude
Latitude
Jorepokhari
Rachela
Phedkhal
Jakholi
Mandal
88.1490
88.7414
78.8704
78.9080
79.4331
26.9877
27.1378
30.1682
30.3862
29.5789
Altitude
(m)
2239
3124
1842
1706
1621
Table 2. Range of Index of Habitat Suitability (IHS) along with
predicted area and the respective percentage of the total suitable
area.
IHS
0.5 - 0.599
0.6 - 0.699
0.7 - 0.799
0.8 - 0.899
0.9 - 1
Predicted area
km2
1,50,316
98,045
76,717
63,697
39,968
% of total suitable
area
35.06
22.87
17.89
14.86
9.32
Modeling species distribution - Modeling method
MaxEnt program version 3.3.3 k (Phillips et al. 2006;
Phillips and Dudik 2008) is a maximum entropy based
general-purpose machine learning method and it was used
for modeling species distribution in geographic space and
creation of habitat suitability maps. Entropy in the context
of probability theory and statistics measures the amount of
information that is contained in a random variable or
unknown quantity. MaxEnt uses the basic set of
information to model the distribution of a species, i.e., a set
of samples (species presence) available from a
geographical region, which is linked to a set of explanatory
variables (e.g. climatic).
Explanatory variables
Climatic variables of monthly precipitation and
monthly mean, minimum and maximum temperature at a
spatial resolution of 30 arc seconds (~1 × 1 km resolution)
were derived from WORLDCLIM (Hijmans et al. 2005)
(tile 18, 19, 28 and 29) which includes interpolation of
climatic records from global network of 4000 climate
stations, with time series of 1950–2000. Current
investigation doesn’t incorporate climatic information of
tile 110 and 210 of WORLDCLIM, hence geographic
space of East Asian countries like Taiwan, Japan etc. are
excluded for potential species distribution. Using DIVAGIS version 7.5 (Hijmans et al. 2001), tile data were
converted into 19 bioclimatic variables i.e., annual mean
temperature [Bio1], mean monthly temperature range
[Bio2], isothermality [Bio3], temperature seasonality
[Bio4], max temperature of warmest month [Bio5], min
temperature of coldest month [Bio6], temperature annual
range [Bio7], mean temperature of wettest quarter [Bio8],
mean temperature of driest quarter [Bio9], mean
temperature of warmest quarter [Bio10], mean temperature
Tmin avg
(˚C)
9.3
1.7
10.3
10.9
9.6
Tmax avg
(˚C)
16.1
12.8
19.4
20.4
19.5
PPT
(mm)
2268
1068
1634
1467
1784
Aspect
351.92 (N)
312.45 (NW)
308.95 (NW)
117.88 (SE)
56.97 (NE)
of coldest quarter [Bio11], annual precipitation [Bio12],
precipitation of wettest month [Bio13], precipitation of
driest month [Bio14], precipitation seasonality [Bio15],
precipitation of wettest quarter [Bio16], precipitation of
driest quarter [Bio17], precipitation of warmest quarter
[Bio18], precipitation of coldest quarter [Bio19] (Busby
1986; Nix 1986; Hijmans et al. 2005) in ESRI ASC format
for use in MaxEnt program. These bioclimatic variables
express spatial variation in annual means, seasonality and
extreme or limiting climatic factors and represent
biologically meaningful parameters for characterizing
species distributions (Saatchi et al. 2008) hence they were
used as explanatory variables.
Model building and evaluation
Presence-only data were used for model building.
However, in order to evaluate models on the basis of error
rates, absence details are needed. To overcome this, 25,000
random points throughout the study area were assumed as
absence ‘pseudo-absence’ (Zaniewski et al. 2002). Linear
regularization feature was used for model building; default
value of 1 for β regularization which gives consistent AUC
peaks (Phillips et al., 2004) was used; prevalence or the
probability of presence was taken to be 0.5.
Threshold-independent analysis
In the threshold-independent analysis, model
performance/strength (the power to discriminate between
sites where a species is present, versus those where it is
absent) was evaluated using the Area under the Curve
(AUC) of the Receiver Operating Characteristic (ROC)
curve (Fielding and Bell 1997; Elith and Burgman 2002).
The AUC statistic ranges from 0 to 1, where a score of 0.5
implies discrimination that is no better than random, and a
score of 1 indicates perfect discrimination (Fielding and
Bell 1997; Pearce and Ferrier 2000).
Explanatory variable importance
The importance of each bioclimatic factors explaining
the distribution of species was determined by Jackknife
analysis of the average gain with training and test data. 500
iterations were conducted for training algorithm, the
increase or decrease in regularized gain was added or
subtracted, respectively, to the input of the corresponding
variable, giving a heuristic estimate of bioclimatic variable
contribution for the model (Phillips et al. 2006).
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B I O D I V E R S IT A S 16 (2): 109-115, October 2015
Model output and Mapping
Logistic format was selected which categorizes
estimates of probability of occurrence or Index of Habitat
Suitability (IHS) within 0-1 (Anderson et al. 2003; Baldwin
2009). In DIVA-GIS, the logistic output grid was
reclassified and exported to raster. IHS regions < 0.5 were
discarded as random prediction, > 0.5 IHS were treated as
suitable areas of species distribution, ≥ 0.7 IHS were
treated as the core areas of species distribution (Kuemmerle
et al. 2010), and prioritized areas for conservation were
taken for IHS ≥0.9.
RESULTS AND DISCUSSION
The potential distribution of Monotropa uniflora in
South Asia have been found to be in the longitudinal range
of 68.94-117.8 decimal degrees and latitudinal range of
6.76-36.81 decimal degrees, covering 1,50,316 square Km
(Table 2). Suitable areas for the growth of the species (IHS
≥0.5) have been found in the countries of Tajikistan,
Afghanistan, Pakistan, India, Nepal, China, Bangladesh,
Myanmar, Srilanka, Laos and Vietnam. Whereas,
conservation areas that could be prioritized (IHS ≥0.9)
have been found in India, Nepal, China, Afghanistan, Sri
Lanka, Myanmar and Vietnam covering are of 39,968
square Km (Figure 3).
The model performed well with the training AUC value
above 0.9 (0.994) with the training omission rate of zero
for evaluation localities. The jackknife test of variable
importance showed the environmental variable with the
highest gain when used in isolation (which has the most
useful information by itself) to be temperature annual range
[Bio7] (maximum temperature of the warmest month minimum temperature of the coldest month). The
environmental variable that decreases the gain the most
when it is omitted is precipitation of driest quarter [Bio17],
which therefore appears to have the most information that
isn't present in the other variables.
Figure 3. Potential distribution of Monotropa uniflora in South Asia with graded Index of Habitat Suitability; warmer colour are
indicative of more suitable areas.
PRADHAN – Potential distribution of Monotropa uniflora in South Asia
Potential conservation areas for M. uniflora (IHS ≥0.9)
in India are predicted in Eastern Kathua in the state of
Jammu and Kashmir; Charua, Chamba, Dalhousie, Kangra,
Brahmaur, Dharmshala, Palampur, Baijnath, Kullu, Mandi,
Thunag, Chachyot, Karsog, Banjar, Nermand, Ani,
Rampur, Rohru, Kotkhai, Theog, Rajgarh, Chaupal,
Renuka, Shilla, Jubbal, Paonta in the state of Himachal
Pradesh; Chakrata, Purola, Rajgarhi, Dunda, Tehri
Garhwal, Dehradun, Northern Lansdown, Pauri, Pauri
Garhwal,
Devprayag,
Narendranagar,
Chamoli,
Karnaprayag, Ranikhet, Naini Tal, Almora, Champawat,
Bageshwar, Southern Joshimath in the state of Uttaranchal;
Darjeeling, Kurseong and Kalimpong subdivisions of
Darjeeling District in the state of West Bengal; South
Eastern Nongstoin area of West Khasi Hills, South Eastern
area of Shillong in the state of Meghalaya.
IHS ≥0.9 have been predicted in the Kuran Wa Munjan
and Wakhan areas of Afghanistan; Chitral of N.W.F.P. in
Pakistan; Central and Northern Matupi, Southern
Thlangtlang areas of Myanmar; Baitadi, Dadeldhura, Doti,
Western Darchula, Bajhang, Bajura, Kalikot, Northern
Jajarkot, South Eastern Jumla, Mugu, South Eastern
Humla, Western Dolpa, Northern Myagdi, Northern Rapti,
Southern Manang, Northern Kaski, Eastern Ilam of Nepal;
Elahera, Thamankadua, Dimbulagala of Sri Lanka; Eastern
Lai Chau and Western Lao Cai areas of Vietnam.
Suitable areas (IHS ≥0.5) in China are predicted mostly
in South-Central and South-Western China with IHS
ranging upto 0.80 in Yunnan, 0.81 in Sichuan and 0.94 in
Xizang provinces. Wenshan, Pingbiang Miao, Hekou Yao,
Maguan, Malipo, Jinping Yao, Miao and Dai areas of
Yunnan province are predicted in continuum of the
potential areas from Northern Vietnam; Zanda, Burang and
Zhongba area of Xizang province are in continuum of
Western and Central Himalaya; Kangding, Luding,
Hongya, Hanyuan, Meishan, Ebian Yi, Meigu, Mabian Yi,
Ganluo, Jinkouhe areas of Sichuan have been predicted as
potential habitats in central China.
Not only is the present prediction takes into account for
high altitude habitats, but also coastal vegetation with IHS
ranging upto 0.82 in Sagar Island, West Bengal, India.
Possibility of suitable habitats of the taxa in coastal areas
(IHS ≥0.5) which await prioritized inventory are presently
predicted along the coast of Western India, Western Ghats,
Bay of Bengal, Gujrat in India; Maungtaw, Buthidaung,
Sitwe, Chaungzon, Mudon, Paung, Moulmein areas of
Myanmar, Patuakhali, Noakhali, Cox’s Bazar, Bandarbon
areas of Bangladesh; Thua Thien - Hue, Nghe An, Thai
Binh, Hai Phong, Quang Ninh area of Vietnam; Hainan,
Haikou, Zhanjiang, Maoming, Yangjiang, Jiangmen,
Zhuhai, Zhangzhou areas of China.
Genus Monotropa was represented by Leake (1994) to
cover Indian, Nepalese and Chinese part of Western,
Central, Eastern Himalaya and coastal Indian Subcontinent
including other parts of India and Bangladesh and Japan in
the east as well. Some of the Monotropoideae reported
from Asia include Cheilotheca malayana from Japan
(Matsuda and Yamada 2003); M. uniflora L. from damp
deciduous or mixed forests of Anhui, Gansu, Guizhou,
113
Hubei, Jiangxi, Qinghai, Shaanxi, Shanxi, Sichuan, Xizang,
Yunnan, Zhejiang regions of China (100-1500 m),
Bangladesh, Bhutan, India, Korea, Myanmar, Nepal,
Sikkim (FOC 2014; Min et al. 2012); Japan (Bidartondo
and Bruns 2001); Murree hills in Pakistan (33.9043˚ N,
73.3942˚ E) (FOP 2014); Monotropa hypopitys L. from
India (Barik et al. 2009), Japan (Bidartondo and Bruns
2001); Monotropastrum humile (D. Don) Hara from
Himalaya (East Asia) to Japan (Kitamura et al. 1975;
Bidartondo and Bruns 2001), Yunnan, China. (Min et al.
2012), M. humile var. humile from Fengshan, Chi-Tou
Region of Taiwan, Mt. Mirokusan of Korea, Shioupuri,
Kathmandu, Nepal (Tsukaya et al. 2008); Monotropastrum
sciaphilum from Yunnan, China (Min et al. 2012);
Monotropastrum macrocarpum from Eastern Himalaya
(Maheshwari 1969) etc. The potential distribution of M.
uniflora is in line with the reported distribution of other
Monotropoids. Large tracts of Central and Western
Himalayas are predicted to be bioclimatically suitable for
M. uniflora, with disjunct distribution in various countries
reaffirming previous reports. However, many areas viz.
Zhejiang, Fujian, Guangdong and Hainan of China are
predicted in addition to earlier reports (E floras 2014a).
Previously, members of Monotropoideae were reported to
grow in coastal dunes in the Pacific North West USA with
prostrate Arctostaphylos mats (Leake 1994). Current
prediction of M. uniflora in many coastal areas of South
Asia therefore necessitates further insight into such habitat
of the species. Though temperature annual range have
singly important role to play in M. uniflora distribution,
precipitation of driest quarter may be important in density
dependent feedback of fungal biomass in the substratum of
mycorrhizal partner of M. uniflora (Krivtsov et al. 2006).
Monotropastrum humile and Cheilotheca malayana
which are distributed in South East Asia from the Himalaya
to Japan (Wallace 1975; Kitamura et al. 1975) are similar
to M. uniflora regarding specialization to fungal family
Russulaceae (Young et al. 2002; Matsuda and Yamada
2003). In fact, M. humile has been shown to be
phylogenetically close to M. uniflora, therefore current
potential distribution of M. uniflora could be functionally
predictive for the said species of Monotropoideae.
However, currently described potential distribution should
be carefully applied to derive cross-continent inference for
the species, as collections of M. uniflora from Asia, North
America, and Central America are indicated to be
molecularly diverged and phylogenetically distinct
(Bidartondo and Bruns 2001; Neyland and Hennigan
2004).
Monotropa uniflora has been reported in association
with Lithocarpus fenestrata (Roxb.) Rehd., from temperate
forests of Meghalaya and adjoining forests of Shillong
plateau, Nagaland, Mizo and Mikir Hills (1800-3500 m.)
during winter season from November to March (Singh et
al. 2002). Report of M. hypopitys L. from North Eastern
India (Barik et al. 2009) has indicated that this belt might
indeed be abode for the Genera. The projected range of
Monotropa in Northeast Hills (Meghalaya and MizoramManipur-Kachin forest zones) has also been proposed for
114
B I O D I V E R S IT A S 16 (1): 115-xxx, April 2015
prioritized conservation of amphibians and reptiles,
highlighting importance of said areas for conservation,
which are yet currently out of protected area network
(Pawar et al. 2007). One of the collection sites
(Jorepokhari) of M. uniflora in the present study is located
within plantation of Cryptomeria japonica (introduced
from Japan), while collection of M. humile by Matsuda et
al. (2011) from ecotone of natural forests of Cryptomeria
japonica in Tateyama, Japan indicates prevalence and
competence
of
Monotropoideae
in
allelopathic
environment.
The climatic relationship of Monotropoid distribution
predicted by earlier workers is in line with the present work
and the present study is able to justify the same by refining
spatial extent of their distribution in climatically suitable
areas. The congruence of the potential range of the M.
uniflora
and
other
Asian
Monotropoids
like
Monotropastrum and Cheilotheca, indicates that the
potential distribution of M. uniflora could act as a surrogate
for understanding the range of the Monotropoideae
(Ericaceae) in South Asia. The identified novel
distributional areas may add to the effective conservation
efforts of the subfamily in the region.
One of the prerequisite of realizing niche of a
mycoheterotrophic species is the range maps of many
ectomycorrhizal host trees, which unfortunately are of
limited availability in public domain of a developing
nation. Furthermore, modeling species distribution with
more occurrence records would be helpful in refining
spatial scale of suitable areas. Therefore the currently
predicted distribution of Monotropoideae in South Asia has
to be further tested with more occurrence records and
calibrated with the range maps of ectomycorrhizal fungi
and their host trees for consolidation.
ACKNOWLEDGEMENTS
Author would like to share his heartfelt gratitude to Dr.
K.C. Semwal (Mekelle University, Ethiopia) and S.
Suratna Sherpa, IFS (Divisional Forest Officer, Research
Division, Darjeeling, India) for providing inputs to the
occurrence records of Monotropa uniflora.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160202
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 116-120
Short Communication:
A new record of plant parasitic green algae, Cephaleuros diffusus
(Trentepohliaceae, Chlorophyta), on Acacia auriculiformis hosts in
Thailand
ANURAG SUNPAPAO♥, MUTIARA K. PITALOKA♥♥
Department of Pest Management, Faculty of Natural Resources, Prince of Songkla University. Hatyai, Songkhla, 90110 Thailand. Tel. +66-7428-6108,
Fax. +66-7428-8806, email: anurag.su@psu.ac.th, mutiarakp@gmail.com
Manuscript received: 12 January 2015. Revision accepted: 13 May 2015.
Abstract. Sunpapao A, Pitaloka MK. 2015. A new record of plant parasitic green algae, Cephaleuros diffusus (Trentepohliaceae,
Chlorophyta), on Acacia auriculiformis hosts in Thailand. Biodiversitas 16: 116-120. Cephaleuros diffusus algae were found to cause
leaf spot disease of the host Acacia auriculiformis. The identification was based on morphological and molecular properties. The
observed morphology identified the algae as C. diffusus. Transverse sections of the host leaves were used to quantify the disease severity
of the host in terms of a four-point necrosis index. The host’s lesions were subcuticular and subepidermal on the upper leaf surfaces. The
identification of the algae was confirmed from molecular analysis, and a phylogenetic tree distinguished our samples from other
Cephaleuros species. This is the first report of C. diffusus on Acacia auriculiformis in Thailand.
Key words: Cephaleuros, disease severity, morphology, necrosis, phylogenetic analysis
INTRODUCTION
The algae in the order Trenthepohliales are subaerial
green algae, which are widespread and of diverse forms
and habits in the tropical and the subtropical regions. This
order is represented by the single family Trenthepohliaceae, which includes six genera: Cephaleuros Kunze,
Phycopeltis Millardet, Physolinum Printz, Stomatochroon
Palm, Printzina Thompson & Wujek, and Trentepohlia
Mauritius. Among these, Cephaleuros is the most common
and widespread algae found on vascular plants as well as
on several economically important plants (Lopez-Bautista
et al. 2002).
In most cases Cephaleuros is mistaken for a fungus,
because the symptoms show erect, yellow to red filaments
and hairs like a fruiting body that is raised on the leaf
surface, which matches the characteristics of rust fungi
(Marlatt and Alfieri 1981). The identification of Cephaleuros
can be based on morphological characteristics, although
they do not provide definitive separation to distinguish
between the species. Cephaleuros grow in the subcuticular,
subepidermal and intramatical leaf tissues, and necrosis is
found beneath the alga thallus infection (Thompson and
Wujek 1997). Thompson and Wujek (1997) concluded that
the features most valuable in determining the species are:
(i) thallus growth habit, (ii) manner of bearing head cell or
sporangiate laterals, and (iii) the kinds of lesions produced.
To confirm identification based on such features, molecular
characterization is needed. However, pathogenicity tests of
Cephaleuros as a plant pathogen have not been completed,
probably due to the difficulties with producing zoospores
for reinoculation on synthetic media (Chapman and Good
1983; Holcomb et al. 1998).
Acacia (Acacia auriculiformis) is a woody plant of the
genus Acacia, originally from Australia, and spread to
several countries including Thailand (Midgley 2007). This
woody plant is now mostly found in commercial
plantations in Southeast Asia. In Thailand acacia is also
common in the wild, and is planted on the road sides to
provide shade. The tropical regions where acacia is found
are also in the spread range of Cephaleuros. The first report
of Cephaleuros on acacia (Marlatt and Alfieri 1981) did
not provide a specific species description. Furthermore,
various pathological and physiological problems associated
with a parasitic Cephaleuros infection still need
confirming. Altogether, there is little prior research on the
identification of Cephaleuros in Thailand. Therefore, the
aim of this research was to characterize the Cephaleuros
species on acacia in Thailand, based both on morphology
and on molecular properties, and to estimate the disease
severity in the acacia hosts using a necrosis index.
Materials and Methods
Sample collection and morphological identification
Algae: Cephaleuros sp., host: acacia (Acacia
auriculiformis); locality: Pest Management field, Prince of
Songkla University, Songkhla Thailand; collection: Dec 12,
2013, by Mutiara K. Pitaloka (PSU-A15). Macroscopic
features of the parasitic algae were measured under a stereo
microscope. The samples were prepared by hand
sectioning, a piece of thallus was peeled from the leaf to
observe the morphological characters of the thallus and
SUNPAPAO & PITALOKA – Cephaleuros diffusus in Thailand
sporangiophores. Fresh thalli which producing gametangia
or sporangia were placed on a drop of sterile water on a
glass slide to observe the gametes and zoospore. All of the
morphological characteristics were characterized under a
light compound microscope. The identification based on
morphological characteristics was referred to the
descriptions in a monograph by Thompson and Wujek
(1997). Specimens were deposited in the Culture Collection
of the Pest Management Department, Prince of Songkla
University, Thailand.
Algal culture and isolation
Algal thalli were isolated from the leaves and cultured
on Bold’s basal medium (BBM) (Bischoff and Bold 1963;
Andersen 2005). The isolation method followed Suto and
Ohtani (2011) with some adaptations. Leaves with fresh
thalli were washed under running water for five minutes
and soaked in water for one hour, wiped with cotton wool,
dipped in 70% ethanol, and rinsed with sterile water three
times. For isolation, a small pieced of thalli was peeled
from the leaf surface and placed on agar medium.
DNA extraction, PCR amplification and DNA sequencing
The filament-like colonies were harvested from BBM
and subjected to DNA extraction by the CTAB method.
The amplification of 18s nuclear small subunit rDNA (18s
nsu rDNA) by PCR used universal primers PNS1-forward
(5’ CCAAGCTTGAATTCGTAGTCATATGCTTGTC 3’)
(Hibbett 1996) and NS41-reverse (5’ CCCG
TGTTGAGTCAAATTA 3’). The PCR reaction mixture
had a final 50 L reaction volume containing 10 pmol of
each primer, 2x DreamTaq Green PCR Master Mix
(Thermo Scientific), and 50 ng of template DNA. The
amplification was carried out in a BIO-RAD T100TM
Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the
following program: one cycle of denaturation step for 3
min at 95C; 35 cycles of denaturation for 30 s at 95C,
annealing for 30 s at 50C, and extension for 1 min at
72C; followed by the final extension step of 10 min at
72C. The PCR products were visualized by agarose gel
electrophoresis. The PCR products of 18s rDNA portion
were sequenced at the Scientific Equipment Center, Prince
of Songkla University, Songkhla, Thailand, by automated
DNA sequencing with ABI Prism 377 (Applied
Biosystems, USA), using the same primers used in the PCR
reaction. The sequences obtained were compared with
known sequences of Cephaleuros available in GenBank
(The National Center of Biological Information, USA) by
using BLAST search. Phylogenetic and molecular
evolutionary analyses sequence query results were
conducted using CLUSTAL W and the software package
MEGA6.
Disease severity
A four-point necrosis index (Brooks 2004) was used to
assess the disease severity in the acacia hosts of the
parasitic algae. The index labeling criteria were: (0) No
necrosis, (1) superficial necrosis of the cell layer beneath
the algae thallus, with or without tissue hyperplasia, (2)
necrosis of > 1 cell layer but not full leaf thickness, with or
117
without tissue hyperplasia, erosion, or suberin formation,
and (3) necrosis from upper to lower leaf surface, including
“shot hole” symptoms.
Results and Discussion
To characterize the algal leaf spot disease causal
organism, infected samples were collected. The most
obvious symptom was orange tufts found on the upper leaf
surfaces, constituting thalli of the green alga. Lesions were
raised spots with no discoloration around the spot on the
upper leaf surface (Figures 1.A and 1.B). The epidermal
cells and palisade cells were necrotic, being brown to dark
brown beneath the thalli (Figures 1.G and 1.H). The thalli
growth was subcuticular and subepidermal on the upper
leaf surfaces, with open narrow filaments. The radial
extension of the thalli ranged from random to periodic
structures, with equal dichotomy of the marginal apical
cell. Filamentous-like ramuli were compacted, raising a
thin layer on the upper leaf surface of 1-5 mm diameter.
The filamentous cells ranged from short cylindrical to
irregularly shaped, 7.5-42.5 µm long and 5-17.5 µm wide
(Figure 1.D). The width to length ratio (W/L) was 1-6, and
as the irregular and open filamentous growth becomes
congested, the larger cells extended from the filament,
while the small cells raised the setae, sporangiophores, or
initial gamentangia. Some of the small cells produced a
small, dense, irregularly shaped expanse by close
dichotomies, which raised one to two celled setae. The
setae grew from short cylindrical filaments, one to five
celled, 16.5-280 µm long and 5-7.5 µm wide.
Gamentangia were spherical in shape, dark orange,
developed in a cluster of 3-7 cells, 12.5-32.5 µm long and
12.5-22.5 µm wide (Figure 1.C). Gametes were obbiconic
to spherical, 5-10 µm long and 5-7.5 µm wide, with
biflagella 15-20 long. Zoospores were obovoid and some
had a bullet shape, 10-16.25 µm long and 5-8.75 µm wide
with biflagella 15-25 µm long (Figure 1.F). The
sporangiophores were sparsely produced on the upper leaf
surface, being cylindrical, erect, solitary or in a tuff of three
or more, 250-440 µm long and 10-12.5 µm wide (Figure
1.E). Three to five of head cells developed terminally on
the sporangiophores bearing two to four sporangia laterally,
with both sporangia and their sulfutory cells. The sporangia
were spherical to elliptical, 12.5-27.5 µm long and 10-20
µm wide, yellow to dark orange. Based on the key species
referred to in the monograph of Thompson and Wujek
(1997), the fifteen collected algal samples were identified
as Cephaleuros diffusus.
A Cephaleuros species with a wide range of hosts is C.
virescens. In this study, we compared C. virescens to C.
diffusus, and their distinguishing characteristics are
summarized in Table 1. The thalli of C. diffusus formed
raised spots, whereas C. virescens forms circular discs
without gaps crenate or entire margin. The thallus growth
habit of C. diffusus was open and filamentous, whereas for
C. virescens this is pseudoparenchymatous. Both species
bear head cells terminally.
118
B I O D I V E R S IT A S 16 (2): 116-120, October 2015
B
A
D
C
E
F
H
G
Figure 1. A. Lesions with C. diffusus parasitic algae on an acacia leaf, B. Thalli on the leaf surface, C. Clusters of gametangia (arrows)
along with sporangiophores (Sp) and setae (Se), D. Filaments of C. diffusus with radial expansion, E. Sporangiophores terminally
bearing the head cell (Hc) with suffultory cells (Sc) and sporangia (S), F. Zoospores with biflagella (arrows), G. Transverse section of C.
diffusus showing thallus growth through to leaf tissue causing necrosis of cuticle and epidermis and of some palisade cells, H. Necrosis
on the leaf tissue and protective corky tissue (arrows), Cu: cuticle, Ep: epidermal, Pm: palisade mesophyll, Th: thallus, Ga: gametangia.
To estimate disease severity, assessment on a four-point
necrosis index was conducted. Ten transverse leaf sections
from different plants were observed. The thalli grew
subcuticularly and subepidermally through the leaf tissue,
and some thalli colonized beneath the cuticle. Necrosis
caused by Cephaleuros only occurred beneath of the thalli,
while some lesions showed tissue hyperplasia without any
necrosis. Due to these observed symptom characteristics,
the necrosis index of a leaf was scored as 2 with necrosis of
> 1 cell layer but not of full leaf thickness, with or without
tissue hyperplasia, erosion, or suberin formation.
SUNPAPAO & PITALOKA – Cephaleuros diffusus in Thailand
119
Figure 2. A phylogenetic tree showing genotypic comparison of a current Cephaleuros diffusus (PSU-A15) sample with highly
sequence-similar species from GeneBank, using segments of 18s rDNA. The bootstrap values are shown on the branches, and the
GenBank accession numbers are shown in parentheses. The comparison indicates that the PSU-A15 sample represents Cephaleuros, as a
species distinct from C. virescens and C. parasiticus.
Table 1. Characteristics for distinguishing between Cephaleuros diffusus (PSU-A15) and C. virescens, based on phenotype and
behavior.
Characters
Host
Thalli
Filamentous cells
Setae
Gametangia
Gametes
Sporangiophores
Sporangia
Zoospores
Lesions
Shape
Diameter (mm)
Growth habit
Shape
Length width (µm)
L/W ratio
Branching manner
Shape
Shape
Length width (µm)
Shape
Length width (µm)
Head cell placement
Shape
Length width (µm)
Shape
Length width (µm)
Discoloration
Cephaleuros diffusus
(PSU-A15)
Acacia auriculiformis
Raised spot
1-5
Open filamentous
Short cylindrical to irregular
7.5-42.5 5-17.5
1-6
Dichotomous
Cylindrical filament
Spherical
12.5-32.5 12.5-22.5
Obbiconic to spherical
250-440 10-12.5
Terminally
Spherical to elliptical
12.5-27.5 10-20
Obovoid
10-16.25 5-8.75
Absent
The Cephaleuros species are known as plant parasitic
algae on several woody plants, attacking leaves, branches,
stems, or fruit of the plant. The Cephaleuros on acacia has
been recorded in Florida (Marlatt and Alfieri 1981),
although while the samples were described as Cephaleuros
no taxonomy of the genus was reported. Those samples
were reported as Cephaleuros sp. on Acacia auriculiformis.
However, in this current study, we describe the species of
Cephaleuros virescens
(Suto and Ohtani 2009)
Magnolia grandiflora & Persea thunbergii
Circular disk, without gaps, crenate or entire margin
1-8
Pseudoparenchymatous
Long cylindrical
22-79 7-24
2.7-4.4
Equal dichotomy
1) Slender filament; 2) short bunt tipped filament
Spherical or elliptical
29-58 18-43
Ellipsoidal to fusiform
70-240 12-14
Terminally
Elliptical
17-27 15-21
Ellipsoidal to broad fusiform
7-11 4.5-6.5
Absent
Cephaleuros found on the leaves of acacia and identified as
C. diffusus.
Thompson and Wujek (1997) concluded that the
morphological features most valuable for determining the
species of Cephaleuros are the thallus growth habit, the
manner of bearing the head cell or sporangia laterally, and
the kind of lesion produced. However, they stated that the
dimensions of filamentous cell, gametangium, were not
always definitive, and the color of algae is unhelpful in
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B I O D I V E R S IT A S 16 (2): 116-120, October 2015
identification since it varies with exposure. This makes
phenotypic identification ambiguous because the
characteristics simply are not definitively distinctive in
general. Therefore, genotypic observations combined with
phylogenetic analysis are important for confidence in the
species identification. One colony sample of C. diffusus
was harvested from BBM and subjected to DNA
extraction, PCR amplification and DNA sequencing.
BLAST searches in GenBank indicated that the present
algae were similar to and grouped along with Cephaleuros.
The sequence of the PCR product was deposited in the
GenBank database with accession number AB972267. A
phylogenetic tree from neighbor joining analysis shows
that C. diffusus (PSU-A15) is closely related to
Cephaleuros, and well separated from C. virescens and C.
parasiticus (Figure 2). This corroborates the tentative
identification from morphological observations with a
genomics based identification that we consider definitive.
The current research is the first to definitively associate C.
diffusus with algal leaf spot on acacia in Thailand. This
report constitutes a new record of occurrence,
symptomatology,
morphology
and
molecular
characterization of C. diffusus associated with algal leaf
spot disease on acacia.
ACKNOWLEDGEMENTS
The authors would like to thank the Center of
Excellence in the Agricultural and Natural Resources
Biotechnology (CoE-ANRB), the Faculty of Natural
Resources, Prince of Songkla University, Songkla,
Thailand for funding and facilities. The copy-editing
service of RDO/PSU and the helpful comments of Dr.
Seppo Karrila are gratefully acknowledged.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160203
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 121-127
Short Communication:
Traditional dye yielding plants of Tripura, Northeast India
1
BISWAJIT SUTRADHAR1, DIPANKAR DEB2,♥, KOUSHIK MAJUMDAR1, B.K. DATTA1
Plant Taxonomy and Biodiversity Laboratory, Department of Botany, Tripura University (A Central University), Suryamaninagar 799022, Tripura, India
2
Agroforestry and Forest Ecology Laboratory, Department of Forestry and Biodiversity, Tripura University (A Central University), Suryamaninagar
799022, Tripura, India. Tel. 0381-237-9093, Fax. 0381-237-4807, e-mail: debdip23@gmail.com
Manuscript received 27 October 2014. Revision accepted: 14 May 2015.
Abstract. Sutradhar B, Deb D, Majumdar K, Datta BK. 2015. Traditional dye yielding plants of Tripura, Northeast India. Biodiversitas
16: 121-127. This present paper deals with the survey of traditional dye yielding plants, their ethnobotanical usage and cultural
practices by the different ethnic communities of Tripura. Field investigation was carried out in different villages and adjacent forest
pockets in South and West district of the State. The ethnobotanical information was collected based on semi-structured questioner,
personal interviews and group discussion among the major ethnic communities of Tripura. The study reports a checklist of 39 species of
dye yielding plants belonging to 35 genera and 26 families documented along with their vernacular name, habit, parts use. The active
coloring agents were also listed for each plant based on earlier reports. Natural dye yielding plants have immense significance in the
socio-economic and socio-cultural life of indigenous ethnic people and if we promote these products in a managed way then efforts
towards preservation of traditional knowledge and local biodiversity will be more fruitfully achieved.
Keywords: Dye yielding plants, indigenous knowledge, natural products, Tripura
INTRODUCTION
The relation between man and plants originated with
the prehistoric human civilization with early human
Neanderthals to modern human civilization. The plants are
used not only for maintaining the basic life sustaining
needs like food, fuel, shelter, but also for making clothes
and natural dye to fabric clothes (Das and Mondol 2012).
Natural dyes occupy an important place in human culture
and dye yielding plants were probably discovered early
through human curiosity, use, reuse and trials (Canon and
Cannon 2003; Dogan et al. 2008).
The first report of natural dye extraction from plant
sources dates back to around 2600 BC in China. The Indus
valley civilization at Mahenjo-Daro and Harappa (3500
BC) traces of dyeing garments with natural madder (Siva
2007). According to Dogan et al 2008, the use of natural
dye stuff in by Phoenicians, Hebrews and Venetians was
also started from the beginning of 13th century (Dogan et
al. 2008). Later the technology passed across regions and
cultures like Greeks, Romans, old world Africans,
Mexicans and was also evident in Peru (TRMIC 1991;
Dogan et al. 2008). According to Zohary and Hopf (1994),
dye yielding plants have been cultivated in southwest Asia
since ancient times and earliest persisting indication of
textile dyeing comes from an over 5,000-year-old piece of
cloth dyed with natural madder (Rubia cordifolia)
discovered at Mohenjo-Daro (Singh 2002; Mahanta and
Tiwari 2005; Das and Mondol 2012). Natural
dyes are colorants
derived
from plants, invertebrates
or minerals, while majority of natural dyes are extracted
from vegetable or plant sources like roots, berries, bark,
leaves, wood and other biological sources such as fungi and
lichens.
The use of natural dyes has been declining since the
discovery of synthetic dyes (Singh 2002). However, many
studies have found that synthetic dyes are harmful to
human health as well as environment (Kwok et al. 1999;
Singh and Singh 2002; Cristea et al. 2003; Mahanta and
Tiwari 2005; Seker et al. 2006). Natural dyes are
environment and skin friendly; for example, turmeric, the
brightest of naturally occurring yellow dye is a powerful
antiseptic and revitalizes the skin, while indigo yields a
cooling sensation. Hence, there exist a significant
justification for the application and promotion of natural
dyes as they are harmless both for human and environment
(Hartl and Vogl 2003; Kumar and Sinha 2004; Kim and
Park 2007). The present study is a comprehensive account
of dye yielding plants of Tripura and gathers information
on traditional knowledge system of extraction and use of
natural dyes by the local ethnic people. Consequently, the
study is an attempt to overcome the paucity of
documentation of dye plants and to create baseline
information on the natural sources of plant based dye.
The significant research on ethnobotany has created a
mass awareness among the scientists in India during the
last two decades (Rashid 2013). A number of publications
have come from different regions of India including the
north eastern states related to the traditional health care
practices, wild edible plants, ethnoveterinary plants and
fiber plants (Borthakur 1976; Tiwary et al. 1978, 1979;
Bhattacharjee et al. 1980; Baruah and Sharma 1984;
Mahanta and Tiwari 2005; Sawian et al. 2007; Jamir 2008;
Kar and Borthakur 2007). In north eastern states some
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B I O D I V E R S IT A S 16 (2): 121-127, Ocotober 2015
pioneer workers have made a brief note on dye plants
(Akimpou et al. 2005; Kar and Borthakur 2007).
Ethnobotanically Tripura has quiet lagged behind rest of
India (Deb et al. 2012). However, few workers have made
a concise note on traditional ethno medicine and wild
edible plants (Deb 1968; Singh 1997; Majumdar and Datta
2007; Das 2009; Roy 2010; Sen et al. 2011, Deb et al
2012). But, none of the workers have made a detailed note
of customary dye yielding plants of Tripura used by the
ethnic peoples of the state and therefore this is the first
such study.
Materials and Methods
Study area
Tripura is one of the seven states in the north eastern
part of India with a geographical area of 10, 491 sq Km. It
is located in the south west extreme corner of the north
eastern region, between 22° 57' to 24° 33' N latitude and
97° 10' to 92° 20' E longitude (Figure 1). The state is
situated between river valley of Myanmar and Bangladesh
on the north, west, south and southeast; in the east it has a
common boundary with states of Assam and Mizoram.
Tripura is a land locked state and its geographical limits
touch both national and international boundary lines.
International boundary with Bangladesh measures 839 km.
Its national boundary with Assam and Mizoram measures
53 km and 109 km respectively. The average minimum
temperature is 15°C and maximum temperature is 34°C.
The average annual rain fall in the state is 2024.4 mm.
Figure 1. Study site in the State of Tripura, India
The major part of the State is covered by dense forests
in which various ethnic groups live close to the forest and
are largely dependent on the wild biological resources for
their livelihood. The ethnic groups inhabiting different
areas of the state have indigenous knowledge systems and
have evolved methods for utilizing the vast plant resources
available. Floral diversity is the main source of raw
materials being used traditionally by the indigenous people
of Tripura state as food supplements and for fodder, fibers,
construction, handicrafts, beverages, coloring agents (dyes)
and more importantly in health care practices. Their
knowledge in utilizing these resources is characteristic and
differs from community to community.
Field survey
Field investigations were conducted during the year
2013-2014 as per well planned schedule and rich pocket of
tribal areas were visited for documentation of dye yielding
plants. Survey was done by standard procedure (Jain and
Rao 1977), through a questionnaire and group discussion
with the community heads, old people, women and also
village local market (Rao and Hazra 1994). The specimens
were collected from the adjacent forest area with the help
of local informant. The plant specimens were made into
herbarium following the standard herbarium techniques.
Herbarium specimens were identified with the help of Flora
of Tripura State (Deb 1983). The reference specimens were
deposited in the herbarium of Department of Botany,
Tripura University, India.
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160203
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 121-127
Table 1. Traditional dye yielding plants of Tripura, India
Scientific name
Family
Local name
Adhatoda vasica (L) Nees.
Acanthaceae
Basak
Alpinia galanga (L) Willd.
Zingiberaceae
Areca catechu Linn.
Arecaceae
Basella alba Linn.
Bauhinia variegata Linn.
Bauhinia purpurea Linn.
Basellaceae
Fabaceae
Fabaceae
Bixa orellana Linn.
Part used
Leaves
Dye color
Dye uses for
Active coloring agent
Yellow
Cotton
Telbanok juknai Root stalk
sam (Kok)
Supari, Gua
Seed
Yellowish
green
Red color
Cotton, wool
Adhatodic acid, carotein, lutolin, quercetin (Singh et al.
2011)
Galangin (Chudiwal et al. 2010)
Cotton
Gallotannic acid (Amudhan et al. 2012)
Fruit
Petal
Petal
Violet
Silk and cotton, food coloring
Light pink Cotton
Pink
Cotton and silk
Gomphrenin-I (Kumar et al. 2013)
Anthocyanin (Jash et al. 2014)
Chalcone, butein (Gokhle et al. 2004)
Bixaceae
Muifrai (Kok)
Kanchan (Beng)
Goondilata
(Kok)
Powassi
Seed
Wool and cotton
Bixin, orellin, beta-carotene (Siva 2007)
Butea monosperma (Lam.)
Taub.
Camellia sinensis Linn.
Celosia argentea Linn.
Clerodendrum philippinum
Schau.
Clitoria ternatea Linn.
Curcuma domestica Valeton.
Fabaceae
Jong-obi (Kok) Petal
Orange/
Red
Orange
Theaceae
Amaranthaceae
Verbenaceae
Cha
Sweet murga
Not known
Black
Red color
Green
Orange dye, which is used for coloring
the clothes and other decorative purposes
Fishing net, cotton, rope
Wool and cotton
Silk and cotton
Fabaceae
Zingiberaceae
Aparajita (Beng) Flower
Haldi
Rhizome
Blue
Yellow
Butein, butin, isobutrin, coreopsin, isocoreopsin
(Sindhia and Bairwa 2010)
Phenolic compound, flavonoids (Reto et al. 2007)
Betalains pigment (Patel et al. 2010)
Phenolic compound (Tiwary and Bharat 2008;
Venkatanarasimman et al. 2012)
Anthocyanin pigment ternatin (Kazuma et al. 2003)
Curcuminoids, curcumin (Revathy et al. 2011)
Curcuma zedoaria Roxb.
Zingiberaceae
Halka
Yellow
Cuscuta reflexa Roxb.
Diospyros peregrina Gürke.
Convolvulaceae
Ebenaceae
Jirai (Kokborok) Whole plant Yellow
Makur (Kok)
Fruit
Black
Duranta repens Linn.
Verbenaceae
Ban mehendi
Eclipta prostrata Linn.
Asteraceae
Hibiscus rosa sinensis Linn.
Malvaceae
Indigofera tinctoria Linn.
Fabaceae
Erythrina variegate Roxb.
Fabaceae
Leaves
Flower
Leaves
Rhizome
Wool, cotton, silk
Wool, silk and cotton and also a food
colorant
For the production of Abir powder used
in Holi
Silk and cotton
Fishing net, cotton
Curcumin, arabins and albuminoids (Hamdi et al. 2014)
Leaves/
Seed
Manathingsabup Leaves
hang (Kok)
Jaba
Flower
petal
Nil
Green crop
Green/
Orange
Black
Cuscutin, quercetin, coumarin (Ramya et al. 2010)
Triterpenes, B-sitosterol, betulin, gallic acid (Sinha and
Bansal 2008)
Silk, cotton and for palm staining of girls Durantosides, c-alkylated flavonoids (Ahmed et al.
2009)
Hair blackening and cotton
Phenols, coumarins, flavones (Lee et al. 2008)
Red
Wool and cotton
Blue
Silk, wool cotton
Mandar
(Beng)
Red
Wool and cotton
Flower
Anthocyanidins, isoflavanol, flavone (Kumar and Singh
2012)
Indigotin (Saraswathi et al. 2012)
Anthocyanin and betalains pigment (Subhashini et al.
2011)
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Lawsonia inermis Linn.
Lythraceae
Mehendi
Leaves
Mallotus philippensis (Lam.)
Muell. Arg.
Euphorbiaceae
Khurchub (Kok) Seed
Yellow red The dye obtained from macerated or
Lawsone (Musa and Gasmelseed 2012)
powdered leaves stains skin. Leaves also
used for coloring skins,
Leather, silk and wool..
Red
Silk, cotton, wool
Rottlerin, isorottlerin (Sharma and Varma 2011)
Melastomataceae Phutki, Ban
Stem/ Seed Red/ Black Cotton and wool
padam
Meliaceae
Ghora neem
Leaves
Blue
Cotton
Rubiaceae
Haldiruk (Kok) Root
Red
The dye is used for coloring cotton cloth
& other printing materials.
Musa acuminata Colla
Musaceae
Thailic
Pseudostem Black
The sap is used for coloring traditional
(Kok)
attire
Nyctanthes arbor-tristis Linn. Nyctanthaceae
Hengra
Flower tube Orange
The dye is used for coloring silk; also
(Kok)
useful in printing purposes.
Parkia javanica Merr.
Fabaceae
Kuki Tetai
Fruit peel Brown/
Silk and cotton
Black
Phyllanthus emblica Linn.
Phyllanthaceae
Amla
Bark/ fruit Black
Cotton, rope, silk
(Kok/ Beng)
Piper betle Linn.
Piperaceae
Fatwi (Kok)
Leaves
Red brown Cotton
Melastoma malabathricum
Linn.
Melia azedarach Linn.
Morinda tinctoria Roxb.
Psidium guajava Linn.
Myrtaceae
Gayam
Fruit
Yellow red Silk and cotton
Rubia cordifolia Linn.
Rubiaceae
Fruit
Red
Solanum nigrum Linn.
Solanaceae
Manjistha
(Beng)
Rummunta
(Halam)
Not known
Terminalia arjuna
(Roxb.) White & Arn.
Terminalia belerica Roxb.
Combretaceae
Arjun
Brown
black
Leaves/
Black &
young buds blue
Young
Red
leaves
Fruit
Brown
Combretaceae
Bahera
Fruit
Black
Silk and cotton
Terminalia chebula Retz.
Combretaceae
Bakhla (Kok)
Fruit
Black
Cotton
Strobilanthes cusia (Nees) O. Acanthaceae
Kuntze.
Tectona grandis (Linn. f)
Lamiaceae
Segun
Fruit
Wool, cotton and silk
Cotton
Not known
Catechin, quercetin (Sen and Batra 2012)
Morindone (Shanthi et al. 2012)
Not known
Nyctanthin, carotenoids (Sah and Verma 2012)
Carotenoids, flavonoids (Chanu et al. 2012)
Flavonoids,kaempferol, ellagic acid and gallic acid
(Habib-ur-Rehman et al. 2007)
Piperitol, piperbetol, eugenol, piperol (Pradhan et al.
2013)
Guajanoic acid,carotenoids, lectins, leucocyanidin
(Kamath et al. 2008)
Manjistin, Purpurin (Siddiqui et al. 2011)
Silk, cotton, wool
Gallic acid, catechin, caffeic acid, epicatechin, rutin, and
niringenin (Gogoi and Islam 2012)
Lupeol, betulin, lupenone, indigo, indirubin (Li et al.
1993)
Tectoleafquinone (Khera and Bhargava 2013)
Wool and cotton
Arjunic acid (Burapadaja and Bunchoo 1995)
Chebulaginic acid, gallic acid, ellagic acid (Meena et al.
2010)
Chebulinic acid (Lee et al. 2006)
Silk and cotton
B I O D I V E R S IT A S
SUTRADHAR et al. – Dye yielding plants of Tripura, India
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Pages: 121-127
Results and Discussion
The study elucidated the 39 dye yielding plants of
Tripura which were traditionally used by the tribal of
Tripura. Among these plants, many were common to all
communities, and their uses were also almost same. The
documented natural dye yielding plants with their
vernacular name, part used, dye color, specific uses and
coloring agent are listed in Table 1.
The listed plant species belong to 3 families of
monocotyledons (12% species) and 23 families of
dicotyledons (88% species). Among the monocotyledons,
Zingiberaceae is represented by 3 species while Musaceae
and Arecaceae are represented by single species each.
Among dicotyledons, 7 species (18%) are Fabaceae; 3
species (8%) Combretaceae followed by Acanthaceae,
Rubiaceae and Verbenaceae with 2 species (5%) each.
Dyes were produced from different parts of the plants i.e.
underground parts (root 5%, rhizome 5%), stem (4%), bark
(3%), leaf (26%), flower (21%), fruit (26%), seeds (13%)
or even whole plant (5%). More than 2 plant parts were
used in7% species of documented plants.
The important dyes extracted from roots or rhizome
include Alpinia galanga (L) Willd., Curcuma domestica
Valeton., Curcuma zedoaria Roxb., Morinda tinctoria
Roxb. Hort. Beng. Stem and bark is the important source of
the species Melastoma malabathricum Linn., Phyllanthus
emblica Linn. etc. Floral dyes includes Bauhinia variegata
Linn., Bauhinia purpurea Linn., Butea monosperma,
Celosia argentea Linn., Clitoria ternatea Linn., Hibiscus
rosa sinensis Linn., Erythrina stricta Roxb., Nyctanthes
arbor-tristis Linn. etc. Commonly used fruit dyes are
obtained from Basella alba Linn., Diospyros peregrina
Gürke., Parkia javanica Merr., Rubia cordifolia Linn.,
Solanum nigrum Linn., Terminalia belerica Roxb.,
Terminalia
chebula
Retz.,
Terminalia
citrina
(Gaertn.) Roxb but in some cases tender fruits of Psidium
guajava Linn. is used for black coour. The leaf dyes are
extracted from Adhatoda vasica (L) Nees., Camellia
sinensis Linn., Clerodendrum philippinum Schau., Duranta
repens Linn., Eclipta prostrata Linn., Lawsonia inermis
Linn., Melia azedarach Linn., Piper betle Linn.,
Strobilanthes cusia (Nees) O. Kuntze., Tectona grandis
(Linn. f) etc. Whole plant is used in case of Cuscuta reflexa
Roxb. Green crop of Indigofera tinctoria Linn. is used
while sap of pseudostem is used for Musa acuminata Colla.
Dyes extracted from the various plant parts are weak in
nature and their permanency varies from plant to plant and
traditional techniques of preparation. Use of multiple plants
parts in a particular ratio may increase the longevity of dye.
Sometimes special techniques viz. heat and cold treatment
may increase dye stability. The selection of plants also
depends on the color choice, product type and purpose.
This present study recorded 14 different colors from 12
different plant parts, where black color was yielded by
maximum number (26%) of plants. The ethnic
communities used their own customary approach to extract
and process the natural dye.
Ethnic communities used the dye yielding plants for
various day to day activities like coloring food and clothes,
making cosmetics and fashion jewelries. The dyes are
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DOI: 10.13057/biodiv/d160203
colored compound capable of being fixed to fabrics which
do not washout with soap and water or fade on exposure to
light. For dyeing purposes, either fresh extract or paste
form were best suited for the communities. Present record
of 38 species was very high compared to 10 species
reported from West Bengal (Das and Mondol 2012) and 15
species from Manipur (Akimpou 2005) and less than the
documentation of 47 species from Assam (Kar and
Borthakur 2007). However, documentation of 37 species
from Arunachal Pradesh and 33 species from Central India
(Tiwari and Bharat 2008) was quite similar to the present
study. Thus, the study presents the potential resources of
dye plants which have immense scope and small-scale
industrial prospects. But the indigenous knowledge of
processing and using the natural dyes from plants has to be
valued and at the same time has to be upgraded or value
added to incorporate with modern product generation.
In conclusion, the indigenous knowledge of extraction,
processing and practice of using natural dyes has declined
to a great extent among the new generation due to easy
availability of cheap synthetic dyes and modern attitude
and life style. It has been observed that the traditional
knowledge of dye making is now practiced by older people
only. Unfortunately, there is lack of fruitful attempts to
promote and conserve this immense treasure of traditional
knowledge. Typically, most commercial synthetic dyes are
attractive in color and are easy and cheap to processes. But,
material collection for preparation of natural dye is closely
dependent on resource availability, seasons and proper
traditional knowledge. Due to lack of precise technical
knowledge on the extraction and dyeing procedure, the
natural dyes could not compete with commercially
successful synthetic dyes. The natural dyes obtained from
plant sources are biodegradable and non-toxic. Indigenous
traditional knowledge on dye yielding plants is very
essential for community based development, future bio
prospecting and eco-friendly products.
ACKNOWLEDGEMENTS
The authors are grateful to Department of
Biotechnology, Government of India for the financial
assistance received through Bioresource network Project
(No. BT/29/NE/2011). They also indebted to Mantosh Roy
and Partha Das for field assistance. Special thanks are due
to all the ethnic informants of different communities for
their active participation and knowledge sharing during the
field investigations.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160204
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 128-131
Short Communication:
Genetic diversity of Rana (Pelophylax) ridibunda and Bufo
(Pseudepidalea) viridis in different populations
TAYEBEH MOSLEHI, MAJID MAHDIEH, ALIREZA SHAYESTEHFAR, SEYED MEHDI TALEBI♥
Department of Biology, Faculty of Science, Arak University, 38156-88138, Arak, Markazi, Iran. Tel.: +98-86-4173317.email:
seyedmehdi_talebi@yahoo.com
Manuscript received: 23 February 2015. Revision accepted: 8 June 2015.
Abstract. Moslehi T, Mahdieh M, Shayestehfar A, Talebi SM. 2015. Genetic diversity of Rana (Pelophylax) ridibunda and Bufo
(Pseudepidalea) viridis in different populations. Biodiversitas 16: 128-131. In present study, genetic diversity of two genus of Anura
order was investigated in Iran. For this reasons, four populations of Rana (Pelophylax) ridibunda (R1-R4) and three populations of Bufo
(Pseudepidalea) viridis (B1-B3) were selected from different regions of Arak province. Random Amplified Polymorphic DNA (RAPD)
marker was used for molecular investigation. Nine random primers were used that only four of them were suitable and revealing
different bands pattern for further analysis. A total of 69 scorable bands (loci) were found. The obtained data were analyzed using
MVSP software for clustering. The studied populations were separated from each other in the UPGMA tree as well as PCA and PCO
plots. In the mentioned diagrams the populations of B. (Pseudepidalea) viridis were closely together, while the populations of other
species were placed far from each other. The results of this study showed that high genetic variations were present among R. ridibunda
populations, but this condition was in contrast to B. viridis populations, where these populations were near each other.
Key words: Bufo, genetic variations, population, Rana, RAPD.
INTRODUCTION
Amphibians have important roles in food chains and are
important as indicators of ecosystem health (Welsh and
Ollivier 1998). The amphibians of Iran consists of 13
species and five subspecies of frogs and toads belonging to
five genera of four families, in addition eight species of
salamanders belonging to four genera of two families
(Rastegar-Pouyani et al. 2008). Despite of worthily studies
of them in Iran by foreign (Schmidt 1952; Blandford 1876;
Anderson 1957; Tuck 1974; Leviton et al. 1992) and native
researchers (Hezaveh et al. 2009; Rastegar-Pouyani et al.
2011: Baloutch and Kami 1995), the amphibians still needs
to study systematically.
Physiological, morphological and genetically variations
were seen in populations of species that occurred in
different habitat, these variations were created in response
to contrasting environmental conditions (Talebi et al.
2014). Random amplified polymorphic DNA (RAPD) is a
multilocus technique which allows obtaining information
on the general polymorphism of a genome. Low expense,
high efficiency in developing a large number of DNA
markers in a short time and requirement for less
sophisticated equipment, the simplicity and applicability,
requirement of small amount of DNA without the
requirement of cloning, sequencing or any other form of
the molecular characterization of the genome has made the
RAPD technique valuable (Bardakci 2001; Williams et al.
1990).
There was no previous study on genetic variation of
amphibians in Arak Province, therefore in this study RAPD
technique was used for investigation of genetic variation in
some populations of two species namely, Rana
(Pelophylax) ridibunda and Bufo (Pseuopidalea) viridis in
Arak Province, because these species are dominant ones
with more populations in this area. In the other hand, Arak
is one of the most important industrial areas that can affect
on migration of amphibians to other locations.
Materials and Methods
The materials for this study consisted of a total of 47
individuals selected from 7 different populations of Rana
(Pelophylax) ridibunda and Bufo (Pseuopidalea) viridis in
Arak province (Table 1). Individuals were sampled by
using triangular ring frame 30-mesh dip nets and manual
picking of substrates with field forceps, then samples
dissected in the field and the liver was removed. Genomic
DNA was extracted from 100% ethanol preserved liver
using a genomic DNA purification protocol (Sambrook and
Russel 2001). The isolated DNA was amplified using nine
primers that were bought from Sinaclon Company (Table
2). PCR reactions were performed in a volume of 25 μ L
containing 2.5 μ L PCR reaction buffer (10x), 1.5 μ L MgCl2
(25 mM), 2.5 μ L dNTPs (10 mM), 2 μ L primer (10μ M), 14
ng of genomic DNA and 0.3 μ L Taq polymerase (1 unit)
(Kohler et al. 2000). Amplification was done with a
programmable thermal cycler (Ependorf, AG, Hamburg,
Germany) under following conditions: 94°C for 1.5 min,
45 cycles of 94°C for 30 sec, 42°C for 1 min and 72°C for
MOSLEHI et al. – Genetic diversity of Rana ridibunda and Bufo viridis
2 min, followed by one cycle of 72ºC for 10 min. (Kohler
et al. 2000). The amplified fragments were separated on
2% agarose gels and stained with ethidium bromide and
photographed under UV light. Fragment sizes were
estimated by comparison with 1 kb DNA ladder. Bands
were distinguished by Labworks software. The data were
used to compute the genetic variation of species. MVSP
software was used for statistical analyses. A dendrogram
was constructed by the Unweighted Pair Group Method of
Arithmetical Average (UPGMA).
Results
In this study, the extracted DNA from four populations
of R. ridibunda (codes R1-R4) and three populations of B.
viridis (codes B1-B3) were amplified by using nine random
primers of RAPD technique. Between the nine tested
primers, we selected four that yielded clear and repeatable
band patterns. Other primers either failed to amplify any
fragment or only amplified a few fragments, making them
inappropriate for a study on variations. These four primers
provided a distinct pattern of amplified fragments. The
numbers of fragments were varied among the primers. In
total 69 scorable bands (loci) obtained that were varying
from 10 to 21 per primer. The primer ROTH-180-08
produced the highest number of bands in comparison with
the other primers. This primer amplified 21 bands with
molecular weight range from 550-10000 bp. Band at 1800
bp was unique band in B2 population from Delijan area.
Primer ROTH-180-02 and ROTH-180-04 produced 19
bands with molecular weight ranged from 900-10000 bp
and 700-8000 bp. In addition primer ROTH-180-05 created
10 polymorphic bands with molecular weight range from
700-7900 bp (Figure 1).
The studied populations of these species were separated
from each other in the PCA and PCO plots (Figures 2 and
3) as well as UPGMA tree (Figure 4). As shown in the
mentioned diagrams three populations of B. viridis were
close together while the populations of R. ridibunda were
placed separately. These showed that the genetic
similarities were present among populations of B. viridis.
But high genetics variations were found among R.
ridibunda populations, so that the R3 population was
placed in separate clade. Species studied were different in
genetic characters and separated from each other in PCA
and PCO plots as well as UPGMA tree (Figures 2 to 4).
129
Table 2. Used RAPD primers with their sequences
Primers
Sequences
ROTH-180-01
ROTH-180-02
ROTH-180-03
ROTH-180-04
ROTH-180-05
ROTH-180-06
ROTH-180-08
ROTH-180-09
ROTH-180-10
5'-GCACCCGACG-3'
5'-CGCCCAAGC-3'
5'-CCATGGCGCC-3'
5'-CGCCGATCC-3'
5'-ACCCCAGCCG-3'
5'-GCACGCCGGGA-3'
5'-CGCCCTCAGC-3'
5'-GCACGGTGGG-3'
5'-CGCCCTGGTC-3'
Figure 1. Band patterns on agarose gel (the right band related to
ladder).
Table 1. Sampled populations from different locations of Arak
province .
Pop.
code
R1
Species/
populations
Rana ridibunda
R2
R3
R4
B1
Rana ridibunda
Rana ridibunda
Rana ridibunda
Bufo viridis
B2
B3
Bufo viridis
Bufo viridis
Location
Markazi Province, north eastern of
Arak
Markazi Province, Delijan
Markazi Province, Shazand
Markazi Province, west of Arak
Markazi Province, north eastern of
Arak
Markazi Province, Delijan
Markazi Province, south eastern of
Arak
Figure 4. UPGMA tree based on the molecular data (details of
symbols were given in Table 1).
130
B I O D I V E R S IT A S 16 (2): 128-131, October 2015
PCO case scores (Euclidean)
4.6
R4
3.7
Axis 2
2.8
1.8
0.9
-1.8
R3
-0.9
0.9
1.8
2.8
3.7
4.6
B1
B3
B2
R1
R2
-0.9
-1.8
Axis 1
Figure 2. PCO plot of populations of R. ridibunda and B. viridis based on the RAPD data (details of symbols were given in Table 1)
PCA case scores
5.5
R4
4.4
Axis 2
3.3
2.2
1.1
R1
B3
B2
B1
-2.2
-1.1
R2
1.1
-1.1
2.2
3.3
4.4
5.5
R3
-2.2
Axis 1
Figure 3. PCA plot of populations of R. ridibunda and B. viridis based on the RAPD data (details of symbols were given in Table 1)
Discussion
In this study the intra-specific genomic variations in
four R. ridibunda and three B. viridis populations from
different regions of Arak province were analyzed by using
RAPD technique. The molecular technique RAPD analysis
is currently used to differentiate between the genomes of
the closely related species in order to determine the genetic
distance and genetic diversity (Williams et al. 1990;
Camargo et al. 2010). The studied populations of these
species were separated from each other in the UPGMA tree
as well as PCA and PCO plots. As shown in the mentioned
diagrams populations of B. viridis were closely together
while the populations of R. ridibunda were arranged
separately.
A low genetic variability was discovered among the
different B. viridis populations from various altitudes and
located at short geographical distances and high similarity
was seen between B1 and B3 populations. We believe that
MOSLEHI et al. – Genetic diversity of Rana ridibunda and Bufo viridis
the explanation for this result is the very short geographical
distances (10 km) among the populations, together with the
fact that all of the habitats were of the unpredictable type
(rain pools), causing the toads to adapt to the wide
ecological variations.
While, high genetic variations were found among R.
ridibunda populations, so that the R3 population was
placed in separate clade. R3 population from Shazand area
was almost different genetically from others that may be
due to presence of mountains between this area and Arak,
different climate and lack of migration make it different
from others populations. So it’s a very important point to
say that ecological and biological conditions are one of
important reasons for genetic differentiation. The results of
the present study are very similar to those of Degani and
Kaplan (1999), who (s) studied the genetic variation of
salamanders from different habitats, using RAPD. They
discovered a very low genetic variation between two
populations from semi-arid habitats (band sharing was
94%), and in contrast, a high genetic variation between
populations from semi-arid and humid habitats (85-86%
band sharing). Their results agree with the present study, in
which ecological conditions affected genetic variation.
Because one of the main ecological factors is altitude,
which differed between populations and this factor
influences other ecological factors such as wind,
temperature as well as moisture. In study on Triturus
vittatus vittatus by Pearlson and Degani (2007) also obtain
similar result about relation between DNA variation and
different site populations.
RAPD genetic analysis showed that there is molecular
variation between different populations of these species in
Arak province, because these samples clustered separately.
These conditions hold true for many other species of
anurans worldwide (Driscoll 1998; Baptista 2001; Marsh
and Trenham 2001; Palo et al. 2004). Although local
populations tend to be slightly different, this population
differentiation is not strongly structured in geographic
space, and the UPGMA tree indicated only a slight
significant and relatively weak spatial structure at short
geographic distances so it may be difficult to find a general
explanation for genetic divergence among local populations
across geographic space (Sokal et al. 1986). It is important
to stress that even some of very close local populations
might tend to be independent in respect to genetic
variability. Thus, beyond micro evolutionary and
ecological processes, human effects may help explaining
patterns of genetic distances in this species (Telles et al.
2007).
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160205
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 132-138
Short Communication:
Genetic diversity of patchouli cultivated in Bali as detected using ISSR
and RAPD markers
1
MADE PHARMAWATI1,♥, I PUTU CANDRA2,♥♥
Biology Department, Faculty of Mathematics and Natural Sciences, Udayana University. Kampus Bukit Jimbaran, Badung 80361, Bali, Indonesia.
Tel./Fax. +62361703137, email: pharmawati@hotmail.com
2
Faculty of Agriculture, Warmadewa University, Jl. Terompong No 24 Tanjung Bungkak, Denpasar 80235, Bali, Indonesia
Manuscript received: 2 April 2015. Revision accepted: 9 June 2015.
Abstract. Pharmawati M, Candra IP. 2015. Genetic diversity of patchouli cultivated in Bali as detected using ISSR and RAPD markers.
Biodiversitas 16: 132-138. Patchouli is a bushy herb that has strong scent. Patchouli's oil is extracted from patchouli leaves and used as
perfumes, incense and traditional medicines. Centre of patchouli cultivation in Bali is in Badung District, however it is also grown in
other area such as Buleleng District. The patchouli plant from Aceh ((Pogostemon cablin (Blanco) Benth) is considered as a better plant
species due to its high quality oil. Java patchouli (Pogostemon heyneanus) has lower quality. Molecular marker was used to detect
diversity of patchouli grown in Bali. Leave samples of patchouli from 12 areas in Badung and Buleleng District were collected. The
areas included Lemukih, Wanagiri, Pupuan, Belok, Mekarsari, Nungnung, Plaga, Sidan, Mengwi, Lukluk, Abiansemal and Jegu.
Patchouli samples of Lhokseumawe, Tapak Tuan, Sidikalang and Java were obtained from Research Institute of Spices and Medicinal
Plants, Bogor, Indonesia. DNA was extracted using CTAB buffer. Seven ISSR primers and five RAPD primers produced scorable bands
and used for diversity and cluster analyses. The dendrogram showed that patchoulis grown in Bali are group together, separated from
Java patchouli. This support observation based on leaf morphology, that they belong to Aceh patchouli. The patchouli grown in Bali
showed low genetic diversity with Nei and Li’s similarity in the range of 0.857 to 0.989.
Keywords: Bali, genetic diversity, ISSR, patchouli, RAPD
INTRODUCTION
Patchouli (Pogostemon cablin Benth.) belongs to mint
family, Lamiaceae. Patchouli is believed to be a native of
the Philippines; however it also grows wildly in Indonesia,
Malaysia and Singapore (Ramya et al. 2013). Patchouli is
also cultivated in India (Kumara and Anuradha 2011). The
main product of patchouli plant is essential oil known as
patchouli oil. This oil is used as perfume, scents and
medicine (Kalra et al. 2006; Yang et al. 2013). Besides
that, patchouli oil can be used as insect repellant (Maia and
Moore 2011).
The patchouli cultivation in Indonesia was initially
developed in Aceh, North Sumatra, West Sumatra and
Bengkulu (Haryudin and Maslahah 2011). Cultivation of
patchouli then developed to other areas of Indonesia
including Java, Kalimantan and Bali (Nuryani 2006). The
productivity of patchouli in Indonesia is 87.20 kg/ha, this is
a relatively low production mean (Setiawan and Rosman
2013). The low productivity of patchouli is caused by
traditional cultivation technology, pest and disease attack,
and the use of unidentified patchouli varieties or the used
of non superior varieties (Setiawan and Rosman 2013).
Patchouli is best grown in humid climate condition, up to
an altitude of 800-1000 m above sea level (m asl.) (Ramya
et al. 2013).
In Indonesia, there are three patchouli species which can
be differentiated by morphological characters, oil quality and
resistance to biotic and abiotic stress. The three species are P.
cablin Benth. or P. patchouli Pellet van Suavis Hook also
known as aceh patchouli, P. heyneatus Benth. also known
as java patchouli and P. hortensis Becker also known as
soap patchouli. Three superior quality of patchouli varieties
(Tapak Tuan, Lhokseumawe and Sidikalang) have been
resealed by Indonesia Research Institute of Spices and
Medicinal Plants, Bogor, Indonesia. The names of those
varieties are based on their provenance. Tapak Tuan is
superior for its production; Lhokseumawe has high oil
content, while Sidikalang is tolerant to bacterial wilt and
nematode (Nuryani 2006).
Patchouli has also been cultivated in Bali. It is
considered that Bali will become a new production center
for patchouli (Setiawan and Rosman 2013). The cultivation
of patchouli in Bali is driven by rapid development of spa
industries, where there is high demand of patchouli oil. The
purity and identity of patchouli varieties is important for
germplasm characterization (Kumara and Anuradha 2011).
Common molecular markers used for detection of genetic
diversity are RAPD (Random Amplified Polymorphism
DNA) and ISSR (Inter Simple Sequence Repeat). The
RAPD marker successfully identified variation of Jatropha
curcas in India (Ikbal et al. 2010), assess diversity of
medicinal plant Catharanthus roseus (Shaw et al. 2009) as
well as determine genetic diversity of spice plant Ocimum
basilicum (Ibrahim et al. 2013). The ISSR marker was
shown to be able to detect genetic variation between Allium
PARMAWATI & CHANDRA – Patchouli’s genetic diversity cultivated in Bali
species (Son et al. 2012), and Capsicum species (Thul et al.
2012).
This study aims to evaluate genetic diversity of
patchouli grown in Bali using PCR-RAPD and PCR-ISSR.
Patchouli cablin does not flower (Bhaskar and
Vasanthakumar 2000), thus it is propagated using stem
cutting or in vitro multiplication (Swamy et al. 2010). The
diversity may have resulted from a long adaptation time to
climatic condition and cultivation system (Chacko 2009).
Information on genetic diversity of patchouli cultivated in
Bali will be useful for breeding of patchouli. Patchouli
plant with good adaptation to environment can be used as
genetic source in patchouli breeding.
Materials and Methods
Sample collection
Leaves of patchouli were collected from cultivations
areas in Bali. Leaf was collected from one plant in each
cultivation area. The areas included Lemukih, Wanagiri,
Pupuan and Belok which are highland areas with altitude of
133
≥1000 m asl, Mekarsari, Nungnung, Plaga and Sidan with
altitude of 500-1000 m asl, Mengwi, Lukluk, Abiansemal
and Jegu wich are lowland areas with altitude of ≤500 m
asl. (Figure 1). As comparisons, Aceh patchouli varieties
(Sidikalang, Lhokseumawe, Tapak Tuan) and Java
patchouli were used and collected from Research Institute
of Spices and Medicinal Plants, Bogor, Indonesia. Voucher
specimens of all samples were deposited at Herbarium
Biology Udayana (HBU) at Biology Department, Faculty
of Mathematics and Natural Sciences, Udayana University,
Denpasar, Bali, indonesia.
DNA extraction
DNA was extracted according to Khanuja et al. (1999).
Leaf samples (0.1 g) were ground using mortar and pestle,
and 600 µL of extraction buffer (2% w/v CTAB, 1.4 M
NaCl, 50 mM EDTA, 100 mM Tris-HCl (pH 8), 2% (v/v)
2-mercaptoethanol) was added and incubated at 60°C for 1
h. After that, 500 µL chloroform : isoamylalcohol (24:1)
was added and vortexed, and centrifuged at 12,000 rpm for
12
11
10
9
8
6
5
7
4
3
2
1
Figure 1. Study sites of sampling collection in Bali Island ( ). Patchouli leaves were collected from 1. Lukluk, 2. Abiansemal, 3.
Mengwi, 4. Jegu, 5. Nungnung, 6. Plaga, 7. Belok, 8. Sidan, 9. Pupuan, 10. Mekarsari, 11. Wanagiri, and 12. Lemukih. Insert is map of
Indonesia. Map of Bali was modified from Lansing and Fox (2011).
134
B I O D I V E R S IT A S
16 (2): 132-138, October 2015
10 min. The supernatant was transferred to a new
microtube and 250 µL of 5 M NaCl was added and mixed.
Then, 0.6 volume of cold isopropanol was added and
incubated at -20ºC for 1 h. Following incubation, the
sample was centrifuge at 12,000 rpm for 10 mins. Pellet
was washed with 500 µL 70% ethanol and centrifuged at
12,000 rpm for 3 mins. Pellet was air dried for 15 mins and
dissolved in 500 µL buffer TE. RNase A (5 µL) was added
and incubated at 37 °C for 30 mins. Then same volume of
chloroform: isoamylalcohol (24:1) was added and
centrifuged. The supernatant was transferred to a new tube
and 2 volume of cold ethanol was added. Sample was then
centrifuged and pellet was washed with 70% etanol. Pellet
was air dried and 100 µL ddH2O was added.
PCR-ISSR and PCR-RAPD
The PCR-ISSR and PCR-RAPD were conducted in 25µL PCR reaction. The mixtures contained 20 ng DNA, 1.5
mM MgCl2, 1·x PCR buffer (MoBio), 0.5 µM primer, 200
µM of each dNTP (Promega) and 1 unit of Taq DNA
polymerase (MoBio). To reduce background amplification,
5% (final concentration) glycerol was added (Grunenwald
2003). Amplifications were carried out using a
thermocycler (MyGenie) with an initial denaturation/
activation step of 4 min at 95oC, followed by 40 cycles of 1
mins at 94oC, 1 min at annealing temperature (36oC for
RAPD, 48oC and 50oC for ISSR) and 2 min extension at
72oC. The 40 cycles were followed by a final extension for
10 min at 72oC. Twelve ISSR primers (University of
Bristish Columbia, Canada) and seven RAPD primers
(Operon Technology, USA and University of British
Columbia, Canada) were tested.
The amplification products were analysed using 1.8%
agarose electrophoresis in 1 x TAE buffer for 50 min at
100 V, and then stained with ethidium bromide at final
concentration of 0.5 μ g/mL for 30 min. As a size marker, 1
kb DNA ladder (Fermentas) or 100bp ladder (GeneAid)
was included in the gel. Visualization was done using
GelDoc UV transilliminator.
Data analysis
The presence of the band was scored 1 and the absent of
band was scored 0. The genetic differences were calculated
using Nei and Li’s coefficient of similarity by Unpaired
Group Method of Average (UPGMA) using Multi-Variate
Statistical Package (MVSP) software version 3.1. and a
dendrogram was developed using the same software.
Results
Patchouli is now grown extensively in Bali. Based on
leaf morphology, the patchouli grown in Bali is similar to
Aceh patchouli. Patchouli samples collected from 12
cultivation areas have leaf shape that is categorized as
ovate with pointed apex and double serrate margin. Leaf is
green-purple color with smooth surface. These characters
are main leaf characters of Aceh patchouli (P. cablin)
(Fatriana 2011). The leaf length varied from 5.75-8.25 cm
and leaf width varied from 4.70-6.75 cm. In shaded area,
the length of leaf is in the range of 9.15-13.70 cm and leaf
width is in the range of 4.70-6.75 cm.
Molecular testing of patchouli grown in Bali using
ISSR and RAPD markers were conducted to evaluate their
genetic diversity. The amplification products of PCR-ISSR
using primer UBC 855 is shown in Figure 2, while
products of PCR-RAPD using OPB 04 primer are
presented in Fugure 3. Among the 12 ISSR primers tested,
seven primers resulted in 32 clear and scorable bands,
while other primers resulted in smear pattern. From seven
scorable primers, 3 primers (UBC 808, UBC 820 dan UBC
888) resulted in monomorphic bands. Among the seven
RAPD primers used, five primers produced 32 scorable
bands, and all bands were polymorphic. The number of
bands, the size of PCR products in each primer as well as
the percentage of polymorphism is presented in Table 1
and 2.
Using PCR-ISSR only, low polymorphism was detected
(Table 1). Therefore, to analyzed genetic diversity of
patchouli cultivated in Bali, combined RAPD and ISSR
markers were used. Based on PCR-ISSR and PCR-RAPD
profiles, the Nei and Li’s similarity coefficient was
performed to group the patchouli samples. The Nei and
Li’s similarities of patchouli grown in Bali are in the range
of 0.857 to 0.989. The matrix of Nei and Li’s coefficient of
patchouli cultivated in Bali as well as Aceh and Java
patchouli is shown in Table 3.
Cluster analysis showed that Java patchouli is separated
from other patchoulis and form group A, while samples of
patchouli grown in Bali and samples of Aceh patchouli are
in group B (Figure 3). Based on Nei and Li’s coefficient of
similarity of 0.92, group B can be further divided into
subgroup B1, B2, B3 and B4. Subgroup B1 consists of
patchouli sample from Lukluk. Subgroup B2 consists of
samples from Belok and Lemukih, and subgroup B3
consists of samples from Jegu and Wanagiri. Sample from
Sidan, Plaga, Mertasari, Tapak Tuan, Pupuan, Sidikalang,
Abiansemal, Mengwi Nungnung, Abiansemal and
Lhokseumawe are clustered into group B4. When similarity
coefficient of 0.94% was used as a baseline, samples
become more separated and form smaller groups.
Discussion
Leaf morphological characters of patchouli cultivated in
Bali were similar to those of Aceh patchouli. However, the
size of leaf differed when they grow in open area and in
shaded area. This indicates that morphological characters
are influenced by environment as stated by Sugimura et al.
(2006) which resulted in difficulty of botanical classification.
Therefore it is hard to determine whether patchoulis
cultivated in Bali belong to the three high quality
patchoulis (Lhokseumawe, Sidikalang and Tapak Tuan).
Both using PCR-ISSR and PCR-RAPD, Java patchouli
showed different banding patterns as compared to other
patchoulis tested. For examples, using primer UBC 855,
band of 320 bp was present in Java patchouli, while using
primer OPB 04, bands of 510 bp and 790 bp were present
in Java patchouli but absent in other patchouli samples.
Java patchouli is a different species than Aceh patchouli.
These results showed that both PCR-ISSR and PCR-RAPD
were able to differentiate the two species In the dendrogram,
Java patchouli was separated from other.
135
1kb ladder
Jegu
Lukluk
Abiansemal
Sidan
Mengwi
Plaga
Nungnung
Mekarsari
Belok
Pupuan
Wanagiri
Lemukih
Java*
Tapak*
*Tuan*
Sidikalang*
Lhoksemawe*
PARMAWATI & CHANDRA – Patchouli’s genetic diversity cultivated in Bali
1000bp
500bp
250 bp
100bp ladder
Lukluk
Abiansemal
Jegu
Mengwi
Plaga
Sidan
Nungnung
Mekarsari
Belok
Wanagiri
Pupuan
Lemukih
Java*
Tapak Tuan*
Sidikalang*
Lhoksemawe*
Figure 2. Amplification products of patchouli using UBC 855. Patchouli samples from Research Institute of Spices and Medicinal
Plants, Bogor (*) and samples from cultivation areas in Bali were shown across the top of figure
1500bp
1000bp
500bp
Figure 3. Amplification products of patchouli using OPB 04. Patchouli samples from Research Institute of Spices and Medicinal Plants,
Bogor (*) and samples from cultivation areas in Bali were shown across the top of figure
Table 1. ISSR primers, numbers and sizes of bands amplified and the polymorphisms produced
Primer name
Primer sequence
Bands size (bp)
Number of band
UBC 810
UBC 848
UBC 855
UBC 891
Average
(GA)8T
(CA)8 RG
(AC)8YT
HVH(TG)7
505-792
500-1508
352-810
436-1000
3
9
4
6
5.5
Number of
polymorphic band
1
4
2
3
2.5
Polymorphism (%)
33
44.4
50
50
44.35
Table 2. RAPD primers, numbers and sizes of bands amplified and the polymorphisms produced
Primer name
Primer sequence
Bands size (bp)
Number of band
OPA 04
OPB 04
OPD 11
OPD 14
UBC 250
Average
AATCGGGCTG
GGACTGGAGT
GTAGACCCGT
TCCGCTCTGG
CGACAGTCCC
150-750
500 -1305
178-1030
245-2140
350-895
6
9
5
8
4
6.4
Number of polymorphic
band
5
6
4
6
3
4.8
Polymorphism (%)
83.3
66.7
80
75
75
76
136
B I O D I V E R S IT A S
16 (2): 132-138, October 2015
Table 3. Matrix of Nei and Li’s Coefficients of patchouli cultivated in Bali as well as Aceh and Java patchouli based on PCR-ISSR and
PCR-RAPD
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1
1.000
2
0.943 1.000
3
0.944 0.909 1.000
4
0.649 0.658 0.667 1.000
5
0.944 0.932 0.889 0.615 1.000
6
0.925 0.891 0.936 0.659 0.894 1.000
7
0.978 0.944 0.954 0.633 0.945 0.947 1.000
8
0.941 0.881 0.884 0.622 0.930 0.867 0.920 1.000
9
0.943 0.930 0.909 0.658 0.909 0.913 0.944 0.881 1.000
10
0.967 0.956 0.935 0.625 0.957 0.938 0.989 0.909 0.956 1.000
11
0.943 0.953 0.886 0.658 0.932 0.913 0.944 0.881 0.977 0.956 1.000
12
0.941 0.929 0.907 0.676 0.884 0.911 0.943 0.878 0.952 0.932 0.929 1.000
13
0.957 0.923 0.946 0.617 0.925 0.928 0.957 0.899 0.945 0.968 0.923 0.921 1.000
14
0.920 0.884 0.909 0.605 0.886 0.870 0.899 0.905 0.860 0.889 0.860 0.857 0.923 1.000
15
0.955 0.897 0.921 0.649 0.899 0.903 0.956 0.894 0.920 0.945 0.897 0.941 0.957 0.920 1.000
16
0.925 0.870 0.936 0.683 0.872 0.939 0.926 0.876 0.891 0.917 0.870 0.889 0.928 0.891 0.925 1.000
Note: 1 = Lhokseumawe, 2 = Tapak Tuan, 3 = Sidikalang, 4 = Java, 5 = Lemukih, 6 = Wanagiri, 7 = Pupuan, 8 = Belok, 9 = Mekarsari,
10 = Nungnung, 11 = Plaga, 12 = Sidan, 13 = Mengwi, 14 = Lukluk, 15 = Abiansemal, 16 = Jegu
Figure 3. Dendrogram of patchouli cultivated in Bali and three superior patchouli varieties based on PCR-ISSR and PCR-RAPD
analyses
patchouli samples. This result supported leaf morphological
observations which found that patchouli grown in Bali has
similar characteristics with Aceh patchouli.
Comparing PCR-ISSR and PCR-RAPD, this study
found that PCR-RAPD resulted in more polymorphism.
This is in discordance with several reports which stated the
robustness and the highest ability of ISSR to detect
variations compared to RAPD (Datta et al. 2010; Yadav et
al. 2014). High efficiency of RAPD in detecting
polymorphism in plant was reported (Sadeghi and
Cheghamirza 2012). According to Sadeghi and
Cheghamirza (2012), this may due to the use of primer
with high GC content that resulted in higher stability. This
is in agreement with our finding.
The Nei and Li’s similarities of patchouli cultivated in
Bali were in the range 0.857 to 0.989. This indicates that
the patchouli grown in Bali had low genetic diversity. A
study of patchouli genetic diversity of Johor variety
(Malaysia), Singapore variety (Singapore) and Bangalore
variety (India) using 10 RAPD primers found that the
varieties have significant diversity (Kumara and Anuradha,
2011). According Kumara and Anuradha (2011), the
varieties studied in their research may have originated from
different regions.
PARMAWATI & CHANDRA – Patchouli’s genetic diversity cultivated in Bali
Samples of patchouli from Bali did not group into the
altitude of their growing locations, for example Mengwi,
Lukluk, Abiansemal and Jegu are lowland areas with
altitude of ≤500 m asl., however, the patchouli samples
from those areas were scattered in the dendrogram.
Furthermore, samples from nearby areas did not cluster
together. In our study, sample from Belok was far
separated with sample from Sidan and Plaga, while those
areas are geographically close to one another. A study
reported by Wu et al. (2011), using RAPD marker, showed
that patchouli from adjacent areas was classified together.
According to Wu et al. (2011), this might have been due to
the possibility that the chosen populations lived in the
regions for rather a long time and were seldom
transplanted. They further explained that patchouli was
introduced to China for long time. However, this is not the
case for patchouli in Bali. The commercial cultivation of
patchouli in Bali only started in 2006 (Bali Post, 8
December 2007).
The diversity of patchouli grown in Bali could be
because of different source of seedling or different
varieties. The patchouli samples from Plaga and Mekarsari
are genetically closer to Tapak Tuan, while patchouli from
Nungnung and Pupuan are closer to Lhokseumawe.
Personal communication with patchouli farmers at each
location obtained information that seedlings of patchouli
grown at Pupuan, Nungnung, Mekar Sari, Mengwi, Sidan
and Abiansemal came from Yogyakarta. Patchouli
seedlings grown at Lemukih, Plaga, Wanagiri and Jegu
came from Bogor, while seedling of patchouli at Belok and
Lukluk were from East Java. The diversity could also be
due to natural mutation caused by biotic or abiotic stress at
plant nursery. Aceh patchouli does not flower, therefore the
diversity does not come from hybridization (Swamy et al.
2010).
This study revealed that patchouli cultivated in Bali is
Aceh patchouli which is genetically similar to those grown
in plantation areas. This study indicates low genetic
variation of patchouli in Bali. Genetic improvement of
patchouli requires wide variation of germplasm which can
be obtained through induced mutation (Rekha et al. 2009)
or somaclonal variation (Swamy et al. 2010; Ravindra et al.
2012).
ACKNOWLEDGEMENTS
We thank Research Institute of Spices and Medicinal
Plants, Bogor, Indonesia, for providing Lhokseumawe,
Sidikalang and Tapak Tuan patchouli varieties leaf
samples.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160206
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 139-144
Short Communication:
Population genetic structure in medicinal plant Lallemantia iberica
(Lamiaceae)
FAHIMEH KOOHDAR1,♥, MASOUD SHEIDAI1, SEYYED MEHDI TALEBI2, ZAHRA NOORMOHAMMADI3
1
Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran, 1983963113. Tel: +98 21 29902111, ♥email: msheidai@yahoo.com
2
Department of biology, faculity of sciences, Arak University, Arak, 38156-8-8349 Iran.
3
Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Manuscript received: 2 April 2015. Revision accepted: 11 June 2015.
Abstract. Koohdar F, Sheidai M, Talebi SM, Noormohammadi Z. 2015. Population genetic structure in medicinal plant Lallemantia
iberica (Lamiaceae). Biodiversitas 16: 139-144. Lallemantia iberica (Bieb.) Fischer and C.A. Meyer (sin. Dracocephalum ibericumM.
Bieb.) also named Dragon’s head” is an annual plant cultivated for its seeds that contain about 30% -38% drying oil (siccative oil). Its
seed oil is used in foods, dye and varnish industry. L. iberica seeds have traditional uses as reconstitute, stimulant, diuretic and
expectorant. L. iberica in an important medicinal plant in our country and grows in various regions with different environmental
conditions. At present no investigation has been reported about population genetic structure of this valuable plant species in Iran.
Therefore, we carried out population genetic analysis of 11 populations of L. iberica by using ISSR molecular markers for the first time.
Genetic diversity analysis revealed high within population genetic variability. AMOVA test produced significant genetic difference
among the studied populations. Mantel test revealed significant correlation between genetic distance and geographical distance of the
populations. STRUCTURE analysis and K-Means clustering revealed population genetic fragmentation and the presence of three gene
pools for this species. The assignment test revealed the occurrence of limited gene flow among the populations. The results suggested
that genetic divergence, limited gene flow, genetic drift and local adaptation have played role in diversification of L. iberica.
Key words: Gene flow, population fragmentation, IBD, Lallemantia iberica.
INTRODUCTION
Lallemantia iberica (Bieb.) Fischer and C.A. Meyer
(syn. Dracocephalum ibericum M. Bieb.) also named
Dragon’s head” is a crop cultivated from the prehistoric
times in Southwestern Asia and Southeastern Europe. It is
an annual plant and has been cultivated for its seeds that
contain about 30% -38% drying oil (siccative oil). The
iodic index of its seeds oil is between 163 and 203. The oil
is used in foods, but especially in dye and varnish industry
(Shafiee et al. 2009; Ion et al. 2011).
Lallemantia iberica seeds have traditional uses as
reconstitute, stimulant, diuretic and expectorant. It is
considered as a linseed substitute in a number of
applications including: wood preservative, ingredient of
oil-based paints, furniture polishes, printing inks, soap
making, and manufacture of linoleum (Samadi et al. 2007;
Shafiee et al. 2009). The plant also has high ornamental
value and it is used in arid landscaping and urban
horticulture at various places in Turkey (Ozdemir et al
2014).
Population genetics analyses can produce important
data on the levels of genetic variation, the partitioning of
variability within/between populations, gene flow,
inbreeding, selfing versus outcrossing rates, effective
population size and population bottleneck. These analyses
may be of help in developing effective management
strategies for endangered and/or invasive species (Ellis and
Burke 2007). Lallemantia iberica in an important
medicinal plant in our country and grows in various regions
with different environmental conditions (Ozdemir et al
2014). This plant forms several local populations. At
present no investigation has been reported about population
genetic structure of this valuable plant species in Iran.
Studying genetic variability and gene flow versus genetic
fragmentation of local populations can provide valuable
information for conservation of this medicinal plant in the
country. Therefore, we carried out population genetic
analysis in 11 populations of L. iberica for the first time in
Iran. Different molecular markers have been used in
population genetic studies. We used ISSR (Inter-simple
sequence repeats) to study genetic diversity of populations
in L. iberica, since these markers are reproducible, cheap,
easy to work and are known to be efficient in population
genetic diversity studies (Sheidai et al. 2012, 2013, 2014).
Materials and Methods
Plant material
Ninety plant specimens were collected from 11
populations of Lallemantia iberica. Details of the studied
populations are provided in Table 1, Figure 1. Voucher
specimens are deposited in Herbarium of Shahid Beheshti
University (HSBU), Tehran, Iran.
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B I O D I V E R S IT A S
16 (2): 139-144, October 2015
Table 1. Populations studied, their locality and ecological features.
Province
Zanjan
Zanjan
West Azerbaijan
West Azerbaijan
West Azerbaijan
West Azerbaijan
Markazi
Qazvin
Kermanshah
Mazandran
Qazvin
Locality
1.
50 km from Qazvin
to Zanjan
2. 23 km from Qazvin
to Zanjan
3. Takab
4. 2km from Takab to
Shahindej
5. 65 km from Takab
to Shahindej
6. Sero
7. Sangak
8. Khereqan
9. Paveh
10. Chalus
11. Abyek
Alt.
(m)
Long. Lat.
1839
3619 4905
1785
3634 4843
1729
1751
3627 4703
3628 4701
2047
3630 4658
1680
1929
1980
1533
1605
1263
3743
3511
3519
3502
3616
3602
4449
4948
4955
4621
5114
5031
DNA extraction and ISSR assay
Fresh leaves were collected randomly in each of the
studied populations and dried in silica gel powder.
Genomic DNA was extracted using CTAB with activated
charcoal protocol (Sheidai et al. 2013). The quality of
extracted DNA was examined by running on 0.8% agarose
gel. Ten ISSR primers; (AGC)5GT, (CA)7GT, (AGC)5GG,
UBC810, (CA)7AT, (GA)9C, UBC807, UBC811, (GA)9A
and (GT)7CA commercialized by UBC (the University of
British Columbia) were used. PCR reactions were
performed in a 25μ l volume containing 10 mM Tris-HCl
buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of
each dNTP (Bioron, Germany), 0.2 μ M of a single primer;
20 ng genomic DNA and 3 U of Taq DNA polymerase
(Bioron, Germany). The amplifications’ reactions were
performed in Techne thermocycler (Germany) with the
following program: 5 min initial denaturation step 94°C, 30
S at 94°C; 1 min at 50°C and 1min at 72°C. The reaction
was completed by final extension step of 7 min at 72°C.
The amplification products were visualized by running on
2% agarose gel, followed by the ethidium bromide staining.
The fragment size was estimated by using a 100 bp
molecular size ladder (Fermentas, Germany). ISSR bands
obtained were coded as binary characters (presence = 1,
absence = 0). The following genetic diversity parameters
were determined in each population: percentage of allelic
polymorphism, allele diversity (Weising et al. 2005), Nei’s
gene diversity (H), Shannon information index (I), number
of effective alleles, and percentage of polymorphism
(Freeland et al. 2011).
Data analysis
Genetic diversity and population structure. ISSR
bands obtained were scored as binary characters. Genetic
diversity parameters were determined in each population.
These parameters were Nei’s gene diversity (H), Shannon
information index (I), number of effective alleles, and
Figure 1. Distribution map of Lallemantia iberica populations.
Note: Population numbers are according to Table 1.
percentage of polymorphism (Weising et al. 2005; Freeland
et al. 2011). Nei’s genetic distance was determined among
the studied populations and used for clustering (Weising et
al. 2005; Freeland et al. 2011). For grouping of the plant
specimens, Neighbor Joining (NJ) clustering method,
Neighbor Net method of networking as well as principal
coordinate analysis (PCoA) were performed after 100 times
bootstrapping/ permutations (Freeland et al. 2011; Huson
and Bryant 2006). The Mantel test was performed to check
correlation between geographical distance and the genetic
distance of the studied species (Podani 2000). PAST ver.
2.17 (Hamer et al. 2012), DARwin ver. 5 (2012) and
SplitsTree4 V4.13.1 (2013) programs were used for these
analyses.
Significant genetic difference among the studied
populations and provinces were determined by: 1AMOVA (Analysis of molecular variance) test (with 1000
permutations) by using GenAlex 6.4 (Peakall and Smouse
2006), and 2- Nei,s Gst analysis of GenoDive ver.2 (2013)
(Meirmans and Van Tienderen 2004). The population
genetic differentiation was studied by G'st_est =
standardized measure of genetic differentiation (Hedrick
2006), and D_est = Jost measure of differentiation (Jost
2008). In order to overcome potential problems caused by
the dominance of ISSR markers, a Bayesian program,
Hickory (ver. 1.0) (Holsinger et al. 2003), was used to
estimate parameters related to genetic structure (theta B
value). Three runs were conducted with default sampling
parameters (burn-in = 50,000, sample= 250,000, thin = 50)
to ensure consistency of results (Tero et al. 2003).
The genetic structure of populations was studied by two
different approaches. First by using Bayesian based model
STRUCTURE analysis (Pritchard et al. 2000), and second
by maximum likelihood-based method of K-Means
clustering. For STRUCTURE analysis, data were scored as
dominant markers (Falush et al. 2007). The Evanno test
was performed on STRUCTURE result to determine proper
KOOHDAR et al. – Population genetic of Lallemantia iberica
number of K by using delta K value (Evanno et al. 2005).
We performed K-Means clustering as done in GenoDive
ver. 2. (2013). Here, the optimal clustering is the one with
the smallest amount of variation within clusters. This is
calculated by using the within-clusters sum of squares. The
minimization of the within-groups sum of squares that is
used in K-Means clustering is, in the context of a
hierarchical AMOVA, equivalent to minimizing the
among-populations-within-groups
sum
of
squares,
SSDAP/WG (Meirmans 2012). Two summary statistics, 1pseudo-F (Calinski and Harabasz 1974), and 2- Bayesian
Information Criterion (BIC) (Schwarz 1978, provide the
best fit for k (Meirmans 2012).
Gene flow. Gene flow was determined by two different
approaches. (i) Calculating Nm an estimate of gene flow
from Gst by PopGen ver. 1.32 (1997) as: Nm = 0.5(1 Gst)/Gst. This approach considers equal amount of gene
flow among all populations. (ii) Population assignment test
based on maximum likelihood as performed in Genodive
ver. in GenoDive ver. 2. (2013).
Results and discussion
Genetic diversity
Genetic diversity parameters determined in the studied
populations are presented in Table 2. The highest values for
gene diversity and percentage of polymorphism occurred in
population 6 (0.162 and 63.89, respectively). The lowest
values for these parameters were observed in population 10
(0.048 and 12.5, respectively).
Population genetic structure
AMOVA test produced significant genetic difference
(PhiPT = 0.49, P = 0.010) among the studied populations.
It also revealed that, 59% of total genetic variability was
due to within population diversity and 41% was due to
among population genetic differentiation. Pairwise
AMOVA produced significant difference among the
studied populations. Hickory test also produced high Theta
B value (0.4) supporting AMOVA. Gst (0.40, P = 0.001),
Hedrick, standardized fixation index (G'st = 0.47, P =
0.001) and Jost, differentiation index (D-est = 0.13, P =
0.001), revealed that the studied populations are genetically
differentiated.
Neighbor Joining (NJ) tree and PCoA plot produced
similar results. Therefore, only PCoA plot is presented and
discussed (Figure 2). The studied population was placed in
separate group which was in agreement with AMOVA
result. It also revealed a higher degree of within population
genetic variability in population 6 (as plants in this
populations were more scattered than the other
populations) which is in agreement with genetic diversity
parameters presented before. PCoA plot revealed higher
genetic affinity between populations 1, 2 and 3, and
between 4, 5 and 6, as well as between 9, 10 and 11.
Neighbor-Net diagram (Figure 3), supported grouping
obtained by PCoA and also revealed the presence of three
distinct genetic groups. The populations 1-3 formed the
first genetic group, populations 4-6 and populations 7-11
comprised second and third genetic groups respectively.
This result was supported by Evanno test based and K-
141
Means clustering that produced the best k = 3 and 2
respectively. Therefore, we have population genetic
fragmentation in L. iberica.
STRUCTURE plot (Figure 4) based on k = 3, identified
three distinct genetic groups (gene pools) for of L. iberica
populations. The first gene pool (populations 1-3) is
distributed from Ghazvin to Zanjan and to Takab (West
Azerbayejan). The second gene pool (populations 4-6) is
distributed from Takab to Shahindej and Orumiyeh, while
the third gene pool (populations 7-11) is distributed mainly
in West and North-West of the country.
Gene flow
The STRUCTURE plot that was based on admixture
model, revealed some degree of genetic admixture in the
studied populations. For example, populations 1-3 had
some alleles from populations 4-6 and vice versa. These
shared alleles might be the possible reason for close genetic
affinity of these populations as revealed by PCoA plot
presented before. Populations 7-11 also had some degree of
shared alleles with the other studied populations.
The population assignment test also revealed some
degree of gene flow or ancestral shared alleles among the
studied populations. This was true particularly for
populations 1 and 6, 1 and 2, 2 and 3, 3 and 4, 4 and 6, 7
and 8, 6 and 9, 6 and 10, as well as 6 and 11. Gene flow
determined by Nm, produced mean value = 0.60, that is not
considered to be high. Therefore, all these results revealed
restricted gene flow and strong population differentiation
among L. iberica populations. The Mantel test produced
significant correlation between genetic distance and
geographical distance of the studied populations (r = 0.36,
P = 0.01, Figure 5). This indicated the occurrence of
isolation by distance (IBD) in L. iberica populations.
LFMM analysis revealed that 20 out of 72 ISSR loci
had >1.3 -log 10 value (P <0.05) and may be considered as
adaptive loci. Some of these loci had low Nm value, for
example ISSR loci 2, 7, 37, 38, 41, 43, 57, 59. 63, and 71
had Nm <1. However, some loci like ISSR loci 14, 18, 22,
26, 34, 47, 51, 65, 66, had nm >1. Therefore, both ISSR
loci that were shared by different populations and loci with
lower admixture value were used by L. iberica plants to
adapt to their environment.
Discussion
The extensive use of natural resources to meet the needs
of expanding human populations, deforestation and habitat
fragmentation could lead to reductions in the rate of gene
flow among populations. This in turn increases the genetic
differentiation among populations and genetic structuring
and reductions in genetic variation within populations due
to genetic drift (Setsuko et al. 2007; Hou and Lou 2011).
However, there are cases in which fragmentation did not
result in reduced genetic diversity (Prober et al. 1990), due
to various reasons, like population size and the time scale
of fragmentation. Templeton (1991) stated that
“Heterozygosity is not always beneficial, nor does
inbreeding always have adverse effects. In some
circumstances, a population may be so well adapted to its
local circumstances. STRUCTURE analysis and K-Means
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B I O D I V E R S IT A S
16 (2): 139-144, October 2015
Table 2. Genetic diversity parameters in the studied populations of Lallemantia iberica
Pop
N
Na
Ne
I
Pop1
10.000
0.931
1.235
0.217
Pop2
10.000
1.208
1.292
0.267
Pop3
10.000
1.083
1.211
0.211
Pop4
10.000
0.944
1.224
0.204
Pop5
10.000
0.875
1.200
0.179
Pop6
10.000
1.306
1.249
0.260
Pop7
10.000
0.639
1.180
0.152
Pop8
10.000
0.583
1.204
0.162
Pop9
3.000
0.472
1.105
0.101
Pop10
3.000
0.417
1.083
0.071
Pop11
4.000
0.486
1.134
0.119
Note: N = Number of populations, Na = No. of different alleles, Ne = No. of effective
Gene diversity, UHe = Unbiased gene diversity, and %P = Percentage of polymorphism.
He
UHe
%P
0.142
0.149
44.44%
0.174
0.183
56.94%
0.133
0.140
50.00%
0.133
0.140
43.06%
0.119
0.125
34.72%
0.162
0.170
63.89%
0.102
0.108
29.17%
0.112
0.118
27.78%
0.066
0.079
19.44%
0.048
0.058
12.50%
0.079
0.090
22.22%
alleles, I = Shannon's Information Index, He =
Figure 2. PCoA plot of Lallemantia iberica populations based on ISSR data. Note: Population numbers are according to Table 1.
Figure 4. STRUCTURE plot based on k = 3
KOOHDAR et al. – Population genetic of Lallemantia iberica
Figure 3. Neighbor-Net diagram of Lallemantia iberica populations
based on ISSR data. Note: Population numbers are according to
Table 1.
143
environmental conditions, and enables change in the
genetic composition to cope with changes in the
environment (Çalişkan 2012; Sheidai et al. 2013, 2014).
High value of within population genetic diversity was
observed in the studied populations (for instance, high
percentage of polymorphism in each population).
Moreover, AMOVA test revealed that 59% of total genetic
variability was due to within population diversity and 41%
was due to among population genetic differentiation. This
is possibly related to outcrossing nature of L. iberica.
Mantel test revealed a pattern of isolation-by distance
across the distribution range of the studied L. iberica
populations. This pattern suggested that the dispersal of
these populations might be constrained by distance and
gene flow is most likely to occur between neighboring
populations. As a result, more closely situated populations
tend to be more genetically similar to one another (Slatkin
1993; Hutchison and Templeton 1999; Medrano and
Herrera 2008). In fact population assignment test revealed
that limited gene flow occurred mostly between
neighboring populations, populations 1-3, 4-6, and 7-11.
LFMM analysis revealed that some of the genetic loci
were adaptive nature and possibly used by local
populations to adapt to their environment. Therefore,
combination of genetic divergence, limited gene flow and
local adaptation have played role in diversification of L.
iberica. In conclusion the present study may provide some
useful information about population genetic structure and
genetic variability of L. iberica that may be used in
conservation of these important species.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160207
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 145-150
Within and among-genetic variation in Asian flax Linum austriacum
(Linaceae) in response to latitude changes: Cytogenetic and molecular
analyses
1
ZAHRA NOORMOHAMMADI1,♥, TINA SHAFAF2, FATEMEH FARAHANI2, MASOUD SHEIDAI2,
SEYED-MEHDI TALEBI3, YEGANEH HASHEMINEJAD-AHANGARANI-FARAHANI 2
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. Tel.: +982144865939, Fax.: +982144865939, ♥email:
marjannm@yahoo.com, z-nouri@srbiau.ac.ir
2
Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran
3
Botany Department, Arak University, Arak, Iran
Manuscript received: 31 May 2015. Revision accepted: 24 June 2015.
Abstract. Noormohammadi Z, Shafaf T, Farahani F, Sheidai, Talebi SM, Hasheminejad-Ahangarani-Farahani Y. 2015. Within and
among-genetic variation in Asian flax Linum austriacum (Linaceae) in response to latitude changes: Cytogenetic and molecular
analyses. Biodiversitas 16: 145-150. Linum austriacum L. (Linaceae) which is known as Asian flax is an herbaceous medicinal plant
species that grow in Iran in different latitudes and forms some local populations particularly in the West and North-West of the country.
Within-and between-genetic diversity in response to altitude changes was investigated in geographical populations of L. austriacum by
using cytogenetic and ISSR molecular markers. These populations were diploid with 2n = 18 and differed significantly (P<0.05) in their
mean chiasma frequency and chromosome pairing. Significant positive correlation (r >0.90, P < 0.05) occurred between latitude and the
mean number of quadrivalents. The highest level of genetic diversity parameters as Shanon Information Index and gene diversity
occurred in Salavat-abad population. Moreover, the STRUCTURE analysis also identified this population as the most varied population.
This population had medium altitude distribution. Mantel test performed between genetic distance and geographical distance showed no
significant correlation (R2 = 0.09, p = 0.39). Pearson coefficient of correlation determined between genetic diversity parameters and
altitude, produced a significant negative correlation (r =-0.85, P<0.01) with the number of effective alleles. The present study revealed
that Linum austriacum populations do have some cytogenetic and molecular variation in response to altitude.
Key words: Chromosome pairing; Fst; genetic variability; ISSR; wild flax.
INTRODUCTION
The genus Linum is the type genus for the flax family,
Linaceae DC. (Dumort) comprising 22 genera and about
300 species distributed worldwide. Linum is the largest
genus (about 200 species) within the family and is found
both in the Mediterranean region and the Americas. It
includes both horticultural plants with various flower
colors and one field crop (Linum usitatissimum L.), and
have been used as a source of fiber (L. usitatissimum), seed
oil, fodder, medicine and as ornamentals (Muir and
Westcott 2003). Many species are cross-pollinated due to
heterostyly. Distyly is widespread and very common in the
genus Linum (about 40 % of the Linum species are distylous) (Sheidai et al. 2015). It occurs in Linum pubescens,
L. grandiflorum and L. mucronatum, L. perenne, L.
grandiflorum, L. alpinum, L. aretioides, L. austriacum, L.
album, and L. glaucum (Talebi et al. 2012).
In recent years researchers has been extensively studied
to conserve and explore germplasm of crop wild relatives.
Crop wild relatives are species closely related to crops,
including their progenitors, which may contain beneficial
traits such as pest or disease resistance and yield
improvement (Sheidai et al. 2014). Genetic diversity
analysis and population structure have been the subject of
several studied in cultivated and wild flax species (see for
example, Fu 2006, Fu and Allaby 2010, Abou El-Nasr and
Mahfouze 2013, Sheidai et al. 2014).
Linum austriacum L. is an herbaceous medicinal plant
containing important lignans such as arylnaphthalene
lignan, 3,4-dimethoxy-3',4'-methylenedioxy-2,7'-cycloligna7,7'-dieno-9,9'-lactone (1,0.03-0.73% dry wt), together with
justicidin B (2, 0.18-1.69% dry wt) (Mohagheghzadeh et al.
2002). Because elevation is a complex factor, altitudinal
gradients comprise an assemblage of environmental
variables that influence the distribution of population
genetic variation of plant species. Along altitudinal
gradients, the genetic differentiation between populations
of some plant species results in rapid, elevation-related
changes in environmental conditions. However, in other
taxa, only a little or no differentiation with respect to
altitude occurs (Di et al. 2014).
Species have three options that may allow them to
survive rapidly changing environments: dispersal
(expansion), phenotypic plasticity, or adaptation (Heather
and Freeland 2011). The plant species expansion of the
core population is associated with reduced withinpopulation genetic diversity (Austerlitz et al. 2000), and
reduced levels of phenotypic variation (Pujol and Pannell
2008).
146
B I O D I V E R S IT A S
16 (2): 145-150, Ocotober 2015
The present study considers cytological and the genetic
diversity analysis of some Linum populations distributed in
different altitudes and investigates degree of within-and
among-population genetic variability and tries to study
genetic variability response to altitude changes. For
molecular study, we used ISSR markers that are powerful
molecular tools to differentiate the species populations and
reveal their genetic diversity (see, for example, Sheidai et
al. 2012, 2013).
MATERIAL AND METHODS
Plant materials
For cytological studies, suitable flower buds have been
obtained in five populations of L. austriacum (Table 1).
Different flower buds were selected randomly from at least
ten plants in each population and used for cytological
preparations. For ISSR studies, 70 plants were randomly
selected from four populations including, Saleh-abad and
Hamekasi Village, from Hamedan Province, Iran, and
Salavat-abad and Abidar-Sanandaj, from Kurdistan
Province (Table 1). The voucher specimens were deposited
in the Herbarium of Shahid Beheshti University (HSBU),
Tehran, Iran.
Cytological study
The young flower buds collected were fixed and used
for cytological investigation by the squash method
according to our previous report (Sheidai et al. 2012).
Meiotic characters including polyploidy level, chiasma
frequency and distribution, as well as chromosome pairing
and segregation were observed in plants collected.
ISSR assay
The genomic DNA was extracted from silica gel dried
leaves by using CTAB activated charcoal protocol
(Krizman et al. 2006). The quality and quantity of extracted
DNA were assessed by running on 0.8% agarose gel and
NanoDrop® spectrometer respectively. Ten ISSR primers:
(AGC)5GT, (CA)7GT, (AGC)5GG, UBC 810, (CA)7AT,
(GA)9C, UBC 807, UBC 811, (GA)9A and (GT)7CA
commercialized by UBC (the University of British
Columbia) were used. PCR reactions were performed in a
25 µl volume containing 10 mM Tris-HCl buffer at pH 8;
50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP
(Bioron, Germany); 0.2 µM of a single primer; 20 ng
genomic DNA and 3 U of Taq DNA polymerase (Bioron,
Germany). Amplification reactions were performed in
Techne thermocycler (Germany) with the following
program: 5 min initial denaturation step 94°C, 30 s at 94°C,
1 min at 50°C and 1 min at 72°C. The reaction was
completed by a final extension step of 7 min at 72°C.
Amplification products as well as No DNA (control
sample) were visualized by running on 2% agarose gel,
following ethidium bromide staining. Fragment size was
estimated by using a 100 bp molecular size ladder
(Fermentas, Germany). The experiment was replicated
three times and constant ISSR bands were used for further
analyses.
Data analyses
ANOVA (Analysis of variance) was performed to reveal
cytogenetic difference among the studied populations.
Pearson correlation determined among geographical
features (longitude and latitude) and meiotic characters.
PCA (Principal Components Analysis) biplot was used to
group populations based on cytogenetic similarity.
ISSR bands obtained were treated as binary characters
and coded accordingly (presence = 1, absence = 0). The
Dentrented correspondence analysis plot was used to check
distribution of ISSR loci and their effects on genotypes
grouping. The role of each locus in discriminating plant
genotypes was checked by Gst analysis performed by
POPGENE program ver. 2. (1999). Genetic diversity
parameters were determined in each population. These
parameters were percentage of allelic polymorphism, allele
diversity (Weising et al. 2005), Nei’s gene diversity (H),
Shannon information index (I), the number of effective
alleles and percentage of polymorphism and polymorphic
information content (PIC) (Weising et al. 2005; Freeland et
al. 2011).
Jaccard similarity index and Nei’s genetic distance
(Freeland et al. 2011), were determined among plants and
used for the grouping of the genotypes. Neighbor Joining
(NJ) tree followed by 100 times bootstrapping, and
Principal Coordinate Analysis biplot (PCoA) (Freeland et
al. 2011, Podani 2000) were used for this purpose.
DARwin ver. 5 (2012), was used for these analyses.
Genetic differences among the studied populations were
determined by: 1-AMOVA (Analysis of molecular
variance) test (with 1000 permutations), and 2-Nei,s Gst
analysis of GenoDive ver.2 (2013) (Meirmans and Van
Tienderen 2004). Moreover, to avoid possible problem
caused by the dominant nature of ISSR markers, Hickory
test (Hickory program ver. 1.0) that is a Bayesian approach
was used to reveal populations, genetic differentiation
(Holsinger et al. 2003).
The genetic structure of geographical populations was
studied by Bayesian based model STRUCTURE analysis
(Pritchard et al. 2000). Data were scored as dominant
markers (Falush et al. 2007).
RESULTS AND DISCUSSION
Cytology
Linum austriacum populations studied showed n = 9
(2n = 2x = 18) chromosome number (Table 1, Figure 1, AE). The mean value for total chiasmata varied from 16.36
in Tehran-Lashkarak population to 20.38 in Tehran
population. These populations had the lowest and highest
values of terminal chiasmata too (15.27 and 19.21
respectively). The lowest value of intercalary chiasmata
(0.28) occurred in Salavat-abad population, while the
highest value of the same parameter occurred in Tehran
population (1.17). Although these populations are diploid
and mostly formed bivalents (Table 1), they formed few
univalent and quadrivalents in metaphase I cells (Figure 1).
The chromosomes mostly showed normal segregation
during anaphase and telophase stages, but laggard
NOORMOHAMMADI et al. – Genetic diversity of Linum austriacum
147
chromosomes and micronuclei were formed in some cases
(Figure 1).
ANOVA test showed significant difference (p<0.05) for
chiasma frequency and chromosome pairing among
populations studied. Pearson correlation determined among
ecological and meiotic characters showed significant
positive correlation between longitude and the mean
number of quadrivalents as well as between latitude and
mean number of quadrivalents. Significant negative
correlation was observed between longitude and the mean
number of univalents.
PCA biplot of meiotic characters (Figure 2), separated
the populations studied in distinct groups, indicating
cytogenetic differences of the populations studied, also
supported by ANOVA test. It also showed that, Tehran
population was differentiated from the other populations
due to its intercalary chiasmata and number of
quadrivalents, while Salavat-abad population was
differentiated from the others due to mean number of total
chiasmata, ring bivalents and terminal chiasmata. The
number of rod bivalents differentiated Tabriz-Ahar
population, while No. of univalents separated Roodbar
population from the other populations studied.
population genetic diversity was investigated by using
Jaccard similarity index. In Saleh-abad population of
Hamedan (Pop. 1), Jaccard similarity among plants of this
population ranged from 0.26-0.94.
Similarly, in Hamekasi Village population of Kordestan
(Pop. 2), Jaccard index ranged from 0.43-0.81. In Salavatabad population (Pop. 3), the range of Jaccard index was
0.34-1.00, while the same value in Abidar population of
Sanandaj (pop 4) was 0.38-0.90. STRUCTURE analysis
(Figure 3) that is based on Bayesian approach also revealed
the occurrence of a higher degree of within-population
genetic variability in Salavat-abad population (Pop. 3).
Pearson coefficient of correlation determined between
genetic diversity parameters and altitude, produced a
significant negative correlation (r =-0.85, P<0.01) with the
number of effective alleles only (Table 3). Longitude did
not show correlation with genetic diversity parameters, but
latitude had a significant negative correlation with Shanon
Information Index (r =-0.90, P<0.01), mean gene diversity
(r =-0.88, P<0.01) and mean unbiased gene diversity (r =0.86, P<0.01 P<0.01; Table 3).
Within-population genetic diversity
Genetic diversity parameters determined among 4
populations studied are presented in Table 2. The highest
level of genetic polymorphism occurred in Hamekasi
Village population of Hamedan Province (Pop. 2)
(42.42%), while the lowest value of the same parameter
occurred in Saleh-abad, Hamedan Province (Pop. 1) and
Salavat-abad population, Kurdistan Province (Pop. 3)
(40.40%). The highest value for effective No. of alleles
(1.326), I (0.258) and gene diversity (0.179) occurred in
Abidar population of Kurdistan Province (Pop. 4). Within-
Pop
Table 2. Genetic diversity parameters in populations studied
N
Na
Ne
I
He
UHe
%P
15.000 0.899 1.256 0.210 0.142 0.147 40.40%
1
19.000 0.990 1.264 0.227 0.153 0.157 42.42%
2
20.000 0.859 1.292 0.242 0.166 0.170 40.40%
3
16.000 0.899 1.326 0.258 0.179 0.185 41.41%
4
Abbreviations: Na = No. of different Alleles, Ne = No. of
Effective alleles, I = Shannon's Information Index, He = Gene
diversity, UHe = Unbiased gene diversity, and % P = Percentage
of polymorphism. Populations 1-4 are: Saleh-abad and Hamekasi
Village from Hamedan Province and Salavat-abad and AbidarSanandaj from Kurdistan Province respectively.
Table 1. Cytogenetic features of L. austriacum populations studied.
Altitude
n TOX IX
(ft)
Tehran-Lashkarak Tehran
35°15´.50″ 51°34´.00″ 2100
9 11.23 1.00
Roodbar
Gilan
36°50´.00″ 49°25´.00″
544
9 10.65 0.15
Salavat-abad
Hamedan
35°08´.00″ 47°52´.00″ 1900
9 12.04 0.82
Tabriz-Ahar
East Azarbayejan 38°09´.00″ 46°39´.00″ 3809
9 10.67 0.75
Abidar-Sanandaj Kurdistan
35°19´.00″ 46°57´.00″ 1645
9 10.5 0.64
Abbreviations: TOX = Total chiasmata, TX = Terminal chiasmata, IX = Intercalary
bivalents, I = Univalents, IV = Quadrivalents.
Population
Province
Longitude Latitude
TX
ROD RB
10.23 6.70
10.5 7.04
11.23 5.91
9.92 7.33
9.86 7.36
chiasmata, RB
I
IV Voucher No.
1.87 0.10 0.20
1.77 0.19 0.04 2011126
2.91 0.14 0.04 2011108
1.58 0.17 0.00 2011136
1.5 0.28 0.00 2011112
= Ring bivalents, ROD = Rod
Table 3. Correlation between geographical features and genetic diversity parameters
N
Na
Ne
I
He
UHe
%P
Altitude Longitude Latitude
N
0
Na
0.09214
0
Ne
-0.01326 -0.41076 0
I
0.22846
-0.28382 0.96518
0
He
0.18355
-0.32895 0.97856
0
0.99813
UHe
0.13186
-0.33415 0.98731
0
0.99446
0.99863
%P
0.21938
0.005495 0.15685
0.10866
0.10047
0
0.90224
altitude
0.5045
0.23777
-0.72148 -0.74822 -0.78166 -0.0634
0
-0.8529
longitude 0.37928
-0.76166 -0.09971
-0.09805 -0.08002 -0.1033
-0.81555 0.429
0
latitude
-0.61235 0.22796
-0.78123
-0.11801
0
-0.90863 -0.88952 -0.86438 -0.18283 0.36649
Abbreviations: Na = No. of different Alleles, Ne = No. of Effective alleles, I = Shannon's Information Index, He = Gene diversity, UHe
= Unbiased gene diversity, and % P = Percentage of polymorphism.
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16 (2): 145-150, Ocotober 2015
Figure 1. Representative meiotic cells in L. austriacum populations studied. A-E = A: metaphase I cell, B: univalent (arrows), C:
laggard chromosome, D and E: metaphase I cells
Figure 2. PCA biplot of meiotic characters. Abbreviations: TOX = Total chiasmata, TX = Terminal chiasmata, IX = Intercalary
chiasmata, RB = Ring bivalents, ROD = Rod bivalents, I = Univalents, IV = Quadrivalents.
NOORMOHAMMADI et al. – Genetic diversity of Linum austriacum
149
Figure 3. STRUCTURE plot showing higher degree of withi-population genetic diversity in population. Populations 1-4 are Saleh-abad,
Hamekasi village, Salavat-abad and Abidar, respectively
Genetic differentiation of populations
AMOVA test showed significant differences among the
studied populations (p<0.01). It showed that 63% of total
variation is due to, among populations and 37% due to
within populations. Irrespective of the good genetic
variability observed within each population, Neighbor
Joining tree of all 70 plants separated the studied
populations from each other (data not shown). The plant
specimens of each population were placed close to each
other and formed a separate group. Moreover, high values
of B (>0.52) showed great genetic difference among pairs
of populations which support AMOVA result. These results
indicated genetic distinctness of the studied populations.
Mantel test performed between genetic distance and
geographical distance showed no significant correlation (R2
= 0.09, p = 0.39). However, Fst values of populations
showed negative significant correlation with the Eastern
distribution (r =-.92, p = 0.05), and high negative (but not
significant) correlation with altitude of populations (r =0.81). Fst values showed high (but not significant) positive
correlation with Western distribution and minimum
temperature.
Nm values obtained for all ISSR loci in the studied
populations ranged from 0.0 to 1.0, with the mean Nm
value of 0.44. This is a moderate value and indicates a
moderate degree of gene flow among the studied
populations.
Discussion
Linum austriacum populations studied showed n = 9
(2n = 2x = 18) chromosome number, supporting Öztürk et
al. (2009) report. Although these populations are diploid
and mostly formed bivalents, they formed a few
quadrivalents in metaphase I cells. Quadrivalent were
formed due to the occurrence of the heterozygote
translocations, which may have adaptive value (Sheidai et
al. 2012). Cytogenetic studies performed by Gill and
Yermanos (1967) in six 18-chromosome Linum taxa and
nine of their interspecific hybrids, also indicated the role of
translocations in Linum species diversification. They
showed that L. altaicum differs from L. alpinum, L.
austriacum, L. julicum, L. narbonense, and L. perenne by
one reciprocal translocation. L. austriacum and L.
narbonense, and L. julicum and L. narbonense also differed
by one translocation, whereas L. perenne and L.
narbonense differed by two translocations.
Variation in chiasma frequency and localization is
genetically controlled and has been reported in several
plant species (Quicke 1993; Sheidai et al. 1999). Such a
variation in species or populations with the same
chromosome number is considered a means for generating
new forms of recombination influencing the variability
within natural populations in an adaptive way (Rees and
Dale 1974).
Therefore, significant difference observed for chiasma
frequency and chromosome pairing among Linum
austriacum populations indicate a change in genetic control
of chromosome pairing during population diversification.
Significant positive correlation between longitude and the
mean number of quadrivalents, as well as between latitude
and mean number of quadrivalents, indicate that
heterozygote translocations may have played a role in
response to these ecological parameters.
ISSR analysis of the studied populations revealed
almost high degree of within population genetic variability
and significant genetic differentiation among populations
(significant AMOVA result). However, in spite of
significant genetic difference among populations, we did
not observe isolation by distance (IBD) (Mantel test
showed no correlation between genetic distance and
geographical distance of the studied populations). This
result suggested some degree of gene flow among
populations that are supported by the fact that Fst values of
populations decreased towards Eastern distribution
(possibly due to limited occurrence of gene flow).
However, the studied populations become genetically more
differentiated towards the western part of the country
where the minimum temperature is lower.
Genetic variability of these populations was reduced in
response to an increase in altitude (Pearson coefficient of
correlation produced a significant negative correlation
between altitude and the number of effective alleles). These
results reveal complex interaction between genetic
diversity distribution of Linum austriacum populations and
environmental features.
In the present study, irrespective of the genetic
variability observed within each population, NJ tree of all
70 plants separated the studied populations from each
other. The plant specimens of each population were placed
close to each other and formed a separate group. This
clearly reveals that each local population has its own
specific genetic contents along the altitude its plants grow.
150
B I O D I V E R S IT A S
16 (2): 145-150, Ocotober 2015
This is particularly true for Salavat-abad population of
Kurdistan Province (Pop. 3) that formed a distinct cluster
and was placed far from the other studied populations.
Evaluation of within and among-population genetic
variations have been considered to prioritize populations
for conservation efforts (Petit et al. 1998), high withinpopulation variation. These populations may have
increased likelihood of persistence over less variable
population and hence the ability of a population to
contribute demographically to the species through time,
and have increased adaptability in the face of future
environmental change.
Genetic diversity within populations can vary along
altitudinal gradients. In many cases the populations at
intermediate altitudes have greater diversity than
populations at lower and higher altitudes (see for example,
Gapare et al. 2005; Ohsawa and Ide 2008; Di et al. 2014).
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160208
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 151-155
Short Communication:
Felids of Sebangau: Camera trapping to estimate activity patterns and
population abundance in Central Kalimantan, Indonesia
1
ADUL1,2, BERNAT RIPOLL1, SUWIDO H. LIMIN2, SUSAN M. CHEYNE1,3,♥
The Orangutan Tropical Peatland Project (OuTrop). Jalan Semeru No. 91, Bukit Hindu, Palangka Raya 73112, Kalimantan Tengah, Indonesia
Center for International Cooperation in Sustainable Management of Tropical Peatland (CIMTROP), University of Palangka Raya. Jl. Yos Sudarso,
Kampus UNPAR Tunjung Nyahu, Palangka Raya 73111, Central Kalimantan, Indonesia
3
Wildlife Conservation Research Unit (WildCRU), Department of Zoology, Oxford University. Recanati-Kaplan Centre, Abingdon Road, Tubney
House, Tubney, Oxfordshire OX13 5QL, Inggris. Tel./Fax.: +44 1865 611100. ♥email: scheyne@outrop.com
2
Manuscript received: 2 Juni 2015. Revision accepted: 27 Juni 2015.
Abstract. Adul, Ripoll B, Limin SH, Cheyne SM. 2015. Felids of Sebangau: Camera trapping to estimate activity patterns and
population abundance in Central Kalimantan, Indonesia. Biodiversitas 16: 151-155. We present data from a seven year camera trapping
project in the Natural Laboratory of Peat-Swamp Forest in the Sebangau Catchment, Central Kalimantan, Indonesia (2008-2015). The
project has identified four of the five felids on Borneo: Sunda clouded leopard (Neofelis diardi; macan dahan), flat-headed cat
(Prionailurus planiceps; kucing tandang), marbled cat (Pardofelis marmorata; kucing batu) and leopard cat (Prionailurus bengalensis;
kucing kuwuk). All of these species are protected by Indonesian Law (PP. 7/1999) and are listed on the IUCN Red List. The four species
have clearly defined activity budgets, especially the smaller cats, to allow niche partitioning. We have identified this forest block as an
important area for numbers of all four species in the global context of cat populations. The bay cat (Pardofelis badia (kucing merah) has
not been found in tropical peat-swamp forest at time of writing.
Keywords: Activity patterns, camera trapping, felids, population abundance, Sebangau
INTRODUCTION
Robust population density estimates or estimates of
total population size of any of the four threatened Bornean
felids are completely lacking and the extent of hunting and
trade of these species and their prey in Indonesian Borneo
is unclear. Peat-swamp forest is the dominant lowland
forest type in Indonesian Borneo and represents 68,000
km2 of land (Page et al. 1999), thus these forests may be of
vital importance for the future of felids, in particular the
Sunda clouded leopards (Cheyne et al. 2013) and flatheaded cats (Wilting et al. 2010). The Sebangau
(sometimes spelled Sabangau) catchment (5,600km2) has a
history of disturbance, selective logging (legal and illegal),
fire and hunting yet the forest remains relatively contiguous
with good forest cover, which is important for the
conservation of felids (Nowell and Jackson 1996). The
effect of different macro-habitat types, micro-habitat
characteristics and disturbance on these felids remains
unstudied. Initial data from Sebangau suggest that there is a
density of 1.81 clouded leopards/km2 in the forest across all
three habitat types (Mixed Swamp Forest (MSF, Low
Interior (LIF) and Tall Interior Forest (TIF), but this
preliminary study suggests that the Sebangau could hold a
substantial population of Sunda clouded leopards (Cheyne
et al. 2013).
This research is a joint venture between the Orangutan
Tropical Peatland Project (OuTrop), CIMTROP and the
Wildlife Conservation Research Unit, University of Oxford
and aims to facilitate the conservation of Borneo’s
endangered wild cats by merging pioneering ecological
research, host country capacity building and environmental
education within Indonesia. Our research activities will
provide an insight into the relative abundance of each
species, and the long-term impacts of various forest
management practices on these little known felids information which is essential to facilitate the development
of effective management and conservation measures. This
initiative is currently the only research project focusing on
the ecology of Borneo’s wild cats in Kalimantan.
Additionally this project is now the longest running felid
and prey project in Kalimantan and we hope that with
funding to continue this important project in the long-term
(>6 years) we can make a significant contribution to the
understanding of these elusive and charismatic species as
well as facilitating training and capacity building for local
scientists and communities.
The objectives of this long-term project are: (i) To
study the status, behavior and ecology of the four felid
species found in Sebangau. (ii) To investigate the density
and population size. (iii) To investigate the community
structure, niche partitioning and intra-guild relationships.
(iv) To assess the impacts of habitat alteration and habitat
requirements of mammals in the study area.
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B I O D I V E R S IT A S 16 (2): 151-155, Ocotober 2015
MATERIALS AND METHODS
Study site
This study aims to identify the felid biodiversity,
population and behavior in the Natural Laboratory for PeatSwamp Forest (NLPSF) in the Sebangau catchment,
Central Kalimantan, Indonesia. The study was initiated in
May 2008 and has been running continuously since then in
the 50km2 NLPSF. Robust population density estimates or
estimates of total population size of any of the threatened
Bornean felids are lacking, particularly in Indonesian
Borneo. Sebangau is a seasonally flooded forest and is
underwater for 9 of 12 months. It is the largest area of
contiguous lowland rainforest remaining in Kalimantan
(5,600km2, Figure 1; Page 2002).
Methods
By using passive infrared camera traps we investigated
the distribution, habitat associations, activity and density of
cat species. Pairs of camera traps were placed along animal
trails, human trails, logging roads and watering areas.
Photographic capture rates of species were used to
calculate a Relative Abundance Index of (RAI) for each cat
species and capture-recapture techniques will be used to
estimate the density of different species in which
individuals can be distinguished from one another due to
their distinctive pelage patterning. Time of the picture and
date were used to describe the daily activity patterns.
Relative abundance index
The capture probability at each location was not
uniform (repeated measures analysis of variance, F = 0.68,
df = 12, P = 0.942). This does not affect the calculation of
the relative abundance index, which discounts all locations
where felids have not been captured. The relative
abundance index was calculated as
Where i is a trap location and tn is a trap night at the ith
location and d is a detection of the species at the ith
location. Detection is one capture per location during one
trap night (Kawanishi and Sunquist 2004, Azlan and
Lading 2006, Azlan and Sharma 2006). This index cannot
account for frequency of trail use and degree of arboreality,
all of which will affect detection (Giman et al. 2007). To
calculate the relative abundance index we assumed that
photographs represent independent contacts between
animal and camera and that the population is closed
(Rowcliffe and Carbone 2008, Rowcliffe et al. 2008). To
ensure the assumptions of a closed population were met
only data from a 90 day period were analyzed.
Figure 1. Location of the Natural Laboratory for the Study of Peat-Swamp Forest (NLPSF) within Sebangau tropical peat-swamp forest
and Borneo. Forest cover is shaded gray, non-forested areas white. Adapted from (Ehlers Smith et al. 2013)
153
ADUL et al. – Felids of Sebangau, Central Kalimantan
RESULTS AND DISCUSSION
Distribution and population
All felids in Sebangau are non-endemic i.e. they have
ranges that also extend outside Borneo (Figure 2; IUCN
2013).
Activity patterns
Data are presented on the % of total photo captures for
each of the four felids. Data are from May 2008 - April
2015.Clouded leopards are active throughout the day
though more captures are obtained at night (1700-0500h)
thus they are predominantly nocturnal (Figure 3).
Flat-headed cats have a more irregular capture rate
though again active throughout the day, more captures are
obtained at night thus they are predominantly nocturnal
(Figure 4).
Leopard cats have a more regular capture rate. They
appear to be active both during the day and night though
appears to avoid the hottest time of the day (1100-1300h,
Figure 5).
Marbled cats have a regular capture rate with the
majority of photos taken during the day (0500-1600h)
suggesting they are diurnal (Figure 6).
Relative abundance index all felids
Clouded leopards are the most commonly captured cat
on the camera traps, followed by leopard cat, flat-headed
cat and marbled cat (Figure 7).
A
B
C
D
Figure 2. Natural distribution of Sebangau felids. A. Sunda Clouded Leopard - Neofelis diardi IUCN Vulnerable, B. Flat-Headed Cat Prionailurus planiceps IUCN Endangered, C. Marbled Cat - Pardofelis marmorata IUCN Vulnerable, D. Leopard Cat - Prionailurus
bengalensis IUCN Least Concern (IUCN 2013)
154
B I O D I V E R S IT A S 16 (2): 151-155, Ocotober 2015
Table 1. Summary of global populations and records from the NLPSF
Clouded leopard
Leopard cat
Marbled cat
Flat-headed cat
Global population
estimates (IUCN 2013)
<2,500
>50,000
<10,000
<2,500
NLPSF
individuals
1-4
~200
~100
~150
Sebangau individuals
(Cheyne et al. 2013)
40-246
NA
NA
NA
Total independent
photos
152
74
41
30
Number of known
individuals
9♂1♀
NA
NA
NA
Figure 3. Activity patterns of clouded leopards in Sebangau.
Figure 7. RAI of all felids in Sebangau.
Figure 4. Activity patterns of flat-headed cats in Sebangau.
Figure 5. Activity patterns of leopard cats in Sebangau
Figure 6. Activity patterns of marbled cats in Sebangau.
Discussion
These data represent almost double the information
presented in (Cheyne and Macdonald 2011). The nocturnal
activity of the clouded leopards is confirmed from the
present dataset but flat-headed cats appear to be more
diurnal than reported in Cheyne and Macdonald (2011).
Leopard cats are avoiding the hottest part of the day and we
present new information on the marbled cat: based on 41
photos they are predominantly diurnal (more discussion for
this information is needed). The clouded leopard is the
largest predator in Borneo and a different activity pattern
would be expected in the absence of tigers (Seidensticker
1976).
This is the first study for these endangered felids in any
tropical peat-swamp forest, although we have been at pains
to emphasize the methodological caveats. There is an
estimated 68,000 km2 in of tropical peat-swamp forest in
Kalimantan (Page et al. 1997, 1999; Cheyne and
Macdonald 2011). We conclude that even with these
preliminary density range estimates that tropicalpeatswamp forest may be more important to cat conservation
than previously supposed. If our evidence is typical then,
by extrapolation, the totality of peat forest in Kalimantan
might harbor a significant population of clouded leopards,
leopard cats, flat-headed cats and marbled cats. No bay cats
have been reported in peat-swamp forest and more work is
needed to determine if bay cats are present in this habitat
type. Local surveys suggest that hunting pressure is
relatively low, and thus that habitat loss and fragmentation
is likely to be the greatest threat.
In conclusions, density of clouded leopards = 0.72 to
4.41 individuals per 100 km2. (No discussion about this in
previous paragraphs); Activity patterns differ for the 4 felid
species, especially significant for the Marbled Cat, niche
partitioning is related to feeding ecology and activity
patterns, but further research and analysis is required to
understand this; Population numbers for the small cats are
ADUL et al. – Felids of Sebangau, Central Kalimantan
estimates only and are the subject of further work; PeatSwamp Forest habitat is critical to preserve populations of
the four felid species.
ACKNOWLEDGEMENTS
RISTEK, CIMTROP and the University of Palangka
Raya for permissions. The people and administrations of
Kereng Bangkirai, Kecamatan Sebangau, Kota Palangka
Raya and Provinsi Kalimantan Tengah. This work was
funded through a grant to the Wildlife Conservation
Research Unit, University of Oxford from Panthera as well
as Point Defiance Zoo and Aquarium Holly Reed
Conservation Fund and the Fresno Chaffee Zoo. We thank
all our collaborators, staff, volunteers, interns, and students,
without whose efforts this work would not have been
possible.
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Wilting A, Cord A, Hearn AJ, Hesse D, Mohamed A, Traeholdt C,
Cheyne SM, Sunarto S, Jayasilan MA, Ross J, Shapiro AC, Sebastian
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PLoS ONE 5(3): e9612. doi: 10.1371/journal.pone.0009612
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160209
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 156-160
The nutritional quality of captive sambar deer (Rusa unicolor
brookei Hose, 1893) velvet antler
GONO SEMIADI♥, YULIASRI JAMAL
Research Centre for Biology, Indonesian Institute of Sciences, Jl. Raya Jakarta-Bogor Km. 46, Cibinong Bogor 16911, West Java, Indonesia.
Tel./Fax.:+62-21-8765056; email: semiadi@yahoo.com
Manuscript received: 7 May 2015. Revision accepted: 30 June 2015.
Abstract. Semiadi G, Jamal Y. 2015. The nutritional quality of captive sambar deer (Rusa unicolor brookei Hose, 1893) velvet antler.
Biodiversitas 16: 156-160. Deer farming has been a well-developed agriculture diversification worldwide since 1970s. To the present
time information concerning the nutrient value of velvet antler of sambar deer (Rusa unicolor brookei Hose, 1893) is limited. Therefore,
a study on the nutritional quality of velvet antler of captive sambar deer was conducted. Velvet antlers were obtained from captive
sambar deer in Penajam Paser Utara, East Kalimantan, Indonesia, and were analyzed for its nutritional quality from the hard and soft
parts. The results showed that fresh weight of a pair of velvet antler (approx. 70 days post hard antler cast) was 523.1 g (SE = 49.99). In
the soft part of the velvet antler, ash content was 25.9% DM (SE= 0.78) as compared to 40.4% DM (SE = 1.07) in hard part, whilst the
lipid and protein contents from the soft part were 3.3% DM (SE = 0.20) and 70.8% DM (SE = 2.07), respectively, higher compared to
those in the hard part being 1.9% DM (SE = 0.12) and 59.5% DM (SE = 1.92), respectively. From the study it can be concluded that the
production of velvet antler from captive sambar deer seemed to be far from its genetic potency, and the nutritional qualities of the velvet
antler contents were not different from the red deer Cervus elaphus.
Key words: nutritional quality, production, Rusa unicolor brookei, sambar deer, velvet antler
INTRODUCTION
Deer farming has been one of the fastest developed new
livestock diversification worldwide since 1970s (van den
Berg and Garrick 1997, Hoffman and Wiklund 2006). At
present, the species being bred are not only limited to
temperate origin species, but also from tropical part, such
as rusa deer (Rusa timorensis) and sambar deer (Rusa
unicolor; Sookhareea et al. 2001; Haigh 2002), although
farming the sambar deer is very limited to Australia,
Malaysia, and Thailand. The main products of the deer
farm are venison and velvet antler, with by-products known
as pitzel, dry tail and sinew are still having high market
values in oriental markets, such as in Korea and China
(Kong and But 1985; Kim 2001; Kim et al. 2004).
The culture of using velvet antler among Chinese
medical practitioners as part of their traditional medicine
has been known for hundred years. Some claims on those
Chinese beliefs on the power of velvet antler in
maintaining health, particularly related to rheumatism and
the improvement of body vitality have been scientifically
proven (Allen et al. 2002; Frolov et al. 2001; Shin et al.
2001; Sim and Sunwoo 2001). Study also showed that the
extract of velvet antler of Formosan sambar deer (Rusa
unicolor swinhoei) has the potential as the anti-infective
activity against pathogenic Staphylococcus aureus (Dai et
al. 2011). This accelerates the development of food
supplement industry from velvet antler origin in the western
part, in the forms of powder, slices, extract or tonic.
Indonesia has three native deer species; the Javan deer,
sambar deer and Bawean deer (Axis kuhlii), besides the
deer introduced from India, spotted deer (Axis axis). The
distribution of sambar deer in Indonesia is limited to
Sumatra and Kalimantan islands (Wilson and Reeder
2013), whereas in Kalimantan the utilization of sambar
venison is very high through poaching activities. From one
district, no less than 120 heads of sambar per month were
poached, providing no less than 234 kg venison that was
sold in traditional market. Average of carcass weight from
stag was 74.99 kg and from hind was 63.06 kg (Semiadi et
al. 2004).
Under the Indonesian Wildlife Law, the native deer
species are protected animals. However, they are possible
to be utilized once has been bred in captivity. In 2002 a
Decree of the Minister of Agriculture was issued
concerning the inclusion of deer as prospective livestock.
However, the execution of this regulation is far from the
reality. The latest Indonesian Animal Husbandry and
Veterinary Law (UU no. 18/2009) has made possible for
wildlife animal that has been bred in captivity, specifically
for production purposes, to be adopted as a domesticated
animal. The development of deer farming in Indonesia was
initiated in 1990 by the establishment of a pilot project of
sambar deer farm (Rusa unicolor brookei, Hose 1893) in
Penajam Paser Utara District, East Kalimantan. At present,
the number of deer being bred reaches 301 deer in an area
of 15 ha paddock (IG Ngurah, pers. comm.).
To the present time, information related to the
production of sambar deer is still very limited (Ismail and
Hanoon 2008), and so does with the velvet antler quality
(Jamal et al. 2005). Therefore, as part of the development
strategy of deer farming in Indonesia, it is necessary that
SEMIADI & JAMAL – The nutritional quality of captive sambar deer
the information related to the quality of velvet antler of
sambar deer and the possibility of utilizing them as a food
supplement is available. The purpose of this study was to
understand the quality of captive sambar velvet antler from
its nutritional values.
MATERIALS AND METHODS
The research was conducted in a sambar deer farm in
Penajam Paser Utara District, East Kalimantan. Velvet
antlers were harvested from eight mature (> 4 years) stags,
placed in a drop floor crush while velvet antler cutting was
conducted. The cutting time for velvet antler was
determined based on the antler physical condition, in which
the main beam has not yet branched.
Method of velvet cutting followed Wilson et al. (2000).
On each of velvet antlers, around its ring block, local
anesthetic injections were administered using Lidocaine
(2% lidocaine hydrochloride) approximately 6-8 ml/antler.
The subcutaneous injections were circled in four to five
places using a syringe of 1" x 20 G. After the injection,
approximately 3-5 minutes later, tourniquet was placed
around each ring block and velvet antler was cut.
Once it had been cut, the harvested velvet antlers were
directly turned upside down and slanted 150 for five
minutes to avoid excessive blood lost. They were cleaned
from dust, weighed, and the length and diameter of the
antler were then measured using polypropylene meter tape
and digital micro-caliper (Yamato, Japan), respectively,
twice each, then were put into plastic bag, labeled and
stored in a freezer (-5oC). Velvet antler diameter was
measured at the mid-point of the antler length. Prior being
transported to the laboratory, the velvet antlers were put
into ice boxes containing blue chips ice cooler to maintain
their freezing level during the traveling. The time of arrival
in laboratory was six hours with the antlers condition were
still frozen, and then put into a deep freezer (-20oC) until
the laboratory analysis process.
Before the velvet antler being processed, the antlers
were thawed and the soft hair was burnt on a pressurized
gas Bunsen burner. Left and right parts of the soft velvet
antler part were then manually sliced thinly with a sharp
knife, with a thickness of 3-5 mm, until it reached the hard
part (semi ossified) that could not be cut manually by
ordinary knife. The hard part was then cut into small pieces
using a chopping knife. All samples that had been cut (soft
and hard parts) were freeze dried for 18-24 hours, then
ground using hammer mill (Retch Muhle, Germany) to
pass through a sieve of 1 mm.
Analyses of nutrient content of antler were only done
on the samples taken from the right side of the antler,
representing the hard and soft parts. Moisture content was
analyzed by putting into a ventilated oven at 105oC for 18
hours. The ash content was determined using a furnace at
550oC for 12 hours (AOAC 2005). All analysis was
conducted in Nutrition Laboratory, Zoological Division of
the Research Center for Biology, Indonesian Institute of
Sciences (LIPI), Cibinong Bogor, West Java.
157
Mineral content analysis was conducted using the
Atomic Absorption Spectrometry (AAS), for amino acids
with chromatography technique (HPLC), fatty acids with
gas chromatography technique (GC), total lipid with
soxhlet technique and total nitrogen with Kjehdhal
technique (AOAC 2005), conducted at the Integrated
Chemical Laboratory, Faculty of Mathematics and Natural
Science, Bogor Agricultural Institute (IPB), Bogor, West
Java. The conversion to crude protein content was done by
a multiplying factor of 6.25 to the nitrogen content.
Analyses of chemical components of antler were done
on the samples taken from the left part of the antler,
representing the hard and soft parts. Each sample was
extracted with ethanol absolute placed in shakers (100 rpm
per minute). The addition of alcohol solvent was done three
times every 24 hours, 100 ml each, until reaching a total
volume of 300 ml for each sample. The result of ethanol
extraction was then evaporated using rotating evaporator at
50oC until it was free from the solvent. This process was
conducted at the Phytochemistry Laboratory, Botany
Division, Research Center for Biology, Indonesian Institute
of Sciences (LIPI), Cibinong, West Java, Indonesia.
The analysis on chemical compounds from the extract
was carried out at the Department of Natural Product
Chemistry, Faculty of Pharmacy and Pharmaceutical
Science at Fukuyama University, Hiroshima, Japan using
Gas Chromatography Mass Spectrometry (GCMS system
electron impact, Shimadzu Qp-5000, Japan), using the TC17 column (L=30 m, = 0.25 mm, GL Science USA). The
injection volume was 0.1 ul. The carrier gas was helium
with a gas speed flow of 1.36 ml/minute and a column
pressure of 90 kPa. The column temperature was
programmed to be 100oC to 270oC with a temperature
increase of 3oC per minute. The detector temperature
(quadruple) was programmed to be constant at 270oC with
energy of 1.25 kV. The injector temperature was 300oC.
Identification of each peak was done using spectrum mass
authentic from the National Institute Standard of
Technology (NIST) library, ver. 6.2. Data were analyzed
using the general linear model procedure of SAS ver. 9.0
(SAS 2002). Whenever, appropriate data were analyzed
either by regression for the antler morphometry, and T-test
or factorial analysis between hard and soft parts against its
nutrient values (Steel and Torrie 1980).
RESULTS AND DISCUSSION
Due to the lack of good management practice in
managing stags for velvet antler production, it was difficult
to obtain accurate data concerning the age of velvet antler
growth when it was harvested. Therefore, observation on
the shape of the main beam which almost to branch was
used as an indicator for the optimum cutting time to obtain
the best quality of velvet antler. On red deer (Cervus
elaphus), cutting age of velvet antler was at 60-65 days
post hard antler drop (Sunwoo and Sim 2001), coincided
with unbranched condition of the main beam. However,
from this study it was predicted that the ages of velvet
158
B I O D I V E R S IT A S 16 (2): 156-160, October 2015
antler of sambar deer that were harvested had never been more
than 70 days old post antler cast (A Trasodiharjo pers. comm.)
The fresh mean weight of a pair sambar deer velvet
antler was 523.1 g (SE = 49.99; n = 8) with the average
overall moisture content of the velvet antler being 74.1 %
(SE = 1.78; n= 8). The soft part of the velvet antler had its
moisture content of 30.1% (SE = 4.0; n= 8) of the total dry
matter weight. The velvet antler dimensions of the right
and left parts showed a symmetrical shape, therefore no
significant differences were found (Table 1). However, one
antler did not have its first tine. The correlation between
the length and diameter of the main beam was much higher
(R2= 0.75; p<0.001) compared to that of the first tine (R2=
0.55; p<0.008). A symmetrical shape of antler was also
showed in majority of wild Javan deer (Semiadi 1997).
The production of velvet antler cut that only reached
0.5 kg fresh weight per cutting time, indicated that the
genotype quality of sambar stags as velvet antler producer
was far from the expected. In Thailand, sambar deer velvet
antler at 74-92 days of harvest age, could weight 820-1,640
g per side, with the length and circumference were 41-63
cm and 11.5-17.2 cm per side, respectively (Liangpaiboon
et al. 1996). While in farmed red deer, the velvet antler
production can reach up to 2.0 kg/cutting time (Haigh and
Hudson 1993; Jeon and Moon 2001) and can be predicted
from the age with an average genetic correlation of 0.74
with heritability estimates age from 2 to 8 years being 0.43
to 0.85 (van den Berg and Garrick 1997). Ismail and Jiwan
(2013) suggested that the best velvet antler harvest time in
sambar deer is at 49-63 days post hard antler casting when
the antler length is 25-30 cm.
The low antler production in this study seems to be
related with the unselected breeding system of sambar stags
being managed in the premises. From the body weight
condition, sambar deer were about 30-40% heavier than red
deer, therefore it was still possible to have a higher velvet
antler production, once the breeding system has been set
up. In red deer industry, grading system of velvet antler
was based on the combinations between the length,
diameter and weight. Similar system must also be started to
be developed for the velvet antler of sambar deer.
The results of ash, lipid and protein contents showed a
very significant differences between parts of the antler (soft
and hard) on the ash and lipid (p<0.001) and protein
contents (P<0.005; Table 2). In the soft part, ash content
was 36% lower, but lipid and protein contents were
approximately 74% and 19%, respectively, higher than
those were in the hard part. Values for protein and ash
contents in this study were close to that of wapiti, sika and
its hybrid deer species velvet antlers, except for ash, the
current study tended to have 45% lower (Wang et al. 2004).
While from the Formosan sambar deer, the protein content
of velvet antler was reported as 64% DM at the harvesting
age of 75 ± 2 days (Yun et al. 2009).
The best grade of velvet antler under the Korean market
system is when the soft part of antler has the ash content
below 25% DM (Kang et al. 2001). In this study, the soft
part contained 25.9% DM ash, in which it was 30.1% of the
antler total dry weight. Therefore, as a starting step, the use
of physical parameter in cutting time on the velvet antler in
this study had a practical good indicator and almost
fulfilled the quality requirement under the Korean standard.
There was no interactive correlation obtained between
parts of antler (hard and soft) and the types of fatty acid,
however, the concentrations of fatty acid was significantly
different (p<0.001) between the two antler part, as well as
the type of fatty acid contents (p<0.1085; Table 3).
Palmitic acid was the highest, while mistiric acid being the
lowest concentration.
In term of amino acid contents, a very strong
correlation was obtained (p<0.001) between the types of
amino acids and antler parts (Table 4). Velvet antler of
sambar deer contained 8 out of 9 essential amino acids,
with only tryptophan group undetected. There was a
tendency that amino acid concentration in the soft part was
higher than that in the hard part. Glycine and glutamate
groups were the ones with the highest concentration,
followed by alanine, arginine and aspartate. Whilst
methionine had the lowest concentration.
From Table 3 it can be seen that all fatty acid in velvet
antler consisted of long chain fatty acid (14-24 carbon
atoms), with the highest concentration was palmitic acid.
Linoleic and linolenic acids are known to function in
maintaining cell structure and cell membrane. These two
fatty acids are also functioned as the principle materials in
synthesizing hormones, for the formation of blood
coagulant, blood pressure, blood lipid concentration,
immune response, responses on wounds and infections
(Mahan and Arlin 1989). In the present study the content of
both compounds were relatively low compared to the other
fatty acid compounds.
Table 1. Fresh sambar deer velvet antler dimension used in the study.
Parts
Right
Left
Main beam (mean SE;
First tine (mean SE; n=7)
n=8)
Length
Diameter
Length
Diameter
(cm)
(mm)
(cm)
(mm)
22.6 (0.55) 29.9 (0.63) 13.0 (0.63)
22.5 (0.70)
22.9 (0.61) 29.8 (0.60) 12.1 (0.89)
22.4 (0.67)
Table 2. Chemical compositions of sambar deer velvet antler.
Part
Hard
Soft
Ash
(%DM SE;
n=16)
40.4 (1.07)
25.9 (0.78)
Fat
(%DM SE;
n=12)
1.9 (0.12)
3.3 (0.20)
Protein
(%DM SE;
n=6)
59.5 (1.92)
70.8 (2.07)
Table 3. Fatty acid composition from sambar deer velvet antler.
Fatty acids
Behenic acid
Lignoseric acid
Linoleic acid
Linolenic acid
Myristic acid
Oleic acid
Palmitic acid
Stearic acid
Hard part
(%DM SE; n=6)
2.49 (0.227)
0.81 (0.033)
0.48 (0.113)
0.35 (0.022)
0.22 (0.033)
2.41 (0.496)
8.39 (0.382)
2.96 (0.491)
Soft part
(%DM SE; n=6)
2.60 (0.227)
0.74 (0.097)
0.74 (0.088)
0.39 (0.032)
0.26 (0.030)
3.64 (0.561)
8.59 (0.315)
3.01 (0.241)
SEMIADI & JAMAL – The nutritional quality of captive sambar deer
The essential amino acids concentration showed that the
soft part had higher content than that in the hard part. Jeon
et al. (2004) showed that feed sources created differences
in velvet protein, lipid, amino acid and mineral
composition. From this study, glycine was the amino acid
with the highest concentration in both parts. Comparing the
amino acid composition in velvet antler from red deer and
sambar deer, it shows the consistency of glycine as the
highest concentration component among all amino acids.
However, the concentration in red deer is higher than in
sambar deer (15.58% vs. 9.07%; Sim and Sunwoo, 2001).
It was further reported that the amino acid group of aspartic
acid, glycine, alanine and glutamic acid were the major
components in temperate deer velvet antler. By observing
cholesterol concentration or its derivates, velvet antler
contained relatively low cholesterol compounds compared
to other saturated fatty acids. The ethanol extract of velvet
antler from both the soft and hard parts, consists of
unsaturated long chain fatty acids, carboxylic acids with
amine chains and one compound of cholesterol derivative.
Minerals content showed that there was a high
significant interaction (p<0.001) between antler parts with
the types of minerals (Table 5). Iron (Fe) was the
microelement with the highest concentration in the velvet
antler, in which the concentration in the soft part was 65%
higher than that in the hard part. Calcium was the macro
mineral with the highest concentration. Bubenik et al.
(2005) had shown that mineralization of temperate deer
velvet antler was affected by the role of 17β estradiol (E2).
The analysis of chemical compound using GCMS
resulted in unsatisfying outcome, as it was only capable of
detecting fatty acid groups, but not the steroid hormone
groups. Analysis of both antler parts, showed that both had
14 identical chemical compounds, but in different
concentrations (Table 6).
Minerals play important roles in oxygen transportation,
nervous system, the formation and maintenance of the bone
itself. The range of minerals content in velvet antler of red
deer is very wide (Table 7), considering that these very
much depend on the calcification level in antler when being
cut. Calcium is the most dominant one, considering its role
in the formation and maintenance of bone. In sambar deer,
159
the mineral content in the hard part almost twice higher
than that in the soft part. However, in general, the minerals
content of the present sambar deer velvet antler is within
that of the red deer (Table 7)(Haines and Suttie 2001).
From this study it can be concluded that the production
of velvet antler of captive sambar deer was still far from its
optimum potency. Whereas, the nutrient contents were
close to that of red deer velvet antler values.
Table 4. Amino acid compositions from sambar deer velvet antler.
Amino acids
Alanine
Arginine
Aspartate
Phenylalanine
Glycine
Glutamate
Histidine
I-Leuisine
Leusine
Lysine
Methionine
Serine
Threonine
Tyrosine
Valine
Hard part
(%DM SE; n=6)
4.24 (0.135)
4.10 (0.122)
3.57 (0.141)
1.57 (0.079)
9.07 (0.226)
5.61 (0.187)
0.86 (0.071)
0.99 (0.039)
2.54 (0.133)
2.37 (0.116)
0.59 (0.022)
2.32 (0.099)
1.86 (0.095)
0.94 (0.052)
1.78 (0.091)
Soft part
(%DM SE; n=6)
4.46 (0.208)
4.54 (0.216)
4.63 (0.173)
2.19 (0.097)
8.40 (0.517)
6.95 (0.249)
1.36 (0.097)
1.43 (0.046)
3.77 (0.148)
3.20 (0.141)
0.93 (0.021)
3.04 (0.125)
2.56 (0.107)
1.55 (0.041)
2.52 (0.106)
Table 5. Mineral compositions from sambar deer velvet antler.
Mineral
Ca (%)
K (%)
Mg (%)
Na (%)
P (%)
Co (ppm)
Cu (ppm)
Fe (ppm)
Mn (ppm)
Se (ppb)
Hard part (DM
SE; n=6)
6.87 (0.504)
0.20 (0.028)
0.35 (0.030)
0.65 (0.04)
0.21 (0.005)
0.05 (0.000)
7.91 (1.082)
147.20 (23.950)
3.72 (0.39)
1.00 (0.289)
Soft part (DM SE;
n=6)
3.97 (0.087)
0.45 (0.039)
0.23 (0.019)
0.94 (0.119)
0.20 (0.005)
0.05 (0.00)
9.16 (1.701)
223.73 (28.328)
3.80 (0.413)
0.85 (0.247)
Table 6. Chemical compositions from ethanol extraction of sambar deer velvet antler.
Components
Methyl ester decanoic acid
Hexadecanoic acid
Palmitic acid
12-methyl-methyl ester tetradecanoic acid
Methyl ester 13-docosenoic acid
Methyl ester 11,14-eicosadienic acid
Oleic acid
Undeconoic acid
2-methyl-2-(dimethylamino) ethyl ester 2-propenoic 1 acid
2-methyl (dimethylamino) ethyl ester 2-propenoic acid
Propyl hexedrine
9-octadecenamide
2-(dimethylamino) ethyl ester
Dicholesteryl sucsinate
Molecular
structure
C11H22O2
C16H32O2
C15H30O2
C16H32O2
C23H44O2
C21H38O2
C18H34O2
C11H20O2
C7H13NO2
C7H13NO2
C10H21N
C18H35NO
C18H35NO2
C58H94O4
Molecular
weight
186
256
256
256
352
322
282
184
157
143
155
281
157
854
Retention
time
18.4
19.6
19.8
21.1
21.2
21.4
22.3
22.6
23.4
25.8
26.0
26.4
28.3
43.0
Proportion (%)
Soft
Hard
2.50
2.47
11.57
5.65
7.93
10.37
7.71
8.22
1.05
0.64
1.86
2.45
17.26
16.86
8.49
10.87
3.86
2.64
7.09
4.85
1.53
1.55
1.42
1.21
1.72
1.94
23.17
25.20
160
B I O D I V E R S IT A S 16 (2): 156-160, October 2015
Table 7. Mineral compositions of red deer velvet antler by infra
red spectroscopy technique from freeze dry sample (Haines and
Suttie 2001).
Components
Mean
Range
Ash (%)
Fat (%)
N (%)
Ca (%)
P (%)
Fe (ppm)
37.0
0.56
8.5
12.2
5.9
347
7.6-61.0
0.01-1.72
5.3-12.6
0.1-22.0
0.3-9.6
33-970
ACKNOWLEDGEMENTS
Authors wish to thank to Dr. Andria Agusta of
Phytochemistry Laboratory, Botany Division, Research
Center for Biology, Indonesian Institute of Sciences (LIPI),
Cibinong Bogor, West Java, for his generous time in
analyzing the samples which could not be done in our
laboratory. We are also grateful to the Animal Husbandry
Division, East Kalimantan Province c/q Sambar Deer
Captive Breeding Project management in Penajam Paser
Utara, East Kalimantan for allowing us to conduct the
study.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160210
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 161-167
Structure of natural Juniperus excelsa stands in Northwest of Iran
FARZAM TAVANKAR
Department of Forestry, Khalkhal Branch, Islamic Azad University, Khalkhal, Ardabil Province, Iran. P.O. Box 56817 - 31367, Tel.: +98 4532451220-2,
Fax: +98 42549050452, email: tavankar@aukh.ac.ir
Manuscript received: 7 April 2015. Revision accepted: 1 July 2015.
Abstract. Tavankar F. 2015.Structure of natural Juniperus excelsa stands in Northwest of Iran. Biodiversitas 16: 161-167. Juniperus
excelsa M. Bieb. stands are important forest ecosystems in mountain areas of Iran. In this research the structure of J. excelsa stands was
studied at altitudes of 1,400 to 1,900 m in northwest of Iran. The results showed that the mean densities of trees and seedling (trees with
height up to 1.3 m) were 99.8 ± 32.0 and 70.5 ± 18.3 stem ha-1, respectively. The juniper trees comprise 52.8% of the total tree density,
while the juniper seedlings comprise 19.3% of total seedling density. The mean of basal area in this stand was 3.12 ± 0.3 m2 ha-1 and the
mean of canopy cover was 42.7 ± 17.9 percent. The mean of trees height was obtained 2.75 ± 1.1 m. The total of 28 woody species
belonging to 14 families was recorded from the study area. The juniper trees had the maximum value of species importance value
(SIV=87.3). Amygdalus lyciodes and Pistacia atlantica were two tree species that have high SIV values, 34.1 and 27.0, respectively.The
distribution of trees density in different tree diameters resemble to a reverse J-shaped indicating uneven-aged structure. The results
indicated juniper seedlings were increased by increasing stand crown cover (P< 0.01). Moreover extreme environmental conditions,
grazing and timber harvesting for firewood are two important socioeconomic problems in these forests. These valuable stands needs to
urgently conservation strategies.
Keywords: Juniperus excelsa, stand structure, species importance value, natural regeneration
INTRODUCTION
Junipers (Juniperus spp.), containing 60 species and
spreading among many different temperature environments
from the northern hemisphere to Southern Africa, are
evergreen trees and shrubs (Assadi 1997; Deligoz 2012).
Although most juniper trees are unisexual (dioecious), but
there are also monoecious individuals. The seed production
is very low in these trees (Javanshir 1981; Ahani et al.
2013). Juniper stands cover an area of 1.3 million ha in Iran
(Marvie-Mohadjer 2006), and the genus Juniperus is
represented by six species: J. sabina L., J. communis L., J.
oxycedrus L., J. foetidissima, J. oblonga M. Bieb and J.
excelsa M. Bieb (Javanshir 1981; Korouri et al. 2011). The
J. polycarpos and J. excelsa are the most common species
among the six juniper species in the Iran (Korouri and
Khosnevis 2000; Ahani et al. 2013). The J. excelsa
(Cupressaceae) grow naturally over most of the
mountainous areas of Iran (south slopes in high mountains
of Elburz, Arassbaran, and Northern parts of Khorassan),
and have a great ecological importance (Korouri and
Khosnevis 2000; Marvie-Mohadjer 2006; Taheri Abkenai
et al. 2012). J. excelsa exhibits growth plasticity and can
adapt and grow in diverse regimes (shade - light), while, in
favorable conditions, it is able to increase its growth rates
even at old ages (Milios et al. 2009). Their vital needs are
limited (Deligoz 2012; Ramin et al. 2012). The juniper
trees can grow from lowland at sea level up to an altitude
of 3,600 m depending on latitude, so elevation has an inreverse relation with latitude (Javanshir 1981; Fisher and
Gardner 1995; Zangiabadi et al. 2012). J. excelsa usually
appears in mountainous areas (Ahmed et al. 1990; Fisher
and Gardner 1995; Sabeti 2006; Milios et al. 2009). J.
excelsa is a cold resistant species but their seedling requires
a high degree of humidity (Aussenac 2002; Ozkan et al.
2010) and shady conditions (Ahani et al. 2013). J. excelsa
can grow not only in harsh abiotic environments such as
shallow and stony soils, cold, hot and dry climates, but also
have ability to grow in extreme biotic conditions like
grazed sites (Ahmed et al. 1989; Fisher and Gardner 1995;
Korouri and Khosnevis 2000; Carus 2004; Stampoulidis et
al. 2013). They are important food sources for wildlife,
several bird species feed on juniper cones (Decker et al.
1991). This species is capable of protecting the soil; it has
high resistance against cold weather and can grows in areas
where the minimum of temperature reaches to -35° C
(Ghahreman 1994; Korouri and Khosnevis 2000; Aussenac
2002; Taheri Abkenai et al. 2012).
In a study by Ahmed et al. (1989) natural regeneration
of J. excelsa was investigated in Balouchistan, Pakistan and
reported regenerating seedlings ranged from zero to 219
stem ha-1 with a mean of 52 stem ha-1 that the highest
seedling density and basal area were recorded on west
facing slopes. The researchers also indicated that the
seedling density was significantly correlated with tree basal
area of the parent trees. An investigation on the distribution
and ecology of juniper genus was conducted as a national
plan in the Iran (Korouri and Khosnevis 2000). According
to previous studies the natural regeneration of juniper trees
is very low and difficult due to grazing and tree felling for
firewood by villagers in Iranian juniper forests
(Ravanbakhsh et al. 2010; Shirzad and Tabari 2011; Ramin
et al. 2012; Taheri Abkenai et al. 2012). Shahi et al. (2007)
studied age structure and seed characteristics of juniper
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B I O D I V E R S IT A S 16 (2): 161-167, October 2015
stands in the northwest of Iran and reported that due to high
rate of seed production there is no extinction risk for
species. The researchers also reported about 13% of
individuals were damaged by wild and domestic
herbivores. They concluded maintenance of natural juniper
regeneration needs to production of good quality seeds.
Juniper is a suitable tree species for afforestation in
semi-arid and arid areas (Javanshir 1981; Esmaeelnia et al.
2006; Ozkan et al. 2010; Deligoz 2012). The best age for
transferring saplings to natural areas is three years; a 86.9%
success rate was achieved in Iran by Koruri et al. (2011).
Ramin et al. (2012) studied properties of J. excelsa stands
in northern Iran and reported that average of canopy cover
was 7.5%, and the average tree height was 4.9 m. There is
some indication from previous studies that Physiographic
factors effects on attributes of juniper stands (Pourmajidian
and Moradi 2009; Khosrojerdi et al. 2010; Ravanbakhsh et
al. 2010; Maghsoudlou Nezhad et al. 2013). Momeni
Moghaddam et al. (2012) studied the impact of some
physiographic and edaphic factors on quantitative and
qualitative characteristics of J. excelsa stands in northeast
of Iran. They reported that the elevation has significant
effect on density of trees and regeneration, stand basal area,
slenderness ratio and crown diameter of trees. It was also
indicated that slope gradient has significant effect on trees
height and diameter, and pH and texture of soil are
important factors in distribution of juniper trees.
Pourmajidian and Moradi (2009) studied site
characteristics and silvicultural properties of J. excelsa
stands in southern slopes of Alborz Mountain in Iran. They
reported site characteristics were different from altitude,
slope direction and soil properties. The researchers also
indicated that the silvicultural properties of J. excelsa
stands such as tree and seedling density, basal area and
canopy cover depended to site characteristics. Their results
indicated that the seedling density of J. excelsa was
increased by increasing stand basal area and canopy cover.
Maghsoudlou Nezhad et al. (2013) studied quantitative
characteristics of J. excelsa stands in Gorgan province in
northern Iran. The researchers reported that landform units
were different significantly in terms of tree density, canopy
cover, tree diameter and stand basal area. They also
reported that the slope aspect and soil wetness indices are
the best predictors for the density of trees. J. excelsa stands
are widespread in Iran, under different environmental
conditions. Cataloging silvicultural properties of juniper
stands is essential as a basis for monitoring and
management of these valuable forests (Gardner and Fisher
1996; Pourmajidian and Moradi 2009).
Preparation and planning for biodiversity conservation
and sustainable management of forest ecosystems is needed
to identify the exact structure of the forest (Zenner and
Hibbs 2000; Haidari et al. 2012; Tavankar 2013). The main
objective of the present study was to investigate structure
of J. excelsa stands, such as tree density, species
compositions, trees height and diameter, stand basal area,
canopy cover, regeneration, and stand structure in
Northwest of Iran and compare these data with silvicultural
properties from other sites.
MATERIAL AND METHODS
Study area
The study area is located in the Ardebil Province in the
northwest of Iran (latitude 37° 27' 14" to 37° 27' 50" N,
longitude 48° 22' 25" to 48° 23' 10" E) (Figure 1). The
elevation of the study area ranges from 1,400 to 1,900 m
above sea level. The mean annual temperature is 12.5°C
and the mean annual precipitation is 380 mm for the years
1990 to 2008. The slope aspect is southwestern and the
slope gradient ranges from 24 to 63% with an average
37%. The soil type is lithic lithosol and texture varies
between clay loam to loam. The original vegetation of this
area is natural uneven-aged mixed stands of Juniperus
excelsa with the companion species.
Figure 1. Study site in in the Ardebil Province in the northwest of Iran
TAVANKAR –Juniperus excelsa in Northwest of Iran
Data collection and analysis
Data were collected in summer 2014, by systematic
sample plots with an area of 400 m2 (20 m × 20 m). The
number of sample plots was 55. The sample plots were
located on the study area (59 ha) through systematic grid
(100 m × 100 m) with a random start point. Diameter at
breast height (DBH) and heights of all trees (height ≥1.3
m) were measured. Individuals of trees with height < 1.3 m
were counted by species as seedling (Stampoulidis et al.
2013). Species importance values determine the dominant
species in an area and at the same time provide an overall
estimate of the influence of these species in the community
(Amoroso et al. 2011). Species importance value (SIV) for
each tree specious was calculated by: SIV= Relative
density (RD) + relative frequency (RF) + relative
dominance (RD). Basal area was considered for dominancy
and relative dominance (RD) calculated by: RD = (basal
area of a species × 100) / total basal area of all species
(Amoroso et al. 2011; Pourbabaei et al. 2013; Tavankar
and Bonyad, 2015). Confidence intervals on the means
calculated as accuracy by: CI% = (
,
where CI is confidence interval,
is standard error, t95% is
t value in 95% confidence level from t table and is mean
(Zobeiri 2007). After checking for normality of data
distributions (Kolmogorov-Smirnov test) and homogeneity
of variances (Levene’s test), regression analysis was
applied to test of the relations between DBH and trees
density and trees height. The means of juniper seedlings in
crown cover classes were compared using Analysis of
Variance (ANOVA) test and multiple comparisons were
made by Tukey’s test (significant at α < 0.05). SPSS 19.0
software was used for statistical analysis; also the results of
the analysis were presented using descriptive statistics.
RESULTS AND DISCUSSION
Results
The structural properties of the studied juniperstand are
shown in Table 1. The mean densities of trees and seedling
(trees with height up to 1.3 m) were 99.8 ± 32.0 (SD) and
70.5 ± 18.3 stem ha1, respectively. Juniper trees comprise
52.8% of total trees density, while juniper regeneration
comprises 19.3% of total seedling density in the study area.
The means of trees diameter (DBH) and trees height were
obtained 10.1 ± 6.5 cm and 2.75 ± 1.1 m, respectively. The
means of juniper trees diameter (8.4 cm) and trees height
(2.53 m) were lower than the mean diameter (13.7 cm) and
height (3.12 m) of other tree species. The mean of basal
area in this stand was obtained 3.12 ± 0.3 m2 ha-1 and the
mean of canopy cover was obtained 42.7 ± 17.9 percent
(Table 1).
A total of 28 woody species belonging to 14 families
were recorded from the study area (Table 2).The family of
Rosaceae with 9 species had the highest number of woody
species in the study area that includes Amygdalus lyciodes,
Crataegus songarica, Prunus divaricata, Sorbus
torminalis, Malus orientalis, Amygdalus scoparia, Cerasus
microcarpa, Cotoneaster nummularia and Rosa canina.
The families of Caprifoliaceae, Rhamnaceae and Aceraceae
163
had 4, 3 and 2 woody species, respectively; and each of
other families had only one species.
The density of different tree species is shown in Table
2. The total density of trees was 99.8 stem ha-1. The density
of juniper trees (J. excelsa) was 52.7 stem ha-1, which
comprise 52.8% of the total tree density. After juniper
trees, the species of Amygdalus lyciodes, Pistacia atlantica
and Acer monspessulanum have the highest density, (9.1,
7.4 and 5.5 stem ha-1, respectively) in the study area. These
species (A. lyciodes, P. atlantica and A. monspessulanum)
comprise 22% of the total tree density. Other tree species
(24 species) comprise 25.2% of the total tree density.
The total basal area in the study area was 3.12 m2ha-1,
that the J. excelsa had the highest value of basal area (0.67
m2ha-1, 21.5%). The basal areas of A. lyciodes, P. atlantica
and A. monspessulanum were 0.32, 0.19 and 0.17 m2ha-1,
respectively. These species comprise 21.8% of the total
basal area. Other tree species (24 species) comprise 56.7%
of the total basal area (Table 2).
The total seedling (regeneration) density was 70.5 stem
ha-1, that the Amygdalus lyciodes with 14.2 stem ha-1
(20.1%), had the highest frequency of seedlings in the
study area. The Pistacia atlantica had 13.8 stem ha-1
(19.6%) and the J. excelsa had 13.6 stem ha-1 (19.3%) of
seedling density. The seedling density of Acer
monspessulanum was 6.3 stem ha-1 (8.9%). Other tree
species (24 species) comprise 32.1% of the total tree
density (Table 2).
Species Importance Value (SIV) of different tree
species in the natural junipers stands is shown in Table 2.
Eight species (J. excelsa, A. lyciodes, P. atlantica, A.
monspessulanum, L. nummulariafolia, R. spathulaefolia, P.
spina Christi, A. campestre) have SIV more than 10 and
formed over 73% of total SIV. The SIV of J. excelsa was
the highest (87.3) in the study area. The SIV of A. lyciodes,
P. atlantica and A. monspessulanum were 34.1, 27.0 and
19.6, respectively.
Relation between diameter (DBH) and height of trees
are shown in Figure 2. According to this figure, the height
of juniper and other tree species were increased by
increasing of their DBH. The heights of juniper trees were
lower than the heights of other tree species in DBH more
than 4 cm. The regression analysis showed that the
correlation coefficient (R) between trees height and trees
DBH are statistically significant (P < 0.01).
The distribution of trees density in different DBH
resemble to a reverse J-shaped indicating uneven-aged
structure (Figure 3), so trees density were decreased with
increasing DBH. The density of juniper trees was more
than the density of other tree species up to DBH of 21 cm.
The regression analysis showed that the correlation
coefficient (R) between trees density and trees DBH are
statistically significant (P < 0.01).
The abundance of sampling plots in crown-cover
classes are shown in Figure 4. According to Fig. 3, the
crown-cover class 20 to 30 percent has the highest
abundance (29.1%) of sampling plots (n=16). The
abundance of sampling plots was decreased by increasing
crown cover class more or less than 20-30 percent.
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B I O D I V E R S IT A S 16 (2): 161-167, October 2015
The results of this study showed that the juniper
seedlings were increased by increasing crown-cover
percentages (Figure 5). The ANOVA test showed the
crown-cover classes had significantly affect on the mean of
juniper seedlings (F=3.67, P < 0.01).
Discussion
The results of this study showed that the density of trees
(height ≥1.3 m) was estimated to be about 100 stem ha-1.
The density of trees in other studies was reported as: 65 to
102 stem ha-1 in different land units of J. excelsa stands by
Zangiabadi et al. (2012) and 60 to 200 stem ha-1 by
Momeni Moghaddam et al. (2012) in southeast Iran; 32
stem ha-1 by Ramin et al. (2012), 188 stem ha-1 by
Pourmajidian and Moradi (2009) and 66 stem ha-1 by
Maghsoudlou Nezhad et al. (2013) in Northern Iran; 592
stem ha-1 in protected juniper stands by Rostami kia and
Zobeiri (2013) in Northwest Iran. Atta et al. (2012) density
of juniper trees (> 6 cm DBH) reported from 29 to 268
stems ha-1 with a mean 176 ± 77 individuals ha-1 in J.
excelsa forests in Balouchestan Pakistan. The density of
seedlings (trees with height up to 1.3 m) in this study was
estimated 70 stem ha-1.
Pourmajidian and Moradi (2009) reported the mean
density of seedlings 71 stem ha-1 in the J. excelsa stands in
the Northern Iran. They reported the highest seedlings
density on Northwestern facing slopes (102 stem ha -1 )
Table 1. Structural properties of a Juniperus excelsa stand in the
northwest of Iran
Stand properties
Max. Min. Mean SD
CI%
-1
Tree density (stem ha )
Juniper
68.3 12.6 52.7 12.8
Other
59.0 14.2 47.1 11.5
All
119.7 23.5 99.8 32.0
Seedling density (stem ha-1)
Juniper
19.4 2.5 13.6 4.8
Other
78.4 23.6 56.9 11.3
All
103.7 26.6 70.5 18.3
Tree diameter (cm)
Juniper
22.5 1.0 8.4
4.4
Other
28.4 1.0 13.7 9.4
All
28.4 1.0 10.1 6.5
Tree height (m)
Juniper
3.07 0.55 2.53 0.7
Other
5.33 0.72 3.12 1.1
All
5.33 0.55 2.75 1.1
Basal area (m2 ha-1)
Juniper
0.47 0.08 0.67 0.2
Other
0.86 0.15 2.45 0.3
All
1.30 0.32 3.12 0.3
Canopy cover (%)
Juniper
35.6 8.0 25.5 13.1
Other
29.3 7.7 17.2 10.5
All
55.2 10.3 42.7 17.9
Note: SD: Standard deviation, CI: Confidence interval
6.5
6.6
8.6
9.5
5.4
7.0
14.2
18.4
17.3
7.5
9.5
10.8
14.6
10.8
7.2
13.8
16.5
11.3
Table 2. Density of trees and seedling, basal area and species importance values (SIV)
Tree species
Family
Juniperus excelsa M. Bieb.
Amygdalus lyciodes L.
Pistacia atlantica F&M.
Acer monspessulanum L.
Lonicera nummulariafolia J.
Rhamnus spathulaefolia F&M.
Paliurus spina christi Mill.
Acer campestre L.
Berberis integerrima L.
Quercus macranthera Fish & Meyer
Carpinus orientalis Mill.
Crataegus songarica C. Koch
Prunus divaricata Ledeb.
Sorbus torminalis(L.) Crantz
Viburnum opulus L.
Viburnum lantana L.
Malus orientalis Ugl.
Eunymus latifolia(L.) Mill.
Amygdalus scoparia Spach.
Colutea persica Boiss.
Cerasus microcarpa(C.A.Mey)
Cotoneaster nummularia Pojark.
Cornus sanguinea L .
Lonicera iberica M.B.
Rhamnus pallasii F. M.
Rosa canina L.
Celtis caucasica Willd.
Jasminum fruticans L.
All species
Cupressaceae
Rosaceae
Anacardiaceae
Aceraceae
Caprifoliaceae
Rhamnaceae
Rhamnaceae
Aceraceae
Berberidaceae
Fagaceae
Corylaceae
Rosaceae
Rosaceae
Rosaceae
Caprifoliaceae
Caprifoliaceae
Rosaceae
Celastraceae
Rosaceae
Papilionaceae
Rosaceae
Rosaceae
Cornaceae
Caprifoliaceae
Rhamnaceae
Rosaceae
Ulmaceae
Oleaceae
-
Tree
(stem ha-1)
52.7
9.1
7.4
5.5
4.0
3.3
3.1
2.4
2.0
1.3
1.0
0.8
0.7
0.6
0.6
0.6
0.6
0.5
0.5
0.5
0.5
0.4
0.4
0.3
0.3
0.3
0.2
0.2
99.8
Basal area
(m2 ha-1)
0.67
0.32
0.19
0.17
0.16
0.14
0.13
0.12
0.12
0.14
0.12
0.10
0.12
0.11
0.08
0.10
0.10
0.07
0.05
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.03
0.01
3.12
Seedling
(stem ha-1)
13.6
14.2
13.8
6.3
2.4
2.0
1.3
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.8
0.8
0.8
0.5
0.5
0.5
0.5
0.5
0.5
0.5
70.5
SIV
87.3
34.1
27.0
19.6
15.7
14.0
12.8
10.3
7.5
9.4
7.5
6.2
4.6
4.2
4.2
4.1
3.0
3.1
4.0
3.1
2.7
2.5
2.5
2.2
2.2
2.2
2.0
2.0
300
TAVANKAR –Juniperus excelsa in Northwest of Iran
Figure 2. Relation between diameter and height of trees
Figure 3. Relation between diameter and density of trees
Figure 4. Abundance of sampling plots in crown-cover classes
Figure 5 . Density of juniper seedlings in crown-cover classes
165
and the lowest on Southeastern facing slopes (48 stem ha1
). The results of this study indicated juniper seedlings were
increased by increasing stand crown cover. Ahmed et al.
(1989) also concluded that juniper tree seedlings need
shady conditions in the early stages of their growth and
development. Reforestation by local tree species in empty
areas is necessary. Many studies have been noted that the
nurse trees have a major role in the success of juniper
regeneration (Ahmed et al. 1989, 1990; Fisher and Gardner
1995; Milios et al. 2007; Stampoulidis et al. 2013). In a
study by Khosrojerdi et al. (2010) reported that
establishment, growth and survival of juniper seedlings that
planted under Cotoneaster sp. and Rosa sp. as nurse trees
were more than the juniper seedling that planted in open
areas.
The mean basal area in the study area was obtained 3.12
m2 ha-1. The other researchers reported of basal area 4.67
m2ha-1 by Rostami Kia and Zobeiri (2013) in the Northwest
Iran, 1.09 m2ha-1 by Momeni Moghaddam et al. (2012) in
the Northeast Iran and 10.55 m2ha-1 by Maghsoudlou
Nezhad et al. (2013) in the northern Iran.
The mean height of trees was 2.53 m in the study area.
The mean tree height in the other J. excelsa sites were
reported 4.51 m by Momeni Moghaddam et al. (2012) in
the Northeast Iran, 4.9 m by Ramin et al. (2012) in the
Northern Iran, 2.9 m by Rostami kia and Zobeiri (2013) in
the Northwest Iran, 4.7 m by Pourmajidian and Moradi
(2009) in Northern Iran.
The results of this research showed that the juniper
stands are rich for tree species, so 28 tree species from 14
families were present in the study area in the northwest
Iran. After the J. excelsa, the three species of Amygdalus
lyciodes, Pistacia atlantica and Acer monspessulanum are
the important trees that have the highest tree and seedling
density and species importance values (SIV) in the study
area. Stampoulidis and Milios (2010) reported that in the
natural J. excelsa stands in the Greece there are also
species such as Quercus macedonica, Juniperus oxycedrus,
Quercus pubescens, Pyrus amygdaliformis, Carpinus
orientalis, Acer monspessulanum and Juniperus
foetidissima. Shirzad and Tabari (2011) reported 16 woody
species from 9 families in the J. excelsa stands in the
Northeast Iran. Rostami kia and Zobeiri (2013) reported 8
woody species from 7 families in J. excelsa stands in the
Northwest Iran. It is widely accepted in forest ecology that
different management practices affect species diversity,
and high diversity of plant and animal species is needed to
ensure more complex forest structure (Bacaro et al. 2014).
Many ecological, pathological, socioeconomic impacts,
increased human population, over- grazing, illegal cutting
for timber and collection for fuel wood, periodic drought,
and effect of climate change have been left adverse affects
on regeneration and structure of these forests. These factors
also reported as the main impacts on species composition,
productivity, structure and dynamics of juniper forests by
Atta et al. (2012) in Pakistan.
Considering to the results of this study and comparing
with previous studies it can be concluded that the J. excelsa
stands are very important forests in Iran. Conservation
166
B I O D I V E R S IT A S 16 (2): 161-167, October 2015
strategies for these stands are urgently needed. The
richness of woody species in these forests is high and this
issue is very important for conservation biodiversity. These
stands are open forests with low regeneration densities.
Forest protection should aim at ensuring that forests
continue to perform all their productive, socio-economic
and environmental functions in the future. It is widely
accepted, that a structurally diverse stand provides living
space for a larger number of organisms (Tavankar and
Bonyad 2015). Iran is a country with low forest cover
(UNFF 2005), so that only 7.3% of it is covered by forest
areas (FAO 2005). Therefore, the main objective of forest
policy is to protect forests in natural ecosystem. Moreover
extreme environmental conditions, grazing and timber
harvesting for firewood are two important socioeconomic
problems in these forests.
ACKNOWLEDGMENTS
This paper is one of the results of a research project that
was carried out in the period 2012-2013 in the Ardebil
province, North West of Iran. I would like acknowledge the
financial support of Islamic Azad University (IAU),
Khalkhal Branch for the research project No. 5/719.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160211
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 168-172
Phylogeny analysis of Colutea L. (Fabaceae) from Iran based on ITS
sequence data
1
LEILA MIRZAEI1,♥, IRAJ MEHREGAN1, TAHER NEJADSATARI1,♥♥, MOSTAFA ASSADI2
Department of Biology, Faculty of Basic Science, Tehran Science and Research Branch, Islamic Azad University, Hesarak-1477893855, Tehran, Iran.
Tel. +98-2147911. ♥email: l.mirzaei_2009@yahoo.com, ♥♥nejadsattari_t@yahoo.com
2
Research Institute of Forest and Rangelands, National Botanical Garden of Iran, P.O. Box 13185-116, Tehran, Iran
Manuscript received: 2 May 2015. Revision accepted: 13 July 2015.
Abstract. Mirzaei L, Mehregan I, Nejadsatari T, Assadi M. 2015. Phylogeny analysis of Colutea L. (Fabaceae) from Iran based on ITS
sequence data. Biodiversitas 16: 168-172. This study was carried out on the species of Colutea L. that growing in Iran. Some of these
species are native to Iran. Internal transcribed spacer (ITS) sequences were obtained from 12 samples representing seven species of
Colutea recognized by recent taxonomic treatments from Iran and we used 20 ITS sequences from GenBank to test the phylogeny of
Colutea. Phylogenetic analyses were conducted using Bayesian inference and maximum likelihood methods. Our results of cladistic
analysis of phylogenetic relationships among Coluteae tribes showed its monophyletic origin and Astragaleae was its sister group. In
addition, C. cilicica was a sister group to C. gifana whose separated of other Colutea genus from Iran. Bayesian inference and maximum
parsimony analyses confirmed the monophyly of three sections of Colutea in Iran. Our results showed that further investigation
including application of larger number of markers and involving all the Iranian Colutea species will be more effective in estimation the
relationships of the genus.
Keywords: Colutea, Fabaceae, Iran, ITS, phylogeny
INTRODUCTION
Fabaceae is the third largest family of angiosperms with
730 genera and more than 19,000 species that is distributed
mainly in the temperate and subtropical parts of the world.
Legumes are second grasses in their agricultural and
economical value, and include many important species
grown for food, fodder, wood, ornamentals, and raw
materials for industry. In addition, they play ecologically
important role in biological nitrogen fixation. This family
of angiosperm without counting the genus Astragalus
contains 429 species is distributed in Iran; among them 173
are rare and 117 species are endemics. About half of the
observations in Iran were from the provinces Fars,
Hormozgan, Tehran, Bushehr and Mazandaran (Yousefi
2006; Lewis et al. 2005; Mousavi and Khosravi 2010).
Colutea L. (Fabaceae L.) is a small genus included
nearly 30 species of shrubs and small trees with inflated
fruits and is distributed throughout the Mediterranean
region, China, Himalaya, Eastern and North-Eastern
Africa, mostly in dry mountains (Mabberley 1997).
Colutea genus includes 13 species from three sections in
the Iranian plateau. Seven species of this genus are growing
in Iran, that five of them are endemic (Browizc 1959). In
current scenario, the DNA markers become the marker of
choice for the study of crop genetic diversity to
revolutionize the plant biotechnology. The internal
transcribed spacer (ITS) of nuclear ribosomal DNA
(rDNA) is one of the most extensive sequenced molecular
markers (Alvarez and Wendel 2003). The region is part of
the rDNA cistron, which consists of 18S, ITS1, 5.8S, ITS2,
26S and present in several hundred copies in most
eukaryotes.
The internal transcribed spacer (ITS) contains the
signals need to process the rRNA transcript and often used
for inferring phylogeny at intra and inter generic levels in
many plant families (Baldwin 1992; Baldwin et al. 1995;
Wojciechowski et al. 1999; Kazempourosaloo et al. 2005;
Ahangarian et al. 2007). The length of the ITS region of
flowering seed plants is highly uniform (Baldwin et al.
1995). By contrast, that of non-flowering seed plants shows
much variation especially the length of the ITS1 region,
which ranges from 630 to 3125 bp, is strikingly more labile
in flowering plants (Liston 1995; Maggini et al. 2000).
ITS1 and ITS2 are not equivalent structures, even though
they are sometimes convergent in length as well as
substitutional patterns; ITS1 evolved from an intergenic
spacer and ITS2 from an expansion segment in the rDNA
large subunit.
The previous phylogenetic studies of Fabaceae by using
analyses of the nrDNA ITS were focused exclusively on
Hedysareae tribe (Ahangarian et al. 2007; Lewke et al.
2013), Vicieae (Fabeae) tribe (Foladi et al. 2013),
Pogonophaca subgenus (Kang et al. 2003), Phyllolobium
genus (Zhang et al. 2012), Genista genus (Hakki et al.
2010), and Astragalus genus (Zarre and Azani 2012) and
presented the monophyly of majority of this clades.
Thereby, ITS sequencing provides valuable phylogenetic
data for resolving relationships among species and genus of
Fabaceae family by molecular markers. Major objective of
this paper is to ascertain the taxonomic and phylogenetic
relationships within species of Colutea genus in Iran.
MIRZAEI et al. – Phylogeny analysis of Colutea from Iran
MATERIALS AND METHODS
Taxon sampling
Accessions in the amount of 12 plant sample belonging
to seven species of Colutea L. were collected by authors
from wild populations in different regions of Iran (see
Table 1). All the specimens examined (or its duplicates) are
deposited in the Islamic Azad University Avicennia
Herbarium (IAUH, without voucher number/in registration
process). In this study we used 20 ITS sequence of 17
species obtained from the GenBank: three species of
Colutea, three species of Astragalus, three species of
Swainsona, Chesneya kotschyi, Lessertia herbacea,
Sutherlandia
frutescens,
Carmichaelia
williamsii,
Clianthus puniceus, Eremosparton flaccidum, Erophaca
baetica subsp. Orientalis and Smirnowia turkestana. The
list of non-Iranian taxa used in our analysis with GenBank
accession numbers are showed in Table 2.
Table 2. List of non-Iranian taxa with GenBank accession number
used in our analysis.
Species
Astragalus vogelii
A. complanatus
A. cysticalyx
Carmichaelia williamsii
Colutea. abyssinica
C. arborescens
C. arborescens
C. atlantica
Chesneya kotschyi
Clianthus puniceus
C. puniceus
Eremosparton flaccidum
Erophaca baetica subsp. orientalis
Lessertia herbacea
Smirnowia turkestana
Sutherlandia frutescens
Swainsona canescens
S. formosa
S. pterostylis
S. pterostylis
ITS GenBank
accession number
U50499.1
EU591995.1
AF121682.1
AF113854.1
GQ246039.1
U56010.1
U56009.1
GQ246040.1
GQ246104.1
L10801.1
L10800.1
GQ246035.1
EU070920.1
AF121752.1
GQ246037.1
GQ246033.1
GQ246042.1
U56008.1
U56007.1
GQ246032.1
169
DNA extraction and ITS sequencing
The DNA extraction was performed from leaves dried
with silica gel by using the NucleoSpin Plant Kit
(Macherey-Nagel GmbH & Co. KG, Du ren, Germany)
after the manufacturer’s protocol. Concentration and
quality of extracted DNAs were checked by 1% agarose gel
electrophoresis. We amplified the ITS region (ITS1-5.8SITS2) of the nuclear ribosomal DNA by using primer
combinations AB101 and AB102 primers: a forward
primer AB101annealing, 5'-ACG AAT TCA TGG TCC
GGT GAA GTG TTC G-3', and a reverse primer (AB102)
annealing, 5'-TAG AAT TCC CCG GTT CGC TCG CCG
TTA C-3' ( Kang et al. 2003).The PCR amplifications were
performed in 25µL reaction volumes containing 1µL
template DNA, 10.5µL ddH2O, 10× buffer + MgCl2 +Taq
polymerase+Tween 20 + dNTP=12.5µLand 0.5µL each of
the both primers. Thermal cycles were performed with
2min denaturation step at 95° C, followed by 35 cycles at
95 °C for 1 min, 51.5 °C for 1 min, and 72 °C for 1.5 min,
followed by 7-10 min final extension at 72° C for
completion of primer extension. PCR products were
resolved by electrophoresis in 1% agarose gel and then
visualized under UV light.
Phylogenetic analysis
Forward and reverse sequences were visually compared
and edited, and then initially aligned using Sequencher 4
software (Gene Codes Corporation, Ann Arbor, MI USA).
All ITS sequences were assembled and aligned using Mac
Clade 4 (Maddison and Maddison 2010).
Maximum parsimony analyses (MP)
Parsimony analyses were implemented by employing
PAUP ver. 4.0 (Swofford 2002) using following criteria:
100 heuristic search replicates, random stepwise addition of
taxa, and tree-bisection reconnection (TBR) branch
swapping. These parsimonious trees were used to calculate
the consensus tree. Bootstrap analyses (BS) were applied to
determine the clade support. BS for clades was calculated
using PAUP with 100 replicates of heuristic searches, and
randomly stepwise addition of taxa. Clades with a
bootstrap value of 70% or more were considered as
robustly supported nodes.
Table 1. List of Colutea species investigated and voucher specimen information (TARI = Herbarium of Research Institute of Forests
and Rangelands, IAUH = Islamic Azad University Avicennia Herbarium).
Species
Origin, Voucher
Colutea buhsei (Boiss.) Shapar.
C. buhsei (Boiss.) Shapar.
C. buhsei (Boiss.) Shapar.
C. buhsei (Boiss.) Shapar.
C. buhsei (Boiss.) Shapar.
C. gracilis Fryen & Sin.f.
C. persica Boiss.
C. persica Boiss.
C. porphyrogramma Rech.f.
C. uniflora G .Beck. ex Stap f.
C. cilicica Boiss. & Balansa.
C. gifana Parsa
Iran, Prov. N. Gorgan, 1400 m, (30871 TARI).
Iran, Prov. E. Khorasan, 1550 m, Foroghi, (50312 TARI).
Iran, Prov. S. Ardebil, Khalkhal to chuli, 1000m (1), Ferguson, (000013619 IAUH).
Iran, Prov. Tehran, 1800 m.Trott, (000013617 IAUH).
Iran, Prov. Gorgan, Aliabad, 600 m, Gauba (88858 TARI).
Iran, Prov. N. Gorgan, 20800 m (000013611 IAUH).
Iran, Prov. Kerman, 2300m, Mussavi and Tehrani (16256 TARI).
Iran, Prov. Fars, Dahte arzhan, 2200 m,Foroghi,(45755 TARI).
Iran, Prov. Khorasam, Bojnord, 1350 m,Resh,(000013614 IAUH).
Iran, Prov. Khorasan, Gazvin, 1600 m (000013621 IAUH).
Iran, Prov. Azerbaijan, Kaleibar,vinag, 1000 m, Assadi & Wdb (000013620 IAUH).
Iran, Prov. E Khorasan, Gifan, 1300 m, Parsa (000013623 IAUH).
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B I O D I V E R S IT A S 16 (2): 168-172, October 2015
Bayesian analysis (BA)
The BA analyses of the ITS datasets were performed
using MrBayes ver. 3 (Huelsenbeck and Ronquist 2001). In
order to find the appropriate model of DNA substitution,
the Maximum Likelihood criteria for datasets were
determined by the Akaike Information Criterion (AIC;
Akaike 1974) as implemented in the software ModelTest v
3.7 (Posada et al. 1998).
Modeltest
For the Maximum Likelihood (ML) and MrBayes
analyses (MB), the best fit of DNA substitution model
should be found. The Akaike information criterion (AIC)
and hierarchical likelihood ratio test (hLRT) were
calculated based on the log likelihood scores of 56 models
using Modeltest 3.7 (Posada et al. 1998). In general, AIC
was chosen (Posada 2008). For ITS spacer dataset of this
paper, Likelihood settings from best-fit model (TVM+G)
were selected by AIC in Modeltest 3.7 with the nucleotide
frequencies A = 0.2072, C = 0.2576, G = 0.2751, T =
0.2601, a gamma shape parameter of 0.5955 and an
assumed proportion of invariable sites of 0.0.
Maximum likelihood
Maximum search was performed on the basis of the
result of Modeltest in PAUP. The parameters of best
model, such as the base frequency, the mean relative
substitution rates, proportion of invariable sites, Gamma
distribution shape, were all employed. The heuristic search
and bootstrap were implemented as in parsimony analysis
in PAUP above mentioned.
Bayesian inference
Bayesian inference of phylogenetic trees was analyzed
by some parameters from the Modeltest, and included in
the analysis. The option was set up using 1,000,000
generations of Markov Chain Monte Carlo (MCMC)
searches and a sample frequency of 1000. Saturation was
reached after a burn-in of 1000 generations. The clade
support was assessed using Bayesian posterior probabilities
employing MrBayes version 3.0 (Huelsenbeck and
Ronquist 2001).
RESULTS AND DISCUSSION
The data set of the ITS region included 432 characters
with 270 including variable positions within the ingroup;
82 were parsimony informative. The Bayesian 50%
majority- rule consensus tree for ITS contained 11 internal
nodes with a posterior probability (PP) of 1.0 (Fig. 2).
Strict consensus phylogeny trees, with 256 steps was
included consistency index (CI) = 0.791, retention index
(RI) = 0.799. Using the data of Figure 1, Clianthus
puniceus and Carmichaelia williamsii taken as an outgroup
form a separate clade, and Swainsona canescens species
form a group which is sister to other Swainsona species.
The ingroup consists of two main clades that labeled as A
and B. In clade A, Swainsona formosa and S. pterostylis
form a separate group. The support was occurred in clade
A (PP=1; BS 96%). Clade B comprises two clades
including BI and BII. Clade BI comprises two subclades
including BI1 (Astragalus complanatus, Chesneya kotschyi
and Erophaca baetica forming a separate group) and BI2
clades included a single species Astragalus vogelii. Clade
BII, recognized in three subclades, BII1, BII2 and BII3,
respectively. Within BII1 subclade Astragalus vogelii
separated of Eremosparton flaccidum + Smirnovia
turkestana subclade. Clade BII2 involves BII2a and BII2b
that consisting of Colutea species. In clade BII2a Colutea
cilicica and C. gifana grouped together separately from the
other Iranian species of Colutea. The species of Colutea
arborescens and C. atlantica recognize in BII2b subclades.
In BII3 subclade, Lessertia herbacea and Sutherlandia
frutescens are grouped together. The support was occurred
in clade B for two sister-group BI and BII (PP=1; BS
98%). Best support relationship between three sister-group
clades BII (PP =1; BS = 93%) and relationship between
two sister group clade BII2a and BII1 was supported by
(PP=1; BS= 99%). Clade BII2b was contain the Colutea
species genus supported with pp=1, BS=74%. Clade BII2b
divided into two sister groups BII2a and BII2b. Within the
Colutea species three major clades were identified and
named as D, E and F clade (12 species; PP = 0.90; BS =
56%). Clade D comprising 10 accessions of C. buhsei, C.
gracilis, C. uniflora and C. porphyrogramma (PP = 0.97;
BS = 74%) constitutes the unresolved group and shows
polytomy. C. gifana in clade F together with its sister C.
cilicica in clade E separated of other species of Colutea
from Iran was sister group to C. gifana in clade E. Actually
sampled species in three clade D, E, F included to Sect.
Rostrata (five accessions of C. buhsei), two species of Sect.
Armata (C. uniflora, C. porphyrogramma) and four species
of Sect. Colutea (C. gracilis, C. persica, C. cilicica) from
Iran that little supported relationships among main clades
were existed. On the other side, relationships among D
clades remained indefinite in our results. So our results
show that Astragalus cysticalyx from Astragaleae tribe is
close to Colutea tribe (Ahangarian et al. 2007).
According to the other studies, all species examined in
our study nested within coluteoid clade in large astragalean
clade; Carmichaelia williamsii and Clianthus puniceus
species were basal group in sub tribe Coluteinae, and
among members of the sub tribe closely relation existed
(Polhill 1981; Lavin et al. 1990; Sanderson and Liston
1995; Wojciechowski 2005; Zhang et al. 2012).
Wojciechowski et al. (2000) represented Colutea istria
which is sister to the Lessertia+Sutherlandia complex plus
Eremosparton + Smirnowia. As can be concluded from the
phylogenetic study performed by Lock and Schrire (2005),
Swainsona formed the monophyletic group sister to
Astragalus cysticalyx, Colutea istria and Colutea
arborescens. The results of the present work are consistent
with those of Wagstaff et al. (1999) showing the genera
Carmichaelia and Clianthus nested within the Swainsona,
and thus the monophyly of Carmichaelia was reported by
these authors. Therefore, our study supported these results.
In the same study based on a matK, trnL and ITS molecular
markers Astragalus epiglottis was shown as the sister group
MIRZAEI et al. – Phylogeny analysis of Colutea from Iran
for Colutea persica (Liston 1995; Amirahmadi et al. 2014).
Our results of cladistic analysis the phylogenetic
relationships among Coluteae tribes showed its
monophyletic origin and Astragaleae was its sister group.
In addition, C. cilicica was a sister group to C. gifana
whose separated of other Colutea genus from Iran.
However, ITS marker could not sufficiently informative to
provide adequate resolution for Colutea genus and the
171
relationships between some of the major groups remained
unresolved. According to our results, we can assume that
further investigation insufficiently resolved nodes within
the Colutea genus will provide important insight into the
relationships between the species and, consequently, the
genera of Coluteae tribe. The combined approach including
application of different markers would increase the
resolutions and supports of the clades in Colutea.
Figure 2. Bayesian consensus phylogram for combined data (Numbers above branches are Bayesian posterior probabilities. Numbers
below branches are maximum likelihood percentage bootstrap values).
172
B I O D I V E R S IT A S 16 (2): 168-172, October 2015
ACKNOWLEDGMENTS
This article is extracted from Ph.D. dissertation of the
first author. We finally thank Islamic Azad University,
Tehran Science and Research Branch for providing the
facilities necessary to carry out the work.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160212
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 173-178
Study of the digestive tract of a rare species of Iranian blind cave fish
(Iranocypris typhlops)
ALI EBRAHIMI
Department of Biology, College of Science, Malard Branch, Islamic Azad University, Malard, Tehran, Iran. Tel./Fax.: +98-21-65672964 email:
ebrahimibal@gmail.com
Manuscript received: 7 April 2015. Revision accepted: 16 July 2015.
Abstract. Ebrahimi A. 2015. Study of the digestive tract of a rare species of Iranian blind cave fish (Iranocypris typhlops). Biodiversitas
16: 173-178. The Iranian blind cave fish (Iranocypris typhlops) is a unique taxon which only lives within a cave in Lorestan Province in
southwest of Iran. Regardless of its enormous genetic interest, this species faces an imminent risk of extinction as no conservation
efforts have been done for its protection. This study aimed to analyze the morphology of the digestive tract of this interesting fish using a
histological approach. Detailed examinations of the fishes showed that the mouth is horseshoe-shaped and located in an inferior situation
(ventral side) of the head. In it, there are three rows of pharyngeal teeth including inner, middle and outer rows, which bear 5, 3 and 3 teeth
respectively. The esophagus is very short and lined with an epithelium containing numerous mucous folds. The stomach is not present.
The anterior segment of the intestine is S-shaped which comprises about one half the gut. In this part, there are mucosal folds which show
different sizes and mucous layer is thicker than other layers (submucosa, muscle layer and serosa). The distal portion of the intestine is
straight (rectal) and terminates to the anal region. In general, the ratio of intestine length is 1.2, as compared to the body length. This
study showed that the liver in this taxon is composed of two lobes, the right lobe being two-parts and bigger than the left one. The gall
bladder is clear and spherical in appearance. The pancreas is red or orange and observed as scattered masses of cells on the mesentery of the
digestive system. To our best knowledge, this is the first study to analyze the morphological characteristics of the digestive tract of the
Iranian blind cave fish, and its unique characteristics here found confirmed its singularity and so, the urgent need for its conservation.
Keywords: Blind cave fish, digestive system, digestive tract, histology, morphology
INTRODUCTION
MATERIALS AND METHODS
The fishes are very diverse taxa; weighing from a few
grams to several tones and with a length of several
centimeters to several meters. As well as, the variety of fish
is abundant in Iran. Some species are not yet fully known.
For example, there are few sources about one of the most
unique species in Iran called the “Iranian blind cave fish” –
Iranocypris typhlops (Figure 1) – which is limited to a cave
in “Lovan” Village located at Lorestan Province, Iran.
This species is threatened to extinction mostly because
no conservation actions were taken to the moment and
virtually no attentions have been paid to it. The Iranian
blind cave fish (its characteristics are the same as the
taxon) was first identified by Bruun and Kaiser (1944).
This is the only blind fish species known in Iran and its
unique morphology, grants it an enormous genetic interest.
The Iranian blind cave fish is mentioned in the 1996 Red
List of IUCN as one of the fish species threatened to
extinction. The information on the species is scarce and
many of its biological aspects are still unknown (IUCN 2015).
The fish’s digestive tracts are as diverse as the species
themselves, sometimes with differences occurring within
the same species (Coad 1996; Sheybani 2005). Knowing
the feeding ecology of a species is a key aspect to
understand its role and positioning in the prey/predator
relationships, therefore, this study aimed to analyze the
morphology of the digestive tract of Iranian blind cave fish
as a step ahead to the knowledge of this evasive species.
After obtaining the necessary permits from the
Environmental Protection Organization of Iran as a
necessity for fishing, ten fishes were fished collected from
the wild habitat and were transferred to laboratory. The
water temperature was about 17◦C at the time of fishing.
The average of total length of fish’s body was 41 mm and
the average of their weight 1.5 g. The fishes were immediately processed and placed in a saline Formalin 10%
fixative solution. Through a processing and embedding
process in Paraffin, tissue sections were then prepared from
the obtained samples. The thickness of sections was about
7 m. The sections were stained through Hematoxylin &
Eosin techniques (Slack 1995). After they were prepared,
tissue sections were examined through Leica microscope
and then the required pictures with various microscopic
magnifications were prepared by a digital “Canon” camera
attached to the microscope. This study included the morphological and histological studies analysis of the digestive tract.
1 cm
Figure 1. A total dorsal view of Iranian blind cave fish
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RESULTS AND DISCUSSION
Morphology of digestive tract
The mouth of fish is located in lower position (Figure
2) and the mandibles have no teeth in it.
Three rows of pharyngeal teeth located at 5th gill arch
(Figure 4) are present. These teeth are extremely small and
can only be seen using a loupe. Pharyngeal teeth are
situated in 3 inner, middle and outer rows and the numbers
of teeth are: 4 in 3 specimens, 5 in 7 specimens on the inner
row, 3 in middle row in all specimens, and 3 in outer row
in all specimens (Figure 3).
The esophagus is quite short between 1 to 2mm in
length of and it is not as thick as the intestine. This species
is similar to carp fishes as they have no stomach.
The intestine consists of three distinct sections of
variable thickness between them. The first section located
next to esophagus is darker in contrast to the other sections
and comprises half of the intestine. This section has an S-
shaped form and the intestine is thicker in this part. The
joint part of bile duct is also located in this part. In the
second third, the intestine is less thick and lighter in color.
In the last third, the intestine becomes thicker. The total
ratio of intestine to body length in this species is about 1.2
and the pylorus appendage is absent.
Liver, gallbladder, pancreas and swim bladder are the
attached and joint parts of digestive tract. The liver consists
of two separate lobes. The right lobe is larger and consists
of two parts. The color of the liver is slightly different in
specimens but its usual color is light cream. Gallbladder
has a spherical shape and has a thin and clear wall. It is
located at right part and under the liver.
Pancreas has a scattered structure located on mesentery
peritoneum. Its color is between orange and red. In
average, 3 to 7 scattered parts of pancreas can be found at
this species locating on mesentery and extends from
esophagus to intestine last part (Figure 7).
Figure 2. Moth and Barbels position in cave fish, (ventral view),
oc (oral cavity), ul (upper lip, ll (lower lip), b 1-4 (barbells)
Figure 3. Pharyngeal tooth, inner row (I), middle row (M), outer
row (O). Tip of tooth is cone or round
Figure 4. Initial part of digestive tract: Pharyngeal tooth (Pt),
Esophagus (E), Intestine (I) and folds of mucous (F)
Figure 5. Cross-section of esophagus; Lumen (L), Epithelium (E),
Parine (P), Longitudinal muscles (M1), Circular muscles (M2),
Serous (S), H & E, ×10
EBRAHEMI – The digestive tract of a rare species of Iranocypris typhlops
175
Digestive tract histology
Regarding histology, esophagus structure is clearly
distinguishable from the other parts of the digestive tract.
The esophagus mucosal contains many longitudinal
mucosal folds that extend into lower cavity (Figure 5) but
esophagus is thinner than other parts of digestive tract. Its
epithelium is simple, stratified, squamous, and the number
of cellular layers differ in various areas.
The longitudinal mucosal folds vary, both in shape and
length.Two flat inner and outer muscle layers can be found
which are longitudinal and circular respectively.
Esophagus muscular layers are thicker than other parts
of digestive tract. Squamous cells of serous membrane
surround the esophagus (Figure 5).The cells located on the
base membrane of the esophagus contain one row of
cylindrical cells with oval nucleus. A row of polyhedral
cells are located on the surface of them. The cells of top
layer are squamous and include elongated nucleus. In some
parts of esophagus, between epithelium cells and the cells
directed to the top of epithelium, there are some round,
large and sub mucosal secretory cells that include foamy
and nuclear cytoplasm near to base. These cells are similar
to goblet cells and are not evenly distributed (Figure 6).
By means of serial sections of intestine, three parts of
tissue can be distinguished. Part 1 is from beginning of
intestine, the part to which the bile duct is attached to end
of dilatation and includes half of intestine length. In this
part, there are intestine mucosal folds in various sizes and
the mucosal layer is thicker than other layers and muscular
layers (inner and circular, outer and longitudinal) are
thinner (Figure 7). Intestine epithelium is simple cylindrical
and Paryn loose connective tissue is clearly recognizable in
the mucosal folds (Figure 8).
In second part, intestine is thinner but there are more
mucosal folds. In this part, the mucosa is quite thick and
there is a thin muscular layer that is present but cannot be
seen as a double layer.
Regarding histology, the distal part of intestine has a
few intestine mucosal folds; the mucosa, sub mucosa and
muscular layer which are thinner. In addition, the diameter
of intestine increases in this part.
In liver, cells are arranged like irregular columns and
form a mixed matrix which is not similar to mammals liver
structure. There are no central veins. Liver exposures are
seen between liver irregular columns in various shapes and
Kupffer cells are located in their wall (Figure 10).
Gallbladder has some distinguished layers including
simple cylindrical epithelium, Paryn connective tissue, very
delicate muscular layer and serous membrane (Figure 9). In
this specimen, the gallbladder is so clear because the
constituent layers of its wall are so thin.
Pancreas is located throughout the digestive tract from
esophagus to end of intestine and next to digestive tract; it
is as scattered mass of cells on the peritoneum (Figure 7).
The cells of pancreas exocrine part include a round nucleus
and acidophilic cytoplasm that imply the presence of
Zymogen granules. Among Acinar cells, there are a mass
of cells containing smaller nucleus which are endocrine
part of the glands (Figure 11).
Figure 6. Cross-section of esophagus; Lumen(L), Epithelium (E),
Parine (P), Gobllet cell (G), H & E,×20
Figure 7. Cross-section of intestine in first part; Lumen(L),
Epithelium (E), parine (P), muscular layer (M), Pancreas (Pa),
Gall bladder (Ga) , H & E,×10
Discussion
The mouth location in the fish is soleues and when
located in a lower and more ventral position, it is a clear
indication that it is a bottom feeding fish. Pharyngeal teeth
in carps are different in various species regarding shape,
number and their location on 5th gill arch. Therefore, they
are used to identify the different carp species (Vosoghi and
Mostajir 1994; Coad 1996; Bastani 1999).
Regarding the number of rows of pharyngeal teeth of
blind cave fish, 2 rows (Bruun and Kaiser 1944) and 3 rows
have been reported. In each row, inner, middle and outer
teeth were reported to vary between 1-3, 3-4, 3-5 teeth
(Humason 1979). The present study agrees with those
finding as 3, 3 and 5 teeth were respectively found in those
rows.
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16 (2): 173-178, October 2015
Figure 8. A plica mucous of midgute; Lumen(L), Epithelium (E),
Parine (P), muscular layer (M), Pancreas (Pa), Gall bladder (Ga),
H & E,×10
Figure 9. Cross - section of Gall bladder; Lumen (L), Epithelium
(E), Parine (P), muscular layer (M), serous (S) , H & E,×40
Figure 10. Liver tissue; cords of liver cells(C) are very erratic and
kupffer cells (K) are seen in sinusoids, H & E, ×40
Figure 11. Pancreas tissue; Acini cells (Ac) and Endocrine cells
(Ec), H & E, ×40
In the carp fishes, esophagus is shorter and
morphologically in some of the species (e.g. Amur fish,
silver carp and common carp) there is no difference
between esophagus and intestine and it is hard to
distinguish between them (Bastani 1999). In the present
study, due to small size of fish, it was impossible to
visually identify the esophagus which was only possible
through histology. In some fish species such as white fish
(Cyprinidae family), esophagus wall includes a thick
muscular layer and it is easy to distinguish between
esophagus and intestine (Bastani 1999). Regarding the
esophagus epithelium cells, large and round secretory cells
are identifiable. The same is true of Acipenser stellatus
esophagus (Acipenser stellatus is one species of sturgeon
that produces the caviar) (Ghavami 2000). The carp fish
esophagus are a simple and twisty tube in the abdominal
cavity (Bastani 1999) a characteristic also seen in Iranian
blind cave fish.
Intestine is in S shaped curve and intestines’ epithelium
is a simple cylindrical structure. The existence of brush
border in intestine epithelium is a main mechanism in
absorbing and consuming food and intestinal absorption
process in fishes and mammals (Kapoor et al. 1975).
Since the body of Iranian blind cave fish is virtually
transparent, intestines can be seen from the outside.
Depending on the fish diet, the length and diameter of the
intestine differ. In carnivorous fishes, the esophagus length
is shorter than herbivorous fishes (they have no stomach)
that have long esophagus including many folds (Coad
1996).
EBRAHEMI – The digestive tract of a rare species of Iranocypris typhlops
The ratio of intestines’ total length to body length varies
in Cyprinidae. For example, this ratio in silver carp is
approximately 6 (Vosoghi and Mostajir 1994), in Aspius
aspius, the ratio is 0.7 to 0.9 (Bastani 1999). In Persian
sturgeon, the esophagus constitutes 40-50% of the digestive
tract length (Sheybani 2005). In blind cave fish, the
average ratio of intestine length to body length was 1.2.
The fish’s intestine is divided into four parts: ventral,
anterior, middle, posterior. The ventral part includes oral
cavity and gill (pharyngeal). Anterior part is from gills’
posterior edge to esophagus, stomach and pylorus (Coad
1996). In Persian Sturgeon, the intestine constitutes two
distinguished parts including anterior intestine and
posterior intestine (Sheybani 2005). In carp fishes which
have no stomach or pylorus (Vosoghi and Mostajir 1994),
middle intestine begins from back of pylorus to posterior
esophagus with no clear border. Middle intestine includes a
number of pyloric caeca which are not present in fishes
which have no stomach. Middle intestine is the longest part
of intestine and since it is longer than body length, is in
shape of complex loops (Coad 1996). This loop is also seen
in S shape in the blind cave fish.
Main function of liver as a digestive gland is producing
and secreting bile. Color of fishes’ liver ranges from dark
brown to light cream (Navarro et al. 2006) and blind cave
fish liver is light cream (Alboghabish and Khaksari 2005).
The liver of blind cave fish is divided into two (right and
left) lobes. Carp species have two-part liver. In the blind
cave fish, like the carp species, a large part of liver is
located in the right part of abdominal cavity (Alboghabish
and Khaksari 2005). Liver parenchyma is surrounded by a
delicate capsule of loose connective tissue (Alboghabish
and Khaksari 2005; Navarro et al. 2006). Liver cells secrete
bile which flow to extracellular capillary tubes. The liver
cells are hepatocytes containing spherical and central
nucleus including different quantities of heterochromatin
(Abbasi and Gharzi 2000).
Herbivorous carps as well as African lungfish have
liver cells with two nucleuses (Alboghabish and Khaksari
2005), but in present study, those were not identified. In
this taxon the liver cells are arranged like columns and
form liver cords a morphological characteristic also
reported in Japanese salmon, too. The fish liver cells
willingness to create liver cords is lower in contrast to
mammals (Alboghabish and Khaksari 2005). Liver cells
including this particular morphology were identified in the
present study, but they do not form regular columns as liver
cords.
The main stored materials in fish’s liver are glycogen
and fat. The fat is present in organelles of cellular cirrhosis
forming small to average drops which occupy the
cytoplasm of these cells. Due to this fact, these cells are
commonly referred as fat storage cells. Since fat and
glycogen are not so chromophil through Hematoxylin and
Eosin staining techniques, many vacuole structures are seen
in the hepatocytes (Poosti and Sedighmarvdasti 2000;
Navarro et al. 2006). These two substances can however be
distinguished regarding the vacuole shape. Fat drops are
spherical, single or in a mass, while glycogens are irregular
177
in form (Poosti and Sedighmarvdasti 2000). These vacuoles
were also identified in the liver of blind cave fish.
Kupffer cells belong to macrophage cells which
comprise reticuloendothelial system (Navarro et al. 2006).
Some researchers believe that fish liver has no Kupffer
cells, but these cells were reported in catfish (Alboghabish
and Khaksari 2005) and carps (Alboghabish and Khaksari
2005). Kupffer cells including triangle nucleus and
heterochromatin were observed among the endothelial cells
of the liver exposure wall of the blind cave fish.
Gallbladder is present in many fishes and is divided into
three parts: mucosa (epithelium and Paryn connective
tissue), muscular part and serous part (Abbasi and Gharzi
2000). In the present study, the blind cave fish gallbladder
appeared to be crystalline, spherical and clearly segmented
in the showing the three tissue parts of gallbladder.
Nonetheless, its muscular part was particularly thin.
In many fishes, pancreas tissue gradually lies next to
portal vein branches and the result is called hepatopancreas
tissue (Abbasi and Gharzi 2000; Dabrowski et al. 2003;
Dyk et al. 2005). A part of pancreas of carp herbivorous
fish is located within the liver (Alboghabish and Khaksari
2005) a singularity that was not observed in blind cave fish.
The pancreas of teleosts is an organ is scattered around the
digestive tract peritoneum or other limbs and is present
among fat tissues (Dyk et al. 2005). Exocrine cells in
immature fish as well as that part of pancreas which
scattered in fat tissue of adult fish have acinar form (Poosti
and Sedighmarvdasti 2000; Dyk et al. 2005).
In the study on blind cave fish it was observed that the
pancreas is present in the mesentery peritoneum as
scattered parts in red to orange. Observing the pancreas
parts through light microscope, it was found that secretory
cells include a strongly basophilic cytoplasm and
acidophilic spherical particles called zymogen (Poosti and
Sedighmarvdasti 2000; Dabrowski et al. 2003;
Alboghabish and Khaksari 2005; Dyk et al. 2005). This can
be easily observed in the tissue section of blind cave fish
pancreas. Islets of Langerhans were identified forming light
cellular masses in exocrine part of pancreas, but for more
accurate identification of these organelles it is necessary to
use specialized staining methods to identify all types of
present cells.
ACKNOWLEDGEMENTS
I have taken efforts for accomplishing this project.
However, it would not have been possible without the kind
support and help of many individuals and organizations. I
would like to express my sincere thanks to all of them, in
particular, the Mallard Branch of Islamic Azad University,
Iran.
REFERENCES
Abbasi M, Gharzi A. 2000. Veterinary Comparative Histology, Preclinical
Relation. Partoovaghee Publisher, Tehran.
Alboghabish N, Khaksari R. 2005. Structural study of liver and pancreas
of Cyprinidea family fishes. Iranian Vet J 11: 25-33.
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Bastani P. 1999. Comparative study of digestive system in Cyprinidea fish
with different diet. [M.Sc. Thesis]. Tarbiat Modares University,
Tehran.
Bruun AF, Kaiser EW. 1944. Iranocypris typhlops n.g., n.sp., the first true
cave fish from Asia., Danish scientific investigations in Iran. Part 4.
Copenhagen, 4: 1-8.
Coad BW. 1996. Threatened fishes of the world: Iranocypris typhlops
Bruun & Kaiser, 1994 (Cyprinidae). Environ Biol Fish 46: 374.
Dabrowski K, Lee K, Rinchard J. 2003. The smallest vertebrate, Teleost
fish, can utilize synthetic dipeptide-based diets. J Nutr 133: 42254229.
Dyk J, Pieterse G, Vuren J. 2005. Histological changes in the liver of
Oreochromis mossambicus (Cichlidae) after exposure to cadmium
and zinc. Ecotoxicol Environ Saf 66: 432-440.
Ghavami SM. 2000. Study of Pharynx, Esophagus and Prestomach, of
Oozoonbooroon Fish. [Ph.D. Dissertation]. Veterinary Department,
Tehran University, Tehran.
Humason GL. 1979. Animal Tissue Techniques. 4th ed. W. H. Freeman,
New York.
IUCN. 2015. Iranocypris typhlops (Zagros cave garra), Status: Vulnerable
D2 ver 2.3. In: The IUCN Red List of Threatened Species. Version
2015.2. www.iucnredlist.org. [13 July 2015].
Kapoor B, Smith H, Verghina A. 1975. The alimentary canal and
digestion in telosts. Adv Mar Biol 13: 109-239.
Navarro M, Lozano M, Agulleiro B. 2006. Ontogeny of the endocrine
pancreatic cells of the gilthead sea bream, Sparus aurata (Teleost).
Gen Comp Endocrinol 148: 213-226.
Poosti I, Sedighmarvdasti S. 2000. Fish Histology Atlas. Tehran
University Publisher, Tehran.
Sheybani M. 2005. Microscopic Study of Iranian Sturgeon Fish,
Acipenser persicus [Ph.D. Dissertation]. Department of Veterinary,
Tehran University, Tehran.
Slack J. 1995. Developmental biology of the pancreas. Development 121:
1569-1580.
Vosoghi G. Mostajir B. 1994. Freshwater Fishes. Tehran University
Publisher, Tehran.
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160213
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 179-187
Bayesian and Multivariate Analyses of combined molecular and
morphological data in Linum austriacum (Linaceae) populations:
Evidence for infraspecific taxonomic groups
FATIMA AFSHAR1, MASOUD SHEIDAI2, SEYED-MEHDI TALEBI3,♥, MARYAM KESHAVARZI1
1
Department of Biology, Alzahra University, Tehran, Iran.
Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran.
3
Department of Biology, Faculty of Sciences, Arak University, Arak, 38156-8-8349 Iran. Tel. +98-863-4173317. ♥e-mail:
seyedmehdi_talebi@yahoo.com.
2
Manuscript received: 10 May 2015. Revision accepted: 31 July 2015.
Abstract. Afshar F, Sheidai M, Talebi SM, Keshavarzi M. 2015. Bayesian and Multivariate Analyses of combined molecular and
morphological data in Linum austriacum (Linaceae) populations: Evidence for infraspecific taxonomic groups. Biodiversitas 16: 179187. Plant specimens of Linum austriacum (Linaceae) were collected from 16 geographical populations of nine provinces in Iran and
used for morphological and molecular (ISSR) analyses. Different multivariate and Bayesian methods were used to study
interpopulations differences. Analysis of variance test and Principal coordinate analysis plots indicated morphological difference of the
populations. Mantel test revealed positive significant correlation between morphological and geographical distance of these populations.
Pearson, coefficient of correlation showed significant correlations between basal leaf length, width and length/width ratio with latitude
and altitude of the studied populations. Bayesian analysis of combined molecular and morphological features revealed divergence of the
studied populations and consensus tree showed separation of 6 populations in different clusters. Canonical Variate Analysis plot of these
populations showed that Sang Sefid and Salmas populations differed greatly from the other populations. New ecotypes are suggested for
these populations.
Keywords: Infraspecific variation, morphology, Iran, ISSR, population.
INTRODUCTION
The presence of intraspecific variation in organisms is a
source of natural variation and is a way of response of
organisms to their environment. Intraspecefic variation
brings about biodiversity and is thought as the main origin
and storage of speciation (Christine and Monica 1999;
Hufford and Mazer 2003).
Many plant species have wide geographical distribution
and face a wide range of climatic and edaphic conditions.
Individuals of these species are able to give appropriate
response to a tremendous variety of different conditions.
These responses or adaptations change the descents of
these individuals of a species, genotypically,
phenotypically and physiologically and led to infraspecific
diversity. Therefore, descents of these individuals tend to
appear as new ecotypes, ecophenes, chemotypes, cytotypes
and even subspecies (Christine and Monica 1999).
Habitat heterogeneity, combined with natural selection,
often results in multiple, genetically distinct ecotypes
within a single species. In addition, the populations of a
given species facing different environmental conditions
may undergo genetic changes to adapt to their local
conditions (Linhart and Grant 1996; Hufford and Mazer
2003).
Infraspecific variation, difference between individuals
or populations of a given species, is responsible for a
relation of functionally relevant niche space occupation in
biological communities (Albert et al. 2010; Fridley and
Grime 2010; Violle et al. 2010). It is confirmed that plants
occupy different space to forbear competitive interactions
by means of variations in morphological and physiological
characteristics. In addition, same plant species elude
competition with each other and variegate their biological
strategies by means of feature variation.
Linum L. genus (Linaceae) contains about 180 species
that are source of fiber (Smeder and Liljedahl 1996), seed
oils (Diederichsen and Raney 2006), and fodder (Bhathena
et al. 2002). Some species are of medicinal value and
contain Omega-3 fatty acids and potential anti-cancer
compounds (Rogers 1982) as well as Lignans (Schmidt et
al. 2010). Linum species grow in temperate and subtropical
regions of the world (Rogers 1982; Muir and Westcott
2003). Linum austriacum L. is an herbaceous medicinal
plant containing important lignans such as arylnaphthalene
lignan and justicidin (Mohagheghzadeh et al. 2002), with
antifungal, antiprotozoal, cytotoxic and piscicidal
properties (Gertsch et al. 2003).
Although extensive biosystematics and phylogenetic
studies have been carried out in the genus Linum (see for
example, Velasco and Goffman 2000; Everaert et al. 2001;
Sharifnia and Albouyeh 2002; Hemmati 2007; Rogers
2008; Schmidt et al. 2010; Soto-Cerda et al. 2011, Talebi et
al. 2012a,b), little is known about the infraspecific diversity
and taxonomic forms of wild Linum species (Sheidai et al.
2014b).
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B I O D I V E R S IT A S 16 (2): 178-187, October 2015
The neutral molecular data have been extensively used
to study the speciation process (see for example, CasselLundhagen et al. 2009; Pampoulie et al. 2011), the genetic
diversity (Sheidai et al. 2012, 2013), the populations,
genetic structure (Sheidai et al. 2012, 2014), and the
genetic drift (Heather and Freeland 2011). In particular,
ISSR (Inter simple Sequence Repeats) markers have been
applied in genetic characterization and taxonomy studies
on cultivated flax as well as on wild flax species.
Recently we reported inter-population genetic diversity
in L. austriacum L. (Sheidai et al. 2014). Furthermore, we
also encountered morphological variability among these
geographical populations. The aim of present study was to
illustrate if genetic variability of populations is associated
with morphological variability and if combined effects of
these variation lead to the formation of infra-specific
taxonomic forms.
Therefore, in the present study randomly collected
plants of L. austriacum from 16 geographical populations
were studied from morphological and molecular (ISSR)
points view. Bayesian and multivariate analyses were
performed on combined data set obtained to identify
potential infraspecific forms. The genetic data employed in
this current manuscript has been entirely taken from the
former Sheidai et al. (2014).
Figure 1. Distribution map of the studied populations in northwest Iran.
Table 1. Locality and herbarium voucher number of the studied
populations.
MATERIALS AND METHODS
Plant materials
Plant specimens of L. austriacum (Linaceae) were
collected from 16 geographical populations of nine
provinces in Iran (Figure 1) and used for morphological
and molecular (ISSR) analyses. In order to acquire data of
the studied populations, four plant samples were selected of
each population.
Samples were identified based on the descriptions
provided in accessible references such as Flora Iranica
(Rechinger 1974) and Flora of Iran (Sharifnia and Assadi
2001). The voucher specimens were deposited in the
herbarium of Shahid Beheshti University (HSBU), Iran
(Table 1). Detail of DNA extraction and molecular study is
provided in our previous report (Sheidai et al. 2014). In
short, genomic DNA was extracted using CTAB activated
charcoal protocol (Križman et al. 2006). Ten ISSR primers;
(AGC) 5GT, (CA) 7GT, (AGC) 5GG, UBC810, (CA) 7AT,
(GA) 9C, UBC807, UBC811, (GA) 9A and (GT) 7CA
commercialized by UBC (the University of British
Columbia) were used. PCR reactions were performed in a
25μ l volume containing 10 mM Tris-HCl buffer at pH 8;
50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP
(Bioron, Germany); 0.2 μ M of a single primer; 20 ng
genomic DNA and 3 U of Taq DNA polymerase (Bioron,
Germany). The amplifications, reactions were performed in
Techne thermocycler (Germany) with the following
program: 5 minutes initial denaturation step 94°C, 30 S at
94°C; 1 minutes at 50°C and 1 minute at 72°C. The
reaction was completed by final extension step of 7
minutes at 72°C.
Habitat address
Zanjan, Abhar, 1785 m
Kurdistan, Sanandaj, Abidar
Mountain, 1645 m
East Azerbaijan, Ahar, 1593 m
Guilan, Darestan Forest, 544 m
Hamedan, Famenin, 1761m
East Azerbaijan, Kalibar, 1460 m
Kermanshah, Kangavar,1564 m
Markazi, Nobaran, 1654 m
Qazvin 1, Highway, 1476 m
Qazvin 2, Cargo Terminal, 1205 m
Hamedan, Razan,1898 m
Guilan, Rudbar, 228 m
Zanjan, Saeen Qaleh, 1805 m
West Azerbaijan, Salmas, 1700 m
Markazi, Arak, Sang Sefid, 2084 m
East Azerbaijan, Tabriz, 1552 m
Collector
Herbarium
No.
Talebi
Talebi
HSBU2011133
HSBU2011112
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
Talebi
HSBU2011134
HSBU2011126
HSBU2011103
HSBU2011135
HSBU2011116
HSBU2011102
HSBU2011124
HSBU2011122
HSBU2011118
HSBU2011125
HSBU2011131
HSBU2011138
HSBU2011151
HSBU2011136
Data analyses
In total thirty two, nine qualitative and twenty three
quantitative, morphological characteristics were studied.
Four plants were measured or scored of each population
and for each characteristic one measurement was taken per
each flowering stem. The used terminologies for qualitative
morphological traits were on the basis of descriptive
terminology provided by Stearn (1983). Qualitative
characters were: petal color, basal leaf shape, floral leaf
shape, apex, margin and base shape of floral and basal leaf
blade. While, basal leaf width, basal leaf length, stem
height, branch number, basal leaf length/width ratio, floral
leaf width, floral leaf length, floral leaf length/width ratio,
AFSHAR et al. – Infraspecific variations in Linum austriacum
calyx length, calyx width, calyx length/width ratio, sepal
length, sepal width, sepal length/width ratio, capsule
length, capsule width, capsule length/width ratio, stem
diameter, leaf diameter, pedicle length, corolla length,
corolla width and corolla length/width ratio were
quantitative features.
Mean as well as standard deviation of the quantitative
traits were determined. The analysis of variance (ANOVA)
test was performed to show significant morphological
difference between the studied populations. Principal
coordinate analysis (PCoA) and Correspondence Analysis
(PCA) were performed to group the plants specimens based
on morphological characters. Morphological data were
standardized (mean = 0, variance = 1) for these analyses
(Podani 2000). Non-metric Multidimensional Scaling
(MDS) and Canonical Variate Analysis (CVA), were used
to illustrate populations, morphological distinctness.
For combined morphological and molecular analyses,
the characters were coded as binary and multistate
characters. Grouping of the populations was done by two
methods. First we carried out structure analysis (Pritchard
et al. 2000). For this analysis, data were scored as dominant
markers (Falush et al. 2007). Second, we performed KMeans clustering as done in GenoDive ver. 2. (2013).
In order to identify the populations that are genetically
and morphologically differentiated from the others, a
consensus tree was constructed from morphological and
genetic obtained trees. The Mantel test was performed to
check correlation between geographical, morphological and
genetic distances of the populations (Podani 2000). PAST
ver. 2.17 (Hamer et al. 2012), DARwin ver. 5 (2012)
programs were used for these analyses.
181
morphological characters.
PCA analysis of morphological features revealed that
the first three PCA components comprised about 73% of
total variability of the studied populations. In the first PCA
axis with about 35% of total variation, morphological traits
like sepal length and basal leaf diameter possessed the
highest correlation (r >0.90) while in the second PCA axis,
characters like stem diameter, sepal length/width ratio, and
pedicel length, possessed the highest correlation (r>0.80).
Therefore, these morphological characters were the most
variable morphological characteristics among the studied
populations. The obtained data showed that characteristics
such as floral leaf width as well as corolla width had lowest
correlation, this mean that the mentioned traits were the
most stable morphological features between the studied
samples.
PCA biplot (Figure 3) revealed that morphological
characters of sepal length and basal leaf diameter separated
Famenin population from the other populations, while
morphological characters like stem diameter, sepal
length/width ratio, and pedicel length separated Sang Sefid
population.
Pearson, s coefficient of correlation determined between
morphological characters and geographic traits of the
studied populations (longitude, latitude and altitude)
produced significant positive correlations between basal
leaf length and latitude, and between basal leaf width and
basal leaf length/width ratio with altitude.
The Mantel test performed between morphological
distance and geographical distance of the studied
populations produced significant correlation (P = 0.01,
Figure 4), indicating that with an increase in geographical
distance of these populations they showed a higher
magnitude of morphological difference.
RESULTS AND DISCUSSION
Morphological analysis
In present study thirty two quantitative and qualitative
morphological features of the both vegetative as well
reproductive organs were investigated (Table 2). Among
the studied qualitative traits, the shapes of blade apex,
margin and also base of basal and floral leaves were stable
among the studied sample, and were in the shapes of acute,
entire and cuneate respectively, furthermore the petal color
was invariable inter-populations and presented as blue.
While other characteristics such as floral and basal leaf
shape varied between populations and were recorded in the
shapes of linear, lanceolate or linear-lanceolate.
In addition, quantitative traits differed between
populations and the performed ANOVA test for these
characters showed significant difference (p = 0.05) for all
the studied traits with the exception of basal and floral leaf
length/width ratio, calyx length/width ratio, capsule length
as well as capsule length/width ratio among the studied
populations (Table 3). Moreover, PCoA plots of all
morphological characters separated these populations from
each other (Figure2). These results indicated that the
studied populations differed significantly in their
Combined molecular and morphological results
A combined data matrix of 44 × 91 was formed from
ISSR and morphological data and used for further analyses.
MDS plot of combined data is presented in Figure 5.
Almost complete separation of the studied population
indicated genetic and morphological distinctness of these
populations. STRUCTURE plot of the combined data that
is based on Bayesian method is presented in Figure 6.
Although it showed some degree of similarity between
Abidar and Darestan populations (populations numbers 2
and 4, respectively), and Rudbar and Salmas populations
(populations numbers 12 and 14, respectively), complete
difference was observed among the other studied
populations. This indicates populations, divergence in both
genetic and morphological features.
Delta K results of Evanno test that is based on
STRUCTURE analysis is presented in Figure 7. It
produced K value of 13 as the optimum K number and
indicated that the studied populations were placed in 13
different groups. Therefore L. austriacum populations are
highly stratified with regard to their morphological and
genetic features.
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B I O D I V E R S IT A S 16 (2): 178-187, October 2015
Table 2. The most important morphological traits of the studied populations (all values were in cm).
Population
Stem Branch Basal leaf
high
no.
shape
Abhar
Mean 43.87
N
4
SD 8.74
Abidar
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Mean
N
SD
Ahar
Darestan
Famenin
Kalibar
Kangavar
Nobaran
Qazvin1
Qazvin2
Razan
Rudbar
Saeen
Qaleh
Salmas
Sang
Sefid
Tabriz
3.00
4
.81
Linear
50.05 2.50 Lanceolate
4
4
3.24
.57
57.25 6.75
Linear
4
4
2.25 1.25
33.50 2.25
Linear4
4
Lanceolate
6.28
.50
38.30 4.50 Lanceolate
4
4
6.27
.57
54.25 10.25
Linear
4
4
8.14 3.30
54.75 6.00 Lanceolate
4
4
4.34 2.44
31.87 12.75
Linear
4
4
8.29 7.41
44.72 9.99
Linear
4
4
6.28
.00
56.00 3.25
Linear4
4
Lanceolate
8.75 1.25
62.12 7.00 Lanceolate
4
4
4.40 3.55
53.62 7.00
Linear
4
4
3.22 1.63
49.37 4.50 Lanceolate
4
4
5.02 1.29
58.00 17.50
Linear
4
4
9.09 4.50
62.25 6.75 Lanceolate
4
4
7.07 1.50
63.62 5.00
Linear
4
4
4.71
.81
Basal Basal
Floral Floral
Floral leaf
Calyx Calyx Pedicle Sepal Sepal Corolla Corolla
leaf
leaf
leaf
leaf
shape
length width length length width length width
length width
length width
1.52
.10 Lanceolate .82
.09
.38
.37
.10
.12
.16
0.74
0.74
4
4
4
4
4
4
4
4
4
4
4
.25
.01
.14
.015
.02
.04
.005
.05
.02
4.98
4.99
1.90
4
.21
1.50
4
.32
1.22
4
.17
1.73
4
.67
1.45
4
.36
1.55
4
.19
1.02
4
.20
1.12
4
.26
2.10
4
.11
1.87
4
.34
1.15
4
.12
1.67
4
.30
1.67
4
.35
1.27
4
.22
1.30
4
.29
.11
4
.01
.197
4
.00
.10
4
.00
.30
4
.08
.16
4
.03
.17
4
.04
.07
4
.04
.12
4
.02
.20
4
.04
.23
4
.04
.14
4
.04
.18
4
.08
.21
4
.06
.15
4
.05
.12
4
.02
Linear
Linear
Lanceolate
Lanceolate
Lanceolate
Lanceolate
Lanceolate
Lanceolate
Linear
Linear
Lanceolate
Lanceolate
Linear
Linear
Lanceolate
Finally, in order to identify those populations that are
differentiated in both morphological and genetic tree, a
consensus tree was obtained which is presented in Figure 8.
It revealed that 6 populations, Sang Sefid, Salmas,
Darestan, Qazvin 1, Abhar, and Razan were separated from
the others and were differentiated in both morphological
and NJ trees. Therefore, although all studied populations
differed in their morphological and genetic content as
indicated by STRUCTURE plot and MDS plot of
.97
4
.12
1.05
4
.10
.85
4
.17
1.05
4
.129
.67
4
.09
.87
4
.09
.80
4
.20
.72
4
.09
1.00
4
.08
.90
4
.29
.82
4
.09
1.12
4
.35
.75
4
.12
.72
4
.15
.90
4
.08
.10
4
.00
.10
4
.00
.07
4
.02
.14
4
.04
.08
4
.01
.08
4
.02
.08
4
.01
.09
4
.02
.12
4
.02
.10
4
.00
.09
4
.01
.12
4
.04
.09
4
.02
.08
4
.00
.10
4
.05
.38
4
.04
.40
4
.01
.40
4
.00
.37
4
.06
.31
4
.03
.40
4
.00
.32
4
.05
.40
4
.00
.35
4
.05
.38
4
.03
.43
4
.04
.46
4
.04
.40
4
.00
.30
4
.08
.38
4
.04
.34
4
.05
.40
4
.00
.38
4
.02
.40
4
.00
.30
4
.00
.39
4
.00
.37
4
.05
.40
4
.00
.33
4
.04
.37
4
.05
.42
4
.05
.49
4
.02
.38
4
.02
.35
4
.05
.47
4
.12
.08
4
.01
.12
4
.05
.08
4
.01
.10
4
.00
.05
4
.01
.10
4
.00
.06
4
.02
.06
4
.01
.09
4
.01
.10
4
.00
.10
4
.00
.10
4
.00
.09
4
.01
.05
4
.00
.27
4
.20
.10
4
.01
.10
4
.01
.16
4
.03
.15
4
.05
.16
4
.04
.10
4
.00
.10
4
.00
.20
4
.08
.12
4
.05
.10
4
.00
.20
4
.00
.13
4
.04
.13
4
.04
.15
4
.05
.42
4
.04
.19
4
.01
.23
4
.04
.20
4
.00
.25
4
.05
.18
4
.00
.21
4
.02
.18
4
.04
.22
4
.05
.19
4
.01
.23
4
.02
.26
4
.02
.20
4
.00
.20
4
.00
.23
4
.04
.26
4
.11
1.05
4
.05
1.47
4
.35
1.32
4
.17
1.22
4
.22
1.20
4
.14
1.41
4
.46
1.20
4
.18
1.53
4
.19
1.35
4
.129
1.60
4
.34
1.25
4
.12
2.15
4
.12
1.40
4
.35
1.09
4
.08
1.10
4
.00
.80
4
.08
1.20
4
.08
.98
4
.19
1.10
4
.20
.82
4
.09
.96
4
.30
.90
4
.15
1.17
4
.27
.82
4
.20
1.10
4
.24
1.05
4
.09
1.15
4
.05
1.05
4
.19
.91
4
.09
1.05
4
.00
combined data, only these 6 populations were placed in a
similar position on the constructed tree in both analyses
and formed a distinct cluster.
Detailed analysis of morphological characters in these 6
populations revealed that Sang Sefid population possessed
highest value of stem diameter, pedicel length, sepal length
and sepal length/width ratio. Similarly, Salmas population
contained the largest basal leaf length and largest floral leaf
length. It had the shortest calyx leaf length, shortest corolla
AFSHAR et al. – Infraspecific variations in Linum austriacum
width, shortest sepal length, and the lowest corolla
length/width ratio.
These two populations with maximum number of
morphological characters which were significantly
different from the other studied populations.
Qazvin 1 population had the shortest calyx width and
the lowest pedicel length, while Abhar population had the
highest floral leaf length, the shortest sepal length and the
lowest pedicel length. Razan population had the longest
calyx length. This suggestion is supported by CVA plot of
combined data in these 6 populations (Figure 9). These
Figures showed that Sang Sefid and Salmas populations
were placed far from the other 4 populations and took
position in different corners of this plot. The other 4
populations were placed close to each other. Therefore
Sang Sefid and Salmas populations are much more
differentiated from the other studied populations. These
populations may be considered as different ecotypes.
Table 3. ANOVA test of quantitative morphological features of
the studied populations
Characteristics
Stem height
Sum of
Squares
df
Mean
Square
BG
5923.677 15 394.912
WG
1953.062 48 40.689
Total
7876.740 63
Basal leaf
BG
5.754
15
.384
length
WG
4.497
48
.094
Total
10.251 63
Basal leaf width BG
.205
15
.014
WG
.103
48
.002
Total
.308
63
Floral leaf
BG
1.050
15
.070
length
WG
1.299
48
.027
Total
2.349
63
Floral leaf
BG
.021
15
.001
width
WG
.031
48
.001
Total
.053
63
Calyx length
BG
.103
15
.007
WG
.079
48
.002
Total
.182
63
Calyx width
BG
.139
15
.009
WG
.106
48
.002
Total
.245
63
Pedicle length BG
.156
15
.010
WG
.139
48
.003
Total
.295
63
Sepal length
BG
.364
15
.024
WG
.083
48
.002
Total
.447
63
Sepal width
BG
.056
15
.004
WG
.080
48
.002
Total
.136
63
Capsule length BG
.127
12
.011
WG
.183
34
.005
Total
.310
46
Capsule width BG
.203
12
.017
WG
.224
34
.007
Total
.427
46
Note: BG = Between Groups, WT = Within Groups
F
Sig.
9.706 .000
4.094 .000
6.357 .000
2.589 .006
2.154 .023
4.194 .000
4.168 .000
3.587 .000
14.042 .000
2.220 .019
1.967 .061
2.569 .015
183
Discussion
Ellison et al. (2004), stated that geographic variations in
morphological characters of plants is a function of changes
in phenotypic characters in response to local ecological
conditions, variations in genetic structure as well as
evolution between populations, and the biogeographic
history of an individual species.
Although some qualitative morphological features such
as petal color as well as leaf apex, margin and base fixed
among the studied populations, but floral and basal leaf
shape varied between the studied populations, so different
shapes of leaves were recorded. Different studies showed
that morphological characteristics such as leaf shapes are
constrained genetically, but they also can be affected
greatly by the local environment in which they develop
(Thompson 1991; Schlichting and Pigliucci 1998).
In addition, most of quantitative morphological traits
varied between populations and ANOVA test as well as
PCA analysis of morphological traits confirmed these
conditions. Some of these variations had either taxonomical
or ecological (adaptive) importance. For example, foliar
features were used in identification keys of this genus in
different references such as Flora Iranica (Rechinger 1974)
and Flora of Iran (Sharifnia and Assadi 2001).
Furthermore, PCA biplot revealed that some quantitative
feature had diagnostic value and were useful in
identification of populations.
The obtained results of Mantel test confirmed that, in
the studied L. austriacum populations, morphological
distance (difference) of these populations was not
correlated to their genetic distance, but morphological
distance was positively correlated with their geographical
distance. It showed that morphological changes of these
populations were not merely under influence of genetic
difference and other factors along with genetic variation
affect morphological differences.
Moreover, some of the morphological characters like
basal leaf length, basal leaf width and basal leaf
length/width ratio were correlated to latitude and altitude.
Because results of this study showed that populations that
grow in higher latitude possess larger basal leaf length,
while populations that grow in eastern parts of the country
have larger basal leaf width, and bigger basal leaf
length/width ratio.
Since the ecological and environmental conditions
varied between population’s habitats, it might be possible
that floristical composition of neighboring plants of L.
austriacum samples, ecological factors, pollinator species
as well as nature of dominant species differed between
populations, these conditions were seen in Linum album
populations (Talebi et al. 2014a). In order to adaptation to
these conditions, different morphological traits of L.
austriacum adapted to ecological factors of habitat,
therefore morphological variations occurred between
populations. Infraspecific studies on various species of this
genus or other genera such as Linum album (Talebi et al.
2014 a), Linum glaucum (Talebi et al. 2015) as well as
Stachys inflata (Talebi et al. 2014b) confirmed these
findings.
184
B I O D I V E R S IT A S 16 (2): 178-187, October 2015
Figure 5. MDS plot of L. austriacum populations based on
combined morphological and molecular data. Note: 1. Abhar, 2.
Abidar, 3. Ahar, 4. Darestan, 5. Famenin, 6. Kalibar, 7. Kangavar,
8. Nobaran, 9. Qazvin 1, 10. Qazvin 2, 11. Razan, 12. Rudbar, 13.
Saeen Qaleh, 14. Salmas, 15. Sang Sefid, 16. Tabriz.
Figure 2. Representative PCoA plots of morphological data
showing distinctness of the studied populations. Note: 1. Abhar,
2. Abidar, 3. Ahar, 4. Darestan, 5. Famenin, 6. Kalibar, 7.
Kangavar, 8. Nobaran, 9. Qazvin 1, 10. Qazvin 2, 11. Razan, 12.
Rudbar, 13. Saeen Qaleh, 14. Salmas, 15. Sang Sefid, 16. Tabriz.
Figure 7. Delta K results of Evanno test based on the combined
data.
Figure 3. PCA biplot of populations based on the morphological
characters. Note: 1. Abhar, 2. Abidar, 3. Ahar, 4. Darestan, 5.
Famenin, 6. Kalibar, 7. Kangavar, 8. Nobaran, 9. Qazvin 1, 10.
Qazvin 2, 11. Razan, 12. Rudbar, 13. Saeen Qaleh, 14. Salmas,
15. Sang Sefid, 16. Tabriz.
Figure 4. Mantel test result between morphological and
geographical distance of the studied populations.
Figure 9. CVA plot of reduced data.
AFSHAR et al. – Infraspecific variations in Linum austriacum
185
Figure 6. STRUCTURE plot of the combined data. Note: 1. Abhar, 2. Abidar, 3. Ahar, 4. Darestan, 5. Famenin, 6. Kalibar, 7.
Kangavar, 8. Nobaran, 9. Qazvin 1, 10. Qazvin 2, 11. Razan, 12. Rudbar, 13. Saeen Qaleh, 14. Salmas, 15. Sang Sefid, 16. Tabriz.
Figure 8. Consensus tree of molecular and morphological characters.
Different studies (for example, Bradshaw 1965; Travis
1994; Schmitt et al. 1999) showed that the phenotypic
responses to various environments may also consist of
highly special physiological, developmental as well as
reproductive adaptive that multiply plant function in those
environments. Sultan (1995), believed that capability for
particular functionally appropriate environmental answer is
called adaptive plasticity, as differentiated from the
inevitable effects of resource restricts and other suboptimal
environments on phenotypic expression. Both adaptive and
inevitable natures of developmental plasticity are basic to
ecological development, because they influence the success
of organisms in their natural contexts. However,
functionally adaptive plasticity is of particular interest
because it allows individual genotypes to successfully grow
and reproduce in many different habitats. Consequently,
such plasticity can play a major pert in both the organism’s
ecological distribution and also its evolutionary
diversifications (Sultan 2003).
In our earlier study (Sheidai et al. 2014 a), we reported
a significant positive correlation between genetic distance
and geographical distance of these populations. However,
Mantel test did not show significant correlation between
morphological and genetic distance of the studied
populations.
Linum austriacum is a widespread species which its
different populations occur in different regions of the word,
and also this species grow naturally in different parts of
Iran in wide ranges of environmental conditions. Sample
collecting by authors as well as herbarium vouchers
mentioned in different flora such as Flora Iranica
(Rechinger 1974) as well as Flora of Iran (Sharifnia and
Assadi 2001) confirmed this condition and showed that this
species finds in various phytogeographical regions. There
are several reasons for such distribution; one of the most
important factors for this ability is plasticity in genomic
structure of this species. Various studies (Such as Baker
1974; Oliva et al. 1993) confirmed that taxa consisting of
adaptively plastic genotypes may inhabit a wide range of
ecological conditions; many widespread generalist species
may upon examination show this property. Williams et al.
(1995), suggested that the adaptive plasticity may also
contribute specifically to species invasiveness by allowing
rapid colonization of diverse new habitats without the need
to undergo local selection. As Sultan and Spencer (2002)
believed, plasticity of individual may influence
evolutionary diversification models at the population level
by precluding selective divergence in environmentally
distinct habitats.
186
B I O D I V E R S IT A S 16 (2): 178-187, October 2015
Losos and Glor (2003), believed that morphological
variation and geographical separation among populations
are important and considered as the prerequisite to the
formation of new taxonomic ranks such as subspecies and
species. It is suggested that phylogeographic analysis can
be used to illuminate the interplay of climate, geographical
history, and evolutionary dynamics in generating new taxa
(Avise et al. 1987; Arbogast and Kenagy 2001). Similarly,
the Bayesian and consensus tree obtained in the present
investigation produced the results that suggested the
presence of potential subspecies and ecotypes in L.
austriacum. Moreover, population studies on the pattern of
variation in many plant species have revealed the existence
of localized populations each adapted to the particular
environmental conditions of their habitat (Christine and
Monica 1999).
“The term ecotype is defined as: distinct genotypes (or
populations) within a species resulting from adaptation to
local environmental conditions; capable of interbreeding
with other ecotypes or epitypes of the same species
(Hufford, and Mazer 2003)”. This definition suits
morphological and genetic discontinuities observed in 4
populations of L. austriacum species.
Ockendon (1968), studied morphological diversity in
several geographical populations of the L. perenne group in
Europe. He reported that most of these characters vary
continuously and no sharp differences existed among
populations. However, significant difference was observed
in some of the quantitative characters and therefore,
different geographical populations were considered to be
ecotypes within each subspecies. In a similar investigation,
Nicholls (1986), carried out multivariate analysis of
morphological characters in populations of L. tenuifolium
and recognized sub-specific taxa in this species. The same
argument holds true for Sheidai et al. (2014b), who based
on molecular and morphological difference of the studied
population in Linum album, considered them as ecotypes
within this species.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160214
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 188-195
Impact of Gujjar Rehabilitation Programme on the group size of Asian
elephants (Elephas maximus) in Rajaji National Park, North-West India
RITESH JOSHI
Conservation and Survey Division, Ministry of Environment, Forest and Climate Change, Indira Paryavaran Bhawan, Jor Bagh Road, New Delhi110003, India. Tel./Fax.: +91-24695359,♥email: ritesh_joshi2325@yahoo.com
Manuscript received: 13 July 2015. Revision accepted: 1 August2015.
Abstract. Joshi R. 2015.Impact of Gujjar Rehabilitation Programme on the group size of Asian elephants (Elephas maximus) in Rajaji
National Park, North-West India. Biodiversitas 16: 188-195. A comparative study has been done to assess the impact of the Gujjar
Rehabilitation Programme on the group’s size of Asian elephants (Elephas maximus) in Rajaji National Park, north-west India. Field
surveys were carried outbefore the Gujjar’s rehabilitation during 1999-2001 and after the Gujjar’s rehabilitation during 2006-2008 in
Chilla and Haridwar forest ranges of the park. A total of 833 groups of elephants were sighted, varying from 2-5 (mean
value±SD=28.5±24.7) to 21-25 animals (mean value±SD=8.2±4.6). The number of groups sighted in Haridwar forest after the Gujjars’
rehabilitation were significantly low in summer and winter as compared before the Gujjars’ rehabilitation. However, the number of
groups sighted in Chilla forest before and after the Gujjars’ rehabilitation in both the seasons was found to be same. Results indicated
that elephant’s group’s size and movement was shrinking/reducing in Haridwar forest, however, in Chilla forest it was found to be
slightly expanding/increasing. The impact of Gujjar Rehabilitation Programme has not brought any drastic change in restoring the larger
population of elephants and in increasing their group’s size, however, this has increased the frequent movement and activities of
elephants within their home range. Restoration of large fragmented forest stretches/corridors for elephant’s migration and habitat
management are of paramount importance in providing elephants a wider way to move across entire landscape in large herds. As
increase in human population in the nearby areas and developmental activities, with increase in vehicle traffic pressure on national
highways and railway track existing across the park were found to be creating negative impacts on the overall movement of large groups
of the elephants.
Keywords: Asian elephant, group size, Gujjar Rehabilitation Programme, Rajaji National Park, Shivalik Elephant Reserve
INTRODUCTION
Group formation is a widespread phenomenon
throughout the social mammals, and the problem of animal
grouping is one of the most fundamental ones in biology
(Durand et al. 2007). Most of the vertebrates, including
humans, are gregarious to a certain degree and tend to form
groups of conspecific individuals, which build up a major
component of their environment thereby influencing major
aspects of their lives, such as predation pressure, pathogen
pressure, aggression, foraging success, metabolism and
sexual selection (Reiczigel et al. 2008). The size and
composition of social groups have diverse effects on
morphology and behavior, ranging from the extent of
sexual dimorphism to brain size, and the structure of social
relationships (Silk 2007). Elephants are known to live in
large herds, which generally depend upon the availability
of feeding grounds and suitable habitats. Besides,
availability of adequate water sources (rivers, reservoirs,
etc.) is another important factor, which influences grouping
in elephants. Elephant’s group sizes are probably
determined by factors such as forage abundance,
seasonality, animal density and numbers, human
disturbance, natural predation pressure, genetic relatedness;
and among all these factors, food resource availability
probably plays important role in determining family or
group size (Sukumar 2008).
A larger group of elephants is generally called herd,
which is a large family unit. In elephants, single groups
generally consists an adult cow and few individuals,
however large herds consists several small groups making
it a larger clan, especially during migrations or adverse
environmental condition. Sukumar (1994) proposed that
the term ‘family’ should be restricted to a single adult
female plus off springs, and the term ‘joint family’ is used
in the Asian tradition to describe groups with more than
one adult female, even if these are reasonably stable.
Elephant populations are composed of several clans, which
represent large extended families; different clans or
members of different clans do not associate with each
other, however, members of a clan can mingle and form
associations with others in the clan (Desai 1997).
North-western Shivalik landscape is one of the most
crucial elephant’s habitats, which holds nearly 1346
elephants, distributed within 14 protected areas (records of
the 2007s elephant census, Uttarakhand Forest
Department). During the last 3-4 decades, rapid rate of
developmental activities, especially after the establishment
of Uttarakhand state in 2000, had disconnected a long chain
of elephant’s habitat, which earlier was known to spread
across river Yamuna in the west to Sharda in the east, as a
result of which elephants were pocketed in smaller forest
patches, and thus their larger herds were converted into
smaller groups. Rajaji National Park (RNP), one of the
JOSHI –Effects of Gujjars’ rehabilitation on elephants in Rajaji, Northwest India
crucial elephant’s habitat in Shivalik Elephant Reserve
serves as the north-western limit to the distribution range of
the Asian elephants. It holds a healthy population and sex
ratio of elephants as well. A total of 416 elephants were
recorded in RNP during the elephant’s census carried out in
2007. In a study carried out in Shivalik Elephant Reserve,
the male-female sex ratio of the elephants in Rajaji and
Corbett National Parks was recorded as 1:1.8 male:females
in RNP and 1:1.5-2.1 male:females in Corbett National
Park (Williams 2002). However, in a study carried out in
Chilla, Motichur and Haridwar forests of the RNP, the
elephant’s sex-ratio was recorded as 100 females:22.4
males (male:female ratio=1:4.4), which revealed on a
healthy elephant’s sex ratio (Joshi et al. 2007).
Gujjars are a nomadic pastoral community, arrived in
the Shivalik hills from Jammu and Kashmir State nearly
200 years ago, as part of the dowry of a princess of Nahan
(at present, a part of the Himachal Pradesh State). In
Shivaliks, they raised domestic buffalos and practiced
pastoralism, spending winter and beginning months of
summer (from October to April) in the Shivalik foothills
and peak summer and monsoon (from May to September)
in the Himalayan alpine pastures. These traditional
migrations generally take 20 days to complete one side
journey from lower to higher altitude areas or from higher
to lower altitude areas. Gujjar’s livelihood is primarily
based on rearing buffalo and cattle, and selling milk in
local markets. In view of achieving the objectives of the
provisions of the Wildlife (Protection) Act 1972, Gujjar
Rehabilitation Programme (GRP) commenced effectively
in 1980s, especially after the establishment of Uttarakhand
State in November 2000. Initial attempts were made in
1984 to resettle Gujjars (by the then Uttar Pradesh State
Government) but the program could not succeed, because
of non-participation of Gujjars in the program. Besides, the
fear of divesting of their traditional rights was another
reason, which encouraged them not to leave the forest.
With the passage of time, program was made more
effective because of sincere and dedicated efforts of
Government and Gujjars’ understanding about the benefits
of urban life and in this way, nearly 93.3% Gujjars were
resettled to two different rehabilitation sites, namely Pathri
and Gaindikhatta.
As a result of effective implementation of the Wildlife
(Protection) Act, out of total nine forest ranges, seven
ranges are completely free from Gujjars so far, which has
reduced anthropogenic pressure from the park. GRP has
motivated Gujjars to live urban life, which considerably
enhanced their livelihood and socio-economic status. Nowa-days, maximum of their children are gaining education
from schools, established by the State Government at
rehabilitation sites. Besides, they are cultivating few of the
cash crops and vegetables in the land provided to each
family at rehabilitation sites. GRP in RNP can be
considered as a model demonstration to showcase effective
conservation programs of the country, which has facilitated
in achieving the objectives of the provisions of the Act on
one hand, and has provided better livelihood options to the
pastoral Gujjars on the other hand. Program has also
ensured our priorities of wildlife conservation and is a
189
milestone to showcase the successful rehabilitation and
ecological restoration model and to share conservation
lessons with other range countries (Joshi and Pande 2007;
Joshi 2012). Overall, substantial increases in the encounter
rates of several species were apparent from 2002 onwards.
This study illustrates the impact of GRP on the group’s size
of elephants in Chilla and Haridwar forest ranges of the
RNP.
MATERIALS AND METHODS
Rajaji National Park (RNP) is located in Uttarakhand,
north-west India at 29º15'-30º31' N 77º52'-78º22' E, and
falls under the Gangetic plains biogeographic zone and
upper Gangetic plains province (Figure 1). Maximum
portion of the park lies under Shivalik’s bio-geographic
sub-division. RNP was established in 1983 with the aim of
maintaining a viable Asian elephant’s population in the
Shivalik landscape and is designated a reserved area for
‘Project Elephant’ by the Ministry of Environment, Forest
and Climate Change. The total geographical area of the
park is 820.21 km2. The dominant vegetation of the area
comprises Sal (Shorea robusta), Kamala (Mallotus
philippensis), Cutch (Acacia catechu), Kadam (Adina
cordifolia), Bahera (Terminalia bellirica), Indian Banayan
(Ficus benghalensis) and Indian Rosewood (Dalbergia
sissoo). However, dominant fauna of the park consists of
Tiger (Panthera tigris), Leopard (Panthera pardus),
Himalayan Black Bear (Ursus thibetanus), Sloth Bear
(Melursus ursinus), Hyaena (Hyaena hyaena), Barking
Deer (Muntiacus muntjak), Goral (Naemorhedus goral),
Spotted Deer (Axis axis), Sambar (Rusa unicolor) and Wild
Boar (Sus scrofa) and among reptilian fauna the Mugger
Crocodile (Crocodylus palustris) and King Cobra
(Ophiophagus hannah) represents Rajaji’s faunal
diverseness.
The datasets for this article have been extracted from
the field studies, which were carried out from 1999 to 2011
on the ecology and behavior of elephants in RNP and
adjoining protected areas. Haridwar and Chilla forests were
selected for this study, because Haridwar forest was the site
where anthropogenic activities were higher, whereas Chilla
forest was the site where anthropogenic activities were
quite lesser, except of the Gujjars’ activities during their
stay in the park area up to 2004. A comparative study on
elephant’s group composition, before the GRP (from June
1999 to May 2001) and after the program (from June 2006
to May 2008) was attempted to address the impact of
Gujjar’s rehabilitation on the grouping pattern of elephants
in RNP.Chi-square test was used to determine differences
in the group’s size of elephants. In addition, information on
the elephant’s group’s size was also collected from the
forest officials, Gujjars residing in HFD and local people to
cross check the datasets. Field binocular (Nikon Action
Series, 10x50 CF) was used to observe the elephants in
forests and Nikon Coolpix 8700 Camera was used to
produce photographic evidence. Geographical coordinates
of each observation were taken (Garmin GPS 72).
190
B I O D I V E R S IT A S
16 (2): 188-195, October 2015
Figure 1. Location map of the Rajaji National Park, Uttarakhand, north-west India
RESULTS AND DISCUSSION
Considering the records of direct sightings of elephants
in between 1999-2001 in Chilla forest, maximum sightings
occurred were of 11-15 animals (24.54%, sighted 54 times
in summer), followed by 2-5 animals (22.27%, sighted 49
times in summer). However, maximum sightings occurred
in between 2006-2008 were of 11-15 animals (26.16%,
sighted 62 times in summer), followed by 6-10 animals
(24.05%, sighted 57 times in summer). On the other hand,
maximum sightings of elephant’s group recorded in
Haridwar forest in between 1999-2001 were of 2-5 animals
(27.46%, sighted 78 times in summer), followed by 6-10
and 16-20 animals, respectively (15.84%, sighted 45 times
in summer). However, maximum sightings occurred in
between 2006-2008 were of 11-15 animals (21.81%,
sighted 24 times in summer), followed by 6-10 animals
(17.27%, sighted 19 times in summer) (Figure 2A-D).
In contrast, minimum sightings occurred in Chilla forest
in between 1999-2001 and 2006-2008 were of 21-25
animals (0.90%, sighted twice in winter), followed by 1620 animals (1.26%, sighted thrice in winter). Similarly,
minimum sightings occurred in Haridwar forest in between
1999-2001 and 2006-2008 were of >25 animals (0.35%,
sighted once in summer), followed by 16-20 animals
(2.72%, sighted thrice in winter) (Table 1). The largest
group of elephants, comprising of 37 individuals was
sighted in May 2007 in Luni river in Chilla forest,
however, another group comprising of 28 elephants was
sighted in June 2006 in Ghasiram water stream, while
moving across Ganges. In respect of Haridwar forest only a
large group, comprising of 26 elephants was sighted in
June 2000 in Kharkhari forest.
A total of 833 groups were sighted in between 19992001 and 2006-2008 in Chilla and Haridwar forests
respectively,
which
varied
from
2-5
(mean
value±SD=28.5±24.7)
to
21-25
animals
(mean
value±SD=8.2±4.6). Significant changes were not observed
in the number of groups sighted in Chilla forest during
summer before the Gujjars’ rehabilitation outside from the
park area (mean value±SD=30.4±20.5) as compared after
the Gujjars’ rehabilitation (mean value±SD=32.2±23.4;
One-sample goodness-of-fit test χ2=7.4; df=5; p=0.19521).
Similarly, during the winter significant changes were not
observed in the number of groups sighted before the
Gujjars’ rehabilitation (mean value±SD=6.4±5.05) as
compared after the Gujjars’ rehabilitation (mean
value±SD=7.4±5.2; One-sample goodness-of-fit test
χ2=14.1; df=4; p=0.00724).
Significant changes were observed in the number of
groups sighted in Haridwar forest during summer, before
the Gujjars’ rehabilitation (mean value±SD=35.0±27.7) as
compared after the Gujjars’ rehabilitation (mean
value±SD=13.2±9.0; One-sample goodness-of-fit test
χ2=13.2; df=4; p=0.01053). Similarly, during the winter
significant changes were observed before the Gujjars’
rehabilitation (mean value±SD=12.7±8.4) as compared
after the Gujjars’ rehabilitation (mean value±SD=5.2±4.9;
One-sample goodness-of-fit test χ2=4.9; df=4; p=0.30432).
Noticeably, the number of groups sighted in Haridwar
forest after the Gujjars’ rehabilitation were significantly
low in summer and winter as compared before to the
Gujjars’ rehabilitation. However, the number of groups
sighted in Chilla forest before and after the Gujjars’
rehabilitation in both the seasons was almost same (Figure
3A-D).
JOSHI –Effects of Gujjars’ rehabilitation on elephants in Rajaji, Northwest India
Population dynamics
Social factors such as territoriality, average group size,
seasonality of breeding etc. have a profound influence on
the population dynamics of many large mammals and are
important in their management (Caughley and Walker
1983). Population dynamics has been widely studied in
African elephants (Loxodonta africana), which has given a
higher insight on their social life, however, only few
studies have been conducted on this aspect in Asian
elephant range countries and more information on their
families’ bond and group’s interaction is still needed to be
studied. GRP has strengthened the frequent movements and
activities of elephants in RNP, however, has not placed any
drastic impact on their grouping pattern. In between 19992001 and 2006-2008, maximum sightings of elephant’s
groups occurred in Chilla forest was of 11-15 individuals.
In contrast, maximum sightings of elephant’s groups
occurred in Haridwar forest was of 2-5 and 6-10
individuals respectively. It is evident from the data sets that
elephant’s group’s sizes were slightly shrinked and their
movements were reduced in Haridwar forest. However,
elephant’s group’s sizes were slightly expanded and their
movements were slightly increased in Chilla forest.
In between 2007-2009, tremendous wildfires destroyed
the natural forest in Haridwar and Chilla ranges and
adjoining habitats. The Kharkhari and Chilla forests were
affected severely by the wildfires and thus disrupted
elephant’s seasonal movement. Wildfires occurred in
between 2006-2009 in Chilla and Haridwar forests and
shrinkage of natural water sources has affected the frequent
movement of free-ranging elephants within the RNP (Joshi
and Singh 2010). Since last 15 years, vehicle’s traffic
pressure on two national highways (Haridwar-Bijnor and
Haridwar-Dehradun) has increased almost two folds and
elephants were observed not in the situation to cross the
road easily, especially during evening hours. It was
exposed in a study that nearly 9,900 and 14,100vehicles
moves across the Haridwar-Bijnor and Haridwar-Dehradun
national highways per day, except only 3 hours, from 12
am to 3 am (Joshi et al. 2010). Besides, train’s traffic has
also increased in the Haridwar-Dehradun railway track. In
2000, establishment of State Industrial Development
Corporation of Uttarakhand in nearly 10 kilometers long
stretch, spread along the Haridwar forest range had
severely affected the movement of elephants, especially in
191
the buffer zone of the park. Anthropogenic activities were
also observed enhanced mainly because of increased
human population along the periphery of the park.
In eastern part of the park, bigger groups of elephants
were observed maximum at the onset of summer (MarchApril), when elephants were observed arriving to RNP
from Lansdowne forest division and when elephant’s
movements were found confined towards the riparian
corridors of Ganges. At the onset of monsoon, elephants
were observed migrating towards Lansdowne forest
division and in some higher ridges of the park in closed
family groups. However, in the south-western part of the
park, bigger groups were observed maximum during
summer and monsoon, when elephant’s movements were
found confined nearer to the natural water sources.
Occasional splitting of herds was also recorded; especially
when elephants reached in the lower patches of the park,
however, composition of most of the herds were observed
remained same. Generally, large herds used to split into
smaller groups, after reaching to the new feeding grounds
and stay there nearly for 3-4 months. Environmental factors
affect elephant’s population dynamics, home range,
migration patterns, diet, group size and composition, all of
which can vary tremendously, in turn influences the
dynamics of elephants and their habitats (Poole 1996). The
dynamics of a population may be affected through changes
in group’s size or other social disruptions such as the
removal of age classes, which are important to the group’s
social structure and functioning (Dublin and Taylor 1996).
In African elephant’s ranges, larger elephant’s groups
were reported during wet season when resources are
abundant (Western and Lindsay 1984; Poole and Moss
1989), however, in Asian elephant ranges, larger groups
were reported during dry season, when elephants
congregate around scarce water sources and chances of
their social contact and association are high (Sukumar
1989). Studies in East Africa revealed that elephants used
to enter forests in large groups, but leave in small groups,
thereby implying that the large groups break up into
smaller units within the forests (Laws et al. 1975). In RNP,
pronounced elephant’s mating season was recorded as
warm period, therefore one of the important reasons behind
such gathering particularly in summer might also be for
mating needs (Joshi et al. 2009).
Table 1.Sighting of elephant’s groups during summer and winter seasons in Chilla and Haridwar forests of the Rajaji National Park in
between 1999-2001 and 2006-2008
Number
of
individual
in groups
2-5
6-10
11-15
16-20
21-25
> 25
Total
Chilla
1999-2001
Summer Winter
49
05
43
11
54
07
11
13
14
02
11
182
38
Total
54
54
61
24
16
11
220
2006-2008
Summer Winter
36
14
57
09
62
11
19
03
12
07
07
193
44
Haridwar
Total
50
66
73
22
19
07
237
1999-2001
Summer Winter
78
18
45
21
30
13
45
17
12
04
210
73
Total
96
66
43
62
16
283
2006-2008
Summer Winter
18
10
19
11
24
07
12
03
06
79
31
Total
28
30
31
15
06
110
192
16 (2): 188-195, October 2015
B I O D I V E R S IT A S
A
B
C
D
Group size
A
Group size
B
Group size
C
>25
21-25
16-20
11-15
2-5
6-10
>25
21-25
16-20
6-10
11-15
2-5
>25
21-25
16-20
11-15
2-5
6-10
>25
21-25
16-20
11-15
2-5
6-10
Figure 2.A. An elephant’s herd during migration from Rajaji National Park to Lansdowne forest division, B. Elephant’s group at Chilla
forest with a newly born infant, C. A small family of elephants, consisting of five members; mother elephants are taking their calves
with care, D. Members of a group greeting each other by inter-twining their trunks in Haridwar forest.
Group size
D
Figures 3 A-B. Number of elephants observed in different group sizes (with standard error) during summer and winter seasons in Chilla
forest in between 1999-2001 and 2006-2008 (before and after the Gujjars’ rehabilitation), C-D. Number of elephants observed in
different group sizes (with standard error) during summer and winter seasons in Haridwar forest in between 1999-2001 and 2006-2008
(before and after the Gujjars’ rehabilitation).
JOSHI –Effects of Gujjars’ rehabilitation on elephants in Rajaji, Northwest India
Sukumar (1994) recorded elephant’s aggregation
ranging from 50 to 200 in southern India. In a study carried
out in the same area, elephant’s group size was recorded
ranging from 1 to 26 individuals with mean group size
4.32±3.2; it was also revealed from this study that
elephants have skewed group size with smaller group sizes
being more common than larger groups (Ashokkumar et al.
2010). However, in a similar study carried out in the same
area, it has been exposed that elephant’s group size varied
from 1 to 22 individuals with mean group size 4.6±0.16
and 6.8 respectively (Ramesh et al. 2012). Vance et al.
(2008) have illustrated on the social networks in African
elephants and pointed out that social groups tend to be less
cohesive and smaller, families are often divided into small
subgroups in dry season, and they rarely fuse with other
families to form larger aggregations, which is mainly due
to scarcity of fodder resources. Further, during wet season,
families often travel in intact groups and whole families
often fuse with other families, constituting a bigger
continuous aggregation, which occurs mainly because of
availability higher resources.
Scientific reasons behind conversion of large groups
into smaller ones
Habitat fragmentation and disconnectivity of large
migratory corridors were observed as prime cause, which
had converted large elephant’s herds into smaller ones.
Four crucial wildlife corridors namely, Motichur-Chilla,
Motichur-Gohri, Motichur-Kansrao-Barkot and RawasanSonanadi, which exists across RNP and connect this
protected area with Corbett Tiger Reserve, were found
almost blocked, mainly because of expansion of human
settlements, national highways/motor roads and agriculture
lands and increasing traffic in Haridwar-Dehradun railway
track. The presence of traffic on the road, construction of
steep retaining walls and the presence of human population
along the entire Rajaji-Corbett wildlife corridor area have
almost restricted the migration of elephants in between the
Rajaji-Corbett National Parks (Johnsingh and Williams
1999).
Nandy et al. (2007) carried out the assessment of
Chilla-Motichur wildlife corridor using temporal satellite
imagery from years 1972, 1990 and 2005, which exposed
that an area of 17.56 Km2 has been lost in between 19722005 mainly because of various developmental activities.
Another study carried out in between June 2009 to May
2011 exposed that a total of 352 individuals of 39 species
were killed on Haridwar-Bijnor and Haridwar-Dehradun
national highways and on an ancillary road (HaridwarChilla-Rishikesh) existing across RNP (Joshi and Dixit
2012). Noticeably, 23 elephants were killed in collision
with train on Haridwar-Dehradun railway track since 1987.
Besides, few cases of collision of elephants with the
speeding vehicles on national highways were also
observed, during the study period.
In 1970s, the establishment of Chilla hydro-electric
power plant/channel had almost divided RNP into two
major parts, the eastern and western fringe. Further, after
the establishment of Uttarakhand state in 2000, vehicle
traffic pressure on Haridwar-Dehradun national highway
193
and railway track, which exists in between the park area
has increased many folds, which restricted elephants to
inter-change the forests frequently. In south-western part of
the park, elephant’s access to river Ganges, which is
flowing in between the park area was found restricted,
mainly because of presence of a national highway, railway
track and human settlements. Some recognized bulls (~2-3)
were observed visiting Ganges occasionally by crossing the
populated area and national highway and railway track,
however group’s movement was found almost restricted in
this part. In eastern part of the park, elephant’s were found
visiting Ganges, especially in summer when their
movements were confined nearer to the riparian corridors.
However, elephant’s frequent movement towards Ganges
was observed affected because of the presence of the Chilla
power channel.
Non availability of large grasslands was recorded
another reason, which compels the elephants to aggregate
in smaller groups. In addition to some small patches of
grasslands, Mundal grassland in Chilla forest is only a vast
patch (tentatively 500 hectares), where large groups of
elephants were recorded in summer. This grassland was
developed across the Mundal river after the relocation of
Gujjars in 2003-2004, consisting mainly Wild Sugarcane
(Saccharum spontaneum) and Kans Grass (Saccharum
munja) species.
Anthropogenic activities were observed affecting
frequent movement of elephants within their home range
and thus their group’s size. More than 20 villages are
situated along the south-western boundary of RNP, 20
villages along the eastern fringe and nearly 15 villages are
situated along the northern axis of the park. Noticeably,
most of the villagers were found dependent on forest
resources for their livelihood, which primarily includes
collection of fuelwood and fodder. Spreading of invasive
species like Lantana Weed (Lantana camara) and
Parthenium Weed (Parthenium hysterophorus), and native
weeds like Malabar Nut (Adhatoda vasica), Indian Hemp
(Cannabis sativa) and Wild Senna (Cassia tora)was also
recorded affecting the regeneration of various species of
grasses. Spreading of these invasive species was found
occurring mainly because of cattle’s grazing across the
boundary of the park. Besides, annual/torrential rivers were
also noted as cause of spreading of these species.
Few previous records
Evidences of sighting of large herds of elephants in the
park were mostly noticed sporadic and specifically during
dry period. In between 1999-2011, I had seen the largest
herd of elephants in May 2000, consisting of ≈50
individuals in Gohri forest of the RNP, while moving
across the Ganges, which embraces the peak of dry season.
In between 2004-2008, largest herd of elephants,
comprising of 47 individuals was sighted in 2005 in Chilla
forest of the RNP in summer (Pande GS 2015, personal
communication) months. Since last two decades, only a
large herd of elephants was sighted in 1997 during summer
in Rawasan river in Chilla forest, which consisted nearly 78
elephants and later on, such a large herd was never sighted
(Negi MS 2015, personal communication).
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16 (2): 188-195, October 2015
Management guidelines
Three small bridges (±3.5 meter wide) were constructed
during 1970s over to the Chilla power channel during the
establishment of Chilla hydro-electric power plant, one in
Chilla forest, named Soni water source bridge, and two
other in Gohri forest, one near to Kaudia village (Kaudia
village bridge) and another at Kunao forest (Kunao water
source bridge). Elephants were found utilizing only two
bridges, one in Chilla and another in Gohri forest (Soni and
Kunao water source bridges), however, not utilizing
Kaudia bridge mainly because of presence of Kaudia and
Ganga-Bhogpur villages across the bridge. Soni water
source and Kaudia village bridges are connected with
Chilla-Motichur and Motichur-Gohri corridor, whereas,
Kunao water source bridge is connected to MotichurKansrao-Barkot corridor. All these corridors link RNP with
Corbett Tiger Reserve area. It is needed to widen all these
bridges tofacilitate the movement of elephants in between
RNP and Corbett Tiger Reserve. Besides, two more bridges
could also be constructed over to the power channel, one at
Ram water source, in between Chilla power house and
Kaudia village and another at Kunao, in between Binj river
and Kunao bridge, which would be feasible approaches to
give elephants wider way to move across Ganges and
corridors as well.
It is not easy to count elephants directly when moving
in large herds or groups, especially in foothills and
vegetation dominant landscapes. Further, there are chances
of ground-based errors during estimating their numbers.
Generally, elephant’s large groups maintain a range while
feeding or drinking, which ranges tentatively from 50-200
meter. Sometimes, some members of the group used to
arrive in open areas or river beds from dense forest to
drink, however some companions remain inside the forest,
which can emerge out of the forest area after a while.
Elephant is a giant animal and if some individual assembles
at a place, it covers large space and looks like a large
group. Similarly, pugmarks impressions of few individuals
give an impression of movement of a big group. These are
some examples which should be taken into account while
estimating elephant’s numbers in forests.
Some villages, which are situated across the crucial
elephant’s corridor, should be rehabilitated outside the
protected area. In addition, Gujjars from Shyampur and
Chiriapur forest ranges of the Haridwar forest division and
Gohri forest of the RNP should also be rehabilitated
outside from the protected area. In addition to the relevant
provisions of the Wildlife Protection Act and Project
Elephant, this could be achieved with the help of
conservation actions, which were identified for
development of site-specific management plans, securing
identified corridors and connectivity areas for the
integration of the protected areas, participatory wildlife
monitoring for strengthening management and conducting
targeted studies on protected areas valuation assessment as
well as on climate change resilience and adaptation
assessment in selected protected areas, under India's Action
Plan for Convention on Biological Diversity's Programme
of the Work on Protected Areas (Pande and Arora 2014).
Some important natural water reservoirs, which spread
across the Bagro, Ranipur, Ravli, Chirak and Harnaul
annual rivers in Haridwar forest and Ghasiram, Mitthawali,
Mundal, Luni and Rawasan annual rivers in Chilla forest
should be restored. Besides, water sources which are
connected with the Chilla power channel should also be
managed.
Riparian corridors of Ganges should be restored from
anthropogenic activities, especially from mining activities.
Scientific studies on elephant’s ecology and behaviour
and its implementation would ensure the future survival of
elephants in entire Shivalik Elephant Reserve.
In conclusion, the impact of GRP has not placed any
drastic change in restoring elephant’s population and
increasing their group’s size, however, has restored the
wilderness of the park, which has increased the frequent
movement and activities of elephants within their home
range, and thus facilitated in ensuring the effective
implementation of Wildlife (Protection) Act 1972.
Catastrophic increase in human population, developmental
activities, and increasing rate of vehicle traffic on national
highways existing across the landscape were observed
reinforcing negative impact on the movement of large
groups and herds of the elephants. Since last 15 years
vehicle-traffic pressure on the national highways and a
railway track existing across RNP has increased drastically.
Besides, developmental and anthropogenic activities have
also increased across maximum of the forest edges and in
some crucial corridors. Moreover, the establishment of
State Industrial Development Corporation of Uttarakhand
in 2000 has affected the frequent movement of elephants,
especially in park’s buffer zone. All these activities have
reduced the connectivity between different protected areas,
thus affected the elephant’s movement across large
undisrupted landscapes in large herds. Understanding how
elephant populations acclimatize to such unwanted changes
in their habitat, is essential for addressing future challenges
in elephant’s management and conservation. Since the
extent to which elephant’s aggregate in a particular area is
relevant to their management, restoring the crucial wildlife
corridors, facilitating the Ganges’ access and ensuring
community participation would strengthen the elephant’s
traditional migration across entire Shivalik Elephant
Reserve.
ACKNOWLEDGEMENTS
The author would like to acknowledge the anonymous
reviewers, who had provided valuable inputs and
comments on the previous versions of the manuscript and
contributed significantly to improve the manuscript to its
present form. The author would like to acknowledge G.B.
Pant Institute of Himalayan Environment and
Development,
Garhwal
Unit,
Srinagar-Garhwal,
Uttarakhand, India and the Doon Institute of Management
and Research, Rishikesh, Uttarakhand, India, as the author
was associated with these institutions during the study
period. Author would also like to acknowledge the
Uttarakhand
Forest
Department,
especially
the
JOSHI –Effects of Gujjars’ rehabilitation on elephants in Rajaji, Northwest India
administration of the RNP for providing permission to
conduct research on elephant’s ecology and behavior. The
author would like to also thank Shanti Prasad, Field
Assistant, field staff of the RNP and various Gujjars and
locals, who had provided assistance and inputs during
collection of the field data.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160215
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 196-204
Diversity of butterflies in four different forest types in Mount Slamet,
Central Java, Indonesia
IMAM WIDHIONO
Faculty of Biology, Jenderal Soedirman University. Jl. Dr. Soeparno No. 68, Purwokerto, Banyumas 53122, Central Java, Indonesia. Tel. +62-281638794, Fax: +62-281-631700, email: imamwidhiono@yahoo.com
Manuscript received: 26 May 2015. Revision accepted: 20 August 2015.
Abstract. Widhiono I. 2015. Diversity of butterflies in four different forest types in Mount Slamet, Central Java, Indonesia.
Biodiversitas 16: 196-204. The study was carried out in four different habitat types (secondary forest, plantation forest, agroforest, and
tourist area) on the southern slope of Mount Slamet, Baturaden Forest, Central Java, Indonesia from July 2009 to August 2010. A total
of 99 species belonging to eight families showed a dominance of Nymphalidae (30 species) followed by Pieridae (17 species),
Lycaenidae (15 species), Papilionidae (13 species), Satyridae (11 species), Danaidae (6 species), Amathusidae (4 species), and
Riodinidae (3 species). From the 99 butterflies species found on the southern slope of Mount Slamet, 32 species (30%) were specific to
the forest, whereas 63 species (60.6%) were common to all habitats sampled, and the last 10 species (9.4%) were endemics species with
one protected species (Troides helena). The present results was showed that butterflies diversity, abundance, and endemism is still
relatively high, representing 18% of all butterfly species found in Java and supporting 71.4% endemic species found in Central Java.
The plantation forest were contributed the highest diversity and abundance of butterfly species, whereas the agroforest showed the
lowest diversity, abundance, and endemism. Among all habitats surveyed, the secondary forest represented the most suitable habitat for
biodiversity conservation and maintenance of rare and endemic species.
Keywords: Butterfly, Central Java, diversity, endemism, Mount Slamet
INTRODUCTION
Mount Slamet is the second largest volcanic mountain
in Java, located in the western region of Central Java
Province, with an altitude of 3,432 m above sea level. A
large area of Mount Slamet is covered by a variety of
forests, including secondary, plantation, agroforests and
tourist areas (SFC 1999). Ecologically, forests on Mount
Slamet are divided into three types of forest, i.e.: lowland,
montane, and subalpine that has diverse vegetation
(Sumarno and Girmansyah 2012). The forest areas on
Mount Slamet are managed by the State Forest
Management Agency (Perum Perhutani). From an
ecological viewpoint, forest areas on Mount Slamet is a
transition from tropical rainforests in western Java to
monsoon forests in eastern Java, which significantly impact
on the conservation of biodiversity in Java.
Secondary and plantation forests are usually
monocultures of exotic tree species, meaning that they
provide poorer habitats than original forests for native
butterfly species. Both secondary and plantation forests are
expected to play positive roles in biodiversity restoration
(Matsumoto et al. 2015), especially when reforestation and
natural regeneration are allowed. These secondary and
plantation forests can be considered additional conservation
areas, as they are in close proximity to natural forests that
are known to house of a large population of butterflies.
Secondary and plantation forests can also act as buffers and
connections between natural forests and other lands, like
agroforests and tourist areas. They may improve
connectivity among forest patches, which is important for
the maintenance of butterfly diversity.
Java island is a suitable places hosts a diverse butterfly
population (583 species, Yukawa 1984; and 629 species,
Whitten et al. 1997) with 46 endemic species (Matsumoto
et al. 2015), most or all of which depend to some extent
upon closed forests (Bonebrake et al. 2010; Sodhi et al.
2010; Vu et al. 2015). From a conservationist viewpoint,
patterns in the richness of geographical restricted or
endemic butterflies are of particular interest. The diversity
of butterfly communities has been studied in different
habitat types in different part of Java, such as Gunung
Halimun National Park, West Java (Ubaidillah 1998),
Mount Tangkuban Perahu, West Java (Subahar et al. 2007),
Gunung Salak, West Java (Tabadepu et al. 2008), Gunung
Halimun-Salak National Park, West Java (Murwitaningsih
and Dharma 2014), and Bromo-Tengger-Semeru National
Park, East Java (Suharto et al. 2005). However, few studies
have been performed on the diversity of butterfly
communities on Mount Slamet. The study was examined
the diversity, abundance, and endemism of butterfly on
Mount Slamet and to address the importance of secondary
and plantation forests for butterfly conservation.
MATERIALS AND METHODS
Study area
The study was conducted in Baturaden Forest, East
Banyumas Forest Management Unit on the southern slope
WIDHIONO – Butterflies of Mount Slamet, Central Java
of Mount Slamet, Central Java, Indonesia. Geographically,
this region lies between 70 18’23 72” S and 1090 14’ 06 51”
at 600-800 m above sea level. The total study area is 267.5
ha, and the forest types are mainly classified as secondary
forest (SF, 50 ha), plantation forest (PF, 50 ha), agroforest
(AF, 50 ha), and tourist area (AF, 117.5 ha) (Figure 1).
Description of the study site
Secondary forest (SF)
Vegetation in SFs are tropical, rainforest-type
vegetation that consists of 19-20 tree species, such as
Palaquium
rostratum,
Turpinia
sphaerocarpa,
Xanthophyllum excelsum, Terminalia catappa, Tarenna
incerta, Sterculia campanulata, Spathodea campanulata,
Semecarpus heterophyllus, Planchonia valida, Macaranga
rhizinoides, Litsea angulata, Hernandia peltata, Helicia
javanica, Gluta renghas, Ficus variegata, F. benjamina,
Fagraea crenulata, Euodia roxburghiana, Erythrina
variegata, Elaeocarpus glaber, Dipterocarpus gracilis,
Dendrocnide sinuata, Artocarpus elastica, Antidesma
tetrandum, and Aglaia elliptica.
197
Plantation forest (PF)
PFs are dominated by resin plants (Agathis dammara);
other tree species are used for secondary products such as
firewood, animal forage, site amelioration, and fodder.
Some are planted as border trees, including Leucaena
leucocephala, L. glauca, Calliandra californica, and
Acacia villosa. Other trees were planted to increase the
heterogeneity of the forest, such as Schima noronhoe,
Pterospermum javanicum, Magnolia blumei, Tarenna
incerta, Antidesma bunius, Macadamia ternifolia,
Swietenia spp., Michelia montana, Mesua ferrea, Machilus
rimosa, Cinnamomum burmanii, and Santalum album.
Agroforest (AF)
AFs were established as soon as the land was clear-cut
and the forest management was performed by Perhutani
(State Forest Company/SFC), adopting the Taungya system
from Myanmar and involving local people (Whitten et al.
1997). Each family of forest farmers received 2 ha of forest
land for a duration 3-5 years. On this land, farmers were to
plant the main tree species (A. dammara) and food plants,
primarily dry land rice, corn, and certain vegetables.
Figure 1. Location of Baturaden Forest, southern slope of Mount Slamet, indicating the sampling sites.
198
B I O D I V E R S IT A S 16 (2): 196-204, October 2015
Tourist area (TA)
In addition to forest management techniques that SFC
created, tourist or recreational areas to increase the quality
of life and prosperity of local people were created.
Originally, TAs functioned as campgrounds, but at present,
more than 50% of all TAs consist of open gardens with
white exotic plant species.
Climatology
The study site receives an annual rainfall of
approximately 4500 mm3, representing one of the highest
precipitation areas in Indonesia. The long rainy season with
precipitation of more than 100 mm3/month ranges from
October to May/June. The very short dry season with
precipitation of less than 60 mm3/month ranges from July
to September. Day light temperatures range from 20 to
28°C.
Sampling procedure
Field surveying of butterfly was conducted from July
2009 to August 2010, following the Modified Pollard Walk
Method with kite netting in four distinct habitats of Mount
Slamet. Five permanent lines of transects (PLTs) (~ 0.5 km
long and 5 m wide) were laid in the four habitat types.
Butterflies were captured during sunny days at a constant
speed in each transect from 8 am to 12 am local time for
four consecutive days. This process was repeated at 30-day
intervals, maintaining the same spatial scale in each of the
five sampling sites. Identification of butterfly species was
used the method described by D’ Abrera (1982, 1985 and
1986). Collected specimens are maintained in the
entomology laboratory at Jenderal Soedirman University,
Purwokerto, Banyumas, Central Java, Indonesia.
Data analysis
The Shannon diversity index was applied to estimate
butterfly diversity along the habitats. This index was
calculated using the equation: Hs =-Σpi In p, where, pi is
the proportion of individuals found in the ith species and
‘In’ denotes the natural logarithm. Species dominance
across habitats was estimated using the Simpson’s
dominance index to determine the proportion of more
A
common species in a community or an area with the
following formula: Ds = Σsi=1 [ni (ni-1)]/[N (N-1)], where
ni is the population density of the ith species, and N is the
total population density of all component species in each
site. Comparisons of butterfly species composition between
different forest habitats was estimated using single linkage
cluster analysis based on Bray-Curtis similarity.
Biodiversity Pro version 2 (McAleece et al. 1997) was used
for data analysis.
To classify the status of species, the rare species, as
defined in this study, are those species represented by
fewer than 10 individuals, while the endemic species
defined as species which is only found in Java and nowhere
else in the world.
RESULTS AND DISCUSSION
The butterfly community at Mount Slamet
A total of 99 butterfly species were recorded at the
study site: the southern slope of Mount Slamet (Table 1).
The species recorded were obtained from July 2009 to
August 2010.
The butterfly species belong to eight families with a
dominance of Nymphalidae (30 species, 28.7%) followed
by Pieridae (17 species, 16.6%), Lycaenidae (15 species,
15,1%), Papilionidae (13 species, 12.3%), Satyridae (11
species, 10.1%), Danaidae (6 species, 5.5%), Amathusidae
(4 species, 3.7%), and Riodinidae (3 species, 3.7%) (Figure
2). This result represents only 18% of the 583 species
recorded from Java (Yukawa 1984); 13 species of
Papilionidae represent 37.4% of the total species in Java
(35 species). Pieridae were represented by 18 species
(30%) of a total of 52, and Nymphalidae were represented
by by 30 species (15%) from 226 species found in Java.
Additionally, Satyridae were represent by 11 species (30%)
from 44 species, Danaidae were represented by 6 species
(16.6%) from 36 species, Amathusidae were represented by
4 species (30%) from 13 species, Riodinidae were
represented by 4 species, and Lycaenidae were represented
by 18 species (10.6%) from 179 species found in Java.
B
Figure 2. A. Species composition of 8 butterfly families. B. Number of individuals of 8 butterfly families
WIDHIONO – Butterflies of Mount Slamet, Central Java
199
Table 1. Butterfly species captured on Mount Slamet from July 2009 to August 2010 in different forest types.
Atrophaneura coon
Atrophaneura nox
Atrophaneura priapus
Graphium sarpedon
Papilio acheron
Papilio demolion
Papilio helenus
Papilio memnon
Papilio paris
Papilio polytes
Chilasa paradoxa
Troides helena
SF
27
8
9
1
1
0
21
15
11
18
9
8
PF
13
6
6
29
7
5
30
12
6
1
1
1
Habitat
AF
1
0
0
3
0
2
0
14
0
0
0
0
TA
3
2
6
19
6
7
0
23
0
0
0
0
Appias lyncida
Appias cardena
Catopsilia pomona
Catopsilia pyranthe
Catopsilia florella
Cepora iudith
Eurema ada
Eurema andersonii
Delias belisama
Delias descombesi
Delias crithoe
Delias hyparete
Delias pasithoe
Delias periboea
Leptosia nina
Prioneris autothisbe
Gandaca harina
5
0
1
2
0
0
0
39
21
2
4
0
12
2
32
1
0
9
5
3
6
13
3
14
119
56
51
13
1
10
19
47
6
107
96
1
152
148
112
115
32
125
11
37
47
0
35
77
17
0
123
84
15
134
113
135
112
98
129
68
57
90
0
3
0
0
0
88
194
21
290
269
260
230
144
412
156
147
154
1
60
98
96
7
318
Amnosia decora
Amnosia decora endamia
Athyma cama
Athyma pravara
Cethosia munjava
Cethosia penthesilea
Chersonesia peraka
Cirrochroa clagia
Cirrochroa emalea
Cupha arias
Cynitia iapsis
Cyrestis lutea
Eulaceura osteria
Rhinopalpa polynice
Rohana nakula
Symbrenthia anna
Symbrenthia hypselis
Tanaecia trigerta
Junonia atlites
Junonia hedonia
Junonia orithya
Junonia almana
Lasippa heliodore
Lasippa tiga
Hypolimnas bolina
Hypolimnas misippus
Stibochiona coresia
Euthalia monina
Vanessa cardui
Neptis nisea
Euploea gamelia
35
42
3
5
3
5
2
12
12
3
8
4
28
2
3
0
0
3
1
2
2
0
46
0
5
1
14
1
0
9
0
72
31
42
12
5
12
52
30
60
21
44
4
23
1
18
1
19
91
21
64
11
0
50
24
48
89
3
84
221
83
0
0
0
19
54
0
0
97
22
22
8
0
0
24
0
0
188
64
12
22
62
0
82
57
100
3
6
0
36
405
0
7
0
0
36
57
0
0
33
1
3
7
8
0
0
0
4
104
51
54
27
23
0
93
124
15
38
20
17
49
0
66
0
107
73
100
128
8
17
184
65
97
39
60
8
75
3
25
293
134
160
71
151
13
175
277
139
94
116
34
170
626
158
7
Family
Genera
Species
Papilionidae
Atrophaneura
Graphium
Papilio
Chilasa
Troides
Pieridae
Appias
Catopsilia
Cepora
Eurema
Delias
Leptosia
Prioneris
Gandaca
Nymphalidae
Amnosia
Athyma
Cethosia
Chersonesia
Cirrochroa
Cupha
Cynitia
Cyrestis
Eulaceura
Rhinopalpa
Rohana
Symbrenthia
Tanaecia
Junonia
Lasippa
Hypolimnas
Stibochiona
Euthalia
Vanessa
Neptis
Euploea
∑individuals
44
16
21
52
14
14
51
64
17
19
10
9
200
B I O D I V E R S IT A S 16 (2): 196-204, October 2015
Danaidae
Danaus aspasia
Danaus vulgaris
Euploea climena
Euploea gamelia
Parantica albata
Parantica pseudomelaneus
5
7
5
3
10
3
15
97
11
3
16
82
0
30
2
7
6
0
0
11
7
103
6
0
20
145
25
116
38
85
Elymnias casiphone
Elymnias ceryx
Elymnias hypermnestra
Elymnias nesaea
Lethe europa
Melanitis leda
Melanitis zitenius
Mycalesis sudra
Ypthima nigricans
Ragadia makuta
Neorina krishna
12
1
6
6
64
45
25
25
111
89
32
25
2
13
15
182
267
199
413
536
45
29
12
5
0
9
48
12
1
78
561
0
0
16
0
78
22
62
54
47
86
296
0
0
65
8
97
52
356
378
272
602
1504
134
61
Zemeros
Abisara kausambi
Abisara savitri
Zemeros flegyas
17
1
2
55
1
8
0
0
0
0
0
0
72
2
12
Amathusiidae
Faunis
Thaumantis
Amathusia
Zeuxidia
Faunis canens
Thaumantis odana
Amathusia taenia
Zeuxidia luxerii
90
37
30
2
314
79
101
32
0
0
0
0
0
0
0
0
407
116
131
149
Lycaenidae
Arthopala
Prosotas
Dacalana
Nacaduba
Arthopala sp.
Prosotas dubiosa
Dacalana vidura
Nacaduba angusta
Nacaduba kurava
Nacaduba sp.
Surendra vivarna
Stiboges calycoides
Heliophorus epicles
Jamides alecto
Jamides celeno
Jamides cunilda
Jamides cyta
Jamides pura
Poritia eryconoides
2
0
2
0
0
0
0
1
28
12
3
6
2
15
0
1203
3
26
8
48
109
4
9
1
41
116
96
20
16
52
59
5088
12
11
3
22
57
17
0
0
0
68
4
8
5
19
21
3469
17
21
2
23
69
3
7
0
44
79
20
5
10
93
28
3234
34
58
15
93
235
24
16
2
113
275
123
39
33
179
108
12994
Danaus
Euploea
Parantica
Satyridae
Elymnias
Lethe
Melanitis
Mycalesis
Ypthima
Ragadia
Neorina
Riodinidae
Abisara
Surendra
Stiboges
Heliophorus
Jamides
Poritia
Table 2. Butterfly on Southern slope of Mount Slamet compared
to other Indo-Malayan region
Region
Oriental Region
Indo-Malayan
Borneo
Java
Krakatau Island
Sumba Island
Buru Island (Molucas)
Halimun-Salak Mountains
National Park (West Java)
Bromo-Tengger-Semeru
Mountains National Park
(East Java)
Mount Slamet (Central Java)
Species
known
number
Reference
4103
1043
937
629
60
50
49
173
Whitten et al.1997
Whitten et al.1997
Whitten et al.1997
Whitten et al.1997
Bush and Whitaker 1991
Hammer et al. 1997
Hill et al. 1995
Ubaidillah 1998
31
Suharto et al. 2005
105
This study
Species richness at Mount Slamet was quite low
compared to the expected richness of butterflies in Java and
that found at Gunung Halimun National Park, West Java
(Ubaidillah et al. 1998) but was quite similar to the results
at Ujung Kulon and nearby islands (New et al. 1987).
Compared to studies done at Mount Tangkuban Perahu,
West Java (Subahar et al. 2007), Gunung Salak, West Java
(Tabadepu et al. 2008), West Java (Murwitaningsih and
Dharma 2014), Bromo-Tengger-Semeru National Park,
East Java (Suharto et al. 2005), the SF in Kendal, Central
Java (Rahayuningsih et al. 2012) and the open area in
Malang, East Java (Khoirun-Nisa et al. 2013), the species
number found on Mount Slamet during this study was
relatively high. The species composition recorded at Mount
Slamet was similar was recorded in Ujung Kulon, West
Java (Tabadepu et al. 2008), Krakatau island, West Java
(Bush and Whitaker 1991), Gunung Halimun National
Park, West Java (Ubaidillah 1998), and Sumba Island
WIDHIONO – Butterflies of Mount Slamet, Central Java
201
(Hammer et al. 1997). The fact indicating that the species
assemblages of butterflies at Mount Slamet are dominated
by common and widely distributed species in the IndoMalayan region (Table 2).
The general low species richness found at Mount
Slamet in comparison to other parts of Java might also due
to the sampling methods used. Net capture methods were
used as described by Corbet (1941). One limitation of this
method is the restriction to capture of understorey
butterflies only, as indicated by the fact that the most
abundant species captured were understorey species within
the families of Satyridae with 3,924 individuals, followed
by Nymphalidae (with 3,737 individuals), Lycaenidae
(with 1.290 individuals), and Amathusidae (with 969
individuals). Some canopy fliers might be present but were
possibly not captured, as shown by the low abundance of
the family Danaidae with 406 individuals and Papilionidae
with only 306 individuals. The higher number of
individuals of species belonging to family Pieridae might
be explained by the fact that several species usually come
down to the ground in open habitats. Tropical butterfly
communities are divide naturally into two adult feeding
guilds (De´ Vries et al. 2012). One guild is composed of
species that obtain the majority of their nutritional
requirements from flower nectar and include most species
of the families Papilionidae, Pieridae, Lycaenidae,
Riodinidae, and some groups within Nymphalidae. The
second guild is composed of certain genera within
Nymphalidae, Satyridae, and Amathusidae, whose adults
gain virtually all of their nutritional requirements by
feeding on juices of rooting fruits and plant sap (Luk et al.
2011). As the numbers of flower-visiting butterflies
increased, fruit-feeding butterflies decreased in abundance
towards the canopy. A significant negative relationship
between trap height and abundance, as well as the number
of recorded species, was found among Satyridae and
Nymphalidae (Houlihan et al. 2013). Both the Satyridae
and Nymphalidae families were showed decreasing
abundance and species number with trap height (Schulze et
al. 2001; Fermon et al. 2000). Compared to the count walk
method, kite netting results in lower species abundance
during research done in Brazil (Caldasa and Robbins
2003).
Habitat preference
From the 99 butterfly species found on the southern
slope of Mount Slamet, 32 species (30%) were specific to
the forest (Houlihan et al. 2013; Majumder et al. 2013),
whereas 63 species (60.6%) were commonly distributed to
all habitats sampled, and the last 10 species (9.4%) were
endemic to the area. Butterfly species richness between
habitats was showed PFs have the highest abundance (97
species) followed by TAs with 71 species, SFs with 64
species, and AFs with only 59 species (Figure 3).
The higher species composition in PFs was due to the
variability in environmental factors that affect butterfly
movement. High species richness of butterflies in PFs
revealed that habitat specificity is directly linked to the
availability of host plants for larvae and adults. This results
was also in agreement with the prediction that highest
diversity should occur in situations of intermediate
disturbances when both climax and pioneer species can
coexistent (Basset, et al. 2011). This finding contradicted
that of Mihindukulasooriya et al. (2014) who found that
SFs have the highest species diversity compared to
regenerative forests in Sri Lanka, but was in line with other
research (Peer et al. 2011; Bergerot et al. 2012; Kumar
2012; Lee et al. 2015). The present study revealed that
although SFs had fewer species than PFs, SFs were
excellent sites for unique species, which i important from a
conservation point of view.
The diversity index (H) was highest in PFs (1.647),
followed by TAs (1.655), SFs (1.52), and AFs (1.441). The
index dominance (1/D) was highest in TAs and lowest in
AFs. The evenness index (J’) was relatively similar in all
habitats (0.814-0.894).
Forested habitats, like SFs and PFs were dominated by
Nymphalidae, Satyridae, and Amathusidae, whereas open
habitats like AFs and TAs were dominated by Pieridae. The
dominance of Nymphalidae, Satyridae, and Amathusidae in
forested areas may be correlated with the availability of
host plants, adult food resources, and microclimate
conditions. Many studies have documented the dominance
shown by members of Nymphalidae in tropical regions,
owing to its polyphagous nature, which helps in all habitats
(Sarkar 2011; Harsh et al. 2015).
A
B
Figure 3. A. Number of species at four habitats type. B. Number of individuals at four habitats type
202
B I O D I V E R S IT A S 16 (2): 196-204, October 2015
A high proportion of nymphalid species indicates high
host plant richness in the study area (Majumder et al.
2013). The dominance of Nymphalidae in SFs and PFs may
also be due to the fact that this family needs a larger
spectrum of food resources in both closed and open
habitats. Fermon et al. (2005) indicated that nymphalid
butterflies have a much higher diversity of phenotypes
when larval food plants are more evenly distributed across
all habitats.
Species of the Satyridae and Amathusidae families,
such as those in subfamilies Morphinae and Satyrinae,
exclusively feed on monocotyledonous food plants (Vu et
al. 2015). In Southeast Asian rainforests, these plants are
restricted to the lower forest layer, which may be one
reason why the abundance of Satyrinae and Morphinae is
highest in the understorey of closed canopy forests (Harsh
et al. 2015). Vu (2007) found that fruit-feeding Satyrinae
and Morphinae with relatively uniform phenotypes and a
comparatively small set of larval food plants are basically
restricted to lower vegetation layers, and many are known
to be sensitive to changes in humidity. In this research, it
was also found that several species of satyrids are restricted
to forested habitats, including Ragadia makuta, Melanitis
leda, M. zitenius, Neorina chrisna, Elymnias casiphone,
and E. ceryx, whereas other species, such as Lethe europa,
Mycalesis sudra, and Ypthima nigricans, are abundant in
both forest and agriculture areas. The former group
primarily feeds upon a small set of larval foods plants, such
as R. makuta, which depends only upon Selaginella and is
distributed in closed forests only and is very sensitive to
humidity (Vu 2007). The latter group exclusively depends
on grasses as food plants, which tend to be abundant in all
habitats, and especially open areas.
The abundance of the family Amathusidae decreased in
SFs and PFs, preferring more heavily disturbed, open areas,
like AFs and TAs. For example, Faunis canens occurred
with similar abundance in all habitats, whereas Thaumantis
odana, Amathusia taenia, and Zeuxidia luxerii occurred at
similar abundances in SF and PF habitats. Elliot (1992)
expected that adult amathusids butterflies to show a
conspicuous preference for the understorey layer of closed
forests (Barlow et al. 2007). In Borneo, most amathusids
species were recorded near the ground, and 87.9% of the
specimens were trapped in the understorey at 0 to 10 m
above ground level (Schulze 2001). Amathusid butterflies
might be constrained to understorey layers of tropical
forests by their food resource requirements. First, their
larva are typically bound to grasses (mainly Poaceae),
palms (Arecaceae), and others monocotyledonous (Ackery
1988; Elliot 1992). Secondly, the adult butterfly
exclusively uses fruits and related food sources that are
generally more common on the closed forest floor.
Butterfly species restricted to undisturbed forests often
have narrower geographical ranges than species found in
disturbed habitats (Posa et al. 2008).
Most Pieridae species showed the highest abundance in
open areas; significant differences were found for the
species Appias cardena, A. libythea, Delias belisama, D.
periboea, Catopsilia pyranthe, C. pomona, C. florella, and
Cepora iudith. Both Pieridae species, Eurema andersonii
and Leptosia nina were equally abundant in all habitats
except for SFs, where lower capture frequencies were
found. Since members of Pieridae are nectar feeding, they
rarely penetrate into the dense forest understorey (Sundufu
and Dumbuya 2008). Both open and disturbed forest
formations that are present in the AFs and TAs appear to
support butterfly species that are more commonly
associated with ruderal habitats, primarily from the family
Pieridae. Widely ranging heliophilous species, which are
typical of ruderal habitats, are most likely to successfully
establish viable populations in open areas than in closed
canopy forests (Chinaru and Joseph 2011). The preference
of Pieridae species for open habitats may be correlated with
the host plant distribution (Sarkar 2015). Records of
species from the Papilionidae, Danaidae, and Pieridae
families, all assumed originally to be canopy fliers, may be
due to the habits of males to come down to moist floor
sites. Many tropical butterfly species (mainly males) take
up water and nutrients at moist ground sites, including a
number of canopy species (Lawson et al. 2014). Almost all
Pierid species found at Mount Slamet were also found at
other sites in the Indo-Malayan region, such as Singapore,
Malaysian, Thailand, and the Philippines (Matsumoto et al.
2015). This finding indicates that most species dominating
open habitats are generalist species distributed throughout
the Indo-Malayan region. Generalist species should be
simultaneously locally abundant and widely distributed, as
a consequence of their ability to exploit a wide range of
resources on both local and regional scales. Species with
wider geographical distributions may be inherently more
adaptable and better able to exploit a wider range of
ecological niches; they may therefore be less sensitive to
land use changes than are species with narrower
distributions (Vu 2007).
Cluster analysis based on the Bray-Curtis single linkage
similarity value revealed the percent similarity between
species composition across the four habitat types. SFs
stood out clearly from the other three habitats and showed a
linkage of 28.23%, which represents the lowest similarity.
PFs were linked at 44.67% similarity to the cluster habitats
of TAs and AFs; a close similarity was found between TAs
with AFs, with a linkage of 63.4298%. This result indicates
that SFs had the highest diversity of butterflies with a
restricted distribution, making it an important butterfly
habitat for future conservation efforts (Figure 4).
Tourist area
Agroforest
Plantation forest
Secondary forest
Figure 4. Bay-Curtis similarity between habitat
WIDHIONO – Butterflies of Mount Slamet, Central Java
Endemic and rare species
The 99 species of butterfly was found on the southern
slope of Mount Slamet, 10 were endemic to Java Island
with 542 individuals from three families: Nymphalidae
(Cynitia iapsis, Cyrestis lutea, Rohana nakula, Tanaecia
trigerta, Neptis nisea, and Euploea gamelia), Satyridae
(Elymnias ceryx, Y. nigricans, and M. sudra) and Pieridae
(Prioneris autothisbe) (Table 3). Species richness of
endemic butterflies in different habitats showed that SFs
have the highest species richness with eight species (80%),
PFs have six species (60%), TAs have five species (50%),
and AFs have the lowest at four species (40%). Abundance
of endemic species in the different habitats showed that the
most abundant were found in PFs (158 individuals) and the
lowest was in SFs (112 species). The 10 endemic species,
five were specific to the forest and very rare, indicating that
endemic species mostly depend upon forest vegetation and
suggesting the need for strict conservation measures. The
other five species found in all habitats and were very
abundance, especially Y. nigricans and M. sudra
(Widhiono 2015). This result indicates that forests on
Mount Slamet support the existence of endemic Java
butterflies (30.43%). Then 14 endemic species found in this
location amount 71.4%.
Rabinowitz (1981) suggested an eight cell model of
abundance or rarity involving large/small habitat range,
wide/narrow habitat specificity, and large/small
populations where present. Rare species, as defined in this
study, are those represented by fewer than 10 individuals.
In total, 10 butterfly species were found with fewer than 10
individuals during the entire period and at a different study
site (Table 3). They are therefore classified in this study as
“rare species”. For example Abisara savitri and Stiboges
calycoides (Riodinidae). The rarity of A. savitri is due to
Table 3. Endemic and rare species of butterfly found on Mount
Slamet.
Endemics species
Number of
individuals
Cynitia iapsis
Cyrestis lutea
Elymnias ceryx
Euploea gamelia
Rohana nakula
Tanaecia trigerta
Mycalesis sudra
Ypthima nigricans
Neptis nisea
Prioneris autothisbe
20
6
8
7
25
160
156
358
8
7
Rare species
Cyrestis lutea
Elymnias ceryx
Prioneris autothisbe
Troides helena (protected species)
Cethosia munjava
Delias hyparete
Euploea gamelia
Rhinopalpa polynice
Abisara savitri
Stiboges calycoides
Number of
individuals
6
8
6
9
8
1
7
3
2
2
203
fact that this species mainly inhabits primary forests and is
most abundant at the height of rainy period, a time when
little collecting is normally done (Callaghan 2009). Only
one species (Troides helena (Papilionidae) is a protected
species based on Government Regulation (Peraturan
Pemerintah) No. 7/1999 and CITES Appendix II.
The result showed that butterfly diversity, abundance,
and endemism on Mount Slamet is relatively high,
representing 18% of butterfly species found in Java and
supporting 71.4% of endemic species and one protected
species (T. helena) found in Central Java. PFs contributed
the highest diversity and abundance of butterfly species,
and AFs showed the lowest butterfly diversity, abundance,
and endemism. Among all forest habitats surveyed, the SFs
represent the most suitable habitats for biodiversity
conservation and the maintenance of rare and endemic
species.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160216
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 205-212
Recovery of plant diversity and soil nutrients during stand development
in subtropical forests of Mizoram, Northeast India
SH. B. SINGH1, B.P. MISHRA2, S.K. TRIPATHI1,♥
1
Department of Forestry, Mizoram University, Aizawl-796004, Mizoram, India. Tel. +91-389-2330394, Fax. +91-389-2330834, email:
sk_tripathi@rediffmail.com
2
Department of Environmental Science, Mizoram University, Aizawl-796004, Mizoram, India
Manuscript received: 4 January 2015. Revision accepted: 28 August 2015.
Abstract. Singh ShB, Mishra BP, Tripathi SK. 2015. Recovery of plant diversity and soil nutrients during stand development in
subtropical forests of Mizoram, Northeast India. Biodiversitas 16: 205-212. The present study assessed the recovery of tree species
diversity and soil nutrient dynamics with stand development in subtropical semi-evergreen forest of Mizoram. The study was carried out
in two regenerating forest stands following disturbance and one undisturbed forest. Schima wallichii was the dominant species in all
stands showing IVI of 63.8, 83.3 and 75.9 in undisturbed, moderately disturbed and highly disturbed stands, respectively. Castanopsis
tribuloides was co-dominant species in the undisturbed and the moderately disturbed but this species was replaced by Sterculia villosa in
the highly disturbed stand. The shift in position of species and families from undisturbed to highly disturbed stands could be linked with
degree of disturbance. Log-normal dominance-distribution curves in the undisturbed and moderately disturbed stand indicating the
stability of community, while short hooked curve in the highly disturbed stand indicates unstable nature of the community. The soil
properties (organic C, total nitrogen, and available phosphorus) increased significantly during the course of stand development, whereas,
decrease with the depth in these forest stands.
Keywords: Community, disturbance, plant diversity, tropical semi-evergreen forest
INTRODUCTION
Biodiversity provides many essential goods and
services to the mankind particularly in densely populated
tropical ecosystems. Rapid decrease in tropical biodiversity
is recorded in many forest ecosystems over the world as a
result of human activities like habitat destruction, over
exploitation, pollution and species introduction (Pimm et
al. 1995; Pragasan and Parthsarthy 2010). These human
activities have led to the conversion of natural forest areas
into different stages of forest degradation that exhibited
significantly
different
structural
and
functional
characteristics at local and regional levels (Sapkota et al.
2010). Therefore, proper species diversity along with other
characteristics need to recorded in different ecosystems to
account multi-dimensional aspect of biodiversity that
should take into account the variation in environmental
factors, taxonomic variations among species, compositional
diversity and functional diversity (Roy et al. 2004). The
extents of deforestation in recent years in tropical regions
draw attention to the urgent need for intervention to restore
and protect biodiversity, ecological functioning and the
supply of goods and services used by poor rural
communities (Lamb et al. 2005; Cayuela et al. 2006).
The North-east India is known for its rich biodiversity
with high level of endemism (Khan et al. 1997). Several
studies have been carried out to quantify plant diversity and
to understand the ecology of forest communities of
different areas (Mishra et al. 2004; Laloo et al. 2006;
Mishra 2010; Tynsong and Tiwari 2010, 2011). The plant
inventory and community characteristics of tropical semievergreen forest of Mizoram have been studied by some
workers (Lalramnghinglova 2003; Singh et al. 2011, 2012).
In Mizoram, one of the states of North-east India, an
extensive sandstone quarry has been carried out for the
many developmental activities like construction of roads
and buildings in the last few decades that converted large
tract of forest area into degraded forest and created an
unfavorable habitat conditions for plant growth and
development. The unfavorable habitat conditions prevailing
in the mined areas have reduced the chances of
regeneration of many species, thereby reducing the number
of species in mined areas.
Anthropogenic disturbance is one of the significant
impacts arising out of mining and quarrying activities and
is mainly in the form of alteration of land structure due to
excavation, stacking of top soil and loss of fertile soil due
to dumping of mined overburden on top soil. Sandstone
mining causes damage to property, depletion of forest land,
adverse effects on the aquatic biodiversity and public
health. The present study aimed to determine the recovery
of plant community characteristics and soil nutrients in
secondary successional forests with stand development in
relation to a reference forest in semi-evergreen tropical
region of Mizoram, Northeast India.
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B I O D I V E R S IT A S 16 (2): 205-212, October 2015
MATERIAL AND METHODS
The study was conducted during 2009 to 2011 in the
forest patches within and outside the Mizoram University
campus situated in Tanhril area (23o45’25” N-23o43’37” N
latitude and 92o38’39’’E-92o40’23”E longitude) of Aizawl
District, Mizoram, India (Figure 1). For detailed
investigation, a total of three study sites were selected
along age gradient after disturbance in terms of sandstone
quarry, representing undisturbed (UD), moderately
disturbed (MD) and highly disturbed (HD) stands. The
natural forest stand where no sandstone mining activity was
done in the past referred to as undisturbed stand. The
moderately disturbed forest stand is a secondary forest
developed naturally after the abandonment of sandstone
quarry (mining operation about 7 years back in 2002). The
highly disturbed forest stand is an open mining area where
sandstone mining is continued. Tree species (≥15 cm girth
at breast height, GBH, i.e. 1.3 m above ground) were
recorded using 70 quadrats of 10 x 10m size at each site.
The plant species were identified with the help of
herbarium of the concerned University Department;
herbarium of the Botanical Survey of India (BSI), Eastern
circle, Shillong, and counter checked with the help of
regional floras (Kanjilal et al. 1934-1940; Haridasan and
Rao 1985). The field data on vegetation was quantitatively
analyzed for phyto-sociological attributes namely,
frequency, density and abundance as proposed by Curtis
and Mclntosh (1950). The Importance value index (IVI)
was determined as per Philips (1959). Species diversity and
dominance indices were determined following the methods
as outlined in Misra (1968); Mueller-Dombois and
Figure 1. Map of Mizoram, India showing the study area
Ellenberg (1974). The soil samples were collected with
the help of soil corer for top-soil (0-10 cm depth) and subsoil (10-20 cm depth). The physico-chemical
characteristics of soil have been analyzed by the methods
outlined by Allen et al. (1976) and Anderson and Ingram
(1993). Correlation was also developed between different
vegetational and soil parameters across different sites.
RESULTS AND DISCUSSION
Plant diversity and community characteristics
Altogether, a total of 71 woody plant species belonging
to 59 genera and 33 families of angiosperms were recorded
from undisturbed, moderately disturbed and highly
disturbed forest stands. Of this, 50 species representing 43
genera and 29 families, 49 species belonging to 41 genera
and 22 families and 24 species belonging to 21 genera and
17 families were reported from the undisturbed, moderately
disturbed and highly disturbed stand, respectively. It was
found that Wendlandia tinctoria (Roxb.) DC., Schima
wallichii (DC.) Korthals., Emblica officinalis Gaertn.,
Callicarpa arborea Roxb., Castanopsis tribuloides DC,
and Sterculia villosa Roxb. ex Smith species were common
in all three stands, and Acacia farnesiana, Debregeasia
velutina, Murraya koenigii, Turpinia nepalensis and
species Cassia laevigata, Melia azedarach, Pterospermum
acerifolium and species Lepionurus oblongifolius, Ficus
virens, Grewia macrophylla were restricted to undisturbed,
moderately disturbed and highly disturbed stand,
respectively. There was shift in dominance of plant species
and families from undisturbed to highly disturbed
SINGH et al. – Plant diversity and soil nutrients during forests development
stand. S. wallichii was the dominant species (IVI 63.76) in
the undisturbed stand. The co-dominant species were C.
tribuloides (IVI 19.99) and C. arborea (IVI 19.44). In the
moderately disturbed stand, S. wallichii was dominant
species (IVI 83.31) and it was followed by co-dominant
species namely, C. tribuloides (IVI 39.81) and C. arborea
(IVI 17.54). In the highly disturbed stand, S. wallichii was
recorded as a dominant species (IVI 75.87), and it was
followed by co-dominant species namely, S. villosa (IVI
39.04) and C. arborea (IVI 24.89). S. wallichii, the
dominant species in all three stands, contributed maximum
tree density in the undisturbed stand (225 ind. ha-1 and
basal area 5.04 m2ha-1), moderately disturbed stand (222
ind. ha-1 and basal area 3.28 m2ha-1) and highly disturbed
stand (105 ind. ha-1 and basal area 1.59 m2ha-1) (Table 1).
The distribution pattern of species differed from
undisturbed to the highly disturbed stand. There was
preponderance of contagious distribution. Whereas, few
species namely, C. arborea and E. officinalis showed
random distribution.
Euphorbiaceae was the dominant family with maximum
number of species in the undisturbed (5) and moderately
disturbed (5) stands. However, it was replaced by
Mimosaceae (3) in the highly disturbed stand. The codominant families were Fagaceae (4), Lauraceae (4),
Mimosaceae (4) in the undisturbed stand; Caesalpinaceae
(4), Fagaceae (4), Lauraceae (4) in the moderately
disturbed stand; Anacardiaceae (2), Rubiaceae (2),
Papilionaceae (2) in the highly disturbed stand (Table 2).
There was sharp decline in tree density from the
undisturbed stand (970±3.2 ind. ha-1) to the moderately
disturbed stand (742±3.4 ind. ha-1) and finally to the highly
disturbed stand (410±3.2 ind. ha-1). A similar trend in
results was also observed for total tree basal area which
was highest in the undisturbed stand (20.8 m2 ha-1)
followed by moderately disturbed stand (9.85 m2 ha-1) and
highly disturbed stand (5.38 m2 ha-1). Shannon-Wiener
diversity index was maximum (3.3) in the undisturbed
stand and minimum in the highly disturbed stand (2.6). The
species richness Index was highest in moderately disturbed
stand followed by undisturbed stand and highly disturbed
stands. The Evenness Index did not show much variation in
results with respect to the degree of disturbance. The
Sorenson’s Index of similarity for tree species was
maximum (0.58) between undisturbed and moderately
disturbed stands. On contrary, value was minimum (0.46)
between moderately disturbed and highly disturbed stands
(Table 3).
Findings on the girth class distribution of trees showed
a decreasing trend in the number of individuals from lower
to higher girth classes, irrespective of stands, except in case
of the undisturbed stand where number of individuals was
highest in girth classes 50-70 cm and 30-50 cm. The
moderately disturbed stand showed maximum number of
adult trees (girth classes 30-50 cm). However, mature trees
(girth classes 50-70 cm) were maximum in the undisturbed
stand. There was a sharp decline in the number of
individuals in the higher girth classes with increase in
degree of disturbance (Figure 2).
207
The log-normal dominance-diversity curve (based on
IVI) was found in the undisturbed and moderately
disturbed stands; however, it was short hooked in case of
highly disturbed stand (Figure 3).
Soil characteristics
The average soil moisture on different stands ranged
from 23.66 to 28.84%. pH ranged from 4.45 to 6.18 which
indicated that the soils on all stands were acidic. Compared
to the moderately disturbed and highly disturbed stands, the
undisturbed stand has the highest values of soil in organic
carbon, total nitrogen, available phosphorus, exchangeable
potassium as given in Table 4.
Correlation between vegetation and soil parameters
The correlation between different vegetation parameters
along disturbance gradient is given in Figure 4. The tree
density was positively correlated with the tree basal area,
tree diversity and tree richness (Figure 4 A, B, D), whereas,
tree basal area was negatively correlated with concentration
of dominance (Figure 4). Tree richness was positively
correlated with diversity and negatively with concentration
of dominance but the relationship was rather weak (Figure
4 E, F).
Figure 2. Distribution of tree species in different girth classes in
the undisturbed (UD), moderately disturbed (MD) and highly
disturbed (HD) forest stands
Figure 3. Dominance-diversity curves of tree species along
disturbance gradient
208
B I O D I V E R S IT A S 16 (2): 205-212, October 2015
A
B
C
D
E
F
Figure 4. Correlation between various vegetational parameters along disturbance gradient
A
B
C
D
E
F
Figure 5. Correlation between various soil parameters for top-soil along disturbance gradient
The correlation between various soil parameters for topsoil and sub-soil along disturbance gradient is given in
Figure 5 and 6. The soil moisture content showed a positive
correlation with soil organic carbon and negative
correlation with pH (Figure 5 A, B). However, soil organic
carbon showed significant and positive correlation with
total nitrogen and exchangeable potassium (Figure 5 C, E).
Available phosphorus showed positive and significant
correlation with exchangeable potassium, and the total
nitrogen showed positive correlation with available
phosphorus (Figure 5 D, F). Correlation among different
nutrients showed similar trends in top-and sub-soil with
varying degree of coefficients (Figure 5, 6).
SINGH et al. – Plant diversity and soil nutrients during forests development
209
A
B
C
D
E
F
Figure 6. Correlation between various soil parameters for sub-soil along disturbance gradient
Discussion
The sandstone mining impacted vegetation composition,
community organization to a great extent, and there was
decrease in species richness, tree density and basal area
from undisturbed to the highly disturbed stand. S. wallichii
was the dominant tree species in all the stands. However,
C. tribuloides, the co-dominant species of the undisturbed
and moderately disturbed stands was replaced by S. villosa
in the highly disturbed stand. Moreover, the species
tolerant to stress showing better growth and survival under
disturbed condition, such species express greater IVI in the
disturbed stand. Similarly, Euphorbiaceae was the
dominant family in both undisturbed and moderately
disturbed stands, whereas, it was replaced by Mimosaceae
that was the dominant family in the highly disturbed stand.
The shift in position of the species and families along the
disturbance gradient could be linked with the levels of
anthropogenic disturbance and similar trends was also
reported by number of workers in the past (Kadavul and
Parthasarathy 1999; Mishra et al. 2003, 2004, 2005; Mishra
and Laloo 2006).
During present investigation, it has been found that
majority of the species showed contagious distribution
pattern and few were distributed randomly. Similar,
distribution pattern has also been reported by other workers
(Singh and Yadav 1974; Metha et al. 1997). The trend in
tree population structure observed during present study is
similar to those reported from the forest at Costa Rica
(Nadakarni et al. 1995), Brazalian Amazon (Campbell et al.
1992), Eastern Ghats (Kadavul and Parthasarathy 1999)
and sub-tropical humid forest of Meghalaya (Mishra et al.
2004). The log-normal dominance distribution curve in the
undisturbed and moderately disturbed stands indicates
stable community. However, short hooked curve in the
highly disturbed stand depicts un-stabile community. The
past workers have also reported a similar trend in results
(Khera et al. 2001; Tripathi et al. 2004; Mishra et al. 2004, 2005).
The soil properties greatly varied from undisturbed to
highly disturbed stand. High pH value in highly disturbed
stand could be due to low accumulation of decomposed
organic matter and extraction of sand stone. The soil
organic carbon, total nitrogen, available phosphorus and
exchangeable potassium varied greatly along disturbance
gradient and decreased from undisturbed to highly
disturbed stand and higher values were reported from topsoil. This could be linked with presence of thick humus
layer on forest floor of the undisturbed stand. Litter
accumulation on forest floor is positively linked with litter
decomposition and plays a significant role in maintenance
of soil moisture content and other micro-environmental
conditions (Arunachalam and Pandey 2003; Reddy 2010;
Mishra 2010; Tripathi et al. 2012). Nayak and Srivastava
(1995) have also reported a similar trend in results from the
humid sub-tropical soils in north east India.
A positive correlation of species richness with diversity
index was observed with few exceptions, and findings are
in conformity with the report of Tripathi et al. (1989) that
the species richness is positively correlated with Shannon
diversity index in majority of cases in western Himalayan
forests. Various diversity indices also followed similar
trend (Jha et al. 2005; Yu-Hai et al. 2009). A positive
correlation was observed between total basal area and
organic carbon in top-soil and sub-soil. The findings are
supported by Sharma et al. (2009).
210
B I O D I V E R S IT A S 16 (2): 205-212, October 2015
Table 1. Phyto-sociological attributes of tree species along disturbance gradient
Species
Acacia auriculiformis A. Cunn. ex Benth.
Acacia farnesiana (L.) Willd.
Albizia chinensis Osb. Merr.
Albizia lebbeck (L.) Benth.
Albizia procera (R.) Benth.
Antidesma diandrum B.Hey. ex (Roxb.)
Aporosa roxburghii Baill
Artocarpus heterophyllus Lam
Bauhinia purpurea L.
Beilschmiedia assamica Meisn.
Bombax ceiba L.
Bridelia cuneata Gehrm
Bridelia stipularis Bl.
Bursera serrata Wall.ex Colebr.
Callicarpa arborea Roxb.
Callicarpa macrophylla Vahl.
Cassia laevigata Willd
Castanopsis hystrix A.DC.
Castanopsis tribuloides A.DC.
Clausena excavata Burn.f.
Colona floribunda (Wall.ex Kurz) Craib
Combretum dasystachyum Kurz.
Crotalaria linifolia L.
Debregeasia velutina Gaud.
Delonix regia (Boj)Rafin.
Derris robusta Roxb.exDC.
Dipterocarpus turbinatus Gaertn.
Emblica officinalis Gaertn.
Engelhardia spicata Lechen ex Bl.
Erythrina stricta Roxb.
Eucalyptus globulus Labill.
Eugenia macrocarpa Schltdl. & Cham.
Eugenia malaccensis L.
Ficus elastica Roxb.ex Hornem
Ficus virens Ait.
Glochidion velutinum Wt.
Grevillea robusta A.Cunn.ex R.Br.
Grewia macrophylla G.Don
Lagerstroemia parviflora Roxb.
Lepionurus oblongifolius (Griff.) Mast
Litsea chinensis (Lour.) C.B.Rob.
Litsea citrata Bl.
Macaranga denticulata (Bl.) Mull. Arg.
Machilus bombycina King ex Hook.f.
Mangifera indica Linn.
Melia azedarach Linn.
Meliosma pinnata Roxb.
Michelia champaca Linn.
Michelia panduana Hook.Thamn.
Micromelum integerrimum (B-Ht.ex Can.)Wt.
Murraya koenigii (L.)Spreng.
Oroxylum indicum Benth.ex Kurz.
Phoebe cooperiana Pc. Kanj. & Das.
Plumeria acutifolia Poir.
Psidium guajava Linn.
Pterospermum acerifolium Linn.
Quercus semiserrata Roxb.
Quercus spicata Sm.
Rhus succedanea Linn.
Sarcochlamys pulcherrima Roxb.
Saurauia napaulensis D.C.
Schefflera wallichiana (Wight & Arn.) Harms
Schima wallichii (DC.) Korthals.
Sterculia villosa Roxb.ex Smith.
Syzygium cumini Linn.
Tapiria hirsuta Hook.f
Taxus wallichiana Zucc.
Terminalia arjuna Roxb.W & A
Toona ciliata M.Roem.
Turpinia nepalensis Wall.ex Wight & Arn.
Wendlandia paniculata (Roxb.) DC.
Wendlandia tinctoria (Roxb.) DC.
Undisturbed
Density (Ind. ha-1) IVI
5
1.17
8
10
18
3
4
3
7
7
15
20
2
58
4
2.37
3.73
6.39
0.88
1.53
1.21
1.53
2.24
4.99
6.3
0.78
19.44
1.32
31
62
10.41
19.99
18
13
11
4.92
3.5
4.96
7
32
2
15
2.26
10.45
0.72
5.17
20
6.05
6
5
2.45
1.53
2
2
8
6
15
0.54
1.17
2.42
1.75
4.73
18
5.04
11
15
9
14
3.8
3.35
2.33
4.98
4
9
14
32
21
1.48
3.38
4.63
8.72
7.44
18
225
57
5.76
63.76
17.07
17
12
3
15
7
26
33
6.18
3.35
1.27
5.24
1.83
7.34
9.53
Moderately disturbed
Density (Ind. ha-1)
IVI
3
1.36
4
2.04
13
4.95
12
6.39
2
0.96
Highly distrubed
Density (Ind. ha-1)
IVI
15
6
12
10.19
4.78
9.38
4
3.48
6
2.56
42
17.54
32
24.89
5
8
102
5
1.73
3.2
39.81
2.95
24
18.05
7
6.23
4
2.45
1
2
0.86
0.93
30
11.87
6
1
20
4.44
1.77
13.78
1
0.75
4
2.96
5
6
2.18
2.83
15
2
6.14
0.75
5
1
4.83
1.77
2
0.91
14
8
2
4
4
5
5
3
5
5
17
5.83
3.63
1.06
1.57
2.29
2.07
2.09
1.36
1.95
1.78
7.11
5
5
8
3.22
4.19
5.09
8
2
5
4.09
1.42
3.08
10
2
22
2
12
2
222
24
2
2
3.84
0.86
8.11
1.29
5.07
1.5
83.31
10.15
0.61
0.86
7
5.04
21
14.67
105
58
75.87
39.04
9
6.7
12
6.02
12
10.33
28
36
10.02
12.49
26
17
17
12.3
SINGH et al. – Plant diversity and soil nutrients during forests development
211
Table 2. Ranking of families of angiosperm in the undisturbed, moderately disturbed and highly disturbed forest stands
Family rank
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Undisturbed
Family
No. of species
Euphorbiaceae
5
Fagaceae
4
Lauraceae
4
Mimosaceae
3
Caesalpiniaceae
2
Combretaceae
2
Moraceae
2
Myrtaceae
2
Verbenaceae
2
Anacardiaceae
2
Papilionaceae
2
Bombacaceae
2
Rubiaceae
2
Rutaceae
2
Tiliaceae
1
Urticaceae
1
Olacaceae
1
Lythraceae
1
Juglandaceae
1
Meliaceae
1
Sapindaceae
1
Dipterocarpaceae
1
Sterculiaceae
1
Taxaceae
1
Theaceae
1
Burseraceae
1
Araliaceae
1
Bignoniaceae
1
Sabiaceae
1
Forest stands
Moderately disturbed
Family
No. of species
Euphorbiaceae
5
Fagaceae
4
Mimosaceae
4
Lauraceae
4
Myrtaceae
4
Caesalpiniaceae
4
Anacardiaceae
3
Rubiaceae
3
Rutaceae
2
Meliaceae
2
Sterculiaceae
2
Papilionaceae
2
Theaceae
1
Lythraceae
1
Magnoliaceae
1
Verbenaceae
1
Apocynaceae
1
Araliaceae
1
Sabiaceae
1
Proteaceae
1
Saurauiaceae
1
Moraceae
1
Highly disturbed
Family
No. of species
Mimosaceae
3
Anacardiaceae
2
Fagaceae
2
Papilionaceae
2
Rubiaceae
2
Tiliaceae
2
Verbenaceae
1
Dipterocarpaceae
1
Euphorbiaceae
1
Sterculiaceae
1
Caesalpiniaceae
1
Bombacaceae
1
Lythraceae
1
Theaceae
1
Meliaceae
1
Olacaceae
1
Moraceae
1
Table 3. Tree community structure in the undisturbed, moderately disturbed and highly disturbed forest stands
Parameter
No. of family
No. of genera
No. of species
Tree density (Indv.ha-1)
Tree basal area (m2h-1)
Shannon-Wiener index
Simpson dominance index
Simpson index of diversity
Simpson reciprocal index
Species richness (Margalef's index)
Evenness index (Pielou)
Forest stands
Moderately disturbed
22
41
49
742±3.4
9.85
2.9
0.12
0.8
8.2
7.7
0.74
Undisturbed
29
43
50
970±3.2
20.83
3.3
0.07
0.9
13.3
7.5
0.84
Highly disturbed
17
21
24
410±3.2
5.38
2.6
0.1
0.8
9.1
4
0.81
Table 4. Physico-chemical characteristics of soil along the disturbance gradient
Stands
Undisturbed
Moderately disturbed
Highly disturbed
Depth ( cm)
0-10
Mean
SE ±
10-20 Mean
SE ±
0-10
Mean
SE ±
10-20 Mean
SE ±
0-10
Mean
SE ±
10-20 Mean
SE ±
Moisture content (%)
30.45
2.47
27.23
2.65
27.28
1.56
24.15
1.57
25.79
2
21.53
2.28
pH (H20)
4.57
0.08
4.34
0.09
5.5
0.04
5.1
0.04
6.35
0.04
6.01
0.05
Org-C (%)
2.5
0.14
1.89
0.15
1.8
0.16
1.55
0.12
1
0.14
0.76
0.06
TN (%)
0.26
0.02
0.2
0.02
0.2
0.02
0.16
0.01
0.29
0.17
0.48
0.23
Avail P µg g-1
26.08
1.36
17.41
1.88
14.41
1.16
12.16
1.15
10.25
1.43
6.75
0.49
K µg g-1
210
12.85
171.58
7.44
163.33
10.56
133.25
11.24
88.33
9.93
75.83
9.39
212
B I O D I V E R S IT A S 16 (2): 205-212, October 2015
Present investigation showed that there is a
considerable reduction in the number of species from
natural forest to disturbed sites due to unfavorable habitat
conditions for plants created by disturbance. The
disturbance adversely affected the juvenile stage, leading to
arrested survival and growth of seedlings and saplings. The
findings of the present study would be an important tool for
formulation of appropriate strategies for management of
abandoned mined areas through re-vegetation with suitable
dominant and co-dominant species of the present study for
effective management strategy. Such site-specific selection
of species will add a new dimension for rehabilitating of
abandoned land and conservation of biodiversity.
ACKNOWLEDGEMENTS
Authors are grateful to the University Grants
Commission, New Delhi, India for financial assistant and
to the various communities for extending facilities to
complete this piece of work desirably.
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BIODIVERSITAS
Volume 16, Number 2, October 2015
Pages: 213-224
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160217
Conservation status of the Family Orchidaceae in Mt. Sinaka, Arakan,
North Cotabato, Philippines
CHERRY LEE T. PANAL1,♥, JENNIFER G. OPISO2,♥♥, GUILLER OPISO3,♥♥♥
1
Agusan del Sur State College of Agriculture and Technology, San Teodoro, Bunawan-8506, Agusan del Sur, Philippines Tel.: +63-099177183449,
♥
email: lengkay22@gmail.com
2
Biology Department, Central Mindanao University, Musuan, Maramag, Bukidnon-8710, Philippines. ♥email: mcgalvz@gmail.com
3
Philippine Eagle Foundation, Davao City, Philippines. ♥email: guilleropiso@gmail.com
Manuscript received: 28 February 2015. Revision accepted: 16 September 2015.
Abstract. Panal CLT, Opiso JG, Opiso G. 2015. Conservation status of the Family Orchidaceae in Mt. Sinaka, Arakan, North
Cotabato, Philippines. Biodiversitas 16: 213-224. This study determines the conservation status of the family Orchidaceae, as present
on Mt. Sinaka, Arakan, North Cotabato, Philippines. A thorough survey and alpha taxonomy was done, from base to peak of the
mountain. Identification of the specimens and assessment of their conservation status was based on the IUCN Red List of Threatened
Plants 2013.2 and National List of Threatened Philippine Plants of Fernando et al. (2008). Based on the result conducted on October
2013-March 2014, out of 59 identified species found in the area, 12 species are widespread, 22 are endemic, 1 vulnerable, 1 critically
endangered (Paphiopedilum adductum), 1 endangered (Corybas sp.), 2 least concerned species, and 20 unassessed species (not yet
assessed by the IUCN). It has been also noted that there are probably some new species, which need thorough study for further
identification. The result calls for a desperate need for conservation. Facts from this study helps in addition to the existing wildlife
conservation of flora in Mt. Sinaka and to the other forested mountains in Mindanao, the Philippines.
Keywords: Alpha taxonomy, threatened, endangered, endemic
INTRODUCTION
The orchid family (Orchidaceae) contains an estimated
25,000 species (Gravendeel et al. 2004). Orchids exist as
epiphytes, terrestrials, lithophytes, and aquatics making
conservation a concern when trying to protect this family.
This is the largest group of flowering plants (Huynh et al.
2009). More than 1,100 species of orchids are found in the
Philippines, 80% of which are endemic (Cootes 2001).
According to the National List of Threatened Philippine
Plants of Fernando et al. (2008), the family Orchidaceae
has 19 species categorized as critically endangered, 35
endangered species, and 3 vulnerable species.
Mt. Sinaka has an elevation of 1,448 meters above sea
level and approximately 3,000 hectares of land area located
at Arakan, North Cotabato, Philippines. This mountain is
part of the Mt. Apo mountain range. Located east of
barangays Marilog of Davao City, San Miguel and west of
barangays Tumanding, Salasang, Lanao Koran, and Datu
Ladayon of Arakan, North Cotabato (Estremera 2011).
This mountain has been threatened by unscrupulous human
activities. The secondary forest of this mountain has been
destroyed by the entry of palm oil and banana plantations.
Both flora and fauna in the area has been destroyed.
Because of the charismatic blooms of orchids they
become one of the economic sources of the natives living
near the mountains. This group of species becomes more
prone to extinction because of their economic importance
(ornamental, medicinal, flavoring and perfume). Ecologically, these species are bio-indicator and it has being
forgotten that they also provide habitat for microscopic
organisms. Many more species await scientific description,
but with the rapid destruction of the remaining forests,
many species will never be known to the scientific
community, or orchid enthusiasts. Thus, there is a need to
assess their conservation status for protection and
conservation of these species. This will enhance the
existing conservation management of the area.
MATERIALS AND METHODS
The sampling site is located on Mt. Sinaka, Arakan
Valley, North Cotabato, Philippines (Figure 1). This study
was done from October 2013 to March 2014. The species
were just photographed for conservation purposes, and the
characteristics (petals, sepals, rachis, pseudobulbs if
present) were measured and described. Only species that
were not identified were collected for further identification.
Species located above 5 meters high on tree trunks were
excluded. A standard alpha taxonomy (5m both sides) was
done from the base to the peak of the mountain.
Preliminary identification of the species was based on the
book Philippine Native Orchid Species by Cootes (2011a)
and other published scientific articles and journals.
Identification of orchids was further verified by Jim Cootes
during his visit to the Central Mindanao University on
February 21-22, 2014. The assessment of the collected
species was based on Fernando et al. (2008), IUCN (2014),
AO-DENR (2007), and from published floristic works and
books like that of Cootes (2011a, b).
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B I O D I V E R S I T A S 16 (2): 213-224, October 2015
Figure 1. Study site in Mt. Sinaka, Arakan Valley, North Cotabato, Philippines
RESULTS AND DISCUSSION
Diversity
The Table 1 reveals that locally, 15 species were found
to be abundant, 14 species are common, 30 species found
to be rare. This means that there is a high rate of rarity
among the orchid species in the area. This might be
because of human intervention. As observed, some natives
living near the mountain have been selling orchids as one
of their economic sources. It has been also observed that
the species in the wild, which captures their attention, has
been brought to their gardens and been grown.
Mt. Sinaka harbored endemic, vulnerable, widespread
and endangered species of Orchidaceae. There are more
endemic species found in this study when compared to the
study done by Buenavista (2014) in the five LTER sites in
Mindanao (Mt. Kitanglad, Mt. Musuan, Mt. Malindang,
Mt. Apo, Mt. Kiamo). But the result of both studies reflects
that the family Orchidaceae is not well studied, though it
harbors a great percentage of the flowering plants. It has
also been noted that some of the species found in the area
have not yet been described, nor cited in the list of
Fernando et al. (2008) and IUCN (2014) which suggests
that there is a need for further identification of these
species.
Table 1. Number of endemic, widespread, and critically
endangered species found in Mt. Sinaka, Arakan, North Cotaboto.
Status
No. of species
I. Local status
A. Abundant
15
B. Common
14
C. Rare
30
II. Conservation status
A. Endemic
22
B. Endangered
1
C. Critically endangered
1
D. Widespread
12
E. Vulnerable
1
F. Least Concerned
2
III. Unassessed
20
Note: Assessment of local conservation status was done by
counting the number of individuals, per species, both in the
sampling plots and in the transect walk. It was categorized using
the following criteria: (i) 1-3 individual/s-rare, (ii) 4-9
individuals-common, (iii) 10 or >10 individuals-abundant.
PANAL et al. – Orchids of Mt. Sinaka, Philippines
Table 2. Conservation Status of each Species found in Mt.
Sinaka, Arakan, North Cotabato, Philippines.
Genus/species
Conservation status
Agrostophyllum inocephallum
Agrostophyllum saccatilabium
Anoectochilus sp.
Vulnerable
Appendicula alba
Widespread
Appendicula malindangensis
Endemic
Appendicula micrantha
Endemic
Appendicula torta
Bulbophyllum acutum
Bulbophyllum alsiosum
Endemic
Bulbophyllum alagense
Endemic
Bulbophyllum dearei
Bulbophyllum flavescens
Bulbophyllum unguiculatum
Calanthe macgregorii
Endemic
Calanthe pulchra
Ceratostylis retisquama
Endemic
Ceratostylis subulata
Widespread
Chrysoglossum ornatum
Widespread
Coelogyne chloroptera
Endemic
Corybas sp.
Endangered
Crepidium ramosii
Cryptostylis sp.
Dendrobium auriculatum
Endemic
Dendrobium diffusum
Dendrobium milaniae
Endemic
Dendrobium stricticalcarum
Endemic
Dendrochilum arachnites
Endemic
Dendrochilum glumaceum
Dendrochilum mindanaense
Endemic
Dendrochilum serratoi
Dendrochilum wenzelii
Widespread
Epipogium roseum
Widespread
Euphlebium josephinae
Endemic
Flickingeria sp.
Goodyera sp.
Habenaria sp.
Liparis condylobulbon
Widespread
Liparis dumaguetensis
Endemic
Liparis terrestris
Oberonia sp.
Octarrhena amesiana
Endemic
Oxystophyllum cultratum
Widespread
Paphiopedilum adductum
Critically endangered
Peristylus spiralis
Phaius tankervilleae
Widespread
Widespread
Phalaenopsis amabilis
Phreatia sp.
Least concerned
Pinalia cylindrostachya
Endemic
Plocoglottis bicallosa
Endemic
Podochilus plumosus
Endemic
Robiquetia compressa
Endemic
Schoenorchis paniculata
Widespread
Spathoglottis plicata
Widespread
Spathoglottis tomentosa
Endemic
Stichorkis amesiana
Endemic
Taeniophyllum gracillimum
Least concerned
Thelasis micrantha
Widespread
Thrixspermum linearifolium
Endemic
Note: (-) not yet assessed by IUCN (2014).
215
Description
Anoectochilus sp. Blume
Growth habit: upright, sympodial, terrestrial. Rhizomes
are succulent. Leaves are petiolate, relatively broad,
brownish purple with coppery veins (Figure 2.A).
Distribution: It was found in the montane and mossy
forest of Mt. Sinaka at an elevation of 1200-1400 meters
asl. It has been recorded in Leyte based on Wenzel 286;
also in Malaysia and Indonesia (Cullen 1992).
Appendicula malindangensis (Ames) Schlechter
Growth habit: upright to semi-pendulous, sympodial
(Figure 2.B).
Inflorescence: pendulous, up to 3 cm long, bearing
numerous 7 mm flowers. Flower is blue to purplish; dorsal
sepal is lanceolate, hooded, about 4.5 mm long by 2mm
wide; petals are oblong, about 4.5 mm long by 1.5 mm
wide; lateral sepals are triangular to lanceolate, about 4.5
mm long by 4 mm wide, forming a short spur; labellum is
oblong, without side lobes, about 5 mm long by 2 mm wide
(Figure 2.C,D).
Distribution: It was found in the mossy forest of Mt.
Sinaka at an elevation of 1400-1500 meters asl. It has been
recorded from Bukidnon, Misamis, Negros and is endemic
to the Philippines (Cootes 2011a).
Appendicula micrantha Lindley
Growth habit: semi-pendulous, sympodial, epiphyte
(Figure 2.E).
Inflorescence: short, bearing up to 6 blooms, 4 mm in
diameter. The flower is creamy white in color; dorsal sepal
and petals are similar, triangular about 2 mm long by 1.5
mm wide; lateral sepals are elliptic-ovate, 2 mm long by
1.5 mm wide; labellum is oblong, about 3 mm long by 2
mm wide.
Distribution: It was found on Mt. Sinaka at an elevation
of 1100-1400 meters asl. It has been recorded from
Agusan, Bukidnon, Davao, Surigao, and Zamboanga,
Leyte, Negros, and Panay, in the Visayas and Albay,
Bataan, Laguna, Mindoro, Pampangga, Polillo, Quezon,
Rizal and is endemic to the Philippines (Cootes 2011a).
Bulbophyllum alagense Ames
Growth habit: sympodial, epiphyte (Figure 2.F) .
Inflorescence: arising from the rhizome; flower is light
yellow to pale orange; dorsal sepal is triangular to
lanceolate, up to 8 mm long by 2 mm wide; petals are
small, triangular, up to 3 mm long by 1.5 mm wide; lateral
sepals: triangular to lanceolate, up to 8 mm long by 2 mm
wide; labellum is curved and three-lobed, 2.5 mm long by
1.5 mm wide (Figure 2.G, H).
Distribution: It was found in the montane forest of Mt.
Sinaka at an elevation of 1200-1300 meters asl. It was also
recorded throughout Mindanao, Leyte, Mindoro, Benguet,
Camarines Sur, Laguna, and Rizal on Luzon (Cootes
2011a; Pelser 2012).
216
B I O D I V E R S I T A S 16 (2): 213-224, October 2015
Bulbophyllum alsiosum Ames
Growth habit: upright, sympodial, epiphytic (Figure
2.I).
Inflorescence: single flowered, appearing from the
rhizome; the color of the outer surface of the dorsal and
lateral sepals is cream to greenish; inner surface is marked
with pink to purple, petals are pink, spotted with purple;
labellum is pink; dorsal sepal is lanceolate, obtuse at the
apex, keeled, to 2.3 cm long by 1.3 cm wide; petals are
broadly ovate, to 1.4 cm long by 1 cm wide; lateral sepals
are oblong to lanceolate keeled, to 2 cm long by 1.1 cm
wide; labellum is thick and fleshy, tongue-shaped, to 9 mm
long by 6 mm wide (Figure 2.J).
Distribution: It was found in the mossy forest of Mt.
Sinaka at an elevation of 1300-1400 meters asl. It has been
found in Leyte, Negros and Rizal and is endemic to the
Philippines (Cootes 2011a).
Calanthe mcgregorii Ames
Growth habit: upright, sympodial, terrestrial.
Inflorescence: upright, up to 1 m in length, flowers
opening successively; each bloom is backed by a green
recurving bract about 1 cm long; flower is white and a
yellow spot on the labellum at the junction of the mid lobe;
dorsal sepal is reflexing, elliptic to lanceolate, 7 mm long
by 3 mm wide; petals are not reflexing; linear to oblong,
6.5 mm long by 2 mm wide; lateral sepals are reflexing; 7
mm long by 3 mm wide; labellum is three lobed; mid-lobe
divided into four spreading lobes; side lobes are wedgeshaped being widest at the apex; spur is straight; without
hairs (Figure 2.K, L, M).
Distribution: It was found in the Dipterocarp forest of
Mt. Sinaka at an elevation of 1100-1200 meters asl. It has
been found in Lanao, Leyte, Mindoro, Polillo, Quezon and
Rizal and is endemic to the Philippines (Cootes 2011a).
Ceratostylis retisquama Reichenbach f.
Growth habit: upright to semi-pendulous, sympodial,
epiphyte (Figure 2.N).
Inflorescence: bear blooms that appear from the base of
the plant and are up to 4 cm in diameter; flower is bright
reddish orange; labellum white; dorsal sepal is lanceolate,
up to 2 cm long by 6 mm wide; petals are oblanceolate,
pointed, up to 2 cm long by 5 mm wide; lateral sepals are
oblong-lanceolate, pointed, up to 2 cm long by 5 mm wide;
labellum is tiny, pointed, 3 mm long by 1.5 mm wide
(Figure 2.O).
Distribution: It was found in dipterocarp and montane
forests in Mt. Sinaka at an elevation of 1200-1300 meters
asl. It has been found in Agusan, Lanao, Bataan,
Camarines, Ilocos Norte, Quezon, Rizal, Zambales and is
endemic to the Philippines (Cootes 2011a).
Coelogyne chloroptera Reichenbach f.
Growth habit: upright, sympodial, epiphyte (Figure
2.Q).
Inflorescence: upright, appear with the new growth and
can reach 25 cm in length. Each inflorescence can carry up
to 12 flowers. Blooms are up to 4 cm in diameter; flower is
apple-green, labellum is white and brown; dorsal sepal is
lanceolate, hooded over the column, up to 2 cm long by 7
mm wide; petals are linear, up to 2 cm long by 2 mm wide.
They reflex towards the pedicel; lateral sepals are
lanceolate, up to 2 cm long by 7 mm wide; labellum: threelobed, about 1.9 cm long by 1.2 cm wide (when flattened),
lateral lobes rounded, mid-lobe semi-circular, three ridges
run lengthways along the labellum (Figure 2.R, S).
Distribution: It was found in the montane forest of Mt.
Sinaka at an elevation of 1200-1300 meters asl. It has been
found in Mindoro, Negros, Bataan, Benguet, Cagayan,
Kalinga-Apayao, the Mountain Province, Nueva Ecija,
Nueva Vizcaya, Pampanga, Pangasinan, Quezon and is
endemic to the Philippines (Cootes 2011a).
Corybas sp. Salisb, Parad, Lond.
Growth habit: sympodial epiphyte. Leaf solitary,
circular to cordate, some are lobed (Figure 2.P).
Distribution: It was found in the mossy forest of Mt.
Sinaka at an elevation of 1200-1300 meters asl. It has also
been recorded from India, South China, Taiwan, Peninsular
Malaysia, Borneo, New Caledonia, Vanuatu, Ponape,
Indonesia, New Guinea, Solomon Islands, Australia, New
Zealand, Tahiti, and Samoa (Pridgeon et al. 2001).
Dendrobium auriculatum Ames & Quisumbing.
Growth habit: upright, sympodial, epiphyte (Figure
2.T).
Inflorescence: single flowered, blooms very showy,
about 3.5 cm in diameter; flower is milky white with a
short green spur when fresh; dorsal sepal is ovate, up to 2.2
cm long by 1.1 cm wide; petals are oblong, up to 2 cm long
by 8 mm wide; lateral sepals are oblong-lanceolate and are
joined at their base to form a spur about 1 cm long, overall
length 3.3 cm by 1.1 cm wide; labellum is simple, 3.4 cm
long by 1.7 cm wide (at the widest point when flattened),
lower third circular, the front portion is broadly heartshaped when flattened (Figure 2.U,V).
Distribution: It was found on Mt. Sinaka at an elevation
of 1200-1300 meters asl. It has been found in Davao,
Mindoro, Bulacan, Mountain Province, Nueva Ecija,
Nueva Vizcaya, Quezon and is recorded as endemic to the
Philippines.
Dendrobium milaniae Fessel & Luckel
Growth habit: semi-pendulous; sympodial, epiphyte
(Figure 2.W).
Inflorescence: pendulous, bearing up to 4 blooms that
are about 1.5 cm in diameter with a short spur; flower is
white; labellum has yellow in the center surrounded by
purple markings; dorsal sepal: linear, acute, 1 cm long by 5
mm wide; petals are linear, tip rounded, 1.1 cm long by 3.5
mm wide; lateral sepals are triangular, 9 mm long, labellum
is about 1.2 cm long with a wavy margin, the middle of the
labellum has three ridges, which are short, central part of
the labellum is round (Figure 2.X, Y).
Distribution: It was found in montane forest of Mt.
Sinaka at an elevation of 1260 meters asl. It is recorded
from Leyte and is endemic to the Philippines (Cootes
2011a).
PANAL et al. – Orchids of Mt. Sinaka, Philippines
Dendrobium stricticalcarum W. Suarez & Cootes
Growth habit: slightly pendulous to upright, sympodial,
epiphyte (Figure 2.Z).
Inflorescence: appear from the leafless pseudobulbs;
usually near the apex; to 1.4 cm long, bearing between 3
and 12 flowers. Flowers are about 5 mm across the lateral
sepals by 2.3cm long from the tip of the mentum to tip of
the dorsal sepal. The flowers of this species do not open
widely; flower is pink, anther cap is purplish, odorless.
Pollinia are brown in color; dorsal sepal is ovatelanceolate, acuminate, reflexing slightly, 7 mm long by 3.2
mm wide; petals is narrowly lanceolate to linear, acuminate
7 mm long by 1.5 mm wide; lateral sepals are triangular,
together with the mentum 2.5 cm long by 3.5 mm wide;
labellum is ovate-elliptic; 1 cm long by 3 mm wide (when
flattened); edges rolling around the column (Figure 2.AA).
Distribution: It was found in montane forest of Mt.
Sinaka at an elevation of 1200-1300 meters asl. It was also
found in Leyte, Mindoro, Laguna and recorded as endemic
in the Philippines (Cootes 2011a).
Dendrochilum arachnites Reichenbach f.
Growth habit: upright, sympodial, epiphyte. This
species is a bit of a rambler having a distance of about 5 cm
between its pseudobulbs (Figure 2.AB).
Inflorescence: arching, appearing with the new growths.
The flowers are about 2 cm across the lateral sepals, which
is their widest point. Each inflorescence can carry up to 30
blooms; flower is yellow; dorsal and lateral sepals are
linear-lanceolate extending into acute tips, up to 1 cm long
by 2 mm wide; petals are lanceolate up to 7 mm long by 3
mm wide; labellum is oblong, 4 mm long by 1.5 mm wide,
three ridges run its length (Figure 2.AC, AD).
Distribution: It was found in both montane and mossy
forest of Mt. Sinaka at an elevation of 1200-1500 meters
asl. It has been recorded from Agusan, Bukidnon, Davao,
Misamis, Zamboanga, Leyte, Mindoro, Benguet, Ifugao,
Mountain Provinces, Nueva Ecija, Nueva Vizcaya,
Pampanga, Quezon, Rizal and is endemic to the Philippines
(Cootes 2011a).
Dendrochilum mindanaense (Ames) L.O. Williams
Growth habit: upright, sympodial, epiphyte (Figure 2.
AE).
Inflorescence: semi-pendulous, bearing minute blooms;
color is cream. Labellum is dark orange; dorsal sepal is
oblong, about 1.5 mm long by 0.5 mm wide; petals are
narrowly elliptic-oblong; lateral sepals are elliptic about 1.5
mm long by 0.5 mm wide; labellum is three-lobed, side
lobes are oblique measures 0.5 mm long by 1 mm wide;
mid-lobe is subquadrate and has three dentate apex (Figure
2.AF, AG).
Distribution: It was found on Mt. Sinaka at an elevation
of >1400 meters asl. It has also been recorded from
Cabadbaran, Agusan del Norte. No other information has
yet been published other than the Philippines localities
(Pedersen 1997).
217
Liparis dumaguetensis Ames
Growth habit: upright, sympodial, terrestrial (Figure
2.AH).
Inflorescence: upright, to 20 cm long, bearing numerous
blooms about 1.3 cm in diameter; sepals and petals are a
dull purple. Labellum is yellow when the flower first
opens. As the flower ages the labellum goes purplish-red;
dorsal sepal is linear to narrowly lanceolate, to 9 mm long
by 3 mm wide, reflexing; petals are linear, to 9 mm long by
1 mm wide; lateral sepals are narrowly lanceolate, to 9 mm
long by 3 mm wide; labellum is heart-shaped; edges erose,
to 1 cm long by 8 mm wide (Figure 2.AI, AJ).
Distribution: It was found in montane forest of Mt.
Sinaka at an elevation of 1200-1300 meters asl. It was also
found on Camiguin, Negros, Panay, Mindoro, Benguet,
Laguna, Nueva Vizcaya, Quezon, Rizal, and recorded is
endemic to the Philippines (Cootes 2011a).
Liparis terrestris J.B. Comber
Growth habit: upright, sympodial, terrestrial (Figure
2.AK).
Inflorescence: upright, peduncle: 14 cm long by 1 mm
in diameter bearing blooms with 1 cm apart. Pedicel: about
9 mm long by 0.5 mm in diameter. Bracts are linear, about
7 mm long by 1 mm wide; flower is green, yellow column;
dorsal sepal is linear, about 5.5 mm long by 0.5 mm wide;
petals are linear, about 6 mm long by 0.5 mm wide; lateral
sepals are linear, about 5.5 mm long by 0.5 mm wide;
labellum is cordate, erose edge, 4 mm long by 5 mm wide
(Figure 2. AL, AM).
Distribution: It was found in the montane forest of Mt.
Sinaka at an elevation of 1200-1300 meters asl. It is also
recorded from Sumatra (Renz 2012).
Paphiopedilum adductum Asher
Growth habit: upright, sympodial, terrestrial (Figure
2.AN).
Inflorescence: upright, bearing up to three blooms;
dorsal sepal is creamy-white with dark red vertical striping.
Petals are yellowish dark red stripes. Labellum is reddishbrown with darker striping. Synsepalum is creamy-white
with dark red vertical striping; dorsal sepal is ovate,
pointed tip, up to 5.5 cm long by 3 cm wide; petals are
narrowly linear, gradually tapering, drooping, about 10 cm
long by 8 mm wide; labellum is up to 4.5 cm long by 2 cm
wide; synsepalum is oblanceolate, up to 6 cm long by 3 cm
wide.
Distribution: It was found in montane forest of Mt.
Sinaka at an elevation of 1200-1300 meters asl. It is also
found in Bukidnon and recorded as endemic to the
Philippines (Cootes 2011a).
Pinalia cylindrostachya (Ames) W. Suarez and Cootes
Growth habit: upright, sympodial, epiphyte (Figure
2.AO).
Inflorescence: arching, up to 18 cm long, and there can
be several per pseudobulb, bearing many. Flowers are
about 7 mm in diameter; flower is pale-yellow, almost
translucent. Labellum is bright yellow. The outer surfaces,
of the dorsal and lateral sepals are covered with very short
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B I O D I V E R S I T A S 16 (2): 213-224, October 2015
reddish-brown hairs. The anther cap is dark red; dorsal
sepal is elliptic-oblong, up to 6 cm long by 3.5 mm wide;
petals are obovate, 6 mm long by 3 mm wide; lateral sepals
are triangular, 7 cm long by 5 mm wide; labellum is threelobed; wedge-shaped; side lobes oblong; mid lobe is
squarish; overall about 7 mm long by 4 mm wide (Figure
2.AP, AQ).
Distribution: It was found in the mossy forest of Mt.
Sinaka at an elevation of >1400 meters asl. It was recorded
from Bukidnon, Davao, Misamis, Negros, Mindoro,
Bataan, Benguet, Rizal and is endemic to the Philippines
(Cootes 2011a).
Plocoglottis bicallosa Ames
Growth habit: upright, sympodial, terrestrial (Figure
2.AR).
Inflorescence: upright, longer than the leaves, hairy,
bearing numerous attractive blooms; sepals and petals are
greenish-yellow, blotched basally with reddish-brown.
Labellum is cream with a deep yellow spot between the
ridges; dorsal sepal is lanceolate, slightly concave, hairy on
the outer surface, 1.6 cm long by 4.5 mm wide; petals are
upright, linear, 1.6 cm long by 2 mm wide; lateral sepals
are lanceolate, falcate, slightly concave, hairy on the outer
surface, to 1.6 cm long by 4.5 mm wide, reflexing
backwards; labellum is to 1 cm long by 1.3 cm wide, fanshaped, edges toothed, apex pointed and curved under, two
distinct ridges (calli) under the column, which is curved
(Figure 2.AS, AT).
Distribution: It was found in montane forest of Mt.
Sinaka at an elevation of 1100-1200 meters asl. It has been
recorded from Camiguin, Mindoro, Negros, Sorsogon,
Rizal and is endemic to the Philippines (Cootes 2011a).
Podochilus plumosus Ames
Growth habit: upright to semi-pendulous, sympodial,
epiphyte (Figure 2.AU).
Inflorescence: appear dorsally near the tip of the stem
bearing 4-5; flower is milky white. Dorsal sepal has a patch
of purple at the apex Lateral sepals have shades of purple
near its apex; dorsal sepal is ovate to lanceolate, up to 2.5
mm long by 1.5 mm wide; petals are oblong to lanceolate,
pointing forward, up to 2 mm long by 1 mm wide; lateral
sepals are ovate to lanceolate, up to 2.5 mm long by 1.5
mm wide; labellum is triangular, 3 mm long by 1.5 mm
(Figure 2.AV, AW).
Distribution: It was found in the montane forest of Mt.
Sinaka at an elevation of 1100-1200 meters asl. It has been
recorded from Agusan, Cotabato, Surigao, Zamboanga,
Basilan in the Sulu archipelago, Bohol, Leyte, Panay,
Samar, Camarines Sur, Laguna, Nueva Ecija, Quezon,
Zambales and is endemic to the Philippines (Cootes
2011a).
Robiquetia compressa (Lindley) Schlechter
Growth habit: upright to semi-pendulous, monopodial,
epiphyte (Figure 2.AX).
Inflorescence: appear opposite the leaf, sometimes
branching, up to 20 cm long, bearing many small flowers;
flower white with patches of red, side lobes of the labellum
are yellow and dark red; dorsal sepal is ovate, concave, up
to 4.5 mm long by 2 mm wide; petals are broadly ovate, 3.5
mm long by 2.5 mm wide; lateral sepals are obovate,
wedge-shaped, tip broad, 4 mm long by 3 mm wide;
labellum is three lobed, side lobes erect, squarish, mid-lobe
fleshy, spur curved forward, about 1 cm long (Figure 2.AY,
AZ).
Distribution: It was found in the mossy forest of Mt.
Sinaka at an elevation of 1300-1400 meters asl. It has been
recorded from Davao, Misamis, Zamboanga, Leyte, Panay,
Mindoro, Bataan, Camarines Sur, Catanduanes, Quezon,
Rizal, Sorsogon and is endemic to the Philippines (Cootes
2011a).
Thrixspermum linearifolium Ames
Growth habit: pendulous, monopodial, epiphyte (Figure
2.BA).
Inflorescence: appear opposite the leaf; 10 cm long;
rachis flattened bearing the short-lived blooms in
succession; sepals and petals yellow, white to cream;
labellum white or cream with brown blotches on the inner
surface; dorsal sepal is elliptic, concave, 8 mm long by 5
mm wide; petals are obovate, 7 mm long by 4 mm wide;
lateral sepals are elliptic, slightly concave, 8 mm long by 5
mm wide; labellum is pouch-shaped; three lobed, side
lobes crescent-shaped, 2.5 mm long by 2 mm wide; midlobe short and fleshy; pouch 3 mm long, hairy on inner
front surface (Figure 2.BB, BC).
Distribution: It was found in the montane forest of Mt.
Sinaka at an elevation of 1200-1300 meters asl. It has been
recorded from Bukidnon, Misamis, Zamboanga and is
endemic to the Philippines (Cootes 2011a).
Habitat, loss and over-collection of species in the wild,
can result in the extinction of species. This suggests that
there is a desperate need to protect the habitat. Habitat loss
is usually the cause of endangerment of species, while
habitat protection is the key to species conservation.
Considering also the economic uses, and the restricted
distribution of endemic species, continuous habitat
degradation somehow imposes a high risk of these species
becoming threatened and even extinct before they are
described. Furthermore, the result suggests that Mt. Sinaka
must be given priority in protection and conservation to
ensure that orchid habitats will remain intact.
The facts presented in this study serve as supplemental
information for the conservation organizations, and
agencies, not only in Mt. Sinaka but throughout the world,
which will save a myriad of species.
PANAL et al. – Orchids of Mt. Sinaka, Philippines
A
C
219
B
E
D
F
G
I
H
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B I O D I V E R S I T A S 16 (2): 213-224, October 2015
220
K
L
M
N
Q
T
O
R
P
S
U
V
PANAL et al. – Orchids of Mt. Sinaka, Philippines
W
AB
AE
221
X
Y
Z
AA
AC
AD
AF
AG
B I O D I V E R S I T A S 16 (2): 213-224, October 2015
222
AH
AI
AK
AN
AO
AR
AJ
AL
AM
AP
AQ
AS
AT
PANAL et al. – Orchids of Mt. Sinaka, Philippines
AU
AV
AX
BA
223
AY
BB
AW
AZ
BC
Figure 2.A. Anoechtochilus sp.Blume, B, C, D. Appendicula malindangensis (Ames) Schlechter, E. Appendicula micrantha Lindley, F,
G, H. Bulbophyllum alagense Ames, I, J. Bulbophyllum alsiosum Ames, K, L, M. Calanthe mcgregorii Ames, N, O. Ceratostylis
retisquama Reichenbach f., P, Corybas sp. Salisb, Parad, Lond,.Q, R, S. Coelogyne chloroptera Reichenbach f., T, U, V. Dendrobium
auriculatum Ames and Quisumbing, W, X, Y. Dendrobium milaniae Fessel and Luckel, Z, AA. Dendrobium stricticalcarum W. Suarez
& Cootes, AB, AC, AD. Dendrochilum arachnites Reichenbach f., AE, AF, AG. Dendrochilum mindanaense (Ames) L.O. Williams,
AH, AI, AJ. Liparis dumaguetensis Ames, AK, AL, AM. Liparis terrestris J.B. Comber, AN. Paphiopedilum adductum Asher, AO, AP,
AQ. Pinalia cylindrostachya (Ames) W. Suarez and Cootes, AR, AS, AT. Plocoglottis bicallosa Ames, AU, AV, AW. Podochilus
plumosus Ames, AX, AY, AZ. Robiquetia compressa (Lindley) Schlechter, BA, BB, BC. Thrixspermum linearifolium Ames. (photos by
J. Opiso)
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B I O D I V E R S I T A S 16 (2): 213-224, October 2015
REFERENCES
AO-DENR. 2007. Rules and Regulations Governing the Issuance of
Permit over Reclamation Projects and Special Patents over Reclaimed
Lands. www.denr.gov.ph. [15 April 2015]
Buenavista DP. 2014. Alpha and Beta Diversity Assessment of
Orchidaceae in Five Long-Term Ecological Research (LTER) Sites,
Mindanao, Philippines. [Thesis]. Central Mindanao University,
Mindanao.
Cootes J. 2001. The Orchids of the Philippines. Times Editions,
Singapore.
Cootes J. 2011a. Philippine Native Orchid Species. Katha Publishing,
Philippines
Cootes J. 2011b. A Selection of Orchid Species of the Philippines.
www.eurobodalla.ord.au. [15 April 2015]
Cullen J. 1992. The Orchid Book: A Guide to the Identification of
Cultivated Orchid Species. Cambridge University Press, Cambridge,
UK.
Estremera SA. 2011. Protecting their land. Sun Star Davao Yearbook,
Aquamarine Protection and Preservation Alliance Inc., Davao City.
Fernando EL, Co D, Lagunzad D, Gruezo W, Barcelona J, Madulid D,
Lapis A, Texon G, Manila A, Zamora P. 2008. Threatened Plants of
the Philippines: A Preliminary Assessment. Asia Life Sci Suppl 3: 152.
Gravendeel B, Smithson A, Slik FJW, Schuiteman A. 2004. Epiphytism
and pollinator specialization: drivers for orchid diversity. Phil Trans R
Soc London 359: 1523-1535.
Huynh TT, Thomson R, McLean CB, Lawrie AC. 2009. Functional and
genetic diversity of mycorrhizal fungi from single plants of Caladenia
formosa (Orchidaceae). Ann Bot 104: 757-765.
IUCN. 2014. IUCN Red List on Threatened Plants. 2014.3.
http://www.iucnredlist.org/search. [15 April 2015]
Pedersen H. 1997. The Genus Dendrochilum (Orchidaceae) in the
Philippines – A taxonomic Revision. Opera Botanica, Denmark.
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160218
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 225-237
Taxonomy and distribution of species of the genus Acanthus
(Acanthaceae) in mangroves of the Andaman and Nicobar Islands, India
1
P. RAGAVAN1, ALOK SAXENA2, P.M. MOHAN1, R.S.C. JAYARAJ3, K. RAVICHANDRAN4
Department of Ocean studies and Marine Biology, Pondicherry University, Brookshabad Campus, Port Blair, A & N Islands, India Tel.: +91-3192213126/261566. Fax +91-3192-261568,email: van.ragavan@gmail.com
2
Indira Gandhi National Forest Academy, Dehradun, Uttarakhand, India
3
Rain Forest Research Institute, Jorhat, Assam, India
4
Department of Environment and Forests, Andaman and Nicobar Administration, Port Blair, A & N Islands, India
Manuscript received: 3 September 2015. Revision accepted: 16 September 2015.
Abstract. Ragavan P, Saxena A, Mohan PM, Jayaraj RSC, Ravichandran K. 2015. Taxonomy and distribution of species of the genus
Acanthus (Acanthaceae) in mangroves of the Andaman and Nicobar Islands, India. Biodiversitas 16: 225-237. A recent floristic survey
revealed the occurrence of three species of Acanthus in mangroves of the Andaman and Nicobar Islands, India. Of these Acanthus
ilicifolius and A. ebracteatus are shrubs, whereas A. volubilis is a climbing shrub. All the three Acanthus species were recorded from the
Andaman Islands, but only A. ilicifolius from the Nicobar Islands. A. volubilis is easily distinguished from other two species by its
unarmed and twining delicate sprawling stems, un-serrated elliptical leaves, white corolla and absence of bracteoles. A. ilicifolius and A.
ebracteatus are differentiated based on the presence and absences of bracteoles, corolla color, and position of Inflorescence and
direction of stem axial spines. A key for the species of Acanthus of the Andaman and Nicobar Islands is also provided.
Keywords: Acanthus, Andaman and Nicobar Islands, India, taxonomy
INTRODUCTION
The genus Acanthus L. belonging to the family
Acanthaceae is an Old World genus native of tropics and
subtropics with about 30 species. It is often distinguished
from the related genera by spiny leaves, spicate terminal
inflorescences, two bracteoles and uniform anthers (Duke
2006). Four species viz., A. ebracteatus Vahl, A. ilicifolius
L., A. volubilis Wall. and A. xiamenensis are known from
mangrove communities and are classified as true mangrove
species (Polidoro et al. 2010). Of these A. xiamenensis is
endemic to China and all the other species are common in
Indo West Pacific (IWP) region. However, the taxonomical
identity of A. xiamenensis in China is not clear; for instance
Wang and Wang (2007) have treated A. xiamenensis and A.
ilicifolius as the same species. In India all the three
Acanthus species are reported in the Andaman and Nicobar
Islands (ANI). Generally Acanthus species occur either as
an under storey in inner mangrove area or frontal thickets
on edges of the tidal creek in middle to upper estuarine
areas, although they do occur in lower estuarine position
(Duke 2006).
Among the three species of Acanthus the taxonomical
distinction between A. ilicifolius and A. ebracteatus still not
clear in India (Kathiresan 2010). For instance, Remadevi
and Binojkumar (2000) contended that many specimens
identified and indexed as A. ilicifolius in Indian herbaria
are actually A. ebracteatus, but their identification has been
questioned by Anupama and Sivadasan (2004). Further
Mandal and Naskar (2008) noted that A. volubilis
considered as extinct in India has been recorded again with
its very limited population from Sundarbans. Kathiresan
(2008) has not included this species in his report. Thus the
taxonomy and distribution of Acanthus spp., is not well
understood in India as well as in the ANI.
Species of Acanthus were first reported from the ANI by
Parkinson (1923). He recognized all the three species
discussed in this account, however he did not provide the
detailed taxonomical description. He noted that A.
ilicifolius was common and that a few A. ebracteatus were
found along with A. ilicifolius and that A. volubilis was
uncommon found in Bomlungta and Yeratilajig. By the
description and the key given by him it is understood that
he identified the Acanthus species based on serration in the
leaves and flower color, but these two characters are
variable in Acanthus spp. After that all the three Acanthus
species were listed in the mangrove floral list of the ANI
by Sahni (1958), Dagar (1987), Mall et al. (1987), Das and
Dev Roy (1989), Dagar et al. (1991), Singh and Garge
(1993), Dagar and Singh (1999), Singh (2003), Debnath
(2004), and Dam Roy et al. (2009). Among them Dagar et
al. (1991) described the species of Acanthus with locality
data. Mall et al. (1987) and Singh and Garge (1993)
described two species and noted that A. volubilis was not
encountered in the ANI but included it based on the reports
of Parkinson (1923) and Thothathri (1962). Thothathri
(1962) reported A. volubilis from Campbell Bay, Great
Nicobar Island but the specimen deposited by him shows
that he had misidentified spineless form of A. ilicifolius as
A. volubilis. Dam Roy et al. (2009) also described all the
three Acanthus species but later it was found that they
misidentified spineless form of A. ilicifolius as A. volubilis
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B I O D I V E R S IT A S 16 (2): 225-236, October 2015
(Goutham Bharathi et al. 2014; Ragavan et al. 2014).
Recently Goutham Bharathi et al. (2014) reported two
species viz., A. ilicifolius and A. ebracteatus, and further
noted that A. volubilis was recorded nearly three decades
ago as a rare species. Ragavan et al. (2014) reported the
occurrence all the three species of Acanthus in the ANI but
they did not provide detailed taxonomical description.
Hence, in the present study Acanthus spp., in the ANI are
described, illustrated and compared to provide the
taxonomical distinction and to document their distribution.
Figure 1. Map showing surveyed sites in the ANI. South Andaman: 1. Chidiyatapu, 2. Burmanallah, 3. Beadonabad, 4. Corbyn’s
Cove, 5. Sippighat, 6. Manjeri, 7. Guptapara, 8. Manglutan, 9. Wandoor, 10. Ograbraj, 11. Bambooflat Creek, 12. Wright Myo creek,
13. Shoal Bay Creek, 14. Jirkatang, 15. Tirur; Baratang: 16. Middle Strait, 17. Wrafter’s Creek, 18. Baludera; Middle Andaman: 19.
Kadamtala Creek, 20. Yerrata Creek, 21. Shyamkund Creek, 22. Dhaninallah Creek, 23. Rangat Bay, 24. Panchawati; Mayabunder: 25.
Austin Creek, 26. Mohanpur Creek, 27. Karmatang Creek, 28. Chainpur Creek, 29. Rampur, 30. Danapur, 31. Tugapur; Diglipur: 32.
Parangara Creek, 33. Kishorinagar Creek, 34. Kalighat Creek, 35. Smith Island, 36. Ariel bay, 37. Radhanagar, 38. Lakshmipur, 39.
Durgapur,, 40. Ramnagar; Havelock: 41. Govindnagar, 42. Radhanagar, 43. Neil Island; Little Andaman: 44. V.K Pur creek, 45.
Dugong Creek, 46. Jackson Creek; Nicobar islands: 47. Car Nicobar, 48. Kamorta, 49. Katchal, 50. Campbell Bay, 51. Trinket island
RAGAVAN et al. – Acanthus species of the Andaman and Nicobar Islands, India
MATERIALS AND METHODS
During 2009-2013 qualitative survey was carried out
randomly over 51 sites in 8 forest divisions of the ANI to
record the species occurrences in the mangroves of the ANI
(Figure 1). All sites have been visited at least once at the
time of flowering of the different species to crosscheck
identification with flower-based diagnostic features. Site
access was achieved using a combination of road plus
small boat transport to gain access to the extensive range of
mangrove area.
Plant collections were made of all Acanthus taxa
encountered from the range of sites visited. For each
Acanthus species both numeric and multistate attributes of
a wide range of vegetative and reproductive morphological
characters were observed from one or two individuals at
each site. All measurements and observations were made
from fresh material for making key for Acanthus spp. The
morphological measures and observations were developed
and standardized during prior assessments of this genus.
For each species 2-5 specimens were sampled with flowers
and fruits for herbarium preparation. Herbarium specimen
has been prepared and deposited to the National Botanical
Collection of Andaman Nicobar Regional Centre,
Botanical Survey of India, Port Blair.
RESULTS AND DISCUSSION
Key to Acanthus species in ANI
1. Bracteoles present ............................................................ 2
Bracteoles absent or minute …......................................…. 3
2. Inflorescences terminal and axial, flowers light blue to
dark or violet (rarely white), stem axial present or absent,
if present always facing upward ......................... A. ilicifolius
3. Inflorescences terminal, flowers white, stem axial spine
absent .................................................................... A. volubilis
4. Inflorescences terminal, flowers white, stem axial spines
facing downwards ............................................ A. ebracteatus
Acanthus ilicifolius L.-Linnaeus C. 1753. Sp. Pl. 2: 639.
(Figure 2)
Acanthus doloarin Blanco-Blanco, F.M. 1837. Fl. Filip. 487.
Acanthus neoguineensis Engl.-Engler, H.G.A. 1886. Bot.
Jahrb. Syst. 7: 474.
Dilivaria ilicifolia (L.) Juss.-Jussieu, A.L. 1789.Gen. Pl. 103.
Shrub: height up to 3 m (Figure 2A). Stem: thick, green,
light green or purple, sparsely branched and stem axial
spine either present or absent, if present always facing
upward (Figure 2E). Roots: occasionally above ground or
prop roots on lower parts of reclining stem (Figure 2D).
Leaves: simple, opposite, lanceolate to broadly lanceolate,
margin either entire or spiny and dentate, leaf base
attenuate, leaf tip acute and narrowly pointed with or
without spiny edge, presence of spines with greater
sunlight and exposure, size variable, 6-30 x1.5-6 cm, ratio
of length to width is greater than 2; petiole short, green,
0.5-2 cm long. Inflorescences: both terminal (Figure 2B)
227
and axial (Figure 2C), terminal inflorescences longer than
axial, up to 15 cm long, axial inflorescence smaller than
terminal, 5-10 cm long. Mature flower bud: ellipsoidal, 33.5 cm long; bract single, 0.8-1 x 0.5-0.7cm (Figure 2H);
bracteoles 2, lateral, 0.5-0.8 x 0.2-0.4 cm (Figure 2H);
calyx four lobed, outer two lager in size, 1.3-1.5 x 0.8-1.2
cm, enclosing the flower bud, inner lateral lobes narrow, 1
by 0.5-0.8 cm, enclosed by upper and lower lobes; corolla
purple or deep purple, rarely white with dark blue median
band (Figure 1B and 1I), 3-4 cm x 2-2.5 cm; stamens 4, 22.5 cm long, sub equal with thick hairy connectives; anther
medifixed each with 2 cells aggregated around the style,
0.8-1cm long, densely ciliated (Figure 2F); ovary bi-locular
with 2 superimposed ovules in each loculus; style enclosed
by stamens, 2.5-3 cm long, capitate to pointed stigma
exposed (Figure 2G and 2I). Mature fruits: 4 seeded
capsule, ovoid, green, shiny, smooth, 3-3.5 x 1 cm (Figure
2J).
Distribution. Found from India to southern China,
tropical Australia and the western Pacific islands, including
New Caledonia and the Solomon Islands. Occurs
throughout Southeast Asia. In India it is common in both
east and west coast. In ANI it is found common in both
Andaman Islands and Nicobar Islands.
Habitat and Ecology. Common in landward edges of
mangroves just above the high tide mark, also occurs in
inner mangroves as understorey. Individuals under shade
are not serrated.
Phenology. Flowering February to March; fruiting April
to May.
Specimen examined. India, Andaman and Nicobar
Islands, South Andaman, Shoal Bay Creek (11° 47′ 58.4″N,
92° 43′ 03.2″E), P. Ragavan, PBL 30965 and 30966.
Acanthus ebracteatus Vahl-Vahl, M. 1791. Symb. Bot. 2:
75. (Figure 3)
Acanthus ilicifolius Lour-Loureiro, J. 1790. Fl. Cochinch. 2:
375.
Dilivaria ebracteata (Vahl) Pers.-Persoon, C.H. 1806. Syn.
Pl. 2: 179.
Acanthus ilicifolius var. ebracteatus (Vahl) Benoist-Benoist,
R. 1910. Acanthacea nouvelle de Madagascar. Notul. Syst. (Paris)
1: 224-225.
Shrub: height up to 2 m (Figure 3A). Stem: thick, grey
color, sparsely branched, stem axial spines always present
and facing downward (Figure 3C). Roots: occasionally
above ground or prop roots on lower parts of reclining stem
(Figure 3B). Leaves: simple, opposite, broadly elliptic to
lanceolate, 10-20 x 3-6 cm, leaf tip acute to obtuse with or
without spiny edge, base attenuate, margin either/ or spiny
and dentate, presence of spines with greater sunlight and
exposure; petiole short 0.5-1.5 cm long. Inflorescence:
terminal always (Figure 3D), never axial. Mature flower
bud: ellipsoidal, 1.8-2.2 cm long; bract single not persistent
(Figure 3E, K), 0.3-0.5 cm, apex obtuse, bracteoles absent
or obscure, 0.3-0.5 cm, apex acute (Figure 3F); calyx four
lobed (Figure 3L), 0.6-0.8 cm long, upper lobe larger than
lower lobe (Figure 3I), enclosing the flower bud, lateral
lobes narrow, enclosed by upper and lower lobes; corolla
white, with purple stripe in middle of lower lip (Figure
3H), 0.9-1.5 cm long; stamens 4, 1.2-1.5 cm long, sub
228
B I O D I V E R S IT A S 16 (2): 225-236, October 2015
equal with thick hairy connectives; anther medifixed each
with 2 cells aggregated around the style, 0.3-0.5 cm (Figure
3M); ovary bilocular with 2 superimposed ovules in each
loculus; style enclosed by stamens, 0.8-1.2 cm long,
capitate to pointed stigma exposed (Figure 3G). Mature
fruit: 4 seeded capsule, ovoid, green, shiny, smooth, 2-2.5 x
1 cm (Figure 3J).
Distribution. From India to tropical Australia, Southeast
Asia and the west Pacific islands (e.g. Solomon Islands). In
Southeast Asia it has been recorded in Cambodia,
Myanmar, the Philippines, Vietnam, Malaysia, Singapore,
Indonesia and Papua New Guinea. In India A. ebracteatus
occurs in Kerala, Puducherry and ANI. In ANI it is
recorded from Sippighat and Shoal Bay Creek in South
Andaman Island.
Habitat and Ecology. Common in landward edges of
mangroves just above the high tide mark, also occur in
inner mangroves as under-storey. Individuals under shade
are less serrated
Phenology. Flowering and fruiting occurs throughout
the year apparently.
Specimen examined. India, Andaman and Nicobar
Islands, South Andaman, Sippighat (11°36′ 50.1″N, 92° 41′
22.2″E), P. Ragavan, PBL 30969 and 30970.
Acanthus volubilis Wall-Wallich, N. 1831. Pl. Asiat. Rar.
2: 56. (Figure 4)
Dilivaria scandens Nees-Nees von Esenbeck CGD.1847.
Prodr. 11: 269.
Dilivaria volubilis (Wall.)Nees-Nees von Esenbeck, C.G.D.
1832. Pl. Asiat. Rar. 3: 98.
Twining shrub: length to 2-4 m (Figure 4A). Stem: thin,
green, branched, smooth, stem axial spines absent (Figure
4I). Roots: occasionally above ground or prop roots on
lower parts of reclining stem (Figure 4G). Leaves: simple,
opposite, without spines, succulent, elliptic or oblong
lanceolate, leaf tip acute to obtuse with spiny edge, leaf
base attenuate, margin entire without spines and dentate
(Figure 4B), 5-10 x 2.5-4 cm, ratio of length to width
greater than 2; petiole short 0.5-2 cm long, green.
Inflorescence: always terminal (Figure 4C), 8-12 cm long;
Mature flower bud: ellipsoidal, 2-2.8 cm long; bract single
not persistent; bracteoles absent (Figure 4H); calyx 1-1.3
cm long, four lobed, upper lobe 1.3 by 0.5 cm, lower lobe 1
x 0.4 cm, enclosing the flower bud, lateral lobes narrow,
0.5-0.7 cm long, enclosed by upper and lower lobes (Figure
4J); corolla white, 1.5-2 x 2-2.5 cm (Figure 4D); stamens 4,
1.5 cm long, sub equal with thick hairy connectives; anther
medifixed each with 2 cells aggregated around the style
(Figure 4E); ovary bilocular with 2 superimposed ovules in
each loculus; style enclosed by stamens, 1-1.5cm long,
capitate to pointed stigma exposed (Figure 4F). Mature
Fruit: 4 seeded capsule, ovoid, green, shiny, smooth, 2-2.5
x 1cm (Figure 4K).
Distribution. Found from South to Southeast Asia.
Recorded in eastern India (Odisha), Sri Lanka and the
Andaman Islands, to Myanmar, Indonesia, Cambodia,
Malaysia, Singapore, Thailand and Papua New Guinea. In
India Acanthus volubilis occurs in ANI and Sundarbans. In
ANI it is recorded from Shoal Bay Creek and Jirkatang in
South Andaman at confined location.
Habitat and Ecology. Observed in landward edges of
mangroves just above the high tide mark flooded during
spring tides
Phenology. Flowering March to April; fruiting May to
June.
Specimen examined. India, Andaman and Nicobar
Islands, South Andaman, Shoal Bay Creek (11° 47′ 58.4″N,
92° 43′ 03.2″E), P. Ragavan, PBL 30967 and 30968.
Discussion
Among the three Acanthus species described in this
account A. volubilis can be easily distinguished from other
two species by its unarmed and twining with delicate
sprawling stems, un-serrated elliptical leaves, white corolla
and absence of bracteoles. However it was found from this
study that A. ilicifolius also exhibits un-serrated leaves,
stem without axial spines but presence of bract and
bracteoles is consistent in A. ilicifolius. So without flower it
is difficult to distinguish A. volubilis and A. ilicifolius in the
ANI. Specimen deposited by Thothathri (1962) and
photographs shown in Dam Roy et al. (2009) for A.
volubilis possess un-serrated leaves and stem without axial
spines but flowering and fruiting was not reported by them.
This supposes that the only character of the observed
specimen used by them to identify the species as A.
volubilis was spineless leaves and stem. Parkinson (1923)
reported A. volubilis from Middle Andaman whereas Dagar
et al. (1991) reported it from Baratang Island. In the present
study A. volubilis was not observed in the above mentioned
sites; instead wide variation in A. ilicifolius was observed
there. In this study it was recorded from Shoal Bay Creek
and Jirkatang in South Andaman, though in past no records
are available for the presence of A. volubilis in these two
sites.
In this study A. ebracteatus was recorded from South
Andaman at two sites viz., Shoal Bay and Sippighat. The
species status of A. ebracteatus is often doubted by the
botanists since the two species viz., A. ilicifolius and A.
ebracteatus have similar vegetative characteristics and the
difference between them is the presence or absence of
bracteoles which are often lost at anthesis, and many
people have been wary when making identifications
(Barker 1986). The taxonomical identity of A. ebracteatus
was mainly based on the absence of bracteoles, white color
corolla and smaller size of flower and fruits. During the
present study it was found that position of inflorescences
and direction of stem axial spines at nodes aid the rapid
differentiation of A. ilicifolius from A. ebracteatus apart
from flower color and presence of bracteoles in the ANI. In
A. ebracteatus inflorescences are always terminal and stem
axial spines face downwards whereas in A. ilicifolius
inflorescences are both terminal and axial and stem axial
spines face upwards. Bract and bracteoles are always
present and persistent in A. ilicifolius whereas in A.
ebracteatus bracteoles are absent or minute and bracts are
present and both are lost before or at anthesis. Diagnostic
characters of Acanthus spp. in the ANI are given in Table
1.
RAGAVAN et al. – Acanthus species of the Andaman and Nicobar Islands, India
A
D
H
C
B
E
229
G
F
I
J
Figure 2. Diagnostic characters of A. ilicifolius. A. Habitat, B. Terminal infloresences, C. Axial inflorescences, D. Stem based with
above ground roots, E. Stem axial spines facing upwards, F. Densely ciliated anther, G. Style, H. Presences of bract and bracteoles, I.
Blue color corolla, J. Fruit
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B I O D I V E R S IT A S 16 (2): 225-236, October 2015
B
A
D
C
E
F
H
G
I
K
J
L
M
Figure 2. Diagnostic characters of A. ebracteatus: A. Habitat, B. Prop roots, C. Stem axial spine faring downwards, D. Terminal
infloresences, E. Flower bud with only bract, F. Flower bud with bract and minute bracteoles, G. Style, H. White corolla with purple
stripe in middle of lower lip, I. Calyx lobes, J. Fruits, K. Removal of bract before anthesis, L. Mature fruit without bract and bracteoles,
M. Small stamens with densely ciliated anther
RAGAVAN et al. – Acanthus species of the Andaman and Nicobar Islands, India
B
F
I
C
D
A
231
E
H
G
J
K
Figure 4. Diagnostic characters of A. volubilis: A. Habitat, B. Leaves, C. Terminal inflorescences, D. White corolla, E. Stamens with
densely ciliated anther, F. Style, G. Stilt root, H. Prominent bract, I. Twining smooth stem, J. Calyx lobes, K. Fruits
232
B I O D I V E R S IT A S 16 (2): 225-236, October 2015
A
B
C
D
E
F
G
H
I
Figure 5. Variation in A. ilicifolius: A. Light blue flower, B. Flower with white margin and median dark blue band, C. Purplish flower,
D. Smooth stem without axial spines, E. Stem with axil spines, F. Reddish bract and bracteoles with acute tip, G. Bract and bracteoles
with obtuse apex, H. Fruit with persistence bracteoles with obtuse tip, I. Large sized flower
Most of the A. ilicifolius population observed in this
study have lanceolate bract and bracteoles with mucronate
tip, ciliated margin and medial vein whereas the population
observed in Great Nicobar Island possessed ovate bract and
bracteoles with ciliated margin and flowers are
comparatively larger than that of others (Figure 5G-I) A.
ilicifolius specimens have been recorded with stems that
are green, purple or of colors in between with or without
stem axial spines (Figure 5D and 5E) and color of bract and
bracteoles also varied (Figure 5F). Flowers and fruits of A.
ebracteatus are smaller than that of A. ilicifolius, but flower
color of A. ilicifolius varied from light to dark blue or violet
with dark blue median band (Figure 4A-C). In both A.
ilicifolius and A. ebracteatus degree of serration in leaves
varied with respect to exposure to sun, individuals under
shade being less serrated particularly in A. ilicifolius, and
wide variation in leaf shape was recorded in the ANI
(Figure 6 and 7).
In Table 2 Characters of A. ilicifolius and A.
ebracteatus described from Malesia, Java, Australia and
New Guinea are compared with characters of A. ilicifolius
and A. ebracteatus observed in the ANI. This indicates that
presence of both terminal and axial inflorescences in A.
ilicifolius and direction of stem axial spine (downward in
A. ebracteatus and upward in A. ilicifolius) are new
observation and not recorded for these species. There are
marked differences in the size of flower and fruit between
A. ilicifolius and A. ebracteatus in the ANI as in Malesia.
Axillary thorns are variable in A. ilicifolius of the ANI
whereas axillary thorns are more consistent in others places
and flower color is variable in all the places. Bract and
bracteole’s apex is acute and mucronate in most of the
RAGAVAN et al. – Acanthus species of the Andaman and Nicobar Islands, India
B
A
C
D
E
F
H
I
K
233
G
J
L
M
Figure 6. Variation in leaves of A. ilicifolius: A. Smooth needle like leaf, B. Smooth lanceolate like leaf, C and D. Smooth broadly
elliptic leaves with acuminate tip, E. Leaves serrated at base only, F. Highly dentate leaves, G. Smooth leaf with acute tip, G. Obovate
leaves with rounded tip, I. Moderately serrated and dentate leaves, J. Highly serrated leaves, K. Stem with axial spines, L. Stem with
axial spine, M. Smooth leaves with spine axial spines
234
B I O D I V E R S IT A S 16 (2): 225-236, October 2015
A
B
D
C
Figure 7. Variation in leaves of A. ebracteatus. A. Leaf with smooth margin, B. Leaf with moderately serrated and denate, C. Leaf with
less serrated, D. L with highly serrated and dentate
Table 1. Diagnostic characters of Acanthus species of the ANI
Components
Characters
A. ilicifolius
A. ebracteatus
A. volubilis
Leaves
Leaf shape
Leaf apex
Highly variable lanceolate to obovate
Acute to acuminate or rounded with or
without spiny edge
Attenuate
Entire to spiny and dentate
Oblong elliptic
Acute with or without spiny edge
Attenuate
Spiny and dentate
Elliptic
Acute with or without
spiny edge
Cuneate
Entire
Thick
Mostly purple, green also
Thin, twine like
Greenish brown
Axial
spines
Position
Bud length
Bract
Thick
Variable green, purple or colors in
between
Present or absent, if present always
facing upwards
Terminal and axial
3-3.5 cm long
Present, single and persistent
Always resent facing downward
Absent
Terminal always
1.8-2.2 cm long
Present, single fall before anthesis
Bracteoles
Two, lateral, persistent
Petal
Mostly purple, blue or dark blue,
rarely white
Four, 2-2.5 cm long
2.5-3 cm long
Capsule like
3-3.5 cm
Four seeded
Absent or minute, lateral, two, fall
before anthesis
White with purple stripe in middle
of lower lip
Four, 1.2-1.5 cm long
0.8-1.2 cm long
Capsule like
2-2.5 cm
Four seeded
Terminal always
2-2.8 cm long
Present, single, fall
before anthesis
Absent
Stem
Inflorescences
Mature flower
Mature fruit
Leaf base
Leaf
margin
Texture
Color
Stamens
Style
Shape
Fruit length
Seed
White
Four 1.2-1.5 cm long
1-1.5 cm long
Capsule like
2-2.5 cm
Four seeded
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160218
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 225-237
Table 2. Comparison of characteristics for A. ilicifolius and A. ebracteatus from Malesia, Australia, New Guinea and ANI
Characters
Axillary thorns
Inflorescences
Bracteoles
Bract size
Bracteole size
Bract/bracteole apex
Acanthus ilicifolius
Java
(Backer and
Malesia
Australia
ANI
Bakhuizen van
(Bremekamp
(Barker
(This study)
den Brink
1955)
1986)
1965)
Present or
Present
Present
Usually
absent, if
present
present always
facing upwards
Both terminal Terminal
Terminal
Terminal
and axial
Present and
Present
Present
Present
persistent
Acanthus ebracteatus
Java
(Backer and
Malesia
Australia
New Guinea
ANI
Bakhuizen van
(Bremekamp
(Barker
(Barker 1986) (This study)
den Brink
1955)
1986)
1965)
Usually
Present always, Present or not
Present or not Absent
present
facing
downwards
0.8-1 cm
0.5-0.8 cm
0.65-0.8 cm
0.6-0.8 cm
0.65-0.82 cm
0.55-0.65 cm
Often spinetipped
Acute to
obtuse
Acute and
mucronate
mostly, obtuse
also
Corolla length
3-4 cm
Filament/stamen length 2-2.5 cm
Anther length
0.8-1 cm
Style length
2.5-3 cm
Calyx length
1.3-1.5 cm
Suture of anther
Densely ciliate
Corolla color
0.7-0.9 cm
0.7-0.9 cm
More or less equal 0.6-0.8 cm
to bracts
Spinose mucronate -
3.5-4 cm
1.5-2 cm
0.8-0.85 cm
2.5 cm
1.3 cm
Densely ciliate
3-4.5 cm
1.3-1.6 cm
2.25-2.5 cm
1.2-1.5 cm
-
2.2-3 cm
1.1-1.3 cm
0.63-0.65 cm
1.7-1.8 cm
1.1-1.2 cm
Ciliate along
one of both
sides
Violet, light to Dark blue or violet, Violet with
Blue, mauve,
dark blue with rarely white
yellow median whitish edged
dark blue
band, rarely
with lilac
median band
white
Present or
not
Terminal
Terminal
Terminal
Terminal
Present
Absent or
minute if
present, not
persistent
0.3-0.5 cm
0..3-0.5 cm
Usually absent
Usually absent Usually
absent
Usually
absent
0.3-0.6 cm
-
0.3-0.4 cm
0.3-0.4 cm
Bract apex is
obtuse and
bracteoles apex
is acute
0.9-1.5cm
1.2-1.5cm
0.3-0.5cm
0.8-1.2 cm
0.6-0.8 cm
Hairy
-
-
0.65-0.82 cm
Only 1 seen
0.4 cm
Obtuse
0.65-8 cm
Only 1 seen
0.33 cm
Obtuse
1.5-3 cm
0.7-1.2 cm
0.4-0.6 cm
1-1.5 cm
0.7-1.1 cm
Densely ciliate
2-3 cm
0.75-1.25 cm
2 cm
0.75-1.25 cm
-
2.2-2.6 cm
1-1.1 cm
0.6 cm
1.2 cm
1-1.05 cm
Glabrous
along both
sides
Blue-purple,
dark blue,
purple, lilac,
2.2-2.4 cm
1-1.1 cm
0.5-0.65 cm
1.2-1.8cm
1-1.05 cm
Ciliate
along one
side
White with
blue
margins:
scented or
not
2.8 cm
c. 1.3 cm
0.62 cm
1.5-1.8
1.1-1.3 cm
Ciliate along
one of both
sides
Bright blue to White
violet,
becoming
white,
internally with
yellow spot
White or pale blue White
Terminal
New
Guinea
(Barker
1986)
Terminal
B I O D I V E R S IT A S
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B I O D I V E R S IT A S 16 (2): 225-237, October 2015
population of A. ilicifolius in the ANI as in other places
however bract and bracteoles with obtuse apex are also
observed in A. ilicifolius from Great Nicobar Islands.
Axillary thorns and flower color in A. ebracteatus are
consistent in the ANI but variable in other places. A.
ebracteatus of the ANI have similar characteristics with
specimen described in Java, but bracteoles are absent in
Java but are minute in the ANI. Bract apex is obtuse in
ANI and apexes of the bracteoles are acute. Most
importantly two subspecies of A. ebracteatus described
from Australia are more similar to A. ilicifolius described in
this account except in the presence of glabrous anthers and
flower color. It was reported that flower color of A.
ilicifolius is rarely white (Jayatissa et al. 2002). In the
present study A. ilicifolius with white flowers was not
observed in the ANI, whereas flowers color in A. ilicifolius
varied between light to dark violet. Recently Debnath et al.
(2004) reported a new mangrove species Acanthus albus
from Sundarbans. This new species is similar to A.
ilicifolius except in flower color and smaller size of leaves,
fruits and flowers. Similar kind of specimen was reported
from Sri Lanka by Liyanage (1997) as A. volubilis, and
later that it was confirmed as whitish flowered form of A.
ilicifolius (Jayatissa et al. 2002). So the species status of A.
albus has to be checked to avoid further complication in the
identity of A. ilicifolius.
Acanthus ilicifolius was recorded at 46 sites out of 51
sites surveyed and observed in both Andaman Islands and
Nicobar Islands whereas A. ebracteatus and A. volubilis
were recorded at confined locations in the Andaman
Islands. So it is reported that A. ebracteatus and A.
volubilis are rare in these Islands. Earlier Dagar et al.
(1991) reported the occurrence of A. ilicifolius and A.
ebracteatus from Nicobar Islands but, in the present study,
A. ebracteatus was not observed there. It has been reported
that 62-70% of the mangrove forests of Nicobar Islands
were destroyed by the submergence of coastal lands by
about 1 m following the massive earthquake that occurred
on 26th December 2004 (Ramachandran et al. 2005;
Sridhar et al. 2006; Nehru and Balasubramanium 2011). It
is suspected, therefore, that permanent submergence might
have led to the extinction of A. ebracteatus from the
Nicobar Islands, although this needs to be validated by
more extensive and thorough surveys for Acanthus species
in the Nicobar Islands.
Acanthus species has long been used as a traditional
folk remedy for treating various ailments in Indian
traditional medicine, traditional Tai medicine and Chinese
traditional medicine (Wostmann and Liebezeit 2008;
Saranya et al. 2015). Various parts of A. ilicifolius have
been used as crude drug for treatment of asthma, diabetes,
dyspepsia, leprosy, hepatitis, paralysis, snake bite,
rheumatoid arthritis and diuretic (Bandaranayake 1998).
The leaves of A. volubilis are used for dressing boils and
wounds whereas powdered seeds are taken with water as a
blood cleansing medicine and against ulcers (Das et al.
2013). The leaves of A. ebracteatus used for making Tai
herbal tea in Thailand and Indonesia, as it has antioxidant
properties (Chan et al. 2012). Capsule from A. ebracteatus,
named Sea Holly Capsule, is a certified product of ministry
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160218
of Public Health Thailand and has been used for relief of
allergies and rashes (Saranya et al. 2015). In addition
Acanthus species possess wide range of anti microbial
activities (Ganesh and Vennila 2010) and various bioactive
components have also been extracted (Bandaranayake
2002).
As per the IUCN Red data list all the three species of
Acanthus are considered as of least concern. However, in
India only A. ilicifolius is known to occur in all mangrove
habitats except Lakshadweep, whereas A. ebracteatus and
A. volubilis have very restricted distribution. A. ebracteatus
was earlier known to occur only from Kerala and ANI
(Kathiresan 2008), but Saravanan et al. (2008) reported its
occurrence in Puducherry. But in recent times A.
ebracteatus is not reported from Kerala. A. volubilis is
present in ANI and Sundarbans, but in both the places its
distribution is confined to a few locations. Hence A.
ebracteatus and A. volubilis are confirmed as rare in India.
So measures have to be taken to conserve these two species
on priority basis.
ACKNOWLEDGEMENTS
We are extremely grateful to the Principal Chief
Conservator of Forests, Andaman and Nicobar Islands for
his guidance and ensuring necessary support from the field.
We appreciate the cooperation and support provided by the
CCF (Research and Working Plan), CCF, Territorial Circle
and all the Divisional Forest Officers and their staff in the
Department of Environment and Forests, Andaman and
Nicobar Administration. Thanks are due to Dr. N.
Krishnakumar, IFS, Director, Institute of Forest Genetics
and Tree Breeding, Coimbatore for his support and
encouragement. Special thanks are extended to DFO
Campbell Bay for constant support.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160219
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 238-246
Isolation and characterization of a molybdenum-reducing and SDSdegrading Klebsiella oxytoca strain Aft-7 and its bioremediation
application in the environment
NORAZLINA MASDOR1, MOHD SHUKRI ABD SHUKOR2, AFTAB KHAN3, MOHD IZUAN EFFENDI BIN
HALMI4,♥, SITI ROZAIMAH SHEIKH ABDULLAH4, NOR ARIPIN SHAMAAN5,MOHD YUNUS SHUKOR6
1
Biotechnology Research Centre, MARDI, P. O. Box 12301, 50774 Kuala Lumpur, Malaysia
Snoc International Sdn Bhd, Lot 343, Jalan 7/16 Kawasan Perindustrian Nilai 7, Inland Port, 71800, Negeri Sembilan, Malaysia.
3
Department of Biochemistry & Health Science, Hazara University Mansehra, KPK, Pakistan.
4
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi,
Selangor, Malaysia. Tel.: +60-3-89216428; Fax: +60-3-89118345, email: zuanfendi@ukm.edu.my, zuanfendi88@gmail.com
5
Faculty of Medicine and Health Sciences, Islamic Science University of Malaysia, 55100 Kuala Lumpur, Malaysia.
6
Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM 43400 Serdang, Selangor,
Malaysia.
2
Manuscript received: 15 August 2015. Revision accepted: 18 September 2015.
Abstract. Masdor N, Shukor MSA, Khan A, Halmi MIE, Abdullah SRS, Shamaan NA, Shukor MY. 2015. Taxonomy and distribution of
species of the genus Acanthus (Acanthaceae) in mangroves of the Andaman and Nicobar Islands, India. Biodiversitas 16: 238246.Pollution as a result of anthropogenic activities is a severe global issue. These activities including inappropriate disposal, industrial
and prospecting activities and unnecessary use of agricultural chemicals have triggered international initiatives to eliminate these
contaminants. In this work we screen the ability of a molybdenum-reducing bacterium isolated from contaminated soil to grow and
reduce molybdenum on various detergents. The bacterium was able to grow on SDS as a carbon source although the compound did not
support molybdenum reduction. The bacterium reduces molybdate to Mo-blue optimally between pH 5.8 and 6.3 and between 25 and
34oC. Glucose was the best electron donor for supporting molybdate reduction followed by sucrose, D-mannitol, D-sorbitol, lactose,
salicin, trehalose, maltose and myo-Inositol in descending order. Other requirements include a phosphate concentration between 5.0 and
7.5 mM and a molybdate concentration between 5 and 20 mM. The absorption spectrum of the Mo-blue produced was similar to
previous Mo-reducing bacterium, and closely resembles a reduced phosphomolybdate. Molybdenum reduction was inhibited by mercury
(ii), silver (i) and copper (ii) at 2 ppm by 62.1, 33.9 and 33.6%, respectively. Biochemical analysis resulted in a tentative identification
of the bacterium as Klebsiella oxytoca strain Aft-7. The ability of this bacterium to detoxify molybdenum and degrade detergent makes
this bacterium an important tool for bioremediation.
Keywords: Bioremediation, isolation, Klebsiella oxytoca, molybdenum, SDS
INTRODUCTION
Pollution due to anthropogenic activities is a serious
global problem. These activities such as improper disposal,
industrial and mining activities and excessive use of
agricultural chemicals have resulted in global efforts to
remove these pollutants (Rieger et al. 2002). Molybdenum
is one of the essential heavy metals that are required at
trace amount and is toxic to a variety of organisms at
elevated levels (Ahmad-Panahi et al. 2014). The estimated
global molybdenum reserve base in 2008 was 19,000,000
metric tonnes. It has many uses in industries such as
molybdenum grade stainless steels, high-temperature steel,
tool and high speed steel, molybdenum grade cast irons,
automobile engine anti-freeze component, component of
corrosion resistant steel and as lubricant in the form of
molybdenum disulphide. Widespread use of molybdenum
in industry has resulted in several soil and water pollution
cases all around the world such as in the Tokyo Bay, Tyrol
in Austria, the Black Sea, where molybdenum level reaches
in the hundreds of ppm (Davis 1991; Neunhäuserer et
al.2001). In addition terrestrially, it has been recognized as
a significant pollutant in sewage sludge (Lahann 1976).
Molybdenum is very toxic to ruminants at levels of several
parts per million being the most affected are cows
(Underwood 1979; Kincaid 1980). Molybdenum toxicity in
inhibiting spermatogenesis and arresting embryogenesis in
organisms such as catfish and mice at levels as low as
several parts per million have been reported (Meeker et al.
2008; Bi et al. 2013; Zhai et al. 2013; Zhang et al. 2013).
Aside from heavy metals, detergents are often present
as co pollutants, especially in water bodies. Detergents
have detrimental effects to aquatic life (Liwarska-Bizukojc
et al. 2005). Anionic surfactants such as Sodium Dodecyl
Sulfate (SDS) and Sodium Dodecyl Benzene Sulfonate
(SDBS) (Figure 1) exhibited toxic effects at concentrations
ranging from 0.0025 to 300 mg/L (Pettersson et al. 2000).
Toxicity towards invertebrates and crustaceans has been
documented in vivo and in vitro due to the massive amount
of anionic surfactants continuously released into water
bodies. For instance, a study on oyster digestive gland
indicated that exposure to SDS has a detrimental effect.
MASDOR et al. –Potential bioremediation of Klebsiella oxytoca
The detrimental effects include perturbation of the
metabolic and nutritional functions. All of these negative
effects have a direct influence on oyster survival (Rosety et
al. 2000). Detergents could also modify the behavior of the
fish such as muscle spasms, erratic movement, and body
torsion (Cserháti et al. 2002).
A
B
Figure 1.The structure of SDS (A) and SDBS (B).
Some microbes are able to degrade a variety of
xenobiotics and detoxify heavy metals at the same time
(Anu et al. 2010; Chaudhari et al. 2013; Bhattacharya et al.
2014) and the versatility of these microbes are in great
demand in polluted sites where the presence of several
contaminants are the norm (Ahmad et al. 2014). Heavy
metals reduction coupled with azo dye decolorization have
been reported (Chaudhari et al. 2013).
In the present work, we screen for the ability of a novel
molybdenum
reducing
bacterium
isolated
from
contaminated soil to grow on several detergents and
hydrocarbons such as diesel and crude petroleum. Here we
report on a novel molybdenum-reducing bacterium with the
capacity to grow on the detergent SDS, isolated from a
contaminated soil. The characteristics of this bacterium
would make it suitable for future bioremediation works
involving both the heavy metal molybdenum and dye as an
organic contaminant.
MATERIALS AND METHODS
Chemicals
All chemicals used were of analytical grade and
purchased from Sigma (St. Louis, MO, USA), Fisher
(Malaysia) and Merck (Darmstadt, Germany).
Isolation of molybdenum-reducing bacterium
Soil samples were taken (5 cm deep from topsoil) from
the grounds of a contaminated land in the province of
Khyber Pakhtunkhwa, Pakistan, in 2013. One gram of soil
sample was suspended in sterile tap water. 0.1 mL aliquot
of the soil suspension was pipetted and spread onto agar of
low phosphate media (pH 7.0), and incubated for 48 hours
at room temperature. The composition of the low
phosphate media (LPM) were as follows: glucose (1%),
(NH4)2.SO4 (0.3%), MgSO4.7H2O (0.05%), yeast extract
(0.5%), NaCl (0.5%), Na2MoO4.2H2O (0.242% or 10 mM)
239
and Na2HPO4 (0.071% or 5 mM) (Yunus et al. 2009). The
formations of blue colonies indicate molybdate reduction
by molybdenum-reducing bacteria. Colony forming the
strongest blue intensity was isolated and restreaked on low
phosphate media (LPM) to obtain pure culture.
Molybdenum reduction in liquid media (at pH 7.0) was
carried out in 100 mL of the above media in a 250 mL
shake flask culture at room temperature for 48 hours on an
orbital shaker set at 120 rpm with the same media above
but the phosphate concentration increased to 100 mM.
Molybdenum blue (Mo-blue) absorption spectrum was
studied by taking out 1.0 mL of the Mo-blue formed from
the liquid culture above and then centrifuged at 10,000 xg
for 10 minutes at room temperature. Scanning of the
supernatant was carried out from 400 to 900 nm using a
UV-spectrophotometer
(Shimadzu
1201).The
low
phosphate media was utilized as the baseline correction.
Morphological, physiological and biochemical
characterization of the isolated strain
Identification of the bacterium was carried out
biochemically and phenotypically using standard methods
such as colony shape, gram staining, size and color on
nutrient agar plate, motility by the hanging drop method,
oxidase (24 h), ONPG (beta-galactosidase), catalase
production (24 h), ornithine decarboxylase (ODC), arginine
dihydrolase (ADH), lysine decarboxylase (LDC), nitrates
reduction, Methyl red, indole production, Voges-Proskauer
(VP), hydrogen sulfide (H2S), acetate utilization, malonate
utilization, citrate utilization (Simmons), esculin
hydrolysis, tartrate (Jordans), gelatin hydrolysis, urea
hydrolysis, deoxyribonuclease, lipase (corn oil),
phenylalanine deaminase, gas production from glucose and
production of acids from various sugars were carried out
according to the Bergey’s Manual of Determinative
Bacteriology (Holt et al. 1994). Interpretation of the results
was carried out via the ABIS online system (Costin and
Ionut 2015).
Preparation of resting cells for molybdenum reduction
characterization
Characterization works on molybdenum reduction to
Mo-blue such as the effects of pH, temperature, phosphate
and molybdate concentrations were carried out statically
using resting cells in a microplate or microtiter format as
previously developed (Shukor and Shukor 2014).Cells
from a 1 L overnight culture grown in High Phosphate
media (HPM) at room temperature on orbital shaker (150
rpm) with the only difference between the LPM and HPM
was the phosphate concentration which was fixed at 100
mM for the HPM. Cells were harvested by centrifugation at
15,000 x g for 10 minutes and the pellet was washed
several times to remove residual phosphate and
resuspended in 20 mls of low phosphate media (LPM)
minus glucose to an absorbance at 600 nm of
approximately 1.00. In the low phosphate media, a
concentration of 5 mM phosphate was optimal for all of the
Mo-reducing bacteria isolated so far and hence this
concentration was used in this work. Higher concentrations
were found to be strongly inhibitory to molybdate
240
B I O D I V E R S IT A S 16 (2): 238-246, October 2015
reduction (Campbell et al. 1985; Shukor et al. 2007, 2008,
2009a, b,2010a, b; Rahman et al. 2009; Yunus et al.
2009;Lim et al. 2012; Ghani et al. 1993; Abo-Shakeer et al.
2013; Ahmad et al. 2013; Halmi et al. 2013; Othman et al.
2013;Khan et al. 2014). Then 180 µL was sterically
pipetted into each well of a sterile microplate. 20 µL of
sterile glucose from a stock solution was then added to
each well to initiate Mo-blue production. A sterile sealing
tape that allows gas exchange (Corning® microplate) was
used for sealing the tape. The microplate was incubated at
room temperature. At defined times absorbance at 750 nm
was read in a BioRad (Richmond, CA) Microtiter Plate
reader (Model No. 680). The production of Mo-blue from
the media in a microplate format was measured using the
specific extinction coefficient of 11.69 mM.-1.cm-1 at 750
nm as the maximum filter wavelength available for
themicroplate unit was 750 nm (Shukor et al. 2003).
Effect of heavy metals on molybdenum reduction
Seven heavy metals namely lead (ii), arsenic (v), copper
(ii), mercury (ii), silver (i), chromium (vi) and cadmium (ii)
were prepared from commercial salts or from Atomic
Absorption Spectrometry standard solutions from MERCK.
The bacterium was incubated with heavy metals in the
microplate format at various concentrations. The plate was
incubated for 24 hours at 30ºC. The amount of Mo-blue
production was measured at 750 nm as before.
Screening of molybdenum reduction using detergents
and hydrocarbon as source of electron donors and for
growth
The ability of detergents such as Sodium Dodecyl Sulfate
(SDS) and Sodium Dodecyl Benzene Sulfonate (SDBS)
and the hydrocarbon diesel to support growth to support
molybdenum reduction as electron donors was tested using
the microplate format above by replacing glucose from the
low phosphate medium with these xenobiotics at the final
concentration of 200 mg/L for detergents. Diesel was
initially added to the final concentration of 0.5 g/L in 10
mL media and sonicated for 5 minutes. Then 200 uL of the
media was added into the microplate wells. The microplate
was incubated at room temperature for three days and the
amount of Mo-blue production was measured at 750 nm as
before. The ability of these xenobiotics to support bacterial
growth independent of molybdenum reduction was tested
using the microplate format above using the following
media at the final concentration of 200 mg/L. The
ingredients of the growth media (LPM) were as follows:
(NH4)2.SO4 (0.3%), NaNO3 (0.2%), MgSO4.7H2O (0.05%),
yeast extract (0.01%), NaCl (0.5%), Na2HPO4 (0.705% or
50 mM) and 1 mL of trace elements solution. The trace
elements solution composition (mg/L) was as follows:
CaCl2 (40), FeSO4·7H2O (40), MnSO4·4H2O (40),
ZnSO4·7H2O (20), CuSO4·5H2O (5), CoCl2·6H2O (5),
Na2MoO4·2H2O (5). The media was adjusted to pH 7.0.
Then 200 uL of the media was added into the microplate
wells and incubated at room temperature for 72 hours. The
increase of bacterial growth after an incubation period of 3
days at room temperature was measured at 600 nm using
the microplate reader (Bio-Rad 680).
Statistical analysis
Values are means ± SE. Data analyses were carried out
using Graphpad Prism version 3.0 and Graphpad In Stat
version 3.05 available from www.graphpad.com. A
Student's t-test or a one-way analysis of variance with post
hoc analysis by Tukey’s test was employed for comparison
between groups. P < 0.05 was considered statistically
significant.
RESULTS AND DISCUSSION
Identification of molybdenum reducing bacterium
strain AFt-7 was a rod-shaped, non-motile, gramnegative and facultative anaerobe bacterium. The
bacterium was identified by comparing the results of
cultural, morphological and various biochemical tests
(Table 1) to the Bergey’s Manual of Determinative
Bacteriology (Holt et al. 1994) and using the ABIS online
software (Costin and Ionut 2015). The software gave three
suggestions for the bacterial identity with the highest
similarity or homology (96%) and accuracy (100%) as
Klebsiella oxytoca. However, more work in the future
especially molecular identification technique through
comparison of the 16srRNA gene are needed to identify
this species further. However, at this juncture the bacterium
is tentatively identified as Klebsiella oxytoca strain Aft-7.
Previously, two molybdenum-reducing bacterium from this
genus; Klebsiella oxytoca strain Dr.Y14 (Halmi et al. 2013)
and Klebsiella oxytoca strain hkeem (Lim et al. 2012) have
been isolated.
Table 1. Biochemical tests for Klebsiella oxytoca strain Aft-7
Motility
‒
Acid production from:
Pigment
‒
Catalase production (24 h)
+ α-methyl-D-glucoside
Oxidase (24 h)
‒
D-Adonitol
ONPG (beta-galactosidase)
+ L-Arabinose
Arginine dihydrolase (ADH) ‒
Cellobiose
Lysine decarboxylase (LDC) + Dulcitol
Ornithine decarboxylase (ODC) ‒
Glycerol
Nitrates reduction
+ D-Glucose
Methyl red
+ myo-Inositol
Voges-Proskauer (VP)
+ Lactose
Indole production
+ Maltose
Hydrogen sulfide (H2S)
‒
D-Mannitol
Acetate utilization
+ D-Mannose
Malonate utilization
+ Melibiose
Citrate utilization (Simmons) + Mucate
Tartrate (Jordans)
+ Raffinose
Esculin hydrolysis
+ L-Rhamnose
Gelatin hydrolysis
‒
Salicin
Urea hydrolysis
+ D-Sorbitol
Deoxyribonuclease
‒
Sucrose (saccharose)
Lipase (corn oil)
‒
Trehalose
Phenylalanine deaminase
‒
D-Xylose
Note: + positive result,− negative result, d indeterminate result
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
MASDOR et al. –Potential bioremediation of Klebsiella oxytoca
In this work using this bacterium, a rapid and simple
high throughput method involving microplate format was
used to speed up characterization works and obtaining
more data than the normal shake-flask approach (Iyamu et
al. 2008; Shukor and Shukor 2014). The use of resting cells
under static conditions to characterize molybdenum
reduction in bacterium was initiated by (Ghani et al. 1993).
Resting cells have been used in studying heavy metals
reduction such as in selenate (Losi and Frankenberger
1997), chromate (Llovera et al. 1993), vanadate (Carpentier
et al. 2005) reductions and xenobiotics biodegradation such
as diesel (Auffret et al. 2014), SDS (Chaturvedi and Kumar
2011), phenol (Sedighi and Vahabzadeh 2014), amides
(Raj et al. 2010) and pentachlorophenol (Steiert et al.
1987).
Molybdenum absorbance spectrum
The absorption spectrum of Mo-blue produced by
Klebsiella oxytoca strain Aft-7 exhibited a shoulder at
approximately 700 nm and a maximum peak near the infrared region of between 860 and 870 nm with a median at
865 nm (Figure 2). The identity of the Mo-blue is not easily
ascertained as it is complex in structure and has many
species (Shukor et al.2007). Briefly It has been suggested
by Campbell et al. (Campbell et al. 1985) that the Mo-blue
observed in the reduction of molybdenum by Escherichia
coli K12 is a reduced form of phosphomolybdate but did
not provide a plausible mechanism. The Mo-blue spectrum
from the phosphate determination method normally showed
a maximum absorption around 880 to 890 nm and a
shoulder around 700 to 720 nm (Hori et al. 1988). This
spectrum is different from other heteropolymolybdates
such as molybdosilicate and molybdosulphate (Figure 3).
We hypothesize that microbial molybdate reduction in
media containing molybdate and phosphate must proceed
via the phosphomolybdate intermediate and the conversion
from molybdate to this structure occurs due to the
reduction of pH during bacterial growth, in other words the
reduction of molybdenum to Mo-blue requires both
chemical and biological processes. The absorption
spectrum of the Mo-blue from this bacterium if it goes
through this mechanism should show a spectrum closely
resembling the phosphate determination method. To be
exact, the spectrum observed showed a maximum
absorption in between 860 and 870 nm and a shoulder at
approximately 700 nm. We have shown previously that the
entire Mo-blue spectra from other bacteria obey this
requirement (Shukor et al. 2007). In this work the result
from the absorption spectrum clearly implies a similar
spectrum and thus provides evidence for the hypothesis.
Exact identification of the phosphomolybdate species must
be carried out using n.m.r and e.s.r. due to the complex
structure of the compound (Chae et al. 1993). However,
spectrophotometric characterization of heteropolymolybdate species via analyzing the scanning spectroscopic
profile is a less cumbersome and accepted method (Glenn
and Crane 1956; Sims 1961; Kazansky and Fedotov 1980;
Yoshimura et al. 1986). Although the maximum absorption
wavelength for Mo-blue was 865 nm, measurement at 750
nm, although was approximately 30% lower, was enough
241
for routine monitoring of Mo-blue production as the
intensity obtained was much higher than cellular absorption
at 600-620 nm (Shukor and Shukor 2014). Previous
monitoring of Mo-blue production uses several
wavelengths such as 710 nm (Ghani et al. 1993) and 820
nm (Campbell et al. 1985).
Effect of pH and temperature on molybdate reduction
Klebsiella oxytoca strain Aft-7 was incubated at
different pH ranging from 5.5 to 8.0 using Bis-Tris and
Tris.Cl buffers (20 mM). Analysis by ANOVA showed that
the optimum pH for reduction was between 5.8 and 6.3.
Inhibition of reduction was dramatic at pH lower than 5
(Figure 4). The effect of temperature (Figure 5) was
observed over a wide range of temperature (20 to 60°C)
with an optimum temperature ranging from 25 to 34°C
with no significant different (p>0.05) among the values
measured as analyzed using ANOVA. Temperatures lower
than 30°C and higher than 37oC were strongly inhibitory to
Mo-blue production from Klebsiella oxytoca strain Aft-7.
Temperature and pH play important roles in
molybdenum reduction, since this process is enzyme
mediated, both parameters affect protein folding and
enzyme activity causing the inhibition of molybdenum
reduction. The optimum conditions would be an advantage
for bioremediation in a tropical country like Malaysia
which have average yearly temperature ranging from 25 to
35oC (Shukor et al. 2008). Therefore, Klebsiella oxytoca
strain Aft-7 could be a candidate for soil bioremediation of
molybdenum locally and in other tropical countries. The
majority of the reducers shows an optimal temperature of
between 25 and 37ºC (Shukor et al. 2008, 2009a,2010a, b,
2014; Rahman et al. 2009; Yunus et al. 2009; Lim et al.
2012; Abo-Shakeer et al. 2013; Othman et al. 2013; Halmi
et al. 2013; Khan et al. 2014)as they are isolated from
tropical soils with the only psychrotolerant reducer isolated
from Antartica showing an optimal temperature supporting
reduction of between 15 and 20ºC (Ahmad et al. 2013).
The optimal pH range exhibited by Klebsiella oxytoca
strain Aft-7 for supporting molybdenum reduction reflects
the property of the bacterium as a neutrophile. The
characteristics neutrophiles are their ability to grow
between pH 5.5 and 8.0. An important observation
regarding molybdenum reduction in bacteria is the optimal
pH reduction is slightly acidic with optimal pHs ranging
from pH 5.0 to 7.0 (Campbell et al. 1985; Ghani et al.
1993; Shukor et al. 2008,2009a,2010a, b,2014; Rahman et
al. 2009; Lim et al. 2012; Abo-Shakeer et al. 2013; Ahmad
et al. 2013; Halmi et al. 2013; Othman et al. 2013; Khan et
al. 2014). It has been suggested previously that acidic pH
plays an important role in the formation and stability of
phosphomolybdate before it is being reduced to Mo-blue.
Thus, the optimal reduction occurs by balancing between
enzyme activity and substrate stability (Shukor et al. 2007).
Effect of electron donor on molybdate reduction
Among the electron donor tested, glucose was the best
electron donor for supporting molybdate reduction
followed by sucrose, D-mannitol, D-sorbitol, lactose,
salicin, trehalose, maltose and myo-Inositol in descending
242
B I O D I V E R S IT A S 16 (2): 238-246, October 2015
order (Figure 6). Other carbon sources did not support
molybdenum reduction. Previous works by Shukor et al.
demonstrated that several of Mo-reducing bacteria such as
Enterobacter cloacae strain 48 (Ghani et al. 1993), Serratia
sp. strain Dr.Y5 (Rahman et al. 2009), S. marcescens strain
Dr.Y9 (Yunus et al. 2009) and Serratia marcescens strain
DRY6 (Shukor et al. 2008) showed sucrose as the best
carbon source. Other molybdenum reducers such as
Escherichia coli K12 (Campbell et al. 1985), Serratia sp.
strain Dr.Y5 (Rahman et al. 2009), Pseudomonas sp. strain
DRY2 (Shukor et al. 2010a), Pseudomonas sp. strain
DRY1 (Ahmad et al. 2013), Enterobacter sp. strain Dr.Y13
(Shukor et al. 2009a), Acinetobacter calcoaceticus strain
Dr.Y12 (Shukor et al. 2010b), Bacillus pumilus strain lbna
(Abo-Shakeer et al. 2013) and Bacillus sp. strain A.rzi
(Othman et al. 2013) prefer glucose as the carbon source
while Klebsiella oxytoca strain hkeem prefers fructose
(Lim et al. 2012). In the presence of carbon sources in the
media, the bacteria could produce electron donating
substrates, NADH and NADPH thorough metabolic
pathways such as glycolysis, Kreb’s cycle and electron
transport chain. Both NADH and NADPH are responsible
as the electron donating substrates for molybdenum
reducing-enzyme (Shukor et al. 2008,2014).
Effect of phosphate and molybdate concentrations to
molybdate reduction
The determination of phosphate and molybdate
concentrations supporting optimal molybdenum reduction
is important because both anions have been shown to
inhibit Mo-blue production in bacteria (Shukor et al. 2008,
2009b, 2010a, 2014; Yunus et al. 2009; Lim et al. 2012;
Ahmad et al. 2013; Othman et al. 2013). The optimum
concentration of phosphate occurred between 5.0 and 7.5
mM with higher concentrations were strongly inhibitory to
reduction (Figure 7). High phosphate was suggested to
inhibit phosphomolybdate stability as the complex requires
acidic conditions of which the higher the phosphate
concentration the stronger buffering power of the
phosphate buffer used. In addition, the phosphomolybdate
complex itself is unstable in the presence of high phosphate
through an unknown mechanism (Glenn and Crane 1956;
Sims 1961; Shukor et al. 2000). All of the molybdenumreducing bacterium isolated so far requires phosphate
concentration not higher than 5 mM for optimal reduction
(Campbell et al. 1985; Ghani et al. 1993; Shukor et al.
2008, 2009b, 2010a, b, 2014; Rahman et al. 2009; Lim et
al. 2012; Abo-Shakeer et al. 2013; Ahmad et al. 2013;
Halmi et al. 2013; Othman et al. 2013; Khan et al. 2014).
Studies on the effect of molybdenum concentration on
molybdenum reduction showed that the newly isolated
bacterium was able to reduce molybdenum as high as 60
mM but with reduced Mo-blue production. The optimal
reduction range was between 5 and 20 mM (Figure 8).
Reduction at this high concentration into an insoluble form
would allow the strain to reduce high concentration of
molybdenum pollution. The lowest optimal concentration
of molybdenum reported is 15 mM in Pseudomonas sp
strain Dr.Y2 (Shukor et al. 2010a), whilst the highest
molybdenum required for optimal reduction was 80 mM in
Escherichia coli K12 (Campbell et al. 1985) and Klebsiella
oxytoca strain hkeem (Lim et al. 2012). Other Mo-reducing
bacteria such as EC48 (Ghani et al. 1993), S. marcescens
strain Dr.Y6 (Shukor et al. 2008), S. marcescens. Dr.Y9
(Yunus et al. 2009), Pseudomonas sp. strain Dr.Y2 (Shukor
et al. 2010a), Serratia sp. strain Dr.Y5 (Rahman et al.
2009), Enterobacter sp. strain Dr.Y13 (Shukor et al. 2009a)
and Acinetobacter calcoaceticus (Shukor et al. 2010b)
could produce optimal Mo-blue using the optimal
molybdate concentrations at 50, 25, 55, 30, 30, 50 and 20
mM, respectively. In fact the highest concentration of
molybdenum as a pollutant in the environment is around
2000 ppm or about 20 mM (Runnells et al. 1976).
Effect of heavy metals
Molybdenum reduction was inhibited by mercury (ii),
silver (i) and copper (ii) at 2 ppm by 62.1, 33.9 and 33.6%,
respectively (Figure 9). The inhibition effects by others
metal ions and heavy metals present a major problem for
bioremediation. Therefore it is important to screen and
isolate bacteria with as many metal resistance capability.
As described previously (Shukor et al. 2002), mercury is a
physiological inhibitor to molybdate reduction. A summary
Table 2. Inhibition of Mo-reducing bacteria by heavy metals
Bacteria
Heavy metals that inhibit reduction
Author
Acinetobacter calcoaceticus strain Dr.Y12
Bacillus pumilus strain lbna
Bacillus sp. strain A.rzi
Enterobacter cloacae strain 48
Enterobacter sp. strain Dr.Y13
Escherichia coli K12
Klebsiella oxytoca train hkeem
Pseudomonas sp. strain DRY1
Pseudomonas sp. strainDRY2
Serratia marcescens strain Dr.Y9
Serratia marcescens strain DRY6
Serratia sp. strain Dr.Y5
Serratia sp. strain Dr.Y8
Cd2+, Cr6+, Cu2+, Pb2+, Hg2+
As3+, Pb2+, Zn2+, Cd2+, Cr6+, Hg2+, Cu2+
Cd2+, Cr6+, Cu2+,Ag+, Pb2+, Hg2+, Co2+,Zn2+
Cr6+, Cu2+
Cr6+, Cd2+, Cu2+, Ag+, Hg2+
Cr6+
Cu2+, Ag+, Hg2+
Cd2+, Cr6+, Cu2+,Ag+, Pb2+, Hg2+
Cr6+, Cu2+, Pb2+, Hg2+
Cr6+, Cu2+, Ag+, Hg2+
Cr6+, Cu2+, Hg2+*
n.a.
Cr, Cu, Ag, Hg
Shukor et al. (2010b)
Abo-Shakeer et al. (2013)
Othman et al. (2013)
Ghani et al. (1993)
Shukor et al. (2009a)
Campbell et al. (1985)
Lim et al. (2012)
Ahmad et al. (2013)
Shukor et al. (2010a)
Yunus et al. (2009)
Shukor et al. (2008)
Rahman et al. (2009)
Shukor et al. (2009b)
MASDOR et al. –Potential bioremediation of Klebsiella oxytoca
3.0
48 h
243
0.5
0.3
C
32 h
B
Abs
Abs
0.25
0.3
0.2
Abs
0.4
24 h
2.0
A
8h
1.0
0.2
0.15
0.1
0.0
600
650
700
750
800
850
900
0.1
500
950
600
700
800
900
1000
Wavelength
(nm)
Wavelength (nm)
Figure 2. Scanning absorption spectrum of Mo-blue from
Klebsiella oxytoca strain Aft-7 at different time intervals.
Figure 3. Scanning spectra of Mo-blue from molybdosilicate (A),
molybdosulphate (B) and molybdophosphate (C) (from Shukor et
al. 2000).
3.0
3.0
2.0
2.0
Abs 750 nm
Abs 750 nm
Wavelength (nm)
1.0
1.0
0.0
0.0
5.5
6.0
6.5
7.0
7.5
10
8.0
20
30
40
50
60
o
Temperature ( C)
pH
Figure 4. Effect of pH on molybdenum reduction by Klebsiella
oxytoca strain Aft-7. Resting cells of the bacterium were
incubated in a microtiter plate under optimized conditions for 48
hours. Error bars represent mean ± standard deviation (n=3).
Figure 5. Effect of temperature on molybdenum reduction by
Klebsiella oxytoca strain Aft-7. Resting cells of the bacterium
were incubated in a microtiter plate under optimized conditions
for 48 hours. Error bars represent mean ± standard deviation
(n=3).
Abs 750 nm
3.0
2.0
1.0
0.0
Control
D-Xylose
Sucrose
Trehalose
Salicin
D-Sorbitol
Raffinose
L-Rhamnose
Melibiose
D-Mannitol
D-Mannose
Lactose
Maltose
D-Glucose
myo-Inositol
Dulcitol
Glycerol
Cellobiose
L-Arabinose
l
l
l
l
l
l
e
e
e
se it o
ro os e si to ose tose ni to os i ose ose os l ic in bito ose los e os e tro
c
os bi o
l
n
t
n
r
l
n
c
r
n
o
ce
n
b
ul
in
o
Sa -So uc e ha -Xy Co
an el i affi am
D Gly Gl u o-In Lac Ma -Ma
ab el l
S
r
M
r
h
M
D
R R
D
C
T
A
D DD my
LL-
Figure 6. Effect of different electron donor sources (1% w/v) on
molybdenum reduction. Klebsiella oxytoca strain Aft-7 was
grown in low phosphate media containing 10 mM molybdate and
various electron donors. Resting cells of the bacterium were
incubated in a microtiter plate under optimized conditions for 48
hours. Error bars represent mean ± standard deviation (n = 3).
Figure 9. Effect of metals on Mo-blue production by Klebsiella
oxytoca strain Aft-7. Resting cells of the bacterium were
incubated in a microtiter plate under optimized conditions for 48
hours. Error bars represent mean ± standard deviation (n = 3).
244
2.0
2.0
1.5
1.5
Abs 750 nm
Abs 750 nm
B I O D I V E R S IT A S 16 (2): 238-246, October 2015
1.0
0.5
0.5
0.0
0.0
0
10
20
30
40
0
50
Figure7. Effect of phosphate concentration on molybdenum
reduction by Klebsiella oxytoca strain Aft-7. Resting cells of the
bacterium were incubated in a microtiter plate under optimized
conditions for 48 hours. Error bars represent mean ± standard
deviation (n = 3).
1.2
1.0
0.8
0.6
0.4
0.2
Crude petroleum
se
l
pe
tr
ol
Diesel
eu
m
de
C chloride
D
Benzalkonium
ru
ie
on
SD
iu
S
m
en
ch
za
loSDS
lk
rid
on
e
iu
m
ch
lo
Benzethonium chloride
ri
de
B
S
B
SDBS
en
ze
th
SD
Witconol 2301
B
on
tr
ol
Te
rg
Control
ito
lN
P
Te
9
rg
ito NP9
Tergitol
l1
5S
W
9
itc
Tergitolon15
ol S9
23
01
0.0
C
20
40
60
80
Molybdate (mM)
Phosphate (mM)
Abs 600 nm
1.0
Figure 10. Growth of Klebsiella oxytoca strain Aft-7 on
xenobiotics independent of molybdenum reduction. Resting cells
of the bacterium were incubated in a microtiter plate under
optimized conditions for 48 hours. Error bars represent mean ±
standard deviation (n = 3).
of the type of heavy metals that inhibited Mo-reducing
bacteria showed that almost all of the reducers are inhibited
by toxic heavy metals (Table 2). Heavy metals such as
mercury, cadmium, silver and copper usually target
sulfhydryl group of enzymes (Sugiura and Hirayama 1976).
Chromate is known to inhibit certain enzymes such as
glucose oxidase (Zeng et al. 2004) and enzymes of nitrogen
metabolism in plants (Sangwan et al. 2014). Binding of
heavy metals inactivated metal-reducing capability of the
enzyme(s) responsible for the reduction.
Detergents and hydrocarbons as electron donor sources
for molybdenum reduction and independent growth
Screening of detergents and hydrocarbons as electron
donors supporting molybdenum reduction failed to give
Figure 8. Effect of molybdate concentration on molybdenum
reduction by Klebsiella oxytoca strain Aft-7. Resting cells of the
bacterium were incubated in a microtiter plate under optimized
conditions for 48 hours. Error bars represent mean ± standard
deviation (n = 3).
positive results (data not shown). However, the bacterium
was able to grow on the detergent SDS (Figure 10). SDSdegrading bacteria are ideal for SDS remediation due to
economic factors. Microbes are known for their ability to
degrade organics including SDS and their use as
bioremediation agents is important for economical removal
of xenobiotic pollutants (Syed et al. 2010). Biodegradation
of anionic surfactant under aerobic conditions by the
bacterium Pseudomonas sp. strain C12B was among the
first to be studied (Payne and Feisal 1963), and to date
quite a number of SDS-degrading bacteria have been
isolated and characterized (Thomas and White 1990; Lee et
al. 1995; Kostal et al. 1998; Roig et al. 1998; Dhouib et al.
2003; Ke et al. 2003; Yin et al. 2005; Wu 2006; Khleifat et
al. 2010; Asok and Jisha 2013; Chaturvedi and Kumar
2013,2014; Cortés-Lorenzo et al. 2013; Halmi et al. 2013;
Venkatesh 2013; Yilmaz and Icgen 2014). Works on coldadapted microbes with ability to degrade SDS are rare, and
were first reported by Margesin and Schinner (1998).The
existence of multitude of bacteria with detergent-degrading
ability makes bioremediation the more ideal method for
detergent degradation. However, very few bacteria have
been reported to be able to degrade xenobiotics and
detoxify heavy metals, and the ability of this bacterium to
do both suggest that this bacterium will be very useful as a
bioremediation agent in polluted sites co-contaminated
with xenobiotics and heavy metals.
In conclusion, a local isolate of Mo-reducing bacterium
with the novel ability to degrade SDS has been isolated.
This is the first report of a molybdenum reducing
bacterium with the ability to degrade SDS. The ability of
this bacterium to detoxify multiple toxicants is a sought
after property, and this makes the bacterium an important
tool for bioremediation. Currently, work is underway to
purify the molybdenum-reducing enzyme from this
bacterium and to characterize the detergent degradation
studies in more detail.
MASDOR et al. –Potential bioremediation of Klebsiella oxytoca
ACKNOWLEDGEMENTS
This project was supported by funds from Snoc
International Sdn. Bhd., Malaysia
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160220
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 247-253
Assessment of genetic diversity among soursop (Annona muricata)
populations from Java, Indonesia using RAPD markers
SURATMAN♥, ARI PITOYO, SRI MULYANI, SURANTO
Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36A Surakarta 57126, Central Java,
Indonesia. Tel./Fax. +62-271-663375, email: suratmanmipauns@yahoo.com
Manuscript received: 21 April 2015. evision accepted: 19 September 2015.
Abstract. Suratman, Ari Pitoyo, Sri Mulyani, Suranto. 2015. Assessment of genetic diversity among soursop (Annona muricata)
populations from Java, Indonesia using RAPD markers. Biodiversitas 16: 247-253. The objective of this study was to determine genetic
diversity of the soursop (Annona muricata L.) populations from Java (Indonesia) using RAPD markers. A total of 70 individuals were
collected from 7 soursop populations, distributed along the geographical range of natural distribution in Java. Genetic diversity was
estimated by RAPD technique using 6 arbitrary selected primers. Those primers produced 151 polymorphic bands with the percentage
of polymorphism for each primer ranged from 95% to 100%. The genetic diversity value (h) within each population ranged from 0.0418
to 0.0525. The highest h value (0.0525) was found in the KRA population whereas the lowest h value was observed in the PCT
population. The highest genetic distance value (0.0410) was observed in SKH-GKD populations pair whereas the lowest genetic
distance value (0.02448) was estimated in KRA-PCT populations pair. Based on the dendogram, the seven soursop populations were
segregated into three major clusters. The first cluster consisted of SKH, KRA, PCT, NGW, and KPG populations. The GKD population
was then grouped into second cluster. In the third cluster, the BGR population was grouped separately and more genetically distant than
the others. However, the relationship dendrogram showed that the grouping did not always indicate the geographical origins similarity,
but possibly showed the genetic similarity.
Key words: assessment, genetic diversity, Java, RAPD, soursop
INTRODUCTION
Soursop (Annona muricata L.) is a species of the
genus Annona belonging to family Annonaceae, which is
known mostly for its edible fruit. Soursop is distinguished
by its conspicuous spiny fruit and its obovate leaves with
domatia on the underside (Kerrigan et al. 2011). Soursop is
native to the Caribbean and Central America but is now
widely distributed in the tropics and subtropics of the
world, so it can be found in the West Indies, North and
South America, lowlands of Africa and Pacific islands
(Badrie and Schauss 2009). Today the soursop has spread
throughout the world so it is also grown in Southeast Asia
included Malaysia and Indonesia (Hasni 2009).
This species was recorded not only useful in the food
industries but also for medical purposes. Soursop can be
eaten freshly and also used as raw material for puree, juice,
jam, jelly, powder fruits bars and flakes. It is also excellent
for making refreshing drinks, sherbets and flavoring/
topping for ice-cream and dessert (Bates et al. 2001; Abbo
et al. 2006; Hasni 2009). For medical purpose, soursop is
used as an antispasmodic, emetic, and sudorific in herbal
medicine. A decoction of the leaves is used to kill head lice
and bed bugs while a tea from the leaves is well known to
have sedative properties. The juice of the fruit is taken
orally for hematuria, liver complaints, and urethritis
(Badrie and Schauss 2009).
Information about the extent of genetic diversity of
soursop in Java (Indonesia) is still incompletely
understood. Better understanding of genetic diversity and
its distribution is essential for rational utilization of
germplasm in crop improvement (Piyasundara et al. 2009).
Evaluation of genetic diversity among various accessions
of crop species is fundamental for plant breeding programs
(Tanksley 1983; Ikbal et al. 2010). This information can
provide a predictive estimation of genetic variation within
crop species thus facilitating breeding material selection
(Qi et al. 2008).
A variety of molecular, chemical and morphological
markers are used to characterize the genetic diversity
among and within crop species (Ozkaya et al. 2006).
Morphological markers are routinely used for estimating
genetic diversity but are not successful due to strong
influence of environment. Hence, the use of molecular
markers has complemented the classical strategies
(Tanksley 1983). Molecular markers provide a better
estimation of genetic diversity than morphological marker
that is dependent of effect of various environmental factors.
Various molecular markers techniques based on
Polymerase Chain Reaction (PCR) amplification have
become increasingly important at the study of genetic
diversity. Random Amplified Polymorphic DNA (RAPD)
is one of various approaches which available for DNA
fingerprinting (Lakshmi et al. 2011).
The development of RAPD markers, generated by the
PCR using arbitrary primers, has provided a rapid and easy
tool for the detection of DNA polymorphism. RAPD assay
is one of the suitable methods for identifying the genotypes
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16 (2): 247-253, October 2015
within a short period. The DNA amplification with RAPD
does not require any previous knowledge of natural target
DNA sequence, any digestion by restriction enzymes or
radioactive probes. This technique also has been widely
used to ascertain the genetic diversity in many crops by
high levels of polymorphism with only limited amount of
genomic DNA (Williams et al. 1990). It has been proved
that RAPD can be used as an efficient tool for genetic
characterization of many plant species (Hu et al. 1999).
The use of RAPD markers in the applied breeding
programs can facilitate plant breeders to identify
appropriate parents involved for crosses (Abd El-Hady et
al. 2010).
The objective of this research was to determine genetic
diversity of the soursop populations in Java (Indonesia)
using RAPD markers. This is the first report on the
assessment of genetic diversity in seven soursop
populations originating from Java (Indonesia) using RAPD
markers. Thus, information about genetic diversity through
RAPD markers obtained in this study could be valuable for
breeding strategies of soursop in Java.
MATERIALS AND METHODS
Plant materials
A total of 70 individuals were collected from 7 soursop
populations, distributed along the geographical range of
natural distribution of A. muricata in Java (Table 1 and
Figure 1). The material collected consisted of young and
healthy leaves of each individual, which were placed in
plastic bags and kept in ice box, while transported to the
laboratory. In laboratory, samples were subsequently stored
at-20°C until DNA extraction.
DNA extraction
Genomic DNA was extracted by the CTAB extraction
procedure (Dellaporta et al. 1983) with some
modifications. The extracted DNA was quantified in a
spectrophotometer measuring absorbance at 260 and 280
nm. The integrity of the DNA was determined by
electrophoresis on a 1% (w/v) agarose gel and
photographed under ultraviolet (UV) light. The extracted
DNA was then kept at-20°C for RAPD analysis.
Table 1. The location of soursop (Annona muricata L.) populations studied in Java with climatic data for each locality.
Population
Code
Sukoharjo, Central Java
Karanganyar, Central Java
Kulonprogo, Yogyakarta
Gunungkidul, Yogyakarta
Ngawi, East Java
Pacitan, East Java
Bogor, West Java
SKH
KRA
KPG
GKD
NGW
PCT
BGR
Latitude and longitude
S 07041’22,1”, E 110054’33,5”
S 07041’28,16”, E 110054’38,5”
S 07043’24,8”, E 110046’28,6”
S 07089’23,2”, E 110071’29,7”
S 07032’67,1”, E 111012’05,7”
S 08008’94,4”, E 111001’38,7”
S 06020’58”, E 106004’68”
Altitude
(m a.s.l.)
148
617
77,3
235
735
581
155
Temp.
(oC)
34
31
32
37,5
32
34
33
Humidity
(%)
58
59
62
49
42
55
60
Light intensity
(lux)
358
5.153
2.592
16.900
4.156
17.240
237
BGR
NGW
KRA
SKH
KPG
GKD
PCT
Figure 1. Map of the collection areas for 7 natural populations of soursop (Annona muricata L.) in Java. Note: SKH: Sukoharjo, KRA:
Karanganyar, NGW: Ngawi, PCT: Pacitan, GKD: Gunung Kidul, KPG: Kulonprogo and BGR: Bogor
SURATMAN et al. – Genetic diversity of soursop based on RAPD marker
RAPD analysis
RAPD reaction and procedures through PCR were
carried out as described by Williams et al. (1990). Each
amplification reaction was conducted with one unique
primer. A total of 15 RAPD primers were purchased from
commercial source (1st BASE Custom Oligos, Singapore)
and screened initially to find specific diagnostic markers in
the tested soursop populations (Table 2).
Electrophoresis
The amplified PCR products were separated by
electrophoresis on a 1.5% (w/v) agarose gel in 1×TAE
buffer at 80 V for 2 h. A 100 bp DNA ladder (Geneaid
Biotech Ltd, Taiwan) was included in all gels as a
reference, to estimate the size of the amplified bands. Gels
were then stained with 0.5 µg/mL ethidium bromide and
amplification products were visualized and photographed
under UV light. The obtained profile image was then saved
on a magnetic disc.
Data analysis
All distinct bands (RAPD markers) were analyzed
according to their position on gel and visually scored on the
basis of their presence (1) or absence (0), separately for
each individual and each primer. The scores obtained using
selected primers initially in the RAPD analysis were then
pooled to create a single data matrix. In order to estimate
polymorphic loci, genetic diversity, and genetic distance
(Nei 1978), a computer program, POPGENE (Version
1.31), was used in this study (Yeh et al. 1999, Ghosh et al.
2009). A dendrogram among populations was constructed
based on the genetic distance matrix by applying an
Unweighted Pair Group Method with Arithmetic Averages
(UPGMA) cluster analysis using a computer program,
Numerical Taxonomy and Multivariate Analysis System
(NTSYS) Version 2.00 (Rohlf 1998).
RESULTS AND DISCUSSION
A total of 15 primers were screened initially for their
ability to generate amplified band patterns and to assess
polymorphism in the tested populations (Table 2).
Arbitrary primers used in the present study were 10 bp in
size, had a GC content of 60% to 70% and did not contain
any palindromic sequence.
Six random primers (A18, A20, P04, P06, P10, P11)
gave optimal results, which yielded comparatively
maximum number of amplification products with high
intensity and minimal smearing, good resolution and also
clear bands. The selected primers were then used to
produce RAPD profiles for the references soursop
populations. DNA bands resulted by PCR amplification
using one of the selected RAPD primers in this study were
shown in Figure 1.
Six random primers that we used generated a number of
amplified DNA bands. A total of 152 bands were
generated, ranging 23 to 31 bands per primer,
corresponding to an average of 25.3 bands per primer. The
size of bands ranged between approximately 200 and 2950
249
bp. Six random primers also produced 151 polymorphic
bands, with an average of 25.2 polymorphic bands per
primer. The degree of polymorphism within each
population varied depending on the primer tested. The
percentage of polymorphism for each primer ranged from
95% to 100% with an average of 99.3% polymorphism per
primer. In all 5 primers (A20, P04, P06, P10, P11)
produced 100% polymorphism while primer A18 showed
least polymorphism (95.8%) (Table 3). The obtained data
indicates that all selected random primers produced high
level of polymorphism.
As a comparison, Cota et al. (2011) used 15 selected
primers to evaluate genetic diversity of Annona crassiflora.
These primers generated 140 bands of which 123 (87.8%)
were polymorphic. In contrast, Ronning et al. (1995)
reported 15 selected primers to analyze genetic variation
between A. cherimola, A. squamosa and their hybrids. A
total of 92 bands were produced while 86 bands (93.5%)
were polymorphic. Although our study used a lower
number of selected primers compared than Ronning et al.
(1995) and Cota et al. (2011) but the total bands produced
(152) and the average of percentage of polymorphism
(99.3%) were comparable. In all cases, both the total bands
produced and the average of percentage of polymorphism
of Ronning et al. (1995) and Cota et al. (2011) were a bit
lower than our results. Thus, the number of random primers
and polymorphic bands generated were not similar range for
species. They can vary significantly in different plant species.
According to Upadhyay et al. (2004), this is
understandable that product amplification depends upon the
sequence of random primers and their compatibility within
genomic DNA. The number of markers detected by each
primer depends on primer sequence and the extent of
genetic variation, which is genotype specific. Poerba and
Martanti (2008) described that each primer has its own
attaching site, so the DNA band resulted by each primer
was different, both in size of multiple basa pairs and in
amount of DNA bands. Due to this fact, the selection of
primers in RAPD analysis then affected the resultant band
polymorphism.
Table 2. Parameters of the random primers used in the present
study for initial screening.
Nucleotide
GC
References
sequences
content
(5’-3’)
(%)
AGTCAGCCAC
60
Ronning et al. 1995
A3
AGGTGACCGT
60
Ronning et al. 1995
A18*
GTTGCGATCC
60
Ronning et al. 1995
A20*
CCACAGCAGT
60
Ronning et al. 1995
B18
AAAGCTGCGG
60
Ronning et al. 1995
C11
GTGTGCCCCA
70
Ronning et al. 1995
D8
AGCGCCATTG
60
Ronning et al. 1995
D11
AGATGCAGCC
60
Cota et al. 2011
P02
ATGGCTCAGC
60
Cota et al. 2011
P03
CAGGCCCTTC
70
Cota et al. 2011
P04*
CTCTTGGGCT
60
Cota et al. 2011
P05
ACCACCCGCT
70
Cota et al. 2011
P06*
ACCCCCACAC
70
Cota et al. 2011
P07
GGCTCATGTG
60
Cota et al. 2011
P10*
GTCAGGGCAA
60
Cota et al. 2011
P11*
Note: * Selected for RAPD analysis for all samples of the seven
soursop populations in Java
Primers
code
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16 (2): 247-253, October 2015
SKH
KRA
M 1 2 3 4 5 6 7 8 9 10
NGW
M 1 2 3 4 5 6 7 8 9 10 M 1 2 3 4 5 6 7 8 9 10
3 kb
1.5 kb
1 kb
0.5 kb
0.1 kb
PCT
GKD
M 1 2 3 4 5 6 7 8 9 10
M 1 2 3 4 5 6 7 8 9 10
KPG
M 1 2 3 4 5 6 7 8 9 10
3 kb
1.5 kb
1 kb
0.5 kb
0.1 kb
BGR
M 1 2 3 4 5 6 7 8 9 10
3 kb
1.5 kb
1 kb
0.5 kb
0.1 kb
Figure 1. Profile of the RAPD bands amplified by primer P04 for soursop (Annona muricata L.) individuals belonging to seven
populations in Java. M indicated as Marker (100 bp Ladder) and the number well (1 to 10) in each population indicated number of
soursop samples. Note: SKH: Sukoharjo, KRA: Karanganyar, NGW: Ngawi, PCT: Pacitan, GKD: Gunung Kidul, KPG: Kulonprogo
and BGR: Bogor
Table 3. Numbers of bands and polymorphic bands, their size
range and percentage of polymorphism detected by 6 selected
RAPD primers
Primers
Number
of bands
A18
A20
P04
P06
P10
P11
Total
Average
24
23
27
31
23
24
152
25.3
Size
range
(bp)
350-2820
500-2950
400-2950
400-2840
200-2040
400-2820
Number of
polymorphic
bands
23
23
27
31
23
24
151
25.2
Polymorphism
(%)
95.8
100
100
100
100
100
595.8
99.3
Results showed that each soursop population collected
from different localities in Java seemed to have variability
in RAPD profiles by using different primers. All RAPD
primers selected in this study also showed more than 90%
of polymorphism. Percent polymorphism reflects the
number of total bands from each primer that distinguish at
least one individual. Polymorphism detected by RAPD was
determined by the different DNA sequence of the sites,
which the primer bound (Lay et al. 2001). It is generally
reported that polymorphism between populations also can
arise through nucleotide changes that prevent amplification
by introducing a mismatch at one priming site; deletion of a
priming site; insertions that render priming site too distant
to support amplification and insertions or deletions that
change the size of the amplified product (Powell et al. 1996).
SURATMAN et al. – Genetic diversity of soursop based on RAPD marker
251
Table 5. Summary of Nei’s (1978) genetic distance values (below diagonal) between soursop population in Java
Population
SKH
KRA
NGW
PCT
GKD
KPG
BGR
SKH
****
KRA
0.0330
****
NGW
0.0367
0.0298
****
PCT
0.0288
0.0260
****
0.02448
GKD
0.0366
0.0391
0.0316
****
0.0410
KPG
0.0347
0.0293
0.0335
0.0269
0.0351
****
BGR
0.0406
0.0389
0.0382
0.0343
0.0400
0.0323
****
Note: SKH: Sukoharjo, KRA: Karanganyar, NGW: Ngawi, PCT: Pacitan, GKD: Gunung Kidul, KPG: Kulonprogo and BGR: Bogor
Cluster I
0.02448
0.0279
0.0333
0.0299
0.03668
Cluster II
0.03738
Cluster III
Figure 3. Dendrogram produced using UPGMA cluster analysis based on Nei’s (1978) genetic distance indicating genetics relatedness
among 7 soursop populations in Java using 6 selected RAPD primers. The average of genetic distance value of the nodes are indicated
Table 4. Genetic diversity on soursop population in Java using 6
selected RAPD primers
Code
Population
h ± SD
SKH
KRA
NGW
PCT
GKD
KPG
BGR
Sukoharjo
0.0454 ± 0.1105
Karanganyar
0.0525 ± 0.1095
Ngawi
0.0440 ± 0.1158
Pacitan
0.0418 ± 0.1079
Gunung Kidul
0.0439 ± 0.1184
Kulonprogo
0.0474 ± 0.1162
Bogor
0.0459 ± 0.1128
Average
0.0458 ± 0.1130
Note: h: gene diversity (Nei 1978); SD: Standard Deviation
Genetic diversity measures were calculated according
to Nei’s index using POPGENE software and results were
depicted in the Table 4. Within each population, the genetic
diversity was limited, as indicated by the genetic diversity
value (h) that ranged from 0.0418 to 0.0525 with an
average of 0.0458 per population. The lowest genetic
diversity value (0.0418) was observed in population
Pacitan (PCT) whereas the highest genetic diversity value
(0.0525) was found in population Karanganyar (KRA).
According to Stansfield (1991), the intra-population
genetic diversity value (h) was considered low if the value
of h was below point two (<0.2). Thus, the soursop
populations originated from Java was exhibited low levels
of genetic diversity within populations. This occurrence
may possibly due to the very limited chances of gene flows
among populations.
The existence of low genetic diversity within soursop
population possibly has been mostly attributed to self
pollination (Archak et al. 2002). Although soursop flowers
are apparently adapted to cross pollination despite being
anatomically hermaphrodite, the bunched arrangement of
stamens does not result in available fertile pollen. There is
a period from 36 to 48 hours in which both sexual organs
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16 (2): 247-253, October 2015
are ripe simultaneously. However, soursop flowers function
as physiologically protogyneous where its pistil and pollen
are not ripen in the same time. No insect genera has any
influence on pollination because insects unlike the scent of
soursop flowers. It is assumed that generally fruits are
formed by autogamy after stigmas get in contact with
stamens retained by lower petals. Due to those facts,
Escobar et al. (1986) argued that pollination would rather
sporadic occurred and sometimes stigmas shed after
pollination.
Inter-population genetic distance value also were
calculated and ranged from 0.02448 to 0.0410 as shown in
Table 5. The highest Nei’s (1978) genetic distance values
(0.0410) was observed in Sukoharjo (SKH)-Gunungkidul
(GKD) population pair whereas the lowest genetic distance
(0.02448) was estimated in Karanganyar (KRA)-Pacitan
(PCT) population pair.
In order to study the relationship between populations,
UPGMA algorithm based on Nei’s (1978) genetic distance
was used to predict a dendrogram for the soursop
populations in Java using NTYSYS software. Based on the
dendogram, it showed distinct separation of the collected
sample from seven soursop populations into three major
clusters (Figure 3). The first cluster consisted of Sukoharjo
(SKH), Karanganyar (KRA), Pacitan (PCT), Ngawi
(NGW), and Kulonprogo (KPG) populations. It was
interested that within first cluster, the KRA population
from Central Java and the PCT population from East Java,
which were separated by geographical position, showed
very closed relationship, with 0.02448 of their genetic
distance. The NGW population from East Java, the KPG
population from Yogyakarta and the SKH population from
Central Java which were originated from different
provinces also grouped in this cluster. The Gunungkidul
(GKD) population was then grouped into second cluster.
Geographically, the GKD population from Yogyakarta was
far away from West Java and East Java. In the third cluster,
the Bogor (BGR) population was grouped separately and
more genetically distant than the others. The grouping in
the third cluster was very good because the geographical
position of the BGR population was separated with another
population in Java. However, the relationship dendrogram
showed that the grouping was inappropriate with
geographical origins in the first cluster. At least, the second
and third clusters were grouped in accordance with the
geographical consideration. In our study, the grouping did
not always indicate the geographical origins similarity, but
possibly showed the genetic similarity.
One of the main applications of these clusters is the
estimation of the genetic distance among populations and
the identification of parents to perform appropriate crosses,
and reaching maximum heterosis in hybridization
programs. In this study, RAPD markers show that this
method is informative and can be used to determine the
relationships among populations. The data obtained here
confirmed the efficiency of the RAPD technique as a
valuable DNA marker for determination and estimation of
genetic similarity among different plant genotypes in some
populations. The information about genetic similarity will
be helpful to avoid any possibility of elite germplasm
becoming genetically uniform (Fadoul et al. 2013).
Information on genetic diversity and relationship among
and between individuals, populations, varieties, and species
of plant are also important for plant breeders in guiding the
improvement of crop plants (Dharmar and De Britto
2011).Thus, information about genetic diversity through
RAPD markers obtained in this study could be valuable for
breeding strategies of soursop in Java. This information
also indicates that RAPD markers are a suitable marker to
assess genetic diversity of crop species.
ACKNOWLEDGEMENTS
The authors acknowledge gratefully to University of
Sebelas Maret (UNS) for the financial support. This study
was supported by a grant from University of Sebelas Maret
through DIPA BLU Grant 2012 (No. Grant
01/UN27.9/PL/2012).
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160221
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 254-263
Molecular phylogeny inferred from mitochondrial DNA of the grouper
Epinephelus spp. in Indonesia collected from local fish market
EDWIN JEFRI, NEVIATY P. ZAMANI, BEGINER SUBHAN, HAWIS H. MADDUPPA♥
Department of Marine Science and Technology, Faculty of Fisheries and Marine Science, Bogor Agricultural University (IPB). Jl. Darmaga Raya,
Bogor 16680, West Java, Indonesia. Tel./Fax. +62 85693558653, ♥email: hawis@ipb.ac.id
Manuscript received: 21 May 2015. Revision accepted: 21 September 2015.
Abstract. Jefri E, Zamani EP, Subhan B, Madduppa HH. 2015. Molecular phylogeny inferred from mitochondrial DNA of the grouper
Epinephelus spp. in Indonesia collected from local fish market. Biodiversitas 16: 254-263. Groupers are widely distributed in the
tropical and subtropical coastal waters, and are globally one of the most commercially important groups of marine fish, commanding
high market price and are being heavily targeted in fisheries. Over fishing in Indonesia becomes a pivotal factor, which is seriously
threatening the grouper biodiversity, as separate catch statistics are not reported for most species, and landings are often summarized as
‘serranids’ or ‘groupers’. This lack of species-specific catch data is due to the difficulty of identifying many of the species. The focus of
this study was the tracking of molecular phylogeny of Epinephelus spp. of the family Serranidae. DNA amplification using
mitochondrial cytochrome oxidase I resulted in 526-base pairs long sequences all samples. A total of seven species were characterized
that are (Epinephelus areolatus, E. merra, E. fasciatus, E. longispinis, E. coioides, E. ongus and E. coeruleopunctatus). All of which
were found to belong to 7 different clades in the constructed phylogenetic tree. E. ongus is genetically closest to E. coeruleopunctatus
with genetic distance 0.091 (9%), whereas the farthest genetic distance was successfully identified between E. ongus and E. merra with
genetic distance 0.178 (18%). Migration activity on spawning and movement of larvae that are affected by Indonesian Through flow
suspected as the cause of the closeness between species grouper Epinephelus spp. in the phylogeny tree from several Indonesian seas,
although information about the location and time of Epinephelus spp. spawning activity sometimes difficult to obtain certainty. Fish
identification using molecular phylogenetic approach has been successfully applied in this study. It seems need further application on
this method to avoid misidentification and due to high variety of species landing at local fish market. Nevertheless, this study would be
an important data in the genetic management for the sustainable conservation and trade of grouper (Epinephelus spp.) in Indonesia.
Key words: Coral triangle, DNA barcoding, phylogeny, taxonomy, seafood
INTRODUCTION
Grouper are generally found on coral and rocky reefs,
but some species (e.g., Epinephelus aeneus) are commonly
found on sandy, silty or muddy bottoms. The subfamily
Epinephelinae includes 159 species in 15 genera (Allen and
Adrim 2003). They can grow up to 2.5 m in length and 400
kg in weight (Heemstra and Randall 1993). Their desirable
taste and high market value make them the most important
mariculture fish species in Asia and around the world (Chiu
et al. 2008). Groupers are also among the most important
resources targeted by coastal fisheries in tropical and subtropical areas and they exhibit behavioral characteristics
that make them vulnerable (Heemstra and Randall 1993).
Since 1980, Indonesia is known as the third largest supplier
of groupers with export destination countries such as
Singapore, Hong Kong and China. The fishermen caught
the groupers in almost all coral reef seas in Indonesia. This
is because the trade of live grouper is highly profitable
(Nuraini and Hartati 2006). However, One-third of the
Epinephelinae, particularly the genera Epinephelus and
Mycteroperca, have been listed as a threatened species,
thereby emphasizing the threat faced by groupers
worldwide (Morris et al. 2000).
The phylogenetic relationships among the fishes in the
perciform tribe Epinephelinae (Epinephelus, Serranidae)
are poorly understood because of the very numerous taxa
that must be considered and the large, circumtropical
distribution of the group. Knowledge of relationships
within the Serranidae has been equally tenuous (Craig and
Hastings 2007). Recently few questions were raised on the
Epinephelus species on their morphological similarities and
the species had extensive phonetic similarities, suggesting
that some species in Epinephelus spp. might belong to a
same species and group (Zhu and Yue 2008).
Over the last decade, the development of fish
identification using molecular phylogenetic approach has
been widely conducted. One of the molecular phylogenetic
approaches that can be used is the mitochondrial DNA
barcoding intended to distinguish species and identify
specimens that are difficult to identify, such as larval stage,
organ pieces or morphologically incomplete materials,
using short gene sequences (Hebert et al. 2003).
Mitochondrial DNA is a crucial marker allowing
researchers to recognize and identify this Serranid species
for the many advantages that it offers, Mitochondrial DNA
has a high mutation rate than nuclear genome, inherited
solely from the mother, present in large numbers in every
cell. In that it allows researchers to elucidate the
evolutionary relationship among species of groupers,
without looking at the entire life cycle of grouper (Waugh
2007). For evaluating genetic diversity and phylogeny,
JEFRI et al. – Molecular phylogeny of the Indonesia grouper
modern molecular biology has enabled comparisons
between nucleotide and amino acid sequences of different
populations. Many studies were carried out in this filed,
such as this of (Ilves and Taylor 2008) on Osmeridae,
(Sembiring et al. 2015) on sharks, (Akbar et al. 2014) on
Thunnus albacares, (Ku et al. 2009) on E. quoyanus, and
(Merritt et al. 1998) on Epinephelus and Mycteroperca
species. Even some countries such as Egypt and South
Africa also have been doing mithocondrial DNA to fish in
some supermarkets. This is done to keep out of concern
because of the high incidence of substitution and regulation
of the circulation of species of fish, including grouper at
the International level (Galal-Khallaf et al. 2014) and
(Cawthorn et al. 2012). However, the Epinephelus spp. are
often incorrectly identified in the field because of their
closely related to the morphological features.
This study was aimed to identify the genetic and
phylogenic structures of Epinephelus spp. collected from
local fish market in Indonesia as inferred from
mitochondrial DNA. By the results of this study we
intended to support Indonesian government in their efforts
in conservation of fish resources, particularly the genetic
diversity, in accordance to the Indonesian Government
Regulation No. 60/2007 (The Government of the Republic
of Indonesia 2007), before groupers complete wiping-out
from the Indonesian seas.
MATERIALS AND METHODS
Tissue sampling
A total of 39 groupers (Epinephelus spp.) muscle
tissues or fin clip were collected from local fishermen and
fish landing sites, or purchased from seven local fish
market in Indonesia since January-April 2014. Lombok
(n=12 samples) in January, Karimunjawa (n=11) in May,
Lampung (n=4) in February, Kendari (n=3) in January,
255
Madura (n=3) in April, Tanakeke (n=3) in February, and
Numfor (n=3) in May (Fig.1). Fish were photographed and
identified at species level and fin clip was sampled and
preserved in 95% ethanol at -20oC for further analysis.
Grouper (Epinephelus spp.) samples were identified
according to Heemstra and Randall (1993) based on
morphometric characters (i.e. shapes, colors and fins).
DNA extraction and PCR reaction
DNA extraction was done based on commercial kit
(DNeasy Blood & Tissue Kit, Qiagen Cat. No. 69504) with
some modification according to the tissues (blood, fin or
liver), or using 10% Chelex solution (Walsh et al. 1991) at
95oC. Some tissue samples are not in good shape, and
sometimes hard in the extraction process so it must be
combine the best of both these techniques. The segment of
mtDNA COI was amplified with the primer Fish F1-5’
TCA ACC AAC CAC AAA GAC ATT GGC AC-3’ and
Fish R1-5’ TAG ACT TCT GGG TGG CCA AAG AAT
CA-3’ (Sachithanandam et al. 2012). Polymerase Chain
Reaction (PCR) was used to amplify approximately 526bp
fragment of the mtDNA CO1 gene. The procedure was
performed in 24 μ L reaction mixture containing 2 μ L 25
mM MgCl2, 2 μ L 8μ M dNTPs, 1.25 μ L each primer pair
10 mM, 0,125 μ L Taq DNA polymerase, 2.5 μ L 10xPCR
Buffer, 3 μ L DNA template, 12.875 μ L deionize water
(ddH2O), The thermo cycler conditions were: predenaturation at 94oC for 5 min, denaturation at 94oC for 30
sec, annealing at 56oC for 60 sec, extension at 72oC for 60
sec and final extension at 72oC for 7 min with 40x cycles
(Sachithanandam et al. 2012). PCR products were
separated on a 1% agarose gel, which had been stained
with ethidium bromide and viewed under UV
Transilluminator and documented. Sequence reactions were
performed in both directions using the BigDye terminator
v3.1 cycle sequencing kit (Applied Biosystems), 8-10 μ L
purified PCR product, and 4-5 μ L of either primer (3 μ M)
Figure 1. The sampling sites in Indonesia; 1. Lampung, 2. Karimunjawa, 3. Madura, 4. Lombok, 5. Tanakeke 6. Kendari 7. Numfor
256
B I O D I V E R S IT A S 16 (2): 254-263, October 2015
per reaction. Sequence-reaction products were loaded into
an ABI 3130xl automated sequencer (Applied Biosystems)
at the Berkeley Sequencing Facility located in the United
States (Sanger et al. 1977).
evolution model and 1000x bootstrap replications (Tamura
et al. 2004). Cephalopholis cyanostigma was used as an
out-group when constructing the phylogenetic tree.
Data analysis
Sequences data were analyzed using MEGA 6.0.5
program edited and aligned using Clustal W to see the
diversity of their nucleotide bases (Tamura et al. 2013).
Sequence analysis was done along with reference
sequences of various species belonging to the family
Serranidae retrieved from NCBI (National Center for
Biotechnology Information) GenBank. Aligned sequences
were also subjected to nucleotide BLAST (Basic Local
Alignment Search Tool) search to know the identity.
Phylogeny tree was constructed using phylogenetic
analysis of Neighbor Joining (NJ) and Maximum
Likelihood (ML) methods with Kimura 2-parameter
RESULTS AND DISCUSSION
Molecular characteristics
The processes of extracting and sequencing were
conducted on 39 Epinephelus spp. Sequences that have
been aligned were then followed by BLAST analysis on the
National Center for Biotechnology Information (NCBI).
The results obtained seven species, namely Epinephelus
areolatus, Epinephelus merra, Epinephelus ongus,
Epinephelus fasciatus, Epinephelus coioides, Epinephelus
coeruleopunctatus and Epinephelus longispinis, each
species showed 99%-100% similarity value (Table 1).
Table 1. Summary of identified species at specific locations after BLAST in National Center for Biotechnology Information (NCBI),
and their IUCN status. Number of samples per location is shown in bracket. LN is Least Concern.
Species
Locations (number)
Code
Epinephelus areolatus
Karimunjawa (5)
EJ-KRM-01-Epinephelus areolatus
EJ-KRM-02-Epinephelus areolatus
EJ-KRM-03-Epinephelus areolatus
EJ-KRM-04-Epinephelus areolatus
EJ-KRM-05-Epinephelus areolatus
EJ-LBK-12-Epinephelus areolatus
EJ-LBK-13-Epinephelus areolatus
EJ-LBK-14-Epinephelus areolatus
EJ-LBK-15-Epinephelus areolatus
EJ-LBK-16-Epinephelus areolatus
EJ-MDR-01-Epinephelus areolatus
EJ-MDR-02-Epinephelus areolatus
EJ-MDR-05-Epinephelus areolatus
EJ-KDR-03-Epinephelus areolatus
EJ-LPG-04-Epinephelus areolatus
EJ-KRM-27-Epinephelus merra
EJ-KRM-28-Epinephelus merra
EJ-KRM-29-Epinephelus merra
EJ-TNK-01-Epinephelus merra
EJ-TNK-03-Epinephelus merra
EJ-KDR-13-Epinephelus merra
EJ-KDR-14-Epinephelus merra
EJ-LBK-01-Epinephelus merra
EJ-LBK-02-Epinephelus merra
EJ-LBK-03-Epinephelus merra
EJ-LBK-11-Epinephelus merra
EJ-NMP-03-Epinephelus merra
EJ-NMP-05-Epinephelus merra
EJ-TNK-02-Epinephelus ongus
EJ-KRM-58-Epinephelus ongus
EJ-LBK-10-Epinephelus ongus
EJ-LBK-08-Epinephelus fasciatus
EJ-LBK-09-Epinephelus fasciatus
EJ-LPG-03-Epinephelus fasciatus
EJ-LPG-05-Epinephelus fasciatus
EJ-KRM-45-Epinephelus coioides
EJ-KRM-46-Epinephelus coioides
EJ-NMP-02-Epinephelus coeruleopunctatus
EJ-LPG-02-Epinephelus longispinis
Lombok (5)
Madura (3)
E. merra
Kendari (1)
Lampung (1)
Karimunjawa (3)
Tanakeke (2)
Kendari (2)
Lombok (4)
Numfor (2)
E. ongus
E. fasciatus
Tanakeke (1)
Karimunjawa (1)
Lombok (1)
Lombok (2)
Lampung (2)
E. coioides
Karimunjawa (2)
E. coeruleopunctatus
E. longispinis
Numfor (1)
Lampung (1)
BLAST (%)
IUCN Status
100
100
100
99
99
100
99
100
99
99
99
99
99
99
100
99
99
100
100
100
100
99
99
99
99
99
99
99
100
100
100
99
99
99
99
100
99
99
100
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
LN
JEFRI et al. – Molecular phylogeny of the Indonesia grouper
Genetic distance
Genetic distance data obtained from seven species
ranged from 0.091 (9%) to 0.178 (18%) (Table 2).
According to Nei (1972), the closer the genetic distance of
a species with other species means that the COI gene
similarity is closer and the value of genetic distance is still
at the middle limits. The results of data analysis showed
that the closest genetic distance was E. ongus with E.
coeruleopunctatus at 0.091 (9%) and the farthest genetic
distance was E. merra with E. ongus at 0.178 (18%).
Phylogeny tree
Phylogeny tree was constructed from 39 sequences
obtained from the Indonesian seas and added with
257
GeneBank sequences of 31 individuals presented in Table 1
and 3. The addition of 31 sequences from other countries
was aimed to strengthen the position of the sequences from
Indonesia in the phylogeny tree. Phylogenetic is a
description of relationship based on DNA sequence
composition or protein which resembles to that of a tree to
estimate the past evolution process (Baldauf 2003). The
reconstruction of Epinephelus spp. phylogeny tree was
conducted using MEGA 6.0.5 software with the bootstrap
NJ and ML methods. Both tree construction methods
showed similar topologies with only minor differences at
deeper nodes. The results showed seven clades;
Epinephelus areolatus, E. merra, E. fasciatus, E. longispinis,
E. coioides, E. ongus and E. coeruleopunctatus.
Table 2. Genetic distance between the 7 species identified in the study
No.
1
2
3
4
5
6
7
Species
Epinephelus areolatus
E. merra
E. coioides
E. ongus
E. fasciatus
E. coeruleopunctatus
E. longispinis
1
2
3
4
5
6
7
0.152
0.163
0.166
0.145
0.160
0.151
*
0.176
0.178
0.148
0.150
0.160
*
*
0.117
0.144
0.123
0.165
*
*
*
0.168
0.091
0.178
*
*
*
*
0.163
0.157
*
*
*
*
*
0.135
*
*
*
*
*
*
-
Table 3. GeneBank data information of the Epinephelus spp. included in this analysis, location and accession number from National
Center for Biotechnology Information (NCBI)
Species
Locations
Epinephelus areolatus
E. areolatus
E. areolatus
E. merra
E. merra
E. merra
E. coioides
E. coioides
E. coioides
E. coioides
E. coioides
E. ongus
E. ongus
E. ongus
E. ongus
E. ongus
E. fasciatus
E. fasciatus
E. fasciatus
E. fasciatus
E. fasciatus
E. coeruleopunctatus
E. coeruleopunctatus
E. coeruleopunctatus
E. coeruleopunctatus
E. coeruleopunctatus
E. longispinis
E. longispinis
E. longispinis
E. longispinis
E. longispinis
Luzon, Philippines
South China Sea, China
South China Sea, China
Luzon, Philippines
Queensland, Australia
French Polynesia
Pangasinan, Philippines
Andaman, India
Andaman, India
Andaman, India
Queensland, Australia
Queensland, Australia
Queensland, Australia
Queensland, Australia
Cuba
Okinawa, Japan
Luzon, Philippines
Queensland, Australia
Arabian Sea
Arabian Sea
India
Pomene, Mozambique
Madagascar
Madagascar
Viti Levu Island, Fiji
Andaman, India
India
Kerala, India
Kerala, India
South Africa
Pomene, Mozambique
Access number
KC970469
FJ237757
FJ237756
KC970471
DQ107898
JQ431721
KF714940
JX674987
JX674982
JX674983
DQ107891
DQ107858
DQ107859
DQ107872
FJ583398
JF952725
KC970470
DQ107874
FJ459562
FJ459561
EU392208
JF493438
JQ349962
JQ349961
KF929848
JX674991
KJ607970
EF609521
EF609522
HM909800
HQ945868
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258
B I O D I V E R S IT A S 16 (2): 254-263, October 2015
The seven clades formed grouping and showed solid
phylogeny tree, each clade indicated the bootstrap value of
100% both on the NJ method and ML method (except; E.
coeruleopunctatus at 99%) (Figures 2 and 3). Each group;
E. merra clade formed from Numfor, Karimunjawa,
Tanakeke, Kendari and Lombok, with additional samples
from Philippines (KC970471), Australia (DQ107898) and
French Polynesia (JQ431721). E. fasciatus clade formed
from Lombok and Lampung as well as additional samples
from Philippines (KC970470), Australia (DQ107874),
India (EU392208) and the Arabian Sea (FJ459561 and
FJ459562). E. areolatus clade formed from Lombok,
Lampung, Karimunjawa, and Madura and additional
samples from Philippines (KC970469) and China
(FJ237756 and FJ237757), there is also a sample from
Lombok and Lampung formed a separate sub-clade (EJLBK-13 and EJ-LPG-04). E. longispinis clade formed from
Lampung with additional samples from India (KJ607970,
EF609522 and EF609521), South Africa (HM909800) and
Mozambique (HQ945868) with bootstrap value of 100%. A
similar case also occurred in E. coioides clade, the sample
comes from Karimunjawa with additional samples from
Philippines (KF714940), Australia (DQ107891) and India
(JX674982, JX674983 and JX674987). Then E. ongus
clade formed from Karimunjawa, Tanakeke and Lombok
with additional samples from Australia (DQ107858,
DQ107859 and DQ107872), Japan (JF952725) and Cuba
(FJ583398). And the latter with bootstrap value 99% in
both methods NJ and ML. The last E. coeruleopunctatus
clade formed from Numfor with additional samples from
Mozambique (JF493438), Madagascar (JQ349961 and
JQ349962), Fiji (KF929848) and India (JX674991).
Discussion
The fragment length from PCR amplification using COI
with Fish R1 and Fish F1 primers from 39 samples was
526bp (basepairs). Previous research has also conducted
studies and obtained a fragment length of 582bp on
Epinephelus septemfasciatus (Guan et al. 2014),
Epinephelus longispinis at 516bp Epinephelus ongus at
522bp
and
Epinephelus
areolatus
at
318bp
(Sachithanandam et al. 2012). The different sequence
length is determined by the difference of quality DNA in
each sample collected, but it does not affect the results of
the sequence analysis in each sample. In fact, several DNA
barcoding studies using fish samples obtained from some
supermarkets also show good sequence results (300-600bp)
as long as the collection and storage processes are well
conducted (Filonzi et al. 2010). Shark tissues were
collected from three fisheries landing site in Java Islands,
Indonesia also showed 600-700bp a total of seven species
from 59 individuals was identified (Prehadi et al. 2014) and
other part in Indonesia (Sembiring et al. 2015). Even, the
tissue from the museum also showed the base pairs length
although shorter than fresh tissue (Zein et al. 2013).
Fish identification is traditionally based on
morphological features. However, in many cases, fish and
their diverse developmental stages are difficult to identify
using morphological characteristics alone. Molecular DNA
identification techniques have been developed and proven
to be analytically powerful. As a standardized and
universal method, DNA barcoding will correct an error in
grouper identification based on morphological analysis
(Zhang and Hanner 2012). In addition to E. merra, this
species has a relatively small body (grow up to 28 cm in
length) and live up to 25 m in depth, while E. ongus can
grow to nearly 1 m in 100 m depth (Heemstra and Randall
1993) (Figure 4). The current classification of the
Epinephelus genera is primarily based on different
morphological traits: the number of anal fin rays (7-10), the
shape of caudal fins (rounded and truncate) and the head
length (2.1-2.5 in standard length) (Table 4). The use of
morphological characteristics to identify grouper species
and then reconstruct phylogenetic relationships is very
complex and not always satisfactory (Maggio et al. 2004).
Morphological analysis of seven species in this study
showed a difference; although there are some species
nearly as visually and size, but with the analysis of
mitochondrial DNA is very helpful correcting genetic
distance between species, especially of each species.
Heemstra and Randall (1993) stated that E. merra can be
distinguished from the other reticulated groupers by its
pectoral-fin pattern of conspicuous black dots that are
largely confined to the rays of the fin. E. areolatus has
often been confused with E. chlorostigma, which is also
covered with brown spots and has a truncate or emarginate
caudal fin with a white posterior margin. E. ongus also
sympatric with E. coeruleopunctatus has a similar color
pattern, but the caudal and anal fins have only a few white
spots (confined mainly to proximal part of these fins).
Genetic distance and phylogenetic tree also showed a
strong proximity between both.
Groupers (Epinephelus spp.) are distributed in the
tropical and subtropical regions of African to Indo-Pacific
oceans. Madduppa et al. (2012) stated that the diversity of
grouper in the reef slope was higher than in the lagoon.
This shows that the characteristics of the habitat were
instrumental in shaping the fish community. Their
distribution territory is limited, they live in solitary,
sedentary and territories in the coral reef ecosystem that
cause the genetic distance of Epinephelus spp. is not too far.
Although sometimes they are found to migrate several
kilometers for the spawning process to a more conducive
seas for 1 to 2 weeks aggregation, Epinephelus spp.
migrate to form massive spawning aggregations at specific
locations and during specific periods (Erisman et al. 2014).
It is not surprising that Epinephelus spp. exhibits
considerable intraspecific variation based on scale counts
and color pattern.
Phylogeny tree
A strong clade indicated by the bootstrap value of 100%
both on the NJ method and on the ML method (except for
E. coeruleopunctatus at 99%) (Figures 2 and 3). In E.
areolatus clade, there were sub-clades with the bootstrap
value of 99% (EJ-LBK-13 and EJ-LPG-04), in which
geographically the species belonged to Lombok and
Lampung seas but has a close phylogeny with a bootstrap
JEFRI et al. – Molecular phylogeny of the Indonesia grouper
259
Figure 2. Neighbor-Joining tree using 39 Epinephelus spp. grouper sequences from Indonesia based on the mtDNA CO1 and added 31
sequences from GeneBank with Cephalopholis cyanostigma as out-group. Note: KDR = Kendari, LPG = Lampung, LBK = Lombok,
TNK = Tanakeke, KRM = Karimunjawa, MDR = Madura, NMP = Numfor.
260
B I O D I V E R S IT A S 16 (2): 254-263, October 2015
Figure 3. Maximum Likelihood tree using 39 Epinephelus spp. grouper sequences from Indonesia based on the mtDNA CO1 and added
31 sequences from GeneBank with Cephalopholis cyanostigma as out-group. Note: KDR = Kendari, LPG = Lampung, LBK = Lombok,
TNK = Tanakeke, KRM = Karimunjawa, MDR = Madura, NMP = Numfor.
JEFRI et al. – Molecular phylogeny of the Indonesia grouper
261
↔ 22 cm adult
A
0.091 (9%)
0.178 (18%).
↔ 43 cm adult
B
↔ 17 cm adult
C
Figure 4. Three species of grouper Epinephelus spp. The closest genetic distance (0.091 or 9%) between Epinephelus ongus (A) and
Epinephelus coeruleopunctatus, and furthest genetic distance (0.178 or 18%) between Epinephelus ongus (A) and Epinephelus merra
(C) (after Heemstra and Randall 1993)
Table 4. The main morphological characters to identify grouper (Epinephelus spp.) based on Heemstra and Randall (1993)
Species
Epinephelus areolatus
E. merra
E. coioides
E. ongus
E. fasciatus
E. coeruleopunctatus
E.longispinis
Head length (inch)
Anal fin rays
2.4 to 2.8
2.3 to 2.6
2.3 to 2.6
2.3 to 2.5
2.3 to 2.6
2.3 to 2.5
2.4 to 2.6
III spines and 8 rays
III spines and 8 rays
III spines and 8 rays
III spines and 8 rays
III spines and 8 rays
III spines and 8 rays
III spines and 8 rays
value of 99%. Other samples from the Philippines
(KC970469) and China (FJ237757 and FJ237756) were
also joined in one large clade, indicating that several
groupers of E. areolatus species from Indonesia, the
Philippines and China were still have a close kinship. No
significant differences from these seas were due to the
limited distribution and territorial-nature of this grouper
species in accordance with the results of (Heemstra and
Randall 1993).
The other clades, E. longispinis showed the existence of
two sub-clades. EJ-LPG-02 and KJ607970 from Lampung
and India were formed their own sub-clade, whereas
EF609522, EF609521, HM909800 and HQ945868 from
India, South Africa and Mozambique formed other subclades. Sachithanandam et al. (2012) stated that E.
longispinis of Andaman India also showed almost similar
character of South African as well as Arabian sea species.
The existence of a large sub-clade is suspected because of
Shape of caudal fins
Truncate or slightly
Rounded
Rounded
Rounded.
Slightly to moderately rounded
Rounded
Convex
the considerable differences in geography, even though E.
longispinis is a geographically distributed species in the
continental areas and islands in the Indian Ocean region
from Kenya to South Africa and the Banda Sea, including
Madagascar, Comoros, Maldives, India to Sri Lanka
(Heemstra and Randall 1993). Spawning migration activity
for a long time and affected by Indonesian Through flow
that suspected for causing the phylogeny tree has proximity
of some of these seas. It is known that grouper species is a
protogynous hermaphrodite (Craig et al. 2011), although
the location and timing of grouper spawning activity is
sometimes difficult to find the information (Golbuu and
Friedlander 2011).
The results of phylogeny tree either using NJ or ML
methods were also strengthened the data from the analysis
of genetic distance, whereas the closest (E. ongus and E.
coeruleopunctatus) were on the same branch (with the
bootstrap values of 72% (ML) and 73% (NJ)) with a
262
B I O D I V E R S IT A S 16 (2): 254-263, October 2015
different clade. Meanwhile, the farthest E. ongus and E.
merra were in a different clade and the same large branch
(with the bootstrap values of 73% (ML) and 82% (NJ)). E.
fasciatus and E. coioides clades also showed no significant
differences in the position of the phylogeny tree from the
results obtained in the analysis of genetic distance, even
though they had merged with several sequences from
outside Indonesian seas. Craig and Hastings (2007) also
corroborate that Epinephelus spp. are monophyly of the
remaining tribes.
The present study suggested that event morphologically
the species Epinephelus spp. are difficult to differentiate
due to key features are quite similar, but they were
confirmed by the molecular analysis results. Mitochondrial
COI gene, as an ideal region for species barcode, DNA
barcode may be used for the rapid analysis for the
commercial purposes especially confirmation for the
particular species. This study would be an important data in
the genetic management for the sustainable conservation
and trade of grouper (Epinephelus spp.) in Indonesia.
ACKNOWLEDGEMENTS
The current research was funded by Directorate General
of Higher Education, Indonesia (DIKTI), and Marine
Biodiversity and Biosystematics Laboratory, Department of
Marine Science and Technology, Bogor Agricultural
University, Bogor, Indonesia.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160222
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 264-268
Short Communication:
Microfungal diversity on leaves of Eusideroxylon zwageri, a threatened
plant species in Sarawak, Northern Borneo
A. LATEEF ADEBOLA1,2,♥, MUID SEPIAH1, MOHAMAD H. BOLHASSAN1, MANSOR WAN ZAMIR1
1
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota
Samarahan-94300, Sarawak, Malaysia. Tel./fax.: +60-146902928, email: lateef.aa@unilorin.edu.ng
2
Department of Plant Biology, Faculty of Life Science, University of Ilorin, Nigeria
Manuscript received: 16 August 2015. Revision accepted: 27 September 2015.
Abstract. Adebola AL, Sepiah M, Bolhassan MH, Wan Zamir M. 2015. Microfungal diversity on leaves of Eusideroxylon zwageri, a
threatened plant species in Sarawak, Northern Borneo. Biodiversitas 16: 264-268. A survey of the microfungal communities on green
leaves and leaf litters of an endangered plant species, Eusideroxylon zwageri Teijsm. & Binn. (belian) was carried out for the first time.
A total of 200 leaf segments were plated on both water agar and malt extract agar. 74 fungal species were identified from both leaf types
with more fungal taxa found on the green leaves, with a Shannon diversity index of 3.85 compared to that on litters, 2.63 and the
similarity between the microfungal communities on both leaf types was low with a Bray-Curtis similarity index of 0.366. The most
dominant species on both leaf types includes Aphanocladium areanarum, Trichoderma koningii, Nectria sp., Chalara pteridina,
Hyphomycetes sp.3, hyaline Mycelia sterilia, Circinotrichum sp., Phoma sp., Acremonium macroclavatum, Chaetopsina sp., Physarum
sp., Beltrania rhombica and Colletotrichum acutatum.
Keywords: Endophytic, green leaves, leaf litters, new record, saprophytic
INTRODUCTION
General knowledge on the microfungal diversity and
distribution is still inadequately understood. More studies
have been done on fungal diversity and their spatial
distribution in the temperate regions as compared to the
tropics (Hawksworth 2001; Hawksworth and Rossman
1997). Many areas and substrates still remain unstudied in
the world, most especially in the tropics and same applies
to many plant species which are not yet studied for their
associated microfungal communities. The most accepted
fungal estimate of 1.5 million by Hawksworth (1991, 2001)
was considered as too small by some authors (Cannon
1997; O’Brien et al. 2005) on the basis that the used plant
to fungus ratio of 1:6 used by Hawksworth, which assumed
the plant diversity as 270,000, was too low, pointing out
that there are about 300,000-320,000 plant species (Prance
et al. 2000), 420,000 spp. (Govaerts 2001) and 117,734575,320 spp. (Wortley and Scotland 2004). An important
area of global fungal diversity which has been often
overlooked is microfungi on vulnerable, threatened and
endangered plant species. There is a wide gap of data on
microfungi associated with many rare plant species, in
terms of host-specific fungi and also fungal disease caused
to the plants (Buchanan et al. 2002).
Eusideroxylon zwageri Teijsm. & Binn. (belian tree), is
a typical case study of unstudied rare plants. E. zwageri is
the only accepted species in the genus Eusideroxylon which
belongs to the family Lauraceae. This plant species, also
called the Borneo Ironwood, is native to the Southeast
Asian forest and has been listed in the IUCN Global Red
List of Threatened Species as an endangered species due to
over logging and habitat destruction (IUCN 1998). To the
best of our knowledge, no studies have been found in
literature on microfungal communities on belian tree. At
the plant family level (Lauraceae), comparatively few
studies have been done on plant species belonging to the
family Lauraceae, an example is the study done by (Paulus
et al. 2006) on Cryptocarya mackinnoniana from which 81
fungal taxa were identified using direct observation method
and on Chlorocardium rodiei by Cannon and Simmons
(2002) in which only 10 endophytic fungi were identified.
This study aims at revealing the microfungal
communities on green leaves and leaf litters of E. zwageri
(belian tree) from Kubah National Park in Sarawak,
Malaysia. The implications of this study will be far
reaching in the understanding, protection and conservation
of the belian tree.
MATERIALS AND METHODS
Sampling
Green leaves and leaf litters were collected in March,
2014 from under a belian tree (N 01o36’760, E
110o11’794), 127 m above sea level, at the base of the
camp in Kubah National Park in Sarawak, Malaysia. In this
Park, this is the only known location of belian tree and this
area has a very rough terrain. Coupled with this, collection
of belian samples is restricted by the Park officials. Leaf
ADEBOLA et al. – Microfungi on Eusideroxylon zwageri
Colletotrichum acutatum
Beltrania rhombica
Physarum sp.
Chaetopsina sp.
Phoma sp.
Circinotrichum sp.
Mycelia sterilia
Hyphomycetes sp.3
Chalara pteridina
Acremonium macroclavatum
The diversity indices such as Shannon and Simpson’s
diversity indices as well as the Bray-Curtis similarity index
were calculated using the software Estimates (Colwell
2013).
Nectaria sp.
Freq. of isolation = Total no. of leaf segments a fungal taxa was present × 100
Total no. of leaf segments observed
Trichoderma koningii
Data analysis
The frequencies of occurrence of each microfungal taxa
observed was calculated and the frequency of isolation was
determined according to (Hata and Futai 1995; Osono
2008) as follows:
The total of 74 taxa were identified from 200 leaf
segments of E. zwageri on WA and MEA, comprising 11
Ascomycetes, 55 anamorphic taxa, 3 Basidiomycetes, and
3 Zygomycetes. Non-sporulating mycelia (Mycelia
sterilia), both hyaline and melanized were also
documented. 67 taxa were identified from green leaves
while 20 taxa were from leaf litters (Table 1). The most
dominant species observed from both leaf types were
Aphanocladium aranearum, Trichoderma koningii, Nectria
sp., Chalara pteridina, Hyphomycetes sp.3, Hyaline
Mycelia sterilia, Circinotrichum sp., Phoma sp.,
Acremonium
macroclavatum,
Beltrania
rhombica,
Chaetopsina sp. and Physarum sp. (Figure 1).
Other
species
identified
includes
Speiropsis
pedatospora (Figure 2.A-C.), Subulispora longirostrata
(Figure 2.E), Isthmotricladia sp., Fusarium solani,
Cylindrocladium sp., Dactylaria obtriangularia and
Monacrosporium sp (Figure 2G-H). The Shannon and
Simpson’s diversity indices were higher for the
microfungal assemblage on green leaves, 3.85 and 35.93
respectively, than on leaf litters, 2.63 and 11.11
respectively (Tabel 2). This result indicates that leaves of
belian support a high diversity of fungi when compared to
that recovered from other endangered plant species (Sadaka
and Ponge 2003; Shanthi and Vittal 2010; Goveas et al.
2011; Grbić et al. 2015). 41 endophytic fungal taxa were
identified from Coscinium fenestratum (Goveas et al.
2011)and 49 taxa from Nepetartanjensis (Grbić et al.
2015). Also, Sadaka and Ponge (2003) and Shanthi and
Vittal (2010) identified 36 and 54 fungal taxa from
Quercus Rotundifolia and Pavetta indica respectively.
Green leaves are usually richer in nutrients than leaf
litters (Lodge et al. 2014), thus supporting a more diverse
species of microfungi. Green leaves of belian are thick and
leathery in texture making it to last longer before
decomposing. The high number of fungal taxa from leaves
of belian tree altogether shows that this plant species
supports the growth of many microfungal species.
Aphanocladium aranearum
Isolation of microfungi
Isolation of microfungi was based on the methods of
Rakotoniriana et al. (2008) and Lateef et al. (2014) for
endophytic fungi and saprobic fungi respectively, with
some modifications. For endophytic fungi, the leaves were
washed under running tap water to remove dust and debris
adhering to them. The leaves were then cut into 1 cm2 with
and without the midribs under aseptic conditions using a
sterile scalpel. They were then surface sterilized with 70%
ethanol for one minute, then in 10 % hydrogen peroxide
(H2O2) solution for five minutes, rinsed with 70% ethanol
for one minute and finally rinsed with deionized sterile
distilled water five times to remove the sterilants and
blotted on sterile filter paper to remove excess water. Five
segments were plated separately on water agar (WA) and
malt extract agar (MEA). The Petri dishes were sealed with
parafilm and incubated at room temperature. Observation
and isolation of the growing microfungi starts from the
third day of incubation for MEA and four weeks for WA.
For saprophytic microfungi, the leaf samples were
washed with double sterilized distilled water, cut into 1 cm2
into a 500 mL conical flask, washed again with sterile
distilled water for 5 times and then blotted on sterilized
filter paper. The leaf segments were then plated as done for
the endophytic microfungi. A total of 200 leaf segments
were plated, 100 each for green leaves and leaf litters. 20
replicate petridishes were used for the green leaves and leaf
litters separately.
Frequencies of occurrence of each microfungi on the
leaf segments were recorded. A Motic stereo microscope
(MZ 168) and an Olympus compound microscope (CX-31)
were used for monitoring of fungal reproductive structures
and pictures were taken with a hand-held Samsung camera
model ES91. Identification of the observed microfungi
were made to genus level, and wherever possible, to
species level.
RESULTS AND DISCUSSION
Number of occurence
samples were collected randomly under the belian tree,
green leaves as just fallen green leaves and litters as
already brown and weak leaves. The belian trees were very
tall and it is practically impossible to detach a leaf from it.
The samples collected were put in plastic bags, labeled and
transported to the laboratory for processing.
265
Figure 1. Most dominant microfungal taxa on both green leaves
and leaf litters of Eusideroxylon zwageri (belian)
266
B I O D I V E R S IT A S 16 (2): 264-268, October 2015
Table 1. Percent dominance of microfungi on green leaves and
leaf litters of Eusideroxylon zwageri (belian)
Microfungal species
Green
leaves
(%)
1.56
3.91
5.86
1.17
Acladium state of Botryobasidium consperum
Acremonium macroclavatum Ts. Watan.
Aphanocladium aranearum (Petch) W. Gams
Aspergillus nomius Kurtzman, B.W. Horn &
Hesselt.
Aureobasidium pullulans (de Bary &
1.17
Löwenthal) G. Arnaud
Aureobasidium sp.
0.39
Basidiomycetes
0.39
Beltrania rhombica Penz.
1.95
Bipolaris sp.
0.39
Bispora sp.
1.17
Melanized Mycelia sterilia
1.17
Botryodiplodia sp.
0.39
Botrytis sp.
0.39
Brachyphoris sp.
1.17
Calonectria pyrochroa (Desm.) Sacc.
0.39
Calosphaeria cyclospora (Kirschst.) Petr.
0.78
Camposporium sp.
0.78
Ceriospora polygonacearum (Petr.) Piroz. &
0
Morgan-Jones
Chaetendophragmia sp.
0.39
Chaetomium sp.
0.78
Chaetopsina sp.
3.91
Chaetosphaeria sp.
0.39
Chalara pteridina Syd. & P. Syd.
3.13
Chrysosporium merdarium (Ehrenb.) J.W. Carmich 0.39
Circinotrichum sp.
4.69
Colletotrichum acutatum J.H. Simmonds
3.52
Cryptosporium tami Grove
1.17
Cylindrocladium camellia Venkataram. &
0.39
C.S.V. Rame
Cylindrocladiella parva (P.J. Anderson) Boesew. 0
Cylindrocladium sp.
1.17
Dactylaria obtriangularia Matsush.
0.78
Didymella effusa (Niessl) Sacc.
1.56
Didymella sp.
0.39
Diplodia sp.
0.39
Drechslera sp.
0.39
Epicoccum nipponicum Matsush.
0.39
Fusarium merismoides Corda
0.39
Fusarium solani (Mart.) Sacc.
1.56
Gonytrichum sp.
0.78
Harpographium sp.
1.17
Hyphomycetes sp.1
0.78
Hyphomycetes sp.2
1.56
Hyphomycetes sp.3
3.13
Isthmotricladia sp.
1.56
Metacapnodium juniperi (W. Phillips & Plowr.) 0.39
Speg.
Monacrosporium sp.
0
Mortierella sp.1
0.39
Mortierella sp.2
1.17
Calonectria colhounii Peerally
2.73
Nectria sp.
1.17
Neottiosporella sp.
0
Oidiodendron sp.
0.39
Periconia sp.
3.13
Pestalotiopsis sp.
0.78
Pestalotiopsis versicolor (Speg.) Steyaert
0.78
Leaf
litters
(%)
3.68
0
11.03
0
0
0
0.74
3.68
1.47
1.47
0
0
0
0
0
0
0
1.47
0
0
0
0
11.03
0
0
0
0
0
2.94
0
0
1.47
0
0
0
0
0
0
0
0
0
0
10.29
0
0
0.74
0
0
0
16.18
2.94
0
0
0
0
Phaeostalagmus sp.
Phoma sp.
Physarum sp.
Piricauda cochinensis (Subram.) M.B. Ellis
Rhinocladiella cristaspora Matsush.
Septonema sp.
Sordaria fimicola (Roberge ex Desm.) Ces. &
De Not.
Speiropsis pedatospora Tubaki
Hyaline Mycelia sterilia
Subulispora longirostrata Nawawi & Kuthub.
Subulispora procurvata Tubaki
Tetrabrunneospora ellisii Dyko
Thielavia sp.
Thozetella sp.
Trichoderma koningii Oudem.
Trichosporiella sp.
Vermispora sp.
Veronaea indica (Subram.) M.B. Ellis
Volutella ciliata (Alb. & Schwein.) Fr.
0.39
0.78
3.91
1.95
3.52
0.78
0.39
0
7.35
0
0
0
0
0
1.17
4.30
0.39
2.34
0
1.56
0.78
6.25
1.95
0
0.78
0
0
3.68
0
1.47
1.47
0
0
9.56
0
2.94
0
4.41
Table 2. Diversity indices and Similarity index of the microfungal
communties on green leaves and leaf litters of Eusideroxylon
zwageri (belian)
Diversity index
Green leaves
Leaf litters
Number of isolates
Observed species
Number of Singletons
Number of Doubletons
ACE species estimate ± SD
Chao 1 species estimate ± SD
Shannon Index ± SD
Simpson Inv Index ± SD
256
67
21
12
83.09
83.09
3.85
35.93
132
20
2
6
20.14
20.14
2.63
11.11
Table 3. Similarity index of microfungi on green leaves and leaf
litters of Eusideroxylon zwageri (belian)
Samples
Index numbers
Species observed on green leaves
Species observed on leaf litters
Shared Species Observed
Sorensen Classic
Morisita-Horn
Bray-Curtis
67
20
14
0.322
0.442
0.366
Fourteen taxa were commonly identified from both
green leaves and leaf litters with a Bray-Curtis similarity
index of 0.366 (Table 3). Consequently, there were a total
of 53 fungal taxa exclusively identified on green leaves
while 7 taxa were from the leaf litters (Figure 3). There
were more species exclusively on the green leaves than on
the leaf litters which further suggests that the green leaves
are more suitable for fungal growth. Furthermore,
Metacapnodium juniperi, Phaeostalagmus sp. and
Monacrosporium sp. were identified from green leaves and
leaf litters respectively, of which these species are not
ADEBOLA et al. – Microfungi on Eusideroxylon zwageri
267
B
C
A
D
E
H
F
G
I
J
Figure 2. Some microfungal diversity on green leaves and leaf litters of Eusideroxylon zwageri (belian). A. Speiropsis pedatospora, B C. Conidia of S. pedatospora, D. Physarum sp., E. Conidia of Subulispora longirostrata, F. Circinotrichum sp.,G. Conidiophores of
Monacrosporium sp., H. Conidia of Monacrosporium sp., I. Conidia of Beltrania sp., J. Chalara sp.
Figure 3. Number of microfungi exclusively observed only on
green leaves and leaf litters of Eusideroxylon zwageri (belian)
usually frequently isolated from other plant species. Many
rare fungal species have been identified on endangered
plant species in New Zealand (Buchanan et al. 2002) and
also in Sarawak (Hughes 1977). This occurrence signifies
the microfungal loss which can be suffered with the
extinction of such endangered plant species including
belian.
Also, comparison of the fungal communities observed
on belian leaves with other plant species in the family
Lauraceae showed some similarity in their microfungal
communities, in which Acremonium sp., Beltrania sp.,
Chaetopsis sp., Chalara sp., Dactylaria sp., Pestalotiopsis
spp, Rhinocladiella cristaspora, Subulispora sp.,
Thozetella sp. and Volutella sp. have been recorded on
leaves of Cryptocarya mackinnoniana (Paulus et al. 2006)
as saprobic species. Colletotrichum acutatum have
previously been isolated from Chlorocardium rodiei
(Cannon and Simmons 2002) and Cinnamomum bejolghota
268
B I O D I V E R S IT A S 16 (2): 264-268, October 2015
(Suwannarach et al. 2012) as an endophyte. Fusarium
solani, Nectria sp., Oidiodendron sp., Periconia sp.,
Pestalotiopsis sp., Phoma sp. and Trichoderma sp. were
also
recovered
from
Cinnamomum
bejolghota
(Suwannarach et al. 2012). Other taxa recorded in this
study are reported for the first time in the family
Lauraceae.
The high demand for wood materials from belian and
its slow growth, coupled with its habitat destruction makes
it vulnerable to extinction. This study is the first report of
microfungal communities on belian leaves in Sarawak.
Green leaves supported a higher number of microfungi than
the leaf litters. This observation contributes to the
understanding of the biology of E. zwageri and can be used
to strengthen its conservation importance.
ACKNOWLEDGEMENTS
The first author is grateful to Universiti Malaysia
Sarawak (UNIMAS) for the Zamalah scholarship awarded.
We are also grateful to the Sarawak government and to
Sarawak Forestry Co-operation (SFC) for permission to
collect samples from the National Park.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160223
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 269-280
Leguminicolous fungi associated with some seeds of Sudanese legumes
SOHAIR A. ABDULWEHAB1, SAIFELDIN A. F. EL-NAGERABI2,♥, ABDULQADIR E. ELSHAFIE3
1
Department of Botany, Faculty of Science, University of Khartoum, P.O. Box 321, Khartoum, Postal Code 11111, Sudan
Department of Biological Sciences and Chemistry, College of Arts and Science, University of Nizwa, Birkat Al Mouz, Nizwa, P. O. Box 33, PC 616,
Oman. Tel.: + 968-9636-5051, Fax.: +968-25443050, email: nagerabi@unizwa.edu.om
3
Department of Biology, College of Science, Sultan Qaboos University, P.O. Box 36, AlKhoudh, Postal Code 123, Oman
2
Manuscript received: 29 July 2015. Revision accepted: 28 September 2015.
Abstract. Abdulwehab SA, El-Nagerabi SAF, Elshafie AE. 2015. Leguminicolous fungi associated with some seeds of Sudanese
legumes. Biodiversitas 16: 269-280. The mycoflora associated with seeds evidently deteriorate seed viability, germination, emergence
and plant growth performance leading to apparent losses in production and productivity. In the present investigation, seedborne fungi of
six legumes were screened. Twenty six species of fungi from 14 genera were isolated from this seeds. Of these isolates, 6 species are
new reports to the mycoflora of Sudan, whereas some species are new records to the mycoflora of these legumes. These include 6
species for Cajanus cajan, Cicer arietinum (10 species), Dolichos lablab (7 species), Medicago sativa (8 species), Phaseolus vulgaris
(10 species), and Vigna unguiculata (11 species). The seeds are obviously contaminated with saprophytic and pathogenic fungi (1764%) which evidently inhibited seed germination (41-86%), and seedling emergence (29-81%). The Alternaria, Aspergillus and
Fusarium (4 species each) were the most prevalent fungi followed by Curvularia, Drechslera (3 species), Fusariella, Ulocladium (2
species) and one species for the remaining genera (Aureobasidium, Acremonium, Memnoniella, and Rhizopus). Hence, there is a high
need for establishment of standard seed testing methods with strong legislations in order to meet the international quarantine regulations.
The use of certified seeds by the farmers is recommended.
Key words: Cajanus cajan, Cicer arietinum, Dolichos lablab, Medicago sativa, Phaseolus vulgaris, Vigna unguiculata, seedborne,
Sudan
INTRODUCTION
Legumes of the family Fabaceae are one of the most
important plants cultivated as pulse or forage crops in
many tropical and temperate regions. They are rich sources
of plant protein and oil as human food and animals feed
(Embaby and Abdel-Galil 2006; Swami and Alane 2013;
Saleem and Ebrahim 2014). In Sudan, several leguminous
crops such as Cajanus cajan, Cicer arietinum, Dolichos
lablab, Medicago sativa, Phaseolus vulgaris, and Vigna
unguiculata are cultivated as green manure for improving
the soil fertility and reduced the amount of the expensive
nitrogen fertilizer. Cajanus cajan (L.) Millsp. belong to the
family Fabaceae and is placed in Papilionaceae of semiarid tropics is grown as grain crops with high levels of
important amino acids and proteins. It is cultivated for
consumption of its dry seeds and the unripe green seeds
serve as a cooked vegetable. The forage of this plant fed to
livestock, and the stems are used for firewood and as
hedgerow for windbreak (Pal et al. 2011; Singh and Kaur
2012). Cicer arietinum L. (Chickpea, Gram) also known as
“Humus, Kabkabeik” in Arabic, is important pulse legume
throughout the globe (Duke 1981).
The green immature pods and dry seeds are used as
green vegetable or dry pulse boiled or fried, where the
green and dried stems and leaves are used for feeding
livestock. Dolichos lablab (Hyacinth lablab, Bonavista, and
Egyptian bean) and “Lubia Afin” in Arabic is grown for
food where young immature pods are cooked and eaten like
green bean. Young leaves are used in salads and older
leaves are cooked like Spinach. Starchy root tubers,
immature and dried seeds can be boiled and eaten (Sarwatt
et al. 1991). Medicago sativa L. (Medic, Alfalfa, Lucerne,
Queen of Forage) which is also known as “Barsim hegazi”
in Arabic was originated near Iran and endemic to
Mediterranean region. It is extensively grown in warm
temperate and cool subtropical regions as the most widely
adapted agronomic crop. It is highly valued as forage
legume having highest feeding values (Duke 1981). The
seeds are used in many folk medicines and as lactigenic.
Phaseolus vulgaris L. is known as common bean, kidney
bean or “Fasulia” in Arabic is one of the five cultivated
species of this genus as major grain legume crop, third in
importance after soybean and peanuts, but first in direct
human consumption (Broughton et al. 2003). It is grown
for its green leaves, green pods, and immature and dry
seeds. The dry bean are eaten in cooked dishes, bean flour,
whereas the dry leaves, threshed pods and stalks are fed to
animals and used as fuel for cooking in Africa and Asia.
Vigna unguiculata (L.) Walp. which is known as cowpea,
black-eyed pea or “Lubia helo” in Arabci is an annual
legume that was domesticated in West Africa.
It is important grain legume and leaf vegetable in much
of Africa and part of Asia. It is used as human diet, forage,
cover, and green manure crop in many part of the world.
Tender shoot tips, leaves, immature pods and seeds can be
consumed as well as dry seeds for making flour.
Nonetheless, the seeds of these legumes are susceptible to
270
B I O D I V E R S IT A S
16 (2): 269-280, October 2015
fungal contamination, resulting in seeds deterioration
(Rathod et al. 2012; Swami and Alane 2013). These fungi
are of saprophytic or pathogenic nature which affects seed
germination, emergence from soil, plant growth vigor and
storability (Saleem and Ebahim 2014). Worldwide, many
studies have reported about the biology of the seedborne
and plant diseases associated with these leguminous crops
(Nath et al. 1970; Deo and Gupta 1980; Nakkeeran and
Devi 1997; Rathod et al. 2012).
The Alternaria, Aspergillus, Curvularia, Drechslera,
Eurotium, Fusarium, Mucor, Penicillium, Rhizoctonia,
Rhizopus, Sclerotium, and Gliocladium were the most
common fungal genera isolated from different legume
seeds: broad bean (Vicia faba), kidney bean (Phaseolus
vulgaris), lupine (Lupinus termis), cowpea (Vigna
sinensis), chickpea (Cicer arietinum) and pea (Pisum
sativum) under different environmental conditions
throughout the World (Tseng et al. 1995; Ruiz et al. 1996;
El-Nagerabi and Elshafie 2000; Kritzinger et al. 2003;
Castillo et al. 2004; Kumar et al. 2004; Domijan et al.
2005; Embaby and Abdel-Galil 2006; Attitalla et al. 2010).
Fusarium
semitectum,
F.
graminearum,
F.
chlamydosporum, F. equiseti, F. proliferatum and F.
subglutinans were the most dominant fungal species in the
seeds of Phaseolus vulgaris and Vigna sinensis from
Argentina and South Africa (Castillo et al. 2004; Kritzinger
et al. 2003). Alternaria alternata, Sclerotinia sclerotiorum,
Rhizoctonia solani, F. semitectum, and Acremonium
strictum were the dominant species in black bean whereas
A. alternata, Lasiodiplodia theobromae, Drechslera
spicifera and F. moniliforme were isolated from cowpea
(Castillo et al. 2004). The most common seedborne fungi
isolated from Phaseolus vulgaris were from Alternaria,
Aspergillus, Penicillium, Rhizopus, Cladosporium, and
Trichothecium genus (Domijan et al. 2005).
In Sudan, many leguminous crops are cultivated under
different climatic conditions. These seeds are locally
produced or imported and stored by the farmers under poor
quarantine regulations and legislations. Therefore, seed
contamination can occur by seedborne fungi which
adversely affect the production and productivity of these
crops. There is a high possibility for isolation of many
saprophytic and pathogenic fungi from various substrates
including seeds (Elshafie 1985, 1986). A few studies were
conducted on some of the seedborne fungi associated with
locally produced leguminous crops such as peas, soybean
(El-Nagerabi et al. 2000a, 2000b), guar, lupine (ElNagerabi and Elshafie 2000, 2001a, 2001b), faba bean (ElNagerabi et al. 2001), and fenugreek (El-Nagerabi 2000).
Therefore, the present study was designed to investigate the
quality and the incidence level of the seedborne fungi from
six leguminous crops namely Cajanus cajan, Cicer
arietinum, Dolichos lablab, Medicago sativa, Phaseolus
vulgaris, Vigna unguiculata and to assess their effect on
seed germination and seedling emergence. These
information may improve knowledge about invading fungi
and control measures and good management practices to
prevent some fungi in legumes.
MATERIALS AND METHODS
Collection of the seed samples
Ninety seed samples from six leguminous crop namely
Cajanus cajan, Cicer arietinum, Dolichos lablab,
Medicago sativa, Phaseolus vulgaris, and Vigna
unguiculata were purchased from seed companies in
Khartoum State, Sudan. The samples were collected and
tested as recommended by the rules of the International
Seed Testing Association (ISTA 1966).
Seed germination
In this study, the blotter method was used according to
the procedure adopted by the International Seed Testing
Association (ISTA 1966). For this, 400 seeds from each
sample were inoculated on sterilized moistened filter paper
in Petri plates (Blotter). The seeds were aseptically spaced
according to their size at equal distance. The inoculated
plates were incubated in Gallenkamp illuminated incubator
at 28ºC under alternating cycle of 12 hours near ultraviolet
light and darkness to enhance sporulation of many of the
seedborne fungi. The seeds were kept moistened by adding
sterile distilled water throughout the incubation period of
two weeks and the percentage of seed germination was
recorded.
Emergence of seeds in soil
The emergence levels of the seeds from the soil was
tested by sowing 200 seeds from each type of the selected
legumes in pots filled with uniform mixture of sand and silt
(2: 1). The seeds were covered with soil layer of 1-3 cm
deep depending on the seed size. The seeds were sown at
the rate of 20 seeds per pot and were kept in the Botanical
garden of the Department of Botany, University of
Khartoum, which is of partial shade and average
temperature of between 27ºC and 29ºC. The average
percentage of seed emergence was recorded for each
vegetable crop.
Isolation and estimation of fungi
The seedborne fungi were isolated using agar plate
method (ISTA 1966). Four hundred seeds from each
sample were surface disinfected with 1% sodium
hypochlorite for 5 min and washed with several changes of
sterile distilled water. The treated seeds were then
inoculated aseptically on Potato Dextrose Agar (PDA) and
incubated at 28ºC ±2ºC for two weeks. The fungal colonies
developed around the seeds were examined, and identified
microscopically. The average levels of contamination and
incidence were reported.
Identification of isolated fungi
The isolated fungi were identified using macroscopic
features based upon colony morphology and microscopic
observations of mycelia and asexual/sexual spores (Barnett
1955; Raper and Fennell 1965; Pitt 1979; Ellis 1971, 1976;
Sutton 1980; Webster 1980; Nelson et al. 1983; Samson et
al. 1995; Barnett and Hunter 1998, 2003; Barac et al.
2004). For non-sporulating fungi, mycelial fragments were
inoculated on Malt Extract Agar (MEA) and incubated at
ABDULWEHAB et al. – Fungi of Sudanese seeds legumes
28ºC ± 2ºC to stimulate their sporulation and were then
identified to species level. Some of these fungi were
illustrated (Figures 1-18).
RESULTS AND DISCUSSION
Twenty six species of fungi which belong to 14 genera
were recovered from 90 seed samples of six leguminous
crops namely Cajanus cajan (pigeon pea), Cicer arietinum
(chickpea), Dolichos lablab (hyacinth), Medicago sativa
(alfalfa), Phaseolus vulgaris (kidney bean), and Vigna
unguiculata (cowpea). From these isolates, 6 species are
new records to the mycoflora of Sudan, whereas different
fungal species were reported for the first time in the seeds
of the tested legumes (Table 1). The seeds were evidently
contaminated with both saprophytic and pathogenic fungi
(17-64%) and displayed variable levels of seed germination
(41-86%), and seedling emergence from the soil (29-81%)
(Table 2). The genera of Alternaria, Aspergillus and
Fusarium (4 species each) were the most dominant species
followed by Curvularia, Drechslera (3 species),
Fusariella, Ulocladium (2 species) and one species for the
remaining
genera
(Aureobasidium,
Acremonium,
Memnoniella, and Rhizopus).
Worldwide, many researchers investigating the biology
of the seedborne mycoflora from numerous crops such as
fruits, vegetables, cereals, and legumes (Nath et al. 1970;
Deo and Gupta 1980; Nakkeeran and Devi 1997; Rathod et
al. 2012). The seeds of legumes were found to be heavily
infested with numerous mycoflora (Swami and Alane
2013). However, published results on seedborne fungi of
leguminous cops are very few to negligible. Many fungi are
serious parasites of the seed primordial, maturing and
stored seeds and grains. Their invasion is associated with
various damages such as seedling growth and yield loss.
Twelve genera of fungi (Alternaria, Aspergillus,
Ascochyta, Chaetomium, Cladosporium, Fusarium,
Geotrichum, Penicillium, Pythium, Rhizoctonia, Rhizopus,
and Verticillium) were isolated from different legume seeds
in Iraq (Sarhan 2009). Twenty four seedborne fungi
belonging to different genera were detected from 145 seed
samples of major legume cops in Pakistan (Rauf 2000). Of
these fungi, Alternaria alternata, Ascochyta spp.,
Colletotrichum spp., Fusarium spp., and Macrophomina
phaseolina were the most frequent and common pathogens
of these crops. Alternaria, Aspergillus, Curvularia,
Drechslera, Eurotium, Fusarium, Penicillium, Rhizoctonia,
Mucor, Rhizopus, Sclerotium, and Gliocladium were the
most common fungal genera isolated from various legume
seeds (Tseng et al. 1995; Ruiz et al. 1996; El-Nagerabi and
Elshafie 2000; Kritzinger et al. 2003; Castillo et al. 2004;
Kumar et al. 2004; Domijan et al. 2005; Embaby and
Abdel-Galil 2006; Attitalla et al. 2010). Fusarium,
Penicillium, Rhizopus, Cladosporium and Alternaria were
the main seedborne pathogens on the surface of five major
seeds of leguminous plants from Ningxia, China (Liu et al.
2002).
In Saudi Arabia, 46 fungal species belonging to 26
genera were isolated from the seeds of 5 legumes (broad
271
beans, chickpeas, cowpeas, kidney beans, and peas)
(Saleem and Ebrahim 2014). The most prevalent genera
were Aspergillus, Emericella, Mucor, Mycosphaerella,
Penicillium, and Rhizopus, whereas the most common
species were A. flavus, A. fumigatus, A. niger, A.
ochraceus, A. terreus, Emericella nidulans, Mucor
racemosus,
Mycosphaerella
tassiana,
Penicillium
chrysogenum, and Rhizopus stolonifer. In the present study,
the seeds of the six legume crops were highly infested with
different types of fungi (17-64%) which evidently affected
the seed germination (41-86%), and seedling emergence
(29-81%) (Table 2). Of the few studies on the seedborne
mycoflora of Cajanus cajan (pigeon peas), Alternaria
tenuissima (seed or seedling rot, pod spot or rot),
Botryosphaeria xanthocephalus, Cercospora cajani, C.
instabilis (spotting), Colletotrichum cajani (seed or
seedling rot, spotting, blight, pod spot or rot), Creonectria
grammicospora, Fusarium semitectum, and Megalonctria
pseudotrichia (seedling rot) were reported (Kaiser 1981).
The diseases of economic importance at the present are
Fusarium wilt (F. udum), and Phytophthora blight (P.
drechsleri f. sp. cajani) (Marley and Hillocks 1996; Kumar
et al. 2010). Cladosporium species has been reported by
Tarr (1963) as the main cause of sooty mould of pigeon
peas in Sudan. In the present study, Alternaria alternata, A.
tenuis, Aureobasidium pullulans, Curvularia brachyspora,
C. pallescens, Drechslera rostrata, and Fusarium solani
were isolated for the first time as seedborne fungi of pigeon
peas (Table 1). Cladosporium spp. (18%) and Alternaria
alternata (4.5%) which were associated with sooty mould
of this crop are encountered in high levels of incidence
comparable to other fungal isolates. The remaining isolates
were recorded before in similar studies carried on the
seedborne fungi of pigeon pea (e.g. Malone and Muskett
1964; Singh and Khapre 1978). Chickpea (Cicer arietinum)
has been found attacked by 172 pathogens including 67
species of fungi (Nene et al. 1996). The plants are infected
with a large number of fungal diseases viz. blight
(Ascochyta rabiei, A. alternata, Colletotrichum dematium,
Stemphylium sarciniforme), wilt (Fusarium oxysporum),
powdery mildew (Leveillula taurica), dry root rot
(Rhizoctonia
bataticola),
stem
rot
(Sclerotinia
sclerotiorum), wet root rot (R. solani), and foot rot
(Operculella padwickii) (Kaur 1995; Singh and Sharma
2005; Dubey et al. 2007).
Twenty six fungal species belonging to 15 genera were
isolated from 14 seed samples of chickpea collected from
different areas of Pakistan (Ahmed et al. 1993; Dawar et al.
2007). In India, Alternaria alternata, Chaetomium spp.,
Penicillium citrinum, A. niger, A. flavus, Rhizopus
stolonifer, and Fusarium oxysporum were isolated from the
seeds of this crop (Agarwal 2011). In the present
investigations, 18 species of fungi belonging to 12 genera
were recovered from the seeds of highly contaminated
chickpea (40%). Of these isolates, A. tenuis, A. tenuissima,
A. nidulans, A. terreus, Acremonium strictum, Curvularia
lunata, Drechslera papendorfii, D. spicifera, Fusariella
aegyptiaca, and Ulocladium atrum are considered new to
the seeds of this crop (Table 1). Some of the previously
reported as seedborne fungi and pathogenic to this plant
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16 (2): 269-280, October 2015
Table 2. Incidence percentage (I%), number of cases isolation (NCI, out of 90 samples), and occurrence remarks (OR) of seedborne
fungi of leguminous plants
Alternaria alternata (Figure 1)
4.5 N1
8.25
1.5
1.0 N
6.25
4.0 N
A. dianthi (Figure 2)
-3
2.5 N
0.25 N
A. tenuis (Figure 3)
1.75 N
2.25 N
1.5 N
3.75 N
2.0
A. tenuissima (Figure 4)
1.25
3.0 N
4.0 N
Aspergillus flavus
2.5
4.5
3.75
0.75 N
2.0
3.0
A. nidulans
1.0
0.25 N
0.5
2.75
A. niger
13.5
22.5
32.5
0.5 N
7.5
2.5
A. terreus
1.5
2.5 N
1.25 N
1.0
1.5
Aureobasidium pullulans (Figure 5)
1.25 N
0.5 N
0.1
Acremonium strictum
1.25 N
2.3 N
2.0 N
Cladosporium spp.
18.0
7.3
2.0
3.5
5.0
3.5
Curvularia brachyspora (Figure 6)
2.75 N
1.5 N
C. lunata (Figure 7)
1.0 N
0.5 N
C. pallescens (Figure 8)
1.25 N
2.5 N
2.0 N
Drechslera papendorfii (Figure 9)
3.0 N
1.5
D. rostrata (Figure 10) 2NS
1.5 N
2.75 N
2.25 N
D. spicifera (Figure 11)
2.0
3.5 N
1.75 N
0.25 N
3.3
1.75
Fusariella aegyptiaca (Figure 12) NS
0.25 N
0.25 N
Fusariella intermedia (Figure 13) NS
2.0 N
Fusarium equiseti (Figure 14)
1.5
3.25N
F. moniliforme
10.25
F. semitectum (Figure 15)
2.5
2.25 N
3.0
2.0 N
F. solani (Figure 16)
2.0 N
0.75
Memnoniella echinata NS
1.0
1.75
Myrothecium roridum
0.25 N
Penicillium spp.
3.0
4.25
5.0
Rhizopus stolonifer
8.25
6.5
4.5
0.5
19.5
30.0
Ulocladium atrum (Figure 17) NS
1.75 N
0.25 N
Ulocladium botrytis (Figure 18) NS
2.25 N
2.75 N
Note: 1N: New record to the crop, 2NS: New record to the mycoflora of Sudan, 3-: Not detected, OR: Occurrence remarks,
samples, H: High, more than 45 samples, M: Moderate, between 30-45 samples, L: Low, between 15-29 samples,
R: Rare, less than 15 samples.
Table 2. The percentage of seed germination and contamination
of different leguminous crops
Legume species
Cajanus cajan (pigeon pea)
Cicer arietinum
Dolichos lablab
Medicago sativa
Phaseolus vulgaris
Vigna unguiculata
Contamination %
31
40
64
17
44
47
Germination %
68
86
41
86
66
57
Emergence%
54
73
29
81
52
45
were recovered from the present seeds (Ahmed et al. 1993;
Kaur 1995; Nene et al. 1996; Singh and Sharma 2005;
Dawar et al. 2007; Dubey et al. 2007; Agarwal 2011). Of
the few studies on the seedborne mycoflora of Dolichos
NCI/OR
Vigna unguiculata
Phaseolus vulgaris
Medicago sativa
Dolichos lablab
Cicer arietinum
Fungal isolates
Cajanus cajan
Incidence percentage (I%)
87H
36M
49H
41H
88H
32M
92H
34M
27L
18L
72H
30M
27L
39M
25L
33M
76H
12R
9R
13R
10R
31M
15L
11R
5R
38M
89H
26L
21L
out of 90
lablab (Lablab purpureus), the genera of Aspergillus,
Fusarium, and Penicillium were the most important fungi
(Chalaut and Perris 1994). In India, Trichothecium roseum,
Alternaria sp., and Fusarium sp. were linked to severe
multiple infections of lablab resulting in discolored pods,
and shrunken disfigured seeds (Siddaramaiah et al. 1980).
Seed germination was suppressed by A. niger while A.
chevalieri, A. flavus, A. candidus, A. niveus, and A.
alternata caused staining and necrosis of 23-37% of
cotyledons and twisting in 19-27% (Prasad and Prasad
1987). According to Tarr (1963), many diseases of
Dolichos lablab were caused by fungal species such as leaf
spot (A. alternata, Cladosporium sp.), wilt (Macrophomina
phaseolina, Phyllosticta spp.). In the present study, 12
species of fungi were isolated from the seeds of this crop
and of these isolates Alternaria dianthi, A. tenuis,
Aureobasidium
pullulans,
Curvularia
pallescens,
ABDULWEHAB et al. – Fungi of Sudanese seeds legumes
Drechslera spicifera, Fusarium equiseti, and F. semitectum
are considered new to the mycoflora of this crop (Table 1).
Some of the previously reported as seedborne and/or
pathogenic fungi were recovered from the current seed
samples of D. lablab (Tarr 1963; Siddaramaiah et al. 1980;
Prasad and Prasad 1987; Chalaut and Perris 1994). For
Lucerne (Medicago sativa), Mycosphaerella pinodes,
Botrytis cinerea, Phoma herbarum (Phoma exigua var.
exigua) var. medicaginis, Fusarium roseum and
Stemphylium botryosum (Pleospora tarda, Pleospora
herbarum) were the most frequently encountered seedborne
fungi (Leach 1960). Fusarium avenaceum was detected for
the first time on the seed of Lucerne by Kellock et al.
(1978). Fusarium acuminatum, F. avenaceum, F. equiseti,
F. fusarioides, F. oxysporum, F. poae, Diaporthe
phaseolorum and Phoma sorghina were proven pathogenic
to a range of pasture legumes including Lucerne (Trenteva
1974; Nik and Parbery 1977). Many fungal species isolated
from the seeds of this plant by many authors in similar
mycological investigations. These include Cercospora
dematium f.sp. truncata, C. zebrina (C. medicaginis),
Colletotrichum trifolii (anthracnose), F. sporotrichoides, F.
oxysporum, F. sambucinum, F. avenaceum, Ascochyta
infectoria, Stemphylium spp. (leaf spot), Pleospora
herbarum, Stemphylium loti, S. sarciniforme, Sclerotinia
sclerotiorum, and S. trifoliorum (crown rot), Verticillium
albo-atrum (wilt) (Leach 1960; Maloy 1968; Trenteva
1974). In the present research, Alternaria alternata,
Aspergillus flavus, A. niger, A. terreus, Curvularia lunata,
Drechslera rostrata, D. spicifera, and Ulocladium botrytis
were newly isolated from the seeds samples of Lucerne
(Table 1). In similar studies on seedborne fungi of common
bean (Phaseolus vulgaris) collected from Riyadh region,
Saudi Arabia, various fungi from 11 genera were isolated
such as A. alternata, A. flavus, A. flavus var. columnaris, A.
niger, A. ochraceus, A. ustus, Botrytis sp., Chaetomium sp.,
Cladosporium
sp.,
Fusarium
spp.,
Penicillium
chrysogenum,
Phoma
sp.,
Rhizopus
stolonifer,
Stemphylium sp., and Ulocladium sp. (El-Samawaty et al.
2014).
The most common seedborne fungal species isolated
from common bean crops grown in 13 counties of the
Republic of Croatia belong to genera of Cladosporium
(98%), Alternaria (75%), Aspergillus (75%), Rhizopus
(72%), Penicillium (69%), Fusarium (38%), Botrytis
(27%), Trichothecium (24%), and Chaetomium (18%)
(Domijan et al. 2005). In Egypt, A. niger (43.2%), A.
ochraceus (2.4%), A. parasiticus (0.8%), A. flavus (0.8%),
Aspergillus spp. (4.8%), Epicoccum sp. (0.8%), Fusarium
oxysporum (2.4%), Fusarium spp. (5.6%), and
Trichoderma spp. (11.2%) were isolated from common
bean seeds (Embaby and Abdel-Galil 2006). Various
seedborne fungi were isolated from the seed of this crop
such as Alternaria brassicicola, Ascochyta phaseolina, A.
boltshauseri (leaf spot), Aspergillus glaucus, Botrytis
cinerea (chocolate spot), Rhizoctonia solani (damping off),
Fusarium solani f.sp. phaseoli (stem rot), F. solani,
Phyllosticta phaseolina, Aspergillus flavus (rot), F.
oxysporum (wilt), A. glaucus, Colletotrichum dematium,
Drechslera tetramera, F. equiseti, F. oxysporum, F.
273
moniliforme, F. semitectum, Phoma solani, Phoma sp.,
Phomopsis sojae, Colletotrichum lindemuthianum,
Macrophomina phaseolina, Sclerotinia sclerotiorum,
Rhizoctonia
sp.,
Botrytis
cinerea,
Uromyces
appendiculatus, Trichothecium roseum, Phytophthora sp.,
Diaporthe sp., Elsinoe phaseoli, Rhizoctonia solani, and
Alternaria alternata, (e.g. Winter et al. 1974; Gomes and
Dhingra 1983; Sesan and Dumitras 1979). In the present
results, 18 species were recovered from the seeds of this
crop. Of these isolates, Alternaria dianthi, A. tenuissima, A.
tenuis, Acremonium strictum, Curvularia brachyspora,
Drechslera papendorfii, Fusariella aegyptiaca, F.
intermedia, Myrothecium roridum, and Ulocladium atrum
were recovered for the first time from the seed of common
bean (Table 1). In similar mycological investigations on the
seedborne fungi of cowpea (Vigna unguiculata), A. niger
(62.2%), A. parasiticus (6.7%), Aspergillus spp. (15.6%),
and Fusarium spp. (4.4%) were the most frequently
isolated from this crop (Embaby and Abdel-Galil 2006).
Cowpea seeds collected from Riyadh region, Saudi
Arabia were found contaminated with Alternaria alternata,
A. flavus, A. flavus var. columnaris, A. niger, A.
parasiticus, A. ustus, Cladosporium sp., Curvularia sp.,
Fusarium sp., Mucor sp., Nigrospora sp., Penicillium spp.,
Rhizopus stolonifer, and Ulocladium sp. (El-Samawaty et
al. 2014). In Benin, West Africa, A. flavus, a fungus that
produces aflatoxins, was the most frequently encountered
(Houssou et al. 2009). Many fungi were isolated from the
seeds of cowpea by several authors such as Myrothecium
leucotrichum, Rhizoctonia solani, Ascochyta sp.,
Colletotrichum lindemuthianum, C. truncatum, Fusarium
oxysporum, Corticium rolfsii, Colletotrichum capsici,
Cercospora canescens, Aspergillus spp., A. flavus, A. niger,
Botrytis cinerea, Cacumisporum sp., Cephalosporium sp.,
Chaetomium sp., Curvularia verruculosa, Diaporthe
phaseolorum, Drechslera hawaiiensis, D. spicifera,
Fusarium equiseti, F. fusarioides, Memniella sp.,
Nigrospora sp., Penicillium spp., Phoma sp., Pithomyces
sp.,
Alternaria
infectoria,
Stachybotrys
sp.,
Cencephalastrum racemosus, Pestalotiopsis nagniferae, A.
terreus, C. lunata, Pleospora infectoria, Rhizoctonia
bataticola, and Rhizopus stolonifer (e.g. Singh and Khapre
1978; Enechebe and McDonald 1979; Sesan and Dumitras
1979).
Different pathogenic fungi were associated with many
devastating diseases of cowpea plant including Ascochyta
phaseolorum (leaf and pod spot), Cladosporium vignae
(Leaf spot), Cercospora canescens, C. cruenta, C. lunata,
Phyllosticta phaseolorum, Sporidesmium bakeri, Uromyces
vignae, Capnodium spp., and Cladosporium herbarum
(sooty mould), Fusarium oxysporum f. sp. tracheiphilum
(wilt), Rhizoctonia solani, Myrothecium leucotrichum,
Aspergillus terreus (brown lesions), Curvularia lunata,
Rhizoctonia bataticola and F. concolor (post-emergence
death and stem lesions), Oidium sp., and Sphaerotheca
fuliginea (powdery mildew), and Macrophomina phaseoli
(wilt) (Singh and Khapre 1978; Enechebe and McDonald
1979; Siddaramaiah et al. 1980). In the present research,
Alternaria alternata, A. terreus, A. flavus, A. nidulans, A.
niger, Acremonium strictum, Cladosporium spp., Curvularia
274
B I O D I V E R S IT A S
16 (2): 269-280, October 2015
A
A
C
B
B
Figure 1. Alternaria alternata (A) Conidia, (B) Conidiophores. Bar =
50 µm
Figure 2. Alternaria dianthi (A) Conidia, (B) Conidiophores,
(C) Chlamydospores. Bar = 50 µm
A
A
B
B
Figure 3. Alternaria tenuis (A) Conidia, (B) Conidiophores. Bar = 50
µm
Figure 4. Alternaria tenuissima
Conidiophores. Bar = 50 µm
(A)
Conidia,
(B)
ABDULWEHAB et al. – Fungi of Sudanese seeds legumes
275
A
A
B
B
Figure 5. Aureobasidium pullulans (A) Conidia, (B) Conidiophores.
Bar = 50 µm
Figure 6. Curvularia brachyspora
Conidiophores. Bar = 25 µm
(A)
Conidia,
(B)
A
A
B
B
Figure 7. Curvularia lunata (A) Conidia, (B) Conidiophores. Bar =
25 µm
Figure 8. Curvularia pallescens (A) Conidia, (B) Conidiophores.
Bar = 25 µm
276
B I O D I V E R S IT A S
16 (2): 269-280, October 2015
A
A
B
B
Figure 9. Drechslera papendorfii (A) Conidia, (B) Conidiophores.
Bar = 25 µm
Figure 10. Drechslera rostrata (A) Conidia, (B) Conidiophores.
Bar = 50 µm
A
A
B
B
Figure 11. Drechslera spicifera (A) Conidia, (B) Conidiophores.
Bar = 50 µm
Figure 12. Fusariella aegyptiaca (A) Conidia, (B) Phialides. Bar
= 25 µm
ABDULWEHAB et al. – Fungi of Sudanese seeds legumes
277
A
A
B
B
Figure 13. Fusariella intermedia (A) Conidia, (B) Phialides. Bar
= 25 µm
Figure 14. Fusarium equiseti
Chlamydospores. Bar = 25 µm
(A)
Macroconidia,
(B)
(A)
Macroconidia,
(B)
A
A
B
B
Figure 15. Fusarium semitectum (A) Primary microconidia, (B)
Secondary macroconidia. Bar = 25 µm
Figure 16. Fusarium solani
Chlamydospores. Bar = 25 µm
278
B I O D I V E R S IT A S
16 (2): 269-280, October 2015
A
A
B
B
Figure 17. Ulocladium atrum (A) Conidia, (B) Conidiophores. Bar
= 25 µm
Figure 18. Ulocladium botrytis (A) Conidia, (B) Conidiophores.
Bar = 25 µm
pallescens, Drechslera rostrata, D. spicifera, F.
semitectum, Rhizopus stolonifer, and Ulocladium botrytis
were recovered from the seeds of cowpea (Table 1). Of
these isolates, 6 species are considered new to this crop
including A. alternata, Acremonium strictum, Curvularia
pallescens, D. rostrata, F. semitectum, Ulocladium botrytis
(Table 1).
Numerous saprophytic fungal genera were linked with
hazardous plant diseases to many plants. The main genera
of Alternaria, Aspergillus, Chaetomium, Cladosporium,
Curvularia, Drechslera, Fusarium, Penicillium, Rhizopus,
Ulocladium, are commonly known as saprophytes,
however, some species of these genera can cause
destructive plant diseases (Kamble et al. 1999; El-Nagerabi
and Elshafie 2002). In our results, many species of these
genera were isolated from the seed samples of the tested
legumes (Table 1) and showed high levels of contamination
(17-64%), which apparently affected seed germination (4186%) and seedling emergence (29-81%) as concluded by
many authors (Kamble et al. 1999; El-Nagerabi and Ahmed
2001; Abdelwehab et al. 2014). In similar pathogenicity
studies on different plants, some of these fungi were
considered pathogenic and caused various plant diseases;
leaf lesion of Poa pratensis (Curvularia pallescens), seed
rot of Sorghum bicolor (Drechslera spicifera), leaf spot (D.
rostrata), seedling blight (C. lunata), and Fusarium solani
wilt (Richardson 1997; Abdelwehab et al. 2014).
The fungal flora invaded the seeds of six leguminous
crops and their evident effect on seed germination and
seedling emergence were investigated. The seeds were
evidently contaminated with both saprophytic and
pathogenic mycoflora (17-64%) which apparently reduced
seed germination (41-86%), and seedling emergence (2981%). These fungal isolates are natural contaminant on
different seeds whereas some of them are new reports to
the mycoflora of these legumes and to the fungal flora of
Sudan. Therefore, their devastating effects on seed
germinations, plant growth and vigor and eventually losses
in production and productivity together with suitable
control measures need further intensive investigations and
evaluation. Setting proper seed testing method, and proper
quarantine regulations and legislations is urgently required.
ACKNOWLEDGEMENTS
We thank the Department of Botany, Faculty of
Science, University of Khartoum, Department of Biological
Sciences and Chemistry, College of Arts and Sciences,
University of Nizwa, and Department of Biology, College
of Science, Sultan Qaboos University for providing space
and facilities to carry this research. We thank the
University of Nizwa Writing Center for proof reading the
English of this article.
ABDULWEHAB et al. – Fungi of Sudanese seeds legumes
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160224
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 281-287
Diversity in antioxidant properties and mineral contents of Allium
paradoxum in the Hyrcanian forests, Northern Iran
SEDIGHEH KHODADADI1, TAHER NEJADSATTARI1,♥, ALIREZA NAQINEZHAD2, MOHAMMAD ALI
EBRAHIMZADEH3
1
Department of Biology, Science and Research Branch, Islamic Azad University, P.O. Box: 14515-775, Tehran, Iran, Tel. +9844865323, email:
nejadsattari_t@yahoo.com
2
Department of Biology, Faculty of Basic Sciences, University of Mazandaran, P.O. Box: 47416-95447, Babolsar, Iran
3
Pharmaceutical Sciences Research Center, School of Pharmacy, Mazandaran University of Medical Sciences, P.O. Box: 48471-93698 Sari, Iran
Manuscript received: 19 July 2015. Revision accepted: 5 October 2015.
Abstract. Khodadadi S, Nejadsattari T, Naqinezhad A, Ebrahimzadeh MA. 2015. Diversity in antioxidant properties and mineral
contents of Allium paradoxum in the Hyrcanian forests, Northern Iran. Biodiversitas 16: 281-287. The knowledge about variation of
antioxidant properties from local medicinal plant can be achieved by investigating all natural habitats of it. To reach this goal, a
comprehensive survey on eight populations of Allium paradoxum (M. B.) G. Don, was conducted in the Hyrcanian forests. A.
paradoxum (Amaryllidaceae) is a perennial, local plant, native to the northern Iran. Different parts of it, is largely used in food
preparation and traditional medicine. Plants were collected randomly from different altitudes and forest sites of Iranian northern
provinces ranging from west to east (Guilan, Mazandaran and Golestan Provinces). Samples were divided into aerial and bulbous parts.
The antioxidant activities of the extracts were investigated with 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and reducing power assays.
Total flavonoid content was determined by a colorimetric aluminum chloride method. The highest antioxidant activities and flavonoid
contents in A. paradoxum were related to Varaki and Zarinabad sites (Mazandaran province) with relatively higher humidity, in the
central part of the Hyrcanian area and in altitudinal range between 462-860 m asl. The content of total phenolics in the extracts was
determined according to the Folin-Ciocalteu reagent method, and calculated as gallic acid equivalents (GAE). With respect to total
phenolic contents, aerial parts of plants of Jahannama, the lowest elevation site (Golestan province), had the highest amount. Elemental
composition (Fe++, Mn++) and total sulphur were also determined using Atomic Absorption Spectroscopy and digestion method,
respectively. Higher contents of two elements, particularly Fe, and total sulphur can be found in bulbous part of Zarinabad and Kiasar
populations (Mazandaran province) compared to other sites. The results of the current study indicated that there is a remarkable and
significant variation of antioxidant activities among different studied populations of A. paradoxum in the Hyrcanian forests.
Key words: Allium paradoxum, antioxidant activity, medicinal plant, mineral contents
INTRODUCTION
The natural antioxidants present in dietary plants have
great importance in favoring health and resistance to
oxidative stress in humans (Dimitrios 2006). The total
antioxidant potential (TAP) in food products is related to
the different molecules present in foods. Antioxidant
capacities are considerably modulated by numerous factors
including biological and environmental factors, as well as
their interaction (Lisiewska et al. 2006; Moore et al. 2006).
Plant materials contain various forms of antioxidants.
Phenolic compounds are found in plants, have multiple
biological effects. Flavonoids and other phenolics have been
suggested to play a protective role against damages caused
by disease (Kähkönen et al. 1999; Mammadov et al. 2011).
Nutrients play a significant role in improving
productivity and quality of plants. Sulphur supply
influences bulb yield, plant dry matter, bulb pungency and
flavor intensity in Allium crops (Durenkamp and De Kok
2004). Sulphur insufficiency will result in the loss of plant
health, the plant’s resistance to environmental stress, and in
decreased food quality and safety (De Kok et al. 2002;
Durenkamp and De Kok 2004). Micronutrients are
involved in numerous biochemical processes and adequate
intake of certain micronutrients related to the prevention of
deficiency diseases (Ebrahimzadeh et al. 2011).
The genus Allium, a member of Amaryllidaceae family,
contains more than 900 species (APG III 2009; Li et al.
2010; Tojibaev et al. 2014). The most important diversity
source of the genus is considered in the mountainous areas
of southwest and central Asia including the territory of Iran
(Fritsch and Friesen 2002). Allium comprises many
economical, ornamental and medicinal species that have
been used as a remedy for treatment of certain diseases
(Fritsch and Friesen 2002; Putnoky et al. 2013). Among the
different medicinal plants, some endemic species may be
used for phytochemicals with significant antioxidant
capacities and health benefits (Exarchou et al. 2002).
Allium paradoxum (M.B.) G. Don is a perennial species
of shady woods in the Caucasus, northern Iran and
neighboring parts of central Asia (Vvedenskii 1935;
Wendelbo 1971; Stearn 1992). This plant is one of locally
vegetables that was known as “Alezi”, and is used as a
healthy vegetable in raw or cooked form for people who
are living in northern provinces of Iran. The populations of
the plant are obvious during late February to early April in
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16 (2): 281-287, October 2015
the Hyrcanian forests. The Hyrcanian forest is a temperate
deciduous forest ecosystem in the southeastern part of the
Caucasus biodiversity hotspot, that are located in three
northern provinces of Iran, namely, Guilan, Mazandaran
and Golestan with a total surface area of 1.84 million hectares
(Bobek 1951; Takhtajan 1986; Naqinezhad et al. 2015).
So far, most studies about Allium relating to cysteine
sulphoxides and alliinase activity (Krest et al. 2000; Fritsch
and Keusgen 2006), antioxidant activity of several Allium
members (Yin and Cheng 1998) or have focused only on
whole species such as A. paradoxum (Ebrahimzadeh et al.
2010; Nabavi et al. 2012; Elmi et al. 2014), A. sativum L.
and A. cepa L. (Benkeblia 2005; Lawrence and Lawrence
2011), A. ursinum L. (Putnoky et al. 2013). Ghasemi et al.
(2015) investigated properties of garlic under selenium and
humic acid treatments. The results of their study show the
positive effects of applied treatments. Ebrahimzadeh et al.
(2010) reported trace elemental analysis in A. paradoxum
and among them, higher contents related to Fe and Mn. There
is no report about total sulphur content in A. paradoxum.
The purpose of this study is to provide the first report
about variation in phytochemical properties of A.
paradoxum and potential sources of natural habitats in
Northern provinces of Iran. Moreover, we aim to estimate
total sulphur and trace element (Fe, Mn) contents in the
studied populations.
MATERIALS AND METHODS
Chemicals
Barium chloride, 1,1-diphenyl-2-picryl hydrazyl
(DPPH), Magnesium nitrate, Perchloric acid and potassium
ferricyanide were purchased from Sigma Chemicals Co.
(USA). Gallic acid, Quercetin and Ferric chloride were
purchased from Merck (Germany).
Study areas, plant material and preparation of extract
We selected eight forest sites (Zarinabad, Varaki,
Kalaleh, Jahannama, Haftkhal, Asalem, Savadkooh and
Kiasar) from northern provinces of Iran namely Guilan,
Mazandaran and Golestan. These sites were located in the
Hyrcanian area along an altitudinal gradient from 198 to
1980 m asl. (Figure 1, Table 1). Three Meteorological
stations, i.e. Astara in Guilan province, Gharakhil in
Mazandaran province and Hashemabad in Golestan
province were the closest stations to the current studied
sites. According to bioclimatic classification of Iran
(Djamali et al. 2011), the climate of area varies from
temperate oceanic in Astara (Guilan) to Mediterranean
pluviseasonal oceanic in Hashemabad site (Golestan).
Samples were randomly collected from aforementioned
sites during March to April 2014. The materials (aerial and
bulbous parts) were dried at room temperature for two
6
3
4
1
7
5
2
8
Figure 1. Studied sites of Allium paradoxum populations in the Hyrcanian forest. Climatic diagrams are shown from the nearest stations
in each of three provinces (from left: Guilan, Mazandaran and Golestan, respectively).
KHODADADI et al. – Antioxidant properties and minerals of Allium paradoxum
Table 1. The characteristics of eight sites for Allium paradoxum
sampling.
Forest site Province
Zarinabad
Varaki
Kalaleh
Jahannama
Haftkhal
Asalem
Savadkooh
Kiasar
Mazandaran
Mazandaran
Golestan
Golestan
Mazandaran
Guilan
Mazandaran
Mazandaran
Longitude
Latitude
E 053°12′ 19.2″
E 53°07′ 29.6″
E 055°36′ 52.1″
E 54°07′ 23.5″
E 53°23′ 59.2″
E 048°49′ 07″
E 52°48′ 29.9″
E 053°36′ 39.5″
N 36°29′ 44.4″
N 36°17′ 13.9″
N 37°24′ 35.2″
N 36°44′ 37.8″
N 36°17′ 22.1″
N 37°39′ 32.8″
N 36°06′ 27.8″
N 36°06′ 27.8″
Altitude
(m a.s.l)
462
860
698
198
897
1200
1980
1825
weeks (bulbs were oven dried at 35°C, for 2 days). Dried
materials were coarsely ground (2-3 mm) before extraction.
Each part (100 g) was extracted by percolation using
methanol/water (80/20 w/w) for 24 h at room temperature.
The extracts were then separated from the sample residues
by filtration through Whatman No.1 filter paper. The
extractions were repeated three times. The resultant
extracts were concentrated in a rotary evaporator until
crude solid extracts were obtained which were then freezedried for complete solvent removal (Ebrahimzadeh et al.
2010). The yields of each sample were presented in Tables
2 and 3.
DPPH radical-scavenging activity
The stable 1, 1-diphenyl-2-picrylhydrazyl radical
(DPPH) was used for determination of free radicalscavenging activity of the extracts (Yamaguchi et al. 1998;
Nabavi et al. 2009). Different concentrations of each
extract (100, 200, 400, 800 and 1600 µg mL-1) were added,
at an equal volume, to a methanolic solution of DPPH (100
μ M). After 15 min at room temperature, the absorbance
was measured at 517 nm. Methanol with DPPH was used
as control. The experiment was repeated three times.
Vitamin C and butylated hydroxyanisole (BHA) were used
as standard controls. IC50 values denote the concentration
of sample which is required to scavenge 50% of DPPH free
radicals. The scavenging activity was estimated based on
the percentage of DPPH radical scavenged as the following
equation: Scavenging effect (%) = [(A0- As)/A0] × 100,
where A0 is the absorbance without extract and As is the
absorbance of extracts or standards.
Reducing power determination
The reducing power of extracts was determined
according to the method of Yen and Chen (1995) (Nabavi
et al. 2008). Different amounts of each extract (50-800 μ g
mL-1) in water were mixed with phosphate buffer (2.5 mL,
0.2 M, pH 6.6) and potassium ferricyanide [K3Fe (CN)6]
(2.5 mL, 1%). The mixtures were incubated for 20 minutes
at 50°C. 2.5 mL of trichloroacetic acid (10%) was added to
the mixture to stop the reaction, then centrifuged at 3000
rpm for 10 min. The supernatant of solution (2.5 mL) was
mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL,
0.1%), and the absorbance measured at 700 nm. Vitamin C
was used as positive control.
283
Total phenol and flavonoid contents
Total phenolic contents were determined by the FolinCiocalteu reagent method (Ebrahimzadeh et al. 2008;
Singleton et al. 1999). The extract samples (0.5 mL) were
mixed with 2.5 mL of 0.2 N Folin-Ciocalteu reagent for 5
min and 2.0 mL of 75 g l-1 sodium carbonate then added.
The absorbance was measured at 760 nm after 2 h of
incubation at room temperature. The standard curve was
prepared by 0, 50, 100, 150, 200 and 250 mg mL-1 solutions of
gallic acid in methanol: water (50: 50, v/v). Results were
expressed as mg gallic acid equivalents (GAE) g-1 of extract.
Total flavonoid content was determined by a colorimetric
method (Chang et al. 2002; Ghasemi et al. 2009). 0.5 mL
solution of each plant extract in methanol was separately
mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminum
chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of
distilled water and left at room temperature for 30 min. The
absorbance of the reaction mixture was measured at 415
nm with a double beam spectrophotometer (Perkins Elmer
AAS 100).The calibration curve was prepared by preparing
quercetin solutions at concentrations 12.5 to 100 mg mL-1
in methanol. The total flavonoids were expressed as mg
quercetin equivalent (QE) g-1 of extract powder.
Determination of total sulphur content and mineral
analysis
Total sulphur of aerial parts and bulbs were measured in
the digest as described by Quin and Wood (1976). Samples
(0.1 g) were analyzed for sulphur after magnesium nitrate
and perchloric digestion. Barium chlorate was added to the
mixture and it was left overnight, following which the
absorbance of the final reaction mixture was measured at
420 nm (Ghasemi et al. 2015). Results are expressed as µg
in g dry matter. Iron and manganese were analyzed in the
samples. Briefly, the properly dried and ground plant
samples were ash-dried overnight at 400-420°C in a
Vitreosil crucible. The inorganic residue was kept in a
desiccator until needed for analysis. Two-tenths of a gram
of ash was dissolved in a 1: 3 mixture of hydrochloric and
nitric acids (Han et al. 2008) diluted to 50 mL with distilled
water and analyzed with an atomic absorption spectrometer
(Perkins Elmer AAS 100) (Wellesley, MA). The results
were expressed in mg g-1 of sample.
Statistical analysis
Experimental results are expressed as means ± SD. All
measurements were replicated three times. The data was
analyzed by one-way ANOVA and the means separated by
Tukeyʼ s post-hoc test (P <0.05).
RESULTS AND DISCUSSION
DPPH radical-scavenging activity
Scavenging of the stable DPPH radical is a widely used
method to evaluate the free radical scavenging ability of
various samples (Ebrahimzadeh et al. 2009). Results of our
study show that scavenging effect of samples is increased
by increasing their concentrations. Among these samples,
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16 (2): 281-287, October 2015
the aerial parts generally had the strongest radical
scavenging activity than bulbous parts (Tables 2 and 3).
The lowest IC50 (the highest activity) for DPPH radicalscavenging activity was 0.96 µg mL-1 for Varaki aerial
parts (Mazandaran). The IC50 values for Vitamin C and
BHA were 3.7 ± 0.1 and 29.3 ± 5.9 μ g mL-1, respectively.
Reducing power of extracts
The reducing power capacity of compounds may serve
as a significant indicator of its potential antioxidant activity
(Meir et al. 1995). This reducing power capacity was
determined using a Fe3+ to Fe2+ reduction system. The
amount of Fe2+ complex can be monitored by measuring
the formation of Perl’s Prussian blue at 700 nm. Increasing
absorbance at 700 nm indicates an increase in reductive
ability. Figures 2 and 3 show the dose-response curves for
the reducing power of extracts (at 50-800 μ g mL-1
concentration). The reducing power of extracts also
increased with increasing their concentrations. There were
significant differences between extracts and vitamin C (P <
0.01). The highest reducing power activity was observed in
aerial parts of Varaki site (1.32, at 800 μ g mL-1). The aerial
part extracts showed higher reducing power than bulb extracts.
Extraction yield, total phenol and flavonoid contents
The extraction yield of different fractions of A.
paradoxum was varied from 4.7 to 26.1% (Tables 2 and 3).
The extraction of aerial parts resulted in the higher amount
of total extractable compounds than bulb extracts. The
amount of total phenolics varied in different populations
and ranged from 5.7±0.5 to 112.4±7.5 mg GAE g-1 of
extract. The highest total phenolic contents were 112.4±7.5
and 111.2±5.4 mg GAE g-1 of extract, bulbs and aerial parts
of “Jahannama” and “Varaki” samples, respectively. The
content of flavonoid varied from 1.3 to 294.33±11.3 mg
QE g-1 of extract powder (Tables 2 and 3). Aerial part
extracts had significantly higher flavonoid contents than
bulb extracts. Plants of “Zarinabad” site (Mazandaran
province) showed the highest amount of flavonoid contents
followed by Varaki (294.33±11.3 and 173±4.4,
respectively).
Total sulphur and mineral analysis
The results of the current study showed that bulbous
parts had higher total sulphur than aerial parts. According
to Tables 2 and 3, total sulphur content in A. paradoxum
ranged from 69-210 g in g dry matter. The highest sulphur
content was found in bulbs of Kiasar (Mazandaran
province). Results of elemental analysis were presented in
Tables 2 and 3. The comparative study showed that the
bulbous part of Zarinabad sample had the maximum value
of Fe and Mn contents.
Discussion
Investigation on antioxidant properties with particular
concern of geographical distribution of plant populations
has become of interest of current relevant studies (Pisoschi
and Negulescu 2011). This kind of research can be also
very important particularly on endemic and other
restricted-range plants.
Figure 2. Reducing power of aerial parts extracts of Allium
paradoxum populations. Vitamin C used as control. Numbers
show studied sites. For names of 1-8 see Figure 1.
Figure 3. Reducing power of bulbous parts extracts of Allium
paradoxum populations. Vitamin C used as control. Numbers
show studied sites. For names of 1-8 see Figure 1.
Antioxidant agents of natural origin have attracted
special interest because of their free radical scavenging
abilities. Because of this, the antioxidant activities of A.
paradoxum populations were determined by two
spectrophotometric methods. The DPPH method rely on
the reaction of 1, 1-diphenyl-2-picrylhydrazyl radical with
an antioxidant molecule. It decolorizes the DPPH solution
and the degree of color change is proportional to amount of
the antioxidants (Saeed et al. 2012). In reducing power test,
the presence of the reductants in the solution causes the
reduction of the Fe3+/ferricyanide complex to the ferrous
form. Therefore, Fe2+can be monitored by absorbance
measurement at 700 nm. In the present study, the results of
two antioxidant tests showed that the aerial part extract of
Varaki population in Mazandaran province had higher
antioxidant action than other populations. Based on
altitudinal range of current survey (198- 1980 m a.s.l)
(Table 1), this site is located in the middle of the cited range.
Although phenolic acids and flavonoids are not
essential for survival, may provide protection against a
number of chronic diseases over the long term consumption
(Bravo 1998). Variation of total phenol and flavonoids in
our study area showed that the plant extracts of all
populations had the potential for accumulating phenol and
flavonoids. Bulbous part of Jahannama population (eastern
part of our study area with low rainfall) possessed the
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160224
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 281-287
Table 2. Yield, total phenolic and flavonoid contents of methanolic extract, total sulphur and trace elements from aerial part of different populations of Allium paradoxum in Iran.
Parameters
Zarinabad
-1
a
Total phenol content (mg GAE g )
64.4 ± 3.2
-1
a
Varaki
b
111.2 ± 5.4
b
Kalaleh
Jahannama
Haftkhal
Asalem
c
54.4 ± 3.2
af
62.6 ± 2.6
d
c
47.4 ± 3.6
56.4 ± 2.8
c
d
e
c
Savadkooh
e
5.7 ± 0.5
f
Kiasar
f
60.6 ± 2.2
g
F
2325.84*
Flavonoid content (mg QE g )
294.3 ± 1.3
173 ± 4.4
80.3 ± 3.5
51.0 ± 1.3
31.7 ± 2.2
77.8 ± 3.4
61.3 ± 1.1
103.7 ± 5.1
29300.50*
DPPH radical cavenging, IC50 (μ g mL-1)
1.50 af± 0.01
0.96 b± 0.00
1.43 acf± 0.01
1.56 adf±0.01
1.38 c± 0.01
1.46 acf± 0.01
1.66 df± 0.01
1.57 adf± 0.00
101.83*
a
81 ± 3.2
Total sulphur (g in g dry matter)
-1
a
b
99 ± 3.1
a
c
110 ± 5.2
bc
d
120 ± 3.7
bc
c
110 ± 5.1
a
e
69 ± 1.9
ab
f
88 ± 2.3
b
d
120 ± 5.8
a
1056.80*
Fe (mg g )
0.93 ± 0.03
0.82 ± 0.03
3.35 ± 0.11
3.19 ± 0.17
0.54 ± 0.01
1.41 ± 0.10
2.40 ± 0.16
0.77 ± 0.02
25.04*
Mn (mg g-1)
0.05 a± 0.01
0.05 a± 0.01
0.07 ab± 0.01
0.08 b± 0.01
0.03 ac± 0.00
0.05 a± 0.01
0.05 a± 0.00
0.04 ac± 0.00
7.50*
Extraction yield (%)
18
21.2
19.8
18.2
21.2
20.1
26.1
23.2
Note: IC50 of BHA was 29.3 ± 5.9 μ g mL-1. Each value in Table 2 is represented as mean SD (n=3) and F-ratio (*) based on one-way ANOVA of studied variables. Values in the same row
followed by a different letter are significantly different (p<0.05).
Table 3. Yield, total phenolic and flavonoid contents of methanolic extract, total sulphur and trace elements from bulbous part of different populations of Allium paradoxum in Iran.
Parameters
Zarinabad
-1
a
Varaki
a
Kalaleh
a
Jahannama
b
Haftkhal
c
Asalem
d
Savadkooh
e
Kiasar
f
F
Total phenol content (mg GAE g )
47.2 ± 2.1
46.8 ± 1.7
49.2 ± 2.2
112.4 ± 7.5
30.2 ± 1.5
36.4 ± 1.9
59 ± 1.6
80.8 ± 4.1
2088.90*
Flavonoid content (mg QE g-1)
4.3 a± 0.2
1.3 ac± 0.0
1.7 ac± 0.0
7.7 b± 1.2
4.0 a± 0.1
5.3 ab± 1.2
3.3 a± 0.0
6.0 ab± 0.2
11.93*
-1
acd
DPPH radical cavenging, IC50 (μ g mL )
2.32
Total sulphur (g in g dry matter)
120 a± 4.4
-1
Fe (mg g )
-1
Mn (mg g )
± 0.01
a
17.30 ± 0.24
a
0.1 ± 0.01
3.19
abcdf
± 0.02
81 b± 1.2
b
2.50 ± 0.09
a
0.05 ± 0.00
2.34
acd
± 0.02
120 a± 4.9
2.25
bcd
± 0.13
a
0.07 ± 0.00
1.78
acde
± 0.01
150 c± 6.6
bd
2.80 ± 0.18
a
0.06 ± 0.01
4.21
bcf
± 0.02
2.89
abcd
± 0.02
180 d± 6.4
110 e± 5.7
c
bc
0.92 ± 0.04
a
0.02 ± 0.00
1.05 ± 0.15
a
0.02 ± 0.00
b
acde
3.51 ± 0.02
1.72
112 e± 4.1
210 f± 6.4
1.95
bcde
± 0.18
a
0.01 ± 0.00
1.43
± 0.01
14.67*
5311.66*
bcde
28.21*
a
2.20
± 0.1
0.05 ± 0.01
Extraction yield (%)
9.8
8.1
6.4
4.7
6.1
11.6
11.0
12.3
-1
Note: IC50 of BHA was 29.3 ± 5.9 μ g mL . Each value in Table 3 is represented as mean SD (n=3) and F-ratio (*) based on one-way ANOVA of studied variables. Values in the same row
followed by a different letter are significantly different (p<0.05).
B
I O D I V E R S IT A S
286
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B I O D I V E R S IT A S 16 (2): 269-280, October 2015
highest amount of total phenol. In most measurements of
the current experimental analysis, aerial parts had higher
contents than bulbs that are compatible with the results of
Ebrahimzadeh et al. (2010).
Minerals are usually divided in two groups macro- and
micro- minerals (or trace elements), and also classified as
either essential or nonessential, depending on whether or
not they are required for human nutrition and have
metabolic roles in the body (Reilly 2002). Sulphur content
of plant varies from 0.03-2 mmol g-1 dry weight (DW) in
different species (Durenkamp and De Kok 2004). In the
current study, total sulphur content of bulbs showed higher
amounts than in aerial parts. Study of Ghasemi et al. (2015)
showed that total sulphur of A. sativum is in higher amount
than in A. paradoxum. Moreover, the results of our study
revealed that iron concentration is higher than manganese.
However, this result can be varied in aerial and bulbous
parts. Fe and Mn play vital roles in biochemical processes
(Ebrahimzadeh et al. 2010, 2011). Element composition
differs strongly between plant organs (Minden and Kleyer
2013). Variability may also come from differences in size,
age, ontogenetic state and reproductive status between
plant individuals (Agren 2008).
The results of this study demonstrated variation of
antioxidant properties and mineral contents in plant organs
of A. paradoxum populations along the Hyrcanian forests.
The results generally indicated that populations of A.
paradoxum in Mazandaran province had good capacity to
utilize in medicine and food industry. Further field studies
are needed to explain precise correlation between variation
of mentioned properties in A. paradoxum and the
environmental factors.
ACKNOWLEDGEMENTS
We thank M. Khalili (Mazandaran University of
Medical Sciences, Iran), Dr. S. Kelij and M. Bordbar
(University of Mazandaran, Iran) for their support and
helps during the laboratory works.
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DOI: 10.13057/biodiv/d160225
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 288-294
Genetic and morphological diversity in Cousinia cylindracea
(Asteraceae) populations: Identification of gene pools
1
AMIR ABBAS MINAEIFAR1,♥, MASOUD SHEIDAI1, FARIDEH ATTAR2
Faculty of Biological Sciences, Shahid Beheshti University, Central Campus, Tehran-1983963113, Iran. Tel.: +98 21 22434501, +98 21 29902721,
♥
email: aaminaeifar@gmail.com
2
Faculty of Biology, Tehran University, Tehran, Iran
Manuscript received: 1 August 2015. Revision accepted: 16 September 2015.
Abstract. Minaeifar AA, Sheidai M, Attar F. 2015. Genetic and morphological diversity in Cousinia cylindracea (Asteraceae)
populations: Identification of gene pools. Biodiversitas 16: 288-294. Cousinia is one of the largest genera in the Asteraceae family. It
contains 600 to 700 species distributed in Southwest and Central Asia. In Iran with 270 species it is the largest genus after Astragalus,.
Cousinia probably is unique in the degree of diversification of all its parts and high numbers of species in restricted area. In this
investigation 90 plant specimens of 10 geographical populations of Cousinia cylindracea Boiss. were studied from morphological and
genetic (ISSR) points of view. Both intra and inter-population morphological and genetic variability was observed in the studied
populations. ANOVA and CVA tests revealed significant morphological difference among these populations. Similarly, AMOVA tests
revealed significant molecular difference among geographical populations. Mantel test produced significant positive correlation between
genetic distance and geographical distance of the studied populations. Networking, STRUCTURE analysis and population assignment
test revealed low degree of gene flow among these populations. The results identified two different gene pools of C. Cylindracea in Iran,
supporting Rechinger suggestion that C. cylindracea might have two varieties in Iran.
Key words: Asteraceae; Cousinia; genetic variability; gene pools; ISSR; morphological diversity
INTRODUCTION
Cousinia Cass. is one of the largest genera in the family
Asteraceae and the largest genus in tribe Cardueae (Frodin
2004). It contains 600 to 700 species in Southwest and
Central Asia. The distribution area of Cousinia is nearly
identical with the Irano-Turanian region (Knapp 1987). In
Iran, after Astragalus, Cousinia is the largest genus with
over than 270 species and 43 sections. Cousinia species are
distributed in mountainous parts of Iran. Some of the
Cousinia species have medicinal values, and used as
diuretic, antiseptic and antibacterial (Joudi et al. 2011).
Cousinia cylindracea Boiss., is distributed in more than
10 provinces of Iran from Alborz and Zagros ranges (Zar et
al. 2012). It is a perennial herb, with decurrent stem leaves,
and small head bearing a few florets (Rechinger 1979).
Cytological study on C. cylindracea shows chromosome
number of 2n=2x=26 (Djavadi 2012).
One of the main concerns of mankind is the
conservation of biodiversity at present time. Increasing
human population and more extensive use of natural
resources and space, cause damage to plant biodiversity. To
conserve biological diversity, we must first understand the
distribution of organisms and how and why these
organisms are geographically distributed as they are
(Sigrist and Carvalho 2008). Population genetic
investigation is one of the main steps for understanding the
population genetic structure and fragmentation, inter-
population gene flow and diversification (Sheidai et al.
2014).
Habitat fragmentation generally is expected to reduce
genetic diversity and to increase inter-population genetic
divergence by restricting gene flow among fragmented
populations, increasing inbreeding and increasing random
genetic drift within populations (Hou and Lou 2011). An
examination of the genetic diversity among populations
within a species is crucial for a better understanding of
evolutionary processes and the nature of the species.
Cousinia cylindracea is a species that grow in different
geographical regions of Iran and forms several local
populations. Therefore, the present study was performed to
identify the population genetic structure, gene flow and
morphological diversity of C. cylindracea populations and
to identify probable gene pools of this species in the
country. This information can be used in conservation
program of this plant species.
Molecular markers that have been used in plants'
investigations in general, consist almost exclusively of
markers deemed to be neutral or nearly neutral. These data
have been used to study the speciation process, genetic
diversity analysis as well as populations' genetic structure
(Sheidai et al. 2014). We used ISSR (Inter simple sequence
repeats) molecular markers for genetic diversity analysis,
as these molecular markers are reproducible, easy to work,
cheap and also provide useful data for evolutionary and
population genetic studies (Sheidai et al. 2014).
MINAEIFAR et al. – Diversity in Cousinia cylindracea populations
MATERIAL AND METHODS
Plant materials
Extensive field visits and collections were undertaken
during 2013-2014 from the North-West to South- West of
Iran and several geographical populations were identified
for Cousinia species including C. cylindracea (Figure 1).
Many plant specimens were randomly collected from 10
geographical populations (Table 1). We used 90 randomly
collected plant specimens for genetic and morphological
studies. The fresh leaves were used for DNA extraction.
The voucher specimens were deposited in Herbarium of
Shahid Beheshti University (HSBU) and Central
Herbarium of Tehran University (TUH).
Morphometry
Forty-seven morphological characters were studied (38
quantitative and 9 qualitative characters) such as: the basal,
apical and stem leaves length and width, length and width
and number of head, number and length of florets, seeds
length and width, bracts shape and etc. (Table 2).
ISSR assay
DNA was extracted from the fresh leaves that were
randomly collected from 9 plants in each population and
dried in silica gel powder. The genomic DNA was
extracted using CTAB-activated charcoal protocol (Murray
and Thompson 1980). The extraction procedure was based
on activated charcoal and Polyvenyl Pyrrolidone (PVP) for
binding of polyphenolics during extraction and on mild
extraction and precipitation conditions. This promoted
high-molecular weight DNA isolation without interfering
contaminants. Quality of extracted DNA was examined by
running on 1% Agarose gels.
289
Ten ISSR primers; (AGC)5GT, (CA)7GT, (AGC)5GG,
(GA)9A, (GA)9C, UBC 807, UBC810, UBC 811, UBC
823 and UBC 834 commercialized by UBC (the University
of British Columbia) were used. PCR reactions were
performed in a 25µL volume containing 10 mM Tris- HCl
buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of
each dNTP ; 0.2 µM of a single primer; 20 ng genomic
DNA and 3 U of Taq DNA polymerase. Amplification
reactions were performed in Techne thermocycler
(Germany) with the following program: PCR was carried
out with an initial denaturing step (94oC/5 min), followed
by 45 cycles of 94oC/30 s, 52-58oC/30 s, 72oC/1 min, and a
final incubation at 72oC for 10 min. the amplification
products were visualized by running on 1.5% Agarose gel,
followed by Ethidium bromide staining. The fragments size
was estimated by using a 100 bp molecular size ladder
(Fermentas, Germany). The experiment was replicated 3
times and constant ISSR bands were used for further
analyses.
Data analyses
Morphological studies
The analysis of variance (ANOVA) test was performed
to show significant morphological difference among the
studied populations. For grouping of the plant specimens,
UPGMA (Unweighted paired group with arithmetic
average) and CVA (Canonical variate analysis) were used.
Morphological data were standardized (mean = 0, variance
= 1) for these analyses (Podani 2000). Principal
components analysis (PCA) was performed to identify the
most variable morphological characters among the studied
populations.
Figure 1. Distribution map of the studied Cousinia cylindracea populations in Iran
290
B I O D I V E R S IT A S 16 (2): 288-294, October 2015
Table 1. Cousinia cylindracea populations and their localities
Pop.
Locality
1
2
3
4
5
6
7
8
9
10
Iran: Hamadan, Alvand
Iran: Hamadan, Asadabad Pass.
Iran: Zanjan, Abhar
Iran: Zanjan, Soltania
Iran: Zanjan, Zanjan
Iran: Tehran, Firoozkooh
Iran: Kohgiluyeh and Boyer-Ahmad, Bijan pass.
Iran: Kohgiluyeh and Boyer-Ahmad, Dejgerd
Iran: Fars, Eqlid
Iran: Isfahan, Semirom
Altitude
(m)
2038
2150
2145
1975
1850
2152
2950
2335
2410
2250
Longitude
Latitude
48o 26' 51"
48o 11' 45"
48o 58' 57"
48o 43' 03"
48o 22' 06"
52o 32' 13"
51o 30' 45"
51o 55' 49"
52o 36' 29"
51o 48' 02"
34o 48' 07"
34o 49' 55"
36o 07' 11"
36o 22' 39"
36o 26' 30"
35o 41' 06"
30o 52' 21"
30o 39' 21"
30o 53' 34"
31o 45' 53"
Temperature
(oC)
10
9
10
9
9
8
9
10
12
11
Rainfall
(mm)
350
400
350
350
350
400
800
700
350
400
Table 2. Morphological characters in the studied populations
R
Characters
R
Characters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Stem length (mm)
Basal leaves length (mm)
Basal leaves width (mm)
Basal leaves length/Width Ratio
Basal leaves petiole length (mm)
Basal leaves terminal spin length (mm)
Basal leaves marginal spins length (mm)
Basal leaves teeth number
Stem leaves number
Stem leaves length (mm)
Stem leaves width (mm)
Stem leaves length/width ratio
Stem leaves terminal spin length (mm)
Stem leaves marginal spins length (mm)
Stem leaves teeth number
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
16
17
Uppermost leaves number
Uppermost leaves length (mm)
40
41
18
19
Uppermost leaves width (mm)
Uppermost leaves length/widthr
42
43
20
21
Uppermost leaves terminal spin length (mm)
Uppermost leaves marginal spins length (mm)
44
45
22
Uppermost leaves teeth number
46
23
24
Head number
Head length (mm)
47
Head width (mm)
Head length/width Ratio
Involucral bracts number
Bracts seriate number
Florets number
Florets length (mm)
Corolla limb length (mm)
Corolla tube length (mm)
Limb length / tube length ratio
Receptacle bristles length (mm)
Seed number
Seed length (mm)
Seed width (mm)
Seed length / Width Ratio
Stem indumentum (0: Glabrous; 1: Low density; 2: Medium
density; 3: High density)
Basal leaves form (0: Dentate; 1: Pinnatifid; 2: Pinnatisect)
Basal leaves indumentum (0: Glabrous; 1: Low density; 2: Medium
density; 3: High density)
Stem leaves form (0: Dentate; 1: Pinnatifid; 2: Pinnatisect)
Stem leaves indumentum (0: Glabrous; 1: Low density; 2: Medium
density; 3: High density)
Uppermost leaves form (0: Dentate; 1: Pinnatifid; 2: Pinnatisect)
Uppermost leaves indumentum (0: Glabrous; 1: Low density; 2:
Medium density; 3: High density)
Head indumentum (0: Glabrous; 1: Low density; 2: Medium
density; 3: High density)
Bract shape (0: Spreading; 1: Curved)
Molecular analyses
Genetic diversity and population differentiation.
ISSR bands obtained were coded as binary characters
(presence = 1, absence = 0). Genetic diversity parameters
were determined for dominant molecular markers in each
population. These parameters were Nei's gene diversity
(H), Shannon information index (I), number of effective
alleles, and percentage of polymorphism (Freeland et al.
2011). Nei's genetic distance was determined among the
studied populations and used for clustering. Significant
genetic differences among the studied populations were
determined by: 1-AMOVA (Analysis of molecular
variance) test (with 1000 permutations) for dominant
molecular markers as implemented in GenAlex 6.4 (Peakall
and Smouse 2006), 2- Nei,s Gst analysis of dominant
markers as implemented in GenoDive ver.2 (2013)
(Meirmans and Van Tienderen 2004). Furthermore,
populations, genetic differentiation was studied by G'st_est
= standardized measure of genetic differentiation (Hedrick
2005), and D_est = Jost measure of differentiation (Jost
2008).
MINAEIFAR et al. – Diversity in Cousinia cylindracea populations
In order to overcome potential problems caused by the
dominance of ISSR markers, a Bayesian program, Hickory
(ver. 1.0) was used to estimate parameters related to
genetic structure (Theta B value) (Tero et al. 2003).
Grouping of the populations. For grouping of the
plant specimens, we used Neighbor Joining (NJ) clustering
and Neighbor Net method of networking after 100 times
bootstrapping (Huson and Bryant 2006). The Mantel test
was performed to check correlation between geographical
distance and genetic distance of the studied populations
(Podani 2000). PAST ver. 2.17 (Hamer et al. 2012),
DARwin ver. 5 (Cirad 2012) and SplitsTree4 V4.13.1
(Huson and Bryant 2013) programs were used for these
analyses.
Population genetic structure. The genetic structure of
geographical populations was studied by two methods;
First we carried out structure analysis (Pritchard et al.,
2000), for dominant markers (Falush et al. 2007). Second,
we performed K-Means clustering as done in GenoDive
ver. 2. (2013). Model-based clustering was performed by
STRUCTURE software ver. 2.3 (Pritchard et al. 2000). The
Markov chain Monte Carlo simulation was run 20 times for
each value of K (2-10) for 20 iterations after a burn-in
period of 105. Evanno test was carried out to identify the
proper number of K (Evanno et al. 2005). We used two
summary statistics to present K-Means clustering; 1pseudo-F (Calinski and Harabasz 1974) and 2- Bayesian
Information Criterion (Schwarz 1978).
Gene flow. The occurrence of gene flow among
populations was checked by different methods. First we
performed indirect Nm analysis of POPGENE ver. 2 for
ISSR loci studied according to the following formulae:
Nm = 0.5 (1 - Gst)/Gst.
Then we used STRUCTURE plot based on admixture
model. Finally, the population, assignment test was
performed by using maximum likelihood method as
implemented in GenoDive ver.2 (2013) (Meirmans and
Van Tienderen 2004). Frichot et al. (2013) latent factor
mixed models (LFMM) was used to check if ISSR markers
show correlation with environmental features of the studied
populations. The analysis was done by LFMM program
Version: 1.2 (2013).
RESULTS AND DISCUSSION
Morphometry
ANOVA test revealed significant difference in quantitative
morphological characters among the studied populations (P
<0.05). Moreover, CVA plot (Figure 2) separated the
studied populations based on all morphological characters
including both quantitative and qualitative characters,
supporting ANOVA result. In general, two major clusters
were formed in UPGMA tree (Figure not given),
populations 1, 2, 7, 8, 9 and 10 showed morphological
similarity and were placed in the first major cluster, while
populations 3, 4, 5 and 6 formed the second major cluster.
PCA plot supported the grouping made by UPGMA tree and
291
also revealed some degree of intra-population
morphological variability. Therefore, combination of
UPGMA and PCA plot indicated morphological divergence
among the studied populations.
Genetic diversity analysis
AMOVA test revealed significant genetic difference
among the studied populations (P = 0.01). It also revealed
that 58% of total genetic variability occurred among
populations while, 42% occurred within populations. These
results indicated the presence of high level of genetic
variability both within and among C. cylindracea
populations. This conclusion was supported by Gst and
Hickory analyses. The Gst value obtained among
populations after 999 permutations was 0.557 (P = 0.001)
and Theta B value = 0.40, which is significant. Population
genetic differentiation was shown by high values obtained
for Hedrick's standardized fixation index after 999
permutation (G'st=0.66, P=0.001) and Jost's differentiation
index (D-est = 0.232, P = 0.001).
Populations grouping based on genetic data
The Population grouping based on ISSR data by NJ tree
and Neighbor-Net diagram produced similar results.
Therefore, Neighbor-Net diagram (Figure 3) is presented
and discussed here. In general two major clusters were
formed. Neighbor Net diagram revealed closer genetic
affinity between populations No. 3-6, and also between
population No. 1, 2, 7-10.
Population's genetic structure and gene flow
K-Means clustering and Evanno test performed on
STRUCTURE analysis produced the best number of gentic
groups as k=2. This genetic grouping is in agreement with
NJ tree result, presented before. Therefore, two gene pools
are identified for C. cylindracea in the country.
STRUCTURE plot (Figure 4) revealed close genetic
affinity between populations No. 3-6 (the first gene pool)
and also between population numbers 1, 2, 7-10 (the
second gene pool). This is in agreement with NeighborNet
diagram. STRUCTURE plot also indicated very low degree
of genetic admixture between the two gene pools.
Moreover, mean Nm value of 0.40 was obtained for the
studied populations that showed low value of gene flow,
supporting STRUCTURE plot result. LFMM analysis
revealed that many of the studied ISSR loci were
significantly (P<0.05) correlated with the environmental
features studied. These may have adaptive values and being
used by plants of either gene pools for local adaptation.
Among these loci, some had low Nm value (<0.50) such as
ISSR loci 3, 5, 6, 7, 11, 12, 14, 16, 17, 24, 31-35, 48, and
50-52. However, some other loci had high Nm value of
>1.00 or even much higher. For example, ISSR loci 22, 23,
27, 49, and 54.
Discussion
Genetic diversity is an important factor for adaptation
of plants to the environmental changes they encounter. In
general those populations that have high level of genetic
variability may have better chance of survival compared to
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B I O D I V E R S IT A S 16 (2): 288-294, October 2015
Figure 2. CVA plot of populations based on morphological characters. numbers indicate the plant specimens (Numbers 1-10) studied
from each geographical population
Figure 4. STRUCTURE plot of in Cousinia cylindracea populations studied
Figure 3. Neighbor Net diagram of ISSR data
the ones with lower degree of genetic diversity (Sheidai et
al. 2012, 2013).
The present study revealed a high level of within
population and among population genetic variability in C.
cylindracea. The occurrence of high within population
genetic diversity has been reported in other plant species
and outcrossing nature of these species has been suggested
to be the reason for that (Bodo-Slotta et al. 2010). The
same may holds true for C. cylindracea populations.
An examination of the genetic diversity among
populations within a species is crucial for a better
understanding of evolutionary processes and the nature of
the species. AMOVA and Hickory tests revealed
significant genetic difference among the studied
populations. Moreover, the low value of Nm (0.40)
obtained for the studied populations showed low value of
gene flow. Due to limited gene flow among populations,
genetic differentiation increases. In situations with
complete absence or very limited amount of gene flow,
genetic drift is a strong evolutionary force and brings about
high degree of within populations genetic homogeneity.
MINAEIFAR et al. – Diversity in Cousinia cylindracea populations
This may lead to adaptation to local habitats (Hou and Lou
2011). In fact, many plant species grow within a range of
different habitats and have developed adaptive strategies
suited to their particular habitat (Schneller and Liebt 2007).
STRUCTURE analysis revealed population genetic
fragmentation in C. cylindracea and identified the presence
of two gene pools for this species in Iran. Amongpopulation differentiation in phenotypic traits and allelic
variation is expected to occur as a consequence of genetic
fragmentation, isolation, drift, founder effects and local
selection (Jolivet and Bernasconi 2007). In fact the
populations of the two gene pools in C. cylindracea
differed in their morphological characters too. The
populations 3-6 are different from other population in some
characters such as: bract shape, stem leaves, uppermost
leaves, head indumentums and head number. This finding
is in agreement with Rechinger suggestion, that the C.
cylindracea may contain two different varieties (Rechinger
1979). Therefore, genetic fragmentation followed by local
adaptation might be the reason for the presence of two
different taxonomic forms (varieties) in C. cylindracea. In
many studies different ecotypes were identified and formed
due to among populations genetic differentiation followed
by population morphological divergence (Sheidai et al.
2012, 2013).
It is interesting to mention that populations 1 and 2 in
close-by geographical locations (Figure 1), but they
showed completely different genetic make up and belonged
to two different gene pools. It seems they are genetically
isolated by ranges of mountains such as: Kharagan
mountain, Cheragi mountain, Ozonbolag mountain. The
presence of strong isolation by distance and genetic
differentiation of the studied populations was evidenced by
significant Mantel test and population differentiation
indices presented before. The populations growing on
mountains not only are differentiated on the mountains
along vertical axes, but genetic changes can also occur
along horizontal axes. For instance, ridges may provide
geographical barriers to gene flow between populations on
their opposite sides, so genetic differentiation may occur
across ridges (Taberlet et al. 1998).
Assessments of levels of within- and among-population
genetic variation have been used to prioritize populations
for conservation efforts (Petit et al. 1998) with, all else
being equal, more weight given to those exhibiting higher
levels of within-population variation, and to those that are
more genetically divergent from others. These populations
may have increased likelihood of persistence over less
variable population and hence the ability of a population to
contribute demographically to the species through time,
and have increased adaptability in the face of future
environmental changes. LFMM result showed that along
with genetic drift, low degree of gene flow and migration,
adaptive loci also helped populations to diverge and adapt
these plants to their local condition. Therefore we have a
new taxonomic group bellow the species level that can be
considered as a new variety for C. cylindracea based on
morphological and genetic data. We shall publish details of
this new variety in future publication.
293
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160226
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 295-302
Threats and conservation of Paris polyphylla an endangered, highly
exploited medicinal plant in the Indian Himalayan Region
1
2
ASHISH PAUL1,♥, PADMA RAJ GAJUREL2, ARUP KUMAR DAS1
Department of Botany, Rajiv Gandhi University, Rono Hills, Doimukh-791112, Arunachal Pradesh, India.
Tel.: +91-9862035885, email: ashishpaul1@gmail.com
Department of Forestry, North Eastern Regional Institute of Science and Technology, Deemed University, Nirjuli-791109, Arunachal Pradesh, India
Manuscript received: 29 August 2015. Revision accepted: 23 October 2015.
Abstract. Paul A, Gajurel PR, Das AK. 2015. Threats and conservation of Paris polyphylla an endangered, highly exploited medicinal
plant in the Indian Himalayan Region. Biodiversitas 16: 295-302. The Indian Himalayan Region is home of numerous globally
significant medicinal plants. Paris polyphylla Smith is an important medicinal perennial herbaceous species used mostly in traditional
medicine, having medicinal properties like anticancer, antimicrobial, antioxidant, anti-tumor, cytotoxicity, steroid saponins etc. The
present study highlights the uses, population status and threats to P. polyphylla in Arunachal Pradesh. P. polyphylla is distributed in
tropical to temperate region of South East Asia, particularly in Bhutan, China, India, Laos, Myanmar, Nepal, Thailand and Vietnam. In
India it is distributed in the state of Arunachal Pradesh, Himachal Pradesh, Jammu and Kashmir, Manipur, Meghalaya, Mizoram,
Nagaland, Sikkim and Uttarakhand. In the Eastern Himalayan state of Arunachal Pradesh, the species found to be occurring with distinct
morphological interspecific variations. In the past 5 years the market demand of the species increased tremendously, which ultimately
led to the over exploitation of the species and traded illegally in heavy quantities. The present study showed very poor population
density, which ranged between 0.42 individuals m-2 to 1.48 individuals m-2. While, Importance Value Index of the species ranged
between 3.37 to 8.45. Because of the unsustainable extraction and poor natural regeneration of the species, wild populations are at risk
of extinction and accordingly it has been listed as an endangered species. The rhizome is the primary mode of regeneration, although it
regenerate from seeds. Because of the commercial demand of the rhizome, the population of the species may entirely be wiped out if
proper conservation initiatives have not been taken. Effective conservation strategies both in situ and ex situ may help to protect the
species from its extinction. Inclusion of the species under the priority species list of both the National and State Medicinal Plant Boards
for cultivation may be helpful for its long term management and conservation. Mass awareness and active involvement of local people
for large scale cultivation may reduce the pressure on wild populations. This will meet the market demand and boost the rural economy
and will also help in conservation of the species.
Keywords: Paris polyphylla, Himalayan region, Arunachal Pradesh, economic value, over exploitation, population status, conservation
INTRODUCTION
Forests play a significant role in life and wealth of
human beings. It provides natural resources like fuel,
timber, industrial forest products, wildlife habitat, animal
products, etc. and also food (fruits, tubers, leaves, meat),
medicines and many other commercial products. However,
increasing anthropogenic disturbances and unsustainable
extraction have caused the extinction of many species with
ecological and economic importance. Conservation and
management of these species have become a major concern
to the scientific community in this 21st century. Human
activities associated with deforestation, fragmentation,
habitat loss, habitat degradation, over exploitation,
unsustainable extraction, etc. have collectively built up the
pressure on existence of many aromatic, medicinal,
ethnobotanical, economically important plant species such
as Aconitum spp., Coptis teeta, Cordyceps sinensis,
Embelia ribes, Gymnocladus assamicus, Gynocardia
odorata, Homalomena aromatica, Illicium griffithii, Panax
spp., Podophyllum hexandrum, Rhododendron spp.,
Swertia chirayita, Taxus wallichiana, etc. in the Himalayan
region which have been regarded as home of many
valuable aromatic and medicinal plant species. Present
rapid loss of genetic resources due to various
anthropogenic activities will not only affect the
local/regional biodiversity but also affect the various
ecosystem services.
Taxus wallichiana Zuccarini provides an example
which, become endangered due to unsustainable extraction
for its anti-cancerous chemical Paclitaxel (Taxol®), the
most effective drug used for a variety of cancers (Cragg et
al. 1993; Goldspiel 1997). In recognition of its anticancerous properties during 1980s the species has suffered
large scale unsustainable extraction, which led to its
present status (Paul et al. 2013). It is the most threatened
species and has been categorized as endangered by the
IUCN (Thomas and Farjon 2011). Today history is again
repeating for many other species, including Paris
polyphylla Smith because of its high commercial demand.
The genus Paris L. belongs to the family Liliaceae of
monocots comprises rhizomatous herbaceous species. The
genus comprises 24 species which are distributed in
Bhutan, China, India, Japan, Korea, Laos, Mongolia,
Myanmar, Nepal, Russia, Thailand, Vietnam and Europe
(Liang and Soukup 2000). China has the highest number of
species (22 species) with 12 endemic species. In India the
genus is represented by 2 species, namely P. polyphylla
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B I O D I V E R S IT A S 16 (2): 295-302, October 2015
and P. thibetica with about 6 intraspecific taxa (Liang and
Soukup 2000). The P. polyphylla has been widely known
specifically with different subspecies and varieties. It is
distributed in Bhutan, China, India, Laos, Myanmar, Nepal,
Thailand and Vietnam (Liang and Soukup 2000). In India
the species have been recorded from the Himalayan states
like Arunachal Pradesh (Chowdhery et al. 2009), Himachal
Pradesh (ENVIS 2010; Goraya et al. 2013), Jammu and
Kashmir (ENVIS 2010), Manipur (Gogoi 2010),
Meghalaya (Mao et al. 2009; Mir et al. 2014), Mizoram
(Lalsangluaii et al. 2013), Nagaland (Jamir et al. 2012),
Sikkim (Maity et al. 2004) and Uttarakhand (ENVIS 2010).
It is a rhizomatous herb grows up to a height ranging from
10-100 cm and distributed in an altitudinal range between
100-3500 m asl. The whorled leaves number ranging from
5-12 with a long petiole (stalk) and flower with leafy bracts
on a long stalk are the main identifying characters. The
fruit is berry in nature, oval or round in shape with many
seeds and become red on ripening. Seeds are enveloped by
red, succulent aril. Flowering and fruiting occur during
March to November (Liang and Soukup 2000). The species
P. polyphylla has got much attention in recent times owing
to its important medicinal properties, biological activities
and pharmaceutical demand.
Rhizome of various species of the genus Paris is the
major source of raw material for ‘Yunnan Baiyao’, a very
famous Yi ethno-medicine used against various diseases
and injuries like back pain, bleeding, fractured bones,
fungal diseases, poisonous snakes or insect bites, skin
allergy, tumors and a variety of cancers (Long et al. 2003).
The rhizome of the species P. polyphylla is used in various
Chinese traditional medicine, including analgesic,
antiphlogistic, antipyretic, antitussive and depurative (Duke
and Ayensu 1984; Yeung 1985) whereas, the whole plant is
used for the treatment of febrifuge, liver and lung cancer
and laryngeal carcinoma (Khanna et al. 1975; Ravikumar et
al. 1979; Sing et al. 1980; 1982). In Nepal, the rhizome is
indigenously used against snake bites, insect bites, alleviate
narcotic effects, internal wounds, external wounds, fever,
food poisoning and are fed to cattle during diarrhea and
dysentery (Dutta 2007; Baral and Kurmi 2006). It is also
used to treat headache, vomiting and worms (Uprety et al.
2010). A drug called as Gong Xue Ning (GXN) capsule has
been developed from the saponin extract of P. polyphylla
var. yunnanensis in China for the treatment of abnormal
uterine bleeding (AUB) (Zhao and Shi 2005; Guo et al.
2008). Rhizome of the species is also used against uterine
contractile effects (Tian et al. 1986; Zhou 1991). P. polyphylla
is a folk medicinal plant in the Indian Himalayan Region,
traditionally used against analgesic, antibacterial, antiphlogistic, antispasmodic, antitussive, any poisonous bites,
burn, cut or injury, depurative, detoxification, diarrhea,
dressing, dysentery, febrifuge, fever, gastric, gastritis,
intestinal wounds, narcotic, poisoning, rashes or itching,
scabies, skin diseases, sleeplessness, snake bite, stomach
pain, typhoid, ulcer and wounds (Farooquee et al. 2004;
Maity et al. 2004; Tiwari et al. 2010; Jamir et al. 2012;
Lalsangluaii et al. 2013; Pfoze et al. 2013; Mir et al. 2014;
Sharma and Samant 2014). Further Shah et al. (2012)
reviewed the various medicinal properties of the P. polyphylla
and categorized the species as the ‘jack of all trades’.
Several pharmacological properties including antibacterial, anticancer/anti-tumor, antimicrobial, antiviral,
antifungal, antioxidant, cytotoxic, steroid saponin etc.
(Khanna et al. 1975; Ravikumar et al. 1979; Singh et al.
1980; 1982; Zhou 1991; Yu and Yang 1999; Mimaki et al.
2000; Lee et al. 2005; Wang et al. 2006; Yun et al. 2007;
Zhang et al. 2007; Guo et al. 2008; Yan et al. 2009; Xuan
et al. 2010; Zhao et al. 2010; Chan et al. 2011; Wang et al.
2011; Zhu et al. 2011; Kang et al. 2012; Li et al. 2012;
Zhao et al. 2012; Shen et al. 2014) have been reported from
the rhizomes of many species of the genus Paris.
Arunachal Pradesh in the Eastern Himalayan Region of
India is very rich in biological and socio-cultural diversity.
However, anthropogenic disturbances in the recent past
have led to the depletion of many life forms. Besides, there
is a lack of adequate information about the status and
importance of the species in this Eastern Himalayan
Region. With the large extent of decreasing green cover,
many species, including the genus Paris are facing the
impact of ecological disturbances. Rampant and reckless
extraction in the Indian Himalayan Region owing to high
market demand has put the pressure on the existence of the
species particularly in Arunachal Pradesh. Many studies
have been carried out around the world while, no study has
been done in Arunachal Himalaya. Therefore, the present
study has been undertaken to enumerate the present status
of uses, population and threats of this important medicinal
plant species.
MATERIALS AND METHODS
During the survey of medicinal plant diversity in the
Eastern Himalayan state of Arunachal Pradesh in the past
few years, specific observation has been made on some of
the selected species which are commercially exploited.
Discussion and consultation with local communities have
been undertaken to find out the uses and market potential
of the species. All the relevant records on the species were
also analyzed. Based on the available secondary informations and primary field data from the state of Arunachal
Pradesh, an attempt has been made to highlight the
taxonomic diversity, distribution, medicinal importance,
status of occurrence, threats and conservation strategies.
The present scenario of harvesting and trade has also been
discussed.
To assess the population status of P. polyphylla, three
study stand viz., Bomdila, Mayudia and Talle Valley were
selected from West Kameng, Lower Dibang Valley and
Lower Subansiri districts, respectively. The study sites
were selected based on the availability of the species. To
study the community characteristics the sampling of the
vegetation was carried out using the quadrate method.
Twenty five quadrates of 1 m x 1 m were laid randomly in
each study stands. Community characteristics of each of the
stands were studied using quantitative analytical methods.
Important ecological parameters like density, frequency,
Importance Value Index (IVI) were worked out by following
Misra (1968) and Mueller-Dombois and Ellenberg (1974).
PAUL et al. – Paris polyphylla of the Indian Himalayan Region
RESULTS AND DISCUSSION
Paris polyphylla in Arunachal Pradesh
In Arunachal Pradesh, the species P. polyphylla is
distributed in subtropical to temperate forests in most of the
districts. It grows mainly in moist and shady areas of
forests, thickets, bamboo forests, grassy or rocky slopes
and nearby water channel in rich humus soil. Total number
of occurrences of taxa of the genus Paris in Arunachal
Himalaya has not been assessed till date. In the present
survey, distribution of four distinct variances of P.
polyphylla has been recorded from Arunachal Pradesh,
which is harvested from the wild (Figure 1 A-E). Authentic
identification of the variability of the species and further
study is under process. Locally the species is known as
Mungong (Monpa dialect), Orpo (Adi dialect) and Aiichangmu (Sherdukpen dialect). The P. polyphylla and other
distinct variety of the species are found to be growing in an
altitudinal range between 1000-3500 m asl. Till date,
except P. polyphylla no detail taxonomic enumeration of
other taxa of the genus has been described in the flora
references of Arunachal Pradesh.
Utilization, harvesting and population status
Indigenous medicinal uses of P. polyphylla are very
limited in Arunachal Pradesh. The rhizome is used against
fever by the Adi tribe in the Upper Siang district. Rhizomes
of the species are also consumed as fresh while ripen fruits
(Figure 2 A, B) are eaten sometime. The fruits are also
preferred by deer. The paste of the rhizome is used against
snake bites by the Sherdukpen tribe in West Kameng
district. Till recent past the commercial value of the species
was not known in the state and the species were found
abundantly in wild. However, mostly after 2005 the
commercial exploitation of the rhizome started in large
scale from most of the districts owing to its high
commercial demand in the international markets. Large
quantity of rhizomes from wild (Figure 3 A) is harvested,
particularly during the month of April to July from various
places like Baishaki, Bomdila, Chaglagam, Dirang, Lumla,
Manigong, Mechuka, Muktur, Mayudia, Pasighat, Senge,
Shakti, Tawang, Zimithang and many other places of the
state. Collected rhizomes are sold either in dry or raw form
(Figure 3 B) to the middlemen. Middlemen who collect the
rhizomes from the local people sell it to outside traders and
finally it goes to international market. The local people
(Figure 3 C) sell fresh rhizomes at a rate ranging from
about Rs. 700-800 kg-1 while dried rhizomes at a rate
ranging from Rs. 3500-4000 kg-1. Although it is an offfarm income resource or livelihood of the local
communities, but they are not aware of its important
297
medicinal properties. Moreover, the species has not much
utilized in the indigenous medicinal systems of the state,
unlike other Indian Himalayan states and also Himalayan
countries. Local people reported that although the rhizome
of the species has high market potential, but they are not
even aware about the importance and uses of the species.
In the present pilot study, the population status of P.
polyphylla was assessed in three selected study stands (i.e.,
Bomdila, Mayudia and Talle Valley) of West Kameng,
Lower Dibang Valley and Lower Subansiri districts,
respectively. Highest density (1.48 individuals m-2) was
recorded in Talle Valley and lowest (0.42 individuals m-2)
in Mayudia. While, maximum frequency (32%) was
observed in Bomdila and minimum (10%) in Mayudia
(Table 1). The present frequency and density of the species
was found to be very low than the reported average
frequency (60.83%) and density (1.78 individuals m-2)
from Nepal (Madhu et al. 2010). The Importance Value
Index showed the highest (8.45) in Talle Valley and lowest
(3.37) in Mayudia (Table 1). The main dominant associated
species which sharing the maximum IVI includes
Anaphalis busua (5.51), Arisaema sp. (5.85), Fragaria
vesca (21.09), Galearis spathulata (6.78), Gnaphalium
affine (9.13), Grass sp. (8.23), Pteris sp. (7.11), Plantago
major (6.78), Rubia manjith (7.34), Rubus calycinus (6.44),
Rubus nepalensis (5.07), Senecio wallichii (5.36) and
Swertia chirayita (11.77) in Bomdila. Species like
Artemisia nilagirica (11.29), Coptis teeta (28.46), Carduus
edelbergii (6.32), Crassocephalum crepidioides (5.93),
Cyperus cyperoides (13.10), Hemiphragma heterophyllum
(9.96), Hydrocotyle asiatica (10.15), Geranium pratense
(6.96), Grass sp. (10.17), Plantago asiatica (11.61),
Polygonatum verticillatum (9.39), Pteris sp. (28.48),
Rumex nepalensis (10.47), Selaginella sp. (5.07) and
Senecio raphanifolius (9.14) were dominant in Mayudia.
While Anaphalis busua (9.29), Arisaema sp. (9.95),
Artemisia sp. (13.29), Centella sp. (19.79), Carduus
edelbergii (10.68), Fragaria nubicola (13.86), Hydrocotyle
himalaica (11.12), Impatiens sp. (9.72), Podophyllum
hexandrum (5.40), Polygonum hydropiper (9.28),
Potentilla plurijuga (11.56) and Viola sp. (6.26) were the
dominant species in Talle Valley. The natural populations
of P. polyphylla are affected because of anthropogenic
activities like extraction and habitat destruction which
leading to its endangerment. Mao et al. (2009) also
reported that the species has become rare due to over
harvesting from the wild. Owing to unsustainable
extraction and illegal trade the species had already been
categorized as endangered in Himachal Pradesh, Jammu
and Kashmir and Uttarakhand (ENVIS 2010).
Table 1. Frequency, density and Importance Value Index of Paris polyphylla in the selected study stands of Arunachal Pradesh
Study sites
Bomdila
Mayudia
Talle Valley
District
West Kameng
Lower Dibang Valley
Lower Subansiri
Frequency (%)
32
10
22
Density (individuals m-2)
1.32
0.42
1.48
IVI
6.07
3.37
8.45
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B I O D I V E R S IT A S 16 (2): 295-302, October 2015
B
A
C
E
D
Figure 1. The genus Paris in Arunachal Himalaya. A. Paris polyphylla, B-E. Distinct variances of Paris polyphylla
A
B
Figure 2. Paris polyphylla fruits. A. Young, B. Ripen fruit
Trade and marketing
Based on the conversation with the local people, all the
harvested rhizomes of the P. polyphylla are traded to
Myanmar and other South East Asian countries illegally
routed through Assam and Manipur. Illegal trading
occurred either at local or directly to the regional level
through middlemen and then outside of the country. The
rhizomes harvested from Dibang and Lower Dibang Valley
district are traded to Tinsukia/Dibrugarh via Roing. Upper
Siang and East Siang district are traded to Dibrugarh/
Tezpur via Pasighat. West Siang district is traded to Tezpur
via Silapathar. While harvested rhizomes from Tawang and
West Kameng district are traded to Tezpur via Bomdila.
Illegal exporting of rhizomes of the P. polyphylla from
PAUL et al. – Paris polyphylla of the Indian Himalayan Region
A
B
299
C
Figure 3. Rhizomes of Paris polyphylla. A. Freshly collected rhizomes, B. Drying, C. Ready to sell
Arunachal Pradesh to China via Myanmar has been
reported by Basar (2014). Illegal trade of the rhizomes to
Myanmar through Indo-Myanmar border by the local
traders have been reported (The Sangai Express
17/08/2008). Trafficking of the rhizomes from the Indian
states of Arunachal Pradesh, Manipur, Meghalaya and
Nagaland to Myanmar have also been reported (Mao et al.
2009).
Major threats to the species
Most of the population of P. polyphylla is depleted due
to rampant and reckless extraction. In Arunachal Pradesh
the species is facing tremendous pressure because of over
exploitation due to its high market demand. Unsustainable
extraction of the species owing to its high commercial
demand has led to decline in populations and becoming
rare in its natural habitat. Developmental activities like
broading of road, urbanization and habitat destruction, etc.
collectively put pressure on the existence of the species.
Other anthropogenic activities like shifting agriculture,
forest resource collection, logging, etc. are affecting habitat
and wild populations of the species. Heavy rainfall leading
to the land erosion/landslides, etc. is also causing the
habitat loss and population depletion of the species.
Grazing is also one of the factors for loss of
habitat/population of the species. The rhizome is the main
mode of regeneration though it regenerates from seeds.
Uncontrolled and indiscriminate harvesting of whole plant
without leaving any part of the rhizome, harvesting before
reproductive/flowering or seed maturity period etc. are
causing regeneration failure in its natural habitat.
Conversely, regeneration of P. polyphylla from seed in
wild, green house, laboratory is very poor because of long
dormancy period and very slow growing nature (Madhu et
al. 2010; Qi et al. 2013) which is affecting the growth and
survival of the species. Over harvesting has caused the
decrease in natural population of the species. The over
exploitation, rampant illegal extraction and trade, wild
populations of P. polyphylla are at risk of extinction from
its natural habitat. Though the species has not yet been
assessed by the IUCN Red List however, if the present
trend continues, the species will be wiped out from the wild
and thus suitable conservation measures/strategies are of
utmost important. Very recently Arunachal Pradesh State
Medicinal Plants Board (APSMPB) expressed concern over
the rampant and reckless extraction of P. polyphylla from
most of the districts of the state. The Board has also urged
the local administration (Deputy Commissioner,
Superintendent of Police and Divisional Forest Officers) of
most of the districts to check the exploitation of the rare,
endangered and threatened species before being extinct
from the state (The Arunachal Times 22/08/2015).
Management and conservation
The present study on the population status revealed
very low population density (Table 1) that warrant
immediate conservation action. For effective management
the following strategies may be adopted: (i) Detailed
exploration and documentation for identification of major
distribution areas with mapping or ecological niche
modeling is needed to save this natural resource. (ii)
Critical studies on population structure and regeneration
status covering both the open and protected areas provide
the useful data for conservation action. Evaluation of
species specific or area specific threat is essential for
management of this species. (iii) Inclusion of the species
under the priority species list of both the National and State
Medicinal Plant Boards for large scale cultivation and
conservation. (iv) Cultivation and conservation through
community involvement. Out of total recorded forest areas,
about 60.22% of the forest land (FSI 2013) of the state are
traditionally controlled by the communities as per the
customary rights called Unclassed State Forest (USF) or
community forests, are ideal for large scale cultivation and
conservation. This will be accomplished only when local
communities are involved. Mass awareness regarding the
declining population of the species, its high medicinal
properties and economic importance is required. (v)
Sustainable harvesting practices must be adopted as the
rhizome of the species is used. The whole plant is uprooted
to collect the rhizome leading to the destruction of the
plant. Hence, appropriate technique should be used for
300
B I O D I V E R S IT A S 16 (2): 295-302, October 2015
B
A
Figure 4. Cultivation of Paris polyphylla in homesteads. A. Monpa villager busy in cultivation, B. Cultivated Paris polyphylla
harvesting the mature rhizome without effecting the
population like viable portion of the rhizome may be left in
its natural habitat, so that it can regenerate naturally. (vi)
Local/regional level management approaches with active
community involvement and benefits sharing should be
undertaken. On-farm and off-farm management and
scientific studies of the species is urgently needed. Legal
restriction on harvesting or trade of rhizome may be
imposed to stop further genetic loss. (vii) Legal protection
of habitat that are not covered in the protected area of the
sate as it has only 11.68% of the total geographical area
(FSI 2013) under protected area network that may not be
enough for conservation. The selected habitat with a good
viable population may be brought under legal protection
like Medicinal Plants Conservation Area (MPCA) for in
situ conservation.
Commercial cultivation for conservation and economic
development
The habitat of P. polyphylla distributed with an
attitudinal range between 100 to 3500 m asl (Liang and
Soukup 2000) which is one of the important characteristics
for large scale cultivation under the suitable climatic
condition. To meet the need of high market demand for its
important medicinal, biological and pharmaceutical
purposes the species can be cultivated commercially with
active community participation. Most of the local
communities in this Himalayan region of Arunachal
Pradesh can be engaged for large scale cultivation (Figure
4 A, B) in community land, nurseries, homesteads, etc. will
boost the economy and income generation of the tribal
people of the state. Like Curcuma longa L. (turmeric),
Elettaria cardamomum (L.) Maton (cardamom) and
Zingiber officinale Rosc. (ginger), P. polyphylla can be
cultivated in large scale through rhizome in its natural
habitat. Domestication and inclusion in Agroforestry and
social forestry program with community participatory
management will further enhance the rural economy, which
contributes towards the conservation and meet the market
demand. For instance, in Yunnan of China, people have
already been started cultivation of P. polyphylla in their
Agroforestry systems to meet the commercial demand
(Long et al. 2003). Departments like Agriculture,
Horticulture, the State Biodiversity Board, State Forest
Research Institute and State Medicinal Plant Board may
take active participation for promotion of large scale
cultivation. A detail taxonomic study on the genus Paris is
utmost important to find out the taxonomic diversity as the
high variability among the populations have been observed
that may lead the discovery of more new species. Although
the occurrence of eight taxa of Paris from India has been
mentioned (Liang and Soukup 2000) while only one
species, i.e., P. polyphylla has been reported from
Arunachal Pradesh (Chowdhery et al. 2009). There is an
urgent need for our scientific community to pay their ample
attention to this valuable herbaceous species for
conservation and further scientific studies in the Indian
Himalayan Region particularly in Arunachal Pradesh.
ACKNOWLEDGEMENTS
The authors wish to express their deepest thanks to all
the local field guides Chenjeer Dree, Dipu Goyiak, Jamba,
Jam Tsering, Kejang Serthipa, Nima Dondu, Pem Dakpa,
Rabi Mekula, Tado, all the village headmen and villagers
for help and support during the field survey. The first
author is thankful to University Grants Commission,
Government of India, New Delhi for UGC-Dr. D.S.
Kothari Post Doctoral Fellowship. Authors deeply
acknowledge the help and support of Dr. Sanjeeb Bharali,
Amal Bawri and Lobsang Tashi during the field study. The
authors also express their sincere thanks to the various
Forest Officials, Department of Environment and Forests,
Government of Arunachal Pradesh for permitting us to
work in forest areas of the state. We are also thankful to all,
those who extended their cooperation during the field
study. The authors are thankful to anonymous reviewers for
PAUL et al. – Paris polyphylla of the Indian Himalayan Region
sparing their valuable times for corrections and
suggestions.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160227
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 303-310
Growth, development and morphology of gametophytes of golden
chicken fern (Cibotium barometz (L.) J. Sm.) in natural media
TITIEN NGATINEM PRAPTOSUWIRYO1,♥, DIDIT OKTA PRIBADI1, RUGAYAH2
Center for Plant Conservation-Bogor Botanical Gardens, Indonesian Institute of Sciences. Jl. Ir. H.Juanda No. 13, P.O. Box 309 Bogor 16003, Indonesia.
Tel. +62-251-8322187. Fax. +62-251-8322187. ♥email: tienpferns@yahoo.com
Herbarium Bogoriense, Botany Division , Biology Research Center, Indonesian Institute of Sciences, Cibinong Sciences Center, Cibinong, West Java,
Indonesia
Manuscript received: 28 July 2015. Revision accepted: 26 October 2015.
Abstract. Praptosuwiryo TNg, Pribadi DO, Rugayah. 2015. Growth, development and morphology of gametophytes of golden chicken
fern (Cibotium barometz (L.) J. Sm.) in natural media. Biodiversitas 16: 303-310. The golden chicken fern, Cibotium barometz (L.) J.
Sm., is an important export commodity for both traditional and modern medicine. To understand the reproductive biology of this
species, spore germination, gametophyte development, morphological variation, and sex expression were studied by sowing spores on
sterilized natural media consisting of the minced roots of Cyathea contaminans and charcoaled rice husks (1: 1) mix. Spores of
C. barometz are trilete, tri-radially symmetrical, non chlorophyllous, and golden-yellow with a perine. Six stages of gametophyte
development (rhizoid stage, rhizoid/protochorm stage, filament stage, spatulate stage, young heart stage, mature heart stage) were
observed between 24-45 days after sowing. Spore germination of C. barometz is Vittaria-type. Prothallial development of C. barometz is
Drynaria-type. Five morphological types of adult gametophyte were recorded: (i) irregular spatulate shape (male), (ii) fan shape (male),
(iii) elongated heart-shape (male), (iv) short heart or butterfly shape (female), and (v) normal heart shape (bisexual). The presence of
morphological variations is presumed to be related to the population density, which significantly affects the sexual expression of
gametophytes. The variation of sex expression in C. barometz also indicates that this species may has a mixed mating systems that
resulted in genetic diversity within population and among populations.
Keywords: Cibotium barometz, gametophytes, golden chicken fern, spore germination, prothallial development
INTRODUCTION
Cibotium is a genus of 12 species (Hassler and Swale
2002) or 11 species of tropical tree fern (Zhang and
Nishida 2013), which is subject to much confusion and
revision. Two species of them occur in Indonesia, namely
Cibotium barometz (L.) J. Sm and C. arachnoideum (C.
CHR.) Holttum (Holttum 1963). The Golden Chicken Fern,
C. barometz, is easily recognized because of the gold
yellowish-brown, smooth and shining hairs covering its
rhizome and basal stipe. The rhizome is usually prostrate or
erect and rarely more than 1 m high.
Cibotium barometz differs from C. arachnoideum by a
number of diagnostic characters as follows: C. barometz
has sori 2-6 or more pairs on each pinnule-lobe of larger
fronds, largest pinnules 20-35 mm wide, pinnules on the
two sides of a pinna not greatly different in length, hairs on
lower surface of costae and costules flaccid and never
spreading. In contrast C. arachnoideum always has two
pairs of sori on each pinnule-lobe of larger fronds, largest
pinnules 15-26 mm wide, pinnules on basiscopic side of
lower pinnae much shorter than those on acroscopic side,
hairs on lower surface of costae and costules rigid and
spreading (Holttum 1963; Praptosuwiryo et al. 2011).
Economically, C. barometz is an important export
commodity for both traditional and modern medicine in
China, Japan and France (Zamora and Co 1986;
Praptosuwiryo 2003). The golden hairs on its rhizome and
stipes have been used as a styptic for treating bleeding
wounds (Dan and Nhu 1989). An extract from the rhizome
(‘gouji’) is also used as an anti-rheumatic, to stimulate the
liver and kidney, to strengthen the spine, as a remedy for
prostatic disease, and to treat various other illnesses,
including flatulence (Praptosuwiryo 2003; Yao 1996; Ou
1992). Although this species has many uses, it has not yet
been cultivated for commercial trade. Therefore this
species has been included in Appendix II of the Convention
on International Trade in Endangered Species (CITES)
since 1976. In order to utilize it, the NDF (Non Detriment
Findings) system must be applied to determine the annual
quotas. Biological aspects, such as reproductive biology,
are some of the most information needed for the NDF.
Reproductive biology determines plant adaptation and
species evolution (Farrar et al. 2008). Many traits are used
to infer the reproductive biology of ferns, such as the
mating system; the number of spores in each sporangium;
sporogenesis; spore size; the lifespan of the gametophyte
generation; gametophyte morphology and development;
and reproductive systems (Manton 1950; Masuyama 1979,
1986; Walker 1979; Haufler et al. 1985; Lin et al. 1990;
Kawakami et al. 1996; Huang et al. 2006, 2009).
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B I O D I V E R S IT A S 16 (2): 303-310, October 2015
Studies on the reproductive biology of C. barometz
have been reported by some researchers from China. Chen
et al. (2007) studied gametophyte development and its
diversity in C. barometz from China by culturing spores in
Knop's solution and solid medium. Li et al. (2010) reported
in vitro culture and plant regeneration of C. barometz using
Murashige & Skoog (MS) media. Deng et al. (2007)
observed the gametophyte development of C. barometz
from China by culturing the spores both in inorganic
medium and in soil from the original habitat.
This paper reports the reproductive biology of Cibotium
from Sumatra, Indonesia. The aims of this study were: (i)
to verify the reproductive characteristics of C. barometz of
Sumatra, including spore germination, gametophyte
development and morphology; (ii) to study morphological
variation and sex expression, and (iii) to show that a
sterilized natural media consisting of the minced roots of
Cyathea contaminans and charcoaled rice husks (1: 1) mix
can be used as an alternative to study the gametophyte
development and morphology of tree fern.
MATERIALS AND METHODS
Spore collection. Spores of Cibotium barometz were
collected from Riau, Sumatra in June 2011. Spore-bearing
pinnae of mature sporophylls were cleaned in running
water to avoid spore contamination from other species.
Spore-bearing pinnulae were dried and folded in a
newspaper and then placed in an envelope (22 x 11 cm2).
The specimens were kept at room temperature in a dry
place. A few days later (7-10 days) most of spores had been
released from their sporangia and were lying in the
envelope. The spores were separated off from the sporangia
by tilting the envelope paper and removing them from the
envelope to a piece of glassine weighting paper which was
folded into a pocket. The spore collections were kept at
room temperature until the sowing day (not more than two
month).
The spores (10 mg) were sown on a sterilized, mixed
natural media of Cyathea contaminans roots and
charcoaled rice husks ( 1: 1) mix in a transparent plastic
box (7 cm height, 11 cm diam.) with the media layer 2.5-3
cm high. Preparation of the media was as follows: the mix
media was boiled in water for 2.5-3 hours for sterilization.
The sterilized media was kept in a sealed transparent
plastic box (7 cm height, 11 cm diam.) for 24 hours at room
temperature before it was used to sow spores. The plastic
boxes were kept in a glasshouse at 25-32.5ºC with 68-85%
relative humidity. Watering was done once every week to
maintain the humidity of the media.
Observations. Periodically, spore germination,
gametophyte growth and differentiation and sex expression
were observed under an Olympus trinocular fitted with
Nikon Camera SMZ 10A 1.5X. Every 4-5 days 100-300
selected prothallus or gametophytes were observed. First
observations using microscope were carried out 24 days
after sowing. Photographs were taken using the microscope
combined with a computer monitored camera (Olympus
CX 31).
RESULTS AND DISCUSSION
Spore germination
Spores of C. barometz are perinate, trilete, tri-radially
symmetric, non chlorophyllous and golden yellow in colour
(Figure 1.A.). After 23 days from sowing, five stages of the
gametophyte development were found (Figure 1-2.), viz.
rhizoid stage, rhizoid/protochorm stage, filament stage,
spatulate stage and young heart stage. The sixth stage of
gametophyte development, mature heart stage, was first
appeared 45 days after sowing. First germination occurred
two weeks after sowing. Deng et al. (2007) also reported
that spores of C. barometz of China germinated about 1-2
weeks after being sowed.
The first cell produced on germination of the spores
was rhizoid (Figure 1.B.). This is called the rhizoid stage.
The rhizoid cell usually does not contain chloroplasts. It is
produced on the upper surface of the basal cell or
sometimes at one corner of the basal cell. The second stage
of gametophyte development is the rhizoid/protochorm
stage in which the spore bears both rhizoid and the first cell
of a filament.
The filamentous phase of C. barometz occurred within
about 10-15 after sowing and is indicated by formation of a
3-12-celled filament. Every cell of the filament contains
chloroplasts. As stated by Nayar and Kaur (1971) spore
germination results in a uniseriate, elongated, germ
filament composed of barrel-shaped chlorophyllous cells
and bearing one or more rhizoids at the basal end.
Spore germination in C. barometz of Sumatra is of the
Vittaria-type, producing a slender, uniseriate germ filament
four to twelve cells long. The Vittaria-type is apparently
common in Cibotium. Chen et al. (2007) reported Vittariatype and Cyathea-type spore germination of this golden
chicken fern from China. The spore germination of C.
barometz reported by Deng et al. (2007) also belonged to
the Vittaria-type, the first division giving rise to the rhizoid
initial is perpendicular to the polar axis of the spore and the
second division yielding the protonema initial that is
perpendicular to the first (Nayar and Kaur 1968) (Figure
1.B-E).
Prothallial development
The young prothallus plate of C. barometz was initiated
in the terminal cell of the filament in about 14-30 days by
perpendicular divisions. It had two phases, namely a
spatulate phase which had 10-21 cells and an early heart
shape stage which had 13-42 cells (Figure 2). The meristematic
cells of the spatulate stage and early heart shape stage are
occurred in margin of the distal gametophytes. After
repeated divisions these became mature thallus (Figure
3.A, 4.C-H.). Typically, the gametophyte of C. barometz is
a heart-shaped monolayer of cells with an apical cell as the
meristem. The spores took about 30-40 days after sowing
to differentiate into a cordate thallus. Usually, a prothallus
plate did not form reproductive organs until 40 days after
sowing. The period of prothallial development of C.
barometz of Sumatra is not different from that observed by
Deng et al. (2007) for C. barometz of China, as its
prothallial plates formed in 25 days after being sowed.
PRAPTOSUWIRYO et al. – Gametophytes of Cibotium barometz
305
B
A
A
C
D
E
Figure 1. A. Spores of Cibotium barometz (SEM photograph). B. Germinated spore, about 5-7 days after showing. C-E. Filamentous
phase, 3-10-celled filament, about 10-15 after showing. Bar = 30 µm for A, 60 µm for B and C, 100 µm for D, 40 µm for E
Cibotium barometz is a homosporous fern species,
producing only one type of spores with both male and
female reproductive organs in the resulting gametophyte.
Nayar and Kaur (1971) showed that the prothallus of
homosporous ferns follows a definite pattern of
development leading ultimately to the characteristic adult
form. This pattern is constant for each species and common
to taxa of higher order under normal conditions of growth.
Nayar and Kaur (1971) recognized seven different patterns
of prothallial development among homosporous ferns, viz.
Adiantum-type,
Aspidium-type,
Ceratopteris-type,
Drynaria-type,
Kaulinia-type,
Marattia-type,
and
Osmunda-type. These types differ in the sequence of cell
divisions; in the stages of development and the region at
which a meristem is established; and in the resultant form
of the adult thallus. Prothallial development of C. barometz
is of the Drynaria-type (Nayar and Khaur (1971): The
spores germinate into a germ filament composed of barrelshaped chlorophyllous cells with one or more rhizoids at
the base cell; One of the cells at the top margin of the
prothallus (the anterior marginal cells) then divides
obliquely when it has 5-10, or more cells across its width;
This results in an obconical meristematic cells; Division by
this type of cell is parallel to each other and perpendicular
to the rest of the cells, forming rows. This results in the
formation of a notch at the anterior edge of the prothallus,
giving it a roughly heart-shaped appearance.
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B I O D I V E R S IT A S 16 (2): 303-310, October 2015
r
r
A
mc
pc
mc
pc
C
pc
B
mc
D
Figure 2. A-B. Young plate phase (spatulate plate), 27 days; B. Enlargement of A. C-D. Young gametophyte in elongated heart shape,
after one month (38 days); D. Enlargement of C. Rhizoid (r), meristematic cells (mc), prothallial cell (pc). Bar = 120 µm for B and D
PRAPTOSUWIRYO et al. – Gametophytes of Cibotium barometz
307
ar
ar
A
ar
B
s
an
ar
C
D
Figure 3. A. Normal heart-shape, adult gametophyte, bisexual (175 days after sowing); B. Archegonium (ar); C. Antheridium (an)
releasing spermatozoid (s); D. Young sporophyte growing from gametophyte (178 days after sowing). Bar = 0.7 mm for A and D, 100
µm for C
Morphological diversity of mature gametophytes and
sex expression of Cibotium barometz
Mature gametophytes first appeared at around 45 days
after sowing. They were male gametophytes. Mature
gametophytes were indicated by the formation of
reproductive organs, viz. male organ or antheridium and
female organ or archegonium (Figure 3). Both unisexual
and bisexual prothalli produced antheridia earlier than
archegonia. The antheridia walls were composed of 5 cells.
Mature archegonia appeared within 150-165 days after
sowing.
Within two to six months after sowing spores we found
the gametophyte populations in the culture consisting of
male, female and hermaphroditic gametophytes. Moreover,
five morphological types of adult gametophyte in C.
barometz were observed as shown in Figure 4 and Table 1.
These morphological types are clearly correlated with the
gametophytes’ sexual expression. Male gametophytes were
of three shapes, namely scoop shaped, fan shaped and long
heart shaped. On the other hand, female and bisexual
gametophytes had only one shape. They were normal heart
shaped.
As reported by Chen et al. (2007), normally the mature
gametophytes of C. barometz are found to be
symmetrically cordate. The presence of morphological
variations of the gametophyte in the Sumatran fern of this
study is presumed to be related to population density,
which significantly affects gametophytes’ sexual
expression. As reported by Huang et al. (2004), the
gametophyte
size
of
Osmunda
cinnamomea
(Osmundaceae) is negatively related to the population
density. A similar case is observed in Woodwardia
radicans (De Soto et al. 2008). Under low density, the
gametophytes of W. radicans mature sexually at a
relatively large size and turn into females and subsequently
into bisexuals. Under crowded conditions, the
gametophytes of this species mature sexually at a smaller
size and turn into males. How gametophyte population
densities effect the shape of gametophyte? Huang et al.
(2004) stated that gametophyte population density is one of
the factors affecting sexual expression and growth in
gametophyte communities. In overcrowded populations
resources are limited by competition, and gametophytes are
often asexual or male, and narrow (usually spathulate
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B I O D I V E R S IT A S 16 (2): 303-310, October 2015
an
an
B
A
ar
ar
ar
an
D
C
ar
an
ar
an
F
E
ar
an
em
G
H
Figure 4. Morphological diversity of adult gametophytes of C. barometz. A. Irregular shape (male, type A); B. Fan shape (male, type
B); C. Short heart shape or butterfly shape (Female, type C); D. Normal heart shape (bisexual, type D); E. Normal heart shape (bisexual,
type D); F. Normal heart shape (bisexual, type D); G. Normal heart shape (bisexual) showing embryo; H. Elongated heart shape (male).
Bar = 0.6 mm for all. an = antheridium. ar: archegonium. em: embryo
PRAPTOSUWIRYO et al. – Gametophytes of Cibotium barometz
309
Tabel 1. Description of mature gametophyte types in C. barometz
Type
A
B
C
D
E
Description
Irregular scoop shape with filamentous branches: antheridia dispersed everywhere (Figure 4.A).
Fan shape: antheridia dispersed one third from margin of wings area (Figure 4.B).
Short heart shape or butterfly shape: archegonia were formed at thick cushion, on the anterior
part of the midrib (Figure 4.C).
Normal heart shape: Archegonia were produced on the anterior part of the midrib as it
approaches the meristem; antheridia were produced on the wings and midrib of the ventral side
(Figure 4.D. and 4.E-G).
Long heart shape: antheridia dispersed irregularly on the ventral side of wings (Figure 4.H).
shape). Female and hermaphroditic gametophytes, on the
other hand, often occur in sparse populations (Klekowski
and Lloyd 1968; Cousens and Horner 1970; Lloyd and
Gregg 1975; Cousens 1979). The effects of high
gametophytes population density were summarized by
Smith and Rogan (1970). Crowded conditions may alter the
normal sequence of development of fern gametophytes.
Increasing population density leads to an increase in
gametophyte length and delay in the transition to two
dimensional growths.
The variation of sex expression in C. barometz also
indicates that this species may has a mixed mating systems
(self-fertilization, intergametophytic selfing and intergametophytic crossing). However, some species of ferns do
show mixed mating systems (Soltis and Soltis 1987, 1988),
and by now several studies have indicated that mating
systems on some species may vary greatly even between
different genotypes within species (Peck et al. 1990; Suter
et al. 2000; Wubs et al. 2010). The existence of mixed
mating systems in this species will result in genetic
diversity within population and among populations. As
reported by You and Deng (2012), C. barometz in China
showed 41.06% of genetic diversity among populations and
58.94% of genetic diversity within populations. Rugayah et
al. (2009) also confirms the existence of genetic variation
in C. barometz of Sumatra three morphological variants of
C. barometz were reported from West Sumatra.
Most studies on gametophyte development and
reproductive biology of tree ferns, including C. barometz,
are carried out in vitro, using agar media in petri dishes
(see Khare and Srivastava 2009; Khare et al. 2005; Chen et
al. 2007; Li et al. 2010). Our study, using sterilized natural
media, shows that all stages from spore germination to
development of gametophyte, and from rhizoid stage to
mature prothallial stage, are not significantly different from
those obtained in studies using agar media in petridishes in
vitro. The results of this study gives proof that a sterilized
natural media consisting of minced roots of Cyathea
contaminans with charcoaled rice husks (1: 1) mix can be
used as an alternative medium for studying the
development and morphology of gametophytes of a tree
fern. This information is very important for ex situ
conservation of ferns
In conclusion, six stages of gametophyte development
(rhizoid stage, rhizoid/protochorm stage, filament stage,
spatulate stage, young heart stage, mature heart stage) were
Average
size (mm)
2 x 1.5
2x2
2.5 x 1.5
Sex
expression
Male
Male
Female
4x4
Female,
bisexual
5x3
Male
observed 24-45 days after sowing. The first cell produced
after spore germination was rhizoid. Spore germination of
C. barometz is Vittaria-type, producing a slender,
uniseriate germ filament, four to twelve cells long.
Prothallial development of C. barometz is Drynaria-type.
The young prothallus plate was initiated in the terminal cell
of the filament, approximately 14-30 days after sowing, by
perpendicular division. Antheridia and archegonia were
first observed at 45 and 150 days after sowing,
respectively. Five morphological types of adult
gametophyte were recorded: (i) irregular spatulate shape
(male), (ii) fan shape (male), (iii) elongated heart-shape
(male), (iv) short heart or butterfly shape (female), and (v)
normal heart shape (bisexual). The presences of
morphological variations on the gametophyte are presumed
to be related to the population density, which significantly
affects gametophytes’ sexual expression. Cibotium
barometz may has a mixed mating systems (selffertilization, intergametophytic selfing and intergametophytic crossing) that resulted in genetic diversity
among population and within population. A sterilized
natural media consisting of the minced roots of Cyathea
contaminans with charcoaled rice husks (1: 1) mix can be
used as an alternative medium for studying the
development and morphology of gametophytes of the tree
fern. The present study contributes to the understanding of
the full life-cycle of C. barometz, providing information for
cultivation, management and conservation of the species.
ACKNOWLEDGEMENTS
This research was funded by a Grant-in-Aid for
Scientific Research from The Ministry of Research and
Technology, GoI, 03/SU/SP/Insf-Ristek/III/11, under The
Program Insentif Peneliti dan Perekayasa LIPI TA 2011
and Program Kompetitif LIPI TA 2013-2014. We thank to
Prof. Dr. I Made Sudiana, Biology Research Center,
Indonesian Institute of Sciences, and Prof. Dr. Ryoko
Imaichi, Department of Chemical and Biological Sciences,
Japan Women’s University, for reading and giving some
comments and suggestion to improve this manuscript. We
would also like to thank Dr. Graham Eagleton and Lyndle
Hardstaff for reading and correcting the English
manuscript.
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B I O D I V E R S IT A S 16 (2): 303-310, October 2015
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160228
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 311-319
Spatial point pattern analysis of the Sumatran tiger (Panthera tigris
sumatrae) poaching cases in and around Kerinci Seblat National Park,
Sumatra
FARID RIFAIE1,♥, JITO SUGARDJITO2, YULI SULISTYA FITRIANA1
1
Research Center for Biology, Indonesian Institute of Sciences, Cibinong Bogor 16911, West Java, Indonesia. Tel.: +62-21-8765056, Fax.: +62-218765068,email: farid.rifaie@lipi.go.id.
2
Centre for Sustainable Energy and Resources Management, UniversitasNasional, Jl. Sawo Manila, PasarMinggu, Jakarta 12520, Indonesia.
Manuscript received: 26 August 2015. Revision accepted: 28 October2015.
Abstract.Rifaie F, Sugardjito J, Fitriana YS. 2015.Spatial point pattern analysis of the Sumatran tiger (Panthera tigris sumatrae)
poaching cases in and around Kerinci Seblat National Park, Sumatra. Biodiversitas 16: 311-319.Wild Sumatran tigers are in a critical
state with around 250 adult tigers remain in their habitat in Sumatra Island. Despite the fact that this subspecies is an elusive animal with
very wide home range, Sumatran tigers are facing two serious threats, the depletion of its habitats and preys in one side and the increase
of tiger hunt for illegal wildlife market. Improving the capacity and effectiveness of law enforcement in reducing poaching of tigers is
an immediate priority protecting the remaining wild populations in their habitat. Enforcement monitoring was established under the
Tiger conservation program. During their patrols, the anti-poaching team recorded various data including poaching incidents. We
analyzed secondary data of Sumatran tiger poaching cases around Kerinci Seblat National Park that have been documented from 2000 to
2012. Georeferencing process was performed to transform locality data of 87 poaching cases into geographic position. The Nearest
Neighbor (NN) analysis suggested a strong clustering pattern with the observed mean distance was 4.9 km, much lower than the
expected mean distance (10.9 km).Similarly, The Ripley's K analysis also showed the aggregation of the points along the observed
distances. On the other hand, about 35.6% of points were located outside the standard deviational ellipse. The pattern indicated that
poaching incidents were spread in all corner of the region but excessive cases were observed in the center of the park.
Keywords: Kerinci Seblat National Park, Panthera tigris sumatrae, poaching, spatial statistic, Sumatran tiger
INTRODUCTION
Sumatran tiger (Panthera tigris sumatrae) is the only
tiger subspecies that live outside the continent (Tilson et al.
1994), following the extinction of the Bali tiger (P. t.
balica) in 1940s and the Javan tiger (P. t. sondaica) in
1980s (Seidensticker et al. 1999). Similar to its relatives in
the mainland, this subspecies existence is threatened and
the International Union for Conservation of Nature (IUCN)
has categorized it as critically endangered (Tilson et al.
1994). Indonesian government has passed Law No.5/1990,
Government Regulation No.7/1999 and Government
Regulation No.8/1999 which stipulated that Sumatran tiger
is a protected animal. Although its presence is still
observed across the Sumatra Island (Wibisono and
Pusparini 2010), but the exact population has never been
revealed since it is almost impossible to census this
solitary, cryptic animal. The latest assessment estimated
that at least there are 250 adult tigers inhabit Sumatra
Island (Wibisono and Pusparini 2010).
The fundamental impediment for Sumatran tiger survey
is the secretive nature of this animal combines with the low
density and very large home range (Wibisono et al. 2011).
This makes the detection of its presence a very difficult
task. The tiger existence was mostly exposed by their scats,
tracts, urine smell and growl (Tilson and Nyhus 2010).
Sumatran tiger population assessments have been
conducted by mean of questionnaire surveys (Wibisono et
al. 2011), occupancy surveys and camera trap surveys
(Linkie et al. 2010).
Twelve Tiger Conservation Landscapes (TCL) have
been recognized across Sumatra Island to determine
habitats that support tiger populations (Wikramanayake et
al. 1999; Sanderson et al. 2010; Seidensticker 2010).
However, there are only two landscapes (Kerinci Seblat
and Bukit Tigapuluh) that are included in global priority
TCL, while three other landscapes, i.e. Leuser, Sibolga and
Berbak, are categorized to class IV TCL (insufficient
information) due to lack of data (Sanderson et al. 2010).
Furthermore, Wibisono and Pusparini (2010) evaluated 33
forest patches in Sumatra to understand their distribution
and revealed that tigers present in 27 forest patches. Those
patches are mainly located in the western parts of the island
that have mountainous terrain. Only several patches are
expanding eastward, reach the east coast of Sumatra Island
in Riau and Jambi Provinces. These forests are covering
area of 140,226 km2, where only 29% are protected
(Wibisono and Pusparini 2010).
Despite their elusive behavior, Sumatran tigers are
facing two serious threats, the depletion of its habitats and
preys in one side and the increase of tiger hunt due to body
parts trade or retaliation of predation cases (Nyhus and
Tilson 2004; Nugraha and Sugardjito 2009; Wibisono et al.
2009). The habitat loss and fragmentation in Sumatra
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B I O D I V E R S IT A S 16 (2): 301-319, October 2015
Island have reached an alarming rate, varied between 0.8%
and 9.8% per year (Wibisono et al. 2011). O’Brien et al.
(2003) found numerous types of poaching signs in Bukit
Barisan Selatan National Park (BBSNP), such as snares,
cartridges, discarded batteries, gunshots, bush meat and
wildlife parts market. One hundred and seventy two snare
traps that were mainly targeted for muntjac, mouse deer,
sambar and serow, were also spotted in Kerinci Seblat
National Park (KSNP) (Linkie et al. 2003).
This top predator actually has no natural enemy, only
human beings that disproportionately hunt and kill them to
sell their body parts or to take revenge. The pressure of
their habitats creates conflicts between human and tigers
across the island. Between 1978 and 1997, the conflicts
caused 146 people died, 30 people injured and more than
870 cattle became prey (Nyhus and Tilson 2004).During
these 20 years period, 265 tigers were killed and 97 others
were captured as a result of the retaliation (Nyhus and
Tilson 2004). Additionally, 16 others became victims of
retaliation between 2000 and 2004 in which later
investigations revealed the involvement of professional
hunters/poachers on some cases (Nugraha and Sugardjito
2009). This big cat is also hunted due to their body parts
value (Plowden and Bowles 1997) and a police officer was
involved in an organized poacher (Tilson and Nyhus 2010).
More importantly, these poaching activities took place
inside national park and almost no law enforcement for the
offenders (Linkie et al. 2003; O’Brien et al. 2003; Tilson
and Nyhus 2010).
This illicit act has long been recognized as the most
imminent threat for tiger survival and appropriate measures
could halt the declining trend of tiger populations (Galster
and Elliot 2000; Damania et al. 2003; Gopal et al. 2010).In
Sumatra, anti-poaching unit has been established in KSNP
since 2000 (Hartana and Martyr 2001; Linkie et al. 2003),
named the Tiger Protection and Conservation Unit (TPCU).
The main objective of this unit is to assist the national park
management to detect, prevent and deter tiger poaching
activities in and around KSNP (Hartana and Martyr 2001).
This unit also records tiger signs, arrests illegal loggers and
wildlife poachers, confiscates chainsaws and dismantles
wildlife traps (Linkie et al. 2003).
Most studies about tiger poaching investigated the
relation of this illicit activity with tiger population or its
survival. There has not been any research about the tiger
poaching incidents related to their spatial context. The
location where poaching cases occur can be plotted into map.
The arrangement of the points can be studied spatially. One
method for the study of spatial configuration of observed
events within a two-dimensional space is point pattern
analysis (Gatrell et al. 1996; Loosmore and Ford 2006).
This analysis has been widely utilized for epidemiology
studies (Gatrell et al. 1996; Siqueira et al. 2004; Ngowi et
al. 2010; Liebman et al. 2012; Simarro et al. 2012). This
technique was successfully applied to understand clumping
pattern of morbidity and mortality, spatial and temporal
dynamic and modeling the raised incidence of diseases
(Gatrell et al. 1996). This method is also popular in studies
of spatial patterns of plant communities (Haase 1995;
Loosmore and Ford 2006; Perry et al. 2002, 2006).
Applications range from seed dispersal (Seidler and Plotkin
2006), plants coexistence (Liu et al. 2014), plants
competition (Gray and He 2009) and plant disease (Dallot
et al. 2003). Even though almost all animals are nonsedentary, but this analysis has been used to study the
occurrences, lethal incidences and stationary constructions
such as ant hills or birds' nests (Ramp et al. 2005; Hengl et
al. 2009; Cogălniceanu et al. 2013; Iosif et al. 2013). Other
disciplines that harness benefits from this method include
soil science (Huo et al. 2012), volcanology (Bishop 2007)
and urban studies (Zhang et al. 2014).
With the increasing awareness of importance of spatial
pattern in biology, a variety of statistical tests have
emerged (Haase 1995, Perry et al. 2006). Researchers with
limited experience must carefully explore suitable
approaches base on the characteristic of their data and the
relevant questions regarding the spatial information (Perry
et al. 2002, 2006). Consequently, it is a good practice
employing different techniques so that they will
complement each other and avoid partial conclusion about
the spatial data (Perry et al. 2002, 2006). Edge effects are
other pitfalls in spatial statistic and needs some form of
correction. The cause of the edge effect is the assumption
of an unbounded area for spatial point distributions, but
observed distributions are calculated from a defined region
(Dixon 2002a; Wiegand and Moloney 2004; Perry et al.
2006). Edge effects should not be neglected because can
lead to overestimation and alter the conclusion (Dixon
2002a). The addition of a buffer zone around the plot is a
popular method to account for edge effects (Dixon 2002a;
Haase 1995).
This paper aimed to investigate the tiger poaching
pattern by analyzing the spatial point pattern of the tiger
poaching incidents in KSNP and surrounded area. The
main objective of this study was to evaluate the capability
of spatial analysis in exploring the pattern of tiger poaching
cases.
MATERIALS AND METHODS
Tiger poaching data
The tiger poaching data were obtained from the Fauna
& Flora International (FFI), a non-governmental
organization focus on biodiversity conservation across the
globe. They compiled tiger poaching incidents in and
around KSNP reported monthly by the field manager of
TPCU to the head of KSNP. The data was stored in a
spreadsheet file format and contains date of the incident,
location, poaching methods, detail of the case and
enforcement actions. There were 87 incidents recorded
between October 2000 and September 2012. The record
only reported the administrative locations of the incidents.
Twenty-six cases revealed the locations up to village level,
59 records only reported the sub district and two others
only mentioned the name of the district.
Georeferencing spatial data
The process of transforming text base locality
information into geographic coordinates is called
RIFAIE et al. – Spatial pattern analysis of Sumatran Tiger poaching
georeferencing (Garcia-Milagros and Funk 2010).
Common sources of reference of geographic coordinates
for georeferencing process include gazetteers and maps.
However, assigning a single point to a locality is
commonly neglecting the quality of the representation of a
point over actual locality (Wieczorek et al. 2004).
Wieczorek et al. (2004) proposed point radius method to
calculate the potential errors or uncertainties adhered to
descriptive localities.
Georeferencing locality data is time consuming
particularly on the error checking and correcting processes.
Numerous tools have been developed to make this process
become less tedious. Garcia-Milagros and Funk (2010)
proposed the use of Google Earth© to georeference
biological specimen locality data. Google Earth displays
high-resolution satellite imagery with WGS84 datum as the
coordinate system and NASA Shuttle Radar Topography
Mission (SRTM) data as Digital Elevation Model (DEM).
The advantage of utilizing this application is that the
interface allows overlying maps, drawing paths, adding
information marks, measuring distances, checking the
elevation of a point and moving a point to another position
(Garcia-Milagros and Funk 2010).
In order to determine the position of a poaching
incident and calculate the uncertainty, administrative map
of the study area was first uploaded into Google Earth. The
administrative map was only up to sub-district level
because the lack of reliable village map. The determination
of the position of a poaching incident was done by
following steps. Firstly, the point was placed in the middle
of an administrative region if the locality was a sub district
or district. Next, the case were located near or on the point
of a village indicated by Google Earth when the locality
indicates the name of a village. Lastly, the position of the
poaching was placed in a forested area when the data
specifies the habitat is the national park or the tiger
conservation landscape.
The uncertainty calculation on the Google Earth
application was adapting Garcia-Milagros and Funk (2010)
based on steps described by Wieczorek et al. (2004). The
points was then exported into shapefile format and
processed into open source GIS software, QGIS (QGIS
Development Team 2014).
Spatial point pattern analysis
Every point process has a basic property called intensity
(λ(s)). It can be described as the expected number of events
per unit area at the point s (Perry et al. 2006; Stoyan and
Penttinen 2000).The simplest spatial process is complete
spatial randomness (CSR), named the homogeneous
Poisson Process with intensity λ. CSR is commonly used as
null model, and a disproving event would exhibit either (i)
clustering (aggregation in the bivariate case), or (ii)
regularity (segregation) (Wiegand and Moloney 2004;
Perry et al. 2006).
The behavior of a general spatial stochastic process can
be characterized in terms of its first-order and second-order
properties (Gatrell et al. 1996; Wiegand and Moloney
2004; Perry et al. 2006). First-order properties are
described in term of intensity of a point pattern, in which
313
intensity is defined as the mean value of the distribution at
locations throughout the region of interest (Gatrell et al.
1996; Zhang et al. 2014). On the other hand, second-order
properties of a spatial point process define the small-scale
spatial correlation structure of the point pattern and they
are based on the distribution of distances of all pairs of
points (Gatrell et al. 1996; Wiegand and Moloney 2004;
Perry et al. 2006; Zhang et al. 2014).
Numerous spatial statistic methods have been
developed to study the point pattern characteristic. Perry et
al. (2006) compared six types of univariate analyses in
order to provide a guidance concerning the appropriate
selection and use of each method. Each analysis has its
own strengths and weaknesses, and the application of these
tests should complement each other (Perry et al. 2006).
They also added that the tests generally could be divided
into first-order and refined for those derived from Nearest
Neighbor (NN) and second-order summary statistics.
NN test is a simple first-order analysis that determined
the intensity based on distances between two closest points
(Stoyan and Penttinen 2000; Perry et al. 2006). This test
represents a logical first step in analysis and useful for
analyzing spatial point patterns (Perry et al. 2006).
However, this method has limitation to the scale in which
events beyond nearest neighbors are ignored (Stoyan and
Penttinen 2000). Nearest neighbor index (r) can be
explained with the following formula:
[1]
Where is the observed mean distance to nearest
neighbor and is the expected mean distance to nearest
neighbor for a random distribution. Furthermore, the
observed and expected mean nearest distances can be
explained as follow:
[2];
[3]
is total distance to nearest neighbor, n is the
Where,
number of points and A is the size of the area. Finally, the
Z score of the observed mean distance to nearest neighbor
is:
[4]
where SE is the standard deviation. The index will
show a statistically significant at level 5% when the Z score
is less than -1.96 or bigger than 1.96.
On the contrary, Ripley's K function is one of most
commonly use second-order statistic which able to detect
mixed pattern due to difference of the distance scale. This
test considers all inter-point distances and produces more
information on the scale of the pattern (Wiegand and
Moloney 2004). The K function represents the expected
number of points within radius r from a randomly chosen
point (Dixon 2002b; Zhang et al. 2014), and is defined as:
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B I O D I V E R S IT A S 16 (2): 301-319, October 2015
[5]
Where, λ is the density (number per unit area) of points,
and E is the expected other points within distance r of a
randomly chosen event. The expected other points within
distance r from a randomly chosen point in a process with
no spatial dependency is also expressed as λπr2, K(r) for a
homogeneous Poisson process can be defined as:
[6]
In addition, the empirical function
is a ratio of
numeration and the density of events, λ. The density of
events is the ratio between the number of events and the
size of the area. The estimation of numerator should
consider the edge effects. These effects appear when the
numerator does not consider points outside the boundary.
One of commonly used estimator that count in the edge
correction (Haase 1995; Dixon 2002b; Zhang et al. 2014)
is:
[7]
This study examined the applicability of these two
spatial statistic tests for tiger poaching incidents. QGIS and
R spatstat (Baddeley and Turner 2005; R Development
Core Team 2012), were used to perform these analyses.
Most freeware are modular where new additional packages
can be attached to the main program as a library, add on or
plug-in. Two different free programs can also be combined
performing a series of operations as has been demonstrated
A
by Hengl et al. (2010). Unlike their work, which optimized
a scripting language, this study used a combination
between QGIS as the main software and R packages as
plug-ins so that all the work can be done in a graphical user
interface (GUI) environment.
RESULTS AND DISCUSSION
There have been 87 poaching cases recorded by the
TPCU between 2000 and 2012. Most cases were dominated
by snare traps (72 cases) in which 166 tiger snares were
found and destroyed. This record revealed that there were
33 tiger casualties because of poaching activities. While
most of the active traps were found and destroyed by the
team, around thirteen traps took victims. In addition, one
tiger were poisoned in 2000 and three other were shot in
2010 and 2012. On the contrary, body parts (smoked flesh,
skeleton, pelts or skins) from 16 tigers were discovered
during investigations, either on poaching sites or in
poachers stash houses, without exact information about
how the tigers were killed. However, when the TPCU
found the tiger body part from a poacher, their
investigation revealed where the location of the poaching
was. For example, on January 2005 the team observed tiger
skins and skeleton from poachers in Batang Merangin,
Kerinci. Nevertheless, the TPCU team revealed that the
poaching took place in Gunung Raya, Kerinci.
Unfortunately, most of the identified poachers could not be
arrested. Table 1 shows the summary of poaching cases
reported by the TPCU team.
B
Figure 1. A. Case number one (October 2000) was placed on the Renah Kayu Embun Village indicated by Google Earth with
uncertainty of 5 km. B. The record only indicates that case number 72 (January 2010) occurred in Malin Deman Sub district and the
habitat is the national park. It was then placed in the forested area of this sub district and shows uncertainty of 9.2 km. The bright lines
are sub district’s boundaries.
RIFAIE et al. – Spatial pattern analysis of Sumatran Tiger poaching
315
Figure 2.The distribution map of tiger poaching incidents between 2000 and 2012 in Kerinci Seblat National Park and surrounding area.
The coordinate reference system (CRS) of the map is WGS 84/UTM zone 47S.
Table 1. Tiger poaching incidents recorded by the TPCU between
2000 and 2012 and compiled by TRAFFIC.
Year
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
Total
Poaching
cases
1
12
3
8
8
8
9
5
4
12
9
5
3
87
Snares
found
0
27
0
4
21
21
31
6
7
18
11
12
8
166
Tiger
poisoned
1
0
0
0
0
0
0
0
0
0
0
0
0
1
Tigers
shot
0
0
0
0
0
0
0
0
0
0
1
0
2
3
Tigers
killed
1
6
4
6
1
3
0
0
0
2
2
6
2
33
The georeferencing of all 87 poaching locations were
successfully done by using the Google Earth. Almost all
locality names listed in the report were easily found and
plotted; only several locations could not be determined
promptly. For example, the report mentioned several
incidents were occurred in Lempur area, Gunung Raya Sub
District, Kerinci. However, there are four localities started
with Lempur found from Google Earth website, i.e.
Lempur Danau, Lempur Tengah, Lempur Hilir and Lempur
Mudik. Since these villages were closely located, the points
were located in the middle of these villages. The inclusion
of a more specific locality detail was another example that
made Google Earth could not identify the exact position.
One case was reported to take place in an area that belongs
to a government institution property in Curup, Bengkulu.
Two other incidents happened in a concession forest belong
316
B I O D I V E R S IT A S 16 (2): 301-319, October 2015
Figure 3. The mean center and standard deviational ellipse of the tiger poaching cases showing that the poaching incidents were
occurred along the national park, and there were many outliers especially in the southeast and northwest of the ellipse. This forest cover
map in 2011 was obtained from the Ministry of Forestry GIS website (http://appgis.dephut.go.id/appgis/download.aspx).
to a private company. When a locality was unsuccessfully
determined based on the most specific location, it would be
plotted based on the higher administrative region. The
uncertainty of points determination were varied between 5
and 20 km and mostly caused by the extent of
administrative boundaries. Uncertainty in mapping is
something that cannot be avoid due to the complexity of
the nature and measurement methods and should be taken
into account (Rocchini et al. 2011). Figure 1 illustrates two
examples of points determination and uncertainty calculation.
Garcia-Milagros and Funk (2010) demonstrated the use
of expedition map to decrease the uncertainty from 7.5 km
to only 0.85 km. unfortunately, such data could not be
obtained because of concerns that tiger occurrence will be
exposed to hunter syndicates. Other spatial data that could
decrease the uncertainty are village maps. However,
accurate village maps have not been readily available in
Indonesia. The uncertainties may seem showing low
accuracy of point mapping, but these values indicate
overestimation (Wieczorek et al. 2004). Moreover, the
uncertainties are relatively small figures when it is
compared to the study area which extent about 39.000 km2.
It is more likely that the statistical analysis would not
change significantly when more precise positions can be
identified. Figure 2 shows the distribution of the points
where tiger poaching incidents in KSNP area occurred.
The tiger poaching incidents took place in 9 out of 13
districts that surround KSNP. The districts where incidents
were found were distributed in three provinces of West
Sumatra, Jambi and Bengkulu. The mean center of the 87
points were located at 101° 43' 21.62"E and 2° 24' 16.45"
S, with the standard distance of 71.7 km. This center point
is located in Jangkat, Merangin, Jambi only about 55 km
South East of Sungai Penuh where the national park office
is situated. Standard deviational ellipse (SDE) calculated
from the poaching incidents indicates the main orientation
RIFAIE et al. – Spatial pattern analysis of Sumatran Tiger poaching
Figure 4.Plot of
versus distance up to 50 km for all poaching
locations. The difference between
values and K(r) rise as the
distance increases.
Table 2. The cumulative poaching cases recorded in each districts
District
Total incidents
Bengkulu Utara
Bungo
Kerinci
Lebong
Merangin
Mukomuko
Pesisir Selatan
Rejanglebong
Solok Selatan
5
5
28
6
20
11
5
6
1
of the poaching distribution (Gong 2002). The bearing of
the major axis of this ellipse was 147° South East with the
length of 184.3 km. There were 56 points or only 64.4% of
total points that were situated inside the SDE area. Most of
outliers (16 points) were located in Bengkulu Province (see
Figure 3).
The NN test applied to this data showed that the
distribution of tiger poaching incidents had the NN index
of 0.451973869542. The observed mean distance was
4,967.646 m, while the expected mean distance was
10,991.003 m, with the Z-score -9.77893987179 far less
than the critical value of -1.96. This suggested that the
cases distribution were significantly clustered. The second
order test, Ripley's K, verified this clustering pattern.
Figure 4shows that the empirical K function (solid line)
was constantly higher than the expected value representing
a homogenous Poisson process (dashed blue line). It can be
viewed that the K value soared significantly from the finest
scale (0-20 km). The slope was then noticeably ascending
as the distance rose. This rise can be observed at the
medium scale (about 25 km) and at the longer scale (35
km).
317
The spatial statistic analysis of secondary data of
Sumatran tiger poaching incidents in and around KSNP
area showed the extent and pattern of the tiger poaching.
This vast extent of areas where this illicit activity took
place can be seen from the outlier points and administrative
distribution. The furthest outlier point in Air Duku, Rejang
Lebong District was 62 km away from the SDE boundary.
The outliers were not only scattered in both ends of the
SDE but also in northwest and northeast of the standard
deviational area. The hunt for tigers by poachers were
stretched from Sangir Sub district, Solok Selatan, West
Sumatra in the north to Air Duku, Bengkulu in the south,
and Muara Sako Village, Pancung Soal, Pesisir Selatan in
the west to Tabir Hulu, Merangin, Jambi in the east. This
vast extent of outliers illustrated how pervasive the threat
of Sumatran tiger in this region.
On the other hand, both NN and Ripley's K tests that
represent first and second-order spatial statistic analyses
indicated that the poaching incidents were significantly
clustered. There were three reasons why this pattern was
appeared in this region. First, the georeferencing procedure
was merely based on the administration data. This made
some data were georeferencing in the same or very close
position. The clustering points were visibly apparent in
some districts with several incidents found. Secondly, the
limited resources forced the TPCU to manage their patrol
carefully (Linkie et al. 2003). The TPCU teams put more
focus on areas that have been recognized as the prime tiger
habitat and area with imminent threat of illegal logging and
encroachment (Fauna & Flora International 2008). Several
sub-districts, Gunung Raya and Jangkat sub-districts in
Jambi for instance, were areas where TPCU teams seized
more poaching activities than any other regions. Finally,
the conspicuous clustering point pattern in this area was as
a result of the poachers activities pattern. They commonly
set tiger traps on the animal trails and on certain time when
they think the anti-poaching team did not guard the park
intensively (Fauna & Flora International 2008). On August
2004, TPCU team recorded two snares have been replaced
by poachers in Tapus, Lebong, Bengkulu. One of
previously found snares (July 2004) in the same area took
one tiger life. Moreover, the aggregation of the poaching
cases was also clearly observed from the districts level.
Table 2 shows the accumulation of the recorded poaching
in each district during this 12 years period. This table
shows that there were three districts, which had high
poaching incidents. There were 28, 20 and 11 cases
recorded in Kerinci, Merangin and Mukomuko districts
respectively. These administrative regions are located in
the center of this national park. In contrast, administrative
regions that are located in the fringe of the protected park
such as Solok Selatan, Pesisir Selatan, Bungo and
Bengkulu Utara had very low poaching cases.
As can be seen, statistical analysis performed in this
paper suggested that rampant poaching activities occurred
in the heart of this national park. Even though the antipoaching team has been established since 2000 and actively
patrolling the park, this has not been stopping poachers
hunting Sumatran tigers. Strengthening the TPCU is an
urgent measure. Two simple mechanisms of the TPCU
318
B I O D I V E R S IT A S 16 (2): 301-319, October 2015
enhancement are adding more personnel and increasing the
frequency of patrols. The addition of patrolling team
members will expand the area covered with regular patrol.
Correspondingly, the intensification of patrols will reduce
the possibility of tigers being trapped, poisoned or shot.
However, it is more critical improving the law enforcement
for any illegal act toward wildlife in Indonesia. Very weak
law enforcement and government commitment to
conservation made Indonesia fell far behind than other
developing countries in term of protecting wildlife
(Meijaard 2014).
This study has shown the applicability of two spatial
point pattern analyses for Sumatran tiger poaching pattern
study. Both first-order (NN) and second-order (Ripley's K)
tests were chosen among other spatial statistics based on
their simplicity and popularity to use (Perry et al. 2006).
Perry et al. (2006) elaborated the strengths and weaknesses
of both techniques. The employment of different tests in a
study is not only to combine various results but also to
avoid false deduction due to the weakness of a method
(Perry et al. 2002, 2006). These two purposes of applying
different tests have been reflected by the congenial results
from the two statistics.
As shown above, spatial statistics are robust tools for
animal ecology study especially in Indonesia. Nevertheless,
the collection of point position is the first major problem
that scientists must deal with (Stoyan and Penttinen 2000).
Enormous species occurrences which can be gathered from
museum collections, published data or field records
(Cogălniceanu et al. 2013) have assorted level of locality
precision. Even with most recent locality data acquired
with GPS devices, certain amount of uncertainty occurred
because of GPS inaccuracy, unknown datum and
coordinate imprecision (Wieczorek et al. 2004). This most
likely happened when biologists with limited GPS
knowledge copy coordinates data manually, ignoring the
datum information.
Correspondingly, the exploration of statistical tests to
answer different questions will advance the adoption of this
method in Indonesia. New approaches have also gained
significant consideration to link the spatial structure to
process (Perry et al. 2006).Perry et al. (2006) suggested
that we should use a priori hypotheses and generate testable
hypotheses as an outcome of the spatial analyses. More
importantly, a good multidisciplinary teamwork between
ecologists and spatial statisticians must be established to
attain the goals (Stoyan and Penttinen 2000).
Two spatial point pattern analyses performed in this
paper showed a significant clustered pattern of Sumatran
tiger poaching. Although the points were generated from
georeferencing process and have some degree of
uncertainty, they represent the position of the poaching
incidents quite well. The aggregation of the tiger poaching
demonstrates the enormous pressure to this subspecies
especially in the central parts of the park. On the contrary,
the extent of the points suggests that poachers were lurking
for any opportunity to catch and kill this majestic animal.
While the subject of this study is the poaching activity
of an endangered species, the occurrence of a species itself
should become the target of investigations in the future. It
would be very valuable to perform point pattern analysis of
Sumatran tiger presence based on their scats, scratch
marks, tracks and other signs. In addition, species
occurrence records from museum collections provide
immense opportunity to be explored. Finally, the point
pattern analysis is multivariate in nature, therefore bivariate
and multivariate analyses are potential approaches to study
inter-species coexistence or competition.
ACKNOWLEDGMENTS
The authors thank Deborah J. Martyr (FFI) for the
Sumatran tiger poaching cases data around Kerinci Seblat
National Park. We also thank Dr. Gono Semiadi for helpful
comments on the manuscript. Two anonymous referees
greatly improved the paper by their constructive comments.
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ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160229
B I O D I V E R S IT A S
Volume 16, Number 2, October 2015
Pages: 320-326
Potential in bioethanol production from various ethanol fermenting
microorganisms using rice husk as substrate
1
WOOTTICHAI NACHAIWIENG1,♥, SAISAMORN LUMYONG2, RONACHAI PRATANAPHON1,
KOICHI YOSHIOKA3, CHARTCHAI KHANONGNUCH1,
Division of Biotechnology, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Mueang, Chiang Mai 50100, Thailand
Tel: +6686-923-5172. ♥email: woot072@hotmail.com, chartchai.k@cmu.ac.th
2
Department of Biology, Faculty of Science, Chiang Mai University, Mueang, Chiang Mai 50200, Thailand
3
Laboratory of Forest Resource Circulatory System, Division of Environmental Science, Graduate School of Life and Environmental Science,
Kyoto Prefectural University, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522 Japan
Manuscript received: 7 August 2015. Revision accepted: 29 October 2015.
Abstract. Nachaiwieng W, Lumyong S, Pratanapol R, Yoshioka K, Khanongnuch C. 2015. Potential in bioethanol production from
various ethanol fermenting microorganisms using rice husk as substrate. Biodiversitas 16: 320-326. Rice husk was investigated as the
potential substrate for bioethanol fermentation. It was collected from five locations in northern Thailand and found that the main
component of rice husk approximately 51-54% (w/w) was holocellulose. The sugar composition in rice husk holocellulose was glucose,
xylose and arabinose in the ratio 66.68, 27.61 and 5.71%, respectively. Before further fermentation, acid and alkali pretreatment of rice
husk were prior investigated and 2% (w/v) NaOH at 130oC for 30 min was proved to be the most suitable pretreatment method without
fermenting inhibitors generation. Then, rice husk hydrolysate obtained by enzymatic saccharification with Meicelase enzyme was used
as carbon sources for ethanol fermentation in comparison among 11 ethanol fermenting microorganisms including 3 strains of
Saccharomyces cerevisiae, 3 strains of Zymomonas mobilis, 3 strains of Kluyveromyces marxianus and 2 strains of pentose sugar
fermenting microbes, Candida shehatae TISTR 5843 and Pichia stipitis BCC 15191. All three strains of Z. mobilis exhibited the best
ethanol fermentation yield, giving the ethanol yield of 0.48 g g-1 available monosaccharides and fermentation profile of each individual
genus was also demonstrated. However, some unutilized sugars still remained in rice husk fermenting medium, therefore, conversion to
valuable products or optimization of co-culture ethanol fermentation needs to be further investigated.
Keywords: bioethanol, pretreatment, ethanol, fermentation, Meicelase, rice husk
INTRODUCTION
Due to rapid increasing of gasoline price and depletion
of readily available oil resource, renewable resources
alternative to oil, such as biomass, wind, solar, geothermal
and hydroelectric energy have gained increasing attention
in recent years. Bioethanol (C2H5OH) is widely accepted as
an important source for transportation fuel and energy. It
reduces CO2 emission and pollutants when replacing
gasoline in modified engine (De Oliveira et al. 2005). Since
ethanol produced from edible materials caused high
production cost and created ethical problem regarding
competitiveness to food supply, bioethanol from
lignocellulosic material has gathered keen attention (Nigam
and Singh 2011). Although the bioethanol from
lignocellulosics could not replace all gasoline, its
importance is still widely recognized. Over the past decade,
the number of bioethanol plant from lignocellulosic
materials have begun to increase (Eisentraut 2010), and its
production level, 21 billion gallons from cellulosic
feedstock by 2022 was described in law of USA (Nigam
and Singh 2011). In addition, as expected by Limayem and
Ricke (2012), one billion tons of various lignocellulosic
feedstocks and an additional cultivation of high yielding
energy crops on Conservation Reserve Program (CRP)
lands that are efficiently managed are expected to meet a
30% petroleum-based gasoline displacement in 2030.
As previously reports, ethanol are widely produces
from various lignocellulosic residues such as agricultural
crops i.e. wheat and paddy straws, corn stover, groundnut
shell, sunflower stalks, alfalfa fiber, cotton stalks and
agricultural by-products i.e. sugarcane bagasse, corncobs,
palm bagasse, barley and sunflower hulls, wheat barn and
especially rice husk (Arora et al. 2015). Rice husk is an
agricultural waste abundantly available in rice producing
countries including Thailand. Thailand was reported to be
the Asia 3th rice production as the productivity
approximately 4% of world rice’s production was gained
(Gadde et al. 2009). The annual world rice production
amounts to approximately 400 million metric tons, of
which more than 10% is husk (Conradt et al. 1992). In
addition, rice husk contains around 50% (w/w) of cellulosic
component (Wannapeera et al. 2008; Mansaray and Ghaly
1999). Industrial use of rice husk is mostly burning as a
source of heat for generation of electricity, which causes
environmental problems owing to a large quantity of CO2
emission (Bharadwaj et al. 2004). Due to the environmental
problem and availability as an agricultural residue in
Thailand, conversion of rice husk into bioethanol is an
important subject.
Due to the presence of lignin and hemicellulose in
lignocellulosic structure, the access of cellulolytic enzymes
NACHAIWIENG et al. – Ethanol production from rice husk
to cellulose for hydrolysis and finally ferment to ethanol is
difficult. Therefore, the lignocellulose needed to be delignified
by pretreatment processes which various available including
physical, physico-chemical, chemical and biological
pretreatment (Sun and Cheng 2002) and all having their
specific advantages and disadvantages. However, the
chemical pretreatment was selected in this study because of
no expensive equipment required and their availability.
Both acid and alkaline pretreatment are able to increase
accessible surface area and alter lignin structure, moreover,
alkaline pretreatment has a strong effect to remove lignin
which lead to easier access of cellulolytic enzyme to
cellulose structure (Mosier et al. 2005).
Until now, various ethanol producing microorganisms
have been discovered. Saccharomyces cerevisiae, a good
brewer yeast, is found to be the most popular and produce
high ethanol yield from 6-carbon atom sugars such as
glucose (Piškur et al. 2006). In contrast to S. cerevisiae,
Zymomonas mobilis is another candidate which capable of
grows and produces high concentration of ethanol from
higher initial sugar and more tolerate to ethanol
concentration with lower biomass formation (Rogers et al.
1979). However, the processes of ethanol fermentation by
both strains described above are favorable working under
30-35ºC which is not suitable for tropical countries and
incompatible when using in Simultaneous Saccharification
and Fermentation (SSF) process. Therefore, Kluyveromyces
marxianus, a thermotolerant ethanol fermenting yeast, has
been chosen for ethanol fermentation at high temperature
condition instead, in particular for SSF (Ballesteros et al.
2004). Not only glucose, various 5-carbon atom sugars
such as xylose and arabinose are consisted in
hemicelluloses component of lignocellulosic residues.
Unfortunately, S. cerevisiae, Z. mobilis and some strains of
K. marxianus could not utilize those sugars as their carbon
source. Potential strains which capable of fermenting these
sugars such as Pichia stipitis and Candida shehatae are
called pentose fermenter. Therefore, to complete and
efficient conversion of both 5- and 6- carbon atom sugars,
co-culture fermentation between both hexose and pentose
fermenter should be carried out, even though few
experiments were succeeded (Fu and Peiris 2008; Fu et al.
2009).
This manuscript describes a rice husk compositions and
the most suitable chemical pretreatment method for
obtaining the highest rice husk sugar yield. Moreover, this
is the first report demonstrated the comparison in ethanol
fermentation profile of various ethanol fermenting
microorganism groups including common yeast,
thermotolerant yeast, pentose fermenter yeast and ethanol
fermenting bacteria. This could be helpful for further strain
selection when using rice husk as substrate for ethanol
production.
MATERIALS AND METHODS
Microorganisms
Fermenting microorganisms, Saccharomyces cerevisiae
TISTR 5088 [S5088], S. cerevisiae TISTR 5169 [S5169],
321
S. cerevisiae TISTR 5339 [S5339], Zymomonas mobilis
TISTR 405 [Z405], Zymomonas mobilis TISTR 548
[Z548], Z. mobilis TISTR 551 [Z551] and Candida
shehatae TISTR 5843 [C5843] were purchased from
Thailand Institute of Scientific and Technological Research
(TISTR). Pichia stipitis BCC 15191 [P15191],
Kluyveromyces marxianus BCC 7025 [K7025] and
Kluyveromyces marxianus BCC 7049 [K7049] were
purchased from Biotech Culture Collection (BCC),
Thailand. Kluyveromyces marxianus CK8 [KCK8] was
previously isolated from rotten fruit in Chiang Mai (Amaiam and Khanongnuch 2015).
Sample preparation
Rice husk samples were randomly collected from
Northern provinces of Thailand including Chiang Mai,
Chiang Rai, Lamphun, Lampang and Nan without rice
variety consideration. Samples were washed thoroughly
with tap water and dried at 60oC for 3 days. Dried samples
were milled with hammer mill and size screened by sieving
through 16 mesh aluminium sieve. All samples were kept
in the desiccators until the experiments.
Analysis of rice husk composition and sugar
components
The ground rice husk samples from all 5 sources were
subjected to composition analyses including holocellulose,
hemicellulose, lignin, ash, protein, lipid and soluble
carbohydrate according to the protocols of acid chlorite
method (Browning 1963), TAPPI T203 om-88 (TAPPI,
1992), TAPPI T222 om-88 (TAPPI, 1988), TAPPI T211
om-85 (TAPPI, 1985), Kjeldahl method (Conklin-Brittain
et al. 1999), Soxhlet extractor method (Wren and Mitchell
1959) and phenol sulfuric method (Dubois et al. 1956),
respectively.
For sugar component analysis, approximately 0.5 g of
rice husk was hydrolyzed with 8 ml of 72% (w/w) sulfuric
acid, gentle mixed and incubated at 30oC for 1 h. The
sample was then diluted with 300 ml of distilled water to
adjust a final concentration of sulfuric acid to be 2% (w/w).
The solution was then autoclaved at 121oC for 30 min and
neutralized by addition of Ba(OH)2. A clear supernatant
was obtained by centrifugation at 4290 x g for 20 min and
subjected to quantitative analysis of neutral sugars by High
Performance Liquid Chromatography (HPLC) Shimadzu
LC 20A system (Shimadzu Corp., Kyoto, Japan) equipped
with Aminex HPX-87P (300 mm×7.8 mm) (BioRad, USA)
using deionized distilled water as an eluent at a flow rate of
0.3 ml min-1 and the column temperature, 58oC.
Glucoheptose was used as an internal standard. Detection
was carried out with a Fluorescent Detector (FLD) at 420
nm by post reaction with a mixture of arginine and boric
acid (1:3, v/v) at 150oC.
Pretreatment of rice husk samples
Dried rice husk sample was pretreated by 0.5, 1.0, 1.5
and 2.0 % (w/v) of diluted sulfuric acid and sodium
hydroxide solution with ratio 1:10. Pretreatment process
was carried out in autoclave machine at 130oC for 30 min
and allowed to cool down overnight. Solid fractions were
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B I O D I V E R S IT A S
16 (2): 320-326, October 2015
obtained by filtration through Whatman No.1 filter paper
and then washed by excess volume of tap water for
neutralization. Pretreated rice husk sample was dried at
60oC for 2 days before enzymatic saccharification.
Enzymatic saccharification of pretreated rice husk
Approximately 0.2 g of chemical pretreated rice husk
samples were hydrolyzed by 40 FPU g-1 substrate of
commercial cellulolytic enzyme “Meicelase” (510 FPU g-1
enzyme powder, from Trichoderma viride, Meiji Seika
Company, Tokyo, Japan) dissolved in sodium succinate
buffer (pH 4.8), in the presence of 0.1% sodium azide. A
saccharification was carried out in a shaking incubator at
45oC, 150 rpm for 48 h and the reducing sugars liberated
were finally quantified by dinitrosalicylic acid (DNS)
method (Miller 1959).
Rice husk fermenting medium preparation
Rice husk hydrolysate was prior prepared by
hydrolyzing the 2.0% (w/v) NaOH pretreated rice husk
sample with Meicelase (40 FPU g-1 of substrate) at 45ºC for
48 h as previously described without sodium azide added.
The liquid portion was obtained by filtered through
Whatman No.1 filter paper and used as a carbon source for
fermenting medium preparation. The sugar concentration
was adjusted to approximately 20 g L-1 by rotary
evaporator. The concentrates were supplemented with yeast
extract, (NH4)2HPO4 and MgSO4.7H2O at the final
concentration of 3.0, 0.25 and 0.025 g L-1, respectively
(Gupta et al. 2009), and sterilization by autoclaving at
121oC for 15 min.
Determination of fermenting inhibitor in rice husk
fermenting medium
Fermenting inhibitors which may generate during
pretreatment process are very important factors for ethanol
yield and need to be investigated before further
fermentation. Furfural, 5-hydroxy methyl furfural (5-HMF)
and acetic acid were determined by HPLC (Shimadzu LC
20A system) equipped with Aminex HPX-87H column
(300mm×7.8mm with guard cartridge). Separation was
carried out at 35oC using 8 mM sulfuric acid as a mobile
phase with flow rate 0.6 mL min-1. Peaks were detected by
Photo Diode Array (PDA) detector. Lignin degradation
products, vanillin, syringaldehyde and 4-hydroxybenzaldehyde, were also analyzed by HPLC (Shimadzu LC
20A system) equipped with Imtakt Unison UK-Phenyl
(150×4.6 mm, 3μ m) without guard cartridge. Separation
was performed at 40oC using gradient between 10mM
ammonium acetate buffer and acetonitrile as mobile phase
with flow rate 1 mL min-1 and peaks were also detected by
PDA detector.
Comparison of fermentative ability of various
microorganisms
The comparative fermentation experiment by
fermenting microorganisms were preliminary carried out
with 3 strains of common ethanol fermenting yeast S.
cerevisiae, 3 strains of thermotolerant ethanol fermenting
yeast K. marxianus, 3 strains of ethanol fermenting bacteria
Z. mobilis and 2 strains of pentose fermenter, C. shehatae
and P. stipitis, by inoculated 5% (v/v) of inoculum (108
CFU) into 125 mL-Duran bottle containing of 50 ml of rice
husk fermenting medium. The fermentation was carried out
at 30oC, except for K. marxianus which carried out at 45oC
for 96 h, and the samples were collected 12-h interval.
Ethanol
concentration
was
analyzed
by
gas
chromatography (GC-17A; Shimadzu, Tokyo, Japan) using
a flame ionization detector and stainless steel column with
15.0 m in length, 0.53 mm of diameter and 0.5 µm of film
thickness. The column, injection and detector temperature
were maintained at 40°C, 230oC and 250oC, respectively.
Nitrogen was used as carrier gas at a flow rate of 30 mL
min-1. Reducing sugar was determined by DNS method.
Concentration of glucose and xylose were measured by
Autokit Glucose (Wako Chemicals, Osaka, Japan) and Dxylose assay kit (Megazyme, Wicklow, Ireland),
respectively.
Cell
density
was
measured
by
spectrophotometer at wavelength 600 nm.
RESULTS AND DISCUSSION
Rice husk composition analysis
There were slightly significant differences in
compositions among 5 rice husk samples at p-value < 0.05.
Variation of rice husk component could occur due to the
varieties of paddy sown, watering, geographical conditions,
fertilizer used, climate, soil chemistry, age of paddy and
growth conditions (Foo and Hameed 2009). Holocellulose,
alpha cellulose, hemicelluloses and lignin content were 5154%, 20-25%, 28-32% and 28-30% (w/w), respectively
Table 1. Compositions of 5 different sources rice husk samples collected from Northern Thailand
Holo
Alpha
Hemi
Soluble
Lignin
Ash
Lipid
Protein
cellulose
Cellulose
Cellulose
Carbohydrate
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
ab**
a
a
a
a
a
a
CM
52.86
20.86
32.00
30.01
18.73
1.63
0.07
2.34a
a
b
c
b
b
b
a
CR
51.28
22.26
29.02
28.92
20.21
2.04
0.10
2.55a
LPo
54.14b
25.50c
28.64d
30.30a
18.79a
1.47a
0.25b
2.49a
ab
a
a
b
a
a
a
LPa
52.73
20.87
31.86
28.81
18.67
1.53
0.09
2.47a
a
a
b
ab
a
a
a
NN
51.16
20.17
30.99
29.72
19.03
1.71
0.12
2.36a
Note: * CM, CR, LPo, LPa and NN were rice husk sample collected from Chiang Mai, Chiang Rai, Lamphun, Lampang and Nan
Provinces. ** Superscript letters represented a significant at p < 0.05
Samples*
NACHAIWIENG et al. – Ethanol production from rice husk
(Table 1). Rice husk sample (LPo) was selected for further
study due to its highest holocellulose content. The
holocellulose content (>50% w/w) of rice husk was
comparable to previous studies (Saha, Badal C. and Cotta
2008; Banerjee et al. 2009; Hsieh et al. 2009). The ash
content was 18-20%, interestingly 95% of ash in rice husk
is silicon (Della et al. 2002) and use for silicon-based
materials after bioethanol production is also attractive. The
neutral sugar composition in rice husk was glucose, xylose
and arabinose in ratio 66.68 ± 0.97, 27.61 ± 1.06 and 5.71
± 0.17%, respectively. This result was similar to previous
result which was analyzed the neutral sugar, glucose,
xylose and arabinose, from rice husk in ratio 61.62, 34.23
and 4.15%, respectively (Nabarlatz et al. 2007). These
results indicated that cellulose and xylan are the major
polysaccharides in rice husk and arabinan is also present in
the sample but only in trace amount.
To optimize a chemical pretreatment process, the ability
of enzymatic saccharification after pretreatment was crucial
in the evaluation of the best pretreatment process for rice
husk. Sulfuric acid and sodium hydroxide were widely
used as pretreatment substances as referred to the previous
reports (Saha, Badal C et al. 2005; Singh et al. 2011).
Concerning to environmental friendly policy and the high
quantity of chemical usage in rice husk pretreatment with
sulfuric acid and sodium hydroxide, the maximum
concentration of each pretreatment substance was assigned
at maximum of 2.0% with implementation by conventional
autoclave. The highest yield of liberated sugars from
enzymatic saccharification based on 2.0% sodium
hydroxide pretreatment process was 37.35 ± 2.11% (w/w)
of rice husk dry weight and significantly different at p <
0.05 from pretreatment by the other concentrations of
sodium hydroxide (Figure 1). In contrast, the highest yield
of liberated sugars from enzymatic saccharification after
pretreatment by 0.5% sulfuric acid was only 0.96 ± 0.41%
of rice husk weight. These indicated that acid pretreatment
was not suitable for rice husk pretreatment and
hemicellulose might be lost during pretreatment process
with the formation of a toxic hydroxymethylfurfural (Lee et
al. 1999).
Moreover, even though, both acid and alkaline
pretreatment could increase accessible surface area and
alter lignin structure, but lignin was only removed in case
of alkaline pretreatment (Mosier et al. 2005) especially
sodium hydroxide pretreatment on rice husk residue
(Nikzad et al. 2015) which allow cellulolytic enzyme to
penetrate and hydrolyze cellulose structure, lead to high
amount of sugar released. Therefore, alkaline pretreatment
process based on 2.0% sodium hydroxide was selected to
be the most appropriate chemical pretreatment for rice husk
in this experiment. In addition, after pretreatment rice husk
by 2% (w/v) NaOH solution, glucose, xylose and arabinose
contents were non-significantly decreased, the total sugar
loss during pretreatment process was calculated as 6.04%
(Table 2) without any detection of fermenting inhibitors
such as furfural, 5-hydroxy methyl furfural (5-HMF),
acetic acid, lignin degradation products, vanillin,
syringaldehyde and 4-hydroxybenzaldehyde. Because of
low removal of hemicellulose and cellulose structure by
323
alkaline pretreatment (Mosier et al. 2005; Ang et al. 2013),
there was slightly sugar released during pretreatment
process as presented in this study. Low sugar released and
no sugar degradation during pretreatment is key factors for
effective pretreatment method as described by Yang and
Wyman (2008). As the result, the alkaline pretreatment was
chosen in this study for further ethanol fermentation
without concerning of fermenting inhibitor and sugar loss
during pretreatment.
Furthermore, our results were
compatible with previous studies, alkaline pretreatment
with 120oC was sufficient to increase the digestibility of
low lignin containing lignocellulosic biomass (Kaar and
Holtzapple 2000) without any fermenting inhibitors
generation (Chang et al. 2001). Even though liquid
fractions from various kinds of biomass from acid
hydrolysis method are widely used for ethanol production,
however, time consuming and complicated detoxification
processes are needed because of the presence of various
fermenting inhibitors (Larsson et al. 1999).
Comparison of ethanol fermentation of rice husk sugar
by various fermenting microorganisms
According to previous study of fermentation, all
fermenting strains could grow and ferment in the presence
of succinic acid but some microorganisms could not grow
in the presence of acetic and citric acid (Nachaiwieng et al.
2015). Therefore, sodium succinate buffer was used in
saccharification process to avoid some inhibitors from acid
mentioned above. With this buffer and under a static
condition, all fermenting strains could grow well except for
K. marxianus. This strain need more oxygen to grow and
ferment, thus 150 rpm shaking condition was then applied
for this strain as previously described by Limtong et al.
(2007). Furthermore, an anaerobic bacteria Z. mobilis,
which could not grow well under aerobic condition needed
a sterilized liquid paraffin to make a layer for absorbing
oxygen (Li et al. 2000; Yoshida et al. 1970). When the
fermentation was terminated, the ethanol yield obtained
from the same genus was similar. The highest ethanol yield
was obtained from Z. mobilis with 0.48 g g-1 available
monosaccharides or 94.12% of theoretical yield (Table 3).
Z. mobilis is found to be the highest ethanol yield
producing strain because of less biomass is produced and a
Figure 1. Percentage of liberated sugars per rice husk weight
from enzymatic saccharification after acid and alkaline
pretreatment of rice husk
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B I O D I V E R S IT A S
16 (2): 320-326, October 2015
Figure 2. HPLC based sugar analysis of rice husk hydrolysate,
before and after ethanol fermentation by hexose fermenting strain
(Glc, Xyl, Ara and Man were glucose, xylose, arabinose and
mannose, respectively)
Table 2. The amount of rice husk monosaccharide (g L-1) loss
after pretreatment rice husk by 2% (w/v) NaOH at 130oC for 30
min
Monosaccharides
Glucose
Xylose
Arabinose
Untreated rice
husk
(g L-1)
2% (w/v) NaOH
pretreated rice husk
(g L-1)
0.356a ± 0.03
0.085a ± 0.01
0.039a ± 0.01
0.347a ± 0.04
0.074a ± 0.01
0.030a ± 0.01
Note: Significant at p < 0.05
Table 3. Ethanol yield production from comparative fermentation
by various fermenting microorganisms on each microorganism
optimum fermenting condition
Microorganisms
Ethanol yield
(g g-1 available
monosaccharides)*
Ethanol yield
(g g-1
available
sugar)
S. cerevisiae TISTR 5088
S. cerevisiae TISTR 5169
S. cerevisiae TISTR 5339
Z. mobilis TISTR 405
Z. mobilis TISTR 548
Z. mobilis TISTR 551
K. marxianus BCC 7025
K. marxianus BCC 7049
K. marxianus CK8
C. shehatae TISTR 5843
P. stipitis BCC 15191
0.45
0.45
0.45
0.48
0.48
0.48
0.43
0.42
0.42
0.40
0.42
0.27
0.27
0.26
0.29
0.28
0.28
0.24
0.24
0.25
0.24
0.25
Note: *Hydrolyzed rice husk sugar contained unidentified
oligomers and could not utilize by microorganisms
higher metabolic rate of glucose is maintained through its
special Entner-Doudoroff pathway (Bai et al. 2008).
Ethanol yield produced by Z. mobilis strains in this study
are corresponding to previous reports that ethanol yield
from Z. mobilis ATCC 10988 and Z. mobilis ATCC 31821
at 0.472 and 0.468 g g-1 available glucose, respectively
(Rogers et al. 1982; Tao et al. 2005). Other fermenting
strains, S. cerevisiae, K. marxianus, P. stipitis and C.
shehatae TISTR 5843, produced ethanol 0.45, 0.42-0.43,
0.42 and 0.40 g g-1 available monosaccharides,
respectively. Interestingly, ethanol yield obtained from all
S. cerevisiae strains in this study were significantly higher
than 0.41 g g-1 obtained from S. cerevisiae ITV-01 (Ortiz‐
Muniz et al. 2010) and also higher than those strains
mentioned by Hahn-Hägerdal et al. (2006) from various
source of agricultural hydrolysate fermenting media.
Moreover, comparing ethanol yield from rice husk
hydrolysate, ethanol yield obtained from this study is better
than 0.43 g g-1 sugar obtained using commercial S.
cerevisiae as fermenter yeast (Dagnino et al. 2013). As
previous results, various strains used in this study,
especially Z. mobilis and S. cerevisiae, seemed to be
suitable strains for producing ethanol from rice husk
hydrolysate although the fermentation process has not yet
optimized in this study. In addition, possible highest
ethanol yield from rice husk in this study was 0.26-0.28 g
g-1 of dry rice husk which is potential substrate when
compared to barley straw, corn stover, oat straw, rice straw,
sorghum straw, wheat straw and bagasse which gave an
ethanol yield at 0.26-0.31 g g-1 of dry biomass (Kim and
Dale 2004). However, this ethanol yield was calculated
from rice husk holocellulose and maximum ethanol yield
(0.51 g g-1 sugar), therefore, further study of optimization
on fermentation process and co-culture fermentation
between hexose and pentose fermenter should be carried
out to reach to expecting target of ethanol yield.
As presented in Figure 3, the fermentation profile was
unique in each genus. Highest ethanol yield from S.
cerevisiae, Z. mobilis and pentose fermenter was obtained
after 24 h and continued constant. In contrast, the highest
ethanol yield from K. marxianus was obtained after 12 h
and gradually decreased as similar due to high temperature
used in fermentation process (Teixeira and Vicente 2013)
as presented in previous study (Signori et al. 2014).
Interestingly, there were some sugars remaining after
fermentation by all microorganisms and were detected by
HPLC as xylose, arabinose and unidentified oligomers
(Figure 2) while all of glucose was utilized within 24 h.
Except for C. shehatae TISTR 5843 and P. stipitis BCC
15191 which could utilized both glucose and a little
amount of xylose (Figure 3). Unfortunately, even both
strains could utilize both glucose and xylose but ethanol
yield obtained from these strains were lower than others.
Therefore, a fermentation of rice husk medium with high
potential ethanol producing strain and the conversion of
remaining pentose and its oligomer to high valued
substances such as xylooligosaccharide or xylitol, or
optimize a co-culture ethanol fermentation between pentose
and hexose fermenter have to be further investigated.
NACHAIWIENG et al. – Ethanol production from rice husk
325
Figure 3. Comparative study of ethanol fermentation from various fermenting microorganism strains, S. cerevisiae (A); Z. mobilis (B);
K. marxianus (C) and Pentose sugar fermenter (D)
ACKNOWLEDGEMENTS
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B IO D IV E RS IT A S
Volume 16, Number 2, October 2015
Pages: 327-354
ISSN: 1412-033X
E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d160230
Review:
Genetic diversity of local and exotic cattle and their crossbreeding
impact on the quality of Indonesian cattle
SUTARNO♥, AHMAD DWI SETYAWAN
Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36A Surakarta 57126, Central Java,
Indonesia. Tel./Fax. +62-271-663375, email: nnsutarno@yahoo.com; volatileoils@gmail.com
Manuscript received: 3 September 2015. Revision accepted: 30 October 2015.
Abstract. Sutarno, Setyawan AD. 2015. Genetic diversity of local and exotic cattle and their crossbreeding impact on the quality of
Indonesian cattle. Biodiversita16: 327-354.Several species of cattle had been domesticated around the world, but only two species were
farmed extensively, zebu cattle (Bos indicus) of the tropics and taurine cattle (Bos taurus) of the subtropical areas. Both of them had
hundreds variety of offspring in the worlds. The third species of cattle that most widely farmed was Bali cattle (Bos javanicus), an
indigenous cattle from Indonesia that was domesticated from wild banteng (Bos javanicus javanicus). Besides Bali cattle, Indonesia had
also some local cattle as direct descendants of or as Crossbreeds of those three cattle. These cattle had been adapted to climatic
conditions, feeds and diseases in Indonesia. Local zebu cattle that relatively pure were Peranakan Ongole (PO) or Ongole breeds and
Sumba Ongole (SO). The main Crossbreed between zebu and Bali cattle was Madura cattle. The other well-known cattle of this were
Aceh cattle, Pesisir cattle, Rancah cattle, Jabres cattle, Galekan cattle and Rambon cattle. Crossbreeds of taurine and zebu cattle
generally produced calf that declining reproductive ability in generations. One fairly successful was Grati cattle or Holstein Friesian
Indonesia (FHI) which was a crossbreed of Holstein Friesian and PO cattle. In recent decades, there were many crossbreed activities
through artificial insemination between local cattle and taurine cattle to produce excellent beef cattle, mainly Simmental and Limousin.
This activity was carried out widely and evenly distributed throughout Indonesia. It was conducted on all local cattle breeds and was
strongly supported by local farmers. This crossbreeding activity was feared to change the genetic diversity of local Indonesia cattle,
where the descendants could not adapt to the climatic conditions, feeds and localized diseases; and the ability of reproduction continues
to decline in generations, there fore the availability of parental cattle should be maintained continuously. This crossbreed had produced
some new breeds, among others Simpo (Simmental x PO), Limpo (Limousin x PO), Simbal (Simmental x Bali cattle), Limbal
(Limousin x Bali cattle), and Madrasin or Limad (Limousin x Madura cattle). Male offsprings were sterile, while female offsprings had
lower reproductive capacity than of the parent’s. This lead to uncertainty over the guarantee of meeting the needs of protein (meat and
milk) of Indonesian in the future, thus there was a need of regulation. On the other hand, in the grasslands of North Australia, the
breeder had produced an eminent cattle breeds, namely Australian Commercial Cattle (ACC), from uncontrolled crossbreeds between
different breeds of taurine and zebu cattle in the pasture, therefore this concerns ignored.
Keywords: Crossbreeding, exotic cattle, genetic, local cattle, quality
INTRODUCTION
Cattle raising activities have been widely practiced in
Indonesia since immemorial time. In the era of Hindu
kingdoms, cattle is commonly awarded by kings to the
Brahmin priests as an expression of gratitude, as shown in
some inscriptions, such as the inscription of Muara Kaman,
Kutai, East Kalimantan (4th century AD), the inscription of
Tugu, Jakarta (mid-5thcenturyAD), and the inscription of
Dinaya, Malang, East Java (760 AD). Cattle have long
been used as draught animals in Indonesia. A relief on the
wall of Borobudur temple, Magelang, Central Java (750
AD) showed a pair of zebu cattle (Bos indicus) is being
used for plowing, while in Sukuh, Karanganyar, Central
Java (mid-15th century AD), it is found a relief of cattle
without humps or Bali cattle (Bos javanicus) with a big
bells on the neck (Java: klonengan) as a characteristic of
draught animals (Sutarno and Setyawan 2015). In the
Islamic era, the need for cattle is increasing at the time of
Eid al-Adha celebration by slaughtering livestock.
Moreover, in some areas the fasting of Ramadan and Eid
al-Fitr were also celebrated by consuming livestock, for
example, Meugang tradition of Aceh which has been traced
back to Sultan Iskandar Muda (1607-1636), thus
encouraging the development of local Aceh cattle (Yunita
2012).
Cattle are the most important livestock commodities as
a source of milk (dairy cattle), meat and leather (beef
cattle), as well as draught animals. Cattle meet most of the
world's needs for meat (50%), leather (85%) and milk
(95%) (Bappenas 2007; Umar 2009). In Indonesia, demand
for meat and dairy cattle cannot be met from domestic
stockbreeding, which can only fulfill approximately 75%
and 20% of overall need respectively, so that Indonesia
becomes a net importer of both commodities. From year to
year, dependence on imports is increasing and without
significant breakthrough, it is predicted that in the next 10
years the production of cattle in the country is only able to
meet the half of needs. In the last three years (2013, 2014,
2015), before and after Eid al-Fitr is always turmoil in the
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domestic market related to the soaring price of beef cattle,
due to lack of supplies. Demand for beef cattle increased
because of the population growth, improved living
standards, changing consumption patterns, and the presence
of expatriates and foreign tourists who demand beef cattle
with certain quality (DGLS 2010a; Khasrad and Ningrat
2010). The government has taken a long-term policy to
achieve self-sufficiency in beef cattle based on domestic
resources, but the effort is failed though it has been
launched three times, in 2000, 2010, and 2014. The lack of
breed quality, limited pastures and limited cattle feed are
considered as the primary reason for the cause of this
failure (DGLS 2010b; Mahbubi 2014).
This paper aims to review the genetic diversity of local
and exotic cattle in Indonesia, the crossbreeds impact and
conservation effort.
WORLDWIDE CATTLE DOMESTICATION
The domestication of wild banteng (Bos javanicus
javanicus) into Bali cattle, which continue to be the main
cattle in Indonesia until now, is a native of Indonesia's
cultural heritage that should be preserved. The yielding of
Bali cattle shows the potential of Indonesia to be an
independent and sovereign country in terms of food. Cattle
domestication process is mostly done in Europe and Asia
but yields no sustainable offspring, except for only two
species namely taurine cattle (Bos taurus) and zebu cattle
(Bos indicus). Both are descendants of wild aurochs cattle
(Bos primigenius), which is widespread in Asia, Europe,
and North Africa at the end of the last glacial period
(12,000 BP) (Felius et al. 2014). Taurine cattle
domesticated between 10,300-10,800 BP at the border
country of Turkey, Syria and Iraq (Helmer et al. 2005;
Vigne 2011; Bollongino et al. 2012), while the zebu cattle
domesticated in the Indus Valley on the desert edge of
Mehrgarh, Baluchistan, Pakistan around 8000 BP (AjmoneMarsan et al. 2010; Chen et al. 2010). Domestication of
species and a long history of migration, selection and
adaptation have created a wide variety of breeds
(Groeneveld et al. 2010). Based on the place of origin of
domestication, the taurine cattle (without the hump) are
sub-tropical cattle, with the main populations in Europe,
North America and Australia. While, the zebu cattle (with
the hump) are tropical cattle, with the largest population in
India, Africa and Brazil. Each species of cattle have had
hundreds of breeds, including the descent of its
crossbreeds.
Bali cattle have been domesticated from wild banteng
since c.a. 5000 BP (Payne and Hodges 1997). Bali cattle
are the most successful domestication of cattle outside
taurine and zebu cattle. When taurine and zebu cattle are
the main world livestock, Bali cattle are the main livestock
in Indonesia. However, the selection process of Bali cattle
is relatively under-developed, that it has relatively same
genetic as a wild banteng. Although, many Bali cattle is
crossed with zebu cattle and, now, with taurine cattle, but
the genetic proportion of its offspring is dominated by the
exotic cattle, so it is no longer classified as Bali cattle,
whilst the Bali cattle is relatively pure (Mohamed et al.
2009), except for Bali cattle in Malaysia which is a mixture
of zebu and banteng with relatively balanced proportion
(Nijman et al. 2003).
Domestication of the other cattle species was also
conducted in Asia (Ho et al. 2008; Achilli et al. 2009). In
Tibet, yak cattle (Bos grunniens) had been domesticated
and able to adapt to high altitudes (Qiu et al. 2012) since
c.a. 4500 BP (Payne and Hodges 1997). Gayal or mithun
cattle (Bos frontalis) had been domesticated from wild gaur
(Bos gaurus) (Uzzaman et al. 2014) on the border of
northeast India, Bangladesh and Myanmar (Mason 1988;
Payne and Hodges 1997). These species of Asia cattle
hybridize with taurine and zebu cattle that are spreading
more widely, resulting mixtures species that give a unique
contribution to the world of livestock resources (Felius et
al. 2014).
INDONESIAN LOCAL CATTLE
Indonesian local cattle have experienced a selection of
various pressures of wet tropical climate, and an adaptation
to low quality of feed, local parasitic and diseases, so it is a
new adaptive phenotype (Sutarno 2006). Besides Bali
cattle, Indonesia has also several local cattle which are
direct descendants of the Indian zebu cattle, the result of
crossbreeding between zebu and Bali cattle, as well as
crossbreeding with taurine cattle which is introduced latter
(Martojo 2003; Johari et al. 2007). Primary crossbreeds
between zebu or taurine cattle with banteng (or Bali cattle)
produce fertile female and male sterile breeds (Lenstra and
Bradley 1999). The Indonesian local cattle are generally a
hybrid of zebu cattle with Bali cattle. Kikkawa et al. (2003)
and Mohamad et al. (2009) found mtDNA of banteng on
Indonesian zebu cattle, especially Madura cattle (56%) and
Galekan cattle (94%). While Nijman et al. (2003), based on
analysis of mtDNA and other genes, showed that Bali cattle
in Indonesia comes purely from a banteng, while Bali cattle
in Malaysia is a mixture of zebu and banteng.
Some local cattle belonging to zebu group are
Peranakan Ongole (PO) cattle in Java, Pesisir cattle in West
Sumatra, Aceh cattle in Aceh, and Sumba Ongole cattle on
the island of Sumba. India is the center of zebu cattle genes
(Nozawa 1979). Local cattle deriving from crossbreeds
between zebu and Bali cattle includes Madura cattle in
Madura and surroundings, Jabres cattle in Brebes, Rancah
cattle in Ciamis and surroundings, Rambon cattle in
Bondowoso and surroundings and the rare Galekan cattle in
Trenggalek. In Indonesia, there are also local cattle which
are considered as a crossbreed of two exotic cattle, zebu
and taurine, namely Grati cattle in Pasuruan and
surroundings, and now are more commonly known as
Holstein Friesian (FH) Indonesia, which is a cross between
FH male and PO female (Blakely and Bade 1998;
Williamson and Payne 1980; Johari et al. 2007). According
to Sutarno et al. (2015), based on studies of DNA
microsatellites, Madura cattle have most different genetic
characteristics than Bali cattle population (of Lombok and
Sumbawa) and zebu cattle population (Aceh cattle and PO
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
cattle).
Bali cattle, PO cattle, and Madura cattle become a
mainstay to meet the needs of meat in Indonesia, while the
Holstein Friesian cattle become a mainstay to meet the
needs of milk (Okumura et al. 2007). Beef cattle population
in Indonesia is currently about 12.3 million (BPS 2014),
and dairy cattle is about 500,000 (DGLSAH 2014). These
cattle consist of Bali cattle (33.73%), PO cattle (23.88%),
Madura cattle (5.16%), and others (13.45%) (DGLS
2010b). The main concentration of the population of cattle
is in Java (45%), Sumatra (22%), Bali and Nusa Tenggara
(13%), Sulawesi (13%), and the rest in other islands (7%).
The population of cattle is slightly decreased after the
financial crisis at the end of 1990s, as many local cattle are
consumed to replace the imported one. The consumption
rate of local cattle exceeds the natural reproduction ability
rate; there is a decrease in the number of calves born in the
following years (Pamungkas et al. 2012).
Local cattle have proved that it can adapt to the local
environment, including feed, water availability, climate and
disease, but it generally has lower productivity than the
exotic cattle (ILRI 1995). Adapted animals have production
and reproduction regulating gene which is superior to
environmental stress. Conservation of local cattle got a lot
of challenges, especially since the rise of improving the
calf quality by crossbreeding using frozen semen of exotic
cattle, mainly Simmental and Limousin. Hybrid offsprings
are high favorite for breeders because it is relatively high in
daily weight gaining, although it requires higher production
costs (Sutarno 2006; Sullivan and Diwyanto 2007). The
efforts of crossbreeds are relatively successful for the same
species of cattle, such as zebu and zebu, or taurine and
taurine, but in crossbreeds of different species, it will
produce calves of sterile males and fertile females with the
ability of reproduction decreasing from generation to
generation, thus the breeders must provide a new breeding
male and female from time to time. The crossbreeding of
local cattle with exotic cattle spreads widely without
evaluation, control and ignoring the importance of local
cattle as unique germplasm. It is feared that it can lead to
erosion of genetic resources towards extinction. Loss of
important genes in cattle that have been locally adapted to
local environmental conditions would be difficult, or even
impossible, to be replaced. This has happened to many
local cattle in India that have become extinct before it has
been identified and utilized due to the many crossbreeds
(Sodhi et al. 2006).
The weakness of cattle development in Indonesia is the
poor quality of cattle genetics, lack of superior bulls, lack
of farmers’ ability in dealing with cattle breeding, and
traditional method of stock raising (Atmakusuma et al.
2014). The development of local cattle is also facing
challenges due to the rise of uncontrolled crossbreeding
especially using artificial insemination methods and the
pressures of other local cattle that are more superior, for
example, the development of Pesisir cattle of West Sumatra
is suppressed by superior Bali cattle. FAO (2000) has
warned that livestock with the risk of extinction is in
developing countries due to the high market demand,
crossbreeding, breeds replacing and mechanization of
329
farming activities where the use of cattle as draught
animals is decreased (Figure 1).
Bali Cattle
Origin and distribution. Bali cattle (Bos javanicus)
are the result of direct domestication of wild banteng in
Bali or Blambangan, East Java (MacHugh 1996; Verkaar et
al. 2002; Martojo, 2003, 2012; Hardjosubroto 2004). Bali
cattle spread widely throughout Indonesia, especially in
South Sulawesi, Bali, East Nusa Tenggara, West Nusa
Tenggara, Southeast Sulawesi and Lampung (Entwistle and
Lindsay 2003; Sutarno 2010). These cattle are major
genotypes in Eastern Indonesia (Pribadi et al. 2014). Bali
cattle is not much raised in Central Java and West Java
where the breeders prefer to raise goats (Capra hircus),
which are an intermediary agent of malignant catarrhal, a
deadly disease in Bali cattle calf. Bali cattle population is
about 4.1 million (DGLS 2010b). These cattle are also
found in northern Australia and Malaysia (Toelihere 2003).
In northern Australia, Bali cattle live in wild as a banteng.
They were from the 20 Bali cattle that were imported from
Bali in 1849, and now the number is around 8,000-10,000
(Bradshaw and Brook 2007). In comparison to Bali cattle,
the genetic purity of these cattle comes near to the genetic
purity of wild banteng in Java, since Bali cattle is allegedly
received genetic mixing of zebu cattle (and now taurine). In
Malaysia, Bali cattle began to be developed on a large scale
and replace the local cattle of Kedah-Kelantan that has low
productivity (Somarny et al. 2015). Bali cattle are the
ancestor of most local cattle breeds in Indonesia. In fact,
PO cattle which were considered as pure zebu cattle also
have a gene introgression of Bali cattle, as well as Pesisir
and Aceh cattle. Cattle that clearly and phenotypically pick
Bali cattle gene are Madura, Rambon, Galekan, Jabres and
Rancah cattle. But these cattle are genetically more close to
the zebu cattle (Mohamed et al. 2009).
Physical characteristics. Bali cattle has similar
physical characteristics to a wild banteng (Handiwirawan
and Subandriyo 2004), but banteng is larger and more
aggressive (Martojo 2003, 2012). The study of genetic
diversity in Bali cattle and banteng is still limited (Kikkawa
et al. 1995, 2003; Namikawa 1981; Nijman et al. 2003;
Verkaar et al. 2003). Bali cattle have a great frame and
solid muscle; adult male can weigh 600-800 kg, while the
female weighs 500-600 kg (Martojo 2003). At the time of
calf, cattle's body is brick red or golden red. Meanwhile,
when adult, female cattle remain in red brick, while the
male cattle change to blackish at the age of 12-18 months.
There are white on all four legs, from the knee to toes,
buttock is white with clear boundaries and oval with black
on tail tip (Williamson and Payne 1980). They have no
humps, a small wattle and compact body. It has wide head,
short, flat forehead and standing ears. The female horn is
short and small, and the male horn is long and large
heading to the front upper side and taper, with a slender
neck. It has deep chest with powerful legs (Pane 1991;
Susilorini 2010).
Advantages and disadvantages. Bali cattle have a very
high reproductive ability, able to give birth every year, able
to adapt to the marginal environment with dry climates,
able to digest low quality of forage for example during the
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B IO D IV E RS IT A S 16 (2): 327-354, October 2015
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
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SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
P
Q
R
S
T
U
V
W
X
Y
Z
AA
AB
AC
AD
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B IO D IV E RS IT A S 16 (2): 327-354, October 2015
AE
AF
AG
AH
AI
AJ
AK
AL
AM
Figure 1. The diversity of cattle in Indonesia. A-C. Banteng (males, females, herds); D-F. Bali cattle (bulls, cows, herds); G-I.
Peranakan Ongole cattle (bulls, cows, herds); J-L. Madura cattle (bulls, cows, herds); M-O. Aceh cattle (bulls, cows, herds); P-R. Pesisir
cattle (male, puppies, herds); S. Jabres cattle; T. Rancah cattle; U. Rambon-Bali cattle; V. Rambon-Madura cattle; W. Galekan cattle; X.
Brahman Cross cattle; Y-AA. Brahman cattle (male, female, herds); AB-AD. Simmental cattle (bulls, cows, herds); AE-AG. Limousine
cattle (bulls, cows, herds); AH-AJ. Holstein Friesian cattle (bulls, cows, herds); AK-AM. The appearance of various types of cattle at
the feedlot enterprise (feedlofter) originating from northern Australia
dry season and able to be immediately restored to its
original state if there is enough fodder; it can be kept as
draught animals or beef cattle. Carcass percentage is higher
than zebu and taurine cattle, with high-quality of low-fat
beef. The quality of skin is good and rather thin. Bali cattle
is the most suitable for farming in the traditional
production system with low-input and high environmental
stress which is widely practiced by Indonesian breeders
(Wirdahayati 1994; Copland 1996; Diwyanto and Praharani
2010; Sutarno 2010; Zulkharnaim et al. 2010; Noor et al.
2011). Bali cattle have various disadvantages, namely slow
growth, low milk production that lead to the high calf
mortality (Susilorini 2010). These cattle are known as
resistant to many diseases and parasites, but there are two
very deadly diseases, namely malignant catarrhal that
attack calves, and Jembrana viral disease that attacks the
brain (Budiarso and Hardjosworo 1976). Malignant
catarrhal is detected in Denpasar, Banyuwangi, Mataram
and Kendari (Damayanti 1995). First, Jembrana viral
disease infected the population on the island of Bali, and is
allegedly as a result of long-term isolation that causes a lot
of inbreeding and produce low resilient offspring (Tenaya
2010; Wilcox et al. 1992, 1995). It is quite alarming, as in
some other places, Bali cattle have lower genetic variability
than the one in Bali, for example, on the island of Lombok
(Winaya et al. 2009). Jembrana viral disease has also been
detected in Bali cattle population in West Sumatra, Jambi,
Riau and Riau Islands. Previously, the disease has been
successfully handled in Bali, East Java and Lampung
(BPPV Bukittinggi 2013).
Breeding and conservation. Bali cattle have not
experienced an advanced selection as zebu and taurine
cattle have. However, the genetic purity of Bali cattle is
now threatened by negative selection and crossbreeding.
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SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
A
B
C
Figure 2. The quality depletion of Bali cattle from Kupang due to negative selection of poor quality cows mating. A. The bulls; B. The
cows; C. Cattle herds.
Negative selection on the Timor island by shipping of highquality cattle and let the low quality ones to breed, causing
the cattle’s weight become progressively low (Wirdahayati
2010; Sudarma 2013)(Figure 2). The crossbreeding begins
to be done even on the island of Bali, especially with
frozen semen of Simmental and Limousin cattle. In the era
of Klungkung kingdom to the colonial period, the island
was set specifically for the development of Bali cattle. But
today, specific areas for Bali cattle development is only on
a small island of Nusa Penida (DGLSAH 2015), while in
Bali Island, crossbreeding of Bali cattle with other cattle
breeds has been allowed. However, there is a Bali
Governor Regulation No. 45/2004 and Regional Regulation
of Bali Province No. 2/2003 which prohibits shipping
female breeds of Bali cattle out of Bali province. Genetic
purity of banteng as source domestication of Bali cattle is
also threatened by interbreeding. Utilization zone of
national parks in Baluran, Alas Purwo and Meru Betiri
which are natural habitat for banteng, are used by local
residents as domestic cattle grazing, particularly in TN
Baluran (Tempo 27.6.2013), so there is possibility of wild
banteng mating to domestic cattle and disturbing the
genetic purity. The Indonesian government has made a
banteng conservation action plan (Regulation of the
Minister of Forestry No. P.58/Menhut-II/2011), but the
results have not been much of an effect. In addition to the
island of Nusa Penida, Bali cattle breeding are also
concentrated in Siak, Central Lombok, Barito Kuala, Barru
and West Pasaman districts (DGLSAH 2015).
Peranakan Ongole and Sumba Ongole cattle
Origin and distribution. Zebu cattle of Indonesia has
been known for centuries as Java cattle, but the quality is
steadily declining due to lack of new genes input, so at the
early of the 19th century, it was brought here different
breeds of the Indian zebu cattle for genetic improvement
and it gave satisfactory results. In the early of the 20th
century, the government took the initiative to bring Ongole
cattle to Sumba Island and were able to breed and adapt
well, they were known as Sumba Ongole cattle
(Hardjosubroto 2004). Peranakan Ongole (PO) (Bos
indicus) or Benggala is a crossbreed of uncontrolled mating
of Java cattle and Sumba Ongole cattle (Suyadi et al.
2014). PO cattle population is estimated at 2,9 million and
almost 90% is in Java (DGLS 2003, 2010b).
Physical characteristics. PO cattle has a white or gray
colored skin, fan tail and fur around the eyes is black, short
curved shape of the head, short horns, long hanging ears,
and a rather large belly. On male cattle, sometimes, there
are black splotches on the knees, big bright eyes encircled
with black spot about 1 cm from the eye; big body, big
hump, short neck, long legs, strong muscle, wattle loose,
hanging from the bottom of the head, neck to stomach.
Male cattle can reach a weight of about 600 kg and female
cattle are 450 kg. PO cattle weight gain range between 0.40.8 kg per day, but in unfavorable conditions only reached
0.25 kg per day (Wiyatna et al. 2012). Meanwhile, SO
cattle had reached the body weight gain of 1.18 kg per day,
the percentage of carcass is more than 50% and meat
production had reached 77% (Ngadiyono 1995).
Advantages and disadvantages. PO cattle are known
as beef cattle and draught cattle. They are suitable as
draught animals due to a big and strong body, docile and
quiet, tolerant to heat, have high adaptability over different
environmental conditions, able to grow in limited forage
conditions, and high reproductive activity. The female
quickly returned to normal condition after giving birth.
However, the percentage of carcass is generally lower than
other Indonesian local cattle.
Breeding and conservation. In addition to PO cattle
and SO cattle, in Indonesia, it is also developed other
species of zebu cattle, particular breeds of Brahman and
Brahman Cross, which is bigger than the PO. Pure PO
cattle began rare because many breeders cross them with
Brahman cattle. Their mating produces fertile calf and
usually is also called PO because of its smaller size. In
Kebumen, Central Java, PO cattle are also known as
Madras cattle which are the origin of zebu cattle in East
India. In Kebumen, zebu cattle genetic improvement efforts
have been done long before Ongolisation program in
1930s, therefore the PO cattle (Madras) in this region are
known to have qualities like pure Ongole cattle (Utomo et
al. 2015). Kebumen, Gunungkidul and South Lampung
have been chosen as the breeding center of PO cattle
(DGLSAH 2015).
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Madura cattle
Origin and distribution. Madura cattle (Bos javanicus
x Bos indicus) are the result of a crossbreed between Bali
cattle and zebu cattle from India in the island of Madura,
but the time is unknown. Some sources say that the process
occurs about 1,500 years ago (Meijer 1962; NRC 1983;
Nijman et al. 2003). Given the Madura cattle also dominant
on the northern coastal region of East Java where there are
many Madurese immigrants, then certainly these cattle
were already present before the migration. The area is
experiencing depopulation of Javanese because of war
continuing from the 15th to 18th centuries. The Madurese
fill these abandoned lands because the previous owners are
victims of war or escape. At the beginning of the 20th
century, Madura cattle are distributed to Flores and East
Kalimantan. But, they are replaced by Bali cattle which are
brought in later for the better quality (Omerling 1957;
Hardjosubroto 2004). In 2002, the total population of
Madura cattle was approximately 900,000 (DGLS 2003).
In 2008 on the island of Madura, the population is
approximately 400,000 (Office of Animal Husbandry East
Java Province 2009), and in 2010, it is about
635,000(DGLS 2010b).
Physical characteristics. Madura cattle is brick red or
brownish red with distinctive white markings on the back
and below. It has small horns, short and heading outside.
The uniformity of breeding is developed through selection
conducted by the breeders.
Advantages and disadvantages. Madura cattle have
good growth in poor quality of forage, the percentage of
carcasses is high with meat quality is good; it has high
adaptability to tropical environments, and can run fast, so it
is usually used for racing (karapan), and have a good body
appearance, so it is also used as displayed cattle (sonok).
Karapan cattle require high energy metabolism to get
physical strength, hard work of skeletal muscle, and the
aggressiveness. On the other hand, sonok cattle need to
withstand muscle framework stretching and emotions
(tamed). Cattle that do not have these properties are
categorized as common beef cattle. Madura cattle breeders
keep them as draught animals, life savings, producer of
organic fertilizer, source of revenue and for the karapan
and sonok cattle (Siswijono et al. 2010). Compared to Bali
cattle, Madura cattle are relatively resistant to Jembrana
viral diseases (Suwitri et al. 2008).
Breeding and conservation. Karapan race and sonok
festival are instrument of Madura cattle selection, which
can only be attended by the selected cattle with excellent
performance and condition. It can be affected by genetic
and environmental factors, including feed and health. The
selection of cattle having performance appropriate to the
tastes of society needs to be considered, if it affects
variations in genes involved in energy metabolism or not
(Siswijono et al. 2010; Febriana et al. 2015). In the colonial
era, Madura Island is specialized for Madura cattle
development and the introduction of other cattle breeds is
prohibited. This was stated in Staatsblad (Gazetted) No.
226/1923, No. 57/1934, and No. 115/1937. Even it implied
in Indonesian Law No. 18/2009, on Animal Husbandry and
Animal Health. However, development of other cattle is
allowed in the later. In 1957, the crossbreeds of Madura
cattle with dairy cattle Red Danish (a taurine cattle) was
carried out, but the calves are less than desirable, and in
recent decades Madura cattle are crossed with Limousin
through artificial insemination with the results of
considerable interest, because have larger body size and
higher selling price (Siswijono et al. 2010). The
crossbreeds of Madura and Limousin cattle are the belle for
traditional breeders. This process is carried out directly in
the field and is not controlled exclusively, so that in the
long term, there is a concern that it will change the genetic
composition of Madura cattle and affect the durability of
the dry climate and limited fodder. Furthermore, it can
disturb the continuing of local culture of karapan and
sonok cattle (Widi et al. 2013) as well as reduce the ability
of self-sufficiency in beef cattle. Sapudi Island is pointed as
pure Madura cattle conservation area in order to avoid the
uncontrolled genetic changes (Decree of the East Java
Governor No. 188/Kpts/013/2010; DGLSAH 2015).
However, on this island are also reared PO cattle and its
breed (Kutsiyah 2012).
Aceh cattle
Origin and distribution. Aceh cattle (Bos indicus) are
a small-sized local cattle developed in Aceh (Martojo 2003;
Dahlanuddin et al. 2003). Aceh cattle allegedly were
imported by Indian merchants in the past (Abdullah et al.
2007).
Physical characteristics. Aceh cattle are predominantly
red-brownish for male and red brick for female; the color
around the eyes, the inner ear and upper lip is whitish, neck
is darker in males; it has a dorsal blackish brownish stripe,
red brick hamstrings, light brownish buttocks, whitish legs,
black tail tip, generally concave face, generally concave
back, horn leads to the side and curved upwards, small ears
leading to the side and do not droop. Body weight is 253 ±
65 kg of male, 148 ± 37 kg of female. Carcass percentage
is 49-51%. Aceh cattle have good adaptability,
employability and disease resistance. They are very
productive, with the parent fertility of 86-90%, birth rate of
65-85%, age of puberty of 300-390 days, estrus cycle of
18-20 days and 275-282 days old pregnancy (Abdullah et
al. 2007).
Advantages and disadvantages. Aceh cattle are used
as beef cattle and draught animals. Most local farmers
utilize Aceh cattle to plow the field. Aceh cattle breeding
business is generally done by individuals, there have been
no industrial-scale livestock enterprises (Abdullah et al.
2007; 2008). Although relatively small in size, some Aceh
cattle have the ability to give birth to twin calf.
Breeding and conservation. In Indrapuri, Aceh, there
is a special institution that develops Aceh cattle breeding.
Raya Island of Aceh Jaya is designated as specific areas for
the breeding and development of Aceh cattle (DGLSAH
2015).
Pesisir cattle
Origin and distribution. Pesisir cattle (Bos indicus)
are local cattle breeds that have the smallest body size.
These cattle are widely kept in Pesisir Selatan district, and
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
on a small portion in the districts of Padang Pariaman and
Agam, West Sumatra (Anwar 2004; Hosen 2006).
Population of Pesisir cattle continues to decline. In Pesisir
Selatan, it only reached 76,111 heads in 2011 (OAHAHP
Pesisir Selatan District 2012). The decline of Pesisir cattle
population is allegedly associated with high slaughtering of
productive livestock, forage limitations, pasture
depreciation, and decreased genetic quality (Adrial 2010).
Physical characteristics. Pesisir cattle have small
bodies, short stature, slender legs, small hump, and benign.
Male cattle has a short head, short and large neck, wide at
the back of the neck, a big hump, short and round steering.
Female cattle has a rather long head and a thin, sloping tail,
short and thin, small horns and head for outside such as
goat horns (Saladin 1983). Adult male cattle (age 4-6
years) has only a weight of 160 kg (Adrial 2010). The high
diversity of skin color with a single pattern and is grouped
into five dominant colors, namely red brick (34.3%) yellow
(25.5%), chocolate (20%), black (10.9%), and white (9.3%)
(Anwar 2004).
Advantages and disadvantages. Pesisir cattle have a
high reproductive efficiency (Sarbaini 2004), high birth
rates, birth weight of 14-15 kg, average daily weight gain
from birth to calf about 0.32 to 0.42 kg per day (Saladin
1983). Carcass percentage is 50.6%. They are able to
survive in adverse environmental conditions and poor
forage. The ability to convert fibrous feed into meat is high
(Saladin 1983). Pesisir cattle traditionally are maintained
by relying on pasture grass, vacant lots, and rain fed; are
resistant to disease and able to adapt to a tropical
environment (Hendri 2013). Improvement in forage quality
can increase the growth rate and carcass percentage,
although it will increase the percentage of fat (Khasrad and
Ningrat 2010).
Breeding and conservation. Development of the
Pesisir cattle is exposed to the genetic deterioration. Body
weight and body size of these cattle are much smaller than
before. For 22 years (1982-2004), the body weight and
body size of Pesisir cattle have decreased around 35%. For
25 years (1980-2005), body weight of adult male of Pesisir
cattle has decreased from 275 kg to 237.5 kg, while female
cattle have decreased from 256 to 172 kg (Sulin 2008).
Today, breeders prefer exotic cattle such as Brahman and
Simmental, as well as Bali cattle, especially since the
government introduces thousands of Bali cattle to West
Sumatra since 1985 (Wilcox et al. 1996; Mariani 2013) and
introduce artificial insemination later. In 2009, Pesisir
cattle population is still dominant, reaching 70% of cattle
population, but in 2011 it is only 25% (Hendri 2013). Since
there are no Pesisir cattle conservation efforts, in the long
term, these breeds will be allegedly replaced by Bali cattle
which are more superior. Pasaman Barat, one of the
original distributions of Pesisir cattle, was chosen to be the
center of Bali cattle breeding (DGLSAH 2015). However,
in Padang Mengatas, District of Limapuluhkota, there are
breeding of various cattle breeds, including Pesisir cattle.
Jabres cattle
Origin and distribution. Jabres cattle (Bos javanicus x
Bos indicus) (Jabres = Java Brebes) are allegedly to be a
335
crossbreed between Madura cattle or Bali cattle with
Peranakan Ongole cattle (Minister of Agriculture Decree
No. 2842/Kpts/LB.430/8/2012). These cattle thrive in the
highlands of southern Brebes, Central Java at an altitude
800 m asl. (Lestari 2012). According to Munadi (2010),
Jabres cattle are found in five sub-districts, namely
Bantarkawung (5,757 heads), Salem (543 heads),
Banjarharjo (1,994 heads), Ketanggungan (2,900 heads),
and Larangan (890 heads).
Physical characteristics. Jabres cattle have similar
characteristics to Bali cattle, but Bali cattle has a white
color on the legs and buttocks that contrast with the body
color of red-brownish, on the Jabres cattle, that color
becomes gradation and has no visible boundary between
red-brownish and white. The color varies from brownish,
white brownish, white, dark-brownish and black; white
feet, white upper lip, white lower lip; in the head, it is often
found signs of a small white rhombus, black tip. The cattle
have rump, white hind legs, and the black stripe from the
back to the tail. Male cattle horns curved upward, the
female curved downward. In general, there is no hump.
Body shape is slim and compact with dense flesh structure.
Male cattle body weight is 350 ± 25 kg, female cattle is
286 ± 20 kg. Fertility of parent is 82-85%, with 40-85% of
birth rate and estrus cycles of 18-24 days, pregnancy period
of 9-10 months, 21-28 months of the first estrus, first birth
age of 30-36 months (Decree of the Minister of Agriculture
No. 2842/Kpts/LB.430/8/2012; Lestari 2012).
Advantages and disadvantages. Jabres cattle have the
ability to work and high adaptability to extreme climate
conditions, are able to utilize lower quality feed, not
susceptible to disease, insect-resistant, and have good
reproducibility. One female of Jabres cattle able to give
birth up to 15-20 times; with calving interval is only 12
months. It can be pregnant by 45 days after birth. The
average birth weight is 16 kg to facilitate calving, whereas
the weight of adult males ranged between 195-269 kg and
females 168-296 kg. Cattle meat has a solid structure;
carcass percentage can reach 52% (Lestari 2012).
Breeding and conservation. Jabres cattle traditionally
are maintained with fellow cattle grazing systems
(Adiwinarti et al. 2011; Lestari 2012). Mating system of
Jabres cattle occurs naturally. Breeding through artificial
insemination with exotic cattle have not done for reasons of
cost, so that the genetic purity is relatively maintained.
Jabres cattle are the only local cattle of Central Java, and
are chosen as a local cattle based on the Decree of the
Ministry of Agriculture No. 2842/Kpts/LB.430/8/2012.
Rambon cattle
Origin and distribution. Rambon cattle (Bos javanicus
x Bos indicus) are local cattle in the eastern part of East
Java, especially in Bondowoso, Situbondo, Jember and
Banyuwangi. In the past, there were three cattle breeds in
this area, namely PO cattle, Madura cattle and Bali cattle.
Rambon cattle are natural crossbreeding of the three breeds
so that their genetic composition is quite diverse
(Susilawati et al. 2002; Susilawati 2004). Rambon cattle
living in Situbondo and Bondowoso have characteristics
which are predominant to Madura cattle and PO, while
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B IO D IV E RS IT A S 16 (2): 327-354, October 2015
Rambon cattle in Jember and Banyuwangi have
characteristics which are predominant to Bali cattle and PO
(Susilawati et al. 2002; Susilawati 2004). It is strongly
associated with the geopolitical history of the region in the
past. Until the 18th century, there was still existing Hindu
kingdom of Blambangan in Banyuwangi which
periodically obtains the influence of Bali (Margana 2007).
The Balinese allegedly bring in Bali cattle from Bali island,
but the other opinion says that Bali cattle were
domesticated in this region since, until now, there is still a
wild banteng, such as in Baluran National Park, Alas
Purwo National Park and Meru Betiri National Park. In the
18th century, the region fell to the kingdom of Mataram
and Madurese began migrating mainly on the north coast,
bringing Madura cattle with them. Bali cattle are dominant
in the south and Madura cattle dominate the north area.
Consequently, the breeds of Rambon cattle in the north
tend to resemble Madura cattle, while the cattle in the
southern part resemble Bali cattle. According to Amin
(2010), Rambon cattle of Banyuwangi experienced natural
crossbreeding with Bali cattle, PO cattle and Brahman, and
underwent cross through artificial insemination with
Limousin, Simmental, Aberdeen Angus and Santa
Gertrudis cattle.
Physical characteristics. Rambon cattle have a weight
of about 300-400 kg. Rambon cattle in Situbondo and
Bondowoso has a dominant varied skin color; brick red,
red-brownish, red without clear color boundary; white
rump; long tail with black fur; diverse foot fur, clear white,
white, red brick; diverse shape of backs, straight or curved,
with or without the back line; varies direction of the horn,
forwards, upwards, sideways and backwards; the existence
of hump varied, with hump, no hump and unclear hump.
On the other hand, Rambon cattle in Banyuwangi and
Jember are predominantly of red brick; thin wattle; black
back line; white leg fur; specific white rump; the horn
direction is to the side; tail with black fur; no hump
(Susilawati et al. 2002; Susilawati 2004).
Advantages and disadvantages. Rambon cattle are
composite that have high resistance to climate, feed and
disease in the eastern of East Java. However, as the
descendants of Bali cattle, these cattle are not resistant to
Jembrana viral diseases, so the local government has
banned the cattle-shipping from Bali to East Java (Circular
of East Java Governor No. 524/8838/023/2010).
Breeding and conservation. Rambon cattle are a
natural crossbreeding and need no efforts for conservation.
Rambon cattle experience a lot of genetic mixing,
including artificial insemination with Simmental and
Limousin as the main stud, so there are dozens breeds of
these cattle (Amin 2010).
Rancah cattle
Origin and distribution. Rancah cattle or Pasundan
cattle (Bos javanicus x Bos indicus) are local cattle in West
Java. The naming is based on the location of initial
development, namely Rancah, Ciamis, West Java. These
cattle are often called kacang (bean) cattle because it is
relatively small in size (Hilmia et al. 2013). Distribution
area includes Ciamis, Tasikmalaya, Pangandaran, Garut,
Cianjur, Sukabumi, Purwakarta, Majalengka and Kuningan
(Indrijani et al. 2012).
Physical characteristics. Rancah cattle have physical
characteristics as Madura cattle and Bali cattle. The
females have no hump; body size is relatively small, mostly
red brick and white on the pelvis and the four lower legs
(tarsus and carpus) with no clear restrictions. There is a
stripe along the back with the older color of the dominant
colors. Male cattle are similar to females, but mostly with
darker body color. Some Rancah cattle male may
experience changes in color from brick red to black
according to sexual maturity (such as Bali cattle).
Rectangular shape with long small legs and has a short
horn but not uniform and varies from small to large. Body
size is with an average shoulder height of 115 cm to 109
cm in males and females. Male cattle body weight on
average 240 kg and 220 kg in females (Payne and
Rollinson 1973; Huitema 1986; Decree of the Minister of
Agriculture No. 1051/Kpts/SR.120/10/2014). In Rancah,
Ciamis, these cattle are relatively small compared to other
breeds which are also kept by cattle breeders, such as PO,
Simpo and Limpo (Derajat 2014).
Advantages and disadvantages. Their behavior is not
wild and easy to adapt to the surrounding environment.
Reproductive ability is quite efficient; it can be re pregnant
within 2.5-5 months after birth (Hilmia et al. 2013). They
have superior carcass percentage reaching 50% of the live
weight, the fat content in meat is low, the meat is more
abrasive, does not contain a lot of water; the meat color is
brighter because of the high pigment content, so the price is
more expensive than imported beef cattle. In addition, these
cattle have high resistant to tropical diseases; practicality in
taking care of; resistant to extreme weather. Rancah cattle
growth is relatively slow, although it can live with a low
quality feed, the fostering costs are much cheaper, about
25-30% of crossbreed cattle.
Breeding and conservation. Pure Rancah cattle
populations have decreased, there were only about 3001000 heads, their existence is marginalized by other cattle.
By 2015, at Rancah Animal Market, 70 cattle are sold
every day, but only 30% which is a Rancah cattle or its
crossbreed. The decreasing of Rancah cattle population
leads to an increase of inbreeding so that it lowers the
quality of these cattle. In 1990, one of Rancah cattle could
produce 500-700 kg of carcass, but at present, it is only
able to produce 300-350 kg of meat carcasses. Genetic
improvement efforts to restore the quality of Rancah cattle
as in the past are relatively difficult, because of the
difficulty in finding good (pure) quality Rancah cattle. In
addition, these efforts require a long time commitment
(about 25 years), high cost and there is no serious
institution that handles Rancah cattle breeding, all
produced naturally to breeders. This genetic decline is
responded by breeders by crossing it with other local cattle,
especially PO cattle. In 2014, there were 52,540 heads of
Rancah cattle, but it is mostly the result of crossbreeding
with other local cattle. Artificial insemination is also
conducted intensively in this region, causing introgression
from other cattle gene (Hilmia et al. 2013). Genetic
improvement can be done by inserting the wild banteng
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
gene back to Rancah cattle. These species still found in
West Java, namely in Leuweng Sancang forest, Garut. The
mating is expected to restore the performance of Rancah
cattle as the original which is more robust and meaty. In
addition, certain areas need to be protected since it is the
source of Rancah cattle breeds, mostly scattered in the
forest area in the district of Ciamis, Pangandaran, Garut
and Cianjur. The protection can be done by, for example,
banning Rancah cattle interbreed with other breeds of
cattle. It should also be supported by breeding policies so
that the quality of cattle is maintained well, such as sperm
banks and embryo transfer. Rancah cattle have been
classified as newest Indonesian local cattle based on
Decree of the Minister of Agriculture No.
1051/Kpts/SR.120/10/2014.
Galekan cattle
Origin and distribution. Galekan cattle (Bos javanicus
x Bos indicus) are one of the cattle germplasm which needs
to be conserved due to a sharp population decline. This
cattle breed is alleged as a crossbreed of Java(PO) cattle
and Bali cattle.
Physical characteristics. Skin color is light brownish,
dark brownish to blackish red brick, and while white or
light brownish on the buttocks and the edge of wattles with
an undefined border, lower legs are white with defined
borders. Tail is long with black hair; has a dark eye circle
and straight back striped with black dorsal stripe. These
cattle are humped and horned with black striped ears and
long little horn which initially came out sideways, then out
onto the front. Habitat is in dry lowland usually grazing in
the seashore, 66-322 kg body weight; performance is 3-5
months of anoestrus post partus; services per conception
are 1.3 times and calving interval is 14-18 months (Aryogi
and Romjali 2009).
Advantages and disadvantages. Galekan cattle have a
larger body size than most of the local cattle, thus it
becomes popular beef cattle that it is to threaten its
sustainability.
Breeding and conservation. Galekan cattle are local
cattle from Trenggalek, East Java. Galekan cattle belong to
type of superior cattle, but its presence is very critical
because of being pressured by the development of PO
cattle breeding and from new species of cattle that come
out from artificial insemination. The continuous natural
processes of crossbreeding with PO cattle cause genetic
purity to be blurred and difficulty to identify the
descendants as Galekan cattle breeds. At this time, the
number of pure Galekan cattle is in estimation of 20-500
heads. These cattle have distribution in coastal area of
Trenggalek district, East Java (Aryogi and Romjali 2009).
Grati cattle (FH Indonesia)
Origin and distribution. Grati cattle (Bos taurus x Bos
indicus) are the only local dairy cattle that are still raised
by breeders. At the beginning of the 20th century, various
taurine dairy cattle were imported into Grati, Pasuruan and
were mated with local cattle, to get dairy cattle that are
resistant to tropical climate (Sudono et al. 2003; Siregar
1995; Soehadji 2009). But from the physical appearance,
337
Grati cattle are the result of a mating of males FH cattle
with PO females. These cattle have gained international
recognition as local dairy cattle of Indonesia (Payne 1970).
These cattle were once widely kept in highland of Pasuruan
and Malang (Pujon, Nongkojajar, Batu and surroundings)
(AAK 1995), but the quality is declining and a back
crossbreed of a pure FH male cattle with PO females needs
to be done. Grati cattle are now known by the name of FH
Indonesia (FHI). Unlike its predecessor whom quality is
steadily declining and is abandoned by many breeders, FHI
cattle are still being developed and the semen is widely
used for artificial insemination program (BBIBS 2015).
The population of old Grati cattle had reached less than
10,000, while the number of new Grati cattle (FHI) has not
been recorded (Sariubang1992; DGLS 2003).
Physical characteristics. Grati cattle has a color
similar to the FH cattle, namely striped black and white
skin, but not as bright as FH, on the forehead are white
triangles and on the chest, lower abdomen, tail and legs are
white; wide long and straight head, small and short horns
heading to the front; body size and milk production is
lower than FH.
Advantages and disadvantages. At first, Grati dairy
cattle were able to produce milk with average of 15 liters
per day, but since there is no further genetic improvement,
milk production capacity has decreased to only 12.3 liters
per day with lactation period of 9 months. These cattle are
able to adapt to the hot tropical environments, and are
easily controlled, docile and quiet. With intensive feeding,
weight can increase to 0.9 kg per day (Syarif and Harianto
2011). Grati cattle on the plateau show better yields than
the one in the lowlands (Ratnawati et al. 2008).
Breeding and conservation. Grati cattle will
experience a loss of quality, especially milk production, in
line with the increasing generation, so FH male cattle is
always needed to maintain the quality.
EXOTIC LIVESTOCK
In 1970-1980, various zebu and taurine cattle breeds
lived or their frozen semen was introduced from Europe,
USA, Australia and New Zealand and crossed with the
local cattle. Zebu cattle breeds imported mainly Brahman
and Brahman Cross, while the breeds of taurine cattle
imported mainly from Simmental, Limousin, Holstein
Friesian, and the Australian Commercial Cross (ACC). The
imported cattle have very high daily body weight gain, but
it is not suitable to be maintained in hot tropical regions,
except for Brahman and Brahman Cross. In Indonesia,
there are also several other exotic cattle, but not much
developed. Since the 1990s, the production of beef cattle in
the country is insufficient and each year continues to
increase imports of beef cattle, mainly from Australia and
the United States. In the end, imported feeder cattle to be
fattened mainly Brahman Cross and the Australian
Commercial Cross breeds of Australia. By 2015, as many
as 650,000 heads of Australian cattle entry in Indonesian
feedlofter, unfortunately, these cattle lasted only 3-6
months in fattening and slaughter, not bred. Today, often
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B IO D IV E RS IT A S 16 (2): 327-354, October 2015
found crossbreeds between taurine cattle breeds of
Simmental and Limousin with local cattle, especially with
Peranakan Ongole, through artificial insemination
(Pamungkas et al. 2012). However, this breeds has not been
naturalized well where pregnancy process almost through
artificial insemination, produce offspring of male sterile
and fertile females, but steadily declining reproduction
from generation to generation. In the long term, this
concern will change the genetic composition of local cattle
Indonesia (Putro 2009).
Friesian Holstein
Friesian Holstein (FH) or Holstein Friesian (Bos taurus)
have been developed since the 13th century in the
Netherlands (North Holland and Friesland) and Germany
(Schleswig-Holstein). A century after the breeding effort
produced the best dairy cattle in the world in typical black
and white colors, this color is preferred than the original
brownish color that is also found in this breeds. In
Indonesia, the first FH cattle imported from the
Netherlands in the 19th century, the next imported cattle
came from Australia, New Zealand, USA, Japan and
Canada. These cattle have a good performance in
producing milk and meat, and have good reproducibility.
These cattle are generally maintained in the highlands of
Java, at an altitude of 700 m asl., but its crossbreeds with
PO can live at an altitude of 300 m asl. In 2002, the
population of FH cattle in Indonesia was approximately
354,000 heads (DGLS2003) and now about 500,000 heads
(DGLSAH 2014). This stock is generally a relatively pure
Holstein Friesian breeds, because in descendant of FH
cattle, the production of milk and beef are far less than of
pure breeds. However, there is still an offer of FH cattle
descendant as the result of crossbreeding between FH cattle
males and PO cattle females. These cattle were formerly
known as Grati cattle, but is now named Indonesian FH
cattle. These breeds have high genetic quality, i.e., high
milk production (about 20 liters per day) and have high
adaptability to tropical environment, birth weight average
of 35 kg and have rapid growth, giving birth ability at the
age of around 25 months, with a calving interval of about
12.6 months. Pure FH cattle are generally black and white
patterns, sometimes red with white streaks and clear color
boundaries. The head is long, wide, and straight, with
relatively short horns and curved toward the front (Sudono
et al. 2003; Siregar 1995); Mouth of FH cattle is wide,
nostrils are wide open, powerful jaws, bright eyes, medium
ears, wide forehead, long and thin neck; the location of the
shoulder is a good deal on the chest wall and forming
relationships neatly with the body, strong back and flat
with vertebrae associated in good way, long and wide
steering, rectangle, short nails with a good circle, low heels
with flat palms, big udder and hanging down at the back of
belly between the thighs (Samad and Soeradji 1990).
Simmental
Simmental cattle (Bos taurus) are originated from
Simme valley, Switzerland. These cattle have been
developed since the 13th century, and became the female
broodstock for various newer breeds of cattle. In 1985,
Indonesia got live cattle and frozen semen of Simmental
from Australia and New Zealand. Simmental cattle are
subtropical cattle. In Indonesia, the pure breeds are only
being kept in government-owned research or large breeder
located in the highlands to take the semen. Stocky and
muscular body shape, a very good muscle development;
high carcass yield with less fat, adult’s body weight can
reach over 1,000 kg. Simmental cattle of the United States
are black due to selection, but the breeds developed in
Indonesia is yellow-brownish or red face, knees down and
the tail tip are white as the original characteristics of this
breeds. In Indonesia, it is only used as beef cattle and many
crossed with the local cattle through artificial insemination,
especially with Peranakan Ongole cattle, but also Madura
cattle, Bali cattle, and even FH cattle. The result of
crossbreeds male is preferred because it grows faster, while
female calf less satisfactory growth and yield little milk. At
the age of 2.5 years the weight has reached 1,000 kg.
Artificial insemination performed directly in the field and
produced offspring with untested ability to adapt to the
climate, feed and local diseases. But breeders like this
business because young cattle are having a larger size and
faster growth than the local cattle, and it continues. In the
long term this needs attention due to genetic changes in
local cattle of Indonesia.
Limousin
Limousin cattle (Bos taurus) that originated from
Limousine and Marche, France have been developed since
the 17th century. These cattle have a long, large, compact
body, as well as a large chest, shallow ribs, thick and
fleshy, with a pattern of meat better than Simmental. Its
eyes are sharp; well-built legs. In males, the horns grow out
and slightly curved. Its skin is dark red, brownish, or
yellow rather gray, but white around the udder and the knee
down and the color around the eyes are lighter. At this
time, it has been developed Limousin cattle without horns
and black. Male cattle weights can reach 1400 kg, while
females 850 kg. The productive period of female cattle is
between 10-12 years old. They are the most rapid cattle in
weight gain, i.e., 1.1 kg per day. Because they come from
the sub-tropical climate, they are only suitable to be
maintained in the highlands that have high rainfall. These
cattle are resistant to attacks of various diseases.
Worldwide, the cattle are widely used for crossbreeding
with various other breeds of cattle. In Indonesia, the cattle
semen is used for insemination in local cattle, especially
PO cattle, even with Brahman cattle.
Brahman and Brahman Cross (BC)
Brahman Cross cattle (Bos indicus x Bos taurus) are the
result of crossbreeds between zebu cattle of Brahman
breeds with some taurine cattle breeds in Australia.
Brahman breeds were first developed in the United States
since 1849 and became genetic sources for some new
breeds. Brahman cattle have a large body, long and deep,
humped over the shoulders; and the loose-skinned, wattle
bark from the lower jaw to the tip of the front chest with
many folds. Elongated head, large ears and a pointed-toe
hang. It has big thighs, thick and loose skin. The skin color
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
is varied, generally white and gray, but there are also black,
brownish, red, yellow, and striped. These cattle are the best
pieces to be developed in the lowlands because it is
resistant to high heat and parasites (Banerjee 1978;
Gunawan 2008). Brahman Cross cattle come from
crossbreeds between Brahman cattle and various taurine
cattle in Australia since 1933, so that the genetic mixture in
every offspring varies widely (Banerjee 1978; Turner 1977;
Friend and Bishop 1978). These cattle have good growth
(1.0 to 1.8 kg per day), high carcass yield (45-55%),
resistant to tropical climates, and resistant to various
diseases, mites or ticks. In 1973, the Brahman Cross cattle
being imported into Sulawesi, Indonesia (Gunawan 2008)
to be used as a draught cattle and cut in old age. In 2006,
they massively distributed throughout Indonesia to support
the acceleration of self-sufficiency in beef cattle program.
Artificial insemination with semen of Brahman or Brahman
Cross with PO cattle is preferred because it produces fastgrowing livestock and able to adapt to local conditions.
Today in Indonesia, especially in West Java, Banten and
Lampung many emerging fattening companies (feedlofters)
are intensively fatten Brahman Cross breeds. Maintenance
is ideal for fattening cattle for 60-70 days for females, and
for 80-90 days for male cattle.
Australian Commercial Cross (ACC)
Australian Commercial Cross (ACC) cattle (Bos indicus
x Bos taurus) have unclear genetic origins, they are from
open crossbreed between various cattle in pastures which
are raised in Northern Australia and Queensland. In the
pasture, Brahman, Shorthorn and Hereford cattle are raised
(Beattie 1990). Thus, allegedly ACC is a cross between
zebu cattle of Brahman breeds with Shorthorn and
Hereford taurine cattle (AMLC 1991; Ngadiyono 1995).
However, in contrast with the Brahman Cross, these cattle
have characteristics more like Hereford and Shorthorn, the
body is shorter and dense, large head, small ears and do not
hang up, do not have a hump and wattle, fur around the
head, the color pattern varies between Hereford and
Shorthorn cattle. These mixed genetic cattle are very
promising for fattening program, because it is easy to adapt
to suboptimal environments like Brahman breeds and has a
rapid growth like Shorthorn and Hereford. If a small and
young ACC cattle are fattened in a short time (60 days) it
would be very beneficial because it produces daily weight
gain ± 1.61 kg per day with a feed conversion 8:22 (Hafid
1998). Along with Brahman Cross, ACC cattle are
excellent for large cattle fattening companies (feedlofters)
in Indonesia.
Therefore, there are indigenous cattle, local cattle and
exotic cattle in Indonesia. The only native cattle of
Indonesia is Bali cattle. Local cattle of Indonesia is an
exotic cattle that has long been nurtured in Indonesia and
even mixed genetically with Bali cattle, namely Peranakan
Ongole cattle, Sumba Ongole and Madura cattle, as well as
Aceh cattle, Pesisir cattle, Jabres cattle, Rancah cattle,
Rambon cattle, Galekan cattle, and Grati cattle (FHI).
Several exotic cattle had been introduced to Indonesia, but
the main cattle include Brahman, Brahman Cross,
Simmental, Limousin, Holstein Friesian, and ACC cattle.
339
Indonesian cattle distribution and density in each province
are shown in Figure 3 and 4, respectively. The relationship
between Indonesian local cattle with several major world
breeds of zebu cattle shown in Figure 5.
QUALITY IMPROVEMENT
Development of cattle in Indonesia has been practiced
for thousands of years and is still being done until now to
improve the quality and quantity of cattle population.
Livestock production is influenced by environmental and
genetic factors. Environmental factors include feed, both
forage and concentrate, water, climate, facilities
maintenance, and is controlled by the gene (Sutarno 2006).
Both can be manipulated, but genetics plays a larger role
because it determines the level of reproduction,
productivity of meat or milk, carcass percentage, growth
rate, feed efficiency, resistance to climate and disease,
physical strength as draught animals, etc. (Frankham et al.
2002). The main obstacles to the sustainable use of cattle
are a lack of information about the population of local
cattle, geographical distribution and genetic characteristics
(Long 2008). Phenotype and genetic characterization in
cattle population are still limited (Hannotte and Jianlin
2005). Almost all cattle in the world are descendants of two
bovine species, namely zebu (humped) and taurine (without
hump). The history of formation of both cattle from wild
aurochs ancestors has been traced through mitochondrial
DNA (mtDNA) (Baig et al. 2005). Bali cattle are the only
other breeds of bovine significantly raised.
Cattle diversities are formed through mutations, genetic
drift and artificial selection of species from wild ancestor
(Long 2008). Genetic studies are necessary to prevent loss
of quality cattle. One threat to the sustainability of
livestock is inbreeding and loss of genetic variation. To
ensure a population can multiply in a sustainable manner,
the level of genetic variation in a population needs to be
known. Genetic variation is often correlated with fitness;
reduced genetic variability may limit the success of
population to respond to environmental changes, such as
climate change, disease or parasites (Frankel and Soule
1981). Inbreeding causes decreased genetic variation
resulting in lower livestock resistance to environmental
change and disease. Inbreeding generally occurs in small
isolated populations without the input of new genes from
the outside. Isolation of Bali cattle in Bali, by preventing
the entry of new gene suspected to have caused decreased
resistance to disease that Jembrana viral disease can infect
them (Tenaya 2010).
Growth is the most important indicator in the meat
production of cattle, so it has important economic value in
livestock raising (Sutarno 2006). The inserting of new
genes through hybridization with other related cattle breeds
may threaten the purity and the specific characteristics of
one breed of livestock. In the colonial era, the island of
Madura declared to be closed to other cattle breeds and
only devoted to the development of Madura cattle; while
Bali Island is devoted to the development of Bali cattle,
even has started since the Klungkung kingdom. However,
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B IO D IV E RS IT A S 16 (2): 327-354, October 2015
K
A
B
I
L
J
H
C
D
E
F
G
Figure 3. Distribution of local Indonesian cattle. A. Aceh cattle; B. Pesisir cattle; C. Rancah cattle; D. Jabres cattle; E. Galekan cattle; F.
Rambon cattle; G. Sumba Ongole cattle; H. Holstein Friesian cattle; I. Grati cattle; J. Madura cattle; K. Peranakan Ongole cattle; L. Bali
cattle (All Indonesia except for Central Java, West Java and Banten)
Livestock density:
≥ 1,650,000
1,070,001-1,650,000
409,001-1,070,000
159,001-409,000
≤ 159,000
Figure 4. Population density of livestock in Indonesia
Figure 5. The relationship of Bali cattle, Indonesian local cattle and main zebu cattle of India (Mohamed et al. 2009)
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
at present these two islands, as well as other places that
became the center of cattle development, became the target
of improving the quality of livestock through artificial
insemination, which is widely used frozen semen from
zebu cattle (Brahman and Brahman Cross) and taurine
cattle (Simmental, Limousin, etc.). This crossing is done on
the fields in an uncontrolled manner and the impact is
difficult to predict in the long term. PO cattle development
is a success story about improving the quality of livestock
in Indonesia, which gained a new hybrid with strong
adaptability to climate, feed availability, and diseases in
Indonesia, and is very suitable as draught animals.
However, due to daily weight gain inferior compared to
other cattle, PO cattle have now also become the target of
crossbreeding. While Madura cattle crossbreeding with
various dairy taurine cattle show reproductive failure which
offspring do not have the endurance and the production of
milk as desired and descendants are no longer found.
Quality improvement through crossbreed between same
cattle species is generally successful, for example, PO (Bos
indicus) and Brahman (Bos indicus) or Brahman Cross
(Bos indicus x Bos taurus). Meanwhile, a cross between
cattle of different species are not always successful,
because it produces male sterility and even if successful
performance of reproductive females will suffer a loss of
quality after a few generations, so it should always supplied
the male semen purely to crossed, for example, Madura
cattle (Bos javanicus x Bos indicus) with Red Danish (Bos
taurus) failed to give offspring that will grow, reproduce
and adapt to the local environment. Similarly, a cross
between FH cattle (Bos taurus) and PO cattle (Bos indicus)
that produce Grati breeds, where after a few generations the
quality is much lower, so it should always be provided new
stocks. Same things happen to gynecological Simpo and
Limpo, where the pregnancy rate of female calf with
Simmental or Limousin semen has a lower success than PO
females. However, in the case of beef cattle, breeders are
generally not too concerned with long-term conditions for
the calves produced that are intended to be cut, not bred, so
it always needs to provide the frozen sperm of pure male.
In the present time, the common males used for
crossbreeding are Simmental and Limousin breeds,
whereas females are from PO cattle breeds, but also has
reported success with female cattle of Bali and Madura
cattle. In some cases, a cross between species of cattle may
also produce offspring that remain qualified after several
generations, such as Madura cattle, Brahman Cross, and the
ACC. Before conservation and management, it is important
to recognize the level of genetic variation in cattle
populations.
Another aspect that is less favorable for the
development of Indonesian local cattle is the lack of effort
to improve descent with the right technology. Efforts to
select and get rid of unsatisfying cattle from its group are
never being done, and the growth rate was never recorded.
This is in addition to less favorable economic terms, can
also worsen next generation. By improving the quality and
quantity of Indonesian local cattle production, it is
expected that the interest of local cattle breeders to
maintain both will be increased, so that the Indonesian
341
local cattle extinction can be avoided and at the same time
dependence of Indonesia for beef cattle from other
countries may be reduced (Sutarno 2006).
Indonesian local cattle, such as Bali cattle, have the
advantage of reproduction capability and high adaptability
to the local environment, but the quality and quantity of
production are lower than the imported cattle (Sutarno
2006). Various government programs to improve the local
cattle population so that it becomes the main source of beef
cattle which include reduction of slaughtering on
productive local cattle, and expand the range of
interbreeding programs of local female cattle through
artificial insemination (DGLS 2010c). However, the latter
program became controversial because it is done directly in
the field, that it triggered an uncontrolled genetic mixing,
and produced offspring that have not been proven their
adaptability to climate, natural feed and local disease as
well as their reproductivity. Artificial insemination also
reduced the period of open days (Siregar et al. 1993).
Genetic studies provide insight about the loss of genetic
diversity due to inbreeding and the implications on natural
populations. Conservation of genetic diversity is very
important because it represents the potential evolution of a
species (Frankham et al. 2002). In the case of Indonesian
local cattle, especially Bali and zebu cattle, Mohamad et al.
(2009) have revealed the origins of Bali cattle, as well as
the level of genetic variation, inbreeding and genetic purity
to determine the sub-populations that are more suitable for
conservation.
GENETIC MARKERS BASED SELECTION
Cattle breeding have now reached a new scene with the
nearly completed genome map of taurine cow (Elsik et al.
2009). QTLs facilitate understanding of the level of
production and the behavior characteristics of beef and
dairy cattle, thus providing a definite guide to selection
(Friedrich et al. 2015). Meat and milk production can be
increased through artificial selection. Improved genetic
quality of cattle can be done with conventional methods of
performance-based selection (PBS) or growth, and direct
selection on the DNA by using marker assisted selection
(MAS) that can recognize certain genes such as growth
hormone gene and mitochondrial DNA (Sutarno 2010).
Until a few years ago, the selection to obtain quality breeds
was generally performed only by external appearance
(phenotype). Individuals having a good phenotype are led
to mate each other to obtain offsprings with good
phenotype. However, this technique is not appropriate
because of the environment factors, such as feed, water
maintenance and management, able to affect the body
appearance, but not inherited. While genetic factors that
codes growth properties are inherited, so it is appropriate
for the selection (Sutarno 2006).
Selection is done with a guide marker gene, which
variations of DNA sequences that characterize variations in
the nature of phenotype, both of which directly affect the
trait (candidate gene marker approach), or indirectly
through a linkage with DNA sequences which affect the
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B IO D IV E RS IT A S 16 (2): 327-354, October 2015
nature of phenotype (random marker approach). Candidate
gene markers approach is based on the knowledge that the
selected genes involved in desirable traits, such as growth
hormone gene. However, this approach is limited to
characteristics known on physiological and biochemical
relationships. Instead, in the random marker approach,
genotypes measurements are performed on a number of
loci of whole genome without knowing the influence of
phenotype, with the hope that there is locus relating to the
desired characteristic, so this approach is less valuable than
the candidate approaches (Sutarno 2006).
Specific genes have a significant influence on growth,
milk production or other specific targets. Edwards and
Page (1994) and Lande and Thompson (1990) stated that
the increase in the genetic trait to 50% will occur with
MAS technique. This increase occurred due to greater
control of the MAS technique in the selection, thereby
reducing the selection time between generations because
the genes can be identified early during birth or still in the
uterus. Gene markers approach have been widely used
successfully for the characteristics of disease resistance, the
quality and quantity of carcasses, fertility and reproduction,
milk production, and the performance of growth (Sutarno
2006).
Growth is controlled by several genes, either gene with
big influence (major gene) or genes with small influence
(minor gene). One of the genes thought to be key genes in
affecting growth is the growth hormone genes. In addition,
mitochondrial DNA which is located outside the nucleus
(the cytoplasm) is also considered affecting the growth
since mtDNA is the controller of energy formation process
(Sutarno 2010).
Growth hormone gene
Growth hormone in cattle (bovine growth hormone) has
a major role in the growth, milk production, animal body
composition, and is associated with a higher average
growth (Winkelmann et al. 1990; Hoj et al. 1993;
Cunningham 1994). Administration of growth hormone
may increase the average growth of cattle (Burton et al.
1994). Increased growth is influenced by the performance
of IGF-I (Armstrong et al. 1995), so the variation of these
genes leads to variations in growth (Ballard et al. 1993), for
example, observed in PO cattle (Sutarno 2003) as well as
Composite and Hereford (Sutarno 1998). According to
Schlee et al. (1994b) polymorphism in the growth hormone
gene (GHG) cause differences in hormone synthesis,
resulting in differences in the concentration/circulation of
these hormones. This difference causes the growth
variation among individuals. Thus, DNA variations on
growth hormone gene can serve as a potential candidate as
cattle growth feature gene markers (Sutarno 2006) (Figure
6).
Variations in the growth hormone gene locus of
Composite cattle in Western Australia significantly affect
variation of the mean growth (Sutarno et al. 1996; Sutarno
1998). Schlee et al. (1994b) found that differences in the
genotype of a growth hormone gene influence circulating
concentrations of growth hormone and IGF-I in the
Simmental cattle. Rocha et al. (1992) found a significant
association between alleles of growth hormone with weight
at birth and the ridge width at birth in Brahman cattle.
These variations have been reported in taurine cattle, but is
still limited to a local Indonesian beef cattle (Sutarno and
Junaidi 2001; Sutarno 2003).
To obtain superior Indonesian local cattle in the
production of meat, it is important to acquire the marker
gene from the population of local cattle thorough analysis
of the combination of phenotype data (growth), genotype
data (allele), as well as all supporting data which may
affect growth (cattle species, sex, age, the concentration of
circulating growth hormone, etc.). Cattle growth hormone
gene which has been mapped is located on chromosome 19
with location-qtr Q26 (Hediger et al. 1990). This gene
sequence consists of 1793 bp which was divided into five
exons and is separated by four introns (Sutarno 2006).
Variations of the gene encoding growth hormone has been
reported in taurine cattle, for example, Red Danish dairy
cattle (Hoj et al. 1993), Simmental beef cattle (Schlee et al
1994a), Hereford and Composite beef cattle (Sutarno et al.
1996; Sutarno 1998) as well as PO cattle, Bali cattle and
Madura cattle (Sutarno et al. 2002, 2003). Such variations
are generally caused by the deletion, substitution or
insertion (Sutarno 1998, 2003).
In Simmental cattle breeds, Schlee et al. (1994a)
showed that individuals which have LV (leucine/valine)
genotype on their growth hormone gene are superior in
achieving weight of carcass and meat quality.
Polymorphisms which were detected by TaqI on its growth
A
B
Figure 6. A. The role of growth hormone in regulating
metabolites material for fuel regulation and growth; B. The
structure of growth hormone gene in cattle; The letters A, B, C
and D show the introns, while the Roman numerals I, II, III, IV
and V show the exons (Sutarno 1998).
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
343
Figure 7. The mitochondrial DNA of mammals, including cattle (St John et al. 2004)
hormone gene was reported to be associated with birth
weight in Brahman cattle (Rocha et al. 1992) and with
growth in Korean cattle (Choi et al. 1997). Research
conducted by Sutarno (1998) against Hereford cattle and
Composite showed that MspI polymorphism in the growth
hormone gene region between exons III and IV
significantly affect the growth, in which individuals that
have allele MspI (-) are superior. At PO cattle, individuals
who have MspI +/- heterozygous genotype has higher
weight, chest circumference and body length than MspI
genotype homozygous +/+ or MspI -/- (Sutarno 2003;
Paputungan et al. 2013). The relationship of genotype
variations in growth hormone locus with a total growth of
cattle is probably caused by differences of growth hormone
circulation as a result of the growth hormone gene variation
(Sutarno 2006). Study of gene polymorphisms of growth
hormone has also been done on some Indonesian local
cattle such as Pesisir cattle (Jakaria et al. 2007), Bali cattle
(Jakaria et al. 2009; Jakaria and Noor 2011), Madura cattle
(Purwoko et al. 2003; Hartatik et al. 2013), PO cattle
(Sutarno et al. 2005; Sutarno 2010; Paputungan et al. 2013;
Rahayu et al. 2014), Sumba Ongole cattle (Anwar et al.
2015), Aceh cattle (Putra et al. 2013, 2014) and Grati dairy
cattle (Maylinda 2011).
Selection to obtain superior descendants based on DNA
markers such as DNA polymorphisms can provide a more
accurate and efficient result (Schlee et al. 1994b). Gene
variations in growth hormone gene are related to variations
in growth hormone and insulin-like growth factor (IGF-I).
Then, variations of growth hormone and IGF-I have caused
differences in growth, so that the encoding growth
hormone gene can be used as a potential starting point as
DNA markers for Indonesian local cattle breeding(Sutarno
2006).
Mitochondrial DNA
Mitochondrial DNA (mtDNA ) is a genetic marker that
is very useful to study the origin, genetic diversity and
descendant differentiation because it has unique
characteristics inherited from the female broodstock, the
fast rate of evolution and the less level of recombination
(Bailey et al. 1996; Liu et al. 2006; Galtier et al. 2009).
Mitochondrial DNA is located outside the nucleus and is
responsible for the energy formation. It affects the growth,
reproduction and production characteristics in cattle
(Schutz et al. 1994). MtDNA gene is a marker that is
efficient, because the segments of genes that evolved
differently (Zardoya and Meyer 1996; Kikkawa et al. 1997;
Hassanin and Ropiquet 2004; Kartavtsev and Lee 2006).
Variations on mtDNA cattle have been reported
(Sutarno and Lymbery 1997; Sutarno 2002a). MtDNA
evolved faster than nuclear DNA, and even though there
are thousands of copies of mitochondrial genome in each
cell, nucleotide substitution occurred about five to ten
times faster than the same mutation in the DNA core.
Modifications and variation in mtDNA will affect the
phenotype. Schutz et al. (1994) reported that there is effect
of various mtDNA sequences on milk production, while
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B IO D IV E RS IT A S 16 (2): 327-354, October 2015
Schutz et al. (1993) found a significant effect of
substitution at nucleotide pairs (bp) no. 169 D-loop
sequences in the percentage of milk fat. MtDNA variations
on D-loop segment significantly affected the reproductive
feature of Hereford and Composite cattle (Sutarno et al.
2002a; 2002b) (Figure 7).
Mitochondrial DNA is a marker based on the maternal
pedigree that may show the family history (Ascunce et al.
2007). Research on the D-loop region of mtDNA on
several breeds cattle have shown that taurine cattle
ancestors came from Syria, and then spread throughout
Europe (Edwards et al. 2007). MtDNA D-loop region in
cattle may show hybridization to the banteng and Madura
cattle (Nijman et al. 2003). Based on the sequence of
mtDNA D-loop region, Aceh cattle is one cluster with
Pesisir cattle and PO is zebu cattle, while Bali cattle and
Madura cattle form its own separate cluster. The base
composition of mtDNA D-loop region nucleotide of Aceh
cattle is different from other local cattle with difference
sequence from smallest to largest, i.e., from Pesisir cattle,
PO cattle, Bali cattle and Madura cattle. It can be used as a
marker for distinguishing and grouping of Indonesian local
cattle. Aceh cattle have similarity base arrangement of
nucleotide for about 94.36% with the zebu cattle, so based
on the maternal line, Aceh cattle is originated from zebu
cattle (Abdullah et al. 2008).
MtDNA analysis can also be used to trace relationship
and genetic purity of the female parent lines. Bali cattle in
Malaysia generally contain genes of other cattle, such as
China's Yellow cattle, Kedah-Kelantan and Brakmas, while
Bali cattle in Indonesia tend to be pure (Somarny et al.
2015). This happens because in Malaysia the Bali cattle is
raised together with other breeds of cattle, while in
Indonesia they are not generally raised together with other
cattle (Nijman et al. 2003). Instead, Chinese Yellow cattle
which are believed to be descendants of crossbreeding of
taurine and zebu (Mao et al. 2006) actually contain Bali
cattle genes or banteng (Chang et al. 1999). An
understanding of the origin, differentiation and genetic
relationships between offspring cattle is very important for
the genetic management, sustainable usage and cattle
conservation (Somarny et al. 2015).
However, cattle with the same genetic traits can have
the performance of a much different because of differences
in the management of maintenance. In Madura, Madura
cattle function as beef cattle, racing cattle (karapan) and
displayed cattle (sonok), each of which has different
physical character and behavior, but all three were taken
from generally same calf. Diversity analysis of BCKDH
(Branched-chain α-ketoacid dehydrogenase), the main
enzyme complex in the inner membrane of mitochondria
that metabolize valine, leucine, and isoleucine, showed no
mutations associated with differences in the allotment of
Madura cattle (Febriana et al. 2015).
Analysis of molecular data from mtDNA and growth
hormone gene, combined with the phenotype data of
cattle’s growth, it is known that genotypes of cattle that is
superior in the production of meat that can be used as a
marker gene. Gene markers approach can be used in animal
breeding to obtain superior breeds through planned
crossbreed that sets parent genotype. Further development
of the marker gene is that it can be used in DNA
recombinant to produce growth hormone. This hormone
can be used temporarily to induce growth, and furthermore
towards the establishment of transgenic animals, early
detection of diseases and other phenotypic traits (Sutarno
2006).
CROSSBREEDING
Crossbreeding is an important part of cattle
improvement because of the variability of environment and
market demand that need to be addressed. Crossbreeds
bring out heterosis effect in which to collect the new
advantageous characteristics, for example, crossbreeds
between zebu cattle that are impervious to hot climates and
taurine cattle that can grow faster. Refinement on breeding
governance can improve the performance of livestock, but
these characteristics are not inheritable, on the other hand,
genetic improvement can be inherited. Cattle productivity
is influenced by genetic and environmental factors; cattle
can reach their genetic potential conditions when supported
by an optimal environment (Talib et al. 2002).
Environmental conditions and the livestock breeds affect
the rate of growth, calving production and reproduction
ability (Nugroho 2012). Cattle which are raised on the
traditional system generally will experience a forage
shortage due to the limited and low quality of feed, and
rarely given additional feed such as concentrates and
grains, so they rarely reach the optimum conditions
(Wiyatna et al. 2012).
Breeders prefer crossbreeding because of the effect of
heterosis where offsprings will have more superior
characteristic than the parents. In crossbreeding through
artificial insemination, the breeder can set all female
broodstocks to give birth at relatively the same time to
obtain calves of the same age. Crossbreeding can be
designed to establish certain cattle breeds, according to
market needs. Cattle crossbreeding cannot be done
haphazardly but it must be done with clear objectives and
will last over the long term from generation to generation.
Because of weak control in crossbreeding, the entry of
additional genetic from other cattle may frustrate the
achievement of goals because of too much variability in the
offspring (Comerford 2014). However, a crossbreed may
lead to the loss of adaptability to the local environment
(Mohapatra 2004), so, ideally, an assessment of the impact
of introduced livestock before it becomes a realization
(Mastuti 2014). Crossbreeding to get superior calf must
begin by characterizing all the characteristic of local cattle
which have adapted to the local environment and predict
the results to be obtained.
Performance of cattle reproduction is influenced by
environmental conditions. In the semi-arid or tropical
climates, zebu cattle show more superior reproductive
performance than taurine cattle (Meirelles et al. 1991), but
in highland or temperate zones taurine cattle have better
reproductive performance (Duarte and Barbosa 1989).
Some studies have reported a close relationship between
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
genotype and environmental factors on the productive
performance and reproduction of beef and dairy cattle
(Mulder and Bijma 2005; Hammack 2009; Hammami et al.
2009). The characteristics of reproduction, first mating age,
number of service per conception, open days and calving
interval are the basis for determining the profitable cattle
ranch (Enyew et al. 1999; Tavirimirwa et al. 2013). Age of
mating and first calving is significantly affected by height
(in relation to the ambient temperature), where cattle age of
mating and first calving is slower in lowland (Suyadi et al.
2014). Heritability of these traits is usually low, so,
including conditions and feed management; environmental
factors play an important role in the variability (Olori et al.
2002).
In Indonesia, crossbreeding cattle are mostly done
through artificial insemination. This activity has been
known in Indonesia since 1953, and since 1980-1990, it has
widely performed using semen of some foreign taurine
cattle that the growth and body weight is relatively higher.
However, until now the purpose of artificial insemination
program is not clear yet, it will be towards the formation of
a composite cattle, terminal cross, or commercial livestock.
Many breeders helped the officer to the quality of livestock
by crossing local cattle with Simmental or Limousin cattle.
Cattle breeders like this, because the price of male
offspring is very high, even though the local cattle turned
into large cattle type that needs a lot of feed. In the forage
shortage conditions, cattle crossbreeding be thin, poor body
condition, and declines in reproductive performance, such
as high number of services per conception, long time of
calving interval, and low quality calf. This condition is
accompanied by low production of milk and high calf
mortality. On the condition of good maintenance,
reproductive performance of crossbreeding cattle remains
good. While on the local cattle, forage shortage conditions
only lead to thin body, but still capable of estrus, ovulation
and pregnant. In quantity and quality, forage is one of the
important keys to the condition of crossbreeding cattle to
remain good and productive (Diwyanto and Inounu 2009).
The interaction of genotype and environmental factors
can be observed from the age of first calving and the first
lactation in dairy cattle (Suyadi et al. 2014; Sahin et al.
2012). Nugroho (2013) reported that PO cattle showed
lower feed intake, lower growth rates and lower body
condition score (BCS) than Limpo crossbreed cattle. It
shows that in tropical conditions, the breeds of cattle affect
the growth rate. On the other hand, the chewing ability of
Limousin crossbreed is better than the PO (Purnomoadi et
al. 2003). In the FH dairy cattle, the age of first lactation
affects total milk production during the second and third
lactation. Earlier age of first calving and first breastfeeding
cause lower milk production in the next lactation (Madani
et al. 2008). Cattle species have no effect on this variable,
even the PO cattle in lowland shows slower age of mating
and age of first calving. This is due to the different levels
of genetic response to environmental conditions which are
critical due to lower feed intake and BCS. Bridges and
Lemenager (2008) states that the low BCS in cattle causing
low reproductive performance including age of first mating
and first calving (Figure 8).
345
Bali cattle
Increased production of Bali cattle has been done
through artificial insemination using frozen semen from
taurine cattle such as Simmental, Limousin, Hereford, and
Charolais as well as zebu cattle of Brahman breeds, up to
now the best result for the rate of growth is Simmental
(Diwyanto and Inounu 2009). Crossbreeding systems to
improve livestock meat production utilize heterosis and
exploit differences among cattle in certain characteristics
(Tang et al. 2011), in the different environmental
conditions (Dadi et al. 2002). However, these efforts may
not have a positive impact if it is not followed by the
environmental improvement. Replacement of local cattle
with exotic cattle could create new problems, such as
obstructed birth due to increased birth weight, low
tolerance to harsh environmental conditions, and increased
forage demand due to higher growth rates and larger body
size (McCool 1992).
Performance of breed of cattle or its crossbreeding is
not always the same in different environmental conditions.
Environmental factors that most affect the livestock
production are heat and humidity (Yeates et al. 1975). In
tropical countries, air temperature varies depending on the
altitude (Williamson and Payne 1980). Lowland is hotter
than highlands. Sub-tropical taurine cattle only produce
well in the highlands, while the highland area in Indonesia
is relatively limited. In West Nusa Tenggara, Bali cattle
calf raise in lowland and highland (> 700 m) had a
relatively same growth rate, while Simbal crossbreeds
(Simmental x Bali cattle) have a higher growth rate in the
highlands. The offspring which backcross with Simmental
has a higher growth rate in the highlands and lower growth
rate in the lowland, while the offspring which backcross
with male Bali cattle have a higher growth rate in the
lowlands (Pribadi et al. 2014).
Therefore, it is important to evaluate the factors that
affect the economical properties of diverse environments to
understand the proper management in hybrid system,
because interaction can affect the productive efficiency
(Pribadi et al. 2014). Growth characteristics such as weight
at birth, weaning and yearling are economically very
important in cattle production systems. Birth weight and
weaned weight are affected by the parent’s genetic (Meyer
1992). Weaning calf and yearling cattle are the main
products of beef cattle, body weight greatly affect the
selling price, as well as an important criterion in cattle
breeding (Bazzi and Alipanah 2011; Ashari et al. 2012).
Peranakan Ongole
Peranakan Ongole (PO) derived from the uncontrolled
crossing between Sumba Ongole cattle with local cattle in
Java since the 1930s. PO cattle are a tropical species that
have adapted in Indonesia, especially in East Java. Since
the 1990s, many PO cattle crossed with taurine cattle,
mainly Simmental and Limousin, through artificial
insemination without considering the genetic composition
of descendant, so it is feared that it will affect their
adaptation, reproduction and growth. But, breeders support
this effort, because it gave them good calves, with faster
346
B IO D IV E RS IT A S 16 (2): 327-354, October 2015
A
B
C
D
E
F
G
H
I
J
K
L
Figure 8. The diversity of cattle crossbreeds from artificial insemination in Indonesia. A-B. Simpo cattle (Simmental bull x PO cow) in
Central Java; C. Simpo cow and its calf that produced from artificial insemination with Simmental bull in Boyolali, Central Java; D.
Simpo calf and its Simpo cow that produced from artificial insemination with Simmental bull in Klaten, Central Java; E. Limbal calf
(Limousin male x Bali cattle female) in Nunukan, North Borneo; F. Limousin crossbreed in Jember, East Java; G. Limad calf
(Limousine bull x Madura cow) in Rote, East Nusa Tenggara; H. Limad bull in the island of Madura or locally known as Madrasin; I.
Limpo cattle in Sukoharjo, Central Java; J. Calf of Simbal (Simmental bull x Bali cattle cow) in Nunukan, North Borneo; K. Calf of
Simpo in Dairi, North Sumatra; L. Crossbreed calf of Brahman and local cattle in South Tapanuli, North Sumatra
growth. At the age of three years, hybrid cattle (Simpo or
Limpo) are able to reach a weight of 800 kg, while the PO
cattle reach less than half of it. Conversely, PO cattle
production costs less than half of the hybrid cattle (Sutarno
2006). Endrawati et al. (2010) showed that consumption of
green fodder and concentrate on Simpo cattle
than the PO’s, but if it is calculated based on
body weight, it makes no different. Digestibility
Simpo and PO is not different, as well as the
estrous cycle.
is greater
metabolic
of feed in
BCS and
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
Limpo and Simpo cattle are widespread across Java, in
the lowlands and highlands. PO cattle’s first mating age is
higher than Limpo cattle’s. In the highlands, service per
conception (S/C) is higher for Limpo and there was no
significant different for open days period (DO) and calving
interval period (CI), making it both more efficient to be
maintained in the lowlands. Yulyanto et al. (2014) showed
that the value of S/C, DO and CI between PO cattle and
Limpo differ significantly, where the reproduction
performance of PO cattle is better than Limpo. At PO cattle
and Limpo, calving interval period and first mating are
around 99-137 days. Environmental conditions and the
breeds of cattle affect calving interval period and first
mating (Suyadi et al. 2014). Cattle species and the average
daily temperature resulted in a different interval between
calving and mating. This interval is longer in the dry
season than the wet season, probably because of the low
quantity and quality of feed during the dry season which
result of low BCS (body condition score) (Kebede et al.
2011). Service per conception ranged from 1.64 to 2.01 is
affected more by climate than by species of cattle that in a
tropical climate it shows higher service per conception
(Kebede et al. 2011). Limpo cattle containing genetic of
zebu and taurine cattle show high service per conception
(Suyadi et al. 2014). The period of open days and calving
interval was not significantly influenced by the breeds of
cattle and environmental conditions (Suyadi et al. 2014).
Performance of reproduction based on the reproduction
cycle (first mating after calving, service per conception,
open days and calving interval) of PO cattle is more
efficient than Limpo. Altitude and breeds of cattle affect
age of first mating and first calving, the first mating after
calving and the number of services per conception, but not
on open days and calving interval. Based on the
reproduction performance, PO cattle and Limpo cattle are
more efficient to be raised in the lowlands than highlands
(Suyadi et al. 2014).
Simpo and Limpo cattle have better growth than PO
cattle’s in traditional breeding. Male PO and Limpo’s
consumption with rice straw and concentrate in the ratio of
60: 40 is 2.8% live weight (DM basis), but the growth of
PO is less than half compared to Limpo (0.47 kg per day)
(Purnomoadi et al. 2003). According to Pamungkas et al.
(2012), feed quality can reduce the possibility of
differences in daily weight gain. For comparison, Moran
(1985) recorded a daily weight gain of 0.65 kg per day for
male PO cattle fed by fiber-based (70% grass and 30%
concentrate, DM basis) and 0.81 kg per day with
concentrates. Cruz de Carvalho et al. (2010) reported lower
growth rate of PO cattle than the Simpo given high
concentrate diet. Simpo cattle have higher carcass weight
and higher carcass percentage and feed cost per gain is
more efficient than PO cattle’s. The low rate of growth is
often due to the high proportion of poor quality of forage in
the diet such as straw (as the main feed, 48% in the dry
season and 78% in the rainy season). Small breeder cannot
afford to give fodder with energy content and high protein.
But actually, legumes, grass, bran and remains of cassava is
a feed of high quality and low cost (Pamungkas et al.
2012). Under the unfavorable fodder conditions, to raise
347
PO cattle is more profitable than Simpo or Limpo cattle
(Hartati et al. 2005).
The difference in average daily gain (weight) of PO
cattle, Simpo and Limpo in wet and dry seasons is not
significant, although the growth rate tends to be higher in
the wet season than the dry season. In contrast, the
thickness of body circumference (girth) and body weight
were higher in the rainy season for all cattle, where the
BCS increased by 5% during the rainy season and
decreased by 9% during the dry season (Pamungkas et al.
2012). Body weight was higher in the rainy season than the
dry season due to provide the feed contains more protein in
the rainy season than the dry season (Evitayani et al. 2004).
On the other hand, the body weight of cattle at the
traditional breeding is much lower than at the research
station, so it has great potential to be increased (Pamungkas
et al. 2012).
Madura cattle
Madura cattle quality improvement is generally done by
crossbreeding between the Madura cattle with stud of
Limousin cattle through artificial insemination (Wijono
2004; Hartatik 2009). These crossbreed cattle have better
quality of meat production and are known locally as
Madrasin or Limad cattle. Madrasin cattle’s size and body
weight is higher than Madura cattle. This condition is
certainly less favorable primarily related to the effort of
maintaining the existence of pure Madura cattle as one of
Indonesian native germplasm cattle (Siswijono et al. 2014;
Decree of the Director General of Livestock No.
18020/Kpts/PD.420/F2.3/02/2013 ).
Madura cattle maintained by small breeder for a variety
of purposes including draught animals, life savings,
producer of organic fertilizer, source of income and means
of cultural celebrations such as cattle races (karapan) and
beauty contest (sonok). Karapan involving livestock cattle
compete with muscle strength and speed, while a beauty
contest in the form of cattle dance to the traditional music
beat on a long catwalk. Since 1993, Madura cattle crossed
with Limousin to increase live weight. Such efforts are
slowly adopted by farmers and supported by localgovernment, especially when decentralization policies
implemented in Indonesia. To increase the income of
farmers, local governments are now trying to stimulate
cross cattle because calves from crossbreeds have high
selling price. Though, it is contrary to the efforts of a
national policy to preserve Madura cattle as indigenous
genetic resources. Also, the result of crossbreeding will
experience the possibility of losing the characteristics of
livestock needed for cultural celebrations such as karapan
and sonok cattle. However, Madura breeders felt that
Limousin cattle crossed with Madura (Limad or Madrasin)
increase body weight and more profitable than Madura
cattle (Siswijono et al. 2010; Rahmawati et al. 2015). At
this time the cattle Limad is excitement for breeders who
have long seen the development of Madura cattle breeding
was stagnant. There is no definite count of Limad cattle
population, but the number is believed to be a lot,
especially in Bangkalan and Sumenep. In every exhibition
of agriculture, Madrasin cattle have always been a star.
348
B IO D IV E RS IT A S 16 (2): 327-354, October 2015
A
B
C
D
E
F
Figure 9.The diversity of cattle on island of Madura, East Java. A. Karapan (race) bulls; B. Sonok cattle (displayed cow); C. Madura
superior cattle; D. Madura superior cattle in festivals; E. Madrasin (Limad) superior cattle in festivals; F. Performance of Madrasin
bulls.
Breeding programs should include controls for crossed
male cattle to avoid the possibility of uncontrolled mating;
especially Madura cattle reproductive performance is
greater than Madrasin, while the production of performance
in Madrasin crossbreed is greater than Madura cattle. Feed
conditions greatly affect calves growth, so that good
quality of local feed and continuity of provision should be
maintained in order to prevent negative effects on growth
(Kutsiyah et al. 2003) (Figure 9).
CONCLUDING REMARKS
Self-sufficiency in beef cattle will be met if the local
production and cattle population is sufficient, but the facts,
the ability of Indonesian cattle population to meet the
demands continues to decline, year by year. Crossbreeding
between exotic cattle and Indonesian local cattle is a
shortcut to increase beef production. These crossbreeding
cattle have size and weight gain higher than local cattle but
need higher cost of maintenance, that the economic
benefits are not much different. On the other hand, the
crossbreeds have not a clear direction, and it is feared that
it is not sustainable because the cattle produced has lower
climate durability and lower reproductive capacity than the
existing local cattle. Scientists believe that the
sustainability of beef and dairy cattle supply is highly
dependent on an understanding of the diversity,
characteristics and use of local genetic resources in
developing countries that have not been developed.
Indonesia has enough local cattle, some of which have the
performance of a very satisfactory and well adapted to the
dry climate, to limited fodder, and to various tropical
diseases, namely Bali cattle, PO and Madura cattle, in
addition there are also local cattle with more limited
population, namely: Aceh cattle, Pesisir cattle, Rancah
cattle, Jabres cattle, Rambon cattle, Galekan cattle and
dairy Grati cattle. However, along with the broader and
easier service of artificial insemination, the pattern of
breeds began to change, which initially the cattle are
traditionally raised as draught animals and as life savings
turned into semi-intensive system with orientation for beef
cattle (meat), so that the performance of crossbreeds
descendant of local cattle crossed with male Simmental and
Limousin cattle are preferred because of the growth rate
and maximum weight is much higher.
Theoretically, crossbreeds between different species of
cattle would produce sterile males, while females would
decrease the ability of reproduction from generation to
generation. This crossbreed cattle descent proved to be less
able to adapt to the tropical climate conditions, natural feed
and local diseases. Thus, allowing the continuation of
crossbreeding with cattle stud taurine, for example,
Simmental and Limousin, against the local female cattle is
the gamble for the future of Indonesian meat supply.
However, proponents of these programs argue that such
concerns are exaggerated, as evidenced in northern
Australia, the uncontrolled genetic mixing in pasture
among the various breeds of taurine and zebu cattle
generate sustainable calf and become one of the main cattle
which were exported in the last 30 years, namely
Australian Commercial Cross (ACC) cattle. The desire to
SUTARNO & SETYAWAN – Genetic diversity of Indonesian cattle
maintain crossbreeding cattle, such as Simpo and Limpo,
continues to rise. Studies in 2008 showed that in Central
Java the population ratio of local PO cattle with
crossbreeding cattle (Simpo and Limpo) is 51.93% and
48.07%, while in Yogyakarta is 25.75% and 74.25%. This
shows that the PO cattle population tends to decrease
drastically, while the crossbreeding cattle population
increased. In terms of meat production, to maintain Simpo
and Limpo cattle is quite positive, but from the aspect of
environmental capacity, increase in population, and the
conservation of local cattle as national germplasm, it is
very detrimental. If this continues, then in 20 years to
come, PO cattle which proved adaptive to climate
conditions and tropical environment are expected to
become extinct. Ironically, PO cattle itself is the result of
uncontrolled crossbreed between Sumba Ongole cattle and
old Java cattle. The presence of SO cattle caused extinction
of the old Javanese cattle, but this mating produces PO’s
offspring sustainable cattle, because both species are zebu.
Extinction of local cattle will cause full dependence of
breeding stock of imported cattle. At this time, the deficit
of beef cattle population in Indonesia is only 25-30%, but
in the next 10 years, it will increase to about 50%.
Currently, feeder of Brahman Cross and the Australian
Commercial Cross cattle imported from Australia dominate
the market for fattening ranch. This leads to the ideals of
Indonesian people to be independent and sovereign in food
demand will be far from reality.
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ISSN: 1412-033X
E-ISSN: 2085-4722
Potential distribution of Monotropa uniflora as a surrogate for range of Monotropoideae
(Ericaceae) in South Asia
PRAKASH PRADHAN
109-115
A new record of plant parasitic green algae, Cephaleuros diffusus (Trentepohliaceae,
Chlorophyta), on Acacia auriculiformis hosts in Thailand
ANURAG SUNPAPAO, MUTIARA K. PITALOKA
116-120
Traditional dye yielding plants of Tripura, Northeast India
BISWAJIT SUTRADHAR, DIPANKAR DEB, KOUSHIK MAJUMDAR, B.K. DATTA
121-127
Genetic diversity of Rana (Pelophylax) ridibunda and Bufo (Pseudepidalea) viridis in
different populations
TAYEBEH MOSLEHI, MAJID MAHDIEH, ALIREZA SHAYESTEHFAR, SEYED MEHDI TALEBI
128-131
Genetic diversity of patchouli cultivated in Bali as detected using ISSR and RAPD
markers
MADE PHARMAWATI, I PUTU CANDRA
132-138
Population genetic structure in medicinal plant Lallemantia iberica (Lamiaceae)
FAHIMEH KOOHDAR, MASOUD SHEIDAI, SEYYED MEHDI TALEBI, ZAHRA
NOORMOHAMMADI
139-144
Within and among-genetic variation in Asian flax Linum austriacum (Linaceae) in
response to latitude changes: Cytogenetic and molecular analyses
ZAHRA NOORMOHAMMADI, TINA SHAFAF, FATEMEH FARAHANI, MASOUD SHEIDAI,
SEYED MEHDI TALEBI, YEGANEH HASHEMINEJAD-AHANGARANI-FARAHANI
145-150
Felids of Sebangau: Camera trapping to estimate activity patterns and population
abundance in Central Kalimantan, Indonesia
ADUL, BERNAT RIPOLL, SUWIDO H. LIMIN, SUSAN M. CHEYNE
151-155
The nutritional quality of captive sambar deer (Rusa unicolor brookei Hose, 1893)
velvet antler
GONO SEMIADI, YULIASRI JAMAL
156-160
Structure of natural Juniperus excelsa stands in Northwest of Iran
FARZAM TAVANKAR
161-167
Phylogeny analysis of Colutea L. (Fabaceae) from Iran based on ITS sequence data
LEILA MIRZAEI, IRAJ MEHREGAN, TAHER NEJADSATARI, MOSTAFA ASSADI
168-172
Study of the digestive tract of a rare species of Iranian blind cave fish (Iranocypris
typhlops)
ALI EBRAHIMI
173-178
Bayesian and Multivariate Analyses of combined molecular and morphological data
in Linum austriacum(Linaceae) populations: Evidence for infraspecific taxonomic groups
FATIMA AFSHAR, MASOUD SHEIDAI, SEYED-MEHDI TALEBI, MARYAM KESHAVARZI
179-187
Impact of Gujjar Rehabilitation Programme on the group size of Asian elephants
(Elephas maximus) in Rajaji National Park, North-West India
RITESH JOSHI
188-195
Diversity of butterflies in four different forest types in Mount Slamet, Central Java,
Indonesia
IMAM WIDHIONO
196-204
Recovery of plant diversity and soil nutrients during stand development in subtropical
forests of Mizoram, Northeast India
SH. B. SINGH, B.P. MISHRA, S.K. TRIPATHI
205-212
Conservation status of the Family Orchidaceae in Mt. Sinaka, Arakan, North Cotabato,
Philippines
CHERRY LEE T. PANAL, JENNIFER G. OPISO, GUILLER OPISO
213-224
Taxonomy and distribution of species of the genus Acanthus (Acanthaceae) in
mangroves of the Andaman and Nicobar Islands, India
P. RAGAVAN, ALOK SAXENA, P.M. MOHAN, R.S.C. JAYARAJ, K. RAVICHANDRAN
225-237
Isolation and characterization of a molybdenum-reducing and SDS-degrading Klebsiella
oxytoca strain Aft-7 and its bioremediation application in the environment
NORAZLINA MASDOR, MOHD SHUKRI ABD SHUKOR, AFTAB KHAN, MOHD IZUAN
EFFENDI BIN HALMI, SITI ROZAIMAH SHEIKH ABDULLAH, NOR ARIPIN SHAMAAN,
MOHD YUNUS SHUKOR
238-246
Assesment of genetic diversity among soursop (Annona muricata) populations from
Java, Indonesia using RAPD markers
SURATMAN, ARI PITOYO, SRI MULYANI, SURANTO
247-253
Molecular phylogeny inferred from mitochondrial DNA of the grouper Epinephelus spp.
in Indonesia collected from local fish market
EDWIN JEFRI, NEVIATY P. ZAMANI, BEGINER SUBHAN, HAWIS H. MADDUPPA
254-263
Short communication: Microfungal diversity on leaves of Eusideroxylon zwageri, a
threatened plant species in Sarawak, Northern Borneo
A. LATEEF ADEBOLA, MUID SEPIAH, MOHAMAD H. BOLHASSAN, MANSOR WAN ZAMIR
264-268
Leguminicolous fungi associated with some seeds of Sudanese legumes
SOHAIR A. ABDULWEHAB, SAIFELDIN A. F. EL-NAGERABI, ABDULQADIR E. ELSHAFIE
269-280
Diversity in antioxidant properties and mineral contents of Allium paradoxum in the
Hyrcanian forests, Northern Iran
SEDIGHEH KHODADADI, TAHER NEJADSATTARI, ALIREZA NAQINEZHAD, MOHAMMAD
ALI EBRAHIMZADEH
281-287
Genetic and morphological diversity in Cousinia cylindracea (Asteraceae) populations:
Identification of gene pools
AMIR ABBAS MINAEIFAR, MASOUD SHEIDAI, FARIDEH ATTAR
288-294
Threats and conservation of Paris polyphylla an endangered, highly exploited medicinal
plant in the Indian Himalayan Region
ASHISH PAUL, PADMA RAJ GAJUREL, ARUP KUMAR DAS
295-302
Growth, development and morphology of gametophytes of golden chicken fern
(Cibotium barometz (L.) J. Sm.) in natural media
TITIEN NGATINEM PRAPTOSUWIRYO, DIDIT OKTA PRIBADI, RUGAYAH
303-310
Spatial point pattern analysis of the Sumatran tiger (Panthera tigris sumatrae) poaching
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FARID RIFAIE, JITO SUGARDJITO, YULI SULISTYA FITRIANA
311-319
Potential in bioethanol production from various ethanol fermenting microorganisms
using rice husk as substrate
WOOTTICHAI NACHAIWIENG, SAISAMORN LUMYONG, RONACHAI PRATANAPHON,
KOICHI YOSHIOKA, CHARTCHAI KHANONGNUCH
320-326
Review: Genetic diversity of local and exotic cattle and their crossbreeding impact on
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SUTARNO, AHMAD DWI SETYAWAN
327-354
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References Author-year citations are required. In the text give the authors
name followed by the year of publication and arrange from oldest to newest
and from A to Z. In citing an article written by two authors, both of them
should be mentioned, however, for three and more authors only the first author
is mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or
(Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and Nijs 2005; Balagadde et
al. 2008; Webb et al. 2008). Extent citation as shown with word "cit" should
be avoided. Reference to unpublished data and personal communication
should not appear in the list but should be cited in the text only (e.g., Rifai MA
2007, pers. com. (personal communication); Setyawan AD 2007, unpublished
data). In the reference list, the references should be listed in an alphabetical
order (better, if only 20 for research papers). Names of journals should be
abbreviated. Always use the standard abbreviation of a journal's name
according to the ISSN List of Title Word Abbreviations (www.issn.org/222661-LTWA-online.php). The following examples are for guidance.
Journal:
Saharjo BH, Nurhayati AD. 2006. Domination and composition structure
change at hemic peat natural regeneration following burning; a case study
in Pelalawan, Riau Province. Biodiversitas7: 154-158.
Book:
Rai MK, Carpinella C. 2006. Naturally Occurring Bioactive Compounds.
Elsevier, Amsterdam.
Chapter in book:
Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization,
biogeography, and the phylogenetic structure of rainforest tree
communities. In: Carson W, Schnitzer S (eds) Tropical Forest Community
Ecology. Wiley-Blackwell, New York.
Abstract:
Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th
annual symposium of the International Association for Vegetation
Science, Swansea, UK, 23-27 July 2007.
Proceeding:
Alikodra HS. 2000. Biodiversity for development of local autonomous
government. In: Setyawan AD, Sutarno (eds) Toward Mount Lawu
National Park; Proceeding of National Seminary and Workshop on
Biodiversity Conservation to Protect and Save Germplasm in Java Island.
Sebelas Maret University, Surakarta, 17-20 July 2000. [Indonesian]
Thesis, Dissertation:
Sugiyarto. 2004. Soil Macro-invertebrates Diversity and Inter-Cropping Plants
Productivity in Agroforestry System based on Sengon. [Dissertation].
Brawijaya University, Malang. [Indonesian]
Information from internet:
Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake
SR, You L. 2008. A synthetic Escherichia coli predator-prey ecosystem.
Mol Syst Biol 4: 187. www.molecularsystemsbiology.com
Front cover: Monotropa uniflora
(PHOTO: SIMON EADE)
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ISSN: 1412-033X
E-ISSN: 2085-4722