Molecular biogeography of the
Bulbophyllum lobbii Lindl. complex
(sect. Sestochilos, Orchidaceae)
in Southeast Asia
Dissertation for obtaining the
Master’s degree in Botany/Plant Biology
at the
Department of Organismic Biology
AG Ecology and Diversity of Plants
Paris-Lodron-University of Salzburg
Presented by
Gerald Hochschartner Bakk. Biol.
Salzburg, August 2006
Dedicated to
Christine, Christine and Simona
Photo at the front page:
Bulbophyllum facetum (left) and Bulbophyllum dearei (right)
Index of contents
Index of contents
1. Introduction
1.1 Paleogeological background
3
5
1.2 Study system
12
1.3 Study questions
20
2. Materials and methods
22
2.1 Plant material
22
2.2 DNA extraction
23
2.3 DNA amplification and sequencing
23
2.4 AFLP fingerprinting
24
2.5 Molecular analyses
26
2.5.1 ITS sequence analyses
2.5.1.1 Phylogenetic reconstructions and molecular dating
26
26
2.5.1.2 Lineages-through-time (LTT)
and diversification analyses
28
2.5.1.3 DIVA (dispersal-vicariance analysis)
29
2.5.2 CpDNA sequence analysis
30
2.5.3 AFLP data analysis
32
2.5.4 Partition homogeneity test
33
2.6. Morphological analysis
33
2.7 Documentation
36
3. Results
3.1 Molecular analyses
37
37
3.1.1 Partition homogeneity test
37
3.1.2 Phylogenetic analyses of ITS sequences
37
3.1.3 Lineages-through-time (LTT) plot of ITS data
47
3.1.4 Phylogenetic analyses of cpDNA sequences
48
3.1.5 AFLP analyses
55
3.1.6 Geographical distribution of AFLP clades/species
59
1
Index of contents
3.1.7 Combined ITS/AFLP analysis
63
3.1.8 Dispersal-vicariance analysis (DIVA)
66
3.2 Morphological analysis
4. Discussion
4.1 Monophyly of the Bulbophyllum lobbii complex
70
77
77
4.2 Delimitation of major clades within
the Bulbophyllum lobbii complex
4.3 On the role of hybridization within the B. lobbii complex
78
79
4.4 Putative ancestral area and age
of the Bulbophyllum lobbii complex
80
4.5 Diversification of the B. lobbii complex, evolutionary
processes and historic-biogeographical scenario
81
4.6 Yellow vs. red flower variation within the B. lobbii complex
83
4.7 The species within the B. lobbii complex
83
5. Conclusion
87
6. Abstract
88
7. Literature cited
90
8. Appendix
101
9. Colour plates of the B. lobbii complex
and extra-complex species
110
Acknowledgements
124
Curriculum vitae
125
2
Introduction
1. Introduction
Oceanic islands such as Hawaii or the Canaries, which have never been
connected to any continental landmass provide opportunities for the study of
speciation
(Darwin,
1859;
Carlquist,
1974).
For
example,
recently
phylogenetic studies of species which are endemic to such islands, and their
relatives allow inferring the geographical origin and routes of colonisation of
the island taxa (Takayama et al., 2005). Because oceanic islands are
relatively simple systems where both patterns of dispersal and natural
selection can be assessed more easily than in continental systems (e.g.
small and new habitats force species fast to adaption or extinction due to
biotic and abiotic factors), many molecular studies concerning speciation and
colonisation of plant organisms of such isolated locations were published
recently (e.g. Francisco-Ortega et al., 1996; Takayama et al., 2005).
By contrast, there is only very little known about differentiationprocesses of natural plant populations on continental island systems.
Continental islands developed recently by rising sea level and, thus, became
disconnected from each other and the mainland. Similar to oceanic islands,
continental ones can provide insights into colonization and radiation of
species. However, continental islands also provide unique systems for
studying the effects of past fragmentation provided the level of over-sea
dispersal is sufficiently low (Bittkau & Comes, 2005). In this context, the IndoMalay Archipelago (the island system between mainland Indochina and
Australia) in Southeast Asia seems to be particularly suited for phylogenetic
and phylogeographic surveys. This archipelago is one of the main centres of
biodiversity worldwide, likely due to its complex geological and climatic
history and ecological diversity, including the presence of large rainforests
(Bänfer et al., submitted).
The Sunda Islands (mainly Borneo, Sumatra and Java), which are part
of the archipelago and lie on a shallow continental shelf (Sundashelf; Fig.
1.3), were periodically connected with each other and the mainland
(Indochina) in the recent history (Pliocene and Pleistocene) and, thus, recent
fragmentation of populations could have contributed to speciation in this
region.
While
the
importance
of
these
intermittent
connections/
3
Introduction
fragmentations of the Sunda islands and the mainland of SE Asia to the
evolution of the Sundashelf biota has been studied in detail for some animal
groups (Heaney, 1986; Schmitt et al., 1995; Ruedi & Fumagalli, 1996; Ruedi,
1996; Karns et al., 2000; Inger & Voris, 2001; Gorog et al., 2004, Lourie et
al., 2005) only a few studies concerning plants are available (Ridder-Numan,
1998; Cannon & Manos, 2003; Bänfer submitted).
The current distribution of animal species in Southeast Asia, especially
in the Indo-Malay Archipelago, is thought to be the result of widespread
faunal migration across the Sundashelf during Pleistocene glacial maxima.
There is a correlation between the current distribution of mammal species
and the boundaries of land areas defined by Pleistocene sea levels (Heaney,
1986; Koopman, 1989), reflecting dispersals over land bridges (Gorog et al.,
2004). However, there are also some studies which provide evidence for
migrations (from Indochina to Sundashelf and vice versa) in the prePleistocene (Schot, 1998; Ridder-Numan, 1998; Gorog et al., 2004), i.e. at
times when also connections between the Sunda Islands and the mainland
(Indochina) existed (Hall, 1998; Gorog et al., 2004).
In this study, I tested the hypothesis of Pleistocene speciation by
focusing on the phylogenetic relationships and divergence times of
submontane to montane-rainforest-restricted orchids (Bulbophyllum lobbii
complex, c. 11 species) from the Sundashelf region (Peninsula Malaysia,
Sumatra, Java, Borneo), the Philippines, Sulawesi and adjacent mainland
areas (Thailand, Myanmar), using nuclear rDNA internal transcribed spacer
(ITS) sequences, chloroplast (cp) DNA sequences, AFLP (amplified fragment
length polymorphism) markers and morphometric analysis.
The ITS region has been widely used in phylogenetic studies of
angiosperms at the species level (Bortiri et al., 2001; Schultheis, 2001). The
substitution rate of cpDNA is generally known to be lower than that of ITS
(Wolfe et al., 1987; Small et al., 1998), so that the elucidation of phylogenetic
relationships below generic level is often difficult (Gielly & Taberlet, 1994;
Sang et al., 1997). However, some intergenic spacer regions of the cpDNA
were used successfully for analysing inter- and intraspecific relationships
(Taberlet et al., 1991; Gielly and Taberlet, 1994; Fujii et al., 1997; Ohsako
and Ohnishi, 2000; Soejima & Nagamasu, 2004). The AFLP technique is
4
Introduction
known to resolve phylogenetic relationships between closely related taxa
(Vos et al., 1995; Wolfe & Liston, 1998; Mueller & Wolfenbarger, 1999;
Ritland & Ritland, 2000; Guo et al., 2005).
As the paleogeological history of Southeast Asia is very complex, here
I give a brief review, because the spatial and temporal distribution and
diversification of species/intraspecific lineages studied might have been
influenced by climatic and tectonic events in the area (Avise, 1994; Avise,
2000; Lourie et al., 2005).
1.1 Paleogeological background
The tetonic history of Southeast Asia shows that the collision of six major
plates (Pacific, Australian, Eurasian, Indian, Indian Ocean and Philippines)
resulted in two waves of Australian microplates moving towards the Eurasian
plate. The oldest microplates formed most of Southeast Asia and the western
part of the Malay Archipelago (up to the Wallace line) approximately 90
million years ago (Mya). The second wave started approximately 15 millions
years ago and has recently formed stepping stones between the Australian
and Eurasian plates (v. Welzen et al., 2003).
Eocene to Oligocene
The Peninsula Malaysia and the major islands of Sumatra, Borneo and Java,
which lie on the shallow continental Sundashelf, existed as a continental
block with Indochina from the early Eocene (approximately 50 Mya) to the
late Oligocene (approximately 25 Mya) (Gorog et al., 2004). The Izu-BoninMariana (IBM; see Fig. 1.1) Arc was formed by outpouring of intra-oceanic
volcanic rocks (Stern and Bloomer, 1992) caused by the subduction of the
Pacific-Northern New Guinea ridge (about 40 Mya). At the same time, the
Philippine Sea plate became a recognizable entity and the West Philippine
basin, Celebes Sea, and Makassar Strait opened as a single oceanic basin
within the Philippine Sea plate.
5
Introduction
Mid-Oligocene to Miocene
In the mid Oligocene (approximately 30 Mya) the global sea level fell about
250 m below present (Haq et al., 1987; Hutchinson, 1989) and thus very
large areas of the Sundashelf were exposed. The climate at that time was
probably tropical monsoon (Ridder-Numan, 1998). The north Australian
margin came into contact with Sulawesi and the Halmahera Arc about 25
Mya, which resulted in a discontinous land connection between the
Halmahera and the Philippine Arcs and Sulawesi (Hall, 1998). Java and
Sumatra were submerged from the late Oligocene (about 25 Mya) until the
Mid-Miocene (approximately 15 Mya; Gorog, et al., 2004).
Early Miocene
At about 20 Mya (Fig. 1.1) the deep water passage between the Indian and
the Pacific Ocean was closed (Kennett et al., 1985) and central Borneo was
uplifted. There was probably always some land in the area of Sulawesi from
that time on. Although there was no direct land connection between Sulawesi
and both Australia and the Sundashelf probably numerous islands
periodically emerged within the broad shallow water zone separating those
areas (Hall, 1998). The northern Makassar Strait (Celebes Sea), a deep
water area northwest of Sulawesi, remained and formed probably a migration
barrier (Wallace line; Fig 1.1) for many plants and animals (Moss & Wilson,
1998). Australia continued moving northward and caused the counter-clock
rotation of Borneo. This rotation was accompanied by the counter-clockwise
motion of West Sulawesi and the adjacent Sundaland blocks. The northern
Peninsula Malaysia rotated clockwise, but remained linked to the southern
Peninsula Malaysia and Indochina and thus widened the basins of the Gulf of
Thailand. North Sumatra rotated counter-clockwise with south Peninsula
Malaysia. There was a period of high sea level of about 220 m above present
sea level, and also of warm and moist climate conditions, in the latest part of
the early Miocene and the early part of the middle Miocene, which allowed
the proliferation of diverse mangroves. Under a mixed warm climate
paratropical forests (palms, mangroves and other tropical families) flourished
and many species migrations may have occurred (Morley, 1998; RidderNuman, 1998).
6
Introduction
Fig.1.1: Postulated distribution of land and sea in SE Asia at 20 Mya. The deep water
passage between the Pacific and the Indian Ocean was closed, central Borneo was uplifted
and permanent land was in the area of Sulawesi from that time on (Hall 1998).
Mid-Miocene to Pliocene
At approximately 10 Mya, Southeast Asia had obtained its largely
recognizable modern formation. The rotation of Borneo was completed and
the collision of the Sulu platform (Sulu pf.; see Fig. 1.2) with the eastern arm
of Sulawesi caused the rotation of the eastern and northern arms of Sulawesi
to their present position.
Between 15 Mya and 5 Mya the area of the emerged Sundashelf was
reduced and deep marginal basins in the east were eliminated or reduced in
size. More of Borneo emerged and the central mountains along the
Kalimantan-Sarawak border (extending into Sabah) widened and were
further uplifted. There was already some highland in Sumatra and Sulawesi
in the Early Pliocene, i.e. at about 5 Mya (Fig. 1.2., Hall, 1998). While the sea
level had dropped up to 220 m below the present sea level in the late
Miocene, it rose up again to 140 m above present sea level in the Pliocene
(Ridder-Numan, 1998).
7
Introduction
There were periods of drier climates from the end of the Middle
Miocene to the Late Miocene, and also in the Plio-Pleistocene. At the
beginning of the Pliocene a seasonal climate ((semi)arid winter, (semi)humid
summer) had developed in Java (Morley, 1998).
Fig. 2: Postulated distribution of land and sea in SE Asia at 5 Mya. SE Asia was largely
recognizable in its present form. Rotation of Borneo was completed and the collision of the
Sulu platform with Sulawesi led to its present position. The mountains of Borneo were
extended, and highland was recognizable in Sumatra and Sulawesi (Hall, 1998). IBM Arc =
Izu-Bonin-Mariana Arc; Sulu pf. = Sulu platform; C. S. = Celebes Sea.
Pleistocene
During the Pleistocene interglacial-periods rising sea-levels periodically
converted mountains into geographically isolated islands, creating ideal
conditions for speciation. Moreover, through lowering of the sea-level during
glacials, the islands of the Sundashelf were repeatly connected with the
Asian mainland and formed a giant block, enabling biotic migrations from the
mainland to the archipelago (Sodhi et al., 2004). At least twice during the
period of the last 250000 years, most recently about 17000 years ago, the
sea level dropped 120 m (Fig. 1.3) for approximately 15000 years in total
8
Introduction
time (Chappell & Shackleton, 1986) exposing an additional land area south
and east of the Isthmus of Kra (along the border of Malaysia and Thailand;
Fig. 1.3) of 1.53 square km, which resulted in a newly connected area of the
Sundashelf (including Sumatra, Java and Borneo) of 3.2 million square km
(Voris, 2000). 190000 years ago, the once continuous subcontinental
rainforest of SE Asia fragmented due to a glacial drought, expanded
afterwards and re-contradicted again during a second drought about 80000
years ago. Rainforest refugia survived in North Sumatra, North Borneo, West
Java, Northeast Indochina and Southern India (Fig. 1.3). Rainforest
communities on islands such as the Philippines and Sulawesi were probably
been entirely eliminated, leaving severely degraded forest or no forest
(Brandon-Jones, 1998).
Fig. 1.3.: Map of SE Asia at about 80000 years ago, when sea level was 120 m below
present. The major Sunda islands (Borneo, Sumatra and Java) were connected via the
Peninsula Malaysia with the SE Asian mainland. Putative rainforest refugia in West Java,
North Sumatra, North Borneo and Northeast Indochina are indicated (green line).
The
black
line
encircles
regions
in
the
Philippines
and
Sulawesi,
where
rainforest communities were degraded or eliminated. Base map was downloaded
and
belongs
to
the
Field
Museum
of
Natural
History,
Chicago,
Illinois.
(http://fmnh.org/research_collections/zoology/zoo_sites/seamaps/).
9
Introduction
About 17000 years ago the Balabac Strait between Borneo and Palawan,
which were not connected by land, was reduced to a width of 12 km. At that
time a narrow deep ocean trench (Makassar Strait) still separated Sulawesi
and Borneo. Until 9500 years ago the major Sunda islands (Borneo,
Sumatra, Java) remained connected with the Peninsula Malaysia and
Indochina (due to a drop in sealevel about 50 m below today). At this time,
the ancient North Sunda River, which had probably provided gallery forest
corridors between Sumatra and Borneo, was largely eliminated (Voris, 2000).
Borneo and Sumatra were finally separated by the Karimata Straits which
formed about 7000 years ago (Moss & Wilson, 1998). The present sea level
was attained approximately 6000 years ago (Fairbanks, 1989; Voris, 2000).
The climate of Southeast Asia at the time of the last glacial maximum
(approximately 17000 years ago) was drier and more seasonal than today
(Whitmore, 1981; Morley & Flenley, 1987; Heanny, 1991; Dawson, 1992;
Shackleton, 1994; Voris, 2000). Rainforest was likely reduced by drought (as
evidenced by plant fossil data, zoological and sedimentological information;
Ray & Adams, 2001; Fig. 1.4), but this reduction was not as dramatic as
about 80000 years ago, due to the slower decrease in temperature (BrandonJones, 1998; see also Kershaw et al., 2001). Grassland plants, characteristic
of savanna or steppe vegetation (Verstappen, 1992, 1997; Morley, 1998,
2000; van der Kaars et al., 2001; Gorog et al., 2004) were predominant in the
emergent paleolandscape. It is feasible that tropical rain forests and
margrove swamps dominated at least the central southern region of the shelf
(Sumatra, Borneo), Sulawesi and on Mindanao/Philippines (Sun et al., 2000;
Kershaw et al., 2001; Ray & Adams, 2001; Gorog et al., 2004). Thailand,
Peninsula Malaysia as well as some parts of the Philippines (Luzon,
Palawan) were covered by monsoon forests and tropical woodland was in the
south of Borneo. Furthermore, tropical montane rainforests have existed on
Luzon, Mindanao, Borneo, Sulawesi, Sumatra and in Thailand (Ray &
Adams, 2001; Fig. 1.4).
10
Introduction
Fig. 1.4: Gis-based vegetation map of Eurasia at the Last Glacial Maximum. Tropical
rainforests dominated the southern core of the Sundashelf (Sumatra and Borneo), Sulawesi
and Mindanao. Thailand, Peninsula Malaysia as well as some parts of the Philippines
(Luzon, Palawan) were covered by monsoon forests, whereas tropical woodland existed in
the south of Borneo and tropical montane rainforests subsisted on Luzon, Mindanao,
Borneo, Sulawesi, Sumatra and in Thailand (Ray & Adams, 2001).
11
Introduction
1.2 Study system
Bulbophyllum lobbii Lindl. belongs to section Sestochilos (Breda) Benth. &
Hook.f. which is currently under revision by Dr. Jaap Jan Vermeulen
(Nationaal Herbarium Nederland, University of Leiden, The Netherlands;
personal communication). Most species in this section (c. 40-50 species,
distributed from Nepal via Indochina and the Sundashelf to the Philippines
and New Guinea, and in China; Seidenfaden, 1990) are large epiphytes with
a creeping rhizome and conspicuous pseudobulbs, each of which bears a
single leaf (Comber, 1990). Those species with more than one flower have a
subumbellate or racemose inflorescence. Furthermore, section Sestochilos
differs from other sections of Bulbophyllum (c. 2000 species) by having larger
petals (more than half as long as sepals) rather than smaller ones (section
Ephippium); moreover, petals are glabrous rather than ciliate (section Hirtula)
and
the
pseudobulbs
are
distinct
rather
than
indistinct
(section
Aphanobulbon; Seidenfaden, 1992). Furthermore, the chambers of the
pollinia of section Sestochilos are separated by a membrane-like structure,
which is distinct from the outside cap. Most flowers of this section have a
pleasant scent, but some smell foul. These various smells attract various
kinds of flies, the predominant pollinators (Comber, 1990), which transfer the
pollinarium (see below) to another flower of likely the same fragance.
Interestingly, during pollination male flies aquire chemical components by
licking on the floral tissues and biotransform them into female-attracting
pheromones (B. cheiri: Nishida et al., 2004). In addition to attraction of
pollinators by smell, the lip may also play an essential role. Some authors
suggest that quick lip movements, triggered by wind, mimic flies shaking their
wings and so attract pollinators (Barth, 1985; Meve & Liede, 1994; Borba &
Semir, 1998). Besides the visual attraction, movement of the lip is also very
important for pollination. This mechanism is well described for B.
macranthum (Ridley, 1890). When a fly lands on the hinged lip of the orchid,
the lip moves down so that the fly has to crawl up. It passes the balance point
of the lip and, thus, the lip returns to its initial position, pressing the fly against
the column/pollinia chambers, whereby the pollinarium is attached to the fly
(Teixeira, 2004).
The first knowledge of Bulbophyllum lobbii goes back to the year 1827
12
Introduction
when Breda described and illustrated one of Kuhl & v.Hasslet’s Javanese
plants and referred to it as Sestochilos uniflorum Breda (Breda, 1827). In
1847 Lindley established Bulbophyllum lobbii on the basis of a Javanese
collection by William Lobb (Lindley, 1847). Thereby he evidently overlooked
Breda’s earlier description of the same species, because he did not use the
specific epithet uniflorum. As the name Bulbophyllum uniflorum was and is,
respectively, used for another species the name Bulbophyllum lobbii Lindl.
has outlived till today. Different authors have added a series of names in
synonymy in many publications (Table 1.1). Nowadays the name
Bulbophyllum lobbii, which is used in herbaria and in the literature, covers a
whole series of plants that might be recognized as separate species. It is also
possible that some synonyms used in the literature are based on
misidentifications (Seidenfaden, 1997).
The current taxonomy of the Bulbophyllum lobbii complex (as inferred
from a detailed literature survey as well as personal communications of
taxonomic experts (Dr. J. J. Vermeulen, G. Fischer, A. Sieder)) is listed
below (Table 1.1). Eleven species (B. cameronense, B. coweniorum, B.
dearei, B. facetum, B. lobbii, B. microglossum, B. orectopetalum, B.
polystictum, B. siamense, B. smitinandii and B. sumatranum) are assigned to
the B. lobbii complex, whereby their phylogenetic relationships among each
other and to closely related taxa remain illusive. The distribution and habitats
of the various species and of the taxa used as outgroup is summarized in
Table 1.2. Within the B. lobbii complex six species are geographically
restricted (B. cameronense, B. polystictum and B. microglossum: Cameron
Highlands/Peninsula Malaysia; B. facetum: Luzon/Philippines; B. smitinandii:
Khao Luang/Thailand, B. sumatranum: Sumatra) and five species are more
or less widely distributed (B. coweniorum: Laos, Luzon/Philippines; B. dearei:
Philippines, Borneo, Peninsula Malaysia; B. lobbii: Peninsula Malaysia,
Borneo, Sumatra, Java, Bali, Philippines, Sulawesi; B. orectopetalum and B.
siamense: Assam, Thailand, Myanmar (Burma)). Figure 1.5 shows the
distribution of the 11 species belonging to the B. lobbii complex.
All species of the B. lobbii complex are restricted to (sub)montane
rainforests, growing almost exclusively on trunks and main branches of trees
(rarely terrestial on limestone; B. dearei) and require more or less shade and
13
Introduction
humidity. All species of the complex as well as of the outgroup taxa are
diploid (2n = 2x = 38), except B. patens which is triploid (3x = 57; Lim &
Jones; 1981; Lim, 1985). Similar to other members of the Orchidaceae, the
cpDNA should be inherited maternally (Trapnell & Hamrick, 2004). Although
most taxa within the complex are interfertile but not selfcompatible, and form
capsules after artificial crossings (greenhouse experiments; G. Fischer), no
evidence of natural hybridisation between the species was found in the
literature.
14
Introduction
Table 1.1: Current taxonomy of the Bulbophyllum lobbii complex. Eleven species are currently recognized and their synonyms are indicated together
with their first description and their current status in the taxonomic literature.
Taxon name
First description
Current status (reference)
B. cameronense Garay, Hamer & Siegerist
Garay et al., 1996
accepted (Garay et al., 1996)
B. coweniorum J. J. Verm. & P. O`Byrne
Vermeulen & O'Byrne, 2003
accepted (Vermeulen & O'Byrne, 2003)
B. dearei (Hort.) Rchb. f.
Reichenbach, 1888
accepted (Seidenfaden & Wood, 1992)
B. godseffianum Weathers
Weathers, 1890
synonym (Vermeulen, 1991)
B. goebelianum Kraenzl.
Kraenzlin, 1921
synonym (Vermeulen, 1991)
B. punctatum Ridl.
Ridley et al., 1908
synonym (Vermeulen, 1991)
B. reticosum Ridl.
Ridley, 1893
synonym (Seidenfaden & Wood, 1992)
Sarcopodium dearei Hort.
Hort, 1883
synonym (Wood & Cribb, 1994)
B. facetum Garay, Hamer & Siegerist
Garay et al., 1996
accepted (Garay et al., 1996)
B. lobbii Lindl.
Lindley, 1847
accepted (Comber, 2001)
B. bataanense Ames
Ames, 1905
synonym (Vermeulen, 1991)
B. claptonense Rolfe
Rolfe, 1905
synonym (Vermeulen, 1991)
B. henshallii Lindl.
Lindley, 1852
synonym (Comber, 1990)
B. lobbii Lindl. var. colosseum Hort
Hort, 1905
synonym (Vermeulen, 1991)
B. lobbii Lindl. var. henshallii (Lindl.) Henfrey
Henfrey, 1852
synonym (Vermeulen, 1991)
B. lobbii Lindl. var. nettesiae Cogn.
Cogniaux, 1901
-
Phyllorchis lobbii (Lindl.) Kuntze
Kuntze, 1891
synonym (Vermeulen, 1991)
Sarcopodium lobbii (Lindl.) Lindl. & Paxt.
Lindley, & Paxton, 1850
synonym (Vermeulen, 1991)
Sestochilos uniflorum Breda
Breda, 1827
synonym (Vermeulen, 1991)
Ridley, 1908
accepted (Seidenfaden & Wood, 1992)
B. microglossum Ridl.
Synonym
15
Introduction
B. orectopetalum Garay, Hamer & Siegerist
Garay et al., 1992
accepted (Seidenfaden, 1997)
B. polystictum Ridl.
Ridley, 1909
accepted (Seidenfaden, 1997)
B. siamense Rchb. f.
Reichenbach, 1867
accepted (Seidenfaden, 1997)
B. smitinandii Seidenf. & Thorut
Seidenfaden & Thorut., 1996
accepted (Seidenfaden, 1997)
B. lobbii Lindl. var. siamense (Rchb. f.) Rchb.f. Reichenbach & Saunders, 1882 synonym (Seidenfaden, 1997)
B. sumatranum Garay, Hamer & Siegerist
B. lobbii Lindl. var. breviflorum J. J. Sm.
Garay et al., 1996
accepted (Garay et al., 1996)
Smith, 1908
synonym (Garay et al., 1996)
Table 1.2: Distribution and habitats of the Bulbophyllum lobbii complex and related taxa of section Sestochilos used as outgroup (Vermeulen, 1991;
Seidenfaden & Wood, 1992; Garay et al., 1996; Seidenfaden, 1997; Vermeulen & O'Byrne, 2003). *personal observation;
** Vermeulen personal communication.
Bulbophyllum taxa
Distribution
Habitat description
Peninsula Malaysia
Submontane and montane forests on
B. lobbii complex
B. cameronense
trunks and main branches of trees
B. coweniorum
Laos, Philippines*
Intermediate to warm growing epiphyte in
submontane and montane forests
B. dearei
Philippines, Borneo, Peninsula Malaysia
Unifoliate epiphyte and occasional
lithophyte in submontane and montane
forests on bare trunks of large trees or in
forests on limestone
B. facetum
Endemic to Philippines in the montainous
On tree trunks in shady locations in
region of Luzon
submontane and montane forests
16
Introduction
B. lobbii
B. microglossum
Peninsula Malaysia, Borneo, Sumatra, Java, Submontane and montane forests on
Bali, Philippines, Sulawesi**
trunks and main branches of trees
Peninsula Malaysia
Submontane and montane forests on
trunks and main branches of trees
B. orectopetalum
Assam, Thailand, Myanmar (Burma),
Warm to cool growing epiphyte in
submontane and montane forests
requiring light shade
B. polystictum
Peninsula Malaysia
Intermediate to warm growing epiphyte in
submontane and montane forests that
needs a slight winter rest
B. siamense
Assam, Thailand, Myanmar (Burma)
Intermediate to warm growing epiphyte in
submontane and montane forests
B. smitinandii
Endemic to Thailand (foothills of Khao
Moderately warm growing epiphyte in
Luang, Nakorn Sritammarat)
submontane and montane forests which
needs half shade
B. sumatranum
Sumatra
Warm to cool growing epiphyte in
submontane and montane forests that
requires shade, good drainage and high
humidity
Used outgroup taxa
B. affine Lindl.
Indian Himalaya, Nepal, Sikim, Bhutan,
Evergreen lowland forests
Assam, Laos, Thailand, Vietnam, China,
Ryukyu
17
Introduction
B. emiliorum Ames & Quisumb.
Philippines, New Guinea, Papua Guinea
On tree trunks in open semi-deciduous
forests
B. hamatipes J. J. Sm.
Endemic to Java
On trunks and main branches of isolated
trees in grass land
B. macranthum Lindl.
Burma, Vietnam, Thailand, Peninsula
Occasionally in Lower montane forests
Malaysia, Sumatra, Java, Bangka, Bali,
Borneo, Philippines, New Guinea
B. membranifolium Hook. f.
B. patens King ex Hook. f.
B. piestoglossum J. J. Verm.
Peninsula Malaysia, Sumatra, Borneo,
Podzolic forests, swampy forests, mixed
Philippines
montane forests, forest on ultrabasic soil
Thailand, Peninsula Malaysia, Borneo,
On tree trunks in lowland swamp forests,
Sumatra, Bangka, Biliton, Java
avoiding detritus
Endemic to Philippines
Hot to cool growing epiphyte on tree
trunks
B. hamatipes J. J. Sm.
Endemic to Java
Warm to cool growing epiphyte
B. monanthum (Kuntze) J. J. Sm.
Thailand, Vietnam
Warm to cool growing epiphyte
B. pileatum Lindl.
Peninsula Malaysia, Borneo, Sumatra
Swamp forests on mature mangrove
trees as a miniature sized, hot to warm
growing epiphyte
B. alsiosum Ames
Philippines
Warm to cool growing epiphyte
18
Introduction
Fig. 1.5: Distribution of species of the Bulbophyllum lobbii complex (see also Table 1.2).
Note that all species are restricted to (sub)montane rainforests. Turquoise = B. siamense, B.
orectopetalum; dark blue = B. microglossum, B. cameronense, B. polystictum; pink = B.
sumatranum; light blue = B. dearei; light green = B. coweniorum; dark green = B. facetum;
violet = B. smitinandii; black = B. lobbii sens lat.. Base map adapted from the maps of the
University of Texas, Austin, USA (www.lib.utexas.edu/maps/asia.html).
19
Introduction
1.3 Study questions
1) The taxonomic position and the monophyly, respectively, of the
Bulbophyllum lobbii complex within the section must be clarified.
2) The temporal and spatial origin of this orchid group should be
determined. Answering these two questions is accomplished by the
sequencing of the ITS (internal transcribed spacer) region of nuclear
ribosomal DNA and the interpretation of the phylogenetic trees
(maximum
parsimony
and
maximum
likelihood)
under
the
assumptions of a molecular clock. Furthermore, a dispersal-vicariance
analysis should help to reconstruct the putative ancestral areas of the
B. lobbii complex and the various clades/species, respectively.
3) By means of molecular markers (ITS and cpDNA sequences, AFLPs)
and by using morphometric analyses, the relationships between the
various clades/species within the B. lobbii complex should be
identified. While ITS and cpDNA markers should elucidate the
relationships along more or less general lines (relationships between
various species and well defined clades, respectively) using maximum
parsimony, distance and maximum likelihood analyses, fast evolving
DNA-fingerprint-markers (AFLPs) should give deeper insights into
more shallow inter- and intraspecific relationships (by the means of
distance and principal coordinate analyses) of the B. lobbii complex.
The morphological analyses (principal component analyses) should be
compared to the molecular data, examining whether the morphological
variablility reflects the genetic one. Taking all these data together a
relative clear picture of the relationships of species/clades within the
B. lobbii complex should be obtained.
4) The onset of diversification of the B. lobbii complex (also that of the
various species/clades) and the corresponding important evolutionary
processes
should
be
discovered.
Additional
to
the
methods
(mentioned above), a lineage-through-time plot and statistical tests
(e.g. gammastatistic; Pybus & Harvey; 2000) might help to answer
20
Introduction
these questions.
5) On the basis of these data a historic-biogeographical scenario for the
B. lobbii complex should be reconstructed.
6) The B. lobbii complex comprises plants with red and yellow flowers.
With the aid of the methods mentioned above it should be possible to
test the hypotheses about a single or multiple origin of either of these
phenotypic variants.
7) Although further taxonomic work is certainly necessary, the molecular
and morphological data presented here, along with personal
observations, should help clarifying how many and which species
actually belong to the B. lobbii complex.
21
Material and Methods
2. Materials and methods
2.1 Plant material
Most of the examined plants of the Bulbophyllum lobbii complex were
collected in Southeast Asia and cultivated in the glasshouses of the Botanical
Gardens at Salzburg and Vienna. Some samples were received as silicagel
dried leaf material from the Botanical Gardens of Singapore and Leiden. The
sources of the samples are detailed in Table 7.1 (appendix) and the sampling
localities for each species/accession are shown in Figure 2.1.
Fig. 2.1: Sampling localities. See also Table 1 for specific locality information. Base map
adapted from online map creation (http://www.aquarius.geomar.de/).
= B. lobbii (l); V = B.
dearei (d); U = B. siamense (sia); « = B. smitinandii (smi); ¹ = B. facetum (fac), B.
coweniorum (cow); ¹ = B. sumatranum (sum); = B. cameronense (cam), B. microglossum
(mic); B. polystictum (pol); = B. orectopetalum (ore). Accessions of the widespread taxa
(B. lobbii and B. dearei) are numbered (identified in Table 7.1). B. facetum, B. coweniorum,
22
Material and Methods
B. cameronense, B. microglossum and B. polystictum (5, 5, 4, 4 and 3 accessions,
respectively) are mapped with a single symbol without accession numbers. Accessions of B.
orectopetalum and B. sumatranum (6 and 7 accesssions, respectively), for which no exact
location (only area) information was available, are mapped on their area with a single symbol
without numbers. Accessions for which no (B. lobbii (16, 20, 24, 31); B. dearei (5)) or only
coarse (B. lobbii (29, 30); “Indonesia”) locality information was available were not mapped.
2.2 DNA extraction
Total DNA was extracted from either silica-dried or fresh leaves. Leaf tissue
was pulverized to a fine powder in a grinding mill using liquid nitrogen.
Because extracts from Bulbophyllum leaves contain high amounts of
polysaccharides, prior to DNA extraction the leaf powder was washed using
sorbitol extraction buffer (100 mM Tris-HCL pH 8.0, 0.35 M sorbitol, 5 mM
EDTA pH 8.0). Total DNA was isolated from a washed leaf pellet using the 2x
CTAB (hexadecyltrimethyl ammonium bromide) extraction method (Doyle &
Doyle, 1987) and purified using PCI (phenol-chloroform-isoamylalcohol;
25:24:1) and CI (chloroform-isoamylalcohol; 24:1). After precipitation with
cooled isopropanol and subsequent centrifugation, the DNA pellet was
washed 2 times in 70% ethanol and air-dried for at least 30 min. This was
followed by resuspension in TE buffer and incubation at 37 °C for 30 min with
RNase. DNA was quantified using a Smart SpecTMPlus photometer (Biorad)
and stored at –20°C.
2.3 DNA amplification and sequencing
The complete ITS (ITS1, 5.8S rDNA, ITS2) region of nuclear ribosomal DNA
and 8 regions of the chloroplast genome were PCR (polymerase chain
reaction)-amplified and sequenced. The cpDNA regions included: trnL-F (trnL
5’exon, intron, 3’-exon, intergenic spacer (IGS) and trnF-gene), trnH-psbA
(trnH-gene, IGS, psbA-gene), trnD-trnT (trnD-gene, IGS, trnY-gene, IGS,
trnE-gene, IGS, trnT-gene), trnD-E (trnD-gene, IGS, trnY-gene, IGS, trnEgene), trnS-trnfM (trnS-gene, IGS, psbZ-gene, IGS, trnG-gene, IGS, trnfMgene), trnL-ndhJ (trnL 3’-exon, IGS, trnF-gene, IGS, ndhJ-gene), rpoB-trnC
(rpoB-gene, IGS, trnC-gene), trnT-trnL (trnT-gene, IGS, trnL 5’-exon) and
trnS-trnG (trnS-gene, IGS, trnG 5’-exon). The following additional cpDNA
23
Material and Methods
regions could not be amplified: trnY-trnT (trnY-gene, IGS, trnE-gene, IGS,
trnT-gene), trnS-trnT (trnD-gene, IGS, rpS4-gene, IGS, trnT-gene), trnS-trnL
(trnD-gene, IGS, rpS4-gene, IGS, trnT-gene, IGS, trnL 5’-exon), rpL16 Intron
(rpL16 3’-exon, intron, rpL16 5’-exon) and rpS16 Intron (rpS16 3’-exon,
intron, rpS16 5’-exon) (see Table 7.2 in appendix for primer information).
Amplifications were carried out in a volume of 50 µl, containing 1 µl
(10-30 ng/µl) of genomic DNA, 5 µl F-Taq polymerase buffer (10x), 8 µl
MgCl2 (25 mM), 1 µl BSA (20 mg/ml), 1 µl dNTP mix (10 mM of each dNTP),
1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 0.25 µl F-Taq
polymerase (Fermentas; 5 U/µl) and 7.45 µl purified water (see also Table
7.3). Reactions were carried out in either a GeneAmp®PCR-System 2700
(Applied Biosystems) or a MyCycler (Biorad) and using the programmes
described in Table 7.4.
PCR products were purified using Cleaning Kit (Promega) and
quantified
by
means
of
a
Universal-Hood
II
gel-electrophoresis
documentation system (Biorad) and a Smart SpecTMPlus photometer
(Biorad). For sequencing 500 ng or 150 ng of purified PCR product was dried
in an Univapo 150 ECH vacuum centrifuge (Uniequip) and sent, together with
10 pmol or 100 pmol of each sequencing primer to Macrogen (Seoul, Korea)
and MWG (Munich, Germany), respectively, for cycle sequencing and the
detection of reaction products on automatic sequencers (ABI 3730xl DNA
Analyzer and LI-COR 4000 sequencer, respectively). All primers used for
amplification and/or sequencing (singly in only forward, or in forward and
reverse reactions) are listed in Table 7.2.
2.4 AFLP fingerprinting
Because of the low level of nucleotide variability detected at the interspecific
level for both, the nuclear and the chloroplast sequences, AFLP analysis was
performed for 93 individuals, representing all species of the B. lobbii complex
(B. cameronense 4 accessions, B. coweniorum 4, B. dearei 15, B. facetum 6,
B. lobbii 31, B. microglossum 4, B. orectopetalum 5, B. polystictum 3, B.
siamense 1, B. smitinandii 1 and B. sumatranum 7 accessions) and some
outgroup species (Table 7.1).
The AFLP procedure followed Gaudeul et al. (2000), to the exception
24
Material and Methods
that reaction volumes in the PCRs were reduced by 50%. After the digestionligation reaction (Table 7.6) two steps of amplification followed, a preselective PCR in which primers with 1 base pair (bp) extension (Table 7.7),
and a selective PCR (Table 7.8) in which primers with 3 bp extensions (Table
7.5) were used, thereby reducing the number of fragments. The respective
PCR programmes used are detailed in Table 7.9.
The Pre-PCR product was checked for the presence of a smear of
fragments (length from 100 to 1500 bp) by agarose gel electrophoresis. For
the selective PCR initially 12 different primer combinations (Table 7.5) with 3
bp extensions were used in a primer-trial with 8 individuals (6 different
species which were representative for the complex; B. cameronense 1, B.
coweniorum 1, B. dearei 1, B. lobbii 3, B. polystictum 1 and B. siamense 1
accessions). The EcoRI-primers were labelled with fluorescent dyes (Table
7.5).
For each individual, 0.6 µl (blue labelled, 6-FAM), 0.9 µl (green
labelled, VIC) and 1.2 µl (yellow labelled, NED) selective PCR products were
combined with 1 µl mixture composed of formamide, loading buffer and size
standard (GenScan ROX 500; Applied Biosystems). This mixture was then
denatured at 95 °C for 2 min prior to loading on a 6% polyacrylamide gel.
Electrophoresis was run for approximately 4 h on an automated sequencer
ABI 377TM (Applied Biosystems) at the Faculty Center Botany of the
University of Vienna. Raw data was collected and aligned with the internal
size standard using ABI PRISM GENESCAN® version 3.7 analysis software
(PE Applied Biosystems). The GeneScan files were imported into
GENOGRAPHER
version
1.6
(http://hordeum.oscs.montana.edu/
genographer/) for scoring. Fragments in the size range of 60 to 500 bp were
scored and the data were exported into PAUP* (version 4.0b5; Swofford
2001) as a presence/absence matrix.
Out of the 12 different primer combinations initially tested, EcoRI AGG
(VIC)-MseI CTG, EcoRI ATC (6-FAM)-MseI CTA and EcoRI AAC (NED)MseI CTG were selected (because these combinations produced the best
scorable and visible fragments) for analysing all individuals (Table 7.5).
25
Material and Methods
2.5 Molecular analyses
2.5.1 ITS sequence analyses
2.5.1.1 Phylogenetic reconstructions and molecular dating
The ITS sequence analyses were performed using 53 accessions of the B.
lobbii complex (ingroup) together with 12 accessions of the same section
(Sestochilos) which were used as an outgroup for the rooting of the trees
(Table 7.1). Sequences were assembled using VECTOR NTI (Invitrogen) and
then visually improved. The alignment of sequences (including outgroup
taxa) was unambiguous and required the introduction of only a few gaps. All
analyses were conducted using the entire ITS region (ITS1, 5.8S rDNA,
ITS2). The boundaries of the various regions were determined by
comparision with the sequence from B. lobbii (GenBank accession number
AF521074; van den Berg et al., 2005). Basic molecular characteristics of the
ITS sequences were examined using the programme MEGA (Molecular
Evolutionary Genetics Analysis) version 3.1 (Kumar, 2004). To assess overall
phylogenetic relationships, an unweighted maximum parsimony (MP)
analysis was initially performed on the whole dataset using PAUP* (version
4.0b5; Swofford 2001). Heuristic MP searches were performed with the
following conditions in effect: 10 replications of random sequence addition,
tree bisection-reconnection (TBR) branch swapping (on best trees only),
MULTREES on. In these searches, the maximum number of trees saved
(MAXTREES) were set to 10000 and gaps (indels) were treated as missing
data. To support individual nodes of the tree bootstrap values (Felsenstein,
1985) were estimated from 100 pseudoreplicates, and using the same
heuristic search settings.
Maximum Likelihood (ML) and ML distance (Neighbour Joining; NJ)
analyses under the model determined by MODELTEST 3.7 (Prosada &
Crandall, 1998) were also performed with PAUP*. Based on MODELTEST,
the best-fitting substitution model was the TIM+G+I “transitional model”
(Prosada & Crandall, 1998) which allows base frequencies to vary and
assigns different substitution rates to purine and pyrimidine transitions as well
as a third rate to all transversions. Furthermore, different rates are permitted
at different sites based on a discrete gamma (γ) distribution with four rate
categories for the variable sites and with estimated shape parameter. Finally,
26
Material and Methods
this model allows for a certain proportion of invariant sites (I).
Tree searches under the ML criterion were performed using 10
replicates of random sequence addition and nearest neighbour interchange
(NNI) branch swapping. MAXTREES were set to 100. Maximum likelihood
bootstrap analysis was conducted using model parameters derived from the
original data matrix (100 replicates, 10 random addition sequences, NNI
branch swapping, MAXTREES = 100).
The NJ-distance analyses (Saitou & Nei, 1987) were conducted also
under the ML criterium (with model parameters derived from the original data
matrix) and bootstrap values were obtained by running 10000 replicates.
The ML tree searches (as mentioned above) were also accomplished
with a molecular clock enforced (mce) (see PAUP* option: maximum
likelihood settings – miscellaneous). Thus, in order to test whether the ITS
dataset follows a “global” (i.e. tree-wide) molecular clock or not, the two log
maximum likelihood (logL) scores (obtained i.e. with and without mce) were
compared to each other using the tree based likelihood ratio test (LRT) of
Felsenstein (1988) which is implemented in MODELTEST 3.7. The test
statistic: -2(logLclock – logLno
clock)
should be distributed as χ2 with (N-2)
degrees of freedom, where N is the number of sequences in the tree
(Felsenstein, 1988; Sanderson, 1998). After confirming rate-constancy of ITS
sequence evolution, the ML-tree with a molecular clock enforced was taken
for estimating both the age and the onset of diversification of the B. lobbii
complex, as well as for dating various subsequent events in the evolutionary
history of the group. As there is neither a fossil record nor an ITS substitution
rate available for orchids, three different, previously published ITS
substitution rates were used for calibration of the clock: 1.72 x 10-9
subst./site/year (slow clock), 3.94 x 10-9 subst./site/year (moderate clock) and
7.83 x 10-9 subst./site/year (fast clock; see Richardson et al., 2001). In
addition the clock was calibrated biogeographically using ITS sequence
information of two sister taxa of Bulbophyllum (G. Fischer, unpublished data),
one endemic to Reunion (B. variegatum) and the other occuring in
Madagascar (B. bathieanum). After confirming rate-constancy of ITS
sequence evolution for the whole section (Alcistachys) these two latter
species belong to, the clock tree was used to calibrate, to the node of their
27
Material and Methods
common ancestor. The node of divergence between the two sister taxa in a
clock-enforced ML tree was dated to the emergence of Reunion
approximately 1.8 Mya (Gillot et al., 1994), thereby assuming that the island
was soon colonized after it breached the surface of the ocean and that
colonization of the island corresponded with the speciation of B. varigatum
(see also Kimball et al., 2003). This analysis resulted in an ITS mutation rate
of 1.297 x 10-9 subst./site/year (G. Fischer, unpublished data). Note,
however, that this approach provides maximum age estimates and therefore
this latter rate should be considered a minimum rate.
2.5.1.2 Lineages-through-time (LTT) and diversification analyses
In lineages-through-time (LTT) plots, the increasing (log) number of lineages
of a reconstructed phylogeny (ultrametric tree) is plotted against their
absolute time of divergence or the (genetic) distance relative time of each
node from the tip of the tree.
The nodes were numbered and afterwards a diagram was constructed
(X-values = distance from the tip; Y-values = log number of lineages). Node
heights (branch lengths) were derived as genetic distances from the clockconstrained phylogenetic (ITS) tree calculated under ML.
To further examine if lineage diversification in the B. lobbii complex
conforms to a constant-rates birth and death process (null-hypothesis), the
programme GAMMASTATISTICS V1.0 (Griebeler, 2004) was used, which
performs the γ-statistic test developed by Pybus and Harvey (2000). If the
internal nodes of the reconstructed phylogeny are closer to the root (γ < 0) or
to the tips (γ > 0) than expected under the pure birth model, the constantrates model is rejected. This method is relatively robust to incomplete taxon
sampling and is conservative with respect to extinction (Pybus & Harvey,
2000).
Each node height (branch length in terms of genetic distance to the
tip) of the calculated ML tree (with mce) was exported to an excel-sheet and
then sorted in ascending order according to the distance to the tip.
Afterwards the distance-differences of the nodes were calculated and loaded
into the programme.
28
Material and Methods
2.5.1.3 DIVA (dispersal-vicariance analysis)
The programme DIVA (version 1.1; Ronquist, 1996) was used for the
examination of the ancestral area(s) of the B. lobbii complex. This method is
based on a three dimensional cost matrix derived from a simple
biogeographic model, which assumes that the current distribution of species
(taxa) and their ancestor can be described by unit areas (the whole
distribution area is splitted in smaller, but distinct areas and these are called
unit areas; e.g. Luzon, Mindanao, Palawan, Borneo, Sumatra, Java/Bali,
Sulawesi, Peninsula Malaysia and the region north of the Isthmus of Kra are
the unit areas of the study region in the first analysis). Four assumptions are
made: (i) speciation by vicariance (no costs); (ii) allopatric speciation (no
costs); (iii) dispersal (costs per unit area +1); (iv) extinction (costs per unit
area –1).
For the reconstruction of ancestral areas a simplified phylogenetic tree
of the ITS dataset was used to reflect relationships among the major clades
and subclades identified within the complex (see Results, Figs. 3.12, 3.13).
The 0/1 data matrix, which was used for the DIVA analysis, is shown in Table
2.1, whereby 0 and 1 refer to the absence and presence of (sub)clades in
predefined biogeographical regions, respectively.
A
0
0
0
0
0
0
1
0
0
B
0
0
0
0
0
0
0
1
0
C
1
0
0
0
0
0
0
0
0
D
1
0
0
0
0
0
0
0
0
E1
1
0
0
1
1
1
0
1
0
E2
1
0
0
0
0
0
0
1
1
E3
1
1
0
1
0
0
0
1
0
E4
0
0
1
1
1
0
0
1
0
of Kra
Above Isthmus
Malaysia
Peninsula
Sulawesi
Sumatra
Borneo
Palawan
Luzon
(Sub)clades
Mindanao
Distribution
Java/Bali
Table 2.1: Data matrix containing all clades and subclades used for DIVA analysis
In a second DIVA analysis, Luzon, Mindanao and Palawan were combined to
one area (Philippines) and the subclades E1 – E4 were reduced to a single
29
Material and Methods
clade, E. According to suggestions by Ronquist (1996) and Oberprieler
(2005), this clade was coded according to its likely ancestral distribution and
not being distributed in all areas where descendants occur presently.
Because of the basal position of a Bornean lineage (phylogenetic evidence)
and the high genetic/subclade diversity within clade E on Borneo
(circumstantial evidence), this area was chosen as the ancestral area of
clade E. In addition, this second DIVA analysis was performed with and
without using B. emiliorum as an outgroup. This resulting data matrix is
shown in Table 2.2.
Table 2.2: Data matrix containing all clades (A-E) with B. emiliorum
used as outgroup.
Thailand
Peninsula Malaysia
Sulawesi
Java/Bali
Sumatra
Borneo
Clades
Philippines
Distribution
A
0
0
0
0
1
0
0
B
0
0
0
0
0
1
0
C
1
0
0
0
0
0
0
D
1
0
0
0
0
0
0
E
0
1
0
0
0
0
0
Outgroup
1
0
0
0
0
0
0
2.5.2 CpDNA sequence analysis
Five distantly related accessions of the B. lobbii complex (as evidenced by
ITS) were initially chosen for a pilot screen using the regions trnH-psbA, trnDtrnE, trnS-trnfM, trnL-trnF and rpoB-trnC. The three other successfully
amplified regions were not sequenced due to their expected lower variability
(Shaw et al., 2005). The regions trnD-trnE and trnH-psbA were finally chosen
for a full screen because (i) the trnS-trnfM region showed the lowest
variability (Table 2.3); (ii) paralogous sequences of the trnL-trnF region
emerged in various Madagassian Bulbophyllum species, posing difficulties
for phylogenetic reconstruction (G. Fischer, unpublished data); and (iii) the
sequencing reaction of the rpoB-trnC region was successfully done for only
30
Material and Methods
one of the five individuals examined.
Table 2.3: Variability of four cpDNA regions. Data were
obtained by examining 5 different accessions of the
B. lobbii complex for each marker.
Length
Variable
Number
Length of
Marker
(bp)
sites
of indels
indels (bp)
trnD-trnE
440
4
2
9&2
trnS-trnfM
900
3
2
1
trnH-psbA
800
8
2
7&6
trnL-trnF
940
11
1
5
The full analyses were performed on 32 accessions of the B. lobbii complex
(ingroup) together with 4 outgroup accessions for the trnH-psbA region and
31 accessions of the B. lobbii complex together with 5 outgroup accessions
for the trnD-trnE region (Table 7.1). B. emiliorum was taken for the rooting of
the trees.
The sequences of each region were aligned and MP, ML, and distance
(NJ) analyses were performed for each dataset separately and also for the
combined dataset (31 accessions of the B. lobbii complex and 4 outgroup
accessions). The selected models determined by MODELTEST for the NJ
and ML analyses were TIM for the trnD-trnE dataset, F81+I+G for the trnHpsbA dataset and GTR+I+G for the combined dataset. The settings of the
MP, ML, and distance (NJ) analyses for the cpDNA dataset analyses are
summarised in Table 2.4. Characteristics of the cpDNA sequences were
examined using MEGA.
31
Material and Methods
Table 2.4: Settings for the phylogenetic analyses of the cpDNA datasets. Note that the same
settings were applied to the ITS dataset.
Type of analysis/settings
Neighbour
Maximum
Parsimony
Bootstrap (MP)
(MP)
likelihood (ML)
Bootstrap (ML)
Joining
Bootstrap
(NJ)
(NJ)
distance
Heuristic
Heuristic
search;
10 randomadditionsequence;
TBR;
MAXTREES
10000
Maximum
Heuristic search;
search;
Heuristic search; Modeltest
10 random-
Modeltest
parameter;
addition-
parameter;
10 random-
sequence;
10 random-
addition-
TBR;
addition-
sequence; NNI;
MAXTREES
sequence; NNI;
MAXTREES
10000;
MAXTREES 100 100;
100 replicates
Modeltest
Modeltest
parameter;
parameter 10000
replicates
100 replicates
Gaps were defined as missing data; all characters were weighted equally
2.5.3 AFLP data analysis
81 accessions (11 species) of the B. lobbii complex (ingroup) and 12
accessions (9 species) of the same section (Sestochilos) were used as
outgroup for the analyses. Genetic distances of all AFLP samples were
calculated using the complementary value of Nei and Li’s (1979) similarity
coefficient: D = 1 – SC = 1 – [2nxy/(nx + ny)] as implemented in PAUP*. D
represents the genetic distance, SC the similarity coefficient, nxy the number
of fragments shared between two samples (x,y), and nx and ny are the
number of fragments of sample x and y, respectively. PAUP* was used to
construct a NJ tree of the Nei-Li distance matrix (with B. hamatipes used for
rooting) and bootstrap analysis was performed using 10000 replicates. In
addition, a PCO (principal coordinates analysis) was performed on the 0/1
matrix using Euclidian distances and PCO scores of all AFLP phenotypes
were plotted in two dimensions using the PAST software package (version
1.40; Hammer et al., 2001).
32
Material and Methods
2.5.4 Partition homogeneity test
The partition homogeneity test (Farris et al., 1994), implemented in PAUP*, is
commonly used to test whether two different molecular datasets of the same
set of taxa contain a similar phylogenetic information or not. First, the
programme calculates the most parsimonious tree for each dataset (partition)
and adds up the number of steps for each tree (original tree). Then, the two
datasets are intermixed randomly and the sum of the steps of the most
parsimonious tree for each partition is calculated again. The latter
(randomization) step is carried out up to 500 times.
As a result, these randomizations and calculations provide a
distribution of the number of steps for trees as expected under the null
hypothesis (data homogeneity). Now, the step number of the original tree is
compared to the distribution to find out if this value falls within a one-tailed
5% rejection region or not. Thus, if less than 5% (P < 0.05) of the randomized
trees have as many or fewer steps than the original tree, then the
phylogenetic signal of the two different molecular marker datasets is
significantly different and the datasets should not be combined (Felsenstein,
2004).
In the present study, the partition homogeneity test was performed for
the two cpDNA data sets (trnH-psbA; trnD-trnE), for ITS and trnH-psbA, for
ITS and trnD-trnE, for ITS and cpDNA data combined (AD) and for ITS and
the AFLP data.
2.6. Morphological analysis
All specimens used in the morphological (multivariate-morphometric) analysis
are presently cultivated in the glasshouse of the Botanical Garden Salzburg.
For each plant, in general, a few flowers (depending on how many were
available), at least two pseudobulbs and at least one leaf were conserved in
“Kew solution” (53 % ethanol, 5 % formaldehyde and 5 % glycerol). For some
plants, however, only flowers were available. Conversely, for some plants
which did not flower during the time of study only vegetative characters could
be measured. Furthermore, photographs were taken of all flowering plants
measured. Most accessions used in the molecular analyses were included in
the morphological study, with few exceptions (because only silica-gel dried
33
Material and Methods
leaves were available).
Almost all flower characters selected for analysis were kindly
suggested by Dr. J. J. Vermeulen (personal communication). The remaining
floral and vegetative traits were chosen by the author given, at least in part,
their likely taxonomic relevance. The initial character matrix was constructed
from 25 floral characters (including 4 categorical ones and 5 ratios) plus 10
vegetative characters (including one categorical trait and two ratios; see
Table 2.5). Measurements were made with a ruler and slide gauge. The floral
measurements were mostly made on one flower, one pseudobulb and one
leaf (measures of more than one flower, pseudobulb or leaf were averaged).
Vegetative characters of 76 accessions could be measured, and both
vegetative and floral measurements were collected from 53 accessions.
Although 17 plants of the latter set could not be scored for all characters
(typically PLE - RAC characters were lacking), the 53 accessions
representing all identified (sub)clades of the complex, except clade A, and all
species of the B. lobbii complex (except B. smitinandii) plus several outgroup
species.
Prior to the multivariate analysis (see below), all quantitative
characters measured were tested for normal distribution using both ShapiroWilk test and χ2 test as implemented in PAST. Those traits deviating from the
normality assumption were either log-transformed or arcsine transformed
(only ratios) before analysis. For two deviating ratios (RAL, RAC), which
could not be arcsine transformed (i.e. with values larger than one), no
transformation was conducted. Finally, 24 characters (17 floral plus 7
vegetative ones) were used for analysis.
Morphological variation within and among taxa/clades was assessed
using multivariate morphometric methods (principal component analysis
(PCA)) based on the trait correlation matrix as implemented in the PAST
programme package.
34
Material and Methods
Table 2.5: Characters used for morphometric analysis.
Morphological characters
Code
floral
1. Peduncle length (mm)
PLE
2. Number of peduncle bracts (units)
NPB
3. Length of the longest peduncle bract (mm)
LPB
4. Distance between inflorescence - upper peduncle bract (mm) PPB
5. Length of floral bract (mm)
LFB
6. Length of basal internode (mm)
LBI
7. Length of median sepal (mm)
LMS
8. Width of median sepal (mm)
WMS
9. Mean length of lateral sepals (mm)
LLS
10. Mean width of lateral sepals (mm)
WLS
11. Mean length of petals (mm)
LEP
12. Mean width of petals (mm)
MWP
13. Length of lip (mm)
LOL
14. Width of lip (mm)
WOL
15. Length of column (m)
LOC
16. Width of column (mm)
WOC
17. Nectar mark (present (1) vs. absent (0))
NEM
18. Blooming time (January (1) to December (12))
BLT
19. Flower color (yellow (0) vs. red (1))
FLC
20. Scent (foul (0) vs. pleasant (1))
SCE
21. Ratio median sepal (width/length)
RMS
22. Ratio lateral sepals (width/length)
RLS
23. Ratio petals (width/length)
RAP
24. Ratio lip (width/length)
RAL
25. Ratio column (width/length)
RAC
vegetative 26. Height of pseudobulbs (mm)
HOP
27. Length of next to last pseudobulb (mm)
LOP
28. Width of next to last pseudobulb (mm)
WOP
29. Length of leaf of next to last pseudobulb (mm)
LLF
30. Width of leaf of next to last pseudobulb (mm)
WLF
31. Surface of pseudobulbs (plain (0) vs. rough (1))
SPB
32. Distance between last and next to last pseudobulb (mm)
ADP
33. Length of rhizome segment (mm)
LRS
34. Mean ratio pseudobulbs [(length/width1+length/width2)/2]
RPB
35. Ratio leaves (length/width)
RLE
35
Material and Methods
2.7 Documentation
All DNA sequences obtained in this study will be deposited in GenBank.
Complete ITS, trnH-psbA and trnD-trnE data matrices are available
electronically on request from the author. Herbarium specimens were only
prepared if enough Kew-conserved samples were in stock. The vouchers of
the plants (herbarium specimens and/or in “Kew solution”) are deposited
either at the University of Salzburg, Austria (analysed fresh material) or at the
University of Leiden, The Netherlands (silica-gel dried samples).
36
Results
3. Results
3.1 Molecular analyses
3.1.1 Partition homogeneity test
According to the various partition homogeneity tests conducted (Table 3.1),
the ITS dataset could not be combined with any of the cpDNA datasets (Pvalues from 0.002 to 0.004). Despite a low P-value of 0.036 for the AD
dataset (trnH-psbA and trnE-trnD combined), indicating a marginally
significant difference in phylogenetic signal, phylogenetic analyses were
performed with both cpDNA datasets combined, which might be justifiable
because the cpDNA genome essentially behaves as a single (nonrecombining) locus. By contrast, the ITS and AFLP datasets could be
analysed together because of a P-value of 0.552.
Table 3.1: Results of the partition homogeneity tests.
Combined datasets
P-values*
ITS/trnH-psbA
0.002
ITS/trnD-trnE
0.004
ITS/trnD-trnE-trnH-psbA
0.002
trnD-trnE/trnH-psbA
0.036
ITS/AFLP
0.552
* Significance level 0.05
3.1.2 Phylogenetic analyses of ITS sequences
Characteristics and variation of the ITS sequences (ITS-1, 5.8S rDNA, ITS-2)
are summarized in Tables 3.2, 3.3 and 3.4. The ITS-1 region within the B.
lobbii complex varied from 327 to 331 bp in length and, thus, was much
longer than both, the 5.8S rDNA region and the ITS-2 region, which varied
from 159 to 162 bp and from 156 to 157 bp, respectively. The fully aligned
ITS-region consisted of 646 positions and included 8 gaps. Four gaps were
found in ITS-1, two in the 5.8S rDNA and two in the ITS-2 region. Including
the outgroup accessions into the alignment, the total number of gaps
increased to 9 due to an additional gap in the ITS-2 region. The number of
parsimony-informative sites within the B. lobbii complex were 41 (ITS-1 = 11;
5.8S rDNA = 12; ITS-2 = 18) of 60 variable sites altogether. Of the remaining
37
Results
19 positions, which were singleton sites, 13 were found in the ITS-1, four in
5.8S rDNA and two in the ITS-2 region. When the accessions of the outgroup
were taken into account, 24 sites (of 49 variable sites) were potentially
parsimony-informative in ITS-1, 14 (of 20 variable sites) in the 5.8S gene and
23 (of 26 variable sites) in ITS-2, i.e. in total 61 (of 95 variable sites).
Since many intraspecific accessions of the B. lobbii complex had
identical ITS sequences, 17 redundant sequences were pruned before
analysing pairwise nucleotide sequence differences (Table 3.2). Within the B.
lobbii complex the pairwise sequence difference of the whole ITS region
ranged from 0.2 to 2.9 % with a mean value of 1.4 %. Between the B. lobbii
complex and the outgroup, the divergence value of pairwise comparisions
ranged from 1.4 to 4.9 % (mean value 2.5 %), among which the distances to
B. patens were the lowest, with values ranging from 1.4 to 3.2 % (mean value
1.9 %), and those to B. alsiosum the highest, with values ranging from 3.9 to
4.9 % (mean value 4.4 %). As two distinct clades were identified within the
outgroup, hereafter called outgroup 1 (B. emiliorum and close relatives) and
outgroup 2 (B. alsiosum and B. hamatipes), pairwise sequence differences
between the complex and these outgroups were also calculated separately
(see Table 3.2).
Table 3.2: Characteristics and variation of the ITS nrDNA region in the Bulbophyllum lobbii
complex and taking into account more closely and distantly related (outgroup) accessions
(outgroup 1 and 2, respectively). Genetic distances were calculated using the Kimura-2
algorithm.
Taxa/sequence characteristics
ITS-1
5.8S rDNA
ITS-2
Total region
Length of range (bp)
327 - 331
159 - 162
156 - 157
642 - 646
Number of gaps
4
2
2
8
GC content (mean %)
57
62.6
55.2
57.9
Ratio of transitions/transversions
6.7
2.2
1.3
2.3
Constant sites*
305
143
137
585
Variable sites*
24
16
20
60
Parsimony-informative sites
11
12
18
41
Singleton sites
13
4
2
19
Length of range (bp)
326 - 331
159 - 162
153 - 157
641 - 646
Number of gaps
4
2
3
9
Within B. lobbii complex
B. lobbii + outgroups (aligned)
38
Results
GC content (mean %)
56.9
62.7
55.2
57.9
Ratio of transitions/transversions
5.4
2.3
1.5
2.6
Constant sites*
282
140
131
553
Variable sites*
49
20
26
95
Parsimony-informative sites
24
14
23
61
Singleton sites
25
6
3
34
Mean percentage pairwise sequence divergence (range)**
Within B. lobbii
0.9 (0.0 - 2.2) 1.3 (0.0 - 2.6) 2.5 (0.0 - 4.1) 1.4 (0.2 - 2.9)
Between B. lobbii + outgroup 1
2.4 (0.3 - 4.5) 1.5 (0.6 - 3.2) 3.7 (2 - 5.5)
Between B. lobbii + outgroup 2
5 (2.8 - 5.8)
2.5 (1.4 - 4.7)
2.2 (1.3 -3.2) 4.7 (2.7 - 7.1) 4.2 (3.7 - 4.9)
Between B.lobbii + both outgroups 3.2 (0.3 - 5.8) 1.7 (0.6 - 3.2) 3.9 (2 - 7.1)
2.5 (1.4 - 4.9)
*Calculations were made after considering gaps as missing data
**Multiple intraspecific accessions with identical sequences were excluded from calculations
Between the major ITS clades identified within the B. lobbii complex (see
below, Figs. 3.1 and 3.2) the mean divergence value of pairwise
comparisions ranged from 1.4 % (between clades E and D) to 2.7 %
(between clades A and B; Table 3.3). The highest mean percentage
divergence was found within clade A with a value of 1.4 %. The mean
percentage divergence between the various clades within the B. lobbii
complex and outgroup1 (B. emiliorum and close relatives) varied from 2.1 to
3.2 %, and with respect to outgroup 2 (B. alsiosum and B. hamatipes) from
3.8 to 4.3 % (Table 3.3).
Table 3.3: Mean percentage pairwise ITS sequence divergence (SE) between and within
clades.
CladeA CladeB CladeC CladeD CladeE Outgroup1 Outgroup2 Both outgroups
CladeA
1.4 (0.4)
CladeB
2.7 (0.6) 0.2 (0.1)
CladeC
2.3 (0.5) 2.1 (0.5) 0.6 (0.2)
CladeD
2.3 (0.5) 1.5 (0.5) 1.2 (0.4) 0.2 (0.1)
CladeE
2.5 (0.5) 1.9 (0.5) 1.6 (0.4) 1.4 (0.4) 0.9 (0.2)
Outgroup1
3.2 (0.6) 2.8 (0.6) 2.2 (0.5) 2.1 (0.4) 2.4 (0.5)
1.4 (0.3)
Outgroup2
4.3 (0.8) 4.3 (0.8) 4.1 (0.7) 3.8 (0.7) 4.3 (0.7)
3.8 (0.7)
1.2 (0.3)
-
-
Both outgroups 3.5 (0.6) 3.2 (0.6) 2.8 (0.5) 2.5 (0.4) 3.0 (0.5)
2.5 (0.4)
Within the species-rich clade E, among-subclade sequence divergence
ranged from 0.7 % (between subclades E3 and E1) to 1.4 % (between
39
Results
subclades E4 and E2), and the highest within-subclade divergence was
observed within subclade E2 with 0.8 % (Table 3.4).
Table 3.4: Mean percentage pairwise sequence divergence (SE)
between and within the subclades of clade E.
SubcladeE1 SubcladeE2 SubcladeE3 SubcladeE4
SubcladeE1
0.2 (0.1)
SubcladeE2
1.0 (0.3)
0.8 (0.2)
SubcladeE3
0.7 (0.3)
0.9 (0.3)
0.3 (0.1)
SubcladeE4
1.2 (0.4)
1.4 (0.4)
1.2 (0.4)
0.5 (0.2)
The maximum parsimony (MP) analysis of the whole 646 character matrix (B.
lobbii complex plus outgroups; 65 accessions) with gaps scored as missing
data resulted in 10000 equally parsimonious trees (Maxtrees = 10000) of 151
steps with a consistency index (CI) of 0.695 (excluding uninformative
characters) and a retention index (RI) of 0.856 (Table 3.5). One of the most
parsimonious trees with the highest -log L value (settings in PAUP*: tree
scores – maximum likelihood; 1893.78315) is shown in Figure 3.1.
Table 3.5: Results of the maximum parsimony analyses. TL = treelength in steps; N =
number of shortest trees; CI = consistency index; RI = retention index; HI = homoplasy
index.
Maximum parsimony analysis
Marker
TL
N
CI
RI
HI
ITS
151
10000
0.695
0.856
0.305
trnH-psbA
41
10000
0.854
0.765
0.146
trnD-trnE
31
11
0.968
0.976
0.032
trnH-psbA/trnD-trnE
81
10000
0.704
0.780
0.296
40
Results
Fig. 3.1: ITS MP tree of the B. lobbii complex and 12 extra-complex individuals (marked by
asterisks). Within the complex five major clades (A-E) are identified, with clade E mainly
comprising four subclades (E1-E4). The tree shown had the highest -logL score
41
Results
(1893.78315) and was rooted by B. hamatipes. Numbers above branches indicate MP
bootstrap values (> 50 %) from 100 replicates (heuristic search, 10 random sequence
additions). Accession numbers (in brackets) are identified in Table 7.1. AF521074 is a B.
lobbii ITS sequence downloaded from GenBank. Note that B. facetum, B. coweniorum and
B. sumatranum (marked red) have red flowers.
The ML-distance analysis (based on the TIM+I+G model) resulted in a tree,
which was topologically most similar to the MP tree (Fig. 3.1), but had almost
always lower bootstrap support values for both the monophyly of the B. lobbii
complex and its various (sub)clades (not shown).
Maximum likelihood (ML) searches under the best fit model
(TIM+I+G), and without a molecular clock enforced, resulted in a single tree
with a log L of -1902.6852 (Fig. 3.2). The topology of this tree is almost
identical with that of the MP tree (Fig. 3.1). One hundred equally likely ML
trees (setting MAXTREES = 100), each with a log L of –1868.7782, were
obtained after the search with a molecular clock enforced. One of the clockenforced trees is shown in Figure 3.3, with ML bootstrap values shown above
branches. Rate constancy of ITS sequence evolution was confirmed after
comparing the log L scores of the ML clock enforced/non-clock trees (2Δ = χ2
= 67.814, P-value = 0.317; Table 3.6).
Table 3.6: Results of the maximum likelihood analyses. M = used model according to
Modeltest; γ = gamma distribution (shape parameter); SWC = -log L score with a molecular
clock enforced; SWOC = -log L score without a molecular clock enforced; R = ratio (2 x
difference); P = P-value.
Maximum likelihood analysis
M
Γ
ITS
TIM+I+G
0.9705
trnH-psbA
F81+I+G
1.0135
-
trnD-trnE
TIM
equal rates for all sites
1.1206
Marker
trnH-psbA/trnD-trnE GTR+I+G
SWC
SWOC
R
P
67.814
0.317
1521.1619
-
-
-
801.7705
-
-
-
2413.305
-
-
1868.7782 1902.6852
Both the MP and the non-clock ML tree (Figs. 3.1, 3.2) strongly supported the
monophyly of the B. lobbii complex (BV MP = 90 %; ML = 100 %), which was
sister to outgroup 1 (MP = 91 %; ML = 90 %), comprising B. affine, B.
emiliorum, B. patens, and B. pileatum. Surprisingly, two outgroup accessions
(B. piestoglossum, B. monanthum) were found within the B. lobbii complex:
42
Results
B. piestoglossum clustered together with B. coweniorum and formed a well
supported clade, C (MP = 100 %; ML = 100 %), whereas B. monanthum
clustered together with B. polystictum and B. lobbii (plus AF521074) in
subclade E1, which is weakly supported by the ML analysis only (ML = 70
%). Interestingly, all three accessions of B. lobbii from Sulawesi formed a
strongly supported clade, A (MP/ML = 100 %). Furthermore, two clades
within the complex, each containing accessions of just one species, were
well supported: clade D of B. facetum sampled from Luzon/Philippines (MP =
100 %; ML = 80 %) and clade B of B. microglossum with accessions
originating from the Cameron Highlands (Penang/Peninsula Malaysia;
MP/ML = 100 %). The species-rich clade E, and also three of its four
subclades (E1, E2, E3), were not supported (MP/ML < 50 %), whereas
subclade E4, containing B. sumatranum and various accessions of B. lobbii
from
the
Cameron
Highlands/Peninsula
Malaysia,
Sabah/Borneo,
Palawan/Philippines and Sumatra received strong support, at least, by the
MP analysis (MP = 88 %).
Notably, there were also B. lobbii accessions present in clade E (all
subclades except E2) as well as in clade A (see above), suggesting that “B.
lobbii” is a highly poly-/or paraphyletic taxon and/or conferring a wrong
taxonomy (see Discussion). Otherwise, almost all intraspecific accessions of
the complex clustered together, to the exception of those of B. polystictum,
which nonetheless grouped in the same subclade (E1).
43
Results
Fig. 3.2: ITS ML tree of the B. lobbii complex and 12 extra-complex individuals of the same
section (marked by asterisks). The tree was rooted by B.hamatipes. Numbers above
branches indicate ML bootstrap values (> 50 %) from 100 replicates (heuristic search, 10
random sequence additions). Accession numbers (in brackets) are identified in Table 7.1.
44
Results
AF521074 is a B. lobbii ITS sequence downloaded from GenBank. Note that B. facetum, B.
coweniorum and B. sumatranum (marked red) have red flowers.
In the ML clock tree (Fig. 3.3) the monophyly of the B. lobbii complex was
less supported (BV 70 %), but all other major clades within the complex were
at least equally supported as in the ML non-clock tree. However, subclades
E2 and E4 received only weak support (BV 70 and 60 %, respectively).
Importantly, clade E was well supported by a bootstrap value of 80 %.
After confirming rate-constancy of ITS sequence evolution (see
above), times of divergence were first inferred from a biogeographically
calibrated ITS mutation rate for Bulbophyllum (see Material and Methods,
2.5.1). Accordingly, the origin of the B. lobbii complex (node A in Fig. 3.3)
was dated to approximately 10.8 million years ago (Mya), and the onset of
first diversification (node B) to about 8.6 Mya. At this latter time, three
lineages arose that gave rise to clades A, B and a third lineage, the latter of
which then further diversified into clades C, D and E about 6.9 Mya (onset of
second diversification; node C). While clades A, B, C, and D consist of only
one to two species and are distributed in restricted areas (A: Sulawesi; B:
Cameron Highlands/Peninsula Malaysia; C and D: Luzon/Philippines), clade
E started diversifying about 4.5 My ago (onset of third diversification; node
D), and thereby became very species-rich (seven of the 11 current species)
and widespread (Luzon, Mindanao, Palawan, Borneo, Sumatra, Bali,
Peninsula Malaysia, Thailand and Myanmar). Using the three alternative
substitution rates (Richardson et al., 2001), the origin of the B. lobbii complex
(node A) was approximately 1.8 Mya (fast clock), 3.6 Mya (moderate clock)
and 8.2 Mya (slow clock), respectively. The age of node B (onset of first
diversification) ranged from 1.4 to 1.6 Mya, of node C (onset of second
diversification) from 1.1 to 5.2 Mya and of node D (onset of third
diversification) from 0.7 to 3.4 Mya.
However, regardless of which clock was used, the origin of well
recognized species within clade E (i.e. B. dearei, B. sumatranum, B.
smitinandii, B. orectopetalum, B.siamense, B. cameronense, B. polystictum)
likely took place at the Plio-/Pleistocene boundary or well within the
Pleistocene. Furthermore, all clocks, except the fast one, showed that certain
geographically restricted lineages arose with high probability in pre45
Results
Pleistocene times such as B. microglossum/Cameron Highlands (clade B), B.
lobbii/Sulawesi (clade A), B. facetum/Luzon (clade D), and probably also B.
coweniorum/Luzon (clade C).
Fig. 3.3: Clock constrained maximum likelihood (ML) tree for the B. lobbii complex and
outgroups based on ITS sequences and estimated under the TIM+I+G model of sequence
46
Results
evolution. The tree was rooted by B. hamatipes (1). Numbers above branches indicate ML
bootstrap values (> 50 %) from 100 replicates (heuristic search, 10 random sequence
additions). Bullets and letters (A-D) indicate relevant branching events (nodes). The scales
represent the time scale in Mya under four different ITS substitution rates (see text).
Accession numbers (in brackets) are identified in Table 7.1. Locations of the accessions are
also provided. Note that B. facetum, B. coweniorum and B. sumatranum (marked red) have
red flowers.
3.1.3 Lineages-through-time (LTT) plot of ITS data
The LTT plot (Fig. 3.4) was generated using the node height information of
the clock enforced ML tree (Fig. 3.3), with a focus on the diversification of the
B. lobbii complex. The nodes of all lineages (as close as a genetic distance
of 0.0005 to the tip of the tree) were taken into account. The nodes of
accessions coinciding with the tip were not included because this would lead
to a strong increase in the number of lineages towards the present and, thus,
would falsely suggest a recent rapid increase in lineage number, albeit
without diversification. According to the gammastatistic test of Pybus and
Harvey (2000) the diversification of the complex as reconstructed above is
compatible with a pure birth model (with birth > 0, and death = 0) or a
constant rates birth-death model (with birth > 0, and death > 0) as the γ-value
was -0.805 and, thus, well within the 95 % acceptance range of the test
statistic (-1.96 < γ < 1.96 for pure birth; -1.645 < γ for constant rates birthdeath). The coloured dots (see also coloured nodes in clock enforced ML
tree) show the relative time (in terms of genetic distance) of the origin node of
the B. lobbii complex (at 0.01613, red dot) and of its three major nodes of
diversification (first onset at 0.01120, orange dot; second onset at 0.00898,
green dot; third onset at 0.00587, yellow dot), as measured from the tip of the
tree.
47
Results
LTT-plot of B. lobbii lineages
10
Number of lineages
100
1
0,018
0,016
0,014
0,012
0,01
0,008
0,006
0,004
0,002
0
Genetic Distance
Fig. 3.4: Lineages-through-time (LTT) plot of all B. lobbii lineages (based on genetic
distances > 0.0005) surveyed for ITS variation. The y-axis is the logarithm of the cumulative
number of lineages and the x-axis represents the genetic distance of relevant nodes from the
tip of a clock constrained phylogenetic (ITS) tree calculated under ML (see Fig. 3.3). Red dot
= origin of the B. lobbii complex; orange dot = onset of 1st diversification; green dot = onset
of 2nd diversification; yellow dot = onset of 3rd diversification. Note that coloured dots
correspond to coloured nodes in the clock enforced ML tree (Fig. 3.3).
3.1.4 Phylogenetic analyses of cpDNA sequences
Characteristics and variation of both the trnH-psbA and the trnD-trnE
sequences (and their combined alignment) in the B. lobbii complex (32, 31
and 31 accessions, respectively) and outgroup accessions (B. emiliorum, B.
affine and B. piestoglossum) are summarized in Table 3.7. Within the B.
lobbii complex the length of the trnH-psbA region varied from 797 to 894 bp
and that of the trnD-trnE region from 361 to 426 bp. Six gaps were found
within trnH-psbA and 16 within trnD-trnE. Combinig the two regions (for all in
total 31 B. lobbii accessions), their length varied from 1148 to 1174 bp and
12 gaps were found. Taking the outgroup accessions into the alignment, the
number of gaps increased to 17, but only for the combined trnH-psbA+trnDtrnE (AD) region (35 accessions). For the ingroup, only 7 parsimonyinformative sites were found within trnH-psbA (of 32 variable sites), 10 within
trnD-trnE (of 24 variable sites) and 25 within AD (of 51 variable sites). When
48
Results
the accessions of the outgroup were taken into account, 17 sites (of 34
variable sites) were potentially parsimony-informative in trnH-psbA, 14 (of 30
variable sites) in trnD-trnE, and 31 (of 58 variable sites) in AD.
As for the ITS dataset, all intraspecifically redundant cpDNA
sequences were pruned for the calculation of pairwise nucleotide sequence
differences. Within the whole dataset (in- and outgroup accessions) the
pairwise sequence difference of the AD region ranged from 0.0 to 1.7 % with
a mean value of 0.7 %, that of the trnH-psbA region from 0.1 to 0.8 % (mean
value 0.7 %) and that of the trnD-trnE region from 0.0 to 2.9 % (mean value
1.0 %).
Table 3.7: Characteristics and variation of the cpDNA sequences in the Bulbophyllum lobbii
complex and taking into account 4 to 5 outgroup accessions (B. emiliorum (1), B. affine (1),
B. affine (2), B. piestoglossum (1); plus B. affine (3) indicated with a and b, respectively).
Genetic distances were calculated using the Kimura-2 algorithm.
trnH-psbAa
trnE-trnDb
Combineda
Length of range (bp)
797 - 894
361 - 426
1148 - 1174
Number of gaps
6
16
12
GC content (mean %)
35.5
36.7
35.4
Ratio of transitions/transversions
0.7
0.6
0.8
Constant sites*
772
399
1117
Variable sites*
32
29
51
Parsimony-informative sites
7
10
25
Singleton sites
25
19
26
Length of range (bp)
797 - 894
358 - 426
1096 - 1188
Number of gaps
6
16
17
GC content (mean %)
35.5
36.7
35.4
Ratio of transitions/transversions
0.7
0.6
0.8
Constant sites*
765
398
1110
Variable sites*
34
30
58
Parsimony-informative sites
17
14
31
Singleton sites
17
16
27
Taxa/sequence characteristics
Within the B. lobbii complex
B. lobbii + outgroups (aligned)
Mean percentage pairwise sequence divergence (range)**
Whole dataset
0.7 (0.1 - 1.8) 1.0 (0.0 - 2.9) 0.7 (0.0 - 1.7)
Within B. lobbii
0.4 (0.0 - 1.4) 0.7 (0.0 - 2.9) 0.6 (0.0 - 1.5)
Between B. lobbii + outgroup
1.0 (0.0 - 1.8) 0.8 (0.0 - 2.9) 1.1 (0.2 - 1.7)
*Calculations were made after considering gaps as missing data
49
Results
**Multiple intraspecific accessions with identical sequences were excluded from calculations
As summarized in Table 3.5 the analysis of the trnH-psbA matrix (908
characters) resulted in 10000 equally parsimonious trees (Maxtrees = 10000)
of 41 steps with a CI of 0.854 and a RI of 0.765. The data matrix of the trnDtrnE region consisted of 449 characters, and 11 equally parsimonious trees
of 31 steps were found (CI = 0.968 ; RI = 0.976). Finally, for the 1287
character matrix of the combined dataset (AD) the MP search resulted in
10000 equally parsimonious trees (MAXTREES = 10000) of 81 steps with a
CI of 0.704 and a RI of 0.780. The heuristic ML searches under the best fit
sequence evolution models (Table 3.6), recovered a single tree (log L = 1521.16194) for the trnH-psbA dataset, a single tree (log L = -801.77052) for
the trnD-trnE dataset and a single tree (log L = -2413.30496) for the AD
dataset.
The resolution of all MP- and ML trees of the various datasets was
very poor, and this also holds true for the respective NJ-ML distance trees
(not shown). Despite the partition homogeneity test result, which indicates
marginal significance for the AD dataset (Table 3.1), only the results of this
combined dataset are described and discussed below. The ML trees of the
trnH-psbA (Fig. 3.5) and trnD-trnE (Fig. 3.6) datasets are nonetheless shown,
and should only give insight into the different tree topologies recovered by
the two cpDNA regions (analysed separately).
50
Results
Fig. 3.5: ML tree of 36 accessions of the B. lobbii complex and four extra-complex individuals
(marked by asterisks) obtained from the trnH-psbA cpDNA dataset. The tree was rooted with
B. emiliorum. Numbers above branches indicate ML-, and below branches MP- bootstrap
values (> 50 %) from 100 replicates (heuristic search, 10 random sequence additions). B.
monanthum was not analysed because no material of this species was available at the time
of the cpDNA study. Accession numbers (in brackets) are identified in Table 7.1.
51
Results
Fig. 3.6: ML tree from 36 accessions of the B. lobbii complex and five extra-complex
individuals (marked by asterisks) obtained from the trnD-trnE dataset. The tree was rooted
with B. emiliorum. Numbers above branches indicate ML-, and below branches MPbootstrap values (> 50 %) from 100 replicates (heuristic search, 10 random sequence
additions). B. monanthum was not analysed because no material of this species was
available at the time of the cpDNA study. Accession numbers (in brackets) are identified in
Table 7.1.
52
Results
The ML tree of the combined dataset is shown in Figure 3.7. While B. affine
(1) was found grouping together with B. lobbii (11) within a cluster comprising
four B. dearei accessions (1, 2, 4, 5) plus B. lobbii (1) and B. microglossum
(BV ML/MP < 50 %), and thus within the ingroup, which is supported by 100
% MP and ML bootstrap values, B. affine (2) clustered together with B. dearei
(3) near the root (MP = 63 %) and therefore close to B. emiliorum, which was
taken as outgroup (only this accession was taken for rooting the tree).
Otherwise, the two accessions of B. coweniorum formed an ingroup-clade,
which was supported by 92 % MP and 90 % ML BV, respectively, though
without showing close affinities to B. piestoglossum (compare e.g. ITS clock
tree in Fig. 3.3). Nonetheless the latter species, again, turned out as a
member of the ingroup. One clade including five B. lobbii accessions (2, 3, 5,
6, 12; MP = 58 %; ML = 70 %) was identified, a topology, however, which is
not congruent with the ITS dataset. Unfortunately B. monanthum was not
included in any of the cpDNA analyses, because no material of this species
was available at the time of the cpDNA study.
53
Results
Fig. 3.7: Combined cpDNA ML tree from 35 accessions of the B. lobbii complex and four
extra-complex individuals (marked by asterisks). The tree was rooted by B. emiliorum.
Numbers above branches indicate ML-, and below branches MP-bootstrap values (> 50 %)
from 100 replicates (heuristic search, 10 random sequence additions). B. monanthum was
not analysed because no material of this species was available at the time of the cpDNA
study. Accession numbers (in brackets) are identified in Table 7.1.
54
Results
In summary, in all three trees B. affine (1) was found within the ingroup, the
two B. coweniorum accessions formed a well supported clade, B. dearei (3)
and B. affine (2) clustered together close to B. emiliorum (1) and the B. lobbii
accessions (2, 3, 5, 6, 12) formed also a group which was more or less
supported.
3.1.5 AFLP analyses
A total number of 259 fragments were generated using the three primer
combinations EcoRI ATC (6-FAM)-MseI CTA, EcoRI AGG (VIC)-MseI CTG,
and EcoRI AAC (NED)-MseI CTG with 124, 78, 57 fragments per
combination, respectively (average 86.3; SD 34.3). 252 of these 259 markers
were polymorphic across the whole dataset of 93 accessions, and 7 markers
(2.7 %) were monomorphic. All 93 individuals surveyed were characterized
by a different AFLP profile. However, three accessions (B. alsiosum (1), B.
pileatum (1) and B. lobbii (21)) had to be subsequently removed from the
dataset because of far too long branches in a preliminary NJ tree topology,
likely reflecting methodological artefacts. The resulting NJ tree, which
consists of 90 distinct phenotypes, is shown in Figure 3.8.
Although more accessions were analyzed for the AFLP fingerprints
than for ITS sequencing, the delimitation of clades in the AFLP tree was
largely congruent with that recovered by the ITS data. However, the
monophyly of the B. lobbii complex was not supported, and also the
relationships among the AFLP clades remaind unresolved. Nevertheless, the
bootstrap values of clades A, B, C, D and E1 were similar to those of the
corresponding clades in the ITS tree (BV 96 %, 97 %, 98 %, 100 % and 71
%, respectively). There was no support, however, for (sub)clades E, E2, E3
and E4. In contrast to the ITS tree topology, clade D formed a subclade of E,
and subclade E2 was no longer member of clade E, but formed an
independent, but not supported clade. The two extra-complex species B.
monanthum (1) and B. piestoglossum (1), which in the ITS tree surprisingly
were found within the B. lobbii complex (in clade E1 and C, respectively),
were now placed together with B. smitinandii (1) in a basal position of the
AFLP tree (Fig. 3.8). Moreover, B. lobbii (12) clustered in clade E1 (rather
than E4), and B. lobbii (33) in E2 (rather than E3). All B. affine accessions (1,
55
Results
2, 3) grouped together (at the base) and B. dearei (3) clustered within the B.
lobbii complex, next to conspecific accessions (within E3, similar to ITS).
These two observations stand in marked contrast to the combined cpDNA
tree topology (Fig. 3.7).
The principal co-ordinates (PCO) analyses of squared Euclidian
distances between 90 AFLP phenotypes of the B. lobbii complex (80
accessions) plus outgroups (10; Fig. 3.9 and 3.10) largely supported the
clades suggested by the NJ-tree (Fig. 3.8). The accessions of clades A, B, C,
D, E2 and E3 formed more or less distinctive groups in the two-dimensional
plot of coordinates 1 vs. 2 (Fig. 3.9), while the distinct grouping of clades E1
and E4 became evident after plotting coordinates 2 vs. 3 (Fig. 3.10).
56
Results
Fig 3.8: Relationships among 80 individuals of the B. lobbii complex and 10 extra-complex
individuals of the same section (Sestochilos; marked by small asterisks) based on NJ
analysis of AFLP variation and Nei & Li (1979) genetic distances. The tree was rooted by B.
hamatipes (1). Numbers above branches indicate bootstrap values (> 50 %) from 10000
replicates. Accession numbers (in brackets) are identified in Table 7.1. The big asterisks
indicate accessions whose position in the AFLP tree conflicts with their position in the ITS
57
Results
trees (Figs. 3.1-3). Sampling localities of the B. lobbii and B. dearei accessions are added
after the species name. The localities of all other species/accessions largely correspond to
their distribution area, except for B. orectopetalum and B. siamense (accessions only
available from Thailand and Myanmar, respectively). The overall distribution of the clades is
indicated in the right-hand panel. Note that B. facetum, B. coweniorum and B. sumatranum
(marked red) have red flowers.
0,3
0,2
Coordinate 2
0,1
0
-0,1
-0,2
-0,2
-0,1
0
Coordinate 1
0,1
0,2
0,3
Fig. 3.9: PCO analysis of 90 AFLP phenotypes of the B. lobbii complex (80 accessions) and
outgroups (10), with the separation of most clades achieved by plotting coordinates 1 vs. 2.
Turquoise triangles = clade A; violet circles = clade B; light green diamonds = clade C; green
crosses = clade D; red crosses = clade E1; blue squares = clade E2; dark blue stars = clade
E3; pink squares = clade E4; black dots = outgroups (1 + 2); olive oval = B. monanthum;
dark brown bar = B. piestoglossum; light brown line = B. smitinandii.
58
Results
0,2
0,1
Coordinate 3
0
-0,1
-0,2
-0,2
-0,1
0
Coordinate 2
0,1
0,2
0,3
Fig. 3.9: PCO of 90 AFLP phenotypes of the B. lobbii complex (80 accessions) and
outgroups (10), with the separation of especially clades E1 and E4 achieved by plotting
coordinates 2 vs. 3. Turquoise triangles = clade A; violet circles = clade B; light green
diamonds = clade C; green crosses = clade D; red crosses = clade E1; blue squares = clade
E2; dark blue stars = clade E3; pink squares = clade E4; black dots = outgroups (1 + 2); olive
oval = B. monanthum; dark brown bar = B. piestoglossum; light brown line = B. smitinandii.
3.1.6 Geographical distribution of AFLP clades/species
The distribution and species composition of the various clades recovered in
the AFLP survey is summarized in Table 3.8 and graphically shown in Figure
3.11. Inferred distribution ranges, where species are known to occur but
which were not sampled are also considered and marked by asterisks.
All species assembled in clade A were B. lobbii from Sulawesi. The
distribution of clade B, in which only B. microglossum was found, is restricted
to the Cameron Highlands (Peninsula Malaysia). Clades C and D consisted
of B. coweniorum and B. facetum, respectively, and all occur in Luzon
(Philippines). B. coweniorum, however, is also reported from Laos*
(Vermeulen & O’Byrne, 2003), but unfortunately no accession from there was
59
Results
available for the present analyses. Clade E is widespread over the whole
region. Subclade E1, comprised of B. polystictum and numerous B. lobbii
accessions (2, 3, 5, 6, 12, 18, 20, 22, 23, 24, 25, 27, 30), is distributed in Bali
(B. lobbii (5, 18, 23)), probably Java* (no accession available), Sumatra (B.
lobbii (12)), the Cameron Highlands/Peninsula Malaysia (B. polystictum, B.
lobbii (22, 25)), Gunung Ledang/Peninsula Malaysia (B. lobbii (27)), Borneo
(B. lobbii (3)) and Luzon (B. lobbii (2)). Subclade E2 contained B.
cameronense, B. orectopetalum, B. siamense and a single B. lobbii (33)
individual, whereby B. cameronense is likely sister to the latter three. This
clade is found in Assam, Myanmar and Thailand (B. orectopetalum and B.
siamense; note that B. siamense from Assam/Thailand was not available,
and that B. orectopetalum was not sampled in Assam/Myanmar; compare
Table 1.3), Cameron Highlands/Peninsula Malaysia (B. cameronense) and
Luzon (B. lobbii (33)). Clade E3 (without the nested clade D), which
consisted of B. dearei and B. lobbii (1, 4, 10, 11), occurs on Tioman
Island/Peninsula Malaysia (B.dearei (3, 9)), Sumatra (B. lobbii (4)),
Sarawak/Borneo
(B.
dearei
(4,
14)
and
B.
lobbii
(10,
11)),
Mindanao/Philippines (B. dearei (1, 2, 6, 7, 8, 11, 12, 13)) and Luzon (B.
lobbii (1)). B. sumatranum and B. lobbii (7, 8, 13, 14, 16, 19, 26, 28, 29)
belonged
to
clade
E4,
which
is
distributed
in
the
Cameron
Highlands/Peninsula Malaysia (B. lobbii (8, 19)), Sabah/Borneo (B. lobbii (13,
14), Kalimantan/Borneo (B. lobbii (26)) Palawan/Philippines (B. lobbii (B.
lobbii (7)) and Sumatra (only B. sumatranum and B. lobbii (28)).
In summary, seven to nine more or less distinct genetic units/taxa are
recognizable that show restricted distribution ranges (see also Table 3.8).
Four of those units correspond to clades, i.e. B. lobbii (clade A: Sulawesi), B.
microglossum (clade B: Cameron Highlands), B. coweniorum (clade C:
Luzon, probably Laos), B. facetum (clade D: Luzon), whereas one additional
restricted taxon occur in each of three of four seemingly widespread
subclades of E, i.e. B. polystictum (subclade E1: Cameron Highlands), B.
cameronense (subclade E2: Cameron Highlands), and B. sumatranum
(subclade E4: Sumatra). To those could be added even B. orectopetalum
and B. siamense (subclade E2), both of which are confined to the area north
of the Isthmus of Kra.
60
Results
Table 3.8: Distribution and species composition of the clades. While clades A, B, C and D
consist of only one species, each confined to a restricted area, the widespread clade E
comprises seven species. Inferred distribution areas (i.e. where species are known to occur
but were not sampled) are marked by asterisks. Superscripts are used to assign species to
areas for subclades E1-E4. Geogrophically restricted endemics are underlined.
Clade Species
Distribution
A
“B. lobbii“
Sulawesi
B
B. microglossum
Peninsula Malaysia
(Cameron Highlands)
C
B. coweniorum
Luzon, Laos*
D
B. facetum
Luzon
E
B. lobbii, B. polystictum, B. siamense, B. dearei
B. orectopetalum, B. cameronense, B. sumatranum
E1
B. lobbiia, B. polystictumb
Peninsula
Highlands,
Malaysiaa,b
(Cameron
Ledang),
Borneoa,
Luzona, Sumatraa, Javaa*, Balia
E2
B. lobbiia, B. orectopetalumb,
Assamb,c*, Myanmarb,c, Thailandb,c,
B. siamensec, B. cameronensed
Peninsula
Malaysiad
Highlands), Luzon
E3
a
b
B. lobbii , B. dearei
Peninsula
Island),
(Cameron
a
Malaysiab
b
Mindanao ,
(Tioman
Borneoa,b,
Luzona, Sumatraa
E4
B. lobbiia, B. sumatranumb
Peninsula
Malaysiaa
(Cameron
a,b
Highlands), Sumatra , Borneoa,
Philippines (Palawan)a
61
Results
Fig. 3.11: Distribution of the various AFLP clades and restricted species (B. sumatranum, B.
cameronense and B. polystictum) of subclades E1, E2 and E4. Yellow = clade A; dark blue =
clade B; light green = clade C; dark green = clade D; red = clade E1; turquoise = clade E2;
light blue = clade E3; violet = clade E4. ▲ = B. polystictum (clade E1) and B. cameronense
(clade E2); ¹ = B. sumatranum (clade E4). Base map adapted from the maps of the
University of Texas, Austin, USA (www.lib.utexas.edu/maps/asia.html).
62
Results
3.1.7 Combined ITS/AFLP analysis
Based on the high P-value (0.552) of the homogeneity test for the ITS and
AFLP data, heuristic MP searches were performed (10 replications of random
sequence addition; TBR branch swapping, MULTREES on; MAXTREES =
10000; gaps scored as missing data). Bootstap values were obtaind by
running 10000 replicates.
The ITS/AFLP dataset consisted of 920 positions of which 308 were
potentially parsimony-informative (350 variable sites). The MP analysis of the
whole character matrix (B. lobbii complex (51 accessions) and 11 extracomplex individuals) resulted in 12 equally parsimonious trees of 2000 steps
(CI = 0.180; RI = 0.527; HI = 0.820). One of the most parsimonious trees is
shown in Figure 3.12.
Notwithstanding
the
exceptionally
high
homoplasy
index,
the
monophyly of the B. lobbii complex was supported by 78 % bootstrap value
and the delimitation of the clades was largely congruent with both the ITS
and AFLP trees (Figs. 3.1-3 (ITS) and 3.8 (AFLPs)). Clade A (B.
lobbii/Sulawesi; BV 100 %) formed a well supported clade at the basis of the
complex, followed by clade C (BV < 50 %), consisting of B. piestoglossum
and B. coweniorum. Although also weakly supported (< 50 %), the remaining
accessions formed two likely sister lineages, one comprising clades E1 (BV
81
%)
and
E2
(not
supported),
and
the
other
clade
B
(B.
microglossum/Cameron Highlands; BV 100 %) and B. monanthum (both in a
basal position), as well as E3 (BV 62 %) and E4 (not supported) plus D (B.
facetum/Luzon; BV 100 %). In contrast to the AFLP tree (Fig. 3.8), B.
smitinandii and B. piestoglossum were now placed into clades E2 and C,
respectively, as suggested by ITS, whereas the enigmatic B. monanthum
was now found close to clade B rather than nested within E1 (see ITS trees,
Figs. 3.1-3). Similar to the AFLP tree, however, nothing can be inferred from
this topology (with confidence) with respect to the phylogenetic relationships
between these (sub)clades. As a consequence, this combined ITS/AFLP
dataset will not be discussed in any further detail.
63
Results
Fig. 3.12: ITS/AFLP MP tree of 51 accessions of the B. lobbii complex and 11 extra-complex
individuals (marked by asterisks). The tree was rooted by B.hamatipes. Numbers above
branches indicate MP bootstrap values (> 50 %) from 100 replicates (heuristic search, 10
random sequence additions). Accession numbers (in brackets) are identified in Table 7.1.
Locations are added after species name. Note that B. facetum, B. coweniorum and B.
sumatranum (marked red) have red flowers. Cameron H: = Cameron Highlands.
64
Results
As stated above, the delimination of clades was largely congruent between
the ITS and AFLP datasets (and also the ITS/AFLP dataset). One major
exeption involed (the two extra-complex species) B. piestoglossum and B.
monanthum, as well as B. smitinandii, all of which were found close to the
outgroup in the AFLP tree (Fig. 3.8), but clearly intermingled with accessions
from the B. lobbii complex (ingroup) in the ITS tree (Fig. 3.3). However, the
arrangement of the clades (i.e. the tree topology) was incongruent between
the ITS and AFLP datasets as shown in a simplified form in Figures 3.13 and
3.14, respectively.
While in the ITS tree (Fig. 3.3) the clades were ordered ((A, B), (C, D),
(E4, (E2, (E1, E3)))), the order of the clades in the AFLP tree (Fig. 3.8) was
(B, (E2, (C, (A, (E1, (E4, E3)))))) with clade D nested within clade E3 (not
shown). Thus, neither the monophyly of clade E nor the successive
branchings of clades A/B vs. C/D from near the base of the tree were
recovered by the AFLP tree. As there was only weak bootstrap support for
internal branches within the AFLP tree, the ITS tree topology was considered
the more likely one and used for the subsequent dispersal-vicariance
analysis.
Fig. 3.13: Simplified tree of the clock constrained ITS dataset.
65
Results
Fig. 3.14: Simplified tree of the AFLP dataset.
3.1.8 Dispersal-vicariance analysis (DIVA)
The first DIVA analysis, whith subclades E1-E4 included, produced no clear
results concerning ancestral areas of separate clades as well as the entire B.
lobbii complex. This was due to the fact that these subclades were coded
according to all their current distribution areas (and not according to their
likely ancestral distributions). The simplified unrooted ITS tree, which was the
basis of the first DIVA analysis, together with the 9 a priori defined
distribution areas of (sub)clades (A-J) and the ancestral ranges inferred for
each node are summarized in Figure 3.15. At least 9 dispersal events
between areas were necessary for the reconstruction of ancestral areas of all
interior nodes as well as the node subtending the whole complex. Moreover,
for several nodes, there were far too numerous equally alternative or
probable ancestral ranges recovered, and for the basalmost node (7), the
reconstructions postulated a relatively widespread ancestor of the complex
distributed across the entire study area.
The results of the second analysis, based on a simplified ITS tree
rooted with B. emiliorum, are shown in Figure 3.16. In this analysis (i) the
number of a priori defined areas were reduced to seven (A-G) by subsuming
subclades into a single clade E, and (ii) the latter was coded according to its
66
Results
likely ancestral distribution (i.e. Borneo). Borneo was chosen because of (i)
the basal position of a Bornean lineage (B. lobbii (11)) within clade E
(phylogenetic evidence) and (ii) a high genetic/subclade diversity within clade
E in that area (circumstantial evidence). Now the reconstruction of ancestral
areas of all clades and the whole complex required only (at least) one
dispersal event, the number of alternative solutions were reduced, and
Borneo, Peninsula Malaysia, Sulawesi and, potentially, the Philippines were
postulated as ancestral areas for the basalmost node (4) of the B. lobbii
complex.
67
Results
Fig. 3.15: Simplified ITS tree, representing the various clades, used for DIVA analysis.
Ancestral areas of each node are listed left below the tree. Note that at least 9 dispersal
events were necessary for the reconstruction of ancestral areas of both all interior nodes as
well as the node subtending the whole B. lobbii complex. Note that the reconstructions
postulated a widespread ancestor of the complex distributed across the entire study area
68
Results
(node 7).
Fig. 3.16: Simplified ITS tree, representing the clades A to E and one outgroup-taxon (B.
emiliorum), used for the DIVA analysis. Ancestral areas of each node are listed left below the
tree. Borneo was postulated for the ancestral area of clade E (see text). At least one
dispersal event was necessary for the reconstruction of ancestral areas. Note that Borneo,
Peninsula Malaysia, Sulawesi and, potentially, the Philippines were postulated as ancestral
69
Results
areas of the whole B. lobbii complex (node 4; two alternative solutions).
3.2 Morphological analysis
As the two tests used for assessing normality produced differing results for 8
characters (nos. 3, 4, 9, 22, 30, 32-34; Table 3.9), PCAs were preliminarily
conducted on separate data matrices with deviant traits transformed
according to either the Shapiro-Wilk or the Chi2 test (Table 3.9). The results,
however, were essentially similar (not shown). Thus, any character, which
deviated from normal distribution according to just one test, was transformed.
The ratio characters RAL (24) and RAC (25) were not (arcsin) transformed
due to values larger than one. Eleven characters that were scored for only
few accessions were not included and, thus, 24 characters (17 floral
characters; 7 vegetative characters) were finally used in the analysis (shaded
grey in Table 3.9).
Table 3.9: Transformation (TF) of morphological data. Examination of the data for normal
distribution was performed using Shapiro-Wilk and Chi2 tests. Any character that deviated
from the assumption of a normal distribution in either test was transformed (except RAL and
RAC; see text). Grey shaded characters were used for the final analysis. See Table 2.5 for
character description. ND = normal distribution.
Shapiro-Wilk test
Type Char.
floral
Chi-square-test
Code W-value p-value ND
TF
Chi2
p-value ND
TF
1.
PLE
0.849
1.04E-04 no
log
19.154
1.50E-05 no
log
2.
NPB
0.835
5.86E-05 no
log
19.835
8.10E-06 no
log
3.
LPB
0.942
4.29E-02 no
log
1.9231
1.66E-01 yes
-
4.
PPB
0.942
4.28E-02 no
log
1.5128
2.19E-01 yes
-
5.
LFB
0.962
2.06E-01 yes
-
0.89744 3.43E-01 yes
-
6.
LBI
0.902
2.47E-03 no
log
6.2308
1.26E-02 no
log
7.
LMS
0.982
7.68E-01 yes
-
3.1538
7.57E-02 yes
-
8.
WMS
0.925
1.21E-02 no
log
8.2821
4.00E-03 no
log
9.
LLS
0.961
1.48E-01 yes
-
11.974
5.39E-04 no
log
10. WLS
0.956
1.27E-01 yes
-
1.9231
1.66E-01 yes
-
11. LEP
0.978
6.19E-01 yes
-
0.0769
7.82E-01 yes
-
12. MWP
0.873
4.07E-04 no
log
8.4872
3.58E-03 no
log
13. LOL
0.953
1.07E-01 yes
-
3.5641
5.90E-02 yes
-
14. WOL
0.905
3.00E-03 no
log
9.5128
2.04E-03 no
log
15. LOC
0.809
1.28E-05 no
log
29.205
6.50E-08 no
log
16. WOC
0.887
9.74E-04 no
log
6.2208
1.26E-02 no
log
70
Results
17. NEM
-
-
-
-
-
-
-
-
18. BLT
-
-
-
-
-
-
-
-
19. FLC
-
-
-
-
-
-
-
-
20. SCE
-
-
-
-
-
-
-
-
21. RMS
0.930
1.80E-02 yes
-
2.333
1.27E-01 yes
-
22. RLS
0.917
7.09E-03 no
arcsin
2.9487
8.59E-02 yes
-
23. RAP
0.932
2.05E-02 no
arcsin
4.7949
2.85E-02 no
arcsin
24. RAL
0.719
2.64E-07 no
-
10.128
1.46E-03 no
nt
25. RAC
0.734
2.31E-08 no
-
18.615
1.60E-05 no
nt
vegetative 26. HOP
0.983
7.93E-01 yes
-
0.69231 4.05E-01 yes
-
27. LOP
0.976
5.47E-01 yes
-
1.5128
2.19E-01 yes
-
28. WOP
0.967
3.06E-01 yes
-
0.69231 4.05E-01 yes
-
29. LLF
0.986
9.19E-01 yes
-
0.076923 7.82E-01 yes
-
30. WLF
0.960
1.76E-01 yes
-
6.0256
31. SPB
-
-
-
32. ADP
0.895
2.46E-03 no
log
2.667
1.02E-01 yes
-
33. LRS
0.900
6.01E-03 no
log
2.25
1.34E-01 yes
-
34. RPB
0.932
2.11E-02 no
arcsin
0.69231 4.05E-01 yes
-
35. RLE
0.979
6.78E-01 yes
-
1.5128
-
-
-
1.41E-02 no
-
-
2.19E-01 yes
log
-
Altogether 53 accessions were analysed using these 24 characters. These
accessions represented all (sub)clades/species of the B. lobbii complex
(except clade A and B. smitinandii) with 48 samples and one individual each
of the outgroup species B. piestoglossum, B. monanthum, B. emiliorum, B.
affine and B.hamatipes. The first two PCA factors accounted for 42.1 % and
14.1 % of the total variance, respectively. PC1 had the highest loadings (>
|0.2|) for PLE (1), LMS (7), LLS (9), WLS (10), LOP (11), MWP (12), LOL
(13), WOC (16), HOB (26), LOP (27), WOP (28) and LLF (29) and all of them
were negative. The highest loadings (> |0.2|) of PC2 were found for PLE (1),
MWP (12), WOC (16), RLS (22), RAP (23), RAL (24), RAC (25) and WOP
(28) (Table 3.10).
71
Results
Table 3.10: Character coefficients (loadings) for the first two principal components of the B.
lobbii complex and outgroup accessions.
No. Character
PC1
PC2
No. Character
PC1
PC2
1.
PLE
-0.2347
0.2847
21.
RMS
0.1269
-0.1653
6.
LBI
0.1264
0.06758
22.
RLS
0.1171
-0.3816
7.
LMS
-0.3188
0.1325
23.
RAP
0.128
-0.3186
8.
WMS
-0.0565
-0.02257
24.
RAL
0.03924
-0.3641
9.
LLS
-0.2589
0.0412
25.
RAC
0.1022
0.3635
10.
WLS
-0.3108
-0.1939
26.
HOP
-0.2449
-0.006407
11.
LEP
-0.3297
0.1632
27.
LOP
-0.2419
-0.1766
12.
MWP
-0.2174
-0.2584
28.
WOP
-0.2458
-0.2284
13.
LOL
-0.2996
0.09354
29.
LLF
-0.2712
0.02037
14.
WOL
-0.1455
-0.1136
30.
WLF
-0.03965
-0.01522
15.
LOC
-0.1895
0.03492
34.
RPB
0.01768
-0.1597
16.
WOC
-0.2089
-0.287
35.
RLE
0.03783
-0.0983
PCA analyses were performed plotting components 1 (c1) vs. 2 (c2) and
marking either (sub)clades (defined according to the AFLP analysis; Fig.
3.17) or species (3.18). Additional to the results I want to give some further
information based on either (i) characters (Table 3.9) which were not
included in the analyses or (ii) personal observations because this could
probably give deeper insights into the morphological variability of the
complex (especially of the various B. lobbii accessions) and likely support the
results of the PCA analyses.
As no living plant material of B. lobbii from Sulawesi (clade A) was
available they could not be included in the morphological analysis. B. affine
was subsequently excluded from the analysis because it fell out of range of
the current two-dimensional plot in a preliminary analysis. The extra-complex
taxa B. emiliorum and B. hamatipes (brown bars) as well as B. piestoglossum
and B. monanthum (dark blue stars), were well distinct of all other
species/clades because of their relatively small flowers and their high RAC
value. Within the B. lobbii complex, the accessions of each (sub)clade (B: 5;
C: 2; D: 4; E1: 11; E2: 12; E3: 9; E4: 7) hardly cluster together and there is
almost
no
distinct
grouping
recognizable
except
for
clade
B
(B.
72
Results
microglossum), clade D (B. facetum) and subclade E1 (mostly B. lobbii and
B. polystictum; Fig. 3.17). In Figure 3.18 the grouping of species appears
more evident, albeit to the exception of “B. lobbii” and B. dearei, with the
former encompassing almost the total morphological variance of the entire
complex, to the exception of B. cameronense, B. siamense and B.
microglossum. B. microglossum (clade B; black dots), which had very small
flowers and very long internodes between the pseudobulbs, was well distinct
in both the morphological and the molecular analysis. B. coweniorum (clade
C; violet circles) was found within clade E close to most accessions of
subclade E4 (Fig. 3.17). B. facetum (clade D; green crosses) was
morphological quite variable and two accessions (B. facetum (1, 2)) were
situated within clade E. Both B. coweniorum and B. facetum as well as B.
sumatranum of subclade E4 (pink squares; Fig. 3.17) have large flowers with
wide petals, relative short peduncles, red lips and a foul scent.
Accessions of subclade E1 (blue squares; Fig. 3.17) have very long
peduncles and also large flowers. B. lobbii (2) from Luzon has the longest
peduncle of all analysed accessions. B. lobbii (24) was found close to clade
E4. This accession from the Cameron Highlands (Peninsula Malaysia)
differed from the other plants found in clade E1 by having at least a shorter
peduncle.
Within subclade E2 (red crosses; Fig. 3.17), B. cameronense was well
separated from B. orectopetalum and B. lobbii (33) by PC2, having a RAL
lower to or equal to one. This ratio was larger in all other accessions of this
clade. B.lobbii (33) was found in clade E3 in the ITS tree and was at the
basis of clade E2 in the AFLP tree. Morphologically it is very similar to B.
orectopetalum, but differs in the form of the lip. B. siamense from Myanmar,
which had larger flowers than all other accessions of this clade, was found at
the margin of clade E1 (Fig. 3.17).
In subclade E3 (olive ovals; Fig. 3.17), almost all B. dearei accessions
(except B. dearei (3)) cluster together. B. dearei (3) from Tioman Island
(Peninsula Malaysia) and B. lobbii (4) from Sumatra were found near the B.
orectopetalum accessions. B. dearei (3) was much smaller than all other B.
dearei found on Borneo and Mindanao and it cannot be excluded that this
sample is of hybrid origin involving B. affine based on the cpDNA evidence.
73
Results
B. lobbii (4) was not part of clade E3 in the ITS tree, but was found at the
basis of clade E3 in the AFLP tree. Although the form of the flower of B. lobbii
(4) was very similar to B. dearei, it differed in size (smaller), colour (not the
typical yellow) and the lip (yellow nectar mark). B. lobbii (1) from Luzon was
found close to clade E4. This accession was only found in this clade in the
AFLP tree, where it was situated close to B. lobbii (4). However, based on
Figure 3.17, B. lobbii (1) and B. lobbii (4) are fairly distant. In fact, B. lobbii (1)
differed in the colour of the peduncle which was red, which otherwise was
seldom found within the B. lobbii complex.
Within subclade E4 (pink squares; Fig. 3.17) all accessions (except B.
lobbii (7)) cluster together. B. lobbii (7) from Palawan had a longer peduncle
and larger flowers than all other plants in clade E4 and, thus, was found
close to B. dearei from Mindanao (clade E3).
B. lobbii (32) from Mindanao and B. lobbii (31), which were not
included in the molecular analysis, were found close to the B. orectopetalum
accessions of clade E2. Both accessions had small flowers, were yellow and,
interestingly, B. lobbii (32) smelt foul.
4
pie1
3
emi2
ham1
mon1
2
Component 2
lob2
1
dea3
lob7
0
lob24
mic1
-1
sia1
lob32
lob1
l33 lob31
-2
-6
-5
-4
-3
-2
-1
0
1
Component 1
lob4
2
3
4
5
6
7
74
Results
Fig. 3.17: PCA analysis of 53 accessions representing 49 of the B. lobbii complex and four
outgroup individuals. Separation of the clades by component 1 vs. 2; black dots = clade B;
violet circles = clade C; green crosses = clade D; blue squares = clade E1; red crosses = E2;
olive ovals = clade E3; pink squares = E4; light brown line = B. lobbii (31, 32) which were not
included in the molecular analyses; brown bars = outgroup; dark blue stars = B. monanthum,
B. piestoglossum which were in no clade in the AFLP analysis; lob = B. lobbii; l33 = B. lobbii
(33); mic = B. microglossum; dea = B. dearei; sia = B. siamense; pie = B. piestoglossum;
mon = B. monanthum; emi = B. emiliorum; ham = B. hamatipes; accession numbers are
identified in Table 7.1.
4
B. piestoglossum
B. emiliorum
3
B. hamatipes
2
B. dearei
B. polystictum
lob2
B. monanthum
B. cameronense
B. lobbii
Component 2
1
dea3
0
lob7
B. coweniorum
B. microglossum
lob32
-1
B. sumatranum
B. siamense
lob33
lob31
-2
lob4
B. orectopetalum
B. facetum
-6
-5
-4
-3
-2
-1
0
1
Component 1
2
3
4
5
6
7
Fig. 3.18: PCA analysis of 53 accessions representing 49 of the B. lobbii complex and four
outgroup individuals. I suppose B. dearei (3) is a hybrid as it contains a B. affine cpDNA. As
a consequence this wide morphological variance of B. dearei should be considered with
caution. Separation of species by component 1 vs. 2; conspecific accessions (>= 3) are
connected by convex hulls; black dots = B. microglossum; violet circles = B. coweniorum;
green crosses = B. facetum; blue squares = B. polystictum; red crosses = B. cameronense;
green diamonds = B. orectopetalum; olive ovals = B. dearei (dea); pink squares = B.
sumatranum; turquoise triangles = B. lobbii (lob); light brown line = B. siamense; brown barrs
= outgroup; dark blue stars = B. monanthum, B. piestoglossum which were in no clade in the
AFLP analysis.
75
Results
Perhaps most importantly, while most species within the B. lobbii complex
could be well separated by morphology (Fig. 3.18), B. lobbii (s. lat.) showed a
wide range of morphological variation and, thus, could be found close to all
species except B. microglossum B. siamense and B. cameronense in the
two-dimensional PCA plot.
76
Discussion
4. Discussion
4.1 Monophyly of the Bulbophyllum lobbii complex
The ITS sequence data show that the B. lobbii complex largely forms a
strongly supported (MP = 90 %; ML = 100 %) monophyletic group within
section Sestochilos (Figs. 3.1, 3.2), with two exceptions. Thus, two additional
species of the same section (B. piestoglossum and B. monanthum), originally
rendered outgroups (Table 7.1), were placed into this complex by the ITS
and (tentatively) the AFLP trees (Figs. 3.1-3 and 3.8, respectively), which
was very surprising due to the different morphology of these two taxa (Figs.
3.17, 3.18). Evidently this implies that these species should be considered
members of the complex. Based on ITS, the complex is sister to a well
supported clade (MP = 91 %; ML = 90 %) comprising B. affine, B. emiliorum,
B. patens, B. macranthum, and B. pileatum (outgroup 1). Taking these two
groups together, they also form a monophyletic clade (MP/ML = 100 %). The
AFLP data show a similar picture but could not support the monophyly of the
B. lobbii complex (Fig. 3.8). The combined cpDNA dataset (Fig. 3.7) does not
fully support the monophyly of the B. lobbii complex either, but the relevant
clade (MP = 96 %; ML = 100 %) nonetheless contains most of the
accessions of this complex (B. monanthum was not included in these cpDNA
analyses because no material was available at the time of the cpDNA study).
While one accession of B. affine (1) was found within the complex (next to B.
lobbii (11)/B.dearei), one accession of the B. lobbii complex (B. dearei (3))
was placed outside this clade next to another accesssion of B. affine (2)
close to the root (Fig. 3.7). As both these deviant accessions (B. affine (1)
and B. dearei (3)) cluster according to expectation in both the ITS and AFLP
trees (Figs. 3.1 – 3; Fig. 3.8) it is feasible that the most probable evolutionary
process accounting for this cpDNA/nuclear incongruency is transfer (or
capture) of cpDNA mediated by (introgressive) hybridization, with B. lobbii
(11)/B. dearei and B. affine (2) acting as the maternal (‘donor’) parents,
respectively. Thus, irrespective of these deviations, I suggest that monophyly
of the B. lobbii complex (including B. piestoglossum and B. monanthum), as
shown by both ITS and AFLPs, is most likely. Nonetheless to completely
ensure the monophyly of the group, more accessions, particularly of the
outgroup 1 around B. affine, which comprises approximately 20 species (Dr.
77
Discussion
J. J. Vermeulen, personal communication), should be sequenced and
analysed. This work is currently underway.
4.2 Delimitation of major clades within the Bulbophyllum lobbii
complex
All 11 species of the B. lobbii complex, as currently placed into this group by
taxonomists (Seidenfaden & Wood, 1992; Garay et al., 1996; Seidenfaden,
1997; Comber, 2001; Vermeulen & O’Byrne, 2003), plus B. piestoglossum
and B. monanthum (see above), are found in a monophyletic clade as
suggested by the ITS and the AFLP datasets. Overall, phylogenetic analyses
of both marker systems (ITS, AFLPs) provide initial evidence of the existence
of five well-supported clades (A-E) comprising this group. In particular, the
ITS tree topologies (Figs. 3.1-3) suggest a near simultaneous origin of two
basal clades (A and B) and a third clade that subsequently diversified into the
remaining clades (C-E). While clades A to D are species-poor and
geographically largely restricted, clade E is species-rich and apparently
widespread.
All B. lobbii accessions of clade A are restricted to Sulawesi
(unfortunately not included in the morphological analyses, because no
material was available). Clade B comprises the morphologically well
delimitated B. microglossum (Figs. 3.1-3; 3.17, 3.18) which is only known
from the Cameron Highlands (Peninsula Malaysia). The species B.
piestoglossum and B. coweniorum of clade C, and B. facetum of clade D, are
mainly distributed in Luzon (Philippines). B. coweniorum, however, was also
reported from Laos (Vermeulen & O’Byrne, 2003), but unfortunately no such
accession was available for analyses. Within clade E only ITS allows
delimitating four subclades (E1 – E4), comprising up to seven species (B.
dearei, B. cameronense, B. polystictum, B. orectopetalum, B. siamense, B.
smitinandii, and B. lobbii) but phylogenetic relationships among the
subclades remain poorly resolved. In contrast to ITS, the AFLP tree (Fig. 3.8)
shows that (i) clade E is not monophyletic (subclade E2 close to the basis of
the complex), (ii) clade D (B. facetum) is nested within clade E3 mainly
composed of B. dearei (rendering the latter species paraphyletic with respect
to the former), and (iii) three taxa (B. smitinandii, B. monanthum and B.
78
Discussion
piestoglossum), found within (sub)clades E2, E1 and C, respectively, in the
ITS trees (Figs. 3.1-3) are located at the basis of the complex. This basal
position is likely reflected by the morphological analyses (B. monanthum and
B. piestoglossum close to outgroup taxa; B. smitinandii not analysed; Figs.
3.17, 3.18) whereby an artefactual basal clustering in the NJ-AFLP tree
cannot be ruled out. However, ITS is still the most likely topology, given 80 %
bootstrap value of a monophyletic clade E (excluding clade D) in the clockenforced ML tree (Fig. 3.3).
4.3 On the role of hybridization within the B. lobbii complex
While the gammastatistic test of Pybus and Harvey (2000) suggests a
constant birth and death or a pure birth model of diversification for the B.
lobbii complex (based on all lineages of the complex), the low level of ITS
variation (0.9 %) within clade E nonetheless provides evidence for a recent
radiation, a phenomenon which was also reported in several other plant
genera (e.g. Chenuil & McKey, 1996; Zhang et al., 2001), and one from the
Southeast Asia/Sundashelf region (Macaranga: Blattner et al., 2001).
There are at least two mutually non-exclusive hypotheses which could
explain this low level of ITS variability within clade E. The low genetic
differentiation of the currently widespread clade E (across the entire study
area except Sulawesi) could be either the result of relatively recent range
expansion followed by fragmentation and associated high morphological
diversification, or could be due to recent interbreeding/hybridization of
previously isolated and well differentiated genetic entities. In fact, most plants
within the B. lobbii complex can interbreed and form seed capsules (B.
lobbii/B. facetum; B. lobbii/B. sumatranum; B. lobbii/B. dearei; B. siamense/B.
coweniorum; B. sumatranum/B. dearei; B. facetum/B.dearei; unpublished
greenhouse experiments, G. Fischer). However, the AFLP tree (Fig. 3.8)
mostly groups intraspecific accessions of species of ITS clade E according to
taxonomy (B. dearei, B. cameronense, B. sumatranum), except B.
polystictum (intermingled with B. lobbii in subclade E1) and the enigmatic B.
lobbii (discussed later). For other species, including those outside clade E,
that are (potentially) sympatric with congeners (e.g. B. microglossum with B.
cameronense and B. polystictum/B. lobbii in the Cameron Highlands; B.
79
Discussion
sumatranum and B. lobbii on Sumatra; B. dearei and B. lobbii on Borneo; B.
orectopetalum and B. siamense in the region north of the Isthmus of Kra; B.
facetum and B. coweniorum in Luzon) it needs to be emphasized that the
AFLPs were also able to delimitate species – leaving little room for pervasive
hybridization within the complex, except for the two deviant instances of likely
cpDNA capture discussed above. Otherwise, the fairly low level of
phylogenetic structure revealed by cpDNA is more likely the result of
insufficient sequence variation and/or incomplete lineage sorting rather than
hybridization.
Because of the highly variable flower morphology is feasible that prezygotic isolation mechanisms, such as different pollinator behaviour, may
probably exist between many (sympatric) species within the complex.
However, for examining in detail whether interspecific hybridization was
involved in the low genetic differentiation within the B. lobbii complex
(especially clade E), more extensive population-level sampling and a broader
survey of both nuclear and cytoplasmic markers would be necessary. At least
based on the present analyses it appears that the expansion/fragmentation
scenario is more plausible than a scenario of pervasive hybridization.
4.4 Putative ancestral area and age of the Bulbophyllum lobbii
complex
According to the second DIVA analysis (with subclades E1-E4 reduced to
clade E; Luzon, Mindanao and Palawan combined to one area (Philippines);
clades coded according to their likely ancestral distribution; and outgroup
taxon B. emiliorum; Fig. 3.16), Borneo, Peninsula Malaysia, Sulawesi and
potentially the Philippines comprise the ancestral area of the B. lobbii
complex, and at least one dispersal event was necessary for this
reconstruction. However, both the Philippines and Sulawesi were never
connected to the Sundashelf and, thus, at least two independent dispersal
events are likely, i.e. from Borneo/Peninsula Malaysia to Sulawesi and to the
Philippines, respectively. Based on the ITS clock-enforced tree (Fig. 3.3), the
two distinct lineages occurring on the Philippines (clades C and D) are
younger than those of Sulawesi (“B. lobbii”; clade A) and Peninsula
Malaysia/Cameron Highlands (B. microglossum; clade B). Thus, the
80
Discussion
Philippines can safely be excluded as an ancestral area. The remaining
areas are therefore Borneo, Peninsula Malaysia and Sulawesi. As there was
only one lineage/species found on Sulawesi (i.e. “B. lobbii” (9, 15, 17)), I
suggest that the ancestral area of the B. lobbii complex should be Borneo
and Peninsula Malaysia which were connected by land during the late
Miocene/early Pliocene (see below).
For calibrating the ultrametric ITS tree (Fig. 3.3), four different
substitution rates (fast, moderate, slow and very slow) were used. The
“Bulbophylum rate” (very slow), which was inferred from a biogeographical
event, is without much doubt too slow, because this rate, if valid, would be
the slowest reported for plants in the literature, except that for Winteraceae
(for rates see Richardson et al., 2001). By contrast, the fast rate from
Robinsonia might be too fast, because this rate is the highest for ITS found in
plants. Thus, the most likely rate for the B. lobbii complex should lie between
the moderate and the slow rate.
4.5 Diversification of the B. lobbii complex, evolutionary processes
and historic-biogeographical scenario
Based on the above rate assumption, the origin of the B. lobbii complex was
between 3.6 and 8.2 Mya (see Fig. 3.3). Furthermore, the ages of origin of all
major clades are inconsistent with Pleistocene events. Rather, clades B and
A appear to have originated between 2.8 and 6.5 Mya and likely arose from,
respectively, the fragmentation of a widespread, most likely BorneoPeninsula
Malaysia
region
–
ancestor
in
the
west
(Peninsula
Malaysia/Cameron Highlands: B. microglossum); and a dispersal event
across the Makassar Strait (“Wallace’s line”) to the east (Sulawesi: B. lobbii
(9, 15, 17)). This dispersal event is not surprising, because in contrast to the
situation observed in animals, this sea barrier appears to be rather
unimportant for most plants (Dransfield, 1981; George, 1981; Moss, 1998).
Especially, the montane flora of Sulawesi is similar to that of Borneo
(Balgooy, 1987; Whitten et al., 1987; Moss & Wilson, 1998). This may be,
because plants – in general – are considered good oversea dispersers
(Briggs, 1987). The fragmentation, which led to the origin of clade B, could
probably have taken place in the Pliocene, due to the rise in sea level to up
81
Discussion
to 140 m above present sea level (Ridder-Numan, 1998). Also the estimated
ages of clades C, D and E appear to be fairly ancient (2.3 – 5.2 Mya) with
clades C and D presumably arising independently following dispersal events,
to Luzon (Philippines), probably across the Sulu/Celebes Seas, which formed
deep water barriers from the Eocene onwards (Hall, 1998). Implicitly, the
origin of B. coweniorum/B. piestoglossum and B. facetum would be a result
of oversea dispersals, at least based on the ITS evidence (Fig. 3.3). By
contrast, the putative Sundashelf ancestor of clade E, which may have
originated in Borneo/Philippines (node 3; Fig. 3.16), started diversifying
approximately 1.5 – 3.4 Mya, and gave rise to several species that clearly
evolved at the Plio-/Pleistocene boundary or well within the (Late)
Pleistocene. During glacials, the extent of rainforest habitats was probably
severely restricted to mountainous regions on the exposed Sundashelf due to
arid climatic conditions (Brandon-Jones, 1998; but see Kershaw et al., 2001).
Opportunities for rainforest expansion were likely (re-)established when the
climate improved during interglacials, but at those times the Sundashelf was
fragmented due to a rise in sea level! Thus, the rainforest was perhaps
permanently disjunct or insular in distribution (Fig. 1.3) and has mainly
dispersed across sea barriers (Brandon-Jones, 1998). Several authors (e.g.
Kershaw et al., 2001), however, also content that despite the generally drier
climate during glacials, the exposure of land/continental shelf likely resulted
in a net increase in the area of rainforest rather than a reduction (see also
Fig. 1.4). As the species of the B. lobbii complex are restricted to
(sub)montane rainforests and probably poor dispersers (due to their
symbiosis with fungi which are probably absent in new habitats; Dressler,
1981), the once widespread ancestor of clade E likely followed the repeated
range expansion/contraction cycles of the rainforests. In fact, it is feasible
that in times of glacial periods, when area of rainforest likely increased (see
above), the range of the common ancestor of clade E also expanded,
perhaps facilitated by the giant riverine systems supporting gallery forests
(see Voris, 2000; Gorog et al., 2004). If correct, interglacial island formation
and continental vicariance events would have mainly led to speciation due to
geographic isolation, whereby a widespread, B. lobbii-like (polymorphic)
ancestor of clade E would have very recently given rise to numerous
82
Discussion
locally/regionally
restricted
derivatives,
such
as
B.
polystictum,
B.
cameronense, B. sumatranum, B. orectopetalum and B. siamense (see p.
60). More detailed hypotheses concerning the Pleistocene history of clade E,
however, must await a robust molecular phylogeography for this group in
general, and still difficult and widespread taxa (B. lobbii, B. dearei) in
particular, ideally at a high level of spatial and temporal resolution.
4.6 Yellow vs. red flower variation within the B. lobbii complex
Within the B. lobbii complex there are species with redish flowers (B.
facetum/clade D, B. coweniorum/clade C and B. sumatranum/within subclade
E4) and yellowish flowers (all other species). Within subclade E4 all red B.
sumatranum plants are placed within a cluster of yellow B. lobbii plants (in
both the ITS and AFLP trees; Figs. 3.3, 3.8). Thus, the redish flower morph
could have originated from the yellowish flower morph at least three times,
i.e. in clades C and D, and within subclade E4. If correct, such parallel
evolution of red flowers could reflect adaptation to various enviroments
and/or pollinators, although, testing this hypothesis requires a more fully
resolved internal tree topology (in both ITS and AFLPs) and a detailed
character mapping approach using, e.g., MACLADE 4 (Madison & Madison,
2003). While redish flowering plants are always associated with a foully
scent, the yellowish flowering plants usually smell fruit-like. However, one
yellow flowering B. lobbii (32) from the Philippines, which unfortunately was
analysed only morphologically, had a foully scent as well. Further studies are
needed to provide deeper insights into how far the evolution of flower scent
and colour are associated within the B. lobbii complex.
4.7 The species within the B. lobbii complex
As mentioned in the introduction (1.2 study system) the name “B. lobbii“
covers a whole series of plants (in herbaria and the literature) that based on
the present ITS/AFLP analyses perhaps should be assigned to different
species. Furthermore, as seen in the morphometric analyses B. lobbii is
morphologically very variable, being present in clade A and all subclades of
clade E. In the discussion below, which is based on the molecular data
83
Discussion
(mostly AFLP for subclades of E; ITS for all other clades), the morphological
analysis and personal observations, I will try to give some information, which
might be useful for further taxonomical work.
Clade A consists of B. lobbii accessions which are restricted to
Sulawesi. Dr. J. J. Vermeulen (personal communication) suggests that this
could be regarded a new species (B. hyalosemoides), and all molecular data
support this view. Unfortunately, no such accessions were available for
morphometric analysis.
The species within clades B, C and D are very well distinguishable
according to the molecular and (largely) the morphological data and, thus,
the following species names should be retained: B. microglossum (clade B),
B. coweniorum (clade C) and B. facetum (clade D).
All morphologically analysed accessions of B. polystictum and B. lobbii
within AFLP subclade E1 (Fig. 3.8) also broadly cluster together in the PCA
(Fig. 3.17) by having very long peduncles and large flowers. Furthermore,
they look very similar (in shape of sepals and petals) to that one from the
collection of Lobb, which was described by Lindley (1847). As the three
accessions of B. polystictum are interspersed within this subclade (Fig. 3.18),
I do not agree with Ridley (1909) and Seidenfaden (1997) that this taxon
should be recognized as a separate species. Thus, I suggest that all plants
within subclade E1 should be called B. lobbii (B. lobbii s. str.; distribution:
Java/Bali, Peninsula Malaysia, Borneo and Luzon).
However, I do agree with Seidenfaden (1997) that B. lobbii is absent
from Thailand and I am quite sure (based on personal communication with G.
Fischer) that B. lobbii is not present north of the Isthmus of Kra.
Four species could be found within AFLP subclade E2 (B. siamense,
B. orectopetalum, B. cameronense and “B. lobbii”). The distribution of B.
siamense and B. orectopetalum is probably restricted to the area north of the
Isthums of Kra (i.e. to Assam, Thailand, Myanmar). The analysed material of
B. siamense had much larger flowers and longer peduncles (see distinct
grouping of this species in Fig. 3.18) than the B. orectopetalum accessions,
while the shape of the flowers of these two species are similar. I agree with
Garay et al. (1992) and Seidenfaden (1997) that B. siamense should be split
in B. siamense and B. orectopetalum. The B. lobbii (33) accession from
84
Discussion
Luzon grouped next to B. orectopetalum in the morphological analysis (Fig.
3.17) and next at the base of the B. siamense/B. orectopetalum cluster in the
AFLP analysis, albeit with little support (53 %). This accession might be
ascribed to B. orectopetalum although it is genetically distinct. By contrast,
the molecular (AFLP and ITS) and the morphological (PCA) data show that
B. cameronense is a well distinct species which should be retained.
In the AFLP tree (Fig. 3.8), subclade E3 consists of three species (B.
dearei, “B. lobbii” and B. facetum), but mostly of B. dearei, and with B.
facetum (clade D) nested within the latter. B. dearei is a well recognizable
species and, therefore, this name should be retained. The different groupings
of B. facetum (forming a separate clade in the ITS tree, Fig. 3.3) is difficult to
explain but there can also be no doubt that this species forms a distinct
genetic entity (the different grouping could be explained by, e.g., differential
lineage sorting of ITS vs. AFLP markers although this is fairly speculative).
Although the B. lobbii accessions (1, 4) from Luzon and Sumatra within
subclade E3 were found to be similar tho B. facetum and B. orectopetalum
(Figs. 3.17, 3.18), respectively, nonetheless differ genetically from these
species and also differ morphologically in flower shape and colour from B.
dearei and in flower shape from B. lobbii s. str. (see above). These “B. lobbii”
accessions (1, 4) can only be named “B. lobbii s. lat.” at this time.
Finally, subclade E4 includes B. sumatranum and “B. lobbii“. B.
sumatranum is well discernable (ITS and especially AFLP evidence), and
should be referred to as a distinct species (despite morphological overlap
with, e.g. B. coweniorum, B. facetum and B. dearei in the PCA; Fig. 3.18)
The numerous B. lobbii (7, 16, 19) accessions from Palawan and the
Cameron Highlands within this subclade differ in scent, flower shape and
colour from B. sumatranum and, therefore can only be named “B. lobbii s.
lat.”. Note that within the taxonomically highly problematic subclades E3 and
E4 there are fairly well delimitated tip clusters comprising B. dearei/B.
facetum and B. sumatranum, respectively, that appear to have evolved
locally (Mindanao, Borneo, Tioman Island/Luzon; Sumatra) from an ancestral
“B. lobbii-like” stock (still widespread) – a process which renders the latter
‘taxon’ highly paraphyletic with respect to these latter taxa. In fact, it appears
from the molecular analyses that “B. lobbii” is a paraphyletic base group (see
85
Discussion
Kadereit et al., 1997) of the entire clade E and most of its constituent taxa
(probably except B. facetum).
According to the above suggestions, the B. lobbii complex consists of
13 mostly well defined species (B. hyalosemoides ined., B. microglossum, B.
coweniorum, B. piestoglossum, B. facetum, B. lobbii, B. siamense, B.
orectopetalum, B. cameronense, B. dearei, B. sumatranum, B. smitinandii
and B. monanthum). At present time, however, it is hardly possible to present
a satisfactory reply to taxonomical questions and further taxonomic and
molecular work should be done.
86
Conclusion
5. Conclusion
The results of this study show a strong influence of the recent (Plio/Pleistocene) history of the Indo-Malay Archipelago on the evolution of the B.
lobbii complex. Especially periodical fragmentation and reconnection of the
Sundashelf islands (to each other and to the adjacent mainland) due to the
rising and falling of sea level, respectively, have triggered diversification and
speciation. But also oversea dispersal and continental mainland vicariance
likely played a fundamental role in the evolutionary history of this plant group.
Thus, the first onset of diversification of the B. lobbii complex took place in
the Late Miocene or Pliocene, when the range of a putative widespread
(Borneo/Peninsula Malaysia) ancestor was fragmented due to rising sea level
leading to clade B in the Cameron Highlands/Peninsula Malaysia. At about
the same time, clade A originated after a dispersal event to Sulawesi, which
was never connected to the Sundashelf. The putative ancestor persisted in
the Sundashelf region and gave rise to (i) two clades (C and D) after
independently colonizing Luzon/Philippines (also never connected to the
Sundashelf) in the Pliocene, and (ii) finally subclades E1-E4 during the
Pleistocene. The ancestor of these subclades likely expanded its range at
times of continuous land connection during glacials (as the net area of
rainforests increased, despite generally drier conditions) and gave rise to
several species most likely in times of inter-glaciation when the Sundashelf
became fragmented due to rising sea levels. However, diversification and
speciation may have also been triggered when rainforests (also on the
mainland) were reduced to refuges during the dry glacials. The putative
ancestor of clade E, however, likely persisted (“B. lobbii” s. lat.) all the time,
and thus forms today a paraphyletic base group, as can be inferred from the
molecular data. It appears, that palaeogeographical circumstances allowed
this base group to evolve at a faster rate than earlier-evolving basal lineages
(A-D) that remained geographically restricted and species-poor at the
margins of the Sundashelf region (i.e. Sulawesi, Luzon/Philippines, Cameron
Highlands/Peninsula Malaysia). In this context, the B. lobbii complex is a
good study system to illustrate the impact of historical contingency on
differential rates of plant diversification and speciation in the Indo-Malayian
Archipelago.
87
Abstract
6. Abstract
Recently, it has been suggested that the distribution and evolution of plants
and animals in Southeast-Asia may owe much more to the last one or two
million years than the preceding 30 million years. In context, there has been
a resurgence of debate among zoologists on whether increased aridification
during Pleistocene glacial periods and/or postglacial vicariance caused by
rising sea levels triggered the radiation of rain forest taxa especially in the
western part of the Malay Archipelago. Here, I test the hypothesis of
Pleistocene speciation by focusing on the phylogenetic relationships and
divergence times of submontane to montane-rainforest-restricted orchids
(Bulbophyllum lobbii complex, c. 12 species) from the Sundashelf region
(Peninsula Malaysia, Sumatra, Java, Borneo), the Philippines, Sulawesi and
adjacent mainland areas (Thailand, Myanmar), using nuclear rDNA internal
transcribed spacer (ITS), chloroplast (cp) DNA sequences, AFLP markers
and morphological analyses.
Phylogenetic analyses of both marker systems provide initial evidence
of the monophyly of the complex and the existence of five well-supported
clades (A–E). ITS tree topologies suggest a near simultaneous origin of (i)
two basal clades (A: B. hyalosemoides/Sulawesi; B: B. microglossum/
Peninsula Malaysia-Cameron Highlands); and (ii) three clades comprising the
crown group, two of which are also species-poor and geographically largely
restricted
(C:
B.
coweniorum/Philippines-Luzon,
Laos
(?),
B.
piestoglossum/Philippines-Luzon; D: B. facetum/Philippines-Luzon), while the
third clade (E) is species-rich and widespread. Within this latter clade both
ITS and AFLPs allow delimitating four subclades (and up to seven species),
but phylogenetic relationships remain poorly resolved.
Based on ITS clock estimates, it is argued that the origins of all five
major clades are inconsistent with Pleistocene events. Rather, clades B and
A appear to have originated approximately 2.8 to 6.5 Mya, and likely arose
from, respectively, (i) the fragmentation of a widespread - most likely BorneoPeninsula Malaysia region – ancestor in the west (Peninsula Malaysia); and
(ii) a dispersal event across the Makassar Strait (“Wallace’s line”) to the east
to Sulawesi. Also, the estimated ages of clades C-E appear to be fairly
ancient (approximately 2.3 - 5.2 Mya), with clades C and D presumably
88
Abstract
arising independently, following dispersal events to the north (Philippines),
i.e. across the Sulu/Celebes Seas, which formed deep water barriers from
the Eocene onwards. By contrast, the putative Sunda-region ancestor of
clade E started diversifying approximately 1.5 to 3.4 Mya, and gave rise to
several species that clearly evolved in the (Late) Pleistocene.
In sum, four of the five major clades identified represent narrow
paleorelicts (A–D) that became isolated in the Late Miocene to Pliocene
when Southeast Asia still experienced a perhumid climate and tropical rain
forest was widespread. In contrast, while also having originated in the Late
Miocene to Pliocene, clade E experienced a radiation. I posit that this species
proliferation could reflect vicariant events in the Sundashelf region (viz
rainforest fragmentation), likely triggered by the deterioration of global
climatic conditions in the early to mid Pliocene followed by a marked increase
in aridification (≤ 2.5 Mya), especially during glacials. However, it appears
equally likely that much of the most recent diversification and speciation
events surrounding clade E also result – at least in the Sundashelf region –
from inter-glacial island formation due to rising sea levels. Thus, barring high
levels of oversea dispersal (which appears to be unlikely for this orchid
group), the high level of genetic similarity observed among geographically
restricted and disjunct species of clade E would reflect common ancestry
from a widespread, polymorphic “B. lobbii-like” population/species that likely
expanded its range at times of continous land connection, i.e. during glacials
(!) when the net area of rainforests increased – as recently suggested by
palaeogeographers – and despite generally drier conditions. Taken together,
the present study illustrates that plant evolution in the Indo-Malay
Archipelago proceeded at different rates at different times, likely dependent
on historical/palaeogeographical circumstances.
89
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100
Appendix
8. Appendix
Table 8.1: In general, material used for molecular (ITS; cpDNA: trnH-psbA, trnE-trnD; AFLP)
and morphological (Morph) analysis was collected and determined by G. Fischer. Living
plants were cultivated in the glasshouses of the Botanical Gardens in Salzburg and Vienna;
herbarium specimens and/or Kew-conserved samples (vouchers) are stored at the University
of Salzburg. Accessions marked by an asterisk were obtained only as silica dried leaves by
Dr. J. J. Vermeulen, who collected and determined the species (vouchers in the Botanical
Garden in Leiden, The Netherlands). Thus these accessions could not be included in the
morphometric analysis. Accessions/species marked by a superscript (a) are shown in
chapter 9 (colour plates of B. lobbii complex and extra-complex species).
Taxon
Locality
Voucher
ITS
no.
trnH-
trnE-
psbA
trnD
AFLP
Morph
B. cameronense Garay, Hamer & Siegerist
Peninsula Malaysia; Cameron Highlands
OR 90/99
+
+
+
+
+
Peninsula Malaysia; Cameron Highlands
OR 64/99
+
-
-
+
+
3
Peninsula Malaysia; Cameron Highlands
OR 132/99
+
-
-
+
-
4
Peninsula Malaysia; Cameron Highlands
OR 92/99
-
-
-
+
+
Philippines; Luzon; Nueva Vizcaya
OR 241/98
+
+
+
+
+
2
Philippines; Luzon; Nueva Vizcaya
OR 219/98
+
+
+
+
-
3
Philippines; Luzon; Nueva Vizcaya
OR 222/98
-
-
-
+
-
4
Philippines; Luzon; Nueva Vizcaya
OR 32/05
-
-
-
+
-
5
Philippines, Luzon; Nueva Vizcaya
OR 294/98
-
-
-
Philippines; Mindanao; Agusan del Sur
OR 136/98
+
+
+
+
+
2
Philippines; Mindanao; Bukidnon
OR 227/98
+
+
+
+
+
3
Peninsula Malaysia; Tioman Island
OR 606/98
+
+
+
+
+
4*
Borneo; Sarawak; Gunung Serapi
932551
+
+
+
+
-
5
No locality information available
SBG713
+
+
+
+
-
6
Philippines; Mindanao; Bukidnon
OR 12 /98
+
-
-
+
+
7
Philippines; Mindanao; Bukidnon
OR 165/98
+
-
-
+
+
8
Philippines; Mindanao; Bukidnon
OR 243/98
+
-
-
+
-
9*
Peninsula Malaysia; Tioman Island
SBG740
+
-
-
+
-
10
Philippines; Mindanao; Bukidnon
OR 398/98
-
-
-
+
+
11
Philippines; Mindanao; Bukidnon
OR 397/98
-
-
-
+
+
12
Philippines; Mindanao; Agusan del Sur
OR 146/98
-
-
-
+
-
13
Philippines; Mindanao; Bukidnon
OR 166/98
-
-
-
+
-
14*
Borneo; Sarawak; Gunung Mulu NP
980433
-
-
-
+
-
15
Philippines; Mindanao; Bukidnon
OR 176/98
-
-
-
+
-
1
2
a
B. coweniorum J.J. Vermeulen & P. O'Byrne
1
a
+
B. dearei Reichb.f.
1
a
B. facetum Garay, Hamer & Siegerist
101
Appendix
Philippines; Luzon; Nueva Vizcaya
OR 215/98
+
+
+
+
+
Philippines; Luzon; Nueva Vizcaya
OR 16/98
+
-
-
+
+
3
Philippines; Luzon
OR 223/99
+
-
-
+
+
4
Philippines; Luzon; Nueva Vizcaya
OR 228/98
+
-
-
+
+
5
Philippines; Luzon; Nueva Vizcaya
OR 218/98
+
-
-
+
+
6
Philippines; Luzon;
OR 619/99
-
-
-
+
-
1
2
a
B. lobbii Lindl.
1
a
Philippines; Luzon; Zambales
OR 390/98
+
+
+
+
+
2
a
Philippines; Luzon
OR 211/03
+
+
+
+
+
3
Indonesia; Borneo
OR 292/99
+
+
+
+
+
4
Indonesia; Sumatra; Sinabung Volcano
OR 620/99
+
+
+
+
+
5
Indonesia, Bali
OR 337/99
+
+
+
+
+
6
Indonesia; Sumatra; Padang
OR 126/05
+
+
+
+
+
Philippines; Palawan
OR 61/05
+
+
+
+
+
8
Peninsula Malaysia; Cameron Highlands
OR 28/99
+
+
+
+
+
9*
Indonesia; Sulawesi; Pendolo Divide
SBG5059
+
+
+
+
-
10*
Borneo; Sarawak; G. Penrissen
970761
+
+
+
+
-
11*
Borneo; Sarawak; Kelabit Highlands
980107
+
+
+
+
-
12
Indonesia; Sumatra; Solok
OR 317/03
+
+
+
+
-
13*
Borneo; Sabah; Upper Matang River
+
+
+
+
-
14*
Borneo; Sabah; Road to Kinabalu
SBGO
3796
SBGO
4758
SBGO
5728
+
+
+
+
-
+
+
+
+
-
7
a
15*
a
Indonesia; Sulawesi; Poso District
16
No locality information available
OR 160/05
+
+
+
+
-
17*
Indonesia; Sulawesi; Kpg. Doda
SBGO
4903
+
+
-
+
-
18
Indonesia; Bali
OR 377/99
+
-
-
+
+
19
Peninsula Malaysia; Cameron Highlands
OR 21/99
+
-
-
+
+
20
No locality information available
OR 145/05
+
-
-
+
+
21
Philippines; Palawan
OR 152/05
+
-
-
+
-
22
Peninsula Malaysia; Cameron Highlands
OR 100/99
-
-
-
+
+
Indonesia; Bali
OR 452/99
-
-
-
+
+
24
No locality information available
OR 86/05
-
-
-
+
+
25
Peninsula Malaysia; Cameron Highlands
OR 602/99
-
-
-
+
+
26*
Borneo; Kalimantan; Apo Kayan
913488
-
-
-
+
-
27*
Peninsula Malaysia; Gunung Ledang
960117
-
-
-
+
-
28
Indonesia; Sumatra
OR 107/01
-
-
-
+
-
29*
Indonesia
-
-
-
+
-
30*
Indonesia
SBGO
1818
SBGO
1840
-
-
-
+
-
31
No locality information available
OR 202
-
-
-
-
+
32
Philippines, Mindanao
OR 485/03
-
-
-
-
+
Philippines; Luzon
OR 22/05
+
-
-
+
+
23
33
a
a
B. microglossum Ridl.
a
102
Appendix
1
Peninsula Malaysia; Cameron Highlands
OR 161/99
+
+
+
+
+
2
Peninsula Malaysia; Cameron Highlands
OR 160/99
+
-
-
+
+
3
Peninsula Malaysia; Cameron Highlands
OR 163/99
-
-
-
+
-
4
Peninsula Malaysia; Cameron Highlands
OR 537/99
-
-
-
+
-
B. orectopetalum Garay, Hamer & Siegerist
1
Thailand
OR 283/99
+
+
+
+
+
2
Thailand
OR 294/99
+
-
-
+
+
Thailand
OR 18/01
-
-
-
+
+
4
Thailand
OR 204/03
-
-
-
+
+
5
Thailand
OR 546/98
-
-
-
+
+
6
Thailand
OR 337/98
-
-
-
Peninsula Malaysia; Cameron Highlands
OR 169/99
+
+
+
+
-
Peninsula Malaysia; Cameron Highlands
OR 603/99
+
-
-
+
+
Peninsula Malaysia; Cameron Highlands
OR 599/99
-
-
-
+
+
OR 237/01
+
+
+
+
+
HBV 3601
+
+
+
+
-
Indonesia; Sumatra
OR 276/99
+
+
+
+
+
2
Indonesia; Sumatra
OR 269/99
+
-
-
+
+
3
Indonesia; Sumatra
OR 148/05
+
-
-
+
+
4
Indonesia; Sumatra
OR 156/05
-
-
-
+
+
5
Indonesia; Sumatra
OR 150/05
-
-
-
+
-
6
Indonesia; Sumatra
OR 154/05
-
-
-
+
-
7
Indonesia; Sumatra
OR 158/05
-
-
-
+
-
No locality information available
OR 209/99
+
+
+
+
-
2
Myanmar; Shan; north of Aungban
OR 228/01
+
+
+
+
-
3
Nepal
OR 488/03
-
-
+
+
+
Philippines; Luzon; Nueva Vizcaya
OR 277/98
-
-
-
+
-
OR 342/98
+
+
+
+
+
Philippines; Mindanao; Surigaro del Norte OR312/98
+
-
-
+
-
OR 442/03
+
-
-
+
-
OR 281/98
+
-
-
3
a
+
B. polystictum Ridl.
1
2
3
a
B. siamense Reichb.f.
1
a
Myanmar; Shan; north of Aungban
a
B. smitinandii Seidenf. & Thorut
1
Thailand
B. sumatranum Garay, Hamer & Siegerist
1
a
Outgroups:
B. affine Lindl.
1
a
B. alsiosum Ames
1
a
B. emiliorum Ames & Quisumb.
1
2
No locality information available
a
B. hamatipes J. J. Sm.
1
Java
B. macranthum Lindl.
1
a
Philppines; Luzon, Nueva Ecicha
-
103
Appendix
B. membranifolium Hook.f.
1
Peninsula Malaysia; Cameron Highlands
OR 575/98
+
-
-
+
-
OR 144/05
+
-
-
+
+
OR 541/99
+
-
-
+
-
OR 103/98
+
+
+
+
+
960106
+
-
-
+
-
a
B. monanthum (Kuntze) J.J. Sm.
1
No locality information available
B. patens King ex Hk. f.
1
Peninsula Malaysia; Pulau Redang
B. piestoglossum J.J. Vermeulen
1
a
Philippines; Luzon; Bicol
B. pileatum Lindl.
1*
No locality information available
104
Appendix
Table 8.2: Primers used for ITS and cpDNA amplification and sequencing (grey).
Marker
Primername Direction Sequence
Literature
nrDNA
ITS
AB 102R
reverse
TAGAATTCCCCGGTTCGCTCGCCGTTAC
AB 101F
forward
ACGAATTCATGGTCCGGTGAAGTGTTCG
ITS4
reverse
TCCTCCGCTTATTGATATGC
ITS5
forward
GGAAGTAAAAGTCGTAACAAGG
TAB C
reverse
CGAAATCGGTAGACGCTACG
TAB F
forward
ATTTGAACTGGTGACACGAG
trnHGUG
reverse
CGCGCATGGTGGATTCACAATCC
Tate et al., 2003
psbA
forward
GTTATGCATGAACGTAATGCTC
Sang et al., 1997
reverse
ACCAATTGAACTACAATCCC
forward
CTACCACTGAGTTAAAAGGG
reverse
ACCAATTGAACTACAATCCC
Demesure et al.,
trnEUUC
forward
AGGACATCTCTCTTTCAAGGAG
1995
UGA
reverse
GAGAGAGAGGGATTCGAACC
trnfM
forward
CATAACCTTGAGGTCACGGG
TabE
reverse
GGTTCAAGTCCCTCTATCCC
Taberlet et al., 1991
ndhJ
forward
ATGCCTGAAAGTTGGATAGG
Vijberg et al., 1999
rpoB
reverse
C(GT)ACAA(CT)CC(CT)AATTG
forward
CACCC(AG)GATT(CT)GAACTGGGG
TabA
reverse
CATTACAAATGCGATGCTCT
TabB
forward
TCTACCGATTTCGCCATATC
trnSGCU
reverse
TTTTACCACTAAACTATACCCGC
trnG2S
forward
GAGAGAGAGGGATTCGAACC
trnYGUA
reverse
CCGAGCTGGATTTGAACCA
trnTGGU
forward
CTACCACTGAGTTAAAAGGG
trnSGGA
reverse
TTACCGAGGGTTCGAATCCCTC
UGU
trnT
forward
AGGTTAGAGCATCGCATTTG
trnSGGA
reverse
TTACCGAGGGTTCGAATCCCTC
TabB
forward
TCTACCGATTTCGCCATATC
Sun et al., 1994
Baldwin et al.,1992
cpDNA
trnL-trnF
trnH-psbA
trnD-trnT
trnD-trnE
trnS-trnfM
trnL-ndhJ
rpoB-trnC
trnD
trnS-trnG
trnY-trnT
trnS-trnT
trnS-trnL
F
trnTGGU
trnD
GUC
F
trnS
CAU
trnC
trnT-trnL
GUC
GCA
CCCTTCATTCTTCCTCTACTATGTTG
Taberlet et al., 1991
Ohsako et al., 2000
Taberlet et al., 1991
Hamilton, 1999
Demesure et al.,
1995
Shaw et al., 2005
Taberlet et al., 1991
rpL16-
rpL16R1516 reverse
Intron
rpL16F71
forward
GCTATGCTTAGTGTGTGACTCGTT
rpS16-
rps16R
reverse
AACATC(AT)ATTGCAA(GC)GATTCGATA
Oxelman et al.,
Intron
rps16F
forward
AAACGATGTGGTA(AG)AAAGCAA
1997
Small et al., 1998
105
Appendix
Table 8.3: Mastermix for cpDNA and ITS amplification
Reagent
Volume in µl
Purified water
7.45
F-Taq polymerase buffer (10x)
5
MgCl2 (25 mM)
8
BSA (20 mg/ml)
1
dNTP mix (10 mM of each dNTP)
1
Primer 1 (10 µM)
1
Primer 2 (10 µM)
1
F-Taq polymerase (Fermentas; 5 U/µl)
0.25
Table 8.4: PCR-programmes for amplification of ITS and cpDNA regions.
Temperature
Region
Cycle
(°C)
Time
1
94
2 min
94
1 min
50
1 min
72
2 min
1
72
7 min
1
94
3 min
94
30 sec
ndhj, trnS-trnfM, 30
55
30 sec
trnH-psbA
72
1 min 40 sec
1
72
7 min
1
96
5 min
96
1 min
57
2 min
72
3 min
1
72
7 min
1
94
5 min
94
45 sec
58
30 sec
72
1 min
72
5 min
ITS
trnL-trnF,
rpoB-trnC,
trnS-trnG
trnD-trnE,
trnT-trnL
35
trnL-
30
25
1
106
Appendix
Table 8.5: Primers used in the primer-trial for AFLP analysis. Primers coloured, according to
their fluorescence, were finally used for analysis.
Labelling
Primer 1
Primer sequence 1
Primer 2
Primer sequence 2
EcoRI
GACTGCGTACCAATTC
MseI
GATGAGTCCTGAGTAA
MseIx
M-C
Preselective PCR
EcoRIx
E-A
Selective PCR
Blue
(6-
FAM)
Yellow
(NED)
EcoRIxxx
E-ACG
M-CAT
E-ACT
M-CAG
E-ATC
M-CTA
E-ACA
M-CAC
E-AAC
M-CTT
E-AAC
MseIxxx
M-CTG
E-AAC
M-CAG
E-ACC
M-CAT
E-AAG
M-CTG
Green
E-AGG
M-CAA
(VIC)
E-ACG
M-CTT
E-AGG
M-CTG
Table 8.6: Restriction-digestion reaction mix using 5.5 µl (500 ng) DNA.
Reagent
Volume in µl
Purified water
0.405
T4 DNA ligase buffer (10x)
1.1
NaCl (500 mM)
1.1
BSA (1 mg/ml)
0.55
MseI restriction endonuclease (50 U/µl)
0.02
EcoRI restriction endonuclease (40 U/µl)
0.125
T4 DNA ligase (5 U/µl)
0.2
MseI adapter pair (10 µM)
1
EcoRI adapter pair (10 µM)
1
107
Appendix
Table 8.7: Pre-selective PCR for 1.5 µl Restriction-digestion solution (10 x diluted).
Reagent
Volume in µl
Purified water
7.45
AmpliTaq polymerase buffer (10x)
1.25
MgCl2 (25 mM)
0.75
dNTP mix (10 mM of each dNTP)
1
EcoRI primer (E.A; 10 µM)
0.25
MseI primer (M.C; 10 µM)
0.25
AmpliTaq polymerase
0.05
(Applied Biosystems; 10 U/µl)
Table 8.8: Selective PCR for 1.5 µl pre-selective PCR product (10 x diluted).
Reagent
Volume in µl
Purified water
5.95
AmpliTaqGold polymerase buffer (10x)
1.25
MgCl2 (25 mM)
1.25
dNTP mix (10 mM of each dNTP)
1
BSA (1mg/ml)
0.1
EcoRI primer (E.AXX; 10 µM)
0.1
MseI primer (M.CXX; 10 µM)
0.25
AmpliTaqGold polymerase
0.1
(Applied Biosystems; 10 U/µl)
108
Appendix
Table 8.9: PCR programmes used for (pre-)selective fragment amplification in the AFLP
analysis.
Preselective
Cycle
Temperature (°C)
Time
1
72
2 min
94
30 sec
56
30 sec
72
1 min
1
72
10 min
1
95
10 min
94
30 sec
65 - 56 decrease by 0.7
1 min
72
1 min
94
30 sec
56
1 min
72
1 min
72
10 min
30
13
Selective
23
1
109
Pictures of the B. lobbii complex and extra-complex species
9. Colour plates of the B. lobbii complex and extracomplex species
Fig. 9.1: Bulbophyllum lobbii (15), Sulawesi; Bulbophyllum hyalosemoides ined. (clade A).
Picture taken by Dr. J. J. Vermeulen
Fig. 9.2: Bulbophyllum microglossum,
Cameron Highlands/Peninsula Malaysia (clade B).
110
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.3: Bulbophyllum coweniorum (1), Luzon/Philippines (clade C).
Fig. 9.4: Bulbophyllum coweniorum, Luzon/Philippines (clade C).
111
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.5: Bulbophyllum facetum (2), Luzon/Philippines (clade D).
Fig. 9.6: Bulbophyllum polystictum (3), Cameron Highlands/Malaysia (subclade E1).
112
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.7: Bulbophyllum polystictum, Cameron Highlands/Malaysia (subclade E1).
Fig. 9.8: Bulbophyllum lobbii (2), Luzon/Philippines (subclade E1).
113
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.9: Bulbophyllum lobbii (23), Bali (subclade E1).
Fig. 9.10: Bulbophyllum lobbii, Borneo (subclade E1).
114
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.11: Bulbophyllum orectopetalum (3), Thailand (subclade E2).
Fig. 9.12: Bulbophyllum cameronense (2), Cameron Highlands/Malaysia (subclade E2).
115
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.13: Bulbophyllum siamense (subclade E2). Picture taken by A. Sieder.
Fig. 9.14: Bulbophyllum lobbii (33), Luzon/Philippines (subclade E2).
116
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.15: Bulbophyllum dearei (1), Mindanao/Philippines (subclade E3).
Fig. 9.16: Bulbophyllum dearei (1), Mindanao/Philippines (subclade E3).
117
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.17: Bulbophyllum lobbii (1), Luzon/Philippines (subclade E3).
Fig. 9.18: Bulbophyllum sumatranum (1), Sumatra (subclade E4).
118
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.19: Bulbophyllum sumatranum, Sumatra (subclade E4).
Fig. 9.20: Bulbophyllum lobbii (7), Palawan/Philippines (subclade E4).
119
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.21: Bulbophyllum smitinandii, Thailand. Picture taken by Suitee.
Fig. 9.22: Bulbophyllum piestoglossum (1), Luzon/Philippines (extra-complex).
120
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.23: Bulbophyllum monanthum (extra-complex).
Fig. 9.24: Bulbophyllum emiliorum (2), Mindanao/Philippines (extra-complex).
121
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.25: Bulbophyllum affine ((1); extra-complex).
Fig. 9.26: Bulbophyllum macranthum (1), Luzon/Philippines (extra-complex).
122
Pictures of the B. lobbii complex and extra-complex species
Fig. 9.27: Bulbophyllum alsiosum (extra-complex). Picture taken by A. Sieder.
123
Acknoledgements
Acknowledgements
I would like to thank all the people supporting me in writing my master thesis,
especially:
Prof. Dr. Hans Peter Comes (supervisor), Department of Organismic Biology,
Study Group Ecology and Biodiversity of Plants, University of Salzburg,
Austria.
Mag. Gunter Fischer (for the possibility to ask all kinds of questions),
Department of Organismic Biology, Study Group Ecology and Biodiversity of
Plants, University of Salzburg, Austria.
Labor of Prof. Dr. Tod F. Stuessy, especially Dr. Peter Schönswetter and
Prof Dr. Rose Samuel (for helping in AFLP-fingerprint technique),
Department of Sytematic and Evolutionary Botany, University of Vienna,
Austria.
Dr. Jaap Jan Vermeulen (for supporting silica dried leaf material and aiding in
morphological analysis), Nationaal Herbarium Nederland, Universit of Leiden,
The Netherlands.
Anton Sieder (for searching in literature and supporting some plants),
Botanical Garden, University of Vienna, Austria.
Elisabeth Egger (for attendance of the plants), Botanical Garden, University
of Salzburg, Austria.
Mag. Birgit Fuchs and Mag. Cornelia Moser (for helping in the lab),
Department of Organismic Biology, Study Group Ecology and Biodiversity of
Plants, University of Salzburg, Austria.
124
Curriculum vitae
Curriculum vitae
Curriculum Vitae
Gerald Hochschartner
Weyerstrasse 15
4810 Gmunden
Austria
Tel.: +43 650 8615198
E-mail:
gerald.hochschartner@sbg.ac.at
Personal Information
Date of birth:
Place of birth:
Nationality:
Marital status:
Children:
April 11, 1974
Gmunden, Austria
Austrian
Single
None
Education
1989 – 1993
1995 – 1999
2000 – 2004
Since February 2004
Since April 2005
Electrician apprenticeship at Steyrermühl
Papier AG, Steyrermühl, Austria; Final exam in
February 1993
Commercial School (part-time) in Vöcklabruck,
Austria; Diploma in June 1999
Studies in Genetics and Molecular Biology at
the University of Salzburg, Austria; Bachelor
degree in Biology (Bakk. Biol) in February
2004
Student of Genetics/Biotechnology and
Botany/Plant Biology at the University of
Salzburg, Austria
Master thesis on molecular biogeography of
the Bulbophyllum lobbii-complex (Sect.
Sestochilos, Orchidaceae) in Southeast-Asia;
Supervisor Prof. H. P. Comes
125
Curriculum vitae
Previous Activities
1993
1993 – 1994
1994 – 1995
1995
1996
1997 – 1999
2000
Summer 2001, 2003, 2004
Military Service, Austria
Assistant at the paper factory in Steyrermühl
Electrician for B.C. Industry Montage at
Jenbacher Werke, Jenbach
Electrician for Dorner & Mayer at the
petroleum factory OMV, Vienna
Electrician at Ebner Productronic, Gmunden
Electrician at Ing. Pesendorfer Engineering,
Gmunden
Electrician
at
Ing.
Rauch
Industrial
Engineering, Gmunden
Electrician for Johann Schmid Ges.M.B.H. &
Co. KG at various companies in Bad Vöslau,
Vienna and Lenzing
Additional scientific experience
August 2002
October 2005
Practical training at the hospital Elisabetinnen
(Department for molecular biology), Linz
Short term mission to the University of Vienna
for AFLP-analysis
Conferences
2005
Participation at XVII International Botanical
Congress, Vienna, Austria
Additional qualifications and Interests
Languages
Computer skills
Sports
Other interests
German (mother tongue), English (fluent) and
French (Basic knowledge)
Basic knowledge in C and Python
Hiking, Mountain climbing (also in winter with
skis), Mountain biking, skiing, running,
swimming, soccer
Reading, travelling, art and culture
126