Journal of Tropical Biodiversity and Biotechnology
Volume 06, Issue 01 (2021): jtbb58635
DOI: 10.22146/jtbb.58635
Research Article
The Use of DNA Barcoding and Phylogenetic Analysis to
Improve Identification of Usnea spp. Based on ITS rDNA
Miftahul Jannah1*, Muhammad Rifqi Hariri2, Rina Sri Kasiamdari3, Niken Satuti Nur Handayani4
1) Biology Department, As-Syafi`iyah Islamic University, Jl. Raya Jatiwaringin No. 12, Jakarta Timur, 17411,Indonesia.
2) Research Center for Plant Conservation and Botanic Gardens – Indonesian Institute of Sciences, Jl. Ir. H.Juanda No 13, Paledang,
Bogor, Jawa Barat, 16122, Indonesia.
3) Laboratory of Plant Systematics, Faculty of Biology, Gadjah Mada University, Jl. Teknika Selatan, Sinduadi, Mlati, Sleman,
Yogyakarta, 55281, Indonesia.
4) Laboratory of Genetics and Breeding, Faculty of Biology, Gadjah Mada University, Jl. Teknika Selatan, Sinduadi, Mlati, Sleman,
Yogyakarta, 55281, Indonesia.
* Corresponding author, email: miftahuljannah.fst@uia.ac.id
Submitted: 14 August 2020; Accepted: 09 February 2021; Published online: 02 March 2021
ABSTRACT
Lichen of the genus Usnea is quite common being used as a traditional herbal
remedy. This genus is characterized by thallus, which is very similar among the
species, leads to some difficulties in distinguishing them. In Indonesia, such
research report on the availability of this genus based on their morphological
characteristic is minimal. This might be due to too high morphological similarities
among them. The molecular character, which is based on the DNA Barcode of
Internal Transcribed Spacer (ITS) rDNA sequences, with its conserved region
(5.8S) and varied region (ITS1 and ITS2), are becoming essential characters on
identifying as well as analyzing the phylogenetic. The current study then proposed
to identify and draw the species dendrogram of species within the Usnea genus
obtained from Mount Lawu Forest of Central Java and Turgo Forest of
Yogyakarta based on their phylogenetic and phenetic analysis. The dendrogram
was constructed with UPGMA using the simple matching coefficient, whereas the
phylogenetic tree was constructed with Maximum Likelihood (ML) using Kimura2 parameter with 1000 bootstrap. The data were unable to draw phenetic
relationships among the subgenus Usnea and Eumitria members. The phylogenetic
tree shows the primary two clades, distinguishing the subgenus Usnea and Eumitria.
The ITS rDNA sequence was able to identify most of the Usnea species.
Keywords: Usnea, DNA Barcode, ITS rDNA, Phylogenetic
INTRODUCTION
Lichen is an outstanding successful group of symbiotic organisms comprised
of algae (phycobiont) and fungal (mycobiont) (Zachariah & Varghese 2018).
Lichen of the genus Usnea is used as traditional herbal remedies in Solo and
Yogyakarta, with of local name of "Kayu Angin". The local people collect the
thallus from the forests of Turgo Hill and Mount Lawu. They use Usnea as
one component of all herbal medicines, such as cholesterol, diabetes, gout,
maternity, high blood pressure, skin, clods, and heart disease (Jannah 2019a).
Usnea (Parmeliaceae) has been distinguished with approximately 600
species worldwide (Hawksworth et al. 1995). Articus (2004) grouped the
Copyright: © 2021, J. Tropical Biodiversity Biotechnology (CC BY-SA 4.0)
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J. Tropical Biodiversity Biotechnology, vol. 06 (2021), jtbb58635
Usnea members as inadequate taxonomy since they have many similarities
among the species. The thallus of this genus is very similar among species. At
this level, there is exceptionally high plasticity on their morphological
characters to respond to environmental factors, which leads to a very
complicated effort in drawing a clear boundary among the species (Clrec
1998). The situation leads to species identification limitation based on their
morphological characters but not on the genus' family.
Divakar et al. (2006) stated there were sister species cases due to high
similarities between two species of U. florida and U. subfloridana. These two
species could only be distinguished on the presence or absence of their
reproductive organs where U. florida has more sexual organs than another.
The reproductive organ, such as apothecia, is an essential character for
species-level identification, but the prolonged growth during the life phase
becomes an obstacle to identification (Clerc 1998; Swinscow & Krog 1978).
Based on the above facts, molecular analysis with DNA Barcodes is needed
to strengthen and support species identification quickly, precisely, and
accurately.
Research on the identification of Usnea in Indonesia using DNA
barcodes from ITS rDNA has not been reported yet. However, the ITS
rDNA region has been used extensively in the study of lichenized fungi
overseas, including in assessing the species boundaries as well as testing the
correlation between genetic and morphological diversity in species
complexity (White et al. 1990; Korabecna et al. 2007; Del-Prado et al. 2010;
Kelly 2011; Jannah 2019b). The molecular characters based on Internal
Transcribed Spacer (ITS) rDNA sequences that have conserved regions
(5.8S) and varied regions (ITS1 and ITS2 ) are needed to strengthen and
support the identification and phylogenetic analysis (Articus et al. 2002;
Ohmura 2002). The objective of this research was to carry out a phylogenetic
analysis of Usnea species.
MATERIALS AND METHODS
Materials
The samples used in this research were fresh thallus obtained through the
explorative method. A total of 16 examined specimens in the present study
were collected at Mount Lawu Forest (LW) (East Java) and Turgo Hill Forest
(T) (Yogyakarta) (Table 1).
Table 1. List of Usnea spp. examined and location of origin.
No.
Species
Accession
Number
Origin
1
U. himalayana
LW1
Mount Lawu Forest
2
U. himalayana
LW2
Mount Lawu Forest
3
U. pectinata
LW3
Mount Lawu Forest
4
U. rubrotincta
LW4
Mount Lawu Forest
5
U. himalayana
LW5
Mount Lawu Forest
6
U. fragilescens
LW6
Mount Lawu Forest
7
U. baileyi
LW7
Mount Lawu Forest
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J. Tropical Biodiversity Biotechnology, vol. 06 (2021), jtbb58635
Table 1. Contd.
No.
Species
Accession
Number
Origin
8
U. nidifica
LW8
Mount Lawu Forest
9
U. fragilescens
LW9
Mount Lawu Forest
10
U. nidifica
LW10
Mount Lawu Forest
11
U. himalayana
LW11
Mount Lawu Forest
12
U. baileyi
T1
Turgo Hill Forest
13
U. bismolliuscula
T2
Turgo Hill Forest
14
U. baileyi
T3
Turgo Hill Forest
15
U. bismolliuscula
T4
Turgo Hill Forest
16
U. bismolliuscula
T5
Turgo Hill Forest
Methods
The Usnea thallus' total DNA was extracted using a modified CTAB method
that we developed by adding High Salt-TE. It is challenging to get pure
DNA from the Usnea thallus since there has never been a manuscript on
DNA isolation from lichens in Indonesia. The following is the DNA
isolation method that we developed. Total DNA of lichen was extracted
from the thallus using a modified CTAB method. As many as 0.05 gr of
Usnea thallus was powderized with additional liquid nitrogen. The powders
are transferred into a 1.5 ml tube, added 400 µl of pre-heated 2X CTAB
buffer, and incubated for 65 ºC for 10 minutes. Then, added 400 µl of
chloroform/isoamyl alcohol solution 24:1, homogenized gently for 5 minutes
at room temperature, then centrifuged at 12,000 rpm for 1 minute. The
supernatant, in turn, was added with 250 μl isopropanol and centrifuged
speed of 12,000 rpm for 1 minute. The supernatant was removed, added 100
µl of High Salt-TE, incubated at 65 ºC until dissolved, then added 800 µl
100% ethanol, incubated at -20ºC for 15 minutes, centrifuged 15,000 rpm 15
minutes at 4ºC and the supernatant removed and the pellet dried. After the
pellet was added with 300-500 µl of 70% ethanol added, centrifuged at
15,000 rpm for 3-5 minutes 4ºC, the supernatant was removed, and the pellet
was drained. A volume of 300-500 µl 70% ethanol was added into the pellet,
centrifuged 15,000 rpm for 3-5 minutes at 4ºC, and the pellet is dried again.
Finally, the pellet was dissolved by adding 50 μl of buffer pH TE 8. The
purified DNA is stored at -20 °C.
Fungal nuclear ITS rDNA was amplified using the primer ITS 1 and
ITS 4 (White et al. 1990). The PCR amplifications using KAPPA 2GTM Fast
ReadyMix (2x) were performed with a program of initial denaturation for 2
min at 95°C, followed by 25 cycles of 0.5 min at 95°C, 0.5 min at 56.3°C, 1
min at 72°C, and a final elongation for 1 min at 72°C. The PCR product was
electrophoresed in 1% agarose gel stained with 1 µl good view at 50 Volt for
40 minutes and visualized through UV-trans illuminator. The sequencing
process was carried out at First Base, Singapore, through the PT Genetika
Science Indonesia service.
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J. Tropical Biodiversity Biotechnology, vol. 06 (2021), jtbb58635
The ITS rDNA sequence was analyzed and edited using Bioedit and
DNA Baser. The homology level was determined through BLAST online.
The sequence alignment was analyzed using Clustal-X and nucleotide
similarity was executed through Phydit. The phylogenetic tree reconstruction
was carried out using Maximum Likelihood (ML) with Kimura-2 parameter
and 1000 bootstrap replications on MEGA-5.05. The Automatic Barcode
Gap Discovery (ABGD) was carried out through http://bioinfo.mnhn.fr/
abi/public/abgd/abgdweb.html using Jukes-Cantor (JC69) distance.
RESULTS AND DISCUSSION
Seven species were found based on morphological characters, namely U.
pectinata (LW3), U. rubrotincta (LW4), U. himalayana (LW1, LW2, LW5, LW11),
U. fragilescens (LW6, LW9), U. nidifica (LW8,LW10), U. baileyi (LW7,T1,T3),
and U. bismolliuscula (T2, T4, T5). These species of Usnea come from
subgenus Usnea (Figure 1) dan Eumitria (Figure 2).
Figure 1. Morphological structure of U. baileyi (subgenus Eumitria). A. thallus, B.
fibril, C papillae, D. isidia, E. soralia, F. apothecia, G. compact medulla & fistulose
central axis. Bar=0.5 mm.
Figure 2. Morphological structure of U. fragilescens (subgenus Usnea). A. thallus, B.
fibril, C. branch, D. white isidia, and black papillae, E-F. soralia, F. apothecia, G.
loose medulla & solid central axis. Bar=0.5 mm.
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The dendrogram generated from morphological characters showed
that all specimens are divided into two large clades (clade A and B),
separating U. baileyi (clade A) and the other Usnea species (clade B) (Figure 3).
Morphologically, U. pectinata is closely related to U. bismolliuscula, U. rubrotincta
is closely related to U. fragilescens, U. himalayana is closely related to U. nidifica,
and U. baileyi is distantly related to those clustered in clade B.
The morphological character distinguishing both clades is the central
axis type. Clade A consists of U. baileyi with a fistulose central axis, while the
clade B member has a solid central axis. The morphological differences
between the subgenus Usnea and Eumitria are not always apparent in some
species. Usnea pectinata and U. baileyi belong to the subgenus Eumitria but both
are separated into distinct clades because U. pectinata having a solid central
axis. The clustering result based on morphological characters in this study
could not reflect the overall relationship among Usnea species. It can not
separate the member of subgenus Usnea and Eumitria accordingly.
Sokal et al. (1963), stated that the phenetic approach in determining the
relationship between individuals is based on the existing similarity of
characteristics without comparing the characters that are homology
(characters inherited from ancestors to their descendants) and homoplation
(characters obtained as a result of the adaptation to the same environment).
The existence of convergent evolution is obtained because of the adaptation
process to the same habitat conditions and divergent evolution that makes
the gains different forms from the same ancestor due to different
environmental habitats resulting in the phenetic method almost unable to
describe the true relationship among species (Campbell et al. 2008).
Figure 3. The dendrogram of Usnea is based on the UPGMA method using Simple Matching Coefficient.
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The identification of Usnea used by the people around Yogyakarta and
Solo, which comes from Turgo Hill forest and Mount Lawu forest, was
carried out based on molecular data inferred from the Internal Transcribed
Spacer (ITS) rDNA sequence. ITS rDNA sequences are widely used for
species-level identification, capable of differentiating inter and intra-species,
and determine the relationship of species through the differences of the
conserved region and the similarity of variable region (White et al. 1990;
Korabecna et al. 2007); Del-Prado et al. 2010; Kelly 2011).
The amplification of ITS rDNA from the Usnea genome was
successful. The result of PCR visualized using agarose shows a clear strong
band, which means that the ITS rDNA has been successfully amplified. ITS
sequence length is ± 559 bp. The results of the PCR products that have been
obtained and are under the target sequence length (ITS rDNA) are then
sequenced to read the ITS rDNA nucleotide sequence from each sample
(Figure 4). The sequencing results were then analyzed with BLAST software
on the NCBI Gene Bank to determine the homology level of the ITS rDNA
sequences obtained from the Gene Bank database, besides being able to
show that the sequence obtained was true ITS rDNA. This can be proven if
the homology level of the ITS rDNA sequence obtained with the ITS rDNA
sequence in Gene Bank shows high compatibility between 94-99%. This high
compatibility means that the target sequence obtained is the ITS rDNA
sequence lichen genus Usnea.
The phylogenetic tree was constructed using Maximum Likelihood
(ML) and 1000x bootstrap methods. This method is used to identify
differences in genetic distance and analyze the similarity between samples.
The ITS rDNA sequence was able to identify most of the Usnea, but do not
support the separation of some species in Usnea. The topology of the
phylogenetic tree that was formed showed that ITS rDNA was able to
separate species in each sub-genus into groups to form one clade. In the
results of the phylogenetic tree reconstruction, it is clear that the species
included in the Usnea subgenus are grouped into one clade (monophyletic),
and separate from the Eumitra subgenus (Figure 4). The results of this study
indicate that the genetic distance between Usnea species is meager. The
lowest genetic distance of 0 is found in U. pectinata and U. baileyi (T1 and T3)
(Table 2).
Figure 4. Results of 496 bp ITS rDNA sequences (1-188 bp for ITS1, 189-346 5.8S, & 347-496 for ITS2) of U. baileyi.
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Table 2. Genetic distances of Usnea spp. based ITS rDNA sequences.
The ABGD species delimitation method retrieved four “initial
partitions” and five “recursive partitions” with all samples of U. bismolliuscula
are monospecific. Only when values of intraspecific divergence (P) were
lower than 0.0046 was a higher number of groups suggested (5) and only by
the recursive analysis resulting in the split of U. nidifica in two different
lineages; both represented the specimens from Lawu (Table 3, Figure 5).
The topology of the phylogeny based on ITS rDNA sequences showed
that within the clade of subgenus Usnea, U. himalayana is closely related to U.
nidifica and U. bismolliuscula is more closely related to U. rubrotincta than U.
fragilescens. Within the clade of subgenus Eumitria, U. pectinata is closely related
to U. baileyi (Figure 6).
In the Usnea subgenus clade, it shows U. himalayana and U. nidifica to
form one clade, which means that among these species, they have the same
ancestor (monophyletic) supported by a very high bootstrap value of 99%.
The bootstrap shows that U. himalayana is very closely related to U. nidifica.
The phylogenetic topology shows U. himalayana (LW 2) separated from its
fellow species and forming a clade with U. nidifica (LW8), which is also
separated from fellow species U. nidifica (LW 10). This indicates that U.
himalayana (LW2) and U. nidifica (LW 10) began to navigate from their fellow
Table 3. The specific composition of the four and five groups hypotheses derived from ABGD analysis.
Group
Four group hypothesis species
composition
Group
Five group hypothesis species
composition
1
U. himalayana (LW1), U. himalayana (LW2), U.
himalayana (LW11), U. himalayana (LW5), U.
nidifica (LW8), U. nidifica (LW10)
1
U. himalayana (LW1), U. himalayana (LW2), U.
himalayana (LW11), U. himalayana (LW5), U.
nidifica (LW8)
2
U. pectinata (LW3), U. baileyi (LW7), U. baileyi
(T1), U. baileyi (T3)
2
U. pectinata (LW3), U. baileyi (LW7), U. baileyi
(T1), U. baileyi (T3)
3
U. bismolliuscula (T5), U. bismolliuscula (T2), U.
bismolliuscula (T4)
3
U. bismolliuscula (T5), U. bismolliuscula (T2), U.
bismolliuscula (T4)
4
U. fragilescens (LW9), U. fragilescens (LW6), U.
rubrotincta (LW4)
4
U. fragilescens (LW9), U. fragilescens (LW6), U.
rubrotincta (LW4)
5
U. nidifica (LW10)
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Figure 5. Results of the Automatic Barcode Gap Discovery (ABGD) analysis showing the four partitions (initial) also 5
and 1 (recursive) recovered. Nearly all partitions suggested the different number of species (≠ No of groups) with all
representatives of U. bismolliuscula rendered monospecific. Only recursive partitions rendered a higher number of species
(5) when P values were ≤ 0.0046.
Figure 6. Phylogram obtained from a Maximum Likelihood analysis, evolution model Kimura-2 parameter with 1000
bootstrap replications, showing the phylogenetic relationship among Usnea based on internal transcribed spacer (ITS)
rDNA.
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species. This is in line with the opinion of Ohmura (2012) which stated that
U. himalayana and U. nidifica have very close genetic relationships both
morphologically and molecularly. The morphology between U. himalayana
and U. nidifica is only distinguished by the absence of soralia in U. himalayana.
The alignment results of 515 bp ITS rDNA of U. himalayana with U.
nidifica showed that there were substitutions, namely ten transitions and three
transversions (72, 486, and 490 base positions) (Table 4). The alignment
results showed the insertion of 12 nucleotide U. himalayana (LW 1)
(TTCTACGTCGGT) in the 79th to 90th base positions (Table 5).
The subgenus Eumitria clade consists of 4 specimens (LW3, LW7, T1,
and T3), which are divided into two species, namely U. pectinata and U. baileyi.
The phylogenetic tree topology formed shows that U. baileyi (T1 and T3)
formed one clade with U. pectinata (LW3) supported by a very high bootstrap
value of 100%. Instead, U. baileyi separated from its fellow species and was
supported by a value high bootstrap of 100%. It can be said that, based on
ITS rDNA, U. baileyi (T1 and T3) is more closely related to U. pectinata, than
to its fellow species (LW7). Ohmura (2002) stated that phylogenetic result
based on ITS rDNA sequences strongly suggests that the close relationship
between U. pectinata and U. baileyi.
Table 4. Nucleotide base substitution in ITS rDNA of U. himalayana and U. nidifica.
Variation of Nucleotide Base
Sample
57
59
68
71
72
103
107
202
237
351
359
486
490
LW 1
(U. himalayana)
C
G
T
A
C
G
T
G
G
C
T
G
G
LW 2
(U. himalayana)
.
.
.
.
.
.
C
.
.
.
.
.
.
LW 11
(U. himalayana)
T
.
.
.
.
.
.
.
.
.
C
.
T
.
.
.
.
.
.
.
C
.
.
.
.
T
.
C
A
A
T
.
.
.
LW 5
.
.
.
.
.
.
(U. himalayana)
LW 8
.
.
.
.
.
.
(U. nidifica)
LW 10
.
A
C
G
G
A
(U. nidifica)
Note: green shows transition and red shows transversion.
Table 5. Insertion in ITS rDNA nucleotide bases of U. himalayana and U. nidifica.
Variation of Nucleotide Base
Sample
79
80
81
82
83
84
85
86
87
88
89
90
LW 1
T
T
C
T
A
C
G
T
C
G
G
T
LW 2
-
-
-
-
-
-
-
-
-
-
-
-
LW 11
-
-
-
-
-
-
-
-
-
-
-
-
LW 5
-
-
-
-
-
-
-
-
-
-
-
-
LW 8
-
-
-
-
-
-
-
-
-
-
-
-
LW 10
-
-
-
-
-
-
-
-
-
-
-
-
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J. Tropical Biodiversity Biotechnology, vol. 06 (2021), jtbb58635
Table 6. Nucleotide similarity in ITS rDNA of U. baileyi and U. pectinata.
U. pectinata(LW3)
U. baileyi (LW7)
U. baileyi (T1)
U. baileyi (T3)
---
15/499
0/499
0/499
U. baileyi (LW7)
96.99
---
15/499
15/499
U. baileyi (T1)
100.00
96.99
---
0/499
U. baileyi (T3)
100.00
96.99
100.00
---
U.pectinata(LW3)
Table 7. Nucleotide Base Substitution in ITS rDNA of U. baileyi and U. pectinata.
Variation of Nucleotide Base
Sample
35
44
51
92
137
334
346
361
378
436
439
443
446
448
U.pectinata(LW3)
G
C
T
A
A
C
T
G
A
T
T
T
T
T
U. baileyi (LW7)
T
T
C
G
G
T
C
A
G
A
C
C
A
C
U. baileyi (T1)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
U. baileyi (T3)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Note: green shows transition and red shows transversion.
This statement is reinforced by the results of the similarity analysis of
nucleotide bases, from the compared 494 nucleotide bases ITS rDNA, which
shows that between U. pectinata and U. baileyi (T1 and T3) there is not a single
difference in nucleotide bases. At the same time, U. baileyi (LW7) and its
fellow species only had a 96.99% similarity of nucleotide bases (15 of 499
differences in compared nucleotide) (Table 6).
Usnea baileyi (LW7), which separates from its fellow species, is indicated
by a rather long branch in the phylogenetic tree topology, indicating that this
species is starting to experience divergence. The alignment result supports
the divergence, showing the existence of substitution in the form of 11
transitions and three transversions (G-T, T-A) at the position of the 35th,
436th, and 446th nucleotide bases (Table 7).
The results of this study are in line with what Ohmura (2002) did, that
U. pectinata and U. baileyi shared a common ancestor to form one clade, which
was supported by a boost value of 99%. A similar case was reported by
Articus et al. (2002), the unclear separation between U. florida and U.
subfloridana also in U. rigida and U. barbata. Based on a phylogenetic approach
using β-tubulin and ITS-LSU, U. floridana and U. subfloridana clustered in one
clade supported with high bootstrap values. This also happened to U. barbata
and U. rigida, so the concept of species pair was proposed among them.
Based on the results of this study, it shows that in some Usnea species
such as U. himalayana with U. nidifica, also in U. pectinata and U. baileyi
indicates the starting of species divergence. It is necessary to carry out further
comprehensive research so that the position of the taxon can be clearly seen.
In the genus Usnea, only a few morphological characters are distinguished
from one species to another, and most of them are only distinguished by one
character. Some of them are only distinguished by their reproductive organs,
whereas the environment very much influences the appearance of
reproductive organs. The determination of the species name in the genus
Usnea follows the concept of species presented by Motyka et al. (1936-1938)
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J. Tropical Biodiversity Biotechnology, vol. 06 (2021), jtbb58635
in Clerc (1998), that was “A species is a strong character that has a little
variation between them (1 character = 1 species)”. The result of this study
needs further and thorough observation related to the nomenclature in the
genus Usnea.
CONCLUSION
The ITS rDNA sequence can be utilized to strengthen the identification and
investigation of relationships within the Usnea genus. The ITS rDNA
sequence was able to identify most of the Usnea spp. However, it cannot
distinguish between U. baileyi and U. pectinata. So that further research is
needed by using more distinguishable sequences with a faster evolutionary
rate so that differences in species of Usnea can be solved.
AUTHORS CONTRIBUTION
Contribution of the author: M.J. designed the research, collected and
analyzed the data, and wrote the manuscript, M.R.H. analyzed the data and
wrote the manuscript, R.S.K. and N.S.N.H. supervised all the processes.
ACKNOWLEDGMENTS
We thank Sayyid Fachrurrazi for providing the transliteration help.
Gratitudes are due to Aisyah Hadi R. and Nurul Istiqomah for their help
during sample collection until this research completion.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest regarding the
publication of this article.
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