1 Strong genetic subdivision in Leptobrachium hendricksoni (Anura: Megophryidae) in
3
4 Gordon Draškića,b*, Sansareeya Wangkulangkula, Iñigo Martínez-Solanoc, Judit Vörösb,d
5 a Department of Biology, Faculty of Science, Prince of Songkhla University, Hatyai 90110,
6 Songkhla, Karnjanavanit Soi 15 Rd., Thailand
7 b Laboratory of Molecular Taxonomy, Hungarian Natural History Museum, Budapest 1083,
8 Ludovika tér 2-6., Hungary
9 c Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias
10 Naturales c/ José Gutiérrez Abascal, 2, 28006 Madrid, Spain
11 d Department of Zoology, Hungarian Natural History Museum, Budapest 1088, Baross u. 13,
12 Hungary
13
*Corresponding author: Gordon Draškić
Email: [email protected]
Type of manuscript: article
Total number of words: 6401
14
15 Abstract
16 Many biodiversity hotspots are located in areas with a complex geological history, like
17 Southeast Asia, where species diversity may still be far underestimated, especially in
18 morphologically conservative groups like amphibians. Recent phylogenetic studies on the
19 frog genus Leptobrachium from Southeast Asia revealed the presence of deeply divergent
1
20 mitochondrial clades in Leptobrachium hendricksoni from Malaysia and Sumatra but
21 populations from Thailand have not been studied so far. In this study, we re-evaluate patterns
22 of intraspecific genetic diversity in L. hendricksoni based on the analysis of combined
23 sequences of mitochondrial 12S and 16S genes (1310 base pairs) including for the first time
24 samples from southern Thailand. Thai populations of L. hendricksoni formed a distinct clade
25 with respect to populations from central and southern Malaysia and Sumatra. High sequence
26 divergence between lineages from Thailand, Malaysia and Sumatra suggests the possible
27 presence of cryptic species in L. hendricksoni. Divergence within L. hendricksoni dates back
28 to the late Miocene, around 6 Mya, when lineages from Thailand, north Malaysia and
29 Sumatra split from a lineage in south Malaysia, at about the same time as rising sea levels
30 isolated the Thai-Malay peninsula. Subsequent splits took place later in the Pliocene, around
31 4.5 and 2.6 Mya. Our results highlight the role of geological history in promoting population
32 divergence and speciation.
33
34 Keywords: Megophryidae, southern Thailand, mtDNA, phylogenetic relationships, genetic
35 differentiation, cryptic species
36
37 Introduction
38 The geological history of a region can have a profound impact on the current distribution of
39 populations and species (Pfrender et al., 2004). Areas with a complex geological history
40 usually have high levels of endemism, because changing patterns of connectivity through
41 time triggered by geological and climatic events promote population differentiation and
42 speciation. One of these biodiversity hotspots is Southeast Asia, which has experienced major
2
43 geological changes in the last 20 million years (Myr) (Corlett, 2009) (Fig. 1). The areal extent
44 of this region has changed dramatically through time because of eustatic changes (Fig. 1) and
45 forests have covered up to twice the area they occupy today, while the average temperature
46 varied from +3C° to –5C° (Woodruff, 2003; Sathiamurthy and Voris, 2006; Woodruff,
47 2010). These events have likely played a major role in shaping the evolutionary history of
48 entire biotic communities.
49 According to Woodruff (2003) sea straits may have cut the Thai-Malay peninsula
50 during two periods in the last 24 Myr: first in the early-middle Miocene for ca 11 Myr
51 beginning at 24 million years ago (Mya), and the second in the early Pliocene for another 1
52 - 1.4 Myr beginning at 5.5 Mya. In the north, a seaway opened from today’s town of Krabi
53 in the west to near Surat Thani on the east, whereas a southern seaway, about 40 - 50 km
54 wide, ran from the towns of Alor Setar and Satun on the Andaman Sea to Songkhla and
55 Pattani on the Gulf (Fig. 1a). Between these two seaways much of the east side of the central
56 peninsula was submerged and forest habitats were greatly reduced to fragments on the
57 Nakhon si Thammarat mountain range and western hills. Woodruff (2003) proposed that
58 these two seaways existed long enough for populations of plants and animals to become
59 isolated on either side to accumulate genetic divergence. This sea level rise did not only have
60 a huge impact on central parts of the Thai-Malay peninsula. Lowlands of southern parts of
61 the Thai-Malay peninsula and Sumatra were submerged as well (Fig. 1a). Indeed, these
62 changes seem to have had a significant impact on the distribution, phylogenetic and genetic
63 structure of species in this region, including amphibians (Zheng et al. 2008; Brown et al.
64 2009; Rao and Wilkinson 2009, Matsui et al. 2010; Hamidy et al. 2011).
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65 The genus Leptobrachium Tschudi, 1838 is a group of frogs in the family
66 Megophryidae characterized by a stocky body with slender, short hind limbs (Inger and
67 Stuebig, 1997). It currently includes 35 species occurring from southern China and India to
68 the islands of the Sunda Shelf and the Philippines (Sondhi and Ohler, 2011; Stuart et al.,
69 2011, 2012; Frost 2016). Matsui et al. (2010) estimated the origin of genus Leptobrachium
70 at around 50 Mya. Later, two major clades of Leptobrachium split around 45 Mya: a China-
71 Indochina clade and a Sundaland-Thailand clade. In the latter, species from Borneo, Sumatra
72 and the Philippines formed a subclade, species from Peninsular Malaysia and Java formed a
73 second one, and finally, species from Thailand and Myanmar formed a third subclade. Due
74 to their wide geographical distribution and long history in the region, Leptobrachium frogs
75 are good model systems to test the effect of particular geological events on speciation and
76 intraspecific diversification.
77 Several groups of widespread frog species from Southeast Asia have been shown to
78 contain cryptic species, including genus Leptobrachium (Evans et al., 2003; Brown et al.,
79 2006a, 2006b; Stuart et al., 2006; Brown and Richards, 2008; Brown et al., 2009). According
80 to Matsui et al. (1999), three species of Leptobrachium occur in Thailand: L. chapaense
81 (Bouret, 1937), L. hendricksoni Taylor, 1962, and L. smithi Matsui, Nabhitabhata and Panha,
82 1999. Leptobrachium hendricksoni was described from Bhethong, Yala, Thailand, and is a
83 medium sized species with snout to vent length of up to 70 mm in females and around 50
84 mm in males. In Thailand, L. hendricksoni is restricted to the lowlands of the extreme south
85 along the Nakhon si Thammarat (1835 m) and Titiwangsa (2183 m) mountain ranges, and is
86 more widely distributed in Peninsular Malaysia (along Titiwangsa in the west and the
87 Banjaran Pantai Timur range (1300 m average elevation) in the east) to western Borneo and
4
88 Sumatra (Taylor, 1962; Matsui et al., 2010) (Fig. 2). Matsui et al. (2010) uncovered high
89 genetic divergence within L. hendricksoni, with intraspecific clades dating back to the
90 Pliocene, about the same time when the rising of the sea level separated the Malay peninsula
91 and adjacent islands. However, they did not study the populations from southern Thailand.
92 The aim of our study was to assess the genetic diversity of populations of L. hendricksoni
93 from southern Thailand and to compare them with available DNA sequence data from
94 populations from Malaysia and Sumatra. We hypothesized that the flooded area between
95 mountain ranges in southern Thailand and Malaysia as well as the presence of the Malacca
96 strait between the mainland and Sumatra could have acted as a barrier to gene flow between
97 local populations resulting in genetic subdivision in L. hendricksoni.
98
99 Materials and Methods
100 Sampling of specimens
101 A total of 27 individuals of L. hendricksoni were sampled from six sites on the Nakhon Si Thammarat and
102 Titiwangsa mountain ranges in southern Thailand between February 2014 and January 2016. We also
103 downloaded mtDNA sequences from eight additional individuals from GenBank. The final dataset for analyses
104 thus comprised a set of 35 individuals from 14 localities from southern Thailand, Malaysia and Indonesia,
105 covering most of the range of the species with the exception of Bornean populations (Fig. 2, Table 1). In
106 addition, we used sequences of L. hasseltii Tschudi, 1838, L. smithi Matsui, Nabhitabhata and Panha, 1999 and
107 L. boringii (Liu, 1945) from GenBank as sequential outgroups (see accession numbers in Table 1). Tissue
108 samples for molecular analyses were obtained from either the liver of dead specimens or toe clips from live
109 individuals. Sampling was authorized by the National Park, Wildlife and Plant Conservation Department,
110 Thailand. All tissue samples were preserved in 95 % ethanol and kept in a freezer. The specimens are stored in
111 the reference collection of Prince Maha Chakri Sirinthorn Natural History Museum at Prince of Songkhla
112 University Hat Yai for future reference.
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113
114 DNA extraction, PCR and sequencing
115 DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) or the phenol-chloroform
116 method of Collins et al. (1987). We amplified with PCR fragments of two mitochondrial genes (12S and 16S)
117 using primers 12Sh (Cannatella et al., 1998) and H1548 (Matsui et al., 2005) and 16SL2021 (Tominaga et al.,
118 2006) and 16H1 (Hedges, 1994), respectively. For both genes, PCRs were run in a total volume of 25 µl or in
119 some cases 50 µl. PCR programs were as follows: initial denaturation at 94ºC for 5 min, 33 cycles at 94ºC for
120 30 sec, 55ºC for 30 sec and 72ºC for 90 sec, and final extension at 72ºC for 5 min. Some of the resulting double
121 strand amplified products were purified using High Pure PCR Product Purification Kit (Roche, Pleasanton,
122 USA) and directly sequenced from both directions following the ABI Prism BigDye Terminator Cycle
123 sequencing protocol on an ABI 3130 Genetic Analyser (Applied Biosystems, Foster City, USA). Other PCR
124 products were purified using Favorgen Gel/PCR Purification Mini Kit (Prima Scientific Co., Ltd, Bangkok,
125 Thailand) and sent to Macrogen Inc., Korea for sequencing.
126
127 Alignment and genetic diversity analyses
128 We used software BioEdit (Hall, 1999) to edit the sequences, which were then aligned taking into account their
129 secondary structure with online software LocARNA (Will et al., 2007; Smith et al., 2010; Will et al., 2012).
130 For all downstream analyses 12S and 16S sequences were trimmed and combined into a single alignment. The
131 number of haplotypes (N) and estimates of haplotype (h; Nei, 1987) and nucleotide diversity (π; Nei and Tajima,
132 1981) were computed using DnaSP 5.1 (Librado and Rozas, 2009).
133
134 Phylogenetic analysis and haplotype network construction
135 We used software jModelTest 2.1.7 (Darriba et al., 2012) to find the best DNA substitution model for each
136 gene, using default settings and the Akaike Information Criterion (AIC). Phylogenetic analyses based on
137 Bayesian inference were run with Mr.Bayes 3.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck,
138 2003). We set the doublet model for the 12S and 16S stem regions and a 4by4 model for loop regions in both
139 genes. Phylogenetic reconstruction was performed running Metropolis-coupled Markov chain Monte Carlo
6
140 sampling with 4 chains for 3x106 generations, sampling every 100th tree. Results of Bayesian phylogenetic
141 reconstruction were used to group sequences into major clades and estimate pairwise distance between clades
142 in MEGA 6 (Tamura et al., 2013) using the Maximum Composite Likelihood model with gamma distribution.
143 Additionally, a Maximum Likelihood (ML) phylogenetic analysis on the combined dataset was
144 performed using MEGA 6 (Tamura et al., 2013). We set the general time reversible nucleotide substitution
145 model with a gamma distribution and proportion of invariant sites (GTR+I+G) as calculated by jModelTest
146 under the AIC. Gaps and missing data were excluded, and 1000 nonparametric bootstrap replicates were
147 performed to evaluate clade support.
148 Finally, a median-joining haplotype network (Bandelt et al., 1999) was constructed using Popart 1.7
149 (Leigh and Bryant, 2015) to visualize relationships among haplotypes, their relative frequencies, and patterns
150 of haplotype sharing and geographical extent.
151
152 Estimation of divergence times
153 Divergence times were estimated using BEAST 1.8.4 (Drummond et al., 2012). The alignment was analyzed
154 as a single partition, with the TIM2+G model of nucleotide substitution selected based on jModelTest results
155 under the Bayesian Information Criterion. We ran the analysis under a strict molecular clock with an
156 uninformative prior with a gamma distribution (shape = 0.01, scale = 100) for the clock rate and with the birth-
157 death model as a tree prior. Analyses under a relaxed (uncorrelated lognormal) molecular clock resulted in 95
158 % Highest Posterior Density estimates of the parameter “coefficientOfVariation” including zero, suggesting
159 good fit to a strict clock model. For the estimation of lineage splitting times we specified priors on selected
160 node ages based on Matsui et al. (2010). These priors were specified as lognormal distributions centered on the
161 desired age with a standard deviation encompassing the full confidence interval estimated by Matsui et al.
162 (2010). Selected nodes included the root (46.4 Mya with a confidence interval (CI) of 31.6 to 61.4), the clade
163 L. smithi + (L. hasseltii + L. hendricksoni) (34 Mya, CI 22.5 - 45.7), and the clade L. hasseltii + L. hendricksoni
164 (18.8 Mya, CI 11.0 - 27.2). We ran the MCMC chain for 10 million generations sampling every 10,000
165 generations for a total of 10,000 tree samples. Convergence of results and adequate effective sample sizes of
166 parameters of interest were visually assessed using software Tracer (Rambaut et al., 2014).
7
167
168 Results
169 Genetic diversity
170 Combined sequences of the mitochondrial 12S and 16S genes consisted of 1310 sites (376
171 bp of 12S and 934 bp of 16S sequences). Excluding outgroups, there were 85 polymorphic
172 sites of which 72 were parsimony informative. We identified 14 haplotypes (H1 - H14)
173 among 35 individuals (Table 1) with haplotype diversity (h) = 0.73 and nucleotide diversity
174 (π) = 0.01.
175
176 Phylogenetic analyses and median-joining haplotype network
177 Bayesian inference and ML analyses resulted in similar tree topologies (Fig. 3) in which L.
178 hendricksoni haplotypes formed a monophyletic group. This monophyletic group was split
179 in four well-supported major lineages including 1) individuals from southern Thailand and
180 northern Malaysia (Lineage A, including all samples from Thailand plus the Malaysian
181 localities of Penang and Pasir Puteh), 2) central Malaysia (Lineage B, localities Hulu
182 Trengganu and Sekayu), 3) Sumatra (Lineage C), and 4) southern Malaysia (Lineage D,
183 localities Kuala Lumpur and Selai). Lineage B was recovered as the sister lineage to Lineage
184 A, and Lineage C was recovered as the sister group to lineages (A + B). Lineage D was in
185 turn recovered as the sister clade to lineages C (A + B). Average p-distances between lineages
186 are presented in Table 2 and range from 2.0 to 4.1 %.
187 Figure 4 shows the median-joining network of combined 12S and 16S mitochondrial
188 haplotypes. Excluding one haplotype from Penang, Malaysia, all other haplotypes were
189 unique for each Malaysian or Sumatran population. From the six localities from southern
8
190 Thailand where multiple sequences were sampled, three localities contained private
191 haplotypes (H3 Thaleban, Satun, H5 Ton Nga Chang, Songkhla and H6 and H7 Hala-Bala,
192 Narathiwat) while two haplotypes (H1 and H4) were shared among populations. Haplotype
193 H1 was found in almost every population in Lineage A (except Hala-Bala, Narathiwat),
194 whereas H4 was shared only in two localities of Lineage A.
195
196 Divergence times
197 The mean nucleotide substitution rate estimated in BEAST analyses was 0.0037
198 subst./site/Myr, (95% Highest Posterior Density Interval: 0.0028-0.0049). Divergence within
199 L. hendricksoni was initiated in the late Miocene, around 6 Mya, when Lineages A, B and C
200 split from Lineage D from south Malaysia (Fig. 3). Later in the Pliocene, around 4.5 Mya,
201 Lineages A and B split from Lineage C from Sumatra. Lineages A and B split from each
202 other around 2.6 Mya. Additional diversification occurred within each of these lineages in
203 the late Pleistocene from 0.7 - 0.02 Mya. It should be noted that, since they are based on a
204 gene tree, these estimates are times to the most recent common ancestor (TMRCAs) rather
205 than split times, which would be more recent.
206
207 Discussion
208 Southeast Asia has a rich amphibian diversity and the exact number of frog species is still
209 unknown since new species are being continuously described (Brown et al., 2009; Hamidy
210 et al., 2011, 2012; Wogan, 2012). The complex geological history of the region has probably
211 had a significant impact on speciation processes and in the present distribution of frogs.
212 Modeling the last million years, Cannon et al. (2009) concluded that today’s Southeast Asian
9
213 rainforests have acted as historical refugia, and that lowland evergreen forests have doubled
214 their area of occupancy, spreading across the emergent Sunda Shelf rather than shrinking
215 during cooler periods. Leptobrachium hendricksoni is widely distributed in southern parts of
216 Thailand, in Malaysia, Sumatra and some parts of Borneo and like other coastal lowland and
217 swamp species (e.g. Limnonectes malesianus (Kiew, 1984), Pseudobufo subasper Tschudi,
218 1838, Polypedates colletti (Boulenger, 1890), or Leptobrachium nigrops Berry and
219 Hendrickson, 1963), it was probably able to disperse throughout the region whenever sea
220 levels dropped at least 30 m compared to those in the present (Inger and Voris, 2001). On the
221 other hand, rising sea levels in the past (Woodruff, 2003) and high mountains could have
222 acted as natural barriers, disrupting potential dispersal routes and isolating populations on
223 different sides of sea straits or mountain ranges.
224 In this study, we investigated the genetic diversity of L. hendricksoni in Peninsular
225 Thailand. High haplotype diversity and low nucleotide diversity in combination with a small
226 number of shared haplotypes between local populations suggest low levels of gene flow
227 among populations of L. hendricksoni. However, this conclusion should be taken with
228 caution due to the small sample sizes in most localities and the fact that sampled localities in
229 Thailand, Malaysia and Sumatra are geographically far from each other. Different analyses
230 consistently recovered four lineages with differences indicative of independent evolutionary
231 histories potentially linked to geographic isolation and perhaps also to ecological divergence.
232 L. hendricksoni occurs in low elevation mountain streams and swampy areas and breeds in
233 very slow-flowing water (Inger and Voris, 2001; personal observations). Inger and Voris
234 (2001) reported that this species is widely distributed and is considered as one of the
235 examples of natural exchange of species among the Malay Peninsula and Greater Sunda
10
236 Islands. However, the limited dispersal abilities of amphibians in combination with
237 geographic barriers often result in high levels of genetic differentiation among populations
238 (García-París et al., 2000; Cabe et al., 2007; Martínez-Solano et al., 2007; Zhang et al., 2010).
239 Considering that in the last 0.8 Myr the sea level changed from –150 m to +20 m (Rohling et
240 al., 1998, Lambeck and Chappell, 2001; Lea et al., 2002; Woodruff, 2003, Woodruff, 2010),
241 it is likely that natural barriers limited dispersal in L. hendricksoni and conditioned the range
242 of potential migration routes between the Thai-Malay Peninsula and Sumatra as well as
243 between the Titiwangsa and Banjaran Pantai Timur mountain ranges in Peninsular Malaysia,
244 in particular the flooded areas between these land masses. These repeated sea level
245 fluctuations could have had a double impact on the local populations of L. hendricksoni.
246 During Pleistocene interglacial stages, rainforest retreated to the hills of Thai-Malay
247 peninsula and Sumatra providing refugia for local populations whereas glacial drops in sea
248 level could have promoted gene flow between these local populations. However, the presence
249 of a southern seaway between the Nakhon si Thammarat and Titiwangsa mountain ranges in
250 southern Thailand seems to have had a lesser effect as a barrier to dispersal in view of the
251 little genetic divergence among populations in these two mountain ranges (see Table 1 and
252 Figs. 3 - 4).
253 Phylogenetic analyses showed the presence of four different lineages in L.
254 hendricksoni from the Thai-Malay peninsula and Sumatra. If a difference of 3 % in
255 uncorrected genetic distances in the mitochondrial 16S gene is taken as measure of species
256 distinction (Fouquet et al., 2007), then Lineage A from southern Thailand can be considered
257 as conspecific with Lineage B from north-east Malaysia (p-distance 2.0 %). In contrast,
258 Lineages A and B are well differentiated from Lineage C from Sumatra (p-distances of 2.9
11
259 % and 2.8 %, respectively) and especially so with respect to Lineage D from south Malaysia
260 (p-distances of 3.9 %, 4.1 % and 3.4 % with lineages A, B, and C, respectively), which can
261 be regarded as a candidate species to be further tested against independent ecological,
262 morphological and molecular evidence. Similar patterns of cryptic diversity in the genus
263 Leptobrachium have been previously reported (L. chapaense: Zheng et al. 2008, Rao and
264 Wilkinson 2009, Matsui et al. 2010; L. hasseltii: Brown et al. 2009; L. montanum: Matsui et
265 al. 2010, Hamidy et al. 2011; L. abbotti: Hamidy et al. 2011; L. nigrops: Hamidy et al. 2012).
266 However, there are also quite a few examples of small genetic distances between otherwise
267 distinct species of frogs (e.g., Matsui et al., 2006; Kuramoto et al., 2011) including two
268 species in the genus Leptobrachium (Hamidy et al., 2011). Thus, even though the uncorrected
269 p-distance between Lineages C and lineages A and B is slightly lower than 3 %, we argue
270 for its consideration as a candidate species and encourage further morphological, bioacoustic
271 and molecular analyses including nuclear markers to test this hypothesis and help identify
272 and delimit potential cryptic species within L. hendricksoni.
273 The diversification of L. hendricksoni involved multiple geological events, with
274 parallels to other groups like the separation of Peninsular and Bornean lineages in L. nigrops
275 (Hamity et al., 2012). Our time estimates suggest most of the intraspecific divergence within
276 L. hendricksoni occurred in the late Miocene and Pliocene (6 - 2.6 Mya). Around 6 Mya
277 Lineages A, B and C (from Thailand, north Malaysia and Sumatra, respectively) split from
278 Lineage D from south Malaysia, at about the same time as rising sea levels isolated the Thai-
279 Malay peninsula according to Woodruff (2003). Inger and Voris (2001) suggested that L.
280 hendricksoni could have dispersed through the Thai-Malay peninsula and Sumatra in the
281 Pleistocene, when a sea level drop provided dispersal avenues between these two land
12
282 masses. However, according to our estimates, the lineages from Thailand and north Malaysia
283 split from the Sumatran lineage in the early Pliocene, around 4.5 Mya, which is more likely
284 connected to the same geological event that separated Thai, north-Malaysian and Sumatran
285 lineages from the south-Malaysian lineage. Additionally, our results showed that minor
286 diversification events occurred within all L. hendricksoni lineages from 0.7 - 0.02 Mya.
287 The separation of Thai Lineage A (Titiwangsa and Nakhon si Thammarat ranges) and
288 north-Malaysian Lineage B (Banjaran Pantai Timur range) corresponds to the Pliocene-
289 Pleistocene transition (2.6 Mya). Following the early Pliocene highstand, sea levels fell to -
290 100 m around 2.75 Mya, followed by a series of fluctuations that continued in the Pleistocene
291 (Woodruff, 2003). Even though the area between Titiwangsa and the Banjaran Pantai Timur
292 ranges was not flooded in this period of geological history, the distance between these
293 mountain ranges could explain the independent evolutionary history of these lineages.
294 In this study, we confirmed the presence of deeply divergent lineages within L.
295 hendricksoni and showed that populations from southern Thailand had an independent
296 evolutionary history from populations from south and north-east Malaysia and Sumatra. High
297 genetic distances between these lineages suggest the possible presence of cryptic species,
298 although this should be further explored with additional data from nuclear markers as well
299 as other characters including advertisement calls and morphology, with features like eye
300 coloration. We found good correspondence between the geographical extent of these lineages
301 and natural barriers operating in the past or in the present, like mountain ranges or sea straits,
302 highlighting the role of geological history in promoting population divergence and
303 speciation.
304
13
305 Acknowledgements
306 We would like to thank Graduate School, Prince of Songkhla University, Hat Yai, Thailand
307 for providing Thesis Financial Support (Graduate School, 2015). This research would not be
308 possible without National Park, Wildlife and Plant Conservation Department from Thailand,
309 who kindly provided the permit for Conducting Study/Research in the Protected Areas. GD
310 was supported by Balassi Insitute Scholarship of the Hungarian Scholarship Board Office,
311 Budapest, Hungary. JV was supported by the Bolyai János Research Scholarship of the
312 Hungarian Academy of Sciences (BO/00579/14/8). Many thanks to O. Márton and M.
313 Tuschek from the Molecular laboratory of the Hungarian National History Museum from
314 Budapest for all suggestions and lab assistance. We would also like to thank Dr. K. Sridith
315 from the Department of Biology, Faculty of Science, Prince of Songkhla University, Hat Yai,
316 Thailand for the support and suggestions during the process of writing the manuscript. Many
317 thanks to Mr. N. Acton-Bond for final proofreading. This work was completed in partial
318 fulfillment of the requirement for the doctoral degree of GD at the Department of Biology,
319 Faculty of Science, Prince of Songkhla University, Hat Yai, Thailand.
320
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500 Table 1. List of samples of L hendricksoni and outgroups used in phylogenetic analyses. The list includes samples from this study and from previous studies, with information on vouchers, GenBank accession numbers and sampled localities. No: sample codes as in Figs. 2, 3 and 4. UN: Unnumbered. Countries: THA: Thailand, MY: Malaysia, IND: Indonesia, CHN: China Provinces: SA: Satun, SO: Songkhla, NA: Narathiwat, KE: Kelantan, TR: Trengganu, SU: Sumatra, SE: Selangor, JO: Johor, TR: Trang, CJ: Central Java, SI: Sichuan
No Species Voucher GenBank Locality Reference Haplotypes Phylogroup 12S 16S 01-05 L. hendricksoni Herp.A 01502-6 MF686827-31 MF686854-58 THA, SA, Ton Pliu This study H1(1), H2(4) Lineage A 06-10 L. hendricksoni Herp.A 01497-501 MF686832-36 MF686859-63 THA, SA, Thaleban This study H1(3), H3(1), H4(1) Lineage A 11-15 L. hendricksoni Herp.A 01508,11,13,17,18 MF686837-41 MF686864-68 THA, SO, Ton Nga Chang This study H1(3), H4(1), H5(1) Lineage A 16-20 L. hendricksoni Herp.A 01471-73,75,85 MF686842-46 MF686869-73 THA, SO, Sadao, Kaichon This study H1(5) Lineage A 21-25 L. hendricksoni Herp.A 01489-93 MF686847-51 MF686874-78 THA, SO, Kho Hong Hill This study H1(5) Lineage A 26-27 L. hendricksoni Herp.A 0742, 01520 MF686852-53 MF686879-80 THA, NA, Hala-Bala This study H6(1), H7(1) Lineage A 28 L. hendricksoni KUHE 15336 AB530411 MY, Penang Matsui et al. (2010) H1(1) Lineage A 29 L. hendricksoni KUHE 52403 AB530412 MY, KE, Pasir Puteh Matsui et al. (2010) H8(1) Lineage A 30 L. hendricksoni UKM HC1 10 AB530413 MY, TR, Hulu Trengganu Matsui et al. (2010) H9(1) Lineage B 31 L. hendricksoni KUHE UN tissue AB530414 MY, TR, Sekayu Matsui et al. (2010) H10(1) Lineage B 32 L. hendricksoni MDK 10 AB530415 IND, SU, Jambi, Bungo Matsui et al. (2010) H11(1) Lineage C 33 L. hendricksoni KUHE UN tissue AB530416 IND, SU, Lahat Matsui et al. (2010) H12(1) Lineage C 34 L. hendricksoni KUHE 15680 AB530417 MY, SE, Kuala Lumpur Matsui et al. (2010) H13(1) Lineage D 35 L. hendricksoni KUHE 52150 AB530418 MY, JO, Endau Rompin, Selai Matsui et al. (2010) H14(1) Lineage D 36 L. smithi KUHE 23342 AB530438 THA, TR, Kaochong Matsui et al. (2010) 37 L. hasseltii KUHE 44535 AB646408 IND, CJ, Mt. Ungaran Hamidy et al. (2011) 38 L. boringii SCUM120630 NC024427 CHN, SI, Emei Mt. Xu et al. (2014) 501
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Table 2. Uncorrected p-distance (%) between lineages of L. hendricksoni and between these lineages and outgroups. Genetic distances were calculated from combined sequences of the mitochondrial 12S and 16S genes of 1310 sites (376 bp of 12S and 934 bp of 16S sequences).
Lineage A Lineage B Lineage C Lineage D L. hasseltii L. smithi Lineage A Lineage B 2.0 Lineage C 2.9 2.8 Lineage D 3.9 4.1 3.4 L. hasseltii 10.2 9.5 9.3 10.0 L. smithi 15.9 15.2 15.4 15.7 15.6 L. boringii 20.5 19.3 19.0 18.9 18.5 23.0 502
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503 Figure legends
504 Figure 1. Map of the Thai-Malay peninsula and Sumatra showing land mass changes through
505 the geological history of the region. Shaded areas represent different land mass extensions
506 when sea level was at a) 100 m and b) 25 m above present level in the Miocene and the
507 Pliocene (adopted from Woodruff, 2003), c) –60 m in the Pleistocene and d) –120 m below
508 present level in the last glacial period (adopted from Sathiamurthy and Voris, 2006).
509 Figure 2. Map of the Thai-Malay peninsula and Sumatra showing sampling localities of L.
510 hendricksoni. Localities 1 - 27 were sampled for the present study and localities 28 - 35
511 were sampled in previous studies with sequences downloaded from GenBank. Sample
512 codes as in Table 1. The shaded area represents the distribution range of L. hendricksoni in
513 Thailand, Malaysia and Sumatra.
514 Figure 3. Time-calibrated gene tree reconstructed with BEAST, based on the analysis of
515 1310 bp of combined 12S rRNA and 16S rRNA mitochondrial genes for samples of L.
516 hendricksoni (including the four major lineages discussed) and three outgroups. Sample
517 codes and localities as in Table 1. Numbers on nodes represent ML bootstrap support values
518 and Bayesian posterior probabilities, respectively (ML/BPP). Node ages are represented by
519 horizontal bars (95 % highest posterior density intervals) and numbers next to the nodes
520 (median estimates). Scale (bottom) in millions of years.
521 Figure 4. Median joining haplotype network of combined mitochondrial 12S and 16S
522 sequences showing the relationships among haplotypes of L. hendricksoni. Circles represent
523 haplotypes, with sizes proportional to the number of the individuals sharing that haplotype.
524 Hatch marks on the branches represent the number of mutations distinguishing haplotypes.
525
26
526 Figure 1
527
528
27
529 Figure 2
530
28
531 Figure 3
532
533
29
534 Figure 4
535
30