1 Strong genetic subdivision in Leptobrachium hendricksoni (Anura: ) in

2

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.,

7 b Laboratory of Molecular , 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 . Recent phylogenetic studies on the

19 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

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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 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 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 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 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 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

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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.

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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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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.

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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).

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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

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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).

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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

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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 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

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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

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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

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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

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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

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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