1 Strong genetic subdivision in Leptobrachium hendricksoni (Anura: Megophryidae) in 2 Southeast Asia 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). 3 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. 5 113 114 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.