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Home , Leiolepis, Mae Hong Son, Mae Hong Son Province

Herpetology Notes, volume 14: 893-897 (2021) (published online on 10 June 2021)

Revised distribution and a first record of Leiolepis peguensis Peters, 1971 (: Leiolepidae) from

Pattarapon Promnun1,*, Jenjit Khudamrongsawat1, Jesse L. Grismer2, Nontivich Tandavanitj3, Chalita Kongrit1, and Preecha Tajakan4

The Butterfly lizard, Leiolepis peguensis Peters, 1971, and collected tissue samples (tail tips). Three specimens is terrestrial, diurnal, and omnivorous. It possesses unique were deposited in the Thailand Natural History Museum, external body patterns (Peters, 1971) that distinguish it Pathum Thani, Thailand (voucher no. THNHM28601– from all other of Leiolepis and has equally been 28603), while other specimens were released after shown to be genetically distinct (Grismer and Grismer, examination. They were identified to species level 2010; Grismer et al., 2014). It was previously reported based on meristic and molecular approaches. in Pegu-Yoma on the eastern side of Irrawaddy River, Meristic characteristics of our specimens were from Arakan-Yoma and Chatthin Wildlife Sanctuary on compared to those of the following Leiolepis species: L. western side of the Irrawaddy River (), and some localities in central and west of (Peters, 1971; Zug et al., 1998; Grismer et al., 2014). Although L. peguensis is common in Myanmar, it has never been reported east of the Tenasserim mountain range which geographically separates Myanmar from the rest of Southeast Asia. Here we present a new record of L. peguensis from east of the Tenasserim range in Province, (Fig. 1). We encountered five adult L. peguensis (Fig. 2) in Pang Mu Sub-district (19.3572°N, 97.9809°E) and Pha Bong Sub-district (19.2394°N, 97.9978°E), Namtok Mae Surin National Park, during two dry seasons (March–May) in 2018–2019. These localities are covered by dry deciduous forests and open areas which are the preferred habitats of this species. We examined meristic characteristics of all specimens

1 Systematics and Molecular Ecology Laboratory, Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, 10400, Thailand. 2 Department of Biology, La Sierra University, Riverside CA, 92505 USA. Figure 1. Previous reports from Myanmar (blue circles) based 3 Department of Biology, Faculty of Science, Chulalongkorn on Grismer et al. (2014), Peters (1971), and Zug et al. (1998) University, Phayathai Road, Pathumwan, Bangkok 10330, and the first report in Thailand (red circles) of L. peguensis. Thailand. Chatthin Wildlife Sanctuary (1), Mandalay (2), Pegu- 4 Namtok Mae Surin National Park, Mueang Mae Hong Son Yoma, eastern Irrawaddy River (3), Araakan-Yoma, western District, Mae Hong Son 58000, Thailand. Irrawaddy River (4), Yangon (5), unspecified areas in Chin * Corresponding author. E-mail: [email protected] State (6), unspecified areas in Shan State (7), and Mae Hong © 2021 by Herpetology Notes. Open Access by CC BY-NC-ND 4.0. Son, Thailand (MH). 894 Pattarapon Promnun ������ belliana (Hardwicke and Gray, 1827); L. guttata Cuvier, 1829; L. ocellata Peters, 1971; L. peguensis (Peters, 1971) and L. reveesii Gray, 1831 (see Table 1). Meristic characteristics of our specimens and L. peguensis were consistent (Fig. 2, Table 1). They showed numerous distinct dorsal spots with bright centres and dark margins and lacked a median dorsal stripe. Some spots in the pelvic region formed a reticulum. Two dorsolateral stripes were present in the pelvic region. Narrow bright and broad dark flank wedges were clearly distinct. The venter was bright-coloured with black markings sometimes present in the pectoral region. In comparison to other Leiolepis species, our specimens differed from the patterns found in L. belliana, given their distinct bright and dark stripes on the flank, and greater scales across the ventral side of the tibia midway between the ankle and the knee (STIB). Our specimens could be distinguished from L. guttata in having relatively smaller snout-vent length (SVL), fewer enlarged subdigital lamellae under the fourth toe (SL4T) and a lower number of femoral pores (FMP). They differed from L. ocellata in having distinct bright and dark stripes on flank and greater STIB. They differed from L. reevesii in having distinct dorsal spots with dark margins, greater supralabials (SL) and infralabials (IL), and greater STIB. These newly discovered populations were not considered as a unisexual (parthenogenetic) species, since both sexes were found indicating they are sexual species (Grismer et al., 2014). Furthermore, we also conducted molecular analyses. Genomic DNA was extracted from our tissue samples using NucleoSpin tissue kit (Macherey-Nagel). We amplified partial ND2 mitochondrial gene using primers Figure 2. Specimens of L. peguensis. (A) Male specimen of and PCR conditions following Promnun et al. (2021). L. peguensis from Mueang Mae Hong Son District, Mae Hong Presence of PCR products was visualy checked using Son Province, Thailand (voucher no. THNHM28601). (B) 1.5% agarose gel electrophoresis and purified using Female specimen of L. peguensis from Mueang Mae Hong NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). Son District, , Thailand (voucher Purified products were sent for sequence analysis no. THNHM02). (C) Male specimen of L. peguensis from with BigDye Terminator Cycle Sequencing Kit. Final Mandalay, Myanmar. Photos by Chatchaiy Chueachat (A and alignments of 658 base pairs were performed using B) and Parinya Pawangkhanant (C). ClustalW in MEGA7 v.7.0.21 (Kumar et al., 2016) and deposited on GenBank (Accessions MN105113– 105117). ND2 sequences of our samples were aligned MrBayes v.3.2.6 (Huelsenbeck and Ronquist, 2001) with those of other Leiolepis species from Grismer and under the Metropolis-coupled Markov chain Monte Grismer (2010) and Grismer et al. (2014). We calculated Carlo (MC-MCMC) approach, started from random mean genetic distance among species using MEGA7 trees. A four-chain analysis was run twice in parallel v.7.0.21 under the Kimura 2-parameter model. We for 5 million generations. Trees were sampled every also constructed phylogenetic trees based on Bayesian 100 generations and 25% of generations were discarded Inference approach (BI). The HYK85 Gamma model as burn-in. Effective Sample Size (ESS) values (>200) was selected for the trees using Kakusan4 (Tanabe, were used to evaluate stationarity using Tracer v.1.7.1 2007). Phylogenetic analyses were carried out using (Rambaut et al., 2018). The results were then visualised Revised distribution and a first record of Leiolepis peguensis from Thailand 895

Table 1. Meristic measurements of the specimens and those of other bisexual species described by Peters (1971). Measurements: snout-vent Tablelength 1.(SVL), Meristic supralabials measurements (SL), of infralabials the specimens (IL), and scales those across of other the bisexual ventral species side of describedthe tibia (STIB),by Peters subdigital lamellae under the fourth(1971). toe Measurements: (SL4T), femoral snout-vent pores (FML). length (SVL), supralabials (SL), infralabials (IL), scales across the ventral side of the tibia (STIB), subdigital lamellae under the fourth toe (SL4T), femoral pores (FML).

L. peguensis L. belliana L. guttata L. ocellata L. peguensis L. reevesii (Thailand) SVL (mm) 114–126 103–156 96–184 118–161 116–162 103–151 (n=5) (n=54) (n=28) (n=20) (n=7) (n=43) SL 9–11 8–11 8–11 8–12 8–12 6–9 (n=5) (n=81) (n=29) (n=24) (n=10) (n=49) IL 10–11 7–11 9–13 7–12 9–11 6–10 (n=5) (n=79) (n=29) (n=24) (n=10) (n=49) STIB 11–16 7–14 13–25 8–12 11–16 7–12 (n=5) (n=84) (n=30) (n=49) (n=9) (n=49) SL4T 33–34 32–41 37–45 34–40 33–40 29–38 (n=5) (n=80) (n=19) (n=19) (n=10) (n=49) FMP 16–17 13–20 20–26 13–17 16–24 12–18 (n=5) (n=82) (n=30) (n=24) (n=10) (n=49)

Table 2. Percentage of mean genetic distance between Leiolepis species based on ND2 and edited in FigTree v.1.4.3 (Rambaut,L. peguensis 2009). L. belliana Since it L. guttatafrom Thailand. L. ocellata L. peguensis L. peguensis populations L. reevesii in Mae Hong was difficult to find appropriate (Thailand)outgroups for Leiolepis, Son are interesting because they were found outside its L. peguensis we avoided the problem by using0 mid-point rooting known distribution range in Myanmar (Peters, 1971; (Thailand) (Grismer et al., 2014). Grismer et al., 2014), crossing the Tenasserim Range Molecular analysesL. belliana were strongly 11.3 concordant 0 to and which are known to be biogeographic TableL. 1. guttata Meristic measurements19.6 of the specimens 15.7 and those 0 of other bisexual species described by Peters the meristic(1971). data. Measurements: Mean genetic snout-vent distance length between (SVL), supralabials our barriers. (SL), infralabials According (IL), to scales the phylogeneticacross the ventral tree, they are samples andside ofL.L. the peguensisocellata tibia (STIB), from subdigital Myanmar13.8 lamellae was under 11.9 2.6%, the fourth 17.5genetically toe (SL4T), 0 distinctfemoral pores from (FML). L. peguensis from Myanmar while mean geneticL. peguensis distances between 2.6 our samples 11.5 and 19.4indicating 12.8there may be 0some geographic sub-structure L. peguensis L. belliana L. guttata L. ocellata L. peguensis L. reevesii L. reevesii 12.5 11.8 18.9 12.9 12.9 0 other Leiolepis species were(Thailand) greater than 11% (Table within this species. Further studies on phylogeographic 2). Moreover, the phylogenetic tree showed reciprocal and systematics are necessary for clarification of SVL (mm) 114–126 103–156 96–184 118–161 116–162 103–151 monophyly of our samples (n=5) and L. peguensis(n=54) samples (n=28)intraspecific (n=20) variation (n=7) found between(n=43) L. peguensis from MyanmarSL with high statistical9–11 supports 8–11 (Fig. 3). 8–11populations. 8–12 Furthermore, 8–12 L. peguensis 6–9 is present only Within this clade, L. peguensis(n=5) formed many(n=81) sub-clades (n=29)in the northern(n=24) part of Mae(n=10) Hong Son(n=49) Province while L. with our samplesIL having no shared10–11 haplotype 7–11 with other 9–13ocellata is found7–12 in the eastern9–11 and southern6–10 regions of L. peguensis from Myanmar.(n=5) (n=79) (n=29)the province(n=24) (Promnun (n=10)et al., 2020). (n=49)Based on our field The consistentSTIB results of1 1–16 meristic and 7–14 molecular 13–25surveys, these8–12 two species11–16 do not occur7–12 in sympatry. (n=5) (n=84) (n=30) (n=49) (n=9) (n=49) approaches indicated that our collected specimens are We hypothesise that the geography of Mae Hong Son SL4T 33–34 32–41 37–45 34–40 33–40 29–38 L. peguensis and thereby form(n=5) a new record of(n=80) Leiolepis (n=19)Province, (n=19) such as north-south-running(n=10) (n=49) mountains FMP 16–17 13–20 20–26 13–17 16–24 12–18 (n=5) (n=82) (n=30) (n=24) (n=10) (n=49) Table 2. Percentage of mean genetic distance between Leiolepis species based on ND2. Table 2. Percentage of mean genetic distance between Leiolepis species based on ND2

L. peguensis L. belliana L. guttata L. ocellata L. peguensis L. reevesii (Thailand) L. peguensis 0 (Thailand) L. belliana 11.3 0 L. guttata 19.6 15.7 0 L. ocellata 13.8 11.9 17.5 0 L. peguensis 2.6 11.5 19.4 12.8 0 L. reevesii 12.5 11.8 18.9 12.9 12.9 0

896 Pattarapon Promnun ������

Figure 3. Phylogenetic tree of ND2 sequences obtained from Bayesian Inference (BI). Percentage of posterior probabilities were shown above branches. Trees were constructed based on the specimens collected in this study and sequences of other species from Grismer and Grismer (2010) and Grismer et al. (2014). and rivers, may act as geographical barriers and play References a significant role in the separation between these two Grismer, J.L., Bauer, A.M., Grismer, L.L., Thirakhupt, K., Aowphol, Leiolepis species. Elsewhere in Southeast Asia where A., Oaks, J.R., et al. (2014): Multiple origins of parthenogenesis, these types of barriers are absent, however, some and a revised species phylogeny for the Southeast Asian butterfly Leiolepis taxa come in contact with one another, as seen lizards, Leiolepis. Biological Journal of the Linnean Society in L. belliana and L. reevesii populations in southern 113(4): 1080–1093. (Grismer et al., 2014). Further intensive field Grismer, J.L., Grismer, L.L. (2010): Who’s your mommy? Identifying maternal ancestors of asexual species of Leiolepis sampling in poorly explored Mae Hong Son Province, Cuvier, 1829 and the description of a new endemic species of and elsewhere, in areas along the Thailand-Myanmar asexual Leiolepis Cuvier, 1829 from Southern . Zootaxa border, could increase our knowledge of the distribution 2433(1): 47–61. patterns of Leiolepis taxa in northern Thailand and may Huelsenbeck, J.P., Ronquist, F. (2001): MrBayes: Bayesian uncover new undescribed species. inference of phylogenetic trees. Bioinformatics 17(8): 754–755. Kumar, S., Stecher, G., Tamura, K. (2016): MEGA7: Molecular Acknowledgements. We thank wildlife rangers from Department evolutionary genetics analysis version 7.0 for bigger datasets. of National Parks, Wildlife, and Plant Conservation of Thailand Molecular Biology and Evolution 33(7): 1870–1874. (permission no. 6110314). The use of animal in research was Peters, G. (1971): Die intragenerischen Gruppen und die permitted by MU-IACUC (no. MU-IACUC 2017/030). We Phylogenese der Schmetterlingsagamen (: Leiolepis). thank Parinya Pawangkhanant, Sunchai Mekchai, and Chatchaiy Zoologische Jahrbücher. Abteilung für Systematik, Geographie Chueachat for photographs of some specimens. We also thank und Biologie der Tiere 98: 11–130. Philip D. Round for his comments and Michael Cota for reviewing Promnun, P., Kongrit, C., Tandavanitj, N., Techachoochert, S., the manuscript. Khudamrongsawat, J. (2020): Predicting potential distribution of an endemic butterfly lizard, Leiolepis ocellata (Squamata: Agamidae). Tropical Natural History 20(1): 60–71. Promnun, P., Tandavanitj, N., Kongrit, C., Kongsatree, L., Kongpraphan, P., Dongkumfu, W., et al. (2021): Phylogeography and ecological niche modeling reveal evolutionary history Revised distribution and a first record of Leiolepis peguensis from Thailand 897

of Leiolepis ocellata (Squamata, Leiolepidae). Ecology and Evolution 2021(11): 2221–2233. Rambaut, A. (2009): FigTree, version 1.4.3. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK. Rambaut, A., Drummond, A.J., Xie, D., Baele, G., Suchard, M.A. (2018): Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology 67(5): 901–904. Tanabe, A.S. (2007): KAKUSAN: A computer program to automate the selection of a nucleotide substitution model and the configuration of a mixed model on multilocus data. Molecular Ecology Notes 7(6): 962–964. Zug, G.R., Win, H., Thin, T., Min, T.Z., Lhon, W.Z., Kyaw, K. (1998): Herpetofauna of the Chatthin Wildlife Sanctuary, northern-central Myanmar with preliminary observations of their natural history. Hamadryad 23(2): 111–120.

Accepted by Lukas Hartmann