Herpetology Notes, volume 14: 485-491 (2021) (published online on 04 March 2021)

Molecular and morphological evidence for a significant range extension of Bronchocela burmana Blanford, 1878 to eastern Thailand

Daniel G. Mulcahy1, Michael Cota2, Sunchai Makchai2, and Bryan L. Stuart3,*

The agamid lizard genus Bronchocela consists of specimens of Bronchocela (Fig. 1) that are geographically arboreal, diurnal species that are distributed from South separated from their congeners by hundreds of kilometres, Asia across Southeast Asia to western New Guinea with the nearest known localities of B. burmana and (Grismer et al., 2015). Bronchocela burmana Blanford, B. rayaensis approximately 360 and 580 air-km to the 1878 was described from Tavoy (= Dawei; Zug et al., southwest, respectively (Grismer et al., 2016; Zug et al., 2017), Myanmar, but was soon after synonymized 2017), and B. smaragdina Günther, 1864 approximately by Boulenger (1885) with the more geographically 530 air-km to the east (Stuart et al., 2006b; Fig. 2A). widespread species B. cristatella (Kuhl, 1820). Zug et Herein, we use a small fragment of mitochondrial al. (2017) noted the distinctiveness of B. burmana from DNA recovered from one formalin-fixed specimen, and B. cristatella, removed it from synonymy, and expanded morphology of four specimens, to show that the Trat and the description using newly collected specimens Chanthaburi populations in Thailand should be referred from southern Myanmar. Zug et al. (2017) also noted to as B. burmana. These records extend the range of B. morphological and genetic similarities of B. burmana burmana eastward across the Gulf of Thailand to near the with the recently described B. rayaensis Grismer et al., Cambodian border (Fig. 2A). 2015 from Peninsular Malaysia and southern Thailand (Grismer et al., 2015, 2016) but recommended treating Materials and Methods B. burmana and B. rayaensis as distinct species pending Fieldwork. Four specimens were collected and further study. As currently recognized, B. burmana photographed in the field at three localities (Table occurs from its type locality at Tavoy (= Dawei) 1) by SM and MC, fixed in 10% buffered formalin, through southern Peninsular Myanmar to the District of transferred to 70% ethanol, and deposited in the Natural Kawthaung, B. rayaensis occurs from southern Thailand History Museum, National Science Museum, Thailand, to Langkawi Island, northwestern Peninsular Malaysia, Pathum Thani, Thailand (THNHM). and B. cristatella occurs from extreme southern Thailand Contemporary DNA. As no DNA sequence data for B. southward into Malaysia and Indonesia (Grismer et al., smaragdina were available in GenBank, a “fresh” liver 2015, 2016; Zug et al., 2017). tissue that had been preserved at the time of collection Fieldwork by MC and SM in Trat and Chanthaburi for molecular analysis was sequenced by BLS from one Provinces of eastern Thailand unexpectedly revealed individual, Field Museum of Natural History (FMNH) 262309, that was collected in Phnom Nam Lyr Wildlife Sanctuary, Pichrada District, Mondolkiri Province, eastern Cambodia (Fig. 2A; Stuart et al., 2006b). Total 1 Global Genome Initiative, National Museum of Natural genomic DNA was extracted using the DNeasy Blood History, Smithsonian Institution, Washington, D.C. 20013, and Tissue Kit (Qiagen). A 1267 nucleotide base pair (bp) USA. fragment of mitochondrial DNA that encodes part of the 2 Natural History Museum, National Science Museum, Thailand, Technopolis, Khlong 5, Khlong Luang, Pathum tRNA Met, the complete NADH dehydrogenase subunit Thani 12120, Thailand. 2 gene, the complete tRNAs Trp and Ala, and part of the 3 North Carolina Museum of Natural Sciences, Raleigh, North tRNA Asn (hereafter, “ND2”) was amplified using the Carolina 27601, USA. polymerase chain reaction (PCR). PCR was performed * Corresponding author. E-mail: [email protected] in a 25-μl reaction containing 2.5 μl of 10X DreamTaq © 2021 by Herpetology Notes. Open Access by CC BY-NC-ND 4.0. Buffer (Thermo Scientific), 1.0 μl of 20 mM dNTP Mix 486 Daniel G. Mulcahy et al.

Trat Province, Thailand (THNHM H-17867), and processed in a clean laboratory facility dedicated to degraded DNA samples at the North Carolina Museum of Natural Sciences. This clean laboratory facility had never contained any samples of Bronchocela, and the aforementioned B. smaragdina was the only “fresh” tissue of Bronchocela that had been previously extracted or sequenced in any facility at the North Carolina Museum of Natural Sciences. Extraction was performed following the historical DNA extraction protocol described in Stuart et al. (2006a). A total of 333 bp of the mitochondrial NADH dehydrogenase subunit 2 gene was obtained by amplifying a 181 bp fragment using the primers L4831 (Macey et al., 1997a) and GR1 (Luu et al., 2015), and a 196 bp fragment using the newly designed primers L-260BronchoND2 (5’-ATCCACCGCCTCCTGCCTGATC-3’) and H-450BronchoND2 (5’-AAATTAGGACTATTGGG GCGA-3’). PCR was performed in a 25-μl reaction containing 12.5 μl of AmpliTaq Gold 360 Master Mix (Applied Biosystems), 1.5 μl of 10 mM dilutions of each primer, 2.0 μl of 20 mg/ml bovine serum albumin (New England BioLabs), 4.5 μl of water, and 3.0 μl of DNA extraction at an initial denaturation of 95°C for 10 min, 40 cycles of 95°C for 15 s, 48°C for 30 s, Figure 1. Adult male Bronchocela burmana (THNHM 72°C for 45 s, and a final extension of 72°C for 7 min. H-17867) from Trat Province, eastern Thailand in life, in (A) Negative controls containing all reagents except DNA profile and (B) lateral views. Photographs by Michael Cota. extraction were run simultaneously. PCR products were cleaned, sequenced in both directions using the amplifying primers, and edited as above. The two (Thermo Scientific), 1.0 μl of 10 mM dilutions of each amplicons overlapped by 44 bp after trimming primer of the primers L4437b (Macey et al., 1997b) and H5934 sequences. The assembled sequence was deposited in (Macey et al., 1997a), 0.2 μl of 5 U/μl DreamTaq DNA GenBank under accession number MT325799. Polymerase (Thermo Scientific), 18.3 μl of water, and Molecular analysis. The two newly generated 1.0 μl of DNA extraction at an initial denaturation of sequences were aligned by DGM to all Bronchocela 95°C for 3 min, 35 cycles of 95°C for 45 s, 48°C for ND2 sequences and seven individuals of outgroup 60 s, 72°C for 90 s, and a final extension of° 72 C for taxa (following Grismer et al., 2015; Zug et al., 2017) 5 min. The PCR product was cleaned using ExoSAP-IT available from GenBank using the default parameters and sequenced in both directions by direct double-strand in the MUSCLE alignment algorithm (Edgar, 2004) cycle sequencing using the BigDye Terminator version implemented in Geneious version 10.0.9 (Biomatters), 3.1 Cycle Sequencing Kit on a 3130 DNA Analyzer except that tRNA secondary structure was aligned (all products by Applied Biosystems). The amplifying manually (Macey et al., 1997a). Maximum-likelihood primers and the internal primers ALAr.2m (McGuire and (ML) analysis was performed using RAxML version Kiew, 2001) and H4980 (Macey et al., 1997a) = ND2r.6 8.2.3 (Stamatakis, 2014) under the GTRCAT model (McGuire & Kiew, 2001) were used in the sequencing for each of four partitions (ND2 codon position and the reactions. Sequences were edited and assembled using tRNATRP–TYR region). A single analysis was conducted Sequencher version 5.4.6 (Gene Codes) and deposited in using a thorough best tree search and 1000 replicates of GenBank under accession number MT325798. the rapid hill-climbing bootstrap analysis (Stamatakis Historical DNA. Liver tissue was removed by et al., 2008). Nodes with bootstrap values ≥ 80 were BLS from one of the formalin-fixed specimens from considered to be moderately supported and those ≥ 95 to Range Extension of Bronchocela burmana to Eastern Thailand 487 488 Daniel G. Mulcahy et al.

Figure 2. (A) Localities of Bronchocela burmana (circles) and B. rayaensis (asterisks) summarized by Zug et al. (2017), with additions of new localities of B. burmana from eastern Thailand (triangles), and the locality of the sequenced sample of B. smaragdina (FMNH 262309; star). (B) Maximum-likelihood phylogeny of Bronchocela based on 1416 characters of the mitochondrial ND2 and flanking tRNA genes. Numbers at nodes are bootstrap values ≥ 50 based on 1000 replicates of the rapid hill-climbing bootstrap analysis. GenBank accession numbers are provided in parentheses. The two newly generated sequences in this study are indicated in bold font.

be strongly supported. Uncorrected pairwise (p) distances from the Philippines, with moderate bootstrap support of of sequences were calculated in Geneious using only the 80% (Fig. 2B). homologous 333 bp of ND2 that was generated from the Morphology. Two preserved males (THNHM H-17867, Trat specimen (THNHM H-17867). H-21064) and two preserved females (THNHM H-17879, Morphology. Measurements and scalation characters H-21063) were examined from Trat and Chanthaburi were taken by DGM on preserved specimens following Provinces of eastern Thailand (Table 2). These were the characters and definitions of Zug et al. (2017), found to be very similar in measurements and proportions except that nuchal spines (NuchalS) were counted as the to the descriptions of both B. burmana from Tanintharyi, number of enlarged nuchal crest scales that are as high as Myanmar (Zug et al., 2017) and B. rayaensis from long (versus those scales that are at least twice as large as southern Thailand and Peninsular Malaysia (Grismer et the enlarged vertebral scales; Zug et al., 2017). al., 2015, 2016; Table 3).

Results Discussion Molecular. The alignment contained 1416 characters The small fragment of mitochondrial ND2 sequence of 74 individuals of Bronchocela and seven individuals of data obtained here assigned the Trat, Thailand, specimen outgroup taxa. The 333 bp ND2 fragment obtained from to B. burmana (Fig. 2B), and extended the range of the Trat specimen (THNHM H-17867) had uncorrected this species approximately 360 air-km eastward from p-distances of 1.8–3.0% to samples of B. burmana from known records in the area of Lenya, Tanintharyi Region, the Tanintharyi Region of Myanmar (Table 2). The ML Myanmar (Zug et al., 2017; Fig. 2A). The proximity of analysis recovered the Trat specimen in a clade with these eastern Thailand records to the Cambodian border all other B. burmana samples, with strong bootstrap suggests that B. burmana probably also occurs in the support of 99% (Fig. 2B). The clade of B. burmana was Cardamom Mountains of southwestern Cambodia. To recovered as the sister taxon to B. rayaensis, with strong date, B. smaragdina in eastern Cambodia (Fig. 2A) is bootstrap support of 100% (Fig. 2B), as also shown by the only species of Bronchocela documented from the Zug et al. (2017). The B. smaragdina sample was found country (Stuart et al., 2006b). The absence of records to be distantly related to the Trat specimen and instead of Bronchocela from the Chao Phraya River Basin recovered as sister to a clade containing B. cf. marmorata of central Thailand, currently over 150 km wide and

Table 1. Localities and sexes of the four examined specimens of Bronchocela burmana from eastern Thailand deposited at the Thailand Natural History Museum.

THNHM Coordinates Elevation Sex Locality Date Collectors No. (approx.) (m) Agroforestry Research Station, 12.3894°N 26 March H-17867 M 66 MC, SM Mueang District, Trat Province 102.6769°E 2010 Khlong Yai Kee Waterfall, Ko Kut 11.6800°N 28 March H-17879 F 82 MC, SM Island, Ko Kut District, Trat Province 102.5453°E 2010 Namtok Phlio National Park, Mueang 12.5294°N 15 March Narongrit H-21063 F 140 District, Chanthaburi Province 102.1842°E 2002 Sukprakran Namtok Phlio National Park, Mueang 12.5294°N 15 March Narongrit H-21064 M 140 District, Chanthaburi Province 102.1842°E 2002 Sukprakran

Range Extension of Bronchocela burmana to Eastern Thailand 489

Table 2. Uncorrected pairwise (p) distances for Bronchocela burmana and B. rayaensis for the homologous 333 bp fragment of the mitochondrial ND2 gene that was generated from the Trat Province, eastern Thailand specimen (bold font).

GenBank Accession No. 1 2 3 4 Comments 1 KR053115 B. rayaensis 0.90 5.71 6.91 B. rayaensis vs. B. burmana 2 KR053116 B. rayaensis 0.90 5.11 6.01 4.51 7.81 (mean = 5.92) 3 KY498356 B. rayaensis 5.71 5.11 5.11 ⎯ 4 MT325799 B. burmana 6.91 6.01 5.11 5 KY366308 B. burmana 6.61 5.71 4.51 1.80 6 KY366309 B. burmana 7.51 6.61 5.41 2.70

7 KY366310 B. burmana 6.61 5.71 4.51 1.80 Within B. burmana 8 KY366311 B. burmana 6.61 5.71 4.51 1.80 0.0 3.0 (mean = 0.73) 9 KY366312 B. burmana 6.91 6.01 4.81 2.10 ⎯ 10 KY366313 B. burmana 6.61 5.71 4.51 1.80 11 KY366314 B. burmana 6.61 5.71 4.51 1.80 12 KY366315 B. burmana 7.81 6.91 5.71 3.00

Table 3. Summary of morphological characters of Bronchocela burmana and B. rayaensis from Thailand, Myanmar, and Peninsular Malaysia. Characters are defined in Zug et al. (2017), except NuchalS, which is defined in the text. Values are presented as means, followed by ranges in parentheses. Measurements are provided in millimetres. Data for B. burmana from southern Myanmar are from Zug et al. (2017), those for B. rayaensis from Grismer et al. (2015, 2016).

B. burmana B. rayaensis Peninsular Malaysia, Characters Eastern Thailand Southern Myanmar Combined Localities Southern Thailand n = 2♂, 2♀ n = 4♂, 5♀ n = 6♂, 7♀ n = 2♂, 3♀

SVL♂ 82.5 (82–83) 88.2 (84–93) 84.1 (82–93) 82

SVL♀ 84.4 (80–88) 80.8 (79–84) 81.8 (79–88) 83.1–85.4 HeadW/HeadL (%) 57 (52–60) 54 (44–62) 54 (44–62) 57 (56–59) HeadH/HeadL (%) 48 (45–51) 47 (45–50) 47 (45–51) 50 (45–56) TympD/OrbD (%) 53 (48–57) 48 (40–54) 49 (40–57) 44 (38–49) ForelL/SVL (%) 54 (53–56) 54 (50–58) 55 (50–58) 57 (53–59) HindlL/SVL (%) 88 (84–91) 92 (86–97) 91 (84–97) 92 (87–99) Nuchal crest height Low Low Low Low NuchalS 8 (8–9) 8 (6–9) 8 (6–9) 10 (8–13) Dorsal1 10 (9–11) 7 (6–8) 8 (6–11) 6 (5–8) Dorsal2 15 (13–19) 17 (15–21) 16 (13–21) Not reported 4HindfLm 30 (29–33) 33 (31–38) 32 (29–38) 33 (one male) Midbody 62 (57–71) 59 (55–67) 61 (55–71) 71 (67–74)

490 Daniel G. Mulcahy et al. approximately five million years in age (Sinsakul, 2000), Geckonidae, Eublepharidae, Uroplatidae, Pygopopdidae, results in a large geographic gap between western and Agamidae. London, United Kingdom, Trustees of the British eastern populations of B. burmana (Fig. 2A). The Trat Museum of Natural History. Edgar, R.C. (2004): MUSCLE: multiple sequence alignment with specimen exhibited relatively low, but noteworthy, high accuracy and high throughput. Nucleic Acids Research 32: uncorrected p-distances of 1.8–3.0% to the Tanintharyi 1792–1797. specimens, perhaps due to limited gene flow across the Grismer, L.L., Wood, P.L., Jr., Lee, C.H., Quah, E.S.H., Anuar, barrier formed by the Chao Phraya River Basin. S., Ngadi, E., Sites, J.W., Jr. (2015): An integrative taxonomic In contrast, B. burmana had uncorrected p-distances of review of the agamid genus Bronchocela (Kuhl, 1820) from 4.5–7.8% to specimens of B. rayaensis (Table 2) across Peninsular Malaysia with descriptions of new montane and the Isthmus of Kra in Thailand but only approximately insular endemics. Zootaxa 3948: 1–23. Grismer, L.L., Wood, P.L., Jr., Aowphol, A., Cota, M., Murdoch, 200 air-km away (Grismer et al., 2016; Fig. 2A). These M.L., Aguilar, C., Grismer, M.S. (2016): Taxonomy, phylogeny, findings suggest that the Isthmus of Kra may represent and distribution of Bronchocela rayaensis (Squamata: a significant biogeographic barrier to Bronchocela, as Agamidae) on the Thai-Malay Peninsula. 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Accepted by Justin Bernstein