4092 (3): 414 420 article of zoology, faculty of science, kasetsart university, bangkok 10900,...

ZOOTAXA

ISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)Copyright © 2016 Magnolia Press

Zootaxa 4092 (3): 414–420

www.mapress.com/zootaxa/Article

http://dx.doi.org/10.11646/zootaxa.4092.3.6

http://zoobank.org/urn:lsid:zoobank.org:pub:57809A4B-65EC-43E1-8F04-D18C23197F75

Taxonomy, phylogeny, and distribution of Bronchocela rayaensis (Squamata:

Agamidae) on the Thai-Malay Peninsula

L. LEE GRISMER1, PERRY L. WOOD, JR2., ANCHELEE AOWPHOL3, MICHAEL COTA4,

MATHEW L. MURDOCH1, CÉSAR AGUILAR2,5,6 & MARTA S. GRISMER1

1Herpetology Laboratory, Department of Biology, La Sierra University, 4500 Riverwalk Parkway, Riverside, California 92515 USA.

E-mail: [email protected], [email protected], [email protected] of Biology, Brigham Young University, 150 East Bulldog Boulevard, Provo, Utah 84602 USA. E-mail: [email protected] of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. E-mail: [email protected] History Museum, National Science Museum, Thailand, Technopolis, Khlong 5, Khlong Luang, Pathum Thani 12120 Thai-

land. E-mail: [email protected] de Herpetología, Museo de Historia Natural de San Marcos (MUSM), Av. Arenales 1256, Jesús María, Lima, Peru.

E-mail: [email protected] de Ciencias Biológicas Antonio Raimondi, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos,

Lima, Peru

Abstract

An integrative taxonomic analysis used to identify a new population of Bronchocela from Phuket Island, Thailand indi-

cates it is conspecific with B. rayaensis from the Langkawi Archipelago of northwestern Peninsular Malaysia. An addi-

tional specimen photographed from Khura Buri District, Phang-nga Province is also considered to be B. rayaensis and

represents a northern range extension of 295 km from the Langkawi Archipelago.

Key words: Peninsular Malaysia, Thailand, Systematics, Bronchocela

Introduction

The agamid genus Bronchocela (Kuhl, 1820) contains 12 species (Grismer et al. 2015; Hallermann 2009) that collectively range from South Asia, southern Indochina and the Philippines, southward and eastward through the Thai-Malay Peninsula and the Indo-Australian Archipelago to at least western New Guinea (Manthey 2008). Bronchocela are attractive, conspicuous, diurnal, arboreal lizards inhabiting open and disturbed areas from sea level to over 1,600 meters in elevation and are often seen perched in open, sunlit areas as high as 30 meters above the ground (Grismer 2011). Based on an integrative taxonomic analysis, Grismer et al. (2015) recognized two new species of Bronchocela from Peninsular Malaysia; B. shenlong Grismer, Wood, Lee, Quah, Anuar, Ngadi, & Sites and B. rayaensis Grismer, Wood, Lee, Quah, Anuar, Ngadi, & Sites. The latter was considered a possible endemic to the Langkawi Archipelago of northwestern Peninsular Malaysia although based on photographs, Grismer et al.

(2015) noted similarities of it to populations from Phuket Island, Thailand—a locality from which Bronchocela has yet to be officially recorded—and suspected the two populations were closely related. We collected a gravid female (ZMKU R 00717) found sleeping in a tree 5 m above the ground at the Kathu Waterfall on Phuket Island (Fig. 1) on 31 August 2015 and report here that based on genetic and morphological analyses, it is conspecific with B.

rayaensis. An additional specimen photographed from Khura Buri, Phang-nga Province 120 km north of the Kathu Waterfall and 295 km northwest of the Langkawi Archipelago, is also considered to be B. rayaensis. The evidence for these decisions is presented below.

414 Accepted by S. Carranza: 10 Feb. 2016; published: 17 Mar. 2016

FIGURE 1. Distribution of Bronchocela rayaensis in Thailand and Peninsular Malaysia.

Materials and methods

Genomic DNA was isolated from liver or skeletal muscle samples stored in 95% ethanol using the Qiagen

DNeasyTM tissue kit (Valencia, CA, USA). ND2 was amplified using a double-stranded Polymerase Chain Reaction (PCR) under the following conditions: 1.0 µl (~10–30 µg of DNA) genomic DNA, 1.0 µl (10 µM) light strand primer, 1.0 µl (10µM) heavy strand primer, 1µl (10 µM) dinucleotide pairs, 2.0 µl (1.5 mM) 5x buffer, 1.0 µl (1.5 mM) MgCl 10x buffer, 2.5 u Taq polymerase, and 7.56 µl ultra-pure H

2O (Table 2). PCR reactions were executed

on an Eppendorf Mastercycler gradient thermal cycler under the following conditions: initial denaturation at 95°C for 2 min, followed by a second denaturation at 95°C for 35 s, annealing at 48°C for 35 s, followed by a cycle extension at 72°C for 35 s, for 31 cycles. All PCR products were visualized on a 1 % agarose gel electrophoresis. Successful PCR products were vacuum purified using MANU 30 PCR plates (Millipore) and purified products were resuspended in ultra-pure water. Purified PCR products were sequenced using the ABI Big-Dye Terminator v3.1 Cycle Sequencing Kit in an ABI GeneAmp PCR 9700 thermal cycler. Cycle sequencing reactions were purified with Sephadex G-50 Fine (GE Healthcare) and sequenced on an ABI 3730xl DNA Analyzer at the BYU DNA Sequencing center. Primers used for amplification and sequencing are presented in Table 2. Sequences were

Zootaxa 4092 (3) © 2015 Magnolia Press · 415BRONCHOCELA RAYAENSIS FROM PHUKET

analyzed from both the 3' and the 5' ends separately to confirm congruence between the reads. Both the forward

and the reverse sequences were uploaded and edited in GeneiousTM version v5.5.6 (Drummond et al. 2011). The protein-coding region of the ND2 sequence was aligned by eye. MacClade v4.08 (Maddison & Maddison 2005) was used to calculate the correct amino acid reading frame and to confirm the lack of premature stop codons.

FIGURE 2. Inferred phylogenetic relationships of Bronchocela from Peninsular Malaysia and its associated islands with

samples from Indonesia, Borneo, and the Philippines. The phylogram is a Maximum Likelihood topology with Bayesian

posterior probabilities and Maximum Likelihood bootstrap values, respectively.

For the phylogenetic analyses we applied two model-based methods, Maximum Likelihood (ML) and Bayesian Inference (BI). We used a pruned data set of 14 individuals from 10 localities ranging across Sundaland from the original data set of Grismer et al. (2015) that contained 55 individuals from 33 localities. The data set was partitioned by gene and the Bayesian Information Criterion (BIC) implemented in IQ-TREE (Nguyen et al. 2015) indicated K3Pu+I+G4 as the best-fit model of evolution for each. The ML analysis was performed using IQ-TREE (Nguyen et al. 2015) with 1000 bootstrap pseudoreplicates using the ultrafast bootstrap approximation algorithm. The Bayesian analysis was carried out in MrBayes 3.2.3. on XSEDE (Ronquist et al. 2012) using CIPRES (Cyberinfrastructure for Phylogenetic Research; Miller et al. 2010) employing default priors. Two simultaneous runs were performed with four chains, seven hot and one cold. The chains were run for 5,000,000 generations and sampled every 500 generations using the Markov Chain Monte Carlo (MCMC). The first 25% of each run was discarded as burn-in using the sumt function. Stationarity was confirmed in Tracer v1.6 (Rambaut et al. 2014). We considered BI posterior probabilities of 0.95 and above and ML bootstrap support values of 95 and above as significant nodal support values (Huelsenbeck et al. 2001).

GRISMER ET AL.416 · Zootaxa 4092 (3) © 2015 Magnolia Press

Results

The phylogenetic analyses strongly indicate (1.00/100) that the Bronchocela specimen from Phuket Island is conspecific with B. rayaensis from Pulau Langkawi (Fig. 2). The ML analysis possibly indicates that one of the individuals from Pulau Langkawi (LSUHC 12061) is actually more closely related to the Phuket Island individual (ZMKU R 00717) than it is to the other Langkawi individual (LSUHC 7535) although bootstrap significance for this (74) is not significant and the BI analysis is uniformative in this regard.

TABLE 1. Variation in measurements, morphometric ratios, scales counts, and color pattern characteristics between

Bronchocela rayaensis from Pulau Langkawi, Peninsular Malaysia and Phuket Island, Thailand.

LSUHC LRCUKM LSUHC

7535 136 12437

Langkawi Langkawi Phuket

holotype paratype

Sex m f f

SVL 82.0 85.4 83.1

TL 308.0 302.97 281

TL/SVL 3.76 3.55 3.04

DT 3.10 3.30 2.30

DO 6.38 7.15 6.11

DT/DO 0.49 0.46 0.38

HL 21.73 20.86 20.35

HW 12.10 12.38 11.38

HD 10.44 11.22 9.14

HW/HL 0.56 0.59 0.56

HD/HL 0.49 0.56 0.45

HL/SVL 0.27 0.24 0.25

HaL 15.74 15.69 13.82

FLL 48.23 49.96 43.95

HaL/FLL 0.34 0.31 0.31

FLL/SVL 0.59 0.59 0.53

FoL 32.10 28.94 26.24

HLL 81.21 78 71.83

FoL/HLL 0.38 0.37 0.37

HLL/SVL 0.99 0.91 0.87

supralabials 11 10 10

infralabials 10 8 9

number of canthal scales 7 5 5

postmentals 3 3 3

upper dorsal scale rows pointing backwards 8 5 5

midbody scales 71 67 74

number of nuchal spines 13 9 8

3rd finger lamellae 30 30 30

4th finger lamellae 33 31 35

3rd finger longer than 4th yes yes same


FIGURE 3. Upper and middle: Bronchocela rayaensis (ZMKU R 00717) from the Khatu Waterfall, Phuket Island, Thailand

(photos by L.L. Grismer). Lower: B. rayaensis (LSUDPC 9813; photo by M. Cota) Khura Buri District, Phang-nga Province,

Thailand.


The morphological analysis supports the phylogenetic analyses in that the squamation and coloration characteristics of the Phuket Island specimen fall within the range of that of B. rayaensis for most characters (Table 1). Additionally, the Phuket Island specimen bears the combination of diagnostic character states that separate B.

rayaensis from all other Bronchocela (fide Grismer et al. 2015) in having a relatively small SVL (83.1 mm); a small tympanum (DT/DO 0.38); head squarish in lateral profile (HD/HL 0.45) and relatively narrow (HW/HL 0.56); forelimbs relatively long (FLL/SVL 0.53); foot relatively short (FoL/HLL 0.37); hind limbs relatively long (HLL/SVL 0.87); five canthal scales; three postmentals; nuchal crest low, longer than diameter of orbit, spines lancolate, and bearing eight scales; 74 midbody scales; no rows of paravertebral scales bearing keels pointing dorsally; five rows of dorsal scales bearing keels pointing posteriorly; ventral scales less than five times the width of dorsal scales; 30 subdigital lamellae on the third finger; 35 subdigital lamellae on the fourth toe; third finger same length or smaller than fourth finger; fourth finger not longer than fifth toe; no white patch between tympanum and orbit; a white patch below tympanum, tympanum green in adults; no white or yellow supralabial stripe; no red gular patch; no white ventrolateral line on body; no postfemoral red line; and faint caudal bands (Fig. 3).

We photographed an additional specimen of Bronchocela from Khura Buri District, Phang-nga Province 120 km north of Phuket Island that has a similarly appearing gracile body stature, a low nuchal crest bearing eight scales, a green tympanum, and a white patch below the tympanum—all diagnostic characters of B. rayaensis (Fig. 3). We thus consider this specimen conspecific with B. rayaensis as well.

Discussion

The lack of a deep genetic divergence and geographic substructuring among the specimens of Bronchocela

rayaensis from Phuket and Langkawi Islands (Fig. 2) indicates that gene flow is highly likely on the Thai-Malay Peninsula between these three populations (Fig. 1) or there has not been enough time for mutations to accumulate. The presence of B. rayaensis in southern Thailand confirms the hypothesis of Grismer et al. (2015) that the Langkawi Island population would have phylogenetic affinities with Thai or Indochinese populations rather than with populations from Peninsular Malaysia. This underscores the fact that the Thai-Malay Peninsula is a biogeographical crossroads between Indochinese and Sundaic taxa (Lohman et al. 2010). Although this crossroads hypothesis has been purported for a broad range of taxa (see Dejtaradol et al. 2015 and references therein), there has been surprising little phylogenetic evidence for the herpetofauna of this region in this regard. We are in the process of developing an extensive phylogenetic data set on taxa ranging throughout the Thai-Malay Peninsula that will further bear out the crossroads hypothesis.

Acknowledgments

Partial funding was provided to LLG by the College of Arts and Sciences, La Sierra University.

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