Herpetology Notes, volume 10: 457-461 (2017) (published online on 06 September 2017)

Morphological and genetic verification of Ovophis tonkinensis (Bourret, 1934) in Hong Kong

Jonathan J. Fong1, Alessandro Grioni2, Paul Crow2 and Ka-shing Cheung3,*

Abstract. Hong Kong lies in the putative ranges of two Ovophis species: O. makazayazaya (Sichuan and Yunnan Provinces, Taiwan, and northern Vietnam) and O. tonkinensis (Guangxi, Guangdong, and Hainan Provinces, and northern Vietnam). It is unclear which Ovophis species is/are present in Hong Kong. Previous studies identified O. tonkinensis using morphology, but without molecular data. In this study, we verified the presence of O. tonkinensis in Hong Kong using molecular (four mitochondrial DNA loci) and morphological data of two specimens. The clade that is currently identified as Ovophis tonkinensis has a large geographic range and relatively deep divergences, hinting that its taxonomy requires further work.

Key words. Hong Kong, Ovophis tonkinensis, Ovophis makazayazaya, reptile, snake.

Introduction makazayazaya (Karsen et al. 1998). Malhotra et al. (2011) included four Ovophis specimens from Hong Kong The Asian pitviper genus Ovophis has had a (BMNH 1983 281–284) in their morphometric analyses. complicated taxonomic history. Previously, Ovophis These four specimens grouped with O. tonkinensis was believed to contain three species (O. chaseni specimens from northern Vietnam and southern China. (Smith, 1931), O. okinavensis (Boulenger, 1892), O. Studies in areas close to Hong Kong also confirmed the monticola (Gunther, 1864; 4 subspecies)) (McDiarmid presence of O. tonkinensis, including Hainan Province et al. 1999). However, Malhotra and Thorpe (2004) (morphology: David (1995), David (2001), Wang et removed the first two species from this genus so that al. (2009); molecular: Malhotra et al. (2011)), western Ovophis referred only to the O. monticola species Guangdong Province (morphology: Zhang et al. (2011)), complex. Malhotra et al. (2011) delineated five species and western Guangxi Province (morphology: Kadoorie in Ovophis by elevating some subspecies to the species Farm & Botanical Garden (2003)). In this study, we use level based on molecular and morphological datasets. molecular and morphological data from two specimens In Hong Kong (and southern China), it is unclear which to clarify the identity of the Ovophis species in Hong Ovophis species is/are present. Previous morphological Kong, and comment on the status of O. tonkinensis. work in Hong Kong identified the species in Hong Kong to be Trimereseurus/Ovophis monticola (Romer Materials and Methods and Bogadek 1983, Zhao et al. 1998, Li et al. 2002), T. monticola monticola (Karsen et al. 1986), or T. m. Samples.—Two Ovophis specimens were captured on the hillside at Kadoorie Farm and Botanic Garden (KFBG) Tai Po, Hong Kong (22.43121°N, 114.11822°E) by the staff of KFBG in February 2011. General anaesthesia was administered prior to collecting blood and scale clippings. All the procedures were previously 1 Science Unit, Lingnan University, Tuen Mun, New Territories, approved by the KFBG ethical committee and were Hong Kong performed by the resident veterinary surgeon. Snake 1 2 Fauna Conservation Department, Kadoorie Farm and Botanic (������������������������������������������������ Garden, Hong Kong 3 Agriculture, Fisheries and Conservation Department, Cheung The total length of each individual was ��������� Sha Wan, Kowloon, Hong Kong 56�������������� cm� Upon recovery, the snakes were * Corresponding author e-mail: [email protected] released back to the site of capture. 458 Jonathan J. Fong et al.

Morphology.—Scalation was used to assign the with bootstrapping. The GTR+G model of nucleotide specimens to species. Ovophis makazayazaya and O. substitution was used for each partition in the 100 tonkinensis can be distinguished from other Ovophis ML tree searches and 1000 bootstrap replicates. For species based on the relative size of the supralabial BI analyses, models of nucleotide substitution were scales—these two species have a larger 4th supralabial, chosen within MrBayes using the reversible jumping while other species have a larger 3rd supralabial (David model choice (nst=mixed) with both rate variation and & Tong 1997). The state of the subcaudal scales and invariable sites (rates=invgamma). Two BI searches number of ventral scales can be used to distinguish O. with random starting trees were run and compared using makazayazaya and O. tonkinensis—O. makazayazaya four Markov Chain Monte Carlo (MCMC) chains, each has paired subcaudal scales and 131–155 ventral chain run for 10 million generations, and sampled every scales, while O. tonkinensis has predominantly single 1000th generation. Stationarity was evaluated by plotting subcaudal scales and 127–145 ventral scales (Malhotra the log-likelihood scores in Tracer v.1.5 (Rambaut et al., et al. 2011). 2013), and data from the first 2 million generations were Molecular.—DNA was extracted from blood and/or discarded before combining to building a consensus scales using the Qiagen DNeasy mericon Food Kit. tree. We evaluated sequence divergence for each locus Following the primer sets and thermocycler conditions by calculating the uncorrected p-distances using MEGA used in Malhotra et al (2011), four mitochondrial DNA v7.0.16 (Kumar et al. 2016). Calculations of uncorrected loci were amplified: 12S rRNA (12S), 16S rRNA p-distances were made with partial deletion of missing (16S), cytochrome b (cytb), and NADH dehydrogenase data and a site coverage cutoff of 95%. subunit 4 (ND4). Polymerase chain reactions (PCR) in a final volume of 25 μL containing 2 μL genomic DNA, PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, and 0.01% gelatin), 0.4 mmol/L of each deoxynucleotide triphosphate (dNTPs), 0.5ul forward and reverse primers and 2.0 U DNA Taq polymerase (Applied Biosystems, Foster City, CA). PCR amplifications were carried out in an Applied Biosystems 9700 automated thermal cycler. PCR products were visualised on a 2% agarose gel and purified using the Qiagen QIAquick PCR purification kit. Products were sequenced in both directions using PCR primers on an Applied Biosystems 3130 Genetic Analyzer using BigDye v3.1. Corresponding sequences for each specimen and locus were visually inspected, assembled in Geneious Pro v5.3.6, and aligned to data from Malhotra et al. (2011) using MUSCLE (Edgar 2004). Alignments for cytb and ND4 were checked for stop codons by translating nucleotides to amino acids. Sequences were submitted to GenBank (KX710068- KX710075). Phylogenetic analyses were performed on five datasets: 12S, 16S, cytb, ND4, and the concatenated dataset. Phylogenetic trees were inferred using Maximum Likelihood (ML) in RAxML v8.2.4 (Stamatakis 2014) and Bayesian Inference (BI) in MrBayes v3.2.5 (Ronquist et al. 2012). The loci coding Figure 1 Scale characters of the Ovophis specimen (MPV1) for gene products (cytb and ND4) were partitioned by collected from Kadoorie Farm and Botanic Garden, Hong codon position, while the concatenated dataset was Kong. Both specimens in this study had the same characters, partitioned by locus and codon position. For ML, we which are unique to identify O. tonkinensis. A) 4th supralabial performed the analysis that combines the ML search larger than 3rd supralabial, and B) unpaired subcaudal scales. Morphological and genetic verification of Ovophis tonkinensis in Hong Kong 459

Figure 2 Maximum Likelihood (ML) phylogeny of the concatenated Ovophis dataset—12S rRNA (12S), 16S rRNA (16S), cytochrome b (cytb), and NADH dehydrogenase subunit 4 (ND4). Support values of major nodes are indicated (ML bootstrap/ Bayesian Inference posterior probability). Asterisks (*) indicate ML bootstrap >90 and Bayesian Inference posterior probability >0.9. Both Ovophis specimens from Hong Kong (MPV1, MPV2), indicated by arrows and larger, bold font, were nested within the O. tonkinensis clade. Clade names and taxonomy follow Malhotra et al. (2011). 460 Jonathan J. Fong et al.

Results and Discussion (Malhotra et al. 2011, M.-O. �������������������������� Comm. 2 Dec 2016). Based on scant records with Our study is the first to include DNA sequence data from morphological data (Zhang et al. 2011, Kadoorie Farm Hong Kong Ovophis specimens. Both morphological & Botanical Garden 2003) we expect O. tonkinensis is and molecular data confer, suggesting that the two present in Guangdong and Guangxi Provinces. Future Ovophis specimens in this study are O. tonkinensis. studies should focus on southern China to determine The deep divergences within O. tonkinensis, combined species presence based on both morphological and with the large geographic range (China including Hong molecular data. In addition to delineating the ranges of Kong to central Vietnam), suggests that O. tonkinensis O. makazayazaya and O. tonkinensis in the region, these is a species complex that requires further study. data will help us understand whether O. tonkinensis Scale characters matched those of O. tonkinensis— is a single genetically diverse species, or a species both specimens had 4th supralabials larger than 3rd complex. supralabials (Figure 1A) and unpaired subcaudal scales (Figure 1B). Ventral scale counts largely overlap Acknowledgements. We would like to thank many people for between O. makazayazaya (131–155 scales) and O. their help with this work: Toni Ngai and David Willott for helpful tonkinensis (127–145 scales) and were not diagnostic discussion, Dr. Gary Ades, Dr. Michael Lau, and ������� ������ for Hong Kong specimens (MPV1= 136, MPV2=131). KFBG for the provision of specimens� ������������������������ Phylogenetic analyses of individual loci (not comments on the manuscript, TH Fung and Wing Tang of the shown) had poor resolution and did not resemble the Agriculture, Fisheries and Conservation Department (AFCD) for consensus tree of Malhotra et al. (2011). Analysing technical assistance, Dr. Anita Malhotra for access to references, Mr. Patrick David for providing references and unpublished data the concatenated dataset, the topologies of the inferred on the taxonomy of O. tonkinensis, Dr. Mark-Oliver ����� ��� phylogenetic trees were identical when using ML and Frank Tillack for information on specimens at the ������ ��r BI (only slight differences in branch lengths and support Naturkunde, and Dr. Yik Hei Sung for reviewing this manuscript. values) (Figure 2). This tree was identical to that of This study was financially supported by the AFCD. Malhotra et al. (2011). Both Hong Kong specimens are genetically highly similar (sequence divergence References for the concatenated dataset: 0–1.2%) and were nested David, P. (1995): Geographic distribution. Ovophis tonkinensis. in O. tonkinensis (Clade B of Malhotra et al. (2011)) Herpetological Review 26: 157. with strong support (ML bootstrap=82, BI posterior David, P. (2001): On the occurrence of the snake genus Ovophis probability=1.0). The O. tonkinensis clade (including (Serpentes: Viperidae: Crotalinae) on Hainan Island, China. the Hong Kong specimens) had medium to high levels of Acta Zoologica Sinica 26: 388–393. intraspecific sequence divergence (maximum sequence David, P., Tong, H. (1997): Translations of recent descriptions divergence: ND4=12.3%, cytb=18.5%, 12S=4.3%, of Chinese pitvipers of the Trimeresurus complex (Serpentes: Viperidiae), with a key to the complex in China and adjacent 16S=2.0%) compared to other species/clades (maximum areas. 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Accepted by Benjamin Tapley