Asian Herpetological Research 2010, 1(1): 1-21 DOI: 10.3724/SP.J.1245.2010.00001

Toward a Phylogeny of the Kukri ,

Marc D. Green1, Nikolai L. Orlov2 and Robert W. Murphy1,3* 1 Department of Natural History, Royal Ontario Museum, 100 Queen’s Park, Toronto, ON M5S 2C6 Canada 2 Zoological Institute, Russian Academy of Sciences, Universitetskaya nab 1, St. Petersburg 199034, Russia 3 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223,

Abstract The South and Southeast Asian genus Oligodon, known for its egg-eating feeding behavior, has been a taxonomically and systematically challenging group. This work provides the first phylogenetic hypothesis for the ge- nus. We use approximately 1900 base pairs of mitochondrial DNA sequence data to infer the relationships of these snakes, and we examine congruence between the phylogeny and hemipenial characters. A hypothesis for the position of Oligodon within the is also proposed. We discuss the implications of the phylogeny for previous taxonomic groupings, and consider the usefulness of the trees in analysis of behavior and biogeography of this genus. Keywords Oligodon, genealogy, Colubridae, hemipenis, mitochondrial DNA, Southeast , South Asia

1. Introduction commenting on their relationships. However, his work predated the formalization of phylogenetic methods The genus Oligodon Fitzinger 1826, the kukri snakes, (Hennig, 1966). Very little progress has been made since contains 70 distributed throughout much of South then and Oligodon has yet to receive a modern phyloge- and Southeast Asia (Green et al., in press). The common netic treatment using either morphology or molecules. name derives from a distinctively shaped Nepalese knife, Leviton (1963a) studied six Philippine taxa and Zhao et the kukri. The hind teeth of these snakes are broad and al. (1998) added new data in their examination of Chi- strongly recurved, much like the shape of the kukri. nese species. Neither study constructed a phylogenetic These non-venomous snakes are usually nocturnal and tree. Most works on Oligodon have not been comparative often brightly colored. They feed primarily on the eggs of and they have focused on small, politically defined geo- and . The morphology of their teeth is very graphic regions. Thus, in the absence of a comparative effective for cutting open eggs. Although some work on framework, some authors have perpetuated errors and this aspect of their life history exists, little can be inferred confusion. about the evolution of this unique feeding strategy wi- Oligodon’s preference for eggs as a source of food is thout a phylogeny. well documented (Broadley, 1979; Coleman et al., 1993; Several attempts have been made to study the phy- Golf, 1980; Hu and Zhao, 1987; Minton and Anderson, logenetic relationships and of Oligodon 1963; Ota and Lin, 1994; Wall, 1921). Their egg-eating (Campden-Main, 1969; David et al., 2008b; Leviton, mechanisms and behavior are known (Coleman et al., 1963a; Pope, 1935; Smith, 1943; Tillack and Günther, 1993; Kiran, 1981; Kiran, 1982; Minton and Anderson, 2009; Wall, 1923; Wallach and Bauer, 1996) and these 1963; Ota and Lin, 1994). The snakes enthusiastically efforts have met with mixed success. Smith (1943) made ingest the contents of opened eggs presented to them, and the most ambitious review, examining 34 species and also ingest small and eggs whole. An in-

teresting and unusual aspect of this natural history is their *Corresponding author: Prof. Robert W. Murphy, holds faculty posi- apparent adaptation to taking eggs too large to swallow. tions at the University of Toronto, the Chinese Academy of Sciences, They use their uniquely shaped teeth and jaw articulation Normal University and East China Normal University, with his to saw a hole in an egg-shell large enough to insert their research focusing on evolutionary biology and conservation genetics. E-mail: [email protected] head, and feed on the contents. Stomach content studies Received: 30 June 2010 Accepted: 20 July 2010 reveal that Oligodon also eat small rodents, tadpoles,

2 Asian Herpetological Research Vol. 1 frogs and insects (Batchelor, 1958; Cox, 1991; David and (1895) drew the hemipenis of O. ancorus for use in the Vogel, 1996; Hu, 2001; Hwang et al., 1965; Meggitt, species’ description, suggesting that it might indicate 1931; Schulz, 1988; Shi and Zheng, 1985; Toriba, 1987; historical relationships. Wall (1923) first looked at the Wall, 1921; Wall, 1923). Juveniles appear to be primarily hemipenes of Oligodon comparatively. Pope (1935) com- insectivorous. It is not known whether they hunt or sca- piled a list of hemipenial characters in an attempt to dis- venge for food. tinguish Holarchus from Oligodon. However, he was The feeding habits of Oligodon have aroused interest unable to do so because O. bitorquatus, the type species in Chinese agriculture. At least one influential Asian re- for the genus, was intermediate in condition. Smith (1943) ference stated they are harmful to crop production (Hu compiled observations on the hemipenes of Oligodon. and Zhao, 1987), presumably because they eat the eggs of Leviton (1963a) used hemipenial data to reconstruct the other beneficial insectivorous reptiles. evolutionary history of the Philippine taxa. The groupings Some other behaviors of Oligodon may have an un- used today within Oligodon have been primarily based on derlying phylogenetic basis. Most species are docile Smith’s work and they have been taken to reflect histori- (David and Vogel, 1996; Pope, 1935; Stuebing and Inger, cal descent. Based on hemipenial morphology, Oligodon 1999; Wall, 1921). However, O. fasciolatus has an was considered to be a member of the (Zaher, aggressive disposition (Smith, 1943; Taylor, 1965). It also 1999), now the Colubridae (Zaher et al., 2009). Herein, displays an interesting defensive behavior in which a the diversity of Oligodon’s hemipenial morphology (Fi- tail-up posture is taken and the bright red hemipenes are gures 1–4) provided a foundation for character coding. extruded repeatedly, contrasting noticeably with the white ventral scales (Wüster and Cox, 1992). Feeding, behavior and the apparent large number of species in Oligodon provoke interest in its phylogenetic position within the Colubridae. Phylogeny is useful for discussing the evolution of oophagy in snakes (de Queiroz and Rodríguez-Robles, 2006) and phylogenetic relationships within Oligodon could provide insights into what drove the radiation within the clade. Phyllorhynchus Figure 1 Photograph of one everted, bifurcate (forked) hemipenis uses a similar egg-contents extracting strategy for feeding of Oligodon chinensis (ROM34581) (Gardner and Mendelson III, 2003), and it and Oligodon might share a close phylogenetic relationship (Dowling, 1974; Dowling et al., 1996; Lawson et al., 2005). How- ever, the position of these snakes within the larger Colu- bridae is unresolved (Dowling, 1974; Dowling et al., 1996; Kraus and Brown, 1998; Lawson et al., 2005; Lopez and Maxson, 1996; Zhang et al., 1984). Although de Queiroz and Rodriguez-Robles discuss Oligodon and Phyllorhynchus, they do not include them in their tree-based character-optimization analysis. The geographic distribution of Oligodon is complex. Several species are widespread and many have overlap- ping ranges. Those ranges are all threatened due to the rapid pace of development in South and Southeast Asia (Dang and Nhue, 1995; de Silva, 1998). Many ranges are now fragmented and it is unclear what impacts this has had on the genetic diversity of isolated populations.

Although hemipenial defensive behaviors have not Figure 2 Photograph of the everted hemipenes of Oligodon for- been extensively surveyed in Oligodon, the hemipenis mosanus (ROM35634), showing the bifurcate (forked) condition itself has been used to infer historical relationships. Cope and large papillae (arrows). No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 3

Mitochondrial DNA (mtDNA) sequences can be used to reconstruct the phylogenetic relationships of snakes (de Queiroz and Lawson, 1994; Kraus and Brown, 1998; Lawson et al., 2005; Lopez and Maxson, 1996; Murphy et al., 2002; Rodríguez-Robles and de Jesús-Escobar, 1999; Slowinski and Lawson, 2002; Vidal et al., 2000). Herein, we present a molecular phylogeny for Oligodon based on mtDNA data and discuss correlation of rela- tionships and hemipenial morphology. Because Oligodon is such a speciose group, and tissue samples are not

Figure 3 Drawing of everted hemipenes of , available for most species, this study suffers somewhat showing the non-bifurcate (non-forked) condition. Small papillae from an under-sampling of taxa. Nevertheless, it forms present in O. albocinctus are not visible on this view. Some apical the foundation for future comparative work for exami- flounces and calyces are shown. Redrawn from Wall (1923; everted ning the evolution of structures, behaviors and biogeog- drawing), and Smith (1943; dissected view). raphy in the group.

2 Materials and Methods

2.1 Specimens examined In total, 48 specimens of Oligodon were sequenced (Table 1), including three uni- dentified specimens, two of which appeared very similar to Oligodon chinensis, but would have represented a sub- stantial and unusual range extension for that species. The sequenced individuals represented 17 of 70 named spe- cies and the collecting localities were mapped (Figure 5).

Figure 4 Drawing of everted hemipenes of , The sequence data for O. modestus were a synthesis of a showing non-bifurcate (non-forked) condition. Spines are typical partial sequence from ROM17490 for 12S rRNA, tRNAval for many of the Indian Oligodon. Redrawn from Wall (1923). and the 16S rRNA data of Lopez and Maxson (1996).

Table 1 A list of specimens chosen for DNA sequencing, their voucher numbers and locality data. For some specimens in the ROM collec- tion, denoted *, field numbers are provided in lieu of a catalogue number. ROM=Royal Ontario Museum; CAS=California Academy of Sci- ence; TNHM=Texas Natural History Museum; UMMZ=University of Michigan Museum of Zoology; USNM=United States National Mu- seum.

Species Voucher reference Locality and GenBank accession numbers Dinodon semicarinatus AB008539 Oligodon arnensis ROM 19155 Assam, HM591499 O. barroni ROM 32464 Krong Pa, Gai Lai, HM591523 O. chinensis ROM 25757* Chi Linh, Hia Duong, Vietnam O. chinensis ROM 34540 Chi Linh, Hia Duong, Vietnam HM591527 O. chinensis ROM 35626 Quang Thanh, Cao Bang, Vietnam HM591526 O. chinensis ROM 26940* Quang Thanh, Cao Bang, Vietnam O. chinensis ROM 31032 Tam Dao, Vinh Phu, Vietnam HM591524 O. chinensis ROM 30824 Pac Ban, Tuyen, Vietnam HM591525 O. sp. ROM 30971 24 km W of Con Cuong, Nghe An, Vietnam O. sp. ROM 30970 24 km W of Con Cuong, Nghe An, Vietnam HM591528 O. cinereus ROM 25755* Chi Linh, Hia Duong, Vietnam O. cinereus ROM 32462 Chi Linh, Hia Duong, Vietnam HM591501 O. cinereus ROM 29552 Tam Dao, Vinh Phu, Vietnam HM591502 4 Asian Herpetological Research Vol. 1

(Continued Table 1)

Species Voucher reference Locality and GenBank accession numbers O. cinereus ROM 37092 Cat Tien, Dong Nai, Vietnam HM591504 O. cinereus CAS 210159 Alaungdaw Kathapa Ntl. Pk., Sagaing Div., HM591505 O. cinereus CAS 215597 Alaungdaw Kathapa Ntl. Pk., Sagging Div., Myanmar O. cinereus CAS 205028 Rakhine Yoma Mtn. Rng., Rakhine State, Myanmar HM591507 O. cinereus CAS 213379 Hlaw Ga Park, Yangon Div., Myanmar HM591506 O. cinereus CAS 215261 Kalaw Tnshp., Shan State, Myanmar HM591508 O. cinereus ROM 30969 24km W of Con Cuong, Nghe An, Vietnam HM591503 O. cruentatus CAS 213271 Hlaw Ga Park, Yangon Div., Myanmar HM591517 O. cruentatus CAS 213408 Hlaw Ga Park, Yangon Div., Myanmar O. cyclurus CAS 204963 Mwe Hauk, Ayeyarwady Div., Myanmar HM591535 O. cyclurus CAS 215636 Alaungdaw Kathapa Ntl. Pk., Sagging Div., Myanmar HM591536 O. formosanus ROM 25827* Chi Linh, Hia Duong, Vietnam O. formosanus ROM 35806 Chi Linh, Hia Duong, Vietnam HM591532 O. formosanus ROM 35629 Quang Thanh, Cao Bang, Vietnam HM591533 O. formosanus ROM 35630 Quang Thanh, Cao Bang, Vietnam O. formosanus ROM 30939 Ba Be, Cao Bang, Vietnam HM591531 O. formosanus ROM 30823 Pac Ban, Tuyen, Vietnam HM591529 O. formosanus ROM 30826 Tam Dao, Vinh Phu, Vietnam HM591530 O. maculatus TNHC 59846 Barangay Baracatan, Mindinao Is., Philippines HM591511 O. modestus ROM 17490 Valencia, Negros Is., Philippines HM591498 O. modestus 16S mRNA from Lopez and Maxson, 1995 O. ocellatus ROM 32261 Yok Don, Dak Lak, Vietnam HM591534 O. octolineatus UMMZ 201913 3 Km E of Tutong, Tutong Dist., Brunei HM591519 O. planiceps CAS 213822 Shwe Set Taw Wldlf. Sanc., Magwe Div., Myanmar HM591514 O. splendidus CAS 204855 Kyauk Se Tnshp., Mandalay Div., Myanmar HM591509 O. splendidus USNM 520626 2 Km WNW Chatthin, Chatthin Wldlf. Sanc., Burma HM591510 O. taeniatus USNM 520625 2 Km WNW Chatthin, Chatthin Wldlf. Sanc., Burma HM591520 O. taeniatus ROM 32260 Yok Don, Dak Lak, Vietnam HM591521 O. taeniatus ROM 37091 Cat Tien, Dong Nai, Vietnam HM591522 O. theobaldi CAS 210710 Naung U Tnshp., Mandalay Div, Myanmar HM591515 O. theobaldi CAS 213896 Shwe Set Taw Wldlf. Sanc., Magwe Div., Myanmar HM591516 O. torquatus CAS 210693 Pakokku Tnshp., Magwe Div., Myanmar HM591512 O. torquatus CAS 210695 Pakokku Tnshp., Magwe Div., Myanmar Min Gone Taung Wldlf Sanc, Mandalay Div., Myan- O. torquatus CAS 215976 HM591513t mar O. venustus ROM 19156 India HM591500 O. sp. ROM 27049* Quang Thanh, Cao Bang, Vietnam HM591518 Phyllorhynchus decurtatus ROM 14153 Ocotillo, Imperial County, California, United States HM591497 Pituophis lineaticollis AF512746

The sequenced specimens of Oligodon were primarily Phyllorhynchus decurtatus, a putative close relative of from Vietnam and Myanmar. Specimens were selected to Oligodon, was selected as the initial outgroup taxon and a maximize species representation in the analysis. We also specimen was sequenced de novo for the equivalent attempted to evaluate inter- and intra-population genetic mtDNA regions used in the ingroup. Sequences from Gen- variation within species when possible by including mul- Bank (http://www.ncbi.nlm.nih.gov/GenBank) for more tiple specimens. distantly related colubrids, including Dinodon semicarinatus No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 5

Figure 5 Composite satellite photograph of Southeast Asia, showing the relief and topographic features and the collection localities of specimens of Oligodon from Vietnam and Myanmar. The localities from Brunei, India and the Philippines are indicated by arrows in their approximate directions. CB=Ba Be & Quang Thanh, Cao Bang, Vietnam; PB=Pac Ban, Tuyen, Vietnam; CL=Chi Linh, Hia Duong, Vietnam; TD=Tam Dao, Vinh Phu, Vietnam; CC=24km W of Con Cuong, Nghe An, Vietnam; KP=Krong Pa, Gai Lai, Vietnam; YD=Yok Don, Dak Lak, Vietnam; CT=Cat Tien, Dong Nai, Vietnam; VP=Valencia, Negros Is., Philippines; BP=Barangay Baracatan, Mindinao Is., Philippines; TB=3 km E of Tutong, Tutong Dist., Brunei; IN=India; MH=Mwe Hauk, Ayeyarwady Div., Myanmar; HG=Hlaw Ga Park, Yangon Div., Myanmar; RY=Rakhine Yoma Mtn. Rng., Rakhine State, Myanmar; KS=Kyauk Se Tnshp., Mandalay Div., Myanmar; KT=Kalaw Tnshp., Shan State, Myanmar; SS=Shwe Set Taw Wldlf. Sanc., Magwe Div., Myanmar; NU=Naung U Tnshp., Mandalay Div, Myanmar; MGT=Min Gone Taung Wldlf Sanc, Mandalay Div,.Myanmar; PT=Pakokku Tnshp., Magwe Div., Myanmar; AK=Alaungdaw Kathapa Ntl. Pk., Sagging Div., Myanmar; CWS=2 km WNW Chatthin, Chatthin Wldlf. Sanc., Myanmar and Pituophis lineaticollis, were also used as additional DNA was purified from the cell lysate using a phe- outgroup taxa. nol-chloroform-isoamyl alcohol (PCI), chloroform- iso- Tissue samples included either skeletal muscle or liver amyl alcohol (CI) extraction. A volume of 450 µL PCI tissue preserved in 95% ethanol at the time of collection was added to the digested DNA, mixed well and centri- and subsequently stored either in ethanol or frozen at fuged at 13000 x g for 5min. The supernatant was trans- −80°C. Voucher specimens are preserved in the collec- ferred to a new tube and the PCI step was repeated tions of the California Academy of Sciences (CAS), the 2–3 times. Subsequently, the DNA supernatant was Royal Ontario Museum (ROM), Texas Memorial Mu- cleared of remaining phenol with a 450 µL CI wash, cen- seum (TNHC), Museum of Zoology, University of trifugation and final supernatant draw-off. Final volume Michigan (UMMZ) and National Museum of Natural of DNA suspension product was 30–120 µL. The pre- History (USNM). sence of unsheared mtDNA was tested by adding 10 µL 2.2 DNA extraction, amplification and sequencing of product to a 1% agarose/TAE buffered electrophoretic Three contiguous mtDNA genes were selected for phy- gel with ethidium bromide. Results were visualized on a logeny reconstruction, including 12S ribosomal RNA ultra-violet light table and recorded with Polaroid film or (rRNA), 16S rRNA and tRNA Valine (tRNAval). Total a digital camera. The DNA extraction procedure was re- DNA was obtained from alcohol preserved and frozen peated if the sample failed to amplify with the poly- tissue samples using a preparation of 500 µL 0.1 M STE, merase chain reaction (PCR). 50 µL 20% SDS and 12.5 µL 20 mg/mL Proteinase K Specimens of O. arnensis, O. modestus and O. venus- (GIBCO) added to 0.2 g of well-macerated tissue. The tus were collected predating the practice of separately preparation was incubated at 37°C for 24–48 h with se- preserving tissues for molecular investigation, and were veral intervals of mixing. fixed with at least some formalin. Consequently, an ex- 6 Asian Herpetological Research Vol. 1 traction protocol modified after Chatigny (2000) was Table 2 Primer sequences used for PCR amplification and se- used. Small pieces (>0.1 g) of loose or free tissue were quencing of mtDNA in this study. See also Figure 6. harvested from the specimen/container. These were Name Sequence 5' to 3' transferred to 1.5 mL tubes and frozen using a mixture of 12S1L CCA ACT GGG ATT AGA TAC CCC ACT AT dry-ice and ethanol, and then ground to a fine powder 12S2LM ACA CAC CGC CCG TCA CCC T using a pestle sized for 1.5 mL conical bottom centrifuge 16S3H GTA GCT CAC TTG ATT TCG GG tubes. These powdered tissues were digested with the 16S4H CCA GCT ATC ACC AAG TTC GGT AGG CTT TTC STE/SDS/Proteinase K buffer above, but with the fol- 16S1LM CCG ACT GTT GAC CAA AAA CAT lowing changes: Proteinase K concentration was doubled; 16S1H GGC TAT GTT TTT GGT AAA CAG DTE (Dithioerythritol) was added as 2.5 µL of 20 mg/mL 16S5H CTA CCT TTG CAC GGT TAG GAT ACC GCG GC stock solution to reverse protein cross-linkages; incuba- CCG GAT CCC CGG CCG GTC TGA ACT CAG ATC 16S2H tion temperature was increased to 55°C. The samples ACG were incubated for 24 h, then an additional volume of 2.5 L of reaction buffer (containing 67 mM Tris, pH 8.8, Proteinase K and DTE was added and the samples were 2 mM MgCl) (Sigma) and 19 µL distilled deionized water. incubated for a further 48 h. The extracted DNA was Thermocycling of the reaction mix was performed on MJ subjected to the PCI/CI clean-up described above. This Research PTL-200 and Perkin-Elmer GeneAmp 9700 produced DNA from which short PCR amplicons could machines. Cycling parameters were set as follows: 38 or be made. 40 cycles; denature at 92°C for 30 s; anneal at 45°C for Primer 12S1L, located at approximately 410 base pairs 45 s; extension at 72°C for 45–90 s, and a final extension (bp) from the beginning of the 12S ribosomal RNA (rRNA) region, and primer 16S2H, located approxi- cycle at 72°C for 5 min. The annealing temperature was mately 200 bp from the terminus of the 16SrRNA region, varied upward or downward to increase or decrease were used for amplification of purified mtDNA. These primer-DNA specificity when using different primer primers flanked a region of approximately 1800 conti- combinations with different samples. The extension time nuous bp and internal primers were used to sequence was increased when amplifying the longer fragments overlapping regions. However, in many cases, and espe- when using the 12s–1L primer to either 16s–4H or cially with the formalin-fixed samples, shorter amplicons 16s–2H. Some samples could not be amplified for the were produced and sequenced leaving some internal se- entire ~1800 bp fragment; these were amplified in three quence corresponding to the priming region unknown. overlapping segments of 12s–1L to 16s–4H, 12s–2LM to The complete list of primers used in this study and their 16s–5H and 16s–1L to 16s–2H. Other less desirable am- relative positions are presented in Figure 6 and Table 2. plicons (12S–2LM to 16S–1H) were also used when the sample DNA proved refractory to amplification owing to variation in the sequence at the priming region. The com- bination of 12S–2LM and 16S–3H proved particularly effective for the formalin fixed samples. Double stranded DNA PCR product was subjected to electrophoresis in a 100 mL 1% agarose gel with 10 µL ethidium bromide for 20–30 min at 120 V and 99 mA,

Figure 6 A representation of a linear double stranded section of and visualized under UV light. The amplified DNA frag- the mitochondrial genome, including 12S rRNA, tRNAval and 16Sr ments were cleaned of agarose and purified using either RNA. Arrows represent relative location of primers used in this GeneClean (Bio101/Can Scientific), a standard QiaQuick study. (Qiagen) protocol, or with a protocol involving centri- fuging the band through an aerosol resistant p20/200 pi- Double stranded mtDNA was amplified in 25 µL PCR pette tip at 13000 g for 5 min. The cleaned DNA was reactions (Saiki, 1989). The reactions were set up using suspended in distilled deionized water. 1 L of DNA (0.05 μg/µL), 1 µL of each primer (10 mM), DNA sequencing was performed using either 33P in- 0.08 µL of a dNTP mix (containing 10 mM of each of corporation and autoradiograph film or with dye-labeled dATP, dCTP, dGTP and dTTP) (Perkin-Elmer Cetus Kit), nucleotides and fluorescent detection during electropho- 0.2 µL of Amplitaq polymerase (Perkin-Elmer Cetus), resis. For radio-sequencing, a Thermo Sequenase 33P- No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 7 labeled cycle sequencing kit (Amersham) was used on tree bisection-reconnection branch swapping and collapse Perkin-Elmer Cetus and Perkin-Elmer GeneAmp 9700 zero length branches. All multistate characters were thermocyclers using the manufacturers’ protocols. The evaluated as nonadditive (unordered) and unweighted. sequencing reaction mix consisted of 8 µL dH2O, 1 µL of Gaps were treated as “missing data”. light or heavy strand primer, 1.5 µL of buffer, 1.5 µL of Bayesian inference (BI) was also used to infer phy- Sequenase, 3 µL DNA and 6 µL termination mix, and logenetic relationships. MrModeltest v.2.3 (Nylander, was divided into four tubes (A, C, G, T). Subsequently, 2004) was used to obtain evolutionary models using the 0.37 µL of the appropriate 33P labeled ddNTP was added Akaike Information Criterion (Akaike, 1974, 1979). BI to each tube and overlaid with a drop of paraffin oil. was performed using MrBayes v.3.1.2 (Huelsenbeck and Upon completion of cycling, 3 µL of stop solution Ronquist, 2001). Two simultaneous runs of four chains (Amersham) was added to each tube and samples were were run for 3 X 106 generations, sampling every 100 stored at –20°C until electrophoresis (usually within 24 hr). generations. Stationarity was assessed when likelihood The products of the isotope-sequencing reactions were scores reached a stable equilibrium. A burn-in value of resolved in a 40 cm long 7M urea-5% polyacryla- 7500 was implemented to discard topologies with low mide-0.6% TBE buffer gel electrophoresed at 65 V for likelihood scores prior to generating a 50% majority rule 2 hr or 6 hr (short run and long run). The gels were blot- consensus tree. Nodal support within the Bayesian con- ted onto filter paper, dried under heat and vacuum, and sensus tree was assessed from the posterior distribution exposed for 2–5 d on Kodak XAR autoradiograph film. of topologies (Erixon et al., 2003). Gels were developed for 5 min and fixed for 2 min with Nodal support was evaluated using non-parametric Kodak developer and fixer. bootstrap proportions (BSP; Felsenstein, 1985) and Alternatively, some sequencing reactions were per- Bremer Decay Indices (Bremer, 1988). BSP was based on formed with the BigDye Terminator v 3.1 Cycle Se- 10000 replicates calculated in PAUP* and nodes recei- quencing Kit (Applied Biosystems) using: 1 µL BigDye, ving >50% support were noted on the most parsimonious 2 µL 5x BigDye Terminator Buffer, 2 µL ddH20, 1 µL of trees (MPTs). DIs were calculated by searching for trees 10 mM primer solution, and 4 µL PCR product. Follow- in PAUP* using run instructions produced by AutoDecay ing the sequencing reactions, samples were cleaned and v4 (Eriksson, 1998) and decay values were noted on the precipitated with sodium acetate and ethanol. These MPT on nodes also having >50% BSP. products were resolved on an ABI377 (Applied Biosys- Hemipenial characters were included in a total evi- tems) automated sequencer using the manufacturer pro- dence approach because of their potential to be phyloge- tocols. netically informative. The data set was reduced to 17 in- 2.3 DNA sequence alignment and phylogenetic analy- group taxa by creating consensus DNA sequences for the sis Sequences were either read from X-ray films ma- nominal species. Variable base pair characters within nually and entered into BioEdit 3.0 (Hall, 1997), or ex- species were coded as multi-state characters. Three un- tracted from ABI pherogram files, and initially aligned ordered, unpolarized hemipenial characters were using ClustalW 1.7 (Thompson et al., 1994) with a appended to the molecular data set. Hemipenial data for gap-opening penalty of 15 and extension penalty of 6.66. Phyllorhynchus were determined from literature (Klauber, The aligned sequences were imported into MacClade 4.0 1935; Savage and Cliff, 1954) to be as follows: hemi- (Maddison and Maddison, 1992) and final alignment was penis with spines, no apical papillae, and partially forked achieved by eye. Nucleotide ratios and proportions in- (or “lobed”, a condition in some Oligodon, but not any cluding transition/transversion ratios were calculated in species for which sequence data were available). Zaher’s MacClade. PAUP* 4.0 (Swofford, 2001) was used to (1999) assignment of both these species to the Colubrinae calculate pairwise divergences among samples using un- (Colubridae) was based on them bearing distal calyces corrected p-distances. and the single sulcus in those Oligodon with undivided, The aligned sequences were analyzed with unlobed (non-quadrapenes) organs. PAUP*4.0b10 (Swofford, 2001) using maximum parsi- Hemipenial characters were mapped on to phyloge- mony (MP) as the criterion for tree selection. Phyloge- netic tress using MacClade. Specimens in Table 1 and netically uninformative sites were excluded, and heuristic literature records were used to determine character states. searches were performed using random addition sequence Hemipenial structure was described as three characters as with 10000 replicates while retaining minimal trees only, follows: hemipenis with a forked division (quadrapenis), 8 Asian Herpetological Research Vol. 1 unforked or partially forked; apical papillae present or lozyme, protein encoding loci shared between Oligodon absent; spines present or absent. and the other colubrids. These sequences were assembled Available sequences from GenBank were used to test into a file creating 48 synthetic genera. Concatenation the relationship of Oligodon with Phyllorhynchus and to produced 6971 aligned nucleotide positions. An un- investigate where Oligodon might belong within the weighted MP analysis in PAUP* with 10000 random ad- Colubridae. Table 3 was constructed to summarize 255 dition heuristic replicates and TBR branch swapping was sequence fragments from eight genes plus known al- applied to the data set.

Table 3 Colubroidea sequences from GenBank used to hypothesize the phylogenetic relationship of Oligodon to other colubrids. “x” de- notes a gene sequence that was not available for use in the sample, or synthetic representative. “+” denotes known protein loci data from Dowling et al. (1996), their coding used. “1” indicates a sequence obtained from Lopez and Maxson, 1995. 12S/16S sequences for denoted “this study” are from specimen CAS205028. 12S/16S sequences for Pseudoxenodon bambusicola “this study” are ROM30825. 12S/16S sequences for Lycodon fasciatus “this study” are ROM25654. Higher taxonomic names follow Zaher et al. (2009). Protein Genus Species 12S 16S c-MOS CytB NADH4 NADH1 NADH2 COI loci Colubridae incertae sedis Grayia ornata AF158434 AF158503 AF544684 AF544663 x x x smythii DQ112080 DQ112077 Calamariidae Calamaria pavimentata x x AF471103 AF471081 x x x x + Colubridae Ahaetulla fronticincta x x AF471161 AF471072 x x x x + Boiga cynodon AF139568 Z46525 x x x + dendrophila AF471128 AF471089 U49303 x x x Cemophora coccinea x 1(RH54131) AF471132 AF471091 AF138754 x x x + Coluber constrictor U96794 L01770 AY486937 AY486913 AY487040 AY486963 AY487001 AY122649 + Coluber dorri x AY188081 AY188001 AY188040 AY487042 AY486964 AY487003 x Coluber zebrinus x AY188084 AY188004 AY188043 AY487058 AY486980 AY487019 x Coronella girondica AY122835 AY643353 AF471113 AF471088 AY487066 AY486988 AY487027 AY122751 Crotaphopeltis tornieri x x AF471112 AF471093 x x AF428016 x Dasypeltis atra x AF471136 AF471065 x x x x scabra x 1(RH64432) x x x x + Dinodon rufozonatum AF471163 AF471063 semicarinatus AB008539 AB008539 AB008539 AB008539 AB008539 AB008539 AB008539 + Dipsadoboa unicolor x x AF471139 AF471062 x x AF428017 x Dispholidus typus x AY188051 AY187973 AY188012 U49302 x x x quatuor- Elaphe AY122798 AF215267 AY486955 AY486931 AY487067 AY486989 AY487028 AY122714 + lineata Eirenis modestus AY039143 AY376780 AY486957 AY486933 AY487072 AY486994 AY487033 AY039181 Gastropyxis smaragdina AF158435 AF158504 DQ112078 DQ112075 x x x x Gonyosoma oxycephalum AY122679 Z46490 AF471105 AF471084 x x x AY122661 + Hemerophis socotrae AY039132 AY188083 AY188003 AY188042 AY487055 AY486977 AY487016 AY039170 Hemorrhois nummifer AY039163 AY376771 AY376800 AY376742 AY487049 AY486971 AY4870010 AY039201 Hierophis jugularis AY039152 AY376769 AY486941 AY486917 AY487046 AY486968 AY487007 AY039190 Lycodon capucinus U49317 x x x fasciatus This study This study x x x laoensis Z46455 Z46485 x x x + zawi AF471111 AF471040 x x x

No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 9

(Continued Table 3)

Protein Genus Species 12S 16S c-MOS CytB NADH4 NADH1 NADH2 COI loci Lytorhynchus diadema AY647229 AY188064 AY187986 AY188025 x x x x Macroprotodon cucullatus AY643296 AY643337 AF471145 AF471087 AY487064 AY486986 AY487025 x Masticophis flagellum AY122668 1(SM) AY486952 AY486928 AY487061 AY486983 AY487022 AY122650 + Oligodon cinereus This study This study AF471101 AF471033 x x x + octolineatus U49316 x x x Oocatochus rufodorsatus AY122800 x AF435015 AF036016 x x x AY122716 Opheodrys aestivus x 1(RH61940) AF471147 AF471057 x x x x + Oxybelis aeneus AF158416 AF158498 AF471148 AF471056 x x x x + Pantherophis obsoletus AY122844 Z46493 AF471140 AF283644 AF138757 x x AY122760 + Philothamnus heterodermus x 1(RH61045) AF471149 AF471055 x x x x + AF471098 & Phyllorhynchus decurtatus AF544783 AF544812 AF471083 x x x x + AF544728 Platyceps rogersi AY039127 AY188082 AY188002 AY188041 AY487052 AY486974 AY487013 AY039165 Pseustes poecilonotus x 1(RH62677) x x x x x x + Ptyas korros AF236680 AY486953 AY486929 AY487062 AY486984 AY487023 AY122652 + mucosus 1(RH56036) Rhinechis scalaris AY122802 x AY486956 AY486932 AY487068 AY486990 AY487029 AY122718 + Salvadora mexicana x AY486958 AY486934 AY487075 AY486997 AY487036 grahamiae AY122847 1(RH56651) AY122662 + Sonora semiannulata x x AF471164 AF471048 x x x x Spalerosophis diadema AY039144 AF471155 AF471049 AY487059 AY486981 AY487020 AY039182 + cliffordi 1(RH58139) Spilotes pullatus x x AF471110 AF471041 x x x x Tantilla melanocephala AF158424 AF158491 x x x x + relicta AF471107 AF471045 x x x x Telescopus fallax x AY188078 AF471108 AF471043 x x x x + Thelotornis capensis x x AF471109 AF471042 x x x x Toxicodryas pulverulenta x x AF471118 AF471047 x x x x Natricidae AF471121 & Natrix natrix AY122682 AF158530 AF471059 AY873736 AY873760 AY870640 AY122664 AF544697 Thamnophis godmani AF471165 AF420135 AF420138 AF420136 AF420137 x sirtalis AF402647 1(LM3071) x + Pseudoxenodontidae Pseudoxenodon bambusicola This study This study x x x x karlschmidti AF471102 AF471080 x x x x Dipsadidae Alsophis cantherigerus AF544694 x x x portoricensis AF158448 AF158517 AF471126 AF471085 U49309 x x x +

3. Results nine cases, multiple individuals of the same species and locality did not vary and, in these cases, only one sample 3.1 Sequence variability in Oligodon The aligned data was selected for phylogenetic analysis. In the case of set contained 1885 bp of homologous DNA sequence O. cinereus CAS205028 from Rakhine Yoma, Myanmar sites from 48 ingroup samples and three outgroup taxa. In and O. cinereus CAS213379 from Hlaw Ga, Myanmar, 10 Asian Herpetological Research Vol. 1 no variation was discovered. Further, O. theobaldi strong support (Figure 7). The heuristic search found the CAS213896 from Shwe Set Taw, Myanmar and tree island with the MPT in 99.4% of all replicates. Oli- O. theobaldi CAS210710 from Naung U, Myanmar godon, as a genus, received very strong support. It is the showed no sequence variation. However, these sequences sister group of Phyllorhynchus + Spilotes. were maintained in the analysis to maximize geographic The GTR+I+G model of nucleotide evolution was coverage. chosen for these data. The well resolved BI tree (Figure 8) DNA extracted from O. arnensis, O. modestus and was very similar to the MPT, differing only in the place- O. venustus proved difficult to PCR amplify. However, ment of O. modestus, O. maculatus and O. octolineatus. high quality sequences were obtained for approximately Almost all species received high support (BS ≥98%, 500 bp (using 12S–LM and 16S–3H). About 400 addi- DI ≥10+) with localities/populations showing mono- tional bp were added to O. modestus from the literature. phyly. Exceptions to this were O. taeniatus and O. The aligned data set includes a contiguous region of chinensis. Both were monophyletic, but O. chinensis only 507 bp from 12SrRNA, 64 bp of tRNA valine (tRNAval) received high support values when the specimens of Oli- and 1314 bp of 16SrRNA for a total of 1875 bp. The godon sp. from Con Cuong, Vietnam were included. number of variable sites for each of the three gene seg- Support for monophyly in O. taeniatus was high (BS = ments is given in Table 4. The average and ranges of the 75%, DI = 4), even though the Chatthin (Myanmar) and nucleotide frequencies across the ingroup taxa are as fol- Cat Tien, Yok Don (Vietnam) specimens represented ex- lows: A, 0.389 (0.380–0.400); C, 0.237 (0.224–0.246); G, treme east-west sampling of the range and the localities 0.159 (0.157–0.172) and T, 0.214 (0.194–0.221). Overall, showed considerable genetic divergence with respect to 713 (38%) sites vary among ingroup and outgroup taxa, each other. of which 513 are potentially phylogenetically informative. An O. cyclurus-group (sensu Smith, 1943) is resolved Within the ingroup, 618 characters are variable, 439 of within the MPT, albeit with moderate support only (BS = which are potentially phylogenetically informative. 62%, DI = 4). It consists of O. cyclurus, O. ocellatus, O. The overall transition:transversion ratio was 1.13:1. formosanus, and O. chinensis, and within this group a Uncorrected p-distances, the pair-wise absolute number clade of three Vietnamese-Chinese species—O. ocellatus, of bp differences, were summarized in Table 4, with O. formosanus and O. chinensis—receives higher support. average and range of sequence divergence within the in- The BI analysis differed in not uniting O. cyclurus with O. group and an intergeneric comparison between outgroups ocellatus + O. formosanus + O. chinensis. and the ingroup samples. Eight species in the analysis An O. taeniatus-group (sensu Smith, 1943), consisting had representatives from multiple localities included as of O. taeniatus and O. barroni (BS = 99%, DI = 12), is terminal nodes. The p-distances for the average in- resolved and this is sister to the O. cyclurus-group in the ter-locality variation for these samples were as follows: MPT. Philippine and Sunda Island’s O. octolineatus and O chinensis 0.027, O. cinereus 0.036, O. cyclurus 0.040, O. modestus show a weakly supported relationship with O. formosanus 0.014, O. splendidus 0.011, O. taeniatus the O. cyclurus-group + O. taeniatus-group. The O. cy- 0.029, and O. torquatus 0.018. clurus-group + O. taeniatus-group is only recovered in 3.2 Phylogeny The unweighted maximum parsimony the BI tree if O. octolineatus is included. analysis using all DNA data resulted in one MPT (2124 Indian O. arnensis, a widespread species, and O. venustus, steps, CI = 0.488, RI = 0.710) with many nodes receiving a restricted-range species, show a well-supported association

Table 4 A list of total sites, variable sites and uncorrected p-distances for the three mtDNA genes in the data set for snakes of the genus Oligodon. Inter-generic measures range and mean of divergences between the ingroup and outgroup samples, intra-ingroup measures range and mean of p-distances among ingroup samples.

Variable sites Range in pairwise divergence (average pairwise divergence) Gene Total bp (potentially parsimony informative), Ingroup Inter-generic Intra-ingroup 12SrRNA 507 161 (116) 0.115–0.266 (0.166) 0.000–0.149 (0.083) tRNAval 64 16 (11) 0.000–0.198 (0.114) 0.000–0.186 (0.063) 16SrRNA 1314 441 (312) 0.110–0.188 (0.141) 0.000–0.149 (0.087) Total 1885 618 (439) 0.121–0.202 (0.147) 0.000–0.125 (0.084) No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 11

Figure 7 Maximum parsimony phylogeny of Oligodon. Values denoted above internodes are bootstrap proportions, numbers below inter- nodes are decay index values. with the O. cyclurus-group, O. taeniatus-group, O. octo- CI = 0.598, RI = 0.577 (Figure 9). The topology of the lineatus and O. modestus. The other major clade reco- tree with the hemipenial characters was very similar to vered in the tree consists mostly of mainland Asian taxa the MPT produced by the DNA data alone (Figure 7). The including O. cinereus (a widespread and highly variable two trees differed only at weakly supported nodes. In one species), O. splendidus, O. maculatus (Philippines), and case, O. splendidus moved from being the sister of O. the following species whose relationships are shown in cinereus to becoming the sister of O. maculatus (as re- parenthetical notation: ((O. torquatus, O. planiceps) (O. solved in the BI tree). In the second case, O. chinensis theobaldi, O. cruentatus)). This latter clade contains three and O. formosanus obtained a sister relationship to O. taxa with quite restricted distributions and O. theobaldi, ocellatus. Tree instability was not due to the addition of which is less restricted in distribution. The ranges of new data, but rather to the deletion of taxa with uncertain these species might overlap in Myanmar. identity and those having sequences from combined indi- 3.3 Hemipenial characters Reduction to 17 taxa viduals. The branching pattern in Figure 9 was obtained allowed the branch-and-bound algorithm to be used for in the molecular-only data set when one taxon of uncer tree building, which resolved a single MPT of 1420 steps, tain identity (ROM27049) and one locality of O. formo- 12 Asian Herpetological Research Vol. 1

Figure 8 Bayesian inference phylogeny for Oligodon. Values denoted behind the nodes are posterior probabilities. sanus (Quang Thanh, Cao Bang, Viet Nam) were deleted. ACCTRAN these become unequivocal for the presence of Regardless, the hemipenial characters had the same papillae, under DELTRAN they are non-papillate. transformations on both trees. 3.4 Position of Oligodon in the Colubridae Optimizing the three hemipenial characters on the Meta-analysis of colubrine sequences recovered a single phylogeny (Figure 9, A–C) reveals that the bifurcate MPT on a tree island found in 40% of the replicates (Figure hemipenis arose once only. Apical papillae and spines 10, length 14314 steps). The tree had a clade consisting of appear to have more instances of parallel, homoplastic Oligodon + (Phyllorhynchus + Spilotes), the same as found state changes, but they are apomorphic and homologous in the MP analysis of Lawson et al. (2005). for some clades. Accelerated (ACCTRAN) and delayed (DELTRAN) transformation optimizations display the 4. Discussion same mapping for the hemipenial characters with one exception. Mapping apical papillae character states pro- 4.1 Molecular variation Slowly evolving 12S and 16S duces two equivocal internodes within the ingroup. Under ribosomal RNA genes are appropriate for resolving older No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 13

Figure 9 Hemipenial character state mapping for Oligodon. A: presence, absence, or partially divided/forked hemipenis. B: presence or absence of apical papillae on hemipenis (under ACCTRAN, equivocal internodes are papillate, under delayed transformation optimization they are non-papillate). C: presence or absence of spines on hemipenis. Several clades have been labeled to facilitate discussion. All charac- ters optimized unordered. divergences (~150 Ma) (Mindell and Honeycutt, 1990). samples of Oligodon. Among 507 aligned nucleotide sites Divergence in the rRNA sequences is moderate for the 48 for 12S, 32% are variable and 23% are potentially infor- 14 Asian Herpetological Research Vol. 1

Figure 10 Maximum parsimony analysis of 48 colubrids including Oligodon and outgroups using published sequences of 12SrRNA, 16SrRNA, nuclear gene c-Mos, Cytochrome b, NADH4, NADH1, NADH2, COI and allozyme, protein-coding loci (Acp-2, Ldh-2, Mdh-1 and Pgm from Dowling et al., 1996). mative. Of 1314 sites in 16S, 34% are variable and 24% Evaluation of mtDNA genes as a single data partition are potentially informative. The ratio of A: C: G: T for is appropriate because mtDNA genes do not freely seg- Oligodon is similar to that of other snakes (Kraus and regate and recombine but instead are inherited as a single Brown, 1998; Rodríguez-Robles and de Jesús-Escobar, linkage group. The data could be partitioned by transcrip- 1999; Slowinski and Lawson, 2002) and typical of tional unit, but it is not necessarily true that these data mtDNA. Analysis of p-distances for each gene reveals a partitions are appropriate or “natural” (Eernisse and similar range and average of variation as described in Kluge, 1993). There are no a priori grounds to believe other studies, such as on Bufo (Liu et al., 2000) and that knowledge of the transcriptional structure indicates snakes (Vidal et al., 2000). Taken together, this suggests that the whole sequence is under the same evolutionary that these characters are useful for reconstructing history forces. The most appropriate scale for data partitioning is in Oligodon at the taxonomic level of species and possi- the individual base pair. bly the haplo-history between populations. The finding of no intra-locality variation within spe- No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 15 cies for these genes is interesting, but can be explained - O. taeniatus-Group Smith (1943) several ways. It may be that faster evolving genes are suggests that O. cyclurus and O. chinensis are most needed to assess genetic variation at this level, that more closely related, that O. taeniatus and O. barroni are most samples need to be sequenced, or that populations may in closely related, and that these two groups are sisters. He fact be quite isolated from each other. Nuclear gene se- places several other species in these groups as well. The quences will be required to assess gene flow. larger grouping, which Smith refers to as the O. cyclurus The p-distances within species of Oligodon with mul- - O. taeniatus complex, is recovered in the MPT but not tiple populations were less than 50% of the average in the BI tree. This study contains a fair sampling of the p-distance among all populations and species. They species that Smith assigns to these groups. Notwith- ranged from being as low as 13% of the variation seen standing, there are several recent taxonomic changes in among the whole ingroup. The p-distances were generally this group. correlated with increasing geographic distance between David et al. (2008b) describe three new species, localities (r2=0.664) indicating that isolation-by-distance O. deuvei, O. pseudotaeniatus, and O. moricei, and asso- was a factor to consider. ciate them with O. taeniatus. The discovery of these new 4.2 Phylogeny and previous hypotheses of relation- species suggests that genetic information might be help- ships Many relationships recovered in this phylogeny ful for identifying cryptic species of Oligodon. Our study agree with prior proposals. Although several species of includes a specimen of O. taeniatus from Myanmar Oligodon were absent from this study, the corroboration (Chatthin Wildlife Sanctuary, USNM520625). This ani- between genetic characters and hemipenial morphology mal is discussed by David et al. (2008b) who report that allowed for sound speculation about the relationships of it was incorrectly identified by Zug (1998; catalogue several other taxa. number incorrect in publication). The voucher specimen Philippine taxa Taylor (1922–25, 1965), Taylor and is lost (David et al. 2008b). However, Zug’s description Elbel, (1958) and Leviton (1963a, 1963b) propose that (Zug, pers. comm.) and the sequence data leave little O. maculatus is most closely related to Philippine O. an- doubt that it is O. taeniatus. Specimen USNM520624, corus, and then to O. purpurascens, which is not included which David et al. refer to as “most likely” being O. in this study. Oligodon maculatus is more closely related theobaldi is not included herein. to O. splendidus and O. cinereus. Oligodon purpurascens Several recent publications (David et al., 2004, is a widespread species that extends onto the Malay Pe- 2008a,b; Pauwels et al., 2002, 2003; Stuart and Emmett, ninsula. Oligodon maculatus, O. splendidus and 2006; Stuart et al., 2006; Teynie et al., 2004) draw atten- O. cinereus all have quite similar hemipenes in their be- tion to an opinion (Wagner, 1975) that O. fasciolatus is a ing papillate, non-spinose and proximally calyculate. The species distinct from O. cyclurus. Although this study relationship of O. purpurascens, which also has large does not include O. fasciolatus, that species occurs in the hemipenial papillae, with either O. cinereus or O. splen- center of what was once the nearly pan-Southeast Asian didus is new. It is possible that the large papillae of range of O. cyclurus. Future studies should include O. purpurascens are homologous with those in a clade populations of this taxon and additional populations of O. that includes widespread and complex O. cinereus. cyclurus to evaluate the validity of this change. This However, another set of relationships is also possible for study includes another taxon historically included in the O. purpurascens. variation of O. cyclurus, i.e., O. ocellatus, as well as O. Leviton (1963a) reports that O. modestus is related to cyclurus sensu stricto. O. ocellatus does not form the O. ancorus and O. purpurascens. However, our phylo- sister group of O. cyclurus. geny refutes a close relationship between O. maculatus (a This study’s sampling of the O. cyclurus - O. taeniatus close relative of O. purpurascens) and O. modestus. group contains two specimens of uncertain identity (Oli- While they share large hemipenial papillae, this condition godon sp., from the vicinity of Con Cuong). Genetically is not a defining synapomorphy. Philippine diversity these are quite similar to O. chinensis, yet the locality is might be best explained by multiple invasions of the is- far outside that species’ known range. These snakes might lands by different clades. A close relationship between represent an undescribed species. A morphological O. purpurascens and O. modestus would allow examination of the specimens is underway. O. maculatus to represent an invasion by an ancestor Oligodon cinereus This species has a considerable more closely allied with the O. cinereus-group. geographic range and is highly variable in coloration. 16 Asian Herpetological Research Vol. 1

Samples of this species are from the western and eastern O. multizonatus. David et al. (2008a,b) note the organ areas of its range, but not the center. Predictably, the when describing new species. p-distance between these samples is moderately high A survey of three museum catalogues (FMNH: Alan (0.036) relative to other species in this study. The sam- Resetar, pers. comm.; CAS: Joseph B. Slowinski, pers. ples used here include some of the color varieties: comm.; and ROM), indicates that a majority of Oligodon ROM37092 from Cat Tien National Park, southern Viet- are not sexed. This situation precludes group assignment nam, represents the “palidocinctus” color form, while the of many species of Oligodon. Unfortunately, this pivotal specimens from Chi Linh, northern Vietnam, represent character is present in males only and its diagnostic fea- the moderately inornate form (Smith’s type II). Other tures have been neglected in authoritative but less aca- specimens are closest to Smith’s form III. Given that O. demically oriented literature (Chan-Ard et al., 1999; Cox taeniatus is a species complex (David et al., 2008b) and et al., 1998; Manthey and Grossmann, 1997). Hemipenial because the Myanmar and Vietnamese haplotypes are not morphology is either neglected or not examined com- sister lineages, it is likely that O. cinereus is also a com- paratively in several important academic treatments plex of cryptic species. Additional sequenced specimens (Campden-Main, 1969, 1970, 1970 (1984); in den Bosch, will be required to solve this problem, especially speci- 1994; Lazell et al., 1999; Mahendra, 1984; Tillack and mens from the central region of the range. Günther, 2009; Yang and Inger, 1986; Zhao et al., 1998). Oligodon torquatus - O. planiceps - O. cruentatus - O. Using hemipenial microstructure, Smith (1943) di- theobaldi Smith (1943) proposes a close relationship vides the spinose hemipenial snakes into four groups. among O. torquatus, O. planiceps, O. cruentatus and Herein, two spinose groups are recovered, consistent with O. theobaldi, but is unsure of where to place O. torquatus. Smith’s system, and there is no refutation for the other His grouping is supported here and O. torquatus is re- two groups, although Smith reports that spines are more solved as the sister taxon of O. planiceps. The autapo- plastic than either papillae or division of the organ into morphic loss of hemipenial spines in O. torquatus serves quadrapenes. Leviton (1963) proposes three groups as to diagnose that species. This group differs from other follows: deeply forked with papillae present or absent; spinose hemipenial Oligodon by the presence of apical unforked, papillae usually present, spines usually absent; papillae. Smith describes additional details of spine size and unforked, spines usually present, papillae usually and placement, which taken together indicates that the absent. Only one of these groups is recovered herein. character coding of spinose/not-spinose might be overly Leviton groups all species with deeply forked hemipenis simplistic. together, but indicates that the individuals of both other One unidentified specimen (ROM27049, Oligodon sp.) groups can have either spines or papillae. Our analysis is associated with this clade, albeit with low measures of suggests that the two clades independently acquired support. Examination of the specimen is ongoing. It spines. The hemipenes of most species in one clade are could represent a major range extension of a known spe- without spines but with papillae and this is comprised of cies, or perhaps represents an undescribed species. Its O. theobaldi, O. cruentatus, O. planiceps, O. torquatus, hemipenes are unknown, but are predicted to be spinose O. cinereus, and other unsampled taxa. The other clade and have papillae. perhaps contains species with spines but without papillae 4.3 Hemipenes and phylogeny Fifty of 70 recognized on their hemipenes, including O. arnensis, O. venustus, species have described hemipenes. Of the remaining spe- and others. However, these two groupings fail to capture cies, most are rare (from a highly restricted range, and/or all species of Oligodon that also have unforked hemipe- not very abundant) or very rare (in some instances known nes. only from the types). Pope (1935) reports hemipenial data Figure 9 shows that taxa tend to undergo subsequent for 12 Chinese species of Oligodon. Smith (1943) de- modification of papillae and spines, especially secondary scribes the condition in 33 species, including nine sur- loss. However, neither character is freely plastic. Secon- veyed by Pope, and divides Oligodon into seven groups dary loss or reduction of these ornamentations may be a based on hemipenial condition. Leviton (1963) adds data key that reinforces speciation. for four Philippine species and proposes recognizing Hemipenial characters appear to be robust and diag- three hemipenial groups of Oligodon. Leviton (1953, nostic (Leviton, 1960; Pope, 1935; Smith, 1943; Wall, 1960) notes the hemipenial condition in O. annamensis. 1923), providing relief from reliance on color and scala- Zhao and Jiang (1981) describe and illustrate the organ of tion for species identification and phylogenetic corrobo- No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 17 ration (Eernisse and Kluge, 1993; Kluge, 1997; Siddall 1998) may be responsible for the isolation of populations and Kluge, 1997). The heritability of color and scalation of widespread species that subsequently accrued mor- characters remains a question in Oligodon in particular phological changes. This is consistent with the idea that (Smith, 1943), and, in general, their use in phylogenetics many of the extant species of Oligodon appear to have a often rests on invalid evolutionary assumptions (Murphy subset of the variation of one of the widespread species. and Doyle, 1998). Oligodon can be found on many Pleistocene landbridge islands. Certainly, glacial cycles can play a role in speci- 4.4 Color, confusion and biogeography Much confu- ation throughout the Philippines and Indonesia (Karns et sion in Oligodon occurs because of extensive intraspeci- al., 2000). fic variation in color and patterning. Most species of Oligodon have a fairly invariant color pattern, but a few 4.5 Taxonomic implications Kukri snakes were origi- species, namely O. cyclurus, O. fasciolatus, O. cinereus nally partitioned into two genera based on dentition. The and O. taeniolatus, are highly variable. The venters are genus Simotes Duméril, Bibron et Duméril, 1854 was described for some species of Oligodon, but not others. In established to include kukri snakes that had palatine and some species, individuals may be checkered or not. The pterygoid teeth (Günther, 1864), whereas Oligodon Fit- dorsal color of Oligodon ranges from brown and black to zinger 1826 was reserved for those lacking this dentition green and orange with highlights in black, white, red and (Günther, 1864; Boulenger, 1894). Holarchus (Cope, yellow. Overall color can change with age and contrast in 1886; Pope, 1935; Stejneger, 1907) was used later to in- pattern can be lost. The overall dorsal pattern can be very clude most of the same species originally assigned to plain with loose, indistinct reticulation confined to scale Simotes (Duméril et al., 1854), but this group was rede- margins, as in one form of both O. cyclurus and O. for- fined (by Pope) based on the absence of hemipenial mosanus. It can also be more complex with varying spines and the highly correlated condition of having a numbers of longitudinal stripes, crossbars, or spots ran- divided anal scale. As late as 1970, Deuve continued to ging in size and shape. Some species combine elements recognize Holarchus and Leviton (1963) speculated that there might be two clades of Oligodon, one that could of all of the above. Taken together, O. cyclurus, carry the name Holarchus. Most of the kukris surveyed O. taeniolatus and O. fasciolatus have five distinct color herein were originally assigned to Simotes based on den- morphs, and O. cinereus has four. Geographic overlap tition. Oligodon modestus was an important exception in and intergradations of the color morphs are poorly under- lacking palatine and pterygoid teeth. Based on hemipenial stood. and anal scale conditions, two clades in this study could The highly variable species are also widespread. Oli- have been assigned to Oligodon: clade A and clade C, godon cyclurus and O. fasciolatus range from India Figure 9. However, these two lineages were not mono- eastward through (at approximately 11°15′N), phyletic, and the clade that would carry the name Oli- to Vietnam and southwestern China (). A separa- godon was uncertain because the type species of Oli- tion of about 500 km on the Thai peninsula separates godon, O. bitorquatus, was not included. Regardless, this O. fasciolatus from O. purpurascens, which continues arrangement precluded a monophyletic Simotes and/or throughout Indonesia. The distribution of O. cinereus Holarchus. The division of Oligodon drawing on any closely matches cyclurus+fasciolatus. Ologodon previous formulation would have required the erection of taeniolatus ranges throughout India to eastern Iran and several additional genera. northward to southern Turkmenistan. Nomenclatorial stability should be a primary concern. Many of Smith’s (1943) seven hemipenial groupings A phylogenetically informed taxonomy contains the most are corroborated here. None are refuted. If his assign- information within a hierarchical system (Brooks and ments are mostly correct, then several clades will contain McLennan, 1991, 2002; Farris, 1967; Wiley, 1980). Until a morphologically variable widespread species with a a far more complete sampling of species is possible, Oli- range that will encompass that of other clade members. godon should not be subdivided into multiple genera. This morphologically variable species will also encom- Oligodon: Position within the Colubridae/Colubrinae pass much of the color and pattern variation found in To determine whether Oligodon is an unusually speciose other clade members. Thus, the sympatric species will genus of snakes, it is necessary to determine its sister have similar colors and patterns. group relationships, and not just an appropriate outgroup Pleistocene ecological changes (Hall and Holloway, (Brooks and McLennan, 1991). It is also important to 18 Asian Herpetological Research Vol. 1 establish the sister group of Oligodon because outgroup tantalizing to speculate that the ornamented organ may choice can be critical in phylogenetic reconstruction play a role in speciation or maintenance of species. (Milinkovitch and Lyons-Weiler, 1998; Tarrío et al., 2000; Speculation on cladistic relationships by Smith (1943), Ware et al., 2008). based on hemipenial morphology, are validated herein, Relationships within the Colubridae are discussed even though challenged by others. Thus, new species of elsewhere (Dowling, 1974; Dowling et al., 1996; Kraus Oligodon should be based on a type series that includes and Brown, 1998; Lawson et al., 2005; Lopez and males with described hemipenes. Maxson, 1996; Zhang et al., 1984) and they will be the Splitting the genus Oligodon is not justified at this subject of future studies. Oligodon is included in several time. This would likely result in taxonomic instability. of these studies, yet no one uses all of the available data The genus Oligodon may be old. Its age might be as- from molecular investigations to hypothesize the position sociated with its species diversity. Burbrink and Lawson of Oligodon relative to other colubrines. Kelly et al. (2007) discuss the possible land bridges between Asia (2003) use much of the available molecular data in a and North America that were traversable by snakes. If meta-analysis of snakes, but they include too few colu- Phyllorhynchus arrived in North America in the early brines (and no Oligodon) to address this question. Most Oligocene along with rat snakes, then its sister group, studies agree that Oligodon falls within the Colubrinae. Oligodon, may be at last 20 Ma old. During this long Two studies (Dowling et al., 1996; Lawson et al., 2005) period of isolation, Oligodon could have experienced suggest that Oligodon is related to New World Phyl- repeated habit fragmentations and reconnections. As mo- lorhynchus, but they disagree on the placement of these lecular clock methods improve, this possibility will be- two genera within the Colubrinae. The close relationship come testable. The inclusion of Spilotes in a clade with between a relatively speciose Asian Oligodon and a rela- Phyllorhynchus and Oligodon should be treated with tively depauperate North American genus (Phyllorhyn- great skepticism. The nucleotide dataset for Spilotes in chus) remains an unintuitive result. the colubrid analysis, is the smallest of all taxa. This analysis (Figure 10) did not refute a close rela- This phylogeny may be applied to future surveys of tionship between Oligodon and Phyllorhynchus feeding and defensive behaviors. Egg-eating and defen- (+Spilotes). However, this clade did not share a close sive displays in this genus may not be uniform. If they relationship with other New World colubrines. It diverged are not ubiquitous, then the diversity can be mapped to early from the remaining Colubrinae. This finding was this phylogeny thus inferring phylogenetically unique consistent with proposal of the Oligodontini by Dowling events. Better defined distributions will facilitate bio- (1974), Dowling and Duellman (1978) and Dowling et al. geographic investigations of Oligodon and these might (1996). The positioning of Oligodon herein was also con- reveal historical connections between areas and identify sistent with that of Zhang and Zhao (1984). barriers to dispersal in South and Southeast Asia. Range overlap between kukri snakes that are relatively distantly 5. Concluding Remarks related might reveal historical biogeographic patterns in sub-clades. Phylogeny and biogeography of Oligodon Oligodon is a genus worthy of future investigation. The could improve our understanding of speciation modes rarity of specimens in collections and in the field across and they could be taken into consideration when planning most, if not all of their distributions, will continue to be a for conservation in South and Southeast Asia. key challenge to future work. There are relatively few Acknowledgements This research formed part of the specimens of Oligodon in museum collections, and even requirements for the fulfillment of a M.Sc. Degree at the fewer have tissues separately preserved for molecular University of Toronto for MDG. Ross MacCulloch care- investigation. Political instability across much of the fully proofread the manuscript. The research was sup- range of these species in the twentieth century often ported by a Natural Sciences and Engineering Research forces researchers to take an artificially geographically Council (Canada) Discovery Grant A3148 to RWM. restricted approach to work. Notwithstanding these limi- Fieldwork in Southeast Asia was supported by NSERC, tations, this study produces several interesting results. the Royal Ontario Museum (ROM) Foundation and the The hemipenial morphology of Oligodon is phyloge- ROM Members Volunteer Committee. All fieldwork was netically constrained and, therefore, informative. The conducted using Use Protocols approved by the hemipenes are useful in species identification, and it is ROM Animal Care Committee. No. 1 Marc D. Green et al. Toward a Phylogeny of the Kukri Snakes, Genus Oligodon 19

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