COLOR PATTERN DIMORPHISM IN THE COLUBRID SNAKE Oligodon purpurascens (SCHLEGEL, 1837) (REPTILIA: SQUAMATA) more

Russian Journal of Herpetology Vol. 18, No. 3, 2011, pp. 215 – 220 COLOR PATTERN DIMORPHISM IN THE COLUBRID SNAKE Oligodon purpurascens (SCHLEGEL, 1837) (REPTILIA: SQUAMATA) Johan van Rooijen,1* Perry L. Wood Jr.,2,3*, Jesse L. Grismer,4,5* L. Lee Grismer,2 and Wolfgang Grossmann4 Submitted February 23, 2011. The Southeast Asian colubrid snake Oligodon purpurascens (Schlegel, 1837) exhibits a strongly bimodal variation in color pattern. Two competing hypotheses can be put forward to explain the coexistence of the two phenotypes. According to the first hypothesis, the two phenotypes are the result of a color pattern polymorphism within Oligodon purpurascens. According to the second, the two phenotypes in fact represent distinct, sympatrically occurring, taxa. In this study, multivariate analyses of morphological data as well as a phylogenetic analysis of a small segment of mitochondrial DNA were used to assess the likelihood of each of these hypotheses. The results strongly support the polymorphism-hypothesis. The incidence of each phenotype in various areas suggests that negative frequency-dependent selection maintains the coexistence of the two phenotypes. Keywords: color pattern polymorphism, Oligodon purpurascens. INTRODUCTION The colubrid genus Oligodon Boie, 1827 is widely represented in Central and tropical Asia, eastwards as far east as the Philippines (Wallach and Bauer, 1996; Malkmus et al., 2002). Members of this genus are called Kukri snakes in popular nomenclature because of the presence of one to three large posterior maxillary teeth which resemble a Ghurkan kukri knife (Smith, 1943; Tweedie, 1983). Those teeth are used to open reptile eggs (Coleman et al., 1993) which constitute the primary food source for many species of this genus. Another conspicuous character of this genus is a rather blunt head terminating in a large rostral shield (Malkmus et al., 2002; Manthey and Grossmann, 1997) which is an adaptation for burrowing. All species are terrestrial, semifossorial snakes. Netherlands Centre for Biodiversity Naturalis, section Zoological Museum Amsterdam, Mauritskade 61, 1092 AD Amsterdam, The Netherlands; e-mail: j1.van.rooijen@hetnet.nl 2 Department of Biology, La Sierra University, Riverside, CA 92515-824, USA; e-mail: perry.wood@villanova.edu 3 Present address: Department of Biology, Villanova University, Villanova, PA 19085, USA. 4 Museum of Natural Sciences, Louisiana State University, Baton Rouge, LO 70820, USA; e-mail: jesse.grismer@villanova.edu 5 Wulfia-Ufer 33, D-12105 Berlin, Germany * These authors contributed equally to this paper. 1 Oligodon purpurascens (Schlegel, 1837) ranges throughout the Sunda region (Tillack and Günther, 2009). The species appears to exhibit a strongly bimodal variation in color pattern (e.g., Tillack and Günther, 2009). One of the two phenotypes is characterized by a highly conspicuous coloration that suggests mimicry of an aposematic signal (Fig. 1 and illustrations in Grismer et al., 2004; Van Rooijen and Van Rooijen, 2007; Tillack and Günther, 2009) whereas the second phenotype exhibits a cryptic coloration (Fig. 1 and illustrations in Manthey and Grossmann, 1997; Chan-ard et al., 1999; Stuebing and Inger, 1999; Malkmus et al., 2002; Tillack and Günther, 2009). Two competing hypotheses can be put forward to explain the coexistence of the two phenotypes. The first hypothesis entails a color pattern dimorphism within Oligodon purpurascens. Color pattern polymorphism is a well-studied phenomenon (e.g., Jones et al., 1977; Gray and McKinnon, 2006; Olendorf et al., 2006) that has been documented for several snake species (e.g., Zweifel, 1981; Kark et al., 1997). According to the second hypothesis, the two phenotypes in fact represent distinct, sympatrically occurring taxa. In this study, multivariate analyses of morphological data as well as a phylogenetic analysis of a small segment of mitochondrial DNA were used to assess the likelihood of each of these hypotheses. In addition, the incidence of each phenotype at various 1026-2296/2011/1803-0215 © 2011 Folium Publishing Company 216 Johan van Rooijen et al. Fig. 1. Oligodon purpurascens, living specimens from Pulau Tioman, West-Malaysia. Left, phenotype 1; right, phenotype 2. locations is analyzed in an attempt to shed light on the mechanism that underlies the maintenance of the two phenotypes. MATERIAL AND METHODS Morphological Analyses Data pertaining to various aspects of morphology were taken from the following 52 alcohol preserved specimens of Oligodon purpurascens. Museum acronyms follow Leviton et al. (1985). Non standard abbreviations are: LSUHC, La Sierra University herpetological collection. Brunei: Kampong Kapok: LSUMZ 55855. East Malaysia: Borneo: RMNH 9006; FMNH 121201. Sampit ZMB 8413. Sarawak: ZMB 7100, South east Borneo: Balik Papan: RMNH 4903. Poeroek Tjahoe RMNH 7499 [3 specimens with the same number]. Indonesia: Java: ZMB 3030; RMNH 242 No Data (syntype). Buitenzorg: ZMB 24559. Sumatra: RMNH 241, 11456; ZMB 15999, 32198, 24047. Indragiro: ZMB 15881. Lakat: ZMB TABLE 1. Sample-Size According to Locality and Phenotype Location West-Malaysia/Thailand Pulau Tioman Pulau Pinang Singapore Borneo Sumatra Pulau Gallang Java Sample size phenotype 1 4 5 0 0 1 0 0 3 phenotype 2 18 1 1 2 9 7 2 0 8460. Pulau Gallang: ZRC 2.3880, 2.3881. West Malaysia: Johor: ZRC 2.3882; Kulang: ZRC 2.3391; Kota Tinggi, ZRC 2.3887. Selangor: FMNH 18378 – 80. Kepong, LSUHC 4401. Pahang: Pulau Tioman: LSUHC 5041, 6426, 7073, 6427; BPRM 14211; DRR 2010. Cameron Highlands: ZMB 52306, 52307, 51037, 51038. Kuala Tahan: ZRC 2.3390. PENANG: Pulau Penang; LSUHC 6781. Singapore: Changi: ZRC 2.3877, 2.3878. Thailand: ZMB 65095, 55190, 53551, 53552; UTACV R-25446, R-25447, R-25603. Pattani: FMNH 179278. Nakkon Si thammarat: FMNH 191102. Some data from a living Bornean specimen were taken from Van Rooijen and Van Rooijen (2007). The total sample included 13 specimens representing phenotype 1 and 40 specimens representing phenotype 2 from various localities (Table 1). For each preserved specimen, the following characters were recorded (definitions according to Peters (1964)): (1) ventral scales counted using the method of Dowling (1951); (2) subcaudals, excluding the terminal spine; (3) supralabials; (4) supralabials in contact with the orbits; (5) infralabials; (6) loreals; (7) preoculars; (8) presuboculars; (9) postoculars; (10) supraoculars; (11) anterior temporals; (12) posterior temporals; (13) anterior chin shields; (14) posterior chin shields; (15) labials in contact with anterior chin shields; (16) labials in contact with posterior chin shields; (17) gular scales; (18) snout vent length–taken by measuring the length of the snout to the vent using a piece of string then measured on a metric ruler, repeated three times and then averaged; (19) tail length–measured by using a piece of string on a metric ruler, repeated three times and then averaged; (20) dorsal scales at one head length posterior from head; (21) dorsal scales at one head Color Pattern Dimorphism in Oligodon purpurascens length anterior the vent; (22) dorsal scales at mid body; (23) nasal divided or not; (24) anal plate divided or not. Statistical analyses of morphological data were run on SPSS 12.0 for Windows. Tail-length was adjusted to a common SVL of 46.5 cm to correct for potential ontogenetic variation between the samples of the phenotypes (e.g., Thorpe, 1975, 1983; How et al., 1996). The following allometric equation was applied: Xadj = = X – â(SVL – SVLmean) where Xadj is the adjusted value of the morphometric variable; X is the original value; SVL is the snout-vent length; SVLmean is the overall mean snout-vent length; â is the coefficient of the linear regression of X against SVL. The variables were subjected to a Principal Components Analysis (PCA, e.g., Cramer, 2003) to reduce the dimensionality of the dataset. Plots of the PC-scores were generated to visualize the phenotypes in morphospace. PC-scores were additionally analyzed in a two way MANOVA (e.g., Maxwell and Delaney, 1990) using phenotype and location as factors. Molecular Analyses Eight tissue samples of Oligodon purpurascens were taken from specimens collected from Endau-Rompin, Johor, Peninsular Malaysia (LSUHC 7715); Kepong, Selangor, Peninsular Malaysia (LSUHC 4401); Pulau Penang, Peninsular Malaysia (LSUHC 6781); Pulau Tioman, Pahang, Peninsular Malaysia (LSUHC 5041, 6426, 7073, 6427); and Santubong, Sarawak, East Malaysia (LSUHC 7735). A tissue sample of O. cyclurus from Pulau Langkawi, Kedah, Peninsular Malaysia (LSUHC 7561) comprised the outgroup. Mitochondrial DNA was isolated from liver tissue and one ventral scale clipping (LSUHC 7735) with a 20 – 25 ìl Proteinase K digestion for 3 – 24 h in a 55°C water bath following standard protocols (Qiagen Inc., Valencia, CA USA). The final product of DNA extractions was stored in elution Buffer at –70°C. All DNA extractions were put into a 1:20 dilution with DNA grade water and stored in a 4°C refrigerator. Polymerase Chain Reaction (PCR) primers were used in the amplification and sequencing of a 375 base pair segment of the cytochrome b region of the mitochondrial genome (Palumbi et al., 1991). 50 ìl PCR reactions were used to amplify cytochrome b segments and contained 5 ìl of DNA template, 5 ìl 10X Ex Taq Buffer, 36.8 ìl of DNA grade water, 1 ìl of DNTP mixture, 1 ìl of each Primer, and 0.2 ìl of Ex Taq. All PCR reactions included a negative control and were performed by a Peltier Thermal Cycler. PCR product for samples LSUHC 6426 and 7561 were amplified at (a) one cycle at 94°C for 3 min, 48°C for 217 1 min, and 72°C for 1 min; (b) 34 cycles at 94°C for 45 sec, 48°C for 45 sec, and 72°C for 1 min; (c) one cycle at 72°C for 6 min. PCR products for samples LSUHC 4401, 5041, 6781, 7073, 7597, 7715, and 7735 were amplified at (a) one cycle at 94°C for 3 min, 50°C for 1 min, and 72°C for 1 min; (b) thirty four cycles at 94°C for 45 sec, 50°C for 45 sec, and 72°C for 1 min; (c) one cycle at 72°C for 6 min. Amplified PCR products were cleaned using PEG Precipitation protocols. The complementary strands of the cleaned PCR product were individually sequenced in 10 ìl reactions containing 1 ìl of primer, 1.5 ìl of 5X Buffer, 1 ìl of DNA grade water, 0.5 ìl of Big Dye 3.0, and 1 ìl of DNA template using a Peltier Thermal Cycler at (a) 96°C for 10 sec, (b) 50°C for 5 sec, (c) 60°C for 4 min, and steps a – c were repeated 25 times then cooled down to 4°C. The products were cleaned using the Sephadex Cleanup protocol and the cleaned sequences were run on an ABI 3100 automated sequencer. Sequences were edited using Sequencher 4.0 and were aligned using Clustal X. Phylogenetic analyses were preformed in PAUP* 4.0b10 (Swafford, 2002) for phylogenetic analysis. The nine individuals were analyzed under maximum parsimony (MP) criteria. We used branch and bound searches, guaranteeing the resolution of the shortest tree, with the initial upper bound computed via stepwise, addition sequence replicates. Nodal support was estimated by bootstrapping with 10,000 replicates. Analyses of Phenotype-Incidence In order to maximize information about the incidence of each phenotype at the various locations, some additional locality records were used: a specimen of each phenotype from Sumatra (ZMA 13655, NMW 25816:2, Tillack and Günther, 2009) as well as three specimens of phenotype 1 from Pulau Tioman (RMNH 37207 and two specimens currently kept in captivity). The relative incidence of each phenotype at the various locations was analyzed by applying a cumulative binomial distribution function, testing the null hypothesis of equal relative frequencies of the phenotypes. As application of this function is not meaningful in case of small sample size due to insufficient statistical power, this analysis was restricted to Thailand/West-Malaysia, Pulau Tioman, Sumatra, and Borneo. 218 Johan van Rooijen et al. Phenotype 1, West Malaysia/Thailand Phenotype 2, West Malaysia/Thailand Phenotype 1, Pulau Tioman Phenotype 2, Pulau Tioman Phenotype 2, Pulau Pinang 0 –1 –2 Phenotype 1, Java –2 –1 0 1 2 3 –3 –2 –1 0 1 2 3 4 5 Phenotype 1, Borneo Phenotype 2, Borneo Phenotype 2, Singapore Phenotype 2, Sumatra Phenotype 2, Pulau Gallang 3 3 Second principal component 1 0 –1 –2 –3 –4 –5 –3 Fourth principal component First principal component 2 2 1 Third principal component Fig. 2. Ordination of phenotype 1 and phenotype 2 along the first and second (left) and third and fourth (right) principal components. Pulau Tioman (phenotype 1) Pulau Penang (phenotype 2) Pulau Tioman (phenotype 2) Pulau Tioman (phenotype 1) specimens cluster according to geographic proximity. In fact, two individuals from Pulau Tioman, one representing phenotype 2 (LSUHC 6427) and one representing phenotype 1 (LSUHC 5041), were found to have a sister relationship with 0.0% sequence divergence. Analyses of Phenotype-Incidence Pulau Tioman (phenotype 1) Peninsular Malaysia (phenotype 2) Peninsular Malaysia (phenotype 2) Borneo (phenotype 1) O. cyclurus Fig. 3. Phylogenetic tree based on a 375 base pair segment of the mitochondrial cytochrome b region. RESULTS Morphological Analyses Plots of the first two as well as the third and fourth principal components are shown in Fig. 2. No morphological differences between the phenotypes are in evidence. Results of a two-way MANOVA of the 6 PC’s having eigenvalues >1 agree with this observation: significant geographic variation (P = 0.00001) was revealed but no differences between the phenotypes (P = 0.9). Phylogenetic Analyses The phylogenetic tree is shown in Fig. 3. A grouping according to phenotype is not in evidence, if anything A striking observation is that, irrespective of location, one of the phenotypes appears to be rare whereas the other is common. For instance, the observed incidence of phenotype 1 in Thailand/West Malaysia, Sumatra and Borneo is 0.18, 0.11, and 0.10, respectively, whereas it is 0.89 on Pulau Tioman. A cumulative binomial distribution function was applied to test whether the observed frequencies are compatible with a null hypothesis that assumes equal frequencies of the two phenotypes in the populations. Phenotype 2 turned out to be significantly more common in Thailand/West-Malaysia, Sumatra, and Borneo (P = 0.002, P = 0.02, and P = 0.01, respectively) whereas phenotype 1 is significantly more common on Pulau Tioman (P = 0.02). These results suggest that negative frequency-dependent selection is the mechanism that maintains the coexistence of the two phenotypes. This type of selection favors a phenotype so long as it remains uncommon (e.g., Cockburn, 1991; Olendorf et al., 2006). DISCUSSION If the two phenotypes currently referred to as Oligodon purpurascens represent a color pattern dimorphism (hypothesis 1) one would expect the phenotypes to be morphologically identical and one would expect specimens to cluster according to geographic proximity in Color Pattern Dimorphism in Oligodon purpurascens stead of according to phenotype. Alternatively, if the two phenotypes in fact represent distinct species (hypothesis 2) one would expect phenotype 1 and phenotype 2 to exhibit appreciable morphological differentiation as well as to constitute separate phylogenetic groups. The results are fully consistent with hypothesis 1: morphological differentiation was not established and individuals were shown to form groups based on geographical proximity rather than on phenotype. The fact that two individuals from Pulau Tioman representing both phenotypes were found to have a sister relationship with 0.0% sequence divergence provides the most compelling molecular support for a color pattern dimorphism. Although the results provided by this study are sufficiently conclusive, the molecular data set would have been more informative if specimens of phenotype 1 from Peninsular Malaysia as well as phenotype 2 from Borneo had been included. However, finding snakes in the tropics is very time-consuming. For example, one of the authors (JvR) invested 450 search-hours in Sarawak, Borneo which yielded a single Oligodon purpurascens. As such, a disproportionate additional investment would have been required to substantially improve the analyses. Acknowledgments. We would like to thank P. David, Terin Martinjak, Rafe Brown, and Renee Johnson, for comments on early versions of this manuscript. We also like to thank Dr. Rainer Günther, Institut für systematische Zoologie der Humbold-Universität zu Berlin, Germany (ZMB), Ivan Ineich, Départment de Systématiques et Évolution, Muséum National d’Histore Naturelle, Paul C. Ustach, the University of Texas at Arlington, Alan Resetar, The Field Museum of Natural History, Lim Kok Peng Kelvin herpetological collections, Raffles Museum of Biodiversity Research, Alison Jennings, Louisiana State University and Agricultural and Mechanical College, Museum of Natural Sciences for the loan of specimens. We thank Christopher C. 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