Conserv Genet DOI 10.1007/s10592-007-9418-8 RESEARCH ARTICLE Ventral coloration differentiation and mitochondrial sequences of the Chinese Cobra (Naja atra) in Taiwan Hua-Ching Lin Æ Shou-Hsien Li Æ Jonathan Fong Æ Si-Min Lin Received: 10 September 2006 / Accepted: 28 August 2007 Ó Springer Science+Business Media B.V. 2007 Abstract Differences in coloration between eastern and mitochondrial sequences on a relatively short evolutionary western populations of the Chinese cobra (Naja atra)in time scale, ventral coloration is potentially a result of local Taiwan have been noted by snake collectors, snake keep- adaptation. Based upon the results of this study, along with ers, and users of Chinese traditional medicine, but have traditional observations, we strongly recommend treating never been verified by scientific research. In this study, we each of the four populations of the Chinese cobra in quantified the amount of black pigment on ventral scales, Taiwan as distinct ESUs. Reintroducing confiscated snakes and found prominent differences in ventral coloration of from the illegal trade back into the wild needs to be halted populations across Taiwan; populations in eastern Taiwan to prevent artifical gene flow. have black ventral scales while populations in the west are predominantly white. Previous studies have shown a Keywords Cobra Á Coloration Á Conservation genetics Á similar east-west population differentiation in regards to ESU Á Mitochondrial Á Naja atra venom components. We supplement these data with mitochondrial control region sequences, which show extremely low nucleotide diversity. Black-ventral and Introduction white-ventral snakes share major haplotypes and show no genetic differentiation. Nevertheless, moderate Fst and low Snakes, especially venomous species, are facing tremen- Nm values between populations indicate low levels of gene dous hunting pressure throughout Asia (Kuntz 1963; flow. With a morphological fixation earlier than Warrell 1995; Whitaker 1997). The main reason for this is their wide use as food and traditional Chinese medicine. In countries with large amounts of snake consumption, such as China, Hong Kong, and Taiwan, overexploitation has H.-C. Lin Á S.-H. Li caused serious population declines of some snake species Department of Life Science, National Taiwan Normal (Warrell 1995; Whitaker 1997). An investigation of the University, 88 Tingzhou Road, Sec. 4, Taipei 116, Taiwan, commercial use and harvesting of snakes indicated that ROC roughly 45,000 venomous snakes are captured and traded H.-C. Lin each year in Taiwan (Lin 1997). The Chinese cobra (Naja Taipei Zoo, 30 Xinguang Road, Sec. 2, Taipei 116, Taiwan, atra) constitutes the highest proportion among all snakes ROC traded in Taiwan; more than 20,500 cobras, or 10,800 kg, are traded each year, representing 46% and 65% of the total J. Fong Musem of Vertebrate Zoology, University of California snake trade figures, respectively (Lin 1997). Although the Berkeley, Berkeley, CA 94720, USA Chinese cobra is listed as a protected species under the Wildlife Conservation Law of Taiwan, little attention has & S.-M. Lin ( ) been paid to its conservation due to the negative image of Department of Life Science, Chinese Culture University, 55 Huagang Road, Taipei 111, Taiwan, ROC snakes by the general public. While this negative image is a e-mail: fi[email protected] problem for all snakes, it is particularly strong in the case 123 Conserv Genet of the cobra, due to the serious public concerns regarding Materials and methods snakebites (Hung 2004). Conflicts between conservation- ists and people who consume snakes are ongoing, but Sample collection and categorizations resolution of these differences are urgently needed (Dodd 1987). From 2001–2003, a total of 127 adult snakes were collected Cobras with various color morphs bearing distinctly from 34 localities throughout the island of Taiwan. All different ventral colorations have been observed in Tai- voucher specimens are now deposited at the Taipei Zoo. wan. Our preliminary observations along with those Samples were categorized into four geographic regions due made by the local hunters and snake dealers (H.-C. Lin, to existing geographic barriers/features (Fig. 1). Specimens personal interviews during the 1990s) hint at geographic obtained in the eastern side of the Central Mountain Range differentiation of color morphs. In the snake markets, were grouped into the ‘‘eastern population’’ (localities ‘‘black-ventraled’’ cobras, harvested from eastern Taiwan, 30–34). The Miaoli Plateau separates the ‘‘northern popu- are more valuable than ‘‘white-ventraled’’ specimens lation’’ (localities 1–10) from the ‘‘central population’’ from western Taiwan. These differences in market price (localities 11–19), while the central population is separated are due to the traditional belief that cobras with darker from the ‘‘southern population’’ (localities 20–29) near ventral scales provide stronger medicinal potency. The where the Tropic of Cancer passes through Taiwan. These Central Mountain Range, which separates the island into biogeographic breaks were chosen because they serve as two major geographic regions, might play the major role geographic barriers for the distribution of terrestrial fauna in such geographic differentiation. The geography of in Taiwan, including birds (Hachisuka and Udagawa 1950), Taiwan as well as observed differences in morphology frogs (Lue and Lai 1991; Lue et al. 1999), lizards (Lin and venom indicate potential for genetic differentiation et al. 2002; Lin 2003), small mammals (Fang and Lee across the geographic range of cobras in Taiwan (Hung 2004). However, patterns of color and genetic differen- tiation of cobras from both sides of the mountain range 10% have never been quantitatively and scientifically evaluated. 30% 60% 2 The difference seen in toxicology and potential differ- 1 3 4 ence in morphology between eastern and western 5 N = 30 6 populations show the necessity for a comprehensive survey 8 7 of N. atra across Taiwan. Due to the severe hunting pres- 9 sure, evolutionarily significant units (ESUs) of N. atra in 10 16% 4% Taiwan need to be clarified before suitable conservation 11 policies can be decided (Ryder 1986; Moritz 1994a; 1994b; 12 14 80% Crandall et al. 2000). Under these concepts, population 24ºN 13 30 15 genetic studies will provide valuable information for con- N = 45 18 16 17 servation, human consumption management, and treatment 31 of snakebites. With conservation in particular, there is 19 controversy surrounding the re-release of snakes illegally 20 100% brought into the markets. Currently, the common practice 22 21 23 is to release seized animals in the vicinity of confiscation. 23ºN Data from this study will determine the impact, if any, of 24 32 N = 22 7% 13% 33 release of market animals on the natural populations of 25 34 N. atra. 26 27 In our study, population differentiation of cobras in 80% 28 N Taiwan is evaluated via morphological and molecular data. The objectives of this investigation are to: (1) quantify the N = 30 22ºN 29 morphological differences of the two color morphs in 0 60km relation to geography; (2) identify the connection and 120ºE 121ºE 122ºE propose hypotheses for patterns between color morph, molecular data, and geographical distribution; and (3) Fig. 1 Sample localities and coloration compositions of the Chinese cobra (Naja atra) in different regions of Taiwan. The populations are apply these data towards the conservation of N. atra in divided into four regions: northern (sample localities 1–10), central Taiwan. (11–19) southern (20–29), and eastern (30–34) 123 Conserv Genet 2002), and several freshwater fishes (Tzeng 1986; Chen black, and mosaic color morphs from all regions being of and Fang 1999). 56:41, and 8 individuals, respectively (Table 1). Ventral coloration of each snake was defined by quan- Genomic DNA was extracted from the blood or muscle tifying the overall coverage of black coloration of the tissues using a LiCl method (Gemmell and Akiyama 1996). ventral scales. The ventral area of a snake was first divided After ethanol precipitation, DNA was suspended in 1X TE into 10 regions. Next, the ratio of the black area was cal- buffer and stored at –20°C. The complete mitochondrial culated by counting the number of black scales from each control region, including two flanking tRNAs (tRNA-Pro region, and combined to obtain the total coverage of the and tRNA-Phe), was amplified by polymerase chain reac- ventral surface (from 0% to 100%). After evaluating all tion (PCR). Primers were designed from the consensus 127 individuals, we divided these snakes into 10 groups at sequences of several Squamata species: 10% intervals. The sample sizes for each interval is rep- PL1: 50-CCACAAAGCATTGTTCTTGTAAACC-30 and resented in Fig. 2, revealing a bimodal distribution. The PH1: 50-GAAGCTTGCATGTATAAGTAGGG-30. highest peak was located between 20% and 30% (35 Reactions (including negative controls) were carried out individuals), representing a typical ‘‘white-ventral morph.’’ in 20-ll reactions using iCycler thermal cyclers (Bio-Rad), The second peak was located between 80% and 90% (24 with the following thermal cycles: 1 cycle at 94°C for individuals), representing a typical ‘‘black-ventral morph.’’ 3 min; 35 cycles at 94°C for 30 s, 55°C for 40 s, and 72°C According to the distribution in Fig. 2, we defined snakes for 70 s; and 1 cycle at 72°C for 10 min. PCR products with ventral coverage of black scales of \40% as ‘‘white- were purified with a PCR Product Pre-Sequencing Kit ventral morphs,’’ and those with coverage exceeding 70% (USB Corporation), and subsequently used as the template as ‘‘black-ventral morphs.’’ Individuals between 40% and for direct DNA sequencing reactions with a DYEnamic ET 70% are categorized as ‘‘mosaic-ventral morphs.’’ Color Dye Terminator Cycle Sequencing Kit (Amersham Phar- composition in each sampled geographic region (Fig. 1) macia Biotech). The same primers for PCR were used for was evaluated and compared to each other using Chi- the sequencing reactions in addition to two internal primers square tests.