Current Herpetology 32(1): 66–70, February 2013 © 2013 by The Herpetological Society of Japan
Extremely Low Genetic Diversity in the Japanese Population of Zootoca vivipara (Squamata: Lacertidae) Revealed by Mitochondrial DNA HIROHIKO TAKEUCHI1*, MIZUKI TAKEUCHI2, AND TSUTOMU HIKIDA1 1
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606–8502, JAPAN 2 Department of Advanced Technology for Transplantation, Graduate School of Medicine, Osaka University, Yamadaoka 2–2, Suita, Osaka 565–0871, JAPAN
Abstract: Genetic diversity in the Japanese population of Zootoca vivipara was investigated using partial sequences of mitochondrial cytochrome b (706 bp) gene for 24 individuals from three sites. The phylogenetic status of the Japanese population was also examined using data from the current and previous studies. Only one haplotype was recognized in the Japanese population, which was different by only one nucleotide from the Russian and Belarusian populations. Individuals from disparate regions (>7000 km apart) quite rarely share such closely related haplotypes. From the results of the current and previous studies, members of the continental population seem to have invaded Hokkaido via the land bridge during the latest Pleistocene glaciation. Key words: Zootoca vivipara; mitochondrial DNA (mtDNA); cytochrome b (cytb); Hokkaido; Japan
INTRODUCTION Distribution of the small lacertid lizard Zootoca vivipara is extremely wide, stretching * Corresponding author. Tel: +81–75–753–4074; Fax: +81–75–753–4114; E-mail address:
[email protected]
doi 10.5358/hsj.32.66
from western and northern Europe through Russia and northern China to Japan (SurgetGroba et al., 2001; Hikida, 2002). This distribution range is the widest and farthest north of any terrestrial reptile. Studies on the phylogeography of Z. vivipara have been conducted using allozyme or DNA data in order to reveal the evolutionary history of viviparity in this species (Guillaume et al., 1997; Heulin et al., 1999; Surget-Groba et al., 2001, 2006). Of these, Surget-Groba et al. (2001) demonstrated the presence of closely related haplotypes in populations from a wide region in the eastern part of Europe and Russia. However, no phylogeographical studies have been focused on the Japanese population of this species. In Japan, the geographical distribution of Z. vivipara is extremely limited, and this lizard is only found on the northern tip of Hokkaido (Hikida, 2002). It has been listed in the Red List of Japan as a vulnerable species since 2006 (Takenaka, 2010). For conservation purpose, information regarding genetic diversity is very important and helpful (McNeely et al., 1990; Frankham, 1995; Frankham et al., 2002). Nevertheless, little is known about genetic diversity in the Japanese population of Z. vivipara. In this study, genetic diversity in the Japanese population of Z. vivipara was examined using partial sequences of the mitochondrial cytochrome b gene. The phylogenetic status of the Japanese population was inferred using data from the present and previous studies (Surget-Groba et al., 2001, 2006) in order to gain insight into its historical biogeography.
MATERIALS AND METHODS In total, 24 samples were collected from three localities: Wakkanai city (n=15), Toyotomi town (n=8), and Sarufutsu village (n=1). Sampling was conducted from 2002 to 2010 outside the nature reserves in these places. As a source of total genomic DNA, tail tips were removed from living lizards and stored in 99.5% ethanol. DNA extraction, polymerase
TAKEUCHI ET AL.—LOW GENETIC DIVERSITY IN A LIZARD
67
FIG. 1. Maximum likelihood (ML) tree based on partial sequences of the mitochondrial cytochrome b (706 bp) gene under the HKY+G model. Bootstrap confidence values >70% are provided at each node. The reference codes of haplotypes and their groups follow Surget-Groba et al. (2006).
chain reaction, and sequencing were performed following the methods of Takeuchi et al. (2012), except for amplifications and cycle sequencing reactions, where another primer set was used: L-14731 (Tanaka-Ueno et al., 1998) and CB3-H (Palumbi et al., 1991). To determine the phylogenetic status of the Japanese population of Z. vivipara, sequence data taken from GenBank (accession numbers AY714882–714929; Surget-Groba et al., 2006)
were used. A homologous sequence of Lacerta bilineata (accession number AY714881; SurgetGroba et al., 2006) was also used as an outgroup. In total, 706 base pairs (bp) in cytochrome b were aligned using CLUSTAL X (Thompson et al., 1997). To infer the mitochondrial DNA genealogy, the maximum likelihood (ML) method was employed (Felsenstein, 1981). The most appropriate sequence evolution model (HKY+G) was selected
68
Current Herpetol. 32(1) 2013
according to the Akaike Information Criterion (AIC: Akaike, 1974; Posada and Crandall, 1998). Initial trees for the heuristic search were generated using an improved version of the neighbor-joining method (BIONJ) with a Markov Cluster distance matrix. Reliability of the tree was assessed by calculating bootstrap probability (BP) (Felsenstein, 1985) with 1000 replications. Selection of the model, construction of the ML tree, and the bootstrap analysis were all performed using MEGA version 5 (Tamura et al., 2011).
RESULTS Partial sequences corresponding to 706 bp of mitochondrial cytochrome b gene were obtained for the 24 samples from the Japanese population of Z. vivipara. In these sequences, only one haplotype was recognized. The ML tree and BP values (>70%) are shown in Fig. 1. The highest log likelihood of the tree was –2721.4535 and the gamma parameter was 0.2965. The haplotype of the Japanese population belonged to the eastern viviparous clade of Surget-Groba et al. (2001), which is distributed in Eastern Europe and Asia (Fig. 1). As shown in the figure, the Japanese haplotype differed by one nucleotide from haplotypes
UV1, UV2, and UV3 of Surget-Groba et al. (2001, 2006), and a very small genetic distance (p-distance) of 0.12% was found between the Japanese haplotype and these haplotypes. These haplotypes are found in the Russian and Belarusian populations (Surget-Groba et al., 2001) as shown in Fig. 2.
DISCUSSION The haplotype of the partial cytochrome b gene from the Japanese population differed by only one mutation from those of the Russian and Belarusian populations (0.12% in pdistance between the Japanese and Belarusian populations: Surget-Groba et al., 2001, 2006). Individuals from disparate regions (>7000 km apart) quite rarely share such closely related haplotypes, particularly in small-bodied terrestrial animals such as Z. vivipara. Previous studies showed that individuals from widely distant regions of the eastern part of Europe and Russia shared closely related haplotypes (Surget-Groba et al., 2001, 2006). These previous data suggest a recent range expansion in the eastern continental population, although this was not addressed in previous studies. During the last glacial period, a land bridge is thought to have existed between the northern tip of Hokkaido and the eastern part of the
FIG. 2. Map of the Eurasian Continent showing the distribution of Zootoca vivipara (dashed line). Dots indicate distribution of the VU1, VU2, VU3 (Surget-Groba et al., 2001), and the Japanese haplotypes.
TAKEUCHI ET AL.—LOW GENETIC DIVERSITY IN A LIZARD
Eurasian continent via Sakhalin Island (e.g., Ohshima, 1990). These lines of information suggest that in the last glacial period, rapid range expansion involving some members of the continental populations may have occurred, and resulted in invasion of the northern tip of Hokkaido via the land bridge. In Squamata, the cytochrome b region is known for its very fast molecular evolution in mitochondrial DNA (Burbrink et al., 2000; Poulakakis et al., 2005; Poulakakis et al., 2008; Brown et al., 2008). However, in spite of the use of this region, an extremely low genetic diversity was observed within the Japanese population in the present study. Such a low genetic diversity may be explained by the following reasons: (i) the Japanese founder population(s) may have originated from the continental population(s), which rapidly expanded in range, and (ii) the date of invasion was too recent for haplotype variation via mutation, although the possibility of artificial introduction is not precluded (Lever, 2003). The Japanese population of Z. vivipara prefers a special habitat, such as wetland areas in a very narrow range, as evidenced by the difficulty in finding this species outside wetland areas in Hokkaido. Therefore, habitat conservation is essential in order to maintain a stable population, since populations with a low genetic variation may have reduced ability to cope with environmental change (e.g., Frankham, 1995).
ACKNOWLEDGEMENTS We thank Dr. Ryo Ito for supporting a part of the sampling works. This research was financially supported in part by the Global Center of Excellence Program ‘Formation of a Strategic Base for Biodiversity and Evolutionary Research: from Genome to Ecosystem’ of the Ministry of Education, Culture, Sports and Technology (MEXT), Japan.
LITERATURE CITED AKAIKE, H. 1974. A new look at the statistical
69
model identification. IEEE Transactions on Automatic Control 19: 716–723. BROWN, R. P., TERRASA, B., PEREZ-MELLADO, V., CASTRO, J. A., HOSKISSON, P. A., PICORNELL, A., AND RAMON, M. M. 2008. Bayesian estimation of post-Messinian divergence times in Balearic Island lizards. Molecular Phylogenetics and Evolution 48: 350–358. BURBRINK, T. F., LAWSON, R., AND SLOWINSKI, B. J. 2000. Mitochondrial DNA phylogeography of the polytypic North American rat snake (Elaphe obsoleta): a critique of the subspecies concept. Evolution 54: 2107–2118. FELSENSTEIN, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution 17: 368–376. FELSENSTEIN, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. FRANKHAM, R. 1995. Conservation genetics. Annual Review of Genetics 29: 305–327. FRANKHAM, R., BALLOU, J. D., AND BRISCOE, D. A. 2002. Introduction to Conservation Genetics. Cambridge University Press, Cambridge. GUILLAUME, C. P., HEULIN, B., BECHKOV, V. 1997. Biogeography of Lacerta vivipara: Reproductive mode and enzyme phenotypes in Bulgaria. Ecography 20: 240–246. HEULIN, B., SURGET-GROBA, Y., GUILLER, A., GUILLAUME, C. P., AND DEUNFF, J. 1999. Comparisons of mtDNA sequence (16S rRNA gene) between oviparous and viviparous strains of Lacerta vivipara: A preliminary study. Molecular Ecology 8: 1627–1631. HIKIDA, H. 2002. Natural History of the Reptiles. University of Tokyo Press, Tokyo. LEVER, C. 2003. Naturalized Reptiles and Amphibians of the World. Oxford University Press, New York. MCNEELY, J. A., MILLER, K. R., REID, W. V., MITTERMEIER, R. A., AND WENER, T. B. 1990. Conserving the World’s Biological Diversity. IUCN, World Resources Institute, Conservation International, WWF-US and the World Bank, Washington, DC. OHSHIMA, K. 1990. The history of straits around the Japanese Islands in the late-Quaternary.
70
Daiyonki Kenkyu (the Quaternary Research) 29: 193–208. PALUMBI, S. R., MARTIN, A., ROMANO, S., MACMILLAN, W. O., STICE, L., AND GRABOWSI, G. 1991. The Simple Fool’s Guide to PCR, Version 2.0. Privately published document compiled by S. Palumbi, Department of Zoology, The University of Hawaii, Honolulu. POSADA, D. AND CRANDALL, K. A. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818. POULAKAKIS, N., LYMBERALIS, P., TSIGENOPOULOS, C. S., MAGOULAS, A., AND MYLONAS, M. 2005. Phylogenetic relationships and evolutionary history of snake-eyed skink Ablepharus kitaibelii (Sauria: Scincidae). Molecular Phylogenetics and Evolution 34: 245–256. POULAKAKIS, N., PAKAKI, V., MYLONAS, M., AND LYMBERAKIS, P. 2008. Molecular phylogeny of the Greek legless skink Ophiomorus punctatissimus (Squamata: Scincidae): The impact of the Mid-Aegean trench in its phylogeography. Molecular Phylogenetics and Evolution 47: 396–402. SURGET-GROBA, Y., HEULIN, B., GUILLAUME, C., PUKY, M., SEMENOV, D., ORLOVA, V., KUPRIYANOVA, L., GHIRA, I., AND SMAJDA, B. 2006. Multiple origins of viviparity, or reversal from viviparity to oviparity? The European common lizard (Zootoca vivipara, Lacertidae) and the evolution of parity. Biological Journal of the Linnean Society 87: 1–11. SURGET-GROBA, Y., HEULIN, B., GUILLAUME, C., THORPE, R. S., KUPRIYANOVA, L., VOGRIN, N., MASLAK, R., MAZZOTTI, S., VENCZEL, M., GHIRA, I., ODIERNA, G., LEONTYEVA, O., MONNEY, J. C., AND SMITH, N. 2001. Intraspecific phylogeography of Lacerta vivipara and the evolution of viviparity. Molecular Phylogenetics
Current Herpetol. 32(1) 2013 and Evolution 18: 449–459. TAKENAKA, S. 2010. Lacerta vivipara Von Jacquin, 1787. p. 4. In: Wildlife Division, Natural Environment Bureau, Ministry of the Environment (ed.), Appendix for The Third Revised Red List of Japan–Amphibians and Reptiles. Wildlife Division, Natural Environment Bureau, Ministry of the Environment, Tokyo. TAKEUCHI, H., OTA, H., OH, H.-S., AND HIKIDA, T. 2012. Extensive genetic divergence in the East Asian natricine snake, Rhabdophis tigrinus (Serpentes: Colubridae), with special reference to prominent geographical differentiation of the mitochondrial cytochrome b gene in the Japanese populations. Biological Journal of the Linnean Society 105: 395–408. TAMURA, K., PETERSON, D., PETERSON, N., STECHER, G., NEI, M., AND KUMAR, S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731– 2739. TANAKA-UENO, T., MATSUI, M., CHEN, S. L., TAKENAKA, O., AND OTA, H. 1998. Phylogenetic relationships of brown frogs from Taiwan and Japan assessed by mitochondrial cytochrome b gene sequences (Rana: Ranidae). Zoological Science 15: 283–288. THOMPSON, J. D., GIBSON, T. J., PLEWNIAK, F., JEANMOUGIN, F., AND HIGGINS, D. 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882.
Accepted: 3 December 2012
