Mammal Study 28: 67–72 (2003) © the Mammalogical Society of Japan Geographic variation of Mus caroli from East and Southeast Asia based on mitochondrial cytochrome b gene sequences Mie Terashima1, Agustinus Suyanto2, Kimiyuki Tsuchiya3, Kazuo Moriwaki4, Mei-Lei Jin5 and Hitoshi Suzuki1,* 1 Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan 2 Research Centre for Biology, Indonesian Institute of Sciences, Cibinong 16911, Indonesia 3 Department of Agriculture, Tokyo University of Agriculture, Atsugi 243-0034, Japan 4 RIKEN, Bioresource Center, Tsukuba, Ibaraki 305-0074, Japan 5 Shanghai Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China Abstract. We examined intraspecific genetic variation in the mitochondrial cytochrome b gene (1140 bp) in the Okinawa mouse, Mus caroli (Rodentia, Muridae), from Okinawa, Taiwan, Hainan, Yunnan (southern China), Thailand, Vietnam and Java, to better understand their evolutionary history and human impact on dispersal and colonization by the mice. Populations from each of the seven localities had distinctive mitochondrial DNA sequences. The genetic distances, ranging from 0.015 to 0.045 (0.033 on average), were comparable with inter-subspecies differences in the house mouse Mus musculus (0.024). Assuming that nucleotide substitutions occur at a constant rate of 0.024/million years/lineage for mitochondrial DNA, the divergence of in M. caroli is estimated to have occurred 0.3–0.9 million years ago. The relatively great extent of intraspecific mitochondrial DNA variation in this taxon, and the spatial mode of variation suggest that M. caroli is genetically structured in space, to a considerable extent. Our data imply that local insular populations of M. caroli have been separated for long evolutionary periods, and that the scattered distribution patterns in the insular domains are due to historical shrinkage of appropriate areas of habitat, rather than to recent establishment of dispersed local populations by inadvertent human introductions. Key words: geographic variation, mitochondrial cytochrome b, Mus caroli, Okinawa mouse. The Okinawa mouse, Mus caroli, is known to occur in distinct morphological features, including coat color grasslands and in areas associated with humans, such as (Corbet and Hill 1992). However, taxonomic subdivi- rice or sugar cane fields. Mus caroli is found on sion based on such morphological variation has been dis- Okinawa Island in the Ryukyu Islands of Japan, and is puted (Musser and Newcomb 1983) and is under debate. also widely distributed across East and Southeast Asia, The patchy geographic distribution of M. caroli from including Taiwan, Hainan, Yunnan (southern China), Japan and Indonesia is not well understood. The popula- Vietnam, Thailand, Malay Peninsula, Sumatra, and Java tions in the continental part, Hainan Island, and Taiwan (Langham and Lam 1977; Musser and Newcomb 1983; is considered to reflect natural habitation, while those Corbet and Hill 1992). This species shows substantial from Indonesia and Japan are possibly introduced by morphological variation among the disparate areas of its humans (Musser and Newcomb 1983; Motokawa 2000; range. The individuals of the Javan population, for Motokawa et al. 2003). In Indonesia, M. caroli is known example, have sometimes been considered as belonging to occur on Flores, Java, and Sumatra, but is absent to a distinct taxa, Mus ouwensi or Mus caroli ouwensi from other islands including Borneo (Musser 1981; (Kloss 1921; Corbet and Hill 1992), owing to their Corbet and Hill 1992; Musser and Carleton 1993). In *To whom correspondence should be addressed. E-mail: [email protected] 68 Mammal Study 28 (2003) Table 1. List of samples used in this study. Locality no. Locality Specimen number Code Accession no. 1. Shuri, Okinawa, Japan HS598 Okinawa-1 AB033698a HS1766 Okinawa-2 AB109792 2. Taichung, Taiwan HS1526 Taiwan AB109793 3. Haikou, Hainan, China MG619 Hainan-1 AB109794 Sanya, Hainan, China MG630 Hainan-2 AB109795 4. Menhai, Yunnan, China MG600 Yunnan-1 AB109796 MG602 Yunnan-2 AB109797 5. Thailand — Thailand AY057812b 6. Vietnam HS2048 Vietnamc — 7. Jogyakarta, Java, Indonesia MZB24350/HS2474 Java-1 AB109798 MZB24351/HS2475 Java-2 AB109799 Bantul, Java, Indonesia MZB24352/HS2476 Java-3 AB109800 MZB24353/HS2477 Java-4 AB109801 MZB24354/HS2478 Java-5 AB109802 MZB24355/HS2479 Java-6 AB109803 a Suzuki et al. (2000), b Lundrigan et al. (2002), c Chinen et al., unpublished data. the Ryukyu Islands, it is found only on Okinawa Island (Motokawa 2000; Motokawa et al. 2003). There are two possible explanations for this scattered distribution. One could be recent exclusion of populations, and shrinkage of the natural range owing to environmental changes (Marshall 1977). Alternatively, the presence of this semi-commensal species in several places on the Sunda Shelf could be explained by inadvertent human introduc- tions (Musser and Newcomb 1983). Mus caroli from the northern end of the Malay Peninsula, northern Sumatra, and Java, are reported to have no differences in the morphological features of the skull and skin (Musser and Newcomb 1983). Another interesting issue is that posed by the distribution of M. caroli on Java. There the spe- cies inhabits the central part of the island, but is entirely absent from the western part (Marshall 1977). Marshall (1977) supposed that M. caroli was absent from western Java because of the unfavorable, extremely wet climate. It can also be explained as being the result of a new intro- duction by humans (Musser and Newcomb 1983). To better understand the evolutionary history and human impact on colonization of M. caroli, we con- ducted molecular phylogenetic analyses using the mitochondrial cytochrome b gene (cyt b) sequences of individuals from seven localities in Okinawa, Taiwan, Hainan, Yunnan, Vietnam, Thailand, and Java. We found considerable genetic variation among mice from these geographic localities. Fig. 1. East and Southeast Asia showing the seven localities where Mus caroli were sampled. The exact location of the sample for Thailand is unknown. Terashima et al., Mitochondrial DNA variation in Mus caroli 69 Material and methods PCR). Both DNA strands of the product of the second PCR were directly sequenced automatically using a Dye Sources of sequences Primer Cycle Sequencing Kit (ABI) and an automated Six M. caroli individuals were trapped in Central Java, sequencer (model 310, ABI). Indonesia (see Table 1, Fig. 1). We prepared skin and The nucleotide sequences reported in this paper appear skull specimens and deposited them in the Museum Zoo- in the DDBJ, EMBL, and GenBank nucleotide sequence logicum Bogriense, Indonesia. One M. caroli individual databases under the following accession numbers was captured in Taiwan. Four DNA samples from AB109792-AB109803 (Table 1). Hainan and Yunnan, with serial number MG, were kindly provided by Dr. Toshihiko Shiroishi, of the Phylogenetic analysis National Institute of Genetics, Japan. The DNA sam- We constructed a phylogenetic tree using the neigh- ples, with the serial number HS, are preserved in the bor-joining (NJ) method (Saitou and Nei 1987) with Laboratory of Ecology and Genetics, Graduate School genetic distances computed by Kimura’s two-parameter of Environmental Earth Science, Hokkaido University. (Kimura 1980), and maximum parsimony (MP) methods The mitochondrial cyt b gene sequences for the indi- using the computer program PAUP* 4.0b10 (Swofford viduals from Okinawa, in the Ryukyu Islands, Japan 2001). The statistical confidence of the sequence clus- (AB033698; Suzuki et al. 2000), Vietnam (Chinen et al. ters in the NJ and MP trees was evaluated by bootstrap unpublished data) and Thailand (AY057812; Lundrigan percentages derived from 1000 replications. Genetic et al. 2002) were obtained from the nucleotide databases distances between localities were calculated using the DDBJ, EMBL, and GenBank. The sequences for the cyt software PHYLTEST (Kumar 1996). b gene of M. musculus domesticus (J01420; Bibb et al. 1981), M. m. musculus (= M. m. molossinus; Suzuki et al. Results 2000) and M. spretus (AB033700; Suzuki et al. 2000) were used as the outgroup. From 15 samples of M. caroli, from seven geographic regions of Okinawa (n = 2), Taiwan (n = 1), Hainan Gene analysis (n = 2), Yunnan (n = 2), Thailand (n = 1), Vietnam (n = We determined sequences for the mitochondrial cyt b 1) and Java (n = 6) (Table 1), 14 haplotypes were iden- gene. Amplification of a fragment of the mitochondrial tified. The individuals from Java, Hainan, and Yunnan cyt b gene was performed using previously described exhibited substantial within-population variation, while methods (Suzuki et al. 1997, 2000). The reactions were the two individuals from Okinawa shared an identical carried out for 35 cycles, each consisting of 30 sec at sequence (Table 2). In particular, the six individuals 96°C for denaturation, 30 sec at 50°C for annealing, and from Java showed marked differences, with three distinct 30 sec at 60°C for extension. The reaction mixtures lineages with a genetic distance of approximately 0.022. (20 l) contained 2.5 mM MgCl2 (1.25 mM for second The two sequences from Hainan were quite similar, with Table 2. Distance of sequence divergence of the cyt b gene within and between the localities.* Okinawa Taiwan Hainan Yunnan Vietnam Thailand Java 1. Within locality n 212211 6 range (mean) 0.000 — 0.006 0.003 — — 0.002–0.024 (0.016) 2. Between localities Okinawa — Taiwan 0.045 — Hainan 0.034 0.038 — Yunnan 0.035 0.033 0.021 — Vietnam 0.033 0.033 0.017 0.018 — Thailand 0.022 0.039 0.029 0.027 0.026 — Java 0.031 0.041 0.032 0.030 0.029 0.030 — *Sequence divergence was computed by Kimura (1980) with all substitutions at all codon positions. 70 Mammal Study 28 (2003) Fig. 2. A neighbor-joining (NJ) tree based on mitochondrial cytochrome b gene sequences (1140 bp). Genetic distances were computed using Kimura’s two parameter method (1980) with consideration of all codon positions and all substitutions.