Animals

A profile of the Japanese giant flying squirrel Petaurista leucogenys examined in the present study is shown in Table 1. Thirty seven specimens of P. leucogenys were collected from 17 localities in Japan (Fig. 1). A female red giant flying squirrel Petaurista petaurista melanotus (PPM) imported from Hong-Kong to Japan in 1990 was used as an out-group.

Table 1

Specimen profiles of Petaurista leucogenys examined in the present study

Fig. 1

Sampling localities in the present study and fossil localities of Petaurista leucogenys. Closed circles, sampling localities; open square, Middle Pleistocene fossil localities; closed squares, Late Pleistocene fossil localities. Sampling locality numbers correspond to those of Table 1 and Fig. 2. Arrow indicates the presumed migration route of P. leucogenys from southern China to Japan.

DNA preparation and sequencing

Total DNAs were extracted from muscle or liver tissues using the phenol/proteinase K/sodium dodecyl sulfate method of Sambrook et al. (1989). The whole control region was amplified using polymerase chain reaction (PCR), with a set of newly designed primers: L15933 5′-CTCTGGTCTTGTAAACCAAAAATG-3′ and H637 5′-AGGACCAAACCTTTGTGTTTATG-3′. Primer names correspond to the light (L) or heavy (H) strand and the 3′end-position of the primers in the human mtDNA sequences (Anderson et al., 1981). The reaction mixture of 50μl contained 100 ng of genomic DNA, 25 picomoles of each primer, 200 μM dNTPs, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, and 2.5 units of rTaq DNA polymerase (Takara). Amplification was carried out for 35 cycles as follows: 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, and then the extension reaction was performed at 72°C for 10 min. PCR products were purified with the Qia-quick PCR purification kit (QIAGEN) and directly sequenced using an automated DNA sequencer (SQ5500L, Hitachi). For sequencing, two PCR primers and another new primer (5′-CCTAATGGATATCCCCTTCCAACG-3′) were used.

Phylogenetic analysis

All sequences were aligned using the computer software GeneWorks (Intelligenetics). The phylogenetic tree was constructed via the neighbor-joining (NJ) method (Saitou and Nei, 1987) in Clustal W (Thompson et al., 1994) and via the maximum parsimony (MP) method using a heuristic search algorithm with the 50% majority-rule consensus in PAUP (Swofford, 1993). In the NJ tree, the numbers of nucleotide substitutions per site were estimated for multiple substitutions by Kimura's (1980) two-parameter method. Using sequences without gap-sites, the MP tree was produced by unweighted parsimony. To assess the branching confidence, bootstrap values (Felsenstein, 1985) were derived from 1,000 replications of the NJ method and 100 replications of the MP tree.

Sequence Divergence of mtDNA Control Region

All mtDNA control regions (1,052–1,054 bases) of P. leucogenys from 17 localities in Japan were successfully sequenced. All sequences of animals from Kyushu had 1,052 bases, while those of populations from Honshu and Shikoku had 1,054 bases with insertions except for three specimens: EH1 (1,052 bases), KT1 (1,052 bases), and HS1 (1,053 bases) (Table 2). Of all sequences obtained, 145 sites were variable: transitions at 117, transversions at 14 sites, and both transitions and transversions at 12 sites (Table 2). In the 37 animals, 24 haplotypes were identified (Tables 1 and 2). The sequence divergence among haplotypes was 0.7–4.8% (Table 3).

Table 2

Sequence variation of the mt DNA control region (1,052–1,054 bases) in Petaurista leucogenys. Dots indicate identical nucleotides or indels with those of haplotype K1

Table 3

Pairwise comparison of mt DNA control region sequences without gap-sites (1,052 bases) between 24 haplotypes from Petaurista leucogenys

The control region of P. leucogenys was divided into three domains: two variable domains (the 5′ and 3′ ends) and one conserved central domain (Table 2). The 5′ end domain was more variable than the 3′ end domain and contained one gap-site (site number 281) in all haplotypes from Kyushu, in H1 and H2 from Honshu, and in H3 from Shikoku. The 3′ end domain contained an additional gap-site (site number 1,052) in all haplotypes from Kyushu, in H2 from Honshu, and in H3 from Shikoku. The sequence of P. petaurista used as an out-group has 1,051 bases (accession number in DDBJ:  AB043816).

Molecular phylogeny based on the mtDNA control region

Phylogenetic relationships reconstructed via NJ and MP methods were similar to each other. In the NJ tree, the Japanese population of P. leucogenys was separated into three major lineages: Group A consisting of K1, Group B consisting of K2, K3, K4, K5, K6, K7, K8, and H1 (95% bootstrap value), and Group C consisting of H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, and H15 (62% bootstrap value) (Fig. 2a). In the MP analysis, only one most-parsimonious phylogenetic tree was obtained by unweighted parsimony, and it had a consistency index of 0.668. The three major groups were also recognized in the MP tree: Group A (K1), Group B consisting of K2, K3, K4, K5, K6, K7, K8, and H1 (88% bootstrap value), and Group C consisting of H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, and H15 (57% bootstrap value) (Fig. 2b). In both trees, H1 from Honshu was clustered with the Kyushu population consisting of K2, K3, K4, K5, K6, K7, and K8 with high bootstrap values (95% in the NJ tree and 88% in the MP tree), and H2, H3, and H16 were not included in the major three lineages. In addition, in both trees, genetic differences between haplotypes of Group C did not correspond to geographic distances. The sequences of Groups A and B had 1,052 bases and those of Group C had 1,054 bases, although there was an exception (H1).

Fig. 2

Phylogenetic relationships reconstructed by (a) the neighbor-joining (NJ) and (b) the maximum parsimony (MP) methods based on the control region sequences of Petaurista leucogenys. The scale bar for NJ tree represents branch length in terms of nucleotide substitution per site. Numbers at nodes indicate bootstrap values higher than 50% derived from 1,000 replications for NJ tree and 100 replications for MP tree. Numbers in parentheses, which correspond to those of Table 1 and Fig. 1, indicate sampling localities.

Characterization of the mtDNA control region in Petaurista leucogenys

In vertebrates, it has been reported that the control region consists of two variable domains (5′ and 3′ ends) and a conserved central domain (Brown et al., 1986; Southern et al., 1988; Saccone et al., 1991). In the same way, the control region of P. leucogenys examined in the present study was also divided into three domains (Table 2). In particular, the 5′ end domain was more variable than the 3′ end domain and contained one gap-site (site number 281) in all haplotypes from Kyushu, in H1 and H2 from Honshu, and in H3 from Shikoku. Another gap-site (site number 1,052) was recognized in the 3′ end domain in all haplotypes from Kyushu and in H2 and H3. The two gap-sites were specific to the control region of the Kyushu population. In addition, repetitive sequences, which were reported in the control region of some mammals (e.g., Hoelzel et al., 1994; Nagata et al., 1998; Kurose et al., 1999; Matsuhashi et al., 1999), were not found in P. leucogenys and P. petaurista.

Phylogeography of Petaurista leucogenys

According to pelage characteristics, Imaizumi (1960) classified P. leucogenys into three subspecies (leucogenys, nikkonis, and oreas), and demonstrated that P. l. leucogenys is distributed throughout the Kyushu and Shikoku islands, that P. l. nikkonis occurs in the eastern part of the Honshu Island, and that P. l. oreas occurs in the western part of the Honshu Island. However, in the present study, phylogenetic relationships among haplotypes did not correspond to subspecies classification and distribution.

Despite of the small number of specimens collected (Fig. 1), the Kyushu population was divided into two lineages: K1 lineage (Group A) and another lineage consisting of K2, K3, K4, K5, K6, K7, and K8 in Group B (Fig. 2). In the NJ and MP trees, K1 was likely to have been isolated from the other haplotypes by the first dichotomy, although the bootstrap values were not so high (< 50% in NJ tree; 59% in MP tree), suggesting that K1 diverged from the other haplotypes at an earlier point. On the other hand, although H1 was closely related to K2, K3, K4, K5, K6, K7, and K8 in both trees, the phylogenetic positions of H2 and H3 in the NJ tree were different from those in the MP tree (Fig. 2). The three haplotypes (H1, H2, and H3) shared the two gap-sites specific to the Kyushu population: site number 281 in H1, H2, and H3 and site number 1,052 in H2 and H3 (Table 2). This indicated that H2 and H3 are more closely related to K2, K3, K4, K5, K6, K7, and K8 or that they are intermediate haplotypes between the Kyushu population and Honshu/Shikoku population. The phylogenetic position of H16 that was not clustered with the three major lineages was not clear in the present study.

In terms of the Honshu/Shikoku population in the NJ and MP trees, it is noteworthy that genetic distances between haplotypes do not always correspond to geographic distances of sampling localities, and exhibit low bootstrap values (Fig. 2). Moreover, six specimens from different localities (Gifu, Kagawa, Nagano, Toyama, Wakayama, and Yamanashi) shared the H4 haplotype, and two specimens from Aomori and Gifu had the H5 haplotype (Fig. 1 and Table 1). In the Honshu/Shikoku population, except for H1, H2, H3, and H16, haplotype H4 seemed to be most common.

Based on some fossil records, Kawamura (1988) reported that P. leucogenys had presumably migrated from southern China to Japan through the land bridge (Fig. 1) which was formed around the area of the present East China Sea in the Early Middle Pleistocene. It was not known that Petaurista had existed in the Korean Peninsula at that time. Accepting Kawamura's hypothesis (1988), inevitably, the first place where P. leucogenys had migrated from southern China could have been the Kyushu Island in Japan, and then it could have extended its distribution toward the Honshu and Shikoku Islands (see Fig. 1). Fossils of Petaurista before the Holocene period are very rare in the Japanese islands. The Middle Pleistocene fossils of P. leucogenys were found in two localities of Japan (Hasegawa, 1966; 1972; Kowalski and Hasegawa, 1976; Kawamura, 1988) which are very close to each other as shown in Fig. 1. Moreover, the Late Pleistocene fossils of P. leucogenys were also recognized in six localities of Japan (Shikama, 1949; Hasegawa, 1966; Kowalski and Hasegawa, 1976; Kawamura, 1980; 1981; 1982; 1988; Kawamura and Sotsuka, 1984; Kawamura et al., 1986), as shown in Fig. 1. Judging from these fossil records, by the Middle or Late Pleistocene, P. leucogenys could already have been distributed in the Honshu and Shikoku Islands. Petaurista leucogenys is an arboreal animal and inhabits the temperate forests (Nowak, 1991). Therefore, during glacial stages in the Pleistocene, the habitats of this animal may have been reduced due to the southward shifting of temperate forests in Japan. The results of the present study show that the genetic distances in the Honshu/Shikoku population were not related to geographic distances of sampling localities. This suggests that P. leucogenys rapidly extended its distribution in a short time during the northward expansion of temperate forests in Japan after the last glacial stage of the Pleistocene.

Although the evolutionary rate of humans may not always correspond to that of the giant flying squirrel because of the differences of generation time between humans and giant flying squirrels, applying the evolutionary rate (approximately 8.4% per million years, Myr) of the human control region reported by Vigilant et al. (1989), the divergence times between Groups A and B, between Groups A and C, and between Groups B and C were estimated to be approximately 0.4–0.5, 0.4–0.5, and 0.4–1.0 Myrs ago, respectively. On the other hand, the divergence times in Group B and in Group C were approximately 0.1–0.3 and 0.1–0.5 Myrs ago, respectively. Accordingly, the divergences among haplotypes of P. leucogenys may have occurred rapidly from the Middle to Late Pleistocene.