Petauristinae (flying squirrels) consists of 44 extant species in 14 recent genera, and their phylogenetic relationships and taxonomy are unsettled questions. We analyzed partial mitochondrial cyto-chrome b gene sequences (1,068 base pairs) to investigate the phylogenetic relationships among six flying squirrel genera (Belomys, Hylopetes, Petaurista, Petinomys, and Pteromys from Asia and Glaucomys from North America). Molecular phylogenetic trees, constructed by neighbor-joining and maximum likelihood methods, strongly indicated the closer relationship between Hylopetes and Petinomys with 100% bootstrap values. Belomys early split from other flying squirrels. Petaurista was closely related to Pteromys, and Glaucomys was most closely related to the cluster consisting of Hylopetes and Petinomys. The bootstrap values supporting branching at the deeper nodes were not always so high, suggesting the early radiation in the evolution of flying squirrels.
Flying squirrels (Petauristinae), which are distributed throughout Eurasia and the North America, consist of 44 extant species in 14 recent genera (Corbet and Hill, 1991). However, phylogenetic study of flying squirrels has been superficial so far. Genetic analyses of intrageneric relathionships of the genus Petaurista (Oshida et al., 1992; 1996; 2000) and of the genus Glaucomys (Nadler and Sutton, 1967; Rausch and Rausch, 1982; Arbogast, 1999) have been reported. From the morphological perspective, the close phylogenetic relathionship between Glaucomys and Hylopetes was indicated by Burt (1960) and Thorington Jr. et al. (1996). Rausch and Rausch (1982) reported the phylogenetic relathionship between Glaucomys and Pteromys based on their chromosomal characterizations. Hight et al. (1974) described the phylogenetic relationships of four flying squirrel genera (Iomys, Petaurista, Petinomys, and Pteromys), based on immunological data. However, the phylogenetic relationships of flying squirrels remain poorly understood.
In the present study, molecular phylogenetic analyses using partial sequences (1,068 base pairs: bp) of the mitochondrial cytochrome b gene was conducted for ten flying squirrel species distributed in various areas of Asia and North America: Belomys pearsonii, Hylopetes phayrei, Glaucomys volans, Petinomys setosus, Petaurista alborufus, Petaurista leucogenys, Petaurista Petaurista, Petaurista philippensis, Pteromys momonga, and Pteromys volans. We here discuss the phylogenetic relationships among the six flying squirrel genera.
MATERIALS AND METHODS
Flying squirrels examined in the present study are shown in Table 1. Muscle tissues of Belomys pearsonii and Hylopetes phayrei and the skin tissue of Pteromys momonga were collected. Samples of Glaucomys volans and Petinomys setosus were commercially obtained. Sequence data of Petaurista alborufus, Petaurista leucogenys, Petaurista philippensis, Petaurista Petaurista, and Pteromys volans were quoted from our previous report (Oshida et al., 2000). These flying squirrels are representative species in each area: Belomys from Taiwan and Southeast Asia, Petinomys and Hylopetes from South and Southeast Asia, Petaurista from southern Eurasia and Japan, Pteromys from northern Eurasia and Japan, and Glaucomys from North America.
Species of flying squirrels examined in this study
From homogenates of muscle or skin tissues, total DNAs were extracted according to the phenol/proteinase K/sodium dodecyl sulfate method of Sambrook et al. (1989). A partial nucleotide sequence (1,068 bp) of the mitochondrial DNA (mtDNA) cytochrome b gene was amplified with polymerase chain reaction (PCR) using the following two primers: L14724 5′-GATATGAAAAACCATCGTTG-3′ and H15910 5′-GATTTTTGGTTTACAAGACCGAG-3′. Primer names correspond to the light (L) or heavy (H) strand and the 3′end-position of the primers in the human mtDNA sequence (Anderson et al., 1981). The former primer was synthesized from the sequence described by Kocher et al. (1989) and the latter was also done from the sequences reported by Oshida et al. (2000). The 50 μl of reaction mixture 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 unirs of rTaq DNA polymerase (Takara). Amplification was carried out for 35 cycles using the following cycle program: 94°C for 1 min, 53°C for 1 min, and 72°C for 2 min. The extention reaction was completed by incubation 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).
After sequence alignment was made using GeneWorks (Intelligenetics), the phylogenetic trees were reconstructed with the neighbor-joining (NJ) method (Saitou and Nei, 1987) in Clustal W (Thompson et al., 1994) and with the maximum likelihood (ML) method using DNAML in PHYLIP package program (Felsenstein, 1993). Numbers of nucleotide substitutions per site were estimated for multiple substitutions using Kimura's two-parameter method (Kimura, 1980). To estimate branching confidence, the bootstrap analyses (Felsenstein, 1985) were performed at 1,000 replications in NJ method and at 100 replications in ML method. Rattus norvegicus (Gadaleta et al., 1989) was employed as an out-group for reconstructing trees. In addition, the transversional divergence rate (0.5%/million years, Myr) at the third codon positions of mammalian cytochrome b gene (Irwin et al., 1991) was used for divergence time estimation.
RESULTS AND DISCUSSION
Phylogenetic relationships among flying squirrels
In the present study, the partial sequences (1,068 bp) of cytochrome b gene were determined for five species of flying squirrels: Belomys pearsonii, Glaucomys volans, Hylopetes phayrei, Petinomys setosus, and Pteromys momonga. Percentage differences corrected by Kimura's two-parameter model (1980) and numbers of transitions and transversions obtained from pairwise comparison are shown in Table 2. Data of transversional substitutions at the third codon positions are present in Table 3. Phylogenetic trees reconstructed using the NJ and the ML methods showed essentially identical branching patterns (Fig. 1a and b), although bootstrap values at the deeper nodes on both trees were not always high enough. The first dichotomy isolated Belomys from the other flying squirrels (bootstrap values: 64% for NJ tree and 42% for ML tree) and then two clusters were constructed: one cluster consists of Glaucomys, Hylopetes, and Petinomys (bootstrap values: 74% for NJ tree and 43% for ML tree) and another was formed by Petaurista and Pteromys (bootstrap values: 89% for NJ tree and 49% for ML tree). However, the low bootstrap values at the deeper nodes, especially in ML tree, indicated that the order of early divergence of flying squirrels was still unclear, suggesting their polychotomy consisting of five clusters: the genus Belomys, the genera Hylopetes and Petinomys, the genus Glaucomys, the genus Petaurista, and the genus Pteromys. Based on the fossil records, Mein (1970) and Black (1972) reported that flying squirrels were dominant in Europe in supplanting other squirrels during the Pliocene. Considering the abundant fossil records of flying squirrels from Europe and the unclear divergence order of flying squirrels in the present study, their divergence might have early occurred in the Eurasian Continent supplying ancestral stock.
Pairwise comparisons of cytochrome b nucleotide sequences (1,068 bp) between eleven flying squirrel specimens
Transversional substitutions at the third codon positions of cytochrome b gene between eleven flying squirrel specimens
Phylogenetic position of Belomys pearsonii
Belomys pearsonii is distributed in southern parts of the Eurasian Continent and Taiwan (Ellerman and Morrison-Scott, 1966; Mitchell, 1979; Lekagul and McNeely, 1988). This has been considered a monotypic genus (Corbet and Hill, 1991; Nowak, 1991), until recently. Whereas Corbet and Hill (1992) reclassified this species into the genus Trogopterus, renaming it Trogopterus pearsonii, the previous scientific name (Belomys pearsonii) seems to be generally adopted (e.g., Yu, 1996) and is used in the present paper. Thus the classification of Belomys pearsonii is still controversial. Phylogenetic trees of the present study suggest an early divergence of Belomys pearsonii from other flying squirrels, although the bootstrap values were not so high (64% for NJ tree and 42% for ML tree). This is the first time that the phylogenetic relationships of this rare species has been examined. There are no analyses of fossil records, protein data, and morphological characteristics with which to compare our molecular data.
It has been reported that Belomys pearsonii inhabits dense forests (Lekagul and McNeely, 1988) and may not be as expert at gliding as some of the other genera of flying squirrels (Nowak, 1991). This behavioral data and our molecular data suggest that Belomys pearsonii is a specialized species in the group of flying squirrels.
Phylogenetic relationships between Hylopetes and Petinomys
There are 11 species in the genus Hylopetes. Flying squirrels belonging to this genus are widely distributed in southern Eurasia (Lekagul and McNeely, 1988; Corbet and Hill, 1991; 1992). Petinomys includes eight species and its distribution is similar to that of Hylopetes. Threfore, the two flying squirrel genera show the sympatric distribution (Lekagul and McNeely, 1988; Corbet and Hill, 1991; 1992). In the present study, Hylopetes phayrei was closely related to Petinomys setosus with high bootstrap iterations (100% for both trees), suggessting the recent divergence between them. Corbet and Hill (1992) described that Petinomys is closely related to Hylopetes, distinguishing the two genera by number of septa in the auditory bullae. Our data strongly supports their hypothesis. Using the available substitution rate at the third codon positions estimated from the mammalian cytochrome b genes (Irwin et al., 1991), the divergence between Hylopetes and Petinomys could have occurred approximately 2.2 Myr ago, suggesting that two flying squirrel genera diverged in Southeast or South Asia during the Pleistocene. Since Black (1972) reported that the Miocene fossil remains of Petinomys had been found in Europe, Petinomys may be considered ancestral to Hylopetes, especially.
Phylogenetic position of Glaucomys
The genus Glaucomys, which includes two species (G. volans and G. sabrinus), is the only flying squirrel genus found in the New World (Corbet and Hill, 1991). Compared with the Old World flying squirrels, Glaucomys has been carefully studied in detail (e.g., Wells-Gosling, 1985). However, the classification and the phylogenetic relationships of two Glaucomys species are still controversial (Burt, 1960; Muul, 1968; Thorington Jr., 1996; Arbogast, 1999). Several questions still remain concerning the phylogenetic position of Glaucomys within flying squirrels and the phylogenetic relationships between New World and Old World flying squirrels. Here we suggest that Glaucomys is most closely related to Hylopetes and Petinomys from Southeast Asia (Fig. 1), although the bootstrap values were low (74% for NJ tree and 43% for ML tree). Based on the immunological data, Hight et al. (1974) reported that Petinomys has a slightly greater affinity to Glaucomys than either Petaurista or Pteromys. Our cytochrome b data support the relationship supposed from the immunological data, and indicates that the divergence between Glaucomys and Hylopetes and that between Glaucomys and Petinomys occurred approximately 28.6 Myr ago and 29.2 Myr ago, respectively. Recently, based on the paleontological data, Marincovich Jr. and Gladenkov (1999) reported that the first break of the Bering Strait occured between 4.8 and 7.3–7.4 Myr ago. Considering the divergence time between Glaucomys and Hylopetes and that between Glaucomys and Petinomys, Glaucomys might have diverged from other flying squirrel genera much earlier than the formation of the Bering Strait. After the Oligocene-Miocene radiation of flying squirrels in Europe, the ancestral stock of Glaucomys may have migrated to North America, and at the same time the ancestor of Hylopetes and Petinomys might have migrated to South and Southeast Asia.
Phylogenetic relationships between Petaurista and Pteromys
The genus Petaurista, which includes ten species, is distributed in the southern parts of the Eurasian Continent, on many islands of Southeast Asia, and main islands of Japan excluding Hokkaido island (Corbet and Hill, 1991; 1992). The phylogenetic relationships within this genus has been studied by Oshida et al. (1992, 1996, 2000) using the mitochondrial DNA and chromosome data. On the other hand, flying squirrels belonging to the genus Pteromys are only two species and are widely distributed throughout the northern parts to the central parts of the Eurasian Continent and main islands of Japan (Corbet and Hill, 1991; Nowak, 1991). Only few attempts (Yanagawa et al., 1996) have been made for elucidating the intrageneric phylogeny of Pteromys.
The NJ tree (Fig. 1a) clearly indicates that Petaurista is closely related to Pteromys with a high bootstrap iteration (89%). In addition, the ML tree (Fig. 1b) confirms this close relationships, although the bootstrap value is not so high (49%). The mitochondrial 12S rRNA sequences data by Oshida et al. (1996) suggested that Petaurista is closely related to Pteromys. From cytochrome b data of the present study, we estimated that the divergence between Petaurista and Pteromys occurred approximately 28.0–36.0 Myr ago. The present geographic distribution of Petaurista does not overlap with Pteromys, except in Japan. Pleistocene fossils of Petaurista have been recorded in Europe (Black, 1972), China (Young, 1934; Young and Liu, 1950; Hsu et al., 1957; Hu and Qi, 1978), and Japan (Hasegawa, 1966; Kowalski and Hasegawa, 1976; Kawamura, 1988). Accordingly, Petaurista may have been a dominant flying squirrel throughout the Eurasian Continent during the Pleistocene. Kawamura (1988) suggested that Petaurista may have expanded northward with the forests in the warmer periods of the middle Pleistocene. Pleistocene fossils of Pteromys have been found only from Japan (Kowalski and Hasegawa, 1976; Kawamura, 1988). Judging from the fossil records and the present distribution of these two genera, their biogeographic history could be described in the following manner. After the radiation in Europe during the Oligocene-Miocene, the ancestor of Petaurista might have occurred throughout the Eurasia until at least the middle Pleistocene. Its subsequent distribution was restricted to the southern parts of the Eurasian Continent by the southward shifting of its forest habitat. At the same time, the ancestor of Pteromys may have extended its distribution to northern and central parts of the Eurasia.
We would like to thank Mr. L-S. Tzen for supplying specimens, and Dr. C. L. Bridgman of the Department of Ecology and Evolutionary Biology, University of Tennessee, for her critical reading the manuscript. This study was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan.