Molecular phylogeny and biogeography of woolly flying squirrel (Rodentia: Sciuridae), inferred from mitochondrial cytochrome b gene sequences

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 33 (2004) 735–744 www.elsevier.com/locate/ympev

Molecular phylogeny and biogeography of woolly flying squirrel (Rodentia: Sciuridae), inferred from mitochondrial cytochrome b gene sequences Fahong Yua,*,1, Farong Yub, Peter M. McGuirec, C. William Kilpatrickd, Junfeng Panga, Yingxiang Wanga, Shunqing Lue, Charles A. Woodsf,* a Kunming Institute of Zoology, Kunming, Yunnan 650223, China School of Life Sciences, Lanzhou University, Lanzhou 730000, China Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA d Department of Biology, University of Vermont, Burlington, VT 05405, USA e Chengdu Institute of Biology, Chengdu, Shichuan 610041, China f Florida Museum of Natural History, Gainesville, FL 32611, USA b

c

Received 18 February 2004; revised 12 May 2004 Available online 11 September 2004

Abstract To investigate the genetic diversity between the populations of woolly flying squirrels (Eupetaurus) from the eastern and western extremes of the Himalayas, partial mitochondrial cytochrome b gene sequences (390–810 bp) that were determined from the museum specimens were analyzed using maximum parsimony (MP) and maximum likelihood (ML) methods. The molecular data reveal that the two specimens that were collected in northwestern Yunnan (China) are members of the genus Eupetaurus. Reconstructed phylogenetic relationships show that the populations of Eupetaurus in the eastern and western extremes of the Himalayas are two distinct species with significant genetic differences (12%) and diverged about 10.8 million years ago. Eupetaurus is significantly different from Petaurista and Pteromys. The level of estimated pairwise-sequence divergence observed between Eupetaurus and Petaurista or Pteromys is greater than that observed between Eupetaurus and Trogopterus, Belomys, Glaucomys, or Hylopetes. Considering the divergence time of the two Eupetaurus groups, the glaciations and the uplift of the Himalayas and Qinghai-Tibet plateau during the Pliocene–Pleistocene period might be the major factors affecting the present distribution of Eupetaurus along the Himalayas.
2004 Elsevier Inc. All rights reserved. Keywords: Sciuridae; Flying squirrels; Eupetaurus; Cytochrome b gene; Phylogeny; Biogeography

1. Introduction Flying squirrels are found in both the Old and New World. Fourteen to 15 forms including 38–52 species have been given generic rank in recent years. The woolly flying squirrel (Eupetaurus), the most unusual and least *

Corresponding authors. Fax: 1-352-392-1445 (F. Yu). E-mail address: [email protected] (C.A. Woods). 1 Present address: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA. 1055-7903/$ - see front matter
2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.05.008

known genus in the world (Chakraborty and Agrawal, 1977; Zahler, 1996; Zahler and Woods, 1997), is considered among the most endangered mammals (Baillie and Groombridge, 1996) and is probably the most threatened of all flying squirrels. It is commonly considered as a monotypic genus with a single species, E. cinereus, and is distributed from Pakistan to southwestern China along the Himalayas (Corbet and Hill, 1992; Wang, 2002). Eupetaurus is a crucial genus in the phylogenetic study of flying squirrels. The cheekteeth of Eupetaurus are hypsodont and share many characteristics with

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rodents that have high-crowned teeth with flat surfaces, such as capromyids (hutias) from the West Indies, thryonomyids (cane rats) from Africa, and New World echimyids (spiny rats) (McKenna, 1962). These highly specialized grinding teeth allow it to live in relatively treeless rocky areas and possibly supplement its diet during winter months by eating some abrasive materials (Zahler and Woods, 1997). Since the dental structure is so divergent from other flying squirrels, Shaub (1958) and Grasse and Dekeyser (1955) placed Eupetaurus in its own rodent family, Eupetauridae. By comparing this dentition with the giant flying squirrel (Petaurista xanthotis), McKenna (1962) proposed that Eupetaurus is a very high-crown flying squirrel and demonstrably a petauristine sciurid on the basis of a large number of characters other than dentition, and returned it to the sciurid subfamily Petauristinae. Eupetaurus had been considered to be very rare or even extinct until it was recently rediscovered in Pakistan by Zahler (1996), which confirmed the present existence of the woolly flying squirrel. Eupetaurus was historically found in the areas of the Himalayas in which high mountain ranges meet the lowlands of Asia in a series of deep, narrow, and often xeric gorges. This region is described as the ‘‘trans-Himalayas.’’ The eastern trans-Himalayas includes southwestern Yunnan, eastern Tibet, southern Yunnan, northern Burma, and India, and the western trans-Himalayas consists of northwestern India, Pakistan, and Afghanistan. They form, in effect, the left and right sides of an open book, with the Tibet Plateau as the center. Currently, there are 15 Eupetaurus specimens available in museums and academic institutes, including 13 specimens in the British Museum of Natural History (London), the Leiden Museum (the Netherlands), the Bombay Natural History Society (India), the Indian Museum (Calcutta), and the Kunming Institute of Zoology (China) (Zahler and Woods, 1997), and two new specimens from Pakistan in the Florida Museum of Natural History (USA). Most specimens are from the general region of Gilgit, the area of the confluence of the Himalayan, Karakoram, and Hindu Kush mountain ranges in northern Pakistan. All specimens were identified as the same species E. cinereus based on pelage characteristics and dental structures. However, it is impossible to establish with certainty that all specimens are the same species, because the majority of the specimens available were collected at the beginning of the last century and some specimens are incomplete. The collecting sites of some specimens are not conclusively associated with individual specimens because the data recorded were based solely on the description made by dealers. In addition, observations in the wild have been precluded by the rarity of Eupetaurus populations. The paucity of Eupetaurus specimens in collections and questionable records have hampered taxonomic and phylo-

genetic studies. The phylogenetic status of Eupetaurus and the phylogenetic relationships between Eupetaurus and other flying squirrels are not yet well understood. In this study, Eupetaurus specimens from different localities were compared by analyzing molecular data from the mitochondrial cytochrome b gene using parsimony and likelihood methods. Here we discuss the taxonomic status of the Eupetaurus populations in Pakistan and southwestern China to determine how much variety exists within the genus Eupetaurus along its extensive distribution. Petaurista, Pteromys, Trogopterus, and Belomys, which are closely related to Eupetaurus (McKenna, 1962; Mein, 1970; Mercer and Roth, 2003; Thorington and Darrow, 2000), were included for reconstructing the phylogeny of Eupetaurus.

2. Materials and methods 2.1. Materials The molecular study was based on partial mitochondrial cytochrome b sequences that were determined from museum specimens, including hair and skin. Of the 15 known Eupetaurus specimens, only seven specimens have reliable collection data associated with them. The remaining specimens were described based mainly on comments from either medicinal dealers or intermediaries who shipped or purchased the specimens from different locations. All Eupetaurus specimens used in this study were confirmed by checking pelage characteristics and/or dental structures. Only the specimens that are in good condition and were recorded by collectors with certainty were used for molecular analysis (Table 1 and Fig. 1). Flying squirrels are a well-supported monophyletic group and could be separated as two major groups (Mercer and Roth, 2003; Thorington and Darrow, 2000). Pteromys and Petaurista share similar morphological characteristics with Eupetaurus (McKenna, 1962; Mein, 1970; Thorington and Darrow, 2000), but Eupetaurus is closely related to Aeromys, Pteromys, Belomys, and Trogopterus according to the combined molecular data of the nuclear gene coding for interphotoreceptor retinoid-binding protein (IRBP) and mitochondrial 12S and 16S ribosomal DNA (Mercer and Roth, 2003). Although we were not able to include Aeromys in our analyses because of its rarity in collections, several other members of each major group were used to reconstruct phylogenetic trees, including Pteromys, Trogopterus, Belomys, Petaurista, Glaucomys, and Hylopetes (Table 1). 2.2. DNA extraction, PCR amplification, and sequencing Because museum skins and hair were primarily used for the sampling, the chance of cross contamination is

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Table 1 Specimens of Eupetaurus and other flying squirrels examined in this study Species

Code

Collecting locality

Museum ida

Accession No.

Eupetaurus cinereus

ECL ECK1 ECK2 ECB ECD ECF1 ECF2 PPF PPB PPM PTX PVO TRX GVO HPH BPE SCA TAH

Tibet, China Yunnan, China Yunnan, China Gilgit, Pakistan Sai Valley, Pakistan Gilgit, Pakistan Gilgit, Pakistan Gilgit, Pakistan Malaysia Southern China Gansu, China Japan Yunnan, China Tennessee, USA Thailand Taiwan Brazil New Mexico, USA

LM19524 KIZ73372 KIZ73921 BNHS7107 BNHS7108 UF26583 UF28620 USMNH353202

AY331673 AY331671 AY331672 AY331674 AY331668 AY331669 AY331670 AY615268 AF063067 AB023909 AY615269 AB023910 AY526354 AF063066 AB030259 AB030262 AJ389530 AF147643

Petaurista petaurista

Petaurista xanthotis Pteromys volans Trogopterus xanthipes Glaucomys volans Hylopetes phayrei Belomys pearsonii Sciurus aestuans Tamiasciurus hudsonicus

NPIB984 KIZ630784

a LM, Leiden Museum, Leiden; KIZ, Kunming Institute of Zoology, Kunming; BNHS, Bombay Natural History Society, Bombay; UF, Florida Museum of Natural History, Gainesville; USMNH, National Museum of Natural History, Washington, DC; and NPIB, Northwest Plateau Institute of Biology, Xining.

Fig. 1. The sampling sites of this study.

high and potentially more difficult to recognize. In this study, these low-copy/low-quality samples were extracted and amplified in a dedicated room. Negative

controls for DNA extractions and PCRs were performed to determine potential contamination in DNA preparation and amplification.

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Total DNA of samples was extracted from dry skins using the DNeasy tissue kit (Qiagen, Valencia, CA) and the protocol for animal tissue was recommended by the manufacturer. Two primer pairs were used to amplify a partial mitochondrial cytochrome b gene sequence (390– 1041 bp) with a polymerase chain reaction (PCR): H15915 and L14841 (Kocher et al., 1989), and H15915 and L14725 (50 -CGA AGC TTG ATA TGA AAA ACC ATC GTT G-30 ). The 25 ll PCR mixture contained 2 ll (10 ng) of genomic DNA, 1.5 ll of each primer (25 pmol), 2.5 ll of 10· PCR buffer (100 mM Tris– HCl, pH 8.3, at 25 C, 500 mM KCl, 0.01% gelatin), 4.0 ll of dNTPs (10 mmol), 3.0 ll MgCl2 (25 mmol), and 0.2 U Taq polymerase (Sigma Chemical, St. Louis, MO). Amplification was carried out using the following program: 5 min at 94 C, followed by 38 cycles of 1 min at 94 C, 1 min and 5 s at 55 C, and 1 min at 72 C. PCR amplification was terminated with a post-extension of 5 min at 72 C. PCR products were purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA). Sequencing was performed with automated DNA sequencers. The variant sites of sequences were rechecked by comparing the four-color electromorph of sequencing data against the computer results. Amino acid translations of the sequences were compared with those of Petaurista in GenBank to ensure that there were no frame shifts or premature stop codons. All sequences determined in this study were deposited in GenBank (Table 1). 2.3. Phylogenetic analysis Cladistic analyses were performed by using the PAUP (version 4.0b10) program package (Swofford, 2002). Quantitative pairwise comparisons between taxa were made for the partial cytochrome b gene sequences (444–810 bp). To obtain reliable estimates of the evolutionary distances between two nucleotide sequences compared, the percentages of sequence divergence between taxa were corrected by KimuraÕs two-parameter model (Kimura, 1980). This method has the merit of incorporating the possibility that ÔtransitionÕ type substitutions may occur more frequently than ÔtransversionÕ type substitutions. The divergence time between taxa was estimated based on 444–810 bp according to the transversional substitution rate at the third codon positions of mammalian cytochrome b genes proposed by Irwin et al. (1991), which was 0.5% per million years. Arbogast (1999) and Oshida et al. (2000a,b) used this rate to estimate the divergence of flying squirrels. In phylogenetic analysis, each nucleotide was treated as an unordered character with four alternative states and gaps were considered as missing data. Maximum parsimony (MP) and maximum likelihood (ML) methods were applied for reconstructing phylogenetic rela-

tionships based on 810 bp of mitochondrial cytochrome b genes. In MP analysis, the phylogenetic tree was generated with unweighted maximum parsimony. A heuristic search algorithm with TBR branch swapping and 100 random-addition sequences was performed. Bootstrap values were derived from 1000 replicates to quantify the relative support for branches of the inferred phylogenetic tree. In ML analysis, the Modeltest 3.06 (Posada and Crandall, 1998) selected GTR + I + G as the most appropriate substitution model for sequence analysis. A heuristic ML search algorithm with 10 random additional sequences and TBR branch swapping reconstructed the ML tree. Tree reliability was assessed using 500 bootstrap replicates with the ÔFaststepÕ search method. Molecular characters indicate that flying squirrels are a well-supported monophyletic group and show a sistergroup relationship with the tribes Sciurini and Tamiasciurini (Herron et al., 2004; Mercer and Roth, 2003; Steppan et al., 2004). In order to increase the reliability of tree topology, the tree squirrels Sciurus aestuans and Tamiasciurus hudsonicus were selected as the outgroup taxa for phylogenetic reconstruction.

3. Results Since some specimens were collected more than 100 years ago and are in very poor condition, only partial sequences (390–810 bp) of the cytochrome b genes were successfully determined for some Eupetaurus and Petaurista samples. Table 2 presents the results of pairwise comparisons based on the partial cytochrome b sequences (444–810 bp) after adjusting for missing data (gap/ambiguities) (Swofford, 2002). The genetic variations obtained from the quantitative pairwise comparison separated the populations of Eupetaurus in Pakistan and southwestern China as two distinct groups. The sequence difference between these two groups was about 12%. The level of estimated pairwise-sequence divergence observed between Eupetaurus and Petaurista (18.7–21.6%) was greater than that observed between Eupetaurus and Trogopterus, Belomys, Glaucomys, or Hylopetes (16.9–19.2%). Pteromys differed from other flying squirrels with 19.3–23.6% sequence variations. Phylogenetic trees constructed under MP and ML criteria generated essentially identical branching orders (Figs. 2 and 3), although the bootstrap iterations at some deeper nodes were not very high. Of the 810 bp of the cytochrome b gene examined in the MP analysis, 254 characters were parsimony informative and 72 are non-informative. In both trees, all flying squirrels analyzed formed one tight clade (93–98% bootstrap values) with Pteromys as the basal branch. The second dichotomy isolated the Petaurista group from the remaining

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Table 2 Percentage differences of Eupetaurus and other flying squirrels based on the pairwise comparisons of the cytochrome b gene (444–810 bp)

Eupetaurus

Petaurista

Hylopetes Trogopterus Pteromys Belomys Glaucomys

ECD

ECK1

ECL

PPF

PPM

PPP

ECD ECK1 ECL

13.8 11.7

3.8

PPF PPM PPP PTX

18.7 20.7 20.3 19.8

19.2 21.2 21.6 19.4

18.9 21.6 20.5 18.7

13.7 14.2 12.1

0.5 14.8

12.5

HPH TRX PVO BPE GVO

18.9 19.2 19.3 19.0 18.7

17.3 19.1 18.5 18.6 18.6

16.9 18.7 19.4 18.5 18.4

19.4 19.3 19.0 20.1 18.4

21.4 22.4 20.5 22.6 20.5

21.5 21.8 20.5 22.6 20.6

PTX

HPH

TRX

PVO

BPE

16.2 20.5 20.9 19.9 16.3

20.1 23.8 21.0 18.2

19.4 14.8 19.4

23.6 21.8

21.2

Fig. 2. Phylogenetic relationships of Eupetaurus and other flying squirrels using the maximum parsimony (MP) method. The numbers above branches indicate the bootstrap values derived from 1000 replicates.

flying squirrels, but the bootstrap values to support this branching pattern were only moderate, with 64% for the MP tree and 65% for the ML tree. The Eupe-

taurus populations formed two distinct groups with high bootstrap iterations (>95% for both trees) in both analyses presented: one group consisting of the samples

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Fig. 3. Phylogenetic relationships of Eupetaurus and other flying squirrels constructed using the maximum likelihood method. Likelihood ratio tests select GTR + I + G (
ln likelihood = 5514.0975) as the most appropriate model for subsequent analyses. Settings for the model were as follows: Rmatrix = (1.8119, 6.2114, 3.3646, 0.1027, and 9.5921); base frequencies = (A = 0.2834, C = 0.3109, G = 0.1272, and T = 0.2785); proportion of invariant sites = 0.4930; and Gamma distribution shape parameter = 1.3793. The numbers above branches indicate the bootstrap values derived from 500 replicates.

from Tibet (ECL) and Yunnan (ECK1 and ECK2) of China, and another group including samples from Chitral (ECB) and Gilgit (ECD, ECF1, and ECF2) of Pakistan. This separation is concordant with pairwise comparison. In consistent with the genetic variations observed in the pairwise comparisons, Trogopterus and Belomys were held together with 90–95% bootstrap support, while the phylogenetic positions of Glaucomys and Hylopetes had moderate support (70–73% bootstrap values). The Belomys–Trogopterus clade showed a closed phylogenetic relationship with Eupetaurus, but this tie was obtained with only 54–72% bootstrap support.

Table 3 presents the estimated divergence times of Eupetaurus and other flying squirrels, based on the transversional substitution rate at the third codon positions of 444–810 bp of cytochrome b genes. The divergence of the eastern and the western trans-Himalayan Eupetaurus populations could have occurred about 10.8 mya (million years ago). Eupetaurus might have diverged from Petaurista (29.2–32.2 mya) much earlier than from Hylopetes and Glaucomys (19.8– 20.0 mya). The divergence time between Trogopterus and Eupetaurus or Petaurista was estimated to be approximately 37.0 mya, while Pteromys diverged from other flying squirrels approximately 27.6– 37.8 mya.

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Table 3 Estimated divergence times of Eupetaurus and other flying squirrels based on a rate of divergence for the third codon positions of mammalian cytochrome b genes of ca. 0.5% · 106 years

Eupetaurus Petaurista Hylopetes Trogopterus Pteromys Belomys Glaucomys

ECD ECK1 PPF PTK HPH TRX PVO BPE GVO

ECD

ECK1

PPM

PTK

HPH

TRX

PVO

BPE

10.8 30.8 32.2 19.8 37.0 27.6 27.6 20.0

29.2 30.8 19.8 37.0 28.6 27.6 20.0

10.8 31.4 37.0 30.4 35.4 29.8

29.2 37.0 29.6 27.6 29.2

29.2 37.8 29.2 28.6

30.8 24.4 24.6

37.0 29.8

36.0

4. Discussion 4.1. Taxonomic implications On the basis of the pelage color, hair density, and structures of feet, the two specimens collected in the deep gorge country of southwestern Yunnan near the Burma and Tibet borders were identified as Eupetaurus (Corbet and Hill, 1992; Wang and Yang, 1986; Zahler and Woods, 1997). The molecular data of this study strongly support this identification. The genetic difference of the Eupetaurus populations in northwestern Yunnan and Tibet (3.8%) is less than the interspecific differences of cytochrome b sequences in rodents (Bradley and Baker, 2001), including squirrels (Arbogast, 1999; Oshida and Masuda, 2000), and, therefore, these two populations are the same species. Eupetaurus in Pakistan is currently limited to the region of the Sai Valley in the central Indus River Valley near Nanga Parbat, the most westerly main massif in the Himalayan Range. It lives in caves of high alpine zones that are characterized as high, cold desert dominated by Artemisia and Juniperus above 2000 m, and apparently shows quite unique ecological adaptations for surviving in regions that are inhospitable to any other flying squirrels (Zahler and Woods, 1997). In these regions, E. cinereus, P. petaurista, and Eoglaucomys fimbriatus occur sympatrically in the Himalayan temperate forest with a mixture of deciduous and coniferous tree species (Roberts, 1997). A similar sympatric distribution of E. cinereus, P. petaurista, and Hylopetes alboniger exists in southwestern China, where the two Eupetaurus skins were collected. In northwestern Yunnan, the elevation ranges from 1500 to 3000 m, and the habitat is mountainous with coniferous forest, such as Yunnan pine, spruce, and Juniperus, which provides an optimum habitat for Eupetaurus. The similar habitat of the Eupetaurus populations in the eastern and western transHimalayas also confirms the presence of Eupetaurus in southwestern China. However, patterns of mtDNA variation observed in this study suggest that the current taxonomic classifica-

tion may not be an accurate reflection of evolutionary relationships within Eupetaurus. The two Eupetaurus populations are significantly different (11.7–13.8%) (Table 2) and are differentiated as two distinct clades in both MP and ML analyses with strong support (Figs. 2 and 3). Because genetic distance values of cytochrome b gene sequences >11% were considered to be indicative of specific recognition in rodents (Bradley and Baker, 2001), the Eupetaurus populations in the western (Pakistan) and eastern (China) trans-Himalayas can be considered to be two distinct species. Their genetic variations correspond to the geographic distances between sampling localities (Fig. 1). The transversional substitution rate at the third codon positions shows that the divergence of the two populations occurred approximately 10.8 mya (Table 3), implying that this differentiation into two Eupetaurus groups is most likely to be the result of isolation by the uplift of the Himalayas and the Qinghai-Tibet plateau during the Pliocene–Pleistocene period, which resulted in speciation. 4.2. Phylogenetic relationships between Eupetaurus and other flying squirrels Flying squirrels are monophyletic and were distinguished as two major groups (Mercer and Roth, 2003; Thorington and Darrow, 2000). This view has led to the widespread acceptance of this relationship, which in turn has had a tremendous influence on the interpretation of flying squirrel evolution in genetics and morphology. The topology of flying squirrels in the present study is extremely well established, with 93– 98% bootstrap support for placing S. aestuans and T. hudsonicus together as the outgroup taxa. The morphological differences between Eupetaurus and other flying squirrels are primarily in dental structures. Eupetaurus has high-crowned teeth and survives in the restricted areas that appear to meet their unique habitat requirements. It is morphologically convergent with capromyids as well as cane rats and New World spiny rats, rather than with other petauristines. A good ecomorph of Eupetaurus is Plagiodontia aedium from

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Hispaniola, which lives in rock crevices and caves at high elevation (3000 m). Both forms probably feed on similar abrasive materials given their similar modifications to the masticatory apparatus (Woods and Howland, 1979). Thomas (1888) believed that woolly flying squirrels fed mainly on lichens, mosses, and other plants associated with rocky areas. Local people in Pakistan believe that Eupetaurus feeds on seeds, needles, and bark of conifers, spruce buds, and some abrasive materials (Zahler and Woods, 1997). These data imply that the unique dental structures of Eupetaurus might be due to isolation in marginal habitat and the strong competitive pressure from P. petaurista, Eo. fimbriatus (Zahler and Woods, 1997) or H. alboniger for their sympatric distribution. For example, Petaurista feeds mainly on leaves and pads all the year round and flowers, various seeds, and fruits in autumn or spring (Kawamichi, 1997), including walnuts, acorns, and even cultivated corn, as I observed in the field in Gongshan, China, in September. Petaurista and Pteromys are considered to be the closest living relatives of Eupetaurus in morphology (McKenna, 1962; Mein, 1970; Thorington and Darrow, 2000), but the sequence-based phylogeny does not support both the conventional assumption and the more recent comparative morphological studies. Our molecular data show convincingly that the genetic discrepancies between Eupetaurus and Petaurista or Pteromys are not consistent with the morphological conclusions. Eupetaurus and Petaurista formed two distinct clades, and all Petaurista species were clustered together with a strong bootstrap support. The level of estimated pairwise-sequence divergence between Eupetaurus and Petaurista is greater than that observed between Eupetaurus and Trogopterus, Belomys, Glaucomys, or Hylopetes. According to McKenna (1962), the differentiation of the recent genus Eupetaurus from a Petaurista-like sciurid provided a significant parallel to the derivation of various high-crowned rodents from sciurid and paramyid stocks in the early and middle Tertiary. P. xanthotis is a Chinese endemic species that occurs from 2000 m in northwestern China to 3300 m in southwestern China and has semi-hypsodont molariform teeth. It shares similar dental structures with Eupetaurus (McKenna, 1962), but its phylogeny has not been studied in detail. The major differences between P. xanthotis and Eupetaurus are the result either of the increased height of crown in the molars of Eupetaurus or of a few minor changes in molar crown pattern acquired by P. xanthotis. However, the findings of the present study reveal that P. xanthotis diverged from Petaurista about 10.8 mya, while Eupetaurus diverged from Petaurista-stock approximately 29.2 mya, much earlier than the formation of P. xanthotis. The similar hypsodont teeth of Eupetaurus and P. xanthotis might be convergent adaptation to similar feeding habits. The adaptive shift of the feeding mechanism of Eupetaurus from Petaurista is analogous to the

shifts that led to the distinctive morphology of the dentition of beavers, mylagaulids, eutypomyids, and numerous other high-crown rodents (McKenna, 1962). Pteromys shares similar morphological characteristics with Eupetaurus, such as the morphology of the pisiform bone and the insertion of the tibio-carpalis muscle, two important phylogenetic indicators (Thorington and Darrow, 2000). However, our molecular analyses show that Pteromys diverged from other flying squirrels approximately 27.6–36.0 mya (Table 3) and is distantly related to Eupetaurus, with 19.2–19.5% genetic variation (Table 2). Pteromys is morphologically different from Petaurista (Johnson-Murray, 1977; Thorington and Darrow, 2000), but the molecular analyses suggest that Pteromys and Petaurista are closely related to each other and show a sisterhood relationship (Herron et al., 2004; Oshida et al., 1996). Our results strongly support their hypotheses. Also, it is noteworthy that in all analyses Pteromys is consistently basal to other flying squirrels, in agreement with the recent findings of Herron et al. (2004), but inconsistent with the results of Oshida et al. (2000b) and Mercer and Roth (2003). These discrepancies suggest that the phylogenetic topology of flying squirrels may be different when different outgroup or genes were used for phylogenetic reconstruction. Trogopterus lives in subtropical forests at elevations of 1300–2750 m (Nowak, 1999), while Belomys inhabits dense, temperate, broad-leaved forests from 1500 to 2400 m in elevation (Mitchell, 1979) and shows special morphological characteristics (Nowak, 1999). The sequence variation between Eupetaurus and Trogopterus or Belomys is less than that between Eupetaurus and Petaurista or Pteromys (Table 2). The phylogenetic trees constructed with MP and ML methods consistently included Belomys and Trogopterus as the nearest relatives of Eupetaurus, although there was overall moderate bootstrap support for this arrangement (Figs. 2 and 3). Agreement of molecular data and morphological data implies that Belomys and Trogopterus share a common ancestry. Our results support the sisterhood of Eupetaurus and the Belomys–Trogopterus clade, but point out that the relationship is not as strong as is often assumed. Glaucomys is the only flying squirrel found in the New World and widely inhabits temperate and subtropical forests in North and Central America. Although questions still remain concerning the phylogenetic relationships between New World and Old World flying squirrels, as a group, Glaucomys and Hylopetes are moderately well-supported (70–73% bootstrap). The grouping of Glaucomys and Hylopetes observed in this study is in consistent with what has been observed by Mercer and Roth (2003). Considering the molecular analyses made by Oshida et al. (2000b) and Herron et al. (2004), it is well established that the Glaucomys–Hylopetes clade is specifically allied with the Belomys–Trogopterus clade. However, the tie between these two

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clades is not universally clear-cut because they were clustered into different groups based on the morphological data (Thorington and Darrow, 2000) and the molecular data (Mercer and Roth, 2003). Eupetaurus is more closely related to Belomys, Trogopterus, Glaucomys, and Hylopetes over to Petaurista and Pteromys (Table 2), but the divergence times within these groups are rather complex. For example, the estimated divergence times show that Eupetaurus diverged from Trogopterus (37.0 mya), much earlier than from Belomys (27.0 mya), and that Eupetaurus diverged from Hylopetes (20.0 mya) earlier than Glaucomys diverged from Hylopetes (29.6 mya). These inconsistent results suggest that the divergence estimation should be interpreted with caution, as they are based on calibration derived from partial cytochrome b sequences. The phylogenetic relationship between Eupetaurus and Trogopterus, and that between Eupetaurus and the Glaucomys–Hylopetes clade are probably more complex than indicated by our present knowledge, and could only be illuminated by further morphological and molecular studies. 4.3. Biogeography of Eupetaurus Geological changes have strongly affected the evolution and distribution of mammals by creating dispersal barriers and corridors. Both morphological and molecular data support flying squirrels a monophyletic group (Herron et al., 2004; Mercer and Roth, 2003; Steppan et al., 2004; Thorington and Darrow, 2000), but it is as yet difficult to confidently date divergence events within flying squirrels due to the highly fragmented fossil record. Considering the early fossil remains of flying squirrels found in Europe (Black, 1972; Mein, 1970) and the hypothesis about the radiation of flying squirrels made by Oshida et al. (2000a,b), the present geographical distributions of flying squirrels might have been associated with the recent tectonic movements during Pliocene–Pleistocene. In the Pliocene–Pleistocene period, the uprising of the Himalayas helped cause the diversification of climate and became an important regulator of the Asian environment (Amano and Taira, 1992; An et al., 2001; Wang, 1984; Xu, 1981; Zheng et al., 2000). As a result, several distinct topographic regions were determined by drainage patterns and the parallel mountain chains in both the western and eastern trans-Himalayan regions, such as the Indus, Yangtze, Salaween, and Mekong river systems. After the Oligocene–Miocene radiation in Europe, flying squirrels migrated towards the south. The ancestral stock of Glaucomys may have migrated to North America, and at the same time the ancestors of Hylopetes, Eupetaurus, Petaurista, and Pteromys may have migrated towards the south. The Eupetaurus-stock migrated to its current geographical distribution when Petaurista was a dominant

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flying squirrel throughout the Eurasian Continent during the Pleistocene (Oshida et al., 2000b). Before the glacial eustacy, eastern and western trans-Himalayan Eupetaurus diverged from the ancestral Eupetaurus and migrated independently to their present localities after the closure of the Tethys Sea at the end of Miocene. During the glacial and inter-glacial periods of the Pliocene, they adapted to the cold habitat of mountain valleys. In the Pleistocene, the glaciations and the further uplift of the Himalayas and Qinghai-Tibet plateau (Amano and Taira, 1992; Xu, 1981; Zheng et al., 2000) led to southwestern China, and possibly northern Pakistan, becoming refuges for Eupetaurus and other specialized mammals. The fact that the eastern and western trans-Himalyan Eupetaurus populations are likely to have diverged after the closure of the Tethyan seaway suggests that the dispersal hypothesis seems more appropriate to explain present-day distribution patterns of Eupetaurus. At the same time, with the retreating of glaciers and the further uplift of the Himalayas, some species of flying squirrels may have extended its distribution to northern or central parts of the Eurasia, such as the ancestor of Pteromys (Oshida et al., 2000b) and P. xanthotis, and some were restricted to the southern parts of the Eurasian continent (i.e., Hylopetes). This inference implies that the divergence of the eastern and western Eupetaurus appears to be subject to the glacial events and the uplift of the Himalayas and Qinghai-Tibet plateau due to tectonic activities of the Cenozoic.

Acknowledgments We thank the Florida Museum of Natural History (Gainesville, USA), the National Museum of Natural History (Washington, DC, USA), the Kunming Institute of Zoology (Kunming, China), and the Northwestern Institute of Biology (Qinghai, China) for supplying tissue samples. We thank Ginger Clark, Joel Ernst (University of Florida), Laurie Wilkins, Candace McCaffery (Florida Museum of Natural History), and M. Tian (Kunming Institute of Zoology, China) for their unselfish help and invaluable suggestions on the phylogenetic analysis. This study was partially supported by a grant from the Laboratory of Cellular and Molecular Evolution of Kunming Institute of Zoology and the key program of the Chinese Academy of Sciences (KSCX2-109) and a grant from the National Natural Science Foundation of China (NSFC 30370194).

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