Molecular Phylogeny of Japanese Leporidae, the Amami Rabbit
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Genes Genet. Syst. (2002) 77, p. 107–116 Molecular phylogeny of Japanese Leporidae, the Amami rabbit Pentalagus furnessi, the Japanese hare Lepus brachyurus, and the mountain hare Lepus timidus, inferred from mitochondrial DNA sequences Fumio Yamada1*, Mika Takaki2 and Hitoshi Suzuki2 1Wildlife Ecology Laboratory, Forestry and Forest Products Research Institute, PO Box 16, Tsukuba-Norin, Ibaraki 305-8687, Japan 2Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan (Received 8 December 2001, accepted 12 February 2002) We determined mitochondrial 12S ribosomal RNA (rRNA) and cytochrome b (cyt b) gene sequences in three leporid species of Japan, the Amami rabbit Pentalagus furnessi from the Ryukyu Islands, the Japanese hare Lepus brachyurus from Honshu, and a Japanese form of the mountain hare Lepus timidus ainu from Hokkaido. We compared the sequences with those of other taxa of leporids avail- able in databases. Phylogenetic trees of the 12S rRNA gene sequences indicated that the lineage of P. furnessi diversified during the generic radiation of the lep- orids at an ancient time, which was estimated to have been the middle Miocene. Cyt-b gene trees revealed that the lineage of L. brachyurus branched off at an early stage in the speciation of Lepus, probably at the beginning of the Pliocene. The cyt b sequences of L. t. ainu were somewhat distinct from those of continental conspecific populations; this lineage divergence is likely to have occurred during the middle or late Pleistocene. The results show that the three regions of the Japanese archipelago, Ryukyu, Honshu-Shikoku-Kyushu, and Hokkaido, now preserve their own leporid taxa, each with a different extent of genetic endemicity. It is possible that the zoogeographic traits of the Japanese leporids are a consequence of the evolutionary dynamics of leporids in East Asia, in that the radiation centers of leporids are likely to have shifted from tropical, through temperate, to arctic zones. conservation biology. INTRODUCTION The family Leporidae (Lagomorpha, Mammalia) is Japanese mammals, especially small species, reflect a composed of 53 species of 11 extant genera (Chapman and considerable amount of endemicity from a taxonomic Flux, 1990). Nine of the genera, however, are mono- viewpoint. Many are endemic species, and some are typic, as in the case of Pentalagus (Amami rabbit) and classified as endemic genera. Meanwhile, through Romerolagus (volcano rabbit), or poorly speciated, as recent progress made in molecular phylogeny, high levels in the case of Pronolagus (rock hare), with three of endemicity have been demonstrated at the molecular species. These lineages are thought to retain primitive level in many taxonomic groups, such as rodents (e.g., morphological characters. In contrast, Lepus (jack- Serizawa et al., 2000; Suzuki et al., 2000), moles rabbits and hares) and Sylvilagus (cottontails) are spe- (Tsuchiya et al., 2000), and mustelids (Hosoda et al., cies-rich genera, comprising 29 and 13 species, 2000). The specific properties of the fauna of Japanese respectively, and are thought to be advanced genera on terrestrial mammals have begun to be understood in the basis of their morphology (Dawson, 1981; Angermann the context of the evolution of mammals in East et al., 1990; Chapman and Flux, 1990). Asia. Japanese taxa of leporids (rabbits and hares), how- The Japanese leporids comprise three taxa: the Amami ever, have not yet been explained, in spite of their contro- rabbit Pentalagus furnessi from the Amami Islands versial status in taxonomy and their importance in (Amamioshima and Tokunoshima islands, in the Ryukyu Islands in southernmost Japan), the Japanese hare L. Edited by Toshihiko Shiroishi brachyurus from the mainland (Honshu, Kyushu, and * Corresponding author. E-mail: [email protected] Shikoku), and the mountain hare L. timidus ainu from 108 F. YAMADA et al. Hokkaido. Phylogenetic information will be useful for occurred during the early stages of speciation within the conservation of these taxa, especially for P. furnessi, Lepus (Halanych et al., 1999). In addition, Halanych which is categorized as endangered (IUCN, 2000). The et al. (1999) used cyt-b gene sequences to examine status of this species is very serious; its habitat has intraspecific relationships in the most variable species of undergone considerable fragmentation and the size of the Lepus, L. timidus. They found that the species L. timi- population has greatly declined (2700–6500 rabbits in dus, L. arcticus, and L. othus form a clade, and that L. 1995, Sugimura et al., 2000; Yamada et al., 2000). timidus displays clear geographic subdivisions. Pentalagus furnessi is considered one of the most prim- The objective of this study was to elucidate the phylo- itive Leporidae (Corbet, 1983; Matsuzaki et al., 1989). It genetic relationships of the three taxa of Japanese lep- has been stated that the Japanese taxon Pentalagus is orids in relation to other leporid lineages of the world, most similar to the African taxon Pronolagus in morpho- based on mitochondrial DNA (mtDNA) sequences. We logical characteristics, and that they evolved from the examined the sequence variation of the mitochondrial common ancestor Pliopentalagus (Fejfar, 1961; Hibbart, 12S rRNA and cyt b genes in the three Japanese species 1963; Daxner and Fejfar, 1967; Dawson, 1981; Tomida, of leporids and compared them with those of other lago- 1997). The mainland taxon L. brachyurus was once morph lineages available in databases. regarded as the same species as that of the continent, L. mandshuricus, but is now classified as a separate species (Angermann et al., 1990; Hoffmann 1993). The MATERIALS AND METHODS Hokkaido form of L. timidus is often treated as a subspe- cies (L. t. ainu) distinct from those of Sakhalin and the Materials All three species of Japanese leporids were continent (Ellerman and Morrison-Scott, 1951; Corbet, examined in this study (Table 1). Tissue samples of P. 1978; Flux and Angermann, 1990). furnessi, which is one of the Natural Monuments of Mitochondrial DNA has been used to examine the evo- Japan, were collected after obtaining permission from the lutionary relationships of leporid genera. The generic Environment Agency and the Agency for Cultural Affairs relationships among seven leporid taxa, Brachylagus, of Japan. Bunolagus, Lepus, Oryctolagus, Pronolagus, Romero- lagus, and Sylvilagus, have been investigated using the PCR and sequencing Polymerase chain reactions variation in the mitochondrial 12S ribosomal RNA (PCRs) and direct sequencing were performed according (rRNA) gene, which evolves at a relatively slow rate to the methods described by Suzuki et al. (1997, (Halanych and Robinson 1999; Surridge et al., 1999). It 2000). A DNA fragment covering the entire target gene has been suggested that a rapid radiation occurred in six region was first amplified in 20-µl reaction mixtures con- genera, and that Pronolagus branched off slightly earlier taining 10 mM Tris (pH 8.3), 50 mM KCl, 0.01% gelatin, from the main leporid current (Halanych and Robinson 0.1% Triton X-100, 2.5 mM MgCl2, each dNTP at 0.2 mM, 1999). The 12S sequences of Nesolagus have been each primer at 0.05 mM (1 pmol of each primer per reac- reported by Surridge et al. (1999) following their discov- tion), 0.5 units of Amplitaq DNA polymerase (Perkin ery of a new species (Nesolagus sp.) from Vietnam that is Elmer), and 0.1–0.5 µg of total genomic DNA as template, related to the Sumatran rabbit, Nesolagus netscheri. with an automated thermal cycler (model TP400; Takara, The interspecies relationships of Lepus have been Japan). The thermal cycling parameters were 35 cycles examined using mitochondrial cytochrome b (cyt b) gene of denaturation at 94°C for 0.5 min, annealing at 50°C for sequences (Halanych et al., 1999; Halanych and Robin- 0.5 min, and extension at 60°C for 0.5 min. A 0.5-µl ali- son, 1999), and revealed several major lineages of Lepus quot of each reaction mixture after PCR was then used as spread across North America, Europe, Asia and template for the second PCR in a 20-µl reaction mixture Africa. These analyses suggest that a rapid radiation with the same reagents and under the same conditions as Table 1. Japanese lagomorphs analyzed in this study Taxon Common name N Individual code Collection locality Pentalagus furnessi Amami rabbit 3 Pfur_Amami1 Sumiyo, Kagoshima, Japan (28° 19´ N, 129° 22´ E) Pfur_Amami2 Sumiyo, Kagoshima, Japan (28° 19´ N, 129° 22´ E) Pfur_Amami3 Uken, Kagoshima, Japan (28° 14´ N, 129° 20´ E) Lepus brachyurus Japanese hare 2 Lbra_Honshu1 Ushiku, Ibaraki, Japan (35° 58´ N, 140° 11´ E) Lbra_Honshu2 Ami, Ibaraki, Japan (35° 58´ N, 140° 11´ E) Lepus timidus ainu mountain hare 2 Ltim_Hokkaido1 Sapporo, Hokkaido, Japan (42° 59´ N, 141° 25´ E) Ltim_Hokkaido2 Sapporo, Hokkaido, Japan (42° 59´ N, 141° 25´ E) Molecular phylogeny of Japanese leporids 109 for the first PCR, apart from the primer pairs and the (MP) analyses using PAUP* 4.0b8 (Swofford, 2001). In concentration of MgCl2 (1.25 mM). To construct the the NJ tree, sequence divergences were calculated by primer pair for the second PCR, the sequence of an 18- Kimura’s (1980) two-parameter method. The MP analyses meric dye-labeled primer, M13RP1(R) or -21M13(U), from were performed using a priori weighting (transversions : Applied Biosystems, was attached to the 5’ end of each of transitions = 2:1). A heuristic search was performed with the gene-specific primers. Both strands of the second 10 random addition replicates and the tree bisection and PCR product were directly sequenced by an automated reconnection option (TBR). The statistical confidence method using a DyePrimer Cycle Sequencing Kit and an of the sequence clusters in the NJ and MP trees was automated sequencer (model 373A; Applied Biosystems). evaluated by bootstrap percentages derived from 500 A 0.9-kb fragment of the 12S rRNA gene was amplified replications.