Local Population Differentiation and Phylogenetic

Local Population Differentiation and Phylogenetic Relationships of Russian Brown Frog, Rana amurensis Inferred by Mitochon- drial Cytochrome b Gene Sequences (Amphibia, Ranidae)

TOMOKO TANAKA-UENO, MASAFUMI MATSUI, TAKANORI SATO, SEN TAKENAKA AND OSAMU TAKENAKA

Abstract: In order to assess local population differentiation and phylogenetic relationships of a Russian brown frog, Rana amurensis, the sequences of 587 base pairs of the mitochondrial cytochrome b genes are compared with seven species of Japanese and one species complex of Taiwanese brown frogs (R. pirica, R. ornativentris, R. dybowskii, R. japonica, R. okinavana, R. tagoi, R. tsushimensis, and the R. sauteri complex). Genetic differentiation between populations of R. amurensis from Sakhalin and the Maritime Territory was found to be minimal. The resultant phylogenetic tree indicates monophyly of brown frogs and earliest divergence of R. amurensis among all the brown frogs studied. For this reason, separation of R. amurensis from the R. japonica group is suggested, but separation of the R. sauteri complex as a distinct genus or subgenus Pseudorana is not supported. Key words: Brown frog; Cytochrome b; Phylogeny; Pseudorana; Rana amurensis; Russia

Rana amurensis Boulenger, 1886 occupies a ships of Japanese brown frogs have been report- very wide range, including western Siberia east ed by Nishioka et al. (1992) and Tanaka et al. to Sakhalin, northern and eastern Mongolia, (1996). In the present study, we first investigat- northeastern China and Korea, and regions ed the degree of genetic differentiation between north to bevond the Arctic Circle to 71°N populations of R, amurensis in Sakhalin and (Frost, 1985). In several previous studies, some continental Russia using the nucleotide sequence brown frog species were proven to be highly of mitochondrial DNA cytochrome b gene. differentiated genetically among conspecific Rana amurensis has 2n=26 chromosomes (Orlo- populations without showing marked morpho- va et al., 1977; Wu and Yin, 1983), but its logical variations (e. g., Sumida, 1996; Tanaka et phylogenetic relationships with other brown al., 1994, 1996, Tanaka-Ueno, Matsui, Sato et frogs are still poorly understood, and only two al., 1998). Because R. amurensis is noted for its studies based on the isozyme data mentioned immense distribution as mentioned above, the above are available (Nishioka et al., 1992; Green presence of considerable genetic differentiation and Borkin, 1993). In both of those studies, R. is expected in this species. However, the amurensis is inferred to be involved in very early populational genetic differentiation in R. divergence among the brown frogs with 2n=26 amurensis has been studied only from the elec- chromosomes. Thus, our second purpose was trophoretic analyses of allozymes and proteins to clarify the phylogenetic relationships of R. between the populations of the Amur Basin and amurensis with respect to seven species of brown Sakhalin Island (Green and Borkin, 1993) or frogs in Japan on the basis of sequence variation among the populations of north Mongolia, of cytochrome b. From our previous study northeastern China, and the Maritime Territory (Tanaka-Ueno, Matsui, Chen et al., 1998), the (Nishioka et al., 1992). The results reported by R. sauteri complex from Taiwan (Chou and Lin, these authors indicate absence of notable genetic 1997), which is occasionally assigned to a dis- differentiations among the populations studied. tinct genus (Fei et al., 1990) or subgenus Analyses of allozymes and DNA nucleotide (Dubois, 1992), has been suggested to form a sequence do not always give congruent results. sister group with some Japanese brown frogs. For example, discordant phylogenetic relation- Our third purpose was to examine how the relationships of these frogs change when R. Accepted 27 Dec. 1997 amurensis is added to them. 92 Jpn. J. Herpetol. 17 (3) 1998

FIG. 1. Aligned sequences of a 572-bp segment of the cytochrome b gene. Dots indicate identity with the sequence of Xen opus laevis. TANAKA-UENO ET AL.-RELATIONSHIPS OF BROWN FROGS 93

FIG. 2. A neighbor-joining tree rooted at the mid- point of the longest path (A), a maximum-likelihood tree (B), and a bootstrapped parsimony tree (C). Nodal values in A and C indicate percent support for branches in 1,000 bootstrap replicates.

method (Saitou and Nei, 1987), using the NJ MATERIALS AND METHODS procedure included in the PHYLIP package DNA was extracted from small amounts (less (Felsenstein, 1993), the maximum-likelihood than 50mg) of frozen liver, muscle, heart, or method using the DNAML procedure of eggs of a total of 12 specimens of R. amurensis PHYLIP, and the parsimony method using the from Russia and referential R. dybowskii from heuristic search algorithm in PAUP (Swofford, Tsushima (see appendix). 1993) with sequences added in a random order The methods used to extract, amplify, and se- for 1,000 repetitions. quence are as described previously (Tanaka et For comparisons, the published sequences of al., 1994, 1996). A part of the mtDNA cyto- seven species of Japanese and one species com- chrome b gene was amplified using the poly- plex of Taiwanese brown frogs (R. pirica, R. or- merase chain reaction (PCR) and approximately nativentris, R, japonica, R. okinavana, R. tagoi, 600 bP of this gene were sequenced. Primers R. tsushimensis, and the R. sauteri complex), newly designated for the amplification and se- and an outgroup taxon, R. catesbeiana were quencing are: L14731 (5'-GAAAAACTATCG- used (Tanaka-Ueno, Matsui, Chen et al., 1998). TTGTTATTCAACTA-3'), L14850 (5'-TCTCA- The published sequence of Xenopus laevis TCCTGATGAAACTTTGGCTC-3'), H15502 (Dunon-Bluteau et al., 1985) was also incorpo- (5'-GGATTAGCTGGTGTGAAATTGTCTGGG rated into the analysis as that for another out- -3'), and H15584 (5'-CCTAGTTTATTAGGGA- group. When a taxon showed some variation in TGGAGCGGAG-3'). The sequence numbering the nucleotide sequence, the most frequently ap- system followed that of the human sequence pearing sequence was considered as the (Anderson et al., 1981). representative sequence for the taxon. Three different methods were employed to in- fer relationships among taxa on the basis of se- RESULTS quence data obtained-the neighbor-joining Nucleotide sequence data of 587 by were con- 94 Jpn. J. Herpetol. 17(3) 1998 stantly obtained for all samples (Fig. 1). Of the (72.3% and 89.6% iterations, respectively). R. amurensis from Sakhalin, two samples from Slepikovskogo had an identical sequence, and DISCUSSION they exhibited a high nucleotide sequence Sequence similarities found between samples similarity of 99.7% with the sample from Aniva. of R. amurensis from Lazo and Sakhalin (98.5- Similarly, two samples from continental Lazo 99.0%) are higher than the values reported for had a high similarity of 99.7%. Between samples populations of Japanese R. tagoi (93.2-96.8%: from Sakhalin and Lazo, similarities varied Tanaka et al., 1994), and interpopulation genetic from 98.5 to 99.0%. variation in this species is judged to be very low. Brown frogs showed a monophyletic relation- Similar low genetic differentiation between ship with high bootstrap iterations (99.2% in populations was reported from the analyses of neighbor-joining and 97.6% in maximum-par- allozymes and proteins by Nishioka et al.(1992). simony analyses: Fig. 2A, C). Maximum-likeli- According to their report, Nei's (1975) genetic hood analysis also resulted in identical grouping distances among populations of R. amurensis (Fig. 2B). from northern Mongolia, the Maritime Territory Samples of R. amurensis from the continent of Russia, and northeastern China range from and Sakhalin were monophyletic with bootstrap 0.005 to 0.008, and are smaller than interpopu- iterationsof 100%in both the neighbor-joining lation distances among the Japanese R, japonica and maximum-parsimony trees. They exhibited that they studied. Green and Borkin (1993) also the earliestdivergence both in neighbor-joining analyzed allozymes and showed low genetic and maximum-likelihood trees,but this diver- differentiation between populations of R. gence was supported only in very low iterations amurensis from the Amur Basin (Khabarovsk) of 38.5% in the maximum-parsimony analysis. and Sakhalin. From these previous results of The order of divergence among the remaining allozyme analyses and our DNA analyses, genet- brown frogs was the same as that reported by ic variation appears to be low at least among Tanaka-Ueno, Matsui, Chen et al. (1998). In populations of R. amurensis occurring from the neighbor-joining tree (Fig. 2A), trichoto- Mongolia and northeastern China to the Far mous relationships were observed; Rana tagoi East Asia. Therefore, R. amurensis currently and the R. sauteri complex from Sanyi formed found in this region is considered to have the first cluster with relatively low iterations diverged in a short time rather recently. (59.9%). The second cluster was supported by However, the distribution of R. amurensis is high iterations of 92.1% and consisted of R. very wide, and several morphologically distinct okinavana and the subcluster containing R. subspecies, such as R. amurensis coreana from tsushimensis and the R. sauteri complex from Korea (Shannon, 1956), have been recognized in Wulai. The sister relationship of the latter sub- some parts of its species range, but these forms cluster was completely supported (itera- have been poorly studied. tions=100%). In the third cluster which was The Strait of Mamiya (Tatal) separating Sak- supported by 88.4% iterations, R. japonica was halin and the Maritime Territory of the conti- separated from the three species with 2n=24 nent with a depth of 25m, is estimated to have chromosomes. The latter group, supported by been about -30±5m deep about 8,500 years B. iterations of 92.5%, consisted of R. ornativen- P.,before the separation of the two areas started tris and the group of R. pirica and R. dybowskii (Ohshima,1990). From this geological infor- that was supported by 92.1% iterations. mation, the distribution of R, amurensis is in- The maximum-likelihood tree (Fig. 2B) slight- ferred to have been continuous in these areas ly differed from the neighbor-joining tree in that until very recently. Gene flow between the two the first and the second clusters of the neighbor- areas would have been interrupted by the forma- joining tree formed a group and split from the tion of the strait. On the other hand, R. third cluster. amurensis has never been found in Hokkaido in The maximum-parsimony analysis resulted in spite of its present wide distribution in northern a polytomous consensus tree encompassing six Asia. But another amphibian species, Salaman- clades (Fig. 2C). As noted above, nearly com- drella keyserlingii, similarly occurring in a wide plete sister relationships were supported in sam- range from Russia and northeastern China to ples of R. amurensis. Similarly, sister relation- Sakhalin (Bannikov et al., 1977), does occur in a ships in each of the second cluster and the sub- small area on Hokkaido (Nakamura and Ueno, cluster containing species with 24 chromosomes 1963). The formation of the Soya Strait, of the neighbor-joining tree were supported separating Hokkaido and Sakhalin, is consi- TANAKA-UENO ET AL.-RELATIONSHIPS OF BROWN FROGS 95 dered to date back to about 12,000 years B. P. that the ancestral form of R. amurensis diverged (Ohshima, 1990). From the pattern of the very early from the common ancestor of other present distribution, S. keyserlingii is thought to brown frogs. have invaded Hokkaido from the northern route In revising classification of the superfamily (Continent-Sakhalin-Hokkaido) before the for- Ranoidea, Dubois (1992) split brown frogs mation of the Soya Strait. On the other hand, chiefly on the basis of larval dentition. Accord- R. amurensis, now absent on Hokkaido, would ing to his thinking, the brown frogs studied in have migrated from the continent to Sakhalin the present survey are classified into the R. later than S. keyserlingii after formation of the japonica group (R. amurensis, R. japonica), R. Soya Strait, although the possibility of popula- temporaria group (R. okinavana, R. tsushimen- tion extinction of R. amurensis after its invasion sis), R. tagoi group (R. tagoi), and R. chen- of Hokkaido is not ruled out. sinensis group (R. pirica, R. dybowskii, R. or- Nakamura and Ueno (1963) suggested that the nativentris) of the subgenus Rana. Rana sauteri ancestral stock of Japanese R. tagoi occurs on is placed in a separate subgenus Pseudorana. the continent, and this ancestral stock implies R. However, R. amurensis cannot be placed either amurensis (Ueno, personal communication). in the R. japonica group or in any other groups, Later, Nishioka et al. (1992) speculated on the because, as shown above, the species is inferred order of divergence among the brown frogs from to have diverged earliest among all the brown East Asia. According to their theory, R. frogs of the subgenera Rana and Pseudorana, tsushimensis first diverged from the other spe- and is considered to have a sister relationship cies, followed by the separation of the remaining with them. forms into two groups, one with 2n=24 and From the study of Taiwanese and Japanese another with 2n=26 chromosomes. Rana brown frogs, we have already reject Dubois' amurensis is supposed to have diverged first (1992) idea regarding R. sauteri from Taiwan as among the latter group. On the other hand, constituting a distinct subgenus Pseudorana. Green and Borkin (1993) estimated that R. We suspected, instead, that the species belonged amurensis diverged after the divergence of two to the subgenus Rana (Tanaka-Ueno, Matsui, species of brown frogs from the Caucasus and Chen et al., 1998). The present results also in- before the divergence of the two groups of Eu- dicate a monophyly of the brown frogs including ropean and Asian brown frogs with a different R. sauteri. Further, the stock diverged earliest number of chromosomes. Although not sup- among the brown frogs studied was inferred to ported by maximum-parsimony analysis, the be the lineage not of R. sauteri, but of R. result of the present neighbor-joining analysis amurensis. From these considerations, R. sau- greatly differed from that of Nishioka et al. teri should not be regarded as representing a dis- (1992), but was consistent with that of Green tinct subgenus. The unique characteristics of R. and Borkin (1993). Namely, R. amurensis is in- sauteri that are divergent from other brown ferred to have diverged earliest among the brown frogs include complicate dentition and the ab- frogs studied, especially before the major dominal sucker of its larvae that are adapted for dichotomy of species with 2n=26 chromosomes life in swift streams (Kuramoto et al., 1984). and those with 2n=24 chromosomes. These characteristics, however, are found in het- This inference, coincidentally drawn from two erogeneous lineages of many genera and families independent studies-the allozymic analysis by of frogs (Duellman and Trueb, 1986), probably Green and Borkin (1993) and the nucleotide se- as results of convergent evolution. This con- quence analysis here presented, is further sideration seems to indicate the robustness of strengthened by the results of chromosome molecular approaches as used here in estimating banding analysis by Lee and Park (1986), in phylogenetic relationships at least among anuran which the karyotype of Korean R. dybowskii amphibians. with 24 chromosomes is inferred to have derived from that of R, amurensis with 26 chromo- Acknowledgments.-First of all, we would like to somes. In the neighbor-joining tree, the genetic thank the late Dr. A. M. Bassarukin for his help and distance between the lineage leading to R. hospitality during field trips in the former Soviet Un- amurensis and that to the other brown frogs was ion. For support in collecting sample materials we inferred to be longer than the distances among thank S. Tanabe and S. Nakabayashi. R. C. Goris lineages that are thought to have diverged early kindly corrected verbal errors. 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APPENDIX Rana dybowskii Gunther, 1876 (N=7): Mit- Material examined.-Sources of tissue sam- sushima-cho, Nagasaki (Tsushima, N=3: ples from a total of 12 frogs are stored at the KUHE 11278, 11279, 11596); Izuhara-cho, Graduate School of Human and Environmental Nagasaki (Tsushima, N=4: KUHE 11598, Studies, Kyoto University (KUHE). 16784, 16785, 16786). Rana amurensis Boulenger, 1886 (N=5):

要旨ミトコンドリアのチトクロームb遺伝子塩基配列から推定されるアムールアカガエルのロシア産個体群における分化と系統関係田中-上野寛子・松井正文・佐藤孝則・竹中践・竹中修アムールアカガエルのロシア産個体群におけから分離すべきだが,ザウターガエル複合種群る分化と系統関係を推定するため,ミトコンドをアカガエル属とは独立の属ないし,亜属とすリアのチトクロームb遺伝子587塩基対の配列べきではないと考えられた.

を日本産7種(エゾ,ヤマ,チョウセンヤマ, ニホン,リュウキュウ,ツシマの各アカガエル (606-8501京都市左京区吉田二本松町京都とタゴガエル)と台湾産のザウターガエル複合大学大学院人間・環境学研究科(田中-上野・種群を含むアカガエル類と比較した.アムール松井;田中-上野の現在の所属:060-0810札幌アカガエルのサハリン産と沿海州産の個体群間市北区北8条西7丁目北海道大学実験生物センには遺伝的分化がほとんど見られなかった.分ター)・632天理市杣之内町1050天理大学お子系統樹はアカガエル類の単系統性と,調べらやさと研究所(佐藤)・005-8601札幌市南区南れたアカガエル類の中でアムールアカガエルが沢5条1-1-1北海道東海大学教育開発センター最も早く分岐したことを示唆した.この結果か (竹中践)・484犬山市官林京都大学霊長類らアムールアカガエルをニホンアカガエル種群研究所遺伝子情報部門(竹中修))