Skip to main content Skip to main navigation menu Skip to site footer
Published: 2011-02-17

Inter- and intra-island divergence in Odorrana ishikawae (Anura, Ranidae) of the Ryukyu Archipelago of Japan, with description of a new species

3-6-15 Hikarigaoka, Munakata, Fukuoka 811-3403, Japan
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan
Section of Agriculture and Forest, Amami City Government, Amami, Kagoshima 894-0048, Japan
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan
Amphibia Odorrana splendida sp. nov. Odorrana ishikawae Ranidae Ryukyu Archipelago Japan


The endangered frog, Odorrana ishikawae (Anura, Ranidae), is a species endemic to the Amami and Okinawa Islands of the Ryukyu Archipelago, Japan. Segmentation of these islands has been considered to occur middle or upper Pleistocene. Our morphometric analyses revealed obvious differences between the Amami and Okinawa populations. Two distinct morphotypes were also recognized from the Amami Island (Amami common and Amami large types). Furthermore, the Amami and Okinawa populations could be distinguished clearly by coloration and dorsal tuberculation. Based on 16S rRNA gene data, the Okinawa and Amami populations were phylogenetically separated but the genetic divergence (1.44– 2.16%) was lower than the value suggested as species threshold in anurans (> 3% in 16S). Individuals of the Amami common and large types were nested within a single clade. Artificial hybridization experiments revealed normal hybrid viability between the two Amami types, with one exception. By contrast, between Okinawa females and two Amami type males, complete hybrid inviability was observed at early embryonic stages in the hybrids contrary to expectations from their low divergence in 16S. The reciprocal hybrids between two Amami type females and Okinawa males were viable, but spermatogenesis in the hybrid males showed some degree of abnormality. These results strongly indicate specific separation of the Amami population from the Okinawa population of O. ishikawae. Thus, we describe the Amami population as a new species, which is readily distinguishable from O. ishikawae by smaller ruggedly edged dorsal spots and an immaculate ventral surface.


  1. Berger, L. (1983) Western Palearctic water frogs (Amphibia, Ranidae): Systematics, genetics and population compositions. Experientia, 39, 127–130.

    Cai, H. -X., Che, J., Pang, J. -F., Zhao, E. -M. & Zhang, Y. -P. (2007) Paraphyly of Chinese Amolops (Anura, Ranidae) and phylogenetic position of the rare Chinese frog, Amolops tormotus. Zootaxa, 1531, 49–55.

    Cracraft, J. (1989) Speciation and its ontology: the empirical consequences of alternative species concepts for understanding patterns and processes of differentiation. In: Otte, D. & Endler, J. (Eds.), Speciation and its consequences. Sinauer Assoc., Sunderland, MA. pp. 28–59.

    de Queiroz, K. & Donoghue. M.J. (1990) Phylogenetic systematics or Nelson's version of cladistics? Cladistics, 6, 61–76.

    Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797.

    Frost, D.R. (2010) Amphibian Species of the World: an Online Reference. Version 5.4. Available at American Museum of Natural History, New York.

    Fouquet, A., Gilles, A., Vences, M., Marty, C., Blanc, M. & Gemmell, N.J. (2007) Underestimation of species richness in Neotropical frogs revealed by mtDNA analyses. PLoS One, 2, e1109.

    Futuyma, D.J. (2005) Evolution. Sinauer Assoc., Sunderland, MA. Gouy, M., Guindon, S. & Gascuel O. (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Molecular Biology and Evolution, 27, 221–224.

    Günther, R. (1979) General remarks on the evolutionary genetics of the European water frog complex. Mitteilungen aus dem Zoologischen Museum in Berlin, 55, 7–11.

    Hikida, T., Ota, H., Kuramoto M. & Toyama, M. (1989) Zoogeography of amphibians and reptiles in East Asia. In: Matsui, M., Hikida, T. & Goris, R. (Eds.), Current Herpetology in East Asia. Herpetological Society of Japan, Kyoto. pp. 278–281.

    Inger, R.F. (1947) Preliminary survey of the amphibians of the Riukiu Islands. Fieldiana: Zoology, 32, 295–352.

    Inger, R.F. (1950) Distribution and speciation of the amphibians of the Riu Kiu Islands. American Naturalist, 84, 95–115.

    IUCN. (2010) Red List of Threatened Species. Available at

    Jobb, G. (2008) TREEFINDER version of October 2008. Munich, Germany. Distributed by the author at

    Katsuren, S., Tanaka, S. & Ikehara, S. (1977) A brief observation on the breeding site and eggs of a frog, Rana ishikawae (Stejneger) in Okinawa Island. In: Ikehara, S. (Ed.), Ecological Studies of Nature Conservation of the Ryukyu Islands. III. University of Ryukyus, Naha, Okinawa. pp. 49–54.

    Kawamura, T. & Nishioka, M. (1986) Hybridization experiments among Rana lessonae, Rana ridibunda and Rana esculenta, with special reference to hybridogenesis. Scientific Report of the Laboratory for Amphibian Biology, Hiroshima University, 8, 117–271.

    Kotaki, M., Kurabayashi, A., Matsui, M., Kuramoto, M., Djong, T.H. & Sumida, M. (2010) Molecular phylogeny of the diversified frogs of genus Fejervarya (Anura: Dicroglossidae). Zoological Science, 27, 386–395.

    Kurabayashi, A., Yoshikawa, N., Sato, N., Hayashi, Y., Oumi, S., Fujii, T. & Sumida, M. (2010) Complete mitochondrial DNA sequence of the endangered frog Odorrana ishikawae (family Ranidae) and unexpected diversity of mt gene arrangements in ranids. Molecular Phylogenetics and Evolution, 56, 543–553.

    Kuramoto, M. (1972) Karyotypes of the six species of frogs (genus Rana) endemic to the Ryukyu Islands. Caryologia, 25, 547–559.

    Kuramoto, M. (1979) Distribution and isolation in the anurans of the Ryukyu Islands. Japanese Journal of Herpetology, 8, 8–21. (In Japanese with English abstract).

    Kuramoto, M. (1980) Acoustic characterization of some Japanese frogs (genus Rana). Bulletin of Fukuoka University of Education, Part III, 29, 93–97.

    Kuramoto, M. (1998) Spermatozoa of several frog species from Japan and adjacent regions. Japanese Journal of Herpetology, 17, 107–116.

    Maeda, N. & Matsui, M. (1999) Frogs and Toads of Japan, Revised ed. Bun-ichi Sogou Shuppan, Tokyo. 223 pp. (In Japanese with English summary).

    Matsui, M. (2005) Geographic diversities of Japanese amphibians. In: Matsuda, R. & Abe, H. (Eds.), Natural History of Faunal Geography. Hokkaido University Press, Sapporo, Japan. pp.63–77. (In Japanese).

    Matsui, M., Shimada, T., Ota, H. & Tanaka-Ueno, T. (2005) Multiple invasions of the Ryukyu Archipelago by Oriental frogs of the subgenus Odorrana with phylogenetic reassessment of the related subgenera of the genus Rana. Molecular Phylogenetics and Evolution, 37, 733–742.

    Mayden, R.L. (1997) A hierarchy of species concepts: the denouemant in the saga of the species problem. In: Claridge, M.F., Dawah, H.A. &Wilson, M.R. (Eds.), Species: the Units of Biodiversity. Chapman & Hall, London. pp. 381–424.

    Mayr, E. (1942) Systematics and the origin of species. Columbia University Press, New York.

    Moriya, K. (1960) Studies on the five races of the Japanese pond frog, Rana nigromaculata Hallowell. III. Sterility in interracial hybrids. Journal of Science of the Hiroshima University, Series B, Div. 1, 18, 125–156.

    Ota, H. (1998) Geographic patterns of endemism and speciation in amphibians and reptiles of the Ryukyu Archipelago, Japan, with special reference to their paleogeographical implications. Researches on Population Ecology, 40, 189–204.

    Rodriguez, F., Oliver, J.L., Marin, A. & Medina, J.R. (1990) The general stochastic model of nucleotide substitution. Journal of Theoretical Biology, 142, 485–501.

    Roe, B.A., Ma, D.P., Wilson, R.K. & Wong, J.F.H. (1985) The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. Journal of Biological Chemistry, 260, 9759–9774.

    Schmid, M., Olert, J. & Klett, C. (1979) Chromosome banding in Amphibia. III. Sex chromosomes in Triturus. Chromosoma, 71, 29–55.

    Seto, T., Utsunomiya, T. & Utsunomiya, Y. (1984) Interpopulation variation of the C-banded karyotypes in Rana ishikawae (Stejneger) from Okinawa-jima and Amami-Oshima in the Ryukyu Islands. Japanese Journal of Herpetology, 10, 67–72. (In Japanese with English summary).

    Stejneger, L. (1901) Diagnoses of eight new batrachians and reptiles from the Riu Kiu Archipelago. Proceedings of the Biological Society of Washington, 14, 189–191.

    Sumida, M. & Ishihara, T. (1997) Natural hybridization and introgression between Rana nigromaculata and Rana porosa porosa in central Japan. Amphibia-Reptilia, 18, 249–257.

    Sumida, M., Kondo, Y., Kanamori, T. & Nishioka, M. (2002) Inter- and intraspecific evolutionary relationships of the rice frog Rana limnocharis and the allied species R. cancrivora inferred from crossing experiments and mitochondrial DNA sequences of the 12S and 16S rRNA genes. Molecular Phylogenetics and Evolution, 25, 293–305.

    Swofford, D.L. (2003) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sunderland, Massachusetts, Sinauer Associates.

    Tanabe, A.S. (2007) Kakusan: a computer program to automate the selection of a nucleotide substitution model and the configuration of a mixed model on multilocus data. Molecular Ecology Notes, 7, 962–964.

    Utsunomiya, T. (1989) Five endemic frog species of the Ryukyu Archipelago. In: Matsui, M., Hikida, T. & Gorris, R. (Eds.), Current Herpetology in East Asia. Herpetological Society of Japan, Kyoto. pp. 199–204.

    Utsunomiya, Y. & Utsunomiya, T. (1983) On the eggs and larvae of frog species occurring in the Ryukyu Archipelago. Journal of Faculty of Applied Biolological Science, Hiroshima University, 22, 255–270. (In Japanese with English summary).

    Utsunomiya, Y., Utsunomiya, T. & Katsuren, S. (1979) Some ecological observations of Rana ishikawae, a rare frog endemic to the Ryukyu Islands. Proceedings of the Japan Academy, Series B, 55, 233–237.

    Vences, M., Thomas, M., van der Meijden, A., Chiari, Y. & Vieites, D.R. (2005) Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians. Frontiers in Zoology, 2, 5.

    Vieites, D.R., Wollenberg, K.C., Andreone, F., Köhler, J., Glaw, F. & Vences, M. (2009)Vast underestimation of Madagascar's biodiversity evidenced by an integrative amphibian inventory. Proceedings of the National Academy of Sciences of the United States of America, 106, 8267–8272.

    Zhao, E.-M. (1999) Distribution pattern of amphibians in temperate Eastern Asia. In: Duellman (Ed.), Patterns of Distribution of Amphibians. A Global Perspective. Johns Hopkins University Press, Baltimore. pp. 421–443.