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Type: Article
Published: 2022-08-08
Page range: 25-45
Abstract views: 2595
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Genetic and morphological variation analyses of Glandirana rugosa with description of a new species (Anura, Ranidae)

Department of Science Education, Aichi University of Education, 1 Hirosawa, Igaya, Kariya, Aichi 448–8542, JAPAN
Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, Kyoto 606–8501, JAPAN
Preservation and Research Center, City of Yokohama, 155-1 Asahi Ward, Yokohama 241-0804, JAPAN
Amphibian Research Center, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, JAPAN
Department of Science Education, Aichi University of Education, 1 Hirosawa, Igaya, Kariya, Aichi 448–8542, JAPAN. Takahama High School, 1-6-1 Hongo, Takahama, Aichi 444-1311, JAPAN
Conservation Genome Resource Bank for Korean Wildlife, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Seoul 08826, SOUTH KOREA
Kitakyushu Museum of Natural History & Human History, Higashida 2-4-1, Yahatahigashi, Kitakyushu, Fukuoka 805-0071, JAPAN
Amphibia eastern Japan Glandirana rugosa larval ventral glands mitochondrial DNA phylogeny nuclear DNA phylogeny


Glandirana rugosa is known to include several geographic groups differing in sex chromosomes, and has been proven to be paraphyletic in mitochondrial phylogeny with respect to G. susurra. By analyzing genetic and morphological variation in a large number of individuals of Glandirana, we studied their taxonomic relationships. A mitochondrial DNA phylogeny, with the G. tientaiensis as outgroup, revealed two major lineages containing respectively (1) the East group of G. rugosa, G. susurra, and the Central and Southeast-Kyushu groups of G. rugosa; and (2) G. emeljanovi, and the North and West groups of G. rugosa. In contrast, in a nuclear DNA phylogeny based on SNP data, lineages of (1) G. susurra and East group, and (2) the remaining groups of G. rugosa and G. emeljanovi, were split, indicating a distinct status of the East group among G. rugosa. In adult morphology, there were only minor differences between the East group and the remaining groups of G. rugosa, but in larvae, the East group had significantly more sparse skin glands than the others. The exact type locality of G. rugosa is most probably in western Japan, not including the range of the East group. From these results, we describe the East group as a new species, G. reliquia, distinct from the remaining groups of G. rugosa. The new species with sexually homomorphic chromosomes is thought to represent a basic stock of Japanese Glandirana, which existed far before G. rugosa originated.



  1. Ashizawa, K., Osawa, S. & Katsuno, T. (2013) Spawning site selection of Rana rugosa in paddy field at bottomland in hilly area. Papers on Environmental Information Science, 27, 33–36.
    Ballard, J.W.O. & Whitlock, M.C. (2004) The incomplete natural history of mitochondria. Molecular Ecology, 13, 729–744.
    Bolger, A.M., Lohse, M. & Usadel, B. (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30, 2114–2120.
    Castresana, J. (2000) Selection of conserved blocks from multiple alignment for their use in phylogenetic analysis. Molecular Biology and Evolution, 17, 540–552.
    Catchen, J., Amores, A., Hohenlohe, P., Cresko, W. & Postlethwait, J. (2011) Stacks: building and genotyping loci de novo from short-read sequences. G3: Genes, Genomes, Genetics, 1, 171–182.
    Chang, T.K. (1933) Two new amphibian records from Chekiang. Peking Natural History Bulletin, 8, 75–80.
    Dubois, A. (1992) Notes sur la classification des Ranidae (Amphibiens anoures). Bulletin Mensuel de la Société Linnéenne de Lyon, 61, 305–352.
    Earl, D.A. & von Holdt, B.M. (2012) STRUCTURE HARVESTER: A website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetics Resources, 4, 359–361.
    Evanno, G., Regnaut, S. & Goudet, J. (2005) Detecting the number of clusters of individuals using the software Structure: a simulation study. Molecular Ecology, 14, 2611–262.
    Fei, L., Ye, C.-Y. & Jiang, J.-P. (2010) Phylogenetic systematics of Ranidae. Herpetologica Sinica, 12, 1–43.
    Fei, L., Ye, C.-Y. & Jiang, J.-P. (2012) Colored Atlas of Chinese Amphibians and Their Distributions. Sichuan Publishing House of Science & Technology, Sichuan.
    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 (10), e1109.
    Frost, D.R., Grant, T., Faivovich, J.N., Bain, R.H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M. & Wheeler, W.C. (2006) The amphibian tree of life. Bulletin of the American Museum of Natural History, 297, 1–370.[0001:TATOL]2.0.CO;2
    Gasso Miracle, M.E., van den Hoek Ostende, L.W. & Arntzen, J.W. (2007) Type specimens of amphibians in the National Museum of Natural History, Leiden, The Netherlands. Zootaxa, 1482 (1), 25–68.
    Gosner, K. (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica, 16, 183–190.
    Hasegawa, Y., Ueda, H. & Sumida, M. (1999) Clinal geographic variation in the advertisement call of the wrinkled frog, Rana rugosa. Herpetologica, 55, 318–324
    Hillis, D.M. & Bull, J.J. (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology, 42, 182–192.
    Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754–755.
    Hurst, G.D.D. & Jiggins, F.M. (2005) Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts. Proceedings of the Royal Society B, 272, 1525–1534.
    Jombart, T. (2008) adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics, 24, 1403–1405.
    Kozlov, A.M., Darriba, D., Flouri, T., Morel, B. & Stamatakis, A. (2019) RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics, 35, 4453–4455.
    Leaché, A.D. & Reeder, T.W. (2002) Molecular systematics of the eastern fence lizard (Sceloporus undulatus): a comparison of parsimony, likelihood, and Bayesian approaches. Systematic Biology, 51, 44–68.
    Leaché, A.D., Banbury, B.L., Felsenstein, J., Nieto-Montes de Oca, A., & Stamatakis, A. (2015) Short tree, long tree, right tree, wrong tree: new acquisition bias corrections for inferring SNP phylogenies. Systematic Biology, 64, 1032–1047.
    Maeda, N. & Matsui, M. (1989) Frogs and Toads of Japan. Bun-ichi Sogo Shuppan, Tokyo.
    Malone, J.H. & Fontenot, B.E. (2008) Patterns of reproductive isolation in toads. PLoS ONE, 2 (10), e1109
    Matsui, M. (1984) Morphometric variation analyses and revision of the Japanese toads (Genus Bufo, Bufonidae). Contributions from the Biological Laboratory, Kyoto University, 26, 209–428.
    Matsui, M. (1997) Call characteristics of Malaysian Leptolalax with a description of two new species (Anura: Pelobatidae). Copeia, 1997, 158–165.
    Matsui, M. & Maeda, N. (2018) Encyclopedia of Japanese Frogs. Bun-ichi Sogo Shuppan, Tokyo, 271 pp.
    Matsui, M., Shimada, T. & Miura, I. (2021) Glandirana rugosa. The IUCN Red List of Threatened Species 2021: e.T58706A118983374.
    Matsui, M., Okawa, H., Nishikawa, K., Aoki, G., Eto, K., Yoshikawa, N., Tanabe, S., Misawa, Y. & Tominaga, A. (2019) Systematics of the widely distributed Japanese clouded salamander, Hynobius nebulosus (Amphibia: Caudata: Hynobiidae), and its closest relatives. Current Herpetology, 38, 32–90.
    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.
    Mawaribuchi, S., Ito, M., Ogata, M., Oota, H., Katsumura, T., Takamatsu, N. & Miura, I. (2016) Meiotic recombination counteracts male-biased mutation (male-driven evolution). Proceedings of the Royal Society B: Biological Sciences, 283, 20152691.
    Miura, I. (2007) An evolutionary witness: the frog Rana rugosa underwent change of heterogametic sex from XY male to ZW female. Sexual Development, 1, 323–331.
    Miura, I., Ohtani, H., Nakamura, M., Ichikawa, Y. & Saitoh, K. (1998) The origin and differentiation of the heteromorphic sex chromosomes Z, W, X and Y of the frog Rana rugosa, inferred from the sequences of a sex-linked gene, ADP/ATP translocase. Molecular Biology and Evolution, 15, 1612–1619.
    Nakamura, K. & Uéno, S. (1963) Japanese Reptiles and Amphibians in Colour. Hoikusha, Osaka.
    Nakamura, M., Nakamura, Y., Oike, A., Tojo, K., Suzuki, T. & Ito, E. (2022) A new frog species of the genus Glandirana from Southeastern Kyushu, Japan (Anura Ranidae). EC Veterinary Science, 7.1, 11–23.
    Nishioka, M., Hanada, H., Miura, I. & Ryuzaki, M. (1994) Four kinds of sex chromosomes in Rana rugosa. Scientific Report of the Laboratory for Amphibian Biology, Hiroshima University, 13, 1–34.
    Nishioka, M., Kodama, Y., Sumida, M. & Ryuzaki, M. (1993) Systematic evolution of 40 populations of Rana rugosa distributed in Japan elucidated by electrophoresis. Scientific Report of the Laboratory for Amphibian Biology, Hiroshima University, 12, 83–132.
    Ogata, M., Hasegawa, Y., Ohtani, H., Mineyama, M. & Miura, I. (2008) The ZZ/ZW sex-determining mechanism originated twice and independently during evolution of the frog, Rana rugosa. Heredity, 100, 92–99.
    Ogata, M., Lee, J.Y., Kim, S., Ohtani, H., Sekiya, K,, Igarashi, T,, Hasegawa, Y., Ichikawa, Y, & Miura, I. (2002). The prototype of sex chromosomes found in Korean populations of Rana rugosa. Cytogenetic and Genome Research, 99, 185–193.
    Ogata, M., Ohtani, H., Igarashi, T., Hasegawa, Y., Ichikawa, Y. & Miura, I. (2003) Change of the heterogametic sex from male to female in the frog. Genetics, 164, 613–620.
    Ogata, M., Suzuki, K., Yuasa, Y. & Miura, I. (2021) Sex chromosome evolution from a heteromorphic to a homomorphic system by inter-population hybridization in a frog. Philosophical Transactions of the Royal Society B, 376, 20200105.
    Oike, A., Watanabe, K., Min, M.-S., Tojo, K., Kumagai, M., Kimoto, Y., Yamashiro, T., Matsuo, T., Kodama, M., Nakamura, Y., Notsu, M., Tochimoto, T., Fujita, H., Ota, M., Ito, E., Yasumasu, S. & Nakamura, M. (2017) Origin of sex chromosomes in six groups of Rana rugosa frogs inferred from a sexlinked DNA marker. Journal of Experimental Zoology, 327A, 444–452.
    Oike, A., Mochizuki, M., Tojo, K., Matsuo, T., Nakamura, Y., Yasumasu, S., Ito, E., Arai, T. & Nakamura, M. (2020) A phylogenetically distinct group of Glandirana rugosa found in Kyushu, Japan. Zoological Science, 37, 193–202.
    Okada, Y. (1930) A Monograph of the Japanese Tailless Batrachians. Iwanami-shoten, Tokyo, 215 pp.
    Pritchard, J.K., Stephens, M. & Donnelly, P. (2000) Inference of population structure using multilocus genotype data. Genetics, 155, 945–959.
    Pyron, R.A. & Wiens, J.J. (2011) A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. Molecular Phylogenetics and Evolution, 61, 543–583.
    R Core Team. (2014) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna (Austria). Available from: (accessed 12 May 2022)
    Rice, W.R. (1989) Analyzing tables of statistical tests. Evolution, 43, 223–225.
    Sekiya, K., Miura, I. & Ogata, M. (2012) A new frog of the genus Rugosa from Sado Island, Japan (Anura, Ranidae). Zootaxa, 3575, 49–62.
    Sekiya, K., Ohtani, H., Ogata, M. & Miura, I. (2010) Phyletic diversity in the frog Rana rugosa (Anura: Ranidae) with special reference to a unique morphotype found from Sado Island, Japan. Current Herpetology, 29, 69–78.
    Shimada, T. (2015) A comparison of iris color pattern between Glandirana susurra and G. rugosa (Amphibia, Anura, Ranidae). Current Herpetology, 34, 80–84.
    Shimada, T. & Matsui, M. (2021) Comments on the article titled “Dr. Siebold and six species of Japanese frogs”, published in number 1, volume 2021 of Bulletin of the Herpetological Society of Japan. Bulletin of the Herpetological Society of Japan, 2021, 217-224.
    Shimada, T., Matsui, M., Yambun, P. & Sudin, A. (2011) A taxonomic study of Whitehead’s torrent frog, Meristogenys whiteheadi, with descriptions of two new species (Amphibia: Ranidae). Zoological Journal of the Linnean Society, 161, 157–183.
    Sokal, R.F. & Rohlf, F.J. (1981) Biometry, 2nd edition. W. H. Freeman and Co., San Francisco.
    Stamatakis, A. (2014) RAxML version 8: A tool for phylogenetic analysis and post analysis of large phylogenies. Bioinformatics, 30, 1312–1313.
    Stejneger, L. (1907) Herpetology of Japan and adjacent territory. Bulletin of United States Natural Museum, 58, 1–577.
    Sugawara, H., Naito, J., Iwata, T. & Nagano, M. (2022) Molecular phylogenetic and morphological problems of the Aki salamander Hynobius akinesis: description of two new species from Chugoku, Japan. Bulletin of the Kanagawa Prefectural Museum (Natural Science), (51), 35–46.
    Suyama, Y. & Matsuki, Y. (2015) MIG-seq: An effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform. Scientific Reports, 5, 16963.
    Tanabe, A.S. (2011) Kakusan4 and Aminosan: two programs for com- paring nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data. Molecular Ecology Resources, 11, 914–921.
    Temminck, C.J. & Schlegel, H. (1838) Fauna Japonica sive descriptio animalium, quae in itinere per Japonianum, jussu et auspiciis superiorum, qui summum in India Batava Imperium tenent, suscepto, annis 1823–1830 colleget, notis observationibus et adumbrationibus illustratis. Volume 3 (Chelonia, Ophidia, Sauria, Batrachia). J. G. Lalau, Leiden.
    Ting, H.P. & T’sai, M. (1979) A new species of frog (Rana minimus) from Fujian Province. Acta Zootaxonomica Sinica, 4, 297–300.
    Yamamoto, K. & Shimada, T. (2021) Geographic and seasonal variations on the density of dermal glands in larvae of Glandirana rugosa and G. susurra. Bulletin of the Herpetological Society of Japan, 2021, 96–97.