Abstract
To clarify the taxonomic status of salamanders from Tsushima Islands, Japan, we examined two species of salamanders, Hynobius tsuensis and Hynobius sp. from Tsushima Islands, and compared them with H. nebulosus from Kyushu. We found that the three taxa differ from each other in nuclear DNA, adult morphology, and egg-sac shape, and consider them to be independent species. Based on the investigation of type specimens of the synonyms, we identified Hynobius sp. as H. tagoi Dunn, 1923 and redescribe it herein. Also, we designate a neotype for H. tsuensis and redescribe H. tsuensis. The distribution areas of the two species partly overlap but they were distinguishable by their body colorations: H. tsuensis has uniformly blackish tail sides and a distinct yellow stripe on the upper tail edge; whereas H. tagoi has brown tail sides with numerous dark stippling and without a distinct yellow stripe on the upper tail edge.
References
- Abe, Y. (1922) On Ambystomidae from Japan. Zoological Magazine, 34, 328–332.
- AmphibiaWeb (2023) Available from: https://amphibiaweb.org/lists/Hynobiidae.shtml (accessed 28 February 2023)
- Aoki, G., Matsui, M. & Nishikawa, K. (2013) Mitochondrial cytochrome b phylogeny and historical biogeography of the Tohoku salamander, Hynobius lichenatus (Amphibia, Caudata). Zoological Science, 30, 167–173. https://doi.org/10.2108/zsj.30.167
- Dunn, E.R. (1923a) New species of Hynobius from Japan. Proceedings of the California Academy of Sciences, 4th Series, 12, 27–29.
- Dunn, E.R. (1923b) The salamanders of the family Hynobiidae. Proceedings of the American Academy of Arts and Sciences, 58, 445–523. https://doi.org/10.2307/20026019
- Dunn, O.J. (1964) Multiple comparisons using rank sums. Technometrics, 6, 241–252. https://doi.org/10.1080/00401706.1964.10490181
- Earl, D.A. & vonHoldt, B.M. (2012) STRUCTURE HARVESTER: A website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetics Resources, 4, 359–361. https://doi.org/10.1007/s12686-011-9548-7
- Evanno, G., Regnaut, S. & Goudet, J. (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology, 14, 2611–2620. https://doi.org/10.1111/j.1365-294X.2005.02553.x
- Hammer, Ø., Harper, D.A. & Ryan, P.D. (2001) PAST: paleontological statistics software package for education and data analysis. Palaeontologia Electronica, 4, 1–9.
- Hara, S. & Nishikawa, K. (2020) Intraspecific variation in the external and skeletal morphology of Hynobius setouchi Matsui, Okawa, Tanabe et Misawa, 2019 (Amphibia: Urodela: Hynobiidae). Zoological Science, 37, 346–357. https://doi.org/10.2108/zs190155
- Hasegawa, M., Kishino, H. & Yano, T. (1985) Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution, 22, 160–174. https://doi.org/10.1007/BF02101694
- Huelsenbeck, J.P., Ronquist, F., Nielsen, R. & Bollback, J.P. (2001) Bayesian inference of phylogeny and its impact on evolutionary biology. Science, 294, 2310–2314. https://doi.org/10.1126/science.1065889
- Ito, J., Baba, A., Niwa, K. & Nishikawa, K. (2023) A note on underground egg sacs of the Tsushima salamander, Hynobius tsuensis (Caudata: Hynobiidae). Herpetology Notes, 16, 211–212.
- Iwasawa, H. & Yamashita, K. (1991) Normal stages of development of a hynobiid salamander, Hynobius nigrescens Stejneger. Japanese Journal of Herpetology, 14, 39–62. https://doi.org/10.5358/hsj1972.14.2_39
- Jakobsson, M. & Rosenberg, N.A. (2007) CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics, 23, 1801–1806. https://doi.org/10.1093/bioinformatics/btm233
- Kanamori, S., Nishikawa, K., Matsui, M. & Tanabe, S. (2022) A new species of lotic breeding salamander (Amphibia, Caudata, Hynobiidae) from Shikoku, Japan. PeerJ, 10, e13891. https://doi.org/10.7717/peerj.13891
- Kramer, C.Y. (1956) Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12, 307–310. https://doi.org/10.2307/3001469
- Kumar, S., Stecher, G. & Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870–1874. https://doi.org/10.1093/molbev/msw054
- Lee, B.H., Kim, J.Y., Song, S., Hur, J.M., Cho, J.Y. & Park, Y.C. (2011) The complete mitochondrial genome sequence of the Kori salamander Hynobius yangi (Caudata: Hynobiidae). Mitochondrial DNA, 22, 168–170. https://doi.org/10.3109/19401736.2011.636437
- Matsui, M., Misawa, Y. & Nishikawa, K. (2009) Morphological variation in a Japanese salamander, Hynobius kimurae (Amphibia, Caudata). Zoological Science, 26, 87–95. https://doi.org/10.2108/zsj.26.87
- Matsui, M., Misawa, Y., Nishikawa, K. & Shimada, T. (2017) A new species of lentic breeding salamander (Amphibia, Caudata) from central Japan. Current Herpetology, 36, 116–126. https://doi.org/10.5358/hsj.36.116
- 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. https://doi.org/10.5358/hsj.38.32
- Matsui, M., Tominaga, A., Hayashi, T., Misawa, Y. & Tanabe, S. (2007) Phylogenetic relationships and phylogeography of Hynobius tokyoensis (Amphibia: Caudata) using complete sequences of cytochrome b and control region genes of mitochondrial DNA. Molecular Phylogenetics and Evolution, 44, 204–216. https://doi.org/10.1016/j.ympev.2006.11.031
- Matsui, M., Tominaga, A., Liu, W.Z. & Tanaka-Ueno, T. (2008a) Reduced genetic variation in the Japanese giant salamander, Andrias japonicus (Amphibia: Caudata). Molecular Phylogenetics and Evolution, 49, 318–326. https://doi.org/10.1016/j.ympev.2008.07.020
- Matsui, M., Yoshikawa, N., Tominaga, A., Sato, T., Takenaka, S., Tanabe, S., Nishikawa, K. & Nakabayashi, S. (2008b) Phylogenetic relationships of two Salamandrella species as revealed by mitochondrial DNA and allozyme variation (Amphibia: Caudata: Hynobiidae). Molecular Phylogenetics and Evolution, 48, 84–93. https://doi.org/10.1016/j.ympev.2008.04.010
- Ministry of the Environment Government of Japan (2020) Red List of Ministry of the Environment Government of Japan, 2020. Available from: https://www.env.go.jp/content/900515981.pdf (accessed 23 April 2023)
- Misawa, Y. (1989) The method of counting costal grooves. In: Matsui, M., Hikida, T. & Goris, R.C. (Eds.), Current Herpetology in East Asia. Herpetological Society of Japan, Kyoto, pp. 129–134.
- Mori, T. (1928) On amphibians and reptiles of Quelpart Island. Chosen Natural History Society Journal, 6, 47–52.
- Nagaoka, S. (2001) Northern and Central Part of Kyushu Island: Tsushima Island and Tsushima Strait. In: Machida, Y., Ohta, Y., Kawana, T., Moriwaki, H. & Nagaoka, S. (Eds.), The Topography of Japan. 7. Kyushu Island and Nansei Islands. University of Tokyo Press, Tokyo, pp. 106–109.
- Nagasaki Prefecture (2022) Prefectural Red List of Nagasaki, 2022. Available from: https://www.pref.nagasaki.jp/shared/uploads/2022/03/1648644060-2.pdf (accessed 23 April 2023)
- Naito, J.I. & Kambayashi, C. (2022) Distribution pattern of Shikoku clawed salamander, Onychodactylus kinneburi, in the Western Chugoku Mountains. Biogeography, 24, 79–83. https://doi.org/10.11358/biogeo.24.79
- Nakamura, K. & Uéno, S.-I. (1963) Japanese Reptiles and Amphibians in Colour. Hoikusha, Osaka, 214 pp.
- Nishikawa, K. (2009) The first specimen of Karsenia koreana (Caudata: Plethodontidae) collected 34 years before its description. Current Herpetology, 28, 27–28. https://doi.org/10.3105/018.028.0104
- Nishikawa, K., Jiang, J.P., Matsui, M., Mo, Y.M., Chen, X.H., Kim, J.B., Tominaga, A. & Yoshikawa, N. (2010) Invalidity of Hynobius yunanicus and molecular phylogeny of Hynobius salamander from continental China (Urodela, Hynobiidae). Zootaxa, 2426 (1), 65–67. https://doi.org/10.11646/zootaxa.2426.1.5
- Nishikawa, K. & Matsui, M. (2014) Three new species of the salamander genus Hynobius (Amphibia, Urodela, Hynobiidae) from Kyushu, Japan. Zootaxa, 3852 (2), 203–226. https://doi.org/10.11646/zootaxa.3852.2.3
- Nishikawa, K., Matsui, M., Tanabe, S. & Sato, S. (2007) Morphological and allozymic variation in Hynobius boulengeri and H. stejnegeri (Amphibia: Urodela: Hynobiidae). Zoological Science, 24, 752–766. https://doi.org/10.2108/zsj.24.752
- Niwa, K., Kuro-o, M. & Nishikawa, K. (2021) Discovery of two lineages of Hynobius tsuensis (Amphibia, Caudata) endemic to Tsushima Island, Japan. Zoological Science, 38, 259–266. https://doi.org/10.2108/zs200149
- Niwa, K., Yoshikawa, N., Nishikawa, K. & Matsui, M. (2022a) Development of eight microsatellite markers for the Tsushima salamander, Hynobius tsuensis, and its close relatives (Amphibia: Caudata) with preliminary analysis of genetic differentiation. Current Herpetology, 41, 125–131. https://doi.org/10.5358/hsj.41.125
- Niwa, K., Tran, D.V. & Nishikawa, K. (2022b) Differentiated historical demography and ecological niche forming present distribution and genetic structure in coexisting two salamanders (Amphibia, Urodela, Hynobiidae) in a small island, Japan. PeerJ, 10, e13202. https://doi.org/10.7717/peerj.13202
- Okada, Y. (1934) A contribution toward a check list of the Urodeles of Japan. Copeia, 1, 16–19. https://doi.org/10.2307/1436428
- Okada, Y. (1935) Summary of taxonomy and distribution of Japanese salamanders. Zoological Magazine, 47, 575–588.
- Oyama, J. (1930) Hynobius tagoi and Hynobius tsuensis. Bulletin of the Biogeographical Society of Japan, 2, 21–35. https://doi.org/10.2307/1436476
- Pritchard, J.K., Stephens, M. & Donnelly, P. (2000) Inference of population structure using multilocus genotype data. Genetics, 155, 945–959. https://doi.org/10.1093/genetics/155.2.945
- Rambaut, A., Suchard, M., Xie, W. & Drummond, A. (2014) Tracer. Version 1.6. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh. [program]
- Raymond, M. & Rousset, F. (1995) An exact test for population differentiation. Evolution, 49, 1280–1283. https://doi.org/10.2307/2410454
- Rice, W.R. (1989) Analyzing tables of statistical tests. Evolution, 43, 223–225. https://doi.org/10.2307/2409177
- Ronquist, F., Teslenko, M., Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539–542. https://doi.org/10.1093/sysbio/sys029
- Rosenberg, N.A. (2004) DISTRUCT: a program for the graphical display of population structure. Molecular Ecology Notes, 4, 137–138. https://doi.org/10.1046/j.1471-8286.2003.00566.x
- Rousset, F. (2008) GENEPOP’007: A complete reimplementation of the genepop software for Windows and Linux. Molecular Ecology Resources, 8, 103–106. https://doi.org/10.1111/j.1471-8286.2007.01931.x
- Sato, I. (1934) Taxonomical study of the salamanders of the nebulosus group, with special reference to the morphology of the skull. Zoological Magazine, 46, 214–224.
- Sato, I. (1937) A synopsis of the family Hynobiidae of Japan. Bulletin of the Biogeographical Society of Japan, 7, 31–45.
- Sato, I. (1943) A Monograph of the Tailed Batrachians of Japan. Nippon Shuppan-sha, Osaka, 520 pp.
- Sparreboom, M. (2014) Salamanders of the Old World. KNNV Publishing, Zeist, 431 pp. https://doi.org/10.1163/9789004285620_017
- Stejneger, L. (1907) Herpetology of Japan and adjacent territory. Bulletin of the United States National Museum, 58, 1–577. https://doi.org/10.5479/si.03629236.58.i
- Sugawara, H., Watabe, T., Yoshikawa, T. & Nagano, M. (2018) Morphological and molecular analyses of Hynobius dunni reveal a new species from Shikoku, Japan. Herpetologica, 74, 159–168. https://doi.org/10.1655/Herpetologica-D-17-00002.1
- Sugawara, H., Iwata, T., Yamashita, H. & Nagano, M. (2021) Taxonomic reassessment of the Izumo lineage of Hynobius utsunomiyaorum: Description of a new species from Chugoku, Japan. Animals, 11, 2187. https://doi.org/10.3390/ani11082187
- Sugawara, H., Naito, J., Iwata, T. & Nagano, M. (2022a) Molecular phylogenetic and morphological problems of the Aki salamander Hynobius akiensis: description of two new species from Chugoku, Japan. Bulletin of the Kanagawa Prefectural Museum (Natural Science), 51, 35–46. https://doi.org/10.32225/bkpmnh.2022.51_35
- Sugawara, H., Fujitani, T., Seguchi, S., Sawahata, T. & Nagano, M. (2022b) Taxonomic re-examination of the Yamato salamander Hynobius vandenburghi: description of a new species from Central Honshu, Japan. Bulletin of the Kanagawa Prefectural Museum (Natural Science), 51, 47–59. https://doi.org/10.32225/bkpmnh.2022.51_47
- Sugawara, H., Tahara, Y., Nakazono, S., Matsukoji, T. & Nagano, M. (2022c) Taxonomic revision of the Yamaguchi salamander Hynobius bakan: Description of two new species from Chugoku and Kyushu, Japan. Science Report of the Yokosuka City Museum, 69, 1–17.
- Suk, H.Y., Kim, D.Y., Cha, S. & Min, M.S. (2019) The complete mitochondrial genome information of Hynobius unisacculus (Amphibia, Caudata, Hynobiidae) and the phylogenetic implication. Mitochondrial DNA Part B, 4, 3705–3706. https://doi.org/10.1080/23802359.2019.1679680
- 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. https://doi.org/10.1038/srep16963
- Tago, K. (1904) A topic of salamanders. Zoological Magazine, 16, 41–46.
- Tago, K. (1907) Study of salamanders from Japan. Zoological Magazine, 19, 229–248.
- Tanabe, A.S. (2011) Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data. Molecular Ecology Resources, 11, 914–921. https://doi.org/10.1111/j.1755-0998.2011.03021.x
- Tavaré, S. (1986) Some probabilistic and statistical problems in the analysis of DNA sequences. In: Miura, R.M. (Ed.) Some Mathematical Questions in Biology: DNA Sequence Analysis. American Mathematical Society, Providence, Rhode Island, pp. 57–86.
- Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673–4680. https://doi.org/10.1093/nar/22.22.4673
- Tominaga, A., Matsui, M. & Nishikawa, K. (2019a) Two new species of lotic breeding salamanders (Amphibia, Caudata, Hynobiidae) from western Japan. Zootaxa, 4550 (4), 525–544. https://doi.org/10.11646/zootaxa.4550.4.3
- Tominaga, A., Matsui, M., Tanabe, S. & Nishikawa, K. (2019b) A revision of Hynobius stejnegeri, a lotic breeding salamander from western Japan, with a description of three new species (Amphibia, Caudata, Hynobiidae). Zootaxa, 4651 (3), 401–433. https://doi.org/10.11646/zootaxa.4651.3.1
- Tukey, J.W. (1953) The problem of multiple comparisons. Mimeographed monograph.
- Yoshikawa, N. & Matsui, M. (2014) Two new salamanders of the genus Onychodactylus from eastern Honshu, Japan (Amphibia, Caudata, Hynobiidae). Zootaxa, 3866 (1), 53–78. https://doi.org/10.11646/zootaxa.3866.1.3
- Yoshikawa, N. & Matsui, M. (2022) A new salamander of the genus Onychodactylus from central Honshu, Japan (Amphibia, Caudata, Hynobiidae). Current Herpetology, 41, 82–100. https://doi.org/10.5358/hsj.41.82
- Yoshikawa, N., Matsui, M. & Inoue-Murayama, M. (2013) Characterization of nineteen microsatellite markers for the Japanese clouded salamander, Hynobius nebulosus, using the NGS. Conservation Genetics Resources, 5, 603–605. https://doi.org/10.1007/s12686-013-9861-4
- Zharkikh, A. (1994) Estimation of evolutionary distances between nucleotide sequences. Journal of Molecular Evolution, 39, 315–329. https://doi.org/10.1007/BF00160155