Division of Speciation Mechanism Sumida Laboratory

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sumida_lab — 2011-06-06T12:12:27+09:00

Welcome to the Homepage of Sumida lab

This page deals with the current research activities and the recent publications by Masayuki Sumida, Ph. D.

See-through frog developed in this laboratory is here(movie)

Masayuki Sumida，D. Sc.
Professor

e-mail:
Postal address:
Division of Diversification Mechanism
Institute for Amphibian Biology
Graduate School of Science
Hiroshima University
Higashihiroshima 739-8526
Japan

Tel: +81-82-424-7482
Fax: +81-82-424-0739

Creation of see-through frogs

sumida_lab — 2008-03-27T18:59:50+09:00

We have created the first see-through four-legged creature by artificial crossing between two recessive color mutants of the Japanese brown frog, Rana japonica, whose back is brown, although some small fish are also see-through (Fig. 18, Fig. 19, Fig. 20, Fig. 21).

We can observe organs of the same frog over its entire life without dissecting it, so it is useful as a new experimental animal.

We can see through the skin how organs grow, how cancer starts and develops, how toxins affect bones, livers and other organs.

Fig. 18. Ovulated female.

Fig. 19. Tadpoles.

Fig. 20. Ventral view.

Fig. 21. Eggs of wild-type and see-through frogs.

Microscopical observation of see-through frogs

The most beautiful frog in Japan and conservation using an artificial breeding technique

sumida_lab — 2008-03-27T18:57:24+09:00

Ishikawa’s frog (Rana ishikawae) is considered to be the most beautiful in Japan, and is designated as a protected species in Kagoshima and Okinawa Prefectures, and also as an endangered species in the Red List of the Ministry of the Environment. We are now attempting to conserve this species by artificial breeding in the laboratory (Fig. 15, Fig. 16, Fig. 17).

Fig. 15. Ovulated eggs.

Fig. 16. Artificial insemination.

Fig. 17. Three-year-old Ishikawa’s frogs produced by artificial breeding.

Hybridization experiments to elucidate reproductive isolating mechanisms

sumida_lab — 2008-03-27T18:51:57+09:00

(a) Reproductive isolating mechanisms among species and populations
Hybridization experiments on various species and local populations collected from Palearctic and Nearctic regions were performed in order to confirm the existence of reproductive isolating mechanisms among these species and populations, based on the definition of the biological species concept. Most Bufo species were found to be reproductively isolated from one another by gametic isolation, hybrid inviability at the embryonic or tadpole stage, and male hybrid sterility. However, several lineages classified as separate species were found not to be reproductively isolated from one another, and in fact seemed to belong to a single species (Fig.9).

(b) Incipient speciation in Rana japonica
The eastern and western groups of Rana japonica were reproductively isolated from each other by incomplete male hybrid sterility due to abnormal spermatogenesis (Fig.10, Fig.11). The differentiation between the eastern and western groups of Rana japonica was considered to be an incipient speciation of this species (Fig.12).

(c) Sex-linked genes and linkage maps
While a good number of linkage groups have been conserved in amphibians, there does not seem to be a common ancestral or conserved sex-linkage group (Fig. 13). Comparisons of amphibian linkage maps with those of fishes and mammals reveal several synthetic associations that apparently have been conserved over a very long period of vertebrate divergence.

(d) Color mutants and their loci in Rana japonica
Three color mutant strains were established in the laboratory, and the mode of inheritance and the linkage relationships of their loci were analyzed (Fig. 14). The black-eyed mutant locus (Blk) was found to link with the AAT-1 locus.

Comparisons of mitochondrial DNA and ribosomal DNA to elucidate the phylogeny and speciation of amphibians

sumida_lab — 2008-03-27T18:48:41+09:00

(a) Comparisons of mitochondrial DNA to elucidate phylogeny and speciation
The nucleotide sequences of the mtDNA genes were investigated using Japanese pond frogs to clarify the phylogenetic relationships at the nucleotide sequence level. Based on a conventional mtDNA clock calibration of 2~4% nucleotide sequence divergence per million years, Rana nigromaculata and Rana porosa are roughly estimated to have diverged about three to five million years ago, and Rana porosa porosa and the two local races of Rana porosa brevipoda are estimated to have diverged about one to two million years ago (Fig. 1). These results imply that the rate of sequence change for the cytochrome b gene is about two or three times greater than that for the 12S rRNA gene at the specific level of the Japanese pond frog. The cytochrome b gene sequences are apparently adequate to resolve phylogenetic relationships among Japanese pond frogs, whereas the 12S rRNA gene sequences are not informative at racial or subspecific level and should be used only at taxonomic levels higher than species (Fig. 2).

(b) Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the Japanese pond frog Rana nigromaculata
The complete nucleotide sequence of the mitochondrial genome of the Japanese pond frog Rana nigromaculata was determined. The length of the sequence was 17,804 bp (Fig. 3). A comparison of gene organization between two amphibian species, Rana and Xenopus, provided evidence that the gene arrangement of Rana differs by four tRNA gene positions from that of Xenopus, a species with a gene arrangement common among vertebrates (Fig. 4). Evolutionary relationships were estimated from the concatenated sequences of the 12 proteins encoded in the H-strand of mtDNA among 37 vertebrate species. A quartet puzzling tree showed, firstly, that three amphibian species form a monophyletic clade, and secondly, that the caecilian is a sister group of the monophyletic Anura (Fig. 5).

(c) Natural hybridization and introgression in a hybrid zone
The pond frogs of the Sengokuhara population from central Japan were analyzed for nuclear and mitochondrial DNA to elucidate the extent of natural hybridization and gene introgression in this population. As the outcome of long-term hybridization, there was some likelihood of introgression of genetic material between Rana nigromaculata and Rana porosa porosa through female hybrids and backcrosses (Fig. 6).

(d) Sequencing and analysis of the ribosomal DNA of the Japanese pond frog Rana nigromaculata
The nucleotide sequence of the EcoR I fragment of the rDNA of Rana nigromaculata was determined by the use of the deletion mutant and primer extension methods. The results showed that the EcoR I fragment was 4,806 bp long and included the ITS 1, 5.8S rRNA, ITS 2, the 3'-terminus of 18S rRNA, and the 5'-terminus of 28S rRNA (Fig. 7). The pylogenetic analysis was carried out using the nucleotide sequences of the 5.8S rDNA gene of various taxa (Fig. 8).

Current Research Activities

sumida_lab — 2008-03-27T18:17:39+09:00

Diversification Mechanism (Institute for Amphibian Biology)
　The Division of Diversification Mechanism engages in experimental research to reveal the process of diversity of genome structures during amphibian evolution, as well as to elucidate the process of speciation and the factors affecting the process and establish the concept of species. Genetic conservation of endangered species is also forwarded by artificial reproduction. The division is currently conducting the following four major research projects.

1) Comparisons of mitochondrial DNA and nuclear DNA to elucidate the genomic diversity of amphibians.

2) Species diversity and reproductive isolating mechanisms based on hybridization experiments.

3) Conservation of and genetic variation in endangered species.

Field-caught Rana ishikawae (Ishikawa's frog) from Amami Oshima

4) Creation of see-through frogs

Wild-type, two color mutant strains, and see-through frogs of Rana japonica established in the laboratory.

Takeshi Igawa

sumida_lab — 2003-10-01T01:06:34+09:00

Assistant Professor (Special Appointment)

Institute for Amphibian Biology
Graduate School of Science, Hiroshima University
Higashi-Hiroshima, Hiroshima 739-8526, Japan
Phone: +81-82-424-4494
Fax: +81-82-424-0739
E-mail: tigawa (atmark) hiroshima-u.ac.jp

Work Experience
2009- Assistant Professor (Special Appointment), Institute for Amphibian Biology, Graduate School of Science, Hiroshima University
2008-2009, Research Associate, Graduate University for Advanced Studies, Advisor: Dr. Y. Satta

Education
2003-2008, Ph.D., Hiroshima University. Advisor: Dr. M. Sumida
1999-2003, Bachelor of Life and Environmental Science, Shimane University.

Research Interests:
Speciation and Evolution of Amphibians
Molecular and Genome Evolution

Honor:
2008, Eligibility for Exemption from Repayment of Scholarship Loan Programs (JASSO) by having achieving particularly outstanding results during the course
2006, The Excellent Student Scholarship, Hiroshima University

Publication:
Igawa T., S, Oumi, S. Katsuren, M. Sumida. (in press) Population structure and landscape genetics of two endangered frog species of Genus Odorrana: different scenarios on two islands. Heredity

Kim, H. L., M. Iwase, T. Igawa, T. Nishioka, S. Kaneko, Y. Katsura, N. Takahata and Y. Satta. (2012) Genomic structure and evolution of multigene families: "Flowers" on the human genome. Int. J. Evol. Biol. 2012: Article ID 917678. doi:10.1155/2012/917678

Sugawara, H., T. Igawa, M. Yokogawa, M. Okuda, S. Oumi, S. Katsuren, S. Kaneko, T. Umino, Y. Isagi, M. Sumida.
Isolation and characterization of ten microsatellite loci of endangered Anderson’s crocodile newt, Echinotriton andersoni. Conser. Genet. Resour., 4: 595-598.

Hasan, M., M. M. Islam, M. M. R. Khan, M. S. Alam, A. Kurabayashi, T. Igawa, M. Kuramoto, and M. Sumida. (2012) Cryptic anuran biodiversity in Bangladesh revealed by mitochondrial 16S rRNA gene sequences. Zool. Sci. 29: 162-172.

Sumida, M., Satou, N., Yoshikawa, N., Kurabayashi, A., Islam, M. M., Igawa, T.,, Oumi, S., Katsuren, S., Ota, H., Shintani, N., Fukuniwa, H., Sano, N., and Fujii T (2011) Artificial production and natural breeding of the endangered frog species Odorrana ishikawae, with special reference to fauna conservation in the laboratory. Zool. Sci., 28: 834-839.

Igawa, T., Okuda, M., Oumi, S., Katsuren, S., Kurabayashi, A., Umino, T., Sumida, M. (2011) Isolation and characterization of twelve microsatellite loci of endangered Ishikawa's frog (Odorrana ishikawae). Conserv. Genet. Res. 3: 421-424.

Kim, H. L.*, Igawa, T.*, Kawashima, A., Satta, Y. and Takahata, N. (2010) Divergence, Demography and Gene Loss along the
Human Lineage. Phil. Trans. Roy. Soc. B, 365: 2451-2457.
*authors equally contributed.

Kurniawan, N., Islam, M. M., Djong, T. H., Igawa, T., Daicus, M. B., Yong, H. S., Wanichanon, R., Khan M. M. R., Iskandar, D. T., Nishioka, M. and Sumida, M. (2010) Genetic divergence and Evolutionary relationship in Fejervarya cancrivora from Indonesia and other Asian countries inferred from allozyme and mtDNA sequence analyses. Zool. Sci., 27: 222-233.

Alam, M. S., Igawa, T., Khan, M. M. R., Islam, M. M., Kuramoto, M., Matsui, M., Kurabayashi, A. and Sumida M. (2008) Genetic divergence and evolutionary relationships in six species of genera Hoplobatrachus and Euphlyctis (Amphibia: Anura) from Bangladesh and other Asian countries revealed by mitochondrial gene sequences. Mol. Phylogenet. Evol., 48: 515-527.

Igawa, T., Kurabayashi, A., Usuki, C., Fujii T. and Sumida M. (2008) Complete mitochondrial genomes of three neobatrachian anurans: A case study of divergence time estimation using different data and calibration settings. GENE , 402: 116-129.

Sumida, M., Kotaki, M., Islam, M. M., Djong, H. T., Igawa, T., Kondo, Y., Matsui, M., De Silva, A., Khonsue W. and Nishioka, M. (2007) Evolutionary relationships and reproductive isolating mechanisms in the rice frog Fejervarya limnocharis complex from Sri Lanka, Thailand, Taiwan and Japan inferred from mtDNA gene sequence analysis, allozyme analysis and crossing experiments. Zool. Sci., 24: 547-562.

Igawa, T., Kurabayashi, A., Nishioka M. and Sumida M. (2006) Molecular phylogenetic relationship of toads distributed in the Far East and Europe inferred from the nucleotide sequences of mitochondrial DNA genes. Mol. Phylognet.Evol., 38: 250-260.