Next-Generation Sequencing of an 88-Year-Old Specimen of the Poorly Known Species Liagora japonica (Nemaliales, Rhodophyta) Supports the Recognition of Otohimella gen. nov.

RESEARCH ARTICLE Next-Generation Sequencing of an 88-YearOld Specimen of the Poorly Known Species Liagora japonica (Nemaliales, Rhodophyta) Supports the Recognition of Otohimella gen. nov. a11111 OPEN ACCESS Citation: Suzuki M, Segawa T, Mori H, Akiyoshi A, Ootsuki R, Kurihara A, et al. (2016) Next-Generation Sequencing of an 88-Year-Old Specimen of the Poorly Known Species Liagora japonica (Nemaliales, Rhodophyta) Supports the Recognition of Otohimella gen. nov.. PLoS ONE 11(7): e0158944. doi:10.1371/ journal.pone.0158944 Masahiro Suzuki1*, Takahiro Segawa2,3,4, Hiroshi Mori5, Ayumi Akiyoshi3,4, Ryo Ootsuki6,7, Akira Kurihara8, Hidetoshi Sakayama9, Taiju Kitayama10, Tsuyoshi Abe11, Kazuhiro Kogame12, Hiroshi Kawai1, Hisayoshi Nozaki13 1 Kobe University Research Center for Inland Sea, Awaji, Hyogo, Japan, 2 Center for Life Science Research, University of Yamanashi, Yamanashi, Japan, 3 National Institute of Polar Research, Midori-cho, Tachikawa-shi, Tokyo, Japan, 4 Transdisciplinary Research Integration Center, Toranomon, Minato-ku, Tokyo, Japan, 5 Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan, 6 Department of Natural Sciences, Faculty of Arts and Sciences, Komazawa University, Setagaya-ku, Tokyo, Japan, 7 Faculty of Chemical and Biological Sciences, Japan Women's University, Tokyo, Japan, 8 Bioresource Sciences, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka, Japan, 9 Department of Biology, Graduate School of Science, Kobe University, Rokkodai, Nada-ku, Kobe, Japan, 10 Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki, Japan, 11 The Hokkaido University Museum, Hokkaido University, Sapporo, Hokkaido, Japan, 12 Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo, Hokkaido, Japan, 13 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan * Editor: Dorothee Huchon, Tel-Aviv University, ISRAEL Received: November 4, 2015 Accepted: June 24, 2016 Published: July 7, 2016 Copyright: © 2016 Suzuki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: MS and HN were supported by Grants-inAid for Scientific Research (grant numbers 26840125 and 24247042) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ Japan Society for the Promotion of Science (JSPS) KAKENHI (https://www.jsps.go.jp/english/e-grants/). Abstract Liagora japonica is a red algal species distributed in temperate regions of Japan. This species has not been collected from its type locality on the Pacific coast of Japan since 1927 and seems to have become extinct in this area. For molecular characterization of L. japonica, we extracted DNA from the topotype material of L. japonica collected in 1927, analyzed seven genes using Illumina next-generation sequencing, and compared these data with sequences from modern samples of similar red algae collected from the Japan Sea coast of Japan. Both morphological and molecular data from modern samples and historical specimens (including the lectotype and topotype) suggest that the specimens from the Pacific and Japan Sea coasts of Japan should be treated as a single species, and that L. japonica is phylogenetically separated from the genus Liagora. Based on the phylogenetic results and examination of reproductive structures, we propose Otohimella japonica gen. et comb. nov., characterized morphologically by diffuse carposporophytes, undivided carposporangia, and involucral filaments initiated only from the cortical cell on the supporting cell. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0158944 July 7, 2016 1 / 17 Next-Generation Sequencing of an 88-Year-Old Specimen of Otohimella japonica Introduction Taxonomic studies are based on type specimens, which are permanently attached to taxonomic names. The type specimens of red algal seaweeds are basically specimens housed in herbaria. To determine the correct names of modern collections, type specimens have been used for comparisons, including habit and anatomy. In many cases, morphological information from type specimens is not sufficient for taxonomic studies, because many type materials are fragmented and lack reproductive structures required for comparison with modern collections. Instead of type materials, detailed morphological observations and DNA sequencing from topotypes newly collected from their type locality have been used for taxonomic studies [1, 2]. However, in some cases the coastal environment of type localities has changed and the species are missing. The most reliable method to determine the correct name of modern collections involves sequencing of type specimens and comparing them to related sequences from fieldcollected material [3, 4]. However, DNA fragments of less than 300 base pairs can be obtained typically [3, 4], and thus complete gene sequences required for phylogenetic analyses are not available. Many type specimens were established more than 50 years ago, and the DNA has become highly fragmented due to deterioration over time [5]; therefore, Sanger sequencing is not reliable with old DNA samples. Recently, next-generation sequencing (NGS) has been used to examine DNA sequences from old samples [6]. For red algal seaweeds, Hughey et al. [7] performed NGS to determine the complete plastid and mitochondrial genomes from 140-year-old type specimens of the bangiophycean species of Pyropia using the published complete organelle genome data from Pyropia spp. for reference mapping and sequence assembly. However, in the case of Florideophyceae, the largest group of red algae, limited genomic data are available as references in NGS, compared with bangiophycean red algae, and thus old DNA sequences have not been determined previously by NGS for molecular phylogenetic analyses. The red algal genus Liagora is the largest genus of Liagoraceae and is widely distributed in warm temperate to tropical regions of the Atlantic, Indian, and Pacific Oceans [8]. Recent molecular analyses suggest that Liagora is polyphyletic, and the generic concept of Liagora sensu stricto has been revised substantially in recent years [9–11]. Although three new genera, Macrocarpus, Neoizziella, and Titanophycus, have since been separated from Liagora sensu lato [10, 12], the generic positions of some species of Liagora remain unresolved. Liagora japonica Yamada was originally described based on plants collected from Misaki, Kanagawa Prefecture, on the Pacific side of Japan in 1903 [13]. According to ou (...truncated)