Identification of a new mating group and reproductive isolation in the Closterium peracerosum–strigosum–littorale complex Yuki Tsuchikane, Hiroka Kobayashi, Machi Kato, Juri Watanabe, Jiunn- Tzong Wu & Hiroyuki Sekimoto Journal of Plant Research ISSN 0918-9440 J Plant Res DOI 10.1007/s10265-018-1043-8 1 23 Your article is protected by copyright and all rights are held exclusively by The Botanical Society of Japan and Springer Japan KK, part of Springer Nature. This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Journal of Plant Research https://doi.org/10.1007/s10265-018-1043-8 REGULAR PAPER Identification of a new mating group and reproductive isolation in the Closterium peracerosum–strigosum–littorale complex Yuki Tsuchikane1 · Hiroka Kobayashi2 · Machi Kato1 · Juri Watanabe1 · Jiunn-Tzong Wu3 · Hiroyuki Sekimoto1,2 Received: 13 February 2018 / Accepted: 5 May 2018 © The Botanical Society of Japan and Springer Japan KK, part of Springer Nature 2018 Abstract Reproductive isolation is essential for the process of speciation. In order to understand speciation, it is necessary to com- pare one mating group with other phylogenetically related but reproductively isolated groups. The Closterium peracero- sum–strigosum–littorale (C. psl.) complex is a unicellular isogamous zygnematophycean alga, which is believed to share a close phylogenetic relationship with the land plants. In this study, we identified a new mating group, named group G, of C. psl. complex and compared its physiological and biochemical characteristics with the mating group I-E, which was closely related to the mating group G. Zygospores are typically formed as a result of conjugation between mating-type plus (mt+) and mating-type minus (mt−) cells in the same mating group during sexual reproduction. Crossing experiments revealed mating groups G and I-E were reproductively isolated from each other, but the release of lone protoplasts from mt − cells of mating group G was induced in the presence of mt+ cells of mating group I-E. In fact, the sex pheromone, protoplast- release-inducing protein of mating group I-E induced the release of protoplasts from mt− cells of mating group G. When mt+ and mt− cells of both mating groups I-E and G were co-cultured (multiple-choice matings), the zygospore formation of mating group G, but not that of mating group I-E, was inhibited. Based on these results, we propose a possible mechanism of reproductive isolation between the two mating groups and suggest the presence of sexual interference between mating group G and mating group I-E. Keywords Alga · Closterium · Mating group · Reproductive isolation · Sex pheromone · Speciation Introduction the isolation barriers, it is better to pay attention to closely related but reproductively isolated organisms rather than Reproductive isolation is essential for the process of spe- to far related organisms because many differences in their ciation. Analyses of isolation barriers have been carried sexual reproductive mechanism could be accumulated in the out using various organisms in order to understand specia- latter as a result of reproductive isolation. tion mechanisms (Widmer et al. 2009). In order to analyze The Closterium peracerosum–strigosum–littorale (C. psl.) complex represents a unicellular isogamous zygne- matophycean alga that is believed to share a close phyloge- Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1026 5-018-1043-8) contains netic relationship with the land plants (Wickett et al. 2014). supplementary material, which is available to authorized users. Heterothallic strains of the C. psl. complex consist of two sexes: a mating-type plus (mt +) and a mating-type minus * Yuki Tsuchikane − (mt ). The presence of at least six reproductively isolated [email protected] mating groups (groups II-A, II-B, II-C, I-D, I-E, and I-F) of 1 Department of Chemical and Biological Sciences, Faculty the heterothallic C. psl. complex has been reported (Wata- of Science, Japan Women’s University, 2-8-1 Mejirodai, nabe 1977; Watanabe and Ichimura 1978). Using strains Bunkyo-ku, Tokyo 112 8681, Japan belonging to the mating group I-E, we have studied the 2 Division of Material and Biological Sciences, Graduate details of the sexual reproduction from a physiological, bio- School of Science, Japan Women’s University, 2-8-1 chemical, and molecular biology perspective (Kanda et al. Mejirodai, Bunkyo-ku, Tokyo 112 8681, Japan 2017; Sekimoto 2000, 2017; Sekimoto et al. 2012). When 3 Research Center of Biodiversity, Academia Sinica, Nankang, the mt+ and mt − cells were mixed in a nitrogen-depleted Taipei 115, Taiwan Vol.:(0123456789)1 3 Author's personal copy Journal of Plant Research medium in the presence of light, they divided once to form it is pre-requested to obtain many sexually isolated and/or sexually competent gametangial cells. This cell division partially isolated strains. is called sexual cell division (SCD) (Ichimura 1971). The In this study, we established novel clonal culture strains gametangial cell of one mating type forms a pair with the from cells isolated from natural fields. Since they had a dif- other type. The paired cells then release their gametic pro- ferent group I intron sequence compared to the other mat- toplasts, and the protoplasts fuse immediately to form a ing groups and were reproductively isolated from them, zygospore. The mt+ and mt− cells recognize each other we named them mating group G. In addition, since mating by chemical communication through mating-type-specific group G was phylogenetically related to the mating group sex pheromones, protoplast-release-inducing protein (PR- I-E, we analyzed the details of sexual interaction between IP) Inducer and PR-IP (Akatsuka et al. 2003, 2006; Seki- them and discussed the possible mechanism of sexual isola- moto et al. 2012; Tsuchikane et al. 2003). PR-IP Inducer tion in C. psl. complex. is a glycoprotein (Nojiri et al. 1995) that is released from the mt− cells and induces both SCD (Tsuchikane et al. 2005) and PR-IP production (Sekimoto 2002; Sekimoto Materials and methods et al. 1994) in mt+ cells. PR-IP is also a glycoprotein that is released from the mt+ cells and induces both the SCD Strains and clonal culture conditions (Akatsuka et al. 2006) and the release of gametic proto- plasts from mt− cells (Sekimoto et al. 1990). The Closterium strains used in this study are listed in The group I-E is completely isolated from mating groups Table 1. The strains NIES-53, 54, 55, 64, 65, 66, 67, 68, II-A and II-B (Watanabe 1977; Tsuchikane et al. 2008). The 261, and 262 of the C. psl. complex used in this study were barrier in the reproductive isolation was attributed to the obtained from the National Institute for Environmental sex pheromone because there is no pheromonal interaction Studies, Environmental Agency (Ibaraki, Japan). Strains between I-E and the two isolated mating groups (Tsuchikane of TW15-5, ASA12-4, ASA13-5, 9, 10, 12, 13, 14, 15, et al. 2008). Till date, strains belonging to both groups I-D 16, 17, 18, 21, 29, 30, 31, 37, and 40 were established the and I-F are not present in the stock center and those of group following way: Three water samples (TW15, ASA12, and II-C are not fertile. Therefore, for further analysis of repro- ASA15) were collected from two localities by a plankton ductive isolation among mating groups of C. psl. complex, net (mesh size; 32 µm, RIGO, http://www.rigo.co.jp). The Table 1 Closterium List of Strain designation Water sample Locality Mating type Mating group strains used in this study NIES-53a IB-6b Ibaraki, Japan + II-A NIES-54a IB-6b Ibaraki, Japan − II-A NIES-64a KAS-4b Ibaraki, Japan − II-B NIES-65a KAS-4b Ibaraki, Japan + II-B NIES-261a IB-8b Ibaraki, Japan + II-C NIES-55a IB-8b Ibaraki, Japan − II-C NIES-66a N-13c Piuthan, Nepal + I-Dd NIES-262a N-13c Piuthan, Nepal − I-Dd NIES-67a N-31c Damchan, Nepal + I-Ee NIES-68a N-31c Damchan, Nepal − I-Ee TW15-5 TW15 Taoyuan, Taiwan − G ASA12-4 ASA12 Shizuoka, Japan + G ASA13-14e ASA13 Shizuoka, Japan − G ASA13-5, -9, 10, 12, -13, -15, -16, ASA13 Shizuoka, Japan + G -17, -18, -21f, -29, -30, -31, -37, -40 a Strains obtained from the National Institute for Environmental Studies (NIES; Ibaraki, Japan) b Watanabe and Ichimura 1978 c Ichimura 1973 d Mating group I-D is denoted as IA in NIES e Mating group I-E is denoted as IB in NIES f Strains used for physiological experiments of this work 1 3 Author's personal copy Journal of Plant Research water sample of TW15 was collected from an artificial of group II-B), NIES-64 (mt− of group II-B), NIES-66 lotus pond (pH 7.9, 33 °C; 25º02′24.6″N 121º07′58.5″E) (mt+ of group I-D), NIES-262 (mt− of group I-D), NIES- at Taoyuan, Taiwan, on July 3, 2006 while the water sam- 67 (mt+ of group I-E), and NIES-68 (mt − of group I-E) to ples of ASA12 and ASA13 were collected from Asahata determine the mating type and mating group in a 24-well Pond (ASA12: pH 9.6, 31.2 °C; ASA13: pH 7.0, 27.1 °C; microplate.