Identification of a New Mating Group and Reproductive Isolation in the Closterium Peracerosum–Strigosum–Littorale Complex

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

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Journal of Plant Research https://doi.org/10.1007/s10265-018-1043-8

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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 peracerosum–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 zygnematophycean 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

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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 protoplasts 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

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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. 35°00′56.7″N 138°24′10.1″E) at Shizuoka-shi, Shizuoka, Japan, on August 6, 2012 and July 20, 2013, respectively. A specimen of Closterium sp. containing the vegetative Induction of conjugation cells was isolated from each water sample and then washed using the pipette washing method (Pringsheim 1946). The Vegetative cells in the late logarithmic phase (at 14 days) isolated single cells were cultured in independent test were collected by centrifugation (1,450×g, 5 min) and tubes (18 mm diameter and 150 mm length) containing washed three times with a nitrogen-depleted mating 15 mL of a nitrogen-supplemented medium (CA medium; medium (MI medium; Ichimura 1971). Ten milliliter of Kasai et al. 2004) at 24 °C under a 16 h light:8 h dark the cell suspension was prepared and 100 µL of the cell regime. Proliferating clonal cells were then inoculated suspension was diluted 100-fold. Cells contained in the into a 300 mL Erlenmeyer flask containing 150 mL of diluted cell suspension were counted using a microscope CA medium under the same culture conditions described equipped with a traditional hemocytometer (1 × 1 mm, grid previously. Light from LED lamps (LT30C; Beamtec, length × grid width) to clarify the cell density of undi- Saitama, Japan) was adjusted to 60 µmol (photons) m −2 s−1 luted cell suspension. A cell suspension assumed to be at the surface of the culture medium. Strains of TW15-5 containing 10,000 cells each was added and the total cell and ASA12-4 were established from the water samples number was adjusted to approximately 2 × 104 in 2 mL of of TW15 and ASA12, respectively. Sixteen clones were MI medium in a test tube (17.5 mm diameter and 130 mm isolated from ASA13, and the clones of two complemen- length). The cells were mixed in various combinations tary mating types were selected as representatives after (ASA13-21 × ASA13-14, ASA13-21 × NIES-68, ASA13- test-crossing against TW15-5 and ASA12-4 (Table 2) in a 14 × NIES-67, NIES-67 × NIES-68) and incubated under 24-well microplate (16 mm diameter wells, Iwaki Micro- continuous light for 72 h. After the incubation, relatively plate, Funabashi, Chiba, Japan) using the conjugation- short cells compared to the vegetative cells (including inducing method (Watanabe and Ichimura 1978). Each gametangial cells formed by sexual cell division (Tsuchi- subclone was also test-crossed with NIES-53 (mt + of kane et al. 2003), zygospores, paired gametic protoplast- group II-A), NIES-54 (mt− of group II-A), NIES-65 (mt+ releasing cells, and lone gametic protoplast-releasing cells) were individually counted using a hemocytometer. As it was difficult to distinguish the gametangial cells from Table 2 Intercrossing between TW15-5 ASA12-4 the cells formed immediately after asexual cell division, strains a small number of cells formed by asexual cell division ASA13 might be included along with the number of short cells. − 5 + − Then, the relative cell number was calculated using the − 9 + − following equation: − 10 + − − 12 + − Relative number of zygospores (%) − 13 + − = (number of zygospores − 15 + − − 16 + − × 2 ∕ number of total cells) × 100. − 17 + − Relative number of paired gametic protoplast − 18 + − − 21 + − − releasing cells (%) − 29 + − = (number of paired gametic protoplast − releasing cells − 30 + − × 2 ∕ number of total cells) × 100. − 31 + − − 37 + − Relative numbers of lone gametic protoplast − 40 + − − releasing cells (%) = (number of lone − 14 − + gametic protoplast − releasing cells +, zygospores formed; −, no ∕ number of total cells) × 100. zygospore formed

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Relative number of short cells (%) (1900 mL) was mixed with 100 mL of 1 M Tris–HCl buffer = (number of short cells ∕ number of total cells) (pH 8.0). This mixture was run through a diethylaminoethyl × 100. (DEAE)-Sepharose CL-6B (GE Healthcare, Buckingham- shire, England, UK) column (25 mm diameter, 100 mm The results presented here are the averages of three inde- long) equilibrated with 50 mM Tris–HCl buffer (pH 8.0). pendent samples. Experiments were performed at two sepa- Proteins were eluted sequentially with the same buffer sup- rate times and the reproducibility was confirmed. plemented with 50 and 1000 mM NaCl at a flow rate of 20 mL × h−1. The 1000 mM NaCl eluate was subjected to Vital staining ultrafiltration (Amicon Ultra Centrifugal Filters Ultracel- 3K; Millipore, Darmstadt, Germany) and the buffer was Vital staining using the Fluorescent Brightener 28 (FB28, changed to MI medium. The protein content was determined Sigma-Aldrich) was performed as described previously using Qubit Protein Assay Kit (Life technologies, Oregon, − (Tsuchikane et al. 2012) to distinguish between mating USA). Ten thousand mt cells were incubated in 2 mL of group I-E and G in multiple-choice mating. The cells stained MI medium containing various concentrations of partially by FB28 were observed under ultraviolet excitation using purified PR-IP, under the same conditions as the mating cul- a fluorescent microscope (IX-83, Olympus) fitted with a ture. After 48 h of incubation, the protoplasts-releasing cells U-FUNA filter (Olympus). were counted.

Multiple-choice mating Phylogenetic analysis Vegetative cells in the late logarithmic phase (at 14 days) were collected by centrifugation (1,450×g, 5 min) and DNA was extracted from each strain, as previously described washed three times with MI medium and incubated sepa- (Abe et al. 2016). The 1506 group I intron interrupting rately in 72 mL of MI medium in 300 mL Erlenmeyer nuclear small subunit rRNA genes was amplified and direct flasks (1.0 × 105 cells mL −1) under continuous light for 24 h sequenced using the protocols described by Tsuchikane et al. (low-density pre-culture). Vital staining was performed (2010). after this pre-culture. The cell number was measured as The sequence with dual peaks was amplified from the described above. The vital stained mt+ and mt− cells of the NIES-66 strain and the amplified products were treated with mating group I-E were mixed with the non-stained mt+ and a 10 × A-attachment mix (TOYOBO, http://www.toyobo.co. mt− cells of the mating group G such that 5 × 103 cells were jp). Then, they were inserted into a pGEM T-easy vector in each test tube (17.5 mm diameter and 130 mm length) (Promega) and transformed into an Escherichia coli (E. coli) and incubated under continuous light for 72 h. Conversely, strain (DH5α) according to the manufacturer’s instructions. the stained mating group G (mt+ and mt−) was mixed with Plasmid DNA was extracted from the E. coli suspension the non-stained mating group I-E (mt + and mt −) and treated using the High Pure Plasmid Isolation Kit (Roche Diagnos- the same way as described above. Intragroup mixings (mix tics GmbH, Mannheim, Germany) and the sequence was of mt+ and mt− cells in the same mating groups) were per- determined using the CEQ8000 Genetic Analysis System formed under the same conditions to serve as controls. (Beckman Coulter, CA, USA) and the DTCS-Quick start Cells showing a conjugative reaction were counted using kit (Beckman). a hemocytometer. For the counting, the stained and non- For the phylogenetic analyses, identical sequences were stained cells were individually counted using a fluorescent treated as a single operational taxonomic unit (OTU). The microscope. sequences isolated in this study were aligned with those of Experiments were performed 3–5 separate times where 10 strains and outgroups using MAFFT ver. 6 (Katoh and three independent tubes were used each time. The results Toh 2008). Gaps were omitted from the aligned sequences presented here are the averages of 9–15 independent for the phylogenetic analysis. samples. For the Bayesian analysis, we applied an HKY + G model, selected by a hierarchical likelihood ratio test (hLRT) using Partial purification of PR-IP MrModeltest v. 2.1 (Nylander 2004). Bayesian analysis was performed using MrBayes v. 3.1.2 (Ronquist and Huelsen- PR-IP was partially purified based on Sekimoto et al. (1990). beck 2003), as described by Tsuchikane et al. (2012). Mixture of 1,800,000 mt+ and 360,000 mt− cells of the mat- Unweighted maximum-parsimony (MP) analyses of the ing groups G or I-E was cultured in 72 mL of MI medium 1506 group I intron sequences were performed and included under continuous light for 48 h. The conditioned medium bootstrapping (Felsenstein 1985) based on 1,000 replica- was filtrated through a filter paper. The conditioned medium tions of full heuristic searches (with the tree bisection and

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Journal of Plant Research reconnection branch-swapping algorithm), using PAUP with sequences from Closterium ehrenbergii (DDBJ acces- v. 4.0b10 (Swofford 2002). We applied the Jukes–Cantor sion number: AY148821). model (Jukes and Cantor 1969) for the neighbor-joining (NJ) analysis (Saitou and Nei 1987). Bootstrap probabilities for Statistical analysis the NJ analysis were calculated based on 1,000 replications using PAUP v. 4.0b10. The phylogenetic trees were rooted Significant differences were calculated using the general- ized linear model with a Poisson regression by R v. 3.1.0 (R Development Core Team 2014). Significant differences are indicated by asterisks; *p < 0.05, **p < 0.01 and ***p < 0.001.

Results

Establishment of strains

TW15-5 (Fig. 1a) and ASA12-4 (Fig. 1b) strains were isolated and established from a pond in Taoyuan (Taiwan) and Asahata Pond at Shizuoka-shi (Japan), respectively. Zygospore formation was observed in the mixture of TW15-5 and ASA12-4 (Fig. S1a). In order to obtain two complementary mating types from the same locality, collection of water samples was performed again from Asahata Pond in 2013 and 16 clonal strains were established. When Fig. 1 Photographs of vegetative cells of mating group G. Vegetative cells of TW15-5 (a), ASA12-4 (b), ASA13-14 (c), and ASA13-21 these strains were incubated with TW15-5 and ASA12- (d). Scale bar 100 µm 4, only one strain (ASA13-14, Fig. 1c) conjugated with

Fig. 2 Sexual morphology of mating group G. Formation of gam- stained mt+ cells of mating group I-E were co-cultured and incubated etangial cells (a), formation of a sexual pair (arrowhead) (b), release (f–h). The arrow points to protoplasts released from a stained mt− cell of gametic protoplasts from each member of a pair (c), formation of of mating group G. f Light microscopy image, g fluorescent bright- zygospore by protoplast fusion (d), and release of lone gametic pro- ener 28 fluorescence image, and h merged fluorescence/light-micro- toplasts (e). The vitally stained mt − cells of mating group G and non- scopic images. Scale bar 100 µm

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ASA12-4 while the remaining fifteen strains conjugated with sequence in the phylogenetic analysis. A Bayesian phylo- TW15-5 (Table 2). As a matter of convenience, TW15-5 and genetic tree was constructed with an alignment of 394 bp 16 strains from Asahata Pond were defined as mt+ cells and combining the 1506 group I intron sequences from the C. ASA13-14, a complementary strain of the mt + cells, was psl. complex. The monophyly of mating group G and mating defined as mt − cells (Table 1). In the sexual reproductive group I-E was confirmed by high bootstrap values (99 and processes, respective cells divided to form gametangial cells 79% for NJ and MP analyses, respectively) and a Bayesian (Fig. 2a) that then formed a sexual pair (Fig. 2b). The paired posterior probability of 1.00 (Fig. 3). For further experi- cells then released their gametic protoplasts (Fig. 2c) and the ments, ASA13-14 (mt −) and ASA13-21 (mt +) were used protoplasts fused immediately to form a zygospore (Fig. 2d). because these strains had been collected more recently and Cells that released protoplasts without pair formation (lone at the same time. protoplast-release) were also observed (Fig. 2e). Four established strains (TW15-5, ASA12-4, ASA13- Reproductive isolation between mating groups G 14, and ASA13-21) and the strains belonging to the other and I-E mating groups of the C. psl. complex were subjected to the mating tests. During intragroup mating, mating group II-C The morphological features of mating groups G and I-E could not be tested because its mt − cells were not properly were analyzed. Compared to mating group II-C, it was maintained in the NIES. Although NIES-66 and NIES-262 impossible to distinguish the cell size of G and I-E (Fig. 4). (mating group I-D) were co-cultured, sexual reproduction In addition, no distinctive morphological differences were was not induced under the conditions of this experiment observed between G and I-E. (Fig. S2). Finally, no zygospore was formed between newly To investigate the reproductive isolation between groups established strains and other mating groups (Fig. S2). This G and I-E in detail, either of mt+ or mt− cells of group G suggested that they were sexually isolated from the other were co-cultured with either of mt + or mt− cells of mating fertile mating groups (II-A, II-B, and I-E). Therefore, we group I-E in MI medium, and the respective conjugation treated these strains as a new mating group, namely mating processes were observed (inter-group mating). In the case of group G. intragroup mating (co-cultures of mt + and mt− cells of same mating group), ratios of zygospores in mating groups G and Phylogenetic analysis I-E were 1.7% ± 1.5 (Fig. 5a) and 2.0% ± 0.3 (Fig. 5b), respectively. No zygospores were formed during inter-group Although the NIES-55 belonged to the mating group II-C, mating (Fig. 5c, d), however, promotion of gametangial cell the sequence of the group I intron was identical to that of formation was observed in the mixture between mt− cells NIES-64 and 65 of mating group II-A (Fig. 2). The sequence of group G and mt+ cells of group I-E. In addition, lone identity of the 1506 group I intron between NIES-53 (mating protoplast-releasing cells were observed in the same com- group II-A) and NIES-64 (mating group II-B) was 92.7%, bination (Fig. 5d). while ASA13-4 and NIES-67 (mating group I-E) was 82.5% Cells of mating group G were vitally stained using a (Table 3). The sequences of 1506 group I introns of ASA12- fluorescent dye. When the stained mt − cells of group G and 4, ASA13-14, and ASA13-21 were identical while that of non-stained mt+ cells of group I-E were co-cultured in MI TW15-5 differed by only one base from the sequence of medium and incubated for 72 h, all lone protoplast-releasing the other strains. However, they were treated as the same cells showed fluorescence (Fig. 2f, arrow). Thus, mating groups I-E and G were reproductively isolated from each other but a partial mating response was retained in both mat- Table 3 Identity of the 1506 II-Aa II-Bb I-Ec Gd ing groups. group I introns II-Aa 100 92.7 76.3 71.5 Exposure of partially purified PR-IP II-Bb 100 73.7 73.2 I-Ec 100 82.5 Mt− cells of mating group G or mt − cells of mating group Gd 100 I-E were exposed to various amounts of partially purified Sequences of the group I intron PR-IP of mating group G or mating group I-E. Partially in each mating group were purified PR-IP of mating group G exerted the protoplast- aligned and showed identity releasing activity on cells of the mating group G at 1 µg a Sequence of NIES-54 and the effect was elevated with increasing concentration of b Sequence of NIES-64 PR-IP. It was also active against mating group I-E, however, c Sequence of NIES-67 a higher concentration was required for the induction of lone d Sequence of ASA13-14 protoplast-release (Fig. 6a). Partially purified PR-IP from

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Fig. 3 Bayesian tree based on 394 bp of the combined aligned 1506 from neighbor-joining (NJ) (bottom left), and maximum-parsimony group I intron. Closterium strains with identical sequences were (MP) (bottom right) analyses. Branch lengths represent nucleotide treated as a single operational taxonomic unit. Numbers indicate pos- substitutions per site. From NIES-66, two sequences were obtained terior probabilities from the Bayesian analysis (top), bootstrap values at sub cloning mating group I-E also showed protoplast-releasing activi- the other hand, the number of paired protoplast-releasing ties against mt− cells of both mating groups, however, the cells remarkably decreased and zygospore formation was required concentration was higher for mating group G than completely inhibited in the presence of mating group I-E that for mating group I-E (Fig. 6b). Thus, partially purified (Figs. 7a, S3a). In the case of mating group I-E, the num- PR-IP from mating groups G and I-E, although limited in bers of protoplast-releasing cells (both lone and paired) activity, were still active against different mating groups. decreased in the presence of mating group G, however, zygospore formation was not affected in the presence of Multiple-choice mating mating group G (Figs. 7b, S3b).

To study the influence of different mating groups on the sexual reproduction within the same mating group, vital Discussion stained mt+ and mt− cells of mating group I-E, and non- stained mating type (mt+ and mt−) cells of G were co- Previously, the presence of six reproductively isolated cultured (Fig. 7). Mixing of vital stained mt+ and mt− cells mating groups (groups II-A, II-B, II-C, I-D, I-E, and I-F) of mating group G, and non-stained mating type (mt + and of the heterothallic C. psl. complex had been reported mt−) cells of I-E were also conducted (Fig. S3). For the (Watanabe 1977; Watanabe and Ichimura 1978). No mating group G, lone protoplast-releasing was not signifi- zygospore formation was observed between NIES-262 and cantly affected in the presence of mating group I-E. On 66 (mating group I-D), and sequences of the group I intron

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group I-E, mt+ and mt− cells recognize mating-type-specific sex pheromones, PR-IP Inducer and PR-IP, which are produced by mt− and mt+ cells, respectively (Sekimoto 2017). In this study, cells of ASA13-14 were defined as mt − cells of mating group G because this strain accepts PR-IP of group I-E (Fig. 6). The sex ratio of mating group G in Asahata Pond was highly biased (mt+:mt− = 15:1, Table 2). Theoreti- cally, mt+ and mt− cells germinate at a ratio of 1:1 from the zygospore in Closterium (Kasai and Ichimura 1987). How- ever, a possible explanation for the sex ratio to be biased in Asahata Pond might be because the frequency of conjugation is low and the proliferation of mt− cells is relatively slow for some reason such as difference in temperature sensitivities. Alternatively, establishment of culture strains of mt− cells may be difficult. In fact, we tried to isolate 40 vegetative cells from water samples, however, 24 cells did not proliferate and their sexes were unknown. It was reported that the mating groups II-A and II-B were partially isolated from each other and zygospores could be formed between mt + cells of group II-A and mt − cells of group II-B (Fig. S2, Tsuchikane et al. 2008; Watanabe and Ichimura 1978). The SCD in both mating-type cells of group II-A was stimulated by pheromone-containing conditioned Fig. 4 Graphic analysis of width and distance variation among the media in which cells of group II-B had been cultured. The strains. Scheme of morphological characters (a). Mean values of each SCD of mt − cells in group II-B was also induced by the strain in width and distance are shown (b). Symbols: triangles; group + conditioned medium derived from group II-A. However, G, circles; group I-E, squares; group II-C, closed symbols; mt , and + open symbols; mt−. Horizontal lines depict SDs of distance (n = 50). mt cells of group II-B did not respond to the conditioned Vertical lines depict SEs of width (n = 50) medium. On the other hand, mating groups G and I-E were completely isolated from each other and no zygospores could be formed between them although some mating inter- of these strains were not located in the same clade (Fig. 3). action was retained in both mating groups (Figs. 5, S4). The This made us consider the possibility that these two strains stages of reproductive isolation between groups G and I-E do not belong to the same mating group and that some would be more progressive than between groups II-A and mistake occurred during the preservation of these strains II-B. In other words, the isolating barrier between groups G in the culture collection. The sequence of the 1506 group and I-E is more complicated than that between II-A and II-B. I intron of NIES-55 of the mating group II-C was identical In fact, the identity of 1506 group I introns between mating to that of NIES-62 and 63 of mating group II-A (Fig. 2), group G and the reproductively isolated mating group I-E suggesting that present NIES-55 is not a primary strain was relatively lower (82.5%, Table 3) than that between mat- belonging to the mating group II-C and that only one strain ing group II-A and the asymmetrically isolated mating group (NIES-261) should be maintained in the culture collection. II-B (92.7%, Table 3). However, the conjugation ability of NIES-261 could not The lone protoplast-release of mating group I-E is known be evaluated because the complementary mt cells are not to be induced by the PR-IP, which is released from mt + maintained anywhere. Also, the mating group I-F could cells during the mating reaction (Sekimoto et al. 1990). The not be used in this experiment because reported strains of release of lone protoplast of mt− cells of mating group G this group (M-10-21 and M-10-25, Ichimura 1973; Watan- was observed in the presence of mt+ cells of the mating abe 1977) have not been maintained anywhere. Therefore, group I-E (Fig. 2f, g, h, 5d, S4), suggests that the PR-IP there is a possibility that mating group G is the same as produced by mt+ cells of mating group I-E is physiologi- mating group I-F. cally active against mt− cells of the mating group G. Indeed, In the genus Closterium, mating-type is not absolutely partially purified PR-IP from mating group I-E showed defined but is, instead, determined by a conjugation test with protoplast-releasing activity against mt− cells of mating reference strains (e. g., Tsuchikane et al. 2012). Therefore, group G (Fig. 6b, S4). In addition, it can be considered that there is no basis for the determination of the mating-types of the PR-IP Inducer released from mt− cells of mating group reproductively isolated mating groups. In the case of mating G retained the physiological activity on the production of

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Fig. 5 Sexual reproductive processes of C. psl. complex using groups G and I-E. Cells were incubated in MI medium. The panels show ASA13-21 and ASA13-14 (a), NIES-67 and NIES-68 (b), ASA13-21 and NIES-68 (c), and ASA13- 14 and NIES-67 (d). The number of short cells (open squares and dotted lines), paired protoplast-releasing cells (open circles and dotted lines), lone protoplast-releasing cells (ﬁlled squares and solid line), and zygospores (ﬁlled circles and solid line) were counted at 24-h intervals. The number of cells is expressed as a percentage. The vertical bars indicate the SD (n = 3). No paired protoplast- releasing cells were observed between ASA13-14 and NIES- 67 (d)

PR-IP from mt+ cells of mating group I-E. On the contrary, observation was not quantitative because numbers of lone protoplast-release of mt− cells of mating group I-E was cells used in the experiments were not equally adjusted. not observed in the presence of mt + cells of mating group In this study, equal numbers (5,000 cells each) of mt + and G (Fig. 5c) although the partially purified PR-IP from mat- mt− cells of both mating groups I-E and G were co-cultured. ing group G showed the protoplast-releasing activity against Zygospore formation of mating group G was inhibited in mt− cells of mating group I-E (Fig. 6a). It has been shown the presence of mating group I-E (Fig. 7a, S3a) while that that the mt − cells of mating group I-E release the PR-IP of I-E was not inhibited by mating group G (Fig. 7b, S3b). Inducer in nitrogen-depleted medium under light condition It has been shown that the addition of excess PR-IP inhibits even if mt+ cells are not present (Sekimoto et al. 1993). the release of protoplasts from mt− cells (Sekimoto et al. Therefore, mt+ cells of mating group G could be exposed to 1990). The medium in intragroup mating contains only one PR-IP Inducer of mating group I-E. We consider that PR-IP type of PR-IP and the medium in multiple-choice mating Inducer released from mt− cells of mating group I-E does not contains PR-IPs of both mating group I-E and G. There is retain activity to stimulate the production of PR-IP from mt + a possibility that mt− cells of group G, in multiple-choice cells of mating group G. We also speculate that the PR-IP mating, excessively received PR-IP from both mating groups Inducer of mating group I-E may act on the group G but the I-E and G. Therefore, in a future experiment, it is neces- activity of PR-IP derived from mating group G is not suf- sary to observe whether conjugation of mating group G is ficient to induce the protoplast-release of mt− cells of mating inhibited by adding purified PR-IP of mating group I-E or group I-E. Since the PR-IPs prepared from the respective by changing the ratio of cells of mating group G and I-E mating groups were only partially purified and might contain in multiple-mating. Reproductive interference refers to the non-PR-IP proteins, effective concentrations of PR-IP for the interaction in which the reproductive activity of one spe- protoplast-release could not be compared exactly. cies inhibits the reproduction of a closely related species Ichimura and Kasai (1987) performed multiple-choice (Gröning and Hochkirch 2008). Several examples are known mating in Closterium ehrenbergii for the first time. This in land plants (Brown et al. 2002; Takakura et al. 2008).

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Fig. 6 Effects of partially purified PR-IP on the release of protoplasts. Mt− cells of mating group G (ASA13-14) or mt− cells of mating group I-E (NIES-68) were incubated in MI medium containing various concentrations of partially purified PR-IP of group G (a) or group I-E (b). The vertical bars indicate the SD (n = 3)

Fig. 7 Multiple-choice mating of vital stained group I-E and non- ured (ﬁlled boxes). At the same time, intragroup mating was tested in stained group G. Vital stained mt+ cells, vital stained mt− cells of the absence of other mating group (open boxes). Relative numbers of mating group I-E, non-stained mt+ cells, and non-stained mt− cells cells in mating for group G and group I-E were shown in (a) and (b), of mating group G were co-cultured. After 72 h, the ratios of stained respectively. The vertical bars indicate the SD (n = 15) or non-stained cells in the mating processes were individually meas-

Inhibition of conjugation in mating group G by mating group isolated groups II-A and II-B in a future experiment. It is I-E, although slightly, suggests the presence of reproductive also necessary to verify that there is no reproductive interfer- interference in algae. To obtain more evidence, it is neces- ence between I-E and II-A (or II-B) which are completely sary to observe reproductive interference using incompletely isolated to each other.

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In this study, we succeeded in finding a new mating Kasai F, Ichimura T (1987) Stable diploids from intragroup zygospores Closterium ehrenbergii group, so called G, whose sexual reproduction could be of Menegh. (Conjugatophyceae). J Phycol 23:344–351 induced under laboratory conditions. Reproductive isola- Kasai F, Kawachi M, Erata M, Watanabe MM (eds) (2004) NIES-col- tion is observed between mating group I-E and G, indicat- lection. List of strains. Microalgae and protozoa, 7th edn. National ing that release of protoplast in mt− cells of mating group G Institute for Environmental Studies, Tsukuba was induced by PR-IP released from the mt+ cells of mating Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–298 group I-E (Fig. S4). Elucidating the mechanism of reproduc- Nojiri T, Fujii T, Sekimoto H (1995) Purification and characteriza- tive isolation and identification of the pre-zygotic isolation tion of a novel sex pheromone that induces the release of another barriers among mating groups would require the cloning of sex pheromone during sexual reproduction of the heterothallic Closterium peracerosum–strigosum–littorale genes for sex pheromones from the available mating groups complex. Plant Cell Physiol 36:79–84 and evaluating the cross activities of pheromones against Nylander JAA (2004) MrModeltest 2.1. Program distributed by the other mating groups. author. Evolutionary Biology Centre. Uppsala University, Uppsala Pringsheim EG (1946) Pure cultures of algae. Cambridge University Acknowledgements We thank Yukako Kurihara and Atsushi Ono Press, London for their support in sample collection in Asahata, Japan. 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