Review

Tansley insight The genus Closterium, a new model organism to study sexual reproduction in streptophytes

Author for correspondence: Yuki Tsuchikane and Hiroyuki Sekimoto Yuki Tsuchikane Tel: +81 3 5981 3674 Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo Email: [email protected] 112-8681, Japan Received: 13 April 2018 Accepted: 4 June 2018

Contents

Summary 99 V. Sexual reproduction and inheritance of mating types in Closterium ehrenbergii 102 I. Introduction 99 VI. mt-determining gene of the C. psl. complex 103 II. Life cycle of Closterium 100 VII. Future perspectives 103 III. Sexual reproductive processes in the heterothallic Closterium peracerosum-strigosum-littorale complex 101 Acknowledgements 103

IV. Homothallism in the C. psl. complex 102 References 103

Summary

New Phytologist (2019) 221: 99–104 Closterium occupies a key phylogenetic position as an ancestor of land plants and is the best- doi: 10.1111/nph.15334 characterized Charophycean alga in terms of the process of sexual reproduction. Zygospores form as a result of sexual reproduction between genetically determined mating type plus (mt+) Key words: charophycean, Closterium, land and mating type minus (mtÀ) cells in heterothallic strains, or between clonal cells in homothallic plants, mating-type, sexual reproduction. strains. Here we review knowledge on the intercellular communication and mating type determination for successful sexual reproduction in Closterium. Using genomic information and transgenic techniques, the genus could be a model organism to study the mechanisms and evolution of sexual reproduction in streptophytes.

Klebsormidiophyceae), sexual reproduction is absent or unknown, I. Introduction whereas it is well known in the Zygnematophyceae (conjugating Sexual reproduction is an essential process for almost all panmictic green algae), Coleochaetophyceae and Charophyceae (McCourt populations of organisms. Although organisms have different et al., 2004), although their life cycles have not been fully methods of sexual reproduction, there is a common basic process: characterized (Haig, 2010). Recent evidence points to the zygne- two specialized sexually competent cells, which are determined matophycean algae, which include the genus Closterium, as the genetically or developmentally, recognize each other and this clade most closely related to early land plants (Wickett et al., 2014). leads to fertilization or conjugation. Land plants are thought Flagellate cells are not produced in the Zygnematophyceae and to have evolved from charophyte algal ancestors (Karol et al., 2001). sexual reproduction occurs via conjugation. Common features of The extant charophyte algae consist of six recognized lin- conjugation (see Box 1 for a glossary of terms) include the physical eages: Mesostigmatophyceae, Chlorokybophyceae, Kleb- pairing of filaments or single cells, the production of mucilage, and sormidiophyceae, Zygnematophyceae, Coleochaetophyceae and the subsequent fusion of nonflagellate gametes (Graham & Wilcox, Charophyceae (Leliaert et al., 2012). In the early diverging 2000). The genus Closterium, belonging to the Zygnematophyceae, groups (Mesostigmatophyceae, Chlorokybophyceae and is the best-characterized charophycean alga in terms of sexual

Ó 2018 The Authors New Phytologist (2019) 221: 99–104 99 New Phytologist Ó 2018 New Phytologist Trust www.newphytologist.com New 100 Review Tansley insight Phytologist

the intercellular communication have been well documented Box 1 Glossary (Sekimoto, 2017). Recently, several genetic and genomic tools have been developed for Closterium. Reverse genetic analysis has been Conjugation: The process consisting of the joining together of the performed by knockdown (Hirano et al., 2015) or CRISPR/Cas9- contents of two haploid cells. In zygnematalean green algae, two based knockouts (Kanda , 2017) using a stable nuclear nonflagellate gametes fuse together to form a zygospore. et al. Gametangial cell: A cell in which gamete (protoplast) is produced. transformation system with particle bombardment (Abe et al., Heterothallic: Two different clones are required for sexual repro- 2011, 2016). duction; self-sterile. Homothallic: Only one clone is required for sexual reproduction; self-fertile. II. Life cycle of Closterium Parthenogenesis: Production of a new individual from an unfertilized gamete. Algal taxonomists have described many species of Closterium based Parthenospore: A dormant spore developed without fertilization. on the morphological characteristics of vegetative cells since Sexual cell division: Mitotic cell division to form gametangial cells. Nitzsch (1817) established the genus (in the nomenclature, it has Sporangial cell: A cell in which a parthenospore is produced. been officially recognized since Ralfs, 1848). These cells proliferate Zygospore : The product of gamete fusion (also known as zygote). through asexual division. Sexual reproduction is often observed Graham & Wilcox (2000) was referred to for the creation of this under stressful conditions, including dryness or nitrogen shortage section. (Graham & Wilcox, 2000). As a result of this sexual reproduction, dormant zygospores are formed. There are two modes of zygospore formation: heterothallism (self-sterile) and homothallism (self- reproduction, since Ichimura (1971) developed an efficient fertile). In heterothallism, zygospores are formed between different method for inducing sexual reproduction. The processes involved clones (Fig. 1a,b). The heterothallic strains have two complemen- have been described morphologically and the molecules involved in tary mating types, mating type plus (mt+) and mating type minus

(a) Twin zygospore type (b) Single zygospore type in heterothallism (c) Homothallism (d) Parthenogenesis in heterothallism

mt+ mt– mt+ mt–

1 1

Gametangial cell 1

2 2 Sporangial cell

N

2N 2 3 3 Cell wall Germination

mt+ mt– mt+ mt–

Fig. 1 Schematic illustrations of the processes involved in zygospore formation in Closterium. (a) Twin zygospore type seen in heterothallic Closterium ehrenbergii: (1) sexual pair formation between complementary cells; (2) sexual cell division (SCD) for the formation of gametangial cells; and (3) zygospores formed by the fusion of nonsister protoplasts (gametes) released from respective gametangial cells. (b) Single zygospore type of the heterothallic Closterium peracerosum-strigosum-littorale (C. psl.) complex: (1) SCD for the formation of gametangial cells; (2) sexual pair formation between complementary gametangial cells; and (3) zygospore formed by the fusion of gametes released from respective gametangial cells. (c) Sister conjugation of the homothallic strain of the C. psl. complex. Sister conjugation proceeds between two sister gametangial cells derived from one vegetative cell. (d) Parthenospore formation in Closterium moniliferum: (1) cell division and (2) parthenospore. Gray shading indicates the diploid phase. Meiosis occurs during germination in heterothallism and homothallism.

New Phytologist (2019) 221: 99–104 Ó 2018 The Authors www.newphytologist.com New Phytologist Ó 2018 New Phytologist Trust New Phytologist Tansley insight Review 101

+ (mtÀ), which are determined genetically. Cells of mt and mtÀ are sexual cell division (SCD; Fig. 1b1) (Ichimura, 1971). The nearly identical in size and shape. It is unknown whether the gametangial cell of one mating type forms a pair with a cell of the inheritance of organelles is linked to the one of the mating types. If other type (Fig. 1b2). The paired cells then release their gametic they are co-incubated in the same place, they fuse with each other protoplasts, which fuse immediately to form a zygospore (Fig. 1b3). + (isogamous sexual reproduction) to form zygospores. The matured The mt and mtÀ cells recognize each other by chemical zygospores are dormant and show resistance to dehydration. By communication through mating-type-specific sex pheromones: some form of stimulation such as rehydration, meiotic cell division protoplast-release-inducing protein (PR-IP) Inducer and PR-IP + occurs and mt and mtÀ progenies originate equally from a (Fig. 2; Akatsuka et al., 2003, 2006; Tsuchikane et al., 2003; zygospore (Kasai & Ichimura, 1983). In homothallism, zygospores Sekimoto et al., 2012). PR-IP Inducer is a glycoprotein (Nojiri et al., are formed within a single clone (Fig. 1c). The two homothallic 1995) that is released from the mtÀ cells and induces both SCD progeny originate from a single zygospore (Graham & Wilcox, (Tsuchikane et al., 2005) and PR-IP production (Sekimoto et al., 2000). The vegetative part of the life cycle is spent in the haploid 1994; Sekimoto, 2002) in mt+ cells. PR-IP is also a glycoprotein that phase, with meiosis taking place upon germination of the is released from the mt+ cells and induces both SCD (Akatsuka et al., zygospore (Kasai & Ichimura, 1983). In natural Closterium 2006) and the release of gametic protoplasts from mtÀ cells populations, there is another mode of dormant spore formation, (Sekimoto et al., 1990). The cDNAs encoding the respective parthenogenesis, in which thick-walled resistant spores pheromones have been cloned (Sekimoto et al., 1994, 1998). (parthenospores) are formed without conjugation (Fig. 1d). On Morphological species of heterothallic Closterium have been germination, the parthenospore produces a single vegetative cell further subclassified into several reproductively isolated groups (Tsuchikane et al., 2014). (biological species; Ichimura, 1981). Seven reproductively isolated mating groups (groups II-A, II-B, II-C, I-D, I-E, I-F and G) of the heterothallic . . complex have been reported (Watanabe & III. Sexual reproductive processes in the heterothallic C psl Ichimura, 1978; Tsuchikane , 2018). The above-mentioned Closterium peracerosum-strigosum-littorale et al. sex pheromones were first identified in mating group I-E (Sekimoto complex et al., 2012; Sekimoto, 2017), which is completely isolated from The Closterium peracerosum-strigosum-littorale (C. psl.) complex is the other mating groups (Tsuchikane et al., 2008). Subsequently, the best-characterized Closterium species. When mt+ (NIES-67) and the pheromones and corresponding genes were identified in other mtÀ (NIES-68) cells (obtained from the National Institute for mating groups (Tsuchikane et al., 2008) and the mechanism of Environmental Studies, Ibaraki, Japan) are mixed in nitrogen- reproductive isolation has been studied in terms of its physiology, depleted medium in the presence of light, they divide once to form biochemistry and molecular biology (Sekimoto et al., 2012; sexually competent gametangial cells. This cell division is called Sekimoto, 2017).

Plus Minus

PR–IP Inducer PR–IP

Fig. 2 Schematic illustration of sexual reproduction of the heterothallic Closterium peracerosum-strigosum-littorale complex. Most sexual reproductive processes are induced by protoplast-release-inducing protein (PR-IP) and PR-IP Inducer.

Ó 2018 The Authors New Phytologist (2019) 221: 99–104 New Phytologist Ó 2018 New Phytologist Trust www.newphytologist.com New 102 Review Tansley insight Phytologist

strains. Recently, we obtained RNA-seq data from various stages in IV. Homothallism in the C. psl. complex the life cycle of homothallic strains (unpublished). Some of the The detailed conjugation process of the homothallic strain (NIES- contigs showed high similarities to sex-specific genes, which had 2666) has been revealed (Tsuchikane et al., 2010b). The first step in been identified in the heterothallic strains by cDNA microarray the conjugation process is mitotic cell division, resulting in the analysis (Sekimoto et al., 2006). We are now attempting to detect formation of two sister gametangial cells from one vegetative cell the transient expression of the genes and corresponding proteins in (Fig. 1c). These two gametangial cells form a sexual pair and then a just-divided sister gametangial cells. zygospore. Approximately 90% of homothallic zygospores origi- It is not clear whether homothallism or heterothallism represents nate as a result of conjugation of two sister gametangial cells derived the ancestral reproductive strategy. To clarify this, the phylogenetic from one vegetative cell. In other words, sister gametangial cells of relationship between homothallic and heterothallic strains of the the homothallic strain recognize each other. Sister conjugation has C. psl. complex should be investigated. In addition, progenies of also been observed in Penium margaritaceum, another unicellular hybrid zygospores between the homothallic cells and phylogenet- isogamous zygnematophycean alga (Tsuchikane et al., 2011). ically related heterothallic mt+ cells (Tsuchikane et al., 2012) must The formation of hybrid zygospores between the homothallic be studied phenotypically and genetically to identify genes involved cells and phylogenetically related heterothallic mt+ cells has been in the control of these reproductive patterns. observed (Tsuchikane et al., 2012). This suggests that at least some homothallic gametangial cells possess the same characteristics as V. Sexual reproduction and inheritance of mating heterothallic mtÀ cells. Because the conjugation-regulating sex types in Closterium ehrenbergii pheromone of the homothallic strain was found to be orthologous to the heterothallic PR-IP Inducer (Tsuchikane et al., 2010a,b), In the genus Closterium, C. ehrenbergii is one of the most commonly homothallic cells and heterothallic mt+ cells may recognize each encountered species in nature. Heterothallic strains of C. + other through their respective sex pheromones. These findings ehrenbergii also have two complementary sexes (mt and mtÀ). + support the idea that the division of one vegetative cell into two The conjugation process consists of pairing between mt and mtÀ sister gametangial cells is a segregative process, capable of producing (Fig. 1a1), SCD (Fig. 1a2) of the paired cells, papilla formation, the complementary mating types characterized in the heterothallic release of protoplasts (gametes) from gametangial cells, and

Telo 1 Telo II Germlings

mt+ mt – mt – mt – – 1 2 1 mt + mt+ 1 mt (a) – – mt2 mt2 + – – mt mt1 mt2

Plus Minus Zygote Plus Minus

– + – – + mt mt mt + mt1 – mt 1 2 mt mt1 – + + – mt (b) mt mt mt2 2 – – – – mt mt2 mt 1 mt2 1 + – – mt mt1 mt2

Minus Minus

(N) (2N) (3N)

+ mt – mt – + + mt 2 1 mt mt – – mt1 (c) mt1 – – mt mt2 2 + – – mt mt2 mt1

Minus Minus

Fig. 3 Closterium mating type allele transmission to F1 progeny through a ‘triploid’ zygospore. Three mating type alleles incorporated into a fused nucleus are distributed in three possible ways (a, b, c). The estimated ratio of germinated mating types in the total population was mating type plus : mating type minus + + (mt : mtÀ) = 1 : 5, and the estimated ratio of the germinated sets from respective zygospores was (mt and mtÀ) : (mtÀ and mtÀ) = 1 : 2, if the mtÀ phenotype is dominant over the mt+ phenotype. We referred to Kasai & Ichimura (1990) for the creation of this figure.

New Phytologist (2019) 221: 99–104 Ó 2018 The Authors www.newphytologist.com New Phytologist Ó 2018 New Phytologist Trust New Phytologist Tansley insight Review 103 zygospore formation (Fig. 1a3) (Lippert, 1967). Hogetsu & CpMinus1-expressing and CpMinus1-disrupted transformants in a Yokoyama (1979) showed that SCD, papilla formation and the homothallic background to dissect the function of the gene for release of protoplasts from gametangial cells depend on cell–cell homothallism. interactions, while Fukumoto et al. (1997) showed that the SCD of + mt cells was induced by a sex pheromone released from mtÀ cells. VII. Future perspectives This pheromone was named SCD-inducing pheromone (SCD- IP). The full-length cDNA was isolated and the deduced amino In this review, we have discussed the sexual behaviors within the acid sequences of SCD-IP in C. ehrenbergii and PR-IP Inducer in genus Closterium, belonging to the Zygnematophyceae. The the C. psl. complex appear to share significant homology (Fuku- means of intercellular communication may be species-specific, moto et al., 2003). The sex pheromones are thought to have evolved although the signaling mechanism would be conserved among from a single ancestral Closterium pheromone (Sekimoto et al., streptophyta. For example, CpRLK1 encoding a receptor-like 2012). protein kinase was isolated as a gene involved in sexual Homozygous mtÀ/mtÀ diploid clones of heterothallic C. reproduction, from the heterothallic C. psl. complex. Phyloge- ehrenbergii were obtained by hypertonic treatment of minus type netic analysis and phenotypes of the knockdown transformants vegetative cells (Kasai & Ichimura, 1990). The mating type support the idea that CpRLK1 belongs to the CrRLK1L-1 segregation ratios in the F1 progeny of ‘triploid’ zygospores, which subfamily, which is known as a sensor of cell wall integrity in + were formed between a wild-type mt haploid and mtÀ/mtÀ Arabidopsis thaliana, and that it functions in regulating osmotic homozygous diploid, were analyzed (Kasai & Ichimura, 1990). pressure in the cell for cytoplasm condensation and gamete release The ratio of germinated mating types within the population was during successful conjugation in the C. psl. complex (Hirano + mt : mtÀ = 1 : 4.8 (154 : 736 cells) and the ratio of the germinated et al., 2015). + + sets from the respective zygospores was (mt and mtÀ) : (mt and In addition to CpRLK1, we have found the presence of many + mt ) : (mtÀ and mtÀ) = 1 : 0 : 2.1 (73 : 0 : 151 pairs). If the mating important genes, which are putative orthologs of land plants + type of mt /mtÀ heterozygous diploid is mtÀ, the theoretical involved in sexual reproduction, although the genomic informa- germinated mating types from three possible ways are tion relating to Closterium has not been fully published. In addition, + mt : mtÀ = 1 : 1 (Fig. 3a), 0 : 2 (Fig. 3b) and 0:2 (Fig. 3c), and we have successfully developed a system for CRISPR/Cas9-based + the total estimated ratio would be mt : mtÀ = 1 : 5. On the other knockout, the only successful example among charophyte algae hand, the theoretical germinated sets from three possible ways are (Kanda et al., 2017). We believe that the C. psl. complex could be a + mt : mtÀ (Fig. 3a), mtÀ : mtÀ (Fig. 3b) and mtÀ : mtÀ (Fig. 3c). model organism to study the mechanisms and evolution of sexual + + In other words, the estimated ratio is (mt and mtÀ) : (mt and reproduction in streptophytes. + mt ) : (mtÀ and mtÀ) = 1 : 0 : 2. Because the experimental ratios closely matched the theoretical ratios, it was concluded that mtÀ Acknowledgements was dominant over mt+. The validity of the assumption that the two mating types are We thank Dr Takashi Nakada for taxonomic advice. The authors determined by one genetic factor (mtÀ allele dominant) was were supported financially by Grants-in-Aid for Scientific Research confirmed in a backcross progeny analysis (Kasai & Ichimura, (nos. JP24370038, JP26650147, JP15H05237, JP16H02518, 1990). In addition, as the mating types (sexes) are not changed by JP16H06378, JP16H06279, and JP16H04836 to H.S., any environmental factors, the presence of a single mt-determining JP26440223 and JP15H04413 to Y.T.) from the Japanese Society gene has been suggested in C. ehrenbergii. for the Promotion of Science.

VI. mt-determining gene of the C. psl. complex ORCID In unpublished transcriptomic analyses, we obtained a contig that Yuki Tsuchikane http://orcid.org/0000-0002-2316-1574 was strongly linked to the mtÀ phenotype of the progeny. The contig contains a gene that encodes a putative transcription factor and was named . CpMinus1 References To evaluate the function of CpMinus1, a construct for ectopic expression was prepared and introduced into mt+ cells. CpMinus1- Abe J, Hirano N, Komiya A, Kanda N, Fujiwara A, Hori S, Tsuchikane Y, Sekimoto H. 2016. Preparation of knockdown transformants of unicellular knockout mtÀ strains were also established. The phenotype of charophycean alga, Closterium peracerosum-strigosum-littorale complex. Bio- transformants was evaluated by a conjugation test with parental protocol 6: e1813. strains and by expression of mt-specific genes such as sex Abe J, Hori S, Tsuchikane Y, Kitao N, Kato M, Sekimoto H. 2011. Stable nuclear pheromone genes. We have not yet published the results but we transformation of the Closterium peracerosum-strigosum-littorale complex. Plant & postulate that CpMinus1 is an mt-determining gene, responsible Cell Physiology 52: 1676–1685. Akatsuka S, Sekimoto H, Iwai H, Fukumoto RH, Fujii T. 2003. Mucilage secretion for both expression of the mtÀ phenotype and suppression of the + regulated by sex pheromones in Closterium peracerosum-strigosum-littorale mt phenotype in the C. psl. complex. complex. Plant & Cell Physiology 44: 1081–1087. Recently, we found a gene orthologous to CpMinus1 in the Akatsuka S, Tsuchikane Y, Fukumoto R, Fujii T, Sekimoto H. 2006. Physiological homothallic strain. We are now trying to establish constitutively characterization of the sex pheromone protoplast-release-inducing protein from

Ó 2018 The Authors New Phytologist (2019) 221: 99–104 New Phytologist Ó 2018 New Phytologist Trust www.newphytologist.com New 104 Review Tansley insight Phytologist

the Closterium peracerosum-strigosum-littorale complex (Charophyta). Phycological Sekimoto H, Abe J, Tsuchikane Y. 2012. New insights into the regulation of sexual Research 54: 116–121. reproduction in Closterium. International Review of Cell & Molecular Biology 297: Fukumoto R, Fujii T, Sekimoto H. 1997. Detection and evaluation of a novel sexual 309–338. pheromone that induces sexual cell division of Closterium ehrenbergii Sekimoto H, Fukumoto R, Dohmae N, Takio K, Fujii T, Kamiya Y. 1998. (Chlorophyta). Journal of Phycology 33: 441–445. Molecular cloning of a novel sex pheromone responsible for the release of a Fukumoto R, Fujii T, Sekimoto H. 2003. Cloning and characterization of a cDNA different sex pheromone in Closterium peracerosum-strigosum-littorale complex. encoding a sexual cell division-inducing pheromone from a unicellular green alga Plant & Cell Physiology 39: 1169–1175. Closterium ehrenbergii (Chlorophyta). Journal of Phycology 39: 931–936. Sekimoto H, Satoh S, Fujii T. 1990. Biochemical and physiological properties of a Graham LE, Wilcox LW. 2000. Algae. Upper Saddle River, NJ, USA: Prentice Hall. protein inducing protoplast release during conjugation in the Closterium Haig D. 2010. What do we know about Charophyte (streptophyta) life cycles? peracerosum-strigosum-littorale complex. Planta 182: 348–354. Journal of Phycology 46: 860–867. Sekimoto H, Sone Y, Fujii T. 1994. Regulation of expression of the genes for a sex Hirano N, Marukawa Y, Abe J, Hashiba S, Ichikawa M, Tanabe Y, Ito M, Nishii I, pheromone by an inducer of the sex pheromone in the Closterium peracerosum- Tsuchikane Y, Sekimoto H. 2015. A receptor-like kinase, related to cell wall strigosum-littorale complex. Planta 193: 137–144. sensor of higher plants, is required for sexual reproduction in the unicellular Sekimoto H, Tanabe Y, Tsuchikane Y, Shirosaki H, Fukuda H, Demura T, Ito M. Charophycean alga, Closterium peracerosum-strigosum-littorale complex. Plant & 2006. Gene expression profiling using cDNA microarray analysis of the sexual Cell Physiology 56: 1456–1462. reproduction stage of the unicellular charophycean alga Closterium peracerosum- Hogetsu T, Yokoyama M. 1979. Light, a nitrogen-depleted medium and cell–cell strigosum-littorale complex. Plant Physiology 141: 271–279. interaction in the conjugation process of Closterium ehrenbergii Meneghini. Plant Tsuchikane Y, Fujii T, Ito M, Sekimoto H. 2005. A sex pheromone, protoplast & Cell Physiology 20: 811–817. release-inducing protein (PR-IP) Inducer, induces sexual cell division and Ichimura T. 1971. Sexual cell division and conjugation-papilla formation in sexual production of PR-IP in Closterium. Plant & Cell Physiology 46: 1472–1476. reproduction of Closterium strigosum. In: Nishizawa K, ed. Proceedings of the 7th Tsuchikane Y, Fukumoto R, Akatsuka S, Fujii T, Sekimoto H. 2003. Sex International Seaweed Symposium. Tokyo, Japan: University of Tokyo Press, 208– pheromones that induce sexual cell division in the Closterium peracerosum- 214. strigosum-littorale complex (Charophyta). Journal of Phycology 39: 303–309. Ichimura T. 1981. Mating types and reproductive isolation in Closterium ehrenbergii Tsuchikane Y, Ito M, Sekimoto H. 2008. Reproductive isolation by sex Meneghini. Botanical Magazine Tokyo 94: 325–334. pheromones in the Closterium peracerosum-strigosum-littorale complex Kanda N, Ichikawa M, Ono A, Toyoda A, Fujiyama A, Abe J, Tsuchikane Y, (Zygnematales, Charophyceae). Journal of Phycology 44: 1197–1203. Nishiyama T, Sekimoto H. 2017. CRISPR/Cas9-based knockouts reveal that Tsuchikane Y, Kobayashi H, Kato M, Watanabe J, Wu J-T, Sekimoto H. 2018. CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium Identification of a new mating group and reproductive isolation in the Closterium peracerosum-strigosum-littorale complex. Scientific Reports 7: 17873. peracerosum–strigosum–littorale complex. Journal of Plant Research. doi: 10.1007/ Karol KG, McCourt RM, Cimino MT, Delwiche CF. 2001. The closest living s10265-018-1043-8. relatives of land plants. Science 294: 2351–2353. Tsuchikane Y, Kokubun Y, Sekimoto H. 2010a. Characterization and molecular Kasai F, Ichimura T. 1983. Zygospore germination and meiosis in Closterium cloning of conjugation-regulating sex pheromones in homothallic Closterium. ehrenbergii Meneghini (Conjugatophyceae). Phycologia 22: 267–275. Plant & Cell Physiology 51: 1515–1523. Kasai F, Ichimura T. 1990. A sex determining mechanism in the Closterium Tsuchikane Y, Sato M, Ootaki T, Kokubun Y, Nozaki H, Ito M, Sekimoto H. ehrenbergii (Chlorophyta) species complex. Journal of Phycology 26: 195–201. 2010b. Sexual processes and phylogenetic relationships of homothallic strain of Leliaert F, Smith DR, Moreau H, Herron MD, Verbruggen H, Delwiche CF, the Closterium peracerosum-strigosum-littorale complex (Zygnematales, Clerck OD. 2012. Phylogeny and molecular evolution of the green algae. Critical Charophyceae). Journal of Phycology 46: 274–284. Reviews in Plant Sciences 31:1–46. Tsuchikane Y, Nakai A, Sekimoto H. 2014. Detailed analyses on the parthenospore Lippert BE. 1967. Sexual reproduction in Closterium moniliferum and Closterium formation in Closterium moniliferum (Zygnematophyceae, Charophyta). ehrenbergii. Journal of Phycology 3: 182–198. Phycologia 53: 571–578. McCourt RM, Delwiche CF, Karol KG. 2004. Charophyte algae and land plant Tsuchikane Y, Tsuchiya M, Hindak F, Nozaki H, Sekimoto H. 2012. Zygospore origins. Trends in Ecology and Evolution 19: 661–666. formation between homothallic and heterothallic strains of Closterium. Sexual Nitzsch CL. 1817. Beitrag zur Infusorienkunde oder Naturbeschreibung der Zerkarien Plant Reproduction 25:1–9. und Bazillarien. Neue Schriften der naturforschenden Gesellschaft zu Halle. Halle, Tsuchikane Y, Tsuchiya M, Kokubun Y, Abe J, Sekimoto H. 2011. Conjugation Germany: Hindel’s Verlag. processes of Penium margaritaceum (Zygnemophyceae, Charophyta). Phycological Nojiri T, Fujii T, Sekimoto H. 1995. Purification and characterization of a novel sex Research 59: 74–82. pheromone that induces the release of another sex pheromone during sexual Watanabe MM, Ichimura T. 1978. Biosystematic studies of Closterium reproduction of the heterothallic Closterium peracerosum-strigosum-littorale peracerosum-strigosum-littorale complex. II. Reproductive isolation and complex. Plant & Cell Physiology 36: 79–84. morphological variation among several populations from northern Kanto area in Ralfs J. 1848. The British Desmidieae. London, UK: Reeve, Benham, and Reeve. Japan. Botanical Magazine Tokyo 91:1–10. Sekimoto H. 2002. Production and secretion of a biologically active Closterium sex Wickett NJ, Mirarab S, Nguyen N, Warnow T, Carpenter E, Matasci N, pheromone by Saccharomyces cerevisiae. Plant Physiology & Biochemistry 40: 789– Ayyampalayam S, Barker MS, Burleigh JG, Gitzendanner MA et al. 2014. 794. Phylotranscriptomic analysis of the origin and early diversification of land Sekimoto H. 2017. Sexual reproduction and sex determination in green algae. plants. Proceedings of the National Academy of Sciences, USA 111: E4859– Journal of Plant Research 130: 423–431. E4868.

New Phytologist (2019) 221: 99–104 Ó 2018 The Authors www.newphytologist.com New Phytologist Ó 2018 New Phytologist Trust