Sexual Plant Reproduction

, Volume 25, Issue 1, pp 1–9 | Cite as

Zygospore formation between homothallic and heterothallic strains of Closterium

  • Yuki TsuchikaneEmail author
  • Miki Tsuchiya
  • František Hindák
  • Hisayoshi Nozaki
  • Hiroyuki Sekimoto
Original Article


Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt+) and mating-type minus (mt) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt+ cells, compared with that in the homothallic strain alone and in a coculture with mt cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt+ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.


Closterium Conjugation Homothallism Heterothallism Sexual reproduction Zygospore 



We are grateful to Dr. Syo Kato (University of Tsukuba) for his technical advice concerning vital staining. This work was supported by Grants-in-Aid for Scientific Research (no. 23770093 to Y.T., nos. 20247032, 22405014, and 23657161 to HS) from the Japan Society for the Promotion of Science, Japan; a Grant-in-Aid for Scientific Research on Innovative Areas “Elucidating common mechanisms of allogeneic authentication” (no. 22112521 to HS) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; a grant from the New Technology Development Foundation to HS; and a research grant (2009–2011) from the Institute for Fermentation, Osaka, Japan.

Supplementary material

497_2011_174_MOESM1_ESM.pdf (401 kb)
Fig. S1 Schematic illustration of the sister and non-sister zygospore formation processes. Sister zygospores originated as a result of the conjugation of two sister gametangial cells derived from one vegetative cell. The non-sister zygospore was formed between gametangial cells of separately adjoined individuals. (PDF 400 kb)
497_2011_174_MOESM2_ESM.pdf (423 kb)
Fig. S2 Effect of vital staining on zygospore formation. Non-stained mating-type plus (mt+) and non-stained mating-type minus (mt), stained mt+ and non-stained mt, non-stained mt+ and stained mt, and stained mt+ and stained mt were cocultured in MI medium, respectively, and the number of zygospores was counted. Vertical bars indicate SD (n = 3). Relative number of zygospores was calculated using the equation. Relative number of zygospores (%) = (number of zygospores X 2/number of cells) X 100. (PDF 424 kb)
497_2011_174_MOESM3_ESM.pdf (30 kb)
Fig. S3. Effect of mating-type plus (mt+) and mating-type minus (mt) cell ratio on the formation of hybrid zygospores. Homothallic cells were cocultured with either stained mt+ (a) or stained mt cells (b). After 72 h, the number of zygospores, hybrid zygospores, and cells were counted. Vertical bars indicate the SD (n = 3). (PDF 29 kb)

Movie S1. Movie of the homothallic cell conjugation test in the presence of heterothallic mating-type plus (mt+) cells. This movie shows 72 h of the conjugation process condensed to 28 s. Images were taken every 15 min (25.6 MB MPEG-1). (MPG 7,290 kb)

Movie S2. Movie of the homothallic cell conjugation test in the presence of heterothallic mating-type minus (mt) cells. This movie shows 72 h of the conjugation process condensed to 28 s. Images were taken every 15 min (19.3 MB MPEG-1). (MPG 7,470 kb)

Movie S3. Movie of the homothallic cell conjugation test. This movie shows 72 h of the conjugation process condensed to 28 s. Images were taken every 15 min (14.6 MB MPEG-1). (MPG 6,598 kb)

Movie S4. Movie of the heterothallic mating-type plus (mt+) and mating-type minus (mt) cell conjugation test. This movie shows 72 h of the conjugation process condensed to 28 s. Images were taken every 15 min (22.2 MB MPEG-1). (MPG 6,440 kb)

497_2011_174_MOESM8_ESM.pdf (46 kb)
Supplementary material 8 (PDF 46 kb)


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Yuki Tsuchikane
    • 1
    Email author
  • Miki Tsuchiya
    • 1
  • František Hindák
    • 2
  • Hisayoshi Nozaki
    • 3
  • Hiroyuki Sekimoto
    • 1
  1. 1.Department of Chemical and Biological Sciences, Faculty of ScienceJapan Women’s UniversityTokyoJapan
  2. 2.Department of Non-Vascular PlantsInstitute of Botany, Slovak Academy of SciencesBratislavaSlovakia
  3. 3.Department of Biological Sciences, Graduate School of ScienceUniversity of TokyoTokyoJapan

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