Biologia 63/6: 778—780, 2008 Section Botany DOI: 10.2478/s11756-008-0098-8 Zygote germination in Pleodorina starrii (Volvocaceae, Chlorophyta)* Hisayoshi Nozaki Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; e-mail: [email protected] Abstract: Zygote germination of the anisogamous/oogamous colonial green flagellate Pleodorina starrii was observed. After the zygotes were transferred to the usual, illuminated conditions from the dark treatment on the agar plate, they began to germinate. The germinating zygotes gave rise to one or two viable biflagellate gone cells. This type of zygote germination is rare in the colonial Volvocales and may characterize a certain lineage within the anisogamous/oogamous members of the colonial Volvocales. Key words: Chlorophyta; Volvocales; Volvocaceae; Pleodorina starrii; morphology; zygote germination Introduction packet formation, conjugation between male and female ga- metes and zygote maturation occurred. Mature zygotes were transferred to 1% agar plates (AF-6 medium, Kasai et al. During our research of the male specific “OTOKOGI” ◦ (PlestMID) gene in the anisogamous/oogamous colo- 2004), and put into darkness for 1–3 months at 20–25 C. nial volvocalean Pleodorina starrii (Nozaki et al. After the dark treatment, the zygotes were transferred to the liquid AF-6 medium under 14:10 h LD and 20–25 ◦C. 2006a, Nozaki 2008) two heterothallic strains (2000- Light microscopy was carried out using an OLYMPUS BX60 602-P14female and 2000-602-P15male) of P. starrii microscope (KS OLYMPUS, Tokyo, Japan), equipped with were used. These two strains were established in June Nomarski interference optics. 2000 from a water sample collected in Lake Sagami, Kanagawa, Japan (Nozaki et al. 2006b). However, the induction of sperm packet formation had markedly de- Results creased after five years (Nozaki, unpublished). In or- der to recover the fertility, F1 progeny (2005-701-F1- Three days after their transfer from darkness to the illu- 5female, 2005-701-F1-1male and 2005-701-F1-3male) minated condition, the zygotes began to germinate. Ini- were produced based on other stains of P. star- tially, part of the outer wall ruptured. The inner wall in rii (2001-608-P26female, 2001-608-P21male, and 2001- this region protruded and swelled, forming a ellipsoidal 608-P17male) that were established from a different col- shape (Fig. 1). lection in Lake Tsukui, Kanagawa, Japan (Nozaki et al. Relatively small zygotes occasionally missed the 2006b). Thus, F2 progenies were obtained from 2005- transverse division (see below) when they germinated. 701-F1-5female X 2005-701-F1-1male. During these In the germinating, ellipsoidal zygote, a small hyaline studies, zygote germination was successively observed body, possibly a meiotic product, budded off from the in P. starrii. In this report the morphological details of protoplast within the protruded wall (Figs 1, 2). The zygote germination in P. starrii are described. protoplast then grew two equal flagella. As the proto- plast squeezed out into the protruding wall, the tip of the wall ruptured and a single biflagellate gone cell was Material and methods released, leaving the empty wall behind (Figs 3, 4). The protoplast within the zygote frequently un- In order to obtain F1 and F2 strains of P. starrii, forma- tion and germination of zygotes were induced as described derwent a transverse division, forming two protoplasts by Nozaki et al. (1989, 2006b). Actively growing male and of approximately equal size within the expanding zy- female cultures in VTAC medium (Nozaki et al. 1989; Ka- gote wall. A hyaline body could be observed within sai et al. 2004) were concentrated and mixed with Pleodor- the zygote wall (Fig. 5). The two protoplasts became ina mating medium. During the subsequent 10 days, sperm separated from each other within the wall. The tip of * Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia. c 2008 Institute of Botany, Slovak Academy of Sciences Zygote germination in Pleodorina starrii 779 Figs 1–9. Nomarski interference microscopy of zygote germination in the anisogamous/oogamous volvocacean Pleodorina starrii.Note a hyaline body or meiotic product (arrow). All at the same magnification throughout. 1–4 – Germinating zygotes producing a single gone cell. 2–4 – Successive stages of a germinating zygote. 5–8 – Germinating zygotes producing two viable gone cells. Initial stage (5), successive stages of two viable gone cells released from a single germinating zygote (5–8). 9 – Gone colony formation. Note developing embryo enclosed by gelatinous envelope. India ink preparation. the protruded wall then ruptured and two biflagellate of the two flagella inherited from the original gone gone cells were released separately, leaving the empty cell. zygote wall behind (Figs 6–8). Each of the two gone cells had the potential to develop into a new colony Discussion (gone colony). Irrespective of these two types of zygote germi- Zygote germination in Pleodorina was previously ob- nation, the liberated gone cells had the same form served only in P. japonica (Nozaki et al. 1989; Nozaki & and exhibited the essentially the same mode of gone Ito 1994). Only a single gone cell was produced from the colony formation. The gone cell was nearly spherical germinating zygote in P. japonica (Nozaki et al. 1989). in shape, measured 20–26 µm in diameter and con- In contrast, a germinating zygote of P. starrii gave rise tained reddish brown granules provided by the former to one or two viable gone cells (Figs 1–8). This differ- zygote (Nozaki et al. 2006b). Before cell division, the ence in mode of zygote germination between these two protoplast formed a gelatinous envelope from which the Pleodorina species may be reflected by its polyphyletic two flagella projected. The protoplast within the gelati- status: P. japonica is phylogenetically separated from nous envelope then divided successively to form a gone P. starrii within the anisogamous/oogamous members colony (Fig. 9) as in the asexual reproduction (Nozaki et of the Volvocaceae (Nozaki et al. 2006b). In most volvo- al. 2006b). During colony formation, the gelatinous en- cacean algae, only a single viable gone is produced velope containing a developing embryo moved by means from a germinating zygote (see Nozaki & Ito 1994). 780 H. Nozaki However, two species of the Volvocaceae, Eudorina illi- References noisnesis (Waters 1960; Goldstein 1964) and E. elegans var. synoica (Nozaki 1986) exhibit two viable gone cells Goldstein M. 1964. Speciation and mating behavior in Eudorina. J. Protozool. 1: 317–334. produced from a single germinating zygote,. Interest- Kasai F., Kawachi M. Erata M. & Watanabe M. M. (eds.) 2004. ingly, these two species of Eudorina and P. starrii oc- NIES-Collection. List of Strains. Microalgae and Protozoa 7th cupy closely related phylogenetic positions (Nozaki et ed. National Institute for Environmental Studies, Tsukuba, al. 2006b), so this attribute may be important for char- 257 pp. Nozaki H. 1986. Zygote germination in Eudorina elegans var. acterizing a lineage within the anisogamous/oogamous synoica (Chlorophyta, Volvocales). J. Jpn. Bot. 61: 316–320, Volvocacae. However, the modes of zygote germina- pl. 8. tion in other related species (such as Volvox gigas and Nozaki H. 2008. A new male specific gene “OTOKOGI ”in Pleodorina indica) have not been observed. Several gen- Pleodorina starrii (Volvocaceae, Chlorophyta) unveils the origin of male and female. Biologia 63: DOI: 10.2478/s11756- era within the ansogamous/oogamous Volvocaceae are 008-0097-9. not considered to be monophyletic because of a lack of Nozaki H. & Ito M. 1994. Phylogenetic relationships within shared morphological attributes (Nozaki et al. 2006b). the colonial Volvocales (Chlorophyta) inferred from cladistic 30: Thus, the mode of zygote germination may contribute analysis based on morphological data. J. Phycol. 353–365. Nozaki H., Kuroiwa H. Mita T. & Kuroiwa T. 1989. Pleodorina to the establishment of a natural taxonomic system at japonica sp. nov. (Volvocales, Chlorophyta) with bacteria-like the genus level. endosymbionts. Phycologia 28: 252–267. Nozaki H., Mori T., Misumi O., Matsunaga S. & Kuroiwa T. 2006a. Males evolved from the dominant isogametic mating 16: Acknowledgements type. Curr. Biol. R1018–R1020. Nozaki H., Ott F. D. & Coleman A. W. 2006b. Morphology, molecular phylogeny and taxonomy of two new species of This work was supported by Grants-in-Aid for Creative Sci- Pleodorina (Volvoceae, Chlorophyceae). J. Phycol. 42: 1072– entific Research Nos 16GS0304 and 17370087 to HN from 1080. the Ministry of Education, Culture, Sports, Science and Waters A. J. 1960. Studies onEudorina. Unpublished thesis, Uni- Technology, Japan. versity of California, Berkeley. Received September 1, 2007 Accepted March 17, 2008.