Biologia 63/6: 772—777, 2008 Section Botany DOI: 10.2478/s11756-008-0097-9 Review

A new male-specific gene “OTOKOGI ”inPleodorina starrii (Volvocaceae, Chlorophyta) unveils the origin of male and female*

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: Eukaryotic sex was initially isogametic and it is assumed that anisogamy/oogamy evolved independently in many lineages including animals, land plants and volvocine green algae. The exact evolutionary mechanisms that were responsible for the evolution of oogamy from isogamy were poorly understood until Nozaki et al. (2006) introduced the use of molecular- genetic data in elucidating the evolutionary origin of oogamy from isogamy in the colonial volvocacean Pleodorina starrii.In the close relative Chlamydomonas reinhardtii, sexual reproduction is isogametic with mating-types plus and minus. Mating type minus represents a “dominant sex” because the MID (“minus-dominance”) gene of C. reinhardtii is both necessary and sufficient to cause the cells to differentiate as isogametes of the minus mating type. No sex-specific genes had been identified in the volvocine green algae until Nozaki et al. (2006a) successfully cloned the MID gene of P. starrii.This “OTOKOGI ”(PlestMID) gene is present only in the male genome, and encodes a protein localized abundantly in the nuclei of mature sperm. Thus, P. starrii maleness evolved from the dominant sex (mating type minus) of its isogamous ancestor. This breakthrough provides an opportunity to address various extremely interesting questions regarding the evolution of oogamy and the male-female dichotomy. Key words: evolution; maleness; mating type; oogamy; sex; Volvocales

Discovery of the male-specific gene from the identified in the closely related isogamous, unicellu- anisogamous/oogamous Volvocaceae lar alga Chlamydomonas reinhardtii (Ferris & Goode- nough 1994, 1997). However, no mating type-specific Both animals and land plants exhibit “oogamy,” that genes had been isolated previously from the Volvo- is, they produce male and female gametes (sperm and caceae until the recent study of Nozaki et al. (2006a). eggs) that differ markedly in size and motility. In Although modes of uniparental inheritance of the plas- contrast, many primitive organisms exhibit “isogamy,” tid and mitochondrial genomes have been characterized having gametes of different mating types that are very in Chlamydomonas reinhardtii/smithii (Kuroiwa et al. similar in size and appearance. The evolution of oogamy 1982; Boynton et al. 1987) and the oogamous volvo- from isogamy (and with it the evolution of male/female cacean Volvox carteri (Adams et al. 1990; Kuroiwa et differences) is surely one of the most interesting biologi- al. 1993), they are essentially different between these cal issues. However, no extant isogamous organisms are two organisms. In Chlamydomonas, the plastid or mi- known that are closely related to animals or land plants tochondrial genome is inherited from the plus or minus (Karol et al. 2001; Rokas et al. 2005), and so there is mating type of the parents, respectively (Kuroiwa et al. no molecular-genetic data that would indicate what the 1982; Boynton et al. 1987). In contast, both the plas- evolutionary relationship may be between the two sexes tid and mitochondrial genomes are inherited from the of oogamous organisms and the mating types of isog- female parental strains of Volvox (Adams et al. 1990; amous organisms until our recent study (Nozaki et al. Kuroiwa et al. 1993). Thus, evolutionary relationships 2006b). between isogamous mating types in Chlamydomonas The anisogamous/oogamous multicellular green al- and female/male in the anisogamous/oogamous volvo- gae in the family Volvocaceae provide an ideal model caceans can not be deduced based on comparison for exploring such evolutionary relationships, because of modes of uniparental inheritance of the organelle evolutionary progression from isogamy to oogamy can genomes, and orthologues of the mating type-specific be observed within the closely related lineage (Nozaki genes of Chlamydomonas reinhardtii (Ferris & Goode- & Ito 1994; Nozaki et al. 2000; Nozaki 2003; Kirk nough 1994; Ferrris et al. 2002) are badly needed. 2006) and several mating type-specific genes have been The difficulties of isolating mating type- or sex-

* 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 A new male-specific gene “OTOKOGI ”fromPleodorina starrii 773 specific genes from the colonial Volvocales appear to male strain, but neither is present in the female strain have two causes. Firstly, evolution of the sex-related (Fig. 1C). genes is rapid. Ferris et al. (1997) tried to isolate ho- RT-PCR experiments using “OTOKOGI ”-specific mologues of the C. reinhardtii dominance (minus) mat- primers demonstrated that the “OTOKOGI ”geneis ing type-determining (MID)genefromGonium pec- expressed in nitrogen-starved, gamete-producing males, torale and Volvox carteri by Southern blotting, but but neither in males that had not been nitrogen- could not obtain the genes which they attributed to the starved to induce gametogenesis, nor in females regard- rapid evolution of sex-related genes. Secondly, the abil- less whether or not those females had been starved for ity to induce sexual reproduction in the colonial Volvo- nitrogen (Fig. 1D). Nor was any PsPlestMID expression cales generally decreases during the long-term mainte- detected when RT-PCR was performed with PsPlest- nance of the cultures (Coleman 1975). Especially in the MID-specific primers in conjunction with RNA from anisogamous/oogamous members of the Volvocaceae, either control or nitrogen-starved male or female cul- the decrease in sexual induction is rapid: male strains tures (Fig. 1D). of the anisogamous/oogamous volvocacean alga Pleodo- Whenspermpacketswerestainedwithananti- rina starrii (Fig. 1A) did not produce sperm packets body directed against the “OTOKOGI” protein, an (bundles of male gametes) five years after their isolation immunofluorescent signal was observed in the vicinity (Nozaki 2008). This problem may be resolved by cry- of the nuclei, and the nuclear location of this signal opreservation of the colonial Volvocales, but favourable was much clearer after the sperm packets had disso- results of cryopreservation treatments have not yet been ciated to release individual sperm (Fig. 1E). The sig- obtained (Mori et al. 2002). nal was prominent especially in the peripheral region In order to isolate sex-related genes from the colo- of the male nuclei (Fig. 1E). No immunofluorescent sig- nial Volvocales, Nozaki et al. (2006a) used the newly nal above background was detected in vegetative males, established, heterothallic strains of Pleodorina starrii even when they had been cultured in nitrogen-deficient (Nozaki et al. 2006b), and degenerate PCR primers. medium. These findings parallel those obtained for Cr- They eventually succeeded in isolating a Pleodorina MID, which is expressed only in nitrogen-starved cells orthologue of the MID gene of Chlamydomonas rein- that are actually producing minus gametes (Ferris et al. hardtii that causes cells to develop as mating type minus 2002). Because the CrMID protein is necessary for dif- gametes. In the following chapters, I report details and ferentiation of minus gametes in C. reinhardtii (Ferris the biological significance of the Pleodorina ortholog of & Goodenough 1997), and because CrMID, CiMID and MID (PlestMID, Nozaki et al. 2006a) that is here called “OTOKOGI” (Fig. 1B) all encode similar RWP-RK do- “OTOKOGI ” (meaning “Japanese male chivalry”). mains and leucine zippers that might be involved in DNA binding and protein dimerization (Schauser et al. A new male-specific gene “OTOKOGI ” 2005), our working hypothesis is that the “OTOKOGI ” gene product plays an important role in male gameto- Nitrogen-starvation of Pleodorina starrii males induces genesis in P. starrii (Nozaki et al. 2006a). the formation of sperm packets that later dissociate into individual sperm capable of fertilizing female ga- Evolution of “OTOKOGI ” and female/male dif- metes, or eggs (Nozaki et al. 2006b). Nozaki et al. ferentiation in the Volvocaceae (2006a) used differential cDNA screening of nitrogen- starved male and female cultures to identify a cDNA A phylogenetic analysis was performed with a that was present only in nitrogen-starved, sexually- 53-residue region spanning the RWP-RK domains induced males. Like products of the MID genes of two of the three MID proteins (CrmMID, CiMID and species of Chlamydomonas (CrMID and CiMID from “OTOKOGI”) and 67 other sequences containing this C. reinhardtii and C. incerta, respectively, Ferris et domain, including 33 from chlorophytes (Chlamy- al. 1997), “OTOKOGI ” has a median G+C content domonas, Volvox, Ostreococcus and various land plants). of 48.4% in the coding region, and encodes a short This group of proteins included a number of nodule ORF (163aa) containing an RWP-RK domain and a inception (NIN) and NIN-like proteins (Schauser et leucine-zipper in the C-terminal region (Fig. 1B) (Fer- al. 2005). Our analysis revealed clearly that the three ris & Goodenough 1997; Ferris et al. 1997; Nozaki et MID/“OTOKOGI” proteins formed a monophyletic al. 2006a). We established the presence of four introns, group with moderate bootstrap values, whereas all of three of which are in positions very similar to those of the other RWP-RK proteins of Chlamydomonas and the three introns that are present in the CrMID and Volvox were separate from the MID/“OTOKOGI” lin- CiMID genes (Fig. 1B) (Ferris et al. 1997, Nozaki et al. eage (Fig. 2). This outcome suggests that MID probably 2006a). A second MID-like sequence, PsPlestMID,was evolved early in volvocalean evolution as a gene regu- found in the male genome, but is probably a pseudo- lating the dominant mating type, and was conserved gene of “OTOKOGI ” (Nozaki et al. 2006a). A South- for such a function in “OTOKOGI ” of the anisoga- ern blot of restriction-digested male and female DNA mous/oogamous Volvocaceae. Thus, males in oogamous probed with a PCR-fragment of “OTOKOGI”thatcov- volvocaceans such as P. starrii are homologous to the ered the second exon indicated that both “OTOKOGI ” dominant, minus mating type of C. reinhardtii.There- and PsPlestMID are present in the genome of the fore, evolution of sperm-producing males in the fam- 774 H. Nozaki

Fig. 1. The anisogamous/oogamous colonial green alga Pleodorina starrii and its male specific genes “OTOKOGI ”(PlestMID)and PsPlestMID. A – Vegetative colonies, female gamete (egg, asterisk) and male gamete (sperm, arrow). B – Alignment of the sequences of “OTOKOGI” and two Chlamydomonas MID proteins (CrMID and CiMID). Blue shading: intron locations; red box: an RWP- RK motif; arrows: a leucine-zipper; asterisks: identical amino acids. C – Southern hybridization of “OTOKOGI ”probewithBamHI (B) and SalI (S) fragments of DNA from female (F) and male (M) P. starrii strains. D – Semiquantitative RT-PCR analyses of “OTOKOGI ”andPsPlestMID genes in female (F) and male (M) strains that were either nitrogen deprived to induce gametogenesis (N) or nitrogen fed (V). The EF-1 like gene was used as an internal control. E – Visualization of “OTOKOGI” protein in mature sperm after release from sperm packets. “OTOKOGI ” expression is obvious in nucleus (arrowheads in DAPI images). Specimens were double-stained with 4’6–diamidino-2–phenylindole (DAPI) and either anti-“OTOKOGI ” antibodies. All panels show identical cells. DAPI (pseudo-coloured) and immunofluorescence images are merged (rightmost panels). Scale bar represents 5 µm (from Nozaki et al. 2006; reproduced by permission of Elsevier Ltd., Licence No. 1961691484312). ily Volvocaceae appears to be based on the genetic 1999) after the establishment of oogamy in the ori- system controlled by the dominant, mating type mi- gin of Metazoa (animals) (∼600 MYA, Rokas et al. nus locus of the family’s isogamous, Chlamydomonas- 2005), isogamous C. reinhardtii and oogamous volvo- like ancestor. This system bears some resemblance caceans such as Pleodorina appear to have shared a to the X/Y sex-determining chromosomal system of common ancestor as recently as 50 ±20 MYA (Kirk mammals, in which the male is the dominant phe- 2006) (Fig. 3). Thus, it appears that male/female dif- notype. But whereas the X/Y system that controls ferentiation evolved very rapidly in these green algae male/female differentiation in the diploid mammals is based on the MID-regulated, sex-determination system thought to have evolved 240–300 MYA (Lahn & Page in the haploid cells. Such rapid evolution of gamete di- A new male-specific gene “OTOKOGI ”fromPleodorina starrii 775

Fig. 2. Phylogenetic analyses suggesting a relationship between sex-specific MID/“OTOKOGI” proteins and other RWP-RK domain- containing proteins. Green, red and yellow colors represent green plants, red algae and oomycetes, respectively. Numbers without or within parentheses indicate bootstrap values of 50% or more, based on 500 or 100 replicates by Maximum Likelihood (based on WAG model of protein evolution) or Minimum Evolution (using p-distances), respectively. Abbreviations used by Schauser et al. (2005), accession numbers, or other forms of gene identification are given after the species names (from Nozaki et al. 2006; reproduced by permission of Elsevier Ltd., Licence No. 1961691484312). morphism may also have occurred in the origin of the the CrMID gene product. However, the marked expres- animals and land plants based on the dominant mating sion of “OTOKOGI” protein in the individual sperm type-controlled, sex-determination system of the hap- that have been released from sperm packets (Fig. 1E) loid ancestral organisms. may indicate that in addition to its presumed role In Chlamydomonas reinhardtii, CrMID is located in gametogenesis, this protein may also contribute to in the minus MT-locus, which consists of an ∼1Mb maintenance or mating behaviour of the male gametes. region of recombinational suppression; it is not found The male-specific gametic traits in the oogamous volvo- in the mating type plus locus (Ferris & Goodenough caceans are more complex than those of mating type 1994). Since both “OTOKOGI ”andPsPlestMID are minus gametes of Chlamydomonas, especially before found only in the male genome (Fig. 1C), we as- syngamy (Nozaki 1996). The differentiation of male sume that they are located in the male mating locus gametes of Pleodorina starrii involves the formation of Pleodorina starrii. Ferris and Goodenough (1997) of specialized male organs called sperm packets that demonstrated that the CrMID gene is necessary and move to the female colony and dissociate into individ- sufficient to convert a plus cell into a minus cell, ual, small, spindle-shaped male gametes that penetrate and suggested that all other genes that encode minus- the female colony in search of fertilizable eggs (Nozaki specific gametic traits are carried by both mating type et al. 2006b). Therefore, essential genes encoding the plus and munus cells. Those genes can be considered mating type minus/male-specific gametic traits proba- “autosomal” genes whose expression is controlled by bly have increased in number during the evolution of 776 H. Nozaki

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