Evolutionary Divergence of the Sex-Determining Gene MID Uncoupled from the Transition to Anisogamy in Volvocine Algae Sa Geng, Ayano Miyagi and James G
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© 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev162537. doi:10.1242/dev.162537 RESEARCH ARTICLE Evolutionary divergence of the sex-determining gene MID uncoupled from the transition to anisogamy in volvocine algae Sa Geng, Ayano Miyagi and James G. Umen* ABSTRACT organism (Matt and Umen, 2016). Members of the smaller, less Volvocine algae constitute a unique comparative model for complex genera such as Chlamydomonas and Gonium have investigating the evolution of oogamy from isogamous mating isogamous mating systems with two mating types, while anisogamy types. The sex- or mating type-determining gene MID encodes a characterizes most intermediate genera such as Eudorina and conserved RWP-RK transcription factor found in either the MT− or Pleodorina, and oogamy is found in the most complex genus male mating locus of dioecious volvocine species. We previously Volvox (Nozaki, 1996, 2003; Nozaki et al., 2000) (Fig. 1). found that MID from the isogamous species Chlamydomonas In the isogamous species Chlamydomonas reinhardtii, cells have reinhardtii (CrMID) could not induce ectopic spermatogenesis when either a minus or plus mating type. minus and plus gametes are expressed heterologously in Volvox carteri females, suggesting morphologically similar, yet express mating type-specific genes that coevolution of Mid function with gamete dimorphism. Here we allow fusion with a partner of the opposite mating type found that ectopic expression of MID from the anisogamous (Goodenough et al., 2007). The differentiation of minus and plus species Pleodorina starrii (PsMID) could efficiently induce gametes in C. reinhardtii is governed by a mating locus (MT) whose − spermatogenesis when expressed in V. carteri females and, two haplotypes, MT+ and MT , are large, rearranged multigenic unexpectedly, that GpMID from the isogamous species Gonium regions that are suppressed for recombination and segregate as pectorale was also able to induce V. carteri spermatogenesis. Neither single Mendelian alleles (De Hoff et al., 2013; Ferris and VcMID nor GpMID could complement a C. reinhardtii mid mutant, at Goodenough, 1994). The C. reinhardtii MID gene (CrMID) least partly owing to instability of heterologous Mid proteins. Our data encodes a putative RWP-RK family transcription factor that is − show that Mid divergence was not a major contributor to the transition found in the MT haplotype and is necessary and sufficient to between isogamy and anisogamy/oogamy in volvocine algae, and specify the minus mating type (Ferris and Goodenough, 1997). instead implicate changes in cis-regulatory interactions and/or trans- Proteins from the RWP-RK family have also recently been shown to acting factors of the Mid network in the evolution of sexual play a key role in the life cycles of plants, where they control dimorphism. gametophyte identity or gametophyte-sporophyte transitions (Koi et al., 2016; Kőszegi et al., 2011; Rövekamp et al., 2016; Waki et al., KEY WORDS: Chlamydomonas, Gonium, Pleodorina, RWP-RK, 2011), and RWP-RK or Mid-like proteins were reported in possible Volvox, Sex determination prasinophyte algal sex-determining regions (Blanc-Mathieu et al., 2017; Worden et al., 2009) and in the mating locus of the ulvophyte INTRODUCTION green alga Ulva partita (Yamazaki et al., 2017). Yet, little is known Gamete size dimorphism (anisogamy or oogamy) is a nearly about how RWP-RK proteins have undergone functional ubiquitous trait in multicellular eukaryotes, and is thought to have diversification within and between lineages in the Viridiplantae, originated from an ancestrally isogamous state that is still found in where they are ubiquitous (Chardin et al., 2014). most unicellular eukaryotes (Bell, 1978; Lehtonen et al., 2016; Vegetatively (asexually) reproducing Volvox carteri spheroids of Togashi and Cox, 2011). Although gametic differentiation plays a either sex are morphologically identical, but upon exposure to the crucial role in the evolution of sex, the molecular evolutionary bases glycoprotein hormone sex-inducer (Kochert and Yates, 1974; Starr for the transitions from isogamy to anisogamy (unequally sized and Jaenicke, 1974; Tschochner et al., 1987), both sexes undergo gametes) and oogamy (small motile sperm, large immotile eggs) modified developmental programs that result in differentiation as have been difficult to study in most extant lineages such as plants egg-bearing females or sperm-bearing males (Kochert, 1968; Starr, and animals owing to the ancient origins of this innovation. 1969). Like the case for C. reinhardtii mating types, sexual Volvocine algae form a monophyletic clade that encompasses the differentiation in V. carteri is under the control of a dimorphic unicellular genus Chlamydomonas and multicellular genera with mating locus with two haplotypes, MTF (female) and MTM (male), different gradations of size and complexity, including Gonium, where the V. carteri MID gene (VcMID) is found only in MTM Pleodorina and Volvox, the latter of which contains a few thousand (Ferris et al., 2010; Umen, 2011). We have previously found that cells and exhibits germ-soma differentiation and other developmental VcMID is sufficient to induce spermatogenesis when expressed in V. innovations that result in a functionally integrated multicellular carteri females, and that in its absence germ cell precursors differentiate as eggs (Geng et al., 2014). Therefore, Mid protein has maintained a homologous function in volvocine algae as a dominant Donald Danforth Plant Science Center, 975 N. Warson Rd., St. Louis, MO 63132, USA. determinant of minus/male sexual differentiation. MID genes have been found in the minus or male mating *Author for correspondence ( [email protected]) haplotypes of other volvocine algae, including several isogamous J.G.U., 0000-0003-4094-9045 Gonium species (Hamaji et al., 2008, 2013; Setohigashi et al., 2011) and in anisogamous Pleodorina starrii (Nozaki et al., 2006), Received 18 December 2017; Accepted 13 March 2018 suggesting that the genetic basis of sex or mating type determination DEVELOPMENT 1 RESEARCH ARTICLE Development (2018) 145, dev162537. doi:10.1242/dev.162537 strain (Eve #15) with the PsMID gene expressed under the control of its own promoter/terminator and fused to a blue fluorescent protein (BFP) and a hemagglutinin (HA) epitope tag at its C-terminus (pPsMID-BH), similar to pVcMID-BH that was used previously for generating the V. carteri MID transgenic strains (Geng et al., 2014) (Fig. 2A, Fig. S2A). Briefly, MID-containing expression plasmids were co-transformed with a nitA plasmid (encoding nitrate reductase) into a nitA− strain, and nit+ transformants were selected and further tested. When wild-type vegetative stage females are exposed to sex- inducer their reproductive cells (gonidia) undergo modified embryogenesis to produce sexual progeny containing 32-48 eggs and ∼2000 somatic cells (Kochert, 1968; Starr, 1969; Umen, 2011) (Fig. 2B). When wild-type vegetative males are exposed to sex- inducer, their gonidia also undergo modified development to Fig. 1. Volvocine algal gamete dimorphism and phylogenetic produce sexual progeny containing 128 somatic cells and 128 sperm relationships. Cladogram of selected volvocine algal species with color DIC packets, with each sperm packet containing 64 or 128 sperm cells images taken from vegetative stage cultures. Illustrated beneath each (Fig. 2C, Fig. S6A). In control experiments with Eve::VcMID-BH species image is the mating system employed and a diagram of gamete types. transformants, we observed 100% conversion of presumptive eggs Scale bars: 10 µm, except 100 µm for V. carteri. into sperm packets after sexual induction (Geng et al., 2014). We identified four Eve::PsMID-BH-containing transformants (#1- is conserved throughout the volvocine lineage (Fig. S1A,B). #4, Materials and Methods), two of which (#2, #3) were examined in Interestingly, a MID gene from the homothallic species V. more detail (Table 1 and see below). All four transformants showed africanus (VaMID) showed expression correlating with the degree normal vegetative development (Fig. S3A), and upon sexual of male differentiation in monoecious versus male sexual spheroids, induction produced sperm packets and eggs in different suggesting that MID is associated with the male-female proportions, which ranged from 95% sperm packets to equal ratios differentiation switch even when sexes are not determined by a of sperm packets and eggs (Fig. 2D,E, Table 1). All four dimorphic mating locus (Yamamoto et al., 2017). Other than the transformants also exhibited self-fertility, which is a phenotype we MID gene, no sex-related genes are universally conserved among previously observed in V. carteri male MID partial knockdown the MT loci of V. carteri, Gonium pectorale and C. reinhardtii strains that had a homothallic monoecious (hermaphroditic) (Hamaji et al., 2016). Even though MID is a rapidly evolving gene, phenotype (Geng et al., 2014) (Fig. 2E, Table 1). We chose the finding that MID from C. incerta [now reclassified as C. globosa transformant line #2, which made mostly sperm packets (95% sperm (Nakada et al., 2010)] can substitute for CrMID indicates that packets, 5% eggs) (Fig. 2D), and line #3, which produced about an functional conservation of Mid proteins can be retained after equal ratio of sperm packets and eggs (Fig. 2E), to assess the speciation (Ferris et al., 1997). However, CrMID was not able to expression of PsMID-BH by semi-quantitative RT-PCR and to assess substitute