Evolutionary Divergence of the Sex-Determining Gene MID Uncoupled from the Transition to Anisogamy in Volvocine Algae Sa Geng, Ayano Miyagi and James G

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

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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 for its ortholog in V. carteri, suggesting that significant PsMid-BH protein levels by immunoblotting. Although expression changes in Mid sequence or in its regulatory network were required of PsMID mRNA in P. starrii males is normally induced by nitrogen for the transition to anisogamy and oogamy in the volvocine lineage starvation (Nozaki et al., 2006), we found that expression levels of (Geng et al., 2014). PsMID-BH in V. carteri were similar to those of VcMID-BH (Fig. 2F) Here we set out to more clearly define the point at which Mid and were not influenced by sexual induction or nitrogen availability acquired its male-determining function in volvocine algae by (Fig. S4A). Full-length PsMid protein was detected in both strains ascertaining whether MID genes from the isogamous species and its levels were higher for line #2 than line #3, correlating with its G. pectorale (GpMID) and/or from the anisogamous species P. starrii higher level of spermatogenesis induction (Fig. 2G, Table 1). (PsMID) were able to functionally substitute for VcMID in inducing However, neither of the PsMID-BH-expressing strains produced as spermatogenesis in V. carteri females. We found that PsMID was able much protein as VcMID-BH controls (Fig. 2G). It is not clear why to induce spermatogenesis when expressed in V. carteri females and, there are similar mRNA levels (detected using BFP primers) but unexpectedly, that GpMID also had this capability, although it was different protein levels for lines #2 and #3, but it is possible that the not as effective as PsMID or VcMID. By contrast, neither VcMID nor upstream portions of the mRNAs in each transgenic strain are slightly GpMID could complement a C. reinhardtii mid mutation, a result different due to position effects at their respective genomic insertion most likely due to instability of the non-native Mid proteins in sites (i.e. transcription start site or 5′ UTR differences) and impact C. reinhardtii. Our data suggest that early functional divergence of translation efficiency. It is also possible that the RT-PCR assay loses Mid proteins between unicellular and multicellular volvocine clades some accuracy at the higher cycle numbers required to detect MID underlies the lack of interspecific compatibility between Mid transgenes compared with the internal control transcript from RPS18. orthologs, and that changes in the sex-determination network Our main interest was in assessing relative protein levels of PsMid- unrelated to Mid protein were mainly responsible for the transition BH, so we did not pursue this observation further. from isogamy to anisogamy and oogamy. Similar to previously described phenotypes for VcMID-BH transgenic females (Geng et al., 2014), both of the characterized RESULTS PsMID-BH-expressing strains had spermatogenesis-related MID from an anisogamous genus, Pleodorina, can activate developmental defects, including sperm cell morphological spermatogenesis in Volvox abnormalities and delayed sexual development (Figs S5 and S6). In order to test whether PsMid is capable of inducing They also showed delayed or incomplete hatching of sperm packets spermatogenesis in V. carteri, we transformed a female V. carteri from their vesicles that was more severe than the delayed vesicle DEVELOPMENT

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Fig. 2. Expression of PsMID in V. carteri females. (A) The PsMID-BH construct. Red boxes, PsMID coding sequence; light blue box, BFP; orange box, 2x hemagglutinin (HA) epitope tag; red lines, non-coding regions. Arrows indicate positions of primers for cDNA amplification in F. (B) Wild-type mature sexual Eve (female) spheroid with ∼45 large green eggs. (C) Wild-type mature sexual AichiM (male) spheroid with 128 sperm packets. Inset shows single sperm packet. (D) Sexually induced Eve::PsMID-BH #2 spheroid with sperm packets, two of which (arrowed) are magnified in the inset. (E) Sexually induced Eve::PsMID-BH #3 spheroid with unfertilized egg that has de-differentiated and begun a program of vegetative embryogenesis (Ei; arrowhead indicates vesicle wall surrounding young spheroid), fertilized egg (Eii; arrow indicates thick wall forming around zygote), intact sperm packet (Eiii) and dissolved sperm packet within its vesicle wall (Eiv, arrowhead). Scale bars: 100 µm in B-E. (F) Expression of VcMID-BH and PsMID-BH transgenes in the indicated strains as assessed by semi- quantitative RT-PCR with BFP-F/BFP-R primers and with ribosomal protein gene S18 as an internal control. Reactions were stopped at the indicated cycle numbers and PCR products visualized in ethidium bromide-stained agarose gels. –, no-template control. (G) Immunoblots of extracts from the indicated strains probed with anti-HA (top) or anti-tubulin antibodies as a loading control (bottom). hatching in VcMID-BH transformants (Table 1, Fig. 2E, Fig. S3B,C, Eve::PsMID-BH versus Eve::VcMID-BH transformants could have Fig. S6). However, unlike sperm from VcMID-BH transformants, been partly due to the impact of the epitope tag on PsMid function. which were eventually released from their mother spheroid (Fig. Therefore, we generated untagged PsMID constructs (Fig. S2B) and S3B, Fig. S6B), most of the PsMID-BH transformant sperm from tested four independent Eve::PsMID transformants identified by both lines #2 and #3 were unable to leave the mother spheroid and PCR genotyping (Geng et al., 2014). Unlike the case for tagged remained trapped within a partially dissolved sperm packet vesicle PsMID-BH constructs, where conversion to sperm packets was (Fig. 2E, Fig. S3C, Table 1). incomplete, in all four untagged Eve::PsMID transformants we Although the BFP-HA tag fused to VcMid had no detectable observed 100% conversion of eggs to sperm packets, indicating that impact on the function of endogenous VcMid-BH protein the BFP-HA tag may weaken Mid activity and/or partially expressed in V. carteri females (Geng et al., 2014) (Table 1), the destabilize the protein. Interestingly, however, the phenotypes of weaker penetrance and more severe sperm hatching defects in incomplete vesicle hatching and inability to release from mother DEVELOPMENT

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Table 1. Sexual phenotypes of female Volvox MID transformants Sperm packet hatching Sperm packet hatching Wild-type or transgenic line Lines Eggs* Sperm packets* Self-fertility‡ from vesicle§ from spheroid¶ Self-induction** AichiM (male) 0 (0/2275) 100 (2275/2275) No ++++ +++ Yes Eve (female) 100 (708/708) 0 (0/708) No N/A N/A No Eve::VcMID-BH #3 0 (0/618) 100 (618/618) No +++ ++ Yes #4 0 (0/631) 100 (631/631) No +++ ++ Yes Eve::PsMID #1 0 (0/581) 100 (581/581) No ++ − Yes #2 0 (0/614) 100 (614/614) No ++ − Yes Eve::PsMID-BH #1 40 (209/546) 60 (328/546) Yes ++ − Yes #2 5 (31/597) 95 (563/597) Yes ++ − Yes #3 50 (310/615) 50 (288/615) Yes ++ − Yes #4 35 (186/538) 65 (348/538) Yes ++ − Yes Eve::GpMID #1 5 (26/528) 95 (500/528) Yes + − No #2 10 (50/504) 90 (451/504) Yes + − No #3 5 (24/482) 95 (456/482) Yes + − No #4 10 (39/520) 90 (475/520) Yes + − No #5 10 (43/514) 90 (465/514) Yes + − No Eve::GpMID-BH #1 60 (301/507) 40 (185/507) Yes + − No #2 60 (273/477) 40 (179/477) Yes + − No Eve::GpMIDcDNA-BH #1 60 (295/481) 40 (177/481) Yes + − No #2 60 (296/503) 40 (186/503) Yes + − No *The approximate percentage of eggs or sperm packets produced, with absolute numbers indicated in parentheses. ‡Yes, mixture of self-fertile eggs and sperm produced by indicated strain; no, spheroid produces either all eggs or all sperm packets. §Speed and completeness of sperm packet hatching represented by the number of ‘+’ symbols. N/A, not applicable. ¶Speed and completeness of sperm packet release from parental spheroid represented by number of ‘+’ symbols, or by ‘−’ if no sperm packets were observed to hatch. N/A, not applicable. **Observation of spontaneously occurring sexual spheroids in non-induced cultures. n=20 for each wild-type and all transgenic lines. spheroids were not alleviated in Eve::PsMID strains (Fig. S3C, weakened the expression or activity of Mid proteins, we found Fig. S6B, Table 1). We note that these hatching-related phenotypes that tagged Eve::GpMID-BH and Eve::GpMIDcDNA-BH strains are unlikely to be just the result of reduced PsMID expression as both had only ∼40% of their eggs converted to sperm they were not observed in V. carteri male strains in which packets (Fig. 3E, Fig. S7A, Table 1). Nonetheless, it was endogenous VcMID expression was partially reduced by RNAi remarkable that GpMid from an isogamous species could largely (Geng et al., 2014). We conclude that PsMid protein is likely to have substitute for VcMid in driving ectopic spermatogenesis in somewhat weaker or altered activity compared with VcMid protein, V. carteri females. and that these differences with native VcMid function led to more Other than the degree to which eggs were converted to sperm severe defects in sperm packet hatching from their vesicles and in packets, strains expressing each of the three different GpMID the release of sperm from the mother spheroid in PsMID-expressing constructs we tested (untagged genomic, tagged genomic, tagged females. Although the BFP-HA tag caused some functional cDNA; Fig. 3A-C) had nearly identical developmental phenotypes impairment of Mid proteins, use of the tag also served as a as described below. Besides the formation of ectopic sperm packets diagnostic tool to help evaluate relative Mid activity, so we and self-fertility (Fig. S7A-C), the additional phenotypes included continued to use it in combination with untagged constructs for sperm cell developmental abnormalities and incomplete or delayed experiments with MID from the isogamous species Gonium hatching from the sperm vesicle that were even more severe than pectorale, as described below. those observed for PsMID transformants: in GpMID transformants, most sperm packets never hatched from their vesicle, but instead MID from an isogamous genus, Gonium, can induce dissolved within it; and we never observed sperm or sperm packets spermatogenesis in Volvox released from parental spheroids (Fig. S6B, Fig. S7A, Table 1). As we did for PsMID, we generated tagged and untagged GpMID Finally, unlike the case for natural V. carteri males or for female transgenic constructs driven by its native promoter/terminator VcMID and PsMID transformants, we never observed spontaneous (Fig. 3A,B, Fig. S2C,D) and assessed their phenotypic effects production of sexual spheroids (i.e. self-induction) with any of the when expressed in female V. carteri transformants. We also GpMID transformants, all of which had to be treated with generated a tagged construct that expressed the GpMID cDNA exogenously applied sex-inducer to initiate sexual development (GpMIDcDNA-BH) (Fig. 3C, Fig. S2E) to control for possible (Table 1). defects in RNA processing that we observed in transformants expressing the native GpMID gene (see below). Five independent Pleiotropic developmental defects caused by GpMID untagged Eve::GpMID transformants (#1-#5) all had self-fertile expression in Volvox hermaphrodite/homothallic phenotypes with 90-95% conversion of Besides the sperm development and hatching abnormalities eggs to sperm packets. However, among all five independent Eve:: observed in GpMID-expressing transformants, all of the different GpMID lines we very rarely saw complete conversion to sperm GpMID constructs we tested caused additional sexual stage and packets (<1% of transformants), in contrast with what we observed vegetative stage phenotypes that were never observed in PsMID or for untagged PsMID or VcMID transgenics (Fig. 3D, Table 1). VcMID transgenic strains. These included smaller vegetative and

Supporting the idea that the BFP-HA epitope tag somewhat sexual spheroids with fewer vegetative gonidia or sexual germ cells DEVELOPMENT

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Fig. 3. Expression of GpMID in Volvox females. (A) Native GpMID genomic construct. (B) GpMID genomic construct fused in frame to C-terminal BFP-2xHA tag. (C) GpMID cDNA fused in frame to C-terminal BFP-2xHA tag. (A-C) Gray boxes, GpMID coding sequence; gray lines, non-coding and intronic sequences; light blue box, BFP; orange box, 2xHA tag. Arrows indicate positions of primers for cDNA amplification in H,I or in Fig. S4. (D) Sexually induced Eve::GpMID spheroid with normal morphology. An individual sperm packet (arrowhead) is magnified in inset. (E) Sexually induced Eve::GpMIDcDNA-BH spheroid. Inset shows sperm packet (arrowhead) and egg (arrow). (F) Normal somatic cell arrangement with even spacing of cells (magnified in inset) from sexual stage parental strain E15. (G) Irregular somatic cell spacing in sexual stage Eve::GpMID-BH spheroid. Scale bars: 100 µm. (H,I) GpMID cDNA amplified by RT-PCR from the indicated strains using primers GpMidGOI.f1 and GpMidGOI.r1, which amplify the entire GpMID coding region. PCR products were separated on agarose gels and stained with ethidium bromide. Arrowheads show position of cDNA from correctly processed full-length GpMID, and asterisk (H) indicates the main isoform amplified from genomic GpMID constructs. Isoform structures are diagrammed in Fig. S9. (J) Anti-HA immunoblot (top) of SDS-PAGE fractionated protein extracts of Eve::GpMIDcDNA-BH and control transformants, and Coomassie Blue-stained gel (bottom) used as a loading control. Asterisks mark likely breakdown products.

than in control strains (Fig. S8, Table S1). About 30-50% of the Fig. S8A), and ∼30-40% of the vegetative-phase spheroids from vegetative and sexual spheroids that expressed GpMID had the same strains were misshapen, which might have been caused by unevenly spaced and disorganized somatic cells (Fig. 3F,G, incomplete or aberrant inversion – the post-cleavage reversal of DEVELOPMENT

5 RESEARCH ARTICLE Development (2018) 145, dev162537. doi:10.1242/dev.162537 embryo curvature that turns the spheroid right-side out (Fig. S8B) with those of ribosomal protein RPL23 (pL23:CrMID-6XFLAG) (Kirk and Nishii, 2001; Sessoms and Huskey, 1973). We classified (Fig. S10A), which we have previously used to generate high-level these abnormal phenotypes as pleiotropic because they did not transgene expression in C. reinhardtii (Li et al., 2016; López-Paz relate to any processes associated with normal sexual development et al., 2017). We co-transformed CC3712 with the pL23:CrMID- or germ cell formation. 6XFLAG construct and a hygromycin resistance (HygR) marker gene The pleiotropic defects in GpMID transformants prompted us to (Berthold et al., 2002) and screened HygRcolonies for presence of the examine whether the expected mRNA and proteins were being transgene (Materials and Methods). Around 24% of the HygR produced. mRNA for GpMID-BH, as detected using BFP primers, colonies tested positive for having the MID transgene (22/91), and was expressed at similar levels to VcMID-BH and to PsMID, and most of these positive colonies (19/22) showed full complementation was not affected by nitrogen availability or presence of sex-inducer of the mid deletion phenotype (100% minus mating phenotype; no (Fig. S4B,C), even though in its native context GpMID is induced self-agglutination) and produced CrMid protein that was detectable by nitrogen starvation (Hamaji et al., 2008). However, most of the by immunoblotting (Fig. 4A, Table 2). GpMID mRNA produced in V. carteri female transformants The high-frequency complementation of the mid deletion expressing GpMID and GpMID-BH was partially processed, with mutation obtained with this control construct facilitated testing of a variety of isoforms, the most abundant of which skipped exon 3 heterologous MID genes for complementation in C. reinhardtii.We and used an internal 3′ splice acceptor within exon 4 to produce a transformed RPL23 promoter-driven FLAG-tagged cDNAs for shorter mRNA containing a predicted frameshift (Fig. 3H, Fig. GpMID and VcMID (Fig. S10B,C) into CC3712 and assessed co- S4D, Fig. S9B). On the other hand, the only message detected for transformation rates, mating type complementation, mRNA GpMIDcDNA-BH transformants was full length (Fig. 3I) and it was expression, and protein levels. Co-transformation rates with these expressed at similar levels to VcMID-BH (Fig. S4E), indicating that constructs were similar to those we observed for pL23:CrMID- altered mRNA processing and the resulting frameshifted 6XFLAG (24/112 and 13/96, respectively, by colony PCR), but in polypeptides are unlikely to underlie the phenotypic effects no case did we see even partial complementation of the mid deletion associated with GpMID expression in V. carteri females. phenotype; nor were we able to detect tagged protein for any of the Immunoblots from strains expressing tagged GpMIDcDNA-BH or GpMID and VcMID transformants despite detecting the expression GpMID-BH detected proteins that migrated as predicted for full of full-length GpMID and VcMID mRNAs in several selected strains length or near full length, but also smaller products that are likely to (Fig. 4B-D). Although in some circumstances codon optimization be stable degradation fragments containing the C-terminal epitope can improve transgene expression (Barahimipour et al., 2015), the tag (Fig. 3J, Fig. S7E). The total amount of GpMid-BH protein signal endogenous CrMID gene shows almost no codon bias relative to detected in transgenic GpMIDcDNA-BH strains was less than for optimal codon usage in C. reinhardtii (Ferris et al., 1997), yet the VcMid-BH (Fig. 3J) and this reduction was most likely attributable endogenous transgenic CrMid protein was easily detectable by to reduced translation or protein stability, as the detectable mRNA for immunoblotting (Fig. 4A). Moreover, the GpMID and VcMID genes this construct was all full length (Fig. 3I) and expressed comparably have C. reinhardtii codon adaptation indices (CAIs) in the same to VcMID-BH mRNA (Fig. S4E). Together, these findings suggest range as the endogenous CrMID gene (CrMID, 0.370; GpMID, either inefficient translation or protein instability as the cause for 0.429; VcMID, 0.324). By contrast, well-expressed genes such as reduced steady-state levels of GpMid-BH in transgenic strains. TUA1 (alpha tubulin) and RPL23 (cytoplasmic ribosomal protein) Importantly, the pleiotropic developmental defects caused by have CAIs of 0.813 and 0.751, respectively. We conclude that GpMID expression in V. carteri females were not influenced by heterologous Mid proteins from G. pectorale and V. carteri are the presence/absence of the epitope tag or by the substitution of a poorly expressed in C. reinhardtii, most likely because of protein cDNA for the genomic version (Table S1). In summary, the instability and possibly inefficient translation, and that poor pleiotropic developmental phenotypes observed when GpMID was expression may have precluded complementation. expressed in V. carteri were likely to be due to neomorphic (i.e. off- target) interactions between native GpMid protein (or truncated DISCUSSION versions) and other developmental regulators in V. carteri. Coevolution of the MID gene and Mid regulatory networks in volvocine algae GpMID and VcMID cannot substitute for CrMID Our prior work on MID in V. carteri and previous studies of MID in We previously found that CrMID and chimeric constructs expressing C. reinhardtii showed that the presence/absence of native MID gene combinations of N-terminal and C-terminal domains of VcMID and expression in sexually induced individuals is necessary and largely CrMID could be expressed in V. carteri females but could not induce sufficient to determine mating type or germ cell differentiation spermatogenesis (Geng et al., 2014). Experiments to test programs (Ferris and Goodenough, 1997; Geng et al., 2014; Lin and heterologous MID genes in C. reinhardtii have only been reported Goodenough, 2007). Further work showing the inability of ectopic for one closely related sister species, C. incerta [now called C. CrMID or chimeras between CrMID and VcMID to induce globosa (Nakada et al., 2010)], where cross-species complementation spermatogenesis in V. carteri females led to a simple hypothesis was observed (Ferris et al., 1997). In control experiments, we found that the evolution of sexual dimorphism and oogamy in the that complementation of a C. reinhardtii mid deletion mutant strain volvocine lineage was due to molecular changes in the Mid protein CC3712 by the endogenous CrMID gene or a C-terminally FLAG (Geng et al., 2014). The experiments described here rule out the epitope-tagged version of CrMID-6XFLAG occurred at low simplest version of this hypothesis because GpMid from the frequency and was often just partial (evidenced by self- isogamous species G. pectorale could induce spermatogenesis agglutination), as was previously reported (Ferris and Goodenough, when ectopically expressed in V. carteri females, albeit at reduced 1997; Lin and Goodenough, 2007) (data not shown). To overcome efficiency (Fig. 3, Table 1). It would still be possible that the problem of low efficiency expression and complementation with anisogamy/oogamy coevolved with Mid if Gonium or related native CrMID, we made a version of the FLAG epitope-tagged MID clades had been ancestrally anisogamous/oogamous but secondarily construct in which the native MID promoter/terminator were replaced lost this trait. If so, then GpMid might still have residual DEVELOPMENT

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Fig. 4. Complementation of Chlamydomonas mid mutant. (A) Anti-FLAG immunoblot of SDS-PAGE fractionated protein extracts from gametes of independent CC3712::L23:CrMID-6XFLAG transformants that tested positive for the CrMID transgene (lanes 1-11) and parental mid mutant strain CC3712 (lane 12). (B,C) (Top) Anti-FLAG immunoblot of SDS-PAGE fractionated protein extracts of CC3712 (lane 1), a positive control CC3712::L23:CrMID-6XFLAG line (lane 2), and independent transformants (lanes 3-12) containing CC3712::L23:GpMID-6XFLAG (B) or VcMID-6XFLAG (C). (A-C) Lower panels are the same blots stained with Ponceau S as a loading control. (D) Ethidium bromide-stained agarose gel of RT-PCR products from CC3712 transformants or control samples used to detect expression of MID transgenes using primers VcMID_ATG_F and VcMid-C-R3 for VcMID, primers GpMID-BamATG and GpMID-XhoTTA for GpMID, and primers CrMID_F and CrMID_R for CrMID. Amplification of 18S rRNA was used as an internal control. Reactions with non-transformed CC3712 cDNA as template, or with template substituted by water, provided negative controls. Primers and transgene constructs are diagramed in Fig. S10. functionality in a dimorphic sex-determining regulatory circuit. functional divergence and loss of interspecific compatibility However, secondary loss of anisogamy/oogamy is unlikely since all between transcription factor (TF) orthologs is not an inevitable species of the genus Gonium and other genera at or near the base of outcome over the time scale of 200-300 million years (MY) during the multicellular volvocine radiation (Astrephomene and which volvocine algae diversified (Herron et al., 2009). For Tetrabaena/Basichlamys) are isogamous (Nozaki, 1996, 2003; example, the well-known metazoan Eyeless/PAX6 TFs retained Nozaki et al., 2000, 1996). function in eye development between fruit flies and mice over 800 Our data on cross-species complementation with Mid instead MY despite the evolution of anatomically completely different eye support a phyletic model in which Mid functional divergence was structures between arthropods and vertebrates (Kumar et al., 2017; not coupled to the evolution of gamete dimorphism, but occurred in Shubin et al., 2009). Similarly, an ortholog of the circadian TF a more gradual manner that was proportional to divergence times CONSTANS (CO) from Chlamydomonas was able to complement between lineages. Under this model, the Mid protein from the an Arabidopsis CO mutant (Serrano et al., 2009), although the two Chlamydomonas lineage has accumulated too many changes for it CO orthologs share significantly less sequence similarity (32% to function in the multicellular volvocine taxa (and vice versa), but identity/38% similarity) than the most diverged Mid orthologs in the core interactions between Mid, its target DNA sequences, and volvocine algae (41% identity/52% similarity) (Fig. S1), and exhibit other transcriptional regulators have been sufficiently conserved a much deeper phylogenetic divergence of ∼1000 MY (Kumar within the multicellular taxa (Volvox, Pleodorina, Gonium) for it to et al., 2017). Ideas about how Mid proteins and the Mid sex- retain basic function across these genera (Fig. 1). We note that determination network might have diverged in function within the volvocine algae are discussed below. Table 2. Complementation of Chlamydomonas mid mutation The ability of GpMid from an isogamous species to partially Hygromycin- function in V. carteri male differentiation means that the transition resistant Co-transformants to anisogamy/oogamy was not driven directly by changes in Mid, Transgenic line colonies (from genotyping) Complemented but mainly by changes in its direct and indirect target genes that CC3712::L23: 91 22 19 evolved to control the developmental programs leading to gamete CrMID:6XFLAG dimorphism. Changes in TF target genes can result from alterations CC3712::L23: 112 24 0 in the DNA binding specificity of a TF (and/or its co-regulators) or GpMIDcDNA:6XFLAG from loss/gain of cis-regulatory elements (Wray et al., 2003). Sexual CC3712::L23: 96 13 0 development and mating behaviors in V. carteri are significantly VcMIDcDNA:6XFLAG more complex than mating type specification and gamete DEVELOPMENT

7 RESEARCH ARTICLE Development (2018) 145, dev162537. doi:10.1242/dev.162537 interactions in C. reinhardtii or Gonium (Nozaki, 1996; Umen, that a single spontaneously produced sexual male can induce the 2011), implying that during the evolution of anisogamy/oogamy remaining spheroids in an entire culture (or, presumably, an entire Mid must have gained control over new genes and/or regulons natural pond environment) to undergo sexual development, a responsible for this additional complexity. In the isogamous, process termed self-induction. Wild-type V. carteri males unicellular C. reinhardtii a few hundred genes exhibit mating type- frequently self-induce in culture (Kirk, 1998; Starr, 1970) and we specific differential regulation (Joo et al., 2017; Lopez et al., 2015; previously observed the same phenomenon in V. carteri females Ning et al., 2013) but very little is known about mating type and sex- expressing VcMID (Geng et al., 2014). We also observed self- regulated genes in multicellular volvocine algae (Ferris et al., 2010; induction in V. carteri PsMID transformants, but never for GpMID Umen, 2011). We predict that many new direct or indirect targets of transformants (Table 1). Although the lack of self-induction in Mid arose in the transition from isogamy to anisogamy/oogamy, GpMID transformants could be attributed to low levels of GpMid and future work aimed at identifying Mid targets in isogamous and protein in transgenic strains, we note that the best sperm packet sexually dimorphic volvocine species will help illuminate how the conversion rates of GpMID transgenics (∼95%) were higher than Mid network changed and/or expanded in this lineage. those of the weakest PsMID transformants (∼50%), yet the PsMID Our data suggest that the largest changes in the Mid network were transformants all showed normal self-induction (Table 1). This unrelated to sexual dimorphism and occurred after the split from the suggests that some aspect(s) of the sex-inducer production/sensing/ last common ancestor shared by C. reinhardtii and the multicellular amplification process are under the control of Mid, and that the volvocine clade (V. carteri, P. starrii and G. pectorale) (Fig. 1). We activity of GpMid protein is unable to support the process of self- hypothesize that, after this split, Mid proteins from the two lineages induction and/or signal amplification. gradually became incompatible due to altered network interactions, During normal male development in V. carteri, sperm must ultimately leading to the instability of heterologous Mid proteins breach three ECM-based barriers in order to be released and fertilize expressed in C. reinhardtii (Fig. 4) and the inability of CrMid to a female. The first barrier is the vesicle in which each sperm packet function in V. carteri (Geng et al., 2014). These incompatibilities is formed, a structure analogous to the vesicle that encapsulates a could have arisen from neutral drift in rapidly evolving MID genes vegetative V. carteri embryo, or to a mother cell wall in a postmitotic (Ferris et al., 1997; Yamamoto et al., 2017) that resulted in altered C. reinhardtii division cluster. The second barrier is the ECM within protein-protein interactions, perhaps similar to what has been the parental spheroid and the sheath material surrounding the observed in TFs that control fungal mating type regulatory circuits parental spheroid (Jaenicke and Waffenschmidt, 1981). The third (Tuch et al., 2008a,b). TFs are often targeted for rapid degradation and barrier is the female mating partner’s ECM, which must be entered may be especially sensitive to changes in structure or conformation through a fertilization pore that forms after a sperm packet contacts a that are coupled to regulated turnover (Kodadek et al., 2006; Yao and sexual female (Kochert, 1968; Starr, 1969). Ndoja, 2012). We speculate that our inability to detect expression of In PsMID-expressing and GpMID-expressing V. carteri female heterologous Mid proteins in C. reinhardtii (Fig. 4) and the transgenic strains, defects in vesicle hatching and parental spheroid prevalence of non-full-length Mid protein fragments for GpMid hatching were both evident to different degrees, suggesting that and CrMid proteins expressed in V. carteri (Geng et al., 2014) production of the relevant sexual stage hatching enzymes was (Fig. 3J, Fig. S7E) reflect the lability of Mid protein and sensitivity to inadequate in these transgenic strains due to weakened or altered changes in its interaction network. For example, if Mid is protected Mid activity (Table 1). By contrast, dissolution of sperm packets, from degradation by binding to a second stabilizing protein with possibly mediated by V. carteri sperm lysin (Waffenschmidt et al., which it has coevolved, then a weakened interaction between these 1990), still occurred in all transgenic MID strains (Fig. 2, Figs S3 and two partners in the case of non-endogenous Mid proteins could lead S8). Release of sperm packets from their vesicle is likely to be mediated to decreased Mid stability. The pleiotropic developmental phenotypes by proteolytic hatching enzymes such as the VHE/lysin/sporangin caused by expression of GpMid in V. carteri might further reflect subfamilyof proteases, which in C. reinhardtii are secreted through the promiscuous interactions of GpMid with off-target partners or cis- daughter cell flagella to enable hatching (Kubo et al., 2009). Once regulatory motifs that are also thought to coevolve with TFs (Baker released from their vesicle, sperm packets are still constrained within et al., 2011; Nadimpalli et al., 2015; Sayou et al., 2014). their parental spheroids by ECM and the outer wall or boundary zone (sheath), which constitutes the second barrier to sperm release New Mid-controlled aspects of male sexual development (Jaenicke and Waffenschmidt, 1981). An analogous process to revealed from cross-species complementation sperm packet release from spheroids is daughter colony release Although the core functions of spermatogenesis were fulfilled by during V. carteri vegetative reproduction, a process that is mediated by PsMid and GpMid proteins when expressed in V. carteri,we specific hatching enzymes VheA and LSG2 (Fukada et al., 2006; highlight below new or enhanced phenotypes caused by Nishimura et al., 2016), the former of which is homologous to C. heterologous MID gene expression that shed light on different reinhardtii vegetative hatching enzyme (Kubo et al., 2009). During aspects of V. carteri sexual development and the Mid pathway: self- their sexual cycle, C. reinhardtii cells produce a second type of induction, sperm packet release from vesicles, and sperm packet hatching enzyme called gamete lytic enzyme (GLE or G-lysin), which release from parental spheroids. belongs to a family of matrix metalloproteases (MMPs) distinct from An interesting property of the V. carteri sex induction pathway is vegetative hatching enzyme (Buchanan et al., 1989; Kinoshita et al., its ultra-sensitivity to the sex-inducer hormone, a glycoprotein 1992; Matsuda et al., 1985). We predict that one or more sexual stage- related to other hydroxyproline-rich glycoproteins that constitute specific GLE-like or Vhe-like activities might be defective in PsMID most of the V. carteri extracellular matrix (ECM) (Gilles et al., 1984; and GpMID transformants, leading to defects in sperm packet release Hallmann, 2003; Mages et al., 1988; Starr and Jaenicke, 1974). from vesicles and parental spheroids, although this remains to be Males and females can produce sex-inducer in response to stress determined. In V. carteri, many ECM genes and ECM MMP-related (Kirk and Kirk, 1986; Nedelcu and Michod, 2003), but sex-inducer gene families are expanded relative to those in C. reinhardtii (Prochnik is also produced spontaneously by males (Starr, 1970). In a poorly et al., 2010), and a subset of these might be dedicated to sperm packet understood process, an initial sex-inducer signal can be amplified so release/hatching and be produced under the control of VcMid. DEVELOPMENT

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MATERIALS AND METHODS amplified with primers GpMid promoter f1 and GpMid3UTR.r1, and Strains and culture conditions ligated into pGEM-T Easy to obtain pGpMID (Fig. S2C). pGpMID-BH Female V. carteri strain Eve (V.c.f.nagariensis UTEX 1885), male plasmid was created by digesting pGpMID and pVcMID-BH with NheI and V. carteri strain Aichi M (V.c. f. nagariensis NIES 398), P. starrii (NIES BamHI, and replacing the VcMID gene from pVcMID-BH with the GpMID 1363) and G. pectorale (NIES 1710) were obtained from UTEX (https:// gene (Fig. S2D). The GpMID cDNA (495 bp) was amplified from G. utex.org) or NIES (http://mcc.nies.go.jp/) stock centers. The female nitA− pectorale cDNA using primers GpMidGOI.f1 and GpMidGOI.r1, which V. carteri strain Eve #15 used for transformations was described introduce flanking NcoI and NheI sites, respectively. The cDNA fragment previously (Geng et al., 2014). All V. carteri strains were cultured in and plasmid pGpMID-BH were digested with NheI and BamHI, and the Standard Volvox Medium (SVM) at 32°C (unless otherwise specified) cDNA fragment was ligated to the backbone of pGpMID-BH to form and synchronized on a 48 h developmental cycle with a 16 h:8 h light: pGpMIDcDNA-BH (Fig. S2E). dark regime (Kirk and Kirk, 1983) with a combination of 100 µE blue Constructs pL23:CrMID-6XFLAG, pL23:GpMIDcDNA-6XFLAG and (465 nm) and 100 µE red (465 nm) LED lights with bubbling aeration. pL23:VcMIDcDNA-6XFLAG were assembled and cloned into the EcoRI Eve::VcMID-BH lines were grown at 28°C for vegetative cultures and site of pGEM-T Easy using Gibson assembly reactions (New England 32°C to obtain sexual cultures. Sexual development was induced by Biolabs, E2611) via ∼20 bp overlapping sequences generated by PCR. adding pre-titered crude sex-inducer to juveniles 24 h prior to embryonic PCRs were performed with Phusion polymerase (Thermo Scientific) cleavage (Starr and Jaenicke, 1974). For the nitrogen (N)-free SVM according to the manufacturer’s protocol. A spacer (GG) and a 6XFLAG growth experiments, V. carteri cultures were first grown in 300 ml SVM sequence (5′-CGCGACTACAAGGACCACGATGGGGACTACAAGG- in flasks with 3000 spheroids/flask, then collected and washed with ACCATGACATCGACTACAAGGACGACGACGACAAGGGCGGCC- 500 ml N-free SVM using a magnetic funnel. The washed cultures were GCGACTACAAGGACCACGATGGGGACTACAAGGACCATGACAT- returned to 300 ml N-free SVM flasks and grown for 24 h prior to RNA CGACTACAAGGACGACGACGACAAGGGCGGCCGC-3′) were inserted isolation. immediately before the Stop codon for all constructs. pL23:CrMID-6XFLAG The C. reinhardtii strains used include the MT− mid deletion mutant was generated as follows. The stretch of C. reinhardtii MID native promoter- CC3712 obtained from the C. reinhardtii Resource Center (www.chlamy. 5′ UTR, coding region and 6XFLAG tag sequence were amplified from org) and the mating testers CC620 (referred to as R3, MT+) and CC621 CrMID-6XFLAG expression plasmid [FLAG Stop #11, a gift of Dr Huawen derivative CJU10 MT− (Umen and Goodenough, 2001). CC3712 Lin (Lin and Goodenough, 2007)] using primers L_CrProEcoRIF and differentiates as a pseudo-plus mating type and agglutinates with minus 6FLAG_AvrIIR1. Another set of primers, 6FLAG_AvrIIF1 and pGEM-T gametes (but cannot fuse) (Ferris and Goodenough, 1997). Strains were Easy EcoRI_p3R2, was used to amplify a 725 bp stretch of the terminator-3′ maintained on TAP agar plates and grown in liquid TAP (Harris, 2009). UTR of the C. reinhardtii RPL23 gene (Cre04.g211800) (López-Paz et al., 2017). The plasmid backbone was prepared by digesting pGEM-T Easy Mid protein alignments and similarity scoring vector with EcoRI. Finally, all three fragments, including the EcoRI-cut Multiple sequence alignments of Mid orthologs from volvocine algae were vector backbone, were assembled using Gibson assembly to yield pCr: generated in MEGA7 (Kumar et al., 2016) using MUSCLE (Edgar, 2004) CrMID-6XFLAG:L23. Next, the promoter-5′bUTR region of pCr:CrMID- and formatted using BoxShade (https://www.ch.embnet.org/software/ 6XFLAG:L23 was replaced with a 1005 bp stretch of RPL23 promoter-5′ BOX_form.html). Identity and similarity were calculated using SIAS with bUTR sequence to yield pL23:CrMID-6XFLAG:L23 (López-Paz et al., default settings (http://imed.med.ucm.es/Tools/sias.html). 2017). The 1005 bp L23 sequence was amplified with primers pGEM-T Easy EcoRI_p3F2 and p3promoter_R. The stretch of C. reinhardtii MID coding Plasmid construction sequence through the L23 terminator-3′ UTR was amplified from pCr: Oligonucleotide primers used for plasmid construction are listed in CrMID-6XFLAG:L23 using primers p3promoter_cN_F and pGEM-T Easy Table S2. EcoRI_p3R2. Finally, these two PCR products and EcoRI-cut pGEM-T Easy Plasmid pPsMID was created as follows. The PsMID promoter and vector backbone were assembled using Gibson assembly. 5′ UTR (−444 bp to −1 bp) were amplified from P. starrii (N1363) pL23:GpMIDcDNA-6XFLAG and pL23:VcMIDcDNA-6XFLAG were genomic DNA using primers PlestMidpromoter f1 and PlestMidpromoter generated as follows. pGEM-T Easy vector backbone containing RPL23 r1, which introduced flanking SacI and NcoI restriction sites, with the ATG promoter-5′ UTR and 6XFLAG-L23 terminator-3′ UTR sequence was start codon of PsMID within the NcoI site (ccATGg). The PsMID gene generated by PCR from pL23:CrMID-6XFLAG:L23 using primers region (coding exons and intervening introns, 1354 bp) was amplified using p3promoter_R and 6FLAG_F2. Next, GpMID and VcMID coding primers PlestMid GOI f1 and PlestMid GOI r1, which introduce flanking sequences were amplified from cDNA-containing plasmids with primers NcoI and NheI sites, respectively. The PsMID 3′ flanking region (378 bp p3promoter_gN_F and GpMID-6FLAGR, and with p3promoter_vN_F and following the Stop codon) was amplified with primers PlestMid3′UTRf1 VcMID-6FLAGR, respectively. Finally, each coding sequence was and PlestMid3′UTRr1, which introduce flanking BamHI and KpnI assembled with the PCR-generated vector backbone using Gibson restriction sites. The above three fragments were digested at the assembly. appropriate flanking restriction enzyme sites, ligated together, and the full-length product amplified with primers PlestMidpromoter f1 and PCR amplification conditions, RNA preparation, cDNA PlestMid3′UTRr1, and ligated into pGEM-T Easy vector (Promega) to preparation and immunoblotting for Volvox-related experiments obtain pPsMID (Fig. S2C). pPsMID-BH was created by digesting plasmids PCR genotyping, PCR amplification conditions, RNA and cDNA pPsMID and pVcMID-BH with NheI and BamHI, and replacing the VcMID preparation and RT-PCR on V. carteri strains were as described gene from pVcMID-BH with the PsMID gene (Fig. S2B). previously (Geng et al., 2014) using primers listed in Table S2. Selected Plasmid pGpMID was created as follows. The GpMID promoter and RT-PCR products were cloned into pGEM-T Easy vector for sequencing. 5′ UTR (−185 bp to −1 bp) were amplified from G. pectorale genomic The V. carteri DGAT1 gene was used as a control for nitrogen starvation DNA using primers GpMid promoter f1 and GpMid promoter r1, which (www.phytozome.org, Volvox carteri V2.1 gene ID Vocar.0001s0624.1). introduced flanking SacI and NcoI restriction sites, with the ATG start codon Primers for amplifying DGAT1 cDNA (DGAT1-F1 and DGAT-R1), BFP of GpMID within the NcoI site. The GpMID gene region (coding exons and cDNA (BFP-F and BFP-R), GpMID-BH (BFP-F and BFP-R; GpMidGOI.f1 intervening introns, 760 bp) was amplified from G. pectorale genomic DNA and GpMidGOI.r1), PsMID-BH (PlestMid GOI f1 and PlestMid GOI r1) using primers GpMidGOI.f1 and GpMidGOI.r1, which introduce flanking and ribosomal protein S18 cDNA (VcS18-1 and VcS18-2) are listed in NcoI and NheI sites, respectively. The GpMID 3′ region (760 bp following Table S2. Stop codon) was amplified with primers GpMid3UTR.f1 and GpMid3UTR.r1, which introduce flanking BamHI and KpnI restriction Volvox immunoblotting sites. The above three fragments were digested at the appropriate flanking Preparation of whole-cell lysates and immunoblotting of V. carteri cultures restriction enzyme sites, ligated together, and the full-length product were performed as described previously (Geng et al., 2014). DEVELOPMENT

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Metrics of Volvox development and differentiation Gametogenesis and immunoblotting of Chlamydomonas The diameters of 20 randomly selected juvenile spheroids were measured Lawns of selected C. reinhardtii strains were spread on TAP plates and 24 h after the start of embryogenesis (i.e. first embryonic cleavage). grown under continuous light for five or six days. A pea-sized loopful of Numbers of germ cells and germ cell differentiation patterns (eggs or sperm cells was scraped into NF-HSM and kept in the light for 2 h. Aliquots were packets) were counted 40 h after the start of embryogenesis. Measurements tested briefly for mating, then gametes were spun down (5 min, 2000 g) and and photomicrographs were made using a Leica DMI6000 microscope with washed in 1 ml lysis buffer [1× PBS supplemented with 1× ProteaseArrest 10×, 20× or 40× objectives using DIC optics, a Leica DFC 450 camera, and (G-Biosciences), 5 mM benzamidine, 1 mM PMSF] and finally Leica LAS v4.0 software. resuspended in 150 µl lysis buffer before flash-freezing in liquid nitrogen. The cells were thawed at room temperature and spun down at full speed Transformation and screening of Chlamydomonas CC3712 (10 min, 13,200 g) to pellet debris. The supernatant was collected and, prior The mid deletion strain CC3712 was co-transformed using the glass-bead to mixing with sample buffer, the protein concentration was determined method (Kindle, 1990) with 2 µg MID expression construct DNA (see using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) with above), and 200 ng hygromycin resistance plasmid pHyg3 (Berthold et al., bovine serum albumin as a standard. Equal amounts of protein (∼30 µg per 2002). After recovery overnight under dim light and gentle shaking, the cells lane) were loaded for each sample and resolved on 12% SDS-PAGE gels. were collected by centrifugation (5 min, 2000 g) and spread onto TAP plates The gels were blotted to Immobilon-P PVDF membranes (Millipore) for supplemented with 35 µg/ml hygromycin B (Gold Biotechnology). Plates immunodetection. The blotting was performed in an XCell II Blot Module were kept under continuous light for 5-6 days, when visible colonies were (Invitrogen) with transfer for 48 min at 45 V in buffer comprising 25 mM picked and tested. Tris pH 8.6, 192 mM glycine, 0.1% SDS, 20% methanol. Blocking was Individual colonies that grew on the hygromycin B plates were transferred performed overnight at 4°C in 9% nonfat dried milk in TBST (TBS with to individual wells of 96-well plates containing 200 µl TAP medium. After 0.3% Tween 20), followed by incubation with primary antibody in blocking 2 days under continuous light, the transformants were spotted onto TAP buffer overnight at 4°C. Anti-FLAG rabbit polyclonal (Rockland 600-401- plates and grown 5-7 days as small spots ∼5 mm in diameter. For genotypic 383) was used at 1:10,000 to detect the FLAG epitope tag. The blots were screening, a tiny amount of cells from each spot was collected using a 10 µl washed three times with TBST at room temperature for 15 min followed by pipette tip and suspended in 100 µl TE (10 mM Tris pH 8.0, 1 mM EDTA). incubation for 1 h with HRP-conjugated goat anti-rabbit secondary antibody The suspensions were boiled at 95°C for 10 min, centrifuged at 3000 g for (Thermo Scientific Pierce PI31460) at 1:20,000 in TBST. Blots were 10 min, and 1 µl supernatant was used as template for PCR to detect the washed three times as described above and signal was detected by presence of the MID transgene using primers CRMID_ATG_F and chemiluminescence (Luminata Forte Western HRP substrate, Millipore) crMID_HA_crR, or p3promoter_gN_F and GPMID-6FLAGR, or using a ChemiDoc CCD camera system (Bio-Rad). p3promoter_vN_F and VCMID-6FLAGR for CrMID, GpMID and VcMID, respectively. Genotyping PCR reaction mixtures contained 1 μM Acknowledgements each primer, 0.2 mM each dNTP, 1× Ex-Taq Buffer (with 2.5 mM We thank the staff of the Tissue Culture and Transformation core facility at Donald Danforth Plant Science Center for technical assistance with biolistic transformation; MgCl2) (Takara), 2% DMSO, and 1-2 U Taq polymerase (Invitrogen). Products were amplified using 40 cycles of 94°C for 15 s, 60°C for 30 s, and Takashi Hamaji for helpful discussion; Takashi Hamaji, Gavriel Matt and Yi-Hsiang 72°C for 30 s. Chou for feedback on the manuscript; and Richard Davenport and Jie Li for excellent technical support. Transformants that tested positive for an MID transgene were randomly selected and tested for mating as follows. About one-quarter of each small spot from a TAP agar plate (see above) was suspended in 200 µl of Competing interests The authors declare no competing or financial interests. nitrogen-free HSM (NF-HSM) (Harris, 2009) in a well of a 96-well plate and kept under continuous light for 2 h. Then, 100 µl each suspension was mixed with gametes of MT+ mating tester R3 to observe mating. Author contributions The other 100 µl was kept to observe potential self-agglutination. Conceptualization: S.G., J.G.U.; Methodology: S.G., J.G.U., A.M.; Validation: S.G.; − Formal analysis: S.G., J.G.U.; Investigation: S.G., A.M.; Resources: J.G.U.; Data Selected non-complemented lines were mixed with MT mating tester curation: S.G., A.M.; Writing - original draft: S.G., A.M.; Writing - review & editing: strain CJU10 and observed for agglutination to confirm that S.G., J.G.U.; Visualization: S.G., J.G.U.; Supervision: J.G.U.; Project administration: gametogenesis had been induced. Mating and agglutination were J.G.U.; Funding acquisition: J.G.U. scored using a Zeiss Axiostar compound microscope with 40× objective and phase optics. Transformants that either mated with MT+ Funding strain R3 or self-agglutinated were scored as positive transgenic lines This research was funded by National Institutes of Health grant GM 078376 to J.G.U. (Table 2). Deposited in PMC for release after 12 months.

Chlamydomonas RNA extraction and RT-PCR Supplementary information Gamete RNA extraction was as follows. About 5×107 cells were harvested Supplementary information available online at and flash-frozen in liquid nitrogen. The pellets were quickly thawed in http://dev.biologists.org/lookup/doi/10.1242/dev.162537.supplemental 3-4 ml Trizol (Invitrogen) by pipetting at room temperature. After 5 min incubation at room temperature, lysates were centrifuged at top speed References (10 min, 13,200 g) to remove debris. The supernatants were used for RNA Baker, C. R., Tuch, B. B. and Johnson, A. D. (2011). Extensive DNA-binding extraction according to the manufacturer’s protocol. cDNA was prepared specificity divergence of a conserved transcription regulator. Proc. Natl. Acad. Sci. from 2-3 µg total RNA according to the manufacturer’s protocol for USA 108, 7493-7498. Superscript III (Thermo Fisher Scientific, 18080–044) using a 10:1 Barahimipour, R., Strenkert, D., Neupert, J., Schroda, M., Merchant, S. S. and mixture of oligo(dT) and random hexamer for priming. The reaction Bock, R. (2015). Dissecting the contributions of GC content and codon usage to conditions were: 25°C 10 min, 42°C 10 min, 50°C 20 min, 55°C 20 min, gene expression in the model alga Chlamydomonas reinhardtii. Plant J. 84, 60°C 20 min, 85°C 5 min. The reactions were diluted 1:7 with TE and 704-717. Bell, G. (1978). The evolution of anisogamy. J. Theor. Biol. 73, 247-270. used as templates for semi-quantitative RT-PCR with the primers Berthold, P., Schmitt, R. and Mages, W. (2002). An engineered Streptomyces indicated in Table S2. The C. reinhardtii 18S ribosomal RNA gene hygroscopicus aph 7” gene mediates dominant resistance against hygromycin B (GenBank KX781336) was used as an internal control. Primers for in Chlamydomonas reinhardtii. Ann. Anat. 153, 401-412. amplifying VcMID cDNA (VcMID_ATG_F and VcMid-C-R3), GpMID Blanc-Mathieu, R., Krasovec, M., Hebrard, M., Yau, S., Desgranges, E., Martin, cDNA (GpMID-BamATG and GpMID-XhoTTA), CrMID (CrMID_F and J., Schackwitz, W., Kuo, A., Salin, G., Donnadieu, C. et al. (2017). Population CrMID_R) and the 18S ribosomal RNA gene (Cr18SrRNA-1 and genomics of picophytoplankton unveils novel chromosome hypervariability. Sci.

Cr18SrRNA-2) are listed in Table S2. Adv. 3, e1700239. DEVELOPMENT

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