Molecular evolutionary analysis of a gender-limited MID ortholog from the homothallic species Volvox africanus with male and monoecious spheroids

Volvox is a very interesting oogamous organism that exhibits various types of sexuality and/or sexual spheroids depending upon species or strains. However, molecular bases of such sexual reproduction characteristics have not been studied in this genus. In the model species V. carteri, an ortholog of the minus mating type-determining or minus dominance gene (MID) of isogamous Chlamydomonas reinhardtii is male-specific and determines the sperm formation. Male and female genders are genetically determined (heterothallism) in V. carteri, whereas in several other species of Volvox both male and female gametes (sperm and eggs) are formed within the same clonal culture (homothallism). To resolve the molecular basis of the evolution of Volvox species with monoecious spheroids, we here describe a MID ortholog in the homothallic species V. africanus that produces both monoecious and male spheroids within a single clonal culture. Comparison of synonymous and nonsynonymous nucleotide substitutions in MID genes between V. africanus and heterothallic volvocacean species suggests that the MID gene of V. africanus evolved under the same degree of functional constraint as those of the heterothallic species. Based on semi quantitative reverse transcription polymerase chain reaction analyses using the asexual, male and monoecious spheroids isolated from a sexually induced V. africanus culture, the MID mRNA level was significantly upregulated in the male spheroids, but suppressed in the monoecious spheroids. These results suggest that the monoecious spheroid-specific down regulation of gene expression of the MID homolog correlates with the formation of both eggs and sperm in the same spheroid in V. africanus.


Introduction
Volvox is a genus of spheroidal, multicellular green algae with a surface layer of hundreds to thousands of biflagellated somatic cells, and a much smaller number of non-flagellated germ cells (gonidia) that develop into asexual progeny. Life cycles of all Volvox species are facultatively sexual with haploid asexual phase; typically many rounds of asexual reproduction occur between rounds of sexual reproduction in which thick-walled diploid zygotes are formed and meiosis occurs during zygote germination to produce haploid progeny (Fig 1). During sexual reproduction, spheroids that contain eggs or sperm packets (bundles of male gametes or sperm) or both are produced. This genus exhibits various types of sexuality and/or sexual spheroids that have been used to define separate taxa within Volvox [1][2][3]. For example, whether the sexual spheroids are dioecious or monoecious is an important criterion for distinguishing species of Volvox; several monoecious species are recognized in Volvox [1][2][3].
Starr [4] recognized four types of sexuality in several strains identified as Volvox africanus originating from locations around the world (S1 Fig); 1) heterothallic, dioecious type: male spheroids (containing sperm packets) or female spheroids (containing eggs) are formed in the male or female strain, respectively; 2) homothallic, dioecious type: separate male and female spheroids are formed in the same strain; 3) homothallic, monoecious type: monoecious spheroids (containing both eggs and sperm packets) are formed; and 4) homothallic, monoecious with males type: monoecious spheroids and male spheroids are both formed in the same strain. Coleman [5] resolved a small clade composed of these four sexual types of V. africanus based on the internal transcribed spacer-2 (ITS-2) of nuclear ribosomal DNA (rDNA) sequences. Thus, these related strains may be very useful for studying the diversity and evolution of monoecy and/or homothallism in Volvox. However, further studies of sexuality in these strains have been lacking except for the heterothallic, dioecious type [6], since strains exhibiting the three types of homothallic sexuality are not available [3]. Recently, new Japanese strains of two V. africanus-like species were isolated from water samples collected in Lake Biwa, Japan [3]. One that corresponds to sexual type 1 (heterothallic, dioecious type) by Starr [4] was renamed as a new species, V. reticuliferus (Fig 1A, S2D and S2E Fig). The other was re-identified as V. africanus, and produces both monoecious and male spheroids in a single strain (sexual type 4 of Starr [4], Fig 1B and S2 Fig).
In the heterothallic isogamous species Chlamydomonas reinhardtii, two sexes or mating types are determined by the presence or absence of the mating type-specific minus dominance gene (MID) [7]. In anisogamous volvocine Pleodorina starrii and oogamous Volvox carteri, a MID ortholog is present only in male strains [8,9]. Although MID is the master gene determining mating type minus of C. reinhardtii [7], the MID ortholog in V. carteri (VcMID) was recently reported as regulating formation of sperm packets, but not formation of male-specific sexual spheroids [8][9][10]. The MID ortholog is present in only one of the two heterothallic mating types in the isogamous volvocine Gonium, but it is present in a homothallic strain of Gonium multicoccum [11].
In the male strain of heterothallic V. carteri, experimental knock-down of VcMID results in sexual spheroids with eggs and sperm packets (similar to monoecious spheroids in wild monoecious species) or female-like sexual spheroids (with eggs and no sperm packets), depending upon the degree of suppression of VcMID expression [10]. This implies MID gene expression may be important for formation of monoecious spheroids in homothallic wild species of Volvox. However, MID orthologs in wild homothallic Volvox species with monoecious spheroids, like sexual type 3 or 4 of Starr [4], have not been previously reported.
To understand the evolution and development of monoecious spheroids in wild Volvox species, comparative analysis of their MID genes with those of closely related heterothallic  [3]. (A) V. reticuliferus (heterothallic, dioecious type). Germination of a diploid zygote yields a single meiotic product: either a female or a male depending on which MT locus is inherited. The algae reproduce asexually and can undergo sexual induction to produce sperm packets (in male lineages) or eggs (in female lineages). (B) V. africanus (homothallic, monoecious with males type). Germination of a diploid zygote yields a single meiotic product that reproduces asexually. Upon sexual induction, a clonal population can produce both spheroids containing sperm packets only (male spheroids) and spheroids containing both eggs and sperm packets (monoecious spheroids).
https://doi.org/10.1371/journal.pone.0180313.g001 dioecious species should be fruitful. In the present study, we examined MID homologs from two closely related species of Volvox sect. Merrillosphaera: V. africanus and V. reticuliferus, identified by Nozaki et al. [3]. Since these two species are heterothallic, dioecious type and homothallic, monoecious with males type (sexual types 1 and 4 by Starr [4], respectively), comparative analyses of MID orthologs from these two species will lead to a greater understanding of the evolution of monoecy or homothallism in Volvox. In addition, in order to elucidate general MID characteristics of monoecious spheroids, the MID homolog from V. ferrisii which produces only monoecious sexual spheroids [2] was also studied. V. ferrisii is belongs to Volvox sect. Volvox that is phylogenetically separated from Volvox sect. Merrillosphaera [3].  (Table 1) were used in the present study. The cultures were maintained in screw-cap tubes (18 × 150 mm) containing 10 ml AF-6/3 medium [3] at 20˚C on a 14-h light: 10-h dark schedule or at 25˚C on a 12-h light:12-h dark schedule under cool-white fluorescent lamps at an intensity of 55-80 μmolÁm -2 Ás -1 .

Strains and culture conditions
To induce sexual reproduction, about 0.5 ml of growing cultures were transferred into 10 ml of USVT medium diluted one to two with distilled water [3] and grown at 25˚C on a 12-h light:12-h dark schedule under cool-white fluorescent lamps at an intensity of 160-180 μmolÁm -2 Ás -1 . Sexual spheroids developed after 4~5 days (V. africanus) or 7~10 days (V. reticuliferus).
To induce sexual reproduction, about 0.5 ml of growing cultures were transferred into 10 ml of VTAC medium [13,14] and grown at 25˚C on a 12-h light:12-h dark schedule under cool-white fluorescent lamps at an intensity of 160-180 μmolÁm -2 Ás -1 . After 7~10 days sexual spheroids developed abundantly. Identification of MID orthologs V. africanus. A full-length sequence of the V. africanus MID (VaMID) mRNA was determined from total RNA using RT-PCR with degenerate primers (S1 Table) as described previously [9,15]. Total RNA was isolated from cultures in which sexual reproduction had been induced as described above, using the RNeasy Mini Kit (Qiagen, Hilden, Germany) after the cells had been homogenized with ceramic beads and a wash brush [9,15]. Production of cDNA was carried out with Superscript III reverse transcriptase (Thermo Fisher Scientific, MA, USA) using 3'-RACE CDS Primer A from the SMARTer™ RACE cDNA Amplification Kit (Clontech Laboratories, Inc., CA, USA). Nested PCR using this cDNA as template with degenerate MIDgene primers (S1 Table) yielded a partial fragment of VaMID. The primers used in in the first PCR were dMT-dF3 [16] (S1 Table) and Nested Universal Primer A (Clontech Laboratories); the primers used in the second PCR were dMT-dF3 and SMID-R6. The PCR reactions were carried out using rTaq polymerase (TAKARA, Shiga, Japan) and the cycling conditions described previously [17]. The resulting fragments were TA subcloned using the TOPO TA Cloning Kit (Thermo Fisher Scientific), and the plasmid insert sequenced on an ABI PRISM 3100 Genetic Analyzer (Thermo Fisher Scientific) using the BigDye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Thermo Fisher Scientific) as described previously [9].
To determine the 3'-terminus sequence of VaMID, 3'-RACE was performed with Nested Universal Primer A and gene specific primers VaMID_F1, VaMID_F2 and VaMID_F3 (S2 Table). The resulting fragments were TA subcloned using the TOPO TA Cloning kit, and sequenced as described above. The 5'-terminus sequence was determined using the GeneRacer kit (Thermo Fisher Scientific) according to the manufacturer's protocol; the antisense gene specific primer was VaMID_5'R1 (S2 Table). Nested PCR was performed with GeneRacer 5' Primer and gene specific primers VaMID5'R1, VaMID5'R2 and VaMID5'R3 (S2 Table). The resulting fragments were TA subcloned and sequenced as described above.
To determine the intron-exon structure of VaMID, genomic PCR using total DNA extracted as described previously [18] was performed, followed by DNA sequencing of the product. The PCR reaction used KOD FX Neo DNA polymerase (TOYOBO, Osaka, Japan) and VaMID-specific primers (VaMID_AR and VaMID_ValR2; S2 Table) with cycling conditions 2 min at 94˚C, followed by 35 cycles of 10 sec at 98˚C and 30 sec at 68˚C. V. ferrisii. A partial sequence of V. ferrisii MID (VfMID) mRNA was obtained by PCR amplification and sequencing as described for VaMID except for the primers used for the nested PCR and determination of 3' and 5' termini (S2 Table). Degenerate primers SMID-F1 and SMID-R5 were used for the first PCR, and SMID-F1 and SMID-R4 for the second PCR (S1 Table). To determine the 3'-terminus sequence of VfMID, 3'-RACE was performed with Nested Universal Primer A and VfMID-specific primers VfMID_F1, VfMID_F2, VfMID_F3, VfMID_F4, VfMID_F5 and VfMID_F6 (S2 Table). Specific primers VfMID_R1 and VfMID5'R (S2 Table) were used for amplifying the 5'-terminus sequence.
The intron-exon structure of VfMID was determined using genomic PCR as described above for VaMID but using VfMID-specific primers (VfMID_Af and VfMID_AR; S2 Table).
V. reticuliferus. Polyadenylated mRNA was isolated from sexually induced cultures using Dynabeads Oligo (dT) 25 (Thermo Fisher Scientific) and reverse transcribed with Superscript III reverse transcriptase (Thermo Fisher Scientific).
The intron-exon structure of VrMID was determined using genomic PCR as described above for VaMID but using VrMID-specific primers (F1-7MID_AF and F1-7MID_AR; S2 Table).
Availability of sequence data. The new sequence data of MID orthologs have been deposited to DDBJ/EMBL/GenBank (accession numbers: LC274875-LC274882; S3 Table).

Phylogenetic analysis of MID orthologs
Phylogenetic analyses were performed using MUSCLE [19]-aligned full-length protein sequences of sixteen Volvocales MID orthologs (S3 Table). The maximum likelihood (ML) method (based on LG model [20] selected by MEGA 6.0 [21]) and the neighbor joining (NJ) method (using JTT model [22]) by MEGA 6.0) were carried out with bootstrap values from 1000 replications.
A molecular evolutionary analysis of nonsynonymous and synonymous substitutions was performed between the MID ortholog of G. pectorale and those of seven other Volvocales by MEGA 6.0, using a modified Nei-Gojobori model [23,24] (assumed transition/transversion bias = 1.55).

Detection of VrMID based on genomic PCR of V. reticuliferus
Genomic PCR was performed in six strains of V. reticuliferus (Table 1) using total DNA extracted as described previously [18], KOD FX Neo DNA polymerase and a pair of VrMID specific primers (F1-7MID_AF and F1-7MID_AR; S2 Table). The ITS2 sequence was amplified as a control, using an ITS2-specific primer pair designed based on the ITS2 sequence of 2013-0703-VO2 (S2 Table). PCR cycles were 2 min at 94˚C, followed by 30 (ITS2) and 35 (VrMID) cycles of 10 sec at 98˚C and 30 sec at 68˚C.

Southern blot analysis
Genomic DNA of V. africanus strain 2013-0703-VO4, V. reticuliferus strains VO123-F1-6 (female) and VO123-F1-7 (male) ( Table 1) was prepared by the "miniprep" method [25]. Restriction enzyme digests of genomic DNA (2 μg) were separated by 1.0% agarose gel electrophoresis and transferred onto a Hybond-N+ nylon membrane (GE Healthcare, UK). A hybridization probe containing part of the VrMID gene (Fig 2) labeled with digoxigenin-11-dUTP was prepared by PCR using a plasmid clone of the VrMID gene as template and the primer pair F1-7_ southMID_F and F1-7_MID_R1 (S2 Table) using PCR DIG Probe Synthesis Kit (Roche Diagnostics, Germany), and hybridized at 42˚C. A hybridization probe containing part of the V. reticuliferus elongation factor 1-like gene (EF1-like) labeled with digoxigenin-11-dUTP was prepared by PCR using a plasmid clone of the EF1-like gene as template and the primer pair (CV_EF1A1-R2 and GpEF1A-INT3-R [16]; S2 Table) using KOD FX Neo DNA Polymerase, and hybridized at 42˚C. The signals were detected using DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics) and Chemidoc XRS (Bio-Rad, Hercules, CA, USA). The resulting image was processed with a median filter (diameter: 1 pixel) in ImageJ (National Institutes of Health, Bethesda, MD, USA) to remove random noise produced by long exposure (2 hr).

Estimation of genome sizes of Volvox africanus and V. reticuliferus
To estimate relative genome size of V. africanus and V. reticuliferus, 4',6-diamidine-2-phenylindole (DAPI)-staining was performed using somatic cells of V. africanus, male and female strains of V. reticuliferus (2013-0703-VO2 and 2013-0703-VO3, respectively), and V. carteri strain EVE (control). One ml of each vegetative sample was fixed with 0.25% glutaraldehyde, followed by postfixation in 100% methanol for reducing autofluorescence, and washed with phosphate-buffered saline. Fixed samples were stained with 0.1μg/μl DAPI overnight. DAPIstained somatic cells of V. africanus and V. reticuliferus male and female were mixed separately with DAPI-stained V. carteri strain EVE and mounted in the same slide. The images were obtained using a BX-60 Microscope and DP Controller 1. 2. 1108 (Olympus, Tokyo, Japan). The image analyses were performed using ImageJ, measuring the mean gray value of 10 nuclei for each exposure time (0.50, 0.67, 1.0, 1.5, 2.0 and 2.5 s).

Semi-quantitative reverse transcription (RT)-PCR analyses
EF1-like genes were used as internal controls. To obtain sequences of EF1-like genes from the three Volvox species, PCR amplifications were performed with full-length cDNA of each Volvox and the primer pair, CV_EF1A1-R2 and GpEF1A-INT3-R [16]. From direct sequencing of PCR-products, we designed EF1-like-specific primer pairs for each of the three Volvox species (S2 Table) for semi-quantitative RT-PCR analyses.
For V. africanus, 30 asexual, male or monoecious spheroids were collected by a micropipette from cultures that were sexually induced or not. Polyadenylated mRNAs were isolated separately from these three sets of spheroids and reverse transcribed as described for V. reticuliferus MID determination. Likewise, cDNAs of the other two Volvox species were obtained by reverse transcription using mRNAs isolated from 30 asexual (in cultures that were sexually induced or not), male or female spheroids in V. reticuliferus, as well as from 30 asexual (in cultures that were sexually induced or not), or monoecious spheroids in V. ferrisii.
PCR analyses were performed using KOD FX Neo DNA polymerase. PCR cycles and primer pairs are described in S4 Table. The amplified products were electrophoresed on 2%  (wt/vol) agarose gels and stained with ethidium bromide. The gel images were captured using a ChemiDoc XRS (Bio-Rad), level adjusted and gradation inverted with Adobe Photoshop CS6 (Adobe Systems Inc., San Jose, CA).

Identification and characterization of MID orthologs
We identified full-length cDNA sequences and intron-exon structures of VaMID, VrMID and VfMID (Fig 2). The genomic sequences of the three genes determined in this study covered the entire DNA sequences of the genes and demonstrated that all three genes contained introns at the same four positions. The deduced protein sequences of the three genes were composed of 163-167 amino acids that contained the DNA binding RWP-RK domain near the C-terminus. RWP-RK domains of seven volvocine MID proteins were highly conserved even among homothallic and heterothallic volvocine species (S3 Fig). Based on the phylogenetic analysis of 14 colonial volvocine MID proteins, a large clade composed of seven genes of the Volvocaceae was resolved with 83-85% bootstrap values in ML and NJ methods (Fig 3). However, phylogenetic relationships of Gonium MID proteins were not well resolved. Within the volvocacean clade, MID proteins from V. carteri, V. reticuliferus, V. africanus, Pleodorina starrii and Eudorina sp. formed a robust monophyletic group (with 98-99% bootstrap values in both analyses) from which Yamagishiella unicocca and V. ferrisii MID proteins were separated. These results were consistent with the phylogenetic relationships of the colonial volvocine algae based on chloroplast genes [26].
A molecular evolutionary analysis of the volvocacean MID genes demonstrated that nonsynonymous and synonymous substitutions of the genes from two homothallic species of Volvox (V. africanus and V. ferrisii) fell within the range of those of heterothallic species (Fig 4).

Genomic PCR and Southern blot analysis of MID genes of Volvox africanus and V. reticuliferus
Results of genomic PCR using VrMID-specific primers (S2 Table) for strains of V. reticuliferus (Table 1)  Estimation of genome sizes of Volvox africanus and V. reticuliferus based on epifluorescence microscopy of DAPI-stained somatic cells.
Since V. africanus or V. reticuliferus might have originated from their common ancestor by duplication of the whole genome, relative genome sizes of these two species were measured based on the degree of fluorescence of DAPI-stained nuclei in somatic cells using epifluorescence microscopy. By using the fluorescence value of nuclei of V. carteri EVE somatic cells as a  (Fig 2). Analyses were conducted with the outgroup Gonium pectorale MID gene (AB353340) using the modified Nei-Gojobori (assumed transition/transversion bias = 1.55) model ( [23,24] by MEGA6 [21]. All positions containing gaps and missing data were eliminated. There were a total of 162 positions in the final dataset. Filled circles (•) indicate the homothallic strain.

Semi-quantitative RT-PCR analyses of expression of MID genes
Results of semi-quantitative RT-PCR analyses of expression of MID genes in the V. africanus homothallic strain, V. reticuliferus male strain and V. ferrisii homothallic strain are shown in Fig 5. In a sexually induced V. africanus culture, VaMID expression was extremely high in male spheroids, whereas the expression was low in monoecious and asexual spheroids (Fig 5A  and 5D). The VaMID expression of asexual spheroids in a sexually uninduced culture was slightly higher than that in a sexually induced culture (Fig 5A). In heterothallic V. reticuliferus, the VrMID level was highly upregulated in male spheroids when compared to that of asexual spheroids in the same culture (Fig 5B and 5E). Three alternative splicing variants of VaMID were also identified. All these variants were identified as due to intron retention [27] (S7 Fig). No alternative splicing variants were identified in VrMID and VfMID. In contrast to V. africanus, VrMID expression of asexual spheroids in a sexually induced culture was slightly higher than that in a sexually uninduced culture (Fig 5E). No alternative splicing variants were detected in VrMID expression.
In contrast to V. africanus, the VfMID level in monoecious spheroids of V. ferrisii was higher than that of asexual spheroids in sexually induced and sexually uninduced cultures. The VfMID transcription level in monoecious spheroids was more than 2.5 times higher than that of asexual spheroids from either culture condition.

MID orthologs in homothallic species of Volvox with monoecious spheroids
The present study demonstrated that two homothallic species of Volvox with monoecious spheroids, V. africanus and V. ferrisii, have MID orthologs (Figs 2 and 3). The MID orthologs (VaMID and VfMID) of these two homothallic species are essentially consistent with those of heterothallic colonial or multicellular volvocacean species [8,9] in containing 5 exons, 4 introns, and a DNA binding RWP-RK domain at the C-terminus. Phylogenetic relationships of MID orthologs within the Volvocaceae (Fig 3) were consistent with those based on chloroplast genes [2,3]. Moreover, comparison of synonymous and nonsynonymous substitutions of MID genes between homothallic and heterothallic volvocacean species suggested that the MID genes of the two homothallic species have evolved under the same degree of functional constraint as those of the heterothallic species. Thus, no signs of altered selection on MID could be detected in the monoecious species. Nozaki et al. [9] reported that MID protein expression is strong in nuclei of the gametes of the male strain of P. starrii. Geng et al. [10] demonstrated that the MID ortholog (VcMID) of the heterothallic species Volvox carteri controls sperm packet formation by sexual reproductive cells (androgonidia). The present study showed that expression of VaMID in homothallic V. africanus is very high in male spheroids (Fig 5A and  5D). Therefore, the MID orthologs of the two homothallic species of Volvox may control sperm packet formation as in the heterothallic species.

VaMID transcription in monoecious and male spheroids
The number of sperm packets in a monoecious spheroid is very small, 1-4 in V. africanus [3] or 3-5 sperm packets in V. ferrisii. [2]. In contrast, the male spheroid of V. africanus contains 100-260 androgonidia that divide to form sperm packets. The semi-quantitative RT-PCR data showed down-regulation of MID expression in monoecious spheroids and extremely high upregulation in male spheroids in V. africanus, suggesting that VaMID transcriptional level is correlated with the quantity of sperm packets in monoecious or male spheroids. It indicates that V. africanus has spheroid type-specific regulation of VaMID. In heterothallic V. carteri, VcMID protein is localized in the sperm nucleus and controls formation of sperm packets [10]. As discussed above, VaMID may also control formation of sperm packets. Thus, V. africanus may determine the fate of reproductive cells in monoecious spheroids by differentially controlling VaMID expression between eggs and androgonidia. Further analyses of the localization of VaMID in the monoecious spheroid at the cellular level is required to confirm this hypothesis.

VfMID transcription in monoecious spheroids
In V. africanus, expression of VaMID in monoecious spheroids is lower than that of asexual spheroids (Fig 5A and 5D). By contrast, VfMID expression in monoecious spheroids is higher than that of asexual spheroids (Fig 5C and 5F). This difference of MID expression in monoecious spheroids may be related to the phylogenetic positions of V. africanus and V. ferrisii. V. ferrisii belongs to Volvox sect. Volvox that is clearly separated from the large monophyletic group (Eudorina group) composed of the other three Volvox sections (including V. africanus, V. reticuliferus and V. carteri), Eudorina and Pleodorina [2,3,27]. The phylogenetic positions suggest that monoecious spheroids might have been acquired independently in the evolutions of Volvox sect. Volvox and the Eudorina group. Further studies of VfMID expression at the cellular level are needed to understand the role of VfMID in the monoecious species V. ferrisii.

Conclusions
Sexual differentiation in heterothallic species of the unicellular and colonial/multicellular volvocine algae is controlled by a sex-determining or mating type locus (MT) containing MID in the minus or male strain [8,28,29]. Comparative analyses of MT loci in volvocine algae are important to elucidate the molecular and genomic basis of evolution of sexual differentiation [8,29]. However, there is, as yet, no genome information from homothallic species in the chromosomal region homologous to the MT locus of their heterothallic relatives, except for the MID genes that we described here in two homothallic species, V. africanus and V. ferrisii.
A homothallic euascomycete fungus, Neosartorya fischeri, has duplicated MT loci, (MAT1 and MAT2) possibly originating from the MT loci of the complementary sexes of the heterothallic ancestral species [30]. In C. reinhardtii, diploid heterozygotes (mt + /mt -) sometimes skip meiosis and produce diploid vegetative cells under experimental conditions [31]. The present study showed that the genome size in homothallic V. africanus is almost the same as that of its heterothallic relative V. reticuliferus (S6 Fig). Thus, the origin of the homothallic V. africanus cannot be explained simply by whole genome duplication via lack of meiosis of a diploid zygote of a possible heterothallic ancestor, although partial duplication of only the male and female MT loci cannot be ruled out based on the present genome measurement (S6 Fig). Homothallic V. africanus has an ortholog of the male-limited gene MID found in heterothallic species. In addition, androgonidia (male reproductive cells) in the heterothallic, male strain of V. carteri may function as eggs by experimental suppression of VcMID expression [10]. The present study demonstrated monoecious spheroid-specific down regulation of gene expression of the MID ortholog in V. africanus (Fig 5). Therefore, the homothallic species V. africanus might have evolved directly from a male strain of the heterothallic ancestor by modification of the regulation system of MID expressions in sexual spheroids. In any case, comparative analyses of the whole genomes of V. africanus and V. reticuliferus would be indispensable for further understanding the molecular and genome bases of evolution of both species.
The present study suggested that the male-specific transcription factor MID is functional in two homothallic Volvox species that produce both eggs and sperm packets in a single sexual spheroid. As discussed above, MID in homothallic species of Volvox may also be a key gene that controls formation of sperm packets. Thus, other unknown factors controlling MID may be crucial for differentiating monoecious or male spheroids in Volvox. Further studies of molecular mechanisms controlling MID in various sexual types of Volvox will improve our understanding of the evolution of monoecious spheroids in Volvox.