Morphology, mating system and taxonomy of Volvox africanus (Volvocaceae, Chlorophyceae) from Thailand

The oogamous green algal genus Volvox exhibits extensive diversity in mating systems, including heterothallism and homothallism with unisexual (male and/or female) and/or bisexual spheroids. Although four mating systems have been recognized worldwide in strains identified as “Volvox africanus”, most of these strains are extinct. However, we previously rediscovered two types of the four mating systems (heterothallic, and homothallic with male and bisexual spheroids within a clone) from an ancient Japanese lake, Lake Biwa. Here, we obtained strains exhibiting the third mating system (homothallic with unisexual male and female spheroids within a clone) from a freshwater area of Kalasin Province, Thailand. When sexual reproduction was induced in the present Thai strains, both male and female unisexual spheroids developed to form smooth-walled zygotes within a clonal culture. Phylogenetic analyses of the internal transcribed spacer region-2 of nuclear ribosomal DNA sequences from all four mating systems, including the extinct strains, resolved the third mating system is basal or paraphyletic within the homothallic clade. The present morphological and molecular data of the Thai strains indicate that they belong to the homothallic species V. africanus. The phylogenetic results suggested that third mating system (homothallic with separate male and female sexual spheroids) may represent an initial evolutionary stage of transition from heterothallism to homothallism within Volvox africanus. Further field collections in geologically stable intracontinental regions may be fruitful for studying diversity and taxonomy of the freshwater green algal genus Volvox.


Background
Evolutionary transitions in the mating systems of a wide range of eukaryotic taxa are a crucial topic in the biological sciences (Wittenberger 1979;Barrett 2010). Freshwater algae are convenient research materials for examining sexual habit, as sexual reproduction can be easily induced under controlled laboratory conditions (Stein 1973). Thus, classical genetic studies of algae have primarily used the unicellular green algal genus Chlamydomonas (Harris 1989), and subsequent extensive molecular genetic studies of C. reinhardtii resolved its mating-type locus and mating-type specific genes Goodenough 1994, 1997). Chlamydomonas species are isogamous without differentiation between males and females, but the closely related multicellular volvocine lineage includes anisogamous genera and the oogamous genus Volvox that are diverse in their mating systems (Harris 1989;Hanschen et al. 2018). Starr (1971) reported four mating systems in worldwide strains of a volvocine alga identified as the single species "Volvox africanus" ("VxAf "). Although Coleman (1999) analyzed the internal transcribed spacer region (ITS)-2 of nuclear ribosomal DNA (rDNA) sequences from the four mating systems of "VxAf ", most of the "VxAf " strains deposited in UTEX (Starr and Zeikus 1993) were unavailable (Nozaki et al. 2015a). This problem was partially resolved when new "VxAf " strains representing the two mating systems were established from field-collected water samples in an ancient Japanese lake, Lake Biwa (Nozaki et al. 2015a). One of the two types is homothallic with male and bisexual spheroids and was identified as Volvox africanus G.S. West, whereas the other has been described as the new heterothallic species Volvox reticuliferus Nozaki (Nozaki et al. 2015a) (Table 1). However, details of morphology and sexual reproduction of "VxAf " strains with the other two mating systems (homothallic with separate male and female sexual spheroids, and homothallic with only bisexual spheroids) have remained unresolved.
Very recently, we obtained "VxAf " strains of the third mating system (homothallic with separate male and female sexual spheroids; Table 1) from a freshwater habitat in Kalasin Province, in the interior continental region of Thailand. Our morphological and phylogenetic data identified these Thai strains as V. africanus, and this type of homothallic mating system may be ancestral within V. africanus. The morphology, mating system, and taxonomy of these homothallic strains of V. africanus are described in this report.

Establishment of cultures and morphological observations
Water samples (pH 6.85; temperature 30.5 °C) were collected from a marsh in Nong Ya Ma, Yang Talat District, Kalasin Province, Thailand (16° 28ʹ 14.55ʺ N, 103° 16ʹ 25.55ʺ E) on 1 November 2019. Clonal cultures of V. africanus (strains 1101-NK-1 and 1101-NZ-11) were established from the water sample using the pipette washing method (Pringsheim 1946). The strains are available as NIES-4467 (1101-NK-1) and NIES-4468 (1101-NZ-11) in the Microbial Culture Collection of the National Institute for Environmental Studies, Japan (Kawachi et al. 2013). Cultures were grown in 18 × 150-mm screw-cap tubes containing 10-11 mL artificial freshwater-6 (AF-6) or Volvox thiamin acetate (VTAC) medium (Kawachi et al. 2013), at 25 °C under a 14:10-h light:dark schedule, under cool-white fluorescent lamps (with a color temperature of 5000 K) at an intensity of 80-130 μmol m −2 s −1 . The cultures were initially maintained in AF-6 medium. For morphological observations, possible contaminant bacteria were removed from the cultures by squeezing a young, unhatched spheroid out of the parental spheroid and washing the spheroid using the pipette washing method. The young spheroid was then grown in 10-11 mL VTAC medium. Asexual spheroids were observed in actively growing cultures in VTAC medium, as described previously (Nozaki et al. 2015a). To induce production of sexual spheroids, 0.3-0.8 mL actively growing culture in VTAC medium (the volume depended on the number and density of spheroids in the culture) at 25 °C were inoculated with 10-11 mL urea soil Volvox thiamine medium (Nozaki et al. 2015b). This culture was then grown at 20 °C under a 14:10-h light:dark schedule under cool-white fluorescent lamps at an intensity of 50-70 μmol m −2 s −1 . Sexual spheroids usually developed within 20 days. Light microscopy was conducted using the BX60 microscope (Olympus, Tokyo, Japan) equipped with Nomarski interference optics. To examine the form of individual sheaths in the gelatinous matrix in spheroids, approximately 20 μL cultured material was mixed with 4-10 μL 0.002% (w/v in distilled water) methylene blue (1B-429 Waldeck GmbH & Co Division Chroma, Münster, Germany). Spheroid cells were counted as described previously (Smith 1944;Nozaki 1988;Nozaki et al. 2019).  Starr (1971), the present study Starr (1971) For transmission electron microscopy, an actively growing cultured sample was pre-fixed with 1% glutaraldehyde and post-fixed with 2% OsO 4 and examined as in the previous study (Nozaki et al. 2015a) except for using 4% samarium chloride solution in distilled water instead of saturated solution of uranyl acetate (Matsuzaki et al. 2014).

Molecular experiments
To infer the phylogenetic position and origin of the Thai strains of V. africanus, we analyzed ITS-2 and 6021 base pairs of the coding regions of five chloroplast genes (large subunit of RuBisCO, adenosine triphosphate synthase beta subunit, photosystem I P700 chlorophyll a apoprotein A1, photosystem I P700 chlorophyll a apoprotein A2, and photosystem II CP43 reaction center protein genes) as in previous studies (Nozaki et al. 2015a;Yamamoto et al. 2021), except that the alignments included an additional operational taxonomic unit of the Thai strains of V. africanus. New sequences from the Thai strains were determined based on direct sequencing of polymerase chain reaction products, as described previously (Nozaki et al. 2015a;Yamamoto et al. 2021), and are available under Accession numbers LC631309-LC631310 (ITS rDNA for 1101-NZ-11 and 1101-NK-1) and LC631311-LC631315 (five chloroplast genes for 1101-NZ-11). The alignments of ITS-2 and the five chloroplast genes are available from TreeBASE (https:// treeb ase. org/ treeb aseweb/ home. html; study ID: 28191). Designation of the outgroup was performed as described previously (Nozaki et al. 2015a;Yamamoto et al. 2021). Maximum-likelihood analyses based on the ITS rDNA and chloroplast multigene alignments were performed using MEGA X , with 1000 replicates of bootstrap analyses F Surface view of gonidium showing radial striations (s). G Pre-inversion embryo or plakea. Note that differentiation of gonidia (g) of the next generation is evident Nozaki et al. Botanical Studies (2022) 63:1 (Felsenstein 1985). In addition, Bayesian inference of ITS-2 was conducted using MrBayes 3.2.7a (Ronquist et al. 2012), as described in a previous study (Nozaki et al. 2015a). Timetree analysis was performed using the chloroplast multigene data set by MEGA X, as in a previous study (Yamamoto et al. 2021). The secondary structures of ITS-2 were predicted as described previously (Nozaki et al. 2015a).

Asexual spheroids
The Thai strains of V. africanus produced asexual spheroids that were ovoid, subspherical, or ellipsoidal in shape (Fig. 1A, B). The spheroid was up to 550 μm in length and consisted of approximately 1500-6000 somatic cells and 2-8 reproductive cells (gonidia). Gonidia were generally arranged in one or two tiers. When arranged in two tiers, one was positioned primarily at the equator of the spheroids and the other within the posterior half of the spheroid (Fig. 1A, B); the gonidia of the posterior tier often showed delayed embryogenesis. The cells lacked cytoplasmic bridges between them and were embedded in individual sheaths at the periphery of the gelatinous matrix (Fig. 1C, D). Individual sheaths were rectangular or hexagonal in shape and compactly arranged without fenestrations between them in the frontal view (Fig. 1E). Somatic cells had two equal flagella and a cup-shaped chloroplast with a single basal pyrenoid and a single eyespot, and they measured up to 8 μm in length (Fig. 1C, D). Gonidia had radial striations on the surface of the chloroplast in the frontal view (Fig. 1E). During the plakeal stages of the developing embryo, gonidia of the next generation were evident (Fig. 1G).

Sexual spheroids
Both male and female sexual spheroids were unisexual and developed within a single clonal culture. Male spheroids were generally 500-1300-celled and subspherical or ovoid in shape, with 60-100 sperm packets distributed almost throughout the surface of the spheroid ( Fig. 2A, B). Sperm packets were hemispherical in shape and were composed of approximately 100-200 spindle-shaped male gametes (sperm) (Fig. 2C). Female spheroids contained 500-1500 cells and usually 7-11 eggs within the posterior three-fifths (Fig. 2D, E). Production of zygotes (thick-walled cells) were rare under the present culture conditions, but they were found even within the single clonal culture. Zygotes measured approximately 30 μm in diameter and had a smooth cell wall (Fig. 2F, G).

Transmission electron microscopy
The entire spheroid of the present Thai strain was surrounded by a tripartite boundary (colonial boundary) of the extracellular matrix as (Fig. 3A) as in other volvocacean algae (Nozaki and Kuroiwa 1992). Just beneath the colonial boundary, cells were observed with profiles of a chloroplast, mitochondria, and a nucleus inside the cell membrane ( Fig. 3A-C). Each cell was tightly enclosed by a thin layer [cellular envelope (Nozaki and Kuroiwa 1992)] of the matrix (Fig. 3B). At the peripheral region of the spheroid, the matrix within the space between the cells formed a fibrillar layer (Fig. 3A) as in V. africanus from Lake Biwa (Nozaki et al. 2015a), representing the individual sheath observed under the light microscopy ( Fig. 1E).

Phylogenetic analyses
Based on the chloroplast multigene data set, phylogenetic relationships within the anisogamous/oogamous members of the colonial volvocine algae [Eudorina group (Nozaki et al. 2015a)] were resolved as described in a previous study (Yamamoto et al. 2021), except for the addition of the Thai strain of V. africanus. Four different isolates of "VxAf " formed a robust monophyletic group ("VxAf " lineage) within Volvox sect. Merrillosphaera (Fig. 4). The "VxAf " lineage was subdivided into two sister groups: one homothallic V. africanus composed of the Japanese and Thai strains and the other two strains of heterothallic V. reticuliferus. Our timetree analysis dated the origin of "VxAf " and the separation between Thai and Japanese strains of V. africanus as approximately 11 MYA and 5 MYA, respectively (Fig. 4). The ITS-2 phylogenetic analysis included five "VxAf " strains of UTEX [Culture Collection of Algae at the University of Texas at Austin (Starr and Zeikus 1993)], in which Starr (1971) observed four mating systems (Fig. 5). As in the chloroplast multigene phylogeny, two sister clades were resolved in the "VxAf " lineage: homothallic V. africanus and heterothallic V. reticuliferus (Fig. 5). However, the most basal position of UTEX 1889 within the homothallic clade was moderately resolved (with only a 73% bootstrap value using the maximum-likelihood Fig. 4 Timetree analysis of advanced members of the colonial volvocine algae including the Thai strains of Volvox africanus. Note that the divergence time between heterothallic Volvox reticuliferus and homothallic V. africanus and the divergence between Thai and Japanese strains of V. africanus are "11.44 (8.48-15.43) MYA" and "5.04 (2.84-8.95) MYA", respectively. Species names in orange or black indicate homothallic or heterothallic sexuality, respectively. Tree topology was inferred by maximum likelihood (ML) analyses of 6021 base pairs of five chloroplast genes from 47 operational taxonomic units [TreeBASE (Vos et al. 2010(Vos et al. , 2012 ID 28191] with a model selected by MEGA X . Asterisks on branches indicate 80% or more bootstrap values (based on 1000 replicates) by the ML analyses. A timetree was inferred by applying the RelTime method (Tamura et al. 2012 to the ML phylogenetic tree whose branch lengths were calculated using the ML method and the General Time Reversible substitution model (Nei and Kumar 2000). The timetree was computed using two calibration constraints [C1 (65-90 MYA) and C2 (50-90 MYA)] based on TimeTree: the Time Scale of Life <http:// www. timet ree. org/> (Liss et al. 1997;Herron et al. 2009). Outgroup was designated as in the previous study (Yamamoto et al. 2021). Evolutionary analyses were conducted in MEGA X  method) (Fig. 5). The strains of two homothallic mating systems with production of bisexual spheroids (Homo I and III, with and without male unisexual spheroids, respectively) constituted a derived lineage, to which two non-sister strains [Thai and Missouri (UTEX 1889;Starr 1971) strains] of the homothallic mating system with male and female unisexual spheroids were basal (Fig. 5).

Compensatory base changes (CBCs) in nuclear rDNA ITS-2
As resolved previously (Nozaki et al. 2015a), CBCs were not detected in the most highly conserved region of nuclear rDNA ITS-2 [helix III (Nozaki et al. 2015a)] among operational taxonomic units within the heterothallic clade identified as V. reticuliferus (Fig. 6). However, the Thai strains or UTEX 1889 had two or three CBCs compared with other strains within the homothallic clade identified as V. africanus (see below) (Fig. 6).

Taxonomy
Volvox reticuliferus is clearly distinguished from V. africanus, based on its morphological traits (reticulate zygote walls and confluent or indistinct individual sheaths), heterothallic mating system and phylogeny (Table 1) (Nozaki et al. 2015a). Even though information regarding zygote morphology and/or mating systems is lacking for UTEX 1890, 1891, and 2907 strains, they were identified as V.
reticuliferus based on vegetative morphology (individual sheaths) and phylogeny (Nozaki et al. 2015a). In the homothallic clade, however, such vegetative morphology has previously been obtained in only one (Homo I) of the three homothallic mating systems of "VxAf " by Starr (1971), due to the extinction of strains belonging to other homothallic mating systems (Homo II and III) (Nozaki et al. 2015a). The present study established new strains of "VxAf " originating from Thailand and revealed their homothallic sexual habit (Homo II) and morphological characteristics (smooth zygote walls and compact and angular individual sheaths; Figs. 1, 2 and 3) that are consistent with those of V. africanus (Table 1) (Nozaki et al. 2015a). In addition, the divergence date between mating systems Homo I (Lake Biwa, Japan) and Homo II (Kalasin, Thailand) is younger than those between sister species within the anisogamous/oogamous members of the multicellular volvocine algae (Fig. 4). Thus, "VxAf " strains of all homothallic mating systems (Homo I-III; Table 1) should be classified as V. africanus based on their monophyletic status (Figs. 4,5) and common morphological/ phenotypical characteristics (Table 1), even though their mating systems vary in the production of sexual spheroids (Table 1), and their ITS-2 rDNA demonstrated the presence of CBCs between them (Fig. 6). This taxonomic conclusion is consistent with the monophyletic morphological species concept in microalgae (Nozaki et al. 1998).  (Table 1) of Volvox africanus (homothallic) and V. reticuliferus (heterothallic), as inferred from internal transcribed spacer region 2 of nuclear ribosomal DNA. UTEX 2907 originating from Australia (https:// utex. org/) lacks information of sexual reproduction (Nozaki et al. 2015a), but it was classified as V. reticuliferus based on vegetative morphology and phylogeny (Nozaki et al. 2015a). The tree was constructed by ML method (based on T92 + G model). The alignment is available in TreeBASE (Vos et al. 2010(Vos et al. , 2012ID 28191). Branch lengths are proportional to the genetic distances, which are indicated by the scale bar above the tree. Numbers on the left or right side at the branches represent bootstrap values (≥ 50%, based on 1000 replicates) obtained with the ML analysis or posterior probabilities (≥ 0.95) of Bayesian inference, respectively

Evolution of homothallic mating systems in Volvox
Volvocine algae, especially Volvox species are an excellent model for studying the evolution of sexual dimorphism and mating systems. Some species of Volvox are heterothallic species, in which different genotypes produce either eggs or sperm, while others are homothallic species with a single genotype producing both eggs and sperm (Hanschen et al. 2018). The "VxAf " lineage is an excellent model for investigating the evolutionary transition between heterothallism to homothallism as it represents both homothallism and heterothallism within a closely related group (Figs. 4, 5).
Based on the genome comparison of sex-determining regions in male and female determining chromosomes of heterothallic V. reticuliferus and the sex-determining region-like region of homothallic V. africanus, the evolutionary transition from heterothallism to homothallism in the "VxAf " lineage was unambiguously resolved (Yamamoto et al. 2021). In addition, the present phylogenetic analyses of ITS-2 demonstrated that two strains of the homothallic mating system with separate or unisexual male and female sexual spheroids (Homo II) are basal and paraphyletic and may represent the ancestral state of the homothallism in the "VxAf " lineage (Fig. 5). The heterothallic mating system of "VxAf " (V. reticuliferus) also produces unisexual male and female sexual spheroids as in Homo II (Table 1). Thus, during the initial stage of the evolutionary transition from heterothallism to homothallism in the "VxAf " lineage, the ancestor of the "VxAf " lineage may have first evolved this type of homothallic mating system (Homo II) from the heterothallic mating system (Hetero) without modification of the form of sexual spheroids (unisexual male and female). Bisexual spheroids Fig. 6 Comparison of the most conserved region [near the YGGY motif of helix III (Coleman 2009)] of nuclear rDNA ITS-2 secondary structure between strains of four mating systems of Volvox africanus and V. reticuliferus (Table 1). Note the modified YGGY motif (boldface). For secondary structures of ITS-2, see Nozaki et al. (2015a). Red and black open boxes indicate compensatory base changes between Homo II strains (from Thailand and USA) and those of other sexual systems Nozaki et al. Botanical Studies (2022) 63:1 may have subsequently evolved in the homothallic ancestor of the "VxAf " lineage.

Conclusions
Algal diversity in Kalasin Province shows that freshwater bodies of this region contain a variety of volvocine and other interesting green algae (Heman 2015). The present re-discovery of the homothallic "VxAf " strains with unisexual male and female spheroids is based on field collections in a freshwater habitat of an intracontinental region of Thailand. We also found a new species of Volvox sect. Volvox, V. longispiniferus Nozaki & Mahakham, from the same field collection (Nozaki et al. 2020). In addition, two species of "VxAf " were collected in an ancient lake, Lake Biwa, Japan (Nozaki et al. 2015a). Thus, freshwater habitats in such geologically stable intracontinental regions may be fruitful for Volvox diversity.