Delineating a New Heterothallic Species of Volvox (Volvocaceae, Chlorophyceae) Using New Strains of “Volvox africanus”

The volvocine algae represent an excellent model lineage in which to study evolution of female and male genders based on comparative analyses of related species. Among these species, Volvox carteri has been extensively studied as a model of an oogamous and complex organism. However, it may have unique derived features that are not present in other species of Volvox. Therefore, information regarding the characteristics of sexual reproduction of other species of Volvox is also important. In 1971, Starr studied four types of sexuality in several global strains identified as Volvox africanus; however, further taxonomic studies of these strains have been lacking, and strains of three of the four sexual types are not available. Here, we studied the morphology, sexual reproduction, and taxonomy of two V. africanus-like species isolated recently from Lake Biwa, Japan. These two species were very similar to two sexual types described by Starr in 1971: one producing dioecious sexual spheroids in heterothallic strains and the other forming both male spheroids and monoecious spheroids in a single strain. The former species produced zygotes with a reticulate cell wall, whereas a smooth zygote wall was observed in the latter species as in V. africanus previously reported from various localities around the world. Our multigene phylogenetic analysis demonstrated that these are sister species to each other. However, the presence of a compensatory base change in the most conserved region of the secondary structure of nuclear ribosomal DNA internal transcribed spacer-2, hybrid inviability demonstrated by intercrossing experiments, and morphological differences in the density of abutment between the gelatinous material of adjacent cells (individual sheaths) in the spheroid supported the recognition of the two species, V. africanus having a smooth zygote wall and V. reticuliferus Nozaki sp. nov. having a reticulate zygote wall.

For observation of asexual spheroids, aliquots of actively growing culture (about 0.5 mL) were inoculated into fresh AF-6 medium or AF-6/3 medium every 10-20 days. A BX60 microscope (Olympus, Tokyo, Japan) equipped with Nomarski interference optics was used for light microscopic examinations. Transmission electron microscopy (TEM) of asexual spheroids was carried out as previously described [22] except using a JEM-1010 electron microscope (JEOL, Tokyo, Japan).
Sexual spheroids developed spontaneously in each strain of the two species when the culture was repeatedly inoculated (every 5-7 days). To enhance sexual induction, the cultures were grown in USVT medium [20] diluted with double-distilled water (USVT/3 medium) at 25°C on a 14-h light:10-h dark schedule under cool-white fluorescent lamps at an intensity of 200-220 μmolÁm -2 Ás -1 . Sexually induced male and female cultures were mixed for formation of zygotes in heterothallic strains. Upon the mixture of sexually induced male and female cultures (in the heterothallic species V. reticuliferus) or just after the formation of sexual spheroids (in the homothallic species V. africanus), a half to equal volume of new USVT/3 medium was added to the culture for zygote maturation.
For intercrossing experiments, 17-28 male spheroids of V. africanus strain VO4-F1-1 induced in USVT/3 medium were isolated with a micropipette and washed twice with new AF-6/3 medium, and only male spheroids were inoculated into the induced female cultures of V. reticuliferus strain VO123-F1-6 in 7-11 mL USVT/3 medium. Then, a half to equal volume of new USVT/3 medium was added to the culture for zygote maturation.
ITS-2 secondary structures were compared to detect compensatory base changes (CBCs) of nuclear rDNA ITS-2.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants; hence, the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

Asexual spheroids
Volvox africanus strains 2013-0703-VO4 and VO4-F1-1, and V. reticuliferus strains 2013-0703-VO1, VO2 and VO3, and VO123-F1-6 and F1-7 can be assigned to V. africanus based on the morphology of the asexual spheroids, as delineated previously [2,5] except for individual sheaths. The asexual spheroids of both species were ovoid or ellipsoidal with a broad anterior face, and contained 800-3000 cells arranged in a single layer at the periphery of the gelatinous matrix, measuring up to 350-400 μm in length ( Fig 1A; S1A Fig). The somatic cells were embedded in individual sheaths of the gelatinous matrix, and nearly spherical in shape, lacking cytoplasmic bridges between them (Fig 1B-1E; S1B-S1D Fig). The cells had two equal flagella, two contractile vacuoles near the base of the flagella, and a cup-shaped chloroplast with a single stigma and a basal pyrenoid, measuring up to 7-8 μm in diameter. There was gradual diminution in stigma size from the anterior to posterior pole of the spheroids. In V. reticuliferus, the number of pyrenoids in somatic cells often increased to two or more (Fig 1C), whereas V. africanus almost always had a single pyrenoid (S1B Fig). Both species generally had two to four gonidia, or sometimes more, in an asexual spheroid (Fig 1A; S1A Fig). Two to four of the gonidia were distributed near the middle of the spheroid, and the remainder, when present, were located in the posterior half. The mature gonidia, measuring up to 60-65 μm in diameter, were spherical in shape and vacuolated, and had numerous contractile vacuoles and a large chloroplast occupying the whole cytoplasm and containing a dozen or more pyrenoids distributed randomly. The surface of the chloroplast showed numerous longitudinal fine striations that were radially arranged in top view ( Fig 1F). Juvenile spheroids developed in pairs within the parent in both species (Fig 1A; S1A Fig).
Individual sheaths, the outermost layer of matrix that abuts that of the adjacent cell, of asexual spheroids of V. africanus were distinct and adhered to one another in such a way as to appear pentagonal, hexagonal, or heptagonal in front view due to mutual compression (S1C Individual sheaths appear to stain with methylene blue or aniline blue (Fig 1D and 1E; S1C and S1D Fig  In asexual reproduction of both species of Volvox, gonidia divided successively to develop a hollow plakea that became a juvenile spheroid after inversion. During the plakeal and inversion stages, the gonidia or gonidial initials of the next generation were evident ( Fig 1G; S1E Fig).
Two types of sexual spheroid, male and monoecious spheroids, were produced within a culture or even in a single parental spheroid of the present V. africanus strains (2013-0703-VO4 and VO4-F1-1) (S1F Intercrossing between V. africanus male spheroids and V. reticuliferus female spheroids Male spheroids of the sexually induced V. africanus strain VO4-F1-1 were isolated by micropipette and mixed with sexually induced V. reticuliferus female spheroids (strain VO123-F1-6). After several days, a small portion (< 20%) of eggs in the female spheroids secreted a cell wall. Such walled eggs were spherical in shape and had an almost smooth cell wall (S4A and S4D Fig). Approximately 2 weeks after mixing of gametes, part of the walled eggs turned reddish brown in color and had a thick cell wall that was smooth or weakly reticulate (S4B and S4E  Fig). However, no reddish brown eggs were observed 3 weeks after mixing; all of the walled eggs lost their reddish or greenish color and almost all disintegrated, with rupture of their walls (S4C and S4F Fig). As no eggs of V. reticuliferus female spheroids developed into a reddish brown cell with a thick cell wall without male spheroids (not shown), the thick-walled reddish brown eggs in the female spheroids were possible hybrid zygotes.

Compensatory base changes (CBCs) of nuclear rDNA ITS-2
Between OTUs within the lineage RT or AF (Fig 3), CBCs were not detected in the ITS-2 sequence encompassing the YGGY motif (boldface in S5 Fig) in helix III, which is the most highly conserved region of nuclear rDNA ITS-2 [35,39]. The nuclear rDNA ITS-2 sequence of "V. africanus" strain UTEX 1889 in this region showed two or three CBCs compared to that of the lineages RT or AF, respectively (Fig 4). However, a single CBC was present in the nuclear rDNA ITS-2 sequence between lineages RT and AF except for ITS-2 sequence comparison between a single OTU (V. reticuliferus strains 2013-0703-VO1, VO2 and VO3) of linage RT and lineage AF (Fig 4).

Discussion
Volvox africanus was originally described by West [40] based on material collected in Lake Albert Nyanza, a shallow lake on the border between Uganda and Congo in the Great Rift Valley, Africa (S2 Table). West [40] observed asexual spheroids of V. africanus and a single male spheroid of an uncertain species of Volvox. He later examined possible female spheroids and zygotes with a smooth wall of V. africanus originating from small ponds in Ussangu Desert in the African region formerly known as "German East Africa" [41]. Rich and Pocock [42] reported asexual spheroids of V. africanus collected in South Africa. Shaw [43] and Starr [14] demonstrated that both male and monoecious spheroids were produced in the same parental spheroid of V. africanus originating from the Philippines and India, respectively. Iyengar [44] observed both dioecious (male and female) and monoecious sexual spheroids of V. africanus originating from India. Although Starr [14] did not report zygotes of his strains of V. africanus, West [41], Shaw [43], and Iyengar [44] observed smooth zygote walls of V. africanus as in our V. africanus material from Japan (S1J and S1K Fig). In contrast, our heterothallic strains of V. reticuliferus formed mature zygotes with a reticulate wall (Fig 1K and 1L).
The asexual spheroids of our new strains of V. africanus and V. reticuliferus are consistent with those of V. africanus characterized by Smith [2] and Nozaki and Coleman [5] with regard to their shape, the number and development of gonidia or juvenile spheroids in them, and the early development of gonidia of the next generation during embryogenesis. However, the structure of the gelatinous matrix in V. reticuliferus is different from that of our Japanese strains of V. africanus. In our new strains of V. africanus, individual sheaths are distinct and appear pentagonal, hexagonal, or heptagonal in surface view due to mutual compression (S1C and S1D Fig). Rich and Pocock [42] observed distinct pentagonal or hexagonal individual sheaths in their South African V. africanus material. According to Iyengar [44], distinct pentagonal or hexagonal individual sheaths were also observed in the Indian material of V. africanus as well as the slide material of V. africanus studied by Shaw [43]. However, individual sheaths of our new strains of V. reticuliferus were confluent or indistinct even when stained with aniline blue (Fig 1D and 1E). This morphological difference was also confirmed by our TEM examinations of both species (S3 Fig), possibly resulting from the difference in density of abutment between the gelatinous material of adjacent cells. Thus, V. reticuliferus can be clearly distinguished from V. africanus by its reticulate zygote wall, and confluent or indistinct individual sheaths of spheroids. In addition, the presence of a single CBC in the most highly conserved region of nuclear rDNA ITS-2 secondary structure [39] between lineage RT (composed of three OTUs of V. reticuliferus, see below) and lineage AF (including V. africanus strain 2013-0703-VO4) ( Fig  4) and lack of production of mature viable hybrid zygotes (representing hybrid inviability) in our intercross between the two species (S4 Fig) suggest sexual isolation between them. Thus, V. reticuliferus and V. africanus are morphologically and genetically different species.
Although zygote morphology is unknown in two heterothallic strains of "V. africanus" [UTEX 1890 and 1891 (Darra 4 and 6, respectively [14])] and "V. africanus" strain UTEX 2907, the three strains have indistinct individual sheaths of gelatinous matrix in the asexual spheroids as in V. reticuliferus strains 2013-0703-VO1, VO2 and VO3 (S1 and S2 Figs). In addition, these three UTEX strains and V. reticuliferus strains 2013-0703-VO1, VO2 and VO3 form a small clade (lineage RT, Fig 3) in which no CBC is detected in the most highly conserved region of the secondary structure of ITS-2 of nuclear rDNA (Fig 4; S5 Fig). Therefore, they can be identified as V. reticuliferus.
Starr [14] reported four types of sexual reproduction in "V. africanus" (S2 Table). Based on our study, the strains of heterothallic, dioecious type [Darra4 (UTEX 1890) and Darra6 (UTEX 1891)] were assigned to V. reticuliferus. However, strains of the other three sexual types exhibiting homothallic sexuality studied by Starr [14] are not available, but the nuclear rDNA ITS-2 phylogeny demonstrated that V. africanus strain 2013-0703-VO4 (homothallic) and strains of the other three sexual types (exhibiting homothallic sexuality) studied by Starr [14] form a clade that is sister to the clade of V. reticuliferus (lineage RT) (Fig 3). Within the homothallic clade, dioecious, homothallic type (UTEX 1889) is sister to the lineage AF (composed of the other three homothallic strains), and these two sister lineages show three CBCs in the most conserved region [39] of secondary structure of nuclear rDNA ITS-2 (Fig 4). Thus, this homothallic clade may be subdivided into at least two cryptic species when considering the ITS-2 sequence and types of sexual spheroids (Figs 3 and 4). However, other phenotypic data including zygote wall and gelatinous matrix morphology as well as other sequence data are now lacking, but are needed to taxonomically identify strain UTEX 1889 and two strains of lineage AF (strains UTEX 1893 and UTEX 1892).
West [41] reported "female" spheroids containing mature zygotes in the African material of V. africanus, but he did not observe male spheroids. Thus, these female spheroids may have actually been monoecious. According to Shaw [43], the Philippine V. africanus material produces both male and monoecious spheroids in the same parental spheroid as in our new strains and monoecious with males type described by Starr [14] (S1 Table). The Indian material of V. africanus examined by Iyengar [43] produces both dioecious (male and female) and monoecious sexual spheroids. Therefore, V. africanus may be a worldwide species with homothallic sexuality and smooth-walled zygotes. Further morphological and molecular studies using living strains of V. africanus-like species are needed to understand the evolution of homothallism and/or monoecism within this group and to more clearly delineate V. reticuliferus and V. africanus.
As in previous multigene phylogenies [5,23,24], our phylogenetic analyses robustly resolved that three volvocacean genera Volvox, Pleodorina, and Eudorina are not monophyletic, and the lectotype species V. globator [45] is robustly separated from Volvox sect. Merrillosphaera sensu Smith (Fig 2). Therefore, V. africanus and V. reticuliferus should not be classified to the genus Volvox when the generic classification is strictly based on the monophyletic concept. As discussed previously [5], however, division of the genus Volvox into four monophyletic genera (Fig 2) would not resolve problems for nonmonophyly of the genera Eudorina and Pleodorina [5,24]. New phenotypic characters are still needed to clearly delineate monophyletic genera proposed in future within the Volvocaceae [5,24]. Thus, division of the genus Volvox was not proposed here as suggested previously [5].
The present study delineates a new species of Volvox (V. reticuliferus) that could be assigned to the section Merrillosphaera sensu Smith [2]. However, this section is non-monophyletic as shown in Fig 2. Thus, in order to avoid discrepancy between phylogeny and section level classification of Volvox in future studies, here we propose a new classification system (four monophyletic sections) of Volvox at section level (S3 Table). Thus, V. powersii and V. gigas should be removed from section Merrillosphaera and assigned to section Besseyosphaera (type species: V. powersii), and section Copelandosphaera (type species: V. dissipatrix) should be synonymized under section Merrillosphaera based on the phylogenetic results (for details, see S3 Volvox reticuliferus Nozaki sp. nov. Asexual spheroids ovoid or ellipsoidal with broad anterior face, composed of 800-3000 cells, measuring up to 400 μm in length, with 2-4 gonidia distributed in the middle portion with or without an additional 1-4 gonidia in the posterior portion. Somatic cells nearly spherical in shape, lacking cytoplasmic bridges, embedded within individual sheaths of gelatinous or extracellular matrix, measuring up to 8 μm in diameter. Individual sheaths confluent or indistinct. Each somatic cell enclosed by a broad secondary boundary layer of gelatinous matrix. Gonidia vacuolated, with a large chloroplast, measuring up to 60 μm in diameter. The surface of the chloroplast of gonidia with fine striations radially arranged. Juvenile spheroids developing in pairs within the parent. Gonidia of the next generation evident during cell divisions of formation of juvenile spheroids. Sexual spheroids dioecious with production of male or female spheroids in a single genetic strain. Male spheroids ellipsoidal or cylindrical, containing 1000-1500 biflagellate somatic cells and 80-120 androgonidia. Androgonidia dividing into plateshaped sperm packets.  Table).
Type locality: Lake Biwa, Shiga Prefecture, Japan. A water sample was collected by FT and HN on 3 July 2013.
Key to the Sections Emended in the Genus Volvox