Morphology
Asexual spheroids of Taiwanese strains were ovoid or spherical in shape and measured up to 920 μm long; each spheroid contained 1400–3000 somatic cells embedded within the periphery of the gelatinous matrix and large reproductive cells or gonidia in the posterior two-thirds (Fig. 1a, b). Each somatic cell was nearly spherical or ovoid in shape, up to 10 μm in diameter, and enclosed by a rectangular to hexagonal space formed by individual sheaths of gelatinous matrix (Fig. 1c, d). The cells lacked adjoining cytoplasmic bridges (Fig. 1c), and exhibited cup-shaped chloroplasts with a single stigma and a basal pyrenoid. The spheroid exhibited a gradual decrease in stigma size from the anterior to posterior pole. Gonidia were spherical in shape and had large vacuoles, measuring up to 92 μm in diameter (Fig. 1e). There were up to 16 gonidia per spheroid (Fig. 1a, b).
Asexual reproduction occurred as previously described (Starr 1969; Nozaki 1988). Successive divisions of each gonidium resulted in formation of a plakea. Gonidia of the next generation were evident outside the plakea because of the unequal cytokinesis (Fig. 1f, g). The plakea then inverted to form a compact, spheroidal daughter spheroid.
Sexual male spheroids were ellipsoidal or ovoid and 128- or 256-celled, containing biflagellate somatic cells and androgonidia that developed into sperm packets (Fig. 2a–d). The number of androgonidia or sperm packets in male spheroids varied even within the same culture. The ratio of somatic cells to androgonidia (sperm packets) in male spheroids was generally 4–12:1 (Fig. 2a, b). However, male spheroids sometimes exhibited a > 50:1 ratio (Fig. 2c) or approximately 1:1 ratio (Fig. 2d) of somatic cells to androgonidia. Female spheroids were ellipsoidal or ovoid in shape, and contained 1000–2000 biflagellate somatic cells and 22–28 eggs (Fig. 2e). The zygotes developed within the female spheroid after possible fertilization (Fig. 2f). The mature zygotes had a reticulate cell wall and were reddish brown in color, measuring 34–40 μm in diameter (Fig. 2g).
Morphology of asexual and sexual spheroids in the present Taiwanese strains was essentially consistent with that of the Japanese strains of V. carteri f. nagariensis except for male spheroids (Starr 1969; Nozaki 1988). The Japanese strains produce male spheroids with only a 1:1 ratio of somatic cells to androgonidia (Starr 1969; Nozaki 1988). In contrast, male spheroids of the present Taiwanese strains exhibited variability in the ratios (Fig. 2).
Molecular phylogeny of nuclear rDNA ITS-2
All 33 strains from Taiwan had the same nuclear rDNA ITS-2 sequences, which were also identical to the V. carteri f. nagariensis strain Eve (UTEX 1885). The phylogenetic analysis clearly demonstrated that the Taiwanese strains belonged to the V. carteri f. nagariensis clade (Fig. 3).
Genomic PCR of 33 strains from Taiwan
We screened the 33 Taiwanese strains for the presence or absence of five female-specific genes, two male-specific genes, the female and male types of the sex-based divergent gene MAT3, and the male type of LEU1S (Ferris et al. 2010) (Tables 1, 2).
Thirty-two of the Taiwanese strains and Eve could be clearly subdivided into female and male types (Table 1). The female type was composed of 13 Taiwanese strains and Eve, which shared the presence of five female-specific genes and MAT3f and the absence of two male-specific genes and male types of two sex-based divergent genes (MAT3m and LEU1Sm) (Table 1, Fig. 4; Ferris et al. 2010). Nineteen other strains belonged to the male type, based on the presence of two male-specific genes, MAT3m and LEU1Sm and the absence of five female-specific genes and MAT3f (Table 1, Fig. 4; Ferris et al. 2010). Thus, there seemed to be no recombination or deletion of such genes between male and female mating-type locus haplotypes in 32 of the 33 Taiwanese strains. However, in the remaining strain (2016-tw-nuk-8-2), one female-specific gene (HMG1f) was not amplified whereas this strain contained four other female-specific genes and MAT3f and showed no amplifications of all four of the male genes examined (Fig. 4). Thus, this strain seemed to be a female strain without amplification of HMG1f in genomic PCR.
Asexual spheroids and sexual induction of the HMG1f-lacking putative female strain
Asexual spheroids of the HMG1-lacking strain (2016-tw-nuk-8-2) were essentially the same as those of other Taiwanese strains, with up to 16 gonidia that developed into daughter spheroids via successive divisions and inversion (Fig. 5a–c). When sexually induced, apparently normal female sexual spheroids developed in this strain. The number of eggs produced was generally 20 or more (Fig. 5d). No morphological differences were recognized between these female spheroids and the female spheroids of other female stains. Attempts to cross 2016-tw-nuk-8-2 with male Taiwanese strains were unsuccessful, because the male Taiwanese strains suddenly lost their ability to form male spheroids during the study (from April to June 2017).