An ulvophycean marine green alga produces large parthenogenetic isogametes as predicted by the gamete dynamics model for the evolution of anisogamy

In eukaryotes, the gamete size difference between the two sexes (anisogamy) evolved from gametes of equal size in both mating types (isogamy). The gamete dynamics (GD) model for anisogamy evolution combines gamete limitation and competition and predicts that, if gametes of both mating types can develop parthenogenetically (i.e. without fusing with the opposite mating type), large isogamy can evolve under gamete-limited conditions. Ulvophycean marine green algae that have been claimed to exhibit various gametic systems from isogamy to anisogamy are important models for testing such theories. However, in most previous papers, whether a species is isogamous or anisogamous has not been examined statistically. Caution is necessary regarding claims of slight anisogamy because of gamete size variation. We reveal (i) that the gametic system of Struvea okamurae is large isogamy using a generalized linear mixed model (GLMM), which accounted for the variation of gamete size among individual gametophytes, and (ii) that gametes of this alga can actually develop parthenogenetically, contrary to a previous report. Its habitat environments and protracted duration of gamete release suggest that this alga might experience gamete-limited conditions. S. okamurae seems to produce large parthenogenetic isogametes following GD model predictions, as an adaptation to deep waters.

Methods
We collected gametophytes and sporophytes of Struvea okamurae in early spring (March 2020) at a depth of about 3 m off the coast of Ginoza, Okinawa, Japan (26°47’N, 127°78’E) (figure 1), and established the uni-algal culture strains of these gametophytes (So-4, So-6, So-14, So-21, and So-27) and sporophyte (So-1) using a protoplast-formation method. Protoplasts were cultured in 1/4 concentration Provasoli’s enriched seawater (PES) medium [20] at 22°C under a long day condition (14 h: 10 h = light: dark) and ca. 15 μmol photons ·m-2 · s-1 in a culture chamber (LH-220S; NK system, Osaka, Japan). We isolated gametophyte/sporophyte Struvea plants developed from a protoplast individually into 12 well plastic plates (IWAKI, Tokyo, Japan) and induced gametogenesis/zoosporogenesis under an eutrophic condition by changing the medium.

We estimated the volume of S. okamurae gametes more accurately than previous studies on other ulvophycean species by eliminating the error due to discrepancies between the calculation of biovolume based on geometric approximation and that based on real shapes. Some gametes were fixed with 1% glutaraldehyde immediately after they were released. We observed them on a cover glass coated with poly-L-lysine (0.01 w/v%) and took a micrograph of each gamete using a differential interference microscope (Axio Imager A1; Zeiss, Oberkochen, Germany). We obtained successive vertical cross-sections through each pear-shaped gamete using the micrograph, calculated the volumes of each section with 1-pixel height, and summed the volumes of all sections.