Kohei Takahashi (2nd Year Doctoral Student, Department of Biological Sciences)

Tetsuya Higashiyama, Professor, Department of Biological Sciences

Hisayoshi Nozaki (Project Researcher, Department of Biological Sciences)

Key points of the presentation

• Long-term field surveys in lakes and marshes of the Sagami River system and culture and mating experiments have revealed that the same species of green alga Pleurodryna has a third sexual phenotype, the hermaphroditic type, in addition to females and males.
• The presence of three sex phenotypes, female, male, and hermaphroditic, in the same species is the first such finding in algae and fungi, which are thought to have a simple sex-determination system.
• This may be an early stage in the evolution from a species with separate females and males to a hermaphroditic species, and there are great expectations for the elucidation of the determinants of the hermaphroditism suggested by the mating experiments.

Summary of Presentation

It is not so unusual for a single species to have three types of sexuality. In terrestrial plants and invertebrates, rare species in which "female," "male," and "hermaphroditic" individuals coexist are recognized. However, sexual reproduction in simple haploid ^{(Note 1)} organisms such as algae and fungi can be dioecious (heterothallic) or dioecious (homothallic) depending on the species ^{(Note 2),} with the former having two types of sex (female and male) and the latter having one type of hermaphrodite.

In this study, a research group led by the Graduate School of Science of The University of Tokyo and others, based on a long-term field survey of lakes in the Sagami River system (Figure 1) and culture and mating experiments, revealed that one species of green alga Pleodrina ^{(Note 3)} of the Volvox family has a third sexual phenotype of both sexes in addition to females and males (Figure 2). The presence of female, male, and hermaphroditic phenotypes in the same species (Fig. 3) is the first finding in algae and fungi, which are considered to have a simple sex-determination system.

The molecular genetic basis of the three sexually dimorphic phenotypes is expected to be elucidated, and similar long-term studies in other species are expected to continue.

Announcement

According to Plato's "The Feast," prehistoric humans were three types of bisexual individuals (male-male, female-female, and male-female) (Ref. 1). The coexistence of these three types of sexual (sex) expression within a single species is recognized in some land plants and invertebrates, and is interpreted as an evolutionary intermediate stage between hermaphroditic and dioecious species. However, no haploid organisms such as algae or fungi are known to have three types of sexual expression in the same species. Sexual reproduction in these organisms can be heterothallic or homothallic, depending on the species, with females and males being genetically determined in the former case (monogametic) and cells of the same genotype producing both female and male gametes in the homothallic case (bisexual). In the green alga Volvox lineage, where evolutionary biology of sex has been actively studied, evolution from heterothallic to homothallic was observed in several lineages, but what happened in the early stages of this evolution was a mystery.

Associate Professor Hisayoshi Nozaki's group at the then Department of Biological Sciences, Graduate School of Science, The University of Tokyo has been continuously studying the green algae of the Volvox lineage for more than 20 years by field surveys in Lake Sagami and Lake Tsukui in the Sagami River system in Kanagawa Prefecture (Figure 1).

Figure 1: A survey of Lake Sagami in Kanagawa Prefecture, Japan, carried out in a rowboat.
The occurrence of Pleodrina spp. was obtained in advance from the Tanigahara water treatment plant, and at this time, the sample yielded amphipathic Pleodrina starryi. photo by Nozaki, June 2013.

During this period, we described Pleodorina starrii (scientific name: Pleodorina starrii) as a new species in 2006, and discovered the male-specific gene " OTOKOGI (PlestMID)^{" (Note 4) in} the male strain of this species, which was an evolutionary genetic study of sex using green alga Volvox series This was a breakthrough in research (Refs. 2-5). Since that time, Pleodrina starry was considered to be a heterotallic species, with only male and female strains. However, in the last 10 years, two strains of hermaphroditic Pleodrina that produced both male and female gametes in a single strain were obtained from the same lake in the Sagami River system.

In this study, the group conducted comparative analysis of morphological and molecular data and crossbreeding experiments between female and male Pleodrina starry strains (Figure 2), which had been considered heterothallic, and a hermaphroditic Pleodrina strain (Figure 2).

Figure 2: Asexual swarming and sexual reproduction of bisexual Pleodrina starry strains.
(A) Asexual clade. No differences from previously reported female or male monogametic strains, with small non-sexual cells (arrows) in the anterior part of the swarm. Scale bar 50 μm.(B-H) Sexual reproduction. (B-H) Sexually reproductive; male and female gametes form in a solitary strain when cultured on nitrogen-starved medium, and union is complete. Scale bars are 50 μm (B-D), 10 μm (E, H), and 5 μm (F, G). (B) Male gametophyte. Germ cells have differentiated into sperm bundles, which are aggregates of male gametes (spermatozoa). Arrows indicate non-germ cells. (C) Spermatogonia ejected from a female gametophyte and a male gametophyte (arrowheads). In the female gametophyte, germ cells have differentiated into female gametes. Arrows indicate non-germ cells. (D) Spermatogonia reaching the female gametophyte separate into a single male gametophyte (arrowhead) and enter the female gametophyte. Arrows indicate non-germ cells of the female gametophyte. (E) Spermatozoon bundle. (F) Single male gametophyte. Arrowheads indicate nuclei. (G) The nucleus (arrowhead) of a male gametophyte is strongly stained by fluorescent staining with DAPI. (H) Fluorescence staining with DAPI shows penetration of the male gametophyte nucleus (arrowhead) into the female gametophyte, confirming the union of male and female gametophytes within the same plant. Modified from this research paper.

The results showed no morphological or molecular differences between these strains. In addition, the results of the crossbreeding experiments between female and male strains and both sexes showed that zygote formation and offspring survival between these strains were almost identical to those observed in the crossbreeding between female and male strains. Thus, it is clear that the hermaphroditic strains are the same Pleodrina starry as the female and male strains, and that this species has three types of sexual phenotypes: "female," "male," and "hermaphroditic" (Figure 3).

Figure 3: Life cycle of Pleodrina staley revealed in this study.
This species has three types of sexual phenotypes [unisexual male, unisexual female, and bisexual]. Both types are haploid (n) under nutritional conditions and reproduce asexually (asexual cycle). When sexual induction by nitrogen starvation induces a shift to sexual reproduction, monogametic males reproduce only in male colonies in which gametes differentiate into sperm packets (sp), which are aggregates of male gametes (male gametes, mg), while monogametic females reproduce in male colonies in which gametes differentiate into spermatozoa (female gametes, sp), which are aggregates of male gametes (female gametes). The monogametic female forms only a female colony in which gametes have differentiated into female gametes (female gametes, fg). Hermaphrodites, on the other hand, form both male and female colonies when sexually induced. Spermatozoa ejected from the monogametic or hermaphroditic male colony swim to the monogametic or hermaphroditic female colony, divide into single male gametes, and join (syngamy) with female gametes in the female colony to form diploid (2n) zygotes. From the zygote, the zygote develops into one of three types of clutches at different rates depending on the mating combination. Reproduced from this research paper.

Furthermore, genetic analysis revealed the presence of " OTOKOGI," which is considered to be a male-specific gene, in the hermaphroditic strain. Genetic analysis of crossbreeding experiments indicated that the hermaphroditic strain has a male sex chromosome region ^{(Note 5)} in which " OTOKOGI " is present, suggesting the presence of a hermaphroditic determinant (BF) in the autosomal region ^{(Note 6)} (Figure 4).

Figure 4: Predicted genotypes (upper box) and sexual phenotypes (lower box) from genetic analysis of Pleurodolina starryi mating experiments.
The three types of sexual phenotypes [unisexual male, unisexual female, and bisexual] are determined by the sex chromosomal determinant (BF) in the autosomal region (gray) and the sex chromosomal region where " OTOKOGI " is located (male MTM or female MTM or female MTM or female MTM). The bisexuality is determined by the sex chromosome region (male MTM or female MTF ) in which the "OTOKOGI" is located. Modified from this research paper.

This study is the result of a field survey of the Sagami River system over a period of more than 20 years. We are very grateful to Mr. Shoji Saito (deceased), Ms. Shizue Arii, Ms. Izumi Tateno, and other members of the Kanagawa Prefecture Tanigahara Water Treatment Plant for providing us with information on algal growth in Lake Sagami and Lake Tsukui during this period. This work was also supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Kouhei Takahashi 21J10259; Hisayoshi Nozaki 19K22446 and 20H03299).

References
1. Dover KJ. Aristophanes' speech in Plato's symposium. J Hellenic Stud. 1966; 86:41-50.
2. Nozaki H, Mori T, Misumi O, Matsunaga S, Kuroiwa T. Males evolved from the dominant isogametic mating type. Curr Biol. 2006; 16:R1018-20.
Graduate School Press Release < https://www.s.u-tokyo.ac.jp/ja/press/2006/22.html>.
3. Ferris P, Olson BJ, De Hoff PL, Douglass S, Casero D, Prochnik S, Geng S, Rai R, Grimwood J, Schmutz J, Nishii I, Hamaji T, Nozaki H, Pellegrini M, Umen JG. Evolution of an expanded sex-determining locus in Volvox. Science. 2010; 328:351-4.
Graduate School Press Release < https://www.s.u-tokyo.ac.jp/ja/press/2010/16.html>.
4. Hamaji T, Kawai-Toyooka H, Uchimura H, Suzuki M, Noguchi H, Minakuchi Y, Toyoda A, Fujiyama A, Miyagishima SY, Umen JG, Nozaki H. Anisogamy evolved with a reduced sex-determining region in a reduced sex-determining region in volvocine green algae. commun Biol. 2018; 1:17.
Graduate School Press Release < https://www.s.u-tokyo.ac.jp/ja/press/2018/5786/>.
5. Yamamoto K, Hamaji T, Kawai-Toyooka H, Matsuzaki R, Takahashi F, Nishimura Y, Kawachi M, Noguchi H, Minakuchi Y, Umen JG, Toyoda A, Nozaki H. Three Proc Natl Acad Sci U S A. 2021; 118 :e2100712118.
Graduate School Press Release < https://www.s.u-tokyo.ac.jp/ja/press/2021/7373/>.
6. Ferris P, Goodenough U. Mating type in Chlamydomonas is specified by mid, the minus-dominance gene. Genetics. 1997; 146:859-69.

Journals

Journal name	Evolution
Title of paper	Three sex phenotypes in a haploid algal species give insights into the evolutionary transition to a self-compatible mating system
Author(s)	Kohei Takahashi, Hiroko Kawai-Toyooka, Ryo Ootsuki, Takashi Hamaji, Yuki Tsuchikane, Hiroyuki Sekimoto, Tetsuya Higashiyama, and Hisayoshi Nozaki* (in Japanese)
DOI Number	10.1111/evo.14306
Abstract URL	https://doi.org/10.1111/evo.14306

Terminology

Note 1 Haploid.

Land plants and multicellular organisms have diploid (2n, biphasic) multicellular bodies with two homologous chromosomes in each cell, while many species of algae and fungi have haploid (n, monophasic) multicellular bodies with only one homologous chromosome in each cell. In the life rings of haploid species, only the unicellular zygote (fertilized egg) is diploid. ↑

Note 2 Heterothallic and homothallic

In haploid algae and fungi, both sexes, such as female and male, are genetically determined, and it was believed that there are species in which both sexes must be mixed for gametogenesis (fertilization) and species in which gametes are produced and joined within the same clonal strain (culture strain consisting of genetically uniform cells). The former mode of sexual reproduction is called heterothallic and the latter homothallic. Until now, only these two types of sexual reproduction have been recognized, but this study reveals for the first time the existence of a species that reproduces sexually in a manner intermediate between the two (Pleurodryna stagnalis). ↑up

Note 3: Pleodrina of the green alga Volvox series

Pleodrina is a member of the Volvox series, a group of green algae that grows in freshwater and is composed of Chlamydomonas, a unicellular organism with two flagella, and multicellular organisms (usually called "groups") in which four or more cells are aggregated. Multicellular species are thought to have evolved from unicellular ones such as Chlamydomonas, which consists of more than 500 cells, with small non-sexual cells (that do not divide and reproduce asexually) that differentiate into male and female Volvox, and Chlamydomonas, with undifferentiated male and female isomorphic mates, Gonium, Yamagiella, Eudolina, and Pleodolina, there are extant organisms that are evolutionarily intermediate stages in terms of regime and sexual reproduction. Since Pleodrina has 32-, 64- or 128-cell spherical groups with non-germ cells and produces large female gametes and small male gametes during sexual reproduction, it has long been considered to correspond to organisms just prior to the evolution of this lineage to the volvox. ↑up

Note 4: Male-specific gene " OTOKOGI (PlestMID) "

In the unicellular green alga Chlamydomonas, the MID gene is known as the gene that determines mating type minus (one of the sexes in an isomorphic marriage in which the sexes are undifferentiated) (Ref. 6). In the male-female differentiated Volvox series ^{(Note 3} ) of Pleodrina starry, a gene (PlestMID) similar in origin to MID was present only in males (Ref. 2) and was therefore called "OTOKOGI". The present study revealed that the gene is also present in the hermaphroditic Pleurodolina starry. ↑up

Note 5: Sex chromosome region

A chromosomal region in which sex-specific genes are located and in which the gene composition and sequence order differ between the two sexes, such as female and male. It is also called the sex-determining region because it determines sex. Chromosomes in this region often do not differ in size or shape between the two sexes, but they are sometimes referred to as sex chromosomes. ↑up

Note 6 Autosomal region

A region of a chromosome ^{other than the} sex chromosome region of an organism ^{(Note 5)} where the gene composition and sequence order of both sexes, such as female and male, are basically identical. ↑up