“Starrii” blessed us twice

Two major discoveries were made in a green alga, Pleodorina starrii. For the first time, a male-specific gene was identified within the volvocine algae. The stable coexistence of male, female, and bisexual types in one species was suggested, implying that more than two sex types may be peaceful.
Published in Ecology & Evolution
“Starrii” blessed us twice
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A new species of the volvocine green algae, Pleodorina starrii, was established in 2006 based on my cultured materials originating from a sample of Lake Sagami, Japan (Nozaki et al. 2006a, doi: 10.1111/j.1529-8817.2006.00255.x). The specific name “starrii” was dedicated to the late Dr. Richard C. Starr (Fig. 1), a pioneer of Volvox cultural studies (Coleman and Zeikus 2001), who welcomed and supported me during the last century in studies of the multicellular volvocine green algae such as Pleodorina and Volvox. When I described P. starrii (Fig. 2), I never dreamed that “Starrii” would lead us to two great discoveries.

Fig. 1. Dr. Richard C. Starr and a Japanese boy (H. Nozaki). 14 August 1988, Australia.
Fig. 1. Dr. Richard C. Starr and a Japanese boy (H. Nozaki). 14 August 1988, Australia. 

 Since newly established cultures of the colonial or multicellular volvocine algae are very active in sexual reproduction when cultured in the nitrogen-deficient medium and abundant sperm packets (bundles of male gametes) were produced even within a single male culture of P. starrii. By using such a sexually active male culture, a new male-specific gene OTOKOGI (PlestMID) was successively identified (Nozaki et al. 2006b, doi: 10.1016/j.cub.2006.11.019). The identification of OTOKOGI was expected a breakthrough for the molecular genetic studies of the evolution of sex using multicellular volvocine green algae (Kirk 2006, doi: 10.1016/j.cub.2006.11.015; Charlesworth 2007, doi: 10.1016/j.cub.2007.01.056). Actually, extensive studies on the evolution of sex/mating type were subsequently carried out as focusing MID/OTOKOGI homologs and their harbored sex-determining regions (SDRs) of sex chromosomes in the multicellular volvocine algae (e, g. Ferris et al. 2010, doi: 10.1126/science.1186222; Hamaji et al. 2018, DOI: 10.1038/s42003-018-0019-5).

Fig. 2. The volvocine green alga Pleodorina starrii originating from Lake Sagami, Japan.

Fig. 2. The volvocine green alga Pleodorina starrii originating from Lake Sagami, Japan. Original photo by Hisayoshi Nozaki.

 Even after the discovery of OTOKOGI, I continued field collections of multicellular volvocine algae from Japanese lakes because I love fieldwork and sightseeing outside laboratories. In addition, I aimed to let young scientists and students understand the interests and scientific importance of the field collections in lakes. Thus, I gathered young people and went to Japanese lakes for seeking multicellular volvocine algae (Fig. 3).

Fig. 3. Young people in the field collection of Lake Sagami, 6 June 2022.

Fig. 3. Young people in the field collection of Lake Sagami, 15 June 2022. Original photo by Hisayoshi Nozaki.

 After the Great East Japan Earthquake occurred in March 2011, young people or undergraduate students did not become interested in our laboratory, possibly influenced by the drastic social changes in Japan. Thus, I decided to take part in UTRIP (The University of Tokyo Research Internship Program) to welcome young undergraduate students worldwide to our lab and activate our lab members. UTRIP is a short-term internship program to provide students with an opportunity to gain the experience of "real graduate-school life" or "research-centered life". In 2013, the first internship in the Nozaki lab welcomed a UTRIP student from the National University of Singapore and he conducted research in Lake Biwa, Lakes Sagami, and Lake Tsukui (Fig. 4). I also conducted the survey with the UTRIP student and established 10 new culture strains of Pleodorina starrii from samples collected in Lake Sagami. Although I thought that the OTOKOGI/MID research in P. starrii was almost over, I kept the 10 strains because the new strains are generally active in inducing sexual reproduction and could be used for something.

Fig. 4. Field collections of multicellular volvocine algae in Japanese freshwater lakes. A. Lake Biwa survey from Miami Beach (5 July 2015). B. Rowing boat survey in Lake Sagami (22 June 2021). C. Collection of water samples from the pier facing the parking lot of Lake Biwa Museum (6 July 2017). Original photos by Hisayoshi Nozaki.

Fig. 4. Field collections of multicellular volvocine algae in Japanese freshwater lakes. A. Lake Biwa survey from Miami Beach (5 July 2015). B. Rowing boat survey in Lake Sagami (22 June 2021). C. Collection of water samples from the pier facing the parking lot of Lake Biwa Museum (6 July 2017). Original photos by Hisayoshi Nozaki.

 In 2017, we welcomed another UTRIP student from Jones Hopkins University, USA. UTRIP students had to present their research results at the end of the program, but it was difficult to decide what kind of research she would be going to do. Then, I remembered 10 strains of P. starrii that I established in 2013 by surveying Lake Sagami with the previous UTRIP student from Singapore. The research program I suggested for the USA UTRIP student included a simple experiment of morphological identification of sexes of the 10 strains of P. starrii. By exchanging medium from growth to nitrogen-deficient mating medium, the culture will produce sperm packets (male) or not (female). We believed that the sex phenotypes of P. starrii culture strains are either male or female because this species was considered to be heterothallic (Nozaki et al. 2006a,b). I explained this simple experiment to the UTRIP student and her teaching assistant (graduate student). After a while, the teaching assistant and the UTRIP student said to me, "Sir, we don't understand. Please take a look." I was surprised at one of the 10 culture strains. Despite the fact that only a single culture strain had been sexually induced, a "zygote-like dormant cell" had been formed. I remembered that such a bisexual (hermaphroditic) strain of Pleodorina, which produces zygotes within a single culture, was established from Lake Tsukui in 2007.

 The year 2017 was only three years away from my official retirement as an associate professor at the University of Tokyo, and I thought there would be no students to thoroughly study this interesting topic about bisexual strains of Pleodorina in their doctoral course. However, in February 2018, “Starrii” blessed us again. A master's graduate student suddenly appeared who wanted to change his lab and finish his master's thesis within one year. He got interested in the bisexual strain of Pleodorina originating from the same lake as P. starrii, and successfully completed his master's thesis then, fortunately, continued his research of Pleodorina in his doctoral program and received his doctoral degree from the University of Tokyo in this March.

 In his master's program, Kohei Takahashi demonstrated that P. starrii is a trioecious species, based on the morphological and molecular data (Takahashi et al. 2021, doi: 10.1111/evo.14306). A mating system in which a single species has three sex phenotypes is called trioecy, and is rarely recognized in species of flowering plants and invertebrates in which "female," "male," and "bisexual" individuals coexist (Roy 2021, doi: 10.1111/evo.14345). On the other hand, mating systems in simple haploid organisms such as algae and fungi were believed to be heterothallic or homothallic depending on the species, with the former having two sexual phenotypes (female and male) and the latter having one bisexual phenotype. However, our long-term field surveys of lakes in the Sagami River water system (Lake Sagami and Lake Tsukui) and culture experiments revealed that P. starrii has a third sexual phenotype with both sexes (bisexual strains) in addition to female and male phenotypes (Takahashi et al. 2021). This is the first finding in algae and fungi of a mating system in which three sex phenotypes (female, male, and bisexual) are present in the same species.

 Very recently, whole genome data from three sex phenotypes of P. starrii were generated to resolve the molecular genetic bases of the trioecy (Takahashi et al. 2023, doi: 10.1038/s42003-023-04949-1; press release). Our genome analyses revealed very unique features of sex-related genes including the male gene OTOKOGI and the female gene FUS1. Both the male and bisexual phenotypes of P. starrii have a male-type SDR with three paralogs of OTOKOGI. The female phenotype harbors a female-type SDR lacking FUS1. FUS1 was surprisingly found in an autosomal region in the three sex phenotypes (Fig. 5). Thus, the trioecy might have evolved based on the drastic reorganization of genes in SDR constituting the sex chromosome of the ancestral species. Trioecy has been sometimes considered an unstable intermediate stage of evolutionary transitions between dioecious species (heterothallic species) and monoecious species (homothallic species). However, our genome study revealed that the three sex phenotypes of P. starrii might have evolved based on a drastic reorganization of genes such as OTOKOGI and FUS1 in SDRs, providing the first support for the stability of the trioecy from genomic data. Thus, the results of our genome study suggest that the trioecious species P. starrii does not represent an intermediate evolutionary stage. The co-existence of more than two sex phenotypes may be peaceful for algae as in the human world (Fig. 5). It is expected that similar studies will be conducted in the future on closely related species of P. starrii to clarify the concrete processes of evolution of the trioecious species.

Fig. 4. Diagram of evolution of the trioecious species Pleodorina starrii. Based on Takahashi et al. (2023).
Fig. 5. Diagram of evolution of the trioecious species Pleodorina starrii. Based on Takahashi et al. (2023).

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