培養株を用いた形態比較と分子系統解析に基づくアオミドロ属及び近縁属(ストレプト植物門・ホシミドロ藻綱)の分類学的研究

概要

The genus Spirogyra and its related ~enera Sirogonium and Temnogyra(hereafter referred to as Spirogyra-like algae, SLA) belong to streptophytes that include land plants. They are found mainly in freshwater, and there are over 570 species described from all over the world, with 84 species known from Japan. The SLA have a filamentous thallus consisting of a series of cylindrical cells with characteristic spiral ribbon-like chloroplasts. They. are referred to as conjugating algae because of their characteristic sexual reproduction, in which gametes without flagella conjugate to form zygospores.

Sirogonium and Temnogyra, differ from Spirogyra in their conjugation characteristics: in Spirogyra,. a progametangial cell divides to form two gametangia, whereas, in these two genera, a progametangial cell divides to form a gametangium and a sterile cell that does not conjugate. In Temnogyra, the gametangia are connected by conjugation tubes as in Spirogyi・a-type, and in Sfrogonium, they are connected directly without conjugation tubes. Recent molecular phylogenetic analyses based on rbcL gene have shown that the SLA are divided into seven clades (Clade I-VII) and that three species of Sirogonium form a clade with some species of Spirogyra. However, the species of Temnogyra have not yet been analyzed. In addition, the phylogenetic relationship between the seven clades were not solved.

In the SLA classification, characteristics such as sexual reproduction and mature zygospores are significant for accurate species-level and genus-level identification. In previous taxonomic studies, species identification has been carried out using field-collected samples with mature zygospores. However, there is a wide range of seasonality in the occurrence of mature zygospores, and it is often difficult to collect samples with mature zygospores. Molecular phylogenetic studies of species identified・ based on field samples with sexual traits have been limited. Therefore, establishing a reproducible method to induce sexual reproduction in the SLA is necessary to develop a taxonomic study. Recently, the inducing conjugation method using agar plates has been established, and species identification of the SLA has been carried out based on the characteristics of the obtained mature zygospores.

In the present stt1dy, I improved the inducing conjugation method and identified more SLA species accurately based on the morphology of sexual reprodu~tion. I also re-examined the taxonomy of the SLA _by clarifying their phylogenetic relationships based on molecular data. Extensive field survey_ in Japan was conducted and 505 culture strain~ were established. I identified'51 species based on conjugating field samples and sexual morphology observed by inducing conjugation. Two species exhibiting Temnogyra-type conjugation and other two species of Sirogonium-type conjugation were identified: Thirteen species were found in Japan for the first time, and three were undescribed species. Some of them were rare species with few reports. It was assumed that they seldom, or rarely, sexually reproduced in ~ature, so our cultivation procedure for inducing conjugation in vegetative cells proved useful f~r species identification (Chapter 1).

Sexuai reproduction in zygnematophycean algae occurs either within a single clone (homothallic strains) or between different clones(heterothallic strains). The SLA are fou_nd in a wide range of freshwater environments, and the process of conjugation has been well studied. However, heterothallic strains of Spirogyra have not been reported because of experimental difficulties in inducing conjugation. In this study, I detected heterothallic strains of Spirogyra by inducing conjugati_on for the first time. These strains were identified as S..iuviatilis, and two genetically different strains were required for conjugation. Crossbreeding experiments with these strains showed that sexuality was genetically determined and that the Fl strain germinated from a zygospore was only conjugated with one parent strain. Molecular phylogenetic analyses conducted in this study showed that these heterothallic strains formed a monophyletic group with several homothallic strains: Only one heterothallic strain was found in this study. More heterothallic strains will need to be discovered using the methods established in this study in order to solve homothallic and heterothallic transitions in Spirogyra (Chapter 2).

A total of 136 strains with different rbcL sequences (1094 hp) were obtained in Japan, and the maximum likelihood phylogenetic analysis was carried out using rbcL sequences. According to previous studies, the SLA was divided into seven clades (Clade I-VII), but 16 strains from Japan were not included in any clade. These strains included two species _exhibiting Temnogyra-type conjugation (S. corrugate and S. punctata) and a strain with se面 i-replicatetransverse walls. The latter was described as S. tertia sp. nov. The clades I, III, IV, V, and VII were monophyletic with high statistical support (98-99% bootstrap value), but Clade II and VI formed a monophyletic clade with lower statistical support (65% bootstrap value). The clade VI and its sister lineage S. micropunctata from Japan formed clade VI'with moderate statistical support(bootstrap value 85%). In addition, Clade III was divided into Clade Illa and Clade IIIb.

To elucidate the phylogenetic relationships and evolutionary processes of morphological traits within the SLA, I sequenced whole chloroplast genomes of the selected 32 strains of the SLA and conducted molecular phylogenetic analyses using a multi-gene dataset. The molecular phylogenetic analyses, based on 73 chloroplast 氏名：高野智之 N0.3 genes, support the phylogenetic relationship between SLA clades (Clade I, II, Illa, IIIb, rv, V, VI', VII) with high statistical support. One Japanese strain (S. crassispina chiA303), which was included in Clade II in the phylogenetic analysis using rbcL, was not included in Clade II and was separated from any of the other cl1:1des. Two species exhibiting Temnogyra-type conjugation and two other Japanese strains of unknown conjugation morphology form a monophyletic group (Clade T). Two species exhibiting Sirogonium-type conjugation species were included in Clade II as previous studies. The results of ancestral state reconstr~ction suggested that the ancestor of the SLA performed Spirogyi鳴.!Z-typeconjugation, and it was・ implied that Sirogonium-type and Temnogyra-type species were evolved from Spirogyra-type ancestor.・ The results of ancestral state reconstruction also suggested that the ancestor of the SLA was approximately 50 μm in a cell diameter and had multiple chloroplasts in each cell. In addition, the ancestor of the SLA had transverse walls with a folded structure. From their common ancestor, Species of Clade VII and Clade II ev_olved with enlarged cell diameters and loss of folded structures at transverse walls, respectively, while S. crassispina chiA303Tetained its folded structure. In previous studies, all species with a folded structure were included in Clade I, but the strain with a folded structure riot included in Clade I was found for the first time in this study. The common ancestor of the other lineages (Clade I, IIIa, IIIb, IV, V, yr•, T, and S. tertia sp. nov. wak305) was estimated to have a cell diameter of about 30 μm, a si~gle chloroplast per cell and a folded structure at the transverse walls. S. tertia sp. nov. wak305 was the sister lineage of the remaining lineages. This species has an incomplete folded structure (semi-replicate), and -the phylogenetic position of the species with this structure was revealed for the first time. In the next diverged lineage, Clade -I, some species had a folded structure and o~hers do not, suggesting multiple losses within this clade. The common ancestor of Clade IIIa, IIIb, Iv, V, VI', T was presumed to have lost the folded structure. The results of ancestral trait reconstruction implied that the conjugation pattern changed from Spirogyra-type to Sirogonium-type twice independentiy in Clade II and from Spirogyra-type to Temnogyra-type in the common ancestor of Clade T. (Chapter 3).

As a result of the establishment of new culture strains and the improvement of inducing conjugation, two Temnogyra-type species and two of Sirogonium-type species were identified, which were included in clades with Spirogyra-type strains. Therefore, I concluded that three genera of the SLA should be assigned to the genus Spirogyra. Further taxonomic studies using more strains, including the type species of the genera 'Sirogonium and TE如 nogyrafrom the type locality, are needed to revise the overall taxonomic system of the SLA. The establishment of culture strains and inducing conjugation will play a significant role is tureSLA classification methods. However, it is not easy to induce conjugation in all SLA species. Therefore, combining this with field collection with mature zygospores will be necessary. Finally, the large number of cultured strains and the knowledge of sexual reproduction established in this study are expected to be used in various future studies, such as the elucidation of the evolution of sex-determination systems in streptophytes using SLA species as a model.