This study determined the transcriptomes of eight Oedogoniales species, including six species from Oedogonium and two species from Oedocladium to conduct phylotranscriptomic and evolutionary analyses. 155,952 gene families and 192 single-copy orthogroups were detected. Phylotranscriptomic analyses based on single-copy orthogroups were conducted using supermatrix and coalescent-based approaches. The phylotranscriptomic analysis results revealed that Oedogonium is polyphyletic, and Oedocladium clustered with Oedogonium. Together with the transcriptomes of the OCC clade in the public database, the phylogenetic relationship of the three orders (Oedogoniales, Chaetophorales, Chaetopeltidales) is discussed. The non-synonymous (dN) to synonymous substitution (dS) ratios of single-copy orthogroups of the terrestrial Oedogoniales species using a branch model of phylogenetic analysis by maximum likelihood were estimated, which showed that 92 single-copy orthogroups were putative rapidly evolving genes. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses results revealed that some of the rapidly evolving genes were associated with photosynthesis, implying that terrestrial Oedogoniales species experienced rapid evolution to adapt to terrestrial habitats. The phylogenetic results combined with evolutionary analyses suggest that the terrestrialization process of Oedogoniales may have occured more than once.

Oedogoniales comprises the three genera Oedogonium, Oedocladium, and Bulbochaete, which include more than 600 described species. The classification of Oedogoniaceae is currently based on morphology, and the complicated morphological characteristics make species identification difficult, with the limited molecular data also restricting the phylogenetic analysis. In the present study, we collected 47 Oedogonium specimens from China and sequenced 18S rDNA, ITS2, ITS (ITS1 + 5.8S + ITS2), and rbcL sequences to conduct phylogenetic analyses. We selected nine morphological characteristics, most of which were considered important in traditional systematics, for comparison with the molecular phylogeny results. All the topologies based on different datasets showed similar results; Oedogonium was a paraphyletic group, and Oedocladium and Bulbochaete clustered with Oedogonium. The morphological characteristics matching the phylogenetic results showed that the types of sexual differentiation, characteristics of the oogonium (including shape, types of aperture, and ornamentation of oospore wall), division types of antheridial, and number of sperm of each antheridial, which are considered the most important morphological characteristics in traditional taxonomy of Oedogonium, did not form monophyletic lineages respectively, indicating that traditional systematics may not reflect the real phylogeny of the genus Oedogonium. In addition, a new taxonomical classification of the genus Oedogonium was presented according to the shapes of basal cells, which matched well with the phylogenetic topologies. In addition, we propose to divide the genus Oedogonium into two sections, section Globosum and section Elongatum, representing the species with spherical or sub-hemispherical basal cells and elongated basal cells, respectively.

Considering the phylogenetic differences in the taxonomic framework of the Chaetophorales as determined by the use of nuclear molecular markers or chloroplast genes, the current study was the first to use phylotranscriptomic analyses comparing the transcriptomes of 12 Chaetophorales algal species. The results showed that a total of 240,133 gene families and 143 single-copy orthogroups were identified. Based on the single-copy orthogroups, supergene analysis and the coalescent-based approach were adopted to perform phylotranscriptomic analysis of the Chaetophorales. The phylogenetic relationships of most species were consistent with those of phylogenetic analyses based on the chloroplast genome data rather than nuclear molecular markers. The Schizomeriaceae and the Aphanochaetaceae clustered into a well-resolved basal clade in the Chaetophorales by either strategy. Evolutionary analyses of divergence time and substitution rate also revealed that the closest relationships existed between the Schizomeriaceae and Aphanochaetaceae. All species in the Chaetophorales exhibited a large number of expanded and contracted gene families, in particular the common ancestor of the Schizomeriaceae and Aphanochaetaceae. The only terrestrial alga, Fritschiella tuberosa, had the greatest number of expanded gene families, which were associated with increased fatty acid biosynthesis. Phylotranscriptomic and evolutionary analyses all robustly identified the unique taxonomic relationship of Chaetophorales consistent with chloroplast genome data, proving the advantages of high-throughput data in phylogeny.