Watanabeales ord. nov. and twelve novel species of Trebouxiophyceae (Chlorophyta).

The Trebouxiophyceae include Chlorellales, Prasiolales, Trebouxiales, Microthamniales, and numerous members for which taxonomic locations have not been determined. The Watanabea clade is a group in the Trebouxiophyceae of Chlorella-like taxa, which are predominantly solitary and terrestrial. With the increase in descriptions of new species within the Watanabea clade in recent years, it is necessary to define the boundary and taxonomic status of the clade. In this study, nineteen strains of terrestrial algae collected from China were investigated based on molecular and morphological data, involving phylogenetic analyses, light and electron microscopy, and ITS-2 secondary structures. These 19 strains were described as five known species and 12 novel ones. Based on the consistent topological structures of the phylogenetic analyses for three taxonomic levels (the core Chorophyta, Trebouxiophyceae based on 18S sequences, Watanabeales and Symbiochloris based on 18S, 5.8S, ITS2 and rbcL gene sequences) and common morphological characteristics, especially the key phenotypic feature of unequally sized autospores, the Watanabea clade was defined as a new order, Watanabeales ord. nov., with one family, Watanabeaceae fam. nov. The boundary of Watanabeales was delineated and included the genera Chloroidium, Calidiella, Jaagichlorella, Kalinella, Massjukichlorella, Mysteriochloris, Polulichloris, Phyllosiphon, Watanabea, and Viridiella. Symbiochloris did not belong to Watanabeales according to the phylogenetic analyses and the reproduction by equal-sized autospores, aplanospores, or zoospores. The Watanabeales were separated into four groups during the phylogenetic analyses, but no regular differences in habitats or morphology could be used as the morphological basis to split Watanabeales into the four groups.