Ultrastructural and molecular phylogenetic delineation of a new order, the Rhizophydiales (Chytridiomycota)

In the order Chytridiales, Rhizophydium is a morphologically defined genus based upon the production of a monocentric, inoperculate, epibiotic sporangium, an endobiotic rhizoidal axis which branches, and an epibiotic resting spore. Despite its simple morphology, over 220 species of Rhizophydium have been described. Recent phylogenetic analyses using nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophydium cultures revealed that the classical genus Rhizophydium is genetically more variable than previously understood and actually represents multiple genera. In the present study, we use zoospore ultrastructural characters and 28 S rRNA and 5.8 S ribosomal gene sequences of 96 isolates in culture to circumscribe the monophyletic Rhizophydium clade as a new order, Rhizophydiales. Correspondingly, zoospores of members of the Rhizophydiales exhibit a unique suite of ultrastructural character states that further define the order and distinguish it from the order Chytridiales. Molecular analyses reveal several strongly supported clades within the Rhizophydiales. Three of those clades encompass a broad range of isolates and are defined as new families Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae. To resolve close relationships within Terramycetaceae, combined 28 S rRNA and ITS1–5.8 S–ITS2 sequences were analysed and details of zoospore ultrastructural character states determined, with two new genera, Terramyces and Boothiomyces, described. Two species formerly classified in Rhizophydium are transferred to the new genera. This work provides a framework for additional taxonomic revisions within the new order Rhizophydiales and compares genetic variation useful in defining genera, species, and populations within this lineage of chytrids. A broader sampling of representatives is needed before taxonomic decisions can be made for remaining clades within the Rhizophydiales.