Phylogenetic relationships among Rhizophydium isolates from North America and Australia

The order Chytridiales is the largest and most diverse of five orders in phylum Chytridiomycota. Rhizophydium is one of two genera in the Chytridiales with more than 220 described species. Because thallus characters used in classical descriptions of Rhizophydium species often intergrade into other species, as well as other genera, species distinctions frequently are unclear. Species often are delimited solely on substrate or host; many described species consequently may be synonymous. On the other hand, because the thallus is relatively simple morphologically similar forms actually may be genetically distinct. As a beginning for the revision of the genus Rhizophydium, this study used molecular and ultra-structural analyses to characterize cultures identified as Rhizophydium species. A broad geographic sampling of Rhizophydium-like organisms from North American and Australian soils was studied as a foundation for enhanced identification of soil chytrids. The first objective was to ascertain the genetic variability of Rhizophydium isolates with spherical to angular sporangia and multiple discharge pores, using nuclear large subunit rRNA gene sequence analysis. Sequences of 45 isolates of Chytridiales, including 29 isolates in the Rhizophydium clade were analyzed. Alignment based on LSU rRNA secondary structure revealed a similar reduced stem and loop structure in the C1_3 helix region that distinguished morphologically similar Rhizophydium clade members from other members of the Chytridiales. In our parsimony analysis, the Chytriomyces clade was sister of the Nowakowskiella, Lacustromyces and Rhizophydium clades. Six subclades within the Rhizophydium clade were resolved. Several closely related isolates appeared geographically widespread because North American and Australian isolates were found together in three of the six subclades. The second objective was to sample zoospore ultrastructure among isolates in the six subclades and an unresolved polytomy group within the Rhizophydium clade, thus evaluating the application of zoospore ultrastructure for lower level taxonomic decisions. All isolates were examined by transmission electron microscopy, and four types of zoospores were found. Thus, within the well-supported Rhizophydium clade, zoospore ultrastructure appeared divergent. Because similar zoospore types also were found in two distinct subclades, zoospore structure might be interpreted superficially as convergent. However, unresolved polytomys indicated molecular divergence among these taxa and the need for a more diverse taxa and gene sampling to resolve relationships. One of the zoospore types characterized represents the most simplified form of zoospore described so far in the Chytridiales. The range in molecular secondary structure composition and in zoospore morphology suggested that isolates we provisionally placed in Rhizophydium actually represent multiple genera.