Ultrastructural and Molecular Phylogenetic Delineation of a New Order, Rhizophydiales ( Chytridiomycota)

1. Ultrastructural and Molecular Phylogenetic Delineation of a New Order, Rhizophydiales(Chytridiomycota) Peter M. Letcher, Martha J. Powell, Perry F. Churchill, and James G. Chambers Dept. of Biological Sciences, The University of Alabama MP 120 INTRODUCTION Rhizophydium is a genus of >220 described species of simple thallus morphology. Recent phylogenetic analyses of NuLSU (28S) rRNA gene sequences (Letcher et al. 2004) reveal that Rhizophydium is genetically variable and actually represents multiple genera. Among isolates in the Rhizophydium clade (James et al. 2000), multiple zoospore types occur. The Rhizophydium zoospore (Barr 1980) is ultrastructurally distinct from other zoospores in the Chytridiales sensu Barr (1980). Through analyses of an extensive and geographically diverse sampling of isolates in the Rhizophydium clade, and on the bases of molecular monophyly and zoospore ultrastructure, the Rhizophydium clade is designated as a new order, the Rhizophydiales, with three new families and two new genera. MATERIALS & METHODS We examined 96 ingroup isolates in the Rhizophydium clade. Partial LSU and complete ITS1-5.8S-ITS2 rRNA gene sequences were generated. For all isolates, partial LSU sequences were combined with 5.8S sequences, because ITS1 and ITS2 were too divergent for alignment across the entire data set. For 44 isolates, partial LSU sequences were combined with ITS1-5.8S-ITS2 sequences. MP and NJ analyses were conducted with PAUP*, and Bayesian analyses conducted with MrBayes. We examined zoospore ultrastructure of 87 of the 96 ingroup isolates. RESULTS For combined LSU + 5.8S analyses of all 96 isolates, MP, NJ, and Bayesian topologies were similar. Within the Rhizophydium clade were numerous strongly supported clades (Fig. 1, clades A-C). For combined LSU + ITS1-5.8S-ITS2 analyses of the 44 isolates in clade A, two strongly supported clades were revealed (Fig. 2,clades A1 and A2). Although ultrastructural character states varied among examined isolates, for all isolates an electron-opaque plug was absent from the base of the flagellum proper (Figs. 3 A-D). Distinctive suites of character states correlated with isolates in clades A1, A2, B, and C. DISCUSSION Correlation of zoospore ultrastructural character states with molecular analyses of nuclear ribosomal genes demonstrates that the monophyletic Rhizophydium clade is clearly distinctive from other clades in the Chytridiales. Specific zoospore ultrastructural features, such as the spur adjacent to the kinetosome, and the fibrillar zone of convergence between the kinetosome and non-flagellated centriole are particularly taxonomically informative. Molecular divergence and ultrastructure divergence are reliably linked; molecular phylogeny predicts zoospore ultrastructure, and vice versa. FIG. 3. Schematic drawings of four zoospore types in Rhizophydiales ord. nov. FIG. 2. NJ phylogram of clade A, from combined LSU + ITS1-5.8S-ITS2 data. A.Terramycetaceae, Terramyces gen. nov. -simple cisterna -zc 0.010-0.025 µm - spur present -no microtubular root FIG. 1. NJ phylogram of Rhizophydiales ord. nov., from combined LSU + 5.8S rDNA data. B.Terramycetaceae, Boothiomyces gen. nov. -fenestrated cisterna -zc 0.010-0.025 µm -spur present -microtubular root C.Kappamycetaceae, Kappamyces -simple cisterna -no zc -no spur -no microtubular root D.Rhizophydiaceae, Rhizophydium -inconspicuous fenestrated cisterna -zc ~0.075 µm -laminated spur -microtubular root