Ultrastructural and molecular phylogenetic delineation of a new order, the Rhizophydiales (Chytridiomycota)
Introduction
Rhizophydium is among the earliest genera of chytrids established. Schenk (1858) proposed the genus for inoperculate members of Chytridium, and Rabenhorst (1868) formally described the genus. Members of the genus occur in aquatic systems primarily as parasites of algae, and in soil primarily as saprotrophs of pollen, and to a lesser extent, keratin and chitin. The morphological concept of the genus based on its type, Rhizophydium globosum (Clements & Shear 1931), is of a relatively simple thallus consisting of a spherical, multipored, epibiotic sporangium bearing a single endobiotic rhizoidal axis that branches and an epibiotic resting spore. The concept of the genus has been problematic because key morphological features intergrade with those of other genera (Letcher et al. 2004b). Despite the simple thallus morphology and plasticity of morphological characters, over 220 species have been described (Karling, 1977, Longcore, 1996, Sparrow, 1960). Through time the concept of the genus has expanded to include sporangia that were oval, oblong, pyriform, cylindrical, and angular and that produced single to numerous inoperculate zoospore discharge areas. To manage this difficult genus, Sparrow (1960) sorted its species into five sections based primarily on sporangial shape. Karling (1977) merged Phlyctidium with Rhizophydium, which broadened the generic concept to include species with an endobiotic, unbranched, haustorial-like rhizoidal axis. Many Rhizophydium species are distinguished based on substrate or host utilization, without studies determining their nutritional ranges. Thus, some species may be identical, others may be members of species complexes in which character states of specific morphological features intergrade with those of other genera, and some may represent new genera.
Rhizophydium has traditionally been classified in the order Chytridiales, but family alliances have varied with authors. Sparrow (1960), separating genera into two series based on operculation versus inoperculation, classified Rhizophydium in the family Phlyctidiaceae and subfamily Phlyctidioideae. However, because the genus (Phlyctidium) on which the family Phlyctidiaceae was based is not valid as a chytrid genus (Karling 1939), having been used earlier as a genus in the Ascomycota, the family Phlyctidiaceae is also not valid (Greuter et al. 1999, Article 18.3). In Karling's 1977 summary of the Chytridiomycota, he returned to the family classification of Gaumann and Dodge (1928) for Rhizophydium and followed Whiffen's (1944) view that operculation versus inoperculation should not be used as a primary taxonomic character. Accordingly Karling (1977) merged Sparrow's (1960) families Phlyctidiaceae and Chytridiaceae into the family Rhizidiaceae, classifying Rhizophydium in the subfamily Rhizidioideae. Accordingly, Karling (1977) combined the invalid genus Phlyctidium with Rhizophydium.
Zoospore ultrastructural characters are considered stable and reliable to reveal relationships among chytrids (Barr, 2001, Powell, 1976, Powell, 1978) and are now vital in determining ordinal and familial relationships. In 1980 Barr revised the order Chytridiales and segregated out a new order Spizellomycetales based upon zoospore ultrastructural characters. In the Chytridiales, Barr (1980) emended Bary and Woronin's (1865) concept of the family Chytridiaceae in which he included Rhizophydium, rather than in Karling's (1977) family Rhizidiaceae. Although zoospore characters, such as location of nucleus, organization of the microbody–lipid globule complex (MLC) (Powell 1976), and aggregation of ribosomes unified the emended order Chytridiales [Barr 1980 as J. Schröt (1892: 64), emended], differences in zoospore kinetid features and microtubular root systems formed the basis for Barr's (1980) nomenclatural system of zoospore ‘Groups’ in the Chytridiales. The Rhizophydium Group III-type zoospore was clearly distinct from other chytridialean zoospores (Barr 1980).
Molecular sequence datasets of chytrids have been used primarily to understand deep phylogenetic relationships (Keeling, 2003, Tanabe et al., 2005), and one study emphasizing the Chytridiales identified four monophyletic lineages in the order (James et al. 2000). The study of Letcher et al. (2005) was the first to integrate ultrastructural and molecular datasets to designate one of the four lineages (the Chytridium/Chytriomyces clade) in the Chytridiales as a new taxon, the Chytridiaceae. In this revision of the Chytridiaceae (Letcher et al. 2005), organisms with a Rhizophydium Group III-type zoospore (Barr 1980) were excluded from the family Chytridiaceae.
Recent phylogenetic analyses combining zoospore ultrastructural characters with nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophydium cultures (Letcher et al. 2004b) revealed that the classical genus Rhizophydium was genetically more variable than previously understood and actually represented multiple genera (Letcher & Powell 2005). The range of character states in Barr and Hadland-Hartmann's (1978) zoospore ultrastructural study of 12 Rhizophydium species presaged the genetic diversity discovered in molecular analyses (Letcher et al. 2004b). Analyses of 28 S rRNA gene sequences (Letcher et al. 2004b) also confirmed results of an earlier study of nuSSU rRNA gene sequences (James et al. 2000), showing that the other clades within the Chytridiales were sister to the Rhizophydium clade.
In the present study we analyse an extensive and geographically diverse sampling of isolates in the Rhizophydium clade using combined nu-rRNA gene sequences and zoospore ultrastructural characters. On the bases of molecular monophyly and zoospore ultrastructure, the Rhizophydium clade is designated as a new order, the Rhizophydiales, in which three new families and two new genera are delineated. The order Chytridiales is emended to reflect this revision.
Section snippets
Taxonomic sampling
We examined 96 ingroup isolates in the Rhizophydium clade and two outgroup isolates (Monoblepharella sp. and Oedogoniomyces sp.), members of the Monoblepharidaceae, Monoblepharidales, which is a sister clade to the Rhizophydium clade (James et al., 2000, Chambers, 2003). DNA was extracted from pure cultures obtained from chytrid collections maintained at the American Type Culture Collection, the Canadian Collection of Fungal Cultures, The University of Alabama, University of Maine, and
Combined LSU + 5.8 S analyses
The partition homogeneity/incongruence-length difference test determined that LSU and 5.8 S data did not have significantly different signals. The combined data had 1542 characters, with 453 parsimony-informative sites. For MP analysis, of 1005 trees derived from PAUPRat, 566 most parsimonious trees [length (L) = 2328 steps, CI = 0.472, and RI = 0.784] were used to compute a majority rule consensus tree (≥70 % branch support). ModelTest indicated the most appropriate model of DNA substitution was the
Taxonomy
Chytridiales J. Schröt. (1892: 64).
Emend. Barr (1980: 2388); and Letcher & M.J. Powell
In the zoospore a microtubular root may or may not be present, but when present extends from one side of the kinetosome in a parallel array to the side of a cisterna that is on the lipid globule surface; ribosomes are enclosed by a system of double membranes; mitochondria are associated with the MLC. The nucleus is not associated with the kinetosome. The non-flagellated centriole and kinetosome lie parallel or
Discussion
Circumscription of Rhizophydiales
This study correlated zoospore ultrastructural character states with molecular analyses of nuclear ribosomal genes, demonstrating that the monophyletic Rhizophydium clade (James et al., 2000, Letcher et al., 2004b) is clearly distinctive from other clades in the Chytridiales. It showed that zoospore ultrastructural features, such as the KAS as a spur and the nature of the fibrillar bridge between the kinetosome and non-functional centriole, are particularly
Acknowledgements
This study was supported by the National Science Foundation through PEET grant DEB-9978094, REVSYS grant DEB-0516173, and AFTOL grant DEB-0228668; The University of Alabama, Department of Biological Sciences Aquatic Ecology and Systematics Graduate Enhancement Program; a Howard Hughes Medical Institute Undergraduate Biological Sciences Program Grant to The University of Alabama, and a scholarship from the Alabama Power Company. We express our appreciation to the Assembling the Fungal Tree Of
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