mycological research 110 (2006) 898–915 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/mycres Ultrastructural and molecular phylogenetic delineation of a new order, the Rhizophydiales (Chytridiomycota) Peter M. LETCHER*, Martha J. POWELL, Perry F. CHURCHILL, James G. CHAMBERS Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487, USA article info abstract Article history: In the order Chytridiales, Rhizophydium is a morphologically defined genus based upon the Received 9 February 2006 production of a monocentric, inoperculate, epibiotic sporangium, an endobiotic rhizoidal Received in revised form axis which branches, and an epibiotic resting spore. Despite its simple morphology, over 18 May 2006 220 species of Rhizophydium have been described. Recent phylogenetic analyses using Accepted 1 June 2006 nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophy- Corresponding Editor: Teun Boekhout dium cultures revealed that the classical genus Rhizophydium is genetically more variable than previously understood and actually represents multiple genera. In the present study, Keywords: we use zoospore ultrastructural characters and 28 S rRNA and 5.8 S ribosomal gene se- Boothiomyces quences of 96 isolates in culture to circumscribe the monophyletic Rhizophydium clade as Kappamycetaceae a new order, Rhizophydiales. Correspondingly, zoospores of members of the Rhizophydiales Ribosomal genes exhibit a unique suite of ultrastructural character states that further define the order Terramyces and distinguish it from the order Chytridiales. Molecular analyses reveal several strongly Terramycetaceae supported clades within the Rhizophydiales. Three of those clades encompass a broad range Zoospore of isolates and are defined as new families Rhizophydiaceae, Terramycetaceae, and Kappamy- cetaceae. 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 spe- cies formerly classified in Rhizophydium are transferred to the new genera. This work pro- vides 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. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Rhizophydium globosum (Clements & Shear 1931), is of a rela- tively simple thallus consisting of a spherical, multipored, epi- Rhizophydium is among the earliest genera of chytrids estab- biotic sporangium bearing a single endobiotic rhizoidal axis lished. Schenk (1858) proposed the genus for inoperculate that branches and an epibiotic resting spore. The concept of members of Chytridium, and Rabenhorst (1868) formally de- the genus has been problematic because key morphological scribed the genus. Members of the genus occur in aquatic sys- features intergrade with those of other genera (Letcher et al. tems primarily as parasites of algae, and in soil primarily as 2004b). Despite the simple thallus morphology and plasticity saprotrophs of pollen, and to a lesser extent, keratin and chi- of morphological characters, over 220 species have been de- tin. The morphological concept of the genus based on its type, scribed (Karling 1977, Longcore 1996, Sparrow 1960). Through * Corresponding author. E-mail address: [email protected]. 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.06.011 New order, Rhizophydiales (Chytridiomycota) 899 time the concept of the genus has expanded to include et al. 2005), organisms with a Rhizophydium Group III-type zoo- sporangia that were oval, oblong, pyriform, cylindrical, and spore (Barr 1980) were excluded from the family Chytridiaceae. angular and that produced single to numerous inoperculate Recent phylogenetic analyses combining zoospore ultra- zoospore discharge areas. To manage this difficult genus, structural characters with nuLSU rRNA (28 S rRNA) gene Sparrow (1960) sorted its species into five sections based pri- sequences of a geographically diverse sampling of Rhizophy- marily on sporangial shape. Karling (1977) merged Phlyctidium dium cultures (Letcher et al. 2004b) revealed that the classical with Rhizophydium, which broadened the generic concept to genus Rhizophydium was genetically more variable than previ- include species with an endobiotic, unbranched, haustorial- ously understood and actually represented multiple genera like rhizoidal axis. Many Rhizophydium species are distin- (Letcher & Powell 2005). The range of character states in Barr guished based on substrate or host utilization, without studies and Hadland-Hartmann’s (1978) zoospore ultrastructural determining their nutritional ranges. Thus, some species may study of 12 Rhizophydium species presaged the genetic diver- be identical, others may be members of species complexes in sity discovered in molecular analyses (Letcher et al. 2004b). which character states of specific morphological features in- Analyses of 28 S rRNA gene sequences (Letcher et al. 2004b) tergrade with those of other genera, and some may represent also confirmed results of an earlier study of nuSSU rRNA new genera. gene sequences (James et al. 2000), showing that the other Rhizophydium has traditionally been classified in the order clades within the Chytridiales were sister to the Rhizophydium Chytridiales, but family alliances have varied with authors. clade. Sparrow (1960), separating genera into two series based on In the present study we analyse an extensive and geo- operculation versus inoperculation, classified Rhizophydium graphically diverse sampling of isolates in the Rhizophydium in the family Phlyctidiaceae and subfamily Phlyctidioideae. How- clade using combined nu-rRNA gene sequences and zoospore ever, because the genus (Phlyctidium) on which the family ultrastructural characters. On the bases of molecular mono- Phlyctidiaceae was based is not valid as a chytrid genus (Karling phyly and zoospore ultrastructure, the Rhizophydium clade is 1939), having been used earlier as a genus in the Ascomycota, designated as a new order, the Rhizophydiales, in which three the family Phlyctidiaceae is also not valid (Greuter et al. 1999, new families and two new genera are delineated. The order Article 18.3). In Karling’s 1977 summary of the Chytridiomycota, Chytridiales is emended to reflect this revision. 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 Materials and methods as a primary taxonomic character. Accordingly Karling (1977) merged Sparrow’s (1960) families Phlyctidiaceae and Chytridia- Taxonomic sampling ceae into the family Rhizidiaceae, classifying Rhizophydium in the subfamily Rhizidioideae. Accordingly, Karling (1977) com- We examined 96 ingroup isolates in the Rhizophydium clade bined the invalid genus Phlyctidium with Rhizophydium. and two outgroup isolates (Monoblepharella sp. and Oedogonio- Zoospore ultrastructural characters are considered stable myces sp.), members of the Monoblepharidaceae, Monoblephari- and reliable to reveal relationships among chytrids (Barr dales, which is a sister clade to the Rhizophydium clade 2001, Powell 1976, 1978) and are now vital in determining ordi- (James et al. 2000, Chambers 2003). DNA was extracted from nal and familial relationships. In 1980 Barr revised the order pure cultures obtained from chytrid collections maintained Chytridiales and segregated out a new order Spizellomycetales at the American Type Culture Collection, the Canadian Collec- based upon zoospore ultrastructural characters. In the Chytri- tion of Fungal Cultures, The University of Alabama, University diales, Barr (1980) emended Bary and Woronin’s (1865) concept of Maine, and University of California at Berkeley. Origins, of the family Chytridiaceae in which he included Rhizophydium, source of isolates in culture, and GenBank accession numbers rather than in Karling’s (1977) family Rhizidiaceae. Although or AFTOL numbers (Assembling the Fungal Tree of Life, Duke zoospore characters, such as location of nucleus, organization University; http://www.biology.duke.edu/fungi/mycolab/) are of the microbody–lipid globule complex (MLC) (Powell 1976), listed in Table 1. and aggregation of ribosomes unified the emended order Chy- tridiales [Barr 1980 as J. Schro¨t (1892: 64), emended], differences in zoospore kinetid features and microtubular root systems Sample preparation formed the basis for Barr’s (1980) nomenclatural system of zoospore ‘Groups’ in the Chytridiales. The Rhizophydium Group DNA was purified and amplified for sequencing as described III-type zoospore was clearly distinct from other chytridialean (Letcher & Powell 2005) from pure cultures of isolates (Table 1). zoospores (Barr 1980). The LROR/LR5 primer pair (White et al. 1990) was used Molecular sequence datasets of chytrids have been used for amplification of nuLSU (28 S) rDNA, and the ITS5/ITS4 primarily to understand deep phylogenetic relationships primer pair (White et al. 1990) for the ITS1–5.8 S–ITS2 (Keeling 2003, Tanabe