A molecular phylogeny of the flagellated fungi (Chytridiomycota) and description of a new phylum (Blastocladiomycota) Timothy Y. James1 Department of Biology, Duke University, Durham, North Carolina 27708 Peter M. Letcher Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama 35487 Joyce E. Longcore Department of Biological Sciences, University of Maine, Orono, Maine 04469 Sharon E. Mozley-Standridge David Porter Department of Plant Biology, University of Georgia, Athens, Georgia 30605 Martha J. Powell Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama 35487 Gareth W. Griffith Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, Wales SY23 3DA, UK Rytas Vilgalys Department of Biology, Duke University, Durham, North Carolina 27708 Abstract: Chytridiomycota (chytrids) is the only phylum of true Fungi that reproduces with motile spores (zoospores). Chytrids currently are classified into five orders based on habitat, zoospore characters and life cycles. In this paper we estimate the phylogeny of the chytrids with DNA sequences from the ribosomal RNA operon (18S+5.8S+28S subunits). To our surprise the morphologically reduced para- sites Olpidium and Rozella comprise two entirely new, and separate, lineages on the fungal tree. Olpidium brassicae groups among the Zygomycota, and Rozella spp. are the earliest branch to diverge in the fungal kingdom. The phylogeny also suggests that Chytri- diomycota is not monophyletic and there are four major lineages of chytrids: Rozella spp., Olpidium brassicae, the Blastocladiales and a ������core chytrid clade������ containing the remaining orders and families and the majority of flagellated fungi. Within the core chytrid group 11 subclades can be identified, each of which correlates well with zoospore ultrastructure or morphology. We provide a synopsis of each clade and its morphological circumscription. The Blastocla- diales appears to be the sister taxon of most nonflagellated fungi. Based on molecular phyloge- netic and ultrastructural characters this order is elevated to a phylum, the Blastocladiomycota. Key words: Blastocladiomycota, Chytridiales, holocarpic, kinetosome, phylogeny, zoospore ultrastructure INTRODUCTION The Chytridiomycota is a phylum of Fungi that reproduces through the production of motile spores (zoospores), typically propelled by a single, poster- iorly directed flagellum. These organisms, often referred to as chytrid fungi or chytrids, have a global distribution with approximately 1000 described spe- cies. Based on biochemical characteristics, including chitin in cell walls, the a-aminoadipic acid lysine synthetic pathway and storage carbohydrates as glycogen, Bartnicki-Garcia (1970) classified the Chy- tridiomycota as true Fungi. Others considered chy- trids as a transitional group between protists and Fungi because of their production of motile zoo- spores (Barr 1990). Phylogenetic studies with ribo- somal RNA genes unified these views and conclusively demonstrated that chytrids were true Fungi that occupied a basal position in the fungal tree (e.g. Fo ��rster et al 1990). Chytrids are essentially ubiquitous, occurring in diverse and unique habitats from the tropics to the arctic regions (Powell 1993). They are found in aquatic systems such as streams, ponds, and estuarine and marine ecosystems primarily as parasites of algae and plankton components. Perhaps the majority of chytrid species occur in terrestrial habitats (Barr 2001) such as forest, agricultural and desert soils, and acidic bogs as saprotrophs of refractory substrata including pollen, chitin, keratin and cellulose. In soil chytrids are obligate parasites of a wide variety of vascular plants, including potatoes (Synchytrium) and curcurbits (Olpidium). The only known chytrid parasite of vertebrates is Batrachochytrium dendrobati- dis (Longcore et al 1999), considered the causative agent of die-offs and population declines of amphib- ian species (Berger et al 1998). In most habitats a few chytrid species are relatively frequent and abundant (i.e. Chytriomyces hyalinus in freshwater and Rhizoph- lyctis rosea in agricultural and perturbed soils), Accepted for publication 6 September 2006. 1 Corresponding author. E-mail: tyj2@duke.edu Mycologia, 98(6), 2006, pp. 860���871. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897 860

whereas most species are infrequent and scarce to rare (Letcher and Powell 2001, Letcher et al 2004a). The most prominent morphological feature of the chytrid body, or thallus, is the sporangium (FIG. 1). The sporangium is a sac-like structure in which internal divisions of the protoplasm result in pro- duction of zoospores. These zoospore-producing sporangia (zoosporangia) are thin-walled (FIG. 1k) whereas resting spores are thick-walled structures (FIG. 1a, l) that may germinate to produce a sporan- gium after a dormant period. Zoosporangia always are produced asexually, but resting spores may be sexually or asexually formed. Eucarpic chytrids are those that consist of a sporangium and filamentous rhizoids (FIG. 1k). In contrast holocarpic chytrids produce thalli that are entirely converted to sporangia during reproduction. Chytrid thalli can be either monocentric, in which an individual produces only a single sporangium (FIG. 1g), or polycentric, in which an individual is composed of multiple sporan- gia produced on a network of rhizoids termed a rhizomycelium (FIG. 1j). Classically chytrids also were described on the basis of whether they grow on (epibiotic, FIG. 1h) or within (endobiotic, FIG. 1a) their substrate. Other characteristics historically used for taxonomy include the presence of a lid-like operculum (FIG. 1e), which opens to allow zoospore release from a sporangium (Sparrow 1960), and the apophysis (FIG. 1i), which is a subsporangial swelling. Zoospores are unwalled cells, usually 2���10 mm diam, which contain a single nucleus and, with the exception of some genera of the Neocallimastigales, are propelled by a single posteriorly oriented whip- lash flagellum (FIG. 1b, m). Considerable effort has been placed on understanding the ultrastructure of the zoospore with electron microscopy these studies have produced a wealth of information about the complexity of the internal contents of the spores. Two character-rich components of the zoospores can be identified: the flagellar apparatus, basal bodies and associated structures (Barr 1981), and the microbody- lipid globule complex (MLC, Powell 1978). The Chytridiomycota is divided into five orders based primarily on the mode of reproduction and ultrastructure of the zoospore. Three groups can be distinguished largely on the basis of life cycle and gross morphology: the oogamous Monoblepharidales the Blastocladiales displaying sporic meiosis and the alternation of sporophytic and gametophytic genera- tions and the Chytridiales, a group characterized by zygotic meiosis. A fourth order, Spizellomycetales, was separated from the Chytridiales on the basis of distinctive ultrastructural character states (Barr 1980), and a fifth order, Neocallimastigales, occurs exclusively as anaerobic symbionts of the rumen. Chytridiales is the largest of the orders (more than 75 genera) and the classification of this group has been variously approached using developmental, sporan- gial and more recently ultrastructural characters. Sparrow (1960) considered the operculum to be a defining characteristic and created two series of families based on whether sporangia were operculate. In contrast Whiffen (1944), Roane and Paterson (1974) and Barr (1978) suggested development could be used to divide the Chytridiales into families. Systems of classification based on morphology and those based on development of the thallus and sporangium both have been shown by molecular phylogenetics to be inaccurate in defining genera and families of Chytridiales (James et al 2000, Letcher et al 2004b). Molecular phylogenies with 18S rDNA have sug- gested that both the Chytridiomycota and Chytri- diales might not be monophyletic (James et al 2000). Phylogenies based on data from entire mitochondrial genomes (Seif et al 2005) have suggested strongly that two main lineages exist within the Chytridiomycota��� the Blastocladiales and the remaining four orders (the ������core chytrid clade������). In this paper we present a new dataset that includes an analysis of most chytrid 18S rDNA data in conjunction with sequence data for the entire rRNA operon (18S, 28S and 5.8S). These data provide a comprehensive view of chytrid phylog- eny and define four major lineages of flagellated Fungi. MATERIALS AND METHODS Our dataset consists of taxa for which most of the rRNA operon was sequenced (18S+28S+5.8S subunits, n 5 54) as well as chytrid taxa represented only by 18S data (n 5 49) and additional fungal, animal and Mesomycetozoae taxa (n 5 21 complete operon with one exception, Diaphanoeca grandis). GenBank accession numbers and strain/voucher data are provided (SUPPLEMENTARY TABLE I). Data gathered for the full operon were obtained by PCR, primarily with primers SR1R and LR12 (Vilgalys and Hester 1990) using LATaq (TaKaRa), with these thermocycling conditions: 94 C for 1 min followed by 35 cycles of denaturing at 94 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 5 min and a 10 min final extension at 72 C. Amplicons generally were cloned into pCR2.1-TOPO (Invitrogen). The three gene regions were aligned by eye with GeneDoc v2.6 (http://www.cris.com/,Ketchup/genedoc.shtml) and combined into one supermatrix with MacClade 4.05 (Maddi- son and Maddison 2002). Regions of ambiguous alignment were excluded from further analysis leaving 4109 included characters, 1388 of which are parsimony informative. We estimated the phylogeny with MrBayes v3.1 (Huelsenbeck and Ronquist 2001) by four independent runs with the GTR+I+C model of evolution, sampling trees every 500 generations for 10 3 106 generations. We also assessed JAMES ET AL: PHYLOGENY OF CHYTRID FUNGI 861