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

mycological research 110 (2006) 898–915

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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 species formerly classified in Rhizophydium are transferred to the new genera. This work provides 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 relatively 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 et al. 2005), and one study emphasizing rDNA region. For LSU analyses, partial nucleotide se- the Chytridiales identified four monophyletic lineages in the quences of the LSU rRNA gene (677–824 bp from the 50 order (James et al. 2000). The study of Letcher et al. (2005) end) and ITS1–5.8 S–ITS2 sequences (563–574 bp from the was the first to integrate ultrastructural and molecular data- 50 end) from 91 taxa (Table 1) were generated; LSU and sets to designate one of the four lineages (the Chytridium/Chy- ITS1–5.8 S–ITS2 sequences for an additional five isolates triomyces clade) in the Chytridiales as a new taxon, the (JEL 151, Rhizophydium haynaldii; JEL 197, Batrachochytrium Chytridiaceae. In this revision of the Chytridiaceae (Letcher dendrobatidis; JEL 299, R. sphaerotheca; JEL 317, Rhizophydium 900 P. M. Letcher et al.

Table 1 – Taxon sampling for phylogenetic analyses of 96 isolates in Rhizophydiales (Chytridiomycota) Culture number/isolate Source Origin GenBank accession no.

28 S ITS1–5.8 S–ITS2

Ingroup: ATCC 24053 Rhizophlyctis harderia ATCC British Columbia, CAN AY349087b DQ485595 Barr 003 Rhizophydium pollinis-pinia CCFC Michigan, USA DQ485532 DQ485596 Barr 004 R. lateralea CCFC Ontario, CAN DQ485533 DQ485597 Barr 089 R. cladea CCFC British Columbia, CAN DQ485534 DQ485598 Barr 100 R. haynaldiic CCFC Quebec, CAN DQ485535 DQ485599 Barr 102 R. chlorogoniia CCFC Ontario, CAN DQ485536 DQ485600 Barr 106 R. haynaldiia CCFC Quebec, CAN DQ485537 DQ485601 Barr 107 Rhizophydium cladec CCFC Ontario, CAN DQ485538 DQ485602 Barr 250 Kappamyces sp.c CCFC New Brunswick, CAN DQ485539 DQ485603 Barr 263 R. littoreuma CCFC Massachusetts, USA DQ485540 DQ485604 Barr 303 R. aestuariia CCFC Germany DQ485541 DQ485605 Barr 316 Kappamyces sp.c CCFC New Brunswick, CAN DQ485542 DQ485606 Barr 436 Rhizophydium cladec CCFC Ontario, CAN DQ485543 DQ485607 Barr 903 Rhizophydium cladec CCFC Kuala Lumpar, Malaysia DQ485544 DQ485608 JEL 001 Kappamyces sp.c UM Maine, USA DQ485545 DQ485609 JEL 008 Rhizophydium cladec UM Maine, USA DQ485546 DQ485610 JEL 055 Rhizophydium cladec UM British Columbia, CAN DQ485547 DQ485611 JEL 134 R. carpophilumc UM Maine, USA DQ485548 DQ485612 JEL 136 R. brooksianuma UM Maine, USA AY349086b DQ485613 JEL 138 Rhizophydium cladec UM Florida, USA DQ485549 DQ485614 JEL 150 Rhizophydium clade UM Maine, USA DQ485550 DQ485615 JEL 151 R. haynaldiic UM Maine, USA AFTOL 30 AFTOL 30 JEL 197 Batrachochytrium dendrobatidisa UM Washington, DC AFTOL 21 AFTOL 21 JEL 222 R. globosumc [epitype] UM Maine, USA DQ485551 DQ485616 JEL 223 Rhizophydium cladec UM Maine, USA DQ485552 DQ485617 JEL 281 Rhizophydium cladec UM Maine, USA AY439032b DQ485618 JEL 291 Rhizophydium cladec UM Maine, USA DQ485553 DQ485619 JEL 292 Rhizophydium cladec UM Maine, USA DQ485554 DQ485620 JEL 294 Rhizophydium cladec UM Arizona, USA DQ485555 DQ485621 JEL 299 R. sphaerothecac UM Oklahoma, USA AFTOL 37 AFTOL 37 JEL 300 Rhizophydium clade UM South Dakota, USA DQ485556 DQ485622 JEL 302 Rhizophydium cladec UM Maine, USA DQ485557 DQ485623 JEL 317 Rhizophydium clade UM Maine, USA AFTOL 35 AFTOL 35 JEL 326 Entophlyctis helioformis UM Maine, USA AFTOL 40 AFTOL 40 JEL 348 Rhizophydium cladec UM Maine, USA DQ485558 DQ485624 JEL 356 Rhizophydium clade UM California, USA DQ485559 DQ485625 JEL 385 Rhizophydium clade UM Maine, USA DQ485560 DQ485626 JEL 393 Rhizophydium cladec UM New Zealand DQ485561 DQ485627 JEL 395 Rhizophydium cladec UM New Zealand DQ485562 DQ485628 JEL 396 Rhizophydium cladec UM New Zealand DQ485563 DQ485629 JEL 400 Rhizophydium cladec UM Maine,USA DQ485564 DQ485630 MP 008 Rhizophydium cladec UA Alabama, USA DQ485565 DQ485631 PL AUS 002 Boothiomyces macroporosumc UA NSW, Australia AY439044b DQ485632 PL AUS 003 Rhizophydium cladec UA NSW, Australia AY439045b DQ485633 PL AUS 006 Rhizophydium cladec UA NSW, Australia AY439046b DQ485634 PL AUS 007 Rhizophydium cladec UA NSW, Australia AY439047b DQ485635 PL AUS 008 Rhizophydium cladec UA NSW, Australia AY439048b DQ485636 PL AUS 009 Rhizophydium cladec UA NSW, Australia AY439049b DQ485637 PL AUS 010 Rhizophydium cladec UA NSW, Australia DQ485566 DQ485638 PL AUS 012 Rhizophydium cladec UA NSW, Australia AY439035b DQ485639 PL AUS 015 Kappamyces sp.c UA NSW, Australia DQ485567 DQ485640 PL AUS 018 Rhizophydium cladec UA NSW, Australia AY439051b DQ485641 PL AUS 021 B. macroporosumc [epitype] UA NSW, Australia AY439040b DQ485642 PL AUS 022 Rhizophydium cladec UA NSW, Australia AY439030b DQ485643 PL AUS 024 Rhizophydium cladec UA NSW, Australia AY439052b DQ485644 PL AUS 025 Rhizophydium cladec UA NSW, Australia DQ485568 DQ485645 PL AUS 038 Rhizophydium clade UA NSW, Australia DQ485569 DQ485646 PL AUS Ad014 Rhizophydium cladec UA NSW, Australia DQ485570 DQ485647 PL AUS R002 Rhizophydium cladec UA NSW, Australia DQ485571 DQ485648 PL AUS R008 Rhizophydium clade UA NSW, Australia DQ485572 DQ485649 PL AUS R013 Rhizophydium cladec UA NSW, Australia DQ485573 DQ485650 New order, Rhizophydiales (Chytridiomycota) 901

Table 1 – (continued) Culture number/isolate Source Origin GenBank accession no.

28 S ITS1–5.8 S–ITS2

PL AUS R014 Rhizophydium clade UA NSW, Australia DQ485574 DQ485651 PL 001 Terramyces subangulosumc UA Vermont, USA AY439042b DQ485652 PL 003 T. subangulosumc [epitype] UA Virginia, USA AY439041b DQ485653 PL 004 Rhizophydium cladec UA Virginia, USA AY439057b DQ485654 PL 005 Rhizophydium cladec UA Tennessee, USA AY439058b DQ485655 PL 008 Rhizophydium cladec UA Alabama, USA AY439059b DQ485656 PL 010 Rhizophydium cladec UA Alabama, USA AY439037b DQ485657 PL 042 Rhizophydium cladec UA North Carolina, USA AY439056b DQ485658 PL 072 Rhizophydium cladec UA Virginia, USA AY439031b DQ485659 PL 073 Rhizophydium cladec UA Virginia, USA AY439039b DQ485660 PL 074 Kappamyces sp.c UA Virginia, USA AY439033b DQ485661 PL 075 Kappamyces sp.c UA Virginia, USA AY439053b DQ485662 PL 076 T. subangulosumc UA Virginia, USA DQ485575 DQ485663 PL 088 Rhizophydium cladec UA Alabama, USA AY439036b DQ485664 PL 097 Rhizophydium cladec UA Virginia, USA DQ485576 DQ485665 PL 098 K. laurelensisa UA Georgia, USA AY439034b DQ485666 PL 102 Rhizophydium cladec UA Georgia, USA AY439072b DQ485667 PL 104 Rhizophydium cladec UA Georgia, USA DQ485577 DQ485668 PL 113 Rhizophydium cladec UA New Hampshire, USA DQ485578 DQ485669 PL 116 Kappamyces sp.c UA NSW, Australia DQ485579 DQ485670 PL 118 Kappamyces sp.c UA Virginia, USA DQ485580 DQ485671 PL 120 Kappamyces sp.c UA Virginia, USA DQ485581 DQ485672 PL 122 T. subangulosumc UA Virginia, USA DQ485582 DQ485673 PL 127 Rhizophydium cladec UA Colorado, USA DQ485583 DQ485674 PL 133 Rhizophydium cladec UA Alabama, USA DQ485584 DQ485675 PL 137 R. aestuariic UA South Africa DQ485585 DQ485676 PL 139 Rhizophydium cladec UA Taipei, Taiwan DQ485586 DQ485677 PL 141 Rhizophydium cladec UA Lake Windermere, ENG DQ485587 DQ485678 PL 143 Rhizophydium cladec UA Virginia, USA DQ485588 DQ485679 PL 144 Rhizophydium cladec UA Virginia, USA DQ485589 DQ485680 PL 147 Rhizophydium cladec UA Virginia, USA DQ485590 DQ485681 PL 149A Rhizophydium cladec UA Texas, USA DQ485591 DQ485682 PL 152 Rhizophydium cladec UA Virginia, USA DQ485592 DQ485683 PL 153 Rhizophydium cladec UA Virginia, USA DQ485593 DQ485684 PL 157 Rhizophydium cladec UA Buenos Aires, Argentina DQ485594 DQ485685

Outgroup: M15Monoblepharella sp. UM Maine, USA AFTOL 25 AFTOL 25 CR 84 Oedogoniomyces sp. UCB Costa Rica AFTOL 298 AFTOL 298

a Isolates for which ultrastructural analysis was from published work. b GenBank accession numbers from Letcher et al. (2004b). c Isolates for which zoospore ultrastructure was examined using TEM.

clade; JEL 326, Entophlyctis helioformis) were obtained from model) analyses were conducted in PAUP version 4b10 (Swof- the AFTOL database with permission of T. Y. James. ford 2002). We used the partition homogeneity/incongruence- length difference test in PAUP to determine whether the LSU Phylogenetic analyses and 5.8 S data had signiﬁcantly different signals. MP phylogenetic trees were constructed using PAUPRat (Sikes & Lewis Contiguous sequences were assembled and aligned as de- 2001) as described (Letcher et al. 2004b). ModelTest (version scribed (Letcher et al. 2004b). Two combinations of sequence 3.06, Posada & Crandall 1998) was used to calculate the most alignments were used in analyses because ITS1–5.8 S–ITS2 appropriate model of DNA substitution, and Bayesian analysis could not be aligned unambiguously among the total sampling used MrBayes 3.0 (Huelsenbeck & Ronquist 2001). Bayesian of organisms. tree inference with MCMC sampling used four simultaneous Combined LSU þ 5.8 S analyses Markov chains run over 1M generations. Trees were sampled For all isolates in the study, partial LSU sequences (677–824 bp) every 100 generations, with an overall sampling of 10,001 were combined with 5.8 S sequences (w120 bp). ITS1 and ITS2 trees. Burnin was calculated when the average standard devi- sequences were excised from the complete ITS1–5.8 S–ITS2 re- ation of split frequencies had declined to <0.01. A consensus gion, because they were too divergent for alignment across of remaining trees was used to compute a majority rule tree the entire data set. MP and NJ (using the HKY85 DNA distance to obtain estimates for PP. 902 P. M. Letcher et al.

Combined LSU þ ITS1–5.8 S–ITS2 analyses NJ, MP, and Bayesian tree topologies were similar; conse- For 44 isolates, partial LSU sequences were combined with quently, only the NJ tree is illustrated (Fig 1), with MP BS and complete ITS1–5.8 S–ITS2 sequences (w600 bp). Partitioned Bayesian PP values. Where a branch collapsed in the Bayesian data was tested for homogeneity, MP and NJ (using the analysis, only BS values are shown. Within the Rhizophydium HKY85 DNA distance model) analyses were performed using clade were numerous strongly supported clades, three of PAUP and PAUPRat, and Bayesian analysis was conducted in which contained at least 12 isolates (Fig 1, clades A–C). Differ- MrBayes. MCMC sampling used four simultaneous chains run ences in the NJ, MP and Bayesian trees occurred with respect over 100K generations. Trees were sampled every 100 genera- to the position of four isolates (Barr 100, Barr 106, JEL 151, tions, with an overall sampling of 1001 trees. Burnin was calcu- and JEL 299), which were basal in the NJ and MP analyses lated when the average standard deviation of split frequencies (Fig 1), but were sister to clade A in the Bayesian analysis. had declined to <0.01. A consensus of remaining trees was Clade A (Fig 1) contained 44 isolates (partial LSU sequences used to compute a majority rule tree to obtain estimates for PP. 732–762 bp), and relationships among many of the isolates were unresolved in polytomies. Clade B (Fig 1) contained 12 Morphology and ecology isolates (partial LSU sequences 714–734 bp), including the type species Kappamyces laurelensis (Letcher & Powell 2005). Ingroup isolates were examined by light microscopy (either Clade C (Fig 1) contained 13 isolates (partial LSU sequences a Nikon Labophot-2 or Zeiss Axioskop) to assess range and 784–824 bp), one of which was identified as Rhizophydium variation in thallus structural features, including sporangial brooksianum (JEL 136, Longcore 2004). size and shape, number of discharge pores, type of discharge, and morphology of rhizoids. The type of habitat and substrata Combined LSU þ ITS1–5.8 S–ITS2 analyses from which each culture was originally recovered were The partition homogeneity test determined that the LSU and analysed. ITS1–5.8 S–ITS2 data did not have significantly different signals. The combined data for 44 isolates had 2373 characters, TEM with 101 parsimony-informative sites. All 1005 trees derived in PAUPRat (L ¼ 512 steps, CI ¼ 0.842, RI ¼ 0919) were used to Fixation for electron microscopy compute a majority rule consensus tree. Bayesian analysis Fixation and observation of zoospores followed procedures used the GTR þ Iþ G model. From an overall sampling of described in Letcher and Powell (2005). We examined zoospore 1001 trees, the first 200 were discarded (average standard de- ultrastructure of 87 of the 96 ingroup isolates (Table 1). viation of split frequencies ¼ 0.008). A consensus of 801 remaining trees was used to compute a majority rule tree to Ultrastructural analysis obtain PP estimates. Zoospores were examined for presence or absence of an electron- NJ, MP, and Bayesian tree topologies were the same; conse- opaque plug in the base of the flagellum, an electron-opaque spur quently, only the NJ tree is illustrated (Fig 2) with MP BS and or shield adjacent to the kinetosome, a vesiculated region at the Bayesian a posteriori values at deeper nodes. The tree con- proximal end of the kinetosome, a microtubular root, and tained two strongly supported clades designated as clades a densely granular cylinder in the core of the kinetosome and/ A1 and A2 (Fig 2). Similarities between ITS1–5.8 S–ITS2 se- or the non-flagellated centriole. Also evaluated were number of quences in clades A1 and A2 were 91 %, among isolates in lipid globules, location of mitochondria relative to the double clade A1 were 95 %, and among isolates in clade A2 were membrane-delineated ribosomal core, and structure of MLC cis- 93 %. In clade A1, ITS1–5.8 S–ITS2 sequences for isolates PL terna (Powell 1978). 001, PL 003, PL 076, and PL 122 were 100 % similar; in clade A2, for isolates PL AUS 002, PL AUS 006, and PL AUS 021 were Results 100 % similar.

Phylogenetic analyses Morphology and ecology

Combined LSU þ 5.8 S analyses Wide variation and range in morphological features were ob- The partition homogeneity/incongruence-length difference served among the 96 ingroup isolates. Isolates were recovered test determined that LSU and 5.8 S data did not have signiﬁ- from both terrestrial and aquatic habitats, and from a variety cantly different signals. The combined data had 1542 charac- of substrata including pollen, keratin, and algae. ters, with 453 parsimony-informative sites. For MP analysis, The sporangia of the 44 isolates in clade A (Figs 1 and 2) of 1005 trees derived from PAUPRat, 566 most parsimonious were spherical (Fig 3A, B, E) or angular (Fig 3C–D, F–G), were trees [length (L) ¼ 2328 steps, CI ¼ 0.472, and RI ¼ 0.784] were multipored (Fig 3F–G), and rhizoids were branched and exten- used to compute a majority rule consensus tree (70 % branch sive (Figs 3B, E). A portion of the zoospores within the sporan- support). ModelTest indicated the most appropriate model of gium initially emerged from discharge pores as a quiescent DNA substitution was the general time reversible model mass and soon began became motile and dispersed. Shortly with invariant sites and rates of substitution among sites ap- thereafter the zoospores remaining in the sporangium proximated by a gamma distribution (GTR þ Iþ G). In the swarmed within the sporangium and emerged individually Bayesian analysis, the ﬁrst 491 trees were discarded (average and dispersed. All isolates came from soil and most were sap- standard deviation of split frequencies ¼ 0.008). A consensus rotrophs primarily of pollen (Fig 3C, F) and to a lesser extent, of 9510 trees was used to compute a majority rule tree. keratin (Fig 3G). New order, Rhizophydiales (Chytridiomycota) 903

Fig 1 – Phylogram (NJ, majority rule) of Rhizophydiales based on analyses of combined (LSU D 5.8 S) data for 96 ingroup isolates. On the bases of molecular and ultrastructural divergence, clades A, B, and C represent new families Terramycetaceae, Kappamycetaceae, and Rhizophydiaceae. Terramycetaceae is further resolved in Fig 2. Isolate PL 098 (Kappamyces laurelensis) is the type for the monogeneric Kappamycetaceae; isolate JEL 222 (Rhizophydium globosum) is the type for the monogeneric Rhizophydiaceae. Values above branches are MP BS (1K replicates), and below branches are Bayesian a posteriori probabilities. Tree length [ 2328 steps, CI [ 0.472, RI [ 0.784. Scale bar below phylogeny indicates substitutions per site (0.01). Members of Monoblepharidaceae used as outgroup.

The 12 isolates in clade B (Fig 1) had morphological, ecolog- vesicle (e.g., PL AUS Ad 014, Rhizophydium clade). Discharge ical and substrate characteristics as described for the genus pores numbered from a few (2–5, e.g., JEL 222, Rhizophydium Kappamyces (Letcher & Powell 2005). These chytrids had small, globosum, Fig 3M, N) or occasionally a single pore (e.g., JEL spherical, single-pored sporangia (Fig 3H–J) and branched but 222, Rhizophydium globosum, Fig 3O) to >20 (e.g., JEL 136, Rhi- compact rhizoids (Fig 3H–I), and were all isolated from soil on zophydium brooksianum). Resting spores, when observed, pollen (Fig 3J). were spherical (e.g., JEL 222, Rhizophydium globosum, Fig 3M, The 13 isolates in clade C (Fig 1) varied in sporangial size, P). Isolates were from aquatic (e.g., JEL 138, Rhizophydium and most were subspherical or spherical during develop- clade) or terrestrial (e.g., PL 149A, Rhizophydium clade) habi- ment (e.g., Fig 3K) and spherical at maturity (e.g., Fig 3L). Rhi- tats, and were cultured from pollen (Fig 3M–N) or on algae zoidal structure was meager and slightly branched (e.g., JEL (Fig 3O–P). 292, Rhizophydium clade), or robust and extensively branched (e.g., JEL 222, Rhizophydium globosum, Fig 3L). Zoospores Zoospore ultrastructure analysis swarmed within the sporangium before discharge, then emerged singly through multiple or occasionally single dis- Ultrastructural analysis indicated that all isolates exam- charge pores (Fig 3M, O), or were discharged in a transparent ined (Table 1) had a ribosomal core enclosed by a system 904 P. M. Letcher et al.

Fig 2 – Phylogram (NJ, majority rule) of Terramycetaceae (clade A, Fig 1) based on combined (LSU D ITS) data for 44 isolates. Tree is unrooted. Values are MP BS/Bayesian a posteriori probabilities. Tree length [ 512 steps, CI [ 0.852, RI [ 0.919. Scale bar below phylogeny indicates substitutions per site (0.005). Zoospores of all isolates have a KAS as a spur (S), zoospores of isolates in clade A1, Terramyces, have a simple cisterna (SC), and zoospores of isolates in clade A2, Boothiomyces, have a fenestrated cisterna (FC).

of double membranes (Figs 4A–D, 5A, 6A, 7A, 8A). The slightly angled (Fig 4C) at its proximal end toward the ki- majority of isolates had a single lipid globule (rarely two netosome (e.g., Kappamyces;seeLetcher and Powell 2005). or more) with a simple or fenestrated cisterna and micro- The kinetosome and non-flagellated centriole were bridged body on the lipid globule surface (Figs 4A–D, 5A, 6A, 7B, by fibrous material (Figs 4A–D, 5E, 6G, 7E) that converged 8A). For many isolates, a microtubular root was present between the two structures (Figs 4A, B, D, 5E, 6G, 7E), (Figs 4B, D, 6A, 7F). Mitochondria were located outside with a zone of convergence of fibrils ranging in width ap- the double-membrane that enclosed the ribosomal mass proximately 0.010–0.075 mm. Zoospores of some isolates (Figs 4A, B, D, 5B, 6B, 7A), or inside the double-membrane contained a kinetosome-associated structure (KAS, Letcher that enclosed the ribosomal mass (Figs 4C, 8C). The non- et al. 2004b), shaped like (1) a bar (McNitt 1974) and re- flagellated centriole and kinetosome were parallel (Figs ferred to as a ‘spur’ (Barr & Hadland-Hartmann 1978), 4A, B, D, 5E, 6F, 7E) or the non-flagellated centriole was with variable morphologies (Figs 4A, B, D, 5C, 6D, 7D), New order, Rhizophydiales (Chytridiomycota) 905

Fig 3 – Morphologies of four isolates in Rhizophydiales. A–D. Terramyces subangulosum, Terramycetaceae. A. Developing thallus on agar. B. Spherical mature thallus on agar. C. Slightly angular mature sporangia on pollen. D. Angular mature sporangium on Oscillatoria sp. E–G. Boothiomyces macroporosum, Terramyceataceae. E. Spherical mature thallus on agar. F. Angular mature thallus on pollen. G. Angular mature thalli on keratin. H–J. Kappamyces laurelensis, Kappamyceataceae. H. Immature thalli on agar. I. Mature thallus and clustered germlings on agar. J. Mature sporangium on pollen. K–P. Rhizo- phydium globosum, Rhizophydiaceae. K. Developing thallus on agar. L. Mature thallus on agar, with extensive rhizoids. M. Multipored sporangium discharging zoospores, and resting spore on pollen. N. Multipored empty sporangium on pollen. O. Single-pored sporangium on Oedogonium sp. P. Resting spore on Oedogonium sp. Scale bars in A [ 10 mm (for A–D), in E [ 20 mm, in F [ 10 mm (for F–G), in H [ 5 mm (for H–J), in K [ 10 mm (for K–P). 906 P. M. Letcher et al.

Fig 4 – Schematic drawings of four zoospore types in the Rhizophydiales (A–D), and the Group I-type zoospore of the Chytridiales emended (E). A. Terramyces subangulosum (Terramycetaceae), isolate PL 003, with a solid spur, a narrow zone of convergence, and a simple cisterna. B. Boothiomyces macroporosum (Terramycetaceae), isolate PL AUS 021, with a solid spur, a narrow zone of convergence, and a fenestrated cisterna. C. Kappamyces laurelensis (Kappamycetaceae), isolate PL 098, with a densely granular core in both kinetosome and non-flagellated centriole and a simple cisterna. D. Rhizophydium globosum (Rhizophydiaceae), isolate JEL 222, with a laminated spur, a wide zone of convergence, and a cisterna with inconspicuous fenestrations. E. Chytriomyces hyalinus (Chytridiaceae), isolate MP 004, with an electron-opaque plug in the base of the flagellum proper, a feature which distinguishes Chytridiales from Rhizophydiales. Below each figure is a schematic drawing of the cross-section of the kinetosome and non-flagellated centriole, illustrating differences in the fibrillar bridge and zone of convergence. F, flagellum; FB, fibrillar bridge; FC, fenestrated cisterna; FP, flagellar plug; GC, granular cylinder; K, kinetosome; L, lipid globule; M, mitochondrion; Mb, microbody; Mt, microtubular root; N, nucleus; NfC, non-flagellated centriole; P, flagellar prop; Pl, plate; R, ribosomes; SC, simple cisterna; Sp, spur; TP, terminal plate; V, vesicle; VR, vesiculated region; ZC, zone of convergence.

or (2) a curved shield and referred to as a ‘shield’ (e.g., Rhi- 6D, 7D). For all isolates, an electron-opaque plug was zophlyctis harderi;seeRoychoudhury & Powell 1992). When absent from the base of the ﬂagellum proper (Figs 4A–D, a KAS as a spur was present, it extended into a vesiculated, 5A, 6A, 7A, 8A), a feature that distinguished the zoospores cup-shaped invaginated region of the endoplasmic reticu- of members of the Rhizophydium clade from the zoospores lum that surrounded the ribosomal aggregation, adjacent of members of the Chytridiaceae (Fig 4E; see Letcher et al. to the proximal end of the kinetosome (Figs 4A, B, D, 5C, 2005). New order, Rhizophydiales (Chytridiomycota) 907

Fig 5 – Ultrastructural features of the zoospore of Terramyces subangulosum (Terramycetaceae). A. Longitudinal section. B. Transverse section. C. Longitudinal section through kinetosome, illustrating the spur protruding into vesiculated region. D. Longitudinal section through kinetosome and base of flagellum, illustrating Golgi apparatus adjacent to vesiculated region. E. Longitudinal section through kinetosome and non-flagellated centriole, illustrating fibrillar bridge and narrow zone of convergence between the two structures. F. Transverse section through kinetosome and non-flagellated centriole, illustrating fibrillar bridge and spur. Scale bars in A [ 0.5 mm (for A–B), in C [ 0.25 mm (for C–D), in E [ 0.25 mm (for E–F). (For list of abbreviations see Fig 4.)

a system of double membranes; mitochondria are associated Taxonomy with the MLC. The nucleus is not associated with the kinetosome. The non-flagellated centriole and kinetosome lie paral- Chytridiales J. Schro¨ t. (1892: 64). lel or at a slight angle and are connected by fibrous material. Emend. Barr (1980: 2388); and Letcher & M.J. Powell There is an electron-opaque plug in the base of the flagellum In the zoospore a microtubular root may or may not be proper. present, but when present extends from one side of the Note: Although Cohn (1879: 279) raised the family concept kinetosome in a parallel array to the side of a cisterna that of chytrids equal to orders of Oomycetes (Saprolegniales, Perono- is on the lipid globule surface; ribosomes are enclosed by sporales), Schroeter (1892) provided a concept for the order. 908 P. M. Letcher et al.

Fig 6 – Ultrastructural features of the zoospore of Boothiomyces macroporosum (Terramycetaceae). A. Longitudinal section. B. Transverse section. C. Face view of fenestrated cisterna. D. Longitudinal section through kinetosome, illustrating spur and terminal plate. E. Longitudinal section through kinetosome and non-flagellated centriole, illustrating the narrow zone of convergence between the two structures, the spur protruding into the vesiculated region, and the microtubular root. F. Longitudinal section through the kinetosome and non-flagellated centriole, illustrating the fibrillar bridge connecting the two structures, and the narrow zone of convergence of the fibres. G. Transverse section through the kinetosome and non- flagellated centriole, illustrating the fibrillar bridge, zone of convergence, and spur. Scale bars in A [ 0.5 mm (for A–C), in D [ 0.25 mm (for D–F), in G [ 0.25 mm. (For list of abbreviations see Fig 4.)

Barr (2001) reviewed the orders in the Chytridiomycota, but array, to a cisterna on the lipid globule surface; ribosomes are the only formal emendatum to the Chytridiales was by Barr enclosed by a system of double membranes; mitochondria, (1980). microbodies, lipid globules, and membrane cisterna are typically associated as a MLC. The non-flagellated centriole Rhizophydiales Letcher, ord. nov. and kinetosome lie parallel or slightly angled toward each Zoosporum exemplum tres; Flagellum obturamentum nullum. other and are connected by fibrillar material. A kinetosome- In the zoospore a microtubular root composed of one or more associated structure, a spur or shield, may or may not be pres- microtubules may or may not be present, but when present, ent, adjacent to the kinetosome. The base of the flagellum extends from one side of the kinetosome, usually in a parallel proper lacks an electron-opaque plug. New order, Rhizophydiales (Chytridiomycota) 909

Fig 7 – Ultrastructural features of the zoospore of Rhizophydium globosum (Rhizophydiaceae). A. Longitudinal section. B. Transverse section, illustrating inconspicuous fenestrated cisterna that covered only a small region of the lipid globule. C. Tangential section through fenestrated cisterna. D. Longitudinal section through kinetosome and base of flagellum, illustrating spur adjacent to kinetosome, protruding into vesiculated region. E. Longitudinal section through kinetosome and non-flagellated centriole, illustrating fibrillar bridge with a thick plate centrally located between the two structures. F. Transverse section through kinetosome and non-flagellated centriole, illustrating fibrillar bridge and thick plate, spur, and microtubular root adjacent to spur. Scale bars in A [ 0.5 mm (for A–C), in D [ 0.25 mm (for D), in E [ 0.25 mm (for E–F). (For list of abbreviations see Fig 4.)

Note: In the following, three new families (Rhizophydia- Rhizophydiaceae Letcher, fam. nov. ceae, Terramycetaceae, and Kappamycetaceae)inRhizophydiales Zoospora cum uno globulo lipoideo ex parte cum cisterna. Kineto- are delineated on the bases of rDNA (LSU þ 5.8S) molecular soma et centriolum non flagellatum adligatum cum materio sequence groupings (Fig 1) and zoospore ultrastructural fea- fibrato adligatum ad asserem crassum medium. [KAS spur] crassum, multi-cumulum, et cuvatum. Sporangium globosum cum tures (Figs 4A–D, 5–8). Within Terramycetaceae fam. nov. two multis poris evacuationis, rhizoidia ramosa. new genera (Terramyces and Boothiomyces) are delineated on Typus: Rhizophydium Schenk 1858. the bases of rDNA (LSU þ ITS1-5.8S-ITS2) sequence group- Zoospore contains a single lipid globule partially covered ings (Fig 2) and zoospore ultrastructural features (Figs 4A, with a cisterna. Kinetosome and non-flagellated centriole are B, 5–6). 910 P. M. Letcher et al.

Fig 8 – Ultrastructural features of the zoospore of Kappamyces laurelensis (Kappamycetaceae). A. Longitudinal section, illustrating densely granular core in the kinetosome. B. Transverse section through kinetosome and non-ﬂagellated centriole, illustrating densely granular core in both structures, diagonal ﬁbrillar bridge connecting the two structures, and three vesicles surrounding the structures. C. Transverse section. Scales bars in A [ 0.5 mm (A, C), in B [ 0.25 mm. (For list of abbreviations see Fig 4.)

parallel and are connected by fibrillar material with a zone of The illustrations from Braun (1855) merely show small convergence approximately 0.075 mm in width centrally lo- round sporangia on Oedogonium fonticola and Eunotia cated between the two structures. Kinetosome-associated struc- amphioxys, with no rhizoids or details of zoospore discharge. ture (KAS) present as a laminated, curved spur. Sporangium Thus, because original material associated with C. globosum spherical at maturity, with multiple discharge pores or papil- ‘is demonstrably ambiguous and cannot be critically identified lae. Rhizoids branched. for purposes of the precise application of the name of the At present there is a single genus, Rhizophydium Schenk taxon’ (Greuter et al. 2000: Art. 9.7), we have designated illus- (1858), in this family, and the type of R. globosum (Braun) trations based on observations of culture JEL 222 to serve as Rabenhorst constitutes the type of Rhizophydium (Clements & the epitype for R. globosum. Shear 1931). Rhizophydium globosum has spherical sporangia 10–30 mm diam (Fig 3K–O) with a small number (1–7) of protruding dis- Rhizophydium globosum (A. Braun) Rabenh., Fl. Eur. Alg. 3: charge papillae (Fig 3M, O), and extensive, branched rhizoids 280 (1868). (Fig 3K–L). The resting spore is spherical (Fig 3M, P), with the Basionym: Chytridium globosum A. Braun, Abhandl. Berlin outer surface smooth when on pollen (Fig 3M), or the outer Akad.: 34 (1855). surface is covered with small spines when on Oedogonium sp. Type: Braun, Abhandi. Berlin Akad. 1855: 34, pl. 2, figs 14–20 (Fig 3P). The zoospore of Rhizophydium globosum (Figs 4D, 7) (1855–holotype); Letcher et al., Mycol Res. 110: 907 figs 3K-P, 911 has a single lipid globule with an inconspicuous fenestrated fig 7 (2006) - epitypus hic designatus from observations of cul- cisterna (Figs 22, 38, 39) that covers only a small region of ture JEL 222, GenBank LSU rDNA sequence DQ485551, ITS1- the lipid globule (Fig 7B). A microtubular root (Figs 4D, 7F) is 5.8S-ITS2 rDNA sequence DQ485616. Culture JEL 222 from present, the kinetosome and non-flagellated centriole are par- which the epitype illustrations of R. globosum were made is be- allel (Figs 4D, 7A, E–F) and connected by fibrous material in ing deposited in ATCC and CBS. which a zone of convergence approximately 0.075 mm in width Because no material appeared to remain from Braun’s orig- is centrally located between the two structures (Figs 4D, 7A, inal collection (Sparrow 1973), we consider his illustrations as 7E–F), and a curved, laminated spur is adjacent to the kineto- the holotype for C. globosum (Greuter et al. 2000, Art. 9.1) some (Figs 4D, 7A, D, F). New order, Rhizophydiales (Chytridiomycota) 911

Specimen examined: United States: Maine: Penobscot County, original description, and thus we designate illustrations 0 Orono, from garden soil, on pollen and keratin substrata, 44 53 from observations of this culture as the epitype in support of 00 0 00 38 N, 68 41 09 W, alt. 10 m,25 Oct 1998, culture JEL 222 isolated the holotype for C. subangulosum. by Joyce Longcore and also grown on algae. The sporangia of Terramyces subangulosum are spherical when immature (Fig 3A) and spherical or angular at maturity Terramycetaceae Letcher, fam. nov. m Zoospora cum uno globulo lipoideo ex parte cum cisterna. Kinet- (Figs 3B–D), 10–35 m diam, with multiple discharge papillae, osoma et centriolum non flagellatum adligatum cum materio and extensive, branched rhizoids (Fig 3B). Resting spores fibrato adligatam ad asserem attenuatum medium. [KAS spur] were not observed. Zoospores of Terramyces subangulosum attenuatum, solidum, et curvatum. Sporangium angulatum ad (Figs 4A, 5) have a single lipid globule partially covered with maturum, cum multis papillarum zoosporicis emissionibus prae- a simple cisterna (Figs 4A, 5A–B), and mitochondria are associ- stans levatum aut sublevatum. Rhizoidia robusta et ramose. Sap- ated with the MLC. The kinetosome and non-flagellated cen- rophyticum ex pollone atque keratine; praecipue ex domis triole are parallel (Figs 4A, 5E, F) and connected by a fibrillar terrestris. bridge with a zone of convergence 0.010–0.020 mm in width Typus: Terramyces Letcher 2006. (Figs 4A, 5E). The KAS is a spur (Figs 4A, 5A, C–D, F). A micro- Zoospore contains a single lipid globule partially covered by tubular root was not observed. a cisterna. Kinetosome and non-flagellated centriole are con- Specimen examined: United States, Virginia, Rockbridge nected by fibrillar material with a zone of convergence of County, 12 miles north of Lexington, State Route 39, Goshen fibrils approximately 0.010–0.025 mm in width centrally Wildlife Management Area, Laurel Run, from forest soil, 37 located between the two structures. Kinetosome-associated 550 3000 NX79 270 3000 W, alt. 400 m, 22 Oct 1993, Culture PL structure (KAS) present as a solid, non-laminar, curved spur. 003 collected by Peter M. Letcher and isolated by Martha Sporangium is spherical to angular at maturity, with multiple, J. Powell on pollen, also grown on algae. slightly or prominently raised discharge papillae. Rhizoids are robust and branched. Saprotrophic on pollen or keratin; pri- Boothiomyces Letcher, gen. nov. marily from terrestrial habitats. Etym.: The generic name honours James Thomas Booth, a Two new genera are described into which two existing spe- pioneer in the ecology of soil chytrids. cies currently classified in Rhizophydium are transferred. Sporangium globosum aut angulatum, cum multis papillis emissionibus praestans sublevatum aut levatum. Zoosporae cum cis- Terramyces Letcher, gen. nov. terna fenestra in globuli lipoidei superficie, et [KAS spur] Etym.: The generic name reflects the primary habitat of these opacum aedificium terminatum cum nodo globoso aut bifurcato. chytrids, which are common in soils and distributed globally.

Sporangium angulatum, cum multis papillis emissionibus suble- Typus: Boothiomyces macroporosum (Karling) Letcher 2006. vatum. Zoosporae cum cisterna simplece in globulo lipoideo Sporangium spherical to angular, with either slightly raised superficie, et opacum aedificium [KAS spur] terminatum cum or prominent, dome-shaped to conical, multiple discharge pa- nodo globoso. pillae. Zoospore contains a single lipid globule partially covered Typus: Terramyces subangulosum (A. Braun) Letcher 2006. with a fenestrated cisterna. KAS a spur terminated with a spherical or bifurcated knob. Sporangium angular, with multiple, slightly raised discharge papillae. Zoospore contains a single lipid globule partially cov- Boothiomyces macroporosum (Karling) Letcher, comb. nov. ered with a simple cisterna. Kinetosome-associated structure (Figs 3E–G, 6) (KAS) is a spur terminated with a spherical knob. Basionym: Rhizophydium macroporosum Karling, Sydowia 20: 76, Terramyces subangulosum (A. Braun) Letcher, comb. nov. plates XII, XIII, figs 9-20 (1967). (Figs 3A–D, 5) Type: Because no material appears to remain from Karling’s Basionym: Chytridium subangulosum A. Braun, Abhandl. Berlin collection of R. macroporosum, we consider Karling’s illustra- Akad.: 44 (1855) tions the holotype (Rhizophydium macroporosum Karling 1967: Type: Braun, Abhandl. Berlin Akad. 1855: 44, pl. 3, figs 27–31 plates XII, XIII, figs 9–20. - holotype); Letcher et al., Mycol Res. (1855–holotype); Letcher et al., Mycol. Res. 110: 907 figs 3A-D, 110: 907 figs 3E–G, 910 fig. 6 (2006) - epitypus hic designatus 909 fig 5 (2006) - epitypus hic designatus from observations of from culture PL AUS 021, GenBank LSU rDNA sequence AY culture PL 003, GenBank LSU rDNA sequence AY439041, ITS1- 439040, ITS1-5.8S-ITS2 rDNA sequence DQ485642. Culture PL 5.8S-ITS2 rDNA sequence DQ485653. Culture PL 003, on which AUS 021 with which the epitype is associated to be deposited the epitype is based, is being deposited in ATCC and CBS. with ATCC and CBS. Because illustrations that serve as the ho- Synonym: Rhinzophydium subangulosum (A. Braun) Rabenh., Fl. lotype of R. macroporosum are ambiguous in distinguishing Eur. Alg. 3: 281 (1868). R. macroporosum from R. elyensis (Sparrow 1957) and cannot be Type: Because no material appeared to remain from critically used to apply the name of the taxon, we have desig- Braun’s original collection (Sparrow 1973), we consider illus- nated illustrations based on observations of culture PL AUS trations as the holotype. The holotype illustrations of C. suban- 021 to serve as the epitype for R. macroporosum. gulosum are demonstrably ambiguous because multiple Sporangia of Boothiomyces macroporosum are spherical on species with angular, multipapillate sporangia are found on agar (Fig 3E) and angular on pollen and keratin (Fig 3F–G), with algae and this species cannot be critically identified for pur- multiple prominent, raiseddischarge papillae (Fig 3F–G), and ex- poses of the precise application of the name of the taxon. Cul- tensive, branched rhizoids (Fig 3E). Resting spores were not ob- ture PL 003 demonstrated the form and habitat of Braun’s served. The zoospore of Boothiomyces macroporosum (Figs 4B, 6) 912 P. M. Letcher et al.

Taxonomic key to families and genera of Rhizophydiales

1 Kinetosome and non-ﬂagellated centriole each with a densely granular core...... Kappamycetaceae: Kappamyces Kinetosome and non-ﬂagellated centriole without a densely granular core...... 2

2(1) KAS is a laminated, curved spur; zone of convergence in fibrillar bridge between kinetosome and non-flagellated centriole approximately 0.075 mminwidth...... Rhizophydiaceae: Rhizophydium KAS is a solid, curved spur; zone of convergence in fibrillar bridge between kinetosome and non-flagellated centriole approximately 0.010–0.025 mminwidth...... 3

3(2) Lipid globule partially covered with a simple cisterna...... Terramycetaceae: Terramyces Lipid globule partially covered with a fenestrated cisterna ...... Terramycetaceae: Boothiomyces

have features as described by Chen and Chien (1996), including in the Chytridiomycetes. Briefly, the small (2.5–3.0 mm diam) a single lipid globule with a fenestrated cisterna (Figs 4B, 6A, C), zoospore contains a ribosomal aggregation (Figs 4C, 8A, C), mitochondria associated with the MLC, the kinetosome and a single lipid globule partially covered with a simple cisterna non-flagellated centriole are parallel (Figs 4B, 6E–G) and con- (Figs 4C, 8A, C) a microbody, a nucleus, and a single mito- nected by a fibrillar bridge with a zone of convergence approxi- chondrion (Figs 4C, 8A), all enveloped in a double-membrane mately 0.025 mm in width (Figs 4B, 6E–G), the KAS is a spur (Figs system (Figs 4C, 8A, C). One to five vesicles with morphologi- 4B, 6A, D, E, G), and a microtubular root (Figs 4B, 6A, E) is present. cally distinct matrices (Figs 4C, 8B) cluster around the kine- Specimen examined: Australia, New South Wales, Wilburforce, tosome and non-functional centriole. The zoospore lacks a south of Dharug NP, north of Hawkesbury River at Valley Center KAS and microtubular root, and the fibrillar bridge that con- for Environmental Education and Research, from wet sclerophyll nects the kinetosome and non-flagellated centriole is unique forest soil, on pollen, 33 24.8250 SX150 54.8830 E, alt. 110 m, 16 and distinct, being positioned diagonally between the two Jan 2003, Culture PL AUS 021 collected and isolated by Peter M. Letcher. structures (Figs 4C, 8B).

Kappamycetaceae Letcher, fam. nov. Discussion Thallus eucarpicus, monocentricus. Sporangium et sporae quiescentes cum cysta zoosporae endogenum. Rhizoidia capillar- iorum composita. Zoosporae molem ribosomalem, unicum globu- Circumscription of Rhizophydiales lum lipoideum, corpusculum parvum [microbody], nucleum, This study correlated zoospore ultrastructural character atque unicum mitochondrium continentes. Kinetosoma et cen- states with molecular analyses of nuclear ribosomal genes, triolum non flagellatum, parallela vel fere parallela. Kinetosoma demonstrating that the monophyletic Rhizophydium clade et centriolum non flagellata cum medulla opaca cincta. (James et al. 2000, Letcher et al. 2004b) is clearly distinctive Typus: Kappamyces laurelensis Letcher & M.J. Powell 2005. from other clades in the Chytridiales. It showed that zoospore ultrastructural features, such as the KAS as a spur and the Thallus eucarpic, monocentric. Sporangium and resting spore nature of the fibrillar bridge between the kinetosome and endogenous with the zoospore cyst. Rhizoids thread-like, non-functional centriole, are particularly taxonomically branched and compact. Zoospores contain a ribosomal mass, informative. a single lipid globule, a microbody, a nucleus, and a single mi- Analysis of zoospore ultrastructural characters has been, tochondrion. Kinetosome and non-flagellated centriole lie par- until recently, the principal basis for phylogeny and taxonomy allel or slightly angled and contain a densely granular core among the chytrids because ultrastructural characters were located centrally in each structure. more conserved than thallus morphology (reviewed in Barr At present there is one genus, Kappamyces Letcher and 1980, 2001). For example, based on zoospore ultrastructure, Powell (2005). Barr (1980) hypothesized a divergence among organisms in Kappamyces laurelensis Letcher & M.J. Powell, Nova Hedwigia the Chytridiales sensu Sparrow (1960) and delineated a new 80: 125 (2005). order, the Spizellomycetales. Currently the five orders in the Type: Kappamyces laurelensis Letcher & M.J. Powell (2005: 125, Phylum Chytridiomycota (Chytridiales, Spizellomycetales, Blasto- figs 1–15, holotype). Holotype illustrations from observations cladiales, Monoblepharidales, and Neocallimastigales; Alexopou- of Culture PL 098. GenBank LSU rDNA sequence AY439034, los et al. 1996, Barr 2001) exhibit characteristic zoospore ITS1–5.8 S–ITS2 rDNA sequence DQ485666. Culture PL 098 on architecture (Barr 2001). Hypotheses of divergence of lineages which the illustrations that serve as holotype of K. laurelensis based on zoospore morphology have been confirmed by mo- is based will be deposited with ATCC and CBS. lecular analyses of ribosomal gene sequences within Chytridio- mycota (Chambers 2003, James et al. 2000, Letcher et al. 2005) Sporangia are spherical (Fig 3H–J), 6–12 mm diam. Zoospores dis- and have revealed reliable constructs of clades that include charge through a single large pore and often germinate in both ordinal (Chambers 2003, James et al. 2000) and familial clusters when on agar (Fig 4I). The zoospore of K. laurelensis levels (Letcher et al. 2005). Insights from these studies are valu- has ultrastructural features (Figs 4C, 8) as described in Letcher able in taxonomic revision of the Chytridiales. In an extensive and Powell (2005) and may be the simplest type of zoospore analysis of combined molecular and zoospore ultrastructure New order, Rhizophydiales (Chytridiomycota) 913

in the Chytridium/Chytriomyces clade, Letcher et al. (2005) ana- flagellum, an ultrastructural feature absent in the Group III lysed LSU and combined SSU/LSU rDNA sequence data and Rhizophydium-type zoospore. The absence of this particular zoospore ultrastructural data for 25 isolates from ten genera. ultrastructural character concisely delineates organisms in the Twenty-one of those isolates representing eight genera Rhizophydium clade from organisms in all other clades in the formed a monophyletic clade in which all isolates had a Group Chytridiales sensu Barr. The Rhizophydium-type zoospore also I-type zoospore (Barr 1980). On the bases of molecular mono- has a suite of characters and character states that, in conjunc- phyly and unity of zoospore type, that clade has been circum- tion with absence of the flagellar plug, characterizes the zoo- scribed at the family level as the Chytridiaceae. spore type. However, because Barr (1980) fashioned the Recent phylogenetic analyses using nuLSU rRNA (28 S Rhizophydium-type zoospore as a composite of zoospores of rRNA) gene sequences of a geographically diverse sampling 12 Rhizophydium isolates (Barr & Hadland-Hartmann 1978), of Rhizophydium cultures revealed that the classical genus Rhi- which exhibited different character states, not all Rhizophy- zophydium is genetically more variable than previously under- dium-type zoospores have the entire suite of features. Thus, stood and actually represents multiple genera (Letcher et al. no single representative zoospore can encompass all charac- 2004b). The monophyletic Rhizophydium clade (Letcher et al. ter states because different zoospore morphologies are pres- 2004b) is more complex than the Chytridium/Chytriomyces ent in the Rhizophydiales. clade, being composed of multiple molecular lineages and Ultrastructural analyses of isolates in the Rhizophydiales multiple zoospore morphologies. On those bases we have cir- have revealed new characters and character states that facil- cumscribed the Rhizophydium clade at the ordinal level as the itate taxonomic delineation. Character states of the KAS as Rhizophydiales with the descriptions of three new families a spur and the fibrillar bridge that connects the kinetosome and two new genera. Each family will require further analyses to the non-flagellated centriole have been used in the circum- to resolve with confidence the taxonomic standings among scription of families, and character states of the cisterna ap- the isolates studied. A broader sampling of representatives pressed to the lipid globule have contributed to delineation is still needed before taxonomic decisions can be made for of genera. remaining clades within the Rhizophydiales. Some species classified as Rhizophydium have been well- The primary conclusion from our study is that molecular studied ultrastructurally, such as R. planktonicum Canter divergence and ultrastructure divergence are congruent and emend (Canter 1969), but clearly are not members of the Rhizo- reliably linked. Molecular phylogeny predicts zoospore ultra- phydiales based on our structural information. This taxon structure, and vice versa. A second conclusion from analyses should be retained in the Chytridiales emended and removed of the molecular data is that the LSU rRNA gene is informative from Rhizophydium, and its generic affinities significantly at the familial level, while the ITS region informs at the ge- reassessed. neric and species levels. Thus, delineating taxonomic units on the basis of molecular clades conjoined with zoospore mor- Circumscription of new families in Rhizophydiales phology is a reliable means of integrating phylogeny and taxonomy to circumscribe divergent groups of organisms in the Rhizophydiaceae Chytridiomycota. The Rhizophydium thallus is relatively simple, consisting of Both molecular and ultrastructural rationalizations sup- a single or multi-pored sporangium and a single tapering rhi- port circumscription of a new order. At the molecular level, zoid that often branches. R. globosum, the type species in the the Rhizophydium clade is a strongly-supported lineage, as genus, has been included in numerous chytrid inventories are the Chytridium/Chytriomyces, Nowakowskiella, and Lacustro- (Gaertner 1954, Karling 1967, Letcher et al. 2004a, Miller 1965, myces clades (James et al. 2000). At the ultrastructural level, the Sparrow 1936). R. globosum was rather sparsely described Rhizophydium-type zoospore is fundamentally different from (Braun 1855) as having a spherical sporangium with 2–4 dis- all other zoospore types in the Chytridiales sensu Barr. In reclas- charge papillae, occurring as a parasite on Closterium, and sifying the Chytridiales, Barr (1980) retained in the order organ- other algal hosts. The formal description of R. globosum isms that possessed a chytridialean zoospore, but placed (Rabenhorst 1868) was expanded to acknowledge observations similar species into groups based on differences in zoospore by Cohn (1853) of a fairly extensive, branched rhizoidal sys- ultrastructure. Thus, although the Group I-, Group II-, and tem, and a half-century later Serbinow (1907) described a mi- Group III-type zoospores (Barr 1980) all possessed a suite of nutely spiny resting-spore for the organism. Thus, common characters, character states were recognized among morphology of the type of Rhizophydium is an inferred and the three zoospore types. TEM analyses of a variety of isolates composite concept. Although originally described as an algal in the Chytridiales sensu Barr (1980) have revealed additional parasite from an aquatic environment, R. globosum also has zoospore morphologies: (1) subtypes within the Group III, been reported from soil samples as a saprotroph of pollen Rhizophydium-type zoospore are distinguished by newly grains (Gaertner 1954, Karling 1967, Letcher et al. 2004a). As recognized character states (Barr 1978, Letcher et al. 2004b, pointed out by Sparrow (1960), R. globosum is a difficult species Letcher & Powell 2005), (2) Group IV-type zoospore for to delimit, and is either a widespread omnivorous organism or Nowakowskiella (Letcher et al. 2005, Lucarotti 1981), (3) Group is a complex of species. V-type zoospore for Chytriomyces angularis (Letcher et al. There is neither a type specimen nor type material remain- 2005, Longcore 1992), and (4) Lacustromyces-type zoospore for ing from Braun’s original collection except for line drawings the Lacustromyces clade (James et al. 2000, Longcore 1993). (Braun 1855), which we consider the holotype for R. globosum Five of these zoospore types (Group I, II, IV, V, and Lacustromy- (Greuter et al. 2000: Art. 9.1; Art. 37.4). Because spherical spo- ces) have a distinct electron-opaque plug in the base of the rangia on the surface of algae are not adequate for critical 914 P. M. Letcher et al.

identification and ‘precise application of the name of the 021, they are considered the same species (but not type taxon’, it was necessary to designate an epitype that supports isolates). the holotype and corresponds to the limited discernable characters that exists for R. globosum (Greuter et al. 2000: Art. 9.7). Kappamycetaceae Within Rhizophydiaceae, we designate our illustrations from observations of the culture JEL 222 as the epitype of The family Kappamycetaceae contained a single genus, Kappa- R. globosum. JEL 222 was saprotrophic on keratin, pollen, and myces (Letcher & Powell 2005), members of which are common Oedogonium sp. (Braun 1855: plate II, fig. 14). Braun (1855) never soil-inhabiting chytrids that are saprotrophs of pollen, with described resting spores for R. globosum, but much later Serbi- a small, spherical sporangium having a single, large discharge now (1907) did. On Oedogonium JEL 222 produced resting spores pore. Ultrastructural configuration of the zoospore of Kappa- as Serbinow (1907) described, but smooth-walled resting myces was distinct, as it is the only zoospore in Chytridiomycota spores on pollen. Its rhizoids were similar to those Cohn to have a densely granular cylinder centrally located in both (1853) illustrated for R. globosum and its discharge papilla sim- the kinetosome and non-flagellated centriole, and a ring of ilar to Karling’s (1977: plate 25, fig. 51) illustration. Clearly JEL distinct vesicles surrounding the kinetosome and non-flagel- 222 embodies characteristics of R. globosum reflecting both lated centriole. the historic and current concept of the species. With our des- Our molecular analyses suggest that the diversity of organ- ignation of an epitype for R. globosum, molecular and ultra- isms in the Rhizophydiales is greater than previously realized, structural characterization of the genus Rhizophydium can with the new order containing multiple families and genera. now be pursued. Ultrastructural analyses indicate multiple subtypes of the Rhi- zophydium zoospore, and newly discovered character states of Terramycetaceae the KAS as a spur and the fibrillar bridge between the kinetosome and non-flagellated centriole permit discrimination Isolates in Terramycetaceae were common inhabitants of among families in the Rhizophydiales. soil, and most commonly were saprotrophs of pollen grains. This work provides a framework for additional taxonomic Angulation of the sporangial wall that results from wall dis- revisions within the new order Rhizophydiales and compares tention at the multiple exit papillae was a characteristic genetic variation useful in defining families, genera, and spe- and diagnostic feature of isolates on pollen bait in water cies within this lineage of chytrids. cultures. Thus, thallus morphology of isolates in Terramyce- taceae clearly was not characteristic of the type species of Rhizophydium. Combined LSU þ 5.8 S data were too conserved to discrim- Acknowledgements inate among the isolates included in Terramycetaceae. How- ever, analyses of combined LSU þ ITS1–5.8 S–ITS2 sequence This study was supported by the National Science Founda- data indicated clear delineation of two strongly supported tion through PEET grant DEB-9978094, REVSYS grant DEB- clades of isolates, each of which was coincidental with a spe- 0516173, and AFTOL grant DEB-0228668; The University of cific zoospore morphology. Correspondingly, two new genera Alabama, Department of Biological Sciences Aquatic Ecol- were described within Terramycetaceae. ogy and Systematics Graduate Enhancement Program; Isolates in Terramyces gen. nov. were characterized by a Howard Hughes Medical Institute Undergraduate Biologi- sporangia that were angular at maturity due to the pres- cal Sciences Program Grant to The University of Alabama, ence of multiple discharge papillae slightly raised above and a scholarship from the Alabama Power Company. We the sporangial wall. Several described species of Rhizophy- express our appreciation to the Assembling the Fungal dium exhibit angular sporangia, among them R. subangulo- Tree Of Life (AFTOL) project, Duke University, for access sum, described by Braun (1855) as parasitic on Oscillatoria, to their database, and to Phillip Dean, Rachel Gillis, Scott and later recorded (Gaertner 1954, Letcher et al. 2004a) Wakefield, and Michael Brooks for assistance with DNA iso- from soil as a saprobe of pine pollen. Isolate PL 003 was lation and sequencing. We are deeply indebted to Joyce cultured from a soil sample as a saprobe on pollen, and Longcore for providing chytrid cultures and to Donald in experimental inoculations PL 003 parasitized a pure cul- Barr, Chiu-Yuan Chien, Frank Gleason, Joyce Longcore, ture of Oscillatoria. Because isolate PL 003 was considered to and Carlos Velez for supplying soil samples for chytrid iso- be an isolate of R. subangulosum, we based the epitype for R. lation for this study. Will H. Blackwell assisted in interpret- subangulosum on this culture. Because ITS1–5.8 S–ITS2 se- ing rules of nomenclature. quences of isolates PL 001, PL 076, and PL 122 were 100 % similar to that of PL 003, they are considered the same spe- references cies (but not type isolates). Isolates in Boothiomyces gen. nov. had sporangia that were either spherical or angular. PL AUS 021 was similar in Alexopoulos CJ, Mims CW, Blackwell M, 1996. Introductory thallus morphology, zoospore discharge, and substrate utili- Mycology, 4th edn, John Wiley & Sons, New York. zation to R. macroporosum Karling (1967), and we used illus- Barr DJS, 1978. Taxonomy and phylogeny of chytrids. BioSystems trations of observations of PL AUS 021 as the epitype for 10: 153–165. R. macroporosum. Because ITS sequences of isolates PL AUS Barr DJS, 1980. An outline for the reclassification of the Chytri- diales, and for a new order, the Spizellomycetales. Canadian 002 and PL AUS 006 were 100 % similar to that of PL AUS Journal of Botany 58: 2380–2394. New order, Rhizophydiales (Chytridiomycota) 915

Barr DJS, 2001. Chytridiomycota. In: McLaughlin DJ, McLaughlin EG, Longcore JE, 1992. Morphology and zoospore ultrastructure of Lemke PA (eds), The Mycota. Vol. VIIA. Systematics and Evolution. Chytriomyces angularis sp. nov.(Chytridiales). Mycologia 84: 442– Springer-Verlag, Berlin, Heidelburg, pp. 93–112. 451. Barr DJS, Hadland-Hartmann VE, 1978. Zoospore ultrastructure in Longcore JE, 1993. Morphology and zoospore ultrastructure of the genus Rhizophydium (Chytridiales). Canadian Journal of Botany Lacustromyces hiemalis gen. et sp. nov. (Chytridiales). Canadian 56: 2380–2404. Journal of Botany 71: 414–425. Bary A, de Woronin M, 1865. Beitrag zur Kenntnis der Chytridieen. Longcore JE, 1996. Chytridiomycete taxonomy since 1960. Myco- Berichte Verhandlungen Natuforschende Gesellschaft Freiburg 3: taxon 60: 149–174. 22–61. Longcore JE, 2004. Rhizophydium brooksianum. sp. nov., a multip- Braun A, 1855. Uber Chytridium, eine Gattung einzelliger ored chytrid from soil. Mycologia 96: 162–171. Schmarotzergewa¨chse auf Algen und Infusorien. Deutsche Lucarotti CJ, 1981. Zoospore ultrastructure of Nowakowskiella Akademie der Wissenschaften 1855: 21–83. elegans and Cladochytrium replicatum (Chytridiales). Canadian Canter HM, 1969. Studies on British chytrids. XXIX. A taxonomic Journal of Botany 59: 137–148. revision of certain fungi found on the diatom Asterionella. McNitt R, 1974. Ultrastructure of Phlyctochytrium irregulare zoo- Botanical Journal of the Linnean Society 62: 267–278. spores. Cytobiologie 9: 307–320. Chambers JG, 2003. Ribosomal DNA, secondary structure, and phylo- Miller CE, 1965. Annotated list of aquatic phycomycetes from genetic relationships among the Chytridiomycota. PhD thesis, The Mountain Lake Biological Station, Virginia. Virginia Journal of University of Alabama, Tuscaloosa. Science 14: 219–228. Chen S-F, Chien C-Y, 1996. Morphology and zoospore ultrastruc- Posada D, Crandall KA, 1998. Modeltest: testing the model of DNA ture of Rhizophydium macroporosum (Chytridiales). Taiwania substitution. Bioinformatics 14: 817–818. 41: 105–112. Powell MJ, 1976. Ultrastructure and isolation of glyoxysomes Clements FE, Shear CL, 1931. The Genera Of Fungi. H.W. Wilson, (microbodies) in zoospores of the fungus Entophlyctis sp. Pro- New York. toplasma 98: 1–27. Cohn F, 1853. Untersuchungen u¨ ber die Entwicklungsgeschichte Powell MJ, 1978. Phylogenetic implications of the microbody– der mikroskopischen Algen und Pilze. Nova Acta Akademie lipid globule complex in zoosporic fungi. BioSystems 10: Leopoldina-Carolina Carol. 24: 101–256. 167–180. Cohn R, 1879. Ueber sein Thallophytensystem. Jahresberichte Rabenhorst L, 1868. Flora Europaea Algarum Aquae dulcis et Sub- Schlesische Gesellschaft Vaterlaendische. Culture 57: 279–289. marinae. Vol. 3. E. Kummer, Leipzig. Gaertner A, 1954. U¨ ber das Vorkommen neiderer Erdphycomy- Roychoudhury S, Powell MJ, 1992. Precise flagellar configuration ceten in Afrika, Schweden und an einigen mitteleuropaïschen of the Rhizophlyctis harderi zoospore. Canadian Journal of Botany Standorten. Archiv fu¨r Mikrobiologie 21: 4–56. 70: 762–771. Gaumann EA, Dodge CW, 1928. Comparative Morphology of Fungi, Schenk A, 1858. Algologische Mittheilungen. Verhandlungen Phys- 1st edn. McGraw-Hill, New York. ikalisch-Medizinische Gesellschaft. Wuerzburg, A.F. 8: 235–259. Greuter W, McNeill J, Barrie FR, Burdet HM, Demoulin V, Schroeter J, 1892. Phycomycetes. Die natu¨rlichen Pflanzenfamilien. Filgueiras TS, Nicolson DH, Silva PC, Skog JE, Trehane P, 1(1): 63–87. Turland NJ, Hawksworth DL (eds), 2000. International Code of Serbinow JL, 1907. Kenntiss der Phycomyceten. Organisation Botanical Nomenclature (Saint Louis Code) adopted by the Sixteenth u. Entwickelungsgeschichte einiger Chytridineen Pilze International Botanical Congress, St. Louis, Missouri, July–August (Chytridineae Schro¨ter). Scripta Botanica Horti Universitatis 1999. Koeltz Scientific Books, Ko¨nigstein [Regnum Vegetabile Imperialis Petropolitanae 24: 1–173. No. 138.]. Sikes DS, Lewis PO, 2001. Beta software, version 1. PAUPRat: PAUP* Huelsenbeck JP, Ronquist F, 2001. MrBayes: Bayesian inference of implementation of the parsimony ratchet. Distributed by the phylogenetic trees. Bioinformatics 17: 754–755. authors. Department of Ecology and Evolutionary Biology, James TY, Porter D, Leander CA, Vilgalys R, Longcore JE, 2000. University of Connecticut, Storrs, CT. Molecular phylogenetics of the Chytridiomycota supports the Sparrow FK, 1936. A contribution to our knowledge of the aquatic utility of ultrastructural data in chytrid systematics. Canadian phycomycetes of Great Britain. Botanical Journal of the Linnean Journal of Botany 78: 336–350. Society 50: 417–478. Karling JS, 1939. A note on Phlyctidium. Mycologia 31: 286–288. Sparrow FK, 1957. A further contribution to the phycomycete Karling JS, 1967. Some zoosporic fungi of New Zealand. III. Phlyc- flora of Great Britain. Transactions of the British Mycological tidium, Rhizophydium, Septosperma, and Podochytrium. Sydowia Society 40: 523–535. 20: 74–85. Sparrow FK, 1960. Aquatic Phycomycetes, 2nd edn. University of Karling JS, 1977. Chytridiomycetarum Iconographia. J. Cramer, Michigan Press, Ann Arbor, MI. Vaduz. Sparrow FK, 1973. The type of Chytridium olla A. Braun. Taxon 22: Keeling PJ, 2003. Congruent evidence for alpha-tubule and beta- 583–586. tubulin gene phylogenies for a zygomycete origin of micro- Swofford DL, 2002. PAUP*: Phylogenetic Analysis Using Parsimony sporidia. Fungal Genetics Biology 38: 298–309. (*and other methods). Version 4.0b4a. Sinauer Associates, Sun- Letcher PM, McGee PA, Powell MJ, 2004a. Zoosporic fungi from derland, MA. soils of New South Wales, Australia. Australasian Mycologist 22: Tanabe Y, Watanabe MM, Sugiyama J, 2005. Evolutionary rela- 99–115. tionships among basal fungi (Chytridiomycota and Zygomycota): Letcher PM, Powell MJ, Chambers JG, Holznagel WE, 2004b. Phy- insights from molecular phylogenetics. Journal of General and logenetic relationships among Rhizophydium isolates from Applied Microbiology 51: 267–276. North America and Australia. Mycologia 96: 1339–1351. Whiffen AJ, 1944. A discussion of taxonomic criteria in the Chy- Letcher PM, Powell MJ, 2005. Kappamyces, a new genus in the tridiales. Farlowia 4: 583–597. Chytridiales (Chytridiomycota). Nova Hedwigia 80: 115–133. White TJ, Bruns T, Lee S, Taylor JW, 1990. Amplification and direct Letcher PM, Powell MJ, Chambers JG, Longcore JE, Churchill PE, sequencing of fungal ribosomal RNA genes for phylogenetics. Harris PM, 2005. Ultrastructural and molecular delineation of In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Pro- the Chytridiaceae (Chytridiales). Canadian Journal of Botany 83: tocols: A Guide to Methods and Applications. Academic Press, San 1561–1573. Diego, pp. 315–322.