Ultrastructural and Molecular Delineation of the Chytridiaceae (Chytridiales)

Home , Chytridiales, Chytridium, Chytriomyces, Rhizoclosmatium

1561 Ultrastructural and molecular delineation of the Chytridiaceae (Chytridiales)

Peter M. Letcher, Martha J. Powell, James G. Chambers, Joyce E. Longcore, Perry F. Churchill, and Phillip M. Harris

Abstract: The Chytridiomycota is in need of taxonomic revision, especially the largest order, the Chytridiales. We analyzed 25 isolates in, or allied to, the Chytridium clade of this order. Isolates were selected based on one or more of the following criteria: (i) having a large subunit molecular sequence similar to that of the type of the genus Chytriomyces,(ii) having specific zoospore morphology, and (iii) currently classified as a species in the genus Chytriomyces. We examined ultrastructural characters and partial sequences of large subunit and small subunit rDNA and generated a phylogenetic hypothesis using maximum parsimony and Bayesian analyses. The sequence analyses strongly supported the Chytridiaceae, Phlyctochytrium, and Chytriomyces angularis clades, and each clade had a specific zoospore type. Developmental morphology of the thallus did not mirror the DNA-based phylogeny. Based on the results of phylogenetic analyses of sequences and ultrastructural characters, we emend the Chytridiaceae by including exogenous and polycentric development and define the family on the basis of a single zoospore type. Species identified as being in the genus Chytriomyces occur in several separate, well-supported clades along with species currently classified in seven other genera (Asterophlyctis, Ento- phlyctis, Obelidium, Physocladia, Podochytrium, Rhizoclosmatium, and Siphonaria), indicating that Chytriomyces as currently defined is polyphyletic. Key words: Chytridiomycota, morphology, ribosomal RNA genes, ultrastructure, zoospore. Re´sume´ : Une re´vision de la taxonomie des Chytridiomycota est nećessaire, surtout l’ordre le plus important, celui des Chytridiales. Les auteurs ont analyse´ 25 isolats du clade Chytridium et entite´s apparenteés, appartenant a` cet ordre. Ils ont se´lectionne´ les isolats sur la base d’un ou plusieurs des crite`res suivants: (i) posse´dent une se´quence molećulaire LSU si- milaire a` celle du genre type Chytriomyces;(ii) posse´dent une morphologie zoosporale spećifique; (iii) couramment classifie´s comme espe`ces du genre Chytriomyces. Ils ont examine´ les caracte`res ultrastructuraux et les se´quences partielles de la grande sous-unite´ et de la petite sous-unite´ de l’ADN ribosomal, et ils ont geńe´re´ une hypothe`se phylogeńe´tique, en utili- sant les analyses de parcimonie maximale et baye´sienne. Les analyses de se´quences supportent fortement les clades Chy- tridiaceae, Phlyctochytrium, et Chytriomyces angularis, et chaque clade posse`de un type spećifique de zoospore. La morphogene`se du thalle ne refle`te pas la phylogeńie baseé sur l’ADN. A` partir de ces re´sultats, obtenus par analyse phylo- geńe´tique des se´quences et des traits ultrastructuraux, les auteurs modifient les Chytridiaceae en incluant le de´veloppement exoge`ne et polycentrique, et ils de´finissent la famille sur la base d’un seul type de zoospore. Les espe`ces identifieés comme e´tant du genre Chytriomyces se retrouvent dans plusieurs clades se´pare´s, bien supporte´s, avec des espe`ces couramment classifieés dans sept autres genres (Asterophlyctis, Entophlyctis, Obelidium, Physocladia, Podochytrium, Rhizoclosmatium et Siphonaria), ce qui indique que les Chytriomyces, tels que pre´sentement de´finis, sont polyphe´tiques. Mots cle´s:Chytridiomycota, morphologie, ge`nes de l’ADN ribosomal, ultrastructure, zoospore. [Traduit par la Re´daction]

Introduction vestigators to seek alternative characters for chytrid systematics. Zoospore ultrastructural characters proved to be more Classically, chytrid taxonomy has been based on thallus stable and conserved than taxonomic characters used previ- morphology and development (Whiffen 1944; Sparrow ously. Thus, for the past 30 years, combinations of zoospore 1960; Roane and Paterson 1974; Karling 1977); however, ultrastructural characters have defined orders in the Chytri- observations of morphological variation in chytrids (Miller diomycota (Barr 1980, 2001), and ultrastructural characters 1968, 1976; Powell and Koch 1977a, 1977b) prompted in- have been used to define genera in the Chytridiales sensu Barr (Longcore 1993; Letcher and Powell 2005a) and Spi- Received 24 May 2005. Published on the NRC Research Press zellomycetales (Barr 1980; Longcore et al. 1995). Web site at http://canjbot.nrc.ca on 11 February 2006. In an attempt to resolve phylogenetic groups within the P.M. Letcher,1 M.J. Powell, J.G. Chambers, P.F. Churchill, Chytridiales, Barr (1980) described zoospore types: Group I and P.M. Harris. Department of Biological Sciences, The type zoospore based on Chytridium olla (Barr and Hartmann University of Alabama, Tuscaloosa, AL 35487, USA. 1976, Barr 1980), Group II type zoospore based on Chytri- J.E. Longcore. Department of Biological Sciences, University dium lagenaria (Barr and Hartmann 1976, Barr 1980), and of Maine, Orono, ME 04469, USA. Group III type zoospore based on Rhizophydium spp. (Barr 1Corresponding author (e-mail: [email protected]). and Hadland-Hartmann 1978, Barr 1980). We consider

Can. J. Bot. 83: 1561–1573 (2005) doi: 10.1139/b05-115 # 2005 NRC Canada 1562 Can. J. Bot. Vol. 83, 2005

Group IV as the Nowakowskiella spp. type of zoospore (Lu- Molecular methods carotti 1981) and Group V as the type of zoospore for Chy- triomyces angularis. Evaluation of zoospore ultrastructure DNA extraction, PCR, and sequencing from a broader range of taxa is needed to clarify the taxo- DNA was purified and amplified for sequencing as de- nomic use of ultrastructural features because some genera scribed in Letcher and Powell 2005a from 22 pure cultures contain species with different zoospore subtypes. Barr and (Table 1). The LROR/LR5 primer pair was used for amplifi- Hartmann (1976) reported variation in zoospore ultrastruc- cation of the LSU (28S) rRNA gene and the NS1/NS4 pri- ture among Chytridium species: Chytridium olla and Chytri- mer pair for the SSU (18S) rRNA gene (White et al. 1990). dium confervae with one type of zoospore and Chytridium For LSU analyses, partial nucleotide sequences of the LSU lagenaria with another type. Batko (1975) transferred Chy- rRNA gene (approx. 900 bp from the 5’ end) from 22 taxa tridium confervae to Chytriomyces because it produced epi- (Table 1) were generated. LSU sequences for an additional biotic resting spores, the location of the resting spore three isolates (JEL 45 Chytriomyces angularis, JEL 347 un- relative to the substratum being the primary feature distin- identified sp. C, and JEL 187 unidentified sp. D) were ob- guishing Chytriomyces from Chytridium (Sparrow 1960). tained from the AFTOL database (Assembling the Fungal Thus, the dilemma is that two types of zoospores are re- Tree of Life, Duke University; http://www.biology.duke. ported in one genus (Chytridium), while the same type of edu/fungi/mycolab/) with the permission of Dr. T.Y. James. zoospore is found in different genera (Chytridium and Chy- For combined SSU and LSU analyses, partial nucleotide se- triomyces). quences of the SSU rRNA gene (approx. 1100 bp from the Phylogenetic hypotheses derived from analyses of gene se- 5’ end) from 14 taxa (Table 1) were generated. quences provide frameworks upon which to test the reliability and taxonomic applications of zoospore characters. A recent Sequence analysis phylogenetic reconstruction of the Chytridiomycota from SSU and LSU rRNA gene sequences were aligned based analyses of the small subunit (SSU) rRNA gene supported on the secondary structure model of Saccharomyces cerevi- the reliability of zoospore characters (James et al. 2000), siae rRNA (sites 4–29 for the SSU rRNA gene (Van de Peer although in that study, only a few of the taxa had been studied et al. 1997) and sites B11–D5 for the LSU rRNA gene (Ben ultrastructurally. For the Chytridiales sensu Barr (Barr 1980), Ali et al. 1999; Letcher et al. 2004)). Outgroup taxa for the James et al. (2000) resolved four monophyletic clades (Chy- LSU and the combined LSU and SSU analyses were Mono- tridium, Rhizophydium, Nowakowskiella, and Lacustromy- blepharis macrandra and Oedogoniomyces sp., members of ces), each corresponding to one of Barr’s (1980), Lucarotti’s the Monoblepharidales, which is a sister clade to the Chytri- (1981), or Longcore’s (1993) zoospore types. dium clade (James et al. 2000; Chambers 2003). The purpose of this study was to use zoospore ultrastruc- We analyzed nuclear LSU rRNA gene sequences alone tural and molecular analyses to resolve relationships among and combined with SSU rRNA sequences. ModelTest (v. a broad sampling of organisms provisionally placed in the 3.06) (Posada and Crandall 1998) was used to calculate the Chytridium clade (James et al. 2000). We included isolates most appropriate model of DNA substitution for both the in this study based on one or more of the following criteria: LSU data set and the combined LSU–SSU data set. For the (i) having a large subunit (LSU) molecular sequence like LSU data set, the general time-reversible (GTR) model with that of members of the Chytridium clade (James et al. invariant sites and rates of substitution among sites approxi- 2000), (ii) zoospore morphology (having a Group I type or mated by a gamma distribution ( = GTR + I + G) was Group II type zoospore (Barr 1980), and (iii) currently clas- chosen. This model and maximum parsimony (MP) and sified as a species in the genus Chytriomyces (Letcher and Bayesian inference algorithms were used to construct trees Powell 2002). We generated a phylogenetic hypothesis for in PAUP* (v. 4.0b10) (Swofford 2002). MP analyses were 25 isolates through analyses of the LSU rRNA gene, with conducted as described in Letcher and Powell (2005a). sequence alignment based on rRNA secondary structure. Bayesian tree inference (MRBayes 3.0b4) (Huelsenbeck and We further investigated the resolution of the LSU gene com- Ronquist 2001) with Markov chain Monte Carlo sampling bined with the SSU rRNA gene in molecular analyses. Us- used four simultaneous Markov chains run over 1 million ing this phylogenetic framework, we examined the generations. Trees were sampled every 100 generations with consistency of zoospore ultrastructure among the included an overall sampling of 10 001 trees. From these, the first isolates to evaluate the significance of zoospore ultrastruc- 491 trees were discarded (burnin = 491, by which time the ture for family-, genus-, and species-level taxonomic deci- average standard deviation of split frequencies had declined sions. to 0.008). A consensus of 9510 remaining trees was used to compute a majority rule tree to obtain estimates for the a Materials and methods posteriori probability of groups of isolates. We used the partition homogeneity/incongruence-length Taxonomic sampling difference test in PAUP to determine if the SSU and LSU We examined 25 isolates in 10 ingroup genera and two data sets had significantly different signals. Because they isolates in two outgroup genera. Cultures are from collec- did not, combined SSU and LSU data sets of representatives tions at the Canadian Collection of Fungal Cultures of clades resolved in LSU rRNA gene analyses were sub- (CCFC), The University of Alabama, University of Maine, jected to MP and Bayesian analyses. Phylogenetic hypothe- and University of California at Berkeley. Origins, sources ses for MP were constructed using PAUP* 4.0b10 (Swofford of isolates, and GenBank accession numbers of sequences 2002), with the same parameters used with the LSU data set. are listed in Table 1. Bayesian tree inference (Huelsenbeck and Ronquist 2001)

# 2005 NRC Canada Letcher et al. 1563

Table 1. Taxon sampling for rRNA gene phylogenetic analyses of 25 members of the Chytridiales.

28S: 28S: GenBank Taxon Culture No. 18S: GenBank ID or AFTOL ID Culture sourcea Culture originb Outgroup Monoblepharis macrandra M 53 B AY349029 AY349061 UM ME, USA Oedogoniomyces sp.c CR 90 (=CR 84) AY349025 AY349056 UBC Costa Rica Ingroup Asterophlyctis sarcoptoidesd JEL 186 AY988500 AY439070e UM ME, USA Chytriomyces angularisc JEL 45 ND AFTOL 630 UM ME, USA Chytriomyces angularisd PL 070 ND AY988507 UA UT, USA Chytriomyces appendiculatusd JEL 91 AY349033 AY439077e UM ME, USA Chytriomyces appendiculatus JEL 165 AY349034 AY439076e UM ME, USA Chytriomyces confervaec Barr 97 (ATCC 24931) AY349032 AY439074e CCFC ONT, CAN Chytriomyces hyalinusd MP 004 AY988501 AY988514 UA AL, USA Chytriomyces sp. JEL 176 ND AY349064 UM ME, USA Chytriomyces sp. PL 115 ND AY988516 UA AL, USA Chytriomyces spinosusd JEL 59 AY988502 AY439073e UM ME, USA Entophlyctis luteolusc JEL 129 (ATCC 90967) AY988503 AY442957 UM ME, USA Obelidium mucronatumd JEL 57 AY988504 AY439071e UM ME, USA Phlyctochytrium planicornec JEL 47 ND AY439028e UM ME, USA Physocladia obscura JEL 137 AY988505 AY439062 UM ME, USA Podochytrium dentatumc JEL 30 AY349030 AY439060 UM ME, USA Podochytrium sp. JEL 161 ND AY988517 UM ME, USA Polyphlyctis unispinad PL AUS 026 ND AY988518 UA NSW, AUS Rhizoclosmatium globosumc JEL 06 AY988506 AY439061 UM ME, USA Rhizoclosmatium globosum PL 006 ND AY439055e UM TN, USA Siphonaria peterseniid JEL 102 AY349036 AY439072e UM ME, USA Unidentified sp. Ad JEL 65 ND AY988508 UM ME, USA Unidentified sp. Bd JEL 103 AY349035 AY439064 UM ME, USA Unidentified sp. C JEL 347 ND AFTOL 20 UM ME, USA Unidentified sp. D JEL 187 ND AFTOL 39 UM ME, USA Unidentified sp. E JEL 220 AY349054 AY439063 UM ME, USA Note: ND, not determined. aUM, University of Maine; UA, The University of Alabama; CCFC, Canadian Collection of Fungal Cultures; UCB, University of California at Berkeley. bONT, CAN, Ontario, Canada; NSW, AUS, New South Wales, Australia. cTaxa for which ultrastructural analysis was from published work. dTaxa for which the zoospores were examined by transmission electron microscopy. eGenBank accession numbers from Letcher et al. (2004). with Markov chain Monte Carlo sampling used the time in- nonflagellated centriole to the kinetosome (0 = parallel, 1 = variant model (TIM + I + G). Four simultaneous Markov angled but less than 908, 2 = orthogonal), (iii) microtubular chains were run over 1 million generations. Trees were root (0 = absent, 1 = in cross section near the kinetosome sampled every 100 generations with an overall sampling of six to eight microtubules visible with no space in between 10 001 trees. From these, the first 100 trees were discarded and in longitudinal section aligned in a cord-like arrange- (burnin = 100, by which time the average standard deviation ment (Barr and Hartmann 1976; Barr 1981) extending from of split frequencies had declined to 0.008). A consensus of the side of the kinetosome to the base of the MLC cisterna, 9901 remaining trees was used to compute a majority rule 2 = microtubules radiate anteriorly from region around the consensus tree to obtain estimates for the a posteriori proba- base of the kinetosome), (iv) kinetosome to nonflagellated bility of groups of isolates. centriole connection (0 = absent, 1 = a fibrous bridge con- nects the kinetosome to the nonflagellated centriole, and the Transmission electron microscopy bridge is most dense in the posterior and anterior regions of We examined the zoospore ultrastructure of 17 isolates the kinetosome with few cross-bridging fibers along the cen- from among the 27 isolates used in this study (Table 1). Fix- tral areas of the kinetosome and nonflagellated centriole ation and observation of zoospores of 10 isolates followed (Barr and Hartmann 1976), 2 = uniformly dense cross bridg- procedures described in Letcher and Powell (2005a). From ing fibers between the long axis of the kinetosome and the these 10 isolates and an additional seven isolates from pub- nonflagellated centriole, 3 = distal ends of cross bridging fi- lished works, 15 characters with multiple character states bers intersect and form a dense band parallel to the long axis were identified: (i) electron-opaque plug in the transition re- of the kinetosome), (v) an electron-opaque veil on the side gion of the flagellum (0 = absent, 1 = present), (ii) angle of of the nonflagellated centriole distal to the kinetosome (0 =

# 2005 NRC Canada 1564 Can. J. Bot. Vol. 83, 2005 absent, 1 = present), (vi) electron-opaque plates adjacent to Chytriomyces, Entophlyctis, Obelidium, Physocladia, Podo- the kinetosome (0 = absent, 1 = two stacks of two or three chytrium, Rhizoclosmatium, and Siphonaria) and five un- plates on each side of the lateral microtubule root, 2 = identified species. Sister to the Chytridiaceae was a group stacks of two or three plates on each side of the kinetosome, that included Phlyctochytrium planicorne and Polyphlyctis 3 = three-layered saddle-shaped structure covers anterior end unispina with 100% support; this clade we designate as of the kinetosome, 4 = striated disc), (vii) cell coat visible the Phlyctochytrium clade (Fig. 1). Sister to the Phlycto- over zoospore body but not flagellum (0 = absent, 1 = chytrium clade were two isolates of a single species, Chy- present), (viii) nuclear location (0 = not associated with the triomyces angularis, which grouped with 100% support and kinetosome, 1 = spatially associated with the kinetosome, 2 = that we designate as the Chytriomyces angularis clade connected to the kinetosome), (ix) ribosomal aggregation (0 = (Fig. 1). The LSU MP/Bayesian phylogeny was congruent absent, 1 = present), (x) organization of MLC (microbody – with the ultrastructure MP/Bayesian phylogeny (Fig. 1). lipid globule complex; Powell 1978) (0 = closely associ- Within the Chytridiaceae clade, several subclades were re- ated organelles, 1 = loosely associated organelles), (xi) lo- solved with strong support. One of these, which we desig- cation of Golgi apparatus and association with the nate the Chytriomyces clade (Fig. 1), contained the type microtubule rootlet (0 = absent, 1 = Golgi apparatus asso- species of Chytriomyces, Chytriomyces hyalinus, plus Chy- ciated with the microtubule root or in the posterior region triomyces confervae and two unidentified isolates of Chy- of the zoospore body, 2 = Golgi apparatus present but not triomyces. Assessment of the taxonomic status of the other associated with the microtubule root or in the posterior re- subclades will require analyses with larger taxon sam- gion of the zoospore body), (xii) paracrystalline inclusion plings. in the peripheral cytoplasm (0 = absent, 1 = present), (xiii) MLC cisterna (0 = absent, 1 = fenestrated cisterna Analyses of combined LSU and SSU rDNA sequences on the side of the lipid globule facing the plasma mem- The LSU and combined LSU–SSU data sets were congru- brane, 2 = simple cisterna on the side of the lipid globule ent, with no significant difference (p = 0.07). From 14 iso- facing the plasma membrane, 3 = fenestrated cisterna lates (Table 1), combined LSU–SSU rRNA gene sequences backed by microbodies), (xiv) position of mitochondria (0 = included 2512 characters of which 532 were parsimony in- absent, 1 = one or more mitochondria or mitochondrial lobes formative. In the MP/Bayesian phylogeny (’Fig. 2), the ma- inside the endoplasmic reticulum that delineates the ribosomal jority rule consensus tree was L = 1420 steps, CI = 0.618, core, 2 = mitochondria outside the endoplasmic reticulum and RI = 0.720. Topology of the MP tree was identical to that delineates the ribosomal core), and (xv) shape of the the topology derived from the Bayesian analysis. The Chy- zoospore (0 = variable, 1 = oval, 2 = spherical). Character tridiaceae clade and several of its subclades that were identi- states were coded for each isolate sampled (Table 2) and fied in the broader LSU analyses (Fig. 1) were also analyzed with MP and Bayesian analyses. Analysis of zoo- recovered with strong statistical support in the combined spore ultrastructural characters was compared with a phy- LSU–SSU trees (Fig. 2). logeny based on sequences of LSU rDNA (Fig. 1). Transmission electron microscopy Morphology For comparative analysis of thallus morphology, organ- Zoospore ultrastructural analysis isms were grown on modified PmTG agar and examined Of the 25 isolates (Table 1), zoospores of 10 isolates were with a Zeiss Nomarski differential interference contrast mi- examined ultrastructurally for the first time, and zoospores croscope. We recorded zoospore shape and size, presence or of six additional isolates had been examined previously absence of a subsporangial swelling, primary nucleus, (Barr and Hartmann 1976; Reichle 1972; Longcore 1992a, vesicle discharge, operculation, type of development, sub- 1992b, 1995; Letcher and Powell 2005b). In addition to strate, and habitat. these 16 isolates, details of the zoospore of an isolate of Rhizoclosmatium globosum (a different culture than our isolates) had been published (Barr and Hartmann 1976). Results We examined an isolate of Chytriomyces hyalinus (MP Molecular data analysis 004) (’Fig. 3a), and the zoospore of this isolate was a Group I type zoospore (Barr 1980). Additionally, JEL 186 Astero- LSU rDNA sequence analyses phlyctis sarcoptoides, JEL 59 Chytriomyces spinosus, JEL From 25 chytrid taxa (Table 1), LSU rRNA gene sequen- 129 Entophlyctis luteolus (ATCC 90967) (Longcore 1995), ces (743–883 residues in length) included 1428 characters of JEL 57 Obelidium mucronatum, JEL 30 Podochytrium den- which 400 were parsimony informative. In the MP phylogeny tatum (Longcore 1992a), JEL 06 and PL 06 Rhizoclosma- (Fig. 1), the majority rule consensus tree had length (L)= tium globosum (after Barr and Hartmann 1976), JEL 102 1571 steps, consistency index (CI) = 0.521, and retention Siphonaria petersenii, JEL 65 unidentified sp. A, and JEL index (RI) = 0.720. The Bayesian tree topology was identi- 103 unidentified sp. B (Miller 1968) had a Group I type zo- cal to the MP tree topology. Twenty-one isolates grouped ospore. Thus, all examined members of the Chytridiaceae in a clade with 100% bootstrap support and Bayesian a produced a Group I type zoospore. posteriori probability; this clade we designate as the Chy- Characteristic ultrastructural features identified in the tridiaceae (Fig. 1). The Chytridiaceae is equivalent to the Group I type zoospore (’Figs. 3a and 4–10) were (i) an elec- Chytridium clade of the Chytridiales (James et al. 2000); tron-opaque plug in the base of the flagellum (Figs. 4 and it included organisms from eight genera (Asterophlyctis, 10), (ii) a fenestrated cisterna ( = rumposome) on the side

# 2005 NRC Canada Letcher et al. 1565

Table 2. Distribution of zoosporic ultrastructural character states (see Material and methods: Transmission electron microscopy) for organisms analyzed as provisional members of the Chytriomyces clade (clades from Fig. 1).

Clade and character state Chytriomyces Oedogoniomyces, Character Chytridiaceae Phlyctochytrium angularis Monoblepharidaceae 11 1 1 1 20 0 1 0 31 1 0 2 41 2 3 2 51 0 0 0 61 2 0 4 71 1 0 0 80 0 0 0 91 1 1 1 10 0 0 0 1 11 1 2 0 0 12 1 1 0 0 13 1 1 2 3 14 1 2 2 2 15 1 0 1 1 of the lipid globule facing the plasma membrane (Figs. 4, 7, members of the Chytridiales and not unique to the Group I and 8), (iii) a microtubule root, which in cross section shows type zoospore. six to eight microtubules aligned in a cord-like arrangement (Fig. 5) (Barr and Hartmann 1976; Barr 1981) extending Morphology from the side of the kinetosome to the base of the fenes- The Chytridiaceae contains species with a range of types trated cisterna, (iv) a fibrillar bridge connecting the kineto- of development and thallus features (’Figs. 11–23), with en- some to the nonflagellated centriole and the bridge most dogenous development predominating (Figs. 11–22), but dense in the posterior and anterior regions of the kineto- also with exogenous development by Entophlyctis luteolus some, with few cross-bridging fibers along the central areas (Longcore 1995) and polycentric development by Physocla- of the kinetosome and nonflagellated centriole (Figs. 5 and dia obscura (Fig. 23). The thallus morphology of Chytrio- 10) (Barr and Hartmann 1976), (v) an electron-opaque veil myces hyalinus, Chytriomyces confervae, and two on the side of the nonflagellated centriole distal to the kinet- unidentified Chytriomyces spp. was similar (e.g., Fig. 11, osome (Fig. 5), (vi) a Golgi apparatus associated with the isolate MP 004), although variation existed in sporangial microtubule root (Fig. 4), (vii) a pair of stacked, flat, elec- size and rhizoid morphology. Isolates of Chytriomyces ap- tron-opaque plates adjacent to the kinetosome and lateral pendiculatus (Fig. 20, isolate JEL 165) had similar thallus microtubule root (Fig. 5), (viii) one or more mitochondria morphology, with a multilobed sporangium. Outside of the inside the endoplasmic reticulum that delineates the riboso- Chytridiaceae, the morphology of the isolates of Chytriomy- mal core (Fig. 4), (ix) a paracrystalline inclusion in the pe- ces angularis was similar (Fig. 26, isolate PL 70). Their ripheral cytoplasm (Fig. 9), and (x) a conspicuous cell coat thread-like rhizoidal system, in which the first branch is al- that covers the plasma membrane but not the flagellar sheath ways at a distance from the sporangium, distinguished them (Figs. 4 and 6) (Dorward and Powell 1982, 1983). from species in the Chytridiaceae and the Phlyctochytrium The two isolates of the Phlyctochytrium clade (Fig. 1), clade. Both Phlyctochytrium planicorne (Fig. 24) and Poly- JEL 47 Phlyctochytrium planicorne and PL AUS 026 Poly- phlyctis unispina (Fig. 25) of the Phlyctochytrium clade had phlyctis unispina, have zoospores similar to the Group II distinct subsporangial swellings, and this character also was type zoospore (Fig. 3b) (Barr and Hartmann 1976; Barr shared by some taxa in the Chytridiaceae (e.g., Fig. 12, As- 1980). The only potential difference in the zoospores of terophlyctis sarcoptoides, and Fig. 15, Obelidium mucrona- these two species and the Group II type is the structure of tum). the electron-opaque plates adjacent to the kinetosome, which We observed a range of morphologies, zoospore discharge will require analyses of serial sections to resolve (Letcher mechanisms, substrates, habitats, and types of development and Powell 2005b). Zoospores of isolates in the Phlyctochy- in the Chytridiaceae. The zoospores of most isolates were trium clade are distinct from those of examined isolates in ovoid when motile and did not vary remarkably in size. the Chytridiaceae, sharing only 60% of the scored character Both operculate and inoperculate sporangia were present. states (Table 2). All members of the family that we examined had vesicular Both isolates of the Chytriomyces angularis clade (Fig. 1) zoospore discharge. We did not observe discharge in Physo- have a Group V type of zoospore (Fig. 3c, isolate PL 70) cladia because, in culture, it does not form zoosporangia. (Longcore 1992b). The Group V type zoospores shared only However, on bait in gross culture, Physocladia obscura also 33% of the character states (Table 2) with Group I type zo- has vesicular discharge (Sparrow 1960). We noted a primary ospores, the majority of which are common characters in nucleus (Karling 1947) in isolates in the subclade of the

# 2005 NRC Canada 1566

Fig. 1. Majority rule consensus tree from maximum parsimony analysis of partial nuclear LSU rRNA gene sequences of 25 taxa in the Chytridiales mapped against a maximum parsimony phylogeny derived from zoospore ultrastructural characters. Values are bootstrap values (1000 replicates)/Bayesian inference estimates for a posteriori probabilities. LSU phylogeny tree length (L) = 1571 steps, consistency index (CI) = 0.521, retention index (RI) = 0.720. Members of the Monoblepharidaceae were used as the outgroup in both LSU and ultrastructure analyses. Ultrastructure features of Rhizoclosmatium globosum from Barr and Hartmann (1976) and of Oedogoniomyces sp. from Reichle (1972). Bars below phylogenies indicate substitutions per site (LSU phylogeny: 0.05; ultrastructure phylogeny: 0.01). a.J o.Vl 3 2005 83, Vol. Bot. J. Can. # 05NCCanada NRC 2005 Letcher et al. 1567

Fig. 2. Majority rule consensus tree from maximum parsimony analysis of combined partial SSU and LSU rRNA gene sequences of 14 taxa sampled from the clades resolved in Fig. 1. First values above branches are bootstrap values (1000 replicates); second values are estimates for a posteriori probabilities. L = 1420 steps, CI = 0.618, RI = 0.720. Outgroup taxa are members of the Monoblepharidaceae.

Chytridiaceae that included Siphonaria petersenii, Obeli- Taxonomy dium mucronatum, Podochytrium dentatum, Podochytrium Chytridiaceae Nowak. (1878, pp. 174, 191); emend. sp., Rhizoclosmatium globosum, and Chytriomyces appendi- D.J.S. Barr (1980, p. 2388); emended as follows. culatus and isolates JEL 65 and JEL 103. In comparison Thallus eucarpic, monocentric or polycentric; sporangium with the Chytridiaceae, both members of the Phlyctochy- endogenous or exogenous to the zoospore cyst, operculate or trium clade were inoperculate, and these isolates also had inoperculate; rhizoids endobiotic or interbiotic. Resting vesicular discharge. Both isolates of Chytriomyces angularis spore endogenous or exogenous; Group I type zoospore were operculate and lacked vesicular discharge. (Barr 1980). Note: Chytridiaceae de Bary & Woronin, Ber. The majority of species in the Chytridiaceae were isolated Verh. Nat. Ges. Freib. 3(2): 45 (1865), nom. inval.; see from material from aquatic habitats, although several species CABI Bioscience databases, http://www.indexfungorum.org/ have been recovered from both aquatic and soil habitats. Or- Names/familyrecord. ganisms that grouped within subclades (Fig. 1) tended to use Molecular analyses separated the Chytridiaceae from the similar substrates; however, in one subclade, the isolates Phlyctochytrium clade (Fig. 1). Our emended circumscrip- varied in their substrate, with Chytriomyces confervae and tion of the Chytridiaceae includes only taxa with the Group two Chytriomyces spp. occurring in water with algae and I type zoospore and excludes taxa with a Group II type or Chytriomyces hyalinus in soil on chitinous substrates and Group III type zoospore. It expands thallus developmental pollen. All members of the subclade containing Chytriomy- types to include polycentric thalli and retains Barr’s (1980) ces appendiculatus, Obelidium mucronatum, Podochytrium expanded emendment that incorporated taxa with sporangia dentatum, Rhizoclosmatium globosum, Siphonaria petersenii, that are operculate or inoperculate. and unidentified spp. A, B, and C were saprobes of chitinous substrates, whereas a subclade consisting of Chytrio- myces spinosus, Entophlyctis luteolus, Physocladia obscura, Discussion and unidentified sp. D were saprobes of cellulosic sub- The Chytridiales (Barr 1980) is the largest order in the strates. Chytridiomycota and is much in need of taxonomic revi-

# 2005 NRC Canada 1568 Can. J. Bot. Vol. 83, 2005

Fig. 3. Schematic drawings of longitudinal sections of a Group I type zoospore (isolate MP 004) (a), Group II type zoospore (Chytridium lagenaria; Barr and Hartmann 1976) (b), and Group V type zoospore (isolate PL 70) (c) illustrating major characters and character states diagnostic of each zoospore type. CC, cell coat; EOF, electron-opaque region of the flagellum; EOP, electron-opaque plates; ER, endoplasmic reticulum; F, flagellum; FB, fibrillar bridge; FC, fenestrated cisterna (=rumposome); G, Golgi apparatus; K, kinetosome; L, lipid globule; M, mitochondrion; Mb, microbody; Mt, microtubule root; N, nucleus; Nu, nucleolus; NfC, nonflagellated centriole; P, flagellar prop; PCI, paracrystalline inclusion; R, ribosomal aggregation; SC, simple cisterna; TP, terminal plate; V, veil; Vac, vacuole; Ve, vesicle.

(a) (b)

(c)

# 2005 NRC Canada Letcher et al. 1569

Figs. 4–10. Ultrastructural features of the zoospore of the type I zoospore shown here in Siphonaria petersenii JEL 102 (Fig. 4) and Chy- triomyces hyalinus (Figs. 5–10). Fig. 4. Longitudinal section illustrating the typical arrangement of organelles. Fig. 5. Cross section through the kinetosome (K) and nonflagellated centriole (NfC) illustrating two stacks of three electron-opaque plates (EOP) each on opposite sides of the microtubule root (Mt); cross section of the microtubule root showing 10 tightly packed microtubules (inset). The fibrillar bridge (FB) in cross-section is composed of intersecting fibers. Fig. 6. Cell coat (CC) (arrow) exterior and adjacent to the plasma membrane. Figs. 7 and 8. Fenestrated cisterna (rumposome) appressed to a lipid globule (L): tangential section (Fig. 7) and cross section (Fig. 8). Fig. 9. Paracrys- talline inclusion: longitudinal section and cross section (inset). Fig. 10. Longitudinal section through a portion of the flagellar apparatus. Notice the characteristic fibrillar bridge connecting the kinetosome to the nonflagellated centriole. The bridge is most dense in the posterior and anterior regions of the kinetosome, with few cross-bridging fibers along the central areas of the kinetosome and nonflagellated centriole, leaving that area electron transparent. Scale bars = 0.5 mm for Figs. 5–9 and 0.2 mm for Fig. 10.

sions. Although increasingly larger data sets of zoospore Letcher and Powell 2005a). Molecular sequence data sets of characters and ribosomal gene sequences have become chytrids have been primarily used to understand deep branch available, authors have made limited use of the information phylogenetic relationships, and one study emphasizing the to change taxonomic classification (Longcore et al. 1995; Chytridiales identified four monophyletic lineages in this or-

# 2005 NRC Canada 1570 Can. J. Bot. Vol. 83, 2005

Figs. 11–26. Illustrations of thalli of 13 isolates placed in the Chytridiaceae and three isolates that are not in this family. Fig. 11. Chytrio- myces hyalinus, MP 004. Fig. 12. Asterophlyctis sarcoptoides, JEL 186. Fig. 13. Siphonaria petersenii, JEL 102. Fig. 14. Unidentified sp. A, JEL 65. Fig. 15. Obelidium mucronatum, JEL 57. Fig. 16. Unidentified sp. B, JEL 103. Fig. 17. Podochytrium dentatum, JEL 30. Fig. 18. Podochytrium sp., JEL 161. Fig. 19. Rhizoclosmatium globosum, JEL 06. Fig. 20. Chytriomyces appendiculatus, JEL 165. Fig. 21. Chytrio- myces spinosus, JEL 59. Fig. 22. Unidentified sp. D, JEL 220. Fig. 23. Physocladia obscura, JEL 137. Fig. 24. Phlyctochytrium planicorne, JEL 47. Fig. 25. Polyphlyctis unispina, PL AUS 26. Fig. 26. Chytriomyces angularis, PL 70. pn, primary nucleus; ss, subsporangial swelling. Scale bar = 10 mm for all figures.

der (James et al. 2000). Our study is the first to integrate ul- Phlyctochytrium and Chytriomyces angularis clades are out- trastructural and molecular data sets to circumscribe one of side of the Chytridiaceae on relatively long branches, and their these lineages (the Chytridium clade) as the Chytridiaceae. positions may be the effect of long-branch attraction (Dacks et al. 2000; Berbee et al. 2000). Determining their position Molecular analyses within the Chytridiales will need additional taxon sampling. Inferred phylogenies derived from molecular and ultrastructural characters were congruent. Our results corroborate Zoospore ultrastructure those of James et al. (2000) who found that zoospore ultrastructure predicts molecular phylogeny and vice versa. Our The Group I type zoospore analyses show with statistical support that members in the All isolates in the Chytridiaceae clade examined had a

# 2005 NRC Canada Letcher et al. 1571

Group I type zoospore. Discovering this zoospore type in a polycentric Physocladia obscura in the Chytridiaceae. broad range of isolates and genera indicates the conservative Phlyctorhiza endogena, which was not represented in our nature of the Group I type zoospore among species with di- study, is also in the Chytridiaceae based on its position in vergent thallus forms. Possession of a Group I type zoospore the Chytridium clade (James et al. 2000). is sufficient to predict that an isolate is a member of the We included the type species for five genera: Astero- emended Chytridiaceae, and in the emendment of the fam- phlyctis sarcoptoides, Chytriomyces hyalinus, Obelidium ily, the Group I type zoospore is diagnostic. mucronatum, Physocladia obscura, and Rhizoclosmatium globosum, in our molecular analyses. Thus, we expect the The Group II type zoospore placement of these genera in the family to be stable. Phlyc- Phlyctochytrium planicorne (Letcher and Powell 2005b) torhiza endogena is also the type of the genus, and on the and Polyphlyctis unispina comprised the Phlyctochytrium basis of its 18S rDNA sequences (James et al. 2000), we ex- clade in the LSU phylogeny, and both had zoospores similar pect this genus also to remain in the family. Placement of to the Group II type zoospore (Barr 1980). Their zoospores other genera now included in the family (Entophlyctis, Po- exhibited minor ultrastructural differences from Barr’s dochytrium, and Siphonaria) is based on molecular analyses Group II type zoospore description, primarily in the mor- of species other than the type and will ultimately be verifi- phology of the electron-opaque saddle-like structure adja- able only after the types are cultured and analyzed molecu- cent to the kinetosome. We could not find a viable culture larly and ultrastructurally. of Barr’s Chytridium lagenaria in any culture repository; Chytridium olla is the type for the genus Chytridium thus, molecular and detailed ultrastructural comparisons are (Sparrow 1973)and for the family Chytridiaceae. Inclusion not possible. Chytridium lagenaria, Phlyctochytrium plani- of Chytridium olla in the Chytridiaceae clade and retention corne, and Polyphlyctis unispina are the only organisms re- of the family name Chytridiaceae are based on Barr and ported thus far to have a Group II type zoospore; however, Hartmann’s (1976) and Lange and Olson’s (1979) reports of further sampling in these and related genera may reveal ad- a Group I type zoospore in Chytridium olla from electron ditional occurrences. Because of its different zoospore mor- microscopic studies of cultures (Barr and Hartmann 1976: phology and the relationships suggested by the molecular IMI 86666; Lange and Olson 1979: CBS 192.69). We are analyses, we separate organisms with a Group II type of zoo- not able to confirm the identity of these cultures or analyze spore out of the Chytridiaceae. these cultures molecularly because these isolates are no lon- ger viable in culture collections and no micrographs of thal- The Group V-type zoospore lus morphology of these isolates were published. However, Two isolates of Chytriomyces angularis (JEL 45 and PL at this time, the published evidence (Barr and Hartmann 70) have a zoospore distinctively different from the Group I 1976; Lange and Olson 1979) is that the emended family type and Group II type zoospores. Chytriomyces angularis characterized by a Group I type zoospore includes Chytri- was classified as a member of the genus by virtue of being dium olla, the type for the family. a monocentric, operculate species in the Chytridiales exhib- Our analyses suggest that some isolates included in our iting endogenous development and an epibiotic resting spore phylogenetic hypotheses represent new genera in the Chytri- (Longcore 1992b). Its distinctive zoospore and its placement diaceae. Members identified as species of Chytriomyces oc- in our phylogenetic trees and those of James et al. (2000) curred in four lineages. Both Chytriomyces appendiculatus segregate Chytriomyces angularis from the Chytridiaceae and Chytriomyces spinosus grouped outside the subclade and additionally suggest that this species needs to be placed that contained Chytriomyces hyalinus, the type species. in a different genus. These organisms are clearly within the Chytridiaceae but may represent new genera. Chytriomyces angularis falls out- Taxonomy side the Chytridiaceae clade entirely. Therefore, the genus We emend Barr’s concept of the Chytridiaceae on the ba- Chytriomyces (Letcher and Powell 2002) is polyphyletic sis of zoospore ultrastructure, molecular analyses, and thal- and in need of revision. lus morphology. We include in the family only taxa with a Evidence now suggests that lineages included in the Chy- Group I type zoospore and exclude taxa with a Group II tridiales exhibit different constraints in the evolution of type or Group V type zoospore. Our molecular analyses character states or reflect differences in the time for evolu- clearly separate the Chytridiaceae (characterized by the tionary change. As a consequence, the level at which ultra- Group I type zoospore) from the Phlyctochytrium clade structural and molecular characters are useful in (characterized by the Group II type zoospore) (see Letcher classification varies in different lineages. In the Chytridia- and Powell 2005b) and the Chytriomyces angularis clade, ceae lineage, evolution of zoospore ultrastructural character the latter clade representing a fifth lineage in the Chytri- states is conserved, whereas thallus morphology is divergent. diales. Species in Rhizophydium were in the Chytridiaceae Therefore, we believe that zoospore ultrastructure is taxo- sensu Barr (Barr 1980); however, published molecular stud- nomically applicable at the family level in this lineage. In ies clearly separate the Chytridiaceae, as we have emended contrast, in the Rhizophydium lineage, evolution of zoospore it, from the Rhizophydium clade, which is characterized by character states is divergent (Barr 1978; Barr and Hadland- a Group III type zoospore (James et al. 2000; Letcher et al. Hartmann 1978; Letcher et al. 2004; Letcher and Powell 2004). Barr (1980) accepted monocentric, endogenously de- 2005a), and evolution of thallus character states is con- veloping members of the Chytridiales into the Chytridia- strained. In the Rhizophydium lineage, we believe that zoo- ceae; however, our results from analyses of LSU sequences spore ultrastructural character states are applicable at the and those of James et al. (2000) with the SSU place the genus level (Letcher and Powell 2005a). It is clear in both

# 2005 NRC Canada 1572 Can. J. Bot. Vol. 83, 2005 lineages that analyses of molecular sequences will be neces- inference of phylogenetic trees. Bioinformatics, 17: 754–755. sary to resolve species. doi: 10.1093/bioinformatics/17.8.754. James, T.Y., Porter, D., Leander, C.A., Vilgalys, R., and Longcore, Acknowledgments J.E. 2000. Molecular phylogenetics of the Chytridiomycota sup- ports the utility of ultrastructural data in chytrid systematics. This study was supported by the National Science Foun- Can. J. Bot. 78: 336–350. doi: 10.1139/cjb-78-3-336. dation through PEET grant DEB-9978094, the Department Karling, J.S. 1947. New species of Chytriomyces. Bull. Torrey Bot. of Biological Sciences Aquatic Ecology and Systematics Club, 74: 334–344. Graduate Enhancement Program, The University of Ala- Karling, J.S. 1977. Chytridiomycetarum Iconographia. Lubrecht bama, a Howard Hughes Medical Institute Undergraduate and Cramer, Monticello, N.Y. Biological Sciences Program Grant to The University of Lange, L., and Olson, L.W. 1979. The uniflagellate phycomycete Alabama, and a scholarship from the Alabama Power Com- zoospore. Dan. Bot. Ark. 33: 41–43. pany. We express our appreciation to the Assembling the Letcher, P.M., and Powell, M.J. 2002. A taxonomic summary of Fungal Tree Of Life (AFTOL) project, Duke University, for Chytriomyces (Chytridiomycota). Mycotaxon, 84: 447–487. access to their database and to Phillip Dean and Rachel Gil- Letcher, P.M., and Powell, M.J. 2005a. Kappamyces, a new genus lis for assistance with DNA isolation and sequencing. in the Chytridiales (Chytridiomycota). Nova Hedwigia, 80: 115– 133. doi: 10.1127/0029-5035/2005/0080-0115. References Letcher, P.M., and Powell, M.J. 2005b. Phylogenetic position of Barr, D.J.S. 1978. Taxonomy and phylogeny of chytrids. Biosys- Phlyctochytrium planicorne (Chytridiales, Chytridiomycota) tems, 10: 153–165. doi: 10.1016/0303-2647(78)90037-0. based on zoospore ultrastructure and partial nuclear LSU rRNA Barr, D.J.S. 1980. An outline for the reclassification of the Chytri- gene sequence analysis. Nova Hedwigia, 80: 135–146. doi: 10. diales, and for a new order, the Spizellomycetales. Can. J. Bot. 1127/0029-5035/2005/0080-0135. 58: 2380–2394. Letcher, P.M., Powell, M.J., Chambers, J.G., and Holznagel, W.E. Barr, D.J.S. 1981. The phylogenetic and taxonomic implications of 2004. Phylogenetic relationships among Rhizophydium isolates the flagellar rootlet morphology among zoosporic fungi. Biosys- from North America and Australia. Mycologia, 96: 1363–1375. tems, 14: 359–370. doi: 10.1016/0303-2647(81)90042-3. Longcore, J.E. 1992a. Morphology, occurrence, and zoospore ultra- Barr, D.J.S. 2001. Chytridiomycota. In The Mycota VII, Part A: structure of Podochytrium dentatum sp. nov. (Chytridiales). My- Systematics and evolution. Edited by D.J McLaughlin, cologia, 84: 183–192. E.G. McLaughlin, and P.A. Lemke. Springer-Verlag, Berlin. Longcore, J.E. 1992b. Morphology and zoospore ultrastructure of pp. 93–112. Chytriomyces angularis sp. nov. (Chytridiales). Mycologia, 84: Barr, D.J.S., and Hadland-Hartmann, V.E. 1978. Zoospore ultra- 442–451. structure in the genus Rhizophydium (Chytridiales). Can. J. Bot. Longcore, J.E. 1993. Morphology and ultrastructure of Lacustro- 56: 2380–2404. myces hiemalis gen. et sp. nov. (Chytridiales). Can. J. Bot. 71: Barr, D.J.S., and Hartmann, V.E. 1976. Zoospore ultrastructure of 414–425. three Chytridium species and Rhizoclosmatium globosum. Can. Longcore, J.E. 1995. Morphology and zoospore ultrastructure of J. Bot. 54: 2000–2013. Entophlyctis luteolus sp. nov. (Chytridiales): implications for Batko, A. 1975. Zarys Hydromikologii. Pan´stwowe Wydawnictwo chytrid taxonomy. Mycologia, 87: 25–33. Naukowe, Warsaw. Longcore, J.E., Barr, D.J.S., and De´saulniers, N. 1995. Powello- Ben Ali, A.B., Wuyts, J., De Wachter, R., Meyer, A., and Van de myces, a new genus in the Chytridiales. Can. J. Bot. 73: 1385– Peer, Y. 1999. Construction of a variability map for eukaryotic 1390. large subunit ribosomal DNA. Nucleic Acids Res. 27: 2825– Lucarotti, C.J. 1981. Zoospore ultrastructure of Nowakowskiella 2831. doi: 10.1093/nar/27.14.2825. elegans and Cladochytrium replicatum. Can. J. Bot. 59: 137– Berbee, M.L., Carmean, D.A., and Winka, K. 2000. Ribosomal 148. DNA and resolution of branching order among the Ascomycota: Miller, C.E. 1968. Observations concerning taxonomic characteris- how many nucleotides are enough? Mol. Phylogenet. Evol. 17: tics in chytridiaceous fungi. J. Elisha Mitchell Sci. Soc. 84: 337–344. 100–107. Chambers, J.G. 2003. Ribosomal DNA, secondary structure, and Miller, C.E. 1976. Substrate influenced morphological variations phylogenetic relationships among the Chytridiomycota. and taxonomic problems in freshwater, posteriorly uniflagellate Ph.D. dissertation, University of Alabama, Tuscaloosa, Ala. phycomycetes. In Recent advances in aquatic mycology. Edited Dacks, J.B., Marinets, A., Doolittle, W.F., Cavalier-Smith, T., and by E.B.G. Jones. Elek Science, London, U.K. pp. 459–487. Logsdon, J.M. 2000. Analyses of RNA polymerase II genes Nowakowski, L. 1878. Przyczynek do morfologii i systematyki from free-living protists: phylogeny, long branch attraction and Skoczko´w (Chytridiaceae). Akad. umiejetnosˇci Krakowie. Wyd- the eukaryotic big bang. Mol. Biol. Evol. 19: 830–840. zı´at mat.-przyo´d., Pamietnik, 4: 174–198. de Bary, A., and Woronin, M. 1865. Beitrag zur Kenntnis der Chy- Posada, D., and Crandall, K.A. 1998. MODELTEST: testing the trideen. Ber. Naturforsch. Ges. Freib. Breisgau, 3: 21–61. model of DNA substitution. Bioinformatics, 14: 817–818. Dorward, D.W., and Powell, M.J. 1982. Cross-linking bridges asso- Powell, M.J. 1978. Phylogenetic implications of the microbody-li- ciated with the microbody – lipid globule complex in Chytrio- pid globule complex.. BioSystems, 10: 167–180. myces aureus and Chytriomyces hyalinus. Protoplasma, 112: Powell, M.J., and Koch, W.J. 1977a. Morphological variations in a 181–188. doi: 10.1007/BF01284092. new species of Entophlyctis. I. The species concept. Can. J. Bot. Dorward, D.W., and Powell, M.J. 1983. Cytochemical detection of 55: 1668–1685. polysaccharides and the ultrastructure of the cell coat of zoos- Powell, M.J., and Koch, W.J. 1977b. Morphological variations in a pores of Chytriomyces aureus and Chytriomyces hyalinus. My- new species of Entophlyctis. II. Influence of growth conditions cologia, 75: 209–220. on morphology. Can. J. Bot. 55: 1686–1695. Huelsenbeck, J.P., and Ronquist, F. 2001. MRBAYES: Bayesian Reichle, R.E. 1972. Fine structure of Oedogoniomyces zoospores,

# 2005 NRC Canada Letcher et al. 1573

with comparative observations on Monoblepharella zoospores. Van de Peer, Y., Jansen, J., De Rijk, P., and De Wachter, R. 1997. Can. J. Bot. 50: 819–824. Data base on the structure of small ribosomal subunit RNA. Nu- Roane, M.K., and Paterson, R.A. 1974. Some aspects of morphol- cleic Acids Res. 25: 111–116. doi: 10.1093/nar/25.1.111. ogy and development in the Chytridiales. Mycologia, 64: 147– Whiffen, A.J. 1944. A discussion of taxonomic criteria in the Chy- 164. tridiales. Farlowia, 1: 583–597. Sparrow, F.K. 1960. Aquatic Phycomycetes. 2nd ed. University of White, T.J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification Michigan Press, Ann Arbor, Mich. and direct sequencing of fungal ribosomal genes for phyloge- Sparrow, F.K. 1973. The type of Chytridium olla A. Braun. Taxon, netics. In PCR protocols: a guide to methods and applications. 22: 583–586. Edited by M. Innis, D. Gelfand, J. Sninsky, and T. White. Aca- Swofford, D.L. 2002. PAUP*: phylogenetic analysis using parsi- demic Press, San Diego, Calif. pp. 315–322. mony (and other methods). Version 4.0b10. Sinauer Associates, Sunderland, Mass.

# 2005 NRC Canada