Dendrochytridium crassum gen. et sp. nov., a taxon inChytridiales with unique zoospore ultrastructure

Peter M. Letcher1

Department of Biological Sciences, University ofAlabama, Tuscaloosa, Alabama 35487

Joyce E. LongcoreSchool of Biology and Ecology, University of Maine,Orono, Maine 04469

Martha J. PowellDepartment of Biological Sciences, University ofAlabama, Tuscaloosa, Alabama 35487

Abstract: A water culture of detritus collected froman Australian tree canopy yielded multiple isolates(designated JEL 352, JEL 353, JEL 354) of anunidentified chytrid that grew on pollen bait andencysted spores of a Dictyuchus sp. oomycete. Mor-phological information from JEL 352 and geneticinformation from JEL 354 of this unidentified chytridhave been in several publications but the organismhas not been named. Because isolates JEL 352 andJEL 354 are no longer viable, we sequenced partialSSU and LSU rDNA of isolate JEL 353, documentedits thallus morphology with light microscopy anddetermined its zoospore ultrastructure via transmis-sion electron microscopy. DNA evidence placed JEL353 in Chytridiaceae, and its genetic composition wasidentical to that of JEL 354. Thallus morphology ofJEL 353 was similar to that of JEL 352. Its zoosporeultrastructure is less complex compared to othermembers of Chytridiaceae. In pure culture, therhizoidal system differed from other members of thefamily in being unevenly broad and not tapering tofine tips. Based on genetic, morphological andultrastructural evidence, we place this chytrid in anew genus in Chytridiaceae and describe it as the newspecies Dendrochytridium crassum.

Key words: chytrid, Chytridiaceae, sampling, sys-tematics, taxonomy

INTRODUCTION

Molecular phylogenetic and transmission electronmicroscopy (TEM) methods have given mycologiststhe tools needed to name and classify chytrids in amanner that reflects their relatedness far better thandid characters determined from light microscopy.

Based on these tools, four orders (Letcher et al. 2006,Simmons et al. 2009, Mozley-Standridge et al. 2009,Longcore and Simmons 2012) have been separatedout of what was once the large and confusingChytridiales (sensu Barr 1980), and one order hasbeen segregated from the Spizellomycetales (Letcheret al. 2008). Only a fraction of described chytridio-mycete species have had their ordinal and genustaxonomy verified on the basis of molecular and TEMcharacters. Although the majority of molecularlyuncharacterized chytrid species are not yet in publicculture collections, some isolates are in culture and inthe literature that have not been ascribed to anexisting genus or species. One such chytrid isrepresented by three putatively identical isolates(JEL 352, JEL 353, JEL 354) that were retrieved frompollen bait placed in a water culture of detrituscollected from a tree canopy (Longcore 2005).

This tree-canopy fungus has been in phylogeneticstudies and has been referred to as ‘‘unknownchytrid’’ (JEL 352; Longcore 2005), and, as JEL 354,as ‘‘Chytriomyces clade’’ (James et al. 2006), ‘‘Uniden-tified sp. H’’ (Picard et al. 2009) and ‘‘Unidentifiedsp. 4’’ (Velez et al. 2011). The organism is in theorder Chytridiales (James et al. 2006), which currentlycontains two families, Chytridiaceae and Chytriomy-cetaceae (Velez et al. 2011). In analyses of DNA datathe tree-canopy isolate occurs in a lineage that is sisterto both families (Picard et al. 2009) or in theChytridiaceae (Velez et al. 2011), although in bothinstances with little support. Herein we (i) analyze theposition of isolate JEL 353 based on partial small(SSU) and large (LSU) subunits of ribosomal DNAsequences, (ii) determine the ultrastructure of thezoospore and (iii) record light microscopic morphol-ogy and development. We also compare its rDNAsequences with those of JEL 354, and its morphologywith that of JEL 352. Based on our investigation, wefind isolates JEL 352, JEL 353 and JEL 354 to beconspecific and place this chytrid in the familyChytridiaceae as the new genus and species Dendro-chytridium crassum.

MATERIALS AND METHODS

Sampling and morphology.—We recovered isolate JEL 353from a sample of tree-canopy detritus from Australia(Longcore 2005) and maintained it on PmTG agar (Barr1986; 1 g peptonized milk, 1 g tryptone, 5 g glucose, 10 gagar, 1 L distilled water). To determine morphology and

Submitted 22 Apr 2013; accepted for publication 5 Jul 2013.1 Corresponding author. E-mail: letch006@bama.ua.edu

Mycologia, 106(1), 2014, pp. 145–153. DOI: 10.3852/13-134# 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

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development we spread zoospores on Cd nutrient agar(0.2 g peptonized milk, 0.4 g tryptone, 2 g cellobiose, 2 gsoluble starch, 10 g agar, 1 L distilled water) and photo-graphed developmental stages daily with a Spot RT digitalcamera mounted on an E400 Nikon microscope.

DNA extraction, purification, amplification and phyloge-netic analyses.—We examined 32 ingroup isolates inChytridiales (sensu Velez et al. 2011) and two outgroupisolates representing Monoblepharidales (sensu James et al.2006) (TABLE I). Partial SSU and partial LSU sequences forall ingroup and outgroup isolates were generated asdescribed by Letcher and Powell (2005a) or obtained fromGenBank. Phylogenetic analyses were performed as de-scribed by Velez et al. 2011. In summary, partial SSUsequences (843–965 bp) were combined with partial LSU

sequences (799–882 bp) and aligned with manual adjust-ments. Maximum parsimony (MP) trees were generatedwith PAUPRat (Sikes and Lewis 2001), and bootstrap valueswere generated as heuristic searches with 500 replicates,each with 10 random-addition replicates. Maximum likeli-hood (ML) phylogenetic trees were constructed as de-scribed in Velez et al. (2011). Modeltest 3.7 (Posada andCrandall 1998) was used to determine the best model ofbase substitution and GARLI 0.951 (Zwikl 2006) was used toassess maximum likelihood. Branch support was assessedwith 500 bootstrapping replicates.

Zoospore ultrastructure.—To induce zoospore discharge, 7 dold PmTG cultures of isolate JEL353 were flooded withsterile distilled water 30 min. For transmission electronmicroscopic observations, zoospore suspensions were fixed

TABLE I. Taxon sampling for phylogenetic analyses of 32 isolates in Chytridiales

GenBank accession nos.

Isolate/taxon 18S 28S

Ingroup:

ARG 066 Chytridium lagenaria FJ822964 FJ822969ARG 095 Odontochytrium milleri KC812606 JX905512ARG 100 Chytridium olla FJ822966 FJ822970ARG 109 Phlyctochytrium planicorne KC812607 JX905514ARG 112 Phlyctochytrium bullatum KC812608 JX905515ARG 113 Delfinachytrium mesopotamicum KC812609 JX905516ARG 116 Delfinachytrium mesopotamicum KC812610 JX905517ARG 117 Delfinachytrium mesopotamicum KC812611 JX905518ARG 123 Avachytrium platense KC812612 JX905521Barr 097 Chytriomyces hyalinus AY349032 AY439074JEL 006 Rhizoclosmatium globosum AY988506 AY439061JEL 030 Podochytrium dentatum AY349030 AY439061JEL 047 Phlyctochytrium planicorne DQ536473 AY439028JEL 057 Obelidium mucronatum AY988504 AY439071JEL 059 Chytriomyces spinosus AY988502 AY439073JEL 102 Siphonaria petersenii AY349036 AY439072JEL 103 Odontochytrium milleri AY349035 AY439064JEL 129 Entophlyctis luteolus AY988503 AY349094JEL 137 Physocladia obscura AY988505 AY439062JEL 165 Chytriomyces appendiculatus AY988034 AY439076JEL 186 Asterophlyctis sarcoptoides AY988500 AY439070JEL 221 Pseudorhizidium endosporangiatum DQ536484 DQ273834JEL 347 Rhizoclosmatium sp. AY601709 DQ273769JEL 353 Dendrochytridium crassum n. gen., n. sp. KC812613 KC812616JEL 378 Rhizidium sp. AY601709 DQ273832JEL 729 Chytriomyces annulatus KC812614 KC812617KP 013 Rhizidium phycophilum KC812615 FJ214802KP 061 Phlyctochytrium aureliae GU358606 GU358607MP 004 Chytriomyces hyalinus AY988501 DQ273836PL AUS 026 Polyphlyctis unispina FJ822965 AY988518PL 167A Chytridium lagenaria FJ804152 FJ804156WB 235A Unidentified sp. DQ536486 FJ822968

Outgroup:

CR 084 Oedogoniomyces sp. AY635839 DQ273804UCB-78-1 Monoblepharella mexicana AF164337 DQ273777

146 MYCOLOGIA

in 2.5% glutaraldehyde in 0.1 M s-collidine buffer, pH 7.4,stained in 1.0% osmium tetroxide in 0.1 M s-collidinebuffer, and in-bloc stained with saturated aqueous uranylacetate (Barr 1981, Letcher and Powell 2005b). Randomsections were stained with saturated uranyl acetate in 70%

ethanol for 8 min and lead citrate for 4 min and wereexamined on a Hitachi 7650 transmission electron micro-scope at 60 kV.

RESULTS

Phylogenetic analyses.—The combined data had 1901characters, with 622 parsimony- informative sites afteruninformative characters were excluded. For MPanalysis, of 1005 trees derived from PAUPRat, 946most parsimonious trees (L 5 2400 steps) were usedto compute a majority rule consensus tree (. 70%

branch support). For ML analysis, Modeltest indicat-ed that the most appropriate model of DNAsubstitution was the Tamura-Nei model with rates ofsubstitution among sites approximated by gammadistribution (TrN + G). MP and ML (2lnL 5

10447.17) phylogenies were identical with similar orequal support values at major nodes. The phylogeny ispresented (FIG. 1) with ML and MP bootstrap values.Within the phylogeny three major clades wererecovered (FIG. 1, Clades A–C). Clades were delineat-ed based on support values and the maximallyinclusive group with similar zoospore ultrastructure.The tree canopy chytrid (JEL 353) was a sister lineageto eight isolates of the Chytridiaceae (Clade A).Chytridiaceae contained the species Chytridium ollaBraun, C. lagenaria Schenk, Phlyctochytrium aureliaeAjello, P. bullatum Sparrow, P. planicorne G.F. Atk.and Polyphlyctis unispina (Paterson) Karling, allcharacterized by a Group II-type zoospore (Barr1980). Clade B, Chytriomycetaceae, contained 18isolates (FIG. 1), which are characterized by a Group I-type zoospore (Barr 1980). Clade C, family incertaesedis, contained Pseudorhizidium endosporangiatum(Karling) M.J. Powell, Letcher & Longcore (Powellet al. 2013) and Delfinachytrium mesopotamicum Velezand Letcher (Velez et al. 2013), each with anundetermined type zoospore.

Morphology.—On Cd nutrient agar, zoospores(FIG. 2A) encyst and begin a rhizoidal system within24 h (FIG. 2B). Development is endogenous andmonocentric with the zoospores encysting and thecyst expanding into a zoosporangium over 4 d(FIG. 2A–G). Uncrowded mature zoosporangia arespherical or oblate and , 25–32 mm diam, with oblateforms being , 20% wider than tall (FIG. 2D). Therhizoidal system extends from one area of thezoosporangium (FIG. 2B–D), and the rhizoidal baseis usually apophysate (a subsporangial swelling up to

10 mm diam; FIG. 2C, D) with smaller catenulations nearthe base. When mature, the length of the rhizoidal systemis slightly greater than the diameter of the zoosporangi-um. Rhizoids are unevenly expanded, producing a zig-zagappearance (FIG. 2C–E) and are , 1 mm diam with littletapering toward the tips (FIG. 2E). Zoospores are releasedthrough one to several inoperculate discharge pores(FIG. 2F), beginning by oozing out as a mass in one ormore evanescent vesicles (FIG. 2G). Fully developedzoospores are 3–3.5 mm diam with a , 15 mm longflagellum and usually possess a single oil droplet; they maybe elongate when first released (FIG. 2H) but arespherical (FIG. 2A) when in motion.

Zoospore ultrastructure.—Zoospores of isolate JEL 353are subspherical to elongate, 2–2.5 mm diam 3 2.5–5.0 mm long (FIGS. 3A, B; 4A, B). Not atypically thezoospores appear eccentric or irregular in profile(FIGS. 3A, 4B). Ribosomes are aggregated (FIG. 3A,B), although not delineated by endoplasmic reticu-lum, and the aggregation encloses the nucleus(FIG. 3A, B). Outside the ribosomal aggregationare multiple mitochondria (FIG. 3A, B) and amicrobody lipid-globule complex (FIG. 3A, C). Asimple cisterna is appressed to a portion of the lipidglobule. A paracrystalline inclusion , 0.13 mm incross section and 0.4 mm long (FIG. 3A, G–I), a Golgiapparatus and vesicles 0.3–0.4 mm diam (FIG. 3B, J)occur in the peripheral cytoplasm. The non-flagel-lated centriole is parallel to, or its anterior endslightly angled toward, the kinetosome (FIG. 3F),and the two are joined by a fibrillar bridge (FIG. 3F).Adjacent to the kinetosome is a kinetosome-associatedstructure (KAS), a spherical to subspherical electron-opaque globule 0.11–0.14 mm diam (FIGS. 3A, D, E; 4A,C, D). A three-layered electron-opaque flagellar plug isin the transition region of the flagellum (FIG. 3A, D,F), in which the anterior and posterior layers are moreelectron opaque than the central layer. No microtu-bular root and no obvious cell coat were observed.

TAXONOMY

Dendrochytridium Letcher, Longcore and M.J. Powell,gen. nov.

MycoBank MB803847Fungus saprobic. Thallus monocentric, eucarpic,

endogenous, white in mass culture; sporangium epibio-tic, rhizoids endobiotic. Sporangium spherical to oblatethroughout development. Rhizoids irregularly broad(0.5–1.0 mm) in culture. Resting spores not observed.Zoospores posteriorly uniflagellate, subspherical, eccen-tric or irregular upon release, becoming spherical;containing one large lipid globule covered by a simplecisterna and microbody; ribosomes aggregated, nucleus

LETCHER ET AL.: NEW CHYTRID FROM AUSTRALIA 147

embedded in ribosomal aggregation, mitochondriaoutside but partially embedded in ribosomal aggrega-tion; paracrystalline inclusion present in peripheralcytoplasm; kinetosome-associated structure spherical;flagellar plug composed of three layers.

Type: Dendrochytridium crassum Letcher, Longcore,and M.J. Powell

Etymology: Greek; dendro 5 tree, in recognition of the origin ofthe type isolate; chytridium recognizes the type genus of the order.

Dendrochytridium crassum Letcher, Longcore, andM.J. Powell, sp. nov. FIGS. 2, 3

MycoBank MB803848Type: Type specimen designated here (FIGS. 2, 3)

(morphology and ultrastructure of isolate JEL 353),

FIG. 1. Phylogram inferred from maximum likelihood analysis of 32 taxa in Chytridiales from a combined dataset of partialSSU and partial LSU rDNA sequences. Numbers at nodes are ML/MP bootstrap support values. ML 2lnL 5 10447.17; MP treelength 5 2400 steps. Clade A is Chytridiaceae with a Group II-type zoospore; Clade B is Chytriomycetaceae with a Group I-typezoospore; Clade C is incertae sedis with undetermined type zoospores.

148 MYCOLOGIA

and GenBank SSU rDNA sequence KC812613, LSUrDNA sequence KC812616.

Etymology: Latin; crassum 5 broad, referring to the widthof the rhizoids.

Specimen examined: AUSTRALIA, QUEENSLAND: Butch-er’s Creek, Australian Tropical Research Centre, 17u249S,145u439E, , 700 m. On pollen grains and encysted spores ofa Dictyuchus sp. oomycete, 15 Jun 2002, from tree-canopydetritus flooded with water.

DISCUSSION

In nature (cf. Longcore 2005, FIG. 3), D. crassumresembles other ‘‘little round chytrids’’. Unlike themany other endogenously developing chytrids with asingle rhizoidal axis and discharge of zoosporesthrough multiple pores, however, this chytrid isidentifiable when in pure culture because of thenature of its rhizoids. Other members of the

FIG. 2. Morphology of Dendrochytridium crassum. A–E, H. Phase contrast microscopy. F, G. Bright field microscopy.A. Spherical zoospores with single oil globule. B. One-day-old germling with single rhizoidal axis. C. Two-day-old thallus;arrow points to apophysis (subsporangial swelling). D. Three-day-old oblate thallus; arrow points to apophysis. E. Broadrhizoidal tips. F. Five-day-old thallus; arrowheads point to zoospore discharge papillae. G. Zoospores oozing from twodischarge sites (arrowheads) on a zoosporangium, in evanescent vesicles. H. Recently discharged zoospores withirregular shape, single lipid globule and shadowy area of aggregated ribosomes. Bars: A, E, H 5 10 mm; B, C, D, F, G 5

10 mm.

LETCHER ET AL.: NEW CHYTRID FROM AUSTRALIA 149

FIG. 3. Ultrastructural features of Dendrochytridium crassum. A. Longitudinal section (LS) illustrating nucleus within theribosomal aggregation, multiple mitochondria and lipid globule with an appressed microbody outside the ribosomalaggregation, a paracrystalline inclusion in the peripheral cytoplasm, a globular kinetosome-associated structure (KAS) (arrow)and an electron-opaque plug in the base of the flagellum. B. Transverse section (TS) with a vesicle and vacuole in theperipheral cytoplasm. C. Section through lipid globule and appressed microbody. D. LS with electron-opaque plug in base offlagellum and globular KAS lateral to kinetosome. E. TS through kinetosome and non-flagellated centriole, with an electron-opaque structure (arrow) adjacent to the kinetosome. F. LS through kinetosome and non-flagellated centriole, the twoconnected by a fibrillar bridge (arrow), and the three-layered electron-opaque plug in the base of the flagellum. G, H, I. LS(G) and TS (H, I) through paracrystalline inclusion; notice fibrillar linkers between structural elements (G, I). J. Vesicle withreticulate content. Bars: A, B, D, F 5 0.5 mm; C, E 5 0.35 mm; J 5 0.2 mm, G, H 5 0.1 mm; I 5 0.075 mm. Abbreviations: 3, 4. EOP5 electron-opaque plug, F 5 flagellum, FB 5 fibrillar bridge, G 5 Golgi apparatus, K 5 kinetosome, KAS 5 kinetosome-associated structure, L 5 lipid globule, M 5 mitochondrion, Mb 5 microbody, N 5 nucleus, NfC 5 non-flagellated centriole,PCI 5 paracrystalline inclusion, R 5 ribosomes, SC 5 simple cisterna, Vac 5 vacuole, Ves 5 vesicle.

150 MYCOLOGIA

Chytridiales and members of the Rhizophydialesin pure culture form rhizoids that taper to fine(# 0.5 mm) tips. The tips of D. crassum rhizoids, incontrast, are broader than 0.5 mm, a cutoff measure-ment suggested by Barr (1980) that helps identifyChytridiales when compared to Spizellomycetales.Unlike the Spizellomycetales, in which the rhizoidsare smoothly broad, D. crassum produces rhizoidsthat appear knotty and contorted.

In a survey of zoosporic fungi cultured fromorganic detritus from rainforest canopy, we recoveredthree putatively identical isolates (JEL 352, JEL 353,JEL 354) from the same baited water culture. IsolateJEL 352 was illustrated (Longcore 2005, FIGS. 47–53)and briefly described, but the culture subsequentlydied. Isolate JEL 354 was included in molecularphylogenies as ‘‘Chytriomyces clade’’ (James et al.2006) and ‘‘Unidentified sp. 4’’ (Velez et al. 2011)and nested in the ‘‘Chytriomyces clade’’ of theChytridiales (James et al. 2006) and as sister ofChytridiaceae (Velez et al. 2011). As with isolate JEL352, isolate JEL 354 subsequently ceased to grow.Consequently for our molecular, TEM and morpho-logical studies we used isolate JEL 353; its LSUsequence is identical to that of JEL 354. In ourphylogeny D. crassum nested as part of the currentlydefined Chytridiaceae, with the same genera (P.planicorne and C. annulatus) as it did in the Jameset al. (2006) phylogeny, which was based on theSSU+LSU+5.8S subunits of the rRNA operon. It mayhave grouped as a sister to Chytridiaceae in Velez etal. (2011) because that analysis included ITS sequenc-es as well as SSU and LSU; divergence of ITSsequences perhaps forced D. crassum out of thefamily.

Ultrastructural considerations.—Based on the molec-ular phylogenetic placement and zoospore ultrastruc-tural description of Chytridium olla (Velez et al. 2011),Chytridiales was established as a monophyletic order.The zoospores of isolates in Chytridiales are charac-terized by a unique suite of ultrastructural charactersand character states, which include a cisterna (oftenfenestrated) appressed to the lipid globule, anelectron-opaque plug in the base of the flagellum, amicrotubular root consisting of a bundle of 5–7microtubules that extends from the kinetosome to thefenestrated cisterna, a paracrystalline inclusion in theperipheral cytoplasm, a kinetosome-associated struc-ture (KAS) of varied morphology and a cell coat(Dorward and Powell 1983). The ‘‘Group I-typezoospore’’ ultrastructure (Barr 1980) characterizesthe Chytriomycetaceae, while ‘‘Group II-type zoo-spore’’ characterizes the Chytridiaceae. Although bothtypes have the overall unique suite of ultrastructural

FIG. 4. A. Schematic of medial longitudinal sectionthrough the zoospore of Dendrochytridium crassum. Perfo-rated circle superimposed on kinetosome indicates positionof kinetosome-associated structure (KAS) relative to thekinetosome, yet out of plane for this section. B. Transversesection through kinetosome and non-flagellated centriole,with KAS adjacent to the kinetosome. C. Longitudinalsection (90u to FIG. 4A) through the kinetosome, withadjacent KAS. D–F. Three typical profiles of elongate, first-released zoospores.

LETCHER ET AL.: NEW CHYTRID FROM AUSTRALIA 151

features characteristic of the Chytridiales, the twozoospore types differ, most notably in the configura-tion of the KAS. In the ‘‘classic’’ (sensu Barr 1980)Group I-type zoospore the KAS is a pair of three plateslateral to the kinetosome, whereas in the ‘‘classic’’Chytridiaceae Group II-type zoospore the KAS is ashield, cap- or half saddle-like structure over the topand one side of the kinetosome.

The family Chytriomycetaceae contains representativesfrom 10 genera (Asterophlyctis, Chytriomyces, Entophlyctis,Obelidium, Phlyctorhiza, Physocladia, Podochytrium, Rhizi-dium, Rhizoclosmatium, Siphonaria) and, with the excep-tion of Rhizidium phycophilum Picard, those taxa have aclassic Group I-type zoospore. Rhizidium phycophilum hasa reduced Group I-type of zoospore, having a simpleMLC cisterna that lacks fenestrations, no microtubularroot and no KAS (Picard et al. 2009).

The family Chytridiaceae currently contains Chytri-dium olla, C. lagenaria, Phlyctochytrium planicorne, P.bullatum, P. aureliae, Polyphlyctis unispina, Chytriomycesannulatus and D. crassum. Of these taxa, all have beenexamined ultrastructurally except C. annulatus. Mostisolates in Chytridiaceae examined for ultrastructuralfeatures (including P. bullatum unpubl data) have aclassic Group II-type zoospore (Barr 1980). Two excep-tions are D. crassum and Phlyctochytrium aureliae, thelatter of which has many Group II-type zoospore featuresbut differs remarkably in its KAS (a mass of electron-opaque material that surrounds the kinetosome andnon-flagellated centriole; Letcher et al. 2012). In D.crassum a microtubular root or cell coat are not present,and the paracrystalline inclusion is smaller than thatfound in other taxa in Chytridiaceae. We consider thezoospore of D. crassum to be a reduced Group II-typezoospore in which certain ultrastructural features havebeen simplified or lost (a process similar to that seen withthe zoospore of Rhizidium phycophilum in Chytriomyce-taceae). Although its zoospore ultrastructural configura-tion differs from that described for the order, weconsider D. crassum to be a member of Chytridiaceaebased on its molecular phylogenetic position and theprobability that its zoospore ultrastructure is reduced.

Two taxa in our phylogeny, Pseudorhizidiumendosporangiatum (Powell et al. 2013) and Delfinachy-trium mesopotamicum (Velez et al. 2013), are consid-ered incertae sedis, because the sampling in thatclade is limited and the ultrastructure of theirzoospores differ. However, the zoospores of Pseudo-rhizidium, Delfinachytrium and Dendrochytridiumshare an ultrastructural synapomorphy, a globularKAS. Because of its prominence in these three taxa,this character state deserves further investigation inChytridiaceae isolates. The zoospores of these incer-tae sedis taxa and other Chytridiaceae taxa differin the morphology of the flagellar plug, which is

two-layered in P. endosporangiatum and D. mesopota-micum, three-layered in D. crassum and one-layered inother Chytridiaceae taxa. Variations in ultrastructure,combined with divergence in genetic constitution,indicate that Chytridiaceae and the incertae sedislineage are under-sampled and further sampling maywell reveal additional diversity.

ACKNOWLEDGMENTS

This study was supported by the National Science Founda-tion through MRI DEB-0500766 and REVSYS DEB-00949305. We thank Dr Steve Stephenson (NSF INT0139547) for furnishing the samples from which Dendrochy-tridium crassum was isolated.

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