Mycologia
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DOI: 10.3852/mycologia.100.2.325
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Mycologia, 100(2), 2008, pp. 325-334.
© 2008 by The Mycological Society of America

Rediscovery of an unusual chytridiaceous fungus new to the order Rhizophydiales


Peter M. Letcher 1
Martha J. Powell

     Dept. of Biological Sciences, The University of Alabama, Tuscaloosa AL 35487

M. Claudia Viusent

     Departamento de Biodiversidad y Biologia Experimental, Universidad de Buenos Aires, C1428EHA Buenos Aires, Argentina

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 

The newest order in Chytridiomycota, Rhizophydiales, shows remarkable genetic divergence and zoospore ultrastructural diversity. From submersed mud from a marsh near an oligotrophic lake in the Patagonia region of Argentina, we isolated a previously undescribed chytrid in Rhizophydiales with unique 28S rRNA and ITS1-5.8S-ITS2 sequences and a distinctive zoospore and thallus. In a combined LSU + 5.8S phylogeny of representative taxa in the order, the nearest relative to this new species is Rhizophlyctis harderi, and their zoospores share several similar character states. A new genus is erected for this chytrid, the first to be brought into pure culture from this region.

Key words: chytrid, ITS rDNA, phylogeny, 28S rDNA, zoospore


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
The order Rhizophydiales (Letcher et al 2006Go) is a monophyletic clade in Chytridiomycota ( James et al 2000Go, 2006Go) with remarkable genetic and ultrastructural diversity. Analyses of nuclear large subunit rRNA (28S rRNA) and ITS1-5.8S-ITS2 sequences in Rhizophydiales have defined numerous well-supported lineages, and zoospore ultrastructural analyses have revealed multiple zoospore morphologies (Letcher et al 2004Go, 2006Go, Letcher and Powell 2005Go) that correlate with these lineages. When the order was established (Letcher et al 2006Go), three clades of primarily soil-inhabiting chytrids were delineated as new families (Kappamycetaceae, Rhizophydiaceae, and Terramycetaceae) on the basis of molecular monophyly and specific zoospore morphologies. A second study of taxon diversity in the order analyzed numerous isolates primarily from aquatic habitats (Letcher et al 2008Go) and revealed additional diversity leading to the delineation of seven new families (Alphamycetaceae, Angulomycetaceae, Aquamycetaceae, Globomycetaceae, Gorgonomycetaceae, Pateramycetaceae, and Protrudomycetaceae) in the Rhizophydiales. These families were also defined on the bases of molecular monophyly and zoospore architecture. In addition to these well-resolved clades on which new families were based, other lineages contained only one or a few isolates. Because it is important to understand more about the ultrastructure and development of organisms in these poorly represented lineages, one chytrid is characterized and described in this study.

As the most recently established order in Chytridiomycota, Rhizophydiales had its origin in an ongoing and extensive sampling for chytrids from geographically diverse and disparate habitats worldwide. The chytrid that is the subject of this paper was isolated from a sample containing mud and organic detritus from a marsh in Argentina. Analyses of 28S and ITS1-5.8S-ITS2 rDNA molecular sequences and zoospore ultrastructure place this chytrid in the Rhizophydiales with a rhizophydialean zoospore (Letcher et al 2006Go) having a unique suite of ultrastructural character states. The chytrid is herein described as a new genus and species, family incertae sedis, in the Rhizophydiales.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
Taxonomic sampling.— – A submersed mud and detritus sample obtained in the vicinity of Lake Muster, Chubut Province, Argentina was baited with pine pollen (Pinus strobus), from which an undescribed chytrid was isolated (isolate PL 163L). The chytrid was initially isolated on NAG plus antibiotics (NAG + A) medium (6 g n-acetyl-d-glucosamine, 8 g agar, 0.5 g streptomycin sulfate, 0.5 g penicillin-B, in 1 L deionized water) by streaking discharged zoospores onto the medium. Isolation occurred at room temperature (~20 C). After initial establishment of an axenic culture, the isolate was maintained on dSS medium slants (2 g peptonized milk, 0.2 g tryptone, 4 g soluble starch, 10 g agar, in 1 L deionized water) at 5 C.

Morphology.— – Isolate PL 163L was examined by light microscopy with a Nikon Labophot-2 and a Zeiss Nomarski differential interference contrast microscope. Examinations assessed thallus structural features, including sporangial size and shape, number of discharge pores, type of discharge, and morphology of rhizoids.

Zoospore ultrastructure.— – Fixation and observation of the zoospore of isolate PL 163L followed procedures described in Letcher and Powell (2005)Go. Zoospores were examined on a Zeiss 10A or Hitachi 7650 transmission electron microscope at 60 kV.

DNA preparation.— – DNA was extracted from a pure culture of isolate PL 163L maintained at The University of Alabama and was purified and amplified for sequencing as described (Letcher and Powell 2005Go). The LROR/LR5 primer pair (White et al 1990Go) was used for amplification of LSU (28S) nuclear ribosomal DNA (rDNA), and the ITS5/ITS4 primer pair (White et al 1990Go) for the ITS1-5.8S-ITS2 rDNA region. For molecular analyses, partial nucleotide sequences of the LSU rRNA gene (818 bp from the 5' end) and ITS1-5.8S-ITS2 sequences (621 bp from the 5' end) were generated.

Phylogenetic analyses.— – We assembled and aligned contiguous sequences of 30 isolates in Rhizophydiales and one isolate in Chytridiales (TABLE IGo) (Letcher et al 2004Go). For this study, sequences for all but two isolates (JEL 142 and PL 163L) were from previous studies (TABLE IGo) or the AFTOL database (http://www.aftol.org/index.php). For all isolates in the study, partial LSU sequences were combined with 5.8S sequences, because the ITS1 and ITS2 regions were too divergent for alignment across the entire data set. The dataset included 954 characters with 361 parsimony informative sites.


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  		TABLE I. Taxon sampling for phylogenetic analysis of 30 isolates in Rhizophydiales

 
Phylogenetic trees were constructed using PAUPRat (Sikes and Lewis 2001Go) as described (Letcher et al 2004Go). Branch support was estimated with nonparametric bootstrapping as implemented in PAUP* with 100 replicates, 10 random addition sequences per replicate, holding 50 trees per replicate, with MAXTREES = 1000. ModelTest (v. 3.06, Posada and Crandall 1998) was used to calculate the most appropriate model of DNA substitution, and Bayesian analysis used MrBayes 3.0 (Huelsenbeck and Ronquist 2001Go). Bayesian tree inference with Markov Chain Monte Carlo sampling used four simultaneous Markov chains run over 106 generations. Trees were sampled every 1000 generations, with an overall sampling of 1000 trees. Burnin was calculated when the average standard deviation of split frequencies had declined to <0.01. Remaining trees were used to compute a majority rule consensus tree to obtain estimates for a posteriori probabilities.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
Habitat.— – Lake Muster is an isolated oligotrophic lake in southern Argentina (FIG. 1AGo) at approximately 45°S and 271 m above sea level, located east of the San Bernardo Hills in the foothills of the Andes, on a harsh, windswept, semi-arid plateau. The lake is 40 km west of the coastal city of Comodoro Rivadavia and about seven km west of the nearest town, Sarmiento, a region rich in grasses and used for grazing cattle. Isolate PL 163L was isolated from a mud and detritus sample collected in shallow water in a juncal, a reed bed or marsh (FIG. 1BGo) 60 m from the lake, in mid-winter (Aug 2005) when the air temperature was below freezing.


Figure 1
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  		FIG. 1. Collection site for Coralloidiomyces digitatus. A. Facing west towards Lake Muster, Argentina (arrow), an oligotrophic lake located at the foothills of the Andes. B. A small lake in the juncal (reed bed or grass-dominated marsh), from which C. digitatus was isolated from a submersed mud sample.

 
Morphology.— – On dSS agar medium, the zoospore cyst of isolate PL 163L was initially spherical (FIG. 2A, BGo), and early in development the incipient sporangium became angular (FIG. 2C, DGo). As individual sporangia matured they became lobed, branched and morphologically diverse (FIG. 2E–PGo), 25–50 µm in length and 20–40 µm diam. Some sporangia had a single or a few short branches, while others had up to eight branches that were bifurcated into short discharge papillae. Zoospores were motile within the sporangium before discharge, and exited singly from one or more inoperculate discharge papillae simultaneously (FIG. 2PGo). The rhizoidal system was composed of a short, thin, isodiametric axis and thin, sparsely divided branches (FIG. 2C, D, L, MGo). Occasionally the rhizoidal axis was apophysate early in development (FIG. 2C, DGo). Resting spores were spherical, hyaline, 12–20 µm diam, with a thick, smooth wall and contained one to several large lipid globules and several smaller lipid globules (FIG. 2QGo).


Figure 2
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  		FIG. 2. Morphology of Coralloidiomyces digitatus on dSS agar. A–B. Germlings. Arrows indicate zoospore cyst. C–N. Developing thalli. C, D. Rhizoidal axis with a subsporangial apophysis. E–N. Illustrations of variable sporangial morphology. O. Mature sporangium with cleaved zoospores, prior to discharge. P. Mature sporangium discharging zoospores. Q. Two resting spores. Scale bar in a = 5 µm (for A–C), 10 µm (for D–F), 15 µm (for G–N, Q), and 20 µm (for O, P).

 
On natural substrates of pine and sweet gum pollen grains (Pinus strobus L. and Liquidambar styraciflua L.), thalli were epibiotic or interbiotic and similar to those on dSS agar medium (FIG. 3A–DGo). Branched rhizoids were visible within the pollen grains. Resting spores were similar to those on agar.


Figure 3
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  		FIG. 3. Morphology of Coralloidiomyces digitatus on pollen grains. A–C. On pine pollen. D. On sweet gum pollen.

 
On dSS agar plates at 20 C the isolate was difficult to maintain without monthly transfer to fresh media. On dSS agar slants the isolate has remained viable at 5 C for up to six months.

Zoospore ultrastructure.— – The zoospore of isolate PL 163L (FIGS. 4Go, 5Go) most often had a single lipid globule (occasionally two or three lipid globules in close association) with a conspicuous fenestrated MLC cisterna (FIG. 5A, BGo). The lipid globule was laterally located in the zoospore body. Associated with the lipid globule was a robust microbody with lobes that extended into and partially around the ribosomal aggregation (FIG. 5C, DGo). One or more mitochondria and the nucleus were located in the peripheral cytoplasm, outside the endoplasmic reticulum that enclosed the ribosomal mass (FIG. 5A, BGo). The kinetosome and non-flagellated centriole were parallel and connected by a fibrillar bridge in which the zone of convergence was approximately 0.075 µm wide (FIG. 5FGo). Adjacent to the kinetosome was a kinetosome-associated structure (KAS) as a solid shield (FIG. 5EGo) and a microtubular root composed of four stacked microtubules that extended from the KAS shield to the fenestrated cisterna (FIG. 5EGo).


Figure 4
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  		FIG. 4. Schematic drawing of longitudinal section through the zoospore and transverse section through the kinetosome and non-flagellated centriole of Coralloidiomyces digitatus. F, flagellum; FB, fibrillar bridge; FC, fenestrated cisterna; K, kinetosome; L, lipid globule; M, mitochondrion; Mb, microbody; Mt, microtubular root; N, nucleus; NfC, non-flagellated centriole; R, ribosomes; Sh, shield; ZC, zone of convergence.

 

Figure 5
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  		FIG. 5. Ultrastructural features of the zoospore of Coralloidiomyces digitatus. A. Longitudinal section. B. Transverse section. C. Transverse section with a portion of the microbody ramifying in the ribosomal mass. D. Transverse section with a portion of the microbody lobed around an adjacent lipid globule. E. Longitudinal section through kinetosome illustrating the KAS shield and transverse section through the microtubular root, containing four microtubules shown in transverse section. F. Longitudinal section through kinetosome and non-flagellated centriole illustrating the zone of convergence in the fibrillar bridge. Scale bar in A = 0.5 µm (for A–D), 0.25 µm (for E–F). (For list of abbreviations see FIG. 4Go).

 
Phylogenetic analysis.— – The sequence alignment will be deposited in TreeBASE. Of 1005 trees derived from PAUPRat, 834 most parsimonious trees (length = 1482 steps, consistency index = 0.509, retention index = 0.655) were used to compute a 50% majority rule consensus tree. Support was assessed using bootstrap analysis with 1000 replicates. ModelTest indicated the most appropriate model of DNA substitution was the general time reversible model with invariant sites and rates of substitution among sites approximated by gamma distribution (GTR + I + G). In the Bayesian analysis the first 274 trees were discarded, when the average standard deviation of split frequencies was ≤0.009. A consensus of 726 trees was used to compute a majority rule consensus tree.

MP and Bayesian tree topologies were congruent (FIG. 6Go) with the exception of the placement of the Gorgonomycetaceae clade. In the parsimony analysis Gorgonomycetaceae was sister to all other isolates, while in the Bayesian analysis Gorgonomycetaceae was sister to a clade composed of Alphamycetaceae and Kappamycetaceae.


Figure 6
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  		FIG. 6. Fifty percent majority rule consensus of 30 taxa in the order Rhizophydiales from combined dataset of partial LSU and 5.8S rDNA sequences with uninformative characters deleted. Isolate ARG 041, Chytriomyces clade, used as outgroup. Isolates JEL 142 and JEL 223 are unidentified isolates in the Rhizophydiales. Tree length = 1482; consistency index = 0.509; retention index = 0.655. Numbers above branches or to the left of slashes (/) indicate support above 50% in 1000 bootstrap replicates with parsimony analysis. Numbers below branches or to the right of slashes represent probability of nodes in Bayesian analysis.

 
Several isolates were lone taxa on branches that were not included in previously described families in Rhizophydiales. Isolate JEL 142 (unidentified sp. #1) was sister to isolate JEL 197, Batrachochytrium dendrobatidis. Isolates ATCC 24053 (Rhizophlyctis harderi), PL 163L, and JEL 223 (unidentified sp. # 2) were basal in a clade sister to Terramycetaceae. Isolate JEL 326 (Entophlyctis helioformis) was sister to all other isolates in the Rhizophydiales.

The nearest relative to isolate PL 163L was ATCC 24053, Rhizophlyctis harderi. LSU rRNA sequences of PL 163L and R. harderi were 84% similar (GenBank accession numbers EF634248 [GenBank] and AY349087 [GenBank] respectively), and 5.8S sequences were 97% similar (Gen-Bank accession numbers EF634250 [GenBank] and DQ485595 [GenBank] respectively).


   TAXONOMY
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 TAXONOMY
DISCUSSION
 LITERATURE CITED
 
Coralloidiomyces Letcher, gen. nov.

Fungus saprophyticus. Thallus monocentricus, eucarpicus, e sporangio sessili aut intervitum. Sporangia 20–40 µm diam, cum multis lobi. Zoosporae cum cisterna fenestra in globuli lipoidei superficie, microbody cum laminare, et [KAS shield] opacum aedificium.

Typus. – Coralloidiomyces digitatus Letcher 2008Go, figurae 2A–Q, 3A–D, 5A–F. Diagnosus cultura PL 163L ex pollinis in aquacultura.

Fungus saprophytic. Thallus monocentric, eucarpic, with sessile or occasionally interbiotic sporangia having multiple lobes. Zoospore with a single lipid globule partially covered with a conspicuous fenestrated cisterna and a microbody with laminated lobes that extend into and partially around the ribosomal core. KAS as a solid shield, from which a microtubular root composed of four microtubules extends to the fenestrated cisterna.

Etymology. – Latin; coralloidium = coral-like.

Type. – Coralloidiomyces digitatus Letcher 2008Go, figures 2A–Q, 3A–D, 5A–F, from observations of culture PL 163L.

Coralloidiomyces digitatus Letcher, sp. nov. (FIGS. 2A–QGo, 3A–DGo, 5A–FGo)

Descriptio ad genus. Sporangium maturum in purum cultum lobosom, 20–40 µm diam, cum multis papillis emissionibus. Sporae remanentes globosae, 12–20 µm diam, unico ad pluro magno globulo repletae.

Typus. – Letcher et al 2008Go, Mycologia 100, figurae 2A–Q, 3A–D, 5A–F (2008). Diagnosis cultura PL 163L ex pollonis esca posita in aquacultura ex terra ex paludis prope Lacus Muster, Provincia Chubut, Argentina, GPS locus 45°35'36''S x 69°13'57''W. GenBank LSU rDNA sequence EF634248 [GenBank] , ITS1–5.8S–ITS2 sequence EF634250.

Description as for genus. In pure culture on nutrient agar sporangium lobed at maturity, 20–40 µm diam, with multiple discharge papillae. Resting spore spherical, 12–20 µm diam, with one to several large lipid globules.

Etymology. – Latin; digitatus refers to the digit-like lobes of the sporangium.

Type. – Letcher et al 2008Go, Mycologia 100, 315–317, 2A–Q, 3A–D, 5A–F (2008) from observations of culture PL 163L from pollen bait placed with water cultures of soil from a marsh near Lake Muster, Chubut Province, Argentina, GPS coordinates 45°35'36''S x 69°13'57''W. GenBank LSU rDNA sequence EF634248 [GenBank] , ITS1–5.8S–ITS2 sequence EF634250. Culture PL 163L, on which the type is based, is being deposited with ATCC and CBS.

On agar, individual sporangia of C. digitatus were lobed, branched and morphologically diverse, some with a single or a few short branches, while others had up to eight branches that were bifurcated into short discharge papillae. Zoospores were discharged from one or more inoperculate discharge papillae. The mature rhizoidal system was a isodiametric rhizoidal axis and sparsely branched, thin rhizoids. Resting spores were spherical, hyaline, with a thick, smooth wall and contained a one to several large lipid globules.

On pollen grains, mature sporangia were similar to those on agar. Coralloidiomyces digitatus colonized pine, spruce, sweet gum, and Typha pollen.

Specimen examined: – ARGENTINA. CHUBAT PROVINCE: Lake Muster, 45°35'36''S x 69°13'57''W, elevation 271 m ASL. On pollen substrate from submersed mud and detritus, 20 Aug 2005, culture number PL 163L collected by Maria Claudia Viusent and isolated by Peter M. Letcher, HOLOTYPE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
LITERATURE CITED
 
Previous research has shown the Rhizophydiales to encompass a remarkable divergence of organisms (Letcher and Powell 2005Go, Letcher et al 2006Go, 2008Go). What was once considered the genus Rhizophydium is now an order currently composed of nine families and eleven genera (Letcher et al 2006Go, 2008Go) and taxa with more than a dozen distinct zoospore morphologies. Members of Rhizophydiales inhabit soils, aquatic systems, the aquatic/terrestrial interface, and the canopy of rainforest trees, and are often specific to a habitat (Powell 1993Go, Letcher et al 2004Go, Longcore 2005Go). Some members of the order are obligate algal parasites, others appear to be facultative symbionts, and many are saprotrophs of a variety of refractory materials. In an extensive sampling for chytrids in Argentina, Coralloidiomyces digitatus is the first chytrid to be isolated from the Lake Muster system in the remote Sarmiento Plateau of central Patagonia.

Molecular sequence and zoospore ultrastructure analyses show Coralloidiomyces digitatus to be a unique taxon in Rhizophydiales. However, its zoospore architecture may indicate a relationship with the organism Rhizophlyctis harderi Uebelmesser. The zoospore of R. harderi has been thoroughly studied (Powell and Roychoudhury 1992Go, Roychoudhury and Powell 1992Go) and the zoospores of both R. harderi and C. digitatus have a fenestrated MLC cisterna, a microtubular root extending from the kinetosome toward the fenestrated MLC cisterna, and perhaps most significantly, a KAS as a shield adjacent to the kinetosome. No other taxa in Rhizophydiales are known to have this type of KAS. However, the zoospores of C. digitatus and R. harderi differ in several respects. Whereas the zoospore of C. digitatus usually has a single lipid globule with a fenestrated MLC cisterna, the zoospore of R. harderi has several lipid globules, with each posteriorly located smaller lipid globule encased by a fenestrated cisterna and each anteriorly located lipid globule clasped by a simple cisterna. The zoospore of C. digitatus has a single microtubular root, while the zoospore of R. harderi has two microtubular roots, the second root extending from the fibrillar bridge between the kinetosome and non-flagellated centriole toward the anterior portion of the zoospore. Finally, the zoospore of R. harderi has a vesiculated region anterior to the kinetosome, a character often associated with the presence of a spur-type KAS; this ultrastructural character is absent in the zoospore of C. digitatus.

We are not the first investigators to notice a chytrid with this morphology. Johnson and Miller (1974)Go described two chytrids with lobed sporangia. One chytrid, from Maine, was observed on sweet gum pollen (Liquidambar styraciflua L.), and although not propagated in culture, was provisionally assigned to Phlyctidium on the basis of thallus morphology. This provisional member of Phlyctidium strongly resembles C. digitatus. (As Phlyctidium has been merged with Rhizophydium [Karling 1977Go, p. 64], placement in this genus is moot). Both chytrids have most commonly a dichotomously lobed sporangium with conspicuous, digitate extensions that ultimately function as inoperculate discharge papillae. Our chytrid differs from other described lobed chytrids, such as Karlingia lobata Karling, which is operculate and has a thick rhizoidal axis and catenulate rhizoids, and Saccomyces endogenus (Nowakowski) Sparrow, which is a parasite of encysted Euglena sp. and has a haustorium of broad distally swollen digitations.

Johnson and Miller (1974)Go were unable to propagate their provisional Phlyctidium in pure culture. Coralloidiomyces digitatus was difficult to isolate, and although it readily discharged zoospores that were streaked onto several common media used in the culture of chytrids, the zoospores germinated only on NAG + A medium. After colonies of thalli were established, it could not successfully be transferred to NAG minus antibiotic medium. Vitality of colonies waned rapidly on NAG + A, requiring frequent transfers, but mature colonies transferred to dSS medium with low agar content (6.4 g/L) survived. Established cultures are currently maintained on normal (8 g agar/L) dSS slants at 5 C, and have maintained vitality for up to six months under these conditions.

This study reinforces previous research that indicates that novel chytrid lineages are present in extreme, stark, or depauperate habitats (Powell 1993Go) such as rainforest canopy (Longcore 2005Go), heath (Letcher et al 2004Go), and nitrogen-depleted soils (unpubl results). Chytrids have also been shown to survive in soils subject to desiccation and high temperatures (Gleason et al 2004Go), and are able to survive different osmotic potentials (Gleason et al 2006Go).

In the course of our research into the systematics of Rhizophydium-like organisms, we have examined terrestrial and submersed soils with broad geographic distribution. A chytrid similar to Coralloidiomyces digitatus has been observed in water cultures of wet soils from Texas and Virginia in the United States, New South Wales, Australia, and Buenos Aires, Argentina (unpubl results). As with Johnson and Miller (1974)Go, these observed chytrids were not isolated or brought into pure culture. Isolation of this taxon from multiple disparate geographic locations and habitats will further our understanding of distribution, dispersal, and genetic divergence of members of this ancient lineage of true fungi.


   ACKNOWLEDGMENTS
 
This study was supported by the National Science Foundation through REVSYS grant DEB-0516173 and MRI grant DEB-0500766. We express our appreciation to the Assembling the Fungal Tree Of Life (AFTOL) Project, Duke University, (NSF DEB-0228668) for access to their database, and to Scott Wakefield for assistance with DNA isolation and sequencing.


   FOOTNOTES
 
1 Corresponding author. E-mail: letch006{at}bama.ua.edu


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
Gleason FH, Letcher PM, McGee PA. 2004. Some Chytridiomycota in soil recover from drying and high temperatures. Mycol Res 108:583–589.[CrossRef][Medline]

———, Midgley DJ, Letcher PM, McGee PA. 2006. Can soil Chytridiomycota survive and grow in different osmotic potentials? Mycol Res 110:869–875.[CrossRef][Medline]

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James TY, Porter D, Leander CA, Vilgalys R, Longcore JE. 2000. Molecular phylogenetics of the Chytridiomycota supports the utility of ultrastructural data in chytrid systematics. Can J Bot 78:336–350.[CrossRef]

———, Letcher PM, Longcore JE, Mozley-Standridge SE, Porter D, Powell MJ, Griffith GW, Vilgalys R. 2006. A molecular phylogeny of the flagellated fungi (Chytridiomycota) and description of a new phylum (Blastocladiomycota). Mycologia 98:860–871.[Abstract/Free Full Text]

Johnson TW, Miller CE. 1974. Two unusual chytridiaceous fungi. Mycologia 66:859–867.[CrossRef]

Karling JS. 1977. Chytridiomycetarum Iconographia. Vaduz: J. Cramer. 414 p.

Letcher PM, McGee PA, Powell MJ. 2004. Distribution and diversity of zoosporic fungi from soils of four vegetation types in New South Wales, Australia. Can J Bot 82:1490–1500.[CrossRef]

———, Powell MJ. 2005. Kappamyces, a new genus in the Chytridiales (Chytridiomycota). Nova Hedwigia 80: 115–133.[CrossRef]

———, ———, Chambers JG, Holznagel WE. 2004. Phylogenetic relationships among Rhizophydium isolates from North America and Australia. Mycologia 96: 1339–1351.[Abstract/Free Full Text]

———, ———, Churchill PF, Chambers JG. 2006. Ultra-structural and molecular phylogenetic delineation of a new order, the Rhizophydiales (Chytridiomycota). Mycol Res 110:898–915.[CrossRef][Medline]

———, Vélez CG, Barrantes ME, Powell MJ, Churchill PF, Wakefield WS. 2008. Ultrastructural and molecular analyses of Rhizophydiales (Chytridiomycota) isolates from North America and Argentina. Mycol Res 112: In press.

Longcore JE. 2005. Zoosporic fungi from Australian and New Zealand tree-canopy detritus. Australian J Bot 53: 259–272.[CrossRef]

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———, Roychoudhury S. 1992. Ultrastructural organization of Rhizophlyctis harderi zoospores and redefinition of the type I microbody-lipid globule complex. Can J Bot 70:750–761.

Roychoudhury S, Powell MJ. 1992. Precise flagellar configuration of the Rhizophlyctis harderi zoospore. Can J Bot 70:762–771.

Sikes DS, Lewis PO. 2001. Beta software, version 1. PAUPRat: PAUP* implementation of the parsimony ratchet. Distributed by the authors. Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT.

Swofford DL. 2002. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods), 4.0b4a. Sunderland, Massachusetts: Sinauer Associates.

White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: a guide to methods and applications. New York: Academic Press. p 315–322.





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