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Rhizophydium brooksianum sp. nov., a multipored chytrid from soil

     Department of Biological Sciences, University of Maine, Orono, Maine 04469-5722

	ABSTRACT

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Rhizophydium136 was isolated from pollen bait placed in a water culture containing garden soil from Penobscot County, Maine. It is an important isolate because its entire mitochondrial genome has been sequenced and it is the representative member of the Chytridiales in a fungal phylogeny based on mitochondrial protein sequences. Also, this isolate is included in an 18S rDNA, chytrid phylogeny. On nutrient agar, many inflated rhizoidal axes extend from the base of the zoosporangium, zoosporangia mature in 3 d and zoospores discharge through numerous, lenticular, discharge pores. Smooth-walled resting spores form in crowded cultures. Zoospores are a variation of the Rhizophydium subtype. This chytrid differs from R. sphaerotheca sensu Barr and because it cannot be placed in a described species it herein is described as Rhizophydium brooksianum sp. nov. Many of the differences between Rhizophydium brooksianum and other multipored Rhizophydium isolates were observed only in pure culture. Attributing a spherical, multipored Rhizophydium to a species that was described without developmental information from pure culture is untenable. Epitypes or neotypes for inadequately characterized species need to be selected, and cultures made available.

Key words: Chytridiales, R. sphaerotheca, ultrastructure, zoospore

	INTRODUCTION

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Members of the orders Chytridiales and Spizellomycetales that form spherical zoosporangia with multiple, discharge pores are common; however, identifying species of such chytrids is difficult because no recent keys delineate differences among species. Difficulties also exist because many species were described from material that was not in pure culture; consequently, descriptions do not contain information about all characters, particularly those of the rhizoidal system. Sparrow's 1960 "Aquatic Phycomycetes" contains descriptions of several species of the genus Rhizophydium (Chytridiales) that possess spherical, unornamented zoosporangia with multiple, discharge pores, e.g., R. globosum (Braun) Rabenhorst, R. sphaerotheca Zopf and R. bullatum Sparrow. Additional Rhizophydium and Phlyctochytrium species that form spherical zoosporangia with multiple, discharge pores have been described since then, e.g., R. venezuelensis Karling, R. macroporosum Karling, R. polystomum Karling, R. littoreum Amon, P. aestuarii Ulken (Longcore 1996).

Barr outlined differences between the Spizellomycetales and he Chytridiales (Barr 1980). Many Spizellomycetales also form spherical thalli with multiple, discharge pores, and some species initially placed in Phlyctochytrium are now in new genera in the Spizellomycetales (Barr 1984, Longcore 1996). On the basis of the ultrastructure of their zoospores, other Phlyctochytrium species with spherical, multipored zoosporangia can be placed in Rhizophydium, e.g., P. aestuarii Ulken, which is now R. aestuarii (Ulken) Amon; McNitt's (1974) study of P. irregulare Koch reveals that this species also should be placed in Rhizophydium.

I contributed a recently isolated Rhizophydium (JEL136) with spherical zoosporangia and multiple, discharge pores to the Fungal Mitochondrial Genome Program. Its entire mitochondrial genome (http://megasun.BCH.UMontreal.CA/People/lang/FMGP/progress.html) has been sequenced and included in phylogenetic analyses based on concatenated mitochondrial proteins (Lang et al 2002, Bullerwell et al in press). As part of another phylogenetic study, James et al (2000) sequenced and compared the nuclear 18S rDNA of JEL136 with that of other chytrids. Because of the importance of JEL136 in these analyses, I attempted to determine its identity beyond the genus level. However, after comparing it with species descriptions and similar Rhizophydium isolates, I could not find a match. Consequently, I describe JEL136 as a new Rhizophydium species, portray its light-level morphology and zoospore ultrastructure and indicate features that distinguish it from similar species.

	MATERIALS AND METHODS

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Isolation and culture – Isolate JEL136 colonized pollen bait in a water culture containing garden soil from Penobscot County, Maine, that had been stored in a freezer 2 yr before use. The letters JEL refer to the chytrid culture collection maintained at the University of Maine. Other isolates recovered from pollen bait and used in this study were JEL08 (Mud Pond, Hancock County, Maine); JEL138 (Visitors' Center, St. Mark's National Wildlife Refuge, Florida); JEL221 and 222 (garden soil, Penobscot County, Maine); JEL294 (garden soil, Arizona State University, Tempe, Arizona) and ATCC # 18786 (neotype culture of R. sphaerotheca). Stock cultures were maintained at 5 C on PmTG agar (1 g peptonized milk, 1 g tryptone, 5 g glucose, 10 g agar, 1 L distilled water; Barr 1986) slants in screw-capped culture tubes and transferred at three-month intervals.

For morphological studies, pieces of stock culture were transferred to mPmTG agar (0.4 g peptonized milk, 0.4 g tryptone, 2.0 g glucose, 10 g agar, 1 L distilled water) in Petri dishes. After several days, sterile water was added to established colonies to spread zoospores. Thalli resulting from the dispersed zoospores were photographed with a Spot RT digital camera attached to a Nikon E400 microscope equipped with phase and brightfield optics. To determine morphology on natural substrates, small pieces of culture on agar were added to dishes containing unsterilized spruce or pine pollen in distilled water. The fungus was incubated in 75 mL of PmTG broth in 125 mL, screw-capped flasks to determine maximum temperature for growth. Three replicates were incubated at each temperature.

Electron microscopy – Zoospores of JEL136 were harvested from 20 dishes of PmTG agar by flooding plates covered with fungal colonies with sterile, distilled water. After 30 min the zoospore suspensions were combined and fixed with a sequential glutaraldehyde-osmium tetroxide method (Barr 1981, Longcore 1992). Serial sections were cut with a diamond knife, placed on carbon-coated, Pioloform-covered slot grids, stained with lead citrate and examined on a Philips CM 10 transmission electron microscope at 80 kV. Kinetosome triplets were numbered following the method described by Barr and Désaulniers (1988).

	TAXONOMY

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Rhizophydium brooksianum Longcore, sp. nov. Figs. 1–21

View larger version (172K): [in this window] [in a new window]   FIGS. 1–10. Development of Rhizophydium brooksianum on mPmTG agar. 1. Germlings. 2–4. 1-day old developing thalli with 2 rhizoidal axes. 5. Developing zoosporangium with 4 visible rhizoidal axes (arrows). Other axes are out of focus. 6. Isodiametric ends of rhizoids. 7. Mature zoosporangium with many discharge papillae and length of rhizoids 1.75x the diameter of zoosporangium. Inset shows individual papilla. 8. Nearly empty zoosporangium. Arrowheads point to open pores with slightly up-turned edges. 9. Zoospores are round when in motion and contain a single, visible lipid globule. 10. Resting spores among crowded zoosporangia. Scale bar in FIG. 3 = 10 µm for all photos except FIG. 7. Scale bar in 7 = 40 µm; bar for inset in 7 = 10 µm

TYPE: Figs. 1–21. Diagnosis based on isolate JEL136 from pollen bait placed with soil collected from a garden, Eddington, Penobscot County, Maine. A culture of the type isolate has been deposited in the American Type Culture Collection (MYA-2891). This species is named for Joan Brooks, who encouraged my return to the study of chytrids and collected the garden soil.

Light microscopy, on nutrient agar: Zoosporangium spherical, with many closely spaced and highly branched rhizoidal axes on the base of the zoosporangium; rhizoids with swollen bases extending to 1.75x the diameter of mature zoosporangium. Multiple, evenly distributed, discharge pores, number depending on size of zoosporangium; pores 4–10 µm diam; appearing as lenticular protrusions before discharge. Swimming zoospores spherical; 4–5 µm diam with one visible lipid globule; flagellum length about 27 µm. Resting spores 12–17 µm diam with one, large, refractive globule. TEM of zoospores similar to other Rhizophydium spp., except hemisphere of vesiculated cytoplasm surrounding the kinetid, spur parallel to triplet 9 arching over the kinetosome, microtubule root consisting of abutting microtubules and leading into aggregated ribosomes, and rumposome lacking. Saprobic on pollen. From soil, Eddington, Maine. 18S rDNA GenBank # AF164268-9; mitochondrial genome NCBI # NC_003053.

	RESULTS

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Development on mPmTG agar – Generation time of Rhizophydium brooksianum is 3 d at ambient laboratory conditions (20–23 C). Within 24 h of zoospore encystment, branched rhizoids form and cysts begin to enlarge (Figs. 1–3). A few young thalli form widely separated rhizoidal axes (Fig. 4). During day two, zoosporangia continue to enlarge and the rhizoidal system becomes more complex. Many rhizoidal axes with swollen bases (up to 4.5 µm diam) arise from the base of the zoosporangium (Fig. 5) and at maturity rhizoids extend to 1.75x the diameter of the zoosporangium (Fig. 7). Near their tips rhizoids are nearly isodiametric and about 0.5 µm diam (Fig. 6). Within 3 d zoosporangia are recognizable as mature by the presence of dome-shaped discharge papillae (Fig. 7). Papillae deliquesce and zoospores escape through pores that have slightly out-turned edges. Empty zoosporangia display numerous, evenly spaced discharge pores (Fig. 8), which vary in size among zoosporangia of differing sizes but frequently are about 5 µm diam. Newly emerged zoospores contain a single, prominent, lipid globule and are variously shaped but become spherical (Fig. 9) when in motion. Colonies on mPmTG agar are off-white becoming buff when older. The fungus grows in PmTG broth at 25 C but not as fast as at 23 C; cultures do not grow at 27 C. Colorless, smooth-walled, resting spores with one globule develop sporadically on crowded culture plates on which several generations of thalli have developed (Fig. 10). Resting spores appeared to be formed asexually.

Development on pollen – Zoosporangia on pine and spruce pollen (Figs. 11–15) were smaller than those that grow on mPmTG agar, and generation time was <2 d. The smaller zoosporangia that develop on pollen have fewer discharge pores than those that develop on mPmTG agar, with the smallest zoosporangia having a single pore (Fig. 14). Smooth or slightly rough-walled, pale amber, resting spores containing a single large globule develop in old cultures on pine and spruce pollen grains (Fig. 15).

View larger version (159K): [in this window] [in a new window]   FIGS. 11–15. Pine and spruce pollen inoculated with Rhizophydium brooksianum; for size comparisons, photos are printed at the same magnification as development on agar (Figs. 1–6, 8–10). 11. Mature zoosporangium on spruce pollen (center) and several smaller zoosporangia on a single pollen grain (right). 12. Empty zoosporangium on spruce pollen grain, showing evenly distributed discharge pores. 13. Mature zoosporangium on pine pollen grain. 14. Empty zoosporangium with single discharge pore on pine pollen. 15. Resting spores on pine pollen. Scale bar in FIG. 14 = 10 µm for all figures

View larger version (155K): [in this window] [in a new window]   FIGS. 16–21. Electron photomicrographs of Rhizophydium brooksianum zoospores. 16. Longitudinal section (No.13 of 32 serial sections) showing lipid globule, L; aggregated ribosomes, R; mitochondria, M, flagellar prop, P; ER bounded (arrowheads) vesiculated area around kinetosome and spur parallel to and arching over kinetosome. 17. Longitudinal section of zoospore showing nucleus, N; nonflagellated centriole (nfc) parallel, and connected to kinetosome (insert is No. 3 of six serial sections through kinetosomal area). 18. Ribosomal inclusion, RI; MLC consisting of lipid, L, microbody, mb, and cisterna of ER. 19. microtubule root extending from kinetosome, K, into aggregation of ribosomes; cross section of spur in membrane-demarcated, vesiculated area. 20. Cross section of zoospore with circular root in shadowed boxes. 21. Part of circular microtubular root in section 8 of 13 serial sections, showing microtubules surrounded by ribosomes. Scale bar in FIG. 16, also for FIG. 17 = 1 µm. Scale bars in inset of FIG. 17 and FIGS. 18–21 = 0.5 µm

Kinetid. The kinetosome and nonflagellated centriole (nfc) lie in the posterior of the zoospore in a pocket of vesiculated cytoplasm separated from the ribosomal mass by ER (Figs. 16, 17, 19). A membrane demarcated, vesiculated area also can be seen around the kinetosome of certain other Rhizophydium spp.; e.g., Phlyctochytrium irregulare Koch (McNitt 1974), P. aestuarii Ulken (= R. aestuarii (Ulken) Amon) (Lange and Olson 1977), R. capillaceum Barr, R. sphaerotheca (sensu Booth), R. subangulosum (Braun) Rabenhorst (Barr and Hadland-Hartmann 1978) and R. macroporosum Karling (Chen and Chien 1996). The nfc is connected to the kinetosome by overlapping fibrils (Fig. 17 inset) that attach two triplets of the nfc to triplets 5 and 6 of the kinetosome (serial sections not shown). A prominent feature of the kinetid is a spur (Figs. 16, 17) that lies parallel to triplet 9 and arches above the kinetosome toward the nfc. A root consisting of abutting microtubules begins near kinetosome triplets 1 and 2 and extends anteriorly into the zoospore surrounded by ribosomes (Fig. 19). A microtubule root also surrounds the mid part of the zoospore (Figs. 20, 21); however, I have not found sections that let me confirm that the circular root attaches to the primary kinetosomal root.

Rhizophydium sphaerotheca sensu Barr – Because R. sphaerotheca frequently is the identity assigned to multipored Rhizophydium species, I compared the neotype culture of R. sphaerotheca (Barr 1969) with R. brooksianum on the same mPmTG agar medium. R. sphaerotheca produces an openly branched system of isodiametric rhizoids that are only slightly enlarged (seldom more than 1.5 µm diam) at their bases (Figs. 22–24). When mature, zoosporangia are about 25–33 µm diam (Fig. 25). Papillae are scattered on the zoosporangia and are narrow (2–3 µm diam) and almost as high as wide (1.5–2.5 µm). Zoospores tend to be small (2–3 µm diam) (Fig. 26). Sizes of zoosporangia and zoospores vary with growth conditions (Paterson 1963), and these measurements should be not be considered as invariant characters.

View larger version (146K): [in this window] [in a new window]   FIGS. 22–30. Rhizophydium isolates for comparison with R. brooksianum, all on mPmTG agar unless otherwise noted. 22–26. R. sphaerotheca (neotype isolate; Barr 1969). 22. Germling. 23. Developing zoosporangium. 24. Immature zoosporangia with open, fine rhizoidal systems. 25. Mature zoosporangium at lower magnification for comparison with other isolates. Inset shows enlarged papilla. 26. Newly emerged zoospores. 27. Mature zoosporangium of Isolate JEL08. Inset shows barely visible papillae. 28. Mature zoosporangium of JEL294; papilla and resting spore in insets. 29. Mature zoosporangium of JEL222; resting spore (on pollen) and papilla in insets. 30. Mature zoosporangium of JEL221. Discharge papilla and resting spore in insets. Scale bars in all insets and in 23 = 10 µm. Scale bar in 23 also for 22, 24, 26. Scale bar in 27 = 40 µm also for 25, 28–30

Other multipored isolates – JEL221, 222 and 294 are larger multipored isolates of Rhizophydium that all have lenticular papillae and form resting spores after several generations have grown on a culture plate (Figs. 28–30 insets). In gross culture, before isolation, they resembled R. brooksianum. In culture on mPmTG agar, these isolates differ from each other and from R. brooksianum in their maximum size, in the relative extent and branching pattern of their rhizoids, and in the size, shape and spacing of their discharge papillae (Figs. 28–30).

	DISCUSSION

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Order – Because species in both the Spizellomycetales and the Chytridiales produce spherical zoosporangia with multiple, discharge pores, identifying unknown spherical chytrids requires consideration of both orders. When grown on nutrient agar most members of the Chytridiales have zoospores with a single oil globule and have rhizoids that taper to <0.5 µm diam (Barr 1980). Conversely, most members of the Spizellomycetales have zoospores that have more than one oil globule, or have globules that are not spherical, and have rhizoids that usually are broader than those in the Chytridiales (tips >0.5 µm diam). Deciding about the order to which an isolate belongs based on light-level zoospore characters alone can be unreliable because some chytridialean species have zoospores with multiple oil globules (e.g., Batrachochytrium; Longcore et al 1999) and some spizellomycetaleans have zoospores with a single oil globule (e.g., Gaertneriomyces; Barr 1984). The tips of R. brooksianum rhizoids are about 0.5 µm diam, but this description also fits the rhizoids of the spizellomycetalean genus Gaertneriomyces (Barr 1984). Under the light microscope, one oil globule was observed in R. brooksianum zoospores, but Barr's (1969) photographs of the zoospore of Gaertneriomyces (as Phlyctochytrium acuminatum) also depict one oil globule. In addition, the presence of inflated rhizoidal bases is a feature associated with members of the Spizellomycetales. Consequently, knowledge of the ultrastructure of the R. brooksianum zoospore was required to determine with certainty the order for this isolate. Placement in the Chytridiales was confirmed by the presence of a nucleus that is not closely associated with the kinetosome, a chytridialean MLC, a microtubule root extending from the side of the kinetosome, an nfc and kinetosome connected along facing sides and the presence of aggregated ribosomes (Barr 2001).

Most of the ultrastructural features of R. brooksianum conform to the description of the Rhizophydium subtype of zoospore (Barr 1980); however, the particulars of the microtubular root differ from those of the described subtype. The several microtubules in the Rhizophydium subtype of zoospore originate from the side of the kinetosome opposite of the connection to the nfc (e.g., from triplet 2 in R. chlorogonii; Barr and Désaulniers 1988) and are positioned one over the other with cytoplasm between (Barr and Hadland-Hartman 1978). The microtubule root in R. brooksianum extends from triplets 1–2 but is surrounded by ribosomes, and the microtubules abut each other to form a cord as they do in the Chytridium zoospore subtype (Barr 1980). Although it has a different root configuration, the lack of an electron-opaque area in the base of the flagellum and the overlapping fibers between the kinetosome and NFC ally the R. brooksianum zoospore with the Rhizophydium subtype. The position of R. brooksianum in the Rhizophydium clade of the Chytridiales, an order and clade whose members usually have a rumposome, is supported by analyses of 18S rDNA sequences (James et al 2000). Consequently, I interpret the lack of a rumposome in this isolate as the loss of this feature

Rhizophydium sphaerotheca and other multipored Rhizophydium spp – With Sparrow's 1960 key to the species of Rhizophydium, the closest match for isolate136 was R. sphaerotheca; however, as chytrid researchers have noted, the description of R. sphaerotheca covers a complex of species. Sparrow (1960:250) recognized R. sphaerotheca as including "forms with multiporous, spherical, or subspherical zoosporangia and a branched rhizoidal system which inhabit the submerged microspores or microgametophytes of pteridophytes and spermatophytes," whereas species with the same morphology but which grew on algae were assigned to R. globosum (Braun) Rabenhorst. When discussing R. bullatum, which can be distinguished by its bullate resting spores, Sparrow (1960:251) suggested: "It is entirely possible that there are a number of spherical multiporous zoosporangial types which differ only in the character of their resting spore." The ability to identify spherical chytrids with multiple, discharge pores from gross cultures was brought further into question when Paterson (1963) reported that a R. globosum-like species that he found on the green alga Pediastrum was not truly parasitic but also grew on pollen and thus could be considered R. sphaerotheca. Barr (1969) isolated a number of chytrids from soil that could have been placed in the broadly defined R. sphaerotheca, but after studying the isolates in pure culture he recognized several different organisms. Barr named three of the multiporous species as new species of Phlyctochytrium (now transferred to genera in the Spizellomycetales; Barr 1984) and one of them as a new Rhizophydium species, R. capillaceum. Of particular importance, Barr (1969) clarified and restricted the use of the R. sphaerotheca epithet when he designated figures depicting an isolate collected from Oklahoma soil as the neotype. Because the R. sphaerotheca epithet has been used for a variety of chytrids, I have included photographs of the type isolate grown on mPmTG agar to supplement the neotype (Barr 1969) and to compare with R. brooksianum. In culture, the rhizoidal system of R. sphaerotheca sensu Barr differs distinctly from the robust rhizoidal system of R. brooksianum. Further, the discharge papillae differ, with R. brooksianum forming broad, lenticular papillae and R. sphaerotheca forming narrower papillae. Finally, R. sphaerotheca lacks resting spores in culture whereas R. brooksianum produces resting spores both in gross and pure culture.

The name R. sphaerotheca has been used for another distinct Rhizophydium sp. Booth's (1971) concept of R. sphaerotheca is similar to JEL08 (Fig. 27); zoospores are released through barely visible papillae and no resting spores are formed. My isolates of this fungus produce more abundant and larger rhizoids than R. sphaerotheca, as typified by Barr but not as robust as those of R. brooksianum.

Rhizophydium136 resembles Zopf's (1887) drawings of Rhizophydium polinis (A Braun) Zopf; however, Sparrow (1943, 1960) considered Zopf's figures of R. polinis (= polinis-pini) to be of R. sphaerotheca and restricted R. polinis-pini for Rhizophydia with a single discharge papilla that grow on pollen. Species concepts for "little round chytrids" are debatable without pure cultures, and Sparrow's (1943, 1960) monographs seem like a reasonable cut-off point for accepting species concepts. Therefore, I will not attempt to change the R. polinis-pini species concept based on my interpretation of Zopf's drawings.

Similar isolates – When considering whether isolate JEL136 was an undescribed species, I searched for similar chytrids on pollen bait placed with collections from other sites. Several spherical chytrids, when first seen in gross cultures, appeared to be the same species; that is they formed zoosporangia with multiple, lenticular discharge pores and formed resting spores on pollen in baited soil cultures. Examination of their development in pure culture, however, revealed differences among these isolates. When grown on mPmTG medium, they differed in the size of mature zoosporangia, in the relative extent of the rhizoidal system as compared to the diameter of the zoosporangium, in the branching pattern of the rhizoids and in the ratio of the number of discharge papillae to the size of the zoosporangium (compare Figs. 7, 27–30). Not pictured is isolate JEL138 of R. macroporosum, which, based on 18S rDNA phylogeny (James et al 2000), is the closest sequenced relative of R. brooksianum. Isolate 138 is similar to photographs of an isolate from Taiwan portrayed by Chen and Chien (1996). The zoospore ultrastructure of their isolate is similar to that of R. brooksianum, except that R. macroporosum has a rumposome and the microtubule root appears to lead to it (Chen and Chien 1996).

Some from this group of isolates that discharge zoospores through multiple, erumpent, discharge pores and form smooth-walled resting spores with a single, large globule, might be closely related, but until mycologists develop a consensus about how much morphological variation to accept within a species in this group, JEL136 is the only isolate to which the name R. brooksianum applies. Most similar to it are photographs of the development of a chytrid that Chen and Chien (1998) reported as Rhizophlyctis hyalina (Karling) Sparrow. In pure culture their fungus also has many rhizoidal axes arising from the base of the zoosporangium and zoospores that are released from multiple, lenticular papillae; however, resting spores were not reported. From my observations of the morphological differences among spherical, multipored Rhizophydium isolates, I conclude that names of spherical Rhizophydium species are not useful unless accompanied by developmental information from pure cultures (and, preferably, access to cultures). If species that have been described without this information are to be reliably identifiable, epitypes or neotypes (and cultures thereof) need to be selected.

	ACKNOWLEDGMENTS

 
This work was supported by National Science Foundation PEET (Partnership for Enhanced Expertise in Taxonomy) grant No. DEB-9978094. I thank Peter M. Letcher and Marilyn R.N. Mollicone for their comments on a draft of this manuscript. I am indebted to Professor Kristina Passman for translating the Latin description.

	FOOTNOTES

 
¹ E-mail: longcore{at}maine.edu

Accepted for publication June 9, 2003.

	LITERATURE CITED

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———. 1980 An outline for the reclassification of the Chytridiales, and for a new order, the Spizellomycetales. Can J Bot 58:2380-2394

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———. 1984 The classification of Spizellomyces, Gaertneriomyces, Triparticalcar, and Kochiomyces (Spizellomycetales, Chytridiomycetes). Can J Bot 62:1171-1201

———. 1986 Allochytridium expandens rediscovered: morphology, physiology and zoospore ultrastructure. Mycologia 78:439-448

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———, Désaulniers NL., 1988 Precise configuration of the chytrid zoospore. Can J Bot 66:869-876

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———. 1996 Chytridiomycete taxonomy since 1960. Mycotaxon 60:149-174

———, Pessier AP, Nichols DK., 1999 Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 91:219-227

McNitt R., 1974 Ultrastructure of Phlyctochytrium irregulare zoospores. Cytobiologie 9:307-320

Paterson RA., 1963 Observations on two species of Rhizophydium from Northern Michigan. Trans Brit Mycol Soc 46:530-536

Powell MJ, Roychoudhury S., 1992 Ultrastructural organization of Rhizophlyctis harderi zoospores and redefinition of the type 1 microbody-lipid globule complex. Can J Bot 70:750-761

Sparrow FK., 1943 Aquatic Phycomycetes. Ann Arbor: University of Michigan Press

———. 1960 Aquatic Phycoycetes, 2nd revised ed. Ann Arbor: University of Michigan Press

Zopf W., 1887 Ueber einige niedere Algenpilze (Phycomyceten) und eine neue Methode ihre Keime aus dem Wasser zu isolieren. Abhandl Naturforsch Gesell Halle 17:77-107, 2 pls

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