Mycologia, 101(5), 2009, pp. 696–706. DOI: 10.3852/08-194 # 2009 by The Mycological Society of America, Lawrence, KS 66044-8897
Rhizidium phycophilum, a new species in Chytridiales Kathryn T. Picard1 Peter M. Letcher Martha J. Powell
and complements the evolutionary hypotheses produced by molecular analyses. This paper is the product of a biodiversity survey of Australian soils, broadly sampled from across the continent. Although a handful of mycologists examined Australian soils for zoosporic fungi in the 20th century (Jeffrey and Willoughby 1964, Willoughby 1965, Karling 1988), species richness and distribution patterns, in addition to chytrid ecology, remained poorly understood (May and Pascoe 1996). It is only within the past decade that the extensive diversity of chytrid taxa in Australian soils has been systematically and specifically explored and characterized. Recent studies combining molecular and morphological analyses (Letcher et al 2004a, b, c, Longcore 2005) have shown that Australian soils exhibit a wide range of chytrid diversity that includes taxa from across Chytridiomycota. From a soil sample collected in a biodiversity survey of chytrid fungi in New South Wales, Australia, a chytrid saprobic on pollen substrate was isolated in culture. Molecular analyses of combined partial nuclear large subunit rRNA (28 S rRNA) and ITS15.8S-ITS2 sequences place this chytrid in Chytridiales. The zoospore is unique among zoospore subtypes known in Chytridiales (Barr 1980), and on the bases of molecular and ultrastructural data this isolate is described as a new species in Chytridiales.
Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487
Abstract: Molecular and ultrastructural investigations are revealing unrealized diversity among chytrids as the taxonomic revision of order Chytridiales (Chytridiomycota) progresses. During a biodiversity survey of soil-inhabiting chytrids in Australia an undescribed chytrid was isolated from a soil sample collected in a cool temperate rainforest of New South Wales, Australia. Combined zoospore ultrastructure analysis and molecular phylogenetic analyses of partial LSU rRNA and ITS1-5.8S-ITS2 sequences demonstrated this chytrid was a new species within Chytridiales and possessed distinctive zoospore architecture previously unknown. Herein we delineate a new Rhizidium species in Chytridiales based on molecular monophyly and unique subcellular organization of the zoospore. Key words: chytrid, phylogeny, 28S rDNA, vesicles, zoospore INTRODUCTION
Unrealized diversity within Chytridiomycota is being revealed as taxonomic surveys are refined with molecular and ultrastructural analyses (Letcher et al 2008a, b). Historically taxonomists relied on thallus development (reviewed in Blackwell et al 2006) and morphological features, such as operculation, sporangial ornamentation and rhizoidal arrangement, for differentiation between families and genera (Sparrow 1960). However numerous studies (Haskins and Weston 1950, Paterson 1963, Miller 1976, Powell and Koch 1977a, b) have recognized morphological plasticity of taxa in axenic culture, resulting from differences in nutrition, pH and light exposure. Relationships among the chytrids are now defined with combined analyses of zoospore ultrastructure and molecular data (James et al 2000, 2006, Letcher et al 2005, 2006, 2008a, b). The subcellular organization of the posteriorly uniflagellate zoospore is largely conserved across genera, families and orders in chytrids (Barr 1980, Letcher et al 2006, 2008a, b)
MATERIALS AND METHODS
Isolation and culture.—A soil sample collected from an Antarctic beech (Nothofagus moorei [F. Muell.] Krasser) forest in the Gloucester Tops sector of Barrington Tops National Park (http://www.environment.nsw.gov.au/NationalParks/ parkSector.aspx?id5N0002&s520080510000000105), New South Wales, Australia, was baited with American sweetgum pollen (Liquidambar styraciflua L.). The sample was collected under NSW National Parks and Wildlife Service research license 11070 (held by PM Letcher). Zoospores discharged from thalli growing on pollen were streaked on a nutritive medium, PmTG plus antibiotics (1 g peptonized milk, 1 g tryptone, 5 g dextrose, 8 g agar, 0.5 g streptomycin sulfate, 0.5 g penicillin-G, 1 L deionized water). Thalli developed on nutrient medium only in the presence of an undescribed coccoid green alga originating from the same soil sample. Single thalli were removed from the nutrient agar, separating them from bacteria, yeast and other chytrid thalli and maintained in culture along with the coccoid alga. No nutrient medium has been found on which this isolate (designated KP 013) would grow as an axenic culture. Mixed cultures of KP 013 and
Accepted for publication 19 March 2009. 1 Corresponding author. E-mail:
[email protected]
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PICARD ET AL: NEW RHIZIDIUM CHYTRID the alga were maintained both in liquid PmTG broth and on solid PmTG medium at , 20 C. Morphology.—Cultures were observed growing in PmTG broth, and on sweetgum pollen and purified shrimp chitin strips (Bills et al 2004). Examination of thallus morphology of isolate KP 013 was performed on Nikon Labphot-2, Zeiss Nomarski differential interference contrast and Philips XL30 scanning electron microscopes. For SEM analysis samples were fixed in 2.5% glutaraldehyde in 0.1 M S-collidine buffer 4 h at 22 C, subsequently rinsed in three 15 min washes with buffer. Samples were postfixed in 4% osmium tetroxide in buffer overnight at 4 C, rinsed once in buffer, three times in DI water and dehydrated in a graded acetone series (10, 30, 50, 75, 85, 95, 100, 100%). Samples were critical-point dried with CO2, mounted on stubs using carbon pads and gold-palladium sputter-coated. Coated specimens were viewed at 20 kV. Zoospore ultrastructure.—Fixation and observation of the zoospore of isolate KP 013 followed procedures described in Letcher and Powell (2005a). Zoospores were examined on a Hitachi 7650 transmission electron microscope at 60 kV. DNA preparation.—DNA was extracted from a culture of isolate KP 013 maintained at the University of Alabama and was purified and amplified for sequencing as described (Letcher and Powell 2005a). The LROR/LR5 primer pair (White et al 1990) was used for amplification of LSU (28S) nuclear ribosomal DNA (rDNA) and the ITS5/ITS4 primer pair (White et al 1990) for the ITS1-5.8S-ITS2 rDNA region. Phylogenetic analyses.—For molecular analyses partial nucleotide sequences of the LSU rRNA gene (799–882 bp from the 59 end) and ITS1-5.8S-ITS2 sequences (847– 859 bp from the 59 end) were generated. We assembled and aligned contiguous partial LSU rDNA sequences of 31 ingroup isolates in Chytridiales and three outgroup isolates, Lobulomyces angularis and Clydaea vesicular in Lobulomycetales (Simmons et al 2009) and Rhizophlyctis rosea in Rhizophlyctidales (Letcher et al 2008a) (TABLE I) as per Letcher et al (2004c). For this study sequences for all isolates except KP 013 and JEL 378 were from previous studies or the AFTOL database (http://www.aftol.org/ index.php) (TABLE I). The LSU dataset included 1651 characters with 447 parsimony informative sites. In addition to the partial LSU sequences ITS1-5.8S-ITS2 sequences were aligned manually for three taxa (KP 013, JEL 378, JEL 116) to assess sequence similarity among closely related isolates. From Modeltest 3.7 (Posada and Crandall 1998) the Akaike information criterion was used to determine the best model of base substitution. The general time-reversible model with invariant sites and gamma distribution (GTR + I + C) was chosen and implemented in both maximum parsimony (MP) and maximum likelihood (ML) analyses. ML analyses were performed in GARLI 0.951 (Zwickl 2006). The run was repeated 10 times from random starting trees with the autoterminate setting. GARLI also was used to generate 100 nonparametric bootstrap replicates from which a majorityrule consensus tree was calculated in PAUP* 4.0b10 (Swafford 2002). For MP analyses a heuristic search (1000 replicates) was carried out in PAUP* with MAXTREES set to 100, random
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taxon addition sequences, and branch-swapping set to tree bisection-reconnection (TBR). Nonparametric bootstrap support was assessed for MP with 1000 pseudoreplicates. RESULTS
Habitat.—The Gloucester Tops (32uS, av. elev. 442 m) are sedimentary and granitic mountains carved from an extinct volcano, , 200 km N of Newcastle, NSW, Australia. Isolate KP 013 was collected in late fall (May 2005) from a pocket of cool temperate rainforest dominated by Antarctic beech and with a thick groundcover of ferns and mosses. Morphology.—In PmTG broth the zoospore cyst of isolate KP 013 was spherical (FIG. 1A), a shape maintained throughout sporangial development (FIG. 1A–F). Mature sporangia were typically 40– 50 mm diam. Under examination by bright field microscopy the wall of the sporangium appeared to be ornamented with small projections and rods (FIG. 1C). SEM analysis of the sporangium revealed a reticulate ornamentation that became increasingly pronounced as the thallus matured (FIG. 2). The monaxial rhizoid system occasionally exhibited a subsporangial swelling in the rhizoidal axis early in development (FIG. 1B) and became increasingly divaricated as the thallus matured (FIG. 1D). As the thallus matured on pollen a gelatinous plug forming proximal to a single discharge pore in the sporangium became visible (FIG. 1E, F). On pollen, thalli were frequently interbiotic but could be epibiotic (FIG. 1E, F). There was typically a stout primary rhizoidal axis (FIG. 1E, F) that entered the pollen grain directly with no branching external to the substrate (FIG. 1F) or that branched distal to the sporangium and entered multiple pollen grains (FIG. 1E). The discharge opening was best observed on empty sporangia on pollen where a torn fragment of the exit papilla remained at the edge of the discharge pore (FIG. 1H). On chitin, thalli were primarily interbiotic although they could develop epibiotically. The rhizoids consisted of a long primary axis with minimal branching (FIG. 1G, J). Zoospore discharge was most easily observed on chitin where a quiescent mass of zoospores emerged surrounded by a mucilaginous vesicle through an inoperculate discharge pore in the sporangium in 2–3 min (FIG. 1G). The mass floated slightly away from the discharge pore and was spherical or citriform. After remaining motionless 2–4 min zoospores began to quiver and move rapidly, eventually swimming away as individual oval zoospores. On both refractory substrates, sporangia could become unusually large (40–80 mm diam) and globose or irregularly shaped when crowded (FIG. 1I).
698 TABLE I. Strain
MYCOLOGIA Taxon sampling for phylogenetic analysis Taxon
28S: GenBank ID
Ingroup Barr 097 JEL 006 JEL 030 JEL 034 JEL 047 JEL 057 JEL 059 JEL 065 JEL 091 JEL 102 JEL 103 JEL 116 JEL 129 JEL 137 JEL 161 JEL 165 JEL 176 JEL 186 JEL 187 JEL 220 JEL 221 JEL 341 JEL 347 JEL 354 JEL 378 KP 013 MP 004 PL 006 PL 115 UCB 81-1 WB 235A
Chytriomyces sp. Rhizoclosmatium globosum Podochytrium dentatum Unidentified sp. C Phlyctochytrium planicorne Obelidium mucronatum Chytriomyces spinosus Unidentified sp. B Chytriomyces appendiculatus Siphonaria petersenii Unidentified sp. D Unidentified sp. A Entophlyctis luteolus Physocladia obscura Podochytrium sp. Chytriomyces appendiculatus Chytriomyces sp. Asterophlyctis sarcoptoides Unidentified sp. F Entophlyctis sp. Rhizidium endosporangiatum Unidentified sp. I Unidentified sp. E Unidentified sp. H Rhizidium sp. Rhizidium phycophilum Chytriomyces hyalinus Rhizoclosmatium globosum Chytriomyces sp. Chytriomyces hyalinus Unidentified sp. G
AY439074 AY439061 FJ861603
Outgroup Barr 186 JEL 045 PL 070
Rhizophlyctis rosea Lobulomyces angularis Clydaea vesicula
AY349079 DQ273815 AY988507
Resting spores were not observed on any substrates or media used. Zoospore ultrastructure.—The zoospore of isolate KP 013 (FIGS. 3, 4) is ovate to subspherical, 3 mm wide and 5 mm long. A cell coat (FIG. 4A) covers the plasma membrane of the zoospore. Ribosomes are aggregated in the center of the zoospore and partially surrounded by endoplasmic reticulum (FIG. 4A). A single lipid globule is located laterally or centrally within the cell and partially within the ribosomal aggregation (FIG. 4A). Appressed to the lipid globule are a microbody (FIG. 4A) and a simple cisterna (FIG. 4E). Multiple mitochondria are within and at the periphery of the ribosomal mass and often cluster in the vicinity of the kinetosome (FIG. 4A). The nucleus is located in the peripheral cytoplasm at the
DQ273813 AY439071 DQ273839 AY988508 AY439077 AY439072 AY349068 DQ273833 AY442957 AY439062 AY988571 AY439076 AY349064 AY439070 DQ273783 AY439063 DQ273834 DQ273831 DQ273769 DQ273785 DQ273832 FJ214802 DQ273836 AY439055 AY988516 AY988510 DQ536493
ITS: GenBank or AFTOL ID
AFTOL 1539 FJ861603
AFTOL 1533
FJ214804 FJ214803
anterior end of the zoospore, outside the ribosomal aggregation (FIG. 4A). A paracrystalline inclusion (FIG. 4C, D) and two distinctive vesicles (FIG. 4A, B) also are in the peripheral cytoplasm in the anterior portion of the zoospores. These vesicles are membrane bound and contain an unidentified granular substance. The kinetosome and nonflagellated centriole are parallel and connected by a fibrillar bridge (FIG. 4F). A biconcave electron-opaque plug is at the base of the flagellum (FIG. 4A, F, G), and nine large (wide) props surround the distal end of the kinetosome attaching it to the plasma membrane (FIG. 4G). Phylogenetic and ultrastructural analyses.—The MP phylogeny was congruent to the ML phylogeny with similar or equal support values at major nodes (FIG. 5). Each phylogeny placed isolate KP 013 sister
PICARD ET AL: NEW RHIZIDIUM CHYTRID of isolate JEL 378, an undescribed taxon (FIG. 5, clade A). Sister of this clade was another undescribed species, isolate JEL 116. Sequence similarities between KP 013 and JEL 378 were 98.5% (LSU rRNA) and 84.6% (ITS1-5.5S-ITS2 rRNA). Sequence similarities between KP 013 and JEL 116 were 95.7% (LSU rRNA) and 63.3% (ITS1-5.5S-ITS2 rRNA). Sister of JEL 116 (FIG. 5) was a clade composed of two Podochytrium isolates (P. dentatum [JEL 030] and Podochytrium sp. [JEL 161]). The LSU rRNA sequences of the Podochytrium isolates were approximately 91% similar to KP 013, and ITS1-5.5S-ITS2 sequences were approximately 58% similar. Twenty-seven isolates including taxa in genera Podochytrium, Obelidium, Siphonaria, Rhizoclosmatium, Chytriomyces, Entophlyctis, Physocladia and Asterophlyctis formed a clade with moderate bootstrap support (FIG. 5, clade B). Three isolates, JEL 221, JEL 354 and JEL 341, were sister of clade B (FIG. 5). Phlyctochytrium planicorne (JEL 047) was sister of all other isolates in the ingroup. All isolates examined in clade B (FIG. 5) with the exception KP 013, and JEL 378 had a Group I-type zoospore (Barr 1980, Letcher et al 2005). Zoospores of isolates JEL 221, JEL 354 and JEL 341 are undescribed. Isolate JEL 047, Phlyctochytrium planicorne, has a Group II-type zoospore (Barr 1980, Letcher and Powell 2005b). TAXONOMY
Rhizidium phycophilum Picard, sp. nov. FIGS. 1A–J, 2, 4A–G MycoBank 513127 Fungus saprophyticus. Thallus monocentricus, eucarpicus, e sporangio sessili aut intervitum, apophysatis vel nonapophysatis. Sporangia 40–50 mm diam, sphericus cum unum papillis emissionibus inoperculatus. Zoosporae cum cisterna simplex in globuli lipoidei superficie, microbody simplex, flagellum obturamentum. Sporis perdurantibus ignotis. Typus. Picard et al 2009, Mycologia 101: 696–706, FIGS. 1A–J, 2, 4A–G (2009). Diagnosis cultura KP 013 ex pollonis esca posita in aquacultura ex terra Barrington Tops NP, New South Wales, Australia, GPS locus 32u039170S 3 151u379 290E. GenBank LSU rDNA sequence FJ214802, ITS1–5.8S– ITS2 sequence FJ214803.
Fungus saprophytic. Thallus monocentric, eucarpic, with sessile or interbiotic spherical or irregular sporangia. Rhizoid consisting of a stout primary axis, which could branch at its extremities and which could be nonapophysate or apophysate. In culture on nutrient agar sporangium spherical at maturity, 40–50 mm diam, with a single inoperculate discharge pore. Zoospore with a single lipid globule partially covered with a simple cisterna and a microbody.
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Flagellum with an electron-opaque plug in the base. Resting spores unknown. Etymology: Latin; phycophilum refers to the association between the chytrid and alga. Type: Picard et al 2009, Mycologia 101: 696–706, 1A–J, 2, 4A–G (2009) from observations of isolate KP 013 from pollen bait placed with water cultures of soil from Gloucester Tops NP, New South Wales, Australia, GPS coordinates 32u039170S 3 151u379290E. GenBank LSU rDNA sequence FJ214802, ITS1–5.8S–ITS2 sequence FJ214803. Culture KP 013, on which the type is based, is being deposited at Centraalbureau voor Schimmelcultures. In broth individual sporangia of R. phycophilum were spherical. Zoospores were discharged from a single inoperculate discharge pore. The mature rhizoidal system was an isodiametric rhizoidal axis with divaricate branched rhizoids. Rhizidium phycophilum colonized pine, spruce, sweetgum and Typha pollen, and chitin. Thalli were interbiotic and sometimes epibiotic on natural substrates. On pollen grains, mature sporangia were similar to those in broth, with globose sporangia and branched rhizoids protruding from a single axis. On chitin, mature sporangia were similar to those in broth and became irregularly shaped when crowded. Rhizoids on chitin and pollen were characterized by a large, trunk-like single axis with markedly less secondary branching than that seen in broth. Specimen examined: AUSTRALIA. NEW SOUTH WALES: Gloucester Tops NP, 32u039170S 3 151u379290E, elevation 907 m ASL. On pollen substrate from soil, 15 May 2005, culture number KP 013 collected by Peter M. Letcher and isolated by Kathryn T. Picard, HOLOTYPE. DISCUSSION
Studies have demonstrated that Chytridiales is a polyphyletic order comprising multiple monophyletic lineages, each of which has a zoospore type with a distinctive suite of ultrastructural characters (James et al 2006). In an effort to achieve monophyly for Chytridiales three of those lineages have been circumscribed at the ordinal level on the basis of molecular monophyly and unique zoospore ultrastructure (Rhizophydiales: Letcher et al 2006, Lobulomycetales: Simmons et al 2009, Cladochytriales: Mozley-Standridge et al 2009), while other lineages are in the process of taxonomic revision. Morphology.—Rhizidium is a member of the Chytriomyces clade of Chytridiales (James et al 2006), a lineage of organisms that are morphologically diverse yet ultrastructurally conserved (Letcher et al 2005). Although several taxa in the Chytriomyces clade are
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FIG. 1. Morphology of Rhizidium phycophilum in PmTG broth and on natural substrates of pollen and chitin. A. Germling in PmTG broth. B. Developing thallus in PmTG broth. C. Collapsed sporangium, exhibiting sporangial ornamentation in PmTG broth. D. Mature thallus illustrating a main primary axis with extensive secondary rhizoidal branching in PmTG broth. E. Interbiotic thallus with single primary rhizoidal axis branching terminally, with branches entering multiple pollen grains. Mature sporangium with hyaline discharge plug (arrow). F. Mature sporangium with cleaved zoospores and discharge plug
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FIG. 2. Scanning electron micrograph showing reticulate ornamentation on the sporangium of Rhizidium phycophilum. Smooth-surface coccoid algae are in the background. Bar 5 20 mm.
morphologically distinct, others have simple thalli that complicate taxon identification. In addition to R. phycophilum in the Rhizidium clade is isolate JEL 378. The thalli of JEL 378 and R. phycophilum have several morphological characters in common, most notably ornamented sporangia and a monaxial rhizoidal system (Longcore 2005). Like R. phycophilum, JEL 378 also has a single, inoperculated discharge pore adjacent to a gelatinous plug of hyaline material (Longcore 2005). In this study Rhizidium is ostensibly paraphyletic (FIG. 5). Isolate JEL 221, identified as Rhizidium endosporangiatum, is sister of an unidentified taxon, isolate JEL 354. Clarification of the generic placement of R. endosporangiatum and JEL 354 will require further study. Sister of Rhizidium clade is JEL 116, which is not available for morphological study. One node further is a clade composed of Podochytrium dentatum (JEL 030) and Podochytrium sp. (JEL 161). Rhizidium and Podochytrium taxa share many common chytridialian characteristics, including a single anterior discharge pore, vesiculate discharge of zoospores and a subsporangial swelling at the base of the rhizoidal axis. Nonetheless Podochytrium dentatum is easily distin-
FIG. 3. Schematic drawing of longitudinal section through the zoospore of Rhizidium phycophilum. CC, cell coat. F, flagellum; FB, fibrillar bridge; FP, flagellar plug; K, kinetosome; L, lipid globule; M, mitochondrion; Mb, microbody; N, nucleus; NfC, nonflagellated centriole; P, prop; PCI, paracrystalline inclusion; R, ribosomes; SC, simple cisterna; V, vesicle.
guished morphologically from R. phycophilum by its bell-shape sporangia, orange pigmentation (Longcore 1992) and sporangial ornamentation, which consists of paired dentate protuberances, as compared to the reticulate surface of Rhizidium sporangia. Further the rhizoids of P. dentatum can assume two morphologies in culture, fine (tips , 0.7 mm) and blunt (tips ,1.6 mm) (Longcore 1992), whereas the rhizoids of most Chytridialian taxa including Rhizidium are slender. Placement of isolate KP 013 within Rhizidium is based on the original description of the genus, the type of which is Rhizidium mycophilum (Sparrow
r (arrow). Thallus growing interbiotically, with single stout rhizoidal axis. G. Thallus on chitin. Vesiculate discharge of zoospores from sporangium bearing a single large discharge pore. H. Thallus on pollen. Torn flap of sporangial wall. I. Irregularly shaped sporangia on chitin when thalli were crowded. J. Thallus growing on chitin demonstrating the long, unbranched primary rhizoidial axis. Scale bar in A 5 10 mm (for A, C, E, H, I), 15 mm (for F) and 20 mm (for B, D, G, J).
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FIG. 4. Zoospore ultrastructure of Rhizidium phycophilum. A. Longitudinal section. B. Longitudinal section through anterior vesicle. C. Longitudinal section through paracrystalline inclusion. D. Transverse section through paracrystalline inclusion, located between two anterior vesicles. E. Longitudinal section through simple cisterna (arrow) appressed to the lipid globule. F. Fibrillar bridge (arrow) between kinetosome and nonflagellated centriole. G. Flagellar prop (arrow). Bars: A 5 1 mm; B, E 5 0.5 mm; C, D 5 0.75 mm; F, G 5 0.87. (List of abbreviations FIG. 3).
1960). The fundamental distinguishing features of the genus that are present in KP 013 include (i) a stout uniaxial rhizoidal system that persists throughout development (FIG. 1B, D, F, J), (ii) the occasional presence of a subsporangial swelling on the primary rhizoidal axis on natural substrate (FIG. 1B, J) and (iii) the method of vesiculate discharge in which
quiescent zoospores are released into a motionless subspherical mass that floats away from the point of discharge before deliquescing. Although more recently delimited Rhizidium species may feature multiple discharge pores or lack a trunk-like primary rhizoidal axis, the three enumerated morphological features were included in the original generic
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FIG. 5. Maximum likelihood tree of 31 taxa in order Chytridiales from dataset of partial LSU rDNA sequences. Isolates JEL 045 (Lobulomyces angularis), PL 070 (Clydaea vesicula) and Barr 186 (Rhizophlyctis rosea) served as outgroups. Isolates JEL 034, JEL 065, JEL 103, JEL 116, JEL 187, JEL 341, JEL 347, JEL 354 and WB 235A are unidentified isolates in Chytridiales. Numbers above branches or to the left of slashes indicate support above 70% in 100 bootstrap replicates with likelihood analysis. Numbers below branches or to the right of slashes represent support above 70% in 1000 bootstrap replicates with parsimony analysis.
concept of Rhizidium and thus served as benchmarks in ascribing R. phycophilum to this genus. R. phycophilum shares both morphological and ecological characteristics with three previously described Rhizidium species. R. verrucosum, R. reniformis and R. elongatum, like R. phycophilum, all are soildwelling, chitinophilic chytrids. Further aspects of sporangial discharge and development are often similar to those found in isolate R. phycophilum, yet R. phycophilum is distinct from any of these delineated species. For example R. phycophilum and R. verrucosum both present spherical, ornamented sporangia. However in the latter sporangia possess small, warty protuberances whereas R. phycophilum sporangia exhibit a reticulated and dimpled surface. R. verrucosum also differs in its manner of zoospore discharge, where zoospores are released from several
tubular discharge papillae (Karling 1944). R. phycophilum and R. reniformis (Karling 1970) exhibit the same vesiculate discharge, including the adherence of the sporangial wall to the edge of the discharge pore, mimicking a small operculum. Yet the sporangium of R. reniformis is neither globose nor ornamented. Although R. elongatum (Karling 1949) and R. phycophilum have similar discharge patterns and structures, such as the hyaline plug adjacent to the discharge pore, R. elongatum has a long, irregular sporangium and thalli often lack the characteristic trunk-like primary rhizoidal axis. Other chitin-degrading Rhizidium species can be differentiated from isolate R. phycophilum according to morphological inconsistencies, such as smaller sporangial size (R. mycophilum, R. chitinophilum), number and type of discharge pores (R. braziliensis)
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and the size of subsporangial swelling (R. laevis). Many remaining Rhizidium species can be dismissed based on the use of different nutrient sources, such as cellulose, and habitat type. Finally, a number of Rhizidium species have been described as colonizing the mucilaginous sheaths of green algae (R. mycophilum and R. nowakowskii) or parasitizing the algal cells themselves (R. braunii and R. windermerense). Although the exact interaction between R. phycophilum and its algal cohort is not yet fully understood, it is not a parasitic relationship (Picard unpubl) and thus would not ally R. phycophilum with these taxa. Based on these morphological and ecological characteristics, we are declaring a new species, Rhizidium phycophilum. Because neither a type specimen nor an illustration of the type exists, and as the limitations of Rhizidium are poorly defined, identifying a new species within Rhizidium is difficult. It is possible that with the recovery of the type or in the course of taxonomic revision that R. phycophilum will be removed from Rhizidium. However at present in the interest of avoiding taxonomic inflation we are including R. phycophilum within this genus. Zoospore ultrastructure.—The Chytriomyces clade currently is represented by two zoospore subtypes, known as Group I and Group II (Barr and Hartmann 1976, Barr 1980, Letcher and Powell 2005b, Letcher et al 2005). The two subtypes have many characters and character states in common, such as aggregated ribosomes, multiple mitochondria, a Type 1 MLC (Powell 1978), kinetosomeassociated structures, a microtubular root, Golgi bodies, a paracrystalline inclusion in the peripheral cytoplasm and an electron-opaque plug in the base of the flagellum. Each subtype has a distinct suite of character states that serve to distinguish it. The zoospore of R. phycophilum has a subcellular organization more similar to that of the Group I subtype, which is found in nine genera of Chytridiaceae (Letcher et al 2005), including Podochytrium (Longcore 1992), sister of Rhizidium (FIG. 5). The characters held in common between the Group I subtype and R. phycophilum’s zoospore include the anterior placement of the nucleus, the biconcave flagellar plug, a single laterally or centrally located lipid globule, the paracrystalline inclusion, the presence of a cell coat (Dorward and Powell 1983, Powell 1994) and placement of the mitochondria within the ribosomal mass. However the ultrastructure of the Rhizidium zoospore has a unique set of characters that differentiates it from both the Group I and Group II subtypes: the MLC has a simple nonfenestrated cisterna, the microtubular root and
its allied structures have been lost (are absent), the paracrystalline inclusion is reduced in size and the kinetosome props are more robust. Rhizidium phycophilum also exhibits distinctive vesicles at the anterior face of the zoospore. These vesicles are unique, not only in Chytridiales but in Chytridiomycota. Other chytrid taxa, such as Zygorhizidium planktonicum (Beakes et al 1988, Canter et al 1992), Rhizophydium planktonicum (Beakes et al 1993), Kappamyces laurelensis (Letcher and Powell 2005a), Alphamyces chaetiferum (Letcher et al 2008b) and Pateramyces corrientinensis (Letcher et al 2008b), also possess prominent vesicles, but in these taxa the vesicles are located laterally and presumably associated with the MLC fenestrated cisterna (Z. planktonicum) or posteriorly and presumably associated with the kinetosome. Cytochemical studies have localized carbohydrates within smaller vesicles in the peripheral cytoplasm of zoospores of Chytriomyces species (Dorward and Powell 1983, Powell 1994) indicative of vesicles capable of forming an encasement layer or releasing adhesion material at encystment. While the function of these vesicles remains unclear, we hypothesize they might play a role in cellular adhesion to a substrate before zoospore encystment (Durso et al 1993). The zoospores of R. phycophilum and JEL 378 are similar (unpubl obs) with both taxa exhibiting these prominent anterior vesicles. Further critical ultrastructural analyses of these two isolates will be necessary to determine both the function and fate of these vesicles. Isolates JEL 030 and JEL 161, the second-nearest neighbors to R. phycophilum and JEL 378, have a Group I subtype zoospore, as do the seven other named genera and several undescribed taxa encompassed by clade B in the phylogeny. Further sampling might locate additional isolates in this clade and clarify ultrastructural ambiguities. Ecology.—The diverse organisms of Chytridiales occupy specific ecological niches, including soils, aquatic systems, the terrestrial/aquatic interface (Willoughby 1961) and epiphytic mats of soil-like material in the canopy of temperate and tropical rainforests (Powell 1993, Longcore 2005). R. phycophilum is a soil chytrid, but its apparent association with a green alga raises questions about potential roles for chytrids in terrestrial habitats. The discovery of R. phycophilum is an indicator of the considerable unrealized molecular and ultrastructural diversity that exists in Chytridiomycota. Additional and extensive sampling from disparate habitats is necessary for a better understanding of the evolutionary history of the basal fungi.
PICARD ET AL: NEW RHIZIDIUM CHYTRID 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 Joyce Longcore for providing chytrid cultures and the Assembling the Fungal Tree Of Life (AFTOL) Project, Duke University, (NSF DEB-0228668) for access to their database. In addition Kathryn Picard thanks the Howard Hughes Medical Institute for a Science Program Grant given to The University of Alabama and the National Science Foundation’s REU and Bridge to the Doctorate programs for both training opportunities and support.
LITERATURE CITED
Barr DJS. 1980. An outline for the reclassification of the Chytridiales, and for a new order, the Spizellomycetales. Can J Bot 58:2380–2394. ———, Hartmann VE. 1976. Zoospore ultrastructure of three Chytridium species and Rhizoclosmatium globosum. Can J Bot 54:2000–2013. Beakes GW, Canter HM, Jaworski GHM. 1988. Zoospore ultrastructure of Zygorhizidium affluens and Z. planktonicum, two chytrids parasitizing the diatom Asterionella formosa. Can J Bot 66:1054–1067. ———, ———, ——— . 1993. Sporangium differentiation and zoospore fine-structure of the chytrid Rhizophydium planktonicum, a fungal parasite of Asterionella formosa. Mycol Res 97:1059–1074. Bills GF, Christensen M, Powell MJ, Thorn G. 2004. Saprobic soil fungi. In: Mueller GM, Bills G, Foster MS, eds. Biodiversity of fungi: inventory and monitoring methods. Elsevier. p 271–302. Blackwell WH, Letcher PM, Powell MJ. 2006. Thallus development and the systematics of Chytridiomycota: an additional developmental pattern represented by Podochytrium. Mycotaxon 97:91–109. Canter HM, Jaworski GHM, Beakes GW. 1992. Formae speciales differentiation in the chytrid Zygorhizidium planktonicum Canter, a parasite of the diatoms Asterionella and Synedra. Nova Hedwigia 55:437–455. Dorward DW, Powell MJ. 1983. Cytochemical detection of polysaccharides and the ultrastructure of the cell coat of zoospores of Chytriomyces aureus and Chytriomyces hyalinus. Mycologia 75:209–220. Durso L, Lehnen LP, Powell MJ. 1993. Characteristics of extracellular adhesions produced during Saprolegnia ferax secondary zoospore encystment and cytospore germination. Mycologia 85:744–755. Haskins RH, Weston WH. 1950. Studies in the lower Chytridiales I: factors affecting pigmentation, growth and metabolism of a strain of Karlingia (Rhizophlyctis) rosea. Am J Bot 37:739–750. James TY, 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.
705
———, 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. Jeffrey JM, Willoughby LG. 1964. A note on the distribution of Allomyces in Australia. Nova Hedwigia 7:505–515. Karling JS. 1944. Brazilian chytrids II: new species of Rhizidium. Am J Bot 31:254–261. ———. 1949. Two new eucarpic inoperculate chytrids from Maryland. Am J Bot 39:681–687. ———. 1970. Some zoosporic fungi of New Zealand. Arch Mikrobiol 70:266–287. ———. 1988. Additional zoosporic fungi from Australian soils. Nova Hedwigia 46:239–253. Letcher PM, Powell MJ. 2005a. Kappamyces, a new genus in the Chytridiales (Chytridiomycota). Nova Hedwigia 80: 115–133. ———, ———. 2005b. Phylogenetic position of Phlyctochytrium planicorne (Chytridiales, Chytridiomycota) based on zoospore ultrastructure and partial nuclear LSU rRNA gene sequence analysis. Nova Hedwigia 80:135–146. ———, McGee PA, Powell MJ. 2004a. Zoosporic fungi from soils of New South Wales. Australas Mycol 22:99–115. ———, ———, ——— . 2004b. Distribution and diversity of zoosporic fungi from soils of four vegetation types in New South Wales, Australia. Can J Bot 82:1490–1500. ———, Powell MJ, Chambers JG, Holznagel WE. 2004c. Phylogenetic relationships among Rhizophydium isolates from North America and Australia. Mycologia 96: 1339–1351. ———, ———, ——— , Longcore JE, Churchill PF, Harris PM. 2005. Ultrastructural and molecular delineation of the Chytridiaceae (Chytridiales). Can J Bot 83:1561– 1573. ———, ———, Churchill PF, Chambers JG. 2006. Ultrastructural and molecular phylogenetic delineation of a new order, the Rhizophydiales (Chytridiomycota). Mycol Res 110:898–915. ———, ———, Barr DJS, Churchill PF, Wakefield WS, Picard KT. 2008a. Rhizophlyctidales—a new order in Chytridiomycota. Mycol Res 112:1031–1048. ———, Ve´lez CG, Barrantes ME, Powell MJ, Churchill PF, Wakefield WS. 2008b. Ultrastructural and molecular analyses of Rhizophydiales (Chytridiomycota) isolates from North America and Argentina. Mycol Res 112: 759–782. Longcore JE. 1992. Morphology, occurrence, and zoospore ultrastructure of Podochytrium dentatum sp. nov. (Chytridiales). Mycologia 84:183–192. ———. 2005. Zoosporic fungi from Australian and New Zealand tree-canopy detritus. Aust J Bot 53:259– 272. May TW, Pascoe IG. 1996. History of the taxonomic study of Australian fungi. In: Fungi of Australia. Vol. 1A. Canberra: ARBS. p 171–206. Miller CE. 1976. Substrate influenced morphological variations and taxonomic problems in freshwater, posteriorly uniflagellate phycomycetes. In: Jones EBG, ed. Recent advances in aquatic mycology. London: Elek Science. p 459–487.
706
MYCOLOGIA
Mozley-Standridge SE, Letcher PM, Longcore JE, Porter D, Simmons DR. 2009. Cladochytriales—a new order in Chytridiomycota. Mycol Res 113:498–507. Paterson RA. 1963. Observations on two species of Rhizophydium from northern Michigan. Trans Brit Mycol Soc 46:530–536. Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818. Powell MJ. 1978. Phylogenetic implications of the microbody-lipid globule complex in zoosporic fungi. Biosystems 10:167–180. ———. 1993. Looking at mycology with a Janus face: a glimpse at Chytridiomycetes active in the environment. Mycologia 85:1–20. ———. 1994. Production and modifications of extracellular structures during development of Chytridiomycetes. Protoplasma 181:123–141. ———, Koch WJ. 1977a. Morphological variations in a new species of Entophlyctis I: The species concept. Can J Bot 55:1668–1685. ———, ———. 1977b. Morphological variations in a new species of Entophlyctis II. Influence of growth conditions on morphology. Can J Bot 55:1686–1695.
Simmons DR, James TY, Meyer AF, Longcore JE. 2009. Lobulomycetales, a new order in the Chytridiomycota. Mycol Res 113:450–460. Sparrow FK. 1960. Aquatic phycomycetes. 2nd ed. Ann Arbor, Michigan: Univ. Michigan Press. 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. Willoughby LG. 1961. The ecology of some lower fungi at Esthwaite Water. Trans Brit Mycol Soc 44:305–332. ———. 1965. A study of Chytridiales from Victorian and other Australian soils. Arch Microbiol 52:101–131. Zwickl DJ. 2006. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion [Doctoral dissertation]. The University of Texas at Austin.
