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Perry F. Churchill. Department of ..... Australia, Frank Gleason Collection; JEL 5 University of Maine, Joyce E. Longcore Collection; MP 5 University of Alabama,.
Mycologia, 102(3), 2010, pp. 596–604. DOI: 10.3852/09-120 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897

A molecular phylogenetic evaluation of the Spizellomycetales William S. Wakefield Martha J. Powell Peter M. Letcher1 Donald J.S. Barr Perry F. Churchill

INTRODUCTION

Chytridiomycota (chytrids) are common in soil (Powell 1993), but because they reproduce with zoospores they sometimes are erroneously considered as occurring only in aquatic habitats. Members of order Spizellomycetales are among the most prevalent soil-inhabiting chytrids (Lozupone and Klein 2002, Waldrop et al. 2006) and are found almost exclusively in soil and near-shore habitats, including harsh environments such as arid grasslands, canopy soils, sand dunes, alpine soils, halomorphic soils, glacial till and Arctic clay (Barr 1969, 1970a, b, c, 1980, 1984b; Booth 1969; Booth and Barrett 1971; Gleason et al. 2004, 2008; Longcore 2005; Meyer 2004; Powell 1993). Because these organisms are readily recovered from crop soils and disturbed habitats (Barr 1984b, Luzupone and Klein 2002) researchers are now focusing on their roles in soil nutrient dynamics (Gleason et al. 2004, Lozupone and Klein 2002, Meyer 2004, Midgley et al. 2006). Moreover, as parasites of arbuscular mycorrhizae (Ross and Ruttencutter 1977), nematodes and oospores of downy mildews (Kenneth et al. 1975, Person et al. 1955), they may be important members of soil microbial communities influencing plant production, both adversely and beneficially. Thus exploration of the diversity of spizellomycetalean chytrids is vital in understanding soil fertility and sustainability. Barr (1980) recognized fundamental differences in zoospore ultrastructure of some cultured members of the Chytridiales and separated out the Spizellomycetales as a new order, initially including eight genera (Spizellomyces, Gaertneriomyces, Kochiomyces, Triparticalcar, Entophlyctis, Rhizophlyctis, Olpidium and Rozella). Members of the Spizellomycetales produce zoospores with a constellation of characters distinguishing them from other chytrids: (i) the nucleus is structurally or spatially associated with the kinetosome; (ii) the nonflagellated centriole is positioned at an angle to the kinetosome; (iii) ribosomes are dispersed in the cytoplasm; (iv) an electron-opaque core in the flagellar transition zone is absent; (v) a microtubule root extends from a kinetosome-associated structure (microtubule-organizing center) near the apex of the kinetosome; (vi) organelles of the microbody-lipid globule complex (MLC, Powell 1978) are loosely organized; and (vii) the MLC cisterna (when present) is not fenestrated. Since the establishment of the Spizellomycetales (Barr 1980) its generic composition has changed with

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

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

Shu-Fen Chen Department of Health and Nutrition, Chia-Nan University of Pharmacy and Science, Tainan 71710, Taiwan

Abstract: Order Spizellomycetales was delineated based on a unique suite of zoospore ultrastructural characters and currently includes five genera and 14 validly published species, all of which have a propensity for soil habitats. We generated DNA sequences from small (SSU), large (LSU) and 5.8S ribosomal subunit genes to assess the monophyly of all genera and species in this order. The 53 cultures analyzed included isolates on which all described species were based, plus other spizellomycetalean cultures. Phylogenetic placement of these chytrids was explored with maximum parsimony and maximum likelihood analyses, both of which yielded comparable topologies. Kochiomyces, Powellomyces and Triparticalcar were monophyletic, while Gaertneriomyces and Spizellomyces were polyphyletic. Isolates, distinct from described species, clustered among each of the five genera, indicating that species diversity in genera is greater than currently recognized. One isolate formed a clade that included no described species, representing a new genus. Zoospore ultrastructural features and architecture seem to be good indicators of phylogenetic relationships, but finer scrutiny of characters such as kinetosome-associated structures (KAS) is needed to understand more clearly the diversity within this order as it is revised. Key words: chytrid, Gaertneriomyces, habitats, Kochiomyces, Powellomyces, Spizellomyces, Triparticalcar

Submitted 22 May 2009; accepted for publication 23 Sep 2009. 1 Corresponding author. E-mail: [email protected]

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WAKEFIELD ET AL.: SPIZELLOMYCETALES genera added and removed. When determining the generic placement of a new species of chytrid with Entophlyctis-type of development (eucarpic thalli with exogenous development) Longcore (1995) noted that the type of Entophlyctis was aquatic and that aquatic species with Entophlyctis-type of development had a chytridialean zoospore. Accordingly Entophlyctis was transferred from the Spizellomycetales to the Chytridiales and a new genus in the Spizellomycetales, Powellomyces, was described for soil-inhabiting species previously considered members of Entophlyctis (Longcore 1995, Longcore et al. 1995). Barr (2001) transferred the genus Caulochytrium to the order based on features of its zoospore (Powell 1981). Recent molecular phylogenetic studies removed four genera from the Spizellomycetales. Barr and De´saulniers (1986) presaged the diversity within Rhizophlyctis rosea, which is the type of Rhizophlyctis (Blackwell and Powell 1999, Clements and Shear 1931), when they discovered four zoospore ultrastructural subtypes among 10 isolates identified as R. rosea. In a molecular and ultrastructural investigation of a wide geographic sampling of 49 isolates of this morphospecies (Bernstein 1968), including representative of all four zoospore types (Barr and De´saulniers 1986), Letcher et al. (2008) demonstrated great genetic diversity and removed the R. rosea clade from the Spizellomycetales, describing it as a new order, the Rhizophlyctidales. A six-gene phylogeny provided evidence that the endoparasites Olpidium brassicae diverged among the Zygomycota, and Rozella was basal to other members of the Chytridiomycota. Because type species of Olpidium and Rozella were not represented in the analysis ( James et al. 2006a), and pending additional sampling of taxa and genes, these two genera are classified incertae sedis (Hibbett et al. 2007). Ribosomal gene sequences of Caulochytrium (AFTOL ID No. 2017 http://www.aftol.org/ data.php) similarly places Caulochytrium protostelioides outside the Spizellomycetales (Karpov et al. 2009), but because this is not the type species the classification of Caulochytrium is incertae sedis. Thus five genera (Kochiomyces, Spizellomyces, Triparticalcar, Gaertneriomyces and Powellomyces) and 14 validly published species currently comprise the Spizellomycetales. The monophyly of these genera and species has never been assessed with molecular analyses. In the present phylogenetic study we analyzed DNA sequences from small (SSU), large (LSU) and 5.8S ribosomal subunit genes of 53 isolates in the Spizellomycetales, including all cultures on which taxa were described, plus four isolates in the Rhizophlyctidales. Our objective was to determine the support for relationships within the order and the genetic variation and monophyly of genera.

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MATERIALS AND METHODS

Sequences.— We used 53 isolates of spizellomycetalean chytrids and four Rhizophlyctidales in this study (TABLE I). Isolates PL 042 (Rhizophydiales) and Barr 097 (Chytridiales), orders in Chytridiomycetes, were used as outgroup taxa. Cultures were grown in PmTG broth (Barr 1986; 0.5 g peptonized milk, 0.5 g tryptone, 5.0 g glucose, 1 L distilled water). DNA was extracted as described by Letcher and Powell (2005). Complete ITS 1-5.8S-ITS 2 (586–722 bp from the 59 end) and partial large subunit rRNA (874–908 bp from the 59 end) and small subunit rRNA (1048–1083 bp from the 59 end) genes were sequenced with these primer pairs: ITS5/ITS4, LROR/LR5 and NS1/NS4 (White et al. 1990) respectively. Combined sequences were assembled and aligned with Clustal X (Thompson et al. 1997) and BioEdit (Hall 1998) with nonalignable ITS 1 and ITS 2 regions removed. Manual adjustments were made to the alignment based on secondary structure that includes variability in loop and stem structures (Ben Ali et al. 1999). Sequence alignment will be deposited in TreeBASE. Phylogenetic analysis.—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 (GTR) plus proportion of invariable sites (I) and gamma distribution (G) model of evolution was indicated as the best fit for the dataset. Maximum parsimony (MP) trees were constructed with PAUPRat (Sikes and Lewis 2001), and branch support was determined as described by Letcher et al. (2004). Tree-space was searched heuristically with TBR, and a majority rule consensus tree was constructed. Support was determined with bootstrap analysis with 1000 repetitions. Maximum likelihood (ML) analyses were performed in GARLI 0.951 (Zwickl 2006). The run was repeated 10 times from random starting trees with the auto-terminate setting. GARLI also was used to generate 100 ML nonparametric bootstrap replicates from which a majority-rule consensus tree was calculated in PAUP* 4.0b10 (Swofford 2002). Trees were rooted with members of the Rhizophydiales (PL 042) and Chytridiales (BR 097), clades sister of the Spizellomycetales. RESULTS

Phylogenetic analysis.—The combined dataset had 3052 characters with 440 parsimony informative sites. MP and ML analyses differed little in topology and were congruent in terminal branch groupings of taxa (FIG. 1). Of 1005 trees derived from PAUPRat 139 most parsimonious (MP) trees (length 5 1480 steps, consistency index 5 0.468, retention index 5 0.796) were used to compute a 50% majority rule consensus tree. The MP tree differed from the ML tree only in the structure of Clade C (ML analysis). Instead of one clade with 58% support as found in the ML tree (FIG. 1), in the MP tree Clade C split into two lineages, each with 100% branch support but with identical subgrouping as in Clade C of the ML tree.

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MYCOLOGIA

FIG. 1. Majority rule consensus maximum likelihood (ML) tree based on rDNA operon sequence data showing phylogenetic positions of 53 isolates in the Spizellomycetales and four isolates in the Rhizophlyctidales. Maximum parsimony/ maximum likelihood bootstrap support values are above branch lines. For MP, L 5 1480 steps, CI 5 0.486, RI 5 0.769; For ML, 2lnL 5 12457.71. ET 5 epitype culture; NTG 5 neotype culture, genus; T 5 type culture; TG 5 type culture, genus.

The ML tree (FIG. 1, log likelihood 5 21245.71) shows parsimony and likelihood bootstrap values for relationships and levels of genetic diversity among and within clades. Clades were defined based on the maximally inclusive group that had greater than 50% bootstrap support. The Rhizophlyctidales (Clade A), which included culture BR 186 on which the epitype for Rhizophlyctis rosea is based, was well supported (97–99%) and sister of the Spizellomycetales (FIG. 1). Monophyly in the Spizellomycetales.—Three genera formed monophyletic groups in all analyses (FIG. 1). Triparticalcar (Clade G) and Kochiomyces (Clade I) are monotypic genera, and Powellomyces (Clade K) has two described species.

Clade G, Triparticalcar. Six isolates grouped with the type isolate of Triparticalcar arcticum (BR 059) with 65–83% support. Three isolates in this clade (PL162, JEL 350 and JEL 355) grouped as a subclade with 100% support, distinct from isolates that clustered with the type culture of T. arcticum. None of the isolates was identical to the type culture, suggesting more species diversity than currently recognized. All isolates in the Triparticalcar clade had origins in soil that experienced extremes in environmental conditions, including herbivore manure, tree canopy soils, Arctic clay and lava detritus (TABLE I). Clade I, Kochiomyces. Our analysis of Kochiomyces included all four of the cultures that Barr (1984b) considered as K. dichotomus. The four isolates formed

WAKEFIELD ET AL.: SPIZELLOMYCETALES a well supported clade (85–90%) with little genetic diversity, although they were from a range of locations (TABLE I) in Canada (BR 035 [neotype], Quebec; BR 269 and BR 279, New Brunswick; BR 356, Nova Scotia). Two isolates, one from California (JEL 371) and one from Michigan ( JEL 568), were sister of the four isolates of K. dichotomus. Clade K, Powellomyces. This clade had 90–93% support and included the type cultures for the two described species in distinct, well supported subclades. The subclade containing P. variabilis (MP 003) had 100% support as did the subclade including P. hirtus (BR 081); however the divergence of isolate MP 001 suggests that this genus has greater genetic variation than previously realized. Polyphyly in the Spizellomycetales.—Gaertneriomyces and Spizellomyces were polyphyletic (FIG. 1). The three described species of Gaertneriomyces were distributed between clades D and H. The eight validly published species of Spizellomyces were distributed among five clades (C, D, E, F and J). Clade H, Gaertneriomyces. This fully supported (100%) clade included two of the three described species of Gaertneriomyces, G. semiglobifer and G. spectabile, and additional isolates from wide geographic locations and soil habitats (TABLE I). Six isolates clustered with the type of the genus G. semiglobifer (BR 043) with 88–89% support (FIG. 1), demonstrating little genetic diversity. Isolate JEL 550 was sister of the G. semiglobifer subclade (68–99% support). The isolate on which G. spectabile was described (CH 067, Chen and Chien 2000) was distinct from other isolates in Clade H (FIG. 1), supporting its distinctiveness from G. semiglobifer. Phlyctochytrium californium (CBS 667.73), which Barr (1984b) placed in synonymy with G. semiglobifer, was divergent from all other isolates in Clade G; however it was well supported as a species within the Gaertneriomyces clade. Clade D. The third described species of Gaertneriomyces, G. tenuis, was divergent from the type of the genus, G. semiglobifer (Clade H). Gaertneriomyces tenuis grouped with one of the eight published species of Spizellomyces (S. dolichospermus). Clade J, Spizellomyces. The type species for genus Spizellomyces, S. punctatus (BR117), was located in this clade, which had 52–66% support and circumscribed three of the eight validly published species of Spizellomyces. The clade comprised two fully supported subclades (100% each). In one of the two subclades two additional species, S. plurigibbosus and S. palustris, clustered with S. punctatus. The second subclade included an isolate Barr (1970c) identified as Phlyctochytrium reinboldtiae (CBS 669.73), which he considered a representative of S. acuminatus (Barr 1984b).

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Clade E. Spizellomyces lactosolyticus (BR 277) diverged as a lone taxon. Clade F. This clade (95–100% support) contained two of the validly published species of Spizellomyces, S. acuminatus (BR 062A) and S. kniepii (BR 351), within a subclade with 57–65% support (FIG. 1). A second subclade contained the non-validly published Phlyctochytrium africanum (CBS 454.65), which Barr (1980) considered synonymous with S. acuminatus. Clade C. Spizellomyces pseudodichotomus (BR 372), isolated from British Columbia, clustered in a well supported subclade (99–100%) with two isolates from Maine (TABLE I). Undescribed isolates from North Carolina and Virginia formed a fully supported (100%) sister subclade ( JEL 148, PL 044, PL 078). Clade C however had marginal support (58%) in the ML analysis, and in the MP analysis the two subclades of C formed two polytomies (Isolates BR 372, JEL 333 and JEL 430 formed a monophyletic group with 99– 100% support, as did isolates JEL 148, PL 044, and PL 078 with 100% support). DISCUSSION

Overview.—The primary goal of this study was to examine the monophyly of genera currently classified in the Spizellomycetales (Barr 1980, 1984b; Longcore et al. 1995). Genera Kochiomyces, Triparticalcar and Powellomyces were strongly supported as monophyletic, but Spizellomyces and Gaertneriomyces were polyphyletic and thus are in need of taxonomic revision. Another goal was to examine the diversity within the Spizellomycetales as it is currently understood. Diversity in this order is greater than was previously realized, and additional genera and species need to be described. Before our study spizellomycetalean chytrids had never been reported associated with horse and cow manure. Thus dung-inhabiting chytrids grouping with Gaertneriomyces, Triparticalcar and unidentified B clades was unexpected; this correlation makes possible targeted collection of samples to explore additional diversity in the Spizellomycetales. Molecular analyses revealed significant variation within several clades, demonstrating the need for additional sampling and analyses to confidently place problematic taxa, especially for clades that were not strongly supported. Zoospore ultrastructural considerations.—When circumscribing the Spizellomycetales Barr (1980) delineated Spizellomyces, Kochiomyces, Gaertneriomyces and Triparticalcar on the basis of variation in zoospore ultrastructure (Barr 1981, 1984a, b; Barr and Allan 1981; Bar and Hadland-Hartmann 1977, 1979; Chong and Barr 1973, 1974), and Longcore et al. (1995) delineated Powellomyces on this basis also. Although

600 TABLE I.

MYCOLOGIA Taxon sampling for phylogenetic analyses of 57 ingroup isolates GenBank accession number Isolate

Straina

Ingroup: Gaertneriomyces sp. Gaertneriomyces semiglobifer Gaertneriomyces semiglobifer Gaertneriomyces semiglobifer Gaertneriomyces semiglobifer Gaertneriomyces semiglobifer Gaertneriomyces semiglobifer Gaertneriomyces semiglobifer Gaertneriomyces spectabile Gaertneriomyces tenuis Kochiomyces clade Kochiomyces clade Kochiomyces dichotomus Kochiomyces dichotomus Kochiomyces dichotomus Kochiomyces dichotomus Phlyctochytrium africanum Phlyctochytrium californicum Phlyctochytrium reinboldtiae Powellomyces clade Powellomyces sp. Powellomyces sp. Powellomyces sp. Powellomyces sp.

JEL 550 BK 91-10 BR 043 BR 386 CC 002 JEL 553 MP 007 MP 009 CH 067 BR 366 JEL 568 JEL 371 BR 035 BR 269 BR 279 BR 356 CBS 454.65 CBS 667.73 CBS 669.73 MP 001 MP 002 PL 142 PL AUS 017 BR 350

Powellomyces hirtus Powellomyces hirtus Powellomyces variabilis Rhizophlyctis rosea Rhizophlyctis rosea Rhizophlyctis rosea Rhizophlyctis rosea Spizellomyces acuminatus Spizellomyces clade Spizellomyces clade Spizellomyces sp. Spizellomyces dolichospermus Spizellomyces kniepii Spizellomyces lactosolyticus Spizellomyces palustris

BR 009B BR 081 MP 003 BR 109 BR 186 PL 132 PL 140 BR 062A AD 020 CC 410 FG MP Dhong BR 346 BR 351 BR 277 BR 041

Spizellomyces plurigibbosus Spizellomyces pseudodichotomus Spizellomyces pseudodichotomus Spizellomyces pseudodichotomus Spizellomyces punctatus Spizellomyces punctatus Triparticalcar arcticum Triparticalcar clade Triparticalcar clade

BR 033 BR 372 JEL 333 JEL 430 BR 117 SW 001 BR 059 JEL 350 JEL 355

Triparticalcar sp. Triparticalcar sp.

JEL 554 JEL 555

Substrate/origin Horse manure, ME, USA Beach sand, Baltic Sea, Germany Beach sand, Baltic Sea, Germany Downy mildew oospore, Israel Crop soil, NSW, Australia Horse manure, VA, USA Lawn soil, OH, USA Downy mildew oospore, Israel Crop soil, Chiungpu, Taiwan Crop soil, NS, Canada Forest soil, MI, USA Soil, CA, USA Decayed wood, QC, Canada Sand dune, NB, Canada Sand dune, NB, Canada Peat soil, NS, Canada Humic soil, Atlas, Algeria Lawn soil, CA, USA Clay soil, ON, Canada Forest soil, NC, USA Bromeliad capsule, Brazil Forest soil, Windermere, UK Rainforest soil, NSW, Australia Temporary pond soil, NB, Canada Garden soil, ON, Canada Garden soil, ON, Canada Lawn soil, NC, USA Soil, CA, USA Compost, Gottingen, Germany Forest soil, Windermere, UK Clay soil, Republic of Colombia Greenhouse soil, ON, Canada Fallow crop soil, NSW Australia Crop soil, NSW, Australia American Type Cult. Collec. Peat soil, South Huberside, UK Soil, Republic of South Africa Crop soil, BC, Canada Peat bog ditch, Bekerkesa, Germany Lawn soil, ON, Canada Glacial till Soil, BC Canada Garden soil, MA, USA Soil, ME, USA Soil, New Guinea Soil, AL, USA Saline clay, NU, Canadian Arctic Tree canopy, QLD, Australia Detritus in canopy, QLD, Australia Horse manure, MI, USA Cow manure, MI, USA

18S

28S

ITS1-5.8S-ITS2

FJ827655 AF164247 AY349043 FJ827660 FJ827645 FJ827656 AY349043 AY349038 FJ827661 FJ827650 FJ827664 GQ499383 FJ827651 FJ804151 FJ827667 FJ827652 FJ827649 FJ827648 FJ827643 AY349042 AY349041 FJ827634 FJ827635 FJ827647

FJ827704 DQ273778 FJ827702 FJ827700 FJ827701 FJ827703 AY349077 AY349072 FJ827705 FJ827675 FJ827699 GQ499380 FJ827696 FJ804155 FJ827695 FJ827697 FJ827693 FJ827706 FJ827689 AY349076 AY349075 FJ827670 FJ827671 FJ827672

FJ827741 AY997051 FJ827739 FJ827737 FJ827738 FJ827740 AY349099 AY349098 FJ827742 FJ827713 FJ827736 GQ499377 FJ827733 FJ827731 FJ827732 FJ827734 FJ827729 FJ827743 FJ827725 AY349100 AY349103 FJ827708 FJ827709 FJ827710

FJ827654 FJ827642 AY349040 GQ160454 AY349044 GQ160455 GQ160456 FJ827640 FJ827669 FJ827666 AY349037 FJ827639 AF164237 FJ827668 FJ827665

FJ827674 FJ827673 AY349074 AY349078 AY349079 EU379194 EU379196 FJ827692 FJ827691 FJ827690 AY349071 FJ827676 FJ827707 FJ827688 FJ827687

FJ827712 FJ827711 AY349104 AY349105 AY349106 EU379237 EU379239 FJ827728 FJ827727 FJ827726 AY349101 FJ827714 AY349130 FJ827724 FJ827723

FJ827638 FJ827653 GQ160453 GQ499384 AY546684 FJ822967 DQ536480 FJ827663 DQ536477

FJ827686 FJ827677 GQ160457 GQ499381 AY546692 EU379198 DQ273826 FJ827685 DQ273821

FJ827722 FJ827715 GQ160458 GQ499378 AY997092 EU379241 AY997096 GQ499376 AY997073

FJ827657 FJ827658

FJ827682 FJ827683

FJ827719 FJ827720

WAKEFIELD ET AL.: SPIZELLOMYCETALES TABLE I.

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Continued GenBank accession number Isolate

Triparticalcar sp. Triparticalcar clade Unidentified Clade B Unidentified 1 Clade C Unidentified 2 Clade C Unidentified 3 Clade C Unidentified Clade F Outgroup: Chytriomyces hyalinus,S Boothiomyces clade, Rhizophydiales

Straina

Substrate/origin

18S

28S

ITS1-5.8S-ITS2

JEL 560 PL 162 JEL 549 JEL 148 PL 044 PL 078 BR 062B

Horse manure, MI, USA Detritus on lava, Hawaii Horse manure, ME, USA Carolina Biol. Supply, NC, USA Forest soil, NC, USA Garden soil, VA, USA Greenhouse soil, ON, Canada

FJ827659 FJ827637 GQ499385 FJ827644 FJ827636 FJ827646 FJ827641

FJ827684 FJ827681 GQ499382 FJ827678 FJ827679 FJ827680 FJ827694

FJ827721 FJ827718 GQ499379 FJ827716 FJ827717 FJ825362 FJ827730

BR 097 PL 042

Marsh, ON, Canada Forest soil, NC, USA

AY349032 AY349049

AY349065 AY439056

AY349119 DQ485658

a Sources of cultures: BK 5 University of California at Berkeley Collection; BR 5 Canadian Collection of Fungal Cultures, D.J.S. Barr Collection; CBS 5 Centraalbureau voor Schimmelcultures, Utrecht, Netherlands; CC or AD 5 University of Sydney, Australia, Frank Gleason Collection; JEL 5 University of Maine, Joyce E. Longcore Collection; MP 5 University of Alabama, Martha J. Powell Collection; PL 5 University of Alabama, Peter M. Letcher Collection; SW 5 University of Alabama, W. Scott Wakefield Collection.

our study did not include new ultrastructural analyses, published descriptions of ultrastructural features of Kochiomyces (FIG. 1 in Barr and Allan 1981), Triparticalcar (FIG. 20 in Barr and Allan 1981) and Powellomyces (FIG. 36 in Chong and Barr 1974; Powell 1976) zoospores corresponded to the monophyly of each of these genera in our molecular study. Similarly zoospore ultrastructural differences documented among Spizellomyces (Barr 1984) and Gaertneriomyces (Barr 1980, 1981, 1984b) support the molecular polyphyly found among these genera in our study. Monophyletic genera.— Kochiomyces was based on Phlyctochytrium dichotomum (Umphlett and Olson 1967). Because the type culture was no longer viable Barr (1984b) recognized Umphlett and Olson (FIGS. 1–9, 1967) as the type and designated isolate BR 035 as the neotype culture. Although showing little genetic diversity, isolates in the Kochiomyces clade were from a range of habitats including sand dunes and peat soil (Barr 1984b). Isolates JEL 371 and JEL 568 were genetically distinct from other isolates in the clade and may represent a new species. Triparticalcar, based on Phlyctochytrium articum (Barr 1970b), was characterized by a distinct rod-like kinetosome-associated structure extending anteriorly through most of the zoospore (Barr 1980, Barr and Allan 1981). Several isolates in the Triparticalcar clade were genetically divergent and thus also might be ultrastructurally diverse. In our analyses isolates PL 162 and JEL 350 diverged from isolate BR 059 T. arcticum, the type species, as did isolate JEL 355.

Consequently these isolates may represent new species or new genera. Our phylogenetic analyses supported Powellomyces variabilis and P. hirtus as separate species and also suggested that this genus is more species rich than currently documented. Three isolates (MP 001, MP 002 and MP 003) that Powell and Koch (1977) used in their analysis of the range of thallus morphological variation were resolved in three distinct subclades in the Powellomyces clade. These isolates might be different species within the genus or the subclades might represent multiple genera. Isolate BR 350 which Longcore et al. (1995) considered as P. variabilis clustered in the P. variabilis subclade, but it was genetically distinct from the type culture and also may represent another taxon. Divergence in the Powellomyces clade predicts that morphological and zoospore ultrastructural differences will be detected among representatives of this clade. Polyphyletic genera.—Barr (1980) established genus Gaertneriomyces based on Uebelmesser’s (1956) nonvalidly published species Phlyctochytrium semiglobifer. Barr described distinct features of its zoospore, including the posterior location of its nucleus with a concave depression just anterior to the kinetosome and a multilaminate kinetosome-associated structure (FIG. 4 in Barr 1980, FIG. 1 in Barr 1981b). G. semiglobifer is a commonly found species, based on our sampling. Our molecular analyses supported the validity of the transfer of Phlyctochytrium spectabile to Gaertneriomyces (Chen and Chien 2000). The distant relationship of Phlyctochytrium californicum (Barr

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1969) to G. semiglobifer indicates that, instead of being a representative of G. semiglobifer as Barr suggested (Barr 1984b), P. californicum is a distinct species within Gaertneriomyces. Conversely G. tenuis likely represents a new genus. Barr (1984b) was tentative when he classified this organism as a species of Gaertneriomyces. Although not illustrated, Barr reported a kinetosome-associated spur similar to that found in many Spizellomyces species he had described instead of a multilayered structure as found in zoospores of the type species of Gaertneriomyces (Barr 1980, 1981). The absence of a nuclear heel extension (typical of genus Spizellomyces, Barr 1984) in the zoospore of G. tenuis however led Barr to place the isolate in Gaertneriomyces (Barr 1984b). The phylogenetic placement of G. tenuis in our molecular analyses suggests a potential relationship with S. dolichospermus. Like that of G. tenuis, the zoospore of S. dolichospermus lacked a nuclear heel and had a spurlike structure instead of a multilaminate kinetosomeassociated structure (Barr 1984). Thus, based on our molecular analyses and similarities in zoospore ultrastructure, G. tenuis and S. dolichospermus may represent a new genus in the Spizellomycetales. Additional ultrastructural analysis is necessary to resolve the taxonomic position of these isolates. Molecular phylogenetic analyses revealed that Spizellomyces contains a complex of species, as could be predicted from the morphological variation and physiological differences Barr (1984b) reported. Our phylogenetic analyses showed that three of the eight validly described species of Spizellomyces are justifiable members of genus Spizellomyces: S. punctatus as the type species (Barr 1980, Koch 1957), S. plurigibbosus and S. palustris. These species all shared the zoospore ultrastructural character states of an anteriorly located nucleus with a heel-like extension posteriorly directed toward the kinetosome, an electron-opaque bridge between the kinetosome and nonflagellated centriole, and a ‘‘very small’’ kinetosome-associated spur, which appeared in some morphologically similar isolates to be absent (FIGS. 1, 3 in Barr 1980, Barr 1984a). Spizellomyces plurigibbosus was genetically distinct but closely related to S. punctatus, which is significant because Barr questioned whether these taxa should be retained as separate species (Barr 1984a, b). Spizellomyces acuminatus and S. kniepii were in a clade separate from that which contained the type species S. punctatus. The molecular distinction of these two species from S. punctatus is supported by ultrastructural differences in the kinetosome-associated spur. Spizellomyces acuminatus zoospores contained a ‘‘slender spur’’ instead of the ‘‘very small’’ spur of S. punctatus and had little to no material joining the kinetosome to the nonflagellated centri-

ole (Barr 1984a). Our analysis revealed that some species considered synonymous with S. acuminatus were instead distinct. Barr (1984b) considered an isolate he identified as Phlyctochytrium reinboldtiae (isolate CBS 669.73, Barr 1970c) as a representative of S. acuminatus, but in our analysis this isolate was weakly supported in a clade sister of that which contained the type species (Clade J). Barr (1984b) considered an isolate of Phlyctochytrium africanum (CBS 454.65) a representative of S. acuminatus, and although it grouped in a clade with S. acuminatus it was genetically distinct from the type of this species (BR 062A). These results suggest that further examination of zoospore ultrastructure is necessary for resolution of the correct taxonomic placement of these isolates. The phylogenetic placement of S. lactosolyticus was problematic and did not receive strong support for location in any clade. The zoospore of S. lactosolyticus was distinct from that of the type species S. punctatus because, instead of having a small spur characteristic of the type, its zoospore had a stout spur (FIG. 16 in Barr 1984a) and no material connected the kinetosome to the nonflagellated centriole (Barr 1984a). The two remaining described species of Spizellomyces are in clades distinct from the type of the genus, and both of these species have zoospores structurally distinct from that of the type species. The zoospore of S. pseudodichotomus contains a compound kinetosome-associated spur but no connecting bridge between the kinetosome and the nonflagellated centriole (FIGS. 19–21 in Barr 1984a). The zoospore of S. dolichospermus lacked the nuclear-heel extension characteristic of the type of Spizellomyces and also had a compound spur instead of a ‘‘very small spur’’ found in zoospores of the type species (Barr 1984a). Conclusions.—Order Spizellomycetales, which was described based on ultrastructural characters (Barr 1980) and revised based on DNA sequence information (Hibbett et al. 2007; James et al. 2000, 2006a, b; Letcher et al. 2008), was monophyletic in our analyses. Based on our sampling, genetic diversity in this morphologically conserved group is much greater than formerly realized and new genera and species need to be described. Kochiomyces and Triparticalcar, each with a single species, have additional species level diversity as does Powellomyces, which currently contains two species. Spizellomyces and Gaertneriomyces are polyphyletic and new genera are needed to accommodate some of the species now in these genera. The presence of unnamed isolates and polyphyletic genera among the monophyletic genera

WAKEFIELD ET AL.: SPIZELLOMYCETALES highlights the need for additional genetic, ultrastructural and morphological investigations. ACKNOWLEDGMENTS

This study was supported by the National Science Foundation through PEET grant DEB-0529694. WSW is appreciative of support through University of Alabama alumni graduate fellowships. We are indebted to Ms Carolyn Babcock, curator of cultures at DAOM, who preserved and revived many of the Barr isolates used in this study.

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