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Molecular phylogeny of Leptosphaeria and Phaeosphaeria

     Department of Plant Pathology, The Pennsylvania State University, University Park 16802; and United States Department of Agriculture (USDA), Agriculture Research Service (ARS), Systematic Botany and Mycology Laboratory, Rm. 304, Bldg. 011A, BARC- West, Beltsville, Maryland 20705-2350

Mary E. Palm ²

     USDA, Animal and Plant Health Inspection Service, Systematic Botany and Mycology Laboratory, Rm. 329, Bldg. 011A, BARC- West, Beltsville Maryland 20705-2350

Peter van Berkum

     USDA, ARS, Soybean Genomics and Improvement Laboratory, Beltsville, Maryland 20705-2350

Nichole R. O'Neill

     USDA, ARS, Molecular Plant Pathology Laboratory, Beltsville, Maryland 20705-2350

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

The objectives of this study were to determine the phylogenetic relationships of species of Leptosphaeria and Phaeosphaeria and evaluate the phylogenetic significance of morphological characters of the teleomorph, anamorph, and host. Sequences of the entire ITS region, including the 5.8S rDNA, of 59 isolates representing 54 species were analyzed and the phylogeny inferred using parsimony and distance analyses. Isolates grouped into three well-supported clades. The results of this study support the separation of Phaeosphaeria from Leptosphaeria sensu stricto. Leptosphaeria bicolor and the morphologically similar Leptosphaeria taiwanensis formed a separate, well-supported clade. We conclude that peridial wall morphology, anamorph characteristics, and to a lesser extent host, are phylogenetically significant at the generic level. Ascospore and conidial morphology are taxonomically useful at the species level.

Key words: ITS sequence, Phaeoseptoria, Phoma, Plenodomus, Septoria, Stagonospora, systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
More than 1.600 taxa have been described in Leptosphaeria Ces. & De Not. (Crane and Shearer 1991). In the broadest sense, the genus is characterized by the production of pale to dark brown, septate ascospores in bitunicate asci, and usually single, ostiolate ascomata with walls of either pseudoparenchyma or scleroplectenchyma. Some authors have maintained a broad concept for Leptosphaeria, often subdividing the genus into sections (Müller 1950, Munk 1957). Holm (1957) proposed a narrower generic concept for Leptosphaeria based on the presence of scleroplectenchyma in the peridial walls, and subsequently transferred many species to Entodesmium Riess, Nodulosphaeria Riess, and Phaeosphaeria I. Miyake.

Leptosphaeria sensu stricto, as accepted by Barr (1987), Eriksson (1967), Hedjaroude (1969), Holm (1957), Shoemaker (1984) and von Arx and Müller (1975), includes species with scleroplectenchymatous ascomata that occur on dicotyledonous plants. Shearer et al (1990) designated a lectotype specimen for L. doliolum, the type species of Leptosphaeria, and provided a detailed description of this species. The majority of species of Leptosphaeria sensu stricto produce anamorphs that belong to Phoma section Plenodomus (Preuss) Boerema, Kesteren & Loer., a taxon treated in detail by Boerema et al (1994). Descriptions of the anamorphs of a number of species of Leptosphaeria have also been provided by Lucas (1963), Lucas and Webster (1967), and Webster (1955).

A number of species producing pseudoparenchymatous ascomata smaller than those of Leptosphaeria sensu stricto and occurring mainly on monocotyledenous plants were placed in the genus Phaeosphaeria (Holm 1957, Barr 1987, Shoemaker and Babcock 1989). Eriksson (1981) broadened the generic concept of Phaeosphaeria to include dictyosporous taxa and noted that many species produce ascospores with a characteristic perispore. Leuchtmann (1984) provided descriptions of the anamorphs of Phaeosphaeria in his extensive treatment of the genus.

Several studies have utilized molecular techniques to determine relationships among and between species of Leptosphaeria and Phaeosphaeria. Morales et al (1995) analyzed sequences of the 18S, 5.8S, and the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene of five species. They concluded that the separation of Leptosphaeria and Phaeosphaeria was not well-supported and that anamorph characters were not phylogenetically informative. Khashnobish and Shearer (1996) drew the opposite conclusion in their study of the phylogenetic relationships between two species of Leptosphaeria, including the type species L. doliolum, and five species of Phaeosphaeria based on a cladistic analysis of morphological data and sequence data (ITS2 and part of the 28S). While that study demonstrated that species of Phaeosphaeria form a group that is distinct from Leptosphaeria, it did not provide support for the division of Phaeosphaeria into subgenera based on ascospore morphology. Those workers also concluded that ascomatal wall anatomy appears to be a good character for delimiting Phaeosphaeria from Leptosphaeria (Khashnobish and Shearer 1996).

Leptosphaeria sensu lato and Phaeosphaeria still are large, heterogeneous taxa that include species sharing the characteristics of both genera. For example, Phaeosphaeria dennisiana and P. silenes-acaulis, originally described in Leptosphaeria, are species with pseudoparenchymatous ascomata that occur on members of the dicot family Caryophyllaceae. In addition, the generic placement of some species, such as L. bicolor, has been controversial because their placement in currently recognized genera is not supported by morphological and molecular data.

This study was initiated to further clarify the phylogenetic relationships of taxa in Leptosphaeria sensu stricto and Phaeosphaeria by sequencing the complete ITS region of a large number of species. Another objective was to evaluate further the phylogenetic significance of ascomatal characteristics, as well as anamorph and host. The significance of anamorph characteristics especially has not been evaluated fully and the anamorphs of species in Leptosphaeria and Phaeosphaeria have been placed in a number of different genera (Rossman et al 1987, Crane and Shearer 1991).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isolates – Fifty-nine isolates representing 54 species of Leptosphaeria and Phaeosphaeria from diverse hosts, habitats, and geographic locations and which varied in anamorph characteristics were chosen for this study (Table I). Paraphaeosphaeria michotii was selected as the outgroup because it is the closest known relative of these genera (Câmara et al 2001). Cultures isolated by M. Câmara (MPSC) and N. O'Neill have been deposited in Centraalbureau voor Schimmelcultures (CBS). All other cultures were obtained from culture collections as noted. Morphological data for species isolated by the authors were obtained from observations of the fungus on host material and in culture. Because many isolates from culture collections did not sporulate, morphological information for most species was obtained from previously published descriptions, many of which were based on those isolates (Leuchtmann 1984).

Polymerase chain reaction (PCR) amplification of the ITS1 and ITS2 including the 5.8S rDNA regions – Primers NS1 and ITS4 (White et al 1990) were used to amplify fragments that included the ITS1, ITS2, and 5.8S, and portions of the 18S and 28S of the rRNA cistron. The reaction mixture was optimized by using a PCR optimizer kit (Invitrogen, Carlsbad, California), and included 10 µL of 5x buffer C [60 mM Tris-HCl, 15 mM (NH₄)SO₄, 2.5 mM MgCl₂ at a final pH of 8.5 (22 C)], 1.25 µL of 10 mM of each dNTP, 5 µL 10 pmol/µL of each primer, 2 µL Perkin Elmer Taq polymerase and 22 µL of sterile water. Amplifications were achieved with an ERICOMP Delta Cycler II ^TM system with the following program parameters: 35 cycles of 94 C for 30 s; 57 C for 1 min; 72 C for 1.5 min, and a final extension at 72 C for 3 min. Amplification products were subjected to electrophoresis in a 0.7% agarose gel containing EtBr and visualized by UV illumination.

DNA sequencing – The PCR products were purified by using QIAquick spin columns (Qiagen Inc., Chatsworth, California) and both strands were sequenced with an ABI 377 DNA sequencer [Applied Biosystems Inc., Foster City, California (ABI)] using a Taq Dye-Deoxy Terminator Cycle Sequencing Kit (ABI). Sequencing of the ITS region was accomplished by using primers ITS3, ITS4, ITS5 (White et al 1990), and a newly designed primer ITS2c (Câmara et al 2001) to provide overlap with the sequence derived from primer ITS3. Sequences were edited and assembled using the software Factura and Autoassembler (ABI) on a Macintosh computer.

Analysis of sequence data – Sequences were aligned using the PILEUP program in the Wisconsin package of the Genetics Computer Group (Madison, Wisconsin). Parameters were empirically adjusted to a gap penalty of one and gap extension penalty of zero. Alignments were manually checked for ambiguities and adjusted when necessary using GeneDoc 2.5 (Nicholas and Nicholas 1997). The ITS1 contained a hypervariable region of 80 base pairs (positions 26–106 in the aligned sequences) which was difficult to align. Two data sets were created, one in which the hypervariable region was left intact (hereafter "the complete alignment"), and another in which the hypervariable region was deleted (hereafter "the pruned alignment"). Parsimony analyses were performed in PAUP* 4.0b2 (Swofford 1999) using ten heuristic searches with random taxon addition sequences and TBR branch swapping, with MAXTREES set to 100. All characters were unordered and equally weighted, and gaps were treated as missing data. A neighbor joining analysis (Saitou and Nei 1987) using the Kimura-2-parameter option was used to derive a distance tree in PAUP*. Topological robustness in both parsimony and distance analyses was estimated using 1000 bootstrap replicates, with characters unweighted and sampled with equal probability using a branch and bound search. Sequences were deposited in GenBank (Table I) and the complete alignment, including the ambiguous regions, was deposited in TreeBase (S680, M1065).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Sequences of the entire ITS region (ITS1, ITS2 and 5.8S) ranged from 468 to 533 bp. The complete alignment was 596 bp with 275 constant characters, 265 parsimony-informative positions and 56 parsimony-uninformative positions. Parsimony analysis of the complete alignment recovered more than 100 equally parsimonious trees (1311 steps, with CI = 0.4863, RI = 0.6964, and RC = 0.3386).

Three major clades labelled A, B, and C were resolved with similar levels of bootstrap support using parsimony (Fig. 1) and distance (Fig. 2) analyses. Clade A includes species of Leptosphaeria sensu stricto which produce scleroplectenchymatous ascomata and Phoma section Plenodomus anamorphs. Phaeosphaeria setosa and L. typharum also are included in Clade A but differ from other members of this group in morphological and biological characters.

View larger version (43K): [in this window] [in a new window]    FIG. 1. Phylogeny of 54 species of Leptosphaeria and Phaeosphaeria based on parsimony analysis of ITS1, 5.8S and ITS2 rDNA sequences. The tree is one of the more than 100 most equally-parsimonious generated from the heuristic search of the complete alignment (length = 1311 steps, CI = 0.4863, RI = 0.6964 and RC = 0.3386). The numbers at the branch node indicate the confidence value obtained from bootstrap analysis using 1000 replications. Those outside the parentheses denote bootstrap values from the pruned alignment; those within parentheses were obtained using the complete alignment. Only confidence values above 50 are indicated. Paraphaeosphaeria michotii was used as the out-group

View larger version (43K): [in this window] [in a new window]    FIG. 2. Distance tree generated using neighbor joining analysis and the Kimura-2-parameter option. The numbers at the branch node indicate the confidence value obtained from bootstrap analysis using 1000 replications. The numbers outside the parentheses denote bootstrap values from the pruned alignment; those inside parentheses were obtained using the complete alignment. Only confidence values above 50 are indicated. Paraphaeosphaeria michotii was used as the out-group

Clade C includes L. bicolor and other species isolated from sugarcane (Saccharum officinarum) and the closely related genus Miscanthus Andersson. The anamorphs of these species are coelomycetous, but their hyaline, several-septate, guttulate conidia have a l/w ratio less than four.

The pruned alignment was 502 bp long, with 265 constant, 192 parsimony-informative, and 45 parsimony-uninformative characters. Parsimony analysis of this dataset recovered more than 100 equally parsimonious trees (984 steps, with CI = 0.4878, RI = 0.7306, and RC = 0.3564). Clades A, B and C were well-supported (bootstrap support >74%) (Figs. 1, 2) in both distance and parsimony analyses using this alignment. Bootstrap support for Clades A, B and C was always greater using the complete alignment (trees not shown). A high level of sequence similarity among taxa, as reflected in the number of trees recovered and the low CI values, was observed in both alignments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The objectives of this study were to clarify the phylogenetic relatedness among species placed in Leptosphaeria and Phaeosphaeria, and to evaluate the taxonomic significance of morphology, anamorphs, and hosts. The results of analyses of sequence data from the 59 isolates indicate that peridial wall characteristics and anamorph, and to a lesser extent host, are taxonomically predictive at the generic level (Figs. 1, 2).

Species of Leptosphaeria, including the type species L. doliolum, cluster in a well-supported group (Clade A) representing Leptosphaeria sensu stricto. This clade includes species that produce relatively large (>300 µm), scleroplectenchymatous ascomata and Phoma anamorphs. Most of these anamorphs are placed in Phoma section Plenodomus because of their scleroplectenchymatous conidiomata that often form a pilose neck (Boerema and DeGruyter 1999, Boerema et al 1994). Leptosphaeria weimeri produces ascomata with scleroplectenchymatous peridial walls but has an anamorph, Stagonospora meliloti (Lasch) Petr., which produces multiseptate, hyaline conidia. For that reason the inclusion of this species in Clade A seemed inconsistent, but L. weimeri also produces a Phoma synanamorph (Phoma meliloti Allesch.) (Boerema et al 1994). These synanamorphs often are produced within the same conidiomata; the Phoma anamorph in cultures grown at 8 C and the Stagonospora anamorph at higher temperatures (Boerema et al 1994).

Leptosphaeria typharum and P. setosa are anomalous members of Clade A. Leptosphaeria typharum produces thick-walled, echinulate ascospores, and ascomata with pseudoparenchymatous walls. The coelomycetous anamorph of this species, Scolecosporiella typhae (Oudem.) Petr., is distinct from all others in this study in producing pale brown, elongate, multiseptate conidia with an apical cell that is attenuated to form a short appendage (Sutton 1980, Webster 1955, Nag Raj 1993). Leptosphaeria typharum also is distinct from the other members of Clade A in that it occurs on the monocot genus Typha L. in freshwater habitats (Kohlmeyer and Volkmann-Kohlmeyer 1991). Our results differ from those of Khashnobish and Shearer (1996) which grouped L. typharum (as P. typharum) with other species of Phaeosphaeria. These differences are likely due to a difference in sample size but they also support the idea that the phylogenetic placement of this species is not yet resolved.

Phaeosphaeria setosa differs from species in Clade A in that it occurs on the monocotyledonous host Agave L. and produces pseudoparenchymatous ascomata. The Phoma-like anamorph of P. setosa is similar to the anamorph produced by other members of Clade A, but the conidiomatal walls of P. setosa are pseudoparenchymatous rather than scleroplectenchymatous. Additionally, the ascomatal and conidiomatal necks of this species are setose. Shoemaker and Babcock (1989) transferred P. setosa to Leptosphaeria but that combination was a later homonym of L. setosa Niessl. Shoemaker and Babcock (1989) indicated that this species did not fit well in either Leptosphaeria or Phaeosphaeria and that a new genus might be warranted. Although our results place P. setosa and L. typharum within Leptosphaeria sensu stricto, their distinctive morphological characteristics and occurrence on monocotyledonous hosts might prove to be phylogenetically relevant when additional species of Leptosphaeria are studied.

Clade B includes species of Phaeosphaeria, including the type species, P. oryzae. These species occur mainly on monocots and produce relatively small ascomata (<250 µm) with pseudoparenchymatous peridia. The results of this study clearly support the separation of Phaeosphaeria from Leptosphaeria sensu stricto as proposed by Barr (1987), Holm (1957), Khashnobish and Shearer (1996), Leuchtmann (1984), and Shoemaker and Babcock (1989). Our analyses position P. nodorum and P. microscopica in Phaeosphaeria (Clade B), and L. doliolum and L. maculans in Leptosphaeria (Clade A), a separation that is consistent with the differences in peridial wall structure and anamorph characteristics seen in the other members of Clade A and Clade B. Phaeosphaeria nodorum and P. microscopica produce pseudoparenchymatous ascomata and Stagonospora-like anamorphs, whereas L. doliolum and L. maculans produce scleroplectenchymatous ascomata and anamorphs belonging to Phoma section Plenodomus (Boerema et al 1994). Our results differ from those of Morales et al (1995) who concluded that P. nodorum (as L. nodorum) and P. microscopica (as L. microscopica) were congeneric with Leptosphaeria doliolum, the type species of Leptosphaeria. Our study supports the observation of Khashnobish and Shearer (1996) that ascospore septation is not predictive of relatedness in these genera.

Anamorphs of species in Clade B are similar in that they produce elongate to filiform, hyaline or pale brown, multiseptate conidia mostly from holoblastic conidiogenous cells in pycnidial conidiomata (Leuchtmann 1984). Some species produce microconidia. These anamorphs have been placed in Hendersonia Berk., Phaeoseptoria Speg., Septoria Sacc., Septoriella Oudem. and Stagonospora (Sacc.) Sacc. (Sprague 1944, Webster and Hudson 1957, Punithalingam 1980), genera that differ mainly in the pigmentation and l/w ratio of the conidia. Leuchtmann (1984) treated these anamorphs of Phaeosphaeria as species of Stagonospora because he found that the color and length of the conidia formed a continuum. Our data support Leuchtmann's (1984) suggestion that there is no phylogenetic basis for separating species of Phaeosphaeria based on pigmentation of the conidia.

With the exception of Phaeosphaeria sp. from Phlox L., the members of Clade B group in four subclades in both parsimony and distance analyses. Subclade B1 includes the type of Phaeosphaeria, P. oryzae, as well as P. juncina, Phaeoseptoria musae, and a Phaeoseptoria sp. from Cyperus L. These taxa occur on monocots, produce ascomata with pseudoparenchymatous peridia, and form pycnidia containing pale, filiform conidia.

Four species of Phaeosphaeria group in subclade B2 and all occur on monocots except P. dennisiana (Caryophyllaceae). The hosts of the members of this subclade occur sympatrically on acidic, rocky soils in the Alps, often in close proximity (Leuchtmann pers comm June 2001). A host jump of one of the grass-inhabiting species to the dicotyledonous Minuartia L. (Caryophyllaceae) may have initiated speciation of P. dennisiana, which retained peridial wall and ascospore morphology similar to the taxa from monocots (Leuchtmann pers comm, June 2001). Similar host jumps may have occurred several times in the course of speciation in Phaeosphaeria. Phaeosphaeria alpina is the only member of subclade B2 with an anamorph, which produces conidia similar to some species of Phaeosphaeria in subclade B3.

The majority of species in Clade B are members of subclade B3, a group that includes taxa from monocots and fern allies. Anamorphs of these species typically produce elongate to filiform, yellowish brown or pale brown conidia. Phaeosphaeria nodorum, P. avenaria, and Stagonospora foliicola differ in producing hyaline conidia, while P. spartinae forms a Microsphaeropsis anamorph. Three members of subclade B3 (P. vagans, P. phragmitis, and P. phragmiticola) also differ from the majority of Phaeosphaeria in Clade B in producing ascospores with irregular, longitudinal septa. The pycnidial anamorphs of these species, which produce pale yellowish brown, elongate, septate conidia, are typical of other anamorphs of taxa in Clade B. Our results support Barr (1990), Eriksson (1967) and Leuchtmann's (1984) inclusion of these dictyosporous species in Phaeosphaeria and suggest that ascomatal and anamorph characters are more predictive of relatedness than ascospore septation within this genus. Another member of subclade B3, P. pleurospora, had been placed in the segregate genus Sulcispora Shoemaker & C.E. Babc. (Shoemaker and Babcock 1989) because of its striately ornamented ascospores. Our results do not resolve P. pleurospora as a separate taxon at the generic level and suggest that the ornamentation is more appropriate as a species character.

Phaeosphaeria spartinae is a member of subclade B3 but it always grouped separately from other species of Phaeosphaeria with strong support. This taxon differs from the other members of subclade B3 in that the anamorph produces small, nonseptate, yellowish conidia (Lucas and Webster 1967) typical of Microsphaeropsis. The two isolates examined in this study have nearly identical sequences, supporting Kohlmeyer and Kohlmeyer's (1979) treatment of P. spartinae and L. albopunctata as synonyms. The name L. albopunctata (Westend.) Sacc. (Saccardo 1883) has priority over Leptosphaeria spartinae Ell. & Ever. (Ellis and Everhart 1885) but the epithet albopunctata has been used in Phaeosphaeria and refers to a different fungus on a different host (Kohlmeyer and Volkmann-Kohlmeyer 1991). Therefore Phaeosphaeria spartinae is the appropriate name for the species called L. albopunctata from Spartina.

Unlike nearly half of the species in Clade B that occur on Poaceae, the members of subclade B4 are from diverse, non-grass, monocotyledonous families such as Juncaceae, Cyperaceae, Juncaginaceae, Liliaceae as well as the dicotyledonous family Caryophyllaceae. Anamorphs are not known for species in this subclade. Leptosphaeria bellynckii is a member of this subgroup and produces ascomata with the pseudoparenchymatous peridial walls and a monocot host typical of Phaeosphaeria (Clade B). It is not related to the species of Leptosphaeria sensu stricto included in this study.

Clade C includes members of the genus Leptosphaeria from sugarcane (L. bicolor and L. taiwanensis) and one species from Miscanthus; both hosts are members of the subtribe Saccharinae, tribe Andropogoneae (Poaceae). The ITS sequence of the isolate of Leptosphaeria sp. from Miscanthus was identical to those of L. taiwanensis from sugarcane, suggesting conspecificity. Additionally, the Miscanthus isolate and others obtained in a study of Miscanthus blight (O'Neill and Farr 1996) were pathogenic to sugarcane.

Leptosphaeria bicolor differs from Leptosphaeria sensu stricto (Clade A) in producing ascomata with a pseudoparenchymatous peridium that becomes carbonaceous and ascospores in which the second cell from the apex is swollen and dark brown (Punithalingam 1983). In previous molecular phylogenetic analyses of Leptosphaeria sensu lato, L. bicolor was consistently distant from all other species (Morales et al 1995, Dong et al 1998, Olivier et al 2000) but no closely related taxa were determined. Olivier et al (2000) found that Helminthosporium solani Durieu & Mont. and H. velutinum Link : Fr. grouped with L. bicolor. In this study we report the close relationship between L. bicolor and morphologically similar taxa that occur on closely related hosts of tropical or subtropical origin.

Although the members of Clade C occur on monocotyledonous hosts, they are distinct from species of Phaeosphaeria (Clade B) in producing hyaline, several-septate, ellipsoidal, guttulate conidia, with a l/w ratio of three (O'Neill and Farr 1996). These anamorphs have been placed in Stagonospora but, based on morphological differences and the results of this study, they are not members of that genus. Although we did not sequence the lectotype of Stagonospora, Stagonospora paludosa (Sacc. & Speg.) Sacc. (Sutton 1977), that species is morphologically similar to the anamorphs of Phaeosphaeria. Sutton (1980) described the conidia of S. paludosa as fusiform, minutely guttulate, 6–8-septate, and 41–67 x 8–11 µm. The lower and upper l/w ratios of the conidia of S. paludosa (5–6) are typical of those of the members of Clade B. Therefore, we consider the l/w ratio of conidia to be a useful character in distinguishing the anamorphs of Clade C (l/w < 4) from the hyaline-spored Stagonospora-like anamorphs of Clade B (l/w > 4).

In conclusion, our results suggest an evolutionary separation of the species included in this study into three main groups, two of which share a more recent common ancestor and are equivalent to Leptosphaeria sensu stricto (Clade A) and Phaeosphaeria (Clade B). The third group (Clade C) includes L. bicolor and other taxa from sugarcane and related hosts that produce a distinctive anamorph. We conclude that peridial characters, anamorphs and hosts are phylogenetically significant in this group of fungi. However, to establish a phylogenetically accurate classification for the species described in Leptosphaeria and Phaeosphaeria, it will be necessary to study additional taxa. It also will be important to utilize all possible characters including anamorph and host since it appears that a great deal of convergent evolution has taken place in the evolutionary history of this group of fungi. Analyses of multiple genes will provide additional information concerning the phylogenetic relationships among these taxa and may help to more accurately establish specific and generic boundaries.

	ACKNOWLEDGMENTS

 
The authors would like to thank Patrick Elia for technical assistance and James Plaskowitz for graphics work. We thank Dr. Elwin L. Stewart for his support during this project and Dr. Amy Y. Rossman for logistical support and constructive comments. We sincerely appreciate the insights of Drs. Margaret Barr, Birgitte Volkmann-Kohlmeyer and Jan Kohlmeyer, and Adrian Leuchtmann. Thanks to Dr. David Hibbett for his review of the molecular analyses, Dr. Ove Eriksson for his comments on taxonomic aspects, and Dr. Wendy Unterreiner for her many helpful editorial suggestions. The first author is thankful for funding provided by a Ph.D. fellowship from the Conselho Nacional de Desevolvimento Científico e Tecnológico (RHAE/CNPq), Brazil, which is part of a fungal taxonomy grant coordinated by Dr. José C. Dianese, Univ. de Brasilia (UnB).

	FOOTNOTES

 
¹ Current address: USDA/ARS, Molecular Plant Pathology Laboratory, Bldg. 9 Rm. 3–3, BARC-West, Beltsville, Maryland 20705-2350

² Corresponding author, Email: mary{at}nt.ars-grin.gov

Accepted for publication January 30, 2002.

	LITERATURE CITED

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