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Using phylogenetic species recognition to delimit species boundaries within Lasiosphaeria

     Botany Department, The Field Museum, 1400 S. Lake Shore Drive, Chicago, Illinois 60605-2496 and University of Illinois at Chicago, Department of Biological Sciences, Chicago, Illinois 60607-7060

Sabine M. Huhndorf

     Botany Department, The Field Museum, 1400 S. Lake Shore Drive, Chicago, Illinois 60605-2496

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

The genus Lasiosphaeria recently has been circumscribed more narrowly to include five mor-phospecies united by tomentose ascomata containing yellow centrum pigments. Species boundaries have not been established and phylogenetic relationships have not been clearly defined for these morphospecies. To delimit species boundaries and determine phylogenetic relationships among species, maximum parsimony, maximum likelihood and Bayesian analyses were conducted on sequence data from four nuclear genes, the ribosomal internal transcribed spacer (ITS) region, 28S large subunit (LSU) rDNA, ß-tubulin and ribosomal polymerase II subunit 2 (RPB2). Representatives of L. glabrata, L. ovina, L. rugulosa and L. sorbina resolved as four highly supported monophyletic groups in almost all analyses and are recognized as well-defined species employing principles of genealogical concordance. These species delimitations are corroborated further by morphology. Representatives of L. lanuginosa were polyphyletic in almost all analyses. Although molecular analyses revealed that this morphospecies comprises several phylogenetic species, formal taxonomic recognition of these lineages is premature, so L. lanuginosa currently is treated as a morphological species complex. Complete species descriptions, including teleomorph, anamorph and culture characteristics, are given for L. glabrata, L. ovina, L. sorbina and the L. lanuginosa species complex along with detailed discussions of significant morphological characters used in recognizing species. These species are compared to five additional morphospecies that also may belong in the genus.

Key words: Ascomycetes, ß-tubulin, Cercophora, genealogical concordance phylogenetic species recognition, ITS, LSU, morphological species recognition, phylogenetics, RPB2, Sordariales, species concepts, systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The genus Lasiosphaeria previously included numerous taxa possessing a broad range of ascomal, asco-spore and anamorph morphologies. However, its circumscription recently has been defined more narrowly to include five morphospecies possessing tomentose ascomata with yellow centrum pigments (Miller and Huhndorf 2004a). The genus presently includes the type, L. ovina (Pers. : Fr.) Ces. & de Not., along with L. glabrata (Fr.) Munk, L. lanuginosa (H. Crouan & P. Crouan) A.N. Mill. & Huhndorf, L. rugulosa (A.N. Mill. & Huhndorf) A.N. Mill. & Huhndorf, and L. sorbina (Nyl.) P. Karst. Five additional morphospecies, Cercophora citrina (Petch) Lundq., C. gossypina Lundq., C. spirillospora (Penz. & Sacc.) Lundq., L. cylindrospora Lar. N. Vassiljeva, and L. libertiana Speg. & Roum., also may belong in the genus because they possess similar tomentose ascomata and yellow centrum pigments (except L. cylindrospora, which has a hyaline centrum). Members of Lasiosphaeria occur worldwide as saprobes on lignicolous and herbicolous substrates and commonly are included in regional field guides and taxonomic treatments of ascomycetes (Munk 1957, Dennis 1977, Breitenbach and Kranzlin 1984, Ellis and Ellis 1987). Although they have been included in several taxonomic works (Seaver 1912, Carroll and Munk 1964, Gnanananthan 1972, Lundqvist 1972, Hilber et al 1987, van Hooff 1993, Candoussau et al 2002), these species never have been treated systematically.

As currently circumscribed, Lasiosphaeria includes two species previously placed in Cercophora, which traditionally has been distinguished from Lasiosphaeria in possessing ascospores that eventually develop a swollen, pigmented head (Lundqvist 1972). While as-cospore morphology recently has been shown to be a poor predictor of generic relationships in the Sordariales (Dettman et al 2001, Miller and Huhndorf 2004b), development of a swollen ascospore head might be useful for distinguishing taxa at the species level. The five morphospecies currently in Lasiosphaeria are distinguished morphologically by differences in the amount and color of the tomentum and the presence or absence of a swollen ascospore head. However, several collections recently have been found that possess intermediate tomentum and as-cospore characters making it difficult to assign them to any one of the five morphospecies. The objectives of this study were to: (i) delimit species boundaries and assess species relationships among taxa using a phylogenetic approach, and (ii) determine which morphological characters, if any, may be phylogenetically informative for grouping taxa at the species level.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Taxon sampling. – Taxa used in this study are listed in TABLE I along with source information, geographical locality and GenBank accession numbers. Multiple representatives from the five morphospecies currently placed in Lasiosphaeria were included in this study. Cercophora areolata Lundq., C. sparsa (Sacc. & Fairman) R. Hilber and C. sulphurella (Sacc.) R. Hilber were used to root trees using the outgroup method based on previous phylogenetic analyses by Miller and Huhndorf (2004b). All voucher specimens are deposited in The Field Museum Mycology Herbarium (F).

DNA extraction, PCR amplification, sequencing and sequence alignment. – Detailed protocols for the extraction, amplification and sequencing of partial DNA sequences from the LSU, ß-tubulin and RPB2 genes along with the methods for the alignment of these sequences are described fully in Miller and Huhndorf (2004b). In this study, a segment of the RPB2 gene between conserved regions 3b and 7 (Liu et al 1999) was PCR amplified in two pieces using primer pairs RPB2AM-3bF (5'-AAGGTSTTGATYGCSCARGAGCG)-RPB2AM-6R and fRPB2-5F-RPB2AM-7R and sequenced using a combination of these primers: RPB2AM-3bF, fRPB2-5F, RPB2AM-5R (5'-CTTGTGVACCATRTAGCC), RPB2AM-6R, RPB2AM-1F, RPB2AM-1R, RPB2AM-1bF, RPB2AM-1bR, RPB2AM-7R (Liu et al 1999, Miller and Huhndorf 2004b). The ITS region was amplified and sequenced with ITS5 and ITS4 (Glass and Donaldson 1995) following the methods for LSU in Huhndorf et al (2004).

Phylogenetic analyses. – Maximum parsimony (MP) and maximum likelihood (ML) analyses were performed on separate and combined datasets using PAUP* 4.0b10 (Swofford 2002). Portions of the 5' and 3' ends in each dataset were excluded from all analyses due to missing data in most taxa. Unequally weighted MP analyses were conducted using the branch and bound search option to find exact solutions with furthest addition sequence and MulTrees option in effect. Constant characters were excluded, and gaps were treated as missing. Twelve and three ambiguously aligned regions were delimited in ITS and LSU, respectively. Characters in these regions and in the single ambiguously aligned intron, which occurred in the ß-tubulin gene, were recoded using the program INAASE (Lutzoni et al 2000). The remaining unambiguously aligned characters were subjected to symmetric stepmatrixes to differentially weight nucleotide transformations. Individual stepmatrixes were generated for each of the three codon positions in the ß-tubulin and RPB2 genes, while all unambiguously aligned characters were subjected to a single stepmatrix in ITS and LSU. Branch support for all MP analyses was estimated by performing 1000 bootstrap replicates (Felsenstein 1985) with a heuristic search consisting of 100 stepwise random addition replicates and tree bisection-reconnection (TBR) branch-swapping for each bootstrap replicate.

Modeltest 3.06 (Posada and Crandall 1998) was used to determine the best-fit model of evolution for each dataset. Maximum likelihood analyses were performed on separate and combined datasets with 100 stepwise random addition replicates and TBR branch-swapping using the best-fit model. Constant characters were included, and ambiguously aligned characters were excluded from all ML analyses. Because ML bootstrap analyses were not computationally possible, Bayesian analyses employing a Markov Chain Monte Carlo (MCMC) method were performed using MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001) as an additional means of assessing branch support. Constant characters were included, the above model of evolution was implemented and four MCMC chains were ran simultaneously for 5 000 000 generations with trees saved every 100th generation resulting in 50 000 total trees. The MCMC chains always achieved stationarity after the first 10 000 generations (= 1000 trees), so the first 10 000 trees, which extended well beyond the burn-in phase of each analysis, were discarded. Posterior probabilities were determined from a consensus tree generated with the remaining 40 000 trees. This analysis was repeated three times starting from different random trees to ensure trees from the same tree space were being sampled during each analysis.

The validity of the incongruence length difference (ILD) test (= partition homogeneity test in PAUP*) for determining whether datasets should be combined recently has been questioned (Yoder et al 2001, Barker and Lutzoni 2002) and, thus, other methods should be explored. One method of assessing combinability of datasets, and the one adopted in this study, is by simply comparing highly supported clades among trees generated from different datasets to detect conflict (de Queiroz 1993, Mason-Gamer and Kellogg 1996). High support typically refers to bootstrap support values 70% and Bayesian posterior probabilities 95% (Alfaro et al 2003). If no conflict exists between the highly supported clades in trees generated from different datasets, this suggests the genes share similar phylogenetic histories and phylogenetic resolution and support could ultimately be increased by combining the datasets.

Species recognition. – Following Taylor et al (2000), the term "species recognition" is preferred over "species concept" when discussing the operational versus the theoretical aspect of species concepts, i.e., morphological species recognition (MSR) and phylogenetic species recognition (PSR). Several types of PSR exist based on diagnosability (Nixon and Wheeler 1990, Harrington and Rizzo 1999), monophyly (de Quieroz and Donoughue 1988, 1990) or both (Mc-Kitrick and Zink 1988), and much debate has occurred over which one is most appropriate for delimiting species (de Queiroz and Donoghue 1990, Wheeler and Nixon 1990, Baum and Donoghue 1995, Luckow 1995). Another type of PSR based on coalescence theory uses the concordance of multiple gene genealogies for recognizing monophyletic groups as species (Avise and Ball 1990, Baum and Shaw 1995). Taylor et al (2000) proposed the genealogical concordance phylogenetic species recognition (GCPSR) and discussed its application in delimiting fungal species. GCPSR has been employed in several fungal studies (Geiser et al 1998, Koufopanou et al 1997, O’Donnell et al 1998, Chaverri et al 2003) and is used in this study for recognizing species of Lasiosphaeria.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Sequence alignment. – Final alignments of the four datasets included 25, 25, 24 and 22 taxa and 622, 1109, 1050 and 1798 bp for the ITS, LSU, ß-tubulin and RPB2 genes, respectively. These four alignments are deposited in TreeBASE (SN1704). The ITS and LSU datasets contained 12 and 3 ambiguous regions representing 195 and 27 characters, respectively, while the single ambiguously aligned intron in the ß-tubulin gene represented 69 characters. The ITS, LSU, ß-tubulin and RPB2 genes contained 383, 1001, 778 and 1123 constant characters and 42, 58, 151 and 478 parsimony-informative characters, respectively. The combined dataset included 22 taxa and 4579 bp and contained 3299 constant characters and 714 parsimony-informative characters.

Phylogenetic analyses and species delimitation. – Maximum parsimony analyses of the ITS, LSU, ß-tubulin and RPB2 datasets generated 3, 1, 5 and 1 most-parsimonious trees, respectively. Multiple trees produced from ITS and ß-tubulin analyses differed only slightly in the placement of members of the L. lanuginosa complex (data not shown). Single most-parsimonious trees from each analysis are shown in FIGS. 1–4. Because very little conflict existed among the highly supported clades in trees generated from each dataset, all four datasets were assumed to be congruent and analyzed simultaneously in a combined analysis. This analysis produced a single most-parsimonious tree (FIG. 5), which was nearly identical in topology to that of the RPB2 tree (FIG. 4), except the European populations of L. ovina formed a well-supported clade in the combined analysis. Four highly supported clades, which correspond to four traditional morphospecies (L. glabrata, L. ovina, L. rugulosa, L. sorbina), occurred in all analyses (FIGS. 1–5). These clades are recognized as four distinct phylogenetic species employing GCPSR. Their species delimitations are supported further by MSR by possessing a unique combination of diagnostic morphological characters (see below, TABLE II). Representatives of L. lanuginosa were polyphyletic in all MP analyses. Although several phylogenetic species are distinguished within the L. lanuginosa morphospecies using GCPSR, it is premature to formally recognize these lineages as species. Specimens of L. lanuginosa from additional populations, along with three additional morphospecies with putative relations to L. lanuginosa, need to be included in further analyses. Therefore, L. lanuginosa currently is treated as a morphological species complex.

View larger version (37K): [in this window] [in a new window]   FIG. 1. Phylogram of one of the three most-parsimonous trees generated from an analysis of 25 ITS sequences; length = 324.71 steps, CI = 0.808, RI = 0.816, RC = 0.660. Bootstrap values 50% are shown above branches. Thickened branches indicate posterior probabilities 95%. Species delimitations are shown along the right side of the tree. Cercophora areolata, C. sparsa and C. sulphurella are outgroups.

View larger version (39K): [in this window] [in a new window]   FIG. 2. Phylogram of the single most-parsimonous tree generated from an analysis of 25 LSU sequences; length = 193.58 steps, CI = 0.786, RI = 0.833, RC = 0.654. Support values, species delimitations, and outgroups as in FIG. 1.

View larger version (35K): [in this window] [in a new window]   FIG. 3. Phylogram of one of the five most-parsimonous trees generated from an analysis of 24 ß-tubulin sequences; length = 569.59 steps, CI = 0.799, RI = 0.745, RC = 0.595. Support values, species delimitations, and outgroups as in FIG. 1.

View larger version (32K): [in this window] [in a new window]   FIG. 4. Phylogram of the single most-parsimonous tree generated from an analysis of 22 RPB2 sequences; length = 1991.09 steps, CI = 0.727, RI = 0.731, RC = 0.531. Support values and species delimitations as in FIG. 1. Cercophora areolata and C. sulphurella are outgroups.

View larger version (32K): [in this window] [in a new window]   FIG. 5. Phylogram of the single most-parsimonous tree generated from the combined analysis of 22 sequences; length = 2970.75 steps, CI = 0.751, RI = 0.740, RC = 0.556. Support values and species delimitations as in FIG. 1. Cercophora areolata and C. sulphurella are outgroups.

Species relationships. – Multiple gene genealogies indicate that L. glabrata and L. rugulosa are closely related taxa that occur at the base of the Lasiosphaeria clade, while L. ovina, L. sorbina and members of the L. lanuginosa complex are more derived taxa. While ITS and LSU suggest L. glabrata is the basal taxon, ß-tubulin and RPB2 indicate L. rugulosa is the basal taxon. The combined tree, which probably is influenced by the large amount of phylogenetic signal derived from the protein-coding genes, also places L. rugulosa at the base of the Lasiosphaeria clade. Lasiosphaeria sorbina is basal to the L. lanuginosa complex, which is basal to L. ovina in all analyses.

Species descriptions. – Complete species descriptions are given below for L. glabrata, L. ovina, L. sorbina and the L. lanuginosa species complex. A complete species description of L. rugulosa, except for culture morphology on PDA which is given below, can be found in Miller and Huhndorf (2001). Species descriptions and illustrations also have been published for C. areolata (Lundqvist 1972), C. sparsa (Hilber and Hilber 1979) and C. sulphurella (Hilber and Hilber 1979, van Hooff 1994, Fallah and Shearer 2001).

Lasiosphaeria glabrata (Fr.) Munk, Dansk Bot Ark 17: 111. 1957. FIGS. 6–23

View larger version (169K): [in this window] [in a new window]   FIGS. 6–23. Lasiosphaeria glabrata. 6, 7. Ascomata (Neotype, TL4529). 8. Longitudinal section through ascoma (1/12/1953 A. Munk). 9. Longitudinal section through ascomal neck (1 Dec 1953 A. Munk). 10. Longitudinal section through ascomal wall showing outer layer composed of sparse, brown hyphae (TL4529). 11, 12. Squash mounts of ascomatal walls showing outer layer composed of sparse, brown hyphae (Neotype, TL4529). 13, 14. Asci (TL4529, Neotype). 15–18. Ascal apices showing poorly developed subapical globules (Neotype, TL4615, TL4615, TL4529). 19. Young ascospore, note the absence of appendages (TL4615). 20–22. Ascospores (Neotype, 4/27/1963 A. Munk, TL4529). 23. Paraphyses (TL4529). Scale bars: 6 = 500 µm; 7, 8 = 100 µm; 9–23 = 10 µm.

Leptospora ovina (Pers. : Fr.) Fuckel var. glabrata (Fr.) Fuckel, Symb Myc 23–24:143. 1870.

Lasiosphaeria ovina (Pers. : Fr.) Ces. & de Not. var. glabrata (Fr.) Sacc., Syl Fung 2:199. 1883.

TYPE: DENMARK. JYLLAND: Staksrode near Juelsminde, District 24, on rotten stump, 10 Dec 1963, A. Munk (C, Neotype, designated here).

Ascomata subglobose to ovoid, papillate, 310–530 µm diam, 350–560 µm high, numerous, gregarious to clustered in large groups, usually superficial, occasionally erumpent; glabrous, shiny, black; neck usually conical, sometimes poorly developed, glabrous, black. Ascomatal wall of textura intricata and textura angularis in surface view, in longitudinal section 3-layered, 33.5–54.5 µm thick, inner layer pseudoparenchymatous, 5–9.5 µm thick, composed of 3–5 layers of elongate, flattened, pale brown cells, middle layer pseudoparenchymatous, 24.5–38 µm thick, composed of 4–10 layers of angular, pale brown cells, outer layer prosenchymatous, 4–7 µm thick, composed of 1–2 layers of sparse, loosely interwoven hyphae, hyphae 1.5–3 µm wide, pale brown to brown, septate, thin-walled. Ascomatal apex with periphyses. Centrum with yellow pigments which quickly diffuse in water. Paraphyses filiform, 2–6 µm wide, longer than asci, hyaline, numerous, septate, unbranched, persistent. Asci cylindrical, 160–190 x 12–18 µm, medium-stipitate, stipe 27–58 x 3–5 µm, numerous, unitunicate, thin-walled, apex rounded to truncate; ring narrow, shallow, refractive; subapical globule small to large, 2–5.5 µm diam, usually poorly developed, smooth, with 8, biseriate to tetraseriate ascospores. Ascospores cylindrical, ends rounded, 33.5–52.5 x 3–5 µm [42 ± 5.0 x 4 ± 0.5], straight when first produced, hyaline, aseptate, bipolar appendages absent; becoming slightly sigmoid or geniculate, lower one-third to one-quarter bent 45° or less; after liberation from the ascus occasionally 1-septate.

Colonies moderately slow-growing on all media, 31–38 mm diam in 21 d, silky on WA and CMA, subfelty on PDA, appressed, hyaline to yellowish white (2A2) on WA and CMA, grayish orange to grayish red (6B3–7B4) on PDA; margin even, appressed, hyaline on WA and CMA, pale yellow to grayish yellow (3A3–4B4) on PDA, distinct on PDA; reverse same as the mat in all media; anamorph not produced on WA, CMA, or PDA.

Hyphae largely undifferentiated, 1–3.5 µm wide, commonly forming hyphal coils, thin-walled, hyaline.

Substrate.. On dead, decorticated deciduous trees.

Distribution.. Seen only from Denmark.

Specimens examined.. DENMARK. FYN: Elsehoved, 4 Sep 1963, G.C. Carroll 447; 4 Oct 1963, G.C. Carroll 448 (G. Carroll Herbarium). JYLLAND: Silkeborg, Lysbro Skov, on Betula wood, 1 Dec 1953, A. Munk (C); Virklund, Knagerne, on 70 cm diam beech log, 24-IX-2001, A. N. Miller & T. Læssøe, SMH4615; on 50 cm diam beech log, SMH4617 (F). MØN: Klinteskoven, (loc. 13), on underside of Fagus wood, 26 Sep 1997, T. Læssøe TL4529 (C). SJAELLAND: Lyngby Mose, District 45a, on rotten wood, 4/27/1963, A. Munk; Lellinge A, on Fagus wood, 5 Mar 1963, A. Munk.

Culture examined.. SMH4617-1, 2, 3, 4, 5.

Lasiosphaeria ovina (Pers. : Fr.) Ces. & de Not., Comm Soc Critt Ital 1:229. 1863. FIGS. 24–45

View larger version (187K): [in this window] [in a new window]   FIGS. 24–45. Lasiosphaeria ovina. 24. Young ascomata covered in bright white tomentum (SMH4664). 25. Older ascomata with remnants of tomentum, tomentum is becoming cream-colored and appears areolate at apex (SMH2670). 26. Older ascomata with tomentum which has formed a thin, waxy, grayish coating (SMH2670). 27. Old ascoma appearing glabrous and black after tomentum has completely worn away (SMH3286). 28. Longitudinal section through ascoma (SMH1538). 29. Longitudinal section through ascomal neck (SMH1538). 30. Squash mount of ascomal wall showing outer layer composed of hyaline to pale brown hyphae (SMH3286). 31. Longitudinal section through ascomal wall showing outer layer composed of hyaline to pale brown hyphae (SMH1538). 32, 33. Asci (SMH1538, TL4634). 34, 35. Ascal apices showing globose, smooth subapical globules (TL4634, Holotype of L. chrysentera). 36. Paraphyses (SMH2670). 37, 38. Young ascospores with awl-like appendages (GC80, AR1890). 39. Hyaline ascospore (TL4634). 40. Old ascospore turning pale brown and forming oil droplets (SMH1538). 41. Phialides produced from old ascospore (SMH2620). 42, 43. Phialides produced in CMA cultures (SMH4605, K. Robinson s.n.). 44, 45. Blastoconidia produced on long and short branches in CMA cultures (SMH3923, SMH4605). Scale bars: 24–28 = 100 µm; 29–45 = 10 µm.

Sphaeria ovina Pers. : Fr., Syst Myc 2:446. 1823.

Leptospora ovina (Pers. : Fr.) Fuckel, Symb Myc 23–24:143. 1870.

= Lasiosphaeria ovina (Pers. : Fr.) Ces. & de Not. var. aureliana Fairman, J Myc 10:229. 1904.

= Lasiosphaeria chrysentera Carroll & Munk, Mycologia 56:83. 1964.

This nomenclature is not complete.

LECTOTY PE: SWITZERLAND (possibly). Hb. Pers., 910.269-84, L10261 No. 12 (as Sphaeria ovina Pers., L).

Ascomata ampulliform to ovoid, rarely obpyriform, papillate, 360–620 µm diam, 380–680 µm high, numerous, scattered to gregarious, superficial; young as-comata tomentose, white, tomentum becoming tightly appressed, crust-like and cream to brownish-gray with age, occasionally areolate, tomentum then partially wearing away and forming a thin, waxy, grayish coating, finally tomentum completely worn away and ascomata appearing glabrous and black; neck conical, glabrous, black. Ascomatal wall of textura angularis in surface view, commonly containing globular, refractive crystals in squash mounts, in longitudinal section 3-layered, 42.5–86 µm thick, inner layer pseudoparenchymatous, 8–9.5 µm thick, composed of 4–6 layers of elongate, flattened, hyaline to pale brown cells, middle layer pseudoparenchymatous, 16.5–35 µm thick, composed of 5–8 layers of polygonal to angular, pale brown cells, outer layer prosenchymatous, 18–41.5 µm thick, composed of several to few layers of hyphae depending on age of ascomata, hyphae 1–3 µm wide, hyaline to pale brown, septate, thin-walled. Ascomatal apex with periphyses. Centrum with yellow to yellowish-orange or rarely greenish-yellow pigments that quickly diffuse in water. Paraphyses filiform, 1.5–5.5 µm wide, longer than asci, hyaline, numerous, septate, unbranched, persistent, contents rarely containing blue pigments in fresh specimens only. Asci cylindrical, 140–250 x 12–23 µm, medium-to long-stipitate, stipe 30–85(–120) x 3–6.5 µm, numerous, unitunicate, thin-walled, apex truncate; ring narrow, shallow, refractive; subapical globule large, 3.5–6 µm diam, smooth to verrucose, with 8, biseriate to tetraseriate ascospores. Ascospores cylindrical, ends rounded, 35–55(–60.5) x 3–6 µm [45.5 ± 4.5 x 4.5 ± 0.5], straight when first produced, hyaline, aseptate, bipolar appendages occasionally present, 5.5–12 µm, gelantinous, spike-like or awl-like, evanescent; becoming slightly sigmoid or geniculate, lower one-third to one-quarter bent 45° or less, appendages rarely present at this stage; after liberation from the ascus up to 7-septate or rarely 9-septate, each cell with a single globose, refractive oil droplet; finally becoming yellowish, yellowish-brown or pale brown with age, sometimes constricted at clamps and appearing worm-like, occasionally producing phialides directly from the ascospore.

Colonies on WA moderately slow-growing, 21–40 mm diam in 21 d, moderately fast-growing on CMA and PDA, covering the CMA plate in 21 d and the PDA plate in 28 d, silky on WA and CMA, wooly on PDA, becoming hispid with long, erect, white hairs composed of agglutinated, thin-walled hyphae, mat appressed on WA and CMA, appressed to aerial on PDA, hyaline on all media, becoming whitish to grayish-yellow (4A1–4B4) on PDA; margin even in all media, appressed, hyaline; reverse same as the mat in all media; anamorph produced abundantly over entire mat on WA and CMA and sparsely at the margin on PDA within 14 d.

Hyphae largely undifferentiated, 1–4 µm wide, thin-walled, hyaline. Conidiogenous cells phialides, produced laterally or occasionally terminally, delimited by a basal septum, monophialidic or sometimes polyphialidic, obclavate to lageniform, 6.5–16.5 x 2.5–3.5 µm at widest part, hyaline to pale brown, constricted below the collarette, 0.5–1.5 µm below collarette; collarette minute, flaring, same color as phialide. Conidia pyriform, truncate at base, 3–4 x 2–3 µm, hyaline, produced enteroblastically, aggregated in slimy heads; slightly larger blastoconidia also commonly produced directly from hyphae or branches, branches produced laterally, not delimited by a basal septum, extremely short to long, cylindrical, 1.5–24 x 1–3 µm at widest part, hyaline, without collarette; blastoconidia subglobose to pyriform or occasionally obclavate, 3–4.5(–6) x 2.5–3.5 µm, hyaline.

Substrate.. On dead, decorticated deciduous trees.

Distribution.. Canada, Costa Rica (as L. chrysentera Carroll & Munk), Denmark, England, France, Germany, New Zealand, Philippines, Russia, Switzerland, USA.

Specimens examined.. CANADA. ONTARIO: Granton, on rotten wood, 23 Aug 1895, J. Dearness, (NY). COSTA RICA. Vulcan Irazu, rough road, second bridge from San Jose, on wood, 22 Jun 1962, G. Carroll 85 (HOLOTY PE of Lasiosphaeria chrysentera, NY; ISOTY PE, G. Carroll herbarium). DENMARK. Copenhagen, Hareskov, 7 Oct 1962, G. Carroll 80 (G. Carroll herbarium). JYLLAND: Silkeborg, Virklund, Knagerne, on 60 cm beech log, 24-IX-2001, A.N. Miller & T. Læssøe, SMH4616; on 70 cm beech log, SMH4623; Tirstrup, Noerupland, on 4 cm branch, 25-IX-2001, A.N. Miller & T. Læssøe, SMH4634; Femmoeller, Strandkjaer at Langemosen, on 10 cm aspen branch, 25-IX-2001, A.N. Miller, T. Læssøe, J. Pedersen, SMH4638. SJAELLAND: Køge, Lellinge Skovhusvaenge, on 6 cm branch, 20-IX-2001, A.N. Miller, T. Læssøe, T. Frøsler, SMH4605. ENGLAND. Hertfordshire, Ayot St. Lawrence, Prior’s Wood, on wood, 9/25/2001, K. Robinson s.n. (F). FRANCE. Bois de Barm, Tarmes, 1902, Pere Duss 173 (NY); Roum. F. sel. Gall. 5857, as Lasiosphaeria ovina var. glabrata, sur les copeaux pourissants du chêne, bois des environs de Saint-Saens (Seine-Inférieure), on wood, August, (UPS). GERMANY. Syd. Myc. Mar. 2144, as Lasiosphaeria ovina var. glabrata, canal by Marienspring by Andow?, on Fagus silvatica wood, F. 1888, P. Sydow, (UPS, NY). NEW ZEALAND. A.Y. Rossman, AR1899, AR1890 (BPI). PHILIPPINES. LAGUNA PROVINCE: Mount Maquiling, 11/4/1963, D. Reynolds 185, (G. Carroll Herbarium). RUSSIA. Kamchatka, Avacha River valley, bridge over Avacha River by Razdolny, 53°13'N, 158°20'E, on cf. Populus suaveolens, 8/1/1997, T. Læssøe, TL4634 (C). SWITZERLAND (possibly). Hb. Pers., J. F. Chaillet?, 910.269-153, L10261 No. 14; Chaill. in Hb. Pers.,? Sphaeria affinis, Sph. ovinae, = J. F. Chaillet?, 910.269-156, L10261 No. 15 (L). USA. CONNECTICUT: Poquonock, 9/8/1893, 8/4/1895, W.C. Sturgis (NY). ILLINOIS: Cook County, Cook County Forest Preserve, Swallow Cliffs, on wood fragment, 3-VII-1996, S.M. Huhndorf, F.A. Fernández, M. Huhndorf, SMH2535; Ogle County, Castle Rock State Park, on 30 cm log, 27-IX-1996, S.M. Huhndorf & F.A. Fernández, SMH2670; SMH2672; on 8 in branch, SMH2679; on branch fragment, SMH2684; White Pines Forest State Park, on 1 in branch, 28-IX-1996, S.M. Huhndorf & F.A. Fernández, SMH2696; SMH2697; on wood fragment, SMH2711; on 7 cm branch, SMH2712; on 6 in branch, SMH2717; Jackson County, Giant City State Park, Devil’s Roundtable, on wood, 10-VIII-1999, A.N. Miller, SMH4124; Vermilion County, Forest Glen County Preserve, on 2.5 cm branch, 30-IX-2000, A.N. Miller, SMH4329. INDIANA: Lake County, Indiana Dunes National Lakeshore, Cowles Bog, on 15 in log, 1-VII-1996, S.M. Huhndorf, F.A. Fernández, M. Huhndorf, SMH2524; Dyer, 9981 Sheffield Ave., on wood fragment, 1-XII-2001, S.M. Huhndorf, SMH4664; Brown County, Yellowwood State Forest, compartment 7, tract 24, on 2 in branch, 26-VII-1996, S.M. Huhndorf, SMH2635 (F). LOUISIANA: 20-VIII-1890, A.B. Langlois 748; Plaquemines Parish, Hebert Center, on perithecia, 3 Nov 1970, Flora Ludoviciani 7539 (NY). MAINE: Oldtown, Aug 1897, F.L. Harvey (NY). MICHIGAN: Berrien County, Warren Woods, south end of trail, through picnic area, up to creek, on 5 cm branch fragments, 8-IX-1998, F.A. Fernández & A.N. Miller, SMH3887; on wood fragment, SMH3894; north end, on wood, 7-X-1999, A.N. Miller, SMH4198; on 12 in maple log, 21-IX-2000, F.A. Fernández & A.N. Miller, SMH4325; SMH4326; Marquette County, Huron Mountain Club, along N side of Middle Pine Lake (Second Pine), [45.8692, –87.854], 210 m, on 8 in birch log, 12-VII-1996, S.M. Huhndorf, F.A. Fernández, M. Huhndorf, SMH2549; along Loop Road, Fisher Creek trail, Trout Lake Trail, Mountain Stream Trail, [46.8657, –87.8844], 270 m, on 20 in hemlock log, 13-VII-1996, S.M. Huhndorf, F.A. Fernández, M. Huhndorf, SMH2576; base and slope of Mount Ives and NW corner of Ives Lake, on 50 cm log, 14-VII-1996, S.M. Huhndorf, F.A. Fernández, M. Huhndorf, SMH2587; on roadside toward Cranberry Bog, [45.8762, –87.8472], 200 m, on 3 in branch, 15-VII-1996, S.M. Huhndorf, F.A. Fernández, M. Huhndorf, SMH2608; on 10 cm branch, SMH2611; on 7 cm branch, SMH2616; on 15 cm log, SMH2618; on 5 in branch, SMH2619; on 30 in maple log, SMH2620; on 3 in branch, SMH2624; S of Howe Lake, road between Ann and Howe Lakes, on 1 in branch, 18-VIII-1997, S.M. Huhndorf & M. Huhndorf, SMH3370; on 1 in branch, SMH3375; on log fragments, SMH3378; on 1 in branch, SMH3382; on 2 cm branch, SMH3387; trail behind boathouses, bottom of ridge to the left, on wood, 5-X-1998, F.A. Fernández, SMH3923; SMH3932; Cranberry Bog, [45.8762, –87.8472], 200 m, on 2 cm branch, 7-X-1998, F.A. Fernández, SMH3955; before the creek on trail to Fisher Creek/Trout Lake, on 70 cm diam log, 8-X-1998, F.A. Fernández, SMH3966; roadside, 2 mi from gate, 30 m to Howe Lake, on inner side of bark, 9-X-1998, F.A. Fernández, SMH4000; west edge of Rush Lake, on wood, 20-IX-1999, A.N. Miller, SMH4171; Neus plot 2, on wood, 22-IX-1999, A.N. Miller, SMH4177 (F). NEW JERSEY: Warren Township, Plainfield, 9 Sage Drive, on Quercus, 9 Oct 1961, G. Carroll 79 (G. Carroll herbarium). NEW YORK: Yalis? on moist bark of rotten log (Tilia?), Oct 1900, C.E. Fair-man, Mycotheca Fairmani 545 (ISOTY PE of Lasiosphaeria ovina var. aureliana); Ridgeway, on prostrate trunk of Tilia, 16 Sep 1904, C.E. Fairman, Mycotheca Fairmani 548, as Lasiosphaeria ovina var aureliana; Lyndonville, on rotten wood, Aug 1905, C.E. Fairman, Mycotheca Fairmani 547; on the ground in woods, on rotten wood, Oct 1905, C.E. Fairman, Mycotheca Fairmani 546; on rotten wood, Sep 1910, C.E. Fairman, Mycotheca Fairmani 3217; Janesville, Oct 1887, O.F. Cooke (NY); Varna, Fries Road Extension, 22 Jul 1960, W.C. Denison 81; Ithaca, RD No. 5, The Briar Patch-W. Ravine, 11 Aug 1965, G. Carroll 987 (G. Carroll herbarium). NORTH CAROLINA: Macon County, west of Franklin, woods along Roaring Fork, Nantahala River, on rotten log, 17 Sep 1960, C.T. Rogerson & R.H. Petersen (NY); Highlands, Highlands Biological Station, on 8 in log, 9-X-1996, S.M. Huhndorf & F.A. Fernández, SMH2784; on 1 in branch, SMH2785; on 10 cm branch, 21-VII-1997, F.A. Fernández, SMH3315; Blue Valley, [35.0192, –83.2736], 1000 m, on 40 cm log, 18-VII-1997, F.A. Fernández, SMH3274 (F). VIRGINIA: above Chain Bridge, 5 Oct 1923, C.L. Shear (NY). WISCONSIN: Dane County, Dawley Park, Fitschburg, along trail, on 24 in log, 13-IX-1995, S.M. Huhndorf, SMH1538; on 6 in branch, SMH1539; Fond du Lac County, Kettle Moraine State Forest, Mantle Lake Recreational Area, wet area scenic trail, on wood, 24-IX-1999, S.M. Huhndorf, SMH4189; Grant County, Wyalusung State Park, on 12 in log, 26-VII-1995, S.M. Huhndorf, SMH1530; Green County, New Glarus State Park, on branch, 13-IX-1995, S.M. Huhndorf, SMH1544 (F).

Cultures examined.. SMH1538-1, 2, 3, 4; SMH3923-1, 2, 3, 4, 5, 6; SMH3286-1, 3; SMH4605-1, 2, 3, 4; CBS958.72, AR1899-1, KR-1, 2, 3, 4, 5.

Lasiosphaeria rugulosa (A.N. Mill. & Huhndorf) A.N. Mill. & Huhndorf Myc Res 108:31.

Cercophora rugulosa A. N. Mill. & Huhndorf, Sydowia 53:215. 2001.

Colonies moderately slow-growing on PDA, 28–34 mm diam in 21 d, silky to subfelty, appressed, hyaline, becoming whitish to yellowish-white (3A2); margin even or plumose, appressed, hyaline, not distinct; reverse same as the mat; anamorph not produced.

Culture examined.. SMH1518-1 (= ATCC MYA-2578), 2 (= ATCC MYA-2579), 3.

Lasiosphaeria sorbina (Nyl.) P. Karst., Bid Kann Fin Nat Folk 23:164. 1873. FIGS. 46–61

View larger version (176K): [in this window] [in a new window]   FIGS. 46–61. Lasiosphaeria sorbina. 46, 47. Ascomata (Holotype, GJS L555). 48. Longitudinal section through ascoma (Holotype). 49. Longitudinal section through ascomal neck (Holotype). 50. Longitudinal section through ascomal wall showing outer layer composed of hyaline to brown hyphae (Holotype). 51. Paraphyses (GJS L555). 52, 53. Asci (GJS L555). 54, 55. Ascal apices, note lack of subapical globules (GJS L555, Holotype). 56–58. Ascospores (GJS L555, EC-556, Holotype). 59. Phialides produced from old ascospore (GJS L555). 60, 61. Phialides produced in CMA cultures (CBS885.85, GJS L555). Scale bars: 46–48 = 100 µm; 49–61 = 10 µm.

Bizzozeria sorbina (Nyl.) Höhn., Ann Mycol 16:76. 1918.

Leptospora sorbina (Nyl.) Munk, Dansk Bot Ark 14(7): 6. 1952.

Additional heterotypic synonyms are listed by von Höhnel (1918), but their synonymy has not been confirmed.

HOLOTY PE: FINLAND. Helsingfors, on Sorbus aucuparia branch, 24 Mar 1863, Nylander (as Sphaeria sorbina Nyl., H).

Ascomata ampulliform to ovoid, papillate, (240–) 320–460 µm diam, (280–)370–600 µm high; numerous, scattered to gregarious, usually superficial, occasionally erumpent; tomentose, tomentum dull gray to brownish-gray or pale pinkish to orange, appressed, waxy, sometimes wearing away with age; neck conical, glabrous, sometimes with a distinct, grayish ring around black ostiole. Ascomatal wall of textura angularis in surface view, in longitudinal section 3-layered, 48.5–76.5 µm thick, inner layer pseudoparenchymatous, 6.5–9.5 µm thick, composed of 3–5 layers of elongate to flattened, pale brown cells, middle layer pseudoparenchymatous, 12.5–22.5 µm thick, composed of 4–6 layers of angular, pale brown cells, outer layer prosenchymatous, 29.5–44.5 µm thick, composed of numerous layers of hyphae, hyphae 1.5–3.5 µm wide, hyaline to yellowish, septate, thin- to slightly thick-walled, becoming melanized and brown in outermost 3–6 layers. Ascomatal apex with periphyses. Centrum with yellow, pinkish, or rarely orange pigments which quickly diffuse in water. Paraphyses filiform, 1.5–4.0 µm wide, hyaline, numerous, septate, unbranched, longer than asci. Asci cylindroclavate to clavate, 125–185 x 13–21.5 µm, medium-stipitate, stipe 26–52 x 4–8.5 µm, numerous, unitunicate, thin-walled, apex rounded to truncate; ring narrow, extremely shallow, refractive, indistinct; subapical globule absent, with 8, biseriate to triseriate ascospores. Ascospores short cylindrical, ends rounded, 21.5–46 x 4.5–9 µm [33 ± 5.5 x 6.5 ± 1.0], straight to slightly curved, hyaline, aseptate to 1-septate, appendages absent; after liberation from the ascus up to 5-septate, occasionally producing phialides directly from the ascospore.

Colonies slow-growing on WA and PDA, 15–28 mm diam and 15–27 mm diam in 21 d on WA and PDA, respectively, moderately slow-growing on CMA, 18–33 mm diam in 21 d, silky on all media, appressed, hyaline, becoming brownish gray to pastel red (7C2–8A5) on PDA; margin even, appressed, hyaline, not distinct; reverse same as the mat in all media; anamorph produced commonly over entire mat on WA, sparsely over entire mat on CMA and sparsely at the margin on PDA within 14 d.

Hyphae largely undifferentiated, 1–3.5 µm wide, thin-walled, hyaline. Conidiogenous cells phialides, produced laterally or terminally, delimited by a basal septum, monophialidic, obclavate to lageniform, 9–18 x 2–4 µm at widest part, hyaline, constricted below the collarette, 1–1.5 µm below collarette; collarette minute, slightly flaring, same color as phialide. Conidia pyriform, truncate at base, 2.5–3.5 2–2.5 µm, hyaline, produced enteroblastically, aggregated in slimy heads; blastoconidia not produced.

Substrate.. On dead, decorticated deciduous trees with remnants of bark.

Distribution.. Ecuador, Finland, France, Netherlands, USA.

Specimens examined.. ECUADOR. PICHINCHA PROV.: 2 km above Toachi, on the Toachi-Palo Quemado Road, 3400 ft, on wood, 19 Jul 1975, K.P. Dumont, S.E. Carpenter, P. Buritica, EC-556 (NY). FRANCE. ARIÈGE: 09 Montseron, Las Muros, Roquebrine-Ruisseau de Peyrau, on Quercus zobur branch, 3 Mar 2002, J. Fournier, JF02053 ( J. Fournier Herbarium). NETHERLANDS. As Sphaeria mutabilis, on wood, Hb. Pers. saltem ei affinis prope parisios, 910.263-1251, L. 10261 No. 10; on wood, 910.269-91, L. 10261 No. 11 (L). USA. LOUISIANA: West Feliciana Parish, St Francisville, Grace Church Cemetary, on branch, 18 Aug 1996, G.J. Samuels & M. Blackwell s.n., GJS L555 (F). OHIO: Morgan s.n., as Lasiosphaeria ovina (NY).

Cultures examined.. GJS L555-1, 2, 3, 4, 5; CBS885.85.

Lasiosphaeria lanuginosa species complex FIGS. 62–81

View larger version (159K): [in this window] [in a new window]   FIGS. 62–81. Lasiosphaeria lanuginosa species complex. 62–65. Ascomata, note older ascomata in Fig. 65 with only a few yellowish hairs remaining (SMH3819, Holotype, SMH4027, SMH2390). 66. Longitudinal section through ascoma (SMH3819). 67. Longitudinal section through ascomal neck (SMH2390). 68. Longitudinal section through ascomal wall showing outer layer composed of hyaline to pale brown hyphae (SMH3819). 69, 70. Asci (SMH3277, SMH2395). 71–73. Ascal apices showing globose, smooth subapical globules (SMH4594, holotype, SMH4027). 74. Paraphyses (SMH3277). 75. Young, cylindrical ascospores (SMH3819). 76. Young ascospores with lash-like appendages (SMH3277). 77, 78. Older ascospores with swollen heads, some of which have turned brown (SMH2390). 79. Phialides produced in WA cultures (SMH4027). 80, 81. Blasto-conidia produced on short and long branches in CMA cultures (both SMH3277). Scale bars: 62, 64–66 = 100 µm; 63 = 500 µm; 67–81 = 10 µm.

Colonies slow-growing on WA and PDA in SMH3277 and SMH3819, 14–22 mm diam and 22–25 mm diam in 21 d on WA and PDA, respectively, moderately slow-growing on WA in SMH4027, 31–35 mm diam in 21 d, (SMH4594 did not grow on WA), moderately slow-growing on CMA in SMH3277, SMH3819 and SMH4594, 31–33 mm diam in 21 d, moderately fast-growing on CMA and PDA in SMH4027, covering the CMA plate in 21 d and the PDA plate in 28 d, moderately slow-growing on PDA in SMH4594, 29–35 mm diam in 21 d on PDA, silky on all media, becoming subfelty to crustose on PDA, hyaline on all media, becoming yellowish-brown to dark brown (5F8–6F8) around the plug on PDA; margin even or plumose in all media, appressed, hyaline on all media, becoming yellowish-gray to brownish-gray (4B2–6C2) on PDA, distinct in PDA; reverse same as the mat in all media except in SMH4594 which was black on PDA; anamorph produced sparsely around the plug in all media within 21 d in SMH3277, SMH4027, and SMH4594, anamorph not produced in SMH3819.

Hyphae largely undifferentiated, 1–3.5 µm wide, thin-walled, hyaline to pale brown. Conidiogenous cells phialides, commonly produced laterally or occasionally terminally from pale brown hyphae, delimited by a basal septum, monophialidic or occasionally polyphialidic, obclavate to lageniform, 6–16 x 2.5–4.5 µm at widest part, pale brown, constricted below the collarette, 0.5–1.5 µm below collarette; collarette minute, slightly flaring, same color as phialide. Conidia pyriform, truncate at base, 2.5–3 x 2–2.5 µm, hyaline, produced enteroblastically, aggregated in slimy heads; slightly larger blastoconidia also commonly produced directly from hyphae or branches, branches produced laterally singly or occasionally terminally singly or in pairs, not delimited by a basal septum, short to long, cylindrical or occasionally bulbulous to lecythiform, 2.5–15 x 1–3 µm at widest part, hyaline, without collarette; blastoconidia pyriform to obclavate, 3.5–5.5 x 2.5–3.5 µm, hyaline.

Substrate.. On the decorticated parts of dead, deciduous branches which still possess remnants of adhering bark, or rarely on dead leaves or grass.

Distribution.. Costa Rica, Denmark, England, France, Peru, USA, Venezuela.

Specimens examined.. COSTA RICA. SAN JOSE: San Gerardo de Dota, Albergue de Montana, Savegre, trail to waterfall along Rio Savegre, [9.5439, –83.8142], 2150 m, on 5 in log, 11-V-1996, S.M. Huhndorf & F.A. Fernández, SMH2350; Sendero la Quebrada, [9.55, –83.8], 2300 m, on 2 in branch, 12-V-1996, S.M. Huhndorf & F.A. Fernández, SMH2389, on old bark, S.M. Huhndorf & F.A. Fernández SMH2390; on 5 cm branch, S.M. Huhndorf & F.A. Fernández, SMH2394. PUNTARENAS: La Amistad Pacifico, Sitio Cotoncito, [8.9403, –82.7892], 1600 m, on wood, 14-I-1999, F.A. Fernández, SMH4027. CARTAGO: Cantón Paraiso, Distrito Orosi, Parque Nacional Tapanti, Oropendula trail, [9.7517, –83.7908], 1300 m, on 4 cm standing stump, 27-VI-2000, F.A. Fernández, SMH4301. DENMARK. Læsø, Kærene, on Molinia caerulea stems, 9-X-1999, T. Læssøe, TL5656. ENGLAND. Northumberland, on the Belsay to Scots Gap road 2 mi N of Belsay, Bolam Lake Country Park, on 3 cm birch log, 13-IX-2001, G. Brand & B. Brand, SMH4594 (F). FRANCE. on Cirsium palustre stems, 5/17/1864, (HOLOTY PE, CO). ARIÈGE: Rimont, Las Muros, Ruisseau de Peyrau, 400 m, on wood, 12-VIII-2001, J. Fournier, JF01167 ( J. Fournier Herbarium); 25-IX-2002, A.N. Miller, J. Fournier, A.M. Stchigel, M. Calduch, SMH4826 (F). PERU. DEPARTAMENTO. CUZCO: along the Cuzco-Pilocopata-Paucartambo Road at a point ca 132 km from the intersection with the Cuzco-Puno Road, on leaf, 19 Jul 1976, K.P. Dumont, S.E. Carpenter, M.A. Sherwood, P. Buritica, PE-1837, PE-1852 (NY). USA. NORTH CAROLINA: Macon County, Otto, Cowieta Hydrological Laboratory, on 4 cm log, 19-VII-1997, F.A. Fernández, SMH3277; Highlands, Highlands Biological Station, on wood piece, 20-VII-1997, F.A. Fernández, SMH3308; Horse Cove, [35.025, –83.1464], 1000 m, on 5 cm diam branch, 27-VII-1998, F.A. Fernández, SMH3819 (F). VENEZUELA. Cerro de Neblina, Neblina base camp, 18-II-1985, A.Y. Rossman, AR2148 (BPI).

Cultures examined.. SMH3277-1, 3; SMH3819-1, 2, 4; SMH4027-1, 2, 3, 4, 5, 6; SMH4594-1, 2, 3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Multiple gene genealogies indicate four of the five Lasiosphaeria morphospecies occur as well-supported phylogenetic species. These species are corroborated further by MSR in possessing a unique combination of morphological characters. Lasiosphaeria lanuginosa forms a genetically variable species complex comprising a single morphological species following MSR and several phylogenetic species employing GCPSR. Although these lineages could be recognized formally as species based on GCPSR, it is premature to do so because additional populations of L. lanuginosa need to be sampled and at least three additional morphospecies, which putatively belong in the L. lanuginosa species complex, should be included in further analyses.

Although species relationships differ slightly among the separate analyses (FIGS. 1–4), they are well-resolved in the combined analysis (FIG. 5). Lasiosphaeria rugulosa and L. glabrata are basal taxa, while L. ovina, L. sorbina and members of the L. lanuginosa complex are more derived. These latter three species possess a well-developed, colored tomentum, whereas L. glabrata and L. rugulosa possess a sparse, brown tomentum. Cercophora sparsa and C. sulphurella possess yellowish hairs, while C. areolata has brown, villose hairs. This suggests that colored vestiture may have been lost in L. glabrata and L. rugulosa and then regained in L. ovina, L. sorbina and members of the L. lanuginosa complex. The out-groups, L. rugulosa, and members of the L. lanuginosa complex possess ascospores that develop swollen heads, while ascospores in L. glabrata, L. ovina and L. sorbina lack swollen heads or the ascospores swell only slightly. The evolution of this character is presently unclear because the ability to develop a swollen ascospore head recently has been shown to have occurred numerous times throughout the Sordariales (Miller and Huhndorf 2004b).

Lasiosphaeria glabrata can be distinguished in possessing nearly glabrous, black ascomata (FIGS. 6, 7) and ascospores that lack a swollen head and appendages (FIGS. 19–22). Ascomata of L. glabrata usually appear shiny due to the outer layer of sparse brown hairs (FIGS. 11, 12). This species closely resembles L. rugulosa in outer ascomal features but can be separated easily by its longer ascospores (33.5–52.5 versus 29.5–37.5), which do not develop a swollen head. Munk (1957) elevated L. glabrata from a variety (as L. ovina var. glabrata) to the species level, but it is not clear whether he actually saw any of Fries’ material. Ascomata of L. glabrata commonly are confused with old ascomata of L. ovina in which the tomentum has worn away (FIG. 27). In fact, two collections of L. ovina var. glabrata distributed in exsiccati (Roum. F. sel. Gall. 5857 [UPS] and Syd. Myc. Mar. 2144 [as Leptospora ovina var. glabrata; UPS, NY]) are unambiguously L. ovina because young ascomata were found that possessed white tomentum and appendages were observed in young ascospores in the NY material. Because Fries’ material of L. ovina var. glabrata could not be located, a neotype for L. glabrata based on Munk’s material is designated here.

Lasiosphaeria ovina is recognized by its white, tomentose ascomata (FIG. 24), asci with globose sub-apical globules (FIGS. 32–35), and ascospores that do not develop a swollen head (FIGS. 39–41) and usually possess short, awl-like appendages (FIGS. 37, 38). The outer ascomal features of L. ovina can be quite variable. Ascomata are densely tomentose and bright white when young (FIG. 24), but as the tomentum breaks down and begins to wear away, it can become areolate and cream or brownish-gray (FIG. 25) and ascomata may appear wax-like (FIG. 26) or even glabrous and black with age (FIG. 27). This species has been described more than once based in part on these environmental variations of the tomentum (e.g., L. chrysentera). There has been much confusion as well concerning the ornamentation and shape of the subapical globule. The globose globule is composed of a cytoplasmic refractive substance and may appear somewhat ornamented, especially in poorly preserved collections. Lundqvist (1972) lectotypified L. ovina based on a Persoon collection (L) and described the subapical globule as verrucose. No spiny-shaped globule as drawn by Carroll and Munk (1964) and Hilber et al (1987) has been seen by the authors. In addition, the oversized ellipsoid subapical globule as illustrated by Réblová et al (1999, FIGS. 4 and 13a) does not accurately represent the globose subapical globule commonly seen in L. ovina (FIGS. 34, 35) but is merely an artifact caused by a slight invagination of the cytoplasm. One striking feature found in some collections of L. ovina is the heterobasidiomycete my-coparasite, Krieglsteinera lasiosphaeriae Pouz., that occasionally infects the ascomata. This mycoparasite most likely is distributed widely throughout Asia, Europe and North America but is found rarely due to its cryptic nature (Miller et al 2003).

The holotype of L. chrysentera was examined; the ascomata possess the full range of tomentum variation from young, white, tomentose ascomata to grayish, areolate ascomata to old, black, glabrous ascomata. Most ascomata were old and appeared black and glabrous. As described by Carroll and Munk (1964), the centrum contents were "bright yellow" to "golden-brown," the subapical globule appeared "golden-brown" (but not "coarsely echinulate" as shown in FIG. 35), and the ascospores were "light yellow to golden-brown" and "finely and irregularly pitted." However, the coloration and ornamentation of the subapical globule and ascospores is merely an artifact of the age, poor condition and method of preservation of the specimen. Carroll and Munk (1964) were unable to culture their fungi, which they believed "may be attributed to the vigorous drying techniques used to preserve the specimens in a tropical climate." These vigorous drying techniques most likely caused the yellow centrum pigments in L. ovina to stain the asci and ascospores a golden color and also might explain the irregular pitting observed in the ascospores. This phenomenon also has been observed to a lesser extent in other specimens of L. ovina that have been poorly preserved due to overdrying. The name L. chrysentera unfortunately has proliferated throughout the literature (Hilber et al 1987, van Hooff 1993, Vasilyeva 1998, Candoussau et al 2002, Hilber and Hilber 2002, Zhang and Zhuang 2003) when referring to specimens of L. ovina in which the tomentum has become areolate, the subapical globule is spiny and the ascospores are slightly yellowish, although these characters either do not exist or fall within the range of variation found in L. ovina. Lasiosphaeria chrysentera is synonymized under L. ovina because it merely represents a poorly preserved specimen of this species.

Fairman (1904) distinguished Lasiosphaeria ovina var. aureliana from L. ovina by the presence of ascospore appendages and the flavovirescent color of the centrum. Although Fairman (1904) and Lundqvist (1972) believed ascospore appendages probably were lacking in L. ovina, structures resembling awl-like appendages were found in lectotype material of L. ovina by the present authors. Hilber et al (1987) examined three collections determined by Fairman to be Lasiosphaeria ovina var. aureliana and believed one was L. chrysentera while the other two were L. ovina. We determined all three collections to be L. ovina. While the centrum in holotype material of Lasiosphaeria ovina var. aureliana does contain a slightly greenish-yellow cast, this hue falls within the range of yellowish centrum colors observed in L. ovina. This variety is synonymized under L. ovina.

Material of Lasiosphaeria ovina var. vagens Chen. was not available from Paris for examination.

Lasiosphaeria libertiana is known from the two isotype specimens (BR and NY), which occur on decaying grass culms. This species is identical morphologically to L. ovina except it occurs on a different substrate (grass versus wood) and the ascospores are slightly longer (51–66 versus 35–55 µm) (TABLE II); the lengths of which fall within the 95% confidence interval of ascospore lengths in L. ovina. Until molecular data can be obtained for L. libertiana, it should be maintained as a distinct morphospecies distinguished from L. ovina by its unique habitat on grass and slightly longer ascospores.

Lasiosphaeria rugulosa is distinguished by the wrinkled appearance of the ascomata upon drying and the short ascospores that form swollen heads and lack appendages. Although not apparent in macroscopic view, squash mounts of the ascomal wall reveal a tightly compressed third wall layer composed of interwoven, sparse, brown hyphae similar to that found in L. glabrata. The holotype was described as possessing paraphyses with blue contents (Miller and Huhndorf 2001). However, the taxonomic value of this character presently is unclear because it now also has been found to occur rarely in L. ovina and in one member of the L. lanuginosa complex (SMH4027). Lasiosphaeria rugulosa is morphologically similar to C. sparsa but has a tropical distribution and rugulose ascomata, whereas C. sparsa is a temperate species with smooth ascomata. These taxa occur as two distinct phylogenetic species in all analyses (FIGS. 1–5).

Lasiosphaeria sorbina is recognized by having as-comata covered in a dense, colored tomentum (FIGS. 46, 47), asci that lack subapical globules (FIGS. 54, 55) and short, slightly curved ascospores that lack appendages (FIGS. 56–58). This species exhibits a broad geographic range and possesses a wide range of tomentum and centrum colors. European specimens (holotype, JF02053) possess a gray tomentum and a yellow or pinkish centrum, North American material (Ohio, Morgan s.n., GJS L555) possess a gray or bright orange tomentum and a yellow or orange centrum and South American material (EC-556) possess a pale pinkish to pale orange tomentum and a yellow centrum. Lasiosphaeria cylindrospora also possesses a gray tomentum but can be distinguished from L. sorbina by its hyaline centrum and longer ascospores (46–56 versus 21.5–46) (TABLE II).

Most members of the L. lanuginosa complex can be distinguished by having ascomata covered with a dense, white to yellowish or rarely pale pinkish tomentum (FIGS. 62–64) and ascospores that usually develop a swollen head (FIGS. 77, 78) and possess lash-like appendages (FIG. 76). This morphospecies grows on the decorticated part of dead branches, which still possess some remnants of bark, or rarely on dead leaves or grass. This is in contrast to L. ovina, which grows on decorticated wood suggesting that members of the L. lanuginosa complex may be earlier colonizers of wood than L. ovina. Although there is a wide range of variation in the amount and color of the tomentum in members of the L. lanuginosa complex, they usually can be differentiated from other species based on ascospore morphology. The tomentum is usually persistent and the ascomata seldom become black and glabrous as in L. ovina. When the tomentum does wear away, only a few sparse yellowish hairs remain (FIG. 65) making it difficult to distinguish this morphospecies from C. sparsa. However, C. sparsa can be separated by its shorter ascospores (29.5–37 versus 33–60) that lack appendages. While most members of the L. lanuginosa complex possess a white or yellow (TABLE II) tomentum, specimens from Peru possess a pale pinkish tomentum similar to that found in Ecuadorian specimens of L. sorbina. However, these specimens readily are distinguished from L. sorbina by their longer ascospores, which develop swollen heads and possess appendages. Ascospores that have formed swollen heads inside asci are usually hyaline and sometimes difficult to find, especially in tropical specimens, in which only one or two asci may contain ascospores with swollen heads. Ascospores in which the swollen heads have turned brown are seldom observed in the ascus, especially in older material, and usually only a few brown heads are found adhered to the outside wall layer of squashed ascomata.

Most of the sampled members of the L. lanuginosa complex possess characters slightly different from those found in the type specimen. Three specimens possess shorter ascospores (e.g., SMH4027, SMH3819, AR2148); SMH2390 has ascomata covered with a sparse, yellow tomentum; SMH4594 contains ascospores that lack a swollen head and possess awl-like appendages; and TL5656 occurs on grass stalks rather than wood (TABLE II). Because only SMH3277 possesses characters similar to those found in the type specimen, it is in this lineage where L. lanuginosa most likely will find its taxonomic placement.

Some members of the L. lanuginosa complex possess combinations of characters making it difficult to assign them to any of the known morphospecies. Two specimens possess characters attributable to L. ovina. Specimen SMH4027 has a white tomentum and the ascospore heads swell only slightly, while specimen SMH4594 possesses ascospores that do not develop a swollen head and possess awl-like appendages (TABLE II). Specimen TL5656 possesses a combination of substrate, tomentum, and ascospore characters that could place it into any one of three morphospecies (TABLE II). It shares a similar substrate (grass stalks) and tomentum color (white) with L. libertiana but possesses ascospores that form a swollen, brown head. This specimen also closely resembles C. gossypina and L. lanuginosa in its white to pale yellowish, tomentose ascomata but differs from both in substrate (TABLE II). These results suggest that certain characters, such as tomentum color, development of a swollen ascospore head and the type of ascospore appendage, form a morphological continuum and are uninformative for delimiting taxa in the L. lanuginosa complex.

Three of the five remaining morphospecies, which putatively belong in Lasiosphaeria, most likely belong in the L. lanuginosa complex. Cercophora citrina, C. gossypina and C. spirillospora possess tomentose as-comata and ascospores that develop swollen heads and possess lash-like appendages. These morphospecies can be distinguished based on their substrate, tomentum color and ascospore length. Cercophora citrina occurs on elephant dung and has a reddish-orange tomentum and extremely long ascospores, C. gossypina occurs on hare dung and has a whitish tomentum, while C. spirillospora occurs on bamboo and has a yellowish-brown tomentum (reported to be reddish when fresh, Penzig and Saccardo 1897) (TABLE II). It is obvious that these morphospecies must be included in additional molecular analyses and much more work is needed in the L. lanuginosa complex before species boundaries can be established.

As previously discussed, a wide range of tomentum and centrum colors exists in L. ovina, L. sorbina and members of the L. lanuginosa complex. Changes in tomentum color are primarily due to the age of the ascomata. For example, young ascomata in L. ovina are bright white but the tomentum quickly becomes dull gray with age, whereas those in the L. lanuginosa complex begin white or pale yellow and sometimes turn orange-brown with age. The tomentum also may wear away with age in all three species, giving the ascomata a glabrous appearance. Climate and/or geography also may affect color in these species. While temperate specimens of L. sorbina have a gray tomentum, subtropical and tropical specimens possess a pale pinkish to orange tomentum. Specimens of L. lanuginosa from North and Central America have a yellow tomentum, while those from South America possess a pale pinkish tomentum. As pointed out by Lundqvist (1972), different substrates also may induce different colors in the tomentum and centrum. The holotype of C. gossypina occurs on hare dung and most ascomata possess a pale yellow tomentum and centrum, whereas the paratype occurs on porcupine dung and ascomata possess a white tomentum and hyaline centrum. Further work is needed to determine the effect of age, climate, geography and substrate on tomentum and centrum colors and their significance in delimiting species boundaries in Lasiosphaeria.

Culture studies mostly were uninformative for delimiting the five included species of Lasiosphaeria. However, these species can be distinguished on PDA by the color of their mycelial mat; L. glabrata is grayish orange to grayish red (6B3–7B4), L. ovina is whitish to grayish-yellow (4A1–4B4), L. rugulosa is whitish to yellowish-white (3A2), L. sorbina is brownish gray to pastel red (7C2–8A5), and members of the L. lanuginosa complex are yellowish-brown to dark brown (5F8–6F8). Growth of isolates of each species was uninformative on all three media because the rates overlapped for most isolates, although those of L. glabrata and L. sorbina tended to grow more slowly than those of the other three taxa.

Lasiosphaeria ovina, L. sorbina and members of the L. lanuginosa complex produced similar Phialophora-type anamorphs in culture (FIGS. 42, 43, 60, 61, 79). Phialides were mostly obclavate to lageniform, hyaline or pale brown and possessed minute, slightly flaring collarettes. Similar phialides also were produced occasionally from old ascospores of L. ovina and L. sorbina (FIGS. 41, 59). This type of anamorph previously has been reported for L. ovina (Gams 1973, Gams and Holubová-Jechová 1976, Gams 2000) and placed in Phialophora Medlar section Catenulatae W. Gams (Gams and Holubová-Jechová 1976). Lasiosphaeria ovina and members of the L. lanuginosa complex also produced blastoconidia directly from the hyphae or from cylindrical, collaretteless branches (FIGS. 44, 45, 80, 81). The production of blasto-conidia previously has been reported in Cercophora atropurpurea A.N. Mill. and Huhndorf (Miller and Huhndorf 2001). Anamorphs were not produced in cultures of L. glabrata and L. rugulosa.

Species boundaries for four of the five morphospecies within Lasiosphaeria have been defined using GCPSR based on multiple gene genealogies. Extensive morphological examinations of teleomorph, anamorph and culture characters reveal that these species can be distinguished morphologically by a combination of diagnostic characters primarily involving those of the teleomorph. Although it is premature to recognize formally the various phylogenetic species discovered in the L. lanuginosa species complex, this study provides important directions for the sampling of additional populations and morphospecies both within this complex as well as within Las