Mycologia, 96(5), 2004, pp. 1106–1127. ᭧ 2004 by The Mycological Society of America, Lawrence, KS 66044-8897

Using phylogenetic species recognition to delimit species boundaries within Lasiosphaeria

Andrew N. Miller1 Key words: Ascomycetes, ␤-tubulin, Cercophora, Botany Department, The Field Museum, 1400 S. Lake genealogical concordance phylogenetic species rec- Shore Drive, Chicago, Illinois 60605-2496 and ognition, ITS, LSU, morphological species recogni- University of Illinois at Chicago, Department of tion, phylogenetics, RPB2, Sordariales, species con- Biological Sciences, Chicago, Illinois 60607-7060 cepts, systematics Sabine M. Huhndorf Botany Department, The Field Museum, 1400 S. Lake Shore Drive, Chicago, Illinois 60605-2496 INTRODUCTION The genus Lasiosphaeria previously included numer- Abstract: The genus Lasiosphaeria recently has been ous taxa possessing a broad range of ascomal, asco- circumscribed more narrowly to include five mor- spore and anamorph morphologies. However, its cir- phospecies united by tomentose ascomata containing cumscription recently has been defined more nar- yellow centrum pigments. Species boundaries have rowly to include five morphospecies possessing to- not been established and phylogenetic relationships mentose ascomata with yellow centrum pigments have not been clearly defined for these morphospe- (Miller and Huhndorf 2004a). The genus presently cies. To delimit species boundaries and determine includes the type, L. ovina (Pers. : Fr.) Ces. & de phylogenetic relationships among species, maximum Not., along with L. glabrata (Fr.) Munk, L. lanuginosa parsimony, maximum likelihood and Bayesian analy- (H. Crouan & P. Crouan) A.N. Mill. & Huhndorf, L. ses were conducted on sequence data from four nu- rugulosa (A.N. Mill. & Huhndorf) A.N. Mill. & Huhn- clear genes, the ribosomal internal transcribed spac- dorf, and L. sorbina (Nyl.) P. Karst. Five additional er (ITS) region, 28S large subunit (LSU) rDNA, ␤- morphospecies, Cercophora citrina (Petch) Lundq., C. tubulin and ribosomal polymerase II subunit 2 gossypina Lundq., C. spirillospora (Penz. & Sacc.) (RPB2). Representatives of L. glabrata, L. ovina, L. Lundq., L. cylindrospora Lar. N. Vassiljeva, and L. lib- rugulosa and L. sorbina resolved as four highly sup- ertiana Speg. & Roum., also may belong in the genus ported monophyletic groups in almost all analyses because they possess similar tomentose ascomata and and are recognized as well-defined species employing yellow centrum pigments (except L. cylindrospora, principles of genealogical concordance. These spe- which has a hyaline centrum). Members of Lasios- cies delimitations are corroborated further by mor- phaeria occur worldwide as saprobes on lignicolous phology. Representatives of L. lanuginosa were poly- and herbicolous substrates and commonly are includ- phyletic in almost all analyses. Although molecular ed in regional field guides and taxonomic treatments analyses revealed that this morphospecies comprises of ascomycetes (Munk 1957, Dennis 1977, Breiten- several phylogenetic species, formal taxonomic rec- bach and Kranzlin 1984, Ellis and Ellis 1987). Al- ognition of these lineages is premature, so L. lanu- though they have been included in several taxonomic ginosa currently is treated as a morphological species works (Seaver 1912, Carroll and Munk 1964, Gnan- complex. Complete species descriptions, including ananthan 1972, Lundqvist 1972, Hilber et al 1987, teleomorph, anamorph and culture characteristics, van Hooff 1993, Candoussau et al 2002), these spe- are given for L. glabrata, L. ovina, L. sorbina and the cies never have been treated systematically. L. lanuginosa species complex along with detailed As currently circumscribed, Lasiosphaeria includes discussions of significant morphological characters two species previously placed in Cercophora, which used in recognizing species. These species are com- traditionally has been distinguished from Lasiosphaer- pared to five additional morphospecies that also may ia in possessing ascospores that eventually develop a belong in the genus. swollen, pigmented head (Lundqvist 1972). While as- cospore morphology recently has been shown to be Accepted for publication May 6, 2004. a poor predictor of generic relationships in the Sor- 1 Corresponding author. E-mail: [email protected] dariales (Dettman et al 2001, Miller and Huhndorf Current address: Center for Biodiversity, Illinois Natural History Survey, 607 E. Peabody Drive, Champaign, Illinois 61820. E-mail: 2004b), development of a swollen ascospore head [email protected] might be useful for distinguishing taxa at the species

1106 MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1107

TABLE I. Taxa used in this study GenBank accession no. Geographical Taxa Sourcea locality ITS LSU ␤-tubulin RPB2 Cercophora areolata UAMH7495 Canada AY587911 AY587936 AY600252 AY600275 Cercophora sparsa JF00229 (a) France AY587912 AY587937 AY600253 — Cercophora sulphurella SMH2531 Illinois AY587913 AY587938 AY600254 AY600276 Lasiosphaeria glabrata TL4529 (a) Denmark AY587914 AY436410 AY600255 AY600277 Lasiosphaeria glabrata SMH4617 Denmark AY587915 AY436411 AY600256 AY600278 Lasiosphaeria lanuginosa SMH2390 Costa Rica AY587916 AY587939 AY600257 AY600279 Lasiosphaeria lanuginosa SMH4027 Costa Rica AY587917 AY587940 AY600258 AY600280 Lasiosphaeria lanuginosa TL5656 (a) Denmark AY587918 AY587941 AY600259 — Lasiosphaeria lanuginosa SMH4594 England AY587919 AY587942 AY600260 AY600281 Lasiosphaeria lanuginosa SMH3277 North Carolina AY587920 AY587943 AY600261 AY600282 Lasiosphaeria lanuginosa SMH3819 North Carolina AY587921 AY436412 AY600262 AY600283 Lasiosphaeria lanuginosa AR2148 (a) Venezuela AY587922 AY587944 — — Lasiosphaeria ovina SMH4605 Denmark AY587923 AY436413 AY600263 AY600284 Lasiosphaeria ovina K. Robinson, s.n. England AY587924 AY587945 AY600264 AY600285 Lasiosphaeria ovina CBS958.72 Germany AY587925 AY587946 AY600265 AY600286 Lasiosphaeria ovina SMH1538 Illinois AY587926 AF064643 AF466046 AY600287 Lasiosphaeria ovina SMH2670 (a) Illinois AY587927 AY587947 AY600266 AY600288 Lasiosphaeria ovina SMH4124 (a) Illinois AY587928 AY587948 AY600267 AY600289 Lasiosphaeria ovina SMH2635 (a) Indiana AY587929 AY587949 AY600268 AY600290 Lasiosphaeria ovina SMH3923 Michigan AY587930 AY587950 AY600269 AY600291 Lasiosphaeria ovina SMH3286 North Carolina AY587931 AY587951 AY600270 AY600292 Lasiosphaeria rugulosa SMH4438 (a) Ecuador AY587932 AY587952 AY600271 AY600293 Lasiosphaeria rugulosa SMH1518 Puerto Rico AY587933 AY436414 AY600272 AY600294 Lasiosphaeria sorbina GJS L555 Louisiana AY587934 AY436415 AY600273 AY600295 Lasiosphaeria sorbina CBS885.85 Germany AY587935 AY436416 AY600274 AY600296 a (a) ϭ DNA extracted from ascomata; all others were extracted from cultures.

level. The five morphospecies currently in Lasio- and Huhndorf (2004b). All voucher specimens are depos- sphaeria are distinguished morphologically by differ- ited in The Field Museum Mycology Herbarium (F). ences in the amount and color of the tomentum and Morphological characterization.—Ascomata were squash- the presence or absence of a swollen ascospore head. mounted in water, and images of micromorphological struc- However, several collections recently have been tures were captured with a Dage DC-330 video system found that possess intermediate tomentum and as- mounted on a Zeiss Axioskop microscope using differential cospore characters making it difficult to assign them interference or phase contrast microscopy. Images were to any one of the five morphospecies. The objectives processed using Adobe Photoshop 3.0 or 5.0 (Adobe Sys- of this study were to: (i) delimit species boundaries tems Inc., Mountain View, California). A minimum of 30 and assess species relationships among taxa using a measurements were taken for most teleomorphic and ana- phylogenetic approach, and (ii) determine which morphic structures for each species and mean and standard morphological characters, if any, may be phylogenet- deviation (shown in brackets) were calculated for asco- ␮ ically informative for grouping taxa at the species lev- spores. Ascomata were sectioned at 5 m according to el. Huhndorf (1991). Cultures of multispore isolates were ob- tained following the techniques of Huhndorf et al (2004). Culture studies were conducted following the methods of MATERIALS AND METHODS Miller and Huhndorf (2001), except Difco potato-dextrose agar (PDA) was used in place of oatmeal agar. Color terms Taxon sampling.—Taxa used in this study are listed in TABLE for cultures are taken from Kornerup and Wanscher I along with source information, geographical locality and (1978). GenBank accession numbers. Multiple representatives from the five morphospecies currently placed in Lasiosphaeria DNA extraction, PCR amplification, sequencing and sequence were included in this study. Cercophora areolata Lundq., C. alignment.—Detailed protocols for the extraction, amplifica- sparsa (Sacc. & Fairman) R. Hilber and C. sulphurella tion and sequencing of partial DNA sequences from the LSU, (Sacc.) R. Hilber were used to root trees using the outgroup ␤-tubulin and RPB2 genes along with the methods for the method based on previous phylogenetic analyses by Miller alignment of these sequences are described fully in Miller and 1108 MYCOLOGIA

Huhndorf (2004b). In this study, a segment of the RPB2 gene random trees to ensure trees from the same tree space were between conserved regions 3b and 7 (Liu et al 1999) was PCR being sampled during each analysis. amplified in two pieces using primer pairs RPB2AM-3bF The validity of the incongruence length difference (ILD) (5Ј-AAGGTSTTGATYGCSCARGAGCG)-RPB2AM-6R and test (ϭ partition homogeneity test in PAUP*) for determin- fRPB2-5F-RPB2AM-7R and sequenced using a combination of ing whether datasets should be combined recently has been these primers: RPB2AM-3bF, fRPB2-5F, RPB2AM-5R (5Ј- questioned (Yoder et al 2001, Barker and Lutzoni 2002) CTTGTGVACCATRTAGCC), RPB2AM-6R, RPB2AM-1F, and, thus, other methods should be explored. One method RPB2AM-1R, RPB2AM-1bF, RPB2AM-1bR, RPB2AM-7R (Liu et of assessing combinability of datasets, and the one adopted al 1999, Miller and Huhndorf 2004b). The ITS region was in this study, is by simply comparing highly supported clades amplified and sequenced with ITS5 and ITS4 (Glass and Don- among trees generated from different datasets to detect aldson 1995) following the methods for LSU in Huhndorf et conflict (de Queiroz 1993, Mason-Gamer and Kellogg al (2004). 1996). High support typically refers to bootstrap support values Ն70% and Bayesian posterior probabilities Ն95% Phylogenetic analyses.—Maximum parsimony (MP) and (Alfaro et al 2003). If no conflict exists between the highly maximum likelihood (ML) analyses were performed on supported clades in trees generated from different datasets, separate and combined datasets using PAUP* 4.0b10 (Swof- this suggests the genes share similar phylogenetic histories Ј Ј ford 2002). Portions of the 5 and 3 ends in each dataset and phylogenetic resolution and support could ultimately were excluded from all analyses due to missing data in most be increased by combining the datasets. taxa. Unequally weighted MP analyses were conducted us- ing the branch and bound search option to find exact so- Species recognition.—Following Taylor et al (2000), the term lutions with furthest addition sequence and MulTrees op- ‘‘species recognition’’ is preferred over ‘‘species concept’’ tion in effect. Constant characters were excluded, and gaps when discussing the operational versus the theoretical as- were treated as missing. Twelve and three ambiguously pect of species concepts, i.e., morphological species recog- aligned regions were delimited in ITS and LSU, respective- nition (MSR) and phylogenetic species recognition (PSR). ly. Characters in these regions and in the single ambigu- Several types of PSR exist based on diagnosability (Nixon ously aligned intron, which occurred in the ␤-tubulin gene, and Wheeler 1990, Harrington and Rizzo 1999), monophyly were recoded using the program INAASE (Lutzoni et al (de Quieroz and Donoughue 1988, 1990) or both (Mc- 2000). The remaining unambiguously aligned characters Kitrick and Zink 1988), and much debate has occurred over were subjected to symmetric stepmatrixes to differentially which one is most appropriate for delimiting species (de weight nucleotide transformations. Individual stepmatrixes Queiroz and Donoghue 1990, Wheeler and Nixon 1990, were generated for each of the three codon positions in the Baum and Donoghue 1995, Luckow 1995). Another type of ␤-tubulin and RPB2 genes, while all unambiguously aligned PSR based on coalescence theory uses the concordance of characters were subjected to a single stepmatrix in ITS and multiple gene genealogies for recognizing monophyletic LSU. Branch support for all MP analyses was estimated by groups as species (Avise and Ball 1990, Baum and Shaw performing 1000 bootstrap replicates (Felsenstein 1985) 1995). Taylor et al (2000) proposed the genealogical con- with a heuristic search consisting of 100 stepwise random cordance phylogenetic species recognition (GCPSR) and addition replicates and tree bisection-reconnection (TBR) discussed its application in delimiting fungal species. branch-swapping for each bootstrap replicate. GCPSR has been employed in several fungal studies (Geiser Modeltest 3.06 (Posada and Crandall 1998) was used to et al 1998, Koufopanou et al 1997, O’Donnell et al 1998, determine the best-fit model of evolution for each dataset. Chaverri et al 2003) and is used in this study for recognizing Maximum likelihood analyses were performed on separate species of Lasiosphaeria. and combined datasets with 100 stepwise random addition replicates and TBR branch-swapping using the best-fit mod- RESULTS el. Constant characters were included, and ambiguously aligned characters were excluded from all ML analyses. Be- Sequence alignment.—Final alignments of the four da- cause ML bootstrap analyses were not computationally pos- tasets included 25, 25, 24 and 22 taxa and 622, 1109, sible, Bayesian analyses employing a Markov Chain Monte 1050 and 1798 bp for the ITS, LSU, ␤-tubulin and Carlo (MCMC) method were performed using MrBayes RPB2 genes, respectively. These four alignments are 3.0b4 (Huelsenbeck and Ronquist 2001) as an additional deposited in TreeBASE (SN1704). The ITS and LSU means of assessing branch support. Constant characters datasets contained 12 and 3 ambiguous regions rep- were included, the above model of evolution was imple- resenting 195 and 27 characters, respectively, while mented and four MCMC chains were ran simultaneously for the single ambiguously aligned intron in the ␤-tu- 5 000 000 generations with trees saved every 100th genera- bulin gene represented 69 characters. The ITS, LSU, tion resulting in 50 000 total trees. The MCMC chains al- ␤ ways achieved stationarity after the first 10 000 generations -tubulin and RPB2 genes contained 383, 1001, 778 (ϭ 1000 trees), so the first 10 000 trees, which extended and 1123 constant characters and 42, 58, 151 and 478 well beyond the burn-in phase of each analysis, were dis- parsimony-informative characters, respectively. The carded. Posterior probabilities were determined from a con- combined dataset included 22 taxa and 4579 bp and sensus tree generated with the remaining 40 000 trees. This contained 3299 constant characters and 714 parsi- analysis was repeated three times starting from different mony-informative characters. MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1109

FIG. 1. Phylogram of one of the three most-parsimonous trees generated from an analysis of 25 ITS sequences; length ϭ 324.71 steps, CI ϭ 0.808, RI ϭ 0.816, RC ϭ 0.660. Bootstrap values Ն50% are shown above branches. Thickened branches indicate posterior probabilities Ն95%. Species delimitations are shown along the right side of the tree. Cercophora areolata, C. sparsa and C. sulphurella are outgroups.

Phylogenetic analyses and species delimitation.—Maxi- Although several phylogenetic species are distin- mum parsimony analyses of the ITS, LSU, ␤-tubulin guished within the L. lanuginosa morphospecies us- and RPB2 datasets generated 3, 1, 5 and 1 most-par- ing GCPSR, it is premature to formally recognize simonious trees, respectively. Multiple trees produced these lineages as species. Specimens of L. lanuginosa from ITS and ␤-tubulin analyses differed only slightly from additional populations, along with three addi- in the placement of members of the L. lanuginosa tional morphospecies with putative relations to L. complex (data not shown). Single most-parsimonious lanuginosa, need to be included in further analyses. trees from each analysis are shown in FIGS. 1–4. Be- Therefore, L. lanuginosa currently is treated as a cause very little conflict existed among the highly morphological species complex. supported clades in trees generated from each data- Modeltest indicated that the Tamura and Nei set, all four datasets were assumed to be congruent (1993) model of evolution was the best-fit model for and analyzed simultaneously in a combined analysis. all datasets. The ITS, LSU and RPB2 datasets pos- This analysis produced a single most-parsimonious sessed equal base frequencies, while the ␤-tubulin tree (FIG. 5), which was nearly identical in topology and combined datasets possessed unequal base fre- to that of the RPB2 tree (FIG. 4), except the Euro- quencies. A proportion of sites were invariable, while pean populations of L. ovina formed a well-support- the remaining sites were subjected to a discrete gam- ed clade in the combined analysis. Four highly sup- ma distribution with six rate categories in all models ported clades, which correspond to four traditional except the ␤-tubulin model, which lacked a signifi- morphospecies (L. glabrata, L. ovina, L. rugulosa, L. cant proportion of invariable sites. Maximum likeli- sorbina), occurred in all analyses (FIGS. 1–5). These hood analyses of each dataset produced single trees clades are recognized as four distinct phylogenetic and recovered the same four monophyletic species as species employing GCPSR. Their species delimita- in the MP analyses, except L. sorbina occurred as tions are supported further by MSR by possessing a polyphyletic in the ␤-tubulin ML tree (data not unique combination of diagnostic morphological shown). Members of the L. lanuginosa species com- characters (see below, TABLE II). Representatives of plex were polyphyletic in all ML analyses, except the L. lanuginosa were polyphyletic in all MP analyses. LSU analysis. 1110 MYCOLOGIA

FIG. 2. Phylogram of the single most-parsimonous tree generated from an analysis of 25 LSU sequences; length ϭ 193.58 steps, CI ϭ 0.786, RI ϭ 0.833, RC ϭ 0.654. Support values, species delimitations, and outgroups as in FIG.1.

FIG. 3. Phylogram of one of the five most-parsimonous trees generated from an analysis of 24 ␤-tubulin sequences; length ϭ 569.59 steps, CI ϭ 0.799, RI ϭ 0.745, RC ϭ 0.595. Support values, species delimitations, and outgroups as in FIG.1. MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1111

FIG. 4. Phylogram of the single most-parsimonous tree generated from an analysis of 22 RPB2 sequences; length ϭ 1991.09 steps, CI ϭ 0.727, RI ϭ 0.731, RC ϭ 0.531. Support values and species delimitations as in FIG.1.Cercophora areolata and C. sulphurella are outgroups.

Species relationships.—Multiple gene genealogies in- Lasiosphaeria glabrata (Fr.) Munk, Dansk Bot Ark 17: dicate that L. glabrata and L. rugulosa are closely 111. 1957. FIGS. 6–23 related taxa that occur at the base of the Lasios- ϵ Sphaeria ovina Pers. var. glabrata Fr., Syst Myc 2:446. phaeria clade, while L. ovina, L. sorbina and mem- 1823. ϵ bers of the L. lanuginosa complex are more derived Leptospora ovina (Pers. : Fr.) Fuckel var. glabrata (Fr.) Fuckel, Symb Myc 23–24:143. 1870. taxa. While ITS and LSU suggest L. glabrata is the ϵ basal taxon, ␤-tubulin and RPB2 indicate L. rugu- Lasiosphaeria ovina (Pers. : Fr.) Ces. & de Not. var. glabrata (Fr.) Sacc., Syl Fung 2:199. 1883. losa is the basal taxon. The combined tree, which TY PE: DENMARK. JYLLAND: Staksrode near probably is influenced by the large amount of phy- Juelsminde, District 24, on rotten stump, 10 Dec logenetic signal derived from the protein-coding 1963, A. Munk (C, Neotype, designated here). genes, also places L. rugulosa at the base of the La- Ascomata subglobose to ovoid, papillate, 310–530 siosphaeria clade. Lasiosphaeria sorbina is basal to the ␮m diam, 350–560 ␮m high, numerous, gregarious L. lanuginosa complex, which is basal to L. ovina in to clustered in large groups, usually superficial, oc- all analyses. casionally erumpent; glabrous, shiny, black; neck usu- Species descriptions.—Complete species descriptions ally conical, sometimes poorly developed, glabrous, are given below for L. glabrata, L. ovina, L. sorbina black. Ascomatal wall of textura intricata and textura and the L. lanuginosa species complex. A complete angularis in surface view, in longitudinal section 3- species description of L. rugulosa, except for culture layered, 33.5–54.5 ␮m thick, inner layer pseudopar- morphology on PDA which is given below, can be enchymatous, 5–9.5 ␮m thick, composed of 3–5 lay- found in Miller and Huhndorf (2001). Species de- ers of elongate, flattened, pale brown cells, middle scriptions and illustrations also have been published layer pseudoparenchymatous, 24.5–38 ␮m thick, for C. areolata (Lundqvist 1972), C. sparsa (Hilber composed of 4–10 layers of angular, pale brown cells, and Hilber 1979) and C. sulphurella (Hilber and outer layer prosenchymatous, 4–7 ␮m thick, com- Hilber 1979, van Hooff 1994, Fallah and Shearer posed of 1–2 layers of sparse, loosely interwoven hy- 2001). phae, hyphae 1.5–3 ␮m wide, pale brown to brown, 1112 MYCOLOGIA

FIG. 5. Phylogram of the single most-parsimonous tree generated from the combined analysis of 22 sequences; length ϭ 2970.75 steps, CI ϭ 0.751, RI ϭ 0.740, RC ϭ 0.556. Support values and species delimitations as in FIG.1.Cercophora areolata and C. sulphurella are outgroups. septate, thin-walled. Ascomatal apex with periphyses. Hyphae largely undifferentiated, 1–3.5 ␮m wide, Centrum with yellow pigments which quickly diffuse commonly forming hyphal coils, thin-walled, hyaline. in water. Paraphyses filiform, 2–6 ␮m wide, longer Substrate. On dead, decorticated deciduous trees. than asci, hyaline, numerous, septate, unbranched, Distribution. Seen only from Denmark. persistent. Asci cylindrical, 160–190 ϫ 12–18 ␮m, me- Specimens examined. DENMARK. FYN: Elsehoved, 4 Sep dium-stipitate, stipe 27–58 ϫ 3–5 ␮m, numerous, un- 1963, G.C. Carroll 447; 4 Oct 1963, G.C. Carroll 448 (G. itunicate, thin-walled, apex rounded to truncate; ring Carroll Herbarium). JYLLAND: Silkeborg, Lysbro Skov, on narrow, shallow, refractive; subapical globule small to Betula wood, 1 Dec 1953, A. Munk (C); Virklund, Knager- large, 2–5.5 ␮m diam, usually poorly developed, ne, on 70 cm diam beech log, 24-IX-2001, A. N. Miller & T. Læssøe, SMH4615; on 50 cm diam beech log, SMH4617 smooth, with 8, biseriate to tetraseriate ascospores. ϫ (F). MØN: Klinteskoven, (loc. 13), on underside of Fagus Ascospores cylindrical, ends rounded, 33.5–52.5 3– wood, 26 Sep 1997, T. Læssøe TL4529 (C). SJAELLAND: ␮ Ϯ ϫ Ϯ 5 m [42 5.0 4 0.5], straight when first pro- Lyngby Mose, District 45a, on rotten wood, 4/27/1963, A. duced, hyaline, aseptate, bipolar appendages absent; Munk; Lellinge A, on Fagus wood, 5 Mar 1963, A. Munk. becoming slightly sigmoid or geniculate, lower one- Culture examined. SMH4617-1, 2, 3, 4, 5. third to one-quarter bent 45Њ or less; after liberation from the ascus occasionally 1-septate. Colonies moderately slow-growing on all media, 31– Lasiosphaeria ovina (Pers. : Fr.) Ces. & de Not., Comm Soc Critt Ital 1:229. 1863. FIGS. 24–45 38 mm diam in 21 d, silky on WA and CMA, subfelty ϵ on PDA, appressed, hyaline to yellowish white (2A2) Sphaeria ovina Pers., Syn Meth Fung 1:71. 1801. ϵ Sphaeria ovina Pers. : Fr., Syst Myc 2:446. 1823. on WA and CMA, grayish orange to grayish red (6B3– ϵ Leptospora ovina (Pers. : Fr.) Fuckel, Symb Myc 23– 7B4) on PDA; margin even, appressed, hyaline on 24:143. 1870. WA and CMA, pale yellow to grayish yellow (3A3– ϭ Lasiosphaeria ovina (Pers. : Fr.) Ces. & de Not. var. 4B4) on PDA, distinct on PDA; reverse same as the aureliana Fairman, J Myc 10:229. 1904. mat in all media; anamorph not produced on WA, ϭ Lasiosphaeria chrysentera Carroll & Munk, Mycologia CMA, or PDA. 56:83. 1964. MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1113 46) 42) 53) 52) 39) 50) 40) 56) 35) 52) 57) 50) 48) 50) 44) 44) 47) 47) 44) 46) 34) 32) 31) 32) 82) 42) 41) 50) 42) ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ length Ascospore 37–53 (x 38–47 (x 48–57 (x 47–56 (x 35–42 (x 48–54 (x 37–43 (x 50–61 (x 32–37 (x 47–56 (x 51–66 (x 43–56 (x 44–54 (x 44–53 (x 36–50 (x 40–48 (x 42–53 (x 42–52 (x 39–50 (x 43–50 (x 31–37 (x 29–34 (x 25–39 (x 22–41 (x 72–89 (x 39–44 (x 37–45 (x 46–56 (x 33–52 (x ages absent absent lash-like lash-like lash-like lash-like lash-like awl-like lash-like lash-like awl-like awl-like awl-like awl-like awl-like awl-like not seen not seen not seen awl-like absent absent absent absent lash-like lash-like lash-like absent absent Append- absent absent present present (brown) slightly swelling present (brown) present (brown) absent present present (brown) absent absent absent absent absent absent absent absent absent absent present (brown) present (brown) absent absent present (brown) present (brown) present (brown) absent absent b color Swollen head Tomentum reddish-orange white to pale yellowish cream to yellowish-brown brown (sparse) brown (sparse) white to pale yellowish yellow (sparse) white pale yellowish white to pale yellowish pale yellowish pale yellowish white to pale yellowish white white white white white white white white white white brown brown gray orange gray brown (sparse) ) were not available for morphological study. Lasiosphaeria L. sorbina elephant dung hare dung bamboo wood wood wood wood wood wood wood wood wood wood wood grass stalks grass stalks wood wood wood wood wood wood wood wood wood wood wood wood wood ) and CBS885.85 ( Sri Lanka Sweden Java Russia Denmark Denmark Denmark France Costa Rica Costa Rica North Carolina North Carolina England Venezuela Denmark New York Switzerland? Denmark England Illinois Illinois Illinois Indiana Michigan North Carolina Puerto Rico Ecuador Finland Louisiana L. ovina Coll. no. Locality Substrate TYPE HOLOTY PE ISOTY PE HOLOTY PE NEOTY PE SMH4617 TL4529 HOLOTY PE SMH2390 SMH4027 SMH3277 SMH3819 SMH4594 AR2148 TL5656 NY ISOTY PE LECTOTY PE SMH4605 K. Robinson, s.n. (Acc.SMH1538 #794) SMH2670 SMH4124 SMH2635 SMH3923 SMH3286 SMH1518 (HOLOTY PE) SMH4438 HOLOTY PE GJS L555 a Taxon II. Morphological characters used in distinguishing putative morphospecies in Reported to be reddish when fresh (Penzig and Saccardo 1897). The voucher specimens for cultures CBS958.72 ( a b ABLE T Cercophora citrina Cercophora gossypina Cercophora spirillospora Lasiosphaeria cylindrospora Lasiosphaeria glabrata Lasiosphaeria glabrata Lasiosphaeria glabrata Lasiosphaeria lanuginosa Lasiosphaeria lanuginosa Lasiosphaeria lanuginosa Lasiosphaeria lanuginosa Lasiosphaeria lanuginosa Lasiosphaeria lanuginosa Lasiosphaeria lanuginosa Lasiosphaeria lanuginosa Lasiosphaeria libertiana Lasiosphaeria ovina Lasiosphaeria ovina Lasiosphaeria ovina Lasiosphaeria ovina Lasiosphaeria ovina Lasiosphaeria ovina Lasiosphaeria ovina Lasiosphaeria ovina Lasiosphaeria ovina Lasiosphaeria rugulosa Lasiosphaeria rugulosa Lasiosphaeria sorbina Lasiosphaeria sorbina 1114 MYCOLOGIA

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 ϭ 500 ␮m; 7, 8 ϭ 100 ␮m; 9–23 ϭ 10 ␮m. MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1115

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 drop- lets (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 ϭ 100 ␮m; 29–45 ϭ 10 ␮m. 1116 MYCOLOGIA

This nomenclature is not complete. composed of agglutinated, thin-walled hyphae, mat LECTOTY PE: SWITZERLAND (possibly). Hb. appressed on WA and CMA, appressed to aerial on Pers., 910.269-84, L10261 No. 12 (as Sphaeria ovina PDA, hyaline on all media, becoming whitish to gray- Pers., L). ish-yellow (4A1–4B4) on PDA; margin even in all me- Ascomata ampulliform to ovoid, rarely obpyriform, dia, appressed, hyaline; reverse same as the mat in all papillate, 360–620 ␮m diam, 380–680 ␮m high, nu- media; anamorph produced abundantly over entire merous, scattered to gregarious, superficial; young as- mat on WA and CMA and sparsely at the margin on comata tomentose, white, tomentum becoming tight- PDA within 14 d. ly appressed, crust-like and cream to brownish-gray Hyphae largely undifferentiated, 1–4 ␮m wide, with age, occasionally areolate, tomentum then par- thin-walled, hyaline. Conidiogenous cells phialides, tially wearing away and forming a thin, waxy, grayish produced laterally or occasionally terminally, delim- coating, finally tomentum completely worn away and ited by a basal septum, monophialidic or sometimes ascomata appearing glabrous and black; neck coni- polyphialidic, obclavate to lageniform, 6.5–16.5 ϫ cal, glabrous, black. Ascomatal wall of textura angu- 2.5–3.5 ␮m at widest part, hyaline to pale brown, con- laris in surface view, commonly containing globular, stricted below the collarette, 0.5–1.5 ␮m below col- refractive crystals in squash mounts, in longitudinal larette; collarette minute, flaring, same color as phial- section 3-layered, 42.5–86 ␮m thick, inner layer pseu- ide. Conidia pyriform, truncate at base, 3–4 ϫ 2–3 doparenchymatous, 8–9.5 ␮m thick, composed of 4– ␮m, hyaline, produced enteroblastically, aggregated 6 layers of elongate, flattened, hyaline to pale brown in slimy heads; slightly larger blastoconidia also com- cells, middle layer pseudoparenchymatous, 16.5–35 monly produced directly from hyphae or branches, ␮m thick, composed of 5–8 layers of polygonal to branches produced laterally, not delimited by a basal angular, pale brown cells, outer layer prosenchyma- septum, extremely short to long, cylindrical, 1.5–24 tous, 18–41.5 ␮m thick, composed of several to few ϫ 1–3 ␮m at widest part, hyaline, without collarette; layers of hyphae depending on age of ascomata, hy- blastoconidia subglobose to pyriform or occasionally phae 1–3 ␮m wide, hyaline to pale brown, septate, obclavate, 3–4.5(–6) ϫ 2.5–3.5 ␮m, hyaline. thin-walled. Ascomatal apex with periphyses. Centrum Substrate. On dead, decorticated deciduous trees. with yellow to yellowish-orange or rarely greenish-yel- Distribution. Canada, Costa Rica (as L. chrysentera low pigments that quickly diffuse in water. Paraphyses Carroll & Munk), Denmark, England, France, Ger- filiform, 1.5–5.5 ␮m wide, longer than asci, hyaline, many, New Zealand, Philippines, Russia, Switzerland, numerous, septate, unbranched, persistent, contents USA. rarely containing blue pigments in fresh specimens Specimens examined. CANADA. ONTARIO: Granton, on only. Asci cylindrical, 140–250 ϫ 12–23 ␮m, medium- rotten wood, 23 Aug 1895, J. Dearness, (NY). COSTA RICA. to long-stipitate, stipe 30–85(–120) ϫ 3–6.5 ␮m, nu- Vulcan Irazu, rough road, second bridge from San Jose, on merous, unitunicate, thin-walled, apex truncate; ring wood, 22 Jun 1962, G. Carroll 85 (HOLOTY PE of Lasio- narrow, shallow, refractive; subapical globule large, sphaeria chrysentera, NY; ISOTY PE, G. Carroll herbarium). 3.5–6 ␮m diam, smooth to verrucose, with 8, biseriate DENMARK. Copenhagen, Hareskov, 7 Oct 1962, G. Carroll 80 (G. Carroll herbarium). JYLLAND: Silkeborg, Virklund, to tetraseriate ascospores. Ascospores cylindrical, ends Knagerne, on 60 cm beech log, 24-IX-2001, A.N. Miller & rounded, 35–55(–60.5) ϫ 3–6 ␮m [45.5 Ϯ 4.5 ϫ 4.5 Ϯ T. Læssøe, SMH4616; on 70 cm beech log, SMH4623; Tir- 0.5], straight when first produced, hyaline, asep- strup, Noerupland, on 4 cm branch, 25-IX-2001, A.N. Miller tate, bipolar appendages occasionally present, 5.5–12 & T. Læssøe, SMH4634; Femmoeller, Strandkjaer at Lan- ␮m, gelantinous, spike-like or awl-like, evanescent; gemosen, on 10 cm aspen branch, 25-IX-2001, A.N. Miller, becoming slightly sigmoid or geniculate, lower one- T. Læssøe, J. Pedersen, SMH4638. SJAELLAND: Køge, Lell- third to one-quarter bent 45Њ or less, appendages inge Skovhusvaenge, on 6 cm branch, 20-IX-2001, A.N. rarely present at this stage; after liberation from the Miller, T. Læssøe, T. Frøsler, SMH4605. ENGLAND. Hert- ascus up to 7-septate or rarely 9-septate, each cell with fordshire, Ayot St. Lawrence, Prior’s Wood, on wood, 9/25/ a single globose, refractive oil droplet; finally becom- 2001, K. Robinson s.n. (F). FRANCE. Bois de Barm, Tarmes, ing yellowish, yellowish-brown or pale brown with 1902, Pere Duss 173 (NY); Roum. F. sel. Gall. 5857, as La- age, sometimes constricted at clamps and appearing siosphaeria ovina var. glabrata, sur les copeaux pourissants du cheˆne, bois des environs de Saint-Saens (Seine-Infe´rieu- worm-like, occasionally producing phialides directly re), on wood, August, (UPS). GERMANY. Syd. Myc. Mar. from the ascospore. 2144, as Lasiosphaeria ovina var. glabrata, canal by Mar- Colonies on WA moderately slow-growing, 21–40 ienspring by Andow?, on Fagus silvatica wood, F. 1888, P. mm diam in 21 d, moderately fast-growing on CMA Sydow, (UPS, NY). NEW ZEALAND. A.Y. Rossman, and PDA, covering the CMA plate in 21 d and the AR1899, AR1890 (BPI). PHILIPPINES. LAGUNA PROV- PDA plate in 28 d, silky on WA and CMA, wooly on INCE: Mount Maquiling, 11/4/1963, D. Reynolds 185, (G. PDA, becoming hispid with long, erect, white hairs Carroll Herbarium). RUSSIA. Kamchatka, Avacha River val- MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1117 ley, bridge over Avacha River by Razdolny, 53Њ13ЈN, F.A. Ferna´ndez, SMH3966; roadside, 2 mi from gate, 30 m 158Њ20ЈE, on cf. Populus suaveolens, 8/1/1997, T. Læssøe, to Howe Lake, on inner side of bark, 9-X-1998, F.A. Ferna´n- TL4634 (C). SWITZERLAND (possibly). Hb. Pers., J. F. dez, SMH4000; west edge of Rush Lake, on wood, 20-IX- Chaillet?, 910.269-153, L10261 No. 14; Chaill. in Hb. Pers.,? 1999, A.N. Miller, SMH4171; Neus plot 2, on wood, 22-IX- Sphaeria affinis, Sph. ovinae, ϭ J. F. Chaillet?, 910.269-156, 1999, A.N. Miller, SMH4177 (F). NEW JERSEY: Warren L10261 No. 15 (L). USA. CONNECTICUT: Poquonock, 9/ Township, Plainfield, 9 Sage Drive, on Quercus, 9 Oct 1961, 8/1893, 8/4/1895, W.C. Sturgis (NY). ILLINOIS: Cook G. Carroll 79 (G. Carroll herbarium). NEW YORK: Yalis? County, Cook County Forest Preserve, Swallow Cliffs, on on moist bark of rotten log (Tilia?), Oct 1900, C.E. Fair- wood fragment, 3-VII-1996, S.M. Huhndorf, F.A. Ferna´ndez, man, Mycotheca Fairmani 545 (ISOTY PE of Lasiosphaeria M. Huhndorf, SMH2535; Ogle County, Castle Rock State ovina var. aureliana); Ridgeway, on prostrate trunk of Tilia, Park, on 30 cm log, 27-IX-1996, S.M. Huhndorf & F.A. Fer- 16 Sep 1904, C.E. Fairman, Mycotheca Fairmani 548, as La- na´ndez, SMH2670; SMH2672; on 8 in branch, SMH2679; siosphaeria ovina var aureliana; Lyndonville, on rotten on branch fragment, SMH2684; White Pines Forest State wood, Aug 1905, C.E. Fairman, Mycotheca Fairmani 547; Park, on 1 in branch, 28-IX-1996, S.M. Huhndorf & F.A. on the ground in woods, on rotten wood, Oct 1905, C.E. Ferna´ndez, SMH2696; SMH2697; on wood fragment, Fairman, Mycotheca Fairmani 546; on rotten wood, Sep SMH2711; on 7 cm branch, SMH2712; on 6 in branch, 1910, C.E. Fairman, Mycotheca Fairmani 3217; Janesville, SMH2717; Jackson County, Giant City State Park, Devil’s Oct 1887, O.F. Cooke (NY); Varna, Fries Road Extension, 22 Roundtable, on wood, 10-VIII-1999, A.N. Miller, SMH4124; Jul 1960, W.C. Denison 81; Ithaca, RD No. 5, The Briar Vermilion County, Forest Glen County Preserve, on 2.5 cm Patch-W. Ravine, 11 Aug 1965, G. Carroll 987 (G. Carroll branch, 30-IX-2000, A.N. Miller, SMH4329. INDIANA: Lake herbarium). NORTH CAROLINA: Macon County, west of County, Indiana Dunes National Lakeshore, Cowles Bog, on Franklin, woods along Roaring Fork, Nantahala River, on 15 in log, 1-VII-1996, S.M. Huhndorf, F.A. Ferna´ndez, M. rotten log, 17 Sep 1960, C.T. Rogerson & R.H. Petersen Huhndorf, SMH2524; Dyer, 9981 Sheffield Ave., on wood (NY); Highlands, Highlands Biological Station, on 8 in log, fragment, 1-XII-2001, S.M. Huhndorf, SMH4664; Brown 9-X-1996, S.M. Huhndorf & F.A. Ferna´ndez, SMH2784;on County, Yellowwood State Forest, compartment 7, tract 24, 1 in branch, SMH2785; on 10 cm branch, 21-VII-1997, F.A. on 2 in branch, 26-VII-1996, S.M. Huhndorf, SMH2635 (F). Ferna´ndez, SMH3315; Blue Valley, [35.0192, Ϫ83.2736], LOUISIANA: 20-VIII-1890, A.B. Langlois 748; Plaquemines 1000 m, on 40 cm log, 18-VII-1997, F.A. Ferna´ndez, Parish, Hebert Center, on perithecia, 3 Nov 1970, Flora Lu- SMH3274 (F). VIRGINIA: above Chain Bridge, 5 Oct 1923, doviciani 7539 (NY). MAINE: Oldtown, Aug 1897, F.L. Har- C.L. Shear (NY). WISCONSIN: Dane County, Dawley Park, vey (NY). MICHIGAN: Berrien County, Warren Woods, Fitschburg, along trail, on 24 in log, 13-IX-1995, S.M. Huhn- south end of trail, through picnic area, up to creek, on 5 dorf, SMH1538 cm branch fragments, 8-IX-1998, F.A. Ferna´ndez & A.N. ; on 6 in branch, SMH1539; Fond du Lac Miller, SMH3887; on wood fragment, SMH3894; north County, Kettle Moraine State Forest, Mantle Lake Recrea- end, on wood, 7-X-1999, A.N. Miller, SMH4198;on12in tional Area, wet area scenic trail, on wood, 24-IX-1999, S.M. maple log, 21-IX-2000, F.A. Ferna´ndez & A.N. Miller, Huhndorf, SMH4189; Grant County, Wyalusung State Park, SMH4325; SMH4326; Marquette County, Huron Mountain on 12 in log, 26-VII-1995, S.M. Huhndorf, SMH1530; Green Club, along N side of Middle Pine Lake (Second Pine), County, New Glarus State Park, on branch, 13-IX-1995, S.M. [45.8692, Ϫ87.854], 210 m, on 8 in birch log, 12-VII-1996, Huhndorf, SMH1544 (F). S.M. Huhndorf, F.A. Ferna´ndez, M. Huhndorf, SMH2549; Cultures examined. SMH1538-1, 2, 3, 4; SMH3923-1, 2, 3, along Loop Road, Fisher Creek trail, Trout Lake Trail, 4, 5, 6; SMH3286-1, 3; SMH4605-1, 2, 3, 4; CBS958.72, Mountain Stream Trail, [46.8657, Ϫ87.8844], 270 m, on 20 AR1899-1, KR-1, 2, 3, 4, 5. in hemlock log, 13-VII-1996, S.M. Huhndorf, F.A. Ferna´ndez, M. Huhndorf, SMH2576; base and slope of Mount Ives and NW corner of Ives Lake, on 50 cm log, 14-VII-1996, S.M. Lasiosphaeria rugulosa (A.N. Mill. & Huhndorf) A.N. Huhndorf, F.A. Ferna´ndez, M. Huhndorf, SMH2587;on Mill. & Huhndorf Myc Res 108:31. roadside toward Cranberry Bog, [45.8762, Ϫ87.8472], 200 ϵ Cercophora rugulosa A. N. Mill. & Huhndorf, Sydowia m, on 3 in branch, 15-VII-1996, S.M. Huhndorf, F.A. Fer- 53:215. 2001. na´ndez, M. Huhndorf, SMH2608; on 10 cm branch, Colonies moderately slow-growing on PDA, 28–34 SMH2611; on 7 cm branch, SMH2616; on 15 cm log, mm diam in 21 d, silky to subfelty, appressed, hyaline, SMH2618; on 5 in branch, SMH2619; on 30 in maple log, becoming whitish to yellowish-white (3A2); margin SMH2620; on 3 in branch, SMH2624; S of Howe Lake, even or plumose, appressed, hyaline, not distinct; re- road between Ann and Howe Lakes, on 1 in branch, 18- VIII-1997, S.M. Huhndorf & M. Huhndorf, SMH3370;on verse same as the mat; anamorph not produced. ϭ 1 in branch, SMH3375; on log fragments, SMH3378;on1 Culture examined. SMH1518-1 ( ATCC MYA- in branch, SMH3382; on 2 cm branch, SMH3387; trail be- 2578), 2 (ϭ ATCC MYA-2579), 3. hind boathouses, bottom of ridge to the left, on wood, 5-X- 1998, F.A. Ferna´ndez, SMH3923; SMH3932; Cranberry Bog, [45.8762, Ϫ87.8472], 200 m, on 2 cm branch, 7-X- Lasiosphaeria sorbina (Nyl.) P. Karst., Bid Kann Fin 1998, F.A. Ferna´ndez, SMH3955; before the creek on trail Nat Folk 23:164. 1873. FIGS. 46–61 to Fisher Creek/Trout Lake, on 70 cm diam log, 8-X-1998, ϵ Sphaeria sorbina Nyl. Flora 21:322. 1863. 1118 MYCOLOGIA

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 show- ing 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 ϭ 100 ␮m; 49–61 ϭ 10 ␮m. MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1119

ϵ Bizzozeria sorbina (Nyl.) Ho¨hn., Ann Mycol 16:76. thin-walled, hyaline. Conidiogenous cells phialides, 1918. produced laterally or terminally, delimited by a basal ϵ Leptospora sorbina (Nyl.) Munk, Dansk Bot Ark 14(7): septum, monophialidic, obclavate to lageniform, 9– 6. 1952. 18 ϫ 2–4 ␮m at widest part, hyaline, constricted be- Additional heterotypic synonyms are listed by von low the collarette, 1–1.5 ␮m below collarette; collar- Ho¨hnel (1918), but their synonymy has not been ette minute, slightly flaring, same color as phialide. confirmed. Conidia pyriform, truncate at base, 2.5–3.5 2–2.5 ␮m, HOLOTY PE: FINLAND. Helsingfors, on Sorbus hyaline, produced enteroblastically, aggregated in aucuparia branch, 24 Mar 1863, Nylander (as Sphaeria slimy heads; blastoconidia not produced. sorbina Nyl., H). Substrate. On dead, decorticated deciduous trees Ascomata ampulliform to ovoid, papillate, (240–) with remnants of bark. 320–460 ␮m diam, (280–)370–600 ␮m high; numer- Distribution. Ecuador, Finland, France, Nether- ous, scattered to gregarious, usually superficial, oc- lands, USA. casionally erumpent; tomentose, tomentum dull gray Specimens examined. ECUADOR. PICHINCHA PROV.: 2 to brownish-gray or pale pinkish to orange, ap- km above Toachi, on the Toachi-Palo Quemado Road, 3400 pressed, waxy, sometimes wearing away with age; neck ft, on wood, 19 Jul 1975, K.P. Dumont, S.E. Carpenter, P. conical, glabrous, sometimes with a distinct, grayish Buritica, EC-556 (NY). FRANCE. ARIE`GE: 09 Montseron, ring around black ostiole. Ascomatal wall of textura Las Muros, Roquebrine-Ruisseau de Peyrau, on Quercus zo- angularis in surface view, in longitudinal section 3- bur branch, 3 Mar 2002, J. Fournier, JF02053 ( J. Fournier layered, 48.5–76.5 ␮m thick, inner layer pseudopar- Herbarium). NETHERLANDS. As Sphaeria mutabilis, on enchymatous, 6.5–9.5 ␮m thick, composed of 3–5 lay- wood, Hb. Pers. saltem ei affinis prope parisios, 910.263- ers of elongate to flattened, pale brown cells, middle 1251, L. 10261 No. 10; on wood, 910.269-91, L. 10261 No. layer pseudoparenchymatous, 12.5–22.5 ␮m thick, 11 (L). USA. LOUISIANA: West Feliciana Parish, St Fran- cisville, Grace Church Cemetary, on branch, 18 Aug 1996, composed of 4–6 layers of angular, pale brown cells, ␮ G.J. Samuels & M. Blackwell s.n., GJS L555 (F). OHIO: outer layer prosenchymatous, 29.5–44.5 m thick, Morgan s.n., as Lasiosphaeria ovina (NY). composed of numerous layers of hyphae, hyphae 1.5– Cultures examined. GJS L555-1, 2, 3, 4, 5; CBS885.85. 3.5 ␮m wide, hyaline to yellowish, septate, thin- to slightly thick-walled, becoming melanized and brown in outermost 3–6 layers. Ascomatal apex with peri- Lasiosphaeria lanuginosa species complex physes. Centrum with yellow, pinkish, or rarely orange FIGS. 62–81 pigments which quickly diffuse in water. Paraphyses Ascomata ampulliform to ovoid, papillate, 360–640 filiform, 1.5–4.0 ␮m wide, hyaline, numerous, sep- ␮m diam, 360–740 ␮m high, numerous, scattered to tate, unbranched, longer than asci. Asci cylindro-cla- gregarious, usually superficial, rarely erumpent; with vate to clavate, 125–185 ϫ 13–21.5 ␮m, medium-stip- cottony tomentum, hairs occasionally spreading at itate, stipe 26–52 ϫ 4–8.5 ␮m, numerous, unituni- base, occasionally whitish to commonly pale yellowish cate, thin-walled, apex rounded to truncate; ring nar- or occasionally pale pinkish, sometimes becoming or- row, extremely shallow, refractive, indistinct; ange-brown with age, occasionally wearing away so subapical globule absent, with 8, biseriate to triseriate that only a few yellowish hairs remain; neck conical, ascospores. Ascospores short cylindrical, ends round- glabrous, black. Ascomatal wall of textura angularis ed, 21.5–46 ϫ 4.5–9 ␮m [33 Ϯ 5.5 ϫ 6.5 Ϯ 1.0], in surface view, commonly containing globular, re- straight to slightly curved, hyaline, aseptate to 1-sep- fractive crystals in squash mounts, in longitudinal sec- tate, appendages absent; after liberation from the as- tion 3-layered, 48.5–71.5 ␮m thick, inner layer pseu- cus up to 5-septate, occasionally producing phialides doparenchymatous, 7.5–9 ␮m thick, composed of 3– directly from the ascospore. 5 layers of elongate to flattened, hyaline to brown Colonies slow-growing on WA and PDA, 15–28 mm cells, middle layer pseudoparenchymatous, 20.5–22 diam and 15–27 mm diam in 21 d on WA and PDA, ␮m thick, composed of 4–7 layers of polygonal to respectively, moderately slow-growing on CMA, 18–33 angular, brown cells, outer layer prosenchymatous, mm diam in 21 d, silky on all media, appressed, hy- 20.5–40.5 ␮m thick, composed of several to few layers aline, becoming brownish gray to pastel red (7C2– of hyphae, hyphae 1.5–3 ␮m wide, hyaline to pale 8A5) on PDA; margin even, appressed, hyaline, not brown, septate, thin-walled. Ascomatal apex with per- distinct; reverse same as the mat in all media; ana- iphyses. Centrum with yellow to yellowish-orange pig- morph produced commonly over entire mat on WA, ments which quickly diffuse in water. Paraphyses fili- sparsely over entire mat on CMA and sparsely at the form to ventricose, 2–5 ␮m wide, longer than asci, margin on PDA within 14 d. hyaline, numerous, septate, unbranched, persistent, Hyphae largely undifferentiated, 1–3.5 ␮m wide, contents with blue pigments in SMH4027 only. Asci 1120 MYCOLOGIA

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 MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1121 cylindrical, 120–230(–300) ϫ 8–20(–22.5) ␮m, me- nute, slightly flaring, same color as phialide. Conidia dium- to long-stipitate, stipe (26–)38–91 ϫ 3–7.5 ␮m, pyriform, truncate at base, 2.5–3 ϫ 2–2.5 ␮m, hya- numerous, unitunicate, thin-walled, apex truncate; line, produced enteroblastically, aggregated in slimy ring narrow, shallow, refractive; subapical globule heads; slightly larger blastoconidia also commonly large, 3.5–6 ␮m diam, smooth, with 8, biseriate to produced directly from hyphae or branches, branch- tetraseriate ascospores. Ascospores cylindrical, ends es produced laterally singly or occasionally terminally rounded, 33–60 ϫ 2.5–5 ␮m [44.5 Ϯ 6.5 ϫ 4 Ϯ 0.5], singly or in pairs, not delimited by a basal septum, slightly sigmoid or geniculate, lower one-third bent short to long, cylindrical or occasionally bulbulous to 45Њ or less, hyaline, aseptate; with bipolar append- lecythiform, 2.5–15 ϫ 1–3 ␮m at widest part, hyaline, ages, 7–30(–41.5) ␮m, gelatinous, lash-like; becom- without collarette; blastoconidia pyriform to obcla- ing differentiated into a swollen head and pedicel vate, 3.5–5.5 ϫ 2.5–3.5 ␮m, hyaline. while inside the ascus, transversely uniseptate; head Substrate. On the decorticated parts of dead, de- ellipsoid to ovoid, 11.5–19 ϫ 6–10.5 ␮m, subacute to ciduous branches which still possess remnants of ad- rounded at the apex, truncate at the base, hyaline to hering bark, or rarely on dead leaves or grass. occasionally brown; pedicel 23.5–32.5 ϫ 3–5 ␮m, hy- Distribution. Costa Rica, Denmark, England, aline to rarely pale brown, 1-septate; head brown and France, Peru, USA, Venezuela. rarely 1-septate after liberation from the ascus, asco- Specimens examined. COSTA RICA. SAN JOSE: San Ger- spore 1- to 2-septate after liberation from the ascus. ardo de Dota, Albergue de Montana, Savegre, trail to wa- Ϫ Colonies slow-growing on WA and PDA in terfall along Rio Savegre, [9.5439, 83.8142], 2150 m, on 5 in log, 11-V-1996, S.M. Huhndorf & F.A. Ferna´ndez, SMH3277 and SMH3819, 14–22 mm diam and 22– Ϫ 25 mm diam in 21 d on WA and PDA, respectively, SMH2350; Sendero la Quebrada, [9.55, 83.8], 2300 m, on 2 in branch, 12-V-1996, S.M. Huhndorf & F.A. Ferna´n- moderately slow-growing on WA in SMH4027, 31–35 dez, SMH2389, on old bark, S.M. Huhndorf & F.A. Ferna´n- mm diam in 21 d, (SMH4594 did not grow on WA), dez SMH2390; on 5 cm branch, S.M. Huhndorf & F.A. Fer- moderately slow-growing on CMA in SMH3277, na´ndez, SMH2394. PUNTARENAS: La Amistad Pacifico, Si- SMH3819 and SMH4594, 31–33 mm diam in 21 d, tio Cotoncito, [8.9403, Ϫ82.7892], 1600 m, on wood, 14-I- moderately fast-growing on CMA and PDA in 1999, F.A. Ferna´ndez, SMH4027. CARTAGO: Canto´n SMH4027, covering the CMA plate in 21 d and the Paraiso, Distrito Orosi, Parque Nacional Tapanti, Oropen- PDA plate in 28 d, moderately slow-growing on PDA dula trail, [9.7517, Ϫ83.7908], 1300 m, on 4 cm standing in SMH4594, 29–35 mm diam in 21 d on PDA, silky stump, 27-VI-2000, F.A. Ferna´ndez, SMH4301. DENMARK. on all media, becoming subfelty to crustose on PDA, Læsø, Kærene, on Molinia caerulea stems, 9-X-1999, T. hyaline on all media, becoming yellowish-brown to Læssøe, TL5656. ENGLAND. Northumberland, on the Bel- dark brown (5F8–6F8) around the plug on PDA; mar- say 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, gin even or plumose in all media, appressed, hyaline SMH4594 (F). FRANCE. on Cirsium palustre stems, 5/17/ on all media, becoming yellowish-gray to brownish- 1864, (HOLOTY PE, CO). ARIE`GE: Rimont, Las Muros, gray (4B2–6C2) on PDA, distinct in PDA; reverse Ruisseau de Peyrau, 400 m, on wood, 12-VIII-2001, J. Four- same as the mat in all media except in SMH4594 nier, JF01167 ( J. Fournier Herbarium); 25-IX-2002, A.N. which was black on PDA; anamorph produced sparse- Miller, J. Fournier, A.M. Stchigel, M. Calduch, SMH4826 (F). ly around the plug in all media within 21 d in PERU. DEPARTAMENTO. CUZCO: along the Cuzco-Pilo- SMH3277, SMH4027, and SMH4594, anamorph not copata-Paucartambo Road at a point ca 132 km from the produced in SMH3819. intersection with the Cuzco-Puno Road, on leaf, 19 Jul 1976, Hyphae largely undifferentiated, 1–3.5 ␮m wide, K.P. Dumont, S.E. Carpenter, M.A. Sherwood, P. Buritica, PE- thin-walled, hyaline to pale brown. Conidiogenous cells 1837, PE-1852 (NY). USA. NORTH CAROLINA: Macon County, Otto, Cowieta Hydrological Laboratory, on 4 cm phialides, commonly produced laterally or occasion- log, 19-VII-1997, F.A. Ferna´ndez, SMH3277; Highlands, ally terminally from pale brown hyphae, delimited by Highlands Biological Station, on wood piece, 20-VII-1997, a basal septum, monophialidic or occasionally poly- F.A. Ferna´ndez, SMH3308; Horse Cove, [35.025, Ϫ83.1464], ϫ phialidic, obclavate to lageniform, 6–16 2.5–4.5 1000 m, on 5 cm diam branch, 27-VII-1998, F.A. Ferna´ndez, ␮m at widest part, pale brown, constricted below the SMH3819 (F). VENEZUELA. Cerro de Neblina, Neblina collarette, 0.5–1.5 ␮m below collarette; collarette mi- base camp, 18-II-1985, A.Y. Rossman, AR2148 (BPI).

← 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 ϭ 100 ␮m; 63 ϭ 500 ␮m; 67–81 ϭ 10 ␮m. 1122 MYCOLOGIA

Cultures examined. SMH3277-1, 3; SMH3819-1, 2, 4; terial. Ascomata of L. glabrata commonly are con- SMH4027-1, 2, 3, 4, 5, 6; SMH4594-1, 2, 3. fused with old ascomata of L. ovina in which the to- mentum has worn away (FIG. 27). In fact, two collec- tions of L. ovina var. glabrata distributed in exsiccati DISCUSSION (Roum. F. sel. Gall. 5857 [UPS] and Syd. Myc. Mar. Multiple gene genealogies indicate four of the five 2144 [as Leptospora ovina var. glabrata; UPS, NY]) are Lasiosphaeria morphospecies occur as well-supported unambiguously L. ovina because young ascomata phylogenetic species. These species are corroborated were found that possessed white tomentum and ap- further by MSR in possessing a unique combination pendages were observed in young ascospores in the of morphological characters. Lasiosphaeria lanugino- NY material. Because Fries’ material of L. ovina var. sa forms a genetically variable species complex com- glabrata could not be located, a neotype for L. gla- prising a single morphological species following MSR brata based on Munk’s material is designated here. and several phylogenetic species employing GCPSR. Lasiosphaeria ovina is recognized by its white, to- Although these lineages could be recognized formal- mentose ascomata (FIG. 24), asci with globose sub- ly as species based on GCPSR, it is premature to do apical globules (FIGS. 32–35), and ascospores that do so because additional populations of L. lanuginosa not develop a swollen head (FIGS. 39–41) and usually need to be sampled and at least three additional mor- possess short, awl-like appendages (FIGS. 37, 38). The phospecies, which putatively belong in the L. lanu- outer ascomal features of L. ovina can be quite var- ginosa species complex, should be included in fur- iable. Ascomata are densely tomentose and bright ther analyses. white when young (FIG. 24), but as the tomentum Although species relationships differ slightly breaks down and begins to wear away, it can become among the separate analyses (FIGS. 1–4), they are areolate and cream or brownish-gray (FIG. 25) and well-resolved in the combined analysis (FIG. 5). La- ascomata may appear wax-like (FIG. 26) or even gla- siosphaeria rugulosa and L. glabrata are basal taxa, brous and black with age (FIG. 27). This species has while L. ovina, L. sorbina and members of the L. been described more than once based in part on lanuginosa complex are more derived. These latter these environmental variations of the tomentum three species possess a well-developed, colored to- (e.g., L. chrysentera). There has been much confu- mentum, whereas L. glabrata and L. rugulosa possess sion as well concerning the ornamentation and shape a sparse, brown tomentum. Cercophora sparsa and C. of the subapical globule. The globose globule is com- sulphurella possess yellowish hairs, while C. areolata posed of a cytoplasmic refractive substance and may has brown, villose hairs. This suggests that colored appear somewhat ornamented, especially in poorly vestiture may have been lost in L. glabrata and L. preserved collections. Lundqvist (1972) lectotypified rugulosa and then regained in L. ovina, L. sorbina L. ovina based on a Persoon collection (L) and de- and members of the L. lanuginosa complex. The out- scribed the subapical globule as verrucose. No spiny- groups, L. rugulosa, and members of the L. lanugi- shaped globule as drawn by Carroll and Munk (1964) nosa complex possess ascospores that develop swollen and Hilber et al (1987) has been seen by the authors. heads, while ascospores in L. glabrata, L. ovina and In addition, the oversized ellipsoid subapical globule L. sorbina lack swollen heads or the ascospores swell as illustrated by Re´blova´ et al (1999, FIGS. 4 and 13a) only slightly. The evolution of this character is pres- does not accurately represent the globose subapical ently unclear because the ability to develop a swollen globule commonly seen in L. ovina (FIGS. 34, 35) but ascospore head recently has been shown to have oc- is merely an artifact caused by a slight invagination curred numerous times throughout the Sordariales of the cytoplasm. One striking feature found in some (Miller and Huhndorf 2004b). collections of L. ovina is the heterobasidiomycete my- Lasiosphaeria glabrata can be distinguished in pos- coparasite, Krieglsteinera lasiosphaeriae Pouz., that oc- sessing nearly glabrous, black ascomata (FIGS.6,7) casionally infects the ascomata. This mycoparasite and ascospores that lack a swollen head and append- most likely is distributed widely throughout Asia, Eu- ages (FIGS. 19–22). Ascomata of L. glabrata usually rope and North America but is found rarely due to appear shiny due to the outer layer of sparse brown its cryptic nature (Miller et al 2003). hairs (FIGS. 11, 12). This species closely resembles L. The holotype of L. chrysentera was examined; the rugulosa in outer ascomal features but can be sepa- ascomata possess the full range of tomentum varia- rated easily by its longer ascospores (33.5–52.5 versus tion from young, white, tomentose ascomata to gray- 29.5–37.5), which do not develop a swollen head. ish, areolate ascomata to old, black, glabrous ascom- Munk (1957) elevated L. glabrata from a variety (as ata. Most ascomata were old and appeared black and L. ovina var. glabrata) to the species level, but it is glabrous. As described by Carroll and Munk (1964), not clear whether he actually saw any of Fries’ ma- the centrum contents were ‘‘bright yellow’’ to ‘‘gold- MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1123 en-brown,’’ the subapical globule appeared ‘‘golden- the lengths of which fall within the 95% confidence brown’’ (but not ‘‘coarsely echinulate’’ as shown in interval of ascospore lengths in L. ovina. Until mo- FIG. 35), and the ascospores were ‘‘light yellow to lecular data can be obtained for L. libertiana, it golden-brown’’ and ‘‘finely and irregularly pitted.’’ should be maintained as a distinct morphospecies However, the coloration and ornamentation of the distinguished from L. ovina by its unique habitat on subapical globule and ascospores is merely an artifact grass and slightly longer ascospores. of the age, poor condition and method of preserva- Lasiosphaeria rugulosa is distinguished by the wrin- tion of the specimen. Carroll and Munk (1964) were kled appearance of the ascomata upon drying and unable to culture their fungi, which they believed the short ascospores that form swollen heads and ‘‘may be attributed to the vigorous drying techniques lack appendages. Although not apparent in macro- used to preserve the specimens in a tropical climate.’’ scopic view, squash mounts of the ascomal wall reveal These vigorous drying techniques most likely caused a tightly compressed third wall layer composed of in- the yellow centrum pigments in L. ovina to stain the terwoven, sparse, brown hyphae similar to that found asci and ascospores a golden color and also might in L. glabrata. The holotype was described as pos- explain the irregular pitting observed in the asco- sessing paraphyses with blue contents (Miller and spores. This phenomenon also has been observed to Huhndorf 2001). However, the taxonomic value of a lesser extent in other specimens of L. ovina that this character presently is unclear because it now also have been poorly preserved due to overdrying. The has been found to occur rarely in L. ovina and in name L. chrysentera unfortunately has proliferated one member of the L. lanuginosa complex throughout the literature (Hilber et al 1987, van (SMH4027). Lasiosphaeria rugulosa is morphological- Hooff 1993, Vasilyeva 1998, Candoussau et al 2002, ly similar to C. sparsa but has a tropical distribution Hilber and Hilber 2002, Zhang and Zhuang 2003) and rugulose ascomata, whereas C. sparsa is a tem- when referring to specimens of L. ovina in which the perate species with smooth ascomata. These taxa oc- tomentum has become areolate, the subapical glob- cur as two distinct phylogenetic species in all analyses ule is spiny and the ascospores are slightly yellowish, (FIGS. 1–5). although these characters either do not exist or fall Lasiosphaeria sorbina is recognized by having as- within the range of variation found in L. ovina. La- comata covered in a dense, colored tomentum (FIGS. siosphaeria chrysentera is synonymized under L. ovina 46, 47), asci that lack subapical globules (FIGS. 54, because it merely represents a poorly preserved spec- 55) and short, slightly curved ascospores that lack imen of this species. appendages (FIGS. 56–58). This species exhibits a Fairman (1904) distinguished Lasiosphaeria ovina broad geographic range and possesses a wide range var. aureliana from L. ovina by the presence of as- of tomentum and centrum colors. European speci- cospore appendages and the flavo-virescent color of mens (holotype, JF02053) possess a gray tomentum the centrum. Although Fairman (1904) and Lund- and a yellow or pinkish centrum, North American qvist (1972) believed ascospore appendages probably material (Ohio, Morgan s.n., GJS L555) possess a were lacking in L. ovina, structures resembling awl- gray or bright orange tomentum and a yellow or or- like appendages were found in lectotype material of ange centrum and South American material (EC- L. ovina by the present authors. Hilber et al (1987) 556) possess a pale pinkish to pale orange tomentum examined three collections determined by Fairman and a yellow centrum. Lasiosphaeria cylindrospora also to be Lasiosphaeria ovina var. aureliana and believed possesses a gray tomentum but can be distinguished one was L. chrysentera while the other two were L. from L. sorbina by its hyaline centrum and longer ovina. We determined all three collections to be L. ascospores (46–56 versus 21.5–46) (TABLE II). ovina. While the centrum in holotype material of La- Most members of the L. lanuginosa complex can siosphaeria ovina var. aureliana does contain a slightly be distinguished by having ascomata covered with a greenish-yellow cast, this hue falls within the range dense, white to yellowish or rarely pale pinkish to- of yellowish centrum colors observed in L. ovina. mentum (FIGS. 62–64) and ascospores that usually This variety is synonymized under L. ovina. develop a swollen head (FIGS. 77, 78) and possess Material of Lasiosphaeria ovina var. vagens Chen. lash-like appendages (FIG. 76). This morphospecies was not available from Paris for examination. grows on the decorticated part of dead branches, Lasiosphaeria libertiana is known from the two iso- which still possess some remnants of bark, or rarely type specimens (BR and NY), which occur on decay- on dead leaves or grass. This is in contrast to L. ovi- ing grass culms. This species is identical morpholog- na, which grows on decorticated wood suggesting ically to L. ovina except it occurs on a different sub- that members of the L. lanuginosa complex may be strate (grass versus wood) and the ascospores are earlier colonizers of wood than L. ovina. Although slightly longer (51–66 versus 35–55 ␮m) (TABLE II); there is a wide range of variation in the amount and 1124 MYCOLOGIA color of the tomentum in members of the L. lanu- strate (TABLE II). These results suggest that certain ginosa complex, they usually can be differentiated characters, such as tomentum color, development of from other species based on ascospore morphology. a swollen ascospore head and the type of ascospore The tomentum is usually persistent and the ascomata appendage, form a morphological continuum and seldom become black and glabrous as in L. ovina. are uninformative for delimiting taxa in the L. lan- When the tomentum does wear away, only a few uginosa complex. sparse yellowish hairs remain (FIG. 65) making it dif- Three of the five remaining morphospecies, which ficult to distinguish this morphospecies from C. spar- putatively belong in Lasiosphaeria, most likely belong sa. However, C. sparsa can be separated by its shorter in the L. lanuginosa complex. Cercophora citrina, C. ascospores (29.5–37 versus 33–60) that lack append- gossypina and C. spirillospora possess tomentose as- ages. While most members of the L. lanuginosa com- comata and ascospores that develop swollen heads plex possess a white or yellow (TABLE II) tomentum, and possess lash-like appendages. These morphospe- specimens from Peru possess a pale pinkish tomen- cies can be distinguished based on their substrate, tum similar to that found in Ecuadorian specimens tomentum color and ascospore length. Cercophora ci- of L. sorbina. However, these specimens readily are trina occurs on elephant dung and has a reddish- distinguished from L. sorbina by their longer asco- orange tomentum and extremely long ascospores, C. spores, which develop swollen heads and possess ap- gossypina occurs on hare dung and has a whitish to- pendages. Ascospores that have formed swollen mentum, while C. spirillospora occurs on bamboo and heads inside asci are usually hyaline and sometimes has a yellowish-brown tomentum (reported to be red- difficult to find, especially in tropical specimens, in dish when fresh, Penzig and Saccardo 1897) (TABLE which only one or two asci may contain ascospores II). It is obvious that these morphospecies must be with swollen heads. Ascospores in which the swollen included in additional molecular analyses and much heads have turned brown are seldom observed in the more work is needed in the L. lanuginosa complex ascus, especially in older material, and usually only a before species boundaries can be established. few brown heads are found adhered to the outside As previously discussed, a wide range of tomentum wall layer of squashed ascomata. and centrum colors exists in L. ovina, L. sorbina and Most of the sampled members of the L. lanuginosa members of the L. lanuginosa complex. Changes in complex possess characters slightly different from tomentum color are primarily due to the age of the those found in the type specimen. Three specimens ascomata. For example, young ascomata in L. ovina possess shorter ascospores (e.g., SMH4027, SMH3819, are bright white but the tomentum quickly becomes AR2148); SMH2390 has ascomata covered with a dull gray with age, whereas those in the L. lanuginosa sparse, yellow tomentum; SMH4594 contains asco- complex begin white or pale yellow and sometimes spores that lack a swollen head and possess awl-like turn orange-brown with age. The tomentum also may appendages; and TL5656 occurs on grass stalks rather wear away with age in all three species, giving the than wood (TABLE II). Because only SMH3277 pos- ascomata a glabrous appearance. Climate and/or ge- sesses characters similar to those found in the type ography also may affect color in these species. While specimen, it is in this lineage where L. lanuginosa temperate specimens of L. sorbina have a gray to- most likely will find its taxonomic placement. mentum, subtropical and tropical specimens possess Some members of the L. lanuginosa complex pos- a pale pinkish to orange tomentum. Specimens of L. sess combinations of characters making it difficult to lanuginosa from North and Central America have a assign them to any of the known morphospecies. Two yellow tomentum, while those from South America specimens possess characters attributable to L. ovina. possess a pale pinkish tomentum. As pointed out by Specimen SMH4027 has a white tomentum and the Lundqvist (1972), different substrates also may in- ascospore heads swell only slightly, while specimen duce different colors in the tomentum and centrum. SMH4594 possesses ascospores that do not develop a The holotype of C. gossypina occurs on hare dung swollen head and possess awl-like appendages (TABLE and most ascomata possess a pale yellow tomentum II). Specimen TL5656 possesses a combination of and centrum, whereas the paratype occurs on por- substrate, tomentum, and ascospore characters that cupine dung and ascomata possess a white tomentum could place it into any one of three morphospecies and hyaline centrum. Further work is needed to de- (TABLE II). It shares a similar substrate (grass stalks) termine the effect of age, climate, geography and and tomentum color (white) with L. libertiana but substrate on tomentum and centrum colors and their possesses ascospores that form a swollen, brown significance in delimiting species boundaries in La- head. This specimen also closely resembles C. gossy- siosphaeria. pina and L. lanuginosa in its white to pale yellowish, Culture studies mostly were uninformative for de- tomentose ascomata but differs from both in sub- limiting the five included species of Lasiosphaeria. MILLER AND HUHNDORF:SPECIES BOUNDARIES WITHIN LASIOSPHAERIA 1125

However, these species can be distinguished on PDA mour Graduate Fellowship from FMNH. Fieldwork for by the color of their mycelial mat; L. glabrata is gray- ANM was supported in part by an ASPT (American Society ish orange to grayish red (6B3–7B4), L. ovina is whit- of Plant Taxonomists) Graduate Student Research Grant, ish to grayish-yellow (4A1–4B4), L. rugulosa is whitish an Explorers Club Research Grant and a UIC Provost to yellowish-white (3A2), L. sorbina is brownish gray Award. Fieldwork for SMH was supported in part by the National Research Council Resident Research Associate to pastel red (7C2–8A5), and members of the L. lan- Post-doctoral Program in co-operation with the USDA For- uginosa complex are yellowish-brown to dark brown est Service, Madison, Wisconsin. The authors are most (5F8–6F8). Growth of isolates of each species was unin- grateful to Bert and Gill Brand, Misericordia Calduch, formative on all three media because the rates over- George Carroll, Fernando Ferna´ndez, Jacques Fournier, lapped for most isolates, although those of L. glabra- Thomas Læssøe, Kerry Robinson, Amy Rossman, Gary Sa- ta and L. sorbina tended to grow more slowly than muels and Alberto Stchigel for providing specimens or cul- those of the other three taxa. tures. The curators of the herbaria listed are thanked for Lasiosphaeria ovina, L. sorbina and members of the material made available on loan. Andrea Adie is thanked L. lanuginosa complex produced similar Phialophora- for sequencing ITS for C. areolata. Sequences were gener- ated in the Pritzker Laboratory for Molecular Systematics type anamorphs in culture (FIGS. 42, 43, 60, 61, 79). Phialides were mostly obclavate to lageniform, hya- and Evolution at FMNH. This work represents a portion of a thesis in partial fulfillment of the requirements for the line or pale brown and possessed minute, slightly flar- doctoral degree at the Graduate College of the University ing collarettes. Similar phialides also were produced of Illinois at Chicago. occasionally from old ascospores of L. ovina and L. sorbina (FIGS. 41, 59). This type of anamorph previ- ously has been reported for L. ovina (Gams 1973, LITERATURE CITED Gams and Holubova´-Jechova´ 1976, Gams 2000) and placed in Phialophora Medlar section Catenulatae W. Alfaro ME, Zoller S, Lutzoni F, 2003. Bayes or bootstrap? A Gams (Gams and Holubova´-Jechova´ 1976). Lasio- simulation study comparing the performance of Bayes- ian Markov Chain Monte Carlo sampling and boot- sphaeria ovina and members of the L. lanuginosa strapping in assessing phylogenetic confidence. Mol complex also produced blastoconidia directly from Biol Evol 20:255–266. the hyphae or from cylindrical, collarette-less branch- Avise JC, Ball RM. 1990. Principles of genealogical concor- es (FIGS. 44, 45, 80, 81). The production of blasto- dance in species concepts and biological taxonomy. conidia previously has been reported in Cercophora Oxford Surveys Evol Biol 7:45–68. atropurpurea A.N. Mill. and Huhndorf (Miller and Barker FK, Lutzoni FM. 2002. The utility of the incongru- Huhndorf 2001). Anamorphs were not produced in ence length difference test. Syst Biol 51:625–637. cultures of L. glabrata and L. rugulosa. Baum DA, Donoghue MJ. 1995. Choosing among alterna- Species boundaries for four of the five morphospe- tive ‘‘phylogenetic’’ species concepts. Syst Bot 20:560– cies within Lasiosphaeria have been defined using 573. , Shaw KL. 1995. Genealogical perspectives on the GCPSR based on multiple gene genealogies. Exten- species problem. 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