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Systematics of Hypocrea citrina and related taxa

¹ The Pennsylvania State University, Department of Plant Pathology, Buckhout Laboratory, University Park, Pennsylvania 16802, U.S.A.: Current address: Lock Haven University of Pennsylvania, Department of Biology, 119 Ulmer Hall, Lock Haven PA, 17745, U.S.A.
² The Pennsylvania State University, Department of Plant Pathology, Buckhout Laboratory, University Park, Pennsylvania 16802, U.S.A.
³ TU Vienna, Institute of Chemical Engineering, Division Applied Biochemistry and Gene Technology, x Getreidemarkt 9/E1665, A-1060 Vienna, Austria.

^* Correspondence: Barrie E. Overton, boverton{at}lhup.edu

	Abstract

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY KEY TO HYPOCREA CITRINA... References

 
Morphological studies and phylogenetic analyses of DNA sequences from three genomic regions – the internal transcribed spacer (ITS) regions of the nuclear ribosomal gene repeat, a partial sequence of RNA polymerase II subunit (rpb2), and a partial sequence of translation elongation factor (tef1) – were used to investigate the systematics of Hypocrea citrina and related species. A neotype specimen is designated for H. citrina that conforms to Persoon's description of a yellow effuse fungus occurring on leaf litter. Historical information and results obtained in this study provide the foundation for selection of a lectotype specimen from Fries's herbarium for H. lactea. The results indicate that (1) Hypocrea citrina and H. pulvinata are distinct species; (2) H. lactea sensu Fries is a synonym of the older name H. citrina; (3) H. pulvinata, H. protopulvinata, and H. americana are phylogenetically distinct species that form a well-supported polyporicolous clade; (4) H. citrina is situated in a clade closely related to H. pulvinata; and (5) H. microcitrina and H. pseudostraminea reside in a highly supported clade phylogenetically distinct from H. citrina. Hypocrea protopulvinata, H. microcitrina, H. megalocitrina, H. pseudostraminea, and a new species, H. aurantiistroma, are reported and described from North America. Variation in rpb2 and tef1 gene sequences suggests geographical subgroupings between European and North American isolates of H. pulvinata. The phylogenies inferred from ITS, rpb2, and tef1 gene sequences are concordant. Hypocrea citrina var. americana is elevated to species status, Hypocrea americana.

Taxonomic novelties: A neotype is designated for Hypocrea citrina (Pers: Fr.) Fr. and a lectotype for Hypocrea lactea (Fr.: Fr.) Fr.; H. pulvinata Fuckel is lectotypified; Hypocrea aurantiistroma Overton, sp. nov., Hypocrea americana, stat. nov.

Keywords Ascomycetes / Hypocrea citrina / Hypocrea lactea / Hypocrea pulvinata / Hypocreales / Hypocreanum / ITS / systematics / rpb2 gene sequences / tef1 gene sequences / Subsection Citrinae / Subsection Pulvinatae / Trichoderma

	INTRODUCTION

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY KEY TO HYPOCREA CITRINA... References

 
Among the most common and conspicuous members of the genus Hypocrea (Hypocreales, Hypocreaceae) are species that form extensively effused, lightly or brightly coloured stromata on leaf litter, soil, wood, and bracket fungi. Names that have been applied to these fungi include H. citrina (Pers.: Fr.) Fr., H. lactea (Fr.: Fr.) Fr., H. pulvinata Fuckel, H. fungicola P. Karst., and H. karsteniana Niessl. Confusion surrounds the application of the name H. citrina (Pers.: Fr.) Fr., and phylogenetically related species have not been determined. The stroma of H. citrina is extensively effuse, typically yellow and asci contain eight 2-celled uniseriate ascospores.

Doi (1972) subdivided Hypocrea into subgenera, sections, subsections, and series based on stroma characters. He recognized H. pulvinata, H. citrina, and H. lactea as distinct species and placed them in Hypocrea subgen. Hypocrea sect. Homalocrea Yoshim. Doi. He included H. citrina and H. lactea in subsect. Citrinae Yoshim. Doi, while H. pulvinata was placed in subsect Pulvinatae Yoshim. Doi. The anamorph of H. citrina has acremonium- or verticillium-like conidiophores with hyaline conidia. Bissett (1991a, b) assigned acremonium- or verticillium-like condiophores to Trichoderma sect. Hypocreanum Bissett. Fourteen species of Hypocrea Fr., with stromata similar to that of H. citrina, or considered as its synonym, and anamorphs assignable to Trichoderma sect. Hypocreanum are newly described or redescribed, type specimens examined, and, where possible, phylogenetic relationships to H. citrina established.

Nomenclature and typification
The name H. citrina has been applied to two different species, one polyporicolous and one lignicolous or terrestrial. Rifai & Webster (1966) considered the terrestrial/lignicolous form of H. citrina to be a synonym of another terrestrial species, H. lactea. Canham (1969) used the name H. citrina, with H. pulvinata as a synonym, for the polyporicolous form. Perspectives on these differing conclusions can be found in Rifai & Webster (1966) and Canham (1969). Application of the current International Code of Botanical Nomenclature (ICBN: Greuter et al. 2000) for the selection of type material provides the mechanism for stabilizing the application of the names, H. citrina, H. lactea, and H. pulvinata.

Persoon (1796) described a yellow terrestrial fungus on leaf litter as Sphaeria citrina Pers. Fries (1816) described Sphaeria lactea Fr. as a white effuse fungus with dark ostioles that occurs on bark and soil. Fries (1823) expanded Persoon's concept of Sphaeria citrina Pers.: Fr. to include a polyporicolous element, listing Scleromyceti Sueciae 31 (SS 31) as representative material. Fries (1849) described the genus Hypocrea (formerly a section of Sphaeria) and transferred S. citrina and S. lactea into Hypocrea. Persoon's (1796) original protologue clearly emphasized a yellow effuse terrestrial species that occurs on leaf litter. Fries's expanded species concept caused confusion. He explicitly ascribed the species, however, to Persoon in the Index to the same volume and to volume 3 of his Systema mycologicum. Dingley (1955) and Canham (1969) followed Fries's (1823) concept of H. citrina to be a fungus that occurs on polypores, even though, historically, others such as Karsten (1873), Niessl (1883) and Saccardo (1883) had recognized the fungicolous element included by Fries as taxonomically distinct from S. citrina. To account for the polyporicolous element that Fries (1823) ascribed to S. citrina, Karsten (1873) published the new name, Hypocrea citrina * fungicola P. Karst., without designating a formal rank. Niessl (1883) raised Karsten's taxon to species rank, H. karsteniana Niessl. Only months later, Saccardo (1883), apparently unaware of Niessl's work, also elevated Karsten's taxon to species rank as H. fungicola (P. Karst.) Sacc. Karsten, Niessl, and Saccardo did not recognize H. pulvinata as a synonym, although it had previously been described from a polypore (Fuckel 1870). Seaver (1910) listed H. karsteniana and H. fungicola as questionable synonyms of H. citrina. Weese (1927) synonymized H. karsteniana and H. fungicola with the oldest available name, H. pulvinata. The synonymy proposed by Weese (1927), which has never been disputed, serves as the basis for the application of the name H. pulvinata to the polyporicolous species.

There is no specimen of S. citrina in Persoon's herbarium (L), and collections that can be directly linked to Persoon cannot be located. Dingley (1955) took Fries's SS 31 (UPS), on a polypore, to be the type collection but did not formally designate the specimen as lectotype. Canham (1969) formally designated SS 31 (UPS) to be the lectotype of S. citrina. Canham (1969) described H. citrina var. americana Canham, with globose part-ascospores from polypores in North America. Fries (1823) adopted Persoon's epithet, thereby sanctioning it, and the name is therefore now cited as Hypocrea citrina (Pers.: Fr.) Fr. Article 7.8 ICBN allows the selection of neotypes among material cited by the sanctioning authority. In this case, the taxonomic circumscription of the name and its type specimen clearly comes from Persoon and Fries's species concept was confused. Fries's interpreation deviates from the protologue and thus we reject Canham's (1969) lectotypification.

Rifai & Webster (1966) rejected Dingley's (1955) [and Canham's (1969)] concept of H. citrina as being fungicolous, because it did not agree with the protologue of S. citrina. Based on the argument proposed by Rifai & Webster (1966), it is evident that they wished to preserve the distinction between the fungicolous and terrestrial/lignicolous species in order to retain the name H. pulvinata for the fungicolous species. Their argument emphasized the original protologues of H. citrina and H. pulvinata. Rifai & Webster (1966) designated Persoon's original description of S. citrina as the lectotype in contravention to Arts. 8.1 and 9.2 of the ICBN that state a lectotype must be a specimen, or, at least, an illustration. By designating a litter-inhabiting specimen as neotype, we intend to stabilize the application of the name H. citrina.

Fries (1816) described Sphaeria lactea as "... latissime effusa subcarnosa lactea nuda, ostiolis punctiformibus immersis prominulisque". He reported that the new species grows on fallen logs and on naked soil in moist places and that the white colour does not change over time. He did not cite a specific collection or collecting locality. There are two specimens in Fries's herbarium (UPS) that can possibly be considered for neotypifying S. lactea, both of which were collected by Fries in "Småland, Femsjö"; one is identified as H. lactea and the other as Sphaeria lactea, but neither specimen has a date of collection. The specimen of H. lactea comprises an effused, yellow Hypocrea stroma that completely covers the leaf of a dicotyledonous plant. Ostiolar openings are visible throughout the specimen but mature ascospores were found only in one region. The specimen of S. lactea comprises an effused Hypocrea on the surface of what appears to be a piece of decorticated wood. The specimen is glued to a piece of stiff paper. The single stroma in this collection is mostly immature with no outward evidence of ostiolar openings or perithecia; most of it is cream or off-white in colour. Perithecial openings are visible in one small part of the stroma; this part of the stroma is darker in colour, more yellow, and the ostioles appear as minute papillae. Perithecia in this region contain asci with developing ascospores, and a few asci were found with apparently mature ascospores. It is unlikely that the specimen identified as H. lactea is part of the original gathering of this species simply because the genus Hypocrea dates from 1849.

The protologues of H. citrina and H. lactea are distinctly different, yet these taxa are now considered synonymous. According to the protologues, H. citrina is yellow and effuse and occurs on leaf litter, while H. lactea is white and effuse and occurs on soil and bark. As early as 1818, Fries's concept of S. lactea began to change as he illustrated S. lactea as a goldish white to greyish orange effuse fungus and suggested that it was closely related to H. citrina. Further confusing the issue, Doi's (1972) concept of H. lactea, which is based on Japanese material, differs from Rifai & Webster's concept of H. lactea. Doi's (1972) description and illustration of H. lactea featuring a yellow-brown stroma with distinct hyphal elements near the surface, likely represents an undescribed species from Japan. The importance that Fries (1816) and Persoon (1796) ascribed to stroma colour in the protologues of H. lactea and H. citrina must be reexamined using current methods including molecular phylogenetics. By designating the specimen in Fries's herbarium labeled S. lactea as neotype, we intend to stabilize the application of the name H. lactea and provide the basis for comparing H. lactea and H. citrina.

Species recognition
Species limits in Hypocrea have been historically based on morphology. Phylogenetic data and genealogical concordance phylogenetic species recognition (GCPSR: Taylor et al. 2000) are now being employed to define species and for systematic studies of Hypocrea (Chaverri et al. 2001, 2003a). GCPSR utilizes the concordance of multiple gene genealogies and the transition from concordance among branches to incongruity among branches to establish species limits (Taylor et al. 2000). Molecular data from RNA polymerase II subunit (rpb2), translation elongation factor 1-alpha (tef1), and ITS1-5.8S-ITS2 (ITS) rDNA, have been used to make crucial anamorph–teleomorph connections in Hypocrea, to establish anamorph relationships at the sectional level in Trichoderma, and to place genera previously described in other pyrenomycete orders within the Hypocreales (Chaverri et al. 2003b, Dodd et al. 2002, Kullnig-Gradinger et al. 2002, Zhang & Blackwell 2002). Data from these gene regions and morphological analyses will be used to address the following objectives: (1) the typification and redescription of H. citrina sensu Persoon and H. lactea sensu Fries using strict interpretations of the protologues for typification; (2) the determination of species related to Hypocrea citrina using GCPSR; (3) the morphological description of phylogenetic species identified in this study; and (4) a discussion of several species treated by Doi (1972) in Hypocrea subsect. Citrinae and subsect. Pulvinatae.

	MATERIALS AND METHODS

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY KEY TO HYPOCREA CITRINA... References

 
Collections and isolates
Species concepts, illustrations, and descriptions from Doi (1972) were used in making identifications except for H. lactea and H. fulva. Table 1 lists the isolates used in this study. Frequently cited collectors are abbreviated, B.E. Overton (B.E.O.) and G. J. Samuels (G.J.S.). All isolates with G.J.S. designations were obtained by isolating single ascospores on CMD with the aid of a micromanipulator. All isolates with B.E.O. designations were obtained from plating the entire contents of individual perithecia. Unless otherwise annotated, host and substrate information were obtained from herbarium labels, and data were entered in specimens examined as it appears on the labels.

Molecular phylogenetic analysis
DNA sequence analysis of the isolates was conducted using three gene sequences: ITS1-5.8S-ITS2 (ITS), a partial sequence of translation elongation factor (tef1), delimited by the primer pair ef1/2, and a partial sequence of RNA polymerase II subunit (rpb2) delimited by the primer pair frpb2-5f/-7cr. Mycelia were lyophilized prior to DNA extraction. The lyophilization protocol is described in Stewart et al. (1999). Extraction of genomic DNA was carried out using the phenol and chloroform extraction outlined in Stewart et al. (1999). A 50 µL polymerase chain reaction (PCR) for ITS and tef1 was performed following the conditions outlined by Chaverri et al. (2001) using the following primer pairs: for ITS, the primers utilized were ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (White et al. 1990); for tef1, the primers were ef-1 (5'-ATGGGTAAGGA(A/G)GACAAGAC-3') and ef-2 (5'-GGA(G/A)GTACCAGT(G/C)ATCATGTT-3') (O'Donnell et al. 1998). A 50 µL polymerase chain reaction (PCR) following the conditions outlined by Chaverri et al. (2003b) was utilized for generating a partial rpb2 product. The following two fungus-specific primers were employed: frpb2-5f (5'-GA(T/C)GA(T/C)(A/C)G(A/T)GATCA(T/C)TT(T/C)GG -3') and frpb2-7cr(5'-CCCAT(A/G)GCTTG(T/C)TT(A/G)CCCAT-3') (Liu et al. 1999). Two percent dimethyl sulfoxide (DMSO) from AMRESCO® was added to each 50 µL PCR reaction. A PCR purification was carried out using a QIAquick® PCR purification kit. The ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit with Amplitaq DNA Polymerase FS (PE Applied Biosystems) was utilized for sequencing cleaned PCR products for each primer direction. Performa® DTR gel filtration cartridges from Edge BioSystems were used for cleaning cycle sequencing products. Sequencing was either conducted on an ABI 377 located in the Plant Pathology Department at The Pennsylvania State University or sent to the Nucleic Acid Facility (Life Science Consortium, The Pennsylvania State University). Sequence data were trimmed at the 5' and 3' ends. The ITS region was sequenced for all isolates and used to screen for replicate isolates. Replicate isolates were removed from subsequent analyses if there were no obvious morphological or locality differences noted from the herbarium specimens. Sequences were assembled using SeqMan® II option and aligned using Clustal W in DNA Star (DNA Star Inc., Madison, Wisconsin), and a phylogenetic analysis was performed using PAUP* v. 4.0b8 (Sinauer Associates, Sunderland, MA). Alignments were manually adjusted in PAUP*. Overton et al. (1999) determined H. sulphurea to be a phylogenetically appropriate outgroup taxon for H. citrina and allies and this species was used as outgroup in all data sets. Maximum parsimony (MP) analyses were completed using the heuristic search option with starting trees obtained via random addition sequence (10 replicates) and TBR branch swapping. Gaps (insertions/deletions) were treated as missing data. Bootstrap analysis was performed in 500 replicates with random sequence addition (10 replicates). The sequences and alignments were deposited in GenBank (Table 1).

Determination of optimal growth temperature
A growth trial using multiple isolates from each phylogenetic species established in the species tree (Fig. 1) was carried out using 20 mL of three media: potato-dextrose agar (PDA, Difco), cornmeal-dextrose agar (CMD, Difco), and SNA with filter paper (Nirenberg 1976). Inoculum was prepared on CMD. A 1-cm-diam disk from the actively growing inoculum colony was placed near the edge of the plate. One plate of each medium for each culture was incubated at 20, 25, 30, and 35 °C in total darkness. The radius of the colony was measured from the edge of the inoculum at 24-h intervals for 4 d. The experiment was replicated three times during subsequent weeks. Optimal growth temperatures are summarized in the species descriptions.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Parsimony analysis of ITS1-5.8S-ITS2. One phylogram of two most parsimonious trees; 48 steps; consistency index: 0.854; retention index; 0.989; homoplasy index: 0.146; numerical values of branch lengths are given above and bootstrap values (500 replicates with 10 random addition replications) are indicated below branches. Outgroup taxon: H. sulphurea.

	RESULTS

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY KEY TO HYPOCREA CITRINA... References

 
The trees generated from ITS are slightly different from those obtained from tef1 and rpb2 (Figs 1, 2, 3). In the ITS gene tree, the proposed neotype of H. citrina from Europe (CBS 894.85) differed from North American isolates of H. citrina in a single nucleotide. The H. citrina isolate G.J.S. 95-183 is identical to other isolates from North America in the ITS tree. The ITS result differed from the tef1 gene tree in which the European CBS 894.85 was identical with the North American isolate B.E.O. 99-29 (Fig. 2) and from the tef1 and rpb2 gene trees, in which isolate G.J.S. 95-183 had a longer branch length than other isolates of H. citrina (Figs 2, 3). Parsimony analysis of the ITS sequences showed a single nucleotide polymorphism separating isolates of H. pulvinata from H. americana supported by a bootstrap score of 63 % (Fig. 1). The tef1 and rpb2 gene trees were more pronounced in placing H. citrina in a highly supported monophyletic group with H. pulvinata, H. protopulvinata, and H. americana with bootstrap scores of 99 % for both trees.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Parsimony analysis of partial sequences of tef1. One phylogram of six most parsimonious trees; 476 steps; consistency index: 0.826; retention index: 0.769; homoplasy index: 0.174. Rest as in Fig. 1.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Parsimony analysis of partial sequences of rpb2. One phylogram of two most parsimonious trees; 172 steps; consistency index: 0.872; retention index: 0.914; homoplasy index: 0.128. Rest as in Fig. 1.

The combined phylogenetic analysis using ITS, partial sequences of tef1 and rpb2 distinguished H. americana from H. pulvinata with a bootstrap score of 100 % (Fig. 4). European and North American isolates of H. pulvinata formed two distinct subclades, each supported by a bootstrap score of 100 %. Hypocrea pulvinata and H. americana formed a monophyletic group supported by a bootstrap score of 87 % (I, II). Hypocrea americana, H. pulvinata, and H. protopulvinata (III) formed a strongly supported monophyletic group with a bootstrap score of 100 % (Clade A). Isolates of H. citrina (including the ex-neotype (IV)) formed a strongly supported monophyletic group with a bootstrap score of 100 % (Clade B). Hypocrea microcitrina (VI) and H. pseudostraminea (V) are situated in an unresolved sister clade to H. citrina supported by a bootstrap score of 100 % (Clade C). Isolate B.E.O. 99-36 is divergent from other isolates of H. pseudostraminea; nevertheless, all isolates of H. pseudostraminea formed a strongly supported monophyletic group (V) with a bootstrap score of 100 %. Isolates of H. microcitrina (VI) formed a strongly supported clade with a bootstrap score of 100 %. The heuristic search of the most parsimonious trees yielded one tree with 678 steps with the minimum possible tree length of 575 (Fig. 4). From 2189 total characters, 1710 characters are constant, 133 variable characters are parsimony-uninformative, and 346 characters are parsimony-informative.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Combined parsimony analysis of ITS, tef1, rpb2. Phylogram of single most parsimonious tree; 678 steps; consistency index: 0.848; retention index: 0.915; homoplasy index: 0.152. Rest as in Fig. 1.

	DISCUSSION

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY KEY TO HYPOCREA CITRINA... References

 
Phylogenetic analysis and establishment of major clades
Despite some variation between the ITSl, tef1, and rpb2 gene trees, it can be concluded that H. citrina is phylogenetically distinct from H. pulvinata. In the combined analysis, Clade A, consisting of H. pulvinata, H. americana, and H. protopulvinata, is monophyletic; Clade B consists of all H. citrina isolates; and Clade C consists of H. pseudostraminea and H. microcitrina (Fig. 4). The H. microcitrina and H. pseudostraminea subclades are separated by long branches (Fig. 4). It is possible, given the large branch length differences, that long-branch attraction may be artificially inflating the bootstrap score uniting these taxa. Future phylogenetic studies that should include H. protocitrina, H. albocitrina, and H. lactea sensu Doi may help to more clearly elucidate the relationship of H. pseudostraminea and H. microcitrina to H. citrina.

Sequences from H. pulvinata and H. americana produced highly concordant gene trees, consistently generating the same subgroupings. ITS data from 3 out of 4 isolates of H. citrina from Europe differed from North American isolates in a single nucleotide. The ex-neotype culture of H. citrina (CBS 894.85) was identical with B.E.O. 99-29 in the tef1 gene tree, but they differed in the rpb2 tree. Furthermore, H. citrina isolate G.J.S. 95-183, which is invariant in the ITS tree from other North American isolates, had significant branch length differences in the tef1 and rpb2 gene trees from all other H. citrina isolates. The differences between isolates of H. citrina can best be explained by sexual recombination. Therefore, the species boundary for H. citrina was determined to be at the point of discordance separating clade B in the gene trees (Fig. 4). Isolates of H. pulvinata show large numbers of nucleotide changes between North American and European isolates in the rpb2 and tef1 data sets. The differences between North American and European isolates in the tef1 and rpb2 may represent cryptic species or simply variations in population structure. North American and European isolates of H. pulvinata are morphologically indistinguishable, and the ITS sequences are identical. Additional sequence analysis and morphological examination of European isolates will be required to determine whether European isolates represent a distinct species. In the ITS gene tree for H. americana, a single nucleotide change separates isolates of H. americana from H. pulvinata, while there are larger differences in the tef1 and rpb2 gene trees between these species.

The optimal temperature for growth was determined for each phylogenetic species. Optimal temperature for growth is reported only when multiple strains with branch length differences within a phylogenetic species could be sampled in order to determine the intra-species variation. Growth temperature optima could not adequately distinguish between any of the phylogenetic species nor were they variable within a phylogenetic species in which strain differences had been established based on branch length differences.

Phylogenetics and classification
The monophyly of many of the infrageneric groupings proposed by Doi (1972) have yet to be tested by DNA sequence analysis. In this study, it was found that subsections Citrinae and Pulvinatae as defined by Doi are not monophyletic assemblages of species. Hypocrea microcitrina and H. megalocitrina (Chaverri et al. 2003b) are not situated in the H. citrina clade. It is more difficult to determine the phylogenetic boundaries of Subsection Pulvinatae because Doi broke it up into several series from which isolates could not be sequenced.

Hypocrea microcitrina, H. pseudostraminea and H. lactea sensu Doi have similar morphological characters in the form of hyphal protrusions and textura intricata near the stroma surface. It is difficult to distinguish between hyphal protuberances and textura intricata. It is unclear at this time if the hyphal protrusions near the surface of the stromata and the textura intricata found in H. pseudostraminea and H. microcitrina are homologous characters, but both of these taxa have similar irregular verticillium-like anamorphs. Doi (1972) classified H. pseudostraminea in subsect. Pulvinatae, series Splendentes, and H. microcitrina in subsect. Citrinae. The results generated here suggest that the subgroupings proposed by Doi (1972) are artificial (Table 2).

At this time, it is unclear how H. albocitrina and H. protocitrina are related to H. citrina, as cultures are not available for sequencing. The overall macromorphology of H. albocitrina and the stroma tissue of H. protocitrina consisting of textura intricata suggest that these taxa are more similar to H. microcitrina and H. pseudostraminea. Hypocrea megalocitrina has been sequenced and is not situated in the same clade as H. citrina (Chaverri et al. 2003b). Molecular data generated by Chaverri et al. (2003b) place H. megalocitrina near a cold-loving species described by Müller et al. (1972) as H. psychrophila. Doi (2001) described a species from Japan, H. mikurajimensis Yoshim. Doi that has characters similar to those of H. fulva, H. megalocitrina, H. aurantiistroma, and H. psychrophila. The strongly warted ascospores, stroma anatomy, and yellow pulvinate stromata of H. mikurajimensis are similar to the characteristics described by Müller et al. (1972) for H. psychrophila, which has a white gliocladium-like anamorph. The nodulose ascospores of H. mikurajimensis, longitudinal elongate ostiolar canals, and regularly verticillate conidiophores are similar to characters found in H. megalocitrina, H. aurantiistroma, and H. fulva. The warted, thick-walled ascospores and long ostiolar canals of Hypocrea psychrophila and H. megalocitrina are the morphological characters uniting these taxa.

Doi (2001) classified H. mikurajimensis in the H. patella group of subsection Pulvinatae, series Splendentes, which has been shown to be paraphyletic (Dodd et al. 2002). The exact phylogenetic position of this species in relation to all other taxa included by Doi (1972) in Series Splendentes cannot be determined at this time. However, Chaverri et al. (2003b) have shown that H. pezizoides Berk. & Broome, also series Splendentes, is situated in the H. rufa clade. These authors also showed that H. megalocitrina and H. psychrophila are phylogenetically related. Based on characters of the teleomorph and anamorph mentioned above, it is possible that H. megalocitrina, H. fulva, H. mikurajimensis, H. aurantiistroma, and H. psychrophila are phylogenetically related, but more taxa have to be sequenced to establish the limits of the "psychrophila" clade.

Typification and nomenclatural conclusions
With the selection of a specimen that agrees with the protologue of S. citrina (Gams 4031, CBS 894.85) as neotype (herein designated), the concept of the species can be firmly attached to a litter-inhabiting fungus. It is clear from the molecular and morphological data that H. pulvinata (= H. citrina var. citrina sensu Canham) and H. citrina, as neotypified here, are not synonyms and that Fries (1823) and later Canham (1969) made a taxonomic error in expanding the species concept of S. citrina to include the polyporicolous element. Hypocrea citrina var. americana is elevated to species status, H. americana, based on morphological characters and a well-delimited phylogenetic species boundary.

Molecular data were compared from many different specimens of H. citrina collected in different geographic locations with variations in stroma colour and substrate. It was determined that the protologue was too narrowly defined for H. citrina. Specimens BPI 1107145, G.J.S. 89-145 and CBS 853.70 (Fig. 5C), which are white to light yellow and occur on soil, were found to be phylogenetically the same as the neotype of H. citrina, which is bright yellow and occurs on leaf litter (Fig. 6). A lemon-yellow specimen, B.E.O. 99-29, on the moss Drepanocladus uncinatus (Hedw.) Warnst., was also found to be the same as the neotype based on molecular results. These findings show that stroma colour and substratum are variable characters, a finding not suggested by the original protologue of S. citrina. Hypocrea citrina was never observed growing on polypores in this study.

View larger version (89K): [in this window] [in a new window]   Fig. 5. A–F. Hypocrea citrina. A. Macromorphology of B.E.O. 99-29; bar = 2 mm. B. Stromatal section showing t. globulosa to t. angularis tissues intermixed with t. intricata in BPI 737759; bar = 20 µm. C. Stroma variable in colour, white to yellow, CBS 853.70 isolated from specimen (photo courtesy of W. Gams). D. Asci with ascospores of neotype CBS 4031; bar = 20 µm. E. Stromatal cross section showing t. globulosa with angular edges due to mutual compaction in BPI 748251; bar = 20 µm. F. Immature asci and ascospores of BPI 748251; bar = 20 µm.

View larger version (134K): [in this window] [in a new window]   Fig. 6. A–C. Hypocrea citrina. A. Neotype specimen Gams 4031, CBS 894.85 on leaf litter. B. Stromatal section of BPI 744475 showing textura angularis below the perithecium and near the stroma surface; bar = 40 µm. C. Variable KOH reaction usually not greater than observed here in BPI 744475; bar = 2 mm.

View larger version (99K): [in this window] [in a new window]   Fig. 7. A–K. Hypocrea citrina (specimens of Hypocrea lactea). A. Illustration of Sphaeria lactea from Fries, Obs. Taf. 8, fig. 4. Compare to B. B–G. Sphaeria lactea, neotype specimen, coll. E.M. Fries, UPS; B. specimen glued to a piece of stiff paper. C. Surface of stroma seen in face view. D. Macroscopic view of stroma surface; bar = 1 mm. E. Ascus with developing ascospores; bar = 20 µm. F. Immature asci showing pore in ascus apex (left, cotton blue in lactic acid, right in 1 % aq. phloxine); bar = 10 µm. G. Mature ascospores in asci (cotton blue in lactic acid); bar = 10 µm. H–K. Hypocrea lactea specimen collected by Fries, Femsjö, UPS. H. Specimen; bar = 1 cm. I. Perithecial papilla, ostiolar opening and stroma surface; bar = 20 µm. J. Stroma surface showing pseudoparenchymatous cells at the surface and loosely disposed hyphae below; bar = 20 µm. K. Mature ascospores in asci (cotton blue in lactic acid); bar = 10 µm.

Some confusion exists around the selection of type material associated with the name H. pulvinata. When Fuckel (1870) described H. pulvinata, he did not cite a specific specimen, but indicated Polyporus sulphureus as the substratum. Rifai & Webster (1966) indicated that the substratum for Fuckel, Fungi Rhenani 2467, preserved at K was "Polyporus resinosus", which recently was identified as Laetiporus sulphureus. This specimen is over-mature, and no asci remain. Two specimens of F. Rhenani 2467 on L. sulphureus in G are also overmature. A specimen at FH, #876, was part of Fuckel's herbarium; the collecting data match those given in the protologue and the substratum also is identified as being Polyporus (= Laetiporus) sulphureus. Moreover, this collection is in good condition and is designated here as the lectotype for H. pulvinata. Measurements obtained from all historical specimens included in this study were consistent with measurements from more recent collections of H. pulvinata used in the molecular analysis. The species concept presented here for the polyporicolous form and the application of the name H. pulvinata is consistent with Weese (1927), Rifai & Webster (1966), and Doi (1972).

	TAXONOMY

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY KEY TO HYPOCREA CITRINA... References

 

1. Hypocrea citrina (Pers.: Fr.) Fr., Summa Veg. Scand.: 383.1849. Figs 5, 6, 7, 8.
   
   • Sphaeria citrina Pers., Obs. Mycol. 1: 68. 1796: Fr., Syst. Mycol. 2: 337. 1823.
     
   • = Sphaeria lactea Fr., K. Svenska VetenskAkad. Handl. II, 37: 141. 1816: Fr., Syst. Mycol. 2: 337. 1823.
     
   • Hypocrea lactea (Fr.: Fr.) Fr., Summa Veg. Scand.: 383. 1849.
     
   
   Anamorph: Trichoderma lacteum Bissett [sect. Hypocreanum Bissett], Canad. J. Bot. 69: 2367. 1991.
   Teleomorph: Stromata effuse, extensive, largest continuous stroma, 140 x 40 mm, smallest continuous stroma, 3 x 2 mm, many stromata not larger than 25 x 10 mm, solitary to confluent, varying in colour, sometimes white to light pastel, usually yellow to greyish yellow, sometimes orange-yellow to brownish yellow (4A4–4B4; 4A6–5A7; 3A4), KOH^+/–, reaction variable, usually very weak with slight browning of stroma; ostiolar canals visible at stroma surface, appearing light brown, giving rise to the greyish yellow overall appearance of the stroma. Stroma surface smooth; tissue immediately below the stroma surface and perithecia of compact to loose pseudoparenchymatous cells consisting of textura globulosa to t. angularis with some compact-vertically arranged hyphae. Perithecia completely immersed except for a slight protrusion of the ostiolar canals, 30 µm high (n = 7), generally widely spaced, compact in some regions, sometimes completely absent near the margins or regions of extensive stroma growth. Perithecia ellipsoidal, (263–)275–332(–360) µm long (including the length of the ostiolar canal, n = 22); width of perithecia near the base (measured from 3/4 total length of the perithecium) (99–)122–192(–214) µm (n = 22); length of ostiolar canal (42–)52–74(–82) µm; width of the ostiolar canal from the outer perithecial wall to the opposing internal perithecial wall (29–)34–54(–65) µm (n = 22); wall KOH^+/–, reaction variable, weak, usually brownish orange. Asci cylindrical, (65–)80–102 (–114) x (3.7–)6.2–7.2(–9.6) µm (n = 149); tip slightly thickened. Part-ascospores hyaline, thick-walled, minutely spinulose, dimorphic; distal part subglobose, sometimes subellipsoidal, (3.2–)4.1–5.2(–6.3) x (2.9–) 3.7–4.8(–5.7) µm, L/W ratio, (0.7–)0.99-1.2(–1.5) (n = 166); proximal part, ellipsoidal, sometimes subglobose to ovate, (3.3–)4.4–6(–7.1) x (2.5–)3.3–4.9(–6.3) µm, L/W ratio, (0.6–)1–1.6(–2.1) (n = 166).
   Anamorph: Colony covering the surface of a 100 mm diam Petri plate with PDA in 10 d, not producing concentric rings or radial rays of mycelium; a layer of cottony aerial mycelium along the entire Petri plate; no distinctive odour or pigmentation. Conidiophores irregularly branched on long hyphal elements, usually verticillium-like; phialides in whorls of 3–4(–5), sometimes solitary; phialides subulate, (8–)14–19 (–26) x (2.3–)2.9–3.7(–4.3) µm (n = 61); conidia variable in size, subglobose to subellipsoidal, (3–)3.3–5.8 (–10.8) x (3–)3.5–4.3(–5.1) µm (n = 61), some conidia asymmetric with a truncate base; chlamydospores cylindrical to subglobose when intercalary and bulbous when terminal, (5–)14-20(–22) x (9–)10–15(–18) µm (n = 16). After 10 d, conidia begin to swell and are more variable in size. Conidiophores not visible at 10 d on SNA or CMD. Conidiophores developed on CMD and SNA at 15–20 d, primarily near the point of inoculation. Optimal temperature for growth is 25 °C on all three media.
   Habitat: On leaf litter and rich soils, especially soil near decaying stumps, less commonly on decorticated wood, and moss-covered bark.
   Known distribution: Europe, Japan, and North America.
   Neotype (designated here): Belgium, Hestreux near Eupen: on leaf litter including pine needles, Oct. 1985, W. Gams 4031 (CBS 894.85).
   Other specimens examined: Austria, Steiermark, Lassnitzhöhe: vic. of Graz, elev. 480 m, on leaf litter and pine needles, 1995, H. Voglmayr (BPI 737697; culture G.J.S. 95-96); Niederösterreich, near Krems at Waldhof, beside Lake Egelsee, on leaf litter of Fagus/Picea, 28 Aug. 2000, W. Klofac, comm. W. Jaklitsch 1617 (BPI 748251; culture G.J.S. 00-161). England, Devon, Budleigh, on ground near base of Picea sitchensis stump, moss present, 22 Oct. 1989, J. Webster (BPI 1107145; culture G.J.S. 89-145). Germany, Pelmer Wald near Gerolstein, Oct. 1970, W. Gams (CBS 853.70). Netherlands, Baarn (Zandheuvelweg), Aug. 1973, W. Gams (CBS 708.73). Sweden, in mixed forest between Heina and Saara, on birch bark, 14 Aug. 1979, Lars Fagerström (BPI 744475); Småland, Femsjö, on wood, date not known, E. M. Fries (UPS, as Hypocrea lactea); Femsjö, on dicot leaf, date not known, E. M. Fries (UPS, as Sphaeria lactea), designated here as lectotype for H. lactea; Uppland, Arentuna parish, Storvreta, Eastern outskirts, on stump of Salix cinerea, 12 Sep. 1987, N. Lundqvist 17028 (UPS). USA, Georgia, Rabun Co., Chattahoochee Natl. Forest, along Little Creek Rd., near Double Bridge Creek, 34°59' N, 83°10' W, 17 Oct. 1990, A. Y. Rossman & G. J. Samuels, G.J.S. 90-137 (BPI 1107189); Kentucky, Laurel Co., Daniel Boone National Forest, elev. 350 m, Laurel River Lake Recreation Area, Cane Creek Wildlife Refuge, on decorticated wood, 27 Sep. 1995, G. J. Samuels (BPI 737759; culture G.J.S. 95-183); New York, Oswego County, Vandercamp Trail, on the moss Dreplanocladus uncinatus, 2 Oct. 1999, B.E. Overton, B.E.O. 99-29 (BPI); Warren County, Pack forest North of Warrensburg, on litter under white pine, 9 Sep. 1963, R.L. Gilbertson 4195 (UPS); North Carolina, Giles Co., off Bullpen Rd., 35°01' N, 83° 08' W, elev. 3000 ft., 14 Oct. 1990, Y. Doi, A.Y. Rossman & G.J. Samuels, G.J.S. 90-104 (BPI 1107172). Ohio: on Salix sp., 3 Oct. 1896, A. P. Morgan No. 46 (BPI 631486).
   Comments: Gams 4031 (herb. CBS, ex-neotype culture CBS 894.85) is designated here as the neotype specimen of H. citrina. This European specimen is well suited to serve as the neotype because it is yellow, effuse, and occurs on leaf litter just as Persoon described. Hypocrea citrina is similar in overall appearance to H. sulphurea, but H. sulphurea is not as effuse near the stroma margin, is intensely yellow throughout, and occurs on Exidia sp. on twigs, branches, and trunks.
   A lectotype is designated here for Hypocrea lactea [Sweden, Småland, Femsjö (as Sphaeria lactea, UPS herb. E.M. Fries)], assuming that this material was collected before 1822 (otherwise it would be neotype). Hypocrea lactea as lectotypified here is therefore a synonym of H. citrina. Doi (1972) illustrated stroma characters and part-ascospore measurements in his composite description of H. lactea that are not present in, or indicative of the lectotype specimen designated here for H. lactea s.s. Doi's (1979) annotation of Fries's historical specimen of H. lactea (cited in his specimens examined) listed part-ascospore measurements as follows: distal part-spores 3.5–4.5 x 3.4–3.9 µm, proximal part-spores 4.5–5.5 x 3.2–3.5 µm. These measurements differ substantially from the measurements given by Doi (1972) in his composite description of H. lactea; distal part-ascospores 4.5–5.2 x 4.5–4.8 µm, proximal part-spores, 5.5–7.1 x 4.2–4.4 µm. The Japanese specimens cited by Doi under the name H. lactea likely represent an undescribed species from Japan, especially given that Doi illustrated hyphal protrusions near the stroma surface, which are not indicative of H. lactea.
   
2. Hypocrea pulvinata Fuckel, Jahrb. Nassau. Ver. Naturk. 23–24: 185. 1870 ["1869"]. Figs 9, 10, 11.
   
   • = Hypocrea citrina * fungicola P. Karst., Mycol. Fenn. 2: 204. 1873.
     
   • Hypocrea karsteniana Niessl in Rehm. Ascomyceten, Hedwigia 22: 53 [Apr.] 1883.
     
   • Hypocrea fungicola (P. Karst.) Sacc., Syll. Fung. 2: 528 [13 June] 1883.
     
   • Protocrea fungicola (P. Karst) Lar.N. Vassiljeva, Nizshie Rasteniya, Griby i Mokhoobraznye Dal'nego Vostoka Rossii, Griby. Tom 4. Pirenomitsety i Lokuloaskomitsety (Sankt-Peterburg): 162. 1998.
     
   • = Hypocrea citrina (Fr.) Fr. var. citrina sensu Cunham, Mycologia 61: 318. 1969.
     
   
   Anamorph: Trichoderma sp. [sect. Hypocreanum].
   Teleomorph: Stromata effuse to subpulvinate, extensive, solitary to confluent, largest continuous stroma 60 x 20 mm, smallest continuous stroma 3 x 2 mm, varying in colour, orangish yellow to greyish orange, sometimes brownish yellow to golden-yellow, or brown-yellow (4B7–4B6; 5C7–5B7; 5C5); KOH⁺, reaction variable, sometimes weak, generally with stromata becoming orange or red; ostiolar openings visible from stroma surface. Warted hairs at surface of stromata, tissue immediately below the stroma surface formed of compact to loose pseudoparenchymatous cells, textura globulosa to t. angularis. Perithecia numerous and completely immersed in the stromata, generally widely spaced but compact near the centre, ellipsoidal, (189–) 206–246(–275) µm long (including length of ostiolar canal, n = 28); width of perithecia near the base (3/4 total length of perithecium) (96–)105–161(–186) µm (n = 28); length of ostiolar canal (36–)48–72(–82) µm; width of ostiolar canal (29–)43–63(–72) µm (n = 28); perithecial wall KOH⁺, reaction variable, usually brownish orange. Asci cylindrical, (44–)66–90(–115) x (2.8–)3.6–5.2(–7.9) µm (n = 321), tip slightly thickened. Part-ascospores hyaline, thin-walled, smooth to minutely spinulose, generally monomorphic and longer than broad; distal part ellipsoidal, sometimes subglobose, (2.8–)3.7–4.7(–6.0) x (2.3–)2.9–3.7(–4.6) µm, L/W ratio (0.8–)1.1–1.5(–2.2) (n = 335); proximal part, ellipsoidal, sometimes subglobose, (2.8–)3.8–5 (–6.7) x (2.3–)2.8-3.8(–4.7) µm, L/W ratio, (0.8–)1.1–1.6(–2.2) (n = 335).
   Anamorph: Colonies covering a 100 mm diam Petri plate with PDA in 10 d; producing concentric growth pattern with radial rays; small white cottony aggregates near the edge of the Petri dish composed of mycelium and acremonium-like conidiophores, no aerial conidiophores; stromata and perithecia produced near the edge of the plate; reddish brown pigment (9E8) produced near the centre of the plate and brown pigment (7E8) near the edge of the plate; no odour. Colonies covering a 100 mm diam Petri plate with SNA also in 10 d; forming a thin layer with small white cottony aggregates of conidiophores (at random places on the plate); no aerial mycelium and only a few scattered conidiophores with conidia; no pigment production, no odour. Conidiophores developed and started producing conidia on SNA at 10 d. Colonies covering a 100 mm diam Petri plate with CMD in 10 d; forming a thin layer with small white cottony aggregates near the edge of the Petri plate; trace of brown pigment (7E8) near the inoculum; no odour. Conidiophores developing on CMD, only a few producing conidia. Optimal temperature for growth on all 3 media is 25 °C.
   Measurements of micro-morphological characters were the same on all three media. A description based on measurements from all three media at 10 d follows: conidiophores variable in branching pattern and phialide number; phialides generally solitary, subulate, variable in length, (7–)24–44(–50) x (2.7–)3.6–4.6 (–5.3) µm (n = 32); below the phialides chlamydospore-like hyphal thickenings sometimes observed; conidia variable in size, many asymmetric, having a truncate base, subglobose to subellipsoidal, some bulb-shaped, (2.7–)4.1–7.1(–11) x (2.1–)3.3–5.6(–9) µm (n = 92). After 10 d conidia began to swell and were more variable in size.
   Habitat: Found on a variety of polypores, including Laetiporus sulphureus, Fomitopsis pinicola, Piptoporus betulinus, and Ganoderma spp.
   Known distribution: Europe, Japan, and North America.
   Lectotype (designated here): Germany, Geis, Hattenheimer Wald, on Polyporus sulphureus (= Laetiporus sulphureus), L. Fuckel, autumn, No. 876 (FH).
   Other specimens examined: Austria, Wachau District, Krems an der Donau, Mosingraben, North of Spitz in mixed forest, elev. 300 m, on undetermined polypore, 2 June 1996, I. Krisai-Greilhuber (BPI 744499; culture G.J.S. 96-142); Waldviertel, Virgin Forest of Dobra, on Fomitopsis pinicola, 10 Oct. 1995, H. Voglmayr (BPI 737803; culture G.J.S. 95-220). Finland, Locality unknown, Fungi Fennici Exs. 264, identified as H. citrina f. fungicola (K); Wasa (= Vassa), on polypore, May 1869, P.A. Karsten s.n. (H, herb. Karsten 4479, as H. citrina f. fungicola); Tammela, Mustiala, on polypore, 25 Sep. 1890, P. A. Karsten s.n. (H, herb. Karsten 4475); Satakunta Tyrvis (= Vammala, Tyrvää), on polypore, date unknown, P.A. Karsten s. n. (H, herb. Karsten 4478; syntype of H. citrina f. fungicola); Karelia Keretina, on Polyporus (Piptoporus) betulinus, 9 Aug. 1861, P.A. Karsten s.n. (H, herb. Karsten 4477, as H. citrina f. fungicola; syntype). Germany, Geis, on "Polyporus resinosus", substrate determined as Laetiporus sulphureus by E. Parmasto (Oct. 1999), Fuckel, Fungi Rhenani 2467 (K 61843); Saarland, Naturpark Saar-Hundsrück, vic. Ritzenberg, 90°40' N, 7°0' W, elev. 600–650 m, on Ganoderma sp., 12 Oct. 1998, G. J. Samuels & H. J. Schroers, G.J.S. 98-104 (BPI 746114); BPI 746115; culture G.J.S. 98-108. Great Britain, Yorks., Wrathe Woods, Pately Bridge, on Piptoporus betulinus, 25 Sep. 1955, C. Booth, IMI 61207 (BPI 631478). Luxembourg, Nassebesh, Hachy, on Piptoporus betulinus, 19 Sep. 1996, Vanhalle S. Mull 40055 (BPI 744722; culture G.J.S. 96-283). Poland, Silesia, Karlsbrunn, on Polyporus pinicola (= Fomitopsis pinicola, conf. E. Parmasto), Aug. 1882, von Niessl, Rehm Ascomyceten 678 (K, 61844). Sweden, on polypore, E.M. Fries, Scleromyceti Sueciae 31 (K). U.S.A., New Hampshire, Chocorua, on Fomitopsis pinicola, 16 July 1906, W.G. Farlow No. 34, identified as H. fungicola (K); New York, Franklin County, Little Green Pond, vic. Paul Smith College, on Ganoderma sp., 17 Aug. 1991, R. Lowen R.L. 886 (BPI 112828; culture G.J.S. 91-55A); Oneida County, on Fomitopsis pinicola, Barrie Overton, B.E.O. 99-34 (BPI); Utah, Beaver County, Tushar Mountains, Fishlake National Forest, on Fomitopsis pinicola, 31 Aug. 1994, C.T. Rogerson (BPI 737831; culture G.J.S. 94-20); Washington, Olympia National Park, Twin Creek, on Fomitopsis pinicola, 18 Oct. 1992, H. Burdsall, H.B. 14889 (BPI 802876; culture G.J.S. 92-127); Olympia National Park, Twin Creek, on Fomitopsis pinicola, 18 Oct. 1992, H. Burdsall, H.B. 14910 (BPI 802877; culture G.J.S. 92-128).
   
3. Hypocrea americana (Canham) Overton, stat. nov. MycoBank MB501053. Figs 12, 13, 14.
   
   • Hypocrea citrina (Fr.) Fr. var. americana Canham, Mycologia 61: 320. 1969.
     
   
   Anamorph: Trichoderma sp. [sect. Hypocreanum].
   Teleomorph: Stromata effuse to subpulvinate, solitary to confluent, extensive, largest continuous stroma 50 x 20 mm, smallest continuous stroma 3 x 2 mm, varying in colour, usually orange to brownish yellow, or orangish yellow to greyish yellow (5B8–5C8; 4B7–4B6); KOH⁺, reaction variable, generally with stromata becoming dark orange or red; perithecial necks or openings barely visible from stroma surface. Warted hairs at stroma surface; tissue immediately below the stroma surface formed of compact to loose pseudoparenchymatous cells composed of textura globulosa to t. angularis. Perithecia numerous and completely immersed in the stromata, generally widely spaced but compact near the centre of the stroma, ellipsoidal, (149–)167–199 (–213) µm long (including the length of the ostiolar canal, n = 15); width of perithecium near the base, measured from 3/4 the total length of the perithecium, (73–)80–106 (–119) µm (n = 15); length of ostiolar canal (44–)54–74 (–84) µm; width of ostiolar canal (41–)45–56(–62); wall KOH⁺, reaction variable, usually brownish orange or red. Asci cylindrical, (49–)59–79(–106) x (3.3–)4–5(–6.2) µm (n = 167), tip slightly thickened. Part-ascospores hyaline, thin-walled, smooth, monomorphic, usually subglobose to rhomboidal by mutual pressure, broader than long; distal part subglobose to rhomboidal, (1.7–) 2.7–3.7(–6.2) x (2.4–)3.2–4.2(–5.3) µm, L/W ratio (0.5–) 0.84–0.89(–1.5) (n = 212); proximal part subglobose to rhomboidal, (1.8–)2.6–3.8(–5.7) x (2.5–)3.2–4.2(–5.4), L/W ratio (0.5–)0.82–0.86(–1.4) (n = 212).
   Anamorph: Colonies covering a 100 mm diam Petri plate with PDA in 10 d; mycelium growing tightly pressed to the surface, appearing wet, no aerial mycelium; conidiophores produced in cottony tufts concentrated near the edge of the plate, tufts thicker and rising further from the agar surface than on CMD; stromata and perithecia emerging from several regions where conidiophores are found in the greatest number; brownish orange (7C8) to reddish brown (8E8) pigment diffusing in the medium, pigments visible within the hyphae. Colonies covering a 100 mm diam Petri plate with SNA in 10 d; mycelium thin, watery in appearance; conidiophores scattered, conidial production poor or absent. Colonies covering a 100 mm diam Petri plate with CMD in 10 d; mycelium tightly pressed to the surface, appearance watery, no aerial mycelium; conidiophores produced in cottony aggregates and tufts concentrated near the edge of the plate; brownish orange (7C8) to light brown pigment (7D8) diffusing into the medium. Optimal temperature for growth on all 3 media is 25°C.
   Measurements of micro-morphological characters were the same on all three media. A description based on measurements from all three media at 10 d follows: conidiophores variable in branching pattern and phialide number; phialides generally solitary, subulate, variable in length, (12–)20–40(–56) x (1.8–)3.3–5.1(–6.4) µm (n = 41), with chlamydospore-like thickenings just below the phialides on CMD and PDA; conidia variable in size and shape, typically subglobose to subellipsoidal, sometimes bulb-shaped, many asymmetric, having a truncate base, (3.2–)3.6–6.8(–10.2) x (2.6–)3.2–5.9 (–8.8) µm (n = 43). After 10 d conidia begin to swell, becoming larger and more variable in size.
   Habitat: On Fomitopsis pinicola and Piptoporus betulinus.
   Known distribution: North America.
   Holotype: USA, New York, Madison Co., Nelson Swamp, on F. pinicola, 9 Oct. 1949, C.T. Rogerson 3284 & Huttleston (NY ex CUP 38045, ex-type culture ATCC 18574).
   Other specimens examined: U.S.A., Arizona, Apache Co., White Mountains, 19.1 miles from forest boundary, on F. pinicola, 21 Aug. 1994, C.T. Rogerson (BPI 737849; culture G.J.S. 94-79); Graham Co., Pin leno Mt., Coronado National Forest, Rt. 366, 4.1 miles south of Riggs Lake, on F. pinicola, 15 Aug. 1996, C.T. Rogerson (BPI 744432; G.J.S. 96-191); New Hampshire, East Monroe, on Piptoporus betulinus, 22 Aug. 1937, C.L. Shear (BPI 631477); New Mexico, Upper Fernando de Taos, forest service map, T25n R15e S15, on Fomes sp., 15 Aug. 1992, R. Lowen 1003 (BPI 802838; culture G.J.S. 92-93); New York, Oswego Co., Vandercamp Trail, on F. pinicola, 2 Oct. 1999, B.E. Overton, B.E.O. 99-30 (BPI); also B.E.O. 99-32 and B.E.O. 99-33 (all BPI); Ulster County, Clinton dale Swamp, on P. betulinus, 11 Oct. 1963, S. J. Smith, R. Detroit, & C. T. Rogerson (NY; paratype).
   Comments: Hypocrea americana is easily distinguished from H. pulvinata by size and shape of the ascospores and degree of visibility of the ostiolar openings. The ascospores of H. americana are globose to rhomboidal with width greater than the length. In H. pulvinata, the ascospores are ellipsoidal, the length exceeding the width. The ostiolar openings in H. pulvinata are visible and the stroma surface has less of an overall rugulose appearance than in H. americana. In the specimen BPI 802838, both H. pulvinata and H. americana occur on the same host.
   
4. Hypocrea protopulvinata Yoshim. Doi, Bull. Natl. Sci. Mus. 15: 695. 1972. Figs 15, 16.
   Anamorph: Trichoderma sp. [sect. Hypocreanum].
   Teleomorph: Stromata effuse, sometimes subpulvinate, solitary to confluent, extensive, varying in colour, usually orangish yellow to light brown, young stromata white, becoming pale yellow where perithecia are developing (4B6–6D7; 4A3); KOH^+/–, reaction variable, weak, generally with stromata becoming darker orange; largest continuous stroma 22 x 15 mm, smallest continuous stroma 5 x 3 mm; ostiolar openings visible from stroma surface. Hyphal protrusions at stroma surface subglobose to pyriform, slightly roughened, not appearing warted like in H. pulvinata. Tissue immediately below the stroma surface variable; cells adjacent to ostiolar canals composed of rather thick-walled t. angularis; cells between perithecia of t. globulosa with some t. intricata intermixed; cells near the base of the perithecia composed of tightly packed irregular pseudoparenchymatous tissue in between t. globulosa, t. intricata, and t. angularis. Perithecia numerous near the stroma centre, scarce near the margin, generally widely spaced, subglobose to ellipsoidal, (151–)180–286(–310) µm long (including the length of the ostiolar canal, n = 11); width of perithecia near the base (99–)106–153(–176) µm, n = 11; length of ostiolar canal (33–)33–57(–68) µm; width of ostiolar canal (27–)30–46(–53) µm, n = 11; wall KOH^+/–, reaction variable, weak, usually brownish orange. Asci cylindrical, (45–)56–72(–76) x (3.7–)4–5(–5.4) µm (n = 52), tip slightly thickened. Part-ascospores hyaline, thin-walled, smooth, sometimes minutely spinulose, dimorphic; distal part subglobose, (3–)3.3–3.9(–4.3) x (2.6–)3–3.5(–4.2) µm, L/W ratio, (0.8–)0.99–1.2 (–1.5) (n = 52); proximal part ellipsoidal, sometimes subglobose, (3.2–)3.6–4.2(–4.8) x (2.4–)2.7–3.3(–3.8), L/W ratio (0.9–)1.1–1.5(–1.8) (n = 52).
   Anamorph: Colonies growing across the surface of a 100 mm diam Petri plate with PDA in 10 d; radial growth with radial rays, mycelium tightly pressed to the surface, sometimes with web-like sectors of rapidly growing mycelium, no aerial mycelium; conidiophores produced near the edge of the colonies in tufts; several large aggregates appear to be stroma initials; other large aggregates consist of clustered conidiophores; agar with a yellowish tint from the reverse, brown pigment (5D7) being produced near the edge of the plate and the agar plugs; sometimes odour (fruity of sour apples) produced when plates are tightly sealed; stromata produced 25–30 d following inoculation in sealed plates of the ex-type strain. Colonies growing across the surface of a 100 mm diam Petri plate with SNA in 10 d; mycelium web-like, random tufts of conidiophores arising from the agar surface, very few mature conidiophores; no pigment production. Colonies growing across the surface of a 100 mm diam Petri plate with CMD in 10 d; mycelium tightly pressed to the surface, appearing wet, no aerial mycelium; conidiophores produced near the edge of the plate, very rarely in tufts arising from the surface, very few mature conidiophores. Optimal temperature for growth on all 3 media is 25–30 °C with cultures growing 3 mm at 35 °C.
   Measurements of micro-morphological characters were the same on all three media. A description based on measurements from all three media at 10 d follows: conidiophores irregular, acremonium-like, variable in branching pattern and phialide number; phialides generally solitary, subulate, variable in length, (8–)20–38(–49) x (3–)3.7–4.7(–6) µm (n = 54); chlamydospore-like swellings below the phialides not as prominent on PDA and CMD; conidia variable in size and shape, generally subellipsoidal, some subglobose to subellipsoidal, most conidia have a well-developed truncate base, (4–)5.9–9.9(–16) x (2.8–)4–7(–9.5) µm (n = 76); chlamydospores not observed after 10 d. After 10 d conidia begin to swell and are more variable in size.
   Habitat: On Fomitopsis pinicola and unidentified polypores.
   Known distribution: Japan and North America.
   Isotype: Japan, Chiba Pref., Fudagou Mt., on unidentified polypore, 24 Oct. 1967, Y. Doi (NY ex TNS.D-365 = TNS-F-223421; ex-type culture CBS 739.83).
   Other specimens examined: U.S.A., Maryland, on unidentified polypore, autumn 2000, Kadri Põldmaa, K.P. 00-56 (BPI); New York, Cranberry Lake, Suckers Trail, on unidentified polypore, 15 Sep. 2001, B.E. Overton & D. Geiser, B.E.O. 00-01 (BPI); Ohio, on unidentified polypore, 11 Nov. 1883, A.P. Morgan No. 29 (BPI 631312; specimen degraded).
   Comments: Hypocrea protopulvinata is easily distinguished from H. pulvinata and H. americana because of its dimorphic ascospores and more effuse stromata with roughened, not warted, hyphal protuberances. The subglobose distal part-spores of H. protopulvinata are similar in size to the monomorphic subglobose part-spores found in H. americana: (3–)3.3–3.9(–4.3) x (2.6–)3–3.5(–4.2) µm in H. protopulvinata versus (1.7–)2.7–3.7(–6.2) x (2.4–)3.2–4.2(–5.3) µm in H. americana; In addition, the ellipsoidal proximal part-spores of H. protopulvinata are similar in size to the monomorphic ellipsoidal part-spores found in H. pulvinata: (3.2–)3.6–4.2(–4.8) x (2.4–)2.7–3.3(–3.8) in H. protopulvinata versus (2.8–)3.8–5(–6.7) x (2.3–)2.8–3.8(–4.7) in H. pulvinata. All three of these species have thin-walled part-ascospores that are smooth to minutely spinulose, characters not common in Hypocrea.
   
5. Hypocrea platypulvinata Yoshim. Doi, Bull. Natl. Sci. Mus. 15: 698. 1972.
   Anamorph: Trichoderma sp. [sect. Hypocreanum].
   Teleomorph: Stromata in aggregates or scattered, disciform, attached to decayed logs via a narrow base, deep yellow-ochre to ochre-brown, 2–6 mm diam, 0.8–1.6 mm thick, cells at surface of stromata roughened; perithecia ellipsoid, 200–240 µm in length; part-ascospores hyaline, warted, generally monomorphic, globose, 2.1–2.3 µm diam or sometimes obovate ellipsoid, 2.0–3.0 µm long (Doi 1972).
   Anamorph: Conidiophores acremonium-like; phialides subulate toward the apices, 15–35 x 1.5–2.0 µm; conidia hyaline, smooth, cylindrical to obovate-ellipsoid, with a truncate base, 4.0–14 x 2.3–5.1 µm; chlamydospore-like, thick-walled cells often produced, 2–12 x 6–8 µm in culture (Doi 1972).
   Habitat: On decayed logs of broad-leaved trees.
   Known distribution: Japan.
   Specimens examined: Type material not available for examination.
   
6. Hypocrea megalocitrina Yoshim. Doi, Bull. Natl. Sci. Mus. 15: 669. 1972. Figs 17, 18.
   Anamorph: Trichoderma sp. [sect. Hypocreanum].
   Teleomorph: Stromata effuse, solitary to confluent, extensive, largest continuous stroma, 40 x 30 mm, smallest continuous stroma 3 x 2 mm, varying in colour, usually light brown (6D7); KOH^–; surface of stromata appearing roughened due to ostiolar canals projecting from the stroma surface, which are covered by a thin layer of t. intricata. Tissue immediately below the stroma surface formed of compact to loose t. intricata and sporadic pseudoparenchymatous cells varying from textura globulosa to t. angularis. Perithecia numerous, generally widely spaced but compact near the centre of the stroma, ellipsoidal-elongate, (207–)240–320 (–345) µm high (including the length of the ostiolar canal, n = 24); width of perithecia near the base, measured from 3/4 the total length of each perithecium, (113–) 127–181(–208) µm (n = 24); length of ostiolar canal (54–)62–94(–130) µm (n = 24); width of ostiolar canal (54–)57–75(–84) (n = 24);