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Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, Getreidemarkt 9-166.5, A-1060 Vienna, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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The new species Hypocrea crystalligena (Hypocreales, Ascomycota, Fungi) is described as a holomorph and characterized based on an integrated phenotypic and phylogenetic approach, using teleomorph and anamorph morphologies, culture studies and analyses of phylogenetic markers including internal transcribed spacer 1 and 2 (ITS1 and 2), two last introns of the translation elongation factor 1-alpha encoding gene (tef1), and a portion of the rpb2 gene, encoding the second largest RNA polymerase subunit. Stromata of H. crystalligena show similarities with those of species from Trichoderma sect. Trichoderma but differ in several respects, including color, presence of white crystals on the surface and small ascospores. Colonies on CMD appear distinct, form colorless to white crystals on isolation, a yellowish to brown pigment and an anamorph with hyaline conidia exhibiting verticillium-like to gliocladium-like structural elements. ITS1 and 2 sequences exhibit all genus-specific features but also contain several unique hallmarks permitting development of a species-diagnostic barcode. Based on the analyses of partial rpb2 and tef1 sequences, H. crystalligena constitutes a separate evolutionary lineage with H. megalocitrina and H. psychrophila as its nearest neighbors. All these species form one phylogenetic clade with the H. pulvinata/H. citrina node.
Key words: Ascomycetes, DNA barcode, Hypocreales, ITS, phylogenetic markers, rpb2, sequence analysis, tef1, systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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, Dodd et al 2002, 2003, Chaverri and Samuels 2003, Druzhinina et al 2004, Lu et al 2004, Samuels et al 2006). The situation is most complex in section Trichoderma (Bissett 1991), which includes the type species of Hypocrea, H. rufa (Pers. : Fr.) Fr. In this section many species exhibit indistinguishable teleomorphs, macro- and microscopically, and even their anamorphs often show only subtle phenotypic variability (Samuels et al 2006). Therefore the application of molecular methods is indispensable for the identification and characterization of most species of Hypocrea.
The corresponding author recently has performed an extensive survey of the biodiversity of Hypocrea in central Europe, from which Hypocrea voglmayrii W.M. Jaklitsch, was described ( Jaklitsch et al 2005). In the course of this investigation specimens of a species that resembles representatives of sect. Trichoderma in the morphology of its teleomorph were collected frequently. However this species differs substantially from typical members of this section in a number of phenotypic characteristics of the teleomorph and anamorph and in its phylogenetic position. It therefore is described as a new species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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, GenBank accession numbers for ITS and tef1 sequences of Hypocrea crystalligena, and rpb2 gene sequences, are listed (TABLE I). Isolates listed as C.P.K. are those maintained in the collection of the institute of the corresponding author. Representative isolates have been deposited at the Centraalbureau voor Schimmel-cultures, Utrecht, The Netherlands (CBS). Specimens of Hypocrea crystalligena, including the type specimen, were deposited in the Herbarium of the Institute of Botany, University of Vienna, Austria (WU).
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. The cultures were maintained at 15 C on CMD agar plates and –80 C as mycelia and conidia scraped from agar plates.
Growth characterization.— –
Optimum temperature of growth and colony characteristics were determined. The strains were on CMD (cornmeal agar, Sigma, St Louis, Missouri, supplemented with 2% [w/v] D[+]-glucose-monohydrate). Agar plugs 0.5 cm diam were cut from the margin of the colonies and transferred to fresh medium, ca 1.5 mm from the edge of the 9 cm diam Petri dish with the mycelium facing down on the surface of the agar. CMD, PDA (potato-dextrose agar, Merck, Darmstadt, Germany) and low nutrient agar (SNA, Nirenberg 1976; pH adjusted to 5.5) were used. The tests were performed at 15 C (alternating 12 h nUV light and 12 h darkness), 25 C (alternating 12 h cool white fluorescent light and 12 h darkness) and 30 C and 35 C (both in darkness). The maximum colony radius was measured once daily at least 7 d or until the plates were covered with mycelium. The data given are ranges obtained from four experiments for all media and temperatures, using strains CBS 118980 and C.P.K. 944. The plates also were examined daily under the compound microscope at low magnification and culture characteristics were noted.
Morphological observations.— – Structures of the anamorph were examined, measured and photographed on a compound microscope from cultures grown on CMD and SNA at 25 C on the plates under low magnification and after mounting in 3% KOH. The size of chlamydospores was measured by examining colonies grown on CMD under the conditions described above with the 40x objective of a compound microscope.
Dry stromata of Hypocrea were rehydrated overnight with water vapor in a TLC chamber at RT, imbedded in Tissue-Tek O.C.T. Compound 4583 (Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) and sectioned at a thickness of 12–15 mm with a freezing microtome. Sections were measured and photographed in lactic acid, asci and ascospores in 3% KOH. Permanent preparations of the sections were made as described by Volkmann-Kohlmeyer and Kohlmeyer (1996). Measurements of asci, ascospores and anamorph characters are reported as maxima and minima in parentheses and the mean plus and minus the standard deviation of a number of measurements given in parentheses. Nomarski differential interference contrast (DIC) was used for observations and measurements. Colors were determined and cited according to Kornerup and Wanscher (1981).
DNA extraction, PCR amplifications and sequencing.— –
Mycelium for DNA extraction was grown on malt-extract agar covered by sterile cellophane. Genomic DNA was extracted with the Plant DNeasy Minikit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. A region of nuclear DNA, containing the ITS1 and 2 region, was amplified by PCR with the primer combinations SR6R and LR1 (White et al 1990), following the protocol of Kullnig-Gradinger et al (2002). A 1.26 kb fragment of the tef1 gene encoding translation elongation factor 1 alpha was amplified with the primer pair EF1728F and TEF1LLErev (Jaklitsch et al 2005). This fragment includes the fourth and fifth intron and a portion of the last large exon. A 0.9 kb fragment of RNA Polymerase II subunit B (rpb2) was amplified with the primer pair fRPB2-5f and fRPB2-7cr (Liu et al 1999). Template DNA (100 mL) was prepared directly from PCR products by purification with the QIAquick PCR Purification Kit (QIAGEN) and sequenced with a capillary sequencer.
Molecular phylogenetic analysis.— –
DNA sequences were aligned visually with Genedoc 2.6 (Nicholas and Nicholas 1997). The interleaved NEXUS file was formatted with PAUP*4.0b10 and manually formatted for the MrBayes v 3.0B4 program. The Bayesian approach to phylogenetic reconstructions (Rannala and Yang 1996, Yang and Rannala 1997) was implemented with MrBayes 3.0B4 (Huelsenbeck and Ronquist 2001). (For details see Jaklitsch et al 2005).
ITS1 and 2 sequences were used to develop the species-specific DNA barcode according to Druzhinina et al (2005). Its integration in the oligonucleotide barcode program led to the release of TrichOKEY v. 1.2 (www.isth.info/molkey). The uniqueness of ITS1 and 2 sequences of H. crystalligena and their similarity to other groups of the genus was visualized on a neighbor-joining cladogram based on the uncorrected "observed" P measure of genetic distance (see Salemi and Vandamme 2003 for references). The corresponding tree was constructed with PAUP* 4.0b10. The reliability of the obtained clades was tested by 500 bootstrap replications. Bootstrap values > 60% have been considered as significant. Sequence similarity analysis of tef1 sequences was performed with the aid of the program TrichoBLAST (Kopchinskiy et al 2005) available online at http://www.isth.info/tools/blast. The multiple sequence alignment file and phylogenetic trees have been deposited in the TreeBASE database (http://www.treebase.org/treebase/submit.html) under submission code SN 2726.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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, www.isth.info/molkey). The result indicated that these isolates might represent a new species of Hypocrea. To test this hypothesis and to find the position of this species on the phylogenetic tree of the genus, we sequenced a fragment of the rpb2 gene. The resulting sequences and vouchered sequences of Hypocrea/Trichoderma spp. from currently established clades were subjected to Bayesian phylogenetic inferences. The rpb2 phylogeny placed the new isolates with significant statistical support (P > 0.94) in the Megalocitrina clade with the Citrina clade as the nearest neighbor (FIG. 1)(see Chaverri and Samuels [2003] for clade definitions). The position of the new species in the Megalocitrina clade was confirmed by an analysis of ITS1 and 2 sequences. We visually examined ITS1 and 2 sequences of eight strains of the new species and detected two alleles, represented by 2 and 6 sequences, which differ only in one nucleotide in the ITS2 area. In addition to great similarity of ITS1 and 2 sequences of the new isolates with those of H. psychrophila and H. megalocitrina (see below), several unique and conserved areas were detected. In diagnostic regions of ITS1 and 2 (with a total length of ca. 250 base pairs), only 86% and 81% of the nucleotides are identical with those of H. psychrophila and H. megalocitrina, respectively (FIG. 2A). In total seven areas of ITS1 and 2 sequences might serve as a hallmark for the barcode design allowing a safe identification of this new species. In general all these point mutations are the basis for the relatively long branch leading to the new species in the ITS1 and 2 phylogram (FIG. 2B). The most diagnostic hallmark 1 (SHM1) area of ITS1 sequences of these isolates has a short sequence identical with SHM1 of H. psychrophila and H. megalocitrina. The relatively long stretch in the variable ITS2 area (SHM4) –3'–CA[G]C4–6G1–2C3T–5' is also unique for these three taxa. Moreover all three species show a number of other essential homologies in the SHM2 area (e.g. a 12–18 nt long "gap" that is not present in other ITS sequences of the genus). Therefore it also was possible to design a clade-specific barcode that differentiates between the Citrina and Megalocitrina clades.
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), the last exon of tef1 yielded poor phylogenetic resolution (data not shown) and therefore was not considered for further comparisons. The similarity searches performed for the most diagnostic fragment of tef1 sequence (i.e. the 4th intron) did not produce any significant alignment (E-value > 0.0001) with the TrichoBLAST database (Kopchinskiy et al 2005, www.isth.info), and only a short region (26 nt) homologous to the corresponding fragment of the tef1 sequence of H. psychrophila Hy 8 was detected in the NCBI BLAST database (GenBank AY737752
[GenBank]
, E-value = 0.001). The result suggests that tef1 sequences of isolates of the new Hypocrea are too divergent from those of any species from other clades to provide for significant alignments and phylogenetic analysis. Thus rpb2, ITS and tef1 data of the studied isolates show that the collections of the unknown Hypocrea form a distinct lineage. Also the morphology of the respective specimens and isolates, characterized below, justifies describing a new species.
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DESCRIPTION OF THE SPECIES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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Stromata in ligno et cortice, solitaria vel gregaria vel dense aggregata, pulvinata ad semiglobosa, 1–6(–8) x 0.5–2 mm; fusce ianthino-brunnea quando sicca, superficies saepe albe crystallina. Asci cylindrici, (68–)72–86(–98) x (3.5–)4.0–4.8(–5.2) µm. Ascosporae bicellulares, verruculosae, hyalinae, ad septum disarticulatae, pars distalis subglobosa, 2.5–3.2(–4.2) x 2.5–3.0 µm, pars proxima cuneata ad oblonga, (2.5–)3.0–3.7(–4.5) x 2.2–2.5(–2.7) µm.
Anamorphosis Trichoderma crystalligenum. Systema hypharum (colonia) (sub-)lobata, formans crystalla hyalina, conidiophora similia ad Trichoderma, Verticillium et Gliocladium in complexibus. Phialides lageniformes vel ampulliformes, in agaro "CMD" (3.7–)5.0–7.5(–10.0) x (2.0–)2.2–3.5(–4.5) µm. Conidia hyalina, ellipsoidea, glabra, (2.3–)2.5–3.4(–4.0) x (1.5–)1.7–2.2(–2.5) µm.
Stromata when fresh (FIG. 3a, b, d, f 
) ca. 1–6(–8) diam, 0.5–2 mm high, single, gregarious to densely aggregated in large numbers, starting as white mycelium, becoming compacted, flat to pulvinate, light (yellowish-, ochraceous- to light reddish-)brown, 4A4, 5–6B5–6 to 6–7D5–6, with white margin, finely tomentose to velutinous; later glabrous, distinctly and thickly pulvinate to semiglobose, less commonly discoid (particularly on bark), broadly attached, outline circular to irregular with often free and undulate margin, surface smooth to somewhat wrinkled, light (reddish-)brown 7CD7–8 to 8CD7–8, dull red, 8–9B4, but mostly dark brown to dark reddish brown 9DE7–8, 8E6–8, 9F5–8. A variable number of stromata usually with white crystalline powder in addition to white ascospore deposits. Ostioles typically macroscopically invisible or sometimes distinct and seen as convex light-colored dots surrounded by dark marginal rings.
Stromata when dry (FIG. 3e, h, i), (0.7–)1.5–3.5 (–4.7) mm long, (0.5–)1.2–3.0(–4.0) mm wide, (0.2–) 0.5–1.0(–1.7) mm high (n = 30), unchanged or slightly darker in 3% KOH, flatter than fresh, pulvinate to discoid, frequently surface finely but distinctly verrucous or tubercular to wrinkled; immature stromata velvety, short-haired, surface of mature stromata usually without hairs, but often with conspicuous water insoluble white powder, not visible after rehydration but immediately re-appearing after superficial drying; color of young, downy to velutinous stromata crème, pale ochraceous to orange, yellowish brown to rust, light to medium brown 5B4–6 – 5CD5–8 – 6CD4–6, 6E5–8, light reddish brown 7CD7–8, more rarely grayish brown 5CD3–4, 5EF2–4, 6EF4–5; older, mature stromata usually dark (violet-) brown 9D7–8, 9EF5–8 (to 10F5–8 or darker), less commonly dark (reddish-)brown 6EF6–8 to 7–8EF5– 8. Ostiolar area (ostioles plus dark margin) in face view, (24–)32–53(–63) µm (n = 30), inconspicuous and only visible under high magnification or conspicuous and semiglobose in some stromata, after addition of water ostioles generally appearing as light and hyaline dots on a bright reddish stroma surface (FIG. 3c). Ostioles in section (FIG. 3g, j) (42–)48–69 (–77) µm long, (20–)22–45(–69) µm at the opening (n = 20), central to eccentric, nearly cylindrical to conical, a palisade of narrow hyaline hyphae terminating in distinctly clavate to subglobose cells (FIG. 3g, k) to ca. 5 µm wide, plane or 3–16(–22) µm (n = 20) projecting beyond the surface. Perithecia (FIG. 3j) (169–)200–231(–243) µm high, (97–)112–159(–211) µm diam (n = 30), flask-shaped, laterally compressed by mutual pressure, often twice as high as broad, mostly broader in upper part, some (mainly in lateral regions of stromata) subglobose. Peridium 8–13(–15) µm thick at base, (14–)15–20 (–22) µm at apex (n = 15), yellowish brown to reddish brown, slightly paler than cortex, apically thickened and directly merging with (sub-)cortical layer, of refractive, glassy, laterally strongly compressed cells. Cortical layer (12–)15–22(–25) µm (n = 15) thick, reddish brown, orange-brown in lactic acid, of small angular thick-walled glassy compressed cells of indistinct outline, (3–)5–10(–11) µm (n = 15) diam in face view (FIG. 3l), 3–6(–7) µm (n = 15) diam in vertical section (FIG. 3g, j). Hairs rare on mature stromata, ca. 10–20 µm long, subhyaline to reddish brown, apically rounded. Subcortical tissue thin, loose textura intricata of thin-walled hyaline hyphae ca. 2–4.5 µm wide, parallel to the surface, branching and running vertical between perithecia, partly intermingled with subglobose to ellipsoidal hyaline cells. Subperithecial tissue (FIG. 3m) dense, hyaline textura angularis to epidermoidea of isodiametric subglobose to angular thin-walled cells directly below perithecia, downward gradually merging into oblong segments of thick, mainly vertically oriented hyphae, becoming smaller toward the base of the stroma, no distinct layers discernible; cells (4–)12–44(–63) x (3.5–)6.5–15.0(–18.5) µm (n = 30). Asci (FIG. 3p) (68–)72–86 (–98) x (3.5–)4.0–4.8(–5.2) µm, including a stipe (5–) 7–20(–28) µm (n = 30) long, emerging in fascicles, cylindrical, stipe often with an inflated base, no croziers seen; apical ring minute. Ascospores (FIG. 3n–p) hyaline, dimorphic with usually slight difference in shape and size, finely verruculose, disarticulating inside asci, distal part ascospore 2.5–3.2(–4.2) x 2.5–3.0 µm (n = 40, three specimens), subglobose, sometimes slightly tapered toward upper end, proximal part ascospore (2.5–)3.0–3.7(–4.5) x 2.2–2.5(–2.7) µm (n = 40, three specimens), wedge-shaped to oblong, broadly rounded at lower end; contact areas of both cells initially prolate.
Anamorph on natural substrate (WU 24044, FIG. 4j, k):
White hairy tufts on wood, partly in close association with stromata, in circular to oblong confluent patches up to 15 mm long, with long sterile elongations when immature. Main axes 3–5 µm wide, with short branches in right angles, loosely disposed, or pachybasium-like (i.e. with dense fascicles of 2–5[–6] phialides on thick 2-celled terminal branches), branched below terminal fascicle, bearing short 1-celled branches with dense fascicles of phialides, resulting in dense structures of conidiation. Phialides (4.2–)4.7–8.2(–12.0) x (2.5–)2.7–3.2(–3.5) µm, l/w = (1.5–)1.6–2.8(–4.5), (1.4–)2.0–2.7(–3.0) µm (n = 30) wide at base, originating from slightly, sometimes strongly thickened cells ca. 3–4(–7) µm thick, plump, short and thick ampulliform to lageniform, neck thin, thickest in the middle. Conidia (2.2–)2.5–3.2(–3.7) x 1.7–2.0(–2.5) µm, l/w = 1.2–1.5(–1.7) (n = 30), in heads and chains, hyaline, smooth, ellipsoidal to oval, with one or few guttules.
Cultures and anamorph.— –
On CMD (25 C) growth slow, colony radius 7–10 mm after 72 h, colony thin, covering plate after 2–6 wk, shape characteristic, usually in silky or iridescent fan-shaped lobes with or without radial gaps (FIG. 4a, b), mainly inside the agar, finely but distinctly zonate, hyphae narrow, central hyphae soon degenerating, remaining hyaline or more commonly developing a diffuse golden yellow to yellow-brownish (e.g. 4B4–7 to 5CD7–8) pigment unevenly distributed across the colony. From ca. 6 d characteristic colorless to white crystals (FIG. 4f) appearing on the surface, growing subsequently to 0.5–1.5 mm diam, sometimes appearing as oily globose drops inside the agar, after several transfers no crystals formed. No odor detected. Aerial hyphae sometimes appearing in loose, irregular, sterile or fertile tufts mostly in distant or lateral regions of the colony on plates entirely covered by mycelium; autolytic activity and coilings variable, usually inconspicuous. Conidiation in freshly isolated strains often after few days, effuse on short single conidiophores, after few transfers absent or scanty, 5–6 d, on scattered aerial hyphae or in white tufts or pustules formed mostly at the distant and lateral margin of the colony, often late (i.e. after 1–2 mo) and with slow development; formation of distant pustules usually strongly enhanced and accelerated by incubation at 15 C for ca. 7 d after incubation at 25 C until the plate is nearly covered by mycelium. Pustules (FIG 4e) 0.4–2 mm diam, mostly as loose tufts, or compact but with a powdery to finely granular surface, confluent to ca. 5 mm, often connected by long aerial hyphae, outline circular, oblong to somewhat irregular, with slow and asynchronous development. Structure determined after 12–13 d at 25 C plus 4–9 d at 15 C (CBS 118980) and 25 d at 25 C plus 23 d at 15 C (CBS 118979). Pustules formed on stipes 6–9 µm wide, sometimes moniliform, with variable branching, branching points sometimes with thickenings to 6 µm; main axes often radial and vertical, with short or long trees (FIG. 4g, i), stout, basal branches long, upper short, 1–3-celled, often paired and in right angles, (2–)3–5(–6) µm wide, becoming multiguttulate; dense structures formed as on the natural substratum. Phialides (3.7–)5.0–7.5(–10.0) x (2.0–) 2.2–3.5(–4.5) µm, l/w = (1.5–)1.7–3.0(–3.5), basal width (1.2–)1.6–2.2(–2.7) µm ( n = 60); lageniform, conical, pointed, to short and thick ampulliform, depending on the strain, thickest in or below middle, neck variable, up to ca. half length, single terminal phialides generally longer and narrower; usually in dense fascicles of 3–5 (–6), originating on often thickened cells 3.0–4.5(–5.7) µm wide; either straight and divergent, or strongly curved and parallel (gliocladium-like arrangement), both types on the same conidiophore. Conidiation scarce or dense, conidia formed in minute heads < 10 µm diam. Conidia (2.2–)2.5–3.5(–4.0) x (1.5–)1.7–2.2(–2.5) µm, l/w = (1.2–)1.3–1.7(–2.2) (n = 90), hyaline, smooth, ellipsoidal-oval, sometimes truncate, usually with few small guttules, finally densely agglutinated in heads or in chains. Chlamydospores (measured after 77 d) absent or rare in aerial hyphae, terminal and intercalary, hyaline, globose, pyriform to irregular, 5–19(–29) x (5–)6–15(–17) µm (n = 10). At 30 C either no growth or similar to 25 C, colony radius 3–10 mm after 72 h, conidiation at 4–5 d, formation of yellow pigment enhanced (3A3–5 to 4B6–7), crystals minute. At 15 C colony radius 5–6.5 mm after 72 h, conidiation from 12–18 d.
On PDA (25 C, FIG. 4c) colony radius 8–10 mm after 72 h, growth often stopping after ca. 1 wk, only rarely mycelium covering the whole plate in 4–5 wk, densely compacted, hyphae thin; thick whitish downy to cottony mat of aerial hyphae, no distinct zonation, no odor, conspicuous diffusing pigment on the reverse or sometimes on surface, starting around the inoculation plug, first yellow (2A4–5 to 3A4–8 to 4A6–7), gradually changing to dark yellowish brown (5CD6–8 to 6CD5–6), autolytic activity inconspicuous, with small brownish excretions from dying hyphae, no crystals formed. Conidiation effuse, whitish, mealy, spreading from the point of inoculation after 2–3 d as numerous densely disposed minute single squarrose or tree-like conidiophores and as fascicles of phialides on long aerial hyphae, phialides often paired. At 15 and 30 C colony radius after 72 h 1–4.5 mm and 0.7–7 mm, and conidiation from 6–7 and 3 d, respectively; diffusing pigment also formed, mycelium not reaching the margin of the plate at these temperatures.
On SNA (25 C, FIG. 4d) colony radius 5–9 mm after 72 h, culture similar to CMD but usually more irregular, often with wide gaps between mycelial lobes, individual lobes reaching margin of the plate within 3 wk, zonation indistinct, aerial hyphae scanty or forming loose sterile tufts of ca. 1.5 mm diam, autolytic excretions more frequent than on CMD, no chlamydospores detected, crystals lacking or inconspicuous, small, polymorphic, often as fine needles along hyphal walls, no pigment formed, chlamydospores rare. Conidiation more reliable than on CMD, effuse, starting after 3 d around the point of inoculation, macroscopically invisible, later formation of small white floccules, tufts or pustules in varying numbers, sizes and arrangements, mostly 0.1–1 mm diam, sometimes large pustules of up to 7 mm diam developing within 2–3(–8) wk in some strains. Structure (determined after 15–16 d) irregularly or often radially arranged main axes with 2–3 fold further branching, bearing mostly regular trees (FIG. 4h) with right-angled and often paired branching. Branches long, with one additional branching in lower parts of trees, shorter and 1–2 celled in apical parts of the trees, paired or not, bearing dense terminal fascicles of phialides. Phialides (FIG. 4l) (4.7–)5.5–9.0(–13.2) x 2.2–2.7(–3.2) µm, l/w = (1.5–)2.0–3.5(–5.7), basal width (1.2–)1.5–2.0(–2.5) µm (n = 30), arising from cells 2–3 µm thick, single or in fascicles of 3–5(–8), narrow, straight or curved upward, thickest mostly below the middle, terminal ones in center of fascicle often longer than the others, divergent or parallel, the latter often inequilaterally thickened. Conidia (FIG. 4m) (2.5–)2.7–3.5(–4.0) x 1.8–2.0(–2.2) µm, l/w = (1.2–)1.5–1.7(–2.0) µm (n = 30), hyaline, ellipsoidal to oblong or tapered toward one end, smooth, abscission scar sometimes distinct. At 15 and 30 C colony radius 2–7 mm after 72 h, conidiation at 8–20 d and 3 d, respectively.
Habit and habitat. – Stromata gregarious to aggregated in large groups, on wood and bark of dead and (medium to) usually well rotted branches of various deciduous trees such as Alnus glutinosa, A. incana, Carpinus betulus, Cornus sanguinea, Corylus avellana, Fagus sylvatica, Quercus petraea, Tilia cordata, on the ground in warm and dry forests and shrubs.
Known distribution: Europe (Austria, Germany, Sweden).
Holotype. – AUSTRIA. STEIERMARK: Weiz, Laßnitzthal, from Arboretum Gundl across the main road, MTB 8959/2, N47°04'17'' E15°38'38'', 420 m s.m., branch of Carpinus betulus, 4–5 cm thick, on the ground, 8 Aug 2003, W. Jaklitsch & H. Voglmayr, WJ 2325 (WU 24041, ex-type culture CBS 118980 = C.P.K. 1600). Holotype of Trichoderma crystalligenum isolated from WU 24041 and deposited as a dry culture together with the holotype of H. crystalligena as WU 24041a.
Selected specimens (from among 21 collections). AUSTRIA. KARNTEN: Klagenfurt Land, St Margareten, Gupf, close to the inn Schuschnig, MTB 9452/4, N46°32'48'' E14°26'57'', 800 m s.m., on base of partly decorticated branch of Cornus sanguinea, 4 cm thick, on the ground in leaf litter, soc. unidentified Corticiaceae, 29 Oct 2005, H. Voglmayr & W. Jaklitsch, WJ 2876 (WU 24060, isolate C.P.K. 2136). NIEDEROSTERREICH: Krems, Krumau, virgin forest at south side of the Dobra-Sperre, MTB 7458/1, N48°35'16'' E15°24'00'', 480 m s.m., on branch of Fagus sylvatica, 3.5–4 cm thick, and partly on old Diatrypella cf. verruciformis, on the ground in leaf litter, soc. old Hypoxylon fragiforme, 28 Sep 2003, W. Jaklitsch, WJ 2433 (WU 24045, isolate C.P.K. 980); Hollabrunn, Hardegg, Semmelfeld, forest between Niederfladnitz and Merkersdorf, MTB 7161/3, N48°48'49'' E15°52'43'', 450 m s.m., partly decorticated branch of Quercus petraea, 5–6 cm thick, on the ground in leaf litter, 21 Jul 2004, H. Voglmayr & W. Jaklitsch, WJ 2532, (WU 24048, isolate C.P.K. 1615); Krems an der Donau, Egelsee, close to Forsthaus Waldhof, MTB 7559/3, N48°25'55'' E15°33'23'', 420 m s.m., branch of Quercus petraea, 2 cm thick, ground, 24.09.2005, H. Voglmayr, WJ 2859 (WU 24059). OBEROSTERREICH: Schärding, St Willibald, Großer Salletwald, MTB 7648/3, N48°20'57'' E13°42'22'', 660 m s.m., corticated branch of Fagus sylvatica on the ground, soc. old Corticiaceae, 26 Oct 2005, H. Voglmayr, WJ 2866 (WU 24061); Melk, Leiben, Weitental, at Hofmühle, MTB 7757/2, N48°14'51'' E15°17'23'', 270 m s.m., partly decorticated branch of Fagus sylvatica, 6 cm thick, bare ground, soc. Tubeufia cerea (on ?Diatrype decorticata, Lasiosphaeria hirsuta, Hypoxylon cohaerens, Lopadostoma turgidum, Orbilia inflatula, Corticiaceae, 25 Jul 2004, H. Voglmayr & W. Jaklitsch, WJ 2539 (WU 24049, isolate C.P.K. 1910). Melk, Sankt Leonhard am Forst, grove ca. 2 km before Großweichselbach toward Melk at the right roadside, MTB 7857/2, N48°09'42'' E15°17'36'', 285 m s.m., partly decorticated branch of Quercus petraea, 3.5–4 cm thick, on the ground in leaf litter, soc. old Diatrypella quercina, Phellinus ferruginosus, 30 Sep 2004, W. Jaklitsch, WJ 2748 (WU 24056, isolate CBS 118979 = C.P.K. 1917). Wien-Umgebung, Mauerbach, right side of the walking path from the cemetery, MTB 7763/1, N48°15'19'' E16°10'13'', 330 m s.m., cut decorticated wedge-shaped part of a log of Carpinus betulus on moist ground in leaf litter, soc. Steccherinum ochraceum, 23.07.2005, W. Jaklitsch, WJ 2820 (WU 24057, isolate C.P.K. 2134). Wien Umgebung, Pressbaum, Rekawinkel, forest path south of the train station, MTB 7862/1, N48°10'47'' E16°02'03'', 360 m s.m., corticated branch of Alnus glutinosa, 5 cm thick, on ground in pile of twigs, holomorph, soc. myxomycete, old ?Diatrypella, 18 Oct 2003, H. Voglmayr & W. Jaklitsch, WJ 2476 (WU 24047, isolate C.P.K. 2133). STEIERMARK: Graz-Umgebung, Mariatrost, Wenisbucher Straße, MTB 8858/4, N47°06'40'' E15°29'11'', 470 m s.m., branch of large dead tree of Fagus sylvatica, 4–4.5 cm thick, on the ground, 20 Aug 2004, W. Jaklitsch, WJ 2611 (WU 24054, isolate C.P.K. 1915). TIROL: Innsbruck-Land, Ampass, Ampasser Hügel, MTB 8734/2, N47°15'31'' E11°27'16'', 720 m s.m., decorticated branch of Alnus incana, 1.5–2.5 cm thick, on ground between moss, moist; holomorph, soc. Nemania serpens, Stereum subtomentosum, moss, 2 Sep 2003, U. Peintner & W. Jaklitsch, WJ 2354 (WU 24043, isolate C.P.K. 944). VORARLBERG: Feldkirch, Rankweil, behind the hospital Valduna, MTB 8723/2, N47°15'40'' E09°39'00'', 510 m s.m., decorticated branch of Fagus sylvatica, 4.5 cm thick, on the ground in leaf litter, soc. Steccherinum ochraceum, 31.08.2004, H. Voglmayr & W. Jaklitsch, WJ 2644 (WU 24055, isolate C.P.K. 1916). GERMANY. BAVARIA: Unterfranken, Landkreis Haßberge, Haßfurt, close to Mariaburghausen, left roadside heading from Knetzgau to Haßfurt, MTB 5929/3, N50°00'33'' E10°31'10'', 280 m s.m., partly decorticated branch of Carpinus betulus, 5.5 cm thick, on the ground in leaf litter, holomorph, soc. Phlebiella vaga, 4 Aug 2004, H. Voglmayr & W. Jaklitsch, WJ 2568 (WU 24050, isolate C.P.K. 1911); same locality, on corticated branches of Tilia cordata, 4 Aug 2004, H. Voglmayr & W. Jaklitsch, WJ 2570 (WU 24052, isolate C.P.K. 1913). Starnberg, Tutzing, Erling, Goaßlweide near Hartschimmelhof, MTB 8033/3, N47°56'33'' E11°11'00'', 730 m s.m., on partly decorticated branch of Fagus sylvatica, 4.5 cm thick, on the ground in grass, soc. Bertia moriformis, Neobarya parasitica, Tomentella sp., 7 Aug 2004, W. Jaklitsch, H. Voglmayr, P. Karasch & E. Garnweidner, WJ 2581 (WU 24053, isolate C.P.K. 1914). SWEDEN. UPPSALA LAN: Sunnersta, forest opposite the virgin forest Vardsätra Naturpark across the road, MTB 3871/2, N59°47'23''E17°37'53'', 15 m s.m., corticated branch of Corylus avellana, 2.5–3 cm thick, on bare, very moist soil, soc. Stereum rugosum, Diatrypella verruciformis, 8 Oct 2003, W. Jaklitsch, WJ 2451 (WU 24046, isolate C.P.K. 1604).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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The ecology of H. crystalligena is difficult to circumscribe, but this species shows a marked preference for dry forests and shrubs and well rotted wood, not usually inhabited by other hyaline-spored teleomorphs of the genus Hypocrea.
It is not clear why this common species has not been described before. For reasons cited above it probably has been collected as H. rufa (sensu lato), although the sizes of ascospores are substantially different. One reason might be that the specific limits of H. rufa are not defined adequately and that type material of H. rufa was not examined by potential collectors. Also stromata of H. rufa usually are accompanied by the anamorph (unpublished observations). Therefore, in the absence of (green) Trichoderma viride on the natural substrate, it can be assumed that teleomorphs with reddish-brown stromata are in fact not H. rufa. On the other hand the teleomorph of H. crystalligena is similar to species of the xylariaceous genus Hypoxylon, particularly H. fuscum, due to the relatively large size and the peculiar color of dry stromata. Identification based on superficial field observations might have been the cause of either not collecting this fungus or depositing specimens in herbaria under different names like H. rufa or Hypoxylon fuscum.
Cultures of H. crystalligena on CMD and SNA are characteristic due to slow growth and the formation of finely zonate radial lobes. The formation of a diffuse yellowish to brownish pigment varies among the strains. Initially after isolation all strains except C.P.K. 1604 formed a water-insoluble substance, appearing as colorless to whitish crystals of varying size on CMD, but after several transfers this ability was lost. Crystals also are formed by other species (e.g. H. voglmayrii, H. aureoviridis; see Jaklitsch et al 2005, Rifai and Webster 1966), but they are usually yellow and exhibit different, mostly needle-like shapes.
The anamorph of H. crystalligena produces hyaline conidia, white in mass, a feature not expected with stromata, morphology of which suggests affiliation with sect. Trichoderma. This white anamorph occurs also in nature together with the teleomorph, which is rare in Hypocrea, where stromata often are accompanied by green Trichoderma.
The thick and stout conidiophores as well as densely disposed, and in some strains, short and broad phialides recall sect. Pachybasium sensu Bissett (1991) and H. neorufa Samuels, Dodd & Lieckfeldt in sect. Trichoderma. The latter also forms similar dark brown stromata. However conidia in H. neorufa (Dodd et al 2002) are green and conidiophores of T. crystalligenum are unusual in the way that they combine trichoderma- (rebranching, right angles), verticillium-(divergent phialides inclined upward in whorls) and gliocladium- (parallel phialides in dense clusters)like morphology on the same conidiophore. This confirms that verticillium- and gliocladium-like arrangements and structures of conidiation are not unusual extremes within Trichoderma but are firmly integrated in the morphologically variable genus. Also Chaverri and Samuels (2003) reported different types of conidiation within species such as H. catoptron Berk. & Broome or H. crassa Chaverri & Samuels, but these occur as gliocladium-like or verticillium-like conidiophores on aerial hyphae separate from pachybasium-like conidiophores in pustules and therefore have been identified as synanamorphs.
The phylogenetic position of H. crystalligena in the Megalocitrina clade was shown by the concordant results obtained from three main phylogenetic markers used in Hypocrea/Trichoderma. The position of the new species on the tree of the whole genus was detected by analysis of the coding region of the rpb2 gene. The species closest to H. crystalligena are H. megalocitrina and H. psychrophila. These species are morphologically different from each other and from H. crystalligena. H. psychrophila shows some resemblance with H. crystalligena in its gliocladium-like anamorph with hyaline conidia, but it has substantially larger ascospores, forms semiglobose golden yellow to orange stromata on twigs of Rhododendron ferrugineum in (sub)alpine habitats and does not grow at 25 C (Müller et al 1972, and unpublished observations). H. megalocitrina on the other hand, deviates even more drastically from H. crystalligena in having thin, widely effused yellow to light brown stromata containing substantially larger and nodulose to coarsely tubercular ascospores, and in its verticillium-like anamorph with long, slender and subulate phialides typical for the former sect. Hypocreanum (Doi 1972, Overton et al 2006).
Thus, although belonging to the same phylogenetic clade, H. crystalligena is morphologically distinct from these species. This can be explained by the relatively long branch leading from the clade node to the new species that suggests an extended period of independent evolution of H. crystalligena. To prove this hypothesis we analyzed two fast evolving phylogenetic markers, ITS1 and 2 and tef1 intron areas, which usually are selected to detect the most adequate genetic distance between the target species and its closest relatives on the intercladal or intersectional levels in this genus. ITS1 and 2 sequences are known to be useful for identification of species in Hypocrea/ Trichoderma (Druzhinina et al 2005). The species-specific hallmarks of ITS1 and 2 sequences for H. crystalligena provided sufficient information to configure hallmarks specific for the whole Megalocitrina clade. The updated master alignment of sequences of these isolates and all other species of Hypocrea/ Trichoderma characterized by molecular data is available at www.isth.info/molkey.
We unfortunately could not perform a complete phylogenetic analysis of the last introns of the tef1 gene because only a limited number of such sequences is available for Trichoderma section Hypocreanum sensu Bissett (1991). At the same time a number of sequences comprising the last exon of tef1 have been available to us for related species in NCBI GenBank and from B. Overton (pers comm). Chaverri and Samuels (2003) showed that the topology of phylogenetic trees obtained by both neighbor joining and Bayesian analyses of tef1 exon sequences is inconsistent with results obtained with rpb2 sequences. In particular the Citrina clade is remote from the Megalocitrina clade in exon-based tef1 trees. The combined tef1 and rpb2 tree represents the rpb2 topology and thus shows clearly that the tef1 exon does not significantly contribute phylogenetic information to the combined tree. For this reason we decided not to use this fragment for phylogenetic analyses. Instead we tested tef1 intron sequences against the TrichoBLAST database and found that based on this fragment H. crystalligena (i) is significantly different from all known Hypocrea/ Trichoderma species and (ii) has H. psychrophila as the closest relative. tef1 intron sequences were not available for H. megalocitrina. Thus the results for three unlinked phylogenetic markers suggest that H. crystalligena is a new species in accordance with the genealogical concordance phylogenetic species recognition criterion (Taylor et al. 2000).
The fact that the Megalocitrina clade currently appears to be monophyletic could be an artifact caused by the small number of representatives. In the future if more species are detected in this part of the phylogenetic tree this group might be subdivided into several clades representing different morphological traits.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: jaklitsc{at}mail.zserv.tuwien.ac.at
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