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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 Wien, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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The holomorph of the new species Hypocrea voglmayrii (Hypocreales, Ascomycota, Fungi) is described by a combined approach, using morphology of the teleomorph, morphology of the anamorph, culture studies and phylogenetic analyses of ITS1 and 2, ech42 and rpb2 gene sequences. Its anamorph Trichoderma voglmayrii is described as a new anamorph species. Unlike most other species of Hypocrea the teleomorph of H. voglmayrii occurs on dry standing trunks and exhibits well defined black ostioles. Although exclusively collected at higher altitudes, this species grows at 35 C in culture. Hypocrea voglmayrii develops pale yellowish to greenish conidia, a yellowish pigment and a coconut-like odor on CMD. Phylogenetically, H. voglmayrii forms a distinct, isolated branch between the section Trichoderma and the H. pachybasioides clade but does not associate with any of these clades in different gene trees.
Key words: Ascomycetes, ech42, Hypocrea, Hypocreales, ITS, phylogenetic analysis, rpb2, systematics, tef1, Trichoderma voglmayrii
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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; Druzhinina et al 2004; Lu and Samuels 2003; Lu et al 2004). For example, many Hypocrea spp. collected worldwide show reddish to brown stromata in varying tints and contain asci with hyaline ascospores. Most mycologists identify such teleomorphs as Hypocrea rufa Pers. : Fr., the type species of Hypocrea. However true H. rufa is actually a rare species and most isolates with this teleomorph morphology likely represent other potentially new species. This underlines the necessity of combining phenetic examinations of the holomorph with molecular and physiological analyses for the description and characterization of new species of Hypocrea (cf. Kraus et al 2004). Most of the Hypocrea holomorphs (i.e. combined anamorph and teleomorph) known today have been described from Japan, South East Asia, Australasia, North and Latin America by Y. Doi and G. J. Samuels and coworkers (Doi 1966, 1968, 1972, 1973, 1975a, b, 1976, 1978; Samuels et al 1990; Chaverri et al 2001a, b; Dodd et al 2002, 2003; Druzhinina et al 2004; Lu et al 2004; Samuels et al 1998; Samuels and Lodge 1996; Seifert and Samuels 1997). In contrast, little is known about Hypocrea spp. from continental Europe. In a project designed to assess the biodiversity of Hypocrea in Central Europe, an apparently undescribed species with peculiar reddish stromata was collected. This species exhibits remarkable ecological, phenotypic and genotypic characteristics, which are described below.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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). 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 voglmayrii including the type specimen were deposited in the Herbarium of the Institute of Botany, University of Vienna (WU).
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Growth characterization.— –
Optimum temperature of growth and colony characteristics were determined. The strains were pregrown on CMD until they reached 55–65 mm diam. Agar plugs 0.5 cm diam were cut from the margin of the colonies and transferred to fresh medium, 1.0–1.5 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) with the pH adjusted to 5.5 were used. The tests were performed at 15 C (with alternating 12 h nUV light and 12 h darkness), 25 C (with alternating 12 h cool white fluorescent light and 12 h darkness) and 30 C and 35 C (both in darkness). For growth at 25 C the Petri dishes were sealed with Parafilm to avoid drying out of the agar caused by the ventilator of the cooling incubator (MIR 153, Sanyo, Gunma, Japan). The maximum colony radius was measured once daily for at least 7 d or until the plates were entirely covered with mycelium. The growth rate was calculated by linear regression of log t versus log r (t = time of incubation, r = radius measured from the edge of the agar plug), using only measurements from the phase where the logarithmic increase of the colony radius was linear over time. The data given are ranges obtained from six experiments for all media and temperatures.
In addition the plates also were examined daily under the compound microscope at low magnification and the time of first appearance of conidia, autolytic behavior of marginal hyphae, presence of chlamydospores, formation of pigments and odor and colony appearance were noted.
Morphological observations.— – Conidiation structures were examined, measured and photographed on a compound microscope from cultures grown 3–7 d on CMD at 25 C (see above) on the plates under low magnification and after mounting in tap water. These characters were measured: length of conidia, width of conidia, length of phialides, width of phialides at the base, width of phialides at the widest point. The size of chlamydospores was measured by examining colonies grown on CMD or on SNA under the conditions described above using the 40x objective of a compound microscope.
Dry stromata of Hypocrea were rehydrated briefly in 3% KOH, 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 µm with a freezing microtome. Permanent preparations of the sections were made as described by Volkmann-Kohlmeyer and Kohlmeyer (1996). These teleomorph characteristics were evaluated: diameter, height, color and shape of the stroma; texture of the surface of the stroma; perithecium shape, length and width; color, width of perithecium wall; length and diameter of ostioles; color and reaction of the stroma surface to 3% KOH; shape, thickness of the surface region, size and wall thickness of cells of the supraperithecial (between the perithecia and the surface region), subperithecial and basal regions of the stroma; ascus length and width; distal and proximal part-ascospore length and width. 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) and bright field microscopy were 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 PDA covered by sterile cellophane. Genomic DNA was extracted with the Plant DNeasy Minikit (QIAgen GmbH, Hilden, Germany) according to the manufacturer’s instructions, using approximately 150 ± 50 mg fresh mycelium. 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 using the primer pair EF1728F (Chaverri and Samuels 2003) and TEF1LLErev (5'-AAC TTG CAG GCA ATG TGG-3'). This fragment includes the fourth and the fifth intron and a significant portion of the last large exon. A 0.7 kb fragment of RNA Polymerase II subunit B (rpb2) was amplified using the primer pair fRPB2-5f and fRPB2-7cr (Liu et al 1999). A 0.5 kb fragment of the endochitinase 42 gene was amplified using the primer pair ech42-1a and ech42-2a as described by Lieckfeldt et al (2000).
Template DNA (100 µL) was directly prepared from PCR products by purification with the QIAquick PCR Purification Kit (QIAgen) and sequenced with a capillary sequencer ABI 3730 XL (Applied Biosystems, Foster City, California). All sequences used in this study are listed (TABLE I
) by their GenBank accession numbers.
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). The model of evolution and prior settings for individual loci were used as estimated by Druzhinina et al (2004) for different taxa of Hypocrea/Trichoderma. Metropolis-coupled Markov chain Monte Carlo (MCMCMC) sampling was performed with four incrementally heated chains that were simultaneously run for 1 000 000 and 3 000 000 generations. To check for potentially poor mixing of MCMCMC, each analysis was repeated three times. The convergence of MCMCMC was monitored by examining the value of the marginal likelihood through generations. Convergence of substitution rate and rate heterogeneity model parameters also were checked. Bayesian posterior probabilities (PP) were obtained from the 50% majority rule consensus of trees sampled every 100 generations after removing the 500 first trees using the "burn-in" command. According to the protocol of Leache and Reeder (2002), PP values lower than 0.95 were not considered significant while values below 0.9 are not shown on phylograms and radial trees.
The MSA file and phylogenetic trees have been deposited in the TreeBASE database (http://www.treebase.org/treebase/submit.html) under the submission code SN 2355. Genetic distance was computed in PAUP*4.0b10 under the GTR + G model.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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, www.isth.info) indicated that they probably represent a new species. All five markers specific for the genus Hypocrea/Trichoderma were found while nucleotide stretches in hallmark areas characteristic for species and/or clades were unique. Therefore it is unlikely that these isolates represent a member of any of the sections or clades known in the genus (Chaverri and Samuels 2003, Druzhinina et al 2004). To prove this hypothesis and to find an exact position of the potentially new species in the phylogenetic tree of the genus we applied the genealogical concordance phylogenetic species recognition (GCPSR) concept (Taylor et al 2000). To do this we amplified and sequenced fragments from three additional phylogenetic markers (i.e. a fragment of the RNA polymerase II subunit B (rpb2) gene, a portion of the last exon of the endochitinase 42 (ech42) encoding gene and a 1260 nt long fragment of the translation elongation factor 1-alpha (tef1) gene which includes sequences of the forth and fifth introns and a portion of the last large exon). The corresponding sequences and vouchered sequences of Hypocrea/Trichoderma spp. from currently established sections and clades were subjected to phylogenetic analyses. Following Chaverri and Samuels (2003) we selected the rpb2 phylogenetic marker to find the exact position of the potentially new species in the generic tree. As shown (FIG. 1) Hypocrea voglmayrii was placed with significant statistical support (P > 0.95) between the species of section Trichoderma (Rufa Clade XII and Pachybasioides "A" Clade XIII, Druzhinina et al 2005) and Pachybasioides Clade IX (Lu et al 2004, Druzhinina et al 2005). This position was confirmed fully by individual ITS1 and 2 and ech42 trees that include only species from neighboring groups (FIGS. 2, 3). Thus our analyses show that the isolates fulfill the criteria of the GCPSR concept, and therefore they represent a new species.
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), confirming that the species under description is neither a member of section Trichoderma nor the Pachybasioides Clade IX.
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DESCRIPTION OF THE SPECIES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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, 5 Anamorph: Trichoderma voglmayrii W.M. Jaklitsch, sp.nov.
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Stromata in ligno Alni alnobetulae et rarius A. incanae in altitudine 1100–1400 m s.m., solitaria vel gregaria, erum-pentia per corticem, discoidea, basi sterili presente an non, (1–)1.3–3(–5.1) x (0.7–)1–2.2(–3.2) x (0.3–)0.3–0.7(–1) mm, lateritia ad ianthino-purpurea, ostiolo fusco ad atro, leviter prominente. Asci cylindrici, (60–)67–85(–94) x (3.3–)3.8–4.6(–5.4) µm. Ascosporae bicellulares, verruculosae, hyalinae, ad septum disarticulatae, pars distalis subglobosa ad late ellipsoidea (3–)3.3–3.9(–4.3) x (2.5–)2.8–3.3(–3.5) µm, pars proxima oblonga ad quasi ellipsoidea, saepe leviter attenuata ad basim (3.9–)4.1–4.8(–5.3) x (2–) 2.3–2.9(–3.2) µm.
Anamorphosis Trichoderma voglmayrii. – Incrementum in agaro "CMD" ad 35 C post 7 d. Phialides lageniformes vel rarius ampulliformes, (6.5–)7.1–11.3(–16.3) x (2.7–)3.1–4.1(–4.7) µm. Conidia hyalina, pallide viridia in acervis, ovoidea usque ad subglobosa, glabra, (3.1–)3.4–4.5(–5.2) x (2.2–)2.6–3.4(–4.2) µm.
Stromata (FIG. 4.1) solitary or in small cespitose groups, breaking through fissures in the bark with the sterile and light-colored margin surrounded by the epidermis of the host (FIG. 4.2), or on wood. Size: length x width x height = (1–)1.3–3(–5.1) x (0.7–) 1–2.2(–3.2) x (0.3–)0.3–0.7(–1) mm (n = 30) in the dry state. Stromata circular to elongate in outline, discoid, with a nearly plane surface and free regular to undulate sharp margins, and often a short sterile constricted stipe (FIG. 4.4) or/and a radiating white basal mycelium. Stromata sometimes annular with the center sunken appearing hollow. Fresh stromata from brick red (7CD6-7) to pinkish or grayish red (9C5-6), and of dry stromata, as usually collected, grayish red or brownish red (9C5-6) to Cuba red (9E7-8) or violet brown (10E7-8), with the margin concolorous or, like the stipe, whitish to yellowish to pale orange. Slight differences between fresh and dry stromata except for wrinkles and fine fissures in dry stromata sometimes forming stellate structures around the ostioles. Ostioles densely arranged with openings visible as conspicuous dark brown to black, well-defined, slightly raised dots (FIGS. 4.3, 4.5) in dry stromata, with an outer basal diameter of (26–)32–49(–55) µm (n = 30), projecting beyond the surface of the stroma by (12–)18–36(–55) µm. Stroma surface discolored dark reddish-brown to nearly black in 3% KOH, consisting of thick-walled compressed angular cells of indistinct outline ca. 3–7 µm diam, and in sections appearing as a thin compact amorphous orange layer, (6–)8–16(–22) µm (n = 20) thick. Cells between this layer and the perithecia (FIG. 4.8) subglobose to angular, (3–)5.4–10.7(–12.4) x (2.5–)4.4–8.3(–9.9) (n = 30), hyaline, but orange to reddish directly below the surface layer. Thickness of the entire tissue above the perithecia (30–)41–67(–77) (n = 20). The subperithecial tissue (FIG. 4.9) of hyphae with strongly constricted septa and hyaline, refractive, elongate to, directly below perithecia, subglobose cells of (7–)12–38(–57) x (6.5–)8.5–17.5(–24) µm (n = 30) with walls approximately 1–2 µm thick. The basal tissue (FIG. 4.10) a hyaline, loose textura intricata of (2.3–)2.7–5.2(–7.5) µm wide hyphae (n = 30). Perithecia (FIG. 1.6) entirely immersed in the upper part of the stroma, ellipsoidal or broadly cylindrical to flask-shaped, height x diam = (186–) 214–258(–283) x (81–)100–167(–228) (n = 31), laterally compressed and approximately by 40% higher than wide on average. Peridia of adjacent perithecia usually in close contact laterally, of refractive, narrow and strongly compressed orange cells. Thickness of the peridium at the base of the perithecia (12–)13–18(–20) µm (n = 20) and at half of their height (5–)6–12(–16) µm (n = 20). Ostioles appearing apically as a palisade of elongate, narrow, strongly compressed orange to reddish cells (FIG. 4.7), resembling those of the peridium; ostioles conical with a length of (50–)60–89(–99) µm (n = 20) and an internal basal diameter of (22–)26–44(–50) µm (n = 20). Asci cylindrical, (60–)67–85(–94) x (3.3–)3.8–4.6(–5.4) µm (n = 30), with a flat subapical ring (FIG. 4.11). Part-ascospores hyaline, uniseriate, finely spinulose, dimorphic. Distal part-ascospores subglobose to ovoid or broadly ellipsoidal, (3–)3.3–3.9(–4.3) x (2.5–)2.8–3.3(–3.5) µm (n = 30). Proximal part-ascospores oblong to nearly ellipsoidal, often slightly attenuated toward the base, (3.9–)4.1–4.8(–5.3) x (2–)2.3–2.9(–3.2) µm (n = 30) (FIG. 4.11).
Cultures and anamorph.— –
Some growth characteristics recorded are provided (TABLE III).
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). After 14 d the yellow pigment sometimes occurring as long needle-shaped crystals on agar surface. Conidiation beginning after 2–3 d as solitary phialides in rows on surface hyphae or as fascicles of 3–5(–6) phialides on very short erect slightly branched conidiophores, effuse between the proximal margin and 2/3 of the growth radius. Within 4–9 d visible as inconspicuous and ill-defined powdery whitish granules mainly in the distant third of the plate, subsequently spreading back nearly across the whole plate. Granules 0.1–0.5(–1.0) mm diam, made up of single or few coalescing conidiophores, bearing conidia in heads of up to 60 µm diam and later sometimes in chains, appearing white to pale yellow. Macroscopically, the color of the granules difficult to determine due to the yellow discoloration of the agar. At the same time conidiation also occurring within the agar (FIG. 5.9). The structure of typical conidiophores determined at 5–7 d. Conidiophores consisting of a main axis up to 200 µm long, simple, forked or with up to 5(–7) nearly pyramidal or tree-like primary branches (FIGS. 5.4, 5.5), later covered by conidial heads, becoming more irregular. Primary branches (FIG. 5.11–14) with short secondary branches, mostly paired or substituted by paired or unpaired phialides (FIG. 5.11, 5.14). Secondary branches short, up to 35 µm long, in the lowest positions at right angles, higher branches including phialides curved upwards, bearing phialides singly, in fascicles of 3–5(–7), or rarely on extremely short tertiary branches, each bearing fascicles of 3–5 phialides, resulting in dense complex and nontransparent structures. Sparse conidial development on long aerial hyphae. Metulae generally absent. Phialides (FIG. 5.13–15) (5.5–)7.2–12.2(–16.5) x (2.7–)3.2–4.1(–4.7) µm, width at the base (1.5–)2.0–3.0(–4.0) (n = 60), terminal phialides often longer than the flanking ones in the fascicle, lageniform to narrowly subcylindrical and sometimes sinuous, less commonly ampulliform or sometimes ventricose, inaequilateral and with a long neck, thickest point median, sub- or supramedian, often multiguttulate. Conidia (FIG. 5.16) hyaline, yellowish in mass, oval to oblong, often attenuated toward one end, smooth, with varying numbers of guttules often in a group at each end, (3.0–)3.5–6.5(–10.5) x (2.2–) 2.6–3.3(–4.2), l/w= (1.1–)1.2–2.2(–3.4) (n = 75). Chlamydospores (FIG. 5.10, measured after 14 d) first noted after 6–8 d, scanty, mainly in the center around the point of inoculation, smooth, terminal on hyphae, oval to subclavate, often truncated at one end, and intercalary in hyphal cells, oval to ellipsoidal, (6.9–)7.5–10.8(–12.9) x (5–)5.6–7.4(–8.2) with l/w = (1.2–)1.2–1.6(–1.7) (n = 12).
Similar but slower culture development was observed at 15 C. At 30 C slightly faster development and more abundant yellowish conidiation structures inside the agar, morphologically indistinguishable from granules on the surface of the agar, were noted. The coconut-like odor also formed at all other temperatures. More abundant chlamydospores and yellow crystals (FIG. 5.8) formed at 30 and 35 C. At 35 C growth occurred for more than a week, but only few hyphae were noted on the surface and sometimes the agar dried out before the colony covered the plate. Scanty effuse simple sporulation without any granulation occurred from 4–5 d.
On PDA (25 C, FIG. 5.2) growth slower than on CMD, with hyphae more thickly and densely arranged than on CMD. Colony thick, dense, surface whitish, finely granular, downy to floccose, with aerial hyphae a net of thick strands and numerous fine branches without any noticeable orientation; 2–3 mm high between the thin, yellowish, finely granular area of inoculation of extremely densely interwoven to substromatically condensed hyphae and the ill-defined, diffuse margin with surface hyphae forming strands. Autolytic activity and coilings (FIG. 5.7) conspicuous at 25 and 30 C. Only inconspicuous zonation noted. Conidiation finely granular, colorless to white, on numerous single phialides or short verticillioid structures seated on surface and aerial hyphae, effuse, spreading across the whole colony. Slight yellowish discoloration of the center and the reverse from 4 d, spreading from the area of inoculation over the whole plate, mainly 3A3 to 3B5-6, finally turning yellowish-brown (4B4-5). Odor indistinct to slightly mushroomy. Slow growth at 35 C, forming small sterile, white, hairy colonies.
On SNA (25 C, FIG. 5.3) colonies thin, hyaline, growth predominantly inside the agar, hyphae loosely arranged and sometimes forming several separated strands rather than a continuous colony, smooth, aerial hyphae scanty, longer and denser at the distant margin, which becomes whitish and downy. Autolytic activity and coilings conspicuous, also at 30 C. Surface mycelium soon degenerating. Conidiation slightly more abundant and denser than on CMD, starting within 3d with phialides sessile or on short conidiophores, mainly at the proximal margin, later also on long aerial hyphae on the distant part of the colony. From 5–7 d conidiation appearing as fine granules containing heads up to 60 µm diam, spreading from the distant margin back nearly across the entire plate, or concentrated in two to three concentric zones, with a light (yellowish-) green color (28CD5-6 to 30CD5-6). Granules more regular than on CMD, remaining small (up to 0.6 mm diam) and appearing waxy to glassy in the stereomicroscope (FIG. 5.6). No pigment formed, no odor detected. At 30 C conidiation denser, granules more regular in 3 concentric zones, with conidial heads up to 100 µm diam. At 35 C colonies irregular, dense, hairy to floccose, sporulation more abundant than on CMD.
Chlamydospores on SNA (measured after 6 d at 35 C): spreading from the point of inoculation, more abundant than on CMD, particularly at higher temperatures (30–35 C). Terminal chlamydospores as on CMD, smooth, oval to subclavate and often truncated at one end, (4.5–)5.6–8.8(–9.4) x (4.2–) 4.5–6.6(–7.7) with l/w = (1–)1–1.6(–1.7) (n = 11); intercalary chlamydospores ellipsoidal to irregularly elongate to sinuous, (7.7–)10.8–17.5(–19.8) x (4–) 4.3–5.7(–6.9) with l/w = (1.6–)2.1–3.7(–4.4) (n = 11).
Habitat.. on dead corticated branches and small trunks of Alnus alnobetula (Ehrh.) C. Koch (=A. viridis (Chaix) DC.) and A. incana (L.) Moench, standing or lying on the ground.
Known distribution.. Austria, upper montane region (1100–1400 m s.m.) of the central Alps.
HOLOTYPE: AUSTRIA: SALZBURG: Böckstein, hiking trail to Böckstein close to the parking lot in front of the Gasteiner Heilstollen, MTB 8944/1, 47°04'58''N 13°06'08''E, 1280 m s.m., on dead partly standing trunk of Alnus alnobetula, 5 Sep 2003, W. Jaklitsch WJ 2378 (WU 25711; ex-type culture CBS 117711 = C.P.K. 948). Holotype of Trichoderma voglmayrii isolated from WU 25711 and deposited as a dry culture together with the holotype of H. voglmayrii as WU 25711a.
Paratype specimens.. AUSTRIA: STEIERMARK: Schladminger Tauern, Kleinsölk, steep wood at the western side of Lake Schwarzensee, MTB 8749/1, 47°17'35''N 13°52'15''E, 1165 m s.m., on dead branch of Alnus incana on the ground, 6 Aug 2003, W. Jaklitsch & H. Voglmayr, WJ 2302 (WU 25712, isolate CBS 117710 =C.P.K. 1592); Schladminger Tauern, Kleinsölk, hiking trail between Schwarzensee and Putzentalalm, MTB 8749/1, 47°16'36''N 13°51'44''E, 1320 m s.m., on dead standing trunk of Alnus alnobetula, 6 Aug, 2003, H. Voglmayr & W. Jaklitsch, WJ 2304, WU 25713; Schladminger Tauern, Kleinsölk, hiking trail between Schwarzensee and Putzentalalm, MTB 8749/1, 47°17'00''N 13°52'02''E, 1190 m s.m., on dead standing trunk of Alnus alnobetula, 6 Aug 2003, H. Voglmayr & W. Jaklitsch, WJ 2305 (WU 25714, isolate C.P.K. 941). KARNTEN: Stappitz, from Gasthof Alpenrose up along the brook parallel to the hiking trail 518, MTB 8945/3, 47°01'07''N 13°11'14''E, 1360 m s.m., on dead trunk of Alnus alnobetula on the ground, 5 Sep 2003, W. Jaklitsch, WJ 2382 (WU 25715, isolate C.P.K. 951).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDESCRIPTION OF THE SPECIESDISCUSSIONLITERATURE CITED |
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) and H. subalpina Petrak (Petrak 1940) have been described from similar altitudes in the Alps. While H. psychrophila develops yellow semiglobose stromata on Rhododendron ferrugineum L. and Vaccinium myrtillus L. in the subalpine zone, H. subalpina is likely to be encountered in the montane zone, because it forms its yellow discoid stromata on dead branches of various coniferous trees.
The occurrence at such altitudes, which are characterized by average summer temperatures of 12–18 C (Central Institute for Meteorology and Geodynamics Vienna personal communication), is in contrast to the ability of this species to grow in culture at 35 C. However this may be related to another conspicuous habit of H. voglmayrii (i.e. the ability to ascend trunks, thereby becoming exposed to climatic influences, such as direct sunshine and potential drought). The ability to grow at 35 C in culture is rare in Trichoderma/Hypocrea. Notable exceptions are members of Trichoderma sect. Long-ibrachiatum, including H. schweinitzii/T. citrinoviride and T. longibrachiatum, which are able to grow and sporulate at 40 C (Samuels et al 1998).
Third, only few species of Hypocrea, such as H. scutellaeformis Berk. & Ravenel described from Acer in North America (Gary Samuels pers comm), or the green-spored H. ceramica Ellis & Everh., show reddish stromata comparable to that of H. voglmayrii. In addition H. voglmayrii is characterized by conspicuous ostioles that are nearly black when dry. Such conspicuous ostioles are not seen in other species with reddish brown stromata, except H. patella Cooke & Peck (Dodd et al 2002) and H. lacuwombatensis B.S. Lu et al (Lu et al 2004). These species, unrelated to each other or to H. voglmayrii, are not known from Europe, have as yet not been collected at high altitudes, do not develop yellow pigments or a coconut-like odor and do not grow at 35 C.
A conspicuous phenotypic character of cultures of H. voglmayrii is the consistent formation of a yellow pigment on CMD, which gives even rise to formation of crystals at 30 and 35 C (and less so at 25 C), a feature also described for H. aureoviridis (Rifai and Webster 1966). Although many strains of Trichoderma form yellow pigments of various shades in cultures on various media (mostly PDA or malt-extract agar), these pigments are often only the result of suboptimal growth (unpublished observations).
H. voglmayrii is unusual in that its conidiophores form a continuous lawn and not in distinct pustules and that the conidia are at most only pale green. Pale-colored conidia occur in the Pachybasioides Clade. The coconut-like odor formed by H. voglmayrii in culture is regarded as typically found only for some species of sect. Trichoderma (Gams and Bissett 1998, Samuels pers comm). However, as shown in FIGS. 1–3, H. voglmayrii cannot be assigned to any major clade of Trichoderma.
An independent intercladal position of this new species already has been shown in the large ITS1 and 2 tree (Druzhinina et al 2005 as "51 vogl" H. sp. WJ 2305) where the largest number of known species (88) was considered. However in the rpb2 tree cited above the location of this species was basal to clades representing the section Trichoderma and this position was not contradicted by other trees. Bayesian trees obtained in the current study based on sequences of the genes ITS1 and 2, rpb2 and ech42 (FIGS. 1–3) consistently place H. voglmayrii in a position between clades of the section Trichoderma and the Pachybasioides clade (cf. Druzhinina et al 2005) with nearly identical genetic distances. Therefore H. voglmayrii represents a well-supported isolated lineage between the section Trichoderma and the Pachybasioides Clade IX and cannot be assigned to any of these clades.
In addition we also have analyzed tef1 sequences of H. voglmayrii. However, while the exon did not yield any useful phylogenetic resolution, sequences of the large intron were poorly alignable with those from species of neighboring clades (viz. FIGS. 1–3), and thus tef1 was not used for phylogenetic analysis (data not shown). When the tef1 large intron or both intron sequences (GenBank DQ086146
[GenBank]
and DQ086147
[GenBank]
) were subjected separately to a similarity search by TrichoBLAST (Kopchinskiy et al 2005, www.isth.info), the most similar species were H. rufa CBS 111094 for the forth large intron (92% of similarity), T. rossicum TUB F-752 for the fifth short intron (97% similarity based on the significant alignment of only 44 nt from 100) and H. sulphurea G.J.S. 95–190 for the last large exon (92% of similarity). Because all three matches belong to different phylogenetic clades of Hypocrea/Trichoderma, this result clearly indicates that H. voglmayrii is different from all other species, also based on the diagnostic parts of the tef1 sequence.
Despite several attempts (Kullnig-Gradinger et al 2002, Chaverri and Samuels 2003), the phylogeny of the genus Hypocrea/Trichoderma is not yet fully understood. Only a part of clades strongly supported in single gene trees coincide with the sections defined by Bissett (1991). However so-called "lone lineages" represented by single species appear in many phylogenetic evaluations (cf. Kullnig-Gradinger et al 2002, Chaverri and Samuels 2003). Hypocrea voglmayrii represents such a new lineage that now stabilizes neighboring clades. The phylogenetic position of the Pachybasioides Clade had been unclear in previous studies, because it was placed from within the large Pachybasium species cluster (Pachybasium B5; Kullnig Gradinger et al 2002), whereas it formed a sister clade of sect. Trichoderma in the rpb2 tree of Chaverri and Samuels (2003). Hypocrea voglmayrii appears to be a missing link because its inclusion in the phylogenetic analysis now stabilizes the phylogeny of the Pachybasioides Clade in the vicinity of the section Trichoderma (Rufa Clade XII and Pachybasioides "A" Clade XIII). Also the position of species of sect. Hypocreanum (Pachybasium B1; Kullnig-Gradinger et al 2002) is now stabilized adjacent to the Pachybasioides Clade. While an overall phylogeny of the genus is still difficult to obtain because of an insufficient number of phylogenetic markers that provide sufficient resolution within the whole genus, it appears that this part of the generic tree can become better understood after inclusion of undescribed species. Several new species collected during the investigation of the diversity of Hypocrea in Central Europe by the corresponding author eventually will reveal further aspects of the phylogeny of Hypocrea/Trichoderma.
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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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LITERATURE CITED |
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