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ETH Zurich, Institute of Integrative Biology IBZ, Forest Pathology and Dendrology, CH-8092 Zürich, Switzerland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Phialocephala fortinii s.l. and Acephala applanata are the dominant dark septate endophytes (DSE) in roots of many trees and shrubs. Population genetic analysis led to the discovery of morphologically indistinguishable but reproductively isolated cryptic species (CSP) within Phialocephala fortinii s.l. In the present study we show that sequence data of two coding (beta-tubulin and translation elongation factor [EF-1alpha]) and three noncoding DNA loci confirm subdivision of P. fortinii s.l. and allow to differentiate seven CSP of P. fortinii. In addition we show that strains collected throughout Europe can be classified correctly based on these sequence markers. Statistically significant differences in growth response on different media were observed among CSP of P. fortinii and A. applanata. Growth inhibition on MEA amended with 100 mgl–1 cycloheximide had the strongest differential effect of all physiological traits examined. In contrast exoenzyme production (laccase, proteinase, pectinase, phenol-oxidase, amylase, cytochrome oxidase and tyrosinase) rarely helped to differentiate CSP of P. fortinii. However A. applanata was a strong producer of amylases, laccases and proteinases. Based on these data we propose to assign species rank to six CSP of P. fortinii: P. turiciensis, P. letzii, P. europaea, P. helvetica, P. uotolensis, P. subalpina spp. nov. and P. fortinii s.s.
Key words: cryptic species, divergence times, DSE, fungal speciation, gene trees, population genetics, root-endopyhte, species boundaries, species tree analysis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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, Jumpponen and Trappe 1998b, Sieber 2002). DSE are characterized by their darkly pigmented and septate mycelia discriminating them from members of the Glomeromycota known to form endomycorrhizae and from other endophytes with hyaline hyphae. The mitosporic taxon Phialocephala fortinii s.l. was shown to be the dominant DSE in roots of species belonging to the Pinaceae but is also known to colonize roots of broadleaf trees, shrubs and herbaceous plants (Ahlich-Schlegel 1997, Ahlich and Sieber 1996, Grünig et al 2006, Sieber 2002, Stoyke et al 1992). New reports suggest that P. fortinii s.l. is not restricted to fine roots of higher plants. Menkis et al (2004) reported isolation of P. fortinii s.l. at low frequencies from (living) stem bases of Pinus sylvestris, Betula pendula and Picea abies.
Of note, P. fortinii s.l. was shown to form ectomycorrhizal structures with Populus tremula x Populus tremuloides clones (Kaldorf et al 2004) but the ecological role of P. fortinii s.l. might be beyond the paradigms of mycorrhizal and pathogenic associations (Addy et al 2005). Conflicting results have been published describing P. fortinii s.l. as beneficial, neutral or pathogenic for different hosts, growing conditions and fungal strains (Haselwandter and Read 1982; Jumpponen and Trappe 1998a, b; Ruotsalainen and Kytoviita 2004; Stoyke and Currah 1993; Vohnik et al 2005; Vohnik et al 2003; Wilcox and Wang 1987) and pronounced strain-specific (Currah et al 1993) as well as experimental design-specific differences (Fernando and Currah 1996, Narisawa et al 2004) were observed. Therefore it is essential to classify P. fortinii s.l. isolates used in ecological experiments properly (Addy et al 2005).
Population genetic studies in Europe suggest that P. fortinii s.l. is composed of several cryptic species (CSP). Species were named CSP because strains of different species were indistinguishable morphologically but reproductively isolated (Grünig 2004, Grünig et al 2006). In addition a morphotype which is known to be closely related to P. fortinii s.l. (Ahlich and Sieber 1996, Grünig et al 2002) was described as Acephala applanata (Grünig and Sieber 2005). The presence of cryptic species in P. fortinii s.l. also was suggested for North America based on AFLP fingerprints (Piercey et al 2004).
Classification of CSP originally was based on single-copy RFLP analysis or inter-simple-sequence-repeat-anchored PCR (ISSR-PCR) (Grünig 2004). Several shortcomings are related, with both marker types rendering a fast and accurate classification difficult. Therefore sequence markers of coding and noncoding loci were successfully developed and applied to characterize CSP (Grünig et al 2007). Assignment of individual strains to different CSP however was complicated due to possible introgression/incomplete lineage sorting (Carbone and Kohn 2004) between CSP. In addition not all sequence loci are equally valuable to separate species and separation of some CSP is impossible with some loci (Grünig et al 2007). Nevertheless population genetic analysis based on sequence data showed that these species are isolated reproductively (Grünig et al 2007). Analysis in the study of Grünig et al (2007) were performed based on sequence data of one single population per CSP. It thus is an open question whether single strains collected at other locations can be assigned to the correct CSP with these sequence markers. In addition it remains to be tested whether they can be differentiated based on physiological traits.
The aim of the present study was to: (i) evaluate the suitability of the previously developed sequence markers to assign single strains of P. fortinii s.l. collected at different locations in Europe correctly to CSP; (ii) to identify physiological markers suitable to differentiate CSP; and (iii) to describe some of the most frequent cryptic species of P. fortinii s.l.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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, Grünig et al 2006). Strains were selected from eight of 17 field populations of P. fortinii s.l. and A. applanata represented by more than 1300 strains analyzed with 11 single-copy RFLP markers. Cluster analysis based on FST values among field populations shows that populations belonging to the same CSP cluster together but clearly are separated from populations of other CSP (see FIG. 1. in Grünig et al 2007). A field population was defined as the strains of a cryptic species collected at 74 grid points within a 14 m x 14 m square of forest floor (Grünig 2004). All field populations were collected in Switzerland except CSP7, which included a population from northern Finland. The study sites were separated by 10–2800 km. At least seven multilocus haplotypes (MLH) of each CSP and A. applanata were chosen from one local population except CSP5. Each MLH was represented by one strain. Strains of CSP1 to CSP4 originated from the study site Zürichberg and strains of CSP6, CSP7 and A. applanata originated from the study site Bödmeren. Six MLH of CSP5 originated from study site Üetliberg and one MLH from the study site Zürichberg 5 km apart. Two coding and three noncoding loci of genomic DNA were sequenced. Sequence data of 82 strains collected in study sites Bödmeren, Zürichberg and Üetliberg were received during earlier studies (Grünig et al 2007). Fifteen additional strains collected in Europe including the type strains of P. fortinii (K93 444, CBS 443.86) and A. applanata (K93 113, CBS 109321) were included in the analysis to test the suitability of molecular markers to classify CSP of P. fortinii and A. applanata (TABLE I). All strains were isolated according to Ahlich and Sieber (1996) except strains from the Lithuanian nurseries (Menkis et al 2004) and the type strain of P. fortinii (Wang and Wilcox 1985). Cultivation and DNA extraction were performed as described by Grünig et al (2003). Additional strains of CSP6, CSP7 and A. applanata were sequenced as described by Grünig et al (2007).
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, Swofford 1993) were observed (Grünig et al 2007). The concatenated approach is thought to produce more accurate results for inferring species phylogenies than the consensus phylogeny approach (Gadagkar et al 2005). Dataset 1 comprised sequence data of the two coding loci EF1-alpha and beta tubulin and the two noncoding loci pPF-018 and pPF-076 of all 97 strains. Dataset 2 comprised strains belonging to CSP1 to CSP5 only but, in contrast to dataset 1, contained the sequence data of an additional locus (pPF-061). Modeltest v3.7 (Posada and Crandall 1998) served to determine the model of substitution that fits the data best for each dataset. Maximum likelihood (ML) analysis using full heuristic searches were performed in PAUP 4.0b10 (Swofford 1993) with the substitution model identified by Modeltest. In addition bootstrapping to generate 100 pseudosamples was used to estimate the phylogenetic support of clades.
Population genetic analysis and comparison between different marker types.— –
Fixed mutations between cryptic species for the two combined datasets as an indicator for DNA divergence (Hey 1991) was analysed with DnaSP 4.1 (Rozas et al 2003). Values of population differentiation (FST) using either the sequence data of the four loci or the single-copy RFLP data were calculated in Arlequin v 3.1 (Excoffier et al 2005). Hierarchical clustering was performed based on pairwise FST values of the sequence and single-copy RFLP dataset with the single-linkage algorithm and trees were compared visually.
Calculation of divergence times and rates of migration based on coalescent theory.— –
Pairwise divergence times and migration rates among CSP of P. fortinii s.l. and A. applanata were calculated based on coalescent theory with the software IM (Hey and Nielsen 2004). First, selective neutrality of sequence loci and possible recombination within loci was tested for each CSP with DnaSP 4.1. (Rozas et al 2003) to ascertain that assumptions of IM were not violated. Second, infinite-site violations in the pairwise combined datasets were excluded with the software SNAP Workbench (Price and Carbone 2005). The divergence time and the migration rate, including 90% confidence intervals, were calculated in IM with a burn-in of 100 000 steps and 2 000 000 updates for the simulation. To improve mixing and thereby convergence simulations, were run with a Metropolis–coupling of five chains and 10 chain swap attempts per step. Every analysis was repeated three times to check for consistency of results. Trees were constructed based on divergence times with cluster analysis.
Search for characters diagnostic for cryptic species of P. fortinii s.l..— –
Nucleotide substitutions, which were unique for a CSP in the present dataset, were regarded as diagnostic characters. The full dataset of each locus was collapsed to unique sequences (haplotypes) with the software Collapse v1.2 (http://darwin.uvigo.es/software/collapse.html). Haplotype datasets were analyzed with TCS v1.21 (Clement et al 2000). TCS output was searched for positions of diagnostic characters, and these were verified in an alignment of all haplotypes.
Growth rate on different culture media.— – Growth of strains was studied with four culture media. Each strain was cultivated on malt-extract agar (MEA; 2% [w/v] malt extract, 1.5% agar), water agar (WA; 1.5% agar), MEA amended with cycloheximide (100 mg/L) and MEA amended with nystatine (100 mg/L). Strains were incubated 20 d at 20 C and culture diameter (d) was determined as the average of measurements along two perpendicular imaginary lines intersecting in the center of cultures. Duplications were prepared for a fraction of strains to test for the stability of growth rates of strains. The proportion of inhibition by fungicides was calculated as
Data were grouped for each CSP and displayed as box blots. Statistical significances between growth responses for the different CSP and A. applanata were determined with the nonparametric Kruskal-Wallis test (Stahel 2002).
Determining presence of extracellular enzymes.— –
Several tests were performed to show the presence of extracellular enzymes including plate tests (protease, polyphenol oxidase, pectinase) as well as droplet tests (cytochrome oxidase, laccase, tyrosinase, amylase). The proteolytic activity was tested according to Petrini et al (1984). Media contained 0.1% (w/v) malt extract (Difco Laboratories, Detroit, Michigan), 2% agar (Difco Laboratories, Detroit, Michigan) and 0.5% (w/v) Promin D (Soya protein, Novartis, Basel, Switzerland). A strain of Idriella bolleyi (ZT 90 254) served as a positive control. Cultures were grown 20 d at 20 C in the dark. Proteolytic activity was evident if the opaque medium got transparent at the edge of the cultures. Proteolytic activity was scored to three categories based on the extent of the clearing zone in relation to the diameter of the culture.
Presence of polyphenol-oxidase was tested with the Bavendamm reaction with gallic acid as substrate (Bavendamm 1928). The medium contained 1.5% (w/v) malt extract, 2% agar and 0.5% (w/v) gallic acid (Sigma, Switzerland). The pH of the medium was adjusted to 6.5 with 10% NaOH before autoclaving. Cultures were grown 20 d at 20 C in the dark. If the medium turned dark brown at the margin of the actively growing culture, strains were classified as positive.
Pectinolytic activity was tested as described by Carroll and Petrini (1983). The medium contained 0.1% (w/v) yeast extract (Difco Laboratories, Detroit, Michigan), 2% agar, 0.5% (w/v) apple pectin (Sigma, Switzerland) and 50% (v/v) mineral salts solution. The medium was adjusted to pH 6.5 before autoclaving. Cultures were grown for 20 d at 20 C in the dark and then flooded with 8 mL 1% (w/v) CTAB. Pectinolytic activity was evident if the opaque medium turned transparent at the edge of the cultures.
In addition four droplet tests were performed to determine presence of cytochrome oxidase, laccase, tyrosinase (Stalpers 1978) and amylase (Taylor 1974). Droplets were applied to the margin of actively growing cultures on MEA. For the amylase test, three inoculi (10 mm diam) from the margin of growing cultures were placed in empty Petri dishes and 200 µL of a 1% (w/v) starch solution dissolved in citric acid buffer (pH 5.22) were pipetted around two inoculi. The third inoculum served as control and 200 µL of the citric buffer without starch was pipetted. Inoculi were incubated 24 h at 20 C. Inoculi were removed and the amylase activity was demonstrated by the addition of 15 µL of Lugol’s solution. Droplet tests were scored subjectively on the basis of size of reaction zone or the degree of color change: (0) = no activity, (1) = slight but definite activity, (2) = intermediate activity and (3) = intense activity.
Comparing the morphology of sporulation within and among CSP.— – To compare the morphology of sporulation between CSP each strain was incubated in two slants containing 2% malt-extract agar. Slants were stored 1 y at 4 C and screened for the presence of conidiophores. Morphology of conidiphores was studied for each sporulating strain and the degree of morphological differences within each CSP was recorded and compared to morphological differences among CSP.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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) substitution model fitted best the nucleotide substitution pattern of the concatenated dataset 1. It was characterized by equal base frequencies, a transition:transversion ratio of 3.064, a shape parameter (
) of the gamma distribution (G) of 0.014 and a proportion of invariable sites (I) of 0.742. The TrNef + I substitution model (Tamura and Nei 1993) fitted best the nucleotide substitution pattern observed in dataset 2. It was characterized by these weights for nucleotide substitutions: 8.08 for A to G or G to A, 5.34 for C to T or T to C; 1.0 for all others. It assumed equal base frequencies and estimated the proportion of invariable sites (I ) to 0.9. ML analyses of the two datasets are presented (FIGS. 1
, 2). Strains that belong to the same species form distinct clusters except strains of CSP6 that were placed at a basal node (FIG. 1) and CSP3, which formed a polyphyletic taxon with the concatenated sequence data of five loci (FIG. 2). In addition 15 strains of CSP6, CSP7 and A. applanata, which had been collected at several locations in Europe and were classified based on single-copy RFLP analysis before sequencing, were assigned correctly to the respective species (FIG. 1).
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). Cluster analysis based on FST values derived from multilocus sequence data or single-copy RFLP data showed a highly similar grouping of the CSP (FIG. 3A, B). In addition pairwise migration rates calculated based on sequence data with the software IM were zero (data not shown). Relative divergence times varied between 0.33 (CSP2 versus CSP3) and more than 50 (A. applanata vs. CSP4) (data not shown). Cluster analysis based on relative divergence times calculated based respectively on four or five sequence loci are presented (FIG. 3C, D).
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). However differences between CSP were often small (MEA and MEA amended with nystatine) and variability was high. Significantly lower growth rates on MEA and WA were observed for A. applanata. In addition significant differences were observed between some of the CSP of P. fortinii. Of note, malt-extract agar amended with cycloheximide showed pronounced differences among closely related CSP such as CSP2 and CSP3 or CSP6 and CSP7 and only four of the 21 pairwise comparisons between species were not statistically significant.
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All strains of A. applanata produced high amounts of laccase. In contrast only some of the strains of P. fortinii s.l. tested positive and the amount of laccase produced was low. Of interest, closely related CSP1, 2, 3 and 5 were significantly more often positive for laccase production then CSP4, 6 and 7.
Amylase activity was observed for all strains belonging to A. applanata except K93_079 and T1_50_2. In contrast no distinct amylase activity was observed for strains belonging to P. fortinii s.l. The majority of strains belonging to CSP6 and 7 showed a weak but clearly positive reaction for cytochrome oxidase and 54% and 92% of the strains respectively tested positive. In contrast CSP3 and 4 were rarely positive for cytochrome oxidase and CSP1, 2 and 5 as well as A. applanata did not produce any cytochrome oxidase. None of the strains showed a positive reaction for the presence of tyrosinases.
Comparing the morphology of sporulation within and among species.— –
Differences in the proportion of sporulating strains per species was pronounced among CSP. Whereas all strains belonging to CSP2 sporulated after 1 y, only seven of the 15 examined strains of CSP6 produced conidiophores. All other CSP including P. fortinii s.s. showed an intermediate proportion of sporulating strains. Variability found in the morphology of conidiophores both within CSP and single strains was pronounced, ranging from well developed melanized conidiophores typically for P. fortinii sensu Wang and Wilcox (1985) to hyaline fertile phialides singly produced directly on vegetative hyphae (FIG. 5). The high variability found within species did not let us identify distinct morphological features for the species. Nevertheless strains of CSP3 often produced "conidiophores" with one to three phialides directly on the mycelium (FIG. 5H). Such structures were observed rarely in other CSP (FIG. 5D, M). Moreover CSP6 tended to produce longer and narrower conidophores (FIG. 5I). However this type of conidiophore also was seen in several other CSP, and some strains of CSP6 also produced "classical" conidiophores similar to those shown in FIG. 5A, E and N.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Phialocephala fortinii s.s. (Wang & Wilcox)
= P. fortinii sensu Wang & Wilcox. Mycologia 77:951–958 (1985).
If a collective species concept is used this fungus best could be addressed as P. fortinii sensu lato (s.l.). If one wishes to emphasize that CSP7 sensu Grünig et al (2006, Fung Genet Biol 43:410–421) is in question, it can be indicated as P. fortinii sensu stricto (s.s.).
Phialocephala fortinii s.s. can be distinguished from other species within the P. fortinii s.l. species complex by molecular characteristics. It differs by these fixed nucleotide characters from other members of the P. fortinii s.l. species complex: beta tubulin positions 57 (C), 206 (C); translation elongation factor (1), pPF-018 and pPF-076 no fixed mutations. Inhibition of cultural growth on MEA amended with cycloheximide (100 mg/L) compared to that on MEA is 72.3 ± 13.8%.
HOLOTYPE: endophytic isolate from a fine root of Scots pine (Pinus sylvestris) originating from Suonenjoki, Finland. A freeze-dried sample is maintained by the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS) (CBS 443.86 
FAP-7).
Phialocephala turiciensis C.R. Grünig et T.N. Sieber, sp. nov.
= P. fortinii, CSP1 sensu Grünig et al Fung Genet Biol 41:676–687 (2004).
Phialocephalae fortinii s.s. morphologia similis, sed ob sequentias nucleotiditum stabilium distincta, viz. ob "beta tubulin" positiones 57 (T), 206 (T), 227 (T), 233 (T), 302 (C), 311 (T), 344 (T), 479 (G), 497 (T); "translation elongation factor (1)" positiones 428 (T); "pPF-018" positiones 72 (A), 202 (T), 274 (C), 297 (C), 337 (G), 355 (T), 364 (A); "pPF-076" positionesque 4 (G), 54 (A), 62 (G), 461 (G), 472 (C), 473 (C). Cultura in malto agaroso antibioticum "Cycloheximide" dictum continente reducta est ad valorem 54.3 ± 8.4%.
HOLOTYPUS: cultura sicca ex radicibus vivis Piceae abietis, Helvetia, Zürichberg, 01.05.2002, C.R. Grünig, herbarium ZT; cultura viva numero CBS 119264 ( 176_1) deposita Centraalbureau voor Schimmelcultures, Utrecht, Batavia.
Phialocephala turiciensis morphologically is similar to Phialocephala fortinii s.s., but the two species differ in some molecular characteristics. It differs by these fixed nucleotide characters from P. fortinii s.s.: beta tubulin positions 57 (T), 206 (T), 227 (T), 233 (T), 302 (C), 311 (T), 344 (T), 479 (G), 497 (T); translation elongation factor (1) position 428 (T); pPF-018 positions 72 (A), 202 (T), 274 (C), 297 (C), 337 (G), 355 (T), 364 (A); pPF-076 positions 4 (G), 54 (A), 62 (G), 461 (G), 472 (C), 473 (C). Inhibition of cultural growth on MEA amended with cycloheximide (100 mg/L) compared to MEA is 54.3 ± 8.4%.
HOLOTYPE: endophytic isolate from a living fine root of Norway spruce (Picea abies) originating from Zürichberg, Switzerland (grid reference 685.000/249.725 according to the National Map of Switzerland, Swisstopo, Federal Office of Topography, Wabern, Switzerland). A dried culture on malt-extract agar was deposited at the herbarium ZT of the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland. A freeze-dried sample is maintained by the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS) (CBS 119264 176_1).
Etymology. – The two study sites Zürichberg and Üetliberg near Zürich were the places of discovery of this species; "Turicum" is the Latin name for "Zürich".
Specimens examined. –
(TABLE I) 124_1, 117_3, 120_2, 124_2, 130_3, 133_3, 154_1, 176_1, 190_4, 197_4
Phialocephala letzii C.R. Grünig et T.N. Sieber, sp. nov.
= P. fortinii, CSP2 sensu Grünig et al Fung Genet Biol 41:676–687 (2004).
Phialocephalae fortinii s.s. morphologia similis, sed ob sequentias nucleotiditum stabilium distincta, viz. ob "beta tubulin" positiones 57 (T), 206 (T), 233 (T), 302 (C), 311 (T), 344 (T), 479 (G), 497 (T); "translation elongation factor (1)" positiones 428 (T); "pPF-018" positiones 119 (A), 160 (T), 337 (G), 355 (T), 364 (A), 415 (A); "pPF-076" positionesque 4 (G), 54 (A), 62 (G), 461 (G), 472 (C), 473 (C). Cultura in malto agaroso antibioticum "Cycloheximide" dictum continente reducta est ad valorem 87.3 ± 9.4%.
HOLOTYPUS: cultura sicca ex radicibus vivis Piceae abietis, Helvetia, Zürichberg, 01.05.2002, C.R. Grünig, herbarium ZT; cultura viva numero CBS 119268 ( 146_1) deposita Centraalbureau voor Schimmelcultures, Utrecht, Batavia.
Phialocephala letzii is morphologically similar to Phialocephala fortinii s.s., but the two species differ in some molecular characteristics. P. letzii differs by these fixed nucleotide characters from P. fortinii s.s.: beta tubulin positions 57 (T), 206 (T), 233 (T), 302 (C), 311 (T), 344 (T), 479 (G), 497 (T); translation elongation factor (1) position 428 (T); pPF-018 positions 119 (A), 160 (T), 337 (G), 355 (T), 364 (A), 415 (A); pPF-076 positions 4 (G), 54 (A), 62 (G), 461 (G), 472 (C), 473 (C). Inhibition of cultural growth on MEA amended with cycloheximide (100 mg/L) compared to MEA is 87.3 ± 9.4%.
HOLOTYPE: endophytic isolate from a living fine root of Norway spruce (Picea abies) originating from Zürichberg, Switzerland (grid reference 685.000/249.725 according to the National Map of Switzerland, Swisstopo, Federal Office of Topography, Wabern, Switzerland). A dried culture on malt-extract agar was deposited at the herbarium ZT of the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland. A freeze-dried sample is maintained by CBS (CBS 119268 146_1).
Etymology. – This species was isolated from the study site Zürichberg near a former defensive fortification of the town of Zürich called "Letzi".
Specimens examined. –
(see TABLE I) 194_4, 120_3, 194_1, 152_2, 147_3, 126_3, 137_4, 146_1, 146_2, 149_5, 168_4
Phialocephala europaea C.R. Grünig et T.N. Sieber, sp. nov.
= P. fortinii, CSP3 sensu Grünig et al Fung Genet Biol 41:676–687 (2004).
Phialocephalae fortinii s.s. morphologia similis, sed ob sequentias nucleotiditum stabilium distincta, viz. ob "beta tubulin" positiones 57 (T), 206 (T), 233 (T), 302 (C), 311 (T), 344 (T), 479 (G), 497 (T); "translation elongation factor (1)" positiones 95 (T), 491 (T); "pPF-018" positiones 119 (A), 160 (T), 337 (G), 355 (T), 364 (A), 415 (A); "pPF-076" positionesque 4 (G), 54 (A), 62 (G), 461 (G), 472 (C), 473 (C). Cultura in malto agaroso antibioticum "Cycloheximide" dictum continente reducta est ad valorem 65.8 ± 18.6%.
HOLOTYPUS: cultura sicca ex radicibus vivis Piceae abietis, Helvetia, Zürichberg, 01.05.2002, C.R. Grünig, herbarium ZT; cultura viva numero CBS 119271 ( 171_5) deposita Centraalbureau voor Schimmelcultures, Utrecht, Batavia.
Phialocephala europea is morphologically similar to Phialocephala fortinii s.s., but the two species differ in some molecular characteristics. P. europea differs by these fixed nucleotide characters from P. fortinii s.s.: beta tubulin positions 57 (T), 206 (T), 233 (T), 302 (C), 311 (T), 344 (T), 479 (G), 497 (T); translation elongation factor (1) positions 95 (T), 491 (T); pPF-018 positions 119 (A), 160 (T), 337 (G), 355 (T), 364 (A), 415 (A); pPF-076 positions 4 (G), 54 (A), 62 (G), 461 (G), 472 (C), 473 (C). Inhibition of cultural growth on MEA amended with cycloheximide (100 mg/L) compared to MEA is 65.8 ± 18.6%.
HOLOTYPE: endophytic isolate from a living fine root of Norway spruce (Picea abies) originating from Zürichberg, Switzerland (grid reference 685.000/249.725 according to the National Map of Switzerland, Swisstopo, Federal Office of Topography, Wabern, Switzerland). A dried culture on malt-extract agar was deposited at the herbarium ZT of the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland. A freeze-dried sample is maintained by CBS (CBS 119271 171_5).
Etymology. – This species seems to be distributed throughout Europe.
Specimens examined. –
(see TABLE I) 117_2, 139_3, 122_3, 137_3, 129_1, 136_1, 136_3, 158_4, 169_2, 171_5, 173_2, 177_3
Phialocephala helvetica C.R. Grünig et T.N. Sieber, sp. nov.
= P. fortinii, CSP4 sensu Grünig et al Fung Genet Biol 41:676–687 (2004).
Phialocephalae fortinii s.s. morphologia similis, sed ob sequentias nucleotiditum stabilium distincta, viz. ob "beta tubulin" positiones 57 (T), 62 (T), 206 (T), 302 (C), 311 (T), 333 (C), 353 (T), 497 (T); "translation elongation factor (1)" positiones 136 (G), 195 (A); "pPF-018" positiones 57 (A), 109 (T), 355 (T), 381 (T), 391 (T); "pPF-076" positionesque 14 (G), 20 (G), 123 (C), 235 (A), 473 (C). Cultura in malto agaroso antibioticum "Cycloheximide" dictum continente reducta est ad valorem 54.3 ± 8.4.
HOLOTYPUS: cultura sicca ex radicibus vivis Piceae abietis, Helvetia, Zürichberg, 01.05.2002, C.R. Grünig, herbarium ZT; cultura viva numero CBS 119273 ( 138_5) deposita Centraalbureau voor Schimmelcultures, Utrecht, Batavia.
Phialocephala helvetica is morphologically similar to Phialocephala fortinii s.s., but the two species differ in some molecular characteristics. P. helvetica differs by these fixed nucleotide characters from P. fortinii s.s.: beta tubulin positions 57 (T), 62 (T), 206 (T), 302 (C), 311 (T), 333 (C), 353 (T), 497 (T); translation elongation factor (1) positions 136 (G), 195 (A); pPF-018 positions 57 (A), 109 (T), 355 (T), 381 (T), 391 (T); pPF-076 positions 14 (G), 20 (G), 123 (C), 235 (A), 473 (C). Inhibition of cultural growth on MEA amended with cycloheximide (100 mg/L) compared to MEA is 54.3 ± 8.4%.
HOLOTYPE: endophytic isolate from a living fine root of Norway spruce (Picea abies) originating from Zürichberg, Switzerland (grid reference 685.000/249.725 according to the National Map of Switzerland, Swisstopo, Federal Office of Topography, Wabern, Switzerland). A dried culture on malt-extract agar was deposited at the herbarium ZT of the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland. A freeze-dried sample is maintained by CBS (CBS 119273 138_5).
Etymology. – The species was found in several study sites in Switzerland; "Helvetia" is the Latin name of "Switzerland".
Specimens examined. –
(see TABLE I) 123_4, 135_1, 136_4, 153_2, 138_5, 140_4, 144_4, 145_2, 160_1, 195_2
Phialocephala uotolensis C.R. Grünig et T.N. Sieber, sp. nov.
= P. fortinii, cluster pPF-076 allele 2 (CSP5) sensu Grünig et al Fung Genet Biol 41:676–687 (2004).
Phialocephalae fortinii s.s. morphologia similis, sed ob sequentias nucleotiditum stabilium distincta, viz. ob "beta tubulin" positiones 57 (T), 206 (T), 233 (T), 302 (C), 311 (T), 344 (T), 479 (G), 497 (T); "translation elongation factor (1)" no positiones fixationibus; "pPF-018" positiones 72 (A), 202 (T), 262 (G), 274 (C), 297 (C), 337 (G), 355 (T), 364 (A); "pPF-076" positionesque 4 (G), 54 (A), 62 (G), 260 (G), 461 (G), 472 (C), 473 (C). Cultura in malto agaroso antibioticum "Cycloheximide" dictum continente reducta est ad valorem 54.7 ± 8.8%.
HOLOTYPUS: cultura sicca ex radicibus vivis Piceae abietis, Helvetia, Üetliberg, 10.02.2004, C.R. Grünig, herbarium ZT; cultura viva numero CBS 119275 ( 264_1r) deposita Centraalbureau voor Schimmelcultures, Utrecht, Batavia.
Phialocephala uotolensis is morphologically similar to Phialocephala fortinii s.s., but the two species differ in some molecular characteristics. P. uotolensis differs by these fixed nucleotide characters from P. fortinii s.s.: beta tubulin positions 57 (T), 206 (T), 233 (T), 302 (C), 311 (T), 344 (T), 479 (G), 497 (T); translation elongation factor (1) no fixed mutations; pPF-018 positions 72 (A), 202 (T), 262 (G), 274 (C), 297 (C), 337 (G), 355 (T), 364 (A); pPF-076 positions 4 (G), 54 (A), 62 (G), 260 (G), 461 (G), 472 (C), 473 (C). Inhibition of cultural growth on MEA amended with cycloheximide (100 mg/L) compared to MEA is 54.7 ± 8.8%.
HOLOTYPE: endophytic isolate from a living fine root of Norway spruce (Picea abies) originating from Üetliberg, Switzerland (grid reference 678.450/246.800 according to the National Map of Switzerland, Swisstopo, Federal Office of Topography, Wabern, Switzerland). A dried culture on malt-extract agar was deposited at the herbarium ZT of the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland. A freeze-dried sample is maintained by CBS (CBS 119275 264_1r).
Etymology. – Üetliberg was the place where this species was isolated most often. Üetliberg derives from "mountain of Uotilo"; "Uotilo" is the diminutive of "Uoto" the allemanic synonym of the German male name "Ulrich".
Specimens examined. –
(see TABLE I) 134_3, 264_1r, 252_7r, 220_1, 234_5, 265_3, 265_4
Phialocephala subalpina C.R. Grünig et T.N. Sieber, sp. nov.
= P. fortinii, CSP6 sensu Grünig et al Fung Genet Biol 43:410–421 (2006).
Phialocephalae fortinii s.s. morphologia similis, sed ob sequentias nucleotiditum stabilium distincta, viz. ob "beta tubulin" positionesque 57 (T), 104 (G), 206 (T). Cultura in malto agaroso antibioticum "Cycloheximide" dictum continente reducta est ad valorem 89.3 ± 7.2%.
HOLOTYPUS: cultura sicca ex radicibus vivis Piceae abietis, Helvetia, Bödmerenwald, 10.09.2003, C.R. Grünig, herbarium ZT; cultura viva numero CBS 119280 ( 78_2) deposita Centraalbureau voor Schimmelcultures, Utrecht, Batavia.
Phialocephala subalpina is morphologically similar to Phialocephala fortinii s.s., but the two species differ in some molecular characteristics. P. subalpina differs by these uniquely fixed nucleotide characters from P. fortinii s.s.: beta tubulin positions 57 (T), 104 (G), 206 (T); translation elongation factor (1) no fixed position; pPF-018 position 355 (T); pPF-076 no fixed positions. Inhibition of cultural growth on MEA amended with cycloheximide (100 mg/L) compared to MEA is 89.3 ± 7.2%.
HOLOTYPE: endophytic isolate from a living fine root of Norway spruce (Picea abies) originating from Bödmeren, Switzerland (grid reference 707.120/203.560 according to the National Map of Switzerland, Swisstopo, Federal Office of Topography, Wabern, Switzerland). A dried culture on malt-extract agar was deposited at the herbarium ZT of the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland. A freeze-dried sample is maintained by CBS (CBS 119280 78_2).
Etymology. – This species was most abundant in the two subalpine forest sites, Scatlé and Bödmeren.
Specimens examined. –
(see TABLE I) 37_6v, 35_6v, 2_7v, 30_4, 50_6v, 8_7v, 16_1, 53_6v, 70_1, 70_4, 9_6v, 22_7v, 78_2, 64-386, 95-460, K93_381, K93_440
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Suitability of sequencing loci to classify species within P. fortinii s.l.— –
Molecular characterization of P. fortinii s.l. using, for example, the ITS regions recently became popular (Addy et al 2000, Allen et al 2003, Ashkannejhad and Horton 2006, Menkis et al 2004) because P. fortinii s.l. often remains sterile or sporulates only after prolonged incubation at low temperatures. However a drawback of ITS sequences is that they might not separate closely related species (Bruns and Shefferson 2004). Indeed sequences of the ITS regions were not adequate to distinguish CSP of P. fortinii s.l. (Grünig et al 2004). However the genealogical concordance species recognition concept (GCPSR) using sequence data of several loci was used successfully to study the species limits of important human (Burt et al 1996, Geiser et al 1998, Koufopanou et al 1997, Pringle et al 2005) and plant pathogens (Carbone et al 1999, Couch and Kohn 2002, Geiser et al 1998, O’Donnell et al 2000, O’Donnell et al 2004). Therefore several coding and noncoding sequence loci were developed for P. fortinii s.l. and A. applanata to show reproductive isolation among species with GCPSR and a population genetic analysis framework (Grünig et al 2007). Recognition of species in the P. fortinii s.l. A. applanata complex however is complicated by introgression/incomplete lineage sorting and loci that do not resolve all CSP. Nevertheless combining up to five loci to form a super gene alignment resulted in clusters congruent with the CSP of P. fortinii and A. applanta found previously based on single-copy RFLP and ISSR-PCR (Grünig et al 2007). So far however CSP were identified by genetic analysis of single field populations from Switzerland, leaving open the question whether single strains collected anywhere can be assigned correctly to these CSP. We therefore included an additional 15 strains of the two closely related species P. subalpina (CSP6), P. fortinii s.s. (CSP7) and A. applanata from various places throughout Europe. All 15 additional strains were assigned to the correct species with four sequence loci demonstrating the suitability of sequence markers to classify CSP of P. fortinii s.l. In addition several strains of P. fortinii s.l. not included in the present study originating from Europe (including Switzerland) and North America were sequenced and could be classified as P. turiciensis, P. europaea, P. subalpina and P. fortinii s.s. (data not shown). However, if some strains show an unexpected placement for some sequence loci, we encourage researchers to use additional markers such as single-copy RFLP or ISSR-PCR to confirm the classification of these strains.
An alternative way to classify whole populations of CSP of P. fortinii s.l. is to calculate the population subdivision among new populations and the dataset published in this study. For example, considering the strains of P. fortinii s.s. sampled in Northern Europe as a new population (eight strains), the FST value between this and the Bödmeren population is 0.017 (p = 0.306) whereas FST values for other pairwise comparisons including CSP1–6 and A. applanata are all >0.66 indicating that the northern European strains and the Bödmeren population of P. fortinii s.s. belong to the same CSP (data not shown). This result is in accordance with previous findings that show that variation is high within populations but small among populations of the same species of P. fortinii s.l. or other ascomycetes (Grünig et al 2006, Linde et al 2002, Salamati et al 2000).
Differences in morphological and phenotypic characters.—.
Morphological differences useful for species differentiation were sought. In contrast to the original description of P. fortinii (Wang and Wilcox 1985), we observed a high variability in the morphology of conidiophores ranging from well developed and melanized conidiophores as seen by Wang and Wilcox (1985) to single hyaline phialides sitting directly on hyphae and producing conidia. The high variability within CSP did not let us identify distinct morphological features among CSP. Nevertheless some types of conidiophores were observed more often in some CSP than others. However the usefulness of these characteristics for the classification of species is limited because they do not let us unequivocally assign strains to species. In addition classification based on morphology is impossible for some strains because they do not sporulate despite extended incubation at low temperature.
Extracellular enzymes and substrate utilization has been tested for a limited number of strains only (Caldwell et al 2000, Currah and Tsuneda 1993, Schulz et al 2002). Ahlich-Schlegel (1997) tested the production of extracellular enzymes and fungicide resistance of 192 strains belonging to A. applanata and P. fortinii s.l. and found statistically significant differences between A. applanata and P. fortinii s.l. However the subdivision of P. fortinii into several CSP was not known when Ahlich-Schlegel (1997) performed her study. In the present study we showed that CSP of P. fortinii statistically differ significantly in phenotypic characters such as growth response on different media and extracellular enzyme production. Differences in cycloheximide tolerance were distinct, and even closely related species such as P. subalpina and P. fortinii s.s. or P. letzii and P. europaea were affected significantly different. In addition significant differences between some of the species were observed also for other phenotypic traits as the proteolytic activity, the production of laccases, amylases and cytochrome oxydases. In contrast to Schulz et al (2002) but in agreement with Ahlich-Schlegel (1997) amylase activity was not observed for P. fortinii s.l. Of note, the strain used by Schulz et al (2002) showed no amylase activity in our laboratory. Significantly more A. applanata strains than P. fortinii s.l. strains were active producers of extracellular enzymes such as lipases, amylases and laccases confirming the finding of Ahlich-Schlegel (1997). In contrast strains of A. applanata were more sensitive to antibiotics and showed a low growth rate on MEA and WA (Grünig and Sieber 2005). Existence of phenotypic differences among CSP of P. fortinii s.l. indicate an advanced stage of speciation (Fisher et al 2002, Taylor et al 2000). Although the morphological and physiological characteristics are not absolutely reliable for the differentiation of CSP, they are suitable for an initial assignment of cultures to species.
Divergence times of species, distribution and diversity.— –
Of interest, diversity measures for CSP of P. fortinii s.l. either calculated based on sequence data or single-copy RFLP data are similar (TABLE IV) and correspond with the known biogeographic distribution of the CSP. Species with small diversity measures such as P. turiciensis (CSP1) and P. uotolensis (CSP5) were found in study sites in a comparatively small area of about 100 km2 around Zürich (TABLE V). In contrast P. europaea (CSP3), P. subalpina (CSP6) and P. fortinii s.s. (CSP7), which possess a higher genetic diversity, seem to be more widely distributed and were found in collections from several European countries (Grünig unpublished). It unfortunately was not possible to transform the relative divergence times into an absolute timescale because calibration was not possible since neither the mutation rates nor a documented historical event are known.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: thomas.sieber{at}env.ethz.ch
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