This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grünig, C. R. Articles by Sieber, T. N. Search for Related Content PubMed Articles by Grünig, C. R. Articles by Sieber, T. N. Agricola Articles by Grünig, C. R. Articles by Sieber, T. N.

Molecular and phenotypic description of the widespread root symbiont Acephala applanata gen. et sp. nov., formerly known as dark-septate endophyte Type 1

     Swiss Federal Institute of Technology, Department of Environmental Sciences, Forest Pathology and Dendrology, ETH-Zentrum, CH-8092 Zürich, Switzerland

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 

Acephala applanata gen. et sp. nov. is described. A. applanata is a dark-septate endophyte (DSE) of conifer roots and belongs to the Phialocephala fortinii species complex. Several genetic markers, including isozymes, inter-simple-sequence-repeat (ISSR) fingerprints, single-copy restriction fragment length polymorphisms (RFLP) and sequences of the internal transcribed spacers (ITS), let us unambiguously separate isolates of A. applanata from isolates of P. fortinii s.l. and other dark-septate endophytes. Alleles at four RFLP loci and two fixed nucleotides in the ITS region were diagnostic for A. applanata. One of the fixed nucleotides resulted in the addition of an Afa I restriction site. PCR amplification with primers prITS4 and the newly developed primer PF_ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and restriction digestion with Afa I yielded three fragments (203 bp, 117 bp, 56 bp) in A. applanata but only two (260 bp and 117 bp) in P. fortinii s.l. Population differentiation (G_ST) between A. applanata and other cryptic species of P. fortinii was pronounced, and the index of association (I_A) did not deviate significantly from zero, showing that recombination occurs or had occurred in A. applanata. Although isolates of A. applanata never were observed to sporulate, it can be distinguished morphologically from P. fortinii s.l. by the scarcity of aerial mycelium, significantly slower growth and denser mycelium on cellophane overlaid on water agar. These phenotypic characteristics, combined with diagnostic RFLP alleles and/or PCR-RFLP of the ITS fragment with the fixed Afa I restriction site, unequivocally allow identification of A. applanata.

Key words: biodiversity, cryptic species, dark-septate endophytes, mycorrhiza, polyphasic taxonomy, recombination, root symbionts, sterile mycelia

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Classical taxonomic classification of fungal species is based on the morphology of sexual or asexual reproductive structures and also is called the morphological species recognition (MSR) (Taylor et al 2000). MSR has several shortcomings. For example morphological differences between closely related species are often small and many species were described based on one or a few isolates only, not considering adequately the within-taxon variability. In addition the reproductive structures of many species show relatively small numbers of morphological characters useful to differentiate species (Brasier 1987). Therefore misclassifications of fungi cannot be excluded when the classical morphological approach is chosen only ( Jumpponen and Trappe 1998b, Sieber 2002). Further complicating the situation is the fact that many fungal isolates or species remain sterile and classification in the classical sense, therefore, is impossible. Nevertheless some sterile fungi were described as species based solely on morphology (e.g. Cenococcum geophilum Fr. [Fries 1829]). Molecular genetic methods today offer additional tools for species definition and recognition as, for example, the biological species recognition (BSR) with mating assays or phylogenetic species recognition (PSR) using the genealogical concordance concept (Taylor et al 2000).

These methods were used successfully to characterize morphologically indistinguishable species (Brasier and Kirk 1993, Brasier and Mehrotra 1995, Fisher et al 2002, Koufopanou et al 2001, Koufopanou et al 1997, O’Donnell et al 2004a). However even these methods will not solve all problems regarding the definition of species. Species are evolving continuously, and sometimes new species may establish and differentiate from old ones (e.g. by selection or genetic drift). On the other hand hybridization between species or even higher taxonomic units may counteract the differentiation processes. It always will be difficult to define species boundaries in an evolutionary continuum independently of the methods used (de Meeus et al 2003). However classification is an important prerequisite to study the ecology and behavior of organisms properly.

Dark-septate endophytes (DSE) are among the most widely distributed fungal endophytes found in roots of plants. So far DSE were isolated from more than 600 plant species ( Jumpponen and Trappe 1998b). DSE are characterized by their darkly pigmented and septate mycelia, which distinguish them from members of the Glomeromycota known to form endomycorrhizae and from other endophytes with hyaline hyphae. Classification of DSE often is difficult because many strains sporulate only under specific conditions or remain sterile (Wang and Wilcox 1985). Nevertheless it was possible to identify Phialocephala fortinii as an important representative of the DSE complex (Ahlich and Sieber 1996, Ahlich-Schlegel 1997, Sieber 2002, Stoyke et al 1992). P. fortinii is the dominant endophyte in roots of species belonging to the Pinaceae and recently was shown to occur also in the rhizoid environment of the liverwort Cephaloziella varians in Antarctica ( Jumpponen et al 2003). The wide distribution of this fungus contrasts with the knowledge of its enigmatic ecological role. P. fortinii may function as a mycorrhizal fungus and mediate nutrient uptake, synthesize secondary metabolites, enhance plant growth and/or play an important role in plant defense against root pathogens (Fernando and Currah 1996, Jumpponen et al 1998, Jumpponen and Trappe 1998a, O’Dell et al 1993, Yu et al 2001). On the other hand it may behave as an opportunistic pathogen (Wilcox and Wang 1987). It is unlikely however that P. fortinii is a primary pathogen in view of its widespread occurrence and abundance. Much of the contradictory results received in previous studies may be explained by the use of undefined isolates. Thus it is advantageous to examine the variability of P. fortinii and, if possible, to classify them before studying the function of root-P. fortinii symbioses.

Ahlich and Sieber (1996) classified DSE collected throughout Europe based on culture morphology. One morphotype, called Type 1, was distinctly different from all other isolates and was characterized by the absence of aerial mycelium and slower growth. Whereas at least some isolates of the other morphotypes sporulated after prolonged cold treatment and were identified as P. fortinii, strains of Type 1 remained sterile. Type 1 was distinctly different from other P. fortinii isolates based on isozyme and ISSR-PCR data (Ahlich-Schlegel 1997, Grünig et al 2001, Sieber 2002). Single-copy RFLP analysis on selected strains of Type 1 and P. fortinii further confirmed these findings (Grünig et al 2003). Subsequent phylogenetic analysis of the ITS regions of the rDNA showed that Type 1 isolates form a separate and well supported clade within P. fortinii strains and have Phialocephala compacta, P. dimorphospora and P. scopiformis as closest relatives (Grünig et al 2002b).

Recent population genetic studies suggest that P. fortinii is composed of several cryptic species that occur sympatrically in the same forest site and even in the same root fragment (Grünig et al 2004, Sieber and Grünig 2005). Sieber and Grünig (2005) estimated that at least eight cryptic species exist within P. fortinii s.l. in Europe based on RFLP data. In contrast to other species found in this complex Type 1 can be identified relatively easily because of its distinct culture morphology. The aim of the present study was to characterize and describe Type 1 as a new species based on morphological and molecular data.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Origin of isolate.— – Two collections of isolates were included. Collection A contained isolates previously collected and classified based on culture morphology by Ahlich and Sieber (1996) and Grünig et al (2002a). In this collection isolates with sparse aerial mycelium typical for Type 1, an isolate with dense cotton-like aerial mycelium typical for P. fortinii and isolates with intermediate aerial mycelium were included (TABLE I). All strains had been collected throughout Europe except for one Canadian isolate from roots of Tsuga heterophylla (Ahlich-Schlegel 1997).

Culture morphology and determination of growth rate.— – Mycelial growth was studied for Collection A as follows: Each strain was cultivated on malt-extract agar (MA; 2% [w/v] malt extract, 1.5% agar) and cellophane overlaid on 1.5% water agar (CW). Strains were incubated 20 d at 20 C and culture diameter was determined as the average of measurements along two perpendicular imaginary lines intersecting in the center of each of two independent cultures of each strain. In addition cultures were classified based on the quality of the aerial mycelium.

DNA extraction.— – Strains of both collections were grown in Erlenmeyer flasks containing 50 mL 2% (w/v) malt extract broth 15–18 d at 20 C. Mycelia were harvested by filtration and lyophilized. Approximately 50–80 mg of lyophilized mycelium was ground and DNA was extracted as described previously (Grünig et al 2003). DNA quality and concentration were estimated by gel electrophoresis (0.8% agarose gels in 0.5 x TBE at 2.5 V cm^–1 for 1 h, stained in a 10 µg mL^–1 ethidium bromide bath and viewed by UV fluorescence).

ISSR-PCR.— – Collection A strains were subjected to ISSR-PCR using primers DHB(CGA)₅ [CGA] and DD(CCA)₅ [CCA] as described previously (Grünig et al 2002a). After an initial denaturation step for 2 min at 94 C, 35 cycles were performed each consisting of a denaturation step at 94 C for 1 min, an annealing step at 53.5 C for 1 min and an extension step at 72 C for 1 min followed by a final extension step for 10 min at 72 C. PCR products were analyzed on 1% agarose 0.5 x TBE gels.

Single-copy RFLP analysis.— – All Type 1 strains from Collections A and B were analyzed with 11 single-copy RFLP probes (Grünig et al 2003). Fungal DNA (5 µg) was digested with 50 units of the restriction enzyme Hind III. Digested DNA was separated on 0.8% agarose 1 x TBE gels for 19 h (2.4 V cm^–1). After partial hydrolysis in 0.25 M HCl for 15 min DNA was blotted onto Hybond^TM-N⁺ membranes (Amersham, Dübendorf, Switzerland) with the capillary transfer method under alkaline conditions (Sambrook and Russell 2001).

Probes were labeled with [–³²P] dCTP using a nick translation kit (Invitrogen, Basel, Switzerland) following the manufacturer’s instructions. Blots were prehybridized 5–8 h at 65 C and hybridized overnight at 65 C before they were washed twice with 1 x SSC, 0.1% SDS, twice with 0.2 x SSC, 0.1% SDS and twice with 0.1 x SSC, 0.1% SDS. Membranes were exposed to X-ray films (Kodak BioMax MS) 24–72 h at –80 C.

Sequencing of ITS1–5.8S-ITS2 region of rDNA.— – rDNA ITS regions were sequenced for selected strains of Collection A. ITS regions were amplified with primers prITS4 and prITS5 (White et al 1990) in a 30 µL reaction volume with ca. 1 ng template DNA. Single bands were gel purified with the QIAquik^® gel extraction kit (QIAGEN, Basel, Switzerland), and sequences were determined with primer prITS4 at Microsynth (Balgach, Switzerland).

Analysis of ISSR-PCR and RFLP data.— – Presence/absence of ISSR fragments with identical length was scored and pairwise distances among strains were calculated with the Jaccard algorithm. The distance matrix was subjected to hierarchical clustering with the single linkage algorithm.

Restriction fragment length polymorphism (RFLP) data were scored based on the presence of RFLP fragments with similar sequence but different size, and each fragment of a particular size was assumed to represent a specific allele at a single genetic locus. The RFLP datasets were subjected to cluster analysis as described previously (Grünig et al 2003). ISSR-PCR and RFLP data were combined for Type 1 strains of Collection A. ISSR fragments that were polymorphic were treated as loci with two alleles (present/absent) and added to the RFLP dataset. Analysis was performed as described for the RFLP data.

For the clone-corrected dataset of collection B, average gene diversity H (Nei 1987), number of polymorphic loci S (frequency of most common allele 95%), potential number of multilocus haplotypes [MLH] (M_pot) and effective number of MLH (M_eff) (Crow and Kimura 1970) were calculated and compared with values of other cryptic species within the P. fortinii complex (Grünig et al 2004). In addition allele frequencies of single-copy RFLP loci were compared with allele frequencies of 196 MLH representing approximately 850 isolates from the P. fortinii complex to identify Type 1-specific alleles or allele combinations. Pairwise G_ST values as estimated by theta (Weir 1996) were calculated between populations of known cryptic species of P. fortinii s.l. and the clone-corrected dataset of collection B with the software Arlequin (Schneider et al 2000).

Associations among loci were analyzed with the index of association (I_A) as implemented in MultiLocus 1.3 (Agapow and Burt 2001). The significance of I_A was tested by comparing the observed I_A with the distribution of the I_A expected under the null hypothesis of random mating from 1000 randomized datasets.

Sequence analysis.— – Sequences were aligned with Clustal W and adjusted manually. Modeltest version 3.06 served to determine the model of nucleotide substitution that best fits the data (Posada and Crandall 1998). Phialocephala compacta (CBS 507.94, AF486125 [GenBank] ) was shown to be the closest relative of P. fortinii s.l. (Grünig et al 2002b) and was used as outgroup. Maximum likelihood (ML) analyses using full heuristic searches and bootstrapping to generate 200 pseudosamples for accuracy estimation were performed in PAUP* 4.0 b10 (Swofford 1998) with the substitution model identified by Modeltest.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
ISSR-PCR analysis of collection A.— – Results of the ISSR analysis of Collection A are presented (FIG. 1A). Variability of ISSR fingerprints using primers CGA and CCA were pronounced. Two main clusters were visible in the dendrogram. The larger cluster comprised 18 strains with sparse aerial mycelium (Cluster I), whereas the smaller cluster contained strains with aerial mycelium intermediate between sparse and dense (Cluster II). Four strains were positioned outside the two main clusters including the Canadian and the Finnish isolate. Within Cluster I three groups of strains showed identical ISSR banding patterns. Of note, two of these groups (K92 078/K92 079/K92 170 and K92 081/K92 082/K93 270) were composed of strains that were sampled from different locations. Examples of ISSR fingerprinting patterns for strains in Cluster I using primer CGA are provided (FIG. 1D).

View larger version (56K): [in this window] [in a new window]   FIG. 1. ISSR-PCR and single-copy RFLP analysis of 28 DSE strains of collection A. Strain number in boldface indicate strains from which the ITS regions were sequenced. A. Result of the cluster analysis of the ISSR-PCR fingerprinting data using the single linkage algorithm. Cluster I represents 18 strains with sparse aerial mycelium. Cluster II contains six strains with "intermediate" aerial mycelium (TABLE I). B. Result of the cluster analysis of the RFLP data of 11 single-copy loci. Clusters I and II correspond to the same clusters in FIG. 1A. C. Result of the cluster analysis of the combined ISSR-PCR and single-copy RFLP dataset of Cluster I strains. Symbols on the right indicate the two ITS haplotypes found among Cluster I strains. ITS haplotype I-2 (black spots) differed by three additional thymine bases at the 5' end of the ITS2 sequence from ITS haplotype I-1 (see text and TABLE I). D. Examples of ISSR-PCR fingerprinting patterns for a selection of strains from Cluster I using primer CGA. Numbers on the right indicate the length of marker fragments (X, Roche Diagnostics) in base pairs.

ITS haplotypes and phylogenetic analysis of Collection A.— – Two ITS haplotypes were observed among the examined strains of Cluster I (FIG. 1C). The two haplotypes (I-1 and I-2, TABLE I) differed from each other by three thymine bases at the 5' end of the ITS 2 sequence. Whereas the three thymine bases were absent in the majority of isolates (eight of 11), they were present in strains K93 079, K93 170 and K93 176. A second poly-T motif of six thymine bases was constantly present at the 3' end of the ITS 2 sequence in all strains of Cluster I. This poly-T motif occurs in P. fortinii s.l. too, but its presence/absence and length varies highly among strains (Grünig et al 2002b). The poly-T motif at the 3' end of the ITS 2 therefore was excluded from phylogenetic analysis. In addition to sequence data received in this study 27 ITS haplotypes detected among 49 P. fortinii s.l. strains in earlier studies were included (Grünig et al 2004, Grünig et al 2002b). The K80+ G model (Kimura 1980) best fitted the observed nucleotide substitution pattern. It is characterized by equal frequencies of each of the four nucleotides, a transition : transversion ratio of 5.3896 and a gamma-shape parameter () of the gamma distribution (G) of 0.2038. Phylogenetic analysis of the ITS rDNA regions confirmed the findings reported above (FIG. 2). Strains of Cluster I formed a well supported clade (90%) within the ITS sequences of other cryptic species of P. fortinii. Strains of ISSR and RFLP Cluster II similarly were separated from other P. fortinii strains (66% bootstrap support). Two transitions were found within the ITS sequence that were shared exclusively by isolates of Cluster I (position 142: A instead of G; position 415: T instead of C; reference sequence GenBank AY078145 [GenBank] , K92 113).

View larger version (24K): [in this window] [in a new window]   FIG. 2. Phylogenetic relationship among 27 ITS haplotypes of Phialocephala fortinii s.l. representing sequences of 49 isolates and six ITS haplotypes derived from this study (boldface). The K80 + G substitution model (Kimura 1980) was used to calculate the 50% majority rule consensus tree (200 replicates). Groups compatible with the 50% majority rule were included. Phialocephala compacta was chosen as outgroup. Bootstrap values >50% are indicated above the branches. ITS haplotypes found among isolates of Clusters I and II (FIG. 1) are indicated on the right.

View larger version (9K): [in this window] [in a new window]   FIG. 3. Variability of colony diameter attained by strains of Clusters I and II (FIG. 1) and selected strains of P. fortinii s.l. after 20 d at 20 C on 2% (w/v) malt-extract agar (MA) (A) and water agar overlaid with cellophane (CW) (B). Box plots according to Stahel (2002).

View larger version (118K): [in this window] [in a new window]   FIG. 5. Comparison of the culture morphology of the type strains of P. fortinii (CBS 443.86) (A, C) and A. applanata (CBS 109321) (B, D). A, B. Colonies on 2% (w/v) malt-extract agar after 20 d at 20 C in the dark (bar = 1 cm). C, D. Density of mycelium of colonies grown 30 d at 20 C in the dark on cellophane overlaid on 1.5% water agar at a distance of 10 mm from the inoculum (Bar = 25 µm).

View larger version (20K): [in this window] [in a new window]   FIG. 4. Frequency distribution of alleles at selected single-copy RFLP loci of 196 MLH of P. fortinii s.l. (white bars) collected in Europe and of 23 MLH of A. applanata (black bars) detected in the population Bödmerenwald. Alleles at loci pPF-018 and pPF-74 were diagnostic for A. applanata.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

Acephala Grünig & Sieber gen. nov.

Hyphomycetibus dematiaceis pertinens. Phialocephalae Kendrick affinis et similis sed sporarum generatio nunquam visa.

Species typica: A. applanata Grünig & Sieber sp. nov.

Dematiaceous hyphomycetes related to Phialocephala Kendrick but sporulation never observed.

Type species: A. applanata Grünig & Sieber sp. nov. (see below)

Etymology: akephalon (Greek): headless, because of the lack of heads of phialides

Acephala applanata Grünig & Sieber, sp. nov.

Phialocephalae fortinii Wang et Wilcox similis sed ab hac specie ob mycelium aereum sparsum, coloniam in malto agaroso lentior crescentem, sporulationem ignotam, transitionem in acido DNA vocato in positione 142 adeninam et in positione 415 thyminam vice guaninae et cytosinae ut in Phialocephalae fortinii sequentia AY078145 [GenBank] in deposito GenBank vocato praebentem differt, thymina in positione 415 novum locum restrictionis enzymatis Afa I formanti. PCR amplificatio nucleotidum cum sequentiis PCR-PF_ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) et PCR-prITS4 et subsequenti digestione enzymatis Afa I vocati fragmenta tria 203 fasciarum, 117 fasciarum et 56 fasciarum praebens. RFLP loci propriis typicisque allelonibus: pPF-008 (allelone: 4250 et 3650 bp vel 4250 et 3750 bp), pPF-018 (allelone: 4900 bp), pPF-036 (allelone: 5150 et 3900 bp), pPF-075 (allelonibus: 3920 et 2140 bp o 4240 et 1730 bp).

HOLOTYPUS: cultura sicca ex radicibus vivis Piceae abietis, Helvetia, Bödmerenwald, 17.9.1992, T.N. Sieber, herbarium ZT; cultura viva numero CBS 109321 deposita Centraalbureau voor Schimmelcultures, Utrecht, Batavia.

Acephala applanata is similar to Phialocephala fortinii, but the two species differ in some phenotypic and molecular characteristics. These phenotypic characteristics diagnostic for A. applanata let us discriminate the two species: aerial mycelium of colonies on malt-extract agar sparse, sporulation absent, slower growth and mycelium denser on cellophane overlaid on water agar (FIG. 5). The two species differ molecularly in two transitions in the DNA of the ITS regions. Nucleotides are an adenine (A) at position 142 and a thymine (T) at position 415 in A. applanata in contrast to guanine (G) and cytosine (C) in P. fortinii s.l., respectively (position numbers correspond to the nucleotide positions in the sequence AY078145 [GenBank] of the type deposited at GenBank). The thymine at position 415 results in the addition of an Afa I restriction site. PCR amplification with primers PCR-PF_ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and PCR-prITS4 followed by digestion with Afa I yields three fragments, one of 203 base pairs, one of 117 base pairs and one of 56 base pairs (FIG. 6). A. applanata has fixed and therefore diagnostic alleles (approximate length of the DNA fragment[s] in base pairs given in brackets) at these RFLP loci (loci as defined in Grünig et al 2003, 2004): pPF-008 (alleles: 4250 and 3650 bp or 4250 and 3750 bp), pPF-018 (allele: 4900 bp), pPF-036 (allele: 5150 and 3900 bp), pPF-075 (alleles: 3920 and 2140 bp or 4240 and 1730 bp).

View larger version (76K): [in this window] [in a new window]   FIG. 6. Detection of the fixed transition in the ITS2 sequence of A. applanata. PCR fragment amplified with primers PF_ITS_F and prITS4 was digested with restriction enzyme Afa I and separated on a 2% agarose gel. Numbers on the right indicate the length of marker fragments (XIII, Roche Diagnostics) in base pairs.

Etymology. applanata; the surface mycelium is clearly visible because the aerial mycelium is sparse or absent, the cultures thus appear to be flat, smooth.

Specimens examined: K92 055, K92 056, K92 078, K92 079, K92 081, K92 082, K92 113, K92 131, K92 169, K92 170, K92 187, K92 188, K93 131, K93 176, K93 240, K93 244, K93 270, K93 321 (TABLE I).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Dark-septate endophytes (DSE) are isolated frequently from surface-sterilized, healthy roots of woody plants. Most DSE from tree and shrub roots belong to P. fortinii, which recently was shown to represent a species complex of several cryptic species (Grünig et al 2004). Whereas cryptic species of P. fortinii are morphologically indistinguishable (N. Nüssli unpubl) other DSE, isolated from the same habitat, are morphologically similar to P. fortinii s.l. but differ distinctly from it in some colonial characteristics. Ahlich and Sieber (1996) made a first attempt to classify DSE according to colonial morphology and defined four different types. Isolates of Types 2, 3 and 4 turned up to represent species of P. fortinii s.l. Type 1 however was distinctly different from P. fortinii because the surface mycelium of colonies on malt-extract agar was visible in contrast to that of P. fortinii, which was covered by a dense olive-green to gray aerial mycelium. Later the distinction of Type 1 and P. fortinii was confirmed by isozyme analysis using seven enzyme systems and ISSR-PCR (Ahlich-Schlegel 1997, Grünig et al 2001, Sieber 2002). Morphotypes with an aerial mycelium intermediate between Type 1 and P. fortinii also occasionally occurred and proved to have distinctly different isozyme and ISSR-PCR fingerprints. Phylogenetic analysis of DNA sequences of several P. fortinii and Type 1 strains and two strains with intermediate morphology suggested that P. fortinii s.l. and Type 1 are more closely related to each other than to the intermediate DSE strains (Grünig et al 2002b). This finding could be confirmed in this study with eight intermediate strains. They were distinctly different from Type 1 and P. fortinii s.l. according to ISSR-PCR, RFLP and phylogenetic analysis (FIGS. 1A, B and 2). Variability was higher among the intermediate strains than among the Type 1 strains as inferred from cluster analysis of the ISSR-PCR and RFLP patterns. In fact, six of the eight strains formed a cluster (Cluster II in FIG. 1A, B) whereas strains K93 333 and K92 035 were outside. K93 333 turned up to be a Cadophora species and K92 035 is special probably because it is the only non-European strain. Variability within Type 1 (Cluster I) was small although the strains originated from various hosts and remote places (TABLE I). Type 1 strains formed a compact and distinct group in all analyses. However on the population-level the variability of Type 1 as measured by M_pot and M_eff was in the range of other cryptic species of P. fortinii (TABLE II). Assignment of species status to Type 1 (i.e. A. applanate) was considered to be justified because: (i) RFLP probes specific for P. fortinii s.l. (Grünig et al 2003) hybridize to DNA fragments diagnostic for A. applanata (FIG. 4) representing fixed alleles; (ii) nucleotides at two positions in the ITS regions of the chromosomal rDNA are fixed in A. applanata; (iii) A. applanata forms recombining populations that are reproductively isolated from sympatric and allopatric populations of other cryptic species of P. fortinii (TABLE III); (iv) A. applanata is characterized by an unique and characteristic ISSR-PCR fingerprint (FIG. 1D); (v) A. applanata differs from P. fortinii by slower growth and sparse aerial mycelium.

RFLP analysis revealed fixed alleles in A. applanata at some loci. These alleles thus are diagnostic for the new species. Alleles at some other loci were shared between P. fortinii s.l. and A. applanata but occurred with significantly different frequencies in populations of the two taxa (FIG. 4). Differentiation of A. applanata populations from sympatric and allopatric populations of cryptic species of P. fortinii was high based on RFLP data (G_ST > 0.65) indicating reproductive isolation (TABLE III). Isolation between A. applanata and P. fortinii s.l. is not complete, however, because the two taxa share several alleles and the probability of these shared alleles being the products of convergence is minimal. Phylogenetic analyses using multigene genealogies could help to resolve this problem (Couch and Kohn 2002, Fisher et al 2002).

RFLPs were used in several other studies to identify genetically distinct subgroups within species. Species status often was assigned to such subgroups during follow-up studies using genealogical concordance phylogenetic species recognition (GCPSR) (Couch and Kohn 2002, Fisher et al 2002, Taylor et al 2000). Borromeo et al (1993) identified four distinct groups of Magnaporthe isolates with host-specificity for certain grass and sedge species using RFLPs and suggested that the lineages from Digitaria and rice represent distinct species. Later species status was assigned to these two lineages based on a multilocus gene genealogy (Couch and Kohn 2002). Zimmerman et al (1994) used RFLPs to study the intraspecific variability of clinical isolates of Coccidioides immitis and identified two divergent groups. These two groups recently were described as separate species, C. immitis and C. posadasii, using phylogenetic analyses of microsatellite loci and gene genealogies (Fisher et al 2002).

Thus the RFLP approach used by Borromeo et al (1993) and Zimmerman et al (1994) proved to be almost as good to discriminate cryptic species as the more sophisticated methods used by Fisher et al (2002) and Couch and Kohn (2002). The combination of three marker types (isozymes, ISSR-PCR and single-copy RFLP), which all separated strains of A. applanata from isolates of P. fortinii s.l., provided strong evidence for the separation of these two taxa.

The DNA sequences of the ITS region of A. applanata and P. fortinii s.l., exclusively the poly-T motif at the 3' end of ITS 2, differed by 4–12 bp depending on the strain of P. fortinii (FIG. 1C). In addition nucleotides at two positions were fixed and therefore diagnostic for A. applanata (FIG. 2, Cluster I). The second polymorphism can be detected by PCR-RFLP analysis using primer pairs Pf_ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and prITS4 (White et al 1990) in conjunction with the restriction enzyme Rsa I or Afa I. The expected fragments will be approximately 260 bp and 117 bp for strains of P. fortinii s.l., whereas strains of A. applanata will show three fragments (203 bp, 117 bp and 56 bp) (FIG. 5).

Acephala applanata is characterized by a unique and easily discernible ISSR-PCR fingerprint (FIG. 1D). In contrast the fingerprints of P. fortinii s.l. and intermediate DSE varied considerably among isolates and were distinctly different from those of A. applanata. However ISSR-PCR fingerprints can be used only for species identification in conjunction with other diagnostic methods because their uniqueness among the mycobiota is not warranted.

In addition to reduced growth rates and sparse aerial mycelium, Acephala applanata differed from P. fortinii and intermediate DSE in substrate-use patterns (Ahlich-Schlegel 1997). Significantly more A. applanata strains were active producers of extracellular enzymes such as lipases, amylases and laccases. Existence of phenotypic differences among A. applanata and P. fortinii s.l. indicate an advanced stage of speciation because phenotypic differences are not expected to occur between species that only recently were isolated genetically (Taylor et al 2000). Although these characteristics are the least reliable ones, growth rates and presence of aerial mycelium are excellent characteristic for an initial classification of DSE colonies and can be used in combination with ISSR-PCR, ITS sequencing or PCR-RFLP analysis of the ITS region using restriction enzyme Rsa I or Afa I to classify A. applanata accurately.

While molecular methods are employed increasingly to detect and describe morphologically indistinguishable or at least very similar species within meiosporic and mitosporic fungal species (Couch and Kohn 2002, Fisher et al 2002, Geiser et al 1998, Koufopanou et al 1997, O’Donnell et al 1998, O’Donnell et al 2004b), assignment of species status to apparently or truly sterile mycelia using these methods is still uncommon. Some sterile mycelia were described as species using classical morphological methods, e.g. Cenococcum geophilum Fr., a dematiaceous ectomycorrhizal ascomycete (Fries 1829), or rhizomorphae which today are known to serve for dispersal and during the pathogenic phase of Armillaria species (Schmitz 1848). Melin (1923) introduced the trinomial Mycelium radicis atrovirens (MRA) for sterile, dark-septate root-colonizing fungi. This name however is not valid according to the International Code of Botanical Nomenclature (Greuter et al 2000). Other examples of descriptions of sterile mycelia were provided by Rabenhorst (1963). Assignment of species status to the sterile A. applanata using molecular methods seemed justified because discrimination from other DSE is easy due to the low variability within the taxon and the availability of distinct diagnostic markers.

In addition to the description of A. applanata as a new species this study provided some other interesting insights into the biology and ecology of DSE. The A. applanata population at Bödmerenwald showed distinct preference for roots of Norway spruce (Picea abies). Only two of 174 strains were isolated from roots of Vaccinium myrtillus. This contrasts with the frequency of isolation of other cryptic species of P. fortinii, which were equally or more frequently isolated from V. myrtillus at the same site.

A. applanata showed a tendency to be more abundant at higher altitudes, at least in Switzerland (Ahlich and Sieber 1996). Acephala applanata, however, was most abundant in a collection from Knesebeck, northern Germany, at an altitude of less than 100 m. Similarly, the hypothesis that the frequency of A. applanata increases with increasing altitude and/or latitude is not tenable because A. applanata did not occur in a representative sample further north at 69°N (Kevo, Finland).

A. applanata strains K92 079 and K92 170 were identical in all characteristics tested in this study. Thus the genotypes of these two strains were assumed to be identical. The strains were isolated from fine roots of Norway spruce at sites more than 100 km distant. More important the sites are separated by the crest of the Alps (i.e. one site is on the northern side of the range the other on the southern side). We suspect that genotype flow had occurred between the two. Occurrence of genotype flow had been inferred from RFLP and ISSR-PCR patterns for other cryptic species of P. fortinii by Grünig et al (2004). However the sites were separated by approximately 10 km only in the latter study. It is not known how genotype flow occurs in these fungi because spores were never detected in A. applanata and conidia were never observed to germinate in P. fortinii. However several alternative explanations exist. Sclerotia or colonized root debris may serve as dispersal units that can be disseminated by wind, animals (birds, insects) or man (silvicultural practices). It is a challenge to discover more of the secret lives of these ubiquitous and ecologically important dark-septate root endophytes.

	ACKNOWLEDGMENTS

 
We thank Orlando Petrini, Comano, Switzerland, for the Latin diagnosis and Angelo Duò, Swiss Federal Institute of Technology, Zürich, Switzerland, for excellent technical assistance.

	FOOTNOTES

 
Accepted for publication February 18, 2005.

¹ Corresponding author. E-mail: thomas.sieber{at}env.ethz.ch

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Agapow PM, Burt A. 2001. Indices of multilocus linkage disequilibrium. Mol Ecol Notes 1:101–102.[CrossRef]

Ahlich K, Sieber TN. 1996. The profusion of dark septate endophytic fungi in non-ectomycorrhizal fine roots of forest trees and shrubs. New Phytol 132:259–270.[CrossRef]

Ahlich-Schlegel K. 1997. Vorkommen und Charakterisierung von dunklen, septierten Hyphomyceten (DSH) in Gehölzwurzeln [Doctoral dissertation]. ETH, Zürich.

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402.[Abstract/Free Full Text]

Borromeo ES, Nelson RJ, Bonman JM, Leung H. 1993. Genetic differentiation among isolates of Pyricularia infecting rice and weed hosts. Phytopathology 83:393–399.[CrossRef]

Brasier CM. 1987. The dynamics in fungal speciation. In: Rayner ADM, Brasier CM, Moore D, eds. Evolutionary biology of the fungi. Cambridge: Cambridge University Press. p 232–260.

———, Kirk SA. 1993. Sibling species within Ophiostoma piceae. Mycol Res 97:811–816.

———, Mehrotra MD. 1995. Ophiostoma himal-ulmi sp. nov., a new species of Dutch elm disease fungus endemic to the Himalayas. Mycol Res 99:205–215.

Couch BC, Kohn LM. 2002. A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea. Mycologia 94:683–693.[Abstract/Free Full Text]

Crow JF, Kimura M. 1970. An introduction to population genetics. New York: Harper & Row.

de Meeus T, Durand P, Renaud F. 2003. Species concepts: What for? Trend Parasitology 19:425–427.[CrossRef]

Fernando AA, Currah RS. 1996. A comparative study of the effects of the root endophytes Leptodontidium orchidicola and Phialocephala fortinii (Fungi Imperfecti) on the growth of some subalpine plants in culture. Can J Botany 74:1071–1078.[CrossRef]

Fisher MC, Koenig GL, White TJ, Taylor JW. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94: 73–84.[Abstract/Free Full Text]

Fries EM. 1829. Systema mycologicum. Gryphiswaldiae: Sumtibus Ernesti Mauritii.

Geiser DM, Pitt JI, Taylor JW. 1998. Cryptic speciation and recombination in the aflatoxin-producing fungus Aspergillus flavus. Proc Natl Acad Sci USA 95:388–393.[Abstract/Free Full Text]

Greuter W, McNeill J, Barrie FR, Burdet H-M, Demoulin V, Filgueiras TS, Nicolson DH, Silva PC, Skog JE, Trehane P, Turland NJ, Hawksworth DL. 2000. International code of botanical nomenclature (Saint Louis Code). Königstein, Germany: Koeltz Scientific Books.

Grünig CR, Sieber TN, Holdenrieder O. 2001. Characterisation of dark septate endophytic fungi (DSE) using inter-simple-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplification. Mycol Res 105:24–32.[CrossRef]

———, ———, Rogers SO, Holdenrieder O. 2002a. Spatial distribution of dark septate endophytes in a confined forest plot. Mycol Res 106:832–840.[CrossRef]

———, ———, ———, ———. 2002b. Genetic variability among strains of Phialocephala fortinii and phylogenetic analysis of the genus Phialocephala based on rDNA ITS sequence comparisons. Can J Botany 80:1239–1249.[CrossRef]

———, Linde CC, Sieber TN, Rogers SO. 2003. Development of single-copy RFLP markers for population genetic studies of Phialocephala fortinii and closely related taxa. Mycol Res 107:1332–1341.[CrossRef][Medline]

———, McDonald BA, Sieber TN, Rogers SO, Holdenrieder O. 2004. Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species. Fung Genet Biol 41:676–687.[CrossRef]

Jumpponen A, Mattson KG, Trappe JM. 1998. Mycorrhizal functioning of Phialocephala fortinii with Pinus contorta on glacier forefront soil: interactions with soil nitrogen and organic matter. Mykorrhiza 7:261–265.

———, Trappe JM. 1998a. Performance of Pinus contorta inoculated with two strains of root endophytic fungus, Phialocephala fortinii: Effects of synthesis system and glucose concentration. Can J Botany 76:1205–1213.[CrossRef]

———, ———. 1998b. Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi. New Phytol 140:295–310.[CrossRef]

———, Newsham KK, Neises DJ. 2003. Filamentous ascomycetes inhabiting the rhizoid environment of the liverwort Cephaloziella varians in Antarctica are assessed by direct PCR and cloning. Mycologia 95:457–466.[Abstract/Free Full Text]

Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120.[CrossRef][Medline]

Koufopanou V, Burt A, Taylor JW. 1997. Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis. Proc Natl Acad Sci USA 94:5478–5482.[Abstract/Free Full Text]

———, ———, Szaro T, Taylor JW. 2001. Gene genealogies, cryptic species, and molecular evolution in the human pathogen Coccidioides immitis and relatives (Ascomycota, Onygenales). Mol Biol Evol 18:1246–1258.[Abstract/Free Full Text]

Lienert S. 2001. Urwaldreservat Bödmeren. In: Gesellschaft SN, ed. Berichte der Schwyzerischen Naturforschenden Gesellschaft. Ensiedeln: p 83.

Melin E. 1923. Experimentelle Untersuchungen über die Konstitution und Ökologie der Mykorrhizen von Pinus sylvestris L. und Picea abies (L.) Karst. In: Falck R, ed. Mykologische Untersuchungen und Berichte 2. Kassel: Druck und Verlag G. Gottheilt. p 73–334.

Nei M. 1987. Molecular evolutionary genetics. New York: Columbia University Press. 512 p.

O’Dell TE, Massicotte HB, Trappe JM. 1993. Root colonization of Lupinus latifolius Agardh. and Pinus contorta Dougl. by Phialocephala fortinii Wang & Wilcox. New Phytol 124:93–100.[CrossRef]

O’Donnell K, Cigelnik E, Nirenberg HI. 1998. Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90:465–493.[CrossRef]

———, Ward TJ, Geiser DM, Kistler HC, Aoki T. 2004a. Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fung Genet Biol 41:600–623.

O’Donnell KO, Ward TJ, Geiser DM, Kistler HC, Aoki T. 2004b. Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fung Genet Biol 41:600–623.

Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818.[Abstract/Free Full Text]

Rabenhorst L. 1963. Kryptogamen-Flora von Deutschland, Oesterreich und der Schweiz. 1. Band: Die Pilze Deutschlands, Oesterreichs und der Schweiz. Weinheim, Germany: Autorisierter Neudruck, von Verlag J. Cramer.

Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Schmitz J. 1848. Beiträge zur Anatomie und Physiologie der Schwämme. 3. Über den Bau, das Wachstum und einige besondere Lebenserscheinungen der Rhizomorpha fragilis Roth. Linnaea 17:487–535.

Schneider S, Roessli D, Excoffier L. 2000. Arlequin: A software for population genetic analysis. Version 2.001. University of Geneva, Switzerland: Genetics and Biometry Lab, Dept. of Anthropology.

Sieber TN. 2002. Fungal root endophytes. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: the hidden half. New York Basel: Marcel Dekker. p 887–917.

———, Grünig CR. 2005. Biodiversity of fungal root-endophyte communities and populations in particular of the dark septate endophyte Phialocephala fortinii. In: Schulz B, Boyle C, Sieber TN, eds. Microbial Root Endophytes. Berlin: Springer (In press).

Stahel WA. 2002. Statistische Datenanalyse. 4th ed. Braunschweig: Vieweg.

Stoyke G, Egger KN, Currah RS. 1992. Characterization of sterile endophytic fungi from the mycorrhizae of subalpine plants. Can J Botany 70:2009–2016.[CrossRef]

Swofford DL. 1998. Phylogenetic Analysis Using Parsimony (*and other methods). Sunderland, Mass.: Sinauer Associates.

Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC. 2000. Phylogenetic species recognition and species concepts in fungi. Fung Genet Biol 31:21–32.[CrossRef]

Wang CJK, Wilcox HE. 1985. New species of ectendomycorrhizal and pseudomycorrhizal fungi: Phialophora finlandia, Chloridium paucisporum, and Phialocephala fortinii. Mycologia 77:951–958.[CrossRef]

Weir BS. 1996. Genetic data analysis II. Sunderland: Sinauer Associates Inc.

White TJ, Bruns TD, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: a guide to methods and applications. New York, NY: Academic Press. p 315–322.

Wilcox HE, Wang CJK. 1987. Mycorrhizal and pathological associations of dematiaceous fungi in roots of 7-month-old tree seedlings. Can J For Res 17:884–899.[CrossRef]

Yu T, Nassuth A, Peterson RL. 2001. Characterization of the interaction between the dark septate fungus Phialocephala fortinii and Asparagus officinalis roots. Can J Microbiol 47:741–753.[CrossRef][Medline]

Zimmermann CR, Snedker CJ, Pappagianis D. 1994. Characterization of Coccidioides immitis isolates by restriction-fragment-length-polymorphisms. J Clin Microbiol 32:3040–3042.[Abstract/Free Full Text]

This article has been cited by other articles:

				C. R. Grunig, A. Duo, T. N. Sieber, and O. Holdenrieder Assignment of species rank to six reproductively isolated cryptic species of the Phialocephala fortinii s.1.-Acephala applanata species complex. Mycologia, January 1, 2008; 100(1): 47 - 67. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grünig, C. R. Articles by Sieber, T. N. Search for Related Content PubMed Articles by Grünig, C. R. Articles by Sieber, T. N. Agricola Articles by Grünig, C. R. Articles by Sieber, T. N.