Population genetic analysis of Phialocephala fortinii s.l. and Acephala applanata in two undisturbed forests in Switzerland and evidence for new cryptic species

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Abstract

The genetic structure of the root endophytes Phialocephala fortinii s.l. and Acephala applanata was analyzed in two undisturbed forests. A total of 606 strains isolated from surface-sterilized, fine roots of Picea abies and Vaccinium myrtillus were examined. Two new cryptic species of P. fortinii were recognized and host specialization of A. applanata was confirmed. This species was almost exclusively isolated from roots of P. abies. The index of association did not deviate significantly from zero within any population, suggesting that recombination occurs or had occurred. Significant gene but no genotype flow was detected among study sites for P. fortinii s.l. In contrast, several isolates of A. applanata with both identical multi-locus haplotype and identical ISSR fingerprint were found in both study sites indicating genotype flow or a recent common history.

Introduction

Dark septate endophytes (DSE) are among the most widely distributed fungal endophytes of plant roots. So far, DSE have been isolated from more than 600 plant species (Jumpponen and Trappe, 1998a). DSE are characterized by their darkly pigmented and septate mycelia, which allow discriminating them from members of the Glomeromycota known to form endomycorrhizae and from other endophytes with hyaline hyphae. Phialocephala fortinii is the dominant root endophyte in roots of species belonging to the Pinaceae but is also known to colonize roots of broad-leaved trees, shrubs and herbaceous plants (Ahlich and Sieber, 1996, Ahlich-Schlegel, 1997, Sieber, 2002, Stoyke et al., 1992). New reports suggest that P. fortinii is not restricted to fine roots of higher plants. Menkis et al. (2004) reported isolation of P. fortinii from (living) stem bases and stumps of Pinus sylvestris, Betula pendula, and Picea abies. Furthermore, Jumpponen et al. (2003) reported P. fortinii from the rhizoid environment of the liverwort Cephaloziella varians in Antarctica showing the broad distribution of this ubiquitous species.

The ecological significance of P. fortinii remains enigmatic. Conflicting results have been published describing P. fortinii as beneficial, neutral, or pathogenic for different hosts, growing conditions, and fungal strains (Jumpponen and Trappe, 1998b, Jumpponen and Trappe, 1998a, Ma et al., 1998, Stoyke and Currah, 1993, Wilcox and Wang, 1987). Interestingly, P. fortinii was shown recently to form ectomycorrhiza with Populus tremula × Populus tremuloides clones (Kaldorf et al., 2004). The occurrence and frequency of P. fortinii on apparently healthy roots indicates that these endophytes are not primary pathogens (Halmschlager and Kowalski, 2004, Sieber, 2002). The ecological role of P. fortinii may be beyond the paradigms of mycorrhizal and pathogenic associations (Addy et al., 2005) and an important prerequisite in this respect will be the proper identification of isolates.

Recent population genetic studies in Europe suggest that P. fortinii is composed of several cryptic species (CSP). They were named “cryptic” because species were morphologically indistinguishable but population subdivision was high between species and no gametic disequilibrium was detected within species (Grünig et al., 2004). Thus, P. fortinii forms a species complex and until the CSP are described as separate species the addendum “sensu lato” (s.l.) should be used. Cryptic species were shown to occur sympatrically in the same forest plot (Grünig et al., 2004) and even in the same root segment (Queloz et al., 2005, Sieber and Grünig, 2006). One CSP of P. fortinii s.l. was recently described as Acephala applanata (Grünig and Sieber, 2005). In contrast to other CSP, A. applanata does not only possess unique alleles at several single-copy restriction fragment length polymorphism (RFLP) loci but differs from all other CSP of P. fortinii by its distinct culture morphology on malt extract agar. Aerial mycelium is sparse to absent and growth rate is reduced in A. applanata. This species never has been observed to sporulate. Therefore, the genus Acephala was introduced to avoid the awkward situation of placing a fungus that lacks heads of phialides in a genus that is defined on the basis of these features. The occurrence of cryptic species in P. fortinii s.l. was suspected for North American isolates based on AFLP fingerprints (Piercey et al., 2004). However, the low number of strains included in that study did not allow a proper analysis of species boundaries. Interestingly, communities of P. fortinii s.l. seem to be spatially and temporally stable at least over a period of three years. Re-sampling of a community of P. fortinii s.l. showed that a comparatively high number of clones can be re-isolated and that for most re-sampled clones a positive spatial correlation exists (Queloz et al., 2005).

Although gene and genotype flow has been thought to be restricted in P. fortinii s.l. because suitable propagules or vectors are missing or unknown (Sieber and Grünig, 2006), high rates of gene flow and genotype flow were detected between populations of CSP of P. fortinii over short distances (Grünig et al., 2004). A possible explanation may be that nursery plants colonized by P. fortinii s.l. were planted in these forests. Several studies showed that nursery seedlings are, indeed, colonized by dark septate endophytes (DSE) including P. fortinii s.l. (Bloomberg, 1966, Danielson and Visser, 1990, Kernaghan et al., 2003) and that new genotypes belonging to different CSP are probably introduced by planting. Therefore, the number of CSP as well as the genotypic diversity is expected to be higher in plantations than in undisturbed forests. Communities of P. fortinii s.l. in undisturbed forests must be examined and compared to those in plantations to verify this assumption. In addition, communities in geographically disjunct undisturbed forests must be studied to estimate natural gene and genotype flow. Unfortunately, undisturbed forests are very rare and often cover small areas in Central Europe.

Thus, a study was designed to test the following hypothesis: (i) communities in undisturbed forests are less diverse regarding the number of cryptic species as well as the genotypic diversity and (ii) gene and genotype flow among populations from disjunct undisturbed forests is absent. In addition, it was tested whether cryptic species within the P. fortiniiA. applanata complex have different host preferences.

Section snippets

Study site, sampling design, and isolation of DSE

Population structure of P. fortinii s.l. and A. applanata was studied in two undisturbed forests in Switzerland. Both forests are difficult to access and are, therefore, supposed to be little influenced by human activities (Kral and Mayer, 1969, Lienert, 2001). The Bödmeren forest (46°58′57.13″N/8°49′23.90″E) is growing on a Karst formation with a very difficult topology including caves and holes. This study site is located 1530 m a.s.l., the mean annual temperature is about 3 °C and the mean

Number of DSE strains in the two study sites

The number of isolates of A. applanata and P. fortinii s.l. was very similar in both study sites. However, the frequency of A. applanata isolates was slightly lower and that of P. fortinii s.l. slightly higher in the study site Scatlé. In total, 322 strains of A. applanata and 284 strains of P. fortinii s.l. were included in the single-copy RFLP analysis (Table 1).

Analysis of population structures within study sites

Cluster analysis of the clone-corrected RFLP data set from Bödmeren resulted in four main clusters (with nine or more MLH) and

Discussion

In the present study, we found evidence for two additional cryptic species within the P. fortiniiA. applanata species complex. Both study sites hosted several species of the P. fortiniiA. applanata species complex as previously observed in managed forests (Grünig et al., 2004). However, the number and species composition of the communities differed clearly between managed and undisturbed forests.

Acknowledgments

We thank Nina Nuessli and Valentin Queloz, Swiss Federal Institute of Technology, Zürich, Switzerland, for their help during the collection and isolation of endophytes in the two study sites.

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