Suitability of methods for species recognition in the Phialocephala fortinii–Acephala applanata species complex using DNA analysis

Abstract

Sequence data of two coding and three non-coding loci were used to study the taxonomic identity within and relatedness among seven previously defined cryptic species (CSP) of Phialocephala fortinii and Acephala applanata using two approaches of species recognition. Identification of taxonomic groups corresponding to CSP was ambiguous in some cases when applying solely the genealogical concordance phylogenetic species recognition (GCPSR) concept. The definition of groups corresponding to CSP using GCPSR was complicated due to shared sequence haplotypes between CSP, unresolved CSP for several loci, and possible introgression. GCPSR in conjunction with a population genetic approach improved resolution significantly and the CSP status could be confirmed for all seven CSP of P. fortinii s.l. The most critical step in both analyses was the definition of groups. The combination of several classes of markers differing in resolution helped to define species boundaries.

Introduction

Species concepts are among the most debated topics in biology (Barton, 2001, de Meeus et al., 2003, Frost and Kluge, 1994, Hey, 2001, Templeton, 1981, Wiens, 2004). Mayden (1997) lists more than 22 species concepts, but no universally accepted concept exists. Probably it is not even possible to establish a conclusive species concept for all organisms (Brasier, 1997, Hull, 1997, Kohn, 2005, Natvig and May, 1996). Besides the problem of choosing the appropriate species concept, researchers are confronted with the problem of how to recognize species. In mycology, species were defined based on morphology of asexual and sexual reproductive structures (morphological species recognition, MSR). Unfortunately, the number of morphological characters is often limited and the variability is pronounced (Brasier, 1997, Burnett, 2003, Petersen and Hughes, 1999). Not surprisingly, an increasing number of morphologically indistinguishable (cryptic) species were described. These species were often recognized by genealogical concordance of different sequence loci (genealogical concordance phylogenetic species recognition, GCPSR) (Dettman et al., 2003b, Fisher et al., 2002, Koufopanou et al., 2001, Koufopanou et al., 1997, O’Donnell et al., 2004). In the GCPSR approach, concordant grouping of species based on several sequences is regarded as evidence for restricted exchange of genetic material and, thus, for the reproductive isolation of taxonomic units indicating speciation (Taylor et al., 2000). Alternatively, reproductive isolation can easily be detected using a population genetic analysis framework (Hartl and Clark, 1989, Templeton, 1981).

The aim of the present study was to compare the genealogical species recognition concept (GCPSR) and a population genetic analysis framework to evaluate species boundaries in the species complex Phialocephala fortinii s.l.–Acephala applanata using several types of markers. The actual knowledge of the population biology of this species complex in conjunction with the high number of cryptic species makes it an ideal system to evaluate different concepts of species recognition. The two concepts were compared using sequence data of five loci. In addition, the assignment to CSP using sequence data was compared with the results received based on single-copy RFLP analysis and inter-simple-sequence-repeat-anchored PCR (ISSR-PCR).

We provide some additional background information about the P. fortinii s.l.–A. applanata species complex in this paragraph. Such information was deemed necessary to underline the significance of this species complex and to appreciate our model system. P. fortinii s.l. and A. applanata are the dominant dark septate endophytes (DSE) in the roots of woody plant species especially in those of species belonging to the Pinaceae (Ahlich and Sieber, 1996, Ahlich-Schlegel, 1997, LoBuglio et al., 1996, Sieber, 2002, Stoyke et al., 1992). P. fortinii s.l. probably are the most abundant fungal colonizers of roots in forest ecosystems of the northern temperate regions (Addy et al., 2005, Jumpponen and Trappe, 1998, Sieber, 2002, Sieber and Grünig, 2006). The ecological significance of P. fortinii s.l. is, however, enigmatic due to the use of genetically different, not clearly defined strains for ecological experimentation (Addy et al., 2005, Sieber, 2002, Sieber and Grünig, 2006). The closest relatives of P. fortinii s.l. are leotialean ascomycetes but a sexual state has never been observed (Grünig et al., 2002, Wang et al., 2006). Classification of P. fortinii s.l. puzzled mycologists because sporulation occurs rarely after prolonged cold treatment at 4 °C for up to 1 year and many strains remain sterile. Therefore, molecular identification of P. fortinii s.l. strains is increasingly common (Addy et al., 2000, Allen et al., 2003, Ashkannejhad and Horton, 2006, Menkis et al., 2004). Population genetic studies in Europe using single-copy RFLP markers showed that P. fortinii s.l. is composed of several cryptic species (CSP) (Grünig, 2004, Grünig et al., 2006). Similarly, presence of several CSP among North American P. fortinii s.l. was assumed using AFLP markers (Piercey et al., 2004). Species were considered “cryptic” because the species were morphologically indistinguishable but population subdivision was high between species and no gametic disequilibrium, i.e. recombination, was detected within species (Grünig, 2004, Grünig et al., 2006). Significant levels of gene flow were detected among populations of the same CSP. In addition, molecular analysis of variance (AMOVA) showed that more than 95% of variance is found within populations and only small proportions were attributable to the variance among field populations as in other ascomycetes (Linde et al., 2002, McDonald et al., 1999, Salamati et al., 2000). An easily recognizable morphotype was recently distinguished from among the P. fortinii species complex and described as A. applanata (Grünig and Sieber, 2005). Up to six CSP of P. fortinii s.l. including A. applanata were shown to occur sympatrically within the same study site (Grünig, 2004, Grünig et al., 2006) and strains belonging to different CSP were isolated repeatedly from the same 5-mm-long root segment (Queloz et al., 2005, Sieber and Grünig, 2006). Whereas no host specificity was evident for CSP of P. fortinii, A. applanata seems to be specific to Norway spruce (Picea abies) (Grünig et al., 2006, Grünig and Sieber, 2005). It will be interesting to explore species boundaries within the P. fortinii s.l.–A. applanata species complex using two different DNA-sequence-based species recognition concepts in the present study.