Presented to Jean-Claude Walser for his thesis entitled: Microsatellites as new markers to investigate population genetic processes in lichens using Lobaria pulmonaria (L.) Hoffm. as a model species
Nuclear microsatellites have become widely used molecular tools to study population processes in animals, plants, and fungi. However, in lichenized fungi, the molecular studies have mostly focused on taxonomic relationships, and the markers available so far showed only little intraspecific variation and were therefore of only limited use for population genetic studies. Greater genetic variation in lichen species has been detected with random amplified polymorphic DNA markers (RAPD), but anonymous DNA-fingerprinting methods are not universally applicable in mutualistic endosymbiotic systems. Hence, the basic requirement for population genetic studies in lichenized fungi was the development of fungal-specific polymorphic molecular markers.
In his PhD, Jean-Claude Walser has selected the epiphytic lichen species Lobaria pulmonaria as a model species for studies in population genetics, and he has established and characterized twelve fungus specific microsatellite loci for this species. The potential resolution of the genetic variation and differentiation detected with these new markers was evaluated at different spatial scales. In addition, a cross-species amplification test demonstrated that the same primers could also be used for genetic studies of other taxa closely related to L. pulmonaria.
Genetic diversity of three L. pulmonaria populations from Switzerland and nine populations from British Columbia (Canada) was investigated by means of fragment length data from six microsatellite loci. The high genetic diversity within the investigated populations and evidence of recombination from the association of alleles indicated that L. pulmonaria was substantially outcrossing. Nevertheless, clonality was also detected in all twelve investigated populations.
However, the presence of recurring multilocus genotypes influenced the spatial genetic structure only within low-density, isolated populations from Switzerland but not in populations of L. pulmonaria from British Columbia, where the species was abundant and widespread. Given that L. pulmonaria has suffered a significant decline in Central Europe within the last few decades, the results could be interpreted as indicative of genetic bottlenecks owing to increased habitat loss or disturbance history. Hence, as in vascular plants, exogenous factors, such as disturbance or fragmentation, might substantially alter population processes and, thus, the genetic structure of lichen populations.
The epiphytic lichen L. pulmonaria vanished almost completely from the Swiss lowlands, and the remnant populations in the Pre-Alps and the Jura Mountains have become increasingly fragmented and isolated from each other. While similar multilocus genotypes were found across different populations from the mainland of British Columbia, the Swiss populations did not have any shared genotypes. Within populations, the maximum distance between identical genotypes was 230 m, and suitable habitat patches at a distance of 350 m from the source tree seemed to be too far away to be colonized. This and the clustered distribution of multilocus genotypes suggested that dispersal of vegetative propagules was spatially limited both within and among populations in Switzerland.
Indeed, many endangered lichens are regarded as organisms with limited dispersal capacity. Lichen propagules showed no species-specific morphological characteristics which made direct experimental assessments of dispersal distances impossible. Therefore, a new and sensitive molecular approach was introduced to study dispersal in L. pulmonaria under natural conditions. The first results showed that a considerable amount of dispersed propagules was found within a radius of 10 m from the source tree and that still a few propagules reached distances of up to 50 m. However, long-distance dispersal over hundreds of meters or even kilometers could not bee demonstrated in L. pulmonaria, although it would be crucial for gene flow among populations.
In lichens, geographic isolation is often regarded as the first step towards differentiation and allopatric speciation. However, in organisms such as lichens, it is not clear whether long-distance dispersal, past range fragmentation or slow evolutionary rates are responsible for the broad, but often scattered, geographic distribution patterns observed today. The non-overlapping allele size distribution in one of the microsatellite loci between samples from two continents, geographically restricted alleles at other loci, low estimated gene flow rates based on private alleles, multivariate analyses of multilocus genotypes, and analyses of molecular variance (AMOVAs) all indicated that L. pulmonaria populations from British Columbia and Switzerland formed two different evolutionary lineages.
This clear genetic differentiation between populations from British Columbia and Switzerland thus questions recent genetic exchange. Within British Columbia, Jean-Claude Walser found also a clear genetic differentiation between populations from Vancouver Island and those from the mainland. Together with several other lines of evidence from the microsatellite data, this suggests that Quaternary glaciation and restricted gene flow substantially influenced the genetic structure of L. pulmonaria populations in British Columbia.
All the above results demonstrate the great potential of microsatellites to study population and microevolutionary processes in lichen species. However, neutral molecular markers do not allow drawing conclusions on ecological adaptation. Classical transplant experiments with thalli from different provenances were thus developed by Jean-Claude Walser to study adaptive variation in L. pulmonaria. The experiments were established in summer 2000 and the first results were promising, though more time will be needed until final conclusions can be drawn from the slowly growing transplants. The occurrence of potentially adaptive traits showed that thalli from different provenances reacted in a similar way to similar ecological conditions. However, some traits also pointed towards distinct provenance effects and, hence, potential adaptation. No evidence was found so far that resident “home” thalli were better adapted than thalli from other provenances.
The present study was among the first to investigate population genetic processes of lichenized fungi using molecular methods. Compared with earlier studies, its strength is the employment of new polymorphic molecular markers and the high number of populations and samples investigated. Microsatellite markers revealed a great potential as new genetic markers in the population biology of lichens and the molecular tools developed by Jean-Claude Walser will open novel fields of lichenological research such as phylogeography, conservation genetics and landscape genetics.
– Christoph Scheidegger, Birmensdorf