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Genetic diversity of cyanobacterial symbionts of lichens and of free-living populations of Nostoc in biological soil crust communities of threatened alvar grasslands

Periodic Reporting for period 1 - Cyanolichens (Genetic diversity of cyanobacterial symbionts of lichens and of free-living populations of Nostoc in biological soil crust communities of threatened alvar grasslands)

Reporting period: 2015-06-15 to 2017-06-14

In Northern Europe habitat loss threatens not only pristine natural habitats (e.g. old-growth forests, dune environments) but also semi-natural habitat types, including calcareous grasslands (alvars). For instance, the gradual cessation of traditional land use practices directly threatens many vascular plants, associated arthropods, and fungi and also terricolous lichens in semi-natural alvar grasslands. Contemporary nature conservation policy in Europe has identified the protection of alvars and other semi-natural grasslands to be important for halting biodiversity loss. Loss of biodiversity implies not only species and communities but also loss of genetic diversity. To design effective conservation strategies for threatened biodiversity, the basic biology of the target organisms needs to be understood. Lichens are symbiotic entities consisting of at least two components, a fungus (the mycobiont) and algae and/or cyanobacteria (the photobionts), living in intimate symbiotic association. This study targeted the very poorly known relationships of symbiont specificity of threatened terricolous crust-forming cyanolichens (e.g. Peltigera, Solorina, Leptogium and Collema) and has been the first of its kind in grassland environments (alvars, dunes). In this project the genetic diversity of both myco- and photobiont of cyanobacterial lichens (cyanolichens) were studied. tRNALeu (UAA) intron sequences for cyanobacteria (Nostoc) and Internal Transcribed Spacer (ITS) for lichen mycobiont were used as genetic markers. Cyanobiont specificity of lichen mycobionts and possible overlap in the cyanobiont spectra of different lichen genera were studied. The sampling was conducted in seminatural grassland communities, in disturbed grasslands and as well as natural undisturbed forest habitats that collectively represented a natural gradient of decreasing atmospheric humidity, increasing soil pH, and increasing anthropogenic and natural disturbance. The diversity of lichen cyanobionts were compared among different habitats to determine the effects of environmental factors and disturbance history on genotype diversity patterns.
In June 2015 the best possible alvar study sites and other habitats in Estonia were selected. Nearly 300 hundred Peltigera samples with both mycobiont and photobiont sequences were characterized. Phylogenetic analyses of ITS sequences revealed previously undetected diversity especially within the Peltigera canina group. Direct ordination methods were used to assess the significance of associations between phylogenetically identified lichen taxa and environmental variables. Multivariate analysis revealed habitat-specific segregation between Peltigera species along a moisture gradient from humid eutrophic forests to dry oligotrophic forests and grasslands. Changes in species composition were also found along a soil pH gradient form alkaline soils of alvar grasslands to acidic soils of conifer forests.
Using nucleotide differences in the tRNALeu (UAA) intron more than 30 different symbiotic Nostoc genotypes were identified. In studied habitats we found the highest number of symbiotic Nostoc haplotypes in alvar grasslands and dunes. However, genetic diversity of Nostoc haplotype was the highest in roadside grassland and on ground of eutrophic forest. Some Peltigera taxa associated with a single or a few closely related Nostoc genotypes, others were generalists, being able to associate with a spectrum of different Nostoc genotypes. The association of different Nostoc haplotypes of studied Peltigera taxa is not necessarily correlated with mycobiont taxonomy instead seems to be correlated to environmental factors.
In addition, environmental factors and ground disturbance affecting the composition of species and functional traits of ground layer lichens on grey dunes and dune heaths of Estonia were analyzed. Several Peltigera species were revealed to be characteristic species for grey dunes and lichens with cyanobacterium as the main photobiont favored grey dune habitat. It was concluded that to protect and conserve the species-rich lichen biota, it is necessary to protect the dune habitats from building activity, to avoid over trampling in recreation areas and to regularly remove shrubs and trees.
Locality data of rare and protected species (Peltigera, Solorina) will be deposited and stored in EELIS (Estonian Nature Information System). Moreover, the evaluation of host tree preferences of red-listed epiphytic lichens, including cyanolichens, was performed. The highest number of red-listed species were accounted for Fraxinus excelsior, Quercus robur and Populus tremula. It was revealed, that several taxa, mainly the cyanolichens, prefer P. tremula to other trees, confirming its high importance as phorophyte species for the threatened epiphytes. Populus tremula can be conisidered as the best possible altervnative phorophyte for the gratest part of threatened lichen species that are growing on temperate broad-leaved trees.
"Recognition of cryptic fungal diversity is important for effective conservation of biological resources. According to the phylogenetic analyses based on ITS sequences it is expected to describe several new species from genus Peltigera. It is expected that those species have distinct morphological characters and ecological preferences. Some putative taxa of P. „neorufescens“ agg. could be confined only to calcareous habitats (grasslands). As calacareous grasslands, especially semi-natural alvar grassland, are seriously endangered habitats in Europe, it is urgent to evaluate the threat of habitat loss or overgrowing for the newly discovered terricolous taxa. The putative taxa P. aff. ""neocanina“, in contrary, prefer sheltered woody habitats and grow on mossy tree bases of large temperate broad-leaved trees. The putative taxa have been found wooded meadows and old deciduous forests. It is possible that those undescribed taxa are confined to biotopes with long habitat continuity as well as large-diameter broad-leaved trees. In Europe, the distribution of old deciduous forest is extremely declined and fragmented due to intensive forest management (especially clear cutting), so the putative species could be seriously endangered.
Species of Peltigera are conspicuous and easily recognizable at genus level. Although the recognition of many species is challenge due to the complicated/unresolved taxonomic problems, this genus includes also some very distinctive and easily recognizable species. Those species have rather distinct habitat preferences and therefore they could use for evaluation of qualities of habitats. As most of the cyanolichens that are already defined as indicator species in hemi-boreal region have declined tremendously due to environmental pollution and habitat degradation, therefore this genus could include applicable species for environmental monitoring or diversity hotspots. Lichens are well known example of bioindicators of the environmental conditions is recognized even beyond the scientific community. A strong decrease in species richness has brought biodiversity on the political agenda on national and international levels. Thus the ""proper"" delimitation of species is of great value for recognize the valuable habitats in the region. This is the basic knowledge to build the governmental policies for nature conservation practices."
Cyanolichen Peltigera rufescens growing in alvar grassland