Lichen endozoochory by snails

Endozoochory plays a prominent role for the dispersal of seed plants. However, for most other plant taxa it is not known whether this mode of dispersal occurs at all. Among those other taxa, lichens as symbiotic associations of algae and fungi are peculiar as their successful dispersal requires movement of propagules that leaves the symbiosis functional. However, the potential for endozoochorous dispersal of lichen fragments has been completely overlooked. We fed sterile thalli of two foliose lichen species (Lobaria pulmonaria and Physcia adscendens) differing in habitat and air-quality requirements to nine snail species common in temperate Europe. We demonstrated morphologically that L. pulmonaria regenerated from 29.0% of all 379 fecal pellets, whereas P. adscendens regenerated from 40.9% of all 433 fecal pellets, showing that lichen fragments survived gut passage of all snail species. Moreover, molecular analysis of regenerated lichens confirmed the species identity for a subset of samples. Regeneration rates were higher for the generalist lichen species P. adscendens than for the specialist lichen species L. pulmonaria. Furthermore, lichen regeneration rates varied among snail species with higher rates after gut passage of heavier snail species. We suggest that gastropods generally grazing on lichen communities are important, but so far completely overlooked, as vectors for lichen dispersal. This opens new ecological perspectives and questions the traditional view of an entirely antagonistic relationship between gastropods and lichens.     

Contrasting changes in palatability following senescence of the lichenized fungi Lobaria pulmonaria and L. scrobiculata

Epiphytic lichens can contribute significantly to ecosystem nutrient input because they efficiently accumulate atmospheric mineral nutrients and, in the case of cyanolichens, also fix nitrogen. The rate at which carbon and other nutrients gained by lichens enters the ecosystem is determined by lichen litter decomposability and by invertebrate consumption of lichen litter. In turn, these processes are driven by the secondary compounds present in senesced lichens. Therefore, we explored how lichen palatability and concentrations of secondary compounds change with tissue senescence for Lobaria pulmonaria, a green-algal lichen with cyanobacterial cephalodia, and Lobaria scrobiculata, a cyanobacterial lichen. During senescence both lichens lost 38–48 % of their stictic acid chemosyndrome, while m-scrobiculin and usnic acid in L. scrobiculata remained unchanged. Snails preferred senesced rather than fresh L. pulmonaria, while senesced L. scrobiculata were avoided. This provides evidence that species with labile secondary compounds will have higher turnover rates, through consumption and decomposition, than those producing more stable secondary compounds.     

Mollusc grazing may constrain the ecological niche of the old forest lichen Pseudocyphellaria crocata

This study reports on mollusc grazing of two epiphytic cyanobacterial lichens (Pseudocyphellaria crocata and Lobaria pulmonaria) transplanted within three Picea abies-dominated boreal rain forest stands (clear-cut, young and old forests) in west central Norway. Grazing was particularly high in transplants located in the old forest and was almost absent in clear-cut transplants. Grazing marks were absent on natural thalli on nearby spruce twigs (required creeping distance for mollusc from the ground >4 m). Transplantation of lichens from twigs to artificial transplantation frames reduced the creeping distance to 1.2 m, and caused a significant increase in grazing damage in P. crocata. Given a paired choice under transplantation, molluscs consistently preferred P. crocata and avoided L. pulmonaria, implying species-specific differences in herbivore defence. Pseudocyphellaria crocata has a much lower content of the medullary depsidones stictic and constictic acid than L. pulmonaria. Heavy grazing occurred in the P. crocata thalli lowest in these two depsidones. The upper part of the medulla hosting the photobiont was the preferred fodder for grazing molluscs. Molluscs avoided the yellow soralia in P. crocata (localised pulvinic acid), suggesting a role for pulvinic acid in preventing grazing of detached soredia and early establishment stages. The preference of P. crocata for thin spruce twigs is probably a result of a lower grazing pressure on twigs compared to e.g. deciduous stems that frequently support the better defended L. pulmonaria. Ongoing climate changes with increased annual rainfall and milder winters have presumably increased mollusc grazing, particularly in SW parts of Norway which have more species of lichen-feeding molluscs than the boreal sites studied. These temperate areas lacking natural spruce populations have recently experienced reported extinctions of the poorly defended P. crocata from rocks and deciduous stems prone to mollusc grazing. Lichen-feeding molluscs have likely played a role in these extinctions, causing spruce twigs in Atlantic boreal forests to be a last strong foothold for P. crocata in Scandinavia.     

Fungal depsidones – an inducible or constitutive defence against herbivores in the lichen Lobaria pulmonaria?

Herbivore-deterrent depsidones in the epiphytic lichen Lobaria pulmonaria were quantified after a 104-day exposure to indigenous lichen-feeding mollusc communities in broadleaved deciduous forests in southeastern Norway. Controls and acetone-rinsed living thalli were transplanted under open and shaded tree canopies. Rinsed thalli had their depsidone concentration reduced to 36% of the pre-rinsing level, which is below the level needed to deter grazing molluscs. Grazing did not raise the concentration of depsidones beyond the level occurring in control to which molluscs had no access. Inducible responses were not detected in controls nor in acetone-rinsed thalli. Depsidone resynthesis was negligible in acetone-rinsed thalli regardless of grazing and/or light regimes. Our results suggest that C-based depsidones represent a constitutive type of herbivore defence in L. pulmonaria. A constitutive defence is probably an advantage for stress-tolerant slow-growing lichens inhabiting habitats with a constant presence of generalist invertebrate herbivores.     

Mollusc grazing limits growth and early development of the old forest lichen Lobaria pulmonaria in broadleaved deciduous forests

This study aims: (1) to quantify mollusc grazing on juvenile and mature thalli of the foliose epiphytic lichen Lobaria pulmonaria, and (2) to test the hypothesis inferring a herbivore defensive role of lichen depsidones in forests with indigenous populations of lichen-feeding molluscs. Lichens were transplanted in shaded and less shaded positions in each of two calcareous broadleaved deciduous forests, one poor in lichens, one with a rich Lobarion community. Preventing the access of molluscs significantly reduced the loss of juvenile L. pulmonaria, particularly in the naturally lichen-poor forest. Molluscs also severely grazed mature thalli in the lichen-poor forest, especially thalli placed under the more shading canopies. Furthermore, reducing the natural concentration of depsidones by pre-rinsing with acetone increased subsequent grazing significantly, showing that lichen depsidones function as herbivore defence in natural habitats. Our results suggest that mollusc grazing may play important roles in shaping the epiphytic vegetation in calcareous deciduous forests, and that recently established juvenile L. pulmonaria thalli seem to be particularly vulnerable.     

Recombination and clonal propagation in different populations of the lichen Lobaria pulmonaria

Propagation, dispersal, and establishment are fundamental population processes, and are critical stages in the life cycle of an organism. In symbiotic organisms such as lichens, consisting of a fungus and a population of photobionts, reproduction is a complex process. Although many lichens are able to reproduce both sexually and asexually, the extent of vegetative propagation within local populations is unknown. We used six polymorphic microsatellite loci to investigate whether recombination is common in natural populations, and to assess if and how clonal reproduction influences the spatial genetic structure within populations of the epiphytic lichen species Lobaria pulmonaria. High genetic diversity within all 12 investigated populations and evidence of recombination, from various tests, indicated that L. pulmonaria is a predominantly outcrossing species. Nevertheless, clonality occurred in all populations, but the presence of recurring multilocus genotypes influenced the spatial genetic structure only within low-density populations. This could be interpreted as indicative of genetic bottlenecks owing to increased habitat loss and disturbance. Consequently, for a predominantly outcrossing lichen species, exogenous factors might be substantially altering population processes and hence genetic structure.     

Bayesian Mapping of Lichens Growing on Trees

Suitability of trees as hosts for epiphytic lichens are studied in a forest stand of size 25 ha. Suitability is measured as occupation probabilites which are modelled using hierarchical Bayesian approach. These probabilities are useful for an ecologist. They give smoothed spatial distribution map of suitability for each of the species and can be used in detecting high‐ and low‐probability areas. In addition, suitability is explained by tree‐level covariates. Spatial dependence, which is due to unobserved spatially structured covariates, is modelled through an unobserved Markov random field. Markov chain Monte Carlo method has been applied in Bayesian computation. The extensive spatial data consist of the occurrences of eight lichen species and one bryophyte on all of the 1253 potential host trees. In addition, coordinates of the trees and several tree characteristics have been recorded. The data have been analysed for four most abundant species: Lobaria pulmonaria, Nephroma bellum, Nephroma parile and Peltigera praetextata. The tree level parameters, subject to estimation, consist of the occurrence probabilities for each tree and for each lichen species. Model validation is discussed in detail and, in addition to Bayesian validation tools, the autologistic model and case‐control design based on logistic regression have been suggested for validation of covariate effects. As a result we present suitability maps for the four lichen species. We observed, that among the observed tree covariates, the diameter at breast height (DBH) correlates with lichen occurrence. Our modelling approach has close connections to disease mapping in spatial epidemiology.     

Sterols of three lichen species: Lobaria pulmonaria, Lobaria Scrobiculata and Usnea Longissima

Sterols were extracted from the lichens Lobaria pulmonaria, Lobaria scrobiculata and Usnea longissima with chloroform-methanol (2:1) (solvent-extractable fraction) followed by saponification of the residual lichen material to give a tightly-bound sterol fraction. The compounds were principally ergosterol, episterol, fecosterol and lichesterol with minor quantities of C₁₇, C₂₈ and C₂₉ monoenes and dienes of the phytosterol type.     

Lichen diversity and red-listed lichen species relationships with tree species and diameter in wooded meadows

This study provides a unique large dataset of total epiphytic lichen diversity (fruticose, foliose and crustose species) and composition on 1,294 trees of 17 tree species in wooded meadows in Sweden and Estonia, the Baltic region. The inventory (25,380 observations and 246 lichen taxa) clearly illustrated that Ulmus minor, Quercus robur and Fraxinus excelsior contributed most significantly to epiphytic lichen richness and number of red-listed species. In Sweden, average single tree α richness was 22.2 on Ulmus (only in Sweden), 21.6 on Quercus (25.0 in Estonia) and 19.8 on Fraxinus (16.7 in Estonia), respectively. Ulmus hosted on average one red-listed species per tree, compared with 0.7 on Fraxinus (0.6 in Estonia), 0.4 on Quercus (0.7 in Estonia) and only 0.05 on Betula (same in Estonia). Lichen species composition and the average number of red-listed lichens were influenced by tree diameter on Fraxinus and Quercus, whilst no such pattern was evident on Ulmus. Randomized species accumulation curves of the dominating tree species illustrated that Fraxinus, Quercus and Ulmus supported α dominated lichen communities where individual trees hosted a substantial part of the total richness. Betula, on the other hand, supported β dominated communities where individual trees tended to be dissimilar and, therefore, more of the total richness existed as species turnover among host trees. Lichen species composition was influenced by tree species, and most notably, lichen species on Ulmus had a strong consistent clumping in ordination graphs, with many rare and red-listed lichens. The broadleaved deciduous trees within the wooded meadows clearly contribute greatly to the biodiversity of the Baltic region.     

Tree and stand level variables influencing diversity of lichens on temperate broad-leaved trees in boreo-nemoral floodplain forests

Tree and stand level variables affecting the species richness, cover and composition of epiphytic lichens on temperate broad-leaved trees (Fraxinus excelsior, Quercus robur, Tilia cordata, Ulmus glabra, and U. laevis) were analysed in floodplain forest stands in Estonia. The effect of tree species, substrate characteristics, and stand and regional variables were tested by partial canonical correspondence analysis (pCCA) and by general linear mixed models (GLMM). The most pronounced factors affecting the species richness, cover and composition of epiphytic lichens are acidity of tree bark, bryophyte cover and circumference of tree stems. Stand level characteristics have less effects on the species richness of epiphytic lichens, however, lichen cover and composition was influenced by stand age and light availability. The boreo-nemoral floodplain forests represent valuable habitats for epiphytic lichens. As substrate-related factors influence the species diversity of lichens on temperate broad-leaved trees differently, it is important to consider the effect of each tree species in biodiversity and conservation studies of lichens. Nomenclature Randlane et al. (2007) for lichens; Leht (2007) for vascular plants.     

Fragment quality and matrix affect epiphytic performance in a Mediterranean forest landscape

Destruction and fragmentation of habitats represent one of the most important threats for biodiversity. Here, we examined the effects of fragmentation in Mediterranean forests on the epiphytic lichen Lobaria pulmonaria (Lobariaceae). We tested the hypothesis that not only the level of fragmentation affects L. pulmonaria populations, but also the quality of the habitat and the nature of the surrounding matrix affect them. The presence and abundance of the lichen was recorded on 2039 trees in a total of 31 stands. We recorded habitat quality and landscape variables at three hierarchical levels: tree, plot, and patch. We found that L. pulmonaria tends to occur in trees with larger diameters in two types of surveyed forests. In Quercus pyrenaica patches, the mean diameter of colonized trees was smaller, suggesting the importance of bark roughness. Factors affecting the presence and cover of the lichen in each type of forest were different. There was a strong positive influence of distance from a river in beech forests, whereas proximity to forest edge positively affected in oak forests. The influence of the surrounding matrix was also an important factor explaining the epiphytic lichen abundance.     

Tree hollows can affect epiphyte species composition

Tree hollows often harbor animals and microorganisms, thereby storing nutritive resources derived from their biological activities. The outflows from tree hollows can create unique microenvironments, which may affect communities of epiphytic organisms on trunk surfaces below the hollows. In this study, we tested whether the species richness and composition of epiphytic bryophytes (liverworts and mosses) and lichens differ above and below tree hollows of Aria japonica and Cercidiphyllum japonicum in a Japanese temperate forest. The species richness of epiphytic bryophytes and lichens did not differ above and below hollows; however, the species composition of bryophytes differed significantly above and below hollows. Indicator species analyses showed that the moss species Anomodon tristis and the liverwort species Porella vernicosa were significantly more common below than above hollows, while the liverwort species Radula japonica and four lichen species, including Leptogium cyanescens, occurred more frequently above than below hollows. Our results highlight that tree hollows can produce unique microenvironments on trunk surfaces that potentially contribute to the maintenance of epiphytic diversity on a local scale.     

Fine spatial pattern of an epiphytic lichen species is affected by habitat conditions in two forest types in the Iberian Mediterranean region

Persistence and abundance of species is determined by habitat availability and the ability to disperse and colonize habitats at contrasting spatial scales. Favourable habitat fragments are also heterogeneous in quality, providing differing opportunities for establishment and affecting the population dynamics of a species. Based on these principles, we suggest that the presence and abundance of epiphytes may reflect their dispersal ability, which is primarily determined by the spatial structure of host trees, but also by host quality. To our knowledge there has been no explicit test of the importance of host tree spatial pattern for epiphytes in Mediterranean forests. We hypothesized that performance and host occupancy in a favourable habitat depend on the spatial pattern of host trees, because this pattern affects the dispersal ability of each epiphyte and it also determines the availability of suitable sites for establishment. We tested this hypothesis using new point pattern analysis tools and generalized linear mixed models to investigate the spatial distribution and performance of the epiphytic lichen Lobaria pulmonaria, which inhabits two types of host trees (beeches and Iberian oaks). We tested the effects on L. pulmonaria distribution of tree size, spatial configuration, and host tree identity. We built a model including tree size, stand structure, and several neighbourhood predictors to understand the effect of host tree on L. pulmonaria. We also investigated the relative importance of spatial patterning on the presence and abundance of the species, independently of the host tree configuration. L. pulmonaria distribution was highly dependent on habitat quality for successful establishment, i.e., tree species identity, tree diameter, and several forest stand structure surrogates. For beech trees, tree diameter was the main factor influencing presence and cover of the lichen, although larger lichen-colonized trees were located close to focal trees, i.e., young trees. However, oak diameter was not an important factor, suggesting that bark roughness at all diameters favoured lichen establishment. Our results indicate that L. pulmonaria dispersal is not spatially restricted, but it is dependent on habitat quality. Furthermore, new spatial analysis tools suggested that L. pulmonaria cover exhibits a distinct pattern, although the spatial pattern of tree position and size was random.     

Landscape-level gene flow in Lobaria pulmonaria, an epiphytic lichen

Epiphytes are strongly affected by the population dynamics of their host trees. Owing to the spatio-temporal dynamics of host tree populations, substantial dispersal rates--corresponding to high levels of gene flow--are needed for populations to persist in a landscape. However, several epiphytic lichens have been suggested to be dispersal-limited, which leads to the expectation of low gene flow at the landscape scale. Here, we study landscape-level genetic structure and gene flow of a putatively dispersal-limited epiphytic lichen, Lobaria pulmonaria. The genetic structure of L. pulmonaria was quantified at three hierarchical levels, based on 923 thalli collected from 41 plots situated within a pasture-woodland landscape and genotyped at six fungal microsatellite loci. We found significant isolation by distance, and significant genetic differentiation both among sampling plots and among trees. Landscape configuration, i.e. the effect of a large open area separating two forested regions, did not leave a traceable pattern in genetic structure, as assessed with partial Mantel tests and analysis of molecular variance. Gene pools were spatially intermingled in the pasture-woodland landscape, as determined by Bayesian analysis of population structure. Evidence for local gene flow was found in a disturbed area that was mainly colonized from nearby sources. Our analyses indicated high rates of gene flow of L. pulmonaria among forest patches, which may reflect the historical connectedness of the landscape through gene movement. These results support the conclusion that dispersal in L. pulmonaria is rather effective, but not spatially unrestricted.     

Microsatellites reveal regional population differentiation and isolation in Lobaria pulmonaria, an epiphytic lichen

Many lichen species produce both sexual and asexual propagules, but, aside from being minute, these diaspores lack special adaptations for long-distance dispersal. So far, molecular studies have not directly addressed isolation and genetic differentiation of lichen populations, both being affected by gene flow, at a regional scale. We used six mycobiont-specific microsatellite loci to investigate the population genetic structure of the epiphytic lichen Lobaria pulmonaria in two regions that strongly differed with respect to anthropogenic impact. In British Columbia, L. pulmonaria grows in continuous old-growth forests, while its populations in the old cultural landscape of Switzerland are comparably small and fragmented. Populations from both British Columbia and Switzerland were genetically diverse at the loci. Geographically restricted alleles, low historical gene flow, and analyses of genetic distance (upgma tree) and of differentiation (amova) indicated that populations from Vancouver Island and from the Canadian mainland were separated from each other, except for one, geographically intermediate population. This differentiation was attributed to different glacial and postglacial histories of coastal and inland populations in British Columbia. In contrast to expectations, the three investigated Swiss populations were genetically neither isolated nor differentiated from each other despite the long-lasting negative human impact on the lichen's range size in Central Europe. We propose that detailed studies integrating local landscape and regional scales are now needed to understand the processes of dispersal and gene flow in lichens.     

Growth and vitality of epiphytic lichens

 We tested the hypothesis that changed microclimate at induced forest edges causes reduced growth of epiphytic lichens. Two foliose, green algal lichens were transplanted to the lower canopy of a mature Picea abies forest at six distances (2, 6.25, 12.5, 25, 50 and 100 m) from a clearcut. The biomass growth in Platismatia glauca (6.2% in 16 months) was 41% higher than in Lobaria pulmonaria (4.4%). We found no growth reduction near the forest edge. In contrast, the highest growth in both species occurred within 12 m from the edge. Further, fluorescence and chlorophyll measurements showed that lichen vitality was unaffected by distance from edge. The light intensity was 4.3 times higher at the edge than in the interior during the growing season, but there were only minor differences in air temperature and relative humidity. Monitoring of thallus water content revealed clear differences in both number and length of wetting and drying cycles. However, the total time with water content sufficient for photosynthetic activity was only slightly higher at the edge. The data thus indicate that our gradient in microclimate was too small to significantly affect lichen growth, and that lichens are largely metabolically inactive when large edge-interior contrasts in microclimate occur. Lichen response to forest edge microclimate results from intricate interactions among several biotic and abiotic factors. Linking data on lichen growth, microclimate and thallus water content with physiological measurements provides a framework for future studies of the mechanisms behind abiotic edge effects. Received: 15 April 1996 / Accepted: 21 June 1996     

Highly variable microsatellite markers for the fungal and algal symbionts of the lichen Lobaria pulmonaria and challenges in developing biont-specific molecular markers for fungal associations

The availability of highly variable markers for the partners of a fungal symbiosis enables the integrated investigation of ecological and evolutionary processes at the symbiotic level. In this article we analyze the specificity of the first and to date only microsatellite markers that had been developed for an epiphytic lichen (Lobaria pulmonaria). We used DNA extracts from cultures of the fungal and of the green algal symbionts of L. pulmonaria as well as total DNA extracts from related Lobaria species associated with the same algal partner, and got evidence that five of the previously described microsatellite markers, proposed to be fungus-specific, are indeed alga-specific. Hence, highly variable microsatellite primer sets available for both, the algal and the fungal symbionts of L. pulmonaria are now at our hands, which allow us to investigate so far unexplored biological processes of lichen symbionts, such as codispersal and coevolution. In a broader sense, our work evaluates and discusses the challenges in developing biont-specific molecular markers for fungi forming close associations with other organisms.     

Hidden crown jewels: the role of tree crowns for bryophyte and lichen species richness in sycamore maple wooded pastures

Tree crowns typically cover the vast majority of the surface area of trees, but they are rarely considered in diversity surveys of epiphytic bryophytes and lichens, especially in temperate Europe. Usually only stems are sampled. We assessed the number of bryophyte and lichen species on stems and in crowns of 80 solitary sycamore maple trees (Acer pseudoplatanus) at six sites in wooded pastures in the northern Alps. The total number of species detected per tree ranged from 13 to 60 for bryophytes, from 25 to 67 for lichens, and from 42 to 104 for bryophytes and lichens considered together. At the tree level, 29 % of bryophyte and 61 % of lichen species were recorded only in the crown. Considering all sampled trees together, only 4 % of bryophyte, compared to 34 % of lichen species, were never recorded on the stem. Five out of 10 red-listed bryophyte species and 29 out of 39 red-listed lichen species were more frequent in crowns. The species richness detected per tree was unexpectedly high, whereas the proportion of exclusive crown species was similar to studies from forest trees. For bryophytes, in contrast to lichens, sampling several stems can give a good estimation of the species present at a site. However, frequency estimates may be highly biased for lichens and bryophytes if crowns are not considered. Our study demonstrates that tree crowns need to be considered in research on these taxa, especially in biodiversity surveys and in conservation tasks involving lichens and to a lesser degree also bryophytes.     

Edge and land-use effects on epiphytic lichen diversity in the forest-steppe ecotone of the Mongolian Altai

Epiphytic lichen diversity was studied in forests of Siberian larch (Larix sibirica) in the forest-steppe ecotone of the Mongolian Altai. These forests are utilized for livestock grazing, fuelwood collection and occasional logging by pastoral nomads. The density of nomad households in the proximity of the forests influences epiphytic lichen diversity more strongly than the position of sample trees in the forest interior or at the forest line to the steppe. This suggests that land use exerts a stronger effect on lichen diversity than the distinct gradient in microclimate between the forest interior and the forest edge. The co-occurrence of nitrophytes with anitrophytic acidophytes on a small spatial scale as well as higher N and Ca concentrations and pH values in the bark of larch trees at the forest edge than in the forest interior indicate that moderate livestock grazing increases the epiphytic lichen diversity due to an increase of the diversity of chemically different microhabitats. Preference of many lichen species (and of rare species in particular) for overmature and decaying trees suggests that logging and fuelwood collection has adverse effects on epiphytic lichen diversity. This adverse effect is likely to be more crucial for lichen diversity than the putatively positive effect of livestock grazing, since more lichens with a preference for old and decaying trees than for nitrogen-enriched bark were found. The present study is the first one investigating the impact of pastoral nomadism in Central Asia on epiphyte diversity.     

Hitchhiking with forests: population genetics of the epiphytic lichen Lobaria pulmonaria in primeval and managed forests in southeastern Europe

Availability of suitable trees is a primary determinant of range contractions and expansions of epiphytic species. However, switches between carrier tree species may blur co‐phylogeographic patterns. We identified glacial refugia in southeastern Europe for the tree‐colonizing lichen Lobaria pulmonaria, studied the importance of primeval forest reserves for the conservation of genetically diverse populations and analyzed differences in spatial genetic structure between primeval and managed forests with fungus‐specific microsatellite markers. Populations belonged to either of two genepools or were admixed. Gene diversity was higher in primeval than in managed forests. At small distances up to 170 m, genotype diversity was lower in managed compared with primeval forests. We found significant associations between groups of tree species and two L. pulmonaria genepools, which may indicate “hitchhiking” of L. pulmonaria on forest communities during postglacial migration. Genepool B of L. pulmonaria was associated with European Beech (Fagus sylvatica) and we can hypothesize that genepool B survived the last glaciation associated within the refuge of European Beech on the Coastal and Central Dinarides. The allelic richness of genepool A was highest in the Alps, which is the evidence for a northern refuge of L. pulmonaria. Vicariant altitudinal distributions of the two genepools suggest intraspecific ecological differentiation.