Local genetic structure in a North American epiphytic lichen, Ramalina menziesii (Ramalinaceae)

Epiphytic lichens possess unique life history traits that can have conflicting effects on genetic structure: symbiotic mutualism between a fungus with its algal or cyanobacterial photobiont, association with a host plant, and ability to reproduce sexually and asexually. Our study species, Ramalina menziesii, has small ascospores that can facilitate long-distance gene movement, and it is capable of clonal reproduction. The goals of this study are to test whether different haplotypes were differentially distributed across host plant species, to look for evidence of asexual vs. sexual reproduction, and to assess the local genetic structure of the population. We sampled individuals from multiple trees of three oak species in four lichen subpopulations within a savanna ecosystem. Using DNA sequence data from four fungal nuclear loci, we found no tendency for host specialization. Alleles were randomly distributed across subpopulations. The frequency of multilocus genotypes was consistent with a randomly mating population. Sexual reproduction involving relichenization appeared to be the predominant mode of reproduction of R. menziesii at this study site. We found no significant local genetic structure suggesting widespread gene flow at the local scale. The genetic structure of this lichen is comparable to that of widely distributed epiphytic plants.     

Genetic diversity and structure of the epiphytic foliose lichen Lobaria pindarensis in the Himalayas depends on elevation

The epiphytic lichen Lobaria pindarensis is a Himalayan endemic species with little information on distribution, genetic diversity and structural complexity. During an intensive survey in the Nepal Himalayas, we collected 1256 thallus fragments from 45 phorophyte species to study their distribution and population genetics along an elevational gradient. We quantified genetic diversity and population structure of each symbiont at 17 fungus specific and 9 alga specific microsatellite loci. The Bayesian clustering identified three and two distinct gene pools for mycobiont and photobiont. We found that genetic diversity, allelic richness and gene pool composition and distribution were significantly influenced by elevation. We discovered both clonally and sexually reproduced repeated genotypes of the symbionts.     

European phylogeography of the epiphytic lichen fungus Lobaria pulmonaria and its green algal symbiont

In lichen symbiosis, fungal and algal partners form close associations, often codispersed by vegetative propagules. Due to the particular interdependence, processes such as colonization, dispersal or genetic drift are expected to result in congruent patterns of genetic structure in the symbionts. To study the population structure of an obligate symbiotic system in Europe, we genotyped the fungal and algal symbionts of the epiphytic lichen Lobaria pulmonaria at eight and seven microsatellite loci, respectively, and analysed about 4300 L. pulmonaria thalli from 142 populations from the species' European distribution range. Based on a centroid approach, which localizes centres of genetic differentiation with a high frequency of geographically restricted alleles, we identified the South Italy–Balkan region as the primary glacial refugial area of the lichen symbiosis. Procrustean rotation analysis and a distance congruence test between the fungal and algal population graphs indicated general concordance between the phylogeographies of the symbionts. The incongruent patterns found in areas of postglacial recolonization may show the presence of an additional refugial area for the fungal symbiont, and the impact that horizontal photobiont transmission and different mutation rates of the symbionts have on their genotypic associations at a continental scale.     

Strong fungal specificity and selectivity for algal symbionts in Florida scrub Cladonia lichens

Symbiosis is a major theme in the history of life and can be an important force driving evolution. However, across symbioses, it is difficult to tease apart the mechanisms that structure the interactions among potential partners. We used genetic similarity and frequency-based methods to qualitatively and quantitatively examine the patterns of association among several co-occurring Cladonia lichen fungi and their algal photobionts in six disjunct Florida scrub sites. The patterns of association were described by the degree of specificity, i.e. the phylogenetic range of associated partners, and of selectivity, i.e. the frequency of association among partners. Six fungal species associated with only one algal internal transcribed spacer clade, with the remaining two fungi being associated with two algal clades. In all cases, the fungi associated in unequal frequencies with the observed algal photobiont genotypes within those clades--suggesting that both specificity and selectivity were higher than expected. Fungal species can be grouped into three significantly different specificity classes: photobiont specialists, intermediates and generalists. In contrast to the pronounced specificity for photobionts among fungal species, the different Florida scrub sites do not harbour distinct photobiont pools, and differential photobiont availability cannot explain the patterning of lichen associations at this spatial scale. Therefore, we conclude that fungal specificity and selectivity for algal photobionts are major factors in determining the local composition of symbiotic partnerships.     

Quantifying the climatic niche of symbiont partners in a lichen symbiosis indicates mutualist‐mediated niche expansions

The large distributional areas and ecological niches of many lichenized fungi may in part be due to the plasticity in interactions between the fungus (mycobiont) and its algal or cyanobacterial partners (photobionts). On the one hand, broad‐scale phylogenetic analyses show that partner compatibility in lichens is rather constrained and shaped by reciprocal selection pressures and codiversification independent of ecological drivers. On the other hand, sub‐species‐level associations among lichen symbionts appear to be environmentally structured rather than phylogenetically constrained. In particular, switching between photobiont ecotypes with distinct environmental preferences has been hypothesized as an adaptive strategy for lichen‐forming fungi to broaden their ecological niche. The extent and direction of photobiont‐mediated range expansions in lichens, however, have not been examined comprehensively at a broad geographic scale. Here we investigate the population genetic structure of Lasallia pustulata symbionts at sub‐species‐level resolution across the mycobiont's Europe‐wide range, using fungal MCM7 and algal ITS rDNA sequence markers. We show that variance in occurrence probabilities in the geographic distribution of genetic diversity in mycobiont‐photobiont interactions is closely related to changes in climatic niches. Quantification of niche extent and overlap based on species distribution modeling and construction of Hutchinsonian climatic hypervolumes revealed that combinations of fungal–algal interactions change at the sub‐species level along latitudinal temperature gradients and in Mediterranean climate zones. Our study provides evidence for symbiont‐mediated niche expansion in lichens. We discuss our results in the light of symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation and niche evolution in mutualisms.     

Congruent genetic structure in the lichen-forming fungus Lobaria pulmonaria and its green-algal photobiont

The extent of codispersal of symbionts is one of the key factors shaping genetic structures of symbiotic organisms. Concordant patterns of genetic structure are expected in vertically transmitted symbioses, whereas horizontal transmission generally uncouples genetic structures unless the partners are coadapted. Here, we compared the genetic structures of mutualists, the lichen-forming fungus Lobaria pulmonaria and its primary green-algal photobiont, Dictyochloropsis reticulata. We performed analysis of molecular variance and variogram analysis to compare genetic structures between symbiosis partners. We simulated the expected number of multilocus-genotype recurrences to reveal whether the distribution of multilocus genotypes of either species was concordant with panmixia. Simulations and tests of linkage disequilibrium provided compelling evidence for the codispersal of mutualists. To test whether genotype associations between symbionts were consistent with randomness, as expected under horizontal transmission, we simulated the recurrence of fungal-algal multilocus genotype associations expected by chance. Our data showed nonrandom associations of fungal and algal genotypes. Either vertical transmission or horizontal transmission coupled with coadaptation between symbiont genotypes may have created these nonrandom associations. This study is among the first to show codispersal and highly congruent genetic structures in the partners of a lichen mutualism.     

Photobiont selectivity for lichens and evidence for a possible glacial refugium in the Ross Sea Region, Antarctica

Lichens are a symbiosis consisting of heterotrophic, fungal (mycobiont) and photosynthetic algal or cyanobacterial (photobiont) components. We examined photobiont sequences from lichens in the Ross Sea Region of Antarctica using the internal transcribed spacer region of ribosomal DNA and tested the hypothesis that lichens from this extreme environment would demonstrate low selectivity in their choice of photobionts. Sequence data from three targeted lichen species (Buellia frigida, Umbilicaria aprina and Umbilicaria decussata) showed that all three were associated with a common algal haplotype (an unnamed Trebouxia species) which was present in all taxa and at all sites, suggesting lower selectivity. However, there was also association with unique, local photobionts as well as evidence for species-specific selection. For example, the cosmopolitan U. decussata was associated with two photobiont species, Trebouxia jamesii and an unnamed species. The most commonly collected lichen (B. frigida) had its highest photobiont haplotype diversity in the Dry Valley region, which may have served as a refugium during glacial periods. We conclude that even in these extreme environments, photobiont selectivity still has an influence on the successful colonisation of lichens. However, the level of selectivity is variable among species and may be related to the ability of some (e.g. B. frigida) to colonise a wider range of habitats.     

Vertical and horizontal photobiont transmission within populations of a lichen symbiosis

Lichens are widespread symbioses and play important roles in many terrestrial ecosystems. The genetic structure of lichens is the result of the association between fungal and algal populations constituting the lichen thallus. Using eight fungus- and seven alga-specific highly variable microsatellite markers on within-population spatial genetic data from 62 replicate populations across Europe, North America, Asia and Africa, we investigated the contributions of vertical and horizontal transmission of the photobiont to the genetic structure of the epiphytic lichen Lobaria pulmonaria. Based on pairwise comparisons of multilocus genotypes defined separately for the mycobiont and for the photobiont, we inferred the transmission mode of the photobiont and the relative contribution of somatic mutation and recombination. After constraining the analysis of one symbiont to pairs of individuals with genetically identical symbiotic partners, we found that 77% of fungal and 70% of algal pairs were represented by clones. Thus, the predominant dispersal mode was by means of symbiotic vegetative propagules (vertical transmission), which dispersed fungal and algal clones co-dependently over a short distance, thus shaping the spatial genetic structure up to distances of 20m. Evidence for somatic mutation generating genetic diversity was found in both symbionts, accounting for 30% of pairwise comparisons in the alga and 15% in the fungus. While the alga did not show statistically significant evidence of recombination, recombination accounted for 7.7% of fungal pairs with identical algae. This implies that, even in a mostly vegetatively reproducing species, horizontal transmission plays a role in shaping the symbiotic association, as shown in many coral and other symbioses in nature.     

Morphological and phylogenetic study of algal partners associated with the lichen-forming fungus <Emphasis Type="Italic">Tephromela atra</Emphasis> from the Mediterranean region

Recent DNA sequence analyses have revealed the diversity of algal partners in lichen symbioses. Although morphologically similar, different genetic lineages of photobionts are detected in wide geographic ranges of the same lichen fungal species. We studied the photobiont of the genus Trebouxia, which are known as partners of diverse lichen-forming fungal species in the Mediterranean region. We studied the phylogeny of these algae with a multilocus dataset including three loci: ITS, rbcL, and actin type I gene. The two lineages found, informally named Trebouxia sp. 1 and Trebouxia sp. 2, are related to Trebouxia arboricola/decolorans. The cultivation under axenic conditions succeeded only for one of them so far. We used light microscopy, confocal laser scanning microscopy and transmission electron microscopy for phenotypic characterisation. The ultrastructural characters currently used to describe species in the genus do not support the segregation of Trebouxia sp.1 from Trebouxia arboricola. The preferential presence in Mediterranean climates of these strains suggests eco-physiological adaptation. Despite their asexuality in long living lichen symbioses, coccoid algal lichen partners have apparently diversified genetically and physiologically.     

Cyanobacterial lichen symbiosis: the fungal partner as an optimal harvester

Lichen symbiosis has been traditionally treated as a model case of mutualism in which both partners, the fungus and the photobiont, gain benefits reciprocally. Some recent evidence, however, supports an alternative view that lichen symbiosis may represent an association largely controlled by the commensal or even parasitic fungal partner. The latter gains photosynthates from the photobiont (algae and/or cyanobacteria) which may not always substantially benefit from the symbiosis. We analyze from this perspective how a lichen fungus may maximize photosynthetic gains in bipartite and tripartite associations. We treat the frequency of nitrogen-fixing cells called heterocysts in cyanobacteria and the relative proportion of green algal cells vs. that of cyanobacteria per unit fungus as the variables to be manipulated for maximal carbon gain. The model predicts that even with a negligible cost of cephalodia (compartments containing cyanobacteria) it is in the interest of the tripartite lichen, first, to increase the heterocyst frequency, and second, keep the relative number of cyanobacteria considerably lower than that of green algae. Hence, the lichen fungus achieves higher fitness by making the cyanobacterial partner to specialize on N fixation. The available empirical data support these predictions as the reported heterocyst frequencies in bipartite lichens range from 2 to 8%, and in tripartite lichens between 10 and 55%. It is concluded that interaction asymmetry (i.e. commensalism or parasitism rather than mutualism) provides a sound basis to understand the high phenotypic plasticity expressed by fungi-forming bipartite and tripartite associations with cyanobacteria and green algae.     

Polymorphic DNA sequences of the fungal honey bee pathogen Ascosphaera apis

The pathogenic fungus Ascosphaera apis is ubiquitous in honey bee populations. We used the draft genome assembly of this pathogen to search for polymorphic intergenic loci that could be used to differentiate haplotypes. Primers were developed for five such loci, and the species specificities were verified using DNA from nine closely related species. The sequence variation was compared among 12 A.?apis isolates at each of these loci, and two additional loci, the internal transcribed spacer of the ribosomal RNA (ITS) and a variable part of the elongation factor 1α (Ef1α). The degree of variation was then compared among the different loci, and three were found to have the greatest detection power for identifying A.?apis haplotypes. The described loci can help to resolve strain differences and population genetic structures, to elucidate host-pathogen interaction and to test evolutionary hypotheses for the world's most important pollinator: the honey bee and one of its most common pathogens.     

Host-related genetic differentiation in the anther smut fungus Microbotryum violaceum in sympatric, parapatric and allopatric populations of two host species Silene latifolia and S. dioica

We investigated genetic diversity in West European populations of the fungal pathogen Microbotryum violaceum in sympatric, parapatric and allopatric populations of the host species Silene latifolia and S. dioica, using four polymorphic microsatellite loci. In allopatric host populations, the fungus was highly differentiated by host species, exhibiting high values of F(ST) and R(ST), and revealed clear and distinct host races. In sympatric and parapatric populations we found significant population differentiation as well, except for one sympatric population in which the two host species grew truly intermingled. The mean number of alleles per locus for isolates from each of the host species was significantly higher in sympatric/parapatric than in allopatric populations. This suggests that either gene flow between host races in sympatry, or in case of less neutral loci, selection in a more heterogeneous host environment can increase the level of genetic variation in each of the demes. The observed pattern of host-related genetic differentiation among these geographically spread populations suggest a long-term divergence between these host races. In sympatric host populations, both host races presumably come in secondary contact, and host-specific alleles are exchanged depending on the amount of fungal gene flow.     

A cyanolichen from the Lower Devonian Rhynie chert

The 400 million-year-old Rhynie chert has provided a wealth of information about various types of fungal interactions that existed in this Early Devonian paleoecosystem. In this paper we report the first unequivocal evidence of a lichen symbiosis from the Rhynie chert. Specimens of a new genus, Winfrenatia, consist of a thallus of superimposed layers of aseptate hyphae and, on the upper surface, numerous uniform depressions. Extending into the base of each depression are hyphae that form a three-dimensional netlike structure. Enclosed within each of the net spaces is a coccoid cyanobacterium, each cell of which is surrounded by a thick sheath. These photobiont cells divide in three planes, resulting in cell clusters of up to perhaps 64 individuals. The photobiont is parasitized by the fungus in the base of each net as new cyanobacterial cells are formed distally. Reproduction is by endospores and soredia. Affinities of the mycobiont appear closest to members of the Zygomycetes, while the photobiont is most similar to coccoid cyanobacteria of the Gloeocapsa and Chroococcidiopsis types. We speculate that this cyanobacterial symbiosis was well adapted to exploit and colonize new ecological niches, especially in the periodically desiccated environment postulated for the Rhynie chert paleoecosystem.     

The scale of genetic differentiation in the Dunes Sagebrush-Lizard (Sceloporus arenicolus), an endemic habitat specialist

The Dunes Sagebrush-Lizard (Sceloporus arenicolus) is a North American species endemic to sand-shinnery oak habitats of the Mescalero and Monahans sand dunes in eastern New Mexico and western Texas. This lizard is listed as Endangered in New Mexico and exhibits habitat specificity at several geographic scales. Dunes Sagebrush-Lizards are only found in topographically complex shinnery oak (Quercus havardii) dominated landscapes within their small geographic distribution and are not found in surrounding human-altered landscapes. Within suitable sand-shinnery oak habitat, individuals predominantly occupy non-vegetated sand dune blowouts and utilize blowouts with particular physical characteristics due to thermoregulatory, reproduction, and foraging requirements. Here, we examined historical and contemporary patterns of genetic differentiation with respect to the current distribution of suitable habitat at multiple spatial scales using mitochondrial DNA sequences and microsatellite data from individuals throughout the entire range. We found three genetic clusters of individuals generally concordant with geographic regions and low sequence divergence at mitochondrial loci suggesting a recent origin of these populations. We also found high levels of genetic structure at microsatellite loci among populations within each of these groups indicating restricted gene flow at intermediate scales. Despite high habitat specificity, we did not detect genetic structure among sand blowouts at finer spatial scales. Within each population, matrices comprised of both sand blowouts and vegetated shinnery oak patches are necessary for genetic connectivity, but the fine scale spatial arrangement of blowouts may not be as critical. We discuss our results with respect to the scale of landscape heterogeneity and habitat connectivity and consider the conservation implications for this threatened taxon.     

Isolation and characterization of polymorphic microsatellite loci in white truffle (Tuber magnatum)

Tuber spp. are fungi that establish symbiosis with several trees and shrubs. Some of these fungal species produce edible ascomata, also known as truffles, which are highly appreciated for their taste and odour. We isolated and characterized eight polymorphic microsatellite loci from Tuber magnatum, the finest white truffle species, and assessed their variability in 370 individuals collected from all over the species range of distribution. Although two to 18 alleles per locus were found, no heterozygous individuals were observed. The availability of simple sequence repeat loci provides valuable tools for assessment of the genetic structure and population dynamics in this species.     

Lichen acclimation to changing environments: Photobiont switching vs. climate‐specific uniqueness in Psora decipiens

Unraveling the complex relationship between lichen fungal and algal partners has been crucial in understanding lichen dispersal capacity, evolutionary processes, and responses in the face of environmental change. However, lichen symbiosis remains enigmatic, including the ability of a single fungal partner to associate with various algal partners. Psora decipiens is a characteristic lichen of biological soil crusts (BSCs), across semi‐arid, temperate, and alpine biomes, which are particularly susceptible to habitat loss and climate change. The high levels of morphological variation found across the range of Psora decipiens may contribute to its ability to withstand environmental change. To investigate Psora decipiens acclimation potential, individuals were transplanted between four climatically distinct sites across a European latitudinal gradient for 2 years. The effect of treatment was investigated through a morphological examination using light and SEM microscopy; 26S rDNA and rbcL gene analysis assessed site‐specific relationships and lichen acclimation through photobiont switching. Initial analysis revealed that many samples had lost their algal layers. Although new growth was often determined, the algae were frequently found to have died without evidence of a new photobiont being incorporated into the thallus. Mycobiont analysis investigated diversity and determined that new growth was a part of the transplant, thus, revealing that four distinct fungal clades, closely linked to site, exist. Additionally, P. decipiens was found to associate with the green algal genus Myrmecia, with only two genetically distinct clades between the four sites. Our investigation has suggested that P. decipiens cannot acclimate to the substantial climatic variability across its environmental range. Additionally, the different geographical areas are home to genetically distinct and unique populations. The variation found within the genotypic and morpho‐physiological traits of P. decipiens appears to have a climatic determinant, but this is not always reflected by the algal partner. Although photobiont switching occurs on an evolutionary scale, there is little evidence to suggest an active environmentally induced response. These results suggest that this species, and therefore, other lichen species, and BSC ecosystems themselves may be significantly vulnerable to climate change and habitat loss.     

No evidence of population structure across three isolated subpopulations of Russula brevipes in an oak/pine woodland

Russula brevipes is common ectomycorrhizal (EM) fungus that is associated with several hosts across temperate forest ecosystems. A previous study has demonstrated that substructuring across large geographic distances (1500 km) occurs in the western USA. To examine genetic structure over a more localized scale, basidiocarps of Russula brevipes from three subpopulations, separated by distances of 230-1090 m, were collected over two consecutive years in an oak/pine woodland. Microsatellite loci were used to test for population differentiation both among subpopulations and by year. No significant population differentiation was detected between subpopulations (theta(ST) = 0.01) or between years (theta(ST) = 0.01). Most loci were consistent with a Hardy-Weinburg equilibrium and 82% of the genets between seasons from similar sampling localities constituted new genotypes. These results indicate that R. brevipes constitutes a randomly mating population with no genetic differentiation between locations or across successive fruiting seasons.     

Random amplified polymorphic DNA markers reveal genetic variation in the symbiotic fungus of leaf-cutting ants

RAPD markers were used to examine the degree of genetic variation within the putatively asexual basidiomycete fungus (Lepiotaceae: provisionally named Leucoagaricus gongylophorus) associated with the leaf-cutting ant species Atta cephalotes. We analyzed fungal isolates from ant nests in two geographically distant sites, two isolates from Panama and five isolates from Trinidad. Ten decamer primers were used to amplify total DNA from these seven fungal isolates, and RAPD banding patterns were compared. Genetic similarity among isolates was determined by pair-wise comparisons of the shared number of DNA bands on an agarose gel. There was considerable genetic variation among isolates of the symbiotic fungus even within sites. Pairs of fungal isolates from the two different sites shared an average of only 36% of the bands in their RAPD profiles, while pairs from the within sites shared an average of 72% of the bands. RAPD markers may be useful for further investigation of the genetic structure of the fungal symbiont within species of leaf-cutting ants.     

Photobiont association and genetic diversity of the optionally lichenized fungus Schizoxylon albescens

The fungus Schizoxylon albescens occurs both as lichen and as saprobe. Lichenized colonies grow on the bark of Populus tremula; saprotrophic morphs grow on dead Populus branches. We wanted to (1) test whether lichenized and saprotrophic S. albescens are genetically distinct, (2) investigate photobiont association and diversity, (3) investigate the interactions between fungi and algae that occur during co-cultivation and (4) test whether Schizoxylon shows algal selectivity during lichenization. Fungal and algal genetic diversity were investigated for three markers. Algae from lichenized thalli were isolated in axenic cultures, and isolate sequence diversity was compared with algae amplified directly from thallus fragments. Co-culture experiments of fungi and algae were performed to study the morphological interaction patterns. Two distinct phylogenetic units are revealed in S. albescens, which are interpreted as phenotypically cryptic species. The algae are related to Coccomyxa and Pseudococcomyxa, and form two distinct sister clades separating samples isolated in cultures from those amplified directly from thallus fragments, indicating that more easily cultured strains of algae are not necessarily major components of the lichens. Schizoxylon albescens interacts with isolated algal strains, similar to fungal-Coccomyxa symbioses in nature. As the system is maintained without difficulty in culture, it can potentially be an easily controlled lichen symbiosis study system under laboratory conditions.