Are there conserved biosynthetic genes in lichens? Genome-wide assessment of terpene biosynthetic genes suggests ubiquitous distribution of the squalene synthase cluster.

Lichen-forming fungi (LFF) are prolific producers of functionally and structurally diverse secondary metabolites, most of which are taxonomically exclusive and play lineage-specific roles. To date, widely distributed, evolutionarily conserved biosynthetic pathways in LFF are not known. However, this idea stems from polyketide derivatives, since most biochemical research on lichens has concentrated on polyketide synthases (PKSs). Here, we present the first systematic identification and comparison of terpene biosynthetic genes of LFF using all the available Lecanoromycete reference genomes and 22 de novo sequenced ones (111 in total, representing 60 genera and 23 families). We implemented genome mining and gene networking approaches to identify and group the biosynthetic gene clusters (BGCs) into networks of similar BGCs. Our large-scale analysis led to the identification of 724 terpene BGCs with varying degrees of pairwise similarity. Most BGCs in the dataset were unique with no similarity to a previously known fungal or bacterial BGC or among each other. Remarkably, we found two BGCs that were widely distributed in LFF. Interestingly, both conserved BGCs contain the same core gene, i.e., putatively a squalene/phytoene synthase (SQS), involved in sterol biosynthesis. This indicates that early gene duplications, followed by gene losses/gains and gene rearrangement are the major evolutionary factors shaping the composition of these widely distributed SQS BGCs across LFF. We provide an in-depth overview of these BGCs, including the transmembrane, conserved, variable and LFF-specific regions. Our study revealed that lichenized fungi do have a highly conserved BGC, providing the first evidence that a biosynthetic gene may constitute essential genes in lichens.

Genome-wide differentiation corresponds to climatic niches in two species of lichen-forming fungi.

Lichens can withstand fluctuating environmental conditions such as hydration-desiccation cycles. Many species distribute across climate zones, suggesting population-level adaptations to conditions such as freezing and drought. Here, we aim to understand how climate affects population genomic patterns in lichenized fungi. We analysed population structure along elevational gradients in closely related Umbilicaria phaea (North American; two gradients) and Umbilicaria pustulata (European; three gradients). All gradients showed clear genomic breaks splitting populations into low-elevation (Mediterranean zone) and high-elevation (cold temperate zone). A total of 3301 SNPs in U. phaea and 138 SNPs in U. pustulata were driven to fixation between the two ends of the gradients. The difference between the species is likely due to differences in recombination rate: the sexually reproducing U. phaea has a higher recombination rate than the primarily asexually reproducing U. pustulata. Cline analysis revealed allele frequency transitions along all gradients at approximately 0°C, coinciding with the transition between the Mediterranean and cold temperate zones, suggesting freezing is a strong driver of population differentiation. Genomic scans further confirmed temperature-related selection targets. Both species showed similar differentiation patterns overall, but different selected alleles indicate convergent adaptation to freezing. Our results enrich our knowledge of fungal genomic functions related to temperature and climate, fungal population genomics, and species responses to environmental heterogeneity.

Circadian clock- and temperature-associated genes contribute to overall genomic differentiation along elevation in lichenized fungi.

Circadian regulation is linked to local environmental adaptation, and many species with broad climatic niches display variation in circadian genes. Here, we hypothesize that lichenizing fungi occupying different climate zones tune their metabolism to local environmental conditions with the help of their circadian systems. We study two species of the genus Umbilicaria occupying similar climatic niches (Mediterranean and the cold temperate) in different continents. Using homology to Neurospora crassa genes, we identify gene sets associated with circadian rhythms (11 core, 39 peripheral genes) as well as temperature response (37 genes). Nucleotide diversity of these genes is significantly correlated with mean annual temperature, minimum temperature of the coldest month and mean temperature of the coldest quarter. Furthermore, we identify altitudinal clines in allele frequencies in several non-synonymous substitutions in core clock components, for example, white collar-like, frh-like and various ccg-like genes. A dN/dS approach revealed a few significant peripheral clock- and temperature-associated genes (e.g. ras-1-like, gna-1-like) that may play a role in fine-tuning the circadian clock and temperature-response machinery. An analysis of allele frequency changes demonstrated the strongest evidence for differentiation above the genomic background in the clock-associated genes in U. pustulata. These results highlight the likely relevance of the circadian clock in environmental adaptation, particularly frost tolerance, of lichens. Whether or not the fungal clock modulates the symbiotic interaction within the lichen consortium remains to be investigated. We corroborate the finding of genetic variation in clock components along altitude-not only latitude-as has been reported in other species.

Lichen holobionts show compositional structure along elevation.

Holobionts are dynamic ecosystems that may respond to abiotic drivers with compositional changes. Uncovering elevational diversity patterns within these microecosystems can further our understanding of community-environment interactions. Here, we assess how the major components of lichen holobionts-fungal hosts, green algal symbionts, and the bacterial community-collectively respond to an elevational gradient. We analyse populations of two lichen symbioses, Umbilicaria pustulata and U. hispanica, along an elevational gradient spanning 2100 altitudinal metres and covering three major biomes. Our study shows (i) discontinuous genomic variation in fungal hosts with one abrupt genomic differentiation within each of the two host species, (ii) altitudinally structured bacterial communities with pronounced turnover within and between hosts, and (iii) altitude-specific presence of algal symbionts. Alpha diversity of bacterial communities decreased with increasing elevation. A marked turnover in holobiont diversity occurred across two altitudinal belts: at 11°C-13°C average annual temperature (here: 800-1200 m a.s.l.), and at 7°C-9°C average annual temperature (here: 1500-1800 m a.s.l.). The two observed zones mark a clustering of distribution limits and community shifts. The three ensuing altitudinal classes, that is, the most frequent combinations of species in holobionts, approximately correspond to the Mediterranean, cool-temperate, and alpine climate zones. We conclude that multitrophic microecosystems, such as lichen holobionts, respond with concerted compositional changes to climatic factors that also structure communities of macroorganisms, for example, vascular plants.

Metatranscriptomics reveals contrasting effects of elevation on the activity of bacteria and bacterial viruses in soil.

Soil microbial diversity affects ecosystem functioning and global biogeochemical cycles. Soil bacterial communities catalyse a diversity of biogeochemical reactions and have thus sparked considerable scientific interest. One driver of bacterial community dynamics in natural ecosystems has so far been largely neglected: the predator-prey interactions between bacterial viruses (bacteriophages) and bacteria. To generate ground level knowledge on environmental drivers of these particular predator-prey dynamics, we propose an activity-based ecological framework to simultaneous capture community dynamics of bacteria and bacteriophages in soils. An ecological framework and specifically the analyses of community dynamics across latitudinal and elevational gradients have been widely used in ecology to understand community-wide responses of innumerable taxa to environmental change, in particular to climate. Here, we tested the hypothesis that the activity of bacteria and bacteriophages codeclines across an elevational gradient. We used metatranscriptomics to investigate bacterial and bacteriophage activity patterns at five sites across 400 elevational metres in the Swiss Alps in 2015 and 2017. We found that metabolic activity (transcription levels) of bacteria declined significantly with increasing elevation, but activity of bacteriophages did not. We showed that bacteriophages are consistently active in soil along the entire gradient, making bacteriophage activity patterns divergent from that of their putative bacterial prey. Future efforts will be necessary to link the environment-activity relationship to predator-prey dynamics, and to understand the magnitude of viral contributions to carbon, nitrogen and phosphorus cycling when infection causes bacterial cell death, a process that may represent an overlooked component of soil biogeochemical cycles.

Virus diversity in metagenomes of a lichen symbiosis (Umbilicaria phaea): complete viral genomes, putative hosts and elevational distributions.

Viruses can play critical roles in symbioses by initiating horizontal gene transfer, affecting host phenotypes, or expanding their host's ecological niche. However, knowledge of viral diversity and distribution in symbiotic organisms remains elusive. Here we use deep-sequenced metagenomic DNA (PacBio Sequel II; two individuals), paired with a population genomics approach (Pool-seq; 11 populations, 550 individuals) to understand viral distributions in the lichen Umbilicaria phaea. We assess (i) viral diversity in lichen thalli, (ii) putative viral hosts (fungi, algae, bacteria) and (iii) viral distributions along two replicated elevation gradients. We identified five novel viruses, showing 28%-40% amino acid identity to known viruses. They tentatively belong to the families Caulimoviridae, Myoviridae, Podoviridae and Siphoviridae. Our analysis suggests that the Caulimovirus is associated with green algal photobionts (Trebouxia) of the lichen, and the remaining viruses with bacterial hosts. We did not detect viral sequences in the mycobiont. Caulimovirus abundance decreased with increasing elevation, a pattern reflected by a specific algal lineage hosting this virus. Bacteriophages showed population-specific patterns. Our work provides the first comprehensive insights into viruses associated with a lichen holobiont and suggests an interplay of viral hosts and environment in structuring viral distributions.

Climate-specific biosynthetic gene clusters in populations of a lichen-forming fungus.

Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen-forming fungus Umbilicaria pustulata. We collected a total of 600 individuals from the Mediterranean and cold-temperate climates. Population genomic analyses indicate that U. pustulata contains three clusters that are highly differentiated between the Mediterranean and cold-temperate populations. One entire cluster is exclusively present in cold-temperate populations, and a second cluster is putatively dysfunctional in all cold-temperate populations. In the third cluster variation is fixed in all cold-temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate-associated BGCs and BGC variations in lichen-forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.

Expanding the mutualistic niche: parallel symbiont turnover along climatic gradients.

Keystone mutualisms, such as corals, lichens or mycorrhizae, sustain fundamental ecosystem functions. Range dynamics of these symbioses are, however, inherently difficult to predict because host species may switch between different symbiont partners in different environments, thereby altering the range of the mutualism as a functional unit. Biogeographic models of mutualisms thus have to consider both the ecological amplitudes of various symbiont partners and the abiotic conditions that trigger symbiont replacement. To address this challenge, we here investigate 'symbiont turnover zones'--defined as demarcated regions where symbiont replacement is most likely to occur, as indicated by overlapping abundances of symbiont ecotypes. Mapping the distribution of algal symbionts from two species of lichen-forming fungi along four independent altitudinal gradients, we detected an abrupt and consistent ß-diversity turnover suggesting parallel niche partitioning. Modelling contrasting environmental response functions obtained from latitudinal distributions of algal ecotypes consistently predicted a confined altitudinal turnover zone. In all gradients this symbiont turnover zone is characterized by approximately 12°C average annual temperature and approximately 5°C mean temperature of the coldest quarter, marking the transition from Mediterranean to cool temperate bioregions. Integrating the conditions of symbiont turnover into biogeographic models of mutualisms is an important step towards a comprehensive understanding of biodiversity dynamics under ongoing environmental change.

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata.

BACKGROUND: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. RESULTS: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. CONCLUSIONS: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

Biosynthetic Gene Content of the 'Perfume Lichens' Evernia prunastri and Pseudevernia furfuracea.

Lichen-forming fungi produce a vast number of unique natural products with a wide variety of biological activities and human uses. Although lichens have remarkable potential in natural product research and industry, the molecular mechanisms underlying the biosynthesis of lichen metabolites are poorly understood. Here we use genome mining and comparative genomics to assess biosynthetic gene clusters and their putative regulators in the genomes of two lichen-forming fungi, which have substantial commercial value in the perfume industry, Evernia prunastri and Pseudevernia furfuracea. We report a total of 80 biosynthetic gene clusters (polyketide synthases (PKS), non-ribosomal peptide synthetases and terpene synthases) in E. prunastri and 51 in P. furfuracea. We present an in-depth comparison of 11 clusters, which show high homology between the two species. A ketosynthase (KS) phylogeny shows that biosynthetic gene clusters from E. prunastri and P. furfuracea are widespread across the Fungi. The phylogeny includes 15 genomes of lichenized fungi and all fungal PKSs with known functions from the MIBiG database. Phylogenetically closely related KS domains predict not only similar PKS architecture but also similar cluster architecture. Our study highlights the untapped biosynthetic richness of lichen-forming fungi, provides new insights into lichen biosynthetic pathways and facilitates heterologous expression of lichen biosynthetic gene clusters.

Environment and host identity structure communities of green algal symbionts in lichens.

An understanding of how biotic interactions shape species' distributions is central to predicting host-symbiont responses under climate change. Switches to locally adapted algae have been proposed to be an adaptive strategy of lichen-forming fungi to cope with environmental change. However, it is unclear how lichen photobionts respond to environmental gradients, and whether they play a role in determining the fungal host's upper and lower elevational limits. Deep-coverage Illumina DNA metabarcoding was used to track changes in the community composition of Trebouxia algae associated with two phylogenetically closely related, but ecologically divergent fungal hosts along a steep altitudinal gradient in the Mediterranean region. We detected the presence of multiple Trebouxia species in the majority of thalli. Both altitude and host genetic identity were strong predictors of photobiont community assembly in these two species. The predominantly clonally dispersing fungus showed stronger altitudinal structuring of photobiont communities than the sexually reproducing host. Elevation ranges of the host were not limited by the lack of compatible photobionts. Our study sheds light on the processes guiding the formation and distribution of specific fungal-algal combinations in the lichen symbiosis. The effect of environmental filtering acting on both symbiotic partners appears to shape the distribution of lichens.

Season-Long Experimental Drought Alters Fungal Community Composition but Not Diversity in a Grassland Soil.

Using terrestrial model ecosystems (TMEs), we investigated how reduced moisture conditions impact soil fungal communities from a temperate grassland over the course of an entire season. Starting at about 65% of the soil's maximum water holding capacity (WHCmax), TME soils were adjusted to three moisture levels for 15 weeks: 70% WHCmax, approximating starting conditions, 50% WHCmax, and 30% WHCmax, representing reduced moisture conditions. Diversity and abundances of soil fungi at the start and at the end of the experiment were characterized using Illumina meta-barcoding. Community diversity at the end of the experiment did not differ between experimental moisture levels and was comparable to diversity measures from the field. However, fungal communities did change compositionally in both abundances and presence/absence of species. Analyzing class-level and individual contributions of fungi to these changes revealed that only a minor portion reacted significantly, indicating that most compositional change was likely driven by many consistent small-scale shifts in presence/absences or abundances. Together, our results show that prolonged reduction in soil moisture conditions will trigger compositional changes in soil fungal communities but not necessarily change overall diversity. We highlight the cumulative contribution of minor but consistent changes among community members, as opposed to significant responses of individual species. We also detected a strong general experimental effect on soil fungi that are moved from the field to experimental TMEs, suggesting the importance of acclimatization effects in these communities under laboratory conditions.

Antimicrobial-producing Pseudoalteromonas from the marine environment of Panama shows a high phylogenetic diversity and clonal structure.

Pseudoalteromonas is a genus of marine bacteria often found in association with other organisms. Although several studies have examined Pseudoalteromonas diversity and their antimicrobial activity, its diversity in tropical environments is largely unexplored. We investigated the diversity of Pseudoalteromonas in marine environments of Panama using a multilocus phylogenetic approach. Furthermore we tested their antimicrobial capacity and evaluated the effect of recombination and mutation in shaping their phylogenetic relationships. The reconstruction of clonal relationships among 78 strains including 15 reference Pseudoalteromonas species revealed 43 clonal lineages, divided in pigmented and non-pigmented strains. In total, 39 strains displayed moderate to high activity against Gram-positive and Gram-negative bacteria and fungi. Linkage disequilibrium analyses showed that the Pseudoalteromonas strains of Panama have a highly clonal structure and that, although present, recombination is not frequent enough to break the association among alleles. This clonal structure is in contrast to the high rates of recombination generally reported for aquatic and marine bacteria. We propose that this structure is likely due to the symbiotic association with marine invertebrates of most strains analyzed. Our results also show that there are several putative new species of Pseudoalteromonas in Panama to be described.

Adaptive differentiation coincides with local bioclimatic conditions along an elevational cline in populations of a lichen-forming fungus.

BACKGROUND: Many fungal species occur across a variety of habitats. Particularly lichens, fungi forming symbioses with photosynthetic partners, have evolved remarkable tolerances for environmental extremes. Despite their ecological importance and ubiquity, little is known about the genetic basis of adaption in lichen populations. Here we studied patterns of genome-wide differentiation in the lichen-forming fungus Lasallia pustulata along an altitudinal gradient in the Mediterranean region. We resequenced six populations as pools and identified highly differentiated genomic regions. We then detected gene-environment correlations while controlling for shared population history and pooled sequencing bias, and performed ecophysiological experiments to assess fitness differences of individuals from different environments. RESULTS: We detected two strongly differentiated genetic clusters linked to Mediterranean and temperate-oceanic climate, and an admixture zone, which coincided with the transition between the two bioclimates. High altitude individuals showed ecophysiological adaptations to wetter and more shaded conditions. Highly differentiated genome regions contained a number of genes associated with stress response, local environmental adaptation, and sexual reproduction. CONCLUSIONS: Taken together our results provide evidence for a complex interplay between demographic history and spatially varying selection acting on a number of key biological processes, suggesting a scenario of ecological speciation.

Fungal-algal association patterns in lichen symbiosis linked to macroclimate.

Both macroclimate and evolutionary events may influence symbiont association and diversity patterns. Here we assess how climatic factors and evolutionary events shape fungal-algal association patterns in the widely distributed lichen-forming fungal genus Protoparmelia. Multilocus phylogenies of fungal and algal partners were generated using 174 specimens. Coalescent-based species delimitation analysis suggested that 23 fungal hosts are associating with 20 algal species. Principal component analysis (PCA) was performed to infer how fungal-algal association patterns varied with climate. Fungi associated with one to three algal partners whereas algae accepted one to five fungal partners. Both fungi and algae were more specific, associating with fewer partners, in the warmer climates. Interaction with more than one partner was more frequent in cooler climates for both the partners. Cophylogenetic analyses suggest congruent fungal-algal phylogenies. Host switch was a more common event in warm climates, whereas failure of the photobiont to diverge with its fungal host was more frequent in cooler climates. We conclude that both environmental factors and evolutionary events drive fungal and algal evolution in Protoparmelia. The processes leading to phylogenetic congruence of fungi and algae are different in different macrohabitats in our study system. Hence, closely related species inhabiting diverse habitats may follow different evolutionary pathways.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.

Relocation, high-latitude warming and host genetic identity shape the foliar fungal microbiome of poplars.

Micro-organisms associated with plants and animals affect host fitness, shape community structure and influence ecosystem properties. Climate change is expected to influence microbial communities, but their reactions are not well understood. Host-associated micro-organisms are influenced by the climate reactions of their hosts, which may undergo range shifts due to climatic niche tracking, or may be actively relocated to mitigate the effects of climate change. We used a common-garden experiment and rDNA metabarcoding to examine the effect of host relocation and high-latitude warming on the complex fungal endophytic microbiome associated with leaves of an ecologically dominant boreal forest tree (Populus balsamifera L.). We also considered the potential effects of poplar genetic identity in defining the reactions of the microbiome to the treatments. The relocation of hosts to the north increased the diversity of the microbiome and influenced its structure, with results indicating enemy release from plausible pathogens. High-latitude warming decreased microbiome diversity in comparison with natural northern conditions. The warming also caused structural changes, which made the fungal communities distinct in comparison with both low-latitude and high-latitude natural communities, and increased the abundance of plausible pathogens. The reactions of the microbiome to relocation and warming were strongly dependent on host genetic identity. This suggests that climate change effects on host-microbiome systems may be mediated by the interaction of environmental factors and the population genetic processes of the hosts.

Meta-analysis of deep-sequenced fungal communities indicates limited taxon sharing between studies and the presence of biogeographic patterns.

High-throughput amplicon sequencing gives new insights into fungal community ecology. Massively generated molecular data lead to the discovery of vast fungal diversity. However, it is unclear to what extent operational taxonomic units (OTUs) overlap among independent studies, because no comparative studies exist. We compared fungal diversity based on the internal transcribed spacer (ITS1) region among 10 published studies. Starting from the raw 454 pyrosequencing data, we used a uniform pipeline to prune the reads. We investigated fungal richness and taxonomic composition among phyllosphere and soil fungal communities, as well as biogeographic signals in the data. We did not find globally distributed OTUs, even when comparing fungal communities from similar habitats (phyllosphere or soil). This suggests that high local fungal diversity scales up to high global diversity. The most OTU-rich classes in the phyllosphere were Dothideomycetes (21%) and Sordariomycetes (14%), and in the soil were Sordariomycetes (13%) and Agaricomycetes (12%). The richness estimates suggest the presence of undiscovered fungal diversity even in deeply sequenced study systems. The small number of OTUs shared among studies indicates that globally distributed taxa and habitat generalists may be rare. Latitudinal diversity decline and distance decay relationships suggest the presence of biogeographic patterns similar to those in plants and animals.

Insights into intrathalline genetic diversity of the cosmopolitan lichen symbiotic green alga Trebouxia decolorans Ahmadjian using microsatellite markers.

Trebouxia decolorans is a widespread and common symbiotic green alga that is found in association with different species of lichen-forming fungi. By applying T. decolorans-specific microsatellite markers, we investigated the within-thallus diversity of T. decolorans in thalli of Xanthoria parietina and Anaptychia ciliaris. We found several algal strains in most of the thalli of both hosts. High genetic differentiation among thalli suggests that algal diversity is generated de novo via mutation in both fungal hosts. Rarefied allelic richness of the algae was higher in thalli of X. parietina. Our results indicate that in X. parietina intrathalline algal diversity is additionally created by environmental uptake of algae either at the start of the symbiotic association or during the lifetime of the thallus. This study indicates that promiscuous host-symbiont associations in lichen symbioses with Trebouxia spp. may be more common than currently recognized.

Integrating coalescent and phylogenetic approaches to delimit species in the lichen photobiont Trebouxia.

The accurate assessment of species boundaries in symbiotic systems is a prerequisite for the study of speciation, co-evolution and selectivity. Many studies have shown the high genetic diversity of green algae from the genus Trebouxia, the most common photobiont of lichen-forming fungi. However, the phylogenetic relationships, and the amount of cryptic diversity of these algae are still poorly understood, and an adequate species concept for trebouxiophycean algae is still missing. In this study we used a multifaceted approach based on coalescence (GMYC, STEM) and phylogenetic relationships to assess species boundaries in the trebouxioid photobionts of the lichen-forming fungus Lasallia pustulata. We further investigated whether putative species of Trebouxia found in L. pustulata are shared with other lichen-forming fungi. We found that L. pustulata is associated with at least five species of Trebouxia and most of them are shared with other lichen-forming fungi, showing different patterns of species-to-species and species-to-community interactions. We also show that one of the putative Trebouxia species is found exclusively in association with L. pustulata and is restricted to thalli from localities with Mediterranean microclimate. We suggest that the species delimitation method presented in this study is a promising tool to address species boundaries within the heterogeneous genus Trebouxia.