The microbiome of the lichen Lobaria pulmonaria varies according to climate on a subcontinental scale.

The Lobaria pulmonaria holobiont comprises algal, fungal, cyanobacterial and bacterial components. We investigated L. pulmonaria's bacterial microbiome in the adaptation of this ecologically sensitive lichen species to diverse climatic conditions. Our central hypothesis posited that microbiome composition and functionality aligns with subcontinental-scale (a stretch of ~1100 km) climatic parameters related to temperature and precipitation. We also tested the impact of short-term weather dynamics, sampling season and algal/fungal genotypes on microbiome variation. Metaproteomics provided insights into compositional and functional changes within the microbiome. Climatic variables explained 41.64% of microbiome variation, surpassing the combined influence of local weather and sampling season at 31.63%. Notably, annual mean temperature and temperature seasonality emerged as significant climatic drivers. Microbiome composition correlated with algal, not fungal genotype, suggesting similar environmental recruitment for the algal partner and microbiome. Differential abundance analyses revealed distinct protein compositions in Sub-Atlantic Lowland and Alpine regions, indicating differential microbiome responses to contrasting environmental/climatic conditions. Proteins involved in oxidative and cellular stress were notably different. Our findings highlight microbiome plasticity in adapting to stable climates, with limited responsiveness to short-term fluctuations, offering new insights into climate adaptation in lichen symbiosis.

The phylogeny and taxonomy of Glypholecia (Acarosporaceae, lichenized Ascomycota), including a new species from northwestern China.

Glypholeciaqinghaiensis An C. Yin, Q. Y. Zhong & Li S. Wang is described as new to science. It is characterized by its squamulose thallus, compound apothecia, ellipsoid ascospores, and the presence of rhizines on the lower surface of the thallus. A phylogenetic tree of Glypholecia species was constructed based on nrITS and mtSSU sequences. Two species G.qinghaiensis and G.scabra are confirmed in China.

A research agenda for nonvascular photoautotrophs under climate change.

Nonvascular photoautotrophs (NVP), including bryophytes, lichens, terrestrial algae, and cyanobacteria, are increasingly recognized as being essential to ecosystem functioning in many regions of the world. Current research suggests that climate change may pose a substantial threat to NVP, but the extent to which this will affect the associated ecosystem functions and services is highly uncertain. Here, we propose a research agenda to address this urgent question, focusing on physiological and ecological processes that link NVP to ecosystem functions while also taking into account the substantial taxonomic diversity across multiple ecosystem types. Accordingly, we developed a new categorization scheme, based on microclimatic gradients, which simplifies the high physiological and morphological diversity of NVP and world-wide distribution with respect to several broad habitat types. We found that habitat-specific ecosystem functions of NVP will likely be substantially affected by climate change, and more quantitative process understanding is required on: (1) potential for acclimation; (2) response to elevated CO2 ; (3) role of the microbiome; and (4) feedback to (micro)climate. We suggest an integrative approach of innovative, multimethod laboratory and field experiments and ecophysiological modelling, for which sustained scientific collaboration on NVP research will be essential.

Phylogeographic analyses of an epiphytic foliose lichen show multiple dispersal events westward from the Hengduan Mountains of Yunnan into the Himalayas.

Lobaria pindarensis is an endemic species of the Himalayas and the Hengduan Mountains. Little information is available on the phylogeography genetics and colonization history of this species or how its distribution patterns changed in response to the orographic history of the Himalayas and Hengduan Mountains. Based on samples covering a major part of the species' distribution range, we used 443 newly generated sequences of nine loci for molecular coalescent analyses in order to reconstruct the evolutionary history of L. pindarensis, and to reconstruct the species' ancestral phylogeographic distributions using Bayesian binary MCMC analyses. The results suggest that current populations originated from the Yunnan region of the Hengduan Mountains in the middle Pliocene, and that the Himalayas of Bhutan were colonized by a lineage that diverged from Yunnan ca. 2.72 Ma. The analysis additionally indicates that the Nepal and Xizang areas of the Himalayas were colonized from Yunnan as well, and that there was later a second dispersal event from Yunnan to Bhutan. We conclude that the change in climate and habitat related to the continuous uplift of the Himalayas and the Hengduan Mountains in the late Pliocene and middle Pleistocene influenced the geographic distribution pattern of L. pindarensis.

Gene flow in a pioneer plant metapopulation (Myricaria germanica) at the catchment scale in a fragmented alpine river system.

River alterations for natural hazard mitigation and land reclamation result in habitat decline and fragmentation for riparian plant species. Extreme events such as floods are responsible for additional local species loss or population decline. Tributaries might provide refugia and subsequent source populations for the colonization of downstream sites in connected riverine networks with metapopulations of plant species. In this study, we analyzed the metapopulation structure of the endangered riparian shrub species Myricaria germanica along the river Isel, Austria, which is part of the Natura 2000 network, and its tributaries. The use of 22 microsatellite markers allowed us to assess the role of tributaries and single populations as well as gene flow up- and downstream. The analysis of 1307 individuals from 45 sites shows the influence of tributaries to the genetic diversity at Isel and no overall isolation by distance pattern. Ongoing bidirectional gene flow is revealed by the detection of first-generation migrants in populations of all tributaries as well as the river Isel, supporting upstream dispersal by wind (seeds) or animals (seeds and pollen). However, some populations display significant population declines and high inbreeding, and recent migration rates are non-significant or low. The genetic pattern at the mouth of river Schwarzach into Isel and shortly thereafter river Kalserbach supports the finding that geographically close populations remain connected and that tributaries can form important refugia for M. germanica in the dynamic riverine network. Conservation and mitigation measures should therefore focus on providing sufficient habitat along tributaries of various size allowing pioneer plants to cope with extreme events in the main channel, especially as they are expected to be more frequent under changing climate.

Population genetics and biogeography of the lungwort lichen in North America support distinct Eastern and Western gene pools.

PREMISE: Populations of species with large spatial distributions are shaped by complex forces that differ throughout their ranges. To maintain the genetic diversity of species, genepool-based subsets of widespread species must be considered in conservation assessments. METHODS: The population genetics of the lichenized fungus Lobaria pulmonaria and its algal partner, Symbiochloris reticulata, were investigated using microsatellite markers to determine population structure, genetic diversity, and degree of congruency in eastern and western North America. Data loggers measuring temperature and humidity were deployed at selected populations in eastern North America to test for climatic adaptation. To better understand the role Pleistocene glaciations played in shaping population patterns, a North American, range-wide species distribution model was constructed and hindcast to 22,000 years before present and at 500-year time slices from then to the present. RESULTS: The presence of two gene pools with minimal admixture was supported, one in the U.S. Pacific Northwest and one in eastern North America. Western populations were significantly more genetically diverse than eastern populations. There was no evidence for climatic adaptation among eastern populations, though there was evidence for range-wide adaptation to evapotranspiration rates. Hindcast distribution models suggest that observed genetic diversity may be due to a drastic Pleistocene range restriction in eastern North America, whereas a substantial coastal refugial area is inferred in the west. CONCLUSIONS: Taken together the results show different, complex population histories of L. pulmonaria in eastern and western North America, and suggest that conservation planning for each gene pool should be considered separately.

Phylogeographic reconstructions can be biased by ancestral shared alleles: The case of the polymorphic lichen Bryoria fuscescens in Europe and North Africa.

Large phylogeographic studies on lichens are scarce, and none involves a single species within which different lineages show fixed alternative dispersal strategies. We investigated Bryoria fuscescens (including B. capillaris) in Europe and western North Africa by phenotypically characterizing 1400 specimens from 64 populations and genotyping them with 14 microsatellites. We studied population structure and genetic diversity at the local and continental scales, discussed the post-glacial phylogeography, and compared dispersal capacities of phenotypes with and without soralia. Our main hypothesis is that the estimated phylogeography, migration routes, and dispersal capacities may be strongly biased by ancestral shared alleles. Scandinavia is genetically the richest area, followed by the Iberian Peninsula, the Carpathians, and the Alps. Three gene pools were detected: two partially linked to phenotypic characteristics, and the third one genetically related to the American sister species B. pseudofuscescens. The comparison of one gene pool producing soredia and one not, suggested both as panmictic, with similar levels of isolation by distance (IBD). The migration routes were estimated to span from north to south, in disagreement with the assessed glacial refugia. The presence of ancestral shared alleles in distant populations can explain the similar IBD levels found in both gene pools while producing a false signal of panmixia, and also biasing the phylogeographic reconstruction. The incomplete lineage sorting recorded for DNA sequence loci also supports this hypothesis. Consequently, the high diversity in Scandinavia may rather come from recent immigration into northern populations than from an in situ diversification. Similar patterns of ancestral shared polymorphism may bias the phylogeographical reconstruction of other lichen species.

Deep divergence between island populations in lichenized fungi.

Macaronesia is characterized by a high degree of endemism and represents a noteworthy system to study the evolutionary history of populations and species. Here, we compare the population-genetic structure in three lichen-forming fungi, the widespread Lobaria pulmonaria and two Macaronesian endemics, L. immixta and L. macaronesica, based on microsatellites. We utilize population genetic approaches to explore population subdivision and evolutionary history of these taxa on the Canary Islands, Madeira, Azores, and the western Iberian Peninsula. A common feature in all species was the deep divergence between populations on the Azores, a pattern expected by the large geographic distance among islands. For both endemic species, there was a major split between archipelagos. In contrast, in the widespread L. pulmonaria, divergent individuals were distributed across multiple archipelagos, suggesting a complex evolutionary history involving repeated migration between islands and mainland.

Low genetic differentiation between apotheciate Usnea florida and sorediate Usnea subfloridana (Parmeliaceae, Ascomycota) based on microsatellite data.

Accurate species delimitation has a pivotal role in conservation biology, and it is especially important for threatened species where decisions have political and economic consequences. Finding and applying appropriate character sets and analytical tools to resolve interspecific relationships remains challenging in lichenized fungi. The main aim of our study was to re-assess the species boundaries between Usnea subfloridana and Usnea florida, which have been phylogenetically indistinguishable until now, but are different in reproductive mode and ecological preferences, using fungal-specific simple sequence repeats (SSR), i.e. microsatellite markers. Bayesian clustering analysis, discriminant analysis of principal components (DAPC), minimal spanning network (MSN), and principal component analysis (PCA) failed to separate U. florida and U. subfloridana populations. However, a low significant differentiation between the two taxa was observed across all populations according to AMOVA results. Also, analysis of shared haplotypes and statistical difference in clonal diversity (M) supported the present-day isolation between the apotheciate U. florida and predominantly sorediate U. subfloridana. Our results do not provide a clear support either for the separation of species in this pair or the synonymization of U. florida and U. subfloridana. We suggest that genome-wide data could help resolve the taxonomic question in this species pair.

Contrasting co-occurrence patterns of photobiont and cystobasidiomycete yeast associated with common epiphytic lichen species.

The popular dual definition of lichen symbiosis is under question with recent findings of additional microbial partners living within the lichen body. Here we compare the distribution and co-occurrence patterns of lichen photobiont and recently described secondary fungus (Cyphobasidiales yeast) to evaluate their dependency on lichen host fungus (mycobiont). We sequenced the nuclear internal transcribed spacer (ITS) strands for mycobiont, photobiont, and yeast from six widespread northern hemisphere epiphytic lichen species collected from 25 sites in Switzerland and Estonia. Interaction network analyses and multivariate analyses were conducted on operational taxonomic units based on ITS sequence data. Our study demonstrates the frequent presence of cystobasidiomycete yeasts in studied lichens and shows that they are much less mycobiont-specific than the photobionts. Individuals of different lichen species growing on the same tree trunk consistently hosted the same or closely related mycobiont-specific Trebouxia lineage over geographic distances while the cystobasidiomycete yeasts were unevenly distributed over the study area - contrasting communities were found between Estonia and Switzerland. These results contradict previous findings of high mycobiont species specificity of Cyphobasidiales yeast at large geographic scales. Our results suggest that the yeast might not be as intimately associated with the symbiosis as is the photobiont.

Microsatellite based genetic diversity of the widespread epiphytic lichen Usnea subfloridana (Parmeliaceae, Ascomycota) in Estonia: comparison of populations from the mainland and an island.

Understanding the distribution of genetic patterns and structure is an essential target in population genetics and, thereby, important for conservation genetics. The main aim of our study was to investigate the population genetics of Usnea subfloridana, a widespread lichenised fungus, focusing on a comparison of genetic variation of its populations amongst three geographically remote and disconnected regions, in order to determine relationships amongst environmental data, variation in lichen secondary chemistry and microsatellite data in genotyped populations. In all, 928 Usnea thalli from 17 populations were genotyped using seven specific fungal microsatellite markers. Different measures of genetic diversity (allelic richness, private allelic richness, Nei's unbiased genetic diversity and clonal diversity) were calculated and compared between lichen populations. Our results revealed a low genetic differentiation of U. subfloridana populations amongst three distant areas in Estonia and also a high level of gene flow. The results support suggestion of the long-range vegetative dispersal of subpendulous U. subfloridana via symbiotic propagules (soralia, isidia or fragments of thalli). Our study has also provided evidence that environmental variables, including mean annual temperature and geographical longitude, shape the genetic structure of U. subfloridana populations in Estonia. Additionally, a weak but statistically significant correlation between lichen chemotypes and microsatellite allele distribution was found in genotyped specimens.

New species and records of Pyxine (Caliciaceae) in China.

In this study, the diversity of Pyxine Fr. in China was assessed based on morphological and chemical traits and molecular data are inferred from ITS and mtSSU sequences. Nineteen species were recognised, including three that are new to science (i.e. P.flavicans M. X. Yang & Li S. Wang, P.hengduanensis M. X. Yang & Li S. Wang and P.yunnanensis M. X. Yang & Li S. Wang) and three records new to China were found (i.e. P.cognata Stirt., P.himalayensis Awas. and P.minuta Vain.). Pyxineyunnanensis is diagnosed by the small size of the apothecia, a white medulla of the stipe and the presence of lichexanthone. Pyxineflavicans is characterised by broad lobes, a pale yellow medulla of the stipe and the presence of atranorin. Pyxinehengduanensis can be distinguished by its pale yellow medulla, marginal labriform soralia and the absence of atranorin. Detailed descriptions of each new species are presented, along with a key to the known species of Pyxine in China.

Unconstrained gene flow between populations of a widespread epiphytic lichen Usnea subfloridana (Parmeliaceae, Ascomycota) in Estonia.

Few studies have investigated the genetic diversity of populations of common and widespread lichenized fungi using microsatellite markers, especially the relationships between different measures of genetic diversity and environmental heterogeneity. The main aim of our study was to investigate the population genetics of a widespread and mainly clonally reproducing Usnea subfloridana at the landscape scale, focusing on the comparison of lichen populations within hemiboreal forest stands. Particular attention has been paid to the genetic differentiation of lichen populations in two geographically distinct regions in Estonia and the relationships between forest characteristics and measures of genetic diversity. We genotyped 578 Usnea thalli from eleven lichen populations using seven specific fungal microsatellite markers. Measures of genetic diversity (allelic richness, Shannon's information index, Nei's unbiased genetic diversity, clonal diversity, the number of multilocus genotypes, the number of private alleles, and the minimum number of colonization events) were calculated and compared between Usnea populations. Shared haplotypes, gene flow and AMOVA analyses suggest that unconstrained gene flow and exchange of multilocus genotypes exist between the two geographically remote regions in Estonia. Stand age, mean circumference of the host tree, size of forest site and tree species composition did not show any significant influence on allelic richness, Shannon's information index, Nei's unbiased genetic diversity, clonal diversity, the number of private alleles, and the minimum number of colonization events of U. subfloridana populations. Therefore it was concluded that other factors of habitat heterogeneity could probably have a more significant effect on population genetics of U. subfloridana populations.

Are species-pairs diverging lineages? A nine-locus analysis uncovers speciation among species-pairs of the Lobaria meridionalis-group (Ascomycota).

In spite of considerable effort to verify the theory of species-pairs, uncertainty still exists about the relationship between sexually or vegetatively reproducing populations of morphologically indistinguishable, sympatric lichen species. The current paper studies putative species-pairs within the Asian Lobaria meridionalis-group, using a nine-locus and time calibrated species-tree approach. Analyses demonstrate that pairs of sexually or vegetatively reproducing lineages split into highly supported monophyletic clades-confirming molecularly the species-pair concept for the L. meridionalis-group. In the broader context of evolution and speciation dynamics in lichenized fungi, this paper attempts to synthesize molecular findings from the last two decades to promote a more modern perception of the species-pair concept. Taxonomically, eight species were found to currently conform to the L. meridionalis-group, which differentiated during the Pliocene and Pleistocene. The coincidence of paleoclimatic events with estimated dates of divergence support a bioclimatic hypothesis for the evolution of species in the L. meridionalis-group, which also explains their current eco-geographic distribution patterns. Greater recognition for species with a long and independent evolutionary history, which merit high conservation priority, will be especially critical for preserving geographically restricted endemics from Southeast Asia, where habitat loss is driving rapid declines.

Discovery of long-distance gamete dispersal in a lichen-forming ascomycete.

Accurate estimates of gamete and offspring dispersal range are required for the understanding and prediction of spatial population dynamics and species persistence. Little is known about gamete dispersal in fungi, especially in lichen-forming ascomycetes. Here, we estimate the dispersal functions of clonal propagules, gametes and ascospores of the epiphytic lichen Lobaria pulmonaria. We use hierarchical Bayesian parentage analysis, which integrates genetic and ecological information from multiannual colonization and dispersal source data collected in a large, old-growth forest landscape. The effective dispersal range of gametes is several hundred metres to kilometres from potential paternal individuals. By contrast, clonal propagules disperse only tens of metres, and ascospores disperse over several thousand metres. Our study reveals the dispersal distances of individual reproductive units; clonal propagules, gametes and ascospores, which is of great importance for a thorough understanding of the spatial dynamics of ascomycetes. Sexual reproduction occurs between distant individuals. However, whereas gametes and ascospores disperse over long distances, the overall rate of colonization of trees is low. Hence, establishment is the limiting factor for the colonization of new host trees by the lichen in old-growth landscapes.

Epiphytes in wooded pastures: Isolation matters for lichen but not for bryophyte species richness.

Sylvo-pastoral systems are species-rich man-made landscapes that are currently often severely threatened by abandonment or management intensification. At low tree densities, single trees in these systems represent habitat islands for epiphytic cryptogams. Here, we focused on sycamore maple (Acer pseudoplatanus) wooded pastures in the northern European Alps. We assessed per tree species richness of bryophytes and lichens on 90 sycamore maple trees distributed across six study sites. We analysed the effects of a range of explanatory variables (tree characteristics, environmental variables and isolation measures) on the richness of epiphytic bryophytes and lichens and various functional subgroups (based on diaspore size, habitat preference and red list status). Furthermore, we estimated the effect of these variables on the occurrence of two specific bryophyte species (Tayloria rudolphiana, Orthotrichum rogeri) and one lichen species (Lobaria pulmonaria) of major conservation concern. Bryophytes and lichens, as well as their subgroups, were differently and sometimes contrastingly affected by the variables considered: tree diameter at breast height had no significant effect on bryophytes but negatively affected many lichen groups; tree phenological age positively affected red-listed lichens but not red-listed bryophytes; increasing isolation from neighbouring trees negatively affected lichens but not bryophytes. However, the high-priority bryophyte species T. rudolphiana was also negatively affected by increased isolation at small spatial scales. Orthotrichum rogeri was more frequent on young trees and L. pulmonaria was more frequent on trees with thin stems and large crowns. The results indicate that local dispersal is important for lichens, whereas long distance dispersal seems to be more important for colonisation by bryophytes. Furthermore, our study highlights that different conservation measures need to be taken depending on the taxonomic and functional species group or the individual species that is addressed. In practice, for the conservation of a high overall richness in sylvo-pastoral systems, it is crucial to sustain not only old and large trees but rather a wide range of tree sizes and ages.

Indigenous knowledge and use of lichens by the lichenophilic communities of the Nepal Himalaya.

BACKGROUND: The aim of the study was to document the prevailing indigenous knowledge and various uses of lichens among the lichenophilic communities in the hills and mountainous settlements of Nepal. METHODS: Ethnic uses were recorded during twelve field trips, each of roughly 15 days in three consecutive years, through direct questionnaires administered to 190 respondents. Lichen samples were identified applying microscopic observation and thin layer chromatography (TLC). Voucher specimens of identified species are deposited at TUCH (Tribhuvan University Central Herbarium) in Nepal. RESULTS: Lichens are being used in several ways by different communities of Nepal. We recorded the ethnic use of seven species of lichens belonging to four families (Parmeliaceae, Physciaceae, Ramalinaceae and Usneaceae) and six genera (Heterodermia, Everniastrum, Parmotrema, Ramalina, Thamnolia and Usnea) among the Limbu, Sherpa, Lama, Gurung, Rai, Dalit, Tamang, Chhetri and Brahman communities. The present study revealed six use values namely; Medicinal value (MV), food value (FV), ritual and spiritual value (RSV), aesthetic and decorative value (ADV), bedding value (BV) and ethno-veterinary value (EVV) from different parts of Nepal. Three lichen species, Everniastrum cirrhatum, E. nepalense and Parmotrema cetratum were consumed by the Limbu and Rai communities. The Limbu and Sherpa ethnic groups are regarded as most lichenophilic communities while respondents from Brahman, Chhetri and Tamang communities showed less interest in lichen uses. CONCLUSIONS: The present study contributes to document traditional knowledge on various uses of lichens among nine communities with three different cultural background, inhabitants of eight different altitudinal levels of Nepal. Regarding the six values as identified from this research, significant difference (p = <0.05) were found along altitudinal gradients or locations of the settlements, cultural groups and ethnicity of the respondents.

Impact of alkaline dust pollution on genetic variation of Usnea subfloridana populations.

Very little is known whether and how air pollution impacts genetic diversity of lichenized fungi that are well-known indicators of environmental quality. We studied the genetic variation of eight Usnea subfloridana populations in Pinus sylvestris-dominated boreal forest stands in southern Estonia, Northern Europe; four of these populations were exposed to long-term dust pollution released from unpaved road. The mean bark pH of lichen phorophyte differed considerably between polluted and unpolluted forest stands. We genotyped 274 Usnea thalli using nine specific fungal microsatellite markers. Genetic variation measures were calculated and compared between populations from different habitats. Allelic richness, Shannon's information index, and genetic diversity of lichen populations were significantly higher in unpolluted forest sites than in polluted forest sites. We conclude that environmental disturbances caused by alkaline dust pollution had negative impact on the genetic variation of U. subfloridana, a common species of lichenized fungi.

Barcoding lichen-forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation.

Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database-currently the most complete database for lichen-forming fungi-can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).