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 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.

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.

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.

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.

As thick as three in a bed.

During the evolution of the lichen symbiosis, shifts from one main type of photobiont to another were infrequent (Miadlikowska et al. ) but some remarkable transitions from green algal to diazotrophic cyanobacterial photobionts are known from unrelated fungal clades within the ascomycetes. Cyanobacterial, including tripartite, associations (green algal and cyanobacterial photobionts in one lichen individual) facilitate these holobionts to live as C- and N-autotrophs. Tripartite lichens are among the most productive lichens, which provide N-fertilization to forest ecosystems under oceanic climates (Peltigerales) or deliver low, but ecologically significant N-input into subarctic and alpine soil communities (Lecanorales, Agyriales). In this issue of Molecular Ecology, Schneider et al. (2016) mapped morphometric data against an eight-locus fungal phylogeny across a transition of photobiont interactions from green algal to a tripartite association and used a phylogenetic comparative framework to explore the role of nitrogen-fixing cyanobacteria in size differences in the Trapelia-Placopsis clade (Agyriales). Within the group of tripartite species, the volume of cyanobacteria-containing structures (cephalodia) correlates with thallus thickness in both phylogenetic generalized least squares and phylogenetic generalized linear mixed-effects analyses, and the fruiting body core volume increased ninefold. The authors conclude that cyanobacterial symbiosis appears to have enabled lichens to overcome size constraints in oligotrophic environments such as rock surfaces. The Trapelia-Placopsis clade analyzed by Schneider et al. (2016) is an exciting example of interactions between ecology, phylogeny and lichen biology including development - from thin crustose green algal microlichens to thick placodioid, tripartite macrolichens: as thick as three in a bed (Scott ).

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).

Multi-gene phylogeny of the genus Lobaria: Evidence of species-pair and allopatric cryptic speciation in East Asia.

PREMISE OF THE STUDY: Accurate species delimitation has critical implications for ecological and conservation studies. The lichen genus Lobaria is widely distributed in old-growth forests. Particularly in East Asia, this genus includes many rare and poorly known taxa that are circumscribed as morpho- or chemospecies, as well as species-pairs. METHODS: To critically examine the relationships between species identified via morphological and chemical criteria, phylogenetic species recognition (PSR) was applied to the genus Lobaria. Morphological and chemical patterns of 87 individuals were examined and three independent nuclear loci were sequenced. The East Asian L. meridionalis-group was additionally studied using split decomposition and haplotype network analysis. KEY RESULTS: The genus Lobaria and most of its species were strongly supported statistically. Split decomposition and haplotype networks suggest complex evolutionary histories of species within the East Asian L. meridionalis-group. CONCLUSIONS: Phylogenetic analyses confirmed the monophyly of the genus Lobaria, including L. anomala. Within Lobaria, three major clades were found. These clades associate with different photobionts and comprise 18 known species and 5 undescribed species. Several chemical compounds were found to be neither stable nor invariant characters. Some taxa of the L. meridionalis-group appear to be monophyletic but remain as allopatric cryptic species. In three clades, this study found evidence for diversification processes between isidiate and nonisidiate specimens (species-pair). These findings are discussed in the context of evolutionary hypotheses for speciation processes.

Molecular phylogeny and symbiotic selectivity of the green algal genus Dictyochloropsis s.l. (Trebouxiophyceae): a polyphyletic and widespread group forming photobiont-mediated guilds in the lichen family Lobariaceae.

Dictyochloropsis s.l. is an ecologically important, common but little-studied genus of green algae. Here, we examined the diversity and host selectivity of algae attributed to this genus at both species-to-species and species-to-community levels. We conducted a molecular investigation of 15 cultured strains and several lichen photobionts, using 18S rRNA, rbcL and ITS sequence data. We further used seven alga-specific microsatellite markers to study algal sharing among fungi of the family Lobariaceae in two populations in Madeira and Taiwan (454 lichens). We found that the genus Dictyochloropsis s.l. is polyphyletic. Dictyochloropsis clade 1 comprises only free-living algae whereas Dictyochloropsis clade 2 includes lichenized algae as well as free-living algae. Fungal selectivity towards algae belonging to Dictyochloropsis clade 2 is high. Selectivity varies geographically, with photobionts being restricted to a single region. Finally, we showed that Dictyochloropsis clade 2 individuals are shared among different fungal hosts in communities of lichens of the Lobariaceae. As for other green algal lineages, there is a high amount of cryptic diversity in Dictyochloropsis. Furthermore, co-evolution between Dictyochloropsis clade 2 algae and representatives of the Lobariaceae is manifested at the community level, with several unrelated fungal species being horizontally connected by shared photobiont clones.

Microclimatic differentiation of gene pools in the Lobaria pulmonaria symbiosis in a primeval forest landscape.

Population genetics of the tree-colonizing lichen Lobaria pulmonaria were studied in the largest primeval beech forest of Europe, covering 10 000 ha. During an intensive survey of the area, we collected 1522 thallus fragments originating from 483 trees, which were genotyped with eight mycobiont- and 14 photobiont-specific microsatellite markers. The mycobiont and photobiont of L. pulmonaria were found to consist of two distinct gene pools, which are co-existing within small areas of 3-180 ha in a homogeneous beech forest. The small-scale distribution pattern of the symbiotic gene pools show habitat partitioning of lineages associated with either floodplains or mountain forests. Using approximate Bayesian computation (ABC), we dated the divergence of the two fungal gene pools of L. pulmonaria as the Early Pleistocene. Both fungal gene pools survived the Pleistocene glacial cycles in the Carpathians, although possibly in climatically different refugia. Fungal diversification prior to these cycles and the selection of photobionts with different altitudinal distributions explain the current sympatric, but ecologically differentiated habitat partitioning of L. pulmonaria. In addition, the habitat preferences of the mycobiont are determined by other factors and are rather independent of those of the photobiont at the landscape level. The distinct gene pools should be considered evolutionarily significant units and deserve specific conservation priorities in the future, for example gene pool A, which is a Pliocene relict.

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.

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.

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.

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.

History matters: ecometrics and integrative climate change biology.

Climate change research is increasingly focusing on the dynamics among species, ecosystems and climates. Better data about the historical behaviours of these dynamics are urgently needed. Such data are already available from ecology, archaeology, palaeontology and geology, but their integration into climate change research is hampered by differences in their temporal and geographical scales. One productive way to unite data across scales is the study of functional morphological traits, which can form a common denominator for studying interactions between species and climate across taxa, across ecosystems, across space and through time-an approach we call 'ecometrics'. The sampling methods that have become established in palaeontology to standardize over different scales can be synthesized with tools from community ecology and climate change biology to improve our understanding of the dynamics among species, ecosystems, climates and earth systems over time. Developing these approaches into an integrative climate change biology will help enrich our understanding of the changes our modern world is undergoing.

Characterization of nuclear microsatellite loci in the calcareous fen specialist Scorpidium cossonii (Calliergonaceae).

PREMISE OF THE STUDY: Nuclear microsatellite markers were developed in the fen specialist moss, Scorpidium cossonii, to study genetic diversity and genetic structure of this species in relation to land-use types, habitat fragmentation, and habitat conservation measures. METHODS AND RESULTS: The polymorphisms of 14 microsatellite markers were characterized. All primers were tested on 140 gametophytes collected from four populations in Switzerland. The primers amplified di- and trinucleotide repeats with three to 26 alleles per locus. The primers worked also in the two other species of the genus: 14 and 12 primers successfully amplified in S. revolvens and S. scorpioides, respectively. CONCLUSIONS: In future studies, our primers have the potential to provide valuable information on genetic diversity, genetic structure, and on historical and recent gene flow in S. cossonii; they should also enable related research in other Scorpidium species.