Reproductive and dispersal strategies shape the diversity of mycobiont-photobiont association in Cladonia lichens.

Ecological preferences, partner compatibility, or partner availability are known to be important factors shaping obligate and intimate lichen symbioses. We considered a complex of Cladonia species, traditionally differentiated by the extent of sexual reproduction and the type of vegetative propagules, to assess if the reproductive and dispersal strategies affect mycobiont-photobiont association patterns. In total 85 lichen thalli from 72 European localities were studied, two genetic markers for both Cladonia mycobionts and Asterochloris photobionts were analyzed. Variance partitioning analysis by multiple regression on distance matrices was performed to describe and partition variance in photobiont genetic diversity. Asexually reproducing Cladonia in our study were found to be strongly specific to their photobionts, associating with only two closely related Asterochloris species. In contrast, sexually reproducing lichens associated with seven unrelated Asterochloris lineages, thus being photobiont generalists. The reproductive mode had the largest explanatory power, explaining 44% of the total photobiont variability. Reproductive and dispersal strategies are the key factors shaping photobiont diversity in this group of Cladonia lichens. A strict photobiont specialisation observed in two studied species may steer both evolutionary flexibility and responses to ecological changes of these organisms, and considerably limit their distribution ranges.

Archaeobotanical evidence for climate as a driver of ecological community change across the anthropocene boundary.

The biodiversity response to climate change is a major focus in conservation research and policy. Predictive models that are used to project the impact of climate change scenarios - such as bioclimatic envelope models - are widely applied and have come under severe scrutiny. Criticisms of such models have focussed on at least two problems. First, there is an assumption that climate is the primary driver of observed species distributions ('climatic equilibrium'), when other biogeographical controls are often reliably established. Second, a species' sensitivity to macroclimate may become less relevant when impacts are down-scaled to a local level, incorporating a modifying effect of species interactions structuring communities. This article examines the role of different drivers (climate, pollution and landscape habitat structure) in explaining spatial community variation for a widely applied bioindicator group: lichen epiphytes. To provide an analysis free of 'legacy effects' (e.g. formerly higher pollution loads), the study focused on hazel stems as a relatively short-lived and recently colonized substratum. For communities during the present day, climate is shown to interact with stem size/age as the most likely explanation of community composition, thus coupling a macroclimatic and community-scale effect. The position of present-day communities was projected into ordination space for eight sites in England and compared to the position of historical epiphyte communities from the same sites, reconstructed using preserved hazel wattles dating mainly to the 16th Century. This comparison of community structure for the late- to post-Mediaeval period, with the post-Industrial period, demonstrated a consistent shift among independent sites towards warmer and drier conditions, concurrent with the end of the Little Ice Age. Long-term temporal sensitivity of epiphyte communities to climate variation thus complements spatial community patterns. If more widely applied, preserved lichen epiphytes have potential to generate new baseline conditions of environment and biodiversity for preindustrial lowland Europe.

Testing the use of ITS rDNA and protein-coding genes in the generic and species delimitation of the lichen genus Usnea (Parmeliaceae, Ascomycota).

In lichen-forming fungi, traditional taxonomical concepts are frequently in conflict with molecular data, and identifying appropriate taxonomic characters to describe phylogenetic clades remains challenging in many groups. The selection of suitable markers for the reconstruction of solid phylogenetic hypotheses is therefore fundamental. The lichen genus Usnea is highly diverse, with more than 350 estimated species, distributed in polar, temperate and tropical regions. The phylogeny and classification of Usnea have been a matter of debate, given the lack of phenotypic characters to describe phylogenetic clades and the low degree of resolution of phylogenetic trees. In this study, we investigated the phylogenetic relationships of 52 Usnea species from across the genus, based on ITS rDNA, nuLSU, and two protein-coding genes RPB1 and MCM7. ITS comprised several highly variable regions, containing substantial genetic signal, but also susceptible to causing bias in the generation of the alignment. We compared several methods of alignment of ITS and found that a simultaneous optimization of alignment and phylogeny (using BAli-phy) improved significantly both the topology and the resolution of the phylogenetic tree. However the resolution was even better when using protein-coding genes, especially RPB1 although it is less variable. The phylogeny based on the concatenated dataset revealed that the genus Usnea is subdivided into four highly-supported clades, corresponding to the traditionally circumscribed subgenera Eumitria, Dolichousnea, Neuropogon and Usnea. However, characters that have been used to describe these clades are often homoplasious within the phylogeny and their parallel evolution is suggested. On the other hand, most of the species were reconstructed as monophyletic, indicating that combinations of phenotypic characters are suitable discriminators for delimitating species, but are inadequate to describe generic subdivisions.

Archaeobotanical evidence for a massive loss of epiphyte species richness during industrialization in southern England.

This paper describes a novel archaeological resource--preserved epiphytes on the timber structure of vernacular buildings--used, to our knowledge, for the first time to quantify a loss of biodiversity between pre-industrial and post-industrial landscapes. By matching the confirmed occurrence of epiphyte species for the pre-industrial period, with a statistical likelihood for their absence in the present-day landscape (post-1960), we robustly identified species that have been extirpated across three contrasting regions in southern England. First, the scale of biodiversity loss observed--up to 80 per cent of epiphytes--severely challenges biodiversity targets and environmental baselines that have been developed using reference points in the post-industrial period. Second, we examined sensitivity in the present-day distribution of extirpated species, explained by three environmental drivers: (i) pollution regime, (ii) extent of ancient woodland, and (iii) climatic setting. Results point to an interacting effect between the pollution regime (sulphur dioxide) and changed woodland structure, leading to distinctive regional signatures in biodiversity loss.

DNA barcoding of lichenized fungi demonstrates high identification success in a floristic context.

• Efforts are currently underway to establish a standard DNA barcode region for fungi; we tested the utility of the internal transcribed spacer (ITS) of nuclear ribosomal DNA for DNA barcoding in lichen-forming fungi by sampling diverse species across eight orders. • Amplification of the ITS region (ITS1-5.8S-ITS2) was conducted for 351 samples, encompassing 107, 55 and 28 species, genera and families, respectively, of lichenized fungi. We assessed the ability of the entire ITS vs the ITS2 alone to discriminate between species in a taxonomic dataset (members of the genus Usnea) and a floristic dataset. • In the floristic dataset, 96.3% of sequenced samples could be assigned to the correct species using ITS or ITS2; a barcode gap for ITS is present in 92.1% of species. Although fewer species have a barcode gap in the taxonomic dataset (73.3% with ITS and 68.8% with ITS2), up to 94.1% of samples were assigned to the correct species using BLAST. • While discrimination between the most closely related species will remain challenging, our results demonstrate the potential to identify a high percentage of specimens to the correct species, and the remainder to the correct genus, when using DNA barcoding in a floristic context.

Climate-woodland effects on population genetics for two congeneric lichens with contrasting reproductive strategies.

Genetic variation is expected to be influenced by the interaction between reproductive mode and dispersal traits on the one hand and environmental and habitat setting affecting establishment success on the other. We evaluated how environmental/habitat setting affects population genetic variation (i.e. variation in genetic diversity and structure) when regulated by contrasting dispersal traits. We used fungus-specific microsatellite markers to examine genetic diversity and structure of two closely related epiphytic lichen fungi that differ in their primary reproductive mode: Nephroma laevigatum (sexually reproducing, n = 191, 10 microsatellites) and N. parile (asexually reproducing, n = 182, 12 microsatellites), along a steep climatic gradient in Scotland. Despite their reproductive differences, we found a high proportion of clones in both species and a background pattern of genetic structure related to climatic gradients. We also demonstrated that woodland connectivity, rather than geographic distance, explained genetic diversity in both species. Environmental/habitat setting, modulated by the reproductive mode of the species, affects genetic diversity and structure, but the putative dissimilarity in their reproductive mode is less important than has been previously assumed. We reinforce the importance of protecting highly connected populations, positioned along a gradient capturing the segregation of gene pool differences in response to climatic variation.

A method for the direct detection of airborne dispersal in lichens.

This study sets out a novel method to determine dispersal distances in lichens. Direct measurement of dispersal often remains difficult for lichens and other small inconspicuous species because of the need to track microscopic reproductive propagules, which even if they can be captured, cannot be identified using traditional morphological approaches. A low-cost device (<£200) was developed to trap the reproductive propagules of lichens, capable of sampling around 0.1 m3 of air per minute. In parallel, molecular techniques were developed to enable species-specific detection of propagules caught by the devices, with identification using novel species-specific primers and optimization of a standard DNA extraction and nested PCR protocol. The methods were tested for both their sensitivity and specificity against a suite of lichen epiphytes, differing in their reproductive mechanisms, dispersal structures and rarity. Sensitivity tests showed that the molecular techniques could detect a single asexual propagule (soredium or isidium), or as few as 10 sexual spores. As proof of concept, propagule traps were deployed into a wooded landscape where the target epiphytes were present. Extractions from deployed propagule traps were sequenced, showing that the method was able to detect the presence of the target species in the atmosphere. As far as we are aware, this is the first attempt to use mechanized propagule traps in combination with DNA diagnostics to detect dispersal of lichens. The tests carried out here point the way for future dispersal studies of lichen epiphytes and other passively dispersed microscopic organisms including fungi or bryophytes.

New insights into classification and evolution of the Lecanoromycetes (Pezizomycotina, Ascomycota) from phylogenetic analyses of three ribosomal RNA- and two protein-coding genes.

The Lecanoromycetes includes most of the lichen-forming fungal species (> 13500) and is therefore one of the most diverse class of all Fungi in terms of phenotypic complexity. We report phylogenetic relationships within the Lecanoromycetes resulting from Bayesian and maximum likelihood analyses with complementary posterior probabilities and bootstrap support values based on three combined multilocus datasets using a supermatrix approach. Nine of 10 orders and 43 of 64 families currently recognized in Eriksson's classification of the Lecanoromycetes (Outline of Ascomycota--2006 Myconet 12:1-82) were represented in this sampling. Our analyses strongly support the Acarosporomycetidae and Ostropomycetidae as monophyletic, whereas the delimitation of the largest subclass, the Lecanoromycetidae, remains uncertain. Independent of future delimitation of the Lecanoromycetidae, the Rhizocarpaceae and Umbilicariaceae should be elevated to the ordinal level. This study shows that recent classifications include several nonmonophyletic taxa at different ranks that need to be recircumscribed. Our phylogenies confirm that ascus morphology cannot be applied consistently to shape the classification of lichen-forming fungi. The increasing amount of missing data associated with the progressive addition of taxa resulted in some cases in the expected loss of support, but we also observed an improvement in statistical support for many internodes. We conclude that a phylogenetic synthesis for a chosen taxonomic group should include a comprehensive assessment of phylogenetic confidence based on multiple estimates using different methods and on a progressive taxon sampling with an increasing number of taxa, even if it involves an increasing amount of missing data.

Scaling up discovery of hidden diversity in fungi: impacts of barcoding approaches.

The fungal kingdom is a hyperdiverse group of multicellular eukaryotes with profound impacts on human society and ecosystem function. The challenge of documenting and describing fungal diversity is exacerbated by their typically cryptic nature, their ability to produce seemingly unrelated morphologies from a single individual and their similarity in appearance to distantly related taxa. This multiplicity of hurdles resulted in the early adoption of DNA-based comparisons to study fungal diversity, including linking curated DNA sequence data to expertly identified voucher specimens. DNA-barcoding approaches in fungi were first applied in specimen-based studies for identification and discovery of taxonomic diversity, but are now widely deployed for community characterization based on sequencing of environmental samples. Collectively, fungal barcoding approaches have yielded important advances across biological scales and research applications, from taxonomic, ecological, industrial and health perspectives. A major outstanding issue is the growing problem of 'sequences without names' that are somewhat uncoupled from the traditional framework of fungal classification based on morphology and preserved specimens. This review summarizes some of the most significant impacts of fungal barcoding, its limitations, and progress towards the challenge of effective utilization of the exponentially growing volume of data gathered from high-throughput sequencing technologies.This article is part of the themed issue 'From DNA barcodes to biomes'.

Phylogenetic Diversity of Peltigera Cyanolichens and Their Photobionts in Southern Chile and Antarctica.

The lichen genus Peltigera has been mainly revised in the Northern Hemisphere, with most species being recorded in Europe and North America. This study assessed the phylogenetic diversity of the mycobionts and cyanobionts of Peltigera cyanolichens collected in Southern Chile and Antarctica, areas in which lichens are extremely diverse but poorly studied. The operational taxonomic units (OTUs) of each symbiont were defined by analyzing the genetic diversity of the LSU and SSU rDNA of the mycobionts and cyanobionts, respectively, and a phylogenetic approach was used to relate these OTUs with sequences previously reported for Peltigera and Nostoc. Among the 186 samples collected, 8 Peltigera and 15 Nostoc OTUs were recognized, corresponding to sections Peltigera, Horizontales, and Polydactylon, in the case of the mycobionts, and to the Nostoc clade II, in the case of the cyanobionts. Since some of the OTUs recognized in this study had not previously been described in these areas, our results suggest that the diversity of Peltigera reported to date in the regions studied using traditional morphological surveys has underestimated the true diversity present; therefore, further explorations of these areas are recommended.

Microsatellite loci in two epiphytic lichens with contrasting dispersal modes: Nephroma laevigatum and N. parile (Nephromataceae).

PREMISE OF THE STUDY: Microsatellite markers were characterized for two epiphytic cyanolichens, Nephroma laevigatum and N. parile (Nephromataceae), and will be used to investigate population structure and estimate gene flow among populations of these two closely related species with contrasting dispersal modes. • METHODS AND RESULTS: Twelve and 14 microsatellite loci were characterized for N. laevigatum and N. parile, respectively. Allele number in N. laevigatum ranged from three to 13 per locus, while in N. parile there were from two to six alleles per locus. As expected, the sexually reproducing N. laevigatum had higher genetic diversity than the predominantly asexual N. parile. • CONCLUSIONS: This new set of markers is suitable for studying population structure and providing insights into gene flow among populations and for understanding processes of diversification. Compared between the species, they will facilitate an understanding of the influence of contrasting reproductive strategies on population and community structure.

Geographic variation in algal partners of Cladonia subtenuis (Cladoniaceae) highlights the dynamic nature of a lichen symbiosis.

Multiple interacting factors may explain variation present in symbiotic associations, including fungal specificity, algal availability, mode of transmission and fungal selectivity. To separate these factors, we sampled the lichenized Cladonia subtenuis and associated Asterochloris algae across a broad geographic range. We sampled 87 thalli across 11 sites using sequence data to test for fungal specificity (phylogenetic range of association) and selectivity (frequency of association), fungal reproductive mode, and geographic structure among populations. Permutation tests were used to examine symbiont transmission. Four associated algal clades were found. Analysis of molecular variation (amova) and partial Mantel tests suggested that the frequency of associated algal genotypes was significantly different among sites and habitats, but at random with respect to fungal genotype and clade. The apparent specificity for Clade II algae in the fungal species as a whole did not scale down to further within-species lineage-dependent specificity for particular algae. Fungal genotypes were not structured according to site and appeared to be recombining. We suggest that ecological specialization exists for a specific lichen partnership and a site, and that this selectivity is dynamic and environment-dependent. We present a working model combining algal availability, fungal specificity and selectivity, which maintains variation in symbiotic composition across landscapes.

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

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

Assembling the fungal tree of life: progress, classification, and evolution of subcellular traits.

Based on an overview of progress in molecular systematics of the true fungi (Fungi/Eumycota) since 1990, little overlap was found among single-locus data matrices, which explains why no large-scale multilocus phylogenetic analysis had been undertaken to reveal deep relationships among fungi. As part of the project "Assembling the Fungal Tree of Life" (AFTOL), results of four Bayesian analyses are reported with complementary bootstrap assessment of phylogenetic confidence based on (1) a combined two-locus data set (nucSSU and nucLSU rDNA) with 558 species representing all traditionally recognized fungal phyla (Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota) and the Glomeromycota, (2) a combined three-locus data set (nucSSU, nucLSU, and mitSSU rDNA) with 236 species, (3) a combined three-locus data set (nucSSU, nucLSU rDNA, and RPB2) with 157 species, and (4) a combined four-locus data set (nucSSU, nucLSU, mitSSU rDNA, and RPB2) with 103 species. Because of the lack of complementarity among single-locus data sets, the last three analyses included only members of the Ascomycota and Basidiomycota. The four-locus analysis resolved multiple deep relationships within the Ascomycota and Basidiomycota that were not revealed previously or that received only weak support in previous studies. The impact of this newly discovered phylogenetic structure on supraordinal classifications is discussed. Based on these results and reanalysis of subcellular data, current knowledge of the evolution of septal features of fungal hyphae is synthesized, and a preliminary reassessment of ascomal evolution is presented. Based on previously unpublished data and sequences from GenBank, this study provides a phylogenetic synthesis for the Fungi and a framework for future phylogenetic studies on fungi.