Environmental drivers and cryptic biodiversity hotspots define endophytes in Earth's largest terrestrial biome.

Understanding how symbiotic associations differ across environmental gradients is key to predicting the fate of symbioses as environments change, and it is vital for detecting global reservoirs of symbiont biodiversity in a changing world.1,2,3 However, sampling of symbiotic partners at the full-biome scale is difficult and rare. As Earth's largest terrestrial biome, boreal forests influence carbon dynamics and climate regulation at a planetary scale. Plants and lichens in this biome host the highest known phylogenetic diversity of fungal endophytes, which occur within healthy photosynthetic tissues and can influence hosts' resilience to stress.4,5 We examined how communities of endophytes are structured across the climate gradient of the boreal biome, focusing on the dominant plant and lichen species occurring across the entire south-to-north span of the boreal zone in eastern North America. Although often invoked for understanding the distribution of biodiversity, neither a latitudinal gradient nor mid-domain effect5,6,7 can explain variation in endophyte diversity at this trans-biome scale. Instead, analyses considering shifts in forest characteristics, Picea biomass and age, and nutrients in host tissues from 46° to 58° N reveal strong and distinctive signatures of climate in defining endophyte assemblages in each host lineage. Host breadth of endophytes varies with climate factors, and biodiversity hotspots can be identified at plant-community transitions across the boreal zone at a global scale. Placed against a backdrop of global circumboreal sampling,4 our study reveals the sensitivity of endophytic fungi, their reservoirs of biodiversity, and their important symbiotic associations, to climate.

Ancient Rapid Radiation Explains Most Conflicts Among Gene Trees and Well-Supported Phylogenomic Trees of Nostocalean Cyanobacteria.

Prokaryotic genomes are often considered to be mosaics of genes that do not necessarily share the same evolutionary history due to widespread horizontal gene transfers (HGTs). Consequently, representing evolutionary relationships of prokaryotes as bifurcating trees has long been controversial. However, studies reporting conflicts among gene trees derived from phylogenomic data sets have shown that these conflicts can be the result of artifacts or evolutionary processes other than HGT, such as incomplete lineage sorting, low phylogenetic signal, and systematic errors due to substitution model misspecification. Here, we present the results of an extensive exploration of phylogenetic conflicts in the cyanobacterial order Nostocales, for which previous studies have inferred strongly supported conflicting relationships when using different concatenated phylogenomic data sets. We found that most of these conflicts are concentrated in deep clusters of short internodes of the Nostocales phylogeny, where the great majority of individual genes have low resolving power. We then inferred phylogenetic networks to detect HGT events while also accounting for incomplete lineage sorting. Our results indicate that most conflicts among gene trees are likely due to incomplete lineage sorting linked to an ancient rapid radiation, rather than to HGTs. Moreover, the short internodes of this radiation fit the expectations of the anomaly zone, i.e., a region of the tree parameter space where a species tree is discordant with its most likely gene tree. We demonstrated that concatenation of different sets of loci can recover up to 17 distinct and well-supported relationships within the putative anomaly zone of Nostocales, corresponding to the observed conflicts among well-supported trees based on concatenated data sets from previous studies. Our findings highlight the important role of rapid radiations as a potential cause of strongly conflicting phylogenetic relationships when using phylogenomic data sets of bacteria. We propose that polytomies may be the most appropriate phylogenetic representation of these rapid radiations that are part of anomaly zones, especially when all possible genomic markers have been considered to infer these phylogenies. [Anomaly zone; bacteria; horizontal gene transfer; incomplete lineage sorting; Nostocales; phylogenomic conflict; rapid radiation; Rhizonema.].

Phylogenetic structure of specialization: A new approach that integrates partner availability and phylogenetic diversity to quantify biotic specialization in ecological networks.

Biotic specialization holds information about the assembly, evolution, and stability of biological communities. Partner availabilities can play an important role in enabling species interactions, where uneven partner availabilities can bias estimates of biotic specialization when using phylogenetic diversity indices. It is therefore important to account for partner availability when characterizing biotic specialization using phylogenies. We developed an index, phylogenetic structure of specialization (PSS), that avoids bias from uneven partner availabilities by uncoupling the null models for interaction frequency and phylogenetic distance. We incorporate the deviation between observed and random interaction frequencies as weights into the calculation of partner phylogenetic α-diversity. To calculate the PSS index, we then compare observed partner phylogenetic α-diversity to a null distribution generated by randomizing phylogenetic distances among the same number of partners. PSS quantifies the phylogenetic structure (i.e., clustered, overdispersed, or random) of the partners of a focal species. We show with simulations that the PSS index is not correlated with network properties, which allows comparisons across multiple systems. We also implemented PSS on empirical networks of host-parasite, avian seed-dispersal, lichenized fungi-cyanobacteria, and hummingbird pollination interactions. Across these systems, a large proportion of taxa interact with phylogenetically random partners according to PSS, sometimes to a larger extent than detected with an existing method that does not account for partner availability. We also found that many taxa interact with phylogenetically clustered partners, while taxa with overdispersed partners were rare. We argue that species with phylogenetically overdispersed partners have often been misinterpreted as generalists when they should be considered specialists. Our results highlight the important role of randomness in shaping interaction networks, even in highly intimate symbioses, and provide a much-needed quantitative framework to assess the role that evolutionary history and symbiotic specialization play in shaping patterns of biodiversity. PSS is available as an R package at https://github.com/cjpardodelahoz/pss.

Comparative transcriptomics of fungal endophytes in co-culture with their moss host Dicranum scoparium reveals fungal trophic lability and moss unchanged to slightly increased growth rates.

Mosses harbor fungi whose interactions within their hosts remain largely unexplored. Trophic ranges of fungal endophytes from the moss Dicranum scoparium were hypothesized to encompass saprotrophism. This moss is an ideal host to study fungal trophic lability because of its natural senescence gradient, and because it can be grown axenically. Dicranum scoparium was co-cultured with each of eight endophytic fungi isolated from naturally occurring D. scoparium. Moss growth rates, and gene expression levels (RNA sequencing) of fungi and D. scoparium, were compared between axenic and co-culture treatments. Functional lability of two fungal endophytes was tested by comparing their RNA expression levels when colonizing living vs dead gametophytes. Growth rates of D. scoparium were unchanged, or increased, when in co-culture. One fungal isolate (Hyaloscyphaceae sp.) that promoted moss growth was associated with differential expression of auxin-related genes. When grown with living vs dead gametophytes, Coniochaeta sp. switched from having upregulated carbohydrate transporter activity to upregulated oxidation-based degradation, suggesting an endophytism to saprotrophism transition. However, no such transition was detected for Hyaloscyphaceae sp. Individually, fungal endophytes did not negatively impact growth rates of D. scoparium. Our results support the long-standing hypothesis that some fungal endophytes can switch to saprotrophism.

Community dynamics of soil-borne fungal communities along elevation gradients in neotropical and palaeotropical forests.

Because of their steep gradients in abiotic and biotic factors, mountains offer an ideal setting to illuminate the mechanisms that underlie patterns of species distributions and community assembly. We compared the composition of taxonomically and functionally diverse fungal communities in soils along five elevational gradients in mountains of the Neo- and Palaeotropics (northern Argentina, southern Brazil, Panama, Malaysian Borneo and Papua New Guinea). Both the richness and composition of soil fungal communities reflect environmental factors, particularly temperature and soil pH, with some shared patterns among neotropical and palaeotropical regions. Community dynamics are characterized by replacement of species along elevation gradients, implying a relatively narrow elevation range for most fungi, which appears to be driven by contrasting environmental preferences among both functional and taxonomic groups. For functional groups dependent on symbioses with plants (especially ectomycorrhizal fungi), the distribution of host plants drives richness and community composition, resulting in important differences in elevational patterns between neotropical and palaeotropical montane communities. The pronounced compositional and functional turnover along elevation gradients implies that tropical montane forest fungi will be sensitive to climate change, resulting in shifts in composition and functionality over time.

Ecological generalism drives hyperdiversity of secondary metabolite gene clusters in xylarialean endophytes.

Although secondary metabolites are typically associated with competitive or pathogenic interactions, the high bioactivity of endophytic fungi in the Xylariales, coupled with their abundance and broad host ranges spanning all lineages of land plants and lichens, suggests that enhanced secondary metabolism might facilitate symbioses with phylogenetically diverse hosts. Here, we examined secondary metabolite gene clusters (SMGCs) across 96 Xylariales genomes in two clades (Xylariaceae s.l. and Hypoxylaceae), including 88 newly sequenced genomes of endophytes and closely related saprotrophs and pathogens. We paired genomic data with extensive metadata on endophyte hosts and substrates, enabling us to examine genomic factors related to the breadth of symbiotic interactions and ecological roles. All genomes contain hyperabundant SMGCs; however, Xylariaceae have increased numbers of gene duplications, horizontal gene transfers (HGTs) and SMGCs. Enhanced metabolic diversity of endophytes is associated with a greater diversity of hosts and increased capacity for lignocellulose decomposition. Our results suggest that, as host and substrate generalists, Xylariaceae endophytes experience greater selection to diversify SMGCs compared with more ecologically specialised Hypoxylaceae species. Overall, our results provide new evidence that SMGCs may facilitate symbiosis with phylogenetically diverse hosts, highlighting the importance of microbial symbioses to drive fungal metabolic diversity.

Climate and seasonality drive the richness and composition of tropical fungal endophytes at a landscape scale.

Understanding how species-rich communities persist is a foundational question in ecology. In tropical forests, tree diversity is structured by edaphic factors, climate, and biotic interactions, with seasonality playing an essential role at landscape scales: wetter and less seasonal forests typically harbor higher tree diversity than more seasonal forests. We posited that the abiotic factors shaping tree diversity extend to hyperdiverse symbionts in leaves-fungal endophytes-that influence plant health, function, and resilience to stress. Through surveys in forests across Panama that considered climate, seasonality, and covarying biotic factors, we demonstrate that endophyte richness varies negatively with temperature seasonality. Endophyte community structure and taxonomic composition reflect both temperature seasonality and climate (mean annual temperature and precipitation). Overall our findings highlight the vital role of climate-related factors in shaping the hyperdiversity of these important and little-known symbionts of the trees that, in turn, form the foundations of tropical forest biodiversity.

Exploring syntenic conservation across genomes for phylogenetic studies of organisms subjected to horizontal gene transfers: A case study with Cyanobacteria and cyanolichens.

Understanding the evolutionary history of symbiotic Cyanobacteria at a fine scale is essential to unveil patterns of associations with their hosts and factors driving their spatiotemporal interactions. As for bacteria in general, Horizontal Gene Transfers (HGT) are expected to be rampant throughout their evolution, which justified the use of single-locus phylogenies in macroevolutionary studies of these photoautotrophic bacteria. Genomic approaches have greatly increased the amount of molecular data available, but the selection of orthologous, congruent genes that are more likely to reflect bacterial macroevolutionary histories remains problematic. In this study, we developed a synteny-based approach and searched for Collinear Orthologous Regions (COR), under the assumption that genes that are present in the same order and orientation across a wide monophyletic clade are less likely to have undergone HGT. We searched sixteen reference Nostocales genomes and identified 99 genes, part of 28 COR comprising three to eight genes each. We then developed a bioinformatic pipeline, designed to minimize inter-genome contamination and processed twelve Nostoc-associated lichen metagenomes. This reduced our original dataset to 90 genes representing 25 COR, which were used to infer phylogenetic relationships within Nostocales and among lichenized Cyanobacteria. This dataset was narrowed down further to 71 genes representing 22 COR by selecting only genes part of one (largest) operon per COR. We found a relatively high level of congruence among trees derived from the 90-gene dataset, but congruence was only slightly higher among genes within a COR compared to genes across COR. However, topological congruence was significantly higher among the 71 genes part of one operon per COR. Nostocales phylogenies resulting from concatenation and species tree approaches based on the 90- and 71-gene datasets were highly congruent, but the most highly supported result was obtained when using synteny, collinearity, and operon information (i.e., 71-gene dataset) as gene selection criteria, which outperformed larger datasets with more genes.

Cyanolichen microbiome contains novel viruses that encode genes to promote microbial metabolism.

Lichen thalli are formed through the symbiotic association of a filamentous fungus and photosynthetic green alga and/or cyanobacterium. Recent studies have revealed lichens also host highly diverse communities of secondary fungal and bacterial symbionts, yet few studies have examined the viral component within these complex symbioses. Here, we describe viral biodiversity and functions in cyanolichens collected from across North America and Europe. As current machine-learning viral-detection tools are not trained on complex eukaryotic metagenomes, we first developed efficient methods to remove eukaryotic reads prior to viral detection and a custom pipeline to validate viral contigs predicted with three machine-learning methods. Our resulting high-quality viral data illustrate that every cyanolichen thallus contains diverse viruses that are distinct from viruses in other terrestrial ecosystems. In addition to cyanobacteria, predicted viral hosts include other lichen-associated bacterial lineages and algae, although a large fraction of viral contigs had no host prediction. Functional annotation of cyanolichen viral sequences predicts numerous viral-encoded auxiliary metabolic genes (AMGs) involved in amino acid, nucleotide, and carbohydrate metabolism, including AMGs for secondary metabolism (antibiotics and antimicrobials) and fatty acid biosynthesis. Overall, the diversity of cyanolichen AMGs suggests that viruses may alter microbial interactions within these complex symbiotic assemblages.

Turnover of Lecanoroid Mycobionts and Their Trebouxia Photobionts Along an Elevation Gradient in Bolivia Highlights the Role of Environment in Structuring the Lichen Symbiosis.

Shifts in climate along elevation gradients structure mycobiont-photobiont associations in lichens. We obtained mycobiont (lecanoroid Lecanoraceae) and photobiont (Trebouxia alga) DNA sequences from 89 lichen thalli collected in Bolivia from a ca. 4,700 m elevation gradient encompassing diverse natural communities and environmental conditions. The molecular dataset included six mycobiont loci (ITS, nrLSU, mtSSU, RPB1, RPB2, and MCM7) and two photobiont loci (ITS, rbcL); we designed new primers to amplify Lecanoraceae RPB1 and RPB2 with a nested PCR approach. Mycobionts belonged to Lecanora s.lat., Bryonora, Myriolecis, Protoparmeliopsis, the "Lecanora" polytropa group, and the "L." saligna group. All of these clades except for Lecanora s.lat. occurred only at high elevation. No single species of Lecanoraceae was present along the entire elevation gradient, and individual clades were restricted to a subset of the gradient. Most Lecanoraceae samples represent species which have not previously been sequenced. Trebouxia clade C, which has not previously been recorded in association with species of Lecanoraceae, predominates at low- to mid-elevation sites. Photobionts from Trebouxia clade I occur at the upper extent of mid-elevation forest and at some open, high-elevation sites, while Trebouxia clades A and S dominate open habitats at high elevation. We did not find Trebouxia clade D. Several putative new species were found in Trebouxia clades A, C, and I. These included one putative species in clade A associated with Myriolecis species growing on limestone at high elevation and a novel lineage sister to the rest of clade C associated with Lecanora on bark in low-elevation grassland. Three different kinds of photobiont switching were observed, with certain mycobiont species associating with Trebouxia from different major clades, species within a major clade, or haplotypes within a species. Lecanoraceae mycobionts and Trebouxia photobionts exhibit species turnover along the elevation gradient, but with each partner having a different elevation threshold at which the community shifts completely. A phylogenetically defined sampling of a single diverse family of lichen-forming fungi may be sufficient to document regional patterns of Trebouxia diversity and distribution.

Molybdenum threshold for ecosystem scale alternative vanadium nitrogenase activity in boreal forests.

Biological nitrogen fixation (BNF) by microorganisms associated with cryptogamic covers, such as cyanolichens and bryophytes, is a primary source of fixed nitrogen in pristine, high-latitude ecosystems. On land, low molybdenum (Mo) availability has been shown to limit BNF by the most common form of nitrogenase (Nase), which requires Mo in its active site. Vanadium (V) and iron-only Nases have been suggested as viable alternatives to countering Mo limitation of BNF; however, field data supporting this long-standing hypothesis have been lacking. Here, we elucidate the contribution of vanadium nitrogenase (V-Nase) to BNF by cyanolichens across a 600-km latitudinal transect in eastern boreal forests of North America. Widespread V-Nase activity was detected (∼15-50% of total BNF rates), with most of the activity found in the northern part of the transect. We observed a 3-fold increase of V-Nase contribution during the 20-wk growing season. By including the contribution of V-Nase to BNF, estimates of new N input by cyanolichens increase by up to 30%. We find that variability in V-based BNF is strongly related to Mo availability, and we identify a Mo threshold of ∼250 ng·glichen-1 for the onset of V-based BNF. Our results provide compelling ecosystem-scale evidence for the use of the V-Nase as a surrogate enzyme that contributes to BNF when Mo is limiting. Given widespread findings of terrestrial Mo limitation, including the carbon-rich circumboreal belt where global change is most rapid, additional consideration of V-based BNF is required in experimental and modeling studies of terrestrial biogeochemistry.

Host availability drives distributions of fungal endophytes in the imperilled boreal realm.

Boreal forests represent the world's largest terrestrial biome and provide ecosystem services of global importance. Highly imperilled by climate change, these forests host Earth's greatest phylogenetic diversity of endophytes, a hyperdiverse group of symbionts that are defined by their occurrence within living, symptomless plant and lichen tissues. Endophytes shape the ecological and evolutionary trajectories of plants and are therefore key to the function and resilience of terrestrial ecosystems. A critical step in linking the ecological functions of endophytes with those of their hosts is to understand the distributions of these symbionts at the global scale; however, turnover in host taxa with geography and climate can confound insights into endophyte biogeography. As a result, global drivers of endophyte diversity and distributions are not known. Here, we leverage sampling from phylogenetically diverse boreal plants and lichens across North America and Eurasia to show that host filtering in distinctive environments, rather than turnover with geographical or environmental distance, is the main determinant of the community composition and diversity of endophytes. We reveal the distinctiveness of boreal endophytes relative to soil fungi worldwide and endophytes from diverse temperate biomes, highlighting a high degree of global endemism. Overall, the distributions of endophytes are directly linked to the availability of compatible hosts, highlighting the role of biotic interactions in shaping fungal communities across large spatial scales, and the threat that climate change poses to biological diversity and function in the imperilled boreal realm.

T-BAS Version 2.1: Tree-Based Alignment Selector Toolkit for Evolutionary Placement of DNA Sequences and Viewing Alignments and Specimen Metadata on Curated and Custom Trees.

The Tree-Based Alignment Selector (T-BAS) toolkit combines phylogenetic-based placement of DNA sequences with alignment and specimen metadata visualization tools in an integrative pipeline for analyzing microbial biodiversity. The release of T-BAS version 2.1 makes available reference phylogenies, supports multilocus sequence placements and permits uploading and downloading trees, alignments, and specimen metadata.

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.

Differential gene expression associated with fungal trophic shifts along the senescence gradient of the moss Dicranum scoparium.

Bryophytes harbour microbiomes, including diverse communities of fungi. The molecular mechanisms by which perennial mosses interact with these fungal partners along their senescence gradients are unknown, yet this is an ideal system to study variation in gene expression associated with trophic state transitions. We investigated differentially expressed genes of fungal communities and their host Dicranum scoparium across its naturally occurring senescence gradient using a metatranscriptomic approach. Higher activity of fungal nutrient-related (carbon, nitrogen, phosphorus and sulfur) transporters and Carbohydrate-Active enZyme (CAZy) genes was detected toward the bottom, partially decomposed, layer of the moss. The most prominent variation in the expression levels of fungal nutrient transporters was from inorganic nitrogen-related transporters, whereas the breakdown of organonitrogens was detected as the most enriched gene ontology term for the host D. scoparium, for those transcripts having higher expression in the partially decomposed layer. The abundance of bacterial rRNA transcripts suggested that more living members of Cyanobacteria are associated with the photosynthetic layer of D. scoparium, while members of Rhizobiales are detected throughout the gametophytes. Plant genes for specific fungal-plant communication, including defense responses, were differentially expressed, suggesting that different genetic pathways are involved in plant-microbe crosstalk in photosynthetic tissues compared to partially decomposed tissues.

Contrasting Symbiotic Patterns in Two Closely Related Lineages of Trimembered Lichens of the Genus Peltigera.

Species circumscription is key to the characterization of patterns of specificity in symbiotic systems at a macroevolutionary scale. Here, a worldwide phylogenetic framework was used to assess the biodiversity and symbiotic patterns of association among partners in trimembered lichens from the genus Peltigera, section Chloropeltigera. We sequenced six loci of the main fungal partner and performed species discovery and validation analyses to establish putative species boundaries. Single locus phylogenies were used to establish the identity of both photobionts, Nostoc (cyanobacterium) and Coccomyxa (green alga). Distribution and specificity patterns were compared to the closely related clade, section Peltidea, which includes mainly Peltigera species with trimembered thalli. For section Chloropeltigera, eight fungal species (including five newly delimited putative species) were found in association with nine Nostoc phylogroups and two Coccomyxa species. In contrast, eight fungal species (including three newly delimited putative species) in section Peltidea were found in association with only four Nostoc phylogroups and the same two Coccomyxa species as for section Chloropeltigera. This difference in cyanobiont biodiversity between these two sections can potentially be explained by a significantly higher frequency of sexual reproductive structures in species from section Chloropeltigera compared to section Peltidea. Therefore, horizontal transmission of the cyanobiont might be more prevalent in Chloropeltigera species, while vertical transmission might be more common in Peltidea species. All Peltigera species in section Chloropeltigera are generalists in their association with Nostoc compared to more specialized Peltigera species in section Peltidea. Constrained distributions of Peltigera species that associate strictly with one species of green algae (Coccomyxa subellipsoidea) indicate that the availability of the green alga and the specificity of the interaction might be important factors limiting geographic ranges of trimembered Peltigera, in addition to constraints imposed by their interaction with Nostoc partners and by climatic factors.

Contemporaneous radiations of fungi and plants linked to symbiosis.

Interactions between fungi and plants, including parasitism, mutualism, and saprotrophy, have been invoked as key to their respective macroevolutionary success. Here we evaluate the origins of plant-fungal symbioses and saprotrophy using a time-calibrated phylogenetic framework that reveals linked and drastic shifts in diversification rates of each kingdom. Fungal colonization of land was associated with at least two origins of terrestrial green algae and preceded embryophytes (as evidenced by losses of fungal flagellum, ca. 720 Ma), likely facilitating terrestriality through endomycorrhizal and possibly endophytic symbioses. The largest radiation of fungi (Leotiomyceta), the origin of arbuscular mycorrhizae, and the diversification of extant embryophytes occurred ca. 480 Ma. This was followed by the origin of extant lichens. Saprotrophic mushrooms diversified in the Late Paleozoic as forests of seed plants started to dominate the landscape. The subsequent diversification and explosive radiation of Agaricomycetes, and eventually of ectomycorrhizal mushrooms, were associated with the evolution of Pinaceae in the Mesozoic, and establishment of angiosperm-dominated biomes in the Cretaceous.

Stable isotope analyses reveal previously unknown trophic mode diversity in the Hymenochaetales.

PREMISE OF THE STUDY: The Hymenochaetales are dominated by lignicolous saprotrophic fungi involved in wood decay. However, the group also includes bryophilous and terricolous taxa, but their modes of nutrition are not clear. Here, we investigate patterns of carbon and nitrogen utilization in numerous non-lignicolous Hymenochaetales and provide a phylogenetic context in which these non-canonical ecological guilds arose. METHODS: We combined stable isotope analyses of δ13 C and δ15 N and phylogenetic analyses to explore assignment and evolution of nutritional modes. Clustering procedures and statistical tests were performed to assign trophic modes to Hymenochaetales and test for differences between varying ecologies. Genomes of Hymenochaetales were mined for presence of enzymes involved in plant cell wall and lignin degradation and sucrolytic activity. KEY RESULTS: Three different trophic clusters were detected - biotrophic, saprotrophic, and a second biotrophic cluster including many bryophilous Hymenochaetales and mosses. Non-lignicolous Hymenochaetales are generally biotrophic. All lignicolous Hymenochaetales clustered as saprotrophic and most terricolous Hymenochaetales clustered as ectomycorrhizal. Overall, at least 15 species of Hymenochaetales are inferred as biotrophic. Bryophilous species of Rickenella can degrade plant cell walls and lignin, and cleave sucrose to glucose consistent with a parasitic or endophytic life style. CONCLUSIONS: Most non-lignicolous Hymenochaetales are biotrophic. Stable isotope values of many bryophilous Hymenochaetales cluster as ectomycorrhizal or in a biotrophic cluster indicative of parasitism or an endophytic life style. Overall, trophic mode diversity in the Hymenochaetales is greater than anticipated, and non-lignicolous ecological traits and biotrophic modes of nutrition are evolutionarily derived features.

Bioclimatic factors at an intrabiome scale are more limiting than cyanobiont availability for the lichen-forming genus Peltigera.

PREMISE OF THE STUDY: Factors shaping spatiotemporal patterns of associations in mutualistic systems are poorly understood. We used the lichen-forming fungi Peltigera and their cyanobacterial partners Nostoc to investigate the spatial structure of this symbiosis at an intrabiome scale and to identify potential factors shaping these associations. METHODS: Ninety-three thalli were sampled in Québec, Canada, along a south-north and an east-west transect of ~1300 km each. We identified the two main partners (Peltigera species and Nostoc phylogroups) using molecular markers and modeled the effects of environmental variables and partner occurrence on Peltigera-Nostoc distributions. KEY RESULTS: Peltigera species showed a high degree of specialization toward cyanobionts, whereas two Nostoc phylogroups dominated both transects by associating with several Peltigera species. Peltigera species had narrower ranges than these two main cyanobionts. Distributions of three Peltigera species were highly associated with precipitation and temperature variables, which was not detected for Nostoc phylogroups at this spatial scale. CONCLUSIONS: For these cyanolichens, factors driving patterns of symbiotic associations are scale dependent. Contrary to global-scale findings, generalist Peltigera species were not more widespread within the boreal biome than specialists. Nostoc availability was not the only driver of Peltigera species' geographic ranges; environmental factors also contributed to their intrabiome distributions. Climatic conditions (especially precipitation) limited the range of some Peltigera species more than the range of their cyanobacterial partners at an intrabiome (boreal) scale.

RNA-based analyses reveal fungal communities structured by a senescence gradient in the moss Dicranum scoparium and the presence of putative multi-trophic fungi.

Diverse plant-associated fungi are thought to have symbiotrophic and saprotrophic states because they can be isolated from both dead and living plant tissues. However, such tissues often are separated in time and space, and fungal activity at various stages of plant senescence is rarely assessed directly in fungal community studies. We used fungal ribosomal RNA metatranscriptomics to detect active fungal communities across a natural senescence gradient within wild-collected gametophytes of Dicranum scoparium (Bryophyta) to understand the distribution of active fungal communities in adjacent living, senescing and dead tissues. Ascomycota were active in all tissues across the senescence gradient. By contrast, Basidiomycota were prevalent and active in senescing and dead tissues. Several fungi were detected as active in living and dead tissues, suggesting their capacity for multi-trophy. Differences in community assembly detected by metatranscriptomics were echoed by amplicon sequencing of cDNA and compared to culture-based inferences and observation of fungal fruit bodies in the field. The combination of amplicon sequencing of cDNA and metatranscriptomics is promising for studying symbiotic systems with complex microbial diversity, allowing for the simultaneous detection of their presence and activity.