Jana M. U’Ren

Community analysis reveals close affinities between endophytic and endolichenic fungi in Mosses and Lichens

Endolichenic fungi live in close association with algal photobionts inside asymptomatic lichen thalli and resemble fungal endophytes of plants in terms of taxonomy, diversity, transmission mode, and evolutionary history. This similarity has led to uncertainty regarding the distinctiveness of endolichenic fungi compared with endophytes. Here, we evaluate whether these fungi represent distinct ecological guilds or a single guild of flexible symbiotrophs capable of colonizing plants or lichens indiscriminately. Culturable fungi were sampled exhaustively from replicate sets of phylogenetically diverse plants and lichens in three microsites in a montane forest in southeastern Arizona (USA). Intensive sampling combined with a small spatial scale permitted us to decouple spatial heterogeneity from host association and to sample communities from living leaves, dead leaves, and lichen thalli to statistical completion. Characterization using data from the nuclear ribosomal internal transcribed spacer and partial large subunit (ITS-LSU rDNA) provided a first estimation of host and substrate use for 960 isolates representing five classes and approximately 16 orders, 32 families, and 65 genera of Pezizomycotina. We found that fungal communities differ at a broad taxonomic level as a function of the phylogenetic placement of their plant or lichen hosts. Endolichenic fungal assemblages differed as a function of lichen taxonomy, rather than substrate, growth form, or photobiont. In plants, fungal communities were structured more by plant lineage than by the living vs. senescent status of the leaf. We found no evidence that endolichenic fungi are saprotrophic fungi that have been “entrapped” by lichen thalli. Instead, our study reveals the distinctiveness of endolichenic communities relative to those in living and dead plant tissues, with one notable exception: we identify, for the first time, an ecologically flexible group of symbionts that occurs both as endolichenic fungi and as endophytes of mosses.

Geopyxins A-E, ent-kaurane diterpenoids from endolichenic fungal strains Geopyxis aff. majalis and Geopyxis sp. AZ0066: structure-activity relationships of geopyxins and their analogues.

Four new ent-kaurane diterpenoids, geopyxins A-D (1-4), were isolated from Geopyxis aff. majalis, a fungus occurring in the lichen Pseudevernia intensa, whereas Geopyxis sp. AZ0066 inhabiting the same host afforded two new ent-kaurane diterpenoids, geopyxins E and F (5 and 6), together with 1 and 3. The structures of 1-6 were established on the basis of their spectroscopic data, while the absolute configurations were assigned using modified Moshers ester method. Methylation of 1-3, 5, and 6 gave their corresponding methyl esters 7-11. On acetylation, 1 and 7 yielded their corresponding monoacetates 12 and 14 and diacetates 13 and 15. All compounds were evaluated for their cytotoxic and heat-shock induction activities. Compounds 2, 7-10, 12, 14, and 15 showed cytotoxic activity in the low micromolar range against all five cancer cell lines tested, but only compounds 7-9, 14, and 15 were found to activate the heat-shock response at similar concentrations. From a preliminary structure-activity perspective, the electrophilic α,β-unsaturated ketone carbonyl motif present in all compounds except 6 and 11 was found to be necessary but not sufficient for both cytotoxicity and heat-shock activation.

Genetic variation in horizontally transmitted fungal endophytes of pine needles reveals population structure in cryptic species

UNLABELLED • PREMISE OF THE STUDY Fungal endophytes comprise one of the most ubiquitous groups of plant symbionts, inhabiting healthy leaves and stems of all major lineages of plants. Together, they comprise immense species richness, but little is known about the fundamental processes that generate their diversity. Exploration of their population structure is needed, especially with regard to geographic distributions and host affiliations.• METHODS We take a multilocus approach to examine genetic variation within and among populations of Lophodermium australe, an endophytic fungus commonly associated with healthy foliage of pines in the southeastern United States. Sampling focused on two pine species ranging from montane to coastal regions of North Carolina and Virginia.• KEY RESULTS Our sampling revealed two genetically distinct groups within Lophodermium australe. Our analysis detected less than one migrant per generation between them, indicating that they are distinct species. The species comprising the majority of isolates (major species) demonstrated a panmictic structure, whereas the species comprising the minority of isolates (cryptic species) demonstrated isolation by distance. Distantly related pine species hosted the same Lophodermium species, and host species did not influence genetic structure.• CONCLUSIONS We present the first evidence for isolation by distance in a foliar fungal endophyte that is horizontally transmitted. Cryptic species may be common among microbial symbionts and are important to delimit when exploring their genetic structure and microevolutionary processes. The hyperdiversity of endophytic fungi may be explained in part by cryptic species without apparent ecological and morphological differences as well as genetic diversification within rare fungal species across large spatial scales.

Interaction type influences ecological network structure more than local abiotic conditions: evidence from endophytic and endolichenic fungi at a continental scale

Understanding the factors that shape community assembly remains one of the most enduring and important questions in modern ecology. Network theory can reveal rules of community assembly within and across study systems and suggest novel hypotheses regarding the formation and stability of communities. However, such studies generally face the challenge of disentangling the relative influence of factors such as interaction type and environmental conditions on shaping communities and associated networks. Endophytic and endolichenic symbioses, characterized by microbial species that occur within healthy plants and lichen thalli, represent some of the most ubiquitous interactions in nature. Fungi that engage in these symbioses are hyperdiverse, often horizontally transmitted, and functionally beneficial in many cases, and they represent the diversification of multiple phylogenetic groups. We evaluated six measures of ecological network structure for >4100 isolates of endophytic and endolichenic fungi collected systematically from five sites across North America. Our comparison of these co-occurring interactions in biomes ranging from tundra to subtropical forest showed that the type of interactions (i.e., endophytic vs. endolichenic) had a much more pronounced influence on network structure than did environmental conditions. In particular, endophytic networks were less nested, less connected, and more modular than endolichenic networks in all sites. The consistency of the network structure within each interaction type, independent of site, is encouraging for current efforts devoted to gathering metadata on ecological network structure at a global scale. We discuss several mechanisms potentially responsible for such patterns and draw attention to knowledge gaps in our understanding of networks for diverse interaction types.

Pervasive Effects of Wildfire on Foliar Endophyte Communities in Montane Forest Trees

Plants in all terrestrial ecosystems form symbioses with endophytic fungi that inhabit their healthy tissues. How these foliar endophytes respond to wildfires has not been studied previously, but is important given the increasing frequency and intensity of severe wildfires in many ecosystems, and because endophytes can influence plant growth and responses to stress. The goal of this study was to examine effects of severe wildfires on endophyte communities in forest trees, with a focus on traditionally fire-dominated, montane ecosystems in the southwestern USA. We evaluated the abundance, diversity, and composition of endophytes in foliage of Juniperus deppeana (Cupressaceae) and Quercus spp. (Fagaceae) collected contemporaneously from areas affected by recent wildfire and paired areas not affected by recent fire. Study sites spanned four mountain ranges in central and southern Arizona. Our results revealed significant effects of fires on endophyte communities, including decreases in isolation frequency, increases in diversity, and shifts in community structure and taxonomic composition among endophytes of trees affected by recent fires. Responses to fire were similar in endophytes of each host in these fire-dominated ecosystems and reflect regional fire-return intervals, with endophytes after fire representing subsets of the regional mycoflora. Together, these findings contribute to an emerging perspective on the responses of diverse communities to severe fire, and highlight the importance of considering fire history when estimating endophyte diversity and community structure for focal biomes.

Sesquiterpenes and other constituents of Xylaria sp. NC1214, a fungal endophyte of the moss Hypnum sp.

Oxygenated guaiane-type sesquiterpenes, xylaguaianols A-D (1-4), an iso-cadinane-type sesquiterpene isocadinanol A (5), and an α-pyrone 9-hydroxyxylarone (6), together with five known sesquiterpenes (7-11), and four known cytochalasins (12-15) were isolated from a culture broth of Xylaria sp. NC1214, a fungal endophyte of the moss Hypnum sp. The structures of all compounds were elucidated by the analysis of their spectroscopic data and relative configurations of 1-5 were determined with the help of NMR NOESY experiments. Cytochalasins C (12), D (13), and Q (14) were investigated for their cytotoxic activity against five tumor cell lines. Cytochalasin D showed significant cytotoxicity against all five cell lines, with IC50s ranging from 0.22 to 1.44 μM, whereas cytochalasins C and Q exhibited moderate, but selective cytotoxicity.

Diversity, taxonomic composition, and functional aspects of fungal communities in living, senesced, and fallen leaves at five sites across North America

Background Fungal endophytes inhabit symptomless, living tissues of all major plant lineages to form one of earth’s most prevalent groups of symbionts. Many reproduce from senesced and/or decomposing leaves and can produce extracellular leaf-degrading enzymes, blurring the line between symbiotrophy and saprotrophy. To better understand the endophyte–saprotroph continuum we compared fungal communities and functional traits of focal strains isolated from living leaves to those isolated from leaves after senescence and decomposition, with a focus on foliage of woody plants in five biogeographic provinces ranging from tundra to subtropical scrub forest. Methods We cultured fungi from the interior of surface-sterilized leaves that were living at the time of sampling (i.e., endophytes), leaves that were dead and were retained in plant canopies (dead leaf fungi, DLF), and fallen leaves (leaf litter fungi, LLF) from 3–4 species of woody plants in each of five sites in North America. Our sampling encompassed 18 plant species representing two families of Pinophyta and five families of Angiospermae. Diversity and composition of fungal communities within and among leaf life stages, hosts, and sites were compared using ITS-partial LSU rDNA data. We evaluated substrate use and enzyme activity by a subset of fungi isolated only from living tissues vs. fungi isolated only from non-living leaves. Results Across the diverse biomes and plant taxa surveyed here, culturable fungi from living leaves were isolated less frequently and were less diverse than those isolated from non-living leaves. Fungal communities in living leaves also differed detectably in composition from communities in dead leaves and leaf litter within focal sites and host taxa, regardless of differential weighting of rare and abundant fungi. All focal isolates grew on cellulose, lignin, and pectin as sole carbon sources, but none displayed ligninolytic or pectinolytic activity in vitro. Cellulolytic activity differed among fungal classes. Within Dothideomycetes, activity differed significantly between fungi from living vs. non-living leaves, but such differences were not observed in Sordariomycetes. Discussion Although some fungi with endophytic life stages clearly persist for periods of time in leaves after senescence and incorporation into leaf litter, our sampling across diverse biomes and host lineages detected consistent differences between fungal assemblages in living vs. non-living leaves, reflecting incursion by fungi from the leaf exterior after leaf death and as leaves begin to decompose. However, fungi found only in living leaves do not differ consistently in cellulolytic activity from those fungi detected thus far only in dead leaves. Future analyses should consider Basidiomycota in addition to the Ascomycota fungi evaluated here, and should explore more dimensions of functional traits and persistence to further define the endophytism-to-saprotrophy continuum.

Delitschiapyrone A, a pyrone-naphthalenone adduct bearing a new pentacyclic ring system from the leaf-associated fungus Delitschia sp. FL1581.

Delitschiapyrone A (1), an α-pyrone–naphthalenone adduct with an unprecedented pentacyclic ring system, was isolated from a solid culture of the leaf-associated fungus Delitschia sp. FL1581. The structure of 1 was elucidated by spectroscopic analysis and X-ray crystallography, and its absolute configuration was defined by experimental and calculated ECD. Biosynthetically, the unique 6/6/5/7/6 pentacyclic core of 1 may be formed by an intermolecular Diels–Alder-type addition of the precursors derived from (1′R)-2′,3′-dihydropyrenocine C (2) and 6-ethyl-2,7-dimethoxyjuglone (3) found to co-occur with 1 in this fungus.

Cytotoxic and Noncytotoxic Metabolites from Teratosphaeria sp. FL2137, a Fungus Associated with Pinus clausa

A new naphthoquinone, teratosphaerone A (1), four new naphthalenones, namely, teratosphaerone B (2), structurally related to 1, iso-balticol B (3), iso-balticol B-4,9-acetonide (4), and (+)-balticol C (5), a new furanonaphthalenone, (3a S,9 R,9a S)-1(9a),3(3a),9-hexahydromonosporascone (6), and the known metabolite monosporascone (7) were isolated from Teratosphaeria sp. FL2137, a fungal strain inhabiting the internal tissue of recently dead but undecomposed foliage of Pinus clausa. The structures of 1-6 were elucidated on the basis of their spectroscopic data including 2D NMR, and absolute configurations of 2, 3, and 6 were determined by the modified Moshers ester method. When evaluated in a panel of five tumor cell lines, metabolites 1 and 7 isolated from a cytotoxic fraction of the extract exhibited moderate selectivity for metastatic breast adenocarcinoma cell line MDA-MB-231. Of these, 1 showed cytotoxicity to this cell line with an IC50 of 1.2 ± 0.1 μM.