A. Elizabeth Arnold

Reconstructing the early evolution of Fungi using a six-gene phylogeny

The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.

Fungal endophytes limit pathogen damage in a tropical tree

Every plant species examined to date harbors endophytic fungi within its asymptomatic aerial tissues, such that endophytes represent a ubiquitous, yet cryptic, component of terrestrial plant communities. Fungal endophytes associated with leaves of woody angiosperms are especially diverse; yet, fundamental aspects of their interactions with hosts are unknown. In contrast to the relatively species-poor endophytes that are vertically transmitted and act as defensive mutualists of some temperate grasses, the diverse, horizontally transmitted endophytes of woody angiosperms are thought to contribute little to host defense. Here, we document high diversity, spatial structure, and host affinity among foliar endophytes associated with a tropical tree (Theobroma cacao, Malvaceae) across lowland Panama. We then show that inoculation of endophyte-free leaves with endophytes isolated frequently from naturally infected, asymptomatic hosts significantly decreases both leaf necrosis and leaf mortality when T. cacao seedlings are challenged with a major pathogen (Phytophthora sp.). In contrast to reports of fungal inoculation inducing systemic defense, we found that protection was primarily localized to endophyte-infected tissues. Further, endophyte-mediated protection was greater in mature leaves, which bear less intrinsic defense against fungal pathogens than do young leaves. In vitro studies suggest that host affinity is mediated by leaf chemistry, and that protection may be mediated by direct interactions of endophytes with foliar pathogens. Together, these data demonstrate the capacity of diverse, horizontally transmitted endophytes of woody angiosperms to play an important but previously unappreciated role in host defense.

Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots?

Fungal endophytes are found in asymptomatic photosynthetic tissues of all major lineages of land plants. The ubiquity of these cryptic symbionts is clear, but the scale of their diversity, host range, and geographic distributions are unknown. To explore the putative hyperdiversity of tropical leaf endophytes, we compared endophyte communities along a broad latitudinal gradient from the Canadian arctic to the lowland tropical forest of central Panama. Here, we use molecular sequence data from 1403 endophyte strains to show that endophytes increase in incidence, diversity, and host breadth from arctic to tropical sites. Endophyte communities from higher latitudes are characterized by relatively few species from many different classes of Ascomycota, whereas tropical endophyte assemblages are dominated by a small number of classes with a very large number of endophytic species. The most easily cultivated endophytes from tropical plants have wide host ranges, but communities are dominated by a large number of rare species whose host range is unclear. Even when only the most easily cultured species are considered, leaves of tropical trees represent hotspots of fungal species diversity, containing numerous species not yet recovered from other biomes. The challenge remains to recover and identify those elusive and rarely cultured taxa with narrower host ranges, and to elucidate the ecological roles of these little-known symbionts in tropical forests.

Filling key gaps in population and community ecology

We propose research to fill key gaps in the areas of population and community ecology, based on a National Science Foundation workshop identifying funding priorities for the next 5-10 years. Our vision for the near future of ecology focuses on three core areas: predicting the strength and context-dependence of species interactions across multiple scales; identifying the importance of feedbacks from individual interactions to ecosystem dynam- ics; and linking pattern with process to understand species coexistence. We outline a combination of theory devel- opment and explicit, realistic tests of hypotheses needed to advance population and community ecology.

Fungal endophytes in dicotyledonous neotropical trees: patterns of abundance and diversity

Fungal endophytes have been found in every plant species examined to date and appear to be important, but largely unquantified, components of fungal biodiversity. Endophytes are especially little known in tropical forest trees, where their abundance and diversity are thought to be greatest. Here, we explore the occurrence of endophytes in a broad diversity of woody, angiospermous taxa in a lowland, moist tropical forest in central Panama. We use similarity indices to assess host preference and spatial heterogeneity of endophytes associated with two co-occurring, but distantly related, understorey tree species in two sites of that forest, and assess the utility of indices based on frequencies of morphospecies occurrence (Morisita-Horn index) and on presenceabsence data (Sorensens index). We suggest that our understanding of fungal diversity will be enhanced by exploring ecological patterns underlying endophyte occurrence in host species, and discuss methods for assessing the proportion of fungal biodiversity represented by tropical endophytes.

Canopy cover and leaf age affect colonization by tropical fungal endophytes: Ecological pattern and process in Theobroma cacao (Malvaceae)

Fungal endophytes inhabit healthy tissues of all terrestrial plant taxa studied to date and are diverse and abundant in leaves of tropical woody angiosperms. Studies have demonstrated that plant location and leaf age influence density of endophyte infection in leaves of tropical forest trees. However, ecological factors underlying these observations have not been explored in detail. Here, we establish that foliar endophytes of a tropical tree (Theobroma cacao, Malvaceae) are transmitted horizontally and that endophyte-free seedlings can be produced for experimental manipulation by protecting aerial tissues from surface wetting. At Barro Colorado Island, Panama, we used transects of endophyte-free seedlings to determine the importance of several factors (canopy cover, abundance of aerial and epiphytic propagules, leaf age, leaf chemistry, leaf toughness and duration of exposure to viable air spora) in shaping colonization by endophytic fungi. Endophytes colonized leaves of T. cacao more rapidly beneath the forest canopy than in cleared sites, reflecting local abundance of aerial and epiphytic propagules. The duration of exposure, rather than absolute leaf age, influenced endophyte infection, whereas leaf toughness and chemistry had no observed effect. Endophytes isolated from mature T. cacao grew more rapidly on media containing leaf extracts of T. cacao than on media containing extracts from other co-occurring tree species, suggesting that interspecific differences in leaf chemistry influence endophyte assemblages. Together, these data allow us to identify factors underlying patterns of endophyte colonization within healthy leaves of this tropical tree.

Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees

Understanding how fungal endophyte communities differ in abundance, diversity, taxonomic composition, and host affinity over the geographic ranges of their hosts is key to understanding the ecology and evolutionary context of endophyte-plant associations. We examined endophytes associated with healthy photosynthetic tissues of three closely related tree species in the Cupressaceae (Coniferales): two native species within their natural ranges [Juniperus virginiana in a mesic semideciduous forest, North Carolina (NC); Cupressus arizonica, under xeric conditions, Arizona (AZ)], and a non-native species planted in each site (Platycladus orientalis). Endophytes were recovered from 229 of 960 tissue segments and represented at least 35 species of Ascomycota. Isolation frequency was more than threefold greater for plants in NC than in AZ, and was 2.5 (AZ) to four (NC) times greater for non-native Platycladus than for Cupressus or Juniperus. Analyses of ITS rDNA for 109 representative isolates showed that endophyte diversity was more than twofold greater in NC than in AZ, and that endophytes recovered in AZ were more likely to be host-generalists relative to those in NC. Different endophyte genera dominated the assemblages of each host species/locality combination, but in both localities, Platycladus harboured less diverse and more cosmopolitan endophytes than did either native host. Parsimony and Bayesian analyses for four classes of Ascomycota (Dothideomycetes, Sordariomycetes, Pezizomycetes, Eurotiomycetes) based on LSU rDNA data (ca 1.2 kb) showed that well-supported clades of endophytes frequently contained representatives of a single locality or host species, underscoring the importance of both geography and host identity in shaping a given plants endophyte community. Together, our data show that not only do the abundance, diversity, and taxonomic composition of endophyte communities differ as a function of host identity and locality, but that host affinities of those communities are variable as well.

Host and geographic structure of endophytic and endolichenic fungi at a continental scale.

PREMISE OF THE STUDY Endophytic and endolichenic fungi occur in healthy tissues of plants and lichens, respectively, playing potentially important roles in the ecology and evolution of their hosts. However, previous sampling has not comprehensively evaluated the biotic, biogeographic, and abiotic factors that structure their communities. METHODS Using molecular data we examined the diversity, composition, and distributions of 4154 endophytic and endolichenic Ascomycota cultured from replicate surveys of ca. 20 plant and lichen species in each of five North American sites (Madrean coniferous forest, Arizona; montane semideciduous forest, North Carolina; scrub forest, Florida; Beringian tundra and forest, western Alaska; subalpine tundra, eastern central Alaska). KEY RESULTS Endolichenic fungi were more abundant and diverse per host species than endophytes, but communities of endophytes were more diverse overall, reflecting high diversity in mosses and lycophytes. Endophytes of vascular plants were largely distinct from fungal communities that inhabit mosses and lichens. Fungi from closely related hosts from different regions were similar in higher taxonomy, but differed at shallow taxonomic levels. These differences reflected climate factors more strongly than geographic distance alone. CONCLUSIONS Our study provides a first evaluation of endophytic and endolichenic fungal associations with their hosts at a continental scale. Both plants and lichens harbor abundant and diverse fungal communities whose incidence, diversity, and composition reflect the interplay of climatic patterns, geographic separation, host type, and host lineage. Although culture-free methods will inform future work, our study sets the stage for empirical assessments of ecological specificity, metabolic capability, and comparative genomics.

Diverse Bacteria Inhabit Living Hyphae of Phylogenetically Diverse Fungal Endophytes

ABSTRACT Both the establishment and outcomes of plant-fungus symbioses can be influenced by abiotic factors, the interplay of fungal and plant genotypes, and additional microbes associated with fungal mycelia. Recently bacterial endosymbionts were documented in soilborne Glomeromycota and Mucoromycotina and in at least one species each of mycorrhizal Basidiomycota and Ascomycota. Here we show for the first time that phylogenetically diverse endohyphal bacteria occur in living hyphae of diverse foliar endophytes, including representatives of four classes of Ascomycota. We examined 414 isolates of endophytic fungi, isolated from photosynthetic tissues of six species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteria using microscopy and molecular techniques. Viable bacteria were observed within living hyphae of endophytic Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes from all tree species and biotic regions surveyed. A focus on 29 fungus/bacterium associations revealed that bacterial and fungal phylogenies were incongruent with each other and with taxonomic relationships of host plants. Overall, eight families and 15 distinct genotypes of endohyphal bacteria were recovered; most were members of the Proteobacteria, but a small number of Bacillaceae also were found, including one that appears to occur as an endophyte of plants. Frequent loss of bacteria following subculturing suggests a facultative association. Our study recovered distinct lineages of endohyphal bacteria relative to previous studies, is the first to document their occurrence in foliar endophytes representing four of the most species-rich classes of fungi, and highlights for the first time their diversity and phylogenetic relationships with regard both to the endophytes they inhabit and the plants in which these endophyte-bacterium symbiota occur.

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.