A target enrichment probe set for resolving the flagellate land plant tree of life.

PREMISE: New sequencing technologies facilitate the generation of large-scale molecular data sets for constructing the plant tree of life. We describe a new probe set for target enrichment sequencing to generate nuclear sequence data to build phylogenetic trees with any flagellate land plants, including hornworts, liverworts, mosses, lycophytes, ferns, and all gymnosperms. METHODS: We leveraged existing transcriptome and genome sequence data to design the GoFlag 451 probes, a set of 56,989 probes for target enrichment sequencing of 451 exons that are found in 248 single-copy or low-copy nuclear genes across flagellate plant lineages. RESULTS: Our results indicate that target enrichment using the GoFlag451 probe set can provide large nuclear data sets that can be used to resolve relationships among both distantly and closely related taxa across the flagellate land plants. We also describe the GoFlag 408 probes, an optimized probe set covering 408 of the 451 exons from the GoFlag 451 probe set that is commercialized by RAPiD Genomics. CONCLUSIONS: A target enrichment approach using the new probe set provides a relatively low-cost solution to obtain large-scale nuclear sequence data for inferring phylogenetic relationships across flagellate land plants.

Diversity and evolutionary origins of fungi associated with seeds of a neotropical pioneer tree: a case study for analysing fungal environmental samples.

Fungi associated with seeds of tropical trees pervasively affect seed survival and germination, and thus are an important, but understudied, component of forest ecology. Here, we examine the diversity and evolutionary origins of fungi isolated from seeds of an important pioneer tree (Cecropia insignis, Cecropiaceae) following burial in soil for five months in a tropical moist forest in Panama. Our approach, which relied on molecular sequence data because most isolates did not sporulate in culture, provides an opportunity to evaluate several methods currently used to analyse environmental samples of fungi. First, intra- and interspecific divergence were estimated for the nu-rITS and 5.8S gene for four genera of Ascomycota that are commonly recovered from seeds. Using these values we estimated species boundaries for 527 isolates, showing that seed-associated fungi are highly diverse, horizontally transmitted, and genotypically congruent with some foliar endophytes from the same site. We then examined methods for inferring the taxonomic placement and phylogenetic relationships of these fungi, evaluating the effects of manual versus automated alignment, model selection, and inference methods, as well as the quality of BLAST-based identification using GenBank. We found that common methods such as neighbor-joining and Bayesian inference differ in their sensitivity to alignment methods; analyses of particular fungal genera differ in their sensitivity to alignments; and numerous and sometimes intricate disparities exist between BLAST-based versus phylogeny-based identification methods. Lastly, we used our most robust methods to infer phylogenetic relationships of seed-associated fungi in four focal genera, and reconstructed ancestral states to generate preliminary hypotheses regarding the evolutionary origins of this guild. Our results illustrate the dynamic evolutionary relationships among endophytic fungi, pathogens, and seed-associated fungi, and the apparent evolutionary distinctiveness of saprotrophs. Our study also elucidates the diversity, taxonomy, and ecology of an important group of plant-associated fungi and highlights some of the advantages and challenges inherent in the use of ITS data for environmental sampling of fungi.

Biogeographic and phylogenetic patterns in diversity of liverwort-associated endophytes.

Liverworts harbor diverse fungi, including endophytes, in their healthy tissues. To address whether patterns of endophyte diversity are correlated with host phylogeny or geography, we designed a broad geographic survey with controlled phylogenetic host sampling. We collected liverworts in North Carolina, Washington, Idaho, British Columbia, Germany, and New Zealand and identified endophytes using culture-based and molecular methods. Of the major lineages of filamentous ascomycetes recovered, 53-88% belonged to the Xylariales. Endophyte accumulation curves did not saturate, and singleton sequences were dominant in each region, suggesting that liverwort endophyte communities are diverse. There was no significant difference in species richness between regional endophyte communities; however, total richness estimators indicated that North Carolina and New Zealand have richer communities than do Germany and the Pacific Northwest. This pattern reflects lower per-host endophyte density and prevalence of a common, shared sequence group in Germany and the Pacific Northwest. Although species richness was relatively low in the Pacific Northwest, the greatest phylogenetic diversity of endophytes was recovered there. Tests for regional and host specificity revealed that endophyte floras of hosts within a geographic area are more similar to one another than to those of closely related hosts. Geographic distance, not host phylogeny, best explains differences among communities.

A novel ascomycetous endophytic association in the rhizoids of the leafy liverwort family, Schistochilaceae (Jungermanniidae, Hepaticopsida).

Liverworts form diverse associations with endophytic fungi similar to mycorrhizas in vascular plants. Whereas the widespread occurrence of glomeromycotes in the basal liverwort lineages is well documented, knowledge of the distribution of ascomycetes and basidiomycetes in derived thalloid and leafy clades is more fragmented. Our discovery that the ramified and septate rhizoids of the Schistochilaceae, the sister group to all other ascomycete-containing liverworts, are packed with fungal hyphae prompted this study on the effects of the fungi on rhizoid morphology, host specificity, the cytology of the association, and a molecular analysis of the endophytes. Two species of Pachyschistochila and their fungi were grown axenically. Axenic rhizoids were unbranched and nonseptate. Reinfected with their own fungus and that from the other species, both Pachyschistochila species produced branched and septate rhizoids identical to those in nature. Woronin bodies and simple septa identified the fungus as an ascomycete referable, according to phylogenetic analyses of ITS sequences, to the Rhizoscyphus (Hymenoscyphus) ericae aggregate, also found in other liverwort-ascomycete associations and in mycorrhizas in the Ericales. Healthy hyphae and host cytoplasm suggest that the Schistochila-fungus association reflects a balanced mutualistic relationship. The recent dating of the divergence of the Jungermanniales from the fungus-free Porellales in the Permian and the origins of the Schistochilaceae in the Triassic indicate that these associations in liverworts predate the appearance of the Ericales.

Endophytic Xylaria (Xylariaceae) among liverworts and angiosperms: phylogenetics, distribution, and symbiosis.

Nuclear ribosomal 18S and internal transcribed spacer (ITS) sequence data were used to identify endophytic fungi cultured from six species of liverworts collected in Jamaica and North Carolina. Comparisons with other published fungal sequences and phylogenetic analyses yielded the following conclusions: (1) the endophytes belong to the ascomycete families Xylariaceae, Hypocreaceae, and Ophiostomataceae, and (2) liverwort endophytes in the genus Xylaria are closely related to each other and to endophytes isolated from angiosperms in China, Puerto Rico, and Europe. Liverwort endophytes are expected to be foragers or endophytic specialists, although little is known about the role of these fungi in symbioses. Features that may indicate a mutualistic role for these endophytes are discussed.