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A survey of the diversity and community composition of culturable endophytic fungi associated with photosynthetic stems of the gnetophyte Ephedra trifurca ( Ephedraceae ) in southeastern Arizona, USA, yielded numerous isolates that are consistent morphologically with members of the genus Preussia ( Sporormiaceae , Pleosporales , Dothideomycetes , Ascomycota ). Preussia (including species until recently classified within Sporormiella ) include dung-, soil-, and plant-inhabiting strains with considerable diversity worldwide. We used morphological evidence and phylogenetic analyses (nuclear ribosomal internal transcribed spacers and 5.8s gene, the adjacent D1-D2 region of the nuclear ribosomal large subunit, and for a subset of strains, the translation elongation factor 1-alpha, EF1-a) to identify distinctive members of the Preussia intermedia clade occurring as endophytes in E. trifurca . These include P. arizonica sp. nov., which also occurs as an endophyte in other plants of the region, and P. elegans sp. nov., which has been found only in E. trifurca to date. We also propose Preussia mariae sp. nov., allied phylogenetically with Preussia lignicola but distinguishable on the basis of morphology and EF1-a data. Our analyses illustrate the potential for several currently recognized species of Preussia to represent species complexes that should be resolved by analyses of additional loci and by further sampling of endophytes, which may provide an ecological connection among strains occurring within living plant tissues and as coprophilous or soil-inhabiting fungi. More broadly, our work expands the known geographic scope, host use, and diversity of Preussia , especially in arid lands. In conjunction with previous work, our study also provides the basis for hypotheses regarding secondary metabolites of the newly described species.