A monograph of the freshwater ascomycete family Annulatascaceae: a morphological and molecular study

Freshwater fungi are important agents decomposing submerged dead plant material. Roughly ten percent of the known teleomorphic (sexually reproducing) freshwater ascomycetes have been referred to or included in the family Annulatascaceae. Placement in this family is based on characters that include perithecial ascomata, unitunicate cylindrical asci with relatively large J- (Melzer’s reagent negative) apical rings, and the presence of long tapering septate paraphyses. However, the large refractive apical apparati are the distinctive feature of the family. As sparse molecular data were available prior to the beginning of this study, a broad survey of freshwater temperate and tropical areas was conducted to collect these taxa for morphological examination, digital imagery, and extraction of DNA for phylogenetic inference. Thirty-five of roughly 70 described species in 21 genera of Annulatascaceae were assessed molecularly, and forty-five illustrated from holotypes and/or fresh collections. Twelve new taxa with relatively large non-amyloid apical apparati were discovered, only one of which, Longicollum biappendiculatum, belongs in Annulatascaceae. Material examined in this study was preserved as permanent slides, dried specimens, and/or living cultures. Phylogenetic analyses of 4 genes alone and in combination revealed a polyphyletic Annulatascaceae, with a sensu stricto clade consisting of the type, Annulatascus velatisporus, and nine other species. The other twenty-five taxa assessed molecularly were located in clades along the Sordariomycetes tree, basal to the Ophiostomatales, the nearest outgroup to Annulatascaceae in these analyses. Annulatascaceae sensu stricto lacks likelihood support at its root node in single and combined gene analyses, but is strongly supported by Bayesian posterior probability in analyses of combined genes. This strongly suggests a familial if not an ordinal relationship of these ten taxa. One limitation of this study was a constraint on the number of genes sequenced. Some taxa only had GenBank sequence data and newly sequenced species did not all provide Mcm7 sequences as the primers are highly degenerate and annealing temperatures were variable. Addition of other genes (e.g. RPB1, TEF1α) to future analyses may increase confidence at both basal and distal nodes for this clade. A second limitation of this study was taxonomic coverage using sequence data. Though great effort was given to collecting, not all Annulatascaceae taxa were encountered. Collecting freshwater ascomycetes entails randomly selecting submerged woody debris and incubating it in moist chambers with the hopes of obtaining these microscopic organism. This is in contrast to collecting macroscopic organisms, which are readily recognizable in the field. Much work remains to be done on taxa thus far known only from the Eastern Hemisphere. Molecular data from these taxa would provide additional resolving power for lineages with large apical rings