Stable isotopes, phylogenetics, and experimental data indicate a unique nutritional mode for <i>Rickenella fibula</i>, a bryophyte-associate in the Hymenochaetales

Fungal-bryophyte interactions have been documented in all lineages of bryophytes, however, many of these relationships are poorly understood. The fungus Rickenella fibula is a known associate of various mosses, but results from previous studies have not yielded conclusive evidence of its relationship to mosses or its trophic mode. The Rickenella clade exhibits a wide variety of nutritional modes, including other bryophyte-associated fungi. Here, I combine a broad range of methods, using phylogenetics, stable isotope analyses, PCR assays, in vitro experiments, and genomics to determine the nutritional mode of R. fibula. First, phylogenetic analysis of a supermatrix of 28S rRNA, 18S rRNA, and rpb2 gene regions affirms the evolutionarily derived position of the Rickenella clade in the Hymenochaetales, although support for this topology is weak. Ancestral state reconstruction analysis supports the hypothesis that ancestral Hymenochaetales were saprotrophic followed by two switches to bryophyte associations. A bryophyte association, however, is not evolutionarily stable as switches to an ectomycorrhizal state and reversals to a free-living state are observed. Stable isotope analysis of δ¹³C [(delta^13)C] and δ¹⁵N [(delta^15)N], indicates that R. fibula has a unique stable isotope signature suggesting a novel biotrophic mode. Assessment of the presence/absence of genes involved in degradation of lignin and cellulose suggests that R. fibula has largely retained these suites of genes involved in plant tissue degradation. However, unlike saprotrophic Hymenochaetales, the R. fibula genome indicates the presence of sucrolytic activity by invertase, consistent with a biotrophic or endophytic habit. PCR assays of the ITS region of R. fibula in moss host tissues support the presence of the fungus throughout the gametophyte tissues, including rhizoids and living and senescent portions of the moss. Lastly, in most cases, inoculation of moss gametophytes with R. fibula does not significantly impact productivity of the moss. Taken together, these results support a unique biotrophic mode in R. fibula with the fungus most likely maintaining a commensal endophytic relationship with its moss host.