Evolution and Diversity of the basidiolichen clade Dictyonema (Agaricales: Hygrophoraceae)

Abstract

Lichenized basidiomycetes represent a small group within Basidiomycota, with Dictyonema s.l. the clade with the highest number of species. This clade occurs world-wide, but has its highest diversity in the tropics and especially in tropical montane regions, such as the endangered paramos. The group has different morphologies, with crustose, filamentous, squamulose and foliose thalli, and the basidiocarps may be cyphelloid, corticioid or steroid. The photobionts are cyanobacteria of the genus Rhizonema, which at present are thought to be entirely lichenized. Recent field observations and laboratory investigations indicate that more than 300 species belong to this clade of lichenized fungi alone, but genus and species concepts are still to be established. Prior to these investigations, taxonomic concepts for the clade were based largely on anatomy, with morphological differences being treated as intraspecific variation, giving an accepted number of only five species. In an attempt to elucidate the evolution of the group and the diversity of species in Dictyonema s.l., morphological, anatomical, ecological and molecular analyses were performed using more than 800 samples from 19 countries. The sequences were generated by standard Sanger and/or next-generation sequencing (NGS) procedures, the later using pyrosequencing with the 454 GS Junior, and then phylogenetic reconstructions were done using the markers ITS, nuLSU and RPB2 for the fungal partner and 16S for the photosynthetic partner. Morphological characters were intensively reanalyzed using features of the thallus, the basidiocarps and numerous additional characteristics (i.e., hairs, sutures, and soredia). Anatomical characters were also collected, including thickness of the thallus and its layers, shape and size of cyanobacteria and hyphae, among others. Results of these molecular, morphological, anatomical and ecological analyses indicated that the group is much more diverse than previously thought, and that anatomical and morphological differences, long thought to indicate forms within the same species, are instead indicative of a far larger number of distinct species. The multilocus and individual marker analyses indicated that the group is monophyletic with five genus-level groups: Cyphellostereum, sister genus to the rest of species, Dictyonema s.str. forming a paraphyletic transitional group, and three additional monophyletic groups representing three genera, Acantholichen, Cora and Corella. So far, Brazil, Colombia and Costa Rica are the only countries in which all genera have been found, although many other countries have been poorly sampled. In the Galapagos Islands, focused sampling during this study augmented collections made previously by local lichenologists and visiting collectors since the 1970’s. Based on previous collections in the Islands, at least eight species of basidiolichens had been reported to occur there, but most were identified using existing names since no molecular data were available. The present study yielded 90 sequenced specimens representing ten species of the Dictyonema clade, most of these from Santa Cruz, Isabela, and Floreana. Molecular, morphological and anatomical investigations indicate that these species are new and mostly endemic to the Islands. Results also demonstrated that the genus Acantholichen, originally thought to be monotypic, is like other genera in the Dictyonema clade, highly endemic and made up of more than a single species. Here I present five new species to this genus, based on differences in squamules and acanthohyphidia (spiny apical cells of hyphae present in lower and upper surfaces), both unique characters to separate this genus from others within the Dictyonema clade. The project also investigated the diversity of the cyanobacterial Rhizonema photobionts of Dictyonema s.l., generating 16S sequences from 560 Dictyonema specimens and 21 additional unrelated cyanolichens in the Ascomycota. Analyses of these sequences indicated that, unlike the diversity of the fungal partner in these lichens, very few Rhizonema lineages are present, suggesting that these photobionts are largely shared among mycobionts (from the same or different species, including some ascolichens). These ecological interactions are similar to those of human domestication of crop species and may explain the unusual distributions of fungal and cyanobacterial symbionts throughout the Neotropics. In conclusion, the results of this study support the idea that Dictyonema s.l, previously considered to represent only few species in a single genus, actually includes a remarkably high diversity of species in several, distinct genera, differing in morphology, anatomy, substrate ecology and geographical distribution.