Resolving relationships at the animal-fungal divergence: A molecular phylogenetic study of the protist trichomycetes (Ichthyosporea, Eccrinida).

Trichomycetes is a group of microorganisms that was considered a class of fungi comprising four orders of commensal, gut-dwelling endosymbionts obligately associated with arthropods. Since molecular phylogenies revealed two of those orders (Amoebidiales and Eccrinales="protist trichos") to be closely related to members of the protist class Ichthyosporea (=Mesomycetozoea), trichomycetes have been considered an ecological association of both early-diverging fungi and protists. Understanding of the taxonomy, evolution, and diversity of the protist trichos is lacking largely due to the difficulties inherent in species collection that have contributed to undersampling and understudy. The most recent classification divides the protist trichos between two families, Amoebidiidae and Eccrinidae (suborder Trichomycina, order Eccrinida). However, there is no comprehensive molecular phylogeny available for this group and major questions about the systematics of protist trichos remain unanswered. Therefore, we generated 18S and 28S rDNA sequences for 106 protist tricho samples and combined them with publicly available Eccrinida sequences for phylogenetic analyses. We also sequenced a conserved protein-coding gene (heat-shock 70 protein) to obtain a multigene data set. We conducted ancestral state reconstruction (ASR) and Bayesian tip-association significance test (BaTS) analyses by mapping six morphological and ecological characters onto the resulting phylogenetic trees. Our results demonstrate: (1) several ecological and morphological character states (habitat, host type, host stage at time of infestation, location within host, spore production, and growth form) are significantly correlated with the phylogeny, and (2) two additional protist tricho families should be incorporated into the taxonomy to reflect phylogenetic relationships. Our data suggest that an integrated strategy that combines morphological, ecological, and molecular characters is needed to further resolve and clarify the systematics of the Eccrinida.