Phylogenetic classification of Laboulbeniomycetes in the context of other entomopathogenic fungi

Laboulbeniomycetes is a class of perithecial ascomycetous fungi consisting of three orders with strikingly different biology and morphology. Two orders – Laboulbeniales and Herpomycetales – are obligate biotrophic ectoparasites of arthropods. They share a number of characters that make them unique among ascomycetes: determinate growth, lack of hyphae and absence of asexual stage. Over 2000 species of Laboulbeniales are known to infect various groups of Arthropoda from all continents except Antarctica making them the most diverse group of entomopathogenic fungi. The recently described order Herpomycetales is monotypic, compromising only the genus Herpomyces with 25 species, all parasitizing on cockroaches Blattodea. The major differences between those two groups is development of fruiting body, especially asci. Significantly less is known about the free-living and mycelial order Pyxidiophorales. Unlike other Laboulbeniomycetes they have ability to reproduce asexually, usually having multiple anamorphs. Because of that, it is difficult to certainly know how many species in this group are valid, as multiple names were used for different morphs. Most probably less than 25 species of Pyxidiophorales are described. They are thought to be mycoparasites based on their uncultivability and a few reports of parasitic Pyxidiophora species. Ephemeral fruiting bodies are rarely recorded in the field and dispersal forms called thaxteriolae are observed on mites in some microhabitats. Intricate arthropod-dependent dispersal strategies of several species have been well documented. Being a difficult to study and mostly unculturable group, Laboulbeniomycetes have been poorly represented or omitted in major molecular phylogenetic studies resolving evolutionary relationships in fungi. One of the main evolutionary questions in Laboulbeniomycetes is the origin of the unique ectoparasitic lifestyle of both Laboulbeniales and Herpomycetales. Since the acknowledgement of Pyxidiophora as a sister group to Laboulbeniales prevailing theory is that of gradual simplification of pleomorphic Pyxidiophora life cycle (with teleomorph and at least two anamorph) paired with morphological reduction. Arthropod hosts for Herpomycetales and Laboulbeniales were mostly collected in Poland using standard entomological collecting techniques and screened for fungal thalli. Samples for Pyxidiophorales were sampled in Białowieża Primeval Forest, and at a few other sites in Poland. Pyxidiophorales strains were isolated on standard mycological media. Additionally, four cultures of Pyxidiophorales were ordered from culture collections. DNA was isolated using standard, previously published methods. When material was scarce whole genome amplification was performed after single-cell DNA isolation. For selection of the best suited novel molecular markers for phylogenetics numerous PCR were tested with various previously published primer pairs on five samples representing all Laboulbeniomycetes orders. Later, selected protocols were optimized and finally two novel markers were amplified and sequenced from available DNA samples in addition to four previously used rDNA markers. Maximum likelihood multilocus phylogenetic trees were generated based on published and newly generated Laboulbeniomycetes molecular data. The main result of this work is a first multilocus molecular phylogeny of Laboulbeniomycetes, including underrepresented Pyxidiophorales. The phylogeny is supporting a theory of gradual simplification of pleomorphic Pyxidiophora-like ancestors into specialized entomoparasites with determined growth. Laboulbeniomycetes seems to be unique among entomopathogenic fungi in that their evolved from mycoparasitic ancestors due to arthropod-dependent dispersal and not from plant parasites like Hypocreales or saprotrophs like Entomophthorales. Current classification within Laboulbeniales is revealed to be incompatible with phylogeny implying that morphological characters such as arrangement of outer cell walls of perithecium and stalk cells are not conserved and cannot serve as diagnostic for family identification. Two novel species of Pyxidiophora, namely Pyxidiophora bialowiezense and Pyxidiophora ips are described from Białowieża Primeval Forest, Poland. All of the new species were isolated from decayed phloem of bark beetle Ips typographus infested Norway spruce Picea abies. Additionally, three species of Pyxidiophora were recorded as new to Poland: P. asterophora, P. microspora and P. corallisetosa. For the last of the species this is the second record worldwide. Detailed description of all Pyxidiophorales species are given, including description of previously unnoticed anamorphs. Analysis of publicly available environmental DNA sequences provided evidence that Pyxidiophorales are ubiquitous and distributed worldwide.