Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

• Keywords
• Laboulbenia, Laboulbeniales, Barcoding, DNA extraction, PCR amplification,
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• Journal Article MeSH

Laboulbeniomycetes is a class of fungi that have obligate associations with arthropod hosts, either for dispersal (order Pyxidiophorales) or as biotrophic parasites (orders Herpomycetales and Laboulbeniales). Here, we focus on Herpomycetales and Laboulbeniales, which include fungi that form thalli, 3-dimensional, multicellular units of 1000 s of cells. Based on recently published data regarding patterns of speciation, we present the One-Host-One-Parasite model (1H1P) for haustorial thallus-forming Laboulbeniomycetes. We hypothesize that taxa with haustoria, rhizoidal structures that make contact with the host's body cavity, have very strict host specificity. For taxa without haustoria, the microhabitat-as selected by the host-governs host shifting, presence or absence of the fungus, abundance, effective host range, and geographic distribution. We make suggestions for future research including fluorescent labeling of waxy lipids and mass spectrometry. These techniques have the potential to generate the data necessary to evaluate the here proposed 1H1P hypothesis for Herpomycetales and Laboulbeniales.

• Keywords
• Ectoparasitic fungi, Haustorium, Herpomyces, Host specificity, Integrative taxonomy, Laboulbeniales, One-Host-One-Parasite,
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• Journal Article MeSH

Laboulbeniales (Ascomycota, Laboulbeniomycetes) are biotrophic microfungi always attached to the exoskeleton of their arthropod hosts. They do not form hyphae or a mycelium; instead, they undergo determinate growth, developing from a two-celled ascospore to form a multicellular thallus. Hesperomyces virescens has been reported on over 30 species of ladybirds (Coleoptera, Coccinellidae); in reality, it represents a complex of species, presumably segregated by host genus association. In this study, we report on Hesperomyces thalli on Hyperaspis vinciguerrae from the Canary Islands and compare them with the Hesperomyces hyperaspidis described on Hyperaspis sp. from Trinidad. We generated the sequences of the internal transcribed spacer (ITS) region, the large subunit (LSU) nuclear ribosomal RNA gene, and the minichromosome maintenance complex component 7 (MCM7) protein-coding gene. Our phylogenetic reconstruction of Hesperomyces based on a concatenated ITS-LSU-MCM7 dataset revealed Hesperomyces sp. ex Hy. vinciguerrae as a member of the He. virescens species complex distinct from He. virescens sensu stricto (s.s.). It also revealed that the Hesperomyces sp. ex Chilocorus bipustulatus from Algeria is different from He. virescens s.s., which is associated with Chilocorus stigma from the USA. This suggests that the species of Hesperomyces are not solely segregated by host association, but that there is also a biogeographical component involved. Based on these data, we refrained from referring our material from Hy. vinciguerrae to He. hyperaspidis. Finally, we discuss the usefulness of MCM7 as a useful marker for species delimitation in Hesperomyces.

• Keywords
• Coccinellidae, DNA barcoding, MCM7, arthropod-associated fungi, integrative taxonomy, nuclear ribosomal DNA, phylogeny,
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• Journal Article MeSH

Harmonia axyridis is an invasive alien ladybird in North America and Europe. Studies show that multiple natural enemies are using Ha. axyridis as a new host. However, thus far, no research has been undertaken to study the effects of simultaneous infection by multiple natural enemies on Ha. axyridis. We hypothesized that high thallus densities of the ectoparasitic fungus Hesperomyces virescens on a ladybird weaken the host's defenses, thereby making it more susceptible to infection by other natural enemies. We examined mortality of the North American-native Olla v-nigrum and Ha. axyridis co-infected with He. virescens and an entomopathogenic fungus-either Beauveria bassiana or Metarhizium brunneum. Laboratory assays revealed that He. virescens-infected O. v-nigrum individuals are more susceptible to entomopathogenic fungi, but Ha. axyridis does not suffer the same effects. This is in line with the enemy release hypothesis, which predicts that invasive alien species in new geographic areas experience reduced regulatory effects from natural enemies compared to native species. Considering our results, we can ask how He. virescens affects survival when confronted by other pathogens that previously had little impact on Ha. axyridis.

• Keywords
• Coccinellidae, Enemy release hypothesis, Fungal entomopathogens, Harmonia axyridis, Hesperomyces virescens, Invasive alien species, Native species, Parasites,
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• Journal Article MeSH

In this paper we present an updated checklist of thallus-forming Laboulbeniomycetes (Ascomycota, Pezizomycotina), that is, the orders Herpomycetales and Laboulbeniales, from Belgium and the Netherlands. Two species are newly described based on morphology, molecular data (ITS, LSU ribosomal DNA) and ecology (host association). These are Hesperomyces halyziae on Halyzia sedecimguttata (Coleoptera, Coccinellidae) from both countries and Laboulbenia quarantenae on Bembidion biguttatum (Coleoptera, Carabidae) from Belgium. In addition, nine new country records are presented. For Belgium: Laboulbenia aubryi on Amara aranea (Coleoptera, Carabidae) and Rhachomyces spinosus on Syntomus foveatus (Coleoptera, Carabidae). For the Netherlands: Chitonomyces melanurus on Laccophilus minutus (Coleoptera, Dytiscidae), Euphoriomyces agathidii on Agathidium laevigatum (Coleoptera, Leiodidae), Laboulbenia fasciculata on Omophron limbatum (Coleoptera, Carabidae), Laboulbenia metableti on Syntomus foveatus and S. truncatellus (Coleoptera, Carabidae), Laboulbenia pseudomasei on Pterostichus melanarius (Coleoptera, Carabidae), Rhachomyces canariensis on Trechus obtusus (Coleoptera, Carabidae), and Stigmatomyces hydrelliae on Hydrellia albilabris (Diptera, Ephydridae). Finally, an identification key to 140 species of thallus-forming Laboulbeniomycetes in Belgium and the Netherlands is provided. Based on the combined data, we are able to identify mutual gaps that need to be filled as well as weigh the impact of chosen strategies (fieldwork, museum collections) and techniques in these neighboring countries. The aim of this work is to serve as a reference for studying Laboulbeniomycetes fungi in Europe.

• Keywords
• Ascomycota, Laboulbeniales, 2 new taxa, Herpomycetales, arthropod-associated fungi, integrative taxonomy, key,
• Publication type
• Journal Article MeSH