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,
• Publication type
• 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,
• Publication type
• 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,
• Publication type
• Journal Article MeSH

Studies of Trochila (Leotiomycetes, Helotiales, Cenangiaceae) are scarce. Here, we describe two new species based on molecular phylogenetic data and morphology. Trochilabostonensis was collected at the Boston Harbor Islands National Recreation Area, Massachusetts. It was found on the stem of Asclepiassyriaca, representing the first report of any Trochila species from a plant host in the family Apocynaceae. Trochilaurediniophila is associated with the uredinia of the rust fungus Ceroteliumfici. It was discovered during a survey for rust hyperparasites conducted at the Arthur Fungarium, in a single sample from 1912 collected in Trinidad. Macro- and micromorphological descriptions, illustrations, and molecular phylogenetic analyses are presented. The two new species are placed in Trochila with high support in both our six-locus (SSU, ITS, LSU, rpb1, rpb2, tef1) and two-locus (ITS, LSU) phylogenetic reconstructions. In addition, two species are combined in Trochila: Trochilacolensoi (formerly placed in Pseudopeziza) and T.xishuangbanna (originally described as the only species in Calycellinopsis). This study reveals new host plant families, a new ecological strategy, and a new country record for the genus Trochila. Finally, our work emphasizes the importance of specimens deposited in biological collections such as fungaria.

• Keywords
• 4 new taxa, Boston Harbor Islands, South America, Trinidad, biological collections, fungarium specimens, fungicolous fungi, taxonomy,
• Publication type
• Journal Article MeSH

The discovery of a new fossil species of the Caribbeo-Mexican genus Proptomaphaginus (Coleoptera, Leiodidae, Cholevinae) from Dominican amber, associated with a new fossil parasitic fungus in the genus Columnomyces (Ascomycota, Laboulbeniales), triggered an investigation of extant species of Proptomaphaginus and revealed the long-enduring parasitic association between these two genera. This effort resulted in the description of the fossil species †Proptomaphaginus alleni sp. nov., and one fossil and two extant species of Columnomyces, selectively associated with species of Proptomaphaginus: †Columnomyces electri sp. nov. associated with the fossil †Proptomaphaginus alleni in Dominican amber, Columnomyces hispaniolensis sp. nov. with the extant Proptomaphaginus hispaniolensis (endemic of Hispaniola), and Columnomyces peckii sp. nov. with the extant Proptomaphaginus puertoricensis (endemic of Puerto Rico). Based on biogeography, our current understanding is that the Caribbean species of Proptomaphaginus and their parasitic species of Columnomyces have coevolved since the Miocene. This is the first occurrence of such a coevolution between a genus of parasitic fungus and a genus of Coleoptera. The phylogenetic relations among Proptomaphaginus species are also addressed based on a parsimony analysis. Fossil specimens were observed by propagation phase-contrast synchrotron X-ray microtomography (PPC-SRμCT) and extant specimens were obtained through the study of preserved dried, pinned insects, attesting for the importance of (i) technological advancement and (ii) natural history collections in the study of microparasitic relationships.

• MeSH
• Ascomycota * MeSH
• Coleoptera microbiology   MeSH
• X-Ray Microtomography * MeSH
• Synchrotrons * MeSH
• Fossils * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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

Thirteen new species are formally described: Cortinarius brunneocarpus from Pakistan, C. lilacinoarmillatus from India, Curvularia khuzestanica on Atriplex lentiformis from Iran, Gloeocantharellus neoechinosporus from China, Laboulbenia bernaliana on species of Apenes, Apristus, and Philophuga (Coleoptera, Carabidae) from Nicaragua and Panama, L. oioveliicola on Oiovelia machadoi (Hemiptera, Veliidae) from Brazil, L. termiticola on Macrotermes subhyalinus (Blattodea, Termitidae) from the DR Congo, Pluteus cutefractus from Slovenia, Rhizoglomus variabile from Peru, Russula phloginea from China, Stagonosporopsis flacciduvarum on Vitis vinifera from Italy, Strobilomyces huangshanensis from China, Uromyces klotzschianus on Rumex dentatus subsp. klotzschianus from Pakistan. The following new records are reported: Alternaria calendulae on Calendula officinalis from India; A. tenuissima on apple and quince fruits from Iran; Candelariella oleaginescens from Turkey; Didymella americana and D. calidophila on Vitis vinifera from Italy; Lasiodiplodia theobromae causing tip blight of Dianella tasmanica 'variegata' from India; Marasmiellus subpruinosus from Madeira, Portugal, new for Macaronesia and Africa; Mycena albidolilacea, M. tenuispinosa, and M. xantholeuca from Russia; Neonectria neomacrospora on Madhuca longifolia from India; Nothophoma quercina on Vitis vinifera from Italy; Plagiosphaera immersa on Urtica dioica from Austria; Rinodina sicula from Turkey; Sphaerosporium lignatile from Wisconsin, USA; and Verrucaria murina from Turkey. Multi-locus analysis of ITS, LSU, rpb1, tef1 sequences revealed that P. immersa, commonly classified within Gnomoniaceae (Diaporthales) or as Sordariomycetes incertae sedis, belongs to Magnaporthaceae (Magnaporthales). Analysis of a six-locus Ascomycota-wide dataset including SSU and LSU sequences of S. lignatile revealed that this species, currently in Ascomycota incertae sedis, belongs to Pyronemataceae (Pezizomycetes, Pezizales).

• Keywords
• 13 new species, 16 new records, Agaricomycetes, Dothideomycetes, Glomeromycota, Laboulbeniomycetes, Magnaporthaceae, Pezizomycetes, Pucciniomycetes, Pyronemataceae, Sordariomycetes, integrative taxonomy,
• Publication type
• Journal Article MeSH

Fungal species identities are often based on morphological features, but current molecular phylogenetic and other approaches almost always lead to the discovery of multiple species in single morpho-species. According to the morphological species concept, the ant-parasitic fungus Rickia wasmannii (Ascomycota, Laboulbeniales) is a single species with pan-European distribution and a wide host range. Since its description, it has been reported from ten species of Myrmica (Hymenoptera, Formicidae), of which two belong to the rubra-group and the other eight to the phylogenetically distinct scabrinodis-group. We found evidence for R. wasmannii being a single phylogenetic species using sequence data from two loci. Apparently, the original morphological description (dating back to 1899) represents a single phylogenetic species. Furthermore, the biology and host-parasite interactions of R. wasmannii are not likely to be affected by genetic divergence among different populations of the fungus, implying comparability among studies conducted on members of different ant populations. We found no differences in total thallus number on workers between Myrmica species, but we did observe differences in the pattern of thallus distribution over the body. The locus of infection is the frontal side of the head in Myrmica rubra and M. sabuleti whereas in M. scabrinodis the locus of infection differs between worker ants from Hungary (gaster tergites) and the Netherlands (frontal head). Possible explanations for these observations are differences among host species and among populations of the same species in (i) how ant workers come into contact with the fungus, (ii) grooming efficacy, and (iii) cuticle surface characteristics.

L’identification des espèces fongiques est souvent basée sur des caractéristiques morphologiques, mais les approches phylogénétiques moléculaires et autres conduisent presque toujours à la découverte d’espèces multiples dans une seule morpho-espèce. Selon le concept d’espèce morphologique, le champignon parasite de fourmis Rickia wasmannii (Ascomycota, Laboulbeniales) est une espèce unique ayant une répartition paneuropéenne et une large gamme d’hôtes. Depuis sa description, il a été signalé chez dix espèces de Myrmica (Hymenoptera, Formicidae), dont deux appartiennent au groupe rubra et les huit autres au groupe scabrinodis, phylogénétiquement distinct. Nous avons trouvé que R. wasmannii était une seule espèce phylogénétique en utilisant les données des séquences de deux loci. Apparemment, la description morphologique originale (datant de 1899) représente une seule espèce phylogénétique. De plus, la biologie et les interactions hôte-parasite de R. wasmannii ne devraient pas être affectées par une divergence génétique entre différentes populations du champignon, ce qui implique une comparabilité entre les études conduites sur des membres de différentes populations de fourmis. Nous n’avons trouvé aucune différence dans le nombre total de thalles chez les ouvrières entre les espèces de Myrmica, mais nous avons observé des différences dans le schéma de distribution des thalles sur le corps. Le locus d’infection est le front de la tête chez Myrmica rubra et M. sabuleti, alors que chez M. scabrinodis, le lieu d’infection diffère entre les ouvrières de Hongrie (tergites abdominaux) et des Pays-Bas (front de la tête). Les explications possibles de ces observations sont les différences entre les espèces hôtes et entre les populations de la même espèce en ce qui concerne (i) le contact des ouvrières avec le champignon, (ii) l’efficacité du toilettage et (iii) les caractéristiques de la surface de la cuticule.

• MeSH
• Ascomycota physiology   MeSH
• Ants microbiology   MeSH
• Phylogeny MeSH
• Host-Parasite Interactions * MeSH
• Mycoses MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Hungary MeSH