title: Bringing Laboulbeniales into the 21st century: enhanced techniques for extraction and PCR amplification of DNA from minute ectoparasitic fungi.
abstract: Laboulbeniales is one of the most peculiar orders of Ascomycota. These fungi are characterized by an ectoparasitic life-style on arthropods, determinate growth, lack of an asexual stage, high species richness, and intractability to culture. The order Laboulbeniales, sister to Pyxidiophorales, has only recently been assigned a separate class, the Laboulbeniomycetes, based on very few ribosomal DNA sequences. So far, DNA isolations and PCR amplifications have proven difficult. Here, we provide details of isolation techniques and the application of commercially available kits that enable efficient and reliable genetic analyses of these fungi. We provide 43 newly generated Laboulbeniales ribosomal DNA sequences, among which are the first published sequences for species in the genera Gloeandromyces, Herpomyces, Laboulbenia, Monoicomyces, and Polyandromyces. DNA extractions were possible using from 1 to 30 thalli from hosts preserved in ethanol (70-100 %). In two cases, we successfully isolated DNA from thalli on dried insect collections. Laboulbeniales molecular systematics could be substantially enhanced through these improved methods by allowing more complete sampling of both taxa and gene regions.
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, 22 Divinity Avenue, Cambridge, MA 02138, USA;; Farlow Reference Library and Herbarium of Cryptogamic Botany, Harvard University, 22 Divinity Avenue, Cambridge, MA 02138, USA.
authors.forename: Danny
authors.initials: D
authors.lastname: Haelewaters
authors.affiliation: Department of Molecular Phylogenetics and Evolution, University of Warsaw, Aleje Ujazdowskie 4, Warsaw, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Gorczak
authors.affiliation: Department of Genetics and Applied Microbiology, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary; Department of Biotechnology and Microbiology, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary; Department of Biotechnology and Microbiology, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary.
authors.forename: Walter P
authors.initials: WP
authors.lastname: Pfliegler
authors.affiliation: Department of Evolutionary Zoology and Human Biology, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Tartally
authors.affiliation: Department of Molecular Phylogenetics and Evolution, University of Warsaw, Aleje Ujazdowskie 4, Warsaw, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Tischer
authors.affiliation: Department of Molecular Phylogenetics and Evolution, University of Warsaw, Aleje Ujazdowskie 4, Warsaw, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Wrzosek
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University, 22 Divinity Avenue, Cambridge, MA 02138, USA;; Farlow Reference Library and Herbarium of Cryptogamic Botany, Harvard University, 22 Divinity Avenue, Cambridge, MA 02138, USA.
authors.forename: Donald H
authors.initials: DH
authors.lastname: Pfister
keywords: Ascomycota; DNA isolation; Laboulbeniales-specific primers; insect collections; ribosomal DNA; unculturable fungi