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Environmental Microbiology |
School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK1
Institute of Grasslands and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK2
Author for correspondence: J. L. Brookman. Tel: +44 161 275 5102. Fax: +44 161 275 5082. e-mail: Jayne.Brookman{at}man.ac.uk
The gut fungi are an unusual group of zoosporic fungi occupying a unique ecological niche, the anaerobic environment of the rumen. They exhibit two basic forms, with nuclear migration throughout the hyphal mass for polycentric species and with concentration of nuclear material in a zoosporangium for monocentric species. Differentiation between isolates of these fungi is difficult using conventional techniques. In this study, DNA-based methodologies were used to examine the relationships within and between two genera of monocentric gut fungi gathered from various geographical locations and host animals. The ribosomal ITS1 sequence from 16 mono- and 4 polycentric isolates was PCR-amplified and sequenced; the sequences obtained were aligned with published sequences and phylogenetic analyses were performed. These analyses clearly differentiate between the two genera and reflect the previously published physiological conclusions that Neocallimastix spp. constitute a more closely related genus than the relatively divergent genus Piromyces. The analyses place two type species N. frontalis and N. hurleyensis together but, contrary to a recent suggestion in the literature, place them apart from the other agreed species N. patriciarum. In situ hybridization and slot-blotting were investigated as potential methods for detection of and differentiation between monocentric gut fungi. DNA slot-blot analysis using ribosomal sequences is able to differentiate between gut fungal genera and thus has considerable potential for use in ecological studies of these organisms.
Keywords: anaerobic gut fungi, molecular phylogeny, in situ hybridization
Abbreviations: DAPI, 4,6-diamino-2-phenylindole; ITS, internal transcribed spacer
The GenBank accession numbers for the sequences determined in this work are given in Methods.
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