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.

Research into the microbiology of the rumen has been undertaken for many years with substantial contributions in bacteriology and protozoology being made during the 1940s, 1950s and 1960s. However, it was not until 1975that Orpin (1975 identified the rumen anaerobic fungi. In fact, zoospores of anaerobic fungi were known since the early part of this century but were mistakenly identified as protozoan flagellates in rumen fluid (Liebetanz, 1910). According to Warner (1966), these flagellates were sequestered on the rumen epithelium, moving to the rumen fluid when the animal was feeding. This hypothesis was necessary to account for the large increase in protozoan flagellates in rumen fluid immediately after feeding. Orpin (1975) showed that the sequestered cells were not on the rumen wall but came from digesta particles, concluding that their abundance after feeding was due to their liberation from digesta-associated fungal zoosporangia. Two reasons can account for our failure to recognize the existence of anaerobic fungi until the mid-1970s. First, there was the persistent belief that all fungi required 0, to grow (Foster, 1949) and second, it was common practice amongst rumen microbiologists to work with strained rumen fluid, discarding the digesta solids and, thus, the digesta-associated fungal thalli (Bauchop, 1983). Life history and survival

[7] sequence data shows that anaerobic chytrids are monophyletic (i.e. they had a single ancestor which was either the ancestral, free-living anaerobic chytrid or the ancestral, free-living aerobic chytrid which invaded the digestive tract of herbivores). In either event, the lack of host specificity [8][9][10] would be sufficient to explain the present day ubiquitous dis