Molecular Characterization of Coprophilous Fungal Communities Reveals Sequences Related to Root-Associated Fungal Endophytes

Abstract

This paper reports the use of molecular methods to characterize the coprophilous fungal communities (CFC) that inhabit the dung of four species of mammalian herbivores at two sites, Sevilleta National Wildlife Refuge (SNWR) in New Mexico and Wind Cave National Park (WCNP) in South Dakota. Results reveal that CFC from domesticated cattle (Bos taurus) at SNWR, and bison (Bison bison) and black-tailed prairie dogs (Cynomys ludovicianus) at WCNP were diverse but dominated primarily by members within eight taxonomic orders, including the rarely cultured and anaerobic order Neocallimastigales. In addition, 7.7% (138 of 1,788) of the sequences obtained from all dung samples were at least 97% similar to root-associated fungal (RAF) sequences previously described from blue grama (Bouteloua gracilis), a common forage grass found throughout North America and growing at both study sites. In contrast, 95.8% (295 of 308) of the sequences and four of the total seven operational taxonomic units obtained from pronghorn antelope (Antilocapra americana) dung belonged to the Pleosporalean order. We hypothesize that some herbivore vectors disperse non-systemic (non-clavicipitaceous) fungal endophytes. These dispersal events, it is argued, are most likely to occur via herbivores that occasionally forage and masticate root tissue, especially in arid regions where aboveground vegetation is sparse. The results of this study suggest that some (possibly many) members of the RAF community can expand their ecological role to include colonizing dung.

Internal Transcribed Spacer 1 Secondary Structure Analysis Reveals a Common Core throughout the Anaerobic Fungi (Neocallimastigomycota)

“…Beside the six known genera of Neocallimastigomycota, several novel groups as identified in previous studies [8]–[11], [41] could be confirmed: AL6, AL8, BlackRhino, DT1, JH1, KF1, MN4, SK1, SK2, SK3, SK4 and UC1 form monophyletic clusters within the tree. In addition, we detected a further novel monophyletic cluster, named DA1, which contains sequences derived from rumen content and faeces of pasture-fed lactating dairy cows in New-Zealand (Al-Halbouni & Jarvis, unpublished data).…”

“…Recently, the internal transcribed spacers 1 and 2 (ITS1 and ITS2) were confirmed to be the best suited marker genes for all classes of fungi [4] and to provide complementary phylogenetic information [5]–[7]. Using these molecular tools, several new groups of anaerobic fungi have been detected [8]–[11]. With the increased rate of discovery of novel sequence types through the application of next-generation sequencing, taxonomic classification of environmental sequences as well as the curation of reference databases are understood to be the next major challenges for fungal taxonomy to enable the description and evaluation of fungal diversity in the environment [12], [13].…”

A Proposed Taxonomy of Anaerobic Fungi (Class Neocallimastigomycetes) Suitable for Large-Scale Sequence-Based Community Structure Analysis

“…Using the primer pair ITS1F and ITS400Rw, we amplified and sequenced 401 ITS1 genes from DNA extracted from 11 samples of rumen contents from 5 different animals on a variety of diets (Table 3). A manual alignment was created from 197 sequences of isolates (6 additional sequences were obtained in this study), 183 representative environmental sequences of recently detected novel groups of anaerobic fungi [21], [22], [26], [28], and 417 clone sequences (401 sequences from the libraries, 16 sequences from excised DGGE bands; this study). Clustering of sequences based on a manual alignment of the primary structure information alone was compared to the clustering based on automatic sequence and secondary structure alignment using the software 4SALE [35].…”

“…However, since these clusters were clearly separated from the true Anaeromyces spp., and potentially represent novel species or genera, we have designated these clusters as SK2 and SK3. Seventeen clusters contained only environmental clone sequences, and with the exception of JH1 [28] all remaining clusters contained sequences from at least two different studies. These were Piromyces 3 and 6, BlackRhino (BlackRhino04IGVMQ, GQ738584; [21]), KF1 [26], SK1 and SK4 (this study), AL1 to AL7 (corresponding to NG1 to NG7 of Liggenstoffer et al [21]), Orpinomyces 3 and 6, and MN3 (corresponding to NG3 of Nicholson et al [22]).…”

“…Here, we constructed ITS1 clone libraries from 11 rumen samples selected based on their distinctive DGGE patterns, and used the 401 clone sequences obtained together with 197 sequences from isolates (including six so far unpublished sequences from three New Zealand isolates), 183 environmental sequences selected from earlier studies [21], [22], [26], [28], and 16 sequences from excised DGGE bands to build an improved taxonomic framework for anaerobic fungi. Due to the limitations of the marker gene, this detailed framework does not try to accommodate all sequences in known genera and species, but instead differentiates novel groups of sequences as groups at approximately species level and applies clustering on genus level only where morphologically described isolates are available.…”