Research Article
Research Article
Buwchfawromyces eastonii gen. nov., sp. nov.: a new anaerobic fungus (Neocallimastigomycota) isolated from buffalo faeces
expand article infoTony Martin Callaghan, Sabine Marie Podmirseg§, Daniel Hohlweck|, Joan Elizabeth Edwards, Anil Kumar Puniya, Sumit Singh Dagar|, Gareth Wyn Griffith
‡ Aberystwyth University, Aberystwyth, United Kingdom
§ University of Innsbruck, Innsbruck, Austria
| Agharkar Research Institute, Maharashtra, India
¶ National Dairy Research Institute Karnal, Karnal, India
Open Access


The novel anaerobic fungus Buwchfawromyces eastonii gen. nov., sp. nov., belonging to order Neocallimastigales (phylum Neocallimastigomycota) is described. Morphologically similar to Piromyces but genetically quite distinct, this fungus (isolate GE09) was first isolated from buffalo faeces in west Wales and then subsequently isolated from sheep, cattle and horse in the same area. Phylogenetic analysis of LSU and ITS sequence confirmed that B. eastonii isolates formed a distinct clade close to the polycentric Anaeromyces spp. The morphology of GE09 is monocentric with monoflagellate zoospores. However, the sporangial stalk (sporangiophore) is often distinctly swollen and the proximal regions of the rhizoidal system twisted in appearance.

Key words

Ruminant, symbiosis, fungal phylogenetics, buffalo, DNA barcoding


At present six genera of anaerobic fungi are recognised, differentiated by thallus morphology and zoospore flagellation (Gruninger et al. 2014). However, the lack of other reliable morphological features, combined with difficulties in the cultivation of these fungi, exchange of cultures between different labs and their morphological variability in culture, has hindered more definitive study of their diversity. As the amount of DNA barcode data (mostly based on the internal transcribed spacer regions, ITS1 and ITS2), both for pure cultures and from environmental DNA sequencing projects has increased, it has become apparent that the six existing clades represent only a subset of the full diversity of the anaerobic fungi (Griffith et al. 2010). For instance, Liggenstoffer et al. (2010) generated 250,000 ITS1 sequences from a diverse range of host species and identified eight potentially novel clades. Furthermore, reconciliation of morphological features and DNA barcode data has led to the reassignment of some taxa (Fliegerová et al. 2012). Following revision of the taxonomy of kingdom Fungi, the anaerobic fungi are now assinged to phylum Neocallimastigomycota (Hibbett et al. 2007). However, the status of the anaerobic fungi as a distinct phylum as opposed to a class within phylum Chytridiomycota remains a matter of debate (Frey 2012; Powell and Letcher 2014).

The genus Anaeromyces was first discovered by Breton et al. (1990), describing Anaeromyces mucronatus. A near-simultaneous publication by Ho et al. (1990) named a morphologically similar fungus as Ruminomyces elegans but in recognition of the rules of priority, Ho and Barr (1993a) later renamed this species as A. elegans. Neither viable cultures nor DNA from these original isolates remains (Prof. Yin Wan Ho and Dr. Brigitte Gaillard-Martinie, pers. comms.), though the >80 ITS sequences submitted to GenBank under this genus name suggests that cultures conforming to the morphological description of Anaeromyces (uniflagellate zoospores, polycentric mycelium) are commonly isolated from the digestive tracts of different types of herbivorous mammals.

The taxonomic status of one isolate (GE09) is addressed, for which data were previously submitted to GenBank (five ITS cloned from this single isolate; EU414755EU414759) under the generic name Anaeromyces (Edwards et al. 2008). More detailed genetic analysis has been conducted to clarify its taxonomic position, along with its morphological characterisation. Microscopic examination and phylogenetic reconstruction, using both the ITS1 and LSU regions of the rRNA locus, showed that GE09 and three other similar isolates (isolated from different host species) were clearly distinct from the main Anaeromyces clade. We therefore conclude that these four isolates represent a new genus, which we name Buwchfawromyces gen. nov.


Enrichment culture from faeces

Anaerobic fungal cultures were enriched using a basal medium (Davies et al. 1993; Orpin 1977) supplemented with milled wheat straw. The basal medium was made up as follows: 150 ml salts solution 1 (3 g.L-1 K2HPO4 in distilled water), 150 ml salts solution 2 (3 g KH2PO4, 6 g (NH4)2SO4, 6 g NaCl, 0.6 g MgSO4.7H2O and 0.5 g CaCl2; dissolved in 1 L distilled water, in that order), yeast extract (3 g; Oxoid, Basingstoke, UK), tryptone (10 g; Fisher Scientific Ltd., Loughborough, UK), resazurin (2 ml of 0.1% solution) and hemin (2 ml of 0.05% solution dissolved in 1:1 ethanol / 50 mM NaOH) were added and the volume made up to 850 ml with distilled water. After boiling (until light red in colour) and cooling, 150 ml centrifuged (clarified) rumen fluid, 6 g NaHCO3 and 1 g L-cysteine-HCl were added (final volume 1 L). The resultant solution was then deoxygenated by gassing with CO2 for 1 h. The medium was dispensed anaerobically in 9 ml aliquots into 15 ml Hungate tubes containing milled wheat straw (5; 0.5% w/v; dry-sieved through 2 mm mesh). Tubes were then capped with a polypropylene bung held in place by a screw cap, and autoclaved (121 °C/15 min).

Freshly voided faecal samples (ca. 20 g) were collected and transported to the laboratory within 1 h. Aliquots (ca.10 g fresh matter) of these samples were then homogenised for 2 min with 90 ml of pre-warmed (39 °C) basal medium (without wheat straw, yeast extract or tryptone) in a pre-sterilised Stomacher 400 Circulator Bag (polythene; 177 × 305 mm) using a Seward Stomacher 400 Circulator Paddle Blender (Seward Ltd., Worthing, W. Sussex, UK). A 10-fold serial dilution of this faecal homogenate was then prepared in pre-warmed basal medium (1 ml transferred to 9 ml basal medium in 15 ml Hungate tube). The 10–4, 10–5 and 10–6 dilutions were used to inoculate (1 ml) the tubes of basal medium (9 ml) supplemented with wheat straw. A mixed solution of penicillin, ampicillin and streptomycin sulphate in 50% (v/v) ethanol (5 of each; 10 ml.L-1 added to give a final medium concentration of 50 µ was also added to tubes before they were recapped, in order to inhibit bacterial growth. Tubes were incubated in the dark at (39 °C), and routine subculture was conducted at 3–5 d intervals. Exposure of the samples to oxygen was prevented by undertaking the manipulations in a box flushed with CO2. Cultures were also grown on basal medium containing cellobiose (5 instead of wheat straw. Purity of the isolates was ensured by three cycles of cultivation in roll tubes (Joblin 1981), with well-separated colonies being excised for subculture with a mycological spear.

For cryopreservation of cultures, a method based on the procedures suggested by Sakurada et al. (1995) and Yarlett et al. (1986) was devised. A 5× cryopreservation solution was prepared by mixing 49.7 g ethylene glycol with 155 ml clarified rumen, 200 µl of 0.1% resazurin, 0.2 g L-cysteine and 1.2 g NaHCO3 under anaerobic conditions (total volume 200 ml; 3.2 M ethylene glycol). This solution was bubbled with CO2 for ca. 3 h, prior to anaerobically dispensing 10 ml aliquots into Hungate tubes which were autoclaved and stored at -20 °C until use. The 5× cryopreservation solution was added to 3–5 d old wheat straw cultures (2.5 ml per 10 ml culture) under anaerobic conditions in Hungate tubes. After mixing, tubes were chilled in ice water for 15 min and then anaerobically dispensed in 2 ml aliquots into sterile 2 ml cryovials. Cryovials were placed at -80 °C overnight, before being transferred to liquid nitrogen for longer-term storage. Storage of cryopreserved cultures at -80 °C is possible for up to a few months but for prolonged storage, liquid nitrogen was found to be much more reliable.


Microscopy was conducted on cultures, grown on either wheat straw or cellobiose, using an epifluorescence microscope (Olympus BX50) with images recorded using a Nikon Coolpix 995 digital camera. For visualisation of nuclei, DAPI (0.3 in 50 mM Tris-HCl, pH 7.2) was added, and for enhanced definition of cell walls and septa, Calcofluor white M2R (100 µM; Day et al. 2002) was used (UV-W filters: 365 nm excitation/420 nm emission).

Phylogenetic analysis

DNA extraction was carried out using the CTAB (hexadecyltrimethylammonium bromide) method of Doyle and Doyle (1987), with modifications as described by Griffith and Shaw (1998). Cultures were harvested after 3–6 d incubation, and the biomass washed three times with sterile distilled water before being freeze–dried and ground to a powder. Ground biomass (ca. 50 mg) was used for DNA extraction, and the purified DNA resuspended in 50 µl TE buffer (pH 8.0) before being stored at -20 °C.

For genetic analysis, the D1/D2 domain of large-subunit (LSU) ribosomal DNA and internal transcribed spacer 1 (spanning ITS1 and ITS2) were amplified, using the primer pairs NL1 (GCATATCAATAAGCGGAGGAAAAG) / NL4 (GGTCCGTGTTTCAAGACGG) (Dagar et al. 2011; Fliegerová et al. 2006) and GM1 (TGTACACACCGCCCGTC) / MN106 (CGTTGTAAAACACTCAWAACC) (Edwards et al. 2008), respectively. Sequence management was conducted within the Geneious (v6.1.6) bioinformatics package (Drummond et al. 2011), using MAFFT (Katoh et al. 2002) for sequence alignment (default settings).

Phylogenetic reconstruction was conducted using TOPALi (v2.5) (Milne et al. 2004). For LSU analysis, maximum likelihood analysis was conducted using PhyML (Guindon et al. 2010) and the TrN+gamma substitution model recommended by TOPALi. For analysis of the ITS, only the ITS1 region was used since the great majority of available sequences cover only this region (and not ITS2). To establish the phylogenetic position of isolate GE09, ITS1 sequences derived from five clones were aligned with environmental and isolate sequences belonging to the four closely related monoflagellate genera. The resulting MAFFT alignment was trimmed to include only 15 bp of the flanking 18S and 5.8S regions, and duplicate sequences were removed (retaining sequences relating to cultured fungi, if present). For ITS1 analysis, maximum likelihood analysis was conducted using PhyML (Guindon et al. 2010) and the GTR substitution model recommended by TOPALi. Both the ITS1 and LSU alignments have been submitted to TreeBase (

Results and discussion


The isolate GE09 was originally isolated from the faeces of a domesticated Asian water buffalo (Bubalus bubalis) on 26th Feb 2004 at Panthwylog Farm, Llanon, Ceredigion, Wales (N52.279; W-4.169). The buffalo was part of a herd kept outdoors, maintained on grass pasture supplemented with grass silage. More recently, three additional pure cultures (each from a different host species have been isolated), all identical in morphology and LSU DNA barcode to GE09. These isolates were obtained as follows: from cow (Bos taurus) faeces (isolate HoCal4.C3.3; Nant yr Arian, Ponterwyd, Ceredigion; 6th Feb 2013; N52.416; W-3.891), sheep (Ovis aries) faeces (HoCal4.B3c, also Nant yr Arian, 6th Feb 2013) and horse (Equus ferus caballus) faeces (isolate HoCal4.D1.2; Aberystwyth University Lluest livery yard; 6th Feb 2013; N52.410; W-4.051).


Thalli of isolate GE09, when grown on wheat straw or cellobiose as a carbon source, were consistently monocentric, with rhizoids radiating from a single developing sporangium. Mature sporangia were spherical to ovoid 30 to 80 µm long and 20 to 60 µm wide (Fig. 1). No apical projections, as found in Anaeromyces mucronatus (Breton et al. 1990) or Piromyces mae (Li et al. 1990) (referred to as a mucro or papilla respectively by these authors), were observed.

Figure 1. 

Morphology of Buwchfawromyces eastonii. Sporangia are ovoid (A) to spherical (B), tending to be more elongate when growing on straw particles (C). Zoospores are uniformly monoflagellate (D). A distinct septum is visible where the sporangium is attached to the sporangiophore (A, E, G, H arrowed) and sporangiophores are often swollen (E–H). Nuclei were not observed in sporangiophores or rhizoids (F, H, I). Scalebar indicates 50 µm.

Zoospores (spherical; diameter 9–11 µm) were readily observed in 3–5 d old cultures grown on wheat straw and consistently bore a single flagellum (30–40 µm long; 3–4× longer than the length of the zoospore body). However, the process whereby zoospores were released from the sporangium was not observed. On a single occasion, a large (30 µm diameter) zoospore-like structure bearing numerous flagella, each emerging from a different point on the zoospore body, was detected (Suppl. material 1A). This is possibly the result of the agglomeration and fusion of numerous zoospores (hence the numerous flagella that are visible), similar to the structure previously observed by Orpin (1975) in N. frontalis (Orpin’s Fig. 2; Suppl. material 1B).

The most distinctive feature of the thalli of GE09 were the swollen sporangiophores (40–80 µm long and 15–50 µm wide), occasionally comparable in volume to the sporangium they supported (Fig. 1E–H). Also visible was a septum at the point where the sporangium joined the sporangiophore (Fig. 1A, E–I). The sporangiophore was contiguous with the rhizoids which tapered (from 20 µm to 5 µm) and branched (Fig. 1B). The proximal rhizoids (within 100 µm of the sporangium) were often contorted (Fig. 1B). DAPI staining was used to observe the location of nuclei within the thalli (Fig. 1F, H, I); these were abundant in sporangia but none were observed in the sporangiophores or rhizoids.

The swollen sporangiophores and twisted rhizoids observed here are very similar to those noted by Ho et al. (1993b) in Piromyces spiralis (Suppl. material 1C, D). Also similar are the swollen sporangiophores reported in P. mae (Li et al. 1990) (isolate PN11 from horse; Fig. 25). It is also noteworthy that Anaeromyces (formerly Piromyces) polycephalus (Suppl. material 1E) (Chen et al. 2002; Kirk 2012), whilst forming multiple rather than single sporangia, also forms a distinctly swollen sporangiophore. It is possible that such structures play some role in physical disruption of the substrate, as is the case for the bulbous holdfasts formed by Caecomyces and Cyllamyces spp.

Cultures of GE09 maintained viability, and could be subcultured, after incubation at 39 °C for periods of several weeks. This raised the possibility that these cultures may form long-term survival structures (McGranaghan et al. 1999; Struchtemeyer et al. 2014). Thick-walled and septate structures (3–4 septa; 30–40 µm long × 10–15 µm wide), very similar to those previously observed in Anaeromyces sp. EO2 (Brookman et al. 2000) were seen in wheat straw cultures incubated for 28 d (Suppl. material 1F, G) and never observed in younger (3–5 d old) cultures. However, detailed examination of the development of these putative resting structures was not undertaken.

Phylogenetic analysis

Detailed examination of isolate GE09 was not undertaken when it was first isolated. However, it was used as a reference sample in a study of the colonisation of forage by anaerobic fungi (Edwards et al. 2008), in the course of which the ITS1/2 spacer regions were amplified and cloned. The sequences of the five clones were submitted to GenBank (EU414755EU414759) under the generic name Anaeromyces, since these sequences clustered close to the Anaeromyces clade at that time. However, more detailed analysis has since suggested that this isolate is quite distinct from Anaeromyces (Kittelmann et al. 2012).

The sequence of the D1/D2 domains (ca. 750 bp) of the LSU of GE09 and the three other isolates were identical (submitted to GenBank as KP205570). These sequences were aligned with 36 other LSU sequences (from GenBank) covering all the known genera of Neocallimastigomycota (700 bp alignment; 188 phylogenetically informative sites), and including the outgroup taxon Gromochytrium mamkaevae (Chytridiomycota). Phylogenetic reconstruction consistently recovered Buwchfawromyces isolates as a distinct clade (85% bootstrap support). LSU sequences from Anaeromyces, Neocallimastix and Orpinomyces were also recovered as distinct clades with strong (≥80%) bootstrap support (Fig. 2). Neocallimastix and Orpinomyces were more closely related to each other than to other genera, consistent with the occurrence of polyflagellate flagella in these genera, a feature not found in other flagellate fungi (James et al. 2006). The genera forming bulbous holdfasts (Caecomyces, Cyllamyces) also formed a distinct clade, and Piromyces isolates occupied a basal position but without strong bootstrap support.

Figure 2. 

Maximum likelihood tree based on alignment of the D1/D2 region of the Large Ribosomal Subunit (700 bp alignment; 37 sequences; 188 phylogenetically informative sites; TrN+gamma model). Bootstrap values over 70% are shown (1000 replicates). Scale bar indicates number of substitutions per site.

Whilst analysis of the LSU proved informative to confirm the distinctiveness of the GE09 clade, there are relatively few LSU sequences available in GenBank for comparison. Therefore, phylogenetic analysis of the ITS1 internal transcribed spacer region, for which there are hundreds of published sequences, was conducted (357 bp alignment; 271 phylogenetically informative sites, 101 sequences) (Fig. 3). Since analysis of the LSU sequences had shown members of the genera Neocallimastix and Orpinomyces to be quite distinct, these were omitted from analyses of the ITS1 region in order to improve the quality of the alignments (fewer gaps). Additionally, ‘environmental’ sequences obtained from clone library studies, (mostly labelled as “uncultured Neocallimastigales”) were also included, as recommended by Nilsson et al. (2011).

Figure 3. 

Maximum likelihood tree based on alignment (357 bp) of the ITS1 region. Midpoint rooting was used to root the tree and bootstrap values over 70% are shown (1000 replicates). Scalebar shows the number of substitutions per site. Clades corresponding to the known genera, the new Buwchfawromyces clade and also the ‘polycephalus’ clade are labelled. Codes in brackets indicate the novel clades identified by Koetschan et al. (2014).

The Buwchfawromyces (GE09) clade was again recovered with high (92%) bootstrap support, as was the Anaeromyces clade. Five environmental sequences, all from New Zealand (from cow or red deer; Fig. 2 and listed in Suppl. material 4) also fall into the Buwchfawromyces clade. This clade was also identified (and denoted as clade SK2) in the recent study by Koetschan et al. (2014), who were able to create a reliable phylogeny by using predictors of ITS1 folding to decrease the effects of gaps in anaerobic fungal ITS1 alignments.

The largest survey of anaerobic fungi conducted to date is that of Liggenstoffer et al. (2010). They obtained ca. 250,000 ITS1 sequences, from the faeces of 30 herbivore species kept in Oklahoma zoos (Liggenstoffer et al. 2010), including three of the four herbivore species from which we cultured Buwchfawromyces. It was surprising that such a large dataset should yield no sequences falling into the Buwchfawromyces clade. However, examination of the primers used by Liggenstoffer et al. (2010) revealed the presence of mismatches between the forward primer (MN100modified; 5’-TCCTACCCTTTGTGAATTTG-3’) and the cognate sequences found in members of the Buwchfawromyces clade (TCCTACCCTTTGTGAATTGT or TCCTTACCCTTTGTGAACTGA) (Suppl. material 2). These mismatches would very likely have caused significant primer bias and thus poor amplification of any Buwchfawromyces spp. present.

Five cloned ITS1/2 PCR amplicons of GE09 were originally submitted to GenBank (EU414755EU414759). These reveal an extremely high level of sequence divergence (<27 polymorphisms within the ca. 200bp ITS1 region; 87.1%–99.5% identity, (Suppl. material 3). High levels of intragenomic variation has also been found in other anaerobic fungi (Ozkose 2001), and also in some other fungal taxa, for instance phylum Glomeromycota (Pawlowska and Taylor 2004; Pringle et al. 2000). For GE09, the most divergent of these clones (EU414756) was more distantly related to the other GE09 clones than were sequences from New Zealand (Fig. 3). A cut-off level of 97% identity is often used to define species or OTUs (operational taxonomic units) in mycology (Nilsson et al. 2008; Yamamoto and Bibby 2014). However, such high levels of intragenomic variation, make it very difficult to generate reliable species hypotheses (Koljalg et al. 2013) for the Neocallimastigomycota based on ITS sequences alone, although the delineation of different genera is still possible.

We consider that isolate GE09 and the three other similar fungi also isolated in the Aberystwyth area represent a new genus Buwchfawromyces within the phylum Neocallimastigomycota. It is not possible for Buwchfawromyces to be placed within the related genus Anaeromyces, since it forms a monocentric thallus not consistent with the circumscription of this genus (Breton et al. 1990) which comprises species with polycentric thalli. Close to Buwchfawromyces and Anaeromyces, is the species currently known as Anaeromyces (formerly Piromyces) polycephalus. This species has a distinctive morphology (Suppl. material 1E), with multiple sporangia arising from a swollen sporangiophore, and with an anucleate rhizoidal system. It was originally isolated and described from buffalo in Taiwan (isolate W-33; (Chen et al. 2002)) and has since been reported from India (isolate CTS-47 from zebra faeces; GenBank EU330178); this clade was denoted DT1 (Fig. 3) by Koetschan et al. (2014). Given that ‘A. polycephalus’ is both morphologically and genetically distinct, this species should be renamed.

The unusually high level of intragenomic variation in ITS1 makes it difficult to nominate a single reference sequence, therefore two are presented (EU414755 and EU414756).


Buwchfawromyces Callaghan, Tony & G.W. Griff., gen. nov.


Strictly anaerobic fungus with determinate, monocentric thallus with single, spherical to ovoid terminal sporangium (often with swollen sporangiophore) and forming uniflagellate zoospores. The clade is defined by the sequences EU414755 and EU414756 (ITS1, 5.8S, ITS2 complete), and also KP205570 (LSU, partial sequence). The most genetically similar genus is Anaeromyces, which is defined as forming a polycentric thallus (Breton et al. 1990. FEMS Microbiol. Lett. 58, p.177), in contrast to the monocentric Buwchfawromyces.

Buwchfawromyces eastonii Callaghan, Tony & G.W. Griff., gen. nov., sp. nov.


An obligately anaerobic fungus with determinate monocentric thallus and spherical to ovoid sporangia. Thalli often with a distinctly swollen sporangiophore and twisted rhizoids. Extensive rhizoidal system but sporangiphore and rhizoids lacking nuclei. Sporangia ovoid to spherical (30–80 µm × 20–60 µm), non-papillate. Zoospores formed abundantly, spherical (9–11 µm diameter) with single flagellum (30–40 µm long). The reference sequences for this species are EU414755 and EU414756 (ITS1, 5.8S, ITS2), and KP205570 (LSU, D1/D2 regions). Since intragenomic variation in ITS1 sequence is present, the ITS1 sequence B. eastonii is defined as the least inclusive clade containing both EU414755 and EU414756. The type culture (isolate GE09) is stored cryogenically in liquid nitrogen at Aberystwyth University. Type material from 3 d old cultures and preserved in 5% glutaraldehyde is lodged in the biorepositories at: Aberystwyth University (code ABS) with isotype material at Royal Botanic Gardens, Kew, London (K); and Friedrich-Schiller-Universität Jena, Germany (JE).


From the Welsh words for large cow (‘buwch fawr’), since the original isolate GE09 was isolated from a buffalo for which there is no Welsh word. The specific epithet in honour of our former colleague Gary Easton who isolated this fungus.


Buwchfawromyces Callaghan, Tony & G.W. Griff., gen. nov. Strictly anaerobic fungus forming a monocentric thallus with a single sporangium, usually borne on a swollen sporangiophore connected to a branching and twisted rhizoidal system. Zoospores are spherical and uniflagellate. “Buwch fawr” means large cow in Welsh.


It has been apparent since the widespread use of DNA sequence data to identify anaerobic fungi that many of the sequences currently lodged with GenBank do not fall into any of the currently recognised genera of the Neocallimastigomycota. The situation is further complicated by the presence of many sequences from isolates which are very likely misidentified, a phenomenon which is also problematic for other groups of Fungi (Schoch et al. 2014).

A new genus is described based on a pure culture which was isolated a decade ago and for which ITS1 sequence has been lodged with GenBank since 2008. The RefSeq project aims to resolve this, and other related issues, by linking ITS and LSU sequences from vouchered reference specimens to accepted names (Schoch et al. 2014). As shown above, ITS sequences for anaerobic fungi can be problematic due to intragenomic sequence variation. Similar problems have not been found for the more conserved LSU region of the rRNA locus (Dagar et al. 2011; Eckart et al. 2010), highlighting the synergy of using both loci. We also note that analysis of LSU data does have the distinct advantage of yielding robust alignments across a wide range of basal fungal taxa (including Chytridiomycetes) and being amenable to direct sequencing of PCR products.


TMC is grateful to the Aberystwyth University Postgraduate Research Studentship Scheme for funding. JEE gratefully acknowledges funding from BBSRC (grant code BBS/E/W/10964A01). SSD and AKP are grateful respectively for the awarding of a Stapledon Memorial Trust Travelling Fellowship and DBT-CREST Award Fellowships, which permitted research visits to IBERS, Aberystwyth University. SP and DH are grateful to Universität Innsbruck and Technische Universität Berlin for the award of travel scholarships. We are also grateful to Dr. Paul Kirk, RBG Kew for nomenclatural advice.


  • Breton A, Bernalier A, Dusser M, Fonty G, Gaillard-Martinie B, Guillot J (1990) Anaeromyces mucronatus nov. gen., nov. sp. A new strictly anaerobic rumen fungus with polycentric thallus. FEMS Microbiology Letters 58: 177–182.
  • Brookman JL, Ozkose E, Rogers S, Trinci APJ, Theodorou MK (2000) Identification of spores in the polycentric anaerobic gut fungi which enhance their ability to survive. FEMS microbiology ecology 31: 261–267. doi: 10.1111/j.1574-6941.2000.tb00692.x
  • Chen Y-C, Hseu R-S, Chein C-Y (2002) Piromyces polycephalus (Neocallimastigaceae), a new rumen fungus. Nova Hedwigia 75: 409–414. doi: 10.1127/0029-5035/2002/0075-0409
  • Dagar SS, Kumar S, Mudgil P, Singh R, Puniya AK (2011) D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp. Applied and environmental microbiology 77: 6722–6725. doi: 10.1128/AEM.05441-11
  • Davies DR, Theodorou MK, Lawrence MIG, Trinci APJ (1993) Distribution of anaerobic fungi in the digestive tract of cattle and their survival in feces. Journal of General Microbiology 139: 1395–1400. doi: 10.1099/00221287-139-6-1395
  • Day JP, Kell DB, Griffith GW (2002) Differentiation of Phytophthora infestans sporangia from other airborne biological particles by flow cytometry. Applied and environmental microbiology 68: 37–45. doi: 10.1128/AEM.68.1.37-45.2002
  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–15.
  • Drummond A, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, A W (2011) Geneious v5.4.
  • Eckart M, Fliegerová K, Hoffmann K, Voigt K (2010) Molecular Identification of Anaerobic Rumen Fungi. In: Gherbawy Y, Voigt K (Eds) Molecular Identification of Fungi. Springer Berlin Heidelberg, 297–313. doi: 10.1007/978-3-642-05042-8_14
  • Edwards J, Huws S, Kingston-Smith A, Jimenez H, Skøt K, Griffith GW, McEwan NR, Theodorou MK (2008) Dynamics of initial colonisation of non-conserved perennial ryegrass by anaerobic fungi in the bovine rumen. FEMS microbiology ecology 66: 537–546. doi: 10.1111/j.1574-6941.2008.00563.x
  • Fliegerová K, Mrazek J, Voigt K (2006) Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP. Folia Microbiologica 51: 273–277. doi: 10.1007/BF02931811
  • Fliegerová K, Voigt K, Kirk PM (2012) Caecomyces hurleyensis (Theodorou & J. Webb) Fliegerová, K. Voigt & P.M. Kirk, comb. nov. IF550013. Index Fungorum 1: 1.
  • Frey W (2012) Syllabus of plant families. Part 1/1: blue-green algae, myxomycetes and myxomycete-like organisms, phytoparasitic protists, heterotrophic heterokontobionta and fungi. Ed. 13. Gebrüder Borntraeger Verlagsbuchhandlung.
  • Griffith GW, Baker S, Fliegerova K, Liggenstoffer A, van der Giezen M, Voigt K, Beakes G (2010) Anaerobic fungi: Neocallimastigomycota. IMA Fungus 1: 181–185. doi: 10.5598/imafungus.2010.01.02.11
  • Griffith GW, Shaw DS (1998) Polymorphisms in Phytophthora infestans: four mitochondrial haplotypes are detected after PCR amplification of DNA from pure cultures or from host lesions. Applied and environmental microbiology 64: 4007–4014.
  • Gruninger RJ, Puniya AK, Callaghan TM, Edwards JE, Youssef N, Dagar SS, Fliegerova K, Griffith GW, Forster R, Tsang A (2014) Anaerobic fungi (phylum Neocallimastigomycota): advances in understanding their taxonomy, life cycle, ecology, role and biotechnological potential. FEMS microbiology ecology. doi: 10.1111/1574-6941.12383
  • Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk WOG (2010) New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Systematic Biology 59: 307–321. doi: 10.1093/sysbio/syq010
  • Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, Eriksson O-E, Huhndorf S, James TY, Kirk PM, Lucking R, Lumbsch T, Lutzoni F, Matheny PB, McLaughlin DJ, Powell MJ, Redhead S, Schoch CL, Spatafora JW, Stalpers JA, Vilgalys R, Aime MC, Aptroot A, Bauer R, Begerow D, Benny GL, Castlebury LA, Crous PW, Dai Y-C, Gams W, Geiser DM, Griffith GW, Gueidan C, Hawksworth DL, Hestmark G, Hosaka K, Humber RA, Hyde K, Ironside JE, Koljalg U, Kurtzman CP, Larsson K-H, Lichwardt R, Longcore J, Miadlikowska J, Miller A, Moncalvo J-M, Mozley-Standridge S, Oberwinkler F, Parmasto E, Reeb V, Rogers JD, Roux C, Ryvarden L, Sampaio JP, Schussler A, Sugiyama J, Thorn RG, Tibell L, Untereiner WA, Walker C, Wang Z, Weir A, Weiss M, White MM, Winka K, Yao Y-J, Zhang N (2007) A higher-level phylogenetic classification of the fungi. Mycological Research 111: 509–547. doi: 10.1016/j.mycres.2007.03.004
  • Ho YW, Barr DJS, Abdullah N, Jalaludin S (1993a) Anaeromyces, an earlier name for Ruminomyces. Mycotaxon 47: 283–284.
  • Ho YW, Barr DJS, Abdullah N, Jalaludin S, Kudo H (1993b) Piromyces spiralis, a new species of anaerobic fungus from the rumen of goat. Mycotaxon 48: 59–68.
  • Ho YW, Bauchop T, Abdullah N, Jalaludin S (1990) Ruminomyces elegans gen. et sp.nov., a polycentric anaerobic rumen fungus from cattle. Mycotaxon 38: 397–405.
  • James TY, Letcher PM, Longcore JE, Mozley-Standridge SE, Porter D, Powell MJ, Griffith GW, Vilgalys R (2006) Chytridiomycota: 14 clades of flagellated Fungi. Mycologia 98: 860–871. doi: 10.3852/mycologia.98.6.860
  • Joblin KN (1981) Isolation, enumeration, and maintenance of rumen anaerobic fungi in roll tubes. Applied and environmental microbiology 42: 1119–1122.
  • Katoh K, Misawa K, Kuma KÄ, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic acids research 30: 3059–3066. doi: 10.1093/nar/gkf436
  • Kirk PM (2012) Anaeromyces polycephalus (Chen YC, Chien CY, Hseu RS) Flieg., K. Voigt & P.M. Kirk. IF550012. Index Fungorum 1: 1.
  • Kittelmann S, Naylor GE, Koolaard JP, Janssen PH (2012) A proposed taxonomy of anaerobic fungi (class Neocallimastigomycetes) suitable for large-scale sequence-based community structure analysis. PLoS ONE 7: e36866. doi: 10.1371/journal.pone.0036866
  • Koetschan C, Kittelmann S, Lu J, Al-Halbouni D, Jarvis GN, Müller T, Wolf T, Janssen PH (2014) Internal Transcribed Spacer 1 Secondary Structure Analysis Reveals a Common Core throughout the Anaerobic Fungi (Neocallimastigomycota). PLoS ONE 9: e91928. doi: 10.1371/journal.pone.0091928
  • Koljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS, Bahram M, Bates ST, Bruns TD, Bengtsson-Palme J, Callaghan TM, Douglas B, Drenkhan T, Eberhardt U, Duenas M, Grebenc T, Griffith GW, Hartmann M, Kirk PM, Kohout P, Larsson E, Lindahl BD, Luecking R, Martin MP, Matheny PB, Nguyen NH, Niskanen T, Oja J, Peay KG, Peintner U, Peterson M, Poldmaa K, Saag L, Saar I, Schuessler A, Scott JA, Senes C, Smith ME, Suija A, Taylor DL, Telleria MT, Weiss M, Larsson KH (2013) Towards a unified paradigm for sequence-based identification of fungi. Molecular Ecology 22: 5271–5277. doi: 10.1111/mec.12481
  • Li JL, Heath IB, Bauchop T (1990) Piromyces mae and Piromyces dumbonica, 2 new species of uniflagellate anaerobic chytridiomycete fungi from the hindgut of the horse and elephant. Canadian Journal of Botany 68: 1021–1033. doi: 10.1139/b90-129
  • McGranaghan P, Davies JC, Griffith GW, Davies DR, Theodorou MK (1999) The survival of anaerobic fungi in cattle faeces. FEMS microbiology ecology 29: 293–300. doi: 10.1111/j.1574-6941.1999.tb00620.x
  • Milne I, Wright F, Rowe G, Marshall DF, Husmeier D, McGuire G (2004) TOPALi: software for automatic identification of recombinant sequences within DNA multiple alignments. Bioinformatics 20: 1806–1807. doi: 10.1093/bioinformatics/bth155
  • Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson K-H (2008) Intraspecific ITS variability in the Kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evolution Bioinformatics Online 4: 193–201.
  • Nilsson RH, Ryberg M, Sjokvist E, Abarenkov K (2011) Rethinking taxon sampling in the light of environmental sequencing. Cladistics 27: 197–203. doi: 10.1111/j.1096-0031.2010.00336.x
  • Orpin CG (1975) Studies on the rumen flagellate Neocallimastix frontalis. Journal of General Microbiology 91: 249–262. doi: 10.1099/00221287-91-2-249
  • Orpin CG (1977) Rumen flagellate Piromonas communis - Its life history and invasion of plant material in rumen. Journal of General Microbiology 99: 107–117. doi: 10.1099/00221287-99-1-107
  • Ozkose E (2001) Morphology and molecular ecology of anaerobic fungi. Ph.D.: University of Wales, Aberyswyth.
  • Pawlowska TE, Taylor JW (2004) Organisation of genetic variation in individuals of arbuscular mycorrhizal fungi. Nature, London 427: 733–737. doi: 10.1038/nature02290
  • Powell MJ, Letcher PM (2014) Chytridiomycota, Monoblepharidomycota and Neocallimastigomycota. In: Esser K (Ed.) The Mycota VII Systematics and Evolution PartA. Springer, 141–175.
  • Pringle A, Moncalvo J-M, Vilgalys R (2000) High levels of variation in ribosomal DNA sequences within and among spores of a natural population of the arbuscular mycorrhizal fungus Acaulospora colossica. Mycologia 92: 259–268. doi: 10.2307/3761559
  • Sakurada M, Tsuzuki Y, Morgavi DP, Tomita Y, Onodera R (1995) Simple method for cryopreservation of an anaerobic rumen fungus using ethylene-glycol and rumen fluid. FEMS Microbiology Letters 127: 171–174. doi: 10.1111/j.1574-6968.1995.tb07469.x
  • Schoch CL, Robbertse B, Robert V, Vu D, Cardinali G, Irinyi L, Meyer W, Nilsson RH, Hughes K, Miller AN (2014) Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi. Database bau061. doi: 10.1093/database/bau061
  • Struchtemeyer CG, Ranganathan A, Couger MB, Liggenstoffer AS, Youssef NH, Elshahed MS (2014) Survival of the anaerobic fungus Orpinomyces sp. strain C1A after prolonged air exposure. Scientific Reports 4: 6892. doi: 10.1038/srep06892
  • Yamamoto N, Bibby K (2014) Clustering of fungal community internal transcribed spacer (ITS) sequence data obscures taxonomic diversity. Environmental Microbiology. doi: 10.1111/1462-2920.12390
  • Yarlett NC, Yarlett N, Orpin CG, Lloyd D (1986) Cryopreservation of the anaerobic rumen fungus Neocallimastix patriciarum. Letters In Applied Microbiology 3: 1–3. doi: 10.1111/j.1472-765X.1986.tb01533.x

Supplementary materials

Supplementary material 1 

SuppFig. 1

Tony Martin Callaghan, Sabine Marie Podmirseg, Daniel Hohlweck, Joan Elizabeth Edwards, Anil Kumar Puniya, Sumit Singh Dagar, Gareth Wyn Griffith

Data type: Adobe PDF file

Explanation note: Putative fused zoospores in isolate GE09 (A) and the similar structure reported by Orpin (1975) (B). Swollen sporangiophores and twisted rhizoids, as found in isolate GE09, were also reported for Piromyces spiralis (C, D) by Ho et al. (1993), whilst Anaeromyces (formerly Piromyces) polycephalus also forms a swollen sporangiophore (E). In older cultures of GE09, thick walled putative spore structures were frequently observed (F, G). Scalebar indicates 20 µm. A video of the putative fused zoospore is found at:

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (6.19 MB)
Supplementary material 2 

SuppFig. 2

Tony Martin Callaghan, Sabine Marie Podmirseg, Daniel Hohlweck, Joan Elizabeth Edwards, Anil Kumar Puniya, Sumit Singh Dagar, Gareth Wyn Griffith

Data type: Adobe PDF file

Explanation note: Alignment of part of the ITS1 region across a range of anaerobic fungi from all the known genera. The sequences of the modified MN100 primer used by Liggenstoffer et al. (2010) (TCCTACCCTTTGTGAATTTG) is indicated (green). For all clades except Buwchfawromyces, there is a good match for this primer. However, for members of the Buwchfawromyces, there are several mismatches at the 3’ end of the primer binding site which are likely to impede PCR amplification.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (884.27 kb)
Supplementary material 3 

SuppFig. 3

Tony Martin Callaghan, Sabine Marie Podmirseg, Daniel Hohlweck, Joan Elizabeth Edwards, Anil Kumar Puniya, Sumit Singh Dagar, Gareth Wyn Griffith

Data type: Adobe PDF file

Explanation note: Intragenomic variation in ITS sequences among the five clones sequenced from isolate GE09. 18S boundary ends with GATCATTA and 5.8S begins with CAACTTT, according to the convention of Hibbett et al. (1995).

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (124.91 kb)
Supplementary material 4 

SuppTable 1

Tony Martin Callaghan, Sabine Marie Podmirseg, Daniel Hohlweck, Joan Elizabeth Edwards, Anil Kumar Puniya, Sumit Singh Dagar, Gareth Wyn Griffith

Data type: Microsoft Excel file

Explanation note: Details of ITS sequences falling into the Buwchfawromyces clade. The first five are all derived from the isolate GE09.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (20.50 kb)