Corresponding author: Gareth Wyn Griffith (
Academic editor: T. Lumbsch
The novel anaerobic fungus
Callaghan TM, Podmirseg SM, Hohlweck D, Edwards JE, Puniya AK, Dagar SS, Griffith GW (2015)
At present six genera of anaerobic fungi are recognised, differentiated by thallus morphology and zoospore flagellation (
The genus
The taxonomic status of one isolate (GE09) is addressed, for which data were previously submitted to GenBank (five ITS cloned from this single isolate;
Anaerobic fungal cultures were enriched using a basal medium (
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 mg.ml-1 of each; 10 ml.L-1 added to give a final medium concentration of 50 µg.ml-1) 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 mg.ml-1) instead of wheat straw. Purity of the isolates was ensured by three cycles of cultivation in roll tubes (
For cryopreservation of cultures, a method based on the procedures suggested by
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 mg.ml-1 in 50 mM Tris-HCl, pH 7.2) was added, and for enhanced definition of cell walls and septa, Calcofluor white M2R (100 µM;
DNA extraction was carried out using the CTAB (hexadecyltrimethylammonium bromide) method of
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) (
Phylogenetic reconstruction was conducted using TOPALi (v2.5) (
The isolate GE09 was originally isolated from the faeces of a domesticated Asian water buffalo (
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.
Morphology of
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
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.
The swollen sporangiophores and twisted rhizoids observed here are very similar to those noted by
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 (
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 (
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
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.
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
The
The largest survey of anaerobic fungi conducted to date is that of
Five cloned ITS1/2 PCR amplicons of GE09 were originally submitted to GenBank (
We consider that isolate GE09 and the three other similar fungi also isolated in the Aberystwyth area represent a new genus
The unusually high level of intragenomic variation in ITS1 makes it difficult to nominate a single reference sequence, therefore two are presented (
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
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
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.
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
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 (
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.
SuppFig. 1
Adobe PDF file
Putative fused zoospores in isolate GE09 (A) and the similar structure reported by
SuppFig. 2
Adobe PDF file
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
SuppFig. 3
Adobe PDF file
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).
SuppTable 1
Microsoft Excel file
Details of ITS sequences falling into the