Neocallimastigales
Anaerobic fungi were formerly classified among the chytridiomycete fungi (Li et al. 1990), later designated to their own phylum Neocallimastigomycota (Hibbett et al. 2007) and subsequently summarized as Chytridiomycota for flagellate fungi (Voigt 2012). Since their recognition as Fungi by Orpin (1974), eight genera of obligately anaerobic fungi, currently classified in the class Neocallimastigomycetes are described (Barr et al. 1989; Breton et al. 1990; Callaghan et al. 2015; Dagar et al. 2015; Gold et al. 1988; Griffith et al. 2010; Gruninger et al. 2014; Orpin 1975; Ozkose et al. 2001).
The first of these was the genus Neocallimastix, originally named as a flagellate protozoan Callimastix frontalis by Braune (1913), renamed by Vavra and Joyon (1966) as Neocallimastix frontalis, and eventually recongised as a fungus by Orpin (1975). It was formally named by Heath et al. (1983) as Neocallimastix frontalis, within the new family Neocallimastigaceae within the chytrid order Spizellomycetales (Fig. 1).
Within genus Neocallimastix, three other species have since been named: N. patriciarum (Orpin and Munn 1986), N. hurleyensis (Webb and Theodorou 1991) and N. variabilis (Ho et al. 1993). However, Wubah et al. (1991) undertook a direct comparison of the type cultures of N. frontalis and N. patriciarum and found them to be morphologically and culturally indistinguishable. Ho and Barr (1995) later synonymised these two species and also N. variabilis as N. frontalis, and also cast doubt on the distinctiveness of N. hurleyensis. The morphological variability of these fungi in culture and the paucity of morphological traits (Gruninger et al. 2014) makes it difficult to assess the validity of this decision. Type material exists only for Orpin’s N. particiarum (IMI 295997 at RBG Kew) and also for N. hurleyensis (IMI 344175 at RGB Kew) and isolate CX, Orpin’s original culture from which the N. patriciarum type material was derived also remains viable, having been cryopreserved in liquid nitrogen at the Rowett Institute (Aberdeen, Scotland).
The genus Piromyces was first observed as flagellate zoospores in rumen fluid by Liebetanz in (1910) and can probably be assumed as ‘the oldest’ known genus of anaerobic fungi, even if at that time it was described as a protozoan: Piromonas communis. Orpin (1977) much later adopted this name for his isolate correctly characterized as a fungus, which was the reason to rename this organism as Piromyces (Gold et al. 1988). The emended description of Piromyces communis was published by Barr et al. (1989) and currently seven other species have been described in the genus including P. mae (Li et al. 1990), P. dumbonica (Li et al. 1990), P. rhizinflata (Breton et al. 1991), P. spiralis (Ho et al. 1993c), P. minutus (Ho et al. 1993b), P. citronii (Gaillard-Martinie et al. 1995), P. polycephal us (Chen and Hseu 2002) and P. cryptodigmaticus (Kirk 2012). This genus thus covers the highest number of species and represents also the most studied anaerobic fungus, especially from the enzymological point of view (Harhangi et al. 2002, 2003a, b, c; Steenbakkers et al. 2001, 2002a, b, 2003, 2008). The recent pyrosequencing analysis of feces samples from 30 animal species has moreover revealed the Piromyces as the most abundant genus being encountered in 28 different animals and representing 36 % of all obtained sequences (Liggenstoffer et al. 2010). Comparing with other anaerobic fungi this genus is also the most heterogenous with some of its member grouping close to Neocallimastix strains and next to Orpinomyces strains (Hausner et al. 2000; Fliegerová et al. 2004) and therefore sometimes wrongly classified (Fliegerová et al. 2010). This heterogenity however can indicate the presence of new hidden Piromyces species. others (Griffith et al. 2010) are in the process of collecting and distributing reference materials and cultures to diverse laboratories engaged in research on these fungi. Here we provide DNA barcode data for Orpin’s isolate CX (GenBank ID: KR920744) and also provide a description for a second species, Neocallimastix cameroonii, which forms a distinct clade within the genus Neocallimastix based on morphological examination (Fig. 2) and phylogenetic analysis of 28S rRNA gene sequence (Fig. 1). No ITS sequence data is provided since within anaerobic fungi there is substantial (up to 13 %) intragenomic variation (Callaghan et al. 2015), which complicates direct sequencing of PCR products and the unambiguous assignment of reference sequences.
Anaerobic fungi were isolated from the faeces of Cameroon sheep (a rare breed of domesticated sheep originally from West Africa), which are kept at Wildpark Poing, Munich, Germany (48.17 N, 11.83 W) and fed on a mixture of grass forage and silage. Faeces were collected on 15th September 2013 and stored frozen during transfer to Aberystwyth, where isolation was undertaken using the methods described by Callaghan et al. (2015). Individual thalli were excised from roll tubes. Three cultures (CaDo3a, CaDo3b, CaDo3d) were isolated into axenic culture with wheat straw as carbon source, and later cryopreserved in liquid nitrogen.
Thalli of CaDo3a were consistently monocentric (Fig. 2c, f), with rhizoids radiating from a single sporangium. Mature sporangia were mostly ovoid to spherical (30 – 50 μm diameter). Zoospores (6 – 9 μm diameter) were observed in most cultures after 3d growth on wheatstraw at 39 °C (Fig. 2c) and these consistently bore multiple (9 – 15) flagella (Fig. 2a, b) 15 – 25 μm long. The rhizoidal system emanating from the base of the sporangium was often observed to be distinctly bifurcated (Fig. 2d, f).
Fig. 1 Maximum likelihood (PhyML) tree based on alignment of the D1/D2 region of the Large Ribosomal Subunit (758 bp alignment; 45 sequences; 96 phylogenetically informative sites; HKY model). Salient bootstrap values are shown (as %; 1000 replicates). Scale bar indicates number of substitutions per site.
Fig. 2 Morphology of Neocallimastix cameroonii (holotype) a, b Muiltiflagellate zoospores c Zoospore formed readily in culture e – f Thalli consistently monocentric and often with bifurcation of main rhizoid at the base of the sporangium (arrowed in d and f). Scale bars a, b = 10 μm, c – f = 50 μm.