A new species of the oligotrophic genus Ochroconis (Sympoventuriaceae)

Ochroconis globalis, a novel species of the melanized genus Ochroconis (Sympoventuriaceae, Venturiales), is described and illustrated and distinguished phenotypically and molecularly from existing ochroconis-like species. Phylogenetic analyses using nuclear ribosomal DNA genes (nuSSU, ITS, nuLSU) and coding gene fragments (ACT1, BT2, TEF1) revealed clustering of all strains as a monophyletic clade which was well separated from known Ochroconis species. Most strains of the new species were obtained from human-made environments, while the natural habitat of the species remains enigmatic. The new combination Ochroconis musae is introduced for one of the most commonly encountered Ochroconis species, and a phenotypic key to all species is provided.


Introduction
The genus Ochroconis, typified by O. constricta, was described by de Hoog and von Arx (1973) for melanized fungi with sympodial conidiogenesis and septate, ellipsoidal conidia. The genus was differentiated from the sister genus Scolecobasidium Abbott (1927) which was originally described with T-or Y-shaped conidia. Samerpitak et al. (2014) showed that the status of Scolecobasidium was doubtful and combined species with forked conidia in Ochroconis on phylogenetic grounds as members of Sympoventuriaceae, although some of the species with elaborate morphology still remain outside the family and are in need of correct classification. Colonies of members of the expanded genus Ochroconis are characteristically rust-to olivaceousbrown and produce small, brownish conidiophores bearing small numbers of mostly rough-walled conidia. All members of the genus share rhexolytic conidial liberation (Ellis 1971). Several species with more elaborate, branched conidia had been added (Matsushima 1971;, and during the following decades more species, such as O. tshawytschae (Kirilenko and All-Achmed 1977) and O. gamsii (de Hoog 1985), were recognized in addition to the classical species O. anellii, O. constricta and O. humicola. Samerpitak et al. (2014) introduced a new genus, Verruconis, for a group of thermophilic species around Ochroconis gallopava. Almost synchronously with the paper of Samerpitak et al. (2014), Hao et al. (2013) proposed a new species of Scolecobasidium, and being unaware of the doubtful status of the generic type species S. terreum opted for maintenance of Scolecobasidium at the expense of Ochroconis for the entire species group. Given the ambiguity of Scolecobasidium this choice is less appropriate and is corrected in the present paper; some necessary name changes are proposed. Machouart et al. (2014) investigated conserved genes (nuSSU, nuLSU, mtSSU, and RPB2) of the species at hand and found that both Ochroconis and Verruconis belonged to the order Venturiales, family Sympoventuriaceae. Detailed taxonomy was elaborated by Samerpitak et al. (2014) using nuclear ribosomal (nuSSU, nuLSU, ITS) and partial coding genes (actin: ACT1, β-tubulin: BT2, translation elongation factor 1-α: TEF1). In this revision, 13 species were recognized in Ochroconis and three in Verruconis. The latter genus comprised the human opportunistic neurotroph, Verruconis gallopava and two related species. Remarkably large phylogenetic distances were noted among and within the species of Ochroconis and Verruconis, which indicated the possible existence of additional, presently unrecognized taxa.
Some basic ecological trends can be observed in Ochroconis and Verruconis. Ochroconis species are mesophilic and often oligotrophic. Ochroconis anellii was isolated from a stalactite (Graniti 1962), O. lascauxensis from ancient drawings on a cave wall, and O. anomala from sediment in the same cave, i.e. the Lascaux Cave in France (Martin-Sanchez et al. 2012). Ochroconis species morphologically similar to O. humicola were reported from wet areas in the domestic environment, such as bathrooms. Several ochroconis-like strains were isolated from soil or water (Lian and de Hoog 2010). Verruconis species differ by being thermophilic and have repeatedly been recovered from hot water and from brains of warm-blooded animals including humans.
The present paper presents a taxonomic study of eleven ochroconis-like strains from various sources. Phenotypic and genotypic characters of the strains were evaluated in view of refined species delimitations and a novel Ochroconis species is introduced.

Phenotypic studies
Eleven strains of an unknown Ochroconis species (Table 1) were cultured on oatmeal (OA) and malt extract agars (MEA), respectively, and incubated at 24°C for 14 days.
Morphological observations were carried out as described by Samerpitak et al. (2014). To investigate the optimal temperature for growth, all strains were cultured on MEA and incubated for three weeks at temperatures varying from 4 to 40°C with 3°C intervals. Colony diameters were measured after 3, 7, 11, 14, 18, and 21 days.
Sequences were concatenated following Samerpitak et al. (2014). Multi-locus analysis was performed using the Bayesian approach with MRBAYES v. 3.1.2 from the CIPRES Science Gateway (Miller et al. 2010). Two parallel runs of 10,000,000 generations were done with a sampling frequency of 1,000 trees. A burnin tree sample of 10 % was discarded. Maximum likelihood (ML) using Tamura-Nei and GTR+I as the best model with 1,000 bootstrap replicates, and maximum parsimony (MP) with 1,000 bootstrap replicates were also carried out in MEGA5 (Tamura et al. 2011). Presented tree was obtained with Bayesian approach. Tree reconstruction, visualization, and editing were done with TREEVIEW v. 1.6.6, FIGTREE v. 1.1.2 and MEGA5.

Results
The overall mean distance of ITS sequences among 15 Ochroconis species and the unidentified Ochroconis was 13.4 %. ITS sequences of the unidentified Ochroconis were 687 bp in length including 317 bp for ITS1, 157 bp for 5.8S, and 213 bp for ITS2. All 11 strains of the unknown species were 99.8−100 % identical in rDNA genes (data not shown). Judging from ITS phylogeny of all Ochroconis and Verruconis species sequenced to date  The dataset of six concatenated sequences contained 4,913 characters of which 1,271 were parsimony-informative. A multi-locus analysis, applying all algorithms mentioned above confirmed that the investigated strains formed a separate, strongly supported cluster at 1/100 %/100 % (BI/ML/MP) and that the 1 strains represented a hitherto undescribed member of the O. tshawytschae species group with smallest distances to O. anellii and O. lascauxensis (Fig. 1). Description based on CBS 119644 at 24°C after 2 weeks in darkness.
On OA, colonies 25-30 mm in diameter after 2 weeks, moderately expanding, smooth, dry, flat, greyish brown to dark brown. On MEA, colonies attaining 18-20 mm in diameter after 2 weeks, flat, velvety to floccose with some shallow radial fissures, brownish olive green to dark olive green with a 0.5 mm submerged colony margin, reverse as dark olive, green and brown in the central portion, on MEA, hyphae subhyaline to pale brown, smooth-and thickwalled; 1.4-3.0 μm wide, coiled hyphae usually present. Conidiophores mostly arising laterally from vegetative hyphae, erect or flexuous, cylindrical with 1-2 septa,  (Table 2) were included in a phylogenetic comparison. Ochroconis atlantica ATCC 32684 (Wellman 1975) was not available for study, but with its muriform conidia and its different habitat, this species was judged to be significantly different from O. globalis. Morphologically, O. globalis resembles O. constricta, but conidia of O. globalis are larger and more variable in shape, i.e. ellipsoidal to cylindrical, 2-to 4-celled, with less rough walls at young age, and with longer, cylindrical conidiophores. Conidia of O. globalis are also similar to those of O. musae and O. cordanae, but have a constriction at the median septum and have more prominently verrucose walls at later stages of development.  Note: Full descriptions of this species were given by Hao et al. (2013) and by Samerpitak et al. (2014) under its synonymous name O. mirabilis. The species was classified in Scolecobasidium (Hao et al. 2013) without consideration of the doubtful identity of the genus Scolecobasidium. The large phylogenetic distances among Ochroconis and Verruconis species were neglected. Samerpitak et al. (2014) described O. mirabilis for the most common Ochroconis species, which mostly had been reported under the name of the phenotypically similar species O. constricta; the authors were unaware of the almost synchronous description of the same species as S. musae by Hao et al. (2013). The ITS sequence of strain GS-2012 (=0HLHKBJ-22) had been deposited at GenBank as Scolecobasidum sp. (JQ364738) and was included by Samerpitak et al. (2014)  from the cuticle of gynes of an ant species, Atta capiguara, and shared a similar habitat with some strains of O. cordanae and O. sexualis . CBS 131956 is the only strain from a tropical climate, while remaining O. globalis strains originated from temperate zones. The tropical ant-associated strain grew optimally at 27 to 30°C. Investigations on fungal diversity and ecology associated with social insects are currently in progress. Given the high diversity of sources of isolation of O. globalis and the scant information on their ecology other than yield in culture, the actual natural habitat of these oligotrophic fungi remains enigmatic.