Three New Species of the Genus Ochroconis

Ochroconis bacilliformis, O. phaeophora and O. robusta, three novel species of the melanized genus Ochroconis (Sympoventuriaceae, Venturiales), are described, illustrated and distinguished phenotypically and molecularly from previously described species in the genus Ochroconis. Their potential significance for infection of cold-blooded vertebrates is discussed.


Introduction
The genus Ochroconis, typified by O. constricta, was morphologically segregated from a genus with lobed conidia, Scolecobasidium by de Hoog & von Arx [1], for melanized fungi with sympodial conidiogenesis and septate, ellipsoidal conidia which were liberated rhexolytically. Machouart et al. [2] elucidated the general phylogenetic position of the genus by investigating highly conserved genes (nuSSU, nuLSU, mtSSU and RPB2) and found that Ochroconis and its relatives belonged to the order Venturiales, family Sympoventuriaceae. Samerpitak et al. [3] studied species diversity by analyzing more variable genes in addition to the partial ribosomal operon, i.e., the partial coding genes, actin (ACT1), b-tubulin (BT2) and translation elongation factor 1-a (TEF1) and recognized thirteen species in Ochroconis. The authors also introduced the genus Verruconis for a group of thermophilic species such as Ochroconis gallopava, an opportunistic neurotropic pathogen, and its sibling, O. calidifluminalis [4]. The taxonomic status of Scolecobasidium was considered to be doubtful because of ambiguity of the type material, S. terreum [3]. The strict morphological parameters to demarcate the genera were abandoned at the expense of a phylogenetic approach. Species with forked conidia similar to S. terreum were added to Ochroconis on phylogenetic grounds as members of Sympoventuriaceae. Some of the species that were morphologically classified in Scolecobasidium are currently not available for sequencing, and their classification remains unresolved.
Relatively large phylogenetic distances were noted among and within species of Ochroconis and Verruconis [3], which indicated the possible existence of additional, presently unrecognized taxa. Giraldo et al. [5] reported three new species, O. icarus, O. ramosa and O. olivacea, during an investigation of Ochroconis and Verruconis strains originating from clinical samples. The first two are sister species of O. minima, while the latter is close to but significantly different from O. verrucosa. Samerpitak et al. [6] proposed a further new species, O. globalis, which was phylogenetically close to but morphologically different from O. tshawytschae. Crous et al. [7] described a new species, O. macozamiae, as a sister species of O. gamsii which has similar morphology but different genotype.
This article presents a taxonomic study of three Ochroconis strains from various sources. Phenotypic and genotypic characters of the strains were evaluated to define species concepts and delimitations, and three novel Ochroconis species are proposed.

Phenotypic Studies
Three  [8][9][10], three markers, viz. nuSSU, nuLSU and ITS, were amplified by PCR using primers and conditions as shown in Table 2, and sequencing was performed by Big Dye Terminator Cycle Sequencing RR mix protocol (Applied Biosystems). BIONUMERICS v. 4.61 (Applied Maths, Sint-Martens-Latem, Belgium) was employed for first iterative alignments. Sequences of nuLSU, nuSSU and ITS were aligned with the web-based program MUSCLE (www.ebi.ac.uk/Tools/msa/muscle). Sequence alignments were adjusted using BIOEDIT v. 7.0.5.2. Guanine-cytosine content (G?C %) of ITS was calculated using BIOEDIT v. 7.0.5.2. Sequences were concatenated [3]. Nuclear ribosomal gene analyses were performed in MEGA6 [18] using maximum likelihood (ML) with Tamura-Nei and GTR?I as the best model with 1000 bootstrap replicates, and maximum parsimony (MP) with 1000 bootstrap replicates was also carried out. These phylogenetic analyses were supported by the Bayesian approach with MRBAYES v. 3.1.2 from the CIPRES Science Gateway [19]. Two parallel runs of 5,000,000 generations were done with a sampling frequency of 1000 trees. A burnin tree sample of 10 % was discarded. The presented tree was obtained with ML approach. Tree reconstruction, visualization and editing were done with TREEVIEW v. 1.6.6, FIGTREE v. 1.1.2 and MEGA6.

Results
The ITS characters of the three investigated strains deviated significantly from those of known species, both in length and in G? With analyses of partial genes and spacers of the nuclear ribosomal operon, the alignment contained 4103 characters (1656 bp from the SSU, 1040 bp from the ITS, 1407 bp from the LSU) of which 788 were parsimony informative. Applying the algorithms mentioned above, the three investigated strains cluster with Ochroconis spp. in a well-supported clade (95 %ML/99 %MP/1PP) but in different branches ( Fig. 1  On OA, colonies 23-24 mm in diameter, moderately expanding, smooth, dry, flat, dark brown; reverse dark brown. On MEA, colonies 17-18 mm in diameter, flat, velvety to floccose, brown; reverse dark brown. Hyphae subhyaline to pale brown, smooth-and thin-walled, 1-2 lm wide; coiled and anastomosing hyphae usually present. Conidiophores mostly arising laterally from vegetative hyphae, conical, flaskshaped to cylindrical, 3-20 9 2-3 lm, pale brown, smooth-walled, with sympodially proliferating conidiogenous cells each bearing one or a few denticles in the apical region; denticles cylindrical, subhyaline, up to 1 lm long. Conidia cylindrical, rounded at both ends or slightly apiculate at the base, 8.8-12.8 9 2.0-2.4 lm, smooth-walled, pale brown, two-celled, becoming verrucose at maturity. Frills remaining visible on denticle and on conidial base. Cardinal temperatures on MEA: minimum at 4°C, optimum at 18-24°C, maximum at 30°C. Note The flask-shaped, rather short conical or cylindrical and pale brown conidiophores of Ochroconis bacilliformis are similar to those of O. constricta and O. minima. Its specific character is the conidial shape which is different to Y-to T-shaped or lobate conidia of O. minima, but somewhat similar to that observed in O. constricta, but conidia of O. constricta are shorter but wider (6-12 9 2.5-4.0 lm) and have constriction at the septum [42].  On OA, colonies 40-41 mm in diameter, moderately expanding, smooth, dry, flat, brown to dark brown; reverse dark brown. On MEA, colonies 37-40 mm in diameter, velvety, brown; reverse dark brown with a dark pinkish pigment exuding into the agar. Hyphae subhyaline to pale brown, smooth-to rough-and thick-walled, 1.2-2.8 lm wide; coiled and anastomosing hyphae usually present. Conidiophores mostly arising laterally from vegetative hyphae, erect or flexuous, short-to long-cylindrical with 1-3 (-5) septa, 6-68 9 2.0-2.8 lm, pale brown to dark brown, initially smooth-and thin-walled, rough-and thick-walled at maturity, with sympodially proliferating conidiogenous cells bearing one or more denticles in the apical region; denticles cylindrical, subhyaline, up to 2 lm long. Conidia cylindrical to slightly fusiform, sometimes constricted at the septum, 7.2-11.6 9 2.0-3.6 lm, smooth-walled, pale brown, two-celled, becoming verrucose at maturity. Frills remaining visible on denticle and on conidial base. Cardinal temperatures on MEA: minimum at 4°C, optimum at 24°C, maximum at 30°C.
Note This species has conidial morphology close to that of O. crassihumicola (CBS 120700), but the conidia of the latter species are longer and wider (7.5-13.0 9 4.2-5.5 lm) and mostly rounded at both ends [34]. Etymology: named after the well-differentiated conidiophores. On OA, colonies 23-24 mm in diameter, dry, flat, dark brown, velvety at the center, smooth at the margin; reverse dark brown. On MEA, colonies 19-21 mm in diameter, velvety with regular shallow radial fissures at the margin, brown; reverse dark brown. Hyphae subhyaline to pale brown, smooth-walled, 1-2 lm wide; coiled and anastomosing hyphae usually present. Conidiophores arising laterally and terminally from vegetative hyphae, erect or flexuous, cylindrical with 1-3 septa, 10-60 9 1.5-2.0 lm, dark brown, smoothand thick-walled, with sympodially proliferating conidiogenous cells bearing one or more denticles in the apical region; denticles cylindrical, subhyaline, up to 1 lm long. Conidia ellipsoidal to cylindrical, rounded at both ends or slightly apiculated at base,     [3] and O. anomala produces short chains of conidia [30].

Discussion
Ribosomal gene analyses were sufficient to recognize the three strains, CBS 112.97, CBS 206.96 and CBS 100442, as separate, undescribed species showing strong support of their taxonomic positions within the genus Ochroconis. Phylogenetic analyses of partial coding genes, actin (ACT1), b-tubulin (BT2) and translation elongation factor 1-a (TEF1) were difficult to apply due to high degrees of variability which interfered with alignment over the entire genus. The large phylogenetic distances between species strongly support their identities as novel taxa in the Sympoventuriaceae and suggest that a possible existence of a large number of as yet unrecognized taxa would be waiting to be discovered in unexplored habitats. With BLAST searches in GenBank, no sequence identical to any of the investigated genes of the new species was encountered. Consequently, although each of the new species is represented by a single strain, their novelty is unambiguous. However, given the morphological variations found upon different culture conditions, identification of these species on the basis of morphology alone remains difficult. Members of the genus have comparable phenotypes despite significant phylogenetic distance between taxa. Nuclear ribosomal sequences of ITS and LSU and even the conserved SSU gene are all usable as diagnostic tools for species identification in Ochroconis. It is remarkable that in the Sympoventuriaceae (including the genera Ochroconis, Verruconis, Veronaeopsis and Sympoventuria), all ribosomal genes, viz. ITS, LSU and the highly conserved SSU, are suitable for identification [3]. This is exceptional in ascomycetous fungi which often share identical ribosomal operons between species, e.g., in Penicillium [43] and Fusarium [44]. ITS sequences with BLAST searches in GenBank directly led to presumptive identification of Ochroconis and Verruconis species. In addition, specific characters in length and G?C % Fig. 3 Ochroconis phaeophora, CBS 206.96. a b Colony on MEA and OA after 3 weeks, respectively. c-k. Conidiophores with conidia. l, m Anastomosing hyphae and hyphal coil. Scale bar 10 lm among the pathogenic species (Table 3) provide potentiality to ITS sequences to be developed as tool for definite diagnosis in the medical laboratory.
During the last decade, several reports of new species members in the genus Scolecobasidiumwhich was considered as of doubtful identification [3]-have appeared such as S. chinense [45], S. qinghaiense [45], S. microsporum [46], S. pallescens [47], S. tuberculatum [48] and S. rostricola [49]. These species all share similar morphology with existing Ochroconis species. Their two-celled conidia may resemble O. robusta and O. phaeophora, but seem to be different in the details and other phenotypic characters. None of them has conidia which are apiculate at both ends, Scolecobasidium chinense has branched conidiophores, S. microsporum has conidia with fine spines at the surface, S. pallescens has caespitose conidiophores, S. qinghaiense has conidia with constricted septa, S. tuberculatum has oblong and tuberculate conidia, and S. rostricola is a fungicolous hyperparasite. The DNA sequences of these Scolecobasidium species are not available in GenBank, and no authentic material was available for study.
Given the limited number of strains per species available, ecological hypotheses are formulated with difficulty. Two species, O. robusta (CBS 112.97) and O. phaeophora (CBS 206.96), are likely to represent plant-associated saprobes, the former having been isolated from leaf litter from Spain and the latter from the leaf under conditions of a tropical rain forest in Papua New Guinea. Ochroconis bacilliformis (CBS 100442) colonized a metal surface submerged in a municipal drinking water network and thus seems to be an oligotrophic saprobe similar to the domestic indoor wet cell colonizers, O. musae, O. constricta, O. globalis [3,6,[23][24][25], to the species occurring on cold moist rock, O. anellii [31], O. anomala and O. lascauxensis [30], and also to the waterborne species, Fig. 4 Ochroconis robusta, CBS 112.97. a, b Colony on MEA and OA after 3 weeks, respectively. c-k Conidiophores with conidia. l, m Anastomosing hyphae and hyphal coil. Scale bar 10 lm O. tshawytschae [3]. Waterborne Ochroconis species have repeatedly been reported as opportunistic agents of disease in cold-blooded animals that complete at least a part of their life cycle in water [22,28,29,50] and may cause superficial infections in humans with impaired blood circulation [5,29]. Given the identification problems when using phenotypic characteristics, the species introduced in this paper may be likely to have similar abilities.