Alignment and phylogenetic reconstruction

For genealogical concordance analysis, four genes ITS, TEF1, BT2 and ACT1 were first analysed separately. Alignment was performed automatically and adjusted iteratively by hand with BioNumerics v. 4.61 (Applied Maths, Kortrijk, Belgium). Topological conflicts were evaluated visually and by using the partition homogeneity test implemented in PAUP v. 4.0b10. ITS and multilocus trees were constructed with maximum likelihood and with 100 bootstrap replicates using RAxML v. 7.2.3 (Stamatakis et al. 2008) as implemented on the Cipres Portal v. 1.10, and edited with MEGA4 software (Tamura et al. 2007).

A phylogenetic approach was used to investigate relationships between 147 waterborne strains of Exophiala and related species (Table 1), with Exophiala castelanii and E. mesophila as outgroups in the SSU tree. For species circumscribed by genealogical concordance, a single strain (usually the ex-type) was selected for sequencing conserved genes. Sequences were compared in a database in BioNumerics containing all described members of Chaetothyriales. The database was regularly updated with recent GenBank and AFTOL submissions. SSU (1 100 comparable sites) trees were generated with the Parsimony option of PAUP after removal of introns.

Taxonomy

Exophiala J.W. Carmich., Sabouraudia 5: 122. 1966. — MycoBank MB8233

The genus Exophiala was described as an anamorph taxon by Carmichael (1966), with E. salmonis as its type species. The genus represents an anamorphic element of the order Chaetothyriales. Note that the type species has among its synonyms the name Aureobasidium salmonis. Aureobasidium, however, is a member of the order Dothideales (Schoch et al. 2009). Teleomorphic relations of Exophiala lie in the genus Capronia, typified by Capronia sexdecimspora. Exophiala is the main genus of black yeasts found as opportunists of vertebrates. It is characterised by annellidic conidiogenesis. Some cultures are entirely yeast-like (synanamorph Phaeococcomyces), or form phialidic collarettes (synanamorph Phialophora), sympodial conidiophores (synanamorph Rhinocladiella) or dry conidial chains (synanamorph Cladophialophora). Chlamydospores or sclerotial bodies may also be formed, occasionally leading to entirely meristematic mutants (synanamorph Sarcinomyces). Several of these morphologies are represented in species of the waterborne salmonis-clade (B).

Thermophilic species of Exophiala have been described from systemic infections in humans, e.g. E. dermatitidis and E. spinifera. In contrast, numerous species of the salmonis-clade (B) are mesophilic or psychrotolerant, and are regularly found as opportunists on cold-blooded vertebrates. Many such cases have been reported in the literature, and these cases have been attributed to a diversity of Exophiala species. However, the identification of etiologic agents in many of these cases must be questioned, since the morphological characters used have proven not to be reliable for species identification. This unreliability is attributable to variable morphologies within species and to overlapping characters among them. Therefore in the present paper we will only reference the case reports where voucher strains have been preserved and sequenced.

Exophiala alcalophila Goto & Sugiy., in Goto et al., Trans. Mycol. Soc. Japan 22: 430. 1981. — MycoBank MB110200

= Phaeococcomyces alcalophilus Goto & Sugiy., Trans. Mycol. Soc. Japan 22: 432. 1981.

Description of CBS 520.82 after 2 wk incubation on MEA, 24 °C.

Colonies restricted, appearing slimy, smooth, soft, jet-black, convex with sharp margin. Initial growth with budding cells, later (after 1 mo) becoming slightly floccose at the centre and remaining slimy at the margin. Reverse brownish black; a rust brown pigment exuded into the agar. Budding cells abundant, smooth- and thin-walled, 1-celled, (sub)spherical to broadly ellipsoidal, 4–8 × 3–6 μm. Germinating cells present, (sub)spherical, 7–9 μm diam. Aerial hyphae smooth-walled, irregularly branched, 1.5–3.0 μm wide. Conidiogenous cells arising from undifferentiated hyphae, terminal or intercalary, with short annellated zones, mostly without discernible annellations; occasionally conidia produced apically in more or less sympodial order. Conidia hyaline, thin- and smooth-walled, 1-celled, spherical, ellipsoidal to slightly reniform, occasionally with truncate base, 3–7 × 2–5 μm, aggregated in slimy heads.

Cardinal temperatures — Minimum 4–9 °C, optimum 24–27 °C, maximum 36–40 °C.

Specimens examined. Japan, Hirose, Wako-shi, Saitama pref., 23 Apr. 1978, K. Horikoshi, from soil, holotype specimen of Exophiala alcalophila, CBS H-19960, culture ex-type IAM 12519 = CBS 520.82; Hirose, Wako-shi, Saitama pref., 23 Apr. 1978, K. Horikoshi, from soil, ex-type culture of Phaeococcomyces alcalophilus, IAM 12520 = CBS 521.82.

Additional material examined. Table 1.

Notes — The species was originally isolated with two morphotypes: one hyphal, and the other purely consisting of budding cells, for which reason the fungus was introduced under two generic names, Exophiala and Phaeococcomyces. This underlines the strongly dimorphic character of the species. Under the growth conditions applied in the present study, the yeast-like phase was pronounced during the first week, while hyphae later became more prevalent. The preponderance of either morphotype is unstable. Yamada et al. (1989) demonstrated that while E. salmonis had a coenzyme Q 10(H₂) ubiquinone system, E. alcalophila had Co-Q 10.

The original cultures were derived from soil on a minimal medium at a pH of 10.4 (Goto et al. 1981). Another strain (GHP R-18; Table 1) came from the soap container of a laundry machine. Nishimura et al. (1987) isolated the fungus repeatedly from bath water. Strain CBS 122256 was isolated from mildly symptomatic human skin in Denmark, but without precise clinical information. Lian & de Hoog (2010) recently noticed a possible link between cutaneous infection and the presence of potentially causal black yeast-like fungi in low-nutrient indoor water systems rich in soap, such as bathrooms. The authors repeatedly isolated several black yeast-like species from bathrooms, which until then had only been known from human skin and nails. They suggested that maceration of skin may provide a portal of entry during hygienic procedures for these fungi having moderate invasive capacity. Exophiala alcalophila apparently belongs to the same ecological group, although at low virulence.

Exophiala angulospora Iwatsu, Udagawa & Takase, Mycotaxon 4: 322. 1991. — MycoBank MB355245

Teleomorph. Capronia coronata Samuels, Trans. Brit. Mycol. Soc. 88: 65. 1967.

Description of CBS 482.92 after 2 wk incubation on MEA, 24 °C.

Colonies restricted, centrally mucous, velvety towards the outside, greyish green to olivaceous black. Germinating cells present, 6–10 × 2.4–4.0 μm. Hyphae pale olivaceous, smooth-walled, 1.5–3.0 μm wide. Budding cells present. Conidiogenous cells intercalary, lateral and terminal and then 1-celled, flask-shaped, 6–16 × 2.5–3.0 μm; conidia produced from a single short annellated zone per cell. Conidia aggregating in slimy heads, 1-celled, smooth- and thin-walled, subhyaline or pale olivaceous, mostly more or less triangular with rounded ends, 2.5–4.0 × 2–3 μm.

Cardinal temperatures — Minimum ≤ 4 °C, optimum 24–27 °C, maximum 30–33 °C. No growth at 37 °C.

Specimens examined. Japan, Yokohama-shi, 18 Apr. 1989, K. Arai, from drinking well water, ex-type culture anamorph, CBS 482.92 = NHL 3101. – New Zealand, Westland County, Nemona State Forest, 5 May 1983, G.J. Samuels, from decorticated wood, ex-type culture teleomorph, CBS 617.96 = ATCC 56201 = PDD 35308.

Additional material examined. Table 1.

Notes — The species was originally repeatedly isolated from cold, low-nutrient drinking water (Iwatsu et al. 1991). The majority of strains sequenced originated from cold water, such as drinking water, aquaria and fish nurseries (Table 1). A teleomorph, Capronia coronata, originating from decorticated wood, was found to be identical in ITS sequence data. Nyaoke et al. (2009) noted a disseminated infection by CBS 119911 in a seawater-dependent weedy sea dragon (Phyllopteryx taeniolatus) in the New England Aquarium in Boston, USA, and also repeatedly in the marine lumpfish (Cyclopterus lumpus). Strain CBS 121503 was isolated from living freshwater fish Scenodus leucichtys in a nursery in Stravropol Kraj near Kislovodsk, southern Russia (V.A. Mel’nik, pers. comm.). Human isolates such as CBS 441.92 and CBS 122264 originated from skin and nail samples, but no proof of infection is available (Saunte et al. 2011). The species has been isolated once from hydrocarbon-polluted soil (Table 1).

In conclusion it appears that this fungus inhabits cold waters worldwide, where it has an invasive potential with fatal dissemination in cold-blooded vertebrates. Human infections have not been proven and may have concerned colonization only. This pathology is likely to be determined by its relatively low maximum growth temperature.

Exophiala aquamarina de Hoog, Vicente, Najafzadeh, Harrak, Badali, Seyedmousavi & Nyaoke, sp. nov. — MycoBank MB515716; Fig. 4, ​,55

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Fig. 4

Exophiala aquamarina, CBS 119918. a. Colony on MEA; b. colony on PDA; c, d. spirally twisted hyphae; e–n. conidial apparatus with conidia; f–j. annellidic conidiogeneses with sympodial conidiophores; o. anastomosis between discrete cells; p–s. conidia; q. budding cells. — Scale bars = 10 μm.

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Fig. 5

Exophiala aquamarina, CBS 119918. Conidial apparatus and conidia. — Scale bar = 10 μm.

Coloniae in agaro maltoso dicto 24 °C lente crescentes, velutinae, griseae; reversum olivaceo-nigrum. In agaro PDA pigmentum brunneum absens. Hyphae leves, dilute olivaeo-brunneae, intervallis regularibus septatae, nonnumquam spiralis. Conidiophora vix distinguenda, ramose vel simplicia, terminalia vel intercalaria; conidium annellidorum vel sympodiorum producens. Conidia late ellipsoidea, levia, 6.7–19.2 × 4.0–4.8 μm. Temperatura maxima crescentiae 36 °C. Teleomorphe ignota.

Etymology. Name refers to sea water, the environment of most of the hosts of this species.

Description of CBS 119918 after 2 wk incubation on MEA, 27 °C.

Colonies restricted, olivaceous black, velvety with aerial mycelium at the centre. Reverse olivaceous black. No diffusible pigment produced. Conidiogenous cells flask-shaped, with short annellated zones, sometimes with sympodial conidiogenesis. Spirally twisted hyphae present. Conidia ellipsoidal to cylindrical, 6.7–19.2 × 4.0–4.8 μm. Yeast cells rarely present.

Cardinal temperatures — Minimum ≤ 4 °C, optimum 24–30 °C, maximum 33–36 °C. No growth at 37 °C.

Specimen examined. USA, Boston, New England Aquarium, S. Frasca, from skin of leafy sea dragon (Phycodures eques), holotype CBS H-19950, ex-type culture CBS 119918 = UTHSC 00-1181 = dH 16401.

Additional material examined. Table 1.

Notes — The species repeatedly caused disseminated infections in several species of fish in the New England Aquarium in Boston, Mass., USA, and in the Adventure Aquarium in Camden, N.J., USA (Nyaoke et al. 2009), particularly in leafy sea dragon (Phycodurus eques) and in weedy sea dragon (Phyllopteryx taebiolatus), but also in winter flounder (Pseudopleuronectes americanus) and little tunnyfish (Euthynnus alletteratus). Necrotic skin lesions were observed with mild inflammatory response, with invasion of blood vessels, and infection of skull and bone, but no brain involvement. Massive amounts of hyphae were observed in tissue. Infections took place over a 5-year period (Nyaoke et al. 2009). Exophiala aquamarina is so far restricted to fish, but is not host specific.

Exophiala brunnea Papendorf, Trans. Brit. Mycol. Soc. 52: 487. 1969. — MycoBank MB330806

Description of CBS 587.66 after 2 wk incubation on MEA, 24 °C.

Colonies developing slowly, with mouse-grey aerial mycelium at the centre; peripheral area depressed, dark olivaceous; reverse greenish black. Hyphae poorly branched, smooth-walled, pale olive-brown, 1–3 μm diam. Budding cells absent. Conidiogenous cells lateral, slightly differentiated from vegetative hyphae, frequently with 1–2 septa, simple or branched, variable in shape, flask-shaped, ovoidal to elongate, pale brown, 8–350 μm long. Annellated zones inconspicuous or occasionally finely fimbriate, 6–20 × 2–4 μm, often inserted on intercalary cells of hyphae and conidiophores. Conidia forming a coherent mass, broadly ellipsoidal or ovoidal, with a broad truncate hilum, continuous or occasionally with a median septum and then slightly constricted, smooth-walled, pale brown, 4.5–10 × 2–3 μm.

Cardinal temperatures — Minimum 4–9 °C, optimum 21–24 °C, maximum 30–33 °C. No growth at 37 °C.

Specimen examined. South Africa, Potschefstroom, from leaf litter of Acacia karroo, specimens CBS H-12618, CBS H-19966, ex-type culture CBS 587.66.

Notes — The species is known from a single strain isolated from topsoil (leaf litter) of an Acacia karroo community (Leguminosae-Mimosoideae). The species was synonymised with Exophiala salmonis on morphological grounds (de Hoog 1977), but was later shown to be distinct based on molecular data.

Exophiala calicioides (Fr.) G. Okada & Seifert, Stud. Mycol. 45: 184. 2000. — MycoBank MB464794

Synonyms listed in Okada et al. (2000).

Notes — This species has fully been described and illustrated by Okada et al. (1998, 2000). It is morphologically remarkable by being synnematous. The fungus resides in rotten wood, eventually in association with bark beetles.

Exophiala cancerae de Hoog, Vicente, Najafzadeh, Harrak, Badali, Seyedmousavi & Boeger, sp. nov. — MycoBank MB515720; Fig. 6, ​,77

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Fig. 6

Exophiala cancerae, CBS 120420. a. Colony on MEA; b. colony on PDA; c, d. spirally twisted hyphae; e, f. short, erect, cylindrical, multi-celled conidiophores; h, i. apical and intercalary chlamydospores; j. budding cells; k, l. intercalary conidiogenous cells; m. hyphae and conidia with anastomoses; n–p. conidia. — Scale bars = 10 μm.

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Fig. 7

Exophiala cancerae, CBS 120420. Conidial apparatus, conidia and torulose hyphae. — Scale bar = 10 μm.

Coloniae in agaro maltoso dicto 25 °C lente crescentes, primum leves, deinde velutinae, griseo-olivaceae; reversum olivaceo-nigrum. In agaro PDA pigmentum brunneum absens. Hyphae leves, dilute olivaeo-brunneae, intervallis regularibus septatae. Cellulae zymoideum quasi absens. Conidiophora vix distinguenda, ramose vel simplicia, terminalia vel intercalaria. Annelloconidia nonnumquam septata, late ellipsoidea vel cylindrea, levia, 4.9–8.0 × 2.7–4.8 μm. Temperatura maxima crescentiae 33 °C. Teleomorphe ignota.

Etymology. Named after the crab, an arthropod host of this species.

Description of CBS 120420 after 2 wk incubation on MEA, 24 °C.

Colonies moderately expanding, circular, initially (on day 3) flat, olivaceous black, slimy with velvety, olivaceous grey centre and flat margin, later (on day 14) becoming velvety, dark olivaceous grey. Reverse olivaceous black, without diffusible pigment. Yeast cells nearly absent. Conidiophores short, erect, brown, cylindrical, multi-celled, poorly differentiated. Conidia 0–1-septate, subhyaline to pale brown, obovoidal to cylindrical, 4.9–8.0 × 2.7–4.8 μm.

Cardinal temperatures — Minimum ≤ 4 °C, optimum 24–27 °C, maximum 30–33 °C. No growth at 37 °C.

Specimen examined. Brazil, Pernambuco State, Goiana City, from diseased Mangrove crab (Ucides cordatus), W. Boeger, holotype CBS-H 20382, ex-type culture CBS 120420 = dH 17409 = Boeger HF16/8.

Additional material examined. Table 1.

Notes — The ex-type strain was isolated from direct culture from tissue of moribund mangrove crabs (Ucides cordatus, Brachyura: Ocypodidae) with Lethargic Crab Disease (LCD). Since 1997 this systemic disease has caused extensive epizootic mortality of crabs along the Brazilian coast (Boeger et al. 2005). The histopathology of crabs in diverse stages of development of the disease shows that the most commonly affected tissues are the epidermis, connective tissues, heart and hepatopancreas. The fungus disseminates hematogenously. Despite the large scale of outbreak, locally causing 50 % of crabs to become diseased, we were unable to isolate E. cancerae from the environment (Boeger et al. 2007). The worldwide occurrence of the species (Table 1) suggests that it may have been present in Brazil prior to the beginning of the epizootic in 1997. Changes in host or in environmental conditions, rather than emergence of a virulent fungal genotype, are thus likely. This is underlined by the fact that also a second black yeast-like fungus was involved in LCD (Boeger et al. 2007). This was a Cladophialophora species close to C. devriesii, which was first described from a fatal disseminated infection in a human at the Caribbean Grand Cayman Island (Gonzalez et al. 1984).

Strain CBS 119920 was derived from the liver of a green toad (Pseudepidalea viridis) in Israel, which was euthanised with clinical signs of systemic mycosis (A. Cunningham, pers. comm.). Additional strains were isolated on separate occasions from water in Germany (Table 1). The species was once isolated from clean water from a CIP (Clean-in-Place) tank (CBS 11749) in the Netherlands and once from fruit drink (CBS 115142) in Australia.

In humans, a skin infection was observed in a patient with diabetes in Germany (GHP 2419; G. Haase, pers. comm.); further human cases concern mild skin and nail infections. Despite its maximum growth temperature of 33 °C, the species possesses intrinsic virulence factors. These may be expressed particularly in external tissues of the extremities patients with reduced blood circulation, e.g. to underlying diabetes.

GenBank contains a number of accessions under the name Exophiala salmonis, which are now reidentified as E. cancerae: a clinical strain in the USA (AY213652; Rakeman et al. 2005), a strain from a bathroom in Japan (AB 456581; Hamada & Abe 2009) and an isolate from a healthy stem of corn in Australia (AM176667, Molnar & Prillinger unpubl. data).

Exophiala equina (Pollacci) de Hoog, Vicente, Najafzadeh, Harrak, Badali & Seyedmousavi, comb. & stat. nov. — MycoBank MB515717; Fig. 8, ​,99

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Fig. 8

Exophiala equina, CBS 121504. Conidial apparatus and conidia. — Scale bar = 10 μm.

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Fig. 9

Exophiala equina, CBS 119.23. a. Colony on MEA; b. colony on PDA; c. spirally twisted hyphae; d. tolurose hyphae; e, g. conidiophore with single conidiogenous cell; f. conidiogenous subcylindrical cells flask shaped with ellipsoidal conidia; h. budding cell; i, j. chlamydospore; k, l. ellipsoidal conidia. — Scale bars = 10 μm.

Basionym. Haplographium debellae-marengoi Pollacci var. equinum Pollacci, Revta Biol. 5: 370. 1923.

= Exophiala tremulae W. Wang, Fungal Planet 70, Persoonia 26: 113. 2011.

Description of CBS 119.23 after 2 wk incubation on MEA, 27 °C.

Colonies restricted, circular, initially (on day 5) flat, olivaceous black, slimy with velvety, olivaceous grey centre and flat margin, later (on day 15) becoming umbonate, felty, greyish black, with velvety, grey centre. Reverse greyish black. No diffusible pigment produced. Yeast cells, when present, consisting of subspherical cells producing conidia. Conidiogenous cells flask-shaped, intercalary or terminal. Conidia ellipsoidal, 2.4–3.3 × 4.8–5.2 μm, with discernible scars. Chlamydospores ellipsoidal, up to 10 μm long and 5 μm wide; spirally twisted hyphae present.

Cardinal temperatures — Minimum ≤ 4 °C, optimum 24–30 °C, maximum 33–36 °C. No growth at 37 °C.

Specimen examined. Italy, Pavia, Dec. 1923, G. Pollacci, subcutaneous infection of a horse, CBS H-19957, ex-type culture CBS 119.23 = dH 15335.

Additional material examined. Table 1.

Notes — The ex-type strain was isolated as the etiologic agent of a subcutaneous infection of the lower leg of a horse (Pollacci 1923). The majority of remaining strains sequenced of this species, however (Table 1), originated from different kinds of cold water, primarily drinking water but also from the cooling system of a packaging machine, the tubing of a instrument using silica gel and from washings of Tilia roots. One isolate came from a bathroom. Strain CBS 115143 was isolated from bottled spring water destined for human consumption in Australia (Avila de la Calle et al. 2006, Crous et al. 2007), while CBS 109789 came from dialysis tubing. Strain CBS 116009 caused a systemic infection in a Galapagos giant tortoise (Geochelone nigra), reported by Manharth et al. (2005). The tortoise presented with ocular lesions, but upon necropsy a widespread granulomatous inflammation was noted, probably resulting from hematogenous dissemination. Exophiala tremulae (from roots of Populus tremuloides seedings in Canada), is identical based on DNA sequence data, and represents a recent synonym (Crous et al. 2011).

Although the species is unable to grow at 37 °C, some superficial infections in humans were noted, particularly from skin of the extremities. Among the infections was a corneal ulcer and an onychomycosis (Table 1). The case caused by CBS 121504 concerned a 1 yr old child with circular, tinea-like lesions from which the fungus was isolated together with Candida guilliermondii. The lesion was successfully treated with ciclipirox. Exophiala equina was judged to be a secondary invader (J. Brasch, pers. comm.). The species was also noted on the skin of patients with diabetes (G. Haase & P. Mayser, pers. comm.), which had a relatively low body temperature due to impaired circulation. This condition allows invasion by species that are unable to grow at temperatures above 37 °C, particularly when infection takes place on the extremities. Further isolates were recovered from skin flakes, stool and sputum. A transmission route from bathing facilities, as hypothesized for other black yeasts (Lian & de Hoog 2010), seems probable for this species as well. The occurrence of the fungus in bottled water is of concern.

Additional strains sequenced originated from soil (CBS 515.76), from washed roots (CBS 160.89, CBS 159.89) and from a twig of Olea sp. (CBS 121502). Neubert et al. (2006) extracted DNA (GenBank AJ875365) from wetland reed in Germany and encountered the same species. All these environments have relatively low temperatures in common. Human infection seems to be coincidental.

Exophiala halophila de Hoog, Vicente, Najafzadeh, Harrak, Badali & Seyedmousavi, sp. nov. — MycoBank MB515715; Fig. 10, ​,1111

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Fig. 10

Exophiala halophila, CBS 121512. a. Colony on MEA; b. colony on PDA; c–e. conidial apparatus with conidia; f–k. budding cells and conidia; i. tolurose hyphae. — Scale bars = 10 μm.

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Fig. 11

Exophiala halophila, CBS 121512. Conidial apparatus, conidia and anastomosing torulose hyphae. — Scale bar = 10 μm.

Coloniae in agaro maltoso dicto 24 °C lente crescentes, primum leves et zymoideae, deinde elevatae, velutinae, griseae; reversum olivaceo-nigrum. In agaro PDA pigmentum brunneum exudens. Hyphae leves, dilute olivaeo-brunneae, intervallis regularibus septatae; mycelium torulosum proferentes. Conidiophora vix distinguenda, ramose vel simplicia, terminalia vel intercalaria; nonnumquam cellulae gemmantes etiam conidiogenae. Annelloconidia haud septata, late ellipsoidea, levia, 1.9–2.5 × 3.0–5.2 μm. Temperatura maxima crescentiae 33 °C. Teleomorphe ignota.

Etymology. Named after one of the sources of isolation of this species.

Description of CBS 121512 after 2 wk incubation on MEA, 24 °C.

Colonies restricted, compact, circular, olivaceous brown, initially (on day 5) moist and slimy, later (on day 14) becoming brownish grey, velvety at the centre. Reverse olivaceous black. Brown pigment produced on PDA. Yeast cells abundant. Torulose hyphae present. Conidiogenous cells intercalary in undifferentiated hyphae, or discrete, then flask-shaped, lateral or terminal, with short annellated zones. Conidia subhyaline, ellipsoidal to subcylindrical 1.9–2.5 × 3.0–5.2 μm.

Cardinal temperatures — Minimum ≤ 4 °C, optimum 24–27 °C, maximum 30–33 °C. No growth at 37 °C.

Specimen examined. USA, Texas, San Antonio, D.A. Sutton, from human skin, holotype CBS H-19967, ex-type culture CBS 121512 = UTHSC 03-2191 = dH 13757.

Additional material examined. Table 1.

Notes — Only three isolates of this species are available. The ex-type strain was isolated from asymptomatic human skin. The nearest phylogenetic neighbour is E. alcalophila at a minimum distance of 8.4 % (ITS), and thus the novelty of the new species is unambiguous (Fig. 3). The species has been isolated from human skin of the armpit, and from human nails, as well as from salt water. Strains were tolerant of 2.5, 5 and 10 % MgCl₂ and of 2.5 and 5 % NaCl_2.

Exophiala lacus de Hoog, Vicente, Najafzadeh, Harrak, Badali & Seyedmousavi, sp. nov. — MycoBank MB515721; Fig. 12, ​,1313

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Fig. 12

Exophiala lacus, CBS 117497. Hyphae with intercalary conidiogenous cells; anastomoses leading network-like hyphae. — Scale bar = 10 μm.

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Fig. 13

Exophiala lacus, CBS 117497. a. Colony on MEA; b. colony on PDA; c. spirally twisted hyphae; d, e. short, erect, cylindrical, multi-celled conidiophores; f, g. hyphae with conidial heads; h. chlamydospores; i, j. budding cells; k. conidia alongside hyphae; l. conidia. — Scale bars = 10 μm.

Coloniae in agaro maltoso dicto 25 °C lente crescentes, velutinae, griseo-olivaceae; reversum olivaceo-nigrum. In agaro PDA pigmentum brunneum absens. Hyphae leves, dilute olivaeo-brunneae, intervallis regularibus septatae. Mycelium torulosum quasi absens. Conidiophora vix distinguenda, brevia, simplicia, preferentia intercalaria. Annelloconidia nonnumquam septata, late ellipsoidea, levia, 1.8–2.6 × 3.1–9.6 μm. Chlamydosporum praesens, maximum 5.2 × 2.6 μm. Temperatura maxima crescentiae 30 °C. Teleomorphe ignota.

Etymology. Named after the lake environment where the species was isolated.

Description of CBS 117497 after 2 wk incubation on MEA, 24 °C.

Colonies moderately expanding, circular, velvety with olivaceous grey aerial mycelium. Reverse olivaceous black, without diffusible pigment. Yeast cells, when present, consisting of subspherical cells producing conidia. Conidiophores short, erect, cylindrical, multi-celled, poorly differentiated with submerged hyphae with conidial heads. Conidia arising alongside the hyphae; conidia 0–1-septate, ellipsoidal to cylindrical, 1.8–2.6 × 3.0–9.6 μm. Chlamydospores ellipsoidal, up to 5.2 × 2.6 μm; spirally twisted hyphae present.

Cardinal temperatures — Minimum ≤ 4 °C, optimum 21–24 °C, maximum 27–30 °C. No growth at 37 °C.

Specimen examined. The Netherlands, Loosdrecht, M.J. Harrak, from fresh-water lake (1 m depth), holotype CBS-H 20407, ex-type culture CBS 117497 = dH 13711.

Notes — Only a single strain of this species is available. It was isolated from a shallow freshwater lake, the lake bottom consisting of a few metres of plant material. The lake is unpolluted, being fed by seepage water from sandy hills.

Exophiala opportunistica de Hoog, Vicente, Najafzadeh, Harrak, Badali & Seyedmousavi, sp. nov. — MycoBank MB515719; Fig. 14, ​,1515

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Fig. 14

Exophiala opportunistica, CBS 109811. Hyphae with mostly intercalary conidiogenous cells with extended annellated zones, and 1–2-celled conidia. — Scale bar = 10 μm.

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Fig. 15

Exophiala opportunistica, CBS 109811. a. Colony on MEA; b. colony on PDA; c. spirally twisted hyphae; d, e. erect cylindrical multi-celled conidiophores; f–l. yeast cells and conidia; i. torulose hyphae. — Scale bars = 10 μm.

Coloniae in agaro maltoso dicto 25 °C lente crescentes, velutinae, griseo-olivaceae; reversum olivaceo-nigrum. In agaro PDA pigmentum brunneum absens. Hyphae leves, dilute olivaeo-brunneae, dense septatae. Mycelium torulosum quasi absens. Conidiophora vix distinguenda, ramose vel simplicia, terminalia vel intercalaria. Annelloconidia nonnumquam septata, late ellipsoidea, levia, 2.4–2.9 × 1.1–1.2 μm. Temperatura maxima crescentiae 30 °C. Teleomorphe ignota.

Etymology. Named after the apparent ability of the species to grow on human nails.

Description of CBS 109811 after 2 wk incubation on MEA, 17 °C.

Colonies restricted, olivaceous grey, velvety with floccose margin and with grey aerial mycelium at the centre. Reverse olivaceous black, without diffusible pigment. Yeast cells, torulose hyphae and spirally twisted hyphae present. Hyphae rather wide, profusely septate and strongly anastomosing. Conidia arising alongside the hyphae or on broadly ellipsoidal, poorly differentiated conidiophores; conidia (0–1)-septate, (sub)hyaline, obovoidal to ellipsoidal, 2.4–2.9 × 1.1–1.2 μm.

Cardinal temperatures — Minimum ≤ 4 °C, optimum 21–24 °C, maximum 27–30 °C. No growth at 37 °C.

Specimen examined. Germany, Duisburg, from drinking water, E. Göttlich, holotype CBS-H 20383, ex-type culture CBS 109811 = dH 12243 = IWW 720.

Additional material examined. Table 1.

Notes — The original strain was derived from drinking water and also from rhizosphere of Triticum aestivum in Western Australia (CBS 660.76). One strain, CBS 637.69 originated from polyvinyl alcohol. We recently also noted presence of E. opportunistica on human nail and foot lesions in Denmark (Table 1).

Exophiala pisciphila McGinnis & Ajello (as ‘pisciphilus’), Mycologia 66: 518. 1974. — MycoBank MB314043

Teleomorph. Unknown.

Description of CBS 537.73 after 2 wk incubation on MEA, 24 °C.

Colonies moderately expanding, dry, floccose, olivaceous black. Yeast cells absent. Conidiogenous cells flask-shaped, mostly in loose clusters or branched systems, with inconspicuous annellated zones. Conidia 0(–1)-septate, (sub)hyaline, ellipsoidal, 6–8 × 2.5–4.0 μm.

Cardinal temperatures — Minimum ≤ 4–9 °C, optimum 24–30 °C, maximum 30–33 °C. No growth at 37 °C.

Specimen examined. USA, Alabama, privately owned freshwater pond, April 1969, N. Fijan, from systemic mycosis in channel catfish (Ictalurus punctatus), ex-type culture CBS 537.73, dried culture CBS H-7135.

Additional material examined. Table 1.

Notes — This species was one of the first Exophiala species described as causing epizootics in cold-blooded vertebrates (Fijan 1969). Eighty percent of a sample of freshwater channel catfish (Ictalurus punctatus) in a pond had cutaneous ulcers. These were 2–15 mm diam and up to 5 mm deep, without inflamed margins. Numerous nodules were found in visceral organs. Hematogenous dissemination led to hemorrhagic peritonitis with purulent exudates. The isolated fungus killed the fish within 13 days after intraperitoneal inoculation, but no neurotropism was noted.

The species was later reported as an opportunistic invader in the marine coastal smooth dogfish (Mustelus canis) in the New York Aquarium (Gaskins & Cheung 1986), but the identity of this now unavailable strain cannot be confirmed by sequencing. Strains causing an epizootic in the captive marine plaice (Pleuronectes platessa), published as Hormoconis resinae (Strongman et al. 1977) were re-identified as Exophiala pisciphila (de Hoog et al. 2009). The fish were maintained in tanks with a continuous flow of pre-filtered seawater. Lesions were mainly cutaneous. The epizootic was thought to have been promoted by a relative high maintenance temperature of up to 15.2 °C. Several isolates originated from disseminated infections in marine potbelly seahorses (Hippocampus abdominalis; Nyaoke et al. 2009). Additional strains sequenced (Table 1) originated from a swimming pool (CBS 121505) and from a water pipe (CBS 101610). The fungus thus occurs on fish living in freshwater as well as seawater, with a low degree of host specificity. In humans, a skin infection in an immunosuppressed patient from Brazil was reported by Sughayer et al. (1991), without sequence confirmation. We also recorded an isolate from the nail of a human patient in Germany (P. Mayser, unpubl. data; Table 1). Yamada et al. (1989) reported a Coenzyme Q10(H₂) system in the species.

Exophiala psychrophila O.A. Pedersen & Langvad, Mycol. Res. 92: 153. 1989. — MycoBank MB135481

Description of CBS 191.87 after 2 wk incubation on MEA, 24 °C.

Colonies initially yeast-like and black, gradually becoming effuse and dome-shaped. After 14 d at 18 °C colonies have a dark centre containing the bulk of the conidial mass surrounded by a felt of mouse grey mycelium. Hyphae pale brown, septate, sparingly branched, hyphae 1–3 μm wide. Moniliform cells very common, 3–6 × 5–15 μm, chains consisting of two to several hundred cells, 4–12 being the most common. Conidiogenous cells with several enteroblastic proliferations at the apices with 2.0–3.5 μm diam. Conidia holoblastic, aseptate, varying in shape from spherical to oblong, sometimes tapered, 1.5–2.5 × 3–6 μm, tending to accumulate in slimy balls at apex of conidiogenous cells. Lipid globules may sometimes give the false impression that the conidia are septate. Conidia may be produced from discrete conidiogenous cells, directly from hyphae, from moniliform cells and from conidia. Yeast-like cells also produce conidia.

Cardinal temperatures — Minimum 0 °C (growth present after 6 mo at 0 °C); optimum 17–21 °C; maximum 23 °C.

Specimen examined. Norway, Mar. 1987, F. Langvad, from Atlantic salmon smolt (Salmo salar), ex-type culture, CBS 191.87 = dH15499 = CBS H-20009 = MBSPEC1293.

Notes — The species was originally isolated from farmed Atlantic salmon smolts (Salmo salar) in a farm in Western Norway. The disease led to very high mortality for four years with correspondingly great economic losses for the farmer (Langvad et al. 1985). Strains from salmon in other countries also became available for this study (Table 1).

Exophiala salmonis J.W. Carmich., Sabouraudia 5: 120. 1966. — MycoBank MB119468

Description of CBS 157.67 after 2 wk incubation on MEA, 24 °C.

Colonies moderately expanding, dry, depressed, hairy, olivaceous black. Yeast cells nearly absent. Conidiogenous cells poorly differentiated, intercalary or flask-shaped; annellated zones short, inconspicuous. Conidia 0–3-septate, subhyaline to pale brown, ellipsoidal to short cylindrical, 5.5–8.5 × 2.0–3.5 μm.

Cardinal temperatures — Minimum ≤ 4 °C, optimum 18–24 °C, maximum 30–33 °C. No growth at 37 °C.

Specimens examined. Canada, Alberta, Calgary, Alberta hatchery; cerebral mycetoma of fingerling trout (Salmo clarkii), J.W. Carmichael, specimens CBS H-12617, CBS H-7136, ex-type cultures CBS 157.67 = BMU 00834 = ATCC 16986 = IHEM 3405 = IMI 124165 = MUCL 10078 = UAMH 34 = VKM F-3000.

Additional material examined. Table 1.

Notes — This is the generic type species. Although isolates have frequently been reported under this name, only very few confirmed isolates are available. The species was originally reported as causing three epidemic episodes of cerebral mycetoma in freshwater fingerling trout cod (Maccullochella macquariensis) at the Provincial Government Fish Hatchery in Calgary, Alberta, Canada (Carmichael 1966). The hatchery drew its water from an underground spring which has a temperature range of 12–14 °C and sometimes had a high nitrate content. We sequenced two further isolates of E. salmonis, both from fresh water in The Netherlands. Otis et al. (1985) ascribed an infection in captive Atlantic salmon (Salmo salar) in the USA to the same fungus, but no isolates were available for sequencing. The authors observed interesting parallels to the Canadian case. Both mycoses were systemic in nature and occurred only in captive or hatchery-raised fishes. It appeared that the fishes were debilitated and hence predisposed to infection by opportunistic pathogens. The Atlantic salmon in this study had not been fed properly in captivity, and had just undergone an unsuccessful spawning period. Infections by cestodes (tapeworm, Proteocephalidea) could have weakened the fish, and provided a route of entry for the fungus with subsequent hematogenous spread to the kidney. Madan et al. (2006) reported on subcutaneous nodules in elbows and knees of a 64 yr old male under cyclophosphamide and prednisolone therapy for non-Hodgkin lymphoma ascribed to E. salmonis, but as no sequence data are available this report has to be regarded as doubtful.

Yamada et al. (1989) demonstrated the presence of a coenzyme Q10(H₂) system in E. salmonis.

Veronaea botryosa Cif. & A.M. Corte, Atti Ist. Bot. Lab. Crittog. Univ. Pavia, Ser. 5, 15: 68. 1958. — MycoBank MB307734

Description of CBS 254.57 after 2 wk incubation on MEA, 24 °C.

Colonies growing rapidly, velvety to lanose, greyish brown or blackish brown. Conidiophores erect, straight or flexuose, unbranched or occasionally loosely branched, sometimes geniculate, smooth-walled, olivaceous brown, up to 250 μm long, 2–4 μm wide. Conidiogenous cells terminal or lateral, often becoming intercalary, cylindrical in the apical part with numerous flat scars. Conidia smooth-walled or slightly verrucose, sometimes cylindrical, rounded at the apex and truncate at the base, pale brown, usually 1-septate, 5–12 × 3–4 μm.

Cardinal temperatures — Minimum 4–9 °C, optimum 24–30 °C, maximum 33–36 °C. No growth at 37 °C.

Specimen examined. Italy, from sansa olive slag, specimen CBS H-19962, ex-type culture CBS 254.57 = IMI 070233 = MUCL 9821.

Additional material examined. Table 1.

Notes — This sympodial species, which is morphologically very different from the annellidic genus Exophiala (Arzanlou et al. 2007), is found amidst the waterborne species in the salmonis-clade. The ex-type strain was isolated from sansa olive slag (olive presscake) in Italy (Ciferri & Montemartini 1957), a substrate rich in phenolic compounds. Additional environmental strains were isolated on separate occasions from Eucalyptus wood treated with creosote in Brazil (Table 1).

Otherwise the species is known to cause moderately severe to highly mutilating human infections. Matsushita et al. (2003) published a chronic disseminated mycosis of a 12-year-old child in China (CBS 102593). The patient did not have any known immune disorder. A deep skin lesion in a 37-year-old male patient from the Philippines (CBS 101462) was reported by Medina et al. (1998). Further cases have been reported from China (Nishimura et al. 1989), Libya (Ayadi et al. 1995), the USA (Sutton et al. 2004) and France (Foulet et al. 1999), mostly in immunocompromised patients. Several of these cases have not been verified by sequencing, but the species is morphologically sufficiently stable and characteristic for reliable classical identification. Strain CBS 121506 was isolated from a lesion on the hand of a female patient and represents the first case in Japan caused by this fungus.

In contrast to most Exophiala members of the salmonis-clade, combining waterborne ecology with an ability to invade cold-blooded animals and possibly cool human body sites, Veronaea botryosa has a strong predilection for human hosts, causing chronic, deep infections. The above-mentioned disseminated case in a Chinese adolescent, which developed over a six-year period (Matsushita et al. 2003), was a particularly impressive example.