A total of 16 strains from different origins were included in the study (Table 1). The fungi were cultured on potato dextrose agar (PDA, Pronadisa, Madrid, Spain) and incubated at 35±1°C for 2–5 days.

DNA was extracted and purified directly from fungal colonies following a slightly modified Fast DNA kit protocol (Bio101, Vista, CA, USA), consisting of a homogenization step repeated three times with a FastPrep FP120 instrument (Thermo Savant, Holbrook, NY, USA). DNA was quantified by the GeneQuant pro (Amersham Pharmacia Biotech, Cambridge, England). The internal transcribed spacer (ITS) region of the nuclear rDNA was amplified with the primer pair ITS5 and ITS4, the D1–D2 domains of the 28S rRNA gene were amplified with the primer pair NL1–NL4, and histone 3 (H3) gene was amplified with the primer pair H3-1a–H3-1b.10

The PCR mix (25μl) included 10mM Tris–HCl (pH 8.3), 50mM KCl, and 2.5mM MgCl2 (10× Perkin-Elmer buffer II plus MgCl2 solution Roche Molecular Systems, Branchburg, NJ, USA), 100μM of each dNTP (Promega, Madison, WI, USA), 1μM of each primer, and 1.5U of AmpliTaq DNA polymerase (Roche). The amplification program for the three DNA fragments included an initial denaturation at 94°C for 5min, followed by 35 cycles of denaturation at 95°C for 30s, annealing for 1min at 54°C, and extension for 1min at 72°C. The products were purified with an Illustra GFX™ PCR DNA and Gel Band Purification Kit (General Electric Healthcare, Buckinghamshire, UK) and stored at −20°C until they were used in sequencing. PCR products were sequenced using the same primers employed for amplification and following the Taq DyeDeoxy Terminator Cycle Sequencing Kit protocol (Applied Biosystems, Gouda, Netherlands). Reactions were run on a 310 DNA sequencer (Applied Biosystems). Consensus sequences were obtained using the Autoassembler program (Perkin-Elmer-Applied Biosystems) and Seqman software (Lasergene, Madison, WI).

The sequences were aligned using Clustal X (version 1.8) computer program, followed by manual adjustments with a text editor. Most-parsimonious tree (MPT) analyses were performed using PAUP* version 4.0b10. One hundred heuristic searches were conducted with random sequence addition and tree bisection–reconnection branch-swapping algorithms, collapsing zero-length branches, and saving all minimal-length trees (MulTrees). Saksenaea vasiformis (FMR 10131) was chosen as the outgroup. Support for internal branches was assessed using a heuristic parsimony search of 1000 bootstrapped data sets. The combined data set of the ITS, D1–D2, and H3 was tested for incongruence with the partition homogeneity test (PHT) as implemented in PAUP*. The Kishino–Hasegawa test was performed to determine whether trees differed significantly. Gaps were treated as missing data.

The strains were subcultured on PDA, Czapek agar (CZA; Difco, Becton Dickinson, France), malt extract agar (MEA; 10g of malt extract, 20g of agar, and 1000ml of distilled water), and starch agar (SA; 5g of soluble starch, 15g of agar, and 1000ml of distilled water), and incubated at 37 and 42°C. The microscopic features were determined on the sixth day in wet mounts on water and on lactic acid, which were examined under a light microscope. The strains were identified using schemes based on morphological characters.8,18

Growth rates at 4, 15, 24, 30, 35, 37, 42, and 50°C were determined on PDA, MEA, CZA, and SA for each of the strains included in the study. The Petri dishes were inoculated in the center, incubated in darkness, and the colony diameters (in millimeters) were measured daily.

Carbon source assimilation profiles were determined with the commercial kit API 50CH (bioMérieux, Marcy, l’Etoile, France), following protocols described previously but with minor modifications.23 To obtain sufficient sporulation, all strains were cultured for 6 days on CZA at 42°C. A final concentration of 5×105CFU/ml was prepared in 20ml of yeast nitrogen base (7.7g/l; Difco), containing 0.5g/l L-chloramphenicol (Sigma-Aldrich) and 0.1% Bacto agar (Difco), and each well of the strips was inoculated with 300μl of medium. The viability of the conidia was verified by plating 100μl of serial dilutions of each inoculum onto PDA and incubating at 42°C for 6 days. The inoculated API 50 CH strips were incubated for 48–72h at 37°C in darkness. After incubation, the strips were read visually and growth or lack of growth was noted. Weak growth was considered as a positive result. For nitrogen source assimilations, we used the same inoculum described above, but the yeast nitrogen base broth was replaced by carbon nitrogen base broth (Difco), and testing was performed in sterile, disposable, multiwell microplates. The medium with the nitrogen sources was dispensed into the wells in 150μl volumes with a multichannel pipette and each well was inoculated with 50μl of the conidial suspension. The microplates were incubated at 37°C in darkness for 48 and 72h. We also determined growth of the strains on NaCl (2%, 5%, 7%, 10%), MgCl2 2%, and cycloheximide 0.1%.9,27 All tests were performed in duplicate.

The production of urease was determined after incubation on Christensen's urea agar slants at 37°C for 8 days.16

Sixteen Apophysomyces strains were grown on CZA plates at 37°C in the dark, and then paired in all combinations, including self-crosses, on CZA. Each strain was streaked onto one half of a CZA plate opposite to the streak of another strain, allowing for a central zone of contact as the strains grew. Plates were incubated at 37°C and examined macroscopically each week for up to 6 months for the presence of zygospores. All tests were performed in duplicate.

The in vitro activity of seven antifungal agents against the 16 strains of Apophysomyces was evaluated according to Clinical and Laboratory Standards Institute guidelines (M38-A2).19 The drugs tested were amphotericin B (USP, Rockville, MD, USA), anidulafungin (Pfizer Inc., New York, NY, USA), caspofungin (Merck & Co., Inc., Rahway, NJ, USA), itraconazole (Jansen Pharmaceutica, Beerse, Belgium), posaconazole (Schering-Plough Ltd., Hertfordshire UK), ravuconazole (Bristol-Myers Squibb Company, New Brunswick, NJ, USA), and voriconazole (Pfizer Inc., New York, NY, USA).

All the sequences obtained in this study were deposited in GenBank database and assigned the accession numbers listed in Table 1.

With the primers used, we were able to amplify and sequence 684–820, 582–683, and 345–382bp of the ITS, D1–D2, and H3 loci, respectively. Of the 1630 nucleotides sequenced, 48 characters were parsimony informative in the different strains, the lowest number was eight for the H3 gene, and the highest was 27 for the ITS region. Sequences of the three region genes were analyzed phylogenetically as separate (data not shown) and combined datasets. The partition homogeneity test demonstrated that the three loci sequence data sets were congruent (P=0.05) and could therefore be combined. A total of 36 MPT was produced from a heuristic search, using the combined dataset from the three loci (Fig. 1). The trees had a consistency index of 0.969, a retention index of 0.956, and a homoplasy index of 0.031. Clustering was similar to that observed in the particular trees of the different genes analyzed. Most nodes in the combined analysis showed increased clade support as measured by bootstrapping (six nodes with ≥70%). Analyses of the combined partitions support the recognition of four well supported clades (Fig. 1), each of which could be considered separate phylogenetic species. Clade 1 (bootstrap support (bs) 73%) was composed of two strains from India, two from Australia, and one strain each from the United States, Netherlands Antilles, and Bahamas. Within clade 2 (bs 100%) were included the type strain of A. elegans and one strain from Indian soil. In clade 3 five strains (bs 96%) were located, four of them of clinical origin from the United States, and one from a dolphin. Finally, clade 4 (bs 98%) consisted of two clinical strains, both from the United States.

Fig. 1.

One of the 36 most parsimonious trees obtained from heuristic searches based on analysis produced from the combined data set. Sequence of Saksenaea vasiformis was chosen as outgroup. Bootstrap support values above 70% are indicated at the nodes. T: Type strain.

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Carbon assimilation profiles of the different strains on API 50CH strips are shown in Table 2. Assimilation patterns of all the strains were positive for 20 tests. Twenty-seven carbon sources were not assimilated by any strain. The profiles of assimilation of two carbon sources, esculin and D-lyxose, were species- and strain-dependent, respectively. Esculin was weakly assimilated only by the strains nested in clade 2. The assimilation of D-lyxose was highly variable among the strains of the different clades. By contrast, the variability in the assimilations of nitrogen sources, and tolerance to NaCl, MgCl2, and cycloheximide was nule among the species (Table 3). All the strains were positive for 11 nitrogen sources. Nitrite was not assimilated by any of the strains. All strains were able to grow at 2% NaCl and at 2% MgCl2, but failed to grow at 5% NaCl and at 0.1% cycloheximide.

In general, all the strains examined displayed the typical features of the genus Apophysomyces described by Misra et al.18 However, a more detailed microscopic study of these fungi showed important and consistent differences, mainly in the morphology of sporangiophores and sporangiospores, which correlated with the different phylogenetic species. The strains included in clade 1 showed some morphological diversity. The sporangiospores ranged from broadly clavate to ellipsoidal, were flattened on one side, and measured 5–14×3–6μm. The strains included in clade 2, which comprises the type strain of A. elegans, showed ovoid, subspherical, broadly ellipsoidal to barrel shaped sporangiospores, although more irregularly shaped spores were also present, measured 6–12×5–8μm, and were the largest for the different species in the complex. The sporangiospores of the strains included in clade 3 were trapezoidal and smaller (5–8.5×3–5μm) while those of strains in clade 4 were thick-walled and clearly biconcave (bone-shaped) in side view, measuring 6–8×3–5.5μm. In addition to differences in spore morphology, the strains in clade 2 also showed two types of sporangiophores: (i) large (up to 540μm), bearing vase- or bell-shaped apophyses and (ii) shorter (up to 400μm), bearing funnel-shaped apophyses. The sporangiophores in strains of clades 1, 3, and 4 are similar to the short ones of clade 2.

Zygospore formation was not observed after 6 months of incubation in all the mating tests assayed.

Based on the described morphological and physiological differences, which correlated with the molecular data, we concluded that clades 1, 3, and 4 represent three species of Apophysomyces, different from A. elegans (clade 2), which are proposed here as new species.

Apophysomyces variabilis Alvarez, Stchigel, Cano, D.A. Sutton et Guarro, sp. nov. (Figs. 2A, B; 3H, I).

Fig. 2.

Morphology of the apophyses and sporangiospores of Apophysomyces: (A, B) A. variabilis CBS 658.93 (A, apophyses funnel-shaped; B, sporangiospores). (C, D) A. ossiformis UTHSC 04-838 (C, apophyses funnel-shaped; D, bone-like shaped sporangiospores). (E) A. trapeziformis UTHSC 08-1425 (E, trapezoid-shaped sporangiospores). (F, G) A. elegans CBS 476.78 (F, apophyses bell- and funnel-shaped; (G) subspherical to broadly ellipsoidal sporangiospores). All bars=10μm.

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Etymology: the epithet refers to the variable morphology of the sporangiospores.

Colonies attaining a diameter of 90mm after 4 days of incubation at 37°C on CZA, whitish, with scarce aerial mycelium; hyphae branched, hyaline, smooth-walled, 3–5.5μm in diameter; reverse concolorous. Sporangiophores erect, generally arising singly, at first hyaline, soon becoming light greyish brown, generally straight, slightly tapered towards the apex, unbranched, 100–400μm long, 2–3.5μm wide, and smooth-walled. Sporangia apophysate, terminal, pyriform, multispored, white at first, becoming light greyish brown when mature, and 15–50μm in diameter. Apophyses short, funnel-shaped, and 15–20×15–20μm. Sporangiospores variable in shape, trapezoid, ellipsoid, subtriangular or claviform, hyaline to light brown in mass, smooth- and thin-walled, and 5–14×3–6μm. Not able to assimilate esculin.

Colonies on SA, PDA, and MEA showed similar features than on CZA, but they were more floccose, white, and with less sporulation. The optimum growth temperature was 35–42°C and the minimum temperature of growth was 15°C. The fungus did not grow at 50°C.

Apophysomyces ossiformis Alvarez, Stchigel, Cano, D.A. Sutton et Guarro sp. nov. (Figs. 2C, D; 3D, E).

Etymology: the epithet refers to the bone-like shape of the sporangiospores.

Colonies attaining a diameter of 90mm after 4 days of incubation at 37°C on CZA, whitish, with scarce aerial mycelium, branched, hyaline, smooth-walled, and 3–5.5μm in diameter; reverse concolorous. Sporangiophores erect, generally arising singly, at first hyaline soon becoming light greyish brown, generally straight, slightly tapered towards the apex, unbranched, 100–400μm long, 2–3.5μm wide, and smooth-walled. Sporangia apophysate, terminal, pyriform, multispored, white at first, becoming light greyish brown when mature, 15–50μm in diameter. Apophyses short, funnel-shaped, 15–20×15–20μm. Sporangiospores mostly bone-like shaped, hyaline to light brown in mass, smooth- and thick-walled, and 6–8×3–5.5μm. Not able to assimilate esculin.

Colonies on SA, PDA, and MEA showed similar features than on CZA, but they were more floccose, white, and with less sporulation. The optimum growth temperature was 35–42°C and the minimum 15°C. The fungus did not grow at 50°C.

Apophysomyces trapeziformis Alvarez, Cano, Stchigel, D.A. Sutton et Guarro sp. nov. (Figs. 2E; 3F, G).

Etymology: the epithet refers to the trapezoid shape of the sporangiospores in side view.

Colonies attaining a diameter of 90mm after 4 days of incubation at 37°C on CZA, whitish, with scarce aerial mycelium, branched, hyaline, smooth-walled, 3–5.5μm in diameter; reverse concolorous. Sporangiophores erect, generally arising singly, at first hyaline soon becoming light greyish brown, generally straight, slightly tapered towards the apex, unbranched, up to 400μm long, 2–3.5μm wide, and smooth-walled. Sporangia apophysate, terminal, pyriform, multispored, white at first, becoming light greyish brown when mature, and 15–50μm in diameter. Apophyses short, funnel-shaped, and 15–20×15–20μm. Sporangiospores mostly trapezoid-shaped in side view, more or less cylindrical in front view, flattened at one side and broadly convex on opposite side, hyaline to light brown in mass, smooth- and thin-walled, and 5–8.5×3–5μm. Not able to assimilate esculin.

Colonies on SA, PDA, and MEA showed similar features than on CZA, but they were more floccose, white, and with less sporulation. The optimum growth temperature was 35–42°C and the minimum 15°C. The fungus did not grow at 50°C.

Based on our morphologic and physiologic studies, the type species of Apophysomyces is redefined as follows:

A. elegans Misra, Srivastava, and Lata (Figs. 2F,G; 3A–C).

Fig. 3.

(A)–(C) Apophysomyces elegans (A, vessel-shaped sporangiophore; B, funnel-shaped sporangiophore; C, sporangiospores). (D, E) Apophysomyces ossiformis (D, sporangiophore; E, sporangiospore, frontal and side views). (F, G) Apophysomyces trapeziformis (F, sporangiophore; G, sporangiospore). (H, I) Apophysomyces variabilis (H, sporangiophore; I, sporangiospores). Bars: 10μm.

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Colonies attaining a diameter of 90mm after 4 days at 37°C on CZA, whitish at first, becoming brownish grey, with scarce aerial mycelium; reverse concolorous. Sporangiophores generally arising singly, emerging from aerial hyphae, straight or curved, mainly unbranched or some times branched at the apex, light greyish brown, with two types of morphology, i.e. (i) those that were large (up to 540μm), bearing vase- or bell-shaped apophyses (15–46×11–40μm) and (ii) those that were shorter (up to 400μm) and bore funnel-shaped apophyses (15–20×15–20μm), of 4–7.5μm wide, and smooth-walled. Sporangia produced terminally, pyriform, with distinct apophyses, and 20–60μm in diameter. Sporangiospores ovoid, subspherical, broadly ellipsoidal to barrel-shaped, frequently irregularly shaped, subhyaline, smooth- and thin-walled, and 6–12×5–8μm. The strains analyzed were able to assimilate esculin.

Similar colonies features as described on CZA were observed on AS, PDA, and MEA, with the exception of lesser production of mycelium in CZA. The optimum growth temperature was 35–42°C and the minimum 15°C. The fungus did not grow at 50°C.

The results of antifungal susceptibility testing for Apophysomyces strains are shown in Table 4. Amphotericin B and posaconazole were the most active antifungal agents. Itraconazole and ravuconazole were more active than voriconazole, and caspofungin and anidulafungin were inactive against all strains.