Figures
Abstract
Wallemia sebi is a xerophilic food- and air-borne fungus. The name has been used for strains that prevail in cold, temperate and tropical climates. In this study, multi-locus phylogenetic analyses, using the internal transcribed spacer (ITS) regions, DNA replication licensing factor (MCM7), pre-rRNA processing protein (TSR1), RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2) and a new marker 3´-phosphoadenosine-5´-phosphatase (HAL2), confirmed the previous hypothesis that W. sebi presents a complex of at least four species. Here, we confirm and apply the phylogenetic analyses based species hypotheses from a companion study to guide phenotypic assessment of W. sebi like strains from a wide range of substrates, climates and continents allowed the recognition of W. sebi sensu stricto and three new species described as W. mellicola, W. Canadensis, and W. tropicalis. The species differ in their conidial size, xerotolerance, halotolerance, chaotolerance, growth temperature regimes, extracellular enzyme activity profiles, and secondary metabolite patterns. A key to all currently accepted Wallemia species is provided that allow their identification on the basis of physiological, micromorphological and culture characters.
Citation: Jančič S, Nguyen HDT, Frisvad JC, Zalar P, Schroers H-J, Seifert KA, et al. (2015) A Taxonomic Revision of the Wallemia sebi Species Complex. PLoS ONE 10(5): e0125933. https://doi.org/10.1371/journal.pone.0125933
Received: November 15, 2014; Accepted: March 25, 2015; Published: May 27, 2015
Copyright: © 2015 Jančič et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was funded by the Slovenian Research Agency (https://www.arrs.gov.si/en/dobrodoslica.asp) through the Infrastructural Centre Mycosmo (http://www.ex-genebank.com/) and the Young Researcher Grant to SJ, 1000-11-310102. This work was partly funded by Slovenian Ministry of Higher Education, Science and Technology (http://www.arhiv.mvzt.gov.si/en/) and European Regional Development Fund through the Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CIPKeBiP, http://cipkebip.org/, OP13.1.1.2.02.0005), and by grants to KAS and HDTN from the Alfred P. Sloan Foundation Program on the Microbiology of the Built Environment (http://www.sloan.org/major-program-areas/). Parts of this work were realized within the frame of FP7 Project CropSustaIn (FP7-REGPOT-CT2012-316205). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The fungal genus Wallemia is based on W. ichthyophaga, which remained the only recognized Wallemia species until von Arx [1] recognized Sporendonema sebi as congeneric. Before the application of nucleic acid sequencing to fungal taxonomy, Moore [2] noted the possible presence of dolipore septa in W. sebi speculating that Wallemia was a basidiomycete. This was confirmed when Zalar et al. [3] applied 18S rDNA sequencing to support the establishment of the higher taxa Wallemiomycetes and Wallemiales. Wallemiomycetes then remained monogeneric until Nguyen et al. [4] added two genera, Geminibasidium and Basidioascus, in a new order Geminibasidiales, which was tentatively classified in Wallemiomycetes, although relatively distant from Wallemia. Zalar et al. [3] clarified species concepts within Wallemia, removing W. ichthyophaga from the synonymy with W. sebi proposed by von Arx [1], and demonstrating that Torula epizoa var. muriae J.J. Kickx should be recognized as a distinct Wallemia species, which they called W. muriae [3].
Wallemia sebi is the most frequently isolated and best-studied species of the genus. It is commonly involved in the spoilage of food with low water activity (aw) [3,5] and its air-disseminated spores [6–9] were associated with allergies, bronchial asthma and farmer’s lung disease [10–15]. Recently, Desroches et al. [16] demonstrated that human antibodies react to compounds produced by W. sebi spores. Metabolites present in spore and mycelial fragments caused inflammation and affected lung biology in an in vivo mouse model [17]. Wallemia sebi produces the metabolites walleminol, walleminone [18], azasteroid UCA1064-B [19], and the highly toxic wallimidione [16]. Walleminol is found in food contaminated by W. sebi [20], has an LD50 of 40 μg/ml for brine shrimp and a minimum inhibitory dose of 50 μg/ml for rat liver cells [21]. The UCA1064-B compound has weak activity against Saccharomyces cerevisae and Gram-positive bacteria and is cytotoxic to HeLa cells [19]. Wallemia sebi produces a cyclopentanopyridine alkaloid that exhibits antimicrobial activity towards Enterobacter aerogenes [22]. Botić et al. [23] showed that salt induces biosynthesis of active compounds in W. sebi that are hemolytic towards mammalian erythrocytes.
Wallemia sebi was recognized as one of the most xerophilic eukaryotes and often lives in diverse, harsh environments. It can grow in a wide range of water activities, whereas W. muriae only grows on media with slightly reduced water activities. Wallemia ichthyophaga is obligately halophilic and requires aw lowering solutes for in vitro growth [3,24–26]. Previous analyses of the highly variable rDNA internal transcribed spacer (ITS) sequences suggested monophyly of all strains identified as W. sebi, but at least two subclades were observed [3]. Recently generated multi-locus DNA sequence data from five genes in a companion study revealed four strongly supported clades among strains, identified as phylogenetic species following the concept of genealogical concordance of phylogenetic species recognition [27]. Accordingly, W. sebi represents a species complex, which we refer to as the W. sebi species complex (WSSC).
Ecophysiological studies and global surveys of house dust [9,27] resulted in the isolation of ~70 new strains since 2005 that conform to the broad concept of the WSSC. The objectives of our study were to redefine W. sebi sensu stricto by focusing on its phenotypic characterization and to delimit the phylogenetic species. Building on the companion study [27], phylogenetic inferences were performed using ITS and partial protein-encoding gene sequences of the DNA replication licensing factor (MCM7), pre-rRNA processing protein (TSR1), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2). Additionally, the protein coding gene for 3´-phosphoadenosine-5´-phosphatase (HAL2) is introduced here and tested as an additional marker for recognizing phylogenetic species within the WSSC. To support the phylogenetic species hypotheses proposed by the companion study, phenotypic variability within the WSSC was examined using culture and micromorphological characters, xerotolerance (tolerance to low aw), halotolerance (tolerance to NaCl), chaotolerance (tolerance to MgCl2) [28,29], growth temperature regimes, extracellular enzyme activity profiling and secondary metabolite patterns. Based on these results, we describe here 3 new species of Wallemia by adopting a “consilient taxonomy” approach.
In mycology and bacteriology, taxonomic studies that combine phenotypic and genotypic data are often referred to as polyphasic [30]. The awkwardness of the root “phase” for this term, which in English tends to refer to phases of matter and not the diverse attributes of living things, led Quaedvlieg et al. to suggest the “consolidated” species concept as an alternative phrase [31]. We prefer “consilient taxonomy” or the “consilient species concept”, because consilience is a central pillar in the development of the modern philosophy of experimental science. Wilson [32] used the word consilience, in a broader sense, however, as a metaphor to examine the relationship between art and science. Instead, we prefer to adopt the original intention of [33], where consilience indicated a scientific situation where “… an induction, obtained from one class of facts, coincides with an induction obtained from another different class.” This describes exactly the application of several different classes of data practiced in modern fungal taxonomy, and we advocate for the replacement of the phrase “polyphasic taxonomy” with “consilient taxonomy”. In our study, the “consilient taxonomy” is based on the convergence of multiple, independent data sets, as a means of delimiting species.
Materials and Methods
Sources of microorganisms
The strains studied (S1 Table) were assembled from previous surveys of fungi in the built environment and other low aw habitats such as food, hypersaline solar salterns and salt lakes, and dry agricultural commodities such as hay, feed, and pollen. These were isolated using either dilution-to-extinction or classical microbiological isolation strategies [3,27,34]. Strains were also obtained from the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands [CBS]; Canadian Collection of Fungal Cultures, Agriculture and Agri-Food Canada, Ottawa, Canada [CCFC/DAOM]; Ex Culture Collection of the Department of Biology, Biotechnical Faculty, University of Ljubljana, Infrastructural Centre Mycosmo, MRIC UL, Ljubljana, Slovenia [EXF]; Mycotheque of the Catholic University of Louvain, Louvain la Neuve, Belgium [MUCL]; and University of Alberta Microfungus Collection and Herbarium, Edmonton, Canada [UAMH]).
DNA extraction, PCR amplification, and sequencing
DNA was extracted as described previously [3,4]. Primer names and sequences are listed in S2 Table. Primers for HAL2 were newly designed, with the help of the online platforms OligoCalc (http://www.basic.northwestern.edu/biotools/oligocalc.html). The ITS, and partial sequences of the five protein-encoding genes RPB1, RPB2, MCM7, TSR1, and HAL2 [35–40] were amplified. Amplification of ITS, RPB1, RPB2, MCM7 and TSR1 was done as described in Nguyen et al. [27]. Amplification of HAL2 was done with 10× Dream Taq DNA polymerase (Fermentas), 1× Dream Taq Buffer (Fermentas), 0.1 mM dNTPs, 0.8 μM forward primer, and 0.8 μM reverse primer. The following PCR profile was used to amplify HAL2: 95°C for 3 min (initial denaturation), then 40 cycles at 95°C for 30 s (denaturation), 55°C for 30 sec (annealing), 72°C for 1 min (extension), followed by 72°C for 5 min (final extension). Amplified DNA fragments were separated by electrophoresis in 1% agarose gels in 0.5× TAE buffer, and visualized using Invitrogen SYBR Safe DNA gel staining. Purified PCR fragments were Sanger sequenced by commercial service providers (Macrogene Europe, Amsterdam, The Netherlands; or Microsynth Vienna, Austria). All newly generated sequences were deposited in GenBank (S1 Table).
Sequence data, alignment and phylogenetic analyses
The sequences of each gene were aligned using MAFFT [41], and concatenated into a single data matrix with SeaView v4.4.2 [42]. PAUP4.10b [43] was used to determine the number of parsimony informative characters for the HAL2 alignment. Appropriate evolutionary models under the Akaike Information Criterion (AIC) were determined with jModelTest 2 [44] (S3 Table).
Bayesian phylogenetic inferences were calculated for each partition and for the combined (ITS + RPB1 + RPB2 + MCM7 + TSR1 + HAL2) sequence data set using MrBayes v. 3.2.2 [45]. Single gene analyses were run for 3.0 ×106 generations. The analysis of ITS sequences also used W. sebi sequences from other studies obtained from GenBank: JX240410, JX317206, JX317199, JX436301 [46], HG764524, FJ524297 [47], FJ820490 [8], KF225873, KF225874, KF225857, KF225858, KF225864 [48], KF800096 [49], EU664486, EU329737, EU329736, EU486095 [50], GU941208, GU931736 [9], GU370753, GU370758, JF497133 [51], GU721563, GU721564, KC460839, FR718458, DQ33856, HQ997370, and HQ997371. The combined analysis ran for 1.0 ×107 generations. Trees were sampled every 500 generations. The first 25% of trees were discarded as burn-in, and from the remaining trees, a 50% majority rule consensus tree was calculated. The alignments and trees were deposited in TreeBASE (http://treebase.org/treebase-web/home.html) under study number 16439.
Consensus trees were imported and visualised using FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) or MEGA5 [52]. To estimate species boundaries, we applied the criteria described by Lutzoni et al. [53]. Internodes were considered strongly supported if they received posterior probabilities ≥0.95. MEGA5 [52] was used to calculate pair-wise distances (p-distances) between sequences. The four clades inferred through phylogenetic analysis are referred to as clades 1 to 4. Each sequence generated from a member of the WSSC was assigned to its respective clade number, which allowed the calculation of p-distances between and within the clades using Microsoft Excel.
Physiological and morphological studies
The following strains were used for physiological and morphological studies: six strains of clade 1 (CBS 818.96, EXF-5860, MUCL 46253, CBS 136841, CBS 136845, CBS 196.56), six of clade 2 (CBS 633.66, EXF-5675, EXF-5677, EXF-8738, EXF-8747, EXF-8745), four of clade 3 (MUCL 15061, DAOM 226642, DAOM 242570, DAOM 242571), and three of clade 4 (EXF-8739, EXF-8744, EXF-8746). Xerotolerance, halotolerance and chaotolerance were determined on malt yeast agar (MYA; 1% malt extract, 1% yeast extract, 0.1% K2HPO4, 2% agar) [54], with aw decreasing from 1.00 to 0.75 in ten steps for sucrose, seven for glycerol and NaCl, and nine for MgCl2. The non-ionic sucrose and glycerol were used as the controlling solutes for the determination of xerotolerance, ionic NaCl for halotolerance, and ionic MgCl2 for chaotolerance. The pH of each medium was adjusted to 6.5 with NaOH or HCl before adding agar.
The aw of media were measured using a water activity meter (AquaLab, model Series 3 TE; Decagon Devices, Pullman, WA, USA). For inoculum, conidia were suspended in sterile saline (0.9% NaCl) with 0.05% Tween 80 and 0.05% agar. Strains were point-inoculated on media in 6-cm Petri dishes in three replicates for each treatment, and incubated at room temperature for 20 d. To determine cardinal growth temperatures, strains were grown on MYA with 40% sucrose (aw = 0.96) and on MYA with 8% NaCl (aw = 0.95), in three replicates for each, and incubated at 4°C, 10°C, 15°C, 20°C, 24°C, 30°C, 34°C, 37°C, and 42°C for 25 d [4].
Colony diameters for all physiological tests were read every 5 d. Mean colony diameters for strains assigned to each of the four clades were calculated. The coefficients of the linear parts of the growth curves generated with sucrose, glycerol, NaCl and MgCl2 represented the colony radial growth rates for the above specified members of each clade.
Colony characters were assessed in 9-cm plastic Petri dishes incubated at 24°C for 14 d in the dark, following three-point inoculation, on the following media: MEA [55]; MYA [54] (aw ≈ 1.00); MYA plus 8% NaCl (aw = 0.95), 16% NaCl (aw = 0.88), 20% sucrose (aw = 0.98), 50% sucrose (aw = 0.94), 70% sucrose (aw = 0.88), 20% glycerol (aw = 0.95), and 40% glycerol (aw = 0.86); and DG18 [56] (aw = 0.953). Characters such as size, color, spreading tendency, structure, texture of colonies, exudate production and sporulation, and color of the colony reverse [3], were assessed with a stereomicroscope (Leica EZ4). Photographs were taken of the colonies using a Canon PowerShot G16 camera.
Micromorphological characters were defined for cultures grown on MYA with 50% sucrose (aw = 0.94) incubated at 24°C for 7 d [3], and included descriptions of the hyphae, conidiophores, conidiogenous cells and conidia. Sporulating material was mounted in 60% lactic acid. Photographs were taken with an Olympus DP73 camera on an Olympus BX51 microscope. For each strain, dimensions of 100 conidia were measured with the image analysis software Cell.
Extracellular enzyme activities
Tests to determine proteolytic (based on casein used as the substrate), amylolytic (soluble starch), cellulolytic (carboxymethyl cellulose), β-glucosidase (easculin), esterase (Tween 80), xylanase (xylan) and urease (urea) activities were carried out on 2% agar media without and with addition of 10%, 17% and 24% NaCl [57–60]. Conidia were suspended in saline (0.9%, 10%, 17%, 24% NaCl) from 7-d-old cultures grown at 24°C on MY50G and used for three-point inoculations. Cultures were incubated in the dark at 24°C for 14 d. Resulting colonies were photographed with a Canon PowerShot G16 camera.
Analysis of secondary metabolites
Selected isolates of clade 1 (CBS 818.96, EXF-5860, MUCL 46253, CBS 110585, EXF-1441, EXF-5860), clade 2 (EXF-5675, EXF-5677, EXF-5918, MUCL 45613, UAMH 6689), clade 3 (MUCL 15061, DAOM 226642, DAOM 242570, DAOM 242571), and clade 4 (EXF-8739, EXF-8744, EXF-8746) were inoculated at three-points on YES (Yeast Extract Sucrose) agar, CYAS (Czapek Yeast Extract Agar) [56,61] plus 5% NaCl, and grown at 24°C for 10 d in the dark. Six colony plugs were excised from each medium, and pooled in 1.5-ml screw-cap vials. A mixture of methanol-dichloromethane-ethyl acetate (1:2:3 [v/v/v]) containing 0.5% formic acid was added (500 μl) for ultrasonic extraction for 60 min [62]. Organic phases were transferred to clean vials and evaporated to dryness during centrifugation under vacuum. Residues were re-dissolved in 500 μl methanol and filtered (0.45 μm filters; Sartorius).
One μl of the solutions was used for HPLC analyses (Chromeleon Dionex UHPLC; Dionex Ultimate 3000 RS Diode array detector) using alkylphenone retention indices and diode array UV/VIS detection from 200–600 nm [63]. Separations were run on a 2 × 100 nm Luna2 OOD-4251-BO-C18 column with a C18 pre-column, both packed with 3 μm particles. A linear gradient from 85% water, 15% acetonitrile was run to 100% acetonitrile over 20 min, then maintained at 100% acetonitrile for 5 min, at a flow rate of 0.4 ml min-1. Both eluents contained 0.005% trifluoroacetic acid. The alkylphenone retention index was calculated for each peak, and compounds were identified by retention times and UV/VIS spectra. All peaks were quantified by height, followed by qualitative and quantitative multivariate statistical analyses [64]. Quantitative secondary metabolite data were analyzed by principal component analysis (PCA) using UNSCRAMBLER (CAMO, Oslo, Norway), with correspondence and the unweighted pair group method with arithmetic mean cluster analysis using NT-SYS (Numerical Taxonomy and Multivariate System, version 2.10; Exeter Software, New York, USA) [65].
Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants [66], and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone.
In addition, the new names introduced here have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank numbers contained in this publication to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from PubMed Central and LOCKSS.
Ethics statement
In this study, no field activities were performed and no endangered and protected species were involved. All strains studied here were obtained from different culture collections and are considered publicly available for research purposes. To the best of our knowledge no specific permissions were required for sampling on the locations specified in S1 Table.
Results
Phylogenetic analyses
The ITS phylogenetic analysis divided the WSSC into two subgroups, with only the subgroup including the ex-neotype strain CBS 818.96 strongly supported. Most strains of the WSSC formed an un-resolved and weakly supported cluster (S1 Fig). Analyses of the rpb2, rpb1, MCM7, and tsr1 sequences resolved the WSSC into four groups, referred to as clades 1–4. The tree topologies based on rpb1, MCM7, and tsr1 (S1 File) were similar to the topology of the combined data set (Fig 1), suggesting a sister-group relationship of clades 1 and 2. Clades 1–4 also received high support in analyses of the hal2 sequences (Fig 2), with a supported sister group relationship for clades 2 and 4. Comparison of pair-wise distance (p-distance), alignment length, and parsimony informative characters obtained from HAL2 sequences are provided in S2 Fig and S3 Table. Further details of the phylogenetic analyses, determination of genetic variability of sampled loci and barcode gap analyses are described in the companion study [27].
The tree from six aligned loci (ITS, rpb2, rpb1, MCM7, tsr1, hal2) provides a resolved structure of the WSSC. Bayesian posterior probabilities are displayed at the nodes of the tree. Labels provide information on strain number and origin. Red T, ex-type strains; red NT, ex-neotype strain; bold, strains included in physiological and morphological studies, and for the determination of extracellular enzyme activities; underlined, strains included in studies of secondary metabolites.
Bayesian posterior probabilities are displayed at the nodes of the tree. Red T, ex-type strains; red NT, ex-neotype strain; bold, strains included in physiological and morphological studies, and for the determination of extracellular enzyme activities; underlined, strains included in studies of secondary metabolites.
Physiology
All strains of the WSSC grew on media (MEA or MYA) with aw ≈ 1.00. Strains from clade 2 had the highest radial growth rate (0.36 mm d-1; R2 = 0.98). Growth rates within clade 1 were 0.30 mm d-1 (R2 = 0.99), within clade 3 they were 0.27 mm d-1 (R2 = 0.99) and within clade 4 they were 0.22 mm d-1 (R2 = 0.98). Members of clades 1 and 2 grew faster than the more distantly related members of clades 3 and 4 (Table 1).
Although all strains of the WSSC grew on media without additional solutes, their growth was optimal on media with low aw, and they grew fastest at aw 0.97 to 0.92, with all of the solutes tested. The growth rates at aw 0.97 to 0.92 were 0.6–0.8 mm d-1 on sucrose, 0.5–0.6 mm d-1 on glycerol and NaCl, and 0.4–0.5 mm d-1 on MgCl2. Accordingly, the tested strains of the WSSC were xerophilic and grew best on MYA supplemented with 20%-50% sucrose. On sucrose media, the minimum aw for all of the tested strains of the WSSC was ca. 0.78 (Fig 3 and S4 Table).
(a-d) Mean colony growth rates (mm d-1) obtained from MYA plus various concentrations of sucrose (a), glycerol (b), NaCl (c), and MgCl2 (d). (e) Mean colony growth rates at temperatures from 4°C to 40°C on MYA with addition of 40% sucrose. Diamonds, clade 1 (W. sebi, 6 strains); squares, clade 2 (W. mellicola, 6 strains); circles, clade 3 (W. canadensis, 4 strains); triangles, clade 4 (W. tropicalis, 6 strains).
Optimal growth occurred at 8% (1.4 M) NaCl, which corresponded to aw ca. 0.95. Accordingly, the tested strains of the WSSC are halophilic. All tested strains from clades 1 and 4 tolerated up to 28% (4.8 M) NaCl (aw = 0.79), whereas the highest tolerated NaCl concentration for clades 2 and 3 was ca. 24% (4.1 M) NaCl (aw = 0.82) (Table 1).
Strains from clade 1 tolerated up to 17% (1.8 M) MgCl2 (aw = 0.85), strains from clades 2 and 4 tolerated up to 13% (1.4 M) MgCl2 (aw = 0.90), and strains from clade 3 tolerated up to 11% (1.2 M) MgCl2 (aw = 0.92) (Table 1). Accordingly, the tested strains of the WSSC are chaophilic or at least chaotolerant. Additional growth parameters of these members of clades 1–4 with different solutes and at different aw are given in the S4 Table.
On MYA with 40% sucrose, the optimum growth temperature for the tested strains of clades 1, 2, and 4 was 30°C (growth minimum, 10°C; growth maximum, 34°C). Clade 3 grew best at 24°C (growth minimum, 10°C; growth maximum, 30°C). Growth rates at the optimum temperature were 0.93 mm d-1 (R2 = 0.99) for clade 1, 0.75 93 mm d-1 (R2 = 0.99) for clade 2, 0.60 93 mm d-1 (R2 = 0.99) for clade 3, and 0.80 93 mm d-1 (R2 = 0.99) for clade 4. No growth occurred at 4°C and 37°C (Fig 3e, Table 1). The cardinal temperatures obtained on MYA with the addition of 8% (aw = 0.95) NaCl were similar to those on 40% sucrose (aw = 0.96).
Micromorphology
Mature conidia were generally spherical, slightly verrucose, thick-walled, and pale brown. On average, the smallest conidia were seen in clade 1 (diameter, 2.1 μm; standard deviation [SD], 0.2 μm; standard error [SE], 0.010 μm), and the largest in clade 2 (diameter, 2.6 μm; SD, 0.3 μm; SE, 0.011 μm). Intermediate values characterized clade 3 (diameter, 2.3 μm; SD, 0.2 μm; SE, 0.012 μm) and clade 4 (diameter, 2.4 μm; SD, 0.3 μm; SE, 0.015 μm). Other micromorphological characters for dimensions of hyphae, conidiophores and conidiogenous cells did not differ among clades 1–4.
Extracellular enzyme activities
Proteolytic, amylolytic, cellulolytic and xylanase activity were not detected in any WSSC strains at any salinity. However, β-glucosidase, esterase and urease activities were seen for members of all four clades, with or without 10% NaCl. No enzymatic activities were detected at 24% NaCl, although β-glucosidase activity was still detected at 17% NaCl for strains of clades 1 and 2. Strains from clade 1 showed strong urease activities only under non-saline conditions (0% NaCl), although they grew in 10% and 17% NaCl. Strains from clade 3 grew and showed urease activity only in 10% NaCl; similarly, clade 4 grew and showed β-glucosidase activity only in 10% NaCl (Table 2).
Secondary metabolites
Forty-six compounds were detected from strains of the WSSC grown on YES agar and CYAS. The provisional identification of these compounds, their retention times, and their characteristic UV/VIS spectra are listed in S5 Table. Clade 2 members produced 28 different compounds, while 23 secondary metabolites were detected for clade 1, and 16 for clade 3. No compounds were detected for clade 4 members grown on YES agar and CYAS. Strains of clades 1 and 2 that are closely related phylogenetically produced similar metabolites, with 20 of the metabolites common to both clades. None of the metabolites detected for clade 3 members were detected for clades 1 and 2. To determine potential groupings suggested by these secondary metabolites, their quantitative amounts detected by HPLC were subjected to PCA, correspondence and cluster analyses. These relative quantitative amounts varied from 3 to 700 absorbance units (mAU) among the isolates examined. Strains of clade 3 were strongly discriminated in the PCA (S3 Fig), correspondence and cluster analyses. PCA also showed that the metabolite profiles from YES agar and CYAS were clearly different, and that NaCl had a strong impact on the production of these secondary metabolites.
Taxonomy
By applying the genealogical concordance phylogenetic species recognition concept [67], we confirmed the results of the companion study [27] and that the WSSC consists of four phylogenetic species. Secondary metabolite profiles support the phylogenetic inference that clade 1 and clade 2 are closely related, and that clade 3 presents a taxon clearly separated from clade 1 and 2 members. Clade 1 members, including the ex-neotype isolate of W. sebi, comprise the most halotolerant and chaotolerant taxon and are physiologically distinguishable from clades 2, 3 and 4. Clade 3 is clearly distinct from the others by optimal and maximal growth temperatures, halotolerance and chaotolerance, and its secondary metabolite profile. These data thus allow a consilient delineation of species within the WSSC and the description of three distinct Wallemia species that are here newly named W. mellicola, W. tropicalis and W. canadensis (see summary of characters in Table 3).
Wallemia sebi (Fr.) von Arx, The Genera of Fungi Sporulating in Pure Culture: 166. 1970. (Fig 4).
(a-l) Colony surface grown on MYA with no additives (a), and MYA with the addition of 20% (b), 50% (c) and 70% (d) sucrose, 20% (e) and 40% (f) glycerol, 4% (g) and 13% (h) MgCl2, and 8% (i), 16% (j), 24% (k) and 28% (l) NaCl. (m) Conidia from MYA plus 50% sucrose. (n) Conidiogenous cell producing conidia in chains. Colonies were incubated for 14 d at 24°C. Scale bars: 5 mm (l) (applies also for a-k), 5 μm (m, n).
Colony characteristics.
Colonies on MEA and MYA: 4–6 mm diam. after 2 weeks; growth rate, 0.30 mm d-1; cerebriform, extending deeply into the agar; compact, pale brown or almost white, without sporulation and exudates; margin pale brown or the same color as the colony, and irregular; gray reverse. Colonies on MY50G or MYA with addition of 50% sucrose: 10–13 mm diam. after 2 weeks; growth rate, 0.73 mm d-1; cerebriform, spreading into the agar; compact, greenish and yellowish brown centrally, dark brown marginally, and powdery because of strong sporulation, without exudates; margin brown, and irregular; dark gray reverse. Colonies on MYA with addition of 70% sucrose: 6–8 mm diam. after 2 weeks; growth rate, 0.46 mm d-1; flat, pale brown, sporulation moderate, without exudates; margin white or pale brown, and regular; gray reverse. Colonies on DG18 or MYA with addition of 20% glycerol: 8–10 mm diam. after 2 weeks; growth rate, 0.59 mm d-1; punctiform to somewhat cerebriform, extending deeply into the agar; compact, dark brown with brighter central part, sporulation weak, without exudates; margin darker than colony, and regular; dark gray reverse. Colonies on MEA or MYA with addition of 16% NaCl: 6–8 mm diam. after 2 weeks, growth rate, 0.35 mm d-1; punctiform, extending deeply into the agar; compact, pale brown or white, sporulation weak, without exudates; margin pale brown or the same color as the colony, and irregular; gray reverse.
Conidial size.
1.5–2.5 μm diam. (mean ±standard deviation, 2.1 ±0.2 μm). For description of hyphae, conidiophores and conidiogenous cells see [3].
Physiology.
Growth at aw ≈1.00 (0% NaCl, 0% MgCl2) positive; optimum at aw = 0.97 to 0.92 (4%-12% NaCl, 6%-9% MgCl2); maximum at aw = 0.78 (28% NaCl, 17% MgCl2).
Extracellular enzyme activities.
β-glucosidase at 0% to 17% NaCl; esterase at 0% to 10% NaCl; urease at 0% NaCl.
Habitat.
Sea salt, hypersaline water in solar salterns and salt lakes, hay; air and dust in indoor environments (house, office, archives); occasionally in pond water, mineral water, and seeds (sunflower, wheat, rye, barley, maize).
Human and animal pathogenicity.
Chronic ulcerative skin lesion in man (one case reported, Groningen, The Netherlands); fatal livestock toxicosis associated with hay contaminated with W. sebi (one case reported, Berkshire, UK) (fide [3]).
Typification.
Sweden, dried MEA culture of CBS 818.96, originating from sunflower seed, collected and isolated by M. Olsen in 1986, neotype designated by Zalar et al. [3], deposited in CBS herbarium. Living ex-type strain: CBS 818.96 = EXF-958.
Diagnostic characters.
Growth positive on media without additional solutes, such as MYA or MEA, conidia 1.5–2.5 μm diam., halotolerance up to 28% NaCl, chaotolerance up to 17% MgCl2, maximum growth temperature 34°C, β-glucosidase activity up to 17% NaCl, no urease activity at 10% NaCl, occurrence worldwide.
Note.
Zalar et al. mentioned Torula minuta Saito as a possible synonym of W. sebi because of its halotolerance, but this species is now commonly referred to as Rhodotorula minuta (Saito) F.C. Harrison [68]. Wallemia sebi was referred to as W. sebi clade 1 in Nguyen et al. [27].
Wallemia mellicola Jancic, Nguyen, Seifert & Gunde-Cimerman, sp. nov. MycoBank MB: 810412 (Fig 5).
(a-k) Colony surface grown on MYA without additions (a), and MYA with the addition of 20% (b), 50% (c) and 70% (d) sucrose, 20% (e) and 40% (f) glycerol, 4% (g) and 13% (h) MgCl2, and 8% (i), 16% (j) and 24% (k) NaCl. (l, m) Conidia (l) and conidiophore and conidiogenous cell (m) from MYA plus 50% sucrose. Colonies were incubated for 14 d at 24°C. Scale bars: 5 mm (k) (applies also for a-j), 5 μm (l, m).
Etymology.
Latin mel, meaning honey and-colo, to reside, referring to the habitat of the type strain.
Colony characters.
Colonies on MEA and MYA: 5-7mm diam. after 2 weeks; growth rate, 0.36 mm d-1; cerebriform, extending deeply into the agar; compact, pale brown or brown, without sporulation and exudates; margin brown or darker than the colony, irregular; dark gray reverse. Colonies on MY50G or MYA with addition of 50% sucrose: 9–12 mm diam. after 2 weeks; growth rate, 0.71 mm d-1; cerebriform in the central part, flat marginally, extending deeply into the agar; compact, greenish brown in the central part, brown marginally, powdery because of strong sporulation, without exudates; margin brown, and irregular; dark gray reverse. Colonies on MYA with addition of 70% sucrose: 8–10 mm diam. after 2 weeks; growth rate, 0.62 mm d-1; flat, pale brown, sporulation strong, without exudates; margin white or pale brown, and regular; gray reverse. Colonies on DG18 or MYA with addition of 20% glycerol: 7–11 mm diam. after 2 weeks; growth rate, 0.60 mm d-1; punctiform, extending deeply into the agar; very compact, dark brown with brighter central part, sporulation weak, without exudates; margin and colony the same color, and regular; dark gray reverse. Colonies on MEA or MYA with the addition of 16% NaCl: 5–7 mm diam. after 2 weeks; growth rate, 0.35 mm d-1; cerebriform, spreading into the agar; compact, pale brown, sporulation weak, without exudates; margin and colony the same color, and irregular; gray reverse.
Physiology.
Growth at aw ≈1.00 (0% NaCl, 0% MgCl2) positive; optimum at aw = 0.97 to 0.92 (4%-12% NaCl, 4%-6% MgCl2); maximum at aw = 0.78 (24% NaCl, 13% MgCl2).
Extracellular enzyme activities.
β-glucosidase at 10% to 17% NaCl; esterase at 0% to 10% NaCl; urease at 0% to 10% NaCl.
Habitat.
Soil, forest plants, hypersaline water of solar salterns, salty food products (peanuts, dried fish), sugary food products (date honey, cakes, jam, maple syrup, chocolate), dried food products (bread, coconut pulp), seeds, straw, pollen; air, dust and surfaces in indoor environments.
Human and animal pathogenicity.
Subcutaneous lesion (phaeohyphomycosis) on foot in an immunocompetent human patient (Varanasi, Uttar Pradesh, India) [69].
Typification.
Unknown origin (possibly Israel), from date honey, 63.5% total soluble solids, collected and isolated by R. B. Kenneth in 1966, holotype, designated here, CBS herbarium H-13315 (originally identified as W. sebi). Living ex-type strain: CBS 633.66 = ATCC MYA-4683 = EXF-956.
Diagnostic characters.
Growth positive on media without additional solutes, such as MYA or MEA, conidia 2.5–3.0 μm diam., halotolerance up to 24% NaCl, chaotolerance up to 13% MgCl2, growth positive at 34°C, no β-glucosidase activity without NaCl, urease activity up to 10% NaCl, occurrence worldwide.
Note.
The genome of the strain CBS 633.66 was sequenced (under the name W. sebi) by the Joint Genome Institute at the Department of Energy, USA [70]. Wheeler et al. [54] studied FRR 3051 (= CBS 110589, EXF-1277) isolated from the dried salted fish Ophiocephalus striatus and concluded that this strain grows optimally at 25°C. Gock et al. [71] observed that conidial germination but no growth occurred in FRR 3051 even at 37°C. This species was referred to as W. sebi clade 2 by Nguyen et al. [27].
Wallemia canadensis Jancic, Nguyen, Seifert & Gunde-Cimerman, sp. nov. MycoBank: MB 810413 (Fig 6).
(a-k) Colony surface grown on MYA without additions (a), and MYA with the addition of 20% (b), 50% (c) and 70% (d) sucrose, 20% (e) and 40% (f) of glycerol, 4% (g) MgCl2 (no growth on MYA with addition of 13% (h) MgCl2), and 8% (i), 16% (j) and 24% (k) NaCl. (l, m) Conidia (l) and conidiogenous cell (m) from MYA plus 50% sucrose. Colonies were incubated for 14 d at 24°C. Scale bars: 5 mm (k) (applies also for a-j), 5 μm (l, m).
Etymology.
The epithet canadensis refers to Canada, where four of the currently five known strains were isolated.
Colony characters.
Colonies on MEA and MYA: 4–5 mm diam. after 2 weeks; growth rate, 0.27 mm d-1; cerebriform, extending deeply into the agar; compact, brown, sporulation weak, without exudates; margin the same color as the colony, and irregular; gray reverse. Colonies on MY50G or MYA with addition of 50% sucrose: 7–12 mm diam. after 2 weeks; growth rate, 0.62 mm d-1; cerebriform, extending deeply into the agar; compact, walnut or pale brown, powdery because of strong sporulation, without exudates; margin white or the same color as the colony, and irregular; dark gray reverse. Colonies on MYA with addition of 70% sucrose: 7–10 mm diam. after 2 weeks; growth rate, 0.51 mm d-1; flat in concentric circles, pale brown, sporulation weak, without exudates; margin white or pale brown, and irregular; gray reverse. Colonies on DG18 or MYA with addition of 20% glycerol: 6–8 mm diam. after 2 weeks; growth rate, 0.48 mm d-1; slightly cerebriform, extending deeply into the agar; compact, pale brown with white central part, sporulation weak, without exudates; margin pale brown, and regular; gray reverse. Colonies on MEA or MYA with the addition of 16% NaCl: 4–5 mm diam. after 2 weeks; growth rate, 0.31 mm d-1; punctiform, extending deeply into the agar; compact, pale brown or white, sporulation weak, without exudates; margin the same color as the colony, and irregular; dark gray reverse.
Physiology.
Growth at aw ≈1.00 (0% NaCl, 0% MgCl2) positive; optimum at aw = 0.97 to 0.95 (4%-8% NaCl, 4%-6% MgCl2); maximum at aw = 0.78 (24% NaCl, 11% MgCl2).
Extracellular enzyme activities.
β-glucosidase at 0% to 10% NaCl; esterase at 0% NaCl; urease at 10% NaCl.
Typification.
Canada (Ontario, Puslinch), from peat soil in a cedar swamp, collected and isolated by G. C. Bhatt in May 1964, holotype, designated here, herbarium CBS H-22007, consisting of a freeze-dried living but metabolically inactivated deposit of spores and mycelium from MY50G. Living ex-type strain: MUCL 15061 = UAMH 2817 = EXF-6149.
Diagnostic characters.
Growth on media without additional solutes such as MYA or MEA, conidia 2.0–2.5 μm, halotolerance up to 24% NaCl, chaotolerance up to 11% MgCl2, no growth at 34°C, no urease activity without NaCl, no esterase activity at 10% NaCl, occurrence in temperate and cold climates.
Note.
Strain BF036 was reported as W. sebi, isolated from house dust of moisture-damaged buildings before and after renovations in Finland (GenBank number FR718458), but ITS sequences are nearly identical with the ex-type ITS sequence of W. canadensis. This species was referred to as W. sebi clade 3 by Desroches et al. [16] and Nguyen et al. [27].
Wallemia tropicalis Jancic, Nguyen, Seifert & Gunde-Cimerman, sp. nov. MycoBank: MB 810414 (Fig 7).
(a-l) Colony surface grown on MYA with no additions (a), and MYA with the addition of 20% (b), 50% (c) and 70% (d) sucrose, 20% (e) and 40% (f) glycerol, 4% (g) and 13% (h) MgCl2, and 8% (i), 16% (j), 24% (k) and 28% (l) NaCl. (m, n) Conidia (m) and conidiogenous cell (n) from MYA plus 50% sucrose. Colonies were incubated for 14 d at 24°C. Scale bars: 5 mm (l) (applies also for a-k), 5 μm (m, n).
Colony characters.
Colonies on MEA and MYA: 3–5 mm diam. after 2 weeks; growth rate, 0.22 mm d-1; punctiform, extending deeply into the agar; compact, pale brown or almost gray, without sporulation and exudates; margin brown or the same color as the colony, and irregular; dark gray reverse. Colonies on MY50G or MYA with addition of 50% sucrose: 9–11 mm diam. after 2 weeks; growth rate, 0.69 mm d-1; cerebriform in the central part and flat marginally, extending deeply into the agar; compact, brown to dark brown, powdery because of strong sporulation, without exudates; margin the same color as the colony, and irregular; gray reverse. Colonies on MYA with addition of 70% sucrose: 6–7 mm diam. after 2 weeks; growth rate, 0.51 mm d-1; flat, pale brown, weak sporulation, without exudates; margin white or pale brown, and irregular; gray reverse. Colonies on DG18 or MYA with addition of 20% glycerol: 7–9 mm diam. after 2 weeks; growth rate, 0.53 mm d-1; slightly cerebriform, extending deeply into the agar; compact, dark brown, sporulation weak, without exudates; margin the same color as the colony, and regular; gray reverse. Colonies on MEA or MYA with the addition of 16% NaCl: 4–5 mm diam. after 2 weeks; growth rate, 0.27 mm d-1; cerebriform, spreading into the agar; compact, pale brown, sporulation weak, without exudates; margin the same color as the colony, and irregular; gray reverse.
Physiology.
Growth at aw ≈1.00 (0% NaCl, 0% MgCl2); optimum at aw = 0.97 to 0.92 (4%-8% NaCl, 4%-6% MgCl2); maximum at aw = 0.78 (28% NaCl, 13% MgCl2).
Extracellular enzyme activities.
β-glucosidase at 10% NaCl; esterase at 0% NaCl; urease at 0% to 10% NaCl.
Typification.
Uruguay (Montevideo), from house dust collected by Z. Torrano in Dec 2008, culture isolated by K. Mwange, holotype, designated here, herbarium CBS H-22006, consisting of a freeze-dried living but metabolically inactivated deposit of spores and mycelium from MY50G). Living ex-type strain: EXF-8739.
Diagnostic characters.
Growth positive on media without additional solutes, such as MYA or MEA, conidia 2.0–3.0 μm diam., halotolerance up to 28% NaCl, chaotolerance up to 13% MgCl2, growth observed at 34°C, no β-glucosidase activity without NaCl, no esterase activity at 10% NaCl, occurrence in (sub)tropical climates.
Note.
Two isolates from the marine sponges Haliclona simulans (ITS, GenBank FJ755832) and Gelliodes carnosa (ITS, FJ770080) from tropical Hainan Island coastal water [72] may represent W. tropicalis. Their sequences are identical or nearly identical with the ITS sequence of ex-type strain EXF-8739. This species was designated W. sebi clade 4 by Nguyen et al. [27].
Discussion
The discovery of Wallemia, named after Mr. Wallem, a fishery inspector who sent Wallemia-infected fish for examination to Johan-Olsen in 1885, dates back to the 18th and 19th centuries, when clipfish was an important part of commercial fishing activities in Norway [73]. Species of Wallemia are among the few osmophilic fungi that can contaminate salted and dried cod. The monotypic genus included only W. ichthyophaga Johan-Olsen 1887 until von Arx in 1970 recognized Sporendonema sebi as a species of Wallemia [1], which provided the epithet for what today is the most frequently cited Wallemia species name.
From previous analyses of ITS sequences, it was suspected that W. sebi represents a genetically heterogeneous group consisting of at least two phylogenetically delineated clades [3]. In the companion study [27], genealogical concordance phylogenetic species recognition [67] based on sequences of the protein-encoding genes rpb2, rpb1, MCM7 and tsr1, clearly resolved the WSSC into four, statistically well-supported groups (clades 1–4). This conclusion was reaffirmed in this study by sampling strains from more diverse ecological niches. The approximately 350-bp partial sequence of HAL2 is used here for the first time as a phylogenetic marker gene. HAL2 contains a comparably high number of informative characters per site, is a single-copy gene [70], and easily amplifiable. Accordingly, HAL2 allows DNA barcode species identifications in Wallemia. Our phylogenetic analysis with HAL2, however, differed in some inferences in comparison to rpb2, rpb1, MCM7 and tsr1.
Our phylogenetic analyses infer that clade 1 members form a monophyletic and statistically supported group. Because this clade includes the ex-neotype strain of W. sebi CBS 818.96, it represents W. sebi sensu stricto. Strains of clade 1 typically originate from sea salt, hypersaline water, and air and dust in indoor environments from locations worldwide. It was also isolated from fresh water, seeds and other substrates with normal aw. Wallemia sebi can be disseminated through air and can colonize various low aw substrates.
Our molecular data suggest that clade 2, described above as W. mellicola, is closely related to W. sebi. Clade 2 representatives are clearly resolved as a strongly supported monophyletic group on the basis of all tested loci but ITS. Because of its relatively large conidia (2.5–3.0 μm diam.), W. mellicola is morphologically distinguishable from W. sebi, which has conidia that are 1.5–2.5 μm diam. Physiologically, W. sebi differs from W. mellicola by its degree of halotolerance and chaotolerance. Its growth characters on MEA or MYA (5–7 mm diameter after 2 weeks; growth rate, 0.36 mm d-1), and the cerebriform colony structure on MYA distinguish W. mellicola from other Wallemia species. Wallemia mellicola was isolated from hypersaline water of solar salterns, salty, sugary and dried food products, and air, dust and surfaces in indoor environments worldwide and occasionally also from soil, forest plants, seeds, straw and pollen.
Wallemia canadensis, formerly referred to as W. sebi clade 3 [16,27], is best distinguished by its lower degree of halotolerance (0%-24% NaCl) and chaotolerance (0%-11% MgCl2), its cardinal growth temperatures, and its unique set of secondary metabolites. Wallemia canadensis is the only species of the WSSC that does not grow at 34°C. It has an optimal growth temperature at 24°C whereas the other species in the WSSC have an optimum growth temperature at 30°C. Strains of W. canadensis were isolated from house dust and soil in temperate and cold climates of North America (Canada).
Wallemia tropicalis is represented by only a few strains in clade 4, and was isolated from house dust and soil in (sub)tropical climates of South America, North Africa, South Asia, and Oceania. Its high degree of halotolerance (0%-28% NaCl) may explain why it could be isolated from marine sponges [72]. Wallemia tropicalis resembles W. sebi, but can grow at salinities of up to 28% NaCl in vitro, and represents the most halophilic species of the WSSC. It is therefore possible that the two isolates from marine sponges also belong to W. tropicalis. Wallemia tropicalis is also characterized by its particularly slow-growing colonies on media that lack additional solutes, such as MEA or MYA.
Secondary metabolite profiles generated provide meaningful support for the taxonomic structure suggested in our study (see also [74]). The secondary metabolites profiles of W. sebi and W. mellicola are hardly distinguishable, and most of the identified or separated compounds are formed by strains of both species. Inventories so far performed clearly indicate that these two species have similar habitats and they both occur relatively frequently in various environments worldwide. Probably they can also co-inhabit similar habitats. Our phylogenetic analyses support this interpretation, because the close phylogenetic relationship of W. sebi and W. mellicola is highly supported. Similarly, the unique secondary metabolite profile of W. canadensis supports its status as a distinct species, and none of its secondary metabolites were encountered in W. sebi or W. mellicola. It is possible that W. canadensis occupies different ecological niches in nature where other secondary metabolites are required or of advantage. The same may apply to W. tropicalis, for which no secondary metabolites were detected with the methods applied here.
With the exception of a few cases, the species of the WSSC are not associated with humans or animals. The few available reports suggest that they may act as opportunistic human pathogens, unless W. sebi strain CBS 196.56, from human skin [3], and W. mellicola strain EXF-8754, isolated from a subcutaneous lesion [69], present accidental airborne contaminants, which also is possible. There is one report of W. sebi as a member of bovine rumen microbiota (GenBank number JX240410), and one of W. mellicola from dog intestine (GenBank number EU486095 [50]).
A few nomenclatural issues concerning the name W. muriae, representatives of which were used as an outgroup in our phylogenetic analyses, warrant comment. The putative synonym Hemispora stellata Vuill. originated in 1906, and the epithet muriae in 1867 as a variety (Torula epizoa var. muriae J.J.Kickx). Names and epithets do not have priority outside of their rank (Art. 11.2, ICN, [66]), and at first glance stellata would have priority over muriae. However, the epithet muriae was raised to species rank in 1902 by Vestergren as Torula muriae (J.J.Kickx) Vesterg [75]. Accordingly, W. muriae (J.J.Kickx) Zalar & de Hoog [3] is still the nomenclaturally correct name for this fungus. The identity of Oidium morrhuae Farlow 1886, included as a tentative synonym of W. muriae by Zalar et al. [3] is unknown, but its description from a fish suggests that it is more likely to be W. ichthyophaga. The type of O. morrhuae has not been re-examined by a modern author. Because of the traction that the name W. muriae has attained in the literature (about 30 citations in Google Scholar, and 280 nucleotide sequence accessions in GenBank, as determined in Feb. 2015), we recommend its eventual inclusion on the lists of protected fungal names being prepared by nomenclatural specialists (Art. 14.13, ICN).
In the present study, we described species that originally were recognized on the basis of multi-locus sequence analyses [27] with phenotypic characters that included micromorphology and macromorphology, xerotolerance, halotolerance and chaotolerance, cardinal growth temperatures, and profiles of extracellular enzyme activities and secondary metabolites profiles. Here, we complete the formal taxonomic process of describing new species within the WSSC and provide a key to all of the proposed Wallemia species, which allows their identification on the basis of physiological, micromorphological and culture characters.
Dichotomous key to Wallemia species
The micromorphological characters used in the key are from cultures grown on MY50G (MYA with addition of 50% sucrose) for 14 d at 24°C and 34°C; culture phenotypes and physiological characters are from colonies grown on MEA or MYA with the addition of sucrose (50%), NaCl (28%) and MgCl2 (15% and 17%) for 14 d at 24°C. For molecular barcode based identifications we suggest the generation of at least one protein-encoding marker in addition to the official fungal barcode ITS. Of the five loci tested here and in a companion study [27], TSR1 and HAL2 allow best DNA barcode based species identifications for the taxa of the WSSC.
1A —Colonies growing only on MYA or MEA with additional solutes (NaCl, glucose): conidia 2.5 μm to 5.0 μm diam.– 2
1B —Colonies growing on MYA or MEA without additional solutes: conidia 1.5 μm to 3.0 μm diam.– 3
2A —Colonies growing on MY50G: dark brown, with a cerebriform surface; conidia 3.5 μm to 5.0 μm diam.—W. ichthyophaga
2B —Colonies growing on MY50G: walnut brown, with a powdery surface; conidia 2.5 μm to 3.0 μm diam.—W. muriae
diam.diam.3A —Colonies growing on MEA or MYA plus 28% NaCl—5
3B —No growth on MEA or MYA plus 28% NaCl—6
4A —Colonies growing on MEA or MYA plus 15% to 17% MgCl2—W. sebi
4B —No growth on MEA or MYA plus 15% to 17% MgCl2—W. tropicalis
5A —Colonies growing at 34°C and on MEA or MYA 13% MgCl2—W. mellicola
5B —No growth at 34°C and on MEA or MYA plus 13% MgCl2—W. canadensis
Supporting Information
S1 Fig. Majority rule consensus tree of Bayesian MCMC sampling inferred from newly generated and GenBank accessed ITS sequences.
Bayesian posterior probabilities are displayed at the nodes of the tree. The tree was rooted to the sequence of W. muriae ex-type strain CBS 116628 (AY302534). Labels provide information on strain numbers, origin and strain status. Red T, ex-type strains; red NT, ex-neotype strain; bold, strains included in physiological and morphological studies, and extracellular enzyme activities; underlined, strains included in studies of secondary metabolites.
https://doi.org/10.1371/journal.pone.0125933.s001
(TIF)
S2 Fig. Comparison of pair-wise distance (p-distance), alignment length, and parsimony informative characters of HAL2.
The grey bars show the distribution range of the p-distance within clades while the blue bars show the range of p-distances between clades. The mean p-distances and the number of observations (N) used to calculate each mean are shown. AL = alignment length in base pairs. PIC = number and percentage of parsimony informative characters in the alignment.
https://doi.org/10.1371/journal.pone.0125933.s002
(TIF)
S3 Fig. Principal component analysis based on the production of secondary metabolites of the groups of strains from clades 1–3.
No compounds were detected for clade 4 members grown on YES agar and CYAS.
https://doi.org/10.1371/journal.pone.0125933.s003
(TIF)
S1 Table. Strains included in the present study, with their original sources and GenBank accession numbers for ITS, MCM7, TSR1, RPB1, RPB2 and HAL2 sequences.
Sequences for MCM7, TSR1, RPB1 and RPB2 from strain CBS 633.66 were extracted from the Joint Genome Institute (JGI) MycoCosm site.
https://doi.org/10.1371/journal.pone.0125933.s004
(XLSX)
S3 Table. Alignment properties, selected nucleotide substitution models, Bayesian analysis settings, and the Bayesian posterior probabilities for clades 1–4.
https://doi.org/10.1371/journal.pone.0125933.s006
(XLSX)
S4 Table. Growth parameters of W. sebi, W. mellicola, W. tropicalis, and W. canadensis on MYA with different water activities.
https://doi.org/10.1371/journal.pone.0125933.s007
(XLSX)
S5 Table. Secondary metabolite patterns detected by HPLC analysis during the growth of W. sebi, W. mellicola, W. tropicalis, and W. canadensis on YES agar and CYAS.
https://doi.org/10.1371/journal.pone.0125933.s008
(XLSX)
S1 File. Trees resulting from single gene phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0125933.s009
(PDF)
Acknowledgments
We thank Karl-Henrik Larsson (Natural History Museum, University of Oslo) for comments on papers written in Norwegian, and the numerous collectors who have submitted their valuable strains to the various culture collections. Special thanks go to Ellen Kirstine Lyhne and Nastja Pirman for technical support.
Author Contributions
Conceived and designed the experiments: SJ HDTN JCF KAS NGC. Performed the experiments: SJ HDTN JCF. Analyzed the data: SJ HDTN JCF. Contributed reagents/materials/analysis tools: SJ HDTN JCF PZ KAS NGC. Wrote the paper: SJ HDTN JCF PZ HJS KAS NGC.
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