Global Molecular Diversity of the Halotolerant Fungus Hortaea werneckii

A global set of clinical and environmental strains of the halotolerant black yeast-like fungus Hortaea werneckii are analyzed by multilocus sequencing and AFLP, and physiological parameters are determined. Partial translation elongation factor 1-α proves to be suitable for typing because of the presence/absence of introns and also the presence of several SNPs. Local clonal expansion could be established by a combination of molecular methods, while the population from the Mediterranean Sea water also responds differently to combined temperature and salt stress. The species comprises molecular populations, which in part also differ physiologically allowing further diversification, but clinical strains did not deviate significantly from their environmental counterparts.


Introduction
Knowledge on fungi living under extreme conditions has increased significantly over the last two decades. Where general hypotheses of survival under hostile conditions concerned dormancy or refractive sporulation stages in the past, we now know of the existence of a wide array of phylogenetic diverse, highly adapted fungi that produce assimilative, growing thallic in the extreme. Examples are found in the cold [1], at high temperatures [2], with toxic hydrocarbons [3] or ultra-low pH [4], at high osmolarity [5], or on rock [6,7]. The halophilic black yeast H. werneckii is one of the most salt tolerant eukaryotic organisms so far described [8]. It is characterized by melanin production, pleomorphism of yeast and filamentous phases, and meristematic development [9,10]-characters we also observe in numerous rock-inhabiting fungi [6]. In culture it reproduces clonally, a sexual state is not known. The fungus has a global distribution in seawater and adjacent habitats, such as sea sponges, marine and salted freshwater fish, corals, microbial mats in salterns, beach soil, salt marsh plants and salted food [11][12][13]. The fungus is consistently encountered in deep Mediterranean Seawater [14].
The primary ecological niche of H. werneckii involves hypersaline waters, the fungus enriched to be dominant in solar salterns [15,16]. Hortaea werneckii has a broad range of growth from zero to 30% NaCl (w/v) [17,18], but with an optimum value of around 15% of salt [19]. The fungus is in use as a model for studying the molecular and physiological basis of salt tolerance in eukaryotes [17,19,20].
Hortaea werneckii has also been described as the cause of human tinea nigra, a superficial infection limited to the dead surface of the skin (stratum corneum), which mostly occurs in warmer climates [21,22]. The infection is mild, but patients tend to be worried because of similarity with serious skin diseases, and therefore the disorder is noteworthy. Questions are whether clinical strains differ from their environmental counterparts, and whether subtypes exist, which might allow pathogenic adaptation. For this reason, we compared growth responses at 25 and 37 • C. The present study was carried out to determine the molecular epidemiology of H. werneckii isolated from different sources (environmental and clinical) on a global scale, including the Mediterranean Seawater strains that were collected at a depth of up to 3402 m during two oceanographic cruises in December 2013 and March 2017. We applied several molecular typing methods, and analyzed physiological parameters.

Fungal Strains
Sixty-seven strains of Hortaea werneckii originating from a wide diversity of geographic localities and sources were considered in this study (Table 1). Twenty-five were collected from the Mediterranean Sea during the oceanographic cruises, DEEP-PRESSURE and VENUS-4, on board of the Research Vessels R/V URANIA (December 2013) and MINERVA 1 (March 2017), respectively, and maintained in the collection of the Department of Chemical, Biological Pharmaceutical and Environmental Sciences of University of Messina (Italy). Seawater was sampled from different stations located in the central and south-east of the Mediterranean Sea from the surface up to 3402 m of depth. Aliquots of sampled seawater were immediately filtered with nitrocellulose filters of a 0.45-µ pore size (Millipore, MI, Italy); filters were then placed face up on the seawater medium (1% glucose, 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 2% agar in filtered seawater) [23] and incubated at room temperature up to two weeks. After growth, enumeration of fungi was carried out as propagules/L of samples and colonies were randomly chosen and isolated on malt extract agar (MEA, Oxoid, Basingstoke, England) at 25 • C and stored on potato dextrose agar (PDA) and MEA (Oxoid) at 4 • C. Genetic and physiological comparisons were done with strains acquired from the reference collection of Centraalbureau voor Schimmelcultures (housed at the Westerdijk Fungal Biodiversity Centre, Utrecht, The Netherlands).

Growth at Different Salinities at 25 • C and 37 • C
Forty-four strains representative of the entire set (14 from the Mediterranean water and 30 from the CBS collection) were used for testing growth at different concentrations of salt and at 25 and 37 • C. A loopfull of colonies grown on MEA was suspended in 500 µL of sterile demineralized water and vortexed for 10-20 s at the maximum speed. The suspension was line-streaked on plates of MEA without NaCl, and with 15%, 20%, and 25% (w/v) NaCl. Colony diameters were measured after 15 days of incubation.

DNA Extraction and Sequencing
Genomic DNA was extracted following the cetyltrimethylammonium bromide (CTAB) protocol [24]. DNA was quantified with a NanoDrop ® ND-1000 Spectrophotometer (Thermo Fisher, Wilmington, NC, USA), and samples were stored at −20 • C until use. Internal transcribed spacer region (ITS) and the partial translation elongation factor-1α (TEF1) were amplified with primer pairs-ITS1 and ITS4 [25] and EF1-728F [26] and EF1-1567R [27], respectively. PCR mixtures were prepared as previously described [28]. Cycling conditions included 95 • C for 5 min, followed by 30 cycles consisting of 95 • C for 45 s, 48 • C for 30 s and 72 • C for 1 min, and a post-elongation step at 72 • C for 8 min for ITS; and one cycle of 5 min at 95 • C, followed by 40 cycles of 45 s at 94 • C, 35 s at 52 • C and 1.20 min at 72 • C and a post-elongation step of 8 min at 72 • C for TEF1. PCR products were visualized by electrophoresis on 1% (w/v) agarose gels. PCR amplicons were sequenced in both directions using standard conditions with a BigDye TM v3.1 terminator cycle sequencing kit (Applied Biosystems, Bleiswijk, The Netherlands). Sequences were edited using SeqMan in the Lasergene package (DNAstar, Madison, WI, USA). GenBank accession numbers are shown in Table 1.

Amplified Fragment Length Polymorphism Genotyping
Amplified Fragment Length Polymorphism (AFLP) genotyping was done as previously described [29]. In brief, genomic DNA was subjected to restriction-ligation with a mixture containing a HpyCH4 IV adapter, a MseI adapter, 2U of HpyCH4 IV, 2U of MseI and 1U of T4 DNA ligase for 1 h at 20 • C. Reaction products were diluted and combined with a size marker, Orange600 (Nimagen, The Netherlands), followed by a heating step for 1 min at 100 • C and cooling down to 4 • C. Fragment analysis was carried out using an ABI3500xL Genetic Analyzer (Applied Biosystems). Data were evaluated using BioNumerics v7.5 (Applied Maths, Sint-Martens-Latem, Belgium) via UPGMA clustering with the Pearson's correlation coefficient. Only DNA fragments in the range of 40-400 bp were taken into account.

Haplotype Networks
The distribution of haplotypes based on sequences data of TEF1 of the studied isolates was determined by PopArt v1.7 [30]. Briefly, a haplotype nexus-file was created with DnaSP 5.10 [31]. Gaps in the alignment were not considered creating haplotypes based on nucleotide differences. The file was manually prepared for PopArt to add the geographical origin of the isolates, if known. A median joining network was created. The lengths of the lines in the network do not correspond with the nucleotide differences, which are indicated by tickmarks and connectors.

Sequencing
Sequencing of the rDNA ITS region and subsequent comparison in GenBank and in an in-house black yeast database maintained at the Westerdijk Institute confirmed all 67 strains belong to Hortaea werneckii, with identity ≥ 99%, e-value: 0, without gaps. Sixteen SNPs were detected in the dataset. Sequences of the partial TEF1 gene were aligned with MAFFT version 7.402 (https://mafft.cbrc.jp) followed by manual adjustment with MEGA v.7. The resulting alignment revealed the presence of one, two or none introns in the partial sequence. Based on the number of SNPs in the introns, strains could be assigned to three main groups.

Physiology
Growth responses at different salt concentrations and at two temperatures are summarized in Table 2. At 25 • C, all strains considered were able to grow on MEA without addition of NaCl. The majority of strains grew up to a NaCl concentration of 25% in the medium, except for CBS 107.67, CBS 116.30, CBS 126.35, CBS 123041, CBS 110352 and CBS 410.51 where limited growth (<4 mm) was observed at 20% NaCl. Excellent growth (≥10 mm/15 d) occurred in all isolates from the Mediterranean Sea on the medium without additional NaCl, with 15% NaCl colony expansion dropped to 4-6 mm/15 d. The majority of clinical strains and strains from environmental habitats grew equally well at 0% and 15% of additional NaCl, with no or little difference between these salt concentrations: 15/30 strains were indifferent on these conditions, 9/30 strains were inhibited, and 6/30 were stimulated at 15% NaCl.
At 37 • C, the majority of all strains investigated was able to grow in the range from 0% to 20% NaCl, while none grew in NaCl with a concentration of 25% (w/v) ( Table 2). Mediterranean Sea strains grew much slower (4-6 mm/15 d) than at 25 • C, but all except one grew equally well with 0% and 15% of additional NaCl. None grew at 37 • C at the concentration of 25% NaCl. Remaining clinical and environmental strains showed poor or no growth on media without additional NaCl, but invariably grew significantly better with 15% salt: in six strains, the stimulus was from zero to ≥ 10 mm diam/15 d. Thirteen strains, among which three clinical strains, i.e., CBS 116.30, CBS 708.76 and CBS 126.35, grew at 37 • C exclusively when incubated on media with 15% or 20% of additional NaCl.

Amplified Fragment Length Polymorphism Genotyping
Frequently cutting restriction endonucleases HpyCH4IV and MseI and two selective primers were used in the AFLP analysis to fingerprint the genomes of 67 strains of H. werneckii (Figure 1). Two main clusters, A and B, were apparent at a profile similarity of 60%. Several subgroups were observed within the two main clusters. All isolates from the Mediterranean Sea grouped in cluster A (subgroup A1), representing the largest part of the strains in that cluster (25/34). Nearly all Mediterranean strains were very close, with similarities ranging from 97% to 100%, but strain MC 857 had a similarity of 90% to its nearest neighbor in group A. Cluster A also comprised seven strains of the environmental origin from Europe, Brazil, and from an unknown origin, plus a tinea nigra strain from Brazil CBS 111.31 (subgroup A2).
Cluster B included all H. werneckii isolates from tinea nigra in Mexico, which were highly similar to each other (97-100%). These strains showed high genetic relatedness (~95%) to the H. werneckii type strain, CBS 107.67 from tinea nigra in Portugal (subgroup B1). Cluster B further comprised 12 environmental strains from Brazil, Greece, Japan, Puerto Rico, Senegal, Slovenia, Spain, Sri Lanka, Sudan, and one from an unknown country, in addition to six clinical strains from France, Italy, Suriname and from unknown countries (subgroup B2).

Haplotype Network
On the basis of SNPs in TEF1 sequence data, 29 haplotypes could be distinguished. These were plotted in a network, where presence/absence of two introns is shown (Figure 2). Two main clusters of closely interrelated haplotypes could be distinguished, I and II, with Hap 11 at a considerable distance, and Haps 1, 9, 10 and 29 loosely affiliated to cluster II. Members of both clusters were geographically diverse, but strains from the Mediterranean Sea in cluster I and from Mexico in cluster II were closely linked with the maximum of one to two mutations between strains. Haplotype 27 showed a high rate of geographical diversity, with isolates from Senegal, Brazil, Surinam, France and an unknown country.
Results obtained from sequencing of partial TEF1 and from AFLP are almost concordant. The two main clusters, A and B, obtained from the AFLP analysis matched, with some exceptions, with the two clusters observed in the Haplotype network of TEF1 (Figure 2). In fact, the strains presented in AFLP cluster A grouped in different haplotypes of cluster I in the network, except for CBS 100456

Discussion
Hortaea werneckii is a halophilic black yeast in the order Capnodiales which is phylogenetically clearly demarcated from its nearest neighbor, Penidiella venezuelensis [33]. Its sexual cycle is still unknown. Whole genome analyses of H. werneckii showed the presence of the mating type idiomorph MAT1-1, while MAT1-2 was absent, suggesting that the species is heterotallic [32,34]. The fungus has an exceptionally large, duplicated genome [32] which interferes with sequencing of most housekeeping genes because of amplification of mutated alleles in a single strain. Among the available epidemiological typing techniques, AFLP stands out as an option for the analysis of genetic diversity between populations. Sequence data of partial TEF1 proved to be significant because of the occurrence of numerous SNPs, and of the absence or presence of two introns, while ITS rDNA contains only a limited number of SNPs. These data are summarized in Figure 2. Combining the applied markers, significant intra-specific diversity was revealed, which may shed light on the species' routes of dissemination.
Hortaea werneckii has a global distribution in haline habitats. The fungus occurs at low density in seawater [35], but emerges exponentially in (sub)tropical coastal areas when salt levels increase until the saturation point is reached [19]. The fungus is extremely abundant in natural and artificial crystallization pans. Another ecological niche of the fungus has been published as human skin. The fungus causes black spots on the palms of human hands, known as 'tinea nigra', and for this reason, Hortaea has been considered as a human pathogen in the past [36]. However, Göttlich et al. [37] noted that the disorder by this non-keratinolytic fungus is nearly confined to hands and sometimes feet of hyperhydrotic individuals after a day at the beach, and thus asymptomatic colonization of exceptionally salty skin was a likely explanation of this phenomenon. Hortaea werneckii thus can be viewed as a fungus unambiguously associated with high salt. Infraspecific diversification might allow adaptation to the habitat of the human skin. In the present study, we observed that most strains, when subjected to temperature stress of 37 • C, grew much better when they were subjected to salt stress of 15% of NaCl, underlining its halophilic nature; apparently, the fungus does not experience the increased salt level as stress, but rather as its optimal condition. This was, however, not the case with the strains from the Mediterranean Sea. A possible explanation is that most strains were derived from deep seawater, which may be less often related to the hypersaline environment of coastal saltpans. Clinical strains were not located in this clade, and did not deviate significantly from their environmental counterparts; no adaptive trend to the human host was revealed.
Global distribution of H. werneckii was studied by combined partial TEF1 sequencing and AFLP profiling. TEF1 proved to be highly suitable to characterize populations, not only by a number of SNPs, but also by two introns that were independently absent or present. AFLP patterns showed intraspecific diversity with two main groups of profiles. This level of diversity is higher than that known in, for example, Cryptococcus, where profiles are nearly monomorphic within sibling species [38], or in anthropophilic dermatophytes [39]. High intraspecific diversity is known, for example, in Aspergillus fumigatus, where strains, even when sampled on a single location, may deviate significantly from each other [40]. In our AFLP dataset, several groups of strains with nearly identical profiles clones and limited geographic distributions were recognizable. Particularly, the strains from the Mediterranean Sea proved to be highly similar. This was corroborated by their response to salt stress, which deviated from that of the remaining strains. In addition, some isolates from Mexico, all from human tinea nigra were highly similar. Intron distribution profiles reinforce similarity of geographically adjacent isolates. This level of molecular similarity and close geographic vicinity of isolates suggests local, possibly clonal dispersal. An important exception is Haplotype 27, containing strains from Europe, Africa and South America.
The group of strains from the Mediterranean Sea consistently deviates from remaining strains, not only molecularly but also by being insensitive to salt stimulation for survival at temperature stress. This group is thus less clearly halophilic. The AFLP dendrogram showed that there is no genetic variability among the strains originating from different stations and depths. This ecologically relevant difference might allow separating the evolution of this cluster of strains, which, however, does not imply any clinical adaptation.