Water is essential to life, and life can only exist within a narrow range of water availability in a particular environment, expressed as water activity (a_w). Water activity is the effective water content expressed as its mole fraction, therefore pure water has a_w = 1, all the other solutions have a_w < 1. Types and amounts of solutes present in the environment lower a_w to various values and exert additional effects on the growth of microorganisms—causing osmotic pressure and/or have toxic effects. The lowest a_w known to support life is 0.61, measured for the xerophilic fungus Xeromyces bisporus grown on sugar-based media (Pitt and Hocking, 2009), and also for some halophilic Archaea and Bacteria (Stevenson et al., 2014). Many fungi are able to thrive at low a_w, especially the numerous xerophilic filamentous fungi and osmophilic yeasts that grow on drying foods or on foods with high concentrations of sugars (Pitt and Hocking, 1977, 2009).

In the past, fungi were not renowned for growth at high salt concentrations. However, after the first record of fungi as active inhabitants of solar salterns were published (Gunde-Cimerman et al., 2000), the study of halotolerant and halophilic fungi expanded. Since that time numerous fungal species thriving in extremely saline environments around the globe have been described, most of them being halotolerant and extremely halotolerant, and few are obligate halophiles (reviewed in Zajc et al., 2012). The most halophilic fungus known to date is Wallemia ichthyophaga as it requires at least 10% NaCl and grows also in solutions saturated with NaCl (Zalar et al., 2005; Zajc et al., 2014).

Fungi have some common characteristics of osmotolerance, for instance they all employ the compatible solutes strategy: they balance the osmotic pressure of the surroundings by accumulating small organic molecules (compatible solutes), most commonly glycerol, and maintain low intracellular concentrations of salt (such as toxic Na⁺ ions) (reviewed in Gostinčar et al., 2011; Zajc et al., 2012). Sensing and responding to turgor stress (either due to organic osmolytes or due to salt) is under the control of the high osmolarity glycerol (HOG) signaling pathway in all halotolerant and halophilic fungi (Gostinčar et al., 2011). The activation of the HOG pathway results in the production of glycerol, which restores the osmotic balance of the cell (Hohmann, 2009). The cells are equipped with channels allowing for a quick expulsion of glycerol, as well as its active intake when required (Luyten et al., 1995; Ferreira et al., 2005). As the concentration of glycerol is carefully regulated, this strategy allows more flexible adaptations to changing salinity. Besides energetically costly synthesis of high concentrations of organic solutes, the cells also use much energy by using different efflux and influx systems to actively eliminate surplus ions, to preserve membrane potential, regulate intracellular pH, and maintain positive turgor of the cell. Hence, the alkali-metal cation transporters are of high importance of the osmoadaptation to extremely saline environments. In fact, the Na⁺- exporting ATPase (EnaA) is the major determinant of salt tolerance in yeasts (reviewed in Ariño et al., 2010). In addition to the above active mechanisms, fungi also employ some strategies for increasing their stress resistance that may be referred as passive—like clustering cells in compact cell clumps (Palkova and Vachova, 2006; Kralj Kunčič et al., 2010), covering the cells with abundant extracellular polysaccharides or increasing the thickness (Kralj Kunčič et al., 2010), and pigmentation/ melanization (Selbmann et al., 2005; Kogej et al., 2006) of the cell wall.

As most hypersaline environments are rich in NaCl, salt tolerance of fungi and other microorganisms, and mechanisms of adaptations were generally tested by using only NaCl as the solute. Therefore, the responses to high concentrations of other chaotropic salts remained unknown. However, other salts such as MgCl₂ are also abundantly present in nature and can be important or even life-limiting. Salts in the environment not only lower the biologically available water and cause toxicity due to the penetration of certain cations into the cell, but they also modify structural interactions of cellular macromolecules. The Hofmeister series of ions (K⁺ > Na⁺ > Mg²⁺ > Ca²⁺; SO₄²⁻ > HPO₄²⁻ > Cl⁻ > NO₃⁻ > Br⁻ > ClO₃⁻ > I⁻ > ClO⁻₄) describes the order of the ability of ions to salt-out or salt-in proteins (Hofmeister, 1888; Kunz et al., 2004). This phenomenon is based on direct interactions between ions and macromolecules and on interactions between ions and water molecules in the first hydration shell of the macromolecule (Zhang and Cremer, 2006). Hofmeister effects of ions on biological structures are either kosmotropic or chaotropic; chaotropes weaken electrostatic interactions and destabilize biological macromolecules, whereas the contrary is true for the kosmotropes (reviewed in Oren, 2013). The difference among the kosmotropic effect of NaCl on one hand and the chaotropic effect of MgCl₂ and CaCl₂ on the other hand might explain why high concentrations of Mg²⁺ and Ca²⁺ are toxic even to the most halophilic microorganisms (McGenity and Oren, 2012). However, to some extent the chaotropic effects of Mg²⁺ and Ca²⁺ can be counteracted by the presence of kosmotropic ions (Williams and Hallsworth, 2009). In fact, few halophilic Archaea can grow at high concentrations of MgCl₂, but only in the presence of significant concentrations of NaCl (Mullakhanbhai and Larsen, 1975; Oren, 1983; Oren et al., 1995). This confirms an early study of interactions among kosmotropic and chaotropic ions on the growth of the halophilic alga Dunaliella salina performed by Baas Becking, who discovered that toxicity of Ca²⁺ ions was diminished in the presence of sodium ions (Baas Becking, 1934; Oren, 2011).

Two types of hypersaline brines are distinguished with respect to their origin of formation; thalassohaline and athalassohaline (Oren, 2002). Thalassohaline waters, such as marine ponds, salt marshes and solar salterns, originate by evaporation of sea water and are therefore dominated by sodium and chloride ions. During the progression of evaporation, ionic composition changes due to the consecutive precipitation of calcite (CaCO₃), gypsum (CaSO₄·2H₂O), halite (NaCl), sylvite (KCl) and final carnalite (KCl·MgCl₂·6H₂O) after their solubilities have been surpassed (Oren, 2002, 2013). The major change in the ratio of divalent and monovalent cations occurs when the total salt concentration exceeds 300–350 g l⁻¹ and most of the sodium (as halite) precipitates. In the remaining brine, so-called bittern, the dominate ion becomes Mg²⁺ (Oren, 2013).

While NaCl-rich (thalassohaline) environments are well known to support a rich biodiversity, including of fungi, very little is known about the occurrence of fungi and other microorganisms in athalassohaline, MgCl₂- and CaCl₂- dominated environments. Several fungi were isolated from the magnesium and calcium-rich water of the Dead Sea (Oren and Gunde-Cimerman, 2012) [~2.0 M and ~0.5 M, respectively; total dissolved salts concentration ~350 g l⁻¹ (Oren, 2013); water activity ~0.683 (at 35°C) (Hallsworth, personal communication)]. However, most frequently isolation media were supplemented with different NaCl concentrations (reviewed in Oren and Gunde-Cimerman, 2012) rather than with chaotropic ions such as magnesium and calcium. Recently fungal strains were isolated from the bittern brines of solar salterns (Sonjak et al., 2010), an environment earlier considered sterile due to the high concentrations of magnesium salts (Javor, 1989). These fungal strains showed elevated tolerance to MgCl₂, a phenomenon not yet reported for fungi. This raised the issue of the existence of chaophiles among extremophilic fungi. To address the question whether chaotolerant/chaophilic fungi may exist, we have examined a range of them both from bitterns, the Dead Sea and other extreme environments, as well as reference strains from culture collections for their ability to grow at high concentrations of various chaotropic as well as kosmotropic salts.

Few studies have addressed the issue of the tolerance of microorganisms to chaotropic conditions over the years (reviewed in Oren, 2013). Searching for the chaophilic strains from the hypersaline deep-sea Discovery Basin, an environment with the highest salinity ever found in the marine environments—the brine is almost at saturated levels of MgCl₂ (5.15 M) (van Der Wielen et al., 2005), did not reveal any prokaryotic representatives (Hallsworth et al., 2007). Instead, a fungus X. bisporus, with the lowest a_w limit so far reported to support life (Pitt and Hocking, 2009), was the first described species of having the preference to chaotropic conditions/solutes as it was able to grow in highly chaotropic media containing up to 7.6 M glycerol (Williams and Hallsworth, 2009) and was markedly intolerant to NaCl (Pitt and Hocking, 1977). Importantly, its growth on chaotropic solutes like MgCl₂ and CaCl₂ was not tested.

Indeed, fungi are promising candidates for chaophiles as they can thrive in the environments, such as crystallizer ponds of solar salterns (Gunde-Cimerman et al., 2000; Butinar et al., 2005a,b), hypersaline water of the Dead Sea (reviewed in Oren and Gunde-Cimerman, 2012) as well as the brine channels of sea ice (Gunde-Cimerman et al., 2003; Sonjak et al., 2006). As these fungi have not previously been examined for their ability to grow in media dominated by chaotropic ions, we have carried out an extensive screening of tolerance to various salts.

Our search for the chaophilic characters of fungi based on their isolation from bittern brines (Sonjak et al., 2010), residual water after the precipitation of NaCl, which is highly enriched with magnesium salts, mostly MgCl₂. These brines were long considered sterile as high concentrations of Mg²⁺ are often toxic for biological systems. However, it was shown recently that bittern brines of the Sečovlje salterns (Slovenia) are not completely free of living microorganisms. They harbor different filamentous fungi, Cladosporium spp., black and other yeasts, albeit their abundance and biodiversity is low when compared to the hypersaline water of the salterns (Sonjak et al., 2010). The lower diversity and abundance might be a consequence of a combination of various factors in situ, such as prolonged exposure to solar radiation and magnesium, its life-limiting effect and nutrient availability.

However, the ionic composition of the bittern brine is not completely unfavorable for microbial growth despite extremely low water activity (0.737); the level of toxic ion Mg²⁺ is compensated by a relatively higher concentration of Na⁺. An outstanding discovery here was that these fungi isolated either from brine rich in MgCl₂ or NaCl were able to grow at high concentrations of MgCl₂(1.5 M) (Sonjak et al., 2010)—higher than previously reported for prokaryotes (1.26 M MgCl₂) (Hallsworth et al., 2007). This observation led to the study of the ability of a list of fungi, composed of the isolates from the Dead Sea and the reference strains from our culture collection, to grow in media with low a_w due to high concentrations of not only kosmotropic salts (NaCl, KCl, MgSO₄) but also chaotropic salts such as NaBr, MgCl₂, and CaCl₂.

Among the extremophilic fungi included in our study, 104 (almost 80% of the strains) were able to grow at concentrations of MgCl₂ higher than 1.5 M, and among these 16 (12%) were able to grow at the highest concentrations of MgCl₂(≥2.0 M). Next, 56 (41.5% of the strains) were capable of growth at concentrations of CaCl₂ higher than 1.5 M, with two of these able to grow at the highest concentration (2.0 M).

The decision trees (more specifically PCTs) obtained by machine learning analysis in various scenarios (Figure 1) revealed key types of salts influencing the ability of growth of fungi. The most important salts for the limitation of fungal diversity turned out to be the chaotropic salts MgCl₂, CaCl₂, and NaBr, whereas KCl and NaCl appeared to be the least limiting and were not present in the nodes of the decision tree. The first decision tree (Figure 1A) revealed that 37 strains, including 11 strains of H. werneckii and W. ichthyophaga can cope with MgCl₂ concentrations higher than 1.8 M. Here, the majority of strains of W. ichthyophaga were unique in their ability to tolerate the highest concentrations of MgCl₂, but not CaCl₂; whereas almost all strains (except for one instance) of H. werneckii could grow at the highest concentrations of all tested salts. Another key variable distinguishing the tested fungal strains is pigmentation, which is at the top of the decision tree using all the variables available. However, melanization is known for its role in UV and other stress responses including in osmoadaptation in halotolerant fungi (Jacobson and Ikeda, 2005; Kogej et al., 2007). Melanin impregnates the outer layer of the cell wall, this decreasing the porosity of the cell wall in order to retain more glycerol, which is most often the main compatible solute (Kogej et al., 2007). Next, cell morphology also appeared high in the decision trees. The ability to form dense clumps of meristematic cells, as observed for W. ichthyophaga and Phaeotheca triangularis, also impacts the ability of fungi to live in stressful conditions (Wollenzien et al., 1995; Palkova, 2004; Palkova and Vachova, 2006).

A simple determination of the type of salt to allow growth of individual strains at the lowest a_w revealed that the largest number of fungi thrived in the media with the lowest a_w when NaCl (52; 38.8%) or KCl (42; 31.3%) were used as the main solutes. On the contrary, less than 10% of strains were able to grow in the presence of chaotropic salts, MgCl₂ (10; 7.5%) and CaCl₂ (2; 1.5%), at their lowest a_w. This again emphasizes the life-limiting effects of chaotropic salts. Whether these fungi have the preference for chaotropic salts is inconclusive, as most of them are able to grow also at the highest concentrations of other—kosmotropic—salts. Nevertheless, the fact that they are not only tolerating but growing at such high concentrations of magnesium and/or calcium salts makes these strains the most chaotolerant organisms described so far.

For comparison, the highest concentration of salts to support growth of X. bisporus given our results (Table 1) were 2.5 M NaCl, 3.5 M KCl, 2.5 M MgSO₄ and 2.5 M NaBr, albeit growth was poor. In addition it was also able to grow at 1.5 M MgCl₂ and 1 M CaCl₂. For the inhibition of growth of X. bisporus the a_w of the medium was clearly not the determining factor—the lowest a_w of media tested in our study was 0.867 for medium containing 3.5 M KCl, which is far above its lowest a_w enabling growth in a glycerol-based medium (Pitt and Hocking, 1977). Here, it seems that high concentrations of salt, regardless of their chao- or kosmotropicity, limit the growth of X. bisporus, which clearly prefers sugar-based media as previously reported. Its chaophilic character on organic solutes such as glycerol (Williams and Hallsworth, 2009) must be reconsidered with caution when addressing ionic chaophilic solutes. Poor growth in the presence of salt might be a consequence of the absence of a gene coding for Na⁺-exporting ATPase (Ena) in the genome of X. bisporus (Leong et al., 2014), whereas this pump is present in multiple copies and/or is differentially expressed in the extremely halotolerant H. werneckii (Gorjan and Plemenitaš, 2006; Lenassi et al., 2013) and the halophilic W. ichtyophaga (Zajc et al., 2013).

Hortaea werneckii is a representative of the polyphyletic group of black (melanized) yeasts that have filamentous and yeast-like growth. It is able to grow across the whole range of NaCl concentrations, from 0 M to saturation, with a broad optimum from 1 M to 2.4 M NaCl (Butinar et al., 2005b), and it is thus considered to be the most extremely halotolerant fungus so far described (reviewed in Gostinčar et al., 2011). Amongst all of the melanized fungi H. werneckii is the most abundant in the hypersaline water of salterns (Gunde-Cimerman et al., 2000). Our screening revealed that H. werneckii strains are able to grow at the highest tested concentrations of salts; in media saturated with kosmotropes (5.0 M NaCl, 4.5 M KCl, 3.0 M MgSO₄) and the highest tested concentrations of chaotropes (2.1 M MgCl₂, 1.7 M CaCl₂, and 4.0 M NaBr) (Table 1 and Figure 2). This exceptional ability might be linked to the redundancy of plasma membrane Na⁺ and K⁺ transporters encoded in its duplicated genome (Lenassi et al., 2013).

The genus Aureobasidium (de Bary) G. Arnaud is a wide-spread osmotolerant (Kogej et al., 2005) representative of black yeast associated with numerous habitats from hypersaline waters, Arctic glaciers, plant surfaces and household dust (reviewed in Gostinčar et al., 2014). In the genus, recently four new species were introduced A. pullulans, A. melanogenum, A. subglaciale and A. namibiae in Gostinčar et al. (2014). All of them are described as polyextremotolerant (Gostinčar et al., 2010, 2011) capable of surviving also hypersaline conditions (Gunde-Cimerman et al., 2000). The maximum concentrations of NaCl supporting growth of A. pullulans was reported to be 2.9 M NaCl (Kogej et al., 2005). Our study confirmed the upper limit of NaCl for Aureobasidium sp. and revealed its ability for growth at high concentrations of KCl (4.0 M) and MgSO₄ (3.0 M), but not extremely high concentration of MgCl₂ (lower than 1.5 M) and CaCl₂ (up to 1.2 M). Aureobasidium spp. can thus be considered kosmophilic. Recent genome analysis uncovered a large repertoire of plasma-membrane transporters in the four Aureobasidium species (Gostinčar et al., 2014). A. melanogenum, which is heavily melanized, is able to grow at the highest concentrations of all salts among the four tested species of the genus Aureobasidium, and the least melanized A. subglaciale on the other hand thrives at the lowest. Here, it seems that melanization is required for the highest salt tolerance. The role of melanin in osmoadaptation was shown previously by modifying the permeability of the cell wall in order to retain the compatible solute glycerol (Jacobson and Ikeda, 2005; Kogej et al., 2006).

Representatives of the cosmopolitan genus Cladosporium are frequently found in habitats characterized by low a_w, like foods preserved with sugar or salt (Samson et al., 2002), salt marshes of Egypt, in the rhizosphere of halophytic plants, and the phylloplane of Mediterranean plants (Abdel-Hafez et al., 1978). They are therefore considered xerotolerant with 0.82 being the minimal a_w for growth of Cladosporium sphaerospermum (Hocking et al., 1994). Cladosporium spp. are among the most abundant melanized fungi throughout the year in the solar salterns in Sečovlje (Gunde-Cimerman et al., 2000; Butinar et al., 2005b) and Cabo Rojo in Puerto Rico (Cantrell et al., 2006). Five species of the genus Cladosporium were isolated from the Dead Sea (reviewed in Oren and Gunde-Cimerman, 2012). The highest concentration of NaCl for in vitro growth of various representatives of the genus Cladosporium was reported to be 2.9 M to 3.5 M (Zalar et al., 2007). Strains of the genus Cladosporium exhibited variable tolerance to different types of salts, ranging from the lowest concentrations used in the study to the highest (Table 1). The highest growth concentrations of kosmotropic NaCl, KCl and MgSO₄ among Cladosporium spp. are respectively 2.5–4.0 M, 2.5–4.5 M and 2.0–3.0 M. Two strains, C. tenuissimum EXF-1943 and C. cladosporoides EXF-1824, were able to grow at 2.0 or 2.1 M MgCl₂ and 1.7 M CaCl₂(Figure 2).

Species of the basidiomycetous genus Wallemia Johan-Olsen can be found in a wide variety of environments characterized by low a_w (Samson et al., 2002; Zalar et al., 2005), such as dried, salty and sweet foods, indoor and outdoor air in urban and agricultural environments, hypersaline water of the salterns on different continents and salt crystals (Zalar et al., 2005). Two species of the genus Wallemia, W. muriae and W. ichthyophaga, are obligate xerophiles with the a_w growth ranges 0.984–0.805 and 0.959–0.771, respectively (Zalar et al., 2005), whereas W. sebi is xerotolerant with the ability to grow in media without additional solutes (a_w growth range: 0.997–0.690) (Pitt and Hocking, 1977). However, in media supplemented with NaCl as the major solute, the lowest a_w for the growth of W. sebi was reported to be 0.80 (Zalar et al., 2005) corresponding to 4.5 M NaCl. W. muriae can grow up to 4.3 M NaCl, while W. ichthyophaga can thrive only in media with NaCl above 1.7 M, has an optimum at 2.6–3.5 M NaCl and can grow up to saturating levels of NaCl (5.2 M) (Zalar et al., 2005; Zajc et al., 2014). Here, we determined that strains of W. ichthyophaga grew well at highest concentrations of NaCl (above 4.0 M), NaBr (4 M) and saturated KCl and MgSO₄, but show quite a variability when cultivated at different concentrations of chaotropes like MgCl₂ and CaCl₂ (Table 1 and Figure 2). A type strain from the hypersaline waters of salterns (W. ichthyophaga EXF-994) grew also at the 2.1 M MgCl₂, whereas it was not able to tolerate high concentrations of calcium (not even 1 M CaCl₂). W. ichthyophaga is indeed the most halophilic fungus ever described. Interestingly, its genome analysis showed that the life in extremely saline environments is possible even with low number of cation-transporter genes, and seems independent of their low transcription and non-responsiveness to variable salinity. In this case, the role of passive barriers against high salinity conditions seems crucial. The cell wall is unusually thick, the cells are joined into thick multicellular clumps and the cell-wall proteins, hydrophobins, are among the highly expressed genes in saline environments (Zajc et al., 2013).

The filamentous fungi of the order Eurotiales, comprised of teleomorphic genera Eurotium and Emericella, and the anamorphic Aspergillus and Penicillium, are commonly found in different salterns around the World (Cantrell et al., 2006; Butinar et al., 2011) as well as in the Dead Sea (reviewed in Oren and Gunde-Cimerman, 2012). Tolerance for high salt concentrations has been known for many food-borne species (Tresner and Hayes, 1971). The representatives of Aspergillus and Penicillium are most abundant at salinities below 1.7 M NaCl in the solar salterns (Butinar et al., 2011); however, the in vitro determined salinity growth ranges of the Eurotium spp. are broad, ranging from 0 up to 4.7 M (Butinar et al., 2005c). Given our results the highest concentrations of salts in which species from the order Eurotiales are able to thrive are highly diverse, ranging from the lowest to highest concentrations tested depending on individual strain (see Figure 2 for Eurotium repens EXF-2132). However, all strains were capable to grow in concentrations higher than 3.0 M NaCl, 3.5 M KCl, 2.0 M MgSO₄, 2.5 M NaBr and even over 1.5 M MgCl₂ and 1.2 M CaCl₂ (except in one incident in the case of MgCl₂ and CaCl₂).

Few halophilic Archaea can grow at high concentrations of MgCl₂, but only in the presence of significant concentrations of NaCl (Mullakhanbhai and Larsen, 1975; Oren, 1983; Oren et al., 1995). For instance, Haloferax volcanii is tolerant to high magnesium as growth is still possible at 1.4 M Mg²⁺ in the presence of 2 M Na⁺ (Mullakhanbhai and Larsen, 1975). Also, Halobaculum gomorrense is moderately tolerant to Mg²⁺ with optimal growth at 0.6–1.0 M Mg²⁺ in the presence of 2.1 M NaCl (Oren et al., 1995). Another archaeon isolated from the Dead Sea, Halobacterium sodomense, has an extremely high magnesium requirement. It grows optimally even at 1.2 M MgCl₂ and 2.0 M NaCl and still grows, albeit poorly, at 1.8 M MgC1₂ and 1.7 M NaCl and at 2.5 M MgC1₂ and 0.5 M NaCl (Oren, 1983). The upper concentration of solely MgCl₂ still supporting life was suggested to be 2.3 M and it based on the presence of specific mRNA indicators of active life, (Hallsworth et al., 2007). However, the highest concentration of MgCl₂ (without compensating kosmotropes) showing microbial growth (after 18 months of cultivation) of deep-sea Discovery brine samples was 1.26 M (Hallsworth et al., 2007). Given the fact that it was not uncommon for fungi to thrive at concentrations of MgCl₂ higher than 1.5 M without compensating NaCl, it is clear that fungi are truly tolerant to magnesium. Some of these were able to grow at 2.1 M MgCl₂, a concentration that is close to the chaotropicity limit of possible life (2.3 M) (Hallsworth et al., 2007).

Fungi from diverse environments (salterns, Dead Sea, ice, freshwater and other) can not only tolerate but also thrive at high concentrations of salts, which are either kosmotropic like NaCl, KCl and MgSO₄ or—to biological systems more toxic—chaotropic like NaBr, MgCl₂, and CaCl₂. A few representatives of various species, such as H. werneckii, E. amstelodami, E. chevalieri and W. ichthyophaga were able to thrive in media with the highest tested salinities of all salts (except in CaCl₂ in case of W. ichthyophaga). In addition, several fungi (Aureobasidium spp., Exophiala spp.) exert a tendency toward kosmotropes, as they are able to grow at relatively high concentrations of NaCl, KCl and MgSO₄, but not at high concentrations of chaotropes, like MgCl₂ and CaCl₂. However, no fungal representatives showed the preference for the highest concentrations of only chaotropic salts but not for the kosmotropic, i.e., being obligately chaophilic. Nevertheless, our study revealed many representatives of the novel group of chaophiles among fungi, which thrive well above the highest previously determined concentration of MgCl₂. The ability to grow in the presence of high concentrations of another potent chaotrope—CaCl₂ was addressed for the first time. This expands our knowledge of possible life performance under diverse and most extreme environmental parameters.