The diversity of filamentous fungi that can grow at high ambient pH values (i.e., 8-11) remains largely understudied. Here we study 100 alkalitolerant and alkaliphilic isolates from the soils around the basin of soda lakes in Asia and Africa to assess the major evolutionary lineages and morphologies pertinent to the alkaliphilic trait in filamentous fungi. The Emericellopsis lineage (Hypocreales, Hypocreomycetidae), along with Plectosphaerellaceae (Hypocreomycetidae), Pleosporaceae (Dothideomycetes), Chaetomiaceae (Sordariomycetidae) families appeared to be overrepresented with strong alkalitolerants and effective alkaliphiles. In particular, Sodiomyces species (Plectosphaerellaceae), Acrostalagmus luteoalbus (Plectosphaerellaceae), Emericellopsis alkalina (Hypocreales), Thielavia sp. (Chaetomiaceae), and Alternaria sect. Soda (Pleosporaceae) grew best at high ambient pH. The pH tolerance of Chordomyces antarcticum, Acrostalagmus luteoalbus and some other species was largely affected by the presence of extra Na + in the growth medium. Moderate alkalitolerants included Scopulariopsis members (Microascales), Fusarium, Cladosporium, and many asexual acremonium-like species from Bionectriaceae. Weak alkalitolerants were represented by sporadic isolates of Penicillium, Purpureocillium lilacinum, and Alternaria alternata species, with the growth optimum at neutral or acidic pH. Weak alkalitolerants develop loose dry chains of spores easily dispersed by air. Their presence at low frequency with the growth optimum at neutral or acidic pH leads us to treat them as transient species in the alkaline soils, as those are also ubiquitous saprobes in normal soils. Our phylogenetic analyses show that the alkaliphilic trait in filamentous fungi has evolved several times. Several lineages harboring strong alkalitolerants derived from the known marine-borne fungi (Emericellopsis, Alternaria sect. Phragmosporae), or fall within the fungi associated with halophytic grasses (Pleosporaceae). Soda soils contain a diversity of fungi that range from weak alkalitolerant to alkaliphilic, which in few cases is associated with darkly pigmented mycelium and formation of microsclerotia. The alkaliphilic trait is spread throughout the Ascomycota, and usually juxtaposes with slime-covered polyphyletic acremonium-, verticillium-, gliocladium-types of asexual morphology, hyphae aggregating in chords, and enclosed fruit bodies.

In this study we reassess the taxonomic reference of the previously described holomorphic alkaliphilic fungus Heleococcum alkalinum isolated from soda soils in Russia, Mongolia and Tanzania. We show that it is not an actual member of the genus Heleococcum (order Hypocreales) as stated before and should, therefore, be excluded from it and renamed. Multi-locus gene phylogeny analyses (based on nuclear ITS, 5.8S rDNA, 28S rDNA, 18S rDNA, RPB2 and TEF1-alpha) have displayed this fungus as a new taxon at the genus level within the family Plectosphaerellaceae, Hypocreomycetidae, Ascomycota. The reference species of actual Heleococcum members showed clear divergence from the strongly supported Heleococcum alkalinum position within the Plectosphaerellaceae, sister to the family Glomerellaceae. Eighteen strains isolated from soda lakes around the world show remarkable genetic similarity promoting speculations on their possible evolution in harsh alkaline environments. We established the pH growth optimum of this alkaliphilic fungus at c. pH 10 and tested growth on 30 carbon sources at pH 7 and 10. The new genus and species, Sodiomyces alkalinus gen. nov. comb. nov., is the second holomorphic fungus known within the family, the first one being Plectosphaerella – some members of this genus are known to be alkalitolerant. We propose the Plectosphaerellaceae family to be the source of alkaliphilic filamentous fungi as also the species known as Acremonium alcalophilum belongs to this group.

Surveying the fungi of alkaline soils in Siberia, Trans-Baikal regions (Russia), the Aral lake (Kazakhstan), and Eastern Mongolia, we report an abundance of alkalitolerant species representing the Emericellopsis-clade within the Acremonium cluster of fungi (order Hypocreales). On an alkaline medium (pH ca. 10), 34 acremonium-like fungal strains were obtained. One of these was able to develop a sexual morph and was shown to be a new member of the genus Emericellopsis, described here as E. alkalina sp. nov. Previous studies showed two distinct ecological clades within Emericellopsis, one consisting of terrestrial isolates and one predominantly marine. Remarkably, all the isolates from our study sites show high phylogenetic similarity based on six loci (LSU and SSU rDNA, RPB2, TEF1-α, β-tub and ITS region), regardless of their provenance within a broad geographical distribution. They group within the known marine-origin species, a finding that provides a possible link to the evolution of the alkaliphilic trait in the Emericellopsis lineage. We tested the capacities of all newly isolated strains, and the few available reference ex-type cultures, to grow over wide pH ranges. The growth performance varied among the tested isolates, which showed differences in growth rate as well as in pH preference. Whereas every newly isolated strain from soda soils was extremely alkalitolerant and displayed the ability to grow over a wide range of ambient pH (range 4–11.2), reference marine-borne and terrestrial strains showed moderate and no alkalitolerance, respectively. The growth pattern of the alkalitolerant Emericellopsis isolates was unlike that of the recently described and taxonomically unrelated alkaliphilic Sodiomyces alkalinus, obtained from the same type of soils but which showed a narrower preference towards high pH.

Alkaliphily, the ability of an organism to thrive optimally at high ambient pH, has been well-documented in several lineages: archaea, bacteria and fungi. The molecular mechanics of such adaptation has been extensively addressed in alkaliphilic bacteria and alkalitolerant fungi. In this study, we consider an additional property that may have enabled fungi to prosper at alkaline pH: altered contents of membrane lipids and cytoprotectant molecules. In the alkaliphilic Sodiomyces tronii, we showed that at its optimal growth pH 9.2, the fungus accumulates abundant cytosolic trehalose (4-10% dry weight) and phosphatidic acids in the membrane lipids, properties not normally observed in neutrophilic species. At a very high pH 10.2, the major carbohydrate, glucose, was rapidly substituted by mannitol and arabitol. Conversely, lowering the pH to 5.4-7.0 had major implications both on the content of carbohydrates and membrane lipids. It was shown that trehalose dominated at pH 5.4. Fractions of sphingolipids and sterols of plasma membranes rapidly elevated possibly indicating the formation of membrane structures called rafts. Overall, our results reveals complex dynamics of the contents of membrane lipids and cytoplasmic sugars in alkaliphilic S. tronii, suggesting their adaptive functionality against pH stress.

No abstract