The extremely halotolerant black yeast Hortaea werneckii - a model for intraspecific hybridization in clonal fungi

Abstract

The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant (growth from 0 to 30% [w/v] NaCl) and has been extensively studied as a model for halotolerance in Eukaryotes for over two decades. Its most frequent sources are hypersaline environments and adjacent sea-water habitats in temperate, subtropical and tropical climates. Although typically saprobic, H. werneckii can also act as a commensal coloniser on human skin, causing tinea nigra on hands and soles. Here, we report that addition of NaCl to culture media expands the growth range of H. werneckii to 37 °C, which explains its colonisation of human skin, with its increased salinity. The morphological and physiological plasticity/ versatility of H. werneckii indicate that a species complex might be involved. This was investigated in this polyphasic taxonomic analysis based on the global diversity of H. werneckii strains collected from hypersaline environments, and from humans and animals. Analysis of D1/ D2domains of 28S and internal transcribed spacer rDNA revealed 10 and 17 genotypes, respectively, that were not always compliant. The genotypes have global distributions. Human and environmental strains with the same genotypes are intermingled. Due to the limited number of phylogenetically informative characters in the ribosomal DNA dataset, the partial genes encoding for β-tubulin (BTB) and mini-chromosome maintenance protein (MCM7) were also sequenced. The use of these genes was hampered by ambiguous sequences obtained by Sanger sequencing, as a consequence of the diploid and highly heterozygous genome of many H. werneckii strains. Analysis of the BTB and MCM7 genes showed that in some cases two copies of the gene from the same genome are positioned in distant phylogenetic clusters of the intraspecific gene tree. Analysis of whole-genome sequences of selected H. werneckii strains generally confirmed the phylogenetic distances estimated on the basis of ribosomal genes, but also showed substantial reticulation within the phylogenetic history of the strains. This is in line with the hypothesis that the diploid genomes of H. werneckii were formed by hybridizations, which have sometimes occurred between relatively divergent strains.

Whole Genome Sequencing and Comparative Genome Analysis of the Halotolerant Deep Sea Black Yeast Hortaea werneckii

“…Comparative analyses of Mediterranean isolates with other H. werneckii strains recovered from different sources evidenced the peculiar genetic and physiological characteristics of the seawater strains compared to the others [ 13 ], but these were not sufficient to assign them to another taxonomic group [ 12 ].…”

“…The genus Hortaea currently also includes the species Hortaea thailandica , isolated from Syzygium siamense leaf spots in Thailand [ 11 ]. The identification at species level is complicated by the fact that some housekeeping genes commonly used for taxonomic purposes, such as β-tubulin, mini-chromosome maintenance protein and translation elongation factor 1, produce ambiguous results in some H. werneckii strains [ 12 , 13 ]. So, the sequencing of ribosomal genes together with morphology, biochemical and physiological characteristics is considered the main useful criteria to identify this species [ 12 ].…”

The beauty and the yeast: can the microalgae Dunaliella form a borderline lichen with Hortaea werneckii?

“…Environmental samples of Dunaliella atacamensis were collected in October 2016 inside a cave located in the Coastal Range of the Atacama Desert (21°15′02.87"S, 70°04′52.33"W). Hortaea werneckii was previously isolated from this cave as EXF-6656 (Zalar et al 2019). The strains H. werneckii EXF-2000 and D. salina EXO-4, were isolated from salterns in Sečovlje at the Adriatic coast in 1996 and 2015, respectively.…”

“…Dunaliella and Hortaea are dominant species of algae and fungi found in hypersaline environments, and have been studied in great detail due to their halotolerance, becoming models of salt-adaptation of eukaryotes ( Oren 2014 ; Petrovič et al 2002 ; Gostinčar et al 2011 ; Plemenitaš et al 2008 , 2014 ; Gunde-Cimerman et al 2018 ; Zalar et al 2019 ). Dunaliella species can withstand NaCl concentrations ranging from about 0.05 M up to saturation, 5.5 M ( Ben-Amotz et al 2009 ), its cells are ovoid to pyriform in shape, lack a rigid cell wall and are enclosed by a thin plasma membrane that causes the cells to round up as the external salinity decreases ( Oren 2005 ).…”

“…This fungal component was later identified as the extremely halotolerant black yeast H . werneckii ( Gostinčar et al 2018 ; Zalar et al 2019 ).…”