DNA Marker Choice Skews Fungal Maps of Europe’s Saltiest Lagoon Sediments

Beneath the shimmering, salt-crusted surface of Salada Grande de Chiprana in northeastern Spain lies one of Western Europe’s most unusual microbial habitats: the region’s only permanent deep hypersaline lagoon. A new study published in the journal Microbial Ecology has now taken a close look at the fungi and bacteria living in its sediments, and the results carry a warning that reaches far beyond this single lagoon. The genetic markers scientists use to catalogue microbial life can shape, and even distort, the ecological stories they tell.

A team of mycologists and microbial ecologists from Rovira i Virgili University in Reus, Spain, led by Alan Omar Granados-Casas, Alberto Miguel Stchigel, Ana Fernández-Bravo and José Francisco Cano-Lira, compared sediment communities from two strikingly different neighbouring water bodies. Salada Grande de Chiprana is a hypersaline lagoon whose waters are far saltier than the ocean, while the nearby La Estanca is an oligohaline lagoon with only low levels of dissolved salts. By sampling both, the researchers could ask a deceptively simple question: does salinity structure the invisible communities of fungi and prokaryotes that live in lagoon sediments, and how reliable are the molecular tools used to detect them?

The team used DNA metabarcoding, a technique that extracts environmental DNA from sediment, amplifies a specific genetic marker with the polymerase chain reaction, and then sequences the resulting mixture to identify which organisms are present. For fungi, the researchers targeted the internal transcribed spacer region of the nuclear ribosomal DNA, the standard barcode for fungal identification. Crucially, they ran two versions of this marker, ITS1 and ITS2, which flank the 5.8S ribosomal RNA gene and are often treated as interchangeable in the literature. For prokaryotes, meaning bacteria and archaea, they sequenced the 16S ribosomal RNA gene, the long-standing workhorse of bacterial diversity surveys.

When the two fungal marker datasets were analysed together, the fungal communities in both lagoons were dominated by three major lineages: the phylum Ascomycota, which contains most described fungal species; the Rozellomycota, a mysterious group of obligate parasites of other microorganisms; and the Basidiomycota, which includes mushrooms and their relatives. The prokaryotic assemblages, by contrast, were composed mainly of Pseudomonadota, a hugely diverse bacterial phylum, along with Cyanobacteriota, the photosynthetic bacteria that often form conspicuous microbial mats in saline environments, plus Bacteroidota and Chloroflexota, both common in sediments rich in organic matter.

The first surprise came from the diversity estimates themselves. When the researchers counted the number of distinct amplicon sequence variants, or ASVs, in each sample, they found no significant difference in fungal alpha diversity between the hypersaline and oligohaline lagoons. In other words, the saltier environment did not appear to host a depauperate fungal community compared with its brackish neighbour. However, the choice of marker mattered: the ITS2 primer pair recovered a significantly higher number of observed ASVs than ITS1, a difference the authors report as statistically significant with a p-value of 0.016. Two markers from the same genomic region, amplified from the same sediments, delivered measurably different inventories of fungal life.

The deeper analysis sharpened that warning. Using Bray-Curtis dissimilarity, a measure of how different community compositions are between samples, combined with PERMANOVA, a permutation-based statistical test for community data, the team found that fungal community composition was significantly associated with two factors: the identity of the lagoon and the molecular marker used. Remarkably, the marker choice explained more of the variation in the fungal data than the lagoon identity itself, and there was also a significant interaction between the two, meaning the apparent effect of the lagoon depended on which marker was being examined. For a field that often draws ecological conclusions from a single marker, that is a sobering result.

Salinity, the environmental variable most obviously differentiating the two lagoons, turned out to be a weaker predictor than expected. The researchers used electrical conductivity as a proxy for salinity and found that it explained only a limited and statistically non-significant fraction of the variation in fungal community composition. The authors are careful to note an important caveat: because the two lagoons differ in more ways than one, conductivity’s independent contribution could not be cleanly separated from other environmental differences between the sites. The hypersaline lagoon is not simply a saltier version of La Estanca; it is a distinct ecosystem with its own chemistry, hydrology and history, and any of those factors could be shaping its fungal residents.

The prokaryotic side of the story unfolded differently. While the bacterial and archaeal communities showed clear taxonomic differences between the two lagoons, the beta-diversity analysis found no statistically significant separation between them at the community level, largely because of substantial variability among samples taken within each lagoon. This contrast between fungi and prokaryotes is one of the study’s most intriguing findings. Two major branches of life, sampled from the same handful of sediments with standard molecular methods, responded in visibly different ways to the same environmental gradient. It suggests that fungal and prokaryotic communities are governed by different ecological rules in these systems, perhaps reflecting differences in dispersal ability, dormancy strategies, or the way each group partitions the sediment environment.

Perhaps the most tantalising result is what the researchers could not identify. A large fraction of the fungal ASVs recovered from the sediments remained taxonomically or functionally unclassified, meaning they did not match any known reference sequence well enough to assign a name or a role. These sediments, it appears, harbour poorly characterised fungal diversity, possibly including lineages new to science or at least new to databases. Inland hypersaline environments are understudied compared with marine and terrestrial habitats, and European examples in particular have rarely been surveyed for fungi with modern molecular methods. Chiprana’s sediments may therefore represent a reservoir of unknown fungal life adapted to some of the saltiest non-marine conditions on the continent.

The broader lesson of the study extends well into the booming world of environmental DNA research. Metabarcoding studies now underpin everything from biodiversity monitoring to climate impact assessments, and most rely on a single marker per organismal group. This work shows that for fungi, ITS1 and ITS2 are not interchangeable windows onto the same community: they recover complementary, partially overlapping views, and conclusions drawn from one may not survive scrutiny with the other. The authors argue that marker choice must be explicitly considered when interpreting metabarcoding-based ecological patterns, and that combining markers, as they did here, offers a more complete picture. For the microbial ecologists charting life in Earth’s extreme environments, the message is clear: before declaring what lives in a hypersaline lagoon, it pays to check which lens you are looking through.

Subject of Research: Fungal and prokaryotic sediment communities in hypersaline and oligohaline lagoons assessed by ITS and 16S rRNA metabarcoding

Article Title: Marker-Dependent Biases and Contrasting Fungal and Prokaryotic Community Patterns in Hypersaline and Oligohaline Lagoons

Article References: Marker-Dependent Biases and Contrasting Fungal and Prokaryotic Community Patterns in Hypersaline and Oligohaline Lagoons. (n.d.). https://doi.org/10.1007/s00248-026-02900-5

Image Credits: AI Generated

DOI: 10.1007/s00248-026-02900-5

Keywords: hypersaline lagoons, fungal ecology, metabarcoding, ITS1, ITS2, 16S rRNA, prokaryotes, salinity, Chiprana, microbial diversity, extremophiles, sediment microbiology