Spring Ecosystems of the Alps: Isolated biodiversity islands with distinctive species assemblages

Freshwater springs are considered distinctive stream sections. Environmental conditions typical of subterranean groundwater habitats such as high temperature stability and nutrient scarcity predominate the spring mouth. Due to the rapidly increasing influence of surface processes such as solar radiation or precipitation downstream, springs exhibit steep environmental gradients and high microhabitat diversity on a small spatial scale. Furthermore, diverse species assemblages composed of crenobiontic (strictly spring-dwelling), stygobiontic (groundwater-adapted), and rhithrobiontic (stream-inhabiting) organisms are characteristic of springs. Due to their topographic distinctiveness and individual species assemblage compositions, particularly alpine springs are considered isolated island-like habitats and freshwater biodiversity hotspots.
Water mites (Hydrachnidia) show many crenobiontic species adapted to different microhabitats such as gravel, moss, lentic, or lotic flow regimes in springs and can be seen as a model system to study crenobiology. Their taxonomy is currently re-investigated intensively and is still mainly morphology-based, although molecular genetic methods can reveal novel insights. Therefore, the a priori morphological species delimitation of the most abundant crenobiontic water mites was assessed with molecular genetic tools. The phylogenetic inferences, including a mitochondrial (cox1) and a nuclear (28S) marker, corresponded to the traditional taxonomy and confirmed the monophyly of Hydrachnidia. Moreover, species putatively new to science were discovered, a genetic species identification reference database was generated, and basic methods for further genetic work were established.
The genomic structure of Partnunia steinmanni Walter, 1906, a strictly crenobiontic water mite species, was then investigated to study degree of spring habitat isolation. Populations were sampled in protected areas across the Alps and restriction site-associated DNA sequencing (RADseq) was performed. The admixture and RAxML analysis revealed a pronounced population genomic structure and distinctiveness of P. steinmanni spring populations between and within the different areas. Combined with the strong isolation by distance that has been found, a high degree of insularity of alpine springs can be concluded. Furthermore, a genetic principal component analysis of individuals between the different protected areas revealed a western genotype extending into eastern populations, likely caused by post-glacial recolonization.
Finally, an environmental DNA (eDNA) bioindication methodology was established to monitor and assess alpine spring ecosystem integrity and detect a potential loss of crenobionts. For that purpose, sequence libraries were generated and novel qPCR primer and probe sets were designed to detect indicator species in eDNA filtered water samples. The final assays targeting spring-dwelling Trichoptera, Plecoptera, and Hydrachnidia species showed to be highly specific and sensitive. Furthermore, equal detection rates were revealed by comparing the qPCR eDNA assays with the conventional approach, which relies on direct sampling and morphological identification of organisms. Due to its non- invasive and time-efficient character, the newly developed spring bioindication method circumvents drawbacks of the conventional techniques and is particularly applicable in protected areas.