The salinity barrier that separates marine and freshwater biomes is probably the most important division in biodiversity on Earth. Those organisms that successfully performed this transition had access to new ecosystems while undergoing changes in selective pressure, which often led to major shifts in diversification rates. While these transitions have been extensively investigated in animals, the tempo, mode, and outcome of crossing the salinity barrier have been scarcely studied in other eukaryotes. Here, we reconstructed the evolutionary history of the species complex Cyphoderia ampulla (Euglyphida: Cercozoa: Rhizaria) based on DNA sequences from the nuclear SSU rRNA gene and the mitochondrial cytochrome oxidase subunit I gene, obtained from publicly available environmental DNA data (GeneBank, EukBank) and isolated organisms. A tree calibrated with euglyphid fossils showed that four independent transitions towards freshwater systems occurred from the mid‐Miocene onwards, coincident with important fluctuations in sea level. Ancestral trait reconstructions indicated that the whole family Cyphoderiidae had a marine origin and suggest that ancestors of the freshwater forms were euryhaline and lived in environments with fluctuating salinity. Diversification rates did not show any obvious increase concomitant with ecological transitions, but morphometric analyses indicated that species increased in size and homogenized their morphology after colonizing the new environments. This suggests adaptation to changes in selective pressure exerted by life in freshwater sediments.

The salinity and humidity barriers divide biodiversity and strongly influence the distribution of organisms. Crossing them opens the possibility for organisms to colonize new niches and diversify, but requires profound physiological adaptations and is supposed to happen rarely in evolutionary history. We tested the relative importance of each ecological barrier by building the phylogeny, based on mitochondrial cytochrome oxidase gene (COI) sequences, of a group of microorganisms common in freshwater and soils, the Arcellidae (Arcellinida; Amoebozoa). We explored the biodiversity of this family in the sediments of athalassohaline water bodies (i.e. of fluctuating salinity that have non-marine origins). We found three new aquatic species which represent, to our knowledge, the first reports of Arcellinida in these salt impacted ecosystems, plus a fourth terrestrial one in bryophytes. Culturing experiments performed on Arcella euryhalina sp. nov. showed similar growth curves in pure freshwater and under 20g/L salinity, and long term survival at 50g/L, displaying a halotolerant biology. Phylogenetic analyses showed that all three new athalassohaline species represent independent transition events through the salinity barrier by freshwater ancestor, in contrast to the terrestrial species, which are monophyletic and represent a unique ecological transition from freshwater to soil environments.

Accurate species delimitation based on an integrative approach is indispensable for biodiversity evaluation. Accordingly, species that were formerly only described morphologically often need to be split into several evolutionary independent units. However, taxonomic actions often lag behind, even when the required data are already available. As a result, invalid species names are carried over the years, with negative implications on ecology, biogeography, and conservation; we designate these entities as ‘shadow species’. This is particularly common in protists, due to the lack of specialized taxonomists and the difficulties of working with microscopic organisms. Here, we resolve the case of the testate amoeba shadow species Cyphoderia ampulla (Rhizaria: Cercozoa: Euglyphida: Cyphoderiidae), a known polyphyletic taxon. Purposely, we integrated the current ecological, genetic, and morphological data on the family Cyphoderiidae with its described evolutionary history. Subsequently, we took the required taxonomic actions to resolve the taxonomy of the family, erecting four new genera (Psammoderia gen nov., Knarr gen. nov., Ichthyosquama gen. nov., and Oleiformis gen. nov.), emending Cyphoderia and describing four new species (Ichthyosquama sanabriensis sp. nov., Ichthyosquama catoirensis sp. nov., Ichthyosquama loricaria sp. nov., and Oleiformis carmelae sp. nov.). Finally, we discuss the extension and relevance of the ‘shadow species’ issue in eukaryotic taxa, and differentiate it from species complex concept.