All Different All Equal: High Polymorphism in Nebela (Amoebozoa: Arcellinida) Revealed by Additional Sampling and Barcoding.

Hyalospheniids are a conspicuous group of testate amoebae whose rich morphology has allowed their classification based on the characterization of their shells. However, recent studies showed the occurrence of convergences and phenotypic plasticity, which complicate their taxonomy. Molecular barcoding is the method of choice to resolve this diversity in an objective manner and accurately delineate species. In the genus Nebela, species diagnosis is based on small differences in shell size and shape, but sampling is scarce and restricted to a few locations, which could underestimate the real phenotypic variability. In this study, we obtained Nebela specimens from a new sampling location in Spain, which were all identified as Nebela collaris through COI barcoding and phylogenetic analysis. We showed through a morphological analysis (PCA and MRA) on 51 collected N. collaris specimens a considerable morphological variation that not only exceeds the diagnosed range for the species but also overlapped with most Nebela species, with only N. rotunda remaining clearly distinct in shape space. This illustrates the importance of intraspecific morphological variability in Arcellinida, and therefore the need for a large sampling to describe species morphology. The results stress the idea that single-cell barcoding is key in Arcellinida taxonomy.