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Background  The sulfide-rich Frasassi caves in central Italy contain a rare example of a freshwater ecosystem supported entirely by chemoautotrophy.  Niphargus ictus , the sole amphipod species previously reported from this locality, was recently shown to host the first known case of a freshwater chemoautotrophic symbiosis. Since the habitat of  N. ictus  is highly fragmented and is comprised of streams and lakes with various sulfide concentrations, we conducted a detailed study to examine the potential genetic diversity of this species within Frasassi.    Results  By sequencing one nuclear (ITS) and two mitochondrial (COI and 12S) regions, we show that four partially sympatric  Niphargus  clades are present in Frasassi. Morphological and behavioral data obtained for three of these clades are perfectly congruent with this molecular delineation and make it possible to distinguish them in the field. Phylogenetic analyses of 28S ribosomal DNA sequences reveal that, among the four clades, only two are closely related to each other. Moreover, these four clades occupy distinct niches that seem to be related to the chemical properties and flow regimes of the various water bodies within Frasassi.    Conclusions  Our results suggest that four distinct  Niphargus  species are present in Frasassi and that they originated from three or four independent invasions of the cave system. At least two among the four species harbor  Thiothrix  epibionts, which paves the way for further studies of the specificity and evolutionary history of this symbiosis.

Unsuspected diversity of Niphargus amphipods in the chemoautotrophic cave ecosystem of Frasassi, central Italy