Polyphyly of the Niphargus stygius species group (Crustacea, Amphipoda, Niphargidae) in the Southern Limestone Alps

A bstract The Niphargus stygius species complex is a groundwater group of large-sized, sexually dimorphic species inhabiting mainly caves and, less frequently, wells and springs. According to the taxonomists of the last century, this species complex was supposed to be present in the whole Southern Limestone Alps of Italy as well as in peninsular Italy, Slovenia, and Croatia. Considering the large, presumed distribution area, we tested the contrasting hypotheses of monophyly versus paraphyly of this subterranean species complex, taking in account the presence of putative cryptic species. For this reason, we sampled the type localities of all the described species in the complex present in the Italian Southern Limestone Alps and neighbouring areas, and used nuclear rDNA (28S, ITS region) and mtDNA (COI) markers to assess the phylogenetic relationships and species richness. Phylogenetic analysis confirmed that the Niphargus stygius complex in the Southern Limestone Alps is polyphyletic comprising an eastern clade (the N. julius clade, present in NW Italy, northern Slovenia, and southern Austria) and two western clades (the N. brixianus and N. montellianus clades). These two clades are not closely related to the eastern one but rather form a monophyletic group together with a widely distributed Apennine clade ( N. speziae clade). None of these clades is closely related to typical N. stygius . Three different molecular species delimitation methods applied to COI and rDNA sequences recognized slighlty different numbers of putative species, suggesting that each clade is a species complex. Bayesian time-calibrated phylogeny revealed that most clades began to split up during Miocene and Pliocene, ruling out the effect of Pleistocene glaciations, evidenced only by the COI marker, in explaining their speciation process and justifying the presence of several putative cryptic or pseudocryptic species.