Abstract
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 and Slovenia. 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 (28S, ITS region) and mtDNA (COI) sequences to assess their phylogenetic relationships and species richness. Phylogenetic analysis confirmed that the Niphargus stygius complex in the Southern Limestone Alps is not monophyletic but comprises an eastern clade (the N. kenki 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 ITS recognized different number of putative species, richer for the COI marker, suggesting that each clade is a species complex in itself. Bayesian time-calibrated phylogeny revealed that most clades began to split up during Miocene and Pliocene, ruling out the effect of Pleistocene glaciations in explaining their evolutionary history and justifying the presence of several putative cryptic species.
Competing Interest Statement
The authors have declared no competing interest.
