AbstractBackgroundMetacrangonyctidae (Amphipoda, Crustacea) is an enigmatic continental subterranean water family of marine origin (thalassoid). One of the species in the genus,Metacrangonyx longipes, is endemic to the Balearic islands of Mallorca and Menorca (W Mediterranean). It has been suggested that the origin and distribution of thalassoid crustaceans could be explained by one of two alternative hypotheses: (1) active colonization of inland freshwater aquifers by a marine ancestor, followed by an adaptative shift; or (2) passive colonization by stranding of ancestral marine populations in coastal aquifers during marine regressions. A comparison of phylogenies, phylogeographic patterns and age estimations of clades should discriminate in favour of one of these two proposals.ResultsPhylogenetic relationships withinM. longipesbased on three mitochondrial DNA (mtDNA) and one nuclear marker revealed five genetically divergent and geographically structured clades. Analyses of cytochrome oxidase subunit 1 (cox1) mtDNA data showed the occurrence of a high geographic population subdivision in both islands, with current gene flow occurring exclusively between sites located in close proximity. Molecular-clock estimations dated the origin ofM. longipesprevious to about 6 Ma, whereas major cladogenetic events within the species took place between 4.2 and 2.0 Ma.ConclusionsM. longipesdisplayed a surprisingly old and highly fragmented population structure, with major episodes of cladogenesis within the species roughly correlating with some of the major marine transgression-regression episodes that affected the region during the last 6 Ma. Eustatic changes (vicariant events) -not active range expansion of marine littoral ancestors colonizing desalinated habitats-explain the phylogeographic pattern observed inM. longipes.