Subterranean fauna provides unique opportunities for the study of evolutionary mechanisms and speciation processes . In recent years, phylogeographic analyses have revealed unprecedented cases of cryptic speciation, restricted distribution and presumed sympatric speciation among different cave-dwelling animal groups . Nevertheless, the occurrence of extensive morphological conservatism in subterranean fauna frequently hampers the establishment of phylogenetic inferences based solely on morphological features. In this context, homoplasy arises from common exposure to the particular selective pressures inherent to cave life (i.e., darkness and oligotrophy) or from the lack of directional selection [3, 4]. Conversely, isolation in caves can lead these morphologically undifferentiated subterranean organisms to display high levels of genetic divergence [4–6].
Geological and hydrological processes, in particular shifts in water tables, can lead to the isolation or connection of aquifers, with consequent effects on gene flow between populations of subterranean aquatic organisms . In the same way, marine regressions are suggested to have played a major role in the isolation of many marine relicts in continental groundwaters [7–10]. Recent molecular phylogenetic and phylogeographic studies on subterranean amphipods emphasize the role played by historical factors (i.e., glacial or drought episodes) in the pattern of genetic diversification and distribution displayed by these animals [5, 6, 11]. Likewise,  considered the influence of contingency, i.e., whether the colonization event involved a single localized surface ancestor or multiple, geographically separated ancestors, on the shaping of these patterns. In addition, larval life history traits, such as feeding mode (planktotrophic vs. lecithotrophic) can play a determinant role in crustacean distribution, as they control the duration of the dispersive phase [13, 14]. However, stygobiont amphipods have a comparatively reduced dispersal potential (as do all peracarid crustaceans), as the females carry offspring in a marsupium and these are brooded and not released into the water column until metamorphosed into diminutive non-natatory adults .
Among the obligate dwellers of subterranean waters (stygobionts), a high number belong to so-called thalassoid lineages, organisms that are derived directly from marine ancestors . Thalassoid forms are known to occur among a vast array of faunistic groups, especially the Crustacea [16, 8]. The ancestors of thalassoid animals presumably inhabited marine transitional habitats, such as submarine fissures, mixohaline submarine karstic springs or the interstitial medium developed in sandy and gravelly coastal sediments, where sharp variations in salinity (i.e., periodical exposure to desalinated waters) and other environmental conditions mimic, in some way, those found in fresh groundwaters . Colonization of inland freshwater aquifers by this preadapted marine fauna might have proceeded as a natural extension of their primary niche, followed by an adaptive shift; this process would be independent of the occurrence of environmental constraints, such as episodes of glaciation, drought or marine regression [17, 18]. This hypothesis provides a plausible explanation for the origin of some freshwater stygobiont ostracods closely related to marine euryhaline taxa . However, most faunistic and biogeographic evidence favours an alternative vicariant scenario by which colonization occurs passively via stranding of ancestral populations during episodes of marine regression [7–10]. Accordingly, sea withdrawal or tectonic uplift at different geological periods could have led to the gradual isolation of populations of ancestral marine taxa in inland groundwaters, triggering their ulterior diversification and speciation. This hypothesis explains satisfactorily the distribution of many stygobiont crustaceans and is testable by collating a phylogenetic framework and molecular-clock-age estimates of relevant clades, with their respective geographic distributions [2, 20].
Here, we studied the phylogeography of Metacrangonyx longipes Chevreux, 1909, a euryhaline stygobiont amphipod crustacean that is endemic to Mallorca and Menorca (Balearic Islands; W Mediterranean). On Mallorca, it occurs in various types of groundwater habitats, from coastal anchialine caves (sensu ) of raised salinity to freshwater inland wells, caves and springs. On Menorca, the species is restricted to coastal anchialine caves and wells and is absent from fresh inland groundwaters. On both islands, the species is limited to lowlands and is absent in apparently suitable habitats located at elevations higher than 125 m above sea level. The Metacrangonyctidae is a strictly inland water subterranean family with no close relatives; however, several lines of evidence strongly suggest its marine origin: (1) its members are known only from continental regions that were covered by ancient epicontinental seas [22, 23]; and (2) several species still maintain ties with the marine environment (i.e., they live in anchialine wells and caves in coastal areas; ).
In this study, we used the sequences of three mitochondrial and one nuclear gene of M. longipes and of several congeneric species to perform a phylogenetic analysis of the species and infer population divergence times. Moreover, we use sequences of the cytochrome oxidase subunit 1 gene from a more comprehensive data set to perform a phylogeographic analysis and to examine the population structure of this taxon. Given the manifested euryhalinity of M. longipes and the absence of any appreciable morphological differentiation between its populations on the two islands, our initial prediction was that the species could have dispersed across the groundwater environment of the islands using the virtually continuous peripheral coastal anchialine pathway, from which it could have colonized inland freshwater habitats recurrently. If this was the case, we could expect a pattern of considerable gene flow and shallow genetic divergences within each island, with genetic signatures of inland populations deriving from coastal ones. However, our study revealed that this amphipod displays a remarkably ancient and highly fragmented population structure, with episodes of cladogenesis that could be related to major sea-level changes that affected the islands during the last 6 Ma.