Species delimitation and mitogenome phylogenetics in the subterranean genus Pseudoniphargus (Crustacea: Amphipoda).

The amphi-Atlantic distributions exhibited by many thalassoid stygobiont (obligate subterranean) crustaceans have been explained by fragmentation by plate tectonics of ancestral shallow water marine populations. The amphipod stygobiont genus Pseudoniphargus is distributed across the Mediterranean region but also in the North Atlantic archipelagos of Bermuda, Azores, Madeira and the Canaries. We used species delimitation methods and mitogenome phylogenetic analyses to clarify the species diversity and evolutionary relationships within the genus and timing their diversification. Analyses included samples from the Iberian Peninsula, northern Morocco, the Balearic, Canarian, Azores and Madeira archipelagoes plus Bermuda. In most instances, morphological and molecular-based species delimitation analyses yielded consistent results. Notwithstanding, in a few cases either incipient speciation with no involvement of detectable morphological divergence or species crypticism were the most plausible explanations for the disagreement found between morphological and molecular species delimitations. Phylogenetic analyses based on a robust calibrated mitochondrial tree suggested that Pseudoniphargus lineages have a younger age than for other thalassoid amphipods displaying a disjunct distribution embracing both sides of the Atlantic Ocean. A major split within the family was estimated to occur at the Paleocene, when a lineage from Northern Iberian Peninsula diverged from the rest of pseudoniphargids. Species diversification in the peri-Mediterranean area was deduced to occur in early Miocene to Tortonian times, while in the Atlantic islands it started in the Pliocene. Our results show that the current distribution pattern of Pseudoniphargus resulted from a complex admix of relatively ancient vicariance events and several episodes of long- distance dispersal.