Persistence of phylogeographic footprints helps to understand cryptic diversity detected in two marine amphipods widespread in the Mediterranean basin

Amphipods of the genus Gammarus are a vital component of macrozoobenthic communities in European inland and coastal, marine and brackish waters of the Mediterranean and the Black Sea. Exceptional levels of cryptic diversity have been revealed for several widespread freshwater Gammarus species in Europe. No comprehensive assessment has yet been made for brackishwater counterparts, such as Gammarus aequicauda and G. insensibilis, which are among the most widely dispersed members of the so-called “G. locusta group” in the Mediterranean and in the Black Sea. Here we probe the diversity of these morphospecies examining the partitioning of mtDNA and nDNA across multiple populations along their distribution range and discuss it within the regional paleogeographic framework. We gathered molecular data from a collection of 166 individuals of G. aequicauda and G. insensibilis from 47 locations along their distribution range in the Mediterranean including the Black Sea. They were amplified for both mitochondrial COI and 16S rRNA as well as the nuclear 28S rRNA. All five MOTU delimitation methods (ABGD, BIN, bPTP, GMYC single and multiple threshold models) applied revealed deep divergence between Black Sea and Mediterranean populations in both G. aequicauda and G. insensibilis. There were eight distinct MOTUs delimited for G. aequicauda (6–18% K2P) and 4 MOTUs for G. insensibilis (4–14% K2P). No sympatric MOTUs were detected throughout their distribution range. Multimarker time-calibrated phylogeny indicated that divergence of both G. aequicauda and G. insensibilis species complexes started already in the late Oligocene/early Miocene with the split between clades inhabiting eastern and western part of the Mediterranean occurring in both species at the similar time. Our results indicate a high cryptic diversity within Mediterranean brackishwater Gammarus, similar to that observed for freshwater counterparts. Moreover, the phylogenetic history combined with the current geographic distribution indicate that the evolution of botThis work was supported by Polish National Science Center (projects no. 2014/15/B/NZ8/00266 and 2015/17/N/NZ8/01628) and partially by the statutory funds of the Department of Invertebrate Zoology and Hydrobiology of University of Lodz. F. Costa and the University of Minho contribution was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript