Multiple transgressions and slow evolution shape the phylogeographic pattern of the blind cave-dwelling shrimp Typhlocaris

Background: Aquatic subterranean species often exhibit disjunct distributions, with high level of endemism and small range, shaped by vicariance, limited dispersal, and evolutionary rates. We studied the disjunct biogeographic patterns of an endangered blind cave shrimp, Typhlocaris , and identified the geological and evolutionary processes that have shaped its divergence pattern. Methods: . We collected Typlocaris specimens of three species ( T. galilea , T. ayyaloni , and T. salentina ), originating from subterranean groundwater caves by the Mediterranean Sea, and used three mitochondrial genes (12S, 16S, COI) and four nuclear genes (18S, 28S, ITS, H3) to infer their phylogenetic relationships. Using the radiometric dating of a geological formation (Bira) as a calibration node, we estimated the divergence times of the Typhlocaris species and the molecular evolution rates. Results: . The multi-locus ML/Bayesian trees of the concatenated seven gene sequences showed that T. salentina (Italy) and T. ayyaloni (Israel) are more closely related than T. galilea (Israel). The divergence time of T. ayyaloni and T. salentina from T. galilea was according to COI – 6.0 [4.5-7.2] Ma and according to 16S – 5.9 [3.6-7.4] Ma. The computed interspecific evolutionary rates for COI – 0.0074 substitutions/Myr and for 16S – 0.0041 substitutions/Myr. Discussion: Two consecutive vicariant events have shaped the phylogeographic patterns of Typhlocaris species. First, T. galilea was tectonically isolated from its siblings in the Mediterranean Sea by the arching uplift of the central mountain range of Israel ca. 7 Ma. Secondly, T. ayyaloni and T. salentina were stranded and separated by a marine transgression ca. 6 Ma, occurring just before the Messinian Salinity Crisis. Our estimated molecular evolution rates were in one order of magnitude lower than the rates of closely related crustaceans, as well as of other stygobiont species. We suggest that this slow evolution reflects the ecological conditions prevailing in the highly isolated subterranean enclosures inhabited by Typhlocaris .