Tethysbaena are small peracarid crustaceans inhabiting extreme environments such as subterranean lakes and thermal springs, represented by endemic species found around the ancient Tethys, including the Mediterranean, Arabian Sea, Mid-East Atlantic, and the Caribbean Sea. Two Tethysbaena species are known from the Levant: T. relicta, found along the Dead Sea-Jordan Rift Valley, and T. ophelicola, found in the Ayyalon cave complex in the Israeli coastal plain, both belonging to the same species-group based on morphological cladistics. Along the biospeleological research of the Levantine subterranean fauna, three biogeographic hypotheses determining their origins were proposed: (1) Pliocenic transgression, (2) Mid-late Miocenic transgression, and (3) The Ophel Paradigm, according to which these are inhabitants of a chemosynthetic biome as old as the Cambrian. Tethysbaena specimens of the two Levantine species were collected from subterranean groundwaters. We used the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear ribosomal 28S (28S rRNA) gene to establish the phylogeny of the Levantine Tethysbaena species, and applied a molecular clock approach for inferring their divergence times. Contrary to the morphological cladistic-based classification, we found that T. relicta shares an ancestor with Tethysbaena species from Oman and the Dominican Republic, whereas the circum-Mediterranean species (including T. ophelicola) share another ancestor. The mean age of the node linking T. relicta from the Dead Sea-Jordan Rift Valley and Tethysbaena from Oman was 20.13 MYA. The mean estimate for the divergence of T. ophelicola from the Mediterranean Tethysbaena clade dated to 9.46 MYA. Our results indicate a two-stage colonization of Tethysbaena in the Levant: a late Oligocene transgression, through a marine gulf extending from the Arabian Sea, leading to the colonization of T. relicta in the Dead Sea-Jordan Rift Valley, whereas T.ophelicola, originating from the Mesogean ancestor, inhabited anchialine caves in the coastal plain of Israel during the Mid-Miocene.
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