The Ayyalon - Nesher Ramla (ANR) system combines hypogenic karst with endemic subterranean fauna. The unique faunal assemblage utilizes chemosynthetic food web based upon H2S within the ascending plumes of hydrothermal water. We attempt to constrain the regional biogeography and the age of the hypogene system using subterranean extant ‘living fossils’, combined with related taxa in the Dead Sea Rift and Mediterranean, as well as geological evidence and previously published molecular phylogenetic data. The molecular evidence of Typhlocaris ayyaloni and T. salentina suggested that ANR aquatic system age is > 5.8 Ma. Evidence from Dead Sea Rift and East Mediterranean biogeographic evolution of aquatic fauna indicates isolation of the rift water bodies from the Mediterranean ~7 Ma. The high endemism of terrestrial troglobites at Ayyalon - Nesher Ramla caves indicate longer isolation age, most probably ~14 Ma – the end of the last transgression which inundated the lower Shefela region. We show that the Ayyalon terrestrial fauna of tropical origin invaded the subsurface warm, humid, and food-rich habitat, escaping middle Miocene surface aridization. Shefela sinkholes preserving middle Miocene sediments share similar features with Nesher Ramla sinkholes, suggesting that they share the same formation processes ~14 Ma, and implying a minimum age of the sulfidic hypogenic aquifer.
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