Abstract Long‐term evolutionary history and demographic processes shape current patterns of genetic variation in natural populations. Genetic diversity is of prime importance for the long‐term survival of species, especially those threatened or endangered, which can be lost in a few generations and can take hundreds to recover. Notably, turtles are among the most endangered vertebrate taxa in the world. The Coahuilan box turtle Terrapene coahuila is the only aquatic species within the genus, microendemic to the desert‐spring ecosystem of the Cuatrociénegas Basin (CCB). The CCB has experienced significant habitat changes during recent decades, fragmenting otherwise interconnected wetlands systems. We aimed to perform a detailed evaluation of the evolutionary and genetic patterns of T. coahuila, both historical and contemporary, linked to the biogeography of the CCB and processes governing the species distribution. We assessed the contributions of historical and contemporary population demographic processes to genetic diversity and structure, by using two mitochondrial DNA genes (cytochrome b and d‐loop), one nuclear gene (GAPD), and 12 nuclear microsatellite loci. We comprehensively analysed these data to address historical patterns of demography, migration, genetic diversity, and times of divergence across the valley for T. coahuila. We also evaluated recent patterns of genetic variation and structure, inbreeding, bottleneck signals, and effective population size. Our results dated the time to the most recent common ancestor for T. coahuila in the CCB at c. 10.1 million years ago, with highest diversification during the Holocene. Structure consistently comprised two genetic clusters, Cuatrociénegas East and Cuatrociénegas West on either side of the main historical barrier, the Sierra San Marcos. Demographic results supported a divergence between western and eastern populations from 47,500 to 110,950 years ago, and colonisation of the southernmost Cuatrociénegas West distribution (Churince) 28,500–66,500 years ago, which experienced a recent bottleneck. All populations showed low values of haplotype and nuclear genetic diversity, migration, effective population size, and relatedness; significant inbreeding and bottleneck signals were also identified within genetic clusters. In accordance with the biogeographic history of the basin, our results show that T. coahuila’s diversification across the Cuatrociénegas valley continued throughout the Pleistocene, peaking during the Holocene, when the valley started to gradually dry. As predicted, T. coahuila exhibited an overall, long‐term, and probably continuing decrement in genetic variation, increased genetic differentiation, and low population sizes, as a consequence of contemporary habitat loss and fragmentation across the CCB. Changes in species demography and population connectivity have the potential to permanently alter the genetic patterns of freshwater species. Hence, given its restricted distribution, high habitat specificity and habitat loss, Terrapene coahuila is at utmost risk of extinction. Captive breeding programmes of the Coahuilan box turtle should consider our results, to prevent further loss of genetic diversity and truly expect reintroduction success. Also, it is crucial to restore connectivity among wetlands, for the conservation of species and the Cuatrociénegas valley altogether.
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