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Understanding genetic and phylogenetic relationships among closely related species is critical for effective conservation planning. In the Rio Conchos basin of northern Mexico, Cyprinodon eximius has a widespread distribution, occurring alongside neighboring microendemic species: C. julimes, C. macrolepis. C. pachycephalus, and C. salvadori. Both the widespread C. eximius and the microendemic congeners face threats from climate change and anthropogenic habitat modification. We utilized 20 nuclear microsatellites and sequenced the mitochondrial cytochrome b region to investigate the evolutionary history and genetic structuring among these taxa. Our analyses revealed multiple genetically distinct groups within C. eximius, including strong divergence in the Devils River, Texas, population. We also found support for recognizing the microendemic populations across the basin as distinct species. However, discordance between mitochondrial and nuclear data indicates historical introgression between many microendemic species and adjacent C. eximius populations in rivers connected to the Rio Conchos riverine habitats. These findings suggest that conservation efforts in the Rio Conchos basin should treat C. eximius populations in Mexico and Texas separately and recognize each microendemic population as an independent conservation unit. The microsatellite data has 12 populations and 5 species. For each of species/population there are close to 30 individuals. For each individual, the length is designated by two numbers for each marker. For the mitochondrial DNA dataset, the sequence is listed under each individual, their population, and the locus number. The metadata associated with the mtDNA sequences are included in a separate text file.