Habitat disturbance leading to the breakdown of ecological barriers has resulted in hybridization between numerous sympatric species and the decline or extinction of the parental species. Despite strong postzygotic selection, hybridization occurs between two normally ecologically isolated toad species, the Gulf Coast Toad (Bufo nebulifer) and Fowler's Toad (Bufo fowleri). This hybridization has potentially contributed to a decline of the rarer species, B. fowleri. Putative hybrids may be morphologically cryptic; therefore, molecular methods were used to identify them. We used single nucleotide polymorphisms (SNPs) to identify hybrids, and mitochondrial sequence variation in a segment of the 12S and 16S rRNA region was also used to identify maternal ancestry of hybrids and seek evidence of directional hybridization and introgression of male B. nebulifer with female B. fowleri. Seventy-two individuals of both species from nine breeding populations were directly sequenced. Twelve species-specific SNPs from a 333 base pair fragment in a nuclear intron of the Rhodopsin gene were used to identify each species, and two male hybrids were identified based on heterozygosity at these sites. The possession of a different mtDNA sequence by each hybrid indicated an alternate maternal lineage from each of the parental species and identified the hybrids as progeny from each reciprocal cross. Use of mitochondrial and nuclear molecular markers allowed unambiguous identification of hybrids and their maternal parent that could not have been performed without the use of both datasets. We also investigated phylogenetic relationships among B. fowleri and its close relatives in the B. americanus species complex using mitochondrial sequence variation.
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