Simple identification of mitochondrial lineages in contact zones based on lineage‐selective primers

A variety of research projects focus on genetic variation among and within maternal lineages as encompassed by mitochondrial DNA (mtDNA). While mtDNA often differs substantially between species, large differences may also be found within species. The evaluation of such divergent lineages, for example in intraspecific contact zones (hybrid zones), commonly involves sequencing numerous individuals. Large‐scale sequencing is both expensive and labour‐intensive. Based on sequences from 15 individuals, we devised a simple and quick polymerase chain reaction assay for identification of divergent mtDNA lineages in a secondary contact zone of the side‐blotched lizard ( Uta stansburiana ). The application uses lineage‐selective primers to amplify a lineage‐diagnostic product, and is based on each group of mtDNA haplotypes being a monophyletic assemblage of haplotypes sharing the same maternal ancestry, deeply divergent from the other group. The assay was tested on a larger sample ( n  = 147) of specimens from the contact zone, confirming its usefulness in quick and reliable identification of mtDNA lineages. This approach can be modified for other species, provided diagnostic lineage variation is available, and may also be performed in simple laboratory settings while conducting fieldwork.