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Heteroplasmy in the mitochondrial genome offers a rare opportunity to track the evolution of a newly arising maternal lineage in populations of non-model species. Here, we identified a previously unreported mitochondrial DNA haplotype while assembling an integrated database of DNA profiles and photo-identification records from humpback whales in southeastern Alaska (SEAK). The haplotype, referred to as A8, was shared by only 2 individuals, a mature female with her female calf, and differed by only a single base pair from a common haplotype in the North Pacific, referred to as A-. To investigate the origins of the A8 haplotype, we reviewed n = 1,089 electropherograms (including replicate samples) of n = 710 individuals with A- haplotypes from an existing collection. From this review, we found 20 individuals with clear evidence of heteroplasmy for A-/A8 (parental/derived) haplotypes. Of these, 15 were encountered in SEAK, 4 were encountered on the Hawaiian breeding ground (the primary migratory destination for whales in SEAK), and 1 was encountered in the northern Gulf of Alaska. We used genotype exclusion and likelihood to identify one of the heteroplasmic females as the likely mother of the A8 cow and grandmother of the A8 calf, establishing the inheritance and germ-line fixation of the new haplotype from the parental heteroplasmy. The mutation leading to this heteroplasmy and the fixation of the A8 haplotype provide an opportunity to document the population dynamics and regional fidelity of a newly arising maternal lineage in a population recovering from exploitation.

mtDNA heteroplasmy gives rise to a new maternal lineage in North Pacific humpback whales (Megaptera novaeangliae)