Genetic analyses are an important contribution to wildlife reintroductions, particularly in the modern context of extirpations and ecological destruction. To address the complex historical ecology of the sea otter (Enhydra lutris) and its failed 1970s reintroduction to coastal Oregon, we compared mitochondrial genomes of pre-extirpation Oregon sea otters to extant and historical populations across the range. We sequenced the first complete ancient mitogenomes from archaeological Oregon sea otter dentine and historical sea otter dental calculus. Archaeological Oregon sea otters (N=20) represent ten haplotypes, which cluster with haplotypes from Alaska, Washington, and British Columbia, and exhibit a clear division from California haplotypes. Our results suggest that extant northern populations are appropriate for future reintroduction efforts. This project demonstrates the feasibility of mitogenome capture and sequencing from non-human dental calculus and the diverse applications of ancient DNA analyses to pressing ecological and conservation topics and the management of at-risk/extirpated species.

Archaeological tooth dentine was sampled at the Laboratories of Molecular Anthropology and Microbiome Research (LMAMR) at the University of Oklahoma, Norman, in the dedicated sample preparation area following standard ancient DNA contamination protocols. Dental calculus was sampled in the Department of Mammals at the Smithsonian Institution National Museum of Natural History and Santa Barbara Museum of Natural History following a calculus-specific sampling protocol designed to reduce contamination. Ancient and historical DNA extraction and library construction was performed in the LMAMR Ancient DNA Laboratory, a dedicated, six-room ISO-6 class clean room custom-built for ancient DNA and microbiome research. DNA was extracted from dental calculus and dentine using a protocol described in Morales et al. (2017). DNA extracts were converted into dual indexed Illumina sequencing libraries and captured using a custom in-solution biotinylated RNA bait set (Arbor Biosciences). Captured libraries were sequenced on an Illumina MiSeq with 2 x 150 bp chemistry. The raw fastq files were quality filtered using the program Adapter Removal2 (2.1.7) and mapped using bwa (0.7.17) with ancient DNA parameters to the published modern sea otter mitogenome (Yonezawa et al. 2007). DNA authenticity was assessed using the program MapDamage2 and fragment length plots. Consensus sequences were called from rescaled bam files in Geneious (11.1.4) and aligned with MAFFT (7.308).

The consensus sequences (ConsensusAlignment_Dryad.fa) contain edits, specifically the addition of "N"s at the ends of the ancient sequences due to low coverage in this region and for ambiguous base calls. We have uploaded the complete consensus sequences. The edited alignment file (ModAlign.fa) used for downstream analysis is also included here. Raw sequence data is publicly available through SRA NCBI (PRJNA550086).