Molecular techniques offer powerful complementary tools for determining the taxonomic identity of fragmented, morphologically non-diagnostic, or otherwise taxonomically indeterminant bone from archaeological and palaeontological contexts. This study focuses on the recovery and identification of ancient DNA (aDNA) from tropical subfossil bone assemblages. Materials from multiple archaeological sites spread across three valleys on Nuku Hiva (Marquesas Islands) in the cultural historical region of East Polynesia are used as a case study. We illustrate how aDNA sampling strategies, laboratory protocols, PCR primer selection, and sequencing methods can be optimised to improve the recovery and taxonomic identification of aDNA from tropical subfossil samples. Sanger sequencing was initially employed as a preliminary approach to assess the quality of aDNA samples. Subsequently, a high-throughput technique, bulk bone metabarcoding (BBM), was utilised for pooled specimens to recover and sequence taxonomically diagnostic ‘barcoding’ regions of DNA. To ensure robust identifications, we constructed a custom reference database tailored to our genetic markers and developed a novel decision tree framework to assign each aDNA sequence to a well-supported taxonomic level. Our application of rigorous bioinformatic tools enabled us to quantify the ability of our genetic markers to identify taxa, and provided a replicable framework for subsequent taxonomic identifications. Our BBM analyses detected a wide range of vertebrate taxa, including birds, fish, mammals, and reptiles. Here we use the archaeological seabird results to illustrate the efficacy of BBM and the outcomes of our iterative approach to enhance aDNA recovery and taxonomic identification. Our analyses provide the first well-dated pre-Western avifaunal records for Nuku Hiva Island, including three seabird taxa that are new to the wider archaeological record of the Marquesas Islands of Polynesia.
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