From fluke to fragment: A multifaceted method for molecular sex identification and mitochondrial haplotyping from environmental DNA samples

Abstract Environmental DNA (eDNA) analyses have become a powerful tool for non‐invasive biodiversity monitoring, yet the applicability of population‐genetic approaches to environmental samples remains largely unexplored. Even when genetic traces originate from a single individual, low target DNA concentrations and amplification or sequencing artefacts can compromise downstream genetic inferences. Here, we present a novel approach for obtaining demographic insights and lineage‐level mitogenomic information from aquatic eDNA samples collected near vertebrate individuals, while assessing the utility of paired tissue sampling for benchmarking eDNA‐based population genetic analyses. Paired eDNA and tissue samples were collected during sperm whale ( Physeter macrocephalus ) encounters in the Azores. Samples were screened for the presence of vertebrate eDNA and analysed with a novel molecular sex identification assay. Additionally, long‐range PCR was used to amplify up to five mitochondrial DNA fragments (~3–4 k bp) before subsequent sequencing on an Oxford Nanopore Technologies platform. A stringent three‐tier filtering framework capable of identifying true mitogenomic variation across eDNA samples was developed for maximum recovery of genetic diversity at the haplogroup level. By validating eDNA samples via their paired tissues, parameter values were optimized to maximize concordance and minimize spurious variant calls. Sexing was successful for 50% of eDNA samples, with 96% concordance to paired tissues and marine vertebrate DNA concentration significantly predicted sexing success. Further, Medaka polishing produced high identity mitochondrial consensus sequences (>16 kb) from eDNA samples. Across filtering regimes in the framework, curated SNP panels comprising up to 453 high‐confidence mitochondrial SNPs resolved 19 haplogroups, with 93% concordance between eDNA and tissue samples. An intermediate bioinformatics filtering strategy maximized biologically accurate haplogroup recovery while minimizing sequencing artefacts, providing the most reliable lineage‐level inferences. This integrative approach demonstrates that targeted nuclear assays combined with long‐range mitochondrial sequencing can recover individual‐level genetic information from aquatic eDNA. By defining analytical thresholds governing success and demonstrating how paired tissue benchmarking can calibrate eDNA‐based population genetic insights for future applications, the framework advances non‐invasive genetic monitoring of populations via eDNA and enables population‐level monitoring and conservation of endangered and genetically‐vulnerable species.