Abstract
Understanding how biological communities are structured across space and time is a fundamental goal in ecology and evolution. Traditional genetic approaches require sampling many individuals, an effort that is often logistically challenging, limited in geographic coverage, and taxonomically biased towards easily sampled species. Environmental DNA (eDNA) offers a transformative alternative, enabling simultaneous characterisation of both community composition and genetic variation across hundreds of species. However, a key challenge for this approach is determining the frequencies of genotypes and haplotypes from mixed environmental samples containing many taxa and individuals. This could be limiting for marine species that have high gene flow, as genetic differentiation is often characterised by changes in gene frequencies, not the presence of unique haplotypes. Here, we directly tested the accuracy of eDNA‐derived mitochondrial DNA diversity and genetic differentiation estimates for 18 Hawaiian marine vertebrates against benchmarks from traditional, individual‐based tissue sampling. eDNA reliably recovered dominant haplotypes at their relative frequencies, and measures of sequence diversity were significantly correlated between approaches. In contrast, population structure metrics were only weakly correlated at this spatial scale, because eDNA more frequently returned positive values of Φ ST than tissue‐based methods. Overall, eDNA successfully converted complex mixed samples into accurate estimates of haplotype composition and relative frequencies, enabling comparative population genetic analyses across entire marine communities and revealing both the strengths and limitations of frequency‐based inference from environmental samples.