Getting to the core of the matter: Assessing the role of replication in sedimentary DNA metabarcoding

Replication is central to most experimental and sampling designs, increasing inferential power and capturing fine-scale data heterogeneity. However, its importance remains poorly evaluated in some ecological and evolutionary settings. This is the case of metabarcoding studies using DNA recovered from sedimentary archives, in which biological signals may integrate ecological information through depositional and burial processes, and are often inferred from a single sediment core per site. Here, we evaluated the effect of different types of replication using sedimentary DNA (sedaDNA) metabarcoding data from two genetic markers (mitochondrial COI and nuclear 18S), under a nested sampling design. The design included three intertidal sites, three spatially separated sediment cores per site (biological replicates), two sediment depth horizons per core, and eight PCR (technical) replicates per sediment sample. Variance partitioning showed that site identity and sediment age group together explained >70% of the variation in beta diversity, indicating that among-site spatial variation and stratigraphic variation were the dominant drivers of community composition. In contrast, variation among different cores within sites was small and non-significant (<5%). Among PCR replicates from the same sediment sample, richness varied substantially, whereas Shannon diversity was more consistent. Despite this variability, differences in community composition among technical replicates remained smaller than among biological replicates and site identity, indicating limited influence on broader ecological patterns. Community composition was highly similar among replicate cores within sites, consistent with stratigraphic coherence. These results indicate limited within-site heterogeneity and suggest that, under stratigraphically coherent conditions, increasing biological replication may yield limited additional information, whereas enhancing technical replication and stratigraphic resolution can improve ecological inference from sedaDNA metabarcoding datasets.