Spatial distribution of environmental DNA in a nearshore marine habitat.

In the face of increasing threats to biodiversity, the advancement of methods for surveying biological communities is a major priority for ecologists. Recent advances in molecular biological technologies have made it possible to detect and sequence DNA from environmental samples (environmental DNA or eDNA); however, eDNA techniques have not yet seen widespread adoption as a routine method for biological surveillance primarily due to gaps in our understanding of the dynamics of eDNA in space and time. In order to identify the effective spatial scale of this approach in a dynamic marine environment, we collected marine surface water samples from transects ranging from the intertidal zone to four kilometers from shore. Using PCR primers that target a diverse assemblage of metazoans, we amplified a region of mitochondrial 16S rDNA from the samples and sequenced the products on an Illumina platform in order to detect communities and quantify their spatial patterns using a variety of statistical tools. We find evidence for multiple, discrete eDNA communities in this habitat, and show that these communities decrease in similarity as they become further apart. Offshore communities tend to be richer but less even than those inshore, though diversity was not spatially autocorrelated. Taxon-specific relative abundance coincided with our expectations of spatial distribution in taxa lacking a microscopic, pelagic life-history stage, though most of the taxa detected do not meet these criteria. Finally, we use carefully replicated laboratory procedures to show that laboratory treatments were remarkably similar in most cases, while allowing us to detect a faulty replicate, emphasizing the importance of replication to metabarcoding studies. While there is much work to be done before eDNA techniques can be confidently deployed as a standard method for ecological monitoring, this study serves as a first analysis of diversity at the fine spatial scales relevant to marine ecologists and confirms the promise of eDNA in dynamic environments.

Genetic signatures of ecological diversity along an urbanization gradient.

Despite decades of work in environmental science and ecology, estimating human influences on ecosystems remains challenging. This is partly due to complex chains of causation among ecosystem elements, exacerbated by the difficulty of collecting biological data at sufficient spatial, temporal, and taxonomic scales. Here, we demonstrate the utility of environmental DNA (eDNA) for quantifying associations between human land use and changes in an adjacent ecosystem. We analyze metazoan eDNA sequences from water sampled in nearshore marine eelgrass communities and assess the relationship between these ecological communities and the degree of urbanization in the surrounding watershed. Counter to conventional wisdom, we find strongly increasing richness and decreasing beta diversity with greater urbanization, and similar trends in the diversity of life histories with urbanization. We also find evidence that urbanization influences nearshore communities at local (hundreds of meters) rather than regional (tens of km) scales. Given that different survey methods sample different components of an ecosystem, we then discuss the advantages of eDNA-which we use here to detect hundreds of taxa simultaneously-as a complement to traditional ecological sampling, particularly in the context of broad ecological assessments where exhaustive manual sampling is impractical. Genetic data are a powerful means of uncovering human-ecosystem interactions that might otherwise remain hidden; nevertheless, no sampling method reveals the whole of a biological community.

Indexed PCR Primers Induce Template-Specific Bias in Large-Scale DNA Sequencing Studies.

Massively parallel sequencing is rapidly emerging as an efficient way to quantify biodiversity at all levels, from genetic variation and expression to ecological community assemblage. However, the number of reads produced per sequencing run far exceeds the number required per sample for many applications, compelling researchers to sequence multiple samples per run in order to maximize efficiency. For studies that include a PCR step, this can be accomplished using primers that include an index sequence allowing sample origin to be determined after sequencing. The use of indexed primers assumes they behave no differently than standard primers; however, we found that indexed primers cause substantial template sequence-specific bias, resulting in radically different profiles of the same environmental sample. Likely the outcome of differential amplification efficiency due to primer-template mismatch, two indexed primer sets spuriously change the inferred sequence abundance from the same DNA extraction by up to 77.1%. We demonstrate that a double PCR approach alleviates these effects in applications where indexed primers are necessary.