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Abstract Studies of stream macroinvertebrates traditionally use sampling methods that target benthic habitats. These methods could underestimate biodiversity if important assemblage components exist outside of the benthic zone. To test the efficacy of different sampling methods, we collected paired reach‐wide benthic and edge samples from up to 10 study reaches in nine basins spanning an aridity gradient across the United States. Edge sampling targeted riparian‐adjacent microhabitats not typically sampled, including submerged vegetation, roots, and overhanging banks. We compared observed richness, asymptotic richness, and assemblage dissimilarity between benthic samples alone and different combinations of benthic and edge samples to determine the magnitude of increased diversity and assemblage dissimilarity values with the addition of edge sampling. We also examined how differences in richness and assemblage composition varied across an aridity gradient. The addition of edge sampling significantly increased observed richness (median increase = 29%) and asymptotic richness (median increase = 173%). Similarly, median Bray–Curtis dissimilarity values increased by as much as 0.178 when benthic and edge samples were combined. Differences in richness metrics were generally higher in arid basins, but assemblage dissimilarity either increased or decreased across the aridity gradient depending on how benthic and edge samples were combined. Our results suggest that studies that do not sample stream edges may significantly underestimate reach diversity and misrepresent assemblage compositions, with effects that can vary across climates. We urge researchers to carefully consider sampling methods in field studies spanning climatic zones and the comparability of existing data sets when conducting data synthesis studies. 

Abstract DNA‐based aquatic biomonitoring methods show promise to provide rapid, standardized, and efficient biodiversity assessment to supplement and in some cases replace current morphology‐based approaches that are often less efficient and can produce inconsistent results. Despite this potential, broad‐scale adoption of DNA‐based approaches by end‐users remains limited, and studies on how these two approaches differ in detecting aquatic biodiversity across large spatial scales are lacking. Here, we present a comparison of DNA metabarcoding and morphological identification, leveraging national‐scale, open‐source, ecological datasets from the National Ecological Observatory Network (NEON). Across 24 wadeable streams in North America with 179 paired sample comparisons, we found that DNA metabarcoding detected twice as many unique taxa than morphological identification overall. The two approaches showed poor congruence in detecting the same taxa, averaging 59%, 35%, and 23% of shared taxa detected at the order, family, and genus levels, respectively. Importantly, the two approaches detected different proportions of indicator taxa like %EPT and %Chironomidae. DNA metabarcoding detected far fewer Chironomid and Trichopteran taxa than morphological identification, but more Ephemeropteran and Plecopteran taxa, a result likely due to primer choice. Overall, our results showed that DNA metabarcoding and morphological identification detected different benthic macroinvertebrate communities. Despite these differences, we found that the same environmental variables were correlated with invertebrate community structure, suggesting that both approaches can accurately detect biodiversity patterns across environmental gradients. Further refinement of DNA metabarcoding protocols, primers, and reference libraries–as well as more standardized, large‐scale comparative studies–may improve our understanding of the taxonomic agreement and data linkages between DNA metabarcoding and morphological approaches.