not-yet-known not-yet-known not-yet-known unknown 1) DNA metabarcoding has been successful for the rapid identification of species in ecological assemblages, including identifying interspecific interactions among species. However, advances in metabarcoding plants have been hampered due to a lack of universal gene regions that work across all taxa, limiting the applications of metagenomics in ecology more broadly. 2) To circumvent these limitations, we propose a spatio-temporal approach that combines multi-gene barcoding with existing plant occurrence databases, species distribution models, and phenological analyses to generate a shortened list of candidate species to increase metabarcoding accuracy. To validate the ecological accuracy of our methodological framework, we compared the results of the DNA metabarcoding from pollen loads of wild bumble bees to long-term field observations of bee-plant interactions, and visual pollen identification. 3) We show that DNA metabarcoding of the plant species included in bumble bee pollen loads was most accurate when combined with a candidate taxa list of plant species flowering in the area when the bumble bees were foraging, which improved the accuracy and taxonomic precision of 77.5% of samples. 4) With the recent proliferation of species occurrence and phenology data in tandem with advances in computing and software development, we believe that spatio-temporal filtering provides a simple approach for interpreting metagenomic studies globally. Additionally, we demonstrate that the Angiosperms 353 probes offer significant promise for metagenomics projects globally, including metabarcoding to reveal species interactions within complex communities. Further, our approach demonstrates that integrating DNA metabarcoding is most accurate and powerful when combined with local ecological data.

Urban growth is occurring rapidly, and the land use changes associated with urbanization may have consequences for pollinators and the plants that rely on them. There is both evidence that urban areas support diverse pollinator communities and evidence that they degrade them. The influence of urbanization on the pollination of urban plants is even less understood. Urban agriculture relies on plant-pollinator interactions for crop production, providing a relevant framework to study pollination in an urban context. We therefore grew 240 plants across six sites at varying levels of urbanization in Chicago, Illinois, to investigate how urbanization relates to pollination in a generalized pollination system in Cucurbita pepo (squash) and a more specialized pollination system in the buzz-pollinated Solanum lycopersicum (tomato). We used a pollen limitation experiment to test whether the reproduction of plants at urban farms is pollen-limited and whether the magnitude of pollen limitation varies with the extent of urbanization, quantified as the percent of impervious surface surrounding each site. We also examined how pollinator visitation rates vary with urbanization. In S. lycopersicum but not C. pepo, the pollen addition treatment had a consistent and significant positive effect on reproductive success, indicating that plants of S. lycopersicum are pollen-limited in our study area. The magnitude of this pollen limitation (the difference in reproduction between paired control and pollen-supplemented plants) increased with greater impervious surface. The limited evidence for pollen limitation in the more generalized C. pepo suggests that plants with more specialized pollination systems are subject to greater pollen limitation in urban environments. Together, our results demonstrate that urban plants are likely experiencing deficits in pollination services, in ways that increase with the level of urbanization in the surrounding area but vary with the type of pollination system.

Most studies of plant--animal mutualistic networks have been temporally static. This approach has revealed many general patterns in the structure of complex webs of mutualistic interactions, but limits our ability to understand the ecological and evolutionary processes that shape these networks, and to predict the consequences of natural and human-driven disturbance on species interactions. The growing availability of temporally explicit data is allowing ecologists to move beyond this static perspective. We review the growing literature dealing with temporal dynamics in plant--animal mutualistic networks including pollination, seed dispersal and ant defence mutualisms. We identify general patterns of temporal variation in these networks across temporal scales. We discuss potential mechanisms underlying variation in interactions, ranging from behavioural and physiological processes at the narrowest temporal scales to ecological and evolutionary processes operating over much broader temporal scales. We conclude by discussing priorities for future research, including an improved understanding of the abiotic and biotic factors driving temporal network change, and further development and refinement of analytical tools. Our review highlights the key role of the importance of considering the temporal dimension for our understanding of the ecology and evolution of complex webs of mutualistic interactions.