Hummingbirds, a highly diverse avian family, are specialized vertebrate pollinators that feed upon carbohydrate-rich nectar to fuel their fast metabolism while consuming invertebrates to obtain protein. Previous work has found that morphologically diverse hummingbird communities exhibit higher diet specialization on floral resources than morphologically similar hummingbird communities. Due to the difficulties of studying avian diets, we have little understanding whether hummingbirds show similar patterns with their invertebrate prey. Here, we use DNA metabarcoding to complete an analysis of floral and invertebrate diets of three species of sympatric North American hummingbirds. We collected fecal samples from 89 Anna’s (Calypte anna), 39 Black-chinned (Archilochus alexandri), and 29 Calliope (Selasphorus calliope) hummingbirds in urban and rural localities as well as across an elevational gradient from sea level to 2500 meters above sea level in California, USA. We found hummingbirds showed high dietary overlap in both invertebrate and plant resources, with few invertebrate and plant families common to most individuals and many families found in only a few individuals. Chironomidae was the most common invertebrate family across all species, and Rosaceae and Orobanchaceae were the most common plant families. Anna’s Hummingbirds had significantly higher invertebrate diet diversity than Black-chinned Hummingbirds when found at the same sites, but we found no difference in plant diet diversity among any of the three species. Hummingbirds in urban sites had higher plant diet diversity than in rural sites, but we found no effect of elevation on dietary richness. Our study shows how DNA metabarcoding can be used to non-invasively investigate previously unknown life-histories of well-studied birds, lending insight to community structure, function, and evolution.

We captured hummingbirds in California, USA and collected fecal samples. We used DNA metabarcoding to examine the floral and invertebrate diet items within each fecal sample. Here we present the metadata regarding each hummingbird, including species, date and location of capture, morphometrics, and total diet richness. Our second dataset is a spreadsheet showing each hummingbird (identified by a unique ID) and a precense/absence list of if a specific diet item was present in the hummingbirds fecal sample.