Evaluating the impacts of metabarcoding primer selection on DNA characterization of diet in an aerial insectivore, the Purple Martin

DNA metabarcoding is a molecular technique frequently used to characterize diet composition of insectivorous birds. However, results are sensitive to methodological decisions made during sample processing, with primer selection being one of the most critical. The most frequently used DNA metabarcoding primer set for avian insectivores is ZBJ. However, recent studies have found that ZBJ produces significant biases in prey classification that likely influence our understanding of foraging ecology. A new primer set, ANML, has shown promise for characterizing insectivorous bat diets with fewer taxonomic biases than ZBJ, but ANML has not yet been used to study insectivorous birds. Here, we evaluate the ANML primer set for use in metabarcoding of avian insectivore diets through comparison with the more commonly used ZBJ primer set. Fecal samples were collected from both adult and nestling Purple Martins (Progne subis subis) at 2 sites in the USA and 1 site in Canada to maximize variation in diet composition and to determine if primer selection impacts our understanding of diet variation among sites. In total, we detected 71 arthropod prey species, 39 families, and 10 orders. Of these, 40 species were uniquely detected by ANML, whereas only 11 were uniquely detected by ZBJ. We were able to classify 54.8% of exact sequence variants from ANML libraries to species compared to 33.3% from ZBJ libraries. We found that ANML outperformed ZBJ for PCR efficacy, taxonomic coverage, and specificity of classification, but that using both primer sets together produced the most comprehensive characterizations of diet composition. Significant variation in both alpha- and beta-diversity between sites was found using each primer set separately and in combination. To our knowledge, this is the first published metabarcoding study using ANML primers to describe avian diet, and also the first to directly compare results returned by ANML and ZBJ primer sets.

Comparative morphology of tongue surface in Neotropical aerial insectivore bats (Mammalia: Chiroptera)

Comparative morphological characters in Neotropical bats are mostly restricted to external and cranio-dentary complexes, and few studies focusing on other morphological complexes have been carried out. In the case of tongue morphology, comparative analyses of the structure have been restricted to the superfamily Noctilionoidea with a wide range of diets, and Molossidae, a strictly aerial insectivore family. In this paper, we studied the morphology of tongue papillae in 10 aerial insectivore Neotropical bat species, representing six families (Emballonuridae, Furipteridae, Thyropteridae, Mormoopidae, Natalidae, and Vespertilionidae), and data from the previous study of Molossidae were compared. We studied tongues in light and scanning electron microscopes following material preparation protocols. We observed two types of sensitive papillae, circumvallate and fungiform, the latter at times presenting a groove surrounding the papillae. Nine mechanic types were observed, one of them, which we called flaky-like, not hitherto described. All Vespertilionoidea families (Vespertilionidae, Natalidae, and Molossidae) presented, as diagnosing characters, fungiform papillae distributed throughout the tongue, as well as anteriorly at the dorsum, and scale-like papillae on the medial lobe directed laterally and anteriorly. Emballonuridae showed the simplest tongue morphology regarding the presence and abundance of some papillae. Families composing the clade Furipteridae + Thyropteridae + Mormoopidae presented small and non-grooved fungiform papillae, and mechanical bifid papillae were absent. In summary, this study has provided additional traits (putative synapomorphies) of the bat tongue to support the clades on the current bat phylogeny.

Phenotypic differences among wild passerine nestlings in relation to early-life rearing environment

Subtle changes in stress physiology during critical developmental stages have been linked to long-term fitness; however, the biological processes and phenotypic responses to early-life rearing environments such as anthropogenic land use conditions, have not been fully evaluated in insectivorous birds. We manipulated Tree Swallow, Tachycineta bicolor (Vieillot, 1808), brood sizes at sites with contrasting agricultural land use to assess phenotypic changes in body condition and genetic and physiological biomarkers of stress during the sensitive nestling growth phase. We predicted that nestling swallows raised on cropland-dominated sites, especially those in enlarged broods, would have lower body condition, shorter telomeres, and higher feather corticosterone than nestlings raised in smaller broods at grassland sites. Body condition was highest among nestlings raised in reduced broods but was unrelated to land use. Telomere lengths tended to be shorter in nestlings from enlarged broods and at cropland sites. Corticosterone was not related to any factor. Locally-abundant insect populations associated with wetlands may have dampened the effects and/or parent swallows assumed higher costs of reproduction rather than passing these costs to nestlings. Results suggest that food stress could reduce fledgling survival via telomere shortening; a hypothesis that requires further investigation due to its potential importance to population viability in multiple declining aerial insectivore species.

Interacting effects of cold snaps, rain, and agriculture on the fledging success of a declining aerial insectivore

Climate change predicts the increased frequency, duration, and intensity of inclement weather periods, such as unseasonably low temperatures and prolonged precipitation. Many migratory species have advanced the phenology of important life history stages, and as a result are likely exposed to these periods of inclement spring weather more often, thus risking reduced fitness and population growth. For declining avian species, including aerial insectivores, anthropogenic landscape changes such as agricultural intensification are another driver of population declines. These landscape changes may affect the foraging ability of food provisioning parents, as well as reduce the probability a nestling will survive periods of inclement weather, through for example pesticide exposure impairing thermoregulation and punctual anorexia. Breeding in agro-intensive landscapes may thus exacerbate the negative effects of inclement weather under climate change. We used daily temperatures related to significant reductions of insect prey availability (cold snaps), combined with measures of precipitation, and assessed their impact on Tree Swallow (Tachycineta bicolor) fledging success, a declining aerial insectivore breeding across a gradient of agricultural intensification. Fledging success decreased with the number of cold snap days experienced by a brood, and this relationship was worsened during periods of prolonged precipitation. We further found the overall negative effects of experiencing periods of inclement weather are exacerbated in more agro-intensive landscapes. Our results indicate that two of the primary hypothesized drivers of many avian population declines may interact to further increase the rate of declines in certain landscape contexts.

The high-energy aerial insectivore lifestyle of swallows does not produce clear thermogenic side effects

Ecological traits related to pace of life, such as foraging strategies and activity levels, influence daily energy expenditure (DEE) and can affect fitness. A fast pace of life tends to be supported by high-energy aerobic activity and is positively correlated with high DEE and basal and maximal metabolic rates in some endotherms. Given that maximal capacities for exercise and thermogenesis are both functions of aerobic muscle output and are often positively correlated with each other, high-energy aerobic lifestyles might be associated with high aerobic capacities, which would be expected to produce high thermogenic capacities as a side effect. We tested whether the high-energy aerial insectivore lifestyle in swallows is correlated with elevated basal and maximal thermogenic metabolic rates. We measured basal (BMR) and summit (Msum = maximum cold-induced metabolic rate) metabolic rates in 6 species of swallows (Hirundinidae) and combined these data with literature data for additional swallows (n = 10 for BMR; n = 8 for Msum) and non-aerial insectivore birds (n = 215 for BMR; n = 64 for Msum) to address the hypothesis that swallows have higher BMR and Msum than non-aerial insectivores. BMR in swallows was significantly higher than for non-aerial insectivore birds for phylogenetically adjusted analyses after correcting for body mass and region of origin (tropical vs. temperate). In contrast, Msum did not differ significantly between swallows and non-aerial insectivores. Thermogenic scope (Msum – BMR), however, was lower in tropical non-aerial insectivore birds compared with tropical swallows and temperate birds. This suggests that the aerial insectivore lifestyle elevates maintenance costs, but maximum thermogenic capacities are not clearly upregulated, despite tropical swallows having higher thermogenic scope than tropical non-aerial insectivores. These data suggest that the high-energy aerial insectivore lifestyle does not produce strong thermogenic side effects in swallows.