Epidemics in native species influence the outcome of a species invasion.

Invasive species can have large effects on native communities. When native and invasive species share parasites, an epidemic in a native species could facilitate or inhibit the invasion. We sought to understand how the incidence and timing of epidemics in native species caused by a generalist parasite influenced the success and impact of an invasive species. We focused on North American native and invasive species of zooplankton (Daphnia dentifera and Daphnia lumholtzi, respectively), that can both become infected with a fungal parasite (Metschnikowia bicuspidata). In a laboratory microcosm experiment, we exposed the native species to varying parasite inocula (none, low, high) and two invasive species introduction times (before or during an epidemic in the native species). We found that the invasive species density in treatments with the parasite was higher compared to uninfected treatments, though only the early invasion, low-parasite and uninfected treatments exhibited significant pairwise differences. However, invasive resting eggs were only found in the uninfected treatments. The density of the native species was lowest with a combination of the parasite present, and the invasive species introduced during the epidemic. Native infection prevalence in these treatments (late invasion, parasite present) was also higher than prevalence in treatments where the invasive species was introduced before the epidemic. Therefore, the timing of an invasion relative to an epidemic can affect both the native and invasive species. Our results suggest that the occurrence and timing of epidemics in native species can influence the impacts of a species invasion.

Two is better than one: Coupling DNA metabarcoding and stable isotope analysis improves dietary characterizations for a riparian-obligate, migratory songbird.

While an increasing number of studies are adopting molecular and chemical methods for dietary characterization, these studies often employ only one of these laboratory-based techniques; this approach may yield an incomplete, or even biased, understanding of diet due to each method's inherent limitations. To explore the utility of coupling molecular and chemical techniques for dietary characterizations, we applied DNA metabarcoding alongside stable isotope analysis to characterize the dietary niche of breeding Louisiana waterthrush (Parkesia motacilla), a migratory songbird hypothesized to preferentially provision its offspring with pollution-intolerant, aquatic arthropod prey. While DNA metabarcoding was unable to determine if waterthrush provision aquatic and terrestrial prey in different abundances, we found that specific aquatic taxa were more likely to be detected in successive seasons than their terrestrial counterparts, thus supporting the aquatic specialization hypothesis. Our isotopic analysis added greater context to this hypothesis by concluding that breeding waterthrush provisioned Ephemeroptera and Plecoptera, two pollution-intolerant, aquatic orders, in higher quantities than other prey groups, and expanded their functional trophic niche when such prey were not abundantly provisioned. Finally, we found that the dietary characterizations from each approach were often uncorrelated, indicating that the results gleaned from a diet study can be particularly sensitive to the applied methodologies. Our findings contribute to a growing body of work indicating the importance of high-quality, aquatic habitats for both consumers and their pollution-intolerant prey, while also demonstrating how the application of multiple, laboratory-based techniques can provide insights not offered by either technique alone.

DNA metabarcoding of nestling feces reveals provisioning of aquatic prey and resource partitioning among Neotropical migratory songbirds in a riparian habitat.

Riparian habitats are characterized by substantial flows of emergent aquatic insects that cross the stream-forest interface and provide an important source of prey for insectivorous birds. The increased availability of prey arising from aquatic subsidies attracts high densities of Neotropical migratory songbirds that are thought to exploit emergent aquatic insects as a nestling food resource; however, the prey preferences and diets of birds in these communities are only broadly understood. In this study, we utilized DNA metabarcoding to investigate the extent to which three syntopic species of migratory songbirds-Acadian Flycatcher, Louisiana Waterthrush, and Wood Thrush-breeding in Appalachian riparian habitats (Pennsylvania, USA) exploit and partition aquatic prey subsidies as a nestling food resource. Despite substantial differences in adult foraging strategies, nearly every nestling in this study consumed aquatic taxa, suggesting that aquatic subsidies are an important prey resource for Neotropical migrants nesting in riparian habitats. While our results revealed significant interspecific dietary niche divergence, the diets of Acadian Flycatcher and Wood Thrush nestlings were strikingly similar and exhibited significantly more overlap than expected. These results suggest that the dietary niches of Neotropical migrants with divergent foraging strategies may converge due to the opportunistic provisioning of non-limiting prey resources in riparian habitats. In addition to providing the first application of DNA metabarcoding to investigate diet in a community of Neotropical migrants, this study emphasizes the importance of aquatic subsidies in supporting breeding songbirds and improves our understanding of how anthropogenic disturbances to riparian habitats may negatively impact long-term avian conservation.

Integrating DNA-Based Prey Occurrence Probability into Stable Isotope Mixing Models.

The introduction of laboratory methods to animal dietary studies has allowed researchers to obtain results with accuracy and precision, not possible with observational techniques. For example, DNA barcoding, or the identification of prey with taxon-specific DNA sequences, allows researchers to classify digested prey tissues to the species-level, while stable isotope analysis paired with Bayesian mixing models can quantify dietary contributions by comparing a consumer's isotopic values to those derived from their prey. However, DNA-based methods are currently only able to classify, but not quantify, the taxa present in a diet sample, while stable isotope analysis can only quantify dietary taxa that are identified a priori as prey isotopic values are a result of life history traits, not phylogenetic relatedness. Recently, researchers have begun to couple these techniques in dietary studies to capitalize on the reciprocal benefits and drawbacks offered by each approach, with some even integrating DNA-based results directly into Bayesian mixing models as informative priors. As the informative priors used in these models must represent known dietary compositions (e.g., percentages of prey biomasses), researchers have scaled the DNA-based frequency of occurrence of major prey groups so that their normalized frequency of occurrence sums to 100%. Unfortunately, such an approach is problematic as priors stemming from binomial, DNA-based data do not truly reflect quantitative information about the consumer's diet and may skew the posterior distribution of prey quantities as a result. Therefore, we present a novel approach to incorporate DNA-based dietary information into Bayesian stable isotope mixing models that preserves the binomial nature of DNA-based results. This approach uses community-wide frequency of occurrence or logistic regression-based estimates of prey occurrence to dictate the probability that each prey group is included in each mixing model iteration, and, in turn, the probability that each iteration's results are included in the posterior distribution of prey composition possibilities. Here, we demonstrate the utility of this method by using it to quantify the prey composition of nestling Louisiana waterthrush (Parkesia motacilla).

Sea Snacks from DNA Tracks: Using DNA Metabarcoding to Characterize the Diet of Green Turtles (Chelonia Mydas).

The green turtle (Chelonia mydas) is a circumglobal species with a wide dietary breadth that varies among regions and life history stages. Comprehensive understanding of foraging ecology over space and time is critical to inform conservation and management of this species and its habitats. Here, we used DNA metabarcoding to test candidate primer sets with 39 gut content homogenates from stranded green turtles (FL, USA) to identify primer sets that maximize detection of food items and specificity of taxonomic classifications. We tested six existing universal primer sets to detect plants, animals, and eukaryotes more broadly (CO1, 18SV1-V3, 18SV4, rbcL, UPA, ITS). The CO1 and 18SV4 primer sets produced the greatest number of dietary amplicon sequence variants (ASVs) and unique taxonomic classifications, and they were the only primer sets to amplify taxa from all three kingdoms relevant to green turtle diet (Animalia, Chromista, and Plantae). Even though the majority of CO1-derived reads were of host origin (>90%), this primer set still produced the largest number of dietary ASVs classified to species among the six primer sets. However, because the CO1 primer set failed to detect both vascular plants and green algae, we do not recommend the use of this primer set on its own to characterize green turtle diet. Instead, our findings support previous research highlighting the utility of using multiple primer sets, specifically targeting CO1 and the V4 region of the 18S gene, as doing so will provide the most comprehensive understanding of green turtle diet. More generally, our results highlight the importance of primer and loci selection and the need to validate primer sets against the study system of interest. The addition of DNA metabarcoding with optimized primer sets to the sea turtle researcher's toolbox will both increase our understanding of foraging ecology and better inform science-based conservation and ecosystem management.

DNA Metabarcoding Across Disciplines: Sequencing Our Way to Greater Understanding Across Scales of Biological Organization.

DNA metabarcoding describes the use of targeted DNA (i.e., amplicon) sequencing to identify community constituents from a complex sample containing genetic material from multiple organisms, such as water, soil, gut contents, microbiomes, or biofilms. This molecular approach for characterizing mixed DNA samples relies on the development of "universal primers" that allow for effective amplification of target sequences across a broad range of taxa. Armed with optimized lab protocols and rigorous bioinformatics tools, DNA metabarcoding can produce a wealth of information about the hidden biodiversity of various sample types by probing for organisms' molecular footprints. DNA metabarcoding has received considerable popular press over the last few years because of gut microbiome studies in humans and beyond. However, there are many other applications that are continually integrating molecular biology with other fields of study to address questions that have previously been unanswerable, for both prokaryotic and eukaryotic targets. For example, we can now sample mostly digested gut contents from virtually any organism to learn about ontogeny and foraging ecology. Water samples collected from different locations can be filtered to extract eDNA (i.e., environmental DNA), revealing the biodiversity of fish and other taxa targeted by carefully selected primer sets. This universal primer metabarcoding approach has even been extended to looking at diverse gene families within single species, which is particularly useful for complex immune system genetics. The purpose of this SICB symposium was to bring together researchers using DNA metabarcoding approaches to (a) showcase the diversity of applications of this technique for addressing questions spanning ecology, evolution, and physiology, and (b) to spark connections among investigators from different fields that are utilizing similar approaches to facilitate optimization and standardization of metabarcoding methods and analyses. The resulting manuscripts from this symposium represent a great diversity of metabarcoding applications and taxonomic groups of interest.

Stream acidification and reduced aquatic prey availability are associated with dietary shifts in an obligate riparian Neotropical migratory songbird.

Streams and their surrounding riparian habitats are linked by reciprocal exchanges of insect prey essential to both aquatic and terrestrial consumers. Aquatic insects comprise a large proportion of total prey in riparian habitats and are opportunistically exploited by terrestrial insectivores; however, several species of songbirds are known to preferentially target aquatic prey via specialized foraging strategies. For these songbirds, reduced availability of aquatic insects via stream acidification may result in compensatory changes in provisioning during the nesting period, thereby influencing both adult and nestling diet composition. In this study, we used DNA metabarcoding to test the hypothesis that an obligate riparian Neotropical migratory songbird, the Louisiana Waterthrush (Parkesia motacilla), expands its diet to compensate for the loss of preferred aquatic prey taxa (primarily pollution-sensitive Ephemeroptera, Plecoptera, and Trichoptera) as a result of stream acidification. Our results revealed that both adult and nestling waterthrush exhibited an increase in dietary richness and niche breadth resulting from the consumption of terrestrial prey taxa in acidified riparian habitats. In contrast, compensatory dietary shifts were not observed in syntopic Neotropical migrant species known to primarily provision terrestrial prey taxa. In addition to providing support for our hypothesis that waterthrush compensate for stream acidification and aquatic prey limitations by expanding their diet, our findings highlight the vulnerability of Louisiana Waterthrush to anthropogenic disturbances that compromise stream quality or reduce the availability of pollution-sensitive aquatic insects.