Traversing the land-sea interface: A climate change risk assessment of terrestrially breeding marine predators.

Terrestrially breeding marine predators have experienced shifts in species distribution, prey availability, breeding phenology, and population dynamics due to climate change worldwide. These central-place foragers are restricted within proximity of their breeding colonies during the breeding season, making them highly susceptible to any changes in both marine and terrestrial environments. While ecologists have developed risk assessments to evaluate climate risk in various contexts, these often overlook critical breeding biology data. To address this knowledge gap, we developed a trait-based risk assessment framework, focusing on the breeding season and applying it to marine predators breeding in parts of Australian territory and Antarctica. Our objectives were to quantify climate change risk, identify specific threats, and establish an adaptable assessment framework. The assessment considered 25 criteria related to three risk components: vulnerability, exposure, and hazard, while accounting for uncertainty. We employed a scoring system that integrated a systematic literature review and expert elicitation for the hazard criteria. Monte Carlo sensitivity analysis was conducted to identify key factors contributing to overall risk. We identified shy albatross (Thalassarche cauta), southern rockhopper penguins (Eudyptes chrysocome), Australian fur seals (Arctocephalus pusillus doriferus), and Australian sea lions (Neophoca cinerea) with high climate urgency. Species breeding in lower latitudes, as well as certain eared seal, albatross, and penguin species, were particularly at risk. Hazard and exposure explained the most variation in relative risk, outweighing vulnerability. Key climate hazards affecting most species include extreme weather events, changes in habitat suitability, and prey availability. We emphasise the need for further research, focusing on at-risk species, and filling knowledge gaps (less-studied hazards, and/or species) to provide a more accurate and robust climate change risk assessment. Our findings offer valuable insights for conservation efforts, given that monitoring and implementing climate adaptation strategies for land-dependent marine predators is more feasible during their breeding season.

Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?

Advances in DNA sequencing technology have revolutionized the field of molecular analysis of trophic interactions, and it is now possible to recover counts of food DNA sequences from a wide range of dietary samples. But what do these counts mean? To obtain an accurate estimate of a consumer's diet should we work strictly with data sets summarizing frequency of occurrence of different food taxa, or is it possible to use relative number of sequences? Both approaches are applied to obtain semi-quantitative diet summaries, but occurrence data are often promoted as a more conservative and reliable option due to taxa-specific biases in recovery of sequences. We explore representative dietary metabarcoding data sets and point out that diet summaries based on occurrence data often overestimate the importance of food consumed in small quantities (potentially including low-level contaminants) and are sensitive to the count threshold used to define an occurrence. Our simulations indicate that using relative read abundance (RRA) information often provides a more accurate view of population-level diet even with moderate recovery biases incorporated; however, RRA summaries are sensitive to recovery biases impacting common diet taxa. Both approaches are more accurate when the mean number of food taxa in samples is small. The ideas presented here highlight the need to consider all sources of bias and to justify the methods used to interpret count data in dietary metabarcoding studies. We encourage researchers to continue addressing methodological challenges and acknowledge unanswered questions to help spur future investigations in this rapidly developing area of research.

High occurrence of jellyfish predation by black-browed and Campbell albatross identified by DNA metabarcoding.

Gelatinous zooplankton are a large component of the animal biomass in all marine environments, but are considered to be uncommon in the diet of most marine top predators. However, the diets of key predator groups like seabirds have conventionally been assessed from stomach content analyses, which cannot detect most gelatinous prey. As marine top predators are used to identify changes in the overall species composition of marine ecosystems, such biases in dietary assessment may impact our detection of important ecosystem regime shifts. We investigated albatross diet using DNA metabarcoding of scats to assess the prevalence of gelatinous zooplankton consumption by two albatross species, one of which is used as an indicator species for ecosystem monitoring. Black-browed and Campbell albatross scats were collected from eight breeding colonies covering the circumpolar range of these birds over two consecutive breeding seasons. Fish was the main dietary item at most sites; however, cnidarian DNA, primarily from scyphozoan jellyfish, was present in 42% of samples overall and up to 80% of samples at some sites. Jellyfish was detected during all breeding stages and consumed by adults and chicks. Trawl fishery catches of jellyfish near the Falkland Islands indicate a similar frequency of jellyfish occurrence in albatross diets in years of high and low jellyfish availability, suggesting jellyfish consumption may be selective rather than opportunistic. Warmer oceans and overfishing of finfish are predicted to favour jellyfish population increases, and we demonstrate here that dietary DNA metabarcoding enables measurements of the contribution of gelatinous zooplankton to the diet of marine predators.

Simultaneous DNA-based diet analysis of breeding, non-breeding and chick Adélie penguins.

As central place foragers, breeding penguins are restricted in foraging range by the need to return to the colony to feed chicks. Furthermore, breeding birds must balance energetic gain from self-feeding with the costs of returning to provision young. Non-breeding birds, however, are likely to be less restricted in foraging range and lack the high energy demands of provisioning, therefore may consume different prey to breeders. We used DNA dietary analysis to determine whether there was a difference in provisioning and self-feeding diet by identifying prey DNA in scat samples from breeding and chick Adélie penguins at two locations in East Antarctica. We also investigated diet differences between breeders and non-breeders at one site. Although previous work shows changing foraging behaviour between chick provisioning and self-feeding, our results suggest no significant differences in the main prey groups consumed by chicks and breeders at either site or between breeding stages. This may reflect the inability of penguins to selectively forage when provisioning, or resources were sufficient for all foraging needs. Conversely, non-breeders were found to consume different prey groups to breeders, which may reflect less restricted foraging ranges, breeders actively selecting particular prey during breeding or reduced foraging experience of non-breeders.

High-throughput real-time PCR and melt curve analysis for sexing Southern Ocean seabirds using fecal samples.

Sex identification of birds is of great interest in ecological studies, however this can be very difficult in many species because their external features are almost monomorphic between the sexes. Molecular methodology has simplified this process but limitations still occur with widely accepted methods using polymerase chain reaction and gel electrophoresis, especially when applied to degraded DNA. Real-time polymerase chain reaction assays are emerging as a more efficient, sensitive, and higher throughput means of identification, but there are very few techniques validated using fecal samples and small target sizes. We present a real-time melt curve analysis assay targeting a small region of the CHD-1 gene allowing for high-throughput, sensitive, specific, and easy-to-interpret sexing results for a variety of Southern Ocean seabirds using fecal and tissue samples.

Searching for the true diet of marine predators: incorporating Bayesian priors into stable isotope mixing models.

Reconstructing the diet of top marine predators is of great significance in several key areas of applied ecology, requiring accurate estimation of their true diet. However, from conventional stomach content analysis to recent stable isotope and DNA analyses, no one method is bias or error free. Here, we evaluated the accuracy of recent methods to estimate the actual proportion of a controlled diet fed to a top-predator seabird, the Little penguin (Eudyptula minor). We combined published DNA data of penguins scats with blood plasma δ(15)N and δ(13)C values to reconstruct the diet of individual penguins fed experimentally. Mismatch between controlled (true) ingested diet and dietary estimates obtained through the separately use of stable isotope and DNA data suggested some degree of differences in prey assimilation (stable isotope) and digestion rates (DNA analysis). In contrast, combined posterior isotope mixing model with DNA Bayesian priors provided the closest match to the true diet. We provided the first evidence suggesting that the combined use of these complementary techniques may provide better estimates of the actual diet of top marine predators- a powerful tool in applied ecology in the search for the true consumed diet.

Adélie penguin population diet monitoring by analysis of food DNA in scats.

The Adélie penguin is the most important animal currently used for ecosystem monitoring in the Southern Ocean. The diet of this species is generally studied by visual analysis of stomach contents; or ratios of isotopes of carbon and nitrogen incorporated into the penguin from its food. There are significant limitations to the information that can be gained from these methods. We evaluated population diet assessment by analysis of food DNA in scats as an alternative method for ecosystem monitoring with Adélie penguins as an indicator species. Scats were collected at four locations, three phases of the breeding cycle, and in four different years. A novel molecular diet assay and bioinformatics pipeline based on nuclear small subunit ribosomal RNA gene (SSU rDNA) sequencing was used to identify prey DNA in 389 scats. Analysis of the twelve population sample sets identified spatial and temporal dietary change in Adélie penguin population diet. Prey diversity was found to be greater than previously thought. Krill, fish, copepods and amphipods were the most important food groups, in general agreement with other Adélie penguin dietary studies based on hard part or stable isotope analysis. However, our DNA analysis estimated that a substantial portion of the diet was gelatinous groups such as jellyfish and comb jellies. A range of other prey not previously identified in the diet of this species were also discovered. The diverse prey identified by this DNA-based scat analysis confirms that the generalist feeding of Adélie penguins makes them a useful indicator species for prey community composition in the coastal zone of the Southern Ocean. Scat collection is a simple and non-invasive field sampling method that allows DNA-based estimation of prey community differences at many temporal and spatial scales and provides significant advantages over alternative diet analysis approaches.