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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.

The evolution of cetaceans, fromtheir early transition to an aquatic lifestyle to their subsequent diversification, has been the subject of numerous studies. However, although the higher-level relationships among cetacean families have been largely settled, several aspects of the systematics within these groups remain unresolved. Problematic clades include the oceanic dolphins (37 spp.), which have experienced a recent rapid radiation, and the beaked whales (22 spp.), which have not been investigated in detail using nuclear loci. The combined application of high-throughput sequencing with techniques that target specific genomic sequences provide a powerful means of rapidly generating large volumes of orthologous sequence data for use in phylogenomic studies. To elucidate the phylogenetic relationships within the Cetacea,we combined sequence capture with Illumina sequencing to generate data for ∼3200 protein-coding genes for 68 cetacean species and their close relatives including the pygmy hippopotamus. By combining data from >38,000 exons with existing sequences from 11 cetaceans and seven outgroup taxa, we produced the first comprehensive comparative genomic data set for cetaceans, spanning 6,527,596 aligned base pairs (bp) and 89 taxa. Phylogenetic trees reconstructed with maximum likelihood and Bayesian inference of concatenated loci, as well as with coalescence analyses of individual gene trees, produced mostly concordant and well-supported trees. Our results completely resolve the relationships among beaked whales as well as the contentious relationships among oceanic dolphins, especially the problematic subfamily Delphinidae. We carried out Bayesian estimation of species divergence times using MCMCTree andcompared ourcomplete data set to a subset of clocklike genes. Analyses using the complete data set consistently showed less variance in divergence times than the reduced data set. In addition, integration of new fossils (e.g., Mystacodon selenensis) indicates that the diversification of Crown Cetacea began before the Late Eocene and the divergence of Crown Delphinidae as early as theMiddle Miocene.

Satellite tags were deployed on 50 east Australian humpback whales (breeding stock E1) between 2008 and 2010 on their southward migration, northward migration and feeding grounds in order to identify and describe migratory pathways, feeding grounds and possible calving areas. At the time, these movements were not well understood and calving grounds were not clearly identified. To the best of our knowledge, this dataset details all long-term, implantable tag deployments that have occurred to date on breeding stock E1. As such, these data provide researchers, regulators and industry with clear and valuable insights into the spatial and temporal nature of humpback whale movements along the eastern coastline of Australia and into the Southern Ocean. As this population of humpback whales navigates an increasingly complex habitat undergoing various development pressures and anthropogenic disturbances, in addition to climate-mediated changes in their marine environment, this dataset may also provide a valuable baseline. At the time these tracks were generated, these were the first satellite tag deployments intended to deliver long-term, detailed movement information on east Australian (breeding stock E1) humpback whales. The tracking data revealed previously unknown migratory pathways into the Southern Ocean, with 11 individuals tracked to their Antarctic feeding grounds. Once assumed to head directly south on their southern migration, five individuals initially travelled west towards New Zealand. Six tracks detailed the coastal movement of humpback whales migrating south. One tag transmitted a partial southern migration, then ceased transmissions only to begin transmitting eight months later as the animal was migrating north. Northern migration to breeding grounds was detailed for 13 individuals, with four tracks including turning points and partial southern migrations. Another 14 humpback whales were tagged in Antarctica, providing detailed Antarctic feeding ground movements. Broadly speaking, the tracking data revealed a pattern of movement where whales were at their northern limit in July and their southern limit in March. Migration north was most rapid across the months of May and June, whilst migration south was most rapid between November and December. Tagged humpback whales were located on their Antarctic feeding grounds predominantly between January and May and approached their breeding grounds between July and August. Tracking distances ranged from 68 km to 8580 km and 1 to 286 days. To the best of our knowledge, this dataset compiles all of the long-term tag deployments that have occurred to date on breeding stock E1.

Antarctic benthic ecosystems support high biodiversity but their characterization is limited to a few well-studied areas, due to the extreme environment and remoteness making access and sampling difficult. Our aim was to compare water and sediment as sources of environmental DNA (eDNA) to better characterise Antarctic benthic communities and further develop practical approaches for DNA-based biodiversity assessment in remote environments. We used a cytochrome c oxidase subunit I (COI) metabarcoding approach to characterise metazoan communities in 26 nearshore sites across 12 locations in the Vestfold Hills (East Antarctica) based on DNA extracted from either sediment cores or filtered seawater. We detected a total of 99 metazoan species from 12 phyla across 26 sites, with similar numbers of species detected in sediment and water eDNA samples. However, significantly different communities were detected in the two sample types at sites where both were collected ( i.e. , where paired samples were available). For example, nematodes and echinoderms were more likely to be detected exclusively in sediment and water eDNA samples, respectively. eDNA from water and sediment core samples are complementary sample types, with epifauna more likely to be detected in water column samples and infauna in sediment. More reference DNA sequences are needed for infauna/meiofauna to increase the proportion of sequences and number of taxa that can be identified. Developing a better understanding of the temporal and spatial dynamics of eDNA at low temperatures would also aid interpretation of eDNA signals from polar environments. Our results provide a preliminary scan of benthic metazoan communities in the Vestfold Hills, with additional markers required to provide a comprehensive biodiversity survey. However, our study demonstrates the choice of sample type for eDNA studies of benthic ecosystems (sediment, water or both) needs to be carefully considered in light of the research or monitoring question of interest.

Incidental mortality in fisheries is a major driver of population declines for albatrosses and petrels globally. However, accurate identification of species can be difficult due to the poor condition of bycaught birds and/or visual similarities between closely related species. We assessed three genetic markers for their ability to distinguish the 36 albatross and petrel species listed in Annex 1 to the Agreement on the Conservation of Albatrosses and Petrels (ACAP) and in Australia's Threat Abatement Plan (TAP) for the bycatch of seabirds during oceanic longline fishing operations. We generated 275 new sequences, from 29 species, to improve the coverage of reference databases for these listed species. The combined use of the selected Cytochrome b and Control Region markers enabled the identification of 31 of 36 listed seabirds to species level and four to sister species. One petrel species could not be evaluated as no reference sequences were available. We tested these markers on 59 feathers from bycaught seabirds and compared these to onboard visual identification. We successfully assigned all procellariiforms to species (n = 58), whereas only two seabirds were correctly identified to species visually onboard, highlighting the difficulty of visual species assignment and the need for alternative methods. We assessed the utility of our two chosen markers for the assignment of all procellariiform species, with 74% of species with reference sequences identified to species or sister species level. However, a precautionary approach is needed for application beyond our listed species due to unvalidated reference sequences. The approach described here provides a streamlined framework for the molecular identification of seabird bycatch. This approach is recommended for use in fisheries within and outside Australian waters to improve the resolution of bycatch reporting and to corroborate logbook entries, observer reports and audits of images captured by electronic monitoring systems as well as help inform conservation efforts. The development of genetic methods and resources to identify seabirds threatened by fisheries bycatch. This has the potential to significantly augment existing bycatch monitoring methods and improve confidence in the understanding of species‐level impacts by specific fisheries.

Antarctic krill (Euphausia superba) is a keystone species in the Southern Ocean ecosystem, and monitoring its distribution and abundance is crucial for the sustainable management of expanding fisheries targeting the species. Environmental DNA (eDNA)‐based monitoring could complement conventional krill surveys, but its applicability is limited by a lack of knowledge on eDNA persistence and decay in the Southern Ocean. We aimed to develop a method that can not only quantify Antarctic krill eDNA, but also estimate a relative time since this eDNA was shed (“recent” vs “older”). Three species‐specific qPCR markers targeting the mitochondrial 16S region were developed, and the eDNA decay characteristics of these markers were determined through tank experiments. Krill eDNA was partially degraded in all samples, even when krill were present. Marker concentrations decreased exponentially at similar rates after krill removal, with initial relative abundances maintained across the three markers. Over time, the concentration of the longest marker decreased faster, changing the relative abundances of the markers, and allowing discrimination of more recent samples from more degraded older samples. We employed this new method to quantify Antarctic krill eDNA collected across a 4800 km Southern Ocean transect, and estimated the age of the eDNA in these samples based on the relative abundance of markers, adding a temporal aspect to a quantitative eDNA survey. We also compared a Euphausiid‐specific metabarcoding marker to the qPCR method to assess sensitivity in detecting Antarctic krill eDNA. While these new eDNA methods should be evaluated against existing non‐molecular survey methods, they could add an important novel, dynamic layer of information to future krill surveys. Our method could not only determine where Antarctic krill eDNA is present but shed light on how they may be using certain habitats, expanding our understanding of this important species’ life cycle and contributing to more accurate abundance and distribution estimates. In this study a species‐specific environmental DNA (eDNA) survey method that can not only quantify Antarctic krill eDNA from samples collected in the Southern Ocean, but also estimate a relative time since this eDNA was shed, was developed. We determined decay characteristics of three species‐specific qPCR markers of different lengths in an aquarium experiment, developed a model that can discriminate recent eDNA from older eDNA in samples based on relative abundances of a long and a short marker, and applied this method to a Southern Ocean field survey covering 4800 km. We also compared a Euphausiid‐specific metabarcoding marker to the qPCR method to assess sensitivity in detecting Antarctic krill eDNA, and these methods could add an important novel, dynamic layer of information to future krill surveys.

Most seabirds do not have any outward identifiers of their chronological age, so estimation of seabird population age structure generally requires expensive, long-term banding studies. We investigated the potential to use a molecular age biomarker to estimate age in short-tailed shearwaters (Ardenna tenuirostris). We quantified DNA methylation in several A. tenuirostris genes that have shown age-related methylation changes in mammals. In birds ranging from chicks to 21 years of age, bisulphite treated blood and feather DNA was sequenced and methylation levels analysed in 67 CpG sites in 13 target gene regions. From blood samples, five of the top relationships with age were identified in KCNC3 loci (CpG66: R2 = 0.325, p = 0.019). In feather samples ELOVL2 (CpG42: R2 = 0.285, p = 0.00048) and EDARADD (CpG46: R2 = 0.168, p = 0.0067) were also weakly correlated with age. However, the majority of markers had no clear association with age (of 131 comparisons only 12 had a p-value < 0.05) and statistical analysis using a penalised lasso approach did not produce an accurate ageing model. Our data indicate that some age-related signatures identified in orthologous mammalian genes are not conserved in the long-lived short tailed shearwater. Alternative molecular approaches will be required to identify a reliable biomarker of chronological age in these seabirds.

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.

Species inventories and biodiversity assessments are critical to conservation. Yet cryptic species or recolonizing species can be challenging to detect. DNA metabarcoding provides an alternative tool to identify species that can be difficult to observe during field surveys. We test the efficacy of DNA analysis to identify burrowing petrel species in a rapidly changing landscape, on a remote sub‐Antarctic island following pest eradication. Discarded feathers and scats provided high quality DNA for species identification, assisting in detection of new species arrivals and new breeding sites across Macquarie Island. We highlight how DNA metabarcoding informs species inventories and is a valuable tool to complement seabird field surveys. DNA metabarcoding provides a rapid assessment tool to identify seabird species presence in a recovering island ecosystem. We test the efficacy of DNA analysis to identify burrowing petrel species on a remote sub‐Antarctic island following pest eradication. This case study highlights how DNA metabarcoding extends our knowledge of species diversity and distribution and provides a valuable tool to complement field surveys.

AIM Management of competition with predators is an important consideration for fisheries, particularly within marine protected areas (MPAs) where conservation is a primary objective. We aimed to test whether static no‐take zones within a large, sustainable‐use MPA prevented overlap between gentoo penguins and a krill fishery during two winters with contrasting prey fields. LOCATION South Georgia, Southwest Atlantic Ocean. METHOD We used satellite tracking (N = 16, June–September 2018) to describe gentoo penguin movements and distribution and quantified their overlap with the MPA’s no‐take zone (NTZ) and the krill fishing grounds. DNA metabarcoding of scats (N = 220, April–September 2018) was used to quantify diet. RESULTS When krill were at moderate densities and evenly distributed in 2001, gentoo penguins would have spent all of their time within the 12 NM NTZ, but when availability was low in 2018, they spent 46.3% of their time outside the NTZ and 9.6% within the krill fishing grounds. The extension of the NTZ to 30 km in response to this finding would have produced a 14.9% increase in protection for penguins and displaced 4% of fishery hauls. Gentoo penguin diet comprised 25.8% krill, which is lower than in the late 1980s but more than in 2009. MAIN CONCLUSIONS Gentoo penguins extend their foraging range when krill is scarce, which increases the potential for spatial overlap with the krill fishery during periods of nutritional stress. Current regulations allow for expansion of both extent and catches by the krill fishery and, should this occur, gentoo penguins may face heightened risks from competition. A dynamic ocean management framework, that extends closed areas in response to near real‐time data on penguin movements and krill density estimates, may reduce the potential for competition in this sustainable‐use MPA while allowing a profitable krill fishery.