Abstract DNA metabarcoding of faeces is a powerful, non-invasive method for assessing predator diets. However, when studying the diet of generalist predators, broad PCR primers are used to amplify the wide range of potential prey species and metabarcoding outputs are often dominated by sequences from the predator. While blocking primers can be used to reduce PCR amplification of predator DNA, they frequently cause partial predator suppression and unintended prey blocking. Peptide nucleic acid (PNA) clamps, offer a promising, underutilised alternative by binding strongly and selectively to predator DNA to block its PCR amplification. In this study we designed and validated a novel PNA clamp targeting the 18S rRNA gene to suppress bird and mammal predator DNA in dietary samples. We tested this clamp on tissue mixtures and faecal samples from three seabird and two seal species across temperate, subantarctic, and Antarctic regions. The PNA clamp substantially increased the proportion of prey reads recovered while maintaining consistent prey community composition across all predator species. Our results demonstrate not only the general effectiveness of PNA clamps over standard blocking primers, but also provide a powerful, broadly applicable new tool to improve the accuracy in DNA diet metabarcoding studies.

Over 15 000 species of fishes are found globally in the marine environment and DNA barcodes are used extensively to describe, catalogue, understand and manage this diversity. The dataset outlined here represents a DNA barcode reference library of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) from 9767 voucher specimens (representing at least 2220 species and 288 families) of marine fishes. This publicly available dataset in the Barcode of Life Data System (BOLD) represents 17 years (2005-2022) of barcoding of marine fishes identified from Australian territorial waters. Tissues targeted for sequencing with their matching physical specimens (and extracted DNA), obtained via a multi-agency sampling effort, are mostly maintained and curated by the CSIRO Australian National Fish Collection (ANFC) in Hobart, Australia. Species-level integrated taxonomy (assigned after combined morphological and genetic assessment) has been determined for 91% of the dataset. The library represents the most complete COI barcode reference dataset for marine fishes from Australian waters and is currently utilised for integrated taxonomy, (meta)barcoding and eDNA studies.

ABSTRACT Environmental DNA (eDNA) is broadly assumed to be highly fragmented (< 600 bp) in seawater. However, several marine eDNA studies that have successfully amplified longer fragments (from 600 up to 16,000 bp) are challenging this notion. We hypothesized that a small, yet amplifiable, proportion of eDNA templates contain fragment lengths exceeding 600 bp. To test this, we designed primers to target a series of mitochondrial fragment lengths between 119 and 15,727 bp for the tiger shark ( Galeocerdo cuvier ) and performed qPCR on seawater eDNA samples collected from the offshore, tropical Kimberley and Roebuck Marine Parks in Western Australia. We observed a steep decrease in eDNA copy number with increasing fragment size between 119 and 1518 bp, beyond which amplification was not successful. Importantly, we demonstrate that fragment sizes larger than conventionally targeted (e.g., 636, 840, and 1518 bp) can still be successfully amplified from seawater eDNA samples. Estimated mean nucleotide damage in seawater eDNA samples was found to be 3.9 breaks per 1000 bp; this equates to a mean undamaged fragment size of 256 bp and is less than damage observed in modern fecal DNA and ancient DNA. Characterizing the extent of eDNA fragmentation in various environmental samples will improve understanding of the genetic material available and enable practitioners to target standard length barcodes and longer hypervariable gene regions. Through the recovery of more informative data, eDNA applications will extend to finer‐scale taxonomic resolution, including complex species and sub‐species discrimination, as well as population analyses.

The Southern Ocean surrounding Antarctica harbours some of the most pristine marine environments remaining, but is increasingly vulnerable to anthropogenic pressures, climate change, and invasion by non-native species. Monitoring biotic responses to cumulative impacts requires spatiotemporal baselines and ongoing monitoring - traditionally, this has been obtained by continuous plankton recorder (CPR) surveys. Here, we conduct a 3000 nautical mile environmental DNA (eDNA) transect from Hobart (Australia) to Davis Station (Antarctica). We evaluate eDNA sampling strategies for long-term open ocean biomonitoring by comparing two water volume and filter pore size combinations: large (12 L with 20 μm) and small (2 L with 0.45 μm). Employing a broad COI metabarcoding assay, we found the large sample/pore combination was better suited to open-ocean monitoring, detecting more target DNA and rare or low abundance species. Comparisons with four simultaneously conducted CPR transects revealed that eDNA detections were more diverse than CPR, with 7 (4 unique) and 4 (1 unique) phyla detections respectively. While both methods effectively delineated biodiversity patterns across the Southern Ocean, eDNA enables surveys in the presence of sea-ice where CPR cannot be conducted. Accordingly, 16 species of concern were detected along the transect using eDNA, notably in the Antarctic region (south of 60°S). These were largely attributed to hull biofouling, a recognized pathway for marine introductions into Antarctica. In a warming Southern Ocean, continued biomonitoring is vital for conserving Antarctic ecosystems. We advocate for the long-term implementation of eDNA metabarcoding alongside CPR surveys to facilitate ecosystem-based management of these vulnerable environments.

Identification of taxonomically cryptic species is essential for the effective conservation of biodiversity. Freshwater-limited organisms tend to be genetically isolated by drainage boundaries, and thus may be expected to show substantial cryptic phylogenetic and taxonomic diversity. By comparison, populations of diadromous taxa, that migrate between freshwater and marine environments, are expected to show less genetic differentiation. Here we test for cryptic diversity in Australasian populations (both diadromous and non-diadromous) of two widespread Southern Hemisphere fish species, Galaxias brevipinnis and Galaxias maculatus. Both mtDNA and nuclear markers reveal putative cryptic species within these taxa. The substantial diversity detected within G. brevipinnis may be explained by its strong climbing ability which allows it to form isolated inland populations. In island populations, G. brevipinnis similarly show deeper genetic divergence than those of G. maculatus, which may be explained by the greater abundance of G. maculatus larvae in the sea allowing more ongoing dispersal. Our study highlights that even widespread, 'high-dispersal' species can harbour substantial cryptic diversity and therefore warrant increased taxonomic and conservation attention.

Knowledge of animal diet is an important part of many fundamental and applied ecological studies. The use of DNA-based methods to identify food remains in stomach contents or faeces has now been applied to study the diet of a wide range of animal species from crustaceans and insects to antelope and whales. In this chapter, we present a brief history of the research field and outline how dietary DNA metabarcoding (i.e. the simultaneous identification of species through amplification of DNA markers and high-throughput sequencing of food DNA in diet samples) is employed to study the diet of animals. Moreover, we identify important technical challenges that researchers may face when carrying out a dietary DNA metabarcoding study, such as unique sampling considerations and dominance of non-target DNA. We also highlight some of the critical questions that need to be considered during study design and data interpretation. Based on our experiences, we describe a few studies that illustrate the diversity of approaches and ecological questions that can be addressed. Overall, we hope to provide a clear view of the complexities associated with this approach as well as a sense of excitement around the large number of opportunities that exist in using DNA metabarcoding to study animal diet.

Antarctic Toothfish are a circumpolar species which are targeted in multiple fisheries around Antarctica covering nine statistical areas within the Convention for the Conservation of Antarctic Marine Living Resources. Despite this, it is still unclear whether the species forms a single stock across its circumpolar distribution, shows a pattern of isolation by distance, or exhibits discrete stock structure between different regions. Recent genetics studies of Antarctic toothfish have shown connectivity between two areas (Ross Sea and Antarctic Peninsula), but earlier studies with smaller number of markers produced inconsistent results with regards to genetic connectivity between other geographic locations. Here we present a range-wide population genetic study of Antarctic toothfish using > 11,000 nuclear single nucleotide polymorphisms from 715 fish collected. Our results indicate that genetic diversity of the Antarctic toothfish is very low, with only 0.1% of genetic variability associated with geographic location. Multiple clustering methods, both supervised and unsupervised, indicated no distinct breeding populations. These results are consistent with current theories of egg and larval dispersal by the predominant Antarctic currents.

Antarctic krill (Euphausia superba) is Earth’s most abundant wild animal, and its enormous biomass is vital to the Southern Ocean ecosystem. Here, we report a 48.01-Gb chromosome-level Antarctic krill genome, whose large genome size appears to have resulted from inter-genic transposable element expansions. Our assembly reveals the molecular architecture of the Antarctic krill circadian clock and uncovers expanded gene families associated with molting and energy metabolism, providing insights into adaptations to the cold and highly seasonal Antarctic environment. Population-level genome re-sequencing from four geographical sites around the Antarctic continent reveals no clear population structure but highlights natural selection associated with environmental variables. An apparent drastic reduction in krill population size 10 mya and a subsequent rebound 100 thousand years ago coincides with climate change events. Our findings uncover the genomic basis of Antarctic krill adaptations to the Southern Ocean and provide valuable resources for future Antarctic research.

Abstract 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.

Marine trophic ecology data are in high demand as natural resource agencies increasingly adopt ecosystem-based management strategies that account for complex species interactions. Harbour seal (Phoca vitulina) diet data are of particular interest because the species is an abundant predator in the northeast Pacific Ocean and Salish Sea ecosystem that consumes Pacific salmon (Oncorhynchus spp.). A multi-agency effort was therefore undertaken to produce harbour seal diet data on an ecosystem scale using, 1) a standardized set of scat collection and analysis methods, and 2) a newly developed DNA metabarcoding diet analysis technique designed to identify prey species and quantify their relative proportions in seal diets. The DNA-based dataset described herein contains records from 4,625 harbour seal scats representing 52 haulout sites, 7 years, 12 calendar months, and a total of 11,641 prey identifications. Prey morphological hard parts analyses were conducted alongside, resulting in corresponding hard parts data for 92% of the scat DNA samples. A custom-built prey DNA sequence database containing 201 species (192 fishes, 9 cephalopods) is also provided.

The bioinformatic code used to process scat DNA sequences and example data. See Readme file.

Summary We investigated the Southern Ocean (SO) prokaryote community structure via zero‐radius operational taxonomic unit (zOTU) libraries generated from 16S rRNA gene sequencing of 223 full water column profiles. Samples reveal the prokaryote diversity trend between discrete water masses across multiple depths and latitudes in Indian (71–99°E, summer) and Pacific (170–174°W, autumn‐winter) sectors of the SO. At higher taxonomic levels (phylum‐family) we observed water masses to harbour distinct communities across both sectors, but observed sectorial variations at lower taxonomic levels (genus‐zOTU) and relative abundance shifts for key taxa such as Flavobacteria, SAR324/Marinimicrobia, Nitrosopumilus and Nitrosopelagicus at both epi‐ and bathy‐abyssopelagic water masses. Common surface bacteria were abundant in several deep‐water masses and vice‐versa suggesting connectivity between surface and deep‐water microbial assemblages. Bacteria from same‐sector Antarctic Bottom Water samples showed patchy, high beta‐diversity which did not correlate well with measured environmental parameters or geographical distance. Unconventional depth distribution patterns were observed for key archaeal groups: Crenarchaeota was found across all depths in the water column and persistent high relative abundances of common epipelagic archaeon Nitrosopelagicus was observed in deep‐water masses. Our findings reveal substantial regional variability of SO prokaryote assemblages that we argue should be considered in wide‐scale SO ecosystem microbial modelling.

We studied the reliability of a series of endpoints in an evaluation of subjects with Duchenne muscular dystrophy (DMD). The endpoints included quantitative muscle tests (QMTs), timed function tests, forced vital capacity (FVC), and manual muscle tests (MMT). Thirty-one ambulatory subjects with DMD (mean age 8.9 years; range 5-16 years) were evaluated at eight sites by 15 newly trained evaluators as a test of interrater reliability of outcome measures. Both total QMT score [intraclass correlation coefficient (ICC) 0.96] and individual QMT assessments (ICC 0.85-0.96) were highly reliable. Forced vital capacity and all timed function tests were also highly reliable (ICC 0.970.99). MMT was the least reliable assessment method (ICC 0.61). These data suggest that primary surrogate outcome measures in large multicenter clinical trials in DMD should use QMT, FVC, or time function tests to obtain maximum power and greatest sensitivity.

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.

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.

Abstract 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.

Antarctic krill (Euphausia superba) are amongst the most abundant animals on Earth, with a circumpolar distribution in the Southern Ocean. Genetic and genomic studies have failed to detect any population structure for the species, suggesting a single panmictic population. However, the hyper-abundance of krill slows the rate of genetic differentiation, masking potential underlying structure. Here we use high-throughput sequencing of bacterial 16S rRNA genes to show that krill bacterial epibiont communities exhibit spatial structuring, driven mainly by distance rather than environmental factors, especially for strongly krill-associated bacteria. Estimating the ecological processes driving bacterial community turnover indicated this was driven by bacterial dispersal limitation increasing with geographic distance. Furthermore, divergent epibiont communities generated from a single krill swarm split between aquarium tanks under near-identical conditions suggests physical isolation in itself can cause krill-associated bacterial communities to diverge. Our findings show that Antarctic krill-associated bacterial communities are geographically structured, in direct contrast with the lack of structure observed for krill genetic and genomic data.

The accuracy of specimen identification through DNA barcoding and metabarcoding relies on reference libraries containing records with reliable taxonomy and sequence quality. The considerable growth in barcode data requires stringent data curation, especially in taxonomically difficult groups such as marine invertebrates. A major effort in curating marine barcode data in the Barcode of Life Data Systems (BOLD) was undertaken during the 8th International Barcode of Life Conference (Trondheim, Norway, 2019). Major taxonomic groups (crustaceans, echinoderms, molluscs, and polychaetes) were reviewed to identify those which had disagreement between Linnaean names and Barcode Index Numbers (BINs). The records with disagreement were annotated with four tags: a) MIS-ID (misidentified, mislabeled, or contaminated records), b) AMBIG (ambiguous records unresolved with the existing data), c) COMPLEX (species names occurring in multiple BINs), and d) SHARE (barcodes shared between species). A total of 83,712 specimen records corresponding to 7,576 species were reviewed and 39% of the species were tagged (7% MIS-ID, 17% AMBIG, 14% COMPLEX, and 1% SHARE). High percentages (>50%) of AMBIG tags were recorded in gastropods, whereas COMPLEX tags dominated in crustaceans and polychaetes. The high proportion of tagged species reflects either flaws in the barcoding workflow (e.g., misidentification, cross-contamination) or taxonomic difficulties (e.g., synonyms, undescribed species). Although data curation is essential for barcode applications, such manual attempts to examine large datasets are unsustainable and automated solutions are extremely desirable.