Cryptic diversity within two widespread diadromous freshwater fishes (Teleostei: Galaxiidae).

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.

The enormous repetitive Antarctic krill genome reveals environmental adaptations and population insights.

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.

Biogeography of Southern Ocean prokaryotes: a comparison of the Indian and Pacific sectors.

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.

Environmental DNA metabarcoding for monitoring metazoan biodiversity in Antarctic nearshore ecosystems.

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.

Bacterial epibiont communities of panmictic Antarctic krill are spatially structured.

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.

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.

A globally distributed Syndiniales parasite dominates the Southern Ocean micro-eukaryote community near the sea-ice edge.

Syndiniales (Dinophyceae, Alveolata) are a diverse parasitic group common in all marine environments, but their ecological role remains poorly understood. Here we show an unprecedented dominance of a single Syndiniales group I operational taxonomic unit (OTU) across 3000 km of Southern Ocean transects near the sea-ice edge. This super-abundant OTU consistently represented >20%, and in some locations >50%, of eukaryote 18S rDNA sequences. Identical 18S V4 sequences have been isolated from seven Northern Hemisphere locations, and the OTU's putative V9 rDNA sequence was detected at every station of the global Tara Oceans voyage. Although Syndiniales taxa display some host specificity, our identification of candidate Southern Ocean hosts suggests this OTU associates with distinct phyla in different parts of the world. Our results indicate Syndiniales are key players in surface waters near the vast and dynamic sea-ice edge in the world's most biologically productive ocean.

Age estimation in a long-lived seabird (Ardenna tenuirostris) using DNA methylation-based biomarkers.

Age structure is a fundamental aspect of animal population biology. Age is strongly related to individual physiological condition, reproductive potential and mortality rate. Currently, there are no robust molecular methods for age estimation in birds. Instead, individuals must be ringed as chicks to establish known-age populations, which is a labour-intensive and expensive process. The estimation of chronological age using DNA methylation (DNAm) is emerging as a robust approach in mammals including humans, mice and some non-model species. Here, we quantified DNAm in whole blood samples from a total of 71 known-age Short-tailed shearwaters (Ardenna tenuirostris) using digital restriction enzyme analysis of methylation (DREAM). The DREAM method measures DNAm levels at thousands of CpG dinucleotides throughout the genome. We identified seven CpG sites with DNAm levels that correlated with age. A model based on these relationships estimated age with a mean difference of 2.8 years to known age, based on validation estimates from models created by repeated sampling of training and validation data subsets. Longitudinal observation of individuals re-sampled over 1 or 2 years generally showed an increase in estimated age (6/7 cases). For the first time, we have shown that epigenetic changes with age can be detected in a wild bird. This approach should be of broad interest to researchers studying age biomarkers in non-model species and will allow identification of markers that can be assessed using targeted techniques for accurate age estimation in large population studies.

Genetic monitoring of open ocean biodiversity: An evaluation of DNA metabarcoding for processing continuous plankton recorder samples.

DNA metabarcoding is an efficient method for measuring biodiversity, but the process of initiating long-term DNA-based monitoring programmes, or integrating with conventional programs, is only starting. In marine ecosystems, plankton surveys using the continuous plankton recorder (CPR) have characterized biodiversity along transects covering millions of kilometres with time-series spanning decades. We investigated the potential for use of metabarcoding in CPR surveys. Samples (n = 53) were collected in two Southern Ocean transects and metazoans identified using standard microscopic methods and by high-throughput sequencing of a cytochrome c oxidase subunit I marker. DNA increased the number of metazoan species identified and provided high-resolution taxonomy of groups problematic in conventional surveys (e.g., larval echinoderms and hydrozoans). Metabarcoding also generally produced more detections than microscopy, but this sensitivity may make cross-contamination during sampling a problem. In some samples, the prevalence of DNA from large plankton such as krill masked the presence of smaller species. We investigated adding a fixed amount of exogenous DNA to samples as an internal control to allow determination of relative plankton biomass. Overall, the metabarcoding data represent a substantial shift in perspective, making direct integration into current long-term time-series challenging. We discuss a number of hurdles that exist for progressing DNA metabarcoding from the current snapshot studies to the requirements of a long-term monitoring programme. Given the power and continually increasing efficiency of metabarcoding, it is almost certain this approach will play an important role in future plankton monitoring.

Antarctic Krill Are Reservoirs for Distinct Southern Ocean Microbial Communities.

Host-associated bacterial communities have received limited attention in polar habitats, but are likely to represent distinct nutrient-rich niches compared to the surrounding environment. Antarctic krill (Euphausia superba) are a super-abundant species with a circumpolar distribution, and the krill microbiome may make a substantial contribution to marine bacterial diversity in the Southern Ocean. We used high-throughput sequencing of the bacterial 16S ribosomal RNA gene to characterize bacterial diversity in seawater and krill tissue samples from four locations south of the Kerguelen Plateau, one of the most productive regions in the Indian Sector of the Southern Ocean. Krill-associated bacterial communities were distinct from those of the surrounding seawater, with different communities inhabiting the moults, digestive tract and faecal pellets, including several phyla not detected in the surrounding seawater. Digestive tissues from many individuals contained a potential gut symbiont (order: Mycoplasmoidales) shown to improve survival on a low quality diet in other crustaceans. Antarctic krill swarms thus influence Southern Ocean microbial communities not only through top-down grazing of eukaryotic cells and release of nutrients into the water column, but also by transporting distinct microbial assemblages horizontally via migration and vertically via sinking faecal pellets and moulted exuviae. Changes to Antarctic krill demographics or distribution through fishing pressure or climate-induced range shifts will also influence the composition and dispersal of Southern Ocean microbial communities.

DNA methylation levels in candidate genes associated with chronological age in mammals are not conserved in a long-lived seabird.

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.

Measuring Animal Age with DNA Methylation: From Humans to Wild Animals.

DNA methylation (DNAm) is a key mechanism for regulating gene expression in animals and levels are known to change with age. Recent studies have used DNAm changes as a biomarker to estimate chronological age in humans and these techniques are now also being applied to domestic and wild animals. Animal age is widely used to track ongoing changes in ecosystems, however chronological age information is often unavailable for wild animals. An ability to estimate age would lead to improved monitoring of (i) population trends and status and (ii) demographic properties such as age structure and reproductive performance. Recent studies have revealed new examples of DNAm age association in several new species increasing the potential for developing DNAm age biomarkers for a broad range of wild animals. Emerging technologies for measuring DNAm will also enhance our ability to study age-related DNAm changes and to develop new molecular age biomarkers.

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.

KrillDB: A de novo transcriptome database for the Antarctic krill (Euphausia superba).

Antarctic krill (Euphausia superba) is a key species in the Southern Ocean with an estimated biomass between 100 and 500 million tonnes. Changes in krill population viability would have catastrophic effect on the Antarctic ecosystem. One looming threat due to elevated levels of anthropogenic atmospheric carbon dioxide (CO2) is ocean acidification (lowering of sea water pH by CO2 dissolving into the oceans). The genetics of Antarctic krill has long been of scientific interest for both for the analysis of population structure and analysis of functional genetics. However, the genetic resources available for the species are relatively modest. We have developed the most advanced genetic database on Euphausia superba, KrillDB, which includes comprehensive data sets of former and present transcriptome projects. In particular, we have built a de novo transcriptome assembly using more than 360 million Illumina sequence reads generated from larval krill including individuals subjected to different CO2 levels. The database gives access to: 1) the full list of assembled genes and transcripts; 2) their level of similarity to transcripts and proteins from other species; 3) the predicted protein domains contained within each transcript; 4) their predicted GO terms; 5) the level of expression of each transcript in the different larval stages and CO2 treatments. All references to external entities (sequences, domains, GO terms) are equipped with a link to the appropriate source database. Moreover, the software implements a full-text search engine that makes it possible to submit free-form queries. KrillDB represents the first large-scale attempt at classifying and annotating the full krill transcriptome. For this reason, we believe it will constitute a cornerstone of future approaches devoted to physiological and molecular study of this key species in the Southern Ocean food web.

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies.

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology- and DNA-based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta-diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer-binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well-developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.

Quantitative DNA metabarcoding: improved estimates of species proportional biomass using correction factors derived from control material.

DNA metabarcoding is a powerful new tool allowing characterization of species assemblages using high-throughput amplicon sequencing. The utility of DNA metabarcoding for quantifying relative species abundances is currently limited by both biological and technical biases which influence sequence read counts. We tested the idea of sequencing 50/50 mixtures of target species and a control species in order to generate relative correction factors (RCFs) that account for multiple sources of bias and are applicable to field studies. RCFs will be most effective if they are not affected by input mass ratio or co-occurring species. In a model experiment involving three target fish species and a fixed control, we found RCFs did vary with input ratio but in a consistent fashion, and that 50/50 RCFs applied to DNA sequence counts from various mixtures of the target species still greatly improved relative abundance estimates (e.g. average per species error of 19 ± 8% for uncorrected vs. 3 ± 1% for corrected estimates). To demonstrate the use of correction factors in a field setting, we calculated 50/50 RCFs for 18 harbour seal (Phoca vitulina) prey species (RCFs ranging from 0.68 to 3.68). Applying these corrections to field-collected seal scats affected species percentages from individual samples (Δ 6.7 ± 6.6%) more than population-level species estimates (Δ 1.7 ± 1.2%). Our results indicate that the 50/50 RCF approach is an effective tool for evaluating and correcting biases in DNA metabarcoding studies. The decision to apply correction factors will be influenced by the feasibility of creating tissue mixtures for the target species, and the level of accuracy needed to meet research objectives.

Antarctic krill population genomics: apparent panmixia, but genome complexity and large population size muddy the water.

Antarctic krill (Euphausia superba; hereafter krill) are an incredibly abundant pelagic crustacean which has a wide, but patchy, distribution in the Southern Ocean. Several studies have examined the potential for population genetic structuring in krill, but DNA-based analyses have focused on a limited number of markers and have covered only part of their circum-Antarctic range. We used mitochondrial DNA and restriction site-associated DNA sequencing (RAD-seq) to investigate genetic differences between krill from five sites, including two from East Antarctica. Our mtDNA results show no discernible genetic structuring between sites separated by thousands of kilometres, which is consistent with previous studies. Using standard RAD-seq methodology, we obtained over a billion sequences from >140 krill, and thousands of variable nucleotides were identified at hundreds of loci. However, downstream analysis found that markers with sufficient coverage were primarily from multicopy genomic regions. Careful examination of these data highlights the complexity of the RAD-seq approach in organisms with very large genomes. To characterize the multicopy markers, we recorded sequence counts from variable nucleotide sites rather than the derived genotypes; we also examined a small number of manually curated genotypes. Although these analyses effectively fingerprinted individuals, and uncovered a minor laboratory batch effect, no population structuring was observed. Overall, our results are consistent with panmixia of krill throughout their distribution. This result may indicate ongoing gene flow. However, krill's enormous population size creates substantial panmictic inertia, so genetic differentiation may not occur on an ecologically relevant timescale even if demographically separate populations exist.

Molecular biomarkers for chronological age in animal ecology.

The chronological age of an individual animal predicts many of its biological characteristics, and these in turn influence population-level ecological processes. Animal age information can therefore be valuable in ecological research, but many species have no external features that allow age to be reliably determined. Molecular age biomarkers provide a potential solution to this problem. Research in this area of molecular ecology has so far focused on a limited range of age biomarkers. The most commonly tested molecular age biomarker is change in average telomere length, which predicts age well in a small number of species and tissues, but performs poorly in many other situations. Epigenetic regulation of gene expression has recently been shown to cause age-related modifications to DNA and to cause changes in abundance of several RNA types throughout animal lifespans. Age biomarkers based on these epigenetic changes, and other new DNA-based assays, have already been applied to model organisms, humans and a limited number of wild animals. There is clear potential to apply these marker types more widely in ecological studies. For many species, these new approaches will produce age estimates where this was previously impractical. They will also enable age information to be gathered in cross-sectional studies and expand the range of demographic characteristics that can be quantified with molecular methods. We describe the range of molecular age biomarkers that have been investigated to date and suggest approaches for developing the newer marker types as age assays in nonmodel animal species.

DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match.

DNA metabarcoding enables efficient characterization of species composition in environmental DNA or bulk biodiversity samples, and this approach is making significant and unique contributions in the field of ecology. In metabarcoding of animals, the cytochrome c oxidase subunit I (COI) gene is frequently used as the marker of choice because no other genetic region can be found in taxonomically verified databases with sequences covering so many taxa. However, the accuracy of metabarcoding datasets is dependent on recovery of the targeted taxa using conserved amplification primers. We argue that COI does not contain suitably conserved regions for most amplicon-based metabarcoding applications. Marker selection deserves increased scrutiny and available marker choices should be broadened in order to maximize potential in this exciting field of research.