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.

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.

Improving accuracy of DNA diet estimates using food tissue control materials and an evaluation of proxies for digestion bias.

Ecologists are increasingly interested in quantifying consumer diets based on food DNA in dietary samples and high-throughput sequencing of marker genes. It is tempting to assume that food DNA sequence proportions recovered from diet samples are representative of consumer's diet proportions, despite the fact that captive feeding studies do not support that assumption. Here, we examine the idea of sequencing control materials of known composition along with dietary samples in order to correct for technical biases introduced during amplicon sequencing and biological biases such as variable gene copy number. Using the Ion Torrent PGM(©) , we sequenced prey DNA amplified from scats of captive harbour seals (Phoca vitulina) fed a constant diet including three fish species in known proportions. Alongside, we sequenced a prey tissue mix matching the seals' diet to generate tissue correction factors (TCFs). TCFs improved the diet estimates (based on sequence proportions) for all species and reduced the average estimate error from 28 ± 15% (uncorrected) to 14 ± 9% (TCF-corrected). The experimental design also allowed us to infer the magnitude of prey-specific digestion biases and calculate digestion correction factors (DCFs). The DCFs were compared with possible proxies for differential digestion (e.g. fish protein%, fish lipid%) revealing a strong relationship between the DCFs and percent lipid of the fish prey, suggesting prey-specific corrections based on lipid content would produce accurate diet estimates in this study system. These findings demonstrate the value of parallel sequencing of food tissue mixtures in diet studies and offer new directions for future research in quantitative DNA diet analysis.

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.

TICI: a taxon-independent community index for eDNA-based ecological health assessment.

Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n = 16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2 = 0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

Stranded dolphin stomach contents represent the free-ranging population's diet.

Diet is a fundamental aspect of animal ecology. Cetacean prey species are generally identified by examining stomach contents of stranded individuals. Critical uncertainty in these studies is whether samples from stranded animals are representative of the diet of free-ranging animals. Over two summers, we collected faecal and gastric samples from healthy free-ranging individuals of an extensively studied bottlenose dolphin population. These samples were analysed by molecular prey detection and these data compared with stomach contents data derived from stranded dolphins from the same population collected over 22 years. There was a remarkable consistency in the prey species composition and relative amounts between the two datasets. The conclusions of past stomach contents studies regarding dolphin habitat associations, prey selection and proposed foraging mechanisms are supported by molecular data from live animals and the combined dataset. This is the first explicit test of the validity of stomach contents analysis for accurate population-scale diet determination of an inshore cetacean.

Who is eating what: diet assessment using next generation sequencing.

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

Variation in the tyrosinase gene associated with a white humpback whale (Megaptera novaeangliae).

Tyrosinase-negative oculocutaneous albinism (OCA1A) is characterized by lifelong white hair and skin, a phenotype that has been described in most mammalian species worldwide. Tyrosinase is the key enzyme in melanin biosynthesis, and mutations in the tyrosinase gene result in OCA1A. We examined sequence variation at exon 1 of the tyrosinase gene in 66 humpback whale samples collected from the east coast of Australia, including an anomalously white humpback whale known as "Migaloo." We identified 3 novel variants, including a cytosine deletion that results in a premature stop codon in exon 1. The deletion truncates the tyrosinase protein including the putative catalytic domains that are essential for tyrosinase enzymatic activity. Migaloo was homozygous for this deletion, suggesting that the albino phenotype is a consequence of inactive tyrosinase caused by the frameshift in the tyrosinase gene.

Analysis of Australian fur seal diet by pyrosequencing prey DNA in faeces.

DNA-based techniques have proven useful for defining trophic links in a variety of ecosystems and recently developed sequencing technologies provide new opportunities for dietary studies. We investigated the diet of Australian fur seals (Arctocephalus pusillus doriferus) by pyrosequencing prey DNA from faeces collected at three breeding colonies across the seals' range. DNA from 270 faecal samples was amplified with four polymerase chain reaction primer sets and a blocking primer was used to limit amplification of fur seal DNA. Pooled amplicons from each colony were sequenced using the Roche GS-FLX platform, generating > 20,000 sequences. Software was developed to sort and group similar sequences. A total of 54 bony fish, 4 cartilaginous fish and 4 cephalopods were identified based on the most taxonomically informative amplicons sequenced (mitochondrial 16S). The prevalence of sequences from redbait (Emmelichthys nitidus) and jack mackerel (Trachurus declivis) confirm the importance of these species in the seals' diet. A third fish species, blue mackerel (Scomber australasicus), may be a more important prey species than previously recognised. There were major differences in the proportions of prey DNA recovered in faeces from different colonies, probably reflecting differences in prey availability. Parallel hard-part analysis identified largely the same main prey species as did the DNA-based technique, but with lower species diversity and no remains from cartilaginous prey. The pyrosequencing approach presented significantly expands the capabilities of DNA-based methods of dietary analysis and is suitable for large-scale diet investigations on a broad range of animals.

Advancing the integration of multi-marker metabarcoding data in dietary analysis of trophic generalists.

The application of DNA metabarcoding to dietary analysis of trophic generalists requires using multiple markers in order to overcome problems of primer specificity and bias. However, limited attention has been given to the integration of information from multiple markers, particularly when they partly overlap in the taxa amplified, and vary in taxonomic resolution and biases. Here, we test the use of a mix of universal and specific markers, provide criteria to integrate multi-marker metabarcoding data and a python script to implement such criteria and produce a single list of taxa ingested per sample. We then compare the results of dietary analysis based on morphological methods, single markers, and the proposed combination of multiple markers. The study was based on the analysis of 115 faeces from a small passerine, the Black Wheatears (Oenanthe leucura). Morphological analysis detected far fewer plant taxa (12) than either a universal 18S marker (57) or the plant trnL marker (124). This may partly reflect the detection of secondary ingestion by molecular methods. Morphological identification also detected far fewer taxa (23) than when using 18S (91) or the arthropod markers IN16STK (244) and ZBJ (231), though each method missed or underestimated some prey items. Integration of multi-marker data provided far more detailed dietary information than any single marker and estimated higher frequencies of occurrence of all taxa. Overall, our results show the value of integrating data from multiple, taxonomically overlapping markers in an example dietary data set.

DNA metabarcoding assays reveal a diverse prey assemblage for Mobula rays in the Bohol Sea, Philippines.

Diet studies provide base understanding of trophic structure and are a valuable initial step for many fields of marine ecology, including conservation and fisheries biology. Considerable complexity in marine trophic structure can exist due to the presence of highly mobile species with long life spans. Mobula rays are highly mobile, large, planktivorous elasmobranchs that are frequently caught either directly or as bycatch in fisheries, which, combined with their conservative life history strategy, makes their populations susceptible to decline in intensely fished regions. Effective management of these iconic and vulnerable species requires an understanding of the diets that sustain them, which can be difficult to determine using conventional sampling methods. We use three DNA metabarcode assays to identify 44 distinct taxa from the stomachs (n = 101) of four sympatric Mobula ray species (Mobula birostris, Mobula tarapacana, Mobula japanica, and Mobula thurstoni) caught over 3 years (2013-2015) in a direct fishery off Bohol in the Philippines. The diversity and incidence of bony fishes observed in ray diets were unprecedented. Nevertheless, rays showed dietary overlap, with krill (Euphausia) dominating their diet. Our results provide a more detailed assessment of sympatric ray diets than was previously described and reveal the complexity that can exist in food webs at critical foraging habitats.

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.

Blocking primers to enhance PCR amplification of rare sequences in mixed samples - a case study on prey DNA in Antarctic krill stomachs.

BACKGROUND: Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA. RESULTS: We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge. CONCLUSION: We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.

Cleaver: software for identifying taxon specific restriction endonuclease recognition sites.

UNLABELLED: Cleaver is an application for identifying restriction endonuclease recognition sites that occur in some taxa but not in others. Differences in DNA fragment restriction patterns among taxa are the basis for many diagnostic assays for taxonomic identification and are used in procedures for removing the DNA of some taxa from pools of DNA from mixed sources. Cleaver analyses restriction digestion of groups of orthologous DNA sequences simultaneously to allow identification of differences in restriction pattern among the fragments derived from different taxa. AVAILABILITY: Cleaver is freely available without registration from its website (http://cleaver.sourceforge.net/) and can be copied, modified and re-distributed under the terms of the GNU general public licence version2 (http://www.gnu.org/licences/gpl). The program can be run as a script for computers that have Python 2.3 and necessary extra modules installed. This allows it to run on Gnu/Linux, Unix, MacOSX and Windows platforms. Stand-alone executable versions for Windows and MacOSX operating systems are available.

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.

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.

Quantification of damage in DNA recovered from highly degraded samples--a case study on DNA in faeces.

BACKGROUND: Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (lambda) can be estimated by determining the rate of decline. RESULTS: The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean lambda(predator) = 0.0106 per nucleotide; mean lambda(prey) = 0.0176 per nucleotide). This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. CONCLUSION: We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will allow researchers to measure template quality in order to evaluate alternate sources of DNA, different methods of sample preservation and different DNA extraction protocols. The technique could also be applied to study the process of DNA decay.

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.

Oligonucleotide primers for PCR amplification of coelomate introns.

Abstract Seven novel oligonucleotide primer pairs for polymerase chain reaction amplification of introns from nuclear genes in coelomates were designed and tested. Each pair bound to adjacent exons that are separated by a single intron in most coelomate species. The primer sets amplified introns in species as widely separated by the course of evolution as oysters (Mollusca: Protostoma) and salmon (Chordata: Deuterostoma). Each primer set was tested on a further 6 coelomate species and found to amplify introns in most cases. These primer sets may therefore be useful tools for developing nuclear DNA markers in diverse coelomate species for studies of population genetics, phylogenetics, or genome mapping.