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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-oflife 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 (nD16). Eight shortamplicon 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. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract 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 (λ) 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 λpredator = 0.0106 per nucleotide; mean λprey = 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.

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. [ABSTRACT FROM AUTHOR]

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. 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 near 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 improvedmonitoring 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. [ABSTRACT FROM AUTHOR]

DNA metabarcoding of food in animal scats provides a non-invasive dietary analysis method for vertebrates. A variety of molecular approaches can be used to recover dietary DNA from scats; however, many of these also recover non-food DNA. Blocking primers can be used to inhibit amplification of some non-target DNA, but this may not always be feasible, especially when multiple distinct non-target groups are present., We have developed scat collection protocols to optimise the detection of food DNA in vertebrate scat samples. Using shy albatross Thalassarche cauta as a case study, we investigated how DNA amplification success and the proportion of food DNA detected are influenced by both environmental and physiological parameters. We show that both the amount and type of non-target DNA vary with sample freshness, the collection substrate, fasting period and developmental stage of the consumer., Fresh scat samples yielded the highest proportion of food sequences. Collecting scats from dirt substrates reduced the proportion of food DNA and increased the proportion of contaminating DNA. Food DNA detection rates changed throughout the albatross breeding season and related to the time since feeding and the developmental stage of the animal. Fasting albatross produced scats dominated by parasite amplicons in universal PCR analysis, with little food DNA recovered. Samples from very young animals also produced reduced food DNA proportions., Based on our observations, we recommend the following procedures for field scat collections to ensure high-quality samples for dietary DNA metabarcoding studies. Ideally, (i) collect fresh scats; (ii) from surfaces with minimal contamination (e.g. rock or ice); (iii) collect scats from animals with minimum time since feeding and avoid fasting animals; (iv) avoid young animals that are not feeding directly (e.g. not weaned or fledged) or target larger/older individuals. The optimised field sampling protocols that we describe will improve the quality of dietary data from vertebrates by focusing on samples most likely to contain food DNA. They will also help minimise contamination issues from non-target DNA and provide standardised field methods in this rapidly expanding area of research. [ABSTRACT FROM AUTHOR]

Many seabird populations are threatened by interactions with commercial fisheries, and climate change. Understanding their prey requirements and dietary flexibility in this context is important for effective conservation and management. However, changes in the methods used to assess diet, as well as the spatial and temporal coverage of monitoring schemes, may reduce our ability to detect and monitor these marine threats. To help assess conservation priorities linked to diet, we performed a systematic review of 109 albatross diet papers published between 1950 and 2016, which corresponded to 296 studies when stratified by sampling year, breeding site, and breeding species. We assessed the methods used, changes over time, and spatial and temporal sampling coverage by species and island group. Most albatross studies have focused on chick-rearing, and diet during other breeding phases is comparatively poorly known. Furthermore, chicks are more commonly sampled than adults and very rarely immature birds, all of which may differ in diet composition. There was a pronounced shift over time in the preferred method of characterising diet, from the morphological examination of prey remains to stable isotope analysis of tissue. This shift has reduced the volume of detailed taxonomic information available from morphological studies. This difference in resolution hinders the ability to detect changes in prey species, with implications for management of threatened albatrosses and for monitoring broader changes in marine ecosystems. In a knowledge gap analysis for important breeding colonies (with>5% of global population), we identified key sites where existing monitoring has provided a foundation for robust longitudinal diet studies. Maintaining and augmenting these long-term research programmes will enable analyses of the impacts of changing climate and fishing practices on seabird populations and facilitate the timely identification and implementation of management options. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 mt DNA 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. [ABSTRACT FROM AUTHOR]

We applied DNA-based faecal analysis to determine the diet of female Australian sea lions (n = 12) from two breeding colonies in South Australia. DNA dietary components of fish and cephalopods were amplified using the polymerase chain reaction and mitochondrial DNA primers targeting the short (~100 base pair) section of the 16S gene region. Prey diversity was determined by sequencing ~50 amplicons generated from clone libraries developed for each individual. Faecal DNA was also combined and cloned from multiple individuals at each colony and fish diversity determined. Diets varied between individuals and sites. Overall, DNA analysis identified a broad diversity of prey comprising 23 fish and five cephalopod taxa, including many species not previously described as prey of the Australian sea lion. Labridae (wrasse), Monacanthidae (leatherjackets) and Mullidae (goat fish) were important fish prey taxa. Commonly identified cephalopods were Octopodidae (octopus), Loliginidae (calamary squid) and Sepiidae (cuttlefish). Comparisons of fish prey diversity determined by pooling faecal DNA from several samples provided a reasonable but incomplete resemblance (55-71%) to the total fish diversity identified across individual diets at each site. Interpretation of diet based on the recovery of prey hard-parts identified one cephalopod beak ( Octopus sp.) and one fish otolith ( Parapriacanthus elongatus). The present study highlights the value of DNA-based analyses and their capabilities to enhance information of trophic interactions. [ABSTRACT FROM AUTHOR]

Age is a fundamental aspect of animal ecology, but is difficult to determine in many species. Humpback whales exemplify this as they have a lifespan comparable to humans, mature sexually as early as 4 years and have no reliable visual age indicators after their first year. Current methods for estimating humpback age cannot be applied to all individuals and populations. Assays for human age have recently been developed based on age-induced changes in DNA methylation of specific genes. We used information on age-associated DNA methylation in human and mouse genes to identify homologous gene regions in humpbacks. Humpback skin samples were obtained from individuals with a known year of birth and employed to calibrate relationships between cytosine methylation and age. Seven of 37 cytosines assayed for methylation level in humpback skin had significant age-related profiles. The three most age-informative cytosine markers were selected for a humpback epigenetic age assay. The assay has an R2 of 0.787 ( P = 3.04e−16) and predicts age from skin samples with a standard deviation of 2.991 years. The epigenetic method correctly determined which of parent-offspring pairs is the parent in more than 93% of cases. To demonstrate the potential of this technique, we constructed the first modern age profile of humpback whales off eastern Australia and compared the results to population structure 5 decades earlier. This is the first epigenetic age estimation method for a wild animal species and the approach we took for developing it can be applied to many other nonmodel organisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Humpback whales undertake long-distance seasonal migrations between low latitude winter breeding grounds and high latitude summer feeding grounds. We report the first in-depth population genetic study of the humpback whales that migrate to separate winter breeding grounds along the northwestern and northeastern coasts of Australia, but overlap on summer feeding grounds around Antarctica. Weak but significant differentiation between eastern and western Australia was detected across ten microsatellite loci ( FST = 0.005, P = 0.001; DEST = 0.031, P = 0.001, n = 364) and mitochondrial control region sequences ( FST = 0.017 and Φ ST = 0.069, P = 0.001, n = 364). Bayesian clustering analyses using microsatellite data could not resolve any population structure unless sampling location was provided as a prior. This study supports the emerging evidence that weak genetic differentiation is characteristic among neighboring Southern Hemisphere humpback whale breeding populations. This may be a consequence of relatively high gene flow facilitated by overlapping summer feeding areas in Antarctic waters. [ABSTRACT FROM AUTHOR]

In understanding the impact of commercial whaling, it is important to estimate the mixing of low latitude breeding populations on Antarctic feeding grounds, particularly the endangered humpback whale populations of Oceania. This paper estimates the degree of genetic differentiation among the putative populations of Oceania (New Caledonia, Tonga, the Cook Islands and French Polynesia) and Australia (western Australia and eastern Australia) using ten microsatellite loci and mtDNA, assesses the power of the data for a mixed-stock analysis, determines ways to improve statistical power for future studies and estimates the population composition of Antarctic samples collected in 2010 south of New Zealand and eastern Australia. A large proportion of individuals could not be assigned to a population of origin (> 52%) using a posterior probability threshold of > 0.90. The mixed-stock analysis simulations however, produced accurate results with humpback whales reapportioned to their population of origin above the 90% threshold for western Australia, New Caledonia and Oceania grouped using a combined mtDNA and microsatellite dataset. Removing the Cook Islands, considered a transient region for humpback whales, from the simulation analysis increased the ability to reapportion Tonga from 86% to 89% and French Polynesia from 89% to 92%. Breeding ground sample size was found to be a factor influencing the accuracy of population reapportionment whereas increasing the mixture or feeding ground sample size improved the precision of results. The mixed-stock analysis of our Antarctic samples revealed substantial contributions from both eastern Australia (53.2%, 6.8% SE) and New Caledonia (43.7%, 5.5% SE) [with Oceania contributing 46.8% (5.9% SE)] but not western Australia. Despite the need for more samples to improve estimates of population allocation, our study strengthens the emerging genetic and non-genetic evidence that Antarctic waters south of New Zealand and eastern Australia are used by humpback whales from both eastern Australia and the more vulnerable breeding population of New Caledonia, representing Oceania. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A goal of many environmental DNA barcoding studies is to infer quantitative information about relative abundances of different taxa based on sequence read proportions generated by high-throughput sequencing. However, potential biases associated with this approach are only beginning to be examined. We sequenced DNA amplified from faeces (scats) of captive harbour seals ( Phoca vitulina) to investigate whether sequence counts could be used to quantify the seals' diet. Seals were fed fish in fixed proportions, a chordate-specific mitochondrial 16S marker was amplified from scat DNA and amplicons sequenced using an Ion Torrent PGM™. For a given set of bioinformatic parameters, there was generally low variability between scat samples in proportions of prey species sequences recovered. However, proportions varied substantially depending on sequencing direction, level of quality filtering (due to differences in sequence quality between species) and minimum read length considered. Short primer tags used to identify individual samples also influenced species proportions. In addition, there were complex interactions between factors; for example, the effect of quality filtering was influenced by the primer tag and sequencing direction. Resequencing of a subset of samples revealed some, but not all, biases were consistent between runs. Less stringent data filtering (based on quality scores or read length) generally produced more consistent proportional data, but overall proportions of sequences were very different than dietary mass proportions, indicating additional technical or biological biases are present. Our findings highlight that quantitative interpretations of sequence proportions generated via high-throughput sequencing will require careful experimental design and thoughtful data analysis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Little quantitative ecological information exists on the diets of most invertebrate feeding reptiles, particularly nocturnal or elusive species that are difficult to observe. In the UK and elsewhere, reptiles are legally required to be relocated before land development can proceed, but without knowledge of their dietary requirements, the suitability of receptor sites cannot be known. Here, we tested the ability of non-invasive DNA-based molecular diagnostics (454 pyrosequencing) to analyse reptile diets, with the specific aims of determining which earthworm species are exploited by slow worms (the legless lizard Anguis fragilis) and whether they feed on the deeper-living earthworm species that only come to the surface at night. Slow worm faecal samples from four different habitats were analysed using earthworm-specific PCR primers. We found that 86% of slow worms ( N = 80) had eaten earthworms. In lowland heath and marshy/acid grassland, Lumbricus rubellus, a surface-dwelling epigeic species, dominated slow worm diet. In two other habitats, riverside pasture and calciferous coarse grassland, diet was dominated by deeper-living anecic and endogeic species. We conclude that all species of earthworm are exploited by these reptiles and lack of specialization allows slow worms to thrive in a wide variety of habitats. Pyrosequencing of prey DNA in faeces showed promise as a practical, rapid and relatively inexpensive means of obtaining detailed and valuable ecological information on the diets of reptiles. [ABSTRACT FROM AUTHOR]

Chronological age is a fundamental and yet elusive variable in studies of many wild animals. Telomeres are nucleoprotein structures on the ends of chromosomes that change size throughout the life of many animals and because of this property have been advocated as a means to estimate age. In this review, we assess the existing and potential application of using telomeres for age estimation. We argue that there are conceptual and statistical inconsistencies in previous studies and that the basis for telomere change over time is not well understood and affected by several intrinsic and extrinsic process unrelated to chronological time. Furthermore, these processes are likely to vary spatially and temporally for animal populations. We conclude that the current data suggest telomeres should not be used for age estimation. If telomere-based age estimation is to be used, more work in understanding variability in key processes affecting telomere dynamics and rigorous substantiation via blind testing is needed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The diet of free-living pinnipeds is most frequently estimated through identification of otoliths, squid mouth-parts and exoskeletons of prey in scats. This is because, although important prey types may not always be detected, sample collection is non-invasive and analysis is easy. Identification of prey DNA in scats is a nascent approach to determining the diet of marine vertebrates that may overcome some of the limitations of hard part analysis. This is the first study to experimentally compare the utility of genetic scatology for identifying consumption of prey types by seals with the occurrence of morphological remains of prey in scats. The occurrences of DNA and hard part remains of one squid and two fish taxa in scats of captive Arctocephalus seals fed mixed prey diets were compared. Both methods detected ingestion of these taxa 7.5–39.5 h prior to defaecation. Although all test prey had robust hard parts, detecting consumption during this period was 1.4 to 5.8 times more likely using genetic analysis than morphological analysis of scats. Based on frequency of occurrence calculations, neither method provided quantitative descriptions of the known diet. Identification of prey using DNA was not compromised by complexity of the diet; each test taxon was unambiguously detected against a background of a multi-species diet. Our results suggest that where diagnostic hard remains of prey are not well represented in scats, or the sample size is small, genetic scatology provides a valuable addition to morphological scat analysis for identifying the recent diet of free-living seals. [Copyright &y& Elsevier]

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 (λ) 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 λpredator = 0.0106 per nucleotide; mean λprey = 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. [ABSTRACT FROM AUTHOR]

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