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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., (© 2019 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

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., (© 2018 John Wiley & Sons Ltd.)

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., (© 2015 John Wiley & Sons Ltd.)

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]

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]