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      Using DNA metabarcoding for simultaneous inference of common vampire bat diet and population structure

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          Abstract

          Metabarcoding diet analysis has become a valuable tool in animal ecology; however, co‐amplified predator sequences are not generally used for anything other than to validate predator identity. Exemplified by the common vampire bat, we demonstrate the use of metabarcoding to infer predator population structure alongside diet assessments. Growing populations of common vampire bats impact human, livestock and wildlife health in Latin America through transmission of pathogens, such as lethal rabies viruses. Techniques to determine large‐scale variation in vampire bat diet and bat population structure would empower locality‐ and species‐specific projections of disease transmission risks. However, previously used methods are not cost‐effective and efficient for large‐scale applications. Using bloodmeal and faecal samples from common vampire bats from coastal, Andean and Amazonian regions of Peru, we showcase metabarcoding as a scalable tool to assess vampire bat population structure and feeding preferences. Dietary metabarcoding was highly effective, detecting vertebrate prey in 93.2% of the samples. Bats predominantly preyed on domestic animals, but fed on tapirs at one Amazonian site. In addition, we identified arthropods in 9.3% of samples, likely reflecting consumption of ectoparasites. Using the same data, we document mitochondrial geographic population structure in the common vampire bat in Peru. Such simultaneous inference of vampire bat diet and population structure can enable new insights into the interplay between vampire bat ecology and disease transmission risks. Importantly, the methodology can be incorporated into metabarcoding diet studies of other animals to couple information on diet and population structure.

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          A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

          Introduction The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. Results We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. Conclusions The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.
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            Environmental DNA for wildlife biology and biodiversity monitoring.

            Extraction and identification of DNA from an environmental sample has proven noteworthy recently in detecting and monitoring not only common species, but also those that are endangered, invasive, or elusive. Particular attributes of so-called environmental DNA (eDNA) analysis render it a potent tool for elucidating mechanistic insights in ecological and evolutionary processes. Foremost among these is an improved ability to explore ecosystem-level processes, the generation of quantitative indices for analyses of species, community diversity, and dynamics, and novel opportunities through the use of time-serial samples and unprecedented sensitivity for detecting rare or difficult-to-sample taxa. Although technical challenges remain, here we examine the current frontiers of eDNA, outline key aspects requiring improvement, and suggest future developments and innovations for research. Copyright © 2014 Elsevier Ltd. All rights reserved.
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              Bias in template-to-product ratios in multitemplate PCR.

              Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.
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                Author and article information

                Contributors
                kbohmann@snm.ku.dk
                Journal
                Mol Ecol Resour
                Mol Ecol Resour
                10.1111/(ISSN)1755-0998
                MEN
                Molecular Ecology Resources
                John Wiley and Sons Inc. (Hoboken )
                1755-098X
                1755-0998
                16 May 2018
                September 2018
                : 18
                : 5 ( doiID: 10.1111/men.2018.18.issue-5 )
                : 1050-1063
                Affiliations
                [ 1 ] Section for Evolutionary Genomics Natural History Museum of Denmark University of Copenhagen Copenhagen Denmark
                [ 2 ] School of Biological Sciences University of East Anglia Norwich Norfolk UK
                [ 3 ] School of Biological Sciences University of Bristol Bristol UK
                [ 4 ] Institute of Biodiversity, Animal Health and Comparative Medicine MRC‐University of Glasgow Centre for Virus Research University of Glasgow Glasgow UK
                [ 5 ] NTNU University Museum Trondheim Norway
                Author notes
                [*] [* ] Correspondence

                Kristine Bohmann, Section for Evolutionary Genomics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

                Email: kbohmann@ 123456snm.ku.dk

                Author information
                http://orcid.org/0000-0001-7907-064X
                http://orcid.org/0000-0002-2004-6810
                http://orcid.org/0000-0002-3718-526X
                http://orcid.org/0000-0002-0554-6550
                http://orcid.org/0000-0002-1904-3735
                http://orcid.org/0000-0001-7475-2705
                http://orcid.org/0000-0002-5805-7195
                Article
                MEN12891
                10.1111/1755-0998.12891
                6120510
                29673092
                2d8eb37d-178b-4789-bed4-75cc02f26314
                © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 17 January 2018
                : 16 March 2018
                : 29 March 2018
                Page count
                Figures: 4, Tables: 2, Pages: 14, Words: 10970
                Funding
                Funded by: Sir Henry Dale Fellowship ‐ jointly funded by the Wellcome Trust and Royal Society
                Award ID: 102507/Z/13/Z
                Funded by: National Science Foundation
                Award ID: NSF‐DEB‐1020966
                Funded by: EU Marie Słodowska‐Curie
                Award ID: WhereWolf 655732
                Funded by: The Danish Council for Independent Research
                Award ID: DFF‐5051‐00140
                Categories
                Resource Article
                RESOURCE ARTICLES
                Molecular and Statistical Advances
                Custom metadata
                2.0
                men12891
                September 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.4.7.1 mode:remove_FC converted:03.09.2018

                Ecology
                diet analyses,ecological genetics,environmental dna,mammals,predator–prey interactions,wildlife management

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