Unexpected bird–feather mite associations revealed by DNA metabarcoding uncovers a dynamic ecoevolutionary scenario

The high relevance of host‐switching for the diversification of highly host‐specific symbionts (i.e., those commonly inhabiting a single host species) demands a better understanding of host‐switching dynamics at an ecological scale. Here, we used DNA metabarcoding to study feather mites on passerine birds in Spain, sequencing mtDNA (COI) for 25,540 individual mites (representing 64 species) from 1,130 birds (representing 71 species). Surprisingly, 1,228 (4.8%) mites from 84 (7.4%) birds were found on host species that were not the expected to be a host according to a recent bird–feather mite associations catalog. Unexpected associations were widespread across studied mite (40.6%) and bird (43.7%) species and showed smaller average infrapopulation sizes than typical associations. Unexpected mite species colonized hosts being distantly related to the set of their usual hosts, but with similar body size. The network of bird–mite associations was modular (i.e., some groups of bird and mite species tended to be more associated with each other than with the others), with 75.9% of the unexpected associations appearing within the module of the typical hosts of the mite species. Lastly, 68.4% of mite species found on unexpected hosts showed signatures of genetic differentiation, and we found evidence for reproduction or the potential for it in many of the unexpected associations. Results show host colonization as a common phenomenon even for these putatively highly host‐specific symbionts. Thus, host‐switching by feather mites, rather than a rare phenomenon, appears as a relatively frequent phenomenon shaped by ecological filters such as host morphology and is revealed as a fundamental component for a dynamic coevolutionary and codiversification scenario.     

DNA barcoding and minibarcoding as a powerful tool for feather mite studies

Feather mites (Astigmata: Analgoidea and Pterolichoidea) are among the most abundant and commonly occurring bird ectosymbionts. Basic questions on the ecology and evolution of feather mites remain unanswered because feather mite species identification is often only possible for adult males, and it is laborious even for specialized taxonomists, thus precluding large‐scale identifications. Here, we tested DNA barcoding as a useful molecular tool to identify feather mites from passerine birds. Three hundred and sixty‐one specimens of 72 species of feather mites from 68 species of European passerine birds from Russia and Spain were barcoded. The accuracy of barcoding and minibarcoding was tested. Moreover, threshold choice (a controversial issue in barcoding studies) was also explored in a new way, by calculating through simulations the effect of sampling effort (in species number and species composition) on threshold calculations. We found one 200‐bp minibarcode region that showed the same accuracy as the full‐length barcode (602 bp) and was surrounded by conserved regions potentially useful for group‐specific degenerate primers. Species identification accuracy was perfect (100%) but decreased when singletons or species of the Proctophyllodes pinnatus group were included. In fact, barcoding confirmed previous taxonomic issues within the P. pinnatus group. Following an integrative taxonomy approach, we compared our barcode study with previous taxonomic knowledge on feather mites, discovering three new putative cryptic species and validating three previous morphologically different (but still undescribed) new species.     

Meta-Fish-Lib: A generalised,dynamic DNA reference library pipeline for metabarcoding of fishes

The accuracy and reliability of DNA metabarcoding analyses depend on the breadth and quality of the reference libraries that underpin them. However, there are limited options available to obtain and curate the huge volumes of sequence data that are available on public repositories such as NCBI and BOLD. Here, we provide a pipeline to download, clean and annotate mitochondrial DNA sequence data for a given list of fish species. Features of this pipeline include (a) support for multiple metabarcode markers; (b) searches on species synonyms and taxonomic name validation; (c) phylogeny assisted quality control for identification and removal of misannotated sequences; (d) automatically generated coverage reports for each new GenBank release update; and (e) citable, versioned DOIs. As an example we provide a ready-to-use curated reference library for the marine and freshwater fishes of the U.K. To augment this reference library for environmental DNA metabarcoding specifically, we generated 241 new MiFish-12S sequences for 88 U.K. marine species, and make available new primer sets useful for sequencing these. This brings the coverage of common U.K. species for the MiFish-12S fragment to 93%, opening new avenues for scaling up fish metabarcoding across wide spatial gradients. The Meta-Fish-Lib reference library and pipeline is hosted at https://github.com/genner-lab/meta-fish-lib .     

lumi: a pipeline for processing Illumina microarray

Illumina microarray is becoming a popular microarray platform. The BeadArray technology from Illumina makes its preprocessing and quality control different from other microarray technologies. Unfortunately, most other analyses have not taken advantage of the unique properties of the BeadArray system, and have just incorporated preprocessing methods originally designed for Affymetrix microarrays. lumi is a Bioconductor package especially designed to process the Illumina microarray data. It includes data input, quality control, variance stabilization, normalization and gene annotation portions. In specific, the lumi package includes a variance-stabilizing transformation (VST) algorithm that takes advantage of the technical replicates available on every Illumina microarray. Different normalization method options and multiple quality control plots are provided in the package. To better annotate the Illumina data, a vendor independent nucleotide universal identifier (nuID) was devised to identify the probes of Illumina microarray. The nuID annotation packages and output of lumi processed results can be easily integrated with other Bioconductor packages to construct a statistical data analysis pipeline for Illumina data. Availability: The lumi Bioconductor package, www.bioconductor.org     

Environmental DNA metabarcoding studies are critically affected by substrate selection

Effective biomonitoring is critical for driving management outcomes that ensure long‐term sustainability of the marine environment. In recent years, environmental DNA (eDNA), coupled with metabarcoding methodologies, has emerged as a promising tool for generating biotic surveys of marine ecosystems, including those under anthropogenic pressure. However, more empirical data are needed on how to best implement eDNA field sampling approaches to maximize their utility for each specific application. The effect of the substrate chosen for eDNA sampling on the diversity of marine taxa detected by DNA metabarcoding has not yet been systematically analysed, despite aquatic systems being those most commonly targeted for eDNA studies. We investigated the effect of four commonly used eDNA substrates to explore taxonomic diversity: (a) surface water, (b) marine sediment, (c) settlement plates and (d) planktonic tows. With a focus on coastal ports, 332 eDNA samples from Australia (Indian and Southern oceans) and Kazakhstan (Caspian Sea) were collected and analysed by multi‐assay DNA metabarcoding. Across study locations, between 30% and 52% of eukaryotic families detected were unique to a particular substrate and <6% of families were found in all four substrates. Taxonomic composition varied significantly depending on the substrate sampled implying that the suitability (and bias) of an eDNA substrate will depend on the focal taxa. These findings demonstrate that single substrate eDNA metabarcoding likely underestimates the total eukaryotic diversity. Future eDNA experimental design should consider incorporating multiple substrates or select substrate(s) best suited to the specific detection of target taxa.     

DNA Pooling: a tool for large-scale association studies

DNA pooling is a practical way to reduce the cost of large-scale association studies to identify susceptibility loci for common diseases. Pooling allows allele frequencies in groups of individuals to be measured using far fewer PCR reactions and genotyping assays than are used when genotyping individuals. Here, we discuss recent developments in quantitative genotyping assays and in the design and analysis of pooling studies. Sophisticated pooling designs are being developed that can take account of hidden population stratification, confounders and inter-loci interactions, and that allow the analysis of haplotypes.     

The topological nature of tag jumping in environmental DNA metabarcoding studies

Metabarcoding of environmental DNA constitutes a state-of-the-art tool for environmental studies. One fundamental principle implicit in most metabarcoding studies is that individual sample amplicons can still be identified after being pooled with others—based on their unique combinations of tags—during the so-called demultiplexing step that follows sequencing. Nevertheless, it has been recognized that tags can sometimes be changed (i.e., tag jumping), which ultimately leads to sample crosstalk. Here, using four DNA metabarcoding data sets derived from the analysis of soils and sediments, we show that tag jumping follows very specific and systematic patterns. Specifically, we find a strong correlation between the number of reads in blank samples and their topological position in the tag matrix (described by vertical and horizontal vectors). This observed spatial pattern of artefactual sequences could be explained by polymerase activity, which leads to the exchange of the 3′ tag of single stranded tagged sequences through the formation of heteroduplexes with mixed barcodes. Importantly, tag jumping substantially distorted our data sets—despite our use of methods suggested to minimize this error. We developed a topological model to estimate the noise based on the counts in our blanks, which suggested that 40%–80% of the taxa in our soil and sedimentary samples were likely false positives introduced through tag jumping. We highlight that the amount of false positive detections caused by tag jumping strongly biased our community analyses.     

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

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

snpqc – an R pipeline for quality control of Illumina SNP genotyping array data

In genome‐wide association studies, quality control (QC) of genotypes is important to avoid spurious results. It is also important to maintain long‐term data integrity, particularly in settings with ongoing genotyping (e.g. estimation of genomic breeding values). Here we discuss snpqc , a fully automated pipeline to perform QC analyses of Illumina SNP array data. It applies a wide range of common quality metrics with user‐defined filtering thresholds to generate a comprehensive QC report and a filtered dataset, including a genomic relationship matrix, ready for further downstream analyses which make it amenable for integration in high‐throughput environments. snpqc also builds a database to store genotypic, phenotypic and quality metrics to ensure data integrity and the option of integrating more samples from subsequent runs. The program is generic across species and array designs, providing a convenient interface between the genotyping laboratory and downstream genome‐wide association study or genomic prediction.     

Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies

The high‐throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged wide use of sample multiplexing. However, this practice results in read misassignment (usually <1%) across samples sequenced on the same lane. Alarmingly high rates of read misassignment of up to 10% were reported for lllumina sequencing machines with exclusion amplification chemistry. This may make use of these platforms prohibitive, particularly in studies that rely on low‐quantity and low‐quality samples, such as historical and archaeological specimens. Here, we use barcodes, short sequences that are ligated to both ends of the DNA insert, to directly quantify the rate of index hopping in 100‐year old museum‐preserved gorilla (Gorilla beringei) samples. Correcting for multiple sources of noise, we identify on average 0.470% of reads containing a hopped index. We show that sample‐specific quantity of misassigned reads depends on the number of reads that any given sample contributes to the total sequencing pool, so that samples with few sequenced reads receive the greatest proportion of misassigned reads. This particularly affects ancient DNA samples, as these frequently differ in their DNA quantity and endogenous content. Through simulations we show that even low rates of index hopping, as reported here, can lead to biases in ancient DNA studies when multiplexing samples with vastly different quantities of endogenous material.     

Optimal sequence similarity thresholds for clustering of molecular operational taxonomic units in DNA metabarcoding studies

Clustering approaches are pivotal to handle the many sequence variants obtained in DNA metabarcoding data sets, and therefore they have become a key step of metabarcoding analysis pipelines. Clustering often relies on a sequence similarity threshold to gather sequences into molecular operational taxonomic units (MOTUs), each of which ideally represents a homogeneous taxonomic entity (e.g., a species or a genus). However, the choice of the clustering threshold is rarely justified, and its impact on MOTU over-splitting or over-merging even less tested. Here, we evaluated clustering threshold values for several metabarcoding markers under different criteria: limitation of MOTU over-merging, limitation of MOTU over-splitting, and trade-off between over-merging and over-splitting. We extracted sequences from a public database for nine markers, ranging from generalist markers targeting Bacteria or Eukaryota, to more specific markers targeting a class or a subclass (e.g., Insecta, Oligochaeta). Based on the distributions of pairwise sequence similarities within species and within genera, and on the rates of over-splitting and over-merging across different clustering thresholds, we were able to propose threshold values minimizing the risk of over-splitting, that of over-merging, or offering a trade-off between the two risks. For generalist markers, high similarity thresholds (0.96–0.99) are generally appropriate, while more specific markers require lower values (0.85–0.96). These results do not support the use of a fixed clustering threshold. Instead, we advocate careful examination of the most appropriate threshold based on the research objectives, the potential costs of over-splitting and over-merging, and the features of the studied markers.     

Management of DNA reference libraries for barcoding and metabarcoding studies with the R package refdb

DNA barcoding and metabarcoding are revolutionizing the study and survey of biodiversity. In order to assign taxonomic labels to the DNA sequence data retrieved, these methods are strongly dependent on comprehensive and accurate reference databases. Producing reliable databases linking biological sequences and taxonomic data can be—and often has been—done using mainstream tools such as spreadsheet software. However, spreadsheets quickly become insufficient when the amount of data increases to thousands of taxa and sequences to be matched, and validation operations become more complex and are error prone if done in a manual way. Thus, there is a clear need for providing scientists with user-friendly, reliable and powerful tools to manipulate and manage DNA reference databases in tractable, sound and efficient ways. Here, we introduce the R package refdb as an environment for semi-automatic and assisted construction of DNA reference libraries. The refdb package is a reference database manager offering a set of powerful functions to import, organize, clean, filter, audit and export the data. It is broadly applicable in metabarcoding data generally obtained in biodiversity and biomonitoring studies. We present the main features of the package and outline how refdb can speed up reference database generation, management and handling, and thus contribute to standardization and repeatability in barcoding and metabarcoding studies.     

Traditional water mite fixatives and their compatibility with later DNA studies

This work compares frozen water, 70% alcohol, Koenike's and Angelier's fluid as preservation media for water mites in terms of their eventual facilitation of DNA extraction and amplification. The time the mites spent in the fixatives ranged between 1 week and 25 years. Two molecular markers were amplified: 16S ribosomal DNA and CoI mitochondrial DNA. DNA was extractable and could be sequenced from specimens fixed in all the above media, although this generally became more difficult as time progressed. In the light of the known characteristics of these media, the results suggest Angelier's fluid to be the most practical, especially on long expeditions.     

Traditional water mite fixatives and their compatibility with later DNA studies

This work compares frozen water, 70% alcohol, Koenike's and Angelier's fluid as preservation media for water mites in terms of their eventual facilitation of DNA extraction and amplification. The time the mites spent in the fixatives ranged between 1 week and 25 years. Two molecular markers were amplified: 16S ribosomal DNA and CoI mitochondrial DNA. DNA was extractable and could be sequenced from specimens fixed in all the above media, although this generally became more difficult as time progressed. In the light of the known characteristics of these media, the results suggest Angelier's fluid to be the most practical, especially on long expeditions.     

DNA metabarcoding reveals changes in the contents of carnivorous plants along an elevation gradient

Resource variation along abiotic gradients influences subsequent trophic interactions and these effects can be transmitted through entire food webs. Interactions along abiotic gradients can provide clues as to how organisms will face changing environmental conditions, such as future range shifts. However, it is challenging to find replicated systems to study these effects. Phytotelmata, such as those found in carnivorous plants, are isolated aquatic communities and thus form a good model for the study of replicated food webs. Due to the degraded nature of the prey, molecular techniques provide a useful tool to study these communities. We studied the pitcher plant Sarracenia purpurea L. in allochthonous populations along an elevational gradient in the Alps and Jura. We predicted that invertebrate richness in the contents of the pitcher plants would decrease with increasing elevation, reflecting harsher environmental conditions. Using metabarcoding of the COI gene, we sequenced the invertebrate contents of these pitcher plants. We assigned Molecular Operational Taxonomic Units at ordinal level as well as recovering species‐level data. We found small but significant changes in community composition with elevation. These recovered sequences could belong to invertebrate prey, rotifer inquilines, pollinators and other animals possibly living inside the pitchers. However, we found no directional trend or site‐based differences in MOTU richness with elevational gradient. Use of molecular techniques for dietary or contents analysis is a powerful way to examine numerous degraded samples, although factors such as DNA persistence and the relationship with species presence still have to be completely determined.     

Environmental DNA metabarcoding as an effective and rapid tool for fish monitoring in canals

We focus on a case study along an English canal comparing environmental DNA (eDNA) metabarcoding with two types of electrofishing techniques (wade-and-reach and boom-boat). In addition to corroborating data obtained by electrofishing, eDNA provided a wider snapshot of fish assemblages. Given the semi-lotic nature of canals, we encourage the use of eDNA as a fast and cost-effective tool to detect and monitor whole fish communities.