Background In light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). Findings Using a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed Nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. Conclusions Sequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost-effective, portable, and universal approach for eukaryote DNA barcoding. Although bulk community analyses using long-amplicon approaches may introduce biases, the long rDNA amplicons approach signifies a powerful tool for enabling the accurate recovery of taxonomic and phylogenetic diversity across biological communities.

Summary Molecular analysis of predator gut content is a popular tool to uncover food web structure and has greatly profited from the emergence of next‐generation sequencing technology. However, the molecular recovery of prey spectra comes with many challenges, particularly the overabundance of predator DNA in extractions. When predator and prey are distantly related, predator‐specific blocking primers can be used to preferentially amplify prey DNA. But this is difficult in the case of arthropods, where prey and predator are often closely related. Here, we present a simple and cost‐efficient protocol for high‐throughput analysis of the gut content of predatory arthropods without the need for blocking primers. We test several factors for their potential to enrich prey DNA from extractions of predators, including size selection of DNA, selection of different body parts for extraction, and variation in extraction lysis time and PCR fragment length. Using a locally abundant spider species (Hololena adnexa, Agelenidae), we show that prey DNA can be significantly enriched from predator extracts by selectively removing high molecular weight DNA and by extracting from the midgut and hindgut only. A comparison of our results with observational data from other agelenid spider species indicates a high efficiency of our approach. Our enrichment protocol allows for the parallel analysis of several hundred predators in a single MiSeq run, reducing the cost per specimen to only a few dollars and requiring a simple and minimal work flow. This will enable large‐scale and ecosystem‐wide analyses of niche differentiation and food web structure.

Large-scale studies on community ecology are highly desirable but often difficult to accomplish due to the considerable investment of time, labor and, money required to characterize richness, abundance, relatedness, and interactions. Nonetheless, such large-scale perspectives are necessary for understanding the composition, dynamics, and resilience of biological communities. Small invertebrates play a central role in ecosystems, occupying critical positions in the food web and performing a broad variety of ecological functions. However, it has been particularly difficult to adequately characterize communities of these animals because of their exceptionally high diversity and abundance. Spiders in particular fulfill key roles as both predator and prey in terrestrial food webs and are hence an important focus of ecological studies. In recent years, large-scale community analyses have benefitted tremendously from advances in DNA barcoding technology. High-throughput sequencing (HTS), particularly DNA metabarcoding, enables community-wide analyses of diversity and interactions at unprecedented scales and at a fraction of the cost that was previously possible. Here, we review the current state of the application of these technologies to the analysis of spider communities. We discuss amplicon-based DNA barcoding and metabarcoding for the analysis of community diversity and molecular gut content analysis for assessing predator-prey relationships. We also highlight applications of the third generation sequencing technology for long read and portable DNA barcoding. We then address the development of theoretical frameworks for community-level studies, and finally highlight critical gaps and future directions for DNA analysis of spider communities.

Stable core microbial communities have been described in numerous animal species and are commonly associated with fitness benefits for their hosts. Recent research, however, highlights examples of species whose microbiota are transient and environmentally derived. Here, we test the effect of diet on gut microbial community assembly in the spider Badumna longinqua. Using 16S rRNA gene amplicon sequencing combined with quantitative PCR, we analyzed diversity and abundance of the spider's gut microbes, and simultaneously characterized its prey communities using nuclear rRNA markers. We found a clear correlation between community similarity of the spider's insect prey and gut microbial DNA, suggesting that microbiome assembly is primarily diet‐driven. This assumption is supported by a feeding experiment, in which two types of prey—crickets and fruit flies—both substantially altered microbial diversity and community similarity between spiders, but did so in different ways. After cricket consumption, numerous cricket‐derived microbes appeared in the spider's gut, resulting in a rapid homogenization of microbial communities among spiders. In contrast, few prey‐associated bacteria were detected after consumption of fruit flies; instead, the microbial community was remodelled by environmentally sourced microbes, or abundance shifts of rare taxa in the spider's gut. The reshaping of the microbiota by both prey taxa mimicked a stable core microbiome in the spiders for several weeks post feeding. Our results suggest that the spider's gut microbiome undergoes pronounced temporal fluctuations, that its assembly is dictated by the consumed prey, and that different prey taxa may remodel the microbiota in drastically different ways.

BackgroundIn light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community, but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). ResultsUsing a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. ConclusionsSequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost effective, portable and universal approach for eukaryote DNA barcoding. Long rDNA amplicons scale up DNA barcoding by enabling the accurate recovery of taxonomic and phylogenetic diversity. However, bulk community analyses using long-read approaches may introduce biases and will require further exploration. MiniBarWe created a de-multiplexing software, called MiniBar. It allows customization of search parameters to account for the high read error rates and has built-in awareness of the dual barcode and primer pairs flanking the sequences. MiniBar takes as input a tab-delimited barcode file and a sequence file in either fasta or fastq format. The barcode file contains, at a minimum, sample name, forward barcode, forward primer, reverse barcode, and reverse primer for each of the samples potentially in the sequence file. The software searches for barcodes and for a primer, each permitting a user defined number of errors, an error being a mismatch or indel. Error count to determine a match can either be a percentage of each of their lengths or can be separately specified for barcode and primer as a maximum edit distance [49]. Output options permit saving each sample in its own file or all samples in a single file, with the sample names in the fasta or fastq headers. The found barcode primer pairs can be trimmed from the sequence or can remain in the sequence distinguished by case or color. MiniBar, written in Python 2.7, can also run in Python 3 and has the single dependency of the Edlib library module for edit distance measured approximate search [50]. MiniBar can be found at...

PCR amplification bias is a well-known problem in metagenomic analysis of arthropod communities. In contrast, variation of DNA degradation rates is a largely neglected source of bias. Differential degradation of DNA molecules could cause underrepresentation of taxa in a community sequencing sample. Arthropods are often collected by passive sampling devices, like malaise traps. Specimens in such a trap are exposed to varying periods of suboptimal storage and possibly different rates of DNA degradation. Degradation bias could thus be a significant issue, skewing diversity estimates. Here, we estimate the effect of differential DNA degradation on the recovery of community diversity of Hawaiian arthropods and their associated microbiota. We use a simple DNA size selection protocol to test for degradation bias in mock communities, as well as passively collected samples from actual Malaise traps. We compare the effect of DNA degradation to that of varying PCR conditions, including primer choice, annealing temperature and cycle number. Our results show that DNA degradation does indeed bias community analyses. However, the effect of this bias is of minor importance compared to that induced by changes in PCR conditions. Analyses of the macro and microbiome from passively collected arthropod samples are thus well worth pursuing.

Food webs form the basis of biological communities, though empirical research has been hindered by difficulties in quantifying interactions. Metabarcoding from predator gut content extractions with universal primers promises to provide simple and rapid insights into food web interactions. However, the highly overabundant predator DNA often completely out‐competes that of the digested prey DNA during PCR, impeding the ability to assess the abundance and diversity of prey items. Focusing on the issue of overabundance of predator DNA amplified by a commonly used COI primer pair, we use predator lineage‐specific SNPs at the 3’‐end of PCR primers to selectively block out predators from amplification. While this approach largely prevents predator amplification, it retains high taxonomic versatility for prey lineages. We introduce a novel multilocus assay, targeting four nuclear and mitochondrial rDNA markers, and test our approach in a diverse set of spiders from 12 families. We estimate the recovered prey DNA proportions and compare the taxonomic composition of prey communities between markers. Using a feeding experiment, we also explore recovery of prey DNA over time. While commonly used COI primers yield low and very unpredictable amounts of prey DNA, our assay allows for a considerable and consistent prey enrichment across all tested species. The recovered prey's taxonomic composition is comparable between markers and supports results acquired by COI. The new marker set can be amplified in a simple multiplex PCR, considerably reducing the necessary workload. Our multilocus approach allows the generation of an unprecedented amount of prey data at low cost and effort. Lineage‐specific PCR is taxonomically versatile and could readily be adapted to any prey–predator interaction, opening up the opportunity for community‐wide studies on food web interactions.

The simplicity and cost efficiency of Illumina amplicon sequencing has greatly contributed to the advancement of DNA barcoding and metabarcoding applications. However, current amplicon sequencing‐based barcoding approaches are usually restricted to short, single‐locus fragments, limiting their taxonomic and phylogenetic resolution. Here, we establish a cost efficient and simple multiplex PCR protocol for arthropod systematics by Illumina amplicon sequencing. We introduce primer sets, including several new, generic primers, to reliably amplify nine loci across a wide range of arthropods. Using a diverse collection of arthropod species from 19 orders, we test loci for amplification efficiency and estimate the effect of cross‐species amplification bias on taxon recovery from bulk community samples. We then explore the taxonomic and phylogenetic utility of the primer sets, focusing on a dataset of spiders that includes both deep and recent divergences. The set of loci provides good phylogenetic support across a wide taxonomic spectrum, making it a useful addition to COI for resolving lineages within a comparative context. All loci recover sequences for the majority of arthropod taxa in separate PCRs. However, cross‐species amplification bias in some primers prevents an exhaustive taxon recovery from bulk community samples. Our protocol makes it possible to generate multilocus datasets for large numbers of arthropod taxa for a fraction of the price and workload of Sanger sequencing. This opens up the possibility for parallel phylogenetic and taxonomic analysis of large collections of arthropods, but also enables rapid exploratory analyses of target lineages. Primers for metabarcoding applications should be carefully evaluated for their performance in bulk community samples and chosen to minimize cross‐species amplification bias.