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.