Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics

Significance Ecological and evolutionary investigations require accurate and high-resolution biodiversity information. Conventional morphological approaches to identifying species in species-rich tropical ecosystems are often unavailable or incapable of timely, cost-effective identification. We show that next-generation sequencing (NGS) of cytochrome c oxidase subunit I (COI) DNA barcodes can accurately detect 83.5% of individually sequenced species (corresponding to 91% of individuals) in a bulk sample of terrestrial arthropods from a Costa Rican species-rich site. Additionally, the 16S and 18S ribosomal DNA gene regions obtained also provide an assessment of the bacteria and protozoa in the bulk sample. This metasystematic approach provides the initial infrastructure for a next generation of biodiversity assessment and environmental monitoring. It can lead to more effective understanding, appreciation, and management of complex ecosystems. Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works.