Metabarcoding and mitochondrial metagenomics of endogean arthropods to unveil the mesofauna of the soil

Biological communities inhabiting the soil are among the most diversified, complex and yet most poorly studied terrestrial ecosystems. The greatest knowledge gaps apply to the arthropod mesofauna (01-2mm body size) because conventional morphological and molecular approaches are in many cases insufficient for the characterisation of these complex communities. The development of high-throughput sequencing (HTS) methodologies is required to solve current impediments and to further advance our understanding of below-ground biodiversity. We propose a flotation-Berlese-flotation (FBF) protocol for sampling and specimen processing to obtain clean' DNA extractions of arthropod mesofauna from the soil. In addition, we developed and tested HTS protocols for the characterisation of arthropod communities from these bulk DNA extractions using cox1 metabarcoding and shotgun metagenomic sequencing on the MiSeq Illumina platform. The FBF protocol provided DNA of soil arthropods from sufficiently large volumes of soil and free from contaminating bacteria and inhibitors. Metabarcoding and metagenomic sequencing on two deep soil samples from Iberian grasslands revealed >100 species of Acari and Collembola from 28 families. Genome assembly straight from shotgun sequencing of bulk specimens produced partial and full mitogenomes for 54 species with average length of >6000bp. Metabarcoding and metagenomic sequencing resulted in closely congruent OTUs, but species numbers were highest with metabarcoding, while approximate to 73% of species were confirmed by matching shotgun sequence reads and approximate to 48% by contig assembly from those shotgun reads. In combination, the FBF protocol together with the PCR-based and shotgun sequencing pipelines addressed most of the challenges of studying soil arthropod mesofauna on the MiSeq Illumina platform. They are powerful, cost-efficient tools for characterising soil diversity in a phylogenetic and community ecology context. These methodological developments of HTS approaches for the study of mesofauna will accelerate ecological and evolutionary studies, biomonitoring of soil arthropods, and progress in both theoretical and applied soil science.