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

Abstract

Short running title:High-throughput sequencing to unveil soil mesofauna Summary 1. Biological communities inhabiting the soil are among the most diversified, complex and yet most poorly studied of terrestrial ecosystems. The greatest knowledge gaps apply to the arthropod mesofauna (0.1-2 mm 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 belowground biodiversity. We propose a Flotation-Berlese-Flotation (FBF) protocol for sampling and specimenprocessing to obtain 'clean' DNA extractions of arthropod mesofauna from the soil. In addition, we developed and tested HTS protocols for the characterization of arthropod communities from these bulk DNA extractions using cox1 metabarcoding and shotgun metagenomic sequencing on the MiSeq Illumina platform. 3.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 >6000 bp. Metabarcoding and metagenomic sequencing resulted in closely congruent OTUs but species numbers were highest with metabarcoding, while ~73% of species were confirmed by matching shotgun sequence reads and ~48% by contig assembly from those shotgun reads. Accepted ArticleThis article is protected by copyright. All rights reserved. 4.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.

Terra incognita of soil biodiversity: unseen invasions under our feet

“…; Arribas et al . ) have found high levels of local diversity and high spatial structure even at local‐regional scales (high species turnover and microendemicity), thus suggesting levels of γ‐diversity in soil mesofauna to a point that could substantially change our estimates on the magnitude of biodiversity on Earth.…”

“…Arribas et al . ). In a noteworthy from the Cover article in this issue of Molecular Ecology, Cicconardi et al .…”

“…; Arribas et al . ), particularly within Collembola where high cryptic diversity and geographical structure have been found (Cicconardi et al . , ; Porco et al .…”

“…Soil mesofauna sampled with the FBF method (Arribas et al . ) in a laurisilva forest from the Anaga region (Tenerife).…”

A simple centrifugation protocol for metagenomic studies increases mitochondrial DNA yield by two orders of magnitude

“…However, different mitochondrial copy numbers per cell can also influence sequencing results (Tang et al., ). Recovery of diverse taxonomic groups from mitogenomic datasets has been shown to work well with samples not enriched for mitochondrial DNA (Arribas, Andújar, Hopkins, Shepherd, & Vogler, ; Gillett et al., ), and efficiency is expected to greatly increase by the application of mitochondrial enrichment. Although our protocol already leads to a high enrichment of mitochondrial DNA, the technique can be further improved in order to make the procedure of extracting mitochondria from tissue more reliable and standardized.…”