Data and code from: Establishing DNA-based strategies for soil biodiversity assessment: Insights from carabid beetles

Molecular-based methods offer valuable opportunities for assessing soil biodiversity in different ecosystems. However, their reliability and large-scale applicability depend on developing, optimizing protocols, and establishing high-quality, curated local reference databases. This study aimed to evaluate key steps in the soil macroinvertebrate metabarcoding workflow, including the sample decontamination process and the efficiency of taxa recovery. Specifically, we sought to: (1) determine the impact of sample decontamination, (2) validate species-level recovery efficiency of the metabarcoding pipeline spiked with a curated mock community of morphologically identified and barcoded carabid beetles, and (3) compare traditional morphological identification and metabarcoding for specimens’ taxonomic assignment and recovery. Our results showed that the commonly used decontamination process did not significantly impact OTU richness, suggesting it is not essential for this fauna. Compared to morphology, metabarcoding provided a more comprehensive taxonomic overview at higher-level taxa. However, validation with the mock community revealed discrepancies in species-level recovery, underscoring that its accuracy is highly contingent on the quality of the reference database. DNA metabarcoding is a currently used and promising technique for macroinvertebrate assessment regarding time, efficiency, and costs, yet reaching greater depth in taxonomic resolution. Yet, its species-level accuracy remains dependent on comprehensive and well-curated barcode reference databases. We recommend an integrative approach, combining molecular data with targeted validation, for the most robust outcomes. For this reason, we recommend the use of integrative methodologies for robust and rapid biodiversity assessments. We found that the common decontamination step is not crucial for soil macrofauna metabarcoding accuracy. Consequently, its removal streamlines sample processing. DNA metabarcoding revolutionizes soil biodiversity assessments by offering unparalleled taxonomic resolution and efficiency compared to traditional methods. Our study shows that the common decontamination step is unnecessary, streamlining workflows without compromising accuracy. However, the reliability of these molecular methods hinges on the development of curated local reference databases, underscoring the importance of integrative approaches for robust biodiversity monitoring. 

Methods

Sample Collection and Processing
This study, conducted under the CULTIVAR project in Idanha-a-Nova, Portugal, monitored soil macrofauna across 24 plots, each containing nine pitfall traps. A total of 216 traps were set up from late November to early December 2022, remaining in the field for 13 to 17 days. Traps were filled with ethylene glycol as a preservative and covered with lids to prevent dilution by rain. Collected specimens were preserved in 96 % ethanol until further analysis. Under low-power microscopy, organisms were counted and identified, per sample, to the lowest possible taxonomic level using morphology-based keys and literature. These community samples were stored at 96 % ethanol.

Establishing a COI Barcode Reference Database
Carabidae specimens were identified to the species level using morphological traits. To crete a local barcode reference base, one to two identified specimens were bleached, washed, and DNA was extracted using a Qiagen DNeasy® kit. For smaller specimens, a non-destructive method was applied, while larger specimens required maceration of appendages. PCR amplification of a 710 bp COI sequence utilized Folmer primers. Amplified products were verified via agarose gel electrophoresis, purified, and sequenced through the Sanger method. Sequences were analyzed against the BOLD and NCBI databases, allowing taxonomic assignments. A mock community of 31 Carabid species was created to validate metabarcoding methods, such as the recovery efficiency.

Bulk Sample Preparation and Metabarcoding

Half of the community samples went through a decontamination process using sodium hypochlorite. All community samples were homogenized into fine powder using a Bullet Blender. DNA was extracted with an E.Z.N.A.® Tissue DNA Kit. Carabid beetles were excluded as they were part of a mock community. PCR amplification targeted a 418 bp COI fragment using primers BF3 and BR2. A two-step PCR process incorporated Illumina adapters and identification indices. Final libraries were quantified, pooled, and sequenced using the Illumina NovaSeq platform. Controls ensured no contamination during amplification.

Bioinformatics Pipeline
The pipeline employed OBITools 4 for sequence processing, VSEARCH for denoising, and LULU for filtering pseudogenes. Paired-end reads were merged, primer sequences removed, and OTUs grouped by 99 % similarity. Taxonomic assignments were made using BOLD and NCBI databases. The script is available as a .qmd document. 

Comparative Analysis and Statistical Validation
Morphological and molecular identification methods were compared at the order and species levels. Statistical analyses were performed using R, (script available as a .qmd document). A barcode gap of Carabid species of 7 % was establishid to define species delimitation. This was accomplished by joining our Carabid sequences with more than 600 sequences retrieved from NCBI (sequence accession number available in the dataset), using MEGA and genetic distances calculated via the K80 model. This allowed the creation of a Venn diagram for comparing species and genus recovery efiency of barcoding and metabarcoding compared to morphology.