Advancing the use of metabarcoding derived nematode-based indices as soil health bioindicators in agricultural and natural environments

Effective frameworks for assessing soil health are a priority, yet the value and adoption of soil biological indicators remain debated. Biological indicators provide essential information on ecosystem functions such as nutrient cycling, population regulation, and organic matter decomposition. Nematode-based indices (NBIs) have long been used by soil ecologists to study biodiversity, the soil food web and ecosystem functions. DNA metabarcoding of soil nematode communities is being increasingly used as a more cost-efficient and high-throughput approach than traditional morphology-based identification. Molecular-based NBIs have great potential as soil health bioindicators if method uncertainties can be addressed. We critically review recent nematode metabarcoding methods for soils and highlight the advantages and limitations associated with different workflow steps including the nematode elutriation method, DNA extraction method, barcode and primer choices, and database selection. Based on an analysis of 95 studies, we propose an updated standardised metabarcoding workflow that includes (a) nematode elutriation from large soil quantities, (b) bulk nematode DNA extraction, (c) the use of the 18S rRNA primers NF1/18Sr2b and (d) curated reference databases for sequence identification. While challenges in concordance between molecular and morphological NBIs remain, improved standardisation of DNA metabarcoding protocols may enable more detailed investigations of nematode distribution across diverse environments and testing of molecular-based NBIs in relation to soil disturbances, management practices, temporal scales, soil functional processes, and soil physicochemical properties. These efforts could strengthen the inclusion of NBIs in models and frameworks supporting soil health management strategies. • Soil eDNA is increasingly being used in metabarcoding protocols compared to bulk nematode extracted DNA; • The 18S rRNA barcode and NF1-18Sr2b primers provide optimal coverage and taxonomic resolution; • New nematode-specific databases show potential for improved identification and nematode-based index (NBI) calculation; • Similar ecological patterns and NBIs shown for metabarcoding and morphological data; • Calibration of molecular-based NBIs by nematode abundance counts needs further investigation.