Estimating belowground plant abundance with DNA metabarcoding

Abstract

Most work on plant community ecology has been performed above ground, neglecting the processes that occur in the soil. DNA metabarcoding, in which multiple species are computationally identified in bulk samples, can help to overcome the logistical limitations involved in sampling plant communities belowground. However, a major limitation of this methodology is the quantification of species’ abundances based on the percentage of sequences assigned to each taxon. Using root tissues of five dominant species in a semi‐arid Mediterranean shrubland (Bupleurum fruticescens, Helianthemum cinereum, Linum suffruticosum, Stipa pennata and Thymus vulgaris), we built pairwise mixtures of relative abundance (20%, 50% and 80% biomass), and implemented two methods (linear model fits and correction indices) to improve estimates of root biomass. We validated both methods with multispecies mixtures that simulate field‐collected samples. For all species, we found a positive and highly significant relationship between the percentage of sequences and biomass in the mixtures (R2 = .44–.66), but the equations for each species (slope and intercept) differed among them, and two species were consistently over‐ and under‐estimated. The correction indices greatly improved the estimates of biomass percentage for all five species in the multispecies mixtures, and reduced the overall error from 17% to 6%. Our results show that, through the use of post‐sequencing quantification methods on mock communities, DNA metabarcoding can be effectively used to determine not only species’ presence but also their relative abundance in field samples of root mixtures. Importantly, knowledge of these aspects will allow us to study key, yet poorly understood, belowground processes.

The role of root community attributes in predicting soil fungal and bacterial community patterns

“…The belowground plant community was assessed following the DNA metabarcoding protocol described in Matesanz et al . (2019). In brief, DNA was extracted from 0.1 g of homogenised root tissue of each root sample using the DNeasy Plant Minikit (Qiagen).…”

“…However, recent advances in DNA metagenomics provide a powerful tool to quantify plant species diversity and biomass partition below ground (e.g. Matesanz et al ., 2019) and, consequently, allows the testing of their effects on soil microbe diversity.…”

A powerful long metabarcoding method for the determination of complex diets from faecal analysis of the European pond turtle (Emys orbicularis, L. 1758)

“…Indeed, the correlation between composition of the sample and sequence reads varies from none to strong. It remains to be shown whether biomass may be linked to read abundance as previously shown in copepods (Clarke et al., 2017; Hirai et al., 2015), in nematode communities (Schenk et al., 2019) and in below‐ground plants (Matesanz et al., 2019), while others failed to assess this link such as in zooplankton assemblages (Harvey et al., 2017). Given that metabarcoding relies primarily on barcode amplification, and that pairs of primers have different affinities for the multitude of targeted gene species amplified within a sample, variable amplification level completely biases the putative quantification of the identified species.…”

Tales from the underground: Soil heterogeneity and not only above‐ground plant interactions explain fine‐scale species patterns in a Mediterranean dwarf‐shrubland

“…This confirms the importance of soil heterogeneity at scales that are assumed as the playground for biotic interactions and neutral processes such as dispersal limitation (Legendre et al, 2009) and its critical role structuring the realized assemblages in these shrublands at fine scale. It may also explain the huge functional diversity found in this type of communities, where very different functional strategies, e.g., grasses, prostrate and erect woody dwarf‐shrubs, forbs and geophytes (Chacón‐Labella, de la Cruz, Pescador, et al, 2016) with different root systems (Matesanz et al, 2019; Illuminati, pers. comm. )…”