Shrublands cover about 7-8% of Earth’s land surface (Lal, 2004; Maestre et al., 2021), and are increasing because of anthropogenic desertification (Eldridge et al., 2011; Huang et al., 2020). Shrubs are an important functional type of vegetation amid largely undervalued by humankind (Tomaselli, 1977; Kemper, Cowling, & Richardson, 1999) and understudied by ecologists, as compared to forest and grassland systems (Wullschleger et al., 2014; Fusco, Rau, Falkowski, Filippelli, & Bradley, 2019; Schrader-Patton & Underwood, 2021). Shrubs grow in resource-poor soils, and allocate much of their biomass to their roots to acquire water and soluble nutrients (Schenk & Jackson, 2002; Bardgett, Mommer, & De Vries, 2014). Through their roots, they absorb and compete for water, nitrogen, phosphorus, and many other soil resources that are subsequently transported to the leaves where plants need them to photosynthesize (Lambers et al. 2008; Kirkham, 2014). However, compared to aboveground plant organs, we know very little about roots, since roots cannot be directly observed and are very difficult to measure (Jones et al., 2011; Lux & Rost, 2012). Additionally, most of what we know about plant roots come from experiments conducted in controlled conditions, yet rooting behavior of plants may be very different in the wild (Poorter et al., 2016). Developing, testing, and standardizing methodologies to measure root traits in the field is a salient pending task for ecologists to address (Addo-Danso, Prescott, & Smith, 2016).
Allometric relationships provide estimates of ecosystem-level above- and belowground biomass from non-destructive and time-efficient biometric measurements, and are very common in forestry science, especially for species with commercial value (Wirth, Schumacher, & Schulze, 2004; Muukkonen, 2007; Chen, Hobbie, Reich, Yang, & Robinson, 2019). Allometric relationships have proven very useful to scale processes from the individual to the landscape in global vegetation models (Tyree & Ewers, 1991; Fischer, Maréchaux, & Chave, 2019). While some shrub allometries from temperate (Smith & Brand, 1983), mediterranean (Usó, Mateu, Karjalainen, & Salvador, 1997), desert (Allen, Pockman, Restrepo, & Milne, 2008; Ma & Wang, 2021), and alpine (Elzein, Blarquez, Gauthier, & Carcaillet, 2011) biomes have been published, they are much less common than published tree allometries (He et al., 2018). Tree allometric relations are usually established using the diameter of the trunk at breast height (dbh, around 1.30 meters)—an easy biometric that has been proven to correlate well with other plant-level variables (Hamilton, 1975; O’Brien et al., 1995; Drexhage & Colin, 2001). However, shrub allometries cannot be based on a metric like dbh, because shrubs are by definition multi-stemmed.
Establishing specific allometric equations for belowground biomass requires obtaining and analyzing direct root measures in the first place. While many root variables can be measured (Freschet, Pagès, et al., 2021; Freschet, Roumet, et al., 2021), the individual-level root density distributions (IRDD) would provide the most comprehensive ecological information about plant foraging strategies (Cabal, De Deurwaerder, & Matesanz, 2021). IRDD maps allow researchers to study both the root density distribution of neighboring plants in the soil and, by integration of such densities in three-dimensional space, the plant allocation of biomass to belowground tissues (Cabal, Martínez-García, De Castro Aguilar, Valladares, & Pacala, 2020). Estimating an IRDD would require samples of roots from known spatial coordinates and depths, which can be accomplished with a core drill, and the assignment of roots in the sample to the surrounding individual plants (Cabal et al., 2021). A few studies have used microsatellite analyses to link root samples to individual plants (Brunner, Brodbeck, Büchler, & Sperisen, 2001; Saari, Campbell, Russell, Alexander, & Anderson, 2005; Lang, Dolynska, Finkeldey, & Polle, 2010). However, dying plant roots with different colors by injecting dye into their above ground stems might be a cheaper and easier alternative to DNA analysis. This technique has been used successfully in several experiments in controlled conditions, in plants grown in pots in the greenhouse (Murakami et al., 2006; Cahill et al., 2010; Cabal et al., 2020) and in tomato plants grown in outdoor containers (Murakami et al., 2011), but has never been used in woody plants in the field.
In this study, we first evaluate three field methods to map IRDD in three mediterranean shrub species growing in central Spain. Firstly, as a method for root extraction, we adapted a diamond core-drilling engine designed for the construction industry. Secondly, we compared two different root identification methods to link root fragments from soil samples to aboveground plants: microsatellite analyses and plant injection with dyes. Based on the results, we estimated root allometries for each species and compared the success of stem diameter at the ground level and aboveground biovolume as allometric predictors.