For this study, sixteen monocotyledonous plant species from Northern Ontario wetlands were selected (Table 1). These species represent two different root overwintering habits. Roots of ten of the species overwinter, and roots of six of the species completely senesce in late autumn (Nieman et al. 2018). Given the differences in root overwintering habit, roots of these species can be considered to be either short-lived (<5 months) or long-lived (>1 year).
Experimental design and growth conditions
The experiment was conducted outdoors as a pot experiment in Sudbury, Ontario, Canada (46°36'N, 81°06'W). Study plants originated in local wetlands, grown in mesocosms in a common garden for a few years. Plants in this experiment were vegetatively propagated in early summer 2019 and planted in 2.7 L pots, 10 cm in diameter and 34 cm in height, on sieved wetland soil (Bainbridge Construction, North Bay, ON, Canada), or, in case of four of the species (D. arundinaceum, A. triviale, S. latifolia, T. palustre), in 2.3 L pots of 15 cm diameter and 15 cm height on sieved artificial blend of loam, peat moss and compost (President’s Choice® Black Earth Topsoil; Brampton, Loblaws, Canada). The pots were kept in pools filled with groundwater, with the water level at the level of the substrate surface. Iron rods placed in some of the pots did not rust below the substrate surface, indicating hypoxic to anoxic conditions (Owens et al. 2008).
The average growing season in Sudbury lasts 125-145 days, defined as the consecutive number of frost-free days (OMAFRA 2016), between May and October. Temperatures during the growing season were the warmest in July with average daily maxima of 26.8°C (Table S1, in Supplementary Information). Between 5 November 2019 and 1 May 2020 the plants were covered with straw and tarps to protect against freezing of the roots. During that time pot temperatures remained mostly between 0.5 and 2°C, similar to soil temperatures in local wetlands during that time of the year (Courchesne et al. 2020).
Seventy-one plants from sixteen species in total were harvested, of which eight species were harvested in September 2019 and eight species in August 2020. For each species, there were three to five replicate plants (Table S2, in Supplementary Information). For each individual plant, ten to twenty root segments consisting of basal root and the connected lateral roots were sampled from a depth of 10 to 20 cm for the taller pots, and 7 to 13 cm for the shallower pots. Basal roots and lateral roots were separated using a razor blade while floated in a water-filled dish. To calculate root porosity and root economics traits, the following variables were measured: root length, root fresh mass (mfresh), root dry mass (mdry), total root volume (Vroot), and the air-excluded root volume (Vair-excluded) (Table 2). All variables were measured separately for basal and lateral roots.
The mfresh was weighed with a microbalance (MX5; Mettler-Toledo, Greifensee, Switzerland), immediately after removing root surface water by carefully blotting with paper tissues (Visser and Bögemann, 2003). The mdry was measured after drying in the oven for 48 h at 70℃. Root length was determined using the grid-intersection method (Newman 1966; Tennant 1975).
The Vroot and Vair-excluded were measured with the pycnometer method (Vernescu and Ryser 2009). This method is a kind of Archimedes’ method as described in Birouste et al. (2014), the most direct measurement to Vroot based on displacement of water by submersed roots. To be specific, we measured the mass of the pycnometer filled with water (m1), filled with water and the roots (m2), and filled with water and roots after a vacuum treatment at about 7 kPa three times for 5 mins in a desiccator (m3). During the vacuum treatment roots were kept submerged with a weighted mesh, resulting in the vacuumed intercellular space of roots to be filled by water when the air pressure was released. The absence of gas bubbles on the surface of vacuumed roots confirmed the efficiency of the air evacuation. Hence, Vroot and Vair-excluded can be calculated with the equations: Vroot = (m1 – m2 + mfresh)/ρ and Vair-excluded = (m1 – m3 + mfresh)/ρ, respectively, in which ρ is the density of water, 1.00 g cm-3 at 25°C.
In addition, to test the values of Vroot obtained with the pycnometer method, diameter of 100 randomly sampled positions along a root system was measured for eight species using a microscope with an ocular micrometer and the root volume calculated assuming a cylindrical form (Ryser and Lambers, 1995). The strong correlation between the two volume measurements validated the pycnometer method as a reliable way to measure Vroot for basal roots (R2=0.78) and for lateral roots (R2=0.73).
Based on the variables measured, root porosity, specific root length (SRL), root average cross-sectional area (RCSA) and root density-associated traits were calculated (Table 2). Root tissue is physically composed of three phases: solid, liquid and air (Roderick et al. 1999a); root cellular tissue mainly contains the solid and liquid phase but root aerenchyma mainly the air phases. Density of root tissue can be calculated as the ratio of mass and volume, based on different combinations of root phases. Accordingly, traits that reflect root density include the commonly-used root tissue density (RTD; Birouste et al. 2014), root tissue density excluding air (RTDA; new variable), fresh root cellular density as the fresh mass of the cellular tissue volume (i.e. the air-excluded volume; Curran et al. 1996), and root dry matter content (RDMC; Birouste et al. 2014) (Table 2).
We also calculated the lateral to basal root length ratio, as a trait describing root architecture in terms of branching density and elongation of the lower-order roots (Maurel and Nacry 2020). Ratios of different root entities have been used to describe root architecture (Freschet et al. 2020b), either in terms of length, mass, or number of the roots, such as root branching density (Postma et al. 2014) or the mass fraction of specific roots within a root system (Ye et al. 2019).
Most statistical analyses were conducted using average values of each of the 16 species. A two-way ANOVA was used to test the interaction between root order (basal/lateral roots) and root life span (short-lived/long-lived roots) on root traits. The difference between RTD and RTDA was examined with a paired t–test separately for basal and lateral roots. Pairwise trait relationships were assessed using Pearson’s correlations for basal and lateral roots respectively. The dominant dimensions of the trait space at the root system level were analyzed with a Principal Component Analysis (PCA), including root porosity and key root economics traits of both basal and lateral roots, as well as the lateral to basal root length ratio. From all measurement data of basal and lateral roots, major axis regression were performed using individual plant values for assessing the relationship of RDMC as proxy to RTDA or RTD (Warton et al. 2006; Birouste et al. 2014). Normality and homoscedasticity of data were tested to satisfy the assumptions of parametric analyses. SRL and RCSA were log transformed for Pearson’s correlation and PCA. Statistical analyses were performed using R version 4.0.3 (R Development Core Team).