Evolutionary Divergences in Root Exudate Composition among Ecologically-Contrasting Helianthus Species

Abstract

Plant roots exude numerous metabolites into the soil that influence nutrient availability. Although root exudate composition is hypothesized to be under selection in low fertility soils, few studies have tested this hypothesis in a phylogenetic framework. In this study, we examined root exudates of three pairs of Helianthus species chosen as phylogenetically-independent contrasts with respect to native soil nutrient availability. Under controlled environmental conditions, seedlings were grown to the three-leaf-pair stage, then transferred to either high or low nutrient treatments. After five days of nutrient treatments, we used gas chromatography-mass spectrometry for analysis of root exudates, and detected 37 metabolites across species. When compared in the high nutrient treatment, species native to low nutrient soils exhibited overall higher exudation than their sister species native to high nutrient soils in all three species pairs, providing support for repeated evolutionary shifts in response to native soil fertility. Species native to low nutrient soils and those native to high nutrient soils responded similarly to low nutrient treatments with increased exudation of organic acids (fumaric, citric, malic acids) and glucose, potentially as a mechanism to enhance nutrition acquisition. However, species native to low nutrient soils also responded to low nutrient treatments with a larger decrease in exudation of amino acids than species native to high nutrient soils in all three species pairs. This indicates that species native to low nutrient soils have evolved a unique sensitivity to changes in nutrient availability for some, but not all, root exudates. Overall, these repeated evolutionary divergences between species native to low nutrient soils and those native to high nutrient soils provide evidence for the adaptive value of root exudation, and its plasticity, in contrasting soil environments.

Fig 1. Phylogeny (based on the most recent phylogeny for Helianthus [47]) and native soil fertility of the study species.

Data are the mean (SE) of five replicates per species. Soil fertility values for four of the species have been previously reported [46]. Different letters indicate significant differences (p < 0.05) between the two sister species within a given clade. Species native to a low nutrient soil (relative to its sister species) indicated by (§). (N) nitrogen; (P) phosphorus; (K) potassium; (OM) organic matter.

Fig 2. Principal components analysis of abundance of 37 metabolites in root exudates of six Helianthus species.

Each point represents an individual seedling. Species native to low nutrient soils are filled symbols, while those native to high nutrient soils are open symbols. Seedlings in the low nutrient treatment (L) are black symbols and seedlings in the high nutrient treatment (H) are red symbols.

Fig 3. Scores of first (a) and second (b) axes (PC1 and PC2, respectively) of the principal components analysis for root exudate composition of six Helianthus species depicted in Fig 3.

H. annuus (H. ann), H. argophyllus (H. arg), H. petiolaris (H. pet), H. praecox (H. pra), H. grossesseratus (H. gro); H. microcephalus (H. mic). Data are the mean of 5–7 replicates (± SE) for seedlings treated with either low (black bars) or high (red bars) nutrient treatments. Species are arranged by clade, with the species native to low nutrient soils (relative to its sister species) indicated by (§). Significant differences between species or treatments as assessed by t-tests indicated by (*).

Fig 4. Normalized peak areas of organic acids, amino acids, and derivatives detected under low (black bars) and high (red bars) nutrient treatments in six Helianthus species.

Data are the mean of 3–7 replicates (± SE) for which those peaks were present. Species are arranged by clade, with the species native to low nutrient soils (relative to its sister species) indicated by (§). γ-guanidobutyric acid (GBA); α-keto-glutaric acid (KGA). Species abbreviations as in Fig 3.

Fig 5. Normalized peak areas of sugars and sugar derivatives detected under low (black bars) and high (red bars) nutrient treatments in six Helianthus species.

Data are the mean of 3–7 replicates (± SE) for which those peaks were present. Species are arranged by clade, with the species native to low nutrient soils (relative to its sister species) indicated by (§).Glycerol-1-phosphate (G-1-P); galacturonic acid (GalA); 2-O-Glycerol-galactopyranoside (GG). Species abbreviations as in Fig 3.