Close and distant: Contrasting the metabolism of two closely related subspecies of Scots pine under the effects of folivory and summer drought

Abstract

Metabolomes, as chemical phenotypes of organisms, are likely not only shaped by the environment but also by common ancestry. If this is the case, we expect that closely related species of pines will tend to reach similar metabolomic solutions to the same environmental stressors. We examined the metabolomes of two sympatric subspecies of Pinus sylvestris in Sierra Nevada (southern Iberian Peninsula), in summer and winter and exposed to folivory by the pine processionary moth. The overall metabolomes differed between the subspecies but both tended to respond more similarly to folivory. The metabolomes of the subspecies were more dissimilar in summer than in winter, and iberica trees had higher concentrations of metabolites directly related to drought stress. Our results are consistent with the notion that certain plant metabolic responses associated with folivory have been phylogenetically conserved. The larger divergence between subspecies metabolomes in summer is likely due to the warmer and drier conditions that the northern iberica subspecies experience in Sierra Nevada. Our results provide crucial insights into how iberica populations would respond to the predicted conditions of climate change under an increased defoliation in the Mediterranean Basin.

Keywords: Pinus sylvestris; drought; evolutionary processes; folivory; herbivorous attack; metabolomics; processionary moth; sympatric subspecies.

Figure 1

Scots pine forests of Collado de Matasverdes in Sierra Nevada National Park. The photograph illustrates part of the study site where Pinus sylvestris ssp. nevadensis (native) coexists with P. sylvestris ssp. iberica (introduced). Photograph by Dr. José Antonio Hódar

Figure 2

Native (green) and planted (blue) populations of Scots pine across Spain (a) and in Sierra Nevada and Sierra de Baza (b). Native populations in Sierra Nevada and Sierra de Baza are Pinus sylvestris ssp. nevadensis. Maps of mean annual temperature (MAT) for Navacerrada, the native habitat of P. sylvestris ssp. iberica planted in southern Spain, (c) and for Sierra Nevada, the study site, (d) and of mean annual precipitation (MAP) for Navacerrada (e) and Sierra Nevada (f) are provided. Native populations of P. sylvestris ssp. iberica in Navacerrada are delimited by the black lines in (c) and (e). The study site is identified by the blue circles in the magnifications of Sierra Nevada in (d) and (f). Maps with the distribution of native and introduced P. sylvestris woodlands were kindly supported by the Spanish network on Genetics and Conservation of Forest Resources (GENFORED). Climatic maps were obtained from the “Atlas Climático Digital de la Península Ibérica” (Ninyerola, Pons Fernàndez, & Roure i Nolla, 2005)

Figure 3

Average maximum (solid dots) and minimum (open dots) temperatures (°C) and accumulated rainfall (mm) (solid squares) for Navacerrada and Sierra Nevada Natural Park for winter and summer. The temperatures are means ± 3 SE. The accumulated rainfalls are means ± SE. Different letters denote significant differences between Navacerrada and Sierra Nevada (p < .05) identified by a t test (see Table S3)

Figure 4

PC1 versus PC2 of the PCAs of the foliar metabolomes of Pinus sylvestris ssp. iberica and P. sylvestris ssp. nevadensis for winter and summer: case (a) and variable (b) plots of the PCA for winter, and case (c) and variable (d) plots of the PCA for summer. The folivory levels (FLs) are represented by different colors: blue, NATs; green, AT.NABs and red, AT.ABs. Crosses represent nevadensis and dots represent iberica. The colored arrows indicate the coordinate averages of PC1 and PC2 of each folivory level (FL) for iberica and nevadensis. Different letters beside the arrows indicate significant differences between FLs for each subspecies detected by Tukey's HSD post hoc tests (p < .05). Different metabolomic families are indicated by different colors: blue, sugars; green, amino acids; cyan, nucleotides; orange, organic acids associated with the tricarboxylic acid cycle (TCA); violet, phenolics; brown, terpenes; dark red, other secondary metabolites. Unidentified metabolites are not represented in the variable plot. Most metabolites are referenced by abbreviations: disaccharides (Disacch), hexoses (Hex), pentoses (Pent), group 1 sugars representing deoxy‐glucose, deoxy‐galactose, and D‐fucose (S1), group 2 sugars representing raffinose and maltotriose (S2), group 3 sugars representing xylitol and arabitol (S3), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), glycine (Gly), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), adenine (Adeni), adenosine (Adeno), cytosine (Cyt), guanine (Gua), uridine (Uri), uracil (Ura), α‐ketoglutaric acid (a.keto.ac), citric acid (Cit.ac), lactic acid (Lac.ac), malic acid (Mal.ac), oxaloacetic acid (Oxa.ac), pyruvic acid (Pyr.ac), succinic acid (Succ.ac), acacetin (Acac), apigenin (Apig), caffeic acid (Caff.ac), catechin, catechol, coumaric acid (Coum.ac), D‐pinitol, epicatechin (Epicat), epigallocatechin (Epigallocat), ferulic acid (Fer.ac), galangin (Gal), kaempferol (Kaemp), myricetin (Myr), quercetin (Quer), quinic acid (Qui.ac), resveratrol (Resve), rhamnetin (Rhamn), robinetin (Rob), sodium salicylate (S.sal), taxifolin (Tax), vitexin (Vit), vanillic acid (Van.ac), 5,7‐dihydroxy‐3,4,5‐trimethoxyflavone (Flavone: Flav), choline, δ‐tocopherol (d.tocoph), eugenol, vitamin B5 (Vit.B5), shikimic acid (Shik.ac), sabinene (Sabi), carvone (Carvo), caryophyllene (Caryo), farnesol (Farn), abscisic acid (ABA), gibberellic acid 1 (GA1), and gibberellic acid 3 (GA3)

Figure 5

Metabolomic distance (Euclidean distances) between iberica and nevadensis metabolomes for each folivory level (FL; NATs, AT.NABs, AT.ABs) and season (winter, summer) (a). Metabolomic distances between NATs versus AT.NABs (circles) and NATs versus AT.ABs (triangles) within each subspecies and for each season (b). Solid and open circles and triangles represent, respectively, to iberica and nevadensis in panel b. Values represent the distance means ± 3 SE. Different letters denote significant differences identified by one‐way ANOVAs and HSD post hoc tests (p < .05). The ANOVA for panel b used both winter and summer data