Recurrent allopolyploidizations diversify ecophysiological traits in marsh orchids (Dactylorhiza majalis s.l.)

Abstract

Whole-genome duplication has shaped the evolution of angiosperms and other organisms, and is important for many crops. Structural reorganization of chromosomes and repatterning of gene expression are frequently observed in allopolyploids, with physiological and ecological consequences. Recurrent origins from different parental populations are widespread among polyploids, resulting in an array of lineages that provide excellent models to uncover mechanisms of adaptation to divergent environments in early phases of polyploid evolution. We integrate here transcriptomic and ecophysiological comparative studies to show that sibling allopolyploid marsh orchid species (Dactylorhiza, Orchidaceae) occur in different habitats (low nutrient fens vs. meadows with mesic soils) and are characterized by a complex suite of intertwined, pronounced ecophysiological differences between them. We uncover distinct features in leaf elemental chemistry, light-harvesting, photoprotection, nutrient transport and stomata activity of the two sibling allopolyploids, which appear to match their specific ecologies, in particular soil chemistry differences at their native sites. We argue that the phenotypic divergence between the sibling allopolyploids has a clear genetic basis, generating ecological barriers that maintain distinct, independent lineages, despite pervasive interspecific gene flow. This suggests that recurrent origins of polyploids bring about a long-term potential to trigger and maintain functional and ecological diversity in marsh orchids and other groups.

Keywords: Dactylorhiza; allopolyploidy; differential expression; ecological differentiation; photosynthesis; soil chemistry.

FIGURE 1

(a) The sibling allotetraploids Dactylorhiza majalis and D. traunsteineri have each originated from allopolyploidizations involving the diploid D. incarnata acting as the paternal parent and D. fuchsii as the maternal. The bubbles give the chromosome number and genome configuration of each species. Plant illustrations modified from Nelson (1976). (b) Curated D. majalis (red) and D. traunsteineri localities (blue) extracted from GBIF (accessed February 2018).

FIGURE 2

(a) Divergent preference for selected soil characteristics between two sibling Dactylorhiza allopolyploids: D. majalis (red) and D. traunsteineri (blue). Soil elemental profiling for available nitrate (NO₃ ⁻), phosphate (P), potassium (K), aluminium (Al), chromium (Cr), nickel (Ni) and soil pH across multiple European populations. (b) Dactylorhiza traunsteineri growing alongside carnivorous sundews (Drosera spp.) at a locality on Gotland island, Sweden. (c) Dactylorhiza majalis, growing in a typical meadow at a site near Örup, Skåne, Sweden. (d) Leaf elemental profiling for wild individuals of D. majalis (red) and D. traunsteineri (blue) at multiple European localities. Data for nitrogen (N), carbon (C), phosphorus (P) and their pairwise ratios in leaf tissues. For (a, d), the data have been normalized to a 0–1 range using feature scaling only for visualization purposes, where x _norm = (x − x _min)/(x _max − x _min). The tabular form summarizes the raw data: Δ, mean difference; M, average; SD, standard deviation. The p‐values are for 1000 permutation tests.

FIGURE 3

Principal component analysis of no‐collinear macro‐environmental variables performed for GBIF localities and background. The distribution of the selected variables loading on the two main axes is given on the left, with inertia explained by colour of the arrows according to the legend. Bioclim codes are explained in the text. On the right, density contour plots encompass occurrence points of the D. majalis (red) and D. traunsteineri (blue) in the 2D environmental space. The occurrence density curves for each axis showed greater environmental divergence in PC1 (Δ = 1.60, p < .001) compared to PC2 (Δ = 0.61, p < .001).

FIGURE 4

Photosynthetic characteristics for the sibling allotetraploids D. majalis (red) and D. traunsteineri (blue) at native localities in the Alps. LEF, linear electron flow; PAR, photosynthetically active radiation (light intensity); Phi2, quantum yield of photosystem II; NPQt, non‐photochemical quenching; PhiNPQ, ratio of incoming light that goes towards non‐photochemical quenching. The boxplots show normalized values only for visualization purposes calculated as in Figure 2. The tabular form gives the details of the likelihood ratio between the null model with time, date and individual measurements as random variables, and the full model with species added to the model as a fixed variable. SD, standard deviation.

FIGURE 5

Examples of leaf stomatal conductance for water vapour (pink line, mmol m⁻² s⁻¹ on the right‐side Y‐axes) and net CO₂ assimilation rate (blue line, μmoles m⁻² s⁻¹ on the left‐side Y‐axes) for an individual of D. majalis (a) and one of D. traunsteineri (b). The levels of stomatal conductance and CO₂ exchange fluctuate with a 12‐h day/night cycle. Stomatal conductance is lower in D. traunsteineri which shows that its leaves do not open stomata as much as D. majalis, resulting in less water loss (higher water use efficiency) for nutrient acquisition via xylem. At night, the net CO₂ exchange is slightly negative for D. majalis (red arrow) meaning that it releases more CO₂ through respiration during nighttime, in contrast to D. traunsteineri (blue arrow), which also features 50% higher CO₂ assimilation rate during daytime. Plant illustrations modified from Nelson (1976).

FIGURE 6

(a) Summary of the enriched gene ontology (GO) categories and the number of differentially expressed genes in each of the GO terms. The length of the bars shows the ‐log(adjusted p‐value) of the category's enrichment, and the bar's colour illustrates the z‐score for the category according to the legend. Blue is for categories where there are more genes highly expressed in D. traunsteineri, and red is for categories where there are more genes highly expressed in D. majalis. Pale colours show that the respective categories have a more balanced ratio of genes that have increased expression in either species. Plant illustrations modified from Nelson (1976). (b) A summary of photosynthetic reactions drawn with BioRender (https://biorender.com), indicating in blue shades, the components higher expressed in D. traunsteineri compared to D. majalis. Fd, ferredoxin; OCE, oxygen‐evolving complex; PC, plastocyanin.