Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species

Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species

Primers used for developing ILs.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.</div

Natural variation at <i>SPL9</i>.

(A) Leaflet number progression of C. hirsuta Ox, the Chspl9 mutant, and the introgression line IL-LLN4_2, all in the Ox genetic background. Differences in leaflet numbers between the 3 genotypes were tested with a Dunn test and the P values, adjusted according to the Bonferroni method, are indicated by asterisks: *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001. (B, C) Genome-wide RNA-seq analyses of entire seedlings. (B) Comparison of C. hirsuta Az1 and C. hirsuta Ox, and (C) the NILs HIF-LLN4_2 (Rec29) with Az1 and Ox alleles at the SPL9 region. Negative log base 10 transformed P values are plotted against fold change of expression and each point is a gene. Red-colored points are significantly differentially expressed, while the black ones are not. The SPL9 gene is indicated in each plot. (D) Phylogeny and homology of SPL9 genes in 16 Brassicaceae. The left panel shows the SPL9 gene tree. The top panel shows the proportion of genes harboring the most common AA. The bottom-middle panel shows the entire SPL9 protein sequence, while the bottom-right panel corresponds to the region around the SPL9 missense SNPE242Q (indicated by asterisk). The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. AA, amino acid; Az1, Azores1; Chspl9, C. hirsuta loss-of-function allele of SPL9; Ox, Oxford; SPL9, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9. (TIFF)</p

Population structure and demography of <i>Cardamine hirsuta</i>.

(A) ADMIXTURE analysis of C. hirsuta strains after filtering for close relatedness (n = 358) reveals 3 major ancestry groups. The number of clusters that best fitted the data was found to be 3 (see also in S1A Fig). Each vertical bar represents a strain, where the colors indicate admixture proportions for the 3 ancestry groups. Strains were assigned to the ancestry group for which the proportion of ancestry was at least 0.5. The ancestry groups were named according to the main sampling location of their respective strains: BAL–Balkan; IBE–Iberia; NCE–Northern Central Europe. Strains with proportions less than 0.5 for all ancestry groups were categorized as ungrouped (top right). (B) The geographical distribution of the C. hirsuta ancestry groups in Western Europe. Each point represents the collection site of a strain and is colored according to the ancestry group it belonged to, with ungrouped strains shown in gray. The Macaronesian islands of the Azores and Madeira are shown at a smaller scale below the map. Map layers were made with Natural Earth and [142]. (C) The distribution of pairwise genetic distances (PGDs) indicates a deep split between groups of C. hirsuta strains. A histogram is shown of PGD between all possible pairs of strains in which the numbers of pairs in each bin are plotted against the PGD. The black outline shows the PGD of all strains in our sample. The presence of 2 major modes in the distribution, of which one at high genetic distance, indicated a group of strains in our sample that is highly differentiated from the others. Hierarchical clustering revealed a group of relict-like strains that was responsible for the second major mode in the distribution. PGDs including 1 or 2 relict-like strains are shown in blue, and PGDs not including those are shown in gray. (D) Identification of groups of C. hirsuta strains that are highly differentiated from each other based on multidimensional scaling and hierarchical clustering of the PGD. The first 2 PCs are plotted against each other where each point is a strain, colored according to the ADMIXTURE ancestry group it belonged to, with ungrouped strains shown in dark gray. Strains with ancestry in only a single ancestry group in the ADMIXTURE analysis are shown by darker shades versus lighter shades for admixed strains. Hierarchical clustering of the PGD matrix revealed that the separation of the strains along PC1 represented the 3 distinct groups of strains shown enclosed by dashed lines. The groups on the left and in the middle were responsible for the second major mode in the distribution of PGD (Figs 1C, S1B and S1C). Those 2 groups are shown here in blue and gray. (E, F) Piecewise constant effective population sizes (Ne) as a function of time for the 3 ancestry groups using MSMC2 (E) and relate (F), and estimates of split times between them considering a mutation rate of 4 × 10−9 mutations per base, per generation. The split times for BAL-NCE and BAL-IBE estimated with fastsimcoal2 (S1I Fig) are indicated by red and blue triangles on the x-axes, respectively. The top panel shows ancestral changes in Ne within the groups plotted against time in years, when considering 1 generation per year. With MSMC2 (E), 20 random sets of 4 strains were analyzed, which are all plotted, while with relate (F), all strains were analyzed jointly, hence a single line. The bottom panels show the RCCRs in BAL vs. NCE (solid lines) and IBE vs. BAL (dashed lines). Light blue shaded areas in the plots show ancient periods of glaciation according to MISs 2–4, 6, 8, 10, 12, 14, 16, and 18 [45], respectively, from left to right. The period of the LGM [46] is likewise indicated by the darker blue shade embedded in MIS2–4. The data underlying the graphs shown in this figure can be found at https://doi.org/10.5281/zenodo.7907435. BAL, Balkan; IBE, Iberian; LGM, last glacial maximum; MIS, marine isotope stage; NCE, Northern Central European; PC, principal coordinate; PGD, pairwise genetic distance; RCCR, relative cross coalescence rate.</p

Population genetic summary statistics in <i>C</i>. <i>hirsuta</i> and <i>A</i>. <i>thaliana</i>.

Population genetic summary statistics in C. hirsuta and A. thaliana.</p

Summary of Illumina read coverage for the 752 resequenced strains.

Summary of Illumina read coverage for the 752 resequenced strains.</p

Collection details of Azorean strains.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.</div

A missense polymorphism in <i>SPL9</i> underlies leaflet number QTL <i>LLN4_2</i>.

(A) Photoperiod shift experiment showing that the Az1 alleles at the QTL LLN4_2 delay the juvenile-to-adult phase transition. Plants of a HIF homozygous for Ox (yellow) or Az1 (blue) alleles at the SPL9 locus were shifted from flowering inducing long photoperiod to a noninductive short photoperiod. The RLN of the plants is plotted against the time spent in long photoperiod. Points show the RLN of individual plants, while the lines show a logistic model fitted to the data. The inflection point of the model is indicated by vertical arrows on the x-axis. (B) Fine-mapping of the leaflet number QTL LLN4_2. The genotype information of the HIF LLN4_2 is shown in the top panel. The graphical genotypes of the homozygous progeny of 4 different recombinant lines segregating in the LLN4_2 genomic region are shown below including the positions (Mb) of the genetic markers in the top axis. The bar chart on the right shows the number of leaflets produced on leaves 1 through 8 for the respective genotypes on the left. The bars show the mean leaflet numbers, and the points the leaflet numbers of the individual replicates. Kruskal–Wallis tests were performed to test for leaflet number differences between the 2 homozygous progenies of the same heterozygous recombinant: *** P LLN4_2 locus (Ox or Az1) inferred from the phenotype of each line is depicted. The LLN4_2 fine-mapped region of 49 kb contains 14 genes shown in the lower part of the panel with wider rectangles indicating exons and narrow rectangles introns and UTRs. The region containing SPL9 is expanded at the bottom with the 2 missense SNPs differing between Ox and Az1 colored in red and other SNPs in blue (see also S4A Fig). (C) Transgenic complementation of the Chspl9 mutant with the genomic constructs of SPL9Ox (gSPL9Ox) and SPL9Az1 (gSPL9Az1). The estimated copy number of the transgene is indicated in parentheses. As a control, the Chspl9 mutant was transformed with an empty vector. Two copies of gSPL9Ox and gSPL9Az1 could complement the phenotype to the level of Ox wt and IL LLN4_2Az1, respectively. Dots correspond to individual T2 transgenic plants derived from 27 independent T1 plants, and their mean and standard error for cumulative leaflet number on the first 8 leaves is shown by the bars. The compact letter display shows significant differences between genotypes according to a Dunn test with a Benjamin–Holm post hoc correction of the P values for multiple pairwise comparisons. (D) Allele swaps for the 2 SPL9 missense SNPs differing between Ox and Az1. The line HIF_LLN4_2 homozygous for the Az1 allele at the SPL9 locus (Fig 4A and 4B) was transformed with the genomic constructs shown in Fig 4C, and with 2 additional chimeric genomic constructs carrying the Ox and Az1 alleles, or the Az1 and Ox alleles for the SNPs (SPL9mixAz1_Ox and SPL9mixOx_Az1). The HIF was transformed with an empty vector as a control. Dots correspond to individual independent T1 transgenic plants, and their mean cumulative leaflet number on the first 8 leaves is shown by the bars. The compact letter display shows significant differences between genotypes according to a Dunn test with a Benjamin–Holm post hoc correction of the P values for multiple pairwise comparisons. The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. Az1, Azores1; HIF, heterogeneous inbred family; Ox, Oxford; RLN, rosette leaf number; wt, wild type.</p