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

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

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

Selection for accelerated developmental progression in Northern and Central Europe.

(A) GWAs for flowering time and leaflet number on leaf 7 using 352 C. hirsuta strains. The negative log base 10 transformed P values for association tests of individual SNPs are plotted against physical position on the 8 chromosomes. Horizontal dashed lines show thresholds of significance with correction for multiple testing according to Bonferroni (magenta) and fdr (cyan; for both α = 0.05). SNPs with transformed P values above the fdr threshold are shown in red, and others in gray. Two regions with strongly associated SNPs were detected on chromosomes 6 and 8 that contained the candidate genes FLC/TPPI and FRI. (B) Close-up view of the locus with the strongest associations showing GWAS for flowering time with the most significant SNP in TPPI used as covariate. Forward regression using a multilocus mixed model GWAS indicated that the highly significant association on chromosome 6 consisted of 2 independent associations. Yellow areas indicate the 2 candidate genes FLC (left) and TPPI (right) where the lighter shades indicate the promoter region (−3,000 bp) and the darker shades indicate the ORF. GWAS without covariates is shown in red, and with the SNP indicated by the blue encircled red point in TPPI as a covariate in blue. This result revealed significant associations for SNPs in the first intron of FLC that were independent of the associations for SNPs linked to TPPI. The associated SNPs in FLC shown in blue were the most significant genome-wide in this analysis. (C) Functional validation of 3 distinct truncated FRI alleles that exist within predominantly European samples of C. hirsuta. In contrast to a significant increase in rosette leaf number in plants transformed with a full-length FRI allele, the truncated FRI alleles showed no effect (Dunn test with Bonferroni adjusted P value, ***: P value FRIstop) and exclusively in NCE strains (see also S2B Fig). (D) Flowering time in DAG until anthesis for all genotype combinations at the 3 candidate genes identified by GWA. The genotypes at the representative SNPs for each gene are shown as either anc or der. The bars indicate the mean flowering time, and the points show the individual observations for each strain. Points are colored according to the ancestry group of strains (Fig 1A). (E, F) Correlation between North–South genetic differentiation (PC2 in S1B Fig) and flowering time (E) as well as leaflet number on leaf 8 (F). The points are observations for individual strains colored according to their ancestry group (Fig 1A) such that strains with ancestry in only 1 group are shown in darker shades vs. lighter shades for admixed. The lines show linear models fitted to the data from the BAL and NCE populations (PPG) Evidence for a selective sweep at the FRI locus (see also S2 Fig). A sliding window analysis of nucleotide diversity (π, top), Tajima’s D (middle), and CLR calculated by SweepFinder2 [59] (bottom) is shown for chromosome 8. The analyses were performed separately in strains with the FRIstop (blue) and the FRIfunc (black) alleles from the NCE group (Fig 1A). Note how the region, which includes the FRI locus (orange dashed line) displays reduced π, reduced Tajima’s D, and high CLR, consistent with a selective sweep, exclusively in strains with FRIstop. The horizontal dashed lines in the top and middle panels indicate the genome-wide averages for the respective groups in blue or gray, and in the lower panel the horizontal dashed line indicates the threshold (α = 0.05) derived from neutral simulations using our best demographic model. (H) The geographic distribution of full-length and truncated FRI alleles on the map and their projection on latitude in A. thaliana (*) and C. hirsuta (triangle) exhibited high similarity. The colors represent distinct truncated FRI alleles. The rectangles represent areas of high sampling density for both species. The pie charts show the proportion of functional and nonfunctional alleles in C. hirsuta (left) and full-length and truncated alleles in A. thaliana (right). The total number of strains inside the respective rectangles is shown inside the pie chart. The histograms on the right side show functional/full-length (black) and nonfunctional/truncated FRI alleles (different colors represent different truncated alleles) along the latitude. FRIstop is the major truncated FRI allele. Note that only one of all mainland European C. hirsuta strains harbors FRIstop2 and none FRIstop3. Map layers were made with Natural Earth and [142]. The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. anc, ancestral; BAL, Balkan; CLR, composite likelihood ratio; DAG, days after germination; der, derived; fdr, false discovery rate; FLC, FLOWERING LOCUS C; FRI, FRIGIDA; GWA, genome-wide association; IBE, Iberian; NCE, Northern Central European; ORF, open reading frame; SNP, single nucleotide polymorphism; TPPI, TREHALOSE-6-PHOSPHATE-PHOSPHATASE I.</p

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

(A) CV errors in ADMIXTURE analysis with different random seeds. The CV error is plotted against the chosen number of populations (K) for 75 independent analyzes. A lower CV error indicates a better fit to the data. In this analysis, K = 3 was unanimously found to be the best number of populations for the data. (B) PCA of SNP data corroborates ADMIXTURE analysis. The first PC separates IBE (blue) from the others, while the second PC separates BAL (red) and NCE (yellow). Strains with ancestry in only a single ancestry group in the ADMIXTURE analysis (Fig 1A) are shown by darker shades versus lighter shades for admixed strains. (C) Hierarcical clustering (hclust) of the PGDs reveals distant relict-like groups. Hclust was used to identify relict-like strains based on PGD, and distant groups among them. The results of hclust are shown as a dendrogram where branch length is a measure of PGD. The first bifurcation separated the strains into 2 groups with high genetic distance between them, one of which predominantly resembled the relict-like group (colored labels) also discovered in Arabidopsis [17] because it contained all IBE strains. In contrast to ADMIXTURE, this analysis also allowed the identification of groups represented by small numbers of strains, which led to the discovery of 2 distinct relict-like groups of strains with relatively large genetic distance among them. A group of 11 lineages (magenta) from North West Spain, Sweden, the Netherlands, and New Zealand was identified. In ADMIXTURE analysis, these strains were located in either BAL or NCE as admixed lines or they were ungrouped. The large genetic and geographic distance between some of the 11 lineages indicated that they may represent a disparate admixed group, possibly with ancestry of underrepresented groups. (D) The 2 relict-like groups discovered with hclust are responsible for the second major mode in the PGD distribution. The PGD distribution of all strains is shown by the black outline. Colors indicate the contribution of the 2 most distant groups of strains identified by hclust of the PGD matrix, and the remaining pairs of strains are shown in gray. In the legend, the PGD groups are assigned to the ADMIXTURE clusters (Fig 1A) to which the contained strains belonged either directly or in parentheses. The colors in the distribution indicate the change caused by progressively dropping the respective hclust groups from the data in the order of the legend. The blue area accounts for PGD between strains of the IBE group and all other strains, and the magenta area for PGD between the other relict-like group and all other strains except IBE. (E) Relict-like C. hirsuta strains predominantly resemble the Iberian relicts discovered in Arabidopsis. The geographic distribution of the C. hirsuta strains is shown with PGD group membership indicated by corresponding colors (S1D Fig). Similar to Iberian relicts in Arabidopsis [17], the C. hirsuta IBE lines (blue) are found at high frequency on the Iberian Peninsula, but also more broadly in Western Europe and on the Macaronesian islands. Map layers were made with Natural Earth and [142]. (F) LD decay in comparable ancestry groups of C. hirsuta (C.h.) and A. thaliana (A.t.). The average squared correlation (r2) between pairs of SNPs is plotted against the average physical distance between them in kilobases. For A. thaliana, ancestry groups comparable to C. hirsuta were included: Relicts (C.h. IBE), NWC_Europe (Western Europe + Central Europe + Germany; C.h. NCE), IBC (Italy, Balkans, Caucasus; C.h. BAL), as well as the Iberian nonrelict population (A.t. Iberia). (G, H) Piecewise reconstruction of ancestral effective population sizes (Ne) in the 3 ADMIXTURE groups using MSMC2 (G) and relate (H), and estimates of split times between them considering a mutation rate of 7∙10−9 mutations per base pair, per generation. The top panel shows ancestral changes in Ne within the ADMIXTURE groups plotted against time in years, when considering 1 generation per year. Red, blue, and yellow lines indicate the BAL, IBE, and NCE genetic clusters, respectively. With MSMC2 (G), 20 random sets of 4 strains were analyzed, which are all plotted, while with relate (H), 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). The split times estimated with fastsimcoal2 (S1I Fig) rescaled for a mutation rate of 7∙10−9 are indicated by triangles below the x-axes. Light blue shaded areas in the plots show ancient periods of glaciation according to marine isotope stages 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. (I) The best demographic model according to fastsimcoal2 with a mutation rate of 4∙10−9 mutations per generation per base pair, and estimated parameters. The 3 populations identified by ADMIXTURE analysis are shown as columns along the y-axis, which indicates time from present at the bottom to more ancient times at the top. The width of the columns is scaled according to the estimated respective Ne, which is also shown in or above the columns themselves. Split times between the populations are shown by arrows connecting the columns, where all lineages from a population merge with its ancestral population. The times at which this occurs are also marked on the y-axis. Bottlenecks are indicated by temporary constrictions but are not to scale. The time at which a bottleneck period starts when looking backwards in time is indicated on the y-axis. Bidirectional arrows below the figure indicate the corresponding modes of migration in the model. See S3 Table for all estimated parameters and their confidence intervals. The data underlying the graphs shown in the figure can be found at https://doi.org/10.5281/zenodo.7907435. BAL, Balkan; CV, cross-validation; hclust, hierarchical clustering; IBE, Iberian; LD, linkage disequilibrium; LGM, last glacial maximum; NCE, Northern Central European; PC, principal component; PCA, principal component analysis; PGD, pairwise genetic distance. (TIFF)</p

Primers used for selecting recombinant HIFs.

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

<i>C</i>. <i>hirsuta</i> strains sequenced from the Azores arranged by island and sampling trip.

C. hirsuta strains sequenced from the Azores arranged by island and sampling trip.</p

Positions for new assembled regions of the reference chromosome 8.

Positions for new assembled regions of the reference chromosome 8.</p

Analysis of <i>FRIGIDA</i> alleles in natural strains of <i>C</i>. <i>hirsuta</i> and <i>A</i>. <i>thaliana</i>.

Analysis of FRIGIDA alleles in natural strains of C. hirsuta and A. thaliana.</p