Figure 1.
Bayesian inference phylogenetic tree of monocot SAD homologs and eudicot outgroup.
Numbers indicate posterior probabilities (where >0.5) above branches. Ophrys SAD homologs and their assignment to different allele groups are highlighted and ω = dN/dS ratios for branches of interest indicated. A black upward triangle indicates significant positive selection; a gray downward triangle indicates purifying selection, and a non-filled triangle indicates relaxed purifying selection. The inset lists the ω-values, associated p-value, and significance (*, p<0.05; **, p<0.01; ***, p<0.001).
Figure 2.
Regression of normalized desaturase gene expression with alkenes.
(A–C) Relative amount of alkenes was used after f(x) = arcsin x0.5 transformation for tissues and floral stages of O. exaltata (A) and O. sphegodes (B,C). (D–F) Absolute amount of alkenes (in µg) was used after f(x) = ln (x+0.01) transformation for population data from three species. For both datasets, normalized SAD expression was used after f(x) = x0.5 transformation. Adjusted R2 is indicated in each graph. (A,D) Correlation of all (C21-C29) 7-alkenes with SAD5; (B,E) Correlation of C27+C29 9-alkenes with SAD2 expression; (C,F), Correlation of all (C25-C29) 12-alkenes with SAD2 expression.
Figure 3.
Distribution of SAD allele occurrence in three species.
Color indicates different allele groups (blue: putatively functional alleles; orange: putatively nonfunctional alleles; gray: alleles not expressed in a given species). Different letters beside pie charts indicate statistical difference (p<0.05; χ2 test).
Figure 4.
Allelic gene expression and alkene production in different species.
Top, gene expression of different SAD alleles; Bottom, alkene levels. Colors indicate species (red, O. exaltata; green, O. garganica; blue, O. sphegodes). Error bars show standard error. Different letters indicate significant differences among species for each allele (p<0.05; ANOVA).
Figure 5.
Effects of alkenes with different double-bond position on pollinator behavior.
(A) Addition of 9- and 12- alkenes (associated with SAD1/2) onto O. exaltata flowers reduced its pollinator (C. cunicularius) response (N = 18). Red bar, control O. exaltata; pinkish bar, scent manipulated O. exaltata. (B) Adding 7-alkenes (associated with SAD5/6) into O. sphegodes floral scent extract reduced its pollinator (A. nigroaenea) response (N = 17). Blue bar, control floral scent of O. sphegodes; light blue bar, manipulated floral scent of O. sphegodes. Y-axis refers to number of counts. Asterisks indicate statistical significance (pairwise Mann-Whitney U test): *, p<0.05.