Distribution of the sister-species M. helenor and M. achilles across South America.

The gray area represents the whole distribution of M. helenor, and the dotted area indicates the localities where M. helenor is in sympatry with M. achilles. Note that the different subspecies of M. helenor are found in different localities and display substantial variations in the proportion of black vs. blue areas on the dorsal sides of their wings. For example, M. h. bristowi and M. h. theodorus are allopatric in Ecuador, with their respective distribution separated by the Andes such that they are never in contact. While M. h. bristowi is found on the Pacific side of the country and has a wide blue band on the dorsal side of its wings, M. h. theodorus is found in Western Amazonia and displays a narrow blue band on the dorsal side of its wings. However, M. helenor is sympatric with M. achilles throughout the Amazonian rainforest. In French Guiana, the subspecies M. helenor helenor and M. achilles achilles display convergent dorsal color patterns with thin blue bands (Llaurens et al., 2021).

Differences of hue and brightness on the proximo-distal plane of Morpho wings

(A) Illustration of the two protocols used to assess the differences in Morpho wing reflectance. The “Specular” set-up allows for the quantification of the wing color variations, while the “tilt” set-up can be used to quantify brightness variation for each Morpho wing (see Appendix for extended methods). Variations of hue (B and C) and brightness (D and E) calculated from the wing reflectance measured with the “Specular” set-up, and variations of brightness calculated from the wing reflectance measured from the “tilt” set-up (F and G). Those hue and brightness parameters were calculated for the sympatric M. h. helenor and M. a. achilles (first column in green and blue) and for the allopatric M. h. theodorus and M. h. bristowi (second column in orange and purple) on the proximal-plane plane of their wings (I= illumination on the internal side of the wings, E= illumination on the external side of the wings). See S1 Table for the PERMANOVA analyses describing those graphs.

Characterization and perception of the iridescent coloration of Morpho butterflies.

(A) and (B): PCAs showing the variation in iridescence for both sexes, (A) in the two sympatric species from French Guiana (M. h. helenor vs. M. a. achilles) and (B) in the two allopatric Ecuadorian subspecies of M. helenor (M. h. theodorus vs. M. h. bristowi). Each point represents the global signal of iridescence of each individual, corresponding to the 21 complete reflectance spectra obtained from the 21 tested angles of illumination. The results of the PERMANOVA are shown on the top left corner of each graph. (C) The chromatic distances (i.e. the visual discrimination rate by a visual model) of the wing reflectance measured with the “Specular” set-up on the proximo-distal plane. Visual modeling was used to calculate the chromatic contrast of blue coloration between allopatric M. helenor subspecies (red) and between the two sympatric sister-species M. helenor and M. achilles (green), as perceived by a Morpho visual system for every angle of illumination measured on the proximo-distal plane. The chromatic contrast likely perceived by UV-sensitive birds is shown in gray. Chromatic contrast of the female wings (top) and male wings (bottom). The threshold of discrimination is shown by the dotted line and set to 1 Just Noticeable Difference (JND).

Tetrad experiment results:

Number of mating events involving the different possible pairs of males and females from the two subspecies of M. helenor (bristowi vs. theodorus) in 30 tetrad experiments

Morpho male preference based on visual cues alone.

Probabilities of (A) approaching and (B) touching a con-subspecific female for M. h. bristowi males (purple) and M. h. theodorus males (orange) from Ecuador, as well as the probabilities of (C) approaching a con-specific female for M. h. helenor males (blue) and M. a. achilles males (green) from French Guiana, measured during Experiment 1. The dotted line indicates the expected probability of approaching/touching a model if no preference is present. The p-values of Wilcoxon tests testing for the significant departure from the 0.5 probability are shown next to the corresponding graphs.

Divergent chemical compounds found on the genitalia of sympatric Morpho males.

nMDS representation of the differences in the (A) C8 to C16 chemical compounds and (B) C16 to C30 chemical compounds found in the genitalia of M. h. helenor and M. a. achilles males and females, calculated using Bray-Curtis distances. M. h. helenor are shown in blue (males are in dark blue and females in light blue), and M. a. achilles are shown in green (males are dark green and females are light green). The result of the PERMANOVA (999 permutations) testing the effect of sex and species on the chemical composition of M. h. helenor and M. a. achilles are shown on each figure.

Experimental design of the male choice experiments

performed between the Morphos originating from Ecuadorian populations of M. helenor (eastern population: M. h. theodorus and western population: M. h. bristowi, experiments 1 to 3) and from French Guiana (M. h. helenor and M. a. achilles, experiment 4), along with the associated preferences and visual cues tested. Note that the same males were used for experiments 1, 2 and 3.