Numerous ecological interactions can impact trait evolution in closely-related species in sympatry, leading to trait convergence or divergence (terHorst et al., 2018). Closely-related species often display similar suites of traits because of their shared evolutionary history (Blomberg et al., 2003). Local selection can also act as a filter and prevent trait divergence (Keddy, 1992), therefore enhancing trait similarity between closely-related species when they occur in sympatry (Chazot et al., 2014). Trait similarity among sympatric species within a given ecological niche can thus stem from retention of locally adapted ancestral traits or from evolutionary convergence (Muschick et al., 2012). Selective pressures promoting the retention of locally adapted traits within species and/or trait convergence among sympatric species can also be due to local ecological interactions: for instance, shared predation pressure may promote the convergence of predator-deterrent traits in sympatry, but allow the trait to differentiate in allopatry (Mallet, 1999).
However, when closely-related sympatric species share a given trait, either as a result of ancestry and/or convergence, they often diverge in other traits because (1) they may be partitioned in different ecological niches (Berlocher and Feder, 2002), or (2) as a result of character displacement due to reproductive interference (Grether et al., 2020) or reinforcement due to poor hybrid fitness (Butlin and Smadja, 2018). As a result, traits involved in sexual competition or mate choice tend to diverge significantly more often between species in sympatry compared to allopatry (Haavie et al., 2004; Marko, 2005).
In this study, we investigate how ecological interactions in sympatry can constrain natural and sexual selection shaping trait evolution. We specifically focus on traits submitted to both natural and sexual selection and compare differences in these traits in allopatric vs. sympatric ranges. Theoretical and empirical studies have shown that sexual selection may favor the evolution of preferences for locally-adapted traits within species (Servedio, 2004; Servedio and Boughman, 2017; van Doorn et al., 2009). For instance, the predator-deterrent coloration of poison frogs is also detected and used as mating cues by females (Reynolds and Fitzpatrick, 2007). Similarly, habitat-dependent coloration of sympatric cichlid fish is also used as a visual cue for mate recognition (Seehausen et al., 2008). Yet, sexual interactions are likely to occur between individuals from closely-related species when they live in sympatry, and similar preferences for adaptive traits may thus result in substantial reproductive interference (Gröning and Hochkirch, 2008; Soni et al., 2025). Hybrids, when produced, can be unfit, thus favoring the evolution of sexual preferences for species-specific cues, rather than locally-adapted traits (Maisonneuve et al., 2024). To determine to what extent ecological interactions shape trait evolution, it is thus necessary to compare patterns of trait evolution in sympatry and allopatry: allopatric populations indeed allow us to estimate background levels of divergence or similarity that arise in the absence of direct ecological interactions (Pfennig and Pfennig, 2009). Comparing variations in adaptive traits in sympatric vs. allopatric populations of recently-diverged species and testing the sexual preference for those traits can shed light on the selective processes targeting traits modulating reproductive isolation and co-existence in sympatry.
In butterflies, the evolution of wing color patterns can be influenced by both natural and sexual selection. The visual discrimination of wing color pattern can enable intraspecific recognition during courtship in many species (Costanzo and Monteiro, 2007; Li et al., 2017). However, the evolution of wing color patterns is also strongly influenced by the risk of detection and/or recognition by predators (Finkbeiner et al., 2014; Oliver et al., 2009). Whether these opposite selective pressures ultimately promote trait convergence or divergence in sympatric species might depend on their relatedness: for instance, a study in Papilionidae showed multiple color pattern convergences between distantly-related species living in sympatry, while divergent colorations are found in closely-related species (Puissant et al., 2023). Divergence in traits involved in species recognition could be favored because of higher reproductive interference in closely related species than in distantly related taxa (Pfennig and Pfennig, 2009). In sympatric species with chemical defenses, such as Heliconinii butterflies, local predation pressures tend to promote the convergence of similar conspicuous warning wing patterns compared to allopatric species (i.e. Müllerian mimicry, Joron et al., 1999; Merrill et al., 2014). But the costs associated with hybrid production, in turn, favor the evolution of alternative divergent mating cues in mimetic butterflies (Estrada and Jiggins, 2008), and divergence in male pheromone bouquets and female attraction has been found among mimetic sister species (González-Rojas et al., 2020). Similarly, the evolution of specific visual mate recognition signals, limiting reproductive interference but indistinguishable by predators, can also be promoted on the wings of mimetic butterflies (Llaurens et al., 2014).
Here, we focus on the evolution of mating cues in the neotropical butterfly genus Morpho, where multiple closely-related species co-exist in sympatry (Blandin and Purser, 2013). In the Morpho species observed in the understory, striking iridescent blue coloration is displayed on the dorsal side of the wings, due to specific wing scale structures (Giraldo et al., 2016; Siddique et al., 2013). The light signal reflected by iridescent surfaces can be very directional, as hue and brightness of iridescent objects can drastically change depending on the light environment or the observer’s position (Doucet and Meadows, 2009). While the iridescent blue color is probably ancestral to the diversification of the understory clade (Chazot et al., 2021), the precise reflectance spectra at different angles likely differ among Morpho species. Directional iridescent signals generated in animals can likely enhance recognition by mates while remaining poorly detected by predators (Endler, 1992). In birds (Simpson and McGraw, 2019) and butterflies (White et al., 2015), the specific directional signal produced by the iridescent trait can be used as a cue during courtship, suggesting that the antagonistic sexual and natural selective pressures may finely tune the evolution of iridescent effects. How much sexual selection shapes the evolution of iridescent properties in sympatric Morpho species is currently unknown, but behavioral experiments carried out in the field in Amazonian Peru highlighted strong visually-based territorial interactions among males from sympatric species and limited species discrimination based on female coloration in males (Le Roy et al., 2021b).
This raises questions on the key visual cues involved in mate choice, given that the iridescent blue coloration shared by closely-related species encountered within the understory is also likely under selection by predators. The iridescent bright blue dorsal coloration of Morpho wings contrasts with the brown and matte ventral side and generates a peculiar visual effect during flight. The combination of the blue flashes produced by the alternate exposure of the bright blue vs. brown sides of the wings during flapping flight, in addition to erratic flight trajectories, makes these Morpho very difficult to catch by bird predators (Young, 1971), potentially enhancing their evasive capabilities (Murali and Kodandaramaiah, 2020). Experimental trials with evasive prey have shown that predators learn to avoid prey they repeatedly fail to catch (Páez et al., 2021). The display of iridescent wings could thus be associated with a higher survival rate in nature because of both (i) direct effects, through successful escape of predator attacks, and (ii) indirect effects, by limiting predation attempts by birds recognizing the blue signals and refraining from attacking, as highlighted by butterfly release experiments investigating the hunting behavior of wild insectivorous birds in Brazil (Pinheiro et al., 2016). Mark recapture experiments in the field with manipulated dorsoventral contrasts in wild Morphos have suggested that dynamic flash coloration can reduce predation rate (Vieira‐Silva et al., 2024). This indirect effect could promote the evolution of convergent blue patterns in sympatric species, similar to the mimicry observed in species with chemical defenses (Joron et al., 1999; Merrill et al., 2014). In line with this hypothesis, repeated local convergence in the proportion of iridescent blue vs. black areas on the dorsal side of the wings has been documented in the sister-species Morpho helenor and Morpho achilles living in sympatry throughout the Amazonian basin (Llaurens et al., 2021). Precise quantification of variations in iridescence is now needed to assess the respective effects of selection by predators and mates that may drive convergent vs. divergent evolution of iridescence in sympatric and allopatric ranges.
First, we quantified iridescence in allopatric vs. sympatric populations of M. helenor subspecies. Since coloration is expected to be more similar within than among species under neutral evolution, we used allopatric populations of M. helenor as a baseline to assess convergence of iridescence between two sympatric species (M. helenor and M. achilles). We then conducted behavioral experiments to test the effect of variation in iridescence on mate recognition, using two subspecies of M. helenor displaying different iridescent phenotypes. This intraspecific comparison allows identifying the visual cues used in mate choice in M. helenor, teasing apart the effects of iridescence and/or wing pattern. We then tested whether those visual cues are used in species recognition between sympatric M. helenor and M. achilles. Finally, we studied variations in the volatile compounds produced by wild males and females from sympatric populations of the two species to explore the evolution of potentially alternative traits, such as chemical cues, possibly acting as a reproductive barrier.