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# Vertical and temporal niche partitioning in Amazonian butterflies: implications for the evolution of thermal tolerance

Titouan Bouinier, Charline Smadi, Violaine Llaurens

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Titouan Bouinier, Charline Smadi, Violaine Llaurens. Vertical and temporal niche partitioning in Amazonian butterflies: implications for the evolution of thermal tolerance. Oikos, 2025, pp.e11567. 10.1002/oik.11567. hal-05348274

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OIKOS

Research article

# Vertical and temporal niche partitioning in Amazonian butterflies: implications for the evolution of thermal tolerance

Titouan Bouinier ID,✉ <fnref n="1" />,<fnref n="2" />, Charline Smadi ID,<fnref n="3" />,<fnref n="4" /> and Violaine Llaurens ID,<fnref n="1" />,<fnref n="2" />

**Correspondence:** Titouan Bouinier (titouan.bouinier@college-de-france.fr)

Oikos

2025: e11567

doi: 10.1002/oik.11567

Subject Editor: Kathryn Stewart

Editor-in-Chief: Pedro Peres-Neto

Accepted 18 July 2025

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Closely related species living in sympatry are often partitioned into divergent ecological niches. Such specialization can be enabled by the evolution of divergent traits enhancing adaptation to different niches. In this study, we investigate the partitioning of closely related butterfly species into different forest strata and daily activity time and test the effects of such spatio-temporal niches on the evolution of thermal traits. First, using experiments in the field in Amazonia, we precisely characterized the daily activity patterns of nine species of *Morpho* butterflies, therefore documenting extensive temporal segregation among species and observing significant variations in temperature between their respective niches. Using controlled experiments in the lab, we then tested the thermal tolerance of wild individuals to both hot and cold conditions. The vertical distribution of species (understory versus canopy micro-habitats) had a significant effect on several thermal traits, even when controlling for the phylogenetic distances between species, suggesting that forest stratification may have shaped thermal adaptation in these tropical butterflies. However, butterfly activity time did not correlate with any thermal traits measured. The extensive temporal segregation observed between these sympatric species might thus stem from ecological interactions observed between species rather than thermal factors.

**Keywords:** butterfly, community, evolutionary ecology, temporal niche, thermal resistance, vertical stratification

Introduction

Community assembly in the wild is strongly shaped by historical factors, but the effect of ecological specialization also play an important role in co-existence between species (Armitage et al. 1994). Such specialization might stem from the evolution of divergent traits enabling the colonization of new microhabitats, favoured by competitive

© 2025 The Author(s). Oikos published by John Wiley & Sons Ltd on behalf of Nordic Society Oikos.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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<footnote n="1">¹Center for Interdisciplinary Research in Biology, CNRS UMR7241, INSERM U1050, Collège de France, Paris Sciences et Lettres University, Paris, France</footnote>

<footnote n="2">²Institut de Systématique, Evolution, Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles CP 50, Paris, France</footnote>

<footnote n="3">³LESSEM, INRAE, Univ. Grenoble Alpes, St-Martin-d'Hères, France</footnote>

<footnote n="4">⁴Institut Fourier, CNRS, Univ. Grenoble Alpes, Gières, France</footnote>
interactions (competition for resources or reproductive interference; Davies et al. 2007). Disentangling the effect of biotic versus abiotic factors shaping the evolution of traits enabling specialization into divergent ecological niches is thus a central question in evolutionary biology and community ecology. The specialization in different niches can be enabled by the evolution of new traits (e.g. key innovations, like the jaw development in Cichlidae fish, granting access to novel food sources; Liem 1973). In turn, adaptation to divergent niches can promote the evolution of new traits. Habitat divergence between species can indeed be followed by the evolution of different traits, like the claw shape in closely related allopatric *Anolis* lizards, which are more or less curved depending on the specific micro-habitat of the island they occupy (Yuan et al. 2020). Identifying feedbacks between phenotypic evolution and niche specialization is required to identify the mechanisms involved in species diversification and community assembly in the wild.

Tropical forests concentrate the highest level of species richness on earth (Beccaloni and Gaston 1995, De Souza Amorim et al. 2022), and the Amazonian rainforest is a relevant example of geographic regions with tremendous animal biodiversity (Myers et al. 2000, Mittermeier et al. 2003). Yet, the factors shaping the evolution and the persistence of such local diversity are still largely unknown. Specialization into multiple micro-habitats (e.g. canopy versus understory habitats for bird species; Marra and Remsen 1997, Walther 2002) appears as an important driver of persistence of multiple species within tropical forest communities. Diversification into different timings of reproductive activities throughout the day (referred to as hourly niche hereafter) within spatial micro-habitats is likely also an important and yet understood dimension of the niche in such environments. Evidence for partitioning of species into divergent hourly niches has been documented in tropical areas, especially in insects (e.g. daily activity patterns in the Hesperiidae butterfly genus; Devries et al. 2008).

The partitioning into different hourly niches may reduce competition and facilitate co-existence of closely related species in sympatry. For instance, differentiated activity timings can be observed between two *Acomys* mice species, but only when they occur in sympatry (Jones et al. 2001) and such differentiation in foraging time is likely due to competitive interactions. The evolution of differences in circadian rhythm between closely related species might thus be influenced by selection generated by antagonistic ecological interactions. Competition for resources might be an important driver of such specialisation into different hourly niches, notably when resources themselves are partitioned throughout the day. For instance, nectar production (Matile 2006) can oscillate throughout a day, and nectar-consuming insect species often display matching foraging time. Such correlations between insect activity and flower opening times have notably been shown in nocturnal moths (Funamoto and Ohashi 2017). Furthermore, sexual interactions between species, by generating substantial reproductive interference (Gröning and Hochkirch 2008) may also promote the evolution of

divergent temporal niches in sympatric species. Mathematical models recently highlight how male-male competition between Lepidoptera species can promote the evolution of divergent hourly niches (Van Doorn et al. 2024). Such divergence in daily temporal niches, as observed for instance in the moth *Spodoptera frugiperda*, can either participate to the speciation process or be promoted by reinforcement (see Taylor and Friesen (2017) for a review on allochronic speciation). Competition for resources and reproductive interference might thus be key ecological interactions driving the evolution of divergent hourly niches in sympatry.

Divergence in hourly niches, together with specialization in different micro-habitats of tropical forests (shadow versus open areas, canopy versus understory), may change the abiotic environment and generate contrasted selective pressures on other traits. Temperature and humidity levels have been documented to differ between different spatio-temporal niches (Montejo-Kovacevich et al. 2020) and these divergent abiotic conditions might promote the evolution of thermal or drought tolerance in species specialized in different niches. Vertical stratification was shown to significantly influence the composition of butterfly communities (Santos et al. 2017), and the evolution of several morphological traits in tropical butterflies is likely influenced by microclimatic differences encountered in different vertical strata (Mena et al. 2020, De Brito Freire et al. 2024), displaying links between habitat specialization and the evolution of flight traits. In particular, temperature influences physiological processes, and tolerance to extreme temperatures is thus likely to set boundaries on where and when individuals from different species can be active (Franzén et al. 2022). The evolution of thermal tolerance in different species might therefore strongly shape community assembly, by constraining physiologically demanding activities in different micro-habitats and at different times of the day. To investigate the thermal traits of wild individuals from different species, we here use a combination of commonly used proxies focused on extreme cold and hot temperatures: heat knock-out time (i.e. time until knocked out under extreme heat stress), heat recovery time, and cold-coma recovery time (i.e. the time after which mobility is recovered after high thermal stress). They are considered relevant proxies to estimate adaptation to climatic cold and heat conditions, and have been extensively used in the past to assess ectotherms thermal traits (Ayrinhac et al. 2004, Sørensen et al. 2005, Karl et al. 2008). Here, we thus aim to characterize the diversification in hourly niches within an Amazonian community of closely related butterflies living in sympatry, and investigate variations in the thermal tolerances within and among these species. We focus on the community of *Morpho* butterfly species in French Guiana. The Amazonian forest in French Guiana is characterized by stable mean temperatures throughout the year (Lavado Casimiro et al. 2013, Adde et al. 2016), with an average daily temperature of approximately 26°C (ranging from 25.8 to 27.1°C). Out of the twenty-nine species of the *Morpho* genus, nine can be observed within localities in French Guiana. Divergence into different temporal niches has already been documented
between two sister species of *Morpho* in Amazonian Peru, where male patrolling hours have been characterized using a capture–mark–recapture experiment (Le Roy et al. 2021b). *Morpho* males typically patrol along trails and rivers, and this behaviour is probably linked to mate searching and territorial activity. It is also probably a metabolically costly behaviour, and might thus be constrained by thermal tolerance (Mattila 2015). Strong variations in wing size and colour, as well as body length, are observed among *Morpho* butterflies, and the evolution of these traits might also impact thermal tolerance. Finally, the flight height strongly differs between *Morpho* species, with species observed mostly in the canopy while others are typically observed in the understory (DeVries et al. 2010, Le Roy et al. 2021). It has notably been shown that daily micro-climatic fluctuations affect the community assembly of butterflies differently between these two forest strata (Richter et al. 2023). This difference in vertical strata might also change the selective regimes acting on the evolution of thermal tolerance.

In this study, we thus aim to: 1) characterize the hourly niche of *Morpho* species in French Guiana by documenting the patrolling behaviour of males throughout the day; 2) characterize variations in resistance to cold (–20°C) and hot (50°C) temperatures within and among species, and their co-variations with morphological traits (wing size and wing colour pattern); and 3) identify the historical and ecological

factors likely influencing the evolution of thermal tolerance, using comparative analyses.

# Material and methods

## Sampling strategy

Individual males were sampled in the Amazonian rainforest within a single site located in French Guiana (Fig. 1), with GPS coordinates 4°33′43.4"N, 52°11′32.8"W. We sampled 91 males from nine species of the *Morpho* genus in July–November 2023 (see Table 1 for more details on the number of samples per species) using entomological nets at the top of a waterfall. Each captured butterfly was identified, kept in a glassine envelope, and fed a mixture of water and honey every morning to keep it in good condition.

A single observer recorded the activity time of each male caught and/or observed in this location throughout 36 days, evenly distributed over the July–November period. We calibrated the time of observation with respect to sunrise time, as it slightly changed over the course of the field experiment. No binoculars were necessary, as all species can be recognized by sight, except for of the locally abundant species *M. helenor* and *M. achilles*, which are visually so similar (Llaurens et al. 2021, Ledamoisel et al. 2025) that they need to be captured for identification. Finally, we recorded the understory

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Figure 1. Map of the density of *Morpho* species throughout Central and South America. Darker colours indicate fewer coexisting species than lighter ones for a given place. All the data were retrieved from the iNaturalist database (www.inaturalist.org) in February 2025, and filtered for only research-grade observations (9652 observations spanning all 30 species of the genus *Morpho*). The size of each cell on the map is 80 km in length/width. The sampling site used in this study is indicated with a star. In this geographic area, nine *Morpho* species can be observed within the same locality.
<table><caption>Table 1. Sample sizes for each of the nine *Morpho* species studied: (1) number of observations used for activity timing characterization and (2) number of individuals used for thermal tolerance tests. The phylogenetic relatedness between species, implied in Chazot *et al.* (2016), is shown on the left-hand side of the table. Understory species are shown in green font, while canopy ones are in red font.</caption><thead><tr><th>Phylogenetic relatedness</th><th>Species</th><th>Number of patrolling behavior observations</th><th>Number of individuals used in thermal tolerance experiments</th></tr></thead><tbody><tr><td></td><td>Morpho eugenia</td><td>89</td><td>4</td></tr><tr><td></td><td>Morpho marcus</td><td>127</td><td>13</td></tr><tr><td></td><td>Morpho helenor</td><td>32</td><td>9</td></tr><tr><td></td><td>Morpho achilles</td><td>32</td><td>12</td></tr><tr><td></td><td>Morpho deidamia</td><td>136</td><td>14</td></tr><tr><td></td><td>Morpho menelaus</td><td>242</td><td>16</td></tr><tr><td></td><td>Morpho telemachus</td><td>165</td><td>3</td></tr><tr><td></td><td>Morpho hecuba</td><td>124</td><td>5</td></tr><tr><td></td><td>Morpho rhetenor</td><td>323</td><td>15</td></tr><tr><td></td><td>TOTAL:</td><td>1270</td><td>91</td></tr></tbody></table>

temperature at this site every 15 min under a shaded area for eight consecutive days using a thermometer (Mastrad classic thermal probe, precision: ±1°C).

## Thermal tolerance test

Thermal tolerance testing was conducted at the LEEISA facilities in Cayenne, French Guiana, using butterflies captured at the same sites as the experiments described above. We tested all nine species, but with various numbers of individuals tested per species (see Table 1 for more details). We used two static extreme temperature (–20 and 50°C) to capture minute variations in thermal tolerances between closely related species. The chosen temperatures are outside the range usually encountered by individuals in the wild but this experimental design was chosen to maximize capturing small-magnitude physiological variations, using a limited number of wild individuals. While the use of such extreme temperatures is still discussed (Lutterschmidt and Hutchison 1997), it has been used in previous studies as a relevant predictor of thermal tolerance in ectotherms (in Lepidoptera, Chidawanyika and Terblanche 2011; in Chrysomelidae beetles, García-Robledo et al. 2016). Such brief experimental protocols reduce stochasticity, hardening and resource depletion in thermal traits measurements (Chown et al. 2009). Here, it allows us to compare several non-model species, using a limited number of wild individuals that we are currently unable to breed or keep in captivity for long periods of time. We used a temperature of 50°C for hot knock-out, as this was within the range of the maximum thermal tolerance recorded by Silva et al. (2020) for *M. helenor*, ensuring a relatively quick knock-out and a great chance of recovery after the heat shock. The temperature of –20°C was inspired by the experimental design reported in Karl et al. (2008), ensuring that all individuals were knocked-out quickly, and would not recover instantly after. The same individuals were used for both hot and cold experiments, allowing us to also

capture individual variation within species. We ensured that individuals would not be killed during the knock-out experiments, as we also studied their recovery timing.

### Cold recovery experiment

Individuals were first acclimatized for 30 min at 27°C, then were put inside a 15 × 15 × 10 cm solid plastic container at –20°C for 5 min. Individuals were then retrieved from the container and put back in the 27°C environment. The time it took them to recover wing motor skills was recorded, and the individuals were then put at rest. Each individual underwent this experiment three times, with a 30 min rest in between each experiment. Recovery time has been defined in the literature as 'the time elapsed between taking the tray out of the cold until a butterfly was able to stand on its legs' (Fischer et al. 2010). Since almost all of our individuals recovered in less than 10 min, 30 min was more than enough time for all of our individuals to fully recover.

### Hot knock-out and recovery experiment

All the individuals already used in the previous experiments were then acclimatized for 30 min at 27°C, and a 1-l glass container was submerged in a hot water bath (Polystat model 86602, precision ± 0.05°C), and temperature inside the container was monitored with a thermometer (Mastrad classic thermal probe, precision ± 1°C). When the temperature reached 50°C, an individual was put in the glass container, and time until loss of motor control was recorded. We ensured that the temperature stayed constant over time, removing any data where a difference higher than 1°C was observed with the experimental temperature. As soon as the individual lost motor control, it was removed and put back in the 27°C environment. The time it took to recover wing motor skills was recorded and they were then laid out at rest. Each individual underwent this experiment only once because further testing frequently resulted in an individual's death, impeding any further recovery time measurement.
## Morphological trait measurements

Because wing size and colouration differ considerably between species and could potentially affect thermal tolerance, we estimated variations in the wing size and colour pattern, as well as in the wing area, among all individuals used in the thermal experiment, by separating the wing from the body of individuals, and taking pictures of each wing separately on their dorsal side (Supporting information).

RGB pictures were taken in a closed box with controlled LED light conditions (similar to the experimental design presented in Brehm (2025)), using a Nikon D90 (camera lens AF-S Micro Nikkor 60 mm 1:2.8G ED). An xRite Mini ColorChecker was also used to colour-correct the images afterwards, by using the R package 'patternize' ver. 0.0.5 (Van Belleghem et al. 2018). We then used the R package 'recolorize' ver. 0.1.0 (Weller et al. 2024), allowing us to segment our images into limited sets of discrete colour categories, and enable comparisons. To assess colour variation between species and quantify the relative area covered by those colours, the 'recolorize' package was used to carry out a k-means clustering to a palette of five selected colours. This palette was handpicked and chosen to be representative of the main colours perceived by humans on the dorsal side for the nine *Morpho* species studied here (blue, black, brown, white and orange). While *Morpho* butterflies do display UV reflectance (Giraldo et al. 2016, Ledamoisel et al. 2025) that are not captured on the wing pictures used in this study, reflectance in the UV range (300-400 nm) is limited to the iridescent areas of the wings. Accounting for the reflectance in the UV range would thus result in the same distribution of distinct colour patches. We finally assessed proximal colours by selecting the third of the wing that was closest from the body, which was shown to account for most of the variation in thoracic temperature (Wasserthal 1975).

The particular multi-layered scale structure of iridescent wing patches could potentially affect heat transfer, and thus thermoregulation capabilities of individuals. In supplementary analyses, we thus analyzed the effect of iridescence wing scale on thermal traits by considering different iridescent phenotypes. We first categorized the *Morpho* species studied here into three phenotypes: full blue iridescence *M. eugenia*, *M. marcus*, *M. menelaus*, *M. rhetenor*; blue-banded iridescence *M. helenor*, *M. deidamia*, *M. helenor*; and no iridescence (*M. hecuba* and *M. telemachus*).

The type of wing scales and arrangements contributing to the structural blue colouration also differ between *Morpho* species; we thus distinguished five categories according to the relative size and overlap of scales, as described in Giraldo et al. (2016): G1 (*M. marcus*, as well as *M. eugenia*, inferred from the phylogeny since it was not part of the studied species in Giraldo et al. (2016)), G2 *M. helenor*, *M. achilles*, *M. deidamia*, *M. menelaus*, G4 (*M. rhetenor*), and none (*M. hecuba* and *M. telemachus*, as they do not have any iridescent blue scales).

## Statistical analysis

To assess how much the neutral divergence among *Morpho* species affects the evolution of the traits measured in this study, we computed the so-called phylogenetic signal of our traits, using Pagel's λ, estimated from phytools (Revell

2012). We used the phylogenetic relationships among *Morpho* species inferred in Chazot et al. (2016). Pagel's λ represents the degree to which differences in trait values among species match the values obtained from a Brownian motion model accounting for the phylogenetic distance between species. When λ=0, no phylogenetic structuration of the evolution of the trait is detected, meaning that phylogenetic relationship between species does not predict similarities in trait values. A value of λ=1 (strong phylogenetic signal) means that trait evolution among species is akin to the Brownian motion model, where trait similarities are likely due to shared evolutionary history between species (e.g. phylogenetically close species are more likely to share similar trait values). p-values for λ indicate whether the λ metric significantly differs from 1.

To test the correlations between thermal resistances, activity timings, micro-habitat and wing colour/sizes, we first built linear models to assess the effect of ecological variables on thermal tolerances. We then built phylogenetic generalized linear mixed models (PGLMM) from the 'phyr' package (Daijang et al. 2020), inserting the phylogenetic tree as a random variable. This allowed us to account for the effect of phylogenetic relatedness on trait variation observed between species. Differences in activity timings or micro-habitat could simply stem from neutral divergence between species, and controlling for phylogenetic effects allows us to test for a putative role of natural selection regimes differing between ecological niches. To distinguish the relative contributions of ecological specialization and phylogenetic structure in the variation of thermal tolerances, we used partial R²'s from the 'rr2' R package (Ives and Li 2018). The partial R²<sub>lik</sub> for each factor was calculated by comparing the full model with reduced models in which a given factor was removed. The reduction in likelihood is then measured, as well as p-values from a χ²-test using likelihood ratio test values. This allows us to test whether the difference between the full and reduced models is significant.

We treated the time in seconds before knock-out and recovery as the response variables, and log-transformation was applied prior to inclusion in the models to meet the normality assumptions required for model validity (Supporting information). Explanatory variables included time of capture, micro-habitat, total wing area, percentage of black colouration, and proximal colouration of black, blue or white. We used only black pigmentation in these models, assuming that these pigments are more likely to play a role in heat absorption (as shown in Liao et al. 2019), and included an interaction term between the wing area and the black pigmentation. All variables were standardized using Z-score normalization in the models to ensure equal contribution in the analysis. We also tested if potential correlations between thermal tolerances existed using Pearson correlation tests.

In supplementary analyses, we replaced black pigmentation by either the iridescent phenotype or scale configuration, and used post hoc Tukey's tests to observe if there were any differences between groups of species.

All statistical analyses were performed in R ver. 4.4.0 (www.r-project.org).
# Results

## Hourly temporal niche evolution across sympatric *Morpho* species

We recorded 1270 instances of male patrolling activity for the nine species of *Morpho* butterflies (see Table 1 for a precise record of observations within each species). *Morpho menelaus* males (242 observations, 19% of all observations) were the most abundantly observed species in the understory, while *M. rhetenor* males were the most frequently observed in the canopy (323 observations, 25% of all observations). Although pseudo-replication likely occurs in these observations, the large sample size and the relatively abundant population size allowed us to confirm that individuals of each species consistently patrolled within the same time window across multiple days.

As seen in Fig. 2, *Morpho* species in French Guiana are strongly partitioned throughout the day. The phylogenetic signal was non-significant for activity timings (Pagel's $\lambda=0.359$, p-value=0.361).

We also note temporally differentiated niches within the species *M. telemachus*, where two distinct colour pattern morphs within populations are observed in French Guiana (Gayman et al. 2016): a grey/blue morph and an orange morph. The two morphs show slightly different activity timings, with the orange morph being active later in the day (Supporting information).

## Thermal tolerance between species

When sampling temperature in 15 min intervals over nine days at the same location, we found significant differences in average temperature encountered in the different time windows occupied by the different species, with a difference of almost 5°C between the temperature conditions encountered by flying males from the species *M. eugenia* versus *M. achilles* (Fig. 3). We then tested the thermal tolerance of 91 male individuals belonging to the nine species (see Table 1 for more details on the sample sizes within each species). We found no significant correlation between the time before heat

knock-out and heat recovery timings (Pearson correlation test: $r=-0.17$, $t(70)=-1.46$, $p=0.15$, 95% CI [-0.39, 0.06]), heat knock-out and cold recovery timings after the first pass ($r=0.18$, $t(77)=1.65$, $p=0.10$, 95% CI [-0.04, 0.39]) or heat recovery and cold recovery timings after the first pass ($r=0.06$, $t(67)=0.51$, $p=0.61$, 95% CI [-0.18, 0.29]). Phylogenetic signals inferred from thermal tolerances were all non-significant, but we nonetheless observed a high phylogenetic signal for the time before heat knock-out ($\lambda=0.84$, $p=0.20$) and the time before cold recovery ($\lambda=0.75$, $p=0.14$). Phylogenetic signal for heat recovery was practically zero ($\lambda < 0.001$, $p=1$). We also documented whether iridescent phenotype or scale configuration had an effect on thermal tolerances (Supporting information), but did not find any significant result.

We then also tested more precisely the spatial arrangement of colouration, by studying the wing colours proximal to the body (shown in the Supporting information). While we found differences in proximal colouration between species, no significant link between any type of proximal colouration and thermal tolerance was found (Supporting information).

## Heat knock-out and recovery

We detected a significant effect of the micro-habitat ($\beta=-1.03 \pm 0.25$, $Z=-4.15$, $p < 0.001$), but not of the time of capture, on the time before heat knock-out (Supporting information): individuals living in the understory were knocked-out earlier than the canopy ones, consistent with a reduced tolerance to heat in understory species. The controlled variables, i.e. the level of black pigmentation ($\beta=0.35 \pm 0.12$, $Z=2.95$, $p=0.004$), and its interaction with the wing area ($\beta=-0.3 \pm 0.14$, $Z=-2.09$, $p=0.039$) also had a significant effect on the resistance to heat. As shown in the interaction plot (Supporting information), individuals with small to medium wing areas tend to be knocked-out later when displaying increased black pigmentation, whereas the effect is weakened for individuals with large wings. Wing area on its own did not significantly explain the time before heat

<figure><img src="image_7.png" /></figure>

Figure 2. Recorded activity times of *Morpho* species in French Guiana. We represent boxplots of the activity timings of the two main recognized clades of *Morpho*: (A) understory species, where individuals fly at 2–5 m above ground, and (B) canopy species, where individuals fly at 8–10 m above ground. Significant differences between groups (using a HSD Tukey test) are indicated with letters on the right of boxplots. We observe highly differentiated temporal niches between understory species, and less so in canopy species, although the mean timings are still significantly differentiated between species.
<figure><img src="image_8.png" /><figcaption>Figure 3. Temperature encountered during patrolling window and thermal traits measured within understory and canopy species. The patrolling windows of each (A) understory and (B) canopy species is shown in coloured bands, with a picture of an individual on top. The black line represents the average temperature at each moment of the day, with the 95% confidence interval in gray. The dotted line is extrapolated temperature data, as we did not sample temperatures this late in the day. Below are the recorded timings for heat knock-out, heat recovery and cold recovery respectively for each species. Dashed lines represent the mean thermal tolerance value between species.</figcaption></figure>

knock-out (β = 0.09 ± 0.12, Z = 0.77, p = 0.44). When adding phylogenetic correction to the model (Supporting information), only the effect of micro-habitat (β = -0.84 ± 0.42, Z = -2.01, p = 0.04) remained significant, while the previously detected effects of other variables were no longer significant, probably because of the strong links between

wing colour, size variations, and phylogenetic distances between species. Phylogenetic effects did explain a significant part of the variation in knock-out timings across species (partial R²<sub>lik</sub> = 10.42%, ΔlogLik = -4.29, p = 0.0034), closely followed by micro-habitat (partial R²<sub>lik</sub> = 7.02%, ΔlogLik = 2.84, p = 0.017).
For recovery times from heat knock-out, we did not detect any effect of the micro-habitat or the time of capture, or of any other controlled variables (Supporting information), with the exception of the interaction between black pigmentation and wing area (β=0.44 ± 0.17, Z=2.51, p=0.014). But this effect became non-significant when correcting for the level of phylogenetic relatedness between species (Supporting information), meaning that differences in recovery time are likely to be related to the evolutionary history of the species.

## Cold recovery

We detected a significant effect of the micro-habitat (β=-0.59 ± 1.40, Z=-4.18, p < 0.001), but not of the time of capture, on the recovery times from the cold knock-out (Supporting information): individuals living in the understory recovered from the cold shock more quickly than the canopy ones, consistent with an increased cold tolerance in understory species. The amount of black pigmentation (β=-0.19 ± 0.07, Z=-2.78, p=0.006) and wing area (β=-0.25 ± 0.07, Z=-3.67, p < 0.001) also had negative effects on recovery to cold shock, where individuals with either higher percentage of black or larger wing area recovered more quickly. However, the effects of black pigmentation and wing area are not independent, as their interaction was significant (β=0.32 ± 0.08, Z=3.86, p < 0.001), indicating that the impact of one depends on the level of the other. As shown in the interaction plot (Supporting information), individuals with small to medium wing areas tend to recover more quickly with increased black pigmentation, whereas those with large wings recover slightly more slowly when melanization is higher. Nonetheless, when controlling for the level of phylogenetic relatedness between species (Supporting information), only the effects of micro-habitat (β=-0.97 ± 0.44, Z=-2.20, p=0.028) and wing area (β=-0.40 ± 0.11, Z=-3.49, p < 0.001) remained significant.

Phylogeny also explained the major part of the variation in cold recovery timings across species (partial R²<sub>lik</sub>=15.1%, ΔlogLik=19.6, p < 0.001), followed by wing area (partial R²<sub>lik</sub>=6.8%, ΔlogLik=8.57, p < 0.001) and micro-habitat explaining a smaller part of the variation (partial R²<sub>lik</sub>=3.3%, ΔlogLik=4.0, p=0.004).

## Discussion

### Vertical stratification in sympatric species shaping the evolution of thermal traits

Our results suggest that the main factor shaping the variations in thermal traits (heat resistance and cold recovery) among individuals was the specialization in the canopy versus understory observed between sympatric species. This effect remained significant even when correcting for the phylogenetic distances between species, consistent with an effect of the divergent selection on thermal tolerance in the canopy versus understory microhabitat. In the Neotropical genus *Heliconius*, thermal tolerance has been shown to differ between populations living at different altitudes within 10

species, and common-garden experiments in *H. erato* revealed a contribution of both plastic and genetic components in the thermal traits measured (Montejo-Kovacevich et al. 2020). Behavioural assays testing the responses of a butterfly facing or dodging sunrise show the effect of solar-heating on the timing of first take-off (Meyer and Sisk 2001), highlighting the plastic response of individuals submitted to microclimatic variations. In our study, we cannot determine the extent to which the observed differences in thermal traits are due to phenotypic plasticity in response to local environmental conditions versus genetic divergence between species specialized in different micro-habitats. Environmental conditions encountered during individual life can lead to changes in thermal tolerance. Such plasticity in thermal tolerance traits has been shown in different ectotherm species, such as *Drosophila melanogaster* (Fallis et al. 2014) and the tropical butterfly *Bicyclus dorothea* (Dongmo et al. 2021). Thermal plasticity in ectotherms is thought to be due to changes in cell size (in *Drosophila*: Verspagen et al. 2020), or up-regulation of certain antioxidant markers (in Nymphalidae: Franke et al. 2019). However, the significant differences between the *Morpho* species studied here suggest that genetic divergence between species may also contribute to the divergence in thermal traits. Our results suggest that both phylogenetic history and selection exerted by difference between micro-habitats have significantly contributed to the evolution of thermal traits in the genus *Morpho*. Using the approach used by De Brito Freire et al. (2024), we measured phylobetadiversity and functional beta diversity metrics, and observed high dissimilarity value for both (0.82 and 0.99, respectively), with most of the dissimilarity coming from the phylogenetic and functional turnover components (0.78 and 0.99). This suggests that the environment is strongly selecting species from different evolutionary backgrounds, which also display divergent traits (Supporting information). Selection generated by different thermal environments could also promote the fixation of different genetic variants controlling thermal tolerance in species evolving in different habitats. In a study on 45 different species of Nymphalidae butterflies from Brazil, Silva et al. (2020) detected significant variation in temperature tolerance between species, with higher maximum temperature tolerated in species found in open as compared to shaded environments. The sensitivity to heat and cold conditions might thus partly depend on the genetic variation across species evolving in different temperature conditions. Many of our previously significant variables lost significance when accounting for the level of phylogenetic proximity between species compared in our analyses. Because canopy species all belong to the same clade, the effect of neutral divergence between the canopy versus understory species can generate significant differences in traits displayed by individuals between these two clades. Nevertheless, independently from their phylogenetic relatedness, individuals with larger wings recovered more quickly after the cold coma. As butterfly wings play a major role in heat absorption (Liao et al. 2019, Tsai et al. 2020), an increased wing area would correlate with faster recovery from cold conditions. In the butterfly communities of the tropical forests of Australia, Xing et al. (2016)
found that cooler environments were inhabited by larger and darker species, and showed that larger wing sizes displayed accelerated heat gain. Recent studies also show increased thermal buffering abilities by large butterflies (Ashe-Jepson et al. 2023), but lower thermal tolerance to high temperatures, contrary to what is observed in our study. Within the genus *Morpho*, species with the largest wings are found in the canopy (DeVries et al. 2010). The gliding flight behaviour in the middle of the day observed in these species might have promoted specific adaptations to intense solar exposition and heat. In our study, individuals from understory species were also found to be less resistant to heat shock and recovered more quickly from cold shock, as compared to individuals from canopy species. The canopy versus understory micro-habitats have been documented to differ in temperature conditions: significantly higher maximum temperatures were measured in the canopy versus understory habitats, in both high and lowland South American rainforests (Montejo-Kovacevich et al. 2020, Richter et al. 2023). This vertical partitioning in temperature conditions could promote divergent evolution of thermal traits in communities of butterflies inhabiting tropical rainforests. In turn, the colonization of the canopy micro-habitat in the *Morpho* genus might have been promoted by the evolution of thermal traits in some ancestral lineage. Our results do not allow us to determine whether the evolution of thermal traits is a cause or a consequence of the divergence between the canopy and understory clades. Nevertheless, the emergence of novel traits fitting with phylogenetic divergence into distinct clades is a commonly observed macro-evolutionary pattern. For instance, in Antarctic fishes, the evolution of antifreeze proteins within a single clade enabled the colonization of environments with cooler temperature conditions (Notothenioidei; Near et al. 2012). Altogether, our results point at a joint effect of selective and neutral processes acting on the divergent evolution of thermal traits in canopy versus understory species of the genus *Morpho*.

Field surveys in temperate areas have detected that butterfly activity patterns (e.g. in *Euphydryas aurinia*, *Parnassius apollo*, *Phengaris arion*) are affected by ambient temperature (Franzén et al. 2022), with a different range of thermal niches between species, leading to variation in seasonal and daily niches. In tropical rainforest, where the average daily temperature is much less variable throughout each year, especially when considering only the dry season (Lavado Casimiro et al. 2013, Checa et al. 2014), seasonal variations in temperature are much less marked. Therefore, specialization into vertical strata might be an important niche dimension in tropical rainforest, with specific selection acting on traits depending on the microclimatic differences between canopy and understory environments.

## Drivers of temporal niche diversification in sympatric species

In this study, we also showed that sympatric *Morpho* species exhibit pronounced temporal niche partitioning throughout the day, with little degree of time overlap between species sharing the same vertical strata within the forest. Ecological

niche diversification can arise from various factors, notably niche availability (Wellborn and Langerhans 2015) and divergent selection (Nosil and Sandoval 2008). Temporal partitioning seems to be common in many species (Taylor and Friesen 2017), especially when those species are closely related (Harris 1971, Shkolnik 1971). Temporal partitioning in particular appears to facilitate species coexistence by reducing competition between individuals from closely related species (Delaval et al. 2005), which are more likely to share resources or exhibit similar mating cues. Nevertheless, temporal segregation between species throughout the day could also stem from divergent selection exerted by temperature variation during the day, promoting different physiology between species. Here we thus tested the correlations between temporal niche and heat and cold thermal tolerances, but no significant effect of activity time on any of the thermal tolerances tested was detected. Nevertheless, the two *Morpho* species with the earliest activity times (*M. eugenia* and *M. marcus*) had poorer resistance to heat, suggesting potential variation in thermal tolerance between species with the largest differences in the temperature conditions encountered in their respective temporal niches.

Overall, the tolerance to extreme temperatures investigated here does not strongly differ between species occupying different temporal niches. The selection potentially exerted by temperature variation across different temporal niches might not be the main driver of temporal partitioning of the *Morpho* species in sympatry, begging the question of the factors shaping the temporal niche partitioning in this community. Experiments carried out in the field provide evidence of intensive heterospecific male-male competitive interactions in wild populations of *M. helenor* and *M. achilles* (Le Roy et al. 2021b). Controlled experiments in cages also highlighted an extensive courtship of wild males towards heterospecific female dummies from these two species (Ledamoisel et al. 2025). Reproductive interference between closely related *Morpho* species living in sympatry is thus likely very high in natural communities, especially between species sharing similar wing colour patterns. We also observed temporal shifts between individuals displaying different colour morphs within the species *M. telemachus*. Whether this activity timing polymorphism is due to genetic or environmental factors is currently unknown, but such genetic modulation of circadian timings has been shown to exist. In *Clunio marinus* midges, for instance, strain-specific genetic timing adaptations results in varying rhythms of adult emergence (Kaiser et al. 2016). Differences in activity timing might result in lower heterospecific interaction and thus favour the evolution and selection of daily temporal segregation in species (Kaiser et al. 2021). The lack of correlation between thermal traits and temporal niches in sympatric species observed in our study nevertheless likely discard the selection exerted by divergent temperature conditions as the main driver of the evolution of divergent temporal niches in this community. Our results are thus more consistent with reproductive interferences between species being a major factor promoting temporal divergence across sympatric species.
## Conclusions

Here, we document the significance of daily temporal niches and vertical stratification as important factors structuring natural communities of closely related species in tropical rain-forest. We found that sympatric species from the same butter-fly genus are strongly partitioned in differentiated temporal niches, but that this temporal niche divergence is poorly asso-ciated with thermal tolerance. We instead highlight that this thermal tolerance is rather shaped by the specialization in dif-ferent micro-habitats, probably because of strong divergent selection exerted by temperature conditions encountered in the canopy versus understory micro-habitats. The evolution of wing morphological traits between species living in dif-ferent micro-habitats might notably have contributed to the variations in thermal tolerance across species. Thus, while the specialization of different sympatric species into different spatial niches could have resulted in differentiated thermal traits, temporal niche specialization might rather result from ecological interactions such as heterospecific competition or reproductive interference.

## Speculations

In this article, we propose that the diversification of ther-mal traits in the *Morpho* genus is primarily linked to micro-habitat specialization. In contrast, daily temporal activity patterns do not appear to be associated with thermal trait variation. We suggest that interspecific differences in daily activity timing might stem from ecological interactions between closely related species living in sympatry and may even fuel divergence between species in sympatry. Such temporal partitioning could arise from factors including competitive interactions between individuals due to male territoriality (Grether et al. 2009), daily variation in resource availability (Matsumoto et al. 2015), or differences in preda-tion pressure across the day (Bosiger and McCormick 2014). Temporal isolation could also act as a reinforcement force in the speciation process, reducing encounters between closely related species, limiting interbreeding and reinforcing reproduc-tive isolation in sympatry. One notable observation of our dataset is the substantially different activity timings of the visually similar sister species *M. achilles* and *M. helenor* (Llaurens et al. 2021, Ledamoisel et al. 2025). However, whether this is due to heterospecific interactions or linked to their evolutionary history is unknown. *Morpho helenor* occurs without its sister species in some part of Central and South America (Blandin and Purser 2013), begging the question of whether its activity timing remains identical in these allopatric areas.

## Alternative viewpoints

Our study has explored the relationship between macro-evolutionary shifts from the understory to the canopy and

the evolution of novel thermal traits. Determining whether one factor drives the other remains unclear and cannot be resolved based on our results alone. However, one of the reviewers prompted us to consider this issue more deeply: specifically, whether canopy species occupy the canopy due to an ancestral key innovation (such as higher thermal tolerance), or whether the canopy environment selected individuals possessing these thermal traits. This question is not trivial, especially in the case of the *Morpho* genus, where the canopy species all belong to the same clade within the genus (Debat et al. 2025), limiting our capacity to estimate the precise relative contribution of 1) neutral processes involved in species and clade divergence, and 2) selective processes linked to adaptation to divergent environments on trait variation among species. *Morpho* species observed in the canopy versus understory differ in multiple traits, including wing shape (Chazot et al. 2016) and flight behaviour (Le Roy et al. 2021a), consistent with an important effect of specialization in divergent environments. Understanding whether ecological opportunity or environmental filtering shaped the diversification of *Morpho* species could provide broader insights on the evolutionary dynamics of environmental transitions and trait shifts.

**Acknowledgements** – The authors would like to thank the LEEISA (Laboratoire Ecologie, Evolution, Interactions des Systèmes Amazoniens, UAR 3456) for granting us access to their facilities, which were invaluable for the thermal tolerance experiments, as well as the help of Melanie McLure, Grégory Genta-Jouve and Mathieu Chouteau. We are grateful for the help of Vincent Debat, Joséphine Ledamoisel and Raphaël Dupillier during our sampling effort, and also thank Marianne Elias and Théo Chavanis for the helpful insights on butterfly thermoregulation. We finally also thank Agathe Puissant for the help in statistical modelling.

**Funding** – This study was funded by the European Union (ERC-2022-COG – OUTOFTHEBLUE – 101088089). Views and opinions expressed are, however, those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.

## Author contributions

**Titouan Bouinier**: Conceptualization (equal); Data cura-tion (lead); Formal analysis (lead); Investigation (lead); Methodology (equal); Resources (equal); Visualization (lead); Writing – original draft (lead); Writing – review and edit-ing (equal). **Charline Smadi**: Conceptualization (equal); Supervision (equal); Writing – review and editing (equal). **Violaine Llaurens**: Conceptualization (equal); Funding acquisition (lead); Methodology (equal); Project administra-tion (lead); Resources (equal); Supervision (equal); Writing – review and editing (equal).

## Data availability statement

Data are available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.pk0p2nh0q (Bouinier et al. 2025).
**Supporting information**

The Supporting information associated with this article is
available with the online version.

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