Supplementary material from "High evolutionary constraints limited adaptive responses to past climate changes in toad skulls"

Interactions among traits that build a complex structure may be represented as genetic covariation and correlation. Genetic correlations may act as constraints, deflecting the evolutionary response from the direction of natural selection. We investigated the relative importance of drift, selection and constraints in driving skull divergence in a group of related toad species. These species distributional range encompasses very distinct habitats with important climatic differences and the species are primarily distinguished by differences in their skulls. Some parts of the toad skull, such as the snout, may have functional relevance in reproductive ecology, detecting water cues. Thus, we hypothesized that the species skull divergence was driven by natural selection associated with climatic variation. However, given that all species present high correlations among skull traits, our second prediction was of high constraints deflecting the response to selection. We first extracted the main morphological direction that is expected to be subjected to selection by using within and between-species covariance matrices. We then used evolutionary regressions to investigate whether divergence along this direction is explained by climatic variation between-species. We also used the quantitative genetics models to test for a role of random drift versus natural selection in skull divergence and to reconstruct selection gradients along species phylogeny. Climatic variables explained high proportions of between-species variation in the most selected axis. However, most evolutionary responses were not in the direction of selection, but aligned with the direction of allometric size, the dimension of highest phenotypic variance in the ancestral population. We conclude that the toad species have responded to selection related to climate in their skulls, yet high evolutionary constraints dominated species divergence and may limit species responses to future climate change.