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Background  While our understanding of the genetic basis of convergent evolution has improved there are still many uncertainties. Here we investigate the repeated evolution of dark colouration (melanism) in eastern fox squirrels ( Sciurus niger;  hereafter “fox squirrels”) and eastern gray squirrels ( S. carolinensis;  hereafter “gray squirrels”).    Results  We show that convergent evolution of melanism has arisen by independent genetic mechanisms in two populations of the fox squirrel. In a western population, melanism is associated with a 24 bp deletion in the melanocortin-1-receptor gene ( MC1RΔ24  allele), whereas in a south-eastern population, melanism is associated with a point substitution in the agouti signalling protein gene causing a Gly121Cys mutation .  The  MC1R∆24  allele is also associated with melanism in gray squirrels, and, remarkably, all the  MC1R∆24  haplotypes are identical in the two species. Evolutionary analyses show that the  MC1R∆24  haplotype is more closely related to other  MC1R  haplotypes in the fox squirrel than in the gray squirrel. Modelling supports the possibility of gene flow between the two species.    Conclusions  The presence of the  MC1R∆24  allele and melanism in gray squirrels is likely due to introgression from fox squirrels, although we cannot completely rule out alternative hypotheses including introgression from gray squirrels to fox squirrels, or an ancestral polymorphism. Convergent melanism in these two species of tree squirrels has evolved by at least two and probably three different evolutionary routes.    Electronic supplementary material  The online version of this article (10.1186/s12862-019-1471-7) contains supplementary material, which is available to authorized users.

Multiple origins of melanism in two species of North American tree squirrel (Sciurus)