The complex effects of demographic history on the estimation of substitution rate: concatenated gene analysis results in no more than twofold overestimation

Our recent estimation of the divergence time and isolation of Death Valley pupfishes, including the iconic Devil's Hole pupfish (DHP), rewrote widespread assumptions about this group. These species were previously assumed to be relic populations isolated over millions of years; our genomic analyses indicated recent colonization of Devil's Hole within the past 105–830 years and frequent gene flow among Death Valley populations [1]. These results understandably attracted substantial attention given the iconic battle for conservation and intense management of DHP [2]; nonetheless, a young age for this species should not diminish its conservation value. Indeed, we argue that the unique natural history of this species makes it a prime candidate for exhibiting one of the fastest mutation rates observed in any vertebrate [3].Saglam et al . [4] argue that we overestimated the substitution rate, the rate at which mutations occur and fix between lineages over time, in pupfishes due to our analysis of a concatenated dataset of RADseq loci and therefore underestimated the age of DHP. Specifically, Saglam et al . argue that a multi-species coalescent analysis would remove our bias and provide strikingly different results. Here, we test this assumption by reanalysing our original RADseq dataset under a multi-species coalescent model and comparing the estimated substitution rates to an analysis of concatenated RADseq loci. It is well known that divergence times estimated from concatenated, multi-gene analyses can overestimate species divergence times due to older gene-tree divergence compared with the true species-tree divergence time, a fact that we both cited [5] and demonstrated in our study: our concatenated gene analysis estimated DHP divergence at 2500–6500 years (fig. 2 of [1]) versus our coalescent analysis using dadi [6 …