Several recent studies have shown that molecular evolutionary rates are correlated with mean temperature in many organisms, such as in mammals , amphibians , and flowering plants [12, 14], providing empirical support for the metabolic theory . These previous studies were in line with the hypothesis that molecular evolutionary rates, through their potential correlation with speciation rates , were responsible for the construction of the latitudinal diversity gradient. In contrast, we show here that, in Squamata, higher molecular evolutionary rates are not associated with higher temperature or lower absolute latitude.
Previously, the few studies that have shown that molecular evolutionary rates and temperature - or latitude - were not associated, were focusing on endothermic taxa (such as birds ). To our knowledge, our study is the first to demonstrate an absence of relationship in ectothermic taxa. According to the predictions of the metabolic theory of ecology there should be a positive relationship between molecular evolutionary rate and temperature (or latitude) in ectothermic species, because their body temperature depends on external temperature . Given the size of the data used here, it is very unlikely that we lack statistical power to detect any significant relationship. It is also unlikely that our conclusions could be affected by methodological artifacts, such as the under-estimation of multiple substitutions in long branches. This artifact may appear when the two members of the species pair belong to clades with large differences in species richness. Because short branches, which are more likely in species-rich clades, are less affected by saturation due to multiple substitutions, the sum of their branch lengths will more accurately represent the true amount of molecular evolution . In contrast, species-poor clades will on average have longer branches, which could be underestimated by phylogenetic reconstruction methods and thus lower our estimates of evolutionary rates. As richness in squamata peaked at high temperature (Fig. 1a), the presence of this node density effect would lead to substantially higher evolutionary rates at high temperatures. As we observe no significant relationship between temperature and evolutionary rates, this possible bias is conservative. Overall, we have greater confidence to affirm that our results cast doubt on the universality of the metabolic theory of ecology and the evolutionary speed hypothesis, as a major explanation to the patterns of species diversity in vertebrates.
There are several other potential explanations for an absence of relationship between molecular evolutionary rates and temperature (or latitude). First, the molecular evolutionary rates of squamates may be particularly high in mid-latitude regions, where richness is higher (Fig. 1). Indeed, high speciation rates – potentially related to increased molecular evolutionary rates, but not necessarily – may be found in low-rainfall regions, e.g. deserts, such as at the boarder of the Hadley cell – between 20 and 30° of latitude. However there are two reasons why we think that this hypothesis is unlikely. First, we did not find any study supporting this hypothesis in the literature and, second, applying a quadratic effect on absolute latitude did not improve the relationship between absolute latitude and molecular evolutionary rates (results not shown).
A second hypothesis for an absence of relationship between molecular evolutionary rates and temperature (or latitude) is that Squamata have a particular biogeographical history that masks the relationship between molecular evolutionary rates and temperature. If dispersal rates have been high from the temperate regions to the tropics, species currently found in tropical areas may have recently colonized the tropics and may have experienced an increase of molecular evolutionary rates only recently. As we measured a mean molecular evolutionary rate from the most recent common ancestor of the sister pairs to the tips of the phylogenetic tree, we might not detect this recent increase of molecular evolutionary rates in tropical lineages (this bias has already explained in ). This hypothesis is consistent with the recent findings of Pyron , suggesting that the diversification of Squamata have been high in temperate regions and that expansions of lineages from the temperate regions toward the tropics have been frequent. These events of dispersal may thus have blurred the relationship between evolutionary rates and temperature (or latitude). This also suggest that present day distributions alone might not be appropriate for testing the validity of the metabolic theory of ecology. This argument is nonetheless unlikely because it must concern a very wide number of lineages.
A third hypothesis is that life history traits (body size or generation time) may be the main drivers of substitution rates of squamata. Indeed, species with short generation times are expected to feature a higher number of replication cycles and mutations per unit times. Such relationships between life history traits and rates of molecular evolution were found in reptiles  and could prevent detection of a temperature/latitude effect.
To our knowledge, our study is the first to show that global diversity of squamata is peaking at mid-latitude. However, it is very difficult to know if this geographical pattern of richness is biased with latitude. It is possible that the number of species in humid tropical areas is underestimated, and this may slightly bias the distribution of species at low latitude. However, the literature provides evidence that squamata have very different distributions than birds and mammals with peaks of diversity in arid regions rather than in tropical wet forest [29, 30]. We have also no reason to think that desert regions (where we detect a high diversity of squamata) were much more sampled than tropical humid areas because both regions have low human population density (and likely low sampling probability). We thus have confidence on our results, and we suggest that further studies will refine the description of the diversity pattern of squamata.