Evaluating the plausibility of this selective scenario is

Evaluating the plausibility of this selective scenario is challenging because of the uncertainties concerning the biological role of calpain-10 and the significance of its variants with regard to gene function and disease susceptibility. However, from a strictly evolutionary standpoint, our findings could be consistent with the “carnivore connection” hypothesis (Miller and Colagiuri 1994; Colagiuri and Brand Miller 2002), which postulates a critical role for the quantity of dietary protein and carbohydrate in the Protease Inhibitor Cocktail (100X in DMSO, EDTA plus) mg of insulin resistance. More specifically, it was proposed that insulin resistance offered survival and reproductive advantages during the Ice Ages, which started ∼2.5 million years ago (mya) and dominated most of human evolution, when a shift to a high-protein, low-carbohydrate diet occurred as a consequence of climatic changes. Notably, when we apply a molecular clock calculation (eq. 3 in Thomson et al. [2000]) to estimate the average time since the most recent common ancestor of the population samples for the 1-kb segment centered on the peak of polymorphism, we obtain age estimates of 2.1–2.8 mya and 2.4–3.1 mya, depending on whether the chimpanzee or the orangutan sequences were used as an outgroup species (we assumed a divergence time of 6 mya and 14 mya for human and chimpanzee and for human and orangutan, respectively). This suggests that, if indeed a balanced polymorphism exists in this location, its age would be consistent with the onset of new selective pressures associated with the Ice Ages. Given the likely role of calpains in insulin secretion and action (Sreenan et al. 2001), these results offer a new working hypothesis for the evolution of intron 13 variation and its role in human metabolic adaptations. Nonuniform rates of recombination or mutation are also possible violations of the assumptions of the standard neutral model. More specifically, a hotspot of both recombination and mutation, with all variation being selectively neutral, may explain the data. However, the absence of a peak of interspecies sequence divergence corresponding to the peak of polymorphism implies that, if a mutational hotspot is present, this must have arisen after the divergence of human and chimpanzee. The coincidence of high π and ρH01 in intron 13 may reflect a relationship between mutation and recombination. Evidence that mutation and recombination may be related processes on a genomewide scale comes from the observation that regions of high recombination in humans tend to have slightly higher levels of divergence between human and mouse (Lercher and Hurst 2002), human and baboon, and human and macaque (Hellmann et al. 2003). On a finer scale, the recombinational hotspot in the human TAP2 gene, which was confirmed by sperm typing (Jeffreys et al. 2000), is associated with an excess of polymorphism relative to divergence (Jeffreys et al. 2000) and rapid decay of LD (Cullen et al. 1995; Jeffreys et al. 2001), just like intron 13 of CAPN10. Interestingly, a recent analysis of LD in the orthologous region of TAP2 in chimpanzees indicates that the recombination hotspot is absent in this species (Ptak et al. 2004). Thus, the rate of both mutation and recombination changed dramatically at the TAP2 gene since the divergence of human and chimpanzee. However, the β-globin recombination hotspot has no corresponding peak of polymorphism or divergence and there is no evidence that the hotspot exists in the orthologous region of macaques and, perhaps, chimpanzees (Wall et al. 2003). Importantly, a recent large-scale study of sequence variation in two population samples detected numerous regions of rapid LD decay, consistent with the presence of recombinational hotspots; however, no association between levels of polymorphism and LD decay was detected in these regions (Crawford et al. ). Thus, an increase in polymorphism levels is unlikely to be a universal feature of human recombination hotspots. Sperm typing and LD analysis in chimpanzees are currently being performed to investigate this alternative neutral explanation for the observations at intron 13 of CAPN10.