Response to Slate and Pemberton

population mean heterozygosity (i.e. 12 data points). Both analyses were statistically significant, indicating that populations with low heterozygosity have relatively abnormal sperm. Two alternatives to inbreeding depression are consistent with this result: a third, unknown factor causes some populations to have low heterozygosity and relatively abnormal sperm; for instance, environmental heterogeneity or genetic drift could be such factors. Alternatively, populations with relatively abnormal sperm could show high rates of infertility and decline in size, thereby causing heterozygosity to decline; i.e., sperm abnormality affects heterozygosity rather than vice versa. Convincing evidence of inbreeding depression requires a significant relationship between heterozygosity and sperm abnormalities across individuals within a population. Most of Gage et al.'s [1] population samples were too small to detect inbreeding depression. There is no relationship between heterozygosity and sperm quality within the largest population (n = 29), although a significant relationship was detected in another population (n = 13). Furthermore, heterozygosity of an individual can be a poor indicator of inbreeding coefficient [4–6]. A recently proposed method [4] was used by Gage et al. [1] to test whether marker heterozygosity reflects the inbreeding coefficient, but it was applied simultaneously to all populations. The relevant question of whether markers can be used as a proxy for the inbreeding coefficient within a population is not addressed. There are several methods to test for inbreeding depression when discrete populations are sampled. If sample sizes permit — Gage et al.'s do not — analyses could be conducted within each population separately or, alternatively, 'population' could be fitted as a categorical term in a model that includes all individuals. There is also a large literature on more refined methods to control for population structure in genetic association studies (reviewed in [7]). We have raised the issue of population structure because we have noticed a new trend in heterozygosity–fitness correlation studies, namely a failure to deal with population structure, e.g. [8]. In general, evolutionary geneticists should consider population stratification as the most likely cause of associations between genetic markers and a focal trait and try to exclude this explanation before testing other explanations. (2006). Reduced heterozygosity depresses sperm quality in wild rabbits, Oryctolagus cuniculus. (1999). From population structure to individual behaviour: genetic analysis of social structure in the European wild rabbit (Oryctolagus cuniculus). Biol. J. (2004). Understanding the relationship between the inbreeding coefficient and multilocus heterozygosity: theoretical expectations and empirical data. In their correspondence, Slate …