Reducing bias in population and landscape genetic inferences: The effects of sampling related individuals and multiple life stages

In population or landscape genetics studies, an unbiased sampling scheme is essential for generating accurate results, but logistics may lead to deviations from the sample design. Such deviations may come in the form of sampling multiple life stages. Presently, it is largely unknown what effect sampling different life stages can have on population or landscape genetic inference, or how mixing life stages can affect the parameters being measured. In this study, we sampled embryos, larvae, and adult Ambystoma maculatum from five ponds in Missouri, and analyzed them at 15 microsatellite loci. We calculated allelic richness, heterozygosity and effective population sizes for each life stage at each pond and tested for genetic differentiation (F_ST and D_C) and isolation-by-distance (IBD) among ponds. We tested for differences in each of these measures between life stages, and in a pooled population of all life stages. All calculations were done with and without sibling pairs to assess the effect of sibling removal. No statistically significant differences were found among ponds or life stages for any of the population genetic measures, but patterns of IBD differed among life stages. There was significant IBD when using adult samples, but tests using embryos, larvae, or a combination of the three life stages were not significant. Further, we found that increasing the ratio of larval or embryo samples in the analysis of genetic distance weakened the IBD relationship, and when using D_C, the IBD was no longer significant when larvae and embryos exceeded 60% of the population sample. Our findings suggest that it may be possible to mix life stages to reach target sample size quotas, but researchers should nonetheless proceed with caution depending upon the goals and objectives of the study.