Data from: Effects of sample size and full sibs on genetic diversity characterization: a case study of three syntopic Iberian pond-breeding amphibians

Accurate characterization of genetic diversity is essential for understanding population demography, predicting future trends and implementing efficient conservation policies. For that purpose, molecular markers are routinely developed for nonmodel species, but key questions regarding sampling design, such as calculation of minimum sample sizes or the effect of relatives in the sample, are often neglected. We used accumulation curves and sibship analyses to explore how these 2 factors affect marker performance in the characterization of genetic diversity. We illustrate this approach with the analysis of an empirical dataset including newly optimized microsatellite sets for 3 Iberian amphibian species: Hyla molleri, Bufo calamita, and Pelophylax perezi. We studied 17–21 populations per species (total n = 547, 652, and 516 individuals, respectively), including a reference locality in which the effect of sample size was explored using larger samples (77–96 individuals). As expected, FIS and tests for Hardy–Weinberg equilibrium and linkage disequilibrium were affected by the presence of full sibs, and most initially inferred disequilibria were no longer statistically significant when full siblings were removed from the sample. We estimated that to obtain reliable estimates, the minimum sample size (potentially including full sibs) was close to 20 for expected heterozygosity, and between 50 and 80 for allelic richness. Our pilot study based on a reference population provided a rigorous assessment of marker properties and the effects of sample size and presence of full sibs in the sample. These examples illustrate the advantages of this approach to produce robust and reliable results for downstream analyses.

准确表征遗传多样性（genetic diversity）对于理解种群动态（population demography）、预测种群未来趋势以及制定高效的保护政策至关重要。为此，研究人员通常会为非模式物种（nonmodel species）开发分子标记（molecular markers），但有关抽样设计（sampling design）的关键问题——例如最小样本量（minimum sample sizes）的计算或样本中亲缘个体的影响——却常被忽视。我们采用累积曲线（accumulation curves）和同胞分析（sibship analyses）探究了这两个因素如何影响遗传多样性表征中的标记性能。随后我们通过分析一组实证数据集（empirical dataset）验证了该方法，该数据集包含针对3种伊比利亚两栖动物新优化的微卫星标记集（microsatellite sets）：Hyla molleri、Bufo calamita及Pelophylax perezi。我们对每个物种的17~21个种群开展了研究（总个体数分别为547、652和516），其中包含一个参考采样点，我们通过设置77~96个个体的更大样本量，探究了样本量的影响。正如预期，固定指数FIS（Fixation Index, FIS）、哈迪-温伯格平衡（Hardy–Weinberg equilibrium）检验以及连锁不平衡（linkage disequilibrium）检验均受到全同胞（full sibs）存在的影响；当从样本中移除全同胞后，绝大多数最初检测到的不平衡不再具有统计学显著性。我们估算得出，为获得可靠的估计结果，针对期望杂合度（expected heterozygosity）的最小样本量（可包含全同胞）约为20，而针对等位基因丰富度（allelic richness）的最小样本量则介于50~80之间。我们基于参考种群的预实验对标记特性、样本量效应以及样本中全同胞的影响进行了严谨评估。上述案例阐明了该方法的优势，可为后续分析提供稳健且可靠的研究结果。