Data from: The effect of geographical scale of sampling on DNA barcoding

Eight years after DNA barcoding was formally proposed on a large scale, CO1 sequences are rapidly accumulating from around the world. While studies to date have mostly targeted local or regional species assemblages, the recent launch of the global iBOL project (International Barcode of Life), highlights the need to understand the effects of geographical scale on Barcoding’s goals. Sampling has been central in the debate on DNA Barcoding, but the effect of the geographical scale of sampling has not yet been thoroughly and explicitly tested with empirical data. Here we present a CO1 dataset of aquatic predaceous diving beetles of the tribe Agabini, sampled throughout Europe, and use it to investigate how the geographic scale of sampling affects 1) the estimated intraspecific variation of species, 2) the genetic distance to the most closely related heterospecific, 3) the ratio of intraspecific and interspecific variation, 4) the frequency of taxonomically recognised species found to be monophyletic, and 5) query identification performance based on six different species assignment methods. Intraspecific variation was significantly correlated with the geographical scale of sampling (R-square=0.7), and more than half of the species with 10 or more sampled individuals (N=29) showed higher intraspecific variation than 1% sequence divergence. In contrast the distance to the closest heterospecific showed a significant decrease with increasing geographical scale of sampling. The average genetic distance dropped from >7% for samples within 1km, to <3.5% for samples up to >6000 km apart. Over a third of the species were not monophyletic, and the proportion increased through locally, nationally, regionally and continentally restricted subsets of the data. The success of identifying queries decreased with increasing spatial scale of sampling; liberal methods declined from 100% to around 90% whereas strict methods dropped to below 50% at continental scales. The proportion of query identifications considered uncertain (more than one species <1% distance from query) escalated from zero at local, to 50% at continental scale. Finally, by resampling the most widely sampled species we show that even if samples are collected to maximise the geographical coverage, up to 70 individuals are required to sample 95% of intraspecific variation. The results show that the geographical scale of sampling has a critical impact on the global application of DNA barcoding. Scale-effects result from the relative importance of different processes determining the composition of regional species assemblages (dispersal and ecological assembly) and global clades (demography, speciation and extinction). The incorporation of geographical information, where available, will be required to obtain identification rates at global scales equivalent to those in regional barcoding studies. Our result hence provides an impetus for both smarter barcoding tools and sprouting national barcoding initiatives – smaller geographical scales deliver higher accuracy.在DNA条形码（DNA barcoding）正式大规模提出八年后，全球范围内的CO1序列正快速积累。尽管迄今的相关研究大多聚焦于局域或区域物种组合，近期启动的全球iBOL（International Barcode of Life，生命条形码国际计划）项目，凸显了探究地理尺度对条形码研究目标的影响的必要性。采样设计始终是DNA条形码讨论的核心议题，但采样地理尺度的效应尚未通过实证数据得到全面且明确的检验。本研究构建了一套覆盖全欧洲的水生肉食性龙虱族（Agabini）CO1数据集，并借此展开分析，以探究采样地理尺度对以下五项指标的影响：1）物种的种内变异估计值；2）与最密切相关的异源物种的遗传距离；3）种内与种间变异的比值；4）被认定为单系群的分类学公认物种占比；5）基于6种不同物种归属鉴定方法的查询序列鉴定性能。研究结果显示，种内变异与采样地理尺度呈显著正相关（决定系数R²=0.7）；在采样个体数≥10的29个物种中，超过半数的种内变异超过了1%的序列分歧阈值。与之形成鲜明对比的是，与最近异源物种的遗传距离随采样地理尺度增大呈显著下降趋势：1公里范围内的样本平均遗传距离大于7%，而间距超过6000公里的样本平均遗传距离则降至3.5%以下。超过三分之一的物种并非单系群，且该比例随数据子集的地理限制范围从局域、国家、区域到大陆尺度逐步升高。查询序列的鉴定成功率随采样空间尺度增大而下降：宽松型鉴定方法的成功率从100%降至约90%，而严格型方法在大陆尺度下的成功率甚至跌破50%。被认定为不确定的查询鉴定结果（即存在多个物种与查询序列的遗传距离小于1%）的占比，从局域尺度下的0%攀升至大陆尺度下的50%。此外，通过对采样最广泛的物种进行重采样分析可知，即便通过采样最大化地理覆盖范围，仍需要至少70个个体才能覆盖该物种95%的种内变异。本研究结果表明，采样地理尺度对DNA条形码的全球应用具有关键性影响。尺度效应源于决定区域物种组合（扩散与生态组装）及全球支系（种群动态、物种形成与灭绝）组成的不同过程的相对重要性差异。若要在全球尺度下获得与区域条形码研究相当的鉴定准确率，需纳入可用的地理信息。因此，本研究结果既推动了更智能的条形码分析工具的开发，也为新兴的国家级条形码计划提供了理论支撑——更小的地理尺度能够带来更高的鉴定准确性。