Data from: A critical evaluation of how ancient DNA bulk bone metabarcoding complements traditional morphological analysis of fossil assemblages

When pooled for extraction as a bulk sample, the DNA within morphologically unidentifiable fossil bones can, using next-generation sequencing, yield valuable taxonomic data. This method has been proposed as a means to rapidly and cost-effectively assess general ancient DNA preservation at a site, and to investigate temporal and spatial changes in biodiversity; however, several caveats have yet to be considered. We critically evaluated the bulk bone metabarcoding (BBM) method in terms of its: (i) repeatability, by quantifying sampling and technical variance through a nested experimental design containing sub-samples and replicates at several stages; (ii) accuracy, by comparing morphological and molecular family-level identifications; and (iii) overall utility, by applying the approach to two independent Holocene fossil deposits, Bat Cave (Kangaroo Island, Australia) and Finsch's Folly (Canterbury, New Zealand). For both sites, bone and bone powder sub-sampling were found to contribute significantly to variance in molecularly identified family assemblage, while the contribution of library preparation and sequencing was almost negligible. Nevertheless, total variance was small. Sampling over 80% fewer bones than was required to morphologically identify the taxonomic assemblages, we found that the families identified molecularly are a subset of the families identified morphologically and, for the most part, represent the most abundant families in the fossil record. In addition, we detected a range of extinct, extant and endangered taxa, including some that are rare in the fossil record. Given the relatively low sampling effort of the BBM approach compared with morphological approaches, these results suggest that BBM is largely consistent, accurate, sensitive, and therefore widely applicable. Furthermore, we assessed the overall benefits and caveats of the method, and suggest a workflow for palaeontologists, archaeologists, and geneticists that will help mitigate these caveats. Our results show that DNA analysis of bulk bone samples can be a universally useful tool for studying past biodiversity, when integrated with existing morphology-based approaches. Despite several limitations that remain, the BBM method offers a cost-effective and efficient way of studying fossil assemblages, offering complementary insights into evolution, extinction, and conservation.当将形态学上无法鉴定的化石骨骼中的DNA混合为批量样本进行提取时，借助下一代测序（next-generation sequencing）技术，可获取具有重要价值的分类学数据。该方法已被提出，可用于快速且经济高效地评估某一遗址中古DNA（ancient DNA）的整体保存状态，并探究生物多样性的时空变化；然而，该方法仍存在若干尚未被充分考量的限制条件。 我们从三个维度对批量骨骼元条形码测序（bulk bone metabarcoding, BBM）法开展了批判性评估：(1) 可重复性：通过嵌套实验设计量化采样与技术差异，该设计在多个阶段设置了子样本与重复样本；(2) 准确性：通过比对形态学与分子生物学的科级鉴定结果；(3) 整体实用性：将该方法应用于两处独立的全新世（Holocene）化石沉积遗址——澳大利亚袋鼠岛蝙蝠洞（Bat Cave）与新西兰坎特伯雷地区的芬斯福莱遗址（Finsch's Folly）。 在两处遗址中，骨骼与骨粉子样本的采集均对分子鉴定得到的科类群组合的差异产生了显著影响，而文库构建与测序环节的贡献则几乎可以忽略不计。尽管如此，总差异幅度仍较小。相较于通过形态学鉴定类群组合所需的骨骼样本量，本研究采集的骨骼样本量仅为其不足20%（即减少了80%以上）；我们发现，分子鉴定得到的科类群是形态学鉴定所得科类群的一个子集，且在绝大多数情况下对应化石记录中丰度最高的科类群。 此外，本研究还检测到了一系列已灭绝、现存及濒危的生物类群，其中不乏化石记录中较为罕见的类群。相较于形态学方法，批量骨骼元条形码测序法的采样工作量相对较低，上述结果表明该方法整体上具备良好的一致性、准确性与灵敏度，因此具备广泛的应用潜力。 此外，我们还评估了该方法的整体优势与限制条件，并为古生物学家、考古学家与遗传学家提出了一套可有效缓解这些局限的标准化工作流程。本研究结果表明，若将批量骨骼样本的DNA分析与现有的形态学研究方法相结合，便可成为研究古生物多样性的通用有效工具。尽管该方法仍存在若干待改进的局限，但批量骨骼元条形码测序法仍为化石组合的研究提供了一种经济高效的途径，可为演化、灭绝与保护生物学研究提供互补性的认知视角。