DNA metabarcoding provides a rapid and effective identification tool of macroinvertebrate species. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries, which, due to incompleteness, are currently a recognized limitation for metabarcoding applications. In this study, we assembled a comprehensive reference library of DNA barcodes for Atlantic Iberia marine macroinvertebrate species, assessed gaps in species coverage and examined data ambiguities. Initially, an Iberian species checklist for the three dominant groups of marine macroinvertebrates was compiled, comprising 2827 species (926 Annelida, 638 Crustacea and 1263 Mollusca). A total of 18162 DNA sequences of the cytochrome c oxidase subunit I barcode region (COI-5P) matching the species checklist were compiled in a BOLD dataset, where taxonomic discordances were evaluated and cases of deep intraspecific divergence flagged. Gap-analysis showed that 63% of the Iberian macroinvertebrate species still lack a DNA barcode. Coverage gaps varied considerably across taxonomic groups with Mollusca displaying the highest sequence representation in the dataset (427 species, 49% of the total number of sequences), and Crustacea the highest species coverage with 338 species barcoded (53% of the checklist). In contrast, Polychaeta displayed the lower levels of completion (288 species, 16% of the total number of sequences). In total, 1545 Barcode Index Numbers (BINs) were assigned to 1053 barcoded species, of which 66% were taxonomically concordant, 26% displayed multiple BINs and 8% were discordant. Overall, results show that there is still a large portion of marine invertebrate taxa in this region of Europe pending barcode coverage, even considering only the dominant groups. However, the most notable finding was the relevant proportion of species flagged for significant intraspecific divergence and possible hidden diversity. The annotated reference library and gap-analysis here provided can therefore contribute to prioritize marine macroinvertebrate taxa for future research efforts and barcode coverage.

DNA元条形码为大型无脊椎动物物种提供了一种快速有效的识别工具。物种级别分配的准确性以及随后的分类学范围依赖于全面的DNA条形码参考文库，由于不完整，目前它们已成为元条形码应用的公认限制。在这项研究中，我们为大西洋伊比利亚海洋无脊椎动物物种组装了一个完整的DNA条码参考库，评估了物种覆盖范围的差距并检查了数据的歧义性。最初，针对三个主要海洋无脊椎动物种群的伊比利亚物种清单进行了编制，其中包括2827种（926 nel科，638甲壳纲和1263软体动物）。匹配物种清单的细胞色素C氧化酶亚基I条码区域（COI-5P）的总共18162条DNA序列被编入BOLD数据集中，在其中评估了分类学不一致并标记了种内差异严重的情况。差距分析显示，伊比利亚大型无脊椎动物物种中仍有63％缺少DNA条码。生物分类的覆盖范围差异很大，其中软体动物在数据集中的序列表示最高（427种，占序列总数的49％），而甲壳纲的物种最高，覆盖了338种带条形码的物种（清单的53％）。相反，Polychaeta的完成度较低（288种，占序列总数的16％）。总共为1553个条形码物种分配了1545个条形码索引号（BIN），其中66％在分类学上是一致的，26％显示多个BIN，而8％是不一致的。总体而言，结果表明，即使仅考虑优势群体，在欧洲这一地区仍有很大一部分海洋无脊椎动物分类群有待条形码覆盖。但是，最显着的发现是标记为明显的种内差异和可能的隐藏多样性的物种的相关比例。因此，此处提供的带注释的参考库和缺口分析可有助于区分海洋无脊椎动物大类的优先级，以用于未来的研究工作和条形码覆盖范围。但是，最显着的发现是标记为明显的种内差异和可能的隐藏多样性的物种的相关比例。因此，此处提供的带注释的参考库和缺口分析可有助于区分海洋无脊椎动物大类的优先级，以用于未来的研究工作和条形码覆盖范围。然而，最显着的发现是标记为明显的种内差异和可能隐藏的多样性的物种的相关比例。因此，此处提供的带注释的参考库和缺口分析可有助于区分海洋无脊椎动物类群的优先级，以用于将来的研究工作和条形码覆盖范围。