Creedy Thomas J, Ng Wui Shen, Vogler Alfried P
Department of Life Sciences Natural History Museum London UK.
Department of Life Sciences Imperial College London Silwood Park Campus Ascot UK.
Ecol Evol. 2019 Mar 4;9(6):3105-3116. doi: 10.1002/ece3.4839. eCollection 2019 Mar.
Metabarcoding of arthropod communities can be used for assessing species diversity in tropical forests but the methodology requires validation for accurate and repeatable species occurrences in complex mixtures. This study investigates how the composition of ecological samples affects the accuracy of species recovery.Starting with field-collected bulk samples from the tropical canopy, the recovery of specimens was tested for subsets of different body sizes and major taxa, by assembling these subsets into increasingly complex composite pools. After metabarcoding, we track whether richness, diversity, and most importantly composition of any size class or taxonomic subset are affected by the presence of other subsets in the mixture.Operational taxonomic units (OTUs) greatly exceeded the number of morphospecies in most taxa, even under very stringent sequencing read filtering. There was no significant effect on the recovered OTU richness of small and medium-sized arthropods when metabarcoded alongside larger arthropods, despite substantial biomass differences in the mixture. The recovery of taxonomic subsets was not generally influenced by the presence of other taxa, although with some exceptions likely due to primer mismatches. Considerable compositional variation within size and taxon-based subcommunities was evident resulting in high beta-diversity among samples from within a single tree canopy, but this beta-diversity was not affected by experimental manipulation.We conclude that OTU recovery in complex arthropod communities, with sufficient sequencing depth and within reasonable size ranges, is not skewed by variable biomass of the constituent species. This could remove the need for time-intensive manual sorting prior to metabarcoding. However, there remains a chance of taxonomic bias, which may be primer-dependent. There will never be a panacea primer; instead, metabarcoding studies should carefully consider whether the aim is broadscale turnover, in which case these biases may not be important, or species lists, in which case separate PCRs and sequencing might be necessary. OTU number inflation remains an issue in metabarcoding and requires bioinformatic development, particularly in read filtering and OTU clustering, and/or greater use of species-identifying sequences generated outside of bulk sequencing.
节肢动物群落的代谢条形码技术可用于评估热带森林中的物种多样性，但该方法需要验证，以确保在复杂混合物中物种出现情况的准确性和可重复性。本研究调查了生态样本的组成如何影响物种恢复的准确性。从热带树冠层实地采集的大量样本开始，通过将不同体型和主要类群的子集组装成越来越复杂的复合池，测试了这些子集的标本回收率。在进行代谢条形码分析后，我们追踪任何大小类或分类学子集的丰富度、多样性，以及最重要的组成是否受到混合物中其他子集的影响。即使在非常严格的测序读数过滤条件下，大多数类群中的操作分类单元（OTU）数量也大大超过了形态物种的数量。当与大型节肢动物一起进行代谢条形码分析时，中小型节肢动物的OTU丰富度恢复没有受到显著影响，尽管混合物中的生物量存在很大差异。分类学子集的恢复一般不受其他类群存在的影响，不过也有一些例外，可能是由于引物错配。基于大小和分类群的亚群落内部存在相当大的组成差异，导致单棵树冠层样本之间的β多样性很高，但这种β多样性不受实验操作的影响。我们得出结论，在复杂的节肢动物群落中，在有足够测序深度且在合理大小范围内，OTU的恢复不会因组成物种的生物量变化而产生偏差。这可能消除了在代谢条形码分析之前进行耗时的人工分类的必要性。然而，分类学偏差的可能性仍然存在，这可能取决于引物。永远不会有万能引物；相反，代谢条形码研究应仔细考虑目标是广泛的周转率（在这种情况下这些偏差可能不重要）还是物种清单（在这种情况下可能需要单独的PCR和测序）。OTU数量膨胀在代谢条形码分析中仍然是一个问题，需要生物信息学的发展，特别是在读数过滤和OTU聚类方面，和/或更多地使用大量测序之外生成的物种识别序列。
