Validating the power of mitochondrial metagenomics for community ecology and phylogenetics of complex assemblages

1. The biodiversity of mixed-species samples of arthropods can be characterized by shotgun sequencing of bulk genomic DNA and subsequent bioinformatics assembly of mitochondrial genomes. Here, we tested the power of mitochondrial metagenomics by conducting Illumina sequencing on mixtures of >2600 individuals of leaf beetles (Chrysomelidae) from 10 communities. 2. Patterns of species richness, community dissimilarity and biomass were assessed from matches of reads against three reference databases, including (i) a custom set of mitogenomes generated for 156 species (89% of species in the study); (ii) mitogenomes obtained by the de novo assembly of sequence reads from the real-world communities; and (iii) a custom set of DNA barcode (cox1-5′) sequences. 3. Species detection against the custom-built reference genomes was very high (>90%). False presences were rare against mitogenomes but slightly higher against the barcode references. False absences were mainly due to the incompleteness of the reference databases and, thus, more prevalent in the de novo data set. Biomass (abundance × body length) and read numbers were strongly correlated, demonstrating the potential of mitochondrial metagenomics for studies of species abundance. 4. A phylogenetic tree from the mitogenomes showed high congruence with known relationships in Chrysomelidae. Patterns of taxonomic and phylogenetic dissimilarity between sites were highly consistent with data from morphological identifications. 5. The power of mitochondrial metagenomics results from the possibility of rapid assembly of mitogenomes from mixtures of specimens and the use of read counts for accurate estimates of key parameters of biodiversity directly from community samples.

1. 节肢动物混合物种样本的生物多样性，可通过对批量基因组DNA进行鸟枪测序（shotgun sequencing）并开展后续线粒体基因组的生物信息学组装来表征。本研究针对来自10个群落的2600余头叶甲科（Chrysomelidae）昆虫个体的混合样本实施Illumina测序，以此检验线粒体宏基因组学（mitochondrial metagenomics）的应用效能。
2. 本研究通过将测序读段与三类参考数据库进行比对，评估了物种丰富度、群落相异性以及生物量的分布模式：(i) 针对本研究中156个物种（覆盖研究中89%的物种）定制构建的线粒体基因组集；(ii) 从真实群落的测序读段中从头组装（de novo assembly）得到的线粒体基因组；(iii) 定制构建的DNA条形码（cox1-5′）序列集。
3. 针对定制构建的参考基因组的物种检出率极高（>90%）。基于线粒体基因组的假阳性检出情况极少，但基于条形码参考集的假阳性略高。假阴性检出主要源于参考数据库的不完备性，因此在从头组装的数据集中更为常见。生物量（丰度×体长）与测序读段数量呈显著相关，这证明线粒体宏基因组学可用于物种丰度的相关研究。
4. 基于线粒体基因组构建的系统发育树与叶甲科已知的系统发育关系高度一致。不同采样点间的分类学与系统发育相异性模式，与形态鉴定得到的数据具有高度一致性。
5. 线粒体宏基因组学的应用优势在于，可快速从混合标本中组装得到线粒体基因组，且可利用读段计数直接从群落样本中准确估算生物多样性关键参数。