Fig. 1

A schematic diagram of mitochondrial metagenomics. The central panel (red) represents the ‘contig-based’ analyses, using a database of complete (circles) or partial (lines) mitochondrial contigs. These are derived from one or more sources: sequencing of taxonomically chosen specimens and/or representative specimens from an ecological study (Voucher MMG; left panel); direct assembly of ecological bulk samples (Bulk MMG; right panel); external databases containing identified mitogenome sequences (superbarcodes), such as NCBI. Specimens for voucher and bulk MMG are shotgun- sequenced in mixtures, assembled with standard assembly pipelines, annotated for each gene, and assigned to known species through matches with cox1 barcodes or other mitochondrial sequences from well-identified specimens where applicable. The ‘contig-based’ analysis concludes with a phylogenetic analysis, with the tree updated as new data become available in an iterative process. This set of mitogenomes can then be used as a reference for a ‘read-based’ analysis (green panel). Here the presence and possible abundance of a given species in the local assemblage is determined by mapping reads from ecological bulk samples against the mitogenome database (dashed arrows). The knowledge of the phylogenetic tree provides an evolutionary perspective to all species in the study