a, (i) Degenerate PCR primers targeting the conserved regions of adenylation domains found in non-ribosomal peptide synthetase genes were used to generate amplicons from an arrayed collection of eDNA isolated from 2,000 unique soils. The reads from these next-generation sequenced amplicons (NPSTs) were analysed by eSNaPD. (ii) A desert soil rich in AD NPSTs from the previously unknown malacidin clade was used to build an arrayed cosmid library. Cosmids harbouring all fragments of a targeted BGC were assembled and integrated into a heterologous host for production, extraction and characterization. b, AD NPSTs identified by the eSNaPD analysis to be evolutionarily related to the conserved Asp4 ADs of known calcium-dependent antibiotics were used to phylogenetically map the unexplored clades of this larger family across all tested soil microbiomes. The subfamilies of calcium-dependent antibiotics and their relative abundance are illustrated on the phylogenetic tree by colour and percentage. Across all sampled soil metagenomes, the malacidin antibiotic-clade represents 19% of the NPSTs, and 59% of calcium-dependent antibiotic tags originate from unexplored branches. c, Geospatial distribution of calcium-dependent antibiotics across sampled US soil metagenomes. States containing at least one soil sample with AD NPSTs from the malacidin clade are indicated in orange. States lacking malacidin tags but still containing calcium-dependent-antibiotic NPSTs are indicated in blue. States with at least one sampled soil but no detected calcium-dependent-antibiotic NPSTs are highlighted in dark grey.