A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine:A case study of Amynthas earthworms as"Guang Dilong"

For wild natural medicine,unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials,which affects the efficacy and safety of clinical medication.DNA barcoding as an effective species identification tool is limited by its low sample throughput nature.In this study,combining DNA minibarcode,DNA metabarcoding and species delimitation method,a novel biological sources consistency evaluation strategy was proposed,and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as"Guang Dilong"and 25 batches of proprietary Chinese medicines.Besides Amynthas aspergillum as the authentic source,8 other Molecular Operational Taxonomic Units(MOTUs)were elucidated.Significantly,even the subgroups within A.aspergillum revealed here differ significantly on chemical compositions and biological activity.Fortunately,this biodiversity could be controlled when the collection was limited to designated areas,as proved by 2796"decoction pieces"samples.This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control,and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.