Insights into traditional Chinese medicine: molecular identification of black-spotted tokay gecko, <i>Gekko reevesii</i> , and related species used as counterfeits based on mitochondrial 16S rRNA gene sequences

Authentication of traditional Chinese medicine (TCM) is challenging due to DNA degradation in Chinese medicinal materials, which are usually processed and stored dry. The standard DNA barcoding length (648 bp) or longer are difficult to amplify, which makes it difficult to identify adulterants in Chinese medicinal materials. In this study, we used the mitochondrial 16S rRNA gene (< 200bp) as a barcode to differentiate black-spotted tokay geckoes (Gekko reevesii) from the related species used as counterfeits. We collected 63 specimens from 17 species of G. reevesii and their counterfeits, and each specimen generated a 189 bp 16S rRNA gene sequence. The average uncorrected p-distances within genuine G. reevesii was 0.9%, while the average uncorrected p-distances between G. reevesii and their counterfeits was 6.3% (at a minimum). According to phylogenetic analysis and genetic distances, the genuine G. reevesii samples collected in this study constitute a monophyly that can be distinguished from its counterfeits in TCM formulations, including G. gecko (red-spotted tokay geckos), which have very similar morphology. Thus, the short 16S rRNA barcode provides an effective tool for distinguishing G. reevesii from its counterfeits, ensuring the safety and efficacy of clinical medications containing components from G. reevesii in TCM.