Pharmacognosy Identification of Hippocampus barbouri Based on the Combination of the Morphological Characteristics and DNA Barcodes / 中国药学杂志

ABSTRACT

OBJECTIVE: To obtain the key identification information of Hippocampus barbouri (H. barbouri), investigate the pharmacognosy of the seahorse adulteration H. barbouri, using the combination of morphological and DNA barcoding technique. METHODS: Typical morphological characteristics of H. barbouri were studied based on the observations according to the previous reports and traditional pharmacognosy methods. Genomic DNAs of H. barbouri and other related seahorse species were extracted and the DNA barcoding genes of COI, 16S and ATP6 sequences were amplified and sequenced, respectively. In order to determine advantages of three barcodes for the identification of hippocampus, MEGA 7 was performed to analyze the intraspecific and interspecific distances and construct the NJ phylogenetic trees based on three different barcoding genes. RESULTS: The most typical distinguishing features of H. barbouri included the streak of the nose, five radial lines in the eyelid, and the long and short intervals of the tail thorns. The lengths of COI, 16S and ATP6 sequences were 649-650, 574 and 603 bp, respectively, with the maximum K2P intraspecific distances were 0.012, 0.003 and 0.003, respectively. The intraspecific distances of the three barcodes were far smaller than the corresponding minimum K2P interspecific distances of H. barbouri and other seven related seahorse species, existing an obvious barcoding gap. The NJ phylogenetic trees based on COI, 16S and ATP6 sequences showed that H. barbouri samples formed of a group indicating that H. barbouri could be distinguished from other seahorse species through the three DNA barcoding markers. In addition, H. barbouri showed close genetic relationship with H. histrix according to the NJ trees. Furthermore, the NJ tree structures of COI and ATP6 were more stable than that of 16S. CONCLUSION: The pharmacognostical investigation bases on the morphological identification and DNA barcodes obtained the key identification information of H. barbouri, providing the possibility for quick and accurate identification of this species. The COI, 16S and ATP6 barcoding genes can be used for further molecular identification markers for seahorse species. This study provides a new technical method for the development and quality control of hippocampus.