[Morphological characteristics identification and molecular DNA barcoding analysis of Hippocampus spinosissimus]

The combination of morphological characteristics and DNA barcodes was used to a systematic study of Hippocampus spinosissimus,laying the foundation for rapid and accurate identification for the medical seahorse species. According to the reported literature and observation on seahorse samples,the typical characteristics of the H. spinosissimus include highly developed spiny,much short nose,single or double cheeks and strongly developed spines bordering pouch. Genomic DNAs of H. spinosissimus and other related seahorse species were extracted using the TIANamp Marine Animals DNA Kit. The COⅠ and ATP6 genes were amplified and sequenced in both directions. After the verification by Blast,the GC content,intraspecific and interspecific genetic distance,and the Neighbor joining( NJ) phylogenetic trees were analyzed by MEGA 7. The lengths of the COⅠ and ATP6 genes were 649 bp and 602-603 bp,respectively,with the average GC content of 39. 96% and 35. 37%. The maximum intraspecific genetic distances in H. spinosissimus based on COⅠ and ATP were both far less than the minimum interspecific genetic distance between H. spinosissimus and other seahorses,suggesting a significant barcoding gap. NJ analysis results of COⅠ and ATP6 exhibited that all H. spinosissimus species clustered together,indicating that the two DNA barcode could identify H. spinosissimus from other seahorses accurately and quickly. In addition,H. spinosissimus shared a close genetic relationship between H. kelloggi according to the NJ tree. Furthermore,there exits three stable subgroup structure of H. spinosissimus,indicating that COⅠ and ATP6 barcodes could be applied the indicator for the geographical ecology research of H. spinosissimus. The results obtained the typical morphological and molecular identification characteristics of H. spinosissimus,which played central roles for the development of species identification. This study provides an important basis data for expanding the medical seahorse resources and ensuring the safety of clinical medicine.