Application Of DNA Barcoding In Diversity Investigation Of Amphibians And Reptiles In Labahe And Wawushan Nature Reserve

In order to investigate the diversity of amphibians and reptiles in two nature reserves of Sichuan province (Wawushan Provincial Nature Reserve and Labahe Provincial Nature Reserve), and further verify the reliability of DNA barcoding and the feasibility of the COI gene sequence as a DNA barcoding marker, in this paper,239 amphibians and reptiles samples were collected in these two reserves, traditional morphological and DNA barcoding approaches were used to identify the taxonomy of the samples, COI sequences were amplified as the DNA barcoding marker by using the DNA barcoding universal primer of amphibians, moreover,16S rRNA and Cyt b sequences were amplified as the complementary sequences by using the universal primers. The identification results are as follows:12 species were identified in Labahe Provincial Nature Reserve, which include 2 orders,5 families,7 species of amphibians and 2 orders,3 families,5 species of reptiles, in addition,26 species were identified in Wawushan Provincial Nature Reserve, which contain 2 orders,6 families,18 species of amphibians and 1 order,3 families,8 species of reptiles. Furthermore, all samples are accurately identified except 3 samples of an Oreolalax species and one sample of Sphenomorphus indicus (it might be a cryptic species). We submit all the accurate identification sequences to the GenBank and COI sequences of 11 species (e.g. Oreolalax major, Rhacophorns chenfui and Hebius metusium) are published for the first time, these may support for further DNA barcoding research. Our research results indicate that the amphibian DNA barcoding universal primer published by Che et al. well solved the universality of COI amplification, and compare to the 16S rRNA sequence, COI is the better marker for DNA barcoding, these are correspond with previous studies. However, we also find that COI sequences in the GenBank arc scanty now and the database has more 16S sequences, for this reason, supplement of 16S or Cyt b sequences might help identification. DNA barcoding has huge potential and can be well applied to investigate diversity of nature reserve species. But at now, because of the incompleteness of database, DNA barcoding still cannot separate from morphological identification.