Sample collection and processing
This study was approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. All operations were executed according to the guidelines for the care and treatment of experimental animals of the Center for Laboratory Animal Care, Tongji Medical College, Huazhong University of Science and Technology. All the original animals were morphologically identified by Professor Jiachun Chen (Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Huazhong University of Science and Technology), and all voucher samples were deposited in the herbarium of School of Pharmacy, Huazhong University of Science and Technology.
For the fresh snake gallbladder samples, a total of 253 fresh gallbladders (Table 1) belonging to 31 snake species in three families were collected from four snake farms in Hubei, Hunan, Jiangxi, and Zhejiang province, respectively. These four provinces were located in the middle and lower reaches of the Yangtze River area where was the main Shedan producing area in China. After dissecting out from the morphologically identified original animals, the fresh snake gallbladders were washed with sterile water, preserved in 95% ethanol, and stored at -20°C until used for DNA extraction.
For the fresh gallbladder bile samples, a total of 68 fresh gallbladder bile samples (Table 2), were collected. Specifically, 51 fresh snake gallbladders belonging to 17 snake species were collected from one snake farm in Hunan province, China. Moreover, 17 fresh gallbladders from five other common adulterated domestic poultry and livestock (cattle, chicken, duck, pig and sheep), were collected from one Animal Husbandry Co., Ltd. in Hubei province, China. The gallbladder bile derived from these 68 fresh gallbladders was freeze-dried into powder using lyophilizer and stored at -20°C until used for DNA extraction.
For the market Shedan samples, a total of 18 batches of market Shedan medicinal materials (Table 3), were collected from Chinese medicinal material markets or commercial companies related to the production of Shedan and its preparations, such as Bozhou herb market, and Deqing Moganshan Snakes Industrial Co., Ltd. In the market, each batch of commercial Shedan contained different numbers of gallbladders, and they were mixed and stored in liquor with an alcohol content of more than 50%. The market gallbladders were divided into three categories according to their size per batch, and then approximately 1/3 of which were randomly selected. Among these chosen gallbladders from the 18 batches of market Shedan, 13 batches of gallbladder bile were used for another purpose for chemical analysis, and the rest gallbladders (gallbladder tissues) were stored at -20 °C until used for DNA extraction, and the other five batches of gallbladder bile were also freeze-dried into powder and stored at -20°C until used for DNA extraction. Altogether, 134 market Shedan gallbladder samples of 13 batches of Shedan medicinal materials and 71 gallbladder bile samples of the other five batches of Shedan medicinal materials were detected through DNA barcoding.
Gallbladder materials were pretreated following the principles of molecular identification using DNA barcoding , and the total genomic DNA was isolated from the gallbladder tissue or gallbladder bile through the phenol-chloroform method  with slight modifications. For the fresh snake gallbladder samples, a small piece of gallbladder tissue was ground into powder in liquid nitrogen, placed in 700 μL of extraction buffer (100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 2% CTAB (pH 8.0), 2% PVP, 2% β-mercaptoethanol, and 10 μL of 20 mg/mL proteinase K), and then incubated at 65°C for about 30 min. DNA was subsequently purified by phenol-chloroform-isoamylalcohol (25:24:1, v/v/v) and ethanol precipitation. Finally, the pellet was dried, dissolved in 50 μL of sterile TE buffer (20 mM Tris-HCl (pH 8.0), 1 mM EDTA), and stored at -20°C before use. For the fresh gallbladder bile samples, the total genomic DNA was extracted from bile (approximately 5 mg) following the DNA extraction method of the foregoing fresh gallbladder samples.
Barcode sequences amplifying and sequencing
Initially, a pair of specific primers (COISNFF: 5'-TCAACAAACCACAAAGAYATYGG-3', COISNFR: 5'-ACTTCYGGRTGKCCRAARAATCA-3') had been originally designed based on COI gene universal primers and their corresponding base sites in snake COI sequences using MEGA 5.0  and Primer 5.0 software (Premier Biosoft International, Palo Alto, CA), and the specificity of these two pairs of primers was tested by partial snake species (see Additional file 1: Table S1). PCR amplification was carried out in a Bio-rad T100 Thermal Cycler (Bio-rad, USA) with a 25 μL reaction mixture, which contained 2.5 μL 10× PCR Buffer, 2.5 μL dNTPs (2 mM), 1.5 μL MgSO4 (1.5 mM), 0.5 U Taq polymerase (1 U/μL) (TOYOBO, Osaka, Japan), 0.75 μL of each forward and reverse primer (10 pmol/μL each), 15.5 μL of sterilized distilled water, and 1 μL of template DNA. The PCR amplification of LCO1490/HCO2198 primers was under the following conditions: 94°C for 2 min, followed by 35 cycles of 98°C for 10 s, 53°C for 1 min, and 68°C for 1 min, and a final extension at 68°C for 5 min. And the PCR amplification of COISNFF/COISNFR primers was under the following conditions: 94°C for 2 min, followed by 35 cycles of 98°C for 10 s, 51°C for 50 s, and 68°C for 50 s, and a final extension at 68°C for 5 min. The PCR products were confirmed on a 1.0% agarose gel, purified with the TIANGel Midi Purification Kit (Tiangen Biotech Co., Beijing, China), and bidirectionally sequenced using an ABI 3730XL DNA Analyzer (Applied Biosystems, USA).
Species identification and data analysis
Consensus sequences and contig generation were accomplished using CodonCode Aligner V 4.0 (CodonCode Co., USA). After trimming the amplification primers, sequences obtained were queried to GenBank and the Barcode of Life Data System (BOLD) for species identification, respectively, and their species would be confirmed based on the best match ≥ 98%, otherwise, the species of the query sequence could not be defined. The average intra- and interspecific genetic distance of the barcodes of the fresh snake gallbladder samples were calculated based on Kimura-2-parameter (K2P) distance model using MEGA 5.0 and they were used to evaluate the DNA barcoding gap. Sequences generated by COISNFF/COISNFR primers were deposited in the GenBank database.
Phylogenetic tree reconstruction
To generate the phylogenetic relationships and ascertain the accuracy of the potential barcode for species identification, a neighbor-joining (NJ) tree was constructed in MEGA 5.0 and the bootstrap values were evaluated based on 1000 replicates. Acrochordus javanicus from the family Acrochordidae (GenBank accession number: KX752053)  was selected as the outgroup in the NJ tree. To provide additional insights about the taxonomic identity of our material: we randomly downloaded one conspecific COI barcode sequence of the 31 snake species previously identified by morphology from GenBank, and then analyzed them together with the barcode sequences obtained from the fresh snake gallbladder samples in the NJ tree analysis.
Investigating the market Shedan medicinal materials
The DNA extraction, PCR amplification with COISNFF/COISNFR primers and sequencing of the market Shedan samples were the same as described above. The sequences obtained were queried to GenBank and BOLD Systems for species determination, respectively, and they were also submitted to the GenBank database. In the process of sequence definition, we also paid attention to the similarities between the query sequences obtained from market Shedan samples and the reference barcode sequences submitted to the GenBank database by this study.