Materials and Methods

To achieve the aims of this study, existing literature on South Africa’s urban traditional medicine markets was reviewed for records of trading in anuran amphibians and reptiles, traditional health practitioners were interviewed to increase understanding of their practices, and herptiles species were identified through visual confirmation during visits to the traditional medicine markets and with DNA barcoding targeting cytochromec oxidase marker 1 (COI) of herptile specimens from those markets.

Literature review

A search of the keywords: animal + traditional medicine + “South Africa” on Google Scholar (https://scholar.google.com/) returned results of literature whose titles and abstracts were pre-screened for mentions of animal use in South African traditional medicine. Following this initial screening, the suitable articles were studied to find records of herptiles sold specifically in South Africa’s urban traditional medicine markets or shops. Subsequently, availability of reference DNA sequences for the herptiles species matching the inclusion criteria of the literature review was verified with searches on the National Center for Biotechnology Information (NCBI) GenBank database (using the search query ((Species name ) AND (CO1[Gene Name] OR COI[Gene Name])) and the Barcode of Life Data Systems (BOLD) database (using the search query “Species name ”).

Fieldwork: interviews, tissue sampling and visual observation

Visual confirmation surveys of herptile specimens available for sale at traditional medicine markets/shops by the first author involved visiting a total of six markets/shops in Polokwane, Pretoria, Johannesburg, Pietermaritzburg and Durban from August to December 2020 (Figure 1). Morphology-based identification of species using visual confirmation was based on wildlife guides for reptiles (Alexander & Marais, 2007; Marais, 2008). In accordance with North-West University Health Research Ethics Committee’s guidelines, the participation of traditional health practitioners at the markets/shops was sought after the first author explained the purpose of this study in SePedi (language spoken by people of Pedi culture) to practitioners from Limpopo and IsiZulu (language spoken by people of Zulu culture) for Gauteng and KwaZulu-Natal practitioners. SePedi was the preferred language for the Limpopo participants, IsiZulu was the most spoken language at the markets/shops in Gauteng and KwaZulu-Natal. Following explanation of the study, 11 traditional health practitioners consented to participation in this study (two in Limpopo, two in Gauteng and seven in KwaZulu-Natal). An informal conversational interview approach was used to collect data about herptiles of traditional medicine value; their Indigenous names, and the collection and preservation methods used for those herptiles. This interview approach relies on continuous participant observation without predetermined questions (Gall et al., 2003). The approach was chosen due to traditional health practitioners expressing apprehension towards researchers based on what they explained as past unpleasant experiences with researchers and conservation practitioners. This interview was guided by the first author’s conversation with participants and questions were introduced to the conversation when participants were forthcoming with details about their practices. Answers to these questions were written in a field book once the practitioners gave permission for their answers to be recorded in that manner. The reasons for the practitioner’s apprehension towards researchers were also noted.
Tissue samples were collected from specimens sold by nine participants at four of the six localities visually surveyed in Gauteng and KwaZulu-Natal markets/shops (Figure 1) as they gave consent for this collection while the two participants in Limpopo said they could not give consent as they did not own the traditional medicine shops. A total of 111 samples were collectively obtained from Gauteng and KwaZulu-Natal (Dataset S1). Practitioners were asked the IsiZulu names for each sampled specimen and notes were made of any morphological features that were still visible on those specimens. Notes about morphology were written as the traditional health practitioners did not allow use of cameras at the markets/shops.
Distinctive morphological traits were not visible on all sampled specimens as sometimes all that remained were ventral scutum, or bones with flesh but no skin. Opting for collection of tissue samples instead of taking entire specimens minimises this study’s environmental impact as removal of entire specimens may prompt traditional health practitioners to acquire replacement specimens to satisfy demand from customers or patients.

DNA extraction and absorbance measurements

From the acquired samples, outside layers of tissue that were most likely exposed to contamination were shaven/scraped off and discarded before taking ~25mg of tissue for DNA extraction. This tissue’s genomic DNA was extracted using the standard extraction protocols for animal tissue provided by the manufacturer in the NucleoSpin®Tissue Genomic DNA Tissue Kit (Macherey-Nagel, Duren, Germany).
To assess the suitability of the extracted DNA samples for downstream applications, (amplification and sequencing) their purity was determined through measures of absorbance using ultraviolet-visible spectroscopy (UV–visible spectrophotometry) where the peak absorbance of pure nucleic acid is 260 nm (Desjardins & Conklin, 2010; Koetsier & Cantor, 2019). These absorbance measurements were carried out on the NanoDrop One Spectrophotometer (Thermo Scientific) according to manufacturer’s instructions. Blank measurements were first performed with 2 μl of the reference solution (elution buffer used during DNA extractions) to minimise this solution’s contribution to the absorbance of the extracted DNA. To be able to make inferences about purity of the extracted DNA, spectrophotometry results from this study’s sample are compared to typical absorbance of pure nucleic acid for DNA; a 260/280 nm (A260/280) absorbance ratio of ~1.8 (1.85 – 1.88) and a 260/230 nm (A260/230) absorbance ratio in the range of 1.8 – 2.3 (Desjardins & Conklin, 2010; Koetsier & Cantor, 2019). Samples with 260/280 nm and 260/230 nm absorbance ratios greater or equal to 1.8 are generally considered suitable for downstream applications (Koetsier & Cantor, 2019), but there are likely to be exceptions to these general guidelines for interpreting absorbance ratios. Negative absorbance ratios could be an indication of contamination that is emitting light instead of absorbing it, absorbance ratios that are minor outliers generally give an indication that DNA extraction procedures need to be improved, while major outlier absorbance ratios suggest presence of impurities in the sample (Desjardins and Conklin 2010). Outliers were determined using the interquartile rule where the minor outliers in the absorbance ratios are lower than the first quartile value minus 1.5 times the interquartile range (i.e., Q1 – 1.5(IQR)), the major outliers are higher than the third quartile plus 1.5 times the interquartile range (i.e., Q3 + 1.5(IQR)), and interquartile range is calculated by subtracting the first quartile value from the third quartile value (i.e., IQR = Q3 – Q1).
Following the absorbance measurements, all extracted DNA samples were used to amplify DNA barcode fragments with a polymerase chain reaction (PCR). In addition to PCR being a step towards obtaining DNA barcodes, it also provides an indication of whether the success of this barcoding conforms to absorbance ratio guidelines. This amplification targeted a region of a length of maximum 664 bp of the COI gene with a primer set from a previous study by Nagy et al., (2012); RepCOI-F (5’-TNT TMT CAA CNA ACC ACA AAG A-3’) and RepCOI-R (5’-ACT TCT GGR TGK CCA AAR AAT CA-3’). For DNA barcoding animals, the mitochondrial 5’ end of the cytochrome c oxidase subunit 1 marker (COI) is proposed as a universal barcode marker (Hebert et al., 2003b). The PCR reactions were performed in total volumes of 25 μl: 12.5 μl Thermo Scientific DreamTaq Green PCR Master Mix (X2) (with DreamTaq DNA Polymerase, 2X DreamTaq Green buffer, dNTPs, at 0.4 mM each and 4 mM MgCl2), 1.25 μl (10 μM) of each of the two RepCOI primers mentioned above, 3 μl of the template DNA elution and 7 μl Thermo Scientific Nuclease-free water (PCR-grade). The reactions were carried out in the Applied Biosystems SimpliAmp Thermal Cycler (Thermo Fisher Scientific Inc) using the following PCR protocol: initial denaturation at 95°C for 3 minutes, 40 cycles of denaturation at 95°C for 30s, annealing at 48.5°C for 30s, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes, and subsequent storage of PCR products at 4°C. The PCR products were visualised on a 1% agarose gel under ultraviolet light on the E-BOX CX5 stand-alone gel imaging system (Vilber Lourmat Deutschland GmbH).

Sequencing protocol

Purification and sequencing of PCR products was outsourced to a commercial sequencing company (Inqaba Biotechnical Industries (Pty) Ltd, Pretoria, South Africa). They cleaned PCR products using the ExoSap Protocol: 10 µl amplified PCR product and 2.5 µl ExoSAP master mix (Exonuclease I 20 U/ul and Shrimp Alkaline Phosphatase 1 U/ul) mixed well and incubated at 37°C for 15 minutes then held at 80°C for 15 minutes. The Nimagen, BrilliantDye™ Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000 was used to sequence fragments according to manufacturer’s instructions. The cycle sequencing protocol provided by the sequencing company was as follows: 10 μl NEB OneTaq 2X MasterMix with standard buffer, 1 μl genomic DNA (10-30ng/μl), 1 μl of forward and reverse primer each (10μM) (using the same primers, RepCOI-F and –R, as in initial amplification), and 7 μl Nuclease free water. The sequencing PCR profile was 94°C for 5 min, 35 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 68°C for 60 seconds, followed by 10 minutes at 68°C and subsequently held at 4°C. Subsequently, products were cleaned with the ZR-96 DNA Sequencing Clean-up Kit and the cleaned products were injected on an Applied Biosystems ABI with a 50cm array (using POP7). Sequence chromatograms were analysed using the FinchTV analysis software.
Sequences obtained from the commercial sequencing company were trimmed with the Decontamination Using Kmers (BBDuk) trimmer, paired, then assembled using De Novo assembly on the Geneious Prime® 2022.0.2 (https://www.geneious.com/prime/) sequence analysis software (Biomatters New Zealand Ltd). The BOLD Identification System (IDS) was used to compare this study’s sequences to reference samples on the BOLD database (https://v3.boldsystems.org/index.php/IDS_IdentificationRequest) for verification of the sequence and species identity using neighbour-joining placement (Ratnasingham & Hebert, 2007). Subsequently, The Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997) was also used for a second comparison of all of this study’s sequences with published sequences on the NCBI Nucleotide collection (nr/nt) database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine sequence and species identity using the MegaBlast (Zhang et al., 2000) algorithm for identifying highly similar sequences. A difference of 2% or less between DNA sequences was used as a limit for discriminating between species (Hebert et al., 2003a; Pereira et al., 2013). Morphology of specimens was used to supplement molecular identification. The sequences obtained from study were deposited in the NCBI GenBank database under the accession numbers [GenBank: XXX-XXX] (accession numbers to be added later).