Results

Literature review

The published sources reviewed here recorded a total 34 herptile species (one anuran and 33 reptile species) with other herptiles only being identified to genus or higher taxonomic ranks from the South African urban traditional medicine markets/shops of Eastern Cape (Simelane & Kerley, 1998), Gauteng (Whiting et al., 2011) and KwaZulu-Natal (Ngwenya, 2001). Eight of those 34 herptile species reported in previous literature did not have COI reference samples available on either BOLD or NCBI GenBank databases at the drafting of this article in June 2022 (Table 1).
From the species recorded in previous literature (Table 1),Kinixys natalensis Hewitt, 1935 and Smaug giganteus(Smith, 1844) have their conservation status as vulnerable, whileEretmochelys imbricata (Linnaeus, 1766) is critically endangered (IUCN, 2022). Furthermore, published literature has records of four species from South Africa’s urban traditional markets that are not native to the country: Cordylus tropidosternum (Cope, 1869) is endemic to Eastern African countries (Broadley & Branch, 2002),Kinixys belliana Gray, 1831 occurs in the north of the Southern African region and beyond (Turtle Taxonomy Working Group, 2021),Naja melanoleuca Hallowell, 1857 is from Central and West African countries (Wüster et al., 2018) and Psammophis phillipsii(Hallowell, 1844) occurs in West African countries (Leaché et al., 2006).

Fieldwork: interviews, tissue sampling and visual observation

Through visual surveys, 9 of the 34 species identified in published literature were confirmed to be on sale among plants and other animal specimens at traditional medicine shops and open markets (Table 2) in the urban areas of three South African provinces (Gauteng, KwaZulu-Natal, and Limpopo). The traditional health practitioners who provided access to these tissue samples explained their apprehension towards conservation practitioners because conservation law enforcement previously confiscated specimens in their possession instead of seeking to collaborate with them to introduce measures that both adhered to environmental laws and respected Indigenous cultural practices. This collaboration is something they were willing to consider.
Traditional health practitioners from the surveyed traditional medicine markets reported obtaining the herptile specimens they use or sell by either hunting the animals themselves, buying from hunters that regularly go on hunts to supply multiple traditional health practitioners, or taking roadkill and animals that died of natural causes. Practitioners specifically target species that they require at the time of their hunts, while the hunters take orders for specific animals from traditional medicine practitioners and will also opportunistically hunt other species they encounter when hunting to fulfil their list of orders. Traditional health practitioners and the hunters that supply them with herptile species employ the same tissue preservation methods and the specimens are either preserved at home or at the open market. They remove visible body fat and internal organs. The body fat has traditional medicine value and is stored in bottles, while the internal organs are usually discarded. Following removal of fat and internal organs, the carcasses are smothered with ash and/or salt and placed in the sun to dehydrate them.
Dried specimens of herptiles and other animals are placed together on display for customers. According to the traditional health practitioners, people usually buy body parts or small pieces (relative to an animal’s size), and it is uncommon for someone to buy an entire carcass. The pricing for each piece of animal that a person wants to buy was noted to be uniform among this study’s participants with the exception being Pseudaspis cana (Linnaeus, 1758) which was priced at 40% higher than the rest of the herptiles being sold by those participants. The reason provided for this pricing difference was because the participants believed that P. cana preyed on other snake species. Due to how they are sold, herptile specimens were sometimes found missing parts of the body or only pieces of bones with muscles and skin remained. Specimens at open traditional medicine markets (in Durban and Johannesburg) were either removed at the end of each business day to be stored together overnight in plastic containers or they were left on the stalls and covered with plastic sheets. All storage of specimens is at ambient temperature; there is no refrigeration.

DNA barcoding of traditional medicine market samples

The absorbance measurements of the extracted DNA suggest that the traditional health practitioners’ preservation of herptiles using salt and/or ash can preserve DNA for molecular identification. Based on the A260/280 absorbance ratio, 20.7% of the 111 extracted DNA samples were not suitable for downstream analysis, while the A260/230 ratio suggested that 29.7% of the samples would be unsuitable due to their absorbance ratios being either negative or outlier values (Figure 2).
The subsequent amplification and sequencing outcomes were partially in line with the interpretation of absorbance ratios as 25 of the extracted DNA samples with negative or outlier absorbance ratios were successfully amplified and sequenced when interpretations of their absorbance suggested they would be unsuitable for downstream applications (Table 3). Conversely, 16 extractions with absorbance ratios that were interpreted as being suitable for downstream applications could not be amplified or sequenced. A total 81.1% (90 of 111) of the extractions were successfully amplified (based on the PCR products visualised using gel electrophoresis) and subsequently sent for sequencing. DNA sequences were successfully obtained from 81 samples (72.9% of original sample) and sequencing reactions failed for the remaining 9 of 90 amplicons. From the 81 DNA sequences, 38 had exact species matches with on the BOLD database with 99.13 - 100% similarity (Table 3). An additional three sequences had species level matches with 99.0% pairwise similarity on the NCBI Genbank database (Ng & Tay, 2004), with e-value of zero suggesting that there is no better match besides that current result (Metzler, 2006). Lists of nearest matches rather than exact species matches were returned for 12 sequences on the BOLD database with 98.12% - 99.05% similarity and one sequence with 98.9% similarity on the NCBI Genbank database (Table 3). The identity of these 13 sequences with lists of nearest species matches were confirmed using morphology as the specimens from which the tissue was obtained had not yet been cut to a point of being difficult to recognise. A 93.8% similarity to P. phillipsii which is not native to South Africa was returned as the highest match for one of this study’s DNA sequences on the NCBI GenBank database. This match to a non-native species was likely due to the sample being obtained from a native species of the same taxonomic group and the morphological traits observed during visual surveys provide confirmation of a genus (Psammophis sp.) level identification (Table 2).
Using molecular identification and observed morphological traits, 55 tissue samples collected during this study were matched to one genus and 12 species of reptiles that had already been recorded in published literature and one additional reptile species that was not recorded in previous literature (Table 2). Twenty-four of the 26 remaining sequences had no matches on the BOLD database, but it could be ascertained that they were DNA fragments of reptiles by comparing them to their NCBI GenBank reference sequence matches of reptiles with a similarity of 81.2 – 86.9%, using 70% similarity to reference sequences as a threshold below which the results would not be meaningful (Baxevanis et al., 2020). Two of the 26 remaining sequences matched with reference sequences from mammal species: Ictonyx striatus (Perry, 1810) with 99.2% similarity on the NCBI GenBank database and Procavia capensis (Pallas, 1766) with 98.14% similarity on the BOLD database. These mammalian tissues were obtained from pieces of bone and muscle that a traditional health practitioner mislabelled as either uxam or imbulu, IsiZulu names for Varanus spp. (Table 3).
Molecular identification of species verified some of the IsiZulu names used by traditional health practitioners to identify reptiles used in Indigenous remedies and also revealed mislabelling of animal tissue with their distinguishing features removed during sale (Table 3). Some IsiZulu names for the specimens were accurate up to species level (e.g.,Dendroaspis angusticeps (Smith, 1849), imamba eluhlaza in IsiZulu) while other Indigenous names were only accurate to higher taxonomic ranks, for example specimens named as unwabu (IsiZulu word for members of Chamaeleonidae) were later confirmed to be Chamaeleo dilepis with molecular identification. Further examples of DNA barcoding as a tool for verification of folk taxonomy include specimens broadly labelled as snakes and monitor lizards in IsiZulu (inyoka and uxam/imbulu respectively) being confirmed up to species level by DNA barcoding as P. cana and Varanus niloticus (Linnaeus, 1766) respectively (Table 3). Mislabelling of tissue meant for use in Indigenous remedies involved herptile tissue (e.g., N. melanoleuca mislabelled as Naja mossambica Peters, 1854, imfezi in IsiZulu), and mammalian bone and muscle which was mislabelled as aVaranus sp. (uxam/imbulu in IsiZulu) (Table 3).