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).