An analysis of the gaps in the South African DNA barcoding library of ticks of veterinary and public health importance.

Ticks transmit pathogens of veterinary and public health importance. Understanding their diversity is critical as infestations lead to significant economic losses globally. Approximately over 90 species across three families have been identified in South Africa. However, the taxonomy of most species has not been resolved due to morphological identification challenges. DNA barcoding through the Barcode of Life Data Systems (BOLD) is therefore a valuable tool for species verifications for biodiversity assessments. This study conducted an analysis of South African tick COI barcodes on BOLD by verifying species on checklists, literature, and other sequence databases. The compiled list represented 97 species, including indigenous (59), endemics (27), introduced (2), invasives (1), and eight that could not be classified. Analyses indicated that 31 species (32%) from 11 genera have verified COI barcodes. These are distributed across all nine provinces with the Eastern Cape having the highest species diversity, followed by Limpopo, with KwaZulu-Natal having the least diversity. Rhipicephalus, Hyalomma, and Argas species had multiple barcode Index Numbers (BINs), suggesting cryptic diversity or unresolved taxonomy. We identified 21 species of veterinary or zoonotic importance from the Argasidae and Ixodidae families that should be prioritised for barcoding. Coordinating studies and defining barcoding targets is necessary to ensure that tick checklists are updated to support decision-making for the control of vector-borne diseases and alien invasives.

DNA barcoding of southern African mammal species and construction of a reference library for forensic application.

Combating wildlife crimes in South Africa requires accurate identification of traded species and their products. Diagnostic morphological characteristics needed to identify species are often lost when specimens are processed and customs officials lack the expertise to identify species. As a potential solution, DNA barcoding can be used to identify morphologically indistinguishable specimens in forensic cases. However, barcoding is hindered by the reliance on comprehensive, validated DNA barcode reference databases, which are currently limited. To overcome this limitation, we constructed a barcode library of Cytochrome c oxidase subunit 1 (COI) and Cytochrome b (Cyt b) sequences for threatened and protected mammals exploited in southern Africa. Additionally, we included closely related or morphologically similar species and assessed the database's ability to identify species accurately. Published southern African sequences were incorporated to estimate intraspecific and interspecific variation. Neighbor-joining trees successfully discriminated 94-95% of the taxa. However, some widespread species exhibited high intraspecific distances (>2%), suggesting geographic sub-structuring or cryptic speciation. Lack of reliable published data prevented the unambiguous discrimination of certain species. This study highlights the efficacy of DNA barcoding in species identification, particularly for forensic applications. It also highlights the need for a taxonomic re-evaluation of certain widespread species and challenging genera.

Evidence of genetic differentiation in cigar wrasse Cheilio inermis (Labridae) within the western Indian Ocean.

Patterns of genetic structure and connectivity of the monotypic cigar wrasse Cheilio inermis within western Indian Ocean (WIO) are poorly understood. Whether the species exists as a single panmictic population across the WIO is unclear. Sequence data were generated from two mitochondrial genes (cytochrome b and ATPase 6) and one nuclear intron (S7 intron I). High levels of haplotype and allelic diversity (h = 0.88-0.98; A = 0.95-0.98), along with low nucleotide diversities were observed across all markers. The pairwise ΦST values indicated differentiation of Tanga from the four WIO localities (Inhaca, Nosy Bé, Gazi, and Shimoni), as well as differentiation between the northernmost WIO localities. AMOVAs indicated high differentiation among defined locality groups, whereas nuclear gene analysis found little differentiation among groups. The observed genetic differentiation in C. inermis could be caused by oceanic barriers, and by limited larval dispersal with the pelagic larvae possibly settling near their parental origin and promoting differentiation.

Forensic application of DNA barcoding for identification of illegally traded African pangolin scales.

The escalating growth in illegal wildlife trade and anthropogenic habitat changes threaten the survival of pangolin species worldwide. All eight extant species have experienced drastic population size reductions globally with a high extinction risk in Asia. Consequently, forensic services have become critical for law enforcement, with a need for standardised and validated genetic methods for reliable identifications. The seizure of three tonnes of pangolin scales, believed to have originated from Africa, by Hong Kong Customs Authorities provided an opportunity for the application of DNA barcoding in identifying scales. Three mitochondrial DNA gene regions (COI, Cyt b, and D-loop) were amplified for a subsample of the confiscated material and compared with taxonomically verified references. All four African species were recovered as monophyletic with high interspecific uncorrected p-distance estimates (0.048-0.188) among genes. However, only three of four African species (Phataginus tricuspis, Phataginus tetradactyla, and Smutsia gigantea, originating from West and Central Africa) and one of four Asian species (Manis javanica from Southeast Asia) were identified among scales. Although the assignment of unknown scales to specific species was reliable, additional genetic tools and representative reference material are required to determine geographic origins of confiscated pangolin specimens.

Development and validation of a novel forensic STR multiplex assay for blue (Anthropoides paradiseus), wattled (Bugeranus carunculatus), and grey-crowned crane (Balearica regulorum).

The blue crane (Anthropoides paradiseus), wattled crane (Bugeranus carunculatus), and grey-crowned crane (Balearica regulorum) are species of concern as their populations are declining and they face several threats including habitat loss, disturbance and illegal trade. In South Africa, these species are bred in captivity for trade purposes which is permitted and regulated globally under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Legal sustainable trade through captive breeding of endangered wildlife species such as cranes has been promoted to counteract the illegal trade of individuals from the wild. Captive breeding independent of wild populations may reduce the harvest pressures on wild bird populations which in turn benefit the recovery of exploited species. This approach is considered to be controversial by some individuals. Although captive breeding of endangered species, for both population sustainability and commercial purposes, is promoted to aid in conserving species, concerns have been raised with regards to breeding facilities being used for laundering of animals. To monitor the legal trade of cranes in South Africa a short tandem repeat (STR) assay following recommendations of the International Society for Forensic Genetics (ISFG) was developed and validated. An STR assay comprising of four multiplexes that include 16 STR markers and two gender determination markers was proven to be highly informative with average polymorphic information content (PIC) values of 0.806, 0.646 and 0.725 for A. paradiseus, B. regulorum and B. carunculatus respectively. In addition, the assay showed sufficient discriminatory power for parentage assignment of closely related individuals in all three species (A. paradiseus: PI = 1.7×10-24, PIsibs = 4.7×10-08, and B. carunculatus: PI = 1.4×10-19, PIsibs = 2.9×10-07 and B. regulorum: PI = 1.7×10-12, PIsibs = 5.0×10-05). Analysis of 251 samples suggested that the validated multiplex assay ensures reliability, reproducibility, and repeatability for applications in forensic case work where illegal trade of offspring is suspected through verifying parentage of captive birds in breeding facilities.

Optimizing waterborne eDNA capture from waterholes in savanna systems under remote field conditions.

Environmental DNA (eDNA) is used for biodiversity assessments in a variety of ecosystems across the globe, whereby different eDNA concentration, preservation and extraction methods can outperform others depending on the sampling conditions and environment. Tropical and subtropical ecosystems in Africa are among the less studied systems concerning eDNA-based monitoring. Waterholes in arid parts of southern Africa represent important agglomeration points for terrestrial mammals, and the eDNA shed into such waterbodies provides a powerful source of information for monitoring mammalian biodiversity in the surrounding area. However, the applied methods for eDNA sampling, preservation and filtering in different freshwater systems vary greatly, and rigorous protocol testing in African freshwater systems is still lacking. This study represents the first attempt to examine variations in eDNA concentration, preservation and extraction methods under remote field conditions using waterborne eDNA in a savanna system. Collected samples were heavily affected by microalgal and bacterial growth, impeding eDNA capture and PCR success. We demonstrate clear effects of the methodological choices, which also depend on the state of eDNA. A preliminary metabarcoding run showed little taxonomic overlap in mammal species detection between two metabarcoding primers tested. We recommend water filtering (using filters with pore sizes >1 µm) over centrifugation for eDNA concentration, Longmire's solution for ambient temperature sample preservation and Qiagen's DNeasy PowerSoil Pro Kit for DNA extraction of these inhibitor-prone samples. Furthermore, at least two independent metabarcoding markers should be utilized in order to maximize species detections in metabarcoding studies.

Metabolomics approach for predicting stomach and colon contents in dead Arctocephalus pusillus pusillus, Arctocephalus tropicalis, Lobodon carcinophaga and Ommatophoca rossii from sub-Antarctic region.

The dietary habits of seals play a pivotal role in shaping management and administration policies, especially in regions with potential interactions with fisheries. Previous studies have utilized various methods, including traditional approaches, to predict seal diets by retrieving indigestible prey parts, such as calcified structures, from intestines, feces, and stomach contents. Additionally, methods evaluating nitrogen and stable isotopes of carbon have been employed. The metabolomics approach, capable of quantifying small-scale molecules in biofluids, holds promise for specifying dietary exposures and estimating disease risk. This study aimed to assess the diet composition of five seal species-Arctocephalus pusillus pusillus, Lobodon carcinophaga, Ommatophoca rossii, and Arctocephalus tropicalis 1 and 2-by analyzing stomach and colon contents collected from stranded dead seals at various locations. Metabolite concentrations in the seal stomach and colon contents were determined using Nuclear Magnetic Resonance Spectroscopy. Among the colon and stomach contents, 29 known and 8 unknown metabolites were identified. Four metabolites (alanine, fumarate, lactate, and proline) from stomach contents and one metabolite (alanine) from colon contents showed no significant differences between seal species (p>0.05). This suggests that traces of these metabolites in the stomach and colon contents may be produced by the seals' gut microbiome or derived from other animals, possibly indicating reliance on fish caught at sea. Despite this insight, the cause of death for stranded seals remains unclear. The study highlights the need for specific and reliable biomarkers to precisely indicate dietary exposures across seal populations. Additionally, there is a call for the development of relevant metabolite and disease interaction networks to explore disease-related metabolites in seals. Ultimately, the metabolomic method employed in this study reveals potential metabolites in the stomach and colon contents of these seal species.

Re-description of two species of the cardinalfish genus Archamia (Teleostei: Apogonidae) from the Red Sea and Western Indian Ocean.

The cardinalfishes Archamia bilineata and A. pallida were originally described from a small number of specimens collected in the Gulf of Aqaba, Red Sea and Oman, respectively. Both species are re-described using specimens collected recently in Yemen and Kenya, including the first known adult of A. pallida. These new collections increased the geographical range of both species considerably. Differences between the two populations now known for each of the species are discussed.

Revalidation and redescription of Serranus knysnaensis Gilchrist, 1904, (Perciformes: Serranidae) with a new distribution record.

A southwestern Indian Ocean (SWIO) percoid fish Serranus knysnaensis Gilchrist, 1904, was long synonymised with the comber, Serranus cabrilla (Linnaeus, 1758), from the eastern Atlantic Ocean, Mediterranean and Black Sea. However, when the species was brought out of synonymy by Heemstra Heemstra (2004), reasons for this decision were not given. This study aims to revalidate the present taxonomic status of S. knysnaensis using morphological and molecular assessments. The two species are distinguished by the number of circumpeduncular scales (2634 in S. knysnaensis versus 3438 in S. cabrilla) and total gill rakers (1822 versus 2224). Serranus knysnaensis is also distinct from S. novemcinctus Kner, 1864, the other SWIO species of Serranus, based on total gill raker counts (1822 versus 3135). Genetic analysis of mitochondrial DNA barcode (COI) sequences for 17 Serranus species revealed three closely-related monophyletic clusters corresponding to S. cabrilla, S. novemcinctus and S. knysnaensis that were supported (P 0.001) by species delimitation methods. Even though the genetic distances among the three species were the lowest in the genus (1.60-1.99%), these species may be ecomorphs or lineages that have only recently diverged from each other. These three species also have allopatric distributions and our morphological and molecular data thus confirm that S. knysnaensis is a valid species.

A taxonomic revision of Gymnothorax undulatus (Anguilliformes: Muraenidae) in the Western Indian Ocean, with description of a new species.

The taxonomic status of the widely distributed Indo-Pacific undulated moray eel, Gymnothorax undulatus, is revised using morphological and genetics features. Ninety-seven specimens previously identified as G. undulatus were examined and their mitochondrial COI and 16S rRNA genes were analysed. The multivariate analysis of eight morphometric characters resulted in separation with little to no overlap among some geographic regions. These groupings explained more than 90% of the total variation, with 86.6% overall classification. Two color morphs were identified, and the South African population was described as new species, Gymnothorax elaineheemstrae n. sp., distinct from G. undulatus in having mottled and faintly reticulated color pattern, 134-136 total vertebrae and further confirmed by the genetic analysis of COI and 16S rRNA with > 0.1 genetic distance. The morphological and genetics results indicate that G. undulatus, previously treated as a single species, consists of more than one species.

Mosquito identification and haemosporidian parasites detection in the enclosure of the African penguins (Spheniscus demersus) at the SANBI zoological garden.

The National Zoological Gardens (NZG) is a facility of the South African National Biodiversity Institute (SANBI) and the largest zoo in southern Africa. Among the 9000 captive animals kept by the NZG, is the endangered African penguin (Spheniscus demersus). There have been several post-mortem reports on deaths of penguins in the NZG due to haemosporidian infections, however, the haemosporidian lineages involved and possible insect vector are unknown. Haemosporidians are apicomplexan parasites that infect vertebrates through blood-sucking dipteran insects. Therefore, the current study aimed to identify mosquitoes that are potential vectors found within the African penguin enclosure as well as to detect the haemosporidian parasites from these insects using nested-PCR and real-time PCR (qPCR) analyses. Mosquito samples were collected using an overnight UV-light trap setup for 3 months. From the 65 pooled samples representing 325 mosquitoes, morphological and molecular analysis showed that Culex pipiens (52.31%) was the dominant species followed by Cx. t heileri (30.77%) and Cx. quinquefasciatus (16.92%). Nested-PCR detected parasite DNA of Leucocytozoon sp. and Plasmodium sp. The Cx. pipiens had the highest minimum infection rate (MIR) of 5.88% by nested-PCR and 9.41% by qPCR whilst Cx. quinquefasciatus had MIR of 3.64% in both assays and no haemosporidian parasites were detected from Cx. t heileri. One Cx. pipiens sample had a co-infection of both Plasmodium sp. and Leucocytozoon sp. detected by nested-PCR. These findings suggest that effective control measures for blood-sucking dipteran insects is required at the NZG and more studies should be conducted to determine the actual prevalence of these haemosporidian parasites among other bird species within NZG.

Molecular systematics and taxonomic status of three latitudinally widespread nototheniid (Perciformes: Notothenioidei) fishes from the Southern Ocean.

The taxonomic status of the three nototheniids, Lepidonotothen squamifrons, L. larseni and Gobionotothen marionensis from different localities in the Southern Ocean is re-evaluated at the DNA level. DNA sequence divergences and phylogenetic relationship were estimated using a combined mitochondrial (mtDNA, ND2 and COI) dataset and data for one nuclear gene (S7 intron 1). Phylogenies of both datasets had Lepidonotothen kempi nested within L. squamifrons lineage, with low sequence divergences (0% to 0.4%) between the two nominal species suggesting that they are populations of one species. Therefore, these results do not support the previous splitting of L. squamifrons into different species. Similarly, the L. larseni specimens also represented a single genetic unit (0.3% to 0.6%) with low geographic variation between Atlantic and Indian Ocean specimens, which does not support the splitting of this species into geographically restricted species. The mtDNA phylogeny clearly separated individuals of G. acuta from Kerguelen, Heard and MacDonald Islands from G. marionensis individuals into different clades, with sequence divergence of 2.9% between these clades supporting they are different species.