Pangolin genomes and the evolution of mammalian scales and immunity.

Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.

Contrasting evolutionary history, anthropogenic declines and genetic contact in the northern and southern white rhinoceros (Ceratotherium simum).

The white rhinoceros (Ceratotherium simum) has a discontinuous African distribution, which is limited by the extent of sub-Saharan grasslands. The southern population (SWR) declined to its lowest number around the turn of the nineteenth century, but recovered to become the world's most numerous rhinoceros. In contrast, the northern population (NWR) was common during much of the twentieth century, declining rapidly since the 1970s, and now only two post-reproductive individuals remain. Despite this species's conservation status, it lacks a genetic assessment of its demographic history. We therefore sampled 232 individuals from extant and museum sources and analysed ten microsatellite loci and the mtDNA control region. Both marker types reliably partitioned the species into SWR and NWR, with moderate nuclear genetic diversity and only three mtDNA haplotypes for the species, including historical samples. We detected ancient interglacial demographic declines in both populations. Both populations may also have been affected by recent declines associated with the colonial expansion for the SWR, and with the much earlier Bantu migrations for the NWR. Finally, we detected post-divergence secondary contact between NWR and SWR, possibly occurring as recently as the last glacial maximum. These results suggest the species was subjected to regular periods of fragmentation and low genetic diversity, which may have been replenished upon secondary contact during glacial periods. The species's current situation thus reflects prehistoric declines that were exacerbated by anthropogenic pressure associated with the rise of late Holocene technological advancement in Africa. Importantly, secondary contact suggests a potentially positive outcome for a hybrid rescue conservation strategy, although further genome-wide data are desirable to corroborate these results.

Mitochondrial genomes of African pangolins and insights into evolutionary patterns and phylogeny of the family Manidae.

BACKGROUND: This study used next generation sequencing to generate the mitogenomes of four African pangolin species; Temminck's ground pangolin (Smutsia temminckii), giant ground pangolin (S. gigantea), white-bellied pangolin (Phataginus tricuspis) and black-bellied pangolin (P. tetradactyla). RESULTS: The results indicate that the mitogenomes of the African pangolins are 16,558 bp for S. temminckii, 16,540 bp for S. gigantea, 16,649 bp for P. tetradactyla and 16,565 bp for P. tricuspis. Phylogenetic comparisons of the African pangolins indicated two lineages with high posterior probabilities providing evidence to support the classification of two genera; Smutsia and Phataginus. The total GC content between African pangolins was observed to be similar between species (36.5% - 37.3%). The most frequent codon was found to be A or C at the 3rd codon position. Significant variations in GC-content and codon usage were observed for several regions between African and Asian pangolin species which may be attributed to mutation pressure and/or natural selection. Lastly, a total of two insertions of 80 bp and 28 bp in size respectively was observed in the control region of the black-bellied pangolin which were absent in the other African pangolin species. CONCLUSIONS: The current study presents reference mitogenomes of all four African pangolin species and thus expands on the current set of reference genomes available for six of the eight extant pangolin species globally and represents the first phylogenetic analysis with six pangolin species using full mitochondrial genomes. Knowledge of full mitochondrial DNA genomes will assist in providing a better understanding on the evolution of pangolins which will be essential for conservation genetic studies.

Advances in canine distemper virus pathogenesis research: a wildlife perspective.

Canine distemper virus (CDV) has emerged as a significant disease of wildlife, which is highly contagious and readily transmitted between susceptible hosts. Initially described as an infectious disease of domestic dogs, it is now recognized as a global multi-host pathogen, infecting and causing mass mortalities in a wide range of carnivore species. The last decade has seen the effect of numerous CDV outbreaks in various wildlife populations. Prevention of CDV requires a clear understanding of the potential hosts in danger of infection as well as the dynamic pathways CDV uses to gain entry to its host cells and its ability to initiate viral shedding and disease transmission. We review recent research conducted on CDV infections in wildlife, including the latest findings on the causes of host specificity and cellular receptors involved in distemper pathogenesis.

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.

SNP discovery and characterisation in White Rhino (Ceratotherium simum) with application to parentage assignment.

The white rhino is one of the great success stories of modern wildlife conservation, growing from as few as 50-100 animals in the 1880s, to approximately 20,000 white rhinoceros remaining today. However, illegal trade in conservational rhinoceros horns is adding constant pressure on remaining populations. Captive management of ex situ populations of endangered species using molecular methods can contribute to improving the management of the species. Here we compare for the first time the utility of 33 Single Nucleotide Polymorphisms (SNPs) and nine microsatellites (MS) in isolation and in combination for assigning parentage in captive White Rhinoceros. We found that a combined dataset of SNPs and microsatellites was most informative with the highest confidence level. This study thus provided us with a useful set of SNP and MS markers for parentage and relatedness testing. Further assessment of the utility of these markers over multiple (> three) generations and the incorporation of a larger variety of relationships among individuals (e.g. half-siblings or cousins) is strongly suggested.

Interspecific hybridization between greater kudu and nyala.

Hybridization of wildlife species, even in the absence of introgression, is of concern due to wasted reproductive effort and a reduction in productivity. In this study we detail an accidental mating between a female nyala (Tragelaphus angasii) and a male greater kudu (T. strepsiceros). The hybrid was phenotypically nyala and was identified as such based on mitochondrial DNA. Further genetic analysis based on nine microsatellite markers, chromosome number and chromosome morphology however, confirmed its status as an F1 hybrid. Results obtained from a reproductive potential assessment indicated that this animal does not have the potential to breed successfully and can be considered as sterile.

Methylation-based markers for the estimation of age in African cheetah, Acinonyx jubatus.

Age is a key demographic in conservation where age classes show differences in important population metrics such as morbidity and mortality. Several traits, including reproductive potential, also show senescence with ageing. Thus, the ability to estimate age of individuals in a population is critical in understanding the current structure as well as their future fitness. Many methods exist to determine age in wildlife, with most using morphological features that show inherent variability with age. These methods require significant expertise and become less accurate in adult age classes, often the most critical groups to model. Molecular methods have been applied to measuring key population attributes, and more recently epigenetic attributes such as methylation have been explored as biomarkers for age. There are, however, several factors such as permits, sample sovereignty, and costs that may preclude the use of extant methods in a conservation context. This study explored the utility of measuring age-related changes in methylation in candidate genes using mass array technology. Novel methods are described for using gene orthologues to identify and assay regions for differential methylation. To illustrate the potential application, African cheetah was used as a case study. Correlation analyses identified six methylation sites with an age relationship, used to develop a model with sufficient predictive power for most conservation contexts. This model was more accurate than previous attempts using PCR and performed similarly to candidate gene studies in other mammal species. Mass array presents an accurate and cost-effective method for age estimation in wildlife of conservation concern.

Potential drivers of samango monkey (Cercopithecus albogularis) population subdivision in a highly fragmented mountain landscape in northern South Africa.

Forests affected by fragmentation are at risk of losing their primate populations over the long term. The impact of fragmentation on primate populations has been studied in several places in Africa, Asia and South America; however, there has been no discernible pattern of how primates react to forest disturbance and fragmentation. In fragmented habitats, the local extinction probability of a species increases due to a decrease in patch area and an increase in genetic isolation. Here we used microsatellite markers and mitochondrial DNA sequences to investigate how habitat fragmentation impacts on the genetic diversity and structure of a samango monkey population inhabiting forest patches in the Soutpansberg mountain range of northern South Africa. We sampled four local populations across the length of the mountain range and an additional outlying population from the Great Escarpment to the south. Our results indicate that local populations along the mountain range were historically more connected and less distinct than at present. In more recent times, a lack of contemporary gene flow is leading to a more pronounced genetic structure, causing population subdivision across the mountain and likely isolating the Soutpansberg population from the escarpment population to the south. Based on our results, we suggest that natural and anthropogenic fragmentation are driving population genetic differentiation, and that the matrix surrounding forests and their suitability for samango monkey utilisation play a role at the local scale. The degree of genetic isolation found for samango monkey populations in our study raises concerns about the long-term viability of populations across the mountain range.

Absence of 2899CPanthera leo) with Polymyopathy.

Polyphasic skeletal muscle degeneration, necrosis and mineralization of skeletal muscle was diagnosed in eight juvenile free-ranging lions (Panthera leo), from five different litters in the Greater Kruger National Park area that were unable to walk properly. A detailed investigation was not possible in free-ranging lions, so the cause could not be determined. The cases resembled hypokalemic polymyopathy in domestic cats with muscle weakness. A candidate-gene approach previously identified a nonsense mutation in the gene coding for the enzyme lysine-deficient 4 protein kinase (WNK4) associated with the disease in Burmese and Tonkinese cats. In this study, we sequenced all 19 exons of the gene in one case, and two control samples, to identify possible mutations that may be associated with polymyopathy in free-ranging lions. Here, no mutations were detected in any of the exons sequenced. Our findings indicate that the WNK4 gene is not a major contributor to the condition in these lions. Further studies into the pathogenesis of this condition are needed to inform conservation policies for this vulnerable, iconic African species.

Monitoring compliance of CITES lion bone exports from South Africa.

From 2008 to 2018, South Africa permitted the export of captive-bred African lion (Panthera leo) skeletons to Southeast Asia under CITES Appendix II. Legal exports rose from approximately 50 individuals in 2008 to a maximum of 1,771 skeletons in 2016, and has led to ongoing concerns over possible laundering of non-lion, multiple-source and wild-sourced bones. South Africa is required under its obligations to CITES to employ mechanisms for monitoring and reporting trade, and to limit the potential for illegal trade and laundering of lion and other large felid bones. Monitoring tools for legal trade are critical to compliance with CITES. Here we evaluate the CITES-compliance procedure implemented by South Africa for export of lion bones and identify six essential general points for consideration in the implementation of animal export quota compliance protocols. We provide specific insight into the South African lion bone export monitoring system through: i) outlining the protocols followed; ii) assessing the utility of cranial morphology to identify species; iii) evaluating skeleton consignment weight as a monitoring tool; and iv) presenting molecular (DNA) species assignment and pairwise-comparative sample matching of individuals. We describe irregularities and illicit behaviour detected in the 2017 and 2018 lion bone quotas. Notably, we report that the compliance procedure successfully identified and prevented the attempted laundering of a tiger (P. tigris) skeleton in 2018. We emphasise the utility of mixed-method protocols for the monitoring of compliance in CITES Appendix II export quota systems.

Population and genetic structure of a male-dispersing strepsirrhine, Galago moholi (Primates, Galagidae), from northern South Africa, inferred from mitochondrial DNA.

The habitats of Galago moholi are suspected to be largely fragmented, while the species is thought to be expanding further into the southernmost fringe of its range, as well as into human settlements. To date, no intraspecific molecular genetic studies have been published on G. moholi. Here we estimate the genetic diversity and connectivity of populations of G. moholi using two mitochondrial gene regions, the cytochrome C oxidase subunit I gene (COI) and the displacement loop of the control region (D-loop). Samples from five localities in northern South Africa were obtained from archived collections. The two mitochondrial DNA gene regions were amplified and sequenced to provide population summary statistics, differentiation [proportion of the total genetic variation in a population relative to the total genetic variance of all the populations (FST), differentiation within populations among regions (ΦST)], genetic distance and structure. There was discernible genetic structure among the individuals, with two COI and six D-loop haplotypes belonging to two genetically different groups. There was population differentiation among regions (FST = 0.670; ΦST = 0.783; P < 0.01). However, there were low levels of differentiation among populations, as haplotypes were shared between distant populations. Adjacent populations were as divergent from each other as from distant populations. The results suggest that genetic introgression, most likely due to past migrations or recent unintentional translocations that include the animal trade, may have led to connectivity among populations.

Isolation and characterization of 30 STRs in Temminck's ground pangolin (Smutsia temminckii) and potential for cross amplification in other African species.

Temminck's ground pangolin (Smutsia temminckii) is one of four species of pangolin, endemic to Africa. Two of the African pangolin species are listed as vulnerable and two are listed as endangered on the International Union for Conservation of Nature Red List of Threatened Species due to their ongoing exploitation for traditional medicine and bushmeat. In this study, we developed 30 species-specific short-tandem repeats (STRs) in Temminck's ground pangolin using next-generation sequencing. The markers were also optimized for crossamplification in other African species. All the markers amplified successfully in Temminck's ground pangolin with allelic polymorphisms observed in 87% of the markers in giant pangolin (S. gigantea) whereas 60% of the markers were amplified polymorphic loci in both whitebellied pangolin (Phataginus tricuspis) and black-bellied pangolin (P. tetradactyla). Analysis of diversity estimates showed moderate levels of variability in Temminck's ground pangolin (Na = 5; Ho = 0.559), giant pangolin (Na = 4.909; Ho = 0.514) and white-bellied pangolin (Na= 2.686; Ho = 0.541) with lower values being observed in black-bellied pangolin (Na = 3; Ho = 0.242). This study provides data of the first available STR markers which was amplified in all four African pangolin species that can now be used in conservation genetic and evolutionary aspects of population histories.

Molecular characterization in the toll-like receptor 9 gene of Cape Mountain Zebra (Equus zebra zebra) from three populations.

Toll-like receptors (TLR) are a family of proteins that signal activation of the innate immune response through the recognition of a variety of pathogen molecular compounds. Here, we characterized the complete TLR9 gene in Cape mountain zebra (Equus zebra zebra) from three populations in South Africa and compared sequences to a variety of horse and donkey breeds. Overall, we identified six single nucleotide polymorpHisms (SNPs). A single SNP (G586S) was non-synonymous, whereas the remaining SNPs were synonymous. The G586S alteration was detected in Cape mountain zebra populations with varying frequency. In addition, adaptive diversity was found to be discordant with variation based on neutral markers. The mutation is unique to the Cape mountain zebra when compared to other equid species. The structure of TLR9 is relatively conserved and the resulting amino acid substitution was found to have minimal interaction with active sites in the protein. Future studies can explore the effects of this potentially functional mutation which will contribute to our understanding of genetic diversity within adaptive sites of the Cape mountain zebra genome.

Occurrence and diversity of avian haemosporidia in Afrotropical landbirds.

Avian haemosporidian infections are widespread and can result in the decline of wild bird populations or in some cases contribute to extinction of species. We determined the prevalence and genetic diversity of avian haemosporidia in 93 samples from 22 landbird species from South Africa (N = 76) and West Africa (N = 17), of which six are intra-African migrants and one is a Palearctic migrant. The samples were analysed for the presence of avian haemosporidian DNA using real-time quantitative PCR (qPCR) and nested PCR assays targeting specific mitochondrial genes of these parasites. The cytochrome b (cytb) gene was sequenced for all samples that tested positive and phylogenetic analysis was conducted in order to determine the relationship of the new sequences with previously published sequences from the MalAvi database. The overall prevalence of avian haemosporidiosis was 68.82% (95% CI: 56.4%-78.87%) and 82.80% (95% CI: 65.68%-86.11%) as determined by qPCR and nested PCR respectively. Eighteen (19.36%; 95% CI; 10.78%-29.97%) samples had mixed infections. Infection prevalence of all haemosporidian spp. were significantly higher (p < 0.05) in samples from West Africa. Forty-six mitochondrial sequences obtained from 14 avian species grouped into three distinct clusters of Haemoproteus (36), Leucocytozoon (8) and Plasmodium (2). These represent eight published and nine new cytb lineages. The most common lineage was Haemoproteus sp. (VIMWE1) which was identified in two bird species from West Africa and seven bird species from South Africa. This study adds to our knowledge of host-parasite relationships of avian haemosporidia of Afrotropical birds.

Canine parvovirus detected from a serval (Leptailurus serval) in South Africa.

Canine parvovirus first emerged in domestic dogs (Canis familiaris), most likely as a variant of the feline panleucopaenia virus. Relatively recently, canine parvovirus-2a and canine parvovirus-2b infections have been identified in both symptomatic and asymptomatic domestic cats, while canine parvovirus infections have also been demonstrated in wild felids. This report documents the first known case of canine parvovirus-2b detected in unvaccinated serval (Leptailurus serval) from South Africa. The serval presented with clinical signs of vomiting, anorexia and diarrhoea that responded to symptomatic treatment. Two weeks later, severe leucopaenia, thrombocytopenia and death occurred. Typical enteric histological lesions of parvovirus infection were not observed on histopathological examination of the small intestine; however, histological lesions consistent with septicaemia were present. Canine parvovirus was detected in formalin-fixed paraffin-embedded small intestine using polymerase chain reaction. Phylogenetic analysis of the sequence of the canine parvovirus viral capsid protein gene showed similarities between the sample from the serval and canine parvovirus-2b isolates from domestic dogs in Argentina and South Africa. A case of canine parvovirus-2b in a domestic dog from South Africa in 2012 that fell within the same clade as the serval sample appears distantly related because of the long branch length. The significance of these findings is explored. More extensive surveys of canine parvovirus in domestic and wild felids and canids are needed to understand the epidemiology of canine parvovirus in non-domestic felids in South Africa.

Lessons for conservation management: Monitoring temporal changes in genetic diversity of Cape mountain zebra (Equus zebra zebra).

The Cape mountain zebra (Equus zebra zebra) is a subspecies of mountain zebra endemic to South Africa. The Cape mountain zebra experienced near extinction in the early 1900's and their numbers have since recovered to more than 4,800 individuals. However, there are still threats to their long-term persistence. A previous study reported that Cape mountain zebra had low genetic diversity in three relict populations and that urgent conservation management actions were needed to mitigate the risk of further loss. As these suggestions went largely unheeded, we undertook the present study, fifteen years later to determine the impact of management on genetic diversity in three key populations. Our results show a substantial loss of heterozygosity across the Cape mountain zebra populations studied. The most severe losses occurred at De Hoop Nature Reserve where expected heterozygosity reduced by 22.85% from 0.385 to 0.297. This is alarming, as the De Hoop Nature Reserve was previously identified as the most genetically diverse population owing to its founders originating from two of the three remaining relict stocks. Furthermore, we observed a complete loss of multiple private alleles from all populations, and a related reduction in genetic structure across the subspecies. These losses could lead to inbreeding depression and reduce the evolutionary potential of the Cape mountain zebra. We recommend immediate implementation of evidence-based genetic management and monitoring to prevent further losses, which could jeopardise the long term survival of Cape mountain zebra, especially in the face of habitat and climate change and emerging diseases.

Assessing introgressive hybridization in roan antelope (Hippotragus equinus): Lessons from South Africa.

Biological diversity is being lost at unprecedented rates, with genetic admixture and introgression presenting major threats to biodiversity. Our ability to accurately identify introgression is critical to manage species, obtain insights into evolutionary processes, and ultimately contribute to the Aichi Targets developed under the Convention on Biological Diversity. The current study concerns roan antelope, the second largest antelope in Africa. Despite their large size, these antelope are sensitive to habitat disturbance and interspecific competition, leading to the species being listed as Least Concern but with decreasing population trends, and as extinct over parts of its range. Molecular research identified the presence of two evolutionary significant units across their sub-Saharan range, corresponding to a West African lineage and a second larger group which includes animals from East, Central and Southern Africa. Within South Africa, one of the remaining bastions with increasing population sizes, there are a number of West African roan antelope populations on private farms, and concerns are that these animals hybridize with roan that naturally occur in the southern African region. We used a suite of 27 microsatellite markers to conduct admixture analysis. Our results indicate evidence of hybridization, with our developed tests using a simulated dataset being able to accurately identify F1, F2 and non-admixed individuals at threshold values of qi > 0.80 and qi > 0.85. However, further backcrosses were not always detectable with backcrossed-Western roan individuals (46.7-60%), backcrossed-East, Central and Southern African roan individuals (28.3-45%) and double backcrossed (83.3-98.3%) being incorrectly classified as non-admixed. Our study is the first to confirm ongoing hybridization in this within this iconic African antelope, and we provide recommendations for the future conservation and management of this species.

Genetic assessment of an isolated endemic Samango monkey (Cercopithecus albogularis labiatus) population in the Amathole Mountains, Eastern Cape Province, South Africa.

The endemic Samango monkey subspecies (Cercopithecus albogularis labiatus) inhabits small discontinuous Afromontane forest patches in the Eastern Cape, KwaZulu-Natal midlands and southern Mpumalanga Provinces in South Africa. The subspecies is affected by restricted migration between forest patches which may impact on gene flow resulting in inbreeding and possible localized extinction. Current consensus, based on habitat quality, is that C. a. labiatus can be considered as endangered as the small forest patches they inhabit may not be large enough to sustain them. The aim of this study was to conduct a molecular genetic investigation to determine if the observed isolation has affected the genetic variability of the subspecies. A total of 65 Samango monkeys (including juveniles, subadults and adults) were sampled from two localities within the Hogsback area in the Amathole Mountains. Nuclear and mitochondrial DNA variation was assessed using 17 microsatellite markers and by sequencing the hypervariable 1 region (HVR1). Microsatellite data generated was used to determine population structure, genetic diversity and the extent of inbreeding. Sequences of the HVR1 were used to infer individual origins, haplotype sharing and haplotype diversity. No negative genetic factors associated with isolation such as inbreeding were detected in the two groups and gene flow between groups can be regarded as fairly high primarily as a result of male migration. This was in contrast to the low nuclear genetic diversity observed (H o = 0.45). A further reduction in heterozygosity may lead to inbreeding and reduced offspring fitness. Translocations and establishment of habitat corridors between forest patches are some of the recommendations that have emerged from this study which will increase long-term population viability of the subspecies.

Isolation and characterization of species-specific microsatellite markers for blue and black wildebeest (Connochaetes taurinus and C. gnou).

The bluewildebeest (Connochaetes taurinus) is distributed throughout southern and east Africa while the black wildebeest (Connochaetes gnou) is endemic to South Africa and was driven to near extinction in the early 1900s due to hunting pressure and disease outbreaks. Extensive translocation of both species throughout South Africa is threatening the genetic integrity of blue and blackwilde beest. To effectively manage these species, genetic tools that can be used to detect hybrid individuals, identify genetically unique subpopulations and determine the levels of genetic diversity are required. In this study, 11 microsatellite markers were developed for wildebeest through next-generation sequencing. The microsatellite loci displayed 2.00-4.14 alleles, unbiased heterozygosity values ranged from 0.32 to 0.60 and observed heterozygosity values ranged from 0.26 to 0.52. The comparatively high level of polymorphism observed in the microsatellite markers indicates that these markers can contribute significantly to our knowledge of population genetic structure, relatedness, genetic diversity and hybridization in these species.