- ItemLandscape and Paleoclimatic influences on the genetic population structure of four forest-dependent passerines in the Eastern Cape of South Africa(Stellenbosch : Stellenbosch University, 2021-12) Mulvaney, Jake Matthew; Cherry, Michael; Matthee, Conrad; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: Anthropogenic activity has placed increasing pressure on the restricted, fragmented forest biome of South Africa. Recent assessments of forest-dependent avifauna strongly indicate the vulnerability of this taxon to deforestation, and ongoing forest exploitation. Half of the forest- dependent bird species in South Africa have reportedly experienced range declines over the past quarter century, most notably within forests of the Eastern Cape province that are incorporated in the Maputaland-Pondoland-Albany Biodiversity Hotspot. These apparent declines have motivated a need to understand the population dynamics, and forest connectivity patterns of forest-dependent avifauna within the country, to better inform conservation efforts seeking to preserve the genetic integrity of these vulnerable bird species. This study investigated the population genetic structures of four range-declining forest- dependent Oscine passerines across the Eastern Cape and southern KwaZulu-Natal provinces of South Africa: Batis capensis (range decline: 1.30%), Cossypha dichroa (range decline: 19.53%), Phylloscopus ruficapilla (range decline 20.69%), and Pogonocichla stellata (range decline 23.02%). These four bird species are small-bodied (<50g) insectivores, that preferentially inhabit the temperate Afromontane forests, and represent a globally important functional group vulnerable to forest fragmentation. The landscape genetics study conducted on these four species revealed that reported range declines did not correspond closely to the genetic responses of these bird species to anthropogenic activity. Batis capensis showed substantial geneflow disruption, and declining effective populations, whereas P. ruficapilla and P. stellata populations appeared comparatively stable, although P. stellata did exhibit geneflow disruption. Only the South African endemic C. dichroa displayed simultaneous decline in distribution and effective population size, alongside geneflow disruptions, highlighting a vulnerability to forest loss and degradation. Landscape resistance modelling revealed the importance of both forest and coastal/mesic thicket for maintaining geneflow within these species. A phylogeographic study investigated the regional species-climate relationships of B. capensis, P. ruficapilla and P. stellata. The effective populations of these species were found not to have been constrained during the last glacial maximum – a climate event speculated to have resulted in drastically restricted forest distributions across South Africa. Furthermore, lowland scarp forests were affirmed as climate refugia for B. capensis and P. stellata, while the Afromontane forests of the Amatole Mountains and central Transkei appear to have harboured regional source populations of P. ruficapilla. Finally, comprehensive mist-netting of regional forests during sampling collection for the genetic studies afforded an opportunity to compare the effectiveness of point counts and mist- netting survey techniques at representing aspects of regional forest bird community structure. These comparisons found that point counts alone were sufficient to reliably assess these bird communities, with mist-netting contributing negligibly towards species detection. Combined survey efforts under-represented forest-edge foragers, woodland and grassland habitat generalists (collectively comprising ~63.6% total diversity), large birds, Palaearctic migrants, and carnivores (raptors), highlighting the potential shortcomings of these survey techniques in determining bird community composition. Overall, this study provided novel insights into forest connectivity; and past forest dynamics of forest-dependent insectivorous passerines within previously poorly investigated forests of the southern Maputaland-Pondoland-Albany Biodiversity Hotspot, and provides recommendations for future field surveys of these forests.
- ItemEvolution and Morphological Shape Ontogeny of the Brown ticks (Acari: Ixodida: Ixodidae: Rhipicephalus)(Stellenbosch : Stellenbosch University, 2021-04) Bakkes, Deon Kahlil; Matthee, Conrad A.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: Rhipicephalus constitute a species-diverse genus of mammal ectoparasites mainly distributed in the Afrotropics that are characterised by generally inornate, uniform brown body colour, short hypostome and palps, basis capituli approximately hexagonal, eyes present and male adanal plates present. They transmit microparasites such as Rickettsia spp., Theileria spp. and Babesia spp. to livestock and humans alike, and some inject neurotoxins during feeding that lead to tick paralysis in livestock. This work infers the phylogeny of Rhipicephalus from molecular lines of evidence (12S, 16S, COI and 28S-D2) and uses this as a basis to infer aspects of their evolutionary history, ecology and evolutionary-development based on geographic distribution data and basis capitulum shape data. Analyses included estimation of divergence times, ancestral area optimisations, ancestral host-use optimisations in immatures and adults, as well as estimations of ancestral climate niches. Basis capitulum morphology is quantified to determine evolutionary- developmental modifications, which are linked to similar patterns in overall body size. Major outcomes of studying Rhipicephalus evolution are 1) radiations coincide with mammal evolution and dispersal, 2) host-use at immature stages partially explain extent of geographic ranges as well as basis capitulum morphology for boring into thick host skin, 3) evolutionary host switches were facilitated by off-host periods and nested connections in predator-prey food webs, 4) speciation partially resulted from niche partitioning along temperature variation gradients, which was reinforced by interspecific competition, and 5) evolutionary-developmental modification (basis capitulum and overall body size) resulted from responses to distinct sets of selection pressure in on- and off-host environments taking into account one-, two- and three-host life cycles. The persistent taxonomic problem of R. turanicus between Palearctic and Afrotropical regions was investigated using integrated lines of evidence to test the species boundary in an iterative framework. This revealed two distinct species in these regions, and the Afrotropical species is described under the name R. afranicus. Another aim of this work was to test the hypothesis of phylogenetic recapitulation in post-embryonic stages of Rhipicephalus basis capituli, where early developmental stages resemble ancestral adults. However, findings indicate no signal for phylogenetic recapitulation is present, most likely due to the action of selection that shape basis capituli over evolutionary timescales. Selection supersedes any possible background action of condensing selection through development that would produce phylogenetic recapitulation. As such, this work serves as a first step for investigating phylogenetic recapitulation using shape data, and suggests future investigation should consider embryonic life stages, alternative features under less selection, or wider phylogenetic comparisons.
- ItemSceletium tortuosum (L.) N.E. Br.: mesembrine alkaloid chemotype signatures in wild type, cultivated and in vitro tissues(Stellenbosch : Stellenbosch University, 2021-04) Glyn-Woods, Christina; Makunga, Nokwanda P.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: The South African indigenous, medicinal succulent Sceletium tortuosum (L.) N.E. Br., has entered the world stage, as increasing evidence of the anxiolytic and human health applications of its mesembrine alkaloid suite emerges. Clinical trials, confirming ethnopharmacogical accounts linked to their bioactivity, has ensured the demand for these metabolites will increase. Concommittal with this global market opportunity, is the need to optimize and tailor existing horticultural production systems, and the parallel development of alternative, high fidelity, phytopharmaceutical production platforms to meet this demand. To accomplish this, greater insight into the fundamental triggers, endogenous and environmental, determining mesembrine alkaloid signatures in planta is needed. The driving rationale behind this research, therefore, was to populate this current gap in knowledge of the in planta aspects of mesembrine alkaloid biosynthesis. Using the resolving power of ultra-high-performance liquid chromatography mass spectroscopy (UHPLC-MS), to quantify; and multivariate principal component analysis (PCA), to visualise, a quantitative and qualitative examination of the factors shaping chemotype signatures in living tissue was undertaken. Firstly, a global view of the seasonal accumulation patterns of the different mesembrine alkaloids was determined. Chemotype profiles of a single, limited range, wild type population were sampled across the 4 seasons of one year. Correlation with physiological plant growth parameters revealed the accompanying growth phase mesembrine alkaloid expression patterns. During summer, the major alkaloids mesembrine (4.01 ±5.62) mg/kg DW, mesembrenol (2.49 ±2.85) mg/kg and mesembranol (52.79 ±51.29) mg/kg DW and the isomers 6-epimesembrenol (2.10 ±2.19) mg/kg DW and 6-epimesembranol (1.45 ±1.41) mg/kg DW exhibited maximal tissue accumulation. This season, of extreme high temperatures and water scarcity, coincided with greatly curtailed plant growth. Conversely during autumn and winter, during which plant growth is initiated and active, respectively, accumulation minima were recorded. This first report of the seasonal accumulation patterns of the mesembrine alkaloids of S. tortuosum, corroborates and explains for the first time the rational of the traditional practice of the summer harvest of these plants. Significant seasonal alkaloid specific accumulation differences were found, however, large within season variation in alkaloid content was also observed. It is proposed that this variation is underpinned by a large, inherent, intra-population chemical heterogeneity occurring amongst individuals. It is conjectured, based on these observations, that additional significant factors, besides seasonality, are determinate in shaping chemotypic variability of the wild growing material and the suggested possibility of inducibility of these alkaloids, awaits further investigations. A potential photoprotective role of the mesembrine alkaloids is proposed. This conjecture is grounded in the high relative amounts of key mesembrines recorded in summer, the spatial accumulation of specific mesembrines in the outer leaves of the bud growth structures and the inherent photochemical UV quenching properties of the mesembrine alkaloids themselves. The high irradiance and incident UV light, inherent to the natural habitats of these plants, would render such a functionality of these compounds an evolutionary advantage. Additionally, in wild type plants an upregulation of betalain pigments and key mesembrine alkaloids over the year, reaching a maximum in summer, was observed. These betalain pigments, made up of the red to maroon betacyanins and yellow betaxanthins groups, occur exclusively in Caryophyllaceae order plants, to which S. tortuosum belongs. As aromatic ring chromophores, these pigments, besides antioxidant and osmotic protectant properties, also possess inherent absorption maxima in both the UV, and visible region, and are functional analogues of the more ubiquitous anthocyanins, with which they share similar photochemical properties. It was interesting to realise, that the nitrogenous mesembrines and betalains share a common primary metabolism pathway precursor, the amino acid tyrosine. While speculative, an interaction of these two metabolite pathways is hypothesised, and may hold clues as to their respective co-evolution, and in planta role, in plants such as S. tortuosum, and the Caryophyllaceae order at large. The second branch of the study examined mesembrine alkaloid accumulation patterns from a tissue developmental perspective. A spatiotemporal study of the chemotypic variation in developing leaves of tunnel grown plants, charted the accumulation of the mesembrine alkaloids to specific leaf age, developmental stages. Mesembrine (5156.7 ±1276.5) mg/kg DW was associated with actively growing leaves, whereas �⁴-mesembrenone (255.9 ±301.15) mg/kg DW, mesembranol (551.52 ±56.4) mg/kg DW and 6-epimesembranol (612.4 ±64.7) mg/kg DW were present at highest levels with the onset and progression of senescent aging of leaves. This new information linked to improved understanding of the developmental and spatial accumulation of these alkaloids will inform biosynthetic pathway investigations and pave the way to defining the possible ecological and/or biological significance of these accumulation patterns. In conclusion, the final branch of the study sought to rapidly and sustainably meet the immediate and future demand for the high value phytochemicals of S. tortuosum, through the development of a suitable in vitro, high throughput, metabolite production technology. Increased scientific and mainstream reports and growing public awareness of the psychoactive properties of S. tortuosum, has exposed wild populations to an increased threat of unscrupulous harvesting. By establishing a biotechnological platform for future production of these compounds, it was envisaged that the current pressure on wild populations could be assuaged. Using in vitro microshoot cultures, a two-phase regimen employing a tissue dehydration pre-treatment step, prior to metabolite extraction, produced commercially exploitable levels of mesembrine (3270.9 ±981.5) mg/kg DW, mesembrenol (333.1 ±36.1) mg/kg DW, mesembranol (4738.8 ±172.9) mg/kg DW and �⁴-mesembrenone (29.9 ±0.5) mg/kg DW in tissue extracts. Additionally, select liquid callus culture lines, once established, accumulated mesembranol (64.6 ±8.5) mg/kg DW as the maJor alkaloid component, and exhibited a retained significant biosynthetic capacity for mesembrine, mesembranol, mesembrenol and �⁴-mesembrenone. These in vitro platforms present a potentially scalable, high throughput alternative, to access mesembrine alkaloids for the various phytopharmaceutical industries. Additionally, the clonal tissue uniformity and exogenous growth parameter control inherent to such technologies have not been previously accessible for this plant and will greatly benefit advances in metabolic pathway and molecular biology investigations. As various key mesembrines are poised to become more prominent in the phytopharmaceutical arena, South Africa, as the home of the Sceletium plant, holds a unique position of responsibility in the global marketplace. The research presented here, on both the seasonal and spatiotemporal chemotype development in this plant, begins to answer for the first-time fundamental question of inherent biological timing and context of mesembrine alkaloid signatures necessary to optimise their harvesting. Sceletium has shared a long history of use within the San culture, and as the industry surrounding this plant grows in this country, and globally, we should remain cognisant of fairness, and of the original knowledge keepers' stake. The production of mesembrine alkaloids using biotechnology as a system, provides a novel manufacturing platform for this species that has the potential to become highly desired in the industry. As the lucrative healing potential of Sceletium unfolds, communities with San heritage are in an opportune position to benefit from their intellectual property and its availability to the world at large, through the commercialisation linked to this work.
- ItemUsing multi-species seascape genomics to conserve areas of evolutionary importance(Stellenbosch : Stellenbosch University, 2021-04) Nielsen, Erica Spotswood; Von der Heyden, Sophie; Henriques, Romina; Beger, Maria; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: Understanding the environmental footprints on species and genetic biodiversity is a key concern in molecular ecology and conservation genetics. As species are increasingly under pressure from anthropogenic climate change, understanding how rapid environmental changes will influence intra- and interspecific diversity is essential if we are to conserve functioning ecosystems. This PhD thesis used the unique environmental backdrop of the South African coastline to infer how environmental variables over space and time shape multiple facets of biological variation. Specifically, this thesis utilised seascape genomic analyses to test the strong environmental gradients within South Africa against the molecular variation of three rocky intertidal species: Cape urchin (Parechinus angulosus), Common shore crab (Cyclograpsus punctatus), and Granular limpet (Scutellastra granularis). The first chapter evaluated which contemporary seascape features most strongly correlate with neutral and adaptive intraspecific diversity across species. Here, the results show that gene-environment relationships are species-specific, with the crab showing less population differentiation, strongly influenced by sea-surface salinity, and the urchin and limpet showing a west-east population differentiation predominantly influenced by sea-surface and air temperature. Chapter Two tested the relative influence of historical climatic stability versus contemporary species distributions in shaping patterns of neutral diversity of the three species. The results from this chapter indicate that historical climatic refugia since the Last Glacial Maximum are potentially stronger predictors of contemporary molecular diversity hotspots than the species’ current distribution. The third research chapter evaluated the vulnerability of the three study species with regards to future climatic change, both at two time-points and under two emission scenarios. Here, the results highlight how future responses to global change will likely differ among species, as well as among populations within each species. In the final chapter, the patterns uncovered in the three data chapters, pertaining to genomic diversity and vulnerability, climatic stability, and adaptive potential, are combined in a conservation planning framework to identify areas of evolutionary importance, which can be thought of as priority areas for forward-thinking conservation action. As a whole, this thesis used novel ecological and evolutionary models to understand the spatio-temporal interplay between species, genes, and environment, and used this information to guide conservation action within South Africa.
- ItemAssessing the evolutionary and physiological resilience of southern African marine species(Stellenbosch : Stellenbosch University, 2021-04) Mertens, Lisa; Measey, John; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: Assessing the genomic basis of local adaptation and critical thermal limits is essential for anticipating species persistence and distribution under climate change. Environmental gradients are associated with genomic and physiological differences between populations. South Africa's two ocean regime creates a thermal gradient, which delimits distinct bioregions between its cold-temperate west coast, warm-temperate south coast and warmsubtropical east coast. Three co-distributed key rocky shore invertebrate species representing different phyla were selected for a multi-species approach. The objectives of this dissertation were to 1) identify selectively neutral genomic loci and neutral population structure, 2) determine putatively adaptive loci and adaptive population structure, 3) explore functional annotations and 4) measure critical thermal limits (CTmin, CTmax). Pooled RAD-Seq (ezRAD) was utilised to identify selectively neutral shared and populationspecific single nucleotide polymorphisms (SNPs) in six populations of shore crab Cyclograpsus punctatus (CP), granular limpet Scutellastra granularis (SG), and Cape sea urchin Parechinus angulosus (PA). Population-specific SNPs were detected in all populations. Nucleotide diversity indices (Tajima.s pi, Watterson.s theta)) appear heightened in PA's northern west coast population compared to the remaining sites. Estimated pairwise FST values range from 0.043-0.055 (CP), 0.044-0.066 (SG) to 0.039-0.089 (PA). Selectively neutral genomic population structure indicates instances of intraspecific subdivisions present in all populations. All species populations harbour unique SNPs, yet increased nucleotide diversity is only detected in PA. The empirical FST-method, BayeScan and BayeScEnv identified overall 1102 outliers under positive selection, of which 69 (CP), 11 (SG) and 27 (PA) could be functionally annotated. Candidate loci are involved in various cellular functions including membrane transport, vesicle signalling, protein folding/modification and cytoskeleton function. Identified loci related to energy cycling might point to selection on metabolic capacity to counter environmental stressors. Environmental differentiation of sea surface temperature (SST), salinity and air temperature could be associated with several putative outliers. There is no isolation-by-distance (IBD), but isolation-by-environment (IBE) suggests salinity variation to account for 48% of genomic variation in P. angulosus and SST and air temperature for 45% in S. granularis. Outlier-based population structure indicates possible intraspecific subdivision in some species. Critical thermal limits (CTmin, CTmax) were investigated with thermal tolerance trials and compared to local min/max environmental temperature for warming and cooling tolerance. Across populations, mean CTmin ranges from -1.5-0.6°C (CP), -0.4-3.2°C (SG) to 5-10.9°C (PA). Mean CTmax ranges from 43.8-46.1°C (CP), 34.4-35.7°C (SG) to 28.9-32.4°C (PA). West coast crabs have significantly higher CTmax than east coast crabs. CTmin is negatively and thermal breadth positively correlated with body mass (CP, PA). Significant regional differences in mass were detected (SG, PA). Warming and cooling tolerance appears sufficient, requiring further investigation within situ microhabitat data. East coast rocky shore populations likely face future warm-edge range contractions, whereas the south coast might experience distributional shifts depending on local thermal conditions. The west coast is an anchor for rocky shore species in South Africa and represents a possible climate change refugium. Strategic recognition in regional marine conservation management is warranted.