Assessing the phylogeography and metabolomic signatures of wild rooibos (Aspalathus linearis) ecological populations

Thesis (MSc)--Stellenbosch University, 2021.ENGLISH ABSTRACT: Aspalathus linearis (Burm. F.) R. Dahlgren, Fabaceae, is an entirely endemic fynbos species, limited to the Cape Floristic Region of South Africa. It is commonly known as rooibos, and is enjoyed as a herbal tea by many South Africans. It is sought after for its health-promoting effects, particularly its antioxidant, antispasmodic and antiaging potential owing to its phytochemical profile. Wild rooibos, unlike the cultivated type, is variable in morphology, biochemistry, ecology and genetics and is classified into ecological types (ecotypes). Ecotypes can broadly be distinguished into two main groups, namely, reseeders and resprouters, based on their fire-survival strategy. These ecotypes are perceived as being variable and distinct by local harvesters and land-user communities, who possess traditional ecological knowledge associated with its rooibos population. Scientific studies have complimented this traditional knowledge to characterise and create a detailed account of morphological, chemical and ecological differences between ecotypes, coupled to their geographical location. Variability among wild rooibos populations were investigated to determine the impact of environmental influences and biochemical differences on genetic variability. Few studies currently exist that address the genetic characterisation and diversity of wild rooibos. The study described here focuses on the metabolomic and genetic variation between six collected wild ecotypes, occurring in the Cederberg Mountains of the Western Cape and the Suid Bokkeveld and Nieuwoudtville area of the Northern Cape. Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) revealed three main metabolomic clusters, yet each population showed distinct chemical profiles. The principal component analysis (PCA) displayed unique chemical profiles between collected wild resprouter populations of the Northern Cape versus the reseeder populations of the Cederberg. The PCA analysis explained 36.79 % of the total variance. Metabolites were identified using a systematic semi-targeted approach and it is also in these plants that several unknown metabolites were detected in this rooibos populations. Overall, the Dobbelaarskop population appeared to have higher concentrations of the metabolites in comparison to the other populations. Dobbelaarskop had the highest relative concentration of aspalathin (165 mg/kg dry weight). Heuningvlei had a relative concentration of 50 mg/kg dry weight of nothofagin, while the rest of the populations contained less than 20 mg/kg dry weight. Overall, plants from the Jamaka region had consistently lower metabolite concentrations than the other populations. Phylogeographical analysis of both chloroplast and newly developed species-specific nuclear markers (microsatellites) was performed on the collected populations. No previous assessment of genetic diversity or population structure using microsatellite markers has been conducted in Aspalathus linearis. From the diversity indices, it is evident that the wild rooibos populations have low-to- moderate genetic diversity. The Jamaka population had the lowest haplotype diversity (0.286), and the lowest nucleotide diversity (0.006) even though haplotype diversity generally varied considerably across the wild populations (h = 0.286 - 0.900). The data revealed large variations in haplotype diversity and nucleotide diversity between populations and between regions. Our data suggests that populations of rooibos become less diverse from the Melkkraal population down towards the Cederberg populations, possibly indicative of clinal variation. The largest genetic differentiation was between Heuningvlei and Jamaka (FST = 0.101), and Blomfontein and Jamaka (FST = 0.101). There was also a significant correlation between geographical distance and genetic distance (R2 = 0.296, P = 0.044). The presence of three main clusters are also clearly reflected in the discriminant analysis of principal components (DAPC) based on the newly developed set of microsatellite markers. This study has successfully established a best practice protocol and provided a highly regenerative plant growth regulator (PGR) regime for shoot multiplication and in vitro seed (cultivated type) germination of Aspalathus linearis. A combination of smoke and sulphuric acid scarification of 20-30 minutes yielded the highest germination rate of cultivated seeds. The PGR ratios, 1:0 and 3:0 [benzyladenine (BA): 1-naphthaleneacetic acid (NAA)], resulted in micropropagules with longer stems and an increase in number of leaves. A phytohormone combination of 5 BA: 3 NAA resulted in callus formation and root growth. The chemical profiles provided by this study are complementary to the novel contribution of genetic characterisation of wild rooibos. These findings may be useful in identifying unique populations for agricultural, phytopharmaceutical or industrial purposes without overlooking conservation and sustainable harvesting priorities.AFRIKAANSE OPSOMMING: Aspalathus linearis (Burm. F.) R. Dahlgren, Fabaceae, is 'n volkome endemiese fynbos-spesie, beperk tot die Kaapse Floristiese streek van Suid-Afrika. Dit wat algemeen bekend staan as rooibos word deur menigte Suid-Afrikaners as 'n kruietee geniet. Dit is besonders gesog weens die gesondheidsbevorderende uitwerking, veral die antioksidant-, antispasmodiese- en antiverouderingspotensiaal vanweë sy fitochemiese profiel. In teenstelling met die gekweekte tipe is wilde rooibos se morfologie, biochemie, ekologie en genetika veranderlik en word dit volgens ekologiese tipes (ekotipes) geklassifiseer. Ekotipes kan breedweg in twee hoofgroepe onderskei word, naamlik hersaaiers en herstellers, gebaseer op hul oorlewingsstrategie teen vuur. Hierdie ekotipes word beskou as veranderlik en afsonderlik deur plaaslike plukkers en grondgebruiker gemeenskappe met tradisionele ekologiese kennis verbonde aan rooibos-populasies. Ondersoeke is gedoen op die veranderlikheid tussen wilde rooibos-populasies, om die impak van omgewingsinvloede en biochemiese verskille op genetiese veranderlikheid vas te stel. Daar bestaan tans ‘n tekort aan studies wat die genetiese karakterisering en diversiteit van wilde rooibos aanspreek. Die studie wat hier beskryf word fokus op die metabolomiese en genetiese variasie tussen ses versamelde wilde ekotipes wat voorkom in die Sederberge van die Wes-Kaap en in die Suid-Bokkeveld en Nieuwoudtville van die Noord-Kaap. Ultraverrigting-vloeistofchromatografie-massaspektrometrie (UPLC-MS) het drie hoofmetabolomiese groepe onthul, maar elke populasie het afsonderlike chemiese profiele getoon. Die hoofkomponentontleding (PCA) het unieke chemiese profiele getoon tussen die versamelde wilde hersteller-populasies van die Noord-Kaap en die hersaaier-populasies van die Sederberge. Die PCA-ontleding het 36,79% van die totale variansie verduidelik. Metaboliete is geïdentifiseer met behulp van 'n sistematiese semi-geteikende benadering. In vergelyking met die ander populasies toon die Dobbelaarskop-populasie ‘n algeheel hoër konsentrasies van metaboliete aan. Dobbelaarskop het die hoogste relatiewe konsentrasie van aspalatien (165 mg / kg droë gewig) gehad. Heuningvlei het 'n relatiewe konsentrasie van 50 mg / kg droë gewig notofagien gehad, terwyl die res van die populasies minder as 20 mg / kg droë gewig daarvan bevat het. Plante uit die Jamaka- streek het konsekwent laer konsentrasies van metaboliete as die ander populasies getoon. Filogeografiese analise van beide chloroplast en nuut ontwikkelde spesie-spesifieke kernmerkers (mikrosatelliete) is op die versamelde populasies uitgevoer. Geen assessering van genetiese diversiteit of populasiestruktuur is vantevore met behulp van mikrosatellietmerkers op Aspalathus linearis uitgevoer nie. Uit die diversiteitsindekse blyk dit dat die wilde rooibos-populasies lae-tot-matige genetiese diversiteit het. Die Jamaka-populasie het die laagste diversiteit van die haplotipe (0.286) en die laagste nukleotieddiversiteit (0.006) gehad, alhoewel die diversiteit van die haplotipe deurgaans grootliks in die wilde populasies gevarieer het (h = 0.286 - 0.900). Die data het groot variasies in diversiteit van haplotipes en diversiteit van nukleotiede tussen populasies en tussen streke onthul. Ons data stel voor dat rooibos-populasies minder divers word van die Melkkraal-populasie af tot by die Cederberg-populasie, moontlik aanduidend van klinale variasie. Die grootste genetiese differensiasie was tussen Heuningvlei en Jamaka (FST = 0.101) en Blomfontein en Jamaka (FST = 0.101). Daar was ook 'n beduidende korrelasie tussen geografiese afstand en genetiese afstand (R2 = 0,296, P = 0,044). Die teenwoordigheid van drie hoofgroepe word ook duidelik weerspieël in die diskriminerende ontleding van hoofkomponente (DAPC) gebaseer op die stel nuut ontwikkelde mikrosatellietmerkers. Hierdie studie het 'n beste praktyk protokol suksesvol gevestig en 'n hoogs regeneratiewe plantgroeireguleerder (PGR) regime verskaf vir die vermenigvuldiging en in vitro-saad (gekweekte tipe) ontkieming van Aspalathus linearis. 'n Kombinasie van rook en skarifikasie met swawelsuur van 20 tot 30 minute het die hoogste ontkiemingskoers van gekweekte sade gelewer. Die PGR- verhoudings, 1: 0 en 3: 0 [bensieladenien (BA): 1-naftaleen-asynsuur (NAA)], het gelei tot mikropropagules met langer stingels en 'n vermeerdering in aantal blare. 'n Fitohormoonkombinasie van 5 BA: 3 NAA het tot kallusformasie en wortelgroei gelei. Die chemiese profiele wat deur hierdie studie verskaf word, is aanvullend tot die nuwe bydrae van genetiese karakterisering van wilde rooibos. Hierdie bevindinge kan nuttig wees om unieke populasies vir landbou-, fitofarmaseutiese- of industriële doeleindes te identifiseer sonder om bewarings- en volhoubare oesprioriteite te verontagsaam.Master