Edaphic Factors and Rhizobia influence the Distribution of Legumes (Fabaceae) in the Core Cape Subregion of South Africa Town Meshack Nkosinathi Dludlu Cape Thesis presented for the Degree of of DOCTOR OF PHILOSOPHY Department of Biological Sciences UniversityUNIVERSITY OF CAPE TOWN February 2018 Supervisors: A/Prof. A. Muthama Muasya & Dr Samson B.M. Chimphango The copyright of this thesis vests in the author. No quotation from it or information derivedTown from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes Capeonly. of Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University Declaration I, Meshack Nkosinathi Dludlu know the meaning of plagiarism and declare that all of the work in the thesis, save for that which is properly acknowledged, is my own. I hereby, a) grant the University free licence to reproduce the above thesis in whole or in part for the purpose of research; b) declare that: i. the above thesis is my own unaided work, both in conception and execution, and that apart from the normal guidance from my supervisors, I have received no assistance except as stated below; ii. neither the substance nor any part of the thesis has been submitted in the past, or is being, or is to be submitted for a degree at this University or any other University, except as stated below. I am now presenting the thesis for examination for the degree of PhD. The thesis contains three papers, which were published in collaboration with my supervisors and research associates, A. Muthama Muasya, Samson B.M. Chimphango; Gemma Walker and Charles H. Stirton: 1. Dludlu, M.N., Chimphango, S.B.M., Stirton, C.H., Muasya, A.M., 2017. Distinct edaphic habitats are occupied by discrete legume assemblages with unique indicator species in the Cape Peninsula of South Africa. Journal of Plant Ecology rtx027. doi: 10.1093/jpe/rtx027. 2. Dludlu, M.N., Chimphango, S.B.M., Stirton, C.H., Muasya, A.M., 2018. Differential preference of Burkholderia and Mesorhizobium to pH and soil types in the Core Cape Subregion, South Africa. Genes 9, 2. doi: 10.3390/genes9010002. ii 3. Dludlu, M.N., Chimphango, S.B.M., Walker, G., Stirton, C.H., Muasya, A.M., (accepted). Horizontal gene transfer among rhizobia of the Core Cape Subregion of southern Africa. South African Journal of Botany. My supervisors have testified that I made substantial contributions to the conceptualization and design of the published papers and that I independently ran experiments and wrote the manuscripts, with their support in the form of comments and suggestions (see Appendix D). Signature: Date: February 2018 iii Dedication I dedicate this thesis to my family, whose unrelenting love and support made this possible. iv Acknowledgements I would like to convey my sincere gratitude to my supervisors, A/Prof. Muthama Muasya and Dr Samson Chimphango for their instrumental guidance, motivation and support from the inception of this project throughout the PhD journey. Likewise, I am indebted to Prof. Charles Stirton for his mentorship, which he has been providing cheerfully from the early days of my Master’s degree studies to the present day. I cannot imagine having better supervisors and a better mentor for my thesis. The technicians of the Biological Sciences Department are gratefully acknowledged for their assistance with the sourcing of equipment and reagents and providing access to the relevant laboratory facilities. I am also thankful to Edward Chirwa for his assistance with fieldwork and some of the laboratory work. Dr Benny Lemaire’s enthusiastic guidance on some laboratory techniques is gratefully appreciated. Thanks to Terry Trinder-Smith, the Curator of the Bolus Herbarium, for providing access to datasets on legume occurrence records for the Cape Peninsula. In the same vein, I would like to acknowledge the South African National Biodiversity Institute (SANBI) for providing access to their database (PRECIS), particularly, data on legume occurrence records for the entire Southern Africa region. I acknowledge the Table Mountain National Parks for providing permits to conduct fieldwork in areas under their management. In the same way, I recognize Mr Giorgio Lombardi, the Warden of the Vogelgat Private Nature Reserve, for granting permission to conduct research on the legumes that occur in their reserve. The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Additional financial support towards this study was provided by the University of Cape Town, the University of Swaziland and the Worldwide Universities Network (WUN); to whom I am very grateful. v Abstract Fabaceae is the second most speciose plant family in the Core Cape Subregion (CCR) of South Africa, a Mediterranean type ecosystem, with mostly nutrient-poor soils. A majority of the legumes occurring in this region belong to the predominantly nitrogen-fixing subfamily Papilionoideae and they employ a variety of strategies for nutrient acquisition. However, legumes are neither uniformly nor randomly distributed in the CCR landscape. Instead, distinct legume species assemblages tend to occupy particular habitats within the landscape. The drivers of this distribution pattern are yet to be determined. In this thesis, it was hypothesized that edaphic factors (soil chemical and physical characteristics) and the distribution of rhizobia have influenced legume distributions in the CCR landscape. The influence of edaphic factors on the distribution of legume species assemblages in the Cape Peninsula (a microcosm of the CCR) is the subject of the second chapter of the thesis. It was hypothesized that the composition of legume species assemblages is correlated with soil physical and chemical properties and that the interaction of Phosphorus (P) and the three cations that often bind P, i.e. Aluminium, Calcium and Iron, making it unavailable to plants, drive legume species assemblages in the landscape. Soils from 27 legume sites, spanning all major soil types of the Cape Peninsula, were analysed for 31 chemical and physical properties. Surveys of legume species present at each site were conducted to generate a presence/absence matrix. Canonical correspondence analysis was used to test for a correlation between legume species composition and edaphic factors. The strength of the association between legume species composition and site groupings based on edaphic properties was assessed using indicator species analysis. A significant correlation between edaphic factors and species composition was found and the key edaphic parameters driving the relationship were clay content, iron (Fe), potassium (K), sulphur (S) and zinc (Zn). Indicator species, characteristic of the various edaphic habitats were also identified. These findings indicate that distinct edaphic habitats are occupied by discrete legume species assemblages, implying a significant influence of edaphic factors on the legume distributions. Chapter three of the thesis sought to determine if the ecological parameters; altitude, pH and soil type influence the distribution of the two main rhizobial genera (Burkholderia and Mesorhizobium) that nodulate various legumes of the CCR, and to determine the diversity and phylogenetic position of rhizobia that associate with the narrowly distributed and rare Indigofera superba in the CCR. The first objective was pursued through molecular characterisation of rhizobial strains isolated from nodules of legume species collected in the field across the Cape Peninsula. DNA sequences for 16S vi rRNA, recA and nodA were combined with data from a previous study that sampled broadly within the CCR and phylogenetic analyses were conducted. Tests for phylogenetic signals for the three ecological parameters were conducted, using the D statistic for soil type and Pagel’s λ for altitude and pH. These analyses were used to test the hypothesis that closely related species occupy similar habitats with respect to each of the three ecological parameters. For the study of rhizobial symbionts of Indigofera superba, field nodules were sampled from multiple populations across its distribution range and a phylogeny of its symbionts was reconstructed in a matrix that included symbionts of diverse legumes from different habitats within the CCR. The results showed that Burkholderia is restricted to acidic habitats, while Mesorhizobium occurs in both acidic and alkaline habitats. Additionally, both rhizobial genera showed significant phylogenetic clustering for pH and most soil types. However, none of the genera showed a phylogenetic structure with respect to altitude. These findings indicate that pH and soil type influence the distribution of rhizobia in the CCR. Implications of these findings for the distribution of legumes in the landscape are discussed. For the narrowly distributed I. superba, the results showed that it associates with diverse strains within the genus Burkholderia and such strains are not phylogenetically distinct from strains isolated from localities outside its distribution range. These findings lead to the hypothesis that I. superba does not exhibit rhizobia specificity at the intrageneric level. Testing of this hypothesis through analysis of its nodulation capability on soils from outside its distribution range is recommended. The fourth chapter of the thesis determined the extent of horizontal gene transfer among rhizobial genera in the