Investigation on selected biotic and abiotic factors in the maintenance of the “fairy circles” (barren patches) of southern Africa by Angelique Joubert Submitted in partial fulfillment of the requirements for the degree Magister Scientiae Department of Plant Science Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria Supervisor: Prof. J.J.M. Meyer Co-supervisor: Prof. M.W. van Rooyen January 2008 i Investigation on selected biotic and abiotic factors in the maintenance of the “fairy circles” (barren patches) of southern Africa by Angelique Joubert Supervisor: Prof. J.J.M. Meyer Co-supervisor: Prof. M.W. van Rooyen Department of Plant Science Faculty of Natural and Agricultural Sciences Degree: MSc ABSTRACT The fairy circles are an intriguing and unexplained feature of the pro-Namib in Namibia and northwestern parts of South Africa. The presence of hundreds of almost circular patches where no plants grow were first mentioned in scientific literature in 1971 and since then scientists have tried to find an explanation for the origin of these circles. Although there are many hypotheses regarding the origin of these circles not one of these can explain the existence of these circles satisfactory. In this study several aspects of the fairy circles were investigated to improve the characterization of the phenomenon. ii Total element analysis of the soil from the different microhabitats (inside the circle, on the edge of the circle and between the circles, referred to hereafter as the matrix) and at different depths in these microhabitats were performed by inductively coupled plasma mass spectrometry (ICP-MS) and analyzed by principal component analysis (PCA). No patterns emerged regarding the concentration of the elements in the respective microhabitats. The occurrence of vesicular arbuscular mycorrhizae (VAM) in the roots of plants collected from the different microhabitats was also investigated. It was shown that VAM occurred in most of the roots of plants collected in the matrix and on the edge but no VAM were found in plants collected inside the circles. The succulent plant Euphorbia damarana has also been implicated in the origin of the circles. The presence of germination inhibiting compounds in this species was investigated. No such compounds were found. The extracts of it inhibited the growth of radicles of lettuce seeds at a concentration of 25 mg/ml. Bio-assays were also performed on soil collected in the different microhabitats using a dominant grass of the area, Stipagrostis uniplumis, as bioindicator. The fresh an dry shoot mass of plants harvested from soil collected on the edge and in the matrix grew much better than the shoots grown in soil collected from the inside of the circles (p = 0.0007). The dry shoot mass showed the same trend as the fresh shoot mass. The fresh root mass showed a marked increase in the roots collected from soil on the edge of circles when compared to plants grown in soil collected from the inside and the matrix (p = 0.013). There was a significant difference in the length of shoots measured in plants grown in the soils collected from the different microhabitats with the shoots measured from plants grown in the soil collected from the edge showing stimulation in growth when compared to the plants grown in soil from the matrix and inside the circles (p = 0.00004). The difference in shoot length between grasses grown in soil collected from the edge and the matrix was also significant (p = 0.00004) with the edge samples showing a stimulation in growth. iii Table of Content Chapter 1: Introduction _____________________________________________ 5 1.1 Introduction_________________________________________________________ 5 1.2 Distribution and characteristics__________________________________________ 7 1.3 Aim of the study ____________________________________________________ 11 Chapter 2: Literature review________________________________________ 12 2.1 Introduction________________________________________________________ 12 2.2 Hypotheses ________________________________________________________ 12 2.2.1 Termite related hypotheses _______________________________________________ 12 2.2.2 Allelopathy hypothesis __________________________________________________ 17 2.2.3 Radioactivity hypothesis _________________________________________________ 18 2.2.4 Vegetation patterns _____________________________________________________ 19 2.2.5 Fungi as causative agents ________________________________________________ 21 Chapter 3: Bioassay of soil using Stipagrostis uniplumis ______________ 23 3.1 Introduction________________________________________________________ 23 3.2 Literature review____________________________________________________ 23 3.3 The genus Stipagrostis _______________________________________________ 25 3.4 Aim of this chapter __________________________________________________ 27 3.5 Materials and methods _______________________________________________ 27 3.5.1 Collection of diaspores and soil ___________________________________________ 27 3.5.2 Seed viability testing____________________________________________________ 28 3.5.3 Seed germination testing_________________________________________________ 28 3.5.4 Bioassay of soil ________________________________________________________ 29 3.6 Results____________________________________________________________ 30 3.6.1 Seed viability testing____________________________________________________ 30 3.6.2 Seed germination testing_________________________________________________ 30 3.6.3. Bioassay of soil _______________________________________________________ 31 3.7 Discussion_________________________________________________________ 36 3.7.1 Viability and seed germination ____________________________________________ 36 3.7.2 Bioassays_____________________________________________________________ 36 3.8 Conclusion ________________________________________________________ 37 2 Chapter 4: The occurrence of vesicular arbuscular mycorrhizae (VAM) in roots collected from plants occurring in and around the fairy circles ___________________________________________________________ 38 4.1 Introduction________________________________________________________ 38 4.2 Anatomy and morphology of VAM _____________________________________ 39 4.3 Factors influencing the occurrence of vesicular arbuscular mycorrhizae_________ 41 4.4 Mycorrhizae in grasslands and arid ecosystems ____________________________ 42 4.4.2 C4 - and C3- photosynthesis _______________________________________________ 43 4.4.3 Techniques for quantifying mycorrhizae colonisation __________________________ 43 4.5 Aim of this chapter __________________________________________________ 44 4.6 Materials and methods _______________________________________________ 44 4.6.1 Field collection of roots _________________________________________________ 44 4.6.2 Bioassay root collection _________________________________________________ 45 4.6.3 Staining of roots for visualisation __________________________________________ 45 4.6.4 Assessment of percentage root colonisation __________________________________ 46 4.7 Results____________________________________________________________ 47 4.8 Discussion_________________________________________________________ 50 4.9 Conclusion ________________________________________________________ 53 Chapter 5: Soil analysis by inductively coupled plasma mass spectrometry (ICP-MS) __________________________________________ 54 5.1 Introduction________________________________________________________ 54 5.2 Literature review____________________________________________________ 54 5.2.1 History of soil research related to the fairy circles _____________________________ 54 5.2.2 Inductively coupled plasma mass spectrometry (ICP-MS) analysis of soil __________ 55 5.2.3 Acid digestion elemental analysis of soils by ICP-MS __________________________ 58 5.3 Aim of this chapter __________________________________________________ 59 5.4 Materials and methods _______________________________________________ 59 5.4.1 Soil collection _________________________________________________________ 59 5.4.2 Extraction ____________________________________________________________ 61 5.4.3 Analysis of data________________________________________________________ 61 5.5 Results____________________________________________________________ 62 5.6 Discussion_________________________________________________________ 81 5.7 Conclusion ________________________________________________________ 83 3 Chapter 6: Investigating the possible allelopathic effects of Euphorbia damarana and the isolation of two compounds ____________________ 85 6.1 Introduction________________________________________________________ 85 6.2 Literature review____________________________________________________ 87 6.2.1 The origin of the allelopathy hypothesis_____________________________________ 87 6.2.2 Euphorbia damarana ___________________________________________________ 88 6.2.3 Proving allelopathic interactions___________________________________________ 89 6.3 Aim ______________________________________________________________ 91 6.4 Materials and methods _______________________________________________ 92 6.4.1 Plant collection ________________________________________________________ 92 6.4.2 Euphorbia damarana extraction____________________________________________ 92 6.4.3 Thin layer chromatography (TLC) agar plate bioassay__________________________ 93 6.4.4 Growth and germination inhibition bioassay _________________________________