GENETIC VARIATION WITHIN AND BETWEEN SOME RARE AND COMMON TAXA OF CAPE PROTEACEAE AND THE IMPLICATIONS FOR THEIR CONSERVATION Thesis submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Faculty of Science of RHODES UNIVERSITY by SUSAN ANN BROWN Department of Botany May 1999 The bright yellow blooms of a male Leucadendron elimense subspecies vyeboomense. This taxon is highly endangered. The showy red-flushed male flowers of Leucadendron elimense subspecies salteri offer great potential for exploitation as cut flowers. ABSTRACT There are 152 rare, threatened or extinct Cape Proteaceae, many of which are found to occur in small numbers, have few populations, or a geographical range of less than 5 km2. This suggests that small nature reserves would be an ideal solution to protect such species provided they contain sufficient levels of genetic variation for long term viability. Genetic variation was measured using RAPD analysis in the rare Leucadendron elimense with its three closely related subspecies, L.e. vyeboomense, L.e. salteri and L.e. elimense. L.e. vyeboomense is restricted to a single endangered population, L.e. salteri has seven known populations and is ascribed the status of vulnerable, and the local endemic of the south coast plains, L.e. elimense, is considered vulnerable. AMOVA analysis of the RAPD results revealed high levels of genetic variation within populations of all three subspecies at 69.8 to 91.4%. The endangered L.e. vyeboomense was genetically most distant from the other two subspecies at a level of 0.40 and 0.39 and also showed the lowest levels of variation within the subspecies at 0.24. Populations of the other two subspecies had levels of variability of about 0.35. Comparable levels were recorded for the ubiquitous Leucadendron salignum. Serruria roxburghii, an endangered species restricted to two populations on a specialised sandy soil, provided another test of the applicability of the method. Both populations of S. roxburghii showed a genetic variability of 0.36 with no significant differences in genetic distances within the two populations. Morphological differences were considered and leaf ratios provided a tool for distinguishing the three subspecies. Total protein electrophoresis and sequencing of the Internal Transcribed Spacer (ITS) region, both showed that L.e. salteri was the least similar of the subspecies, and should be considered for possible taxonomic revision to species level. The implications of the thesis for conservation are that RAPD data is directly of use in conservation management decisions. It has shown that despite the small population sizes of the study taxa they have i adequate to high levels of genetic diversity and would be adequately protected in small nature reserves. Small, private reserves should be considered as a potential economical and ecological long term viable option. ii ACKNOWLEDGEMENTS I would like to acknowledge the supervision, advice and constructive comments I received from Professor CEJ Botha , Dr NP Barker and Professor R Kirby. Various people kindly helped and provided support in the form of laboratory space, use of equipment, field assistance, the collection of leaf material, and help with statistical analysis. In this respect I would like to express my appreciation to Professor HR Hepburn, Dr RA Dorrington and Dr JA Goodwin of Rhodes University, R Pool of Cape Nature Conservation and Dr R Peakall of Australian National University. Assistance with the automated sequencing was most expertly provided by Mrs MD James of the University of Cape Town. Financial assistance from the Foundation for Research Development, Rhodes University Joint Research Committee, the registrar’s office of Rhodes University, and the Chairman's Fund Educational Trust of Anglo American Corporation - De Beers is gratefully acknowledged. Special thanks are due to my husband Dr Christopher Brown, who kindly provided laboratory space, computer facilities, endless encouragement, criticism and help. He patiently read and reread all drafts of the thesis. Thanks are also due to my daughters, Robynne and Caitlin for their support and patience. iii TABLE OF CONTENTS ABSTRACT .......................................................... i ACKNOWLEDGEMENTS .............................................. iii Chapter 1. General introduction .........................................1 Chapter 2. Description of study taxa and their morphology, with particular reference to the leaf morphology of Leucadendron elimense E. Phillips. (Proteaceae) .....................................13 Chapter 3 Assessment of genetic diversity in the three subspecies of the rare and endangered Leucadendron elimense E. Phillips (Proteaceae) using RAPD analysis, and the implications for their conservation ...........32 Chapter 4. RAPD profiling in conservation management: an application to estimate levels of genetic variability in individuals and populations in the endangered Serruria roxburghii R.Br. (Proteaceae) ................76 Chapter 5. Population differentiation of the three subspecies of the rare Leucadendron elimense E. Phillips (Proteaceae) using polyacrylamide gel electrophoresis (PAGE) of total protein ......................95 Chapter 6. A comparison of the DNA sequences of the Internal Transcribed Spacers in the three subspecies of Leucadendron elimense E. Phillips. (Proteaceae) .............................................107 Chapter 7 Summary and conclusions ...................................119 Literature Cited ..........................................135 iv CHAPTER 1 General introduction Introduction Genetic variation and rare plants A major role of conservation is to preserve genetic diversity of species, populations and evolutionary processes in order to prevent genetic deterioration, and hence extinction in the long term (Soulé & Simberloff 1986, Avise 1994). Genetic diversity is generally expected to be low in small isolated populations (Gaston & Kunin 1997, Karron 1997) and endemic species also typically exhibit low levels of genetic diversity (Hamrick & Godt 1989). Because rare species of plants or local endemic species of plants are often found in small isolated populations with a restricted distribution, they might also be expected to exhibit limited genetic variation. Management decisions for such rare taxa ideally necessitate an understanding of their biology and other factors, including genetic variability, that influence their survival. Conservation management decisions for rare taxa, however, often have to be made quickly without adequate ecological or genetic data (Gaston & Kunin 1997). To compensate for this, heavy reliance has been placed on theoretical techniques and empirical generalizations, often taken from island biogeography theory (Doak & Mills 1994). This particularly applies to predictions on genetic variation and inbreeding depression. Such theoretical predictions are often found in practice to be simplistic and untestable (Shaffer & Samson 1985, Soulé 1987). One would therefore expect genetic studies to be a priority in conservation management, especially of rare species. Indeed, Peakall & Sydes (1996) suggest that immediate short term goals in rare plant studies are prevention of extinction, while longer term goals are maintenance of genetic diversity. In order to formulate suitable conservation strategies, a measure of genetic variation and its distribution in rare plant species is therefore essential (Falk 1992). In terms of rare plant species, genetic diversity is best defined as the sum of genetic variation within a population or a species (Peakall & Sydes 1996). Having identified the need for genetic studies, there are a few cautions. Avise (1994) points out that it is a common assumption that high levels of genetic variability within rare or threatened populations enhance the probability of the population surviving over time. This is because the loss of genetic variation is thought to lead to a potential decline in a species' ability to survive environmental changes and stress, both in the short- and long-term (Ellstrand & Elam 1993, 1 Milligan et al. 1994). Ryan & Siegfried (1994) indicate that in birds there is a link between small population size (with presumed low genetic diversity) and the risk of extinction. An example of causal links is provided by the prairie chicken, Tympanuchus cupidopinnatus, which exhibited a decline in Illinois from 2000 individuals in 1962 to 50 in 1994 with an accompanying decline in genetic diversity, measured as mean heterozygosity (Westermeier et al. 1998). Many authors, however, caution that no causal link has yet been proven between the level of genetic diversity and a population's long term viability (see, for example, Avise 1994, Milligan et al. 1994, Schemske et al.1994). Again, using the example of observations of natural populations of birds, there are numerous examples in the literature describing the persistence of small populations (see, for example, Terborgh & Winter 1980, Soulé et al. 1988). It is, however, uncertain as to what constitutes a high or low level of genetic diversity, and the maintenance of a constant level of genetic variation is therefore generally considered essential for long-term protection of a taxon (Frankel & Soulé 1981, Simberloff 1988). Any change in the level of genetic variation should be monitored to provide a critical guide for assessing current status and future prospects (Palacios & González-Candelas 1997). Extinction is a normal evolutionary process and