! "##$ %"&# "" #'($)'"' "*'$#' "*" +,,- Dissertation for the Degree of Doctor of Philosophy in Conservation Biology presented at Uppsala University in 2003 ABSTRACT Gustafsson, S. 2003. Population genetic analyses in the orchid genus Gymnadenia – a conservation genetic perspective. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 799. 43 pp. Uppsala ISBN 91-554-5517-4. Small populations are facing a particular risk of extinction due to a lack of appropriate genetic diversity and associated negative effects, factors dealt with in the discipline of conservation genetics. Many orchid species exhibit characteristics that make them a perfect study object in the scope of conservation genetics. The aim with this thesis was to investigate genetic structure at different levels in two orchid species Gymnadenia conopsea, geographically widespread, although diminishing and G. odoratissima with a long history of being rare. Microsatellite markers, developed in and used in studies of G. conopsea were also used in the study of G. odoratissima. Populations of G. conopsea expressed high levels of genetic variation and a certain amount of gene flow, although investigated mating pattern in a small population indicated non-random mating among individuals, with the majority of pollen exchange between near neighbours, and noticeable levels of geitonogamous pollinations. Further a pronounced year to year variation in flowering frequency among individuals was found. It was also discovered that flowering time variants (early and late) within the species G. conopsea were highly differentiated and seem to have had a more ancient historical separation than the separation between the two different species, G. conopsea and G. odoratissima. Levels of genetic variation in the rare congener, G. odoratissima differed between island and mainland populations where the more numerous island populations expressed larger levels of genetic variation and were less differentiated compared to the few remaining and genetically depauperated mainland populations. Key words: Orchidaceae, Gymnadenia, conservation, microsatellites, genetic structure, mating pattern. Susanne Gustafsson, Department of Conservation Biology and Genetics, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-752 36 Uppsala, Sweden. © Susanne Gustafsson 2003 ISSN 1104-232X ISBN 91-554-5517-4 Printed in Sweden by Uppsala University, Print & Media, Uppsala 2003 PAPERS INCLUDED IN THE THESIS This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I Gustafsson, S. and Thorén, P. (2001). Microsatellite loci in the Gymnadenia conopsea, the fragrant orchid. Molecular Ecology Notes, 1:81-82. II Gustafsson, S. Mating pattern in a small population of the Lepidopteran pollinated orchid, Gymnadenia conopsea, as revealed by microsatellites. Manuscript. III Gustafsson, S. (2000). Patterns of genetic variation in the fragrant orchid (Gymnadenia conopsea). Molecular Ecology 9: 1863-1872. IV Gustafsson, S. and Sjögren-Gulve, P. (2002) Genetic diversity in the rare orchid, Gymnadenia odoratissima and a comparison with the more common congener, G. conopsea. Conservation Genetics 3: 225-234. V Gustafsson, S. Flowering frequency and genetic diversity in a small population of Gymnadenia conopsea - a five year study. Manuscript. VI Gustafsson, S. and Lönn, M. Genetic differentiation and habitat preference of flowering-time variants within Gymnadenia conopsea. Manuscript. Papers I and III are reproduced with kind permission of Blackwell Publishing. Paper IV are reproduced with kind permission of Kluwer Academic Publishers. Contents Introduction.....................................................................................................2 Conservation genetics ................................................................................2 Population genetics ....................................................................................3 Mating systems and gene dispersal ............................................................5 Orchids.......................................................................................................6 Material and methods......................................................................................9 Genetic markers, microsatellites ................................................................9 Development of microsatellite loci ..........................................................10 Genetic and statistical analyses................................................................11 Population structure.............................................................................12 ҏAssignment methods ...........................................................................13 Genetic differentiation within species .................................................13 Study species............................................................................................14 The genus Gymnadenia .......................................................................14 Results and discussion ..................................................................................17 Microsatellite loci in Gymnadenia conopsea (I) ......................................17 Mating pattern (II) and flowering frequency (V).....................................20 Genetic structure in rare and diminishing orchid species (III), (IV) .......22 Restricted gene flow within species (VI).................................................26 Conclusions...................................................................................................32 Acknowledgements.......................................................................................34 References.....................................................................................................36 1 Introduction Conservation genetics During the 20Th century, mankind has had a widespread and devastating impact on the survival of many species. Factors directly or indirectly associated to different human activities, like pollution, habitat destruction, over-exploitation and introduced species have, besides a rapid and direct extinction of numerous species, also through fragmentation events and reduced population size, deteriorated the conditions for long-time survival in an even larger number of species. The area of conservation biology, concerning preservation of biodiversity, has therefore been a huge and rapidly growing discipline of research in the last decades. In preserving biodiversity, the strategy of conservation biology has customarily been to treat the species as the unit of conservation. However, even though the emphasis has been and still will be on rare species, more recently the view has widened to also involve other levels of organization, ranging from genes to ecosystems. Together with demographic and environmental factors, one part of conservation biology therefore includes the influences of genetic factors. Even though the significance of the first two is undisputed, the importance of genetic factors in conservation biology has been questioned (Frankham et al, 2002 and references therein). Responsible for raising the subject of conservation genetics was Sir Otto Frankel, who, in the beginning of the 1970s wrote important papers covering the issue (Frankel 1970, 1974). The area of conservation genetics deals with genetic factors affecting a species risk of extinction and with management regimes that could be acquired to minimize these risks (Frankham et al, 2002). Small populations are, due to their reduced population size, supposed to also express reduced genetic diversity (Frankham, 1995). Different studies, both in plants and animals, have found support for the fact that small populations face a particular risk of extinction due to a lack of appropriate genetic diversity and associated negative effects (Young & Clarke, 2000, Frankham et al, 2002 and references therein). A primary element in conservation management has therefore been preservation of genetic diversity and minimizing inbreeding. However, the significance of genetic factors contributing in species extinction has not been without debate and some will instead emphasize demographic and environmental factors whereas genetic factors have been 2 suggested to be more a symptom than a cause to species extinction (Lande, 1988; Caughley, 1994; Holsinger & Vitt, 1997; see also Lande, 2000). As stressed by Hedrick (2000a) genetic processes do not operate in isolation but influence extinction probabilities indirectly by their effect on reproductive success, behaviour, viability, disease resistance etc. Frankham et al (2002) also point at the difficulties in isolating the effects of genetic processes and imply that genetic threats form one component of the endangering process, and population extinction is due to interactions between genetic, demographic and environmental factors. Quite a number of plant species have been studied in the area of conservation genetics and examples of some genera are:– Caesalpinia (Cardoso et al, 1998), Brighamia (Gemmill et al, 1998), Saxifraga (Hollingsworth et al, 1998), Vicia (Black-Samuelsson & Lascoux, 1999), Goodyera (Wong & Sun 1999), Rutidosis (Brown & Young, 2000), Gymnadenia (Gustafsson & Sjögren-Gulve, 2002), Leucopogon (Zawko et al 2001), Triunia (Shapcott, 2002) Epipactis (Squirrell et al, 2002). The studies describe demographic and genetic status