!"!# $%$""## & ' &(& & )*%! Plant Ecology, &'( University, )*+-./ “Writing a book is an adventure. To begin with, it is a toy and an amusement; then it becomes a mistress, and then it becomes a master, and then a tyrant. The last phase is that just as you are about to be reconciled to your servitude, you kill the monster, and fling him out to the public” Winston Churchill List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Boberg, E., Alexandersson, R., Jonsson, M., Maad J., Ågren, J. and Nilsson, L.A. Pollinator shifts and the evolution of spur length in the moth-pollinated orchid Platanthera bifolia. (Manuscript) II Boberg, E., Xu, L. and Ågren. J. Phenotypic selection on floral traits in divergent populations of the moth-pollinated orchid Platanthera bifolia. (Manuscript) III Boberg, E. and Ågren, J. Reproductive isolation among divergent populations of the moth-pollinated orchid Platanthera bifolia. (Manuscript) IV Boberg, E. and Ågren, J. (2009) Despite their apparent integration, spur length but not perianth size affects reproductive success in the moth-pollinated orchid Platanthera bifolia. Functional Ecology, 23:1022–1028 Paper IV is printed with permission from the publisher. Contents Introduction.....................................................................................................9 The mechanical fit......................................................................................9 Divergent and disruptive selection...........................................................10 Reproductive isolation..............................................................................10 Selection on correlated floral traits ..........................................................11 Aims of this thesis....................................................................................12 Methods ........................................................................................................13 Study species............................................................................................13 Pollination mechanism .............................................................................13 Study populations and site descriptions ...................................................15 Population differentiation and pollinators (I)...........................................16 Reciprocal translocation experiment (I) ...................................................17 Phenotypic selection study (II).................................................................17 Estimating the strength of reproductive isolation (III).............................17 Experimental manipulation of flower morphology (IV) ..........................18 Results and discussion ..................................................................................19 Population differentiation and pollinator shifts (I)...................................19 Disruptive and divergent selection (II).....................................................21 Intraspecific reproductive isolation (III) ..................................................22 The adaptive significance of spur length and perianth size (IV)..............23 Conclusions ..............................................................................................23 Summary in Swedish ....................................................................................25 Långa sporrar och matchande tungor .......................................................25 Acknowledgements.......................................................................................30 References.....................................................................................................31 Introduction Flower morphology and phenology of flowering vary extensively in animal- pollinated plants (Harder and Barrett 2006), and the relative importance of pollinator-mediated selection and random processes for this variation remains a central question in plant evolutionary biology. Animal pollinators have, like all organisms, restricted ranges limited by abiotic or biotic factors. Spatial and temporal variation in pollinator availability may result in divergent selection between plant populations and differentiation of floral traits that affect pollinator attraction and efficiency (Herrera et al. 2006; Johnson 2006). A number of studies of pollinator-driven floral differentiation in plants have focused on large-scale patterns of relationships between pollinators and closely related plant species (Schemske and Bradshaw 1999; Castellanos et al. 2003; Whittall and Hodges 2007). Plant species with large intraspecific variation in floral traits may be in the initial stages of divergence. As Grant and Grant (1965) stated, it is in these plant systems we can study floral evolution as an “ongoing process rather than a historical event”. In this thesis I explore the evolutionary mechanisms responsible for the evolution and maintenance of intraspecific differentiation in floral traits. The mechanical fit Flower parts may be subject to pollinator-mediated selection due to their effects on the mechanical fit between the plants reproductive organs and the pollinator’s body (Darwin 1862). Some plant species produce floral spurs, a tubular structure that contains nectar as a reward to animal pollinators. The length of the spur is expected to affect the mechanical fit to pollinators and is thus essential for effective pollination (Darwin 1862; Nilsson 1988; Johnson and Steiner 1997; Pauw et al. 2009). If the spur is too short to match the length of the proboscis of the pollinator, the pollinator will not come into contact with the flower’s reproductive organs. A long spur will force the pollinator to probe deeper into the flower to reach the nectar at the bottom of the spur and hence cause more effective removal and deposition of pollen. Consequently, selection will favour plant individuals that have a spur, which is longer than the proboscis of the primary pollinator (Nilsson 1988). 9 Divergent and disruptive selection Divergent and disruptive selection may explain the evolution and maintenance of intraspecific floral variation (Schluter 2000). The relationship between phenotypic traits and relative fitness within a population can be visualized as a surface, an adaptive landscape, where the horizontal axes represent trait variation and the height of the surface represents the fitness of a given trait combination. The adaptive landscape can include valleys and peaks, which indicate trait combinations associated with fitness minima and optima, respectively. Divergent selection is the process by which natural selection pulls trait means of populations towards different adaptive peaks. Divergent selection among habitats may be the result of stabilizing selection for different optima (Benkman 2003) or linear selection in opposite directions (Caruso et al. 2003; Hall and Willis 2006). In a contact zone between two divergent populations, floral traits may have a bimodal distribution. Bimodal traits may be subject to disruptive selection that results from the inferior fitness of individuals with an intermediate phenotype (Schluter 2000; Hendry et al. 2009). Disruptive selection may maintain high levels of genetic variation and contribute to the evolution of reproductive isolation and speciation (Coyne and Orr 2004; Rueffler et al. 2006). There is a lack of studies exploring the pattern of current selection in relation to population differentiation in floral traits, and surprisingly few studies have attempted to investigate disruptive selection on these traits in contact zones between divergent populations. Reproductive isolation The evolution of reproductive isolation between divergent plant populations may contribute to the maintenance of floral variation (Grant 1949, 1994; Coyne and Orr 2004). Reproductive isolation barriers reduce gene flow among populations, and may arise as a side-effect of divergent selection or by reinforcement, i.e., selection against hybridization (Coyne and Orr 2004; Rueffler et al. 2006). In plants, prezygotic isolating barriers, which prevents mating and fertilization between species, are generally stronger than postzygotic isolating barriers, which result from the inviability or sterility of hybrids (Lowry et al. 2008a). Populations that differ in their spatial distribution, flowering time or in their primary pollinators may be reproductively isolated due to limited pollen transfer (Grant 1994; Coyne and Orr 2004). Differences in spatial distribution and flowering time may enforce reproductive isolation due to the reduction in among-population encounters (Rice and Salt 1990; Johnson et al. 1996; Levin 2006). Differences in flower morphology may enforce reproductive isolation via pollinator preferences 10 (Chittka et al. 1999; Kennedy et al. 2006), or when pollen transfer among morphologically different flowers are physically impossible, for example when pollen is deposited on different