DOCTORAL THESIS 2017 Universitat de les Illes Balears RESPONSES AND VULNERABILITY OF THE GENUS VIOLA TO ELEVATIONAL GRADIENTS AND ALIEN INVASIVE SPECIES IN MOUNTAIN SYSTEMS Jaime Seguí Colomar DOCTORAL THESIS 2017 Doctoral programme of Plant Biology RESPONSES AND VULNERABILITY OF THE GENUS VIOLA TO ELEVATIONAL GRADIENTS AND ALIEN INVASIVE SPECIES IN MOUNTAIN SYSTEMS Jaime Seguí Colomar Thesis supervisors: Anna Traveset Vilaginés Manuel Nogales Hidalgo Doctor by the Universitat de les Illes Balears Als meus pares i al meu germà A n’Elena, la sal de la meva vida Quería movimiento, no una existencia sosegada. Quería emoción y peligro, así como la oportunidad de sacrificarme por amor. Me sentía henchido de tanta energía que no podía canalizarla a través de la vida tranquila que llevábamos. León Tolstoi, Felicidad familiar No permitas que tu trabajo sea tu forma de ganarte la vida, sino tu diversión. Cada día deberíamos regresar al hogar de lejos, de aventuras, peligros y descubrimientos, con experiencias nuevas y el carácter renovado. Henry David Thoreau, Walden Contents List of Figures 4 List of Tables 6 Outline and publications 7 Resumen 8 Resum 10 Abstract 12 Acknowledgements 14 A. PRÓLOGO 18 Introducción general 19 La ecología reproductiva de las plantas alpinas y su vulnerabilidad al cambio global. 19 Los gradientes altitudinales como laboratorios naturales. 21 Efectos sinérgicos de las especies invasoras y el cambio climático en los sistemas de montaña. Especial vulnerabilidad en las islas oceánicas. 24 Particularidades del género Viola. El porqué de su elección como modelo de estudio. 25 Objetivos generales de la tesis 28 B. MAIN CHAPTERS 30 Chapter 1. Species-environment interactions changed by introduced herbivores in an oceanic high-mountain ecosystem 31 1.1 Introduction 33 1.2 Materials and methods 35 1.3 Results 39 1.4 Discussion 41 1.5 Conclusions 45 1.6 Acknowledgements 45 1.7 Figures 46 1.8 Tables 51 1.9 Supplementary material 54 1 Contents Chapter 2. Invasive rabbits disrupt the selection that pollinators and abiotic conditions exert on floral traits of a high-mountain oceanic plant 55 2.1 Introduction 57 2.2 Material and methods 58 2.3 Results 61 2.4 Discussion 63 2.5 Acknowledgments 66 2.6 Figures 67 2.7 Tables 73 2.8 Supplementary material 74 Chapter 3. Phenotypic and reproductive responses of an Andean violet to environmental variation across an elevation gradient 75 3.1 Introduction 77 3.2 Materials and methods 79 3.3 Results 83 3.4 Discussion 85 3.5 Conclusions 88 3.6 Acknowledgements 89 3.7 Figures 90 3.8 Tables 93 3.9 Supplementary material 94 Chapter 4. What explains the maintenance of outcrossing in an almost exclusively cleistogamous species? The case of the cliffhanger Viola jaubertiana 98 4.1 Introduction 100 4.2 Material and methods 101 4.3. Results 105 4.4 Discussion 108 4.5 Acknowledgments 111 4.6 Figures 112 4.7 Tables 117 4.8 Supplementary material 120 2 Contents C. EPÍLOGO 121 Discusión general 122 Conclusiones generales 129 Ideas para investigaciones futuras 132 REFERENCES 135 3 List of Figures Figure 1. A; study sites selected on the SE slope of the Teide stratovolcano. B; Plant located inside the herbivore fence exclusion at 3500 m. C; Plant with the individual exclusion cage. D; Image of a whole non-protected plant. Figure 2. Plant traits studied along the elevational gradient during 2012 and 2013. Figure 3. Autofertility and pollen addition treatments accounting for fruit set and seed set. Figure 4. Flower visitation rate among elevational localities and between rabbit-excluded and non-excluded plants (2014-2015). Figure 5. Plant traits of rabbit-excluded individuals along the elevational gradient and in the 3500 m fence exclusion during 2014 and 2015. Figure 6. Images of (A) plants rabbit-excluded and non-excluded, (B) the three elevational study sites, and (C) floral traits measured. Figure 7. Mean and standard deviation of floral traits measured in V. cheiranthifolia across study sites. Figure 8. Spur and upper petal length in response to specific leaf area in V. cheiranthifolia. Figure 9. Plant reproductive success in response to spur width across the elevational gradient, and between excluded and non-excluded pants. Figure 10. (A) Fruit set in response to floral display, and (B) seed viability in response to floral display and conspecific flower density. Figure 11. Interactive effect of rabbit exclusion on the response of spur width to specific leaf area. Figure 12. Bar plots of the physiological variables measured in Viola maculata at each site. Figure 13. (A) Mean ± SE fruit set and (B) seeds per fruit in chasmogamous and cleistogamous flowers. 4 List of Figures Figure 14. (A) Partial residual plots showing the relationships between flower area and leaf area at each elevational site. (B) Patterns of correlations among plant traits in the three study populations of Viola maculata. Figure 15. Examples of (A) a vertical cliff habitat type of Viola jaubertiana, (B)(C) two chasmogamous flowering individuals, showing an endemic snail (Iberellicus balearicus) with some damaged leaves, and (D) details of a cleistogamous flower and fruit. Figure 16. Flowering phenology of V. jaubertiana presented as the mean number of flowers (CH and CL) per plant observed at each census and elevational site during 2014. All round unfilled marks indicate dates of observation. Figure 17. CH:CL flower ratio in response to mean specific leaf area and herbivory index. The lines represent the estimates of each variable in the model whereas points are the empirical data. Figure 18. Mean CL seed set per plant as a function of its CH:CL flower ratio. Line represents the estimate of the model, whereas points are the empirical data. Figure 19. Mean and standard deviation of fruit set (proportion of flowers setting fruits) obtained in each treatment and control. Experiments were carried out at Sóller Botanical Garden. Figura 20. Resultados del metaanálisis realizado para analizar el efecto de la altitud sobre el éxito reproductivo de 122 especies de plantas en 49 gradientes altitudinales estudiados. Figura 21. Diagrama de los diseños de cruzamiento para producir descendencia de autofecundación y descendencia procedente de cruzamientos. Figura 22. Polimorfismo floral en Viola cotyledon; morfo violeta y morfo blanco. 5 List of Tables Table 1. Temperature and edaphic data at each elevational site in Teide’s National Park. Table 2. Results of GLMM for A) response variables in non-protected individuals, B) autofertility and pollen addition treatments, and C) response variables in caged plants Table 3. Floral visitors of V. cheiranthifolia observed at each altitudinal site during 2014-15. Table 4. Microclimatic and soil data at each study site in Teide’s National Park. Table 5: Statistics and mean and standard errors of the floral and leaf traits measured at each elevation in V. maculata. Table 6. Mean and standard errors of the traits measured in Viola jaubertiana during 2014 and 2015 at the three altitudinal sites. Table 7. Estimates of the relative flower cost, and relative reproductive success of both CH and CL flowers in V. jaubertiana. Table 8. Performance seed traits of each flower type in the field, and in each treatment performed in the botanical garden with V. jaubertiana. Inbreeding and outbreeding depression estimates are presented. 6 Outline and publications This thesis is a compendium of publications, organized in four parts: Prologue, Main chapters, Epilogue, and References. The prologue presents an overview of plant ecology in alpine ecosystems, and their special vulnerability to global change, as well as of the particularities of the genus Viola, study model in this thesis, and finally highlights the general objectives. The second part consists of four independent chapters, each one corresponding to a different research article. One is already published, one is under review, one is resubmitted, and one is in preparation for submission. The epilogue contains a general discussion of the main findings of the thesis, the final conclusions, and ideas for future investigation in this research line. Finally, a list of references is included. List of publications included in this thesis: Accepted: Seguí, J., López-Darias, M., Pérez, A. J., Nogales, M., & Traveset, A. (2017). Species- environment interactions changed by introduced herbivores in an oceanic high- mountain ecosystem. AoB Plants. 9(1) plw091. DOI: https://doi.org/10.1093/aobpla/plw091 Impact factor: 2.079 Submitted: Seguí, J., Lázaro, A., Traveset, A., Salgado-Duarte, C., & Gianoli, E. (2017). Phenotypic and reproductive responses of an Andean violet to environmental variation across an elevation gradient. Under review in Alpine Botany. Seguí, J. & Traveset, A. (2017). What explains the maintenance of outcrossing in an almost exclusively cleistogamous species? The case of the cliffhanger Viola jaubertiana. Resubmitted to International Journal of Plant Sciences. In preparation for submission: Seguí, J., López-Darias, M., Nogales, M., & Traveset, A (2017). Invasive rabbits change the selection that pollinators and abiotic conditions exert on floral traits of a high- mountain oceanic plant. Prepared for submission. 7 Resumen Los ecosistemas de montaña son centros de biodiversidad mundial, debido a su aislamiento geográfico, a su papel como refugio de especies durante los cambios climáticos del pasado y a la gran variedad de condiciones ambientales que presentan a lo largo de sus gradientes altitudinales. Los gradientes ambientales que presentan las montañas permiten testar respuestas ecológicas de las plantas a diferentes condiciones ambientales a través de distancias cortas, por lo que pueden funcionar como laboratorios al aire libre, especialmente útiles para entender las respuestas de las plantas al cambio global. Por ejemplo, la presencia de especies invasoras en estos sistemas puede crear efectos sinérgicos con los del cambio climático, teniendo consecuencias dramáticas para especies nativas ya amenazadas, o para ecosistemas frágiles, como los presentes en la alta montaña de islas oceánicas.