The effects of soil warming on flowering phenology, reproductive strategy and attractiveness to pollinators in the herb Cerastium fontanum (Caryophyllaceae) Julia M. Johner Department of Biology Education Masters Degree Project 45 hp Plant Ecology Ecology & Biodiversity (120 hp) 2018-2020 Spring-Fall term 2019 Supervisor: Johan Ehrlén PhD The effects of soil warming on flowering phenology, reproductive strategy and attractiveness to pollinators in the herb Cerastium fontanum (Caryophyllaceae) Julia M. Johner Abstract Phenotypic plasticity plays an important role in organisms’ adaptability to environmental change such as global warming caused by greenhouse-gas emissions. One plastic response to increased temperatures is for organisms to shift their phenology. It is of great concern that the phenologies of interacting species, such as plants and pollinators, may be shifting at different rates, causing temporal mismatches, which for plants can lead to unsuccessful reproduction. The “reproductive assurance hypothesis” states that plants capable of self-pollination should be under high selection to employ this as their main reproductive strategy in the event of pollinator scarcity to ensure reproduction, and consequently invest less in attracting pollinators. This study examines how soil warming in the Hengill geothermal area in Iceland affects the flowering phenology, reproductive strategy and investment in attractiveness to pollinators in the self-compatible herb Cerastium fontanum (Caryophyllaceae), when grown in a common garden in Stockholm, Sweden. Previous research showed that C. fontanum from warmed soils flowered earlier in situ than plants from colder soils, and later when grown in a common environment. In this study, C. fontanum plants collected along a temperature gradient followed the same counter-gradient pattern, where plants from warmer soils flowered later than plants from colder soils. Soil temperature at site of origin positively affected flower number but had no effect on flower size, seed production from autogamous self-pollination or visitation rate. Based on my findings it does not appear that C. fontanum, despite having an earlier flowering phenology in situ, is under any selection to alter its reproductive strategy or investment in attractiveness to pollinators when grown in a common temperature, and therefore it seems unlikely that plants are experiencing a temporal mismatch with insect pollinators. However, it would be worthwhile to conduct a similar experiment in Iceland to better understand how an earlier flowering affects pollination systems. Keywords: Climate change, soil warming, phenological mismatch, phenotypic plasticity, counter-gradient variation, plant-pollinator interactions, reproductive assurance, autogamous self-pollination, flower size, common-garden experiment. Popular summary One adaptation to increased temperatures is for organisms to shift their phenology, the timing of key life events, such as flowering, nesting, migration etc. However, if the phenologies of interacting species, such as plants and their pollinators, shift at different rates, these species can mismatch with one another and this can have severe consequences for both species’ survival and reproductive success. Most plants rely on insect pollinators for cross-pollination but some are capable of self- pollination, a useful back-up strategy for plants in unpredictable environments or when pollinators are scarce. Cross-pollination is costly for plants, as they produce extravagant flowers and nectar to attract insects. Therefore, if plants are under selection to self-pollinate due to a lack of pollinators, they should invest less in large flowers or being attractive to pollinators. In this study, I examine how Cerastium fonatnum plants growing on volcanically heated soils in the Hengill geothermal area in Iceland respond to soil warming and what this means for their pollination strategy. Previous research showed that plants growing in warm soils flowered earlier than plants in colder soils. This led to my prediction that plants from warm soils would mismatch with their insect pollinators and therefore switch to self-pollination as their main reproductive strategy, and invest less into attracting pollinators. For this study, C. fontanum seeds collected along a temperature gradient in Iceland were grown under common temperature conditions in an outdoor garden in Stockholm, Sweden. I recorded first flowering date, number of flowers per plant, flower size and visitation rate by pollinating insects, and conducted pollination experiments to see if there was a difference in how many seeds plants could produce from self-pollination along a temperature gradient at site of origin. When grown under a common temperature, plants from warm soils flowered later than those from cold soils, demonstrating that they had genetically adapted to their home soil temperatures. Plants from warmer soils had more flowers than plants from colder temperatures but there was no difference in flower size, attractiveness to pollinators or seed production from self-pollination. From these findings it is unlikely that plants are mismatching with their pollinators, but it would be worthwhile to conduct a similar field study in Iceland to better understand these patterns. Ethical and social aspects Transplant studies have the potential to introduce new species or genes into the local ecosystem or population. Introduced species and the pathogens they may carry can pose a severe threat to the flora and fauna of some, particularly isolated, ecosystems. The plant material used in this study consisted of Cerastium fontanum seeds, which were collected in Iceland and then grown in an outdoor garden in Stockholm, Sweden. No non-native species were introduced, as C. fontanum grows naturally in both locations. This study may have contributed to some gene flow between Icelandic and Swedish populations although the impact of this is likely negligible. In addition, care was taken to isolate plants from the actual soil. All plants were individually potted and resting on elevated beds made of sand/gravel, covered in fiber-cloth. C. fontanum can host the Cucumber mosaic virus, a plant virus with a worldwide distribution and a very broad host range. CMV causes deformation to the leaves, flowers and fruits of many agricultural crops and therefore has large economic implications. To the best of my knowledge none of the plants I worked with showed signs of being infected, and it is therefore unlikely that any diseases were spread through this study. Contents Introduction…………………………………………………………………………………..5 Research questions……………………………………………………………….…….….6 Predictions…………………………………………………………………………………....7 Materials and methods ……………………………………………………………………7 Study system, species and sites.………………………………………………………7 Experimental design……………………………………………………………………….8 Data collection……………………………………………………………………………….8 Experiment 1: Flowering phenology, number & size of flowers ……….…..8 Experiment 2: Pollination………………………………………………………………...9 Experiment 3: Visitation ………………………………………………………………….9 Statistical analyses…………………………………………………...……………………9 Experiment 1: Flowering phenology, number & size of flowers ……………9 Experiment 2: Pollination…………………………………………………………….…10 Experiment 3: Visitation rate………………………………………………………….10 Results…………………………………………………………………………….....………10 Experiment 1: Flowering phenology, number & size of flowers…….….…10 Experiment 2: Pollination……………………………………………………………….12 Experiment 3: Visitation………………………………………………………………..12 Discussion………………………………………………………..………………………….13 Synchrony with pollinators…………………………………………………………….14 Number of flowers……………………………………………………………………..…14 Future directions…………………………………………………………………………..15 Conclusion…………………………………………………….……………………………..15 Acknowledgements.………………………………………………………………………16 References……………………………………………………………………………………16 Introduction Today’s ecosystems are undergoing rapid change on a global scale. Ongoing anthropogenic greenhouse-gas emissions are causing global temperature increases, which are drastically affecting biodiversity (IPCC 2018). Temperatures are expected to continue rising over the next century, with the most prominent heating occurring at high latitudes (IPCC 2018). Organisms in these regions are adapted to harsh environmental conditions such as long winters, extreme light regimes and resource limitation, and may be particularly vulnerable to changes in temperature (Totland 1999). Heating in these areas will necessitate adaptations to new prevailing conditions (Totland 1999), first and foremost through phenotypically plastic responses (Van Etten & Brunet 2013) such as spatial (Pyke et al. 2016) or temporal shifts (Parmesan & Yohe 2003). If selection pressures remain high, such plastic responses can lead to long-term evolutionary change (Totland 1999). One common plastic response to changing temperatures is for organisms to shift their phenologies (key life-history events such as spring flowering, emergence from diapause, nesting etc.). Advanced spring phenologies in connection with rising temperatures have already been observed for many groups of organisms, including plants, insects, birds, and amphibians (Parmesan & Yohe 2003). It is of great concern that shifting phenologies may cause asynchronies between interacting species (Hegland et al. 2008; Bartomeus et al. 2011; Pyke et al. 2016; Olliff-Yang & Mesler 2018). Phenological mismatches can result either when the phenologies of interacting species shift at different rates, or when historically