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 correlated environmental cues (such as day length and air temperature) of interacting species become decoupled due to warming (Hegland et al. 2018). Typically, plants and their pollinators respond to similar cues (air or soil temperature) for spring flowering and emergence (Forrest 2014; Pyke 2016), although some interacting species may use different combinations of cues (Olliff-Yang & Mesler 2018) or different cues altogether (Hegland et al. 2018). Plants, for example, may respond to other cues such as snowmelt (Walker et al. 1995; Thórhallsdóttir 1998), precipitation and photoperiod (Olliff-Yang & Mesler 2018). Temporal shifts due to climate change have been reported for terrestrial plants as well as insect pollinators, with earlier spring flowering in plants (Hegland et al. 2008), and both earlier (Bartomeus et al. 2011) and later emergence in insects (Forest & Thompson 2010). Asynchronies between plants and pollinators could have severe fitness costs for both parts (Parmesan 2007; Hegland et al. 2009; Forrest 2014; Pyke et al. 2016). However, research on the matter is limited and while there is some evidence for phenological mismatches (Kudo & Ida 2013), most studies have found evidence for maintenance of synchrony between interacting species (Bartomeus et al. 2011; Forrest 2014).

Cross-pollination, where external vectors including wind, water or organisms such as insects transfer pollen from anther to stigma between individuals, is the most common strategy for sexual reproduction in flowering plants. Cross-pollination offers the benefit of maintaining genetic diversity and limiting the accumulation of maladaptive mutations in a population (Peterson & Kay 2015). Insect-mediated cross-pollination, while highly effective, is costly to maintain (Solbrig 1976). Plants have evolved traits such as extravagant floral displays and fragrant compounds to attract insects, as well as a rewarding system to maintain a mutualistic relationship with their pollinators by trading nutritious nectar in exchange for pollination services (Solbrig 1976; Klinkhamer & de Jong 1993; Ye et al. 2010). However, relying on another species for reproduction can be risky, especially if environmental conditions are unpredictable (Baker 1955). The “reproductive assurance hypothesis” states that selection should favor self-pollination in the event that pollinators are scarce, for example due to environmental variability (Darwin 1876; Baker 1955, Fausto et al. 2001). In self-pollination, pollen is transferred between flowers of the same individual (geitonogamous self-pollination, often insect-mediated) or between anther and stigma of the same flower (autogamous self-pollination, self- mediated) (Llyod & Schoen 1992; Schoen et al 1996). While this “backup” strategy, common in alpine and high latitude plants (Totland & Schulte-Herbrüggen 2003) including C. fontanum, ensures reproduction, it can lead to inbreeding depression and loss of fitness in the long run (Herlihy & Eckert 2002; Van Etten & Brunet 2013). Therefore, as long as environments remain stable, selection should

5 favor cross-pollination as the best reproductive strategy for maintaining genetically diverse populations.

Recently, Valdés et al. (2018) studied the spring flowering phenology of the hermaphroditic, self- compatible, perennial herb Cerastium fontanum (Caryophyllaceae) along a temperature gradient in the Hengill geothermal area in Iceland. The study area has extreme variation in soil temperature (0 - 50°C) over very small spatial scales due to continuous volcanic activity (> 50 years), making the long-term effects of temperature easy to study. Valdés et al. found that C. fontanum growing in warmer soils responded to the higher temperatures by flowering earlier than plants growing in colder soils. In addition to their research in Iceland, they studied C. fontanum’s flowering phenology experimentally. Seeds were collected from plants spanning the temperature gradient on Iceland and grown in a common garden in Stockholm, Sweden, where the plants flowered in counter-gradient fashion; plants that flowered earlier in Iceland due to warmer soils flowered later under common temperature conditions, and vice versa. This pattern suggests that C. fontanum’s once plastic response to higher temperatures has led to long-term genetic changes in phenology. However, plastic responses are not always adaptive and can lead to reduction in fitness by introducing new environmental challenges, in which case we could expect to see selection acting against plasticity via genetic compensation (Grether 2005). For C. fontanum, an earlier flowering phenology can be a maladaptive plastic response in several ways, either by introducing constraints on growth from a suboptimal light regime, water or nutrient availability, or by causing a phenological mismatch with insect pollinators. While plants are capable of growing in warmer soils despite cold air temperatures, insects, being incapable of regulating their own body temperature, are dependent on suitable air temperatures for flight (Totland 1999). A loss in synchrony with its insect pollinators could have severe consequences for C. fontanum’s reproduction and survival (Hegland et al. 2009; Forrest 2014; Pyke et al. 2016).

If an earlier flowering phenology due to warmer soil temperatures causes C. fontanum to temporally mismatch with their insect pollinators, we would, following the “reproductive assurance hypothesis” expect plants in warmer soils to be under high selection to employ autogamous self-pollination as their main reproductive strategy (Baker 1955; Lloyd 1992; Schoen et al 1996; Herlihy & Eckert 2002; Kalisz & Vogler 2003; Totland & Schulte-Herbrüggen 2003; Moeler 2006; Hegland et al. 2008) and to invest less in attracting pollinators, for example by having smaller flowers (Charlesworth & Morgan 1991; Elle & Carney 2003; Maad et al. 2013) and fewer flowers (Klinkhamer & de Jong 1993), and to have a lower visitation rate by insect pollinators (Totland & Schulte-Herbrüggen 2003) than plants growing in colder soils. With this project I would like to investigate whether soil temperature affects C. fontanum’s reproductive strategy and investment in attractiveness to pollinators.

Research Questions

1. Does C. fontanum exhibit the same counter-gradient phenological pattern as observed by Valdés et al. (2018) when grown under common environmental conditions? 2. Does soil temperature at site of origin affect the number of flowers produced per plant under common environmental conditions? 3. Does soil temperature at site of origin affect flower size under common environmental conditions? 4. Does soil temperature at site of origin affect C. fontanum’s ability to produce seeds via autogamous self-pollination under common environmental conditions, and is there a difference in the amount of

6 seeds C. fontanum can produce after hand-mediated cross-pollination, geitonogamous self-pollination and autogamous self-pollination? 5. Does soil temperature at site of origin affect visitation rate by pollinating insects under common environmental conditions?

Predictions

1. C. fontanum will exhibit a counter-gradient phenological pattern similar to that observed by Valdés et al. (2018) when grown under common environmental conditions. 2. C. fontanum individuals from warmer soils at site of origin will have fewer flowers per plant than individuals from colder soils, when grown under common environmental conditions. 3. C. fontanum individuals from warmer soils at site of origin will have smaller flowers than individuals from colder soils, when grown under common environmental conditions. 4. C. fontanum individuals from warmer soils at site of origin will have more successful autogamous self-pollination (higher seed yield) than individuals from colder soils, when grown under common environmental conditions. Autogamous self-pollination will yield more seeds than cross-pollination or geitonogamous self-pollination. 5. C. fontanum individuals from warmer soils at site of origin will have a lower visitation rate by insect pollinators than individuals form colder soils, when grown under common environmental conditions.

Material and methods

Study system, species and sites Cerastium fontanum, also known as the common mouse-ear or the mouse-eared chickweed, is a short, (10-30 cm), perennial, mat-forming herb belonging to the family Caryophylaceae. Its hermaphroditic flowers are small (4-8 mm in diameter), 5-petalled, have 10 stamens and 5 styles. Its leaves are tear- shaped and grow opposite one another along the stalk, and the entire plant is covered in small, white hairs. C. fontanum is native to Europe and can be found in most habitats, including grasslands, roadsides, forests and gardens. In its first year, the plant grows only vegetatively, and flowers for the first time in its second year. Flowering occurs between April and August, depending on location. C. fontanum is cross-pollinated by flies and small bees but is also highly self-compatible (Den virtuella floran, Naturhistoriska riksmuseet 1999). Plant material for this study consisted of 1851 individually potted C. fontanum plants grown from seeds collected in 2017 along a temperature gradient at the Hengill geothermal area, approximately 40km east of Reykjavik, Iceland (64°3’11”N, 21°18’16”W; 360 m.a.s.l.), at the foot of the Hengill volcanic system. Soil temperature was recorded for the mother plants from which the seeds were collected and was measured at a depth of 10cm, within 2m of the mother plants. Given that plants flowered at different times at their site of origin, and as C. fontanum has a limited seed dispersal ability, it is safe to assume that gene flow between populations is limited (Valdés et al. 2018).

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Experimental design Seeds were planted indoors in individual plastic pots in Stockholm in the fall of 2017 and were placed in an outdoor garden at Stockholm University (59°21’53.1”N, 18°03’00.9”E, 11 m.a.s.l.) in the spring of 2018. From these 1851 plants, smaller experiment groups were formed by randomly selecting plants so that each group spanned the entire temperature gradient (306 plants for Experiment 1. Flowering phenology, number & size of flowers; 102 plants for Experiment 2. Pollination; and 51 plants for Experiment 3. Visitation.) For each experiment group, plants were randomly arranged in 10 x 5 (+1) staggered matrices in elevated beds in the outdoor garden (Figure 1). For visitation observations, plants were carried to a nearby flat patch of grass and randomly arranged in matrices.

Data collection

Experiment 1. Flowering Figure 1. Cerastium fontanum plants randomly phenology, number & size of arranged into matrices in the common garden. flowers To test the prediction that soil temperature at site of origin has an effect on phenology, first flowering date was recorded for 195 plants spanning the temperature gradient between 4 - 41°C, between the 15th of May and the 14th of June 2019. Both fully and partially open buds were recorded as having flowered. Dates were converted to day number (15th of May = day 1) for statistical analyses. To test the prediction that soil temperature has an effect on flower size, corolla length and diameter were measured using manual calipers with a precision of 0.01mm for 76 of these plants (Figure 2).

Figure 2. Measurements for corolla length and diameter were taken as shown above.

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Three flowers from each plant were measured to calculate mean corolla length and diameter. Measurements were taken on days with good weather, when flowers were completely open. To test the prediction that soil temperature at site of origin has an effect on flower number, the maximum number of open flowers at any given time was recorded for 178 of these plants and was log-transformed for data analyses.

Experiment 2. Pollination To test the prediction that soil temperature at site of origin has an effect on how well C. fontanum autogamously self-pollinates, and whether autogamous self-pollination leads to a higher seed yield than geitonogamous self-pollination or cross-pollination, three pollination treatments (A) autogamous self-pollination, (G) geitonogamous self pollination, and (C) cross-pollination were conducted on 102 plants spanning the temperature gradient between 5.9 - 33.9°C, between the 15th of May and the 19th of June 2019. Three unopened buds from each plant were covered with 6x8cm parchment hybridization/selfing bags (YupGoods, Le Taun Hai, 615 HL2 Street, Binh, Tri Dong Ward, Binh Tan District, Ho Chi Minh City, 7000000, Vietnam) and were each assigned one of the three treatments. Bags were folded at the base and fastened with metal paperclips. Bagging date and treatment type was written on each bag. For treatment (A), unopened buds were bagged and left to self-pollinate on their own. For treatments (G) and (C), unopened buds were bagged and bags were frequently removed to see if flowers had opened. When flowers had opened, pollen was transferred with a small paintbrush (AMI, Junior brush A400 No.8: Ø 8.0 mm) from another flower on the same plant to the one receiving the treatment, for treatment (G), and from another flower from a randomly selected flowering plant to the one receiving the treatment, for treatment (C). Brushes were cleaned with a 10:1 chlorine bleach solution following each pollination treatment. After hand-pollination, flowers were re-bagged and the date for pollination was written on each bag. All bags were removed when a fruit had set, and seeds were immediately collected and stored in new parchments bags of the same type. Unfortunately, many of the bags blew away, were removed by birds, or ended up weakening or breaking the flowers, and therefore only 46 plants received treatment (A), 9 received treatment (G) and 5 received treatment (C). Seeds were manually counted in the lab on the 6th and 7th of August 2019.

Experiment 3. Visitation rate To test the prediction that soil temperature at site of origin has an effect on C. fontanum’s attractiveness to pollinators, visitation by pollinating insects was observed on 28 plants spanning the temperature gradient between 5.6 – 35.2°C. Observations were carried out in a flat patch of grasses and forbs of varying size, with a strong presence of pollinators, located behind the common garden at Stockholm University. All plants with open flowers were selected and randomly arranged in a matrix on the ground, with a distance of 20-30cm between pots. Observations were conducted in good weather in the middle of the day for approximately one hour, on 9 days. The number of pollinating insects was recorded for the following 4 categories: 1) Hover: insect hovered over plant for at least 2 seconds, 2) Land on flower: insect landed on an open flower, 3): Drink/Pollinate: insect actively drank from a flower, 4) Land on plant: insect landed on any part of the plant (other than a flower), its soil or its pot.

Statistical analyses Experiment 1. Flowering phenology, number & size of flowers To assess whether soil temperature at site of origin affected flowering phenology, number of flowers corolla length or corolla diameter, the data were examined using the lm model (general linear model) in R, with soil temperature at site of origin as the independent variable, and first flowering date, (log) number of flowers, mean corolla length and mean corolla diameter and as dependent variables. The error distribution for all variables was normal and all models followed a link identity. All variables

9 were also regressed against each other. In addition, mean corolla length and mean corolla diameter were examined in a multiple regression analysis using the glm function (generalized linear model) in R, with soil temperature and number of flowers as independent variables.

Experiment 2. Pollination To assess whether soil temperature at site of origin had an effect on seed production for treatment (A), data were examined using the lm model (general linear model) in R, but first an outlier value was removed from the dataset, with soil temperature at site of origin as the independent variable, and seed count via autogamous self-pollination as the dependent variable. The error distributions for both variables were normal and the model followed a link identity. Mean seed production for the three treatments was compared using ANOVA and Tukey HSD tests, but as sample sizes for (G) geitonogamous self-pollination and (C) cross/pollination were so low, this experiment has very low statistical power.

Experiment 3. Visitation To assess whether soil temperature at site of origin had an effect on visitation rate by pollinating insects, data were examined in a multiple regression analysis using the glm (generalized linear model) in R. Soil temperature at site of origin, date for observation, and the interaction (soil temperature x date) were the independent variables, and number of visits for all four observation categories combined was the dependent variable. The variables followed a Poisson distribution and the model followed a log identity.

Flowering Phenology for Cerastium fontanum

Results 40 35 30 Experiment 1. 25 Flowering phenology, 20 number & size

15 of flowers 10 The general linear Adjusted R2: 0.213 5 model showed that P value: 7.15E-12 soil temperature at site 0 of origin had a -5 5 15 25 35 45 significant effect on Temperature °C (in mother plant's location) first flowering date in date(daynumber) flowering First the common garden. Plants from warmer soils Figure 3. The general linear model showed a significant effect of soil temperature in Iceland flowered later at site of origin on the flowering phenology of C. fontanum. Plants from warmer when grown in a common temperature in soils flowered later than plants from colder soils when grown in a common temperature. 10

Stockholm, and plants from colder soils flowered earlier (Figure 3 & Table 1). Soil temperature had an effect on the maximum number of flowers produced per plant. Plants from warmer soils in Iceland had a higher maximum number of flowers than plants from colder soils. Number of flowers was positively correlated with mean corolla length, and had a nearly significant positive correlation with mean corolla diameter. Soil temperature had no effect on mean corolla length or mean corolla diameter (Table 1). The multiple regression analysis showed a tendency for mean corolla length to be shorter in plants from warmer soils when number of flowers was added as a factor, but this was not significant (Table 2).

Table 1. General linear model for flowering phenology, number & size of flowers Adjusted Dependent variable Predictor variable Estimate SE t p df R2

First Flowering Date Soil temperature °C 0.4001 0.0548 7.30 7.15E-12 0.213 193 (day number)

Mean corolla length Soil temperature °C -0.0206 0.0132 -1.56 0.124 0.019 74 (mm)

Mean corolla Soil temperature °C 0.0114 0.0223 0.513 0.610 -0.0101 73 diameter (mm)

(log) Maximum Soil temperature °C 0.0106 0.00338 3.14 0.00200 0.048 176 number of flowers

Mean corolla length (log) Maximum 0.474 0.271 1.75 0.0846 0.0270 73 (mm) number of flowers

Mean corolla (log) Maximum 1.650 0.412 4.00 0.000152 0.170 72 diameter (mm) number of flowers

Table 2. Multiple regression analysis for mean corolla length and diameter Dependent variable Predictor variable Estimate SE t p Adjusted df R2

Mean corolla length Soil temperature °C -0.0257 0.0132 -1.95 0.0546 0.0632 72 (mm)

(log) Maximum number 0.551 0.269 2.05 0.0443 of flowers

Mean corolla Soil temperature °C -0.00268 0.0206 -0.13 0.897 0.159 71 diameter (mm)

(log) Maximum number 1.65 0.418905 3.95 0.000183 of flowers

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Experiment 2. Pollination Seed count from autogamous self Soil temperature at site of pollination origin had no effect on 50 seed yield for autogamous self-pollination (Figure 45 4). The ANOVA showed 40 a difference in mean seed 35 count for the three pollination treatments 30 (Table 3). The Tukey 25 HSD pairwise comparison 20

showed no difference in Seedcount 15 seed count between any 10 treatments, although the 2 difference between cross- Adjusted R : -0.022 5 P value: 0.822 pollination and 0 autogamous self- 0 5 10 15 20 25 30 35 40 pollination approached significance (Table 4). Temperature °C (in mother plant's location)

Figure 4. The general linear model showed no effect of soil temperature at site of origin on seed count via autogamous self-pollination.

Table 3. The ANOVA showed a difference in mean seed count for the three pollination treatments.

Df Sum Sq F Vaue P-Value Treatment 2 944 471.8 0.0353 Residuals 57 7577 123.9

Table 4. The Tukey HSD pairwise comparison showed no difference in seed count between pollination treatments: (A) autogamous self-pollination, (G) geitnogamous self-pollination and (C) cross-pollination.

Diff Lwr Upr P adj C-A -12.02 -25.09 1.043 0.0773 G-A -7.355 -17.47 2.76 0.196 G-C 4.667 -10.81 20.14 0.749

Experiment 3. Visitation The multiple regression analysis showed no effect of soil temperature at site of origin, date or the interaction (soil temperature x date) on visitation rate by pollinating insects for the four combined observation categories (Table 5).

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Table 5. The multiple regression analysis showed no effect of temperature at site of origin on visitation rate for all four categories combined (hover, land on flower, drink/pollinate, land on plant). Dependent variable Predictor variable Estimate SE z p

All categories combined Soil temperature °C -0.0505 0.0624 -0.81 0.418

Date: 5/29/19 1.78 0.939 1.89 0.0584 Date: 5/30/19 0.552 1.03 0.536 0.592 Date: 5/31/19 0.763 0.991 0.77 0.442 Date: 6/04/19 -1.23 1.21 -1.02 0.310 Date: 6/05/19 -0.0530 1.04 -0.051 0.960 Date: 6/10/19 -1.81 1.34 -1.36 0.175 Date: 6/14/19 0.273 0.982 0.278 0.781 Date: 6/19/19

Temp x Date: 5/29/19 -0.0276 0.0712 -0.387 0.699 Temp x Date: 5/30/19 0.0520 0.0705 0.737 0.461 Temp x Date: 5/31/19 -0.00877 0.0738 -0.119 0.905 Temp x Date: 6/04/19 0.0639 0.0776 0.824 0.410 Temp x Date: 6/05/19 0.0433 0.0705 0.613 0.540 Temp x Date: 6/10/19 0.110 0.0798 1.37 0.170 Temp x Date: 6/14/19 0.0407 0.0685 0.594 0.552 Temp x Date: 6/19/19 0.0650 0.101 0.641 0.521 .

Discussion

Valdés et al. (2018), found that warmer soil temperatures in the Hengill geothermal area in Iceland corresponded with an earlier flowering phenology for Cerastium fontanum and that when transplanted to a common temperature, plants exhibited a counter-gradient phenological pattern, providing evidence that this once plastic response had led to genetic change. However, as an earlier flowering phenology can negatively affect fitness in several ways, it is likely that plants have compensated for this by flowering later than they would have via the plastic response alone. I observed the same counter-gradient pattern in my common-garden study, which led to my hypothesis that early bloomers

13 would lose synchrony with their pollinators, and thereby my predictions that, in accordance with the “reproductive assurance hypothesis” they should be under high selection to self-pollinate and invest less in reproductive traits to attract pollinators. I predicted that plants from warmer soils would have fewer and smaller flowers and have a lower visitation rate by insect pollinators. However, I did not find support for any of these predictions. Soil temperature did not affect flower size or visitation rate, and contrary to my prediction, plants from warmer soils had more flowers than plants from colder soils. Additionally, soil temperature had no effect on seed yield via autogamous self-pollination and there was no difference in seed yield between the three pollination treatments.

Synchrony with Pollinators The findings of this study can be interpreted in several ways. The first is that C. fontanum growing in warmer soils, despite their earlier flowering phenology, have not lost synchrony with insect pollinators and are thereby not under selection to change their investment in reproductive structures. While phenological mismatches between plants and pollinators are feared, and though research on the matter is limited, most field studies have found that insects seem to be keeping up with plants’ earlier spring flowering (Forrest 2014). This is not strange, considering that mutualistic plant-pollinator interactions should be under high selection to stay synchronized (Pyke et al. 2016). Additionally, C. fontanum is a generalist, and is pollinated by many different insects. It is unlikely that all insects respond to temperature in the same way and therefore a moderate level of redundancy in the pollinator community could safeguard against a phenological mismatch (Hegland et al. 2008). Alternatively, C. fontanum from warmer soils could be experiencing a slight phenological mismatch with pollinators but not enough for the benefits of shifting to self-pollination to outweigh the costs of inbreeding depression, and therefore plants may increase their number of flowers as a sort of “reproductive assurance” to attract more pollinators.

Number of flowers In the event that plants have not lost synchrony with insect pollinators and therefore are not under selection to switch to self-pollination, the observed difference in number of flowers per plant could be a direct effect of temperature (Arft et al. 1999; Totland 2001; Meineri et al. 2014). In the international tundra experiment (ITEX) conducted in the late 90’s, a large collaboration of researchers studied the effects of increased temperatures on various arctic plants using open temperature chambers. They found that plants from the high arctic increased reproductive effort (number of flowers) as a result of higher temperatures (Arft et al. 1999) but that this was not in effort to increase attractiveness to pollinators (Totland 1999). Such plastic responses to higher temperatures may have led to long-term local adaptations in C. fontanum plants growing in warmed soils if selection pressures for this adaptation remained high enough. The difference in flower number could also be due to other environmental factors such as water (Spigler & Kalisz 2013; Meineri et al. 2014) or nutrient availability (Spigler & Kalisz 2013) which Valdés et al. did not present any data for, and is unfortunately outside the limits of this study. Furthermore it could be a result of trans-generational non-genetic effects (Valdés et al. 2018).

Alternatively, plants from warmer soils might, rather than shifting to self-pollination, be investing more into reproductive structures to attract pollinators to ensure successful cross-pollination and maintain genetic diversity (Fabbro & Körner 2004; Forrest & Thomson 2010; Arroyo et al. 2013). While this is quite contrary to my prediction and goes against the “reproductive assurance hypothesis,” it would not be the first time such as pattern has been observed. In their study of the highly autogamous alpine plant Chaetanthera euphrasioides, Arroyo et al. (2013) expected plants from higher altitudes to experience pollen limitation due to harsh environmental conditions and therefore switch to self-pollination and invest less into reproductive structures. However, they found that plants from high altitudes invested more into floral structures (biomass) than plants from lower altitudes and

14 suggest that this could be an effort to ensure genetic diversity via cross-pollination in the chance encounter with a pollinating insect (Arroyo et al. 2013).

Future directions In addition to altering the size of their floral display (number of flowers), plants can alter the duration of their flowering period in a growing season to maximize their chances of attracting a pollinator (Forrest & Thomson 2010; Dai et al. 2017). In this study I measured the maximum number of open flowers per plant at any given time (peak flowering) and not the absolute number of open flowers, although this and the duration of the flowering period would have been interesting to examine.

Based on Valdés et al.’s (2018) and my findings, C. fontanum appear to be genetically adapted to the different soil temperatures in the Hengill geothermal area and may therefore also be adapted to the local insect fauna, which could significantly differ from the fauna found in Stockholm, Sweden. Such a difference may have played a role in the lack of difference in visitation rate found in this study. A future study could examine visitation rate by pollinating insects on C. fontanum in situ to gain insight on the nature of this result. In addition, it would be interesting to conduct an observational study on the seasonal changes in abundance and species composition of insect pollinators in the Hengill geothermal area to better understand the level of functional redundancy within the insect community. It would also be of interest to study the emergence phenology of insects in the area, to better understand whether C. fontanum from warmer soils are actually losing synchrony with pollinators due to earlier flowering. Additionally, it would be worthwhile to repeat the hand-pollination experiments in a greenhouse, preferably in the Hengill geothermal area, to avoid issues with pollination bags and to better understand how pollen-limitation affects reproductive strategy.

Conclusion

While our climate continues to change, it will be become increasingly important to understand how rising temperatures affect organisms, interactions between organisms, and the ecosystems they are part of. This project utilized geothermal heating as a natural way of studying the effects of temperature on the flowering phenology of the herb Cerastium fontanum. Plants growing in heated soils in the Hengill geothermal area in Iceland responded plastically to higher temperatures by flowering earlier in the field than plants growing in colder soils. When transplanted to a common temperature, C. fontanum flowered in a counter-gradient fashion, demonstrating that this plastic response has led to long-term genetic change. An earlier flowering can have several fitness drawbacks, and therefore it is likely that genetic compensation counteracts this plastic response and causes plants from warmer soils to flower slightly later in the field than they would from the plastic response alone. In this study I examined if an earlier flowering phenology could lead to a loss of synchrony with insect pollinators by examining floral traits, pollination strategy and attractiveness to insects along a temperature gradient, and whether this could be a possible driver of genetic compensation. I found that plants from geothermally heated soils had more flowers than plants from cold soils but that temperature had no effect on flower size, pollination strategy or visitation rate by pollinating insects, when examined in a common environment. Based on my findings it does not appear that a phenological mismatch is the main driver of this genetic compensation. It is likely other environmental factors such as daylight or water availability that put a constraint on C. fontanum’s earlier flowering. This study demonstrates the many forces at play when organisms undergo large-scale environmental changes. Plastic responses can lead to evolutionary changes but these can in turn be counteracted by selection if responses are maladaptive. Disentangling these and thereby understanding and predicting how organisms will respond to climate

15 change is difficult, but nonetheless necessary if we are to mitigate large-scale, climate-driven loss of biodiversity.

Acknowledgements

I would like to thank my supervisor Johan Ehrlén for giving me the opportunity to create a research project based on my own interests in pollination and climate change, for providing me with plant material, and for guidance and counsel throughout the entire process. I would also like to thank gardener Anna Petterson for maintaining the sprinkler systems in the outdoor garden and providing advice on how to care for C. fontanum. Thank you, Caitlin Wilkinson, Johannes Falk, and Stefan Bjursäter for reviewing and giving feedback on this thesis. Thank you, Tanya Strydom for helping me with R and statistics. A warm thank you to all my friends and family for your endless encouragement and moral support! Last but not least, I would like to thank Peter Hambäck and the faculty at DEEP for giving me the opportunity to conduct this research project.

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