Context dependency of plant animal interactions. Malin A. E. König

Context dependency of plant animal interactions Malin A. E. König

Malin A. E. König, Stockholm University 2014 Cover illustration: E. Stuckey-Lundgren Illustration: L. Ström Back cover photograph: L. Ström ISBN 978-91-7447-857-0 Printed in Sweden by US-AB, Stockholm 2014 Distributor: Department of Ecology, Environment and Plant Sciences, Stockholm University

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Doctoral dissertation Malin A. E. König Department of Ecology, Environment and Plant Sciences Stockholm University SE-106 91 Stockholm Sweden Abstract The strength and direction of interactions between organisms vary spatially across the landscape. Traditionally, the focus has been on how trait variation affects the interactions between species. However, differences in abiotic and biotic environmental factors may also alter the distribution, phenology and behavior of the interacting species. To be able to understand why an interaction varies across the landscape, the effects of trait variation has to be separated from the effects of the environmental context. In this thesis, I try to separate the effects of context and trait differences on plant resistance against herbivory, through experimental and observational studies conducted with two cytotypes of the perennial herb Cardamine pratensis and its main herbivore, Anthocharis cardamines. The results show that differences in plant resistance against oviposition under controlled conditions were mainly mediated by flower size; larger flowers were more attractive to the female butterfly. However, among-populations differences in oviposition under natural conditions were not related to the resistance observed under controlled conditions, or to ploidy type, flowering phenology or plant size. Within populations under natural conditions the oviposition patterns by A. cardamines was affected by the plant traits plant size and flowering phenology. This thesis conclude that among-population differences in intensity of plant-herbivore interactions were caused by differences in environmental context rather than by herbivore preferences for any phenotypic plant traits, while host plant selection within population was based on plant traits. This suggests that biotic and biotic context can have important effects on the intensity of plant-herbivore interactions. Although genetic traits influenced the outcome of the interaction within populations, it was the environmental context of the populations that determined largely if the interaction took place or not. Keywords Anthocharis cardamines, attack intensity, Cardamine pratensis, cytotype, herbivory, larval fitness, oviposition, phenology, plant-animal interactions, plant resistance, plant tolerance, polyploidy, spatial variation, trait variation

List of papers This thesis is based on the following papers, which are referred to by their roman numerals: I. König M. A. E., Wiklund C., Ehlrén J. (2014) Contextdependent resistance against butterfly herbivory in a polyploid herb. Oecologia doi: 10.1007/s00442-013-2831-4 II. III. IV. König M. A. E., Lehtilä, K., Wiklund C., Ehlrén J. Amongpopulation variation in tolerance to larval herbivory by Anthocharis cardamines in the polyploid herb Cardamine pratensis. Plos One in review. König M. A. E., Wiklund C., Ehlrén J. Among-population variation in butterfly oviposition preference and larval performance on a polyploid herb. Ecological Entomology in review. König M. A. E., Wiklund C., Ehlrén J. Timing of flowering influences intensity of attack by a butterfly herbivore in a polyploid herb. Manuscript Paper I is reprinted in this thesis by the kind permission of the copyright holder. The original publication is available at link.springer.com/article/10.1007/s00442-013-2831-4

Contents Introduction... 11 Aim of the thesis... 14 Method... 15 Study system... 15 Experimental design... 17 The cage experiment... 17 Larval performance... 17 The tolerance experiment... 18 Oviposition patterns in the population of origin... 19 Data analyses... 19 Plant resistance against oviposition (Paper I)... 19 Plant tolerance, resistance and attack intensity (Paper II)... 20 Larval performance vs. female preferences (Paper III)... 21 Phenology and ovipostion patterns (Paper IV)... 22 Results and Discussion... 23 The effect of trait vs. context on resistance... 23 Relationship between tolerance, resistance and attack intensity... 24 Mother knows best?... 25 Phenology and oviposition patterns... 27 Conclusion... 29 References... 30 Svensk sammanfattning... 36 Tack!... 42

Introduction The strength and direction of interactions between organisms vary spatially throughout the landscape. Traditionally, the focus has been on how trait variation affects the interactions between species (Morand et al. 1996; Agrawal et al. 2007, Gross et al. 2009). However, differences in abiotic and biotic factors may alter the cooccurrence of the interacting species as well as affect their phenology and behavior (Michalakis et al. 1992, Lehtonen et al. 2005, Thompson 2005, Cleland et al. 2007). To be able to understand the underlying causes of variation in interaction strength across the landscape, the effects of trait variation has to be disentangled from the effect of environmental context (Agrawal et al. 2007). This can only be done by combining observations in natural systems with controlled experiments where contextual factors can be controlled for. An important type of interaction is between plants and herbivores. Herbivores cause fitness losses in plants, either by consuming reproductive parts or by feeding on vegetative parts and thereby reducing the available resources (Marquis 1984, Pilson 2000, McCall & Irwin 2006). To avoid fitness reductions due to herbivory plants have two main strategies, resistance and tolerance (Núñez-Farfán et al. 2007). Resistance is defined as the ability to reduce the amount and intensity of an attack, either through morphological defenses e.g. by producing spines or chemical defenses, by avoiding the interaction temporally i.e. altering phenology, or spatially, i.e. growing in habitats which are non-preferred or sub-optimal for the herbivore (Belsky et al. 1993). Studies of resistance have often focused on plant traits related to defense mechanisms rather than on temporal and spatial avoidance. However, recent studies suggest that avoiding herbivores in space and time seem to play an important part in plant resistance against herbivory (Pilson 2000, Weinig et al. 2003, Carmona et al. 2011, Shouldt et al. 2012, Pearse et al. 2013). For example Carmona et al. (2011) showed that in 19 plant families variation in phenology had a stronger effect on the degree of herbivory than the production of secondary metabolites. Tolerance is defined as the ability to reduce fitness losses from herbivory after an attack e.g. through increased growth, assimilation or relocation of stored resources (Strauss & 11

Agrawal 1999, Stowe et al. 2000, Tiffin 2000). The efficiency of the different tolerance mechanisms has been shown to depend on the type of herbivore damage (Sadras 1996, Rosenheim et al. 1997). Both resistance and tolerance are believed to be costly in the absence of herbivory, since to increase resistance and tolerance the plants has to invest resources which otherwise could have been used for reproduction (Koricheva 2002, Stinchcombe 2002). Therefore, the degree of investment into the two strategies should depend on the cost of defenses and attack intensity within the population (Mauricio et al. 1997). Resistance and tolerance have also often been assumed to be redundant strategies, since a resistant plant will not need to be tolerant and vice versa, a tolerant plant will not have to avoid herbivore damage (van der Meijden et al. 1988, Herms & Mattson 1992). However, plants which are attacked by several herbivore species seems to be more prone to experience selection for mixed defense strategies (Carmona & Fornoni 2013). Phenology, i.e. when different developmental and life cycle events of an organism take place, affect the interaction between plants and herbivores since the phenology of an organism determines how and when it will interact with the biotic and abiotic environment. Insect herbivores have to time their emergence and reproduction to the plants phenology, otherwise they might suffer from reduced survival and reproduction (Feeny 1970, Tikkanen & Julkunen-Tiitto 2003). The synchrony of herbivores with their host plants is thus important for their success. Since plant phenology varies among years due to variations in the abiotic conditions, the optimal period for herbivores also varies among years. Immobile herbivores experience strong selection on their reaction norms to match their phenology with that of their host plant populations (van Asch & Visser 2007, van Dongen et al. 2007). A more mobile herbivore which is able to move among plant populations, should experience weaker selection since it is able to switch between plant population. To synchronize with plants phenology, the herbivore has to be able to respond plastically to environmental cues. The reaction norm describes the relationship between the phenotype and an environmental cue for an organism (Scheiner 1993). Although a plant and its herbivores are synchronized in one habitat, the degree of synchrony often varies among years due to variations in weather conditions (van Asch & Visser 2007), causing variation in temperature and other environmental cues used for phenological onset. Plants should thus experience different selection pressures from herbivore on phenology among years and among populations depending on their timing relative to their herbivores (Mahoro 2002, Tarayre et al. 2007). 12

From a butterfly s perspective, the host plant choice for oviposition is important since the nutritional quality and plant defenses can vary greatly among plant species and among individuals within species (reviewed in Awmack & Leather 2002). The suitability of a plant individual as host plant is also affected by the light availability, temperature, and the level of parasitation and predation in the microhabitat where the plant grows, which will affect the survival and performance of the offspring (Scriber & Slansky 1981). Offspring of females which can correctly evaluate the quality of a host plant should thus have an advantage over offspring from females which cannot evaluate host plant quality, resulting in selection on the female s ability to estimate plant quality. This is summarized in the preferenceperformance hypothesis, or the mother knows best hypothesis, which predicts that the females oviposition choice should be based on how good the host plant is for larval growth and survival (Jaenike 1978, Gripenberg et al. 2010). However, although a majority of studies finds a positive relationship between female preferences and larval performance, an increasing number of studies find no relationship (Scheirs et al. 2000, Scheirs 2002, Refsnider & Janzen 2010). The lack or positive correlations between female preferences and larval performance has been suggested to be the result of optimal host plants being rare, optimal host plants growing in unfavorable habitats with high levels of predators or parasitoids, the optimal host plant varying between years due to weather variations, the females preferring habitats which increases their own survival rather than the fitness of their offspring, or time limitation for the female i.e. producing more offspring to compensate for the reduced fitness of individual larvae (Courtney 1981, Thompson 1988, Scheirs et al. 2000, Mayhew 2001, Rosenheim et al. 2008, Wiklund & Friberg 2009, Singer & McBride 2012). Also, generalist herbivores are considered to be less apt at predicting the quality of individual host plants, since they need to recognize several host species (Wiklund 1981, Janz 2003, Liu et al., 2012). Although the mother knows best hypothesis is applicable both among and within host plants species, studies have mostly focused on among species rather than within species (but see Singer & McBride 2012). 13

Aim of the thesis The aim of this thesis is to try to disentangling the effects of environmental context and genetically based trait differences on plant resistance and tolerance against herbivory, using two cytotypes of the polyploid perennial herb cuckoo flower (Cardamine pratensis L.) and its main herbivore, the orange tip butterfly (Anthocharis cardamines L.), through experimental and observational studies. I will address the following questions: 1) To what extent is variation in resistance against butterfly oviposition among populations due to trait variation and contextual differences in environmental contexts? (Paper I & IV) 2) How are among populations variation in plant tolerance, resistance against oviposition and attack intensity in the field related to each other? (Paper II) 3) Are female butterfly oviposition preferences correlated to larval performance? (Paper III) 4) Is variation in oviposition preferences among and within populations linked to plant flowering phenology? (Paper IV) 14

Method Study system The cuckoo flower, Cardamine pratensis L. (Brassicaceae), is a perennial herb which is distributed throughout Europe, northern North America, Asia and Africa (Lövkvist 1956, Lihová et al. 2003). Cardamine pratensis is a polyploid complex and diploid up to dodecaploid cytotypes has been found within the group (Lövkvist 1956). In Sweden only three polyploid subspecies can be found, the tetraploid spp. pratensis (2n = 4x =30), the octoploid spp. paludosa (2n = 8 12x = 56-96), and the octoploid to dodecaploid spp. polemonides (2n = 8 12x = 60-90). Both spp. paludosa and spp. polemonides are derived from spp. pratensis via autopolyploidization (Franzke and Hurka 2000). Whereas spp. polemonides is limited to artic areas and is only found in the northern parts of Sweden, both spp. pratensis and spp. paludosa can be found throughout Sweden, with spp. pratensis preferring sunny meadows whilst spp. paludosa generally grows in more shaded and damper meadows, ditches and forests (Lövkvist 1956, Arvanitis et al. 2007). However, their distributions partly overlap and they can occasionally be found growing in sympatry. The two ploidy types differ in their morphology and can be visually separated from one another; spp. paludosa grows larger, produces larger but fewer flowers and has a different leaf morphology (Lövkvist 1956). Both flower from mid-may until mid- June and spp. paludosa generally begins flowering slightly later than spp. pratensis (Arvanitis et al. 2007). This phenological difference seems to be due to environmental differences between habitats, as spp. paludosa begin to flower simultaneously with spp. pratensis in syntopic populations (Arvanitis et al. 2008). The orange tip butterfly, Anthocharis cardamines L. (Pieride), is univoltine and flies during May-June. It is a Brassicaceae specialist and utilizes several Brassicaceae species as host plants for their larvae. They often show local specialization to one or a few host plants (Wiklund 1984), and C. pratensis is one of its main host plants. The female prefers to oviposit on plants growing in sunny areas and flies over large areas in search for host plants (Wiklund & Åhrberg 1978, 15

Arvanitis et al. 2007). Anthocharis cardamines is a phenological specialist and only utilizes flowering individuals of its host plant (Dempster 1997, Arvanitis et al. 2008). The first recognition of potential host plants seems to be mostly visually, and females are attracted to brightly colored objects within the size range of a Brassicaceae inflorescence (Wiklund & Åhrberg 1978, Courtney 1982). The female lands on the inflorescence, and if the flower stalk bends under its weight the female abandons the plant, most likely since the plant will not contain enough food for the larva to complete its development (Wiklund & Åhrberg 1978). If the plant is suitable the female bends her abdomen and oviposits a single egg together with an oviposition deterrent pheromone in the inflorescence (Dempster 1992). The pheromone prevents other female from ovipositing, upon the plant, a behavior that has been selected for presumably because the plants usually only can support the development of one larva (Wiklund and Åhrberg 1978). The egg hatches within 7-10 days, and the first instar larva begins to feed on the buds, flowers and young siliquae. The larva will consume all seeds and siliquae and a large amount of the vegetative parts of the host plant before pupation. If the plant has flowered for more than a week before oviposition the death rate of first instar larvae typically increases, most likely as an effect of the siliquae having become too tough for the larvae to consume (Dempster 1997). If the host plant does not contain enough plant material for the larva to complete its development, the older larva can relocate to a plant close by (Wiklund & Friberg 2009). However, the success rate of relocation is unknown, and if a plant avoids becoming oviposited upon it will most likely avoid larval herbivory during the rest of the flowering season. Oviposition avoidance should thus be an important part of plant resistance in this system. This thesis is based on three experiments, conducted in a common garden with standardized environmental conditions, and one observational study, conducted in the field over five years. All experiments are based on plant material from two to four plant C. pratensis individuals in 25 tetraploid populations and 28 octoploid populations collected in Ludgo parish, 100 km south of Stockholm in 2009. By potting leaf material from each individual (genet) the plants were cloned, creating several ramets of the same genet. The ramets were overwintered in a common garden before being used in three experiments at Stockholm University: an enclosed ovipostion preference experiment (Paper I), a plant tolerance experiment (Paper II) and a larval performance experiment to investigate the relationship between female oviposition preferences and larval performance (Paper III). In 21 of the 53 populations used for the experiments, an 16

observational study was carried out during 2009-2013. The information from the observational study was used both as a base line to calculate attack intensity (Paper I-III) and to investigate how phenology affected oviposition patterns among and within populations under natural settings (Paper IV). Together these four studies were used to disentangle the effects of environmental context and genetically based trait differences for plant resistance and tolerance against herbivory. Experimental design The cage experiment In 2010 one to five flowering clonal replicates of a genet, hereon ramets, which had just begun to flower were randomly divided into sets of 16 plants, eight tetraploid and eight octoploids. A set contained maximum one ramet of a genet, but genets from the same population were allowed in the same trial. Each set were placed in a circle in a 2.5 x 2.5 x 1.8 m sunlit cage and a mated A. cardamines female was released in the middle. The time it took until all plants were oviposited upon by the female was recorded. The experiment was continued until all plants had been oviposited upon, or until the sun began to descend and cage was no longer completely sunlit. If less than six plants were oviposited upon, the experiment was continued on the following day. The recorded time until oviposition for each plant was considered as an estimate of plant resistance; the longer it took until a plant became oviposited upon by the female the more resistant it was considered to be. To be able to compare the resistance amongst the cage trials, the time was standardized within each cage by subtracting the mean oviposition time and divided by the standard deviation of that trial. In addition to the resistance estimate, the total number of flowers, the diameter of the second open flower, the first day of flowering, and the height and basal diameter of the flower shoot was estimated for each plant. Larval performance The plants oviposited upon were kept in the common garden sheltered from oviposition by wild butterflies under a thin fabric and placed in trays filled with water. The trays kept the plants moist and hindered larvae from moving between the plants. The plants were checked 17

every week for larvae and the size of a larvae and the percentage plant material consumed was noted. This was continued for each plant until the larva was no longer present. Twenty-six of the ramets in the cage experiment were not oviposited upon. To investigate if larvae performed worse on these ramets compared to the preferred plants, a single first instar larva hatched in the laboratory was added to each ramet. The plants were then placed in the common garden and treated the same way as the ramets with naturally hatched larvae. The tolerance experiment Tolerance is commonly estimated as the ability to compensate fitness losses after herbivory, and can be described as a reaction norm where fitness represents the phenotype and damage the environmental cue (Bardner & Fletcher 1974). One way to calculate the reaction norm for a genotype is to use multiple ramets of a genet, and subject half of the clones to a standardized level of damage while leaving the other half of the ramets undamaged as controls. By comparing the fitness between the damaged and undamaged plants of the same genotype it is possible to calculate the slope of the reaction norm using the linear equation. The slope represents how fitness responds to damage, i.e. how tolerant the genotype is. A positive slope indicates that the plant genotype gains fitness after damage, a negative slope indicates that the genotype suffers from fitness reduction after damage, and a flat reaction norm indicates that the genotype is able to perfectly compensate the damage (Stowe et al. 2000). As a control group one to two flowering ramets of each genet included in the cage experiment was randomly picked and left undamaged in the common garden. These plants were used to calculate the genet s fitness in the absence of herbivory. In total 332 ramets were included in the control group. To calculate tolerance, the damage caused by the A. cardamines larvae was standardized by reducing the remaining plant tissue after the larvae disappeared to 5 % of the original plant mass via clipping. The 95 % damage level was chosen since this was the largest observed amount of plant tissue consumed by larvae in the experiment. The clipped and control plants were left sheltered from herbivory in the common garden until the next flowering season. 18

The year after damage the survival, tendency to re-flower, the first day of flowering, the total number of flowers produced, the height and the basal diameter of the flowering shoot was recorded for all the plants in the treated and control group. Oviposition patterns in the population of origin During the years 2009-2013, 21 of the 53 populations, ten tetraploid and eleven octoploid, used in the common garden were followed each flowering season. Up to 30 flowering individuals were marked and visited once a week throughout the flowering season. At each visit the total number of buds, flower and siliques were counted for each plant, and the plant was searched for A. cardamines eggs and larvae. The number of buds, flowers and siliques were used to estimate first day of flowering. Once all marked plants in all population had begun to flower, the basal diameter and height of the flower shoot was measured in all populations. Data analyses Plant resistance against oviposition (Paper I) Number of flowers (square root transformed) and flower shoot mass (calculated as the volume of a cylinder using stem height and diameter and then log-transformed) was highly correlated in the cage experiment. Therefore the first principle component between the two parameters was calculated, and will hereon be referred to as plant size. Of the 177 genets included in the cage experiment, only genets represented by at least two ramets were used, and only populations represented by at least two genets were included; leaving 173 genets and 52 of the 53 populations for the following analyses. General mixed linear models were used to investigate if resistance, plant size and flower size differed between the two ploidy levels, using genets nested in populations as random factor. Resistance differed between the ploidy types, and to examine how the difference in resistance between tetraploids and octoploids was mediated by the plant traits a path analysis was used. Phenology might affect the outcome of the cage experiment, and although plants of similar phenological stage were used, some variation was still apparent. Days since first open flower was therefore at first included in all models, but never had a significant effect. 19

To compare the results in the cage experiment with no variation in environmental context to patterns of oviposition under natural conditions, data on oviposition frequencies in the field populations from 2009-2012 were used. The percentage of plants not oviposited upon was relativized within year by removing the yearly mean proportion of plants oviposited upon from each population. The population mean was calculated from these relativized population values. The among year mean attack rate was used to test if attack intensity differed between tetraploid and octoploid populations. Plant tolerance, resistance and attack intensity (Paper II) Tolerance was calculated as the slope between damaged and undamaged plants, using the formula: (W(damaged) - W(control)) / (0.95-0), where W equals average fitness of damaged and control plants of a specific genet, and the denominator represents the difference in degree of damage between damaged (with 95 % aboveground biomass removal) and control plants (Boalt et al 2010). Tolerance was calculated for four fitness estimates, probability to survive, probability to re-flower in surviving individuals, number of produced flowers in flowering individuals and total number of flowers in all individuals, i.e. the plants which did not survive or flowered are represented by 0 flowers. Usually seed set was also used as a fitness estimate, but due to settings in the common garden the seed set was not a reliable fitness estimate. Cardamine pratensis is selfincompatible but can to some degree cross pollinate between ploidy types (Lövkvist 1956). Since pollinators were free to visit ramets of the same genet as well as travel between tetraploids and octoploids, the seed set could be highly affected by pollen limitation and unsuccessful hybridization among ploidy types. For the following analyses population means of each of the four tolerance estimates were used. To investigate the relationship between tolerance, resistance against oviposition under controlled conditions and attack intensity in the populations of origin, the four tolerance estimates were first correlated against population mean resistance achieved from the cage experiment. Secondly the four tolerance estimates were correlated against the estimate of attack intensity in the 21 field populations from the years 2009-2012. Lastly, population mean resistance was correlated with attack intensity in the field. 20

Larval performance vs. female preferences (Paper III) To examine if the probability of egg hatching (0 or 1) differed between ploidy types, a generalized mixed effect linear model with binomial response was used. Ploidy type was used as fixed factor and plant genets nested within populations and butterfly female identity were used as random factors. For analyses of egg hatching, a genet had to be represented by at least two ramets, a population by at least two genets and a female by at least two eggs, leaving 419 ramets, representing 163 genets, 51 populations and 21 females. To examine if final larval size differed between ploidy types a general mixed effect linear model was used, with ploidy type as fixed factor and plant genets nested within populations and butterfly female identity as random factors. To examine if plant traits affected final larval size a general mixed effect linear model was used with ploidy type, plant size, flower diameter and phenology as fixed effects and plant genets nested within populations and butterfly female identity as random effects. For both analyses the same criteria for hierarchical nesting among plants as in the egg hatching analysis was used, but a female had to be represented by at least two hatched larvae, leaving 173 ramets, representing 75 genets, 30 populations and 18 females. To examine if female preferences under controlled conditions were related to probability of an egg hatching, a generalized mixed effect model with binomial response was used, with ploidy type, plant resistance from the cage experiment and the interaction term as fixed factors and plant genets nested within populations and butterfly female as random effect. To examine if female preferences under controlled conditions were related to larval success, final larval size was used as response variables in a general mixed effect linear model. Ploidy type, plant resistance achieved from the cage experiment and the interaction term were set as fixed factors and plant genet nested within populations and butterfly female identity as random factors. Twenty-six ramets did not receive an egg in the cage experiment but had first instar larvae hatched in the laboratory added to them. To test if larvae had inferior development on these rejected plants compared to the ones chosen for oviposition, a general mixed model with final size as response variable, larval type (oviposited upon or added), ploidy type and the interaction term as fixed factors, and plant genets nested within population as random factors was used. Lastly, to examine if larval performance was related to female oviposition preferences under natural settings, final larval size was 21

related to attack intensity in the 21 field populations during 2009-2013. Instead of the relativized mean population value used in the resistance and tolerance analysis, the proportion of plants oviposited upon in a population was only averaged across years. Phenology and oviposition patterns (Paper IV) To investigate how differences in first day of flowering affected among and within population patterns of oviposition data from 21 field populations during 2010-2013 were used. The year 2009 was excluded since the data were too sparse to be able to estimate first open flower for plant individuals. In the flowering seasons 2010-2013 first flowering day, the total number of flowers, shoot mass (calculated as the volume of a cylinder) and the presence of eggs of A. cardamines were recorded. Shoot mass and total number of flowers were still highly correlated after being log-transformed to achieve normal distribution. The first principle component between the variables was used instead and referred to as plant size. Each year was analyzed separately. Among population patterns were examined by using the proportion of plants oviposited upon as response variable and population mean first day of flowering, population mean plant size, population mean plant resistance observed in the cage experiment and ploidy type as predictor variables. Plant resistance under controlled conditions was included to partly control for the effect of among population variation in traits coupled to resistance which were not estimated in the populations. Within-population patterns were examined using generalized models with binomial response. Attack (0 or 1) was used as response variable, and first day of flowering, plant size and population identity as predictor variables. To examine if the among year variation in the relationship between phenology and probability to become oviposited upon was due to differences between A. cardamines and C. pratensis in their reaction norm to temperature, the monthly mean temperature in April and May was calculated using data on the daily temperatures during 2010-2013 provided from the Swedish Meteorological and Hydrological Institute s weather station in Södertälje. The yearly mean temperatures for each month, respectively, correlated with median egg observation, median first day of flowering and the slope coefficient of the probability of oviposition on first flowering day. 22

Results and Discussion The effect of trait vs. context on resistance Resistance against oviposition was higher in tetraploids than in octoploids in the cage experiment, but attack intensity did not differ between ploidy types in the populations of origin (Paper I). The difference in resistance observed in the experiment was mediated by flower diameter, where an increase in flower diameter led to a decrease in resistance (Paper I). However, within ploidy types an increase in flower diameter only had an effect among tetraploid individuals whereas an increase in flower diameter in octoploids did not affect the resistance levels. The difference in oviposition preferences under controlled conditions and the lack of difference between ploidy types under natural conditions suggests that environmental context has a large effect on the outcome of the interaction between C. pratensis and A. cardamines; where octoploids possess traits which are more attractive to the butterfly than tetraploids. A previous study has shown that increased shading decreases the attack intensity by A. cardamines and octoploids generally grow in more shaded environments, (Arvanitis et al. 2007). The attractiveness of octoploids under controlled conditions might thus be counterbalanced by the fact that octoploids grow in habitats which are less preferred by A. cardamines females. Anthocharis cardamines is believed to mainly use visual cues in the search of host plants (Wiklund & Åhrberg 1978). Tetraploids generally have smaller flowers than octoploids (Lövkvist 1956) and the cage experiment shows that increased flower size leads to a decreased resistance against oviposition. Larger flowers thus seem to make it easier for the butterfly to locate the host plant. However, an increase in flower size in octoploids was not related to a decrease in resistance. This indicates that there is a threshold level for flower size after which an increase will not lead to any further decrease in resistance. A possible response to selection is that tetraploid flower size will decrease over time to avoid oviposition by A. cardamines, while octoploids do not experience any selection pressure on flower 23

size. This herbivore mediated selection difference could thus in the long run lead to an increased divergence in flower size between the two ploidy types (cf. Segraves & Thompson 1999, Arvanitis et al. 2010). Divergence in flower morphology between ploidy types has been observed in other systems (Segraves & Thompson 1999, Husband and Schemske 2000, Husband & Sabara 2003), but this is to my knowledge the first time that this difference is demonstrated to affect the interaction with a herbivore. Relationship between tolerance, resistance and attack intensity Tolerance levels did not differ between tetraploid and octoploid populations (Paper II). Tolerance and resistance were estimated under controlled conditions (Paper II). Resistance was not correlated to the previous attack intensity (Paper II, IV). Tolerance in terms of the tendency to re-flower, however, was related to the previous attack intensity (Paper II), i.e. individuals from populations with higher attack intensity were more likely to re-flower the year after damage than individuals from populations with lower attack intensity. Tolerance and resistance are commonly regarded as redundant strategies (van der Meijden et al. 1988, Herms & Mattson 1992), but there was no negative correlation between tolerance and resistance estimated under controlled conditions in this study (Paper II). Previous studies in other systems show mixed results, where some found a trade-off between resistance and tolerance (Fineblum & Rausher 1995, Fornoni et al. 2004) whilst others found no trade-off (Mauricio et al. 1997, Weinig et al. 2003, Puustinen et al. 2004). It has been argued that tolerance and resistance are complementary rather than redundant strategies (Tiffin 2000). This seems to be the case in systems consisting of multiple herbivores (Stinchcombe & Rausher 2001, Carmona & Fornoni 2013). This could be the case for C. pratensis since it is attacked by both a flea beetle (Phyllotreta sp., Coleoptera, Chrysomelidae) and the larvae of green dock beetle (Gastrophysa viridula, Coleoptera, Chrysomelidae, pers. obs.). Since tolerance, resistance and attack intensity might be linked in natural population, a simultaneous investigation of the three parameters is needed to disentangle the relationships among them. Tolerance and attack intensity are expected to be correlated to reduce the negative effect of herbivory. Tolerance was positively correlated 24

to attack intensity in the population of origin in this study (Paper II), but resistance could have interfered with the observed relationship. First, resistance could be negatively correlated to tolerance and attack intensity. Female A. cardamines could conceivably prefer populations with lower resistance which also would be more tolerant. However, resistance was not correlated with either of the four tolerance estimates (Paper II) or attack intensity (Paper II, IV). Secondly, if resistance is positively correlated to tolerance and attack intensity, we would detect a spurious positive correlation between tolerance and attack intensity. However, there was no positive correlation between any of the four tolerance estimates and resistance (Paper II) or between resistance and attack intensity (Paper II, IV). Based on these results, tolerance and attack rates does not seem to be linked to resistance levels against oviposition, and the most likely scenario in our system is that A. cardamines only selects for increased tolerance. Few studies have previously investigated the relationship between tolerance and resistance estimates under controlled conditions to levels of herbivory under natural conditions. But the relationship seems to vary depending on the type of damage (Tiffin 2000) and how well adapted the herbivore is to the host plant (Bustos-Segura et al. 2014). If the herbivore is well adapted to the plant s resistance mechanism, tolerance levels will increase instead since the fitness gained by further increase of resistance is less than the cost (Garrido et al. 2012). The main defense in C. pratensis is glucosinolates like all Brassicaceae-plants, and A. cardamines larvae are specialized to handle glucosinolates (Courtney 1982). The increased tolerance levels in populations with high attack intensities could thus be caused by A. cardamines being a highly adapted herbivore. Mother knows best? There was no difference in egg survival or larval development between tetraploids and octoploids (Paper III). Egg survival and larval performance were not correlated to female oviposition preferences under controlled environmental conditions or in the natural environment (Paper III). Larvae added to plants rejected for oviposition grew as large as larvae feeding on plants chosen for oviposition (Paper III). Although ploidy level is known to affect oviposition preferences by herbivores due to differences in morphology (Janz & Thompson 2002, Halversson et al. 2008), no study has investigated if the offspring 25

performs better on the preferred ploidy level. Anthocharis cardamines oviposits mainly upon tetraploids in the field (Arvanitis et al. 2007), but prefers octoploids when the two ploidy types are presented under similar environmental context (Arvanitis et al. 2008, Paper I) since octoploids are easier to locate due to their larger flower size (Paper I). However, there was no apparent difference due to ploidy type in egg survival or larval development. The preference for octoploids over tetraploids thus seems to be a case of indirect effect of super stimuli rather than an active choice done by the female to increase the fitness of the offspring. The only factor affecting final larval size was plant size; the larger the plant was the larger the larvae grew before abandoning the plant (Paper III). This pattern is common amongst butterfly species (Boggs & Freeman 2005). An increase in flower size was not related to increased final larval size (Paper III), although large flowers are more attractive to A. cardamines (Paper I). A lack of or weak correlation between female preferences and larval performance in other systems has been observed to be due to female time limitations, that generalist species are worse at evaluating plant quality than specialist species; larval survival on a specific host species may be hard to predict among years or the optimal host plants may be very rare or grow in unfavorable habitats for the female or larva (Thompson 1988, Rosenheim et al. 2008, Wiklund & Friberg 2009, Liu et al. 2012, Singer & McBride 2012). Anthocharis cardamines has a lifetime fecundity of an average of 170 eggs (Wiklund et al. 2001) and only oviposits a single egg per plant. The butterfly is also a generalist and has to recognize a broad range of Brassicaceae species and mainly uses visual cues to locate potential host plants (Wiklund & Åhrberg 1978). Locating enough host plants before she dies is thus a critical step. If the female rejected plants to maximize the individual larva s fitness, the female risks lowering her own total fitness by reducing the number of eggs laid (Rosenheim et al. 2008). Flower size may therefore not be a predictor of food quality for the growing larvae, but rather the most efficient way to locate host plants suitable for oviposition. The A. cardamines larvae are also mobile in the later stages, and can relocate later on if they run out of food. The success rate of relocation is unknown, but the possibility decreases the importance of the female s first host plant choice. Other butterfly species with low correlation between female preferences and larval performance often have mobile offspring (Tammaru et al. 1995, Janz & Nylin 1997). But mobile genotypes have a tendency to suffer from reduced fitness compared to more sedentary genotypes (Gu et al. 2006). We can therefore not conclude that A. cardamines is a case of 26

bad motherhood under natural conditions, since several factors which might affect larval development in the field are controlled for in our experiment. Interestingly, larval performance was not correlated with attack intensity in the population of origin. There were two potential scenarios that could have been expected. Either the female would prefer populations with low defense levels in which larva would develop well, or populations with high levels of larval herbivory would have developed high levels of defenses suppressing larval growth and survival. But even though an attacked C. pratensis loses all seeds (Arvanitis et al. 2007) and suffers from reduced fitness the following flowering season (Paper II), the larva develops equally well independent of previous attack history (Paper III). It may be that environmental context plays an important role in the outcome of the interaction. Increased canopy cover reduces the probability of a plant to become oviposited upon in both ploidy types (Arvanitis et al. 2007; 2008), but whether this patterns is coupled to reduced female or larval fitness is unknown. It does, however, seem likely that both gain from avoiding shady habitats, since females flight are dependent on temperature (Courtney & Duggan 1983) and the larvae grow faster in higher temperatures (Bryant et al. 1997). If the sunny environment favors increased larval fitness, the oviposition choice under natural conditions is fairly in line with the mother knows best hypothesis even though the smaller tetraploids will not maximize larval fitness in terms of food availability. Phenology and oviposition patterns Mean first day of flowering of individuals had no effect on the among population patterns of oviposition in any of the four study years (Paper IV). Mean population plant resistance under controlled conditions was also never correlated to the proportion of plants oviposited upon. Plant size had an effect in 2012; populations with larger plants were more oviposited upon than populations with smaller plants. Within populations, earlier flowering individuals were always more likely to become oviposited upon than late flowering individuals, but the effect was only significant in 2010 and 2013 (Paper IV). Plant size had a significant effect in all four years, where large individuals were more likely to become oviposited. This indicates that the among population oviposition pattern is caused by contextual differences among populations rather than by active choices by the female A. cardamines based on genotypic or 27

phenotypic differences in mean plant traits among populations. Within population host plant selection on the other hand seems to be based on plan traits rather than being an effect of the surrounding context. Both C. pratensis and A. cardamines are believed to react to increased photoperiod and temperature during spring, but A. cardamines is most likely more sensitive to an increase in temperature and may react stronger to warmer spring periods (Posledovich et al in prep.). The among-year variation in the effect of phenology on oviposition patterns indicates that C. pratensis and A. cardamines differ in their reaction norms to temperature. The synchrony between A. cardamines and C. pratensis may thus be dependent on the temporal variations in temperature among years. However, this was not supported in this study since the relationship between the mean temperatures in April or May with median first day of flowering, median first egg observation or the slope of the probability to become oviposited upon was not significant, and the effect of first day of flowering on the oviposition pattern was similar in all four years (Paper IV). Resistance against herbivory is often claimed to be a key explanation for why herbivores are unevenly dispersed throughout the landscape (Sturgeon 1979, Coley & Barone 1996), but flowering phenology and spatial escape is not always included when investigating plant resistance. Instead, the focus has mainly been on secondary metabolites and morphological defenses, although life history traits have been shown to have a great impact on resistance (Carmona et al. 2011). This study shows that phenology and context can affect host plant preferences more than resistance in terms of genetic traits. 28

Conclusion This thesis shows that abiotic and biotic environmental context can have a great impact on interaction patterns. Although genetic traits influence the outcome of the interaction within populations, it is the context that determines if the interaction will take place or not. The thesis also highlights the importance of combining results from controlled conditions like green-house or common garden settings with observational data obtained under natural conditions. Traits which have high impact under standardized conditions may not be relevant under natural conditions when several other abiotic and biotic conditions interfere, and vice versa, important differences among populations in the field disappear under controlled conditions. 29

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