Wageningen University Laboratory of Entomology
Plant genotype affects the herbivore community of
evening primrose (Oenothera biennis ).
Camille Ponzio
Msc Plant Science
April- August 2010
Report no. 010.22
Thesis ENT-80424
1st examiner : Marcel Dicke
2nd examiner : Jennifer Thaler
Summary
Intraspecific variation within a single plant species plays a key role in determining its associated arthropod diversity and community composition. In a field experiment I replicated 6 genotypes of evening primrose ( Oenothera biennis ) on which 5 treatments were equally applied (including herbivory from two leaf chewers) and looked at the abundance of the 10 most common insect species. While the effects of genotype on the herbivore community associated to evening primrose had already been in part documented in previous studies, the novelty of this study is that it showed that genotypic variation in evening primrose had differential effects on the generalist and specialist herbivores. Generalists tended to prefer genotypes which had been previously shown to be highly attractive to herbivory to the generalist Japanese beetle ( Popillia.japonica ) Specialists on the other hand avoided these genotypes and were highly abundant on the genotypes which are usually avoided by Japanese beetles. Variation in several plant traits could in part explain the variation in abundance, notably for the specialists. The phenolic compound Oenothein B, nitrogen content, water content and trichome characteristics accounted for 73 to 93% of the variation in the abundance of specialists. While no significant effects could be seen on the generalists, several trends indicated that the plant traits may correlate in the opposite direction with generalists. While the original goal of this study was to investigate the effects of herbivore induced plant responses on the insect community, remarkably the treatments had very little significant effects on a majority of the insects, with only the specialist primrose weevil (Acanthoscelidius acephalus ) showing a preference for plants damaged by conspecifics.
Table of contents
Introduction ...... 1
Research aims and questions ...... 3
Materials and methods ...... 4 Study System ...... 4 Common garden experiment ...... 5 Measurements ...... 6 Statistics ...... 8
Results ...... 11
Discussion : ...... 15 Individual and community -level effects of genetic variation ...... 15 Treatment effects ...... 16 Future research: ...... 17
Conclusions ...... 18
Acknowledgements ...... 19
References ...... 20
Introduction
Plants frequently display genotypic variation that can influence the host plant preference and abundance of individual arthropod species (Karban 1992, Underwood and Rausher 2000) and ultimately regulate the structure of the herbivore community (Maddox and Root 1987, Fritz and Price 1988, Dungey et al. 2000, Tovar-Sánchez and Oyama 2006, Whitham et al. 2006, Poelman et al. 2008). Several studies have shown that not only can herbivorous insects discriminate between hybrid plants and the parent species that compose them (Dungey et al. 2000, Fritz et al. 2003, Hochwender and Fritz 2004), they can also differentiate between different genotypes of a single host plant species (Fritz and Price 1988, Maddox and Root 1990, Shuster et al. 2006). The role of plant genotype in structuring arthropod communities is such that high heritability has been found in several model systems. It has been that it estimated that 50-63% of the variation in arthropod community composition in a single cottonwood species could be explained by plant genotypic variation (Shuster et al. 2006). Likewise, in evening primrose ( Oenothera biennis ) differences among the genotypes accounted for up to 41% of the variation in arthropod diversity and abundance (Johnson and Agrawal 2005).
The notion that insect communities will strongly vary according to the genotypic differences in their host plant is not surprising in view of the wide array of host plant traits, herbivore induced or not, that can affect arthropods. Intraspecific genotypic variation has been found in an array of plant traits, such as morphological traits, (Inbar and Gerling 2008) phenological traits (Hunter et al. 1997), trichomes (Lambert et al. 1995, Luo et al 2010), plant chemistry (Dungey et al. 2000, Walling 2000, Osier and Lindroth 2001) and plant secondary chemical defense compounds (Hamilton et al. 2002), all of which have been shown to have effects on arthropods. Further work on evening primrose by Johnson et al. (2009) showed that a large proportion of variation (73%) in herbivory by Japanese beetles, a common and abundant herbivore, could be explained by genetic variation in several secondary compounds and life history traits.
Genetic variation in plant resistance and induced responses can strongly affect the population of individual herbivore species (Underwood and Rausher 2000, Poelman et al 2009) and the structure of the arthropod community (Fritz and Price 1988, Maddox and Root 1990). These induced defenses are known to differentially affect arthropods according to their host specificity. While generalists herbivores are generally negatively affected or deterred by chemical defenses (van Dam et al. 1995, Lankau 2007), many studies show that secondary compounds may make a plant highly attractive to specialist herbivores that have become adapted to these specific compounds (van Dam et al. 1995, Siemens and Mitchell- Olds 1996). Much evidence indicates that specialists use the unique secondary compounds as cues to locate host plants and to assess the suitability of the plant for feeding and oviposition, and thus will have a preference for host plants having higher concentrations of the compound (Da Costa and Jones 1971, Raybould and Moyes 2001, Macel and Vrieling 2003). Differing levels of secondary compounds between plant genotypes could be expected to lead to differences the ratio between generalists and specialists on a given genotype.
1 While herbivores of the same feeding guild elicit the same general type of responses in the host plant, it is often the case that these induced defenses will show some degree of variation from one herbivore to another (Viswanathan et al. 2005), as the elicited responses can largely depend on the nature of damage inflicted and the biochemical composition of the attacker’s saliva. (Musser et al. 2002) The effects triggered in the plant affect not only the herbivore and its conspecifics ( Denno et al . 1995), but also other herbivore species both across space and time. Indeed, an increasing number of studies are demonstrating the specificity of induced responses, in that the identity of the initial damaging herbivore can differentially affect the host plant preferences of subsequent herbivores and modify the community structure throughout the season (Zandt and Agrawal 2004, Viswanathan et al. 2005, 2007, Poelman et al. 2008).
2 Research aims and questions
The main objective of this research was to discover if arthropod abundance and diversity varied among the different genotypes of evening primrose ( Oenothera biennis ). A second aim was to determine the effects of early season herbivory on the same insect community. More specifically, this research addresses the following questions:
1) Does plant genotype affect individual arthropod species and their community structure?
2) Are generalists and specialist herbivores equally affected by plant genotype? If so, can differences in effects be explained by variation in certain plant traits?
3) Does early season herbivory by the generalist Japanese beetle ( Popillia japonica ) or the specialist primrose weevil (Acanthoscelidius acephalus ) similarly affect the nature of the subsequent colonizers as well as the arthropod community structure and diversity?
As previous studies on evening primrose have shown that evening primrose genotypes strongly varied in their associated herbivore abundance and richness, this study first attempts to confirm these earlier findings on a small subset of arthropods that naturally colonize the species. It is hypothesized that the effects of genotype can go beyond simply affecting the insect community as a whole and also affect herbivores according to their degree of host specificity. For several other plant species it is known that unique chemical compounds will confer resistance to generalist herbivores yet lead to increased attractiveness for specialist herbivores. This study also attempts to test the hypothesis early season herbivory by two different leaf chewers will have different effects on the plant’s induced responses and thus have an effect on the arthropod community. Such effects have been shown in other systems, but not in evening primrose as of yet.
3 Materials and methods
The experiment was conducted at the Freese Road field site of Cornell University, located in Ithaca, New York, USA. The field site is enclosed by tall fencing in order to exclude mammalian herbivores.
Study System
Evening Primrose ( Oenothera biennis L., Onagraceae) is a facultative biannual flowering plant native to eastern North America and is particularly found growing in dry soils of abandoned fields and lots, as well as in recently disturbed soils. Plants produce a rosette the first year, and a single, tall (50-160cm) blooming stalk the following year. However in freshly plowed fields it is not uncommon for plants to bloom in the first year, and they will usually grow larger and produce multiple stalks, acquiring a bush like appearance.
Evening primrose is particularly interesting to use from an ecological perspective due to its mode of reproduction. O.biennis primarily self pollinates and its permanent translocative heterozygote genetic system usually leads to the production of clonal seed (Cleland 1972). Due to this system it is possible to grow out from seed many replicates of a single clonal family, each of which can be considered as a different genotype.
O. biennis is a host plant for a large and widely varying community of arthropod species, including generalist and specialist herbivores, omnivores and predators. Previous studies have identified over 100 different arthropod species on O. biennis (Dickerson and Weiss 1920, Johnson and Agrawal 2007). In the field experiment two of the most common primrose herbivores found in upstate New York were used as the experimental herbivores (Figure 1). The Japanese beetle ( Popillia japonica Newman; Scarabaeidae) is a polyphagous chewing insect that feeds on a wide range of host plants, feeding upon over 300 species from 79 different families (Potter and Held 2002). Introduced from Asia in the early 20 th century it has now colonized most of the north-eastern part of North America and is considered a major pest of fruit and vegetable crops as well as many ornamental plants. The beetle feeds by skeletonizing leaves, leaving only the veins intact. One of its preferred host plants in upstate New York is O. biennis, where it can be found aggregating in large numbers. Acanthoscelidius acephalus (Say; Curculionidae) is a small, 3mm weevil which is thought to be a specialist of the Onagraceae, and feeding almost exclusively on Oenothera sp . (Bloem et al. 2002). However, relatively little is known about this weevil.
A B C Figure 1 : The plant used in this study, Onothera biennis (A). The herbivores used for initial induction are Popilla japonica (B) and Acanthoscelidius acephalus (C).
4 Common garden experiment
The seeds were germinated in the greenhouse in potting soil (Metro-Mix) filled flats in late March, then thinned out to leave one plant per well. The plants were kept in a greenhouse at Cornell University under artificial light until they were placed outside early May in order to be hardened off once they had developed into strong rosettes. They were then transplanted in a freshly plowed field plot two weeks later (May 19th ).
Based on observations and measurements from previous years, six genotypes were selected that either had high or low damage from herbivory by P.japonica (Scott McArt, unpublished data). Genotypes 21, 33 and 44 had ‘high’ damage whilst genotypes 5, 6 and 20 had ‘low’ damage. A total of 240 plants were used, with 40 plants per genotype. Genotypes and treatments were equally divided into four blocks due to a gradient in soil moisture in the field and each block was completely randomized. Plants were spaced 125cm in each direction in order to avoid competition for nutrients and light.
As each genotype bolted a 60cm tall fine weave mesh sleeve was placed over each plant and supported by a 120cm stake in order to exclude unwanted arthropods and to later contain the experimental herbivores (figure 2). The sleeve was tied off at the base of the plant and at the top of the stake. As the plants grew the sleeve was adjusted by bringing it further up the plant, and for plants having outgrown the sleeve they were covered with a second, 120cm sleeve. The bags could not be placed at the rosette stage as strong direct sunlight is crucial for bolting. With the exception of one genotype (21) which bolted two weeks prior to transplanting in the field, the majority of the plants bolted between the second and fourth week of June.
A B
Figure 2 : Common garden experiment before application of the treatments (A) and after removal of sleeves at the end of the treatment induction period (B).
One week before the first treatment was applied the plants were sprayed with a solution of baking soda in order to slow down the development of the powdery mildew which appeared shortly after transplanting, and which was likely due to contamination while the plants were still in the greenhouse.
5 Five treatments were applied in early July when the majority of the bolted plants had begun blooming. The treatments were equally divided among the genotypes, giving 8 replicates of each genotype-treatment combination. The treatments which were applied to the plants were a control, mechanical damage, 0.5mM Jasmonic Acid (JA) spray, herbivory by P.japonica and herbivory by A. acephalus. The JA spray was prepared by making a solution of 0.105g of JA in 1ml of acetone, which was then diluted in 1L of water to achieve the desired concentration. The plants were evenly sprayed until near run off. Mechanical damage was applied by using scissors to damage the leaves, so that approximately 15% of the plant had damage.
The weevils and beetles were collected from natural primrose populations (and other host plants in the case of the beetle) in the week before the application of the treatments and were then contained in cages in the lab on primrose until needed for the experiment.
As the weevils and beetles are of greatly differing sizes, 4 P.japonica or 6 A.acephalus were placed on each plant . To attempt to even out the amount of damage caused by the insects during the induction period, the beetles were released into the bags 6 days after the weevils with the damaged monitored every 2 days. The number of herbivores was adjusted as necessary in order achieve similar amounts of damage on all of the plants and replace beetles which had died by entanglement in mesh sleeve. The targeted amount of damage was 15% (which was not attained), however as the insects ate varying amounts on the different genotypes, the actual amount of damage on each plant was between 3 and 10%.
Two days before the removal of the bags (bag removal was 11 days after starting the first treatment with the weevils) the mechanical damage and JA spray treatments were applied so that plants would be fully induced once the bags were removed. After the two days the sleeves were removed from all of the plants, and the experimental herbivores were removed from the plants and dispersed in the field plot.
Measurements
After removal of the mesh sleeves from the plants, the damage caused by the beetles and weevils was estimated, as well as the percentage of each plant that was infected by powdery mildew. The number of stems per plant was then counted, as well as the maximal height of each plant. Additionally, leaves and buds of 10 plants per genotype were sampled in order to measure the trichome density and score the buds according to how glandular their trichomes were.
Three surveys of the naturally colonizing arthropod species on each plant were conducted a week apart, starting one week after the removal of the mesh sleeves. Working alone and under time constraints, it was not possible to survey and identify all of the species present, so the surveys focused on the 10 most abundant species (Coccinellidae were analyzed as one species due to very low numbers of each individual species). These 10 species (Table 1) were determined and identified by an inspection of the plants a few days prior to the first survey. The top 10 species are a high percentage of the total numbers of arthropods and therefore the important ecology is captured by using this small subset of species. The surveying was done by a careful visual inspection of the stems and both upper and lower leaf
6 surfaces of each plant. Additionally at each survey the presence or absence of blooms (open or in bud) on each plant was recorded.
At the plot level the arthropod diversity was assessed by counting the total number of arthropods of the 10 surveyed species, the species richness (number of species) and evenness of the insect population (relative proportion of those species). Biodiversity was also assessed by using the Shannon-Weiner index (H’), which is one of the most common indices for measuring biodiversity. This index describes arthropod diversity by incorporating both the richness of the species present and how evenly they are distributed. Rare species and high evenness will increase the value of the index. Values close to zero represent low community complexity, while values close to H’max (ln(S), with S being the number of species) indicate high community complexity.
In order to determine what may be some of the contributing factors to genotypic effects, data generated from an experiment in 2007 conducted by Anurag Agrawal and Marc Johnson at a nearby field site was used. This data set denotes the mean values of several plant traits for 39 primrose genotypes. Total phenolics, Oenothein B phenolic, water content and nitrogen content were chosen in order to be fitted to the abundance data collected in this study. Abundance data was also fitted to trichome densitites which were measured in this experiment
Table 1 : Top 10 aprthropods found on O.biennis , with their degree of host-specificity Host Species Order Family Feeding type specificity Popillia japonica Coleoptera Scarabaeidae Leaf chewer generalist
Acanthoscelidius acephalus Coleoptera Curculionidae Leaf chewer specialist
Lygus lineolaris Hemiptera Miridiae Leaf chewer generalist
Unknown mirid Hemiptera Miridiae Leaf chewer generalist
Systena frontalis Coleoptera Chrysomelidae Leaf chewer generalist
Schinia florida Lepidoptera Noctuidae Bud chewer specialist
Mompha stellella Lepidoptera Momphidae Flower borer specialist
Philaenus spumarius Hemiptera Aphrophoridae Phloem feeder generalist
Cochylis oenotherana Lepidoptera Tortricidae Stem apex feeder specialist
Coccinellidae :
Coleomegilla maculata Coleoptera Coccinellidae Predator and generalist pollen feeder Harmonia axyridis Coleoptera Coccinellidae predator generalist
Coccinella Coleoptera Coccinellidae predator generalist septempunctata
7 Statistics
Non bolted plants (56) were excluded from analysis since they generally did not attract any insects. Plants likely did not bolt largely in part due to the high soil moisture in one portion of the field plot, as O.biennis is a plant which prefers dry and well draining soils. Thus, in the analysis the group sizes per genotype were the following:
Genotype 21 33 44 6 20 5 count 35 37 35 32 19 23
Statistical analysis was done using JMP 8.0 (SAS Institut Inc). Repeated measurements MANOVA were used to determine whether genotype, the initial treatment, the spontaneous mildew infection and plant morphology (plant height and the number of stems per plant) had effects on the different arthropod species. When means proved to be significantly different a Tukey-Kramer post hoc test was used, which is adapted to unequal sample sizes and thus gives less conservative results than a standard Tukey HSD would. Post hoc tests were run on the maximal abundance of each species per plant, as it is not possible to do so in a repeated measures context. Repeated measures could not be used to analyze the abundance of Cochylis oenotherana , as the presence of this herbivore is noted only by the damage to the plant (stem tips are damaged causing a characteristic rosette of offshoots) and so was counted only once at the end of the experiment. The abundance of this insect was thus measured by using a Poissons distribution with a log link function in a Generalized Linear Model.
The Shannon-Weiner diversity index, arthropod abundance and species richness were calculated by using the maximal abundance of each species, as the number of individuals in each survey was too low to be able to calculate a meaningful diversity index. These were then analyzed using a Kruskal-Wallis Test, as the data could not be suitably transformed as to meet the assumptions of normality. However JMP does not contain non parametric post hoc tests, so it was not possible to accurately know which groups differed significantly. Thus box plots were used to get some visual information as to which groups were likely to be significantly different. Linear regression was used to fit the trait data to the abundance data. The coefficient of determination (r²) was calculated to give an indication of how well the line fit the data points.
8 Results
Genetic variation among the six genotypes of O. biennis affected arthropod abundance, which significantly varied among the plant genotypes for 6 of the 10 common species surveyed (Table 2). Variation in abundance according to genotype has already been shown in a past study for M.stellella , S.florida , and L.lineolaris (Johnson and Agrawal 2007). The initial treatments imposed on the plants had no affect on arthropod abundance, with the exception of A. acephalus , which had significantly less individuals on the control plants (P= 0.036) and had a tendency to prefer plants damaged by conspecifics (Table 2). No other treatment effects could be found. The non experimental infection of the plants by mildew in the field also had a significant effect on the abundance of 4 of the 10 insect species, with abundance decreasing (with the exception of P.japonica ) as the severity of the mildew infection increased.
Table 2: F - test for repeated measures analysis of variance for the effect of genotype, initial treatment and percentage of each plant affected by mildew on the abundance of herbivores. χ²- statistic from a Generalized Linear Model with Poisson distribution for the main effects on C. oenotherana , with the interaction genotype x mildew not being calculated due to lost degrees of freedom (*).
Interaction Interaction Interaction Genotype(1) Treatment(2) Mildew(3) (1 x 2) (1 x 3) (2 x 3) df= 5 df= 4 df= 1 df= 20 df= 4 df= 4 F/ χ² P F/ χ² P F/ χ² P F P F P F P Popillia japonica 6.98 <0.001 0.06 0.99 5.11 0.025 0.61 0,90 0,95 0,43 0,76 0,55
Acanthoscelidius 3.77 0.003 2.63 0.036 1.92 0.16 1.08 0,37 2,97 0,02 2,10 0,08 acephalus
Lygus lineolaris 7.53 <0.001 0.60 0.66 10.96 0.001 1.14 0.31 1 0.40 0,26 0,90
Unknown mirid 1.68 0.14 0.18 0.95 0.59 0.44 0.97 0,5 0,73 0,56 1,47 0,21
Systena frontalis 4.87 <0.001 0.24 0.91 11.11 <0.001 0.68 0,83 0,98 0,41 1,12 0,34
Schinia florida 29.02 <0.001 0.14 0.96 1.69 0.19 0.81 0,68 0,87 0,48 0,34 0,85
Mompha stellella 1.79 0.11 2.36 0.06 1.57 0.21 1.77 0,02 0,03 0,99 0,84 0,49
Philaenus 0.81 0.54 1.23 0.29 0.004 0.94 0.71 0,81 0,65 0,62 0,66 0,61 spumarius
Cochylis 150.8 <0.001 0.72 0.12 10.39 0.001 24.7 0.21 7.18 0.12 * * oenotherana
Coccinellidae 1.97 0.08 0.66 0.61 1.76 0.18 0.94 0,53 2,96 0,02 1,04 0,38
Values in boldface type represent significant effects at P < 0,05
The effects of differences in plant morphology were quantified by the height of each plant as well as the number of stems, which gives some indication of the total biomass of the plant. Plant morphology had a strong influence on the plant preference of many of the surveyed insects (Table 3). The abundance of P.japonica and L.lineolaris was significantly higher on the taller plants (P ≤0.001 for both) as well as the plants that were bushier (P ≤0.001 for both). Other insects’ abundance were either only affected by height (unknown mirid P= <0.001) or by the number of stems per plant (Coccinellidae P =0.04), and here also the species were more abundant on the taller or bushier plants.
11
Table 3: F - test for repeated measures analysis of variance for the effect of plant height, the number of stems per plant and their interactions with genotype (see table 3) χ²- statistic from a Generalized Linear Model with Poisson distribution for effects on C. oenotherana .
Number of Interaction Interaction Plant height (1) stems (2) (1 x genotype) (2 x genotype) F/ χ² P F/ χ² P F/ χ² P F/ χ² P Popillia japonica 10.04 0.001 13.37 <0.001 1.034 0.40 0.58 0.71
Acanthoscelidius acephalus 0.80 0.37 0.69 0.40 4.56 <0.001 2.13 0.06
Lygus lineolaris 23.98 <0.001 50.69 <0.001 3.43 0.005 1.50 0.19
Unknown mirid 13.73 <0.001 0.70 0.40 1.64 0.15 0.56 0.73
Systena frontalis 0.23 0.63 5.39 0.02 0.62 0.67 1.09 0.36
Schinia florida 0.03 0.86 0.94 0.33 2.02 0.08 3.21 0.008
Mompha stellella 0.01 0.96 1.23 0.26 0.01 0.98 1.06 0.38
Philaenus spumarius 0.02 0.86 0.08 0.92 1.05 0.41 0.12 0.98
Cochylis oenotherana 0.04 0.83 0.62 0.43 4.72 0.44 6.99 0.22
Coccinellidae 3.84 0.06 4.27 0.04 1.89 0.09 0.85 0.51
Values in boldface type represent significant effects at P < 0,05
Genotypes of O .biennis differed substantially in the Shannon-Weiner index (H’), total arthropod abundance and in species richness (Table 4). In all cases, treatments had no effect. Genotypes 5 and 20 showed significantly higher diversity and species richness than genotype 21, while in term of insect abundance only 5 and 21 significantly differed from each other. Genotype 21 consistently had the lowest values.
Table 4: Genotype and treatment effects for the Shannon-Weiner diversity index, mean arthropod abundance, species richness and species evenness. For each, a χ² statistic from a Kruskal-Wallis test was calculated. Shannon-Weiner arthropod index (H’) abundance Species richness Species evenness df χ² P χ² P χ² P χ² P
Genotype 5 20.12 0.001 29.14 <0.001 27.17 <0.001 7.88 0.16
Treatment 4 3.03 0.55 1.83 0.77 4.74 0.31 3.47 0.48
Values in boldface type represent significant effects at P < 0,05
When herbivores were classified as either being specialists or generalists of O.biennis , strong differences appear in their distribution over the six genotypes (Figure 3). The three genotypes (21, 33 and 44) that were chosen due to their high P.japonica herbivory in previous experiments (Anurag Agrawal and Scott McArt, unpublished data) harbored 3.1 to 4.9 times more generalists than specialists, with the generalists preferring genotypes 33 and 44. In contrast, specialists showed a strong preference for genotypes 20 and 5, and to a lesser extent genotype 6.
12
A A A)P= 0.002 B) P<0.001
66 A A 6
B B B B B 44 4
C 22 2 C C
abundance per plant mean maximum abundance mean maximum abundance 00 0 211 33 2 44 3 6 4 20 5 5 6 211 33 2 44 3 6 4 20 5 5 6
Genotype Figure 3 : mean abundance (±SE) per genotype of generalists (A) and specialist (B) herbivores. Mean abundance was calculated from the maximal abundance of each species over all three surveys.
In order to help determine what may be the driving forces behind the genotype dependent differences in arthropod abundance, the influence of several plant traits was looked at. When the mean abundance per genotype of generalists (excluding the Coccinellidae as they are predators) and specialists are fitted against the mean values per genotype of five different plant traits, several correlations emerged (Figure 4), more specifically with the specialists. While the total phenolics had little effect on the abundance of specialist herbivores (p = 0.131) one specific phenolic, oenothein B, explained 87% (p = 0.006) of the total variation in abundance among the genotypes, with the specialists being more abundant on genotypes having high levels of the phenolic. Similarly strong correlations could be seen with water content (r² =0.89, p= 0.004) and the C:N ratio (r² = 0.93, p= 0.002) with specialists being more abundance on genotypes having higher water content or a lower C:N ratio. The correlation with leaf trichome density was somewhat weaker than for the other traits although the coefficient of determination is still high (r² =0.73, p= 0.03).
When the abundance of the generalist herbivores is fitted against the same traits, no statistically significant effects could be seen, with the different traits explaining 7 to 50% of the variation in abundance. It is interesting to note however that the generalists show a trend towards a correlation which is the opposite of that observed for the specialists. While specialist abundance shows positive (or negative) correlations with Oenothein B and water content, the trend with the generalists is a negative (or positive) correlation.
13
300 300 300 A
200200 200
100100 100 phenolics phenolics Phenolics r² = 0.075 P = 0.599 r² = 0.47 P = 0.131 (mg/g dry weight) 0 0 0 2 3 4 5 6 0 2 4 6 8 R2 = 0,075 B generalistsr² = 0.5 P = 0.1142 specialists 7575 R = 0,472875
5050 50
2525 25 Oenotherin B B Oenothein B r² = 0.87 P = 0.006 (mg/g dry weight) 00 0 2 3 4 5 6 0 2 4 6 8 8080 2 80 C generalistsr² = 0.44 P = 0.154R = 0,8678 specialists R2 = 0,504
75 75 75
%water 70 %water water 70 70 Water content dry(% weight) r² = 0.89 P = 0.004 6565 65
2 3 4 5 6 0 2 4 6 8
30 generalists 30 specialists 2 30 2 R = 0,435 D R = 0,8949
2020 20
ratio C:N 1010 10
r² =0.34 P = 0.228 r² = 0.93 P = 0.002 00 0 22 3 4 4 5 6 6 00 2 2 4 4 6 6 8 8 150015 15 R2 = 0,3351 generalists specialists E R2 = 0,9286 Abundance generalists Abundance specialists 100010 10
500 5 5 (per (per cm²)
leaf trichomes leaf trichomes r² = 0.5P = 0.114 r² = 0.73 P = 0.030 0 0 0 Leaf trichome density 22 3 3 4 4 5 6 6 00 2 4 66 8 R2 = 0,5035 generalists specialists Abundance of generalistsR2 = 0,7306 Abundance of specialists
Figure 4: Correlations between the abundance of generalist and specialist herbivores, and the total phenolics concentration (A), concentration of the phenolic Oenotherin B (B), water content (C), the C:N ratio (D) and the number of leaf trichomes per cm² (E). Trait data A through D are from means fro m data collected in 2007, while E and herbivore abundance are from the same experiment in 2010.
14 Discussion:
Individual and community -level effects of genetic variation
This study corroborates previous findings on O.biennis (Johnson and Agrawal 2005) and in other systems (Fritz and Price 1988, Fritz 1990, Maddox and Root 1990) which showed that phenotypic variation between genotypes of a same plant species affect the structure and diversity of its associated arthropod community. Indeed, on an individual species level, 6 of the 10 insect species surveyed in this experiment significantly varied in abundance over the six genotypes. When looking at the effects of genotype at the community level, the overall abundance, species richness and value of the Shannon-Weiner index also significantly varied between genotypes. While non parametric post hoc tests were not available, box plots showed that genotype 20 had the highest values for all three community measurements, while genotype 21 had the lowest.
More interestingly, when the species were divided according to their host specificity (excluding the Coccinellidae as they are predators) strong differences in genotype preference appeared. While generalists were most abundant on genotypes 33 and 44 and did not differentiate between the remaining genotypes, specialists on the other hand preferred genotypes 20 and 5 and to a lesser extend genotype 6, while strongly avoiding those genotypes that harbored more generalists. This may in part explain the differences in diversity index and species richness, as genotypes 20 and 5 harbor both specialists and generalists.
It is interesting to probe further into the possible mechanisms of the genotype based differences in the abundance between generalists and specialists. Using plant trait means from a previous unpublished experiment, insect abundance was fitted against some of these plant traits and several interesting correlations arose. Generalist herbivores were not significantly affected by any of the traits, which may be due to the fact –unlike for the specialists- that the magnitude of the effects varied from species to species, and may have cancelled each other out when combined. However, in the case of the specialists several interesting correlations can be noted.
While the total phenolics content of each genotype does not affect the abundance of herbivores, one specific phenolic, Oenothein B, appears to strongly influence the abundance of specialists. Genotypes 5, 6 and 20 have 40 to 70 times more Oenothein B than genotypes 21, 33 and 44. However the generalists are not deterred by the higher concentrations, as has been shown in other studies (van Dam et al. 1995, Lankau 2007), indicating that Oenothein B may serve more as host finding or oviposition cue for specialists. The fact that specialists avoid the genotypes that happen to have lower concentrations of the compound brings further weight to this hypothesis.
Nitrogen and water content both explain a large part of the variation in abundance of the specialists, which is consistent with expectations. Water content and nitrogen play an important role in herbivory, as their combination will strongly determine the palatability and suitability of the plant for the herbivore, especially during development. Nitrogen is an important nutrient for herbivores and often one of the limiting factors for their development, while water content will affect the digestibility of the plant tissue (Scriber 1977, Scriber and Slansky 1981). The fact that generalists show a tendency to prefer plants with lower amounts
15 of each is unexpected, as nitrogen is equally important for all herbivorous insects, irrespective of host specificity. Interestingly, the three genotypes that have the highest levels of Oenothein B also appear to have the highest water content and amount of nitrogen. It may be that the phenolic has the strongest influence on the generalists, and thus neutralizes the effects of water content and nitrogen.
Trichomes also appear to have an effect on the plant preference of the specialists, and they have long been considered as an important plant defense against herbivorous insects (Valverde et al. 2001, Dalin and Bjorkman 2003, Hanley et al. 2007), although the effects can vary among herbivore species (Andres and Connor 2003, Kaplan et Al. 2009) which may in part explain the differences between specialists and generalists. Alternatively, while specialists may be adapted to the secondary compounds in O.biennis , they may also be more sensitive to the physical barriers against herbivory than the generalists are. This may explain why specialists prefer plants having low pubescence, while the generalists’ abundance tends to correlated with higher trichome densities.
However such correlations must be interpreted with caution for several reasons. The first is that data from two separate years and locations is being compared, with the trait data being from 2007 (except for trichome data, which is from 2010) and the abundance data from 2010. Expression of plant traits as well as arthropod abundance and diversity can vary in space (Johnson and Agrawal 2005, 2007, Tack et al. 2010) and in time (Wimp et al. 2005, Johnson and Agrawal 2007); this is the effect of the environment. Johnson and Agrawal (2007) showed that primrose genotype and habitat interacted to affect arthropod abundance, their diversity and community composition. Certain genotypes could harbor a high diversity and abundance of arthropods while in one habitat, but display low values while in another habitat. Furthermore, when variation in habitat was on a large scale (Johnson and Agrawal 2005), the effects of the environment took precedence over those of genotype, and explained a large part of the variation in arthropod community measurements. Thus if there is any genotype x environment effects in the 2010 abundance data, the correlations between traits and herbivore abundance could fall apart. However the fact that the genotypes preferred by Japanese beetles in 2007 are also the genotypes that are preferred in 2010 (as well as in a 2009 experiment by Scott McArt) gives some support to the assumption that the genotypes show phenotypic consistency between years.
Genetic variation in other traits also had an effect, but on an individual species level. Differences in plant height and architecture influenced the preference of three of the herbivores. In the case of Japanese beetles, while host plant preference can depend largely on plant volatiles and aggregation pheromones (Loughrin et al 1995), plant height and the presence of flowers are also used as a visual clue for host plant selection (Held and Potter 2004). Thus the taller or bushier plants may in part be selected by arthropods.
Treatment effects
Although induced responses have a strong impact insect communities, limited evidence of this could be seen from my data, with the exception of the weevil A.acephalus . The weevil colonized all the damage treatments equally, and avoided the control plants. A.acephalus doesn’t appear to differentiate between herbivore damaged plants and artificially damaged plants (JA and mechanical damage) treatments, although it showed a tendency to
16 prefer plants damaged by conspecifics. Prior studies looking at the effects of early season herbivory showed early season herbivory had the potential to affect the diversity of herbivore communities.
However, the lack of response to the treatments may in part be explained by the presence of powdery mildew on a majority of the plants. The herbivores that had their abundance affected by the presence of mildew, generally avoided the more infected plants. The herbivory by leaf chewers and the JA spray treatments would be expected to lead to a wound induced response in the plant, which is generally thought to be regulated by Jasmonic Acid (JA). The presence of a fungus on the other hand will primarily induce the Salicylic Acid (SA) dependent defenses. The SA and JA pathways play a key role in mediating plant defenses, though mounting evidence indicates that the induction of the SA pathway strongly interferes with the JA dependent responses and vice versa (Thaler et al. 2002; Beckers & Spoel 2006; Pieterse et al. 2009). Therefore cross-talk between the signaling pathways could imply a conflict in plant defenses as the presence of a SA inducing mildew infection could mitigate the induced responses of the plant in reaction to the JA inducing treatments. Due to time constraints it was not possible to analyze the leaf samples from the experiment, thus it is not known if there were any differences in the phytohormone levels between treatments on the six genotypes.
Future research:
Fitting insect abundance to plant trait data opened several interesting perspectives for further research that may confirm the relation between abundance and several plant traits. First of all, the correlations observed with the 2007 trait data could be confirmed by measuring the traits in plants of this experiment. Plant tissue had been collected immediately after the treatment induction period and frozen for future use. The phenolic oenothein B apparently plays a role in structuring the herbivory community, and it would be interesting to know how it affects the insects. While specialists prefer plants with higher levels and generalists show a strong tendency towards preferring the opposite, bioassays could help determine if performance is affected by this phenolic.
17
Conclusions
This study established that intraspecific variation among evening primrose genotypes results in phenotypic differences that structure the composition of the associated herbivore community. Not only did genotype lead to differences in the diversity index, species richness, and overall abundance, it also has a strong influence on the host plant preference generalists and specialists, which each group preferring different genotypes. Several plant traits have been shown to have strong correlations with the abundance of the specialists across the 6 genotypes, as they show a strong preference for plants having high levels of the phenolic compound Oenothein B, water and nitrogen. While the original intent of this study was to investigate the effects of early season herbivory on the insect community, only limited evidence was found with only one herbivore being affected by induced defenses. As plants were affected by mildew, the lack of early season herbivory effects can not be affirmed with certainty.
18 Acknowledgements
I would like to thank Jennifer Thaler and Scott McArt as well as the rest of the Thaler Lab for welcoming me into their group at Cornell University for over 4 months. Working with primrose gave me my first real taste of field work, and it was a thoroughly enjoyable way to spend the summer despite the blistering heat and humidity in July and the few weeks spent hobbling around on crutches. Before starting this project I knew nothing about primrose other than its status as a weed, and didn’t even know what it looked like. Scott quickly fixed that, although I was a bit overwhelmed by how much there was to know about a common weed!
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