Ecology Letters, (2014) 17: 229–238
LETTER
Aino Kalske,1* Pia Mutikainen,2 Anne Muola,1 J. F. Scheepens,1 Liisa Laukkanen,1 Juha-Pekka Salminen3 and Roosa Leimu4
doi: 10.1111/ele.12223
Simultaneous inbreeding modifies inbreeding depression in a plant–herbivore interaction Abstract Because inbreeding is common in natural populations of plants and their herbivores, herbivoreinduced selection on plants, and vice versa, may be significantly modified by inbreeding and inbreeding depression. In a feeding assay with inbred and outbred lines of both the perennial herb, Vincetoxicum hirundinaria, and its specialist herbivore, Abrostola asclepiadis, we discovered that plant inbreeding increased inbreeding depression in herbivore performance in some populations. The effect of inbreeding on plant resistance varied among plant and herbivore populations. The among-population variation is likely to be driven by variation in plant secondary compounds across populations. In addition, inbreeding depression in plant resistance was substantial when herbivores were outbred, but diminished when herbivores were inbred. These findings demonstrate that in plant–herbivore interactions expression of inbreeding depression can depend on the level of inbreeding of the interacting species. Furthermore, our results suggest that when herbivores are inbred, herbivore-induced selection against self-fertilisation in plants may diminish. Keywords Abrostola asclepiadis, experimental inbreeding, insect herbivory, performance, phenolic compounds, plant mating system, resistance, secondary chemistry, self-fertilisation, Vincetoxicum hirundinaria. Ecology Letters (2014) 17: 229–238
Inbreeding is increasingly common in natural populations of plants and their insect herbivores, because of the widespread destruction of natural habitats that has led to population declines (e.g. Saccheri et al. 1998; Keller & Waller 2002; Foley et al. 2005). Negative effects of inbreeding arise mainly through expression of deleterious recessive alleles in the inbred individuals (Charlesworth & Charlesworth 1999). This inbreeding depression has been observed in several plant and insect species as increased mortality and decreased reproductive success, fecundity, germination success, resistance and egg hatching rate (Husband & Schemske 1996; Saccheri et al. 1996; Carr & Eubanks 2002). Furthermore, inbreeding and consequent inbreeding depression may significantly modify herbivore-induced selection on plants, and vice versa, across a wide range of species (Ashman 2002; Carr & Eubanks 2002; Ivey et al. 2004). Plant populations may differ in their level of inbreeding because of variation in pollinator behaviour, population size, self-fertilisation rate and connectivity (Ellstrand & Elam 1993; Aguilar et al. 2008; Whelan et al. 2009). In species interactions, especially when the relationship is specialised, patterns of population genetic structure of one species are likely to be reflected also in the associated species, and consequently, inbreeding has been observed to occur simultaneously in coexisting populations of plants and their herbivores (Magalhaes
et al. 2011). However, because herbivore populations are prone to large fluctuations in size, they may go through population bottlenecks followed by increased inbreeding independently of host plant population size (Hanski et al. 1995). Alternatively, if herbivore populations are more connected than the associated plant populations, because herbivores are in general more mobile, they would be less likely to suffer from inbreeding than plants (Michalakis et al. 1993; Salle et al. 2007). Therefore, the level of inbreeding and changes in it in one of the interacting species does not necessarily correlate with the level of inbreeding in the other species at a particular site. This can lead to a situation where inbreeding occurs either only in the plant or the herbivore, or to simultaneous inbreeding of both counterparts, scenarios that can all alter the dynamics of the coevolutionary interaction. It is known that plant inbreeding reduces herbivore resistance (Carr & Eubanks 2002; Hayes et al. 2004), but the influence of herbivore inbreeding and possible simultaneous inbreeding of plants and herbivores on plant resistance and the ability of herbivores to exploit the plant have received much less attention (but see Strauss & Karban 1994). As inbreeding frequently occurs in both plant and herbivore populations, it is important to explore potential interactions between inbreeding in plants and inbreeding in their herbivores in more detail (I 9 I; Leimu et al. 2012b). Next, we will introduce four potential outcomes of this interaction (Fig. 1). First, inbreeding of plants can reduce the expression of
1
4
INTRODUCTION
2 3
Section of Ecology, University of Turku, Turku, Finland € rich, Zu € rich, Switzerland Institute of Integrative Biology, ETH-Zu
Department of Plant Sciences, University of Oxford, Oxford, UK
*Correspondence: E-mail: [email protected]
Laboratory of Organic Chemistry and Chemical Biology, Department of
Chemistry, University of Turku, Turku, Finland
© 2013 John Wiley & Sons Ltd/CNRS
230 Aino Kalske et al.
Letter
inbreeding depression in herbivores, if the inbred host plant causes lower stress in the herbivore due to reduced resistance (Dudash 1990; Fig. 1a). Inbred plants are known to have lowered resistance that has been explained by altered plant chemistry or altered emission of induced plant volatiles (Delphia et al. 2009; Kariyat et al. 2012; Campbell et al. 2013). Second, inbreeding depression in herbivores may also be more pronounced on an inbred vs. outbred host plant, if the outbred herbivores benefit more from the increased quality of the inbred host plant than the inbred herbivores (Fig. 1b). An outbred herbivore may benefit from the increased quality of the host plant mediated by altered nutritional quality, defensive chemistry or stress-related enzymes (Ridley et al. 2011; Leimu et al. 2012a; Campbell et al. 2013), whereas an inbred herbivore may be restricted by its overall lower viability (Greenberg & Crow 1960; Fern andez et al. 1995). Third, the I 9 I interaction may also apply to the host plant: inbreeding depression in plant resistance may be reduced under an attack by inbred herbivores, if the adverse effects of inbreeding of
(b)
(a)
Herbivore fitness
Outbred herbivore Inbred herbivore
Outbred
Inbred
Outbred
Plant (d)
Less resistant Plant damage
(c)
Resistant
Inbred
Plant
Outbred plant Inbred plant
Outbred
Inbred
Herbivore
Outbred
Inbred
Herbivore
Figure 1 Theoretical representation of four potential outcomes of the interactive effects of host plant and herbivore inbreeding (I 9 I) on herbivore performance (a & b) and on plant resistance measured as damage (c & d). Inbreeding depression refers to the difference between the fitness/resistance of the outbred and inbred individuals, when the outbred individuals are outperforming the inbred individuals. Dashed lines in panel a indicate the level of inbreeding depression for the respective circumstances. Note that damage represents an inverse measure of resistance, and thus lower damage refers to higher resistance. (a) Inbreeding depression in the herbivore decreases when host plant is inbred. (b) Inbreeding depression in the herbivore increases when the host is inbred. (c) Inbreeding depression in the host plant decreases when the herbivore is inbred. (d) Inbreeding depression in the host plant increases when the herbivore is inbred.
© 2013 John Wiley & Sons Ltd/CNRS
the herbivore attenuate its ability to overcome plant defences of the outbred plant (Fig. 1c). Finally, inbreeding depression in plant resistance may also be stronger when the herbivore is inbred if the outbred plant is more effectively protected against inbred herbivores than the inbred plant (Fig. 1d). The interactive effects of host-plant and herbivore inbreeding on plant resistance and herbivore performance are also likely to vary among populations with different inbreeding loads (I 9 I 9 P), e.g. due to variation in historical population size and population history of inbreeding (Leimu et al. 2008; Angeloni et al. 2011). We examined the effects of experimental inbreeding both of a long-lived plant, Vincetoxicum hirundinaria Med. (=Cynanchum vincetoxicum (L.) Pers.) (Apocynaceae), and its specialist herbivore, the moth Abrostola asclepiadis Schiff. (Noctuidae), on plant resistance and herbivore performance respectively. We specifically studied whether inbreeding in one of the interacting species affects the expression of inbreeding depression in the other species (I 9 I) and the variation among populations in this effect (I 9 I 9 P). We also studied whether inbreeding alters plant secondary chemistry (phenolic compounds) as well as among-population variation in these compounds. Finally, we aim to explain the variation in the effects of simultaneous inbreeding on herbivore performance and plant resistance with the variation in plant secondary chemistry. In our study area, V. hirundinaria has a mixed mating system, and populations harbour variable frequencies of individuals that are capable of self-fertilisation (Leimu 2004). Populations occur on different islands and are thereby separated by water, which forms a relatively strong barrier to gene flow between the different populations. The level of inbreeding in the Finnish V. hirundinaria populations varies from 0.025 to 0.597 (FIS; Leimu & Mutikainen 2005). Vincetoxicum hirundinaria also suffers from inbreeding depression in resistance to A. asclepiadis (Muola et al. 2011). Population sizes of the plants are relatively constant, whereas those of the herbivores fluctuate (AK, unpublished data). The damage caused by the larvae has significant negative effects on plant reproduction (Muola et al. 2010a) and population growth (Leimu & Lehtil€ a 2006). Vincetoxicum hirundinaria contains many phenolic and other secondary compounds that are presumed to act as chemical defence against herbivores (Muola et al. 2010b). Our results demonstrate that herbivore-induced selection on plants, and vice versa, may be significantly modified by simultaneous inbreeding. Such modifications are likely to be driven by altered plant secondary chemistry that acts as anti-herbivore defence. The effects of simultaneous inbreeding may change our view of the effects of inbreeding on the evolution of plant–herbivore interactions and plant mating systems.
MATERIALS AND METHODS
Study system
In Sweden and Finland, V. hirundinaria occurs on the coastline and islands of the Baltic Sea. For this study we used plants and herbivores from one Swedish (M€ ork€ o) and three Finnish sites (Lammasluoto, Naantali, Seili) harbouring populations
Letter
of both V. hirundinaria and A. asclepiadis. Self- and withinpopulation cross-pollinated plants from the four populations were obtained from 20 maternal plants per population by hand pollination. Inbred and outbred herbivores were derived from a laboratory population of A. asclepiadis that was established in June 2010. Further details on study system, the study populations, and the crossing protocols are provided in Appendix S1 in Supporting Information. Experimental set-up
F1 generation inbred and outbred plants included in the study were potted in 0.9 L pots. When possible, we used a minimum of eight inbred and eight outbred plants from each maternal plant. However, because of problems with pollination and germination, the number of seedlings per maternal plant ranged from 2 up to 61, as the lack of seedlings from some maternal plants was compensated for by using more seedlings from a maternal plant with extra seedlings to obtain similar numbers of seedlings at the population level. On average we had 11 and 10 seedlings from each maternal plant in the inbred and outbred cross treatments respectively. For the Seili population, one-third of the inbred plants used in this experiment were derived from a single maternal plant. This lack of offspring produced by self-fertilisation from some maternal plants may have been due to the variable levels of individuals capable of self-fertilisation among the maternal plants (Leimu 2004). Plants were randomly assigned to 15 blocks with 64 individuals (eight plants from each plant population and plant cross combination) in each block, which makes altogether 960 plants. The experiment was conducted in a greenhouse in the Ruissalo Botanical Garden (University of Turku) in the spring 2012. Temperature and light conditions in the greenhouse were adjusted to resemble summer conditions in the study area: temperature regime of 21/17 °C day/night and an 18 h photoperiod (4.30–22.30 hours). F1 generation pupae of A. asclepiadis were taken to room temperature to hatch. As the F1 adults emerged they were paired to obtain inbred (brother–sister mating) and outbred (between families, within-population mating) F2 families. The pairing was done in the same manner as in the P generation (see Appendix S1). Pairs from F1 generation were allowed to mate until the female had laid a sufficient number of eggs (minimum of 20 eggs), or until the female or the male died. Leaves with eggs on them were removed from the plants 2–5 days after the eggs had been laid and they were placed in plastic containers, the eggs from each family in a separate container. Eggs were monitored daily and once the larvae hatched they were assigned to experimental plants, one larva per plant. Each larva was carefully placed on a leaf of the assigned plant that was covered with a mesh bag. One to three larvae from each family were haphazardly assigned to each of the eight plant treatments (inbred and outbred plants from all four populations, 8–24 larvae from one family), and altogether 12–16 replicates per herbivore origin and cross were used in the experiment. From one outbred herbivore family from Seili up to six replicates per plant treatment were used because of several unsuccessful matings. In the end, we had altogether 927 larvae in the experiment, and thus 33 of the
Inbreeding in plants and herbivores 231
960 plants were left without a larva and left out of the experiment. After 9 days in the experiment, the larvae were weighed to get an estimate of their performance. Larval biomass represents larval performance and is correlated with pupal mass (AK, unpublished data, r = 0.127, P = 0.001, n = 640), which is known to reflect the fitness of adult Lepidoptera (Haukioja & Neuvonen 1985). Larval biomass also correlates negatively with the length of the larval period, i.e. the smaller the larvae are on the eighth day after hatching, the more time it takes for them to complete development (AK, unpublished data, r = 0.524, P < 0.001, n = 662). The damage sustained by the plants was estimated as the proportion of damaged leaves: the number of leaves with any damage divided by total number of leaves in the plant. This is an appropriate measure of damage for this system and species, because plants in the experiment tended to prematurely lose the partly eaten leaves to early senescence. The proportion of damaged leaves therefore accounts for this damage-induced loss of leaves more accurately than, for instance, the percentage of leaf area eaten. To obtain an additional measure of plant resistance, we collected leaf samples from a subset of the plants (463 individuals) for secondary chemistry analysis. The sampling was done by collecting 2–4 leaves from each plant on one of two different days that were ca. 2 weeks apart. Phenolic compounds from each sample were analysed using ultra-performance liquid chromatography with an electrospray triple quadrupole mass spectrometry detector (UPLC-MS/MS, Waters Acquity Xevo, Milford, MA, USA) (see Appendix S1). Data analysis
The data on larval biomass (herbivore performance) were analysed using a mixed model with larval biomass as response variable and herbivore cross (inbred or outbred) and population, plant cross and population of the plant the larvae consumed, and block as fixed factors. Block was included to eliminate the effects of environmental variation due to the different positions of the plants in the greenhouse. All the possible interactions between the fixed factors (except for block) were included in the model. Herbivore family was included in the model as random factor. Plant damage was estimated as the proportion of damaged leaves that inversely reflects plant resistance: the more leaves eaten the less resistant the plant is to herbivore damage. These data were analysed with a mixed model with the same fixed factors as in the previous model: plant cross and population, herbivore cross and population of the larva feeding on the plant, and block as fixed factors. Plant family, i.e. maternal plant, was included in the model as random factor. Plant cross was included in the random residual/group option which specifies a different residual variance for plant cross treatments to correct for heteroscedasticity of residuals. All analyses were conducted using the GLIMMIX procedure in SAS (SAS Enterprise Guide 4.3/SAS 9.2, Cary, NC, USA) and normal distribution with identity link function in both plant damage and herbivore performance data. A total of 26 phenolic compounds were quantified from the leaves out of which 23 were flavonol glycosides (quercetin and © 2013 John Wiley & Sons Ltd/CNRS
232 Aino Kalske et al.
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kaempferol glycosides) and three were caffeoyl quinic acids (chlorogenic acids). To investigate the effects of plant inbreeding on the concentrations of phenolic compounds and variation in this effect among plant populations, we conducted mixed models analysis using GLIMMIX procedure in SAS and normal distribution with identity link function or gamma distribution with log link function, depending on the residual distribution of the data. Data on eight compounds were square root transformed, and data on five compounds had a value 0.01 added in order to improve the fit of the data. We analysed all chemical compounds that were present in 50 or more plants (23 compounds; Table S2), and the total concentrations of quercetin, kaempferol and flavonol glycosides, caffeoyl quinic acids and phenolic compounds. Plant population, plant cross, plant population 9 plant cross and sampling date were treated as fixed factors, and maternal plant as a random factor. Initially, interactions with sampling date and other fixed factors were included in the model, but were removed because they were non-significant. Model assumptions of normality and homogeneity of variances were assessed by visual examination of diagnostic plots, and the significance of the random factors was tested with likelihood ratio tests in all analyses. To assess the defensive role of the secondary compounds, correlations with larval biomass and 11 chosen compounds and five compound groups were conducted using the biomass of the outbred larvae that had been reared on outbred plants. These compounds were chosen because their concentrations differed between inbred and outbred plants according to the results of the first analysis of the chemical data (Table S2),
and they could therefore account for the observed effects of plant inbreeding (see Results).
RESULTS
Herbivore performance
The biomass of the larvae ranged from 0.6 to 107.0 mg with the average of 15.8 0.3 mg (mean SE). We found significant inbreeding depression in herbivore performance (Table 1): inbred larvae had on average 13.6% lower biomass than the outbred larvae (inbred: 14.6 0.5 mg, outbred: 16.9 0.5 mg). Likewise, plant inbreeding had an effect on larval biomass: larvae feeding on inbred plants were 11.9% heavier than the larvae feeding on outbred plants (inbred plants: 16.8 0.5 mg, outbred plants: 14.8 0.4 mg). The larval biomass also varied among their population of origin and depending on the plant population they consumed, which is indicated by the statistically significant main effects of herbivore population and plant population respectively (Table 1). In addition, there was significant variation among herbivore families in herbivore performance (Table 1). Plant inbreeding had an effect on the expression of inbreeding depression in herbivore performance, and the effect varied among herbivore populations (herbivore population 9 herbivore cross 9 plant cross; Table 1, Fig. 2). In some herbivore populations, plant inbreeding led to an increase in herbivore performance: in M€ ork€ o and Seili the outbred herbivores were heavier on inbred than on outbred plants (Fig. 2b,d). However, plant inbreeding did not have a positive effect on herbivores
Table 1 Results (F-values) of analysis of variance testing for effects of inbreeding and population of origin of host plant (Vincetoxicum hirundinaria) and herbivore (Abrostola asclepiadis) on herbivore performance and plant resistance
Herbivore performance Fixed factors Herbivore population Plant population Herbivore cross Plant cross Herbivore population 9 Plant population Herbivore population 9 Herbivore cross Herbivore population 9 Plant cross Plant population 9 Herbivore cross Plant population 9 Plant cross Herbivore cross 9 Plant cross Herbivore population 9 Plant population 9 Herbivore cross Herbivore population 9 Plant population 9 Plant cross Herbivore population 9 Herbivore cross 9 Plant cross Plant population 9 Herbivore cross 9 Plant cross Herbivore population 9 Plant population 9 Herbivore cross 9 Plant cross Block
d.f.
Plant resistance d.f.
F
P
4.12 13.45 5.47 13.07 0.77 0.97 1.06 1.71 2.22 5.54 0.34
** *** * *** n.s. n.s. n.s. n.s. n.s. * n.s.
734.3 735.0 734.4 734.4
0.95 3.07 0.47 0.94
n.s. * n.s. n.s.
14, 801.8
1.32
n.s.
3, 75.0 3, 744.8 1, 74.9 1, 735.1 9, 744.8 3, 75.0 3, 735.0 3, 744.9 3, 734.4 1, 735.0 9, 745.1 9, 3, 3, 9,
Random factors
v2
P
Herbivore family Maternal plant
51.55
***
d.f. = numerator, denominator d.f. for F-values. *P < 0.05; **P < 0.01; ***P < 0.001. © 2013 John Wiley & Sons Ltd/CNRS
F
P
1.84 7.66 6.57 26.46 0.91 0.25 1.31 0.22 3.24 3.89 1.06
n.s. *** * *** n.s. n.s. n.s. n.s. * * n.s.
732.0 736.5 730.6 736.0
2.16 0.93 0.44 1.59
* n.s. n.s. n.s.
14, 746.0
1.00
n.s.
3, 752.8 3, 31.4 1, 756.9 1, 657.0 9, 752.9 3, 760.2 3, 732.1 3, 755.9 3, 623.3 1, 730.7 9, 759.3 9, 3, 3, 9,
v2
P
12.45
***
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Inbreeding in plants and herbivores 233
Larval biomass (mg)
(a) 22
22
20
20
18
18
16
16
14
14
12
12
(c)
Mörkö
*
(d) Naantali
Larval biomass (mg)
The effect of herbivore population on plant resistance varied from inbreeding benefit to inbreeding depression; inbreeding was beneficial for plants from M€ ork€ o when they were consumed by herbivores from Lammasluoto. In all other combinations of plant population and herbivore population, inbred plants were either equally or less resistant than the outbred plants (Fig. 3). In addition, plant inbreeding depression in resistance was statistically significant when plants were consumed by outbred herbivores, but not when they were consumed by inbred herbivores (herbivore cross 9 plant cross; Table 1, Fig. 4). This difference was accounted for by a change in damage in the inbred plants: damage in the inbred plants consumed by outbred herbivores was higher than the damage in the inbred plants consumed by inbred herbivores (closed squares, Fig. 4). The resistance of outbred plants did not differ in response to outbred vs. inbred herbivores (open squares, Fig. 4).
(b) Lammasluoto
Seili
22
22
20
20
18
18
16
16
14
14
12
12
**
Plant secondary chemistry Outbred
Inbred
Outbred
Plant
Inbred
Plant Outbred herbivore Inbred herbivore
Figure 2 Effect of inbreeding of host plant, Vincetoxicum hirundinaria, on performance of inbred and outbred herbivores, Abrostola asclepiadis (larval biomass at 9 days; mean SE). Different panels represent different herbivore populations. Inbred and outbred plants were obtained by selffertilisation and within-population outcrossing respectively. Inbred and outbred herbivores originated from brother–sister matings and withinpopulation crosses respectively. Statistical significance levels have been obtained from Tukey’s test. *P < 0.05; **P < 0.01.
from Naantali or Lammasluoto, as the performance of inbred and outbred herbivores on inbred and outbred plants did not differ statistically significantly (Fig. 2a,c). Plant resistance
The proportion of damaged leaves per plant was on average 0.191 0.004 with variation from 0.020 to 0.900. Inbred plants were less resistant, as their proportion of damaged leaves was on average 19.8% higher compared with outbred plants (inbred: 0.212 0.006, outbred: 0.170 0.004). In addition, outbred larvae caused on average 9.0% higher damage than did the inbred larvae, i.e. the plants were less resistant against the outbred than inbred herbivores (inbred herbivore: 0.182 0.005, outbred herbivore: 0.200 0.006). The proportion of damaged leaves also varied among plant populations but not among herbivore populations (Table 1). In addition, variation among maternal plants in proportion of damaged leaves was statistically significant (Table 1). Inbreeding depression in plant resistance differed among plant and herbivore populations (herbivore population 9 plant population 9 plant cross; Table 1, Fig. 3). Inbred plants were more damaged, i.e. less resistant against the herbivores, than outbred plants in three of the four populations (Lammasluoto, Naantali and Seili), whereas in M€ ork€ o inbred and outbred plants did not differ (dashed lines, Fig. 3).
We found differences in the plant secondary chemistry among populations and between inbred and outbred plants (Table S2, Fig. 5). Concentrations of seven compounds and four compound groups differed among populations (Table S2). Effects of inbreeding on the phenolic compounds were mostly negative; inbred plants had significantly lower concentrations of seven compounds than outbred plants, and in one other compound there was a similar trend (Table S2). Four compounds (quercetin glycoside 596 and 464B, kaempferol glycoside 786A and 610B) were affected differently by inbreeding in different populations (Table S2, Fig. 5a–d). In addition, the concentrations of all compounds varied among the maternal plants, and all but one compound differed between the sampling dates (Table S2). Correlations between larval biomass and the selected plant compounds (i.e. compounds affected by plant inbreeding) revealed that associations between herbivore performance and different phenolic compounds depend on the herbivore population, as the strength, occurrence and direction of the correlations differed among populations (Table S3). Associations between herbivore performance and the compounds were mostly negative: the higher the concentration of the compound in the leaf, the poorer the herbivore performance. The overrepresentation of plants from one maternal plant and of herbivores from one outcrossed family in the Seili population may bias our results, if inbreeding influences resistance in these families differently compared to others in this population. Outbred plants from Seili lacked three of the phenolic compounds present in the other populations (Fig. 5b,d), but this seems to be a common attribute of this particular population based on a previous study (AM, unpublished data). Furthermore, the mean level of damage of the overrepresented family (0.246 for the outbred and 0.161 for the inbred plants) falls well within the variation observed for the other families within this population that varied between 0.118 and 0.289 (min and max) for the outbred plants and between 0.133 and 0.174 for the inbred plants. The same is true for the herbivore performance (mean biomass of the overrepresented family 20.3 mg, min 11.4 mg, max 26.9 mg). © 2013 John Wiley & Sons Ltd/CNRS
234 Aino Kalske et al.
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(a)
(b)
Mörkö
Lammasluoto
Proportion of damaged leaves
Resistant
Less resistant
0.30
0.30
***
0.25
0.25
0.20
0.20
0.15
0.15
0.10
0.10
(c)
(d)
0.30
Naantali
0.30
Seili
0.25
0.25 0.20
0.20
***
0.15
0.15
0.10
0.10 Outbred
**
Inbred
Plant
Outbred
Inbred
Plant Herbivore population: Lammasluoto Naantali Mörkö Seili Population mean
Figure 3 Variation in inbreeding depression of Vincetoxicum hirundinaria in resistance to herbivores among the four studied plant populations (a–d) and among herbivore populations measured as proportion of damaged leaves per plant (mean SE). Note that lower damage reflects higher resistance. Asterisks indicate statistically significant differences (Tukey’s test) between inbred and outbred plants within each plant population (**P < 0.01; ***P < 0.001). Inbred and outbred plants were obtained by self-fertilisation and within-population outcrossing respectively.
Thus, our results are not biased due to the overrepresentation of these families. DISCUSSION
This study is among the first to provide evidence for interactive effects of both host-plant and herbivore inbreeding on herbivore performance and plant resistance. Our results suggest that the expression of inbreeding depression in plant–herbivore interactions depends on the level of inbreeding of the associated species for both counterparts of the interaction. We demonstrate that simultaneous inbreeding can modify herbivore-induced selection on plants, and vice versa. The observed variation in these effects across populations is likely to be driven by corresponding variation in the concentrations of plant secondary chemistry that are altered due to inbreeding. Outbred host plants with effective herbivore resistance represent a higher environmental stress to the herbivores, which is known to increase the expression of inbreeding depression (Dudash 1990; Armbruster & Reed 2005; Mena-Ali et al. 2008; © 2013 John Wiley & Sons Ltd/CNRS
Kariyat et al. 2011). Accordingly, we predicted stronger inbreeding depression in herbivore performance on outbred compared with inbred plants. However, in two of the populations (M€ ork€ o, Seili) plant inbreeding had contrasting effects on herbivore performance: inbreeding depression of the herbivore increased on inbred compared with outbred plants. This effect was in particular due to a significant increase in the performance of the outbred herbivores on inbred plants. Inbreeding of the host plant, therefore, increased its quality as food for the herbivore probably due to reduced levels and altered composition of defensive chemicals (this study; Campbell et al. 2013) or changes in foliar elemental composition (Ridley et al. 2011). The presumed low general viability of the inbred herbivores (Greenberg & Crow 1960; Fern andez et al. 1995) may restrict them from benefiting from the increased quality of inbred host plants, which would explain the stronger inbreeding depression of the herbivores on inbred compared with outbred plants. Strauss & Karban (1994) also discovered increased inbreeding depression on inbred host plants (Erigeron glaucus) in their parthenogenetically reproducing thrips (Apterothrips apteris).
Inbreeding in plants and herbivores 235
Resistant Less resistant Proportion of damaged leaves
Letter
Outbred plants Inbred plants
0.24 0.22 0.20
** ***
0.18 0.16
Outbred
Inbred
Herbivore Figure 4 Effect of inbreeding of herbivore Abrostola asclepiadis, on damage measured as proportion of damaged leaves of inbred and outbred host plants, Vincetoxicum hirundinaria (mean SE). Note that lower damage reflects higher resistance. Inbred and outbred plants were obtained by self-fertilisation and within-population outcrossing respectively. Inbred and outbred herbivores originate from brother–sister matings and within-population crosses respectively. Statistical significance levels have been obtained from Tukey’s test. **P < 0.01; ***P < 0.001.
However, the thrips performed worse on selfed compared to outcrossed plants, which was not the case in our study. Intriguingly, we found variation in the effect of plant inbreeding on herbivore inbreeding depression among herbivore populations (i.e. I 9 I 9 P interaction) as in only two of the studied herbivore populations inbreeding depression in herbivore performance was different on inbred and outbred plants. Because the studied plant populations vary in their leaf chemistry (see also Muola et al. 2010b), and the studied compounds are known to affect local adaptation of A. asclepiadis to their sympatric plant populations (Laukkanen et al. 2012), it is likely that the different herbivore populations have adapted to the specific plant defensive compounds of their sympatric host-plant population. Accordingly, we found variation among the herbivore populations in the association between herbivore performance and those phenolic compounds that were affected by plant inbreeding: there were zero to four negative correlations between herbivore performance and leaf compound concentration per herbivore population. These correlations were mainly with different compounds in different herbivore populations. Taken together, we believe that the differences in the overall phytochemical composition among the host-plant populations resulted in variable associations of herbivore performance with the phenolic compounds, which was reflected in the variation in the interactive effects of plant–herbivore inbreeding among herbivore populations. In other words, depending on whether plant inbreeding modified defensive compounds focal in the interaction in a particular population, plant inbreeding had or did not have an effect on herbivore inbreeding depression through plant defensive chemistry. Variation in phytochemical composition among plant populations thus contributed to the observed variation
in the effect of plant inbreeding on herbivore inbreeding depression among herbivore populations. The observed differences in inbreeding depression in plant resistance among plant and herbivore populations (I 9 P 9 P) were also likely to be derived from the variation in defensive compounds among the plant populations. Plants from the M€ ork€ o population showed no inbreeding depression in resistance, but had relatively high concentrations of defensive compounds some of which were altered by inbreeding. Although concentrations of some compounds were strongly reduced in inbred plants, this reduction was probably compensated by corresponding increases in the concentrations of other compounds, because inbreeding did not markedly reduce the total concentration of phenolic compounds. It is also possible that plant nutritional quality or other resistance mechanisms unaccounted for in this study may explain why the observed effect of inbreeding in defensive compounds did not correspond with leaf damage in M€ ork€ o. Overall, we did not find a single compound that would have explained all observed among-population variation in the effects of plant inbreeding on resistance or on herbivore inbreeding depression, suggesting that the phytochemical diversity and the relative concentrations of different compounds are more important to the interaction than any particular compound alone (Berenbaum et al. 1991). Finally, in plant resistance the significant plant population by herbivore population interaction is suggestive for local adaptation, which has also previously been observed in V. hirundinaria (Kawecki & Ebert 2004; Kalske et al. 2012), but it was not detected here when examining the differences in plant resistance between the sympatric and allopatric plant–herbivore combinations (data not shown). The observed among-population variation in the effects of inbreeding both in plant resistance and herbivore performance could potentially also be explained by variation in the history of inbreeding of the herbivore and plant populations (Ouborg & Van Treuren 1994; Carr & Eubanks 2002; Leimu et al. 2008). In general, there is a negative relationship between inbreeding depression and history of inbreeding (Husband & Schemske 1996; Saccheri et al. 1996). For example, small populations with a long history of inbreeding can be expected to suffer less from inbreeding depression, because the deleterious mutations causing inbreeding depression may have been purged by selection (Crnokrak & Barrett 2002; Angeloni et al. 2011). This explanation seems not to apply to the variation we observed among the plant populations, as the only population where we did not find inbreeding depression in plant resistance was the largest population M€ ork€ o. We have previous data on inbreeding from the three other study populations: the FIS values vary between 0.47 and 0.58, suggesting only minor variation in past inbreeding among the populations, despite the large variation in population size (100–5200 individuals; Appendix S1; Leimu & Mutikainen 2005). Here, the level of inbreeding depression was similar for these populations. Therefore, it seems that the history of inbreeding does not explain current level of inbreeding depression. We do not currently have data on the population sizes of the herbivores. If plant population size roughly predicts the size of the herbivore populations at the same site over years (Burdon 1993; Colling & Matthies 2004), the her© 2013 John Wiley & Sons Ltd/CNRS
236 Aino Kalske et al.
Letter
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 5 Concentrations of phenolic compounds (mg g 1 dry mass; mean SE) in the leaves of Vincetoxicum hirundinaria in different plant populations and in inbred and outbred plants. Panels represent concentrations of (a) quercetin glycoside 596, (b) quercetin glycoside 464B, (c) kaempferol glycoside 786A, (d) kaempferol diglycoside 610B, (e) total quercetin glycosides, (f) total flavonol glycosides (quercetin and kaempferol glycosides), (g) total caffeoyl quinic acids (chlorogenic acids) and (h) total phenolic compounds. Letters that differ between two populations denote statistically significant differences between plant populations at level P < 0.05. Asterisks denote differences between inbred and outbred plants within a population. Statistical significance levels have been obtained from Tukey’s test. *P < 0.01; **P < 0.01; ***P < 0.001.
bivore populations on Naantali and Seili would be the two smallest populations. However, because herbivores from these populations experienced inbreeding depression, small population size does not seem to correlate with low inbreeding depression. Variation in the history of inbreeding could also have caused an additional bias for the herbivores. If the © 2013 John Wiley & Sons Ltd/CNRS
parental individuals were related in some of the populations, because of small population sizes, the difference in the level of inbreeding achieved by our experimental crosses (i.e. brother–sister matings vs. matings between families) would be less than expected. Consequently, the difference in the impact of inbred and outbred herbivores on plant resistance could
Letter
have been diminished. However, the non-significant interaction between herbivore population and herbivore cross for plant resistance suggest that this was not the case; the impact of inbred and outbred herbivores on plant resistance did not vary among the herbivore populations. Therefore, it seems that variation in the history of inbreeding among either the herbivore or plant populations is an unlikely explanation for the among-population variation observed in this study. When the herbivores were outbred, plants from all populations suffered from inbreeding depression in resistance. Conversely, when also the herbivores were inbred and presumably less vigorous, the less well defended inbred plants with lower concentrations of phenolic compounds did not sustain significantly higher damage compared with outbred plants, i.e. there was no inbreeding depression in resistance. Inbreeding depression in plant fitness and growth has repeatedly been found to be stronger under herbivory (Carr & Eubanks 2002; Hayes et al. 2004; Ivey et al. 2004; Campbell et al. 2013), and thus, herbivory can act as an impediment to selection for self-fertilisation (Ashman 2002; Steets et al. 2006). Reduced plant resistance due to inbreeding has commonly been observed in conjunction with reduced growth and fitness of inbred plants ~ez-Farf (Carr & Eubanks 2002; Bello-Bedoy & N un an 2011; Muola et al. 2011; Campbell et al. 2013). In addition, leaf damage by A. asclepiadis correlates negatively with fitness measured as pod production in V. hirundinaria (Leimu & Lehtil€ a 2006). Therefore, we suggest that when herbivores are inbred and plant resistance between inbred and outbred plants does not differ considerably, herbivore-induced selection against self-fertilisation in the host plant may be diminished. However, the importance of this effect depends on inbreeding depression in other fitness-related traits. CONCLUSIONS
Previously, variation in the strength and occurrence of inbreeding depression in plant populations has been explained by population history and among-population variation in abiotic factors (Ouborg & Van Treuren 1994; Husband & Schemske 1996; Leimu et al. 2008). We provide a novel additional explanation to this variation as inbreeding depression in plant resistance can be diminished if the herbivores are inbred. Conversely, host-plant inbreeding can either strengthen or have no effect on inbreeding depression in herbivore performance depending on herbivore population. Our results show that the strength of inbreeding depression in one species can be modified depending on the level of inbreeding in an interacting species, but the effect may vary across populations. Population variation in these effects is partly driven by differences in plant phenolic compounds and by the variation among herbivore populations in their association with these compounds. The results of our study emphasise the importance of inbreeding resulting from either natural or human-induced causes for studies examining specific plant–herbivore interactions as well as any interactions between a host and its natural enemies. Furthermore, simultaneous inbreeding can alter herbivoreinduced selection against self-fertilisation, which can have implications for plant mating system evolution.
Inbreeding in plants and herbivores 237
ACKNOWLEDGEMENTS
We thank T. Honkola, T. Koivisto, N. Palin, A. Syrj€ anen, A. Michelsson and the staff at the Ruissalo Botanical garden for assistance in carrying out the experiment and plant maintenance; The Archipelago Research Institute in Seili and Ruissalo Botanical Garden of University of Turku for providing the facilities during field work and the experimental work respectively; A. Koivuniemi, J. Kim, and T. Buss for conducting the chemical analysis; J. Katajisto at the Centre of Statistics in University of Turku for statistical advice; and N. Mutikainen and four anonymous referees for constructive comments on previous versions of the manuscript. This study was financially supported by the Academy of Finland (grant 138308 to RL and grant 258992 to JPS) and Turku University Foundation (grant to AM). Chemical analyses on an ultra-performance liquid chromatography-mass spectrometry system were made possible by a Strategic Research Grant of University of Turku (Ecological Interactions). AUTHORSHIP
AK, PM and RL conceptualised the research and designed the study. AK conducted the experiments and collected the data with AM, JFS and LL. AK analysed the data with the assistance of AM, JFS, PM and RL. JPS designed and conducted the chemical analyses. AK wrote the first version of the manuscript, all authors contributed substantially to revisions. REFERENCES Aguilar, R., Quesada, M., Ashworth, L., Herrerias-Diego, Y. & Lobo, J. (2008). Genetic consequences of habitat fragmentation in plant populations: susceptible signals in plant traits and methodological approaches. Mol. Ecol., 17, 5177–5188. Angeloni, F., Ouborg, N.J. & Leimu, R. (2011). Meta-analysis on the association of population size and life history with inbreeding depression in plants. Biol. Conserv., 144, 35–43. Armbruster, P. & Reed, D.H. (2005). Inbreeding depression in benign and stressful environments. Heredity, 95, 235–242. Ashman, T.-L. (2002). The role of herbivores in the evolution of separate sexes from hermaphroditism. Ecology, 83, 1175–1184. ~ez-Farfan, J. (2011). The effect of inbreeding on Bello-Bedoy, R. & N un defence against multiple enemies in Datura stramonium. J. Evol. Biol., 24, 518–530. Berenbaum, M.R., Nitao, J.K. & Zangerl, A.R. (1991). Adaptive significance of furanocoumarin diversity in Pastica sativa (Apiaceae). J. Chem. Ecol., 17, 207–215. Burdon, J.J. (1993). The structure of pathogen populations in natural plant communities. Annu. Rev. Phytopathol., 31, 305–323. Campbell, S.A., Thaler, J.S. & Kessler, A. (2013). Plant chemistry underlies herbivore-mediated inbreeding depression in nature. Ecol. Lett., 16, 252–260. Carr, D.E. & Eubanks, M.D. (2002). Inbreeding alters resistance to insect herbivory and host plant quality in Mimulus guttatus (Scrophulariaceae). Evolution, 56, 22–30. Charlesworth, B. & Charlesworth, D. (1999). The genetic basis of inbreeding depression. Genet. Res., 74, 329–340. Colling, G. & Matthies, D. (2004). The effects of plant population size on the interaction between the endangered plant Scorzonera humilis, a specialized herbivore, and a phytopathogenic fungus. Oikos, 105, 71–78.
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SUPPORTING INFORMATION
Additional Supporting Information may be downloaded via the online version of this article at Wiley Online Library (www.ecologyletters.com).
Editor, Ted Turlings Manuscript received 19 August 2013 First decision made 26 September 2013 Manuscript accepted 28 October 2013
LETTER
Aino Kalske,1* Pia Mutikainen,2 Anne Muola,1 J. F. Scheepens,1 Liisa Laukkanen,1 Juha-Pekka Salminen3 and Roosa Leimu4
doi: 10.1111/ele.12223
Simultaneous inbreeding modifies inbreeding depression in a plant–herbivore interaction Abstract Because inbreeding is common in natural populations of plants and their herbivores, herbivoreinduced selection on plants, and vice versa, may be significantly modified by inbreeding and inbreeding depression. In a feeding assay with inbred and outbred lines of both the perennial herb, Vincetoxicum hirundinaria, and its specialist herbivore, Abrostola asclepiadis, we discovered that plant inbreeding increased inbreeding depression in herbivore performance in some populations. The effect of inbreeding on plant resistance varied among plant and herbivore populations. The among-population variation is likely to be driven by variation in plant secondary compounds across populations. In addition, inbreeding depression in plant resistance was substantial when herbivores were outbred, but diminished when herbivores were inbred. These findings demonstrate that in plant–herbivore interactions expression of inbreeding depression can depend on the level of inbreeding of the interacting species. Furthermore, our results suggest that when herbivores are inbred, herbivore-induced selection against self-fertilisation in plants may diminish. Keywords Abrostola asclepiadis, experimental inbreeding, insect herbivory, performance, phenolic compounds, plant mating system, resistance, secondary chemistry, self-fertilisation, Vincetoxicum hirundinaria. Ecology Letters (2014) 17: 229–238
Inbreeding is increasingly common in natural populations of plants and their insect herbivores, because of the widespread destruction of natural habitats that has led to population declines (e.g. Saccheri et al. 1998; Keller & Waller 2002; Foley et al. 2005). Negative effects of inbreeding arise mainly through expression of deleterious recessive alleles in the inbred individuals (Charlesworth & Charlesworth 1999). This inbreeding depression has been observed in several plant and insect species as increased mortality and decreased reproductive success, fecundity, germination success, resistance and egg hatching rate (Husband & Schemske 1996; Saccheri et al. 1996; Carr & Eubanks 2002). Furthermore, inbreeding and consequent inbreeding depression may significantly modify herbivore-induced selection on plants, and vice versa, across a wide range of species (Ashman 2002; Carr & Eubanks 2002; Ivey et al. 2004). Plant populations may differ in their level of inbreeding because of variation in pollinator behaviour, population size, self-fertilisation rate and connectivity (Ellstrand & Elam 1993; Aguilar et al. 2008; Whelan et al. 2009). In species interactions, especially when the relationship is specialised, patterns of population genetic structure of one species are likely to be reflected also in the associated species, and consequently, inbreeding has been observed to occur simultaneously in coexisting populations of plants and their herbivores (Magalhaes
et al. 2011). However, because herbivore populations are prone to large fluctuations in size, they may go through population bottlenecks followed by increased inbreeding independently of host plant population size (Hanski et al. 1995). Alternatively, if herbivore populations are more connected than the associated plant populations, because herbivores are in general more mobile, they would be less likely to suffer from inbreeding than plants (Michalakis et al. 1993; Salle et al. 2007). Therefore, the level of inbreeding and changes in it in one of the interacting species does not necessarily correlate with the level of inbreeding in the other species at a particular site. This can lead to a situation where inbreeding occurs either only in the plant or the herbivore, or to simultaneous inbreeding of both counterparts, scenarios that can all alter the dynamics of the coevolutionary interaction. It is known that plant inbreeding reduces herbivore resistance (Carr & Eubanks 2002; Hayes et al. 2004), but the influence of herbivore inbreeding and possible simultaneous inbreeding of plants and herbivores on plant resistance and the ability of herbivores to exploit the plant have received much less attention (but see Strauss & Karban 1994). As inbreeding frequently occurs in both plant and herbivore populations, it is important to explore potential interactions between inbreeding in plants and inbreeding in their herbivores in more detail (I 9 I; Leimu et al. 2012b). Next, we will introduce four potential outcomes of this interaction (Fig. 1). First, inbreeding of plants can reduce the expression of
1
4
INTRODUCTION
2 3
Section of Ecology, University of Turku, Turku, Finland € rich, Zu € rich, Switzerland Institute of Integrative Biology, ETH-Zu
Department of Plant Sciences, University of Oxford, Oxford, UK
*Correspondence: E-mail: [email protected]
Laboratory of Organic Chemistry and Chemical Biology, Department of
Chemistry, University of Turku, Turku, Finland
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inbreeding depression in herbivores, if the inbred host plant causes lower stress in the herbivore due to reduced resistance (Dudash 1990; Fig. 1a). Inbred plants are known to have lowered resistance that has been explained by altered plant chemistry or altered emission of induced plant volatiles (Delphia et al. 2009; Kariyat et al. 2012; Campbell et al. 2013). Second, inbreeding depression in herbivores may also be more pronounced on an inbred vs. outbred host plant, if the outbred herbivores benefit more from the increased quality of the inbred host plant than the inbred herbivores (Fig. 1b). An outbred herbivore may benefit from the increased quality of the host plant mediated by altered nutritional quality, defensive chemistry or stress-related enzymes (Ridley et al. 2011; Leimu et al. 2012a; Campbell et al. 2013), whereas an inbred herbivore may be restricted by its overall lower viability (Greenberg & Crow 1960; Fern andez et al. 1995). Third, the I 9 I interaction may also apply to the host plant: inbreeding depression in plant resistance may be reduced under an attack by inbred herbivores, if the adverse effects of inbreeding of
(b)
(a)
Herbivore fitness
Outbred herbivore Inbred herbivore
Outbred
Inbred
Outbred
Plant (d)
Less resistant Plant damage
(c)
Resistant
Inbred
Plant
Outbred plant Inbred plant
Outbred
Inbred
Herbivore
Outbred
Inbred
Herbivore
Figure 1 Theoretical representation of four potential outcomes of the interactive effects of host plant and herbivore inbreeding (I 9 I) on herbivore performance (a & b) and on plant resistance measured as damage (c & d). Inbreeding depression refers to the difference between the fitness/resistance of the outbred and inbred individuals, when the outbred individuals are outperforming the inbred individuals. Dashed lines in panel a indicate the level of inbreeding depression for the respective circumstances. Note that damage represents an inverse measure of resistance, and thus lower damage refers to higher resistance. (a) Inbreeding depression in the herbivore decreases when host plant is inbred. (b) Inbreeding depression in the herbivore increases when the host is inbred. (c) Inbreeding depression in the host plant decreases when the herbivore is inbred. (d) Inbreeding depression in the host plant increases when the herbivore is inbred.
© 2013 John Wiley & Sons Ltd/CNRS
the herbivore attenuate its ability to overcome plant defences of the outbred plant (Fig. 1c). Finally, inbreeding depression in plant resistance may also be stronger when the herbivore is inbred if the outbred plant is more effectively protected against inbred herbivores than the inbred plant (Fig. 1d). The interactive effects of host-plant and herbivore inbreeding on plant resistance and herbivore performance are also likely to vary among populations with different inbreeding loads (I 9 I 9 P), e.g. due to variation in historical population size and population history of inbreeding (Leimu et al. 2008; Angeloni et al. 2011). We examined the effects of experimental inbreeding both of a long-lived plant, Vincetoxicum hirundinaria Med. (=Cynanchum vincetoxicum (L.) Pers.) (Apocynaceae), and its specialist herbivore, the moth Abrostola asclepiadis Schiff. (Noctuidae), on plant resistance and herbivore performance respectively. We specifically studied whether inbreeding in one of the interacting species affects the expression of inbreeding depression in the other species (I 9 I) and the variation among populations in this effect (I 9 I 9 P). We also studied whether inbreeding alters plant secondary chemistry (phenolic compounds) as well as among-population variation in these compounds. Finally, we aim to explain the variation in the effects of simultaneous inbreeding on herbivore performance and plant resistance with the variation in plant secondary chemistry. In our study area, V. hirundinaria has a mixed mating system, and populations harbour variable frequencies of individuals that are capable of self-fertilisation (Leimu 2004). Populations occur on different islands and are thereby separated by water, which forms a relatively strong barrier to gene flow between the different populations. The level of inbreeding in the Finnish V. hirundinaria populations varies from 0.025 to 0.597 (FIS; Leimu & Mutikainen 2005). Vincetoxicum hirundinaria also suffers from inbreeding depression in resistance to A. asclepiadis (Muola et al. 2011). Population sizes of the plants are relatively constant, whereas those of the herbivores fluctuate (AK, unpublished data). The damage caused by the larvae has significant negative effects on plant reproduction (Muola et al. 2010a) and population growth (Leimu & Lehtil€ a 2006). Vincetoxicum hirundinaria contains many phenolic and other secondary compounds that are presumed to act as chemical defence against herbivores (Muola et al. 2010b). Our results demonstrate that herbivore-induced selection on plants, and vice versa, may be significantly modified by simultaneous inbreeding. Such modifications are likely to be driven by altered plant secondary chemistry that acts as anti-herbivore defence. The effects of simultaneous inbreeding may change our view of the effects of inbreeding on the evolution of plant–herbivore interactions and plant mating systems.
MATERIALS AND METHODS
Study system
In Sweden and Finland, V. hirundinaria occurs on the coastline and islands of the Baltic Sea. For this study we used plants and herbivores from one Swedish (M€ ork€ o) and three Finnish sites (Lammasluoto, Naantali, Seili) harbouring populations
Letter
of both V. hirundinaria and A. asclepiadis. Self- and withinpopulation cross-pollinated plants from the four populations were obtained from 20 maternal plants per population by hand pollination. Inbred and outbred herbivores were derived from a laboratory population of A. asclepiadis that was established in June 2010. Further details on study system, the study populations, and the crossing protocols are provided in Appendix S1 in Supporting Information. Experimental set-up
F1 generation inbred and outbred plants included in the study were potted in 0.9 L pots. When possible, we used a minimum of eight inbred and eight outbred plants from each maternal plant. However, because of problems with pollination and germination, the number of seedlings per maternal plant ranged from 2 up to 61, as the lack of seedlings from some maternal plants was compensated for by using more seedlings from a maternal plant with extra seedlings to obtain similar numbers of seedlings at the population level. On average we had 11 and 10 seedlings from each maternal plant in the inbred and outbred cross treatments respectively. For the Seili population, one-third of the inbred plants used in this experiment were derived from a single maternal plant. This lack of offspring produced by self-fertilisation from some maternal plants may have been due to the variable levels of individuals capable of self-fertilisation among the maternal plants (Leimu 2004). Plants were randomly assigned to 15 blocks with 64 individuals (eight plants from each plant population and plant cross combination) in each block, which makes altogether 960 plants. The experiment was conducted in a greenhouse in the Ruissalo Botanical Garden (University of Turku) in the spring 2012. Temperature and light conditions in the greenhouse were adjusted to resemble summer conditions in the study area: temperature regime of 21/17 °C day/night and an 18 h photoperiod (4.30–22.30 hours). F1 generation pupae of A. asclepiadis were taken to room temperature to hatch. As the F1 adults emerged they were paired to obtain inbred (brother–sister mating) and outbred (between families, within-population mating) F2 families. The pairing was done in the same manner as in the P generation (see Appendix S1). Pairs from F1 generation were allowed to mate until the female had laid a sufficient number of eggs (minimum of 20 eggs), or until the female or the male died. Leaves with eggs on them were removed from the plants 2–5 days after the eggs had been laid and they were placed in plastic containers, the eggs from each family in a separate container. Eggs were monitored daily and once the larvae hatched they were assigned to experimental plants, one larva per plant. Each larva was carefully placed on a leaf of the assigned plant that was covered with a mesh bag. One to three larvae from each family were haphazardly assigned to each of the eight plant treatments (inbred and outbred plants from all four populations, 8–24 larvae from one family), and altogether 12–16 replicates per herbivore origin and cross were used in the experiment. From one outbred herbivore family from Seili up to six replicates per plant treatment were used because of several unsuccessful matings. In the end, we had altogether 927 larvae in the experiment, and thus 33 of the
Inbreeding in plants and herbivores 231
960 plants were left without a larva and left out of the experiment. After 9 days in the experiment, the larvae were weighed to get an estimate of their performance. Larval biomass represents larval performance and is correlated with pupal mass (AK, unpublished data, r = 0.127, P = 0.001, n = 640), which is known to reflect the fitness of adult Lepidoptera (Haukioja & Neuvonen 1985). Larval biomass also correlates negatively with the length of the larval period, i.e. the smaller the larvae are on the eighth day after hatching, the more time it takes for them to complete development (AK, unpublished data, r = 0.524, P < 0.001, n = 662). The damage sustained by the plants was estimated as the proportion of damaged leaves: the number of leaves with any damage divided by total number of leaves in the plant. This is an appropriate measure of damage for this system and species, because plants in the experiment tended to prematurely lose the partly eaten leaves to early senescence. The proportion of damaged leaves therefore accounts for this damage-induced loss of leaves more accurately than, for instance, the percentage of leaf area eaten. To obtain an additional measure of plant resistance, we collected leaf samples from a subset of the plants (463 individuals) for secondary chemistry analysis. The sampling was done by collecting 2–4 leaves from each plant on one of two different days that were ca. 2 weeks apart. Phenolic compounds from each sample were analysed using ultra-performance liquid chromatography with an electrospray triple quadrupole mass spectrometry detector (UPLC-MS/MS, Waters Acquity Xevo, Milford, MA, USA) (see Appendix S1). Data analysis
The data on larval biomass (herbivore performance) were analysed using a mixed model with larval biomass as response variable and herbivore cross (inbred or outbred) and population, plant cross and population of the plant the larvae consumed, and block as fixed factors. Block was included to eliminate the effects of environmental variation due to the different positions of the plants in the greenhouse. All the possible interactions between the fixed factors (except for block) were included in the model. Herbivore family was included in the model as random factor. Plant damage was estimated as the proportion of damaged leaves that inversely reflects plant resistance: the more leaves eaten the less resistant the plant is to herbivore damage. These data were analysed with a mixed model with the same fixed factors as in the previous model: plant cross and population, herbivore cross and population of the larva feeding on the plant, and block as fixed factors. Plant family, i.e. maternal plant, was included in the model as random factor. Plant cross was included in the random residual/group option which specifies a different residual variance for plant cross treatments to correct for heteroscedasticity of residuals. All analyses were conducted using the GLIMMIX procedure in SAS (SAS Enterprise Guide 4.3/SAS 9.2, Cary, NC, USA) and normal distribution with identity link function in both plant damage and herbivore performance data. A total of 26 phenolic compounds were quantified from the leaves out of which 23 were flavonol glycosides (quercetin and © 2013 John Wiley & Sons Ltd/CNRS
232 Aino Kalske et al.
Letter
kaempferol glycosides) and three were caffeoyl quinic acids (chlorogenic acids). To investigate the effects of plant inbreeding on the concentrations of phenolic compounds and variation in this effect among plant populations, we conducted mixed models analysis using GLIMMIX procedure in SAS and normal distribution with identity link function or gamma distribution with log link function, depending on the residual distribution of the data. Data on eight compounds were square root transformed, and data on five compounds had a value 0.01 added in order to improve the fit of the data. We analysed all chemical compounds that were present in 50 or more plants (23 compounds; Table S2), and the total concentrations of quercetin, kaempferol and flavonol glycosides, caffeoyl quinic acids and phenolic compounds. Plant population, plant cross, plant population 9 plant cross and sampling date were treated as fixed factors, and maternal plant as a random factor. Initially, interactions with sampling date and other fixed factors were included in the model, but were removed because they were non-significant. Model assumptions of normality and homogeneity of variances were assessed by visual examination of diagnostic plots, and the significance of the random factors was tested with likelihood ratio tests in all analyses. To assess the defensive role of the secondary compounds, correlations with larval biomass and 11 chosen compounds and five compound groups were conducted using the biomass of the outbred larvae that had been reared on outbred plants. These compounds were chosen because their concentrations differed between inbred and outbred plants according to the results of the first analysis of the chemical data (Table S2),
and they could therefore account for the observed effects of plant inbreeding (see Results).
RESULTS
Herbivore performance
The biomass of the larvae ranged from 0.6 to 107.0 mg with the average of 15.8 0.3 mg (mean SE). We found significant inbreeding depression in herbivore performance (Table 1): inbred larvae had on average 13.6% lower biomass than the outbred larvae (inbred: 14.6 0.5 mg, outbred: 16.9 0.5 mg). Likewise, plant inbreeding had an effect on larval biomass: larvae feeding on inbred plants were 11.9% heavier than the larvae feeding on outbred plants (inbred plants: 16.8 0.5 mg, outbred plants: 14.8 0.4 mg). The larval biomass also varied among their population of origin and depending on the plant population they consumed, which is indicated by the statistically significant main effects of herbivore population and plant population respectively (Table 1). In addition, there was significant variation among herbivore families in herbivore performance (Table 1). Plant inbreeding had an effect on the expression of inbreeding depression in herbivore performance, and the effect varied among herbivore populations (herbivore population 9 herbivore cross 9 plant cross; Table 1, Fig. 2). In some herbivore populations, plant inbreeding led to an increase in herbivore performance: in M€ ork€ o and Seili the outbred herbivores were heavier on inbred than on outbred plants (Fig. 2b,d). However, plant inbreeding did not have a positive effect on herbivores
Table 1 Results (F-values) of analysis of variance testing for effects of inbreeding and population of origin of host plant (Vincetoxicum hirundinaria) and herbivore (Abrostola asclepiadis) on herbivore performance and plant resistance
Herbivore performance Fixed factors Herbivore population Plant population Herbivore cross Plant cross Herbivore population 9 Plant population Herbivore population 9 Herbivore cross Herbivore population 9 Plant cross Plant population 9 Herbivore cross Plant population 9 Plant cross Herbivore cross 9 Plant cross Herbivore population 9 Plant population 9 Herbivore cross Herbivore population 9 Plant population 9 Plant cross Herbivore population 9 Herbivore cross 9 Plant cross Plant population 9 Herbivore cross 9 Plant cross Herbivore population 9 Plant population 9 Herbivore cross 9 Plant cross Block
d.f.
Plant resistance d.f.
F
P
4.12 13.45 5.47 13.07 0.77 0.97 1.06 1.71 2.22 5.54 0.34
** *** * *** n.s. n.s. n.s. n.s. n.s. * n.s.
734.3 735.0 734.4 734.4
0.95 3.07 0.47 0.94
n.s. * n.s. n.s.
14, 801.8
1.32
n.s.
3, 75.0 3, 744.8 1, 74.9 1, 735.1 9, 744.8 3, 75.0 3, 735.0 3, 744.9 3, 734.4 1, 735.0 9, 745.1 9, 3, 3, 9,
Random factors
v2
P
Herbivore family Maternal plant
51.55
***
d.f. = numerator, denominator d.f. for F-values. *P < 0.05; **P < 0.01; ***P < 0.001. © 2013 John Wiley & Sons Ltd/CNRS
F
P
1.84 7.66 6.57 26.46 0.91 0.25 1.31 0.22 3.24 3.89 1.06
n.s. *** * *** n.s. n.s. n.s. n.s. * * n.s.
732.0 736.5 730.6 736.0
2.16 0.93 0.44 1.59
* n.s. n.s. n.s.
14, 746.0
1.00
n.s.
3, 752.8 3, 31.4 1, 756.9 1, 657.0 9, 752.9 3, 760.2 3, 732.1 3, 755.9 3, 623.3 1, 730.7 9, 759.3 9, 3, 3, 9,
v2
P
12.45
***
Letter
Inbreeding in plants and herbivores 233
Larval biomass (mg)
(a) 22
22
20
20
18
18
16
16
14
14
12
12
(c)
Mörkö
*
(d) Naantali
Larval biomass (mg)
The effect of herbivore population on plant resistance varied from inbreeding benefit to inbreeding depression; inbreeding was beneficial for plants from M€ ork€ o when they were consumed by herbivores from Lammasluoto. In all other combinations of plant population and herbivore population, inbred plants were either equally or less resistant than the outbred plants (Fig. 3). In addition, plant inbreeding depression in resistance was statistically significant when plants were consumed by outbred herbivores, but not when they were consumed by inbred herbivores (herbivore cross 9 plant cross; Table 1, Fig. 4). This difference was accounted for by a change in damage in the inbred plants: damage in the inbred plants consumed by outbred herbivores was higher than the damage in the inbred plants consumed by inbred herbivores (closed squares, Fig. 4). The resistance of outbred plants did not differ in response to outbred vs. inbred herbivores (open squares, Fig. 4).
(b) Lammasluoto
Seili
22
22
20
20
18
18
16
16
14
14
12
12
**
Plant secondary chemistry Outbred
Inbred
Outbred
Plant
Inbred
Plant Outbred herbivore Inbred herbivore
Figure 2 Effect of inbreeding of host plant, Vincetoxicum hirundinaria, on performance of inbred and outbred herbivores, Abrostola asclepiadis (larval biomass at 9 days; mean SE). Different panels represent different herbivore populations. Inbred and outbred plants were obtained by selffertilisation and within-population outcrossing respectively. Inbred and outbred herbivores originated from brother–sister matings and withinpopulation crosses respectively. Statistical significance levels have been obtained from Tukey’s test. *P < 0.05; **P < 0.01.
from Naantali or Lammasluoto, as the performance of inbred and outbred herbivores on inbred and outbred plants did not differ statistically significantly (Fig. 2a,c). Plant resistance
The proportion of damaged leaves per plant was on average 0.191 0.004 with variation from 0.020 to 0.900. Inbred plants were less resistant, as their proportion of damaged leaves was on average 19.8% higher compared with outbred plants (inbred: 0.212 0.006, outbred: 0.170 0.004). In addition, outbred larvae caused on average 9.0% higher damage than did the inbred larvae, i.e. the plants were less resistant against the outbred than inbred herbivores (inbred herbivore: 0.182 0.005, outbred herbivore: 0.200 0.006). The proportion of damaged leaves also varied among plant populations but not among herbivore populations (Table 1). In addition, variation among maternal plants in proportion of damaged leaves was statistically significant (Table 1). Inbreeding depression in plant resistance differed among plant and herbivore populations (herbivore population 9 plant population 9 plant cross; Table 1, Fig. 3). Inbred plants were more damaged, i.e. less resistant against the herbivores, than outbred plants in three of the four populations (Lammasluoto, Naantali and Seili), whereas in M€ ork€ o inbred and outbred plants did not differ (dashed lines, Fig. 3).
We found differences in the plant secondary chemistry among populations and between inbred and outbred plants (Table S2, Fig. 5). Concentrations of seven compounds and four compound groups differed among populations (Table S2). Effects of inbreeding on the phenolic compounds were mostly negative; inbred plants had significantly lower concentrations of seven compounds than outbred plants, and in one other compound there was a similar trend (Table S2). Four compounds (quercetin glycoside 596 and 464B, kaempferol glycoside 786A and 610B) were affected differently by inbreeding in different populations (Table S2, Fig. 5a–d). In addition, the concentrations of all compounds varied among the maternal plants, and all but one compound differed between the sampling dates (Table S2). Correlations between larval biomass and the selected plant compounds (i.e. compounds affected by plant inbreeding) revealed that associations between herbivore performance and different phenolic compounds depend on the herbivore population, as the strength, occurrence and direction of the correlations differed among populations (Table S3). Associations between herbivore performance and the compounds were mostly negative: the higher the concentration of the compound in the leaf, the poorer the herbivore performance. The overrepresentation of plants from one maternal plant and of herbivores from one outcrossed family in the Seili population may bias our results, if inbreeding influences resistance in these families differently compared to others in this population. Outbred plants from Seili lacked three of the phenolic compounds present in the other populations (Fig. 5b,d), but this seems to be a common attribute of this particular population based on a previous study (AM, unpublished data). Furthermore, the mean level of damage of the overrepresented family (0.246 for the outbred and 0.161 for the inbred plants) falls well within the variation observed for the other families within this population that varied between 0.118 and 0.289 (min and max) for the outbred plants and between 0.133 and 0.174 for the inbred plants. The same is true for the herbivore performance (mean biomass of the overrepresented family 20.3 mg, min 11.4 mg, max 26.9 mg). © 2013 John Wiley & Sons Ltd/CNRS
234 Aino Kalske et al.
Letter
(a)
(b)
Mörkö
Lammasluoto
Proportion of damaged leaves
Resistant
Less resistant
0.30
0.30
***
0.25
0.25
0.20
0.20
0.15
0.15
0.10
0.10
(c)
(d)
0.30
Naantali
0.30
Seili
0.25
0.25 0.20
0.20
***
0.15
0.15
0.10
0.10 Outbred
**
Inbred
Plant
Outbred
Inbred
Plant Herbivore population: Lammasluoto Naantali Mörkö Seili Population mean
Figure 3 Variation in inbreeding depression of Vincetoxicum hirundinaria in resistance to herbivores among the four studied plant populations (a–d) and among herbivore populations measured as proportion of damaged leaves per plant (mean SE). Note that lower damage reflects higher resistance. Asterisks indicate statistically significant differences (Tukey’s test) between inbred and outbred plants within each plant population (**P < 0.01; ***P < 0.001). Inbred and outbred plants were obtained by self-fertilisation and within-population outcrossing respectively.
Thus, our results are not biased due to the overrepresentation of these families. DISCUSSION
This study is among the first to provide evidence for interactive effects of both host-plant and herbivore inbreeding on herbivore performance and plant resistance. Our results suggest that the expression of inbreeding depression in plant–herbivore interactions depends on the level of inbreeding of the associated species for both counterparts of the interaction. We demonstrate that simultaneous inbreeding can modify herbivore-induced selection on plants, and vice versa. The observed variation in these effects across populations is likely to be driven by corresponding variation in the concentrations of plant secondary chemistry that are altered due to inbreeding. Outbred host plants with effective herbivore resistance represent a higher environmental stress to the herbivores, which is known to increase the expression of inbreeding depression (Dudash 1990; Armbruster & Reed 2005; Mena-Ali et al. 2008; © 2013 John Wiley & Sons Ltd/CNRS
Kariyat et al. 2011). Accordingly, we predicted stronger inbreeding depression in herbivore performance on outbred compared with inbred plants. However, in two of the populations (M€ ork€ o, Seili) plant inbreeding had contrasting effects on herbivore performance: inbreeding depression of the herbivore increased on inbred compared with outbred plants. This effect was in particular due to a significant increase in the performance of the outbred herbivores on inbred plants. Inbreeding of the host plant, therefore, increased its quality as food for the herbivore probably due to reduced levels and altered composition of defensive chemicals (this study; Campbell et al. 2013) or changes in foliar elemental composition (Ridley et al. 2011). The presumed low general viability of the inbred herbivores (Greenberg & Crow 1960; Fern andez et al. 1995) may restrict them from benefiting from the increased quality of inbred host plants, which would explain the stronger inbreeding depression of the herbivores on inbred compared with outbred plants. Strauss & Karban (1994) also discovered increased inbreeding depression on inbred host plants (Erigeron glaucus) in their parthenogenetically reproducing thrips (Apterothrips apteris).
Inbreeding in plants and herbivores 235
Resistant Less resistant Proportion of damaged leaves
Letter
Outbred plants Inbred plants
0.24 0.22 0.20
** ***
0.18 0.16
Outbred
Inbred
Herbivore Figure 4 Effect of inbreeding of herbivore Abrostola asclepiadis, on damage measured as proportion of damaged leaves of inbred and outbred host plants, Vincetoxicum hirundinaria (mean SE). Note that lower damage reflects higher resistance. Inbred and outbred plants were obtained by self-fertilisation and within-population outcrossing respectively. Inbred and outbred herbivores originate from brother–sister matings and within-population crosses respectively. Statistical significance levels have been obtained from Tukey’s test. **P < 0.01; ***P < 0.001.
However, the thrips performed worse on selfed compared to outcrossed plants, which was not the case in our study. Intriguingly, we found variation in the effect of plant inbreeding on herbivore inbreeding depression among herbivore populations (i.e. I 9 I 9 P interaction) as in only two of the studied herbivore populations inbreeding depression in herbivore performance was different on inbred and outbred plants. Because the studied plant populations vary in their leaf chemistry (see also Muola et al. 2010b), and the studied compounds are known to affect local adaptation of A. asclepiadis to their sympatric plant populations (Laukkanen et al. 2012), it is likely that the different herbivore populations have adapted to the specific plant defensive compounds of their sympatric host-plant population. Accordingly, we found variation among the herbivore populations in the association between herbivore performance and those phenolic compounds that were affected by plant inbreeding: there were zero to four negative correlations between herbivore performance and leaf compound concentration per herbivore population. These correlations were mainly with different compounds in different herbivore populations. Taken together, we believe that the differences in the overall phytochemical composition among the host-plant populations resulted in variable associations of herbivore performance with the phenolic compounds, which was reflected in the variation in the interactive effects of plant–herbivore inbreeding among herbivore populations. In other words, depending on whether plant inbreeding modified defensive compounds focal in the interaction in a particular population, plant inbreeding had or did not have an effect on herbivore inbreeding depression through plant defensive chemistry. Variation in phytochemical composition among plant populations thus contributed to the observed variation
in the effect of plant inbreeding on herbivore inbreeding depression among herbivore populations. The observed differences in inbreeding depression in plant resistance among plant and herbivore populations (I 9 P 9 P) were also likely to be derived from the variation in defensive compounds among the plant populations. Plants from the M€ ork€ o population showed no inbreeding depression in resistance, but had relatively high concentrations of defensive compounds some of which were altered by inbreeding. Although concentrations of some compounds were strongly reduced in inbred plants, this reduction was probably compensated by corresponding increases in the concentrations of other compounds, because inbreeding did not markedly reduce the total concentration of phenolic compounds. It is also possible that plant nutritional quality or other resistance mechanisms unaccounted for in this study may explain why the observed effect of inbreeding in defensive compounds did not correspond with leaf damage in M€ ork€ o. Overall, we did not find a single compound that would have explained all observed among-population variation in the effects of plant inbreeding on resistance or on herbivore inbreeding depression, suggesting that the phytochemical diversity and the relative concentrations of different compounds are more important to the interaction than any particular compound alone (Berenbaum et al. 1991). Finally, in plant resistance the significant plant population by herbivore population interaction is suggestive for local adaptation, which has also previously been observed in V. hirundinaria (Kawecki & Ebert 2004; Kalske et al. 2012), but it was not detected here when examining the differences in plant resistance between the sympatric and allopatric plant–herbivore combinations (data not shown). The observed among-population variation in the effects of inbreeding both in plant resistance and herbivore performance could potentially also be explained by variation in the history of inbreeding of the herbivore and plant populations (Ouborg & Van Treuren 1994; Carr & Eubanks 2002; Leimu et al. 2008). In general, there is a negative relationship between inbreeding depression and history of inbreeding (Husband & Schemske 1996; Saccheri et al. 1996). For example, small populations with a long history of inbreeding can be expected to suffer less from inbreeding depression, because the deleterious mutations causing inbreeding depression may have been purged by selection (Crnokrak & Barrett 2002; Angeloni et al. 2011). This explanation seems not to apply to the variation we observed among the plant populations, as the only population where we did not find inbreeding depression in plant resistance was the largest population M€ ork€ o. We have previous data on inbreeding from the three other study populations: the FIS values vary between 0.47 and 0.58, suggesting only minor variation in past inbreeding among the populations, despite the large variation in population size (100–5200 individuals; Appendix S1; Leimu & Mutikainen 2005). Here, the level of inbreeding depression was similar for these populations. Therefore, it seems that the history of inbreeding does not explain current level of inbreeding depression. We do not currently have data on the population sizes of the herbivores. If plant population size roughly predicts the size of the herbivore populations at the same site over years (Burdon 1993; Colling & Matthies 2004), the her© 2013 John Wiley & Sons Ltd/CNRS
236 Aino Kalske et al.
Letter
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 5 Concentrations of phenolic compounds (mg g 1 dry mass; mean SE) in the leaves of Vincetoxicum hirundinaria in different plant populations and in inbred and outbred plants. Panels represent concentrations of (a) quercetin glycoside 596, (b) quercetin glycoside 464B, (c) kaempferol glycoside 786A, (d) kaempferol diglycoside 610B, (e) total quercetin glycosides, (f) total flavonol glycosides (quercetin and kaempferol glycosides), (g) total caffeoyl quinic acids (chlorogenic acids) and (h) total phenolic compounds. Letters that differ between two populations denote statistically significant differences between plant populations at level P < 0.05. Asterisks denote differences between inbred and outbred plants within a population. Statistical significance levels have been obtained from Tukey’s test. *P < 0.01; **P < 0.01; ***P < 0.001.
bivore populations on Naantali and Seili would be the two smallest populations. However, because herbivores from these populations experienced inbreeding depression, small population size does not seem to correlate with low inbreeding depression. Variation in the history of inbreeding could also have caused an additional bias for the herbivores. If the © 2013 John Wiley & Sons Ltd/CNRS
parental individuals were related in some of the populations, because of small population sizes, the difference in the level of inbreeding achieved by our experimental crosses (i.e. brother–sister matings vs. matings between families) would be less than expected. Consequently, the difference in the impact of inbred and outbred herbivores on plant resistance could
Letter
have been diminished. However, the non-significant interaction between herbivore population and herbivore cross for plant resistance suggest that this was not the case; the impact of inbred and outbred herbivores on plant resistance did not vary among the herbivore populations. Therefore, it seems that variation in the history of inbreeding among either the herbivore or plant populations is an unlikely explanation for the among-population variation observed in this study. When the herbivores were outbred, plants from all populations suffered from inbreeding depression in resistance. Conversely, when also the herbivores were inbred and presumably less vigorous, the less well defended inbred plants with lower concentrations of phenolic compounds did not sustain significantly higher damage compared with outbred plants, i.e. there was no inbreeding depression in resistance. Inbreeding depression in plant fitness and growth has repeatedly been found to be stronger under herbivory (Carr & Eubanks 2002; Hayes et al. 2004; Ivey et al. 2004; Campbell et al. 2013), and thus, herbivory can act as an impediment to selection for self-fertilisation (Ashman 2002; Steets et al. 2006). Reduced plant resistance due to inbreeding has commonly been observed in conjunction with reduced growth and fitness of inbred plants ~ez-Farf (Carr & Eubanks 2002; Bello-Bedoy & N un an 2011; Muola et al. 2011; Campbell et al. 2013). In addition, leaf damage by A. asclepiadis correlates negatively with fitness measured as pod production in V. hirundinaria (Leimu & Lehtil€ a 2006). Therefore, we suggest that when herbivores are inbred and plant resistance between inbred and outbred plants does not differ considerably, herbivore-induced selection against self-fertilisation in the host plant may be diminished. However, the importance of this effect depends on inbreeding depression in other fitness-related traits. CONCLUSIONS
Previously, variation in the strength and occurrence of inbreeding depression in plant populations has been explained by population history and among-population variation in abiotic factors (Ouborg & Van Treuren 1994; Husband & Schemske 1996; Leimu et al. 2008). We provide a novel additional explanation to this variation as inbreeding depression in plant resistance can be diminished if the herbivores are inbred. Conversely, host-plant inbreeding can either strengthen or have no effect on inbreeding depression in herbivore performance depending on herbivore population. Our results show that the strength of inbreeding depression in one species can be modified depending on the level of inbreeding in an interacting species, but the effect may vary across populations. Population variation in these effects is partly driven by differences in plant phenolic compounds and by the variation among herbivore populations in their association with these compounds. The results of our study emphasise the importance of inbreeding resulting from either natural or human-induced causes for studies examining specific plant–herbivore interactions as well as any interactions between a host and its natural enemies. Furthermore, simultaneous inbreeding can alter herbivoreinduced selection against self-fertilisation, which can have implications for plant mating system evolution.
Inbreeding in plants and herbivores 237
ACKNOWLEDGEMENTS
We thank T. Honkola, T. Koivisto, N. Palin, A. Syrj€ anen, A. Michelsson and the staff at the Ruissalo Botanical garden for assistance in carrying out the experiment and plant maintenance; The Archipelago Research Institute in Seili and Ruissalo Botanical Garden of University of Turku for providing the facilities during field work and the experimental work respectively; A. Koivuniemi, J. Kim, and T. Buss for conducting the chemical analysis; J. Katajisto at the Centre of Statistics in University of Turku for statistical advice; and N. Mutikainen and four anonymous referees for constructive comments on previous versions of the manuscript. This study was financially supported by the Academy of Finland (grant 138308 to RL and grant 258992 to JPS) and Turku University Foundation (grant to AM). Chemical analyses on an ultra-performance liquid chromatography-mass spectrometry system were made possible by a Strategic Research Grant of University of Turku (Ecological Interactions). AUTHORSHIP
AK, PM and RL conceptualised the research and designed the study. AK conducted the experiments and collected the data with AM, JFS and LL. AK analysed the data with the assistance of AM, JFS, PM and RL. JPS designed and conducted the chemical analyses. AK wrote the first version of the manuscript, all authors contributed substantially to revisions. REFERENCES Aguilar, R., Quesada, M., Ashworth, L., Herrerias-Diego, Y. & Lobo, J. (2008). Genetic consequences of habitat fragmentation in plant populations: susceptible signals in plant traits and methodological approaches. Mol. Ecol., 17, 5177–5188. Angeloni, F., Ouborg, N.J. & Leimu, R. (2011). Meta-analysis on the association of population size and life history with inbreeding depression in plants. Biol. Conserv., 144, 35–43. Armbruster, P. & Reed, D.H. (2005). Inbreeding depression in benign and stressful environments. Heredity, 95, 235–242. Ashman, T.-L. (2002). The role of herbivores in the evolution of separate sexes from hermaphroditism. Ecology, 83, 1175–1184. ~ez-Farfan, J. (2011). The effect of inbreeding on Bello-Bedoy, R. & N un defence against multiple enemies in Datura stramonium. J. Evol. Biol., 24, 518–530. Berenbaum, M.R., Nitao, J.K. & Zangerl, A.R. (1991). Adaptive significance of furanocoumarin diversity in Pastica sativa (Apiaceae). J. Chem. Ecol., 17, 207–215. Burdon, J.J. (1993). The structure of pathogen populations in natural plant communities. Annu. Rev. Phytopathol., 31, 305–323. Campbell, S.A., Thaler, J.S. & Kessler, A. (2013). Plant chemistry underlies herbivore-mediated inbreeding depression in nature. Ecol. Lett., 16, 252–260. Carr, D.E. & Eubanks, M.D. (2002). Inbreeding alters resistance to insect herbivory and host plant quality in Mimulus guttatus (Scrophulariaceae). Evolution, 56, 22–30. Charlesworth, B. & Charlesworth, D. (1999). The genetic basis of inbreeding depression. Genet. Res., 74, 329–340. Colling, G. & Matthies, D. (2004). The effects of plant population size on the interaction between the endangered plant Scorzonera humilis, a specialized herbivore, and a phytopathogenic fungus. Oikos, 105, 71–78.
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SUPPORTING INFORMATION
Additional Supporting Information may be downloaded via the online version of this article at Wiley Online Library (www.ecologyletters.com).
Editor, Ted Turlings Manuscript received 19 August 2013 First decision made 26 September 2013 Manuscript accepted 28 October 2013
