WAGENINGEN UNIVERSITY LABORATORY OF ENTOMOLOGY

Asclepias syriaca – , One host-specialist ecosystem at intercontinental scale

C. Rogé, Msc sciences

Minor and Major Entomology thesis, No: 010.10

No ...... 85 09 06 701 060 Name ...... Cyril Rogé Study program ...... Msc Sciences Period ...... 2010-2011 Thesis/Internship ...... ENT 80436 + ENT 80424 (+ 6) Thesis number ...... 010.10 1e Examinator ...... M. Dicke 2e Examinator ...... R. Gols

Thesis title : syriaca – Aphis nerii, one host-specialist ecosystem at intercontinental scale

Key words : Milkweed – Danaus plexippus – Induction – Enemy release hypothesis – Local adaptation – performance

Supervisors & advisors Martijn Bezemer Jeff Harvey Arjen Biere Tibor Bukovinszky Rieta Gols

Thesis (major and minor) submitted in partial fulfillment of the requirements for the degree of Master of Sciences with the specialization "plant pathology and entomology".

Research subject was originally proposed by Tibor Bukovinszky, Netherlands Institute for ecology (NIOO). Experimentations were done at the NIOO, Heteren, Netherlands, under the supervision of Arjen Biere, Jeff Harvey and Martijn Bezemer. Thesis process was supervised by Rieta Gols, Entomology dpt, Wageningen. This project started in September 2009 and finished in August 2010.

3 4 Acknowledgment

Thanks Tibor to have given me (again) the opportunity to work on your projects

Thanks to all my supervisors (Arjen, Jeff, Martijn and Rieta) and the NIOO people (Gregor, Roel and Vanes) for the help and all the fruitful advices throughout this past year.

Thanks to K. Veltman and the Artis royal zoo have kindly provided Danaus plexippus individuals.

Thanks to A.A.A. Agrawal to have provided the North American experimental- materials.

Thanks to Olaf, Vanes, Floris and Sotos for all this interesting breaks.

I had to say sorry to all “greenhouse workers” that I disturbed with loud heavy music but it was part of Jeff supervision!

Thanks to the French team, The Black Belgian, my big sister and my tinkerbell for all them supports and consideration.

Special thanks to all living beings that give their lives for this study.

5 6 Abstract

Several theories have been developed to explain why a plant is becoming invasive in its new range. The enemy release hypothesis (ERH) links plant fitness optimization (gain) with a decrease in regulation by herbivores and pathogens. ERH is subject to discussion because herbivore pressure is usually not the largest threat to plants. Nevertheless it can be an important hypothesis when plants escape from specialist herbivores with whom they have co- evolved. The milkweed, , is a toxic lactiferous plant from North America (NA) that has become invasive in Europe (EU). It is a highly specialized trophic system in NA where only 12 specialist herbivores are feeding on it, whereas in EU plants were released free from 11 native enemies. In EU A. syriaca is only attacked by a specialist phloem-sucking herbivore, the aphid Aphid nerii. We chose the A. syriaca- A. nerii system because it is occurring in two different continents. In this study, the performance of Aphis nerii from North America and Europe (i.e. US and HUN) was tested on milkweed populations from NA and EU. First, we conducted aphid performance bioassays on six different plant populations. The objective was to access the degree of continental aphid adaptation by determining whether aphid populations perform better on the plant populations originating from the same continent (i.e. home), compared to plant populations from the other continent (i.e. away). Second, we measured aphid performance on plants that had been induced by the Monarch-butterfly caterpillars, Danaus plexippus, to access the degree of plant induction and the subsequent impact on aphid performance. No local adaptation was demonstrated in the “home vs away” comparison. were not achieving higher fitness on their home plant. However, US aphids had a faster development time, higher fecundity and built larger populations than HUN aphids. Furthermore US aphids did better on EU plants than on NA plants, whereas HUN aphid performance was heterogeneous among NA and EU plants. Induction treatment differed between NA and EU milkweed. On the Quebec population it positively affected HUN aphid development time, whereas it negatively affected aphid performance on the Hungarian milkweed population. Our results suggests that the A. syriaca- A. nerii interaction differs from both the plant’s and the herbivore’s perspective. As suggested by ERH, our study pointed at a change in Milkweed metabolism.

7 8 Table of contents

Introduction 13

The milkweed system as a story between invasiveness and specialism 17

Research questions and hypothesis 21

Materials and methods 23 Plants 23 Aphid 25 Lepidoptera 25 Experimentations 27 Bioassay 1 25 Bioassay 2 29 Measurement of various plant traits 29 Statistics 29

Results 33 Bioassay 1: Home versus Away comparison 33 Measurement of various plant traits 33 Measurement of Aphid performances 29 Bioassay 2: induction by monarch caterpillar 35 Dry-weight measurement 35 Measurement of Aphid performances 35

Discussion 37 References 45

9

10 Table of contents, figures and annexes

Figure 1: Asclepias syriaca (drawing) 15

Figure 2: Some North American Milkweed-herbivores (pictures) 16

Figure 3: Herbivores feeding on A. syriaca in Europe (pictures) 18

Figure 4: Milkweed populations and related coding (table) 22

Figure 5: Danaus plexippus caterpillar (picture) 24

Figure 6: Aphid settling procedure 26

Figure 7: Bioassay 1, Aphid development time (graphic) 32

Figure 8: Bioassay 1, Aphid fecundity after three days (graphic) 32

Figure 9: Bioassay 1, Aphid population densities after two weeks (graphic) 34

Figure 10: Bioassay 2, Aphid development time (graphic) 34

Figure 11: Bioassay 2, Aphid fecundity after three days (graphic) 35

Figure 12: Bioassay 2, Aphid survival (graphic) 36

Figure 13: Bioassay 2, Aphid logarithmic dynamics on three weeks (graphic) 36

Figure 14: Statistics tables from SPSS 49

Table 1: Bioassay 1, aphid development time, general linear model (GLM) Table 2: Bioassay 1, aphid fecundity over 3 days, GLM repeated measure of variances Table 3: Bioassay 1, aphid population dynamics, GLM repeated measure of variances Table 4: Bioassay 2, stem dry weight, Table 5: Bioassay 2, aphid development time, GLM Table 6: Bioassay 2, aphid survival, GLM Table 7: Bioassay 2, aphid population dynamics

11 12 Introduction

Alien species invading new ecosystems are becoming a worldwide problem, affecting biodiversity and leading to economic issues when these exotics become a plague. Numerous hypotheses taking into account abiotic and/or biotic conditions have been formulated to explain how an exotic becomes invasive e.g. allelopathy, competition, human activity etc.

The enemy release hypothesis (ERH) postulates that introduced plants experience in the new ecosystem a decrease in regulation by local herbivores and pathogens. Therefore, they can spread more easily and rapidly in its new range than in its native range (Keane and Crawley, 2002; Muller-Scharer et al., 2004). ERH is subject to discussion (Collautti et al., 2004) because herbivore pressure is not ultimately the largest threat to plants, but it remains an important hypothesis when plants escape from host-specific herbivores (i.e. specialist) with whom they have co-evolved (review in Thompson 1989, 1999). For example, if an exotic plant species experiences a significant decrease in herbivore-pressure, i.e. when it is free of specialist, these plants may survive and disperse, leading to a possible outbreak in its new range.

Successful establishment of exotic plants can be explained by two mutually non-exclusive hypotheses 1) plants harbor pre-adapted traits, to the new range context, insuring successful colonisation and 2) after establishment, plants rapidly evolve traits contributing to successful expansion (Muller-Scharer et al., 2004). It is usually assumed that plants are able to allocate resources in a way that maximizes individual inclusive fitness (Optimal defense hypothesis 1, Stamp, 2003). It is commonly stated that defenses (e.g. secondary metabolites) are costly and are allocated (constitutively and inducible) in proportion to risks and costs with regard to plant fitness (Stamp, 2003). At the same time, defense processes divert resources from other needs e.g. growth and reproduction, resulting in a tradeoff between defense and other plants traits, especially if no crosstalk occurs between defense metabolites and primary chemistry.

The most prominent change with respect to enemies experienced by the exotic in its new range is a shift towards an assemblage dominated by generalist foe, although generalist pressure can vary tremendously (Muller-Scharer et al., 2004). Subsequently, an alien species released in a range free from its specialist foe is expected to decrease investment in defenses against specialists to maximize growth and dispersal; (i.e. Evolution of Increased Competitive Ability (EICA) Muller-Scharer et al., 2004), especially if defenses are toxic at low doses and costly to produce.

13

14 Asclepias syriaca (Figure 1) is a toxic lactiferous plant from North America (NA) that has become invasive in Eastern Europe (EU). The milkweed trophic system is highly specialized with only 12 specialist herbivores that are able to feed on it in NA; whereas in EU, plants were released free from 11 enemies occurring in NA. Milkweed appears to be an ideal model plant to test ERH effects on invasion success, because in the new range 1) plants were released free of specialists, especially those driving the milkweed arm-race and inducing CG secondary metabolites in NA and 2) NA and EU plants are attacked by different feeding guilds i.e. various herbivorous feeding guilds in NA and one feeding guild, i.e. sap-sucking aphids, in EU.

Figure 1: Asclepias syriaca, drawing of an upper plant-part with flowers; seed and silk are also represented.

15 Figure 2: Herbivores feeding on A. syriaca in North America

Coleoptera: (left) Milkweed beetle, Tetraopes tetraophthalmus and (right) stem weevil, Rhyssomatus lineaticollis

Lepidoptera: (left) Tiger moth, Euchaetes egle and (right) Monarch butterfly, Danaus plexippus

Homoptera: (left to right) Oleander aphid, Aphis nerii, Milkweed aphids, Aphis asclepiadis and Myzocallis asclepiadis

16 The milkweed system as a story between invasiveness and specialism

Asclepias ’ well-known toxicity comes from cardiac (CG), which are phloem-mobile allelochemicals present constitutively throughout the plant and especially in latex. CG production is also known to be rapidly induced following damage by several species of foliage-chewing herbivores (Malcolm and Zalucki, 1995, 1996; Agrawal unpubl. cited in Mooney and Agrawal, 2008).

In eastern North America, the herbivore community associated with A. syriaca (Figure 2) is highly specialized (Agrawal and Malcolm 2002), comprising 12 species (Smith et al., 2008) mainly in the orders of the Lepidoptera, Coleoptera and Homoptera (Agrawal and Malcolm, 2002; Agrawal, 2005; Blackman and Eastop, 2006; Smith et al., 2008). The two beetles Tetraopes tetraophthalmus and Rhyssomatus lineaticollis were identified as the main species driving the co-evolutionary arms race in the milkweed system in North America, imposing selection on the plants’ defensive traits such as CG, latex, trichomes, toughness and nitrogen content (Agrawal, 2005).

In Europe, A. syriaca is little attacked by specialists and was released free of specialist enemies including the main co-evolutionary arms race drivers and all chewing-herbivores. In the new range, mainly aphids were recorded to feed on A. syriaca (Blackman and Eastop, 2006). The aphids include species such as Aphis fabae, Aphis gossypii Aphis helianthi, Aphis spiriecola, Aulacorthum solani, Myzus persicae and the specialist Aphis nerii, which is present in both continents. These aphids are not known to induce CG metabolites in A. syriaca.

In Europe, Aphis nerii, is feeding on oleander, a species which also produces CG and is closely related to milkweed. The aphid is typically living in large dense colonies on growing shoots and along leaf veins of plants in the Asclepiadaceae and (Blackman and Eastop, 2006). The aposematic coloration of Aphis nerii (bright yellow) has been linked with CG sequestration from its host plant (Rothschild et al. 1970, Malcolm 1986 Smith et al., 2008). Martel and Malcolm (2004) showed CG induction from A. nerii on A. curassavica but Mooney and Agrawal (2008) and Zehnder and Hunter (2007) failed to show this process on A. syriaca (and four other Asclepias species) even in response to high aphids densities.

In this study, the performance of A. nerii collected from both continents (hereafter US for American aphid line and HUN for Hungarian line) is tested on milkweed populations from

17 Figure 3: Herbivores feeding on A. syriaca in Europe.

Aphid fabae Aphis helianthi

V. Molnár W. Cranshaw

Aphis gossypii Aphis spiriecola,

A. jensen

Aulacorthum solani, Myzus persicae

J.K. Lindsey

18 North America (NA) and Europe (EU). First, we conducted aphid performance bioassays on six different plant populations to assess how well these milkweed populations supported growth of both A. nerii populations. The second objective of this study was to access the degree of aphid adaptation by determining whether the two aphid populations performed better on the plant populations originating from the same continent (i.e. home) compared to the populations not from the same continent (i.e. away). Third, we measured aphid performance on plants that had been induced by D. plexippus caterpillars feeding, to access the degree of plant induction and the subsequent impact on aphid performance.

The ERH is an important hypothesis explaining the success of invasion. By assuming changes in metabolism with regard to optimal defense theory and EICA (Stamp, 2003; Muller-Scharer et al., 2004; Blossey and Notzold, 1995), EU plants experienced a decreased selection pressure by herbivores and are released-free from 11/12 of the specialist fauna. Subsequently, through adaptation, EU plants might contain lower concentrations of constitutive and inducible secondary metabolites that act as herbivores defense traits. The induction pattern is expected to change considering the different herbivore guilds associated with milkweeds in NA and EU. In NA, different herbivores guilds (i.e. chewing and sap- sucking ) are feeding from all plant organs. In EU, only sap-sucking herbivores feed on milkweed. A. nerii is the only specialist occurring in EU and it is not known to induce CG metabolites in A. syriaca. In EU, aphids are assumed to represent the main source of herbivore pressure. We expect EU milkweed populations to possess defense traits adapted to sap-sucking herbivores, whereas in NA plants induction is assumed to be strongly mediated by chewing herbivores.

In A. syriaca CG amounts are known to be in the low-medium toxicity range of the milkweed genus (Malcolm 1990). Moreover A. syriaca is known to exhibit phenotypic and genotypic variation among populations (van Zandt and Agrawal 2004, Agrawal 2005, Malcolm and Zalucki 1995, A.A. Agrawal unpublished data cited in Smith et al., 2008 and cited in Mooney and Agrawal, 2008). Subsequently, the variation among populations from a continent may be similar as the variation among populations between continents. It might be difficult to discriminate NA from EU milkweed without large plant-sample size.

A. nerii populations are expected to differ with regard to the trophic-context differences. In NA, aphids are feeding among different 130 milkweed species (Woodson 1954 cited in Agrawal, 2004) offering a enormous heterogeneity in phenotype and genetype both within and among host-plant species. In EU, aphids feed primarily on Nerium oleander and on

19 20 invasive milkweeds that spread sporadically through the continent. We expect US aphids to have a higher fitness on milkweeds populations without CG induction compared to European aphids; because US aphids are more used to feed on milkweed and are used to cope with intra and inter guild competition and the subsequent impact of these interactions on plant quality (e.g. chewing herbivore inducers).

Research questions and hypothesis

1. Do NA milkweed populations differ in quality compared to European milkweed populations when subjected to A. nerii infestation?

We expect NA milkweeds to sustain slower aphid development, lower fecundity and slower aphid-populations growth than EU populations.

2. Do differences exist in aphid performance between US and HUN aphid populations?

We expect US aphids to have a higher fitness on milkweeds compared to HUN aphids.

3. Does plant-herbivore adaptation occur locally in NA and EU?

We do not expect local adaptation to aphid infestation in NA because aphids are a small threat in comparison to chewing insects. We expect that US aphid populations perform better on EU milkweed because NA plants are globally better defended against herbivore enemies. Inversely, in EU we expect co-adaptation because aphids are assumed to represent the main herbivore pressure. We expect that HUN aphids populations perform homogenously on EU milkweed and heterogeneously on NA plants.

4. Does D. plexippus feeding-damage and subsequent induction alter HUN aphid performances similarly on NA and EU plant populations?

We expect NA milkweed induction to be quick and to reflect an adaptive defensive trait against chewing insects. Subsequently we expect that induction of NA milkweed will have a no effect on aphid performances. In addition, CG metabolites seem not to dramatically impede A. nerii performances (e.g. Agrawal 2004). In contrast, we expect a different induction pattern in EU populations affecting negatively aphid performances, because in EU aphids are the main herbivore pressure subsequently we expect the EU plant populations to exhibit adaptation to aphid infestation.

21 Figure 4: Milkweed populations and related coding. Populations in bold were used in the study.

Country City Code Country City Code

Canada Quebec CQU Hungary Erd HER Canada Frederic town CFR Hungary Kisszékely HLI United states Ithaca, bird reserve CBR Hungary Neszűr HNE United states Ithaca, Neimi CNE Slovakia Malacky Slovakia Vojany Slovakia Cierna Voda Austria Hungarian border France Troyes

22 Materials and methods Plants

Asclepias syriaca (Asclepiadaceae) is a perennial herbaceous plant that grows in abandoned fields throughout eastern North America (Smith et al., 2008). In Europe, A. syriaca was introduced into Italy 350 years ago for its medicinal, culinary (Gaertner, 1979) and ornamental uses. More intense re-introduction occurred during the two world wars in order to use A. syriaca in textile industry and beekeeping (T. Bukovinszky pers. com.). Nowadays, this invasive plant is frequently seen in Eastern Europe growing in dense populations in abandoned fields and along roads

Twelve populations from North America (NA) and Europe (EU) were collected in 2008 and 2009 (see Figure 4). Seeds from the United States and Canada were collected by Prof. A.A. Agrawal at Cornell University (N.Y., USA) and European seeds were collected by T. Bukovinszky (NIOO). Several pods within a population area were collected and pooled. Plants were germinated on glass pearls (Glassrot, Assistant, Germany) at 25˚C and a 16L/8D photoperiod after disinfection with 5 % bleach solution and scarification (seeds’ tip cut). Early seedlings were kept at a lower temperature (10°C) until later seedlings grew to similar sizes (i.e. 1 to 2 roots expanded). Because of the low germination rate of the seeds (CNE and CBR respectively around 10% and 20%), only 3 American populations were used i.e. Ithaca, Quebec and Frederic town. The three Hungarian populations, i.e. Neszűr, Kisszékely and Érd, germinated better (80-90%) and were all used.

Seedlings were transferred to 13x13x13 pots with potting soil (Lentse potground, Hortimea group) covered with ± 1cm of sand in order to limit Fungus gnat flies (Diptera, Sciaridae) proliferation. Plants were watered twice a week and after 8 weeks of growth, plants were fertilized weekly with 100 ml Hoagland solution (strength 0.5). To avoid thrips (Thysanoptera,Thrips tabaci) infestation, Amblyseius cucumeris and A. swirskii (Acarina, Phytoseiidae) obtained from Koppert, were released continuously until the start of the experiments. At the start of the experiments, the plants were approximately 7 weeks old. Two days before the start of bioassays, plants were individually placed inside a cylindrical meshed cage equipped with a zip to allow entrance (Marris house nets, England). The experiments took place in a greenhouse at 21˚C (± 2°C) during the 16-h photoperiod and at 18°C (± 2°C) during the 8-h dark period.

23

Figure 5: Danaus plexippus, Monarch caterpillar (L3) on A. syriaca after herbivore- defoliation.

24 Aphids

Aphis nerii is bright yellow with dark appendices (antennas, legs, siphunculi and cauda), and the 5 instars only differ in size; adults length is ca. 1.5-2.6 mm. American aphid individuals were collected in autumn 2009 in Ithaca (N.Y., USA) and were provided by Cornell University. European populations were collected in Hungary during the summer of 2009 by T. Bukovinszky (NIOO). The American aphid line (US) was kept on the Ithaca milkweed population (CBR) and three European aphid lines (HUN) were kept on the three Hungarian milkweed populations (one line per plant population). All lines were reared in cages (50x30x30 cm) with a 16L8D photoperiod and at 21˚C (± 2°C) during the light period and at 18°C (± 2°C) during the dark period. The aphids were starved for 24h before they were introduced onto the experimental plants.

Lepidoptera

We used Danaus plexippus (Figure 5) caterpillars as plant-defence inducers. The monarch butterfly is a well-known species because of its remarkable annual mass migration from North America to central Mexico and along the Californian coast. This herbivore feeds on 25 different Asclepias species and prefers to oviposit and develops better on plants with low to medium CG concentration e.g. Asclepias syriaca (van Zandt and Agrawal 2004, Agrawal 2005, Malcolm and Zalucki 1995, A.A. Agrawal unpublished data cited in Smith et al., 2008 and cited in Mooney and Agrawal, 2008). Caterpillars-feeding behavior decreases CG concentration in leaf tissues: larvae reduce latex flow by severing leaf petioles and veins, and then feed on cut-leaf tissues (Figure 3). In response, milkweed secondary metabolites such as CG are quickly induced (reaching a maximum after 1-2 days) and return to constitutive levels 6 days after damage (Malcolm and Zalucki 1996).

D. plexippus caterpillar individuals were kindly provided by Artis Zoo, Amsterdam, Netherlands. Butterflies were provided to the Zoo by 2 suppliers: London pupa supply (England) and the Costa Rica Entomological Supply. D. plexippus were reared in cages (50x50x50 cm) and A. syriaca plants were provided ad libitum. Adults were maintained on sucrose solution, and were allowed to mate and to lay eggs on “oviposition plants”. Rearing conditions were a photoperiod of 16L8D at respectively 28˚C (±1°C) and 25˚C (±1°C) during the photo and scotophase, respectively, and at 75% (±5%) relative humidity. Eggs were collected daily on (2 cm Ø) leaf discs cut from oviposition plants and, kept on moist paper in etri dishes. Eggs maturity was controlled by placing eggs from 25 ˚C (±1°C) to 10 ˚C (±1°C).

25 Figure 6: Aphid settling procedure. A0

Apical shoot

A0 Settling

Upper part A1

A1 Development Middle part

A2 Offspring production

Lower part A2

Release

A2

A1

26 Experimentations

Bioassay 1 - Aphid performance (October 2009-january 2010)

We wanted to compare whether plant origin affected performance of aphid populations with different origins. We investigated whether the herbivores had developed local adaptation to its food plant. We wanted to investigate if performance of the two aphid lines differed in “home” and “away” situations. American aphids (US) on North American plants (NA) and European Aphids (HUN) on European plants (EU) were considered in a “home” situation; inversely, US aphids on EU plants and HUN aphid on NA plants were classified as ”away” situation. Plant sample sizes were unequal: CBR (n: 10), CQU (n: 11) and CFR (n: 12). HNE (n: 11), HLI (n: 13), HER (n: 14). Each plant population received independently the two aphid populations (½ n for US and ½ n for HUN). We used large cylindrical meshed cages (height 80 m, diameter 35 cm), hereafter called microcosms, with one plant per microcosm.

Two clip-cages (2.5 cm Ø) were placed on the first couple of mature in each microcosm (N: 92) and every clip-cages received two late instars aphids (A0) (see right side figure 6). When A0 individuals had established and started to produce offspring (A1). One new born A1 was left in each clip-cage; the mother A0 and the older progeny A1 were removed. Development time of A1 individuals was recorded; it was defined as the number of days from birth to first offspring production, and A2 is referred to as the progeny of A1. Offspring production of A2 was recorded during the first 3 days only. After which the clip- cages were removed and the aphids were free to settle on the plant wherever they preferred to. Settling preference was also recorded (see below).

Twenty days after A0 introduction, we measured aphid-population dynamics: aphids were counted weekly during 3 consecutive weeks by carefully checking each plant part starting at the primary leaves moving upwards. We measured aphid settling sites by counting the amounts of aphids on different plant parts as follows (see left part of Figure 6): Apical (A) i.e. new shoot; Upper part (U) i.e. not fully developed pairs of leaves; Middle part (M) i.e. mature and fully developed pairs of leaves (where clip-cages were placed); Lower part (B) i.e. from soil to senescent leaves.

Figure 6: (previous page). Aphid settling procedure. On the left side, the four distinct plant- parts used to take in account aphid settling sites. On the right side, the Aphid settling procedure.

27 28 Bioassay 2 - Aphid performance after plant induction by a chewing specialist (February – June 2010).

As in the previous experiments, we used cylindrical meshed cages (height 110 m, diameter 25 cm) to confine aphids on each plant. We only used plants from one Canadian population (CQU) and one Hungarian population (HER), both with European aphids. The plants were divided into two treatment groups: induced plants (IP) and non-induced plants (NP).

Because of the specific pattern of CG secondary metabolites induction (Malcolm and Zalucki 1996); we planned to induce plants 6 times (once every 6 days) to see the effect of plant induction throughout the aphid cycle (i.e. individual development, offspring production and population dynamics). Nevertheless caterpillars got infected by the protozoan Ophryocystis elektroscirrha and plants were only induced twice. The first induction-period was applied at the start of the experimentation when plants received both herbivores at same time on the same ramet of leaves. Aphids were allowed to settle as in experiment 1 and a single L1 or L2 D. plexippus caterpillar was placed in a clip cage. The caterpillar was left on the plant, between 12 and 24 hours, until it had eaten the clip-cages’ leaf area (Ø 1.5 cm). The second induction-period was applied 6 days after the first. All measurements were done similarly as experiment 1. In total 40 plants/microcosms divided in 4 treatments (n=10).

Measurement of various plant traits

Plant traits were measured at the beginning and the end of the bioassay. Leaf size (in cm) of a mature leaf was defined as the maximum length time the maximum width of a fully developed and mature leaf. Plant height was defined as the length (in cm) from the plant apex to the pot rim. Number of leaves and nodes were defined as the number of leaves and nodes visible per stem. Plant dry mass (in gram) were measured at the end of the bioassay. Leaves and stem were harvested separately and both dried in paper bags at 70°C during five days.

Statistics

We investigated the performance of the two aphid lines on a selection of six milkweed populations that came from two continents. Performances were measured as: a) number of days from birth to offspring production, b) offspring production over the first 72 hours, c) population dynamics during 3 weeks. Performance parameter measured on individuals plants (or microcosm) were used as experimental units in the analysis. Explanatory variables were plant population origin (six populations in bioassay 1 and two populations in bioassay 2) and

29 30 aphid line origin (US or HUN). In bioassay 2, we only used the HUN aphid line. Induction treatments were induced (IP) and non-induced plant (NP). In bioassay 1, aphid population dynamics were recorded over 3 weeks (T1, T2 and T3), however in some aphid populations the density decreased during the third week of the bioassay. Apparently, resources were limited in some of the treatments from the second week to the end of the bioassay (T2 to T3), this fact was correlated with a high number of winged forms on these populations at T3 (personal observations). Because aphid densities peaked earlier on some of the populations, aphid densities measured in the third week were excluded from the statistical analysis.

Statistics were done using SPSS (V.16) software We used a general linear model with repeated analyses of variances for repeated measurements (offspring production and aphid dynamics) and a General linear model with multivariate for punctual measurement.

31 populations clusteredby Figure 8: aphid (US)andblueisE (HLI) andNesz Ithaca (CBR),Frederictown(CFR),Quebec Figure 7:

Mean offspring production after 3 days Mean development time (in days) Meanaphiddevelopm MeanAphidfecundity(number ofoffs ű r (HNE).Eachpopulationisclustere u the 2aphidlines. ropean (H Milkweed populations ent tim Milkweed populations UN) aphid. e on6m (CQU). European(EU):Érd(HER),Kisszékely i l pring producedin3days) on6m k weed populations.NorthAm d byaphidlines:GreenisAm erican (NA): ilkweed erican 32 Results

Bioassay 1: Home versus Away comparison

Measurement of various plant traits in North American and European milkweed populations

It was not possible to differentiate between American and European milkweeds with respect to plant traits, even though the measured plant traits seemed more homogenous among the European than among the North American populations. Dissimilarities between continental populations were as variable as variation within continents.

Measurement of Aphid performances

Aphid settling during the experiment was correlated with aphid density and time. The common pattern was similar among all treatments and was as follow: 1) Establishment on the settling leaf i.e. where clip cages were placed. 2) Falling and dispersion, some individuals fell down and started to settle at the bottom part of the plant. 3) When infested leaves are crowded some winged forms are produced (and/or individuals are walking) and then start to settle on the upper parts of the plant; typically along the stem, on apical and axillaries shoots. 4) Infestation is generalized; aphids fully cover stem and upper leaves. Plants become less tolerant to water stress. American aphids (US) tended to develop faster than European aphids (HUN), especially on European (HER, HLI, HNE) and Ithaca (CBR) milkweed populations (Figure 7). However statistically aphid development time was similar on all milkweed populations (Table 1:

F1;59=4.21; P=0.06). No interactions occurred between aphid lines and plants populations.

Aphid fecundity measured over three days differed significantly among the milkweed populations (Table 2: F5;59=2.39; P=0.05; Figure 8). US aphids produced significantly more offspring than HUN aphids (Table 2: F2;59=4.22; P≤0.05). No interactions occurred between aphid lines and plant populations.

Aphid population dynamics were different across milkweed populations (Table 3: F5;59=2.59; P=0.04; Figure 9). Densities of US aphids increased faster than HUN aphid densities, especially on European plants. Furthermore, American milkweeds (NA) supported fewer aphids than European ones (EU). In addition, densities of the HUN aphids were more variable on EU plants than US aphid densities on these populations.

33

2

Mean aphid density at T

Milkweed populations Figure 9: Aphid population densities (in number of individuals) at T2 on the 6 milkweed populations clustered by aphid lines: Green is American aphid (US) and blue is European (HUN) aphid.

Mean development time (in days)

Milkweed populations

Figure 10: Aphid development time (in days) among CQU and HNE milkweed population clustered by induction treatment. Green is induction treatment and blue is control treatment

34 Bioassay 2: induction by monarch caterpillar

Dry-weight measurement of Quebec and Nezur milkweed populations

Dry-weight plant measurements of leaves and stem did not significantly differ between the two milkweed populations (Table 4). However, the induction treatment had an effect on dry- weight of the leaves, induced plant produced less leaves than non induced plant (Table 4:

F1;14=5.52; P=0.04). No interaction occurred between plants population origin and treatment (Table 4).

Measurement of Aphid performances

Aphid development time differed in interaction between plant populations and induction treatment (Table 5: F1;15=4.87; P=0.05). Induction affected aphid development time positively on CQU plants but negatively on HNE plants (Figure 10). The same trend was found for the results on fecundity measurement (table 6 ; Figure 11) but no significant differences occurred because of low number of replicates due to high aphid mortality. Most of the aphids died during the first steps of the bioassay i.e. aphid settling, development time, offspring production (personal observations). Especially on CQU population (Table 7:

F1;19=8.72; P=0.01; Figure 12).

Measurement of aphid densities over three weeks revealed that the HNE plants supported higher aphid densities than CQU plants (Table 8: F1;14=4.67: P=0.05; Figure 9). Induction of plants had no effect on aphid dynamics (Table 8). Nevertheless, induction treatment on HNE seemed to negatively affect aphid density (Figure 12). No interaction appeared during population dynamics (Table 8).

Figure 12: Aphid survival (in % of individuals) among CQU and HNE milkweed population clustered by induction treatment. Green is induction treatment and blue is control treatment .

35

Mean offspring production after 3 days

Milkweed populations

Figure 11: Fecundity (in number of offspring produce after 3 days) among CQU and HNE milkweed population clustered by induction treatment.

Figure 13: Aphid dynamics (logarithmic scale) during three weeks on the HNE and CQU milkweed population on induced and non-induced plants.

36 Discussion • Do NA milkweed populations differ in quality compared to EU milkweed populations when subjected to A. nerii infestation?

First, we hypothesized that variabilities among plant populations within each continent were similar as variabilities between continental populations. As expected plants traits measured (i.e. leaf size, plant height, number of leaf and node) and plant dry weights were heterogeneous among all populations. Nevertheless, milkweed populations differed dramatically in germination and growth rates causing problems to obtain homogeneous populations.

Second, we expected NA milkweeds to sustain slower aphid development, lower fecundity and slower aphid-populations growth than EU populations. As hypothesized, NA populations were in general less good host plants for aphids than the EU populations. NA populations sustained slower aphid development time and lower aphid densities. As was found in other studies (i.e. Agrawal 2004), aphid fecundity was the performance trait that was affected the most by differences in plant quality. Fecundity of both US and HUN aphids was heterogeneous among the six milkweed populations studied. Milkweed appears to affect aphid reproduction (mainly parthenogenetic) and/or juvenile survival.

• Do differences exist in aphid performance between US and HUN aphid populations?

We hypothesized that US aphids have a higher fitness on milkweeds compared to HUN aphids. As expected, US aphids had a shorter development time (tendency), higher fecundity and built larger populations than HUN aphids. Indeed, these two populations are different with respect to various ecological aspects, and several clues indicate that US aphids might out-compete HUN aphids on milkweed hosts. 1) Aphid populations in NA and EU, are assumed to migrate annually from warmer areas i.e. southern America and throughout the (Groeters, 1989; Agrawal pers. com.) where host availability and quality vary drastically. In NA, aphids are assumed to feed mainly on the 130 different milkweed species (Woodson 1954 cited in Agrawal, 2004) offering a generous heterogeneity, both in phenotypes and genotypes, among and within plants species. In EU, A. nerii feed primarily on Nerium oleander and on invasive milkweeds that spread sporadically among the continent. 2) NA and EU trophic systems are dramatically different. In NA, different herbivores guilds (leaf chewing and sap-sucking) are feeding from all plants organs and are mediating constitutive and inducible plant traits (Malcolm and Zalucki, 1995, 1996; Agrawal unpubl.

37 38 cited in Mooney and Agrawal, 2008). In EU, only sap-sucking herbivores feed on milkweed and A. nerii is the only specialist occurring here and it is not known whether aphids induce the production of secondary metabolites in A. syriaca. In Europe, aphids and especially A. nerii, are pointed out as the main herbivore pressure on European milkweeds.

• Does plant-herbivore adaptation occur locally in NA and EU?

We expected 1) US aphid populations to perform better on EU milkweed and 2) HUN aphids populations to perform homogenously on EU milkweed and heterogeneously on NA plants.

As expected, US aphids performed better on EU plants than on NA plants. Nonetheless, HUN aphid performances were heterogeneous among both NA and EU plants. It is possible that HUN aphids are more sensitive to small change in milkweed quality than US aphids. Alternatively, milkweed defense-traits may affect HUN aphids more effectively than US aphids.

Local adaptation in a bi-trophic-system is a difficult question to assess especially with regard to the ecological complexity. Adaptation is the response to frequency dependent selection exerted by a range of selection pressures, e.g. soil biota, abiotic condition, herbivore community etc. Nevertheless the European A. syriaca- A nerii system is susceptible to exhibit local adaptation (or co-adaptation) to aphids because 1) aphid are the only herbivore pressure on EU milkweeds and 2) EU milkweed were released free of the herbivore imposing selection on the plants’ defensive traits such as CG, latex, trichomes, leaf toughness and nitrogen content (Agrawal, 2005).

• Does D. plexippus feeding-damage and subsequent induction alter HUN aphid performances similarly on NA and EU plant populations?

In bioassay 2, we studied the HUN aphid performance on a milkweed population from both NA and EU and we applied an induction treatment using D. plexippus caterpillars. Various experimental problems such as the death of the inducers by a protozoan parasite and the low aphid survival during this bioassay impeded the experimental procedure to yield strong data. Moreover CQU plants exhibit higher aphid mortality and sustained lower aphid density than HNE plants.

We expected that induction of NA milkweed would have little effect on aphid performance. Contrastingly, we expected that induction of EU populations would have a negative effect on aphid performance. Interestingly, induction of the CQU plants had a positive effect on HUN

39 40 aphid development and induction of HNE had a negative influence on HUN aphid performance.

It is possible that induction on Quebec plants was strong and expressed as an adaptive defensive trait against chewing insects. Although induction enhanced aphid performances, CQU plants altered aphid-survival more than HNE plants. The induction effect was different in the EU population altering negatively HUN aphid performance. It is possible that induction in EU plants may have changed from a defense against chewing insects to a defense affecting sap-sucking herbivores. This adjustment might have taken place as an adaptation to the only herbivore pressure present in the new range.

These results were obtained with the HUN aphid population. Testing caterpillar-feeding damage and the subsequent induction impact on the US aphid line may yield new insights to the system, i.e. does induction of EU and NA milkweed affect US and HUN aphid populations similarly? If yes, the induction pattern has changed between NA and EU populations, consequently providing evidence to a change in milkweed metabolism between the native and the introduced range. If not, it will be a strong indication that European- milkweed have developed adaptation to European herbivore infestation. Alternatively, characteristics related to food plant utilization have diverted in US and HUN aphids.

It is usually assumed that plants are able to allocate resources in a way that maximizes individual inclusive fitness (Optimal defense hypothesis 1, Stamp, 2003). It is commonly stated that defenses (e.g. secondary metabolites) are costly and are allocated (constitutively and inducible) in proportion to risks and costs with regard to plant fitness (Stamp, 2003).

The enemy release hypothesis (ERH) postulates that introduced plants experience in the new ecosystem a decrease in regulation by local herbivores and pathogens. As a consequence of lower herbivore pressure, exotics plants do not have to invest in potentially expensive defenses and subsequently will reallocate resources from defensive traits towards growth and dispersal in the new range. Therefore an exotic plants released free of enemies, is expected to spread more easily and rapidly in its new range in comparison to conspecific from the native range (Keane and Crawley, 2002; Muller-Scharer et al., 2004). For milkweed, European populations were released-free from 11 out of its 12 specialist herbivores, including two strong evolutionary drivers, all chewing insects and all CG inducers. The decrease of herbivores’ selection pressure might allow populations with lower defense-levels to sustain in the new range. Furthermore as a consequence of lower herbivore pressure, exotic plants do

41 42 not have to invest in potentially expensive defenses and subsequently might reallocate resources from defensive traits towards growth and dispersal in the new range. As suggested by ERH, our study points at a change in Milkweed metabolism.

Future studies using this system following the same approach will contribute to yield new insights in this plant-herbivore system; especially by testing other milkweed populations, with both aphid lines and with induction treatments. Increasing the total number of Milkweed populations seems obligatory to compare properly NA and EU milkweed populations. It is necessary to increase the experimental power (i.e. number of milkweed populations from both continents) to obtain more reliable prediction of plant variability. In addition both NA and EU plant populations were sampled close to each other; a better spread of the sampling sites on each continent may further contribute to explain variability in relation to distance.

Comparison of a specific system occurring simultaneously in two different ranges is a new and interesting approach of co-adaptation study. It is especially exciting to compare such systems because it allows to unravel plant- adaptation by means of comparing similar interactions that differ geographically. Nevertheless, the study of bi-trophic system is limited to describe ecological changes. Two additional research directions can be envisaged.

Firstly, we can extend the host-plant range by means of comparing aphid performances on various CG-producing plants i.e. within milkweed taxa and/ or Nerium oleander. In addition, such performance results should be combined with aphid CG-sequestration measurements in order to correlate herbivore fitness (and metabolites sequestration) with host-plant quality.

Secondly, we can extend the trophic complexity by adding predators and/or parasitoid to the system. Similarly as above (and despite the amount of work) it should be challenging to correlate aphid fitness (and sequestration) with carnivore fitness (and performance) and with plant quality.

Our study suggests that A. syriaca-A. nerii system occurring simultaneously in different ranges may differ from both on the plant and herbivore side. As the predecessors of Galilee (with planet earth) we shall put the plant in the middle of the ecological picture. As numerous theories are articulated and focused around the plant, we propose that similar (or not) hypotheses should be developed for second (and higher) trophic levels.

43 44 References

Agrawal AA and Malcolm SB. Once upon a milkweed - In this complex community, one insects poison may be another meal. (2002) 111: pp. 48-53. Agrawal AA. "Plant defense and density dependence in the population growth and herbivores. The american naturalist (2004) Vol. 164; 1: pp. 113-120. Agrawal AA. Natural selection on common milkweed (Asclepias syriaca) by a community of specialized insect herbivores. Evolutionary Ecology Research (2005) 7: pp. 651-667. Blackman RL and Eastop VF. Aphids on the World's Herbaceous Plants and , 2nd edition (2006). Blossey B and Notzold R. Evolution of increased competitive ability in invasive non indegenous plants - A hypothesis. Journal of Ecology (1995) 83: pp. 887-889. Colautti RI, Ricciardi, A, Grigorovich IA and MacIsaac HJ. Is invasion success explained by the enemy release hypothesis? Ecology Letters (2004) 7: pp. 721-733. Callaway RM and Ridenour WM. Novel weapons: invasive success and the evolution of increased competitive ability Frontiers in ecology and the environment (2004) Vol. 2; 8: pp. 436-443 Gaertner EE. The history and use of milkweed (Aslepias syriaca L.). Economic botany (1979) 33: pp. 119-123. Groeters, FR. Geographic and clonal variation in the milkweed-oleander aphid, Aphis nerii (Homoptera, ), for winged morph production, life-history, and morphology in relation to host plant permanence. Evolutionary Ecology (1989) 3: pp. 327-341. Keane RM and Crawley MJ. Exotic plant invasions and the enemy hypothesis. Trends in Ecology and Evolution (2002) Vol. 17; 4: pp164-170. Müller-Schärer H, Schaffner U and Steinger T. Evolution in invasive plants: implications for biological control. Trends in Ecology and Evolution (2004) Vol. 19; 8: pp. 417-422. Malcolm SB. Aposematism in a soft-bodied insect: a case for kin selection. Behavior ecology sociobiology (2008) 18: pp. 387-393. Malcolm SB. Chemical defence in chewing and sucking insect herbivores: Plant-derived cardenolides in the monarch butterfly and oleander aphid Chemoecology (1990) 1: pp. 12-21. Malcolm SB and Zalucki MP. Milkweed latex and cardenolide induction may resolve the lethal plant defence paradox. In: 9th International Symposium on Insect-Plant Relationships (1995) Gwatt, Switzerland: Kluwer Academic Publication, pp. 193-196. Malcolm SB and Zalucki MP. Milkweed latex and cardenolide induction may resolve the lethal plant defence paradox. Entomologia Experimentalis et Applicata (1996) 80: pp. 193-196. Martel JW and Malcolm SB. Density-dependent reduction and induction of milkweed cardenolides by a sucking insect herbivore. Jounal of Chemical Ecology (2004) 30: pp. 545-561. Mooney KA and Agrawal AA. Plant genotype shapes ant-aphid interactions: Implications for community structure and indirect plant defense. American Naturalist (2008) 171: pp. 195- 205.

45 46

Rothschild M, von Euw J and Reichstein T. Cardiac glycosides in oleander Aphid, Aphis nerii. Journal of insect physiology (1970) 16: pp. 1141-1145. Thompson JN. Concepts of coevolution. Trends in Ecology and Evolution (1989) Vol. 4; 6: pp. 179-183. Thompson JN. The evolution of species interactions. Science, new series (1999) Vol. 284; 5424: pp. 2216-2118. Smith RA, Mooney KA and Agrawal AA. Coexistence of tree specialist aphids on commom milkweed, Asclepias syriaca. Ecology (2008) 89: pp. 2187-2196. Stamp N. Out of the quagmire of plant defense hypotheses. Quarterly review of biology (2003) Vol. 78; 4: pp. 23-55. Van Zandt PA and Agrawal AA. Specificity of induced plant responses to specialist herbivores of the common milkweed, Asclepias syriaca. Oikos (2004) 104: pp. 401–409. Zehnder CB and Hunter MD. Interspecific variation within the genus Asclepias in response to herbivory by a phloem-feeding insect herbivore. Journal of Chemical Ecology (2007) 33: pp. 2044-2053.

47 48

Figure 10: Statistics tables from SPSS

Table 1: Bioassay 1, aphid development time, general linear model (GLM)

Tests of Between-Subjects Effects Measure: Aphid development time Type III Sum Source of Squares df Mean Square F Sig. Corrected 49,441a 11 4,495 1,325 ,234 Model Intercept 6230,945 1 6230,945 1,837E3 ,000 Aphid 12,636 1 12,636 3,725 ,058 Plant 21,751 5 4,350 1,282 ,284 Aphid * Plant 15,428 5 3,086 ,910 ,481 Error 200,137 59 3,392 Total 6591,000 71 Corrected Total 249,577 70 a. R Squared = ,198 (Adjusted R Squared = ,049)

Table 2: Bioassay 1, aphid fecundity over 3 days, GLM repeated measure of variances

Tests of Between-Subjects Effects Measure: Offspring production over 3 days Type III Sum Source of Squares df Mean Square F Sig.

Intercept 7640,059 1 7640,059 467,385 ,000

Plant 195,870 5 39,174 2,396 ,048

Aphid 69,018 1 69,018 4,222 ,044

Plant * 108,056 5 21,611 1,322 ,267 Aphid Error 964,438 59 16,346

49

Table 3: Aphid population dynamics, GLM repeated measure of variances

Tests of Between-Subjects Effects Measure: Population dynamics over 2 weeks Type III Sum Source of Squares df Mean Square F Sig. Intercept 3453372,633 1 3453372,633 121,963 ,000 Plant 538112,156 5 107622,431 3,801 ,005 Aphid 342119,476 1 342119,476 12,083 ,001 Plant * 366471,017 5 73294,203 2,589 ,035 Aphid Error 1670575,560 59 28314,840

Table 4: Bioassay 2, stem dry weight, Tests of Between-Subjects Effects Measure: Leaf and plant dry weight Dependen Type III Sum Source t Variable of Squares df Mean Square F Sig. Corrected LeafDW 9,695a 3 3,232 2,872 ,074 Model StemDW ,904b 3 ,301 1,707 ,211 Intercept LeafDW 527,859 1 527,859 469,053 ,000 StemDW 68,715 1 68,715 389,264 ,000 Plant LeafDW 1,370 1 1,370 1,217 ,289 StemDW ,788 1 ,788 4,463 ,053 Treatment LeafDW 6,218 1 6,218 5,525 ,034 StemDW 1,042E-5 1 1,042E-5 ,000 ,994 Plant * LeafDW ,278 1 ,278 ,247 ,627 Treatment StemDW ,203 1 ,203 1,150 ,302 Error LeafDW 15,755 14 1,125 StemDW 2,471 14 ,177 Total LeafDW 639,585 18 StemDW 81,001 18 Corrected Total LeafDW 25,451 17 StemDW 3,376 17 a. R Squared = .381 (Adjusted R Squared = .248) b. R Squared = .268 (Adjusted R Squared = .111)

50

Table 5: Bioassay 2, aphid development time, GLM

Tests of Between-Subjects Effects Measure: Aphid development time Type III Sum Source of Squares df Mean Square F Sig. Corrected 15,213a 3 5,071 1,664 ,217 Model Intercept 1580,813 1 1580,813 518,772 ,000 Plant 1,136 1 1,136 ,373 ,551 Treatment ,329 1 ,329 ,108 ,747 Plant * 14,845 1 14,845 4,872 ,043 Treatment Error 45,708 15 3,047 Total 1911,250 19 Corrected Total 60,921 18 a. R Squared = .250 (Adjusted R Squared = .100)

Table 6: Bioassay 2, Aphid survival, GLM

Tests of Between-Subjects Effects Measure: Aphid survival Type III Sum Source of Squares df Mean Square F Sig. Corrected 1,142a 3 ,381 3,487 ,036 Model Intercept 10,032 1 10,032 91,861 ,000 treat ,048 1 ,048 ,440 ,515 Porigin ,953 1 ,953 8,724 ,008 treat * Porigin ,143 1 ,143 1,312 ,266 Error 2,075 19 ,109 Total 13,000 23 Corrected Total 3,217 22 a. R Squared = .355 (Adjusted R Squared = .253)

51

Table 7: Bioassay 2, aphid population dynamics,

Tests of Between-Subjects Effects Measure: population dynamics over 3 weeks Type III Sum Source of Squares df Mean Square F Sig. Intercept 3,179E7 1 3,179E7 11,878 ,004 Treat 2203594,756 1 2203594,756 ,823 ,380 Porigin 1,251E7 1 1,251E7 4,673 ,048 Treat * 870348,735 1 870348,735 ,325 ,578 Porigin Error 3,747E7 14 2676148,289

52