Unraveling the ecology of the dune aphid Schizaphisrufula (Hemiptera: Aphidoidea):
Ecological preferences and Parasitoids (Hymenoptera)
Charlotte Van Moorleghem Promotor: Eduardo de la Peña
1 Thesis submitted to obtain the degree of Master of Science in Biology
Image on front cover: Schizaphis rufula on its host plant Ammophila arenaria. © Eduardo de la Peña
2 Table of contents
Abstract ……………………………………………………………………………………………………………………………...... 2 Introduction 1. Sand dune landscape …………………………………………………………………………………………………… 2 2. Sand dune community …………………………………………………………………………………………………. 4 3. Schizaphis rufula ………………………………………………………………………………………………………….. 6 4. Parasitoids …………………………………………………………………………………………………………………… 7 Aims …………………………………………………………………………………………………………………………………………. 8 Material and methods 1. Study design a. Ecological preferences Study sites …………………………………………………………………………………………… 9 Study design ……………………………………………………………………………………….. 9 b. Plant suitability experiment Preparations ………………………………………………………………………………………… 11 Experiment ………………………………………………………………………………………….. 12 c. Identification of parasitoids Morphological identification ……………………………………………………………….. 12 Molecular identification ………………………………………………………………………. 13 2. Statistical analysis a. Ecological preferences …………………………………………………………………………………….. 14 b. Plant suitability experiment …………………………………………………………………………….. 14 Results 1. Ecological preferences a. Probability of aphid encounter ………………………………………………………………………… 15 b. Aphid population dynamics …………………………………………………………………………….. 17 2. Plant suitability experiment …………………………………………………………………………………………. 17 3. Identification of parasitoids Morphological identification ……………………………………………………………………………. 18 Molecular identification …………………………………………………………………………………… 21 Discussion 1. General findings ………………………………………………………………………………………………………….. 22 2. Ecological preferences: underlying mechanisms …………………………………………………………. 22 3. Ecological preferences and their meaning for the functioning of a dune ecosystem…… 25 4. Parasitoids: the importance of finding new associations …………………………………………….. 27 5. Overall conclusions ………………………………………………………………………………………………………. 29 Summary …………………………………………………………………………………………………………………………………. 29 Acknowledgements …………………………………………………………………………………………………………………. 31 References ………………………………………………………………………………………………………………………………. 31 Appendix 1. Tables …………………………………………………………………………………………………………………………. 37 2. Glossary ……………………………………………………………………………………………………………………… 41
1
Abstract Schizaphis rufula is an aphid species which is highly abundant in coastal sand dune landscapes across Europe. In these habitats it is usually found on sand stabilizing dune grasses like Ammophila arenaria and Leymus arenarius. These species have a large impact on their surroundings through their ability of fixating large sand masses. Despite S. rufula being a potential candidate for influencing local populations of such sand stabilizing grasses, the ecology of this herbivore is relatively unknown. Our aims were to determine 1) the influence of the surrounding plant community on population densities of S. rufula associated with A. arenaria, 2) the specificity of the aphid’s host choice within and between species and 3) the parasitoid community associated with S. rufula. Through a field study and a plant suitability experiment it was found that the presence of S. rufula on its host plant A. arenaria is mainly determined by biotic environmental factors that also indicate dune fixation, namely species richness and percentage cover by Festuca rubra within a one meter radius from the monitored plant individual. S. rufula is more often present on A. arenaria in a species poor environment with a low presence of the dune grass F. rubra. These conditions are characteristic for mobile dunes where only specialized plants occur that can endure the extreme circumstances linked to these kind of habitats. In addition to these findings, a preference of the aphid for dune stabilizing grasses, like A. arenaria and L. arenarius, was found. These mobile dune species also showed a decline in biomass when occupied by aphid populations in the plant suitability experiment. Both the result of the field survey as those from the plant suitability experiment indicate the potential of S. rufula to impact population dynamics of primary dune fixating grasses and consequently also dune dynamics. The parasitoid wasps Aphidius rhopalosiphi and A. avenae (Hymenoptera: Aphidiinae) where for the first time observed to parasitize on S. rufula. Also hyperparasitoids from the genus Apoanagyrus sp., Dendrocerus sp. and Pachyneuron sp. (Hymenoptera) were found.
Introduction Studying species ecology does not only give us greater knowledge of the species itself, but also of the functioning of the environment and habitat quality (Memmott, 2009). When studying species ecology, however, there must also be attention for the abiotic components of the environment next to the biotic component (e.g. interactions between species). This is particularly the case when the environment has very specific characteristics, like in a coastal sand dune landscape (Bertels et al., 2005). The specific abiotic factors which shape coastal dune systems contribute to its uniqueness through their effect on species ecology and evolution. However, this uniqueness makes coastal dune systems also very vulnerable (Provoost et al., 2011a & 2011b). Research within coastal dunes and on coastal dune species with special focus on dune landscape structure and dynamics is thus important to better understand and protect this environment. Further on in this introduction, more information will be given about a sand dune landscape, its functioning and the community it harbors to eventually end up with elucidating the species of focus within this study.
1. Sand dune landscape
A coastal sand dune landscape is a dynamic system with specific abiotic characteristics which shape the biological community it harbors (Bigot et al., 1982). The main factor that distinguishes it from most other habitats is the great importance of wind dynamic for the appearance and functioning of
2 a.
Yellow dune
b. Beach Embryonic dunes
Yellow dunes Dune scrub
Fig. 1: a. Scheme of successional relations between coastal dune vegetation types within a landscape ecological framework. (altered from Bertels et al., 2005); b. pictures that illustrate the main dune habitats that were considered during this study with the frames being color codes that will be maintained in further graphs. the habitat. It determines whether or not there is soil development. This soil development is largely responsible for the way in which plant succession in a dune landscape occurs. When the soil is not well developed or fixated dry sand is continuously repositioned and can bury whole plant communities. This creates a self regenerating landscape which contains a mosaic of several plant successional stages, each successional stage representing a so called dune habitat (Bonte & Provoost, 2005; Crevits, s.d.). Between these dune habitats there can be occurrences of transitional areas. Scientific research within a dune system must be performed taking into account the specific dune landscape structure (Bertels et al., 2005). Figure 1.a shows a scheme of coastal dune structure and in what follows the different habitats will be discussed. Mainly Belgian dune habitats will be considered, but the general lines are applicable to similar dune habitats in Atlantic Europe. When sand is repositioned from the beach more land inwards either by wind dynamics or the working of tides, it is first captured by Elymus farctus. This species, through limited vertical spread of rhizomes, can endure moderate sand accumulation and it aids in the formation of embryonic shifting dunes (Crevits, s.d.; Bertels et al., 2005; European Commission, 2013). These embryonic shifting dunes are the first step in dune formation and are mostly situated on strandlines. Other vegetation that can occur here are salt-tolerant pioneer species like Cakile maritima. Embryonic shifting dunes form a harsh and unpredictable habitat that can easily be blown apart again by the wind or be washed away during high tides. When these embryonic dunes do manage to become higher and when freshwater is present in the underground E. farctus gets replaced by Ammophila arenaria. This
3 grass species has a more developed system of vertically growing rhizomes which allows it to prevail on large sand masses (Huiskes, 1979). At this stage, the habitat is called a yellow dune habitat or even a mobile dune habitat (also called dynamic or shifting dune habitat), when sand is not fixated entirely by A. arenaria so that large masses of sand can still be relocated by wind dynamics (European Commission, 2013). In Belgium these kind of mobile dunes are only present in the area between the French-Belgian border and Nieuwpoort, being particularly remarkable in extension and natural value in the Westhoek (Doody, 2008). Leymus arenarius can be present in both embryonic shifting dunes and yellow dunes at the Belgian coast, but becomes a more important dune grass in regions north of latitude 63°N, where A. arenaria becomes less dominant (van der Meijden, 2005; Huiskes, 1979). The more sand gets fixated by A. arenaria, the more plant species can settle that are not adapted to dynamic yellow dune conditions. The environment becomes more species rich and Carex arenaria and Festuca rubra are more often encountered while A. arenaria populations deteriorate (European commission, 2013; Bertels et al., 2005; Crevits, s.d.). It is in these more species rich conditions that build up of organic matter and nutrients is promoted. This leads to the establishment of strong competitors such as Calamagrostis epigejos (Maun, 2009b). Eventually the habitat can become a dune scrub with the main species being Hippophae rhamnoides. External factors such as trampling or natural blow outs can again lead to soil destruction and initiate secondary vegetation patterns (Bertels et al., 2005). The dune habitats mentioned above are not the only possible habitats occurring at the Belgian coast. Under specific conditions, other succesional stages can develop. Where sand is blown out up until groundwater level, moist or even flooded dune slacks are formed (Crevits, s.d.). Or when extended rains follow extreme dry conditions the soil is acidified, giving rise to a vegetation cover of acid-loving species like Viola canina, Potentilla palustris and Carex arenaria (Maun, 2009b). Moss dunes are formed at the lee slope of mobile dunes were the environment is less dynamic. This dune habitat is however very sensitive to trampling and must be managed closely at the Belgian coast to prevent further succession towards dune scrub (Crevits, s.d.; Maun, 2009b). These are some examples of other dune habitats. However, this study’s main focus is on embryonic dunes, yellow dunes and transitional zones between them and towards dune scrub.
2. Sand dune community
Insect biodiversity in a dune landscape is surprisingly high and stands in contrast with the harshness and relatively limited diversity in vegetation. This insect diversity is however highly vulnerable because of the restriction of these organisms to a dune habitat. Many dune species exhibit evolutionary adaptations to dune conditions. Some species for example switch to a nocturnal lifestyle to avoid the high summer temperatures, while A. arenaria rolls up its leaves during warm periods to retain water (Maun, 2009b; Bonte & Provoost, 2005; Huiskes, 1979). The evolutionary adaptations that make living within a dune environment possible, however, at the same time constrain their distribution to other habitats (Bonte & Provoost, 2005). This is partly because of the large investment they have to put in these adaptations. An investment that makes it impossible for these organisms to put energy in certain other traits. These other traits may not be important in a dune environment, but they can be essential for living in other habitats. Besides this, the large amount of energy that flows to adaptations to coastal sand dunes also impacts insect development. It takes a much longer time span for dune species to become an adult compared to closely related species from other habitats. The dune beetle Polyphylla fullo has for example a larval development of
4 two to three years (Bonte & Provoost, 2005). This makes them more vulnerable to the high rate of tourism at the Belgian coast nowadays. In the light of the recent deteriorations of European sand dune landscapes, the vulnerability of dune organisms and the restriction to their environment makes the urge of protecting and managing coastal sand dune systems particularly clear (Provoost et al., 2011b). In Belgium it is even more compelling because its coastline is almost 50% urbanized and the remaining dune habitats are very fragmented and damaged (Doody, 2008). Because of extensive landscape changes, mobile dunes become fixated and the regenerating character of the system is lost (Provoost et al., 2011a). Eventually this makes that the whole mobile sand dune ecosystem disappears and becomes replaced by dune scrub or forest.
Various environmental factors impact sand dune communities. The previously mentioned factor of wind dynamics can influence sand dune fauna directly by causing stress through the lashing of overblown sand (Bonte & Provoost, 2005). Also an indirect impact is present from its influence on the vegetational succession. Covering with sand of older successional stages takes the vegetation back to a pioneer stage. Other plant species will occur on these places with other specialist herbivores and their predators. Another indirect effect is through the prevention of soil development. This causes nutrient scarcity which also influences the sand dune community. Although these factors are of major importance within the system, there are still other factors that shape a dune community. The sea influences the ecosystem by buffering seasonal temperatures, causing salty conditions through sea spray and flooding and supplying the calcareous remains of marine organisms which increase soil pH. Also soil surface temperatures can reach extremes of up to 70°C on warm days during the summer months, while at night there is a very rapid cooling due to the low heat retention capacity of sand (Bonte & Provoost, 2005). Living in these harsh and dynamic habitats is thus challenging. Still, many species seem to cope with this challenge often through their specific evolutionary adaptations to dune environmental conditions. A dune community is thus a very specialized community, but above all a variously changing community, especially in and near the most dynamic dune habitats such as mobile sand dunes (Bigot et al., 1982). Major taxa of dune herbivores are aphids (Hemiptera: Aphidoidea), leafhoppers (Homoptera: Ciccadellidae), certain beetles (Coleoptera), grasshoppers (Orthoptera: Caelifera) and snails (Gastropoda) (Maun, 2009a; Bonte & Provoost, 2004). In a yellow dune environment, with A. arenaria being the main plant species, a diverse herbivore community is rather remarkable. Different species can however live next to one another by the aid of different life strategies. Niche segregation is an example of this. An example of this can be found when considering the aphids Schizaphis rufula, Laingia psammae and Metopolophium sabihae which are all associated with A. arenaria. Schizaphis rufula is mostly present on the leaves, while L. psammae can be found living between the flowers or fruits. These aphids are relatively closely related species. It is not surprising that species that are more distantly related show an even larger array of feeding strategies. The larvae of the dune beetle Otiorrhynchus atroapterus (Coleoptera: Curculionidae) feed on the roots and larvae of the moth Apatetris kinkerella are leaf miners of A. arenaria (Weeda et al., 2003c). Herbivores can also cope with competition for a certain plant species by extending their host range to other plant species. Schizaphis rufula is besides on A. arenaria also found on Leymus arenarius and L. psammae is also known feeding on C. epigejos. Metopolophium sabihae was first only known from the species F. rubra and Vulpia membranacea (Vandegehuchte et al., 2010). Aphids are predated on by Syrphid larvae and Coccinellidae but also by other predators present in the vegetation like certain dune spiders
5 (Araneae). Another kind of predators within dune habitats are ground dwelling arthropods like wolf spiders (Araneae: Lycosidae) and carabids (Coleoptera: Carabidae). The dune predator community is like the herbivore community surprisingly diverse.
3. Schizaphis rufula
Aphids (Aphidoidea) are an economically important group of herbivores because they are a major pest on a wide range of crops. Some examples are the cherry tree aphid Myzus cerasi, the cereal aphids Sitobion avenae and Rhopalosiphum padi which are major insect pests in northern Europe and Nasonovia ribisnigri (Mosley), specific to lettuce (Stutz & Entling, 2011; Hansen, 1995; Höller et al., 1993; Barrière et al., 2014). In natural systems aphids do not develop into a pest species. The maintenance in diversity of the predator community in these more complex systems is usually put forward as the main reason for this observation but it remains a subject of discussion (Chaplin- Kramer & Kremen, 2012; Lithourgidis et al., 2011, Müller & Godfray, 1999; Altieri & Letourneau, 1982). Although aphids and insect herbivores in general do not often show outbreaks in natural systems, this does not take away their importance in ecosystem functioning. Chronic herbivory can for example significantly influence forest ecosystems (Weisser & Siemann, 2004; Hunter, 2001). Indeed, leaf area loss to insects in general can reduce tree growth (Wagner et al., 2008; Marquis & Whelan, 1994) and redirects primary production into the herbivore food chain (Cebrián, 1999; McNaughton et al., 1989), altering material flows from the canopy to the forest soil (Metcalfe et al., 2014; Hartley & Jones, 2004; Hunter, 2001)(Mazía et al., 2012). Other examples are the influence of herbivory on interspecific competition of grasses (Ibanez et al., 2013), pollination success (Strauss et al., 2001; Lehtila & Strauss, 1999) and seed production and seedling recruitment (Maron et al., 2002), which can all influence vegetative composition within the habitat. The study by Maron et al. (2002) was conducted in coastal and continental dunes but mainly focused on moths (Lepidoptera). Aphids in natural systems are often overlooked within ecological research when not being a pest species or they are regarded within a broader community of herbivores, e.g. in general herbivore exclusion experiments. Neglecting aphids in plant herbivore research can however be unjustified. Allan and Crawly (2011) put forward the aphid Diuraphis holci as a potential promoter of vegetative diversity in an acid, mesotrophic grassland and even more aphid species are expected to be important actors within the herbivore community. Within a dune habitat an important extra factor is added to the aphid’s potential range of influence. Namely, influence on dune vegetative structure manifests itself further into dune dynamics, causing more drastic changes of landscape appearance and functioning.
Within the European dune landscape, several aphid species have been observed feeding and reproducing on the important dune grasses that form a dune habitat, Schizaphis rufula (Walker 1849) being most abundantly present (Vandegehuchte et al., 2010). The genus Schizaphis contains mainly grass inhabiting aphids which in some cases can be deleterious for food crops like the much more investigated S. graminum, which is a severe pest species on small grains all over the world (Evidente et al., 2009; Pettersson, 1971a & b; Börner & Heinse, 1957). The distribution of S. rufula is restricted to Europe and ranges from northern countries like Finland, Sweden and Denmark to more southern regions such as Corsica and Sicily. In central and western Europe it is known to occur in Germany, Poland, the Netherlands, Britain and Ireland (Nieto Nafría, 2007). It was only until recent, in the summer of 2007, that S. rufula was for the first time reported in Belgium indicating the relatively low
6 amount of taxonomical research conducted in Belgian dune systems (Vandegehuchte et al., 2010). It can be found from June or even earlier up until October feeding and reproducing on dune grasses including A. arenaria, E. farctus and L. arenarius. In contrast with the species’ large range and potential importance for the ecosystem, little is known about its ecology. In articles published around 1970, Pettersson made first attempts to clarify the species biology based on research conducted in Sweden. He found S. rufula feeding on L. arenarius which is more abundantly present at Swedish than at Belgian coasts (Pettersson, 1971a; van der Meijden, 2005). He stated that superficially there was no pronounced damage observed on L. arenarius plants infested by the aphid, which is in contrast with observations on other plants from the genus Hordeum were S. rufula seems to reduce the survival of plants considerably, implying that the aphid’s effect on plants is species specific. To understand the way in which S. rufula influences dune grasses or the dune ecosystem, its host plants and environmental preferences need to be clarified. Pettersson (1971c) already did a host choice experiment with four Schizaphis species, among which S. rufula, investigating sixty different host species coming from a very diverse range of habitats. In this study he did not focus on dune habitats when choosing plant species to be tested. In our study, dune species and the successional stage or dune habitat they represent get a more pronounced position.
4. Parasitoids
Parasitoids can play a major role in controlling aphid populations (Thies et al., 2005; Schmidt et al., 2004). That is why they are often used as biocontrol agents in agricultural crops. Parasitoids regarded in this study are insect parasitoids. These are insects that live a part of their lives at the expense of other organisms and nearly always kill their host. This is opposed to true parasites that keep their host alive (Raper, 2001). Insect parasitoids are spread over various orders, but are very well represented within the Hymenoptera. All together they attack a very large range of hosts spread over numerous taxa worldwide (Sanz & Leverton, 2010; Salvo & Valladares, 2007; Guerrieri, 2006; Kopelke, 2003; Huang & Polaszek, 1998; Hedqvist, 1998; Godfray, 1994; Hàgvar & Hofsvang, 1991). A special case of parasitoids are hyperparasitoids, which parasitize other parasitoids within their host. Parasitoids can be subdivided into two groups according to their lifestyles, namely koinobionts and idiobionts, the biggest difference being that in koinobionts the host is (at most) only partially paralyzed by the wasp’s venom and soon recovers whereupon it continues to develop and is only killed when the parasitoid reaches maturity. Idiobionts on the other hand totally paralyze their host with their venom, terminating its development (Raper, 2001). For the dune aphid S. rufula there are indications of parasitism by koinobionts. In a field survey conducted in 2007 by Vandegehuchte et al. (2010) parasitized S. rufula individuals called mummies were found. These parasitized aphids have a swollen, brown appearance which is caused by parasitoid wasps (Hymenoptera) that lay their eggs inside a healthy aphid by penetrating through its surface with an ovipositor. The egg develops into a larva, which stays in the aphid until it is fully grown. When developed into an adult, it emerges (Hàgvar & Hofsvang, 1991). There are only two families of Hymenoptera that contain parasitoids of aphids, namely the Aphelinidae (only two genera) and the Braconidae (27 genera from the subfamily Aphidiinae) (Turpeau et al., 2010a). The family of Braconidae is the second largest family in the Hymenoptera containing approximately 40 000 species, the largest being its sister family Ichneumonidae with approximately 60 000 species (Goulet & Huber, 1993). The identity of the parasitoids found on S. rufula as well as the way in which they influence aphid population dynamics is not yet clear.
7 Aims It is known that the dune aphid S. rufula uses host plants that are of high importance for the functioning of a dune ecosystem. The main aims of this master thesis are to determine which biotic factors impact population dynamics of S. rufula present on these host plants and under which conditions and on which plant species this aphid’s populations do best. By investigating this, it can be determined whether S. rufula has the potential to impact ecosystem dynamics through its interaction with its host plant and under which circumstances this impact is the most pronounced. Research questions were tackled while giving coastal sand dune structure a pronounced place within the study. More specifically we determined 1) the influence of the surrounding plant community on population dynamics of S. rufula associated with A. arenaria in the field, 2) the specificity of the aphid’s host choice within and between plant species and 3) the parasitoid community associated with S. rufula. Two different research strategies were used. First of all a more observational strategy was conducted during the course of a field survey. Secondly, this field data was complemented with the more experimental setup of a host suitability experiment. Field survey: The main aim of the field survey was to determine how S. rufula populations behave on A. arenaria host plants with varying intrinsic plant characteristics and located in different dune habitats. It was expected that S. rufula populations would be more abundantly present on young plants of A. arenaria, due to the higher nutritional value of younger compared to older plants (Pettersson, 1971c; Kennedy, 1958). The aphids should also do better in dune habitats where stress from predation and overblowing sand is minimal. These conditions are probably more prominently present in areas with a more dense vegetational cover. This vegetation can be mainly composed by A. arenaria itself like in yellow dunes, but can also include other dune plants in transitional zones towards more fixated dune habitats. Hypotheses were based on field observations and studies from de la Peña (2011), Vandegehuchte et al. (2010) and Pettersson (1971c). During the field survey, the species composition of the parasitoid community associated with S. rufula was also determined to have a better view on the community of natural enemies surrounding this aphid species. Similar studies on parasitoids of S. rufula have to our knowledge not yet been performed. Plant suitability experiment: In previous field observations S. rufula is besides being abundantly present on A. arenaria also seen on other dune grasses like E. farctus, F. rubra, L. arenarius and even on the sedge species Carex arenaria. As stated in the introduction under subheading “Sand dune landscape” these plants are important players in the vegetative succession within a dune landscape. In the plant suitability experiment, it was tested if there were observable differences in aphid population dynamics considering mainly the variable “plant species”. In doing so, there is a decoupling of the aphid-plant interaction from any other habitat specific influence. This gives a better view of the aphid’s host preference. The broadness of the aphid’s host choice was also tested by putting plant species from other habitats under experiment. It is expect that S. rufula will not be a true specialist species regarding the fact that it is already observed feeding on various host plants in the field. Still pronounced differences between plant species are expected because of their various identities and observed variation in aphid population dynamics from previous field observations. This experiment complements the field survey in a way that the aphid’s potential impact can be shown within other dune habitats besides the ones containing host species A. arenaria. Embryonic shifting dunes are an example of a dune habitat which could not be investigated in the field survey because A. arenaria is not present in this kind of environment. By following aphid population
8 dynamics on the embryonic dune species E. farctus it can be shown whether or not there is the potential for an impact from the aphid on the functioning of this dune habitat.
Material and methods 1. Study design
a. Ecological preferences
Study sites: When selecting the study sites for conducting the field survey, it was assured that certain criteria were met. First of all, study sites had to comprise the dune habitats that are typical to a European dune landscape. This means that natural dune dynamics had to be influenced as little as possible by human influences. Due to the urbanization of the Belgian coast this already reduced our options significantly (Doody, 2008). Most dunes that are still more or less intact have to be closely managed in order to maintain dune dynamics (Provoost et al., 2011a; Herrier & Killemaes, 1998; Crevits, s.d.). Eventually three nature reserves were selected where sand is still sufficiently mobile to maintain the specific character of the dune landscape. These nature reserves were situated along the Belgian coast with the third one being just across the border with the Netherlands. These three sites are included in the Natura 2000 network and have been designated as Sites of Community Importance (figure 2; Herrier & Van Nieuwenhuyse, 2005). One of the sites chosen was located in nature reserve Westhoek in De Panne, Belgium, near the French border. It is the oldest nature reserve in Flanders. Mobile sand masses are still present in this area to a large extent, although they are significantly declining over the years (Provoost et al., 2011a). The nature reserve is accessible to the broader public, be it only on fenced hiking trails. Another nature reserve that still contains mobile sand dunes is Ter Yde in Oostduinkerke, Belgium (Bonte & Provoost, 2005). Here, public access should also be restricted to hiking trails, but because of the fences that are occasionally overblown by sand, people sometimes do not know that they stray from the paths (own observation, 2012). The last location was situated just across the border with the Netherlands in Retranchement and is part of nature reserve Zwin of which the biggest part is present at the Belgian side of the border. Our study location in this area was fully accessible for the broader public. All three nature reserves are managed by the Agentschap voor Natuur en Bos (ANB) or the Forest and Nature Agency which is an agency of the Flemish government.
Study design: In July 2012, a field study was conducted on the ecological preferences of S. rufula in nature reserves Westhoek and Ter Yde in Belgium and the part of nature reserve Zwin in the Netherlands (figure 3). In each of the three reserves, twenty individuals of the dune grass species A. arenaria were marked with little numbered flags and GPS-coordinates were noted in order to easily find back each individual plant in the weeks that followed (figure 3e). The relative occurrence of bare soil and plant species present within a radius of one meter around the marked A. arenaria individual and plant characteristics of this individual were determined once at the beginning of the study. These plant characteristics include the diameter at bottom and top of the plant, the length of the longest leave and the amount of leaves. Population densities of S. rufula were determined on a weekly base for a period of four weeks.
9
Fig. 2: Situation map of sites of community importance SCI’s on land (1=De Panne, 2=Koksijde, 3=Nieuwpoort, 4=Middelkerke, 5=Oostende, 6=Bredene, 7=De Haan, 8=Zuienkerke, 9=Blankenberge, 10=Zeebrugge, 11= Knokke- Heist) (from Herrier and Van Nieuwenhuyse, 2005). Study sites from the field survey are indicated with arrows.
Fig. 3: a. Nature reserve Westhoek in De Panne, Belgium; b. Nature reserve Ter Yde in Oostduinkerke, Belgium; c. Study location at nature reserve Zwin in Retranchement, the Netherlands; d. Schizaphis rufula Walker 1949 (Hemiptera: Aphidoidea); e. One of the numbered flags is seen on the right side of the picture, standing between A. arenaria plants; f. several S. rufula aphids on a leave of A. arenaria.
10 b. Plant suitability experiment
Preparations: To complement the results from the field survey, where focus was only on host plant A. arenaria, a plant suitability experiment was performed. This kind of experiment has as a main aim to decouple environmental influences from host plant – aphid interactions and purely focus on the suitability of the plant species to maintain populations of S. rufula. Both dune species and grass species from other habitats were tested. Seeds of the embryonic shifting dune species E. farctus as well as the yellow dune species A. arenaria and representatives of more fixated dunes like C. arenaria and F. rubra and a species of coastal tall grasslands and dune scrubs C. epigejos were collected in nature reserve Westhoek in De Panne, Belgium. Seeds of the mobile dune species L. arenarius originated from nature reserve Zwin in Retranchement, the Netherlands. Seeds from non- dune species were obtained from the Dutch company Cruydt-hoeck, which is a distributer of wild plant seeds. The species Deschampsia cespitosa was used as a representative of moist, nutrient rich grassland, Lolium perenne mostly situated on moist, fertilized or highly treaded soils and Poa pratensis on nutrient rich, mostly treaded soils, on walls and between pavers. Also Deschampsia flexuosa was used as a representative of heathland habitat which more closely resembles a dune system. All seeds were first subjected to a surface sterilization treatment. This involves a procedure of washing the seeds in 30 % EtOH for 2 minutes, than in a 10 % bleach solution for 5 minutes and finally with a large amount of sterile dH2O. After this treatment, seeds were kept overnight in a sufficient amount of distilled water to soak of any remaining bleach residues before they were plated onto petri-dishes containing a 1% agar growth medium. These petri-dishes were then put before a window, subjecting them to the prevailing night/day rhythm during the month of October. When seeds germinated, seedlings were planted in polypropylene cups containing sand collected from nature reserve Westhoek, De Panne. Because all plants were raised in similar Westhoek soils a potential variation in soil biota was eliminated. For five weeks these were maintained under fluorescent lamps with a summer night:day regime of 8:16 hours with day temperatures reaching 22°C . By our own observations in the field and also supported by results published by Pettersson (1971c) based on a survey conducted by Kennedy (1958), five to six weeks is a plant age that allows optimal feeding by the aphids. This is linked to a higher intensity of amino acid transport at this stage in the plant’s life. Schizaphis rufula individuals taken from nature reserve Westhoek in De Panne, Belgium, were further reared in the lab on A. arenaria plants under the same conditions as the re- potted plants.
Fig. 4: study design of the plant suitability experiment. On each experimental plant individual, a single adult S. rufula individual from our own aphid culture was placed. To prevent aphids from escaping, plastic long cocktail glasses that were punctured at the bottom were placed upside down over each plant individual.
11 Experiment: Of each species (dune species E. farctus, A. arenaria, C. arenaria, F. rubra and C. epigejos as well as non-dune species D. flexuosa, D. cespitosa, L. perenne and P. pratensis) twenty individuals were randomly chosen from all successfully lab grown plants. Ten were assigned to being a control plant and ten would undergo the experiment. Because of the low amount of successfully grown L. arenarius individuals, only 7 replicas of this species could be put under experiment and no plants were available to set up a control. All plants were put under fluorescent lamps which were connected to a timer that kept a summer night:day rhythm of 8:16 hours. On each experimental plant individual, a single adult S. rufula individual from our own aphid culture was placed. To prevent aphids from escaping, plastic long cocktail glasses that were punctured at the bottom were placed upside down over each plant individual (figure 4). No aphids were put on the control plants. First counts of the amount of aphids per plant replica were performed two days after manually adding aphids to the experimental plants. From then on every two days a counting took place for a period of four weeks. On a regular basis, plants with and without aphids were randomly repositioned under the fluorescent lamps. After the four week period, the experiment was terminated and each plant was weighed. Also dry weight was determined after putting all plants for 24 hours in an oven set to 60°C.
c. Identification of parasitoids
Morphological identification: Within the field survey and during extra field trips in August of the same year and from July up until August in 2013 parasitized aphids were collected from A. arenaria as well as F. rubra and C. arenaria. Most parasitoid wasps (Hymenoptera) were found in nature reserve Westhoek in De Panne, Belgium, with only sporadic observations in nature reserves Ter Yde in Oostduinkerke, Belgium, and Zwin in Retranchement, the Netherlands. Parasitized aphids, called mummies (see figure 5), were taken to the lab in eppendorf tubes together with part of the attached plant. These eppendorf tubes were put in a quiet environment in front of a window in order to minimize disturbance and to maintain a natural night/day rhythm. Between two to five days after collection, the parasitoid wasps emerged. They were preserved in eppendorf tubes containing ethanol with a purity of 99,9%. A first identification was performed using morphological and morphometric characteristics of the wasps. To examine the organisms an Olympus stereomicroscope system SZx7 with the magnification being 57x, was used at the Terrestrial Ecology Unit of Ghent University. Measurements of the specimens were taken with an ocular micrometer and with ImageJ version 1.47 (Wayne Rasband, National Institutes of Health, USA) when appropriate photo’s were available. These pictures were taken using a digital camera attached to the stereomicroscope with a trinocular tube. More detailed images were made using a Hitachi Tabletop Scanning Electron Microscope TM-1000 located at the VIB Department of Plant Systems Biology (UGent) in Zwijnaarde, Belgium. For the identification, keys were used from Goulet and Huber (1993), Tomanović et al. (2012), Kavallieratos et al. (2013), Gibson et al. (1997) and also the sites of Encyclop’APHID (http://www4.inra.fr/encyclopedie-pucerons), BugGuide.Net and Phylogenetics of Ceraphronoidea (http://ceb.csit.fsu.edu/ronquistlab/PCCP/ceraphronoidea/index .html ) were consulted.
12
Fig. 5: an aphid mummy with the parasitoid larvae still inside (darker part).
Molecular identification: DNA barcoding was conducted to support the morphological identifications. First the DNA was extracted from each individual wasp using the NucleoSpin®Tissue (Macherey-Nagel) kit and following the support protocol for purification of genomic DNA from insects (January 2010/Rev. 11). DNA amplification and further analysis was performed based on the 16S ribosomal RNA mitochondrial gene and the long wavelength rhodopsin (LWRh) nuclear gene. There are two ways of amplifying these DNA regions differing only in the identity of the reverse primer. The first method is focused on amplifying DNA from adult wasps that have already emerged out of their aphid host. Primers used are 16S-F (forward) 5’-CGCCGTTTT ATCAAAAACATG T-3’, developed by Simon et al. (1994), and 16S-R (reverse) 5’-TTACGCTGTTATCCCTAA-3’ by Kambhampati & Smith (1995) for the amplification of the 16S gene. The primers LWRh-F (forward) 5’- AATTGCTATTAYGARCANTGGGT-3’ and LWRh-R (reverse) 5’-ATATGGAGTCCANGCCATRAACCA-3’, both developed by Mardulyn & Cameron (1999) were used for the amplification of the LWRh region. The second method aims on amplifying the DNA of immature parasitoids still inside their aphid host (Derocles et al., 2012). Forward primers used here were identical to the ones from the first method. The reverse primers, however, were developed by Derocles et al. (2012) and are specific to the Aphidiinae subfamily. For the amplification of the 16S region 16S-Rspe (reverse) 5’- TCTAWAGGGTCTTCTCGTCT-3’ was used and for LWRh LWRh-Rspe (reverse) 5’- GATGCAACATTCATTTTTTTAGCTTG-3’. PCR amplifications were carried out as described in Derocles et al. (2012) using a 25 µL reaction volume which contains 18.5 µL of H2O, 2.5 µL of buffer (final concentration, 1x), 1 µL of MgCl2 (final concentration, 1 mM), 0.5 µL of dNTPs (final concentration, 0.2 mM), 0.175 µL of each primer (final concentration, 0.07 µM), 0.125 µL of Taq (final concentration, 0.63 U/µL) and 2 µL of extracted DNA. PCR amplification conditions for both genes were also as described by Derocles et al. (2012): 94 °C for 180 s; 40 cycles of 94 °C for 30 s, 56 °C for 60 s, 72 °C for 90 s, with a final elongation of 72 °C for 10 min. A volume of 3 µL of PCR product was run through a 3.6% agarose gel through gel electrophoresis (100V, 35min) and afterwards stained using the fluorescent nucleic acid stain GelRedTM. The eventual product was visualized using the Gel DocTM 2000 from BIO-RAD Laboratories, Inc. (Hercules, CA, U.S.) and the Quantity One® software package. Before performing any sequencing, PCR products were purified with Exonucleas I + fastAPTM (Fermentas Molecular Biology Products, Thermo Fisher Scientific Inc., U.S.) and send to Macrogen Inc., Europe. Service performed was Macrogen’s EZ-seq service. Sequences were edited using BioEdit
13 version 7.2.5 (Hall, 1999). Specimen identification was done by performing a standard nucleotide BLAST of the sequences on the website of the National Center for Biotechnological Information (NCBI) (National Library of Medicine, U.S.; http://blast.ncbi.nlm.nih.gov/Blast.cgi?). Felsenstein- Tajima-Nei distances were calculated between our specimens and specimens from GENBANK to resolve specimen identities that stayed unclear after running the BLAST procedure.
2. Statistical analysis
All Statistical analyses were conducted using the statistical package SAS® version 9.4 (SAS Institute Inc., Cary, NC, U.S.). Models were built based on sequential removal of variables with non-significant Wald statistics (SAS Institute Inc., 2014). To avoid putting covariant independent variables in the models a correlation matrix was made which showed the Pearson correlation coefficients between all variables in our dataset (see table 1 in Appendix).
a. Ecological preferences
From the plant community data the biodiversity index “richness” (R) was calculated for the vegetation in a one meter radius from each studied plant individual to be integrated with all other variables in statistical analyses. Intrinsic plant characteristics and surrounding plant community composition (incl. richness R) were analyzed separately. Generalized linear mixed models, GLMM’s, were used to identify independent variables that influence the probability of aphid encounter and population dynamics of S. rufula on its A. arenaria host plant. Location (Westhoek, Ter Yde and Zwin), plant individual and date were included in the models as random effects. Probability of aphid encounter: An extra variable was added to our dataset indicating the presence or absence of S. rufula with the values one and zero, respectively. This was implemented in further analyses as being binomially distributed. Aphid population dynamics: Also population dynamics on plants where S. rufula was present was considered. In doing so, plant individuals that had no aphids present on their leaves were left out. The eventual data was Poisson distributed and was as such implemented in the analysis. The relative amount of aphids was calculated as being the amount of aphids divided by the amount of leaves the host plant individual had. This was also implemented in models as being Poisson distributed.
b. Plant suitability experiment
A plant individual was regarded as accepted by S. rufula when minimally one aphid prevailed up until the second counting date, that is for four days. Persistence was defined as the amount of days that minimally one aphid individual could still be seen alive. This was not necessarily on the plant individual itself, but could also be on the soil surface or the side of the cup. To determine if the plant species has a significant impact on the rate of acceptance by S. rufula, the longitude of persistence or the amount of aphids present on the plant a GLMM was again used with the dates on which aphids were counted put in as a random effect. A Tukey test was performed in order to pairwise compare plant species means of the amount of aphids present on each plant on a given date. To see whether or not S. rufula had an impact on plant fitness, biomass and dry weight of plants with and without aphids, measured at the end of the experiment, was analyzed using a General Linear Model or GLM. Here, the maximum amount of S. rufula on the host plant was put in the model as a random effect.
14 Results 1. Ecological preferences
a. Probability of aphid encounter
It was found that the chance of encountering S. rufula on its host plant A. arenaria is influenced by certain biotic environmental factors. The presence of aphids on A. arenaria is negatively correlated with the percentage of F. rubra situated within a one meter radius from the A. arenaria host plant
(F1,180 = 11.89, P = 0.0007) and also with plant species richness R within that same area (F1,180 = 21.08, P < 0.0001; figure 6). These two independent variables are mutually covariant and were therefore analyzed separately in SAS® version 9.4.
90
(%) 80 Trendline 70 Datapoint
60 rufula
. . Trendline formula: 50 S Y = 83.77 - 15.85X 40 30 p < 0.05 20 R² = 0.85 10 Presence of Presence 0 0 1 2 3 4 5 6 7 Plant species richness
100
(%) 90 Trendline formula 80 y = 201.18e-1.638x 70
rufula 60
50 p < 0.05 S. 40 R² = 0.83 30 20 10 0 Presence 0 1 2 3 4 5 6 7 Festuca rubra (%) less more Dune fixation
Fig. 6: Both graphs show the trend in chance of finding S. rufula on A. arenaria along gradients in variables that indicate dune fixation, namely plant species richness (above) and percentage plant cover of F. rubra within a one meter radius (below). The trendlineformula, the goodness of fit to this trendline(R²) and the p-valueare indicated. Regarding intrinsic plant characteristics of the A. arenaria host plant it is found that the more leaves the host plant had the more chance there was to encounter S. rufula feeding on the plant (F1,179 = 5.16, P = 0.0243). However, the estimate for the fixed effect “amount of leaves” was relatively close to zero (0.01096) and as seen in figure 7, that shows a box-whisker plot of the variation in plant sizes for plants that carried (1) as well as lacked (0) the aphid on their leaves, variability in both groups is rather high. These results should therefore be regarded with care. Further elucidation on this subject can be found in the section “Discussion”.
15 p < 0.05 Fig. 7: Box plot that shows the variation in . plant sizes (measured in amount of leaves/A.
ind arenaria indivdual) within the group of plants on which S. rufula was absent (0) as well as plants on which the aphid was present (1). The
arenaria length of the box represents the interquartile
range (the distance between the 25th and 75th /A. percentiles). The diamond shape in the box interior represents the group mean. The horizontal line in the box interior represents of of leaves the group median. The whiskers issuing from
No. No. the box extend to the group minimum and maximum values. Group means differ significantly, but variation is equally wide spread. Presence of S. rufula 90 Embryonic dunes Yellow dunes Dune scrub Other environments
80 E. farctus .)
A. arenaria ind
( 70 L. arenarius C. arenaria 60
rufula F. rubra
S. S. 50 C. epigejos D. cespitosa 40 D. flexuosa
density L. perenne 30 P. pratensis
20 Population 10
0
Fig. 8: The variation in population densities of S. rufula is shown during the four week duration of the plant suitability experiment for each plant species that was tested. Color codes are as indicated in figure 1 and summarized in the upper right corner. Error bars represent standard error (SE) around each plant mean on a given date.
Embryonic dunes Yellow dunes Dune scrub Other environments
35 b
) b
30 days ( 25 a a 20 d d,e 15 e e
Persistence f 10 c 5
0
Fig. 9: The mean persistence is shown (amount of days that minimally one aphid persisted on the plant) for every plant species. Which habitat each species represents is indicated through the color of the bars. Letters situated above each bar illustrate the outcomes of a tukey-test for comparing mean persistence between plant species. When bars are indicated with the same letter, they showed no significantdifference in aphid persistence time. Error bars represent standard error (SE) around each mean.
16 b. Aphid population dynamics
Aphid population densities were not influenced by both surrounding plant community as intrinsic plant characteristics when plants with no aphids on their leaves were left out of the analysis. However, when going from plants with a low amount to plants with a higher amount of leaves a slightly deteriorating trend was found in the relative amount of aphids (intercept = 0.1855; estimate
= - 0.02297; F1,93 = 9.39; P = 0.0029). Out of the sixty plant individuals that were studied there was one individual situated in nature reserve Zwin were there was an association of an ant species (Formicidae) with S. rufula in week four of the survey. The aphid population counted 68 individuals which was the biggest observed population at Zwin during the four week span with the second biggest comprising only 26 individuals. There thus seems to be a positive influence from this aphid-ant interaction. Although this is an interesting observation, in this study, we could not go further on the matter.
2. Plant suitability experiment
The amount of aphids present on a host plant at a given moment was dependent on the host plant species (F9, 1 = 951.41, P = 0.0252) with the highest amounts of aphids found on L. arenarius and A. arenaria (see figure 8). When taking these two species out of the analyses, significance is lost (F7,1 = 36.67, P = 0.1265). Also the persistence of the aphids on the plant individual is highly dependent on the plant species (F9,77 = 8.75, P < 0.0001). A longer persistence was seen on species A. arenaria and
L. arenarius, with no significant differences between the two (t77 = -1.03, P = 0.3065, see figure 9). When however comparing plant least squares means of these two with all other plant species, significant differences were seen (all P-values < 0.0002). These results illustrate a clear separation of A. arenaria and L. arenarius from all other plant species within our analysis. Considering the non- dune grasses, aphids on D. flexuosa persist significantly longer compared to all others and even compared to C. arenaria, F. rubra and C. epigejos. No significant differences were found between D. flexuosa and E. farctus (t77 = 1.27, P = 0.2066). When comparing the dry weight of the control plants with the dry weight of the grasses that underwent the experiment, only a significant decrease in biomass was seen for A. arenaria (F1,8 = 18.21; P = 0.0027, see figure 10). For all other plants, a negative impact from S. rufula could not be proven.
Embryonic dunes Yellow dunes Dune scrub control 0.05 experiment
) 0.04 * (g
0.03 weight
0.02 Dry
0.01
0 Elymus farctus Ammophila arenaria Festuca rubra Calamagrostis epigejos
Fig. 10: The graph shows the comparison between the means in dry weight of the control plants versus the plants under experiment per dune species (without L. arenarius, for which we had no control plants). The asterisk indicates a significant difference (p<0.05) within mean dry weight of the control versus the experimental plants. Significance could only be proven for yellow dune species A. arenaria. Color codes are according to figure 1 with blue shades representing an embryonic dune species, yellow shades a yellow dune species and green shades more fixated dune or dune scrub species.
17 3. Identification of parasitoids
Morphological identification: Parasitoids found on S. rufula belonged to the genus Aphidius, which is a genus within the family of Braconidae. Figure 11 shows the most important morphological characters that support our identification to genus level. A glossary can be found in the Appendix. Two Aphidius species were found, namely A. rhopalosiphi and A. avenae, which are shown in figure 12 along with their morphological and morphometrical characteristics. For the species of A. avenae two individuals showed a remarkable difference in the appearance of the anterolateral part of the petiole which was distinct from all other specimens belonging to this species and from what was described in literature. Besides the parasitoids, also hyperparasitoids of Aphidius sp. were found. Through morphological study, specimens could be identified to generic level. Three genera were found: Apoanagyrus sp. (Hymenoptera: Encyrtidae), Dendrocerus sp. (Hymenoptera: Megaspilidae) and Pachyneuron sp. (Hymenoptera: Pteromalidae). These genera could however not be verified through molecular analysis because the protocols used here for amplification of DNA did not support these taxa. The morphological characteristics that support the identifications are shown in figures 13, 14 and 15. The family of Pteromalidae, to which the genus Pachyneuron belongs, is not defined by any unique attribute or combination of attributes. Membership is largely determined by elimination (Goulet & Huber, 1993). Braconidae a. b.
cp C+Sc+R RS+M absent
lb
2m-cu absent
Aphidiinae d. tergum 2 & 3 sec. flexible e. c.
Aphidius sp.
See figure 11b. - brown mummy - pupation inside aphid
Fig. 11: (bottom of previous page) Characters that assign specimens extracted from S. rufula to the family of Braconidae are the concave apical margin of the clypeus (cp) with the anterior surface of the labrum (lb) concave and exposed (a.), the absence of the C cell caused by fusion of the C and R vein and the absence of the 2m-cu vein in the forewing (b.) and a greatly reduced hind wing venation (c.). Metasomal tergum 2 and 3 are secondarily flexible (d.), which is only the case in subfamily Aphidiinae (Goulet & Huber, 1993), a subfamily of parasitoids parasitizing specifically on aphids. Aphidiinae specimens gathered within this study all had normally developed wings, pupated within the aphids skin and caused the aphid to develop into a brown mummy (e.). Together with the absence of the RS + M vein (b.) this indicates that the specimens belong to the genus Aphidius (Tomanović et al., 2012).
18
Aphidius rhopalosiphi Aphidius avenae Haliday 1834 De-Stefani Perez 1902 a. Antenna 16-17 Dark body segments
b.
c. 50 µm Costate (broad ribs) Variation in petiole type
- Costulate (fine ribs) - Petiole 3.5-4 x as long as wide
100 µm e. stigma R1 shorter than stigma 100 µm narrow
500 µm
Fig. 12: Pictures of the three different morphologies of parasitoid wasps retrieved from S. rufula mummies, the second and third column being the same species, namely A. avenae, according to molecular research (see also table 1 and glossary in appendix). Most important characteristics are indicated in the figures. a. female parasitoids. b. head in lateral view. c. petiole or metasomal tergum 1 in lateral view. e. wing venation.
19 Apoanagyrus sp. Dendrocerus sp. a. a. 1 1 2
b. c. b. 4 c. d. 4 2 5
3 3
Fig. 13: Microscopic and scanning electron photographs of specimens Fig. 14: Microscopic and scanning electron photographs of specimens identified as identified as the hyperparasitoid Apoanagyrus sp. a. Female in dorsal Dendrocerus sp. a. Female in lateral view. b. Distal part of the mesotibia with basal part of view. b. Head and part of the thorax in ventrolateral view. c. Head in the mesotarsus. c. Head in dorsal view. d. Thorax in dorsolateral view. Features that anterior view. Features that supported our identification are indicated supported our identification are indicated with numbers and are as follows: (1) forewing with numbers and are as follows: (1) head and body are steel-black with large stigma, (2) mesotibia with two spurs, (3) antenna with nine flagellomeres, (4) with greenish lustre, (2) all segments of the funicle are longer than mesoscutum with three longitudinal grooves, (5) anterior margin of metasoma in dorsal broad, (3) mesoscutum and scutellum are dark, (4) forewings are view with neck-like constriction (see Appendix for a glossary). hyaline. There is also a reticulate structure on the frontovertex but this is not indicated in the figure (see Appendix for a glossary).
20
Pachyneuron sp. a. b. tg no1 1 3
c.
2
Fig. 15: Microscopic and scanning electron photographs of specimens identified as Pachyneuron sp. a. tibia and tarsi. b. head and thorax. c. female in lateral view. The family of Pteromalidae is not defined by any unique attribute or combination of attributes. Membership is largely determined by elimination (Goulet & Huber, 1993). Some important characteristics are indicated with numbers and are as follows: (1) tarsi with five tarsomeres, (2) stigma reduced, (3) ring-like flagellomeres and the pronotum (no1) is separated from the tegula (tg) (see Appendix for a glossary).
Molecular identification: DNA sequences could be retrieved from 21 specimens of which sequences of 15 specimens were appropriate to run in BLAST. From seven specimens, both the 16S and the LWRh region could be used. Table 2 (see Appendix) gives an overview of the specimens from which DNA could be successfully retrieved and amplified and shows the percent of similarity and Felsenstein-Tajima-Nei divergence distances with sequences of other specimens retrieved from Genbank. With these sequences the presence of A. rhopalosiphi and A. avenae within the studied ecosystem could be supported.
21
Discussion Knowing how dune biota function and interact with one another will aid in our understanding of the unique and dynamic dune system. Greater knowledge can contribute to better management. Within the light of Belgium’s very fragmented and damaged coastline and also the deteriorating trends on European scale, the importance of dune ecosystem research is stressed (Provoost et al., 2011a & 2011b). From the results stated above it can be concluded that we have succeeded in our aim to assign factors that impact population dynamics of a dune grass herbivore which is prominently present within European dune landscapes. These influential factors are tightly linked to dune landscape structure, elucidating the importance of taking this landscape structure into account when performing research or management in a coastal dune environment. Also parasitoids were found that were to our knowledge not yet known to be associated with S. rufula. This knowledge reveals a previously unknown trophic level within the network surrounding S. rufula and in the dune ecosystem in general. There is still much to be learned about parasitoid impact on S. rufula and in general, but here a first step has been taken. In what follows, results will be discussed in more detail.
1. General findings
It can be deduced from the outcomes of the field survey that S. rufula on A. arenaria host plants has a preference for a yellow dune habitat. When a dune becomes more species rich and thereby fixated, the presence of the aphid declines. This is the case for transitional dune habitats towards dune scrub and also tall grassland with main species C. epigejos (see figure 1.a). However, because in the field survey’s main focus lay on the host plant A. arenaria, certain dune habitats could not be investigated. This is because A. arenaria does not occur in such environments. Aphid population dynamics could therefore not be followed for habitats like moss dunes, embryonic dunes and their transitional zones. From the experiment it is however known that S. rufula prefers primary dune fixating grasses. It is thus expected that patterns seen for aphids on host plant A. arenaria, which is such a primary dune fixater, can be generalized and occurrence of the aphid in (transitional zones towards) moss dunes should be rather low. This is firstly because moss dunes are highly fixated dunes and secondly because primary dune fixating grasses do not usually occur in this kind of environment. A proper host plant for S. rufula is thus absent in such a moss dune habitat. For embryonic dunes, however, it would have been more interesting to investigate aphid population dynamics. This is especially so because of the occurrence of important host plant L. arenarius in both embryonic as yellow dunes. To compare aphid population dynamics between embryonic and yellow dune habitats another field survey could be done, but this time with main focus on host plant L. arenarius. Results from such a survey could be especially interesting for regions north of latitude 63°N where L. arenarius replaces A. arenaria as most important dune fixating grass. It is expected that in embryonic dunes the even more pronounced presence of the sea should have an impact on aphid population dynamics.
2. Ecological preferences: underlying mechanisms
Why S. rufula has a preference for yellow dune habitats is not yet clear. As stated in the introduction, such a dune habitat forms a very harsh and unpredictable habitat. Why should herbivores settle here instead of taking cover within nearby more fixated dune habitats? When considering the outcomes of the field study, a decrease can indeed be seen in the percentage of plants harboring S. rufula individuals when going from a mobile sand dune habitat, with the presence of A. arenaria, to fixated
22 dunes where more plant species can persist and F. rubra is more abundantly growing (see figure 6). Because population densities themselves do not differ significantly along gradients of plant species richness or amount of F. rubra in the proximity of the host plant, it seems that plants in more fixated parts of the dunes are not necessarily of a lesser food quality. When aphids manage to colonize a plant, they are equally able to develop relatively high population densities in different dune habitats and thus appear to be equally fit when comparing these different habitats. The observation that fewer plants are infested in more fixated regions could be due to the lower abundance of A. arenaria and a potentially higher isolation of individual plants. Schizahis rufula could have problems with locating A. arenaria host plants within fixated dunes where A. arenaria individuals are less aggregated. The effect that specialist insects are more likely to find, remain and reproduce on their hosts when these plants grow in dense patches in pure stands is called a resource concentration effect (Otway et al., 2005). This effect could prevent aphids from seeking cover on host plants in more fixated dunes. It can also be that other more divers and complex vegetation types harbor a greater community of natural enemies, putting aphids on host plants nearby more at risk (Straub et al., 2013; Altieri & Letourneau, 1982). This does not take away that the host plant’s intrinsic food quality could still have an impact on S. rufula. Indeed, no direct measurements for host fitness or host plant quality were at hand. Also, the decreasing trend in chance to find S. rufula on A. arenaria when going to more fixated dunes occurs parallel with the observation that A. arenaria exhibits strongly suppressed growth as sand accretion ceases, generally called “the Ammophila problem” (Marshall, 1965). Knowing the underlying mechanisms supporting the Ammophila problem could thus give us an indication of which factors prevent S. rufula of being abundantly present in more fixated dune habitats and thus influence dune ecosystem dynamics. Various possible solutions for the Ammophila problem have been proposed. These are based on both abiotic and biotic factors. Vandegehuchte et al. (2010) even stated that the underlying cause could be region specific, giving the Ammophila problem a more complex twist. On the one hand you have the biotic component of soil biota like nematodes, bacteria and fungi which can have either a mutualistic or antaganostic impact on plant growth. On the other hand there are still abiotic factors that seem to have an influence which can even suppress the effect of soil biota. This was for example the case in nature reserve Westhoek (Vandegehuchte et al., 2010). Which abiotic factor dominated plant growth in Westhoek soils could, however, not be stated. For nature reserve Ter Yde, the soil biota were the most important factor. Suppose these soil biota have an antagonistic relationship towards A. arenaria. Antagonist soil biota can be biomass reducing soil biota or biota that influence aboveground plant nutritional quality. When these features are not significantly altered, the soil organism can still trigger plant defense mechanisms. Herbivores feeding on plants that interact with antagonists mostly show a reduced performance (Vandegehuchte et al., 2010; Bezemer et al., 2005; Hanounik & Osborne, 1977; Bardgett et al. 1999; Wardle, 2002; Van Loon et al., 1998). The “escape hypothesis” (Van der Putten et al., 1988) states that in dynamic sand dunes the continuing supply of fresh sand triggers vertical growth of A. arenaria and allows it to escape harmful soil biota. Aphids could benefit greatly from this escape through better food quality. They don’t have the indirect negative impact of the antagonist soil biota anymore. This could explain the finding why S. rufula leaves the protection of a more dense vegetation cover in fixated dunes. Under fixated circumstances, the aphid’s food source is poor. It is thus better to move to mobile dunes to get a sufficient amount of nutrients to survive and reproduce. It can be stated that studying factors causing the Ammophila problem can give us a better view of how the aphid-plant interaction works.
23 When looking at the results of the plant suitability experiment it appears that mobile dune grasses A. arenaria and L. arenarius are the most suitable hosts for supporting S. rufula populations. The importance of this result should be stressed, because this is found independent of any other factor that could have had an influence on population dynamics of S. rufula in the field. Schizaphis rufula thus also shows greatest proliferation on plant species that are characteristic for this environment when external environmental influences like soil biota are controlled for. This suggests that these plants have evolved a similar characteristic that not only link them to a yellow dune or similar habitats, but also make them more attractive to S. rufula. The observation that of the non-dune species D. flexuosa does best considering the persistence of the aphids could be an extra indication for this. Results for persistence on D. flexuosa were comparable with the embryonic dune grass E. farctus. As stated above, D. flexuosa is found in heathland habitats that more closely resemble a dune environment with its dry, sandy but also acidic soils. This apparent impact of the environment on plant traits differs from the short term impact causing the Ammophila problem. It is not yet clear which plant characteristics cause the preference of S. rufula for representatives of sandy and dry environments. Either way, a remarkable feature of the aphid is that it has a relatively long rostrum which is rather unique within the genus Schizaphis (Heie, 1986). This long rostrum could be an adaptation to the dense covering of the leaves of host plant A. arenaria with trichomes. These hairlike structures make it easier for the plant to prevent dehydration under dry circumstances by retaining a thin layer of moist air on the leave’s surface. That is the reason why the Mediterranean subspecies A. arenaria ssp. arundinaceae has a higher density of trichomes on its leave surface then the Atlantic subspecies A. arenaria ssp. arenaria (de la Peña, 2011). Trichomes can take many different forms depending on their function for the plant. In the case of Galium aparine they are for example hooked to prevent stems from slipping from their support or in the carnivorous plant genus Drosera they secrete a sticky substance to catch insects (Weeda et al., 2003a; Weeda et al., 2003b). Studying differences in morphology of these trichomes for the plant species investigated in this master thesis could give more insight in what way S. rufula is linked to psammophytic plant species, i.e. species that grow on sandy soils.
It seems that both the field survey and the plant suitability experiment indicate independent from one another that the aphid has the greatest potential of influencing dune dynamics in yellow dune habitats. The result that D. flexuosa does better than other non-dune species could, however, also be a consequence of our study design. For all plants sandy soils were used from nature reserve Westhoek to rear them on. D. flexuosa could thus have been of a better quality as a feeding source in comparison with all other non-dune plants that are not linked to sandy soils, because it was the most adapted to these soils. Even in this case, however, the overall conclusion that S. rufula has the most potential of influencing dune dynamics in yellow dune habitats will still hold because the soil is evenly optimal for all dune grasses among which the same tendency could be seen.
24 3. Ecological preferences and their meaning for the functioning of a dune ecosystem
The observation that S. rufula was mostly found on A. arenaria when it stood in dynamic yellow dunes implies that the aphid has the greatest potential of impacting its surroundings in these kind of environments. From the results found in the plant suitability experiment it could also be concluded that primary fixating grasses supported the largest aphid populations. Interestingly, these grasses are also the ones that suffered the most when S. rufula populations were present on their leaves. Ammophila arenaria showed a significant decrease in dry weight when comparing the control with the experimental plants in the plant suitability experiment. There was no control for L. arenarius but the same results as for A. arenaria are expected because at the end of the experiment these plants had a clearly withered appearance similar to that observed with the A. arenaria individuals under study (own observation, 2013). This observation indicates that S. rufula has the potential of having a significant impact on important dune grasses which could result in an impact on the dune landscape as a whole. However, although these dune grasses seem to suffer the most from the presence of the aphid within the plant suitability experiment, these observations were not yet seen in the field. This must not come as a surprise because interactions in the field are much more complex compared to those artificially put up under lab conditions. Examples of additional factors that have to be taken into account are predation rate, physical stress by windblown sand and as previously stated the impact of below ground fauna on above ground herbivory. Considering for example the predation rate. Syrphid larvae and Coccinellidae have been seen predating on S. rufula, but it is expected that also other generalistic predators, like e.g. certain dune spiders (Araneae), occasionally feed on S. rufula. Similar with the parasitoid wasps, it is not known for certain what the impact is of these organisms on population dynamics of S. rufula. It was hypothesized that aphids on shoots of dune plants are more easily detected by these predators and thus more easily subjected to predation than aphids in more concealing tussocks of grass. This implies that the chance of finding aphids in the latter is higher. Although a greater chance was indeed found of encountering aphids on plants with more leaves during the field survey these results must be considered with care. As seen within the results from the field survey, the variation in plant sizes within the group of plants that contained aphids as well as the group of plants that did not contain aphids was very large and the ranges of both groups almost completely overlapped (see figure 7). There is thus no clear indication of an optimal host plant size. The result of a significant difference between the group means could have been the result of a bias in the amount of smaller versus larger plants. There were 42 plants monitored with an amount of leaves smaller than fifty versus 18 plants with an amount of leaves higher than fifty. The largest plant individual had 236 leaves. When plants with a large amount of leaves are underrepresented within the field survey, it could be that by chance alone the few bigger plants within the field survey had aphids present on their leaves. The relative amount of bigger plants with aphids encountered during the field survey could thus be much higher than is the case for the smaller plants while this is not necessarily true for the real situation. From this, the wrong conclusion could be drawn that the chance is higher of encountering aphids on bigger plants compared to smaller plants. Moreover, in some respects the results that show greater encounter rates on bigger plants are not as logical as for our “concealment from predators” hypothesis. Older and therefore also bigger plants tend to be a less appropriate food source for S. rufula. As stated by Pettersson (1971c) and Kennedy (1958) and supported by our own observations an optimal plant age for aphid feeding
25 is approximately five to six weeks. This is rather young for A. arenaria which is a perennial plant. Also, in a study by Hacker and Bertness (1995) it was shown that salt-marsh aphids were even more predated when present on larger host plants due to ladybird beetles landing on tall structures more often than on shorter structures. Another outcome from the field survey shows however that the bigger the host plant is, the lower the relative abundance of S. rufula becomes. This is more in line with what is expected from the studies by Pettersson (1971c), Kennedy (1958) and Hacker and Bertness (1995). When combining both outcomes from the field survey it is seen that although aphids are apparently more encountered on bigger plants, their populations stay relatively small compared to the (seemingly) larger availability of food resources. This seems to be contradictory but can be explained by stating that bigger plants have a bigger chance to be noticed by S. rufula when it is searching for a host plant. This is an application of the resource concentration effect (Otway et al., 2005). The aphid will, however, experience higher predation rates or lower nutritional values when feeding on these bigger plants. This could eventually result in the smaller relative amount of aphids present on these plants.
There is another remarkable difference when comparing results from the plant suitability experiment with field observations. Although aphid population growth during the experiment was rather limited on dune species other than L. arenarius and A. arenaria, there are numerous observations in the field of S. rufula feeding and reproducing on F. rubra and other species. Mummies were even encountered on C. arenaria. When sampling locations were visited earlier in the year (April 2014) it seemed that there were more aphids present on F. rubra than on A. arenaria. When looking at the outcomes of the plant suitability experiment most plant species could still maintain a small number of aphids for a certain period, with an average of 14.125, 14.8 and 15 days for the dune grasses F. rubra, E. farctus and C. epigejos respectively. This shows that aphids can still retrieve to some extent a sufficient amount of nutrients from these plants for their survival. According to the phenology of A. arenaria, April is the month in which shoot growth and seed germination is initiated (Huiskes, 1979). Because S. rufula prefers young shoots (Pettersson, 1971c) the aphid could be forced towards other dune grass species when shoots of A. arenaria are not yet available, that is, earlier in the year. These other dune grasses, like F. rubra, could thus function as a kind of reservoir from which the aphids can emerge when shoots of the more suitable host plants, L. arenarius and A. arenaria, become more abundant. For these plant species, however, no large impact on population dynamics is expected because aphids can probably not reach sufficiently high population densities for this.
Although no statistically supported conclusions could be drawn from the one observed interaction between S. rufula and a certain ant species (Formicidae), this is still an interesting sighting. Our observation is in concordance with that of Pettersson (1971a) who also reported occasional visits by ants to S. rufula populations. The aphid-ant interaction is a food-for-protection mutualism in which the ants receive food in the form of honeydew produced by the aphids and in return, they protect the aphids against predators and parasitoids (Styrsky & Eubanks, 2007). Besides this it is also shown that even in the absence of predators and parasitoids ants can still influence aphid life history traits such as live span, age of maturation and reproduction rate (Flatt & Weisser, 2000). Intuitively it can be expected that the host plant should thus suffer from this interaction through the positive effect ants have on aphid population densities. However, many studies show that plants actually benefit indirectly. In these studies, increased predation or harassment of other more damaging herbivores by hemipteran-tending ants resulted in decreased plant damage and/or increased plant growth and
26 reproduction (Styrsky & Eubanks, 2007). It is not expected that ants have a significant impact, be it positive or negative, on overall aphid or dune grass populations at the three locations studied in this master thesis because the interaction was encountered only once. It cannot be said, however, that these kind of interactions are not important for other areas where S. rufula is present.
The influence of aphids on the vegetation in a species poor, natural system such as a yellow dune habitat is an interesting topic. Besides giving important insights in the functioning of a dynamic coastal dune landscape it can also be used outside the context of this coastal ecosystem. Despite yellow dunes being a natural system, its simplicity in vegetation structure with a dominant presence of A. arenaria, for example resembles that of an agricultural monoculture. Investigating aphid dynamics in yellow dune habitats thus can give a view on fundamental differences between agricultural and natural systems in general. Indeed, when the natural and agricultural ecosystem is more similar, fundamental differences can more easily be distinguished. These differences could be the key to knowing why aphids can develop into pest species in an agricultural system, while populations are more balanced in natural ecosystems. Although agricultural systems were not the main focus in this study certain differences between natural and agricultural systems can already be shown. It was for example seen that aphids were less present on their host plant A. arenaria when it stood in a species rich environment. Various possible underlying mechanisms were put forward. Depending on which proposed mechanism is the true one, our results may or may not be used in aphid pest management within an agricultural system. When the escape hypothesis is true and aphids largely thrive in dynamic systems where plants are not negatively impacted by soil biota (Van der Putten et al., 1988) no applications for agricultural systems can be found because these systems do not harbor such dynamics. However, when the hypothesis holds that vegetatively richer environments harbor a more diverse community of natural enemies it could be opted that pest species are easier to control when agricultural fields are located near natural, more biodiverse areas. The importance of habitat complexity for pest control was already pointed out by several studies such as the one performed by Chaplin-Kramer and Kremen (2012), Müller & Godfray (1999) and Altieri and Letourneau (1982).
4. Parasitoids: the importance of finding new associations
Parasitoids were found on S. rufula that were to our knowledge not yet known to be associated with this aphid. This observation clarifies another link within the ecological dune network and opens new doors for ecological research in European dune landscapes in general. It is indeed so that Aphidius rhopalosiphi and A. avenae are both very widespread and found in countries across Europe (Nieto Nafría, 2007). This makes that results from this study apply to a geographically wide range of dune landscapes. Interestingly, A. rhopalosiphi and A. avenae are not usually linked to dune ecosystems. Aphidius rhopalosiphi is known from grass infesting aphids like Diuraphis noxia, Rhopalosiphum padi and Sitobion avenae in agricultural systems (Turpeau et al., 2011b). This could however be a reflection of the fact that parasitoids are mainly studied in agricultural systems due to their economical importance. Actual habitat range could be much wider. Because species like S. avenae can also develop on rushes (Juncaceae) and sedges (Cyperaceae), it is also expected that natural habitats containing these plant species have potential of harboring A. rhopalosiphi populations (Blackman & Eastop, 2006). Aphidius avenae also parasitizes on S. avenae, making it already known that distributions of both parasitoid species show overlap (Turpeau et al., 2011a). Aphidius avenae is
27 however also seen to associate with aphid species belonging to the genus Dysaphis sp. which has as primary hosts apple and pear species (Rosaceae: Pyrus sp. & Malus sp. respectively) (Turpeau et al., 2010b). This makes its range of suitable habitats very divers. It can thus be stated that both species are widely distributed which makes that these species are most likely also simultaneously associated with S. rufula beyond Belgian borders. Although very widespread, parasitoids were found only occasionally in Ter Yde and Zwin. This could be due to the fact that these nature reserves are more accessible for tourists, while in the Westhoek accessibility is restricted to a few hiking trails. This is interesting because this means that there could be an indication of habitat deterioration within the way aphids and parasitoids interact in a dune ecosystem. Jane Memmott (2009) is a fierce proponent of using species interactions and ecological networks in general as a habitat quality assessment tool. Next to more traditional interactions like predator-prey and pollinator-plant interactions, she also elucidated on parasitoid-host interactions. Parasitoids are remarkably common in food webs (Lafferty et al., 2006) and their loss could have a profound effect on community structure and function (Lafferty et al., 2008). This, linked to the wide distribution both host species S. rufula and parasitoids A. rhopalosiphi and A. avenae have, gives them the potential of being used as a standard for comparing the quality of similar dune habitats across a wide geographical range.
When coming back on the Ammophila problem, factors influencing A. arenaria and indirectly S. rufula may also influence parasitoids through their interactions with these aphids (Harvey et al., 2003; Godfray, 1994). Bezemer et al. (2005) found that parasitoid mortality and the proportion of males were significantly lower when nematode and/or microorganisms communities were introduced to the soil and in a study by Masters et al. (2001) it was found that root feeding insects can increase the parasitism rate of seed feeding insects. Other studies with varying identities of soil biota and the organisms they influence can be found on this subject where some proved to have found an impact (Gange et al., 2003) and others not (Wurst & Jones, 2003). This indicates that observed patterns are highly species dependent (Bezemer et al., 2005). Studying gradients within parasitism rate or efficiency within a dune ecosystem has potential to give some interesting results when linking them to gradients within S. rufula population dynamics and vegetation composition. Schizaphis rufula is more found in yellow dune habitats, but are they also more often parasitized in this environment? In other words do the aphids in these environments have a different impact on parasitoid fitness and population densities than in more fixated areas? It has already been shown that bottom-up control is important for parasitoid food webs (Petermann et al., 2010; Bukovinszky et al., 2008; Hawkins, 1992). Host fitness can for example influence parasitoid oögenesis (Cicero et al., 2012) or the rate of encapsulation of the parasitoid egg inside the host which is a defense mechanism against parasitism (Klemola et al., 2008). It would be interesting to see in what way plant fitness impacted by soil biota or abiotic factors influences higher trophic levels. This would give us a more complete image of how S. rufula interacts with and influences its environment.
When looking at the aphid-parasitoid relationship in reverse way, that is, when considering the fact that a new aphid host species was found for A. rhopalosiphi and A. avenae, another interesting aspect of our study results is shown. New host discovery is important for assessing the effectiveness of biological control by parasitoids in agricultural systems. Highly specialized predators and parasitoids usually have the largest impact on herbivores compared to generalists (Müller & Godfray, 1999). However, the presence of other parasitoids and hyperparasitoids of the targeted pest species,
28 be it from natural populations or multiple introductions, can affect the effectiveness of an introduced biocontrol agent (Mills, 2002). Knowing parasitoid ecology and the complete host range aids in finding the optimal (combination of) parasitoid species for a certain agricultural system.
5. Overall conclusions
It is concluded that studying the dune aphid S. rufula and its interactions with both lower trophic levels (the dune grasses such as A. arenaria and L. arenarius) as higher trophic levels (parasitoids) resulted in a wider knowledge of species interactions and composition within a dune landscape. This knowledge has the potential of being used in various study domains such as dune management and pest control. Knowing that what has already been described regarding dune community composition could only be the tip of the dune biodiversity iceberg (Bonte & Provoost, 2004), still many interesting and useful facts about dune community structure are to be discovered.
Summary The ecology of a species and ecological interactions with other species tells us much about its habitat and habitat functioning. This knowledge can help us in managing certain ecosystems. European dune landscapes belong to the ecosystems that can benefit from this knowledge. Still, little is known about dune biota and their interactions. Dune biota are highly influenced by their environment because of the very specific and harsh conditions that prevail in dune habitats. This makes them more vulnerable to habitat changes. In the light of the recent deteriorations of European sand dune landscapes, the urge of protecting and managing these systems is particularly clear.
Schizaphis rufula is an aphid which feeds on important dune grasses like Leymus arenarius and Ammophila arenaria and is abundantly present in European dune landscapes. Although this species has potential to significantly impact the dune ecosystem through its influence on primary dune fixating grasses, little is known about its ecology and natural enemies. In previous studies, parasitized aphids called mummies were found. The identity of these parasitoids was however not known. This master thesis has as its main aims to determine which biotic factors impact population dynamics of the dune aphid S. rufula and under which conditions this aphid’s populations do best. More specifically we tried to determine 1) the influence of the surrounding plant community on population dynamics of S. rufula associated with A. arenaria in the field, 2) the specificity of the aphid’s host choice within and between plant species and 3) the parasitoid community associated with S. rufula. By doing so a better view can be made on what impact the aphid has on its environment.
Two different research strategies were used. First of all a more observational strategy was conducted during the course of a field survey in dune complexes of nature reserves the Westhoek, Ter Yde and Zwin. Secondly, field data was complemented with an experimental setup during a host suitability experiment. In the field survey aphid population dynamics on A. arenaria were studied. It was seen that the presence of aphids on A. arenaria is significantly negatively influenced by the percentage of F. rubra situated within a one meter radius from the A. arenaria host plant and also by plant species richness R within that same area. These two factors covary along a dune fixation gradient with a lower chance of finding S. rufula on A. arenaria when dunes become more fixated (amount of F. rubra and plant species richness is higher). What the underlying mechanism of this decline in encounter rate is, could
29 not be determined. It is however remarkably similar to the decline in growth of A. arenaria when going towards more fixated dunes. This is often called the Ammophila problem and the underlying cause is still largely debated. Both abiotic as biotic factors like soil biota have been suggested. Whatever the cause may be, it seems to also influence S. rufula population dynamics and places the highest potential of influence of S. rufula on dune dynamics in yellow and mobile dune habitats. Regarding intrinsic plant characteristics of the A. arenaria host plant it is found that the more leaves the host plant had the more chance there was to encounter S. rufula feeding on the plant. This observation must however be considered with care because of a bias towards a larger amount of smaller plants during the sampling for host plants. It was previously shown that aphids feed most optimally on younger plants and therefore smaller plants (Pettersson, 1971c). However, because it is only the presence of the aphid on the host plant that was significantly influenced and not the population density, this result could still be explained by the resource concentration effect (Otway et al., 2005) which states that specialist insects are more likely to find, remain and reproduce on their hosts when these plants grow in dense patches in pure stands. Bigger plants thus have a bigger chance to be noticed by S. rufula when it is searching for a host plant. The aphid will, however, experience difficulties when feeding on these bigger plants. This could eventually result in the smaller relative amount of aphids. In the plant suitability experiment it was found that aphid populations grew largest on the primary dune fixating grasses L. arenarius and A. arenaria. Also the persistence (amount of days that S. rufula was present on the plant individual) was longest. These species suffered the most from the presence of S. rufula. This result is interesting because it is found independent of any other factor that could have had an influence on population dynamics of S. rufula in the field. Consequently, both the field survey and the plant suitability experiment indicate independent from one another that the aphid has the potential of influencing dune dynamics. Combining the outcomes of the field survey and the plant suitability experiment and especially when focusing on the differences between the two can give a hint of which aspects of population dynamics are affected by plant species alone and what the effect is of additional environmental influences on these population dynamics in the field. Although primary dune fixating grasses seem to suffer the most from the presence of the aphid within the plant suitability experiment, these observations were not yet seen in the field. Consequently, there must be an additional factor in the field that limits proliferation of aphid populations. This additional factor can be predation, parasitism, physical stress by the overblowing sand, interactions with soil biota or a combination of these. The exact cause could however not be determined.
During the field survey, two Aphidius species could be identified, namely A. rhopalosiphi and A. avenae (Hymenoptera: Braconidae), and three hyperparasitoids, Apoanagyrus sp. (Hymenoptera: Encyrtidae), Dendrocerus sp. (Hymenoptera: Megaspilidae) and Pachyneuron sp (Hymenoptera: Pteromalidae). These species were to our knowledge not yet known to parasitize on the dune aphid S. rufula. The importance of finding new associations cannot be underestimated and the knowledge that comes from it can be applied in a wide array of research fields. Jane Memmott (2009) is a fierce proponent of using species interactions and ecological networks in general as a habitat quality assessment tool. Considering the fact that almost all parasitoids were found in nature reserve Westhoek, which is the most monitored and the least accessible to the broader public, it seems that a diverse parasitoid community associated with S. rufula indicates a
30 better quality habitat. Because both the aphid as its parasitoids are widespread in Europe, this gives a high potential of using this interaction for habitat quality assessment. Finding a new host species associated with A. rhopalosiphi and A. avenae could also benefit agriculture. These species are often used as biocontrol agents. Knowing parasitoid ecology and the complete host range aids in finding the optimal (combination of) parasitoid species for a certain agricultural system.
Studying the dune aphid S. rufula and its interactions with both lower trophic levels (the dune grasses such as A. arenaria and L. arenarius) as higher trophic levels (parasitoids) resulted in a wider knowledge of species interactions and composition within a dune landscape. Knowing that what has already been described regarding dune community composition could only be the tip of the dune biodiversity iceberg (Bonte & Provoost, 2004), still many interesting and useful facts about dune community structure are to be discovered.
Acknowledgements I want to sincerely thank Eduardo de la Peña who aided me in finding my own way of accomplishing this master thesis without withholding guidance and clear counseling. I also want to thank Viki Vandomme for getting me through my struggle with the PCR-machine and for staying positive when another week ended with the diagnosis “no bands”. I want to thank Maria Njo and Tom Beeckman from the VIB for giving me the opportunity of working with the electron microscope and Joachim Moens and Patrick De Clercq for providing keys and advice for the identification of the parasitoids. I also want to thank Floris Van Laere for editing the electron microscopy images, Wendy Vrydag and Steven Goossens for reviewing my thesis and fellow thesis buddies at TEREC Eline Vermote, Karen Bisschop, Jelle, Emily Veltjen, Margaux Boeraeve, Alexander Boffin, Stefan Vandamme, Rieneke Vanhulle, Willem Proesmans, Hannah Volckaert and Judith for their support and company.
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36 Appendix 1. Tables
Table 1: Matrix that shows the Pearson correlation coefficients for all pairwise comparisons between the variables from the field survey. For each pairwise comparison three values are given. The first value represents the Pearson correlation coefficient itself. The second value is the p-value and the third value is the number of observations. The first ten columns represent the independent variables. The variables “Diameter at bottom” to “No. of leaves” are intrinsic plant characteristics. The unit of all measurements is centimeter. The variables “Sand” to “Species richness (R)” represent the composition of the vegetation within a one meter radius. In the field each of these variables was measured in percent. The last three columns are the dependent variables. The number of S. rufula individuals and parasitoids were counted per plant. Parasitism rate is the number of parasitized aphids or mummies per plant devided by the total number of S. rufula individuals (including mummies) on that plant.
Pearson Correlation Coefficients Prob > |r| under H0: Rho=0 Number of Observations
Diameter at Diameter at Length of Species No. of No. of leaves Sand A. arenaria F. rubra E. farctus C. arenaria No. of S. rufula Parasitism rate bottom top longest leave richness (R) parasitoids Diameter at 1 0.8215 0.35492 0.70354 0.22604 -0.1968 0.06584 -0.05841 -0.01983 0.13617 0.10434 -0.05374 -0.0507 bottom <.0001 <.0001 <.0001 0.0005 0.0024 0.3139 0.3717 0.7618 0.0366 0.1099 0.4112 0.4382 236 236 236 236 236 236 236 236 236 236 236 236 236 Diameter at 0.8215 1 0.58741 0.80309 0.25783 -0.24804 -0.05274 0.05439 0.02029 0.04049 0.11732 -0.08293 -0.04845 top <.0001 <.0001 <.0001 <.0001 0.0001 0.416 0.4015 0.7544 0.5324 0.0696 0.2004 0.455 236 240 240 240 240 240 240 240 240 240 240 240 240 Length of 0.35492 0.58741 1 0.54562 0.09196 -0.02863 -0.18615 0.12319 0.15539 0.05038 0.1127 -0.02072 -0.03807 longest <.0001 <.0001 <.0001 0.1555 0.659 0.0038 0.0567 0.016 0.4373 0.0814 0.7495 0.5572 leave 236 240 240 240 240 240 240 240 240 240 240 240 240 No. of leaves 0.70354 0.80309 0.54562 1 0.20204 -0.17696 -0.12244 0.15915 0.03077 -0.04745 0.18089 -0.07328 -0.07183 <.0001 <.0001 <.0001 0.0017 0.006 0.0582 0.0136 0.6353 0.4644 0.0049 0.2581 0.2677 236 240 240 240 240 240 240 240 240 240 240 240 240 Sand 0.22604 0.25783 0.09196 0.20204 1 -0.60439 -0.33943 -0.09786 -0.18503 -0.23983 0.20283 0.11921 0.0538 0.0005 <.0001 0.1555 0.0017 <.0001 <.0001 0.1306 0.004 0.0002 0.0016 0.0652 0.4067 236 240 240 240 240 240 240 240 240 240 240 240 240 A. arenaria -0.1968 -0.24804 -0.02863 -0.17696 -0.60439 1 -0.04644 -0.1278 -0.14985 -0.3276 -0.06562 -0.0707 0.01999 0.0024 0.0001 0.659 0.006 <.0001 0.4739 0.048 0.0202 <.0001 0.3113 0.2753 0.758 236 240 240 240 240 240 240 240 240 240 240 240 240
37
F. rubra 0.06584 -0.05274 -0.18615 -0.12244 -0.33943 -0.04644 1 -0.17389 -0.03162 0.53895 -0.15852 -0.04498 -0.10331 0.3139 0.416 0.0038 0.0582 <.0001 0.4739 0.0069 0.6259 <.0001 0.014 0.488 0.1104 236 240 240 240 240 240 240 240 240 240 240 240 240 E. farctus -0.05841 0.05439 0.12319 0.15915 -0.09786 -0.1278 -0.17389 1 -0.06175 0.06733 0.02934 -0.02453 -0.01988 0.3717 0.4015 0.0567 0.0136 0.1306 0.048 0.0069 0.3408 0.2989 0.6511 0.7053 0.7593 236 240 240 240 240 240 240 240 240 240 240 240 240 C. arenaria -0.01983 0.02029 0.15539 0.03077 -0.18503 -0.14985 -0.03162 -0.06175 1 0.25135 -0.0685 -0.02452 -0.0359 0.7618 0.7544 0.016 0.6353 0.004 0.0202 0.6259 0.3408 <.0001 0.2906 0.7054 0.5799 236 240 240 240 240 240 240 240 240 240 240 240 240 Species 0.13617 0.04049 0.05038 -0.04745 -0.23983 -0.3276 0.53895 0.06733 0.25135 1 -0.2632 -0.06676 -0.11289 richness (R) 0.0366 0.5324 0.4373 0.4644 0.0002 <.0001 <.0001 0.2989 <.0001 <.0001 0.303 0.0809 236 240 240 240 240 240 240 240 240 240 240 240 240 No. of 0.10434 0.11732 0.1127 0.18089 0.20283 -0.06562 -0.15852 0.02934 -0.0685 -0.2632 1 0.65481 0.14432 S. rufula 0.1099 0.0696 0.0814 0.0049 0.0016 0.3113 0.014 0.6511 0.2906 <.0001 <.0001 0.0254 236 240 240 240 240 240 240 240 240 240 240 240 240 No. of -0.05374 -0.08293 -0.02072 -0.07328 0.11921 -0.0707 -0.04498 -0.02453 -0.02452 -0.06676 0.65481 1 0.48204 parasitoids 0.4112 0.2004 0.7495 0.2581 0.0652 0.2753 0.488 0.7053 0.7054 0.303 <.0001 <.0001 236 240 240 240 240 240 240 240 240 240 240 240 240 Parasitism -0.0507 -0.04845 -0.03807 -0.07183 0.0538 0.01999 -0.10331 -0.01988 -0.0359 -0.11289 0.14432 0.48204 1 rate 0.4382 0.455 0.5572 0.2677 0.4067 0.758 0.1104 0.7593 0.5799 0.0809 0.0254 <.0001 236 240 240 240 240 240 240 240 240 240 240 240 240
38 Table 2: This table shows a list of all parasitoid specimens belonging to the genus Aphidius (Hymenoptera: Braconidae) for which DNA has been successfully amplified and sequenced. The table shows the results of the morphological identification which was done with the key from Tomanović et al. (2012) and information from the website Encyclop’Aphid (http://www4.inra.fr/encyclopedie-pucerons). A unique specimen code was given to all specimens found during the study. The letters represent sampling site (WH= Westhoek, TY = Ter Yde, R= Retranchement) following two or three numbers are the date and the last number represents the number of the specimen found on that date. Primers that were used to amplify and sequence the DNA are given in the third column. –F indicates a forward primer and –R the reverse. When neither is indicated, both were of such a quality that a contig could be made. Sequence length is given in number of base pares (bp) and most plausible hits in BLAST are shown with the query cover, similarity and Felsenstein-Tajima-Nei distances.
Sequence Felsenstein- Specimen Query Morphological identification Primers length Hits indicated by BLAST Similarity Tajima-Nei code cover (bp) distance
A. avenae WH472 16S 415 A. avenae (JQ240492.1) 0.93 0.99 0.0078 WH472 LWRh 754 A. avenae (JN620702.1) 0.52 1 0 A. avenae WH985 16S-R 264 A. avenae (JQ240492.1) 0.94 0.99 0.00407 A. avenae WH1581 16S 419 A. avenae (JQ240492.1) 0.92 1 0 A. rhopalosiphi WH271 16S 416 A. rhopalosiphi (JQ240518.1) 0.93 1 0 WH271 LWRh 554 A. rhopalosiphi (JN620727.1) 0.58 0.99 0.00626 A. rhopalosiphi WH273 16S-R 195 A. rhopalosiphi (JQ240518.1) 0.9 0.93 0.01809 WH273 LWRh-F 406 A. rhopalosiphi (JN620727.1) 0.79 0.98 0.0095 A. rosae (JN620738.1) 0.96 0.98 0.01596 A. rhopalosiphi WH474 16S 416 A. rhopalosiphi (JQ240518.1) 0.93 1 0 WH371 LWRh-F 464 A. urticae (JN620747.1) 0.84 0.99 0.01071 A. rhopalosiphi or A. urticae A. sonchi (JN620745.1) 0.6 0.98 0.03639 A. rhopalosiphi (JN620727.1) 0.69 0.97 0.03269 A. rhopalosiphi or A. urticae WH473 16S-F 200 A. rhopalosiphi (JQ240518.1) 0.98 0.99 0.01016 A. uzbekistanicus (JQ240542.1) 0.98 0.99 0.01016 A. funebris (JQ240506.1) 0.98 0.99 0.01016 A. urticae (JQ240540.1) 0.98 0.98 0.02052 WH473 LWRh-F 329 A. urticae (JN620747.1) 0.98 0.98 0.01213 A. eadyi (JN620707.1) 0.98 0.98 0.01626 A. rhopalosiphi (JN620727.1) 0.77 0.95 0.03297 larvae WH47para1 16S 390 A. rhopalosiphi (JQ240518.1) 0.99 1 0 WH47para1 LWRh 563 A. rhopalosiphi (JN620727.1) 0.57 0.98 0.02535 A. urticae (JN620747.1) 0.67 0.98 2.54471 larvae WH47para2 16S 393 A. rhopalosiphi (JQ240518.1) 0.98 0.99 0 A. sonchi (JQ240538.1) 0.99 0.99 0.00287 A. ervi (AF174310.1) 0.99 0.99 0.00575 A. microlophii (JQ240514.1) 0.99 0.99 0.00575 A. funebris (JQ240506.1) 0.99 0.99 0.00575 A. matricariae (JQ240510.1) 0.99 0.99 0.00867 A. urticae (JQ240540.1) 0.98 0.99 0.00867 A. uzbekistanicus (JQ240542.1) 0.98 0.99 0.00865 WH47para2 LWRh 562 A. rhopalosiphi (JN620727.1) 0.57 0.97 0.0287 A. sonchi (JN620745.1) 0.5 0.97 0.03652 A. microlophii ( JN620725.1) 0.69 0.98 0.01258 A. urticae (JN620747.1) 0.69 0.98 0.01256 larvae WH475 LWRh 550 A. urticae (JN620747.1) 0.71 0.98 0.00955 A. rhopalosiphi (JN620727.1) 0.58 0.97 0.01278
39 larvae R774 16S-F 192 A. rhopalosiphi (JQ240518.1) 1 0.99 0 A. uzbekistanicus (JQ240542.1) 1 0.99 0 A. microlophii (JQ240514.1) 1 0.99 0 A. funebris (JQ240506.1) 1 0.99 0 A. ervi (JQ240500.1) 1 0.99 0 larvae R775 16S-F 191 A. rhopalosiphi (JQ240518.1) 1 0.99 0 A. uzbekistanicus (JQ240542.1) 1 0.99 0 A. microlophii (JQ240514.1) 1 0.99 0 A. funebris (JQ240506.1) 1 0.99 0 A. ervi (JQ240500.1) 1 0.99 0 R775 LWRh-F 466 A. urticae (JN620747.1) 0.84 0.98 0.01888 A. eadyi (JN620707.1) 0.84 0.98 0.0189 A. microlophii (JN620725.1) 0.84 0.98 0.01893 A. ervi (JN620710.1) 0.84 0.98 0.02214 A. uzbekistanicus (JN620749.1) 0.84 0.98 0.02865 A. funebris (JN620715.1) 0.84 0.98 0.02856 A. rhopalosiphi (JN620727.1) 0.69 0.98 0.02535 egg WH47_ei1 LWRh 531 A. urticae (JN620747.1) 0.72 0.98 0.02209 A. eadyi (JN620707.1) 0.72 0.98 0.02213 A. microlophii (JN620725.1) 0.72 0.98 0.02214 A. ervi (JN620710.1) 0.72 0.98 0.02538 A. rhopalosiphi (JN620727.1) 0.61 0.97 0.02862 undevelloped due to WH47onb1 LWRh 522 A. rhopalosiphi (JN620727.1) 0.61 0.99 0.00626 hyperparasitoid A. ervi (JN620710.1) 0.73 0.99 0.0094 A. rosae (JN620738.1) 0.73 0.99 0.01261
40 2. Glossary figures and definitions for terms used in section “Results” under “Identification of parasitoids”. The wing venation is illustrated according to that of Dolichurus sp. (Apocrita: Ampulicidae). All definitions and figures are taken from Goulet& Huber (1993).
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