DEPARTAMENTO DE CIÊNCIAS DA VIDA Pollinator Preference in A

2014 DEPARTAMENTO DE CIÊNCIAS DA VIDA

FACULDADE DE CIÊNCIAS E TECNOLOGIA

UNIVERSIDADE DE COIMBRA

2014 2014 DE PARTAMENTO DE CIÊNCIAS DA VIDA

FACULDADE DE CIÊNCIAS E TECNOLOGIA UNIVERSIDADE DE COIMBRA

Pollinator preference in a hybrid zone between

two generalist plant species.

Pollinator preference in a hybrid zone between

two generalist plant species.

Pollinator preferencein a hybrid zone between two generalist plant species. plant generalist two between zone hybrid a preferencein Pollinator

José Cerca de Oliveira

2014

José Cerca de Oliveira

José Cerca de Oliveira de Cerca José

2014 José Cerca de Oliveira de Cerca José

DEPARTAMENTO DE CIÊNCIAS DA VIDA

FACULDADE DE CIÊNCIAS E TECNOLOGIA UNIVERSIDADE DE COIMBRA

Pollinator preference in a hybrid zone between two generalist plant species.

Dissertação apresentada à Universidade de Coimbra para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Ecologia, realizada sob a orientação científica do Professor Doutor João Loureiro (Universidade de Coimbra), da Doutora Sílvia Castro (Universidade de Coimbra) e do Doutor Rubén Torices (Universidade de Coimbra).

José Cerca de Oliveira

2014

This study was supported by the CGL2010-18039 Project from Spanish Ministry of Science and Innovation.

Ao Nuno Lopes,

“De vez em quando a eternidade sai do teu interior e a contingência substitui-a com o seu pânico. São os amigos e conhecidos que vão desaparecendo e deixam um vazio irrespirável. Não é a sua ‘falta’ que falta, é o desmentido de que tu não morres.”

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Index

Abstract 1

Resumo 3

Introduction 5

Materials and Methods 9

Study species 10

Study site 11

Experimental Design 12

Statistical Analysis 14

Results 17

Floral visitors 18

How do the floral phenotype, floral display and neighbourhood context affect 18 pollinator attraction?

How does capitulum size affect pollinator attraction? 28 Is there a facilitation or competing pattern between rayed and rayless phenotypes? 28

Discussion 34

Conclusion 38

References 40

Appendix 45

Abstract

The evolutionary mechanism behind flowers and its pollinators is generally understood to be a gradual co-adaptive process where the plant specializes to its most efficient pollinator, which exerts selective pressures on specific traits, driving floral evolution. Still, most flowering plants in nature are visited by a wide array of pollinator species, i.e. are generalist plants. However, the role of pollinators as significant drivers of floral evolution in generalist plants has been questioned due to the potential conflicting selection regimes exerted by different pollinators. Taking this into account, using a combination of observation and manipulative experiments, we assessed pollinator preference in a natural contact zone where the generalist rayed species Anacyclus clavatus and the rayless A. valentinus co-exist and hybridize, forming intermediate phenotypes. These contact areas show a remarkably high phenotypic variation, with the intermediate phenotype bridging both phenotypes and forming an exceptional micro-evolutive framework to explore how generalist pollinators could be driving the evolution of floral phenotypes. We found that the production of rays influenced the probability of being visited by specific insect groups, in particular by Dipteran groups; whereas bees showed no preference for rayed phenotypes and their visitation patterns were mainly driven by the number of capitula simultaneously blooming in the plant. In addition, we found support for the importance of the neighbours’ phenotype when assessing pollinator preference on a focal individual. Rayed plants benefited from having other conspicuous neighbours, whereas rayless and intermediate phenotypes significantly competed for pollinators. In conclusion, all these differential behavioural patterns of floral visitors might affect gene flow within the hybrid zone between A. clavatus and A. valentinus influencing the degree of reproductive isolation and floral evolution between both species.

Keywords

Anacyclus; Asteraceae; Discoid capitulum; Neighbourhood context; Pollinator behaviour;

Rayed capitulum;

Resumo

Os mecanismos evolutivos que atuam nas flores e respetivos polinizadores são normalmente descritos como processos de co-adaptação gradual onde a planta se especializa no seu polinizador mais eficiente, que por sua vez, exerce pressões evolutivas em características específicas e dessa forma guia a evolução da flor. Ainda assim, a maior parte das plantas com flor são polinizadas por um leque diversificado de espécies de polinizadores, denominando-se assim plantas generalistas. No entanto, em plantas generalistas o papel dos polinizadores na evolução floral tem sido questionado devido a potenciais conflitos na selecção exercida pelas diferentes espécies de polinizadores que visitam a flor. Tendo isto em conta, abordagens observacionais e manipulativas foram utilizadas para avaliar as preferências dos polinizadores numa zona de contacto onde as espécies generalistas Anacyclus clavatus (com lígulas) e a espécie A. valentinus (sem lígulas) coexistem e hibridizam, formando fenótipos intermédios. Estas áreas possuem uma variação fenotípica notável, com o fenótipo intermédio a representar o cruzamento entre as duas espécies, garantindo um cenário microevolutivo excecional para estudar de que forma os polinizadores conduzem a evolução fenotípica em espécies generalistas. Os resultados obtidos revelaram que a produção de lígulas influenciou a probabilidade das plantas serem visitadas por grupos específicos de insetos, em particular por dípteros; por sua vez, as abelhas não revelaram preferências por um fenótipo em particular, preferindo maioritariamente plantas com um elevado número de capítulos em flor. Além disto, os nossos resultados evidenciaram também que a composição fenotípica da vizinhança poderá desempenhar um papel importante na atração de uma planta focal específica; em particular, plantas liguladas beneficiaram em ter outros vizinhos com lígulas, enquanto que os fenótipos sem lígulas e intermédios competiram significativamente por polinizadores. Os diferentes padrões de comportamento diferentes por parte dos visitantes florais observados neste estudo podem afetar o fluxo genético na zona híbrida entre A. clavatus e A. valentinus, influenciando o grau de isolamento reprodutivo e evolução floral entre as duas espécies.

Palavras-chave

Anacyclus; Asteraceae; Capítulo ligulado; Capítulo sem lígulas; Comportamento dos polinizadores; Contexto gerado pelos fenótipos vizinhos;

Introduction

Animal pollinated plants rely on pollinators for pollen removal and reception, depending strongly on the behavior and effectiveness of floral visitors for its reproductive success. Consequently, pollinators mediate floral evolution by exerting selective pressures on several floral traits (Wesselingh & Arnold 2000; Sánchez-Lafuente 2002; Campbell 2008; Gómez et al. 2008a; Parachnowitsch & Kessler 2010; Penet, Marion & Bonis 2012; van der Niet, Peakall & Johnson 2014). Most works focusing on flower evolution by pollinator pressures describe this evolutionary process as a gradual co-adaptive mechanism in which the plant evolves in response to its most efficient pollinator, assuming an unidirectional co- evolution that leads to the specialization for a specific pollinator or to a particular group of pollinators (Cope’s rule of specialization; Cope 1896; Johnson & Steiner 2000; Aigner 2003; Gómez et al. 2007, 2014; Vereecken et al. 2012; Van der Niet et al. 2014). However, in generalist plants, some authors doubt that pollinators can act as significant drivers of floral evolution (Waser 2001; Kay & Sargent 2009), mostly because these plants can be visited by a numerous and diverse assemblage of pollinators (Herrera 2005), and because different types of floral visitors have shown distinct trait preferences and attributes as pollinators (Thompson 2001; Sánchez-Lafuente 2002; Castro et al. 2013), thus imposing conflicting selection regimes (Sahli & Conner 2011). Despite of that, recent studies on generalist plant species have found that inter-population variation on pollinator faunas can still exert selection patterns on corolla shape (Sánchez-Lafuente 2002; Gómez et al. 2008b, 2014), suggesting that floral traits of generalist plants may have been also shaped by the selection driven by pollinators. Nevertheless, with a few exceptions (e.g. see Sánchez-Lafuente 2002; Vereecken et al. 2012; Gómez et al. 2014), little is known about how and which pollinator groups select floral traits in generalist plant species.

The largest family of flowering plants, the sunflower family (Asteraceae), is a known example of a highly diverse lineage composed mainly by species with a broad assemblage of pollinators (Lane 1996). Plants of this family are easily recognised by its particular inflorescence, the capitulum (Funk et al. 2009), which functions as a single flower and serves as the basic unit of the plant’s visual display to attract pollinators (Andersson 2001). Indeed, it is frequent that the main pollinators of a particular species are the most abundant floral visitors in that year or spatial area (Ollerton et al. 2007). However, some species with specialist pollination syndrome such as bird pollination are also known in this family (Lane 1996), including its oldest known fossil (Barreda et al. 2012).

Most of the research on pollinator interactions in Asteraceae has focused on understanding the ecological significance of the highly zygomorphic corolla produced by the flowers located on the outermost position of some capitula, i.e. ray florets in rayed capitula (Marshall & Abbott 1984; Stuessy et al. 1986; Celedón-Neghme, Gonzáles & Gianoli 2006; Nielsen, Siegismund & Hansen 2007; Andersson 2008). The presence of rays was shown to have significant consequences on pollination, primarily enhancing the attractiveness of capitula and consequently influencing the levels of outcrossing (Lack 1982; Marshall & Abbott 1984; Sun & Ganders 1990; Celedón-Neghme et al. 2006; Andersson 2008). Thus, petaloid rays seem to provide an advantage for attracting pollinators, although the pollination context, including pollinator’s abundance and floral display, can reduce this effect (Andersson 1996; Nielsen, Philipp & Siegismund 2002).

Introduction

Despite the observed advantage of rayed capitula in attracting more insects, rayless species are frequent in Asteraceae. The several independent reversals towards rayless capitula in the evolution of this family, suggest that rayless capitula could also be adaptive (Bremer & Humphries 1993; Torices, Méndez & Gómez 2011). The production of rays might entail a cost by reducing available resources for fruit and seed production (Andersson 1999, 2001, 2008; Celedón-Neghme et al. 2006) and/or by attracting more seed predators (Fenner et al. 2002). Furthermore, as capitula are usually visited by a large number of pollinators, rayed capitula could be visited by a larger amount of less efficient pollinators than rayless capitula, reducing the amounts of pollen donation. Still, whether specific functional groups of pollinators show different preferences to rayed versus rayless phenotypes remains poorly explored (but see Stuessy et al. 1986).

Hybrid zones represent natural laboratories to understand ecological and evolutionary processes of reproductive isolation and selection on phenotypic traits (Barton & Hewitt 1985; Aldridge & Campbell 2006; Campbell & Waser 2007). These areas present a striking profusion of flower morphologies, allowing a better evaluation of pollinator preferences under natural conditions. Pollinator-mediated selection requires phenotypic variation, and studies in plant hybrid zones have already provided strong evidences on pollinated-mediated selection of floral traits (Hodges & Arnold 1994; Campbell, Waser & Melendez-Ackerman 1997; Campbell 2003, 2008). So far these studies were performed in plants with contrasting pollination syndromes such as bird versus insect plant pollinated species (Aldridge & Campbell 2006) in which pollen transference between related taxa was prevented by large differences in floral morphology, leading to a strong reproductive isolation due to pollinator behaviour (Schemske & Bradshaw 1999; Emms & Arnold 2000). However, little is known about hybrid zones involving generalist plant species, whose pollinator faunas highly overlap reducing the expected ethological isolation, as well as, about the role that generalist pollinators may have as selective agents in floral evolution and ethological isolation.

Within the Circum-Mediterranean genus Anacyclus, along the Western Mediterranean basin the rayed species A. clavatus co-exists with the rayless A. valentinus (Humphries 1979, 1981). In the sympatric areas, a large phenotypic diversity in the number and size of ray florets has been observed revealing the existence of a dynamic hybrid zone (Bello et al. 2013). In addition, a preliminary survey of floral visitors indicated that both plant species were visited by a large array of insects, most of them shared between both plant species (R. Torices, unpublished data). Hence, this hybrid zone provides an exceptional micro-evolutionary framework to explore whether different pollinator groups can preferentially select rayed versus rayless phenotypes and to explore whether hybrid zones between generalist plant species can be influenced by the behaviour of their floral visitors. Using a combination of observational and manipulative experiments we assessed the preferences of floral visitors in a contact zone between the two generalist plant species, Anacyclus clavatus (rayed phenotype) and A. valentinus (rayless phenotype). We performed phenotypic manipulations, simulating the rayed phenotype on an exclusive rayless site, and the rayless phenotype on an exclusive rayed site. In addition, to assess potential facilitation or competition effects between rayed and rayless phenotypes, we explored the effect

Introduction

of the intra-specific surrounding context by both characterizing quantitatively and manipulativing the neighborhoods. Specifically, we aimed to determine: (i) how does the floral phenotype, floral display and surrounding context affect pollinator’s attraction; (ii) how does capitulum size affect pollinator’s attraction; and (iii) if there is an effect (facilitative or competitive) of any phenotype in the pollinator’s visitation rates.

Materials and Methods

Study species

The genus Anacyclus L. (Anthemideae, Asteraceae) is composed of about 12 species of weedy annual herbs found in dry and disturbed habitats throughout the Mediterranean basin (Humphries 1981). This genus shows an extraordinary variation in reproductive traits and sexual expression within their capitula and among species, suggesting different evolutionary trends and hybridization events between some of its recent species (Figure 1; Humphries, 1981). One example is the species complex formed by Anacyclus clavatus (Desf.) Pers. and A. valentinus L. (Figure 1A and 1B). These two species present notable differences in floral morphology, however in areas where the two species coexist, morphological variation of the flowers is remarkably higher (e.g. in number and size of the rays; Bello et al. 2013), suggesting hybridization between both species.

A. clavatus is usually found in disturbed habitats, coastal beaches, fields and waste inland places, within the Circum-Mediterranean Basin (Humphries 1981). This plant has gynomonoecious capitula, with two types of flowers varying both in sex expression and morphology: rayed female florets with creamy-white ligules displayed in the outermost position of the capitulum and yellow bisexual disc- florets with a campanulate corolla and a narrow basal tube displayed in the central part of the capitulum (Figure 1A; Bello et al. 2013). A. valentinus is found in the Western part of the Mediterranean Basin (Morocco, Spain, Algeria and Tunisia), occurring in disturbed grounds, sandy and rocky places, lowlands, river banks, fields and roadsides (Humphries 1981). As A. clavatus, this species bears gynomonoecious capitula, however the female flowers displayed in the outermost positions usually lack rays and are fewer and inconspicuous, being the capitulum mostly represented by bisexual yellow disc-florets (Figure 1B; Humphries, 1979).

Both species are self-incompatible (I. Álvarez, personal communication) and commonly bloom from February to July. This long flowering season allows several generations of capitula to develop on the same individual. However, unsuitable conditions for flowering may often restrain the number of developing branches resulting in high variability in the number of capitula between individual plants (Humphries 1979).

Materials and Methods

Figure 1. The studied phenotypes.

A. Anacyclus clavatus, B. Anacyclus valentinus, C. Hybrid phenotype, D. Fake rays phenotype.

Study sites

This study was conducted during the spring of 2013 in the contact zone between A. clavatus and A. valentinus, nearby Torre del Mar (Spain), at three different sites. The three selected populations included: 1) an open field, 1 m a.s.l., 210 m distance from the sea, where both species grow jointly and where an intermediate phenotype has been previously observed (hereafter sympatric site; +36° 43' 48.875" N, -4° 6' 8.154" W); 2) a road verge, 1 m a.s.l., 160 m away from the sea, being separated from the sympatric site by 100 m with buildings, only with A. clavatus (hereafter rayed site; +36° 45' 4.186" N, -4° 5' 58.289" W); 3) an open field area with planted palm trees, 16 m a.s.l., 1 km distance from the sea, only with A. valentinus (hereafter rayless site; +36° 43' 50.516" N ,-4° 6' 4.697" W). The vegetation in the three sites was very similar, being characterized by ruderal herbaceous species such as Leontodon longirostris (Vill.) Mérat (Asteraceae), Hirschfeldia incana (L.) Lagr.-Foss. subsp. incana (Brassicaceae), Chrysanthemum coronarium L. (Asteraceae), and Echium cretium subsp. granatense (Coincy) Valdés (Boraginaceae). All populations had clear indications of the presence of livestock (J. Cerca de Oliveira, 2013, pers. obs.) and were chosen because they presented a high number of individuals of the desired study species growing in the same conditions.

Materials and Methods

Experimental design

Pollinator preferences in the sympatric site

To assess floral visitor’s preferences under natural conditions, we randomly selected and tagged 107 plants, including rayed, rayless and intermediate phenotypes (Figures 1A-C; Supplementary Table A). In order to maximize the efficiency of field observations, plants were monitored in groups of 2-7 individuals.

The selected plants were characterised phenotypically focusing on the individual plant characteristics (plant size and floral display), the capitulum traits (capitulum size, disk size, ray presence and ray number), and the intra-specific neighbourhood context. In particular, plant size was estimated as: (i) plant height, considering the distance from the ground to the tallest part of the plant, and as (ii) the plants’ dimension, defined by a circular area, whose diameter was calculated by dividing the plants’ largest diameter together with its perpendicular axis) by two. Floral display was defined as the total number of open capitula per individual at each observation day (quantified repeatedly through the field season). Capitula were characterized by: (i) the total diameter of the capitula (from the tip of a ray to the tip of the opposite ray), (ii) diameter of the disk and (iii) number of rays. Ray length was estimated by the following formula: (diameter of the capitulum – diameter of the disk) / 2. Finally, we measured intra- specific neighbourhood context using two proxies: (i) pollination context: the number of open capitula of Anacyclus within a 0.5 m radius, and (ii) neighbour density: the number of Anacyclus individuals within a 0.5 m radius. Neighborhood traits were surveyed at three different periods during the whole study. Floral visitors were monitored as described below in the Floral visitor observations section.

Phenotypic manipulations at single-species sites

To get further insights of the role of the rayed phenotype on the pollinator’s preferences we performed two experiments of phenotype manipulation, one in the rayed site involving the removal of rays, and the other in the rayless site involving the addition of artificial rays to the capitula. Plants were characterized as described above. Floral visitors were monitored in all the experimental plots as described below in Floral visitor observations section.

Materials and Methods

Rayed site: Ray removal experiment

For this experiment we selected 30 pairs of nearby plants. The plants from each pair were carefully chosen to be similar in size, habit and number of capitula. One individual was set as the control and served as a rayed phenotype, while the other served as the rayless phenotype, with its rays being removed using tweezers. To maintain the paired individuals as similar as possible, we removed buds produced after the beginning of the experiment. Neighbourhood effects were studied using two approaches: First, pollination context and neighbourhood density were characterized in 20 pairs of plants to assess for its potential effects on pollinator’s attraction using the variation in natural populations. For that, the number of surrounding Anacyclus plants and open capitula were counted in a radius of 0.5 m (see above). Second, in the remaining subset of 10 randomly selected pairs of plants we performed a manipulative experiment to assess the potential effects of the neighbourhood context on floral visitors’ attraction, by manually removing all surrounding Anacyclus plants within a 1 m radius of the focal individuals.

As a procedural control for the ray addition experiment (see below), a third individual was selected near each pair and equipped with fake rays (Figure 1D). Visitation rate comparisons between the manipulated and naturally rayed phenotypes was performed by means of a Kruskal-Wallis test, and showed that fake rays marginally decreased the visitation rate to the capitula (χ2 = 3.06, P = 0.08) compared to naturally rayed phenotypes.

Rayless site: Ray addition experiment

We carefully selected 30 pairs of individuals with similar characteristics, manipulating the individuals (adding fake rays and removing extra capitula buds) and the neighbourhood context (presence vs. absence of other Anacyclus plants) as described above. Within each pair, one individual served as the rayless individual (control) while the other was equipped with fake rays. Fake rays were made with synthetic paper and they were added to the capitula to mimic the rayed phenotype as realistic as possible (Figure 1 D; see statistical details in above sub-section), similarly to the approach by Nielsen and colleagues in the endemic Scalesia from the Galapagos islands (Nielsen et al. 2002). As in the rayed site, pollination context and neighbourhood density was characterized in 20 pairs of plants and in the remaining subset of 10 pairs of plants, all surrounding Anacyclus neighbours were removed within a 1 m radius.

Materials and Methods

Floral visitor observations

A preliminary survey of pollinators was performed during the spring of 2012 in the contact zone of this study, to get insights about the pollinator fauna that was visiting A. clavatus and A. valentinus, and to collect insects for a reference collection of Anacyclus spp. floral visitors. In 2013, floral visitor observations were carried during the main flowering period of the study species, more specifically, during the central hours (from 10:30 to 18:00, GMT+1) of warm and sunny days from 30th of March to 26th of April. These observations were conducted similarly in the three studied sites. With the aid of small range-binoculars, plant groups were observed during intervals of five minutes, with the observer positioned at a considerable distance (1-2 m apart) from the plant group, to avoid disturbing the foraging activity of the insects, while recording all the insects that visited the tagged plants. A floral visit was only taken into account when there was a direct contact between the insect visitor and the sexual organs (anthers or stigmas) of the capitulum. Considering that these species are self-incompatible, the number of capitula visited per individual plant was not accounted. During observation intervals the overall weather conditions, the hour of the day and the surrounding insect activity were recorded for data quality assessment. A total of 1338 census were performed, corresponding to a total of 111.5 hours of net observation evenly divided by site. Insect identification was based on the reference collection gathered in 2012; still, whenever a new taxon was observed, it was collected with a capture net or a vacuum container for subsequent identification at the laboratory. Smaller insects were conserved in ethanol 70%, while bigger insects were air dried. All insects are being kept at the Centre for Functional Ecology (Faculty of Science and Technology, University of Coimbra). The pollinators were grouped into “functional groups” to facilitate the detection of general patterns. ‘Functional group’ was defined as a group of pollinators that tend to interact with flowers in a similar way. Following the methodology employed in Gomez et al. (2008b) we used criteria of similarity in size, proboscis length, foraging behaviour and feeding habits rather than taxonomic relationships. Also, given the low number of visits of each bee group, bees were grouped in the same group. In the end, the following 6 functional groups were established: ants, bees, beeflies, big flies, hoverflies and small flies.

Statistical analyses

The effects of floral phenotypes, floral display and neighbourhood traits on pollinator attraction were assessed using general linear mixed models (GLMM). Overdispersion was calculated using Pearson residuals (Zuur et al. 2009), and is displayed for each model. All analyses were conducted using the lme4 package of the R 3.0.1 software. The statistical analyses were organized following our three main objectives:

Materials and Methods

1) How do the floral phenotype, floral display and surrounding context affect pollinator’s attraction?

We assessed the effect of floral phenotype (rayed, rayless and intermediate phenotypes), floral display and neighbourhood context on floral visitor rate fitting GLMMs for sympatric, rayed and rayless sites. We analysed the visits of all pollinators in one global model. Additional analyses were performed independently for each functional group. Each site had its own independent functional groups, established depending on the frequency and abundance of pollinator taxa.

Visitation rate was modelled with a Poisson distribution and a log link function. Floral phenotype, floral display and pollination context were included as explanatory variables, while plant identity was included as a random factor. Non-collinearity between explanatory variables was previously checked (Supplementary Table B). Differences between floral phenotypes were tested using least square means differences with the ‘lsmeans’ package. Models for rayed and rayless sites only considered the 20 selected pairs without manipulated neighbourhood conditions.

2) How does capitulum size affect pollinator’s attraction?

The previous section allowed to investigate the effect of rayed versus rayless phenotypes in the attraction of floral visitors. In this section we explored, within each phenotype from the sympatric population, which capitulum traits had an impact on floral visitors’ attraction. First, capitulum size of rayed individuals including intermediate phenotypes was assessed in an exploratory analysis (Supplementary Table C). Afterwards, both capitulum components, disk size and ray length (this last one only for rayed and intermediate phenotypes), were analysed separately due to correlations between these variables (Supplementary Table B). In rayless individuals, only capitulum size, which is equivalent to disk size, was analysed. We fitted GLMMs for each phenotype using floral display, pollination context and capitulum traits as explanatory variables. Visits of all floral visitors were modelled with a Poisson distribution and analysed in a global model for all pollinator groups together. Additional models were fitted to the main pollinator groups of this site. Plant identity was set as a random factor.

Materials and Methods

3) Is there a facilitation or a competing effect between rayed and rayless phenotypes?

After disentangling individual characteristics of phenotypes, we sought to understand how the ecological context of neighbours affected rayed and rayless phenotypes. For that, we followed two approaches: one based on the observational assessment in the sympatric site and the other based on the experimental manipulation of neighbourhoods on rayed and rayless sites. Firstly, on the sympatric site we assessed the effect of having rayed, intermediate or rayless neighbours on floral visitor rate on each phenotype separately, by means of GLMM models. Visitation rate was modelled with a Poisson distribution and a log link function. Floral display and the variable pollination context (either rayed, intermediate or rayless neighbours), were used as explanatory variables. In every model plant identity was set as a random factor. As for the previous analyses, visits of all pollinators were fitted in a global model and additional models were performed for main pollinator groups. Non-collinearity between explanatory variables has been previously assessed.

Secondly, in the rayed and rayless sites, we selected 20 and 18 pairs of plants (each pair including one rayed and one rayless plant), respectively. Half of these pairs had all Anacyclus neighbours removed, while the other half corresponded to the pairs with the highest pollination context (mean ± SD (range): rayed site = 47 ± 17 (25 - 102) neighbouring capitula; rayless site = 89 ± 58 (19 - 201)). GLMM models included floral phenotype (rayed vs. rayless), pollination context (control versus neighbourhood removed) and its interaction as explanatory variables. Each pair of plants was included as a random factor. Visitation of floral visitors was modelled with a Poisson distribution and a log link function.

Results

Floral visitors of Anacyclus species

We observed 128 different morphospecies, which accounted for a total of 640 interactions between Anacyclus capitula and its visitors within all three sites. There was a clear prevalence of Dipteran visitors, which accounted for almost two thirds of these visits (409 interactions; Table 1). The sympatric site was the site where most interactions were registered (408), with the rayed phenotype being the most visited out of the three phenotypes (Table 1). With respect to the rayless site, capitula with fake rays were visited 94 times while control plants were visited 81 times (Table 1). Hymenopteran and Dipteran visitors cover for around 92% of the interactions, with a high prevalence of ants, by far the most abundant Hymeropteran visiting Anacyclus capitula in the rayless site (52 interactions out of 82). This site was the only one where beeflies interacted with monitored plants, with 14 interactions in total. Finally, the rayed site had the lowest number of interactions (57 interactions), with Hymenoptera and Lepidoptera being responsible for 67% of the total number of visits (21 and 18, respectively), and only 10 visits were performed by Dipterans (Table 1).

How do the floral phenotype, floral display and neighbourhood context affect pollinator attraction?

Sympatric site

Floral phenotype significantly affected the total number of visits when the overall assemblage of floral visitors was considered, whereas floral display and pollination context did not show significant effects (Table 2). Rayed phenotypes were visited at a significantly higher rate than rayless phenotype (Figure 2). The intermediate phenotype received less visits than rayed phenotypes but more than rayless one, not differing statistically from any of the phenotypes (Figure 2). The preference for rayed plants was also observed for each of the specific pollinator groups. Dipteran groups (hoverflies, small flies and big flies) were the main insects responsible for the differences in visitation rates according to the floral phenotype (Table 2). When analysed separately, in general, these Dipteran groups showed significant higher visitation rates to rayed phenotype in comparison with the rayless one (Table 2; Figure 2). Apart from the different preferences on floral phenotypes, when analysing pollinators groups separately, floral display and pollination context were relevant factors for specific pollinators groups. For example, floral display seemed to be an important factor driving the foraging behaviour of bees, which visited preferentially plants with larger floral displays (Table 2). On the other hand, hoverflies were impacted by the three variables, showing preference for rayed plants with a large floral display and with high number of Anacyclus capitula in the surrounding (Table 2; Figure 2)

Rayless site: rayed phenoty rayed site: Rayless respectively. 1. Table

Rayless site: rayed phenoty rayed site: Rayless respectively. 1. Table Coleoptera Total Lepidoptera Hymenoptera Hemiptera Diptera Coleoptera Coleoptera Total Lepidoptera Hymenoptera Hemiptera Diptera Coleoptera

Absolute and relative frequencies of floral visitors on visitors floral of frequencies relative and Absolute Absolute and relative frequencies of floral visitors on visitors floral of frequencies relative and Absolute

Malachiidae Cantharidae Cetoniidae Total Others Dermestidae Oedemeridae Malachiidae Cantharidae Cetoniidae Total Others Dermestidae Oedemeridae id. non Attagenus Oedema simplex abdominalis Clanoptilus Rhagonichafulva Oxythyrea funesta id. non Attagenus Oedema simplex abdominalis Clanoptilus Rhagonichafulva Oxythyrea funesta pe (phenotypic manipulation) (phenotypic pe pe (phenotypic manipulation) (phenotypic pe sp. sp.

Sympatricsite Sympatricsite 251 251 203 251 203 36 36 10 36 10 1 1 1 1 10 10 10 8 0 1 1 0 0 8 0 1 1 0 0

14.3 80.9 14.3 80.9 100 100 100 71.6 57.2 71.6 57.2 0.4 0.4 0.4 0.4 0.4 and the rayless phenotype (control) phenotype rayless the and and the rayless phenotype (control) phenotype rayless the and % % 4 4 7.1 7.1 7.1 7.1 7.1 0 0 0 0 0 0 Anacyclus Anacyclus

64 64 11 51 64 11 51 0 0 2 0 0 2 2 1 1 0 0 0 0 2 1 1 0 0 0 0

Results 14.2 17.2 79.7 14.2 17.2 79.7 100 100 100 3.1 3.1 3.1 7.1 7.1 7.1 7.1 7.1 % % spp. capitula in each studied site. Sympatric site: ray site: Sympatric site. studied each in capitula spp. spp. capitula in each studied site. Sympatric site: ray site: Sympatric site. studied each in capitula spp. 0 0 0 0

0 0 0 0 0 0 0 0

93 93 22 63 93 22 63 5 1 2 5 1 2

Table 1. Absolute and relative2 frequencies1 0 of floral0 visitors1 on0 Anacyclus0 spp. capitula in each studied2 1 site. Sympatric0 0 site:1 rayed0 phenotype,0 intermediate phenotype and rayless phenotype, respectively.

23.7 67.7 14.2 23.7 67.7 14.2 100 100 100 Rayless site: rayed phenotype (phenotypic manipulation) and the rayless phenotype 5.4 (control) 2.2 , respectively. Rayed site, rayed phenotype (control) 5.4 and the rayless 2.2 phenotype (phenotypic manipulation), 7.1 7.1 7.1 7.1 7.1 % % 1 1 0 0 0 0 0 0 respectively. 0 0

Total Total , respectively. Rayed site, rayed ph rayed site, Rayed respectively. , , respectively. Rayed site, rayed ph rayed site, Rayed respectively. ,

408 408 317 (Page 1/4) 408 317 69 69 14 69 14 (Page 1/4)(Page (Page 1/4)(Page 6 2 6 2 14 14 10 14 10 2 0 0 1 1 2 0 0 1 1 14.2 16.9 77.7 71.6 14.2 16.9 77.7 71.6 100 100 100

1.5 1.5 0.5 3.4 1.5 0.5 3.4 100 100 100 7.1 7.1 7.1 7.1 7.1 % % 0 0 0 0 Sympatric site Rayless site Rayed site

Rayless site Rayless site

94 94 42 43 94 42 43 % % 3 0 % 6 Total % % % Total 3 % 0 6 % % Total % 0 2 1 6 3 0 0 0 2 1 6 3 0 0 10 4 2 3.1 2 2.2 14 3.4 6 6.4 1 1.2 7 4 2 7.1 0 0 2 3.5 Coleoptera % % 44.7 45.7 44.7 45.7 100 100 100 3.2 3.2 6.4 3.2 6.4 85.7 42.8 28.6 14.3 85.7 42.8 28.6 14.3 Diptera 203 80.9 51 79.7 63 67.70 317 77.7 43 45.7 39 48.2 82 46.9 0 6 21.4 4 13.8 10 17.6 0 0 0 0 Hemiptera 0 0 1 0.4 0 0 1 1 2 0.5 0 0 1 1.2 1 0.6 2 7.1 4 13.8 6 10.5

Hymenoptera 36 14.3 11 17.2 22 23.7 69 16.9 42 44.7 37 45.7 79 45.1 11 39.4 10 34.4 21 36.8 81 81 37 39 81 81 37 39 ed phenotype, intermediate phenotype and phenotype intermediate phenotype, ed ed phenotype, intermediate phenotype and phenotype intermediate phenotype, ed 3 1 1 0.4 0 3 1 5.4 1 1.5 3.2 3.7 3.4 25 38 31.6 0 1 0 0 Lepidoptera 1 0 0 0 1 1 0 0 0 5 1 6 0 0 3 3 6 7 11 18 enotype (control) and the and (control) enotype enotype (control) and the and (control) enotype

% % 45.7 48.2 100 100 45.7 10048.2 100 100 100 100 100 100 100 14.3 14.3 14.3 14.3 100 100 251 64 100 93 408 94 81 175 28 29 57 3.7 3.7 1.2 1.2 Total 3.7 1.2 1.2 0 0 0 Coleoptera 0 0 0 0 0 0 0

Cetoniidae Total Total 175 175 Oxythyrea funesta 0 0 0 0 175 0 0 0 0 1 14.3 0 0 1 14.3 0 0 0 0 0 0 79 79 82 79 79 82 6 1 7 6 1 7 0 1 2 1 Cantharidae 7 3 0 0 1 2 1 7 3 0 % % 45.1 46.9 45.1 46.9 14.3 28.6 14.3 42.8 14.3 28.6 14.3 42.8 100 100 0 0 100 0 0 28.6 0 28.6 0 0 0 3.4 3.4 0.6 Rhagonicha fulva 0 0 3.4 0 0.6 0 2 0 2 0 0 0 100 100 100 4 4

0 Malachiidae 0 0 0

Clanoptilus abdominalis 1 7.1 0 0 1 7.1 2 14.2 0 0 1 14.3 1 14.3 0 0 0 0 0 0 Rayed site Rayed site 28 28 11 28 28 11 rayless phenotype (phenotypic manipulation),(phenotypic phenotype rayless rayless phenotype (phenotypic manipulation),(phenotypic phenotype rayless 7 2 6 2 Oedemeridae 7 2 6 2 0 0 0 0 2 2 0 0 0 0 0 2 2 0

7.1 0 0 7.1 0 0 0 0 0 0 % % 39.4 21.4 Oedema simplex 1 0 39.4 0 21.4 1 0 0 0 0 0 0 100 100 100 100 7.1 7.1 7.1 7.1 7.1 7.1 100 100 100 100 100 25 25 25 25 0 0 0 0 Dermestidae 0 0 0 0 0 0

Attagenus sp. 0 0 1 7.1 0 0 1 7.1 0 0 0 0 0 0 0 0 0 0 0 0

rayless phenotype, respectively phenotype, rayless Others respectively phenotype, rayless 29 29 11 10 29 29 11 10 4 4 0 57.2 7.1 4 7.14 0 71.6 42.8 0 42.8 100 0 100 0 0 0 0 non0 id. 0 0 0 0 8 0 0 1 1 0 10 0 0 3 0 3 2 0 2

% % 34.4 13.8 13.8 34.4 13.8 13.8 100 100 100 100 38 38 38 0 0 0 0 0 0 Total 0 0 0 0 100 71.60 0 2 14.2 2 14.2 0 14 0 100 0 6 85.7 1 14.3 7 100 2 100 0 0 2 100

Total Total 57 57 18 21 10 57 57 18 21 10 6 2 6 2 0 0 0 0 2 2 0 0 0 0 0 2 2 0 % % 31.6 36.8 10.5 17.6 31.6 36.8 10.5 17.6 100 100 100 3.5 3.5 3.5 100 100 100 100 100 Results Results Results

0 0 0 0 0 0 0 0 0 0 19 19 19 . .

Diptera Diptera Total Others Anthomyzidae Calliphoridae Scathophagidae Tachinidae Bombyliidae Total Others Syrphidae Anthomyzidae Calliphoridae Scathophagidae Tachinidae Bombyliidae Syrphidae Small id. non Diptera Dipteraid. non id. non caesarLucilia vomitoriaCalliphora Miltogramminaeid. non stercoraria Scathophaga feraTachina id. non Conophurussp. id. non Chrysotoxum Syritta pipiens Sphaerophoria Episirphus Small id. non Diptera Eupeodes Dipteraid. non Eristalisarbustorum Eristalistenax id. non caesarLucilia vomitoriaCalliphora Miltogramminaeid. non stercoraria Scathophaga feraTachina id. non Conophurussp. id. non Chrysotoxum Syritta pipiens Sphaerophoria Episirphus Eupeodes Eristalisarbustorum Eristalistenax sp. sp. sp. sp.

sp. sp. sp. sp.

Sympatricsite Sympatricsite 203 203 203 51 51 52 17 31 23 12 51 17 23 52 52 31 12 1 3 0 1 0 0 0 8 1 2 0 1 1 3 0 1 0 0 0 8 1 2 0 1 Results 16.6 16.3 16.6 16.3 % % 8.7 8.7 3.9 0.3 0.3 2.6 0.3 0.6 5.6 8.7 0.3 7.5 3.9 0.3 0.3 2.6 0.3 0.6 5.6 0.3 7.5 64 64 64 1 0 0 0 0 0 1 0 0 0 0 0

51 51 11 51 15 11 15 8 2 0 2 1 0 1 0 0 0 0 0 0 8 6 1 4 2 0 2 1 0 1 0 0 0 0 0 0 6 1 4

(Page 2/4) 2.6 2.6 3.4 0.6 0.6 0.3 0.3 4.8 2.6 1.8 3.4 0.3 1.3 0.6 0.6 0.3 0.3 4.8 1.8 0.3 1.3 % % 16 16 16 16 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

63 63 12 Sympatric site 63 19 12 Rayless site 19 Rayed site 5 5 7 0 8 3 2 0 0 0 1 0 0 1 0 0 5 5 0 8 7 3 2 0 0 0 1 0 0 1 0 0

0.3 0.3 0.3 1.6 3.8 1.6 2.2 2.6 0.6 0.3 0.3 1.6 1.6 2.6 3.8 3.8 2.2 0.6 % % % %% % % % % % % % 20 20 20 Total Total Total 0 0 0 0 0 6 0 1 0 0 0 0 0 0 0 6 0 1 0 0

Diptera

Syrphidae Total Total 317 317 317 (Page 2/4)(Page (Page 2/4)(Page 72 72 49 17 85 72 28 49 35 17 85 28 35 1 2 0 0 9 1 2 5 2 3 5 1 1 2 0 0 9 1 2 5 2 Eristalis tenax3 5 1 23 7.5 4 1.3 8 2.6 35 11.4 3 3.7 0 0 3 3.7 2 20 2 20 4 40 27.1 14.3 11.4 27.1 11.4 14.3

100 100 100 100 Eristalis arbustorum 1 0.3 1 0.3 0 0 2 0.6 1 1.2 0 0 1 1.2 0 0 0 0 0 0 5.5 5.5 0.9 1.6 0.3 0.3 0.6 2.9 0.3 0.6 1.6 0.6 5.5 0.9 1.6 0.3 0.3 0.6 2.9 0.3 0.6 1.6 0.6 % % 23 23 23 Eupeodes sp. 17 5.60 0 6 1.8 5 9 1.6 28 9 6 7.2 0 3 0 3.7 9 11 9 0 0 0 0 0 0

0 0 1.6 1.6 1.2 1.2 2.5 0 0 0 Rayless site Episirphus sp. 0 0 5 5 Rayless site 1 1 2 0 0 0 Sphaerophoria sp. 51 16.3 15 4.8 19 6 85 27.1 6 7.2 7 8.6 13 15.9 1 10 0 0 1 10 43 43 43 2 0 4 6 4 0 0 6 1 2 6 6 1 3 0 1 1 0 2 0 4 6 4 0 0 6 1 6 1 3 2 6 0 1 1 0

Syritta pipiens 2 0.6 0 0 0 0 2 0.6 0 0 0 0 0 0 0 0 0 0 0 0 % %

52.4 Chrysotoxum sp. 1 0.3 0 0 52.4 0 0 1 0.3 0 0 0 0 0 0 0 0 0 0 0 0 2.4 2.4 4.9 7.2 7.2 1.2 2.4 7.2 7.2 1.2 3.7 1.2 1.2 2.4 4.9 7.2 7.2 1.2 7.2 1.2 3.7 2.4 2.4 7.2 1.2 1.2 0 5 0 0 0 0 0 5 0 0 non0 id. 0 8 2.6 0 0 1 0.3 9 2.9 4 5 3 3.7 7 8.7 0 0 0 0 0 0

Bombyliidae 0 0 0 0 7.2 3.7 11 0 0 0 39 39 10 Conophurus sp. 0 0 39 0 10 0 6 3 9 0 0 0 0 1 0 1 3 3 0 0 7 1 3 0 0 4 1 3 2 0 1 0 1 3 3 0 0 7 1 4 3 0 0 1 3 2

non id. 0 0 0 0 0 0 0 0 4 4.9 1 1.2 5 6.1 0 0 0 0 0 0 % % 47.6 12.4 47.6 12.4 1.2 1.2 1.2 3.7 3.7 8.6 1.2 3.7 Tachinidae 1.2 3.7 2.4 1.2 1.2 3.7 3.7 8.6 1.2 3.7 1.2 3.7 2.4 0 0 0 0 0 0 5 Tachina fera 0 0 0 1 0.30 0 1 5 0.30 0 2 0.6 0 0 0 0 0 0 0 0 0 0 0 0

Scathophagidae Total Total Scathophaga stercoraria 1 0.3 0 0 0 0 1 0.3 2 2.4 1 1.2 3 3.6 0 0 0 0 0 0 13 13 82 82 16 82 13 16 0 3 0 5 9 7 0 0 2 9 1 3 6 non1 id. Miltogramminae4 3 0 3 0 05 9 17 0.30 0 2 0 6 9 01 3 1 1 0.34 3 0 0 0 0 0 0 0 0 0 0 0 0 % % 15.9 19.2 15.9 19.2 100 100 100 3.6 3.6 6.1 8.7 2.5 1.2 3.7 7.4 7.4 1.2 4.9 3.6 3.6 6.1 8.7 2.5 7.4 1.2 3.7 1.2 4.9 3.6

11 11 11 Calliphoridae 11 11 0 0 0 0 Calliphora vomitoria0 30 1 2 0.60 0 0 0 5 1.6 1 1.2 2 2.4 3 3.6 0 0 0 0 0 0

Rayed site Lucilia caesar 1 0.3 0 0 2 0.6 3 0.9 Rayed site 1 1.2 3 3.7 4 4.9 1 10 0 0 1 10 0 0 0 0 0 0 0 0 1 0 0 0 2 6 Anthomyzidae1 1 0 1 0 0 0 0 0 0 0 0 0 6 1 0 1 0 1 0 2 0 1 0

% non id. 12 3.9 2 0.6 3 1 17 5.5% 0 0 1 1.2 1 1.2 0 0 0 0 0 0 10 10 20 60 60 10 10 10 60 10 10 10 20 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Others

non id. Diptera 31 8.7 11 3.4 7 2.2 49 14.3 6 7.2 10 12.4 16 19.2 1 10 2 20 3 30 non id. Small Diptera 52 16.6 8 2.6 12 3.8 72 23 2 2.4 4 5 6 7.4 1 10 0 0 1 10 0 0 0 0 0 0 0 0 0 0 0 0 2 4 0 2 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 2 0 2 0 0 0

% % 20 20 40 Total 20 203 64 51 16 40 63 20 20 20 317 100 43 52.4 39 47.6 82 100 6 60 4 40 10 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Total Total 10 10 10 0 0 0 0 0 0 0 0 1 0 0 0 4 1 3 0 1 0 0 0 0 0 0 0 0 0 1 0 1 0 3 0 4 0 1 0 20 % % 100 100 100 Results Results Results

10 10 30 10 10 10 30 40 10 10 40 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 20 20 20

Hymenoptera Hemiptera Hymenoptera Hemiptera

Halictidae Megachilidae Apidae Formicidae Total Total Others Others Sphecidae Halictidae Megachilidae Apidae Formicidae Total Others Total Others Sphecidae Hemiptera id. Non id. non id. non Lasioglossum id. non id. non Ammobates longicornisEucera Anthophora sp. Apis mellifera id. non Hemiptera id. Non id. non id. non Lasioglossum id. non id. non Ammobates longicornisEucera Anthophora sp. Apis mellifera id. non sp. sp. sp. sp.

Sympatricsite Sympatricsite 36 36 19 12 36 19 12 0 3 1 0 0 0 0 1 0 3 1 0 0 0 0 1

1 1 1 1

52.2 27.5 17.4 52.2 27.5 17.4 4.3 4.3 1.5 1.5 4.3 1.5 1.5 % % 0 0 0 0 0 0 0 0 0 0

50 50 50 50 50 50

Results 11 11 11 2 0 5 0 0 0 0 0 4 0 2 0 5 0 0 0 0 0 4 0

0 0 0 0

15.9 15.9 7.1 7.1 5.8 7.1 5.8 % % 0 3 0 0 0 0 0 0 0 3 0 0 0 0 0 0

0 0 0 0

(Page 3/4) 22 22 11 22 11 1 6 0 2 0 2 0 0 0 1 6 0 2 0 2 0 0 0

1 1 1 1

15.8 31.9 15.8 31.9 8.6 8.6 1.5 8.6 1.5 % % 0 3 0 0 0 0 3 0 3 0 0 0 0 3 50 50 50 50 Sympatric site Rayless site Rayed site

Total Total 69 69 27 10 27 69 27 10 27 (Page 3/4) (Page 3/4) 1 2 0 0 0 2 0 1 2 0 0 0 2 0 % % % Total % % % Total % % % Total %

2 2 2 2 39.1 14.4 39.1 14.4 100 100 100 1.5 1.5 1.5

Hemiptera 100 100 100 100 39 39 39 % % 3 0 0 0 3 0 3 0 0 0 3 0 Others

Non id. Hemiptera 1 50 0 0 1 50 2 100 0 0 1 100 1 100 2 33.3 4 66.7 6 100 Rayless site Rayless site

42 42 30 42 30 0 0 0 1 0 1 0 0 7 0 3 0 0 0 1 0 1 0 0 7 0 3 50 0 50 100 0 100 100 33.3 66.7 100 Total 1 0 1 2 0 1 1 2 4 6 % % Hymenoptera 53.2 37.9 53.2 37.9 1.3 1.3 1.3 8.9 3.8 1.3 1.3 8.9 8.9 3.8 0 0 0 0 0 0 0 0 0 0 0 0 0 Formicidae 0

non id. 1 1.5 0 0 1 1.5 2 3 30 37.9 22 27.8 52 65.7 0 0 1 4.8 1 4.8

22 22 Apidae 37 22 37 4 0 0 0 0 0 5 1 1 6 0 4 0 0 0 0 0 5 1 1 6 0

Apis mellifera 12 17.4 4 5.8 11 15.8 27 39 1 1.3 5 6.3 6 7.6 6 28.6 7 33.2 13 61.8 % % 27.8 46.8 27.8 46.8 100 100 100 100 100 100 5.2 5.2 6.3 Anthophora 7.5 sp. 5.2 0 0 0 6.3 0 0 0 0 7.5 0 0 0 0 0 0 0 2 9.5 0 0 2 9.5 0 0 0 0 0 Eucera longicornis0 0 0 0 0 00 0 0 0 0 0 0 1 1.3 0 0 1 1.3 0 0 0 0 0 0

Ammobates sp. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 0 0 3 3 Total 0 0 0 0 0 0 52 52 non 79 id. 13 0 0 52 2 79 2 13 0 0 0 0 0 0 7 0 0 0 1 0 6 1 1 Megachilidae 0 7 0 0 0 1 0 6 1 1 0 % % 65.7 16.4 65.7 16.4 100 100 100 100 100 100 100 100 1.3 1.3 7.6 1.3 1.3 7.6

non id. 1 1.5 0 0 0 0 1 1.5 0 0 0 0 0 0 0 0 1 4.8 1 4.8 9 0 0 0 0 Halictidae 0 9 0 0 0 0 0

Rayed site Lasioglossum sp. 3 4.3 5 7.1 2 3 10 14.4 Rayed site 3 3.8 4 5.2 7 9 0 0 0 0 0 0 11 11 11 0 0 0 0 0 2 6 0 2 2 Sphecidae 3 0 0 0 0 0 0 2 6 0 2 2 3 0

% % 28.6 33.3 33.3 52.4 14.3 0 28.6 0 33.3 0 52.4 33.3 14.3 0 0 0 0 0 4.8 4.8 9.5 9.5 non id. 0 0 9.5 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Others

non id. 19 27.5 2 3 6 8.6 27 39.1 7 8.9 6 7.5 13 16.4 3 14.3 0 0 3 14.3

10 10 10 0 1 0 0 0 0 7 1 4 4 0 1 0 1 0 0 0 0 7 1 4 4 0 1

Total 36 52.2 11 15.9 22 31.9 69 100 42 53.2 37 46.8 79 100 11 52.4 10 47.6 21 100 % % 33.2 66.7 66.7 47.6 33.2 66.7 47.6 66.7 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 0 0 0 0 0 0 0 0 0 0 0 0

Total Total 13 13 21 21 13 21 0 1 0 0 0 2 1 6 6 3 1 0 1 0 0 0 2 1 6 6 3 1 % % 14.3 61.8 14.3 61.8 100 100 100 100 100 100 100 4.8 4.8 4.8 9.5 4.8 4.8 4.8 9.5 4.8 Results Results Results

0 0 0 0 0 0 0 0

21 21 21 21 21

Lepidoptera Lepidoptera Total Others Nymphalidae Pieridae Total Others Nymphalidae Pieridae Pararge aegeria croceusColias id. non Pararge aegeria croceusColias id. non Sympatricsite Sympatricsite 1 0 1 0 1 0 1 0

% % 17 17 17 17 17 17 0 0 0 0

0 0 0 0 0 0 0 0

% % 0 0 0 0 0 0 0 0

5 4 0 1 5 4 0 1

Results % % 83 83 66 17 83 66 17 0 0

Total Total (Page 4/4) (Page 4/4)

6 4 1 1 (Page 4/4) 6 4 1 1

100 100 100 % % 66 66 17 17 66 17 17

Rayless site Rayless site Sympatric site Rayless site Rayed site

3 3 0 0 3 3 0 0

% % % Total % % % Total % % % % Total %

50 50 50 50 50 Lepidoptera 0 0 0 0

Pieridae Colias croceus 0 0 0 0 1 17 1 17 0 0 0 0 0 0 0 0 0 0 0 0 3 3 0 0 3 3 0 0

Nymphalidae % 17 0 0 % 17 0 0 0 0 0 0 50 50 50 Pararge aegeria 1 0 50 50 0 1 0 0 0 0 0 0 0 0 Others 0 0

non id. 0 0 0 0 4 66 4 66 3 50 3 50 6 100 7 38.9 11 61.1 18 100 Total Total 6 6 0 0 Total 1 17 0 6 0 6 5 0 83 0 6 100 3 50 3 50 6 100 7 38.9 11 61.1 18 100 % % 100 100 100 100 100 100

0 0 0 0

Rayed site Rayed site 7 7 0 0 7 7 0 0

Table 2. Table fl Small overdispersion. Statistical significances ( (Bees, groups functional pollinator different the for % % 38.9 38.9 38.9 38.9 0 0 0 0

The effects of floral phenotype (rayed, intermediate and rayle and intermediate(rayed, phenotype floral of effects The 11 11 11 11 11 11 0 0 0 0

% % 61.1 61.1 61.1 61.1 0 0 0 0

Total Total 18 18 18 18 18 18 0 0 0 0 % % 100 100 100 100 100 Overd Random Fixed Variables Results Results Results Plant

Pollination context Floral display phenotypeFloral 0 0 0 0

ispersion 22 22 22 22

P < P

0.05) are shown in bold. Significantly

index index

1 2

22 Variance Variance e, i fis n Hoverflies) and flies Big ies, 21.43

0.40 0.96 1.11 χ 2

All groupsAll tor attraction for the entire pollinator entire the for attraction tor pollina on context pollination and display floral phenotypes), ss 1.036 <0.0001 <0.0001 0.329 0.293 0.63 SD SD P positive effects are signed with(+).

o te ypti st. ln iden Plant site. sympatric the for Variance Variance (+) 8.93 0.39 0.590 0.29 0.729 0.63 χ 2

0.838 Bees 0.003 0.003

0.62 SD SD P

Variance Variance 13.55 0.78 0.07 0.76 χ Small flies 2

0.879 iy a ue a a adm variable random a as used was tity 0.001 0.789 0.383 0.88 SD SD P

Variance Variance <0.01 0.83 3.37 7.65 χ 2

Big flies

0.733 0.022 0.066 0.986 0.96 SD SD P

Oedseso in Overdispersion . Variance Variance (+) (+) 4.69 12.99 0.36 χ 4.29 Hoverflies 2

0.977

assemblage (All assemblage 0.038 0.030 0.002 0.60 SD SD P e: maue of measure a dex: groups) andgroups) Results Results 23 23

Table 2. Table Table 2. Table fl Small overdispersion. Statistical significances ( (Bees, groups functional pollinator different the for fl Small overdispersion. Statistical significances ( (Bees, groups functional pollinator different the for The effects of floral phenotype (rayed, intermediate and rayle and intermediate(rayed, phenotype floral of effects The The effects of floral phenotype (rayed, intermediate and rayle and intermediate(rayed, phenotype floral of effects The Overd Random Fixed Overd Random Fixed Variables Variables Plant Plant Pollination context Floral display phenotypeFloral Pollination context Floral display phenotypeFloral

ispersion ispersion

P < P P < P 0.05) are shown in bold. Significantly 0.05) are shown in bold. Significantly

index index index

Df Df 1 2 1 2 1 1

Variance Variance Variance e, i fis n Hoverflies) and flies Big ies, e, i fis n Hoverflies) and flies Big ies, 21.43 21.43 0.40 0.96 1.11 0.40 0.96 1.11 χ χ 2 2

Results

All groupsAll groupsAll tor attraction for the entire pollinator entire the for attraction tor pollina on context pollination and display floral phenotypes), ss tor attraction for the entire pollinator entire the for attraction tor pollina on context pollination and display floral phenotypes), ss 1.036 1.036 <0.0001 <0.0001 <0.0001 <0.0001 0.293 0.329 0.293 0.329 0.63 0.63 SD SD SD P P positive effects are signed with(+). Table 2. The effects of floral phenotype (rayed, intermediate and rayless phenotypes), floral display and pollination context on pollinator attraction for the entire pollinator assemblage positive effects are signed with(+). (All groups) and for the different pollinator functional groups (Bees, Small flies, Big flies and Hoverflies) for the sympatric site. Plant identity was used as a random variable. Overdispersion index: a measure of overdispersion. Statistical significances (P < 0.05) are shown in bold. Significantly positive effects are signed with (+).

o te ypti st. ln iden Plant site. sympatric the for o te ypti st. ln iden Plant site. sympatric the for Variance Variance Variance (+) 8.93 (+) 8.93

0.39 0.590 0.29 0.729 0.63 0.39 0.590 0.29 0.729 0.63 χ χ 2 2

0.838 0.838 Bees Bees

0.003 0.003 0.003

0.62 0.62

SD SD All groups Bees Small flies SD Big flies Hoverflies P P

2 2 2 2 2 Variables Df χ P χ P χ P χ P χ P

Fixed Variance Variance Variance 13.55 13.55 0.78 0.07 0.76 Floral phenotype 0.78 21.43 0.07 0.76 <0.0001 0.63 0.729 13.55 0.001 7.65 0.022 12.99 0.002 χ χ Small flies Small flies 2 2 2

0.879 Floral display 0.879 1.11 0.293 (+) 8.93 0.003 0.76 0.383 <0.01 0.986 (+) 4.69 0.030 iy a ue a a adm variable random a as used was tity 1 variable random a as used was tity 0.038 0.001 0.001 0.789 0.383 Pollination context 0.96 0.789 0.383 0.329 0.29 0.590 0.07 0.789 3.37 0.066 (+) 4.29 0.88 0.88 SD SD 1 SD P P

Variance SD Variance SD Variance SD Variance SD Variance SD

Random Variance Variance Variance Variance <0.01 <0.01 0.83 3.37 7.65 Plant 0.83 0.40 3.37 0.637.65 0.39 0.62 0.78 0.88 0.83 0.96 0.36 0.60 χ χ 2 2

Big flies Big flies

0.733 Overdispersion index0.733 1.036 0.838 0.879 0.733 0.977

0.022 0.022 0.066 0.986 0.066 0.986 0.96 0.96 SD SD SD SD P P

Oedseso in Overdispersion . Oedseso in Overdispersion . Variance Variance Variance Variance (+) (+) 4.69 (+) (+) 4.69 12.99 12.99 0.36 0.36 χ χ 4.29 4.29 Hoverflies Hoverflies 2 2

0.977 0.977

assemblage (All assemblage assemblage (All assemblage 0.038 0.030 0.002 0.038 0.030 0.002 0.60 0.60 SD SD SD SD P P e: maue of measure a dex: e: maue of measure a dex: groups) andgroups) groups) andgroups) Results Results Results 23 23 23 23 23

Results

0.5

0.4 a

ab 0.3

0.2 b a ab a 0.1

Visitation rate (Visits/5 minutes) rate (Visits/5 Visitation ab b a ab a a a b b

0.0 Total Bees Small flies Big flies Hoverflies Floral visitors group

Figure 2. Least square means (± confidence intervals) of visitation rate (number of visits per 5 min intervals) in the sympatric site given for the entire pollinator assemblage (Total) and for each pollinator group observed (bees, small flies, big flies and hoverflies), according with the phenotypes present in the site: rayed (white bar), intermediate (grey bar) and rayless phenotypes (black bar). Means sharing the same letter were not significantly different at P < 0.05.

Rayed site

Due to the low number of interactions (Table 1), we could only fit a model with all groups of floral visitors together. Neither the ray removal factor, nor the pollination context had a significant effect on pollinator attraction in this site (floral phenotype: χ2= 0.30, P = 0.59; pollination context: χ2= 0.88, P = 0.882; Figure 3). Nevertheless, floral display showed a positive and significant effect on total visitation rates (χ2= 22.88, P < 0.0001).

Results

0.10

0.08

0.06

0.04

0.02 Visitation rate ( Visits/5 minutes) rate ( Visits/5 Visitation

0.00 Total

Floral visitors group

Figure 3. Least square means (± confidence intervals) of visitation rate (number of visits per 5 min intervals) in the rayed site given for the entire pollinator assemblage, according with the phenotypes present in this site: rayed (control; white bar) and rayless phenotypes (rays removed; grey bar). No statistically significant differences between phenotypes were found.

Rayless site

The rate of visits of the overall assemblage of pollinators was significantly affected by floral display, only, being unaffected by floral phenotype and pollination context (Table 3, Figure 4). The fake- rayed phenotype did not attract significantly more floral visitors than rayless ones in this population, affecting positively beeflies, only (Table 3, Figure 4). Besides this ray preference, beeflies and ants were significantly affected by floral display, preferring plants with a higher number of capitula (Table 3). Conversely, bees in this population showed a preference for plants with a more dense pollination context (Table 3).

Results

0.35

0.30

0.25

0.20

0.15

0.10

a Visitation rate (Visits/5 minutes) 0.05 b

0.00 Total Ants Bees Big flies Beeflies Small fliesHoverflies

Floral visitors group

Figure 4. Least square means (± confidence intervals) of the visitation rate (number of visits per 5 minutes intervals) in the rayless site given for the entire pollinator assemblage (Total) and for main pollinator groups (ants, bees, big flies, beeflies, small flies and hoverflies), according with the present phenotypes: rayless (control; white bar) and fake-rayed phenotype (grey bar). Significantly different LSmeans at P < 0.05 are signalled with different letters.

Statistical significances (<0.05) are sh Bees, (Ants, groups functionalpollinator 3. Table Statistical significances (<0.05) are sh Bees, (Ants, groups functionalpollinator 3. Table The effects of floral phenotype (rayless vs. fake-rayed), floral fake-rayed), vs. (rayless phenotype floral of effects The floral fake-rayed), vs. (rayless phenotype floral of effects The

Fixed index Overdispersion Random Fixed index Overdispersion Random context phenotype context phenotype Variables Variables Plant Pollination Floral display Ray Plant Pollination Floral display Ray

own in bold. Significantly posit own in bold. Significantly posit

Big flies, Beeflies, Small flies and Ho and flies Beeflies,Small flies, Big Ho and flies Beeflies,Small flies, Big Df Df 1 1 1 1 1 1

Variance Variance Variance (+) 4.25 (+) 4.25 0.36 0.34 0.03 0.36 0.34 0.03 χ χ 2 2

All groups groups All groups All 0.957 0.957 0.039 0.039 0.596 0.563 0.855 0.596 0.563 0.855

SD SD SD Results P P

ive effects are signed with (+). ive effects are signed with (+). ipa ad olnto otx o pliao atato fr t for attraction pollinator on context pollination and display t for attraction pollinator on context pollination and display

Variance Variance Variance (+) 10.95 (+) 10.95 rayles the for verflies) rayles the for verflies) <0.01 <0.01 3.32 1.66 3.32 1.66 χ χ 2 2

0.589 Table 3. The effects of floral phenotype (rayless0.589 vs. fake-rayed), floral display and pollination context on pollinator attraction for the entire pollinator assemblage (All groups) and for the different Ants Ants pollinator functional groups (Ants, Bees, Big flies, Beeflies, Small flies and Hoverflies) for the rayless site. Plant identity was used as a random variable. Overdispersion index: a measure of overdispersion. <0.001 <0.001

Statistical significances (<0.05) are shown in bold. Significantly posit0.198 ive effects0.982 are signed with (+). 0.198 0.982 1.82 1.82 SD SD SD P P

Variance Variance Variance s site. Plant identity Plant site. s identity Plant site. s

(+) 4.03 (+) 4.03 4.41 0.11 0.11 4.41 0.11 0.11 χ χ 2 2 0.327 0.327

Bees Bees

All groups Ants Bees Big flies Beeflies Small flies Hoverflies

0.040 0.737 0.742 0.040 0.737 0.742 2.10 2.10 SD SD SD

2 2 P 2 2 2 P 2 2 Variables Df χ P χ P χ P χ P χ P χ P χ P

Fixed Variance Variance Variance

Ray Overdispersvariable. random a usedas was Overdispersvariable. random a usedas was 0.03 0.855 0.55 0.98 0.88 <0.01 0.982 0.11 0.742 0.88 0.349 0.55 0.98 5.01 0.88 0.025 2.01 0.156 0.01 0.909 χ χ 0 0 Big flies Big flies 2 phenotype 1 2 1.011 1.011

Floral display 1 (+) 4.25 0.039 (+) 10.95 <0.001 0.11 0.737 0.98 0.322 (+) 5.07 0.024 0.37 0.543 0.59 0.443 0.458 0.322 0.349 0.458 0.322 0.349 SD SD Pollination SD P P 0 0.34 0 0.563 1.66 0.198 (+) 4.03 0.040 0.55 0.458 0.05 0.816 0.04 0.838 1.95 0.163 context 1

e nie olntr sebae Al rus ad o te different the for and groups) (All assemblage pollinator entire he different the for and groups) (All assemblage pollinator entire he Variance Variance Variance Variance (+) 5.07 Variance SD (+) 5.07 Variance SD Variance SD Variance SD Variance SD Variance SD Variance SD 0.30 0.05 5.01 0.30 0.05 5.01 χ χ Beeflies Random Beeflies 2 2 1.050 1.050

Plant 0.36 0.596 3.32 1.82 4.41 2.10 0 0 0.30 0.55 0 0 0.85 0.92 0.816 0.024 0.025 0.816 0.024 0.025 0.55 Overdispersion 0.55 SD SD SD SD P 0.957 0.589P 0.327 1.011 1.050 0.985 0.770 index

Variance Variance Variance Variance 0.04 0.37 2.01 0.04 0.37 2.01 Small flies Small flies χ χ 0 0

2 2

Table 4.Table 4.Table individuals. Plant identity was used as a pollinator assemblage (All groups), andof different pollinator groups individuals. Plant identity was used as a pollinator assemblage (All groups), andof different pollinator groups 0.985 0.985

overdispersion.measureof a index: ion overdispersion.measureof a index: ion 0.838 0.543 0.156 0.838 0.543 0.156 SD SD SD SD P P 0 0

The effect of disk size on floral visi The effect of disk size on floral visi

Variance Variance Variance Variance 0.85 1.95 0.59 0.01 0.85 1.95 0.59 0.01 Hoverflies Hoverflies χ χ

2 2 0.770 0.770

0.163 0.443 0.909 0.163 0.443 0.909 0.92 0.92 SD SD SD SD P P

RAYLESS IND. RAYED IND. RAYLESS IND. RAYED IND. Overdisp. index Random Variables Overdisp. indexOverdisp. Random Overdisp. index Random indexOverdisp. Variables Random Capitulum size Fixed Plant Pol. context Floral display Disk size Fixed Plant Pol. context Floral display Capitulum size Fixed Plant Pol. context Floral display Disk size Fixed Plant Pol. context Floral display

Results Results Results

27 27 27 27

random variable. Overdisp. index: a measure of overdispersion. Statistical significan random variable. Overdisp. index: a measure of overdispersion. Statistical significan

tor attraction for the sympatric site. tor attraction for the sympatric site.

1 1 1 1 1 1 1 1 1 1 1 1 Df Df

Variance Variance Variance Variance Variance

0.05 0.61 1.80 1.66 0.52 1.11 0.26 0.42 0.05 0.61 1.80 1.66 0.52 1.11 0.26 0.42 χ χ χ χ All groupsAll groupsAll

2 2 2 2

1.031 1.093 1.031 1.093

0.436 0.180 0.187 0.292 0.612 0.516 0.436 0.180 0.187 0.292 0.612 0.516 (Bees, Small flies, Bigflies, Hoverflies). Weanalysed (Bees, Small flies, Bigflies, Hoverflies). Weanalysed 0.22 0.72 0.22 0.72 SD SD SD SD P P

P P

Disk size, floral display and pollination Disk size, floral display and pollination Variance Variance Variance Variance Variance

(+) 7.73 (+) 7.73 <0.01 <0.01 2.83 2.74 1.61 0.68 0.70 2.83 2.74 1.61 0.68 0.70 0 0 χ χ

χ χ 2 2 2 2 1.144 0.864 1.144 0.864 Bees Bees

0.093 0.097 0.205 0.986 <0.01 0.403 0.093 0.097 0.205 0.986 <0.01 0.403

0.82 0.82 SD SD SD SD 0 0 P P

P P

Variance Variance Variance Variance Variance (-)2.87 (-)2.87 <0.01 <0.01 2.05 1.84 1.14 0.24 0.63 2.05 1.84 1.14 0.24 0.63 0 0 χ χ χ χ Small flies Small flies

2 2 2 2

0.985 0.951 0.985 0.951

0.152 0.175 0.945 0.621 0.428 0.090 0.152 0.175 0.945 0.621 0.428 0.090 1.07 1.07 SD SD SD SD 0 0 P P

P P

context were considered fixed explanatory vari context were considered fixed explanatory vari

separately rayed individuals (including intermediate individuals) and rayless separately rayed individuals (including intermediate individuals) and rayless

ces (<0.05) are shown in bold. Positive significant effects with (+). are signed ces (<0.05) are shown in bold. Positive significant effects with (+). are signed Variance Variance Variance Variance Variance

(+) 4.32 (+) 4.32 0.08 0.64 0.16 0.99 0.07 0.35 0.08 0.64 0.16 0.99 0.07 0.35 0 0 χ χ

χ χ 2 2 Big flies Big flies

2 2 1.005 0.739 1.005 0.739

0.424 0.694 0.787 0.556 0.424 0.694 0.787 0.556 0.77 0.99 0.04 0.77 0.99 0.04 SD SD SD SD 0 0 P P

P P

Variance Variance Variance Variance Variance

0.14 0.97 0.51 0.49 3.26 2.95 0.16 0.14 0.97 0.51 0.49 3.26 2.95 0.16 0 0 χ χ

2 2 Hoverflies Hoverflies

χ χ

2 2 0.999 0.999

1.04 1.04

0.705 0.325 0.477 0.071 0.086 0.685 0.705 0.325 0.477 0.071 0.086 0.685 0.70 0.70 SD SD SD SD SD 0 0 P P P P

ables ofthe visits rate of ofthe enti ables ofthe visits rate of ofthe enti

Results Results Results re re 29 29 29

Results

How does capitulum size affect pollinator attraction?

Larger capitulum sizes significantly increased visitation rates on rayed and intermediate individuals (Supplementary Table C), but not on rayless ones (Table 4). This increase in visitation rates was exclusively due to an increase in ray length (Table 5) and not due to disk size (Table 4). Longer rays significantly increased the visit of small flies and hoverflies, but not of bees and big flies (Table 5). Small flies were the functional group that showed a marginally negative significant relationship with larger disk sizes on rayed individuals (Table 4).

Is there a facilitation or a competing pattern between rayed and rayless phenotypes?

Natural variation in neighbourhood composition

The impact of the pollination context varied according with the floral phenotypes. Two distinct patterns were observed in this study: a positive, facilitative pattern regarding the rayed phenotypes, and a negative, competitive pattern for the intermediate and rayless phenotypes. Rayed plants were significantly more visited by big flies and hoverflies when surrounded by rayed and intermediate neighbours (Table 6). By contrast, intermediate and rayless plants, showed no significant positive effects in visitation rates when grown surrounded by other plants (Table 6). Indeed, bees showed significantly lower visitation rates to intermediate plants in the presence of other intermediate phenotype plants, and to rayless plants when those were surrounded by neighbours of the same phenotype (Table 6).

Results Results 29 re

Results

Table 4. The effect of disk size on floral visitor attraction for the sympatric site. Disk size, floral display and pollination context were considered fixed explanatory variables of the rate of visits of the entire pollinator assemblage (All groups), and of different pollinator groups (Bees, Small flies, Big flies, Hoverflies). We analysed separately rayed individuals (including intermediate individuals) and rayless Table 3. The effects of floral phenotype (rayless vs. fake-rayed), floral display and pollination context on pollinator attraction for the entire pollinator assemblageindividuals. (All Plant groups) identity and forwas the used different as a random variable. Overdisp. index: a measure of overdispersion. Statistical significances (<0.05) are shown in bold. Positive significant effects are signed with (+). pollinator functional groups (Ants, Bees, Big flies, Beeflies, Small flies and Hoverflies) for the rayless site. Plant identity was used as a random variable. Overdispersion index: a measure of overdispersion.

Statistical significances (<0.05) are shown in bold. Significantly positive effects are signed with (+).

ables of the rate of visits of the enti of the of rate visits of the ables

Variables All groups Bees Small flies Big flies P Hoverflies 0 SD SD 0.70 0.071 0.685 0.086 0.477 0.325 0.325 0.705 Df

All groups Ants Bees Big flies Beeflies Small flies Hoverflies 2 2 2 2 2 1.04

χ χ χ χ 0.999 χ 2

RAYED IND.

P P P P P χ

Hoverflies Hoverflies 2 2 2 2 2 2 2 2

χ Variables Df χ P χ P χ P χ P χ P χ P χ P Fixed 0 3.26 0.16 2.95 0.49 0.51 0.97 0.14

Variance Fixed Variance

Disk size 0.42 0.516 0.70 0.403 (-) 2.87 0.090 0.35 0.556 0.16 0.685

Ray 0.03 0.855 <0.01 0.982 0.11 0.742 0.88 0.349 5.01 0.025 2.01 0.156 0.01 0.909 <0.01 Floral display 1 0.26 0.612 (+) 7.73 0.63 0.428 0.07 0.787 2.95 0.086

phenotype 1 P

0.04

Pol. context 1 1.11 0.292 <0.01 0.986 0.24 P 0.621 (+) 4.32 3.26 0.071 0.039 <0.001 0.024 0 SD Floral display 1 (+) 4.25 (+) 10.95 0.11 0.737 0.98 0.322 (+) 5.07 0.37 0.543 0.59 0.443 SD 0.04 0.04 0.77 0.99 0.424 0.424 0.556 0.787 0.694

Pollination Results Variance SD Variance SD Variance SD Variance SD Variance SD Results 0.040 Random 0.34 0.563 1.66 0.198 (+) 4.03 0.55 0.458 0.05 0.816 0.04 0.838 1.95 0.163

context 1 1.005 1.005 0.739 0.739 2

Plant 0.52 0.72 0.68 0.82 flies Big 1.14 1.07 0.99 0.99 0.49 0.70 2 χ

χ 0 0.64 0.08 Overdisp. index 1.093 0.864 0.951 0.35 0.07 0.99 0.16 0.999 Variance SD Variance SD Variance SD Variance SD Variance SD Variance SD Variance SD 0.739 (+) 4.32 (+)

Variance Variance gnificant effects are signed with (+). signedare (+). with effects gnificant si Positive bold. in shown are (<0.05) ces

Random 2 2 2 2 2

RAYLESS IND. χ χ χ χ χ P and rayless individuals) intermediate (including individuals rayed separately P P P P Table 4. The effect of disk size on floral visitor attraction for the sympatric site. Disk size, floral display and pollination context were considered fixed explanatory variables of the rate of visits of the entire Plant 0.36 0.596 3.32 1.82 4.41 2.10 0 0 0.30 0.55 0 0 0.85 0.92

context were considered fixed explanatory vari explanatory fixed considered were context

Fixed

pollinator assemblage (All groups), and of different pollinator groups (Bees, Small flies, Big flies, Hoverflies). We analysed separately rayed individualsP (including intermediate individuals) and rayless

P Overdispersion 0 SD SD Table 3. The effects of floral phenotype (rayless vs. fake-rayed),0.957 floral display and0.589 pollination context0.327 on pollinator attraction1.011 for t he entire1.050 pollinator assemblageindividuals.0.985 (All Plant groups) identity0.770 and forwas the used different as a random variable. Overdisp. index: a measure of overdispersion. Statistical significances (<0.05)1.07 are shown in bold. Positive significant effects are signed with (+). 0.621 0.621 0.175 0.175 0.152 0.090 0.428 0.945 index pollinator functional groups (Ants, Bees, Big flies, Beeflies, Small flies and Hoverflies) for the rayless site. Plant identity was used as a random variable. Overdispersion index: a measure of overdispersion.Capitulum size 1 1.66 0.187 1.61 0.205 <0.01 0.945 0.16 0.694 0.51 0.477 Floral display 1 1.80 0.180 2.74 0.097 1.84 0.175 0.64 0.424 0.97 0.325

0.985 0.985 Statistical significances (<0.05) are shown in bold. Significantly positive effects are signed with (+). 0.951

2 2

Pol. context 1 0.61 0.436 2.83 0.093 Small flies 2.05 0.152 0.08 0.77 0.14 0.705 χ χ 0 0.24 1.84 2.05 0.63 1.14 <0.01 SD Variance SD Variance SD 2.87 (-) Variance SD Variance SD Variance Variance Random Variance

Plant 0.05 0.22 0 0 0 0 0 0 0 0 Variables All groups Bees Small flies Big flies Hoverflies

Overdisp. index 1.031 1.144 0.985 1.005 1.04 P

P 0 SD SD

0.82 Df 0.986 0.986 0.403 0.403 <0.01 0.205 0.097 0.093 All groups Ants Bees Big flies Beeflies Small flies Hoverflies 2 2 2 2 2

RAYED IND. χ P χ P χ P χ P χ P

2 2 2 2 2 2 2 Bees Bees 1.144 1.144 0.864 χ χ χ χ χ χ χ Fixed 2 Variables Df 2

P P P P P P P χ

χ 0 0.70 0.68 1.61 2.74 2.83 Fixed <0.01 (+) 7.73 (+)

Variance Variance Disk size 1 0.42 0.516 pollination and display floral size, Disk 0.70 0.403 (-) 2.87 0.090 0.35 0.556 0.16 0.685 Ray

0.025 0.03 0.855 <0.01 0.982 0.11 0.742 0.88 0.349 5.01 2.01 0.156 0.01 0.909Floral display 1 0.26 0.612 (+) 7.73 <0.01 0.63 0.428 0.07 0.787 2.95 0.086 phenotype 1

Pol. context 1 1.11 0.292 <0.01 0.986 0.24 0.621 (+) 4.32 0.04 3.26 0.071

Floral display (+) 4.25 0.039 (+) 10.95 <0.001 0.11 0.737 0.98 0.322 (+) 5.07 0.024 0.37 0.543 0.59 0.443

1 P

P SD SD 0.72 0.22 (Bees, Small flies, Big flies, Hoverflies). We analysed Hoverflies). Big flies, flies, Small (Bees, 0.292 0.292 0.516 0.612 0.187 0.180 0.180 0.436

Pollination 0.34 0.563 1.66 0.198 (+) 4.03 0.040 0.55 0.458 0.05 0.816 0.04 0.838 1.95 0.163Random 27 Variance SD Variance SD Variance SD Variance SD Variance SD 29 context 1 Plant 0.52 0.72 0.68 0.82 1.14 1.07 0.99 0.99 0.49 0.70

1.093 1.093 1.031

2 2

All groups χ

Overdisp. index 1.093 0.864 0.951χ 0.999 Variance SD Variance SD Variance SD Variance SD Variance SD Variance SD Variance SD 0.739 1.11 0.42 0.26 0.52 1.66 1.80 0.61 0.05

Variance Random 2 2 2 2 2 Variance χ χ χ χ χ

RAYLESS IND. P P P P P Plant 0.36 0.596 3.32 1.82 4.41 2.10 0 0 0.30 0.55 0 0 0.85 0.92

Fixed

Overdispersion 0.957 0.589 0.327 1.011 1.050 0.985 0.770 Df index 1 1 1 1 1 1 Capitulum size 1 1.66 0.187 1.61 0.205 <0.01 0.945 0.16 0.694 0.51 0.477

Floral display 1 1.80 0.180 site. sympatric the for attraction tor 2.74 0.097 1.84 0.175 0.64 0.424 0.97 0.325

of overdispersion. Statistical significan Statistical overdispersion. of measure a index: Overdisp. variable. random Pol. context 1 0.61 0.436 2.83 0.093 2.05 0.152 0.08 0.77 0.14 0.705

SD Variance SD Variance SD Variance SD Variance SD Random Variance

Plant 0.05 0.22 0 0 0 0 0 0 0 0 Pol. context context Pol. Overdisp. index 1.031 1.144 0.985Fixed size Disk Floral display context Pol. 1.005Plant Fixed Capitulum size Floral display 1.04 Plant Variables Random Overdisp. index Overdisp. index Random Random RAYED IND. IND. RAYLESS

The effect of disk size on floral visi floral on size disk of effect The

27 29

pollinator assemblage (All groups), and of different pollinator groups pollinator different and of groups), (All assemblage pollinator individuals. Plant identity was used as a as used was identity Plant individuals. Table 4. Table 4.

le, i fis Hvrle) o te ae idvdas (including individuals rayed overdispersion. Statistical significances (<0.05) are shown in b the for Hoverflies) flies, Big flies, 5. Table le, i fis Hvrle) o te ae idvdas (including individuals rayed overdispersion. Statistical significances (<0.05) are shown in b the for Hoverflies) flies, Big flies, 5. Table The effects of ray length, floral display floral length,ray of effects The The effects of ray length, floral display floral length,ray of effects The

Overd Random Fixed Overd Random Fixed Variables Variables Plant Pollination context Floral display Ray Plant Pollination context Floral display Ray length length

ispersion ispersion

and pollination context on pollinator attrac pollinator on context pollination and and pollination context on pollinator attrac pollinator on context pollination and index index index index

Df Df 1 1 1 1 1 1

old. Positive significant old. Positive significant Results nemdae niiul) in individuals) intermediate nemdae niiul) in individuals) intermediate

Variance Variance Variance Variance All groupsAll groupsAll (+) 6.64 (+) 6.64

1.107 1.107 0.43 1.71 0.54 0.43 1.71 0.54 χ χ

2 2

Table 5. The effects of ray length, floral display and pollination context on pollinator attraction for the entire pollinator assemblage (All groups) and for different pollinator functional groups (Bees, Small

<0.01 <0.01 0.191 0.461 0.191 0.461 0.65 0.65

flies, Big flies, Hoverflies) for the rayed individuals (including SD intermediate individuals) in the sympatric site. Plant identity SD was used as a random variable. Overdispersion index: a measure of P P overdispersion. Statistical significances (<0.05) are shown in bold. Positive significant effects are signed with (+).

effects are signed with (+). effects are signed with (+).

Bees Bees Variance Variance Variance (+) 7.62 (+) 7.62 tion for the entire pollinator assemblage pollinator entire the for tion tion for the entire pollinator assemblage pollinator entire the for tion <0.01 <0.01

0.70 0.26 0.70 0.26 χ χ 0.849 0.849

2 2

h smarc ie Pat ident Plant site. sympatric the h smarc ie Pat ident Plant site. sympatric the

<0.010 <0.010 0.950 0.614 0.950 0.614 0.84 0.84 SD SD SD

P P All groups Bees Small flies Big flies Hoverflies

2 2 2 2 2 Df χ χ χ χ χ Variables P P P P P

Fixed Small flies Small flies Variance Variance Variance (+) 9.91 (+) 9.91

0.91 0.28 0.31 0.91 0.28 0.31 <0.01 χ 0.002 χ 0.027

Ray length 0.967 (+) 6.64 0.26 0.614 (+) 9.910.967 1.72 0.189 (+) 4.91 2 2 1

Floral display 1 0.54 0.461 (+) 7.62 <0.010 0.31 0.580 0.04 0.834 (+) 3.64 0.056

0.002 0.002 0.600 0.580 0.600 0.580 t ws sd s rno variable. random a as used was ity variable. random a as used was ity 0.95 0.95 0.030 SD SD Pollination context SD 1.71 0.191 <0.01 0.950 0.28 0.600 (+) 4.71 (+) 3.74 0.053 P P

1 (All groups) and for different poll different for and groups) (All poll different for and groups) (All

Variance SD Variance SD Variance SD Variance SD Variance SD

Big flies Random Big flies Variance Variance Variance Variance (+) 4.71 (+) 4.71 0.89 0.04 1.72 Plant 0.89 0.43 0.04 0.651.72 0.70 0.84 0.91 0.95 0.89 0.95 0.39 0.63 χ χ 0.755 0.755

2 2

Overdispersion index 1.107 0.849 0.967 0.755 1.021

0.030 0.834 0.189 0.030 0.834 0.189 SD SD SD SD 0.95 0.95 P P

Small flies,Big flies, Hoverflies). Statistical 6.Table Small flies,Big flies, Hoverflies). Statistical 6.Table Hoverflies Hoverflies Variance Variance Variance Variance (+) 3.74 (+) 3.64 (+) 4.91 (+) 3.74 (+) 3.64 (+) 4.91 vriprin ne: maue of measure a index: Overdispersion vriprin ne: maue of measure a index: Overdispersion inator functional grou functional inator inator functional grou functional inator χ χ 1.021 1.021 2 2

0.39 0.39 The effect of particular neighbourhoods for different focal in The effect of particular neighbourhoods for different focal in

Focal rayless individuals individuals intermediateFocal rayedFocal individuals Focal rayless individuals individuals intermediateFocal rayedFocal individuals

0.053 0.056 0.027 0.053 0.056 0.027 SD SD SD SD Neigbourhoods of Neigbourhoods of Neigbourhoods Neigbourhoods of Neigbourhoods of Neigbourhoods of Neigbourhoods of Neigbourhoods 0.63 0.63 P P

ps (Bees ps ps (Bees ps Results Results Results , Small Small , , Small Small , 30 Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate 30 30 30 30 Rayless Rayless Rayless Rayless Rayless Rayless Rayed Rayed Rayed Rayed Rayed Rayed

significances (<0.05) are shown in bold. Ns: significances (<0.05) are shown in bold. Ns:

All groupsAll groupsAll ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

dividuals (rayed, intermediate and dividuals (rayed, intermediate and

b b b b b b b b = -0.51, = -0.65, P = -0.51, = -0.65, P = -0.04, = -0.19, P = -0.04, = -0.19, P Bees Bees P P ns ns ns ns ns ns ns ns ns ns

> 0.10; > 0.10; P P P P = = = = =

= 0.071 = 0.055 = 0.071 = 0.055 0.022 0.023 0.022 0.023 b b

= regression coefficient. = regression coefficient.

b b = -0.09, = -0.09, rayless), for the entire polli rayless), for the entire polli Small flies Small flies ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

P P

= 0.109 = 0.109

b b b b = 0.05, = 0.05, =0.06, =0.06, Big flies Big flies nator assemblage (A nator assemblage (A ns ns ns ns ns ns ns ns ns ns ns ns ns ns P P P P

= =

= 0.090 = 0.090 0.045 0.045

b b b b = = 0.02, = 0.02, = 0.12, = 0.12, Hoverflies Hoverflies ll groups), and for different pollinator (Bees, guilds ll groups), and for different pollinator (Bees, guilds ns ns ns ns ns ns ns ns ns ns ns ns ns ns P P

P P = = = = 0.011 0.011 0.023 0.023

Results Results Results 31 31 31

Small flies,Big flies, Hoverflies). Statistical Table 6.Table Small flies,Big flies, Hoverflies). Statistical Table 6.Table The effect of particular neighbourhoods for different focal in The effect of particular neighbourhoods for different focal in Focal rayless individuals individuals intermediateFocal rayedFocal individuals Focal rayless individuals individuals intermediateFocal rayedFocal individuals Neigbourhoods of Neigbourhoods of Neigbourhoods of Neigbourhoods of Neigbourhoods of Neigbourhoods Neigbourhoods of Neigbourhoods Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Rayless Rayless Rayless Rayless Rayless Rayless Rayed Rayed Rayed Rayed Rayed Rayed significances (<0.05) are shown in bold. Ns: significances (<0.05) are shown in bold. Ns:

All groupsAll groupsAll ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

dividuals (rayed, intermediate and dividuals (rayed, intermediate and

Results b b b b b b b b = -0.51, = -0.65, P = -0.51, = -0.65, P = -0.04, = -0.19, P = -0.04, = -0.19, P

Bees Bees P P ns ns ns ns ns ns ns ns ns ns

> 0.10; > 0.10; P P P P ======

Table 6. The effect of particular neighbourhoods for= 0.071 different focal= 0.055 individuals (rayed, intermediate and rayless), for = 0.071 the entire polli= 0.055 nator assemblage (All groups), and for different pollinator guilds (Bees, 0.023 0.022 0.023 0.022 Small flies, Big flies, Hoverflies). Statistical significances (<0.05) are shown in bold. Ns: P > 0.10; b = regression coefficient. b b

= regression coefficient. = regression coefficient.

b b = -0.09, = -0.09, rayless), for the entire polli All groups Bees Small flies Big flies Hoverflies rayless), for the entire polli Small flies Small flies ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

P P

Focal rayed individuals = 0.109 = 0.109

Rayed ns ns ns b = 0.05, P = 0.045 b = 0.02, P = 0.023

Intermediate ns ns ns ns b = 0.12, P = 0.011 Neigbourhoods of b b b b = 0.05, = 0.05, =0.06, Rayless ns ns =0.06, ns b = 0.06, P = 0.090 ns

Big flies Big flies nator assemblage (A Focal intermediate nator assemblage (A ns ns ns ns ns ns ns ns ns ns ns ns ns ns P P P P

= =

= 0.090 individuals = 0.090 0.045 0.045 Rayed ns ns ns ns ns

0.023 Neigbourhoods of Intermediate ns b = -0.65, P = ns ns ns b b b b = = 0.02, = = 0.02, = 0.12, Rayless ns b = -0.19, P = 0.055 = 0.12, ns ns ns Hoverflies Hoverflies ll groups), and for different pollinator (Bees, guilds ll groups), and for different pollinator (Bees, guilds ns ns ns ns ns ns ns ns ns ns ns ns ns ns P P

P Focal rayless individuals P = = = = 0.011 0.011 0.023 0.023 Rayed ns b = -0.04, P = 0.071 b = -0.09, P = 0.109 ns ns

Intermediate ns ns ns ns ns Neigbourhoods of Rayless ns b = -0.51, P = 0.022 ns ns ns

Results Results Results 31 31 31 31

Results

Experimental manipulation of neighbour’s density

The removal of all Anacyclus neighbours did not significantly reduce the total visits of the floral visitors in both sites (Figure 5 and 6). However, in the rayless site, the removal significantly decreased the visitation rate of hoverflies to rayless plants (Figure 7). The other pollinator’s functional groups were not affected by the experimental removal of the neighbourhood (results not shown).

0.14

0.12

0.10

0.08 Visits/ 5 minutes) 0.06

0.04 Visitation rate ( 0.02

0.00 Neighbourhood Control removed

Figure 5. The effect of removing the neighbourhood on floral visitors within the rayed site. Least square means (± confidence intervals) of the visitation rate (number of visits per 5 minutes intervals) in the rayed site, given for the entire pollinator assemblage, with plants divided according with their surrounding context: neighbours removed and control. The black dots represent the rayed phenotype while the white dots represent the rayless phenotype. No statistically significant differences were found between groups.

Results

0.5

0.4

0.3

0.2

0.1 Visitation rate (Visits/ 5 minutes) rate (Visits/ Visitation

0.0 Neighbourhood Control removed Figure 6. The effect of removing the neighbourhood on floral visitors within the rayless site. Least square mean (± confidence intervals) of the visitation rate (number of visits per 5 minutes intervals) in the rayless site, given for the entire pollinator assemblage, with plants divided according with their surrounding context: neighbours removed and control. The black dots represent the rayed phenotype while the white dots represent the rayless phenotype. No statistically significant differences were found between groups.

0.16 ab b 0.14

0.12 ab 0.10

0.08

0.06 a X Data 0.04

0.02 Visitation rate (Visits/5 minutes)

0.00

Neighbourhood Control removed

Figure 7. The effect of removing the neighbourhood on hoverflies within the rayless site. Least square mean (± confidence intervals) of the visitation rate (number of visits per 5 minutes intervals) in the rayless site, given for the pollinator group hoverflies, with plants divided according with their surrounding context: neighbours removed and control. The black dots represent the rayed phenotype while the white dots represent the rayless phenotype. Different letters refer to statistically significant differences at P < 0.05.

Discussion

The two species of Anacyclus studied in this work are generalists that can be pollinated by a vast array of pollinators from different functional groups. Our observations have shown that Anacyclus capitula were visited by insects from at least 17 different families, from several orders (Table 1). However, three functional groups of diptera (hoverflies, big and small flies) and the bees (considering different functional groups together) were the most frequent floral visitors. Interestingly, diptera and bees showed different patterns of visitation and could be exerting contrasting patterns of selection, affecting the dynamics of the hybrid zone between these two species. Below, the implications of different preferences of the main floral visitors in the evolution and maintenance of floral rays and in the degree of reproductive isolation in the A. clavatus and A. valentinus contact zone are discussed.

Similarly to what was observed before in other studies, our results have shown that rays presented a positive effect in visitation rates. For example, diptera visited preferentially rayed individuals and, among them, individuals with larger rays, regardless of the size of the central yellow disk. This suggests that the production of conspicuous structures could be an adaption to enhance pollinator attraction and guarantee high outcrossing rates between rayed phenotypes (Marshall & Abbott 1984; Stuessy et al. 1986; Sun & Ganders 1990; Nielsen et al. 2002; Celedón-Neghme et al. 2006; Penet et al. 2012). However, not all functional groups of pollinators showed a clear preference for rayed phenotypes. In the particular case of bees, this group of insects was indifferent to the presence of rays and visited preferentially larger plants that presented a higher number of capitula blooming simultaneously. In the absence of other forms of selection, visitation patterns of bees and dipteran visitors might cause a contrasting genetic flow in the hybrid zone, with insect abundance regulating this effect. Although the role of ethological isolation in speciation remains controversial (Aldridge & Campbell 2006), our results from the sympatric site suggest contrasting effects of pollinator’s behaviour, with dipteran pollinators promoting assortative mating between rayed individuals, ultimately leading to an isolation pattern. By opposition, bees, by visiting plants independently of the phenotype, will mix pollen from both species, diminishing the pollinator-mediated effect of diptera (Schmid-Hempel & Speiser 1988; Thompson 2001). Since hybrids between the two plant species are able to produce viable seeds (I. Álvarez 2013, personal communication), this non-discriminated visitation pattern mediated by bees may ultimately cause introgression from one species to the other. Gómez and colleagues found a similar effect in Erysimum mediohispanicum, with some pollinator groups selecting for different corolla shapes, whereas some other groups visited flowers indiscriminately, leading to an attenuate effect and causing a contrasting selection (Gómez et al. 2008b). In this way, the levels of reproductive isolation and ultimately the dynamics of the hybrid zone will be mostly dependent on pollinator abundance (Emms & Arnold 2000; Thompson 2001). In order to better understand the potential selective role of these functional groups, we are developing studies focused in assessing the efficiency of these pollinators and their impact in plant fitness. Furthermore, future studies of fine-scale genetic patterns and parentage identification are needed to assess if differential behaviour between dipteran and bees are driving different matting patterns.

The different visitation patterns of floral visitors observed in the sympatric site were only partially supported by the phenotypic manipulations of rayed phenotypes. This phenotypic manipulations might

Discussion

have failed maybe due to our simplistic ray models, which might not completely mimic the real shapes (i.e., manipulated rays had a straight cut shape, while real rays are elipsoid; see Figure 1 A and D; Gómez et al. 2008b). Furthermore the manipulated rays showed some UV reflection, whereas natural ones do not reflect UVs (results not shown).

Regardless of being visited by one of the main pollinator groups, bees, rayless plants still received a lower number of visits than rayed ones. The lower visitation rate of rayless plants in the sympatric site suggests that rayed plants could be successfully competing with rayless plants for pollination services. Could we then expect that rayless phenotypes would slowly disappear in this contact zone? Considering the preliminary results of other studies that we are performing in the same contact zone, it seems that this is not probable, as both rayed and rayless plants were not pollen limited and both types of capitula showed similar levels of fruit set and absence of seed predation (R. Torices and J. Cerca de Oliveira, unpublished data). In addition, not every insect preferred rayed phenotypes. For example, bees in the sympatric site, and general pollinators in the remaining populations selected larger floral displays, independently of the floral phenotype. Floral visitors are opportunist insects with labile preferences that compete for floral rewards, thus they might bypass a crowded conspicuous flower for a less conspicuous one, if the flower has more resources (Wesselingh & Arnold 2000; Dilley, Wilson & Mesler 2000). Additionally, bigger plants guarantee a higher density of inflorescences in a small area, resulting in a bigger concentration of resources for pollinators, attracting pollinators regardless of their phenotype. Finally, beyond pollinator preferences, rayless plants might bear advantages in “stressful conditions”, as they do not have the cost to produce and subsequently maintain the extra structures (Chaplin & Walker 1982; Charlesworth & Charlesworth 1987; Galen, Sherry & Carroll 1999; Andersson 2001), reallocating resources for seed production (Andersson 1999).

Our observations also revealed that the phenotype of the neighbours plays an important role in driving pollinator’s behaviour. Pollinator attraction did not depend on the focal plant phenotype, only, with the phenotypes of the neighbouring plants affecting the visitation rate of the main floral visitors. When generalist co-flowering plant species occur in the same area they are often obliged to share pollinators, and inter-specific plant-plant interactions arise. These interactions can be neutral, competitive or facilitative (Landry 2013; Ye et al. 2013) and two opposing views emerged: 1) the struggle for existence should be greater between closely related species than between distantly related species, due to similarity in habits and constitution, causing a high overlapping and therefore a direct competition (Violle et al. 2011; Beltrán, Valiente-Banuet & Verdú 2012); 2) facilitation can happen between related species as a by-product of the similarities in the pollinator groups that they attract (Ghazoul 2006; Sargent & Ackerly 2008; Devaux & Lande 2009; Beltrán et al. 2012). Broadly, our results confirm the existence of the three types of interaction when analysing distinct functional pollinator groups. In the sympatric population, rayed phenotypes received more visits from big flies and hoverflies when in the presence of conspicuous rayed neighbours. By opposition, rayless and intermediate plants significantly competed for pollinators (bees). Using the Asteraceae species, Lasthenia fremontii, Sargent and colleagues (Sargent et al. 2011) found that this species would be less pollen limited when occurring in

Discussion

communities composed of close relatives than when occurring in communities composed of more distant relatives. Therefore, disentangling the adaptive role of floral polymorphism should be assessed considering the potential interactions between the focal plant’s phenotype and the phenotypes of their neighbours, which may affect pollinator behaviour and, most likely, plant fitness.

Conclusion

In conclusion, we found that the production of rays influenced the probability of being visited by insects in the studied sympatric site. However, not all groups of floral visitor showed a preference for the rayed phenotype. Also, contrasting selection was found between pollinator groups for the presence of rays, demonstrating that pollinators might be important agents of selection on floral traits for generalist plants. Finally, we found support for the importance of the neighbours’ phenotype when assessing pollinator preference on a focal individual. Rayed plants benefited from having other conspicuous neighbours, whereas rayless and intermediate phenotypes significantly competed for pollinators. All these differential behavioural patterns of floral visitors might affect gene flow within the hybrid zone between A. clavatus and A. valentinus influencing its future dynamics.

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Appendix

individuals. A. TableSupplementary Supplementary Table A. TableSupplementary individuals.

Plant, capitulum and surrounding traits of thetraitssurroundingcapitulum andofPlant, Plant, capitulum and surrounding traits of thetraitssurroundingcapitulum andofPlant, n density Neighbour Pollination context Neighbourhood Ray length (mm) Disk diameter (mm) diameterCapitulum (mm) Number of rays Capitulum Plant dimension (cm Height(mm) Floral display Plant Traits n density Neighbour Pollination context Neighbourhood Ray length (mm) Disk diameter (mm) diameterCapitulum (mm) Number of rays Capitulum Plant dimension (cm Height(mm) Floral display Plant Traits

2 2 ) )

All phenotypes All phenotypes 516.41 ± 79.02 220.53 ± 10.07 516.41 ± 79.02 220.53 ± 10.07 13.22 0.54± 21.20 0.87± 13.22 0.54± 21.20 0.87± Anacyclus AppendixAnacyclus 4.16 ± 0.64 7.44 ± 1.19 3.99 ± 0.51 5.78 ± 0.52 5.99 ± 0.58 4.16 ± 0.64 7.44 ± 1.19 3.99 ± 0.51 5.78 ± 0.52 5.99 ± 0.58 Mean SE ± Mean SE ±

89 89

plants in the sympatricStatisticthe site. plantsin plants in the sympatricStatisticthe site. plantsin

Supplementary Table A. Plant, capitulum and surrounding traits of the Anacyclus plants in the sympatric site. Statistical significances (<0.05) obtained by a Kruskal-Wallis Test are shown in bold. n = 89

individuals. 568.96 ± 135.06 568.96 ± 135.06 220.17 ± 15.01 220.17 ± 15.01 13.24 ± 0.93 28.89 0.60± 13.24 ± 0.93 28.89 0.60± 6.41 ± 0.89 6.41 ± 0.89 4.24 ± 1.16 6.25 ± 1.62 7.83 ± 0.57 9.40 ± 0.37 4.24 ± 1.16 6.25 ± 1.62 7.83 ± 0.57 9.40 ± 0.37 Mean SE ± Mean SE ± Rayed Rayed 41 41 All phenotypes Rayed Intermediate Rayless

Traits Mean ± SE Mean ± SE Mean ± SE Mean ± SE P Df

Plant

Floral display 5.99 ± 0.58 6.41 ± 0.89 7.91 ± 2.04 4.70 ± 0.62 0.39 2 Height (mm) 220.53 ± 10.07 220.17 ± 15.01 223.57 ± 29.57 219.71 ± 15.38 0.10 2 506.26 ± 144.96 506.26 ± 144.96 223.57 ± 29.57 223.57 ± 29.57 Intermediate Intermediate al significances (<0.05) obtained by a Kruskal-Wallis Test are shown in bold. n = 89= n bold. inshownKruskal-WallisTest are a by significances(<0.05)obtainedal al significances (<0.05) obtained by a Kruskal-Wallis Test are shown in bold. n = 89= n bold. inshownKruskal-WallisTest are a by significances(<0.05)obtainedal 13.39 4.07± 14.50 2.03± 19.26 1.27± 13.39 4.07± 14.50 2.03± 19.26 1.27±

4.93 ± 1.42 2 2.38 ± 1.33 9.20 ± 0.63 7.91 ± 2.04 4.93 ± 1.42 2.38 ± 1.33 9.20 ± 0.63 7.91 ± 2.04 Plant dimension (cm ) 516.41 ± 79.02 568.96 ± 135.06Mean SE ± 506.26 ± 144.96 457.23 ± 115.34 0.95 Mean SE ± 2 14 14

Capitulum

<0.0001 Number of rays 5.78 ± 0.52 9.40 ± 0.37 9.20 ± 0.63 0.00 ± 0.00 2

Capitulum diameter (mm) 21.20 ± 0.87 28.89 ± 0.60 19.26 ± 1.27 12.72 ± 0.31 <0.0001 2

Disk diameter (mm) 13.22 ± 0.54 13.24 ± 0.93 14.50 ± 2.03 12.68 ± 0.30 0.74 2 457.23 ± 115.34 457.23 ± 115.34 219.71 ± 15.38 219.71 ± 15.38 <0.0001 12.68 0.30± 12.72 0.31± Ray length (mm) 12.68 0.30± 3.9912.72 0.31± ± 0.51 7.83 ± 0.57 2.38 ± 1.33 0.00 ± 0.00 2 3.74 ± 0.74 6.44 ± 1.70 0.00 ± 0.00 0.00 ± 0.00 4.70 ± 0.62 3.74 ± 0.74 6.44 ± 1.70 0.00 ± 0.00 0.00 ± 0.00 4.70 ± 0.62 Mean SE ± Mean SE ± Rayless Rayless 34 Neighbourhood34

Pollination context 7.44 ± 1.19 6.25 ± 1.62 13.39 ± 4.07 6.44 ± 1.70 0.22 2

Neighbour density 4.16 ± 0.64 4.24 ± 1.16 4.93 ± 1.42 3.74 ± 0.74 0.74 2

with data from all phenotypes (n = 89); while for correlation coeffic highlighted in bold. All the Supplementary Table B: with data from all phenotypes (n = 89); while for correlation coeffic highlighted in bold. All the Supplementary Table B: <0.0001 <0.0001 <0.0001 <0.0001 n <0.0001 <0.0001 89 <0.0001 41 14 34

0.74 0.22 0.74 0.95 0.10 0.39 0.74 0.22 0.74 0.95 0.10 0.39 P P

Df Df Df Df 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Pearson correlation coefficients of plant, capitulum and surrounding traits for all plants in the sympatric Statisticallysite. Pearson correlation coefficients of plant, capitulum and surrounding traits for all plants in the sympatric Statisticallysite.

P P Traits Traits Capitulum Plant Neighbourhood Capitulum Plant Neighbourhood values were adjusted for multiple tests. values were adjusted for multiple tests. Appendix Appendix Appendix Neighbour density Neighbour Pollination context Ray length Disk diameter diameterCapitulum Dimension Height Floral display density Neighbour Pollination context Ray length Disk diameter diameterCapitulum Dimension Height Floral display Number of rays Number of rays

46 46 46 46

Floral display Floral display - 0.19- 0.04- 0.02- 0.19- 0.04- 0.02- 0.79 0.79 0.00 0.14 0.06 0.02 0.00 0.14 0.06 0.02

ients of the of number raysan ients of the of number raysan Correlation coefficients for all plant Correlation coefficients for all plant

Plant Plant Height Height - 0.04- 0.04- -0.15 -0.15 0.42 0.64 0.42 0.64 0.29 0.08 0.02 0.29 0.08 0.02

Dimension Dimension - 0.20- 0.05- 0.20- 0.05- 0.06 0.01 0.10 0.09 0.06 0.01 0.10 0.09

d ray length variables only d ray length variables only

Nº of rays of Nº rays of Nº - 0.31- 0.31- 0.31 0.31 0.04 0.06 0.15 0.04 0.06 0.15

traits and neighbourhood traits, additionall traits and neighbourhood traits, additionall

Capitulum Capitulum diameter diameter 0.74 0.74 0.12 0.02 0.01 0.12 0.02 0.01

Capitulum Capitulum intermediate and rayed phenoty intermediate and rayed phenoty diameter diameter -0.78 -0.78 0.10 0.13 0.10 0.13 Disk Disk

Ray Ray - 0.15- 0.15- 0.02 0.02

length length

significant Pearson c significant Pearson c y for capitulum and disk diameter were performed y for capitulum and disk diameter were performed

Neighbourhood Neighbourhood pes datawere used(n=55). pes datawere used(n=55). Pollination Pollination context context 0.61 0.61

orrelation coefficients are orrelation coefficients are

Appendix Appendix Appendix 47 47 47

P P Traits Traits Neighbourhood Capitulum Plant Neighbourhood Capitulum Plant values were adjusted for multiple tests. values were adjusted for multiple tests. Neighbour density Neighbour Pollination context Ray length Disk diameter diameterCapitulum Dimension Height Floral display density Neighbour Pollination context Ray length Disk diameter diameterCapitulum Dimension Height Floral display Number of rays Number of rays

Floral display Floral display - 0.19- 0.04- 0.02- 0.19- 0.04- 0.02- 0.79 0.79 0.00 0.14 0.06 0.02 0.00 0.14 0.06 0.02

Appendix ients of the of number raysan ients of the of number raysan Correlation coefficients for all plant Correlation coefficients for all plant

Plant Plant Height Height - 0.04- 0.04- -0.15 -0.15 0.42 0.64 0.42 0.64 0.29 0.08 0.02 0.29 0.08 0.02

Supplementary Table B: Pearson correlation coefficients of plant, capitulum and surrounding traits for all plants in the sympatric site. Statistically significant Pearson correlation coefficients are highlighted in bold. All the P values were adjusted for multiple tests. Correlation coefficients for all plant traits and neighbourhood traits, additionally for capitulum and disk diameter were performed

with data from all phenotypes (n = 89); while for correlation coefficients of the number ofDimension rays and ray length variables only intermediate and rayed phenotyDimension pes data were used (n = 55). - 0.20- 0.05- 0.20- 0.05- 0.06 0.01 0.10 0.09 0.06 0.01 0.10 0.09

d ray length variables only d ray length variables only

Nº of rays of Nº rays of Nº - 0.31- 0.31- 0.31 0.31 0.15 0.04 0.06 0.15 0.04 0.06

Plant Capitulum Neighbourhood traits and neighbourhood traits, additionall traits and neighbourhood traits, additionall Traits Capitulum Disk Pollination Floral display Height Dimension Nº of rays Ray length diameter diameter context

Capitulum Capitulum Plant diameter diameter 0.74 0.74 0.01 0.12 0.02 0.01 0.12 0.02

Floral display

Capitulum Capitulum Height 0.02 intermediate and rayed phenoty intermediate and rayed phenoty diameter Dimension 0.79 0.02 diameter -0.78 -0.78 0.10 0.13 0.10 0.13 Disk Disk

Capitulum Number of rays 0.06 - 0.04 0.09 Ray Ray - 0.15- 0.15- 0.02 0.02

length Capitulum diameter 0.14 0.08 0.10length 0.15

Disk diameter - 0.02 0.29 0.01 - 0.31 0.01 significant Pearson c significant Pearson c y for capitulum and disk diameter were performed 0.31 0.74 -0.78 y for capitulum and disk diameter were performed

Ray length 0.00 -0.15 0.06

Neighbourhood Neighbourhood pes datawere used(n=55). Neighbourhood pes datawere used(n=55). Pollination Pollination context context

Pollination0.61 context - 0.04 0.64 - 0.05 0.61 0.06 0.02 0.13 - 0.15

Neighbour density - 0.19 0.42 - 0.20 0.04 0.12 0.10 0.02 0.61

orrelation coefficients are orrelation coefficients are

Appendix Appendix Appendix

47 47 47 47 47

Supplementary Table C. Table Supplementary

Supplementary Table C. Table Supplementary provided. Statistical significances (<0.0 and individuals rayed the for Hoverflies) flies, Big flies, Small provided. Statistical significances (<0.0 and individuals rayed the for Hoverflies) flies, Big flies, Small

on context on pollinator attraction pollinator on context pollinati on and display floral size,capitulum of effect The on context on pollinator attraction pollinator on context pollination and display floral size,capitulum of effect The index Overdispersion Random Fixed index Overdispersion Random Fixed context size context size Variables Variables 5) are shown in bold. Positive and neg 5) are shown in bold. Positive and neg Plant Pollination Floral display Capitulum Plant Pollination Floral display Capitulum

1 1 1 Df 1 1 1 Df

Variance Variance (+) 11.04 (+) 11.04 intermediate individuals for the sympatric population. Plant Plant population. sympatric the for individuals intermediate intermediate individuals for the sympatric population. Plant Plant population. sympatric the for individuals intermediate groups groups groups 0.38 1.54 0.83 0.38 1.54 0.83 χ χ

2 2

All All

1.120 1.120 Appendix <0.001 <0.001 0.215 0.362 0.215 0.362 ative significances are signed with (+). ative significances are signed with(+).

0.62 0.62 SD SD SD P P

Supplementary Table C. The effect of capitulum size, floral display and pollination context on pollinator attraction for the entire pollinator assemblage (Total), and for different pollinator guilds (Bees,

Small flies, Big flies, Hoverflies) for the rayed individuals and intermediate individuals for the sympatric population. Plant identity was used as a random variable. A measure of overdispersion is also

provided. Statistical significances (<0.05) are shown in bold. Positive and negative significances are signedBees with (+). Bees Variance Variance Variance (+) 7.62 (+) 7.62 <0.01 <0.01 0.68 0.03 0.68 0.03 χ χ

2 2

0.859 0.859

All

<0.01 <0.01

0.936 0.865 Bees Small flies Big flies 0.936 Hoverflies0.865 0.83 0.83

groups SD SD P P 2 2 2 2 2 Variables Df χ P χ P χ P χ P χ P

Fixed Small flies Small flies Variance Variance Variance

Capitulum (+) 7.69 (+) 7.69 for the entire pollinator assembl pollinator entire the for for the entire pollinator assembl pollinator entire the for (+) 11.040.92 <0.0010.18 0.29 0.03 0.865 (+) 7.69 <0.01 0.92 1.98 0.1600.18 0.29 (+) 9.23 0.020

1 χ χ

size 2 2

0.982 0.982

Floral display 1 0.83 0.362 (+) 7.62 <0.01 0.29 0.588 0.03 0.871 (+) 4.24 0.040 identity was used as a random variable. A measure of overdispersion is also is overdispersion of measure A variable. random a as used was identity also is overdispersion of measure A variable. random a as used was identity

Pollination 0.673 0.588 <0.01 0.673 0.588 <0.01 0.035 0.96 1.540.96 0.215 <0.01 0.936 0.18 0.673 (+) 4.44 (+) 3.11 0.078 SD SD 1 SD P context P

Variance SD Variance SD Variance SD Variance SD Variance SD

Variance Variance Big flies Variance Variance Big flies (+) 4.44 Random (+) 4.44 0.28 0.03 1.98 0.28 0.03 1.98 χ χ

2 Plant 0.38 0.62 0.68 2 0.83 0.92 0.96 0.28 0.53 0.35 0.59

0.760 0.760

Overdispersion 1.120 0.859 0.982 0.760 1.014

0.035 0.871 0.160 index 0.035 0.871 0.160 SD SD SD SD 0.53 0.53 P P age (Total), and for different for and (Total), age different for and (Total), age

Hoverflies Hoverflies Variance Variance Variance Variance (+) 3.11 (+) 4.24 (+) 9.23 (+) 3.11 (+) 4.24 (+) 9.23 χ χ 2 2 0.35 0.35

1.014 1.014

0.078 0.040 0.020 0.078 0.040 0.020 SD SD SD SD 0.59 0.59 P P

pollinator guilds (Bees,guilds pollinator pollinator guilds (Bees,guilds pollinator Appendix Appendix Appendix 48 48 48