BACKGROUND
the relationship between species number and island area, namely, that species number increases with increasing island area, is a fundamental rule in ecology
<cit> .
macarthur and wilson
<cit>  hypothesized that this relationship results from a dynamic equilibrium between opposing immigration and extinction rates, which depend on island isolation and size, respectively.
because the same trend occurs in continental environments
<cit> , the theory of island biogeography has been applied to the conservation of continental habitats
<cit> .
species–area relationships suggest that the number of interaction links among species, such as prey–predator and plant–pollinator species, increases with island or habitat area
<cit> .
however, surprisingly, only a few studies have tested this prediction, including one that examined plant–ant interactions on oceanic islands
<cit> , and another that studied plant–pollinator interactions on continental habitats
<cit> .
additionally, the network structure of species interactions is empirically known to relate to the total number of species interacting
<cit> , which raises the expectation that there may be a close relationship between area and network structure
<cit> ; however, only one study has tested this prediction at the landscape scale
<cit> .
species–area relationships differ among spatial scales, with the shapes and slopes of the relationships differing among local, regional, and global scales
<cit> .
in addition, the effects of spatial scale on species–area relationships may depend on taxonomic group; for example, responses to habitat area differ between animals and plants
<cit> .
given that networks of species interactions usually involve different taxonomic groups
<cit> , different spatial scales may affect the relationships between these networks and habitat area.
however, how habitat area influences interaction networks at different spatial scales at the landscape level has remained unexplored.
biological invasions impact the interactions among native species
<cit> , and island communities are more likely to be invaded and affected by exotic species than continental ones
<cit> .
however, exotic species may invade continental communities disturbed by human activities
<cit> .
therefore, exotic species may impact the original relationship between habitat area and interaction networks even in continental environments.
here we analysed plant–pollinator networks, both including and excluding exotic species, in temperate continental forests at different spatial scales to clarify scale-variable effects of habitat area on interaction networks.
plant–pollinator interactions provide excellent model systems for investigating the structure of species interactions
<cit> .
plant–pollinator interactions have recently been targeted for studying the network structure of plant–animal mutualistic interactions
<cit> .
for example, network metrics such as connectance and nestedness are often used to clarify the structure of mutualisitic interaction networks
<cit> .
as network metrics are correlated with the total number of interacting species
<cit> , and the number of species is related to habitat area
<cit> , we expect that network structure is also related to habitat area.
reports of such area-related effects on network structure are rare
<cit> , although the mathematical consequences of changing species numbers for network structure have been discussed
<cit> .
the main goal of this study was to test the following hypotheses:  the total number of species increases with forest area,  the network metrics of plant–pollinator interactions are related to the total number of species and forest area,  the relationships between forest area and plant–pollinator networks are scale-dependent, and  exotic species impact the relationships between forest area and plant–pollinator networks.
although the effects of island and habitat area on interaction networks have recently been reported
<cit> , the scale-dependencies have never been examined.
methods
study system
we analysed a data set that was in part used by taki and kevan
<cit>  to examine the effects of forest loss on the degree of specialization/generalization for plant and insect communities.
our analyses focused on the effects of forest area on the size and structure of the whole network in terms of scale-dependency and biological invasion.
the study was conducted in norfolk county, ontario, canada .
the study forests were located in southern ontario’s deciduous carolinian forest zone.
the landscape is flat and characterized by distributed fragments of forest within intensively managed agricultural fields of crops such as corn, soybean, and tobacco.
the forests were composed of deciduous trees such as oak and maple
<cit> .
small shrubs and herbs dominated the forest floors.
the associations of flower-visiting insects with understory plants that flowered prior to leaf flush of the forest crown trees were examined  by observing the interactions between  <dig> flowering plants and  <dig> flower-visiting insect species .
although it is possible that not all of the flower-visitors were legitimate pollinators, we considered them to be pollinators to decipher the general patterns between habitat area and plant–pollinator networks.
garlic mustard alliaria petiolata and honeybee apis mellifera, both of which originated in europe, are exotic species in this region .
both species are widely acknowledged to impact native communities
<cit> .
however, it is unlikely that a. mellifera has become established in the study forests, and the small numbers of individuals captured in this study likely escaped from domesticated colonies.
alliaria petiolata, on the other hand, has become established in these areas, and it invades forest edge and understory.
study design
the study was conducted in seven forest sites
<cit>  , which were selected using a geographical information system  with arcview .
seven geographical points that fell within the forest polygons in the study region were randomly chosen.
the criteria for accepting a selected point included a minimum distance of 40 m from all edges of the forest polygons and a minimum separation distance of 4500 m from any other chosen geographical points.
the geospatial data of forest coverage were produced using aerial photography  obtained from the ontario base map series in  <dig> .
each of the seven selected sites consisted of a hexagonal transect with 20 m sides with the chosen geographical point marking the centre .
in constructing the hexagonal transects, the direction of the first radial arm of each transect was randomly chosen using a  <dig>  m stick thrown into the air.
the axes were marked with bamboo poles and a 120 m section of rope demarcated the perimeter.
although a better choice for transect shape would be a circle, hexagonal transects were more practical.
a belt transect method
<cit>  was applied to the hexagonal transects.
we sampled before canopy closure because most understory plant species bloom during this short season
<cit> .
flower-visiting insects were sampled on sunny days when the temperature was at least  <dig> °c.
sampling started at 11: <dig> and at 14: <dig>  times at which flower visitations by insects are relatively frequent.
two sites were sampled each day in most cases.
four sampling cycles were conducted to ensure that sampling was carried out twice at 11: <dig> and twice at 14: <dig> for each of the seven sites .
all samplings were conducted from late april to late may.
at each sampling, the same two researchers walked the perimeter of the hexagons five times at a slow pace.
one person walked clockwise and the other walked counter-clockwise.
each of these samplings took 80–100 min.
all insects visiting the flowers within 2 m on either side of the perimeter rope were sampled with insect nets and aspirators.
the species of the flowering plants visited by each sampled insect were recorded.
the species richness and abundance of all flowering plants in bloom at each site were recorded using a quadrate method with  <dig> x 1 m quadrates placed every meter along each side of the perimeter of each hexagon, creating  <dig> quadrates along the outer perimeter of each side and  <dig> along the inner row .
the cataloguing and census of plants took place four times for each site, coinciding with the first, second, third, and fourth insect sampling cycle .
to quantify the amount of forest area at different spatial scales, circles of  <dig> radii from  <dig> to 2020 m  with 100 m intervals were created using arcview on maps around each of the hexagonal transects.
the scales followed previous studies on foraging ranges and scale-dependent effects in flower-visiting insects such as bees
<cit> .
the forest area at each of the scales was estimated by the amount of forest coverage  within the circles.
the environment surrounding the forests, i.e., intensively managed annual agricultural fields, was not habitat for most of native plant or pollinator species recorded in the study forests.
therefore, we can consider the forest coverage as habitat area for most plant and pollinator species .
the degree of connectivity among forest patches has been considered one of the most important factors for evaluating the impacts of forest fragmentation on biodiversity [e.g.,
<cit> .
in this study area, taki et al.
<cit>  showed that bee abundance and diversity increased with increasing forest area but not increasing forest edge.
this suggests that connectivity has a lesser influence on plant-pollinator networks than the overall forested area in this study region.
therefore, our analyses did not focus on the effects of forest connectivity but rather the effects of forest area on plant-pollinator networks.
data analysis
the numbers of species and interaction links were pooled at each forest site for the analyses.
to determine the impacts of exotic species on the relationships between forest area and plant–pollinator networks, two data sets were analysed; the first included exotic species  and the second excluded associations with exotic plant and pollinator species .
the numbers of plant species, pollinator species, total species, and occupancy by exotic garlic mustard a. petiolata were analysed in relation to forest area using pearson’s correlation coefficient at each spatial scale.
prior to the analysis, forest area and species numbers were log transformed; occupancy of flowering a. petiolata  was square root-arcsine transformed.
to examine how the network metrics were related to the total number of species and forest area, the number of interaction links, connectance, and nestedness were calculated.
on the basis of previous studies
<cit> , the number of observed interaction links between plant and pollinator species  can be predicted to increase with increases in the total number of species and habitat area.
connectance , or the proportion of interactions actually observed amongst all possible interactions, usually represents the degree of redundancy in a system, with consequences for community stability
<cit> .
it was calculated as follows
<cit> :
c=i/,
where pl and po are the numbers of plant and pollinator species, respectively.
on the basis of previous studies
<cit> , connectance can be predicted to decrease with increases in the total number of species and habitat area.
nestedness is a pattern composed of asymmetrical interactions between generalists and specialists and symmetrical interactions among generalists.
nestedness is frequently detected within plant–pollinator networks
<cit> , although the concept of nestedness was originally used to analyse patterns of species occurrence and absence on a set of islands or in habitat fragments
<cit> .
nested networks are characterized by specialist species that tend to interact only with generalists; generalists that all interact with each other, forming a core of interacting species; and the absence of specialists that interact only with other specialists
<cit> .
we used the software aninhado  <dig> .3
<cit>  to calculate nestedness .
relatively large values of nodf indicate a high degree of nestedness.
two null models  were used to test the degree of nestedness expected from the basic network features.
the first null model assumes that each randomly assigned plant and pollinator pair interacts with a constant probability, c .
therefore, it tests whether the observed nodf is higher than expected for random networks with a similar number of interactions.
the second null model assumes that the probability of a plant, i, interacting with a pollinator, j, depends on the observed number of interactions of both species, such that
c=kipo+kjpl <dig>
where k is the observed number of interactions for the species, pl is the number of plant species  and po is the number of pollinator species .
therefore, null model ii tests whether the observed nodf is higher than expected for random networks with similar heterogeneities of species interactions.
each community was compared to  <dig> replicates generated by each null model.
on the basis of the previous study
<cit> , nestedness in mutualistic networks is predicted to increase with increases in the total number of species and habitat area.
the network metrics, link numbers, connectance, and nestedness were also analysed in relation to forest area using pearson’s correlation coefficient at each spatial scale .
before the analysis, c and nodf/ <dig> were square root-arcsine transformed while link numbers and forest area were log transformed.
linear regression models were used to examine the relationships between the total number of species and network metrics .
we also used linear regression models to investigate the relationships between forest area and network metrics.
as the scale for linear regressions, we used the radius at which the highest correlation coefficient was detected.
RESULTS
relationships between forest area and species numbers
among study sites, the numbers of plant species, pollinator species, and total species ranged from 3– <dig>  10– <dig>  and 14– <dig>  respectively .
the relationships between forest area and numbers of species were dependent on the spatial scale, and differed among plant and pollinator species .
the number of plant species was not related to forest area , while the number of pollinator species and total species were negatively correlated with forest area, with the highest correlation coefficient at radii of  <dig> and 1520 m, respectively .
these results are not concordant with the patterns predicted by species–area relationships.
occupancy by exotic a. petiolata was negatively correlated with forest area, with the highest correlation coefficient at a radius of 1320 m .
when exotic species, and the native species only observed interacting with exotics, were excluded, the numbers of plant species, pollinator species and total species were positively correlated with forest area, with the highest correlation coefficient at a radius of 820 m .
§ the numbers in parentheses indicate the numbers of native plant and native pollinator species, interaction links between native plants and native pollinators, and individuals of native pollinators visiting native plants.
£ the numbers in parentheses indicate connectance and nodf calculated from the native plant–pollinator interactions .
* p <  <dig>  ; other nodf values, p >  <dig>  .
<dig>  lines represent linear regressions:  number of plant species , log =  <dig>  +  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ;  number of pollinator species , log =  <dig>  –  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ;  log =  <dig>  +  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ;  total number of species , log =  <dig>  –  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ;  log =  <dig>  +  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> .
<dig>  correlation coefficients in the shaded grey areas are statistically significant at p =  <dig>  level.
line represents a linear regression: occupancy by a. petiolata , arcsin
ap=  <dig>  –  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> .
relationships between species numbers and interaction networks
the numbers of interaction links between plant and pollinator species ranged from 11– <dig> among the study sites .
of these, the percentage involving exotic species ranged from 0– <dig> % among the study sites .
the numbers of links were positively correlated with the total number of species .
this pattern did not change when associations with exotic species were excluded from the analysis .
furthermore, connectance was not related to the total number of species when exotics were included .
however, when associations with exotic species were excluded from the analysis, connectance was negatively correlated with the total number of species .
most of the plant–pollinator networks were not nested .
the degree of nestedness was not related to the total number of species regardless of whether the associations with exotic species were excluded from the analysis .
<dig>  lines represent linear regressions:  number of interaction links , log = − <dig>  +  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ;  log = − <dig>  +  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p <  <dig> ;  arcsin
c=  <dig>  –  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> .
relationships between forest area and interaction networks
the numbers of interaction links were negatively correlated with forest area, with the highest correlation coefficient at a radius of 1620 m , which is not concordant with the patterns predicted by species–area relationships.
however, when associations with exotic species were excluded from the analysis, the number of interaction links was positively correlated with forest area, with the highest correlation coefficient at a radius of 820 m .
connectance was not related with forest area .
however, when associations with exotics were excluded from the analysis, connectance was negatively correlated with forest area .
nestedness was positively correlated with forest area regardless of whether the associations with exotic species were excluded from the analysis, with the highest correlation coefficients at radii of 520 m including exotics and 620–720 m excluding exotics .
<dig>  lines represent linear regressions:  number of interaction links , log =  <dig>  –  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ; ; log =  <dig>  +  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ;  connectance , arcsin
c=  <dig>  –  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ;  arcsin
c=  <dig>  –  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ;  nestedness , arcsin
nodf/100=  <dig>  +  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> ; , arcsin
nodf/100=  <dig>  +  <dig> *log, r2 =  <dig> , f <dig>  =  <dig> , p =  <dig> .
discussion
some of our results did not support our hypotheses when exotic species were included .
however, almost all of the hypotheses were verified when associations with exotic species were excluded from the networks .
in particular, we determined that the relationship between forest area and plant–pollinator network depended on spatial scale .
to the best of our knowledge, this is the first study to demonstrate scale-dependent effects of habitat area and exotic species on interaction networks.
scale-dependent effects of forest area on plant–pollinator networks
the species–area relationships were vastly different between plant and pollinator species when exotic species were included in the analyses .
additionally, the spatial scale at which the highest correlation coefficient was found differed between plant and pollinator species .
responses to habitat area are generally different between plant and pollinator species
<cit> .
however, the scale-dependent relationship was similar among the number of plant species, pollinator species, and interaction links, when exotic species as well as native species only observed interacting with exotics were excluded .
the similarities among the relationships between native plant and pollinator species are not likely to have been caused by our sampling scheme , because the results using a different sampling method  also showed that native bee abundance and diversity increased with increasing forest area .
the study
<cit>  was conducted in the same region, but in different study forests .
therefore, the similar responses of native plant and native pollinator species to forest area caused a positive relationship between forest area and the numbers of links at the same spatial scale .
previous studies have indicated that network metrics, connectance and nestedness are related to the total number of species involved in mutualistic interactions
<cit> .
our results partly supported this pattern .
although previous studies have used data sets composed of mutualistic networks at different sampling areas as well as different geographical regions
<cit> , all of our data were collected from within the same sampling area  in the same region.
because the total number of species was related to the forest area , so was the network structure .
more redundant networks with highly asymmetric interactions were found in relatively large forest areas , suggesting that the stability of plant–pollinator networks might increase with forest area.
however, these relationships may partly be explained by non-biological factors.
for example, connectance may decrease with increasing numbers of possible interaction links, as the number of observations per species declines when the same absolute effort is made to sample networks of different sizes
<cit> .
in this study, however, connectance did not decrease with the total number of species when exotic species were included .
also, nestedness was not related to the total number of species , although it increased with increasing forest area .
these results suggest that the effects of forest area on connectance or nestedness were caused not only by changes in the total number of species, but also by other factors.
for example, changes in the population densities of some species might affect the structure of interaction networks relative to habitat area because the population densities of various species are known to be related to habitat area
<cit> .
further studies are needed to clarify the mechanism driving the relationships between habitat area and network structure.
impacts of exotic species
the scale-dependent relationships among forest area, total number of species, and interaction networks were different when interactions were considered with and without exotic species , suggesting impacts of exotic species.
plant–pollinator interactions included only two exotic species, garlic mustard a. petiolata and the honeybee a. mellifera .
although the contribution of a. mellifera to the interactions was not insignificant , a. petiolata was central to the interactions in at least two forests .
although exotic plants are only rarely thought to invade temperate natural forests
<cit> , a. petiolata has frequently been reported to invade the forest edge and understory in north america
<cit> .
flowers of a. petiolata produce rich nectar, attracting a variety of native bees and flies
<cit> .
nectar-rich flowers of invasive plants can disturb native plant–pollinator interactions
<cit> .
in the study areas of the present study, a. petiolata, which has invaded relatively small forest patches surrounded by agricultural fields , may have supported more native pollinator species than initially expected .
the presence of exotic species strongly influenced the scale-dependent relationships between forest area and the number of interaction links and connectance .
thus, this invasive plant may have altered the original relationships between forest area and plant–pollinator networks and their scale-dependency.
however, nestedness showed the same trend for both networks, with and without exotics .
vilá et al.
<cit>  hypothesised that invasive plants by being supergeneralists, both interacting with generalists and specialists, would increase the nestedness of the plant–pollinator network.
our results showed that excluding associations with exotic species increased the values of nestedness in some study sites .
however, this increase did not change the relationships between forest area and nestedness .
CONCLUSIONS
since the original publication of the equilibrium theory of island biogeography
<cit> , species–area relationships have been extensively studied for various groups of organisms
<cit> .
furthermore, the theory has been applied to the conservation of focal species in continental habitats
<cit> .
sugiura
<cit>  analysed the relationships between island area and plant–ant mutualistic interactions and suggested an extension of this basic species–area relationship to more specifically address “species interactions–area relationships”.
interaction networks on continental habitats as well as oceanic islands can be considered with a view to the species interactions–area relationships.
indeed, sabatino et al.
<cit>  indicated that the number of interaction links among flowering plants and their pollinators increases with habitat area in the continental environment.
valladares et al.
<cit>  also indicated that the network structure of plant–leafminer–parasitoid webs is related to fragmented forest area.
in this study, we determined that network metrics of plant–pollinator interactions are related to forest area and the relationships depend on spatial scale.
each species has a particular spatial scale of habitat area that most strongly affects the abundance because body size and mobility differ among species
<cit> .
therefore, the spatial scales of habitat area that most strongly affected the abundance generally differed among species
<cit> .
in this study, we determined the spatial scales that most strongly affected the structure of plant–pollinator interactions, which suggests the presence of a spatial scale that most strongly influences the structuring and maintenance of the species interaction network.
finally, exotic species appear to alter the relationship between habitat area and interaction network.
although many researchers have reported that exotic species impact interaction networks, particularly on oceanic islands
<cit> , we suggest that a single exotic plant species can impact this relationship, even in temperate continental habitats.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ss and ht generated the original idea.
ht designed the research.
ht collected field data.
ss and ht analysed data.
ss wrote the paper.
all authors read and approved the final manuscript.
supplementary material
additional file 1
seven study sites with  <dig> radii ranging from 120 m to 2020 m  used to obtain forest area.
all sites were in norfolk country, ontario, canada.
shaded areas represent forests, and the bar indicates a 2000 m scale.
the geospatial data were obtained from the ontario base map series in  <dig> , code numbers indicate study site codes, which are provided in table
<dig>
click here for file
additional file 2
list of plant species at each forest site.
click here for file
additional file 3
list of pollinator species at each forest site.
click here for file
additional file 4
hexagonal transect with 20 m sides, with the chosen geographical point marking the centre .
the axes were marked with bamboo poles and a 120 m section of rope demarcated the perimeter .
click here for file
acknowledgements
we thank long point conservation authority personnel, m. armstrong, j. decloet, j. knack, f. saul and v. sinnave for allowing us to access our study sites.
we also thank p. kevan, c. fuss and a. and j. boone for support and field assistance.
we thank o. honnay and anonymous reviewers for helpful comments on an earlier version of the manuscript.
while conducting the field work, ht was supported by a scholarship from the rotary foundation.
financial support was provided by the forestry and forest products research institute.

 the relationship between species number and island area, namely, that species number increases with increasing island area, is a fundamental rule in ecology. macarthur and wilson hypothesized that this relationship results from a dynamic equilibrium between opposing immigration and extinction rates, which depend on island isolation and size, respectively. because the same trend occurs in continental environments, the theory of island biogeography has been applied to the conservation of continental habitats. species–area relationships suggest that the number of interaction links among species, such as prey–predator and plant–pollinator species, increases with island or habitat area. however, surprisingly, only a few studies have tested this prediction, including one that examined plant–ant interactions on oceanic islands, and another that studied plant–pollinator interactions on continental habitats. additionally, the network structure of species interactions is empirically known to relate to the total number of species interacting, which raises the expectation that there may be a close relationship between area and network structure; however, only one study has tested this prediction at the landscape scale. species–area relationships differ among spatial scales, with the shapes and slopes of the relationships differing among local, regional, and global scales. in addition, the effects of spatial scale on species–area relationships may depend on taxonomic group; for example, responses to habitat area differ between animals and plants. given that networks of species interactions usually involve different taxonomic groups, different spatial scales may affect the relationships between these networks and habitat area. however, how habitat area influences interaction networks at different spatial scales at the landscape level has remained unexplored. biological invasions impact the interactions among native species, and island communities are more likely to be invaded and affected by exotic species than continental ones. however, exotic species may invade continental communities disturbed by human activities. therefore, exotic species may impact the original relationship between habitat area and interaction networks even in continental environments. here we analysed plant–pollinator networks, both including and excluding exotic species, in temperate continental forests at different spatial scales to clarify scale-variable effects of habitat area on interaction networks. plant–pollinator interactions provide excellent model systems for investigating the structure of species interactions. plant–pollinator interactions have recently been targeted for studying the network structure of plant–animal mutualistic interactions. for example, network metrics such as connectance and nestedness are often used to clarify the structure of mutualisitic interaction networks. as network metrics are correlated with the total number of interacting species, and the number of species is related to habitat area, we expect that network structure is also related to habitat area. reports of such area-related effects on network structure are rare, although the mathematical consequences of changing species numbers for network structure have been discussed. the main goal of this study was to test the following hypotheses: the total number of species increases with forest area, the network metrics of plant–pollinator interactions are related to the total number of species and forest area, the relationships between forest area and plant–pollinator networks are scale-dependent, and exotic species impact the relationships between forest area and plant–pollinator networks. although the effects of island and habitat area on interaction networks have recently been reported, the scale-dependencies have never been examined. methods study system we analysed a data set that was in part used by taki and kevan to examine the effects of forest loss on the degree of specialization/generalization for plant and insect communities. our analyses focused on the effects of forest area on the size and structure of the whole network in terms of scale-dependency and biological invasion. the study was conducted in norfolk county, ontario, canada. the study forests were located in southern ontario’s deciduous carolinian forest zone. the landscape is flat and characterized by distributed fragments of forest within intensively managed agricultural fields of crops such as corn, soybean, and tobacco. the forests were composed of deciduous trees such as oak and maple. small shrubs and herbs dominated the forest floors. the associations of flower-visiting insects with understory plants that flowered prior to leaf flush of the forest crown trees were examined by observing the interactions between flowering plants and flower-visiting insect species. although it is possible that not all of the flower-visitors were legitimate pollinators, we considered them to be pollinators to decipher the general patterns between habitat area and plant–pollinator networks. garlic mustard alliaria petiolata and honeybee apis mellifera, both of which originated in europe, are exotic species in this region. both species are widely acknowledged to impact native communities. however, it is unlikely that a. mellifera has become established in the study forests, and the small numbers of individuals captured in this study likely escaped from domesticated colonies. alliaria petiolata, on the other hand, has become established in these areas, and it invades forest edge and understory. study design the study was conducted in seven forest sites, which were selected using a geographical information system with arcview. seven geographical points that fell within the forest polygons in the study region were randomly chosen. the criteria for accepting a selected point included a minimum distance of 40 m from all edges of the forest polygons and a minimum separation distance of 4500 m from any other chosen geographical points. the geospatial data of forest coverage were produced using aerial photography obtained from the ontario base map series in. each of the seven selected sites consisted of a hexagonal transect with 20 m sides with the chosen geographical point marking the centre. in constructing the hexagonal transects, the direction of the first radial arm of each transect was randomly chosen using a  m stick thrown into the air. the axes were marked with bamboo poles and a 120 m section of rope demarcated the perimeter. although a better choice for transect shape would be a circle, hexagonal transects were more practical. a belt transect method was applied to the hexagonal transects. we sampled before canopy closure because most understory plant species bloom during this short season. flower-visiting insects were sampled on sunny days when the temperature was at least °c. sampling started at 11: and at 14: times at which flower visitations by insects are relatively frequent. two sites were sampled each day in most cases. four sampling cycles were conducted to ensure that sampling was carried out twice at 11: and twice at 14: for each of the seven sites. all samplings were conducted from late april to late may. at each sampling, the same two researchers walked the perimeter of the hexagons five times at a slow pace. one person walked clockwise and the other walked counter-clockwise. each of these samplings took 80–100 min. all insects visiting the flowers within 2 m on either side of the perimeter rope were sampled with insect nets and aspirators. the species of the flowering plants visited by each sampled insect were recorded. the species richness and abundance of all flowering plants in bloom at each site were recorded using a quadrate method with x 1 m quadrates placed every meter along each side of the perimeter of each hexagon, creating quadrates along the outer perimeter of each side and along the inner row. the cataloguing and census of plants took place four times for each site, coinciding with the first, second, third, and fourth insect sampling cycle. to quantify the amount of forest area at different spatial scales, circles of radii from to 2020 m with 100 m intervals were created using arcview on maps around each of the hexagonal transects. the scales followed previous studies on foraging ranges and scale-dependent effects in flower-visiting insects such as bees. the forest area at each of the scales was estimated by the amount of forest coverage within the circles. the environment surrounding the forests, i.e., intensively managed annual agricultural fields, was not habitat for most of native plant or pollinator species recorded in the study forests. therefore, we can consider the forest coverage as habitat area for most plant and pollinator species. the degree of connectivity among forest patches has been considered one of the most important factors for evaluating the impacts of forest fragmentation on biodiversity [e.g.,. in this study area, taki et al. showed that bee abundance and diversity increased with increasing forest area but not increasing forest edge. this suggests that connectivity has a lesser influence on plant-pollinator networks than the overall forested area in this study region. therefore, our analyses did not focus on the effects of forest connectivity but rather the effects of forest area on plant-pollinator networks. data analysis the numbers of species and interaction links were pooled at each forest site for the analyses. to determine the impacts of exotic species on the relationships between forest area and plant–pollinator networks, two data sets were analysed; the first included exotic species and the second excluded associations with exotic plant and pollinator species. the numbers of plant species, pollinator species, total species, and occupancy by exotic garlic mustard a. petiolata were analysed in relation to forest area using pearson’s correlation coefficient at each spatial scale. prior to the analysis, forest area and species numbers were log transformed; occupancy of flowering a. petiolata was square root-arcsine transformed. to examine how the network metrics were related to the total number of species and forest area, the number of interaction links, connectance, and nestedness were calculated. on the basis of previous studies, the number of observed interaction links between plant and pollinator species can be predicted to increase with increases in the total number of species and habitat area. connectance, or the proportion of interactions actually observed amongst all possible interactions, usually represents the degree of redundancy in a system, with consequences for community stability. it was calculated as follows: c=i/, where pl and po are the numbers of plant and pollinator species, respectively. on the basis of previous studies, connectance can be predicted to decrease with increases in the total number of species and habitat area. nestedness is a pattern composed of asymmetrical interactions between generalists and specialists and symmetrical interactions among generalists. nestedness is frequently detected within plant–pollinator networks, although the concept of nestedness was originally used to analyse patterns of species occurrence and absence on a set of islands or in habitat fragments. nested networks are characterized by specialist species that tend to interact only with generalists; generalists that all interact with each other, forming a core of interacting species; and the absence of specialists that interact only with other specialists. we used the software aninhado.3 to calculate nestedness. relatively large values of nodf indicate a high degree of nestedness. two null models were used to test the degree of nestedness expected from the basic network features. the first null model assumes that each randomly assigned plant and pollinator pair interacts with a constant probability, c. therefore, it tests whether the observed nodf is higher than expected for random networks with a similar number of interactions. the second null model assumes that the probability of a plant, i, interacting with a pollinator, j, depends on the observed number of interactions of both species, such that c=kipo+kjpl where k is the observed number of interactions for the species, pl is the number of plant species and po is the number of pollinator species. therefore, null model ii tests whether the observed nodf is higher than expected for random networks with similar heterogeneities of species interactions. each community was compared to replicates generated by each null model. on the basis of the previous study, nestedness in mutualistic networks is predicted to increase with increases in the total number of species and habitat area. the network metrics, link numbers, connectance, and nestedness were also analysed in relation to forest area using pearson’s correlation coefficient at each spatial scale. before the analysis, c and nodf/ were square root-arcsine transformed while link numbers and forest area were log transformed. linear regression models were used to examine the relationships between the total number of species and network metrics. we also used linear regression models to investigate the relationships between forest area and network metrics. as the scale for linear regressions, we used the radius at which the highest correlation coefficient was detected.  relationships between forest area and species numbers among study sites, the numbers of plant species, pollinator species, and total species ranged from 3– 10– and 14– respectively. the relationships between forest area and numbers of species were dependent on the spatial scale, and differed among plant and pollinator species. the number of plant species was not related to forest area, while the number of pollinator species and total species were negatively correlated with forest area, with the highest correlation coefficient at radii of and 1520 m, respectively. these results are not concordant with the patterns predicted by species–area relationships. occupancy by exotic a. petiolata was negatively correlated with forest area, with the highest correlation coefficient at a radius of 1320 m. when exotic species, and the native species only observed interacting with exotics, were excluded, the numbers of plant species, pollinator species and total species were positively correlated with forest area, with the highest correlation coefficient at a radius of 820 m. § the numbers in parentheses indicate the numbers of native plant and native pollinator species, interaction links between native plants and native pollinators, and individuals of native pollinators visiting native plants. £ the numbers in parentheses indicate connectance and nodf calculated from the native plant–pollinator interactions. * p < ; other nodf values, p > . lines represent linear regressions: number of plant species, log =   +  *log, r2 = , f  = , p = ; number of pollinator species, log =  – *log, r2 = , f  = , p = ; log =   +  *log, r2 = , f  = , p = ; total number of species, log =  – *log, r2 = , f  = , p = ; log =   +  *log, r2 = , f  = , p = . correlation coefficients in the shaded grey areas are statistically significant at p =  level. line represents a linear regression: occupancy by a. petiolata, arcsin ap= – *log, r2 = , f  = , p = . relationships between species numbers and interaction networks the numbers of interaction links between plant and pollinator species ranged from 11– among the study sites. of these, the percentage involving exotic species ranged from 0– % among the study sites. the numbers of links were positively correlated with the total number of species. this pattern did not change when associations with exotic species were excluded from the analysis. furthermore, connectance was not related to the total number of species when exotics were included. however, when associations with exotic species were excluded from the analysis, connectance was negatively correlated with the total number of species. most of the plant–pollinator networks were not nested. the degree of nestedness was not related to the total number of species regardless of whether the associations with exotic species were excluded from the analysis. lines represent linear regressions: number of interaction links, log = −  +  *log, r2 = , f  = , p = ; log = −  +  *log, r2 = , f  = , p < ; arcsin c= – *log, r2 = , f  = , p = . relationships between forest area and interaction networks the numbers of interaction links were negatively correlated with forest area, with the highest correlation coefficient at a radius of 1620 m, which is not concordant with the patterns predicted by species–area relationships. however, when associations with exotic species were excluded from the analysis, the number of interaction links was positively correlated with forest area, with the highest correlation coefficient at a radius of 820 m. connectance was not related with forest area. however, when associations with exotics were excluded from the analysis, connectance was negatively correlated with forest area. nestedness was positively correlated with forest area regardless of whether the associations with exotic species were excluded from the analysis, with the highest correlation coefficients at radii of 520 m including exotics and 620–720 m excluding exotics. lines represent linear regressions: number of interaction links, log =  – *log, r2 = , f  = , p = ;; log =   +  *log, r2 = , f  = , p = ; connectance, arcsin c= – *log, r2 = , f  = , p = ; arcsin c= – *log, r2 = , f  = , p = ; nestedness, arcsin nodf/100=  +  *log, r2 = , f  = , p = ;, arcsin nodf/100=  +  *log, r2 = , f  = , p = . discussion some of our results did not support our hypotheses when exotic species were included. however, almost all of the hypotheses were verified when associations with exotic species were excluded from the networks. in particular, we determined that the relationship between forest area and plant–pollinator network depended on spatial scale. to the best of our knowledge, this is the first study to demonstrate scale-dependent effects of habitat area and exotic species on interaction networks. scale-dependent effects of forest area on plant–pollinator networks the species–area relationships were vastly different between plant and pollinator species when exotic species were included in the analyses. additionally, the spatial scale at which the highest correlation coefficient was found differed between plant and pollinator species. responses to habitat area are generally different between plant and pollinator species. however, the scale-dependent relationship was similar among the number of plant species, pollinator species, and interaction links, when exotic species as well as native species only observed interacting with exotics were excluded. the similarities among the relationships between native plant and pollinator species are not likely to have been caused by our sampling scheme, because the results using a different sampling method also showed that native bee abundance and diversity increased with increasing forest area. the study was conducted in the same region, but in different study forests. therefore, the similar responses of native plant and native pollinator species to forest area caused a positive relationship between forest area and the numbers of links at the same spatial scale. previous studies have indicated that network metrics, connectance and nestedness are related to the total number of species involved in mutualistic interactions. our results partly supported this pattern. although previous studies have used data sets composed of mutualistic networks at different sampling areas as well as different geographical regions, all of our data were collected from within the same sampling area in the same region. because the total number of species was related to the forest area, so was the network structure. more redundant networks with highly asymmetric interactions were found in relatively large forest areas, suggesting that the stability of plant–pollinator networks might increase with forest area. however, these relationships may partly be explained by non-biological factors. for example, connectance may decrease with increasing numbers of possible interaction links, as the number of observations per species declines when the same absolute effort is made to sample networks of different sizes. in this study, however, connectance did not decrease with the total number of species when exotic species were included. also, nestedness was not related to the total number of species, although it increased with increasing forest area. these results suggest that the effects of forest area on connectance or nestedness were caused not only by changes in the total number of species, but also by other factors. for example, changes in the population densities of some species might affect the structure of interaction networks relative to habitat area because the population densities of various species are known to be related to habitat area. further studies are needed to clarify the mechanism driving the relationships between habitat area and network structure. impacts of exotic species the scale-dependent relationships among forest area, total number of species, and interaction networks were different when interactions were considered with and without exotic species, suggesting impacts of exotic species. plant–pollinator interactions included only two exotic species, garlic mustard a. petiolata and the honeybee a. mellifera. although the contribution of a. mellifera to the interactions was not insignificant, a. petiolata was central to the interactions in at least two forests. although exotic plants are only rarely thought to invade temperate natural forests, a. petiolata has frequently been reported to invade the forest edge and understory in north america. flowers of a. petiolata produce rich nectar, attracting a variety of native bees and flies. nectar-rich flowers of invasive plants can disturb native plant–pollinator interactions. in the study areas of the present study, a. petiolata, which has invaded relatively small forest patches surrounded by agricultural fields, may have supported more native pollinator species than initially expected. the presence of exotic species strongly influenced the scale-dependent relationships between forest area and the number of interaction links and connectance. thus, this invasive plant may have altered the original relationships between forest area and plant–pollinator networks and their scale-dependency. however, nestedness showed the same trend for both networks, with and without exotics. vilá et al. hypothesised that invasive plants by being supergeneralists, both interacting with generalists and specialists, would increase the nestedness of the plant–pollinator network. our results showed that excluding associations with exotic species increased the values of nestedness in some study sites. however, this increase did not change the relationships between forest area and nestedness.  since the original publication of the equilibrium theory of island biogeography, species–area relationships have been extensively studied for various groups of organisms. furthermore, the theory has been applied to the conservation of focal species in continental habitats. sugiura analysed the relationships between island area and plant–ant mutualistic interactions and suggested an extension of this basic species–area relationship to more specifically address “species interactions–area relationships”. interaction networks on continental habitats as well as oceanic islands can be considered with a view to the species interactions–area relationships. indeed, sabatino et al. indicated that the number of interaction links among flowering plants and their pollinators increases with habitat area in the continental environment. valladares et al. also indicated that the network structure of plant–leafminer–parasitoid webs is related to fragmented forest area. in this study, we determined that network metrics of plant–pollinator interactions are related to forest area and the relationships depend on spatial scale. each species has a particular spatial scale of habitat area that most strongly affects the abundance because body size and mobility differ among species. therefore, the spatial scales of habitat area that most strongly affected the abundance generally differed among species. in this study, we determined the spatial scales that most strongly affected the structure of plant–pollinator interactions, which suggests the presence of a spatial scale that most strongly influences the structuring and maintenance of the species interaction network. finally, exotic species appear to alter the relationship between habitat area and interaction network. although many researchers have reported that exotic species impact interaction networks, particularly on oceanic islands, we suggest that a single exotic plant species can impact this relationship, even in temperate continental habitats. competing interests the authors declare that they have no competing interests. authors’ contributions ss and ht generated the original idea. ht designed the research. ht collected field data. ss and ht analysed data. ss wrote the paper. all authors read and approved the final manuscript. supplementary material additional file 1 seven study sites with radii ranging from 120 m to 2020 m used to obtain forest area. all sites were in norfolk country, ontario, canada. shaded areas represent forests, and the bar indicates a 2000 m scale. the geospatial data were obtained from the ontario base map series in, code numbers indicate study site codes, which are provided in table  click here for file additional file 2 list of plant species at each forest site. click here for file additional file 3 list of pollinator species at each forest site. click here for file additional file 4 hexagonal transect with 20 m sides, with the chosen geographical point marking the centre. the axes were marked with bamboo poles and a 120 m section of rope demarcated the perimeter. click here for file acknowledgements we thank long point conservation authority personnel, m. armstrong, j. decloet, j. knack, f. saul and v. sinnave for allowing us to access our study sites. we also thank p. kevan, c. fuss and a. and j. boone for support and field assistance. we thank o. honnay and anonymous reviewers for helpful comments on an earlier version of the manuscript. while conducting the field work, ht was supported by a scholarship from the rotary foundation. financial support was provided by the forestry and forest products research institute.
the positive relationship between habitat area and species number is considered a fundamental rule in ecology. in the study area, the exotic plant species alliaria petiolata, which has invaded relatively small forest patches surrounded by agricultural fields, may have supported more native pollinator species than initially expected. we analysed understory plant–pollinator networks in seven temperate forest sites at spatial scales to clarify scale-associated relationships between forest area and plant–pollinator networks. the total number of species and the number of interaction links between plant and pollinator species were negatively correlated with forest area, with the highest correlation coefficient at radii of and 1620 m, respectively. however, when associations with exotic species were excluded, the total number of species and the number of interaction links were positively correlated with forest area. the network structure, i.e., connectance and nestedness, was also related to forest area, when associations with exotics were excluded. therefore, this invasive plant may have altered the original relationships between forest area and plant–pollinator networks. our results demonstrate scale-dependent effects of forest area on the size and structure of plant–pollinator networks. alliaria petiolatabiological invasionforest areamutualistic networksplant–pollinator interactionsspecies–area relationships we also suggest that a single exotic plant species can impact plant–pollinator networks, even in temperate continental habitats. these results are not concordant with the pattern predicted by species–area relationships. however, how these relationships change at different spatial scales has remained unexplored. this relationship predicts that the link number of species interactions increases with habitat area, and structure is related to habitat area. biological invasions can affect species interactions and area relationships.
 the positive relationship between habitat area and species number is considered a fundamental rule in ecology. this relationship predicts that the link number of species interactions increases with habitat area, and structure is related to habitat area. biological invasions can affect species interactions and area relationships. however, how these relationships change at different spatial scales has remained unexplored. we analysed understory plant–pollinator networks in seven temperate forest sites at spatial scales to clarify scale-associated relationships between forest area and plant–pollinator networks.  the pooled data described interactions between plant and pollinator species. the total number of species and the number of interaction links between plant and pollinator species were negatively correlated with forest area, with the highest correlation coefficient at radii of and 1620 m, respectively. these results are not concordant with the pattern predicted by species–area relationships. however, when associations with exotic species were excluded, the total number of species and the number of interaction links were positively correlated with forest area. the network structure, i.e., connectance and nestedness, was also related to forest area, when associations with exotics were excluded. in the study area, the exotic plant species alliaria petiolata, which has invaded relatively small forest patches surrounded by agricultural fields, may have supported more native pollinator species than initially expected. therefore, this invasive plant may have altered the original relationships between forest area and plant–pollinator networks.  our results demonstrate scale-dependent effects of forest area on the size and structure of plant–pollinator networks. we also suggest that a single exotic plant species can impact plant–pollinator networks, even in temperate continental habitats. alliaria petiolatabiological invasionforest areamutualistic networksplant–pollinator interactionsspecies–area relationships 