Diversified Floral Resource Plantings Support Bee Communities after Apple Bloom in Commercial Orchards

Natural habitats, comprised of various flowering plant species, provide food and nesting resources for pollinator species and other beneficial arthropods. Loss of such habitats in agricultural regions and in other human-modified landscapes could be a factor in recent bee declines. Artificially established floral plantings may offset these losses. A multi-year, season-long field study was conducted to examine how wildflower plantings near commercial apple orchards influenced bee communities. We examined bee abundance, species richness, diversity, and species assemblages in both the floral plantings and adjoining apple orchards. We also examined bee community subsets, such as known tree fruit pollinators, rare pollinator species, and bees collected during apple bloom. During this study, a total of 138 species of bees were collected, which included 100 species in the floral plantings and 116 species in the apple orchards. Abundance of rare bee species was not significantly different between apple orchards and the floral plantings. During apple bloom, the known tree fruit pollinators were more frequently captured in the orchards than the floral plantings. However, after apple bloom, the abundance of known tree fruit pollinating bees increased significantly in the floral plantings, indicating potential for floral plantings to provide additional food and nesting resources when apple flowers are not available.


Results
Abundance. Over the three years of the study in multiple sites (Table 1) Table 2). Out of the ten most common species in floral plantings established with different plant species mix (Fig. 1) and apple orchards, six species were found in both floral plantings and apple orchards (Fig. 2). However, the single most abundant species for each site type -Eucera hamata (floral plantings) and Augchlora pura (apple orchards) -were not among the most abundant in the other site type (Fig. 2). Based on Pielou's evenness index, community evenness was higher in the floral plantings (J' = 0.559) than in the apple orchards (J' = 0.477). Across all samples collected, the known tree www.nature.com/scientificreports www.nature.com/scientificreports/ fruit pollinators were significantly more abundant in the orchards compared to the floral plantings (Z = 3.479, P = 0.0005). However, after apple bloom the abundance of known tree fruit pollinating bees in the floral plantings increased significantly (Z = −6.436, P = 1.23e −10. ). Overall, there was no significant difference in rare species abundance between the orchards and the floral plantings (Z = − 0.137, P = 0.891).

Richness.
Over the course of the study, 138 bee species were identified, of which 100 species were recorded in the floral plantings and 116 species in the orchards ( Table 2). Fifty-eight species were identified as tree fruit pollinators and six species were categorized as rare (Table 2). There was no difference in bee species richness between floral plantings and apple orchards for all species collected (Fig. 3a) and for known tree fruit pollinating species (Fig. 3b), as species accumulation occurred at almost identical rates in both site types. A higher rate of species www.nature.com/scientificreports www.nature.com/scientificreports/ accumulation was observed in the apple orchards as compared to floral plantings during the apple bloom time (Fig. 3c), although 95% confidence intervals still overlapped.
Diversity. Over the course of the entire study, bee communities in floral plantings were more diverse than those in the orchards based on Shannon index values (Z = −3.219, P = 0.0013) (Fig. 4). The floral plantings    www.nature.com/scientificreports www.nature.com/scientificreports/ also had a more diverse community of known tree fruit pollinators than the orchards (Z = −2.056, P = 0.0397) (Fig. 4). However, during bloom time only the bee communities in apple orchards were more diverse than in floral plantings (Z = 8.083, P = 6.66e-16) (Fig. 4).
Community assemblages. The bee communities visiting orchards and floral plantings were not differentiable in the non-metric multidimensional scaling (NMDS) ordination (Stress = 0.1136, P = 0.816) (Fig. 5). However, when the data were limited to known tree fruit pollinators, the communities visiting the orchards and the floral plantings were significantly different (Stress = 0.1395, P = 0.001) (Fig. 6). Among these known tree fruit pollinators, there were distinct community assemblages (F = 8.51, P = 0.002) between orchards and floral plantings, with most species being more closely associated with the orchards (Fig. 7). The first axis of the RDA biplot, which delineated the community gradient between the two site types, explained 22.7% of the variance in species data.

Discussion
Assuring sufficient pollination services is a critical component of commercial apple production. Due to the increasing rental costs of honey bees, growers may rely more on pollination services from wild, non-Apis, bees 9,29 . In addition, in certain cropping systems and landscapes, wild bees have been shown to visit crops at higher rates than honey bees 27,30,31 . Growers relying on pollination services from wild bees would thus benefit from adopting practices that actively promote abundant and diverse bee communities as increased pollinator density and diversity has been linked to improve crop yields [32][33][34] . Our study examined the contribution of season-long floral resource plantings to the wild bee communities in apple orchard agro-ecosystems, and to our knowledge, it is the first multi-year study documenting such season-long utility of perennial floral plantings in commercial apple orchards.
Over the course of the study, we found a diverse array of bees in both the floral plantings and the orchards. An analysis of species assemblages found that the community composition was not different between the habitat types, when the bee community was sampled over the entire season. Similarly, species richness, based on individual-based rarefaction analysis, was also not different between the orchards and floral plantings when  www.nature.com/scientificreports www.nature.com/scientificreports/ compared seasonally. However, diversity indices suggested that the overall diversity and evenness of the bee community (based on Shannon Index and Pielou's Index, respectively) were greater in the floral plantings. This was likely due the increased plant diversity and availability of season-long floral resources -traits that are often associated with increased pollinator diversity [35][36][37] . Although the pre-existing orchard/forest interface habitat in Pennsylvania has been found to support a large population of plant species 28 , the floral plantings used in this study were designed to bloom for a much longer period of time than the orchard/forest interface (see Fig. 1). Many of the plants in the orchard/forest interface in this region are early season perennials 28 . It is also possible that the orchard/forest interface is impacted by orchard pesticide application drift that could reduce both plant diversity and bee diversity.
Since the seed mixes of the floral plantings were constructed to minimize bloom phenology overlap with apples ( Fig. 1), we conducted additional analyses to see if the bee communities responded to these temporal and spatial shifts in floral resources as documented in other settings, such as landscapes with co-blooming crops 38 . When analyses were restricted to pollinators collected during the tree fruit bloom period only, apple orchards supported a greater diversity of bees and tended to have higher species richness levels. The species assemblage patterns differed significantly between orchards and floral plantings during bloom period, as well. In addition, most of the bees characterized as known apple pollinators (based on previous net collections from flowers), also showed a close association with orchards in this study (see Fig. 7). Of the species most highly associated with apples, three species -Augochlora pura, Ceratina calcarata, and Bombus vagans -comprised more than half of the total bee abundance found in orchards. These species are all known tree fruit pollinators that are commonly found in the orchards in this area [39][40][41] . A. pura and C. calcarata nest in rotting wood, stems, or pith, which makes the orchard-forest interface an ideal habitat for them and allows these species to thrive in this landscape. In addition, some of the other species associated with the orchard, such as Osmia spp. and Andrena spp., are early season univoltine species, whose foraging activities coincide with apple bloom, thus making them less reliant on the later-blooming flowers in the pollinator plantings. Among the list of known tree-fruit pollinators, only Xylocopa virginica, Bombus griseocollis, and Lasioglossum pilosum were noticeably associated with the floral plantings. All three of these species are generalists, which may be utilizing the late season blooming resources in the floral plantings.
The different diversity and assemblage patterns between whole season and apple bloom period analyses suggests that floral plantings, if properly designed, may serve as an important reservoir for some known tree fruit pollinators when the crop is not in bloom and can contribute to overall pollinator conservation in agricultural landscapes. The diverse plant community in the floral plantings attracted a diverse set of tree fruit pollinating species. Such diversified floral plantings may be acting as a nest site resource, as well as a reservoir for tree fruit pollinators, as we recorded an increase in the abundance of tree fruit pollinating species in the pollinator plantings after fruit bloom. In addition, as introduced landscape elements, the floral plantings seem to work in a complementary fashion with apple orchards in terms of promoting pollination services: known tree fruit pollinators readily moved into the apple orchards during bloom and the floral plantings did not seem to act as a pollinator 'sink' during this time period. There was no difference in the abundance of rare species in the orchards and the floral plantings, although detecting differences would likely always be difficult due to the inherently low abundance of these species. Triepeolus concavus was the only rare species found in the pollinator plantings and not the orchards, and is a known nest parasite of the bee genus Colletes.
The floral plantings met the design goals of providing season-long support to pollinator communities in apple orchards in this region. Anecdotally, we did not observe plant species in the pollinator plantings becoming weeds in the orchards, or an increase in pests that used the pollinator planting species as hosts. Furthermore, when the www.nature.com/scientificreports www.nature.com/scientificreports/ data were limited to only bees collected during fruit bloom, the orchards were significantly more diverse than the floral plantings, indicating that these plantings were not attracting pollinators away from the orchards during fruit bloom. However, we did witness problems with weed management and sustainability of the floral plantings. Canadian thistle (Cirsium arvense), poison ivy (Toxicodendron radicans), Virginia creeper (Parthenocissus quinquefolia) and field bindweed (Convolvulus arvensis) were abundant in many of the plantings and required spot treatment with herbicides.
Overall, the floral plantings attracted a more diverse community of pollinators, and in order to act optimally as a pollination service supplementation, they must be tailored more specifically to the crop and the species that pollinate it. Only a small minority of common bee species provide most of the crop pollination services 42 , so those bees must be targeted when creating pollinator habitat. For example, it would have been beneficial to have more early season plants in floral plantings, blooming before the crop in the pollinator habitats, which may better support apple pollinating species most of which emerge several weeks before apple bloom 27 , which include early season bees such as Andrena spp. and Osmia spp. 43 . These floral plantings increase populations of wild bees and other flower visiting insects in apple orchards 27 , but other strategies such as developing management practices for hedgerows and woods-edge 43 could also benefit pollination supplementation and bee conservation in commercial apple orchards.

Study sites.
Multi-year field studies were conducted at six separate apple orchards and eight floral plantings (often referred to as pollinator strips) on commercial tree fruit farms in Adams County, PA from 2012-2014 (Table 1). In this region of Pennsylvania, which is the leading tree fruit producing county in the state, orchards have steep slopes, well drained soils and a landscape matrix of approximately 8% orchards, 24% arable and pasture land, 9% developed area and 56% forests 44 . Orchards are generally small and bordered by undeveloped scrub, forest, or fence rows. This orchard-forest interface is home to a diverse plant community, supporting at least 146 plant species 28 and 188 species of bees from a collection of 80,000 field collected bees (DJB unpublished data). Because of the high bee diversity in this complex landscape, and a tripling in the cost of honey bee colony rentals since 2006, the majority of growers in this region do not rent honey bee colonies for pollination and rely completely on wild bees for pollination 29 . The study orchards ranged in age from 15-25 years, and contained primarily 'York' , 'Golden Delicious' , and 'Honey Crisp' apple varieties, and none of these orchards used managed honey bees for pollination, nor were within 1 km of managed honey bee hives.  Table 1.
The seed mixtures for floral plantings were supplied by Ernst Conservation Seed (884 Mercer Pike, Meadville, PA 16335) to best support a diversity of local bees throughout the season (see Fig. 1). Working cooperatively with USDA-NRCS and the fruit growers we established all floral plantings next to apple orchards, with a focus on: (a) providing blooming wild plants throughout the growing season, while minimizing overlap with apple bloom time; (b) including plant species that would not become weeds in apple or other fruit crops; and (c) including plant species that would not serve as alternate hosts of crop diseases or pests, such as brown marmorated stink bug. Orchards were maintained similarly by conventional IPM practices, which included both conventional and reduced risk pesticides as well as biological control. Floral plantings were maintained with yearly mowing and spot spraying for noxious weeds with selective herbicides, in accordance with the USDA-NRCS/Xerces Society recommendations.
Bee sampling in floral plantings and apple orchards. We sampled bee populations in the orchards and the floral plantings using blue vane traps (model Z-BVT, SpringStar Inc., Woodinville, WA), which are highly effective in sampling pollinators 46,47 . The blue vane trap (BVT) consists of a fluorescent yellow plastic container (0.71 L volume capacity) with a blue plastic funnel and two blue plastic cross vanes. Each floral planting was monitored with two BVT traps, while the larger orchard blocks were monitored with five or ten traps. Traps were suspended approximately 1.5 m above the ground with nylon rope from a metal pole. Supertech ® antifreeze (a mixture of ethylene glycol and diethylene glycol, Wal-Mart Stores, Inc., Bentonville, AR) diluted with tap water (60:40) was used as drowning media and specimen preservative for these traps. Trapped bees were collected weekly, from early April to first frost (~mid-October) each year.
Bees were collected from traps using a stainless steel strainer and a pair of flathead forceps, and immediately placed in a clear plastic vial containing 70% ethanol for further processing. In the laboratory, bee samples were dried, pinned, labeled, and identified to species level. Identifications were done by J. Gibbs at Michigan State University (Lasioglossum spp.), R. Jean at Saint Mary-of-the Woods College (Andrenidae samples), and K. Wright at the University of New Mexico (Melissodes spp.). The remaining bees were identified by R. Donoval (USDA-APHIS) and D. Biddinger (Penn State University) using keys [48][49][50] , and Discover Life ID guides (www. discoverlife.org).

Statistical analysis.
Prior to analysis, data were combined across dates and traps at each site within a year.
In addition, some analyses were performed on subsets of the bee community, specifically: known tree fruit pollinators (determined by previous net collections in Pennsylvania orchards at pome and stone fruit bloom), rare Species assemblages. Differences in species composition of the bee communities in the orchards versus those in the floral plantings were visualized with non-metric multidimensional scaling (NMDS) using the metaMDS function in the vegan package in RStudio 52,56 . NMDS is an ordination technique used to visualize the similarity between data points (in this case, each site/year combination) based on the dissimilarity of any number of variables (in this case, the abundance of each species) 57 . We conducted two NMDS analyses; the first analysis included all bee species collected in this study, and the second analysis only included the subset of bees that were known to be tree fruit pollinators. Statistical significance was determined by a PERMANOVA using the adonis function in the vegan package in RStudio 52,56 .
Associations in community assemblage patterns between individual tree fruit pollinating species and site types (orchard and floral plantings) were further examined using redundancy analysis (RDA), a constrained ordination technique, in CANOCO 4.5 58 . Species data were centered and standardized, and year was included as a covariate in the analysis. The significance of site type was determined using Monte Carlo simulations with 499 permutations of the data. Species associations with orchard or floral planting were then visualized using biplots developed in CanoDraw 4.5 58 .