Connectivity and edge effects increase bee colonization in an experimentally fragmented landscape

Though landscape corridors increase dispersal of many animals and plants, it remains unknown whether these positive effects extend to the process of colonization and establishment of new populations in fragments. Working in experimentally fragmented landscapes, we tested how two aspects of habitat fragments altered by corridors – connectivity and edge-to-area ratio – determine patterns of colonization by a solitary, cavity-nesting bee Megachile rotundata . We found that though connectivity initially affected rates of nest-site occupation, edge-to-area ratio ultimately determined the final patterns of patch occupation and nest building, likely due to habitat selection by our focal species. Bee colonization was also higher in patches with higher abundances of their preferred food resources, flowers from the Fabaceae family. Our results show the importance of considering the effects of both connectivity and edge on population dynamics in habitat-based conservation.


Introduction
Habitat fragmentation, which decreases habitat amount and simultaneously increases habitat isolation, is among the most serious threats to animal and plant populations worldwide (Haddad et al. 2015, Hanski 2015. At a landscape scale, fragmentation causes declines in metapopulation persistence by reducing the survival of local populations and providing a significant barrier to immigration between habitat patches (Gonzalez et al. 1998). Colonization is a key driver of these metapopulation dynamics; increasing rates of recolonization in habitats where local populations have been lost can increase metapopulation viability across fragmented landscapes (Hanski 1998). Theory predicts that colonization rates may be affected by spatial aspects of fragments such as connectivity to other habitat patches, patch shape and patch area (Weins 1997, Klok and DeRoos 1998, Moilanen and Hanski 1998, Moilanen and Nieminen 2002, Ewers and Didham 2007. However, there has been little study of how habitat-focused conservation practices, like corridors, alter the ability of dispersing organisms to establish local populations. One justification for the use of landscape corridors (thin strips of habitat used to connect isolated habitat fragment) is that they are expected to promote colonization of fragments and thereby increase population viability. Increased connectivity due to corridors has been shown to effectively increase the movement of plants and animals across landscapes (Gilbert-Norton et al. 2010, Resasco 2019. In turn, increased movement caused by corridors increases abundance Baum 1999, Resasco et al. 2014) and species richness in connected fragments (Gilbert et al. 1998, Damschen et al. 2006. It is unclear whether the positive effects of corridors on abundance and species richness are due to increased population persistence within connected habitats, or to increased rates of colonization and establishment by dispersing organisms. Although the higher dispersal caused by corridors is expected to positively affect rates of colonization, few studies have explicitly studied the effects of corridors on population-level processes like establishment (Beier and Gregory 2012).
In addition to their primary function of restoring connectivity, corridors can have an unintended consequence: they increase edge-to-area ratio of fragments Baum 1999, Tewksbury et al. 2002). Higher edge-to-area ratio due to the long, thin shape of corridors may alter abiotic conditions within connected patches (Menz et al. 2011, Evans et al. 2012, Damschen et al. 2014) and increase the amount of suitable habitat for edge-associated species Didham 2007, Haddad et al. 2014). The addition of edgy habitat may also raise the probability that dispersing organisms will encounter the habitat when moving through the landscape Didham 2006, 2007). Though both connectivity and edge effects of landscape corridors have been found to affect movement of organisms between fragmented habitats, their relative impacts on colonization are unknown.
Habitat-based conservation is commonly used for one group, the wild bees, with little understanding of how spatial factors may affect the ability of bees to establish new populations. Wild bee populations have been devastated by the destruction of natural habitat and other human activities (Potts et al. 2010, Winfree 2010, Goulson et al. 2015, causing these important pollinators to become a major focus of conservation efforts worldwide (Byrne andFitzpatrick 2009, Wilson et al. 2017). Conservation practices generally center on the creation of high-flowering habitats (Winfree 2010, Scheper et al. 2015, Vaudo et al. 2015, Williams et al. 2015, with the hope that bees will quickly colonize these new habitats and diverse bee communities will persist over time. Thus, strategies that increase the likelihood and success of bee colonization may be vital for meeting conservation goals. Bees are mobile organisms with relatively strong flight ability, but female bees are central-place foragers limited in their movement by the energetic costs of traveling back and forth from their nests (Murray et al. 2009, Zurbuchen et al. 2010). In addition, bee flight ability is related to body size, with smaller bees exhibiting smaller flight distances (Greenleaf et al. 2007). Thus, many bees forage relatively close to their nests (Gathmann andTscharntke 2002, Greenleaf et al. 2007) and exhibit reduced reproductive success (Peterson andRoitberg 2006, Zurbuchen et al. 2010) and lower abundance with increasing distance from natural, resource-rich areas (Garibaldi et al. 2011, Kennedy et al. 2013. Landscape corridors may benefit bees in isolated and fragmented habitats by increasing bee movement and their ability to access resources , Townsend and Levey 2005, Cranmer et al. 2012. Foraging bumblebees follow hedgerows and other linear habitat elements through agricultural fields, essentially using them as corridors (Cranmer et al. 2012). In addition, higher connectivity between habitats leads to higher pollen transfer, indicating longer travel distances and increased movement by foraging bees and other pollinators (Tewksbury et al. 2002, Townsend and Levey 2005, Geert et al. 2010, Cranmer et al. 2012, Kormann et al. 2016. However, these and other studies of bee response to landscape fragmentation generally measure foraging bees rather than dispersers (Pyke 1984, Palladini andMaron 2014). It is unknown whether the positive effects of landscape corridors on bee movement extend to populationlevel processes such as dispersal and colonization.
To test the effects of connectivity and edge-to-area ratio on habitat colonization by bees, we conducted a large-scale release experiment with solitary, cavity-nesting bees within a set of experimentally fragmented landscapes. This bee species was not previously found in our landscapes, so any bees we observed originated from the location of our releases. This allowed us to examine patterns of colonization without the confounding effects of pre-existing populations. We tested for effects of patch connectivity and edge-to-area ratio on three aspects of solitary bee colonization: initial bee occupation of nests, nest-building and reproductive output. We also included floral availability as a covariate in our analysis based on the known, positive relationship between bee abundance and floral abundance (Potts et al. 2003, Roulston and Goodell 2011, Hopwood et al. 2015, Herrmann et al. 2017). Due to previous findings that corridors increase pollinator movement (Tewksbury et al. 2002, Townsend and Levey 2005, we hypothesized that corridors would increase bee colonization across our fragmented landscapes and that these effects would be primarily driven by connectivity rather than edge effects.

Experimental landscapes
To test the effects of corridors and habitat edges on patterns of bee colonization, we used a long-established set of experimentally fragmented landscapes: the Savannah River Site Corridor Project.  ('connected' patch). Three unconnected patches in each block are also separated from the center by 150 m and are the size of the connected patch plus the corridor. The additional area is added either as a blind-ended corridor ('winged' patches) or an extended footprint ('rectangular' patches), to control for the added area of the corridor (Fig. 1C). Within each block, one unconnected patch is winged, one is rectangular and the third is either winged or rectangular. The relative position of all patches within each block is randomized to control for any effect of directionality and patch orientation. All habitat patches are actively restored to longleaf pine savanna through prescribed burning and hardwood removal, and contain a diversity of flowering forbs that serve as floral resources for bees. In contrast, the matrix around the habitat patches is predominantly composed of dense plantations of pine trees and is largely devoid of flowering resources.
Our experimental design lets us separate out the effects of connectivity and edge-to-area ratio. Comparison between connected and winged patches provides a test of the effect of connectivity because the two patch types have similar shapes and edge-to-area ratios but differ in connectivity. Comparison between winged and rectangular patches provides a test of the effect of edges, because both patch types are disconnected, but winged patches have ~50% more edge than rectangular patches. Finally, comparison between connected and rectangular patches provides a test of the additive effects of connectivity and edge, because connected patches have more edge and connectivity than rectangular patches.

Experimental bee release
We studied the alfalfa leafcutting bee Megachile rotundata, a small solitary bee commonly managed as a pollinator of alfalfa and other commercial crops. Although this bee originated in Europe, its current range extends throughout the entire United States (Strange et al. 2017). Megachile rotundata was an ideal model organism for our landscape-level experiment for several reasons. First, because this bee species is commercially available and overwinters as larvae within cocoons made from leaves, it was possible to obtain sufficient numbers of bees and carefully control their emergence timing for our experiment (Pitts-Singer and Cane 2011). Second, as M. rotundata is a cavity-nesting species, we were able to manipulate nesting resources across each block to study patterns of dispersal and fragment colonization (McCorquodale and Owen 1997). Third, though this bee is widespread and found in the region surrounding SRS, it has not been previously found in our experimental landscapes, giving us confidence that any M. rotundata found within our sites originated from our release points. Finally, because body size predicts foraging range and dispersal ability in bees (Greenleaf et al. 2007), M. rotundata is a relevant model for the dispersal and colonization behavior of other small-bodied, solitary bees. Megachile rotundata also exhibits similar cavity-nesting behaviors to native Megachilids, a common group in our study region (Horn and Hanula 2004).
In early April 2018, we obtained M. rotundata cocoons from a commercial bee provider, JWM Leafcutters Inc. (Nampa, ID, USA). We stored all cocoons at 4°C until late April when spring temperatures and floral resources at the experimental blocks were approaching suitable levels for M. rotundata. We then incubated the cocoons at 30°C for ~17 days until the first of the male bees started emerging, after which we placed 40 g of cocoons (approximately 500 individuals) into each release box. Release boxes consisted of white cardboard boxes (10.16 by 10.16 by 5.08 cm) with small holes cut in one side, placed inside empty wooden nest boxes and mounted on heavy metal fenceposts one meter above the ground. Wooden boxes were built using 1.5 cm pine plywood, and were 36 cm tall, 24 cm wide and 18 cm deep, with an extra 6 cm overhang to protect bee nests from rain and direct sun. Four of these wooden nest boxes were placed in the center patch of each experimental block (36.77 m from the two closest edges), for a total of approximately 2000 M. rotundata individuals released per block. Because female bees generally make up about 1/3 of all M. rotundata (Pitts-Singer and James 2005), our releases constituted approximately 667 reproductive female bees released per experimental block. All cocoons in release boxes were placed in the field on 10 and 11 May, and full emergence likely occurred within one to two weeks following this initial placement.
Prior to bee release, we placed standardized nesting resources in all peripheral patches to allow observation of bee colonization and brood rearing. Within nest boxes (identical to the wooden boxes described above), we used two types of nesting resources: bundles of Phragmites reeds ~0.8 cm diameter and 15.24 cm long, and polystyrene blocks with holes 0.6 cm diameter and 7.62 cm long. Two bundles of 30 reeds were zip-tied to the sides of each nest box, and one 15.24 by 15.24 cm polystyrene nesting block with approximately 210 holes was glued to the back of each nest box (Supporting information). Nest boxes were provisioned with this overabundance of nesting resources to increase our chances of colonization and so remove nesting resource availability as a factor that could limit bee colonization. We placed four nest boxes in each peripheral patch, positioned in every corner 14.85 m from the two closest edges. Nest boxes were mounted on heavy metal fence posts approximately one meter from the ground and placed facing the south-east to standardize directionality and allow for morning sun exposure. Chicken wire was also attached to the front of all nest boxes as protection from disturbance by birds and mammals.

Bee dispersal and colonization
To study initial M. rotundata occupation of habitat patches, we counted individual bees within nest boxes soon after release. To do this, we visited nest boxes at night when all occupying bees were present in their nests. Shining a bright flashlight into the reeds and nest blocks, we counted every female bee present in nests. We were able to reliably distinguish M. rotundata from other species of wood nesting Megachile by their small size and white scopa. We started our first round of observation on 20 May, 10 days after release, to give female bees adequate time to emerge, mate and search for nests. We continued to visit all nests until no additional nesting M. rotundata were found in nest boxes (13 June, 34 days after release), at which time we removed all nest materials from the field.
In the lab, we dissected all reeds and occupied nesting blocks to count total number of nests and brood cells produced. As a measure of nests produced by M. rotundata, we counted all nesting resources (individual reeds or holes in polystyrene blocks) that had one or more fully formed brood cells. To measure the number of cells per nest, we counted the number of fully formed cells within each individual reed or hole, pulling apart attached cells to gain an accurate count. Brood cells were reared to adulthood and all emerged bees were confirmed as M. rotundata with a dissecting microscope.

Floral resources
To measure floral resources available to nesting bees during our experiment, we counted flowers within all peripheral patches of each experimental block. We created 50 m transects within each peripheral patch at three distances from the nearest edge (1, 14.85 and 36.77 m). The mid-point of each transect was measured from a randomly selected corner toward the center of the patch. The transect formed a right angle, with each extending 25 m parallel to the nearest patch edge (see the Supporting information for diagram of floral transects). Within 1 m of each side of the transect, we counted all flowering plant individuals per species. For ten individual plants of each species, we counted the number of inflorescences per plant. We then multiplied the average inflorescences for these 10 individuals by the number of individuals of that species to obtain an estimated number of inflorescences per species per transect. We then added together the estimated number of inflorescences in a patch to obtain a relative count of inflorescences per species per patch. Because our focal species M. rotundata is known to prefer legume flowers from the plant family Fabaceae, we added together the estimated counts per plant species to create two measures of floral abundance for our analyses: the relative count of Fabaceae inflorescences per patch and the relative count of all non-Fabaceae inflorescences per patch. We conducted two rounds of floral sampling, with the first round between 25 and 29 May and the second between 10 and 13 June. Over these two sample rounds, we identified and counted 11 Fabaceae species and 42 other species of flowering plants. There was no correlation between Fabaceae and non-Fabaceae abundances (r = 0.1, p = 0.63), and patch type did not affect either Fabaceae abundance (χ 2 = 0.82, df = 2, p = 0.66) or the abundance of non-Fabaceae flowering plants (χ 2 = 0.62, df = 2, p = 0.73) over the two rounds of sampling.

Analysis
We used generalized linear mixed models to test for the effects of patch type and floral resources on three measures of M. rotundata colonization: initial nest occupation after 10 days since release, final number of nests created and final number of cells per nest. Due to the large distances between patches and the few points with experimental nest boxes, we expected an overall low rate of nest-site occupation. Therefore, we calculated all response variables per patch by combining counts from both types of nesting materials (reeds and nest blocks) and each of the four nest boxes within a patch. This gave us a total sample size equivalent to the number of peripheral patches in our study (n = 28) per response variable. For each of our three models, we used patch type (connected, winged and rectangular) and floral abundance as our main effects and included block as a random effect. We only used floral abundances from the first round of sampling for the initial nest occupation model to better match the timing of floral counts to the response variable. We summed floral abundances from both rounds of floral sampling for the other two models. For the cells per nest response variable, we removed one block from the model because bees did not build any nests in that block. We used GLMMs with Poisson distribution for initial nest occupation and number of nests, and a normal distribution for the number of cells per nest (package lme4, Bates et al. 2014). Test statistic (χ 2 value) and significance for fixed effects in all models were estimated using type III ANOVAs. We then conducted Tukey-Kramer contrasts (package lsmeans, Lenth 2016) to determine the effect of patch type. All analyses were performed in R (<www.rproject.org>).

Results
In our first survey starting 10 days after bee release, we observed 88 nesting female Megachile rotundata. We found an effect of patch type (χ 2 = 7.8, df = 2, p = 0.02) on initial occupation rates. Connected patches had higher, albeit nonsignificant, initial occupation than winged patches (35% more bees, Fig. 2) and significantly higher initial occupation than rectangular patches (111% more bees, Fig. 2), indicating an additive effect of connectivity and edge-to-area ratio. We also found a significant positive effect of Fabaceae abundance (χ 2 = 4.19, df = 1, p = 0.04, Fig. 3A) but no effect of overall non-Fabaceae floral resources (χ 2 = 0.85, df = 1, p = 0.36, Supporting information) on initial patch occupation.

Discussion
By introducing a new species to experimentally fragmented landscapes and studying its occupation of empty habitat Figure 2. Arithmetic means (± SE) of counts of M. rotundata bees initially occupying nesting resources in each patch type, measured during the first round of observation started 10 days after bee release. Letters denote significant differences (p < 0.05) between patch types, calculated using Tukey-Kramer contrasts on generalized linear mixed models. patches, we show that landscape corridors increase patch colonization and likely increase metapopulation viability. Bee colonization of fragmented habitats responded strongly to both spatial aspects altered in our experimental design by corridors, increased connectivity and increased edgeto-area ratio. There was a temporal component to these effects: landscape corridors increased initial bee occupation of nest boxes in connected habitats due to the additive effects of increased connectivity and increased edge-to-area ratio, whereas higher edge-to-area ratio was the ultimate determinant of nest establishment across habitat fragments. Thus, landscape corridors affect not only individuals' movement and dispersal behaviors as previously shown (Resasco 2019), but also population-level processes within fragmented habitats.
The effects of corridors on colonization observed in our study may have important consequences for metapopulation dynamics. Because viability in the long-term depends on the overall size of the metapopulation and its ability to replace extirpated local populations, metapopulation persistence across landscapes is a function of both local population viability and colonization rates of unoccupied habitats (Hanski 1998). To date, population-focused work with corridors has largely focused on local population viability. Previous studies have shown that higher connectivity can increase dispersal between habitat fragments (Gilbert-Norton et al. 2010, Resasco 2019) and increase population sizes (Gonzalez et al. 1998, Hudgens and Haddad 2003, Resasco 2019. Our study instead focused on the second factor that affects metapopulation viability, colonization, and found positive effects of both increased connectivity and edge. Thus, higher rates of metapopulation survival in landscapes connected by corridors (Gonzalez et al. 1998) may be attributable to both increased local population viability and higher rates of recolonization. Further, these population-level responses can be expected to scale up to affect entire communities, providing a possible mechanism for increased species richness in habitat fragments connected by landscape corridors (Damschen et al. 2006(Damschen et al. , 2019. Our study shows that increases in insect movement that have been observed in response to corridors (Townsend and Levey 2005) result in more rapid colonization of patches. Connectivity had an additive effect on initial rates of nestsite occupation, with the highest initial occupation of nest boxes occurring in patches connected by a landscape corridor. Corridors likely channeled dispersing bees from the center patches of the experimental blocks towards connected patches, allowing them to quickly find suitable nesting habitats and establish new populations. Such behavior may have been due to edge-following from release patches into the corridors (Haddad 1999, Joyce et al. 1999, Berggren et al. 2002, Holzschuh et al. 2009, Cranmer et al. 2012) and a preference for flying through the open habitat of the corridors rather than through the forest matrix. An effect of connectivity was evident in our fragmented habitats separated by only 150 m; differences in colonization rates may be even more pronounced when fragments are more isolated or smaller in size (Haddad 1999).
Although connectivity initially affected occupation of nest-sites, edge-to-area ratio had an additive effect on these occupation rates and ultimately drove patterns of nest building. Almost double the number of nests were produced in high-edge connected and winged patches than in low-edge rectangular patches. Because most nests were incomplete during the final count, we believe that most female bees only produced one nest each within our study; thus, patterns of nest building were likely driven by dispersal by individuals rather than differences in reproductive success between patches. One explanation for our findings is that dispersing bees may have eventually been able to fly anywhere at the scale of our experimental blocks, and preferentially settled in high-edge patches. Such habitat selection for high edge habitat seems likely given that the bee used in our experiment, Megachile rotundata, is a cavity-nesting species that would naturally nest in holes in trees (Pitts-Singer and Cane 2011). Dispersing bees may also prefer the abiotic conditions of high-edge habitats, which are cooler than rectangular patches (Evans et al. 2012). Bees are known to be sensitive to temperature in terms of their foraging (Rader et al. 2013) and nesting Willmer 1997, Weissel et al. 2006), so differences in temperature between patches differing in edge:area ratio could affect bee brood production and nesting. Another explanation for Figure 4. The effect of patch type on M. rotundata nesting and brood production, measured through nest dissection at the end of the flight period. Arithmetic means (± SE) of (a) M. rotundata nests produced in each patch type and (b) M. rotundata cells created per nest. Letters denote significant differences (p < 0.05) between patch types, calculated using Tukey-Kramer contrasts on generalized linear mixed models. these patterns is that the corridors in connected patches and blind-ended corridors (wings) of the winged patches may have functioned to intercept and channel dispersing bees towards the interior of the habitat patches (Fried et al. 2005). Highedge habitats are known to have higher rates of colonization than more compact, low-edge fragments due to the increased chance of encounters by dispersing organisms (Hamazaki 1996, Bevers and Flather 1999, Collinge and Palmer 2002, Ewers and Didham 2006, 2007, but this effect has rarely been shown in relation to habitat corridors (Tewksbury et al. 2002, Townsend and Levey 2005, but see Fried et al. 2005).
Megachile rotundata colonization was higher in patches with more Fabaceae flowers. This is unsurprising, given this species' well-known preference for alfalfa and other species of legumes (Horne 1995). Patches with more Fabaceae flowers had higher initial occupation, nest building and even cell production, indicating that the availability of their preferred food resources affected not only female bees' decision to nest but also their ultimate reproductive success. In fact, once bees settled in patches, female fecundity was affected only by Fabaceae availability, not by patch connectivity or edge.
Megachile rotundata are small bees with limited foraging ranges (Greenleaf et al. 2007), meaning that they may have foraged solely in the patch in which they nested. Thus, connectivity with other patches may have been irrelevant from a foraging perspective.
In our study, the released M. rotundata only produced an average of fewer than three cells per nest, which represents low brood production for this species (Peterson 2006). Floral abundance is known to strongly affect both M. rotundata nesting success and brood production (Peterson 2006, Pitts-Singer andBosch 2010), and previous studies have found similarly low numbers of cells per nest when M. rotundata are placed in fields with insufficient floral resources (Peterson 2006). Though the longleaf savannah system contains high plant diversity, floral abundance was generally low throughout the study season, especially for legume species. Therefore, it is likely that bees were seriously limited by floral abundance across our experimental landscapes. In addition, the highly variable, harsh environmental conditions of our study sites in South Carolina may have further impacted the ability of bees to establish and provision nests. Regardless, despite low establishment across our study, our experimental design allowed us to compare colonization between replicated treatments in an ecologically-relevant system.
Conservation efforts for bees generally focus on improving the quality of food and nesting resources within new and restored habitat patches (Winfree 2010, Scheper et al. 2015, Vaudo et al. 2015, Williams et al. 2015. Our results show that while these factors are essential, spatial aspects of habitats should also be an important consideration for bee conservation. We show that bee colonization of pollinator habitats may be facilitated through increases in habitat connectivity and edge. Given the importance of colonization for metapopulation persistence, habitat-based conservation practices such as landscape corridors that increase these qualities should be implemented widely to support bee populations in fragmented landscapes.