Understanding the multiple resource needs of leaf-cutter bees to inform pollinator

conservation and the restoration of reclaimed mines

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science

in the Graduate School of The Ohio State University

By

John Peter Ballas II, BS

Graduate Program in Evolution, Ecology, and Organismal Biology

The Ohio State University

2020

Thesis Committee:

Karen Goodell, Advisor

Frances Sivakoff

Rachelle Adams

Rebecca Swab

Copyright by

John Peter Ballas II

2020

Abstract

Loss of habitat is one of the drivers of bee declines worldwide. However, conservation and habitat recreation often solely focus on the establishment of forbs as a pollen and nectar source while disregarding other resources that bees need to gather from their landscapes. The family Megachilidae is a large and diverse assemblage of bee species, the majority of which require non-floral resources for nest building. Leaf-cutter bees (genus Megachile) are perhaps the most well-known bees within the family, using cut pieces of leaves to build their nests. Like all bees, Megachile are central place foragers, requiring all of their food plants, as well as suitable leaves for nest construction, within a limited foraging range of their nest. Each female builds her nest within a tunnel in wood or a hollow stem where she constructs a linear series of brood cells lined with cut discs of leaves that protect her offspring and pollen provisions from parasites and desiccation. Poor quality leaves incur fitness costs for the female bee, including longer processing time and excessive mandibular wear. These costs drive strong preferences for specific species of leaves. I investigated the leaf traits that influence preference in

Megachile to better understand how variation in vegetation surrounding the nest might limit bee success through the availability of high-quality leaves. This study focuses on reclaimed coal surface mines in Eastern Ohio because they exhibit variation in vegetation

i at a landscape scale relevant to bee foraging. Reclaimed mines also present novel ecological conditions that can be used to establish habitat for bee conservation.

First, I described the physical characteristics of leaves found on a reclaimed mine in order to determine traits associated with leaf choice. This study focused on a representative species of leaf-cutter bee, Megachile rotundata, because of its abundance at the study site and the availability of published data on the species of leaves it uses for nesting. The 59 surveyed species of leaves were divided into two groups: those which are used by M. rotundata at the study site or in the literature, and those that are not used. By measuring the toughness, trichome density, useable area, and other traits of each leaf, species used and not used by M. rotundata were compared via ordination. The physical characteristics of used and unused leaves differed, but no one trait completely distinguished the two groups. Other untested traits may contribute to preference. The interactions of traits are complex, but toughness is a large factor determining usability.

By ordinating leaf species from their traits, differences were clearly visualized. This study also documented species of plants used by M. rotundata that have not yet been observed, including the petals of multiple forbs.

In order to learn how the availability of leaves affects leaf-cutter bee nesting behavior, I tested how variation in landscapes and plant composition affected the leaf foraging durations of M. rotundata. Building on prior observations about M. rotundata’s preferences for specific leaves and higher rates of reproduction closer to forest edges, I tracked bee foraging duration at different distances from the forest to compare the time investment towards leaf collection in different landscapes. The prediction that bees

ii nesting closer to forest would spend less time foraging for leaves was supported, likely a result of the forest offering a large and apparent source of suitable leaves. Bees nesting far from the forest would be required to search the complex matrix of their surrounding grassland to find suitable leaves, leading to long foraging trip durations. The study also provides rigorous documentation of foraging patterns mentioned anecdotally in earlier literature. These observations verify that bees consistently foraged for leaves by making a series of “short” trips, interspersed by occasional “long” trips. Regardless of site location,

“short” trips varied little in duration. However, the “long” trips lasted more than 7 times longer on average and were more variable in duration for bees nesting further from the forest. The abundance of floral resources did not predict the duration of long foraging bouts, refuting previous hypotheses that long trips represented combined leaf and nectar foraging bouts. These results are consistent with the idea that bees are limited by leaf choices in large reclaimed grasslands and that the forest edge is a particularly valuable habitat for Megachile.

iii

Acknowledgments

First and foremost, I must thank my advisor Karen for providing guidance throughout this entire project. Her enthusiasm and expertise were instrumental in helping me complete this research. I’d also like to pay regards to my lab mates Andrew Lybbert,

Keng-Lou James Hung, and Jessie Lanterman Novotny for their helpful discussions. This work would not have been possible without the research previously done by Megan

Varvaro towards her thesis with Karen. I am grateful to the wonderful researchers and staff at the Wilds, with a special thank you to Anna Starkey for her assistance with fieldwork. My committee’s unique contributions were important in guiding this research from the start. I’d like to thank Frances for our insightful discussions while driving to and from the Wilds and while doing fieldwork; Rachelle for being so welcoming when I was beginning at OSU and for offering an enlightening research perspective; and Rebecca for her seemingly never-ending knowledge on all things related to the Wilds. While the OSU

Chapter of The Society for Ecological Restoration took a decent chunk of my time away from working on my thesis, I am very grateful for the wonderful people that made the organization so great and for the fulfilling work we were able to do. And finally, I’d like to express sincere gratitude to my partner Ian for his continued support and his unwavering attention every time I need to share a fun fact about bees.

iv

Vita

2018 BS Biology, University of Cincinnati

2018-2019 University Fellow, Department of Evolution, Ecology, and

Organismal Biology, The Ohio State University

2019-2020 Research Associate, Department of Evolution, Ecology,

and Organismal Biology, The Ohio State University

Publications

Ballas, J. P., & Matter, S. F. (2020). UV-induced anthocyanin in the host plant Sedum lanceolatum has little effect on feeding by larval Parnassius smintheus. Alpine Botany,

130, 25–30.

Fields of Study

Major Field: Evolution, Ecology, and Organismal Biology

v

Table of Contents

Abstract ...... i Acknowledgments ...... iv Vita ...... v List of Tables ...... vii List of Figures ...... viii Chapter 1: Relating the physical characteristics of leaves to leaf-cutter bee use ...... 1 Abstract ...... 1 Introduction ...... 2 Methods ...... 5 Results ...... 10 Discussion ...... 12 Chapter 2: Landscape scale effects of resource availability on leaf-cutter bee foraging .. 17 Abstract ...... 17 Introduction ...... 18 Methods ...... 21 Results ...... 26 Discussion ...... 26 Tables ...... 32 Figures ...... 34 Literature Cited ...... 41 Appendix A: Plant species used and not used by Megachile rotundata at the study site . 51 Appendix B: Unused linear mixed effects models modeling “long” trip foraging duration and a correlation matrix for the “long” trip dataset ...... 53

vi

List of Tables

Table 1: Results from a GLM correlating each leaf trait to the likelihood of use by Megachile rotundata ...... 32

Table 2: Results from a perMANOVA comparing the use of leaf species used by Megachile rotundata and those not used ...... 32

Table 3: Results from multiple LMEM’s correlating time spent forging with the distance from the forest ...... 33

vii

List of Figures

Figure 1: Examples of LCCD calculations for a variety of leaves ...... 34

Figure 2: Plots showing the distribution of traits from leaf species used and not used by Megachile rotundata ...... 35

Figure 3: NMDS plot of leaf species ordinated by their physical characteristics ...... 36

Figure 4: Locations of artificial domiciles at the Wilds ...... 37

Figure 5: Images of an artificial domicile ...... 38

Figure 6: A sample time budget from a bee included in the study demonstrating the pattern of "long" and "short" trips ...... 38

Figure 7: Leaf foraging trip distributions as fitted from a mixture model ...... 39

Figure 8: Association between distance from the forest and leaf foraging trip duration for both “long” and “short” trips ...... 40

viii

Chapter 1: Relating the physical characteristics of leaves to leaf-cutter bee use

Abstract

Leaf-cutter bees (genus Megachile) are a subset of the diverse family

Megachilidae, sharing the unique of behavior of building nests from foraged materials.

Most leaf-cutter bee species use cut pieces of leaves to line their nests and brood cells, protecting each egg and pollen provision. This behavior has interesting implications for selecting a nest site. Individual female bees must find a location with suitable leaves for nest building and adequate floral resources for pollen and nectar, and thus nesting success depends on the dual availability of these required resources. In this study, I investigated how different leaf characteristics influence leaf choice in a representative species of leaf- cutter bee, Megachile rotundata, the alfalfa leaf-cutter bee. This study focused on multiple grasslands across a reclaimed coal surface mine in Eastern Ohio and tested the physical characteristics of leaves available in the landscape. Instead of examining characteristics of leaves known to be used by M. rotundata, all leaves in the habitat were analyzed to better understand what makes a leaf suitable or unsuitable for use in the nest.

Observations of bees at the site and observations from previous studies were used to construct an index of used and unused leaves for the study area. Using non-metric multidimensional scaling, I visualized different leaves based on their traits (including toughness, trichome density, and size) and found that leaves that are used by bees are

1 distinct from those that are not used. These preferences seem to be driven by leaf toughness but also appear to be moderated by the complex interactions among traits. For example, leaves that might have suitable toughness and trichome densities might simply be too small for a leaf-cutter bee to use. This approach allows for a more universal understanding of the suitability of leaf species, regardless of region and habitat.

Introduction

Megachilidae [Hymenoptera: Apiformes] is a widespread and diverse family of solitary bees (Cane, Griswold, & Parker, 2007; Gonzalez, Griswold, Praz, & Danforth,

2012; Litman, Danforth, Eardley, & Praz, 2011). Megachilid bees are united by their unique behavior of building nests from foraged materials including leaves, petals, mud, resin, and gravel (Cane et al., 2007; MacIvor, 2016). A typical nest of a Megachilid bee consists of a series of brood cells separated by some type of material (Cane et al., 2007;

Pitts-Singer & Cane, 2011). The need to forage for these nesting materials along with pollen and nectar makes the habitat requirements for these bees particularly specific

(Cane, 2001; Requier & Leonhardt, 2020). The species diversity, specialized pollen carrying structures on their abdomens, and wide range of functional niches occupied by this family allow them to contribute uniquely to pollination services and makes them good targets for conservation (Cane et al., 2007; Fründ, Dormann, Holzschuh, &

Tscharntke, 2013; Gonzalez et al., 2012; Hoehn, Tscharntke, Tylianakis, & Steffan-

Dewenter, 2008; Martins, Gonzalez, & Lechowicz, 2015; Winfree, Williams, Dushoff, &

Kremen, 2007).

2 Leaf-cutter bees (genus Megachile) use their mandibles to cut roughly circular pieces of leaf material with which they line their nests, with some exceptions (Gonzalez,

2008; Gonzalez et al., 2012; Litman et al., 2011). Megachile are among the most well- studied solitary bees given the alfalfa leaf-cutter bee’s (Megachile rotundata) value to humans as a managed pollinator (Horne, 1995; Peterson & Roitberg, 2006; Pitts-Singer &

Bosch, 2010; Pitts-Singer & Cane, 2011). Most research has focused on the crops and wild plants used by leaf-cutter bees in order to increase agricultural yields and to understand which wild plants rely on these bees for pollination (Cusser & Goodell, 2013;

Fründ et al., 2013; Vanbergen & The Insect Pollinators Initiative, 2013; Vázquez et al.,

2012). Apart from flowers, the importance of resources like nesting sites and materials are understudied (Cane, 2001; Morato & Martins, 2006; Potts et al., 2005; Requier &

Leonhardt, 2020).

Access to nesting sites and materials can limit reproduction and overall success of solitary bees (Cane, 2001; Grundel et al., 2010; Morato & Martins, 2006; Potts et al.,

2005; Sedivy, Praz, Müller, Widmer, & Dorn, 2008). Suitable habitat for bees must contain adequate pollen and nectar sources as well as nesting sites and materials within a small foraging range (Cane, 2001; Zurbuchen et al., 2010). A better understanding of the specific required nesting materials can guide conservation for bees by allowing for more specific and beneficial habitat creation and augmentation (MacIvor & Packer, 2015).

While little is known about the species of leaves used by leaf-cutter bees, even less is known about the traits that influence these preferences. The use of foreign materials in nesting is important because it prevents desiccation and microbial infection

3 of pollen and nectar provisions (Litman et al., 2011). Analysis of the physical characteristics of leaves that bees are known to use certainly provides insight to their suitability as nesting materials but does little to differentiate suitable from unsuitable species. Studies documenting leaf use in a single area may not be fully representative of the behavior of a bee throughout its entire range or in every circumstance. Our current understanding of preferences for specific leaf characteristics is limited to a few controlled enclosure experiments comparing different crops (Horne, 1995) or horticultural varieties of a single species (Eigenbrode, White, & Tipton, 1999; Nugent & Wagner, 1995). Some have attempted to identify the species used by comparing dried leaf discs from dissected brood cells to pressed plants (Soh, Soh, Ascher, & Tan, 2019). Research has been expanded with innovations like DNA barcoding. This technology is promising, but so far has only been used to identify leaves used by bees in an urban and semi-urban landscape and does not itself offer insights into the leaf traits that drive preference among bees

(Kamblı̇ et al., 2017; MacIvor, 2016). Leaf toughness, trichome density, and leaf chemistry are among the main factors that leaf-cutter bees consider when choosing leaves to use for nesting (Eigenbrode et al., 1999; Horne, 1995). However, no study to date has documented the entire set of species that are and are not used by leaf-cutter bees in a natural habitat and compared the characteristics of each species. Preferences are likely to be modulated by the total pool of available leaves, along with each species’s abundance and proximity to the nest site.

To study the impacts of different leaf characteristics on leaf choice in Megachile,

I tested a suite of physical characteristics from the leaves of 59 plant species present in a

4 grassland-reclaimed coal surface mine in the Eastern United States. By comparing the traits of leaves documented to be used by leaf-cutter bees regionally and observed to be used by leaf-cutter bees locally at the study area, I evaluated the traits that likely drive leaf preference. Generally, I hypothesized that leaves used by bees will have similar physical characteristics, but that interactions between individual characteristics, rather than values of a single trait would be associated with use by Megachile. Finally, I predicted that certain traits like small usable area and extreme toughness would have threshold values beyond which leaves would be unusable, regardless of the values of other traits.

Methods

Study site

I conducted the study at the Wilds, a 37 km2 reclaimed mine in Eastern Ohio

(39.829277, -81.724098). The site was used to surface mine coal from the 1940’s to the

1980’s and is now a non-profit conservation center. The Wilds is made up of a series of grasslands with some young restored forests and a few un-mined forests. An abundance of exotic legumes like Lotus corniculatus supports large populations of the introduced alfalfa leaf-cutter bee, M. rotundata (Pitts-Singer & Cane, 2011; Varvaro, 2018). I selected 11 sites at the Wilds where M. rotundata was known to be abundant. These sites were a mix of large reclaimed grasslands, restored “pollinator prairies”, and forest edge habitats. To further ensure that bees would be present at surveyed locations, each site was stocked with approximately 500 M. rotundata cocoons from Mason Bees for Sale

(masonbeesforsale.com) and artificial domiciles for adult females to nest in. The artificial

5 domiciles were constructed from 1.5 by 2 by 10-cm hardwood lumber that had a semi- circular groove running lengthwise. Two such pieces were fixed together with staples to create a circular cavity with a diameter of 0.25-0.5 cm. The seams and back end were sealed with construction caulk to prevent parasites from entering. The nest tunnels were bundled together in sets of 12 and secured to fenceposts with electrical tape facing eastward so that the rising sun would warm the nest entrances each morning. I secured a small piece of plastic tarp over each bundle of nests to protect them from rain.

Leaf availability surveys

To quantify the leaf materials available for bees nesting at each site, I conducted a series of vegetation surveys. Both the grasslands surrounding each nest and the closest adjacent forest were surveyed. Because the physical characteristics of the leaves from young saplings and mature trees can vary greatly, I chose to treat young encroaching woody growth in grasslands as separate from the mature individuals of the same species present in the adjacent forests (Basset, 2001; Ishida, Yazaki, & Hoe, 2005; Joesting,

2005).

To survey each nest’s surrounding grassland, I selected three random angles more than 30 degrees apart with a random number generator. In each direction from the nest, I placed 1-m2 quadrats at 4 randomly generated distances between 1 and 25 m from the nest such that no quadrats were adjacent. Within each quadrat, I estimated the absolute percent cover of all species of forbs, graminoids, and woody plants (Daubenmire, 1959).

All absolute cover estimates were done by a single person to prevent observer bias.

Absolute cover was defined as the proportion of ground within the quadrat that would be

6 blocked by a given species’ leaves, ignoring all other species. As plants within a quadrat frequently overlapped, the sum of all species’ absolute cover could sum to more than

100%. I choose absolute cover as an estimate of plant abundance over other methods, like counts of individuals, as absolute cover better captures the size variation in plants and the amount of leaves they are contributing to the landscape. Plants were identified using

Newcomb and Morrison (1977) when possible, although some species that were not flowering at the time of surveying were identified using Gleason and Cronquist (1991) or were flagged in the field and identified later in the season upon flowering.

I also surveyed the vegetation in the adjacent forest closest to each nest site. I was not able to calculate percent cover for this woody vegetation in a way comparable to how herbaceous cover was sampled. Instead, I tallied the total number of individuals of all woody plants greater than 1 m tall in a 30 m by 3 m area. For some sites, the 30 m survey area did not follow a straight line but was contoured to match the natural curve of the forest edge.

Physical characteristics

I tested the physical properties of leaves for all plant species that accounted for more than 0.5% of the absolute cover of at least one site (n = 59). All forest species were included in testing. For each plant species to be tested, I collected approximately 30 average aged leaves from as many individuals as possible and stored them in 1-gal plastic zip-top bags in a cooler. Plant samples were collected from late June to early July, during the flight period of M. rotundata when the bee would be actively collecting leaves. I also collected the petals of four species of forbs (Heliopsis helianthoides, Lotus corniculatus,

7 Ratibida pinnata, and Rudbeckia hirta var. pulcherrima) that M. rotundata has been observed using at the Wilds (pers. observation).

Within 2 hours of collection, I tested the toughness of each species’ leaves with a penetrometer fashioned from a 0.125-in (3.175-mm) diameter brass rod cut to a length of

5 cm. This rod was then inserted into a 3-cm cube of styrofoam such that the rod could be held perpendicular on the tray of a balance (Mettler Toledo PB403-S). I measured the toughness in g / 0.125 in of six samples per species by pressing the leaf through the rod by hand with slow and even force until the rod punctured the leaf, at which time I recorded the maximum force registered. This design is based on other simple penetrometers used in studies comparing leaf toughness (Eigenbrode et al., 1999; Horne,

1995). Each leaf was tested so that the edge of the penetrometer closest to the leaf margin was 3.175 mm from the margin, the most likely location of a leaf-cutter bee to cut to the leaf. I also observed and recorded the presence or absence of latex in the leaves of each species.

Within 8 hours of collection, I counted the trichomes on 6 leaves of each species using a dissection microscope with field of view area of 28.27 mm (Leica EZ4, 8x). I counted trichomes on the adaxial side of 3 leaves and abaxial side of the other 3 leaves. I restricted counts to stiff trichomes, defined as those that could not be easily bent with the tip of an entomology pin, for two reasons. First, I propose that the smaller, softer trichomes are less likely to influence a bee’s use of the leaf and second, they are more difficult to count accurately. Trichome densities were scaled to an area of 1 cm2.

8 Within 48 hours of collection, I scanned 10 leaves from each species to measure area. Image analysis was done using ImageJ (Schneider, Rasband, & Eliceiri, 2012). I defined a functional area measure from a bee’s perspective called the largest cuttable circle diameter (LCCD). The metric was defined as the diameter of the largest circle that can be placed on a leaf without crossing any major midribs or veins that add considerable thickness to a leaf (Figure 1). For smaller leaves with a low profile/slender midrib (e.g.

Lotus corniculatus, Securigera varia, Robinia pseudoacacia), the LCCD was placed on a leaf so that the diameter was 75% of the width of the leaf in order to mimic the cutting patterns of M. rotundata. In species with compound leaves, leaflets were treated as individual leaves, as that is how they would be handled by bees. Most grass blades could not be scanned due to their size; their LCCD was recorded as 75% of the width of each blade at its widest point.

Statistical analysis

I compiled the species of plants that M. rotundata use when nesting from relevant literature and observations in the field at the study site. Horne (1995) compared the use of numerous forage crops (some of which have established at our study site) and MacIvor

(2016) used DNA barcoding to identify the species being used by M. rotundata in

Toronto, Canada. I excluded studies that took place outside of eastern North America or that focused on plant species that did not occur at the study site (Eigenbrode et al., 1999;

Kamblı̇ et al., 2017; Nugent & Wagner, 1995; Osgood, 1964; Soh et al., 2019). It is worth noting that M. rotundata is not the only species of leaf-cutter bee in the genus Megachile present at the site (Cusser & Goodell, 2013; Varvaro, 2018). Given that I stocked the

9 artificial domiciles at each survey area with M. rotundata cocoons and that this species has been the most abundant in other trap-nesting studies at the site that used 0.25-0.5 cm diameter domiciles (Varvaro, 2018), I am confident in assuming that M. rotundata was the primary species foraging for nesting materials at our selected locations. Another common species at these sites, Megachile campanulae, uses resin rather than leaves for its nests (Varvaro, 2018). Observations of the artificial domiciles and active foraging bees confirm this assumption.

All analyses were conducted in RStudio using the “tidyverse” and “vegan” packages (Oksanen et al., 2019; RStudio Team, 2016; Wickham et al., 2019). To visualize how leaves used by M. rotundata may differ based on physical characteristics, I used a 2 axes non-metric multidimensional scaling ordination (NMDS, metaMDS,

“vegan” package). I fitted biplot vectors representing each trait on the plot to visualize the similarities between leaf species (envfit, “vegan” package). In order to test if the species that are and are not used by M. rotundata are distinct groups, I conducted both an analysis of similarities (ANOSIM, anosim, “vegan” package) and a permutational multivariate analysis of variance (perMANOVA, adonis, “vegan” package). Finally, to test the ability of attributes and their interactions to predict the use of a plant species by

M. rotundata, I fit a binomial generalized linear model (GLM, glm, “stats” package).

Results

I assessed the toughness, hard trichome density, LCCD, and the presence/absence of soft trichomes and latex for leaf samples of 59 plant species (Appendix A). On average, leaves that are used by M. rotundata were less tough (M = 87.411 g / 0.125 in,

10 SD = 36.059, n = 42) than those not used (M = 191.325 g / 0.125 in, SD = 236.077, n =

17) (Figure 2). Used leaves also had lower average trichome densities (M = 12.482 / cm2,

SD = 37.082, n = 42) than species that were not used (M = 27.171 / cm2, SD = 65.301, n

= 17) (Figure 2). The useable area of leaves was not inherently important in determining bee use, but the species with the smallest average LCCD in the study was Lotus corniculatus, with a mean largest cuttable circle diameter of 3.715 mm (Figure 2). Of the species used by M. rotundata (n = 17), 23.5% had soft trichomes present and none had latex as defense mechanism. Of the species avoided by M. rotundata (n = 42), 28.6% had soft trichomes present and 4.8% had latex within their leaves. None of the individual traits or their interactions were significant in explaining bee use when modeled with a

GLM, but some appear to be marginally significant (Table 1).

Species were ordinated by these 5 traits to visualize how the physical characteristics separate used and unused species and shed light on how multiple variables interact to determine leaf suitability (Figure 3). Considering all traits together, I found that the species of leaves used by M. rotundata were distinct from the rest of the available species when compared with a perMANOVA (F = 3.312, p = 0.021) (Table 2). However, the two groups are not significantly different when compared with an ANOSIM (R = -

0.084, p = 0.904). This disparity in significance is explained by key differences in the way that each test compares groups; ANOSIM compares the similarities between groups and perMANOVA compares the centroids and dispersal of each group. When a group is nested within another, like how used species are nested within species that are not used, these results are expected (Anderson & Walsh, 2013).

11 My observations of foraging bees revealed that M. rotundata uses leaves of two plant species not previously documented, Amorpha fruiticosa and Senna hebecarpa, both members of Fabaceae. In addition, M. rotundata also used petals from Heliopsis helianthoides, Lotus corniculatus, Ratibida pinnata, and Rudbeckia hirta var. pulcherrima. The use of petals was common at sites dominated by grasses and tough- leaved forbs. Petals were not used at sites with preferred leaves, even when present in high abundances.

Discussion

In this study, I asked whether specific physical traits drive leaf choice in leaf- cutter bees. By surveying and collecting available leaves at 11 reclaimed grasslands with populations of M. rotundata, I was able to test the physical characteristics of those used and not used by the species and document sources of nesting materials not yet described in the current literature. This approach is an improvement on previous attempts to characterize usable leaves because it compares the characteristics of leaves used by M. rotundata to all of the leaves available across multiple habitats. In doing so, I avoid a biased result that might identify key traits that overlap with the subset of leaf species not used.

It is apparent that the physical characteristics measured in this study do not completely predict whether a species of plant is a suitable source of leaves for M. rotundata, but the ordination and results from the ANOSIM and perMANOVA do suggest that species used are different from those not used and form a cohesive cluster in multivariate space. The inclusion of other traits that are predicted to be important in leaf

12 choice, like the presence and types of surface waxes, volatile compounds, and secondary defenses, would likely further distinguish the leaves used by M. rotundata (Eigenbrode et al., 1999). The latter two characteristics are particularly understudied in regard to

Megachile use.

Not surprisingly, toughness appears to be one of the primary drivers in M. rotundata leaf preference. This preference has been established in trials comparing a limited set of species, but never when considering an entire landscape of available leaves

(Eigenbrode et al., 1999; Horne, 1995; Kokko, Schaber, & Entz, 1993). Leaf-cutter bee mandible shape is adapted to cutting leaves with teeth and blade surfaces, but leaf cutting wears the cusps of the mandibles, with older individuals exhibiting considerable mandibular wear (Williams & Goodell, 2000). This wear is thought to be accelerated by tough leaves (Kokko et al., 1993). The effect of toughness on mandibular wear has clear implications for the rate of brood cell construction and fitness, and could limit bee success at a site with poor leaf availablity. The rate of cutting from leaves of individual plants can vary from bee to bee; observations of alfalfa found M. rotundata cutting time can be anywhere from 6 seconds to 2.5 minutes (Osgood, 1964). This variation might be a result of differential mandibular wear across individuals, demonstrating the dramatic cost of poor cutting ability. Leaf-cutter bee behavior appears to even moderate mandibular wear. For example, some individuals have been observed selectively choosing senescing leaves with decreased turgidity over tougher leaves of the same plant when given limited species to choose from (Osgood, 1964).

13 Other traits did not appear to influence leaf choice independently but were helpful in defining leaf species in ordination space. The available leaf area does not elicit an inherent preference, but bees were not recorded using any plants with an average LCCD less than 3.715 mm, suggesting a threshold size of leaves. Previous observations predict that bees will avoid leaves with an area of less than 1 cm2 if possible, but the preference was determined in enclosure trials with a wide range of suitable species including

Trifolium spp., multiple varieties of Medicago sativa, and other forage crops (Horne,

1995). The inclusion of Lotus corniculatus in this study lowered the estimate of LCCD as the plant’s leaves can be quite variable in size (pers. observation). When selecting leaves for testing, I aimed to pick average sized leaves. However, leaf-cutter bees would likely search for larger leaves of a species like Lotus corniculatus that exhibits such variability.

The role of trichomes is hard to disentangle from other defenses, as plants may be using a suite of chemical and physical traits in order to deter herbivory. Some plants appear to invest resources into single types of defenses (trichomes, toxic glands), but this is not always the case (Agrawal, 2007). Plants with no trichomes might be using other less apparent defenses to deter damage, and plants with trichomes might rely more heavily on their physical defenses than chemical ones. Generally, many species of leaves used did not have hard trichomes. I also measured the presence or absence of two other apparent traits, latex and soft trichomes. Only two plants had latex within their leaves to deter damage (Apocynum cannabinum, Asclepias syriaca), and it made for a simple and effective deterrence to M. rotundata and other defoliators. More species had soft trichomes, but I conclude that no species present at the site has enough soft trichomes to

14 outwardly prohibit use by M. rotundata. Unused species with soft trichomes present tended to have other traits that were likely prohibiting use. Together, all of these traits allow us to create a hypothetical leaf that is largely useable by M. rotundata: relatively tender with minimal physical defenses and a useable area to cut discs with a dimeter of at least approximately 4 mm.

I extended the list of usable leaf species documented in the literature for M. rotundata to include Senna hebecarpa and Amorpha fruiticosa (Appendix A). Given that their leaves were similar in trait space to many other members of Fabaceae that M. rotundata use and that they were prevalent around certain nests at the study site, it is not surprising that bees used these plants. I also documented the use of the flower petals of 4 different forbs: Heliopsis helianthoides, Lotus corniculatus, Ratibida pinnata, and

Rudbeckia hirta var. pulcherrima. In our ordinations, the petals of these species fall out alongside leaves that are used by M. rotundata. The use of these petals seemed to happen most often at sites with low abundances of the most commonly used leaf species; I hypothesize that petals are only used when more preferred nesting materials are not available, even though they are physically similar to leaves. This is supported by observations at certain sites with these petal species and suitable leaf materials cooccurring; petals were not observed being used by bees in these habitats. The fitness cost of using petals, whether in the form of increased handling time for nesting females or in decreased larval survivorship, is not known; additional research on petal use would improve our understanding of nesting material preferences in Megachile. Other

Megachilid species have been documented to use petals (Requier & Leonhardt, 2020),

15 and it would be interesting to know whether petal material is preferred over leaf material or whether it is used because suitable leaves are in low abundance. The use of flower petals suggests that M. rotundata preference is plastic and this may be one of the features of the species that makes it a highly successful species outside of its native range. Further supporting the plasticity of this species, M. rotundata has even been documented cutting discs from plastic bags in urban landscapes, demonstrating significant flexibility in the species when nesting (MacIvor & Moore, 2013).

Some of the herbaceous species know to be preferred sources of leaves with M. rotundata include Medicago sativa, Trifolium spp. and Lotus corniculatus (Horne, 1995).

The species also prefers woody sources, including members of Rosaceae and Fabaceae

(MacIvor, 2016). While it is hard to recommend specific plant species to promote leaf- cutter bee abundance and diversity in Eastern North America based on this study given

M. rotundata’s exotic status and the prevalence of exotic plants at the study site, herbaceous legumes in prairies/grasslands and tender-leaved woody species on prairie/grassland borders would appear to be useful to a range of leaf-cutter bees. It is also worth noting that plants are known to behave differently on the highly stressed soils of serpentine and reclaimed mine sites; the traits of plant species in this study likely would vary when growing in different, more suitable conditions (Kazakou,

Dimitrakopoulos, Baker, Reeves, & Troumbis, 2008). Nonetheless, preferences for woody species appear to be broad, and forest edges are likely a valuable habitat for the selection of leaf sources.

16

Chapter 2: Landscape scale effects of resource availability on leaf-cutter bee foraging

Abstract

Megachilid bees are unique in their need for non-floral resources used in nesting; they can be limited by nesting resource availability in addition to pollen and nectar availability. The leaf-cutter bees (genus Megachile) are no exception, requiring suitable leaves to build their brood cells. As solitary bees, they have to balance foraging for leaves, pollen, and nectar, along with building and protecting their nest. Specifically, limitations to bees have been documented in the grass-dominated reclaimed mine lands of the Eastern United States. These novel sites provide the opportunity to test how leaf choice may be limited on a landscape scale. Here, I monitored the duration of leaf foraging trips made by Megachile rotundata at 10 different grassland sites within a large reclaimed coal surface mine in Eastern Ohio to determine if bees nesting further from the forest experience lower availability of suitable leaves. The prediction that bees nesting far from the forest will spend more time searching for suitable leaves than those nesting close to the forest was supported. Bee’s foraging bouts were observed to take on two distinct durations: “long” and “short” trips. The distance from the forest was a strong predictor of the duration of “long” trips, while “short” trips varied little in duration and were on average similar for bees regardless of nesting location. I posit these “long” trips

17 represent periods where bees are required to search for new sources of leaves for nesting.

These results suggest leaf-cutter bee nesting productivity may be limited in large, reclaimed grasslands and that forest habitat offers a valuable resource for bees that enhances grassland habitats within a bee’s foraging range.

Introduction

Pressures like agricultural intensification, pesticide use, and the introduction of pathogens have resulted in bee declines (Biesmeijer, 2006; Cameron et al., 2011;

Goulson, Nicholls, Botías, & Rotheray, 2015; Potts et al., 2010; Vanbergen & The Insect

Pollinators Initiative, 2013). These threaten the pollination services offered both to crops and natural systems (Cusser & Goodell, 2013; Hoehn et al., 2008; Koh et al., 2016;

Kremen, Williams, & Thorp, 2002; Martins et al., 2015; Pitts-Singer & Cane, 2011). The effects of anthropogenic stresses on bee success are hard to generalize; different taxa can respond differently to the same stresses (Cameron & Sadd, 2020; Cariveau & Winfree,

2015; Roulston & Goodell, 2011). Solitary bees, which account for the vast majority of bees, are generally thought to be more vulnerable than other commonly studied social bees like the honey bee (Apis mellifera) and bumble bee (Bombus spp.) (Cane, 2001;

Klein, Cabirol, Devaud, Barron, & Lihoreau, 2017). This is in part because solitary bee females have to balance foraging for resources, building their nests, and defending their offspring by themselves – tasks which are made more complex by their short foraging range (Cane, 2001; Cresswell, Osborne, & Goulson, 2000; Klein et al., 2017; Zurbuchen et al., 2010).

18 In addition to being particularly sensitive to stress because they lack the ability to share tasks, solitary bees can also be limited by the floral and non-floral resources they require from the landscape. The is especially true of Megachilid bees and more specifically, the leaf-cutter bees in the genus Megachile that use perishable, seasonal materials like leaves when nest building (Cane, 2001; Pitts-Singer & Bosch, 2010). Like all bees, they require nectar and pollen to forage from, but also require suitable leaves within a short foraging range of their nest (Klein et al., 2017; Zurbuchen et al., 2010).

While conservation efforts for bees most often focus on the establishment of forbs for nectar and pollen, the availability of other nesting resources like adequate leaves in the landscape is often overlooked (Cane, 2001; Grundel et al., 2010; Morato & Martins,

2006; Potts et al., 2005; Requier & Leonhardt, 2020). Anthropogenic stresses like habitat disturbances and agriculture can reduce the quality of the landscape from which a bee is foraging by reducing the pool of available resources (Roulston & Goodell, 2011).

However, anthropogenic disturbance might not equally disadvantage all taxa. The reclamation and restoration of mined lands can take advantage of the unique ecological conditions created after mining to create novel grasslands in areas like the Appalachian

United States (Lanterman & Goodell, 2018). The process of surface mining and subsequent reclamation results in land with shallow and compacted soil that is poor in nutrients (Skousen & Zipper, 2014). It is often impossible to restore mined lands back to their historic conditions; they are reclaimed by planting vegetation that can withstand the poor soil conditions (Skousen & Zipper, 2014). Recent years have seen the rise of newer reclamation and restoration techniques focusing on establishing more native vegetation,

19 and even creating “pollinator prairies” with high abundance and diversity of forbs that aim to support robust bee populations (Bauman, Cochran, Chapman, & Gilland, 2015;

Cusser & Goodell, 2013; Skousen & Zipper, 2014; Swab, Lorenz, Byrd, & Dick, 2017).

In some ways, these novel grasslands provide prime habitat for leaf-cutter bees with large areas of open fields and relatively high densities of flowers (Cusser & Goodell,

2013; Swab et al., 2017). However, other required resources like suitable leaves may be lacking (Cane, 2001; Requier & Leonhardt, 2020). The leaf preferences of leaf-cutter bees in natural habitats are understudied. Some evidence suggests they prefer woody and herbaceous members of Fabaceae and some other woody plants (Horne, 1995; Kamblı̇ et al., 2017; MacIvor, 2016; Soh et al., 2019). On a landscape scale, leaf-cutter bees have been shown to have higher rates of reproduction when nesting closer to the forest in reclaimed grasslands (Varvaro, 2018). If leaves needed for nesting are scarce and hard to find, bees will experience a tradeoff, spending less time foraging for pollen and nectar, building brood cells, and defending their nests.

To study the tradeoffs of leaf-cutter bees nesting in reclaimed grassland sites that vary in local vegetation quality, and presumably the availability of suitable leaves for nest-building, I asked how nest placement relative to the forest edge affected bee foraging durations. I compared the duration of Megachile rotundata leaf foraging bouts at sites adjacent to the forest and at varying distances from the forest edge to estimate the investment made in nest building across landscapes. I predicted that bees nesting closer to the forest would make shorter leaf foraging trips than those nesting far from the forest

20 due to the greater apparentness and abundance of suitable leaves in forests compared to those in grasslands.

Methods

Study system

The alfalfa leaf-cutter bee (M. rotundata) is a solitary cavity nesting bee. Females will find a suitable nesting site, within which they will construct a series of brood cells.

Each brood cell is made up of about 15 cut pieces of leaf (Klostermeyer & Gerber, 1969).

A cell is then provisioned with a mixture of pollen and nectar, upon which the female lays an egg (Cane, Gardner, & Harrison, 2011). The species is native to Eurasia, but has been introduced all over the world where is it has escaped managed nesting sites on agricultural properties to establish feral populations (Pitts-Singer & Cane, 2011). The species is facultatively multivoltine, with some bees completing their development from an egg to an adult in a single growing season and others entering an early diapause stage and overwintering as immatures (Tepedino & Parker, 1986).

The Wilds is a nearly 37 km2 reclaimed mine in Eastern Ohio (39.829277, -

81.724098). The site was mined for coal from the 1940’s to 1980’s and different areas were reclaimed following different regulations as laws changes throughout the site’s operation. Reclamation before 1973 generally involved reforestation, while post-1973 reclamation focused on established grasslands. Additionally, certain reclaimed areas have also been restored by removing invasive exotic species planted in the original reclamations. Some of these areas have been seeded with different mixes of native grasses and forbs. However, some restored portions of the site still have high abundances

21 of exotic legumes like Lotus corniculatus, Lespedeza cuneata, and Melilotus officinalis, all of which were planted during the reclamation process. The study species M. rotundata relies heavily on these plants, contributing to its local success (Pitts-Singer & Cane,

2011; Varvaro, 2018)

Artificial domiciles

At 10 locations at the Wilds (Figure 4), I placed artificial domiciles for M. rotundata (Figure 5). In addition, I stocked each nest with approximately 500 bee cocoons from Mason Bees for Sale (masonbeesforsale.com). Each site received 2 bundles of 12 wooden bee nests, made from two pieces of grooved lumber held together with staples and sealed with construction caulk to prevent parasites from entering. Each nest had a cavity with a diameter of 0.25-0.5 cm and external dimensions of 3 by 2 by 10 cm.

These were attached to fenceposts facing eastward to allow the morning sun to the heat up the nests. Each bundle was covered with a piece of plastic tarp to keep the nests dry. I placed nests at the 10 sites ranging from 7 to 286 m away from the nearest forest. Female alfalfa leaf-cutter bees rarely fly further than 200 m, excluding their initial dispersal upon emergence (Pitts-Singer & Cane, 2011). In this study, a forest was defined as any cluster of woody vegetation taller than 1 m with a total area greater than 30 by 3 m.

Vegetation surveys

As the activity of a bee is likely to vary with changes in the abundance of food resources, I surveyed the available floral resources at each site during the flight period of

M. rotundata. I established three 25-m transects originating from each nest at randomly generated angles more than 30 degrees apart. I then placed 1-m2 quadrats at 4 randomly

22 generated locations along each transect such that no two quadrats were adjacent. Within each quadrat, I counted the total number of open floral units from each plant species. A floral unit was defined as any flower or cluster of flowers that a honey bee could traverse by walking, not flying. I completed three rounds of surveying during the flight period of

M. rotundata.

Using the same transect and quadrat design, I also surveyed the absolute percent cover of leaves within each quadrat. Absolute cover of a species was defined as the percentage of ground all individuals of that species would obscure when viewed from an aerial perspective, ignoring other species (Daubenmire, 1959). This surveying was done by a single person, once for each site at the beginning of the flight period for M. rotundata. A species was designated as suitable for M. rotundata nest construction if it 1) has been observed being used at the study area, 2) has been observed being used in the region by other studies (MacIvor, 2016), or 3) has been used by bees in other trials testing preference (Horne, 1995). See Appendix A for a list of species. The total absolute cover of suitable plants was scaled by dividing by the total absolute cover of all species surveyed, as the total cover varied considerably by site.

In addition to this pooled measure of available leaf materials, I also separately recorded the cover of Lotus corniculatus. This abundant plant is known to be M. rotundata’s main source of nectar and pollen at the study site (Cusser & Goodell, 2013).

This variable acts as an additional and more specific measure of nectar and pollen availability than the previously described floral unit metric.

23 Bee foraging

I monitored bee foraging by video recording the nests at each site for approximately 2.5 hours, spread out over 3 rounds of monitoring from 8 July 2019 to 30

August 2019. Recordings were made with Sony Handycams on tripods placed at least 2 m away from each nest. I collected a total of 25.6 hours of video across all 10 sites.

Video was only collected on days with no rain, minimal wind (M = 2.612 km/hr, SD =

2.165), high temperatures (M = 31.009 °C, SD = 1.959), and low percent cloud cover (M

= 26.667%, SD = 22.765). I coded each video in BORIS (Friard & Gamba, 2016). I assumed that each nesting female was M. rotundata, which is likely because I placed cocoons at each site, the bee is the most abundant species in the genus at the site, and the bee prefers the selected cavity size (Cusser & Goodell, 2013; Varvaro, 2018). A congener that also nests in this size block at the site, Megachile campanulae, uses resin, and therefore is distinguishable from the target species (Varvaro, 2018). I also assumed each cavity was only being used by an individual nesting female. I recorded the time each bee entered and exited her cavity and recorded whether she was carrying pollen or a leaf disc upon returning from a foraging trip. A bee carrying a leaf disc was easily distinguishable from a bee carrying pollen. As a second measure of certainty, bees carrying pollen enter the nest, then exit and re-enter backwards to deposit pollen from the scopa (Pitts-Singer

& Cane, 2011). Cleptoparasites (Coelioxys spp.) could easily be distinguished from M. rotundata due to their more erratic flight pattern and unique behavior of entering and exiting many cavities while searching for a host. After all videos were coded, entrances and departures from each individual were matched up to give a series of foraging trips for

24 each bee, including the total time spent on a trip and the purpose of the trip - either to collect leaves (n = 158) or pollen (n = 15). I only consider leaf foraging trips in this study.

Statistical analysis

All analyses were conducted in RStudio using the “tidyverse”, “mixtools”, and

“afex” packages (Benaglia, Chauveau, Hunter, & Young, 2009; RStudio Team, 2016;

Singmann, Bolker, Westfall, Aust, & Ben-Shachar, 2020; Wickham et al., 2019).

Regardless of bee or site, bee foraging trips fit a consistent pattern of a series of short foraging trips followed by one long foraging trip variable in length (Figure 6). This pattern has been casually documented previously, but attributed to bees foraging for nectar during leaf foraging trips (Klostermeyer & Gerber, 1969; Osgood, 1964). Using a mixture model (normalmixEM, “mixtools” package), I was able to separate the two distributions and assign each foraging trip to one of the two types from posterior probabilities. I will refer to these as “short” and “long” trips.

I modeled trip time as a function of distance from the forest, trip type, and the interaction between the two with individual as a random effect using a linear mixed effects model (LMEM, mixed, “afex” package). This interaction was strong enough to warrant modelling both the “long” and “short” trips separately. Each trip type was similarly modeled with distance from the forest as the explanatory variable, trip duration as the response variable, and individual as a random effect. Other unused models and a correlation matrix for the “long” trip dataset are provided in Appendix B. These models attempted to correlate measures of floral and leaf availability, but these metrics were poor

25 predictors of foraging duration. I used Satterthwaite approximations to calculate the p- values of variables.

Results

Each of the 10 sites had an average of 4.7 individuals (SD = 2.669) that made an average of 15.8 foraging trips (SD = 11.123). The foraging trip durations were bimodally distributed, falling into two normally distributed groups: “short” trips (n = 94, M = 1.892 minutes, SD = 1.119) and “long” trips (n = 64 , M = 14.360 minutes, SD = 7.355) (Figure

7). The distance from the forest was a strong predictor of the duration of “long” trips in this study system (Table 3, Figure 8). Distance from the forest was not significantly associated with the duration of “short” trips. Weather covariates did not help explain trip time, as I controlled for favorable weather for when selecting times to record nests and there was little variation in these environmental variables. Measures of floral abundance,

Lotus corniculatus abundance, and suitable leaf cover were poor predictors of leaf foraging trip duration (Appendix B).

Discussion

Novel, anthropogenically disturbed habitats can offer both challenges and opportunities to wildlife such as bees. Grassland-reclaimed coal surface mines replace forested areas with persistent open field habitats that may provide substantial foraging grounds for native bees in a matrix of otherwise forested land (Lanterman & Goodell,

2018) but when large, they may offer limited woody and tender-leaved herbaceous vegetation that some bees use for nesting materials. I aimed to determine if leaf-cutter bees are limited by access to suitable leaf material in reclaimed grassland habitats distant

26 from forest edges. I tested the hypothesis that leaf-cutter bees would spend more time foraging for leaves when nesting further from the forest edge. Overall, data on M. rotundata leaf foraging durations supported this hypothesis. Longer foraging durations further from the forest may result from the lower abundance or the lower apparency of suitable leaves in a complex matrix of vegetation. Bees nesting close to the forest spend less time searching for leaves and forage more efficiently, with the forest acting as a large and apparent source of preferred leaf material that requires less searching. In large grasslands, forest edge habitat could be a valuable resource for leaf-cutter bees. Bee abundance might be particularly low further into large grasslands, limiting the pollination services offered to vegetation (Cusser & Goodell, 2013).

This study is the first to explore the pattern of “long” and “short” trips that leaf- cutter bees make when foraging for leaves. Other studies have mentioned this pattern anecdotally, but only speculated that the explanation involved foraging for nectar while also collecting leaves (Klostermeyer & Gerber, 1969; Osgood, 1964). While these previous investigations of foraging patterns proficiently tracked individual bees for long periods of time to establish the order of trip types, my study goes further by compiling multiple individuals across a landscape gradient to assess how foraging patterns played out on a larger scale in response to relevant vegetation variables. A strength of this approach is that it aids in making predictions about how interactions between bee behavior and environmental variables may impact bee reproduction across landscapes.

Studies support the contention that solitary bees can remember locations of important resources (Amaya-Márquez et al., 2008; Nieberding, Van Dyck, & Chittka,

27 2018). I predict that these “short” trips represent foraging trips where a bee is recalling a specific plant or cluster of plants that have suitable leaves and directly foraging from them. This retention of the locations of suitable leaves would minimize search time and encourage efficient nest building. I pose two hypotheses for the occurrence of the “long” trips.

I posit “long” trips are a result of bees searching for new sources of leaves to build nests with. Bees nesting closer to the forest have low trip durations during their

“long” trips, a pattern I predict is due to the forest acting as a nearby and apparent source of many suitable leaves (Figure 8). Therefore, further from the forest, bees are increasingly required to search complex landscapes for suitable leaves. This is consistent with the decreased foraging durations for leaves observed in bees nesting closer to the forest in previous studies at the site (Varvaro, 2018). Investing time to search for high- quality leaves can save a bee time they would otherwise spend on processing less suitable leaves. Previous observations demonstrate that different species of leaves can vary dramatically in the time it takes a bee to cut and process (Osgood, 1964). Some leaves are documented taking more than 20 times longer to cut than more efficiently processed leaves (Osgood, 1964). This variation in foraging trip length has been documented in M. rotundata nesting in enclosures (Artz & Pitts-Singer, 2015; Klostermeyer & Gerber,

1969) and agricultural settings (Osgood, 1964). By spending time searching for more easily cut leaves, bees might be able to invest in other behaviors like nest guarding and foraging for pollen and nectar.

28 An alternative hypothesis suggested by previous studies that bees are simply foraging for nectar on these long trips seems less likely, at least in the context of this experiment (Klostermeyer & Gerber, 1969; Osgood, 1964). In our models including available floral units and Lotus corniculatus cover, floral availability was a poor predictor of “long” trip time. If long trips are a result of bees collecting nectar while foraging for leaves, I would predict these trips to be correlated with the availability of nectar sources.

It is also worth noting that the study site has few summer-flowering trees and shrubs in their forests; the only prevalent one during the flight period of M. rotundata is Ailanthus altissima. Megachilid bees are not known to forage on this tree for pollen or nectar

(Thompson, 2008). Therefore, it is unlikely that bees are flying to the forest for nectar when they are nesting far from edge habitat. Together, these observations suggest that

“long” trips relate more to leaf foraging than nectar foraging.

Regardless of the reasons for longer foraging duration when farther from the forest, this increased time investment has clear fitness implications. Bees that invest more time in foraging for leaves sacrifice time they could be collecting pollen and nectar or defending their nest from parasites and predators. Our results find that bees are less efficient foragers far from the forest in large grasslands, an expected result due to the lack of suitable resources in grass-dominated habitats. Based on estimates from the models created, a bee nesting 200 m from the forest would spend about 6.5 more minutes foraging during these “long” trips compared to a bee nesting along the forest edge.

Assuming bees make 3 “long” trips per brood cell and produce 57 brood cells over the course of their lives (Klostermeyer & Gerber, 1969; Pitts-Singer & Cane, 2011), a bee

29 nesting 200 m from the forest is predicted to spend an extra 18.5 hours over the course of her 7-8 week adult life foraging on these “long” trips. It is worth mentioning that the goals of the models in this study are not to predict foraging time, but to correlate landscape context to foraging trips. Foraging duration variance increased dramatically as bees nested further from the forest. Foraging duration is not a direct measure of fitness, unlike metrics such as rate of brood cell production and brood survivorship used in other studies (Pitts-Singer & Bosch, 2010; Varvaro, 2018).

This decline in leaf-cutter bee foraging efficiency has implications for the management of reclaimed and restored grasslands. The results of this study support the need to ensure adequate non-floral resources, such as suitable leaves, that are essential for bee reproduction to achieve the goal of increasing bee abundance and diversity with

“pollinator prairies”. Even if supporting robust bee communities is not a main goal of a reclamation or restoration, the loss of the unique pollination services offered by leaf- cutter bees and other Megachilid bees can affect the success of a project. Megachile is a very efficient pollinator of many plants in Fabaceae, a plant family valued in mine reclamations for its species’ ability to fix nitrogen and restore soil nutrients (Cusser &

Goodell, 2013; Pitts-Singer & Bosch, 2010; Pitts-Singer & Cane, 2011; Skousen &

Zipper, 2014). Megachile and other Megachilids are some of the only pollinators that reliably “trips” the flowers of these plants in order to expose the reproductive structures and successfully pollinate individuals (Pitts-Singer & Bosch, 2010). On a broader scale, the issue of providing adequate nesting resources for bees to ensure robust pollinator communities extends past planting species that are used as a leaf source for leaf-cutter

30 bees. Many bees - especially Megachilid bees - require specific habitats to be successful in a landscape, and if those requirements are not met, we will continue to see bees decline to the detriment of natural and agricultural systems (Cane et al., 2011; Requier &

Leonhardt, 2020; Roulston & Goodell, 2011).

31 Tables

Table 1: Results from a GLM correlating each leaf trait to the likelihood of use by Megachile rotundata

Coefficient Estimate SE p Intercept 2.452 1.462 0.094 LCCD -0.645 0.104 0.113 Toughness -0.024 0.014 0.079 Hard Trichome Density -0.062 0.081 0.441 LCCD × Toughness 0.001 0.001 0.244 LCCD × Trichome 0.003 0.006 0.592 Toughness × Trichome 0.001 0.001 0.373 LCCD × Toughness × Trichome >0.001 >0.001 0.487

Table 2: Results from a perMANOVA comparing the use of leaf species used by Megachile rotundata and those not used

Source df SS F p Used by Megachile? 1 0.417 3.312 0.021 Residuals 57 7.179 Total 58 7.596

32 Table 3: Results from multiple LMEM’s correlating time spent forging with the distance from the forest, with individual as a random effect. The results from three models are shown; the first demonstrating the interaction between trip type and the effect of distance from the forest and the remaining two showing how each individual trip type duration responds to distance from the forest

Trip Type Fixed Effects Estimate SE df p “Short” and “Long” Intercept 6.624 2.031 31.35 0.003 Distance from Forest 0.023 0.011 32.64 0.030 Trip Type 2.875 0.690 131.0 <0.001 Distance × Type 0.007 0.004 131.7 0.058 “Short” Intercept 1.913 0.392 16.38 <0.001 Distance from Forest 0.001 0.002 19.62 0.600 “Long” Intercept 10.073 2.510 26.32 <0.001 Distance from Forest 0.032 0.013 28.08 0.018

33 Figures

Figure 1: Examples of LCCD calculations for a variety of leaves (a: Liquidambar styraciflua, b: Amorpha fruticosa, c: Helianthus giganteus, d: Panicum virgatum, e: Cirsium arvense, f: Echinacea purpurea, g: Rudbeckia hirta var. pulcherrima petal, h: Clinopodium vulgare, i: Solanum carolinense, j: Rosa multiflora)

34

Figure 2: Plots showing the distribution of traits from leaf species used and not used by Megachile rotundata. Leaf area is depicted with a dot plot to illustrate the threshold area of useable species, while toughness and trichome density are represented with smoothed density plots. Means are depicted as vertical lines

35

Figure 3: NMDS plot of leaf species ordinated by their physical characteristics. Biplot vectors represent each physical attribute’s association with the ordination space. The plant type “shrub” refers to all encroaching woody vegetation within each grassland while “tree” refers to all woody vegetation within each adjacent forest

36

Figure 4: Locations of artificial domiciles at the Wilds

37

Figure 5: Images of an artificial domicile. Two bundles of nests can be seen on top, with the stocked Megachile rotundata cocoons below

Figure 6: A sample time budget from a bee included in the study demonstrating the pattern of "long" and "short" trips

38

Figure 7: Leaf foraging trip distributions as fitted from a mixture model. The mixture model detects overlapping distributions and assigns points to each from posterior probabilities

39

Figure 8: Association between distance from the forest and leaf foraging trip duration for both “long” and “short” trips designated by a mixture model. The fit lines and greyed bands represent a simple linear regression and 95% confidence interval; refer to models for more detailed associations. Points are horizontally jittered to prevent overplotting

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50 Appendix A: Plant species used and not used by Megachile rotundata at the study site

MacIvor Horne Family Species The Wilds 2016 1996 Apiaceae Daucus carota Apocynaceae Apocynum cannabinum Asclepias syriaca Asteraceae Achillea millefolium Cirsium arvense Echinacea purpurea Erigeron annuus Helianthus giganteus Helianthus mollis Heliopsis helianthoides Heliopsis helianthoides (petal) X Leucanthemum vulgare Ratibida pinnata Ratibida pinnata (petal) X Rudbeckia hirta var. pulcherrima Rudbeckia hirta var. pulcherrima (petal) X Rudbeckia triloba X Solidago canadensis X Solidago juncea Solidago rigida Caprifoliaceae Lonicera tatarica var. morrowii X Cyperaceae Carex lurida Dipsacaceae Dipsacus fullonum Elaeagnaceae Elaeagnus umbellata Fabaceae Amorpha fruticosa X Lespedeza cuneata Lotus corniculatus X X Lotus corniculatus (petal) X X Medicago lupulina Melilotus officinalis X Robinia pseudoacacia X Securigera varia X Senna hebecarpa X Trifolium pratense X 51 Fagaceae Fagus grandifolia Hamamelidaceae Liquidambar styraciflua (young) Lamiaceae Clinopodium vulgare Monarda fistulosa Prunella vulgaris Pycnanthemum tenuifolium Oleaceae Fraxinus americana X X Fraxinus americana (young) X X Platanaceae Platanus occidentalis Platanus occidentalis (young) Poaceae Andropogon gerardi Bromus inermis Dichanthelium acuminatum Panicum virgatum Tridens flavus Tripsacum dactyloides Rosaceae Rosa multiflora X X Rubus allegheniensis Rubus occidentalis Salicaceae Populus deltoides Populus deltoides (young) Salix interior Simaroubaceae Ailanthus altissima Solanaceae Solanum carolinense Typhaceae Typha angustifolia

52 Appendix B: Unused linear mixed effects models modeling “long” trip foraging duration and a correlation matrix for the “long” trip dataset

Fixed Effects Estimate SE df p Intercept 9.396 2.679 25.69 0.002 Distance from Forest 0.030 0.013 27.26 0.032 Total Floral Units 0.104 0.137 29.58 0.454 Intercept 0.442 7.241 25.52 0.951 Distance from Forest 0.072 0.034 26.46 0.045 Total Floral Units 1.472 1.038 24.00 0.169 Forest × Floral -0.006 0.005 24.32 0.196 Intercept 9.396 3.499 25.64 0.011 Distance from Forest 0.030 0.013 27.47 0.020 Percent Suitable Leaf Cover 2.618 11.275 23.98 0.818 Intercept 9.139 4.493 24.39 0.053 Distance from Forest 0.036 0.025 25.69 0.172 Percent Suitable Leaf Cover 5.966 27.120 24.21 0.828 Forest × Leaf -0.026 0.193 26.49 0.893 Intercept 8.147 3.162 31.08 0.015 Distance from Forest 0.035 0.013 32.58 0.014 Percent Lotus corniculatus Cover 0.011 0.188 30.70 0.953 Intercept 8.623 4.680 29.83 0.075 Distance from Forest 0.032 0.024 30.58 0.203 Percent Lotus corniculatus Cover -0.055 0.508 29.83 0.914 Forest × L. corniculatus <0.001 0.002 30.90 0.888

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