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Home , Cucurbitaceae, Peponapis pruinosa, Squash bee

An assessment of pollinator abundances and environmental relationships at cultivar, field, and landscape scales in cultivated pumpkins on the Texas High Plains

Christopher T. Jewett, B.S.

A Thesis

Plant and Soil Science

Submitted to the Graduate Facility of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Approved

Scott Longing, Ph.D. Chair of Committee

Robert Cox, Ph.D.

Charles West, Ph.D.

Mark Sheridan, Ph.D. Dean of Graduate School

December, 2017

Texas Tech University, Christopher Jewett, December 2017

Acknowledgments

I want to first start by thanking my academic advisor Dr. Scott Longing; none of this would have been possible without his guidance and support. I also want to thank my committee members Dr. Robert Cox and Dr. Chuck West for providing guidance and reviewing my writing and work. Next, I want to thank the faculty and staff of the

Department of Plant and Soil Science and Texas Tech University for the knowledge and academic accommodations. My thesis work was funded in part through a USDA

Conservation Innovation Grant involving the establishment of pollinator habitat on farms.

Next, I owe a special thanks to the people who helped me with research along the way. Samuel Discua, Bianca Rendon, Stephanie Freeman, and Daria McKelvey all provided field assistance in collecting data in pumpkin fields. I give a special thanks to

Samuel Discua for assisting with data analysis and providing technical advice.

I want to thank our pumpkin farmers for allowing us to conduct research in their fields: Jason Pyle, Mathew Rainwater, and Tim Assister. These farmers were generous with their time and in assisting us in locating pumpkin fields and in compiling cultivar data during the 2016 and 2017 growing seasons.

Lastly, I would like to thank my family and friends for their love and support. My wife, Ashley Jewett, provided love and support through the ups and downs of graduate school. Without her and our dog Norah, none of this would have been possible. Also, I would like to give a special thanks to my parents. Without their love and guidance over the last 26 years I would not be where I am today, and because of that I consider this accomplishment as one of yours.

Texas Tech University, Christopher Jewett, December 2017

Table of Contents

Acknowledgments ...... ii

Abstract ...... iv

List of Tables ...... vii

List of Figures ...... viii

I. Pollinator Value and Pollination Services in Agriculture

Economic Value of Pollination ...... 1

Managed Pollinators in Agriculture ...... 1

Pollinator Habitat Conservation in Agriculture ...... 4

Pollinator Decline ...... 6

Pumpkin Production in the U.S...... 9

Pumpkin Production and Pollination ...... 9

Literature Cited ...... 13

II. An assessment of pollinator abundances and environmental relationships at cultivar, field, and landscape scales in pumpkins (Texas, USA)

Abstract ...... 24

Introduction ...... 25

Materials and Methods ...... 27

Study Area ...... 27

Field Methods ...... 28

Habitat and Landscape Variables ...... 30

Data Analysis ...... 31

Results ...... 32

Discussion ...... 34

Literature Cited ...... 38

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Texas Tech University, Christopher Jewett, December 2017

Abstract

In Texas, pumpkins (Cucurbita pepo) are alternative crops with 5,000 to 8,000

acres grown annually. Common practices for pumpkin production include hive rentals

for supplemental pollination services, usually from managed honey bees (Apis mellifera)

and bumble bees (Bombus impatiens). For production of cucurbits, native squash bees can supplement managed bees or be the sole facilitator of pollination. During production, pumpkin flowers are a pulse resources of approximately four-five weeks in duration, providing nectar, pollen, and oils to squash bees and other insects. Native squash bees in the genera Peponapis and Xenoglossa have co-evolved with the large tubular flowers of cucurbits, while honey bees have been associated with cucurbits in North America only since the 17th century.

Although western Texas produces most of the pumpkins in the state, and require

sufficient pollination by insects to sustain yields, little is known of the status of native

squash bee populations in the region. Pumpkin producers could benefit from knowledge

of native, wild resources that could be used for crop production. Moreover, improving

our understanding of the dispersion of squash bees across agricultural landscapes could

guide strategies for the conservation of ground nesting resources for native bees.

Understanding how individuals survive during most of the non-cropped year,

underground, remains a key question, and is supported by this first study of native squash

bees in regional farming systems.

In this study, twenty-three pumpkin fields on the Southern High Plains of Texas

were visited and pollinator communities were sampled to estimate densities and describe

relative abundances and variation across different bee taxa among growing-season weeks,

Texas Tech University, Christopher Jewett, December 2017

fields and cultivars. In addition to comparisons of bee densities, a binary logistic

regression was used to determine relationships of flower occupancy by squash and honey

bees to field, cultivar and land cover factors. In 2016, we collected 1952 bees from 12

fields, with 73.4 percent of the total number of bees collected native squash bees, 25.8

percent honey bees, and 0.1 percent bumble bees. Squash bees were more abundant than

managed honey bees in all fields including those with managed honey bee and/or bumble

bees at the field edge. Year 2 (2017) mirrored the 2016 numbers with 82 percent squash

bees and 15.2 percent honey bees (out of 3511 total number of individuals). Forty

percent (2016) and 60 percent (2017) of the total number of bees were recorded in the

first week of both years. Declining numbers of bees across weeks could indicate natural

seasonal mortality, environmental stressors causing mortality, or sampling bias associated

with enclosed pumpkin canopies and edge effects (i.e. higher squash bee densities at

margins as pumpkin canopies enlarge over the 4-5-week flowering period). In the 2017

analysis, honey bees showed preference for the Turban pumpkin cultivar, and this was

the only cultivar where honey bees were more abundant than squash bees across both

seasons. The results of binomial logistic regression showed that mean bee-occupancy of flowers varied by week and by field in 2016, and in 2017 varied significantly by these and additionally by cultivar, mainly attributed to the Turban-honey bee relationship.

Percent wildland within 250 meters of pumpkin fields, the presence of field-level managed hives, pumpkin field area, nor cultivar richness were significant predictors of flower-occupancy across fields by squash bees or honey bees, emphasizing a need to further investigate the frequency of soil nesting behaviors within pumpkin fields and among field rotations.

Texas Tech University, Christopher Jewett, December 2017

This study provides baseline information to develop hypotheses and to support further studies on native squash bees and their contributions to pumpkin production on the Texas High Plains. Overall, across annual crop rotations benefits from squash bee foraging could be offset by environmental stressors resulting from production practices that affect soil nesting or pollinator health, yet the effects of field management practices on native squash bee populations are unknown. Further information on foraging and nesting resource requirements and densities or number of bees required for pollination would support a better understanding of the biology and value of native bees in pumpkins, to ultimately guide conservation actions for native squash bees on farms.

Texas Tech University, Christopher Jewett, December 2017

List of Tables

2.1. Sample dates for 2016 and 2017 pumpkins. ………………………..…………...40

2.2. List of pumpkin cultivars that were replicated in 2017. ………………..……….40

2.3. Total bee counts in 2016 and 2017 pumpkins. ………………..………………...40

2.4. Summary of Least Square Means Logistic Regression Estimates for 2016, Using Forward Model Selection, only significant variables in the model are presented. …..………………………………………………………..………41

2.5. Summary of Least Square Means Logistic Regression Estimates for 2017, Using Forward Model Selection, Only significant variables in the model are presented. ………………………………………………………..…………..42

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Texas Tech University, Christopher Jewett, December 2017

List of Figures

1.1 Pumpkin field during the third week of sampling in 2017. The photo shows the heavy canopy of the pumpkins. ...…………………………...….……18

1.2. Staminate (male) pumpkin flower. ……………………………….……………..19

1.3. Squash bees foraging on a pistillate (female) flower. Photo was taken in the field and in the early season when flower density was low. ..………….…....20

1.4. Female squash bee emerging from nest in soil. Further observation revealed the individual was deceased. ……….…………………….…...……….21

1.5. Male squash bee (Peponapis pruinosa) resting in a pumpkin flower. ……..…....22

1.6. Chris Jewett, walking pumpkin transects on the third sample round of 2017. Note the biomass of the pumpkin canopy during this sampling event in week 3. ………………...... ………………………………………………...….23

2.1. Pumpkin fields surveyed on the Southern High Plains of Texas, during 2016 and 2017. ……………………………………………………………...…...43

2.2. Example of how percent wild land area with 250m buffer was determined in ArcMap 10.4.1 with USDA – CropScape land use data. …….………...……..44

2.3. Average number of squash bees, honey bees, and bumble bees across 2016 farms. Each sample represents total bee numbers in each class divided by 4, the number of transects in each field. Error bars represent standard deviations. ……………………………………………………………..45

2.4. Average number of squash bees, honey bees, and bumble bees across 2016 farms. Each sample represents total bee numbers in each class divided by (n), the number of transects in each field. Error bars represent standard deviations. …...... ……..46

2.5. Average number of squash bees across 2016 sample season. Each week represents the average number of squash bees for each sample period. Averages were figured by the total number of squash bees counted each week divided by 12, the number of transects walked each sample period. Error bars represent standard deviations...... …………………………………..47

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Texas Tech University, Christopher Jewett, December 2017

2.6. Average number of squash bees across 2017 sample season. Each week represents the average number of squash bees for each sample period. Averages were figured by the total number of squash bees counted each week divided by (n), the number of transects walked each sample period. Error bars represent standard deviations. ………………………...... …...…...…..48

2.7. Average number of squash bees, honey bees, and bumble bees across 14 pumpkin cultivars. Each sample represents total bee numbers in each class divided by (n), the number of transects for each cultivar. Error bars represent standard deviations. …..………………………………...……………..49

Texas Tech University, Christopher Jewett, December 2017

Chapter I

Pollinator Value and Pollination Services in Agriculture

Economic Value of Pollination

Insect pollination is an important ecosystem service that contributes to the production of approximately 75 percent of the crops humans consume (Potts et al. 2010).

In addition to services to agriculture, insect pollinators support reproduction of most of the 295,383 flowering plant species known (Christenhusz and Byng, 2016). Worldwide,

35 percent of agriculture production relies on pollinators (Klein et al. 2007). Gallai et al.

(2009) estimated that the total economic value of pollination services worldwide in 2005 was approximately $190 billion. In Europe, 84 percent of cultivated crops rely on insect pollination (Gallai et al. 2009). In the United States it is estimated that 90-130 crops benefit, directly or indirectly, from pollination services, and the value of pollination services related to managed bees (i.e. migratory beekeeping) totaled $655.6 million in

2012 (Bond et al. 2014).

Managed Pollinators in Agriculture

To meet pollination requirements in crops, farmers have primarily relied on migratory beekeepers and managed honey bees. The western honey bee (A. mellifera), arriving in North America sometime in the 17th century (vanEngelsdorp et al. 2010), has been used in agriculture production from that time, with historical beekeeping dating back to ancient Egypt before 2600 BCE. In the U.S., beekeeping has grown into a valuable practice benefiting beekeepers through the production of hive products (e.g. honey, wax, pollen, bees) and farmers by providing crop pollination services. Honey

Texas Tech University, Christopher Jewett, December 2017 bees have been an effective pollinator for many U.S. crops, with some crops such as almonds (Prunus dulcis) 100% reliant on honey bees for pollination.

Honey bees are social insects and therefore build up abundant and localized and manageable colonies that are conducive to transport. In the U.S., migratory beekeeping begins annually with the movement of hives from over 1,500 beekeepers across the southern U.S. to California in February for almond production. Pollination in almonds is followed by trips to crops in upper northwestern and midwestern states in the U.S. before returning south to overwinter hives. In 2010, the average cost for a bee colony rental in the U.S. Pacific Northwest was $70 (averaged across 10 different crops), with pumpkin producers in the PNW paying an average of $48 per hive (Burgett 2011). Costs per hive were the highest on almonds at $137 and the least on berries at $32. In 2014, contracts for almond pollination ranged from $170-$200 per 8-10 frame colony (Traynor 2014).

Other bee species that are managed for crop pollination services include bumble bees (Bombus spp.), leafcutter bees (Megachile spp.) and mason bees (Osmia spp.).

Bumble bees are commonly used in crops such as greenhouse tomato (Solanum lycopersicum) production because individuals readily pollinate flowers and hives can be easily maintained in controlled and protected environments. In a field study in cucumber

(Cucumis sativus) and watermelon (Citrullus lanatus), B. impatiens was shown to be more efficient at pollinating Cucurbitaceae species than honey bees because of diurnal foraging patterns; B.impatiens was observed to start working early in the day when

Cucurbit flowers opened, and foraged longer during the duration of diel “flowering,” or open-bloomed (Stanghellini 2002). Megachile rotunda, the alfalfa leafcutter bee, was introduced to North America by the 1940’s for alfalfa seed production

Texas Tech University, Christopher Jewett, December 2017

(Pitts-Singer and Cane 2011) and continues to be the primary pollinator in this crop.

Mason bees are commonly used in orchards for the production of apples (Malus pumila) and other orchard crops, where they were shown to be better pollinators because of their ability to forage earlier in the day than other bees (Bosch and Kemp 2002). The managed bee O. lignaria was shown to increase cherry (Prunus avium) yields in Utah orchards

(Bosch et al. 2006). This bee has a high preference for fruit tree pollen and nectar, and is relatively easy to manage through the commercial production of pupae. Mason bees have relatively small foraging ranges, so maintaining colonies in orchards and supplying bees to the orchard before the onset of flowering is a common practice which can have impacts on fruit yield (Bosch et al. 2006).

Using managed bees for crop pollination presents challenges to farmers because hives are costly to rent. Managed bees are sometimes difficult to acquire and they are vulnerable to numerous diseases and parasites because hives are kept in close quarters

(vanEngelsdorp et al. 2010). Some diseases from managed bees can be spread to native pollinators (Fürst et al. 2014), thus potentially impacting wild bee populations.

Native Pollinators in Agriculture

Native bees have been shown to benefit yields of crops such as alfalfa (Medicago sativa) (for seed), almonds, apples, blueberries (Vaccinium spp.), cherries, coffee (Coffea spp.), pumpkins (Cucurbita pepo), strawberries (Fragaria × ananassa) , and watermelon

(Garibaldi et al. 2013, Tepedino 1981, and Watson et al. 2011). Garibaldi et al. (2013) found than wild insects are more efficient at pollination, for fruit set, than honey bees in

41 crop systems.

Texas Tech University, Christopher Jewett, December 2017

A study in northern California showed that tomato size and yield significantly increased when cross-pollinated by wild bees (Greenleaf and Kremen 2006). The study showed that 34 percent of bees observed were B. vosnesenskii and that the visitation was significant with natural habitat distance. In Central Sulawesi, the pollination efficiency of solitary bees was significantly higher than social bees in coffee (Klein et al. 2003). In the study, solitary bees accomplished fruit set on 87 percent of flowers visited, while social bees only accomplished fruit set on 75 percent of flowers visited.

Pollinator Habitat Conservation in Agriculture

Native pollinators occur naturally across agroecosystems and wild lands, where varying proportions of land cover types involving crops and grasslands dominate the landscape. Clusters of these counties were in areas of the conterminous U.S. involved in intensive agricultural production: Upper Midwest, Mississippi delta, California central valley, and southern High Plains (Koh et al. 2015). While many of the effects of land cover change and effects of large scale agriculture in the study region on native pollinator biodiversity are unknown, the native biodiversity as well as the pollination services provide by wild pollinators are continuously exposed to environmental stressors from agricultural production.

Numerous studies have provided evidence that maintaining portions of wild land on farms benefits wild pollinators. Maintaining only 30 percent wild land within a 1-2.5 km buffer of farms was shown to support diverse and abundant pollinator communities that provide sufficient pollination without the need for managed bees (Kremen et al.

2004). Wild habitat benefits pollinators by providing relatively undisturbed areas for nesting and acquiring floral resources, which can in turn benefit crop production on farms

Texas Tech University, Christopher Jewett, December 2017

(Morandin and Winston 2006). Ideally, crop margins should be planted in perennial native vegetation that blooms through the regions full growing season. Blaauw and

Isaacs (2014) planted 15 native perennial wildflower species that provided season-long bloom adjacent to blueberry fields in southwest Michigan. After three years of establishment, there was a significant increase in native bees and hoverflies, with native bee populations doubling. In cotton fields in southern Texas, greater habitat heterogeneity around cotton fields that included a portion of wild land was correlated with visits by wild bees to flowers. In these fields, outcross pollination resulted in larger seed cotton weight and with pollen limitation decreasing with increases in bee richness and abundance (Cusser et al. 2016). Morandin et al. (2016) demonstrated that hedgerow restoration to enhance pollinators benefited tomato production by increasing pest control and pollination services. The study not only suggested that pollinators benefit from hedgerows, but hedgerows also provide habitat resources for predatory insects that help control crop pests. Studies show that enhancing habitat for pollinators could also benefit broader insect community biodiversity including beneficial insect predators and parasites of agricultural pests (Potts et al. 2010).

Enhancement of habitat for pollinators can benefit from wild land conservation, but also from supplying natural or artificial nesting sites for pollinators. For example, in early-flowering crops such as fruit orchards, artificial nesting boxes are used for either wild or commercially-raised bees such as the mason bee. Stubbs et al. (1997) provided evidence that blueberry fields showed potential nesting-site limitations by placing artificial nesting boxes near fields, which were readily colonized by wild mason bees.

They found that nest blocks increased mason bee densities in two of the three

Texas Tech University, Christopher Jewett, December 2017 experimental fields, while they observed no increase in three control fields. The nest blocks not only provided nest habitat for mason bees, but also for leafcutter bees.

Pollinator Decline

The issue of pollinator decline and the environmental threats attributed to pollinator decline has been reported extensively in recent years. Losses of pollinators have the potential to disrupt plant-pollinator networks in addition to disrupting the pollination of crops globally. Burkle et al. (2013) showed that over 120 years a plant- pollinator community lost a significant amount of biodiversity because of specialist relationships. Impacts to pollinator biodiversity have occurred and are likely to continue to occur with increased population human growth and land-use intensification, which has the potential to directly and indirectly diminish the food industry and human quality of life (Aizen and Harder 2009). Kopec and Burd (2017) recently reported on the state of pollinators and found that in North America and Hawaii, 52 percent of native bee species are threatened with population declines. If pollinator declines continue, ecosystem services for agriculture and natural biodiversity could be at risk (Aizen and Harder 2009,

Burkle et al. 2013).

In recent years, declines in managed honey bees sparked the phenomenon known as Colony Collapse Disorder (CCD). Ellis et al. (2010) listed all hypothesized factors contributing to CCD; 1) common bee pests and pathogens, 2) bee management, 3) queen genetics and source, 4) chemicals used in management, 5) toxins in the environment, 6)

Varroa destructor, 7) poor bee fitness, 8) unknown pests or pathogens, and 9) a combination of these. In 2006-2007 beekeepers reported losing 50-90% of overwintering hives in the U.S. due to CCD (Cox-Foster et al. 2007). Declines have been persistent

Texas Tech University, Christopher Jewett, December 2017 since the middle 20th century; from the 1940’s to 2008 hive numbers decreased in the

U.S. by 3.7 million (Pettis and Delaplane 2010).

Multiple interacting factors have been implicated directly or indirectly in pollinator decline, attributed to several co-occurring environmental stressors related to habitat loss, compromised nutritional resources, pests and disease, and management practices in migratory beekeeping (Klein et al. 2007, Koh et al. 2016, Kremen et al.

2002).

Pollinator decline has also received considerable attention regarding the migratory monarch butterfly (Danaus plexippus). In part because of continuing declines in the area occupied by overwintering adults in central Mexico and other imminent threats to these butterflies migrating over 4000 km, the monarch was recently petitioned for listing by the

USFWS as a threatened or endangered species, with a ruling scheduled for 2019.

With a focus regarding pollinator decline related to honey bees and specifically

CCD, it is important to consider that sustaining pollination services for agriculture is not necessarily the same as sustaining pollinator biodiversity. In many cases, agriculture

(e.g. almond production) must rely on managed honey bees for pollination, so there could be little input in these types of systems from a diverse community of native bees.

Moreover, it has been shown that relatively few common and locally abundant pollinators visit crops (Kremen et al. 2002). Understanding the relationships of seasonal pulse floral resources of crops and wild plants is an important step for sustaining pollination via pollination services and pollinator biodiversity. Conservation practices adopted on farms should consider the resource requirements for their common pollinators or flower visitors, which might only be a small subset of the total community (Kremen et al. 2002).

Texas Tech University, Christopher Jewett, December 2017

In all cases, pollinator biodiversity and pollination service conservation should consider

the spatial and temporal floral pulses of the crop + wild land habitat when developing

pollinator conservation actions.

Some agrichemicals used in crop production have been shown to have indirect and direct effects on foraging behaviors of pollinators (Dively and Kamel 2012).

Cresswell (2011) reviewed 13 peer-reviewed honey bee articles on the effects of imidacloprid, an insecticide in the neonicotinoid class. Across the articles, data from

7073 individual honey bees and 36 colonies were tested. Based on field realistic doses,

mortality of honey bees was not at risk. The studies determined honey bee LD =4.5 ng

and an LC =1760 μg L−1, which are volumes much higher than realistic field exposure.₅₀

While there₅₀ was no risk for mortality in field exposure, there was a performance decline in 6-20 percent of honey bees at field doses (Cresswell 2011). Scott-Dupree et al. (2009)

tested lethal concentrations (LC ) of clothianidin, imidacloprid, deltamethrin, spinosad,

and novaluron on other managed₅₀ bees; B. impatiens, M. rotunda, and, O. lignaria.

Results showed that while all bees had lethal effects from these insecticides, LC

volumes correlated with bee size. Megachile rotunda required less volume than₅₀

B. impatiens to result in mortality. Other agrochemicals such as fungicides have been

shown to alter bumble bee workers, hive bee biomass, and the production of smaller

queens when exposed to field doses of fungicides (Bernauer et al. 2015).

Declines in population numbers and range extents has been documented for

bumble bees in the U.S., with B. affinis, B. occidentalis, B. pensylvanicus, and

B. terricola showing declines (Cameron et al. 2011). Changes in pollinator biodiversity

(loss of native species) has been documented for regions of Britain and the Netherlands

Texas Tech University, Christopher Jewett, December 2017

(Biesmeijer et al. 2006). Native bumble bees have shown declines in recent years in countries such as North America and Western Europe (Goulson et al. 2008). In the U.S., the most recent arthropod listing on the USFWS Threatened & Endangered was

B. affinis, the rusty patched bumble bee, currently occupying only a small portion of its historical range.

Pumpkin Production in the U.S.

Pumpkins in the United States are primarily grown for human food and as an ornamental used during the Halloween holiday in the United States. In 2014, over 90,000 acres of pumpkins were planted in the United States, producing approximately 1.5 billion pounds (AgMRC 2015). In 2014, the top producing state for pumpkins in the U.S. was

Illinois, with over 50,000 acres of pumpkins grown. Other top producers of pumpkins in that year included California, Ohio, Pennsylvania, Michigan, and New York, with these states each producing over 6,500 acres (USDA 2015).

The focal area for my thesis research was on the Southern High Plains of Texas.

West Texas growers plant approximately 90 percent of the states pumpkin acres

(Figure 1.1). In total, Texas producers plant up to 9,000 acres, of which 5,000-8,000 is grown in West Texas (Pumpkins in Texas, 2000). Farmers in Floydada, Texas, grow approximately 1,500 acres of pumpkin cultivars each year, producing 20,000 to 50,000 pounds per acre (Floydada Chamber of Commerce and Agriculture, 2016).

Pumpkin Production and Pollination

The pumpkin plant, C. pepo, is in the family Cucurbitaceae and is closely related to cucumbers, melons, and gourds. The genus Cucurbita contains five cultivated species and 27 wild species (Hurd et al. 1971). Cucurbita pepo is split into summer and winter

Texas Tech University, Christopher Jewett, December 2017 squash, with eight cultivar categories; pumpkins, scallops, acorns, crooknecks, straightnecks, vegetable marrows, cocozelles, and zucchinis (Paris 1989). Pumpkins are a distinct cultivar of C. pepo which typically are round and orange in color. Pumpkin vines produce large showy flowers early in the day to attract pollinators, and it is typical for wild pollinators to begin foraging at flowers before dawn. The plants are monoecious and require pollination from the staminate (male) flowers (Figure 1.2) to the pistillate

(female) flower (Figure 1.3) for fruit set (Hurd et al. 1971). Across vines, staminate flowers outnumber pistillate flowers, and early in the growing season plants usually produce several staminate flowers before producing a pistillate flower. Tepedino (1981) showed an average ratio for C. pepo staminate to pistillate flowers of 5.3/1.

Cucurbita pepo flowers are entomophilous (i.e. insect-loving), and it is vital that insects perform the role of pollination. When pollination does not occur, seed count in the fruit can be low, and when insufficient pollen grains are deposited to the stigma, fruit can have an unpleasing shape, making it is less marketable (Nepi and Pacini 1993).

Wild cucurbits have co-evolved with the native squash bee genera Peponapis and

Xenoglossa. Foraging individuals of these genera overlay their diel patterns of foraging with the opening of pumpkin flowers during the morning hours and closing during afternoon hours. Female squash bees collect Cucurbita pollen to provision for developing larvae located in brood chambers in the soil (Figure 1.4), while male squash bees spend their lives out of the ground nests and in flowers (Figure 1.5) (Tepedino,

1981). Julier and Roulston (2009) concluded that P. pruinosa prefer to nest within the cultivated crop areas in both tilled and no-till fields directly below plants. Females dig

Texas Tech University, Christopher Jewett, December 2017 holes at depths of 9-69 cm to lay larval cells, with the highest density of cells at 16-30 centimeters (Mathewson 1968, Julier and Roulston 2009).

Agricultural practices that could induce stress to native pollinator populations include soil tillage and agrichemical treatments on crops. Fungicides are commonly sprayed on pumpkins to fight powdery mildew, Plectosporium blight, black rot, and other fungal diseases pathogenic to pumpkins. In the area of our study, farmers typically apply fungicides every 14 days on pumpkins. Native bees also combat other agrichemical treatments on crops planted adjacent to pumpkins, chemicals such as plant defoliants and pesticides used to combat weeds and insects in crops such as cotton. Another route of agrichemical exposure to bees could come from confined animal operations, where agrichemicals including veterinary medicines have the potential to accumulate on native vegetation adjacent to feedlots (Peterson et al. 2017).

Cucurbit farmers had three times as many squash bees in fields that were no-tilled compared to tilled fields (Shuler et al. 2005). Squash bees lay cocoons directly below host plants, and therefore tilling after pumpkin harvest or prior to spring planting could disturb overwintering pupae or nesting adults. However, the influence of soil conditions and disturbances on wild bee behaviors and populations has not been studied in our region. No-till practices can reduce the need for fungicide applications, because residue and structure help combat powdery mildew, Plectosporium blight, and black rot (Everts

2002). Furthermore, Julier and Roulston (2009) suggested that fields with irrigation could increase presence of ground-nesting species because soils are easier for adult bees to excavate. The resources used by squash bees within and outside of production

Texas Tech University, Christopher Jewett, December 2017

(i.e. cultivated fields and wild lands) are unknown and should be better understood to help in sustaining squash bee populations.

While honey bee and bumble bees are proven pollinators for pumpkin production, understanding the contributions of native bees in pumpkins could help reduce production costs. A first step in understanding how these beneficial insect populations are supported on agricultural lands includes an initial assessment of the variation in occurrences and abundances across production fields. Accordingly, we partnered with regional pumpkin farmers and investigated bee abundances across 16 pumpkin fields and two growing seasons (Figure 1.6). The objectives of my research were to 1) determine relationships of different bee groups with environmental and farm management factors, and 2) investigate spatial and temporal attributes of bees groups across two growing seasons. A goal of the research was to provide basic information on squash bees and other pollinators in pumpkin production fields and to use this information to address further questions aimed at resources requirements of wild pollinators in pumpkins.

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Literature Cited

Aizen, M.A. and L.D. Harder. 2009. The global stock of domesticated honey bees is growing slower than agriculture demand for pollination. Current Biology. 19:915- 918.

Bernauer, O.M., H.R. Gaines-Day, and S.A. Steffan. 2015. Colonies of bumble bees (Bombus impatiens) produce fewer workers, less bee biomass, and have smaller mother queens following fungicide exposure. Insects. 6:478-488.

Biesmeijer, J.C., S.P.M. Roberts, M. Reemer, R. Ohlemüller, M. Edwards, T.Peeters, A.P. Schaffers, S.G. Potts, R. Kleukers, C.D. Thomas, J. Settele, and W.E. Kunin. 2006. Parallel declines in pollinators and insect pollinated plants in Britain and the Netherlands. Science. 313:351-354.

Blaauw, B.R. and R. Isaacs. 2014. Flower plantings increase wild bee abundance and the pollination services provided to a pollination-dependent crop. Journal of Applied Ecology. 51:890-898.

Bond, J., K. Plattner, and K. Hunt. 2014. U.S. pollination-service market. USDA. Economic Research Service.

Bosch, J. and W.P. Kemp. 2002. Developing and establishing bee species as crop pollinators: the example of Osmia spp. (Hymenoptera: Megachilidae) and fruit trees. Bulletin of Entomological Research. 92:3-16.

Bosch J., K. Kemp, and G.E. Trostle. 2006. Bee population returns and cherry yields in an orchard pollinated with Osmia lignaria (Hymenoptera: Megachilidae). Journal of Economic Entomology. 99:408-413.

Burgett, M. 2011. Pacific northwest honey bee pollination economics survey 2010. National Honey Report. 12.

Burkle, L.A., J.C. Marlin, and T.M. Knight. 2013. Plant-pollinator interactions over 120 years: loss of species, co-occurrence, and function. Science. 339:1611-1615.

Cameron, S.A, J.D. Lozier, J.P. Strange, J.B. Koch, N. Cordes, L.F. Solter, and T.L. Griswold. 2011. Patterns of widespread decline in North American bumble bees. Journal of Applied Ecology. 49:723-732.

Christenhusz, M.J.M. and J.W. Byng. 2016. The number of known plants in the world and its annual increase. Phytotaxa. 261:201-217.

Texas Tech University, Christopher Jewett, December 2017

Cox-Foster, D.L., S. Conlan, E.C. Holmes, G. Palacios, J.D. Evans, N.A. Moran, P.L. Quan, T. Briese, M. Hornig, D.M. Geiser, V. Martinson, D. vanEngelsdorp, A.L. Kalkstein, A. Drysdale, J. Hui, J. Zhai, L. Cui, S.K. Hutchison, J.F. Simons, M. Egholm, J.S. Pettis, and W.I. Lipkin. 2007. A metagenomic survey of microbes in honey bee colony collapse disorder. Science. 318:283-287.

Cresswell J.E. 2011. A meta-analysis of experiments testing the effects of a neonicotinoid insecticide (imidacloprid) on honey bees. Ecotoxicology. 20:149-157.

Cusser, S., J.L. Neff, and S. Jha. 2016. Natural land cover drives pollinator abundance and richness, leading to reductions in pollen limitations in cotton agroecosystems. Agriculture, Ecosystems and Environment. 226:33-42.

Dively, G.P. and A. Kamel. 2012. Insecticide residues in pollen and nectar of a cucurbit crop and their potential exposure to pollinators. Journal of Agriculture and Food Chemistry. 60:4449-4456.

Ellis, J.D., J.D. Evans, and Jeff Pettis. 2010. Colony losses, managed colony population decline, and colony collapse disorder in the United States. Journal of Agriculture Research. 49:134-136.

Everts, K. L. 2002. Reduced fungicide applications and host resistance for managing three diseases in pumpkin grown on a no-till cover crop. Plant Disease. 86:1134- 1141.

Floydada Chamber of Commerce and Agriculture. Punkin Day History. http://www.floydadachamber.com/punkin-days-history. (Accessed 30 September 2017).

Fürst, M.A., D.P. McMahon, J.L. Osborne, R.J. Paxton, and M.J.F Brown. 2014. Disease associations between honeybees and bumblebees as a threat to wild pollinators. Nature. 506:364-366.

Gallai, N., J.M Salles, J. Settele, and B.E. Vaissière. 2009. Economic valuations of the vulnerability of world agriculture confronted with pollinator decline. Ecological Economics. 68:810-821.

Texas Tech University, Christopher Jewett, December 2017

Garibaldi, L.A., I. Steffan-Dewenter, R. Winfree, M.A. Aizen, R. Bommarco, S. A. Cunningham, C. Kremen, L.G. Carvalheiro, L. D. Harder, O. Afik, I. Bartomeus, F. Benjamin, V. Boreux, D. Cariveau, N.P. Chacoff, J.H. Dudenhöffer, B.M. Freitas, J. Ghazoul, S. Greenleaf, J. Hipólito, A. Holzschuh, B. Howlett, R. Isaacs, S. K. Javorek, C.M. Kennedy, K.M. Krewenka, S. Krishnan, Y. Mandelik, M.M. Mayfield, I. Motzke, T. Munyuli, B.A. Nault, M. Otieno, J. Petersen, G. Pisanty, S.G. Potts, R. Rader, T.H. Ricketts, M. Rundlöf, C.L. Seymour, C. Schüepp, H. Szentgyörgyi, H. Taki, T. Tscharntke, C.H. Vergara, B.F. Viana, T.C. Wanger, C. Westphal, N. Williams, and A.M. Klein. 2013. Wild pollinators enhance fruit set of crops regardless of honey bee abundance. Science. 339:1608-1611.

Goulson, D., G.C. Lye, and B. Darvill. 2008. Decline and conservation of honey bees. Annual Review of Entomology. 53:191-208.

Greenleaf, S.S. and C. Kremen. 2006. Wild bee species increase tomato production and respond differently to surrounding land use in Northern California. Biological Conservation. 133:81-87.

Hurd, P.D., E.G. Linsley, and T.W. Whitaker. 1971. Squash and gourd bees (Peponapis, Xenoglossa) and the origin of cultivated Cucurbita. Society for the Study of Evolution. 25:218-234.

Julier, H.E. and T.H. Roulston. 2009. Wild bee abundance and pollination service in cultivated pumpkins: farm management, nesting behavior and landscape effects. Journal of Economic Entomology. 102:563-573.

Klein, A-M., I. Steffan-Dewenter, and T. Tscharntke. 2003. Fruit set of highland coffee increase with the diversity of pollinating bees. The Royal Society. 270:955-961.

Klein, A-M., B.E. Vaissiere, J.H. Cane, I. Steffan-Dewenter, S.A. Cunningham, C. Kremen and T. Tscharntke. 2007. Importance of pollinators in changing landscapes for world crops. Proceedings of the Royal Society B: Biological Sciences 274:303– 313.

Koh, I., E.V. Lonsdorf, N.M. Williams, C. Brittain, R. Isaacs, J. Gibbs, and T.H. Ricketts. 2015. Modeling the trend and status, trends, and impacts of wild bee abundances in the United States. PNAS Early Edition. 113:140-145.

Kopec, K and L.A. Burd. 2017. Pollinators in Peril: A systematic status review of North American and Hawaiian native bees. Center of Biological Diversity.

Kremen, C.A., N.M. Williams, and R.W. Thorp. 2002. Crop pollination from native bees at risk from agricultural intensifications. PNAS. 99:16812–16816.

Texas Tech University, Christopher Jewett, December 2017

Kremen, C., N.M. Williams, B.L. Gugg, J.P Fay, and R.W. Thorp. 2004. The area requirements of an ecosystem service: crop pollination by native bee communities in California. Ecological Letters. 7:1109-1119.

Marketing Resource Center (AgMRC). 2015. Pumpkins. http://www.agmrc.org/commodities-products/vegetables/pumpkins/. (Accessed 11 October 2016).

Mathewson, J.A. 1968. Nest construction and life history of the eastern cucurbit bee, Peponapis pruinosa (Hymenoptera: Apoidea). Journal of the Kansas Entomological Society. 41:255-261.

Morandin, L.A. and M.L. Winston. 2006. Pollinators provide economic incentive to preserve natural land in agroecosystems. Agriculture, Ecosystems & Environment. 116:289-292.

Morandin, L.A., R.F. Long, and C. Kremen. 2016. Pest control and pollinator cost – benefit analysis of hedgerow restoration in a simplified agriculture landscape. Journal of Economic Entomology. 109:1020-1027.

Nepi M. and E. Pacini. 1993. Pollination, pollen viability and pistil receptivity in Cucurbita pepo. Annals of Botany. 72:527-536.

Paris, H.S. 1989. Historical records. origins, and development of the edible cultivar groups of Cucubita pepo (Cucurbitaceae). Economic Botany. 43:423-443.

Peterson, E.M., K.J. Wooten, S. Subbiah, T. Anderson, S. Longing and P. Smith. 2017. Agrochemical mixtures detected on wildflowers near cattle feed yards. Environmental Science and Technology Letters. 4:216-220.

Pettis, J.S and K.S. Delaplane. 2010. Coordinated responses to honey bee decline in the USA. Apidologie. 41:256-263.

Pitts-Singer, T. H. and J.H. Cane. 2011. The alfalfa leafcutter bee, Megachile rotundata: the worlds most intensively managed solitary bee. Annual Review of Entomology. 56:221-237.

Potts, G.P., J.C. Biesmeijer, C. Kremen, P. Neumann, O. Schweiger, and W.E. Kunin. 2010. Global pollinator declines: trends, impacts and drivers. Trends in Ecology & Evolution. 25:345-353.

Pumpkins in Texas – Vegetable Resources. 2000. Aggie Horticulture. https://aggie- horticulture.tamu.edu/vegetable/guides/crop-briefs/pumpkins-in-texas/. (Accessed 29 September 2017).

Texas Tech University, Christopher Jewett, December 2017

Scott-Dupree, C.D., L. Conroy, and C.R. Harris. 2009. Impact of currently used or potentially useful insecticides for canola agroecosystems on Bombus impatiens (Hymenoptera: Apidae), Megachile rotundata (Hymenoptera: Megachilidae), and Osmia lignaria (Hymenoptera: Megachilidae). Journal of Economic Entomology. 102:177-182.

Shuler, R.E., T.H. Roulston, and G.E. Farris. 2005. Farming practices influence wild pollinator populations on squash and pumpkins. Journal of Economic Entomology. 98:790-795.

Stanghellini, S.S., J.T. Ambbrose, and J.R. Schultheis. 2002. Diurnal activity, floral visitation and pollen deposition by honey bees and bumble bees on field-grown cucumber and watermelon. Journal of Apiculture Research. 41:27-34.

Stubbs, C.S., F.A Drummond, and S.L. Allard. 1997. Bee conservation and increasing Osmia spp. in Maine lowbush blueberry fields. Northeastern Naturalist. 4:133-144.

Tepedino, V.J. 1981. The pollination efficiency of the squash bee (Peponapis pruinosa) and the honey bee (Apis mellifera) on summer squash (Cucurbita pepo). Journal of the Kansas Entomological Society. 54:359-377.

Traynor, J. 2014. 2015 almond pollination. American Bee Journal. http://beesource.com/point-of-view/joe-traynor/2015-almond-pollination/. vanEngelsdorp, D. and M.D. Meixner. 2010. A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them. Journal of Invertebrate Pathology. 103:80-95.

Watson, J.C., A.T. Wolf, and J.S. Ascher. 2011. Forested landscapes promote richness and abundances of native bees (Hymenoptera: Apoidea: Anthophila) in Wisconsin apple orchards. Environmental Entomology. 40:621-632.

United States Department of Agriculture (USDA). 2015. Pumpkins: background and statistics. USDA, Economic Research Service.

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Figure 1.1. Pumpkin field during the third week of sampling in 2017. The photo shows the heavy canopy of the pumpkins.

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Figure 1.2. Staminate (male) pumpkin flower.

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Figure 1.3. Squash bees foraging on a pistillate (female) flower. Photo was taken in the field and in the early season when flower density was low.

Texas Tech University, Christopher Jewett, December 2017

Figure 1.4. Female squash bee emerging from nest in soil. Closer observation and contact revealed the individual was deceased

Texas Tech University, Christopher Jewett, December 2017

Figure 1.5. Male squash bee (Peponapis pruinosa) resting in a pumpkin flower.

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Figure 1.6. Chris Jewett, walking pumpkin transects on the third sample round of 2017. Note the expansive canopy created by the pumpkin plants in Week 3.

Texas Tech University, Christopher Jewett, December 2017

Chapter II

An assessment of pollinator abundances and environmental relationships at cultivar, field, and landscape scales in pumpkins (Texas, USA)

Abstract

Twenty-three pumpkin (Cucurbita pepo) fields on the U.S. Southern High Plains

were visited and pollinator communities were sampled to estimate densities and describe

relative abundances and variation across different bee taxa among growing-season weeks, fields and cultivars. In addition to comparisons of bee densities, a binary logistic regression was used to determine relationships of flower-occupancy by squash bees

(Peponapis and Xenoglossa) and honey bees (Apis mellifera) to field, cultivar and land

cover factors. In 2016, we collected 1952 bees from 12 fields, with 73.4 percent of the

total number of bees collected native squash bees, 25.8 percent honey bees, and 0.1

percent bumble bees. Squash bees were more abundant than managed honey bees in all

fields including those with managed honey bee and/or bumble bees at the field edge.

Year 2 (2017) mirrored the 2016 numbers with 82 percent squash bees and 15.2 percent

honey bees (out of 3511 total number of individuals). Forty percent (2016) and 60

percent (2017) of the total number of bees were recorded in the first week of both years.

Declining numbers of bees across weeks could indicate natural seasonal mortality,

environmental stressors causing mortality, or sampling bias associated with enclosed

pumpkin canopies and edge effects (i.e. higher squash bee densities at margins as

pumpkin canopies enlarged over the 4-5-week flowering period). In the 2017 analysis, honey bees showed preference for the Turban pumpkin cultivar, and this was the only cultivar where honey bees were more abundant than squash bees across both seasons.

The results of binomial logistic regression showed that mean bee-occupancy of flowers

Texas Tech University, Christopher Jewett, December 2017 varied by week and by field in 2016, and in 2017 varied significantly by these and additionally by cultivar, mainly attributed to the Turban-honey bee relationship. Percent wildland within 250 meters of pumpkin fields, the presence of field-level managed hives, pumpkin field area, nor cultivar richness were significant predictors of flower-occupancy across fields by squash bees or honey bees, emphasizing a need to further investigate the frequency of soil nesting by squash bees within fields.

Introduction

Pollinators provide a huge ecological service for crop production in agriculture systems and in natural systems by maintaining flowering plant communities (Potts et al.

2010). Farmers rely directly or indirectly on pollinators for approximately 30% of the world’s food production (Kremen et al. 2002), with pollinators providing an essential service in 8-9% of the total crop production globally and contributing over $190 billion to the global economy (Gallai et al. 2009). Many crop species such as almonds, apples,

Texas Tech University, Christopher Jewett, December 2017 blueberries and pumpkins require the transfer of pollen by outside sources, and insects are often the key pollinators providing this ecosystem service.

For crops depending on insect pollinators to bear seed or fruit, producers across the United States often rely on managed bees such as the western honey bee A. mellifera, bumble bees (Bombus spp.) and others. Honey bee pollination services are currently at an all-time high due to crop demand and numerous environmental stressors that have threatened both pollinators and migratory beekeeping (Aizen and Harder 2009). Honey bees are proven pollinators for many crops, but increasing rental costs of hives and threats to honey bee health could negatively impact farmers that require insect pollinators. The average cost of managed bee rentals for crop production in 2016, across

Arkansas, Florida, Louisiana, Missouri, Mississippi, New Mexico, Oklahoma, and Texas, was approximately $50 per acre (NASS-USDA, 2016). Because of inherent monetary risks in migratory beekeeping, disease potential and CCD occurrence, and an improved understanding of native bee contributions in agricultural production, farmers could benefit from using inexpensive natural pollinating agents (i.e. native bees and other pollinating insects).

In Texas each year farmers plant up to 9,000 acres of pumpkins per year, with

5,000-8,000 of them grown in West Texas (Pumpkins in Texas, 2000). Approximately

1,500 acres are grown by farmers near Floydada, Texas, producing 20,000 to 50,000 pounds per acre each year (Floydada Chamber of Commerce and Agriculture, 2016). For the production of pumpkins in the region, producers typically rent or buy managed bees

(i.e. honey bees and bumble bees) annually, and the contributions of native bees to pumpkin production in the region are unknown.

Texas Tech University, Christopher Jewett, December 2017

Understanding the occurrences of pollinators across pumpkin production fields is an important piece of information to sustain pollination services. Knowledge of the spatial and seasonal dynamics of pumpkin production systems and integrated wildlands as a resource for pollinators could help sustain regional agriculture. The primary objective of this study was to determine the temporal and spatial characteristics of the community of native and managed bees visiting pumpkins across multiple fields on the

High Plains of western Texas. I hypothesized that the native squash bees would maintain larger abundances across fields than managed bee numbers, but that abundances would vary in relation to field and adjacent landscape characteristics and in the presence of managed commercial bees. Based on field observations in the first year, I also hypothesized that different pumpkin cultivars would attract different relative numbers of pollinators. My overarching goal was to develop basic information on densities, temporal relative abundances and field-level variation of pollinators as a baseline study to support further work on pollinators in regional pumpkin agroecosystems.

Materials and Methods

Study Area

The study area consisted of pumpkin farms in Floyd and Crosby counties in western Texas (Figure 2.1). Both counties occur in a region recently identified as an area of concern because of potential mismatch in pollinator-dependent crops and insect pollinator abundance (Koh et al. 2015). Across the 2016 and 2017 growing seasons, pumpkin fields selected for the study occurred in an area of approximately 320 km2.

Pumpkin growers rotated their production fields annually, therefore a pumpkin field one year was a different crop (i.e. cotton, corn, or sorghum) in the preceding year. For

Texas Tech University, Christopher Jewett, December 2017

example, once farmers are finished planting cotton in one year, they convert their crop

planters to plant pumpkin seeds. This usually occurs at the end of May to early June,

depending on weather patterns. In 2016 pumpkin growers rented 181 commercial honey

bee hives and purchased 13 B. impatiens Quad boxes (Koppert Biological Systems,

Howell, Michigan). In 2017 pumpkin farmers purchased 64 honey bee hives and 35 B.

impatiens Quad boxes across 11 fields. Hives were placed across fields depending on the

farmer’s preference and pollination needs. Each field was managed conventionally, with

insect and fungal pests controlled with scheduled applications of pesticides through the

growing season. Across both years, pumpkins were planted from early to mid-June. At approximately three weeks after planting (around July 15th), pumpkins began flowering and continued to flower for approximately five weeks. Sampling intervals were designated as weeks one through four, from the beginning of flowering (Table 2.1).

Pumpkins were harvested during September of both production years.

Field Methods

In 2016, a total of 46 cultivars were planted, with an average of 7 cultivars per

field and a range in field size from 3.8 to 20.3 ha (9.5 to 50.1 ac). In 2017, 50 cultivars were planted, with an average of 13 cultivars per field and a range in field size from 9.0 to 32.6 ha (22.2 to 80.6 ac). Our first sampling date was based on vine growth and flower abundance across pumpkin fields. We initiated sampling after the field most recently planted (in sequence) started flowering. Once the youngest field started flowering the oldest field had been in bloom for no longer than 2 weeks. In 2016, sampling events consisted of two consecutive days of the 4 sampling weeks (Table 2.1). Four, 50 meter transects were walked in each field between the time of 0700 and 1100, and the number

Texas Tech University, Christopher Jewett, December 2017 of bee taxa per flower was recorded. Bee taxa consisted of native bees in the genus

Xenoglossa or Peponapis, or commercial bees A. mellifera and B. impatiens. We did not exclude the possibility that honey bees observed in our transects were wild honey bees and not directly from the rented hives. Observers counted flowers and foraging bees along each 1 m transect, with transects located down the middle of two crop rows spaced

2 meters apart. To aid in counting, surveyed flowers were observed in two rows but only the side of the observer-row was used to count flowers and bees. The number of individuals of the different bee taxa occurring in flowers within each of the four transects was recorded. Counts of bees were recorded for each transect per field, and the total number of bees per field was calculated as the sum of the four transect counts. The ratio of the number of occupied to unoccupied flowers per transect was selected as the dependent variable in logistic regression.

In 2017, bees were counted per field according to cultivar type. For this second growing season, we replicated sampling by cultivar using an incomplete block design, but abandoned the blocking factor and further included it as a random factor for investigation of variation in the proportion of occupied flowers. In 2017, bee counts were made across

14 cultivars (Table 2.2) occurring across 11 fields. Within the rows of one of 14 cultivars across 11 fields, we used four 25-flower subsamples to record the number and types of bees occurring in 100 flowers per cultivar in each field. We repeated these sampling methods across every cultivar that could be replicated four times across four fields. As a result, we sampled from 100 flowers to 1000 flowers per field in 2017. Sampling events consisted of two consecutive days of the 4 sampling weeks (Table 2.1). Transects across cultivars were walked in each field between the time of 0700 and 1100.

Texas Tech University, Christopher Jewett, December 2017

Starting locations of transects were randomly located at a distance at least 25 m from the field edge. Each flower observed along the transect was observed and the number and types of bees occupying the flower were recorded. At least 100 individual flowers were observed; when 25 flowers were counted, the sampler paced 10 m forward and across rows (within a cultivar) before recording a new 25 flowers. This procedure was repeated four times to yield four, 25 flower subsamples of bee counts per cultivar, for a total of 400 flower observation per cultivar across at least four of the eleven fields.

Habitat and Landscape Variables

We identified environmental variables describing the production and land cover associated with pumpkin fields that might influence bee abundances and flower occupancy. A 250 m buffer was delineated around each field for the 2 years and selected proportional land cover classes as crop land or wildland (i.e. uncultivated land) were calculated for each field using ArcMap v. (ESRI, Redding, CA). Crop GIS data was obtained from CropScape (USDA – National Agriculture Statistics Data 2016).

Field scale variables included field size (ha) area, number of flowers per standard transect, number of pumpkin cultivars per field, type of pollination service (0 = no managed bees, 1 = commercial honey bees, 2 = commercial honey bees and bumble bees), and the number of consecutive years that pumpkins were grown on that farm.

Field polygons were created for both production years, to create 250 m buffer and to calculate field size and land cover classes. Figure 2.2 demonstrates how percent wild land area was determined in ArcMap 10.4.1 with USDA – CropScape land use data.

Texas Tech University, Christopher Jewett, December 2017

Data Analysis

To calculate relative abundances across pumpkin production weeks, cultivars and

fields, we used raw bee counts (counts per four standard transects per field) for 2016

fields and total bee counts per cultivar (2017 fields). For comparisons at the field level,

we divided the total number of bees per field by the number of cultivar-transects to get an average number of bees per 100 flowers.

The numbers of bees clustered in flowers and across flowers per unit area or number of flowers is an estimate of densities of bee groups. From bee counts across flowers or transects, we calculated both the average number of bee groups across transect or cultivar-transect, and additionally calculated relative abundances of each bee group.

For binomial regression, we calculated flower occupancy using presence-absence data

(the flower was occupied by a bee or not), and further calculated the number of flowers occupied by bees per cultivar-transect using 2017 data.

The average densities and relative abundances of bees were assessed graphically across weeks, fields and cultivars. From a total of 35,000 flower observations, flower occupancy was used to model the probability of finding bees in a flower given the predictor variables (cultivar, field, week, area, pollination, number of cultivars, wildland) using a binary logistic regression model:

(1) Model P(bees|x) = model the probability of finding bees on a flower given

(2) Predictor variables: x1 = Cultivar, x2 = Field, x3 = Area, x4 = Pollination, x5 =

wildland, x6 = week,

T (3) w0 + w1x1 + …. w6x6 = w x = linear combination of variables (vector w transpose

Texas Tech University, Christopher Jewett, December 2017

(4) Model P(bees|x) = σ(wTx), where:

(5) σ(a) = 1/(1 + e-a), Logistic function

We used the LOGISTIC procedure in SAS 9.4 (Cary, NC) to select and find an

appropriate binary logit regression model. We used the Event/Trial notation for binary

outcomes in SAS, where the number of bees found (Event) was divided by the total

number of flowers (Trial). Week, Field, and Cultivar (2017 data only) were coded as

CLASS variables. The FORWARD effect-selection procedure was used to fit the best

model given the data. The LSMEANS statement was used to compute least squares

means (LSmeans) of the fixed effects, and differences among LS-means were estimated

using the Tukey-Kramer adjustment.

To assess goodness of fit of the models we compared Akaike Information Criterion

values, Max-rescaled R-squared values, and the Hosmer and Lemeshow goodness of fit

Test. The Williams rescaling method was used to accommodate for over-dispersion.

Results

We surveyed 23 pumpkin fields in 2016 and 2017 that varied in size from 3.8 to

32.6 hectares. The total number of bee groups observed across 2016 and 2017 in 23 pumpkin fields was 4342 squash bees, 1040 honey bees, 25 bumble bees, and 56 other native bees (Table 2.3). In 2016, 1952 bees were observed across 12 fields, with an average of 163 bees per field. Average bee counts across farms in 2016 ranged from 9.3 to 96.3 individuals per transect (Figure 2.3). Squash bees dominated total relative abundances with 73.4 percent of the total number of individuals. The order of numerical dominance of the other bees included honey bees (25.8 percent), other native bees (0.7 percent) and bumble bees (0.1 percent). Transect averages of total bees across 11 farms

Texas Tech University, Christopher Jewett, December 2017 in 2017 ranged from 4.3 to 32.8 individuals (Figure 2.4). Similar to 2016, squash bees dominated total relative abundance with 82.9 percent, with honey bees (15.2 percent), other native bees (1.2 percent), and bumble bees (0.7 percent) similar to the low relative abundances of these groups in 2016.

Across the four week growing period in both years, total bee abundances declined sharply from week 1 to week 4. In 2016, 40 percent of the total bees recorded over the growing season occurred in week 1. The average number of bees per transect ranged from 65.3 in the first sample week to 44.4, 31.1, and 12.5, across weeks 2, 3, and 4, respectively (Figure 2.5). In 2017, 60 percent of the total bees recorded across weeks was recorded in week one. Total bee averages began at 43.9 bees in the first sample week and then dropped to 11.7, 7.9 and 5.9, across weeks 2, 3, and 4, respectively (Figure

2.6). Therefore, most of the information in our relative bee numbers per field and cultivar was contained within week 1 sample data. The reason for this relatively high number of bees in week 1 is unknown, but some possibilities for the declining numbers include 1) bee populations dispersed across field as flowers increased in abundance, 2) as foliar biomass increased, squash bees became clumped in dispersion at field edge, and 3) a sharp increase in mortality after week 1.

Squash bee averages per cultivar (number of bees per 100 flowers) were higher than honey bee averages for 13 of the 14 pumpkin cultivars. In the Turban cultivar, the average number of honey bees were twice the average number of squash bees (Figure

2.7).

For 2016 and 2017 data, only field and week were significant explanatory factors in binomial regression models of the occupancy of bee groups within flowers. In the

Texas Tech University, Christopher Jewett, December 2017

2017 analysis, these variables plus cultivar was significant in predicting occupancy of flowers by bees (p<0.05). No other explanatory habitat variables (field area, pollination management, number of cultivars or percent wild land/grassland) were statistically significant or contributed to the model. Only models for total bees, squash bees and honey bees converged. Because of low frequencies of occurrences, there were too few observations of bumble and other bees to find a model solution. Differences in flower occupancy across weeks and field are shown in Tables 2.4 and 2.5 for year 2016 and

2017, respectively.

Discussion

This study documents pollinator occurrences and relative abundances in

pumpkins on the Southern High Plains of Texas. Based on our findings of high relative

abundances of native squash bees across fields and two growing seasons, it is likely that

pumpkin pollination in the region is dependent on these native squash bee populations.

Overall, squash bees comprised 75 percent of the total number of all bees collected in

2016 and 2017. Native squash bees have been shown to be frequent visitors to pumpkin

flowers and to contribute to improved pumpkin yields (Tepedino 1981). Squash bees in

the genera Peponapis and Xenoglossa are common in pumpkin production regions, yet

information on resource requirements and environmental stressors of these populations in

the Southern High Plains region do not exist.

The statistical model predicts that managed pollination had no significant effect

on bee numbers in pumpkin fields. Even models that ignored the effect of cultivar and

week, but included pollination only, had a 0.08 r-squared value at best. Based on these

regression results and the overall relative numbers of bees in the surveyed pumpkin

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fields, the additions of managed hives to pumpkin fields might not increase the

frequencies of flower visitation by managed bees in pumpkins. Our data show that honey bees are occurring on pumpkin flowers at much lower densities when compared to native squash bees.

In 2017, across 14 cultivars surveyed we observed twice the average number of honey bees than squash bees on the cultivar Turban. Turban was the only cultivar honey bees or any other bee class outnumbered squash bees. The reason for honey bee preference on the specific cultivar is unknown. Based on field observations we can hypothesize that honey bees prefer the cultivar because of its morphology. The cultivar canopy is less dense than all other cultivars, while the flower reaches above the vine canopy. This might allow honey bees easier access because of fewer obstacles, while also making them feel more secure to forage. All other cultivars have dense vine canopies that cover flowers, which could be why honey bee numbers are low across all other cultivars.

At the field-level, sharp declines occurred over week 2-4 flowering period for squash bees. While the cause of this decline is unknown, some factors that might have contributed to sharp declines in numbers of bees include: 1) natural causes of mortality,

2) changes in dispersion of squash bees over time, as canopies close during the growing season. Bees were observed in relatively high numbers at the field edges as pumpkin canopies grew and enclosed flowers during the latter weeks of the growing season.

Furthermore, nesting in ditches adjacent to crop margins could influence the dispersion of bees towards clumping at field edges, and 3) exposure to environmental stressors. It is not known if spraying agrichemicals on pumpkins has affected local squash bee

Texas Tech University, Christopher Jewett, December 2017 populations. The first sampling date in 2017 was after the first scheduled spraying of an insecticide-fungicide mixture. Further studies should address exposure of native squash bee pollinators to agrichemical mixtures used in regional pumpkin production.

Understanding how cultivation and other farm practices affect overwintering habits or seasonal recruitment in pumpkins would help ensure pollinator populations are available annually. Differences in soil compaction, disturbance and substrate composition are factors shown to affect ground-nesting bees (Cane 1991), therefore farming practices adopted to protect ground-nesting native squash bees in the region regarding soil conditions could be a beneficial conservation action. Julier and Roulston

(2009) found a negative correlation between squash bee numbers and clay content of soil.

Furthermore, because quash bees likely nest within the pumpkin crop or in adjacent habitats, irrigation, tillage and ditch management within or adjacent to the field could be important drivers of populations. A study across Virginia, West Virginia, and Maryland showed that abundances on no-tilled fields were three times higher than on tilled fields

(Shuler et al. 2005), suggesting that soil disturbance could influence mortality rates of nesting bees and developing brood because of shallow locations of bee nests in the soil.

Texas Tech University, Christopher Jewett, December 2017 would support a better understanding of the biology and value of native bees in pumpkins, to ultimately guide conservation actions for native squash bees on farms.

Texas Tech University, Christopher Jewett, December 2017

Literature Cited

Aizen, M.A. and L.D. Harder. 2009. The global stock of domesticated honey bees is growing slower than agriculture demand for pollination. Current Biology. 19:915- 918.

Cane, J.H. 1991. Soils of ground nesting bees (Hymenoptera: Apoidea): texture, moisture cell depth and climate. Journal of the Kansas Entomological Society. 64:406-413.

Floydada Chamber of Commerce and Agriculture. Punkin Day History. http://www.floydadachamber.com/punkin-days-history. (Accessed 30 September 2017).

Gallai, N., J.M Salles, J. Settele, and B.E. Vaissière. 2009. Economic valuations of the vulnerability of world agriculture confronted with pollinator decline. Ecological Economics. 68:810-821.

Kremen, C.A., N.M. Williams, and R.W. Thorp. 2002. Crop pollination from native bees at risk from agricultural intensifications. PNAS. 99:16812–16816.

Morandin, L.A. and M.L. Winston. 2006. Pollinators provide economic incentive to preserve natural land in agroecosystems. Agriculture, Ecosystems & Environment. 116:289-292.

National Agricultural Statistics Service (NASS) Agricultural Statistics Board, United Sates Department of Agriculture (USDA). 2016. Cost of Pollination.

Potts, G.P., J.C. Biesmeijer, C. Kremen, P. Neumann, O. Schweiger, and W.E. Kunin. 2010. Global pollinator declines: trends, impacts and drivers. Trends in Ecology & Evolution. 25:345-353.

Pumpkins in Texas – Vegetable Resources. 2000. Aggie Horticulture. aggie- horticulture.tamu.edu/vegetable/guides/crop-briefs/pumpkins-in-texas/. (Accessed 29 September 2017).

Texas Tech University, Christopher Jewett, December 2017

Shuler, R.E., T.H. Roulston, and G.E. Farris. 2005. Farming practices influence wild pollinator populations on squash and pumpkins. Journal of Economic Entomology. 98:790-795.

Texas Tech University, Christopher Jewett, December 2017

Table 2.1. Sample dates in 2016 and 2017 pumpkins.

Week 1 Week 2 Week 3 Week 4

2016 July 13th & 14th July 21st & 22nd July 28th &29th August 10th & 11th

2017 July 20th & 21st July 27th & 28th August 8th & 9th August 17th & 18th

Table 2.2. List of pumpkin cultivars that were replicated in 2017. Cultivar Varieties Blue Hubbard Mini White Choga Mystic Cinderella One Too Many Cronus Phat Jack Fairy Tale Pumpkemon Flat Boer Turban Mini Orange W&W Gourds

Table 2.3. Total bee counts across 2016 and 2017 pumpkins. Squash Bees Year (Peponapis & Honey Bees Bumble Bees Other Total Bee Xenoglossa) (A. mellifera) (B. impatiens) Native Bees Counts 2016 1433 504 2 13 1952 2017 2909 536 23 43 3511

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Table 2.4. Summary of Least Square Means Logistic Regression Estimates for 2016, Using Forward Model Selection, only significant variables in the model are presented. Standard Effect Estimate Error z Value Pr > |z| Field 1 -1.6056 0.4764 -3.37 0.0008

3 -2.5504 0.5733 -4.45 <.0001 4 -1.28 0.417 -3.07 0.0021

5 -2.2015 0.5135 -4.29 <.0001 6 -0.658 0.3817 -1.72 0.0847 7 -1.1099 0.513 -2.16 0.0305

8 -1.8785 0.4696 -4 <.0001 9 -0.3547 0.4263 -0.83 0.4053

10 -2.797 0.6767 -4.13 <.0001 11 -1.1974 0.4172 -2.87 0.0041

12 -2.467 0.5655 -4.36 <.0001 Week 1 0.09349 0.2498 0.37 0.7082

2 -1.6714 0.2695 -6.2 <.0001

3 -2.3887 0.3361 -7.11 <.0001

4 -2.6153 0.3653 -7.16 <.0001 Wald Pr > ChiSq Chi-Square

Week 3 48.6253 <.0001 Field 10 35.7607 <.0001 Max-rescaled R Square 0.6995

Texas Tech University, Christopher Jewett, December 2017

Table 2.5. Summary of Least Square Means Logistic Regression Estimates for 2017, Using Forward Model Selection, Only significant variables in the model are presented.

Effect Estimate Standard Error z Value Pr > |z| Cultivar 1 -1.3198 0.2336 -5.65 <.0001 2 -1.9066 0.2723 -7 <.0001 3 -0.9596 0.2271 -4.22 <.0001 4 -1.6039 0.2546 -6.3 <.0001 5 -2.5044 0.2636 -9.5 <.0001 6 -2.4694 0.2777 -8.89 <.0001 7 -2.7956 0.2962 -9.44 <.0001 8 -2.7071 0.2959 -9.15 <.0001 9 -2.9189 0.3017 -9.68 <.0001 10 -3.0846 0.3143 -9.81 <.0001 11 -3.2189 0.3255 -9.89 <.0001 12 -3.107 0.3309 -9.39 <.0001 13 -3.3129 0.3473 -9.54 <.0001 14 -3.4113 0.3432 -9.94 <.0001 4 -2.6153 0.3653 -7.16 <.0001 Week 1 -0.9885 0.1155 -8.56 <.0001 2 -2.6181 0.1538 -17.02 <.0001 3 -3.0771 0.1732 -17.76 <.0001 4 -3.4079 0.1926 -17.69 <.0001 Field 1 -2.8947 0.2272 -12.74 <.0001 2 -1.6675 0.2951 -5.65 <.0001 3 -1.8086 0.2032 -8.9 <.0001 4 -2.5434 0.3935 -6.46 <.0001 5 -3.3898 0.5128 -6.61 <.0001 6 -3.8612 0.4656 -8.29 <.0001 7 -2.4501 0.2393 -10.24 <.0001 8 -1.2597 0.1725 -7.3 <.0001 9 -3.186 0.2685 -11.87 <.0001 10 -2.5328 0.2703 -9.37 <.0001 11 -2.1579 0.2392 -9.02 <.0001 Wald Pr > ChiSq Chi-Square Week 3 4209.5899 <.0001 Field 10 101.2059 <.0001 Cultivar 89.827 <.0001 Max-rescaled R Square 0.6075

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Figure 2.1. Pumpkin fields surveyed on the Southern High Plains of Texas, during 2016 and 2017.

Texas Tech University, Christopher Jewett, December 2017

Figure 2.2. Example of how percent wild land area with 250m buffer was determined in ArcMap 10.4.1 with USDA – CropScape land use data.

Texas Tech University, Christopher Jewett, December 2017

250

Squash Bees Honey Bees Bumble Bees 200

150

100

50 Agerage Number of Bees Agerage

2106 Fields

Figure 2.3. Average number of squash bees, honey bees, and bumble bees across 2016 farms. Each sample represents total bee numbers in each class divided by 4, the number of transects in each field. Error bars represent standard deviations.

Texas Tech University, Christopher Jewett, December 2017

100 90 Squash Bees Honey Bees Bumble Bees 80 70 60 50 40 30 20 10

Average Number ofAverage Bees 0

2017 Fields

Figure 2.4. Average number of squash bees, honey bees, and bumble bees across 2016 farms. Each sample represents total bee numbers in each class divided by (n), the number of transects in each field. Error bars represent standard deviations.

Texas Tech University, Christopher Jewett, December 2017

160 140 120 100 80 60 40 20

Average NumberSquash of Bees Average 0 Week 1 Week 2 Week 3 Week 4 Sample Week

Figure 2.5. Average number of squash bees across 2016 sample season. Each week represents the average number of squash bees for each sample period. Averages were figured by the total number of squash bees counted each week divided by 12, the number of transects walked each sample period. Error bars represent standard deviations.

Texas Tech University, Christopher Jewett, December 2017

120

100

Average NumberSquash of Bees Average 0 Week 1 (n=48) Week 2 (n=54) Week 3 (n=56) Week 4 (n=56) Sample Week

Figure 2.6. Average number of squash bees across 2017 sample season. Each week represents the average number of squash bees for each sample period. Averages were figured by the total number of squash bees counted each week divided by (n), the number of transects walked each sample period. Error bars represent standard deviations.

Texas Tech University, Christopher Jewett, December 2017

100

90 Squash Bees Honey Bees 80 Bumble Bees 70 60 50 40 30 20 10 Average Number ofAverage Bees 0

Cultivar

Figure 2.7. Average number of squash bees, honey bees, and bumble bees across 14 pumpkin cultivars. Each sample represents total bee numbers in each class divided by (n), the number of transects for each cultivar. Error bars represent standard deviations.