Dasineura Oxycoccana (Diptera: Cecidomyiidae) Populations on Cranberry and Blueberry in British Columbia: Same Species, Host Races Or Sibling Species?

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DASINEURA OXYCOCCANA (DIPTERA: CECIDOMYIIDAE) POPULATIONS ON CRANBERRY AND BLUEBERRY IN BRITISH COLUMBIA: SAME SPECIES, HOST RACES OR SIBLING SPECIES?

Melissa Ashley Cook B. Sc. (Biology Co-op), Simon Fraser University, 2008

THESIS

MASTER OF SCIENCE

in the Department of Biological Sciences Faculty of Sciences

SIMON FRASER UNIVERSITY

Summer 2011

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Approval

Name: Melissa Ashley Cook Degree: Master of Science Title of Thesis: Dasineura oxycoccana (Diptera: Cecidomyiidae) Populations on Cranberry and Blueberry in British Columbia: Same Species, Host Races or Sibling Species?

Examining Committee: Chair: Dr. D. J. Green, Associate Professor

______Dr. B. D. Roitberg, Professor, Senior Supervisor Department of Biological Sciences, S.F.U.

______Dr. S. M. Fitzpatrick, Research Scientist Pacific Agri-Food Research Centre, Agriculture and Agri- Food Canada

______Dr. G. Gries, Professor Department of Biological Sciences, S.F.U.

______Robert G. Bennett Public Examiner Independent Conservation Biology Consultant; Research Associate, Royal British Columbia Museum

Date Defended/Approved: ______

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Abstract

The gall-inducing midge, Dasineura oxycoccana Johnson (Diptera:

Cecidomyiidae), is a pest of cranberry, Vaccinium macrocarpon, and highbush blueberry,

V. corymbosum, in British Columbia. Dasineura oxycoccana was initially found on highbush blueberry and more recently on cranberry. Given the close proximity of many cranberry and blueberry farms in British Columbia, it was hypothesized that D. oxycoccana was moving from highbush blueberry onto cranberry. I investigated whether

D. oxycoccana populations from these two crops were the same species, host races or sibling species. I examined two mechanisms that could contribute to reproductive isolation between these populations: temporal isolation and behavioural isolation.

Phenological data show that D. oxycoccana populations were not temporally isolated, because several generations were active at the same time on both crops. Behavioural isolation data from mating experiments show that these populations are completely reproductively isolated and most likely represent cryptic species.

Keywords: gall midge; host race; cryptic species; sibling species; phenology; assortative mating; host shift; Dasineura oxycoccana; Vaccinium corymbosum; Vaccinium macrocarpon

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Dedication

To my Dad for opening the door to the world of biology

Acknowledgements

I would like to thank my supervisor Dr. Bernie Roitberg for his support, guidance and expertise. You showed me that my project could be both applied and theoretical.

You always knew when to let me figure it out myself and when to guide me. Thank you to the Roitberg laboratory colleagues for their helpful comments and suggestions on my project and presentations and allowing me to bounce ideas off you when I was stuck.

I would also like to thank my „unofficial‟ co-supervisor Dr. Sheila Fitzpatrick. I have known you since I began working with you as an undergraduate co-op student at

SFU. You helped me to become the scientist that I am today and gave me my first opportunity to work in agriculture. Your expertise, support and guidance both inside and outside of school have been invaluable.

I would also like to thank my committee member Dr. Gerhard Gries for his helpful suggestions and support during the course of this research and reviewing the thesis. I will always remember your enthusiasm and dedication in BISC 204 and 317 when I was an undergraduate student at SFU. You significantly impacted my future career path. Thank you to my external examiner Dr. Robb Bennett for reviewing my thesis and providing helpful and constructive comments.

Thank you to Dan Peach, Sneh Mathur and Ringa Erio during my first field season for the countless hours spent staring through microscopes dissecting cranberry and blueberry shoots and processing sticky traps. Thank you to Sasha Ozeroff, Jordan Scheu and Kiran Ranganathan during my second field season for the never-ending hours and

days spent watching insects mate and not mate, misting of young cranberries vines and bagging and de-bagging blueberry bushes. Your humour during these field seasons was greatly appreciated.

There‟s an African proverb that says “it takes a village to raise a child”, well in this case it took a village to get me through my Master‟s degree.

To my Aunt Barb, Uncle Ken and Cousin Brittany Tunshell, thank you for your continuing love and support. A huge thank you to my Grandma Dagg for giving me a place to stay during my summer field seasons! Over the past two years of my Masters, plus three years of Co-op terms during my undergrad, you have opened your heart and home to me putting up with my voracious appetite.

Natalie McCarthy and Dr. Sean Flynn thank you for everything you‟ve done.

We‟ve been through so much over the last couple of years. Natalie you‟re the big sister I never had, your love and support since I was just a kid are invaluable. I always know that you‟ve got my back. To Barbara Best and Dr. Bill Hartwick, thank you for your love, support and encouragement. Bill the chiropractic treatments have saved my back and neck from all those years spent hunched over a computer. To Rose, Greg and Kim

Morris, you‟ve become family to me over the last couple of years. Thank you for your support, all of the afternoon tea and cookies and continued interest in my project. Thank you to Jim Lanzo for your continued interest in my project and always asking when I was going to be finished and what was next. I always looked forward to tell you about my progress.

To Kelly Ablard, thank you for all of your advice, helpful comments and pep talks before presentations. Your friendship is invaluable; I can‟t imagine surviving the last couple of years without your guidance and support. Mireille Kramer and Juliana Yeung we have been friends since we were undergrads at SFU. Thank you for your support and guidance during the last decade at SFU. Thank you to all of my friends at SFU that I have met along the way. To all of my field hockey friends- Erin, Grace, Karen, Elaine,

Shelley, Tabby, Michelle and many more for putting up with all of my rants when I couldn‟t put my project away on the weekends.

To my Mom and Dad. You always encouraged my interest in biology; from cats, dogs, hedgehogs, snakes, mice, earthworm farms, walkingsticks and cockroaches

(admittedly the latter was a bit of a stretch!). In hindsight saying no to a horse was probably the right choice. You both encouraged and guided me in my studies as an undergraduate student. To Mom, the last couple of years have been really challenging for both of us without Dad and I know that I would not have been able to finish my Masters without you. You always know what to say when things were rough and always told me to keep going and that I was almost there. Well I‟m there know. To Dad, I owe my interest in biology to you. I still continue to grow your dahlia‟s and maintain your discus- filled aquariums. You always said “I was the only one who didn‟t know I could do it” and you know what you were right. Dad I finished my Masters!

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Table of Contents

Approval ...... ii Abstract ...... iii Dedication ...... iv Acknowledgements ...... v Table of Contents ...... viii List of Figures ...... x List of Tables...... xi Glossary...... xii

1: Introduction ...... 1 1.1 Gall-Inducing Insects ...... 1 1.2 Host Shifts and the Speciation Gamut ...... 3 1.3 Reproductive Isolating Mechanisms ...... 4 1.4 Dasineura oxycoccana Biology ...... 7 1.5 Cranberry and Blueberry Cultivation ...... 9 1.5.1 Cranberry ...... 9 1.5.2 Highbush blueberry ...... 10 1.6 Research Focus ...... 11 1.7 List of References ...... 13 2: Phenology of Dasineura oxycoccana (Diptera: Cecidomyiidae) on cranberry and blueberry indicates potential for gene flow ...... 18 2.1 Abstract ...... 18 2.2 Introduction ...... 19 2.3 Materials and Methods ...... 21 2.3.1 Field sites...... 21 2.3.2 Sampling D. oxycoccana eggs, larvae and pupae...... 21 2.3.3 Phenology of cranberry and blueberry shoots...... 22 2.3.4 Sampling D. oxycoccana adults...... 23 2.3.5 Temperature recording...... 24 2.3.6 Statistical analysis...... 25 2.4 Results ...... 25 2.5 Discussion ...... 30 2.6 Acknowledgements ...... 35 2.7 Figures ...... 36 2.8 Tables ...... 43 2.9 List of References ...... 45

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3: Host-associated differentiation in reproductive behaviour of cecidomyiid midges on cranberry and blueberry ...... 49 3.1 Abstract ...... 49 3.2 Introduction ...... 50 3.3 Materials and methods ...... 54 3.3.1 Insect collection...... 54 3.3.2 Test of mating between and within cranberry and blueberry populations of Dasineura oxycoccana ...... 55 3.3.3 Statistical analysis ...... 56 3.4 Results ...... 56 3.5 Discussion ...... 58 3.6 Acknowledgements ...... 62 3.7 Figures ...... 63 3.8 Tables ...... 66 3.9 List of References ...... 67 4: Conclusion: The Speciation of Dasineura oxycoccana ...... 72 4.1 The Speciation of Dasineura oxycoccana ...... 72 4.2 Applications ...... 73 4.3 Suggestions for Future Research ...... 74 4.4 List of References ...... 76 5: Appendix ...... 78 5.1 Additional Frequency Data ...... 78 5.2 Oviposition Experiment ...... 83 5.2.1 Cranberry and blueberry cultivation ...... 83 5.2.2 Oviposition experiment ...... 85 5.2.3 Results ...... 85 5.2.4 Conclusion ...... 86

List of Figures

Figure 1: Map of the six field sites, Pitt Meadows, British Columbia. BB= blueberry, Vaccinium corymbosum. CB= cranberry, V. macrocarpon...... 36 Figure 2: Crop phenology from three blueberry and three cranberry fields throughout the season. BB= blueberry, Vaccinium corymbosum, and CB= cranberry, V. macrocarpon...... 37 Figure 3: Mean number of Dasineura oxycoccana stage per shoot collected each week throughout the season from two host plants. BB= blueberry, Vaccinium corymbosum, and CB= cranberry, V. macrocarpon. n=18 sampling dates...... 39 Figure 4: Mean number of Dasineura oxycoccana adults per trap collected from low traps (vine height), high traps (122 cm from ground) and yellow cards (vine height) each week throughout the season from cranberry, Vaccinium macrocarpon (CB1). n=18 sampling dates...... 40 Figure 5: Total number of Dasineura oxycoccana stages collected from galled and ungalled blueberry, Vaccinium corymbosum, shoots from BB1, BB2, and BB3. χ2 =911.30, df=3, p<0.0001...... 41 Figure 6: Total number of Dasineura oxycoccana stages collected from galled and ungalled cranberry, Vaccinium macrocarpon, shoots from CB1, CB2, and CB3. χ2 =1214.06, df=4, p<0.0001...... 42 Figure 7: Matings between Dasineura oxycoccana males (M) and females (F) collected from two host plants, cranberry (C) and blueberry (B) ...... 63 Figure 8: Time until mating between Dasineura oxycoccana males (M) and females (F) collected from two host plants, cranberry (C) and blueberry (B), including four C × C outliers...... 64 Figure 9: Proportion of eclosion of male (white bar) and female (gray bar) Dasineura oxycoccana from field-collected shoots of cranberry (C) and blueberry (B) ...... 65 Figure 10: Frequency (%) of shoots containing Dasineura oxycoccana life stages from cranberry (CB2), Vaccinium macrocarpon, shoots...... 79 Figure 11: Frequency (%) of shoots containing Dasineura oxycoccana life stages from cranberry (CB3), Vaccinium macrocarpon, shoots...... 80 Figure 12: Frequency (%) of shoots containing Dasineura oxycoccana life stages from blueberry (BB2), Vaccinium corymbosum, shoots...... 81 Figure 13: Frequency (%) of shoots containing Dasineura oxycoccana life stages from blueberry (BB3), Vaccinium corymbosum, shoots...... 82

List of Tables

Table 1: Percentage of shoots that did not contain Dasineura oxycoccana stages. Shoots were collected from highbush blueberry, Vaccinium corymbosum, farms BB1, BB2, and BB3; and cranberry, V. macrocarpon, farms CB1, CB2, and CB3. Farm CB2 was not sampled on July 21...... 43 Table 2: Comparison of Dasineura oxycoccana life stages in 272 cranberry and 346 blueberry shoots that contained at least one egg, larva or pupa. Shoots were collected in 2009 from BB1 and CB1...... 44 Table 3: Mean (± SE) time (s) until mating and in copula between Dasineura oxycoccana males (M) and females (F) collected from cranberry (C) and blueberry (B) ...... 66 Table 4: Oviposition results from matings between Dasineura oxycoccana males and females from cranberry, Vaccinium macrocarpon, on cranberry plants...... 87

Glossary

Species a group of individuals that are capable of interbreeding and producing viable, fertile offspring and are reproductively isolated from other populations

Cryptic species species that are morphologically similar and reproductively isolated from each other but, often remain hidden and as such they are incorrectly classified into one species

Sibling species morphologically similar, reproductively isolated from other species, and are each other‟s closest relatives

Host race two populations that are partially reproductively isolated from each other as a direct result of adaptation to a different host

Gall abnormal plant growth that results from the interaction between a plant and a foreign organism

Host plant the plant species that an insect feeds, develops, mates and/or oviposits on

Mating the pairing of a male and female; successful or unsuccessful fertilization

Copulation the physical act of mating and transfer of sperm resulting in fertilization

Sex a chemical cue that can act over long distances to bring the sexes pheromone together for mating and stimulates behaviour in the opposite sex

Courtship a chemical cue that acts over short distances and stimulates courtship pheromone and mating behaviours

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1: Introduction

In chapter 1, I will review the biology of gall-inducing insects (1.1) focusing on gall midges (Cecidomyiidae). I will then review host shifts within gall-inducing insects and how host shifts can result in the formation of host races, cryptic species, and sibling species (1.2). Following this I will review the main prezygotic and postzygotic reproductive isolating mechanisms (1.3) that can arise from host shifts to facilitate host race, cryptic species, and sibling species formation. Focusing on Dasineura oxycoccana,

I will describe its biology (1.4) and its host plants: cranberry (1.5.1) and highbush blueberry (1.5.2). Finally, I will discuss my research objective (1.6) which was to determine whether D. oxycoccana populations within British Columbia represent a single species, host races or sibling species.

1.1 Gall-Inducing Insects

A gall is an abnormal plant growth that results from the interaction between a plant and a foreign organism (Dreger-Jauffret and Shorthouse 1992). Galls can be induced by viruses, bacteria, fungi, protozoa, nematodes and arthropods (Dreger-Jauffret and

Shorthouse 1992). An estimated 13,000 insects, found in seven orders are known to induce galls: Thysanoptera, Hemiptera, Homoptera, Lepidoptera, Coleoptera,

Hymenoptera and Diptera (Dreger-Jauffret and Shorthouse 1992). Galls can be induced on stems, roots, fruits, flowers and leaves, the latter being targeted by most gall-inducing insects (Dreger-Jauffret and Shorthouse 1992). Gall morphology ranges from simple leaf

curls to swollen plant organs to complex structures with hairs and thorns (Dreger-Jauffret and Shorthouse 1992).

Gall induction is a complex and intimate process that requires an insect to be highly host specific to control and manipulate the plants cells that form the gall (Dreger-Jauffret and Shorthouse 1992, Shorthouse et al. 2005). Most phytophagous insects can move within and between their host plants, but gall-inducing insects are unique in that they are essentially sessile endophages feeding exclusively on induced nutritive cells surrounding the inner gall (Dreger-Jauffret and Shorthouse 1992, Craig et al. 1994).

Of particular interest are gall-inducing midges (Diptera: Cecidomyiidae) many of which attack agricultural and ornamental plants, such as the Hessian fly, Mayetiola destructor, and the sorghum midge, Contarinia sorghicola (Barnes 1948, Gagné 1989).

Gall-inducing midges are characterized by four life stages: egg, larva, pupa and adult.

The larval stage is responsible for gall induction. The small (< 3 mm), short-lived adults do not feed and are weak fliers (Gagné 1989). The majority of gall-inducing midges are highly host specific and can only induce galls on either one or a few plants within a particular genus, but some can induce galls on plants in different families (Gagné 1989).

For example, several species within the genus Dasineura are known to induce galls on several closely related plant hosts. Dasineura brassicae, D. affinis, and D. dielsi are each known to induce galls on seven or more plant species within genera Brassica, Viola and

Acacia, respectively (Åhman 1985, Birch et al. 1992, Post et al. 2010).

1.2 Host Shifts and the Speciation Gamut

Host shifts are common in phytophagous insect species, including gall-inducing insects, and can facilitate the evolution of host races, cryptic species and sibling species (Wood

1980, Bierbaum and Bush 1990, Craig et al. 1993, Feder and Filchak 1999, Rossi et al.

1999, Tabuchi and Amano 2003a, Price 2005). In sympatric speciation, via host shifts, host races provide an intermediate step between panmictic populations with little or no reproductive isolation versus species with complete reproductive isolation (Jaenike 1981,

Tauber and Tauber 1989, Via 1990, Via 2001, Berlocher and Feder 2002, Drès and

Mallet 2002).

The biological species concept defines a species as a group of individuals that are capable of interbreeding, producing viable, fertile offspring and are reproductively isolated from other populations (Mayr 1963). Cryptic species are morphologically similar and reproductively isolated from other species but, often remain hidden and as such they are incorrectly classified into one species (Bickford et al. 2007). Sibling species are morphologically similar, reproductively isolated from other species, and are each other‟s closest relatives (Bickford et al. 2007).

Several definitions have been proposed for host races. The most frequently used is

Diehl and Bush (1984: 472) where a host race is defined as a “population of a species that is partially reproductively isolated from other conspecific populations as a direct consequence of adaptation to a specific host”. Drès and Mallet (2002: 474) have recently defined a host race as “genetically differentiated, sympatric populations of parasites that use different hosts, and between which there is appreciable gene flow”. Two well-known examples of host races are Rhagoletis pomonella on hawthorn, Crataegus spp., and on

apple, Malus pumila (Bush 1969, Feder and Filchak 1999); and the stem-gall-inducing tephritid Eurosta solidaginis on the goldenrods Solidago altissima and S. gigantea (Craig et al. 1993, Craig et al. 2000). Here, mating takes place on hosts and as such exacerbates assortative mating (see below).

Determining whether a phytophagous insect is monophagous or polyphagous is important, as many insects can be polyphagous, feeding on a variety of plant species over their geographical range, but can be mono- or oligophagous at the community level (Fox and Morrow 1981). In agriculture, whether an insect pest species is polyphagous and expanding its host range or is a monophagous and undergoing a host shift is important as this will have implications for how the pest is monitored and managed in each situation.

1.3 Reproductive Isolating Mechanisms

Phytophagous insect populations specializing on different host plants with divergent ecologies can become reproductively isolated through a variety of prezygotic isolating mechanisms (including habitat isolation, temporal isolation and behavioural isolation) and postzygotic isolating mechanisms such as host-related fitness trade-offs (Diehl and

Bush 1984, Berlocher and Feder 2002, Funk et al. 2002). Some or all of the above mechanisms can contribute to the reproductive isolation necessary for host race formation and speciation in phytophagous insects.

The first mechanism of prezygotic isolation is habitat isolation that results when mating occurs on the host plant, as is the case with many phytophagous insects (Diehl and Bush 1984, Craig et al. 1994, Drès and Mallet 2002, Funk et al. 2002). When mating occurs on the host-plant, an insect‟s host-plant preference will indirectly select for assortative mating, reducing gene flow between the host-plant populations (Bush 1969,

Diehl and Bush 1984, Craig et al. 1994, Caillaud and Via 2000, Drès and Mallet 2002).

Strong host-plant fidelity can also reduce gene flow between host-plant populations because insects are more likely to associate and mate with individuals of the same host race (Craig et al. 1993, Funk et al. 2002).

A second mechanism of prezygotic isolation is temporal isolation resulting from insect populations adapting to host plants with different phenologies. Phytophagous insect development is often dependent on particular plant organs such as young vegetative shoots, flowers and developing fruit, requiring an insect to time emergence and oviposition in synchrony with these stages (Yukawa 2000, Yukawa and Akimoto

2006). Gall midges are particularly sensitive to host plant phenology because the short- lived adults have limited time to find suitable oviposition sites and because larvae must complete their development within the gall within a narrow phenological window

(Yukawa 2000). Differences in timing of emergence, maturation and mating between insect populations on host plants with different phenologies can result in reproductive isolation that facilitates host-race formation and speciation (Wood and Keese 1990, Craig et al. 1993, Feder and Filchak 1999, Groman and Pellmyr 2000, Funk et al. 2002, Cooley et al. 2003, Tabuchi and Amano 2003a). For example, apple fruit, M. pumila, matures three to four weeks earlier than hawthorn fruit, Crataegus spp. resulting in R. pomonella host-race populations that are partially temporally isolated (Bush 1969, Feder et al. 1993,

Feder and Filchak 1999). The gall midge, Asteralobia sasakii, on Ilex crenata and I. integra has adapted to differing leafing phenology on the two Ilex hosts resulting in complete temporal isolation and potentially the formation of incipient species (Tabuchi and Amano 2003a, Tabuchi and Amano 2003b). In the Enchenopa binotata sibling-

species complex, timing of egg hatch is regulated by spring sap flow in the host plants black walnut, Juglans nigra, butternut, J. cinerea, blackhaw, Viburnum prunifolium, hoptree, Ptelea trifoliata, black locust, Robinia pseudoacacia, bitter sweet, Celastrus scandens, and red bud, Cercis canadensis, each differing in phenology, leading to divergent life histories on the seven host plants (Wood 1980, Wood and Keese 1990).

A third mechanism of prezygotic isolation that can result in assortative mating and reproductive isolation is sexual-behaviour isolation resulting from the use of different host plants (Funk et al. 2002). Pheromones play an important role in insect communication and changes in pheromone composition can result in reproductive isolation and diversification of phytophagous insects (Cardé 1986, Tauber and Tauber

1989, Hardie and Minks 1999, Ayasse et al. 2001). Most examples of host plants affecting sex pheromone production come from the order Lepidoptera because many are agricultural pests and because larvae of some species sequester plant chemicals for use as sex pheromone in the adult stage (reviewed in Landolt and Phillips 1997, Reddy and

Guerrero 2004). The male Arctiid moth, Utetheisa ornatrix, on Crotalaria spp. uses host plant pyrollizidine alkaloids acquired during larval feeding as precursors for their courtship pheromone (Conner et al. 1981). Pheromones in Scolytid bark beetles are also derived from host plant chemicals (Byers 1981, 1982). The lack of attraction between populations of the midge, A. sasakii, on host plants I. crenata and I. integra has been attributed to a change in sex pheromone (Tabuchi and Amano 2003a).

Postzygotic isolating mechanisms such as host-related fitness trade-offs can favour assortative mating and reproductive isolation between host-plant populations. In E. solidaginis, the stem-gall-tephritid fly found on S. gigantea and S. altissima, host races

preferentially oviposited on their natal hosts where survival was highest (Craig et al.

1997). Survival of hybrids was lower than survival of pure host-races on natal and on non-natal hosts (Craig et al. 1997). The gall midge, Asphondylia borrichiae, on

Borrichia frutescens, Iva imbricata and I. frutescens, experienced decreased fitness across its hosts (Rossi et al. 1999). Galls on B. frutescens were larger and less crowded, yielding heavier pupae that eclosed as larger adults with more eggs.

1.4 Dasineura oxycoccana Biology

Dasineura oxycoccana (Johnson) (Diptera: Cecidomyiidae) is a gall-inducing midge found in North America on cranberry, Vaccinium macrocarpon, highbush blueberry, V. corymbosum, lowbush blueberry, V. angustifolium and rabbiteye blueberry, V. ashei

(Gagné 1989, Lyrene and Payne 1992, Sampson et al. 2002, Sarzynski and Liburd 2003,

Dernisky et al. 2005, Mahr 2005, Yang 2005). In British Columbia, several wild

Vaccinium species could also host D. oxycoccana including alaskan blueberry, V. alaskaense, oval-leaved blueberry, V. ovalifolium, dwarf blueberry, V. caespitosum, bog blueberry, V. uliginosum, bog cranberry, Oxycoccus oxycoccos, black huckleberry, V. membranaceum, red huckleberry, V. parvifolium, and evergreen huckleberry, V. ovatum

(Pojar and MacKinnon 2004). Dasineura oxycoccana is called cranberry tipworm and blueberry gall midge on cranberry and blueberry, respectively. On highbush blueberry in

British Columbia, Canada, D. oxycoccana was first recorded in 1991 (Fitzpatrick, personal communication). In British Columbia, many cranberry and blueberry farms are in close proximity to one another; despite this D. oxycoccana was not recorded on cranberry until 1998 (Fitzpatrick, personal communication). Dasineura oxycoccana is now widely distributed on both Vaccinium crops in British Columbia.

Dasineura oxycoccana overwinters in the soil in both cranberry and blueberry and ecloses in early spring (Gagné 1989). Adults are approximately 2.0 mm long, short-lived and weak fliers. Females are easily identified by their orange, pointed abdomen.

Following mating, female D. oxycoccana oviposit between growing vegetative or floral bud scales. The oblong eggs are 0.35 mm long and translucent orange, often with a red pigmented spot (Mahr 2005).

Eggs hatch and larvae develop through three instars that change from clear to milky white to orange as they mature. Mature larvae are 1.5 to 2.0 mm long, eyeless and legless (Mahr 2005). In both cranberry and blueberry, D. oxycoccana larvae kill shoots by piercing through meristematic tissue to feed on plant juices. In cranberry, galled shoots become cup-like while in blueberry, galled shoots become curled and puckered.

In both cranberry and blueberry, shoot death results in secondary or side branching of stems below the gall. In cranberry, shoots galled late in the growing season are thought to result in crop loss as shoots may not have enough time to form flower buds for the following year (Mahr 2005). In British Columbia, highbush blueberry bushes attacked by

D. oxycoccana can be difficult to train for machine-harvesting due to the numerous side branches formed. In Florida, both floral and vegetative rabbiteye blueberry buds are galled resulting in significant crop losses and economic damage (Dernisky et al. 2005).

Following maturation, larvae form pupae 2.0 mm long (Mahr 2005). In cranberry, pupation occurs in the shoots between generations. In rabbiteye blueberry, V. ashei, larvae have been observed pupating in the soil between generations (Dernisky et al.

2005). In highbush blueberry, the site of pupation between generations is unreported.

Determining the number of generations is difficult because generations often overlap and vary depending on the geographic region and crop type. In cranberry, there are three to five generations in Wisconsin (Dittl and Kummer 1997, Mahr 2005) and four to five in

Massachusetts (Averill and Sylvia 1998). In rabbiteye blueberry, V. ashei, multiple generations have been recorded (Sampson et al. 2002). Typically each generation lasts between two to four weeks depending on temperature and day length (Averill and Sylvia

1998, Sampson et al. 2002).

Dasineura oxycoccana is difficult to treat with contact insecticides because larvae develop protected within the galled cranberry and blueberry shoots. In British Columbia, registered insecticides such as the organophosphate diazinon are applied to cranberry fields to kill adult D. oxycoccana, but there are no insecticides registered against D. oxycoccana in highbush blueberry. In cranberry, growers spread 13 to 19 mm of sand onto cranberry beds to target overwintering populations by making spring emergence from the soil difficult for the fragile adults (Mahr 1991). Natural enemies of tipworm in cranberry and blueberry include species within the families Syrphidae, Eulophidae and

Proctotrupidae (Voss 1996, Sampson et al. 2002, Sampson et al. 2006).

1.5 Cranberry and Blueberry Cultivation

1.5.1 Cranberry

Cranberry, V. macrocarpon (Aiton), cultivation occurs in climatically suitable areas of western, central and eastern North America in the Canadian provinces British Columbia,

Ontario, Quebec, Nova Scotia, New Brunswick, Prince Edward Island and the American states Washington, Oregon, Wisconsin, Maine, Massachusetts, Michigan and New

Jersey. Cranberry cultivation in British Columbia began in the mid-1940‟s (Fitzpatrick,

personal communication). In British Columbia, cranberry beds (fields) range in size from a few ha to more than 20 ha. British Columbia currently leads Canada in cranberry production (Statistics Canada 2011). Cranberry is British Columbia‟s second-largest berry crop with a value of $49 million from 2500 ha in 2010 (Statistics Canada 2011).

Cranberry is a low, creeping, woody perennial that spreads by vine-like vegetative runners (Eck 1990). Mature runners produce short vertical branches, or uprights, on which flowering and fruiting occur (Eck 1990). Cranberry cultivation requires moist, organic and acidic soils (pH 4.0-5.5) (Eck 1990). In British Columbia, cranberry bloom begins in mid to late June. Commercial honeybees and wild bees, particularly bumblebees, are responsible for pollination. Berries develop during July and August, and are harvested from late September to late November by flooding the fields and machine harvesting or by dry harvesting.

1.5.2 Highbush blueberry

Highbush blueberry, V. corymbosum (Linnaeus), cultivation occurs in climatically suitable areas of western, central and eastern North America in the Canadian provinces

British Columbia and Ontario, and the American states Washington, Oregon, Michigan,

New Jersey and Georgia. Eastern Canada primarily grows lowbush or wild blueberry.

British Columbia leads Canada in highbush blueberry production (US Highbush

Blueberry Council 2010). Blueberries have been grown in British Columbia at least as long as cranberries (Fitzpatrick, personal communication). Blueberry is British

Columbia‟s largest berry crop with a value of $91 million in 2010 from 7600 ha

(Statistics Canada 2011).

Highbush blueberry is a perennial shrub or bush ranging from 1-3 m tall. Flower buds occur in clusters at the ends of branches while vegetative buds form below flower buds closer to the bush or on separate branches (Eck 1966). Highbush blueberry typically has several “flushes” of vegetative shoots in a growing season. Blueberry cultivation requires acidic, organic soils (Eck 1966). In British Columbia, highbush blueberry blooms from late April to late May, depending on the cultivar. Commercial honeybees, mason bees and wild bees, particularly bumblebees, are used for pollination.

Berries develop through June to August and are harvested from July through to

September depending on the cultivar.

Cranberry and highbush blueberry differ considerably in biology and architecture. As

D. oxycoccana midge populations adapt to differing selective pressures on cranberry and highbush blueberry such as host plant phenology, shoot architecture and biochemistry, differences in these two crops could select for different behaviours such as changes in midge phenology, courtship and pupation sites resulting in assortative mating and partially or completely reproductively isolated midge populations on cranberry and on highbush blueberry.

1.6 Research Focus

The initial objective of my thesis was to determine whether D. oxycoccana was moving from highbush blueberry onto cranberry. While examining this, my research focus evolved to how the different host-plant biologies of cranberry and highbush blueberry may have influenced the behaviour of D. oxycoccana. The ultimate objective of my thesis was to determine whether D. oxycoccana populations on cranberry and highbush blueberry in British Columbia represent a single species, host races or sibling species. I

focused on two prezygotic isolating mechanisms: temporal isolation and behavioural isolation. I asked whether these mechanisms were generating ecological divergence and reproductive isolation in D. oxycoccana populations on the two crops.

In chapter 2, I focus on the prezygotic isolating mechanism of temporal isolation. I hypothesized that D. oxycoccana on highbush blueberry and on cranberry are two host races due to the differing host-plant phenologies of cranberry and blueberry; particularly since highbush blueberry blooms several weeks earlier than cranberry. I collected phenological data from three cranberry and three highbush blueberry farms and phenological data on all life stages of D. oxycoccana throughout the growing season.

This allowed me to assess the potential for gene flow between cranberry and highbush blueberry populations by determining whether D. oxycoccana populations were active at the same time on the two crops.

In chapter 3, I focus on the prezygotic isolating mechanism of behavioural isolation.

Given the behavioural differences observed in chapter 2, I hypothesized that D. oxycoccana adults from cranberry would discriminate against D. oxycoccana adults from highbush blueberry, and vice versa. I conducted mating experiments between D. oxycoccana individuals from cranberry and highbush blueberry. Mating experiments were conducted without host plants present. Four combinations of virgin females (F) and males (M) were tested: C(cranberry)F + CM, B(blueberry)F + BM, CF + BM, and BF +

CM. I recorded whether mating occurred and mating behaviors during each mating combination. I expected that D. oxycoccana populations on cranberry and highbush blueberry would be partially reproductively isolated, i.e., that some discrimination against

mates from non-natal hosts would occur and that host plant presence would be necessary for discrimination to occur.

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2: Phenology of Dasineura oxycoccana (Diptera: Cecidomyiidae) on cranberry and blueberry indicates potential for gene flow

2.1 Abstract

Dasineura oxycoccana (Johnson) (Diptera: Cecidomyiidae) is a pest of both cranberry,

Vaccinium macrocarpon (Aiton) (Ericales: Ericaceae), and highbush blueberry, V. corymbosum (Linnaeus) (Ericales: Ericaceae), in British Columbia. Dasineura oxycoccana was first found on highbush blueberry and then on cranberry approximately ten years later. Since many cranberry and highbush blueberry farms are adjacent to one another, we hypothesized that D. oxycoccana was moving from highbush blueberry onto cranberry. Cranberry and highbush blueberry differ in phenology, and adaptation to these different phenologies may result in host races and cryptic species on these two crops. We recorded the phenology of D. oxycoccana and the development of shoots from three cranberry and three highbush blueberry farms to determine whether the opportunity exists for successful movement between the two crops. Our results show that D. oxycoccana from cranberry and highbush blueberry overlap in phenology for much of the season, indicating a high potential for movement and gene flow. However, important behavioural differences were observed indicating the potential for host race and cryptic species formation on these two crops. More recent data from mating experiments show that D. oxycoccana from cranberry and highbush blueberry represent cryptic species.

2.2 Introduction

Dasineura oxycoccana is a multivoltine, gall-inducing midge found in North America on cranberry, Vaccinium macrocarpon (Gagné 1989, Mahr 2005), highbush blueberry, V. corymbosum (Gagné 1989, Sampson et al. 2002, Yang 2005), rabbiteye blueberry, V. ashei (Lyrene and Payne 1992, Sarzynski and Liburd 2003) and lowbush blueberry, V. angustifolium (Dernisky et al. 2005). Dasineura oxycoccana on cranberry is commonly called cranberry tipworm; on blueberry, it is called blueberry gall midge (Fitzpatrick

2009). Female D. oxycoccana oviposit between growing vegetative or floral bud scales.

Larvae develop through three instars and feed by piercing the plant tissue; the plant responds by forming a gall that eventually kills the shoot. Galled shoots protect the developing larvae from pesticides and predators. In British Columbia, Canada, highbush blueberry bushes attacked by D. oxycoccana form numerous side branches, which make machine-harvesting difficult. In Florida, D. oxycoccana attacks both floral and vegetative rabbiteye blueberry buds, causing significant economic damage (Dernisky et al. 2005). Cranberry shoots infested late in the growing season are unlikely to form flower buds for the following year, resulting in crop loss (Mahr 2005).

In southwestern British Columbia, D. oxycoccana was first recorded from highbush blueberry in 1991, but experienced blueberry growers had seen the characteristic damage for years before the first record. Although many highbush blueberry fields are adjacent to cranberry fields, D. oxycoccana was not seen on cranberry in British Columbia until

1998. During the 1990‟s, cranberry and blueberry growers in British Columbia were expanding production, often importing plant material from Vaccinium-growing regions in other parts of North America. By the early 2000‟s, D. oxycoccana was widespread on

highbush blueberry and cranberry in southwestern British Columbia. We hypothesized that D. oxycoccana was moving from highbush blueberry to cranberry.

Quantifying movement of this tiny insect is difficult, therefore we chose to record and compare its phenology on highbush blueberry and nearby cranberry. We tracked potential oviposition and development sites throughout the season and asked whether the opportunity exists for successful movement of midges between the crops.

Synchronization with host-plant phenology is crucial for gall-inducing midges, because the adults are short-lived and have limited time to locate oviposition sites, and because larvae are confined to the gall for the duration of their development (Yukawa 2000).

We expected to find that D. oxycoccana on highbush blueberry and D. oxycoccana on cranberry are two host races, i.e., two populations that are partially reproductively isolated from each other as a direct consequence of adaptation to their specific hosts

(definition from Diehl and Bush 1984). We expected that D. oxycoccana on highbush blueberry would become reproductively active in synchrony with blueberry plants, which begin to bloom in April, and that D. oxycoccana on cranberry would become reproductively active several weeks later, when cranberry shoots begin to grow and prepare to bloom. Our study is the first step in determining whether D. oxycoccana populations on cranberry and blueberry in British Columbia are partially or completely reproductively isolated.

2.3 Materials and Methods

2.3.1 Field sites.

Three cranberry and three blueberry farms with a history of D. oxycoccana infestation were selected as field sites for this study. All six sites were located in the vicinity of Pitt

Meadows city, British Columbia, Canada (49o13‟15” N, 122o41‟25”W) (Fig. 1). The cranberry cultivar Stevens was grown on all three cranberry farms (CB1, CB2 and CB3).

Blueberry farm one (BB1) grew Bluecrop, which bears fruit in mid-season. Blueberry farm two (BB2) grew Duke, an early season cultivar, and blueberry farm three (BB3) grew the late season cultivar Elliott. These three blueberry cultivars were selected because the time when they produced fruit covered different phenological windows. All cranberry farms and BB1 applied at least one insecticide to the crop during the study period. The date and type of insecticide was recorded, and re-entry intervals to the farm were respected.

2.3.2 Sampling D. oxycoccana eggs, larvae and pupae.

From April until mid-August 2009, samples of cranberry and blueberry shoots were collected weekly from each farm (unless insecticide re-entry regulations prohibited entry). All farms were sampled on the same day. The number of shoots collected was the maximum number that could be collected on one day and processed within 48 h.

From CB1, CB2 and CB3, 60 upright shoots (approx. 50% flowering and 50% vegetative) were collected per farm per week. Cranberry shoots were clipped from vines between the sprinkler rows, at the rate of one tip every 10 m. From BB1, 30 shoots were collected per week from 14 April to 9 June, then 60 were collected per week from 16

June to 11 August; from BB2, 40 shoots were collected per week; from BB3 20 shoots were collected per week from 14 April to 9 June, then 30 per week from 16 June to 11

August. Blueberry shoots were collected by walking between the rows and randomly selecting several bushes from each row, then clipping one shoot from each bush. At all sites, the most phenologically advanced shoots were selected because they were most likely to host a life stage of D. oxycoccana. As the season progressed, cranberry or blueberry shoots hosting later instars were easily seen when sampling so, to increase the probability of collecting eggs and early instars, we collected galled (= visibly infested) and ungalled shoots. In blueberry, the proportion of flowering to vegetative shoots changed through the season. Prior to 19 May, flowering and vegetative shoots were collected but, by 19 May, all blueberry shoots collected were vegetative because flowering shoots were no longer available. By June, only galled blueberry shoots were available. Shoots from all six sites were placed in plastic bags in a cooler within minutes of collection, and kept cool for up to 48 h until they could be examined. Shoots were rigorously examined through dissecting microscopes (25X magnification) to count eggs, first, second and third instars, and pupae. To generate voucher specimens, third instars from blueberry and pupae from cranberry were reared to adult in the laboratory. Voucher specimens were deposited in the Canadian National Collection of Insects (Ottawa, ON,

Canada).

2.3.3 Phenology of cranberry and blueberry shoots.

The phenology of each collected shoot was recorded prior to dissection in the laboratory.

Cranberry shoots were categorized into eight developmental stages: tight bud, bud swell, cabbagehead, bud break, bud elongation, roughneck, hook, and bloom (Workmaster et al.

1997). When shoots reached the stage of bud elongation, they could be clearly identified in the field as flowering or vegetative. We continued recording the phenology of flowering cranberry shoots with four additional categories used by cranberry growers: petal drop, pinhead-sized fruit, pea-sized fruit and grape-sized fruit. For blueberry, the earliest shoots were categorized into three developmental stages: tight bud, bud swell, bud break. Flowering blueberry shoots were further categorized into six developmental stages: tight cluster, early pink bud, late pink bud, early bloom, full bloom and petal fall

(Michigan State University 2003). Vegetative shoots were further categorized into early green tip, late green tip and shoot expansion. We continued recording blueberry phenology based on fruiting stage: green fruit, fruit coloring, 25% blue and 75% blue

(Michigan State University 2003).

2.3.4 Sampling D. oxycoccana adults.

To detect adult D. oxycoccana in cranberry, sticky traps were placed on CB1. Eight clear

Plexiglas traps (30 cm x 30 cm) thinly coated on one side with Tangle-Trap® were placed on the western margin of the field at two heights from the ground: 20 cm (vine height) and 122 cm. Four traps of each height faced east and the other four faced west to account for changes in wind direction through the day. Because some IPM consultants use yellow sticky cards to detect D. oxycoccana adults, four sticky yellow cards were placed at vine height to compare effectiveness to clear Plexiglas traps. Four clear

Plexiglas traps were placed on BB1 at mid-bush height (119 cm) for three weeks. Traps were collected and refreshed with new Plexiglas or yellow cards each week. Traps were examined under dissecting microscopes (25X magnification) to count D. oxycoccana adults.

2.3.5 Temperature recording.

To calculate degree days accumulated between eggs and subsequent stages, two

HOBOTM temperature loggers were placed on each farm. One logger was placed at soil level and the other at vine height (20 cm) on cranberry farms and at below mid-bush height (approx.75 cm) on blueberry farms. The temperature data from BB2 were used for

BB3 given the close proximity of the fields. Styrofoam discs placed above the temperature sensors prevented direct sunlight from heating the probes and causing inaccurate temperature readings. Temperature was recorded every hour. We used the

University of California Integrated Pest Management online degree day calculator

(2007). For degree day calculation we used the minimum and maximum vine (for cranberry) or mid-bush (for blueberry) temperatures within a 24h period and the single- sine curve calculation method with no upper cutoff. The developmental threshold of D. oxycoccana from egg to larva is currently not known. Roubos and Liburb (2010) recently calculated the developmental threshold for pupation of D. oxycoccana in soil at

8.9°C. Degree day modelling has been studied for the blackcurrant leaf midge, D. tetensi, and a developmental threshold of 7.0°C was calculated for post-diapausing larvae in the soil (Hellqvist 2001). The pod gall midge, D. brassicae, developmental threshold in winter rape shoots was calculated at 6.7°C (Axelsen 1992). We chose to use the developmental threshold of 6.7°C to calculate degree days for D. oxycoccana stages in both cranberry and blueberry because the developmental stages being examined occurred in the shoots.

2.3.6 Statistical analysis.

To evaluate seasonal temporal overlap between D. oxycoccana populations on cranberry

(CB1) and on blueberry (BB1) we used Feder et al. (1993) formula:

∑( )

where CBi is the proportion of the total cranberry population present on collecting date i and BBi is the blueberry population. We used the egg stage in this comparison because adults were not collected from blueberry, and because the number of eggs present should correlate positively with the number of ovipositing females.

Galled and ungalled shoot data from cranberry and blueberry were analyzed using

Chi-square tests. Comparison of life stages present in cranberry and blueberry shoots were analyzed using student t-test. Statistical tests were done in JMP 8.0 statistical software (SAS Institute, Cary, NC, USA).

2.4 Results

Crop phenological stages on each farm are shown in Fig. 2. Blueberries on all three farms had vegetative shoots in the shoot expansion stage from 21 April until sampling ceased in mid-August. There were differences due to cultivar in the timing of fruit development, particularly on BB3 where fruit colouring and development were delayed and prolonged. On cranberry, the progression of stages from tight bud through roughneck, hook, bloom and fruit sizing occurred at the same rate on each farm. On all cranberry farms, from 28 July onward, the terminal buds of many sampled shoots were forming or “setting” for the next year. Fruiting buds appeared pointed while vegetative buds appeared round and rosette-like.

Dasineura oxycoccana eggs were first collected in blueberry in expanding vegetative shoots when flowering shoots reached early to late pink stage on 28 April at BB1 and on

12 May at BB2 and BB3 (Fig. 3: Eggs). At BB1, expanding vegetative shoots were available for oviposition by 21 April, but the pymetrozine spray applied on 20 April for aphids may have delayed the first appearance of eggs by killing adult D. oxycoccana.

Eggs were not laid in flowering blueberry shoots. On all three cranberry farms, eggs were first collected on 19 May in uprights at the roughneck stage (Fig. 3: Eggs).

Diazinon was sprayed on CB1 on 28 May, 12 June and 10 July; on CB2 on 13 June and

21 July; and on CB3 on 29 May, 17 and 24 July. A carbaryl spray was applied to CB3 on

28 July. No insecticide sprays were recorded for BB2 and BB3. Due to the protective structure of the gall, it is unlikely that these contact insecticide treatments influenced the larval stages present within the shoots. However, insecticide sprays in cranberry fields may have reduced the number of adults and subsequently the number of eggs. The mean number of eggs per shoot in cranberry and blueberry increased over the season and through the stages of fruit development (Fig. 3 compare with Fig. 2). The majority of eggs in blueberry and cranberry were laid in July.

First instar D. oxycoccana were first collected from BB1 on 19 May, BB2 on 26 May and BB3 on 12 May, corresponding with early to full bloom (Fig. 3: 1st instars). First instars were first observed on 19 May on CB1 and CB3 and 26 May on CB2, when uprights were in roughneck and hook stages (Fig. 3: 1st instars). On all cranberry farms and on BB2 and BB3, the mean number of first instars per shoot increased throughout the season. Second instars were first collected on 19 May from BB1 and BB3 during early to full bloom and 2 June from BB2 during petal fall (Fig. 3: 2nd instars). Second instars

were first collected from all cranberry farms on 26 May when uprights were primarily in roughneck or hook stage (Fig. 3: 2nd instars). The mean number of second instars per shoot on all blueberry and cranberry farms increased as the season progressed. Third instars were first collected on 19 May from BB3, on 26 May from BB1 and 2 June from

BB2 during early to full bloom, full bloom and petal fall, respectively (Fig. 3: 3rd instars).

Third instars were first collected on 26 May from CB1 and on 2 June from CB2 and CB3 during hook stage (Fig. 3: 3rd instars). During fruit development, the mean numbers of third instars per shoot increased on blueberry, while on cranberry, shoot infestation levels remained at approx. two third instars per shoot. Pupae were first collected from all cranberry farms on 2 June when uprights were at the hook stage (Fig. 3: Pupae). Pupae were not found in any of the 1,890 collected blueberry shoots. Pupae were expected to be present in blueberry shoots by 2 June because D. oxycoccana on blueberry were ahead several weeks compared to cranberry. Pupae were found in cranberry shoots during fruit development. On blueberry and cranberry, D. oxycoccana life stages appear to be structured but overlapping generations. Eggs and larvae collected from cranberry and blueberry appeared similar in size, shape and colour, but morphometric data were not collected.

Adult D. oxycoccana were first collected on 5 May on low traps, 12 May on yellow cards and 9 June on high traps (Fig. 4). High traps and yellow cards were discontinued after 30 June due to low trap counts. The few adults trapped on BB1 appeared morphologically similar to those trapped on CB1. Diazinon sprays on CB1 on 28 May,

12 June and 10 July temporarily decreased trap counts the week following each spray.

Despite diazinon sprays, the mean number of adults per trap increased until 4 August.

Because two stages were often found for the first time on the same date (e.g., eggs and first instars on CB1; first and second instars on BB2), it was not possible to calculate degree days for each stage. Degree days accumulated from first egg to first pupa on cranberry were 124.4 on CB1, 143.3 on CB2 and 149.7 on CB3. On blueberry, degree days accumulated from first egg to first third instar were 165.4 on BB1 and 172.9 on

BB2. On BB3, all stages were found for the first time between 12 and 19 May, so degree days were not calculated. On cranberry, mean degree-day accumulation between first egg and first pupae was significantly less than mean degree-day accumulation between first egg and first third instar on blueberry (t = 3.5, df = 2.8, P = 0.04). On blueberry, additional degree days would be needed to complete development of pupae in the soil.

Our data suggest that D. oxycoccana requires more degree days to develop from egg to pupa on highbush blueberry than on cranberry.

To calculate the percentage of seasonal temporal overlap (Feder et al. 1993) between cranberry and blueberry populations of D. oxycoccana, we used the proportion of the total egg population on blueberry (BB1) and on cranberry (CB1) (Fig. 3: Eggs). The seasonal temporal overlap of the two populations of eggs (and, therefore, of ovipositing females) was 47.7%.

Early in the growing season, all shoots collected from the six farms were uninfested, i.e., contained no D. oxycoccana life stages (Table 1). On the three cranberry farms, the percentage of uninfested shoots decreased to approx. 40% in mid-June then increased to approx. 65% in late July and early August. On the blueberry farms, the percentage of uninfested shoots decreased until mid-June, then increased briefly before steadily decreasing until mid-August. There are two major differences between the cranberry and

blueberry farms that might account for the season patterns of uninfested shoots. Each cranberry farm received at least two insecticide applications targeting D. oxycoccana, whereas only one blueberry farm received one insecticide application in April. Blueberry plants produced vegetative shoots throughout the growing season and these shoots were used by D. oxycoccana. Cranberry plants, however, began in mid-July 2009 to initiate bud formation for 2010; these buds stopped growing and were not used by D. oxycoccana. These differences in farm management and plant growth habit would impose selection pressure on D. oxycoccana.

Our sampling strategy of collecting galled and ungalled shoots from each crop revealed that ungalled shoots were likely to contain eggs and first instars, while galled shoots were likely to contain second instars (especially in blueberry), third instars and, in cranberry, pupae (Fig. 5: BB Chi-square test, χ2 =911.30, df=3, P<0.0001 and Fig. 6: CB

Chi-square test, χ2 =1214.06, df=4, P<0.0001).

Shoots infested with D. oxycoccana often contained more than one life stage per shoot (Fig. 3). Cranberry shoots were about four-fold smaller than blueberry shoots, and contained approx. half as many D. oxycoccana individuals as blueberry shoots (Table 2:

Total). Blueberry shoots sometimes contained clusters of eggs (up to 41), and cranberry shoots typically contained single eggs, but the mean number of eggs per infested shoot did not differ (Table 2). The range and mean number of first instars in cranberry and blueberry shoots did not differ, but blueberry shoots contained more second and third instars than did cranberry shoots (Table 2). Cranberry shoots usually contained one or two pupae (rarely three or five), whereas blueberry shoots did not contain pupae (Table

2).

2.5 Discussion

Reproductive isolation between insect host-plant populations can result from differential host synchronization. Allochronic life histories in emergence, maturation, and mating between the host-plant populations resulting from differing host-plant phenologies can reduce gene flow to facilitate host race formation and speciation (Wood

1980, Wood and Keese 1990, Craig et al. 1993, Feder and Filchak 1999, Groman and

Pellmyr 2000, Funk et al. 2002, Cooley et al. 2003, Tabuchi and Amano 2003a, Tabuchi and Amano 2003b). Dasineura oxycoccana on blueberry and cranberry were initially temporally separated by three weeks. Egg congruence data showed moderate overlap in eggs from BB1 and CB1. Despite this, populations on both cranberry and blueberry overlapped throughout much of the season. While we only collected data on adults from cranberry, where sharing of gametes between the cranberry and blueberry populations would be potentially occurring, our data of eggs, larvae and pupae overlap between BB1 and CB1, indicating a high potential for movement and gene flow between D. oxycoccana populations on cranberry and blueberry. Intra-crop variation in insect phenology, particularly between blueberry cultivars, further decreases the chance of assortative mating arising between blueberry and cranberry populations. This limited temporal separation would likely not contribute to the reproductive isolation necessary for host race formation if it is occurring on cranberry and blueberry.

Dasineura oxycoccana oviposition on cranberry continued to increase throughout the season and into fruit set, which contrasts with reports from Wisconsin where oviposition decreased and ended shortly after flowering (Cockfield and Mahr 1994). Continued

oviposition in British Columbia could be attributed to there being more overgrowth and more vegetative than flowering uprights in British Columbia compared to Wisconsin.

More accumulated degree-days between first egg and first third were required on blueberry compared to cranberry first egg and first pupae. Since pupation occurs in the soil for blueberry, we could not calculate degree days for this stage. However, Roubos and Liburd (2010) found that 134 accumulated degree days were required for pupation in the soil for D. oxycoccana in rabbiteye blueberry, V. virgatum. From this we can infer that D. oxycoccana on highbush blueberry would require additional accumulated degree days to develop from egg to pupae. Differences in degree days required for insect development have been found in other crops. For example, the silverleaf whitefly,

Bemisia argentifolii, required more degree days to develop on cotton, Gossypium hirsutum, compared to cantaloupe, Cucumis melo (Nava-Camberos et al. 2001).

Differences in the development rate and distribution of eggs of D. oxycoccana on cranberry and blueberry may be due to nutritional and chemical defensive differences between the two crops and not temperature alone. Host plant quality is known to influence larval development rate, fecundity and distribution of eggs on host plants in phytophagous insects (Scriber and Slansky 1981, Rossi et al. 1999, Tikkanen et al. 2000,

Awmack and Leather 2002). For example the herbivorous ladybird, Epilachna pustulosa, had shorter development time on blue cohosh, Caulophyllum robustum compared to thistle, Cirsium kamtschaticum. The gall midge, Asphondylia borrichiae, on Borrichia frutescens, Iva imbricata and I. frutescens yielded adults from B. frutescens with more eggs compared to I. imbricata and I. frutescens (Rossi et al. 1999). Significant differences in development time and number of eggs oviposited were observed in

whitefly, Bemisia argentifolii, populations on eggplant, Solanum melongena, tomato,

Lycopersicon esculentum, sweet potato, Ipomoea batatus, cucumber, Cucumis sativus, and garden bean, Phaseolus vulgaris (Tsai and Wang 1996). Differences in egg distribution could also be attributed to blueberry shoots being four times larger than cranberry shoots.

Differences in development rate between the blueberry cultivars could also be due to nutritional and chemical defensive differences. Differences in phenolic glycoside levels between Quaking aspen, Populus tremuloides, tree clones accounted for differences in growth, survival and development in gypsy moth, Lymantria dispar, and forest tent caterpillars, Malacosoma disstria, populations (Hwang and Lindroth 1997).

Dasineura oxycoccana were not collected from blueberry floral buds in British

Columbia. In Florida, however, D. oxycoccana attacks both vegetative and floral buds of rabbiteye blueberry beginning in early January and February, resulting in significant economic losses (Sampson et al. 2002, Dernisky et al. 2005). In British Columbia, differences in the timing of highbush blueberry phenology and shorter growing season may limit D. oxycoccana from attacking floral buds, decreasing its economic impact on

British Columbia‟s highbush blueberry industry.

Dasineura oxycoccana immatures were not collected from cranberry shoots that had initiated bud formation for the following year. Dasineura oxycoccana infesting shoots later on in the season is thought to decrease flower bud formation for the following year; a concern expressed by many cranberry growers. While we still do not know whether this is the case, once bud formation has been initiated, D. oxycoccana does not infest these shoots.

Pupae were not collected from blueberry shoots and therefore it is inferred that pupation occurs in the soil between generations throughout the season, as is thought to occur in Florida (Dernisky et al. 2005). Pupae in both cranberry and blueberry overwinter in the soil. Differences in shoot architecture may explain why D. oxycoccana do not pupate in blueberry shoots. In cranberry, each leaf becomes progressively more galled as the insect matures, so a fully mature third instar and subsequent pupa can fit snuggly in an individually galled leaf. However, in blueberry, shoots are approximately four times larger than cranberry. Several effects can be observed as a result of the change in shoot size: 1) galled leaves become curled and puckered; 2) each shoot can support more larvae; and 3) multiple larval stages are often present; all of which could make spinning a silken cocoon quite difficult. Dasineura oxycoccana develops within galled shoots in both cranberry and blueberry protected from contact insecticides. Recognizing that pupation occurs in the soil in blueberry may help to manage this pest by providing an additional stage to target outside of the gall, aside from adults.

Intraspecific competition within blueberry and cranberry shoots may occur. In cranberry shoots, there was a trend towards a reduction in the mean number of D. oxycoccana per shoot as larvae hatched and matured. Experiments using Eurosta solidaginis (Diptera: Tephritidae) on Solidago altissima also found a reduction in the number of surviving larvae which they attribute to intraspecific competition (Craig et al.

2000). In blueberry, multiple larval stages and crowding were observed, especially later in the season when over 80% of shoots collected were infested with eggs and/ or larvae, and fewer uninfested shoots were available in the field (M. A. Cook, personal observation). In blueberry, eggs were frequently oviposited in large clusters within each

shoot, versus singly in cranberry, which could further contribute to intraspecific competition in blueberry. The sessile nature of the larval stages of gall-inducing insects and being confined to their gall may make gall-inducing insects more prone to intraspecific competition. Intraspecific competition could also result in cannibalism within the crowded blueberry and cranberry shoots.

Reduced interspecific competition associated with ancestral and novel host plants can help to offset fitness costs associated with host shifting. Insects adapting to a new host may initially be poorly adapted but fitness costs can be offset due to decreased interspecific competition on the new under-utilized resource (Tauber and Tauber 1989,

Craig et al. 1994, Berlocher and Feder 2002). Strong intraspecific competition in E. solidaginis on S. altissima and reduced interspecific competition on S. gigantea compared to S. altissima are hypothesized to have facilitated a host shift from S. altissima to S. gigantea (Craig et al. 2000). Differences in intra- and interspecific competition may have helped to facilitate a host shift of D. oxycoccana from a common ancestral

Vaccinium onto blueberry and cranberry.

Our data suggest that D. oxycoccana populations on cranberry and blueberry overlap in insect phenology for much of the season and the potential for unrestricted gene flow between insect populations on these two crops. However, important behavioural differences were observed suggesting the potential for host race or cryptic species formation. More recent data from mating experiments show that D. oxycoccana represent cryptic species (Cook et al. in press). Future research on D. oxycoccana should include reciprocal transplant experiments to test for fitness differences between natal and non-natal host plants and testing additional Vaccinium species as potential hosts.

2.6 Acknowledgements

We thank Gerhard Gries (Simon Fraser University) and the Roitberg lab for their helpful suggestions and comments, Daniel A. H. Peach (Simon Fraser University) for help collecting the larvae used in this study and processing samples, Sneh Mathur

(Agriculture and Agri-Food Canada), Ringa Kurniawan (University of British Columbia) for help processing samples, Alberta Dagg (Grandma) for accommodations and the berry farms for access. Funding was provided by Natural Sciences and Engineering Research

Council (M. A. C.), Agriculture and Agri-Food Canada Growing Forward Initiative (S.

M. F.), B. C. Cranberry Marketing Commission and B. C. Cranberry Growers

Association (S. M. F.), Natural Sciences and Engineering Research Council of Canada

Discovery Grant (B. D. R).

2.7 Figures

Figure 1: Map of the six field sites, Pitt Meadows, British Columbia. BB= blueberry,

Vaccinium corymbosum. CB= cranberry, V. macrocarpon.

Figure 2: Crop phenology from three blueberry and three cranberry fields throughout

the season. BB= blueberry, Vaccinium corymbosum, and CB= cranberry, V.

macrocarpon.

Figure 3: Mean number of Dasineura oxycoccana stage per shoot collected each week

throughout the season from two host plants. BB= blueberry, Vaccinium

corymbosum, and CB= cranberry, V. macrocarpon. n=18 sampling dates.

Figure 4: Mean number of Dasineura oxycoccana adults per trap collected from low

traps (vine height), high traps (122 cm from ground) and yellow cards (vine

height) each week throughout the season from cranberry, Vaccinium

macrocarpon (CB1). n=18 sampling dates.

Figure 5: Total number of Dasineura oxycoccana stages collected from galled and

ungalled blueberry, Vaccinium corymbosum, shoots from BB1, BB2, and

BB3. χ2 =911.30, df=3, p<0.0001.

Figure 6: Total number of Dasineura oxycoccana stages collected from galled and

ungalled cranberry, Vaccinium macrocarpon, shoots from CB1, CB2, and

CB3. χ2 =1214.06, df=4, p<0.0001.

2.8 Tables

Table 1: Percentage of shoots that did not contain Dasineura oxycoccana stages.

Shoots were collected from highbush blueberry, Vaccinium corymbosum,

farms BB1, BB2, and BB3; and cranberry, V. macrocarpon, farms CB1,

CB2, and CB3. Farm CB2 was not sampled on July 21.

Cranberry Blueberry Mean ± Mean ± Date SEM SEM Apr. 15 100 ± 0 100 ± 0 Apr. 21 100 ± 0 100 ± 0 Apr. 28 100 ± 0 98.9 ± 1.1 May 5 100 ± 0 98.9 ± 1.1 May 12 100 ± 0 95.6 ± 0.6 May 19 90.0 ± 6.7 86.7 ± 3.3 May 26 86.1 ± 1.5 64.2 ± 27.1 June 2 66.7 ± 2.9 43.3 ± 12.0 June 9 66.1 ± 12.6 54.7 ± 8.9 June 16 37.2 ± 8.7 34.3 ± 7.3 June 23 39.4 ± 7.5 65.0 ± 2.5 June 30 47.8 ± 2.9 70.0 ± 7.7 July 7 46.7 ± 1.7 56.7 ± 6.7 July 14 54.4 ± 7.8 24.4 ± 8.9 July 21 75.8 ± 9.2 16.1 ± 9.6 July 28 65.6 ± 5.6 11.1 ± 6.7 Aug. 4 65.0 ± 0 30.0 ± 23.2 Aug. 11 67.2 ± 2.3 18.3 ± 8.5

Table 2: Comparison of Dasineura oxycoccana life stages in 272 cranberry and 346

blueberry shoots that contained at least one egg, larva or pupa. Shoots were

collected in 2009 from BB1 and CB1.

Cranberry Blueberry Life stage Mean ± SE Range Mean ± SE Range t P df Egg 1.1 ± 0.1 0 - 12 1.3 ± 0.2 0 - 41 1.07 0.29 520 1st Instar 1.0 ± 0.1 0 - 19 0.9 ± 0.1 0 - 14 0.95 0.34 520 2nd Instar 0.7 ± 0.1 0 - 14 3.0 ± 0.3 0 - 27 8.18 <0.001 461 3rd Instar 0.4 ± 0.1 0 - 5 0.9 ± 0.1 0 - 20 3.37 <0.001 453 Pupa 0.4 ± 0.04 0 - 5 0.0 ------Total 3.6 ± 0.2 1 - 23 6.1 ± 0.4 1 - 46 5.74 <0.001 527

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3: Host-associated differentiation in reproductive behaviour of cecidomyiid midges on cranberry and blueberry

A previous version of this chapter is in press in Entomologia Experimentalis et

Applicata

Melissa A. Cook, Sasha N. Ozeroff, Sheila M. Fitzpatrick and Bernard D.

Roitberg

3.1 Abstract

In British Columbia, Canada, Dasineura oxycoccana Johnson (Diptera: Cecidomyiidae) was initially found on highbush blueberry, Vaccinium corymbosum L. (Ericaceae) and has recently become a pest of cranberry, Vaccinium macrocarpon Aiton, a crop which is often found in close proximity with blueberry. Previous work has shown no temporal isolation and a potential for gene flow between these two D. oxycoccana populations.

However, important behavioural differences were observed suggesting the potential for host races or cryptic species. Host races and cryptic species differ in their degree of assortative mating and reproductive isolation from partial to complete. We assessed whether populations of adult D. oxycoccana on these two crops would discriminate against mates from different natal hosts. Mating experiments were conducted within the

greenhouse in 2010 using small glass vials without host plants present. Our results show

D. oxycoccana from cranberry and blueberry hosts displayed complete assortative mating in the absence of their host plants. Behavioural data collected from the different crosses suggest these two D. oxycoccana populations differ in sex pheromones and close range

„courtship pheromones‟. We conclude that D. oxycoccana populations on cranberry and blueberry in British Columbia are reproductively isolated and probably represent cryptic species.

Key words: gall midge, host race, assortative mating, host shift, cryptic species,

Dasineura oxycoccana, Vaccinium corymbosum, Vaccinium macrocarpon, Diptera,

Cecidomyiidae, Ericaceae

3.2 Introduction

Phytophagous gall-inducing insects are known to undergo host shifts which can result in host races and cryptic species (Akimoto, 1990; Craig et al., 1993, 1994; Tabuchi &

Amano, 2003; Price, 2005; Stireman III et al., 2005). Host races are the intermediate step in the sympatric speciation process and have been the focus of many articles over the last several decades (Bush, 1969; Jaenike, 1981; Diehl & Bush, 1984, 1989; Futuyma, 1986;

Tauber & Tauber, 1989; Via, 2001; Berlocher & Feder, 2003). Diehl & Bush (1984) define a host race as a „population of a species that is partially reproductively isolated from other conspecific populations as a direct consequence of adaptation to a specific host‟. Two well-known examples of species that have host races are apple maggot,

Rhagoletis pomonella Downes (Bush, 1969; Feder & Filchak, 1999), and Eurosta solidaginis Fitch (Craig et al., 1993, 2000). Examples of sibling species include the

Enchenopa binotata (Say) tree hopper species complex (Wood & Guttman, 1983), and R.

pomenella and Rhagoletis mendax Curran (Bierbaum & Bush, 1990).

Several mechanisms of pre- and post-zygotic isolation can generate ecological divergence and reproductive isolation needed for host race formation in phytophagous insects. During host shifts, reduction in gene flow can be achieved when mating occurs on the host plant, as is the case with many phytophagous insects (Bush, 1969; Caillaud &

Via, 2000; Abrahamson et al., 2001; Drès & Mallet, 2002). Strong host fidelity can also result in assortative mating and reduced gene flow between the host races (Craig et al.,

2001; Funk et al., 2002). As such, adaptation to different host plant phenologies can result in temporal isolation and assortative mating (Wood & Keese, 1990; Feder &

Filchak, 1999; Tabuchi & Amano, 2003). Gall inducing insects, in particular midges, need to be synchronized with host plant phenology because adults are short lived and the larvae are confined to the gall until emergence as adults (Yukawa, 2000). Using different host plants may also change mating behaviour via pheromone production (Tauber &

Tauber, 1989; Landolt & Phillips, 1997; Tabuchi & Amano, 2003; Reddy and Guerrero,

2004). Decreased performance on a non-natal host plant and lower hybrid fitness on natal and non-natal host plants will favour assortative mating between host plant populations (Craig et al., 1997; Rossi et al. 1999; Filchak et al., 2000).

Dasineura oxycoccana Johnson (Diptera: Cecidomyiidae) is a multivoltine, gall- inducing midge found in North America on cranberry, Vaccinium macrocarpon Aiton

(Ericaceae) (Gagné, 1989; Mahr, 2005), highbush blueberry, Vaccinium corymbosum L.

(Gagné, 1989; Sampson et al., 2002; Yang, 2005), rabbiteye blueberry, Vaccinium ashei

Rehd., and lowbush blueberry, Vaccinium angustifolium Aiton (Lyrene & Payne, 1992;

Sampson et al., 2002; Sarzynski & Liburd, 2003; Dernisky et al., 2005; Yang, 2005). In

British Columbia, Canada, D. oxycoccana was first recorded on highbush blueberry in

1991. Despite the proximity of cranberry and blueberry fields in British Columbia, D. oxycoccana was not observed on cranberry until 1998. Dasineura oxycoccana is now widely distributed on both Vaccinium crops in British Columbia.

Female D. oxycoccana oviposit between growing vegetative or floral bud scales, and larvae develop through three instars protected within the galled shoot. In both cranberry and blueberry, D. oxycoccana larvae kill shoots by piercing through meristematic tissue to feed on plant juices. Galled cranberry shoots become cup-like, whereas galled blueberry shoots become curled and puckered. On cranberry, D. oxycoccana is called cranberry tipworm; on blueberry, it is called blueberry gall midge (Fitzpatrick, 2009).

Dasineura oxycoccana from cranberry and from blueberry appear similar in size, shape, and colour at all life stages, but there are differences in placement of eggs and location of pupae. Eggs are generally laid singly on cranberry but in large clusters on blueberry (MA

Cook, unpubl.). During the growing season, pupae are found in the shoots of cranberry but never on blueberry shoots; it is inferred that pupation in blueberry fields occurs in the soil (MA Cook, unpubl.).

Cranberry and highbush blueberry differ considerably in plant architecture and biology. Cranberry is a low-growing vinelike woody perennial that spreads by vegetative runners that produce short vertical branches on which flowering and fruiting occur (Eck,

1990). Highbush blueberry is a woody perennial shrub or bush of 1-3 m tall. Blueberry flower buds occur at the ends of branches; vegetative buds are formed below flower buds or on separate branches (Eck, 1966). In British Columbia, highbush blueberry flowers several weeks before cranberry. These and other differences in growth habit could select

for different life histories and/or behaviours such as changes in midge phenology, courtship, and pupation sites resulting in assortative mating among midge populations on each host plant. Phenological data in 2009 and 2010 showed that, at the beginning of the growing season, D. oxycoccana populations on cranberry and blueberry were separated by 3 weeks (MA Cook, unpubl.). However, subsequent generations of this midge were active at the same time on cranberry and blueberry, indicating potential for unrestricted gene flow between midges on the two crops (MA Cook, unpubl.).

Dasineura oxycoccana on cranberry and highbush blueberry in British Columbia may represent a single undifferentiated species because phenological overlap and a lack of temporal isolation have been observed. Given the differences in egg placement and location of pupae, midges from the two hosts may represent two host races or cryptic species.

The purpose of the present study was to test the hypothesis that evolution in this species in British Columbia has reached the point where D. oxycoccana adults now discriminate against mates from different natal hosts, i.e., that D. oxycoccana from cranberry would discriminate against D. oxycoccana from blueberry, and vice versa. We tested whether midges from each population recognize and are attracted to each other without host plants present. We chose to test without host plants present as this would be the initial step in determining whether assortative mating was occurring and whether this could be ascribed to host race or cryptic species formation. We define assortative mating as cranberry midges disproportionately mating with cranberry midges, and blueberry midges disproportionately mating with blueberry midges. In our experiments without host plants, a single undifferentiated species would be indicated by a lack of mate

preference between populations from cranberry and blueberry, two host races would be indicated by weak assortative mating, and two separate species would be indicated by complete assortative mating (per Craig et al., 1993).

3.3 Materials and methods

3.3.1 Insect collection

From May through June, 2010, shoots infested with D. oxycoccana were collected once per week from one blueberry and two cranberry farms in Pitt Meadows, British

Columbia, Canada (49°13‟15”N, 122°41‟25”W). The blueberry cultivar was Bluecrop and the cranberry cultivar was Stevens. Two hundred infested shoots from both blueberry and cranberry were collected weekly (total 400 per week). Vegetative blueberry shoots were placed individually into scintillation vials with autoclaved, composted bark mulch that provided substrate for pupation; vials were capped with a fine mesh. Cranberry shoots were placed individually into scintillation vials filled with distilled water and sealed with Parafilm®. The upper part of the shoot was covered with an inverted scintillation vial and sealed onto the vial by Parafilm®. Rearing D. oxycoccana in individual vials helped to ensure that adult D. oxycoccana emerged singly and were not exposed to potential mates before the experiment. Infested blueberry and cranberry shoots were placed in separate growth chambers at 70% r.h. and L16(21

°C):D8(18 °C). Lights came on at 09:00 hours and went off at 01:00 hours. Voucher specimens were submitted to the Canadian National Collection (CNC) of Insects,

Agriculture and Agri-Food Canada, Ottawa, ON, Canada.

3.3.2 Test of mating between and within cranberry and blueberry populations of Dasineura oxycoccana

Mating experiments were conducted in the greenhouse under natural light and humidity conditions during May and June 2010. Emergence of D. oxycoccana was checked twice per day for both cranberry and blueberry. Time of emergence, sex, and number of D. oxycoccana were recorded for each vial. Vials that yielded both males and females were excluded from mating experiments. If multiple, same-sex individuals emerged from any one vial, only one of those adults was used. Earlier work showed that female calling

(pheromone-release behaviour, indicating female receptivity) normally occurs between 1-

6 hours into photophase (SM Fitzpatrick, unpubl.). Therefore, mating experiments were conducted between 10:30 and 14:30 hours in May and June, using 1- and 2-day-old males and females.

Mating experiments were run in small glass vials (7 × 4 cm). One end of the vial was sealed with fine mesh and the other end with a mesh-covered lid, to aid in removing insects. Mesh on both ends of the mating jars helped to reduce putative pheromone build up by facilitating airflow through the vial. Insects were added to the mating vial through a hole plugged with a removable cork on the side of container. Each mating vial also contained a water-soaked, wooden landing platform in the shape of a „fork‟ attached to the lid. After each replicate of the experiment, mating vials were washed with

SparkleenTM and placed in a drying oven. New wooden landing platforms were added to each clean vial.

Mating experiments began by guiding two virgin females through the uncorked hole into the vial. Once the females had settled, two virgin males were added. Two individuals of each sex were used to decrease the chance of selecting pairs that were not

compatible. Four combinations of females (F) and males (M) were tested: C(cranberry)F

+ CM, B(blueberry)F + BM, CF + BM, and BF+ CM. At least 20 replicates of each combination were conducted. An additional 11 replicates of CF + CM and one replicate of BF + BM were included. The additional replicates were intended to generate data on oviposition but, due to difficulties synchronizing the development of Vaccinium shoots with the mating experiments, oviposition data were not collected.

For each replicate, the insects were observed for 1 h. We recorded time until first mating, time in copula, and the following behaviours: calling (extended ovipositor), waving of ovipositor, male-to-female contacting then pushing away from each other, and male flight. As female D. oxycoccana are monandrous, only mating once, copulation was considered successful if the female‟s ovipositor remained retracted for more than 15 min (Voss, 1996).

3.3.3 Statistical analysis

Mating and eclosion data were analyzed using χ2 test. Time until mating for the various cross types and time in copula were analyzed using a Wilcoxon test as data were left and right censored and left censored, respectively. Statistical tests were done in JMP 8.0.2

(SAS Institute, Cary, NC, USA).

3.4 Results

Dasineura oxycoccana adults discriminated against mates from different natal hosts. All crosses between females and males from the same host (CF + CM and BF + BM) mated, while females and males from different hosts (CF + BM and BF + CM) did not mate with each other (χ2 = 105.92, d.f. = 3, P<0.0001; Figure 7). Time until mating for cranberry

pairs was more variable than that for blueberry pairs (cranberry: Coefficient of Variation

= 141.99, blueberry: Coefficient of Variation = 119.37; Figure 8). Time until mating was longer between D. oxycoccana from cranberry than between D. oxycoccana from blueberry (Wilcoxon test, outliers included: Z = –4.151, P<0.0001; outliers removed: Z =

–3.757, P = 0.0002; Table 3). Time in copula did not differ (Wilcoxon test, outliers included: Z = –0.0661, P = 0.95; outliers removed: Z = –0.147, P = 0.88; Table 3).

In all four combinations, females remained almost motionless on the wall of the glass vial with ovipositors in apparent pheromone-release posture. Blueberry and cranberry females were observed waving their ovipositors. Males spent most of their time in flight seeking females. Most behaviours were observed without magnification; however, if insects were close together, a 2.75× magnifier (Donegan OptiVisor®; Lenexa, KS, USA) was used.

During the combinations CF + CM and BF + BM, we observed males repeatedly attempting to mate with females until mating was achieved. A single male was capable of mating with each female in the vial. Once mated, females remained motionless and displayed avoidance behaviours such as walking away from the male, pushing away from the male and moving to a different location in the vial, and dislodging the male if mating was attempted again.

During the combinations CF + BM and BF + CM, only two mating attempts were observed. In both instances, the female‟s ovipositor was extended immediately after the mating attempt and remained extended until the end of the replicate. When a male approached (within a few mm) or contacted a calling female, the female retracted her ovipositor. Males on approach to a calling female were observed abruptly changing

direction and walking away from the female. Males and females would push away from each other to another location within the vial once contact was made. Males and calling females were also observed clinging to the inside of the glass vial, side-by-side, without making contact.

More newly eclosed male D. oxycoccana were found in the morning, and more females were found in the afternoon, in both blueberry and cranberry populations (χ2 =

352.55 and 127.98, respectively, both d.f. = 1, P<0.0001; Figure 9).

3.5 Discussion

Determining whether a phytophagous insect is a specialist or a generalist is important because many insects can be generalists feeding on a variety of related plant species over their geographical range, but yet can be specialists at the community level (Fox &

Morrow, 1981). Gall-inducing insects, at the species level, are generally viewed as specialist feeders on one host plant (Shorthouse et al., 2005). Specialization is attributed to the intimate relationship between the plant and insect that is needed for gall induction.

Among the gall-inducing midges (Cecidomyiidae) some are able to feed on closely related plants and even fewer are able to feed on plants in different families (Gagné,

1989). Within the genus Dasineura, several species, including D. affinis Kieffer, D. brassicae Winnertz, and D. dielsi Rübsaamen, are each known to induce galls on seven or more hosts within genera Viola, Brassica, and Acacia, respectively (Åhman, 1985;

Birch et al., 1992; Post et al., 2010). In agriculture, whether an insect pest species is a generalist and expanding its host range or is a specialist and undergoing a host shift is important because these two different situations will require different monitoring protocols. Here, we showed that rather than evolving into a generalist that could utilize

two hosts, the gall-inducing midge D. oxycoccana has diverged into two specialist species on two congeneric host plants.

Dasineura oxycoccana adults from cranberry and blueberry hosts displayed complete assortative mating in the absence of their host plants. The mating behaviour observed in

D. oxycoccana on both cranberry and blueberry is characteristic of Dasineura species.

Female D. folliculi Felt, D. mali Kieffer, and D. carbonaria Felt all remain motionless on the sides of their mating cages and exhibit the „calling‟ behaviour seen in D. oxycoccana

(Dorchin et al., 2007; Suckling et al., 2007). Males of these species actively sought females and were capable of mating multiple times. The mean time in copula for D. oxycoccana was similar to that for D. mali (Suckling et al., 2007). When mated D. oxycoccana females were given access to natal host shoots of the appropriate developmental stage, oviposition occurred, indicating that copulation had been successful

(Cook, 2011).

On cranberry and on blueberry, males emerge in the morning and females in the afternoon, similar to D. folliculi on Solidago gigantea Aiton and Solidago rugosa Aiton and D. affinis on Viola spp. (Birch et al., 1992; Dorchin et al., 2007). In the context of reproductive isolation, males are present at the same time on cranberry and blueberry and are available to mate with females from either host plant population should the opportunity occur.

Assortative mating in host races is often strong in the presence of their respective host plants but very weak in their absence. Weak assortative mating in the absence of host plants in E. solidaginis allows gene flow and prevents complete reproductive isolation

(Craig et al., 1993). In D. oxycoccana assortative mating occurred in the absence of

cranberry and blueberry plants, which suggests that reproductive isolation is complete.

Note, however, that host plant presence was not required for mating between individuals from the same natal plants thus the absence of mating between cranberry and blueberry individuals cannot be attributed to the experimental protocol.

The general lack of attraction between D. oxycoccana individuals from cranberry and blueberry suggests that host shifting from a common ancestral Vaccinium host onto cranberry and blueberry has altered the sex pheromone(s) produced. Host plants affect sex pheromone production in Lepidoptera when larvae sequester plant chemicals as precursors for sex pheromone use by adults (Landolt & Phillips, 1997; Reddy &

Guerrero, 2004). Male larvae of the Arctiid moth, Utetheisa ornatrix L., sequester pyrollizidine alkaloids from their host plants Crotalaria spp. to derive their courtship pheromone (Conner et al., 1981). Scolytid bark beetles are also known to use host plant chemicals sequestered during feeding as precursors for pheromones (Byers 1981, 1982).

Tabuchi & Amano (2003) suggested that a change in sex pheromone due to adaptation to different host plants may explain the lack of attraction between Asteralobia sasakii

Monzen midge populations on Ilex crenata Thunberg and Ilex integra Thunberg. There is some suggestion that the pheromone may be different between D. oxycoccana on cranberry and on blueberry (GJ Gries, pers. comm.).

In arthropods, both females and males are known to produce close-range „courtship pheromones‟ that are used in mate selection and to stimulate copulation (Ayasse et al.,

2001). Several species of Drosophila use cuticular hydrocarbons as pheromones for species and mate recognition and to stimulate copulation (Cobb & Jallon, 1990; Blows &

Allan, 1998). Several observations point towards D. oxycoccana from cranberry and

blueberry differing in close-range sex pheromones as well as possible differences in close-range auditory, vibrational, and visual cues. Males approaching calling females from non-natal host plants were rejected by females. Females were observed walking away, pushing males away and moving to another location in the vial, and retracting their ovipositors when males from non-natal plants approached. Males were observed walking towards calling females and then abruptly changing direction.

Results from the mating experiments lead us to conclude that D. oxycoccana populations on cranberry and blueberry in British Columbia are reproductively isolated and represent cryptic species. This conclusion is further supported by recent genetic work, which found 9.0-10.6% divergence between a 780-bp region of the mitochondrial cytochrome oxidase I (COI) gene in D. oxycoccana from cranberry and from blueberry (S

Mathur, pers. comm.). These results imply that pest management of D. oxycoccana on blueberry should be independent of management on cranberry in British Columbia.

Several factors could result in strong divergent selection for reproductive isolation between D. oxycoccana populations on cranberry and blueberry such as architectural and biochemical differences and temporal separation, but research has shown that the latter does not contribute to reproductive isolation. Fitness differences between the two host plants may also contribute to strong assortative mating and reproductive isolation. Future research should include reciprocal transplant experiments to test for oviposition preferences as well as examining whether differences exist in gall induction or larval development between cranberry and blueberry. Testing native Vaccinium species for the presence of D. oxycoccana along with further genetic work may help to determine the direction of the host shift and new potential hosts. Ongoing research in this system

includes examining the parasitoids of D. oxycoccana on cranberry, testing for fitness differences, and elucidating pheromone differences between D. oxycoccana populations found on cranberry and blueberry.

3.6 Acknowledgements

We thank Dr. Gerhard J. Gries (Simon Fraser University), Kelly M. Ablard, Jeffrey B.

Joy, the Roitberg lab, and Sneh Mathur (Agriculture and Agri-Food Canada) for their helpful suggestions and comments, Daniel A. H. Peach (Simon Fraser University) for help collecting the larvae used in this study, Kiran Ranganathan and Jordan Scheu for help in experiments, and the berry farms for access. Funding was provided by Natural

Sciences and Engineering Research Council (MAC), Agriculture and Agri-Food Canada

Growing Forward Initiative (SMF), British Columbia Cranberry Marketing Commission and British Columbia Cranberry Growers Association (SMF), and Natural Sciences and

Engineering Research Council of Canada Discovery Grant (BDR).

3.7 Figures

Figure 7: Matings between Dasineura oxycoccana males (M) and females (F) collected

from two host plants, cranberry (C) and blueberry (B)

Figure 8: Time until mating between Dasineura oxycoccana males (M) and females (F)

collected from two host plants, cranberry (C) and blueberry (B), including

four C × C outliers

Figure 9: Proportion of eclosion of male (white bar) and female (gray bar) Dasineura

oxycoccana from field-collected shoots of cranberry (C) and blueberry (B)

3.8 Tables

Table 3: Mean (± SE) time (s) until mating and in copula between Dasineura

oxycoccana males (M) and females (F) collected from cranberry (C) and

blueberry (B)

Cross type n Time until mating Time in copula CF × CM 27 186.7 ± 31.1a 33.5 ± 3.6a BF × BM 21 40.0 ± 10.4b 30.7 ± 3.3a CF × BM 20 no mating - BF × CM 20 no mating -

Different letters following means within a column indicate significant differences

(Wilcoxon test: P = 0.0002)

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4: Conclusion: The Speciation of Dasineura oxycoccana

4.1 The Speciation of Dasineura oxycoccana

The initial question of whether D. oxycoccana from blueberry was moving onto cranberry evolved significantly to address the possibility of host race and sibling species formation on these two crops, a question with both evolutionary and pest management implications. Related to this question was whether D. oxycoccana was evolving into a generalist pest species that was expanding its host range or was it a specialist pest species undergoing a host shift and as such, specializing on blueberry and cranberry. To address these questions, I examined two prezygotic isolating mechanisms that can result in host race, cryptic species, and sibling species: temporal isolation and behavioural isolation.

In chapter two I reported that D. oxycoccana populations from blueberry and cranberry were not temporally isolated, thus I concluded that temporal isolation would not contribute to reproductive isolation between these two populations (if reproductive isolation existed). Dasineura oxycoccana requires more accumulated degree days to develop on blueberry compared to cranberry. Pupae were not found within blueberry shoots, unlike cranberry. Dasineura oxycoccana does not infest cranberry shoots that had initiated bud formation for the following year.

In chapter three, I reported that D. oxycoccana populations from blueberry and cranberry displayed complete assortative mating in the absence of their respective host plants. Behavioural data from the crosses indicate that blueberry and cranberry D. oxycoccana likely differ in sex pheromones and close range courtship pheromones.

To revisit the title of the thesis - Dasineura oxycoccana (Diptera: Cecidomyiidae) populations on cranberry and blueberry in British Columbia: same species, host races or sibling species? -we can now answer the question. They are cryptic species. While we were unable to develop a phylogeny to answer whether they represent sibling species, recent work examining the pheromone of cranberry and blueberry D. oxycoccana populations suggests that they likely represent sibling species (Gries, personal communication).

4.2 Applications

The specialization and cryptic species formation in D. oxycoccana on cranberry and blueberry in British Columbia highlights the importance of conducting behavioural studies to determine whether a pest species is generalist across its geographical range or is a specialist at the community level, potentially forming host races, cryptic species or sibling species (Fox and Morrow 1981). More research needs to be conducted in agricultural systems to look for specialization and host race, cryptic species or sibling species formation. I speculate that host race, cryptic species and sibling species formation in agricultural systems is probably a common occurrence.

One of the most important findings of this study is that pest management of this insect on cranberry should be independent of management on blueberry in British

Columbia. This result is particularly important for cranberry growers who apply insecticides to control this insect. Another important finding of this study is that D. oxycoccana was not able to induce galls in cranberry shoots that had initiated bud formation for the following year. Therefore flowering bud and thus fruit production would likely not be compromised for the following year. We cannot, however, rule out

the possibility of buds infested late in the season that have yet to initiate bud formation producing vegetative instead of flowering buds for the following year. Since pupation occurs in the soil for blueberry during the growing season and not in the shoots, like in cranberry, an additional stage is available to target outside of the gall, aside from adults, which may help in managing this pest on blueberry.

4.3 Suggestions for Future Research

Given how recently research on this system began to be studied compared to other systems such as R. pomonella on apple and hawthorn which has been studied for well over a century and several decades for the Enchenopa binotata tree hopper species complex, numerous questions still remain. For example, what was the direction of the host shift and when did it occur? Speculating about how the current pattern occurred on cranberry and blueberry in British Columbia, four possibilities exist: 1) D. oxycoccana on cranberry and blueberry are cryptic species originating from a common wild ancestral

Vaccinium host and were brought into British Columbia separately when each crop began to be cultivated; 2) D. oxycoccana individuals shifted hosts from blueberry to cranberry in British Columbia; 3) D. oxycoccana shifted from a wild Vaccinium host onto blueberry and then onto cranberry or 4) D. oxycoccana individuals shifted hosts from blueberry to cranberry elsewhere. Agricultural crops are unique in that there are often records about when crops were first cultivated as well as when particular insect species begin to infest these crops, as was the case with R. pomonella (Schneider and Roush 1986, Feder et al.

1993). In British Columbia, we can determine approximately when cranberry and blueberry were first infested by this insect. However, this may not reflect when this insect was first found on these two crops in North America. Genetic work comparing

British Columbia‟s D. oxycoccana populations to other populations throughout North

America could help to resolve these questions.

Related to determining the direction of the host shift would be testing other

Vacciniums for the presence of D. oxycoccana, or if D. oxycoccana is not present on these Vacciniums, conducting experiments to test whether successful gall induction is possible. Rhagoletis pomonella has been recorded on at least eleven species of native hawthorn, Crataegus (Berlocher and Enquist 1993). Species within the genus Dasineura are known to induce galls on closely related plant taxa (Axelsen 1992, Post et al. 2010,

Birch et al. 1992). I expect that D. oxycoccana is present on wild Vacciniums in British

Columbia. However, phenology and shoot morphology may limit which Vaccinium species can be infested.

Another possible area of research would be to examine whether D. oxycoccana from cranberry can induce galls on blueberry and vice versa. Such research might be conducted in the field by taking eggs and placing them on non-natal shoots in a no choice experiment. If gall induction is possible reciprocal transplant experiments could be conducted to test for fitness differences and whether behavioural differences are maintained between cranberry and blueberry plants. Behavioural differences could include whether blueberry D. oxycoccana continue to pupate in the soil when reared on cranberry and whether differences in egg placement are maintained on the opposite host plant. Fitness differences may contribute to the reproductive isolation present in these populations. I conducted oviposition experiments on natal hosts with limited success due to difficulties in synchronizing gravid females with shoots of appropriate phenological

stage needed for gall induction. Specialization on cranberry and blueberry may have progressed to the point that gall induction may not be possible on the opposite host plant.

The presence of cryptic species in British Columbia on cranberry and blueberry is also important as it raises the question as to whether cryptic species are present in other locations in North America where the two crops are grown in close proximity to each other. Researchers in other regions of North America should conduct mating experiments to determine whether their populations of D. oxycoccana are reproductively isolated on blueberry and cranberry.

4.4 List of References

Axelsen J. 1992. The developmental time of the pod gall midge, Dasyneura brassicae Winn. (Dipt., Cecidomyiidae). Journal of Applied Entomology 114:263-267.

Berlocher S. H., and M. Enquist. 1993. Distribution and host plants of the apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae) in Texas. Journal of the Kansas Entomological Society 66:51-59.

Birch M. L., J. W. Brewer, and O. Rohfritsch. 1992. Biology of Dasineura affinis (Cecidomyiidae) and influence of its gall on Viola odorata. Pages 171-184 In J. D. Shorthouse and O. O Rohfritsch, editors. Biology of insect-induced galls Oxford University Press, New York, USA.

Fox L. R., and P. A. Morrow. 1981. Specialization: species property or local phenomenon? Science 211:887-893.

Schneider J. C., and R. T. Roush. 1986. Genetic differences in oviposition preference between two populations of Heliothis virescens. Pages 163-171 In M. D. Huettel, editor. Evolutionary genetics of invertebrate behavior: progress and prospects, Plenum, New York.

5: Appendix

5.1 Additional Frequency Data

Data displaying the mean number of D. oxycoccana per shoot per stage was calculated for CB1 and BB1 in Chapter 2: Table 2. The remaining data for cranberry farms CB2 and CB3 (Figs. 10 and 11) and blueberry farms BB2 and BB3 (Figs. 12 and

13) are provided in this section as frequency graphs. Methodology is provided in chapter 2.

Figure 10: Frequency (%) of shoots containing Dasineura oxycoccana life stages from cranberry (CB2), Vaccinium macrocarpon, shoots.

Figure 11: Frequency (%) of shoots containing Dasineura oxycoccana life stages from cranberry (CB3), Vaccinium macrocarpon, shoots.

Figure 12: Frequency (%) of shoots containing Dasineura oxycoccana life stages from blueberry (BB2), Vaccinium corymbosum, shoots.

Figure 13: Frequency (%) of shoots containing Dasineura oxycoccana life stages from blueberry (BB3), Vaccinium corymbosum, shoots.

5.2 Oviposition Experiment

As part of the mating experiments from chapter 3, oviposition experiments were conducted to test the hypothesis of decreased performance of hybrids from CF + BM and

BF + CM crosses compared to CF + CM and BF + BM crosses. Mated females from all four mating combinations were to be placed on their natal host plant for oviposition.

However, since CF + BM, and BF + CM crosses did not mate, decreased hybrid performance could not be tested. Crosses of CF + CM and BF + BM were, however, used to test for successful mating.

5.2.1 Cranberry and blueberry cultivation

In early March 2010, eighty two-year-old blueberry bushes, variety Bluecrop, were purchased from JRT Nurseries Inc., Aldergrove, British Columbia and brought to

Simon Fraser University, Burnaby, British Columbia. To remove any potential overwintering pupae, the root system was cut back by JRT and most of the soil was removed. The roots of each bush were rinsed and lightly agitated in water to remove any extra soil prior to planting. Each bush was planted in an eight liter pot with autoclaved, composted bark mulch (pH 6) and placed outdoors, away from the greenhouse where experiments were conducted. Plants were fertilized weekly with a dilute 200ppm 20-20-

20 fertilizer. For the oviposition experiment each bush had several microperforated bags placed around its branches and sealed with parafilm to prevent infestation of the shoots by D. oxycoccana. All bags were later removed once they began to constrain vegetative shoot growth.

In early March, 2010, cranberry vine clippings, variety Stevens, were obtained from Cranwest Farm, Delta, British Columbia. Vines were moved to Agassiz, British

Columbia, and kept moist, outdoors, by sprinkler 24 h per day until potting. On March

10, uprights or shoots were potted by Dr. Fitzpatrick and staff at the Pacific Agri-Food

Research Center, in Agassiz, British Columbia. Healthy-looking uprights were trimmed from the vines, surface-sterilized in 1% bleach and rinsed twice in Reverse Osmosis water. Uprights were dipped in rooting hormone and then planted into tree plugs with autoclaved peat mixed with perlite (75% peat: 25% perlite). Plugs were sprinkled with water then covered with humidity domes and white plastic to maintain high relative humidity and limit direct sunlight, respectively. For one week, domes were removed daily every 30 min for gas exchange. Temperature in the greenhouse ranged from 20-

30°C and mercury vapour lights were on at 06:00 and off at 22:00. To maintain a high humidity (>70%) and prevent desiccation, uprights were misted hourly.

In April 2010, 300 cranberry plugs were returned to the SFU greenhouse. The greenhouse was kept at 20°C with natural lighting. Plants were misted every hour to maintain high humidity (>70%) and fertilized weekly with a dilute 200ppm 20-20-20 fertilizer. Groupings of four plugs were placed in microperforated bags and tied with twist-ties to prevent infestation of the shoots. Misting was reduced to three times per day as the bags tended to keep high humidity. In mid-May, cranberry vines were individually repotted in 10 cm pots with 75% peat: 25% perlite and a small amount of composted bark mulch on the top and bottom of the pot. Microperforated bags were placed around two pots and sealed. Plants were monitored weekly for aphids (Aphididae) fungus gnats

(Sciaroidea) and fireworm, Rhopobota naevana; pests were killed upon discovery.

5.2.2 Oviposition experiment

Plants were randomly selected and from that collection only the healthiest plants were used. The first female to successfully mate from each vial was removed and released into a fine mesh bag surrounding her natal host plant. Two mating combinations were tested: CF + CM and BF + BM. Females were given one shoot for oviposition; all other oviposition shoots were removed. For cranberry, the shoot was in roughneck stage or later, and for blueberry the shoot was in late vegetative expansion. Since cranberry shoots were kept in the greenhouse in the same location as the mating experiments and there were two pots in each microperforated bag, as an extra control one pot in the bag was kept as a control and the other reserved for the mated female. Since the blueberry bushes had their bags removed, each bush had two shoots selected on it one for the mated female and the other for the control. Each tip was checked for galling and dissected after one week. A total of 23 replicates of CF + CM and 21 replicates of BF + BM were conducted. Statistical analysis of the oviposition experiment was not conducted due to lack of oviposition in the shoots.

5.2.3 Results

Oviposition experiments yielded few galled shoots. Only six of 23 treatment cranberry plants were galled (Table 4). Two mated cranberry females died upon introduction. Thirteen mated blueberry females were introduced to blueberry shoots, but no galls were observed. Eight mated blueberry females could not be introduced as no blueberry oviposition shoots were available.

5.2.4 Conclusion

Oviposition experiments yielded some galled shoots, providing evidence of successful mating between individuals from natal host plants. A full complement of oviposition data could not be gathered because it was difficult to obtain shoots of the appropriate stage at a time that coincided with gravid D. oxycoccana females from the mating experiment. The galled shoots from the oviposition experiments resulted from females being presented with the correct stage that was needed for gall induction. In the field, D. oxycoccana females seeking oviposition sites are probably very sensitive to the developmental stage of the bud within the shoot.

Table 4: Oviposition results from matings between Dasineura oxycoccana males and females from cranberry, Vaccinium macrocarpon, on cranberry plants.

Cranberry gall (one gall per shoot) Insect #1 #2 #3 #4 #5 #6 Stage Egg 13 14 - - - -

1st instar 9 8 - - - -

2nd instar - - 4 19 2 3

3rd instar ------pupa ------

Total 22 22 4 19 2 3