California State University, Northridge Host-Specificity and Its Effect On

California State University, Northridge

Host-Specificity and its Effect on Mate Choice in a Plant-Eating Beetle

A thesis submitted in partial fulfillment of the requirements

For the degree of Master of Science in Biology

Katherine Gould

May 2014

The thesis of Katherine Gould is approved:

______Dr. David Gray Date

______Dr. James Hogue Date

______Dr. Paul Wilson, Committee Chair Date

California State University, Northridge

iii

Dedication

For Samantha and Jocelyn

Acknowledgements

First and most importantly, I would like to thank my advisor, Dr. Paul Wilson, for his advice, answers, encouragement, and occasional well-aimed prodding. Also instrumental in helping me through this process were my committee members, Dr. Dave

Gray and Dr. Jim Hogue, who were always available to answer questions and assist in whatever way I needed.

Thank you to Cindy Hitchcock, whose wonderful drawings of beetle mating illustrate this work, and whose beautiful watercolor of a mating pair of beetles concludes this work.

I could not have completed all the lab work necessary without the loyal and consistent help of my lab assistants. My "Minions" fed, watered, counted, and observed thousands of beetles over the summer of 2013. Thank you, thank you, thank you to

Victoria Amran, Elias Atri, Jamie Carrafa, Dona Cherian, Amanda Fitzpatrick, Liz

Hamel, Amaya Mendez-Molina, Alexus Merino, Joshua Muñoz, Lara Parsekhian, Lela

Remington, Joyce Theilig, and Dominique Zatarain.

Thanks also to my lab mates, Nickte Mendez, Lena Coleman-Ayala, and Dani

Amorosa, who were an invaluable resource and sounding board. To my parents, Phil and

Mary Ann Andrews, and my brother, Scott Andrews, who supported my decision to go back to school at age 38, thank you, thank you, thank you. And to my wonderful daughters, Samantha and Jocelyn Gould, thank you for not freaking out too much about beetles in the refrigerator, a car filled with the smell of Eriodictyon, and a mom who talks way too much about insects.

Table of Contents

Signature Page ...... iii

Dedication ...... iv

Acknowledgements ...... v

List of Tables and Figures...... vii

Abstract ...... viii

Introduction ...... 1

Materials and Methods ...... 8

Study Organisms ...... 8

Experiments ...... 14

Statistics ...... 19

Results ...... 25

Discussion ...... 43

References ...... 53

vii

List of Tables and Figures

Figure 1: Trirhabda eriodictyonis ...... 8

Figure 2: Distinguishing the sexes ...... 10

Figure 3: Trirhabda eriodictyonis mating...... 11

Figure 4: Leaves of Trirhabda eriodictyonis’ host plants ...... 12

Figure 5: Study site ...... 14

Figure 6: Choice feeding trials, larvae ...... 25

Figure 7: Larval growth and development ...... 26

Figure 8: Time to pupation ...... 28

Figure 9: Lifetime survival, host-plant switch as larvae ...... 29

Figure 10: Choice feeding trials, adults ...... 30

Figure 11: Adult survival, by sex ...... 31

Figure 12: Adult survival, by treatment ...... 32

Figure 13: Average date of death ...... 33

Figure 14: Mating preferences, without plant switch ...... 34

Figure 15: Mating behavior ...... 35

Figure 16: Mating preferences, with plant switch ...... 36

Figure 17: Potential fecundity ...... 37

Figure 18: Realized fecundity, by female treatment ...... 39

Figure 19: Realized fecundity, by mating treatment ...... 41

Figure 20: Realized fecundity, by mating treatment ...... 42

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Abstract

Host-Specificity and its Effect on Mate Choice in a Plant-Eating Beetle

A thesis by Katherine Gould

Master of Science in Biology

The beetle Trirhabda eriodictyonis lives on two shrubs with different plant defenses:

Eriodictyon crassifolium has hairy leaves; E. trichocalyx has sticky leaves. The relationship between these plants and the leaf-eating beetles that depend on them has been unstudied until now. In choice tests, larvae and adults showed unexpected feeding preferences, with larvae from E. crassifolium showing no preference and those from E. trichocalyx preferring E. crassifolium. Adults all strongly preferred eating E. trichocalyx.

Larvae and adults that I switched from E. trichocalyx to E. crassifolium died younger than beetles that I continued to feed the original host species. Mating trials showed that the only difference in preference involved males from E. trichocalyx, which were far more attractive to females on E. crassifolium than males on the same host. Finally, females laid more eggs if they ate E. trichocalyx than E. crassifolium, even if they had started life on the latter. It is clear that E. trichocalyx provides benefit to both males and females and these beetle populations are not differentiating based on host plants. Neither the differentiation hypothesis nor the preference-performance hypothesis are validated by this plant-insect interaction. Instead, it appears that the best explanation of this relationship is phylogenetic conservatism. The plant defenses, which appear dramatically different to humans, are unimportant to the beetles.

Introduction

Chrysomelid beetles lead a simple life. Birth to death, they live on and eat their host plant. Some beetles spend their whole life on a single individual plant. While great for the beetles, this lifestyle is problematic for the plants they feed on, and the conflict between plants and herbivores shapes both organisms. Plants have adapted to defend themselves against attackers, and herbivores have evolved ways around those defenses.

Relationships between plants and insect herbivores have been widely studied, yet new studies continue to reveal surprises and unexpected interactions. I investigated an unstudied relationship of two plant species with one insect herbivore, a specialized chrysomelid beetle, to determine how well this relationship is explained by prevailing theories of plant-insect interactions.

To understand the relationship between plants and their herbivorous adversaries, we must first appreciate the wealth of different plant defenses against herbivores that have evolved over eons. Some plants, many annuals for example, don’t grow when the insects that would eat them are around (Feeny 1976). Plants that grow all year often produce poisonous or bad-tasting chemicals, or grow spines, hairs, or tough tissues that make stems and leaves hard to eat (Feeny 1976, Rhoades 1979, Bottrell et al. 1998,

Jolivet 1998, Lucas et al. 2000). Some plants release chemicals that attract predators to eat the herbivores (Jolivet 1998). Plants frequently respond in real time to herbivory by increasing the energy they put toward defenses, making more chemicals or hairs to slow or harm their attackers (Jolivet 1998, Agrawal et al. 1999).

Occasionally, evolution takes a different tack. A plant that finds a way to put up with a certain level of herbivory may do better than one that invests in a costly defense.

Plants that exhibit this strategy tolerate herbivory rather than defend against it in real time. They grow thin inexpensive leaves that photosynthesize quickly before they are eaten, or produce so many leaves that they overwhelm the appetite of the herbivores

(Agrawal et al. 1999). This induced tolerance enables the plants to make a photosynthetic profit because they produce a lot of cheap photosynthetic machinery.

Of course, insects also have a variety of adaptive strategies for dealing with diverse plant defenses. Some insects ignore heavily defended plants and instead eat from a wide variety of lightly defended plants (Feeny 1976). Others have adapted their life cycles to coincide with the season when the plant is likely to be least defended (Feeny

1970). Still others have developed the ability to break down toxic chemicals, tolerate the flavor of noxious chemicals, navigate spines and hairs, and hide from predators (Levin

1973, Feeny 1976, Jolivet 1998). Some insects have developed the ability to sequester the plant’s chemicals in their bodies and use them for their own defense (Rhoades and Cates

1976, Jolivet 1998). And some remarkable insects get so good at detoxifying or using the plant’s chemicals that the chemicals that defend the plant against generalist herbivores become an attractant to specialized herbivores. The plant’s defenses stop saying, “Keep away,” and start saying, “Dinner is served” (Jolivet 1998).

This ability to thwart one plant’s defenses frequently comes at the expense of the ability to deal with the defenses of other plants. When this happens, the insects become specialized feeders, dependent on the few plant species that produce the chemicals to which they have become adapted (Gilbert 1971, Eigenbrode and Jetter 2002). This phenomenon is particularly common in the beetle family Chrysomelidae, in which numerous species become oligophagous, eating plants in only a few families or genera, or

2 even monophagous, feeding on just one genus or even one species (Cates 1980, Futuyma and Moreno 1988, Ananthakrishnan 1994, Rausher 1996). This appears to be the case with Trirhabda eriodictyonis, the beetle I studied, which feeds almost exclusively on

Eriodictyon crassifolium and E. trichocalyx.

Although these two plant species are very closely related, they appear markedly different to humans in their external defenses, and these defenses would seem to require different feeding strategies from the beetles. Eriodictyon crassifolium has leaves covered in a velvety layer of trichomes, and E. trichocalyx leaves are coated with a thick, sticky, resinous secretion. Studies of other organisms have found different ways that insects deal with distinctly different physical plant defenses, and this has led to several testable hypotheses predicting how natural selection will shape the relationship between herbivore and plant. My research was aimed at understanding the relationship between T. eriodictyonis and its host plant species, and determining which hypothesis best explains the development of this relationship.

Differentiation Hypothesis: The first hypothesis that seemed particularly well suited to this beetle and its host plants is the differentiation hypothesis, which predicts that the distinct defenses of the plants will require different feeding strategies by the beetles. Gradually, beetle populations will become adapted to one feeding strategy over the other and will thrive best on the plant where they hatched. They will mate preferentially with others on the same host plant, increasing the divide between populations feeding on the two host plant species.

The differentiation hypothesis is integral to Thompson’s (2005) theory of the geographic mosaic of coevolution, which argues that species are made up of populations of organisms experiencing slightly different environments. These various environments produce a geographic mosaic of populations that are evolving at different rates and in different ways because local populations experience adaptation to and coevolution with the species with which they interact. This is especially true of sessile organisms and those that have small ranges or short dispersal distances, such as my beetles. Because of their limited ability to move to a different environment, these organisms must constantly adapt to the species around them.

Thompson’s theory predicts that in a situation such as that of T. eriodictyonis, the plants and beetles will coevolve, producing better defended plants and more host-specific beetles. I expected to find that the beetles were adapting to the two different host plant species in ways that differentiated them into separate populations, even though the range of the host plant species overlap. Similar differentiation has been seen in the past, sometimes over very short evolutionary time spans, such as with pea aphids feeding on alfalfa and red clover. Over less than 400 years these aphids have diverged into ecotypes that mate assortatively based on host plant species and have low hybrid viability (Via

1999, Via et al. 2000).

Numerous researchers have argued that such differentiation can proceed all the way to speciation, even when host populations are sympatric, because the close dependence of the herbivore on the plant causes a barrier to gene flow between one herbivore species and another (Bush 1969, Rice and Salt 1990, Feder et al. 1994, Via

2001, Drès and Mallet 2002, Thompson 2005, Servedio et al. 2011, Caillaud & Via

2012). The system I researched seems to be, at first glance, the sort in which such a barrier could arise, creating ecotypes or even cryptic species out of a single population of specialized beetles.

To test the differentiation hypothesis, I conducted choice feeding trials and mating trials to see if feeding preferences existed and whether beetles favored the “natal” plant on which they hatched.

Preference-Performance Hypothesis: On the other hand, just because a beetle hatched on a certain plant does not mean that plant is the best possible environment for it.

The preference-performance hypothesis predicts that individuals will prefer to eat the plant on which they perform best: grow largest, live longest, have the best mating success, or lay the most eggs. For example, if larvae do better on one host, then females might prefer to live on that plant so their eggs can hatch in the best environment, even if adults perform better on a different plant. This hypothesis has been supported in numerous studies in other species (Gripenberg et al. 2010, Balagawi et al. 2013) and is particularly likely to be supported in species that have some capacity for dispersal (at least as adults) and live close enough to different hosts to be able to choose one over another. This is the case with T. eriodictyonis and its host plants.

If the preference-performance hypothesis applies, all beetles will have a similar preference for one plant over the other, and will experience longer lifespans and better mating success on that preferred plant. While the preference-performance hypothesis may at first seem obvious, it does not always hold true. The hypothesis assumes that organisms directly or indirectly choose food based on nutrition and that they eat the food

5 that provides the best nutrition and leads to the greatest growth and reproductive potential. However, host plant selection has also been shaped by the presence of predators, parasites, competition, and other environmental factors. If the most nutritious plant leaves a beetle susceptible to predators, the beetle would likely opt for a less- nutritious plant that offers better protection (Singer et al. 2004). In this study, I have not attempted to incorporate this third trophic level of interaction and have investigated only to the beetles’ preferences and performance as it relates to their host plant species.

The preference-performance hypothesis may not be wholly applicable in this system because the host plants have an overlapping range, but are not interspersed in most of their range. And although the beetles’ dispersal range has not been studied, I have not found them to be adventurous or able fliers. I find it more likely that any individual beetle lives its whole life on or close to the individual plant where it hatched.

A switch to a different host species may simply be outside the ability or experience of most beetles, so even if they were going to prefer a different host species, that option isn’t readily available to them. More likely, beetles continue feed on a plant that is not nutritionally superior simply because they hatched on a less nutritious plant. The natal plant may not be perfect, but it’s home, and it’s good enough. The preference- performance hypothesis predicts that given the choice, beetles will choose the more nutritious plant, but it does not insist they will. I tested this hypothesis with survival and fecundity analyses in addition to choice feeding trials and mating trials.

Phylogenetic Conservatism Hypothesis: The final hypothesis that may explain the relationship between Trirhabda eriodictyonis and its host plant species is phylogenetic

6 conservatism, which predicts that the relationship will be based more on the evolutionary history of the beetles and the plants than on recent adaptation. The ancestor to T. eriodictyonis and its sister species made a shift from members of the family Asteraceae, used by all other Trirhabda species, to Eriodictyon 5-7 million years ago (Swigonova &

Kjer 2004). The Eriodictyon species studied here likely developed their defenses prior the host switch by Trirhabda, although the radiation of Eriodictyon into five species is also very recent, plausibly during the same several million years (Ferguson 1998). No research has investigated the defensive chemicals in these plant species, beyond noting that they contain flavonoids, so the chemical differences between these E. crassifolium and E. trichocalyx are unknown.

If phylogeny is the most important aspect of this relationship, then beetles may not show different preference or performance on the two host plants. They may simply eat these plants because these are the Eriodictyon species in their environment. Having made the shift to Eriodictyon, the beetles may be able to survive just as well on any species in the genus. This would explain similarities between T. eriodictyonis and its close relative T. diducta. The latter is specialized on three Eriodictyon species that live in its environment, mountain ranges in central California, north of the range of T. eriodictyonis. In a sense, the phylogentic conservatism hypothesis is a null model that postulates no special evolution.

In the following pages, I first explain the methods and materials used for the research, then elucidate the results of those tests, explain the inferences we can make from this research, and describe future directions research could take to further illuminate this plant-insect interaction.

Materials and Methods

Study Organisms

The Beetles: Trirhabda eriodictyonis (Chrysomelidae subfamily Galerucinae,

Figure 1) has been virtually unstudied, probably because it does not eat a plant of any commercial importance, even though it is found in abundance in the Los Angeles foothills, within commuting distance of several major universities. No one has studied its host specificity or anything about its mating behavior, although some research has been done on other members of the genus Trirhabda (Tilden 1953; Hogue 1970; O’Brien and

Atsatt 1982; Palmer 1986; Boldt 1989; Palmer & Haseler 1992; Herzig 1995; Blatt et al.

1999). This previous research has guided me, however the vast majority of information

Figure 1: Trirhabda eriodictyonis. From left, eggs (after hatching), second-instar larva with shed exoskeleton, adult (~10 mm long). about T. eriodictyonis behavior reported here is based on my own observations of populations in the field and in the lab. Help from my lab assistants has been invaluable, but I verified with my own eyes all novel observations reported here.

The life history of Trirhabda eriodictyonis begins with eggs hatching in February and March at the base of plants. The newly hatched larvae are about 2 mm long. They

8 climb up the stem of the host plant to the leaves, which is their sole food source.

Although tiny, these larvae are extremely mobile. In the lab, they thoroughly explore the

Petri dishes in which they are kept, and in one experiment, several climbed a stem 43 cm long in less than half an hour.

Larvae go through three instars over about 90 days (Hogue 1970), growing to 10 mm in length on average. The first two instars are black, and the third is metallic green.

Pupation happens in the soil, where each larva uses bodily secretions to glue together a loose shelter of leaves and soil. In the lab, beetles pupated under leaves or paper towels placed in the Petri dishes. After two weeks, the adult emerges.

Adults are mustard-yellow with grayish-brown elytra and have three black spots equally spaced across the head and three across the pronotum. Individuals vary in the size of the spots, and some individuals have yellow streaks on the elytra, especially at the anterior ends. Although Hogue (1970) suggested that the shape of the central head spot was different on males and females, I observed various shapes of spots on both sexes.

Adults live for about 90 days. Mating occurs on the leaves of the host plant.

Females descend to the soil to lay eggs at the base of the host plant, and those 1-mm-long eggs remain in the soil over the winter. The same size and color as grains of sand, these eggs have not been observed in the field. O’Brien and Atsatt (1982) were unsuccessful in attempts to locate the eggs of other Trirhabda species in the wild. Other species of

Trirhabda lay eggs in the soil, and eggs are never seen on the leaves or stems. In the lab, females lay eggs under leaves or paper towels, usually at the edge of the Petri dish.

Figure 2: Distinguishing the sexes. A gravid female with distended abdomen (left); female posterior sternite (center); male posterior sternite.

Females are larger than males on average, but this difference is not large enough or consistent enough to be used in sexing individuals. Females can easily be distinguished once they are gravid because they develop distended abdomens larger than the abdomens ever grow in males. At other times, males and females can only be distinguished by examining the ventral side of the posterior-most segment of the abdomen under a dissecting microscope. The section is gently concave at the end on males, whereas in females it is notched (Figure 2).

Mating in T. eriodictyonis is prefaced by no obvious courtship. O’Brien and

Atsatt (1982) observed in T. sericotrachyla that receptive females stood on a leaf with their head facing toward the stem waiting for males to approach. No other courtship has been noted. In Petri dishes in the lab, the male approaches the female and mounts, grasping the edges of her elytra with his tarsi (Figure 3A). He strokes her head and pronotum with his antennae while extending his aedeagus. If the female accepts him, she allows him to insert his aedeagus through the notch at the posterior end of her abdomen

(Figure 3B). Once his aedeagus is inserted, the pair stops moving and remains still for 10 minutes on average before the female starts twisting her body quickly back and forth in what appears to be an attempt to dislodge the male. She will continue this “waggle” behavior until he removes his aedeagus and dismounts, on average 9 more minutes.

(A)

(B)

(C)

Figure 3: Trirhabda eriodictyonis mating. (A) Male initiating mating; (B) pair successfully mating; (C) female rejecting male with upturned tip of abdomen, preventing the male from inserting his aedeagus.

If a female chooses not to mate with a male, she will curl up the posterior tip of her abdomen, preventing him from inserting his aedeagus, and waggle her body (Figure

3C). O’Brien and Atsatt (1982) reported that females of T. sericotrachyla dislodged unwanted males in a few minutes. This is generally the same for T. eriodictyonis; most males dismounted in two to three minutes. However, a few males in the lab were able to hang on for far longer, with one male hanging on for four hours, during which time the female intermittently waggled and rested, and the male repeatedly stroked her with his

11 antennae and attempted to insert his aedeagus. Both males and females will mate with multiple partners.

Females lay multiple clutches of eggs over the course of the summer. In the lab, they lay eggs regardless of whether they have mated.

The Plants: Trirhabda eriodictyonis lives almost exclusively on two plants with ranges that overlap. In the study area Eriodictyon crassifolium is found at elevations up to

1300 m, and E. trichocalyx is seen from 1100 m to above 1400 m (Calflora 2014; Jepson

2014). The two species are occasionally adjacent to one another. Presumably because E. crassifolium lives at a lower elevation, eggs hatch on that plant about three weeks earlier on average than on E. trichocalyx.

(A) (B)

Figure 4: Leaves of Trirhabda eriodictyonis’ host plants. (A) Eriodictyon crassifolium, (B) E. trichocalyx

These host plant species are very different to the human observer (Figure 4).

Eriodictyon crassifolium has leaves that are dusty gray and covered with a thick layer of wooly hairs and no obvious surface resins (Hannan 1988a & 1988b; Jepson 2014). The

12 leaves are ovate with crenate margins. Eriodictyon trichocalyx has dark green leaves coated with a thick sticky resin (Hannan 1988a & 1988b; Jepson 2014). Its leaves are lanceolate with an irregularly wavy margin. Both plant species have fragrant leaves, although E. trichocalyx leaves smell stronger and more like camphor. Although the resin of E. trichocalyx is a distinctive and dramatic trait, it is unlikely to affect the health of the beetles. When T. diducta beetles were fed high-resin leaves of E. californicum, they ate more, but ingested the same amount of nitrogen, most likely because the resin dilutes the nutritional value of the leaves (Johnson et al. 1985).

Trirhabda eriodictyonis larvae are sometimes found on a third plant species,

Eriodictyon parryi, the range of which overlaps with the ranges of both other host species in the study area. However, I failed to find the study beetles as adults on E. parryi despite many searches. Because the adults apparently avoid E. parryi, I excluded it from this study. In some cases, two or three species of Eriodictyon are found within a few meters of each other. Eriodictyon parryi benefits from fire, and following the 2009 Station Fire it is now profuse across the study area. However, it usually only grows for a few years following a fire (Horton & Kraebel 1955), so I expect that in the next few years E. parryi numbers will again decrease and it will not be available as a host for T. eriodictyonis beetles. Therefore, from an evolutionary perspective, E. parryi is not important to the beetles.

The Study Site: In the San Gabriel Mountains, Eriodictyon crassifolium and E. trichocalyx have a narrow zone of overlap at 1200-1300 m elevation (Figure 5). My study site extends from about 1050 m to about 1450 m along Angeles Crest Highway (CA-2) on the south side of the San Gabriel Mountains (Los Angeles County, California, USA).

Figure 5: Study site. The study site extends along Angeles Crest Highway (Highway 2) from an elevation of 1066 m to 1487 m. Collection locations for E. crassifolium are shown in purple; sites of E. trichocalyx are in green.

Both plant species grow in large numbers in the study area, in some places blanketing hillsides.

Experiments

I collected larvae and adults weekly from February through July 2013. I also cut leaves from plants in the field and stored them in a refrigerator to ensure a constant supply of fresh food for the beetles. In many areas, larvae were so numerous that it was impossible to collect food without collecting more larvae. These larvae were added to the study.

I collected larvae from plants that were outside the overlap zone, to ensure that larvae from E. crassifolium had not been exposed to E. trichocalyx and vice versa.

However, halfway through the season I discovered two E. crassifolium shrubs growing amongst the E. trichocalyx in one of the primary collection sites. Larvae were not collected from plants adjacent to these E. crassifolium plants, and larvae are not able to

14 move to from one plant to another unless the plants are touching, so I am confident that larvae tested in the lab had experience only with their natal plant.

Wild-caught and lab-reared larvae were maintained in Petri dishes kept in growth chambers with conditions set to mimic conditions in their natural habitat. Spring and summer conditions were set as 25°C and lights on for 14 hours, 17°C and lights off for 10 hours. Fall and winter conditions (for the eggs) were set at 18.9°C and lights on for 11 hours; 7.2°C and lights off for 13 hours. Each dish contained moistened circles of paper towel as well as a leaf or leaves, as appropriate. Larvae used for feeding tests were kept individually in Petri dishes; larvae not used in feeding tests were kept five per Petri dish until reaching the prepupal or pupal stage, at which point they were separated into individual dishes. Each dish was numbered and the treatment, species of leaf fed, date of collection, and life stage of the individual(s) written on the dish.

Each week, my lab assistants and I used digital calipers to measure the length of larvae housed individually. We recorded when molting occurred. Unfortunately, not all molts were recorded because shed exoskeletons of first and second instar larvae can be mistaken for feces, so it was not always obvious when a larva had molted. The prepupal stage was defined as the time when a third-instar larva was found curled up and not moving. We recorded the dates when a larva appeared to be in the prepupal stage, the date they pupated, and the date they emerged into adulthood. Because dishes were checked every other day, these observations may be off by one or two days, but that error is consistent across treatments. We did not measure adults until they died, at which point we used digital calipers to measure the length of the body from the crown of the head

(between the eyes) to the posterior tip of the elytra. Individuals that were discarded from the study before they died were measured before they were released.

In the case of dishes that started with multiple larvae, individual deaths were not recorded. However, I was able to infer deaths based on the dates when larvae or pupae were moved to individual dishes, because the number of larvae remaining in the dish was recorded. These inferred deaths were used for a categorical “survival to pupation” analysis, but eliminated from quantitative survival analyses.

Feeding Studies: In preliminary studies in 2012, individuals either ate copious amounts or nothing; therefore, in this study we recorded feeding as either 1 (feeding) or 0

(no feeding). If an individual took just one or only a few bites (total area smaller than the size of its head), it was recorded as 0. I also recorded whether an individual in choice tests ate more of one plant than the other. Other studies have measured the amount eaten by weighing (Matsubayashi and Katakura 2012) or measuring (Boldt 1989) leaves. These methods seem to me overly taxing and highly prone to error, especially because leaves quickly desiccate and shrink. My protocol, while perhaps crude, nonetheless gives a clear result efficiently and enabled me to have more replicates. Large sample sizes should outweigh any benefit I might have gained from having an unreliable quantitative dependent variable.

Feeding Tests—Choice: Hatchlings (from eggs laid in the 2012 season) and wild- caught first- and second-instar larvae were offered leaves of both E. crassifolium and E. trichocalyx. We carefully checked the leaves to make sure that no bite marks existed before the trial. Leaves were checked after four days and feeding noted. First- and

16 second-instar larvae are so small that their bites are sometimes undetectable with the naked eye, and half of the larvae showed no signs of eating either plant after three days, but ate on the fourth day. Choice feeding tests were done with lab-reared larvae whose natal plant was E. crassifolium, and with wild-caught larvae collected from E. trichocalyx, because there were insufficient eggs laid by E. trichocalyx females from the previous research year.

I used wild-caught adults for choice feeding tests because too few lab-reared larvae survived to adulthood to conduct these tests with adults of known feeding history.

Individuals were offered leaves of both plants and I checked their feeding after one or two days as logistics allowed.

After completion of the choice tests, individuals were given only their non-natal plant, becoming part of the no-choice feeding trials.

Feeding Trials—No Choice: Field-collected larvae and adults were offered a different plant from the one on which they were collected (i.e., larvae collected on E. crassifolium were offered E. trichocalyx and vice versa). We recorded the date on which these individuals entered the prepupal, pupal, and adult stage, as well as when they died.

Mating Trials: Prior to mating tests, I determined the sex of each individual. To ensure adequate sample sizes, I included individuals collected as larvae and as adults. All mating trials were no-choice trials involving virgin females. Because of low numbers of males available for some treatments, a few males that did not attempt to mate in their first trial were re-used in subsequent trials. In two instances, males that had successfully

17 mated were accidentally re-used. The second matings for both these males were excluded from analysis.

For each mating trial, a pair of adults was placed together in a Petri dish and observed constantly. In previous observations I had determined that almost all matings occurred within 75 minutes of the two individuals being placed together. Therefore, observations were stopped after 75 minutes, unless a pair was still actively mating, in which case they were allowed to continue and separate on their own. Pairs in which the male was unsuccessfully trying to mate at the end of the trial time were gently separated.

For each trial, the time from the start of test to the initiation of a mating attempt was recorded as “latency.” If the mating attempt was successful, the time of mating while both adults were still was noted (“still mating interval”). The duration of mating while the female waggled, before the male dismounted, was recorded as “waggle mating interval.”

If the attempt was unsuccessful, the time the male persisted in his attempt was recorded as “rejection waggle interval.”

Mating Trials—No Plant Switch: Control pairs included two individuals both raised on the same plant species. Experimental pairs included one individual from E. crassifolium and a second from E. trichocalyx. Males and females raised on each plant species were assigned to these treatments and paired haphazardly.

Mating Trials—With Plant Switch: In experimental cross pairs a female raised and maintained from hatching through adulthood on a focal plant was paired with a male that was raised as a larva on the other plant but switched as an adult to the focal plant. For example, if the focal plant was E. trichocalyx, a male fed only E. crassifolium as a larva was fed only E. trichocalyx after metamorphosis for at least two weeks, then paired with

18 a female raised and maintained on E. trichocalyx. Males were used for the plant-switch portion of this treatment because females grow such large, distended abdomens when they are sexually mature that it seems less likely that they would fly long distances to visit another plant species. This set-up also simplified statistical analysis. Females were randomly assigned to no-switch and with-plant-switch mating treatments.

Fecundity: At each feeding, we noted whether a female had laid eggs and how many eggs she had laid. All eggs laid by females that mated successfully were saved and transferred to their own Petri dishes marked with the mother’s dish number, feeding treatment, and the date the eggs were laid. We also saved eggs from females that had refused male mating attempts, to determine if males are able to transfer some sperm during these apparently unsuccessful mating attempts. Eggs from females that had not mated were discarded.

In February and March 2014, when eggs hatched, we noted how many larvae hatched from each egg mass.

Statistics

General Info: I compared male and female size first with a two-sample t-test of untransformed data. Then I ran an ANOVA to test for an interaction between sex and host plant, first comparing only beetles that had not switched plants. Finally, an additional ANOVA was run to determine if there was an interaction between plant switch treatments and sex in the size of the beetles.

Feeding Tests—Choice: Beetles that did not eat either plant or that died before the end of five days from the start of the trial were excluded from analyses. Four larvae on E.

19 trichocalyx and one adult from E. crassifolium that ate equal amounts of both plant species were also excluded from the analysis. An exact sign test showed that feeding preferences between first and second-instar larvae were so similar that they could be pooled (P = 0.5), so I combined first and second instar larvae data for each treatment. I ran separate exact sign tests on beetles from E. crassifolium and beetles from E. trichocalyx. I calculated preference by adding the number of beetles that ate only a given plant with the number that ate both plants but ate more of the focal plant. The tests compared preference for E. crassifolium to preference for E. trichocalyx. I ran separate tests for larvae and adults.

Feeding Trials—No Choice: I calculated larval growth rates by subtracting a larva’s initial size from its size after the final molt and dividing that measurement by the number of days between the first and final measurement. Data were log-transformed to be normally distributed. I ran an ANOVA to test whether growth rates were different among treatments.

To determine whether larvae were more or less likely to survive to pupation in different treatments, I gave each individual a score of 1 if they pupated and 0 if they died before pupation. I then ran a test of independence.

To test survival to adulthood, I considered only individuals that reached pupation.

They received a score of 1 if they emerged as adults and 0 if they did not. Again, I ran a test of independence to determine if there were any differences among the treatments.

Finally, I used nonparametric survival analysis to determine whether individuals in different treatments took longer to enter the pupal stage. (For all survival analyses, I used Systat 13 software, version 13.1, Systat Software 2009). This test was done

20 separately for larvae from E. crassifolium and larvae from E. trichocalyx because larvae from E. crassifolium were estimated to be about three weeks older than E. trichocalyx larvae at the time of plant switch. Because all individuals in this test reached pupation, censoring was recorded as 1 for all test subjects.

To compare similar groups of beetles, I assigned the same food-switch dates to individuals that had not switched food plants as to individuals that did switch plants. This provided similar sample sizes for each treatment and a start date to compare switch to non-switch individuals. I used this same process in all survival analyses, so that for each group of beetles that switched host plant species, there was a similar group of non-switch individuals.

After testing time to pupation, I ran a test on overall survival to determine if life spans were different in different treatments. Survival analysis enables inclusion of data for participants that are lost or still alive at the end of the trial, so individuals that died or were lost (and assumed dead) were assigned a censor number of 1, and those that were still alive at the end of the trial were given a censor number of 0.

I conducted separate analyses for larvae from E. crassifolium and larvae from E. trichocalyx to clarify differences in survival. For all survival analyses, I report the

Tarone-Ware test statistic because this test places less emphasis on later event times

(Systat Software 2009). This seemed appropriate given that beetles in the lab live far longer than beetles in the field, so earlier death dates are more similar to those seen in the field and therefore more relevant to the study.

To test adult survival, I first used a nonparametric survival analysis to compare male versus female survival for all treatments. This was significant, so I ran subsequent

21 tests on males and females separately for the two host plant species. I also ran tests separately on individuals from E. crassifolium and E. trichocalyx, again because beetles on E. crassifolium were older at the start of the plant switch trials than those from E. trichocalyx. Unfortunately, for the adult survival tests, I compared adults collected in the field that had been used in choice feeding experiments and then moved to their non- native host plant versus adults that had been reared in the lab. I’m afraid this population of lab-reared adults was not an accurate representation of the field population at the time adults were collected for the switch tests. I calculated the average date of death for the beetles collected as larvae and compared it to those collected as adults using a test of independence to determine if the two groups were similar.

Mating Trials—No Plant Switch: First I ran a test of independence to compare the number of mating attempts (including successful and unsuccessful matings) to the number of trials in which no attempt was made. This test looked at male mate choice, separate from female mate choice, because initiation of a mating attempt is up to the male. A second test of independence compared the number of successful versus unsuccessful mating attempts to examine female mate choice, because mating success is under female control.

Mating Trials—With Plant Switch: Only eight trials were held using males that switched from E. trichocalyx to E. crassifolium because so few of these males survived to the start of mating trials. Only one of these attempts resulted in a successful mating.

Because of these low sample numbers, this treatment was eliminated from analysis.

To compare mating trials with a plant switch to trials without a plant switch, I used the same tests of independence that I had used to analyze the mating trials without a

22 plant switch. However, I only included those using males that had switched from E. crassifolium to E. trichocalyx. I calculated Tukey-like pairwise comparisons of proportions to compare each treatment to every other treatment (Wilson 2011).

I ran ANOVAs on log-transformed data to compare all treatments based on time to start of mating (latency), time of mating while the female was still (still mating), time of mating while the female waggled (waggle mating), and the amount of time a female waggled during unsuccessful mating attempts (rejection waggle). These all showed no significant difference, so I did not follow up with pairwise comparisons.

Fecundity: I calculated the total number of eggs laid by each female, then ran an

ANOVA to determine if switching a female's food source affected the number of eggs she laid. This was not significant, probably because most females were switched soon after they started laying eggs, so I used cumulative egg numbers for all females in subsequent tests. I was not able to transform total egg data to be normally distributed, so I used a Kruskal-Wallis nonparametric test to compare treatments. This was followed by a

Conover-Inman test for all pairwise comparisons using Systat 13, version 13.1 (Systat

Software 2009).

I defined a clutch as a mass of eggs, distinctly separate from other masses.

Average clutch size data was normally distributed after square-root transformation, so an

ANOVA was run on transformed data, followed by Tukey pairwise comparisons using

Systat 13, version 13.1 (Systat Software 2009).

Finally, I used Kruskal-Wallis nonparametric tests with Conover-Inman tests for all pairwise comparisons to test the effect of mating on total egg production.

After eggs had hatched, I further examined fecundity by analyzing the number of eggs that hatched. Data could not be normally transformed, so I performed Kruskal-

Wallace tests. I examined the proportion of potentially fertilized eggs (those laid after mating trials) that hatched, the time after mating until a female lay her first fertilized egg, the time after mating until the female laid the last fertilized egg, and the amount of time that elapsed between the laying of the first and last fertilized eggs. All of these analyses were run first with mating treatment as the grouping variable and next with female host plant as the grouping variable because factorial ANOVA is not appropriate for data not normally distributed. Tests with significant differences were followed by Dwass-Steel-

Chritchlow-Fligner tests for all pairwise comparisons (Systat Software 2009).

For all graphs, error bars are standard errors. No error bars are provided on graphs where the dependent variable is categorical.

Results

Size: Females are, on average, larger than males (females: 8.1 +/- 0.06 mm, n = 171; males 7.3 +/- 0.10 mm, n = 72; t = 7.397, df = 241, P < 0.001). This difference was independent of natal plant (ANOVA, F = 0.109, df = 1, P = 0.741) and independent of feeding treatment (F = 0.472, df = 3, P = 0.702).

100% Feeding Trials—Choice, Larvae:

80% Larvae did not show the preference for Ate Et only 60% their natal plant that would be expected Preferred Et 40% Preferred Ec under the differentiation hypothesis. 20% Ate Ec only 22 46 Those collected or hatched on E. 0% Larvae Larvae crassifolium ate similar amounts of both from Ec from Et

Figure 6: Choice feeding trials, larvae. Ec = E. plants (Figure 6, P = 0.5). Of the 22 crassifolium; Et = E. trichocalyx. Larvae collected on E. crassifolium showed no feeding preference larvae tested, 13 ate only E. (P = 0.5), but larvae collected on E. trichocalyx preferred eating E. crassifolium (P < 0.001). crassifolium, one ate both but ate more

E. crassifolium, and eight consumed only E. trichocalyx. Larvae from E. trichocalyx, on the other hand, showed a significant preference for E. crassifolium over their natal plant (P < 0.001). Almost three-quarters of the larvae tested (34 out of 46) ate only E. crassifolium. Three ate both but consumed more E. crassifolium; two ate both but preferred E. trichocalyx, and seven ate only E. trichocalyx.

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Feeding Trials—No-choice, Larvae: Although larvae from E. trichocalyx preferred to feed on E. crassifolium, feeding on that plant did not lead to higher growth rates, as would be expected in the preference-performance hypothesis (Figure 7A:

ANOVA, F = 1.432, df = 3, P = 0.24). In fact, all larvae grew at similar rates, regardless of whether they had switched plants or remained on their natal plant.

Similar growth rates did not, however, translate into similar demographics.

Larvae that switched from sticky E. trichocalyx leaves to velvety E. crassifolium leaves were less likely to reach pupation compared with the other treatments (Figure 7B: test of independence, χ2 = 28.386, df = 3, P < 0.001). Almost all beetles that entered the pupal stage made it through transformation and emerged as adults, regardless of treatment

(Figure 7C: test of independence, χ2 = 3.852, df = 3, P = 0.347). This difference in survival is not predicted by any of the hypotheses discussed above. Because larvae from E. trichocalyx prefer eating E. crassifolium, differentiation is not supported. And because larvae from E. trichocalyx prefer to eat E. crassifolium but perform poorly on that plant, the preference-performance hypothesis is not supported. The results may coincide with phylogenetic conservatism, but it provides no explanation for why larvae would have reduced survival on E. crassifolium after starting on E. trichocalyx.

Although switching host plants affected the likelihood that a beetle would make it to pupation, it did not a change the amount of time it took larvae to reach that milestone

(Figure 8A: larvae from E. crassifolium: log-rank test, Tarone-Ware χ2 = 0.887, df = 1,

P = 0.346; Figure 8B: larvae from E. trichocalyx: log-rank test, Tarone-Ware χ2 = 3.429, df = 1, P = 0.064). Larvae that had been switched from one host plant species to the other reached pupation in about the same time as those that remained on their natal plant.

Of course, the ultimate test is whether an individual lives long enough to find a mate and reproduce. From this standpoint switching host plants was not detrimental to larvae from E. crassifolium (Figure 9A: log-rank test, Tarone-Ware χ2 = 0.004, df = 1,

P = 0.948), but was disadvantageous to larvae from E. trichocalyx, which died younger

(A) 1 0.9 0.8 0.7 0.6 0.5 0.4 E. crassifolium only 0.3 E. crassifolium to 0.2 E. trichocalyx

Probability of Pupation of Probability 0.1 0 0 10 20 30 40 50 Days after Plant Switch

(B) 1 0.9 E. trichocalyx to 0.8 E. crassifolium 0.7 E. trichocalyx only 0.6 0.5 0.4 0.3 0.2

0.1 Probability of Pupation of Probability 0 0 20 40 60 80 Days after Food Switch

Figure 8: Time to pupation. Beetles reached pupation in about the same time, whether they stayed on their natal plant or not (A) larvae from E. crassifolium: (P = 0.346), E. crassifolium only: n = 38, E. crassifolium to E. trichocalyx: n = 35; (B) larvae from E. trichocalyx: (P = 0.064), E. trichocalyx only: n = 45, E. trichocalyx to E. crassifolium: n = 33.

1 0.9 (A) 0.8 0.7 E. crassifolium only 0.6 0.5 E. crassifolium to 0.4 E. trichocalyx 0.3 0.2

Probability of Survival of Probability 0.1 0 0 50 100 150 200 Days after Plant Switch

(B) 1 E. trichocalyx only 0.9 0.8 E. trichocalyx to 0.7 E. crassifolium 0.6 0.5 0.4 0.3 0.2

Probability of Survival of Probability 0.1 0 0 50 100 150 200 Days after Plant Switch

Figure 9: Lifetime survival, host-plant switch as larvae. (A) Switching to E. trichocalyx caused no change in lifetime survival for larvae from E. crassifolium (P = 0.948), E. crassifolium only, n = 38, E. crassifolium to E. trichocalyx, n = 26. (B) The reverse switch, from E. trichocalyx to E. crassifolium caused a shortening of lifespan (P < 0.001), E. trichocalyx only, n = 44, E. trichocalyx to E. crassifolium, n = 28. than beetles in other treatments (Figure 9B: log-rank test, Tarone-Ware χ2 = 14.444, df = 1, P < 0.001). Although larvae that hatched on E. trichocalyx preferred to eat E. crassifolium, such a change in diet was harmful over the long term. This result is counter to what is expected from the differentiation hypothesis, but also confusing for the preference-performance hypothesis.

Larvae should favor the plant 100% that is most beneficial to them 90% 80% in the long-term, but these 70% 60% Ate Et only larvae did not. 50% Preferred Et Feeding Trials—Choice, 40% Preferred Ec 30% Ate Ec only Adults: All adults showed a 20% 10% preference for eating E. 9 21 0% Adults From Ec Adults From Et trichocalyx rather than E.

Figure 10: Choice feeding trials, adults. Adults from crassifolium (Figure 10). Like both host plants greatly preferred the leaves of E. trichocalyx (adults from E. crassifolium P = 0.02; adults the results of the larval study, from E. trichocalyx P < 0.001). this is counter to what is expected if the differentiation hypothesis were true. Of the nine adults tested that came from E. crassifolium, only one tried E. crassifolium and it still ate more E. trichocalyx

(exact sign test, P = 0.02). Twenty-one adults from E. trichocalyx were tested; nine tried

E. crassifolium, but all of those ate more E. trichocalyx, the rest (12) ate only E. trichocalyx (exact sign test, P < 0.001). This result supports the first aspect of the preference-performance hypothesis: E. trichocalyx is the favored plant. These data do not support either the differentiation hypothesis or the phylogenetic conservatism hypothesis.

Feeding Trials—No-Choice, Adults: Males generally died sooner than females

(Figure 11: pooled treatments, log-rank test, Tarone-Ware χ2 = 4.50, df = 1, P = 0.034).

The switch of host plant species had no appreciable effect on adults from E. crassifolium (Figure 12A: females from E. crassifolium: log-rank test, Tarone-Ware

χ2 = 1.015, df = 1, P = 0.314; Figure 12B: males from E. crassifolium: log-rank test,

0.8

0.6

0.4 Males

0.2 Females Probability of Survival of Probability 0 0 50 100 Days after Plant Switch

Figure 11: Adult survival, by sex. Males had shorter overall life spans than females in all treatments (P = 0.034), male: n = 93, female: n = 169.

Tarone-Ware χ2 = 0.4, df = 1, P = 0.527). However, as with larvae, adults switched from the sticky leaves of E. trichocalyx to the hairy leaves of E. crassifolium had shorter survival times than those left on the original host plant (Figure 12C: females from E. trichocalyx: log-rank test, Tarone-Ware χ2 = 9.365, df = 1, P = 0.002; Figure 12D: males from E. trichocalyx: log-rank test, Tarone-Ware χ2 = 13.445, df = 1, P < 0.001). The effect of switching from E. trichocalyx to E. crassifolium was similar for males and females (log-rank test, Tarone-Ware χ2 = 1.385, df = 1, P = 0.239); both died sooner than those left on E. trichocalyx. Again, the differentiation hypothesis is not supported, but the preference- performance hypothesis is, with E. trichocalyx appearing to have some beneficial effect on beetles that ate it. However, the preference-performance hypothesis is not completely supported because there is no increase in performance for beetles switched from E. crassifolium to the favored E. trichocalyx.

Unfortunately, these results are not as definitive or reliable as I would like them to be. In choosing adults to compare to field-caught adults in the host-switch experiment, I

(A) Females from E. crassifolium (B) Males from E. crassifolium 1 1 0.9 0.9 E. crassifolium 0.8 0.8 only 0.7 0.7 0.6 0.6 E. crassifolium E. crassifolium to 0.5 0.5 only E. trichocalyx 0.4 0.4 0.3 0.3 0.2 E. crassifolium to 0.2

E. trichocalyx Probability of Survival of Probability Probability of Survival of Probability 0.1 0.1 0 0 0 50 100 0 50 100 Days after Plant Switch Days after Plant Switch

(C) Females from E. trichocalyx (D) Males from E. trichocalyx 1 1 E. trichocalyx 0.9 0.9 only 0.8 0.8 E. trichocalyx to E. crassifolium 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 E. trichocalyx only 0.3 0.3 0.2 0.2

E. trichocalyx to E. Survival of Probability 0.1 Probability of Survival of Probability 0.1 crassifolium 0 0 0 50 100 0 50 100 Days after Plant Switch Days after Plant Switch

Figure 12: Adult survival, by treatment. (A and B) The switch from E. crassifolium to E. trichocalyx did not lead to early death (A—females from E. crassifolium: P = 0.314, Ec only: n = 42, Ec to Et: n = 38; B—males from E. crassifolium: P = 0.527, Ec only: n = 29, Ec to Et: n = 16). (C and D) However, the reverse switch did (C— females from E. trichocalyx: P = 0.002, Et only: n = 44, Et to Ec: n = 45; D—males from E. trichocalyx: P < 0.001, Et only: n = 25, Et to Ec: n = 23). unwittingly chose long-lived lab-reared adults that did not accurately represent the population at large. When the average date of death is calculated for all beetles that survived to adulthood, all beetles that ate E. crassifolium died sooner than those that ate

E. trichocalyx (Figure 13: Kruskal-Wallace test statistic = 37.192, df = 3, P < 0.001).

This result might actually make sense for the preference-performance hypothesis, if the difference between performance on the plants is that the trichomes of E. crassifolium are hard to eat. Beetles on that plant would be expected to die sooner, and those on the preferred plant, E. trichocalyx, would be expected to live longer. However, because these samples are poorly randomized, these tests need to be repeated.

9/8/2014 b b 8/29/2014 8/19/2014 8/9/2014 7/30/2014 7/20/2014 a 7/10/2014 a

Date of Death of Date 6/30/2014 6/20/2014 6/10/2014 396 98 148 317 5/31/2014 Ec-Ec Ec-Et Et-Ec Et-Et

Figure 13: Median date of death. Beetles feeding on E. crassifolium died earlier than those eating E. trichocalyx. A shared similar letter signifies non-significance by Conover-Inman tests.

Mating Trials—No Plant Switch: Males tried to mate with about half of the females they were paired with, regardless of treatment (Figure 14A: χ2 = 1.914, df = 3,

P = 0.119). However, females were not so catholic in their preferences (Figure 14B:

χ2 = 10.305, df = 3, P = 0.007). Females feeding on E. trichocalyx showed no preference for males from one plant species or the other. But females living on E. crassifolium were almost twice as likely to accept the mating advances of a male if he had lived and fed on

E. trichocalyx than if he had fed on E. crassifolium (Tukey-like comparison, Q = 3.635, df = ∞, 4 groups, P = 0.005). Of the pairs in which the male and female were both from

E. crassifolium, there were 19 unsuccessful attempts and 11 successful matings out of 51

33 trials (including 21 trials in which no mating attempt took place). However, when the female was from E. crassifolium and the male from E. trichocalyx, there were just 7 unsuccessful attempts and 25 successful matings in 51 trials. This result again supports the preference-performance hypothesis and it appears that at least for adults, E. trichocalyx is favored.

(A) (B)

100% 100% 90% 90% 80% 80% 70% 70% 60% 60% 50% 50% 40% 40% 30% 30% 20% 20% 10% 10% 36 32 30 30 36 32 30 30 0% 0% Ec Male Et Male Ec Male Et Male Ec Male Et Male Ec Male Et Male Ec Female Et Female Ec Female Et Female

Mating Attempts (Successful and Unsuccessful)

No Mating Attempts Successful Matings Unsuccessful Attempts

Figure 14: Mating preferences, without plant switch. (A) Males attempted to mate in about half the mating trials, with no difference among treatments (P = 0.119). (B) Females on E. trichocalyx did not show a preference in potential mates, but females on E. crassifolium accepted mating advances from far more E. trichocalyx males than E. crassifolium males (P = 0.005).

Similar differences were not seen in behavior during mating. Regardless of the host plants of the male and of the female, pairs took the same time to start mating (Figure

15A: ANOVA, F = 0.059, df = 4, P = 0.981), and in all treatments females remained still for the same time during mating (Figure 15B: ANOVA, F = 0.908, df = 4, P = 0.443), and waggled for the same time (Figure 15C: ANOVA, F = 1.397, df = 4, P = 0.255).

There was also no difference among treatments in the amount of time a female waggled when rejecting a male (Figure 15D: ANOVA, F = 2.157, df = 4, P = 0.180).

Mating Trials—With Plant Switch: Analysis could only be done on males that switched from E. crassifolium to E. trichocalyx because there were too few surviving males in the group that switched from E. trichocalyx to E. crassifolium. While unfortunate, this high death rate would be expected if E. trichocalyx is a favored plant.

(A) Latency to Mating (B) Still Mating 3 3.5 3 2.5 2.5 2 2 1.5 1.5 1 1 0.5

(log(min)) 0.5

0 (log(min)) Still 0

Ec Male Et Male Ec Male Et Male Ec Male Et Male Ec Male Et Male

Time of Mating, Female Mating, of Time Time to Start of Mating of Start to Time Ec Female Et Female Ec Female Et Female

Waggling (log(min)) Waggling Ec Male Et Male Ec Male Et Male

Time of Mating Attempt, Mating of Time Ec Male Et Male Ec Male Et Male

Time of Mating, Female Mating, of Time Female Waggling (log(min)) Waggling Female Ec Female Et Female Ec Female Et Female

Figure 15: Mating behavior. There was no difference among treatments in (A) the latency to mating (P = 0.981, Ec male/Ec female: n = 13, Et male/Ec female: n = 24, Ec male/Et female: n = 14, Et male/Et female: n = 16), (B) time of mating while the female remained still (P = 0.443), (C) time of mating while the female waggled (P = 0.255), or (D) the amount of time a female waggled when rejecting a male (P = 0.118, Ec male/Ec female: n = 6, Et male/Ec female: n = 4, Ec male/Et female: n = 7, Et male/Et female: n = 5).

(A) 100% (B) 100% 80% 80% 60% 60% 40% 40% 20% 20% 50 58 28 30 30 15 0% 0% Ec Male Et Male Ec to Et Ec Male Et Male Ec to Et male male Et Female Et Female Mating Attempts (Successful and Successful Mating Unsuccessful) Unsuccessful Mating Attempt No Mating Attempt

Figure 16: Mating preferences, with plant switch. (A) Males switched from E. crassifolium to E. trichocalyx were just as likely to attempt mating as those that had not been switched (P = 0.673). (B) Females on E. trichocalyx did not show a significant preference for males switched from E. crassifolium over males that had not switched plants (P = 0.108).

Males that made the switch from E. crassifolium to E. trichocalyx were no more or less likely to initiate mating than those that stayed on E. crassifolium or those that stayed on E. trichocalyx (Figure 16A: χ2 = 0.793, df = 2, P = 0.673).

Females on E. trichocalyx were similarly likely to accept the advances of males regardless of what host plant they ate or whether they had switched plants (Figure 16B:

χ2 = 4.456, df = 2, P = 0.108).

Fecundity: Switching plants had a large effect on the number of eggs laid by females from both host plants (Figure 17A: Kruskal-Wallis test statistic = 30.623, df = 3,

P < 0.001). Females from E. crassifolium laid more eggs when switched to E. trichocalyx than those left on the natal plant (Conover-Inman test for all pairwise comparisons,

P < 0.001) and females from E. trichocalyx laid fewer eggs when switched to E. crassifolium (Conover-Inman test for all pairwise comparisons, P < 0.001). In fact,

(A) (B) 180 b 3.9 b 160 b 3.8 3.7 140 a,b 3.6 120 3.5 a 100 a a 3.4 a 80 3.3 60 3.2 3.1

Total Eggs/Female Total 40 Clutch Size/Female Clutch 3 20 (square root eggs/clutch) root (square 2.9 126 51 48 129 126 51 48 129 0 2.8 Ec only Ec to Et Et to Ec Et only Ec only Ec to Et Et to Ec Et only

(C ) (D) 16 Females from E. crassifolium Females from E. trichocalyx 16 a 14 a 14 12 a 12 a b 10 10 b 8 8 6 6

4 4 Total Eggs/Female Total 2 Eggs/Female Total 2 11 19 31 15 12 33 0 0 Ec Male Et Male Not Mated Ec Male Et Male Not Mated

Figure 17: Potential fecundity. (A) Eating E. trichocalyx caused females to lay far more eggs than eating E. crassifolium, both for females that started on E. trichocalyx and for those that switched to E. trichocalyx. (P < 0.001). (B) Females that had switched plants laid larger clutches than females that had not switched plants (P < 0.001). Mated females laid more eggs than unmated females for beetles on (C) E. crassifolium (P = 0.001), and on (D) E. trichocalyx (P = 0.010). females that switched plants laid nearly the same number of eggs as if they had never lived on their original host plant.

Plant switch had a different effect on clutch size, with females switched from E. crassifolium to E. trichocalyx laying larger clutches than females that lived entirely on E. trichocalyx (Figure 17B: ANOVA, F = 6.417, df = 3, P < 0.001). Mating also had a distinct impact on egg-laying, with mated females laying far more eggs than unmated

37 females (Figure 17C: females from E. crassifolium: Kruskal-Wallis test statistic = 14.407, df = 2, P = 0.001; Figure 17D: females from E. trichocalyx: Kruskal-

Wallis test statistic = 9.255, df = 2, P = 0.010). Mating resulted in more egg-laying regardless of the plant the male had been eating, so even though E. crassifolium females preferred males from E. trichocalyx, they did not lay more eggs as a result of this choice.

Hatching rates were extremely low, probably as a result of bad husbandry rather than the inherent biology of T. eriodictyonis. Still, because eggs from different treatments were randomly placed in growth chambers, the same husbandry was applied across treatments. Hatching rates were not significantly different for females eating E. trichocalyx than for females eating E. crassifolium (Figure 18A: Kruskal-Wallis test statistic = 0.856, df = 1, P = 0.355), however E. trichocalyx females laid more clutches that produced hatchlings than females eating E. crassifolium (Figure 18B: Kruskal-Wallis test statistic = 5.224, df = 1, P = 0.022) and produced more hatchlings because they laid significantly more eggs (Figure 18C: Kruskal-Wallis test statistic = 4.233, df = 1,

P = 0.040). Females eating both plants waited about three weeks after mating before laying fertilized eggs (Figure 18D: Kruskal-Wallis test statistic = 0.791, df = 1,

P = 0.374). However, females eating E. trichocalyx laid fertilized eggs for a longer time span (Figure 18E: Kruskal-Wallis test statistic = 11.262, df = 1, P = 0.001). This extended duration of fertile egg-laying could be an advantage to females on E. trichocalyx if they lay eggs at the base of different individual plants. With offspring on numerous plants, E. trichocalyx females would increase the probability that some offspring would survive and thrive.

(A) 0.07 0.06 0.05 0.04 0.03 Hatched 0.02

ProportionEggsof 0.01 34 37 0 Ec Female Et Female

(B) 4 (C) 14 12 3 10 8 2 6

1 4 NumberClutchesof ProducingHatchlings 34 37 2 NumberHatchlingsof 34 37 0 0 Ec Female Et Female Ec Female Et Female

(D) 30 (E) 35 25 30 20 25 20

15 Laying - 15 10 Egg 10 21

Fertilized EggFertilizedLaid 5

Days, Mating Days, toFirst 5 21 26 Duration Fertilizedof 26 0 0 Ec Female Et Female Ec Female Et Female

Figure 18: Realized fecundity, by female treatment. (A) Females eating E. trichocalyx did not see a larger proportion of eggs hatch (P = 0.355); but (B) did realize more hatchlings than females eating E. crassifolium (P = 0.040); (C) and laid more clutches of eggs that produced hatchlings (P = 0.022) (D) Females on both plants started laying fertilized eggs on average three weeks after mating (P = 0.374); (E) E. trichocalyx females laid fertilized eggs for a longer duration than those on E. crassifolium (P = 0.001).

Mate choice also had an interesting effect on fecundity. The preference of females from E. crassifolium for males from E. trichocalyx led to decreased, not increased fecundity. These females saw a low proportion of their eggs hatch, although not significantly lower than females in other mating treatments (Figure 19A: Kruskal-Wallis test statistic = 4.263, df = 4, P = 0.372). They produced fewer fertilized clutches than other females, although this was not a significant difference (Figure 19B: Kruskal-Wallis test statistic = 7.675, df = 4, P = 0.104), and they produced fewer hatchlings than other females, although again this difference was not significant (Figure 19C: Kruskal-Wallis test statistic = 6.335, df = 4, P = 0.176). All females waited about the same amount of time to begin laying fertilized eggs (Figure 20A: Kruskal-Wallis test statistic = 6.572, df = 4, P = 0.160). However, E. crassifolium females mated with E. trichocalyx males laid fertilized eggs for a shorter duration than females in all other mating treatments

(Figure 20B: Kruskal-Wallis test statistic = 14.471, df = 4, P = 0.006). This lack of fecundity benefit for females from E. crassifolium mated with males from E. trichocalyx is surprising and is contrary to the preference-performance hypothesis.

One Final Surprise: I found that numerous hatchlings emerged from eggs laid after unsuccessful matings, indicating that even if they cannot perform full intromission, the males can transfer some sperm. Five males fathered a total of 17 hatchlings from

“unsuccessful” matings.

(A) 0.12 0.1 0.08 0.06 0.04 0.02 10 24 14 13 10 0 Ec Male Et Male Ec Male Et Male Ec to Et

Male ProportionHatchedEggsof Ec Female Et Female

(B) 6 5 4 3 2 24

Total Clutches Clutches Total 1 10 14 13 10

0 ProducingHatchlings Ec Male Et Male Ec Male Et Male Ec to Et Male Ec Female Et Female

Total Hatchlings ProducedHatchlings Total Male Ec Female Et Female Figure 19: Realized fecundity, by mating treatment. (A) Hatching rates were similar for all females (P = 0.372), (B) as was the number of clutches producing hatchlings was also similar across mating treatments (P = 0.104), (C) and the total number of hatchlings produced (P = 0.176).

(A) 45 40 35 30 25 20 15

10 FertilizedEggLaid

Days, Mating to First Mating Days, to First 5 7 14 9 9 8 0 Ec Male Et Male Ec Male Et Male Ec to Et Male Ec Female Et Female (B) 50 a 40 a

30 a laying - 20 a Egg 10 b Duration of Fertile Duration Fertile of 0 Ec Male Et Male Ec Male Et Male Ec to Et Male Ec Female Et Female Figure 20: Realized fecundity, by mating treatment. (A) Females on both plants started laying fertilized eggs on average three weeks after mating (P = 0.160). (B) The only significant difference was that females from E. crassifolium mated to males from E. trichocalyx laid all their fertilized eggs quicker than other females (P = 0.006).

Discussion

Differentiation Hypothesis: It is obvious that the differentiation hypothesis is not supported by the data. None of the host plant fidelity occurred that is expected if this population of Trirhabda eriodictyonis were diverging into two populations according to food type. Instead, larvae living on E. crassifolium have no preference, larvae from E. trichocalyx prefer to eat E. crassifolium, and adults on both host species prefer to eat E. trichocalyx. No preference for the natal plant was seen. The differentiation hypothesis would also predict a disadvantage of switching a beetle from its natal plant species to the other plant species, but the data reveal that a switch is harmful only from E. trichocalyx to E. crassifolium, not vice versa. The final signal of differentiation would be a preference for mates from the same host plant, but again, this is not what the data reveal.

Males from both plants and females from E. trichocalyx show no preference for the host plant of their mates. It is only females on E. crassifolium that show any preference, and they prefer males from the other host plant.

Clearly these beetles are not exhibiting the local adaptation that Thompson’s theory of geographic mosaic of coevolution predicts. This study system seemed at first glance to be the perfect candidate for local adaptation and coevolution. However, this may simply be a result of looking at the system from the wrong perspective. Thompson

(2005, p. 57) argues that “if populations are connected by high levels of gene flow... coevolutionary adaptation may be evident only at the overall metapopulation level rather than at the local population level.” The population of beetles under study here is likely to have gene flow among beetles across the two plant species, and therefore any adaptation

43 may not be seen in this population. It would be interesting to study the difference between this population and a population in an area where E. trichocalyx does not grow, such as in the Santa Clarita area (Los Angeles County, CA). Perhaps beetles without a nearby supply of E. trichocalyx would be reluctant to try the novel host plant and more differentiation would be evident. This study does not disprove Thompson, but it does provide a note of caution against applying theories at the wrong scale.

Preference-Performance Hypothesis: The preference-performance hypothesis does a better job at explaining my results. It predicts that one host species will be preferred by all beetles, at least in a certain life stage, and will provide an associated increase in performance for the beetles that feed on it. Preference-performance, however, does not explain all the data. Larvae from E. trichocalyx show a preference for E. crassifolium, but this preference produces worse, not better performance: decreased survival to adulthood and shorter overall lifespan. Likewise, larvae from E. crassifolium do no better if they stay on their host species or if their food is switched to E. trichocalyx.

There is no benefit to staying on E. crassifolium, which would be expected if the preference-performance hypothesis. This result holds even with my poorly randomized sampling; individuals in my choice-feeding experiments lived no longer, and the general lab population on E. crassifolium died sooner than beetles fed E. trichocalyx. The detrimental effect of switching from E. trichocalyx to E. crassifolium was clearly seen in every analysis, so I conclude that this effect is much stronger than the reverse.

For the adults, the picture is just as murky. All adults prefer to eat E. trichocalyx, but they realize only partial benefits from this preference. There is no performance

44 benefit to beetles that eat only E. trichocalyx, which we would expect to see because feeding preference was so strong for E. trichocalyx. The only decrease in performance is for beetles that were switched from the preferred food, E. trichocalyx, to E. crassifolium.

These died much younger than those that stayed on their original host, regardless of whether we look at the individuals chosen for the choice-feeding trials or the larger lab population. This effect is strong enough to be clear even in my poorly randomized sample.

Perhaps once individuals become accustomed to the resinous leaves of E. trichocalyx, it is more difficult for them to manipulate and digest the dense trichomes of

E. crassifolium and they eat less, leading to an early death. This might be because of physiological responses to consuming the resinous coating, which could be easy to turn on in a beetle’s lifetime, but difficult to turn off, or because of the mechanical challenges of chewing the trichomes. I did not measure how much each individual ate, so I do not know if individuals that were switched to E. crassifolium actually ate less than those that started on E. crassifolium. More research would be needed to determine why the switch from E. trichocalyx to E. crassifolium leads to lower performance but not vice versa.

There are two benefits for individuals that eat E. trichocalyx instead of E. crassifolium. The first is increased male attractiveness, but only to females eating E. crassifolium. Eriodictyon trichocalyx males that mated with E. trichocalyx females were accepted in about 50% of their attempts, whereas E. trichocalyx males that attempted to mate with E. crassifolium females were accepted 78% of the time. So a male gets a 28% increase in mating success by choosing a female from E. crassifolium. Females eating E. trichocalyx showed no preference for males from either plant, therefore a male only

45 benefits if he lives on E. trichocalyx then seeks out mates on E. crassifolium. However, females feeding on E. crassifolium lay fewer eggs and produce fewer offspring than females on E. trichocalyx, so males probably would not benefit very much from these matings. Eriodictyon crassifolium females that mated with E. trichocalyx males laid an average of 94 eggs, about half of the 196 eggs laid on average by E. trichocalyx females mated with E. trichocalyx males. These same E. crassifolium females produced just three hatchlings each, compared with nine for E. trichocalyx females mated with E. trichocalyx males. So a male from E. trichocalyx would have to mate with three E. crassifolium females to get the same reproductive success as if he had stayed and mated with a female on E. trichocalyx.

If a male from E. trichocalyx is lucky enough to find a female from E. crassifolium that has traveled to his E. trichocalyx plant, he would definitely benefit from increased attractiveness, but so would all the other males on the plant. The lower offspring production by females on E. crassifolium seems to outweigh any benefit the male gets from his increased attractiveness to E. crassifolium females, especially considering that T. eriodictyonis adults mate repeatedly throughout the summer and with multiple partners. A male who stays on E. trichocalyx has numerous opportunities to mate with females who will produce more offspring than females on the other plant.

Further, if a male were to move from E. trichocalyx to E. crassifolium and stay there, perhaps to take advantage of his increased attractiveness to those females, he would suffer significantly in overall lifespan. Mean survival for males switched from E. trichocalyx to E. crassifolium was just 42 days, compared with 87 days for males that stayed on E. trichocalyx. Because so many males that I switched from E. trichocalyx to

E. crassifolium died early, I was able to conduct only eight trials with this treatment, and in seven of those trials the males did not even attempt to mate. These males may not have been healthy enough for sexual activity. The only male in this treatment that tried to mate was successful. Could attractiveness carry over from time spent eating E. trichocalyx? Or is it possible that adults that have made the switch to E. crassifolium and managed to eat and survive are extremely successful individuals and in very good condition? The survival analysis shows a steep die-off early after the host switch, then a leveling off.

Perhaps only a few very strong individuals are able to make this transition, and those individuals are in such good condition that they are extremely attractive as mates. It would be interesting to repeat the experiment with more individuals to better measure the attractiveness of beetles that were able to switch plants and survive long-term.

The attractiveness of males from E. trichocalyx to females on E. crassifolium is difficult to understand, especially given that these same males are no more attractive to females on E. trichocalyx. If males from E. trichocalyx were in better condition, we would expect that females from E. trichocalyx would also find them more attractive than males from E. crassifolium, but that wasn’t seen. Further, we would expect that females on E. trichocalyx might be healthier than their counterparts on E. crassifolium and therefore be in a position to be choosier about mates, as has been seen in some crickets

(Hunt et al. 2005). Why these females are not choosy about their mates is unclear and deserves further investigation.

The only beetles that see a definite and dramatic performance boost from eating the preferred food are females that fed on E. trichocalyx. Whether they started life on E. trichocalyx or switched to eating it later, females laid more eggs (on average 130 eggs,

47 compared with just 82 for females that ate E. crassifolium) and produced more offspring

(just four for females from E. crassifolium versus 10 for females from E. trichocalyx).

Apparently, something about E. trichocalyx is better for females than E. crassifolium, but what this might be is not clear.

Phylogenetic Conservatism Hypothesis: The best explanation of my results is phylogenetic. Trirhabda eriodictyonis and its sister species, T. diducta, are the only members of the genus that feed on Eriodictyon species. All other Trirhabda species feed on plants in the family Asteraceae. The ancestor of T. eriodictyonis and its sister species

T. diducta switched from Asteraceae species to Eriodictyon recently (Ferguson 1998); therefore, the beetles likely did not coevolve with the plants, but made a host switch after the Eriodictyon species had established their basic chemistry and defense mechanisms.

Also parsimonious is the explanation that the divergence between the beetles species followed the host switch to Eriodictyon. Under this scenario, the beetles would not be adapted specifically to these species of Eriodictyon, but to Eriodictyon in general, and would be able to eat any Eriodictyon species they encounter. It just happens that T. eriodictyonis encounters E. trichocalyx and E. crassifolium in the southern California mountains where it lives. This hypothesis also supports the appearance of T. eriodictyonis larvae on E. parryi. To a female searching for a place to oviposit, E. parryi may seem to be an adequate host, close enough to her usual plant. Adults, able to fly to different plants, may disperse to other hosts after growing up on E. parryi. The adults have not become well adapted or accustomed to E. parryi simply because it is usually not available as a host because it follows fires.

It is interesting to note that there are multiple Asteraceae species within the host range for T. eriodictyonis, including genera used by other Trirhabda species, yet I have never seen T. eriodictyonis beetles on anything but Eriodictyon. Perhaps once the switch was made from Asteraceae to Eriodictyon, the beetles lost the ability to digest their former host plants or lost the appetite for those plants. Once adapted to Eriodictyon, the beetles found the path back to eating Asteraceae was blocked. Again, it would be interesting to test whether the beetles can eat the Asteraceae that exist in their environment, or whether they have evolved to not be able to do so.

Future Research Directions: Unfortunately, this phylogenetic hypothesis still does not explain the adult beetle’s preference for E. trichocalyx, and the scope of this research is not broad enough to answer that question. Something about E. trichocalyx is better for adults than E. crassifolium, resulting in increased egg-laying and offspring production by females. I think the difference may lie not with any advantage in E. trichocalyx, but with the disadvantage of the trichomes of E. crassifolium. The resin on E. trichocalyx does not provide a nutritional benefit itself (Johnson et al. 1985). If the trichomes of E. crassifolium provide no benefit—if they are, for example, made of tough epithelial cells without much cytoplasm (Levin 1973)—then they might dilute the nutritional value of the leaves even more than the resin does for E. trichocalyx. If so, the trichomes would provide a more effective defense against herbivores than the resin, since it decreases fecundity, leaving fewer larvae to attack the plants the next growing season (Levin 1973).

There are many examples of plant species or strains with thick hairs being less beneficial for insects than plants lacking hairs. Levin cites research into hairiness of soybeans,

49 cotton, red clover, and wheat leading to lower levels of feeding, egg-laying, hatching, and larval survival in insect herbivores. The beetles may live on E. crassifolium because it is the plant on which they hatched and it is widespread across their range not generally growing right next to E. trichocalyx, even though E. trichocalyx offers some benefits when the choice is immediate.

It would be interesting to compare the feeding and performance of T. diducta on its two main host species, the resinous E. californicum and the velvety E. tomentosum, to see if it has a similar response to its distinct host plants. They prefer to eat less-resinous leaves (Johnson, et al. 1985), but do they prefer resin over trichomes as T. eriodictyonis adults do? Also worthy of study is whether these two Trirhabda beetle species prefer their own species of Eriodictyon over those of the sister Trirhabda species. The answer to this question would help fill in gaps in our understanding of the allopatric speciation of these sister species. Mating trials might show that they reproduce interspecifically, drawing into question their classification as separate species.

It might also be worthwhile to examine the effect of prior herbivory on plant responses. Some plants respond to herbivory by increasing production of defenses such as resins and trichomes (Agrawal et al. 1999). Plants may also respond with induced tolerance, in which the plant grows thin, cheap, expendable leaves that can quickly photosynthesize. Both species of Eriodictyon I studied are likely to already display inducible responses by the time T. eriodictyonis larvae hatch because they are attacked first by aphids that appear with the first warming of spring, then by a flea beetle, before

T. eriodictyonis hatch. Although such a response has not been studied in Eriodictyon, the two plants seem to react differently to the herbivore damage. Eriodictyon crassifolium

50 appears to put its resources into stronger defense; the young, tender, newly grown leaves are so thick with trichomes on the top and bottom they look and feel like velvet, whereas older leaves have sparser trichomes. Eriodictyon trichocalyx on the other hand, appears to balance efforts between defense and tolerance; the new leaves are markedly thinner and lighter green than older leaves (signs of probable tolerance) and are dramatically stickier than leaves from the previous growing season (signs of probable defense). Whether this is a product of induced tolerance and defense would be interesting to investigate, as would the beetles’ response to plants that have and have not experienced herbivory. In other plants, including E. californicum, the amount of resin and nitrogen can change dramatically during the season, leading to varying herbivore response (Johnson et al.

1984).

Also unclear in this system is how males and females evaluate potential mates.

Neither sex considered longer or shorter mates (length was used in this study as a measure of size) more or less desirable. Perhaps weight might be given more consideration and this should be examined in a future study. But other aspects should be considered as well, such as the way the male approaches the female and strokes her with his antennae. The spotted cucumber beetle (Diabrotica undecimpunctata:

Chrysomelidae) has similar mating behavior to T. eriodictyonis, with the male mounting the female and stroking her with his antennae. Females discriminate among males based on the speed of this antennae stroking; males that stroke faster are accepted more often

(Tallamy et al. 2002). Perhaps T. eriodictyonis females assess males in a similar way.

Conclusion: Trirhabda eriodictyonis and its host plants offer an interesting and unusual window into plant-insect interactions, and one that does not easily lend itself to clear explanation. There is a great deal left to explore and understand about plant-insect interactions in this system. Their proximity to the Los Angeles area and the ease of husbandry make them excellent study organisms whose interactions can illuminate the interwoven ecology around us. It remains fascinating that two plants can be so seemingly different and yet not engender any specialization in this chrysomelid when the family is so well known for near monophagy. This system is another reminder that nature always has another surprise for us and that natural selection is a constant puzzle, a Gordian knot whose details we may never cut through.

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