Sexual behavior in North American cicadas of the genera Magicicada and Okanagana
by
John Richard Cooley
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biology) The University of Michigan 1999
Doctoral Committee:
Professor Richard D. Alexander, Chairman Professor George Estabrook Professor Brian Hazlett Associate Professor Warren Holmes John R. Cooley © 1999 All Rights Reserved To my wife and family
ii ACKNOWLEDGMENTS
This work was supported in part by the Horace H. Rackham School of Graduate Studies, the Department of Biology, the Museum of Zoology (UMMZ), the Frank W. Ammerman Fund of the UMMZ Insect Division, and by Japan Television Workshop Co., LTD. I thank the owners and managers of our field sites for their patience and understanding:
Alum Springs Young Life Camp, Rockbridge County, VA.
Horsepen Lake State Wildlife Management Area, Buckingham County, VA.
Siloam Springs State Park, Brown and Adams Counties, IL.
Harold E. Alexander Wildlife Management Area, Sharp County, AR.
M. Downs, Jr., Sharp County, AR.
Richard D. Alexander, Washtenaw County, MI.
The University of Michigan Biological Station, Cheboygan and Emmet Counties, MI. John Zyla of the Battle Creek Cypress Swamp Nature Center, Calvert County, MD provided tape recordings and detailed distributions of Magicicada in Maryland. Don Herren, Gene Kruse, and Bill McClain of the Illinois Department of Natural Resources, and Barry McArdle of the Arkansas State Game and Fish Commission helped obtain permission to work on state lands. Keiko Mori and Mitsuhito Saito provided funding and high quality photography for portions of the 1998 field season. Laura Krueger performed the measurements in Chapter 7. Artwork in Chapters 7 and 8 is by Melissa Anderson, Dan Otte, and John Megahan.
iii I thank Charles L. Remington for introducing me to the complexities of Magicicada, Oren Hasson, Tom Moore, Mark O’Brien, and Dan Otte for helpful discussion, and Chris Simon, for her pioneering genetic surveys of 13- year cicadas and our conversations about cicadas. Andrew Richards has been instrumental in the project to map the distributions of periodical cicada broods in central Illinois. Although little of that information is included in this thesis, Andrew’s meticulous work and careful thinking have been an important component of all our various and ongoing cicada projects. My development and maturation as a graduate student were strongly influenced by the members of my doctoral committee, Richard D. Alexander, George Estabrook, Brian Hazlett, and Warren Holmes. None of this work could have been possible without the nearly 40 years of effort of Richard D. Alexander. I thank Dick for reigniting my early interest in periodical cicadas. Most of the techniques, approaches, and questions in this work can be traced back to Dick, and the discovery of the new Magicicada species and the female wing flick signal are the legacies of his pioneering bioacoustical studies and his admonition to watch and measure everything. It was at Dick’s suggestion that we followed the behaviors of individually marked females kept separate from males except while under direct observation, allowing us to observe the complete male-female rapprochement sequence. What makes Dick uniquely unique is that he has an uncanny ability, and a proven track record, for being right about how things work. Dick has been tireless in his support, profound in his suggestions, and unwavering in his commitment to seeing that this work meets high quality standards. My friends David C. Marshall and Deborah Ciszek have also provided irreplaceable support, input, and collaboration. The fieldwork in this thesis is the joint collaborative effort of David and myself. The pronoun “I” in this thesis is but a formality; all “I’s” are truly “We’s.” As a collaborative team, we have accomplished far more than we could have separately, and it has been a joy to work with as insightful,
iv careful, and thorough a partner as David. This collaboration has been an important part of my own intellectual development. My thanks would not be complete without considering my family. I thank my parents, Rickie and Bill Cooley, for valuing education above all else, my brothers Bill and Jamie for their interest and technical expertise, my grandmothers, Frances S. Cooley for her determination, and Jane H. Yager for her creativity and patience, and my grandfathers, William H. Cooley for teaching me practical ways to construct things, and Richard S. Yager, for instilling in me a love of the strange, the natural, and the absurd. My wife, Louanne Reich Cooley, is a constant source of support. She designed an inexpensive and portable flight cage, contributed her knowledge of northern Michigan to the studies of Okanagana, and has been incredibly patient and understanding throughout this process. Last, I thank my study organisms. They lead bizarre lives—but then, so do we!
v PREFACE
Sexual behavior in North American cicadas of the genera Magicicada and Okanagana
by John Richard Cooley
Chairman: Richard D. Alexander.
The dense, raucous, mass emergences of North American periodical cicadas (Magicicada spp.) have attracted scientific study since the 17th century. Periodical cicadas are unique in their combination of long life cycles (13- or 17- years) and synchronous development, leading to periodical emergences of adults. Although their long life cycles make it difficult to conduct a longitudinal study of any one periodical cicada population, because populations in different regions of eastern North America are divided into asynchronous “broods,” or year classes, in almost any year, it is possible to study Magicicada. Over four years, I collected natural history information and studied the mating behaviors of 13- and 17- year Magicicada and two species in a non-periodical genus, Okanagana.
vi Chapter 1 is a review chapter, and it owes a major intellectual debt to my experiences as a co-author of a book chapter1 about the evolution of insect mating systems. As I became involved in that project, and as I started to construct my thesis, I realized that I would have difficulty discussing mate choice unless I clarified for myself, at least, the nature of mate choice. In this chapter, I review the reasons, starting with asymmetries in parental investment, why mates should be choosy. I continue with discussions of how sexual conflicts of interest shape mating systems, and the kinds of choice mechanisms and criteria invertebrates are most likely to employ. I conclude with some predictions about possible mechanisms and functions of mate choice in periodical cicadas.
Chapter 2 is largely a methodological chapter. I present data demonstrating that the methods I used to mark and confine cicadas were not likely to cause mortality or behavioral changes that would bias my studies.
Chapter 3 is a brief examination of “seminal plugs” in Magicicada. These structures are usually left behind in the female genital opening after mating. I demonstrate that these structures contain DNA and are likely composed of dried ejaculate, not other specially constructed or secreted materials. Seminal plugs do not prevent future mating attempts, but I suggest that they may be the products of antagonistic coevolution between females who are time-limited and males who can reduce threats to their paternity by imposing time-costs on females’ future mating attempts.
1 Alexander, R. D., D. C. Marshall, and J. R. Cooley. 1997. Evolutionary Perspectives on Insect Mating. Ch. 1 in B. Crespi, J. Choe, eds. The evolution of mating systems in insects and arachnids. Cambridge University Press.
vii Chapter 4 tests two hypotheses for female multiple mating in Magicicada: Females remate either to replenish depleted supplies or to effect postcopulatory mate choice. I demonstrate that interrupting a female’s mating tends to promote remating, while matings with an inappropriate, conspecific mate do not. Further, mated females do not actively seek additional mating opportunities, nor are they sexually attractive to males. Interrupted females, however, solicit second matings. These results are most consistent with the hypothesis that females remate for replenishment, and they disprove the hypothesis that females use postcopulatory choice mechanisms to make species-level mating decisions.
Chapter 5 documents a previously unknown female wing flick signal, used by females to signal sexual receptivity. No such signal has previously been reported in North American cicadas, although similar signals are known in some Australian and New Zealand species. This chapter documents the signal in different Magicicada species and also describes a bizarre effect (behavioral bisexuality) of a fungus disease in males, as well as a specialized, competitive signal males use to “jam” the acoustical signals of interloping rivals. The newly described behaviors allow formulation and testing of a hypothesis for the two-part calling songs of Magicicada species such as M. septendecim . The two-part structure of these songs facilitate female discrimination of an individual male’s call against a background chorus.
Chapter 6 describes a new 13- year species, Magicicada neotredecim. M. neotredecim male calling songs and female mate acceptance criteria are distinct from those of the similar, synchronic and partially sympatric species, M. tredecim. Perhaps the most compelling piece of evidence that M. neotredecim and M. tredecim are separate species is that M. neotredecim has undergone reproductive character displacement as a result of its interactions with M. tredecim. The existence of this new species suggests two general hypotheses for speciation in Magicicada, the “nurse brood” hypothesis, and
viii the “canalization” hypothesis. Each could explain the origin of M. neotredecim, and each leads to specific predictions that may someday clarify how M. neotredecim came to be. This chapter ends with a prediction, based on the “nurse brood” hypothesis and patterns of reproductive character displacement, suggesting what other undiscovered Magicicada species might be like and where they might be found.
Chapter 7 explores the possibility that females discriminate among conspecifics on the basis of phenotypic symmetry. Symmetry has been promoted as a universal and reliable mate choice criterion. I begin by reviewing asymmetry theory in order to clarify the nature of the relationship between symmetry and mating success. I conclude that asymmetry has no special status as a mate choice criterion, but rather, it is like any other condition-dependent trait. I present the results of a study of symmetry and mating success in periodical cicadas demonstrating that males with more symmetrical forelegs are most likely to mate. However, the differential success of some males is more likely due to indirect mate discrimination than to evolved female mate choice mechanisms.
Chapter 8 compares the behaviors of Magicicada to members of another genus, Okanagana. I present the results of a 3- year biogeographic survey of the distributions of Okanagana canadensis and O. rimosa in northern Michigan. I then describe the mating behaviors of both species, which involve females approaching stationary calling males, and contrast them to those of Magicicada, which involve males searching for females. From this contrast, I suggest hypotheses for how the unique mating system of Magicicada may have evolved from mating systems such as those of Okanagana.
ix TABLE OF CONTENTS
DEDICATION...... ii
ACKNOWLEDGMENTS ...... iii
PREFACE...... vi
LIST OF TABLES ...... xii
LIST OF FIGURES ...... xv
LIST OF APPENDICES ...... xviii
CHAPTER
I. MATE CHOICE IN INVERTEBRATES, WITH SPECIAL REFERENCE TO PERIODICAL CICADAS (MAGICICADA SPP.) ...... 1
II. EXPERIMENTAL METHODS IN STUDIES OF PERIODICAL CICADAS: EXAMINATIONS OF POSSIBLE METHODOLOGICAL BIASES IN THE STUDY OF PERIODICAL CICADAS (MAGICICADA SPP.) ...... 30
III. MATE GUARDING AND THE NATURE AND POSSIBLE FUNCTIONS OF “SEMINAL PLUGS” IN PERIODICAL CICADAS (MAGICICADA SPP.)...... 53
IV. FEMALE MULTIPLE MATING AND MATE CHOICE IN A PERIODICAL CICADA, MAGICICADA SEPTENDECIM ...... 70
V. SEXUAL SIGNALING IN PERIODICAL CICADAS, MAGICICADA SPP...... 97
x VI. REPRODUCTIVE CHARACTER DISPLACEMENT AND ALLOCHRONIC SPECIATION IN PERIODICAL CICADAS WITH A NEW SPECIES, MAGICICADA NEOTREDECIM...... 152
VII. ASYMMETRY AND MATING SUCCESS IN A PERIODICAL CICADA, MAGICICADA SEPTENDECIM (HOMOPTERA: CICADIDAE) ...... 202
VIII. THE GEOGRAPHIC DISTRIBUTION AND MATING BEHAVIORS OF TWO CICADA SPECIES (OKANAGANA SPP.) IN NORTHEAST MICHIGAN ...... 235
xi LIST OF TABLES
Table
1.1. Mate discrimination and its alternatives ...... 27
2.1. Reported effects of methods for marking individual insects...... 46
2.2. Results of “seminal plug” analysis ...... 48
2.3. Cage density resampling results...... 49
2.4. Results of pretarsal clipping experiment...... 50
2.5. Summary of resampling results comparing remating frequencies in small bag cages to remating frequency in large flight cage...... 51
3.1. Study sites 1995- 1997...... 65
3.2. Mating durations in Magicicada septendecim, Okanagana rimosa, and O. canadensis...... 66
3.3. Results, female mating interruption experiment...... 67
3.4. Results, resampling algorithm to evaluate whether females with seminal plugs were unlikely to remate ...... 68
4.1. Mating durations in Magicicada septendecim...... 90
4.2. Mating interruption experiment: Likelihood of remating first or ovipositing first, and presence/absence of sperm plug ...... 91
4.3. Counts of eggnests in mating interruption experiments ...... 92
4.4. Results of playback/ mating status experiment...... 93
xii 4.5. Mating interruption and female wing flick response to playbacks of male calls ...... 94
4.6. Census data from female attractiveness experiment...... 95
5.1. Traits distinguishing Magicicada species...... 129
5.2. The sexual sequence in Magicicada species...... 130
5.3. Study Sites, 1995- 1998 ...... 131
5.4. Length of teneral period, in days ...... 132
5.5. Mated and unmated female M. septendecim responses to 2- minute playbacks of male calls...... 133
5.6. Wing flick responses of females to a series of alternating con- and hetero- specific calls ...... 134
5.7. Male M. septendecim responses to simulated wing-flick signals ...... 135
5.8. Male M. –decim movement direction following artificial wing flick signal...... 136
5.9. Experiments on male chorusing behavior...... 137
5.10. M. septendecim wing-flick signals responses to frequency-modified playbacks of male song...... 138
5.11. Contrasts of female wing flick responses to playbacks of complete artificial calling songs and partial artificial calling songs ...... 139
5.12. Effects of background chorus intensity on female wing flick responses to playbacks of complete artificial calling songs and partial artificial calling songs ...... 140
5.13. Effects of M. -decim “interference buzz.”...... 141
6.1. Traits distinguishing Magicicada neotredecim and other Magicicada species...... 184
6.2. Relationship of abdomen color to male call pitch and female pitch preference...... 185
xiii 6.3. Dominant frequencies of Magicicada septendecim chorus recordings in the UMMZ sound library...... 186
7.1. Tests for normality of signed, scaled asymmetries...... 224
7.2. Tests for measurement error and handedness ...... 225
7.3. Numbers of cicadas used in analysisof symmetry and mating success ...... 226
7.4. Pearson pairwise correlation matrix for character asymmetry...... 227
7.5. Pearson pairwise correlation matrix for character size...... 228
7.6. Pearson pairwise correlation matrix for character sizes and asymmetry....229
7.7. Comparison of asymmetry of mated and unmated males for each character...... 230
7.8. Results of tymbal rib asymmetry analysis ...... 231
7.9. Results of resampling algorithm to evaluate whether males with wing asymmetries were as likely to mate as normal males...... 232
8.1. Lengths of Okanagana canadensis calling and silent bouts...... 256
8.2. Mating durations in Okanagana and Magicicada septendecim...... 257
8.3. Responses of male cicadas to presentation of model cicada...... 258
xiv LIST OF FIGURES
Figure
1.1. Distribution of male traits in relation to female threshold acceptance value...... 28
1.2. Comparison of threshold acceptance criteria and best-of-n choice ...... 29
2.1. Survivorship of marked and unmarked cicadas...... 52
3.1. Photograph of gel showing RAPD-PCR amplification...... 69
4.1. Numbers of eggnests laid by female M. septendecim...... 96
5.1. Stylized sonogram of male call/female wing flick coutship duet for M. -decim ...... 142
5.2. Sonogram of male M. -decim call and female wing flick response...... 143
5.3. Sonogram of male M. -cassini call and female wing flick response ...... 144
5.4. Sonogram of male M. -decula call and female wing flick responses ...... 145
5.5 Comparison of male responses to a model female that moves only...... 146
5.6. Male responses to actions of model female that moves and makes sounds ...... 147
5.7. Sonogram of response of M. -decim male with Stage I Massospora cicadina infection to an M. -decim call ...... 148
5.8. Sonogram of response of M. -cassini male with Stage I Massospora cicadina infection to an artificial M. -cassini call...... 149
xv 5.9. Responses of female M. septendecim to playbacks of artificial, pure tone calls and portions of calls against an artificial background chorus ....150
5.10. Sonogram of male M. -decim call and interference buzz of nearby male...... 151
6.1. Geographic distribution of the seven periodical cicada species ...... 187
6.2. Comparison of typical 17- year Magicicada chorus with 13- year chorus containing M. tredecim and M. neotredecim...... 188
6.3. Spectrogram showing a two-banded, mixed-species chorus ...... 189
6.4. Air temperature effects on M. -decim chorus frequency ...... 190
6.5. Sonogram of male M. neotredecim calls illustrating consistency of individual male calling song pitch...... 191
6.6. Power spectrum of mixed M. neotredecim and M. tredecim chorus with accompanying frequency histograms of male song pitches and female pitch preferences ...... 192
6.7. Relative proportions of M. neotredecim and M. tredecim estimated from chorus recordings of the 1998 emergence of Magicicada 13-year Brood XIX...... 193
6.8. Geographic variation in dominant chorus pitch of M. neotredecim, showing reproductive character displacement in and near the region of overlap with the low-pitched M. tredecim...... 194
6.9. Schematic representation of calling song displacement in Brood XIX M. tredecim and M. neotredecim ...... 195
6.10. Geographic variation in dominant chorus pitch of M. tredecim, suggesting weak reproductive character displacement...... 196
6.11. Box plots of male calling song pitch, right wing length, thorax width, and first sternite width of M. tredecim (Sharp Co., AR) and M. neotredecim (Sharp Co., AR. and Piatt Co., IL)...... 197
6.12. Formation of an incipient Magicicada species by “nurse brood facilitation” of life cycle mutants...... 198
xvi 6.13. A model of Magicicada speciation using nurse brood facilitation and reinforcement of premating isolation...... 199
6.14. Two hypotheses for the origins of Midwestern 13- year cicadas ...... 200
6.15. A model of Magicicada life cycle evolution via canalization of a climate-induced life cycle shift...... 201
7.1. Characters used in analysis of phenotypic symmetry...... 233
7.2. Comparisons of cumulative asymmetry scores for mated and unmated males ...... 234
8.1. Locations of study sites, Okanagana canadensis, O. rimosa 1996- 1998 ...... 259
8.2. 1996 distribution map of O. rimosa and O. canadensis ...... 260
8.3. 1997 distribution map of O. rimosa and O. canadensis ...... 261
8.4. 1998 distribution map of O. rimosa and O. canadensis ...... 262
8.5. Location of O. rimosa study site in 1998...... 263
8.6. Sonogram of male Okanagana canadensis call...... 264
8.7. Sonogram of male Okanagana rimosa call ...... 265
8.8. Sketches of lateral view of typical Okanagana male genitalia...... 266
8.9. Sketches of lateral view of typical Magicicada male genitalia ...... 267
xvii LIST OF APPENDICES
Appendix
A. C++ code for mating system simulation...... 269
B. Pascal program code for comparing experimental cage densities to densities of free cicadas observed on cage-sized branches...... 287
C. 1995 Cage mating experiments...... 290
D. C++ code for resampling program to evaluate remating frequencies in large and small cages ...... 291
E. C++ code for seminal plug resampling algorithm ...... 302
F. Male frequency collection, 1998 Brood XIX ...... 313
G. Playback experiments, 1998 Brood XIX ...... 316
H. Locality, chorus pitch data, 1998...... 317
I. Holding jig for symmetry measurements...... 319
J. Symmetry and size measurements for mated and unmated cicadas ...... 320
K. Code for resampling algorithm to evaluate whether males with asymmetrical wing venation were over- or under-represented among males that did not mate in mating experiments ...... 322
L. Miscellaneous field notes on Okanagana...... 326
xviii CHAPTER I
MATE CHOICE IN INVERTEBRATES, WITH SPECIAL REFERENCE TO PERIODICAL CICADAS (MAGICICADA SPP.)
Abstract
Most invertebrates have limited adult experiences and mating opportunities. Thus, studies of invertebrate mate choice require a different approach from many vertebrate-based mate choice models, which implicitly allow animals to make use of past experiences and multiple mating opportunities. Careful consideration of the benefits realizable from mate discrimination as well as the ways in which discrimination may take place can reveal whether observed mating patterns are manifestations of evolved mate choice mechanisms or whether they are incidental effects of other behaviors. For example, for those insects lacking male parental investment and multiple mating opportunities, female choice criteria would not include material benefits, and mate choice mechanisms would be unlikely to involve comparisons. Alternatively, if existing male variation has minimal fitness consequences for females, then the costs of discrimination may outweigh the benefits, and any observed mating biases may be incidental rather than
1 the results of mate choice. With this in mind, I describe a series of studies designed to evaluate whether female periodical cicadas (Magicicada spp.) choose mates and if so, what choice criteria they are likely to use.
Introduction
Every aspect of behavior, life history, and ecology provides unique raw materials for the operation of sexual selection. Many vertebrates have extended, juvenile periods in social contact with conspecifics, multiple opportunities for observing all aspects of adult behavior before becoming sexually mature, multiple breeding opportunities, and the possibility of gaining proficiency at acquiring mates. Insects, in contrast, often have single breeding opportunities (i.e., they are semelparous), no experience with conspecifics prior to mating, and few opportunities for rectifying errors. Life history patterns common among invertebrates often have no vertebrate parallels; thus, vertebrate- based models of sexual selection and mate choice (Bradbury 1981, 1985, Höglund and Alatalo 1995) may not accurately characterize invertebrate mating systems.
Rather than revise or modify vertebrate mate choice models, this review provides a general discussion of how the opportunities for mate choice and the benefits from choosing create the selective pressures shaping a given species’ mating system. This general discussion of mate choice leads to specific predictions for how invertebrates should be expected to choose mates or mating opportunities and concludes with a discussion of mate choice in periodical cicadas (Magicicada spp.), whose lek-like mating aggregations suggest a history of strong sexual selection.
A caveat
Mate choice itself may be difficult to observe directly, so behaviorists usually measure correlates of choice, such as polygyny. The hazards of assuming that observed mating patterns such as extreme polygyny necessarily result from choice are illustrated
2 by the following example: Suppose that in a population of 50 males and 50 females, males are always sexually receptive, females become receptive sequentially (thus the operational sex ratio is always 50 males:1 female), and females mate with the first male encountered. This scenario, written as a C++ computer program (Appendix A) and run for 10,000 iterations, leads to the following results: First, counting the maximum number of matings achieved by any male in each iteration, the 95th percentile is 5 matings. That is, in 9,500 of the iterations, at least one male achieved 5 matings and was notably more polygynous than others. In rare cases, males received up to 9 matings, and invariably, some males received no matings. At first glance, the population appears to be characterized by a 50:50 sex ratio, because a random sample of individuals drawn from the population would contain equal numbers of males and females. Thus, it would be tempting to conclude that the mating skew results from some form of mate choice. However, the operational sex ratio is male-biased, because at any given time only one female is receptive. The skewed operational sex ratio causes some males, by chance, to be much more successful than others even though females mate indiscriminately (see Boggs 1995 for a similar scenario). Some males were more successful than others simply because they were, by accident, in the right place at the right time.
This example, in which a strongly male-biased operational sex ratio leads to polygyny, highlights what should be the first question in any study of mate choice: Are the animals involved behaving such that they receive fitness benefits from participating in mate choice, or are some patterns better explained as spurious or incidental effects? The answer to this question lies in understanding the benefits possible from choice and the ways in which animals might obtain them.
Mate choice
The distinction between mate choice and its alternatives corresponds to Darwin’s original distinction between sexual and natural selection (Table 1.1; Darwin 1859, 1871,
3 Arnold 1983). As with Darwin’s forms of selection, mate choice and its alternatives are not easily separable. Mate choice results from individual actions that reduce the probability of mating with some members of the opposite sex (Parker 1983a). Mate choice includes both direct and indirect choice (see below), or all circumstances in which the identity of a mating partner affects fitness, either through genetic materials provided, or through potential mates’ differential ability to deliver resources or appear at times or locations most appropriate for mating (see Halliday 1983). In choosy species, members of the chosen sex can take actions that increase their own chances of mating success. The alternatives to mate choice are context choice and haphazard mating. Context choice occurs when fitness benefits result from restricting mating to particular contexts, but potential mates have no differential abilities to appear in the appropriate context and cannot take actions to increase their mating success; thus mating is independent of partner identity. Under haphazard mating, differences in mating context or partner identity have no influence on fitness.
Why choose mates at all?
Each sex strives to defend its post-zygotic investment against preemption or usurpation by members of the other sex (see Wiley and Poston 1996). Post-zygotic investment is the “parental investment” of Trivers (1972; also Alexander and Borgia 1979) and refers to any investment that is irretrievably committed to a particular zygote or collection of zygotes. In contrast, pre-zygotic investment is mating effort or resources committed to gametes (or collections of gametes) or to the production and/or delivery of gametes before fertilization. As with any artificial dichotomy, it is not always easy to distinguish pre- and post-zygotic investment. Even the term “post-zygotic investment” is misleading; the terms “investment in zygotes,” or “zygotic investment” are more accurate, because they refer to investment that has its most profound effects after fertilization, although it may be dispensed at any time before or after zygote formation
4 (Alexander and Borgia 1979). Variations in zygotic investment directly affect offspring quality. Thus, parental care by either sex is investment in zygotes, because it is delivered to particular offspring. Such care may actually be dispensed before the zygote is formed; for instance, egg yolk is investment by a female in her zygotes, because variation in the amount or quality of the yolk affects a particular zygote’s likelihood of success. In contrast, male courtship is mating effort, or investment in gametes, not zygotes, because different levels of investment affect only the likelihood that a male will, by delivering gametes, produce a zygote, not the success of any particular zygote. Like any resource, the zygotic investment of one sex is available for use and exploitation by members of the other.
Control of fertilization
Each sex strives to control the act of fertilization in order to defend its zygotic investment against preemption or usurpation. The costs of both finding additional mates and of zygotic investment influence sexual choosiness, but these factors are not independent. As members of one sex increase their zygotic investment, members of the other sex seek them out, so high zygotic investment is correlated with low mate search costs (Parker 1983a). Thus, the sex with the greater zygotic investment has the greater incentive to protect its investment and avoid mating indiscriminately, because its reproductive output is limited more by cost of the investment than by the cost of finding mating partners, while the sex with the lesser zygotic investment is limited by its ability to find willing mates and cannot afford to forego mating opportunities.
In many sexual species, females initially have the greater zygotic investment; they produce large, well-provisioned gametes specialized for post-zygotic success (Parker et al. 1972, Alexander and Borgia 1979). Delivery of resources to gametes makes the resources available to zygotes immediately after fertilization (Alexander and Borgia 1979). Associated with gamete provisioning is physical control and protection of the
5 gametes, probably for the following reasons: Females that evolved to dedicate greater and greater amounts of resources to zygotes also evolved to protect their investment by increasing physical control over them, such as by retaining them within the body after fertilization. As females increased their control, they gained confidence of the maternity of the zygotes they invested in and benefited by investing even earlier, in gametes. Thus, for many females, the distinction between prezygotic and zygotic investment is blurred, because investment in gametes translates directly into investment in zygotes. As females gained control over gametes, such as by retaining them inside their bodies for as long as possible, they also increased their control over the fertilization process.
Conflicts of interest between the sexes
The sexes’ relative levels of post-zygotic investment (which, as noted above, are related to confidence of parenthood) affect the degree of conflict over mating decisions. If the strategies of the sexes are identical, e.g., if both sexes have substantial zygotic investment, or if neither sex invests substantially and therefore neither discriminates, then there is little conflict over investment (Trivers 1972, Alexander and Borgia 1979, Parker 1979). When one sex invests more in zygotes than the other invests, the sexes will be in conflict over mating decisions (Parker 1979). Sexual conflict will, in each sex, favor the evolution of adaptations for controlling fertilization and thwarting the ability of the other sex to do so (see Rice 1996). Because the optimal mating strategy for each sex is contingent upon the other’s strategy, the sexes evolve in response to each other, with both sexes using a variety of strategies to gain control over fertilization (Parker 1979, 1983b, Alexander et al. 1997). Females with internal fertilization have an immediate control advantage; male responses to this advantage include genitalia that place sperm in close proximity to a female’s eggs (Parker 1970, Alexander et al. 1997), contributed substances that alter female physiology (see Lum 1961; Riemann and Thorson 1969; Burnet et al. 1973; Leopold 1976; Scott 1986; Chen et al. 1988; Kingan et al. 1993; Gromko,
6 Newport, and Kortier 1984; Gromko, Gilbert, and Richmond 1984, Wolfner 1996; Eberhard 1997 pp. 37- 40), or any number of other physiological or behavioral adaptations. Each sexes’ adaptations manipulate the other’s mating costs and benefits to effect control over fertilization. For instance, a male might restrict access to some valuable resource (such as a nesting site), trading resource access for matings. If the cost to a female of losing control of fertilization is less than the cost to her of losing access to the resource, then she will accept the trade. Each sexes’ adaptations to gain control may promote counter adaptations, leading to evolutionary races in which a wide range of strategies and possible counter strategies contribute to the complexity of the mating system (Parker 1983b). By inference, any species characterized by complex sexual interactions during sexual rapprochement likely exhibits some form of mate choice, because elaborate sexual interactions suggest that members of the chosen sex can take actions to improve their odds of mating. It is the mutual contingency of the sexes’ mating strategies, a consequence of mate choice, that leads to complex sexual adaptations (Alexander et al. 1997).
Selection and mate choice
Mate choice is an adaptation for producing genetically superior offspring, acquiring direct phenotypic benefits convertible into reproductive output, or both. Genetic benefits from choice fall into two non-exclusive categories: (1) Arbitrary benefits or (2) Offspring quality benefits. Arbitrary benefits consist of the genes coding for ornaments or other traits enhancing sexual attractiveness. Offspring quality benefits include the genetic materials necessary for producing offspring well equipped to survive and reproduce.
For example, in a given habitat, male Colias butterflies with the most appropriate enzyme variants not only gain from efficiencies in flight and foraging, but they are also more able to produce long and successful courtship displays (Watt et al. 1986). Females
7 mating with the most actively displaying males produce offspring that realize flight and foraging benefits and sons that produce more attractive sexual displays (Weatherhead and Robertson 1979; but see Kirkpatrick 1985). In this case, choosy females gain genetic benefits with aspects of both arbitrary and offspring quality benefits.
Females may also discriminate among males in order to receive phenotypic benefits. Such benefits include time and effort conserved by mating with easily located males, acquisition of male-provided nuptial resources, protection from injury or other harm, and reduced costs, or at least minimization of losses, associated with acquiescence to persistent male harassment. To the extent that male ability to provide phenotypic benefits is heritable, female choice for such benefits will impose selection on males, and thus phenotypic and genetic benefits are not sharply distinct.
Mechanisms of mate choice: Direct vs. indirect
Mate choice involves either direct assessment of potential mates or indirect biases (Wiley and Poston 1996, Sullivan 1989). In species with female choice, direct choice requires individual recognition and memory of male characteristics, and it requires females to perceive their different mating options. Indirect choice requires that the highest quality males have stereotypical characteristics, and that females act in ways that discriminate against males not exhibiting such qualities even though they may not have perceived the different mating options available to them.
Females may favor a particular male indirectly, rather than directly, on the basis of several possible cues: 1. Females may be attracted to the loudest or most easily located signals apart from any comparisons among those signals (The “passive attraction” hypothesis; Parker 1983a). 2. Females may accept only those males that have dominated in a contest with others. Male-male contests, in effect, provide résumés of the competitors’ lifetime achievements (Borgia 1979). A female may be absent for the contest itself, but the behavior of the contestants after the contest restricts her mating options. For
8 example, Burk (1983) discusses how losers in male-male contests in the field cricket Teleogryllus oceanicus reduce their calling; thus, females are more likely to mate with the victors. Moore and Moore (1988) present similar results for the cockroach Nauphoeta cinerea. 3. Females may favor males on the basis of some trait or resource. A female may feed on a copulatory gift or secretion, breaking off the mating when the gift is gone and thus disfavoring males with inadequate or poor-quality gifts. Thornhill (Thornhill and Alcock 1983) discusses how male scorpionflies providing inferior nuptial gifts are less successful as mates, because females stop mating once they have consumed the gift. 4. Females may engage in behaviors that discriminate among males according to their relative performance with respect to some environmental cue. Females may accept only males who, because of particular sensory, locomotory, or thermoregulatory abilities, are able to be in the right place at the right time. Honeybee mating swarms may be an example of such mate discrimination (see Winston 1987).
Others have classified these possibilities differently; for instance, Lloyd’s “passive filtering” would include aspects of 1 and 4 above; his “passive selection” is most similar to 2; “active filtering includes 1 and 4; while “active selection” includes aspects of 2, 3, and 4 (Lloyd 1979).
At the crudest level, all females exercise some degree of “indirect” mate choice by successfully mating only with living, sexually mature males (Parker 1979). It is trivial to point out that selection thus favors females who make this distinction, but this distinction is hardly the result of any specially evolved or elaborated adaptation. To the extent that more subtle male variation is heritable and has fitness consequences for females, selection can reinforce both sexes’ phenotypic cues and predictable behaviors involved in pairing, leading to evolved mate choice mechanisms.
Mate choice criteria
Mate choice criteria fall on a continuum of two extremes: (1) Females may, actively or passively, make relative comparisons among available mating partners (“best- of-n” choice, Janetos 1980), or (2) Females may accept as mates males who meet or exceed internally generated criteria (“threshold” or “minimal criterion” choice; Janetos
9 1980). Each of these models leads to distinct predictions about the kinds of behaviors males and females should exhibit (Janetos 1980, Alexander et al. 1997). Note that mate choice criteria do not constrain the evolution of mate choice mechanisms (either direct or indirect), and all four permutations of mate choice mechanisms and choice criteria are theoretically possible.
Direct relative comparisons: Best-of-n choice. Females exercising best-of-n choice seek to identify and mate with the best available male in a group of n- males. Best-of-n choice criteria are relative, open-ended, and require a comparison of potential mates. As long as such relative comparisons do not exact prohibitive costs, and as long as female fitness covaries with incremental changes in the resources or genetic materials provided by mates, selection will favor females using best-of-n choice. Best- of-n choice is functionally equivalent to threshold choice (see below) in which the acceptance criteria are changeable and influenced by the kinds of males a female encounters. The mechanisms involved in best-of-n choice need not be complex: Females could choose the best mate of those available by setting their acceptance threshold to the value of the first male encountered, raising it after encountering a higher quality male, and mating with the highest quality male when the threshold becomes fixed or remains stable after repeated encounters. Male-male contests could also restrict sexual access of all but the best males present. Females may require potential partners to simultaneously perform tests of skill to reveal relative quality. High costs, in terms of time and effort, and limited opportunities for making comparisons make short-lived, semelparous animals such as insects unlikely to exercise best-of-n choice (Alexander et al. 1997).
“Indirect” or “non-simultaneous” relative comparisons. Males, through active, intrasexual competition, can sort themselves in ways that limit sexual or resource access by lesser quality individuals (Cox and LeBoeuf 1977) and thus females’ mating options will be restricted as if they were exercising best-of-n choice. Females
10 making indirect, relative choices do not require simultaneous tests of skill; instead, they create situations in which males compete with each other before mating, and they will mate readily even if only one male is present.
Some “lekking” insects may provide examples of indirect best-of-n choice: If male insects form aggregations through which receptive females fly, and if males within an aggregation chase, catch, and mate with the females, these behaviors place premiums on male flight and perceptual capabilities and cause male mating success to covary with male quality. Lloyd (1979) points out how, by imposing sexual selection on such traits, females will cause males to compete to demonstrate their prowess (p. 337). If more than one male is present in such a “lek,” it provides an arena in which male competitive interactions sort males on the basis of quality. Insects that engage in multi-male courtship chases, such as honeybees (see Winston 1987 p. 207), midges (McLachlan and Cant 1995), some Lepidoptera (Sullivan 1981, Pliske 1975) and syrphid flies (see Chapman 1954, Cooley “hotrod” model, unpublished) may all have mating systems characterized by this kind of choice.
Threshold or minimal criterion choice. Females exercising threshold choice discriminate against a class of potential mates that fail to meet minimal acceptance criteria (Janetos 1980). Males meeting the criteria are all equally acceptable, regardless of differences among them. Females acquire the choice criteria without regard to the characteristics of the male population, and once acquired, the criteria are fixed and unchangeable. The feature most distinguishing best-of-n from threshold choice is that under threshold choice, all acceptable males are equally acceptable independent of other qualities, and differences among acceptable males have no appreciable fitness consequences for females, while under best-of-n choice, in a given group of males, there is one best mate, because each male’s acceptability as a mating partner depends both on his own qualities and on the characteristics of the population at large. Some mate choice
11 mechanisms, such as “one step” or movable threshold mechanisms may appear to involve threshold choice, but from the standpoint of sexual selection they are more similar to best-of-n than to threshold choice because they involve relative comparisons and “thresholds” generated only after females sample the male population (Janetos 1980).
If threshold choice criteria exclude a large proportion of the males in the population, then females with lower acceptance thresholds will locate mates more readily than females using arbitrarily high criteria (Fig. 1.1). This increased efficiency may provide phenotypic benefits compensating any loss in mate quality, promoting an increase in the proportion of females with lower criteria in future generations. Thus, arbitrarily high criteria are unlikely to persist, and threshold choice is unlikely to lead to the extremes of sexual selection possible under best-of-n choice.
If male variation with respect to mate choice criteria is heritable, and if female choice criteria are unchanging and uniform across all females, then over the course of several generations, unless mutation rates are extremely high, threshold-based mate choice should diminish the proportion of unacceptable males, because all offspring will have fathers that met or exceeded the choice criteria. The exception, in which a long history of threshold choice would not lead to the elimination of unacceptable variants, is the case of species discrimination by threshold choice, because all the unacceptable mates would belong to a different species. Within a species, males subjected to threshold choice would be selected to exceed the acceptance threshold to the smallest extent that ensures that they will mate and that their offspring also exceed the threshold. After several generations of threshold choice, females would face a pool of potential mates most of whom slightly exceed the acceptance threshold, but among whom there would be few consequential differences. Thus, differences in male quality could be trivial in the face of the direct advantages females might gain from choosing mating times and locations that maximize their reproductive output. In species for which this is true, there
12 would be little evidence of mate choice; instead, females would mate so as to maximize direct benefits (such as associated with time or place of mating) to themselves while the identity of their mate would be largely irrelevant.
Many studies of insect behavior seem to suggest threshold choice. Scorpionflies (Hylobittacus apicalis ) obtaining nuptial gifts above a certain size threshold are accepted by females, while those offering smaller gifts are rejected or allowed only brief copulations with little or no chance of paternity (Thornhill 1980, Thornhill and Alcock 1983 p. 367). Females do not differentiate among males providing acceptable gifts, but appear to reject mates on the basis of a fixed gift size criterion or threshold.
Moore and Moore (1988) demonstrated that female roaches (Nauphoeta cinerea ) use absolute, rather than relative, preferences when mating. Male roaches formed stable dominance hierarchies, and females appeared to make best-of-n choices, because they decreased courtship time and increased copulation time as male rank increased. However, female behavior was independent of the number of males present, and females deprived of any previous experience with males mated with the first male presented. Thus, females could not have made relative comparisons among dominant and subordinate males, and their mating behavior appears to involve an acceptance threshold, which all males in the experiment met or exceeded.
The action of an absolute mate acceptance threshold in Nauphoeta cinerea does not explain fully why female behavior was different for males of different dominance rank. Some aspects of male behavior provide clues: For example, subordinate males generally delay longer before initiating courtship (Moore and Breed 1986), courtship and mating duration are influenced by quantity and quality of male pheromone (Breed et al. 1980, Moore 1988), and these effects are independent of male-male competition. Furthermore, when females are presented with mating partners sequentially instead of simultaneously, they do not appear to discriminate (Breed 1983). The behavior of
13 females presented with solitary males is evidence for threshold choice; however, superimposed on this choice is another factor: Inherent qualities of dominant males apparently cause them to be more easily accepted or perceived by females, independent of the action of female acceptance criteria.
Passive attraction. “Passive attraction” (Parker 1983a) blurs the distinction between relative and threshold choice mechanisms. Under the passive attraction hypothesis, females may have an acceptance threshold, and males act in ways that ensure they will meet or exceed that threshold. If males employ mate attraction signals or complex courtship, the more readily females perceive such signals, the more likely they are to mate (Parker 1983a). Thus, females may tend to approach signaling males who are more obvious without directly making a relative comparison. For example, if two males were to sing at equal volume, one near a female and one far, then we would predict that the female should approach the nearest one. Similarly, if two males were at equal distance from a female, but one produced a louder signal than the other, the female should approach the louder male. This “passive attraction” can appear to be best-of-n choice if two males court simultaneously, because the female will respond only to the more apparent male. In reality, the female perceives only one of the males, and is not actually choosing. Animals assorting by passive attraction can appear as though they are selecting mates, but any choice is indirect at best. It is possible that the mate sorting qualities of female perception are incidental and provide no selective benefits over and above the benefits of being able to detect mates at all. Males, however, will be subject to selection, to the extent that male ability to make use of appropriate advertisement times, locations, channels, and intensities is heritable (Alexander et al. 1997). Although passive attraction in some ways sorts males relatively, it does not necessarily lead to male-female antagonistic coevolution because the sexes do not evolve strategies in response to each other. Instead, one sex evolves strategies to cope with the unchanging problem of being distinguishable from its surroundings or with the problem of prevailing in competition
14 with members of its own sex. However, if increased male display intensity thwarts female interests, intersexual competition becomes a more important selective force, and, rather than passive attraction, the mating system may be better characterized by a sexual conflict-based model such as chase-away selection (Holland and Rice 1998).
Jang and Greenfield (1996) demonstrated that female wax moths (Achroia grisella) were most likely to orient towards louder male signals, male signals with more rapid pulses, and male signals with longer pulses. Thus, the more evident a male’s signal, the greater the chance of a female response. Sometimes both a mate acceptance threshold and “passive attraction” seem to be involved in mate choice, as suggested by the example of Nauphoeta cinerea, (see above), as well as by male wing amputation experiments in fruit flies (Drosophila melanogaster ; Robertson 1982), in which the greater the area of wing removed, the longer the courtship duration before copulation. Male Drosophila without wings rarely mated, but as long as males had some wing area remaining and were able to produce some song, they eventually mated. A threshold, in which females require males to produce some song before mating, could explain why wingless males failed to mate, and female difficulty in perceiving quiet males could explain why partially amputated males courted longer before copulating.
Conflicts of interest and timing of mate choice
Mate choice could occur at any stage of the sexual sequence, including after copulation. It is in the interests of the choosy sex (usually the female) to effect mate choice as early in the mating sequence as possible. Early in the sequence, she retains substantial control over the mating decision; however, she also has little information on which to base her actions (Alexander et al. 1997). Thus, females should seek to cause males to reveal information about themselves before males have an opportunity to obtain a mating by force. Because the effectiveness of forcing increases as male and female come into closer proximity and more intimate contact, it is in the male’s interests either
15 to: (1) convince the female to commit to him early in the sequence, or (2) delay the female’s final mating decision until he approaches and has a high probability of compelling a mating in the event the female rejects him. Males should be selected, in so far as is possible, to refrain from including in calling songs or other sexual advertisements any information that could jeopardize their ability to approach or be approached closely by females. Thus, there is a conflict of interest between male and female over the nature and timing of information to be transmitted, and there is also a conflict over when in the sequence the female should make her final mating decision.
Multiply-mating females could take actions that affect paternity after they have engaged in one or more matings. However, if a female must copulate in order to gather information to make her mating decision, she places copulating males in a strong position to force fertilization upon her (Sivinski 1984). Because of the threat of forcing, postcopulatory mate choice should be restricted to rare cases in which females can, with certainty, separate copulation success and fertilization success (Eberhard 1985). Females exercising such choice should posses special adaptations for storing, manipulating, or discarding sperm (see Hellreigel and Ward 1998), or they should exhibit a strict pattern of last-male sperm priority so that they can retain control over fertilization by restricting sexual access.
General predictions about mate choice mechanisms
Females choosing the best-of-n available mates can favor rare, novel mutants for no reason other than their novelty or extremeness. The sexual selection advantages of such traits can cause them to spread even if they are costly in terms of natural selection. In contrast, sexual selection under threshold choice sets a lower limit for mate acceptability, and there is little or no mating advantage to exceeding that limit; for equally acceptable males facing threshold choice, novelty is at best irrelevant, and, since any costs are unopposed by sexual selection, novelty may be a liability (Fig. 1.2). One
16 exception is that threshold choice can favor novel traits that enhance a male’s ability to be detected or noticed (passive attraction); females favoring such traits are usually not favoring novelty per se, but augmentation of existing mate location and recognition mechanisms instead. For similar reasons, in species where the only benefits from mate choice are genetic, threshold choice is unlikely to evolve because only best-of-n choice can grant rare, novel mutant males a selective advantage allowing the spread of the mutation. Thus, best-of-n choice is a likely prerequisite of “runaway” evolution and the evolution of sexual ornaments.
Invertebrates’ general lack of male resource investment would seem to place a premium on females’ abilities to discriminate among potential mates (Emlen and Oring 1977, Alexander and Borgia 1979, Andersson 1994, Höglund and Alatalo 1995). Any benefits from discrimination are likely to be genetic, and obtainable only through some form of relative comparison; further, invertebrate life history characteristics make it likely that such comparisons are direct. Even if males provide no nuptial resources, and even if females obtain no genetic advantages for their offspring by selecting mates, females could still materially benefit by mating at a time or location optimal for their own interests, or choosing a mating context.
Mate choice in periodical cicadas
13- and 17- year periodical cicadas (Magicicada spp.1) provide an unparalleled opportunity to examine mechanisms of mate choice. Male Magicicada join mixed- species singing aggregations attractive to sexually receptive females. Males in these aggregations engage in chorusing (or “sing-fly”) behavior (Alexander 1968, 1975), in which they produce a small number of calling phrases (1- 3 in the M. –cassini and M.
1 For convenience the Magicicada cognate species groups are referred to using the following shorthand: M-decim: M. septendecim (17), M. tredecim (13), and M. neotredecim (13); M -cassini: M. cassini (17) and M. tredecassini (13); M. -decula: M. septendecula (17) and M. tredecula (13).
17 –decim species or a short calling bout in the M. –decula species) and then fly to another calling perch. Upon hearing a male’s calling phrase, a receptive female flicks her wings, producing a visual and acoustical signal similar to that reported in other cicada species (Chapter 5). Females signal only when they are mature and ready to mate, most signaling females copulate within several minutes, and most male mating attempts in the absence of a signal fail. A male perceiving a wing-flick signal ceases sing-fly behavior and begins courtship, engaging in an acoustical duet with the signaling female while moving towards her. Most females mate only once (Chapter 4).
Alexander (1975) considered periodical cicada male singing aggregations to be non-resource based leks, in which females have opportunities to discriminate among mates. Because unmated female Magicicada eventually oviposit normal-appearing, presumably unfertilized eggs (Graham and Cochran 1954, pers. obs.), it is unlikely that females visiting male aggregations receive male donated resources (or gain access to resources other than sperm) necessary for reproduction. Magicicada females could choose among mates at several stages of the sexual sequence: (1). They could choose during early stages of courtship when males call and females answer. (2). They could choose during late stages of courtship when males and females approach and achieve physical contact. (3). They could exercise postcopulatory choice by remating with higher quality males should they become available.
Any benefits females receive as a direct consequence of discriminating among potential mating partners must be genetic (because males provide no material resources), or must include phenotypic benefits other than male-donated gifts, such as reductions in the immediate costs of copulation by reducing time and effort wasted in locating appropriate mates or reducing risks of disease, damage, or predation. Alternatively, instead of choosing among potential partners, female cicadas may gain more from mating to
18 maximize direct phenotypic benefits to themselves, such as by selecting an appropriate time and safe location for mating independent of male identity.
Female discrimination against heterospecific males. The Magicicada species M. -decim, M. -cassini, M. –decula are sympatric over most of their ranges (Alexander and Moore 1962). Because females respond sexually only to conspecific male calls (Chapters 5, 6), hybrid matings are unlikely. Females avoiding hybrid matings conserve time and resources that would otherwise be wasted in fruitless matings. If female Magicicada mate choice behaviors evolved in the context of hybrid avoidance, and if the differences in the Magicicada species’ sexual behaviors result in part from past episodes of character displacement (Chapter 6), threshold choice mechanisms, based upon species-typical behaviors such as courtship, would be the simplest and least costly means for females to discriminate against inappropriate mates. Because there is little or no sexual conflict of interest over species identity if individuals of neither sex realize appreciable fitness benefits from cross-species matings, if females discriminate only against heterospecifics, males should be selected to cooperate instead of deceiving females.
Female discrimination against diseased males. Magicicada are vulnerable to an entomophagous fungal infection (Massospora cicadina Peck; Soper 1974, Soper et al. 1976). It is possible that the tendency or predisposition to become infected is inheritable. Infected cicadas are contagious and, in advanced stages of the infection, sterile. One simple way that females could reject infected mates is to fail to respond to their calls, or, if the fungus does not become contagious until males cease normal calling, to refuse to mate with silent males.
Even if there are no genetic differences among males concerning ability to resist or survive infection, the direct phenotypic benefits to females of avoiding infected males are substantial. Females should avoid contagious, infected males at all costs and should
19 reject infected males as early in the mating sequence as possible. Even if males exhibit no heritable genetic variation for susceptibility, females could benefit from discriminating against infected males because the fungus is invariably fatal, it reduces infected individuals’ fitness, it is avoidable, and females thus receive fitness payoffs from avoiding it. If this is an important influence on the Magicicada mating system, then females can avoid a high phenotypic cost by forcing males to engage in behaviors, such as joining singing aggregations and participating in complex courtship, likely to reveal male infection status and help females identify risky matings.
Unless female choice criteria are such that they exclude only sterile, fungus- infected males (who, on their own behalf, have no reproductive interest and thus no conflict of interest with the females) then male and female interests will conflict. Specifically, until infected males become sterile, they should strive to disguise their infection, while females should seek to cause males to court in ways that expose infections (Alexander et al. ms). For example, if females use male song as an “uncheatable honest indicator” of male quality, then they should be reluctant to mate unless their suitors sing, they should place a premium on song power output and the endurance of singing males, and they should discriminate against males with unusual or atypical songs characteristic of infected males. If infected and contagious males remain acceptable to females, then, perhaps because of the low frequency of infection, it is likely that females gain no net selective advantages from expending any effort to discriminate against rare infected males.
Female choice of mating time and location. Females may receive direct phenotypic benefits by restricting mating to optimal times and places. For instance, females may minimize exposure to predation by avoiding small populations and visiting only the densest aggregations (see Hamilton 1971; Alexander and Moore 1962, Williams et al. 1993). Females may also minimize risks by mating early in the day and terminating
20 copulation before cooler evening temperatures force them to remain relatively immobile until morning, or females may avoid mating until they have found a secure perch from which they are unlikely to fall; copulating pairs on the ground are relatively immobile and likely exposed to greater risk of predation from nocturnal rodents (pers. obs.). As long as males and females face different costs for finding mature, healthy mates, their interests over timing and location of mating will differ. If male periodical cicadas typically encounter only one or a few potential mates during their lifetimes, then if they encounter one at a suboptimal time or location, they will persist in courtship. Females, in turn, will be selected to resist forced matings. The net result will be that male and female daily activity patterns will differ, with females tending to become unreceptive before males stop chorusing, and with decreases in activity related to perceived costs of locating future mates. If female choosiness is related primarily to mating context, partner identity will be relevant only if males have heritable differences in their abilities to appear in the appropriate context.
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25 Watt, W. B., P. A. Carter, and K. Donohue. 1986. Females’ choice of “good genotypes” as mates is promoted by an insect mating system. Science (Wash., D. C.) 233: 1187- 1190. Weatherhead, P. J., and R. J. Robertson. 1979. Offspring quality and the polygyny threshold: “The sexy son hypothesis.” Am. Nat. 113: 201-208. Wiley, R. H., and J. Poston. 1996. Indirect mate choice, competition for mates, and coevolution of the sexes. Evolution 50: 1371- 1381. Williams, K. S., K. G. Smith, and F. M. Stephen. 1993. Emergence of 13-yr periodical cicadas (Cicadidae: Magicicada): phenology, mortality, and predator satiation. Ecology 74: 1143- 1152. Winston, M. L. 1987. The biology of the honey bee. (Harvard University Press: Cambridge, MA). Wolfner, M. F. 1996. Tokens of Love: Functions and regulation of Drosophila male accessory gland products. Insect Biochem. Molec. Biol. 27: 179- 192.
26 Table 1.1. Mate discrimination and its alternatives. Identity of Kind of choice mate matters Benefits Selection mate choice yes genetic, material sexual context choice no material natural none no none none
27 Number of males
n, number of 28 males excluded
Male quality, q
t, threshold value
Figure 1.1. Distribution of male traits in relation to female threshold acceptance value, t. Females do not accept males of quality q < t. As t decreases, number of males excluded from mating (n) also decreases.
l, limit of male variation
t1, female acceptance threshold
t2, female acceptance threshold 29
A B C
number of males q, male quality
Figure 1.2. Comparisons of threshold mate acceptance criteria (A, B), and best-of-n choice criteria (C). Increasing width of line (A, B, or C) indicates increasing probability that a male of quality q will be accepted as a mate. (A) Under threshold choice, females do not accept males with quality q < t1 , excluding some males from mating. (B) At a lower value of t (t2), number of males excluded also decreases. Under threshold choice, all males of quality q > t have an equal probability of mating. (C) Under best-of-n choice, as male quality q increases, so does probability of being accepted as a mate. Best-of-n choice can lead to runaway evolution, because a mutant male with quality q > l (limit of male variation), is potentially more attractive than any other male in the population. CHAPTER II
EXPERIMENTAL METHODS IN STUDIES OF PERIODICAL CICADAS: EXAMINATIONS OF POSSIBLE METHODOLOGICAL BIASES IN THE STUDY OF PERIODICAL CICADAS (MAGICICADA SPP.)
Abstract
Periodical cicadas (Magicicada spp.) are ideal subjects for studies of mating behavior because adults can be kept and mated in captivity. However, for the results of captive studies to be applicable to natural populations, the methods used must not bias or alter mating behavior. For instance, marking allows focal-individual studies, but chemical effects of marking could cause premature death or reduced likelihood of mating. Further, cages could bias sexual behaviors, since confined females are limited in their abilities to escape persistent, harassing males. In this paper we demonstrate that the methods we use to mark and cage periodical cicadas are harmless. Our marking methods have no measurable effects, the densities in our cages are not unrealistically higher than densities of free cicadas on branches, and we uncover no evidence that matings observed in cages result
30 from forcing, or that smaller, denser cages increase males’ abilities to force matings upon sexually unreceptive females.
Introduction
Our long-term studies of periodical cicada (Magicicada spp.) mating behavior include experiments and observations in which we confine individually marked periodical cicadas in closed mesh cages (Chs. 3, 4, 5, 6, 7, 8). Increased mortality or morbidity are possible undesirable effects of such manipulation. Other effects could be more subtle; in mate choice studies, cage and marking effects could create false positive results by causing cicadas to be unusually active. Conversely, if manipulation disturbs cicadas or causes them to expend significant effort attempting to escape cages or remove marks, mate choice experiments could yield false negative results. Perhaps the most insidious problem of cage studies is that they may prevent or at least alter normal sexual rapprochement behaviors by placing members of both sexes in close, unsolicited proximity. To assess the seriousness of these problems in our studies of periodical cicadas, we present data addressing changes in mortality and sexual behavior due to marking or cage confinement artifacts and discuss ways of minimizing biases.
Methods and Results
Individual marking
The ease of use of different marking methods and their appropriateness for particular study designs are well reviewed (Southwood 1978, Walker and Winewriter 1981, Kearns and Inouye 1993). Above and beyond logistical concerns, the possible effects on the study subjects further constrain the choice of marking method. Commonly, studies making use of marking techniques assume no ill effects. Table 2.1 summarizes literature reporting effects of individual marking (not mass-marking) methods on study organisms. We located sources using published reviews and electronic databases
31 (Zoological Record, Wilson Indexes to Journal Articles, and Biological Abstracts) and included only papers containing data specifically addressing possible mortality or morbidity due to marking. Of several hundred papers examined, we found only 20 fitting these criteria. Table 2.1 is neither exhaustive nor prescriptive, but rather supplements earlier reviews (Southwood 1978, Walker and Winewriter 1981, Kearns and Inouye 1993). Few general trends are apparent, except that marks applied topically to sclerotized parts seemed to have few deleterious effects, while dusts, powders, or, in the case of soft-bodied insects, some solvents, must be used cautiously. The lack of general trends means that controlled testing of a marking method is the only way to ensure its appropriateness for any given study.
Individual marking has proven effective in our research on periodical cicadas (Magicicada septendecim (L.)), allowing us to track the behaviors of individual insects or classes of insects as they emerge, mature, mate, and die (Chs. 3, 4, 5, 6, 7, 8). One marking technique we have used is to apply colored enamel paint dots to the dorsal thoracic surface. Because published reports contain no clear information on the toxicity of the solvents in these paints (xylene, xylol and n- propoxy propanol) we conducted experiments to determine whether this marking method has adverse effects on our study animals.
Methods. We compared the survivorship and mating activity of marked and unmarked adult Magicicada septendecim in a recently logged clearing along Route 639 in the Horsepen Lake State Wildlife Management Area, Buckingham County, VA, in mid- May 1996. We placed two 1 x 2 m black fiberglass mesh bags over living vegetation and stocked each cage with 10 unmarked and 10 marked mature, unmated female cicadas. The cicadas were marked or handled before being put in the cages, and were assigned to the cages in no particular order.1 We marked cicadas by using a flat toothpick to apply one
1In this design, the 40 female cicadas, not the cages, are replicates; the females were divided into two cages for convenience and to prevent severe overcrowding. The experiments do not test for a cage effect, but rather test for effects of two different treatment regimes on the females. The two treatment regimes are entirely independent of the cages and cage assignments.
32 yellow and two blue dots of Testors’ lead-free gloss enamel (Testors Corp., Rockford IL, 61104) to the dorsal surface of the thorax. The primary solvents in Testors’ enamel paints are xylol and n- propoxy propanol, which are toxic to humans in high doses. We used no anesthetic during marking, and we were careful to prevent paint from contacting the wing articulations or the articulation between the head and thorax. The total surface area covered by paint was between 6 and 10 mm2. To allow matings, we placed ten sexually mature adult males collected from an active chorus into each cage. Cicada densities in these cages were thus high (see below), but intentionally so, to expose any differential survival ability of marked and unmarked cicadas. From May 23- June 4, we censused the cages 8 times by counting and removing dead cicadas and recording whether or not each was marked or showed evidence of a mating sign (or “seminal plug;” White 1973). We scored females exhibiting seminal plugs as having mated; those lacking the mark as not having done so. We preserved all cicadas in 70% ethanol, and at the final census, we removed all remaining cicadas.
Results. We compared the survivorship of the marked and unmarked cicadas using Cox regression analysis, implemented with the PHREG function in SAS (SAS Institute Inc., 1996). This technique allowed us to include individuals who survived through to the end of our observations. The small difference in survivorship curves for painted and unpainted individuals was not statistically significant (Wald Chi-Square-2.32, 1 d.f., P £ 0.13). If anything, marked cicadas seemed to survive longer than unmarked cicadas, although this pattern is not statistically significant. The two cages do appear to have different mortality rates (Wald Chi-Square=14.02, 1 d.f., P £ 0.0002), attributable to the death of substantial amounts of vegetation in one cage, although the increased mortality in this cage was distributed uniformly among marked and unmarked cicadas.
33 A chi-square goodness-of-fit analysis of seminal plug presence/absence data reveals no significant pattern. Marked and unmarked females were equally likely to exhibit this evidence of mating. Data are summarized in Table 2.2 and Figure 2.1.
Cage artifacts
In our studies of periodical cicada behavior, we make extensive use of mesh bag cages. Cages allow recovery of marked individuals, facilitate experiments with manipulated individuals, and permit isolation from free cicadas so that observations may be restricted to cicadas whose entire adult histories are known. Our cages consist of fiberglass hardware cloth folded over living vegetation suitable for feeding or oviposition, giving an approximate volume of 200 liters. We are careful to construct cages so that the highest points, in which cicadas tend to congregate, have few corners or narrow spaces that might trap or cause injury. In experiments where we watch and record cicada behaviors, we typically stock cages with 10- 20 cicadas; when we are less concerned about the details of behavior (such as our paint marking experiments), or for storing cicadas, we typically place 20- 40 cicadas per cage.
Each sex appears subject to sex-specific cage mortality. Females, whether mated or not, oviposit in cage branches, sometimes to a degree that kills or weakens the vegetation. This problem can be alleviated by moving ovipositing females to fresh branches on a regular basis. Males, especially those caged near unmated females, tend to damage themselves by struggling with each other or by attempting to escape. The severity of this effect can be reduced by housing males at low densities somewhat away from females. Since both of these problems could severely compromise long-duration experiments, we have kept cage experiments short and maintained and stocked cages in a manner that minimizes these effects.
34 Of greater concern are the ways in which cages may alter cicada sexual behavior by unnaturally confining males and females in close proximity, perhaps promoting male sexual coercion. For example, females confined with males in cages are denied the opportunity to participate in normal rapprochement behaviors. If critical components of the mating sequence occur during rapprochement, such as if females normally accept or reject a courting male before he comes within one meter of her, females in small cages would not be able to engage in normal mate rejection behaviors. Observations do not support the view that male forcing behavior is generally successful in periodical cicadas; of approximately 100 courtships of caged cicadas observed in 1996, three appeared to involve male forcing, and only one of these three resulted in copulation. Nevertheless, it is still possible that cages may facilitate male forcing behaviors, and thus confinement in cages may compromise mating experiments.
We used three approaches to evaluate the possibility of a cage effect. First, to determine whether our experimental cage densities are unrealistically high, we compared the densities in our cages to densities of natural cicada aggregations. Because of the difficulties of sexing uncaged cicadas, our analysis concerns only cicada densities and not sex ratios within cages. Second, we caged both normal and physically handicapped males with receptive females and examined the relative mating success of each class of male. Comparing the success of normal and handicapped males provides information about forcing behaviors and whether such behaviors might be exacerbated by cage studies. Last, we compared female remating frequencies in a large 4m x 4m x 4m screen “flight cage” to frequencies in small bag cages. Mated females are generally unreceptive (Chapter 4) so extreme polyandry in this experiment would indicate that confinement reduces the effectiveness of female resistance. If cages affect rapprochement or escape behaviors, then the severity of a cage effect should depend on the size of the cage. Demonstrating similar remating frequencies in large and small cages would suggest the absence of a cage effect.
35 Cage density surveys; methods. On May 14 and 17, 1996, an observer (JRC) walked along Virginia Route 639, an unpaved road with little traffic, and counted the numbers of cicadas visible on 39 branches of approximately the same size as those inside our mesh bag cages. We developed a computer resampling algorithm (Appendix B) to evaluate the extremeness, relative to our branch scan data, of specified cage densities. Given a hypothetical cage density, the algorithm randomly picks, with replacement, a value from the 1996 branch scan data and calculates whether the picked value is more or less extreme than the specified density by comparing the distances to the scan data mean. The algorithm repeats these calculations 10,000 times and reports the percentage of iterations in which the picked value was more extreme than the specified value. This percentage is analogous to a P-value indicating the likelihood of, by chance alone, picking a value from the data set more extreme than the specified cage density. The algorithm calculates such P- values for many hypothetical cage densities, generating 95% confidence intervals for the 1996 scan data.
Results. Our branch survey counts ranged from 3 to 27 (mean 12.61, ± 5.6). The resampling algorithm suggests that densities of up to 25 cicadas per caged branch are not extreme in comparison to the 1996 scan data (Table 2.3) and are comparable to natural cicada densities on similar branches. Greater densities are atypical of 1996 scan data, and behavioral data from such cages may be suspect.
Pretarsal clipping experiment; methods. Cicadas have a pair of sharp terminal claws (or pretarsal ungues; Snodgrass 1993) on each leg. Claw damage reduces cicadas’ ability to grasp vegetation or other cicadas. Damage to the foreleg claws appears to increase mortality, because cicadas apparently use their front legs almost as antennae to investigate the characteristics of a surface before venturing onto it; cicadas with damaged front claws tend to remain motionless, perpetually clawing at the surface in front of them.
36 Cicadas with claws other than the front pair removed appear to have reduced gripping abilities but are still mobile.
When a male cicada attempts to force a copulation, he climbs on the back of a female and must resist her attempts to dislodge him. A female so threatened flaps her wings vigorously, and is usually successful in preventing mating, unless the male is able to immobilize her wings or insert his genitalia before being dislodged (JRC pers. obs.). Given the chance, males will attempt to mount and copulate, but successful forced matings require substantial male effort. In contrast, in a mating that involves normal courtship and apparent mate acceptance by the female, the male meets little or no resistance when he attempts to mount.
If males are manipulated by removing some of their claws, they should be less successful at some of the behaviors required for forcing matings. If handicapped and normal males have equal access to females, but normal males mate more frequently, forcing behaviors are likely an important male tactic, perhaps encouraged by cage studies. Alternatively, no difference in mating frequencies would suggest that male forcing behaviors are not generally successful.
To evaluate whether bag cages allow males to trap females and force matings upon them, we placed twelve mature unmated female cicadas into three hardware cloth cages, four females per cage. Cages enclosed living vegetation, providing access to live branches for feeding and oviposition. Into each cage we also placed eight mated males obtained by separating mating pairs. Males can mate more than once; we used mated males to establish that all males were acceptable as mates prior to the manipulations. Four of these eight males had their two midleg pretarsal claws removed with a fingernail clipper, while the other four were handled similarly but left intact. We censused the cages regularly and recorded the identities of copulating males. Copulations are distinguishable from other
37 postures, because even though body orientations vary, the male genitalia are clearly engaged.
Claw removal had observable effects on the cicadas’ mobility and agility, but no evident immediate effects on mortality. If forcing behaviors are prevalent in bag cages, then unmanipulated and fully agile cicadas should obtain a greater proportion of matings than should males whose claws have been removed and who are thus at a disadvantage.
Results. The experiment ran for approximately 71 hours, or until all but two females copulated. The first matings occurred 40 hours after the experiment began, and the last matings at 68 hours. No rematings were allowed. Data were pooled across replicates, and pooled results were analyzed using a two- tailed Fisher’s Exact Test. Manipulated (clipped) and unmanipulated cicadas were statistically equally likely to obtain matings (Table 2.4). For comparison, a data set of this size showing a significant treatment effect would need to have two or fewer males mate in one treatment and eight or more in the other. With the caveat that this experiment involves small sample sizes, it does not support the view that male forcing behaviors are generally prevalent or successful, because males whose ability to force matings was impaired were at no disadvantage compared to normal males. Thus, we have no evidence that male forcing behaviors are a significant complication in cage experiments.
Cage size effects; methods. Unconfined, singly mated female Magicicada do not exhibit normal female behaviors indicating sexual receptivity (Chapter 5). If such females are generally able to avoid encountering males and remating, then a cage effect may have its most observable impact on female remating frequencies. A cage effect would also likely involve a relationship between the cage volume, or cage surface-to -volume ratio, and the severity of the effect, because in the smallest cages, females should be least able to escape unsolicited courtships and males should be most able to find females.
38 One way to test for the operation of a cage effect, then, is to compare remating behavior in small cages with behavior in large cages. In 1995, we stocked one large cage (the “flight cage”) and many smaller cages (“bag cages”) with cicadas. To determine whether remating frequencies were higher in the bag cages than expected from flight cage data we used a Monte-Carlo computer resampling algorithm to compare remating frequencies in the flight cage and bag cage data.
During the 1995 Brood I periodical cicada emergence at Alum Springs, Rockbridge County, Virginia, we stocked a 4m x 4m x 4m nylon mesh cage (our “flight cage”) with 79 adult female Magicicada septendecim, 86 adult male M. septendecim, 14 adult female M. cassini, and 10 adult male M. cassini. Magicicada nymphs tend to emerge from the ground and undergo their final molt in the evening (Maier 1982); the morning after ecdysis, newly emerged cicadas (“teneral” cicadas) are readily identifiable by their pale color, soft bodies, and by their position low in the vegetation. We captured teneral cicadas, individually marked them, placed them in the cage, and recorded the dates, times, and individuals’ identities for all matings. Based on day of tenerality (capture), we assigned each cicada to a “day-cohort” useful in estimating the onset of sexual maturity. We recorded all observed mating pairs in the cage. Out of 79 female M. septendecim, only one mated with a male M. cassini ; in addition, one female M. cassini mated with a M. septendecim male. Because cross-species interactions appear minimal, M. cassini will not be considered further in discussions of this experiment. Of the 78 female M. septendecim that mated, most did so by the eighth day of their adult lives, and 13 mated more than once before ovipositing.
Although the flight cage experiments ran for 20 days, because cicadas in the cage belonged to several day-cohorts, their behavior is best recorded with respect to day from tenerality, not day of first cage stocking. Therefore, only information on the behavior of 78 female cicadas for a period from tenerality to 12 days of adulthood was available. Not all of the cicadas belonging to later cohorts have information available about the later days
39 (days 8-12) of their adult lives, because the experiment was terminated less than 12 days after the latest cohorts were collected. It is not known whether cicadas in later cohorts would have remated, because they may only have had enough time to mate once. Thus, calculations of remating frequency in the flight cage are conservative, because any errors in the collection or interpretation of the flight cage data can lead only to underestimates of remating frequencies. This conservative figure for female remating provides the maximum potential contrast to cicada behavior in the smaller cages.
Of the five bag cage experiments conducted in 1995, only two allowed remating and are appropriate for this analysis. The design and results of each are summarized in Appendix C. Although the flight cage data covers a period of 12 days, and these experiments ran for 11 days, these time periods are comparable, since the number of days is less important than day quality (in terms of weather, etc.). In any case, all but one mating in the flight cage occurred before the mating cicada’s tenth day from tenerality, well before the end of the flight and bag cage experiments.
A cage effect that scales with cage size should manifest itself as higher remating frequencies in the small bag cages than in the large flight cage. We compared mating frequencies using a C++ resampling algorithm (Appendix D). The algorithm resamples the flight cage data on singly-and doubly- mated females to create sets of sizes comparable to the bag cages (n= 8 or n= 4 as appropriate), and it tallies the number of females in the resampled data that mated twice. The program iterates this resampling 10,000 times to generate a frequency distribution, and thus percentiles, of the outcomes of the resampling.
Results. The second replicate in the first experiment I may show a cage effect, since half of its 8 females remated, which is outside the 95th percentile of the resampling algorithm, and more that twice the remating frequency in any other cage (Table 2.5). Interpretation of these results requires caution, because two of the rematings in this cage are unusual, and unlike any others in these experiments. One of the four rematings in this cage
40 occurred after a mating involving conflict between two males. In this particular instance, two males struggled with each other, both attempting to insert their genitalia into the female. It was not possible to determine whether either male was successful in copulating. When the cage was next observed, one hour later, a period too short to be considered a normal mating (Chapter 4), the female was alone and not mating. Approximately 20 hours later, she engaged in a normal mating.
Another of the rematings in this replicate was the only post-oviposition remating in this study. This study was not designed to evaluate post-oviposition remating, which may occur for reasons distinct from pre-oviposition rematings (Chapter 4). If pre- and post- oviposition remating are treated as different phenomena, and if the post-oviposition remating is therefore eliminated from consideration, the results in this replicate are not significantly different from flight cage results. We scored both of these unusual situations as matings, but neither would have met the stricter criteria typical of our other experiments. If either one of the unusual matings in replicate 2 is discounted, then this replicate does not have a remating frequency higher than that in the flight cage.
Conclusions
We have uncovered no evidence that careful marking with enamel paints affects cicadas’ mortality or likelihood of mating, nor is there any evidence that cicada densities in our experimental cages are unrealistically high. The pretarsal clipping experiment suggests that males prevented from forcing are no less successful than normal males, and that male forcing strategies are not a significant factor in bag cage experiments. The cage size resampling algorithm demonstrates the absence of a cage effect whose severity scales with cage size. We have thus uncovered no evidence that our methods significantly alter cicada behavior in ways that would generate false or misleading results.
41 From observing cicada behavior in cages, however, we can add some notes of caution. In 1997, we placed female M. septendecim and male M. cassini in cages away from a male M. septendecim chorus. Under normal circumstances, we observed few sexual interactions among these cicadas. When we played male M. septendecim song, however, female M. septendecim responded with a receptivity signal (Chapter 5). This signal, which is similar in all Magicicada species, apparently incited the male M. cassini males in the cages to increase their sexual activities, and in some cases, a heterospecific male successfully engaged in a cross-species mating by mounting and copulating with a female who had responded to conspecific playbacks.
In another experiment, we constructed several dozen model cicadas from blocks of wood, painted them black, and attached them to the branches of a tree enclosed in a large flight cage. We stocked the cage with male M. septendecim and M. cassini. Normally, the males paid no attention to the models, but if we produced a simulated female wing-flick signal in time with a male call, males directed increased attention to the blocks, in some cases mounting and copulating. Male M. cassini in particular seemed to increase their sexual activity after we produced a simulated female wing-flick signal, and one male’s excitement seemed to have a synergistic effect on the others.
The experimental results in this paper demonstrate the effectiveness of female resistance, and that cages do not cause unreceptive females to mate. However, close confinement of several males and females in cages might compromise mate choice experiments, because if one female in a cage produces a wing-flick signal, indicating sexual receptivity, all the males in the cage may increase their sexual activity. In small, densely stocked cages, males can reach a mating frenzy, in which they interfere with and attempt to replace other courting males. Our observations indicate that these problems are more severe in M. cassini than in M. septendecim, so it appears advisable to avoid using the former species in mating experiments in which several males are confined in one cage.
42 Cage experiments in general require close observation and careful design to ensure that male-male competition does not unduly influence mating. Criteria for scoring matings must be designed so that unusual situations, such as post-oviposition mating, male-male competition, and interloping attempts, do not contribute to false positive results.
43 Literature Cited Brussard, P. F., 1971. Field techniques for investigations of population structure in a “ubiquitous” butterfly. Journal of the Lepidopterists’ Society . 25: 22- 29. Caprio, M. A., D. Miller, and E. Grafius. 1990. Marking adult Colorado potato beetles, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), using paper labels. Great Lakes Entomologist 23: 13-18. Davey, J. T. 1956. A method of marking isolated adult locusts in large numbers as an aid to the study of their seasonal migrations. Bull. Ent. Res . 46: 797-802. Dobson, R. M., J. W. Stephenson, J. R. and Lofty. (1958). A quantitiative study of a population of wheat bulb fly, Leptohylemia coarctata (Fall.), in the field. Bull. Ent. Res . 49: 95-111. Dobson, R. M., and M. G. Morris. 1961. Observations on emergence and life-span of wheat bulb fly, Leptohylemia coarctata (Fall.), under field cage conditions. Bull. Ent. Res. 51: 803- 821. Dreyer, H., and J. Baumgartner. 1997. Adult movement and dynamics of Clavigralla tomentosicollis (Heteroptera: Coreidae) populations in cowpea fields of Benin, West Africa. J. Econ. Entomol. 90: 421-426. Fales, J. H., O. F. Bodenstein, G. D. Mills, Jr., and L. H. Wessel. 1964. Preliminary studies on face fly dispersion. Ann. Ent. Soc. Amer . 57: 135-137. Gallepp, G. W. and A. D. Hasler. 1975. Behavior of larval caddisflies (Brachycentrus spp.) as influenced by marking. Am. Midl. Nat. 93: 247-254. Gangwere, S. K., W. Chavin, and F. C. Evans. 1964. Methods of marking insects, with especial reference to Orthoptera (Sens. Lat.). Ann. Ent. Soc. Amer . 57: 662-669. Garcia, M. A., and L. M. Paleari. 1990. Marking cassinidae (Coleoptera: Chrysomelidae) larvae in the field for population dynamics studies. Ent. News 101: 216-218. Greenslade, P. J. M. 1964. The distribution, dispersal, and size of a population of Nebria brevocillis (F.) with comparative studies on three other Carabidae. J. Anim. Ecol . 33: 311- 333. Hager, B. J., and F. E. Kurczewski. 1986. Nesting behavior of Ammophila harti (Fernald)(Hymenoptera: Sphecidae). American Midland Naturalist 116: 7- 24. Hart, D. D. and V. H. Resh. 1980. Movement patterns and foraging ecology of a stream caddisfly larva. Can. J. Zool . 58: 1174-1185. Hunter, P. E., 1960. Plastic paint as a marker for mites. Ann. Ent. Soc. Amer. 53: 698.
44 Kearns, C. A. and D. W. Inouye, 1993. Techniques for pollination biologists . (University Press of Colorado: Niwot, CO). Maier, C. T. 1982. Obervations on the seventeen-year periodical cicada, Magicicada septendecim (Hemiptera: Homptera: Cicadidae). Ann. Ent. Soc. Amer. 75: 14- 23. Mascanzoni, D., and H. Wallin. 1986. The harmonic radar: a new method of tracing insects in the field. Ecol. Ent . 11: 387-390. Mead-Briggs, A. R. 1964. Some experiments concerning the interchange of rabbit fleas, Spilopsyllus cuniculi (Dale), between living rabbit hosts. J. Anim. Ecol . 33: 13-26. Paulson, G. S., and R. D. Akre. 1991. Role of predaceous ants in pear Psylla (Homoptera: Psyllidae) management: estimating colony size and foraging range of Formica neoclara (Hymenoptera: Formicidae) through a mark-recapture technique. J. Econ. Entomol. 84: 1436-1440. Rieske, L. K. and K. F. Raffa, 1990. Dispersal patterns and mark-and-recapture estimates of two pine root weevil species, Hylobius pales and Pachylobius picivorus (Coleoptera: Curculionidae), in Christmas tree plantations Environmental Entomology 19: 1829- 1836. SAS Institute Inc., 1996. SAS/STAT® Software: Changes and Enhancements through Release 6.11. Cary, NC: SAS Institute Inc. Sempala, S. D. K. 1981. The ecology of Aedes (Stegomyia) africanus (Theobald) in a tropical forest in Uganda: mark-release-recapture studies on a female adult population. Insect Sci. Application 1: 211-224. Snodgrass, R. E. 1993. Principles of Insect Morphology (Cornell University Press: Ithaca, NY). Southwood, T. R. E., 1978. Ecological Methods, with particular reference to the study of insect populations. (Chapman and Hall: London). Stuart, R. J. 1986. Use of polyester fibers to mark small leptothoracine ants (Hymenoptera: Formicidae). J. Kans. Ent. Soc . 59: 566-568. Walker, T. J., and S. A. Winewriter. 1981. Marking techniques for recognizing individual insects. Florida Entomologist 64: 18- 29. White, J. 1973. Viable hybrid young from crossmated periodical cicadas. Ecology 54: 573- 580.
45 changes None (Hemiptera) (Coleoptera) (Coleoptera) None (Coleoptera) None (Coleoptera) None (Diptera) (Diptera) None Toxic (Homoptera) (Orthoptera) None (Trichoptera) None (Coleoptera) None (Orthoptera) (mite) None (Hymenoptera) None (Diptera) Toxic (Coleoptera) None† (Lepidoptera) None (Hymenoptera) None§ (Coleoptera) spp. (Trichoptera) Behavioral Erebia epipsodea Erebia Hylobius pales Pachylobius picivorus Leptinotarsa decemlineata Dicosmoecus gilvipes Laelaspis georgiae Ammophila harti Nebria brevicollis Formica neoclara Lepthohylemia coarctata Lepthohylemia coarctata Brachycentrus Musca autumnalis Charidotis punctatostriata Clavigralla tomentosicollis Leptinotarsa decemlineata Magicicada septendecim Melanoplus sanguinipes Leptinotarsa decemlineata gregaria Schistocerca paper labels affixed with cynoacrylate gluepaper labels affixed None Monolite Yellow in wood rosin and kerosene)Yellow Monolite None 1958 1961 Oil-based paint paint (nitrocellulose) Lacquer-based 1990 by Acetone wash, followed 1990 with cynoacrylate glue Paper labels affixed 1990 based) Enamel paint (xylol, propanol Reported effects of methods for marking individual insects, grouped by general method and organized chrono- organized method and grouped by general individual insects, for marking of methods effects Reported . 1964 Fingernail polish (acetone based) et al. et al. et al. et al. et al. et al Rieske and Raffa 1990Rieske and Raffa Acrylic paint Caprio Hart and Resh 1980 with wire or elastic band Plastic tags affixed Brussard 1971Caprio “Sharpie” marker (xylol solvent) Fales Dreyer and polish (acetone based) Fingernail Greenslade 1964Akre 1991Paulson and based) Enamel paint (petroleum-solvent Dobson Enamel paint (petroleum-solvent based) Dobson Gallepp and Hasler 1975 correction fluid Typewriter Garcia and Paleari 1990 Fingernail polish (acetone based) Baumgartner 1997 Hunter 1960Hager and Kurczewski 1986 Enamel paint (xylol, propanol based) Enamel paint (xylol, propanol based) Table 2.1. Table each grouping. logically within ReferenceThis study Marker propanol based) Enamel paint (xylol, Taxon Effect* Caprio 1956Davey, or Toner (Rubine Oil-based paint
46 Toxic None‡ moderate treatment spp. (Coleoptera) None (Blattaria) None for (Siphonaptera) None (Orthoptera)(Orthoptera) (Orthoptera)(Orthoptera) None‡ None‡ in Toxic Harpagoxenus (Diptera) spp., Spilopsyllus cuniculi Harpalus niger, P. melanarius, Pterostichus rufipes, Carabus granulatus Aedes africanus Acheta domesticus Leptothorax (Hymenoptera)Acheta domesticus Acheta domesticus Acheta domesticus Periplaneta americana None Radar tags combined with Duco paint combined with Duco spray oxide Titanium dry pigmentOil and Cellulose paints, doses large 1964Aniline dyes, or Melted crayon, colored ink, 1964 Metallic dust 1964 or clipping amputation Wing 1964 with glue Colored thread or paper attached 1964 pigment in oil spray, Fluorescent powder, et al. et al. et al. et al. et al. * “None:” no effects reported; “Toxic:” increased mortality reported; “Injury:” unspecified damage or mortality noted. increased mortality reported; “Injury:” reported; “Toxic:” * “None:” no effects § Pers. Comm. noted. † Dispersal attributable to handling effects of limited use. ‡ Method proved to be impermanent and Gangwere Mead-Briggs 1964 1986 Wallin Mascanzoni and clipping Tarsal Gangwere Gangwere Table 2.1 (Continued) Table Sempala 1981Gangwere powder Fluorescent Stuart 1986 tied around body Polyester fiber Gangwere
47 Table 2.2. Numbers of marked and unmarked females showing a “seminal plug,” or evidence of mating, and c 2 comparison of seminal plug frequency in marked and unmarked cicadas. Equal numbers of marked and unmarked females were assigned to two cages, with an excess of males. Total females: Total number of females of each category present. Seminal plug present: total number of females at end of experiment with seminal plug. Marking does not affect likelihood of mating.
Total “seminal plug” females present c 2(2- tail) P
Cage A Marked 10 6 Unmarked 10 7 Total 20 13 0.038 P>>0.1 (n.s.)
Cage B Marked 10 5 Unmarked 10 6 Total 20 11 0.045 P >>0.1 (n.s.)
Combined cages Marked 20 11 Unmarked 20 13 Total 40 24 0.083 P >>0.1 (n.s.)
48 Table 2.3. Cage density resampling results, 10,000 iterations. Algorithm determines whether a specified cage density is greater than densities of cicadas on actual branches by resampling, with replacement, branch density data and generating percentiles. Cage densities above 25 are significantly greater than naturally-occurring densities on branches of similar size.
Hypothetical cage density P 12 0.85 14 0.82 16 0.51 25 0.053 26 0.028*
* indicates significance
49 Table 2.4. Results of pretarsal clipping experiment. Mating likelihoods of normal males and males with center tarsal claws removed were compared by confining 4 unmated females in each of 3 cages, and adding 4 normal and 4 clipped males to each cage. Counts are of normal and tarsal clipped males mating in each cage. 10 total matings occurred. In the combined replicates, the number of normal and tarsal clipped males mating do not differ (Fisher’s Exact 2- Tail P £ 0.656). Males mating Cage Normal Clipped 1 3 1 2 2 1 3 2 1 Combined 6 4
50 n size equal 0.029 0.130 0.396 0.396 0.515 0.515 0.515 P ≤ ≤ ≤ ≤ ≤ ≤ ≤ # Iterations with Observed # Resampling = rematings is the size of the population in small bag cages (8 for experiment 1; 4 for experiment 2). The algorithm 2). for experiment 1; 4 for experiment population in small bag cages (8 is the size of the n Summary of resampling results comparing remating frequencies in small bag cages to remating frequency in large flight to remating frequency in large frequencies in small bag cages results comparing remating Summary of resampling Experiment 1: (A)†(A)§BCExperiment 2: D 8E 8 8 are included as first matings (see text). A † If both questionable matings in replicate 8 is included as a first mating (see text). A § If only one questionable mating in replicate 4 3 4 2 4 2 10,000 10,000 1 1 10,000 10,000 10,000 290 10,000 1299 3960 3960 5150 5150 F 4 1 10,000 5150 Replicate # Females rematings iterations observed rematings Table 2.5. Table draws samples of The algorithm cage. flight mated females from large starts with data on singly- and doubly- Algorithm cage. from this population, where from this population, than or mated females in the sample is greater which the number of multiply of resampling iterations in then tallies the number bag cages. observed rematings in the small to the number of
51
12
10
8
6
Number Living 4 52
2
0 6/1/96 6/2/96 6/3/96 6/4/96 5/27/96 5/28/96 5/29/96 5/30/96 5/31/96 5/11/96 5/12/96 5/13/96 5/14/96 5/15/96 5/16/96 5/17/96 5/18/96 5/19/96 5/20/96 5/21/96 5/22/96 5/23/96 5/24/96 5/25/96 5/26/96 Date
Figure 2.1. Survivorship of marked and unmarked cicadas in two cages. Triangular and square markers indicate different cages, filled markers indicate marked treatment, and open markers indicate unmarked treatment. Cicadas in the two cages differed in survivorship, but within a cage, marking did not affect survivorship. CHAPTER III
MATE GUARDING AND THE NATURE AND POSSIBLE FUNCTIONS OF “SEMINAL PLUGS” IN PERIODICAL CICADAS (MAGICICADA SPP.)
Abstract
After mating, a female periodical cicada (Magicicada spp.) usually exhibits a mass, or “seminal plug,” blocking her genital opening. This mass contains DNA and appears to be composed of dried seminal fluid rather than being specially secreted or constructed. Although the “seminal plug” does not prevent additional matings, it may be part of a male strategy to make remating so costly, in terms of time, that mated females will be sexually unreceptive. Evidence that time is valuable to female Magicicada includes the limited nature of oviposition space, the monopolization of oviposition space by the first females to find it, and the apparent space-conserving nature of Magicicada eggnests. Thus, “seminal plugs” may be part of a male paternity-assurance strategy even though they do not prevent matings.
53 Introduction
As long as a given male’s sperm remain in a female’s reproductive tract, it is not in his interests to facilitate future mating by that female. However, because females may seek opportunities to remate in order to acquire resources or manipulate offspring paternity (Chapter 4), the sexes may be in conflict over remating. In species where males’ paternity is threatened by opportunities for polyandry, selection will favor males that evolve methods for blocking or preventing female remating, such as by inserting a “mating plug,” or barrier, into the female genital opening. Even a current tendency for females not to remate could be the direct result of past male efforts to devalue females’ remating incentives. For example, by inserting a mating plug, a male imposes costs on his mate and reduces her potential benefits from remating by forcing future mating efforts to include time spent removing or circumventing the plug. If females are time-limited, even if mating plugs are not absolute barriers to remating, females with such plugs may benefit from avoiding costly second matings by becoming sexually unreceptive after their first mating.
Mated female periodical cicadas (Magicicada spp.) usually exhibit a whitish or yellowish ovoid mass lodged in their genital openings (White 1973). Although the name applied to this structure, “seminal plug,” implies that it is an adaptation for mate-guarding, there are no detailed studies of its function. Magicicada seminal plugs could be the incidental results of ejaculate desiccation upon exposure to air. However, it is also possible that males use seminal plugs to protect their paternity, because under certain conditions female Magicicada remate (Chapter 4).
In other insect species, seminal plugs may function in biasing paternity by causing ejaculate to be retained within the spermatheca, by excluding additional sperm during the period that previously acquired sperm may be used (or stored for future use), or by permanently excluding additional sperm (Parker 1970, Boorman and Parker 1976, Leopold
54 1976, Ehrlich and Ehrlich 1978, Thornhill and Alcock 1983: 339, Alcock 1994, Polak et al. 1998). Extended copulation may serve similar functions (Alcock 1994, Parker 1970). Functional seminal plugs are often constructed of materials other than ejaculate. For example, honeybee (Winston 1987) and harvester ant (Hölldobler 1976) males leave their genitalia as plugs. In some Diptera, “sperm plugs” appear to be derived from spermatophore materials (see Neilsen 1959, Lum 1961, Kotrba 1996, Yasui 1997), while in many Lepidoptera, males apply sealing substances to the female genital opening after inserting a spermatophore (Labine 1964, Matsumoto 1987, Ehrlich and Ehrlich 1978).
Determining whether “seminal plugs” are male paternity-assurance adaptations or whether they are instead functionless consequences of matings is not a simple task. Two general predictions distinguish between functional and nonfunctional explanations for seminal plugs. (1) If a female insect can be caused to mate and acquire a seminal plug, and if she is prevented from acquiring a normal sperm supply but does not remate, the plug itself may be an effective deterrent to matings; alternatively, if such females show no decrease in likelihood of remating, remating propensity is likely controlled by factors such as spermathecal fullness. (2) If males use special materials or processes to construct or insert seminal plugs, the costs of doing so must be offset by some functional benefit, such as the ability of plugs to block matings; if, on the other hand, seminal plugs are incidental results of ejaculate drying, their functional significance and adaptive nature would be less certain.
I used several approaches to assess whether male Magicicada behave in ways that protect their paternity and whether seminal plugs are part of a paternity-assurance strategy. First, I evaluated whether mating durations are far in excess of what would be required for complete insemination; an excess would imply that males use mating duration as a form of mate-guarding. I also interrupted matings to determine whether seminal plugs are deposited at the conclusion of the normal mating sequence and whether they are produced
55 when males are removed suddenly and without warning. I determined whether seminal plugs deter or prevent subsequent matings, and I analyzed the substances contained in them to evaluate whether they are specially constructed or secreted. I confined my research to the 17- year species Magicicada septendecim, but since M. cassini and M. septendecula, as well as the 13- year species M. tredecim, M. neotredecim, M. tredecassini, and M. tredecula have similar mating systems and exhibit seminal plugs, the results are likely general to the genus Magicicada.
Methods and Results
Mating duration
In 1995 (study sites listed in Table 3.1), I captured and individually marked (Cooley et al. 1998, Chapter 2) teneral females and allowed them to mature in ca. 200 liter single-sex storage cages. Cages were constructed by folding a piece of hardware cloth over living hardwood vegetation. When the females were mature, I placed 5- 10 in newly constructed cages with fresh vegetation, and to each cage I added males captured from those active in nearby choruses. To establish starting and stopping times for each observed copulation, I monitored cages continuously or scanned them every 10-15 minutes, or ad lib scanned when I heard male courtship sounds. Mating durations are thus, in some cases, estimates ± 15 minutes. In 1996- 1998, I conducted similar experiments on 8 individuals belonging to two species of the genus Okanagana to provide comparative data (4 each O. canadensis and O. rimosa). I observed these cicadas continuously during courtship and mating and examined females for the presence of “seminal plugs” after mating.
Results. For the 61 Magicicada mating pairs I observed, mating duration averaged 838.8 (± 1074) minutes. Since poor weather may extend matings over days (unpub. data), if 17 matings lasting more than 10 hours are excluded, the remaining 43
56 matings had an average duration of 243.4 (± 121.48) minutes (Table 3.2). Matings in Okanagana were much shorter, with average durations under 30 minutes (Table 3.2).
Seminal plug deposition
In 1996, I collected 65 unmated, teneral female Magicicada septendecim and allowed them to mature as described above. When they were mature, I allowed them to mate with males caught from those present singing and flying in nearby choruses. I interrupted 38 of the resulting matings after 1 or 2 hours by suddenly, grasping the base of the male’s genitalia, gently working male and female genitalia apart, and noting the presence or absence of a “seminal plug.”
Results. Although interrupted matings could result in seminal plugs, indicating that the plug may be formed if the male transfers at least some ejaculate, interruption significantly decreases the likelihood that a seminal plug will be present (Table 3.3). All seminal plugs were clearly distinguishable from the absence of a plug, but plugs resulting from an interrupted copulation were more likely to have a soft, glossy appearance than plugs from uninterrupted matings. Because some females involved in terminated copulations exhibited sperm plugs, the insertion or creation of this plug requires either (1) no special actions or processes, or (2) that the male engage in processes that he can initiate and complete with little or advance preparation.
When I separated copulating pairs, in several cases I noted a small, whitish drop of ejaculate clinging to the males’ genitalia. In one case, the male continued ejaculating after separation, and the ejaculate quickly dried into a solid, whitish mass reminiscent of seminal plugs. This dried mass had the same color and consistency a typical seminal plug, except that it was of a more uniform consistency; the distal end of seminal plugs (the end visible in the genital opening) tended to be drier and more solid than the soft, moist proximal end.
57 These observations of drying ejaculate demonstrate that it can harden into a solid mass without additions or manipulations by the male.
Female remating
I allowed the 36 of the mating-interrupted females the opportunity to remate with males recently captured from the nearby chorus. Of the 36 females, 11 exhibited seminal plugs. When given the opportunity to remate, 20 of the 36 females in our mating interruption experiment remated; 4 of these 20 females exhibited seminal plugs. I compared remating frequencies of females with and those without seminal plugs using both a Chi-squared goodness of fit test and a computer resampling algorithm (Appendix E). The resampling algorithm randomly draws 20 “remating” samples from a population composed of 11 females with mating plugs and 25 without (total 36). The algorithm tallies the number of females in each sample with seminal plugs and repeats the process 100,000 times to generate percentiles. These percentiles can be used to determine whether the number of remating females with seminal plugs is smaller than expected by chance, as would be predicted if the seminal plug prevented copulation.
Females with seminal plugs were not underrepresented among remating females (Table 3.4). The seminal plug is therefore not a barrier to remating.
Seminal plug composition
In 1997, I captured approximately 25 mated female M. septendecim and, using forceps, removed seminal plugs from their genital openings. I preserved the plugs in 70% ethanol. To isolate high molecular weight DNA, I used Qiagen tissue preparation columns (Qiagen corp., Cat. 29304) to extract DNA from preserved plugs or from 70% ethanol preserved adults collected in 1996. I used a modified version of the tissue protocol included with the kit:
58 1. I removed and macerated a leg from each adult cicada, or I combined several seminal plugs into one microfuge tube. 2. I added 180 ul Qiagen buffer ATL and 20 ul Proteinase K stock solution without vortexing. I incubated the solution for a minimum of 24 hours at room temperature. 3. Without vortexing, I added 200 ul Qiagen buffer AL and incubated at 70°C for at least 10 min. 4. Without vortexing, I added 210 ul 100% ethanol.
5. I washed and eluted the DNA twice with warm distilled water as per instructions.
I amplified extracted DNA using a Randomly Amplified Polymorphic DNA oligonucleotide primer (RAPD; Haymer 1994, Hadrys et al. 1992) obtained from the University of British Columbia, with adult cicadas as positive controls and distilled water as our negative control in all amplifications. After screening approximately 200 other 10- base primers, I found that the primer sequence (CCT GCG CTT A) reliably produces a characteristic double or single band in M. septendecim with a minimum of amplification artifacts. I visualized amplified DNA on 1% agarose gels stained with ethidium bromide, photographed the gels, and noted the presence or absence of bands.
Results. The DNA extraction and amplification demonstrates that seminal plugs contain DNA (Fig. 3.1), a finding consistent with the hypothesis that they are formed of dried ejaculatory material instead of special-purpose substances. Qualities of the ejaculate alone can account for the properties of seminal plugs. As I extracted the seminal plugs, I noted that the proximal ends of most plugs were bifurcated, suggesting that they extended into paired spermathecae. The texture of the plugs was consistent with the hypothesis that they were formed by progressive drying of ejaculatory fluid, starting from the more exposed, distal end.
59 Discussion
The properties of seminal plugs can be accounted for by the properties of ejaculate alone. Males do not appear to employ special substances or structures other than ejaculate to construct seminal plugs; thus, they are not unambiguously male paternity assurance adaptations, although the hardening properties of ejaculate may be the product of selection for ejaculate to form barriers to remating. The remarkably long mating durations and “seminal plugs” of Magicicada may be parts of a male paternity-protection strategy. Mating interruption experiments (Chapter 4) have demonstrated that females allowed to mate for more than 2 hours tend to be sexually unreceptive and unlikely to remate; the longer average mating durations (ca. 5 hours) typical of Magicicada may be a form of male mate guarding (see Thornhill and Alcock 1983:340, Alcock 1994). Furthermore, the hooks and complex structure of male Magicicada genitalia (Chapter 8) may allow males to extend mating duration contrary to female interests, allowing the ejaculate to harden and making future matings costly or difficult. In contrast to Magicicada, Okanagana have short mating durations, simple genitalia, lack a seminal plug, and, presumably, have a greater confluence of interest between the sexes over mating duration.
Paradoxically, although Magicicada have long lifespans, time may be the adults’ greatest enemy. For example, since females appear to avoid constructing new eggnests directly over pre-existing ones, oviposition space in dense emergences may be limited and available only to the first finder. The ideal eggnest density probably represents a complex balance between overly dense eggnests, leading to “flagging,” or death of twigs (and the eggs therein), and overly sparse eggnests, which might be vulnerable to predators or parasitoids (Marlatt 1923). Of these competing sources of egg mortality, flagging is certain to occur on densely oviposited branches, while the risks from ovipositing in sparsely used branches are uncertain; thus females may be better off to err on the side of ovipositing in an area of sparse eggnests than to risk having their eggnests “flag.” The prevalence of
60 flagging may thus be weak evidence suggesting the scarcity of suitable oviposition space and the lack of oviposition options for many females.
The structure of Magicicada eggnests also supports the claim that oviposition space is limited. Magicicada eggnests are bifurcated, unlike those of other cicadas ovipositing in live wood, such as Okanagana. Bifurcated eggnests take up approximately as much linear space on a branch as do single eggnests, so Magicicada eggnests contain approximately twice as many eggs as Okanagana nests. Magicicada’s bifurcated eggnests may be an adaptation to ensure complete oviposition and monopolization of unused twigs when limited oviposition space is available on a “first come, first serve” basis.
Female- female competition for finding oviposition space may place a premium on efficient use of time and ability to locate and take maximum advantage of unused twigs. Males may use the currency of time to coerce females to acquiesce to male interests. Even without being an absolute barrier to copulation, seminal plugs may make future matings costly in terms of time, because future mates would have to remove or circumvent the seminal plug. By engaging in lengthy copulations, obstructing the female reproductive tract, and thereby ensuring that any future copulations would also be lengthy, males may reduce females’ potential benefits from remating.
One hypothesis for how Magicicada male and female interests in mating duration have evolved is as follows: Because males that monopolized the paternity of their mates’ offspring sired more offspring and were more successful than males whose sperm was diluted or discarded because of remating, some males increased their control of paternity by increasing mating duration. Over evolutionary time, as males first began to extend matings, the sexes may have been in conflict over the time costs of mating. As males escalated these costs beyond a certain point, and as males began to employ more and more effective forcible restraining devices, such as complex genitalia and ejaculate that forms seminal plugs (or any other strategy that functions to increase intermating interval), male
61 and female interests would have become more congruent, in that females could not afford to permit additional matings. Those females acceding to male interests in monopolizing paternity would have had an advantage over those engaging in multiple lengthy matings, so females would have evolved to be unreceptive to multiple matings. Thus, by the process described above, a seminal plug that did not prevent mating may have been part of a male strategy to negate any advantages females could realize from remating. This hypothesis would be disproven by findings that (1) seminal plugs provide no impediment to future matings and males have no adaptations for evading or removing them; or (2) seminal plugs are found in similar cicada species (such as Okanagana spp.) in which there is no evidence of male-female conflict over mating duration.
62 Literature Cited Alcock, J. 1994. Postinsemination associations between males and females in insects: The mate-guarding hypothesis. Ann. Rev. Entomol . 39: 1-21. Boorman, E., and G. A. Parker. 1976. Sperm (ejaculate) competition in Drosophila melanogaster, and the reproductive value of females to males in relation to female age and mating status. Ecological Entomology 1: 145- 155. Cooley, J. R., Hammond, G. S., and D. C. Marshall. 1998. The effects of enamel paint marks on the behavior and survival of the periodical cicada, Magicicada septendecim (L.) (Homoptera) and the lesser migratory grasshopper, Melanoplus sanguinipes (F.) (Orthoptera). Great Lakes Entomologist 31: 161-168. Ehrlich, A. R., and P. R. Ehrlich. 1978. Reproductive strategies in the butterflies: I. Mating frequency, plugging, and egg number. J. Kans. Ent. Soc. 51: 666- 697. Hadrys, H., M. Balick, and B. Schierwater. 1992. Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Molecular Ecology . 1: 55-63. Haymer, D. S. 1994. Random amplified polymorphic DNAs and microsatellites: What are they, and can they tell us anything we don’t already know. Ann. Ent. Soc. Amer. 87: 717- 722. Hölldobler, B. 1976. The behavioral ecology of mating in harvester ants (Hymenoptera: Formicidae: Pogonomyrmex ). Behav. Ecol. Sociobiol . 1: 405- 423. Kotrba, M. 1996. Sperm transfer by spermatophore in Diptera: new results from the Diopsidae. Zool. J. Linn. Soc . 117: 305- 323. Labine, P. A. 1964. Population biology of the butterfly, Euphydryas editha I. Barriers to multiple inseminations. Evolution 18: 335- 336. Leopold, R. A. 1976. The role of male accessory glands in insect reproduction. Ann. Rev. Emtomol. 21: 199-221. Lum, P. T. M. 1961. The reproductive system of some Florida mosquitoes. II. The male accessory glands and their role. Ann. Ent. Soc. Amer. 54: 430- 433. Marlatt, C. L. 1923. The periodical cicada. U.S.D.A. Bureau of Entomology Bulletin 71, 183 pp. Matsumoto, K. 1987. Mating patterns of a sphragis-bearing butterfly, Luehdorfia japonica Leech (Lepidoptera: Papilionidae), with descriptions of mating behavior. Res. Pop. Ecol. 29: 97- 110. Nielsen. E. T. 1959. Copulation of Glyptotendipes (Phytotendipes) paripes Edwards. Nature (London) 184: 1252- 1253. Parker, G. A. 1970. Sperm competition and its evolutionary consequences in the insects. Biological Reviews 45: 525- 567.
63 Polak, M., W. T. Starmer, and J. S. F. Barker. 1998. A mating plug and male mate choice in Drosophila hibisci Brock. Anim. Behav. 56: 919- 926. Thornhill, R. and J. Alcock. 1983. The evolution of insect mating systems . (Harvard University Press: Cambridge, MA) White J. 1973. Viable hybrid young from crossmated periodical cicadas. Ecology 54: 573- 580. Winston, M. L. 1987. The biology of the honey bee . (Harvard University Press: Cambridge, MA) Yasui, Y. 1997. Sperm competition and the significance of female multiple mating in the predatory mite Parasitus fimetorum. Experimental and Applied Acarology 21: 651- 664.
64 199519961997 I II III1996- 1998 Siloam Springs Lake SWF Horsepen Siloam Springs SP UMBS BuckinghamAdams Brown, Rockbridge VA IL VA Logged site Emmet Cheboygan/ Clearing MI Logged site Open field Study sites 1995- 1997. Table 3.1. Table SpeciesMagicicada septendecim YearOkanagana canadensis/ O. rimosa Brood Location County State Characteristics
65 Table 3.2. Mating durations in Magicicada septendecim and Okanagana. O. canadensis mating lasted approximately 4 times as long as the average of the others, so two averages, one with this long mating, and one without, are calculated for this species.
Species Duration (min: sec) O. rimosa 19:00 17:00 22:09 17:04 Average 18:48 ± 2:25
O canadensis 80:00 19:06 15:09 23:18 Average 34:23 ± 30:35 Average w/o outlier 19:11 ± 4:05
Magicicada septendecim 195 315 195 330 295 442 167 297 235 235 235 195 310 500 60 124 305 111 354 295 124 331 440 440 310 64 220 444 263 295 354 360 360 250 263 310 65 526 148 91 310 60 405 Average (n= 43) 243:24 ± 121:48
66 Table 3.3. Results, female mating interruption experiment. For each treatment (disrupt 1 hr., disrupt 2 hr., combined 1 and 2 hr disruption, and no disrupt), numbers of females without seminal plugs (No SP), with firm seminal plugs (Firm SP), and with soft seminal plugs (Soft SP) were counted. There were no statistical differences between 1 and 2 hour mating disruptions, so these treatments were combined. Females whose matings were interrupted were less likely to exhibit seminal plugs than were females with uninterrupted matings (Fisher’s Exact 2- tail, P < 0.001). Soft seminal plugs were more prevalent in interrupted matings than in uninterrupted matings (Fisher’s Exact 2- tail P < 0.001).
Mating Total interruption females No SP Firm SP Soft SP Firm + Soft 1 hr. 13 8 1 4 5 2 hr. 25 17 6 2 8 £ 2 hr. (combined) 38 25 7 6 13
Not interrupted 27 2 25 0 25
67 Table 3.4. Numbers of females with and without seminal plugs (SP) remating when given free access to males. The presence of a seminal plug did not affect female likelihood of remating: If rematings were expected to be evenly distributed between females with and without plugs, 6 females with plugs would be expected to remate. 4 did : X 2 P > 0.10. The data were also analyzed with a resampling algorithm, which drew a sample of 11 females from the “without SP” data: In 100,000 iterations of the algorithm, the proportion of iterations in which 4 or fewer plugged females were included in the sample was 0.2214. Remating Not remating Females with SP 4 7 Females without SP 16 9
68 Seminal Plug Seminal Plug Adult Adult Adult
69 H 2 O
Figure 3.1. Photograph of gel showing RAPD-PCR amplification with primer (CCT GCG CTT A). Adults and seminal plugs show characteristic amplification products, demonstrating that seminal plugs contain DNA. If plugs did not contain DNA, they would show no amplification product. Gel is 1% agarose stained with EtBr, negative control is distilled water, positive controls are adult cicadas. CHAPTER IV
FEMALE MULTIPLE MATING AND MATE CHOICE IN A PERIODICAL CICADA, MAGICICADA SEPTENDECIM
Abstract
Explanatory hypotheses for polyandry in the periodical cicada Magicicada septendecim fall into two categories: 1. Females remate because of inadequate insemination; and 2. Females remate to effect mate choice. I gave mated females the opportunity to remate after manipulating both mating duration and mate quality. Females whose first matings were terminated prematurely tended to remate, while females allowed complete, undisturbed matings with inappropriate, heterospecific males did not. After participating in a complete, undisturbed mating, females were no longer sexually receptive nor were they attractive to males. Thus, when placed in situations where they were either inadequately inseminated or mated with low-quality mates, only females in the former manipulations mated multiply, suggesting that polyandry in M. septendecim is best understood as the result of sperm limitation rather than as an evolved mate choice strategy.
70 Introduction
Multiple mating is sometimes considered to be evidence that females exercise postcopulatory mate choice (Eberhard 1985, 1996, 1997). Selection favors the evolution of mate choice mechanisms when individuals discriminating among potential mates produce genetically superior offspring, acquire phenotypic benefits specific to a given mate and convertible into reproductive output, or both. Postcopulatory mate choice is a possibility for females that mate multiply and for which insemination and fertilization are separated in time (e.g.; Eberhard 1985, Birkhead and Møller 1993, LaMunyon and Eisner 1993, Eberhard 1996, 1997), although some models of postcopulatory choice have been subjected to strong criticism (Alexander et al. 1997). Alternatively, females may remate to acquire sufficient sperm (or other resources) to complete their reproduction, in which case, mate choice need not be invoked. Magicicada septendecim 1 females sometimes remate (Chapter 2, Alexander 1968), although anecdotal evidence suggests that remating is not common: During the 1998 emergence of periodical cicadas, mating pairs became increasingly rare as the emergence progressed, the opposite of the pattern expected if females typically remate. To evaluate whether female M. septendecim remating is an evolved mate choice mechanism, I first discuss the general benefits females might obtain by remating and whether such benefits involve mate choice. Although, in order to identify selective contexts of remating, the benefits of polyandry are presented as if they belong to strict categories, this is undoubtedly an oversimplification, because the different kinds of benefits are likely not exclusive. I conclude by presenting the results of manipulations designed to evaluate the causes of polyandry in M. septendecim.
Remating as bet-hedging. Multiply mating females may gain from potential increases in offspring diversity or from bet hedging against genetically incompatible sires
1 This paper specifically concerns Magicicada septendecim. However, its findings may be applicable to other species in this genus since the sexual behaviors of all Magicicada species share general similarities.
71 (Walker 1980, Smith 1984, Watson 1991, Zeh and Zeh 1996, 1997, Zeh 1997, Brown 1997a, Tregenza and Wedell 1998). If females remate for diversity or bet-hedging, most or all females in a population should mate multiply, mate quality should not necessarily increase with each successive mating (see Watson 1991), nor should mating with a lower- quality male necessarily trigger remating. If females remate only to increase offspring diversity, sperm displacement must be incomplete, or, if complete, females must oviposit between matings to allow multiple paternity.
Remating for replenishment. Limited female sperm storage capacity, variable ejaculate volumes, limits to males’ ability to fully inseminate in a single mating, and disturbances during mating may promote serial polyandry because remating allows females to replenish or supplement inadequate sperm supplies (Boorman and Parker 1976, Walker 1980, Gromko et al. 1984, Ward et al. 1992). Females remating to replenish depleted supplies are expected to oviposit or engage in other sperm- or resource- depleting activities between successive matings. Some female dipterans, including Drosophila (Pyle and Gromko 1978, Gromko and Markow 1993), Ceratitis (Nakagawa et al. 1971, Whittier and Shelley 1993) and Rhagoletis (Neilson and McAllan 1965), homopterans (Nilaparvata lugens; Oh 1979), and heteropterans (Riptortus, Sakurai 1996, 1998a, b) experience reversible decreases in rates of ovipositing normal, fertilized eggs (considered an index of fecundity and fertility), several days after mating. In Riptortus and Nilaparvata, coincident with decreasing oviposition rate is a return to sexual behaviors typical of unmated females. In each of the above examples, fertility and fecundity returned to high levels subsequent to remating.
Remating and phenotypic benefits. Just as females may remate to replenish depleted sperm, if remating increases female resource access, females may remate to obtain direct phenotypic benefits. Mating males may provide nuptial gifts, spermatophores, (Morris 1979, Sakaluk and Cade 1982, Thornhill and Alcock 1983, Thornhill 1984,
72 Gwynne 1986, Goulson et al. 1993, Kaitala and Wiklund 1995, Brown 1997a, b, 1999) increased predator protection, or access to limited resources (as in resource-based leks; Alexander 1975, Hoglund and Alatalo 1995). Promiscuous females could collect phenotypic benefits in the same manner as any acquirable resource; here, the price of acquisition is permitting male sexual access. Female “foraging” for male-provided resources may be at least partially responsible for polyandry in some Lepidoptera (Rutowski 1984, Svärd and McNeill 1994, Karlsson 1996,) and perhaps in some Orthoptera (Sakaluk and Cade 1982, Brown 1997b) and Hymenoptera (Alcock et al. 1977). Phenotypic benefits from polyandry also include reduced costs, or at least minimization of losses, associated with acquiescence to persistent male harassment (Trillmich and Trillmich 1984, Clutton-Brock and Parker 1995, Jormelainen and Merilaita 1995, Arnqvist 1997, Crean and Gilburn 1998), although an effective female response to this male tactic is to leave the harassing male’s vicinity (see Alexander and Moore 1962, Stone 1995). Females remating only to replenish depleted supplies, and not to effect mate choice, would be expected to remate when they run low on supplies and not necessarily whenever a high quality male becomes available.
If male variations affecting ability to produce, acquire, or defend high-quality resources or nuptial gifts are heritable, then direct phenotypic benefits from choosing males with superior resources could be accompanied by genotypic benefits through jointly- produced offspring, and female remating could be a form of postcopulatory mate choice.
Remating and genetic benefits. If females remate to improve the genetic constitution of their offspring, then they are exercising mate choice. Genetic benefits from multiple mating include the advantages of cuckolding a genetically inferior parental male (Westneat et al. 1990, Birkhead and Møller 1993, Kempenaeres et al. 1992), and, under restricted conditions, benefits from sperm competition within the female, such that male offspring sired by especially competitive sperm might themselves produce competitive
73 sperm (Harvey and May 1989, Curtsinger 1991). If females remate to improve the genetic quality of their offspring, no complex mechanisms need be invoked; for instance, females with “last male” sperm priority using “threshold criteria” (Janetos 1980; Alexander et al. 1997) to choose mates could, by raising their choice threshold after each mating, “revise” their previous choices by mating with successively higher quality males (see Gabor and Halliday 1997). If males are readily available and not costly to acquire, then choosy females first mated with inferior males should be most likely to remate, and females should grant all their paternity to their best mate, wasting none on inferior ones. If remating tendency is independent of mate quality, replenishment seems a more likely explanation.
Bias against heterospecifics. Bias against heterospecifics is often an example of indirect mate choice (Chapter 1) rather than an evolved mate choice mechanism. Females benefit from such biases by obtaining appropriate genetic materials for their offspring. One form of such bias is conspecific sperm precedence, in which, regardless of mating order, few hybrid offspring are produced when a female is mated with a series of con- and hetero- specifics. Conspecific sperm precedence is reported in flour beetles (Wade et al. 1994), grasshoppers (Bella et al. 1992), sunflowers (Rieseberg et al. 1995), ground crickets (Howard and Gregory 1993, Gregory and Howard 1994; Howard, Gregory, Chu, and Cain 1998; Howard, Reece, Gregory, Chu, and Cain 1998), and several Drosophila species (Price 1997). If precopulatory behaviors allow a significant proportion of heterospecific matings, females could recover from such mistakes by remating (see Jamart et al. 1995). However, this pattern is not necessarily evidence of an evolved post-copulatory mate choice mechanism. Instead, it may result from the inferior competitive ability of heterospecific sperm and seminal products in the environment of the female reproductive tract, especially if the interactions between female and ejaculate are shaped by a long history of coevolutionary antagonism over control of fertilization (Rice 1996, Price 1997, Alexander et al. 1997, Rice 1998).
74 Cross-mated females capable of postcopulatory mate discrimination would be expected to remate before ovipositing unless sperm priority was first in/first out, there was no sperm mixing, and there was no way to remove stored spermathecal sperm except through fertilization. Otherwise, the sperm dilution or replacement caused by subsequent matings prior to oviposition would be to the female’s advantage, since it would minimize the production of hybrid offspring, each of which represents a loss of potential fitness. If females mated to heterospecific males do not actively seek subsequent matings with conspecifics, then it is unlikely that they have evolved to exercise postcopulatory mate choice.
Polyandry in M. septendecim
Precopulatory behaviors may provide female periodical cicadas an efficient first defense against heterospecific males; precopulatory species discrimination based on song characteristics has been demonstrated (Chapter 6), and, in addition, 78 of 80 female M. septendecim in one of our cage experiments avoided heterospecific matings, a figure comparable to data reported in White (1973). However, if, in spite of precopulatory discrimination, heterospecific matings are a significant threat to females, then it is possible that remating behaviors evolved to allow mate choice revision. Females could also remate to discriminate among conspecific sires. To evaluate whether female remating is likely to function in mate choice, I conducted mating experiments to determine the contexts in which females remate, playback experiments to evaluate female sexual receptivity after mating, and experiments to evaluate female sexual attractiveness after mating.
Female remating experiments
For M. septendecim, at the present time, we are unable to identify a class of inferior conspecifics or determine whether, within a species, males vary in ways likely to promote female mate choice. One way to cause females to remate is to interrupt their first matings.
75 Interruption causes remating either because it simulates a mating attempt by an inferior male or because it results in incomplete insemination and an inadequate sperm supply. The mate choice through remating hypothesis predicts that females should remate immediately in response to mating with an “inferior” male and grant paternity only to later mates and that the length of time allowed before interruption should not influence likelihood of remating. In contrast, the replenishment hypothesis predicts that interrupted females will oviposit, and as they deplete their sperm stores, they will remate; thus females allowed the least amount of time for their first matings should be the most likely to remate immediately. Furthermore, if females have an evolved mechanism for improving their circumstances after mating with an inappropriate mate, then females mated with heterospecifics should have high pre-oviposition remating frequencies.
Methods. I studied Brood II periodical cicadas in a large open field near Horsepen Lake State Wildlife Management Area, Buckingham County, VA, during an emergence lasting from late April through early June 1996. Vegetation in the field consisted primarily of oak, maple, and tulip tree stump sprouts from a logging operation several years before. The field was surrounded by a mixed forest composed of mature hardwoods, primarily oaks and maples, and planted pines.
The morning after they molt, newly emerged cicadas (“teneral” cicadas) are readily identifiable by their pale color, soft bodies, and by their position low in the vegetation. I collected unmated, teneral female cicadas daily and placed them into fiberglass mesh storage cages one meter in length and approximately 60 cm in diameter placed over living vegetation. After females had matured in the storage cages without access to males, I marked them with either dots of enamel paint or with symbols drawn onto their wings with laboratory markers. Neither marking method has adverse effects on cicadas (Chapter 2; Cooley et al. 1998). I caught males from those present singing and flying in the area of the experiments and marked them in the same manner as the females.
76 I placed 5- 10 mature females and fewer males (to discourage usurpation or harassment) in nylon tulle cages similar to the storage cages. I stocked these mating cages between 8 A. M. and 12 noon, and most matings occurred shortly thereafter. To establish a starting time for each observed copulation, I monitored cages continuously or scanned them every 10-15 minutes, stopping the scan only when I had seen every cicada in the cage. Alternatively, I ad lib scanned when I heard male courtship songs. Starting times are thus, in some cases, estimates ± 15 minutes. Matings whose beginning times could not be established within this degree of accuracy were used for the “no interruption” treatment (see below). Any cicadas failing to mate the same day they were placed in mating cages were discarded.
Mated female periodical cicadas usually exhibit a mating sign or “seminal plug” in their genital opening (White 1973), although this structure is not always present and does not prevent subsequent matings (Chapter 3). A seminal plug appears as a whitish or yellowish ovoid mass when the female’s ovipositor sheath is gently pulled open. The plug appears to be composed of dried seminal fluid (Chapter 3), and its presence is thus an indicator that the male has transferred at least some ejaculate. In all experiments, I noted the presence or absence of a seminal plug after each mating.
I interrupted conspecific matings after 15 min., 1 hour, 2 hours, or not at all by, at the appropriate time, quickly removing mating pairs from the mating cages, grasping the base of the male genitalia, and gently working male and female genitalia apart. Once separated, males were immediately preserved in 70% ethanol. In addition, I mated female M. septendecim with male M. cassini by confining them together in small cages. I left mating pairs not to be disrupted undisturbed until they stopped mating, at which time I preserved the males. I checked females for the presence of a seminal plug immediately after disruption or cessation of mating.
77 After they had mated once, or after their first mating had been disrupted, females were placed into cages where they had access to conspecific males and appropriate oviposition space. I surveyed these cages at least once per hour and immediately placed females observed ovipositing into separate oviposition cages, one female per cage. In addition, I subjected some females to a treatment in which their mating was interrupted after two hours and they were placed directly into oviposition cages. Once placed in oviposition cages, females were permitted no further mating opportunities.
Oviposition cages were either surplus mating cages or fiberglass screen cages approximately one meter in length and 30 cm in diameter. I placed all oviposition cages over oak, maple, or beech vegetation that had been thoroughly inspected for the absence of eggnests and that had a minimum of one meter of woody branch suitable for oviposition. I distributed females among oviposition cages haphazardly with respect to tree species, cage type, and cage location and removed each female from its oviposition cage when it died, became moribund, or at the end of the experiment. I left all oviposition cages intact and closed until the branch broke due to excessive oviposition (“flagging”) or until the end of the experiment, at which time I removed, counted, labeled, and preserved all eggnests in 70% ethanol.
Results. Normal, uninterrupted matings lasted an average of 243.4 minutes (Table 4.1). Females whose first matings were interrupted after 15 minutes or one hour were most likely to remate before ovipositing, those interrupted after two hours were equally likely to remate or not, and those not interrupted usually did not remate before ovipositing (Table 4.2). Females whose matings were disrupted within 2 hours of starting also tended to lack a seminal plug. Female M. septendecim first mated with M. cassini males did not remate before ovipositing. One female whose mating was uninterrupted and apparently normal did remate before ovipositing.
78 There is a statistical difference in number of pre-remating eggnests constructed by females interrupted at two hours and females who were not interrupted (Table 4.3, Fig. 4.1). However, there was no difference between the total number of eggnests produced by heterospecific-mated, mating-interrupted females allowed to remate, and uninterrupted females.
Female sexual receptivity
Upon hearing a male’s calling phrase, a receptive female flicks her wings with a quick motion (Chapter 5); this signal causes the male to continue courtship and attempt to copulate. If females solicit second matings as a form of mate choice, then we would expect them to employ many of the same courtship behaviors, such as signaling, as in first matings, because non-signaling females would have difficulty obtaining mates.
Mating status and receptivity. I identified a high quality recording of Magicicada septendecim song in the University of Michigan Museum of Zoology Sound Library. I imported this recording into a Macintosh IIcx computer using Mac Recorder hardware and Sound Edit software. I removed extraneous background noise, isolated a single male song, and confirmed that females signal in response to playbacks of this song (Chapter 5).
In 1997, at Siloam Springs State Park, Brown and Adams Counties, Illinois, (Brood III), I collected 44 unmated teneral female M. septendecim and allowed them to mature in single-sex storage cages. Half (22) were mated to males captured from the chorus, half were left unmated. Mated and unmated females into two cages, and in each of 4 subsequent days, I played recorded M. septendecim calling songs to the surviving females for 2 minutes and recorded the number mated and unmated females responding.
Results. None of the mated females responded to playbacks, while at least half of the unmated females responded each day with wing flicks (Table 4.4).
79 Mating completeness and receptivity. In June 1997, I conducted experiments combining the approaches of the female remating experiments and the playback experiments to determine how mating interruption would affect the likelihood that a female would solicit a second mating. As before, I allowed the cicadas to mature in fiberglass cages and then marked them individually prior to use in experiments. After the cicadas were mature, I allowed them to mate, disrupting the matings after 15 min, 1 hour, 2 hours, or not at all. I discarded the males and placed the females in cages without access to males. I visited female cages at least twice daily, played artificial male calling songs, and recorded female responses.
Results. Females whose matings were disrupted were likely to respond positively to male song, while normally mated females never responded. All disruption treatments resulted in an equal likelihood of female response (Table 4.5). These results are compatible with the 1996 experiments, in which all disruption treatments led to an elevated incidence of remating.
Female sexual attractiveness
Our playback experiments demonstrate that normally mated females are not sexually receptive to courting males. Males’ willingness to court mated and unmated females is potentially a separate issue (for a possible example see Gromko and Markow 1993). If there is even a slight chance that a mated female will remate, such as if she were to remate to effect mate choice, then males should find mated females sexually attractive and court them. If, on the other hand, female remating is an unusual event, one that happens only under special circumstances, males should seek unmated females instead of wasting time and effort courting mated and unreceptive females, and they should not behave as though mated females are sexually attractive.
80 Methods. Unmated female M. septendecim confined in cages signal to courting males outside. Cages containing signaling females thus are attractive to males, and males seeking mates congregate on the outsides of such cages. I evaluated the relative sexual attractiveness of mated and unmated females by confining them in cages and censusing the males observed on the outsides of the cages.
I erected three large, cylindrical, white tulle cages 1.5 m in diameter and 2 m tall over stump sprouts, all at the same distance from the forest edge in our Horsepen Lake, VA study site. I collected unmated and mated females in the same manner as for the playback experiments and placed 13 mated females in one cage, 13 unmated females of the same age in another, and nothing in the third cage. The cages were scanned at intervals no greater than one hour over the course of a two-day period and recorded the number of males on the exterior surface of each cage. At each census, all males were colllected from the exterior of the cages and removed to a location at least 15 m distant.
Results. Because the males at each census time were unlikely to be the same, each census is treated as an independent data point. Significantly greater numbers of males were observed on the cage containing unmated females than on the empty cage, demonstrating that it was the females in the cage, not the cage itself, that attracted courting males. Significantly fewer males visited the cage containing mated females than the cage containing unmated females, indicating that males are not strongly sexually attracted to mated females. (Table 4.6).
Discussion
Mating aggregations suggest the action of sexual selection (Emlen and Oring 1977, Alexander and Borgia 1979, Andersson 1994, Höglund and Alatalo 1995). Alexander (1975) considered periodical cicada male singing aggregations to be non-resource based leks. In such leks, males neither control nor have exclusive access to resources (other than
81 gametes) required by females, and thus females visit the lek only for the purpose of mating. Although female periodical cicadas occasionally remate, and although the situations faced by females in these experiments would seem to promote postcopulatory mate choice, the data provide scant evidence of a postcopulatory mate choice mechanism2. If females were able to accomplish mate choice by remating, they might be expected to detect the inappropriateness of a heterospecific mate or the inferiority of an abbreviated ejaculate and remate as quickly as possible. However, in our experiments, only females whose first matings were extremely brief were attractive to males, actively solicited matings, and reliably remated before ovipositing.
The readiness of mating interrupted or cross-mated females to oviposit makes evolved postcopulatory mate choice doubtful. The failure of cross-mated females to remate before ovipositing suggests that females are unable to evaluate male quality after the mating act has commenced or that they realize no benefits from doing so. Thus, any species- identification mechanisms of periodical cicada mating behavior must be precopulatory. Magicicada’s complex, species-specific courtship makes it unlikely that females are routinely in close proximity to heterospecifics or that they normally mate with them. The failure of female postcopulatory behavior to discriminate against mates as unsuitable as heterospecifics and the demonstrated post-mating change in both female receptivity and sexual attractiveness indirectly support the importance of precopulatory discrimination in Magicicada.
For periodical cicada females, mating is not known to provide any phenotypic benefits, either through ejaculatory nutrients or nuptial gifts, nor do males restrict female access to resources (other than sperm). Phenotypic benefits through reduced costs from
2 One female whose mating was uninterrupted and apparently normal did remate before ovipositing; this observation has no real significance, but it is consistent with female remating behavior being an adaptation to discriminate against poor quality mates, because at least some females who were randomly paired with males would be expected to remate because at least some would have been presented with a better alternative to their first mating.
82 acquiescence to male harassment may be applicable, but as noted elsewhere, females can simply fly away from persistent courting males. Furthermore, because the only male behavior that effectively blocks the access of other males is copulation (pers. obs), mating with a persistent male would only immunize female cicadas from courtship during the actual mating act, would give the female no additional feeding time (feeding may be carried out alone or simultaneously with many other behaviors, including copulation; pers. obs. and similar to ladybird beetles, see Obata 1988) and could only cost oviposition time. Another direct cost may concern male health; in Magicicada, contagious and fatal infections of Massospora cicadina Peck, an entomophagous fungus, produce a class of potential mates best avoided and with whom any interactions carry a risk of infection (Soper 1974, Soper et al. 1976, Alexander et al. ms.). Yet female polyandry cannot be explained as an infection-avoidance tactic or as a tactic to avoid genetically susceptible males because females should be selected to identify and avoid infected males early in the courtship sequence and avoid excessive intimate contact. Lastly, the fact that few females mate multiply suggests that there are not likely to be significant direct phenotypic benefits promoting remating.
Females must evidently require some threshold quantity of sperm before ovipositing; once they use up a substantial quantity of stored sperm, they may remate. Evidence in support of this statement includes the results that females interrupted at two hours produced, on average, fewer eggnests before remating than the total number produced by females who were not interrupted (Table 4.3). However, the experiments also indicate that females interrupted at two hours and deprived of further mating opportunities are capable of producing as many eggnests as uninterrupted females. Possibly, when a female’s sperm stores fall below some threshold volume, she will remate if given the opportunity; yet regardless of whether she has access to mating opportunities or not, she will continue to oviposit for as long as possible. This pattern would be consistent with the existence of an evolved mechanism that caused females to replenish sperm
83 supplies well before exhausting their spermathecal contents, perhaps a form of bet-hedging against difficulties of efficiently finding a mate. Alternatively, if it is never difficult for females to find mates, females may have no evolved mechanisms to stop oviposition. Females may maladaptively oviposit unfertilized eggs (Graham and Cochran 1954) after exhausting their sperm stores because the ready availability of receptive mating partners means that females are never in the position of lacking sperm; thus we would expect no history of selection on females for suspension of oviposition pending sperm replenishment.
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87 Obata, S. 1988. Mating refusal and its significance in females of the ladybird beetle, Harmonia axydris . Physiol. Entomol . 13: 193- 199. Oh, R. J. 1979. Repeated copulation in the brown planthopper, Nilaparvata lugens Stål (Homoptera: Delphacidae). Ecological Entomology 4: 345- 353. Price, C. S. C. 1997. Conspecific sperm precedence in Drosophila. Nature (London) 388: 663-666. Pyle, D. W., and M. H. Gromko. 1978. Repeated mating by female Drosophila melanogaster: The adaptive importance. Experientia 15: 449- 450. Rice, W. R. 1996. Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. Nature (London) 381- 232- 234. Rice, W. R. 1998. Intergenomic conflict, interlocus antagonistic coevolution, and the evolution of reproductive isolation. Pp. 261-270 in D. J. Howard and S. H. Berlocher, eds. Endless Forms: Species and Speciation . (Oxford University Press: New York). Rieseberg, L. H., A. M. Desrochers, and S. J. Youn. 1995. Interspecific pollen competition as a reproductive barrier between sympatric species of Helianthus (Asteraceae). Am. J. Bot. 82: 515-519. Rutowski, R. L. 1984. Production and use of secretions passed by males at copulation in Pieris protodice (Lepidoptera: Pieridae). Psyche 91: 141- 152. Sakaluk, S. K., and W. H. Cade. 1982. Orthopteran Mating Systems: sexual competition in a diverse group of insects. (Westview Press: Boulder, CO). Sakurai, T. 1996. Multiple mating and its effect on female reproductive output in the bean bug Reptortus clavatus (Heteroptera: Alydidae). Ann. Ent. Soc. Amer. 89: 481- 485. Sakurai, T. 1998a. Variation in time to sperm depletion and oviposition patterns in females of Riptortus clavatus (Heteroptera : Alydidae). Ann. Ent. Soc. Amer. 91: 737-740. Sakurai, T. 1998b. Receptivity of female remating and sperm number in the sperm storage organ in the bean bug, Riptortus clavatus (Heteroptera : Alydidae). Res. Popul. Ecol. 40: 167- 172. Smith, R. L. 1984. Human sperm competition. Ch. 19 in: Smith, R. L., ed. Sperm competition and the evolution of animal mating systems . (Academic Press: New York). Soper, R. 1974. The genus Massospora entomopathogenic for cicadas. Part I. Taxonomy of the genus. Mycotaxon 1:13-40. Soper, R., A. J. Delyzer, and L. F. R. Smith. 1976. The genus Massospora entomopathogenic for cicadas. Part II. Biology of Massospora levispora and its host Okanagana rimosa, with notes on Massospora cicadina on the periodical cicadas. Ann. Ent. Soc. Amer . 69:89-95.
88 Stone, G. N. 1995. Female foraging responses to sexual harassment in the solitary bee Anthophora plumipes. Anim. Behav . 50: 405- 412. Svärd, L., and J. N. McNeil. 1994. “Female benefit, male risk” polyandry in the true armyworm, Pseudaletia unipunctata. Behav. Ecol. Sociobiol . 35: 319- 326. Thornhill, R. 1984. Alternative female choice tactics in the scorpionfly Hylobittacus apicalis (Mecoptera) and their implications. Amer. Zool. 24: 367- 383. Thornhill, R. and J. Alcock. 1983. The evolution of insect mating systems . (Harvard Unversity Press: Cambridge, MA). Tregenza, T., and N. Wedell. 1998. Benefits of multiple mates in the cricket Gryllus bimaculatus. Evolution 52: 1726-1730. Trillmich, F. and K. G. K. Trillmich. 1984. The mating systems of pinnipeds and marine iguanas: convergent evolution of polygyny. Biol. J. Linn . Soc. 21: 209- 216. Wade, M. J., H. Patterson, N. W. Chang, and N. A. Johnson. 1994. Postcopulatory, prezygotic isolation in flour beetles. Heredity 72: 163- 167. Walker, W. F. 1980. Sperm utilization strategies in nonsocial insects. American Naturalist 115: 780- 799. Ward, P. I., J. Hemmi, and T. Roosli. 1992. Sexual conflict in the dung fly Sepsis cynipsea. Functional Ecology 6: 649-653. Watson, P. J. 1991. Multiple paternity as genetic bet-hedging in female sierra dome spiders, Linyphia litigiosa (Linyphiidae). Anim. Behav. 41: 343- 360. Westneat, D. F, P. W. Sherman, and M. L. Morton. 1990. The ecology and evolution of extra-pair copulations in birds. Current Ornithology 7: 331- 369. White J., 1973. Viable hybrid young from crossmated periodical cicadas. Ecology 54: 573- 580. Whittier, T. S., and T. E. Shelley. 1993. Productivity of singly vs. multiply mated female Meditteranean fruit flies, Ceratitus capitata. J. Kans. Ent. Soc. 66: 200- 209. Zeh, J. 1997. Polyandry and enhanced reproductive success in the harlequin-beetle-riding pseudoscorpion. Behav. Ecol. Sociobiol . 40: 111- 118. Zeh, J. A., and D. W. Zeh. 1996. The evolution of polyandry I: Intragenomic conflict and genetic incompatibility. Proc. R. Soc. Lond. B. 263: 1711- 1717. Zeh, J. A., and D. W. Zeh. 1997. The evolution of polyandry II: Post-copulatory defences against genetic incompatibility. Proc. R. Soc. Lond. B. 264: 69-75.
89 Table 4.1. Mating durations (minutes) in Magicicada septendecim.
195 315 195 330 295 442 167 297 235 235 235 195 310 500 60 124 305 111 354 295 124 331 440 440 310 64 220 444 263 295 354 360 360 250 263 310 65 526 148 91 310 60 405 Average (n= 43) 243:24 ± 121:48
90 Table 4.2. Mating interruption experiment: Likelihood of remating first or ovipositing first, and presence/absence of seminal plug. All P-values Bonferroni corrected for 18 comparisons. £ 1hr category includes combined data from 15 minute/ 1 hr. disruption; £ 2 hr category includes combined data from all disruption treatments.
Treatment Remate first Oviposit first SP No SP Disrupt at 15 min 14 0 0 14 Disrupt at 1 hr. 11 1 1 9 Disrupt at 2 hr. 14 11 6 19 Not disrupted Conspecific (M. septendecim) mate 1 31 26 2 Heterospecific (M. cassini) mate 0 5 5 0
Comparison P (Fisher’s Exact 2- Tail) Remating Frequency 15 min. vs. not disrupt < 0.001* 1 hr. vs. not disrupt < 0.001* 2 hr. vs. not disrupt < 0.001* £1 hr. vs. not disrupt < 0.001* £2 hr. vs. not disrupt < 0.001* 15 min. vs. 1 hr. < 1.000 15 min. vs. 2 hr. < 0.040* 1 hr. vs. 2 hr. < 0.220 M. cassini vs. not disrupt < 1.000 Presence of seminal plug 15 min. vs. not disrupt < 0.001* 1 hr. vs. not disrupt < 0.001* 2 hr. vs. not disrupt < 0.001* £1 hr. vs. not disrupt < 0.001* £2 hr. vs. not disrupt < 0.001* 15 min. vs. 1 hr. < 1.000 15 min. vs. 2 hr. < 1.000 1 hr. vs. 2 hr. < 1.000 M. cassini vs. not disrupt < 1.000 * indicates statistically significant difference.
91 Table 4.3. Counts of female M. septendecim eggnests in mating interruption experiments. Conspecific mates are M. septendecim; heterospecific mates are M. cassini. Eggnests Additional after first eggnests after Total Mate mating Remated second mating eggnests Uninterrupted mating: Conspecific 29 no - 29 Conspecific 31 no - 31 Conspecific 14 no - 14 Conspecific 26 no - 26 Conspecific 34 no - 34 Conspecific 22 no - 22 Conspecific 23 no - 23 Conspecific 34 no - 34 Conspecific 1 no - 1 Average 23.8 ± 10.7 23.8 ± 10.7
Heterospecific 45 no - 45 Heterospecific 32 no - 32 Heterospecific 29 no - 29 Heterospecific 97 no - 97 Average 50.8 ± 31.6 50.8 ± 31.6
Mating interrupted at 2 hr: Conspecific 9 yes 0 9 Conspecific 23 yes 3 26 Conspecific 0 yes 7 7 Conspecific 10 yes 20 30 Conspecific 9 yes 20 29 Conspecific 0 yes 20 20 Conspecific 14 yes 12 26 Average 9.29 ± 8.0 11.7 ± 8.6 21.0 ± 9.5
Conspecific 5 no - 5 Conspecific 35 no - 35 Conspecific 41 no - 41 Average 27.0 ±19.3 27.0 ± 19.3
Conspecific 39 no possibility - 39 Conspecific 51 no possibility - 51 Conspecific 35 no possibility - 35 Conspecific 10 no possibility - 10 Conspecific 87 no possibility - 87 Conspecific 30 no possibility - 30 Conspecific 33 no possibility - 33 Average 40.7 ± 23.8 40.7 ± 23.8
Comparison vs. uninterrupted conspecific mating (Wilcoxon Signed Ranks): Pre-remating eggnests, conspecific mating interrupted at 2 hours with possibility of remating: P £ 0.018 Total eggnests, conspecific mating interrupted at 2 hours with subsequent remating: P £ 0.128 Total eggnests, conspecific mating interrupted at 2 hours without subsequent remating: P £ 0.593 Total eggnests, conspecific mating interrupted at 2 hours without possibility of remating: P £ 0.090 Total eggnests, heterospecific mating interrupted at 2 hours with possibility of remating: P £ 0.068
92 Table 4.4. Mated and unmated female M. septendecim responses to 2 minute playbacks of male calling song. Females that produced at least one wing flick in response to playbacks of male songs were scored as responding positively. Day Female n Response P (+) (-) (Fisher’s Exact 2- tailed) 1 Mated 22 0 22 Unmated 17 9 8 £ 0.001 2 Mated 22 0 22 Unmated 16 8 8 £ 0.001 3 Mated 20 0 20 Unmated 15 7 8 £ 0.001 4 Mated 18 0 18 Unmated 14 7 7 £ 0.001
93 Table 4.5. Mating interruption and female M. septendecim wing flick responses to playbacks of male calls. Mating-interrupted females were likely to signal in response to male calls, and all disruption treatments were equally likely to cause females to remain responsive. Number of Number of females responding females Treatment with wing flick signal not responding Disrupt at 15 min. 8 1 Disrupt at 1 hr. 8 3 Disrupt at 2 hr. 5 4 Not disrupted 0 17
Comparison P (Fisher’s Exact 2- tail)
15 min. vs. not disrupt £ 0.001* 1 hr. vs. not disrupt £ 0.001* 2 hr. vs. not disrupt £ 0.002* 15 min. vs. 1 hr. £ 0.591 15 min. vs. 2 hr. £ 0.294 1 hr. vs. 2 hr. £ 0.642
* indicates statistically significant difference.
94 Table 4.6. Census data from female attractiveness experiment. The cage containing unmated females had a significantly greater number of visiting males than did the empty cage (Wilcoxon Signed Ranks test, Z= -3.320, P£ 0.001), and significantly fewer males visited the cage containing mated females than the cage containing unmated females (Wilcoxon Signed Ranks test, Z= 3.331, P£ 0.001).
Males found on Males found on Males found on cage containing cage containing empty Time unmated females mated females cage Day 1 1:08 1 0 0 1:37 2 0 0 2:27 2 0 0 2:39 0 0 0 3:20 4 0 0 4:09 3 0 0 4:31 2 0 0 5:15 0 0 0 Day 2 9:00 5 0 1 9:31 5 0 0 10:01 6 2 1 10:30 4 0 0 10:50 5 0 0 11:07 2 0 0 11:17 0 0 0 11:30 2 0 0 11:42 2 0 1
95 with 0.068 0.090 0.128 0.018 ² ² ² ²
P P P P ) M. cassini Heterospecific ( Mate (control), mating-interrupted females allowed to Not Permitted To remate Total M. septendecim gned Ranks): nt remating: ossibility of remating: terrupted females not premitted to remate, and mated bility of remating: bility of remating: Conspecific mating Permitted to remate Pre-remating Control ). 0 70 60 50 40 30 20 10 M. cassini . Numbers of eggnests laid by normally-mated female Figure 4.1 remate (pre-remating eggnests and total eggnests), mating in heterospecifics ( Comparison vs. uninterrupted conspecific mating (Wilcoxon Si Pre-remating eggnests, conspecific mating interrupted with p Total eggnests, conspecific mating interrupted with subseque Total eggnests, conspecific mating interrupted without possi Total eggnests, heterospecific mating interrupted with possi
96 CHAPTER V
SEXUAL SIGNALING IN PERIODICAL CICADAS, MAGICICADA SPP.
Abstract
We describe the nature and timing of a previously unknown female sexual receptivity signal in Magicicada. Females produce “wing-flick” signals in response to male calls with a timing that is species-specific. Males perceive both acoustical and visual components of wing-flick signals. Chorusing males perceiving female signals alter their behavior by increasing the number of calls between flights and localizing their mate search. A male receiving repeated nearby responses to his calls approaches the signaling female while calling and begins late-stage courtship behaviors leading to copulation. Using the wing-flick signal as an assay of female receptivity, we demonstrate the functional significance of the complex, two-part structure of male calls in M. septendecim, M. tredecim, and M. neotredecim. These species’ calls include a main element and a downslur; calls without downslurs are unlikely to provoke female responses against a background chorus, but such call fragments may elicit responses in the absence of a background chorus. Normally, only receptive females produce wing-flick signals; however, a fungal pathogen evidently increases its odds of transmission by causing infected males to signal. We also report a novel form of male-male acoustic competition:
97 Males engaged in close-range or protracted courtships obscure the downslurs of arriving potential interlopers with “interference buzzes,” reducing the likelihood of a female response and thus increasing the likelihood that interlopers will depart and search elsewhere.
Introduction
Detailed studies of insect communication have provided valuable insights into topics as diverse as insect systematics (e.g. Alexander 1957a; Walker 1962, 1963), speciation (e.g. Henry 1985, Otte 1992, Butlin and Ritchie 1994, Alexander et al. 1997), development and inheritance (e.g. Fulton 1933, Bigelow 1960, Olvido and Mousseau 1995, Shaw 1996), conflicts of interest and deception (e.g. Lloyd 1966, Lloyd 1979, Spooner 1968, Otte 1974; Alexander et al. 1997), and sexual selection (e.g. Alexander 1975, Thornhill and Alcock 1983, Ritchie 1996). The “singing insects” (sound-producing members of the Orthoptera and Cicadidae) have played a central role in this research. Many key contributions have resulted from long-term synthetic studies of individual species or species groups (e.g. Alexander and Moore 1962, Walker 1964, Lloyd 1966, Spooner 1968, Lloyd 1979, Otte and Alexander 1983, Otte 1994). One such group, the periodical cicadas of eastern North America (Magicicada spp.1, Table 5.1), has attracted interest because of its extraordinary combination of unique traits, including long life cycles (13 or 17 years), synchronized development, periodical adult emergences, near-perfect sympatry and synchrony of multiple related species, dense populations and loud, conspicuous male sexual behaviors. Periodical cicada aggregations served as the primary model for the concept of the non-resource-based lek (Alexander 1975), and they offer an important opportunity to compare and contrast mating aggregations in insects and vertebrates
1 Because the songs and behaviors of the 13- and 17- year cognate species are generally similar, they are abbreviated herewith as M. –decim, M. –cassini, and M. –decula.
98 (Bradbury 1981, 1985). Although their remarkable characteristics have made Magicicada a focus of research on ecology, life cycle evolution, and behavior (Williams and Simon 1995), central questions about Magicicada mating behavior and communication remain unanswered.
Calling male periodical cicadas aggregate to form choruses that attract sexually receptive females (Alexander and Moore 1958, 1962). Teneral (adult immature) and ovipositing females are also found in these aggregations, although they may not be specifically attracted to them. Magicicada chorusing consists of “sing-fly” behavior, or short (ca. 3-15 s) bouts of singing alternated with local flights of variable length (up to several meters; Alexander and Moore 1962, Alexander 1975, Dunning et al. 1979). Under some conditions males appear to engage in local mate search behavior involving extremely short flights or walking between calls. Upon locating a potentially receptive female, a chorusing male may begin courtship, which involves a stereotypical series of behaviors and distinctive acoustical and visual signals (Alexander and Moore 1962; Alexander 1968, 1975; Dunning et al. 1979; Table 5.2, Fig. 5.1). Courtship may be followed quickly by copulation or may last hours and include long periods of waiting (Alexander 1968). These observations have led to the suggestion that periodical cicada females may be extremely choosy, imposing strong sexual selection on males (Alexander 1975) and that males locate females by progressively narrowing their search as they shift from chorusing to local mate search to courtship. The cues causing males to identify a suitable courtship target and switch from chorusing (not directed at a specific individual) to courtship behavior (directed at an individual female), or from extended courtship to copulation, have until now remained unknown (Alexander 1968). The function of male calling song has also remained poorly understood; Alexander (1975) noted that, because females are attracted to choruses of many individuals yet no males within choruses employ silent searching tactics, the calling song must play a role in individual males’ mating success.
99 We describe a visual and acoustical female receptivity signal, hereafter referred to as a “wing-flick.” Receptive Magicicada females flick their wings with a quick motion in timed response to male calls; males perceiving the signal respond by immediately dropping out of the chorus and engaging in courtship behaviors directed at the signaling female. The discovery of this signal has facilitated the development and testing of new hypotheses for the adaptive nature of Magicicada chorusing behavior and the evolution of male calling and courtship songs. The discovery also provides new information about the transmission of a specialized fungal parasite of Magicicada, Massospora cicadina Peck. Although normal males have never been observed making wing-flick signals, males infected with the fungus sometimes produce wing-flick signals in response to the calls of other males; this behavioral modification is presumably a fungal adaptation to increase spore transmission (see also Alexander et al. ms). In addition, we report acoustical jamming signals produced by Magicicada males in close-range competition for receptive females, a form of intrasexual mating competition not previously reported in insects.
General materials and methods
Periodical cicadas in different regions emerge in different years; the populations of different regions are called “broods.” We first observed female Magicicada signaling to males in Brood I in 1995. We continued our studies of sexual signaling in 1996-1998 (Table 5.3) on both 13-and 17-year cicadas, concentrating on the M. -decim species but including the others when sufficient numbers were available. We used a Macintosh computer and Canary software (Cornell Bioacoustics Laboratory) for acoustical analyses, and Sound Edit software (MacroMedia) for modification of songs and synthesis of artificial sounds. Playback equipment consisted of a Sony WM-D6C cassette player or a Macintosh powerbook computer connected to a Radio Shack SA-10 amplifier driving a 3” midrange speaker for M. -decim calls or a tweeter for M. -cassini calls. We maintained playback call intensity at natural levels, ca. 75 db at 20 cm. In all years, we kept cicadas in captivity, but
100 within their natural environment, by placing them in ca. 200 liter cages made by enclosing living vegetation with black fiberglass screen or white nylon tulle. Some observations were completed in larger 4x4x4 m “flight cages” placed over living woody vegetation. Many playback experiments were conducted using 22x24x22 cm screen test chambers.
Magicicada nymphs usually emerge and undergo their final molt in the evening (Maier 1982). The next morning, newly emerged “teneral” adults are identifiable by their dull color, soft bodies (especially diagnostic in the ovipositor), and by their position low in the vegetation. Although mated females commonly have a hardened white seminal plug in the genital opening (White 1973), this plug is occasionally absent in mated females and is therefore an unreliable indicator. To ensure that we used only unmated females in our playback experiments, we collected teneral females early each morning and stored them in single-sex cages.
Female responses to male calls
The nature of the female signal and the “rapprochement duet”
In 1995-1998, we observed interactions between male and female Magicicada. We noted whether females produced wing-flick signals and how the responses were timed in relation to male calls, using sonograms from audio-and videotape to measure timing. We also recorded female M. –decim and M. -cassini responses to playbacks of male calling song and how males responded to natural and simulated wing-flick signals.
Results. We have documented female wing-flick signals in all Magicicada species except M. tredecula, which was not present in sufficient numbers to study in 1998. In all species studied, a female signals in response to a calling conspecific male by moving her wings in a single, quick motion which produces a broad-frequency sound of approximately < 0.02 s duration. The motion and sound are similar to wing flutters produced in response to disturbance, except that wing flutters consist of multiple wing movements. The signal’s
101 visual and acoustical components appear unspecialized but the timing of the signal in relation to the male’s call is species-specific; the signal therefore consists of both the wing- flick and the timing of its delivery. In M. septendecim, females produce the signal an average of 0.387 ± 0.106 s after the end of the male calling phrase (Fig. 5.2). The delay in M. cassini, 0.705 ± 0.112 s, is nearly twice as long (Fig. 5.3). These species’ 13-year counterparts appear to have similar or identical signal timing. M. septendecula females produce multiple wing-flicks in the brief silences between subphrases during the second part of a male’s two-part call (Fig. 5.4).
In all species, a receptive female produces wing-flick signals in response to each of the male’s calls as he approaches, engaging in a “rapprochement duet” with the calling male (Fig. 5.1). This duet continues until the male switches to CII courtship calling. We have not determined what stimuli cause the male to switch from the duet to CII calling; he usually does so once within approximately 1-15 cm of the female, perhaps upon making close visual contact. In M. -decim the female does not respond during the male’s CII song, which is a continuous stream of shortened calling phrases without silent gaps; if the male stops calling, the female responds with a wing-flick after the appropriate delay. M. -decim males continue CII calling until reaching the female; once positioned next to the female, the male switches to CIII courtship calling and attempts to copulate. The rapprochement duet in M. -cassini is similar to that in M. -decim, except that females sometimes signal during male CII calling. In M. septendecula there may not be a homologous CII courtship song; after the rapprochement duet the male may proceed directly to CIII courtship and mounting. Females do not signal during CIII in any Magicicada species studied. The context of the signal, and male reactions to it, suggest its purpose: Sexually receptive females signal to obtain mates.
102 The relationship of the female response to sexual receptivity in M. septendecim
To demonstrate that immature females are not sexually receptive and do not signal, in 1998, we six daily collections of approximately 25 teneral females each. Each day, we played recordings of male song to each of the day-cohorts and watched for wing-flick signals. To demonstrate that mated females do not wing-flick, in the 1997 study, we allowed 22 individually-marked (Chapter 2) female M. septendecim to mate once and divided them between two cages along with 22 marked, unmated females of the same age. In each of four consecutive days we played recorded M. septendecim calling songs to the females for two minutes and recorded the number of mated and unmated females responding.
Results. Females first signaled 5-8 days after emerging (Table 5.4). This period of nonresponsiveness is consistent with Magicicada “teneral periods” reported elsewhere (Maier 1982; see also Karban 1981, Young and Josephson 1983) and with similar periods in nine species of phaneropterine katydids (Spooner 1968). Variable weather during maturation probably causes the inexactness of the relationship between age and maturity. Since sclerotization involves chemical processes such as protein cross-linking, it is affected less by time than by temperature and humidity (Chapman 1971 pp. 443-446). In the experiment comparing mated and unmated females, none of the mated females responded to playbacks, while at least half of the unmated females responded each day with wing-flick signals (Table 5.5). This difference was significant in each of the four days (P £ 0.001; Fisher’s Exact Two-Tailed Test).
Response of M. septendecim females to playbacks of heterospecific song
Magicicada aggregations contain as many as four different species (Alexander and Moore 1962; Chapter 6), yet interspecific hybrids are rarely observed (Alexander and
103 Moore 1962; Dybas and Lloyd 1962, White 1973). To demonstrate the species-specific nature of the wing-flick response, in 1996 we played recorded M. septendecim calling phrases alternating with M. cassini calling phrases to 25 caged, unmated M. septendecim females and noted their responses. We also included three unmated M. cassini females as controls; we could not collect enough to include more. We tested the females in groups of five, playing a series of 15 alternating M. septendecim and M. cassini calls (30 calls total) and recording female responses.
Results. M. septendecim females routinely responded to the M. septendecim call phrases in each trial. Only one of 25 M. septendecim females ever responded to a heterospecific playback (Table 5.6). The three M. cassini females in the experiment responded to conspecific calls and never responded to heterospecific playbacks.
Male responses to female signals
Effects of the signal on male behavior in M. septendecim
In 1996 and 1997, to document the effects of the female signal on male behavior, we produced sounds and movements imitating female wing-flick signals for chorusing male M. septendecim. Before we identified a suitable device for producing simulated wing-flicks, we used a strip of paper that we flicked with our fingers. Later, we discovered that toggling an ordinary household electric light switch was more convenient for the experimenter. Because male responses to both artificial stimuli appear indistinguishable, we combined trials with both methods in our statistical analyses.
A single M. -decim call phrase consists of a 1.5-4.0 s buzz (the “main element”) of constant and nearly pure pitch followed by a brief (ca. 0.35 s) “terminal downslur” ending about 500 Hz lower than the main element pitch. To document the importance of correct timing in eliciting male M. septendecim courtship, we produced simulated wing-flicks in response to the calls of males that had landed and begun calling on nearby vegetation,
104 timing our signals either (1) during the main element, (2) during the downslur, or (3) after the downslur. We placed the flicking device within 25 cm of the male along the branch on which he had just landed. We included control trials in which the experimenter approached in the same fashion but did not produce simulated wing-flicks. We scored a male as responding positively if he moved toward the stimulus and began late-stage courtship behaviors such as CII or CIII calling, foreleg-vibrate, or mounting behavior. Each trial ended when the male flew or walked away from the stimulus, or when the male remained motionless longer than 20 seconds.
The above experiment simulated a scenario in which the male alights near a signaling female. To examine male responses in a scenario involving weaker and/or inconsistent female responses, we conducted trials in which we presented individual chorusing males with either a single nearby or repeated distant simulated wing-flicks. We produced single, nearby simulated wing-flicks in response to the first call following a flight at a distance of approximately 25 cm from the male. For multiple distant simulated wing- flicks, we produced the signals after each of the male’s calls from a distance of 1.3 m. We again included control trials in which the experimenter approached in the same manner but did not produce simulated wing-flick signals. In each trial we recorded the number of calls the male made in his current bout, the nature of his next action (sit, walk, fly), and the direction and distance of movement. We stopped monitoring males that paused for longer than 20 seconds.
Results. Only wing-flicks produced after the downslur caused males to respond positively. Males usually responded to such stimuli by walking toward the stimulus while calling. In this behavior, termed “call-walking,” males stopped walking for ca. 1 second immediately following each downslur. This pattern is distinct from chorusing behavior which involves bouts of calling alternated with flights or silent walks. Males were equally
105 unresponsive to the control treatment and to simulated wing-flicks produced during the main element or during the downslur (Table 5.7).
Males responded to single nearby and multiple distant simulated wing-flick signals in a manner suggesting an attempt to localize the stimulus (Table 5.8). Both kinds of stimuli caused males to increase the number of calls in the current calling bout compared to control males; most males then flew to a new calling perch instead of call-walking toward the stimulus. Whether walking or flying after the calling bout, males presented with the single nearby or multiple distant simulated wing-flicks were more likely to move in the direction of the stimulus than control males were. In control trials, males were more likely to move away from the stimulus than toward it, suggesting that the presence of the experimenter probably disturbs the cicada.
Components of the female signal
Because female wing-flick signals are often audible, we assumed early in our investigation that males locate receptive females primarily by listening for wing-flick signals. However, the importance of investigating separately the visual and acoustical components of the female signal became apparent when we substituted an audiotaped recording of a female wing-flick for the mechanically produced simulated flicks of our earlier experiments. Male M. -decim did not give consistent or strong responses to these playbacks, suggesting that they must perceive the visual component of the female signal (actual signals and signals simulated with paper strips and electrical switches involve a synchronized sound and quick movement, while the speaker produces only a sound). Spectral analysis of wing-flick sounds indicates that they are broad-frequency sounds with most of the energy above 4 kHz. It is possible that the different Magicicada species perceive different aspects of female signals; although such high-frequency sounds are within the range of maximal hearing sensitivity of M. -cassini, female wing-flicks may be above the range of maximal hearing sensitivity for male M. –decim (Simmons et al. 1971;
106 Huber et al. 1980). We conducted experiments addressing the effects on male M. –cassini and M. –decim of visual signals, acoustical signals, and signals containing both acoustical and visual information on male chorusing behavior and male courtship behavior.
To examine the effects of timed flick sounds alone on male chorusing behavior, we constructed a clicking device by attaching a 12 volt relay to the end of a 1-meter wooden pole, covering all wires and dark (cicada-colored) parts of the relay with white masking tape. In test trials, we identified an actively chorusing male M. septendecim or M. cassini, placed the relay within 15 cm of him along the same branch, and clicked the relay in time to his calls, following him as he moved and taking care not to make any timed movements observable by the male. For controls, we placed the device in the same manner, but did not click the relay. For each male, we measured the number of calls in each of two calling bouts and the distance moved between the two calling bouts.
To investigate the effects of timed visual signals alone and the significance of other visual cues such as the presence of a cicada-colored object, we approached chorusing male M. septendecim with a model consisting of a black ballpoint pen cap or another model made from an identical cap painted white. Without making sounds, we moved the model once rapidly back and forth about 2 cm at the appropriate time for a female signal, or we held it still. The black pen cap was similar in size and color to a real cicada, while the white cap lacked only the appropriate color stimulus.
To determine whether combined visual and acoustical stimuli are more potent than either alone, we used the following experimental design: From the surrounding chorus, we selected 48 male M. septendecim and 48 male M. cassini one at a time and placed each in a 22x24x22 cm test chamber. Two walls of the chamber consisted of three layers of dark, opaque cloth; the remaining walls consisted of fiberglass window screen. In one treatment, we suspended a motionless model cicada inside the test chamber and responded to the male’s calls with clicks produced behind the opaque chamber sides. In the second
107 treatment, the experimenter held the model inside the chamber and responded to the calling male by moving the model slightly with the appropriate timing, without making clicks. In the third treatment, the experimenter responded by producing clicks and by moving the model. After the start of each trial, we recorded the male’s behaviors for the next two minutes. Male M. septendecim and M. cassini were scored as responding positively to the model if they exhibited any of the following behaviors during the 5 minute trial: CII or CIII call, extrude genitalia, climb onto the model, foreleg vibrate, or attempt to copulate.
Results. Timed click sounds did not affect male M. septendecim behavior, but male M. cassini given click sounds flew significantly shorter distances between calls (Table 5.9). Male M. septendecim courted plastic pen caps that moved in time with their calls, but were less likely to engage in late-stage courtship with white colored caps than with black colored caps (Fig. 5.5), indicating that visual stimuli alone are sufficient to provoke male responses and that the stimulus includes the presence of an appropriately colored cicada- like object. The model that moved and clicked simultaneously was most attractive to M. septendecim and M. cassini (Fig. 5.6). These results suggest that even though sounds or movements may provoke courtship under certain circumstances, in both M. septendecim and M. cassini, synchronized sounds and movements are most likely to be perceived as female signals.
Behavioral bisexuality of males infected by the fungal pathogen Massospora cicadina
Magicicada are sometimes infected by the fungus Massospora cicadina Peck (Speare 1921, Soper 1974, Soper et al. 1976, Lloyd et al. 1982, White and Lloyd 1983). Stage I (conidial phase) of this fungus is asexual; individual Magicicada so infected lose their abdominal terminalia, exposing a chalky mass of conidiospores. Infected cicadas tend to walk around on vegetation, spreading the spores by rubbing the exposed mass on surfaces (Alexander et al. ms.). Individuals contacting these infective conidiospores in the
108 same emergence develop a Stage II (resting spore) infection. These individuals also lose their abdominal terminalia, but they tend to make long flights, during which the friable resting spores spill from the abdomen (Alexander et al. ms.). These resting spores are believed to infect cicada nymphs of the next generation (Soper 1974, Soper et al. 1976).
In 1996, while observing free-flying Magicicada, we observed a Stage I Massospora-infected male M. septendecim respond to a courting male twice with wing- flick signals (Fig. 5.7). The courting male responded as expected by attempting to copulate with the infected, signaling male, who rebuffed the courter by flapping his wings. These observations were striking because, in hundreds of hours spent to that point observing Magicicada in natural and controlled circumstances, we had never observed normal, uninfected males making wing-flick signals in response to other males’ calls. The observation of signaling by infected males led us to use playback experiments to compare the infected males’ responsiveness to that of unmated adult females.
We placed M. septendecim four at a time in a cage and played 15 calling song phrases to each group of cicadas. Trials began with a playback of male calling song at normal pitch (1.39 kHz); then we first increased, then decreased the pitch in steps, and trials ended with a playback of the calling song at normal pitch. We scored cicadas as giving a positive response to a calling phrase if they responded with wing-flick signals to a minimum of two phrases. We played normal and frequency-altered M. septendecim calling songs to 8 wild-caught uninfected males, 6 Stage I Massospora-infected males, and 32 unmated females. We later tested the responsiveness of 5 Stage II Massospora-infected male M. septendecim to playbacks of unaltered M. septendecim calling song.
Results. Stage I fungus-infected M. septendecim responded to playbacks with appropriately timed wing-flicks, while no normal male or Stage II infected male ever responded. Stage I infected males were generally less responsive than mature females (Table 5.10). We have also observed Stage I infected M. tredecassini responding with
109 wing-flick signals to other males’ calls (Fig. 5.8). We expect that fungus-induced bisexuality will be found in all Magicicada species.
Components of male calls
Alexander (1975) noted that “failure to find evidence of nonsinging males searching through cicada choruses implies that the male’s song remains an essential part of his ability to acquire females.” The discovery of the female sexual signal and its timing in relation to the male’s call confirms this prediction and suggests a more detailed functional hypothesis regarding the structural elements of M. –decim and M. -cassini calls, which we have developed and tested primarily in M. septendecim.
The call phrases of M. -decim males are 1.5-4.0 seconds in length, ending with a frequency downslur (Fig. 5.2). The nearly pure-tone main elements of M. -decim calling songs overlap to produce a uniform chorus drone; as male density and activity increases, it becomes difficult to perceive individual calls of individual males. In such situations the downslur is the only portion of the male’s call that stands out from the background chorus. Because females must be able to respond with a signal timed from the end of a male’s call, we suspected that the downslur might function to enhance the end of the male’s call and improve a calling male’s odds of being perceived by a nearby receptive female. Because males have never been observed producing downslurs only, producing a “main element” must also improve a male’s likelihood of provoking a female response. This hypothesis leads to several predictions. First, if the main element alone is ever sufficient to elicit a signal, its effectiveness should decrease as the background chorus increases in intensity. Second, downslurs alone should not be sufficient to elicit wing-flick signals, unless the background chorus is loud enough to sufficiently stimulate the female. Finally, neither the downslur nor the main element alone should be as effective as the intact call in eliciting the female response at any background level.
110 To determine which components of male M. septendecim calls most effectively elicit a female response under different chorus conditions, we constructed a pure-tone artificial calling phrase, a pure-tone phrase lacking a downslur, a pure-tone downslur alone, and a simulated pure-tone background chorus. In previous experiments, we found that females respond similarly to playbacks of recorded and pure-tone artificial calls. We noted the responses of individually marked females in 19 groups of females during 146 playbacks of complete calls, calls lacking slurs, and slurs only at an intensity of 72-79 db (intensity varied within the test chamber) over a background chorus of four different intensities (0 db, 58-62 db, 63-77 db, 65-80 db).
Results. Although call fragments did elicit female responses under certain conditions, females were more likely to respond to whole calls than to partial calls at all background chorus intensities (Table 5.11). As the background chorus intensity increased, female responsiveness to whole calls and main elements of whole calls declined, while females became more responsive to slurs (Table 5.12; Fig. 5.9), such that at the highest intensity, females were more responsive to slurs alone than to main elements alone.
Inter-male acoustical interference behavior
In 1996-1998 we observed a previously undescribed male sound in M. -decim and M. -cassini species; this sound is composed of a short (ca. 0.25 s) “buzz” with a frequency spectrum similar to that of the main element of the calling song but somewhat less pure- toned. This sound is always produced during the downslur of another male’s call (Fig. 5.10). In part because the downslur is important in eliciting female wing-flick signals, we suspected that a male engaged in close-range courtship uses this sound to obscure a newly- arrived competitor’s downslurs, thereby decreasing the likelihood that the courted female will reveal her presence with a wing-flick signal before the competitor completes his short calling bout and departs.
111 Alternatively, the sounds could be aborted calls caused by interruption or when the stimuli inducing the call are insufficient to stimulate a complete call phrase. This hypothesis does not predict that the buzzes should be consistent in length or timing in relation to another male’s call, nor does it predict that the buzz should be produced under limited circumstances. Another explanation for this behavior is that males might use this sound to signal their sex to other males; males often court other males, so the sound may discourage misdirected sexual attention. A similar explanation has been proposed for a “flick-tick” signal produced by M. cassini males (Dunning et al. 1979). This hypothesis predicts that the signal should be observed most often when males are crowded and encounter one another commonly in the chorus.
We conducted three experiments to evaluate these hypotheses in M. septendecim. In the first, designed to determine if crowded chorus conditions alone can induce buzzes, we placed 30 chorusing males together in a 1x1x1 m cage, watched them interact, and listened for the male buzzes. After 20 minutes of watching, we produced simulated wing- flick signals in response to males on one side of the cage for one minute and then observed the males for another five minutes. We repeated this experiment twice.
In the second experiment, intended to simulate the events involved in the appearance of a potential interloper during courtship, we presented 22 individual males with the following series of stimuli: First, we confined each male in a small screen test chamber. We then played one to five minutes of recorded calling song at an intensity designed to simulate a male calling at a distance of approximately 10 cm. Males often began to call during this treatment, but if the playbacks were not sufficient to stimulate calling, we produced simulated wing-flick signals in response to the playbacks using an electrical switch held outside the chamber within view of the male; signals from the switch always induced call-walking from the male. Once the male had begun calling, we stopped the playbacks and responded to the male’s calls for 10-60 seconds, after which we ceased
112 responding. Once the male stopped his calling or courtship songs (some males had begun CII or CIII calling during this duet), we resumed playbacks of calling song from the speaker at the same volume and distance. We noted the context of any interference buzzes produced by the male during the trial.
In the third test, we examined the effects of artificial pure-tone buzzes on female responsiveness by creating a model call and buzz such that the call could be played with or without the buzz. We confined four unmated female M. septendecim in the test chamber and played a sequence of 30 call pairs; each pair consisted of (1) a normal call played from one speaker followed by (2) a call played from the same speaker with a buzz played from a second speaker and superimposed over the slur. We recorded the number of females responding to each call and repeated the experiment 6 times, using different females. We compared the number of females responding positively to each call pair using a Friedman Two Way analysis of variance.
Results. In trial 1 of the first test, no males produced buzzes prior to the production of simulated wing-flick signals, but at least 2 buzzes were heard once we began to respond to the males with simulated wing-flick signals. In trial 2, one buzz was heard during the first 20 minutes, but over 20 were heard once we began responding to the males. Males called often and frequently landed within centimeters of each other during the trials, and although some males harassed or attempted to court other males, these interactions did not lead to male buzzes.
In the second test, males never produced buzzes in response to the initial series of playbacks, never produced buzzes during artificial duets between the playbacks and the experimenter, and never produced buzzes while duetting with the experimenter. However, in 16 of the 22 trials, males began producing buzzes once the playbacks had resumed following the termination of the male’s duet, usually in response to the first or second playback call, but sometimes not until 3 or 4 calls had played. Males producing buzzes did
113 so only during the downslur of the recorded calling song phrases. In five of the trials, the experimenter again began producing simulated wing-flick signals to the playback while the male buzzed; in four of these five cases this caused the male to cease buzzing and begin call-walking near the simulated wing-flick stimulus. In the fifth case the male walked while buzzing after each playback call.
In the third test, the presence of artificial buzzes significantly decreased the likelihood that a female would respond with wing-flick signals to an artificial calling song (Table 5.13). Under the conditions of these experiments, buzzes halved the likelihood of a female response.
Discussion
Wing-flick signals in Cicadidae
Communicative wing-flicking (sometimes called wing-tapping, -banging, - clapping, -clacking, or -clicking) appears to be widespread in cicadas. We use the term “wing-flick” for Magicicada because it connotes movement and sound, both of which are perceived by males; other terms emphasize only the acoustic component of the signal. Male wing-flicking during close-range courtship interactions with females has been reported in Australian and New Zealand Kikihia, and Amphipsalta, (Dugdale and Fleming 1969, Lane 1995) North American Okanagana (Davis 1919, Alexander 1957b, Chapter 8), and European Tibicina (Fonseca 1991), while males combine wing-flicks with long-range calling song in Asian Cicadetta (Popov 1981), Australian and New Zealand Amphipsalta (Dugdale and Fleming 1969, Lane 1995), and Western North American Platypediinae (Moore 1968). Female wing flick signaling is known in North American Magicicada and Okanagana, (Davis 1919, Chapter 8), Australian Cystosoma (Doolan 1981), Cicadetta (Gwynne 1987), and Amphipsalta (Dugdale and Fleming 1969), and New Zealand Amphipsalta, Kikihia, Maoricicada, Notopsalta, and Rhodopsalta (Lane 1995, Dugdale
114 and Fleming 1969), with the most detailed published reports of female wing-flick signaling from Kikihia spp. (Lane 1995), Amphipsalta cingulata (Lane 1995), Cystosoma saundersii (Doolan 1981), and Cicadetta quadricinctata (Gwynne 1987). In each species studied in detail, female wing-flick signals elicit male courtship behavior and appear to have a specific temporal relationship to the male’s song. In a few cases female wing-flick signals, while present, are apparently not always prerequisites for mating: Although sometimes both sexes of Okanagana canadensis and O. rimosa appear to use wing-flicks to signal their presence, females most often signal receptivity simply by approaching stationary calling males (Chapter 8).
Wing-flick signals and the Magicicada courtship sequence
In Magicicada, female wing-flick signals in response to male calling song cause males to cease chorusing and begin courtship. A female Magicicada wing-flick signal consists of a quick, timed movement of the wings that makes a broad-frequency sound varying from a faint rustle to a loud snap; the signal’s timing in relation to the male’s call is species-specific. A chorusing male perceiving a single wing-flick signal or repeated, distant wing-flick signals increases his number of calls in the current bout and then resumes chorusing behavior while moving in the direction of the stimulus, apparently to elicit further responses and obtain better information about the female’s location. If the male receives repeated nearby responses that allow him to localize the female, he ceases chorusing behavior (calls alternated with flights) and call-walks toward the responding female; the male may use short flights to cross gaps in the vegetation, but such short, irregular flights are distinct from chorusing flights. In M. -decim and M. -cassini species, the male may begin CII calling once within approximately 15 cm. of the female. Female M. -decim do not wing-flick during the CII call, which contains no silent gaps; female M. - cassini occasionally respond during males’ CII calling. After contacting the female or while preparing to mount, males of all species begin CIII calling, which they continue until
115 the genitalia are engaged. Receptive females of all species do not wing-flick during CIII and remain still throughout the entire courtship sequence. In nearly every mating that we have observed, the cicadas have performed, in order, each of the behaviors of this courtship sequence; exceptions include rare forced copulations that occur when females are trapped by vegetation or in aggregations of males.
Males respond to both the acoustical and visual components of the signal. We do not know to what extent male Magicicada can localize sound alone without a visual component, but the wing-flick sounds alone do not elicit later courtship stages as rapidly or consistently as do visual cues. The acoustical and visual stimuli sufficient to cause male responses in all Magicicada species must be quite general, because the click and movement of an ordinary electric light switch are highly attractive to courting males. Since male cicadas directed the full repertoire of normal sexual behaviors towards active, signaling models and ignored these same models when no signals were perceptible, male courtship songs and the female wing-flick signals appear to be sufficient for sexual rapprochement in these species.
Male-male acoustic interference competition
The discovery of the rapprochement duet in Magicicada improves our understanding of male-male competitive interactions in choruses. Most significant is the finding that male M. -decim and -cassini appear to use “interference buzzes” to acoustically jam rival males’ signals in competition for mates. Interference buzzes are produced specifically in the context of a close-range male-female duet or a prolonged courtship that has been interrupted by the arrival of a calling, and potentially interloping, male competitor. The courting male emits short buzzes coincident with the downslurs of his rival’s calls; these buzzes obscure the downslur of the rival’s call and reduce the likelihood that the female will perceive and respond to them. If the rival male does not elicit responses from a female, he is likely to continue chorusing and depart. One potential objection to this
116 hypothesis is that the buzz itself could reveal the presence of the nearby receptive female. For the buzz to function to reduce interloping, calling males either must be unable to discern the buzz from the background chorus or they must be unable to hear sufficiently while calling; otherwise, males should have evolved to respond to interference buzzes by intensifying their local search. Male M. septendecim and M. cassini hearing sensitivity is reduced 5-15 dB during calling by the action of muscles that reduce tension on the tympana (Pringle 1954, Simmons et al. 1971); thus calling males may have difficulty perceiving that their signals are being jammed by buzzing males. Also, buzzing males terminate the signal precisely at the end of the interloper’s song, suggesting that buzzing behavior has been selected to be produced only when it is undetectable by the potential interloper.
Other insects are known to make use of specialized male-male competitive signals. For example, some male katydids produce sounds that appear to mimic female responses to their own calling songs, apparently to confuse potential interlopers (Alexander 1975, Grove 1959). A similar function could be served by male wing-clicking during calling in cicadas such as Amphipsalta cingulata, in which males wing-click to their own calling songs with a timing identical to that of female wing-flick responses (Lane 1995). The Magicicada interference buzz appears uniquely complex, however, in that the signal apparently deceives the courted female in order to achieve a competitive advantage over a rival male.
The intense nature of Magicicada male mating competition likely results in few females remaining in the mating pool long after the onset of sexual receptivity; the wing- flick signal is so potent, and searching males so abundant, that newly-receptive females may be detected almost immediately by one of the many chorusing males, in which case wing-flick rapprochement duets may be brief. In addition, unless a teneral female’s readiness to mate is activated all at once, fully formed, a newly adult female may be expected to pass through a period of partial receptivity in which (1) she produces the wing-
117 flick signal very weakly and/or inconsistently and (2) she is more likely to reject males as a result of fluctuations in her mating readiness. If so, males likely have evolved to respond to very weak or intermittent signs of timed wing-flicking in females. Such a period of partial or weak sexual receptivity could be involved in observations of lengthy courtships involving repeated rejections before eventual copulation (Alexander 1968, Dunning et al. 1979) and the apparent coyness of Magicicada females. Subtlety of wing-flick responses in newly-matured females, in addition to rapid detection of signaling females by chorusing males, could explain why the Magicicada wing-flick signal has for so long remained undiscovered.
Acoustic synchrony in M. –cassini .
The ability of M. –cassini males to synchronize their calls may be an adaptation allowing individual males to maximize their probability of perceiving female responses. All available evidence (Simmons et al. 1971; Huber et al. 1980; this study) suggests that male M. –cassini can detect the acoustical component of female wing flick signals. Unlike M. –decim calls, M. –cassini calls are broad frequency, and overlap the frequency range of female wing flick signals (Fig. 5.3). We propose that male M. -cassini have evolved to delay beginning calling phrases until a nearby conspecific begins, so that each male maximizes his chances of hearing nearby female wing flicks without acoustical interference. The net effect of an entire chorus pursuing this strategy is for males to synchronize calling and flying; thus, acoustic detection of female signals may be an explanation for the well-documented phenomenon of synchronized chorusing behavior in M. –cassini (Alexander and Moore 1958).
Wing-flick signals and the parasitic fungus Massospora cicadina
Alexander et al. (in prep.) show that the fungus Massospora cicadina alters Magicicada behavior in ways that likely increase spore transmission, and that the alterations
118 in behavior are different for the two fungal life history stages in ways that are appropriate for the stage-specific modes of transmission. Our observations suggest that the first stage of the fungus has apparently evolved to increase the odds of spore transmission between males by causing infected males to produce wing-flick signals in response to the calls of other males even while retaining normal male courtship behaviors. Thus, while females are in jeopardy from sexual contact primarily with infected males, males are in jeopardy from sexual contact with infected individuals of both sexes. Second stage Massospora-infected males do not produce wing-flick signals, consistent with the theory that spores of this stage do not infect cicadas of the same generation (Soper 1974, Soper et al. 1976). We do not know the physiological mechanism or timing of infection by Stage I fungal spores, but the behavioral bisexuality of infected males strongly supports the argument that spore transmission between individuals of the same generation is crucial for the Stage I fungus.
The evolution of Magicicada chorusing behavior
Alexander (1975) noted that, in Magicicada, it is apparently the sound of the entire chorus that attracts females, rather than the song of an individual male. Therefore, an individual male Magicicada with chorusing behavior that is less effective in long-range attraction but more likely to be detected by stationary receptive females would realize a fitness advantage relative to those males with chorusing behaviors optimal for long-range attraction. For Magicicada, the discovery that receptive females respond to the calls of individual males with timed wing-flicks resolves the question, raised by Alexander (1975), of why males do not silently parasitize the mate-attracting abilities of others by searching without calling: Because male calls provoke female responses, males who search without signaling sacrifice the opportunity to detect females by provoking responses.
Magicicada chorusing behavior lies at one extreme of the range of rapprochement behavior found in cicadas. Males alternate unusually brief bouts of calling (and unusually short call phrases, especially in M. -decim and M. -cassini) with short flights, a
119 rapprochement strategy that is centered around searching for responding females who have moved to the chorus and become stationary. Only one other well-studied cicada species, the Australian Tick-tock cicada (Cicadetta quadricinctata) has a similar rapprochement system, although calling bouts in this species are still three to four times longer in duration than those of Magicicada (Gwynne 1987, Alexander and Moore 1962). Gwynne (1987) noted the similarities of male behavior in Magicicada and Cicadetta quadricincta and all but predicted that female wing-flick signals would be found in Magicicada. At the other extreme are species in which males produce a continuous song (with or without separate phrases) from a single location for relatively long periods, such as in North American Okanagana canadensis and O. rimosa (Chapter 8). This strategy emphasizes stationary male advertisement over searching, and in such species females approach males for mating. A similar range of rapprochement strategies is found in other singing insects such as Phaneropterine katydids (Spooner 1968, Heller and von Helversen 1993). The Magicicada rapprochement system is similar to those of fireflies (see Lloyd 1966, 1979) and substrate- vibrating leafhoppers (Hunt and Nault 1991, Hunt et al. 1992, Claridge 1985) and lacewings (Wells and Henry 1992, Henry 1994).
Understanding the causes of taxonomic variation in rapprochement requires an understanding of the relative costs and benefits of alternative signaling and searching behaviors for males and females in each species under consideration (Alexander et al. 1997). Males, especially in species such as insects with little or no paternal investment, are expected to be more risk-tolerant than females in rapprochement since their reproductive success is most directly influenced by the number of mates obtained (Trivers 1972, Alexander and Borgia 1979, Parker 1979) and hence by the effectiveness of mate-locating activities. Because of the sexes’ unequal risk tolerance, male mate-locating activities are influenced primarily by patterns of female dispersion (Emlen and Oring 1977), while females are influenced by selection for effective parental investment. The relative costs and benefits of alternative rapprochement behaviors for individuals of each sex will be affected
120 by interrelated factors including the kinds and pattern of predation, the spatial distribution of appropriate habitats and resources (Emlen and Oring 1977, Cocroft and Pogue 1996, Heller and von Helversen 1993), the sex-specific costs and benefits of searching vs. remaining stationary, and the current strategies of the opposite sex.
Periodical cicada rapprochement behavior involves a remarkable combination of related unusual features which have likely resulted from adaptation to high population density. Because the phylogenetic relationship of Magicicada to other cicada genera is poorly known, little information is available on the rapprochement behavior of the most recent non-periodical ancestor of Magicicada. However, the uniqueness of the Magicicada rapprochement system suggests that Magicicada must have evolved from ancestors with relatively stationary, advertising males and relatively mobile females. The following general hypothesis explains the unique features of Magicicada; similar selective pressures have likely shaped related attributes of rapprochement systems of other organisms (e.g. Heller and von Helversen 1993).
Upon the evolution of partial or complete periodicity (see Lloyd and Dybas 1966a, b; Cox and Carlton 1991, Long 1993, Yoshimura 1997) high population density arising from lack of ecological control by predators and parasites became a consistent feature of Magicicada ecology. This presumably led to two important effects with dramatic consequences for the Magicicada mating system: First, males and females began to experience reduced risks associated with movement and/or signaling. Second, the greater density of receptive females increased the chances that a male could encounter a receptive female while moving through his environment. These factors together would have improved the potential payoff to individual males of increasing time spent searching at the expense of advertisement; increased male searching would have caused females to benefit most from moving to areas of loud male chorus sound and remaining still once there, in part because increased movement by males would reduce the ability of females to approach
121 individual males before their next flights. This strategy of moving to the chorus and becoming stationary could have resulted in selection favoring a tendency in males to be attracted to the choruses of other males, completing the evolution of the basic elements of the Magicicada rapprochement system.
It is probably true that for any individual of a species in which females approach calling males, unreceptive females can avoid calling males, and, by voluntarily approaching calling males, sexually receptive females are able to mate on demand. For species in which males locate females, however, females can reduce their rapprochement time by making it easier for males to find them. Although in dense Magicicada emergences, it would seem as though males would have little difficulty finding females, any individual female is likely surrounded by a collection of immature cicadas, cicadas of the same sex, and mated cicadas, as well as a small number of sexually receptive members of the opposite sex. Female Magicicada may have evolved to signal their receptivity to ensure timely mating in confusing, dense aggregations. Males would then have been selected to become efficient at detecting these signals and minimizing their chances of mistakenly courting unreceptive cicadas or other inert objects.
It is not difficult to imagine the advent of stationary females resulting in the evolution or refinement of a female wing-flick signal to reduce rapprochement time, although the female signal may have existed from the start in the nonperiodical ancestor. An environment of locally aggregated, stationary females who respond to the call terminus with a wing-flick would select males further for a local search strategy emphasizing short calling bouts, short calling phrases, and short flights. The density of male aggregations would allow the chorus sound itself to assume the female-attraction function of the male’s call, allowing males to modify their calling and searching behaviors to be most effective for local mate searches, possibly resulting in (1) quieter calls, (2) more pure-tone calls in the
122 M. –decim species, and (3) calls with terminal downslurs in the M. –decim and M. cassini species or their common ancestor.
The best tests of the above scenario will likely come from comparative study using data from pair-forming systems in other Cicadidae, but at present few species have been studied in significant detail. One pattern within Magicicada offers initial support for the theory: Two Magicicada species, M. septendecula and M. tredecula, are consistently less abundant than other Magicicada; these species have longer calling bouts and a longer call unit that contains multiple temporal “windows” for female wing-flick responses. Our playback experiments confirm a different aspect of the hypothesis, that male calling song has evolved under selection for distinctiveness in loud choruses. The two-part structure of M. –decim and M. –cassini calling songs may provide a record of the evolution of song distinctiveness from an ancestral call without a downslur: The downslur functions at least in part to mark the end of a call against a background chorus dominated my main elements.
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128 Table 5.1. Traits distinguishing Magicicada species. "Pronotal extensions" are lateral extensions of the pronotum between the eyes and wing bases. For additional descriptions see Alexander and Moore 1962.
Species Life Cycle Abdominal sternites Dominant call Pronotal Length of call Range Habitat Peak chorus (yrs.) pitch (kHz) extension (seconds) activity color
orange with black lateral band § midwestern, M. neotredecim Marshall and Cooley 13 1.35 - 1.90 orange 1.5 - 4 upland morning
129 or center Ozark
§ midwestern, M. tredecim (Walsh and Riley) 13 mostly orange 1.00 - 1.25 orange 1.5 - 4 mixed morning southern
orange with black lateral band § M. septendecim (L.) 17 1.25 - 1.50 orange 1.5 - 4 eastern, plains upland morning or center
black, rarely with weak †† 17 > 3.00 2 - 6 §§ eastern, plains lowland afternoon M. cassini (Fisher) orange lateral band black
black, rarely with weak †† midwestern, 13 > 3.00 2 - 6 §§ lowland afternoon M. tredecassini Alexander and Moore orange lateral band black southern
† M. septendecula Alexander and Moore 17 black with orange lateral band > 3.00 black 7 - 14 eastern, plains upland midday
midwestern, 13 > 3.00 † M. tredecula Alexander and Moore black with orange lateral band black 7 - 14 southern upland midday
§ Roughly pure-tone, musical buzz terminating in a noticeable drop in pitch; no ticks. Usually 2-3 calls between flights.
§§ Rapid series of ticks followed by high-pitched, broad-spectrum buzz that rises and then falls in intensity and pitch. Usually 1-2 calls between flights.
† Repeated, rhythmic high-pitched, broad-spectrum tick-buzz phrases, followed by repeated phrases containing only ticks. Usually 1 call between flights.
†† Orange band, if present, often interrupted medially. Table 5.2. The sexual sequence in Magicicada species. Female Female Female Female Chorus Wing Courtship Wing Wing Wing Species Calling Flick Synchrony CI Calling Flick Behaviors CII Calling Flick Behaviors CIII Calling Flick Behaviors
phrases similar to walking, repeated, shortened phrases of the same Foreleg 2-3 phrases walking repeated those used in calling flying type as in court I, with greater emphasis onAt end vibrate, M. -decim between Yes No Yes between staccato No 130 and separated by between the slur, or terminal portion, and without of CII mount, flights phrases buzzes silent gaps phrases intervening silence copulate
repeated, shortened phrases of the same phrases similar to walking, Foreleg 1 phrase type as in court I, with greater emphasis on walking repeated Under some those used in calling flying vibrate, M. -cassini between Yes Yes the slur, or terminal portion, without Yes between staccato No conditions and separated by between mount, flights intervening silences and with additional ticks phrases buzzes silent gaps phrases copulate after the downslur
phrases similar to walking, Foreleg 1 calling bout repeated those used in calling flying vibrate, M. -decula between Yes No Yes none known - none known staccato No and separated by between mount, flights buzzes silent gaps phrases copulate Table 5.3. Study Sites, 1995- 1998.
Year Brood Life Cycle Location County State Characteristics 1995 I 17 Alum Springs Rockbridge VA Logged site 1996 II 17 Horsepen Lake SWF Buckingham VA Logged site 1997 III 17 Siloam Springs SP Brown, Adams IL Clearing 1998 XIX 13 Harold Alexander WMA Sharp AR Cleared field
131 Table 5.4. Length of teneral period, in days, as measured by first female M. septendecim responses to playback of male songs. Females were divided into groups containing approximately 25 cicadas and placed in single-sex cages on living vegetation. Females were exposed daily to playbacks of male song, and the day on which at least one female in the cage responded with wing flick signals was recorded.
Group First response (days after emergence) A 6 B 7 C 8 D 7 E 6 F 5 Average 6.5
132 Table 5.5. Mated and unmated female M. septendecim responses to 2- minute playbacks of male calls. Females responding positively produced at least one wing flick signal in response to playbacks of male songs.
Day Mating n Response P Status (+) (-) (Fisher Exact Test) 1 Mated 22 0 22 Unmated 17 9 8 £ 0.001
2 Mated 22 0 22 Unmated 16 8 8 £ 0.001
3 Mated 20 0 20 Unmated 15 7 8 £ 0.001
4 Mated 18 0 18 Unmated 14 7 7 £ 0.001
133 Table 5.6. Wing flick (WF) responses of 25 female M. septendecim and 3 female M. cassini to a series of 135 alternating male M. septendecim and M. cassini calls.
Number responding Species n to M. septendecim call to M. cassini call M. septendecim 25 25 1 M. cassini 3 0 3
134 Table 5.7. Male M. septendecim responses to artificial wing flicks. We simulated female wing flick signals by flicking a strip of paper or by clicking a heavy-duty light switch at the appropriate time in relation to male call. We scored males as responding positively to the artificial wing flicking if they moved toward the stimulus and began late- stage courtship behaviors such as CII or CIII call, forleg-vibrate, or mounting behavior. Fisher’s Exact 2- tailed tests are used to compare treatments to 46 controls in which the clicking device was presented to the male, but no click was made; only 6 males responded positively to such controls.
n (+) (-) vs. Timing n (+) (-) P Control 46 6 40 End of call 83 66 17 £ 0.001 During Call 41 3 38 £ 0.498 During Slur 13 0 13 £ 0.326
135 e, rpreta- 0.003 (Fisher’s Exact Test) Exact 0.003 (Fisher’s Test) Exact 0.02 (Fisher’s 0.355 (Wilcoxon) 0.005 (Wilcoxon) 0.221 (Wilcoxon) 0.223 (Fisher’s Exact Test) Exact 0.223 (Fisher’s 0.001 (Wilcoxon) ≤ ≤ ≤ P ≤ ≤ ≤ P ≤ 29.8 (n = 47) 15.14 (n= 15) 1.26 (n= 19) ± ± 2.46 (n = 53) ± ± 26.55 (n= 13) 20.98 19.4 (n = 40) 22.60 0.83 (n= 17) 3.53 ± ± 1.16 (n = 43) 3.70 ± ± movement direction (flights and walks) following artificial wing flick signal or control (no signal) or control (no wing flick signal following artificial and walks) direction (flights movement Control signal With Control signal With M. septendecim Male Toward ObserverToward from ObserverAway 16 3Fly after signalDo not fly after signal 4 7 13 13 ObserverToward from ObserverAway 7 0 5 14 2 4 Fly after signalDo not fly after signal 7 36 15 38 Multiple distant simulated wing-flick signals Multiple distant simulated wing-flick Direction Likelihood of flight Distance of flight (cm)Direction 35.76 Distance of flight (cm) 25.75 Call Number 1.94 Likelihood of flight Table 5.8. Table on a clock fac representing the directions as a value from 1-12 with the numbers of movement was recorded treatment. Direction Single simulated wing-flick signal Single simulated with only the directions 11, 12, and 1 considered “toward stimulus” and 4, 5, and 6 “away from stimulus,” to avoid biased inte 4, 5, and 6 “away from stimulus,” “toward stimulus” and 12, and 1 considered 11, with only the directions were not monitored further. paused for longer than 20 seconds lateral movements. Males that tion of ambiguous Call Number 2.28
136 Table 5.9. Experiments on male chorusing behavior. Clicking device placed near chorusing male and clicked or not clicked (control). Number of calls in first and second call bouts and length of first and second flights after treatment or control were measured. Results analyzed with Kruskal-Wallis one-way analysis of variance.
M. septendecim Click vs. control P First call bout (64/46) £ 0.277 First flight (50/37) £ 0.573 Second call bout (45/34) £ 0.712 Second flight (39/32) £ 0.117
M. cassini Click vs. control P First call bout (24/26) £ 0.001 First flight (23/25) £ 0.000 Second call bout (18/22) £ 0.399 Second flight (15/19) £ 0.001
137 Table 5.10. Numbers of M. septendecim responding with wing flick signals to frequency-modified playbacks of male song. Mature females made most reponses to unmodified playback songs, as did Stage I Massospora cicadina infected males. Normal males and Stage II infected males never responded to playbacks.
Song Mature Stage I Stage II Normal Frequency (kHz) Female (32) Male (6) Male (5) Male (8) 2.35 0 0 - 0 1.91 2 0 - 0 1.65 13 1 - 0 1.48 16 2 - 0
1.39 (unmodified) 22 3 0 0
1.30 20 1 - 0 1.13 18 0 - 0 0.956 8 0 - 0 0.869 1 0 - 0
138 Table 5.11. Contrasts of female wing flick responses to playbacks of complete artificial calling songs and partial artificial calling songs (main portion only, slur portion only) at four background chorus intensities. P- values are for Fisher’s Exact 2- tailed tests. Whole artificial calling song is always more effective in eliciting a female response than either partial call. Background is a continuous 1.39 kHz pure tone; background levels: 0db, 58- 62 db, 63- 77 db, 65- 80 db, where maximum value is approximately equal to acoustic intensity of playback call. Background Contrast 0 db 58- 62 db 63- 77 db 65- 80 db Whole vs. Slur 0.001* 0.001* 0.001* 0.005* Whole vs. Main 0.001* 0.001* 0.001* 0.001* Main vs. Slur 0.001* 0.004* 0.465 0.012*
139 Table 5.12. Effects of background chorus intensity on female wing flick responses to playbacks of complete artificial calling songs and partial artificial calling songs (main portion only, slur portion only) at four background chorus intensities. Increasing background chorus intensity significantly decreased the likelihood that whole calls and the main portions of calls would elicit female responses and generally increased the likelihood that slur alone would elicit responses. Background is a continuous 1.39 kHz pure tone; background levels: 0db, 58- 62 db, 63- 77 db, 65- 80 db, where maximum value is approximately equal to acoustic intensity of playback call. Effect of increasing chorus intensity, Kruskal-Wallis one way analysis of variance: Playback type Z P Whole 420 £ 0.001 Main portion only 64 £ 0.001 Slur portion only 44 £ 0.001
140 Table 5.13. Effects of M. -decim “interference buzz.” We confined four, unmated female M. septendecim in a test chamber and played a sequence of 60 calls, alternating normal calls and calls with buzzes, recording the number of females responding to each call. We repeated the experiment 6 times.
Average number of P (Friedman Females responding Two-Way Replicate Buzz to each call analysis of variance.) A Yes 1.13 ± 0.86 £ 0.001 No 2.23 ± 0.77 B Yes 0.57 ± 0.57 £ 0.00 No 2.43 ± 0.68 C Yes 0.80 ± 1.00 £ 0.001 No 1.73 ± 1.08 D Yes 1.27 ± 1.93 £ 0.006 No 0.82 ± 0.58 E Yes 0.93 ± 0.87 £ 0.045 No 1.53 ± 0.78 F Yes 0.47 ± 0.57 £ 0.00 No 2.07 ± 0.98
141
female female female answers answers answers
male CI male CI male CII male CIII
142 male approaches male wf wf wf female, foreleg "sing-fly" male lands male male approaches vibrates, mounts, behavior and calls calls female copulates
Figure 5.1. Stylized sonogram of male call/female wing flick coutship duet for M. -decim. Male begins with cout I (CI) calls; female answers each call with a wing flick. After several such interactions, male begins court II (CII) calling, which consists of repeated phrases of the same general type as CI calling, but shortened and without intervening silence. After the male ceases CII calling, the female wing flicks in response, and the male begins cour III (C III) calling, which consists of repeated staccato buzzes. M. -cassini and M. -decula courtship sequences are similar, except that M. -decula lack clearly defined CII calls. 10
8
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143 4
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kHz 0 0 0.5 1.0 1.5 2.0 2.5 S *
Figure 5.2. Sonogram of male M. -decim call and female wing flick response. Female response (marked with asterisk) is a broad-frequency sound. Female produces signal 0.387 s after end of male call (n= 235 examples, ± 0.110s). Wing flick sound enhanced and extraneous background noise removed for clarity. 10 144 8 6 4 2 kHz 0 0 1 2 3 4 S *
Figure 5.3. Sonogram of male M. -cassini call and female wing flick response. Female response (marked with asterisk) is a broad-frequency sound. Female produces signal 0.705 s after end of male call (n= 16 examples, ± 0.112s). Wing flick sound enhanced and background chorus removed from 2 to 4 seconds for clarity
10
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0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 S * * Figure 5.4. Sonogram of male M. -decula call and female wing flick responses. Female responses (marked with asterisks) are broad-frequency sounds. Female produces signals during silent portions of male call. Wing flick sound enhanced and extraneous background noise removed for clarity.
Figure 5.5. Comparison of male responses to a model cicada constructed from a pen cap. Cap was either black or white, and experimenter either moved at the appropriate time for a female wing flick signal, or held it still. Male responses, including call-walk towards, CII call, CIII call, and Foreleg vibrate were noted. Fisher's Exact 2- tailed Test P-values for pairwise comparisons of treatements plaed below a stylized sonogram of courtship sequence in M. -decim.
Artificial Artificial Artificial signal signal signal 146
(male CI) Call-Walk towards CII CIII Foreleg vobrate
Black/move (22) vs. black/still (21) 0.001 0.004 0.108 0.001 White/move (21) vs. white/still (20) 0.001 0.233 1.00 1.00 Black/move (22) vs. white/move (21) 0.129 0.045 0.345 0.001 Black/still (21) vs. white /still (20) 0.233 1.00 1.00 1.00 18
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147 6
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0 Move Click Move and Click Move Click Move and Click M. septendecim M. cassini
Figure 5.6. Male responses to actions of model. Males were scored as responding positively if they produced Court II or Court III courtship songs, or if they attempted to mount and copulate with the model. Positive responses marked with shading, negative responses unshaded. Within M. septendecim, the model that moved and clicked was more effective than the model that clicked only (P ² 0.001, Fisher's Exact 2- tailed Test) or the model that moved only (P ² 0.001, Fisher's Exact 2- tailed Test). For M. cassini, the results were similar; the model that moved and clicked was more effective than the model that clicked only (P ² 0.001, Fisher's Exact 2- tailed Test) or the model that moved only (P ² 0.001, Fisher's Exact 2- tailed Test). Responses to click only and move only treatements did not differ in wither species. 6
4 148
2
kHz 0 0 0.5 1.0 1.5 2.0 S * Figure 5.7. Sonogram of response of M. -decim male with Stage I Massospora cicadina infection to an M. -decim call. Male response (marked with asterisk) is a broad-frequency sound similar in timing and acoustical properties to female wing flick signals. Wing flick sound enhanced and extraneous background noise removed for clarity. 10
5 149
0 kHz 0 0.5 1.0 1.5 2.0 S * Figure 5.8. Sonogram of response of M. -cassini male with Stage I Massospora cicadina infection to an artificial M. -cassini call. Male response (marked with asterisk) is a broad-frequency sound similar in timing and acoustical properties to female wing flick signals. Wing flick sound enhanced and extraneous background noise removed for clarity. 0.45
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0.20 Whole call Main portion only Proportion responding 0.15 Slur portion only 0.10 150 0.05
0.00 0 58- 62 63- 77 65- 80
Background intensit (db)
Figure 5.9. Responses of female M. septendecim to playbacks of artificial, pure tone calls and portions of calls against an artificial background chorus. Females were scored as responding positively if they produced wing flick signals in response to playback. No actual chorus of M. septendecim was audible from the test chamber. Intensity of background chorus increased in increments from 0 db to 80 db. Data presented as number of positive responses for a given call and background condition given the total number of trials with those experimental conditions. Whole calls (square markers) were always more likely to elicit responses than either main portion only (circles) or slur portion only (triangles). As background intensity increased, the effectiveness of main portion only decreased, while the effectiveness of slurs alone increased. 4
3 male interference call buzz 2 151 1
kHz 0 0 1 2 3 4 5 S
Figure 5.10. Sonogram of male M. -decim call interference buzz of nearby male. Note how interference buzz overlaps and acoustically obscures calling male's terminal downslur. CHAPTER VI
REPRODUCTIVE CHARACTER DISPLACEMENT AND SPECIATION IN PERIODICAL CICADAS, AND A NEW 13- YEAR SPECIES, MAGICICADA NEOTREDECIM
Abstract
Acoustic mate-attracting signals of related sympatric, synchronic species differ, but those of related allopatric species sometimes do not, suggesting that these signals may evolve to “reinforce” premating species isolation when similar species become sympatric. This model also predicts divergences restricted to regions of sympatry in overlapping species, but such “reproductive character displacement” has rarely been confirmed. We report such a case in the acoustic signals of a previously undetected, new 13-year periodical cicada species, Magicicada neotredecim, here described. Where M. neotredecim overlaps M. tredecim in the central U.S., the dominant chorus pitch (frequency) of M. neotredecim increases from ca. 1.40 to 1.75 kHz, while that of M. tredecim slightly decreases from ca. 1.15 to 1.08 kHz. Female cicadas from sympatry (both species) and allopatry (only M. neotredecim studied) respond best to song pitches similar to those of conspecific males. M. neotredecim differs from 13-year M. tredecim in abdomen coloration, mtDNA, and song pitch, but does not differ consistently from 17-year M. septendecim; thus, like other
152 Magicicada species, M. neotredecim appears most closely related to a geographically- adjacent counterpart with the alternative life cycle. Speciation in Magicicada may often be initiated by life cycle changes that create temporal isolation, and reinforcement could play a novel role by fostering divergence in premating signals prior to speciation. The geographic and phylogenetic relationships of the M. -decim species, and the pattern of reproductive character displacement, suggest two theories of Magicicada life cycle evolution: “nurse brood facilitation” and “canalization of climate-induced shifts.”
Introduction
The periodical cicada species (Magicicada spp.) of eastern North America have extraordinary life histories and phylogenetic relationships (Marlatt 1923; Alexander and Moore 1962; Lloyd and Dybas 1966a, b; Simon 1988; Williams and Simon 1995). Magicicada live underground juvenile lives of either 13 or 17 years, after which they emerge for a brief adult life of about three weeks. Six Magicicada species have been described: In northern and plains states, three morphologically and behaviorally distinct species coexist and emerge together once every 17 years (Fig. 6.1). These species are reproductively isolated in part by distinctive male acoustic signals and female responses (Alexander and Moore 1958, 1962). In the Midwest and South, three similar 13-year species coexist. In this paper, we describe a fourth 13-year species, M. neotredecim. The 13- and 17-year populations are divided regionally into several temporally isolated “broods” that emerge on different schedules. Each 13-year species is probably most closely related to its 17-year counterpart; some of these species pairs can be distinguished only by life cycle (Table 6.1). This pattern suggests that speciation in Magicicada may involve a combination of geographic isolation and life cycle changes that create temporal, or allochronic, isolation (Alexander and Moore 1962; Lloyd and Dybas 1966b; Lloyd and White 1976). Allochronic speciation has been proposed for other organisms (e.g.
153 Alexander 1968; Alexander and Bigelow 1960; Tauber and Tauber 1977a,b), although one case has recently been challenged (Harrison 1979; Harrison and Bogdanowicz 1995).
The male calling songs of sympatric Magicicada species are strikingly different, while those of parapatric life cycle siblings (e.g. 17-year M. cassini and 13-year M. tredecassini) are similar or indistinguishable (Alexander and Moore 1962). This pattern, common in groups with long-range sexual signals, suggests a process in which costly heterospecific sexual interactions lead to selection reinforcing trait differences that promote premating isolation (Dobzhansky 1940; Blair 1955). The hypothesis of reinforcement also predicts greater signal divergence in sympatry, a pattern termed “reproductive character displacement,” when two species’ distributions partially overlap (Brown and Wilson 1956), but this second pattern is only rarely observed (Alexander 1967; Walker 1974; Howard 1993). For example, only one case is known in the singing Orthoptera and Cicadidae (Otte 1989). Our use of these terms, in which reinforcement is a process and reproductive character displacement a resulting pattern, is similar to that of Loftus-Hills and Littlejohn (1992) and Howard (1993), except that in our usage reinforcement can involve selection against wasteful heterospecific sexual interactions of any kind, not merely heterospecific mating as in Howard (1993). Reinforcement of premating isolation is of interest as a mechanism by which barriers to interspecific gene flow are enhanced as a direct result of species interactions in sympatry rather than as incidental outcomes of evolution in allopatry (Dobzhansky 1940; Blair 1955; Howard 1993).
Here we report that the newly described 13-year periodical cicada species, Magicicada neotredecim, shows reproductive character displacement in male song pitch and female song pitch preferences in the central U.S. where it overlaps the 13-year species M. tredecim (Fig. 6.1). M. neotredecim appears to have been derived by a life cycle change from its 17-year counterpart M. septendecim (see also Martin and Simon 1988, 1990; Simon et al. submitted). We propose new hypotheses of life cycle evolution and speciation
154 in Magicicada suggested in part by the geographic and phylogenetic relationships of M. neotredecim to the remaining Magicicada species.
Methods and Results
Background
The Magicicada mating system involves male acoustical signals, female responses, and complex acoustical courtship (Chapter 5). Periodical cicada males aggregate, forming singing choruses attractive to sexually receptive females. Most Magicicada choruses contain three species, a M. –decim1 species that produces a narrow band of sound frequencies with a single dominant pitch between 1 and 2 kHz, and M. -cassini and M. –decula species that each produce broad-spectrum sounds above 3 kHz (Fig. 6.2). While observing 13-year Magicicada in northern Arkansas in 1998, we found choruses with two peak frequencies in the M. -decim range (ca. 1.1 and 1.7 kHz), suggesting a new M. - decim species (Figs. 6.2, 6.3). The pitch differences, easily detectable by ear, are unlikely to result from temperature variations because the song types coexist in the same choruses, because chorus pitch did not vary with air temperature (Fig. 6.4), and because song pitch is not related to tymbal muscle temperature in the closely-related M. septendecim (Young and Josephson 1983). The nature and location of this discovery suggested that the two sympatric M. -decim might correspond to two forms of M. tredecim identified using mitochondrial DNA (mtDNA) and abdomen coloration (Martin and Simon 1988) and found to overlap in a zone reaching from Arkansas to Indiana (Simon et al. submitted). We conducted acoustical, behavioral, and morphological analyses in this region of overlap to test this possibility and to determine the role of song pitch in reproductive isolation of the
1For convenience we refer to Magicicada sibling species groups using the following shorthand: M-decim: M. septendecim (17), M. tredecim (13), and M. neotredecim (13); M -cassini: M. cassini (17) and M. tredecassini (13); M. -decula: M. septendecula (17) and M. tredecula (13).
155 two species. Analysis of geographic song pitch variation led to the discovery of reproductive character displacement in the new species, M. neotredecim.
Documenting Sympatric M. –decim Species with Songs and Morphology
Martin and Simon (1988) identified two forms of 13-year M. tredecim partly on the basis of the amount of orange coloration on the abdominal sternites. To determine if the sympatric M. -decim song types corresponded to these morphs, we measured the calling songs of a random sample of 151 males from a mixed chorus in Sharp Co., AR, and tested for association of song pitch and abdomen color. We collected the males from privately- owned woods on County Rd. 51 approximately 0.25 mi. S. of County Rd. 62 (Lat. 36.256, Lon. -91.462), at a powerline right-of-way, just outside the northwest boundary of the Harold E. Alexander Wildlife Management Area, Sharp Co., AR; we will refer to this location as the “powerline” site.
We recorded male songs using a Sony Professional Walkman cassette recorder and a Sony microphone mounted in a plastic parabola, or a Sony 8mm videocassette recorder with built-in microphone. Because the songs of an individual male do not vary significantly in dominant pitch (Fig. 6.5), we isolated a single call of each individual for spectral analysis. For each recording we generated a power spectrum (plot of sound intensity vs. frequency) and spectrogram (power spectrum vs. time) using Canary 1.1.1 (Cornell Bioacoustics Laboratory) on a Macintosh computer. Individual male M. –decim songs consist of a 1-3 second steady-pitch and nearly pure-tone (sine wave) “main element” followed by a quieter 0.5 second frequency “downslur” that ends approximately 500 kHz lower than the main element pitch (Fig. 6.3; Alexander and Moore 1958; Weber et al. 1987). Because the comparatively minor sound energy in the downslur is distributed across many frequencies, it contributes little to the spectral profile of the call; thus the main element produces the dominant call pitch.
156 We scored the abdomen color of each individually-recorded male using the method of Martin and Simon (1988), in which each male is assigned a value from 1 (50% black) to 4 (all orange). Because male calling song pitches fell into two distinct categories with just one intermediate male out of 151 sampled, while the morphological traits were more continuously distributed, we tested for association between song pitch and coloration by dividing the male sample through an arbitrary intermediate value of 1.5 kHz and examining abdominal color differences between the resulting groups using a Mann-Whitney U.
Results. Within the Sharp Co., AR, powerline site, M. -decim males producing songs with low dominant pitch (mean 1.11 kHz) had the orange abdomen color (Table 6.2; Appendix F) characteristic of Martin and Simon’s (1988) “mtDNA lineage B,” now recognized to be the previously described Magicicada tredecim (Alexander and Moore 1962). The M. -decim males producing higher-pitched songs (mean 1.71 kHz) had the darker abdomen color of Martin and Simon’s “mtDNA lineage A,” and constitute a new species here named Magicicada neotredecim. The mixed chorus produced little sound at pitches intermediate to those of M. tredecim and M. neotredecim (Figs. 6.2, 6.3, 6.6); this and the strong correlation of song and abdomen color suggest that interbreeding is rare (see also Simon et al. submitted). Among approximately 200 cicadas observed during our study, we found just three unambiguous intermediates: One high-pitch male with a yellow abdomen (category 4), one low-pitch male with a darker abdomen (category 2), and one male with an intermediate calling song (1.43 kHz). These could be hybrids or merely phenotypic outliers.
Magicicada neotredecim , new species: Holotype (male): Collected by Cooley and Marshall on 18 May 1998 in Sharp Co., AR., in a powerline right-of-way on County Rd. 51 approximately 0.25 mi. S. of County Rd. 62 (Lat. 36.256, Lon. -91.462), outside the Harold E. Alexander Wildlife Management Area. Dominant pitch of male song phrase 1.68 kHz. Deposited at University of Michigan Museum of Zoology (UMMZ), Ann
157 Arbor, MI with a recording of the male’s call stored on diskette. Allotype: Collected by Cooley and Marshall on 18 May 1998 at same location as holotype. Deposited at UMMZ. For additional description see Table 6.1.
Measuring Female Song Pitch Preferences in Sympatry
Sexually receptive female Magicicada produce “wing-flick” signals in response to male songs (Chapter 5, Cooley and Marshall in prep.). We used this signal as an assay of female mating receptivity to determine if female preference for song pitch was correlated with abdomen color. Using Sound Edit Pro software(MacroMedia), we produced 14 pure- tone model calling phrases differing only in pitch (1.0 kHz to 2.3 kHz, in 0.1 kHz increments) and lacking temporal structure. We modeled artificial songs after field recordings of M. –decim songs (see example using artificial frog calls in Gerhardt 1992). In previous experiments, we have found that females respond similarly to playbacks of recorded and artificial songs. We estimated song pitch preferences of 91 M. -decim females collected from the Sharp Co., AR, powerline site by playing the model songs to individually-marked caged females in both haphazard and ordered sequences using a Macintosh Powerbook computer connected to an amplified portable speaker. Females that did not respond to any songs were dropped from the analysis. We positioned the speaker 25 cm away from the test cage and played the model songs at a volume simulating the intensity of a calling male, as determined by measuring several males’ call intensities with a sound level meter. The playback experiments were carried out between 11:00 and 16:00 in bright overcast or sunny conditions against an acoustic background of a Magicicada chorus containing M. neotredecim, M. tredecim, M. tredecassini and M. tredecula located in woods ca. 8 meters away. For each female, we recorded whether she responded with wing-flick signals and then calculated the weighted average of her responses to the 14 model songs using the following formula:
158 14 å (kHzcall i )(responses to call i ) i =1 (total responses)
We scored female abdomen color using the method described above for males. We tested for association between weighted average pitch preference and abdomen color by dividing the female sample through the same pitch as the male sample (1.5 kHz) and examining the difference in abdomen color between the resulting groups using a Mann- Whitney U.
Results. The playback experiments showed evidence of two classes of females (Fig. 6.6, Appendix G). At the powerline site, females responding maximally to lower pitch songs (M. tredecim) were significantly more orange-colored than females responding maximally to higher pitch songs (M. neotredecim; Table 6.2). About a third (26/91) of the females did not respond to any call. This response rate is similar to that observed in previous work with 17-year M. septendecim females in Virginia and Illinois (Chapter 5; Cooley and Marshall in prep.), where only one M. –decim species is known.
Because the model songs contained no within-phrase temporal structure, differed only in pitch, and accurately distinguished the species, we conclude that song pitch differences may be an important cause of species-specificity in 13-year M. –decim mate recognition. However, natural calls have a pattern of amplitude-modulation that results from individual tymbal pulses (Young and Josephson 1983; Weber et al. 1987); we have not ruled out a role for this temporal structure. Differences in such temporal patterning are probably not the cause of pitch differences in M. -decim cicadas because variations in tymbal contraction rate do not influence dominant pitch (Young and Josephson 1983). We did not analyze temporal call structure because most of our field recordings contained too much background noise, although this did not affect analysis of dominant pitch. Little is
159 known of the relative roles of temporal patterning and frequency content in cicada song in general, although both function in Australian bladder cicadas (Cystosoma; Doolan and Young 1989). In the singing Orthoptera, species-specificity of calling songs usually results from differences in temporal patterning rather than pitch (but see Toms and Otte 1988 for an example in which pitch may be important), while dominant call pitch is important in many Anuran amphibians (Littlejohn 1977; Gerhardt 1988).
Using chorus recordings to estimate species abundance.
For mixed choruses, we developed a method for using the relative intensities of the two species-specific M. –decim dominant chorus pitches to estimate relative proportions of the species. This approach assumes that (1) M. neotredecim and M. tredecim are uniformly distributed at a given site and (2) both species show the same relationship between male abundance and chorus intensity. To test the first assumption, we recorded a continuous chorus sample along a 200m woodside trail in the Harold E. Alexander Wildlife Management Area, Sharp Co., AR, while pointing the parabola/ microphone assembly into the treetops along one side at a 45° angle. We recorded one side while walking in one direction and then recorded the other side while returning. From portions of this recording taken at seven meter intervals, we generated power spectra and measured the intensities of the M. neotredecim and M. tredecim frequency bands; this yielded 47 samples because of gaps in the forest on one side (350m total). If the M. neotredecim and M. tredecim at the site were not uniformly distributed with respect to one another on a local scale, we predicted that this series of samples would show significant variation among locations in the relative intensities of the two species’ chorus bands. To test the second assumption, we compared the distribution of call pitches of a random sample of males collected from the mixed-species Sharp Co., AR chorus to the distribution of song frequencies in the chorus power spectrum, using a Kolmogorov-Smirnov test to determine if the shapes of the two distributions differed significantly.
160 Results. Although the proportions of the two M. –decim species vary on a scale of miles (e.g. Fig. 6.7 insets), the species do not appear to assort significantly within a location. In the 350m continuous recording, the proportion of M. -decim chorus sound produced by the rarer species (M. neotredecim) remained between 10% and 36% (mean = 19.0%, SD = 6.0, n = 47), and the chorus intensities of the two species were not significantly negatively correlated (Pearson coefficient = -0.229, P= 0.121) as would be expected if the species formed exclusive aggregations. Therefore a recording taken at a given position is likely to give a reasonable estimate of the local chorus sound. This result matches our subjective impressions; we never detected by ear variations in the M. -decim chorus sound within a location. If males of the two species sing with similar consistency, intensity, and call duration, the sound intensity at a given species-specific pitch should be related to the local density of males producing that pitch. The random sample of the Sharp Co., AR powerline population verified this relationship: The standardized histogram of call pitches of individually-recorded males was indistinguishable from the standardized quadratic chorus power spectrum (Kolmogorov-Smirnov test, P > 0.05.; Fig. 6.6). Therefore the power spectrum of a mixed chorus can be used to estimate the relative proportions of M. tredecim and M. neotredecim present.
Although the downslurs of the higher-pitched M. neotredecim songs sometimes overlap the higher-frequency portions of the lower-pitched M. tredecim songs, downslurs from even a dense M. neotredecim chorus do not obscure sparse populations of M. tredecim because M. -decim downslurs are brief and their sound energy is distributed across a broad band of frequencies. M. tredecim at the Sharp Co., AR, powerline site produced a distinct peak in the chorus power spectrum (Fig. 6.6) despite constituting just 8% of the study population. However, extremely rare M. tredecim could be overlooked in a dense M. neotredecim population; we attempted to minimize this possibility by recording several minutes at each location and by sweeping the parabola in different directions. In contrast, rare M. neotredecim are always easily detectable in a M. tredecim chorus because
161 M. tredecim males do not produce sounds at frequencies as high as those of M. neotredecim.
Estimating Species Distributions using Geographic Variation in Songs
Once we had demonstrated that the two 13-year M. -decim forms are species differing in song pitch and that the species’ relative abundance can be estimated from chorus recordings, we analyzed the species’ distributions and geographic variation in song pitch using recordings taken from 80 locations distributed throughout the 1998 Magicicada emergence. Periodical cicadas in different regions emerge in different years, and the cicadas of a region are referred to as a “brood” (Marlatt 1923; see individual brood maps in Simon 1988). The 13-year cicadas emerging in 1998 belonged to the largest 13-year brood, Brood XIX, which reaches from Maryland to Oklahoma. The widespread range of this brood allowed a thorough characterization of 13-year M. -decim calling song biogeography. The recording dates were as follows: Sharp, Fulton and Lawrence Co., AR, 12-25 May; remaining Arkansas sites, 28 May; Missouri sites 1-7 June; Randolph, Monroe, Jersey, Sangamon and Piatt Co., IL 29-30 May; remaining Illinois sites 9-14 June; remaining central and southeastern state sites 31 May - 3 June; Maryland sites 29 May - 1 June.
Results. We found M. neotredecim in Missouri, Illinois, western Kentucky, and northern Arkansas (Fig. 6.7, Appendix H; see also Simon et al. submitted). The southernmost M. neotredecim populations overlap M. tredecim in a zone 50-150 km wide reaching from northern Arkansas into southern Missouri, southern Illinois, and eastern Kentucky. The remainder of Brood XIX contains M. tredecim and not M. neotredecim. Simon et al. (submitted) document a similar pattern of overlap using abdomen coloration and mtDNA and describe additional mixed populations in southern Indiana.
162 Geographic variation in dominant chorus pitch of M. neotredecim shows a pattern of reproductive character displacement, a predicted outcome of reinforcement of premating isolation (Dobzhansky 1940; Blair 1955; Howard 1993). M. neotredecim males have the highest song pitch (ca. 1.7 kHz) where they coexist with M. tredecim. North of the overlap zone, M. neotredecim dominant song pitch decreases to approximately 1.4 kHz in Illinois and 1.5 kHz in Missouri (Fig. 6.8, 6.9). Most of the song change occurs over a short distance immediately north of the zone of sympatry, and little variation occurs elsewhere in allopatry. Only song pitch varies across the range of M. neotredecim; abdomen color and mtDNA haplotype are comparatively uniform (Martin and Simon 1988, 1990).
Song pitch variation in M. tredecim is more subtle, only about 1/4th of that observed in M. neotredecim. M. tredecim populations in deep sympatry with M. neotredecim have very low-pitched songs, and M. tredecim peak song pitch slightly increases south and east of the overlap zone from ca. 1.11 to 1.15 kHz (Figs. 6.9, 6.10). However, the significance of this pattern is not clear because some allopatric M. tredecim choruses in the southeast have songs as low-pitched as those in the overlap zone.
While most of the chorus samples included the songs of thousands of males, many of the populations from Missouri and Alabama were recorded late in the emergence when relatively few males remained. For these locations the chances of overlooking a rare type were greater, although it seems unlikely that rare M. tredecim could have been missed in each of the many samples from central and northern Missouri.
Additional Tests of Reproductive Character Displacement
To determine if female song pitch preferences change with male song pitch in M. neotredecim, we measured weighted average song pitch preferences of 33 Magicicada neotredecim females collected from a woodlot off Rt. 1300E 0.8 miles south of White
163 Heath, IL (Piatt Co.), beyond the northern limit of M. tredecim. We completed the playback experiments at Lodge Park Co. Forest Preserve with a nearby background chorus containing M. neotredecim, M. tredecassini, and M. tredecula. We used a Wilcoxon signed-ranks test to determine if the average pitch preference of the Piatt Co. females differed from that of the Sharp Co., AR females. Because of time constraints, we were unable to study allopatric M. tredecim females.
To test the hypothesis that song pitch variations could be related to geographic variation in body size, we compared the song pitches and body sizes of M. neotredecim and M. tredecim males from sympatry at the powerline site with those of 17 M. neotredecim males collected in Allerton Park, Piatt Co., IL, where no M. tredecim are present. We used three characters to approximate size: right wing length, thorax width between the wing articulations, and first abdominal sternite width between the sutures that join it to the first abdominal tergite. We conducted pairwise comparisons among populations using Mann-Whitney-U tests. For each population, we tested for an association between size and song pitch using linear regressions.
Results. Female M. neotredecim preferences change with male song pitch: In sympatry with M. tredecim, (Sharp Co., AR) female M. neotredecim were most responsive to an average pitch of 1.75 kHz (n = 35), while in allopatry (Piatt Co., IL) female preference averaged 1.31 kHz (n = 13; Wilcoxon signed ranks test, P < 0.002).
Size differences do not explain patterns in calling song variation because (1) M. tredecim and M. neotredecim in sympatry overlapped broadly in size but not in song pitch, (2) M. neotredecim populations from Illinois and Arkansas differed in song pitch but not in size, and (3) we found no significant correlation between song pitch and any measure of body size within species in any population (linear regression; Fig. 6.11).
164 Discussion
Reinforcement and Reproductive Character Displacement in M. neotredecim
Reinforcement is often viewed as a model of species formation (e.g. Butlin 1987, 1995; Liou and Price 1994; Kelly and Noor 1996), the only such model in which prezygotic isolation evolves as a direct outcome of natural selection against hybridization. In keeping with this view, Butlin (1987, 1989) suggests that the term “reinforcement” should apply only to cases in which assortative mating is enhanced despite gene flow and that the term “reproductive character displacement” should be used to refer to the strengthening of prezygotic isolation when hybrids are sterile, because speciation is already completed if gene flow is not possible. However, there are potential problems with this distinction (see also Howard 1993). If the fundamental criterion for species status is evolutionary independence (de Queiroz 1998), or low probability of future amalgamation, then populations that have acquired sufficient postzygotic isolation to undergo reinforcement are species even before they come into contact; the occurrence of reinforcement is itself evidence of evolutionary independence. Furthermore, abundant evidence of natural hybridization (Arnold 1997; Arnold and Emms 1998) shows that species should not be identified, nor the completeness of speciation determined, by degree of hybrid sterility. Practical difficulties with Butlin’s definitions arise because uncertainty about past gene flow prevents the application of either term; for example there is little or no evidence that M. neotredecim and M. tredecim currently hybridize, but this does not necessarily mean that gene flow did not occur in the past. Consequently, we use the term reinforcement to refer to a general process defined without respect to gene flow, speciation, or relatedness of interactants, and we use the term reproductive character displacement to identify the resulting pattern.
165 The process of reinforcement is the elaboration of differences between species’ sexual signals in response to selection against heterospecific sexual interactions (Grant 1972, Waage 1979, Howard 1993). If newly established contact between species involves only slight range overlap, differences resulting from reinforcement may be manifest in the form of clines, in which the two species are most extremely different in sympatry and less distinct in allopatry (Brown and Wilson 1956). The clines reflect the relative contributions of conspecific and heterospecific interactions to local optima of sexually-selected characters. Individuals within the zone of contact face net directional selection for distinctiveness from the other species, while conspecifics away from the zone of contact face net selection for compatibility with their own species, with no benefit, and perhaps some costs, to extreme individuals. Thus, in species undergoing reinforcement of premating isolation, differences in species-specific sexual traits will become less extreme away from the zone of contact where individuals face decreased pressures for distinction from heterospecifics and increased selection for compatibility with conspecifics having no history of contact with the other species involved.
A variety of observations support the hypothesis that the high song pitch of M. neotredecim in sympatry with M. tredecim results from reinforcement with M. tredecim. First, M. neotredecim in or adjacent to the area of overlap all have high-pitch songs (>1.6 kHz), the highest M. -decim dominant chorus pitch observed anywhere, while allopatric M. neotredecim never do. Second, the dominant chorus pitches of M. tredecim and M. neotredecim remain comparatively stable in allopatry, as does that of 17-year M. septendecim, which varies only about 0.15 kHz in archived UMMZ recordings taken throughout its widespread range (Table 6.3). Third, the typical song pitch of 17-year M. septendecim, the likely sister group of M. neotredecim (see below), is indistinguishable from that of allopatric M. neotredecim in Illinois (ca. 1.4 kHz); these species are also indistinguishable in behavior, morphology, and mtDNA (Alexander and Moore 1962; Simon et al. submitted). Fourth, song pitch apparently plays an important role in mate
166 recognition of both species. Fifth, M. neotredecim and M. tredecim songs in sympatry differ just enough for average songs of each species to avoid frequency overlap, with M. neotredecim downslurs ending at about the dominant song pitch of M. tredecim (Fig. 6.3). Finally, the change in song pitch does not appear to be an incidental effect of a latitudinal cline in body size: M. neotredecim populations in Arkansas and Illinois produce different song pitches yet are indistinguishable in size, and song pitch does not correlate significantly with body size within any population.
A potential challenge to the hypothesis of reinforcement in M. neotredecim arises from the existence of some Missouri populations apparently well outside the range of M. tredecim with a partially elevated dominant chorus pitch (ca. 1.5 kHz). This pattern could be explained by the reinforcement model if (1) M. neotredecim colonized Missouri from populations in Illinois that were themselves adjacent to the range of M. tredecim, or (2) undiscovered M. tredecim populations exist in Missouri near the locations we sampled. Future surveys should investigate the latter possibility. Because M. tredecim appears to reach its northern limits on Mississippi River and Wabash River lowlands, it may be found only in the vicinity of rivers elsewhere in the northern part of its range, increasing its chances of being overlooked.
Also of interest is that the dominant chorus pitch of M. neotredecim does not appear to be related to the relative abundance of the two 13-year M. -decim species in mixed populations (linear regression, r2 = 0.053, P £ 0.317, n = 21); under certain conditions the reinforcement model predicts this correlation because the strength of reinforcing selection on one species should depend on the abundance of the other (Howard 1993; Noor 1995). However, there are several possible reasons for this correlation to be absent even under the reinforcement model. First, most M. neotredecim in populations sympatric with M. tredecim have calls that are displaced on average just high enough to avoid frequency overlap, suggesting that reinforcing selection ceases after the M. neotredecim song
167 phenotype reaches that point. If only a minor representation by M. tredecim is necessary to drive this change in M. neotredecim, then a correlation between relative abundance and degree of displacement would be detectable only in populations with very rare M. tredecim; unfortunately we recorded few such populations. Second, the anticipated correlation between relative abundance (in sympatry) and degree of displacement would be weakened if mating signals are usually subject to stabilizing selection when not undergoing reinforcement (e.g. Paterson 1993; Alexander et al. 1997); such selection would maintain song displacement upon later changes in relative species abundance. This hypothesis could be falsified in this case by finding evidence of lowered M. neotredecim song pitch where M. tredecim is very rare and the weakly displaced M. neotredecim are unlikely to be immediately descended from populations with undisplaced songs. Third, rapid, continuous fluctuations in relative species abundance would also tend to weaken any correlation between abundance and displacement.
A notable feature of the pattern of song displacement in Magicicada is its asymmetry, although this phenomenon is not unusual in cases of reproductive character displacement (e.g. Littlejohn 1965; Littlejohn and Loftus-Hills 1968; Fouquette 1975; Waage 1979; Noor 1995). In general, because the strength of selection on each species depends on factors that can differ between the species, symmetrical displacement is probably unlikely (Grant 1972; Howard 1993). The selection each species imposes on the other will be a function of (1) the benefits realizable from hybrid sexual interactions (if hybrid offspring are fertile, the cost of hybridization may differ depending on which species is which parent), and (2) the relative abundance of each species (the likelihood that any individual will ever have a cross-species sexual encounter and face the danger of hybridization; but see above). The selection for divergence imposed by heterospecifics (through interspecific gene flow or the threat of it) should be stronger, and the stabilizing selection imposed by conspecifics (through intraspecific gene flow) should be weaker for the rarer of the species. Possible explanations for asymmetrical displacement in Magicicada
168 include: (1) greater numerical abundance of M. tredecim relative to M. neotredecim during critical stages of the interaction, (2) greater M. tredecim female selectiveness upon initial contact, and (3) greater constraints on evolution of lower song pitch.
The pattern of character displacement in M. neotredecim is similar to an example from Gastrophryne (Anura: Blair 1955; Loftus-Hills and Littlejohn 1992) in east Texas. Like M. neotredecim and M. tredecim, Gastrophryne carolinensis and G. olivacea in sympatry are distinguishable by only average differences in morphological characters and show the greatest nonoverlapping differences in dominant song pitch, a trait relevant to species-specific mate recognition. As in the Magicicada example, evidence of hybridization is weak and allopatric populations of the species show nearly or completely nonoverlapping differences, a key observation suggesting that male calls were distinct, and mating errors rare, even prior to sympatry. Like Loftus-Hills and Littlejohn (1992), we note that reinforcement may occur even in the absence of interspecific mating as long as enhancement of differences in male song or female preference increases the efficiency of advertisement or mate-location.
The rarity of reproductive character displacement is a striking paradox (Alexander 1967; Walker 1974; Howard 1993; Alexander et al. 1997) given the ubiquity of song distinctiveness of sympatric species and given other evidence of reinforcement (Coyne and Orr 1989, 1997). Reproductive character displacement has been found in only one other group of singing insects, crickets of the genus Laupala in Hawaii (Otte 1989). The paucity of convincing examples has been interpreted differently by various authors; some point to a lack of adequately studied cases (Walker 1974; Howard 1993), while others suggest that sexual signal evolution may be driven mainly by within-species processes (West-Eberhard 1983; Paterson 1993). A somewhat neglected explanation for the paradox is that character displacement may be quickly obscured by species range changes that disrupt the association of song divergence with range overlap (Waage 1979): As discussed above, song changes
169 due to reinforcement might remain even after species’ ranges cease to overlap if mate- attracting signals and female preferences for them are usually subject to stabilizing selection (Paterson 1993; Alexander et al. 1997). Patterns of character displacement will also be lost if sympatry becomes complete or if allopatric populations with the pre-contact song types are lost.
Another possible explanation of the paradox is that, when male signals differ prior to the establishment of sympatry, reinforcement may involve changes only in the stringency of female mate acceptance criteria (Waage 1979; Gerhardt 1994; Kelly and Noor 1996) as long as the costs of having a novel, minority receiver phenotype are offset by the advantages of avoiding hybrid sexual interactions (Butlin and Ritchie 1994; see Halliday 1983). Such cases might tend to remain undiscovered because of an overemphasis on male signals. This explanation is based in part on the premise that reinforcing selection should occur initially and/or most strongly on females, who usually have the most to lose from mating errors (Alexander et al. 1997). However, reinforcement of female traits alone should be rare because any change in female behavior will tend to cause concerted evolution in male signals: If selection on male signals derives mainly from efficiency in mate attraction, males benefit from adaptations that increase their likelihood of securing a female response, and male signals would evolve in concert with changes in female preferences. Even if females in sympatry evolve only greater stringency of preferences, with no change in the mean preference, variation among males in sympatry should be reduced (relative to males in allopatry) as a result of stronger stabilizing selection.
Life Cycle Differences and Species Status
Designating M. neotredecim and 17-year M. septendecim as separate species may provoke debate because many populations of M. neotredecim differ from M. septendecim only in life cycle length and in geography (e.g. Lloyd 1984). However, if based upon genetic differences, life cycle differences may provide genetic isolation and facilitate
170 evolutionary independence of population lineages, the fundamental criterion for species status (Alexander and Moore 1962; de Queiroz 1998). The following evidence indicates that Magicicada life cycle differences have a genetic basis: (1) in transplant experiments, 13-year cicadas reared north of 17-year cicadas emerged after a 13-year juvenile period (Lloyd 1987); (2) the border between 13- and 17-year species appears historically stable (but see Lloyd et al. 1983); and (3) the failure of small numbers of cicadas emerging off- schedule to satiate avian predators and establish new populations (Marlatt 1923; Dybas 1969; Williams and Simon 1995) suggests persistent selection favoring additional genes canalizing the majority life cycle phenotype. Life cycle differences substantially restrict opportunities for gene flow, because M. neotredecim and M. septendecim emerge simultaneously just once every 221 years. Moreover, during such co-emergences, opportunities for interbreeding are limited further by the minimal geographic overlap between 17- and 13-year populations and the fragmented nature of Midwestern populations (Alexander et al. in prep.). Although some authors have suggested that 13- and 17-year cicadas are widely sympatric (Marlatt 1923; Cox and Carlton 1991), more recent studies (e.g. Lloyd et al. 1983) have found only limited sympatry. Alexander et al. (in prep.) found that 17-year Brood III and 13-year Brood XIX overlap in Illinois in limited regions, by no more than a few kilometers, and are most often separated by prairie gaps. Such limited opportunities for gene flow are unlikely to prevent divergence of M. neotredecim and M. septendecim, especially if differences in life cycle and geography impose divergent selective pressures (Endler 1977; Rice and Hostert 1993). Absence of known life cycle intermediates where M. neotredecim and M. septendecim meet and the stable position of their contact zone suggest that gene flow is sufficiently restricted to permit divergence.
Simon et al. (submitted) discuss life cycle changes in Magicicada and note that this process could bring M. neotredecim populations back into temporal synchrony and panmixis with M. septendecim. Although life-cycle switching from M. septendecim is a credible explanation for the origin of M. neotredecim, amalgamation of M. neotredecim and
171 M. septendecim by this process appears unlikely because (1) permanent life cycle changes are apparently rare, (2) a random switching event has a low probability of establishing synchrony between M. neotredecim and M. septendecim, and (3) a switching event that synchronizes only part of M. neotredecim with M. septendecim will not affect the remaining M. neotredecim.
The Origin of Magicicada neotredecim and Speciation in Periodical Cicadas
Cladistic analysis of M. –decim phylogeny and determination of ancestral life cycles is not yet possible because the informative traits (morphological and molecular) within the group are not shared with other Magicicada species or with other cicada genera; the same problem obscures relationships among the Magicicada sibling groups. However, because M. neotredecim and M. septendecim are in many locations distinguishable only in life cycle and geography, and share the same mtDNA haplotype (Martin and Simon 1988, 1990), one of these two species probably originated from ancestral populations of the other. Together, these apparent cognates (descendants of an exclusive common ancestor or speciation event) differ from M. tredecim in song pitch, abdomen coloration, and in mtDNA by an estimated 2.6% of nucleotides, suggesting that their common ancestor diverged from M. tredecim 1-2 million years ago (Martin and Simon 1990).
Biogeographic evidence suggests that populations of the M. septendecim lineage gave rise to M. neotredecim, rather than vice versa, probably in the time since the last glacial maximum. First, the comparatively restricted distribution of M. neotredecim, surrounded on the west, north, and east by M. septendecim, suggests that M. neotredecim may be the newer lineage; otherwise rapid and implausible range changes would be required to explain the widespread distribution of M. septendecim. Second, the pattern of M. neotredecim song variation, in which most M. neotredecim have undisplaced songs, probably could not have persisted through range changes associated with Pleistocene glacial cycles. As a species’ range shifts southward during glacial cooling, the populations
172 surviving in refuges are most likely founded from the southern margin of the earlier distribution, while the most northern populations simply go extinct (Hewitt 1996). Had M. neotredecim ever experienced such a range shift, all undisplaced M. neotredecim would likely have been lost, because (1) M. neotredecim is completely bounded on the south by M. tredecim, (2) southern M. neotredecim today all have displaced song pitches as a result of overlap with M. tredecim, and (3) all northern, undisplaced M. neotredecim are found in a region that 18,000 years ago may have contained spruce-fir forest and cooler, drier conditions presently found only north of the range of Magicicada (Webb et al. 1993). Thus, the pattern of reproductive character displacement, together with the similarity of northern M. neotredecim and 17-year M. septendecim calling songs, is consistent with the hypothesis that M. neotredecim originated recently via a permanent life cycle change from a 17-year ancestor (Martin and Simon 1988, 1990; Simon et al. submitted), although the reverse hypothesis (17-year M. -decim evolving from Midwestern 13-year M. -decim) cannot yet be ruled out.
Abdomen color and mtDNA data (Simon et al. submitted) indicate that the other large 13-year brood (Brood XXIII), which inhabits most of the lower Mississippi Valley, contains both M. neotredecim and M. tredecim, with M. neotredecim limited to northern populations and partially overlapping M. tredecim. Archived UMMZ recordings from this brood suggest that M. neotredecim has a displaced song pitch (ca. 1.7 kHz) where the two species overlap; unfortunately we do not have recordings from any of the few populations of pure M. neotredecim in Brood XXIII. Archived tapes further suggest that northern populations of the remaining 13-year brood (Brood XXII), found only in southern Mississippi and Louisiana, contain M. tredecim alone (dominant chorus pitch ca. 1.08 kHz; Warren Co., MS, and Catahoula Parish, LA), although additional sampling will be necessary to confirm the apparent absence of M. neotredecim from the brood. Simon et al. (submitted) note that the presence of M. neotredecim or similar cicadas in Brood XXIII could be explained in three ways: (1) by a second derivation from M. septendecim, (2) by a
173 single origin of Brood XIX M. neotredecim followed by the temporal migration of four- year delayed M. neotredecim stragglers to Brood XXIII, or (3) by the formation of Brood XXIII in part from four year delayed Brood XIX populations containing both M. -decim species. Evidence that the developmental plasticity required by hypotheses #2 and #3 occurs in Magicicada includes observations of off-schedule cicadas emerging in numbers difficult to explain by mutation (e.g. Dybas 1969; White and Lloyd 1979; Kritsky and Simon 1996) and by the existence of temporally isolated broods of the same life cycle, presumably formed as a result of short-lived regional climatic events that caused most cicadas to delay or accelerate their emergence once (Alexander and Moore 1962; but see Moore 1993).
Life cycle changes and speciation may be linked in Magicicada (see also Simon et al. submitted). Each species appears most closely related to a cognate with the alternative life cycle; it is therefore inescapable that 13- and 17-year life cycles have evolved repeatedly in the group (Alexander and Moore 1962). The discovery that 17-year cicadas differ from 13-year cicadas in having a 4-year dormancy period (White and Lloyd 1975, Lloyd and White 1976) increases the plausibility of the argument that Magicicada speciation often involves permanent four year life cycle changes, if such a dormancy period could be induced or deleted by few genetic changes. However, identifying plausible evolutionary mechanisms for such life cycle evolution is a challenge because Magicicada populations apparently must number many thousands per hectare to avoid being destroyed by avian predators (Marlatt 1923; Beamer 1928; Alexander and Moore 1962; Dybas 1969; Williams et al. 1993). In considering possible scenarios for the origin of M. neotredecim, we have developed two models of life cycle evolution that may have general relevance for Magicicada speciation, “nurse brood facilitation” and “canalization of climate-induced plasticity.” These models are not exclusive alternatives; rather, the models are intended to convey different ways in which the unique life-history attributes of Magicicada could be involved in species formation.
174 “Nurse brood” facilitation and life cycle changes: Recent confirmation of microgeographic overlap between 13- and 17-year broods in the Midwest (Alexander et al. unpublished data; Lloyd et al. 1983), along with repeated observations of straggler cicadas, suggests a mechanism in which speciation is initiated by temporal migration of life cycle mutants from one brood to an overlapping brood of a different life cycle (Fig. 6.12). For example, if mutations affecting life cycle length sometimes cause 17-year cicadas to emerge with 13-year life cycles, such mutants could escape predation and establish an incipient species (1) if they emerged in the life cycle overlap zone during an emergence of the sympatric 13-year brood and (2) if the sympatric 13-year brood contained only dissimilar 13-year species in the area of overlap. Absence of a confusingly similar species in the 13- year brood would probably be necessary to avoid loss of the temporal immigrants to interspecific hybridization. The existing brood functions as a “nurse brood,” providing predator protection for rare temporal mutants (Alexander amd Moore 1962); this process has also been termed “induction” of one species into a brood lacking that species (Lloyd and Dybas 1966b). One potential difficulty with this hypothesis is that multiple life cycle mutants must appear in the same generation and location, but this problem may be mitigated by the large size of periodical cicada populations, which can reach 3.7 million per hectare (Dybas 1969), and by the possibility that minor genetic changes may influence life cycle length (see above).
Nurse brood facilitation of life cycle mutants should lead to the formation of a new incipient species whenever a given species in one brood does not already have a counterpart in an overlapping brood of a different life cycle; this suggests an explanation for the surprising tendency of Magicicada to repeatedly evolve the same life cycles (Alexander and Moore 1962). In addition, reinforcement of premating isolation among sympatric species of the same life cycle could interact with the nurse brood mechanism to produce new species pairs. For example, reinforcement in M. neotredecim has incidentally caused some populations of this species to differ from M. septendecim in dominant chorus pitch by at
175 least 300 Hz. If future 17-year life cycle mutants from song-displaced M. neotredecim were to co-emerge with 17-year cicadas in the life cycle overlap zone (such as where 17- year Brood X adjoins 13- year Brood XIX), these preexisting song pitch differences might facilitate assortative mating and establishment of a new 17-year species (Fig. 6.13). The general processes of life cycle change, nurse brood facilitation, and reinforcement could explain patterns in Magicicada speciation including (1) pairs of cognate species differing primarily in life cycle length and (2) initial synchronization of different species of the same life cycle.
The hypothesis of nurse brood facilitation has the potential to explain the origin of M. neotredecim from M. septendecim and its synchronization with the 13-year Brood XIX (Fig. 6.14). However, for this hypothesis to be correct, a large region of Midwestern Brood XIX must have contained only M. tredecassini and/or M. tredecula prior to the arrival of M. neotredecim; otherwise most Midwestern M. neotredecim today would have displaced (high-pitched) calling songs from past ecological interaction with M. tredecim. Such a large region of prior range discordance between the 13-year species seems unlikely because their distributions today are coincident throughout the rest of the 13-year range; however such discordance does exist in 17-year cicadas in Oklahoma where the 17-year brood (IV) contains only M. cassini (Dybas and Lloyd 1974). This hypothesis for the origin of M. neotredecim predicts that Midwestern populations of M. tredecassini and/or M. tredecula, which have not yet been adequately examined, are not more closely related to their 17-year counterparts than to southeastern populations.
Canalization of climate-induced life cycle shift: The nurse brood mechanism can explain only instances of life cycle evolution that occurred after broods of both life cycles were already present. Unless 13- and 17-year life cycles appeared prior to the evolution of periodicity and the need for predator satiation, additional mechanisms must be considered. Without the nurse brood mechanism, life cycle change seems unlikely to be initiated by
176 mutation because life cycle mutants are unlikely to appear simultaneously in numbers sufficient to satiate predators. We suggest a second model of Magicicada life cycle evolution based on the assumption (discussed above) that unusual climatic stimuli can trigger the expression of life cycle plasticity.
Developmental plasticity could facilitate life cycle evolution and speciation in Magicicada if extreme climatic conditions sometimes induce periodical cicadas to switch life cycle in numbers sufficient to satiate predators and if the conditions persist for generations. Persistence of such extreme conditions would promote continued expression of the new cycle, leading to selection that would (1) remove genes tending to cause expression of the old phenotype and (2) favor alleles canalizing the new phenotype. Furthermore, if the climate gradually returned to the initial conditions, canalizing selection could lead to the evolution of cicadas that expressed the new life cycle even under the original conditions (Fig. 6.15; Waddington 1953). The process would succeed only if the climate change was substantial, sudden, and persistent. A sudden shift of lesser magnitude could create overlapping broods or lead to local extinction if no populations remained large enough to satiate predators. Gradual climate change could lead to canalization of the original cycle and reduce the probability of a life cycle switch. A sudden change followed by an equally sudden return to the original conditions would lead only to the formation of a temporally- isolated brood of the original life cycle. A general treatment of the role of phenotypic plasticity in facilitating speciation can be found in West-Eberhard (1989).
If different Magicicada species possess similar life cycle plasticity, a climate shift causing a change in the life cycle of one species will likely change sympatric species in synchrony. In this manner, simultaneous, parallel speciation events could occur in Magicicada (Figs. 6.13, 6.14). The hypothesis that different Magicicada species share similar developmental plasticity is supported by the observation that both species (M.
177 septendecim and M. cassini) present in Brood XIII near Chicago produced millions of four-year premature stragglers in the same year, 1969 (Lloyd 1984).
If M. neotredecim was formed from populations of M. septendecim by canalization of a climate-induced life cycle, we might expect to find that the 13-year M. -cassini and M. -decula sympatric with M. neotredecim are also more closely related to their 17-year counterparts, because their ancestors would have been subjected to the same climate fluctuations. There should also be paleoclimatological or other data consistent with recent, dramatic climate change in Missouri and Illinois. The M. neotredecim / M. tredecim overlap zone is concordant with the hypothesized southern boundary of the Prairie Peninsula (Cox and Carlton 1991); perhaps prior to the climate change the border between 13- and 17-year cicadas was located there, in which case prior discordance between the 13- year species’ ranges is not necessary to explain the undisplaced calling song of northern M. neotredecim. One weakness of the canalization hypothesis is that it offers no explanation other than chance for the synchronization of the new Midwestern 13-year cicadas with the remainder of Brood XIX. Simon et al. (submitted) propose a related hypothesis in which successive life cycle changes occur, each one farther north, and each facilitated by the existence of the adjacent Brood XIX by a mechanism similar to the nurse brood process discussed above.
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183 Table 6.1. Traits distinguishing Magicicada neotredecim and other Magicicada species. "Pronotal extension" is the lateral extension of the pronotum behind the eye. For additional description and color photographs see Alexander and Moore (1962)
Species Life Cycle Abdominal sternite color Dominant call Pronotal Length of call (years) (each) pitch (kHz) extension color (seconds)
M. neotredecim Marshall and Cooley (new 13 orange with black lateral band 1.35 - 1.90 § species) or center orange 1.5 - 4 184 M. tredecim (Walsh and Riley) 13 mostly orange 1.00 - 1.25 orange 1.5 - 4 §
17 orange with black lateral band 1.25 - 1.50 § M. septendecim (L.) or center orange 1.5 - 4
†† 17 black, rarely with weak > 3.00 §§ M. cassini (Fisher) orange lateral band black 2 - 6
†† 13 black, rarely with weak > 3.00 §§ M. tredecassini Alexander and Moore orange lateral band black 2 - 6
M. septendecula Alexander and Moore 17 black with orange lateral band > 3.00 black 7 - 14 †
M. tredecula Alexander and Moore 13 black with orange lateral band > 3.00 black 7 - 14 †
§ Roughly pure-tone, musical buzz terminating in a noticeable drop in pitch; no ticks. Usually 2-3 calls between flights. §§ Rapid series of ticks followed by high-pitched, broad-spectrum buzz that rises and then falls in intensity and pitch. Usually 1-2 calls between flights. † Repeated, rhythmic high-pitched, broad-spectrum tick-buzz phrases, followed by repeated phrases containing only ticks. Usually 1 call between flights. †† Orange band, if present, often interrupted medially. Table 6.2. Relationship of abdomen color to male call pitch and female pitch preference in M. -decim at the Sharp Co. AR “powerline” site. The male and female M. -decim samples were each divided on the basis of calling song pitch and pitch preference (at 1.5 kHz). The abdomen colors of these groups are significantly different.
Males Average male dominant call Abdomen Call pitch in kHz color score Species Category n (mean ± SD) (mean ± SD) designation < 1.5 kHz 27 1.11 (±0.07) 3.67 (±0.55) M. tredecim > 1.5 kHz 124 1.70 (±0.07) 2.00 (±0.44) M. neotredecim Abdomen color differs significantly in each group (Mann-Whitney U= 3348.0; P< 0.001)
Females Average female call pitch Abdomen Preference preference in kHz color score Species Category n (mean ± SD) (mean ± SD) designation < 1.5 kHz 13 1.22 (±0.11) 3.23 (±0.83) M. tredecim > 1.5 kHz 35 1.75 (±0.14) 2.20 (±0.72) M. neotredecim Abdomen color differs significantly in each group (Mann-Whitney U= 366.5; P< 0.001)
185 Table 6.3. Dominant frequencies of Magicicada septendecim chorus recordings in the UMMZ sound library.
Brood State Year Frequency I VA 1995 1.41 II CT 1962 1.39 II VA 1996 1.40 III IL 1997 1.35 IV KS 1998 1.34 VIII PA 1976 1.32 XIII IL 1973 1.30-1.31 XIV OH 1957 1.29-1.32
186 M. septendecim (17) - abdomen black and orange - call pitch 1.25-1.50 kHz - mtDNA lineage A
M. cassini (17) M. septendecula (17)
M. neotredecim (13) M. tredecim (13) - abdomen black and orange - abdomen mostly orange - call pitch 1.25-1.90 kHz - call pitch 1.00-1.25 kHz - mtDNA lineage A - mtDNA lineage B
M. tredecassini (13) M. tredecula (13)
Figure 6.1. Distribution of the seven periodical cicada (Magicicada) species, summarized from county-level maps in Simon (1988) and from 1993-1998 field surveys. The 17-year species are sympatric except in peripheral populations: M. cassini alone inhabits Oklahoma and Texas, while only M. septendecim is found in some northern populations (Dybas and Lloyd 1974). Two 13-year species (M. tredecassini and M. tredecula) are sympatric across the entire 13-year range, while the remaining 13-year species, M. tredecim and the new species M. neotredecim, overlap only in the central US. County-level maps overestimate distribution limits, hence the overlap between the 13- and 17-year populations is probably exaggerated. We have found only limited 13/17 overlap during recent surveys in Illinois (1997-1998 emergences of Broods III and XIX, respectively) and have adjusted this map accordingly. The overlap of M. tredecim and M. neotredecim is plotted from recent field surveys (this paper, Simon et al. submitted). Characters distinguishing M. Ðdecim siblings are noted; the M. -cassini and M. -decula siblings are distinguishable only by life cycle. "Call pitch" is dominant pitch of male call phrase; mtDNA lineage refers to types described in Martin and Simon (1990).
187 M. septendecim M. cassini + M. septendecula A.
Power kJ/Hz
1 2 3 4 5 6 7 Pitch (kHz)
B. M. tredecim M. tredecassini + M. tredecula
M. neotredecim
Power kJ/Hz
1 2 3 4 5 6 7 8 Pitch (kHz)
Figure 6.2. Power spectra of typical 17- year chorus containing M. septendecim, M. cassini, and M. septendecula (A); and 13- year chorus containing M. tredecim, M. neotredecim, M. tredecassini, and M. tredecula (B). Note that M. -cassini and M. -decula frequency characteristics differ little in the two choruses, but that the presence of two M. -decim species in the 13- year chorus is readily apparent. 17 - year chorus recording S. of Fandon, McDonough Co., IL, 7 June 1980 (Brood III); 13- year chorus along US 51 near county line in southern Jackson Co., IL 1 June 1976 (Brood XXIII).
188 2.5
2.0
M. neotredecim Pitch 1.5 (kHz) 189 1.0 M. tredecim
0.5
0.0 2.0 4.0 6.0 Time (s)
Figure 6.3. Spectrogram (power spectrum vs. time) showing a two-banded, mixed- species chorus of male calls with one call of each species standing out against the background chorus. Individual calls end with a downslur. Comparatively faint slurs of background chorus males overlap and are not visible. Intervening time between calls has been removed. 1.8
1.7 M. neotredecim
1.6
1.5
1.4 190
1.3 Chorus pitch (kHz)
1.2 M. tredecim 1.1
1 71.1 79.9 81 81.5 81.5 81.7 82 83 83.3 84.9 85.5 87.1 87.4 Air temperature, ¡F
Fig. 6.4. Dominant pitch of a mixed M. neotredecim/M. tredecim as a function of air temperature. No effect of temperature is evident. 191
2 Pitch (kHz) 1
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (seconds)
Figure 6.5. Sonogram of 30-second recording of an individual male M. neotredecim in Sharp County, AR, illustrating consistency of individual male calling song pitch.
0.6 600
0.5 500
0.4 400
0.3 300 Proportion 192
0.2 200 Power (nJ/Hz)
0.1 100
0 0 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 Pitch (kHz)
Figure 6.6. Power spectrum (shaded area) of mixed M. neotredecim and M. tredecim chorus at "powerline" study site, Sharp Co., AR, with accompanying frequency histograms of male song pitches (black bars) and female pitch preferences (white bars) of individuals collected randomly at the site. M. tredecim (low song pitch) constitute only 8% of the population. IL 100 km
MD
MO
KY VA 193
TN NC AR
N SC
AL GA
Figure 6.7. Proportions of M. neotredecim (black) and M. tredecim (white) estimated from chorus recordings of the 1998 emergence of Magicicada 13-year Brood XIX. IL
MO N
AR
Dominant pitch of chorus (kHz) 1.68 - 1.77 1.58 - 1.67 1.48 - 1.57 1.38 - 1.47 100 km
Figure 6.8. Geographic variation in dominant chorus pitch of M. neotredecim, showing reproductive character displacement (higher pitch songs) in and near the region of overlap with the low-pitched M. tredecim. Lighter shaded circles indicate higher pitched songs. Shaded region is approximate M. tredecim range. Weak choruses not plotted.
194
Song pitch (kHz)
1.8
1.7
1.6
1.5
1.4 M. neotredecim
1.3
1.2
M. tredecim 1.1
1.0
N S
Figure 6.9. Schematic representation of calling song displacement in Brood XIX M. tredecim and M. neotredecim. Light portions of pie graphs represent proportion of M. tredecim present in choruses; dark portions represent proportion of M. neotredecim. In the north, choruses contain only M. neotredecim, while in the south, choruses contain only M. tredecim. Song pitch is displaced in the relatively narrow zone of overlap betweeen these species.
195 N IL 100 km MD
MO
KY VA 196
TN NC
AR
Dominant pitch of chorus (kHz) SC 1.07 - 1.09
AL GA 1.10 - 1.11 1.12 - 1.14 1.15 - 1.17
Figure 6.10. Geographic variation in dominant chorus pitch of M. tredecim, suggesting weak reproductive character displacement. Lighter shaded circles indicate more displaced (lower pitched) songs. Shaded area is approximate M. neotredecim range. Note that range of variation is only 1/4th of that shown in Fig. 6.7. Weak choruses not plotted. A. B. 1.9 40 1.8 * 1.7 1.6 * 35 1.5 * * 1.4 *
1.3 Right wing length (mm) Call pitch (kHz) * 30 1.2 *
1.1 * 1.0 0.9 25 Arkansas Illinois Arkansas Illinois
M. tredecim M. neotredecim M. tredecim M. neotredecim
C. D. 12 8.0
7.5 11
7.0 * Thorax width (mm)
10 First sternite width (mm) 6.5 * * 9 6.0 Arkansas Illinois Arkansas Illinois
M. tredecim M. neotredecim M. tredecim M. neotredecim
Figure 6.11. Box plots of male calling song pitch (A), right wing length (B), thorax width (C), and first sternite width (D) of M. tredecim (Sharp Co., AR; n = 26) and M. neotredecim (Sharp Co., AR, n = 61; and Piatt Co., IL, n = 17). Asterisks indicate outliers; open circles indicate extreme outliers. Song pitch of M. neotredecim is significantly different in Arkansas (sympatry with M. tredecim) and Illinois (allopatry with M. tredecim; P < 0.001, Mann-Whitney-U), indicating reproductive character displacement, while size measurements show no significant differences within M. neotredecim. M. tredecim is different from M. neotredecim in all size traits (for each, P < 0.001; Mann-Whitney-U).
197
* Life cycle North South overlap zone
17 A 17 B 17 C
13 A' 13 A
198 13 B 13 C
Figure 6.12. Formation of an incipient Magicicada species (13-year A') by "nurse brood facilitation" of life cycle mutants. Three 17-year species each have a similar 13-year counterpart, but the 13-year counterpart of species 17 A is not present where the life cycle types overlap geographically. In this situation 13-year life cycle mutants from 17 A (curved arrow) can establish a new species (13 A') in the overlap zone if they emerge synchronously with the 13-year "nurse" brood (13 B + 13 C). Success of life cycle mutants from 17 A is facilitated because 13 B and 13 C provide the rare mutants with numerical protection from predators. Rare life cycle mutants from 17 B and 17 C are likely to be lost to interspecific hybridization with the similar and abundant 13 B and 13 C. Degree of prior divergence of 13 A and the parent species 17 A will influence the evolutionary outcome (mixture, hybrid zone, or reinforcement) of later contact between 13 A' and 13 A. I II III IV
17 13 17 13 17 13 17 13