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The adaptive function of male genital spines in the fruit fly Drosophila ananassae [Doleschall] (Diptera: Drosophilidae)
A thesis submitted to the
Division of Research and Advanced Studies of the University of Cincinnati
In partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
In the Department of Biological Sciences of the McMicken College of Arts and Sciences
9 July 2012
by
Karl Grieshop
B.S., University of Cincinnati June 2009
Committee Chair: Dr. Michal Polak
1 ABSTRACT
Chapter 1: That male genital morphology evolves via postcopulatory sexual selection is a widely held view. In contrast, the precopulatory sexual selection hypothesis for genital evolution has received less attention. Here, we test the hypothesis that male genital spines of Drosophila ananassae promote competitive male copulation success. Using laser surgery to manipulate trait size, we demonstrate that incremental reductions of spine length progressively reduce male copulation success: males without spines failed entirely to copulate because of an inability to couple the genitalia together, whereas males with halfway ablated and blunted spines suffered reductions in copulation success of 87% and 13%, respectively. The decrease in copulation success resulting from spine length reduction was markedly stronger in sexually competitive environments than in non-competitive environments, and females expressed resistance behaviors similarly toward competing male treatments, demonstrating directly the role of genital spines in promoting competitive copulation success. Because these spines are widespread within Drosophila, and because genital traits with precopulatory function are being discovered in a growing number of animal taxa, precopulatory sexual selection may have a more pervasive role in genital evolution than previously recognized.
Chapter 2: The contemporary explanation for the rapid evolutionary diversification of animal genitalia is that such traits evolve via postcopulatory sexual selection. The most common debate within this framework has been over the relative importance of three non-mutually exclusive evolutionary mechanisms: sperm competition, cryptic female choice, and sexual conflict. The first two of these are strictly postcopulatory mechanisms, whereas sexual conflict could operate before, during or after copulation. We investigate the potential for male genital spines in Drosophila ananassae to function in postcopulatory sexual selection. Whereas previous work on two Drosophila species shows that these spines function in precopulatory sexual selection to promote male competitive copulation success, the postcopulatory function(s) of Drosophila genital spines have not yet been thoroughly investigated. Using a precision micron-scale laser surgery technique we test the effect of spine length reduction on male competitive fertilization success, female remating behavior, fecundity, and copulation duration. We find no evidence that male genital spines in this species have a postcopulatory adaptive function. However, partial genital spine ablation had an unexpected positive effect on the probability that males would fertilize at least one gamete of a previously mated female. This effect is discussed in terms of the possibility that Drosophila genital spines are harmful to females as a pleiotropic side effect of evolving to promote competitive male copulation success.
2 COPYRIGHT NOTICE
Regarding Chapter 1, published in Evolution (The International Journal of Organic Evolution):
AUTHORS - If you wish to reuse your own article (or an amended version of it) in a new publication of which you are the author, editor or co-editor, prior permission is not required (with the usual acknowledgements). However, a formal grant of license can be downloaded free of charge from Rightslink if required. http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1558-5646/homepage/Permissions.html
3 GENERAL INTRODUCTION
Evolutionary biologist are interested in traits that vary across closely related taxa, because this indicates that such traits have undergone some type of rapid divergent evolution relative to the traits that do not vary across those taxa. The most famous example of this is the disparate beak morphologies exhibited by finches of the Galapagos Islands, which aided Charles
Darwin’s formulation of the modern theory of evolution (Darwin, 1859). Darwin identified these various beak morphologies as being adaptive within particular evolutionary lineages because their distinct, specialized functions (e.g. foraging for large seeds versus small seeds) allowed individuals bearing the optimal foraging hardware to out-compete those with more generalized morphological equipment in the life or death competition for survival. He termed this process by which nature provides individuals of a population with differential fitness (defined as survival and reproduction) natural selection, where individuals with relatively greater fitness are better able to pass on the heritable components of their success to their offspring, which in turn share such success. The result, over evolutionary time, is the formation of distinct species that occupy different ecological niches.
The most variable type of morphological trait exhibited across even closely related species of the animal kingdom is that of male genitalia (Eberhard, 1985). However, contrary to the beak morphologies of Darwin’s finches that have evolved to exploit specialized niches, male genital traits and their functions are variable across closely related taxa that occupy the same or similar niches. The inference here is that natural selection would not operate on such closely related, similar species in such a variety of ways, whereas sexual selection—a type of natural selection concerned specifically with differential reproductive fitness of individuals (Darwin,
1871)—could account for such a pattern of evolutionary diversification under similar naturally
4 selective pressure(s) (Lloyd, 1979; Eberhard, 1985). Darwin (1871) reserved sexual selection for explaining only the bizarre and elaborate secondary sexual traits of animals (e.g. the peacock’s tail), and evolutionary biologists have only recently begun implicating sexual selection in the evolution of primary sexual traits, such as male genital morphology (Eberhard, 1985; Shapiro &
Porter, 1989; Hosken & Stockley, 2004; Leonard & Córdoba-Aguilar, 2010).
Since this latest paradigm shift in the understanding of genital trait evolution, most studies of genital traits have focused on a particular type of sexual selection that ensues after the onset of copulation—postcopulatory sexual selection. This is understandable considering most genital traits do not contact the opposite sex until copulation has begun. But lately a growing body of evidence for genital traits functioning prior to copulation has emerged, despite its counterintuitive plausibility (Bertin & Fairbairn, 2005; Langerhans et al., 2005; Kahn et al.,
2010; Polak & Rashed, 2010; Miller, 2010).
This thesis investigates the adaptive function of a specific genital trait, male genital spines, of the fruit fly Drosophila ananassae. Though insects offer abundant opportunity to study the adaptive function of animal genitalia because they are easily maintained and manipulated in a laboratory, and typically yield high sample sizes, most insect genitalia are microscopic in size—a practical research impediment to the manipulative experimentation that reveals the causal bases, or selective pressures, responsible for the evolution of these traits. To address this dilemma, a novel micron-scale laser ablation technique is used to perform precision surgeries, generating discrete experimental treatments that can be used to answer specific questions regarding the function of male genital spines in this species. The experiments described in the following two chapters contribute to an underrepresented area of the literature: the role of precopulatory sexual selection in the evolution of genital trait form and function.
5 REFERENCES
Darwin, C. 1859. On the origin of species by means of natural selection. London, UK: John
Murray.
Darwin, C. 1871. The descent of man, and selection in relation to sex. London, UK: John
Murray.
Eberhard, W. G. 1985. Sexual selection and animal genitalia. Cambridge, MA: Harvard
University Press.
Hosken, D. J. & Stockley, P. 2004. Sexual selection and genital evolution. Trends Ecol. Evol.
19:87-93.
Kahn, A. T., Mautz, B., & Jennions, M. D. 2010. Females prefer to associate with males with
longer intromittent organs in mosquitofish. Biol. Letters. 6:55-58.
Langerhans, R. B., Layman, C. A., & DeWitt, T. J. 2005. Mal genital size reflects a tradeoff
between attracting mates and avoiding predators in two live-bearing fish species. Proc.
Natl. Acad. Sci. USA. 102:7618-7623.
Leonard, J. L. & Córdoba-Aguilar, A. (eds) 2010. The evolution of primary sexual characters in
animals. New York, NY: Oxford University Press.
Lloyd, J. E. 1979. Mating behavior and natural selection. Fla. Entomol. 62: 17-34.
Miller, E. H. 2010. Genitalic traits of Mammals. In: The evolution of primary sexual characters
in animals (J. L. Leonard, A. Córdoba-Aguilar, eds), pp. 471-493, New York, NY:
Oxford University Press.
Polak, M. & Rashed, A. 2010. Microscale laser surgery reveals adaptive function of male
intromittent genitalia. Proc. R. Soc. Lond. B. Biol. Sci. 277:1371-1376.
Shapiro, A. M. & Porter, A. H. 1989. The lock-and-key hypothesis: evolutionary and
biosystematic interpretation of insect genitalia. Annu. Rev. Entomol. 34:231-245.
6 ACKNOWLEDGEMENTS
The completion of this study would not have been possible without the assistance of several people, to whom I am incredibly grateful. First and foremost, I am deeply appreciative toward my undergraduate and graduate research advisor, Dr. Michal Polak, who provided the initial idea for this project, invaluable research guidance/expertise, and constructive criticism and advice that will echo in my mind for years to come—undoubtedly enhancing my career trajectory and future success. Secondly, I would like to thank the remainder of my research advisory committee, Dr. George Uetz and Dr. John Layne, for their contribution to my development as a scientist and a person throughout my degree program. Lastly, there are several other people to whom I must express my appreciation, whether having helped with experimental procedures, intellectual support, or both; those are (in no particular order) Dr. Arash Rashed, Dr. Necati
Kaval, Dr. Jorge Hurtado-Gonzalez, M.S. Brooke Hamilton, M.S. Beth Cortright, B.S. Liz
Brown, B.S. Alexandra Warner, B.S. Scott Licardi, Roger Ruff, a host of my peers and elders from the Department of Biological Sciences at the University of Cincinnati, my friends, and family—thank you all. This study was supported by the University of Cincinnati, the McMicken
College of Arts and Sciences, the Department of Biological Sciences, and the National Science
Foundation.
7 CONTENTS
List of Figures: ...... 9
List of Tables: ...... 10
Chapter 1: 11
Abstract: ...... 12
Introduction: ...... 13
Methods: ...... 15
Results: ...... 23
Discussion: ...... 26
References: ...... 30
Tables: ...... 34
Figure captions: ...... 36
Figures: ...... 38
Chapter 2: 45
Abstract: ...... 46
Introduction: ...... 47
Methods: ...... 51
Results: ...... 58
Discussion: ...... 60
References: ...... 66
Tables: ...... 72
Figure captions: ...... 74
Figures: ...... 75
General Conclusions: 76
8 LIST OF FIGURES
Chapter 1
1 Scanning electron micrograph of male genital spines ...... 38
2 Profile images of two species of male genital spines (pulled free) ...... 39
3 Scanning electron micrograph of laser ablated male genital spine ...... 40
4 Probability of copulation across several surgical treatments ...... 41
5 Probability of copulation for tips-cut and control males in two social contexts ...... 42
6 Probability of copulation for partial-cut and control males in two social contexts . . . . . 43
7 Histogram of copulation attempts for two treatments in two social contexts ...... 44
Chapter 2
1 Probability of P2 being > 0 for cut and control males ...... 75
9 LIST OF TABLES
Chapter 1
1 Copulation latencies and durations for all treatments and experiments ...... 34
2 ANCOVA on copulation latency for Experiment 2 ...... 35
Chapter 2
1 Logistic regression on the probability of P2 being > 0 for Experiment 1 ...... 72
2 ANCOVAs on female fecundity for Experiment 2 ...... 73
10 CHAPTER 1:
An expanded version of the Brief Communication published in: Evolution (The International Journal of Organic Evolution) (doi:10.1111/j.1558-5646.2012.01638.x)
The precopulatory function of male genital spines in Drosophila ananassae
[Doleschall] (Diptera: Drosophilidae) revealed by laser surgery
Karl Grieshop*
and
Michal Polak
Department of Biological Sciences, University of Cincinnati, Cincinnati OH 45221-0006, USA
Running title: The precopulatory function of genital spines
*Author for correspondence:
Karl Grieshop
Department of Biological Sciences
University of Cincinnati
Cincinnati, OH 45221-0006, USA
Tel: +1 (513) 646-2467
Fax: +1 (513) 556-5299
Email: [email protected]
11 1 ABSTRACT
2 That male genital morphology evolves via postcopulatory sexual selection is a widely
3 held view. In contrast, the precopulatory sexual selection hypothesis for genital evolution has
4 received less attention. Here, we test the hypothesis that male genital spines of Drosophila
5 ananassae promote competitive male copulation success. Using laser surgery to manipulate trait
6 size, we demonstrate that incremental reductions of spine length progressively reduce male
7 copulation success: males without spines failed entirely to copulate because of an inability to
8 couple the genitalia together, whereas males with halfway ablated and blunted spines suffered
9 reductions in copulation success of 87% and 13%, respectively. The decrease in copulation
10 success resulting from spine length reduction was markedly stronger in sexually competitive
11 environments than in non-competitive environments, and females expressed resistance behaviors
12 similarly toward competing male treatments, demonstrating directly the role of genital spines in
13 promoting competitive copulation success. Because these spines are widespread within
14 Drosophila, and because genital traits with precopulatory function are being discovered in a
15 growing number of animal taxa, precopulatory sexual selection may have a more pervasive role
16 in genital evolution than previously recognized.
17
18 Key words: Adaptive function, animal genitalia, copulation success, functional morphology,
19 laser ablation, precopulatory sexual selection
20
12 20 INTRODUCTION
21 The leading hypothesis to explain the remarkable diversification of male genital traits is
22 that such complexity evolves in response to sexual selection (Eberhard, 1985; Hosken &
23 Stockley, 2004; Leonard & Córdoba-Aguilar, 2010). Specifically, postcopulatory mechanisms of
24 sexual selection, including sperm competition (Parker, 1970; Simmons, 2001), cryptic female
25 choice (Thornhill, 1983; Eberhard, 1985, 1996), and sexual conflict (Parker, 1979; Andersson,
26 1994; Arnqvist & Rowe, 2005), have received the most attention (Hosken & Stockley, 2004).
27 Indeed, the last quarter century has seen considerable growth in the number of studies addressing
28 the postcopulatory function of genitalia (Leonard & Córdoba-Aguilar, 2010). In contrast, fewer
29 studies have focused on the potential for precopulatory sexual selection to drive genital evolution
30 (Bertin & Fairbairn, 2005; Eberhard, 1993, 2010a,b; Arnqvist, 1997; Simmons, 2001; Hosken &
31 Stockley, 2004). Though it is admittedly counterintuitive that genital traits would function prior
32 to copulation, such traits do occur in a variety of animal taxa (e.g. flatworms: Michiels, 1998;
33 insects: Bertin & Fairbairn, 2005; Polak & Rashed, 2010; fishes: Langerhans et al., 2005; Kahn
34 et al., 2010; mammals: see Miller, 2010), and thus they deserve greater empirical and theoretical
35 consideration.
36 An effective way to study the adaptive function of a trait, and test ideas about the causal
37 bases of its evolution, is to employ manipulative experimentation (Sinervo & Basolo, 1996;
38 Arnqvist, 1997; Eberhard, 2011). Although insects offer abundant examples of the rapid
39 divergent evolution of animal genitalia (Eberhard, 1985; Leonard & Córdoba-Aguilar, 2010), the
40 microscopic size of most insect genitalia impedes their phenotypic manipulation. The present
41 study uses a precision laser surgery system, capable of ablating and altering the shape of micron-
42 scale structures with little or no damage to surrounding structures (Polak & Rashed, 2010), to
13 43 study the adaptive function of male genital spines in the cosmopolitan fruit fly Drosophila
44 ananassae (Tobari, 1993). Specifically, we test the hypothesis that genital spines in D.
45 ananassae function to promote competitive male copulation success. Male genital spines in
46 Drosophila are a rapidly evolving and widespread trait within the melanogaster species group
47 (with over 40 species expressing them), ranging from species that do not express them (e.g. D.
48 melanogaster) to those exhibiting one to five pairs of spines (Hsu, 1949; Bock & Wheeler, 1972;
49 McEvey et al., 1987; Schiffer & McEvey, 2006). Polak and Rashed (2010) employed laser
50 ablation in a study of the male genital spines of D. bipectinata, and found support for the
51 hypothesis that genital spines promote competitive male copulation success. In the wild,
52 members of both sexes of these species aggregate on fermenting fruit, where males chase, court
53 and attempt to copulate with females that come to the fruit to feed, mate and oviposit. Typically,
54 there are many males and females at these sites, where there is a premium on males to locate and
55 mate with receptive females before they are usurped by rival males; the mating system of these
56 flies is best described as scramble competition (Thornhill & Alcock, 1983).
57 In D. ananassae, the genital spines are a single pair of hard, sclerotized, claw-like
58 structures that are external at rest, extending from the ventral cercal lobe (or secondary claspers)
59 (Figure 1). These spines move independently of the aedeagus and other genital structures, and
60 insert into the female’s external genitalia (not the gonopore) during copulation. Although similar
61 in appearance, the spines of D. ananassae are 21% longer (controlling for body size variation)
62 than in D. bipectinata (Grieshop and Polak, unpublished data—reported below). Thus, not only
63 was it our intent to examine the function of the spines in D. ananassae in their own right, and
64 hence to begin to assess the generality of the findings concerning Drosophila genital spine
14 65 function reported in Polak and Rashed (2010), but also to compare their relative functional
66 importance for copulation between these two species.
67 Experiment 1 of the present laboratory study investigates the effect of incremental
68 reductions in spine length on male copulation success in a non-sexually competitive context.
69 Experiment 2 investigates the effect of spine length reduction on male copulation success in two
70 social contexts—non-competitive and competitive—simultaneously. This experiment
71 specifically tests the prediction that the negative consequence of spine reduction on male
72 copulation success should be more pronounced in a competitive environment than in a non-
73 competitive environment. Experiment 3 repeats the test for the effect of spine length reduction
74 on copulation success in these two social environments, but does so in smaller arenas to facilitate
75 assessment of the potential role of female behavior in driving differential male copulation
76 success between the surgical treatments.
77
78 MATERIALS AND METHODS
79 Experimental flies
80 The base population of Drosophila ananassae [Doleschall] (Diptera: Drosophilidae) was
81 initiated with 100 inseminated females collected in February 2009 on the South Pacific island of
82 Moorea (17°32'58.78''S, 149°52'59.29''W), Society Islands. Flies were mass cultured in the
83 laboratory on a 12:12 h L:D photoperiod and a 24°C (L): 22°C (D) temperature regime in 240 ml
84 glass milk bottles (N = 6) with 6 g of Formula 4-24 Instant Drosophila Medium (Carolina Supply
85 Co., Burlington, NC), 20 ml water, 8 ml of banana/live yeast slurry (50 ml water : 25 g banana :
86 1.5 g live yeast), and autoclaved tissue paper. The base population was acclimated to these
87 laboratory conditions for 7 generations over 4 months before use in the experiments.
15 88 Virgin males and females were collected from the base population simultaneously within
89 4 h of eclosion, maintained separately as virgins in 35 ml disposable polystyrene shell vials lined
90 with cornmeal-agar food medium (N~25 per vial), and allowed to age for 6 days until use in a
91 given experiment. Experimental flies were transferred to fresh food vials every other day until
92 experimentation, and live yeast was added to vials containing females.
93 After each block of all experiments, males were preserved in 95% ethanol and later
94 examined under an Olympus SZX12 stereomicroscope to verify treatment identity and the
95 integrity of the surgical manipulation (without knowledge of copulatory status). Male thorax
96 length, an estimate of body size (Robertson & Reeve, 1952), was measured (in rehydrated
97 specimens) from the tip of the scutellum to the anterior edge of the thorax using an ocular
98 micrometer; independent repeated measurements of thorax lengths in a random sample of 10
99 males were highly repeatable among males (1-way ANOVA: F9,10 = 516.7, P < 0.0001). In
100 Experiment 3, observation chambers were cleaned with water, and cover-slips and filter paper
101 were replaced between blocks.
102
103 Preliminary interspecific comparison of trait size
104 Male D. ananassae (N = 5) were randomly selected from the base population, and male
105 D. bipectinata (N = 5) were randomly selected from the base population used in Polak and
106 Rashed (2010). Males were briefly preserved in 95% ethanol, their thorax lengths were measured
107 as described above, and their genital spines were pulled free of the male with fine forceps under
108 a dissecting microscope, and placed flat onto transparent two-sided taped adhered to a glass
109 microscope slide. Profile images of the right and left genital spines of each male were taken
110 using a Hitachi KP-F100 digital camera mounted on an Olympus BX60 light microscope (Figure
16 111 2a,b); the length (mm) of each genital spine was measured using ImageJ (rsbweb.nih.gov/ij)
112 (Figure 2a’,b’), and averaged between right and left values. All images were captured at the same
113 magnification. Spine length between species was analyzed using AN(C)OVA, with and without
114 male thorax length as the covariate.
115
116 Laser manipulation
117 Virgin males were laser-treated within 22 h (± 2 h) of eclosion using the protocol
118 described in Polak and Rashed (2010) (Figure 3). Briefly, males were placed one at a time in a
119 Plexiglas surgical chamber while lightly anesthetized with humidified CO2. Pulsed laser light
120 was used to administer precision cuts to genital spines with little or no collateral damage to
121 surrounding structures or bristles (Figure 3). Experimental males had their spine lengths
122 surgically reduced in a bilaterally symmetrical fashion, producing the surgical treatments as
123 follows: full-cut, spines excised at the base; half-cut, half of spines excised; partial-cut,
124 approximately one third of spines excised; and tips-cut, tips of spines excised (blunted). The
125 control treatments were: surgical control, 2 bristles on the seventh sternite of the ventral
126 abdomen excised at the base; and sham control, subject to the same conditions as all other
127 treatments but not actually contacted with the laser light (laser pulse shot just next to the
128 specimen). Laser surgeries for all treatments, including both controls, took approximately the
129 same amount of time to perform (< 1 minute per individual). Following surgery, experimental
130 males were held separately (and without females) in food vials (N~20 per vial) until
131 experimentation. Across the 3 experiments described below (involving over 700 individual
132 surgeries), a negligible number (< 1%) of experimental males died prior to experimentation: 2
17 133 half-cut males (Exp. 1), 2 tips-cut males (Exp. 2), 1 partial-cut male and 1 surgical control male
134 (Exp. 3).
135
136 Experiment 1
137 To investigate the effect of spine length reduction on copulation success in a non-
138 competitive context, observation vials lined with cornmeal-agar were placed in a row along a
139 table. A female was aspirated into each of the vials at 8:00 pm, and the experiment commenced
140 the following morning at 8:00 am (23°C) when males were individually aspirated into the vials.
141 Cut and control males were interspersed among the row of vials such that the following sequence
142 repeated itself five times to constitute the 25 vials of each block: full-cut, half-cut, tips-cut,
143 surgical control, and sham control. The time at which each male was introduced into a vial with a
144 female was recorded, and observers continually scanned the vials in successive order for 2 h, or
145 until a copulation occurred. The start and stop times of all copulations were recorded. Copulation
146 latency refers to the amount of time (s) elapsed between a male’s introduction and the start time
147 of copulation; and copulation duration refers to the amount of time (s) elapsed between the start
148 and stop times of a copulation. Males that were not actively courting (< 1%) were replaced.
149 Three blocks of this experiment were conducted for a total N = 75 vials.
150 Copulation frequency data were pooled across blocks, as the heterogeneity χ2 was non-
151 significant (P > 0.9) (Zar, 1999). The probability of copulation across treatments was analyzed
152 using a subdivided χ2 approach (Zar, 1999): a χ2 test including all treatments was first
153 conducted, followed by χ2 tests on subsets of the data to assess which treatments differed from
154 each other. Log-transformed copulation latency and duration across treatments were analyzed
155 separately using analysis of covariance (ANCOVA), with block and surgical treatment as factors
18 156 and log-transformed male thorax length as the covariate. In this and the two following
157 experiments, block had non-significant effects on copulation latency and duration (all Ps > 0.05),
158 so it was removed from reported models.
159
160 Experiment 2
161 Here we investigated the effect of spine length reduction on copulation success in two
162 social environments simultaneously. In the non-competitive environment either a tips-cut or a
163 surgical control male was placed individually in a vial with a female (N = 15 vials with a cut
164 male and 15 vials with a control male, per block). In the competitive environment each vial
165 contained a tips-cut male and a surgical control male with one female (N = 15 vials per block).
166 The following sequence of vials repeated itself fifteen times along the desktop to constitute the
167 45 vials of each block: non-competitive (cut), non-competitive (control), and competitive (cut
168 plus control). Three blocks of this experiment were conducted for a total N = 135 vials.
169 Males were aspirated into vials at 8:00 pm, and the experiment commenced the following
170 morning at 8:00 am (23°C) when females were individually aspirated into the vials. The time at
171 which each female was entered into a vial was recorded, and observers continually scanned the
172 vials in successive order for 2 h, or until a copulation occurred. The start times of all copulations
173 were recorded. Treatment identities of males were unknown to observers, so copulating pairs
174 were aspirated out of vials as they formed; thus, while copulation latency was calculated as
175 described in Experiment 1, copulation duration was not calculated here.
176 Fourteen vials were removed from relevant analyses for the following reasons: the
177 copulating pair in a competitive vial separated before retrieval (N = 2); flies were accidentally
178 killed or injured (N = 2 competitive vials); males of the tips-cut treatment were deemed (without
19 179 knowledge of copulatory status) not to have enough spine length removed to constitute the
180 treatment designation of “tips-cut” (N = 7 non-competitive, and 3 competitive vials).
181 Copulation frequency data were pooled across blocks, as the heterogeneity χ2 was non-
182 significant (P > 0.9) (Zar, 1999). χ2 was used to analyze the effect of surgical treatment on
183 copulation frequency separately for the two environment types. Log-transformed copulation
184 latency across treatments was analyzed using ANCOVA, with block, treatment, environment,
185 and treatment X environment interaction as factors, and log-transformed male thorax length as
186 the covariate.
187
188 Experiment 3
189 This experiment was designed to investigate the potential influences of male and female
190 behavior on any effect of surgical treatment and social environment on copulation success. Cut
191 and control males were observed with females in non-competitive and competitive environments
192 simultaneously as in Experiment 2, with three major exceptions: (1) to facilitate behavioral
193 observations, the experiment was conducted in small-cell mating chambers, which consisted of a
194 plexiglass rectangle (75mm X 25mm X 6mm) with a 12.5mm diameter arena, a 2.5mm diameter
195 entrance tunnel in the side, a glass cover-slip ceiling fastened to the top with two-sided tape, a
196 filter-paper floor fastened to the bottom with Scotch tape, and fine mesh plugging the entrance
197 tunnel; (2) partial-cut males were used as the cut treatment in this experiment as opposed to the
198 tips-cut males used in Experiment 2 (see Laser manipulation) to help ensure that treatment
199 effect(s) would be detectable in these different mating arenas; and (3) males in both the non-
200 competitive and competitive chambers were distinguished by treatment with a small dot of
20 201 colored paint on their dorsal thorax, which was randomly assigned to treatments. Three blocks of
202 this experiment (45 chambers per block) were conducted for a total N = 135 chambers.
203 Males were aspirated into the mating chambers at 7:00 am the morning of the
204 experiment, which commenced at 8:00 am (23°C) when females were individually aspirated into
205 the chambers. The time at which each female was entered into a chamber was recorded, and
206 observers continually scanned the chambers in successive order for 2 h, or until a copulation
207 occurred, recording the start and stop times of all copulations, as well as the total number of
208 times each chamber was scanned. Chambers were scanned on average 43 times (median: 54.5,
209 range: 1-116). Male identities with respect to treatment were unknown to the observers.
210 During each scan of a given chamber, any occurrence of the following behaviors was
211 recorded: male lunging, and female kicking, fleeing, decamping, and abdominal bending. Male
212 courtship and female behavior in our laboratory population of D. ananassae is similar to that
213 described by Spieth (1952). Males bend/curl their abdomens underneath themselves, then lunge
214 at a female, thrusting the tip of the abdomen forward in an attempt to bring the genitalia together.
215 During lunges, the male probes the female’s genitalia with his own, and after successfully
216 coupling his genitalia to hers, completes the mounting process by climbing forward onto her
217 abdomen between her wings. Failed copulation attempts involve males lunging only to achieve
218 very brief and passing contact with the female’s genitalia, sometimes lunging multiple times in
219 rapid succession. Female resistance behaviors include bending/curling their abdomens
220 underneath themselves away from the courting male, kicking with their hind limbs, fleeing while
221 grounded, and decamping via flight.
222 Because female behaviors were exhibited infrequently, the frequencies of kicking,
223 fleeing, decamping, and abdominal bending were summed for each female to yield a composite
21 224 frequency of female “resistance behaviors.” Behavioral frequencies, including male lunges and
225 female resistance behaviors, were converted to behavioral rates by dividing them by the total
226 number of scans per chamber. The resultant values closely estimated behaviors per unit time, as
227 the amount of scans per chamber was highly correlated with the total amount of time each
228 chamber was under observation (r2 = 0.96, d.f. = 127, P < 0.0001).
229 Seven chambers were removed from relevant analyses for the following reasons: flies
230 were accidentally killed or injured (N = 3 non-competitive chambers); the copulation began
231 before any behaviors were recorded (N = 2 non-competitive, and 1 competitive chamber); the
232 stop time of the copulation was unknown (N = 1 competitive chamber).
233 Copulation frequency data were pooled across blocks, as the heterogeneity χ2 was non-
234 significant (P > 0.4) (Zar, 1999). χ2 was used to analyze the effect of surgical treatment on the
235 frequency of copulation separately for the two environment types. Likewise, behavioral
236 frequencies and rates were analyzed separately for the two environment types: a two-tailed
237 Wilcoxon signed-rank test was used to analyze differences between treatments, and a Welch’s
238 ANOVA was used when variances between treatments were statistically unequal (Zar, 1999).
239 Log-transformed copulation latency and duration across treatments were analyzed using the
240 ANCOVA model described in Experiment 2. JMP (v. 8, SAS Institute Inc., 2009) statistical
241 software was used throughout. Data archived in the Dryad repository:
242 doi:10.5061/dryad.g0v6h003.
243
22 243 RESULTS
244 Preliminary interspecific comparison of trait size
245 ANCOVA revealed that male D. ananassae have significantly longer genital spines than
246 male D. bipectinata, after accounting for differences in male thorax length (F2,7 = 15.25, P =
247 0.006). Least-squares mean spine length (± 1 s.e.) of male D. ananassae was 0.062 ± 0.0016mm,
248 compared to 0.051 ± 0.0016mm in D. bipectinata. Uncorrected mean spine length (± 1 s.e.) for
249 male D. ananassae was 0.064 ± 0.0012mm, and for D. bipectinata was 0.049 ± 0.0012mm
250 (ANOVA: F1,8 = 63.35, P < 0.0001).
251
252 Experiment 1
253 When males were placed individually with females, there was a highly significant effect
2 254 of treatment on copulation frequency (χ 4 = 60.56, P < 0.0001), attributable to a sharp reduction
255 in copulation success of the full-cut and half-cut treatments. No male D. ananassae with their
256 genital spines fully removed achieved copulation, and only 13% of half-cut males copulated
257 (Figure 4). In contrast, 87% of tips-cut males, and 100% of both control treatments, copulated
2 258 (Figure 4); copulation frequency did not differ among these three treatments (χ 2 = 4.19, P =
259 0.12). ANCOVA revealed no significant differences in copulation latency (F3,40 = 0.03, P = 0.99)
260 or duration (F3,40 = 0.28, P = 0.84) among treatments (Table 1).
261
262 Experiment 2
263 When placed individually with females in the non-competitive social environment, tips-
264 cut males exhibited a probability of copulation not significantly different from that of surgical
2 265 control males (χ 1 = 0.44, P = 0.51), consistent with the results of Experiment 1; however, tips-
23 266 cut males did suffer a significantly reduced probability of copulation in the competitive
267 environment when competing directly against surgical control males for access to individual
2 268 females (χ 1 = 5.63, P = 0.02) (Figure 5). Thus, whereas blunted genital spines did not reduce a
269 male’s probability of copulation in the non-competitive context, this subtle manipulation did
270 significantly reduce a male’s probability of copulation when there was another male present to
271 usurp the female, indicating social context interacts with spine manipulation to affect male
272 copulation success.
273 ANCOVA revealed that tips-cut males exhibited a significantly greater latency to
274 copulation than surgical control males (Table 2). That is, when control males gained copulations,
275 they did so, on average, significantly sooner than tipless males (Table 1).
276
277 Experiment 3
278 When assayed in small-cell mating chambers, partial-cut males exhibited a significantly
2 279 lower probability of copulation than surgical control males in both non-competitive (χ 1 = 15.75,
2 280 P < 0.0001) and competitive environments (χ 1 = 35.87, P < 0.0001) (Figure 6). In the absence
281 of sexual rivals, partial-cut males were 61% less likely to copulate than controls, but when the
282 two treatments competed directly for the same female partial-cut males were 93% less likely to
283 copulate than controls. ANCOVA revealed no significant difference in copulation latency (F1,69
284 = 1.89, P = 0.17) or duration (F1,68 = 2.47, P = 0.12) between treatments; likewise, treatment X
285 environment interaction had no significant effect on copulation latency (F1,69 = 0.01, P = 0.91) or
286 duration (F1,68 = 0.76, P = 0.39) (Table 1). This lack of an effect on latency is contrary to that
287 found in Experiment 2.
24 288 A two-tailed Wilcoxon signed-rank test revealed that partial-cut and surgical control
289 males performed a similar number of copulation attempts per unit time (i.e., lunge rate) in non-
290 competitive (Z43,42 = -0.66, P = 0.51) and competitive chambers (Z44,44 = 1.13, P = 0.26). When
291 lunges were analyzed without converting them to a rate, Welch’s ANOVA revealed that partial-
292 cut males exhibited a significantly greater total number of lunges than surgical control males in
293 both environment types (non-competitive: F1,48.4 = 9.38, P = 0.004; and competitive: F1,50.8 =
294 6.35, P = 0.015). In non-competitive chambers the mean number of lunges exhibited by partial-
295 cut males (N = 43) was 2.88 (median: 5.5, range: 0-16), compared to 0.69 (median: 2.5, range: 0-
296 6) for controls (N = 42); and in competitive chambers the mean number of lunges exhibited by
297 partial-cut males (N = 44) was 2 (median: 5, range: 0-13), compared to 0.68 (median: 1.5, range:
298 0-3) for controls (N = 44). Figure 7 shows the frequency distributions of lunges for both
299 treatments in the non-competitive and competitive social environments.
300 Females in non-competitive chambers that were paired with partial-cut males expressed
301 resistance behaviors at a significantly greater rate (Wilcoxon: Z42,43 = -2.85, P = 0.004) and
302 frequency (Welch’s: F1,46.8 = 4.3, P = 0.044) than females paired with surgical control males.
303 The mean number of resistance behaviors expressed toward partial-cut males (N = 43) was 2.12
304 (median: 5, range: 0-28), compared to 0.38 (median: 2, range: 0-7) for controls (N = 42).
305 In contrast, females in competitive chambers did not express a significantly different rate
306 (Z44,44 = 0.39, P = 0.69) or frequency (Z44,44 = 0.41, P = 0.69) of resistance behaviors toward
307 either treatment. The mean number of female resistance behaviors expressed toward partial-cut
308 and control males (N = 44 each) was 0.61 (median: 2, range: 0-9) and 0.48 (median: 1.5, range:
309 0-8), respectively. This result did not change when the analysis was restricted to only those
310 competitive chambers in which both males had been observed to lunge at least once (rate: Z11,11
25 311 = -0.08, P = 0.94; frequency: Z11,11 = -0.12, P = 0.91). Additionally, in all 74 copulations,
312 females were never observed to exhibit resistance behaviors while in copula. Thus, females do
313 not appear to discriminate between cut and control males.
314
315 DISCUSSION
316 The data from the three experiments support the hypothesis that genital spines in D.
317 ananassae function to promote competitive male copulation success. Experiment 1 revealed that
318 incremental reductions in male genital spine length progressively reduced copulation success in a
319 non-competitive context, where one male was paired with one female. Whereas removing only
320 the tips of the spines had a non-significant effect on male copulation success, removing half of
321 both spines reduced male copulation success by 87% relative to controls, and full excision of the
322 spines eliminated entirely the ability of males to copulate. Experiment 2, in turn, evaluated the
323 effects of the “tips-cut” surgical manipulation on male copulation success simultaneously in
324 competitive and non-competitive contexts. The results indicate that only in the competitive social
325 context was there a negative effect of this manipulation on male copulation success. Experiment
326 3 verified the existence of this apparent synergism between social environment and surgical
327 treatment using much smaller observation chambers: partial-cut males suffered a significant
328 reduction in copulation success compared to controls in both social contexts, but this effect was
329 stronger in the competitive context. Furthermore, these reductions in the copulation success of
330 partial-cut males occurred despite similar rates of male copulation attempts (lunges per scan)
331 between the two treatment groups, indicating that the surgical reduction in spine length per se,
332 and not any potential side effects of laser contact such as reduced male motivation to mate,
333 caused the observed reduction(s) in copulation success. Thus, the results from Experiments 2 and
26 334 3 provide support for the prediction that the effect of spine reduction on male copulation success
335 should be stronger in an environment where males compete for access to mates.
336 The behavioral observations conducted in Experiment 3 yield further insight into the
337 function of male genital spines in D. ananassae. A first consideration is that in both social
338 contexts of this experiment the partial-cut males exhibited a sharp increase in the frequency of
339 failed copulation attempts (lunges) with virgin females. In other words, cut males exhibited a
340 strong reduction in the efficiency with which they were able to couple their genitalia to that of
341 the female’s, which translated to longer copulation latency (although only significantly so in
342 Experiment 2), and a significant loss of copulation success. These data indicate that the genital
343 spines of male D. ananassae function in the mechanics of genital coupling, to which even subtle
344 alterations have significant reproductive consequences in the face of direct sexual competition.
345 Subtle variations in spine size and/or shape are therefore likely to have pronounced
346 consequences for male reproductive fitness in natural populations of D. ananassae and other
347 Drosophila species that exhibit a scramble competition mating system (Thornhill & Alcock,
348 1983), where competing males search for receptive females on the surface of fruits and where
349 efficient genital coupling is paramount for male copulation success.
350 A second consideration is that females exhibited statistically similar levels of resistance
351 behaviors expressed toward partial-cut and control males in the competitive context, suggesting
352 that female rejection of potential mates is not the cause of the impaired copulation success of cut
353 males in sexually competitive contexts. We also checked for differences in resistance behaviors
354 in the subset of cases in which both males of a competitive chamber were observed to lunge at
355 the female, because such cases would have provided the female with a better opportunity to
356 sense both males’ genital spines. However, we likewise found no significant differences in the
27 357 rate or frequency at which females expressed resistance behaviors toward cut and control males
358 in this subset of the data.
359 Yet, the existence of female resistance behaviors suggests that sexual conflict (Parker,
360 1979; Andersson, 1994; Arnqvist & Rowe, 2005) may help explain the evolution of male genital
361 spine morphology within Drosophila, as discussed in Polak and Rashed (2010). If the present
362 size and shape of male genital spines reflect their effectiveness at overcoming resistance
363 behaviors exhibited by females during courtship and/or mating (Spieth, 1952), then inter-specific
364 differences in spine morphology could at least in part represent differences in the intensity or
365 form of female resistance across species (Arnqvist & Rowe, 1995, 2002a,b). Indeed, the genital
366 spines of male D. ananassae are 21% longer than that of male D. bipectinata (Grieshop & Polak,
367 unpublished data—reported above), and spine removal has a stronger detrimental effect on male
368 copulation success in D. ananassae than it does in D. bipectinata (Polak and Rashed, 2010).
369 Whereas relative spine size matches the trait’s functional importance between these species, it
370 remains unclear whether they also differ in the intensity of female mating resistance. Clearly, a
371 broad range of species will need to be surveyed in terms of spine size, shape and function, and of
372 the intensity of female resistance, for a robust test of this idea.
373 For D. ananassae, further investigation of the adaptive function of male genital spines
374 will require a thorough test of the postcopulatory sexual selection hypothesis (Eberhard, 2011).
375 Though such tests were not the focus of our study, we found no indication in the admittedly few
376 variables we examined that spine reduction elicited any female behavioral responses during
377 mating. Females almost invariably were motionless and exhibited no detectable resistance
378 behaviors during copulation, and we consistently found no significant effect of spine
379 manipulation on copulation duration. Similarly, Polak and Rashed (2010) found that while
28 380 genital spines in D. bipectinata function to promote competitive male copulation success, spine
381 length reduction had no detectable effect on sperm transfer, fertilization success, competitive
382 fertilization success, fecundity, fertility, or copulation duration. Nevertheless, Polak and
383 Rashed’s (2010) study still was not an exhaustive test of the postcopulatory sexual selection
384 hypothesis (see Eberhard, 2011), so more work is needed to fully test this hypothesis with regard
385 to Drosophila genital spines.
386 There are over 40 species of Drosophila with genital spines (Hsu, 1949; Bock &
387 Wheeler, 1972; McEvey et al., 1987; Schiffer & McEvey, 2006), and genital traits that function
388 prior to copulation are taxonomically widespread outside of Drosophila as well (e.g. flatworms:
389 Michiels, 1998; insects: Bertin & Fairbairn, 2005; Polak & Rashed, 2010; fishes: Langerhans et
390 al., 2005; Kahn et al., 2010; mammals: see Miller, 2010). Therefore, the precopulatory sexual
391 selection hypothesis for genital trait evolution likely applies widely among animal taxa, and
392 should weigh more heavily on future research into the remarkable diversification of male
393 genitalia.
394
395 ACKNOWLEDGEMENTS
396 The research was supported by a University Research Council (URC) Graduate Student
397 Research Fellowship (to KG), the McMicken College of Arts and Sciences and the Department
398 of Biological Sciences at the University of Cincinnati, and the National Science Foundation
399 (DEB-1118599 to MP). We thank Brooke Hamilton, Beth Cortright, and Alexandra Warner for
400 assistance with experiments, and Dr. Necati Kaval of the University of Cincinnati’s Department
401 of Chemistry for the SEMs. We also thank Dr. Alex Córdoba-Aguilar and reviewers for helpful
402 comments on the manuscript.
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33 TABLES
Table 1: Least-squares mean ± 1 s.e. (N) copulation latency and duration (s) across surgical treatments from ANCOVAs for Experiments 1, 2, and 3. Data were loge(y+1)-transformed prior to analysis. Surgical Sham Response Variable Full-cut Half-cut Tips-cut Part-cut control control Experiment 1 5.99 ± 0.9 6.11 ± 0.35 6.13 ± 0.33 6.01 ± 0.33 Copulation Latency N/A -- (2) (13) (15) (15) 5.55 ± 0.18 5.44 ± 0.07 5.49 ± 0.07 5.52 ± 0.07 Copulation Duration N/A -- (2) (13) (15) (15) Experiment 2 6.39 ± 0.19 5.87 ± 0.18 *(NC) Cop. Latency ------(29) (33) 6.13 ± 0.31 5.56 ± 0.23 *(C) Cop. Latency ------(11) (21) Experiment 3 6.37 ± 0.38 5.55 ± 0.24 (NC) Cop. Latency ------(12) (31) 7.11 ± 0.96 6.31 ± 0.25 (C) Cop. Latency ------(12) (31) 5.64 ± 0.06 5.71 ± 0.04 (NC) Cop. Duration ------(2) (29) 5.45 ± 0.15 5.67 ± 0.04 (C) Cop. Duration ------(2) (28) (NC) Non-competitive social environment. (C) Competitive social environment. * Significant differences revealed by ANCOVA (see Table 2). -- Treatment not included in experiment. N/A Zero copulations for that treatment (see Figure 2a).
34 Table 2: Results of ANCOVA on copulation latency for Experiment 2. Term d.f s.s.† F P
Male thorax length 1 2.6489 2.44 0.12
*Treatment (Trt) 1 5.9274 5.46 0.02
Environment (Env) 1 1.6249 1.49 0.22
Trt X Env 1 0.0111 0.01 0.92
Error 89 96.6232 * Significant terms. † sum of squares.
35 FIGURE CAPTIONS
Figure 1: Scanning electron micrographs of male D. ananassae genitalia. (a) (50X) Male with
legs removed, i) head, ii) thorax, iii) abdomen. (b) (350X) Terminal segment of the
male abdomen, i) genital spines (external at rest) in crossed orientation, ii) everted
aedeagus, iii) dorsal abdomen.
Figure 2: Example profile images of male genital spines taken with a Hitachi KP-F100 digital
camera mounted on an Olympus BX60 light microscope. (a) D. ananassae raw image,
(a’) that same image with a line drawn down the center of the spine using ImageJ
measurement software; (b) D. bipectinata raw image, (b’) that same image with a line
drawn down the center of the spine using ImageJ measurement software.
Figure 3: Scanning electron micrograph of male D. ananassae genital spines (1200X),
illustrating the results of the micron-scale laser ablation technique: i) partially ablated
spine with no collateral damage to surrounding structures, ii) opposing spine left intact
for reference.
Figure 4: Results of Experiment 1, the effect of genital spine manipulation on copulation
success in a non-sexually competitive social context. Numerals above bars represent
sample sizes, pooled across three replicate blocks.
Figure 5: Results of Experiment 2, the effect of blunting genital spine tips in both non-
competitive and competitive social contexts. Numerals above bars represent sample
sizes, pooled across three replicate blocks.
36
Figure 6: Results of Experiment 3, the effect of partial spine ablation in both social contexts
performed in small-cell mating chambers. Numerals above bars represent sample
sizes, pooled across three replicate blocks.
Figure 7: Frequency distribution of failed copulation attempts (lunges) in the non-competitive
and competitive environments of Experiment 3.
37 Fig. 1
38 Fig. 2
39 Fig. 3
40 Fig. 4
41 Fig. 5
42 Fig. 6
43 Fig. 7
44 CHAPTER 2:
Formatted for: Evolution (The International Journal of Organic Evolution)
Do the male genital spines of Drosophila ananassae [Doleschall] (Diptera:
Drosophilidae) have a postcopulatory adaptive function?
Karl Grieshop*
and
Michal Polak
*Department of Biological Sciences, University of Cincinnati, Cincinnati OH 45221-0006, USA
Running title: Do genital spines function post-copula?
Correspondence:
Karl Grieshop
Department of Biological Sciences
University of Cincinnati, Cincinnati
OH 45221-0006, USA
Tel: +1 (513) 646-2467
Fax: +1 (513) 556-5299
Email: [email protected]
45 1 ABSTRACT
2 The contemporary explanation for the rapid evolutionary diversification of animal
3 genitalia is that such traits evolve via postcopulatory sexual selection. The most common debate
4 within this framework has been over the relative importance of three non-mutually exclusive
5 evolutionary mechanisms: sperm competition, cryptic female choice, and sexual conflict. The
6 first two of these are strictly postcopulatory mechanisms, whereas sexual conflict could operate
7 before, during or after copulation. We investigate the potential for male genital spines in
8 Drosophila ananassae to function in postcopulatory sexual selection. Whereas previous work on
9 two Drosophila species shows that these spines function in precopulatory sexual selection to
10 promote male competitive copulation success, the postcopulatory function(s) of Drosophila
11 genital spines have not yet been thoroughly investigated. Using a precision micron-scale laser
12 surgery technique we test the effect of spine length reduction on male competitive fertilization
13 success, female remating behavior, fecundity, and copulation duration. We find no evidence that
14 male genital spines in this species have a postcopulatory adaptive function. However, partial
15 genital spine ablation had an unexpected positive effect on the probability that males would
16 fertilize at least one gamete of a previously mated female. This effect is discussed in terms of the
17 possibility that Drosophila genital spines are harmful to females as a pleiotropic side effect of
18 evolving to promote competitive male copulation success.
19
20 Key words: Adaptive function, animal genitalia, fecundity, female remating, fertilization
21 success, functional morphology, laser ablation, postcopulatory, precopulatory, sexual selection
22
46 22 INTRODUCTION
23 The remarkable elaboration and diversification of male genital morphology (as opposed
24 to female genital morphology) exhibited among even closely related animal taxa (e.g. Tuxen,
25 1970) indicates that such traits likely evolve via sexual selection (Lloyd, 1979; Eberhard, 1985).
26 Theoretical and empirical evidence over recent decades largely supports this inference (Shapiro
27 & Porter, 1989; Hosken & Stockley, 2004; Simmons et al., 2009; Leonard & Córdoba-Aguilar,
28 2010; but see Cordero-Rivera & Córdoba-Aguilar, 2010). Yet, the precise mechanism(s) by
29 which sexual selection operates to drive the evolution of genital form and function remain
30 unclear after more than a century of debate (Darwin, 1871; Mayr, 1963; Eberhard, 1985,
31 2010a,b, 2011; Leonard & Córdoba-Aguilar, 2010; Rowe & Arnqvist, 2012).
32 This debate is most often with regard to the relative importance/contribution of three non-
33 mutually exclusive hypotheses (mechanisms) of sexual selection (Arnqvist, 1997; Hosken &
34 Stockley, 2004; Eberhard, 1993, 2006, 2010a,b, 2011). Those are sperm competition:
35 competition between male gametes for access to female gametes, potentially involving other
36 components of the male ejaculate (Parker, 1970; Waage, 1979; Simmons, 2001); cryptic female
37 choice: female control over the fate of male gametes according to male stimulation of (or
38 influence on) female sensory perception, neurology or physiology (Thornhill, 1983; Eberhard,
39 1985, 1996); and sexual conflict: agonistic fitness optima, and hence selection pressures,
40 between the sexes, which drives coevolutionary adaptations and counter-adaptations (Trivers,
41 1972; Parker, 1979; Arnqvist & Rowe, 1995, 2005). Sperm competition and cryptic female
42 choice are strictly postcopulatory processes, meaning they ensue after the onset of copulation. By
43 contrast, sexual conflict can operate before, during and/or after copulation.
47 44 In addition to the three (most popular) models described above, there are two other
45 noteworthy postcopulatory sexual selection mechanisms of genital trait evolution. Those are the
46 holdfast mechanism: derived originally from the sperm competition model (but potentially
47 operating within any of the above three hypotheses), male traits serve to anchor the male
48 securely to the female during copulation to prevent being displaced by rival male competitors or
49 female attempts to terminate copulation (Thornhill & Alcock, 1983; Simmons, 2001; Rönn &
50 Hotzy, 2012); and traumatic insemination: males hypodermically inject (or otherwise transmit)
51 their gametes through the female body wall where they migrate to the site of fertilization, thus
52 bypassing the ‘designated’ route and potentially avoiding the ability of females to control the fate
53 of male gametes (Michiels, 1998, Stutt & Siva-Jothy, 2001; Morrow & Arnqvist, 2003;
54 Reinhardt et al., 2003; Siva-Jothy, 2006; Kamimura, 2007; Řezáč, 2009)—similar processes
55 could involve other components of the male ejaculate making their way into the female
56 circulatory system, but these would most likely represent one or more of first three mechanisms
57 described above. It is important to note that none of the five hypotheses described are mutually
58 exclusive processes, and furthermore that there is considerable overlap among them, with many
59 of them fitting into the broader context of sexual conflict (Arnqvist & Rowe, 2005).
60 The large majority of the field has been focused on postcopulatory mechanisms of genital
61 trait evolution (e.g. Eberhard, 1985, 1993, 1996, 2009, 2010a,b, 2011; Hosken & Stockley, 2004;
62 reviewed in: Leonard & Córdoba-Aguilar, 2010). However, a recently emerging body of
63 evidence for the precopulatory adaptive function of animal genitalia (either as mechanical
64 armaments or sexual signals) poses new insights (Michiels, 1998; Langerhans et al., 2005; Bertin
65 & Fairbairn, 2005; Moreno-García & Cordero, 2008; Kahn et al., 2010; Miller, 2010; Polak &
66 Rashed, 2010; Grieshop & Polak, 2012). The genital spines of male fruit flies in the genus
48 67 Drosophila are one such trait with a precopulatory function—aiding males in coupling the
68 female genitalia and initiating copulation (Polak & Rashed, 2010; Grieshop & Polak, 2012).
69 These spines are rapidly evolving and widespread within the melanogaster species group (with
70 over 40 species expressing them), and consist of 1-5 pairs (depending on species) of hard,
71 sclerotized, claw-like structures that are external at rest, extending from the ventral cercal lobe
72 (or secondary claspers) (Hsu, 1949; Bock & Wheeler, 1972; McEvey et al., 1987; Schiffer &
73 McEvey, 2006). Their precopulatory function, revealed by phenotypically manipulative
74 experimentation, seems to be governed predominantly by intrasexual competition among males
75 for access to females, where females (at least in D. ananassae) apparently do not discriminate
76 between competing males with surgically ablated versus intact genital spines (Grieshop & Polak,
77 2012). Yet, the existence of female resistance behaviors (though not expressed at differential
78 rates toward males of differing surgical treatments in D. ananassae) may imply a role for
79 precopulatory sexual conflict in the function/evolution of male genital spines—if the spines serve
80 to overcome some female resistance behavior, variation in male genital spine size and shape
81 across species could represent variation in the intensity and/or form of female resistance
82 behavior across species (Grieshop & Polak, 2012; and expanded upon in the present Discussion
83 section), but this concept has not been explicitly tested.
84 While the precopulatory function of Drosophila genital spines is similar to the genital
85 claspers of many insects (e.g. Arnqvist & Rowe, 2002; Bertin & Fairbairn, 2005; Moreno-García
86 & Cordero, 2008), they also share qualities with genital traits having postcopulatory functions
87 (e.g. Kamimura, 2007; Hotzy & Arnqvist, 2009) in that they come to a sharp, potentially
88 injurious point, and insert into the female’s external genitalia (though not the gonopore) during
89 copulation (Grieshop & Polak, 2012). However, the postcopulatory sexual selection hypothesis
49 90 for genital spine function/evolution remains to be thoroughly investigated (Eberhard, 2011;
91 Grieshop & Polak, 2012).
92 The present study tests the postcopulatory sexual selection hypothesis for the adaptive
93 function of male genital spines in the cosmopolitan fruit fly D. ananassae (see Chapter 1, Figure
94 1; Tobari, 1993) using a precision micron-scale laser surgery system (Polak & Rashed, 2010;
95 Grieshop & Polak, 2012) as a means of phenotypic manipulation (see Chapter 1, Figure 3).
96 Possible postcopulatory adaptive functions of this trait include all five of the above-mentioned
97 hypotheses. We designed a set of experiments to evaluate several mechanisms by which the
98 genital spines of male D. ananassae could influence male or female reproductive fitness after the
99 onset of copulation. Experiment 1 is a competitive fertilization success assay, designed to assess
100 the effect of surgical reduction in male genital spine length on the proportion of a previously
101 mated female’s clutch of eggs that is sired by her second mate (known as P2). Experiment 2 is a
102 female remating and fecundity experiment, designed to assess the effect of surgical reduction in
103 male genital spine length on female remating and egg laying behavior after copulation. The
104 specific predictions—and their rationales—for these experiments are that surgical reduction in
105 male genital spine length should:
106 i) decrease male offensive sperm competitive ability (competitive
107 fertilization success), measured as P2—male genital spines could
108 enhance the ability of the male ejaculate to outcompete that of other
109 males’ for access to female gametes, thus enhancing male competitive
110 fertilization success;
111 ii) increase female likelihood to remate, or decrease female latency (in days)
112 to remate, with a second male—females that are adapted to replenish
50 113 their sperm reserves with novel (ideally superior) sperm would be
114 more likely, and/or quicker, to remate with another male if their most
115 recent mate was of perceived inferior or substandard quality;
116 iii) decrease female egg deposition after copulation, measured both as short-
117 term (first 24 h) and long-term (6 days) fecundity—females that are
118 adapted to using their finite, expensive gametes so as to maximize
119 their (indirect) reproductive fitness would oviposit fewer eggs sired by
120 males of perceived inferior or substandard quality in order to reserve
121 gametes for fertilization by males of perceived superior quality;
122 iv) decrease copulation duration—male genital spines could serve to anchor
123 the male to the female during mating in order to avoid being displaced
124 by rival male competitors or female attempts to terminate copulation
125 (e.g. to transfer more sperm or further stimulate female reproduction).
126 Empirical support for any of the above predictions would warrant further investigation into the
127 precise mechanism(s) underlying the effect(s).
128
129 METHODS
130 Experimental flies
131 The base population of Drosophila ananassae [Doleschall] (Diptera: Drosophilidae) was
132 initiated with 100 inseminated females collected in February 2009 on the South Pacific island of
133 Moorea (17°32'58.78''S, 149°52'59.29''W), Society Islands. Flies were mass cultured in the
134 laboratory on a 12:12 h L:D photoperiod and a 24°C (L): 22°C (D) temperature regime in 240 ml
135 glass milk bottles (N = 6) with 6 g of Formula 4-24 Instant Drosophila Medium (Carolina Supply
51 136 Co., Burlington, NC), 20 ml water, 8 ml of banana/live yeast slurry (50 ml water : 25 g banana :
137 1.5 g live yeast), and autoclaved tissue paper. The base population was acclimated to these
138 laboratory conditions for 30 generations over 12 months before use in the experiments.
139 Virgin males and females were collected from the base population simultaneously within
140 4 h of eclosion, maintained separately as virgins in 35 ml disposable polystyrene shell vials lined
141 with cornmeal-agar food medium (N~25 per vial), and allowed to age for 5 days until use in a
142 given experiment, of which there were two (see below). Experimental flies were transferred to
143 fresh food vials every other day until experimentation, and live yeast was added to vials
144 containing females. For both experiments, molasses-agar oviposition medium was used to
145 monitor egg laying/hatching because the dark, homogeneous color of the substrate provided a
146 desirable contrast for counting eggs and finding larvae.
147 After each block of both experiments, males were preserved in 95% ethanol and later
148 examined under an Olympus SZX12 stereomicroscope to verify treatment identity and the
149 integrity of the surgical manipulation (without knowledge of copulatory status). Male thorax
150 length, an estimate of body size (Robertson & Reeve, 1952), was measured (in rehydrated
151 specimens) from the tip of the scutellum to the anterior edge of the thorax using an ocular
152 micrometer; independent repeated measurements of thorax lengths in a random sample of 10
153 males were highly repeatable among males (1-way ANOVA: F9,10 = 516.7, P < 0.0001).
154
155 Laser manipulation
156 Virgin males were laser-treated within 22 h (± 2 h) of eclosion using the protocol
157 described in Polak and Rashed (2010). Briefly, males were placed one at a time in a plexiglass
158 surgical chamber while lightly anesthetized with humidified CO2. Pulsed laser light was used to
52 159 administer precision cuts to genital spines with little or no collateral damage to surrounding
160 structures or bristles (Figure 3). Experimental males had their spine lengths surgically reduced in
161 a bilaterally symmetrical fashion. The surgical treatment used for ‘cut’ males throughout the
162 present study entailed the removal of approximately ¼ of the length of males’ spines. It was
163 inferred from Grieshop and Polak (2012) that this amount of spine length removal would provide
164 a sufficient number of mated cut males to conduct postcopulatory experimentation. The control
165 treatment was a surgical control (Grieshop & Polak, 2012)—2 bristles on the seventh sternite of
166 the ventral abdomen were ablated at the base. Laser surgeries for all treatments, including the
167 control, took approximately the same amount of time to perform (< 1 minute per individual).
168 Following surgery, experimental males were held separately (and without females) in food vials
169 (N~20 per vial) until experimentation. Throughout both experiments described below (involving
170 over 300 individual surgeries), a negligible number (< 1%) of experimental males died prior to
171 experimentation: 2 cut males (1 from each experiment), and 1 control male (from Experiment 2).
172
173 Experiment 1
174 In each of two blocks a total of 63 (block 1) and 75 (block 2) virgin females from the
175 base population were individually mated in vials to virgin irradiated ‘donor’ males from the
176 base-population. These donor males were irradiated with a 150 Gy dose from a 60Co source
177 (Polak & Simmons, 2009) when they were two days old so that their sperm could fertilize female
178 eggs but the zygote would die before hatching due to lethal mutations (Simmons, 2001). Within
179 1 h of copulation females were transferred to fresh oviposition vials to lay eggs, and then were
180 transferred to fresh oviposition vials every other morning for the next 7 days. On the morning of
181 the seventh day females were randomly paired with either a cut or a control male; if they did not
53 182 remate within 2 h, females were transferred to fresh vials for an additional two days and then
183 placed with a different male from the same treatment category on the morning of the 9th day after
184 the first copulation, and again on the 11th day after the first copulation. Females that did not
185 remate by the 11th day (50% of the mated females, N = 69) were discarded. The durations of all
186 copulations were recorded and the thorax lengths of doubly mated females’ first and second
187 mates were measured.
188 Doubly mated females were transferred to fresh oviposition vials to lay eggs within 1 h of
189 the second copulation, and then were transferred to fresh oviposition vials every other morning
190 until a minimum of 10 eggs were laid (mean ± s.d.: 19.2 ± 6.1, range: 10-38; N = 47 females).
191 P2, as noted above, is the proportion of a female’s clutch of eggs sired by her second mate (the
192 non-irradiated treatment male in this case), which was calculated as the number of eggs that
193 hatched into larvae divided by the total number of eggs laid after remating (Boorman & Parker,
194 1976; Polak & Simmons, 2009). The “inter-copulation interval” refers to the amount of time
195 elapsed (in days) between a female’s first and second copulations. The variable “pre-P2 eggs”
196 refers to the number of eggs laid by females during this inter-copulation interval. The pre-P2 eggs
197 of a random sample of 10 females from the first block were monitored continuously (~every 6-8
198 h) for any larvae to assess the integrity of the irradiation technique at sterilizing the donor
199 males—no larvae hatched from these females’ pre-P2 eggs. All counts of eggs and larvae were
200 conducted blind with respect to treatment.
201 The probability that a female would remate according to which treatment she was being
202 paired with for her second mating was analyzed using logistic regression, with female thorax
203 length as the covariate. The effect of male treatment on copulation duration of females’ second
54 204 matings was analyzed using analysis of covariance (ANCOVA) with treatment and male thorax
205 length as independent terms.
206 P2 data were first analyzed as a logistic regression on the presence/absence of evidence
207 for second-male fertilizations (0 when P2 = 0, 1 when P2 > 0 ), with treatment of the second male
208 and the inter-copulation interval (7, 9, or 11 days) as independent terms. Arcsine-square root
209 transformed P2 data for which P2 > 0 were analyzed using ANCOVA with treatment and pre-P2
210 eggs as independent terms. Both original full models (for the logistic regression and the
211 ANCOVA) contained eight independent terms that could explain variation in P2 (block,
212 treatment, thorax length of male 1, thorax length of male 2, copulation duration of male 1,
213 copulation duration of male 2, pre-P2 eggs, and inter-copulation interval). Least significant terms
214 were sequentially eliminated from the model; none of the removed terms had alpha values < 0.1.
215 Interactions between remaining terms in the final reported models were non-significant. For the
216 final ANCOVA model we report, P2 residuals were normally distributed (W = 0.96, P = 0.48)
217 and homoscedastic across treatment categories (Levene’s test: F1,17 = 0.05, P = 0.82) (Zar, 1999).
218
219 Experiment 2
220 In each of three blocks a total of 12 (block 1), 9 (block 2), and 11 (block 3) virgin
221 females from the base population were each mated to a cut male, and 10 (block 1), 11 (block 2),
222 and 10 (block 3) virgin females were each mated to a control male, in vials. Within 1 h of
223 copulation females were transferred to fresh oviposition vials to lay eggs. The following morning
224 females were transferred to fresh vials containing 2 randomly selected males from the base
225 population that had been sexually isolated for the prior 2 days. Vials were continuously scanned
226 for any females remating, in which case remated females were preserved for later measurement.
55 227 If females did not remate within 2 h they were transferred to fresh oviposition vials to lay eggs.
228 The number of eggs each female laid each day (including the first day after the first mating) was
229 counted prior to any larvae hatching, and oviposition vials were discarded after eggs were
230 counted. This procedure (attempting to remate females every day and recording the number of
231 eggs they laid each day until they remated) was repeated every morning for 6 days. The
232 durations of all copulations were recorded and the thorax lengths of females and treatment males
233 (females’ first mates) were measured. Four females (2 mated to cut males, and 2 mated to control
234 males) were removed from the analysis because they died in their oviposition vials without
235 remating.
236 The effect of genital spine reduction on copulation duration of females’ first matings was
237 analyzed using ANCOVA, with treatment and male thorax length as independent terms.
238 The probability that a female would remate was analyzed as a logistic regression (0 = not
239 remated, 1 = remated) with male treatment (females’ first mates) and the number of eggs females
240 laid in the first 24 h period after mating as independent terms. The original full model contained
241 six independent terms that could explain the likelihood of female remating (block, male
242 treatment, thorax length of the treatment (first) male, female thorax length, copulation duration
243 of the first mating, and the number of eggs laid in the first 24 h after mating). Of the females that
244 did remate, the inter-copulation interval, measured in days (mean ± s.d.: 3.56 ± 1.93, range: 1-6;
245 N = 16 females) was analyzed non-parametrically using a two-tailed Wilcoxon signed rank test
246 with male treatment as the independent variable (Zar, 1999).
247 Female fecundity was analyzed as four separate ANCOVA models. Block had a non-
248 significant effect in all four ANCOVA models described below (all alpha values > 0.1), and so
56 249 was removed from the final reported analyses. For all four ANCOVAs, interactions between
250 remaining terms in the final reported models were non-significant.
251 The first ANCOVA was on the number of eggs laid in the first 24 h period after the first
252 mating (mean ± s.d.: 23.19 ± 17.59, range: 0-69; N = 59 females); the original full model
253 included six independent terms that could explain variation in female egg laying behavior (block,
254 male treatment, thorax length of the treatment (first) male, female thorax length, copulation
255 duration of the first mating, and whether or not females remated).
256 The second ANCOVA, performed on only those females that remated, was also on the
257 number of eggs laid in the first 24 h period after the first mating (mean ± s.d.: 19.5 ± 18.92,
258 range: 0-69; N = 16 females); the original full model included five independent terms (block,
259 male treatment, thorax length of the treatment (first) male, female thorax length, and copulation
260 duration of the first mating).
261 The third and fourth ANCOVA models were for those females that did not remate. The
262 dependent variable for the third model was (again) the number of eggs laid in the first 24 h
263 period after the first mating (mean ± s.d.: 24.56 ± 17.09, range: 0-67; N = 43 females). The
264 dependent variable for the fourth model was the total number of eggs females laid over six days
265 (total fecundity) (mean ± s.d.: 73.42 ± 27.08, range: 9-135; N = 43 females). Both the original
266 full models for these third and fourth ANCOVAs were identical in structure to the second
267 ANCOVA above.
268 For the ANCOVA models we report, listed in the order they appear above, residuals were
269 approximately normally distributed (W = 0.98, P = 0.57; W = 0.96, P = 0.68; W = 0.94, P = 0.03;
270 and W = 0.96, P = 0.16; respectively) and homoscedastic across treatment categories (Levene’s
57 271 test: F1,57 = 2.58, P = 0.11; F1,14 = 1.72, P = 0.21; F1,41 = 0.32, P = 0.57; and F1,41 = 0.45, P =
272 0.51; respectively). JMP (v. 8, SAS Institute Inc., 2009) statistical software was used throughout.
273
274 RESULTS
275 Experiment 1
276 Logistic regression revealed no significant differences in the likelihood of female
277 remating between those that we attempted to remate to cut males (N = 64, remated/not = 31/33)
2 278 and those that we attempted to remate to control males (N = 74, remated/not = 38/36) (χ 2 =
279 0.46, P = 0.49). Similarly, ANCOVA revealed no significant effect of male surgical treatment on
280 copulation duration (F1,61 = 0.04, P = 0.84).
281 Logistic regression revealed that male D. ananassae with their genital spines partially
282 ablated were significantly more likely to have a P2 score > 0 (i.e. to fertilize at least one gamete
283 of a previously mated female) than control males (Table 1, Figure 3). Of the females with a P2
284 score of zero, the mean ± s.d. number of eggs laid after remating was 18.71 ± 6.87 (range: 10-38;
285 N = 28 females), and ANCOVA (with pre-P2 eggs as the covariate) revealed no significant effect
286 of treatment (F1,44 = 0.05, P = 0.82) or whether P2 was > or = 0 (F1,44 = 0.97, P = 0.33) on the
287 total amount of eggs laid after remating. Thus, whereas male surgical treatment did significantly
288 influence the likelihood that a male would fertilize at least one gamete of a previously mated
289 female (i.e. P2 > 0), neither male treatment nor the likelihood of P2 > 0 had a significant
290 influence on total female fecundity post-remating.
291 Of the females that did produce at least one larvae (meaning their second mate’s P2 > 0),
292 ANCOVA revealed no significant effect of surgical treatment on male offensive sperm
293 competitive ability (F1,16 = 2.75, P = 0.12), measured as P2 (mean ± s.d.: 0.21 ± 0.15, range:
58 294 0.03-0.48; N = 19 females). Thus, male offensive sperm competitive ability was not influenced
295 by partial genital spine ablation.
296
297 Experiment 2
298 ANCOVA revealed no significant effect of male surgical treatment on the copulation
299 duration of the first mating (F1,61 = 0.04, P = 0.84). Logistic regression revealed no significant
300 differences in the likelihood of female remating between those that were mated first by cut males
301 (N = 32, remated/not = 9/23) and those mated first by control males (N = 31, remated/not = 7/24)
2 302 (χ 2 = 0.22, P = 0.64). Of the females that did remate, a two-tailed Wilcoxon signed rank test
303 revealed no significant difference in the inter-copulation interval (i.e. on which day, 1-6, females
304 remated) between females that were first mated to cut males versus control males (Z9,7 = -0.97, P
305 = 0.33). Thus, partial ablation of male genital spines in this species does not appear to influence
306 the likelihood that, or speed with which, females will remate.
307 When including all females that mated at least once, females did not oviposit a
308 significantly different number of eggs in the first 24 h after mating to a cut male (N = 30)
309 compared to after mating to a control male (N = 29), but the covariate female thorax length was
310 significantly positively associated with the number of eggs laid during that time (Table 2). When
311 only including the females that did remate, females likewise did not oviposit a significantly
312 different number of eggs in the first 24 h after mating to a cut male (N = 9) compared to after
313 mating to a control male (N = 7), but this time the covariate male thorax length was significantly
314 positively associated with the number of eggs laid during that time (Table 2). When only
315 including the females that did not remate, of which N = 21 females were first mated to cut males
316 and N = 22 females were first mated to control males, again females did not oviposit a
59 317 significantly different number of eggs in the first 24 h after their first matings, or in total over the
318 6 days following their first matings, with regard to the surgical treatment of their first mates—
319 female thorax length was again significantly positively associated with the number of eggs laid
320 during both of those timeframes (Table 2). Thus, partial ablation of male genital spines in D.
321 ananassae does not appear to influence female short-term egg laying activity (regardless of
322 whether or not those females eventually remate), or long-term fecundity after mating once and
323 being given the opportunity to remate (yet not doing so) each day afterward.
324
325 DISCUSSION
326 The data from the two experiments above do not support the postcopulatory sexual
327 selection hypothesis for the adaptive function of male genital spines in D. ananassae. The
328 evidence indicates that surgical ablation of approximately ¼ of male genital spine length in this
329 species has no significant effect on male competitive fertilization success (P2), female remating
330 behavior, female fecundity, or copulation duration. These findings are congruent with those of
331 Polak and Rashed’s (2010) study regarding genital spine function in D. bipectinata. Thus,
332 despite a more thorough investigation of the postcopulatory sexual selection hypothesis for
333 Drosophila genital spine function/evolution, as advised by Eberhard (2011), there remains no
334 empirical support for a postcopulatory adaptive function of Drosophila genital spines. In light of
335 the evidence presented by Polak and Rashed (2010), Grieshop and Polak (2012), and the present
336 study, we can be relatively confident that the primary adaptive function of Drosophila genital
337 spines is to promote the competitive copulation success of males in a scramble competition
338 scenario (Thornhill & Alcock, 1983), where competing males search for receptive females on the
339 surface of fruits and where efficient genital coupling is paramount for male copulation success.
60 340 Of the hypotheses outlined in the Introduction, sexual conflict remains (by process of
341 elimination) best suited to explain the function and evolutionary diversification of male genital
342 spines in Drosophila. Grieshop and Polak (2012) briefly describe a mechanism by which male
343 genital spine evolution in Drosophila could be subject to the evolutionary conflict of interest
344 between the sexes (Arnqvist & Rowe, 2005). If genital spines are selected in males to promote
345 efficient coupling of the female genitalia, then females would be selected to increase their efforts
346 to resist copulation (e.g. via kicking, fleeing, decamping, abdominal bending or vaginal
347 extruding—Grieshop & Polak, 2012; Spieth, 1952) so as to ensure reproduction with only the
348 most capable males and avoid mating with inferior or behaviorally phenodeviant males (Markow
349 & Gottesman, 1993; Polak, 2008). Thus, variation in the size and shape of male genital spines
350 across species may represent variation in the intensity and/or form of female resistance behavior
351 across those species. Indeed, a preliminary analysis of the relationship between genital spine size
352 and function across two Drosophila species revealed that the size of the trait is directly
353 proportional to its functional importance for male copulation success between D. ananassae and
354 D. bipectinata (Grieshop & Polak, 2012). A thorough evaluation of the contribution of sexual
355 conflict to the evolution of Drosophila genital spines would require a detailed investigation of
356 genital spine size, shape, and function, and female resistance behavior, across a broad range of
357 species.
358 The potential for a sexually antagonistic coevolutionary struggle for control over mating
359 outcomes may lend insight to a puzzling result produced by this investigation—the finding that
360 ¼ genital spine ablations in male D. ananassae did actually increase the likelihood that males
361 would successfully fertilize at least one gamete of a previously mated female (i.e. have a P2 score
362 > 0). Though the effect of partial genital spine ablation on male likelihood to have a P2 score > 0
61 363 does not speak to any of the predictions assessed by this study, it may fit into the context of a
364 particular type of sexual conflict—pleiotropic harm (Arnqvist & Rowe, 2005; Hotzy & Arnqvist,
365 2009). There are two main hypotheses to explain why males, who accrue their fitness via the
366 females they mate with, would harm their mates. Adaptive harm: male traits harm females
367 because female response(s) to such harm directly promote male reproductive fitness (Michiels,
368 1998; Johnstone & Keller, 2000; Morrow et al., 2003; Hosken et al., 2003; Lessells, 2005); and
369 pleiotropic harm: male traits harm females as a consequential side effect of their adaptive
370 function (Morrow et al., 2003; Hotzy & Arnqvist, 2009). Under the pleiotropic harm hypothesis,
371 positive direct selection on males for traits that happen to be harmful to females should outweigh
372 the direct fitness costs to males of the lowered fecundity it produces in their mates (Morrow et
373 al., 2003; Arnqvist & Rowe, 2005; Hotzy & Arnqvist, 2009). Thus, a prediction of the
374 pleiotropic harm hypothesis (which was assessed by the present study, though not intentionally)
375 is that harmful male traits should be associated with reduced female fecundity. This predicted
376 effect is presumably attributable to the damage females accrue by mating with males harboring
377 harmful traits—and therefore surgical reductions to harmful male traits should be associated with
378 increased (i.e. rescued) female fecundity. But no significant relationship between genital spine
379 manipulation and female fecundity was found in the present study or in Polak and Rashed
380 (2010). Additionally, there were no significant differences in female fecundity between those
381 whose second mates’ P2 scores were = 0 and those whose second mates’ P2 scores were > 0. This
382 absence of an effect on female fecundity, however, does not rule out the pleiotropic harm
383 hypothesis, as the predicted association between pleiotropic harm and reduced female fecundity
384 nearly lacks empirical support (with Hotzy & Arnqvist, 2009 being the first to demonstrate such
385 an effect).
62 386 Applying this conceptual relationship between harm and direct fitness costs to our finding
387 that genital spine reduction in D. ananassae promotes the likelihood of those males’ P2 scores
388 being > 0, one could formulate the following hypothesis: the mechanism underlying the positive
389 relationship between male genital spine reduction and the likelihood of male P2 scores being > 0
390 is attributable to females only being able to withstand a finite amount of damage inflicted by
391 their mates before successful fertilizations become compromised. After all, these were females’
392 second matings, the first of which was with a donor male having intact genital spines, these
393 spines do come to a sharp, potentially injurious point that inserts into the female’s external
394 genitalia during copulation, and (virgin) males of this species do tend to contact the external
395 female genitalia with their genital spines during unsuccessful copulation attempts several times
396 before being successful (Grieshop & Polak, 2012)—all of which could increase the estimated
397 damage females accrue from a given mating. Of course, this hypothesis would require explicit
398 testing, for example by investigating whether or not Drosophila genital spines do actually harm
399 females (e.g. by testing for melanized scar tissue—Kamimura, 2007), and then investigating the
400 relationship between the number of female matings and subsequent female fecundity and male
401 fertilization success as a sort of dose-response assessment (where the ‘dosage’ would be the
402 number of matings). If pleiotropic harm is operating in the evolution of Drosophila genital
403 spines, there would exist an interesting evolutionary fitness trade-off between precopulatory
404 sexual selection on males for genital spines that promote competitive male copulation success
405 (Grieshop & Polak, 2012) and direct postcopulatory selection in the opposite direction on males
406 and females for spines that reduce the fitness costs (mainly to females) of mating multiply. That
407 is, one could investigate the pre- and post-copulatory intra- and inter-sexual evolutionary
408 dynamics of a single genital trait.
63 409 The holdfast and traumatic insemination hypotheses were not investigated more
410 thoroughly by the present study for the following reasons. Regarding holdfast mechanisms, most
411 sexually reproducing organisms (especially insects) tend to be fastened together during
412 copulation so securely that it is unlikely conspecific males could generate the force necessary to
413 separate a copulating pair, and such attempts, especially successful ones, are rarely
414 observed/documented (Eberhard, 2010b)—this was the case for Grieshop & Polak (2012), where
415 only 3 instances (none successful) of males attempting to separate, or mate with, a copulating
416 pair were observed in over 1600 observations of 45 competitive-context mating trials (Grieshop
417 & Polak, unpublished data, available in the Dryad repository: doi:10.5061/dryad.g0v6h003).
418 Additionally, the genital spines of many Drosophila, including D. ananassae, appear not to be
419 appropriately designed as holdfast devices (Eberhard, 2010b)—they are structurally much less
420 formidable than other larger structures that do actually insert into the female gonopore, and
421 hence are likely not the trait responsible for anchoring males to their mates during copulation, if
422 there is a trait devoted to such a function in this species. Regarding traumatic insemination,
423 Drosophila genital spines are essentially modified bristles, which have no openings anywhere on
424 them through which seminal products could be dispensed (personal SEM observations); and
425 considering they do not enter the female gonopore during copulation (Grieshop & Polak, 2012),
426 it is unlikely that the spines are capable of producing wounds in/on the female (in close enough
427 proximity to the release of the ejaculate) through which male gametes could enter the female
428 circulatory system, unless acting in conjunction with other male genital traits that may release
429 (part of) the ejaculate in closer proximity (see Kamimura, 2007).
430 At presently, the overwhelming majority of evidence for genital spine function/evolution
431 in the only two species of Drosophila for which experimental investigations of this trait have
64 432 been conducted (D. bipectinata and D. ananassae) remains in support of the hypothesis that this
433 trait promotes competitive male copulation success (Polak & Rashed, 2010; and Grieshop &
434 Polak, 2012; respectively). Intrasexual competition between males for efficiently coupling
435 females’ genitalia in the presence of rival competing males is likely the driving force behind the
436 evolutionary origins of this trait. The subsequent rapid evolutionary diversification of this trait
437 across species is potentially attributable to sexual conflict, as described above—but this would
438 require explicit testing. There remains no convincing evidence of a postcopulatory adaptive
439 function of Drosophila genital spines. An exhaustive test of the postcopulatory sexual selection
440 hypothesis for Drosophila genital spine evolution is now only lacking an investigation of the
441 potential for theses spines to injure females during copulation or copulation attempts. As was the
442 case for the present study, potential future empirical evidence for these spines inflicting injury
443 upon their mates would warrant further investigation into postcopulatory mechanisms of genital
444 trait evolution, for example an investigation of the potential for these spines to create open
445 wounds through which (part of) the male ejaculate could enter into the female circulatory
446 system, which would, in that case, render the harm as adaptive rather than pleiotropic.
447
448 ACKNOWLEDGEMENTS
449 The research was supported by a University Research Council (URC) Graduate Student
450 Research Fellowship (to KG), the McMicken College of Arts and Sciences and the Department
451 of Biological Sciences at the University of Cincinnati, and the National Science Foundation
452 (DEB-1118599 to MP). We thank Elizabeth Brown and Scott Licardi for assistance with
453 experiments, and Dr. Necati Kaval of the University of Cincinnati’s Department of Chemistry
454 for the SEMs.
455
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71 TABLES
Table 1: Results of the logistic regression on the probability of successfully fertilizing at least one gamete of a previously mated female, measured as the presence (1) or absence (0) of at least one successfully hatched larva in the male competitive fertilization success experiment. Term α* s.e.† χ2 P
Treatment -0.76 0.34 5.05 0.025
Inter-copulation interval 0.42 0.24 2.92 0.088 2 χ values testing H0: α = 0. * regression coefficients. † standard errors. Significant terms bold.
72 Table 2: Results of ANCOVAs performed for female egg laying behavior in Experiment 2—headers specify response variable (and data used). First 24 h (all females) First 24 h (remated females) Term d.f. s.s.† F P d.f. s.s.† F P Treatment 1 0.01 0.001 0.98 1 2.20 0.65 0.44 Male thorax 1 3.48 0.89 0.35 1 20.62 6.07 0.031 length Female thorax 1 36.30 9.37 0.004 1 4.00 1.18 0.30 length Copulation 1 4.04 1.04 0.31 1 8.08 2.38 0.15 duration Remate? 1 8.89 2.29 0.14 ------Error 53 205.31 11 37.39
First 24 h (not remated) Total 6 days (not remated) Term d.f. s.s.† F P d.f. s.s.† F P Treatment 1 0.67 0.19 0.67 1 3.18 1.11 0.29 Male thorax 1 1.20 0.34 0.56 1 1.77 0.62 0.44 length Female thorax 1 33.53 9.46 0.004 1 15.39 5.39 0.026 length Copulation 1 1.96 0.55 0.46 1 0.03 0.01 0.91 duration Remate? ------Error 38 134.66 38 108.54 -- term not included in that analysis. † sum of squares; Significant terms bolded.
73 FIGURE CAPTIONS
Figure 1: Results of the competitive fertilization success experiment, the effect of genital spine
manipulation on the probability of successful offensive male competitive fertilization.
Numerals above bars represent sample sizes, pooled across two blocks.
74 Fig. 1
1
0.8 > 0 2 0.6 24
0.4
23 0.2
Probability of P 0 Surgical control 1/4 cut Treatment
75 GENERAL CONCLUSIONS
• The male genital spines of Drosophila ananassae promote competitive male copulation
success, congruent with the findings of Polak and Rashed (2010) for genital spine
function in D. bipectinata, thus supporting the hypothesis that genital spines in the genus
Drosophila have evolved to promote competitive male copulation success.
• Specifically, the genital spines of D. ananassae seem to have evolved via intrasexual
selection (competition between males) to promote the efficiency with which males are
able to couple the female genitalia and initiate copulation in the face of nearby sexual
rivals in a scramble competition scenario—females do not adjust their resistance
behaviors according to experimentally manipulated male genital spine morphology.
• The larger size of the genital spines of D. ananassae compared to D. bipectinata may
reflect an evolutionary history of relatively more intense sexual selection in D. ananassae
because these males suffer a considerably greater detriment to their copulation success
when the trait is removed completely—they cannot mate without genital spines. In
contrast, complete removal of the spines in D. bipectinata does not eliminate entirely
their probability of copulation. Future studies should aim to assess the relationship
between genital spine size, shape, function and female mating resistance across a wide
range of species within the genus Drosophila.
• There is no convincing evidence of a postcopulatory adaptive function of genital spines
in Drosophila, but a follow-up investigation of the pleiotropic harm hypothesis is needed.
• Considering all evidence produced by this study, the rapid evolutionary diversification of
male genital spine morphology in Drosophila is best explained by sexual conflict, aiding
males in the intrasexual competition to overcome female resistance to copulation.
76