PRECOPULATORY MATE GUARDING BEHAVIOR IN CLAM SHRIMP:
A CASE OF INTERSEXUAL CONFLICT
A dissertation submitted to Kent State University
in cooperation with The University of Akron
in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
by
Chiara Benvenuto
December 2008
i Dissertation written by
Chiara Benvenuto B.Sc./M.Sc. (Laurea), Università degli Studi di Firenze, 1999 Ph.D., Kent State University, 2008
Approved by
______, Chair, Doctoral Dissertation Committee Dr. Stephen C. Weeks ______, Co advisor, Doctoral Dissertation Committee Dr. Walter R. Hoeh ______, Members, Doctoral Dissertation Committee Dr. Mark W. Kershner ______, Dr. Lisa E. Park ______, Dr. David C. Riccio Accepted by ______, Chair, Department of Biological Sciences Dr. James L. Blank ______, Dean, College of Arts and Sciences Dr. John R. D. Stalvey
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TABLE OF CONTENTS
LIST OF FIGURES...... vi
LIST OF TABLES ...... viii
ACKNOWLEDGMENTS...... ix
CHAPTERS
I. GENERAL INTRODUCTION ...... 1
Sexual selection and sexual conflict ...... 1
Components of sexual conflict...... 5
The outcome of the war of the sexes: who wins? ...... 9
Testing sexual conflict ...... 13
Mate guarding and sexual conflict...... 20
Precopulatory mate guarding in Crustacea ...... 22
Models of mate guarding ...... 25
Parker’s model (1974)...... 26
Wickler and Seibt’s models (1979 1981) ...... 27
Grafen & Ridley’s model (1983) ...... 28
Hunte, Myers, and Doyle’s model (1985)...... 29
Yamamura’s model (1987) ...... 30
Elwood and Dick’s model (1990) ...... 30
Jormalainen, Tuomi, and Yamamura’s model (1994) ...... 31
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Yamamura and Jormalainen’s model (1996)...... 32
Jormalainen’s model (1998)...... 32
Härdling, Jormalainen, and Tuomi’s model (1999)...... 35
Härdling, Kokko, and Elwood (2004)...... 36
An empirical approach: the economics of mate guarding...... 37
Mate guarding in an androdioecious species: how hermaphrodites
fit into the equation ...... 40
The study system...... 42
Overview of research objectives...... 43
II. MATE RECEPTIVITY ASSESSMENT AND PRECOPULATORY MATE
GUARDING BEHAVIOR IN THE CLAM SHRIMP EULIMNADIA TEXANA ...... 46
Abstract ...... 46
Introduction...... 47
Materials and methods ...... 54
Project 1 Tracking focal animals ...... 56
Project 2 Molting hormone as a soluble cue...... 57
Project 3 Molting hormone as a direct contact cue...... 58
Results...... 61
Project 1 Tracking...... 61
Project 2 Molting hormone as a soluble cue...... 63
Project 3 Molting hormone as a direct contact cue...... 64
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Discussion ...... 70
Acknowledgements...... 79
III. INTERSEXUAL CONFLICT DURING MATE GUARDING IN THE
ANDRODIOECIOUS CLAM SHRIMP EULIMNADIA TEXANA ...... 80
Abstract ...... 80
Introduction...... 81
Materials and methods ...... 85
Mate guarding costs ...... 85
The effect of size...... 86
Compromised and optimal guarding times ...... 87
Results...... 90
Mate guarding costs ...... 90
The effect of size...... 92
Compromised and Optimal Guarding Time...... 93
Discussion ...... 96
Acknowledgements...... 105
IV. MATE GUARDING BEHAVIOR IN CLAM SHRIMP: THE INFLUENCE OF
MATING SYSTEM ON INTERSEXUAL CONFLICT...... 106
Abstract ...... 106
Introduction...... 107
Materials and methods ...... 112
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Rearing in the laboratory...... 112
Mate guarding in Limnadia badia ...... 113
Compromised mate guarding time across species ...... 114
Statistical analyses ...... 114
Results...... 115
Mate guarding in Limnadia badia ...... 115
Compromised mate guarding time across species ...... 117
Discussion ...... 120
Acknowledgements...... 128
V. MATE GUARDING BEHAVIOR IN CLAM SHRIMP: A FIELD APPROACH.129
Abstract ...... 129
Introduction...... 130
Materials and methods ...... 135
Results...... 140
Mate guarding time ...... 140
Male time budgets...... 145
Male takeovers ...... 147
Discussion ...... 148
Acknowledgements...... 154
VI. GENERAL DISCUSSION AND CONCLUSIONS ...... 156
REFERENCES ...... 164
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LIST OF FIGURES
Figure 1.1 Variation in maximum acceptable conflict costs for males and females in relation to time (female molt cycle)...... 33 Figure 2.1 Scheme of the arena used in Project 1 and 2...... 55 Figure 2.2 Mean distance from origin of coordinates (control) and the chamber with opposite sex (stimulus)...... 62 Figure 2.3 Total distance traveled by each sex in absence (control) and presence of other clam shrimp (stimulus) ...... 63 Figure 2.4 Number of mate guarding attempts performed by males on receptive and non receptive hermaphrodites...... 65 Figure 2.5 Number of mate guarding attempts performed by males on hermaphrodites performing different behaviors...... 66 Figure 2.6 Interaction between hermaphrodite kicking behavior and receptivity state on number of mate guarding attempts ...... 67 Figure 2.7 Interaction between hermaphrodite kicking behavior and treatment on number of mate guarding attempts...... 67 Figure 2.8 Number of mate guarding attempts performed by males on hermaphrodites performing different behaviors (analyzed separately)...... 69 Figure 2.9 Scheme of the interactions between the sexes during mate guarding...... 78 Figure 3.1 Reduction in gut fullness as a function of mate guarding duration...... 91 Figure 3.2 Correlation of size difference between the sexes with mate guarding time...... 92 Figure 3.3 Mean mate guarding time for three size categories...... 93 Figure 3.4 Mean mate guarding time for each type of experiment ...... 95
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Figure 3.5 Mean guarding time for each type of experiment accounting for the receptivity state of the hermaphrodite...... 95 Figure 3.6 Variation in hermaphrodites’ length of receptivity among treatments ..... 96 Figure 4.1 Mean guarding time for each type of treatment in Limnadia badia ...... 116 Figure 4.2 Mean guarding time for four species characterized by two mating systems ...... 119 Figure 4.3 Mean guarding time for mating systems...... 119 Figure 5.1 Leverage plot of mean male size and length of mate guarding...... 143 Figure 5.2 Leverage plot of mean relative female density and length of mate guarding...... 143 Figure 5.3 Correlation between population density and size for males and females...... 144 Figure 5.4 Relationship between male and female size for couples observed in mate guarding behavior ...... 145 Figure 5.5 Effect of female receptivity on mate guarding duration ...... 146 Figure 5.6 Effect of mating result on mate guarding duration ...... 146 Figure 5.7 Male time budget...... 147 Figure 5.8 Male and female size in trios...... 148 Figure 6.1 Flowchart of possible interactions between sexes...... 157
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LIST OF TABLES
Table 1.1 Traditional models of sexual selection...... 3
Table 1.2 Predictions from different models of sexual selection...... 16
Table 1.3 Costs of mating...... 17
Table 1.4 Data of mate guarding from literature...... 24 25
Table 2.1 Repeated measures MANOVA results for mean distance from reference side and total distance traveled...... 62
Table 2.2 Full factorial ANOVA for mate guarding attempts ...... 65
Table 2.3 Two way ANOVA for mate guarding attempts...... 69
Table 3.1 Blocked one way ANCOVA on variation of gut fullness between the sexes...... 91
Table 4.1 One way ANOVA on mate guarding time...... 116
Table 4.2 Comparison among the four species for type of mating system ...... 117
Table 4.3 Two way ANCOVA on mate guarding time among species...... 118
Table 4.4 Analysis of the strength of intersexual conflicts during mate guarding in dioecious vs . androdioecious species...... 125
Table 5.1 Summary of data from the field ...... 136
Table 5.2 Statistical analyses...... 141
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ACKNOWLEDGEMENTS
This thesis would have not been possible without the encouragement, support, and help of many people. It is a pleasure for me to thank all of them here.
First and foremost, I would like to thank my advisor, Dr. Stephen Weeks. Steve, thank you so much for your inspiration, for your continuous support, for finding always the time for my never ending lists of questions, and for reading this thesis over and over.
You have been much more than an advisor to me, you have been a real mentor. I wish to thank also all my committee members. Dr. Randy Hoeh, Dr. Mark Kershner, Dr. Lisa
Park, thank you so much, for all your time, your comments, and helpful feedback.
I am deeply indebted to Dr. Brenton Knott and all the people at the School of
Animal Biology at the University of Western Australia, in Perth. Brenton, thanks for the wonderful hospitality, your expertise, the wine and Brie cheese, all the dinners, and the endless conversation on crustaceans. Thanks for sharing your love for classical music. I ca not hear a piece now, without thinking of you. Your help moved beyond my permanence Down Under: I am very grateful for all the reviews on my papers and thesis.
Kerry, Debra, Danny, Magdalena, Wally, Bonnie, Rob, you all made my time spent in
WA an invaluable experience.
I was very lucky to spend three summers working in Las Cruces, NM. I and the rest of the “clam shrimp crew” were exceptionally welcomed by Dr. Naida Zucker and
Dr. Richard Spellenberg, and adopted in Dr. Michele Nishiguci’s lab. Naida and Rich,
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thank you so much for your help, your humor, your great parties, the trips to Mexico, and for coming to the seminar where I presented my data (that was the best surprise ever!)
Nish, you and your lab assisted us so much with all the field trouble and your wonderful company helped us survive all the frustrations when things were not working right.
Vinod, Bryan, TuShun, Will, thank you so much for your friendship. And thanks are due to the rest of the friends, to all the Department of Biology at NMSU (thanks for the lab space) and to Eddie Garcia and all the people at the Jornada LTER.
In these years I have also benefited a lot from the extensive knowledge and expertise of faculty members at Kent State University and the University of Akron. I would like to thank them all, and in particular Dr. Joel Duff, Dr. Francisco Moore, Dr.
Peter Niewiarowski, Dr. Brian Bagatto, Dr. Todd Blackledge, Dr. Patrick Lorch, which were a great help for many of my statistical, behavioral, and methodological questions.
Thanks also to Dr. Robert Huber, “Lobsterman,” at Bowling Green State University, for showing me the possibilities of Java.
My life would have been miserable and even more complicated without the help, support, and friendship of Sadie and Alissa, precious companions in the lab and field adventures. Sadie, we have shared so many adventures from the very beginning. You are a true and wonderful friend. I wonder what I would have done without you. Alissa, I will never forget the crazy last month of our adventure in Australia! I would like to express my thanks to the other members, past and present, of the clam shrimp crew. Tom, thanks for all your great advice to rear clam shrimp; Christine, thanks for your help with the
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behavioral observations; and Beth, thank for your dedication and you cheerful mood in the lab.
I enjoyed the company of great friends in the Department of Biology at the
University of Akron, every day, from early morning to late night, during the weekends and holidays. Thank you all so much, in particular to the students (residents or visiting) of the Fifth Floor. Kb, Drew, Cecilia, Jackie, Steph, Chris, Jarod, Ben, Hope, Lara, Tim,
Mike, Pete, Stephanie, Brian, Jen, Erin, and all the others; we have drunk endless cups of coffee together, and laughed so much and complained about everything! Thank you, I will miss you all very much. Ashley, thanks for all your help with teaching; I was so scared when I started!
My roommates have been incredibly patient with me. Taneisha, thanks for being the wonderful friend you are, on top of being my personal analyst! Alissa and Bryn your company have been such a relief during weeks of stress, and when I was tired and worried. And thanks to the Running group, who helped me to release my stress in the best way. Carol, without your training I would have not ever thought of finishing a marathon!
You have been at the finish line of all my marathons, including the last one (the writing marathon): thanks!
This thesis was improved by conversations with a large number of faculty members and fellow students. I would like to acknowledge all of them and all the many undergraduate students that have helped with many experiments (and are reported throughout the thesis in specific chapters). I might not have included every single name
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here, but I have great appreciation and recognition of all the people who helped in one way or another during my time spent in the US preparing and writing my dissertation. All of you have played a decisive role in my life, and not just in my academic career.
Finally, but most importantly I wish to thank my entire family in Italy, whose constant encouragement and love, telephone calls, e mails, letters, postcards, parcels, and videos have helped me to continue this endeavor. Grazie di cuore. In particular I would like to thank my parents and my sister for their incredible support over all these years.
My grandma Pia is not here to see the end of this adventure, but she has always been supportive and happy for me even if I was far away from home.
To you all my most heartfelt thanks.
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I. GENERAL INTRODUCTION
Sexual selection and sexual conflict
The theory of sexual selection was proposed by Darwin (1859) in order to address the evolution of extreme male secondary sexual characters and displays that are costly to males but important for successful mating. Darwin clearly differentiated the concept of sexual selection from that of natural selection:
“[Sexual Selection] depends, not on a struggle for existence, but on a struggle between the males
for possession of the females; the result is not death to the unsuccessful competitor, but few or no
offspring. Sexual selection is, therefore, less rigorous than natural selection. Generally, the most
vigorous males, those which are best fitted for their places in nature, will leave most progeny. But
in many cases, victory will depend not on general vigour, but on having special weapons, confined
to the male sex” (Darwin, 1859 chapter IV page 88).
Since then, sexual selection has been characterized as a selective process based on variance in reproductive success (Darwin, 1871; Wade, 1979; Arnold & Wade, 1984a;
Arnold & Wade, 1984b; Shuster & Wade, 2003).
“[Sexual selection] depends on the advantage which certain individuals have over others of the
same sex and species solely in respect of reproduction” (Darwin, 1871 chapter VIII page 224).
Darwin’s original analysis of sexual selection focused on direct intra male competition and female choice. The second of these two mechanisms especially encountered an initial formal resistance (e.g., Wallace, 1889; and see review in O'Donald, 1980; Andersson,
1994), and criticism continued over time (e.g., Huxley, 1938a; Huxley, 1938b). Fisher
(1930) supported the idea of female choice and postulated a mechanism a runaway
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process that can explain the evolution and maintenance of exaggerated phenotypes in males merely by female preference. He never theoretically formalized the concept
(O'Donald, 1980), but his insight was fundamental for the development of modern sexual selection theories (Andersson, 1994). Another important milestone was a study by
Bateman (1948) where he considered anisogamy and the difference in energetic costs sustained by males and females in producing gametes. Trivers (1972) extended this idea beyond gametic production to total parental investment (gestation, nourishment, protection, etc.), recognizing an asymmetry between sexually reproducing parents when one sex provides more parental care than the other.
Differences between the sexes are not limited to traits linked to general parental effort or size, energy, and motility of gametes. Other traits, including behavioral traits, can differ between the sexes. Moreover, sexual selection models (which until recently considered female choice as marginal and confined to mate choice) recently started to confer more importance to the role of females in shaping the evolution of morphological and behavioral secondary sexual characters. Female input also includes mating resistance
e.g., studies on fruit flies (Wigby & Chapman, 2004) and crustaceans (Jormalainen &
Merilaita, 1995; Sparkes et al., 2000) and “cryptic female choice” (Eberhard, 1996), wherein the choice is performed by the female after copulation, selecting which sperm to use for fertilizing eggs. In this latter case, a sexual partner not rejected during copulation can be later rejected to sire the offspring.
Through time, many models of sexual selection have been proposed, each one addressing the topic from a different perspective but focusing on single parameters at a
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time. This is not surprising, due to the complexity and extreme variability of the subject.
Table 1.1 presents a list of some classic models of sexual selection (for more detailed reviews see Kirkpatrick, 1987; Kirkpatrick & Ryan, 1991; Andersson, 1994).
TABLE 1.1 TRADITIONAL MODELS OF SEXUAL SELECTION
SELECTION SELECTION MODEL MECHANISM ON ♀ ON ♂ REFERENCES PREFERENCE TRAITS Material benefits (nuptial gift, parental care, protection, high Direct increase in Direct (Thornhill, 1976; fertilization success, survival and fecundity Direct positive Price et al., 1993) avoidance of for females parasites/disease transmission) Arbitrary preference (Fisher, 1930; by females / offspring Run away process and Indirect Weatherhead & attractiveness – Direct “sexy son hypothesis” positive Robertson, 1979; linkage disequilibrium Lande, 1981) – genetic correlation (Zahavi, 1975; Good Genes: Handicap – Zahavi, 1977; Indirect Parasite – Honest Offspring quality Direct Hamilton & Zuk, positive indicator 1982)
Brief summary of some traditional models of sexual selection, with a short outline of the evolutionary mechanisms and explicit mention of the direction of selection on female preference and the resulting selection on male traits. Direct selection: selection on female preference is influenced by benefits to the individual under selection herself; indirect selection: selection on female preference is influenced by benefits to the offspring of the individual under selection.
In Table 1.1, the terms “direct” and “indirect” are used to specify if selection on female preference is influenced by benefits to the individual under selection herself or to her offspring (Kirkpatrick, 1996). In this second category are included mechanisms of choice for arbitrary traits that results in increased male offspring attractiveness to females
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(“sexy son”), possibly started by sensory exploitation processes (Ryan, 1990), or traits indicating high quality of males (“good genes”). In all these models it is assumed that there is a positive correlation between male and female benefits.
The brief and incomplete summary outlined herein is meant to point out the development of sexual selection theory and to underscore an historical change in perspective. Nevertheless a pluralistic approach is necessary to identify, characterize, and define sexual selection more completely at different levels (behavioral, morphological, and molecular). It is likely that the hypotheses formulated thus far are not sufficient to explain all cases of sexual selection, and possibly these hypotheses are not mutually exclusive.
Recently, increased attention had been devoted to sexually antagonistic selection, determined by the recognition of “intersexual conflicts.” This is not a novel idea, but the recent theoretical models and the use of empirical innovative tools have renewed interest in the topic. Interestingly, Fisher (1930), in his analysis of sexual selection, pointed out that the two mechanisms identified by Darwin (the battle of the males and the “ bold hypothesis” of female preference) involve competition “ confined to members of a single sex ” (page 132). Competition can also arise between the two sexes. Bateman (1948) introduced an analysis of sexual differences (at least for gamete production), Williams
(1966) stated that “ inevitably there is a kind of evolutionary battle of the sexes ” (page
184), and Trivers (1972) considered parental conflicts over investment on the production/care of offspring. Dawkins (1976) reinforced the idea, in his theory of gene
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selfishness, and Parker (1979) formalized the concept defining sexual conflict as “ a conflict between the evolutionary interests of individuals of the two sexes. ”
Although the concept is not new, intersexual conflict has recently become the subject of much debate (Arnqvist & Rowe, 2002; Cameron et al., 2003; Chapman et al.,
2003; Cordero & Eberhard, 2003; Pizzari & Snook, 2003; Arnqvist, 2004; Pizzari &
Snook, 2004; Arnqvist & Rowe, 2005; Eberhard, 2005; Hosken & Stockley, 2005;
Chapman, 2006; Parker, 2006). This debate focuses on how to test intersexual antagonistic selection and how to untangle it from other forms of sexual selection. In the next sections I will define intersexual conflict, analyze its outcome, and review the proposed methods to test it.
Components of sexual conflict
Sexual selection was initially developed to address the evolution of extreme male secondary sexual traits and extravagant displays (Darwin, 1859; Darwin, 1871;
Andersson, 1994). Classical models (Table 1.1) focused on this evident result of sexual selection, looking for evolutionary mechanisms to explain the development of such traits, which are often costly to the male but important for successful mating. The general idea is that females who are attracted to these males are going to gain a benefit (direct or indirect). There is a positive feedback that enhances male and female fitness. The alternative view of antagonistic selection considers a different scenario: the increase in fitness of one sex does not necessarily mean an increase in fitness in the opposite sex
(Daly, 1978; Parker, 1979). Conflicts are then expected between mates. In general, even
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related individuals who share genes are prone to experience conflicts (e.g., parent offspring, Trivers, 1974; Wade & Shuster, 2002). Conflicts are even greater between unrelated mates.
A quantitative paradox appears when comparing sexual selection to natural selection: how can sexual selection, acting at the microevolutionary level, produce such strong macroevolutionary results, when it opposes natural selection, which is a stronger evolutionary process? This “quantitative paradox of sexual selection” ( sensu Shuster &
Wade, 2003) can be solved by analyzing fitness variance between the sexes: “ the greater the variance in fitness, the stronger the force of selection ” (Shuster & Wade, 2003;
Shuster, 2007). Similarly, sexual conflicts can be addressed by analyzing the fitness covariance between the sexes (Price, 1970): sex allows for a negative covariance between the sexes (the fitness of one sex increases at the expenses of the other). This, then, introduces antagonistic selection (Partridge & Hurst, 1998) and the resulting antagonistic coevolution. The uncoupling of fitness between the sexes can increase variance, so that sexual selection can have a stronger influence than natural selection.
Sex enhances conflict because sexually reproducing organisms combine their genes during syngamy while in asexually reproducing organisms there is no mixing of genes but, rather, genes are perpetuated unaltered (with the exception of mutations) and thus there is less potential for conflict (Partridge & Hurst, 1998). Moreover, genetic recombination, through crossing over, further changes the relationships between genes on the same chromosome. In this way, alleles on the same chromosome are not persistently associated with one another or with non homologous alleles; this flexibility enhances
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genetic variation in the offspring, creating different genetic combinations and allowing alleles to coevolve in opposite directions (Rice & Holland, 1997; Partridge & Hurst,
1998). Genes can thus diverge in response to different selective pressures.
Genetic conflicts can occur in the same individual (intragenomic conflicts between alleles or non allelic genes) or between different individuals (intergenomic conflicts). One can identify an intragenomic conflict when “ the spread of one gene creates the context for the spread of another gene, expressed in the same individual, and having opposite effect ” (Hurst et al., 1996, page 325). There is a long series of studies on this topic, ranging from meiotic drive, segregation distorters, and uniparentally transmitted genes (excellent review in Hurst et al., 1996) to recent studies on genomic imprinting (Haig, 2000), where certain genes are expressed differently depending on their parental origin, either maternal or paternal (carrying an imprint from the previous generation).
As far as intergenomic conflicts are concerned, intralocus conflicts will emerge in alleles carried by both sexes but with sex limited expression, or with different optima in the two sexes; interlocus conflicts manifest in genes at different loci, specific for sexual behaviors, differential expression in genital organs, or gametes (Rice & Holland, 1997;
Rice, 1998). With few exceptions (e.g., Chippindale et al., 2001), recent studies on sexual selection have focused on Interlocus Contest Evolution (Rice & Holland, 1997). The mechanism postulated is similar to a Red Queen process (Rice & Holland, 1997; Rice,
1998) involving an arms race with an antagonistic coevolution of molecules and receptors, possibly initiated by sensory exploitation. The end result resembles the
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Fisherian run away process: males take advantage of a sensory bias in females, females evolve resistance to stimulation in order to counteract this manipulation, males need to increase their signal to overcome female resistance, in a chase away sexual selection scenario (Rice & Holland, 1997; Holland & Rice, 1998). In this model, direct costs and their avoidance, rather than direct or indirect benefits, are driving selection. In support of their thesis of “ antagonistic seduction ,” Holland and Rice (1998) provided a review of studies encompassing a variety of taxa, from spiders, to frogs, to fish, to birds. Since then, many more studies have taken into consideration the hypothesis of a coevolutionary arms race between sexes (Arnqvist & Rowe, 2002; Rowe & Arnqvist, 2002; Pizzari &
Snook, 2003; Arnqvist, 2004; Eberhard, 2004; Pizzari & Snook, 2004; Wigby &
Chapman, 2004; Eberhard, 2005; Friberg, 2005; Friberg et al., 2005; Rice & Holland,
2005; Rowe et al., 2005).
Intersexual antagonistic coevolution is thought to be particularly strong in sexual organisms with a promiscuous mating system and internal fertilization (Rice, 1998). In this situation a three way interaction is sometimes identified between “male offence,” female resistance (which has been described before), and “male defense” or inhibition of the female propensity to mate again with other males (Rice & Holland, 1997; Rice, 1998;
Rice, 2000). Herein the runaway process should, by definition, produce an accelerated rate of evolution. This has actually been observed, for example, in the evolution of male genitalia in species with internal fertilization (Eberhard, 1985; Eberhard, 1996) and in proteins in gametes (e.g., Landry et al., 2003: sperm egg binding proteins in sea urchins).
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Evolutionary conflicts are thought to be ever present and they can play a role in speciation (Parker & Partridge, 1998; Rice, 1998; Martin & Hosken, 2003), the evolution of genetic systems (Hurst et al., 1996), and the evolution of mating strategies (Eberhard,
2005). This antagonism can influence the evolution of courtship displays, the rate of mating, fertilization processes, precopulatory and postcopulatory interactions, the choice of mating time and place, the risk of predation, and/or transmission of parasites/diseases.
Of course this antagonistic coevolution affects the interactions between the sexes and can result in an arms race between males and females.
The outcome of the war of the sexes: who wins?
Intersexual conflicts present all the elements for an arms race: is there a winner?
Conflict resolution is not always possible. Sometimes the arms race will continue in cycles of temporary success for one sex, until a counter adaptation spreads. This cycling is predicted to depend on the frequency of the appearance of new mutations (Partridge &
Hurst, 1998).
Theoretically there are different possible outcomes for intersexual conflicts.
Traditionally, two outcomes have been considered. Outcome 1 Resolution of the conflict: either there is a winner (one sex overcomes the other) or a balance (or more likely a compromise) is reached. In this case, an evolutionarily stable strategy ( sensu
Maynard Smith, 1982) is achieved. Outcome 2 Escalation (runaway process), with adaptations and counter adaptations: the limit is reached when the trait or behavior under
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selection results in fitness disadvantage for at least one sex. This is the mechanism involved in the evolution of extreme male secondary sexual characters.
It is not easy to differentiate between outcomes 1 and 2 because the apparent resolution of the conflict could merely be a moment of stasis during the escalation trajectory. The term “resolution” itself does not imply that the conflict is over. It may only refer to the evolution of an adaptation in one sex and its relative efficacy compared to an adaptation in the other sex (Arnqvist & Rowe, 2005). A possible way to distinguish the two outcomes is to look at the morphological characters under selection: usually extreme male secondary sexual characters evolve under the runaway process. Also, resolution (Outcome 1) is sometimes reached through the evolution of a novel trait while escalation (Outcome 2) often results in a continuous modification of the male and female traits under selection.
Recently an alternative result of the “battle of the sexes” has been proposed
(Rowe et al., 2005). Instead of an ongoing arms race determined by female biases, in the form of preference (as hypothesed in classical models) or resistance (as postulated in the antagonistic coevolution model), Rowe et al . (2005) proposed the evolution of
“indifference” as a female response to male signals. Female sensitivity to male traits can change with time. In this case there is no longer a correlated response to variation in signal and escalation is then less likely to evolve. Their view of male female interaction is more complex than previously postulated: female sensitivity (and thus the possibility of being manipulated) can increase or decrease, as can female resistance, male persistence, and the expression of certain male traits (Rowe et al., 2005). In this case the escalation
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can be arrested (since females are no longer positively feeding back into the process), can progress (if males try to obtain the desired response, exceeding the female threshold of susceptibility), or can shift to different traits (until female indifference evolves for that trait as well).
Males usually invest less than females in offspring and attempt to fertilize many females while trying to inhibit re mating of their mates. Therefore, they often start the cycle of coevolving antagonism: they evolve adaptations that enhance their own fitness at the expenses of female fitness (Rice, 2000). Females then need to evolve counter adaptations and in the meantime males are gaining more fitness.
Females sometimes can (at least temporarily) win the battle. This can happen when sexual selection is “less rigorous than natural selection” (Darwin, 1859 chapter VI, page 88). The relative strength of natural vs . sexual selection probably fluctuates, but when natural selection is stronger, it will act differently on the two sexes: the extravagance of male characters will be restrained, while a weaker control by natural selection will act on female attraction or resistance (Holland & Rice, 1998). Exaggerated male ornaments can be highly costly to males and can reduce male viability and therefore are under strong selective pressure. Female choosiness is not as deleterious to females, and selection is not as strong. When this happens, it is easier for females to “win” the battle of the sexes.
Females can gain a partial advantage even when they lose, in a form of Fisherian payoffs, via her sons (Eberhard, 2005): their male offspring gain fitness at the expense of female offspring’s fitness. This is because the male offspring of a female will inherit the
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traits that were detrimental to the female herself, but that now are going to be advantageous to her sons, for mating success. The process is similar to the sexy son hypothesis or the Fisherian model (i.e., the indirect benefit models), but starts with an initial direct reduction in female fitness. A net balance between the costs involved in mating and the direct and indirect fitness benefits through offspring need to be calculated.
A partial gain for females is not always achieved. In some cases, females cannot possibly win: the gain in fitness through the offspring still cannot compensate the loss of fitness due to male mating behavior, as in the case of sexual harassment or sexual coercion. In this situation the best strategy for the females is to try to minimize costs
(Wilcox, 1984; Clutton Brock & Parker, 1995).
According to game theory and optimality models, the outcome of such a war of attrition is determined by relative “power” (Parker & Partridge, 1998), i.e., how much one sex is “superior” to the other and how much difference in costs of contest there is between the sexes. Together with relative “power,” the “value of winning” (Parker &
Partridge, 1998) plays a major role. This value represents how important victory is, calculated in fitness differences between a mating success or failure. Ability of assessment of the other side’s armament is presupposed. On the contrary, runaway processes are unpredictable: the direction of the process depends on the counter adaptations taken from the contenders involved, so it is difficult to make predictions on who the winner will be (Kirkpatrick & Ryan, 1991).
The major problem in addressing intersexual conflict with the same tools used in any other type of conflict (i.e., looking for a winner or applying game strategy rules) is
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that this conflict is different from any other interaction because of the mixing of the genomes of the parents in the offspring. This mixing produces a unique situation not found in other interactions. In reality, the battle is at the genetic level and no single sex can actually win; but alleles related to male persistence or female resistance can (Rice,
2000; Arnqvist & Rowe, 2005; Cordero & Eberhard, 2005).
The recognition of conflicts can be particularly hard if we are observing the situation in a moment of temporary compromise, when fitness effects of adaptations and counter adaptations balance one another. Residuals of past conflicts can still be present and a phylogenetic approach is recommended to detect variations between closely related species (Holland & Rice, 1998; Arnqvist & Rowe, 2002). When the process becomes extraordinarily costly and reduces the total fitness of one sex, a resolution is advantageous for both sexes because the average fitness of the two sexes is not independent. Diversification of morphological traits is easy to perceive and assess, but many other traits can be under sexual selection pressure. At the molecular level, costs are probably lower and arm races are expected to last longer than at the morphological or behavioral level (Hosken & Stockley, 2005). Behavioral traits are particularly important and they can vary from courtship displays to mating strategies.
Testing sexual conflict
Sexual conflicts are ubiquitous, but do they play a role as a selective force and if so, how big a role (Partridge & Hurst, 1998; Hosken & Snook, 2005)? Testing the models is the real challenge. There is a lack of resolving power between different models.
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Moreover, different mechanisms depicted by different models can act concomitantly.
Also, it is hard to test sexual selection models because it is not easy to measure fitness,
“the effective design for reproductive survival ” (Williams, 1966 page 158), and net fitness in particular (inclusive of the fitness of the offspring, as implied in the “sexy son” hypothesis).
Sexual selection with direct benefits involves a material contribution by the male
(in terms of food, energy, protection, parental care, etc.) that increases female reproductive output; indirect benefits are those which enhance female fitness through the fitness of offspring. Direct selection is considered easier to test (Kirkpatrick & Ryan,
1991): direct benefits are easier to measure and often involve costs for males and fitness increases for females. Indirect selection hypotheses predict that male traits and female preference coevolve together (either in a mutualistic or antagonistic fashion) in a way that enhances offspring survival and reproduction. The separation between the Fisherian model and all the good genes models is somewhat arbitrary. There is a forced distinction in indirect benefits through sexy characteristics (arbitrary traits that are attractive to females) and indirect benefits through traits favoring survival. Since both models assume an increase in reproductive fitness in the offspring, there should be no reason to differentiate the processes (Eshel et al., 2000; Kokko et al., 2003) even though not everybody agrees (Cameron et al., 2003). Also, sensory bias (an exploitation of the female sensory system by exaggerated male traits) can initiate any of the indirect models, as well as the antagonistic coevolution model, and runaway processes can be consistent with different hypotheses of indirect selection.
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In a very simplistic and misleading way, direct and good genes models are seen as adaptationist models, while the Fisherian runaway model starts with an “arbitrary” preference (and for this reason has been considered a “non adaptationist” model) and continues because of genetic correlation (by linkage disequilibrium, physical linkage, or pleiotropic effects; Kokko et al., 2003). In reality, the two hypotheses are not mutually exclusive, especially in the view of sexual selection as a continuum (Kokko et al., 2002).
As traditionally posed, sexy son and Fisherian models could be at the end of a spectrum, but in reality there are many intermediate situations that assume variable levels of viability versus attractiveness of the offspring (Kokko et al., 2002; 2003).
In order to test models, there is the need to identify a clear null hypothesis that can separate outcomes due to sexual conflicts versus other processes. In a coevolving chain of development, each trait influences an adjustment of another trait in the opposite sex. Therefore the rate of evolution in genes involved in reproductive aspects is expected to be faster than other genes not involved in reproductive aspects (Rice, 1998). Holland and Rice (1998) differentiated their chase away model from other, similar interacting mechanisms (such as the Fisherian run away model) because of the antagonistic nature of the interaction: adaptations in the two sexes evolve in opposition, rather than in self corroboration. Because of this contrast, they predict a stationary or slow progression in the development of traits, rather than a reinforcing, almost exponential sequence.
Phylogenetic reconstructions need to be employed to distinguish the chase away model, because both male and female adaptations are dynamic and studying them at a static point of time can confound the overall coevolutionary process (Holland & Rice, 1998; Arnqvist
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& Rowe, 2002). Also, such a war of attrition should result in a decrease of female fitness, rather than the increase assumed by other models.
It is important to state clearly the predictions expected from the model (Table
1.2). Problems with this approach have been outlined recently (Rowe et al., 2005): if there is a weak selection on female sensory systems, female fitness might be left unaltered even in the presence of sexual conflicts. Other approaches have been used to test the predictions of sexually antagonistic models: measuring costs, using a comparative approach, using artificial selection, analyzing different populations and population crosses. I will outline these briefly below.
TABLE 1.2 EXPECTED PREDICTIONS FROM DIFFERENT MODELS OF SEXUAL SELECTION
MODEL MECHANISM COSTS OF BENEFIT TO ♀ REPRODUCTION COST TO ♂ Direct benefits Direct positive selection Direct > increase in COST TO ♀ fitness Run away process, COST TO ♂ Indirect “sexy son Indirect positive selection. Self > increase in hypothesis,” Good reinforcing mechanism. Fast process COST TO ♀ fitness Genes hypothesis Indirect Direct negative selection. Antithetic COST TO ♂ possible Antagonistic selection mechanism. Stationary or slow < decrease in process COST TO ♀ fitness Brief summary of differences between traditional models of sexual selection and antagonistic selection.
1) Measuring costs The main difficulty for testing and separating traditional sexual selection from sexual conflict lies in the problem with identifying the costs involved in reproduction and assigning their relative value between the sexes.
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TABLE 1.3 COSTS OF MATING
COSTS ♂ ♀ ♂ REFERENCES ♀ REFERENCES (Chapman et al., 1995; Seminal fluid Gems & Riddle, 1996; Rice, Physiological Y toxicity 1996; Civetta & Clark, 2000) (Manning, 1975; Elwood (Parker, 1979; Arnqvist, Energy expenditure Y Y & Dick, 1990; 1989; Rowe, 1994; Jormalainen et al., 2001) Jormalainen et al., 2001) Risk of (Daly, 1978; Kirkpatrick & transmission of Y Y Ryan, 1991; Chapman et al., parasites, diseases 1995) (Rowe, 1994; Watson & Behavioral Loss of time or Lighton, 1994; Clutton (Clutton Brock & energy caused by Y Y Brock & Parker, 1995; Langley, 1997) persistent courtship Clutton Brock & Langley, 1997; Watson et al., 1998) (Ridley & Thompson, Takeovers by other Y 1979; Jormalainen et al., males 1994a) Carrying a partner Y Y (Watson et al., 1998) (Rowe et al., 1994) Reduction in fecundity Y (Jormalainen et al., 1994a) Post copulatory refractory period Y (Hunte et al., 1985) Fecundity Loss due to (Parker, 1974; Hunte et withdrawal for Y al., 1985) further searching Restricted feeding (Robinson & Doyle, Y suboptimal diet 1985) (Grafen & Ridley, 1983; Jormalainen & Merilaita, 1993; Jormalainen et al., Energetic cost of Resistance Y 1994a; Jormalainen & resistance Merilaita, 1995; Sparkes et al., 2000; Sparkes et al., 2002) Survival Y (Friberg & Arnqvist, 2003) Restricted feeding Y Y (Sparkes et al., 1996) (Rowe et al., 1994) sub optimal diet Survival (Strong, 1973; Parker, 1979; (Strong, 1973; Ridley & Wickler & Seibt, 1981; Increased predation Y Y Thompson, 1979; Wickler Arnqvist, 1989; Rowe, risks & Seibt, 1981) 1994; Rowe et al., 1994; Arnqvist & Nilsson, 2000) Schematic list of possible costs incurred by the two sexes, with a few selected references.
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In general, mating behavior is costly (Daly, 1978). Table 1.3 presents a list of possible costs of mating assigned to each sex. Identifying the relative costs is the first step in the analysis of a possible asymmetry of the contest and its probable outcome
2) Comparative approach As stated above, antagonistic coevolution can be hard to assess at a specific point in evolutionary time. Comparative approaches across multiple species can reveal the change in relative male female morphological adaptations
(Arnqvist & Rowe, 2002). This type of approach has been applied to water striders.
These insects are considered a model system (Rowe et al., 1994) for antagonistic coevolution. Their reproductive behavior, in the form of intersexual conflicts, has been investigated in detail (Wilcox, 1984; Arnqvist, 1989; Arnqvist, 1994; Rowe, 1994; Rowe et al., 1994; Rowe & Arnqvist, 1996; Amano & Hayashi, 1998; Watson et al., 1998;
Arnqvist & Rowe, 2002; Sih et al., 2002). Water striders engage in pre mating struggles where males harass females and have evolved morphological adaptations to grasp their partners. Because these interactions are particularly costly to females, females have evolved morphological counter adaptations to reduce the efficiency of the grasping devices of males. Male persistence adaptations and female resistance counter adaptations can balance each other, so a behavioral comparative study was performed on 15 species of water striders (Arnqvist & Rowe, 2002). The authors compared different behavioral traits related to pre mating struggles among species, in terms of morphological armaments between the two sexes, and found a marked variation in the relative level of arms (related to the antagonism), but not in the absolute level of arms (due to the
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balancing effect of adaptations and counter adaptations). Thus the comparative approach was able to reveal the relative variation in arms race otherwise not easily detectable.
3) Artificial selection Elegant manipulative experiments have been carried out in
Drosophila melanogaster (Rice, 1992; Rice, 1996; Holland & Rice, 1999; Rice &
Holland, 2005). The promiscuous mating system of fruit flies had been experimentally manipulated in order to either control and arrest female counter adaptations in response to male offense/defense strategies (Rice, 1996) or to enforce monogamy (Holland &
Rice, 1999; Rice & Holland, 2005). Male seminal fluid products are toxic to females
(Chapman et al., 1995). Using artificial selection, females can be restrained to counter adapt to male adaptations. A “static female phenotype” is created by removing female offspring of experimental crossings and substituting them with females of the original female stock which have not undergone selection. Meanwhile, males are artificially selected. The result is that male fitness increases while female fitness and survival decreases (Rice, 1996). In forced monogamy, both sexes lessen the strength of antagonism and the result is reduced male persistence and reduced female resistance with an increased reproductive rate in the population. In both cases, the experimental manipulation interferes with the ongoing arms race between male and female Drosophila .
4) Analysis of different populations and population crosses These studies follow the rationale that antagonistic coevolution produces high rates of evolution that are variable in different populations (see in Idotea baltica , Jormalainen et al., 2000). Populations expressing different sex ratios, sexual dimorphism, and synchrony in reproduction present different degrees of aggressiveness and different dynamics in the mating contest.
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Adaptations and counter adaptations vary according to the framework of the population and the social environment. Analysis in allopatric populations has been suggested, with a similar function of phylogenetic comparisons, by Holland and Rice (1998).
The four approaches outlined above can be used to assess and measure sexual conflicts. They can be used individually or in a combined fashion, depending on the study system, and they provide a nice framework of methodologies to test the predictions of theoretical models. Some of these methods are used in this thesis (measuring costs, comparative approach, and analysis of different populations) together with manipulative experiments to measure not only the costs but also the optimal behaviors to maximize benefits for each sex.
Mate guarding and sexual conflict
Conflicts between mates may occur at different levels of biological organization: molecular (with interactions between signals and receptors), morphological, or behavioral
(courtship displays, mating strategies, etc.). Exaggerated traits in males or conspicuous costs for females are usually given as examples of sexual conflicts and arms races between the sexes. In particular, morphological adaptations that allow persistence for males and resistance for females are usually chosen to address the problem. Nevertheless, different behaviors can also evolve in response to antagonistic coevolution between the sexes. Many conflicts can arise over mating decisions other than mate choice. For example, a reduction in fitness can depend on a reduced possibility of mating, high energetic costs of mating, increased risk of predation, or suboptimal use of time.
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Even before the recent renewed interest in the topic, mate guarding has been considered an interesting case of intersexual conflict (Jormalainen et al., 1994b;
Yamamura & Jormalainen, 1996; Jormalainen, 1998; Watson et al., 1998; Härdling et al.,
1999; Jormalainen et al., 2000; Jormalainen et al., 2001). Mate guarding is a male strategy to monopolize mates. It ensures paternity for males when females are rare
(unbalanced sex ratio) or difficult to find, in the presence of high male male competition
(in the form of sperm competition or acquisition of females), or when female receptivity is restricted in time. Mate guarding applies to a broad variety of taxa, from invertebrates to vertebrates, including rotifers (Schröder, 2003), ants (Foitzik et al., 2002), water striders (Amano & Hayashi, 1998), weevils (Harari et al., 2003), beetles (Saeki et al.,
2005), dungflies (Parker & Simmons, 1994), dragonflies (Schenk et al., 2004), damselflies (Zeiss et al., 1999), spiders (Schneider, 1997; Prenter et al., 2003), crickets
(Bateman et al., 2001), isopods (Ridley & Thompson, 1979; Jormalainen et al., 1999), amphipods (Hunte & Myers, 1988; Cothran, 2004), blue crabs (Jivoff & Hines, 1998b), hermit crabs (Wada et al., 1999), lizards (Olsson, 1993; Cooper & Vitt, 2002), birds
(Hasselquist & Bensch, 1991; Komdeur, 2001; Davis, 2002), and primates (Henzi et al.,
1998; Matsubara, 2003).
Mate guarding can be performed before mating in order to ensure a mate
(precopulatory mate guarding), or after mating, in order to prevent the female from re mating (postcopulatory mate guarding). Precopulatory mate guarding involves a conflict between the sexes over the timing and duration of mating.
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Interestingly, Parker, who provided the first theoretical approach to sexual conflict (Parker, 1979), initially analyzed mate guarding almost purely as a “ male time investment strategy ” (Parker, 1974). He defined the strategy as the “ optimum allocation of time ” spent with a female versus searching for other females, in order to maximize the reproductive output of males (Parker, 1974), even though he considered that “ female rejection responce ” can be effective in reducing the optimal persistence duration (page
165). Parker’s analysis took into consideration different types of females (i.e., mated females vs . virgins), different encounter rates, changes in the probability of copulation with time, and costs sustained by males, in order to answer two main questions: when should a male start guarding and for how long? However, the maximization of male reproductive success can result in a reduction of female reproductive success.
Precopulatory mate guarding thus can be a case of inter sexual conflict (Jormalainen et al., 1994b; Rowe, 1994; Yamamura & Jormalainen, 1996; Amano & Hayashi, 1998;
Jormalainen, 1998; Watson et al., 1998; Härdling et al., 1999; Zeiss et al., 1999;
Jormalainen et al., 2000; Plaistow et al., 2003; Cothran, 2004; Härdling et al., 2004).
Precopulatory mate guarding in Crustacea
Precopulatory mate guarding is common in crustaceans because females are often receptive only for a short time after molting (excellent review in Jormalainen, 1998).
Thus females nearing molting are valuable resources to be guarded by males. Many of the initial theoretical models of mate guarding have been based on experiments in crustaceans, especially in amphipods (Jormalainen et al., 1994b; Jormalainen &
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Merilaita, 1995; Hatcher & Dunn, 1997; Dunn, 1998; Hume et al., 2002; Plaistow et al.,
2003; Cothran, 2004) and isopods (Jormalainen & Merilaita, 1993; Jormalainen &
Merilaita, 1995; Jormalainen & Shuster, 1999; Jormalainen et al., 1999; Jormalainen et al., 2000; Sparkes et al., 2000; Jormalainen et al., 2001; Sparkes et al., 2002).
There is high variation in mate guarding modes among crustaceans. Different taxa present variation in precopulatory guarding duration, the possibility of male takeovers
(i.e., when males dislodge other guarding males), the possibility of sperm competition and sperm storage, influence of previous experience (mating history) in the decision of how long to guard, high risk of predation on guarding pairs, effect of male size, and efficiency of female resistance. Some parameters can vary not only between species but also among populations due to variation in environmental conditions (Jormalainen et al.,
2000). Table 1.4 lists mate guarding duration and the above mentioned parameters influencing mate guarding behaviors for various orders of Crustacea (with references from the literature).
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TABLE 1.4 DATA OF MATE GUARDING FROM LITERATURE ISTORY H KEOVERS ATING ALE SIZE RDER EFERENCE REDATION ON COUPLES EMALE RESISTANCE PECIES PERM COMPETITION PERM STORAGE P M TA F M O MG TIME R S S S Hyalella azteca 3 5 days N N N Y (Strong, 1973; Borowsky, 1984; Cothran, 2004; Pascoe et al., 2004; Wellborn & Cothran, 2007; Cothran, 2008b) Gammarus 100 h Y N (Jormalainen & Merilaita, zaddachi 1995) Gammarus 4 days Y Y (Hunte et al., 1985; Dunham et lawrencianus al., 1989; Dunham & Hurshman, 1990) Gammarus pulex Just over a week Y Y N (Ward, 1984b; Ward, 1986; Amphipoda to almost a Bollache & Cezilly, 2004a; month Bollache & Cezilly, 2004b) Gammarus 6 to 28 days Y (Hartnoll & Smith, 1980) duebenii Paracalliope Days N (Sutherland et al., 2007) fluviatilis Artemia salina Hours to days (Belk, 1991; Rogers, 2002)
Artemiopsis sp. Hours to days (Rogers, 2002)
Branchinecta sp. Seconds (Rogers, 2002)
Branchipodopsis Seconds (Rogers, 2002) sp. Chirocephalus sp. Hours to days (Rogers, 2002) Anostraca Eubranchipus sp. Hours to days (Rogers, 2002) Linderiella sp. Hours to days (Rogers, 2002)
Streptocephalus sp. Seconds (Rogers, 2002)
Thamnocephalus Seconds (Rogers, 2002) sp. Harpacticoid and Refs in (Lonsdale et al., 1998) parasitic spp. Copepoda
25 ISTORY H KEOVERS ATING ALE SIZE RDER EFERENCE REDATION ON COUPLES EMALE RESISTANCE PECIES PERM COMPETITION PERM STORAGE F M O MG TIME S S S P M R TA Hymenocera picta* 140 530 h (Seibt & Wickler, 1979; Wickler & Seibt, 1981) Callinectes sapidus Y Refs in (Bauer, 1996; Jivoff & Hines, 1998a)
Decapoda Rhyndocinetes 23 180 min Y Y Y Y (Van Son & Thiel, 2006) typus** Lirceus fontinalis 1 3 days Y N N Y (Sparkes et al., 2000)
Idotea baltica 37 h Y Y Y (Jormalainen et al., 1994b; Jormalainen & Merilaita, 1995) Jaera istri; 3 days or more (Veuille, 1980) J. italica; Isopoda J. nordmanni Asellus aquaticus; 100h; 6 12 days Y N Y Y/N (Manning, 1975; Ridley & A. meridianus N Thompson, 1979; Manning, 1980; Jormalainen & Merilaita, 1995) Eulimnadia texana From minutes N (Weeks et al., 2004; Weeks & up to 2 hours Benvenuto, 2008) this thesis Eulimnadia dahli From minutes This thesis up to 3.5 hours Limnadia badia From minutes Y This thesis
Spinicaudata up to 20 hours Limnadopsis tatei 6 23 hours This thesis * Modified extended MG; ** “Cage stage” (males keeps the female between their pereopods) Scheme of mate guarding parameters from different crustacean species from the literature. MG time: duration of mate guarding; Takeover: possibility for males to dislodge other males during a guarding event; Sperm competition: possibility for competition in the female genital tract; Mating history: influence of previous mating on successive mating behaviors; Predation on couples: higher risk of predation on mate guarding couples than single individuals; Sperm storage: possibility of sperm storage by females; Male size: effect of male size on mating interactions; Female resistance: undertaking various behaviors in attempts to dislodge guarding males. Models of mate guarding
Mate guarding is a complex behavior that involves the interactions of multiple
parameters. Many of these parameters change through time during a single mating
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event and thus the system is highly dynamic. If we look at it from the male perspective, mate guarding represents a complicated decision making process. Moreover, each decision (made by males and females) influences the decisions of the opposite sex. For all these reasons, mate guarding has been investigated using a variety of different approaches including game theory, optimality models, and evolutionarily stable strategies (Parker, 1974; Seibt & Wickler, 1979; Wickler & Seibt, 1981; Grafen &
Ridley, 1983; Yamamura, 1987; Jormalainen et al., 1994b; Yamamura & Jormalainen,
1996; Jormalainen, 1998; Härdling et al., 1999; Härdling et al., 2001; Härdling et al.,
2004).
A brief summary of some models of precopulatory mate guarding in crustaceans is reported below, which is presented in order to focus attention on changes in perspectives and in methodological approaches to the issue of mate guarding as an optimal mating strategy.
Parker’s model (1974)
As previously mentioned, Parker (1974) was the first to investigate mate guarding from a theoretical point of view. He approached the behavior in an evolutionary framework, since “ time waste (i.e., where an individual spends longer than other individuals to complete a given activity) can constitute a considerable selective disadvantage ” (page 157). The main focus of his paper is male reproductive success and the parameters taken into account are encounter frequency with mates, types of mates, and the possibility of takeovers (one male displacing another during mate guarding). The
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model is based on the assumptions that females are not always receptive and males can assess female receptivity (even just in the form of a “ probabilistic estimate ”).
Parker developed a graphical optimality model involving a game theoretical approach (the successful guarding time depends on the guarding time of other males in the population). According to this model, males should optimize their guarding time, especially in competitive situations, deciding the optimal time to invest in a female instead of searching for another. With higher competition, longer guarding times are expected unless takeovers are common (in this case the investment gained with guarding can be lost if another male is able to takeover a “guarded” female).
Wickler and Seibt’s models (1979 1981)
Seibt and Wickler (Seibt & Wickler, 1979; Wickler & Seibt, 1981) focused their model on the female life cycle (i, interspawn interval, in days) and the frequency of female encounters per day (x). The latter parameter is influenced by population density and operational sex ratio, the “average ratio of fertilizable females to sexually active males at any given time” (Emlen, 1976; Emlen & Oring, 1977). Their elegant and simple model used only those two variables (i and x) to separate three alternative male mating strategies: partner fidelity, mate guarding, and pure searching. Their model requires some assumptions: both sexes breed continuously, female receptivity is not synchronous, and there is only one copulation per spawn. Mate guarding is then advantageous with intermediate encounter rates (low encounter rates favor partner fidelity, while high encounter rates favor a pure searching strategy).
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When male male competition is taken into account, the male tactic changes: with more males competing for access to mates, the encounter rate with females decreases and a mate guarding strategy is more successful than pure searching. Also male ability to detect receptive females can influence the mate guarding tactic: males able to assess the time left to female receptivity can chose specific females (the ones closer to receptivity) in order to decrease guarding time.
Grafen & Ridley’s model (1983)
Following Parker’s approach (1974), Grafen and Ridley (1983) proposed a mathematical model to optimize male fitness. In their model, the influence of male size is taken into consideration. In general, size is important because it is directly related to fecundity in females and to competitive superiority in males (both for male male competition and/or male female conflicts). The authors considered two different scenarios: one where all males are identical and the other where males differ in size and compete directly, usurping females from smaller males. Takeovers, already postulated by
Parker (1974), are explicit examples of direct male male competition: larger males do not experience the same operational sex ratio as smaller males because they can gain access to more receptive females. As a result, when male size enters into the model it can actually influence the available sex ratio. The main hypothesis of Grafen and Ridley’s
(1983) model is that, when takeovers occur, larger males should guard for a shorter period than smaller males. Using the same terminology of the authors, the males’
“guarding criterion ” is optimal when the “ turnover time ” (searching time plus guarding time) is minimal and the rate of copulation is maximal (Grafen & Ridley, 1983). The
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optimization of the guarding period is a prerogative of the bigger males, not influenced at all by the presence of the small ones (takeovers are considered costless in the model) or by the behavior of females (resistance is not included in the model).
Hunte, Myers, and Doyle’s model (1985)
Mate guarding is a decision making process. Hunte et al. (1985) analyzed this process using Bayesian decision theory, an approach based on prior and current probabilities (McNamara et al., 2006). The first piece of information available to males in a population using mate guarding behavior is, according to Hunte et al. (1985), the distribution of non paired females (prior probability). This distribution can be a stable characteristic of the population (and its values innately known by males) or the result of sampling by males in the local area. The second piece of information is the female state
(time to receptivity). The male cannot be certain about this, but this information can be gathered through physical contact with a female (current probability). Moreover, males consider the time already invested in mate guarding. Both past investment and female state change with time in the same direction: the female becomes more valuable
(approaching receptivity) while the male becomes committed to that specific mate because of the time he has already invested. Hunte et al. (1985) thus added the importance of past investment and female distribution in the population to the previous models. Both these variables change the overall mate guarding time in amphipods, and males that have guarded their mates longer will be more determined to persist than males who just started the guarding phase. In fact, during the guarding phase, the time left to
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female receptivity diminishes while the male investment in that particular female increases.
Yamamura’s model (1987)
Yamamura (1987) used the Grafen and Ridley’s model (1983) as a starting point.
Yamamura developed a model for an evolutionary stable strategy (ESS) of males, minimizing searching and guarding time and optimizing the guarding criterion (using sex ratio, encounter rates, costs, and female receptivity as parameters). Mate guarding time is expected to decrease when costs are not high and encounters with receptive females are frequent, in agreement with previous models.
Elwood and Dick’s model (1990)
The Grafen and Ridley (1983) hypothesis, that larger males guard females for a shorter time compared to smaller males, was tested by Elwood and Dick in Gammarus
(1990). In their experiment, a positive correlation between time spent guarding and male size was found: contrary to the hypothesis, larger males spent a longer time in precopula compared to smaller males. The theoretical model hypothesized that, if males are not equal in size, then larger individuals can easily (with no costs) dislodge smaller males and guard females. However, takeovers are not common in Gammarus and they present some costs for the males. Also, guarding itself requires expenditure of energy. The empirical data collected by Elwood and Dick (1990) did not support the previous model, but instead showed that bigger males can better sustain mate guarding costs. In this way large males can perform mate guarding longer than smaller males. The authors then proposed a new model (verbal and graphical), considering the influence of energetic costs
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for large and small animals during mate guarding and postulating size assortative mating and prolonged mate guarding for larger males. Their model was different from the previous models because it explicitly included an energetic cost of guarding for males, and the importance of size (as a measure of available energy) to sustain those costs. With these additions, Elwood and Dick (1990) were able to explain their empirical results better than by using the previous model.
Jormalainen, Tuomi, and Yamamura’s model (1994)
All the models I have discussed thus far focus on the notion that males maximize their reproductive success by optimizing their time (time spent guarding a female instead of searching for another mate). Males should consider the frequency of encounters with females, analyze the female state, compete with other males (in the form of takeovers or guarding time decisions), evaluate energetic costs (based on size), and keep in mind their past investment. In all of these models, the female’s role had been ignored or just briefly mentioned and the conflict over optimal timing strategy between males and females during the guarding phase had not been considered.
Jormalainen et al. (1994b) were the first to explicitly treat mate guarding as a case of intersexual conflict. They observed precopulatory struggles in the aquatic isopod
Idotea baltica , and they interpreted female resistance as a form of mate choice or a conflict on mate guarding duration. Sexes differ in survival and in ability to find mates.
In their analysis, Jormalainen et al. (1994b) developed different fitness gain functions for males and females, using fecundity and survival costs. The comparison between the gain curves implied a conflict. Males tend to sustain higher costs if they lose the chance
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to fertilize a female when she is sexually receptive. For males, long guarding periods reduce this risk. Females, on the contrary, sustain a high cost while being guarded
(Table 1.3). The optimal guarding time does not coincide in the two sexes. From the female point of view, short guarding periods are preferable. Males are willing to sustain longer guarding and their optimal guarding criterion will change due to interaction with females and other males.
Yamamura and Jormalainen’s model (1996)
Yamamura and Jormalainen (1996) used a game theoretical approach to solve a compromised ESS. The evolutionary stable guarding time depends on the different power among the sexes (i.e., size), guarding costs, and sex ratio. The novelty of this result appears in populations where the female receptive cycle is asynchronous. In this case it is not always true that mate guarding time decreases in female biased situations (where males should have high encounter rates). This is because the more common sex (females in our example) is prepared to pay higher costs than usual in order to obtain a mate (and thus females are willing to be guarded for a longer time). Thus, the variation of a single parameter allows for different predictions from the initial models.
Jormalainen’s model (1998)
The results of the previous model (Jormalainen et al., 1994b) were presented in graphical form (Fig. 1.1) in an excellent review of precopulatory mate guarding in crustaceans (Jormalainen, 1998). In this model, costs for each sex are represented in relation to mate guarding duration (in female molt cycle).
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Figure 1.1 Variation in maximum acceptable conflict costs for males and females in relation to time (female molt cycle). t c: compromised guarding time; t f: optimal guarding time for female; t m: optimal guarding time for male; t’ c: new compromised guarding time when one sex overpowers the other. From Figure 2 page 294 in Jormalainen (1998) “Precopulatory mate guarding in crustaceans: male competitive strategy and intersexual conflict”The Quarterly Review of Biology, 73(3): 275 304 ©1998 The University of Chicago; used with permission of the publisher, The University of Chicago Press.
Since costs and benefits of increased mating duration are often asymmetrical, male and female optima may not coincide. In theory, both sexes should gain from a short guarding time, but since males undergo a high risk of losing the mating opportunity, they are willing to guard longer. In crustaceans, timing of female molting determines receptivity for mating (time 0, Fig. 1.1), and as this time approaches, males are maximally willing to incur costs to guard females. Because females also benefit from mating, they do not expend energy to resist males when they are close to molting. As
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time before molting increases, costs to females of being mate guarded increase (i.e., females might experience energetic costs by carrying guarding males, might be limited in their feeding behavior, or be susceptible to higher predation while guarded), and at some point (t f) females are willing to expend energy to avoid being guarded because prolonged guarding is expected to be costly to them. Males are motivated to fight as the cost of losing a female increases (i.e., closer to the female molt;
The time between t f and t m is the time period during which intersexual conflicts over mate guarding is most likely to manifest (Fig. 1.1).
The actual starting time for mate guarding (t c) is a compromise between male and female interests, and is predicted to occur when the curve depicting the male’s willingness to expend energy to guard females crosses the curve depicting the female’s willingness to resist such guarding. Essentially there are three different phases to mate guarding (moving from right to left on the horizontal axis in Fig. 1.1): (a) no initial conflict, (b) initiation of the contest, when males first attempt guarding, but females resist strongly, and (c) a reduction in female resistance leading to eventual contest conclusion as the female molt approaches and copulation takes place (Jormalainen et al.,
1994b; Jormalainen, 1998).
Mate guarding represents an interesting case of behavioral conflict because it is a dynamic system sensitive to environmental conditions, such as male encounter rates with females, contender size, and the possibility of male takeovers. For example, if one sex is smaller than the other (e.g., a smaller female, represented by a dashed line in Fig. 1.1),
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the compromised guarding time is shifted toward the optimal guarding time of the larger sex (t’ c). An opposite shift is predicted when females are larger and thus resistance is higher. These examples can be useful when testing the model in the laboratory or in the field. Moreover, the power asymmetry between the sexes is particularly helpful when using game theory (see Yamamura & Jormalainen, 1996).
Härdling, Jormalainen, and Tuomi’s model (1999)
An ESS may not always be reached. Härdling et al. (1999) developed a model using asymmetric game theory. The tendency to fight (male persistence and female resistance) is considered as a continuous variable, not with fixed constant values. Costs are allowed to change continuously as well. When costs are low and linearly increasing with aggressiveness (symmetrically for both males and females), there is no ESS, but rather an escalation of aggressiveness, with a cyclic tendency for one sex to win
(alternation of males and females winning). In a situation where costs are asymmetric between sexes, a locally stable strategy can be found. When one sex is more aggressive
(asymmetry in fighting), the other sex should be passive. But this situation is just locally stable because aggressiveness is presupposed to change. A huge change in aggressiveness in the passive sex is not expected, but minor changes can occur. If aggressiveness increased considerably, the stability is disrupted and a new cyclic dynamic can start.
When costs are exponentially increasing with fights, both sexes continue to be aggressive
(because they would have no advantage to retreat at that point of the fight). However, since aggressiveness is costly, an ESS is reached. Both sexes are fighting but they are under the control of fitness costs: the aggression cannot escalate to levels too
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disadvantageous for either sex. Again, costs are found to determine the solution of the contest, and this model adds increased complexity, allowing behaviors and costs to change continuously.
Härdling, Kokko, and Elwood (2004)
Another complex model was formulated to take into account numerous variables that vary through time (Härdling et al., 2004). The model reveals a continuum of optimal strategies, in accordance with the combination of many parameters, and takes into account behavioral plasticity as a successful key to resolving mating conflicts. The model also can be used to understand the conflicting results of the above mentioned empirical studies, explaining why big males can either guard females for a shorter (Grafen &
Ridley, 1983) or a longer (Elwood & Dick, 1990) period of time, compared to smaller males. This outcome depends upon the possibility of takeovers and the frequency of encounters. When takeovers are the rule, competition between males is physical, with bigger males displacing smaller ones in order to start guarding later in the female molt cycle, and the Grafen and Ridley’s (1983) model is confirmed. When takeovers do not occur, competition is not direct, but bigger males can afford a longer guarding time because they can better sustain the costs. In addition, by allowing variation in intensity and success of takeovers, a more detailed scale of size classes can be detected (not restricted to “large” and “small”) and males of intermediate size turn out to have different optimal strategies compared to smaller or bigger males.
In general, in all these models, it is apparent how, with time, each model builds on a preceding model and parameters first ignored (for simplicity or for lack of relevance in
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the individual system studied) are added, making the assumptions and the predictions more general, and thus applicable to more taxa (allowing for a comparative approach to mate guarding). The following step consists in testing these theoretical models in the laboratory or in a field setting.
An empirical approach: the economics of mate guarding
What influences the optimal guarding time in the two sexes? The main cost for the male is lost opportunity to fertilize receptive females, but both males and females undergo other costs. To address the “negative fitness covariance” assumed in models of intersexual conflicts there is the need to measure costs and benefits for the two sexes. In studies of mate guarding behavior the essential comparison is between the costs of guarding for the male and the costs of being guarded for the female. These two types of cost are not necessarily equal. Measuring costs is not easy, especially if costs need to be quantified and assigned in relative proportion to the two sexes, because of asymmetry of energy expenditure and fitness consequences. Even when both sexes experience the same type of costs, the magnitude might differ in males and females. Graphically, the fitness lines can change both in slope and intercept (Cordero & Eberhard, 2003).
Increased risk of predation is a cost incurred by both sexes during guarding.
Higher rates of predation during mating (Table 1.3) can be due to increased activity while searching to locate mates (Van Son & Thiel, 2006), changes in microhabitat, reduced possibility of movement during mating, and the fact that two individuals are more conspicuous than a single one (Strong, 1973; Ward, 1986). Mate guarding behavior can
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last for a long time, limiting movements and enhancing conspicuousness, and thus increasing the risk of predation through time. Depending on the species, males or females can be more exposed to predation risk during the guarding phase (Table 1.4).
Mate guarding is costly also from an energetic point of view. Jormalainen et al.
(2001) evaluated energetic costs in terms of energetic demand of storage compounds
(glycogen and lipids) for both sexes, in the isopod Idotea baltica . No energetic costs of mating in terms of energetic compounds were found in males. In contrast, females experienced energetic costs (decreased glycogen levels) and reproductive costs (reduction of egg mass) with longer guarding. In the amphipod Gammarus pulex , energy storage reserves are correlated with pairing in the field, and the cost of guarding for males seems related to female size (larger females are able to resist more effectively) and not by restriction on feeding or guarding duration (Plaistow et al., 2003). Another approach to measuring energetic costs had been used by Watson et al. (1998). In this case, energetic costs of mate carrying and mate resistance behaviors were measured in the water strider
Aquarius remigis using respirometric recordings. Again, being guarded (i.e., carrying a male) and attempting to resist are energetically costly behaviors to females.
Mate guarding may result in changes in food consumption, representing a cost for one or both sexes. For example, the amphipod Gammarus lawrencianus uses gnathopods to manipulate food: during mate guarding, the male uses its gnathopods to hold the female and this reduces the rate of food intake, which results in reduced male growth. In contrast, female feeding is not influenced by mating status (Robinson & Doyle, 1985). In
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this case, the cost to males is not represented by the energetic expenditure of carrying a female, but rather in reduced feeding opportunities.
Female behavior can be costly to males: in Gammarus , size assortative mating is common (Elwood et al., 1987) and the major cost seems to be overcoming female resistance (and not the cost of carrying a female). Female resistance influences mating interactions also in the isopods Lirceus fontinalis (Sparkes et al., 2000; Sparkes et al.,
2002), Idotea baltica (Jormalainen & Merilaita, 1995), and Thermosphaeroma thermophilum (Jormalainen & Shuster, 1999). In particular I. baltica and T. thermophilum females were experimentally manipulated using osmotic stress and a muscular blocking chemical in order to reduce female resistance. As a result, mate guarding was significantly longer than in the control (Jormalainen & Merilaita, 1995;
Jormalainen & Shuster, 1999).
Females themselves expend much energy to resist males (Watson et al., 1998), so much that in Gerris remigis , females can cease resistance and allow a male to guard in order to avoid harassment by other males (Wilcox, 1984), and thus minimize overall costs.
In conclusion, there is a variety of costs involved in a mate guarding event. As shown above, many empirical studies attempted to assess and quantify costs in order to detect the magnitude and strength of intersexual conflicts during mate guarding behavior.
Some costs are sex specific, other are not. However, when both sexes experience the same kind of costs, these costs can still be asymmetric, differentially influencing fitness of males and females.
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Mate guarding in an androdioecious species: how hermaphrodites fit into the equation
The type of mating system can influence the occurrence and strength of intersexual conflicts. In truly monogamous species, theory suggests that there should be no conflicts because the positive fitness covariance results in a mutualistic coevolution.
The genetic interests of males and females coincide, and there is cooperation in reproduction (Andersson, 1994). True monogamy is not very common: molecular tools helped to reveal that even species considered monogamous often perform extra pair copulation (see review in Griffith et al., 2002). Multiple mating can have genetic advantages not only to males but also to females (Jennions & Petrie, 2000).
Intersexual antagonistic selection is theorized to be stronger in sexual organisms with a promiscuous mating system and internal fertilization because of the direct interaction of proteins and seminal fluid between males and females and possibly between multiple mates of the same female (Rice, 1998). This antagonism can influence the evolution of courtship displays, the rate of mating, the fertilization process, precopulatory and postcopulatory interactions, the choice of mating time and place, the risk of predation, and transmission of parasites/diseases. Thus, intersexual conflicts have been analyzed mainly in dioecious and polygamous species. Hermaphrodites represent a unique situation because they can combine, in a single individual, male and female evolutionary interests. In most cases, hermaphrodites are not self compatible and thus still need mates. Thus, costs and benefits related to reproduction will vary depending
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upon whether the animal acts as sperm donor or sperm recipient. When a specific role is preferred, conflicts can arise (Michiels, 1998).
To my knowledge, intersexual conflict has never been specifically tested in mixed breeding systems, such as gynodioecy (coexistence of hermaphrodites and females) or androdioecy (coexistence of hermaphrodites and males). Gems and Riddle
(1996) observed in the androdioecious Caenorhabditis elegans a reduction of hermaphrodite life span up to 50% by outcrossing with males, but not during selfing.
They found costs associated with copulation, but not with egg production, male sperm interaction, or internal hatching of larvae. This excellent example should be further investigated.
Eberhard (2005) suggested using facultative hermaphrodites (organisms where the “ female’s strict need of males to reproduce ” is removed) as a possible test to investigate sexually antagonistic coevolution. Because hermaphrodites do not necessarily need males to reproduce (and thus are less constrained by male counter adaptations), female traits under antagonistic selection should evolve more freely.
Facultative hermaphrodites can avoid “ male imposed costs ,” if they are too high, completely avoiding any interactions with males without reducing their fitness to zero
(which would happen in females under similar circumstances).
Androdioecy is a rare mating system which might allow us better insight into sexual conflicts because of the intermediate nature of this breeding system to that of hermaphroditism or dioecy (Weeks et al., 2006a). Comparing the responses of females to males relative to those of hermaphrodites to males can help to discern differences in
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mate guarding strategies. This represents a unique method to explore the ideas of intersexual conflicts. The comparison of dioecy and androdioecy allows a different approach to this area of study and has the potential to make a valuable contribution to our understanding of conflict over mate guarding.
The study system
Clam shrimp Class Branchiopoda, Subclass Phyllopoda, Order Diplostraca,
Suborders Laevicaudata, Spinicaudata, and Cyclestherida (Martin & Davis, 2001) represent an excellent opportunity to test the predictions of models of mate guarding in laboratory and field settings. Clam shrimp present a variety of mating systems, unisexual or bisexual, including parthenogenesis, cyclic parthenogenesis, hermaphroditism, androdioecy, and dioecy (Sassaman, 1995; Weeks et al., 2008).
My overall dissertation project focuses on precopulatory mate guarding in clam shrimp, investigating differences between dioecious and androdioecious species.
Sediment containing encysted eggs can be easily brought into the laboratory and hydrated. There are many advantages to using this system in the lab: the simple protocol to breed animals, the large number of animals that can be raised, the quick life cycle (a few weeks total), and the possibility to manipulate densities and sex ratios easily. Also, there is high sex ratio variation in natural populations (Sassaman, 1989; Weeks et al.,
2008) which can be used as a factor influencing mate guarding strategies (i.e., natural changes in mate encounter rates). Due to the short life cycle and the high frequency of egg clutch formation, the average mate guarding time is short, from a few minutes to a
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couple of hours in Eulimnadia texana (Weeks et al., 2004; Weeks & Benvenuto, 2008).
Mate guarding time varies among crustaceans and can range from a few hours to up two weeks in amphipods and isopods (Table 1.4). Time spans of days to weeks can be problematic for studying mate guarding under laboratory and field conditions.
Moreover, the clam shrimp system allows me to investigate sexual conflicts in different taxa from those commonly studied so far (isopods, amphipods, and water striders), which has been suggested as being essential for a more complete understanding of these phenomena (Chapman et al., 2003). For all these reasons, clam shrimp could be considered a novel and uniquely informative model system for the study of intersexual conflicts.
Overview of research objectives
My project investigates mate guarding behavior as a case of intersexual conflict. I had four specific objectives, addressed with four different projects, performed in the lab and in the field.
My objectives were to:
I. assess the role of the molting hormone on mate guarding behavior
II. analyze mate guarding time as a compromise between mates’ interests
III. perform a comparison of mate guarding strategies in different species
characterized by different mating systems
IV. study mate guarding behavior in a field setting
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Objective I was developed to address the mechanism that allows males to perform mate guarding in an “optimal” way. This objective was achieved using the molting hormone
20 Hydroxyecdysone and investigating its role as a cue for Eulimnadia texana males to assess hermaphrodite receptivity (Chapter II).
Objective II was developed to investigate whether mate guarding time in E. texana is a real case of intersexual conflict. This objective was addressed in the lab where, under manipulative conditions, I was able to disentangle male and hermaphrodite optimal mate guarding time. I also assessed differential costs for the sexes during the guarding phase and tested variation in mate guarding time under conditions of an asymmetry in power between the sexes by matching animals of different size (Chapter III).
Objective III was developed to investigate the role of the mating system in influencing mate guarding behaviors. I analyzed mate guarding behavior in a dioecious species,
Limnadia badia , to assess whether this species was experiencing mate guarding as a form of intersexual conflict in a similar way to the androdioecious E. texana . I was then able to measure optimal mate guarding time and compare responses of males and females/hermaphrodites of four different species (belonging to the family Limnadiidae) characterized by two mating systems (Chapter IV).
Objective IV was developed to analyze mate guarding behavior in a natural setting, where all parameters act simultaneously (Chapter V). This study was submitted to
Behavioral Ecology by Chiara Benvenuto, Brenton Knott and Stephen C. Weeks.
My overall goal was to integrate experiments performed under controlled laboratory conditions with field observations and to incorporate these into a broader
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evolutionary context by comparing different species characterized by different mating systems. The final chapter (Chapter VI) presents a general overview of my thesis with the concluding remarks of this broad study of mate guarding behavior in clam shrimp as a case of intersexual conflict.
II. MATE RECEPTIVITY ASSESSMENT AND PRECOPULATORY MATE
GUARDING BEHAVIOR IN THE CLAM SHRIMP EULIMNADIA TEXANA
Abstract
Female crustaceans are receptive only for a short period after molting. In order to maximize mating opportunities, males usually guard females until copulation occurs.
Mate guarding is an adaptive behavior for males. Males can optimize their time, and thus maximize their fitness, by guarding near to molt females instead of those that are far from receptivity. To decide whether to stay with a female or search for another closer to receptivity, males must be able to assess the receptive state of their mates. This is the first step of a complex set of interactions between males and females to determine the duration of guarding. When male and female optimal guarding times do not coincide, an intersexual conflict begins. This conflict was analyzed in the androdioecious clam shrimp
Eulimnadia texana , wherein males guard hermaphrodites rather than females. Three experiments were designed to determine the mode of mate searching and detection of receptivity by males. To do so, I analyzed 1) the reaction of focal animals to the presence of individuals of the same or opposite sex; 2) whether males can detect molting hormone dispersed in water; and 3) whether males use molting hormone as a receptivity cue when clasping a hermaphrodite. Hermaphrodites, but not males, were attracted to members of the opposite sex, supporting the hypothesis that different sexes in different receptive states use different searching tactics to find a mate. Males did not detect molting hormone
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as a soluble cue in the water and the presence of hormone on the carapace was not used by males as a contact cue to assess receptivity. Instead, molting hormone reduced the attempts of mate guarding when combined with specific hermaphrodite behaviors.
Keywords : Branchiopoda, molting hormone, androdioecy, intersexual conflict, resistance behaviors.
Introduction
“A given cue can yield only a probabilistic estimate of time from receptivity. Because a) females
will vary in the time they take between the onset of the cue and the onset of receptivity, b) the
time after onset of the cue and male encounter will vary, and c) some females may show the cue,
but for some reason fail to accept insemination ” (Parker, 1974, page 171).
In many animals, males guard their mates in order to ensure a mating event.
Precopulatory mate guarding is a strategy used by males when females are rare (because
of unbalanced sex ratios) or difficult to find, when there is high male male competition
for direct acquisition of females, or when female receptivity is restricted in time (e.g., in
crustaceans and anurans). In aquatic crustaceans, receptivity lasts for only a short time
after the female molts. This reduced receptivity occurs because the carapace hardens
after molting (or cuticular plates, termed oostegites, will obstruct the female gonopores)
making it impossible for the male to fertilize his mate (Ridley, 1983; Jormalainen,
1998). Mate guarding strategies include those in which a male stays close (mate
attender) or maintains direct physical contact (mate carrier) with a female so that the
male will not miss the moment when the female is receptive, and thus the possibility to
mate (Parker, 1974; Conlan, 1991; Jormalainen, 1998).
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In monogamous species, the male remains with his mate throughout his
reproductive life (e.g., Mathews, 2003). Other species follow a pure searching tactic
wherein males will randomly assess females in order to find one that is receptive (e.g.,
Bauer & Abdalla, 2001). These are the two extremes of a spectrum, from extended
guarding, to total absence of guarding. Between these two extremes, there is a more
complex situation that requires “decisions” to optimize guarding time. Males should not
expend too much time with any one female because they risk losing other mating
opportunities. On the other hand, males also should not expend too little time or they
will risk leaving right before the female becomes receptive.
Mate guarding is more complicated than a simple optimal male strategy. Female
resistance plays a major role in guarding decisions and mate guarding thus represents a
case of intersexual conflict (Jormalainen et al., 1994b; Yamamura & Jormalainen, 1996;
Jormalainen, 1998; Watson et al., 1998; Jormalainen et al., 2000). A short guarding
phase would be most advantageous to both sexes, but males can prolong the guarding
phase more (not to miss a mating opportunity) while females would do best when
guarded for only the minimum amount of time possible (not to sustain the various costs
of being guarded; see chapter I).
We can begin addressing this problem from the male’s point of view, like Parker
(1974) did initially: how do males decide whom to guard and how long to prolong the
guarding phase? There are numerous evaluations to make when encountering a female:
1) is the female receptive or close to receptivity?; 2) how many other females are there
in the population nearer to receptivity than the encountered female?; 3) is the guarding
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male’s size sufficient to overcome female resistance and/or the attempts to be dislodged
by other males (takeovers)?; and 4) how long are other males guarding? All these
questions may influence the decision to leave a female to search for another, or stay
(guard) the encountered female. This is a typical optimal strategy problem and, if we
consider question 4, an example of game theory in which males should “make
decisions” based on their individual judgment and comparison with what the rest of the
male population is doing.
The above questions do not include the entire range of parameters a male could
evaluate. Mating history, for example, can be another relevant factor (Sparkes et al.,
2002; Ortigosa & Rowe, 2003; Van Son & Thiel, 2006). On the other hand, some of
these questions might not be important for some species. In this chapter I investigate
mate searching and I focus on the very first question which is essential for optimal mate
guarding to evolve: can males detect whether an encountered female is receptive or
close to receptivity? Without the capability to assess the receptive state of the female,
males cannot assess the relative “values” of various females and thus the guarding
decision can not be considered in an optimality context.
Mate guarding models (Parker, 1974; Grafen & Ridley, 1983; Yamamura, 1987;
Jormalainen et al., 1994b; Yamamura & Jormalainen, 1996; Jormalainen, 1998; Härdling et al., 2004) assume a certain knowledge by males of the timing of female receptivity
(time until molt). In crustaceans, the cues that males can use to assess female receptivity can be: 1) visual; 2) tactile, such as texture or stiffness of carapace (Hunte et al., 1985) or through palpation of the brood pouch (Dunham, 1986); or 3) chemical (e.g., Sparkes et
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al., 2000). Visual recognition is important to identify members of the opposite sex in some species, including individual recognition in fiddler crabs (Detto et al., 2006).
However its role in assessment of receptivity has been found to be the least likely of the three methods, at least if not combined with other types of cues. Tests with receptive and unreceptive female shrimp Palaemonetes pugio kept in glass containers elicit no differential responses by males (Caskey & Bauer, 2005) and visual communication of female receptivity had been excluded in the hermaphroditic shrimp Lysmata wurdemanni
(Zhang & Lin, 2006) and the blue crab Callinectes sapidus (Gleeson, 1980; Bushmann,
1999). Nonetheless visual information can be important as an indirect cue. For example, male rock shrimp Rhynchocinetes typus can visually find receptive females by locating the aggregation of other males around females (Diaz & Thiel, 2004). The texture of the carapace (soft vs . hard) had been postulated as a possible cue of female receptivity, associated with molting (Hunte et al., 1985), but this hypothesis has not been confirmed
(Caskey & Bauer, 2005). Tactile information on the presence/absence of embryos in the brood pouch of the amphipod Gammarus lawrencianus (Dunham, 1986) seems to play a role in mate guarding decisions, but not exclusively. Pheromones have been the cue most commonly found in studies of crustaceans (Dunham, 1978; Hartnoll & Smith, 1978;
Hartnoll & Smith, 1980; Thompson & Manning, 1981), even if they might not be the only stimulus involved in receptivity assessment (e.g., Dunham, 1986; Diaz & Thiel,
2004).
Hormones (chemical cues) can be soluble or insoluble in water. Soluble (distance) pheromones can be used to attract/locate mates. Insoluble (contact) hormones need to
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react directly with male chemoreceptors on contact and thus are not effective at a distance. In many species, males appear to need to physically contact their mates before engaging in mate guarding. For example, contact with antennal flagella or pereopods have been described in P. pugio (Caskey & Bauer, 2005) and calceoli (situated in the antennae) have been found to be involved in the reproductive assessment process in
Gammarus (Dunn, 1998). Additionally, insoluble hormones are considered responsible for male recognition of female maturity stage (Dunham, 1978; Hartnoll & Smith, 1978;
Ridley & Thompson, 1979; Thompson & Manning, 1981).
My goal in this project was to get some insight as to the mode of detection of
receptivity by male clam shrimp, Eulimnadia texana (Branchiopoda: Spinicaudata:
Limnadiidae). Eulimnadia texana (Packard, 1871) males guard their mates, as many
other crustaceans do. They do not guard females though, but rather hermaphrodites,
since the genus Eulimnadia is androdioecious: male and hermaphrodites coexists in
populations where true females are not present (Sassaman & Weeks, 1993; Weeks et al.,
2006a). Both selfing and outcrossing occur in a population, with hermaphrodites
producing primarily eggs and few sperm (Zucker et al., 1997). Thus they can undergo
self fertilization, but they can also outcross with males. Males are the only means of
outcrossing because they have the first two pairs of thoracic appendages modified into
“claspers” allowing them to gain and then maintain physical contact with the
hermaphrodites to mate guard and eventually to transfer sperm at the end of the
guarding phase (Knoll, 1995).
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It was previously shown that mate searching is not restricted to males.
Hermaphrodites also actively search for mates, moving closer to males and swimming
faster in their presence (Medland et al., 2000). The mechanisms that influence the
encounter rate between sexes are not clear, so I decided to examine searching behaviors
in depth. The basic assessment of hermaphrodite receptivity has also been previously
investigated (Weeks & Benvenuto, 2008) where laboratory experiments were performed
on multiple males in the presence of multiple hermaphrodites. It is easy to observe the
presence or absence of a clutch of eggs through the transparent carapace of a
hermaphrodite, and this allows a rough estimate of its receptive state. Hermaphrodites
with eggs visible in the brood chamber are not receptive whereas hermaphrodites with
no eggs visible (but instead having eggs in the ovotestis ready to be extruded) are closer
to receptivity (Weeks & Benvenuto, 2008). Weeks and Benvenuto (2008) suggested
that males require physical contact with their mate in order to assess receptivity, and
they found that males spend significantly more time clasping receptive (or close to
receptivity) hermaphrodites. However, these authors did not identify any specific cues
nor did they test for the possibility of soluble receptivity cues. Since dropping the eggs
from the brood chamber is closely associated with a hermaphrodite’s molt (Weeks &
Benvenuto, 2008), I decided to investigate the potential role of molting hormone as
either a soluble or direct contact cue for receptivity. A similar study had been performed
in the isopod Lirceus fontinalis and the results supported the predictions that males were
able to discriminate the molting status of their mates using molting hormone as a
contact cue (Sparkes et al., 2000).
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Three separate experiments were designed to investigate: 1) the reaction of focal
males and hermaphrodites to the presence of other individuals of the same and opposite
sex; 2) whether males can detect the presence of molting hormone in the water; and 3)
whether males use molting hormone as a cue when clasping a hermaphrodite. The first
experiment was an extension of the experiment performed by Medland et al. (2000)
where they investigated the behavior of focal clam shrimp (males and receptive
hermaphrodites) in the presence or absence of the opposite sex. Although males are the
searching sex, they did not show any change in behavior in the presence of the other
sex: they did not swim closer to the segregated hermaphrodites, nor did they decrease
their swimming speed. Hermaphrodites, on the contrary, were found more often next to
males and swam slower in their presence (Medland et al., 2000). Unfortunately, same
sex individuals were not used as controls to note whether the focal animals were
attracted to conspecifics generally or the opposite sex specifically. I expanded this
previous experiment by testing the behavior of both receptive and non receptive
hermaphrodites while in presence of individuals of the same (control) and opposite
(treatment) sex. The second and third experiments assessed the effect of molting
hormone as a possible guarding cue. I tested the effectiveness of the hormone as a
mating cue in the water (as a water soluble cue) or on the surface of the hermaphrodite’s
carapace (through direct contact).
The results from these experiments can provide us with better insight into the
processes that lead to the beginning of the guarding phase and allow for the possibility
of optimal mate guarding in clam shrimp. The duration of the guarding phase will then
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be determined by subsequent interactions by the male and his mate, as a result of the
conflict between the two sexes (Chapter III).
Materials and methods
Three separate projects were performed to investigate mate receptivity assessment. Specimens were obtained by hydration of sediment collected in 2000 and
2004 near Portal, Arizona from Wallace Tank (reported in other experiments as WAL;
Sassaman & Weeks, 1993), containing encysted eggs of E. texana . Sediment (~ 500 ml) was added to 37 liter aquaria then flooded with de ionized water, attempting to simulate a natural filling during a monsoon rain event. Aquaria were equipped with air stones and kept under Durotest sunlight simulating fluorescent lights. Water temperature was maintained at a constant 27°C. Upon reaching sexual maturity (approximately 6 7 days after hydration), individuals were randomly selected for the experiments.
Projects 1 and 2 used video tracking software developed by Dr. Robert Huber
(Bowling Green State University) for the Apple Macintosh. The swimming behavior of focal animals was recorded using a video camera (Panasonic CCD), suspended on top of a plexiglass arena placed on a light table. The arena was composed of three chambers
(Fig. 2.1): a central chamber (24.5 cm x 13.7 cm x 2 cm) where the focal animal was placed, and two lateral chambers (4 cm x 13.7 cm x 2 cm). All three chambers were filled with water up to 1 cm. The three chambers were separated by two plexiglass barriers drilled with ten equally spaced 1 mm holes (Fig. 2.1). The barriers were painted white with non toxic and non water soluble glass paint to prevent visual cues. The
55
computer program captured a reference frame of the empty arena initially and then, when the experiment started, captured new frames, tracking a dark object (the focal clam shrimp) on a light background. Light from the light table was diffused from the bottom of the arena. Movements were recorded as x y pair coordinates per unit time and analyzed with a collection of public domain Java Applets for the analysis of behavioral data
(available at http://caspar.bgsu.edu/~software/Java/ ). The Java Applets were used to analyze distance from the side where the opposite sex was located and total distance traveled. Due to the small size of animals (around 5 mm), sometimes particles in the water were mistakenly read as data points. The java program recognizes readings exceeding the acceptable movement of animals and substitutes those with intermediate readings between the previous and subsequent true data points.
Figure 2.1 – Scheme of the arena used in Project 1 and 2. On the side, representation of one barrier (1.5 times enlarged).
Projects 2 and 3 used the molting hormone 20 Hydroxyecdysone C 27 H44 O7 (20
HE). This is the biologically active form of the arthropod steroid molting hormone. The working solution of the hormone was obtained by dissolving 1 mg 20 HE (Sigma H5142)
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in 1 ml distilled water and was stored at 20°C. The stock solution was thawed and diluted in distilled water to 100ng/ml; a similar concentration (120ng/ml) of the 20 HE mimic nonsteroid RH 5849 was used by Sparkers and colleagues (2000) on female isopods to test the effect on male guarding behavior.
Statistical analyses were performed using JMP 7.0 (SAS Institute, Inc.) and
G*Power 3 (Faul et al., 2007). Details are given below for each individual project.
Project 1 Tracking focal animals
The experimental protocol was based on Medland et al. (2000). Sexually mature shrimp were isolated in separate containers (males and hermaphrodites) right before the start of the experiment. Fifty seven focal animals (male, hermaphrodite with eggs, or hermaphrodite without eggs) were individually placed in the central chamber of the arena while the two lateral chambers were maintained empty (control trial). After half an hour, each focal animal was tested again in a stimulus trial: five males were added to one lateral chamber and five hermaphrodites were added to the other (right or left side was decided using a randomized design). The arena was filled with water obtained by hydrating clam shrimp free sediment, from the same area of the population sampled, to assure the water contained all the appropriate minerals and nutrients, but excluded any cue from conspecifics. The focal and stimulus animals were left in their chambers to acclimate for 10 minutes before starting the recording. Each trial lasted 8 minutes, and the camera recorded data at 10 frames per second. After each trial, the arena was emptied, cleaned with ethanol, rinsed with de ionized water, dried, and then filled again for the subsequent trial.
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The main chamber measured 318 x 178 “pixels” (picture elements of the grid of reference, each one approximately 1/13 cm diameter). I tested the central mean distance of males and females from the chamber B (coordinates 0,0) for control and stimulus trials, and then repeated the analysis calculating the distance from the side where the individuals of the opposite sex (males in the case of a focal hermaphrodite and hermaphrodites in the case of a focal male) were placed (either left or right) when stimuli animals were present (“reference side”). In order to calculate distance traveled, “pixels” were transformed into cm.
Multivariate analyses of variance (MANOVA), with the same individuals tested in controlled and experimental trials as repeated measures, were performed to assess the effect of sex (male vs . hermaphrodite) and trials (stimulus vs. control) on mean distance from the reference side (distance from opposite sex) and total distance traveled. The same analysis was repeated taking into account the receptive state of the hermaphrodites and thus analyzing the effect of sex (this time subdivided in the three categories: males, receptive hermaphrodites, and non receptive hermaphrodites) and trials (stimulus vs. control) on mean distance from the reference side (distance from opposite sex) and total distance traveled.
Project 2 Molting hormone as a soluble cue
Fifteen focal males were individually placed in the central chamber and three trials were run for each male: one with no hormone, one with two drops of 20 HE in the right chamber and normal water in the left chamber, and one with the reverse (two drops of 20 HE in the left chamber and normal water in the right chamber) to control for
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possible position preferences within the chamber. The hormone was used at a concentration of 10 g/ml. Each trial lasted 10 minutes, at 10 frames per second. In between trials, the arena was emptied, cleaned with ethanol, rinsed with de ionized water, dried, and then filled again. I used “reconstituted” water (de ionized water with mineral salts: 48 mg NaHCO 3, 15 mg CaSO 4, and 15 mg MgSO 4 per liter of water) to avoid the influence of cues other than the added hormone.
Preliminary data analysis showed a strong position effect: males were attracted to
(or repelled from) one side of the chamber, regardless the hormone treatment. Since natural light or the presence of the observer could have influenced the male swimming behavior, I repeated the experiment with fifteen more males, using a plastic black screen positioned to surround the arena completely. Light was still diffused from the light table below the arena (as in the previous experiment), but the effect of natural light and potential disturbance by the observer were prevented by the black screen. Each male was tested twice: one time with no hormone and one time with two drops of 20 HE in one chamber (right or left, randomized throughout the experiment).
To assess variation in mean distance from the chamber (empty in control and with hormone in treatment), I used t paired tests.
Project 3 Molting hormone as a direct contact cue
I prepared 41 couples pairing a receptive (H +) or a non receptive hermaphrodite
(H ) with a male. Animals were individually isolated prior to pairing and measured with a caliper (maximum carapace length, in millimeters). Half H + and H received hormone treatment (a drop of approximately 0.05 ml of the working solution of 20 HE), applied
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with a glass microcapillary pipette onto the carapace, which had been previously blotted dry. The remaining hermaphrodites received a drop of distilled water (control). This protocol followed the one described by Sparkers et al . (2000) for the isopod L. fontinalis .
For video recordings, each hermaphrodite was introduced into the observation chamber (a
50 ml graduated glass beaker), positioned under a black and white video camera
(Panasonic CCD) that was connected to a time lapse video recorder (Samsung SSC 1280
Real Time Lapse Recorder). The bottom of the beaker was filled with dark sand (to enhance the contrast with the clam shrimp body, allowing better scoring of mating behavior from playback of the videos) and the beaker was filled with 40 ml of
“reconstituted” water to avoid the influence of possible cues from other individuals, variation in nutrients other than the food added, or a build up of microorganisms in the aged water. Clam shrimp were fed by adding 0.2 ml food (2.3 g baker’s yeast + 2.5 g grounded fish flakes in 500 ml de ionized water) to the water at the beginning of the experiment.
Single pair mating behavior assays were recorded. On the screen it was easy to recognize the two sexes, since the hermaphrodites presented the white mass of eggs most of the time and the males swam faster and constantly tried to clasp the hermaphrodites.
The hermaphrodite was introduced first, and given a 2 minute acclimatization period.
Only at this point was the male introduced so that he did not receive the information that another individual was added to the beaker (males are the sex that can attain physical contact clasping the hermaphrodites).
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Hermaphrodites were paired with males of similar size and the couples were recorded for 24 hours. I recorded number and length of encounters (brief contacts, less than 30 seconds) and mate guarding attempts (prolonged contacts, more than 30 seconds) in the first hour (the male should be able to detect the hormone during this amount of time: after an hour the hormone might degrade in the water; Andersen et al., 2001). I also counted the number of “kicks” by the hermaphrodite (bending of the abdomen) considered as a possible form of physical resistance to being guarded. Finally, I noted the result of mating (selfing or outcrossing).
This project aimed to investigate the variation in guarding time after application of 20 HE in unreceptive hermaphrodites. The hormonal manipulation would provide the male with false information (i.e., the male should assume that the hermaphrodite is closer to molt than it actually is), if the male uses the molting hormone as a cue.
I performed a full factorial, three way analysis of variance (ANOVA). I considered the effect of 20 HE (treatment and control), receptive state of hermaphrodite
(with and without eggs), hermaphrodite’s response (kicking and not kicking), and all the possible interactions of these parameters on number of guarding events and number and duration of encounters in the first hour of observation. The ANOVA was then performed again, this time separately analyzing kicking and non kicking hermaphrodites in an attempt to untangle hermaphroditic response (kicking behavior) from treatment
(hormonal application). I also ran contingency analyses on the effect of treatment on hermaphrodite kicking behavior and mating outcome (selfing or outcrossing). In order to
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meet the parametric assumptions of normality, I used a square root transformation for the number of mate guardings.
Results
Project 1 Tracking
A total of 57 focal animals were tested (20 hermaphrodites with eggs, 11 hermaphrodites without eggs and 26 males), but 6 hermaphrodites (3 with and 3 without eggs) were excluded from the analysis since they changed their receptive status from control to stimulus trial (by either dropping or moving eggs).
Overall, hermaphrodites (H) tended to swim closer to the side where the males were kept (Table 2.1a), regardless of their receptivity state, even though hermaphrodites with eggs, which were not yet receptive (H ) appeared to be more responsive (although this response was not quite significant: p = 0.0621) to the presence of members of the opposite sex than receptive hermaphrodites (Fig. 2.2; Table 2.1b).
Males covered a greater distance (Fig. 2.3, Table 2.1c, d) than hermaphrodites, regardless of trial type and receptive state of the hermaphrodites (contrast analysis was run as a post hoc test on male vs . the other two groups: receptive and non receptive hermaphrodites: P = 0.0028).
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TABLE 2.1 – MULTIVARIATE ANALYSIS OF VARIANCE
Value F test Exact F NumDF DenDF Prob>F a) Sex (M, H) a 0.003 0.1367 1 49 0.7132 Dist from opposite 0.040 1.9758 1 49 0.1661 Dist from opposite*Sex (M, H) 0.084 4.1090 1 49 0.0481 b) Sex (M, H +, H )a 0.004 0.0992 2 48 0.9058 Dist from opposite 0.043 2.0702 1 48 0.1567 Dist from opposite*Sex (M, H +, H ) 0.123 2.9459 2 48 0.0621 c) Sex (M, H) a 0.248 12.1315 1 49 0.0011 Total distance 0.002 0.1007 1 49 0.7523 Total distance*Sex (M, H) 0.003 0.1409 1 49 0.7091 d) Sex (M, H +, H )a 0.260 6.2357 2 48 0.0039 Total distance 7.886e 5 0.0038 1 48 0.9512 Total distance*Sex (M, H +, H ) 0.005 0.1238 2 48 0.8838 a between subject contrast Repeated measures MANOVA results for (a,b) mean distance from reference side (distance from opposite sex) and (c,d) total distance traveled between sexes. The groups compared in the analyses were either males (M) with combined hermaphrodites (H) or males with the two receptive states separated (H + = receptive hermaphrodites; H = non receptive hermaphrodites).
Figure 2.2 Mean distance from origin of coordinates (control) and the chamber with opposite sex (stimulus).
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Figure 2.3 – Total distance traveled by each sex (male, hermaphrodite with eggs, hermaphrodite without eggs) in absence (control) and presence of other animals (stimulus) during the 10 minute trials.
Project 2 Molting hormone as a soluble cue
When testing the effect of the hormone as a soluble cue in the water, the preliminary analysis detected that males were consistently attracted to the one side of the test chamber, regardless of the presence or absence of the hormone on that side (paired t test, p < 0.0001, two tail; Wilcoxon Signed Rank test on matched pairs, p < 0.0001; distance from side B = 7.09 cm; distance from side A = 20.04 cm; 95% confidence interval: from 16.137 to 9.7606). This could be interpreted as either a complete lack of response to the hormone in favor of some other stimulus (e.g., natural light) or that this other stimulus was merely swamping any response to the hormone. Thus, I repeated the experiment with the black screen to remove any effect of this added stimulus and again noted no significant difference between the average distance of males from the side where the hormone was added compared with the side with the no hormone control
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(paired t test, p = 0.3043, one tail; Wilcoxon Signed Rank test on matched pairs, p =
0.2106; distance from source of hormone = 11.64 cm; distance from control = 12.2872;
95% confidence interval: from 1.9836 to 3.2656). The combined data from both iterations of this experiment suggest that males are not attracted to the molting hormone as a soluble cue.
Project 3 Molting hormone as a direct contact cue
I recorded 41 couples, 21 treated with 20 HE (12 couples with the hermaphrodite not receptive and 9 receptive) and 20 control (9 with the hermaphrodite not receptive and
11 receptive). Seven couples were excluded from the analysis because the hermaphrodites changed status in the first hour (receptive hermaphrodites moved their eggs into the brood chamber: four in the hormone treatment and three in the control).
Because I wanted to study only cases where males were able to outcross, to avoid considering behaviors of inbred males (inbreeding depression is high in this species, ranging from 0.5 and 0.7; Weeks & Zucker, 1999; Weeks et al., 2000a; Weeks et al.,
2001; Weeks & Bernhardt, 2004), I also excluded five more couples where the hermaphrodite selfed (one in the hormone treatment and three in the control, all receptive at the beginning of the experiment).
The number of mate guarding attempts in the first hour of the experiment was not influenced by the hormone treatment (Table 2.2). Contrary to a priori expectations, the interaction between hormone treatment and receptive state of the hermaphrodite was not significant (Table 2.2).
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TABLE 2.2 – FULL FACTORIAL ANOVA
Source DF SS F Ratio Prob > F Square root NUMBER OF MG ATTEMPTS Treatment 1 1 1.5882 0.2214 H receptivity state 1 4 12.3896 0.0020 Treatment * H receptivity state 1 0 0.1280 0.7241 H kicking behavior 1 9 26.4890 <.0001 Treatment * H kicking behavior 1 5 13.7065 0.0013 H receptivity state* H kicking behavior 1 2 5.8482 0.0248 Treatment * H receptivity state* H kicking behavior 1 0.2 0.5533 0.4652 Error 21 7 Full factorial ANOVA results for mate guarding (MG) attempts in first hour of experiment (square root transformed).
In fact, hermaphrodite receptivity state was correctly assessed by males, regardless of the treatment, and males started more guarding events with hermaphrodites closer to receptivity than with non receptive hermaphrodites (Figure 2.4; Table 2.2).
Figure 2.4 – Number of mate guarding attempts (squared root transformed) in the first hour of the experiment performed by males on receptive (H+, no eggs) and non receptive (H , with eggs) hermaphrodites. Error bars represent two times the standard error.
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The number of mate guarding attempts was also significantly correlated with hermaphrodite behavior: the kicking response by hermaphrodites was associated with more frequent mate guarding contacts (Figure 2.5; Table 2.2). I detected an interaction between kicking behavior and hermaphroditic receptivity state, affecting the number of mate guarding attempts (Figure 2.6; Table 2.2): receptive hermaphrodites performed proportionally more often kicking behaviors and elicited more guarding attempts than non receptive hermaphrodites.
Figure 2.5 – Number of male mate guarding attempts (squared root transformed) in the first hour of the experiment on hermaphrodite performing different behaviors: kicking vs . not kicking. Error bars represent two times the standard error.
Also, a complex interaction was found for the effect of kicking behavior and hormone treatment on mate guarding frequency (Figure 2.7; Table 2.2).
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Figure 2.6 – Interaction between hermaphrodite kicking behavior and receptivity state (H+, receptive, no eggs; H ,non receptive, with eggs) on number of mate guarding attempts (square root transformed). Levels connected by the same letter are not significantly different.
Figure 2.7 – Interaction between hermaphroditic kicking behavior and treatment on (a) number of mate guarding attempts (square root transformed) and (b) mate guarding time (Fifth root transformed). Levels connected by the same letter are not significantly different.
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Without the hormone, there were higher numbers of mate guarding attempts on hermaphrodites that kicked. When the hormone was added, I observed a lower number of mate guarding attempts on hermaphrodites that did kick and a higher (even though not in a significant manner) in the hermaphrodites that did not kick, compared to the control (no hormone).
Since this interaction is difficult to interpret, I repeated the ANOVA (Table 2.3) on mate guarding attempts, again considering the effect of the treatment and the receptive state of the hermaphrodite, but this time separately analyzing the hermaphrodites that responded to males (kicking) and the ones that did not (no kicking). In this way I was able to untangle the effect of the hermaphroditic response from the effect of the treatment. From the new analysis, it appears that a behavioral communication between the sexes might play an important role in pre mating interactions. When hermaphrodites were responsive to males (kicking behavior), there was a significantly higher number of mate guarding attempts on hermaphrodites closer to receptivity (without eggs; Figure
2.8a) and on hermaphrodites not treated with the molting hormone 20 HE than on non receptive hermaphrodites and hermaphrodites without hormone, respectively (Figure
2.8b). These differences were not found in non responsive hermaphrodites, regardless of the receptive state or the hormone treatment (Figure 2.8c,d). The molting hormone elicited a few more mating attempts (marginally significant; Table 2.3) only on non responsive hermaphrodites (no kicking behavior; Figure 2.8d).
Finally, I repeated the full factorial ANOVA to assess the effect of the same predictor parameters (treatment, receptivity state, kicking behavior, and their interactions)
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on three other dependent variables: 1) guarding duration (in minutes), 2) number of encounters (clasping less than 30 seconds), and 3) duration of encounters (in minutes).
TABLE 2.3 – ANOVA TABLE
KICKING NO KICKING Source DF SS F Ratio Prob > F DF SS F Ratio Prob > F Treatment 1 3 11.2122 0.0441 1 1 4.2928 0.0529 H receptivity state 1 4 16.0547 0.0279 1 0 0.8735 0.3623 Treatment * H receptivity state 1 0 0.0679 0.8113 1 0 0.8735 0.3623 Error 3 1 18 6 Two way ANOVA results for mate guarding attempts in the first hour of the experiment (square root transformed), separately analyzing hermaphrodites that responded to males (kicking) and those that did not (no kicking).
Figure 2.8 – Number of male mate guarding attempts (squared root transformed) in the first hour of the experiment on hermaphrodites performing different behaviors: kicking hermaphrodites are represented in white (panels a,b); hermaphrodites not kicking are represented in gray (panels c,d). Error bars represent two times the standard error.
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For the guarding duration, I observed a trend similar to the one observed for number of mating attempts reported above, but I was not able to normalize the residuals so I could not analyze these data parametrically. Number and duration of encounters did not differ among treatments, receptivity states, or kicking behavior (p value > 0.05 for all; analyses not reported). Thus, encounter rates and encounter duration were not influenced by any of the independent variables studied.To make sure that the addition of hormone was not affecting the hermaphrodite’s mating behavior, I analyzed the difference in kicking response and the result of mating (i.e., selfing vs . outcrossing) in the two groups (treated and untreated hermaphrodites). There were no evident effects of the application of 20 HE on kicking behavior (χ 2 = 0.011, p = 0.9156) or on mating result
2 (either selfing or outcrossing; χ = 1.487, p = 0.2226).
Discussion
The first requisite for a successful mating is the encounter of receptive individuals of the opposite sex. In crustaceans, females are often receptive only for a restricted period after molting. For this reason, males of many crustacean species adopt a mate guarding strategy. Nonetheless, males need to 1) find a mate and 2) assess her receptivity, in order to decide to guard a particular female or search for another one which is closer to receptivity.
Male E. texana swim faster than their mates (Medland et al., 2000) and thus cover a greater distance than hermaphrodites in the same amount of time. Their apparent goal is to encounter the maximal number of mates in the shortest amount of time. In many
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dioecious species, males are the searching sex because they are competing with other males to find mates (but see cases of role reversal; Gwynne, 1991). In androdioecious species, the overall sex ratio is highly skewed towards hermaphrodites (Pannell, 2002;
Weeks et al., 2006a). Usually the more abundant sex searches for mates, while the other sex has abundant mating opportunities (Hammerstein & Parker, 1987; Gwynne, 1991;
Kokko & Wong, 2007). Nevertheless, the operational sex ratio, the “average ratio of fertilizable females to sexually active males at any given time” (Emlen, 1976; Emlen &
Oring, 1977), differs from the general sex ratio since not all hermaphrodites are receptive at the same time. Population densities are often high in nature (Benvenuto et al., in press) and males can encounter multiple conspecifics (the majority of the opposite sex) in just a few minutes. However, many of these encounters are with unreceptive hermaphrodites.
Eulimnadia texana males thus maximize their encounter rates by increasing their swimming speed (Medland et al., 2000). The male strategy appears to be a fast screening of possible mates, through direct contact by clasping conspecifics (Weeks & Benvenuto,
2008).
Hermaphrodites are capable of self fertilization, but in order to favor outcrossing they can approach males. I confirmed the results of Medland et al. (2000) of active mate searching by hermaphrodites and determined that hermaphrodites are indeed attracted to males but not to the presence of other hermaphrodites. This was not clear from the experiment by Medland et al. (2000), where the only choice possible was members of the opposite sex. An additional layer of complexity in the current study is that this responsiveness to males was marginally more significant in hermaphrodites with eggs
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still in the brood chamber (i.e., non receptive hermaphrodites) than in receptive hermaphrodites. I used hermaphrodites with and without eggs in my trials, while
Medland et al. (2000) tested only the second category of hermaphrodites, which they obtained by isolating non receptive hermaphrodites in a small amount of water to force them to drop their eggs. Dropping eggs in stressful environments may be useful for egg dispersion or protection from predators, but the receptive status of the hermaphrodite should not be altered by this forced release of their eggs. The fact that hermaphrodites in the current study performed similarly to hermaphrodites forced to drop their eggs in the
Medland et al. (2000) study suggests that the behavior of dropping eggs in situations of stress (i.e., lower levels of water) does not recalibrate the hermaphrodites’ internal receptivity status. Thus, the hermaphrodites used in the initial experiment (Medland et al.,
2000) can be considered a mixture of receptive and non receptive hermaphrodites. Here, using hermaphrodites obtained from rearing tanks and not kept under stressful conditions,
I found that receptivity state might alter the hermaphrodite’s behavior.
My result is opposite from what is expected after the preliminary study of
Medland et al. (2000): hermaphrodites with eggs (and thus not yet receptive) are also attracted to males and might also be the ones that swim closer to males. A possible explanation is that hermaphrodites are assessing the presence of males in a highly hermaphrodite biased population. It seems that hermaphrodites can slow down their receptive cycle in the absence of males and postpone the moment of self fertilization
(Zucker et al., 2002 and see Chapter III). It would thus be important for them to be able to discern if there are any males in proximity. Once the hermaphrodites have dropped
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their eggs and are approaching the molting phase, it appears that they will need to be in the presence of males (via attraction to them from the earlier stage) or they might have waited as long as possible and thus just proceed to self fertilization. It would be interesting to test this behavior in dioecious species to see if females are similarly attracted to males and whether this attraction is similarly altered by their receptive state, given that females do not have the option of self fertilization.
An alternative explanation could be that hermaphrodites in an androdioecious system lose their interest in males because they do not necessarily need males for fertilization of their eggs. There might be selection to not perform normal mating behaviors, particularly in the moment when the behavior would be most important (when hermaphrodites are reaching receptivity) if mating costs are too high, resulting in receptive hermaphrodites not searching for males. In androdioecious nematodes, there is evidence that the hermaphrodites are not responding to male mating behavior and are not releasing the same amounts of pheromones that dioecious species are (Chasnov & Chow,
2002; but see Simon & Sternberg, 2002). Again, a comparison of this attraction behavior in dioecious clam shrimp would be helpful to determine whether the androdioecious hermaphrodites are behaving similarly to dioecious females or not.
Males do not seem attracted to the presence of molting hormone (in the form of
20 HE) in the water. This is in agreement with the results of Medland et al. (2000). Other studies question the active role of this hormone as a sex pheromone or mate guarding elicitor (Gleeson et al., 1984; Kamio et al., 2000; Kamio et al., 2002; Atema & Steinbach,
2007). This result is also consistent with a previous report by Weeks and Benvenuto
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(2008) on the necessity of direct contact for assessment of a hermaphrodite’s receptivity.
Once populations are established in ephemeral pools, the number of individuals can be extremely high due to simultaneous hatching of a portion of the abundant eggs bank in the sediment. In high density situations (resulting from the large number of individuals in relatively small, depressed areas capable of maintaining water), a long distance hormone might be less effective than a contact hormone. Since the chemical is produced by a swimming organism in still water, there should be a “uniform flow” of diffusion (Zimmer
& Butman, 2000), but I imagine that it would be disturbed by the swimming behavior of other clam shrimp. On the other hand, there should be a high probability of direct encounters with conspecifics for males in such densities.
Both males and hermaphrodites need the presence of the other sex in order to conclude a successful outcrossing event, but they employ different searching techniques.
Males are faster (this allows them to cover greater distances), they can use direct contact cues to determine the presence of receptive mates, and seem not to use soluble cues in the water. Hermaphrodites, on the other hand, cannot use direct contact cues (lacking claspers) but they appear to be able to perceive the presence of males through some as yet unidentified distance chemical communication. In project 1, hermaphrodites were attracted to males even though they were separated by a painted barrier that prevented both visual and contact cues. The barriers were perforated, thus soluble chemical cues were allowed to move from one side of the barrier to the other. It is not known what distance cues might be perceived by hermaphrodites, but determination of sex is probably based on different cues than the ones involved in the assessment of the receptive stage.
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Sex determination from a distance it is not uncommon in crustaceans. For example, female lobster, Homarus americanus , use chemicals released in the urine to assess the sex and the dominance status of conspecifics (Bushmann & Atema, 2000; Johansson &
Jones, 2007). This type of chemical communication without direct contact, and with hermaphrodites being the receiver and not the signalers, could represent an interesting area for future research.
In the last part of this study, I focused on contact cues. In many mate guarding models (e.g., Grafen & Ridley, 1983; Yamamura, 1987; but see Hunte & Myers, 1988), males are assumed to be able to detect and assess the receptive state of females. Mate guarding is a very complex behavior that involves multiple interactions between the sexes. During their mate search, male E. texana perform two behaviors: a) encounter
(i.e., rapid clasping and release, when the overall interaction lasts less than 30 seconds because the male has clasped either a non receptive hermaphrodite or a male; Weeks &
Benvenuto, 2008); and b) mate guarding (i.e., prolonged clasping, longer than 30 seconds because the male has clasped a hermaphrodite close to receptivity; Weeks & Benvenuto,
2008). In this last case, the male is starting a guarding phase. At this moment, the clasped hermaphrodite can react (accept or resist being guarded). Thus, the behavior of the hermaphrodite can support, or not, the initial male decision to guard.
I added the molting hormone 20 HE to the carapace of receptive and non receptive hermaphrodites in an attempt to provide males with false information. If males were using the hormone as a cue for receptivity, they should have guarded non receptive, hormone treated hermaphrodites longer than controls (hermaphrodites not receptive and
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not treated with molting hormone). I did not find the expected result, which indicates that molting hormone might not be the cue the male is actually assessing. Instead I noticed an increase in mate guarding activity (measured as number of mate guarding attempts) when hermaphrodites were reacting to males (through kicking behavior).
The hermaphroditic kicking behavior could be the logical result of being harassed multiple times by males. Nevertheless I left this parameter as a possible explanatory variable because I observed that even receptive hermaphrodites (and not just non receptive ones) were exhibiting this behavior (contrary to expectations). “Kicking” (a fast and repeated movement of the telson) can be interpreted as a resistance behavior, or as a pre ecdysis behavior (both sexes “kick” when they molt, in order to dislodge the old exuvia), or could be used by receptive hermaphrodites to assess the “quality” of males
(higher quality males can persist longer than lower quality males). The identical behavior
(kicking) could therefore convey different information depending on the hermaphrodite’s receptivity state. Males might be able to interpret these combined cues. Thus, together with a chemical communication, there seem to be behavioral communications between the sexes and the latter might play an important role in the male’s decision to continue the guarding phase.
The molting hormone, instead of increasing the mate guarding attempts (as expected at the beginning of the experiment), did not induce males to mate guard longer.
Thus the molting hormone does not seem to be an important cue of receptivity (see also
Gleeson et al., 1984; Kamio et al., 2000; Kamio et al., 2002; Atema & Steinbach, 2007)
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and may, in fact, mask any natural cues, or possibly give the male information suggesting that receptivity is not imminent (maybe because this hormone is released after the molt).
To conclude, I can confirm that mate guarding in clam shrimp it is not determined only by the male’s decisions. The hermaphroditic role is important starting from the initial interactions (Figure 2.9). There is an active search for males using chemical cues in the water column by hermaphrodites (including not yet receptive ones). Also there appears to be a continuous exchange of information between the sexes.
Molting hormone might not be the key clue of receptivity, at least not through direct external contact nor with diffusion in the water. This fact does not exclude a possible role of molting hormone metabolites or other chemical compounds involved in ecdysis. For example, some precursor or degraded compounds of the molting hormone can be released in the urine (Kamio et al., 2002). Alternatively, some proteins or peptides in the exoskeleton can undergo modification before and during molting, and these changes could be used by males as a receptivity cue, as shown in copepods (Kelly &
Snell, 1998). Further experiments are needed to understand the nature of the chemical communication between the sexes during mate guarding in these shrimp.
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SWIMMING SWIMMING MALE HERMAPHRODITE
(swims fast always) (changes behavior with reproductive state)
ENCOUNTER
MALE ASSESSES HERMAPHRODITE HERMAPHRODITE “KNOWS” ITS REPRODUCTIVE STATE
RELEASES STARTS GUARDING RESISTS ACCEPTS GUARDING GUARDING Kicks when not Might kick to receptive assess male quality or when approaching molt CONFLICT
NO CONFLICT
NO CONFLICT
Figure 2.9 - Interactions between the sexes from the moment of encounter. Male assessment of hermaphrodite receptivity is integrated with hermaphrodite behavioral response. The interaction can result in a conflict or not, depending on the value of costs and benefits for the two sexes.
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Acknowledgements
I am grateful to Dr. Robert Huber for his help with the video tracking software and the
Java analysis and to Dr. Francisco Moore and Dr. Peter H. Niewiarowski for useful comments on statistical analyses. Many undergraduate students helped greatly with collecting preliminary and initial data on the various projects and I would like to thank all of them: Danielle Maleski and Namrata Jena (tracking project), Anne Clough, Gihyun
Myung, and Jennifer Brunn (hormone tracking project). This study was partially funded with a research grant from the Animal Behavioral Society.
III. INTERSEXUAL CONFLICT DURING MATE GUARDING IN THE
ANDRODIOECIOUS CLAM SHRIMP EULIMNADIA TEXANA
Abstract
A conflict arises when a specific behavior generates different costs and benefits to the participants involved in the behavior. Intersexual conflicts during reproduction are widespread but difficult to detect and assess. Precopulatory mate guarding generates a sexual conflict on the duration of mate guarding prior to fertilization. This study proposes a manipulative approach to assessing such a conflict using androdioecious branchiopod crustaceans. Possible costs to male and hermaphroditic Eulimnadia texana during mate guarding were analyzed. Hermaphrodites were less able to feed during mate guarding than males. I also paired large and small animals to assess the influence of size as a form of differential “power” between the sexes: the bigger the male compared to the hermaphrodite, the longer the mate guarding. Under laboratory conditions I was able to measure the optimal (preferred) mate guarding time for each sex, manipulating the other sex in order to reduce its influence on the duration of the mate guarding. I compared these optimal guarding times to the observed (compromised) mate guarding time when the sexes were not manipulated, testing theoretical models of optimal mate guarding duration. These results confirm that mate guarding in this crustacean is a case of intersexual conflict. Long mate guarding was costly to hermaphrodites, who would prefer shorter guarding times. Males, on the contrary, were willing to guard longer in
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order to avoid losing mating opportunities. Hermaphrodites can outcross with males or self fertilize their own eggs. Interestingly, selfing time was longer than the optimal guarding time of hermaphrodites, suggesting that hermaphrodites might delay self fertilization, waiting for a good quality male to approach. Keywords : Branchiopoda, mate guarding model, female resistance, male persistence, cost of guarding, size.
Introduction
Precopulatory mate guarding represents a complex case of intersexual conflict in that the mate guarding duration produces differential costs and benefits to the sexes involved in the behavior. Mate guarding males attempt to maximize their fitness by monopolizing females. This behavior applies to a broad variety of taxa, from invertebrates (e.g., crustaceans and spiders) to vertebrates (e.g., anurans), characterized by restricted female receptivity (Ridley, 1983; Jormalainen, 1998). During precopulatory mate guarding, the male stays close (or attached) to his mate, waiting for the moment when fertilization can occur (Conlan, 1991; Jormalainen, 1998). In crustaceans this is usually when the female molts. The starting of guarding is an active behavior of the male. For example males can clasp their mate with their antennae (e.g., anostracans; Belk, 1991), with the second pair of gnathopods (e.g., gammarid amphipods; Borowsky, 1984), or join a female in a tube (e.g., ischyrocerid and tanaid amphipods; Borowsky, 1983). At this point, females can accept or attempt to resist the guarding efforts of the males.
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According to mate guarding theories (Jormalainen, 1998), if the female is receptive (or very close to receptivity) there will be no resistance, and thus no conflict.
If the female is not close to receptivity, the male himself might depart (and again there will be no conflict). Another alternative is that the male will continue to guard, even if the female is not yet receptive, which could lead to a conflict because the male persists in guarding while the female attempts to resist. The actual duration of mate guarding will then be the result of this interaction between the sexes (Yamamura & Jormalainen,
1996; Jormalainen, 1998). Both males and females need the guarding phase in order to conclude a successful mating. Because female receptivity is restricted in time, mate guarding will assure the co presence of both sexes at the right moment. Different optimal mate guarding times between the sexes can cause a conflict because a longer guarding phase has different costs and benefits for the two sexes.
Many theoretical models had been proposed to analyze the parameters involved in precopulatory mate guarding (Parker, 1974; Grafen & Ridley, 1983; Yamamura,
1987; Yamamura & Jormalainen, 1996; Jormalainen, 1998; Härdling et al., 2004).
Jormalainen (1998) used a simple and clear graphical model of mate guarding. In his model, the male “optimal guarding criterion” is assumed to be longer than the female optimal guarding criterion. A compromised guarding time is derived by superimposing the function describing the male’s willingness to persist in guarding and the female’s willingness to resist guarding. The intersecting point represents the compromised mate guarding duration (Jormalainen, 1998). This compromised time is influenced by the relative “power” of the two sexes (Parker & Partridge, 1998): when one sex is
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“stronger” than the other, it can exercise some control over the other sex, and thus it can shift the compromised guarding time towards its own optimal guarding time
(Jormalainen, 1998).
In recent years, long after Parker (1979) emphasized the occurrence of reproductive conflicts between the sexes, a substantial amount of empirical and theoretical studies on the topic has been collected. There is general agreement that intersexual conflicts are widespread, but nevertheless difficult to detect and assess (e.g.,
Chapman et al., 2003; Arnqvist & Rowe, 2005). This current study proposes a manipulative approach to detect and measure intersexual conflict using precopulatory mate guarding behavior in branchiopod crustaceans.
My study organism is the androdioecious clam shrimp Eulimnadia texana
(Packard, 1871). Male E. texana guard their mates using the second pair of their thoracic appendages, modified into “claspers.” They clasp the border of the hermaphrodite’s carapace and hold onto it, attaining the physical contact necessary to (1) gather information on the receptive state of the hermaphrodite (Weeks & Benvenuto, 2008;
Chapter II), and (2) transfer sperm at the end of precopula (Knoll, 1995; Weeks et al.,
2004). This species is particularly advantageous in studies of mate guarding for multiple reasons: it has a fast life cycle and short guarding phases (compared to other crustaceans), it is easy to breed in the lab, and it has been successfully used in behavioral experiments (Medland et al., 2000; Zucker et al., 2002; Weeks & Benvenuto,
2008). Moreover, in this species males are coexisting with hermaphrodites, a rare condition among animals (Weeks et al., 2006a). Such a mixture of sexes allows me to
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test for mate guarding conflicts in a situation where both the costs and benefits are different between the sexes (see below).
There are a variety of possible costs involved with mate guarding: survival costs, such as differential predation on couples vs . individuals (e.g., Ward, 1986; Cothran,
2004), energetic costs (e.g., Watson et al., 1998; Jormalainen et al., 2001; Plaistow et al., 2003), and reduced feeding (e.g., Robinson & Doyle, 1985). In this paper I focus on a possible cost for E. texana : reduced feeding during precopula. This cost is easily measurable in clam shrimp because the gut is visible through the transparent carapace.
Thus, I can quantify the level of fullness of the digestive tract in both sexes to assess feeding rates during guarding.
The benefit of mate guarding is similar for males and females in dioecious
(separate male and female = “gonochoristic”) species: the acquisition of a mate during the window of receptivity in the female cycle. In androdioecious species, the benefit is likely higher for the male, who would not reproduce at all if the window of opportunity was missed, than for the hermaphrodite, who still has the option to self fertilize, even though outcrossing would be more favorable in order to reduce inbreeding depression, which is quite high in this species (ranging from 0.5 to 0.7; Weeks & Zucker, 1999;
Weeks et al., 2000a; Weeks et al., 2001; Weeks & Bernhardt, 2004).
My goal was to empirically test the graphical mate guarding model proposed by
Jormalainen (1998) in a comprehensive way. I considered at least one cost resulting from the mate guarding behavior (reduction of food intake). I also introduced a power difference between sexes: size is an easily measured character that can influence the
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physical interaction (persistence vs . resistance) between the sexes during mate guarding.
Finally, I measured the mate guarding time, both as optimal mate guarding time for each sex (by inhibiting the response of the other sex), and as compromised guarding time.
Materials and methods
I designed three experiments to investigate mate guarding using an intersexual conflict perspective. In the first experiment, I analyzed different costs between males and hermaphrodites during precopula. With the second two experiments, I tested a theoretical mate guarding model (Jormalainen, 1998), manipulating the relative power of each sex (second experiment), and measuring optimal mate guarding time for males and hermaphrodites (third experiment). All statistical analyses described below were performed using JMP 6.0 (SAS Institute Inc.).
Mate guarding costs
Feeding can be restricted during the guarding phase for one or both sexes. I analyzed the level of emptiness of the gut in mate guarding couples. I used individuals from three different E. texana populations from the Jornada Experimental Range, Las
Cruces, New Mexico (SWP3, SWP5, and JT4). Animals were raised in semi natural conditions (sediment containing encysted eggs was moved to plastic pools and hydrated on site during July, 2005). I isolated guarding couples and took digital photographs: one at the beginning of the observation and one at the end (when the male released the hermaphrodite). In this way I analyzed couples at different stages of the guarding cycle.
I was interested in the difference of gut fullness between the sexes during mate
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guarding. The digestive trait is fully visible through the transparent carapace of clam shrimp. It is a clear, linear tube, slightly curved on the head and on the telson, and lacking an enlarged stomach. Therefore the gut can be easily observed and measured.
Images were analyzed using GIMP 2.4.7 (GNU Image Manipulation Program). I measured the length of the carapace of each animal, the full length of the gut (as the sum of three or more straight lines to obtain the convex shape of the gut), and finally the filled portion of the gut at the beginning and at the end of the observational period. An index of gut fullness, in percentage, was calculated as the length of the full gut divided by the total gut length. I calculated the variation in gut fullness (index of gut fullness at the end minus at the beginning of the observation) for each sex. I used a blocked, one way analysis of covariance (ANCOVA) on the variation in gut fullness considering sex as a fixed effect and population as a random block effect. The influence of age, size difference between the sexes (as the ratio: male size/hermaphrodite size), and length of mate guarding in minutes (log 10 transformed) were considered as covariates. Tukey's
Honestly Significant Differences (HSD) Test was used to detect pairwise differences between the three populations.
The effect of size
In this experiment I used individuals raised in the laboratory, obtained from encysted eggs in samples of sediment collected in 2004 from Wallace Tank (referred to in other experiments as WAL; Sassaman & Weeks, 1993) near Portal, Arizona. I raised immature clam shrimp at two different densities (low and high) in order to obtain mature individuals of different size. It is commonly observed in the lab and field that
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populations with higher density have smaller individuals (Weeks & Bernhardt, 2004). I prepared similarly sized pairs and also differently sized pairs (matching one small male with a large hermaphrodite and vice versa) in order to assess the effect of relative size on mate guarding duration. I housed the pairs in 50 ml graduated glass beakers filled with water and with black sand as a contrasting background. Clam shrimp were video recorded for 24 hours using a black and white video camera (Panasonic CCD) connected to a time lapse video recorder (Samsung SSC 1280 Real Time Lapse Recorder).
Hermaphrodites were chosen with eggs visible in their brood chamber at the beginning of the recording. Both individuals were measured with a caliper (maximum carapace length, in millimeters).
I performed a linear regression on the size difference in each couple (male length minus hermaphrodite length in millimeters) relative to mate guarding duration (in minutes). I also categorized the data into three groups: male bigger than hermaphrodite
(difference between the sexes > 0.5 mm), male smaller than hermaphrodite (difference between the sexes > 0.5 mm), and male equal to hermaphrodite (difference between the sexes ≤ 0.5 mm), and ran a one way ANOVA on mate guarding duration (minutes) in these three categories. In order to meet the assumptions of normality, mate guarding time was log 10 transformed.
Compromised and optimal guarding times
I experimentally assessed the “optimal guarding time” for each sex in the laboratory as well as the “compromised guarding time” (theoretically this should be a
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compromise between the optimal guarding time of the male and the optimal guarding time of the hermaphrodite; Jormalainen, 1998).
After reaching sexual maturity, clam shrimp were treated according to the specific protocol for each project (see below). The same recording protocol described above for the second experiment (on the effect of size) was used here, this time pairing animals at random from the ones available in the rearing tanks. At the end of the recording, all individuals were measured with a caliper (maximum carapace length, in millimeters).
Experiment 1 The optimal guarding criterion for hermaphrodites was assessed by restricting the movement of males with a cotton thread glued to the carapace using the following procedure. Individual males were removed from the water and placed on a
Petri dish. The outer carapace was dried with Kimwipes ® and one end of a fine cotton thread, dipped in a small dot of non toxic superglue, was positioned on the carapace.
The glue was then allowed to dry and the shrimp was quickly returned to the water. The other end of the thread was fixed to the rim of the beaker with a paper clip, leaving just enough length for the male to swim up and down on the water column, but not far from the point were he was tied. The procedure was fast and harmless to the shrimp, but was effective in limiting male mobility. Males were given a five minute acclimation period before introducing two hermaphrodites into the beaker. I used two hermaphrodites for each male in order to increase the chances of outcrossing (hermaphrodites can still self fertilize, even in the presence of males). For each beaker, only data obtained from the first outcrossing were used.
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Experiment 2 The optimal guarding criterion for males was assessed treating hermaphrodites with a 10 mg/ml solution of magnesium sulphate (MgSO 4). This solution had been successfully used as a muscular relaxant in the freshwater amphipod
Paracalliope fluviatilis (Sutherland et al., 2007). Hermaphrodites were moved to a beaker full of MgSO 4 solution for 75 minutes. At the end of this period, hermaphrodites were lying on the bottom of the beaker, beating their pleopods, but were unable to swim.
Treated hermaphrodites were then moved to clean water for five minutes to wash away any residual of MgSO 4. They were then individually moved to the beaker for recording, where they were paired with a male.
Experiment 3 In order to obtain data on compromised mate guarding time, one male and one hermaphrodite were moved to a beaker to be recorded. None of the individuals were subject to any kind of treatment.
Following these protocols, I collected data on mate guarding time when males were restrained (Experiment 1), when hermaphrodites were treated with MgSO 4
(Experiment 2), and with no treatment (Experiment 3). I also recorded selfing time from experiments 1 and 3 (measured as the time from the hermaphrodite molting to movement of eggs into the brood chamber). Initially, to make sure that the treatment with MgSO 4 was not affecting the reproductive physiology of the hermaphrodites, I compared the time from dropping of the eggs from the brood chamber to molting across treatments (one way ANOVA). I ran a one way analysis of covariance (ANCOVA) to assess the influence of the type of experiment on mate guarding duration, using size differences between the mates as a covariate (I tried to match similar sized animals, but
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there was always a slight difference of size in the couple) to check for the possibility of an interaction between size difference and experimental type. I also accounted for the reproductive state of the hermaphrodite at the beginning of precopula, performing an
ANCOVA as previously described and separating hermaphrodites with and without eggs for each experiment. Again, size difference was used as a covariate. Mate guarding time, selfing time, and the time difference between dropping the eggs and molting were log 10 transformed to meet the assumptions of normality. Finally, I compared the duration of receptivity and selfing time across treatments. In all cases, receptivity was measured as time from molting to the movement of the eggs into the brood chamber
(either as a result of outcrossing or selfing). I ran a non parametric Wilcoxon/Kruskal
Wallis test since I was not able to normalize the residuals.
Results
Mate guarding costs
I observed a total of 69 couples from three separate populations. I was interested in the difference in gut fullness between the sexes during the observations. The
ANCOVA detected a significant interaction between gut fullness of males and females over time guarded (Figure 3.1, Table 3.1): the longer the guarding phase, the larger the decrease in fullness for hermaphrodites, but not for males. I did not observe any influence of age (P > 3.5, analysis not shown) or size (P = 0.0748, analysis not shown) and thus these potential covariates were not used in the final model. Population origin affected the difference in gut content: of the three populations, shrimp from SWP3 were
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significantly different from the others (Tukey’s HSD Test), with the highest decreases in gut fullness.
Male Hermaphrodites
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0
-20
-40
-60
-80 Difference Difference in gut fullness (%)
-100 0 0.5 1 1.5 2 2.5 Log MG time (min)
Figure 3.1 –Reduction in gut fullness from the beginning to the end of the observational period (in percentage) as a function of mate guarding duration (log 10 transformed). Best fit: continuous line (hermaphrodite) and dotted line (male). TABLE 3.1 STATISTICAL ANALYSIS
SOURCE SS MS Num DF F Ratio Prob > F Sex 21191 21190.6 1 75.4692 <.0001 Population 4714 2357.2 2 8.3950 0.0004
Log 10 MG time 4150 4150.22 1 14.7808 0.0002 Log 10 MG time*Sex 2770 2769.76 1 9.8644 0.0021 Error 37064 132
Blocked one way ANCOVA (with population as a randomized block effect) on variation of gut fullness between the sexes. Mate guarding time (log10 transformed) was added as a covariate.
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The effect of size
I analyzed a total of 43 couples. I detected a general increase in mate guarding time as males were increasingly bigger than hermaphrodites (Figure 3.2). The same trend was found when categorizing data into three size classes (Figure 3.3). This time, however, I found a significant decrease in mate guarding time only when the hermaphrodite was larger than the male. I did not find a significant difference when males were equal to or larger than hermaphrodites, although the trend is in the same direction as noted in Figure 3.2.
2.5
1.5
0.5 Log10 MG timeLog10 (min)
-0.5 -2.5 -1.5 -0.5 0.5 1.5 2.5 Size difference M-H (mm)
Figure 3.2 – Correlation of size difference between the sexes in mate guarding couples (male size – hermaphrodite size) with mate guarding time (log transformed). The linear regression indicates an increase in guarding duration as males are proportionally larger than hermaphrodites ( y = 0.2279x + 1.0487, p = 0.002, n = 43, r 2 = 0.2105, best fit: dotted line).
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Figure 3.3 – Mean mate guarding time (log 10 transformed) for three size categories: couples with male smaller than hermaphrodite (M
Compromised and Optimal Guarding Time
For experiment 1 (males restrained) I recorded a total of 64 tapes. In many tapes, neither hermaphrodite approached the male, but instead self fertilized their eggs (data used for determining selfing time below); only in 13 cases did at least one hermaphrodite outcross with the male. For experiment 2 (hermaphrodites treated with
MgSO 4), I recorded a total of 21 tapes. Five tapes were not used in the statistical analyses because the hermaphrodites did not move their eggs, and two more tapes were excluded because the hermaphrodites did not outcross with males but instead self fertilized. For experiment 3 (compromised guarding time) we recorded a total of 39 tapes. Three tapes were excluded from the analyses because it was not clear whether the result of mate guarding was outcrossing or selfing. Eight more tapes were not used in
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the calculation of compromised guarding time because the hermaphrodites selfed.
However, these eight tapes were used to calculate selfing time (noted below).
There was no significant difference across treatment categories in the time from dropping the eggs to molting (ANOVA: F 3,67 = 0.8421; p = 0.4756). Mate guarding time was significantly different across the three treatments (males restrained, hermaphrodites treated with MgSO 4, and compromised time; F 2,54 = 17.2242; p < 0.0001; Figure 3.4). I initially used size differences between the sexes (male size minus female size, in millimeters) as a covariate, but since I did not detect any influence of the covariate (F 1,51
= 1.2034, p = 0.2778) and no interaction between treatment and covariate (F 2,51 =
0.0508; p = 0.9505) on mate guarding time, I removed size difference between the sexes from the analysis. The optimal guarding time for males (obtained when hermaphrodites were treated with MgSO 4) was significantly longer than the compromised guarding time
(none of the sexes treated) and the compromised guarding time was longer than the optimal guarding time for hermaphrodites (obtained when males were restrained).
I also considered the reproductive state of the hermaphrodite at the beginning of mate guarding, finding an overall difference in mate guarding time (ANCOVA: F 5,50 =
25.2477; p < 0.0001; Figure 3.5). In each treatment the mate guarding time was significantly shorter when the hermaphrodites were closer to receptivity (no eggs in the brood chamber) than when the hermaphrodites had not yet dropped their eggs at the beginning of the guarding phase (Figure 3.5). Again I did not detect a significant interaction between size and treatment (ANCOVA: F 5,45 = 0.5390; p > 0.7) and thus we removed this parameter from the analysis. Size difference between male and
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hermaphrodite did not influence mate guarding time (ANCOVA: F 1,50 = 2.9098; p =
0.0942).
Figure 3.4 Mean guarding time (log 10 transformed) for each type of experiment (see text for detailed explanations). Error bars represent two times the standard error.
Figure 3.5 Mean guarding time (log 10 transformed) for each type of experiment accounting for the receptivity state of the hermaphrodite. Error bars represent two times the standard error.
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In the final analysis, I compared the duration of receptivity across treatments
(including selfing). Interestingly, receptivity for the selfing hermaphrodites was longer than receptivity of the hermaphrodites in all the other treatments (Wilcoxon/Kruskal
Wallis: χ 2 = 14.0853; n = 111; df = 3; p = 0.0028; Figure 3.6).
Figure 3.6 – Variation in hermaphrodites’ length of receptivity (time from molt to egg movement to the brood chamber), in minutes, among treatments. Error bars represent two times the standard error.
Discussion
Precopulatory mate guarding is a complex behavior that can be analyzed using an intersexual conflict perspective. According to theory (Jormalainen, 1998), costs and benefits of mate guarding durations are different between the sexes. Males are willing to invest more time than females in a guarding event to reduce the probability of losing a mating opportunity. Females, on the other hand, are more sensitive to costs and would prefer to be guarded for a short amount of time. A compromise is usually reached with an intermediate guarding time between male and female optimal guarding time
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(Jormalainen, 1998). Additional parameters can influence the guarding duration, including a power asymmetry between the sexes (Jormalainen, 1998).
The goal of these series of experiments was to perform a comprehensive study, considering at least one possible cost (restricted feeding during mate guarding), changing the relative power between sexes, measuring the optimal guarding time for each sex (in order to compare it to the compromised guarding time i.e., the resolution of the conflict), and evaluating the different benefits for the sexes. Below I will discuss each of these in turn.
MATE GUARDING COST Hermaphrodites suffered from reduced feeding during mate guarding. The level of gut fullness decreased with increasing guarding duration: the higher reduction in food intake for the hermaphrodites corresponded to the longer time they spent being guarded. Mate guarding couples alternate from phases when the animals are resting on the bottom to phases where animals are swimming in the water column (personal observation). Clam shrimp are filter feeders and while engaged in mate guarding, males can still actively swim. However, hermaphrodites’ swimming activity is reduced during guarding (hermaphrodites are often “pushed” through the water by males when the couple is moving). My first experiment recorded the amount of food in the gut, visually, as an estimate of ingestion rate, and thus is a quantitative assay of diet. I did not consider the assimilation efficiency (qualitative aspect), but a reduction in food intake will likely result in energetic costs.
I also detected variation among populations: in one population there was a significantly greater decrease of food in the gut than in the other two. In this experiment,
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populations were raised under natural conditions, and thus I can expect variation among populations in densities and amount of nutrients in the water, which can explain the significant variation observed among populations.
Other studies have investigated differential feeding costs between the sexes during mate guarding events. In the amphipod Gammarus lawrencianus , males use their gnathopods to guard females. Gnathopods are also used to manipulate food, and thus males cannot feed while guarding (Robinson & Doyle, 1985). In this species males incur higher mate guarding costs than females. Similarly, being guarded is not too costly to female water striders Gerris remigis . Guarded females are more successful in capturing prey than single females, which are constantly harassed by multiple males (Wilcox,
1984). Unlike these species, Eulimnadia texana hermaphrodites suffered higher feeding costs than males during mate guarding and this can differentially influence male and female behaviors and guarding time preferences, as I will outline below.
POWER ASYMMETRY BETWEEN THE SEXES When a male encounters a possible mate, he can decide if it is favorable to start a guarding phase or not. Once the male starts guarding, the chosen mate can accept or try to resist the mate guarding attempt. Persistence and resistance should be proportional to the balance of costs and benefits determined by the duration of the mate guarding phase (Jormalainen, 1998). I manipulated couples, matching individuals of different size. Larger size can provide physical power to partially overcome the contender’s persistence or resistance. I observed, in fact, a significant decrease of mate guarding time when hermaphrodites were larger than males (and thus hermaphrodite resistance was more effective than male
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persistence) and a significant increase in mate guarding time when males where larger than hermaphrodites (and thus male persistence was more effective than hermaphrodite resistance). Similar results have been reported for the isopod Idotea baltica
(Jormalainen & Merilaita, 1993; Jormalainen et al., 1994a; Jormalainen & Merilaita,
1995). In E. texana male persistence seems to be quite strong, even when males are the same size as hermaphrodites. I did not detect any substantial increase in mate guarding duration when males were larger than their mates compared to couples of the same size.
Male persistence is thus quite effective, maybe due to the morphological adaptations of claspers (it is difficult to separate a guarding pair, even when forcefully trying to separate them via agitation; personal observation).
While E. texana presents comparable sizes for males and hermaphrodites, a sexual size dimorphism has been reported for clam shrimp species belonging to the genera Limnadia and Limnadopsis (Weeks et al., 2006b). In the two dioecious genera, males are larger than females and the mate guarding time is significantly longer than in
E. texana (see Chapter IV). These data might suggest that males in some species have been selected to grow larger than their mates to better control the guarding process and skew the compromised guarding time towards their optima. Moreover, both Limnadia and Limnadopsis experience the 50:50 sex ratios typical of dioecious species. An increase in male size might also be selected in response to higher male male competition in dioecious vs . androdioecious species. The competition could be either indirect
(exploitation of females) or direct (i.e., takeovers, when one male can dislodge another mate guarding male). At a population level, the mating advantage for larger males has
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been reported in the mate guarding isopod, Asellus aquaticus , at high population densities, or when the sex ratio is male biased (resulting in increased male male competition), but not at low population densities (Bertin & Cezilly, 2005). Similar results were found in the amphipod Gammarus pulex in the laboratory with increased male: female sex ratio (Bollache & Cezilly, 2004a). Thus, larger male size could be selected to increase male control on mate guarding duration (intersexual conflict) and/or to respond to high male male competition (intrasexual conflict).
OPTIMAL AND COMPROMISED MATE GUARDING TIME
Hermaphrodites and males have reasons to fight over mate guarding duration: according to theory, males should prefer a longer mate guarding time than their mates because of different costs and benefits in the two sexes (Jormalainen, 1998). This notion is consistent with the compromised guarding time noted above; when the guarding time is shorter or longer depending on whether the hermaphrodite or male is the larger sex in the guarding pair, respectively. Additionally, I detected different optimal guarding times when I allowed one sex to make the guarding decision by reducing the ability of the other sex to respond. Mate guarding duration was longer when hermaphrodites were treated with a solution of a muscular relaxant agent, and thus their response was inhibited. Mate guarding was shorter when males were restrained, and thus they could not actively seek females to start a guarding phase, but were forced to wait for hermaphrodites to approach them. Hermaphrodites are capable of assessing male presence (Medland et al., 2000 and see Chapter II) and thus can decide to swim within
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reach of males. I also recorded intermediates (“compromised”) guarding times when neither of the sexes was manipulated.
These results fit perfectly with Jormalainen’s theoretical model (1998). Mate guarding represents a complex case of intersexual conflict over mate guarding duration, when males would like to guard as long as it takes not to miss a mating opportunity
(optimal mate guarding criterion for males), whereas females (or hermaphrodites in this case) would like to be guarded for as short as possible (optimal mate guarding time for females) since mate guarding is costly to them (their feeding costs increase with the guarding duration). A compromise (the resolution of the conflict) is reached with a mate guarding duration somewhere in between the two optimal mate guarding times. In particular, the closer the hermaphrodites are to receptivity (hermaphrodites with no visible eggs in the brood chamber) the shorter should be the compromised guarding time, with both sexes benefiting.
Inhibition of female resistance had been used in other studies: Jormalainen and
Merilaita (1995) used alloferin (a muscle relaxant) in two isopod species ( I. baltica and
Asellus aquaticus ) and one amphipod species ( Gammarus zaddachi ). They also subjected the marine I. baltica females to osmotic stress (placing them in freshwater) to reduce their ability to resist mate guarding attempts by males. Of the three species examined, only I. baltica females were affected by the treatments and males were able to guard them for a significantly longer time than untreated controls. Jormalainen and
Shuster (1999) treated thermal spring female isopods Thermosphaeroma thermophilum with alcohol and recently Cothran (2008b) treated female amphipods Hyalella with CO 2
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or a clove oil solution (depending on the size of the amphipod ecomorphs) and recorded mate guarding time. As found herein, anesthetized females were guarded longer than the control.
Reduction of male persistence is more difficult to obtain. Osmotic stress and clipping of nails or dactyls (to reduce male efficiency in guarding a female) were used on I. baltica and T. thermophilum males (Jormalainen & Merilaita, 1995; Jormalainen
& Shuster, 1999) but did not result in decreased mate guarding duration. The authors interpreted this as an implication of the role of the female in the mate guarding decisions in this species. In E. texana instead, I did find a significant influence of male persistence on mate guarding duration.
MATE GUARDING BENEFIT The goal of mate guarding is to favor the presence of both mature and receptive males and females. This will result in successful mating. In dioecious species, neither sex can reproduce without the other, so the general benefit is to acquire a mate in order to reproduce. In androdioecious species, hermaphrodites have the option of self fertilization. In the clam shrimp E. texana , inbreeding depression is high in natural populations (Weeks & Zucker, 1999; Weeks &
Bernhardt, 2004) and thus outcrossing is beneficial in order to produce fitter offspring.
In this case the benefit of mate guarding is still high, even for hermaphrodites. I unexpectedly found a longer receptivity state in selfing hermaphrodites than in outcrossing hermaphrodites. A possible explanation is that hermaphrodites are able to control their reproductive cycle and possibly increase the time from molt to self fertilization, waiting for a good quality male to approach. A similar increase in
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receptivity was also found in E. texana in a previous study (Zucker et al., 2002). In both the current and previous studies, delayed self fertilization can be interpreted as a method to allow for an increased possibility of encounters with males. My results were obtained from hermaphrodites isolated with a single male, and thus there was no opportunity for interactions with different males. If hermaphrodites are able to assess the quality of their mates, and they do not consider the one available in the experiment of good quality
(which may be quite likely in highly inbred populations), then hermaphrodites might postpone self fertilization while waiting for another higher quality male to approach.
In conclusion, I used the clam shrimp E. texana as a manageable study organism to analyze mate guarding behavior with a comprehensive approach. I evaluated a potential cost of mate guarding for each sex (reduced food intake), created an asymmetry in power by manipulating the size assortment of couples to see the resulting effect on the duration of mate guarding, and assessed the optimal and compromised mate guarding criteria for the two sexes. Jormalainen’s model (1998) assumes that at least one sex should incur higher costs during mate guarding. I found that E. texana hermaphrodites experienced reduced feeding while being guarded and this cost is directly proportional to the guarding duration. Higher costs for one sex have been also reported in other species. Female amphipods of Hyalella azteca undergo higher predation risk by fish when paired (Cothran, 2004), while paired males of Gammarus pulex suffer higher energetic costs than paired females (Plaistow et al., 2003). Asellus aquaticus males also incur higher costs during mate guarding than females, at a point that affects their subsequent fitness and survival (Benesh et al., 2007). As predicted by
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the model (Jormalainen, 1998), a power asymmetry should influence the result of the mate guarding conflict, favoring the more “powerful” sex. I supported this prediction in
E. texana by analyzing an asymmetry in size: the larger sex shifted the compromised guarding duration towards its own optimal guarding time. A similar result was found in the isopod I. baltica (Jormalainen & Merilaita, 1993; Jormalainen et al., 1994a;
Jormalainen & Merilaita, 1995). Finally, I measured the optimal and compromised guarding time for the two sexes. As expected (Jormalainen, 1998), the optimal guarding time for hermaphrodites was shorter, while the optimal guarding time for males was longer than the compromised guarding time. Few other empirical studies have tested this in the laboratory and, similar to the current study, all have obtained longer mate guarding times when female resistance was reduced (Jormalainen & Merilaita, 1995;
Cothran, 2008b).
The current chapter and the above reported empirical studies support
Jormalainen’s model (1998), at least in crustaceans. Evidence for a more general test of this model might be found in other systems employing precopulatory mate guarding
(e.g., spiders; Bel Venner & Venner, 2006).
Using an androdioecious clam shrimp, I was able to study mate guarding in a species where one sex (hermaphrodite) is capable of self fertilization. I was convinced that this ability allows the potential to decrease substantially the benefit of mating with a male, and thus the benefit of being guarded. In reality, I measured a longer receptive window in the selfing hermaphrodites than in the outcrossing hermaphrodites. I am
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interpreting this result as an indication of the importance of outcrossing for hermaphrodites in an androdioecious population.
Acknowledgements
I would like to thank Dr. Brian Bagatto for helpful suggestions on the use of muscular relaxants and Beth Wallace for her help in rearing clam shrimp in the laboratory.
IV. MATE GUARDING BEHAVIOR IN CLAM SHRIMP: THE INFLUENCE OF
MATING SYSTEM ON INTERSEXUAL CONFLICT
Abstract
Mate guarding is a behavioral strategy used by males to gain access to receptive females.
This complex behavior can result in antagonistic interactions between the sexes and can be influenced by social, morphological, and physiological parameters. The mating system of a particular group of animals should also influence the strength of the conflict and thus the guarding duration. The conflict is, in fact, determined by differential costs and benefits of guarding to both sexes; specifically, self compatible hermaphrodites may have reduced benefits of outcrossing with males relative to the benefits of such outcrossing to strict females. Using branchiopod crustaceans as my study organisms, I was able to investigate mate guarding behavior in dioecious (co presence of males and females) and androdioecious (co presence of hermaphrodites and males) species. Hermaphrodites are facultatively unisexual (they can self fertilize in the absence of males). Both sexes in androdioecious systems should shift their guarding times to lower values relative to dioecious species. Males are always present in lower percentages in androdioecy than in dioecy and thus encounter rates with receptive hermaphrodites are relatively higher, which should cause males to guard for shorter durations. Hermaphrodites should also have less incentive to incur high costs for mate guarding, having the alternative of self fertilization. I analyzed mate guarding behavior in a dioecious species, Limnadia badia ,
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measuring compromised and optimal (for males and females separately) mate guarding times. I then compared the compromised guarding time with three other species
(characterized by different mating systems) and was able to confirm that mate guarding times are lower in androdioecious than in dioecious species. This result represents a powerful test of mate guarding theory, while introducing a new parameter (mating system) not previously investigated in mate guarding models. Moreover, this new approach proposes a unique method to explore the ideas of conflict over mate guarding: the analysis of species with different costs and/or benefits than the “standard.” Clam shrimp species allow this approach by using an inter mating system comparison between closely related species that differ in mating system. This approach also validates the idea that the use of facultative hermaphrodites has the potential to make a valuable contribution to our understanding of intersexual conflict. Keywords : Eulimnadia texana ,
Eulimnadia dahli, Limnadia badia , Limnadopsis tatei , androdioecy.
Introduction
Precopulatory mate guarding is a behavioral strategy used by males to gain access to receptive females. It is commonly found is species in which female receptivity is limited to a brief and specific period of time, or the breeding season is particularly short
(Ridley, 1983; Jormalainen, 1998). Precopulatory mate guarding has been described in a variety of taxa, from invertebrates (e.g, crustaceans and spiders) to vertebrates (e.g., anurans). This behavior is more complex than was initially thought in that it is determined by interactions between the sexes and can result in an interesting case of
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intersexual conflict when males and females are experiencing different costs and benefits
(Jormalainen et al., 1994b; Yamamura & Jormalainen, 1996; Jormalainen, 1998).
Mate guarding time can be influenced by a variety of parameters such as physiological parameters (e.g., female molting in arthropods), morphological parameters
(relative size between the sexes and variation in male size), social parameters (including sex ratio and density, which primarily determine mate encounter rate), and mating history. All these parameters can also vary temporally in a single mating event (e.g., time to molt) or locally in a single population (e.g., encounter rates in different microhabitats).
Due to this complexity, mate guarding has been the subject of many empirical studies
(e.g., Jormalainen et al., 2000; Jormalainen et al., 2001; Hume et al., 2002; Bertin &
Cezilly, 2003; Plaistow et al., 2003; Benesh et al., 2007; Cothran, 2008b) and theoretical models (Parker, 1974; Wickler & Seibt, 1981; Grafen & Ridley, 1983; Hunte et al., 1985;
Yamamura, 1987; Elwood & Dick, 1990; Jormalainen et al., 1994b; Yamamura &
Jormalainen, 1996; Jormalainen, 1998; Härdling et al., 1999; Härdling et al., 2004). Mate guarding models in particular have tried to incorporate many different parameters to predict their influence on guarding time, such as the receptive state of females, encounter rates and size difference between the sexes, and the possibility of takeovers (see Chapter
I for an historical account on various models and the parameters investigated). As a result of the interaction among these parameters, the guarding duration is predicted to be quite variable. In fact, variability has also been documented across species (e.g., Jormalainen &
Merilaita, 1995) and across populations of the same species (Jormalainen et al., 2000 and see Chapter V).
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The mating system of a particular group of animals should also influence the strength of the conflict and thus the guarding duration. “Mating system” is a broad term.
Emlen and Oring (1977) defined it in this way: “ The term ‘mating system’ of a population refers to the general behavioral strategy employed in obtaining mates. It encompasses such features as (i) the number of mates acquired, (ii) the manner of mate acquisition, (iii) the presence and characteristics of any pair bonds, and (iv) the patterns of parental care provided by each sex ” (note on page 222). Following this definition, we can group mating systems in categories as (i) polygamy, polygyny, polyandry, or harem breeding; (ii) pure searching, mate guarding, partner fidelity, and alternative mating strategies (e.g., sneaking behaviors); (iii) pure monogamy or serial monogamy; and (iv) maternal, paternal, biparental care, or helper at the nest. Besides using this approach, mating systems can also be investigated focusing on the processes, utilizing an
“ecological framework,” like Emlem and Oring (1977) have done, by analyzing the role of females’ distribution (spatial and temporal) in shaping mating systems, as in Shuster and Wade (2003).
Here I am referring to another group of mating systems (or sexual systems) that categorizes organisms on the basis of the presence of sexual types: hermaphroditism
(sequential hermaphrodites or simultaneous hermaphrodites), dioecy (co presence of males and females), androdioecy (co presence of males and hermaphrodites), and gynodioecy (co presence of females and hermaphrodites). The last two groups commonly represent mixed mating systems and are particularly informative for the study of mating
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behaviors. To my knowledge, mate guarding has never been investigated via comparisons of a dioecious system with a mixed mating system.
Androdioecy is a rare mating system in animals (see Weeks et al., 2006a and references therein). Only a few animal groups are composed of males and hermaphrodites: one fish ( Rivulus marmoratus ), some nematodes (with Caenorhabditis elegans the most well known representative), some cirriped crustaceans, and some branchiopod crustaceans. Thanks to its intermediate state (between dioecy and hermaphroditism), androdioecy is a system that could be extremely valuable in studies of mating behaviors. It allows for the opportunity to compare mating behaviors in males, females, and hermaphrodites in androdioecious and dioecious populations. This, then, allows for the assessment of different strategies used by the different sexes.
The first goal of this chapter is to describe the mate guarding behavior in the dioecious species Limnadia badia (Wolf, 1911), following the same protocol used for
Eulimnadia texana (Packard, 1871; see Chapter III). In this way I can assess whether this dioecious species of clam shrimp is experiencing mate guarding as a form of intersexual conflict in a similar way as in the androdioecious clam shrimp. The second goal of this chapter is to compare mate guarding in four species of clam shrimp (Branchiopoda:
Spinicaudata: Limnadiidae). In order to do this I will use data obtained from L. badia
(present chapter) and E. texana (Chapter III) plus data collected from two more species: the dioecious Limnadopsis tatei (Spencer & Hall, 1896) and the androdioecious
Eulimnadia dahli (Sars, 1896). These last two species will help make the comparison more robust, since limitations of two species comparative studies are well known
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(Garland & Adolph, 1994). I can thus compare four closely related species (clam shrimp in the family Limnadiidae) belonging to two mating systems (androdioecy and dioecy) that commonly mate guard.
Dioecious and androdioecious species differ for a variety of parameters other than the mating system, including sex ratio of populations, size, and length of the receptive cycle (see below). I can predict that both males and females belonging to the dioecious system should guard longer than the corresponding sexes in the androdioecious system.
This expectation is derived by applying the graphical model proposed by Jormalainen
(1998) to each sex (females and males in the dioecious species and hermaphrodites and males in the dioecious species). Males in dioecious population should experience lower encounter rates with receptive mates (due to a higher male percentage in these populations than in androdioecious population; see below). This should cause males to guard for longer durations. Females should also be willing to accept higher costs of being guarded than hermaphrodites, the former not having the option of self fertilization. As a result, combining the optimal guarding time of the sexes to predict a compromised guarding time (see Chapter I) and comparing between mating system, I expect the compromised mate guarding time to be longer in dioecious than in androdioecious species.
Eberhard (2005) suggested the use of facultative hermaphrodites as a possible test of the strength of intersexual conflicts. The difference in mate guarding duration between two mating systems can represent a measure of the different strength of the conflict involved during precopula. Such a comparison on mate guarding decisions and strength
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of intersexual conflicts between species characterized by different mating systems has never been proposed nor investigated before, and clam shrimp present an ideal system with which to test this hypothesis. This new approach represents a unique method to explore the ideas of conflict over mate guarding.
Materials and methods
I used individuals from four different species, all belonging to the family
Limnadiidae:
Eulimnadia texana : sediment collected in 2000 and 2004 in Portal (31°57.387’N;
109°08.998’W; Wallace Tank), Arizona, USA. This collection site has been
referred as WAL in other studies (e.g., Sassaman & Weeks, 1993; Weeks et al.,
1999; Weeks et al., 2000b; Weeks et al., 2008).
Eulimnadia dahli : sediment collected in 2008 in The Humps (Shire of Kondinin,
32°0.558’S; 118°36.321’E; pool 3), Western Australia;
Limnadia badia : sediment collected in 2008 in Dingo Rock (Shire of Lake Grace,
33°19.009’S; 118°57.534’E; pool 1 2) and Puntapin Rock (Shire of Wagin,
33°19.495’S; 117°23.941’E; pool 1 4 6) both in Western Australia;
Limnadopsis tatei : sediment collected in 2003 in Kadji Kadji (Shire of Morawa,
29°8.233’S; 116°24.833’E; claypan), Western Australia.
Rearing in the laboratory
For all species, ~ 500 ml of sediment was added to 37 liter aquaria which was then filled with a strong jet of de ionized water (to simulate natural fillings). Aquaria
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were aerated via air stones (on low) and illuminated with Durotest sunlight simulating fluorescent lights. Water temperature was maintained constant at 27°C. Sexual maturity was reached sooner in androdioecious species (approximately 5 7 days after hydration) than in dioecious species (approximately 10 12 days after hydration for L.badia and 15 days for L. tatei ).
Mate guarding in Limnadia badia
I used the same methodology described in Chapter III for E. texana with minor variations:
The optimal guarding criterion for females was assessed by tying males with a cotton thread glued to the carapace. One female was introduced into the beaker with the restrained male;
The optimal guarding criterion for males was assessed by treating hermaphrodites with a 10mg/ml solution of magnesium sulphate (MgSO 4), a muscular relaxant, for 75 minutes
(preliminary results showed a reduced swimming activity in female L. badia left in the solution for this amount of time);
The compromised mate guarding time was measured by recording one male and one female L. badia not subjected to any treatment.
All tapes were recorded for 72 hours to document a complete guarding cycle per pair. Clam shrimp were fed at the beginning of the experiment and again every 24 hours
(a couple of drops of a solution of yeast and ground fish flake food). All animals were measured with calipers after the trial (maximum carapace length, in millimeters).
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Compromised mate guarding time across species
Compromised mate guarding time was also measured for the other three species: the androdioecious species ( E. texana and E. dahli ) were recorded for 30 hours, L. tatei was recorded for 72 hours. All tapes were scored for mate guarding time, recording the start of clasping time, the end of clasping, time that eggs were dropped, and the time of hermaphrodite/female molt (these last two measures were used to calculate the receptive cycle of hermaphrodites and females). Clam shrimp were measured with calipers after the trial (maximum carapace length, in millimeters).
Statistical analyses
I ran a blocked one way analysis of covariance (ANCOVA) to analyze variation in mate guarding time in the three treatments applied to L. badia : male restrained,
MgSO 4 treated, and compromised guarding time. I used treatment as a fixed effect and outcrop as random effect (since I used animals coming from different populations). I did not run a crossed designed across populations because I did not use all the populations for all the treatments. I used difference in size between sexes (male size minus female size) as a covariate. Since both the blocked effect and the covariate were not statistically significant (P = 0.1515 and P = 0.2498, respectively), I then ran a simpler, one way
ANOVA on variation in mate guarding time in the three treatments. Tukey's Honestly
Significant Differences (HSD) test was used to detect significant pairwise differences between the treatments.
Initially, I performed a nested, one way ANCOVA to analyze mate guarding time among species and mating system. Species were considered random factors nested in
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mating system. Male size, female/hermaphrodite size, and the time between molting and movement of the eggs (measure of the reproductive cycle of females and hermaphrodites) were tested as covariates. Since none of these were significant they were removed from the analysis and a simpler, nested, one way ANOVA was then used.
All analyses were performed using JMP 6.0 (SAS Institute Inc.). To meet criteria of normality, mate guarding time was log 10 transformed.
Results
Mate guarding in Limnadia badia
A total of 23 tapes were recorded for the compromised guarding time (Dingo rock population). Unfortunately, in four tapes, either the male or the female died during the trial, and in two, eggs were not fertilized and moved into the brood chamber, leaving only
17 tapes for comparisons. Twenty four tapes were set up to analyze the optimal mate guarding time of females (tying males) using animals from Dingo (nine tapes) and
Puntapin (15 tapes) populations. Unfortunately, some animals did not interact with each other (four from Dingo and seven from Puntapin), or females did not move eggs in the brood chamber (two from Puntapin), or one animal died during the trail (two from
Puntapin) reducing the number of usable tapes to eight. Twenty four more tapes were set to measure the optimal guarding time for males (treating females with MgSO 4), using
Dingo (11 tapes) and Puntapin (13 tapes) populations. Again the sample size was reduced to 10 because of death of one of the two animals (eight from Dingo and three from
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Puntapin) or no egg movement into the brood chamber (two from Dingo and three from
Puntapin).
Treatment (male constrained, female treated with MgSO 4) and unmanipulated
(control) had a significant effect on mate guarding duration (Table 4.1). The optimal mate guarding time for females (obtained when males were restrained) was significantly lower than the compromised guarding time (Figure 4.1). On the other hand, Tukey's HSD Test did not find any significant difference among the optimal male guarding time and the optimal female guarding time or compromised time (Figure 4.1).
TABLE 4.1 –STATISTICAL ANALYSIS
Source SS DF F Ratio Prob > F Treatment 6.23 2 4.2528 0.0230 Error 23.45 32
One way ANOVA on mate guarding time (log 10 transformed).
Figure 4.1 Mean guarding time (log 10 transformed) for each type of treatment in Limnadia badia (see text for details). Error bars represent two times the standard error.
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Compromised mate guarding time across species
The four species analyzed in this chapter do not just differ for the mating system
(which allows hermaphrodites to self fertilize or outcross with males while females can only outcross), but for other parameters as well. Table 4.2 summarizes some of these parameters. Dioecious species live longer than androdioecious ones (personal observation), present a longer receptive cycle (i.e., time from molt to the moment eggs are moved into the brood chamber) for females compared to the receptive cycle of hermaphrodites, both sexes are larger than in androdioecious species, and the sex ratio is different: in androdioecious species males are a smaller portion of the population, while in dioecious species there is usually a 50:50 sex ratio (Table 4.2).
TABLE 4.2 – SPECIES COMPARISON
% MEAN ♂ SIZE MEAN ♀ SIZE ♀ RECEPTIVE SPECIES MATING SYSTEM MALES (mm) (mm) CYCLE (min) Eulimnadia Androdioecious 26.9** 3.1±2.5 3.1±2.6 18.47±30.40 dahli Eulimnadia Androdioecious 17.9* 4.6±1.7 4.5±1.6 10.91±21.01 texana Limnadia Dioecious 44.7** 7.1±0.4 6.4±0.3 223.57±272.89 badia Limnadopsis Dioecious 52.1** 10.5±0.5 9.7±0.6 74.89±154.06 tatei *(Weeks et al., 2008); **(Weeks et al., 2006b) Comparison among the four species for type of mating system; percentage of males in the population (data from literature); mean ( ± standard deviation) male and female/hermaphrodite size (in millimeters); reproductive cycle (time from molt to the moment eggs are moved to the brood chamber, mean ± standard deviation in minutes). ♂: males; ♀: females or hermaphrodites depending on mating system. I was able to analyze a total of 102 couples (19 for E. dalhi , 59 for E. texana , 17 for L. badia , and 7 for L. tatei ). The statistical analysis revealed a significant difference in
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mate guarding time among species nested within mating system (Table 4.3; Fig. 4.2).
Moreover, there was a considerable overall difference between mating systems, with dioecious species guarding significantly longer than androdioecious species (Table 4.3;
Figure 4.3). I was concerned about factors other than mating system driving this last result. Dioecious species are bigger than androdioecious species (so they could guard longer having higher energy available) and the females’ reproductive cycle is longer than the hermaphrodites’ reproductive cycle (so a longer mate guarding could be expected just based on physiological differences). However, a nested one way ANCOVA did not find any influence of size (male size: P = 0.1229; female or hermaphrodite size P = 0.1186) or receptive cycle (P = 0.7533) on mate guarding duration. I also considered size difference between the sexes. A size dimorphism, with males significantly larger than females, had been reported for L. badia and L. tatei , but not in clam shrimp in the genus Eulimnadia
(Weeks et al., 2006b). I thus added size difference as a covariate for mate guarding time and observed an increase in mate guarding duration when males were larger than their mates (Table 4.3).
TABLE 4.3 STATISTICAL ANALYSIS
SOURCE SS MS Num DF F Ratio Prob > F Species [Mating system] 2.46 1.22949 2 3.8165 0.0271 Mating system 16.76 16.7582 1 18.0237 0.0329 Male size – Female/hermaphrodite size 1.53 1.53099 1 4.7524 0.0329 Error 29.96 98
Two way ANCOVA on mate guarding time (log 10 transformed) among species (nested within mating system). Size difference between sexes was used as a covariate.
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Figure 4.2 Mean guarding time (log 10 transformed) for four species characterized by two mating systems. Androdioecious species are reported in white; dioecious species are reported in gray. Error bars represent two times the standard error.
Figure 4.3 Mean guarding time (log 10 transformed) for mating systems. Error bars represent two times the standard error.
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Discussion
Mate guarding is a complex behavior that has gained the interest of scientists working on different taxa. One motivation of the increasing attention to this behavior is that mate guarding represents a case of intersexual conflict. Clam shrimp are an excellent study system with which to analyze intersexual conflict during mate guarding. As pointed out in the previous chapters, clam shrimp are easily bred in the laboratory, their guarding cycle is relatively short, and they have been successfully studied in manipulative laboratory experiments (Medland et al., 2000; Zucker et al., 2002; Weeks & Benvenuto,
2008) as well as in a field setting (Weeks & Bernhardt, 2004). One more important reason to employ clam shrimp in studies of intersexual conflict is that they allow for the unique opportunity to test the strength of the conflict in different mating systems, namely dioecy and androdioecy. To my knowledge, mate guarding has never been analyzed by comparing different sexual systems and this type of study has the potential to make a valuable contribution to our understanding of conflict over mate guarding.
The simple and elegant graphical model proposed by Jormalainen (1998) is not easy to test. It requires the capability to measure the optimal mate guarding time for each sex to allow a comparison with the compromised guarding time of the couple (see
Chapter III). Another level on which this model can be assessed is by creating a power asymmetry (such as differential male and female size; see Chapter III) and measuring the shift of the compromised guarding time towards the optimal guarding time of the more
“powerful” sex (e.g., the larger sex). An even more sophisticated approach consists of altering costs and benefits of the mate guarding pairs in order to shift the compromised
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time in a predictable direction. Species with different costs and/or benefits than the
“standard” represent a great opportunity to test the theoretical model from this new perspective. Clam shrimp species allow this approach by using an inter mating system comparison between closely related species that differ in mating system (see below).
Since a good amount of information had been accumulated on the mate guarding behavior of the androdioecious clam shrimp, Eulimnadia texana, I was interested in the analysis of the same behavior in a dioecious species of clam shrimp and in the subsequent comparison in mate guarding strategies between dioecious and androdioecious species. I investigated mate guarding duration in the dioecious Limnadia badia , following the theoretical framework proposed by Jormalainen (1998) and the same protocol utilized for
E. texana (Chapter III). The results of the first experiment emphasize that female L. badia
“prefer” to be guarded for a shorter mate guarding time than the compromised guarding time typical of the species. This is in agreement with the general theory that shorter guarding time is optimal in females (relative to males) because being guarded is costly to females (Jormalainen, 1998). Contrary to the expectations, and to what was found in E. texana , males are not guarding females treated with MgSO 4 longer than untreated controls. I can interpret this result as an indication that in dioecious species, males are more capable of forcing females into longer mate guarding because female resistance is not efficient, or that females are more willing to be guarded longer in order not to lose mating opportunities. Anaesthetized females of the amphipod Paracalliope fluviatilis were not guarded longer than the non treated control (Sutherland et al., 2007) and a similar result was reported for the isopod Asellus aquaticus and the amphipod Gammarus
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zaddachi (Jormalainen & Merilaita, 1995). If female L. badia actually are willing to be guarded longer in order to attain a mating, then the level of the intersexual conflict over mate guarding duration should be lower in L. badia relative to E. texana .
It is also possible, though, that the MgSO 4 treatment (which was effective on E. texana hermaphrodites, see Chapter III) might not have been sufficient to reduce female resistance for an adequate amount of time to be useful for this experiment. The treatment is temporary, and the chemical has a sufficiently lasting action in a species characterized by a very short receptive cycle (Table 4.3). In L. badia the effect of the muscular relaxant might have been significantly reduced by the time the female was approaching receptivity and the male was interested in guarding. To avoid this problem, two studies on two crustacean species (the isopod, Idotea baltica , and the amphipod, P. fluviatilis ) that guard longer than L. badia (average time: 37 hours. in I. baltica ; 4 5 days in P. fluviatilis ; seven hours in L. badia ), used daily application of treatment (alloferin in the first case and
MgSO 4 in the second) until the guarding started (Jormalainen & Merilaita, 1995;
Sutherland et al., 2007). In this way the authors were sure to maintain the female in a constant relaxed status. Instead, I applied the treatment only once and the effect of
MgSO 4 could have been too low to be effective by the time males started guarding.
In order to address the differences in mate guarding strategies between dioecious and androdioecious species, I analyzed the compromised mate guarding time of four species, characterized by two different mating systems. The expectations derived from the Jormalainen’s model (1998) are that each sex in the dioecious species should sustain longer mate guarding times than in androdioecious species, but for different reasons:
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males should have longer mate guarding because they experience greater male male competition (due to the sex ratio being less favorable to them than the sex ratio in androdioecious populations), and females should accept longer mate guarding because they must mate to produce offspring (unlike hermaphrodites that can self fertilize). As a result of differences in optimal mate guarding times, the overall compromised time should be longer in the dioecious than in the androdioecious species.
The four species expressed significantly different mate guarding times and, more importantly, differences in mate guarding duration were found between the two mating systems. As expected, mate guarding was significantly longer in dioecious than in androdioecious species. I assessed the importance of male and female size as covariates to account for the possibility of increased mate guarding duration determined by higher energy available to larger animals. Empirical studies had supported the prediction that larger males can actually mate guard longer than small ones (Ridley & Thompson, 1979;
Ward, 1983; Jormalainen et al., 1994b; Hatcher & Dunn, 1997). I also added one additional covariate, the length of the female receptive cycle, to account for physiological variation among species (males might make their guarding decisions at the beginning of the female’s receptive cycle and thus guard their mates longer because they have a longer receptive phase). None of these covariates significantly influenced the mate guarding duration, so I can support the hypothesis that the mating system itself is playing a key role in mate guarding strategies.
I also added one additional covariate, the size difference between male and female
(or hermaphrodite depending on the mating system), as a potential parameter affecting
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mate guarding time. I recorded an increase in mate guarding duration when males were larger than their mates. Size asymmetry had been experimentally manipulated in E. texana and resulted in a better control by the larger sex on mate guarding duration
(Chapter III). In dioecious species in particular, an increase in male size could have been selected as a male strategy to prolong mate guarding duration (intersexual competition), or as a response to direct or indirect male male competition (intrasexual competition). In the first scenario, males might obtain a great advantage with larger size because they could shift the mate guarding duration towards their optimal guarding time (Chapter III).
In the second scenario, larger size might be advantageous in male male competition, which is higher in dioecious than in androdioecious species (androdioecious species present a more favorable sex ratio to males, with high abundance of hermaphrodites).
Males might compete indirectly for access to females (exploitation) or directly (with larger males dislodging smaller guarding males through takeovers). These two scenarios are not mutually exclusive.
In conclusion, I analyzed the precopulatory mate guarding behavior of the dioecious species L. badia and confirmed that it represents a case of intersexual conflict.
Potentially, the conflict here could be weaker than in the androdioecious E. texana , since females do not have the opportunity to self fertilize, and thus might be willing to accept longer guarding phases (as implied in the experiments with MgSO 4, but this result requires further experimental confirmations). Moreover, dioecious males undergo higher male male competition, and less frequent encounter rates than dioecious males (due to the sex ratios typical of the two mating systems). This high male persistence, combined
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with possibly low female resistance, should result in a weaker intersexual conflict on the
duration of mate guarding in dioecious than in androdioecious species (Table 4.4).
TABLE 4.4 – STRENGTH OF INTERSEXUAL CONFLICT
MATING ♂ ♀ ♂ ♂ ENCOUNTER ♀ BENEFITS OF SYSTEM PERSISTENCE RESISTANCE COMPETITION * RATES * OUTCROSSING ** Androdioecious < > < > <
Dioecious > < > < >
Androdioecy ______ Dioecy ______ Strength of intersexual conflict Schematic analysis of the strength of intersexual conflicts during mate guarding in dioecious vs . androdioecious species. Symbols > (higher than) and < (lower than) are a measure of comparison between the two mating systems; ___ represents the strength of intersexual conflict (not to scale); *influenced by sex ratio; **influenced by mating system; ♂: male; ♀: female in dioecious system, hermaphrodite in androdioecious system.
Comparing four species characterized by two mating systems, I was able to test
and support a prediction that indirectly follows from the graphical model of Jormalainen
(1998): dioecious species should guard longer than androdioecious species. This
prediction was not directly made explicit in the model, since the specific case of a
comparison between mating systems has never been proposed nor investigated before.
The inter mating system comparison performed here is a valuable extension of
Jormalainen’s model that validates the theoretical assumptions and predictions of this
graphical model.
Numerous empirical studies have used Jormalainen’s model (1998) as a
theoretical framework for the study of mate guarding, with particular attention to
crustaceans. Many studies have confirmed one of the basic assumption of the model, i.e.,
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the existence of differential costs between sexes, either as predation costs (e.g., Cothran,
2004), energetic costs (e.g., Plaistow et al., 2003), feeding costs (e.g., Robinson & Doyle,
1985 and see Chapter III), or survival costs (e.g., Benesh et al., 2007). The role of intersexual power asymmetry, in the form of size difference between the sexes, has also been investigated and verified to be important in influencing the guarding duration.
Larger individuals are able to persist (males) or resist (females) more than smaller individuals, and thus they can better control the compromised mating time (e.g.,
Jormalainen & Merilaita, 1993; Jormalainen et al., 1994a and see Chapter III). Finally, manipulative experiments have measured, in accordance to theory, longer optimal guarding times for males when female resistance was reduced (Jormalainen & Merilaita,
1995; Jormalainen & Shuster, 1999; Cothran, 2008b and see Chapter III) in species where female resistance plays an important role during the mate guarding conflict (but see Jormalainen & Merilaita, 1995; Sutherland et al., 2007 for examples of species where this is not the case). All these studies supported and validated Jormalainen’s model
(1998). The current study added a new and different test of the model, using a comparison of costs and benefits between species characterized by different mating systems, which adds a new dimension of validation to the original theory.
The mating systems defined on the basis of number of mates involved in reproduction (i.e., monogamy and polygamy) can be manipulated experimentally in the laboratory. This approach had been successfully used in some species of insects with the goal of testing theoretical predictions of intersexual conflicts. For this reason, artificial selection was applied to Drosophila melanogaster to induce monogamy (Holland & Rice,
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1999; Rice & Holland, 2005) or control and arrest female’s counter adaptations to deleterious strategies employed by males (Rice, 1996). In this way the authors were able to measure the strength of antagonism between males and females in this species.
Monogamous or polyandrous yellow dung fly lines, Scathophaga stercoraria , were produced in the laboratory and compared for changes in a morphological character: testis size (Hosken et al., 2001). Monogamous species, where males do not engage in sperm competition, presented males with smaller testes and females with smaller accessory sex glands (the female counter adaptation to sperm competition: these glands produce spermicide and could be used by females to control paternity). The experiment was used to assess the level of interaction between the sexes over conflict on paternity. Also, sex ratio had been experimentally modified in D. melanogaster to create different mating frequencies in order to assess variation of intersexual conflict (Wigby & Chapman,
2004). The results confirmed a female adaptational response in lines with male biased sex ratios. More recently, a study on the monoandrous Musca domestica (where females mate with only one male) used two different strains of flies and subjected them to forced polyandry to test costs and benefit of copulation (Arnqvist & Andres, 2006). In this comparison, it appeared that the costs of monoandry (no benefit of multiple mating to females and use of male accessory seminal substances to reduce female propensity to mate) are reduced by the nutritional value of male accessory seminal fluids to female.
Possible faults with some of the approaches outlined above have been suggested, including a possible problem with “effective population size,” which might be reduced in the artificially selected lines (Wigby & Chapman, 2004; but see response by Rice &
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Holland, 2005). Thus, manipulations of mating systems are advantageous but complex, and could present potential problems.
The current study has the advantage of using closely related species (belonging to the same family), naturally characterized by different mating systems. No manipulative treatments were thus imposed on these populations. Moreover the difference in the sexual system (presence of hermaphrodites instead of females) results in a differential benefit to outcrossing with a male (lower in hermaphrodites compared to females) and thus a different acceptance of mate guarding costs (if costs increase, hermaphrodites have the option to self fertilize). This study thus suggests that the use of mixed mating systems could be incredibly helpful in studies of mating behaviors and intersexual conflicts.
Acknowledgements
I would like to thank and Dr. Brian Bagatto for helpful suggestions on the use of muscular relaxants, Alissa Calabrese, Sadie K. Reed, and Beth Wallace for their help in rearing the shrimp in the laboratory and Jennifer Brunn for helping with the scoring of tapes.
V. MATE GUARDING BEHAVIOR IN CLAM SHRIMP: A FIELD APPROACH
Abstract
Precopulatory mate guarding is a complex behavior: it is influenced by social and physiological parameters, it changes with time during a single mating event, and it represents a case of intersexual conflict. Mate guarding has often been analyzed with the aid of theoretical models. A common assumption of these models is that mate guarding time is influenced by encounter rates between males and females, contenders’ size, and the possibility of one guarding male displacing another (takeovers). The parameters influencing male and female guarding decisions have been measured in laboratory experiments but never under natural field conditions. In this field based study, I observed mate guarding couples of the clam shrimp Limnadia badia in ephemeral pools on top of granite rock outcrops in Western Australia. I recorded guarding duration, focusing on the parameters considered important in mate guarding models: male and female size, population density, sex ratio, operational sex ratio, and female receptivity status. I also estimated time budgets for males, the possibility of male takeovers, and the potential role of female resistance. I found that female receptive status (being close to molting), small male size, and low female relative density are key factors in decreasing mate guarding duration. This study adds a field dimension to manipulative laboratory projects and theoretical models of mate guarding. I was able to observe the simultaneous interactions of multiple parameters in the field and to make a robust examination of the
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ideas of intersexual conflict during mate guarding behavior in these crustaceans.
Keywords : Branchiopoda, Limnadia badia , intersexual conflict, female resistance, male size, sex ratio, density, field observations, Western Australia.
Introduction
In order to increase the probability of paternity, males from across a wide range of taxa guard a potentially receptive female (commonly termed “precopulatory mate guarding”) and defend her against other males (Parker, 1974). Indeed, males maximize their chances of reproduction with females that are receptive to copulation for only a specific and brief period by closely following them (mate attenders) or maintaining physical contact (mate carriers), waiting for the female to become receptive (Parker,
1974; Conlan, 1991; Jormalainen, 1998).
Ridley (1983) analyzed mate guarding behaviors in the Anura and Arthropoda using a comparative approach. In anurans, where female receptivity is restricted to a particularly short breeding season, “amplexus” (the "lengthy associations of the sexes before actual mating," Ridley, 1983) is an adaptive behavior. In many arthropods, copulation is possible for only a brief period after a female molts (Ridley, 1983;
Jormalainen, 1998), when the exoskeleton is still soft enough to allow sperm transfer
(e.g., in some amphipod species) or before the oostegites cover the gonopores, thereby impeding copulation (e.g., in some isopod species). In Crustacea, mate guarding is extremely common and the behavior has evolved and been lost multiple times.
Consequently, crustaceans provide useful working models to test theoretical concepts in
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evolutionary and behavioral ecology in general, and mate guarding in particular (Duffy &
Thiel, 2007).
Precopulatory mate guarding is not just a mating strategy for males: the role of females also needs to be considered (e.g., Jormalainen et al., 1994a; Jormalainen, 1998;
Sparkes et al., 2000). A short guarding time is beneficial to both sexes, but males may invest more time with one female rather than searching for others if, for example, the encounter rate with receptive females in the population is low (Parker, 1974). Longer guarding time can be detrimental to females who would benefit from being guarded for a short time just before molting. Females can struggle by kicking and contorting their bodies to dislodge early guarding males (Jormalainen & Merilaita, 1993; Sparkes et al.,
2000). Mate guarding therefore introduces the possibility of intersexual conflict where male and female optimal guarding times might not coincide (Jormalainen et al., 1994b;
Yamamura & Jormalainen, 1996; Jormalainen, 1998; Watson et al., 1998; Jormalainen et al., 2000). In crustaceans this conflict is regulated by the female reproductive physiology (i.e., time to molt) and thus it is a dynamic interaction where the contest changes during a single mating event. The strength of the sexual conflict is sensitive to socio environmental conditions such as differences in sex ratio (Parker, 1974; Manning,
1980; Dick & Elwood, 1996), density (Parker, 1974; AlonsoPimentel & Papaj, 1996), contenders’ sizes (Elwood & Dick, 1990; Jormalainen et al., 1994a), and the possibility of takeovers (i.e., dislodgements of a guarding male by another male, Ridley &
Thompson, 1979).
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Given its inherent evolutionary significance, mate guarding behavior has been analyzed through a variety of theoretical models (Parker, 1974; Grafen & Ridley, 1983;
Yamamura, 1987; Jormalainen et al., 1994b; Yamamura & Jormalainen, 1996;
Jormalainen, 1998; Härdling et al., 1999; Härdling et al., 2004). Guarding time generally is predicted to be negatively correlated with female encounter rates (Parker,
1974; Jormalainen, 1998). As the probability of encounters increases in the population, males should guard for shorter durations because of the higher likelihood of encountering receptive mates. This prediction is supported by studies on density
(AlonsoPimentel & Papaj, 1996), sex ratio (Dick & Elwood, 1996), and operational sex ratio, OSR (Iribarne et al., 1995; Vepsäläinen & Savolainen, 1995; AlonsoPimentel &
Papaj, 1996), defined as the “average ratio of fertilizable females to sexually active males at any given time” (Emlen, 1976; Emlen & Oring, 1977).
Size is another important parameter. Larger males can sustain longer guarding times (Ridley & Thompson, 1979; Ward, 1983; Hatcher & Dunn, 1997). Alternately, larger females can resist more vigorously male attempts to guard (Ward, 1984a;
Jormalainen & Merilaita, 1993). Male size can have a strong effect on both male female interactions and also on male male competition (Elwood & Dick, 1990; Jormalainen et al., 1994a; Jormalainen, 2007). In the latter case, male size may affect guarding time in differing ways depending on the possibility of takeovers. Takeovers are explicit examples of direct male male competition, with larger males experiencing a higher OSR than smaller males because they can gain access to a higher proportion of receptive females than the latter. Since larger males can expect to be able to displace smaller
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males, they should mate guard for shorter times than smaller males (Grafen & Ridley,
1983). When male takeovers are not prevalent, mate guarding again should be related strictly to relative size and larger males should guard for longer times than smaller males (Elwood & Dick, 1990).
In mate guarding models it is assumed that males should be able to detect different stages of female receptivity, otherwise mate guarding cannot be analyzed in an optimality context. When males can detect receptivity differences among females, they can decide whether to guard one female or search for others of more advanced receptive stage. In crustaceans, unguarded females closer to molting should be more valuable since the overall guarding time will be shorter (Parker, 1974; Jormalainen, 1998). If all receptive females are guarded, and takeovers are not successful, males with a longer guarding criterion ( sensu Grafen & Ridley, 1983) can obtain a fitness advantage. So the guarding time can be adjusted differently among species (because of variation in reproductive cycles) and among populations (because of variation in sex ratio and density).
Many predictions of mate guarding models have been tested through manipulative laboratory experiments on amphipods and isopods, predominantly (e.g.,
Dunham et al., 1986; Jormalainen, 1998; Cothran, 2004; Jormalainen, 2007; Cothran,
2008b). I use clam shrimp, branchiopod crustaceans, as my study organism, which have the same advantages of other crustaceans, while also being easier to breed under laboratory conditions, having a fast life cycle with a short mate guarding duration, and having a female receptivity state that can be easily assessed by the researcher (see
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Material and Methods). All of these qualities enhance their usefulness in laboratory based studies of mating behavior (Knoll, 1995; Knoll & Zucker, 1995; Weeks et al.,
2004; Weeks & Benvenuto, 2008) . Their short guarding duration also makes them ideal for field based research. Because the precopulatory guarding phase in many other crustaceans can last from days to weeks, only a few studies have analyzed natural amphipod populations (Ward, 1986; Dick & Elwood, 1996; Bollache & Cezilly, 2004a;
Sutherland et al., 2007). In all these amphipod studies, mate guarding couples were sampled in the field and then were either preserved or moved to the laboratory for mate guarding analyses. Clam shrimp, on the other hand, present a much shorter guarding time, from minutes to a few hours, enabling direct behavioral observations to be made in natural environments.
To test the validity of various assumptions of mate guarding models, I recorded precopulatory mate guarding times of clam shrimp of the species Limnadia badia Wolf
1911 (Spinicaudata: Limnadiidae) inhabiting weather pits (termed “gnammas” by
Aborigines) on granite rock outcrops in southwestern Western Australia. The pools, varying from less than a meter to tens of meters in diameter, present an opportunity to test the predictions of mate guarding models (Grafen & Ridley, 1983; Yamamura, 1987;
Härdling et al., 1999; Härdling et al., 2004). In this field based study, I focused on the parameters important in mate guarding models: male and female size, population density, sex ratio, OSR, and female receptivity status and I estimated time budgets for males, the possibility of male takeovers, and the potential role of female resistance.
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Materials and methods
Limnadia badia is a dioecious species characterized by a pyriform dorsal organ on the head, no spines on the telson, and male “claspers” bearing a sucker like projection (Wolf, 1911; Richter & Timms, 2005). Claspers are a dimorphic, secondary sexual character, missing in females and not developed in juveniles, used by males to obtain and maintain physical contact with their mates. This contact is necessary for copulation to occur. Once fertilized, the eggs are moved to the brood chamber (located dorsally in females) where they are visible through the transparent carapace. Throughout this study, females were divided into two categories: with and without visible eggs in the brood chamber. This distinction is an easy way for the observer to roughly estimate the receptivity status of the female: females will drop their eggs at the bottom of the pool during or immediately before molting, which is a sign of receptivity to mating
(Weeks & Benvenuto, 2008).
I conducted this field study of mate guarding behavior in L. badia on four granite rock outcrops, following localized rain events in April and May 2007, in the wheatbelt area east of Perth, southwestern Western Australia: Holland Rock (Shire of Kent,
33°21.259’S; 118°44.639’E), Dingo Rock (Shire of Lake Grace, 33°0.558’S;
118°36.321’E), Wave Rock (Shire of Kondinin, 32°26.712’S; 118°53.836’E), and
Puntapin Rock (Shire of Wagin, 33°19.495’S;117°23.941’E). Pools varied in diameter, depth, population size, and sex ratio, providing a range of natural replicates (Table 5.1).
The minimal presence of substrate at the bottom of the pools meant that these pools were crystal clear, which allowed me to easily conduct focal behavioral observations.
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TABLE 5.1 SUMMARY OF DATA FROM THE FIELD
OUTCROP POOL COUPLES VOLUME SEX OSR DENSITY MEAN MALE MEAN FEMALE (dm 3) RATIO 1 MALE SIZE FEMALE SIZE SIZE RANGE SIZE RANGE (mm) (mm) (mm) (mm) Dingo 2D 2 460 0.27* 0.27* 0.226 7.6 7.5 7.6 7.2 6.5 7.9
Dingo 6D 2 253 0.08* 0.09* 1.468 6.7 6.5 6.9 6.0 5.6 6.4
Dingo 8D 11 106 1.23 2.27 0.926 7.0 6.5 7.5 6.6 6.0 7.6
Holland 4H 25 446 1.06 1.37 0.975 6.5 5.8 7.3 6.0 5.1 6.8
Holland 10H 22 123 1.02 1.30 4.610 6.4 6.1 7.0 5.7 4.5 6.5
Holland 16H 15 62 0.92 4.55 0.770 7.8 7.0 8.6 6.9 6.3 7.4
Puntapin 9P 5 150 0.75 1.39 1.556 8.0 7.8 8.3 7.6 7.3 7.8
Puntapin 13P 13 399 1.04 1.92 0.133 7.1 6.7 7.5 6.6 6.3 6.8
Wave 8W 8 439 0.95 1.54 0.645 7.3 7.1 7.5 7.0 6.5 7.2
Wave 9W 11 1009 1.33 4.00 0.116 8.3 7.8 8.6 8.1 7.1 8.6
Wave 11W 8 18 1.10 8.33 4.499 8.7 8.2 9.2 7.6 7.2 8.1
* denotes outliers in the sex ratio estimates (observations excluded from the analysis) 1 males/females Summary of the samples taken for behavioral observations, size ranges, and physical parameters per pool.
The winter rainfall pattern across southwestern Western Australia shows a marked
north eastwards gradient across the hinterland, but during the past two decades, summer
rains from a northern origin have become more prominent; these rains emanating from
tropical lows result in a much more heterogeneous rainfall distribution across the study
area. This study in April May, 2007, focused on pools inundated from rainfall
depressions with tropical origins. The pools were visited at subsequent phases of an
inundation cycle, from 6 25 days after rainfall (Holland Rock and Dingo Rock) to 27 36
days (Wave Rock and Puntapin Rock). Pools were refilled by more raining events during
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the study period, and I might have observed the first cohort of the season in some pools, and subsequent cohorts in other pools.
Observations to assess mate guarding duration were performed on 1 – 11 (mean
± SE = 5.6 ± 0.35) couples per pool per day, which we repeated for 4 6 days (mean ±
SE = 21.9 ± 2.76). As soon as a male clasped a female, the couple was gently moved, using a plastic pipette, to a transparent container filled with water from the pool. Once the guarding phase was started, males held strongly onto their mates and did not release them, even when slightly disturbed via pipetting (personal observation). Each couple was transferred to a color coded container, and checked every 15 minutes until the male released the female. Confining pairs in individual containers allowed for repeated observations through time and quick detection of the end of the guarding phase. Clam shrimp were then measured with calipers (maximum carapace length, in millimeters), marked with a permanent marker (to avoid repeated observations of the same individuals), and returned to the pool. Hence, sex ratio and density were not altered throughout the course of the observations. At the conclusion of the mating observations,
I measured the area of the pools and the average water depth. All clam shrimp were then removed from the pools using a fine mesh net, counted and sexed, to assess density and sex ratio. In order not to stress the animals and to avoid altering their behaviors, this sampling of the population was performed just once per pool after mate guardings were measured. After completion of the sampling, all specimens were returned to the pool.
OSR was calculated as the number of males relative to receptive females, considering females without eggs as “receptive” (Weeks & Benvenuto, 2008). I
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combined sex ratio and density into one variable: the relative gender density ( sensu
AlonsoPimentel & Papaj, 1996). This density adjusted sex ratio is a better indicator of mate availability than either density or sex ratio individually. Relative female density is the density of females in the overall population (expressed in percentage) and represents my best estimate of the real number of females that a male may encounter in the population. Clam shrimp age was deduced by pool age, estimated by the date of the rain events in the area.
Focal observations were performed on unpaired males to assess male time budgets. Males were followed for 5 minutes. I focused on social interactions (i.e., interactions with other conspecifics): the number of encounters with other individuals was recorded, as well as the sex of the interacting individuals. I also estimated the likelihood of male takeovers by isolating groups of three clasping animals (one female with two males clasping her, which always occurred with each male on opposite sides of the female’s carapace). Since the trio was already formed before observations began, I did not measure when the third male joined the couple, and therefore I do not have precise data on total mate guarding time. I gently placed the trios into isolation containers and followed the same protocol as for the regular couples noted above, checking them every 15 minutes. Once one of the two males “released” the female (or was displaced by the competing male), I measured his size (male 1, “loser”). I waited for the couple to separate and then measured the sizes of male 2 (“winner”) as well as the female.
Statistical analyses were performed using JMP 6.0 (SAS Institute Inc.). To meet criteria of normality, mate guarding time was natural logarithm transformed. A nested,
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one way analysis of variance (ANOVA) was used to analyze mate guarding time among outcrops and pools nested within outcrops. Additionally, we used a two way ANOVA to assess the influence of receptive female state and result of mate guarding (positive: successful egg fertilization and movement to the brood chamber; negative: the couple broke with no fertilization and no egg movement) on mate guarding duration. I used a stepwise multiple regression on the raw data (both the mixed and forward models returned the same results) to identify which of nine parameters affected mate guarding duration. I used these significant independent variables to run a multiple regression.
Since the input variables were grouped by population, I repeated the multiple regression analysis with mean values for populations for these variables. In order to graph individually each term of the regression model, I used two partial leverage plots (Sall,
1990). In this way I can visualize the effect of just one parameter on mate guarding time while correcting for the effect of the other parameter.
I performed linear regressions to address the possible interactions between: a) body size and density; b) male vs . female size; and c) size of guarding vs . non guarding males. A nominal logistic regression was used to interpret variation in time budgets among the different social behaviors. Finally, I investigated the influence of relative male size on clasping duration when two males were clasping the same female. Since I was not able to normalize residuals, I performed a non parametric Wilcoxon/Kruskal
Wallis test on “winner” size, “loser” size, and female size to detect any differences among the three groups. To further identify which of the three groups differed from the others, I performed a pairwise Mann Whitney test, adjusting the α level using a
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sequential Bonferroni correction for multiple comparisons (Holm, 1979). Comparing the size of all groups, I was able to test for the possibility that size affects intra sexual competition (takeovers: male vs . male size) and/or inter sexual conflict (males vs . female size).
Results
Mate guarding time
A total of 241 couples were followed in 11 pools on four granite outcrops. For
108 couples, the guarding phase was not concluded by the end of the daily observations and these data were excluded from the analysis. Two pools had estimated sex ratios that were highly female biased. Both pools had high numbers of juveniles, which can sometimes be mistaken for females with no eggs. I thus performed Grubb’s test (extreme
Studentized deviate test) for sex ratio values. Both pools were detected as outliers (P <
0.05) and thus they were excluded from analyses concerning sex ratios (Table 5.1).
Guarding durations varied from a few minutes up to 10 hours (mean ± SE: 163.60
± 15.10 minutes). I used a nested, one way ANOVA (mixed effect model) on mate guarding time for clam shrimp in outcrops and pools nested within outcrops (Table 5.2a).
I did not find any statistical differences between the four outcrops but a significant difference among pools nested within outcrops (Table 5.2a). Thus for all additional comparisons, I did not consider variation among outcrops.
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TABLE 5.2 STATISTICAL ANALYSES
SOURCE SS MS DF F Ratio Prob > F Num a) Outcrop 9.06 3.02 3 0.8665 0.4938 Pool[Outcrop] 27.39 3.91 7 2.1144 0.0477 Error 205.45 111 b) Male size (mm) 16.88 1 11.0459 0.0012 Relative female density 9.60 1 6.2794 0.0137 Error 160.46 105 c) Mean Male size (mm) 1.36 1 12.6522 0.0120 Mean Relative female density 1.42 1 13.1570 0.0110 Error 0.65 6 d) Result of MG 10.49 1 5.8934 0.0167 Female receptivity state 10.33 1 5.8066 0.0175 Result of MG * Fem receptivity 0.68 1 0.3836 0.5369 Error 208.23 117 Results of the statistical analyses performed (see materials and methods section for details): (a) One way analysis of variance (mixed effect model) on mate guarding time in outcrops and pools nested in outcrops; (b) multiple regression on the predictors selected by stepwise regression for mate guarding time; (c) multiple regression on means of significant predictors; (d) two way analysis of variance on mate guarding time considering the receptive state of female and the result of the guarding phase.
As an initial exploratory analysis of all the measured variables across all pools, I combined all data and performed a stepwise regression. I considered the independent variables of size [male size, female size and relative size (i.e., size differences between sexes)], sex ratio, OSR, clam shrimp age, and population density as related to the dependent variable of guarding time. Density was considered as: (a) total population density (juveniles included), (b) relative male and female densities (male density/total density and female density/total density, in percentages), and (c) density differences between sexes (male density female density). Of all these variables, only male size and
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relative female density were significantly related to guarding time, and thus were included in the final model, either when considering all raw data (Table 5.2b) or when considering means for populations in different pools (Table 5.2c). Increased male size was correlated with longer mate guarding durations (Figure 5.1). In contrast with what was expected, relative female density was positively correlated with guarding duration
(Figure 5.2). The significant predictors in the model were sex specific: male size played a more important role than female size, while relative female density was more important than relative male density.
As predicted, size and density values were correlated: both average male size and average female size significantly decreased as density increased (Figure 5.3). I performed the same analysis without Holland Pool 10 and Wave Pool 11, which were characterized by extremely high densities, to note whether these pools alone were driving the correlation, but I obtained similar results without these two pools included (male: P =
0.0034, n = 87; female: P = 0.0020, n = 86).
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5.5
5
4.5
4
3.5
Mean Mean (Ln MG time) 3 6 6.5 7 7.5 8 8.5 9 Mean Male size (mm)
Figure 5.1 Leverage plot of mean male size (mm) and length of mate guarding (ln transformed), controlling for the effect of relative female density.
6
5.5
5
4.5
4
Mean Mean (Ln MG time) 3.5
3 40 45 50 55 60 Mean Relative female density (%)
Figure 5.2 Leverage plot of mean relative female density (%) and length of mate guarding (ln transformed), controlling for the effect of male size.
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9
8
7
6 Size (mm) Size
5
4 0 1 2 3 4 5 Density (individuals/dm 3)
Figure 5.3 Correlation between population density (total adults/dm3) and size (mm) for males (black circles) and females (white circles). The linear regressions indicate a negative relationship between the variables for both sexes (male: y = 0.0982x + 7.3231, P = 0.0277, n = 112, r2 = 0.0433, best fit: continuous line; female: y = 0.1669x + 6.8708, P = 0.0003, n = 111, r2 = 0.1132, best fit: dotted line).
Male and female sizes were positively correlated: larger males usually guarded larger females (Figure 5.4a). This is not a sign of size assortative mating, since there was significant pool to pool variation in size (Figure 5.4b), and after analyzing size data within each pool, I did not find any significant correlation in size between the pairs, with the exception of one pool out of the 11 investigated: Dingo pool 8 (P = 0.019, n = 11).
Finally I examined the length of mate guarding time relative to the receptive state of females and the result of the guarding phase. I did not detect significant pool to pool variation, and so I combined data from all pools (Table 5.2d).
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8.5 8.5
8 8
7.5 7.5
7 7
6.5 6.5
6 6 Female size (mm) Female size (mm)
5.5 5.5
5 5 6 6.5 7 7.5 8 8.5 9 9.5 6 6.5 7 7.5 8 8.5 9 9.5 Male size (mm) Male size (mm)
Figure 5.4 Relationship between male and female size (mm) for couples observed in mate guarding behavior. a) All data together; b) pool by pool variation (mean ± 1 SD).
The female receptive state (estimated by the observer checking for presence/absence of eggs in the brood chamber) reflected the physiological state of the female. Receptive females were closer to molting, so the guarding duration was shorter (Table 5.2d, Figure
5.5). Also the mating result was correlated to the guarding duration: successful mating events (i.e., ones in which females moved eggs to their brood chamber) took longer than unsuccessful mating events (i.e., ones where the male left prior to fertilization, Table
5.2d, Figure 5.6). There was no significant interaction between female receptivity state and the result of mate guarding in these pools (Table 5.2d).
Male time budgets
I observed 116 focal males in 11 pools, for a total of 580 minutes. I recognized seven types of social interactions: clasping a male, clasping a female with eggs, clasping a female without eggs, clasping a molt, clasping a dead carcass, clasping a juvenile, interacting with a paired couple, and being clasped by a male.
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Figure 5.5 - Effect of female receptivity on mate guarding duration (mean ± 2 SE).
Figure 5.6 Effect of mating result on mate guarding duration. Successful mating: eggs fertilized and moved to the brood chamber; non successful mating: eggs not fertilized (mean ± 2 SE).
Male time budgets varied from pool to pool (denoted with an asterisk in Figure 5.7): a nominal logistic regression found differences among pools for clasping males (P =
0.0041, n = 116), clasping females with eggs (P = 0.0126, n = 116), interacting with
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couples (P < 0.001, n = 116) and clasping dead carcasses (P = 0.0169, n = 116). I did not find a significant difference between the size of non guarding males (mean ± SE: 7.53 ±
0.09 mm) and guarding males (mean ± SE: 7.39 ± 0.05 mm; P = 0.4440, df = 1, all pools combined; also no significant differences were found when analyzing each pool individually).
Figure 5.7 Male time budget (mean frequency of behavior/min ± 2 SE). Asterisks denote significant difference from pool to pool. Male takeovers
I also observed and isolated a total of 19 trios. The Wilcoxon/Kruskal Wallis test on non transformed data revealed a size difference between the three groups (female:
6.57 ± 0.17 mm; male winner: 7.09 ± 0.17 mm; male loser: 6.99 ± 0.17 mm; 2 = 7.8879, df = 2; P = 0.0194). Pairwise Mann Withney tests detected that female size was significantly different from male winner size (P = 0.0129, df = 1, significant after sequential Bonferroni correction), but male loser size was not significantly different from male winner (P = 0.3553, df = 1) or female size (P = 0.0280, df = 1, non significant after sequential Bonferroni correction) (Figure 5.8).
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Figure 5.8 Males and female size (mm) in trio (mean size ± 2 SE). Levels connected by same letter are not significantly different.
Discussion
Precopulatory mate guarding exemplifies an intersexual interaction where males and females attempt to optimize their investment in reproduction through minimizing time and energy spent prior to copulation. The case for time investment is the main focus in this study; there may be a greater female active involvement than resistance during the mating interaction (i.e., mate choice), but that has not been assessed in the present study.
The dynamic, intersexual interaction is particularly complex, changing with the reproductive cycle of the female and influenced by many socio environmental conditions.
In this study of natural populations of the clam shrimp L. badia, I investigated the effect of parameters identified in mate guarding models (e.g., Jormalainen et al., 1994b;
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Yamamura & Jormalainen, 1996; Jormalainen, 1998; Jormalainen et al., 2000) to be important in influencing mate guarding time: male and female size, sex ratio, OSR, age, and population density. The age of the pool was not recognized as a significant parameter in determining the guarding strategy recorded in the pools. The influence of male size, relative female density, OSR, as well as receptive state of females and whether the mating event was successful, are discussed below, together with an analysis of male time budgets and male male competition.
Male Size Size (specifically relative size between the sexes, and variation in male size) is predicted to influence the guarding duration in mate guarding models (e.g., Grafen &
Ridley, 1983; Jormalainen et al., 1994b; Jormalainen, 1998; Härdling et al., 2004).
Relative size differences between the sexes influence the mate guarding period in a range of Crustacea (Elwood & Dick, 1990; Jormalainen et al., 1994a; Jormalainen, 2007), but I did not find evidence for a similar influence in the L. badia populations that I studied.
Indeed, I detected neither strong size dimorphism between sexes in each pool nor evidence of size assortative mating as reported for amphipods (Dick & Elwood, 1996;
Bollache & Cezilly, 2004a), even though, if female fecundity is size related, there would be a benefit for larger males to guard larger females. Size, however, was strongly correlated with density, consistent with competition for limited resources in such pools, as concluded by Weeks and Bernhardt (2004). The significantly longer guarding time by larger males reported here is consistent with both their ability to overcome female resistance and/or dislodge smaller guarding males (Grafen & Ridley, 1983).
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Relative female density Density and sex ratio affect mate guarding because both are highly correlated with encounter rates (Parker, 1974). Consequently, shorter guarding durations should occur in populations with a female biased sex ratio (males would have a high encounter rate with females, enhancing their chance of finding females close to receptivity). However, these results on L. badia in populations with higher female relative density, contradict this prediction: mate guarding was significantly longer. This unexpected result may result from one or more alternative processes that will require further studies to elucidate, stemming from the fact that under higher female relative densities, the rate of male females encounters should increase. First, females might decrease the strength of resistance to being guarded under conditions of fewer males being available for mate guarding in a pool (to increase the possibility of being fertilized). On the other hand, females might “search” for males when females experience fewer contacts with males per unit time, essentially expressing their choice to be guarded thereby enhancing female induced bonds with a male. These two hypothesized interactions may be alternative statements of the same basic process, with both pointing to the phenomenon of active positive female selection.
A decrease in female resistance has been postulated in an opposite situation (high male density) in water striders. In the genera Aquarius and Gerris (Arnqvist, 1992a;
Arnqvist, 1992b; Lauer et al., 1996) where the attention of males towards females can be very persistent, it may be less detrimental for a female to be guarded longer by one male than resist the continuous harassment of multiple males (Amano & Hayashi, 1998).
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Some authors use OSR (instead of sex ratio) to underline the importance of the abundance of receptive females (Iribarne et al., 1995; Vepsäläinen & Savolainen, 1995;
AlonsoPimentel & Papaj, 1996). Although I did not find evidence of variation of mate guarding time with variation of OSR, I calculated OSR just once during the observation, assuming overall OSR does not change significantly through time. This assumption might have been wrong, or there might be no effect of OSR due to asynchrony of female receptivity in the population because female clam shrimp start a new reproductive cycle as soon as the previous one is completed and thus reproduction is continuous during their short life cycle. If OSR is continuously changing in an unpredictable way, it could be difficult for males to gauge the likelihood of the number of receptive females in the population.
Female receptivity Mate guarding has been analyzed initially using optimality models from the male perspective to maximize male reproductive success (Parker, 1974), assuming that males can assess the receptivity state of females. In L. badia , females closer to receptivity (i.e. with no eggs in the brood chamber) were guarded for shorter periods of time. Both sexes would gain advantage from a short mate guarding phase: females because of a reduction in costs of being guarded (e.g., less interruption to their feeding) and males because paternity of the brood is guaranteed and their energy expenditure in guarding would be reduced. Thus the conflict between the two sexes is reduced when mate guarding duration is low.
Consequences of guarding Failure to fertilize eggs resulted in shorter guarding time than in successful mating. Whether cessation of mate guarding is male or female driven
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needs to be elucidated. The longer the period that a male spends guarding a female, the more valuable she should be. However, males might have a specific amount of time they will allot per mating, beyond which they will assume failure and move on to seek another mate. From the female perspective, being guarded by a reproductively incompetent male will reduce her genetic fitness, so there might be some sort of female choice, communicated by a specific cue or the lack of a cue (maybe behavioral), that might convince the male to start looking for another female.
Male time budgets In clam shrimp, males are the searching sex and in so doing demonstrated a variety of encounters, ranging from clasping other males, carapaces, dead clam shrimp and juveniles. In some pools where overlapping cohorts were present, males clasped immature conspecifics at length (Benvenuto et al. in press). What the cues for establishing a guarding phase detected through physical contact by males might be, is still unclear. Again, although males may be the searching sex, this does not preclude the possibility of choice being exerted by females, post contact. Some aquatic crustaceans , like the stream dwelling isopod Lirceus fontinalis , key on chemicals released during molting to assess the receptivity state of their mates (Sparkes et al., 2000). Experiments performed on E. texana do not support a role of the molting hormone 20 HE (Chapter II), but this does not exclude an important role of other metabolites involved in the molting process. If this is the case, it could explain the clasping of other males, juveniles (which molt frequently), and empty carapaces (where possible traces of the molting hormone could still be present).
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Male male competition I am convinced of the importance of female resistance, but male male competition can still be relevant in this species. In a population with a male biased sex ratio, guarding duration should increase since once a female is found by a male, it is important for him to remain in contact with her, both to avoid losing the mating opportunity as well as to prevent “exploitation” competition by other males
(Grafen & Ridley, 1983; Dick & Elwood, 1996). I also attempted to quantify “direct” competition observing male male interactions in clasping trios, implying the possibility of male takeovers. Two males clasping a female kick each other vigorously, attempting to dislodge the rival. Contrary to expectations, there were no significant size differences between “loser” and “winner” males. Thus, it appears that size is not the determining factor in deciding which male wins the contest. However, I observed few trios in this field study and I cannot exclude the possibility of a sampling bias: I may have only found trios that had persisted for a comparatively prolonged time. If equally size matched males are more likely to remain in trios for longer periods, then I may have missed the cases where larger males quickly dislodged smaller males in trios (even though I did not detect a significant difference between mate guarding and non mate guarding males). Future quantifications of male takeovers should emphasize observing trios from the initial pairings to remove this bias.
Summary Precopulatory mate guarding is a complex behavior, involving interactions between males and females with objectives that both overlap and conflict. Males need to ensure paternity and also maximize fitness; females, when receptive, need to ensure a male for fertilization and maximize fitness. In order to increase the fitness of each brood,
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the decision making about guarding/being guarded changes with time during a single mating event. This decision making has been the focus of several theoretical models
(Parker, 1974; Grafen & Ridley, 1983; Yamamura, 1987; Jormalainen et al., 1994b;
Yamamura & Jormalainen, 1996; Jormalainen, 1998; Härdling et al., 1999; Härdling et al., 2004) and this field study was undertaken to assess the applicability of the modeling predictions to natural populations of clam shrimp. My results generally are consistent with these predictions, with mate guarding duration in L. badia decreasing when females were closer to receptivity, and when males were small. I found shorter mate guarding duration when female density was low. This last result possibly provides evidence for an active female role in promoting or maintaining the guarding relationship or it may provide evidence of a modulation of female resistance with variation in density and sex ratio. I also recorded male male competition for females, although the underlying reasons for which male “wins” such competitions have yet to be determined. The results presented here point toward planning manipulative, laboratory based studies designed to unravel the contribution to successful reproduction for each of these parameters in this crustacean.
Acknowledgements
I am extremely grateful to Alissa Calabrese, Rob Davis, Wally Gibb, Debra
Judge, Kerry Knott, Sadie K. Reed, Danny Tang, Magdalena Zofkova, the School of
Animal Biology at The University of Western Australia for their invaluable help in the organization of the field study, and most of all to Brenton Knott. Many thanks are due to
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the employees of many shire offices in Western Australia for precious information and
Danny Stefoni at the Department of Conservation and Land Management of Western
Australia for all the permits required to perform the field work. I would like to thank
Francisco Moore for his help with statistical analysis. This work was supported by the
National Science Foundation [grant number IBN 0213358]; the Kent State University
Graduate Senate InTraGrant; and the Kevin E. Kelleher Memorial Fund.
VI. GENERAL DISCUSSION AND CONCLUSIONS
Precopulatory mate guarding is a complex behavior, characterized by a continuous interaction between the sexes. This interaction is dynamic because it varies during a single mating event as a response to changes in female receptivity. Mate guarding involves i) mate receptivity assessment by the male, ii) a male decision to continue or stop guarding, and iii) a female response to this decision, either as acceptance or resistance of the guarding male (Figure 6.1). One of the key factors that influences the starting of a guarding event is the receptivity state of the female (e.g., the time left to molt in crustaceans, see Jormalainen, 1998) when copulation will be possible. Other parameters can also affect the guarding decisions of both males and females (see Chapter
I for a detail account on parameters), one of the most influential being the encounter rate
(which depends on density and sex ratio) of mates in the population (e.g., Parker, 1974;
Wickler & Seibt, 1981; Jormalainen, 1998).
When females are receptive, or close to receptivity, males will start guarding and females will accept it. At this point, there will be a short guarding phase which quickly concludes in a mating event. Both males and females are “in agreement” that the guarding phase will be short, with low costs (in terms of energy or use of time) and a high benefit (a successful mating) for both of them. When females are not receptive (or not close to receptivity), males will release them directly after a quick encounter and there will be no guarding. Again both sexes are “in agreement” that the guarding phase
156 157
will be too long, and thus costly in terms of energy and time. In these instances, it is more beneficial for both sexes if the male leaves to search for another, more receptive female.
Depends on density and sex ENCOUNTER ration in the population
Depends on ♀ ♂ RELEASES ♂ STARTS GUARDING receptivity state (Ch .II)
Depends on ♀ receptivity state ACCEPTANCE BY ♀ RESISTANCE BY ♀ and mating system (Ch .II; Ch. IV)
CONFLICT ♂ RELEASES No conflic t Influenced by multiple SHORT MATE parameters, i.e., sex ratio, GUARDING density, size, mating system (Ch. III; Ch. IV)
Depend on MATE GUARDING different “power” DURATION MATING among sexes (Ch. II)
MATING
Figure 6.1 – Flowchart describing the possible interactions between sexes from encounter to mating; ♂: male; ♀: female in dioecious system, hermaphrodite in androdioecious system.
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A conflict is initiated when the sexes are in disagreement on the duration of mate guarding (Yamamura & Jormalainen, 1996; Jormalainen, 1998). This happens when males decide to start guarding, because their cost (i.e., time investment) is lower than their benefit (mating event), but females are resisting because their cost (in term of energy) is higher than their benefit (mating event). Mate guarding is a thus a challenging example of intersexual conflict.
As outlined in Chapter I, mate guarding has been investigated in many different crustacean taxa, with multiple approaches. I analyzed mate guarding in one specific crustacean group, commonly called clam shrimp, in the suborder Spinicaudata (class
Branchiopoda). I already described throughout this thesis the advantages of working with this study system: short life cycle and short mate guarding phases, easy breeding in the laboratory, the possibility of performing manipulative experiments in the lab as well as observations in the field, and the possibility to observe (through the transparent carapace) the level of gut fullness (as an estimate of rate of food intake) and the presence or absence of eggs (as a rough estimate of receptivity). Moreover, clam shrimp allow for a unique opportunity among animals to compare the same behaviors among species characterized by different mating systems. I was able to study mate guarding behaviors in dioecious and in androdioecious species, which allowed me to test an extension of a mate guarding model (Jormalainen, 1998), which would not be possible in most other systems.
I used the androdioecious species Eulimnadia texana in the majority of my projects. This species has been successfully used in many laboratory experiments (e.g.,
Medland et al., 2000; Zucker et al., 2002; Weeks & Benvenuto, 2008) as well as field
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studies (e.g., Weeks & Bernhardt, 2004). I also used another androdioecious species,
Eulimandia dahli , from Western Australia and I compared the behavior of these two species with the Australian dioecious Limnadia badia and Limnadopsis tatei .
I analyzed mate guarding in clam shrimp starting from the proximate mechanism of this behavior, i.e., the cues males are using to assess their mate’s receptive state and the mate searching strategies employed by each sex in E. texana . I confirmed that mate searching tactics differ between the sexes, as already proposed by Medland et al. (2000).
Males are not chemically attracted to the opposite sex, while hermaphrodites are.
Moreover this sex difference changes with the hermaphrodite’s receptive state: non receptive hermaphrodites appear to be more attracted to males than receptive hermaphrodites (Chapter II). Males do not react to molting hormone as a soluble cue in the water or to the application of molting hormone on the carapace of hermaphrodites.
Other studies have questioned the role of molting hormone as a cue of receptivity
(Gleeson et al., 1984; Kamio et al., 2000; Kamio et al., 2002; Atema & Steinbach, 2007).
Possibly, other metabolites produced during ecdysis could act as a cue for males to assess the receptive state of their mates. Further studies are needed to understand the nature of the chemical communication between the sexes during mate guarding.
I then moved to the ultimate causes of mate guarding behavior in E. texana . I analyzed costs and benefits for each sex, I created a “power” asymmetry between males and hermaphrodites (pairing individuals of different sizes) and I measured the optimal mate guarding time of each sex in order to compare it with the compromised mate guarding time (Chapter III). Mate guarding is more costly to hermaphrodites than males,
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at least considering food intake, during mate guarding. This cost is significantly correlated with the guarding duration. Differential costs to the sexes during mate guarding have been reported in multiple studies [e.g., food manipulation in amphipods
(Robinson & Doyle, 1985); energetic costs in amphipods (Plaistow et al., 2003); predation risks in amphipods (Cothran, 2004); survival costs in isopods (Benesh et al.,
2007)].
As predicted by theory (Jormalainen, 1998), the more powerful sex (in this case the larger one) can have more control over the length of mate guarding. Longer mate guarding times, when males were larger than their mates, have been reported for the isopod Idotea baltica (Jormalainen & Merilaita, 1993; Jormalainen et al., 1994a;
Jormalainen & Merilaita, 1995). With manipulative experiments, I was able to measure the optimal guarding time of males and hermaphrodites. The results perfectly matched the theoretical model proposed by Jormalainen (1998): hermaphrodites “prefer” to be guarded less than the compromised guarding time, while males are willing to guard longer. This reflects the intersexual conflict on the optimal duration of mate guarding that differs in the two sexes. A similar approach, using manipulation of female resistance, has been used in isopods (Jormalainen & Merilaita, 1995; Jormalainen & Shuster, 1999) and amphipods (Cothran, 2008a), and the results are in agreement with what was found in E. texana : longer mate guarding times are achieved when males can control the mate guarding duration. Male manipulations did not result in decreased mate guarding duration
(contrary to what I reported) in the isopods I. baltica (Jormalainen & Merilaita, 1995) and
Thermosphaeroma thermophilum (Jormalainen & Shuster, 1999). Variations across
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species, in fact, need to be taken into consideration (i.e., social environment, different costs and benefits, etc.). Overall, there is a general agreement with the theory and the existence of the conflict over mate guarding duration has always been confirmed, with different strengths in different species (and even populations belonging to the same species; Jormalainen et al., 2000).
Thanks to my unique study organism, I was able to investigate mate guarding behaviors beyond the conflict between males and hermaphrodites. I was, in fact, able to analyze and compare the optimal guarding time of males and hermaphrodites in androdioecious species and males and females in dioecious species (Chapter IV). Thus I was able to measure the strength of the conflict among the sexes and the influence of the mating system on mate guarding strategies. The comparison between females and hermaphrodites is particularly interesting. The benefit of outcrossing is much higher for females than for hermaphrodites, who have the option of self fertilization if the costs of being guarded are too high. Also males experience different social environments in the two mating systems. Males in dioecious species suffer higher relative proportions of males in dioecious relative to androdioecious mating systems (Pannell, 2002; Weeks et al., 2006a). As a result, males in dioecious species are guarding longer than males in androdioecious species. Comparing four species, two dioecious and two androdioecious,
I analyzed the overall effect of the mating system on the mate guarding conflict. I was careful to account for differences other than the sexual system, such as size and length of the receptive cycle. As predicted, dioecious species guarded longer than androdioecious species irrespective of these potentially confounding differences.
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I started my analyses in the laboratory, using manipulative experiments (Chapter
II, III, and IV) and then I moved to the field to verify these results in a natural setting. I studied one dioecious species, Limnadia badia , in its natural environment (Chapter V).
This allowed for a powerful verification of the results obtained in the laboratory. In the field, multiple parameters are acting simultaneously. Consistent with predictions, female receptive status influences mate guarding duration: the closer the female is to molting, the shorter the mate guarding time. Small males guarded for shorter periods than large ones, highlighting the importance of size. Also, high female relative density was found to be a key factor in increasing mate guarding duration. I postulated a reduction of female resistance when females are at risk of reaching receptivity without having a guarding male.
From the field there is then the opportunity to go back to a more controlled setting, to further clarify individual parameters. In my opinion there is a need for a continuous exchange of information from lab to field to lab again in order to strengthen our understanding of mate guarding behavior in crustaceans. The next step, which I would like to pursue in the future, would be to organize these results using a theoretical approach. I built my overall project on the basis of existing theoretical models (Parker,
1974; Wickler & Seibt, 1981; Grafen & Ridley, 1983; Yamamura, 1987; Hunte & Myers,
1988; Elwood & Dick, 1990; Jormalainen et al., 1994b; Yamamura & Jormalainen, 1996;
Jormalainen, 1998; Härdling et al., 1999; Härdling et al., 2004). The uniqueness of the study system, though, allows for the proposal of an innovative model that encompasses all the variables previously considered, plus the influence of mating system type.
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I think that from the analysis of a particular group of crustaceans, characterized by a rare mating system, it is possible to extrapolate conclusions that go beyond the specific system. Eberhard (2005) proposed the use of facultative hermaphrodites as a conceptual example that could increase our understanding of sexual selection. Androdioecious clam shrimp bring this proposition to a reality. This unique study system permits the investigation of mating interactions among sexes and the analysis of the influence of the mating system on a complex behavior, such as precopulatory mate guarding in crustaceans.
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