Sexual selection and the evolution of sex-role reversal in honeylocust beetles

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UNIVERSITATISACTA

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1240

Sexual selection and the evolution of sex-role reversal in honeylocust beetles

KAROLINE FRITZSCHE

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Dissertation presented at Uppsala University to be publicly examined in Zootissalen, Evolutionary Biology Centre, Norbyvägen, Gamla Zoologen (Hus 1), Uppsala, Wednesday, 13 May 2015 at 10:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Adam Jones ( Texas A&M University).

Abstract

Fritzsche, K. 2015. Sexual selection and the evolution of sex-role reversal in honeylocust beetles. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1240. 46 pp. Uppsala: Acta Universitatis Upsaliensis.

ISBN 978-91-554-9209-0.

Sexual selection is the prime evolutionary force that makes males and females different. This process has long been viewed as one where male compete with one another and where females choose. However, since the discovery that multiple mating by females is common in animals, sexual selection theory has been expanded to include mate competition between females and mate choice by males. However, empirical studies addressing these themes are scarce. In my thesis, I explore the evolution of sex role reversed mating systems using the honey locust beetles (Megabruchidius dorsalis and M. tonkineus). I used these species to shed light on (1) how closely sexual selection in females resembles its better#studied male counterpart, (2) the implications of male mating costs for mating system evolution and (3) the effects of reproductive competition between females on the evolution of female courtship behaviour. By manipulating male mating rate, I found that males that mated more lived shorter lives, showing that mating is costly for males. I also demonstrated that males are choosy about whom they mate with and prefer vigorously courting females (Paper II). In contrast to males, previous studies suggested that female honey locust beetles benefit nutritionally from mating due to the large ejaculates provided by males. I manipulated male condition to show that male adult feeding had significant effects on female reproduction. Females that mated with males of good condition lived longer and produced more offspring than females whose mates were in poor condition (Paper III).

When mating is costly for males, theory predicts that sexual selection in females can be strong. I compared sexual selection in honey locust beetles to that in two other species of seed beetles with conventional sex roles. I found substantial sexual selection in honey locust beetle females, which was comparable in strength to that in males (Paper I). I also measured the evolutionary effects of altered sex ratios on mating system parameters in both honey locust beetle species, using an experimental evolution design. Under female-biased sex ratios, representing strong sexual selection in females, females of M. dorsalis rapidly evolved elevated courtship intensity, thereby intensifying the reversal of sex roles (Paper V). In M. tonkineus, males evolved under male- biased sex ratios to transfer larger ejaculates, demonstrating the role of male-male reproductive competition for the evolution of male provisioning (Paper IV). My thesis highlights the essential, and often overlooked, role that females play in mating system evolution and that their contribution cannot simply be reduced to mate choice.

Karoline Fritzsche, Department of Ecology and Genetics, Animal ecology, Norbyvägen 18 D, Uppsala University, SE-752 36 Uppsala, Sweden.

ISBN 978-91-554-9209-0

urn:nbn:se:uu:diva-246715 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-246715)

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Für Petra und Peter

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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Fritzsche, K., Arnqvist, G. (2013) Homage to Bateman: Sex roles predict sex differences in sexual selection. Evolution, 67:1926–1936 II Salehialavi, Y., Fritzsche, K., Arnqvist, G. (2011) The cost of mat- ing and mutual mate choice in two role-reversed honey locust bee- tles. Behav. Ecol., 22:1104–1113

III Fritzsche, K., Arnqvist, G. (2015) The effects of male phenotypic condition on reproductive output in a sex-role reversed beetle. Anim.

Behav., 102:209–215

IV Booksmythe, I. *, Fritzsche, K. *, Arnqvist, G. (2014) Sperm com- petition generates evolution of increased paternal investment in a sex role-reversed seed beetle. J. Evol. Biol., 27:2841–2849

V Fritzsche, K., Booksmythe, I., Arnqvist, G. (2015) The experimental evolution of sex roles in beetles. Manuscript

* joint first author

Reprints were made with permission from the respective publishers.

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The following papers were written during the course of my doctoral studies but are not part of the present dissertation.

Fritzsche, K., Delobel, A. (2012) Megabruchidius dorsalis (Fåhraeus, 1839), Bruche nouvelle pour la faune française (Col., Chrysomelidae, Bruchinae).

Bulletin de la Société entomologique de France., 117: 389-390

Fritzsche, K., Booksmythe, I. (2013) The measurement of sexual selection on females and males. Curr. Zool., 59: 558-563

Fritzsche, K., Timmermeyer, N., Wolter. M., Michiels, N. K. (2014) Female, but not male, nematodes evolve under experimental sexual coevolution.

Proc. R. Soc. B., 281: 20140942

*Henshaw, J. M., Kahn, A., *Fritzsche, K. (2015) Measuring sexual selec- tion: a more holistic model of reproductive life histories. Manuscript

* joint first author

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Acknowledgements

This ride was unique, a one of a kind experience!

Nadine, Leila und Bea, vielen lieben dank für die großartige Zeit während meines Diploms und dafür, dass ihr mich mit so viel Liebe zu meiner Dok- torarbeit nach Schweden gesendet habt. The things I learned from you Na- dine were invaluable during my PhD.

First, I want to thank my supervisor Göran for explaining and teaching me how to pursue science without getting disillusioned. Thank you for sharing your thoughts and knowledge with me, and giving me the freedom I needed.

Alexei, my second supervisor, thank you for all the signatures. :)

Ingrid, you’ve been an absolute sweetheart. Several times, during my PhD I came to you to ask you for advice and also to find support. Your door was always open. You have done a great job in helping me when it was difficult and you always made me feel welcome.

When I started my PhD in Uppsala, I was a little terrified, all the new people in the department. But then there was you Hwei-yen. You made the beginning very enjoyable. Thank you for being such a nice, supportive friend and flying with me to Zurich, we had a couple of gorgeous days there.

Murielle, dear Murielle, thank you so much for being the great friend you are! You will always have a place in my heart. Thank you for the many fikas we had and welcoming me the uncountable times I stood in your office door.

The road-trip we took around Gotland was stunning, beautiful and unique;

traveling with you is a delight! I also want to thank you for supporting me during the times when everything turned dark around me and I couldn’t find a way out. This was one of the hardest experiences in my life and without you and many other people I would not know if I have had the strength to carry on.

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Thank you Cosima, Isobel and Vytautas, for saying the right words and being there when I needed it most. I still remember the moment in my favour- ite café Linné with you Cosima! Thank you for giving me that push!

Isobel, thank you for caring about me hanging out with me! I hope you will forgive me one day for making you take that diving license in Egypt. :) I had a great time with you.

Ana, you’re the one stopping by my office with a smile and a hug every single day. The department is just not the same without you. Thank you for all the positive words and fikas! “Manos frías, corazón caliente!” and yes you are very beautiful, sexy and wise! ;)

My long-term office mate, Erem, I miss you! The two of us commiserating about science and life… I think one could have called us the “emo-office”, I am happy to have shared that time with you. I found a great friend in you and I want to thank you for the great time in Salt Lake City!

Kuba, my new office mate, thank you for feeding me, when I was starving, giving me an extra hug when I needed it most and helping me with all the stats. I won’t forget our uncountable chats about whether the really clever people persist in science or have already left.

Ellie and Leanne, my British/Aussie friends. Thanks for the knitting nights with delicious supper in front of the TV. Also, Ellie, I didn’t forget that you made me run that “Tjurruset” and just got pregnant :) One day we will have to run together!

David W, Alex K, Arild, Jossan, Björn, Mirjam, Fernando, Teddy, Karl and Elina and Simon thank you for the crazy bunch you are! It always cheered me up hanging out with you guys and having awkward, funny and mind- blowing conversations. Also, thank you David W for a lovely trip through Portugal, certainly one of the times I enjoyed most during my PhD (You made me change the way I think about goats).

I should also thank my gym and diving buddies! Thank you for being crazy and doing all those classes and heavy work out with me. Murielle, Martyna and Brian, I really enjoyed getting all soaked in sweat with you. Everything was always so much more fun with you guys! Also, thank you Stefan for the great dives we did in Sweden and Egypt and for making me a better diver. I won’t ever forget our dolphin-dive. Yep, we were quite lucky!

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Joshka, thanks for walking some of the path with me and being there. You helped me with so many statistic questions and I liked all our discussions about scientific ideas. You have become a great friend! Thank you.

During my PhD I had the great opportunity to go to Australia, thanks to the generous funding of Zoologiska Stiftelsen and the EBC graduate school on genomes and phenotypes. On this trip I met a truly awesome bunch of people. First, I want to thank Mike for hosting me. Megan, Andrew, Anna, Re- gina, David, Marta, Jared, Lyam, Nina, Katherine, Josh, Daniela, Thomas, Dan S, Dan H, Jono, Ana, Damien and Conny you created a unique envi- ronment at RSB for me. You made me feel welcome the second I stepped through that door of the battery. David, thank you for sharing your candy with me and for procrastinating by watching funny animal videos. Anna, thank you for always having some food when I was disorganized and for being such a great friend in times of trouble!

Thanks to you Meaghan and Daniel for being such lovely housemates and for listening to me whining about my PhD. I dearly miss the great discussions we had and the evenings we spent killing aliens in the living room.

Birds!

Yes, birding is something I really started enjoying in Australia and this is not only because the birds are fantastic! No, it is because of you Dan Hoops, Thomas, Josh, Viv and Jono. Thank you for all the great trips, I remember the plains-wanderer like it was yesterday. You could almost lick it Dan!

Also, the bad puns we made will stick with me forever! Thomas, here I have to quote you:

“Karo! Karo, ask me what time it is!”

“What time is it?”

“It is hummus four thirty.” Man, that is a bad pun! :)

I also want to thank you Cathy, Tony, Cal and Jono! You created a lovely live/work balance for me. I loved the weekly dinner over at your house Cathy and Tony. Thank you for taking me into your family with love and care and sharing Christmas and other wonderful times with me!

Jono, you gave life a different colour, everything seems so much brighter and much more radiant with you. Thank you for sharing my hunger for ad- venture and for always staying one step ahead and having another great des- tination in mind. Five continents in one year, we can top that! :) Thank you for all the wonderful birding and getting me into climbing. You are the voice of reason in times when I lose sight. Thank you for being by my side and coming to Sweden for me to support me during the finish of my PhD.

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You’ve done a tremendous job! I am looking forward to our next chapter, I am sure it won’t disappoint!

Danke, liebe Mutti und Peti. Ich weiß, die Zeit während meiner Doktorarbeit war nicht immer leicht für euch. Ich hatte meine Höhen und Tiefen und ihr wart immer für mich da ohne viele Fragen zu stellen. Danke, dass ihr immer zugehört habt und nicht das Vertrauen in mich verloren habt, wenn ich ab und an doch etwas merkwürdige Entscheidungen in meinem Leben getroffen habe. Ohne euch wäre ich nicht so weit gekommen. HESL!

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Introduction

My thesis

The study of sex role reversed species allows us to broaden our perspectives on sexual selection in females and increase our understanding of the univer- sal patterns of mating system evolution, thereby providing novel insights into sexual selection

The definition of sexual selection

Sexual selection is a major generator of diversity in all species that repro- duce sexually, both in terms of trait diversity (Darwin 1871; Andersson 1994) and speciation (Ritchie 2007). In 1871 Darwin defined sexual selection as follows:

“The sexual struggle is of two kinds; in the one it is between individuals of the same sex, generally the males, in order to drive away or kill their rivals, the females remaining passive; whilst in the other, the struggle is likewise be- tween the individuals of the same sex, in order to excite or charm those of the opposite sex, generally the females, which no longer remain passive, but se- lect the more agreeable partners.”

(Darwin 1871) In recent years, however, a renewed debate over how sexual selection is best defined and quantified caused some controversy amongst researchers (Car- ranza 2009, Clutton-Brock 2009, Shuker 2010, Klug et al. 2010, Krakauer et al. 2011). Although Darwin’s original definition of sexual selection did not completely exclude sexual selection in females, subsequent research focused almost entirely on males. This view has recently been re-evaluated. Since the discovery that multiple mating in females is abundant in animal taxa, sexual selection theory has expanded to include mate competition between females and mate choice by males (Bonduriansky 2009, Rosvall 2011). These mech- anisms nevertheless remain understudied and it is unclear how similar sexual section in females is to better-studied processes in males. In particular, it is

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controversial whether females typically respond to strong sexual selection by evolving costly traits to attract mates and whether male mate choice is pri- marily driven by direct cues of female fecundity.

Although it is clear that sexual selection is linked to variation in mating systems (Trivers 1972; Shuster and Wade 2003), the causal evolutionary rela- tionships between mating system parameters and the strength of sexual selection is much less obvious (Emlen and Oring 1977; Arnqvist and Rowe 2002). The majority of research on the evolution of mating systems and how these are linked to sexual selection has focused on species with so-called

‘conventional’ sex roles, in which males make no investment in offspring other than their sperm. In these species, sexual selection is stronger in males than in females as an indirect consequence of anisogamy (Bateman 1948, Schärer et al. 2012). Female gametes (eggs) are typically larger, costlier and produced at a slower rate than male gametes (sperm). The reproductive rate of females is thus limited by egg production, while males are limited by the number of eggs they fertilize (Bateman 1948, Trivers 1972).

Studying species with conventional sex roles has provided us with a better understanding of how sexual selection shapes the evolution of female mate choice and male-male competition, including numerous strategies increasing mating success, of which exaggerated ornaments are just one example (e.g.

Andersson 1994, Andersson and Iwasa 1996, Hunt et al. 2009). However, little is known about the importance of sexual selection in females for mating system evolution, particularly in species that diverge from conventional sex roles (Clutton-Brock 2009, Rosvall 2011).

In many species, male investment is not limited to their small sperm: in- stead, sizeable male investment in offspring limits their mating rate to the extent that females compete for mates. In some insects, males produce costly, nutritious ejaculates or provide females with nuptial gifts (e.g. Gwynne 2008, Perry 2011, Perry and Tse 2013, Lewis et al. 2014). These can be very beneficial for females and females increase reproductive success and lifespan by mating multiply (Paper III). In the family Syngnathidae, males even provide for fertilized eggs in a specialized brood pouch (e.g. Vincent et al. 1992). In these species it is males that can be the limiting sex and females thus actively court for matings.

Studying systems where investment in reproduction is greater in males despite anisogamy opens great opportunities to answer new questions on how sexual selection operates and how mating systems evolve. For instance, can sexual selection act in females as much as the same ways as in males? Re- search on when and how sexual selection operates in females is slowly ac-

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cumulating and its importance is beginning to be widely recognized (Clut- ton-Brock 2007, 2009; Rosvall 2011).

Measuring sexual selection

Over the last decades several approaches to quantify the strength of sexual selection have been proposed (Bateman 1948, Arnold and Duvall 1994;

Jones 2009; Klug et al. 2010, Krakauer et al. 2011, Collet et al. 2012, McDonald et al. 2013). However, comparisons of sexual selection across sexes and species are made problematic by the fact that there is little consen- sus over the relative utility of different measures of sexual selection (Table 1; Shuster and Wade 2003; Jones 2009; Klug et al. 2010; Fitze and Le Gal- liard 2011; Krakauer et al. 2011). Three points, in particular, are debated.

First, is there a single sufficient quantitative measure of sexual selection or do we need multiple measures to fully characterize it? Second, are measures based on variance in mating and reproductive success across individuals preferable to measures based on particular phenotypic traits? Finally, how important is it to measure sexual selection in both sexes?

To date, the few studies that have compared different measures of selection within and across species (e.g. Bjork and Pitnick 2006; Mills et al. 2007;

Paczolt and Jones 2010; Fitze and Le Galliard 2011; Munroe and Koprowski 2011) have done so within a single mating system and have provided some- what contrasting answers to these questions. Empirical studies that systemat- ically compare sexual selection in both sexes across species with distinct sexual selection regimes and/or mating system parameters, e.g. including sex role reversed and conventional mating systems, could help settle these issues (Jones et al. 2000).

As noted above, studies of sexual selection in females are comparatively rare. Estimates of the strength of sexual selection in females with ‘conventional’ sex roles are particularly lacking, possibly underestimating the role of sexual selection in females generally. Very few empirical studies have directly compared the strength of sexual selection in females and males (e.g.

Lorch et al. 2008; Fitze and Le Galliard 2011; Aronsen et al. 2013). Fur- thermore, until Paper I in this thesis no one had provided comparable estimates of sexual selection in both sexes across species with divergent mating systems and sex roles. Thus despite the expectation that sexual selection acts differently on males and females, we lack the direct comparisons essential for an explicit understanding of the parameters responsible for these differences. These data are by no means easy to collect, but as a starting point I

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suggest that wherever possible, studies estimating the strength of sexual selection in one sex should gather the same data for the other.

Research objectives

In my thesis I explore the evolution of sex role reversed mating systems by using the honey locust beetles Megabruchidius dorsalis and M. tonkineus.

These species are uniquely suited to address gaps in sexual selection research and have allowed me to shed light on the following themes: (1) as- sessing various measures of sexual selection, (2) comparing sexual selection in females and males (3) understanding the implications of costly male mating for mating system evolution and (4) identifying the driving forces (environmental and demographic) of selection leading to the evolution and maintenance of sex role reversal.

Paper I

In the first paper of this thesis I evaluated the overall strength of sexual selection in females and males and compared the utility of several widely used measures of sexual selection. My main goals were (1) to test classic theory (Shuster and Wade 2003) by comparing the strength of sexual selection in males and females in role reversed and conventional mating systems and (2) to assess how well various measures of sexual selection reflect the sex roles of the organisms studied.

Paper II

In this study I examined mating costs for males and mutual mate choice in the two honey locust beetles. I had three goals: (1) to estimate the cost of mating to males and assess whether such costs are dependent on male resource availability; (2) to examine mutual mate choice in both sexes, and to identify phenotypic traits in both sexes that are the target of mate choice; and (3) to test for differences in the pattern of mutual mate choice between the two closely related species.

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Paper III

Here I assessed how body size of both females and males interacts with adult male food provisioning to influence mating success and reproductive output in sex role reversed mating systems. I hypothesized that male size is under fecundity selection (larger males produce larger ejaculates) and that the size (and hence nutritional value) of a male's ejaculate is condition dependent, and should thus be influenced by both juvenile and adult resource acquisition. I investigated whether male and female condition can influence: (1) the size and nutritious value of ejaculates transferred during mating, (2) reproductive success for both females and their mates, (3) mating behaviour of both sexes and (4) whether the absolute reproductive benefits to females from receiving nutritious ejaculates depend on their own body size.

Paper IV and Paper V

In these two papers I used experimental evolution to find out whether changes in the adult sex ratio (demographic selection pressure) and in food provisioning (environmental conditions) can influence the evolution of sex role reversed mating systems. In paper IV I assessed the relative roles of fecundity enhancement and male-male reproductive competition in the evo- lution of direct resource provisioning by males in M. tonkineus. In paper V, I focused on whether increased intra-sexual competition in females can lead to the evolution of increased investment in female-specific courtship traits.

The model system: Megabruchidius dorsalis and Megabruchidius tonkineus

The main model systems in all of my studies are the sex role reversed honey locust beetles Megabruchidius dorsalis and M. tonkineus (Figures 1 and 2) belonging to the family Chrysomelidae (Coleoptera, Bruchidae). In many species in this family, males provide females with a large and nutritious ejaculate. This places significant constraints on male ejaculate production and provides females with direct benefits (Moya-Larano and Fox 2006;

Rönn et al. 2008). Yet, seed beetles generally show conventional sex roles.

The only known exceptions are the two honey locust beetles M. dorsalis and M. tonkineus. M. dorsalis and M. tonkineus show several typical hallmarks of sex role reversed taxa, with predominantly female-female competition for access to mates (Vincent et al. 1994; Berglund and Rosenqvist 2003).

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First, as in some pipefish (Berglund and Rosenqvist 1993), females show active and extended courtship of males (Takakura 1999). In M. dorsalis and M. tonkineus, females initiate mating by antennating the male’s head and thorax in a face-to-face position. The female then turns 180° and presents her abdominal plate (i.e. the pygidium, Figure 3). The male antennates and palpates the pygidium and may accept or reject the female. If not accepted, the female generally turns back 180° and resumes antennation. Typical courtships last from less than a minute to several minutes and consist of multiple repetitions (on average 10 times; Takakura 1999).

Second, females are equipped with a sex-limited secondary sexual character that is employed during courtship, something that is rare even in role reversed taxa (Funk and Tallamy 2000; Clutton-Brock 2009). The enlarged female pygidium has two patches (oval depressions; Tuda and Morimoto 2004, Figure 3) that are presented to males during courtship and which carry numerous pores that may emit pheromones.

Third, as in many role reversed insects (Vahed 1998), females derive direct benefits from mating. Female M. dorsalis that mated 10 times lay as much as 8 times as many eggs as females that mated only once (Takakura 1999).

The fact that females mate more often when kept on a low-quality diet (Takakura 2004a, 2004b) suggests that females can even be though of as

‘foraging’ for matings (Kaitala and Wiklund 1994).

Fourth, as in role reversed pipefish (Berglund and Rosenqvist 2003), male reproductive investment is considerable. Male honey locust beetles transfer a large and nutritious ejaculate to females (more than 7% of male body weight) and a male’s weight decreases with successive matings (Takakura 1999).

Fifth, as has been found in a few other insects (Gwynne 1990; Simmons 1992) and pipefish (Vincent et al. 1994; Berglund and Rosenqvist 2003), the estimated male reproductive investment exceeds that of females in M. dorsalis (Takakura 2006). Males and females of this species show long refractory periods after mating (Takakura 2001) and males pursue fewer mating possibilities than females (Takakura 2006). After, mating, males must replenish their ejaculate to be ready for a new mating, whereas females produce eggs. If this leads to a difference in the length of refractory periods, then the operational sex ratio may actually be female biased.

All this makes Megabruchidius spp. an ideal model system to study sexual selection in females and its adaptive consequences for mating system evolution.

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Figure 1. Male (left) and female (right) of Megabruchidius tonkineus – honey locust beetles. Photo: Lech Borowiec.

Figure 2. Mating pair of Megabruchidius dorsalis – honey locust beetles. The male (left) is on top of the female (right) and his aedegus is inserted. Photo: Dr. Janos Bodor.

Figure 3. Female pygidium with two oval depressions of M. dorsalis (left) and M.

tonkineus (center). Electron-microscopic close-up of one oval depression with visi- ble pores (right). Photos: Göran Arnqvist

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Methods

Maintenance of study species

Honey locust beetles of the genus Megabruchidius are a genus of seed bee- tles (Coleoptera, Bruchidae) with three known species (Tuda and Morimoto 2004). The two sister species M. tonkineus and M. dorsalis (Kergoat et al.

2007, Figure1, 2) are originally found East Asia. In recent years, both species have spread throughout much of Europe as a result of the cultivation of their host plants as an ornamental tree (honey locusts; Gleditsia spp., Fritzsche and Delobel 2012). Although the biology of M. dorsalis has been studied in some detail (e.g. Kurota and Shimada 2002; Takakura 2004a, 2006) virtually nothing is known about M. tonkineus (György 2007).

I established large (>500 individuals) laboratory stock populations of both species (26 °C, 16:8 light:dark, 70% relative humidity) reared on seeds of Gleditsia triacanthos, from original field samples of M. dorsalis (Inogashira Park, Tokyo, Japan; ≈ 3000 adults, June 2009) and M. tonkineus (Orczykert, Budapest, Hungary; > 300 adults, May 2009) at Uppsala University. Stock populations were reared in multiple 1L containers and were fed 20% sucrose solution, pollen, and water. For all my experiments, I obtained virgin beetles by isolating single seeds into 24-well tissue plates, collecting individual beetles as they hatched, and keeping them isolated until the onset of the experiment.

Experimental procedures

Paper I

In order to evaluate the utility of different measures of sexual selection and to compare the strength of sexual selection between females and males I used four related seed beetle species of two genera (Coleoptera, Bruchidae) that differ strikingly in sexual dimorphism and sex roles (Fox 1993; Eady 1994; Fox et al. 1995; Takakura 1999, 2001; Sakurai and Kasuya 2008; Pa- per II). I compared the two beetle species with sex role reversed mating sys- tems (Megabruchidius spp.) to two closely related seed beetles with conven- tional sex roles (Callosobruchus spp.). The mating system of the genus Cal-

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losobruchus spp. is ‘conventional’, in the sense that males actively search for, court and compete for females (Rönn et al. 2006) whereas females are reluctant to mate and are choosier (Maklakov and Arnqvist 2009).

I created separate mating populations for each species and recorded the total number of copulations by direct observation and reproductive success per individual. Successful intromission can be recognized by a characteristic backwards-leaning posture of the mounted male and almost complete lack of motion by either partner for a duration of 2–7 min. I estimated sexual selection both for males and females. I chose to measure sexual selection on body size. I measured the mean length of the left and right elytra of each individual as an estimate of body size, as elytra length correlates very closely with body weight (Wilson and Hill 1989).

Paper II

To investigate mutual mate choice and whether multiple mating is costly for males of honey locust beetles I used careful observations of staged encoun- ters between males and females with experimentally manipulated phenotypes (virgin vs. non-virgin) and I exposed males to different mating rates.

In order to evaluate the cost of mating to males, defined as those costs that derive from courtship and copulation, I studied the effects of mating rate and food provisioning on male life span (Paukku and Kotiaho 2005; Pomian- kowski et al. 2005; Hall et al. 2009; South et al. 2009; Wedell 2010). For this experiment all males were kept individually. I varied their exposure to virgin females, and food (food vs. non-food) and measured male life span, determined by daily inspections.

I employed a no-choice experimental design (Coyne et al. 2005) to test for mate choice. This was deemed most realistic because males and females do not tend to aggregate in the field (Takakura 2004a). Our basic approach was to carefully record the reproductive behaviour of male-female pairs of which half of the females were virgin and the other half non-virgin. I ensured that all experimental males had experienced female courtship and mating at the time of the mating trial. Mating trials were performed by placing pairs of males and females (virgin or mated) together for 60 min. I recorded and observed indicative behaviours for courtship and choice and noted which sex rejected their mate in cases where courtship did not result in copulation.

After the mating trials we measured the length of both antennae and both elytra in both sexes and the length of the pygidium including the pygidial patches in females.

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I was also interested in whether male preference to mate with a virgin or non-virgin female is adaptive. This depends on how soon after the first copulation female egg production commences (Alonzo and Pizzari 2010). In order to characterize this, I set up male-female groups that were housed together and recorded egg production over several days.

Paper III

In this paper I studied the effects of phenotypic condition on mating behav- iour, ejaculate size and reproductive output in honey locust beetles, M. dor- salis. To assess the effect of larval feeding on ejaculate production and fit- ness I divided individuals of both sexes into two weight classes of ‘large’

and ‘small’ individuals. I also divided adult males into two food regimes (fed versus non-fed) in order to analyse whether juvenile and/or adult resource acquisition influence phenotypic condition.

Individuals were weighed before and after mating to estimate ejaculate weight (Edvardsson & Tregenza, 2005). Females were then paired with males of each weight class (large, small) and feeding treatment for mating.

Each pair was placed together in a petri dish and observed until a mating was completed, or for a maximum of 30 min if no mating commenced during this time. I repeated this procedure for each pair for three consecutive days, meaning that each pair could mate a maximum of three times. To estimate the amount of absolute and relative ejaculate transferred during mating, both males and females were weighed immediately before and after each mating.

After the third mating trial, females and males were kept in their individual petri dishes until death and their life span was recorded. Females were kept with 100 g of Gleditsia beans as an ad libitum substrate for oviposition. The beans were kept until all offspring hatched and the number of offspring of each pair was recorded. I kept additional virgin individuals of both sexes of each food treatment and size group to compare the lifespan of mated beetles to virgin beetles.

Paper IV and Paper V

During the course of my Thesis I performed long-term experimental evolution to study how sex-role reversed honey locust beetles adapted to different sexual selection regimes. Populations evolved under two adult sex ratios (female-biased or male-biased) and two food regimes (abundant food or no food in adulthood). The experimental lines (N = 16) were allowed to evolve in the laboratory for 20 generations. In every generation, 150 virgin adults

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per line were placed at the desired sex ratio together and were allowed to mate and lay eggs for about two weeks.

The experimental conditions were terminated at generation 20 and lines were maintained under common garden conditions of equal sex ratios without access to food or water during adulthood for four subsequent generations prior to our assays. This ensured that trans-generational parental effects did not confound the results of our assays.

After experimental evolution I performed slightly different behavioural es- says to answer two distinct questions in Paper IV (M. tonkineus) and Paper V (M. dorsalis). The evolution of large male ejaculates is thought to be driv- en by male reproductive competition and selection for female fecundity enhancement. In Paper IV I investigated the relative roles of these adaptive advantages of male provisioning in M tonkineus. Virgin adults from genera- tion 24 were collected from each of the 11 experimental evolution lines. I conducted matings within and between all four treatments. I ensured that, to the extent it was possible, every line was crossed with every other line and that each cross was replicated using both sex pairings (i.e. crosses between lines A and B would include matings of A males with B females and B males with A females). The weight of every individual was recorded before and after mating to calculate the weight of the transferred ejaculate. Pairs were placed together and observed until they had mated once. Pairs that did not mate within 3 h were separated overnight and placed together again the following day. I recorded the number of offspring of each pair to provide a measure of female lifetime reproductive success and also female lifespan.

In paper V, I asked whether increased intra-sexual competition in females leads to the evolution of increased investment in female-specific courtship traits. I also was interested in whether direct food availability had evolutionary effects on mating interactions. I collected virgin adults from generation 24 and separately paired females and males from all lines with opposite-sex individuals from the stock population to provide a standard references allow- ing me to disentangle evolutionary changes in females and males (i.e. line females were crossed with stock males and line males with stock females). I observed all pairs for 30 min and subsequently analysed female courtship behaviour, time until mating, whether pairs mated or not and the mating duration. Before and after mating I weighed every individual to provide an indirect measure of ejaculate weight. After mating I kept males and females individually and checked both sexes daily until death to record life span. I also recorded the number of emerged offspring in each female dish.

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Results and Discussion

My thesis shows that mating can bear costs for males and highlights the essential role that females play in mating system evolution and that their contribution cannot simply be reduced to mate choice.

Paper I – Sexual selection in females: How to measure the strength of sexual selection

My work in this paper yielded a series of novel insights into differences in the strength of sexual selection between the sexes and across mating systems. I found that sexual selection was sizeable in females and that the strength of sexual selection was similar in females and males of the role reversed species.

Relative reproductive success increased overall with mating success (F_49,1 = 143.9, P < 0.001, β_ss). Bateman gradients (a measure of the strength of selection on mating rate) were steeper in males than in females, as evi- denced by a significant interaction between sex and mating success on re- productive success (sex × mating success: F49,1 = 4.5, P = 0.034). Mating system had an even stronger effect, such that the Bateman gradients were steeper in role reversed species (Figure I-1A; mating system × mating suc- cess: F_49,1 = 5.99, P < 0.001).

A graphical inspection of the results suggests that the opportunity for sexual selection was higher in males than in females in species with conventional sex roles (Figure I-1B). In contrast, in role reversed species, the opportunity for sexual selection was similar across sexes or higher in females than in males. However, variance in mating success differed significantly between the sexes in only one species (C. maculatus: F49,19 = 0.48, P = 0.042).

For trait-based measures, body size was positively related to mating success (m’) and reproductive success (s’) in both sexes. Variance in mating success and reproductive success was overall higher in males of species with con- ventional sex roles (mating system × body size: F_49,1 = 0.81, P = 0.037, m’;

mating system × sex × body size: F49,1 = 0.83, P = 0.038, s’).

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Figure I-1. Two sex-specific measures of sexual selection across four different seed beetle species (CM = Callosobruchus maculatus; CC = Callosobruchus chinesis;

MD = Megabruchidius dorsalis; MT = Megabruchidius tonkineus; white bars = males; black bars = females). SRR+ denotes species that are sex role reversed and SRR− denotes species with conventional sex roles. Error bars represent SE.

Previous comparisons of the correspondence between variance- and trait- based measures of sexual selection within species have yielded mixed results (e.g. Jones et al. 2000, 2004; Bjork and Pitnick 2006; Mills et al. 2007;

Paczolt and Jones 2010; Fitze and Le Galliard 2011; Munroe and Koprowski 2011). In this study, I found that measures of sexual selection based on variance in mating success and reproductive success (Bateman gradient and selection opportunities, Jones et al 2009) were better predictors of the reproductive behaviour and morphology of the four beetle species than were measures that only use phenotypic traits (selection differentials, m’ and s’).

Sexual selection is by definition generated by competition for matings or fertilizations (Darwin 1871; Andersson 1994). The strength of sexual selection should thus reflect the intensity of competition for matings or fertilizations. The Bateman gradient is the most relevant measure of the strength of sexual selection in this study, due, in part, to the fact that it reflects latent properties of the mating system representing both male and female adaptations to mating. Furthermore, the explicit purpose of the Bateman gradient is to provide a measure of the strength of competition for matings (Arnold and Duvall 1994; Wade and Shuster 2005).

Overall, sexual selection was stronger in males than in females (Figure 1A, 1B). Our results are consistent with the view that sexual selection is typically stronger in males than in females (Bateman 1948; Trivers 1972; Anders- son 1994; Arnold and Duvall 1994; Shuster and Wade 2003). Sexual selection was, however, sizeable in females and the relative strength of selection in males and females varied with mating system. In particular, I found that the strength of sexual selection in females was stronger in role reversed mating systems compared to conventional ones. Because role reversal is typical-

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ly rooted in male mate provisioning or male parental investment, our observation accords with theory (Trivers 1972).

Paper II – The cost of mating for males and mutual mate choice

Experimental tests of mutual mate choice are essential but rare (e.g. Jones and Hunter 1993; Hunt et al. 1999; Aquiloni and Gherardi 2008; Kemp 2008). Our experimental dissection of mutual mate choice in honey locust beetles yielded several novel insights. First, the cost of mating to males was substantial in these beetles and was independent of food availability (mating:

χ² = 80.54, P < 0.001; food regime: χ² = 619.65, P < 0.001). Frequent mating reduced survival and life span by about 30% compared with unmated males.

Although the existence of male mating costs is crucial for our understanding of sex roles in these beetles, theory makes no quantitative predictions re- garding the absolute size of costs required to favour male mate choice. Alt- hough absolute life span was much increased by feeding, the proportional cost of mating was unaffected by resource availability. This suggests that a large component of the cost of mating to adult males derives from producing components of the ejaculate from resources that males recruit during their juvenile stage in the host seed and that they do not refuel as adults (Conner et al. 2000).

Second, male honey locust beetles consistently showed a mating preference for females that delivered a more intense courtship display and for females that were large (no. of female courtship turns, deviance ratio: F47.99,1 = 83.02, P < 0.001; courtship duration: F_33.52,1= 58.00, P < 0.001; species × female size: F6.60,1 = 11.42, P < 0.001). Male mating preferences for large females are common (Bonduriansky 2001) and preferences for female sexual signals that are expressed by both sexes (i.e., sexually homologous signals, sensu Arnold 1985) have been documented in a range of taxa (Jones and Hunter 1993; Hunt et al. 1999; Aquiloni and Gherardi 2008; Kemp 2008), but our study is a rare example of a male preference for a secondary sexual signal present only in females (i.e. courtship behaviour; Funk and Tallamy 2000;

Amundsen and Forsgren 2001).

Third, female mate choice was much less obvious in our experiments and was restricted to a negative relationship between male size and female mate rejection behaviour in M. tonkineus (β’ = -0.271, t₅₈ = 2.11, P = 0.040). In sex-role reversed species, such as honey locust beetles, one might argue that we would predict female choice to be weak or absent for the same reasons that we by convention expect little male mate choice in species showing

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conventional sex roles. Yet, several factors may lead to the evolution of mate choice also in the sex experiencing the most intense intra-sexual competition (Owens and Thompson 1994; Johnstone et al. 1996; Reinhold et al.

2002; Servedio and Lande 2006; Rowell and Servedio 2009).

Fourth, males also preferred virgin females in one species but non-virgin females in the other species (species × female mating status, deviance ratio:

F7.67,1 = 13.27, P < 0.001), and in Paper II I provide data suggesting that this choice is adaptive.

Paper III – Parental investment is affected by phenotypic condition

In Paper III I show that male phenotypic condition has sizeable effects on ejaculate quality. Male adult feeding had significant effects on almost all aspects of male and female reproduction, in the latter case through the nutritional effects of ejaculates in females. For instance, females that mated with males of good condition lived longer and produced more offspring than females that mated with males of poor condition. This also confirms the sizeable direct benefit females gain by mating multiply.

Our results show that large body size and high adult food availability are both beneficial to males. On average, well-fed males (1) mated more often, (2) transferred larger ejaculates (Figure III-1; male size: F_65.45,1 = 30.38, P < 0.001: mating no. × male feeding regime: F97.15,2 = 11.92, P < 0.001), (3) produced more offspring (total no. of matings × male feeding regime:

F58,2 = 4.01, P = 0.023) and (4) lived longer than non-fed males (male size:

F_67,1 = 8.04, P = 0.006; F_67,1 = 88.82, P < 0.001).

Figure III-1. Mean ± SE total ejaculate weight of males over consecutive matings in relation to whether they were fed (grey bar) or non-fed (white bar).

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The production of nutritious ejaculates carries costs in terms of acquiring food resources and replenishing an ejaculate takes time, especially when food resources are scarce (Svärd and Wiklund 1989; Gwynne 1993; Perry and Tse 2013), which would explain the above results for males of different condition.

I also found that (1) large females produced more offspring than small females dependent on male feeding regime (Figure III-2; female size × male feeding regime: F58,1 = 10.25, P = 0.002) and (2) multiple mating increased the life span of all females (Figure III-2; total no. of matings: F_69,3 = 10.50, P < 0.001). Thus, females gain direct benefits by mating multiply, as has been found in many other species (Arnqvist & Nilsson 2000; Jennions &

Petrie 2000; Fedorka and Mousseau, 2002a,b). However, taking female size into account, small females showed an increase in life span but not in offspring production this suggests that large and small females may allocate ejaculate resources differently.

Figure III-2. Mean ± SE fitness components. (a) Total number of offspring of large and small females in relation to whether males were fed (grey bar) or non-fed (white bar). (c) Life span of females in relation to the number of times they mated.

Paper IV – Sperm competition increases paternal investment

In paper IV I aimed to directly measure evolved differences in the ejaculate invested per mating and to study the effects of elevated male mating rate (female-biased sex ratios) and increased male-male reproductive competition (male-biased sex ratios).

The sex ratio experienced by the male line significantly affected the weight of ejaculates. Males from male-biased lines transferred significantly larger ejaculates during mating than did males from female-biased lines (Fig-

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ure IV-1, F136.3,1 = 9.50, P = 0.003). Thus, lines experiencing intensified male-male reproductive competition evolved a larger body mass-specific ejaculate size. The number of offspring produced increased with female weight and with the weight of the ejaculate transferred by the male (female weight: F136.4,1 = 7.32, P = 0.000; ejaculate weight: F132.2,1 = 5.36, P = 0.022).

Figure IV-1. Mean (± SE) percentage weight of the ejaculate transferred during a single mating by males from experimental evolution lines with female-biased and male-biased sex ratios. Males from male-biased lines evolved to transfer considera- bly larger ejaculates.

Male ejaculates evolved to a larger size under conditions of intense male-male competition. In contrast, the fecundity-enhancing effects of provisioning females with ejaculate resources had no apparent effect on the evolution of ejaculate size, as ejaculate size did not differ between lines experiencing conditions of food limitation and abundance. Ejaculate size was positively related to female offspring production. This is consistent with previous results that showed that the size of the ejaculate provided by males is indeed key to female fitness in this species (Takakura 1999, 2006). Thus, males evolved a higher degree of paternal investment under conditions of intense male-male competition, but not under conditions where such investment is of higher direct value to females.

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Paper V – Female-female competition accelerates the evolution of role reversal

In Paper V, I found that male adaptations were less pronounced in M. dorsa- lis and I could not observe a significant change in ejaculate size. In contrast, I found rapid evolution of female courtship. Females evolving under female- biased sex ratios (representing strong female mate competition) significantly outperformed those from male-biased lines in courtship and mating success.

My results add to the growing body of evidence that females can be under strong selection to achieve matings (Clutton-Brock 2007, 2009; Shuker 2010; Stockley and Bro-Jørgensen 2011, Rosvall 2011). Females from female-biased lines made first contact with new males more quickly, and achieved successful mating sooner and after fewer male mounting attempts (Figure V-1, Table V-1). Interestingly, I also found a significant effect of the interaction of sex ratio and feeding regime on female courtship success (Ta- ble V-1). Females from female-biased lines were highly successful in achieving mating after only one courtship bout, regardless of the food regime they evolved under.

Figure V-1. Female courtship behaviour by sex ratio treatment (female line " stock male). Left axis (white and light grey bars): time to first encounter and time to mating in seconds. Right axis (dark grey bars): mounting attempts before successful mating. Mean ± SE.

Time to first encounter Time to mating

Mounting attempts before mating

Female-biased Male-biased

0 100 200 300

0 1 2 3

TimeHsL Mountingattempts

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Table V-1. Female courtship behaviour (female line × stock male). Analyses of variance/covariance of the effects of sex ratio and food treatment on courtship traits in female line × stock crossings.

Source ndf ddf F P

Time to first encounter Feeding regime 1 13 0.84 0.377

Sex ratio 1 13 5.81 0.032

First courtship = mating Feeding regime 1 12 3.78 0.076

Sex ratio 1 12 9.55 0.009

Feeding regime × Sex

ratio 1 12 13.40 0.003

Mounting attempts Feeding regime 1 11 0.13 0.721

Sex ratio 1 11 10.69 0.007

Female weight 1 11 10.28 0.008

Male weight 1 11 5.51 0.039

Time to mating* Feeding regime 1 12 2.02 0.181

Sex ratio 1 12 5.78 0.033

Male weight 1 12 11.01 0.006

My results suggest that the evolution of sexually selected traits in females need not always signal fecundity, as is sometimes assumed (Paper II, Ros- vall 2011). Females with successful courtship displays (females from female-biased lines) neither produced more offspring nor lived longer than less attractive females. I also showed that female traits can evolve more rapidly than male traits in response to corresponding changes in sex ratio. Two other experimental evolution studies found significant female trait evolution in response to sex ratio manipulation without a measurable response in males (Wigby and Chapman 2004, Fritzsche et al. 2014).

My results suggest that the evolution of sexually selected traits in females need not always signal fecundity, as is sometimes assumed (Paper II, Ros-

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vall 2011). Females with successful courtship displays (females from female-biased lines) neither produced more offspring nor lived longer than less attractive females. I also showed that female traits can evolve more rapidly than male traits in response to corresponding changes in sex ratio. Two other experimental evolution studies found significant female trait evolution in response to sex ratio manipulation without a measurable response in males (Wigby and Chapman 2004, Fritzsche et al. 2014).

In contrast to the clear evolution of female courtship, male adaptations were less pronounced. This contrasts with my results in Paper IV for the closely related M. tonkineus, in which males adapted to strong mate competition by significantly increasing ejaculate size.

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Conclusion

The results of my thesis reveal striking interspecific variation in sexual selection and the resulting adaptions of females and males. I provide evidence for mutual mate choice in Megabruchidius spp., and show that sexual selec- tion in females can act on much the same types of traits that are commonly considered sexually selected in males, such as body size and courtship vig- our. Under experimental evolution with manipulated sex ratios, females adapted rapidly to increase mating success under strong sexual selection and this intensified the reversal of sex roles. The traits selected in females provided no significant fecundity benefits to males and thus male mate choice might have evolved due to indirect selection or sensory exploitation in this species. My thesis highlights the essential, and often overlooked, role that females play in mating system evolution and that their contribution cannot simply be reduced to mate choice.

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Sammanfattning på svenska

Sexuellt urval är en betydande källa till biologisk variation, både i termer av mångfald av egenskaper och artbildning, hos alla arter som förökar sig sexuellt. Darwin definierade sexuellt urval som följer:

“The sexual struggle is of two kinds; in the one it is between individuals of the same sex, generally the males, in order to drive away or kill their rivals, the females remaining passive; whilst in the other, the struggle is likewise be- tween the individuals of the same sex, in order to excite or charm those of the opposite sex, generally the females, which no longer remain passive, but se- lect the more agreeable partners.”

(Darwin 1871) Under de senaste åren har en förnyad debatt över hur sexuellt urval bäst skall definieras och kvantifieras skapat en viss turbulens inom fältet. Trots att Darwins ursprungliga definition på sexuellt urval var bred och inte exklude- rade sexuellt urval hos honor, så har forskningen sedan dess varit nästan helt fokuserad på hannar, och denna tolkning har nyligen blivit omvärderad.

Sedan upptäckten att det är mycket vanligt hos de flesta djur att honor parar sig med flera hannar, har teorin om sexuellt urval utvidgats till att inkludera konkurrens honor emellan och partnerval hos hanar. Studier av sexuellt urval hos honor och partnerval hos hannar är dock ,tyvärr, fortfarande bristfälligt studerat. Det är oklart hur mycket sexuellt urval hos honor liknar den hos hannar, som är bättre studerad. Det är speciellt kontroversiellt huruvida ho- nor vanligen svarar på kraftigt sexuellt urval genom att utveckla kostsamma egenskaper för att attrahera hanar, och om hanarnas partnerval är primärt drivet av signaler om honornas fruktbarhet.

I min doktorsavhandling framlägger jag bevis för att sexuellt urval hos ho- nor av flera arter är betydande och jämförbar med samma urval hos hannar, samt att det för hannar kan vara kostsamt att para sig många gånger. I min forskning använde jag mig främst av de könsrollsomvända skalbaggarna Megabruchidius dorsalis och M. tonkineus (se artbeskrivning). Hos dessa båda skalbaggsarter förser hannarna honorna med ett stort näringsrikt ejakulat. Dessa ejakulat har direkt nytta för honorna och honorna tävlar om hanarnas uppmärksamhet. Hanarna, å sin sida, är noga med vilken hona de parar

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sig med. Därför är Megabruchidius spp. ett närmast idealt system för att studera sexuellt urval hos honor och dess evolutionära konsekvenser.

I den första artikeln i min doktorsavhandling undersökte jag styrkan av det sexuella urvalet hos honor och jämförde användbarheten av flera vitt an- vända mått på sexuellt urval. Jag jämförde de redan nämnda två skalbaggsar- terna med två närbesläktade arter (Callosobruchus chinensis och C. maculatus) som har könsroller som mer liknar det sexuella urvalet såsom Darwin beskrev det. Jag fann att det sexuella urvalet var betydande hos honor och att det sexuella urvalet hos arter med omvända könsroller var av likvärdig styrka hos honor och hannar. Dessutom var det sexuella urvalet starkare hos honor av könsrollsomvända arter (Megabruchidius spp.) jämfört med honor av arter med konventionella könsroller (Callosobruchus spp.). Dessutom fann jag att mått på sexuellt urval som är baserade på variansen av parnings- framgång och reproduktiv framgång (”Bateman gradient” och ”selection opportunities”) var bättre på att förutse de fyra skalbaggsarternas fortplan- tingsbeteende och morfologi, jämfört med metoder som endast använder fenotypiska egenskaper (”selection differentials”, artikel I).

Sexuellt urval är ovanligt kraftigt hos honor inom arter där hannarna gör en betydande investering i form av näringsrika ejakulat eller fadersomvårdnad av avkomman. Med andra ord, när hannar förser honor med resurser vid parningen kan hanar bli det begränsande könet vid reproduktionen. I extrema fall leder detta till att de typiska uppvaktningsrollerna omvänds och att styrkan av det sexuella urvalet hos honor och hannar förändras. I min forskning har jag funnit att näringsrika ejakulat, som i fallet med skalbaggarna, är kostsamma för hannarna att producera och kan begränsa hur ofta hannen kan para sig. Hannar som parade sig oftare levde kortare än hanar som parade sig mer sällan. Jag fann också belägg för att hannar är kräsna med vilken hona de parar sig med. I en serie av partnervalsförsök manipulerade vi honornas parningsstatus och hannarnas födotillgång och fann att hannar generellt visade preferens för stora honor och honor som gav en mer ihärdig och aktiv uppvaktning. Hannar föredrog också oparade honor hos en art men redan parade honor hos en annan art, och vi framför data som antyder att det här hanliga valet är adaptivt (artikel II).

I artikel III visar jag att fenotypernas status hade betydande effekter på eja- kulatkvalité. Födointaget hos vuxna hannar hade signifikanta effekter på nästan alla aspekter av hanarnas och honornas reproduktion genom den nut- ritionella effekten av ejakulatet hos honor. Honor som parade sig med hannar som var i gott skick levde till exempel längre och fick fler avkommor än de honor som parade sig med hannar i sämre skick. Det här bekräftar också

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den betydande omedelbara nyttan honor får genom att para sig med flera hannar (artikel III).

Dessa experiment gav viktiga insikter i de korrelativa förhållandena mellan variabler i olika parningssystem. Det är dock än mer kraftfullt att använda sig av experimentell evolution för att förstå orsakssamband. Under mitt dok- torandarbete använde jag mig utav långsiktig experimentell evolution för att studera hur båda arterna av könsrollsombytta skalbaggar anpassade sig till experimentella förhållanden där olika typer av sexuellt urval rådde. Populat- ioner av båda arterna utvecklades under två olika könskvotsförhållanden för de vuxna djuren (överskott honor eller hanar) och två olika födoregimer (mycket mat eller ingen mat för vuxna djur). Efter 20 generationer av evolution observerade jag två något olika typer anpassningar hos de två skal- baggsarterna.

För det första, som visat i experimentet från artikel IV, så uppvisade M. ton- kineus hannar som utvecklades under könsförhållanden med ett överskott av hannar ett större ejakulat än hannar som kom från populationer med ett över- skott på honor. Jag menar att detta utgör bevis för en betydande roll för reproduktiv tävlan för evolutionen av ”utfodring” av honorna hos hannar av denna art. Mina resultat antyder att den ”utfodring” som har orsakat köns- rollsomvändningen i det här modellsystemet är ett resultat av tävlan hannar emellan snarare än ett resultat av ett direkt urval av hannar för att mata honorna och därigenom öka deras fruktbarhet.

För det andra, i artikel V visade jag att hanarnas anpassningar var mindre uttalade hos M. dorsalis och jag kunde inte upptäcka någon signifikant för- ändring i ejakulatstorleken. Istället fann jag en tydlig utveckling av uppvakt- ningsbeteendet hos honorna. Honor som utvecklades under könsförhållanden som hade en överskott av honor (vilket representerar en starkare konkurens om partners honor emellan) uppvisade ett mer aktivt uppvaktningsbeteende, var mer attraktiva för hanar och var mer framgångrika i termer att uppnå parningar jämfört med honor från populationer med ett öveskott av hannar.

Överlag så utvecklade honor kraftiga uppvaktningsuppvisningar antingen när parningar med hannar var en begränsande resurs, eller när hanarnas ejakulat var deras enda näringskälla under vuxenlivet.

Resultaten från min doktorsavhandling visar en stor variation i det sexuella urvalet mellan arter och i de anpassningarna hos honor och hannar som re- sulterar from denna form av naturlig selektion. Jag framlägger bevis för att båda könen väljer partner hos Megabruchidius spp., och visar att sexuellt urval hos honor kan verka på samma typer av egenskaper som vanligtvis anses sexuellt utvalda hos hannar, såsom storleksrelaterade egenskaper och uppvaktningsvigör. Anpassningar hos honor som ökade framgången vid

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parning utvecklades snabbt under starkt sexuellt urval bland honor och detta intensifierade omkastningen av könsroller. Egenskaperna som selekterades hos honor gav dock inga uppenbara fördelar till hanarnas fruktbarhet och därför verkar det möjligt att hanarnas partnerval är ett resultat antingen av s.k. sensorisk exploitering eller av indirekt selektion hos denna art. Min forskning påvisar den viktiga och ofta förbisedda rollen som honor spelar i parningssystemens evolution och att deras bidrag inte bara kan reduceras till partnerval.

(Översatt från engelska av / Translated from English by Johan Ålund)

Sexual selection and the evolution of sex-role reversal in honeylocust beetles