Pre- and Post-Copulatory Sexual Selection in the Fowl, Gallus Gallus

Pre- and post-copulatory sexual selection in the fowl, Gallus gallus

Hanne Løvlie

Department of Zoology Stockholm University 2007

Pre- and post-copulatory sexual selection in the fowl, Gallus gallus Doctoral dissertation 2007

Hanne Løvlie Department of Zoology Stockholm University SE-10691 Stocholm Sweden [email protected]

ISBN 978-91-7155-433-8

Printed in Sweden by US-AB, Stockholm 2007 Distributor: Dept. of Zoology, Stockholm University

2 Abstract

The evolutionary goal of individuals is reproduction and sexual selection favours traits improving reproductive success. When males invest less than females in offspring, males have potentially a higher reproductive rate than females. This typically results in sex-specific reproductive strategies of male-male competition and female choice of mating partner. Under polyandry, sexual selection can continue after copulation as sperm competition and cryptic female choice. This thesis focuses on male and female pre- and post-copulatory reproductive strategies in the promiscuous red junglefowl, Gallus gallus ssp., and its domestic subspecies the domestic fowl, Gallus gallus domesticus. Males impose high re-mating rates on females, which triggers female resistance in copulations. In addition, when sexual harassment increases, females re- mate at times of day when male mating propensity is lower, to avoid intense sexual harassment. Males allocate sperm supplies differentially according to (i) variation in female polyandry and own competitive ability, (ii) earlier sperm investment in a female, and (iii) female reproductive quality, signalled by female comb size. Males also perform ‘aspermic’ copulations (i.e. copulations with no semen transfer), which inhibit polyandry and in turn reduce sperm competition. In mating opportunities with relatives, males do not avoid inbreeding. However, females avoid inbreeding before copulation through kin recognition and after copulation by selecting against related males’ sperm. These results show that selection on males to re-mate at higher rates than females and copulate indiscriminately according to partner relatedness, trigger counteracting female responses, creating the potential for sexual conflict over fertilisation. Teasing apart pre- and post-copulatory strategies and the contribution of each sex therefore becomes crucial in order to understand the evolution of reproductive strategies and the mechanisms affecting paternity.

Key words cryptic female choice, Gallus, inbreeding avoidance, mate choice, sexual conflict, sexual selection, sperm competition

3 4 List of papers

The thesis is based on the following papers, which I will refer to in the text by their roman numbers (I-V):

I. Løvlie, H., & T. Pizzari. Sex in the morning or in the evening? Females adjust daily mating patterns to the intensity of sexual harassment. American Naturalist, in press.

II. Pizzari, T., C. K. Cornwallis, H. Løvlie, S. Jakobsson, & T. R. Birkhead, 2003. Sophisticated sperm allocation in male fowl. Nature 426:70-74.

III. Løvlie, H., T. Pizzari, & C. K. Cornwallis, 2005. Male mounting alone reduces female promiscuity in the fowl. Current Biology 15:1222- 1227.

IV. Pizzari, T., H. Løvlie, & C. K. Cornwallis, 2004. Sex-specific, counteracting responses to inbreeding in a bird. Proceedings of the Royal Society of London B 271:2115-2121.

V. Løvlie, H. & T. Pizzari. Mechanisms of pre- and post-copulatory inbreeding avoidance by female red junglefowl. Evolution, in review.

Copyright notice: paper I: The University of Chicago Press, paper II: The Nature Publishing Group, paper III: Elsevier Inc., paper IV: The Royal Society.

5 Contents

Introduction...... 7 Competing males and choosy females ...... 7 Post-copulatory mechanisms...... 8 Aim of this thesis ...... 10 Study species...... 10 The wild red junglefowl and its domesticated relative ...... 10 Sexual selection in the fowl...... 11 Study populations...... 12 Tovetorp ...... 12 Götala ...... 12 Some useful techniques...... 13 Controlled copulations ...... 13 Hydrolysis points on PVL ...... 13 Results...... 14 Male strategies...... 14 Female strategies ...... 20 Discussion...... 24 When interests differ ...... 24 Behaviours as a predictor of post-copulatory processes ...... 25 Conclusions ...... 26 References...... 27 Acknowledgements...... 34 Sammanfattning på svenska...... 36

6 Introduction Competing males and choosy females Reproduction is the evolutionary goal of all individuals and the selection pressure caused by variation in individuals’ reproductive success is called sexual selection. Sexual selection is therefore a strong selection pressure promoting traits that confer a competitive advantage in reproduction. Reproductive success is increased through two main mechanisms; (i) competition for access to mating partners and (ii) choice of a sexual partner (Darwin 1871; Andersson 1994). Both sexes can be found as the competing and the choosy sex. Nevertheless, we typically find competing males and choosy females. Males produce a vast number of sperm, compared to the number of eggs a female produces and that are available to a male. As a result, males have potentially higher reproductive rates than females and their reproductive success is mainly constrained by the number of eggs that a male fertilises (e.g. Bateman 1948). Males are therefore sexually selected to increase their number of sexual partners, leading to male-male competition over females and mating opportunities (Darwin 1871; Andersson 1994). When copulation reduces number of females available for reproduction, for example during pregnancy, the ratio of males and females ready to mate (i.e. the operational sex ratio, Emlen & Oring 1977) becomes more male-biased and male competition for access to females increases further. Females potential reproductive rate, on the other hand, is mainly constrained by the number of eggs a female produces (Trivers 1972). Beyond securing sufficient amount of sperm to fertilise the whole clutch, multiple sexual partners (i.e. polyandry) often are unlikely to increase the number of zygotes produced by a female (Jennions & Petrie 2000). Because copulation may impose costs on females (e.g. Chapman et al. 1995; Gems & Riddle 1996; Stutt & Siva-Jothy 2001; Martin et al. 2004; Le Galliard et al. 2005), increased copulation rates may therefore increase these costs and, as a result, are not likely to contribute to increased female reproductive success. Instead of multiple copulations, a careful choice of a reproductive partner of high quality may increase female fitness (Anderson 1994). Benefits of choosing a high quality parner can be direct (i.e. the female her self benefits from her choice), or indirect (i.e. the female’s offspring benefit from the female’s choice). Indirect benefits are typically genetic benefits gained from mating with a partner with high genetic quality that increase the reproductive value of an offspring, either because the offspring inherit genes for enhanced survival or increased reproductive success (Fisher 1930; Zahavi 1975; Hamilton & Zuk 1982; Kokko et al. 2002), or offspring have increased fitness because the males genotype match the female genotype (Zeh & Zeh 1996; 1997; Jennions & Petrie 2000; Tregenza & Wedell 2000; Mays & Hill 2004; Neff & Pitcher 2005). Preference for a male whose genotype is compatible and matches the female genotype can increase offspring survival due to within-species heterozygosity. Increased survival of heterozygote offspring can be explained by dominance (i.e. when deleterious recessive alleles at specific loci are expressed in homozygote, but in heterozygote individuals are masked by the allele’s dominant counterpart) or overdominance (i.e. when a more genome-wise heterozygosis is beneficial and homozygosity reduces this benefit). A classic source of reduced heterozygosity is

7 inbreeding, reproduction between close relatives (Charlesworth & Charlesworth 1987; Keller & Waller 2002; but see Bench et al. 1994). Reproduction between related partners can therefore cause incompatibility and reduce offspring fitness. However, inbreeding can have positive effects on individuals’ fitness. Because relatives have more genes in common than unrelated individuals, allowing relatives to reproduce or gain additional reproductive opportunities by inbreeding can therefore increase inclusive fitness (Hamilton 1964). However, when the direct cost of inbreeding exceeds inclusive fitness gains, inbreeding should be avoided (Parker 1979; 2006; Lehmann & Perrin 2003; Kokko & Ots 2006).

Under the classic roles of competing males and choosy females, both sexes’ reproductive optima can often not be met, generating sexual conflict over mating and fertilisation. Manipulation of the other sex to increase own reproductive success and counteracting strategies to retain control of paternity may be triggered (Parker 1979; Arnqvist & Rowe 2005).

Post-copulatory mechanisms Despite females’, compared to males’, reduced benefits from multiple mating it is more the rule than the exception in the animal kingdom that females are polyandrous (e.g. Birkhead & Møller 1998). Explanations for female polyandry have frequently been discussed (Arnqvist & Nilsson 2000; Jennions & Petrie 2000; Simmons 2005). Mainly in insects has polyandry been explained by females gaining direct benefits, and in vertebrate species females are typically polyandrous to obtain indirect benefits (Zeh & Zeh 1996; 1997; Jennions & Petrie 2000; Tregenza & Wedell 2000; Mays & Hill 2004; Neff & Pitcher 2005), or because males force female to copulate with them (Clutton-Brock & Parker 1995). Female polyandry nevertheless results in profound effects on sexual selection. When females receive sperm from multiple males sexual selection may continue after copulation, through the competition between ejaculates of two or more males over fertilisation of a female’s eggs (i.e. sperm competition, Parker 1970; 1998) and female bias in sperm utilisation in favour of ejaculates of certain males (i.e. cryptic female choice, Eberhard 1996; Birkhead 1998). Males’ uncertainty of paternity increases under polyandry, selecting males to avoid polyandry (e.g. Birkhead & Møller 1998). Males prevent females from mating with other males, for example by mate guarding (e.g. Birkhead 1979), application of copulatory plugs (e.g. Shine et al. 2000), removing earlier inseminated ejaculates (e.g. Gage 1992), inseminating large ejaculates (e.g. Cook & Wedell 1999) and/or inseminating specific seminal fluid proteins delaying female re-mating (e.g. Chapman et al. 1995; Wolfner 2002). When males face sperm competition, the intuitive response is for a male to inseminate more sperm than his competitors in a female, in order to improve his fertilisation success. However, sperm production can be costly for males (Dewsbury 1982; e.g. Olsson et al. 1997) and male sperm supplies are limited (e.g. Birkhead 1991; Nakatsuru & Kramer 1982). Increased sperm investment in one female is therefore likely to reduce a male’s investment in other females and in turn constrain male reproductive success (e.g. Preston et al. 2001). In order to maximise reproductive success under sperm competition and with limited sperm supplies, males are therefore

8 predicted to allocate sperm supplies differentially to improve fertilisation success and according to the reproductive return of a copulation (Parker 1998; Wedell et al. 2002). Males should allocate sperm differentially according to the probability of facing sperm competition (i.e. the risk of sperm competition) by increasing the number of sperm inseminated when the risk of sperm competition increases (Parker et al. 1997; 1998; for review see Wedell et al. 2002). Males should also allocate sperm differentially according to the intensity of sperm competition (i.e. the number of males inseminating the same female) and counter-intuitively decrease the number of sperm invested into a female when the number of males inseminating the same female increases above two, to save sperm for less competitive mating opportunities (Parker et al. 1996; Parker 1998; e.g. Simmons & Kvarnemo 1997; Schaus & Sakaluk 2001; Pilastro et al. 2002). When males have an increased probability of obtaining future copulations they are selected to allocate sperm prudently and save sperm also for future copulations (Parker 1982; 1998; Reinhold et al. 2002). Males can also bias probability of fertilisation by targeting reproductively active females as copulation partners or copulate at times in a female’s oviposition cycle where insemination is most likely to result in fertilisation (Birkhead & Møller 1992; Birkhead & Pizzari 2002). Male choice of and higher investment in females of higher reproductive quality may also increase male reproductive success (Parker 1983; Reinhold et al. 2002; for review see Wedell et al. 2002).

Despite decreased control of paternity from the males’ point of view, polyandry may allow females to increase control of paternity, particularly in internally fertilising species where female pre-copulatory mate choice is constrained (e.g. Eberhard 1995; Pizzari & Birkhead 2000; Snow & Andrade 2005). Through cryptic female choice females may counteract male pre-copulatory strategies such as sexual coercion. The mechanisms of cryptic female choice is still scarcely studied, mainly due to the, at least in internal fertilisers, cryptic nature. However, cryptic female choice is known to cover a range of mechanisms (Eberhard 1996). From removal of spermatophores before sperm have been transferred (Eberhard 1996), ejection of ejaculates directly after copulation (e.g. Pizzari & Birkhead 2000; Wagner et al. 2004), further to involve differential sperm:female reproductive tract, or sperm:egg interactions biasing fertilisation success (e.g. Clark & Dell 2006). There is some evidence of cryptic female choice against aged sperm (Wagner et al. 2004), sperm of low-ranking males (Pizzari & Birkhead 2000), and several examples of cryptic female choice refer to bias in favour of genetically compatible partners (e.g. Olsson et al. 1996; Tregenza & Wedell 2002; Bretman et al. 2004; Simmons et al. 2006). Cryptic female choice may in these cases improve offspring quality and result in polyandry be adaptive for females (Reinhold 2002; for review see Jennions & Petrie 2000; Simmons 2005).

To conclude, the sexes have sex-specific reproductive optima and strategies to reach them. These strategies often differ, triggering counteracting responses and creating a potential for sexual conflict that, when females are polyandrous, may continue also after copulation. Knowledge of mechanisms of both sexes’ pre- and post-copulatory reproductive strategies are therefore crucial in order to understand what affects paternity.

9 Aim of this thesis

The aim of this thesis is to increase our understanding of cues and mechanisms affecting male and female reproductive strategies, both before and after copulation. Specifically this thesis focused on: • Male and female re-mating rates and daily re-mating patterns • Male ability to allocate sperm strategically • An adaptive explanation to the puzzling male behaviour ‘aspermic’ copulations • Male pre- and post-copulatory responses to inbreeding • Female pre- and post-copulatory inbreeding avoidance mechanisms

Study species The wild red junglefowl and its domesticated relative The red junglefowl, Gallus gallus, is native to Southeast Asia, and the subspecies Gallus g. gallus, the sole ancestor of all today’s domesticated chickens (Fumihito et al. 1994). Domestication probably occurred through one single domestication process and is dated about 8000 years back (Fumihito et al. 1994; 1996). Red junglefowl and domestic fowl interbreed, are morphologically and behaviourally similar (see ‘study populations’), and considered different subspecies. Throughout this thesis I will refer to both red junglefowl and domestic fowl as ‘fowl’. Both wild and feral fowl live in groups of 2-15 individuals, with a sex ratio of 1:1-1:4 males to females (Collias & Collias 1996; Sullivan 1991). Social hierarchies are sex-specific and typically linear. Social status predicts individuals’ behaviours and access to resources, for example are high-ranking males more vigilant than low-ranking males, and have increased access to females as mating partners (Banks 1956; McBride et al. 1969; Pizzari 2003).

Flocks of red junglefowl are small and relatively isolated in the wild. In addition, young birds seems to stay in their birth flocks even after sexual maturation (Collias & Collias 1996), and no difference in the movements of males and females between flocks been observed (Collias et al. 1966). This suggests that the fowl is prone to inbreeding. Consistent with this idea, 4% of matings observed in a captive, free- ranging red junglefowl population were between 1st order relatives (n=135, Collias & Collias 1996). In domestic fowl inbreeding reduces competitive ability (Craig & Bahruth 1965), mating frequency (Cheng et al. 1985), but also reduce hatchability of eggs and chick survival (Shoffner et al. 1953; Cheng et al. 1985; Abplanalp et al. 1992). Inbreeding is therefore likely to be costly. Nevertheless, no study has investigated potential inbreeding avoidance mechanisms in the fowl. Some species of galliforms are able to recognise kin (Petrie et al. 1999) and also avoid close kin as mating partners (Bateson 1982), suggesting the possibility of kin discrimination as a potential inbreeding avoidance mechanism also in the fowl.

10 Sexual selection in the fowl The sexes follow the classic roles of competing males and choosy females. Males are larger and more ornamented than females, including having a larger comb (a fleshy head ornament, Johnsgard 1999). Both sexes are sexually promiscuous. Polyandry can be initiated by females (McBride et al. 1969; Ligon & Zwartjes 1995), but is mainly due to male coercion of females into copulation (McBride et al. 1969; Collias & Collias 1996; Etches 1996; Pizzari et al. 2002). Females can resist copulations directly, and are able to indirectly exert some control of copulation by uttering a distress call (Collias 1987) which attracts other males, and thus increases the likelihood that the copulation is disrupted (Pizzari 2001; Pizzari & Birkhead 2000). However, costs associated with copulation, like physical injuries (Pizzari 2001) and disrupted foraging, are likely to remain despite insemination may have been prevented.

The fowl has a bimodal daily copulation pattern with copulations occurring early in the morning and especially in the evening (McBride et al. 1969; Craig & Bhagwat 1974; Cheng & Burns 1988; Pizzari & Birkhead 2001) that can be explained by female daily oviposition pattern. Oviposition occurs in early and mid-half of the day (Christensen & Johnston 1977; Johnson 2000; Pizzari & Birkhead 2001). Copulations occurring few hours before and after oviposition have a decreased likelihood of resulting in fertilisation (Moore & Byerly 1942; Parker 1945; Johnston & Parker 1970; Christensen & Johnston 1977), selecting individuals to copulate outside the time of oviposition. The higher copulation peak in evenings can be explained by inseminations in evenings, being more likely to result in fertilisation than inseminations in the morning (Moore & Byerly 1942; Parker 1945; Christensen & Johnston 1977). Copulation in evenings may therefore be more efficient, selecting both sexes to preferentially copulate at this time of day.

Following insemination females store viable sperm in tens of thousands sperm storage tubules (SSTs) in the uterovaginal junction anterior to the vagina in the reproductive tract (Etches 1996). After insemination of a large ejaculate the production of fertile eggs declines rapidly after 10-14 days (Warren & Kilpatrick 1929; Etches 1996). Such prolonged sperm storage in addition to female polyandry creates potential for intense sperm competition (Pizzari et al. 2002). Male sperm supplies are limited and it takes 48 hours to replenish when exhausted (Parker et al. 1942). Males may therefore miss future mating opportunities if the amount of sperm inseminated into a female is increased, in attempt of improving probability of fertilisation. Male fowl are thus predicted to allocate sperm supplies differentially to be competitive in sperm competition and inseminate sperm to several females. Prolonged female sperm storage and polyandry also create the potential for cryptic female choice (Pizzari et al. 2002). One cryptic female choice mechanism is known in the fowl, whereby females eject ejaculates form subordinate males when these force females to copulate with them (Pizzari & Birkhead 2000). A study of domestic fowl detected a female bias in sperm utilisation after artificial insemination of males ejaculates (Birkhead et al. 2004). However, the cue and mechanism behind this bias remains unclear, nevertheless suggests the possibility of female influence of paternity occurring even later in the reproductive event, than differential sperm ejection.

11 Study populations Tovetorp In paper I-III, a population of an old Swedish game breed, ‘Gammal svensk dvärghöna’ (Gallus g. domesticus, Harrison 1987) kept at Tovetorp Zoological Research station (Stockholm University), was used. This population is random-bred under relaxed artificial selective pressures and birds marked with numbered leg-rings have been kept free-ranging in mixed-age, mixed-sex groups since the late 60’s. As a result, birds show strong similarities in morphology and behaviours to the red junglefowl (Schütz & Jensen 2001; Pizzari & Birkhead 2001). Götala In studies of responses to inbreeding, papers IV-V, a population of red junglefowl (Gallus gallus spp.) kept at SLU (Swedish University of Agricultural Sciences) in Skara, was used. This population originated from birds originally captured in Thailand and likely to be of the subspecies G. g. gallus or G. g. spadiceus (for further details, see Schütz & Jensen 2001). To know individuals’ relatedness, individually marked eggs from 18 pairs of sexually rested males and females for the first generation (used in paper IV-V) and 22 pairs for the second generation (used in paper V) were hatched in incubators. All chicks were marked at hatching with wing tags or neck labels. (Later were coloured leg-rings temporarily used to facilitate observations.) Siblings were randomly assigned to groups, resulting in socially familiar (i.e. that had been raised together) and unfamiliar individuals (i.e. that had not been raised together) both being full-sibs and unrelated individuals. Birds were kept in same-age, mixed-sex groups in indoor pens under standard housing condition according to Swedish welfare standards.

12 Some useful techniques In addition to the promiscuous nature, the fowl is a suitable species for studies of pre- and post-copulatory sexual selection for at least two reasons. Firstly, birds are easily habituated to people and handling, enabling an experimental approach. Secondly, the use of certain techniques facilitates studies of otherwise cryptic post-copulatory mechanisms. The combination of those two resulted in techniques that have been useful for this thesis: Controlled copulations Copulations under controlled conditions are set up by first isolate a male and a female (at least physically) from the rest of the group. The female is held, initially facing the male to facilitate familiarisation between them. After a set amount of time, the female is turned around and presented to the male in a soliciting position allowing the male to copulate with her. The technique allows natural copulations to occur (in comparison to artificial insemination), but with more control of number of copulations and insemination success compared to free-ranging copulations. Insemination success can be quantified by looking for traces of semen around the female cloaca (paper III-V), or by the use of video cameras recording the copulation focusing on the female cloaca (paper V). Further can insemination be prevented by fitting the female with a harness covering her cloaca (paper III), facilitating collection of ejaculates allocated to the female (paper II, IV-VI). Volume of collected ejaculates were measured with a pipette, and if sperm numbers later were to be counted (according to Bakst & Cecil 1997) ejaculates were stored in 10% formalin. Hydrolysis points on PVL From the poultry industry it is known that in freshly laid eggs, the number of sperm that reached an ovum during the period of time available for fertilisation (i.e. following ovulation when the ovum is in the body cavity and upper infundibulum, Olsen & Neher 1948; Etches 1996) can be quantified by counting the number of hydrolysis points made by live sperm on the outer periviteline layer (PVL, i.e. the layer around the yolk) around the blastodisc of the egg (Wishart 1987; 1997). The pattern of variation in sperm numbers on eggs laid over successive days following an insemination provides an accurate measure of the amount of sperm a female store in her sperm storages (Brillard 1993). Sperm reaches the ovum 48 hours after fertilisation, and amount of hydrolysis points found on PVL of eggs typically drop over successive days as sperm is lost from SSTs (Lodge et al. 1971; Froman et al. 2002). Since the number of hydrolysis points observed depends on the area of PVL observed, the magnifications of the microscopes used are likely to give different number of observed and counted sperm. In this thesis, two different microscopes have been used, explaining why sperm numbers counted in paper IV are higher than in paper V.

13 Results Male strategies As predicted from the lesser investment in offspring than females, males re-mate at higher rates than females (paper I), and copulate with both related and unrelated partners (paper IV). In addition, as predicted from their limited sperm supplies and faced sperm competition, males allocate sperm supplies differentially according to (i) earlier sperm investment in a female (paper I-II), (ii) variation in polyandry and own competitive ability (paper II) and (ii) female reproductive quality (paper II, IV).

Copulation rates and copulation history Male and female re-mating rates were studied in free-ranging groups of fowl with three different sex ratios (2:8, 4:6, 6:4, males to females) and over two time scales; cumulative time of exposure to the opposite sex following sexual rest (i.e. days), and time of day (i.e. mornings and evenings). In all sex ratios males adopted much higher re-mating rates than females and initiated more than 90% of all observed copulation attempts (n=3568). However, male re-mating rates were reduced after the first day after sexual rest (paper I, figure 1).

2,5

1,5

0,5 rate of copulations initated copulations of rate

0 1234 day Figure 1. Male and female re-mating rates in free-ranging groups, across days. Re- mating rates (number of copulations initiated by an individual, per hour observed) were sex-specific. Males (empty) initiated more copulations than females (filled) and initiated more copulations on the first day after sexual rest (day: F3,752=9.46, p≤0.0001). Females solicited fewer copulations over successive days (day: F3,748=59.46, p≤0.0001, for further details and statistics, see paper I). Columns represent means, bars represent standard error.

A reduction in male sexual interest with increased exposure to and sexual familiarity with a female can be explained by increased cumulative number of inseminations and/or reduced male sexual activity because of exhausted sperm supplies. Because only pre-copulatory behaviours were observed in this study, these two explanations can not be disentangled. However, in paper II, male sexual interest and also sperm allocation over successive copulations across females was quantified by Cornwallis and Pizzari, showing that males lost sexual interest in a female with increased number

14 of copulations with her, after controlling for sperm depletion. One male at the time was allowed to copulate with a female until he lost interest in her, whereby the male was presented a new female. Male propensity to copulate with a particular female declined with time, but was renewed by replacing the female with a new female. Similarly, sperm numbers in males’ ejaculates declined over successive copulations with a female, and even resulted in copulations without semen transfer at all. However, sperm numbers were restored when the male copulated with a new female, to once again decline over successive copulations (paper II, figure 2).

Figure 2. Relative sperm investment over successive copulations and across females. Relative sperm investment of male fowl declined over successive inseminations (circles) with the same female (circles in same colour) and was renewed by the presence of a new female. Males tend to invest more sperm in the first ejaculate with a new female than in the last ejaculate with the familiar female (for statistics and further details, see paper II, figure 3). Circles represent means, bars represent standard error.

This copulation pattern show two things, (i) that males allocate sperm differentially over successive copulations across females, and (ii) that the lack of sperm in ejaculates late in the copulation sequence with a female is not due to sperm depletion. Firstly, males allocate sperm reserves in a way saving sperm for future copulation opportunities. Sæther and co-workers (2001) showed a similar male behaviour in the sense that males did not re-mate with females they already had mated with. This suggests that males use sexual familiarity with a female as a cue to reduce investment in her to save sperm for future copulations (Wedell et al. 2002). Secondly, the observed copulations without any semen transfer (i.e. ‘aspermic’ copulations, also observed in other taxa: Parker et al. 1942; Dewsbury 1972; Adkins 1974; Birkhead et al. 1988; Birkhead 1991; Hunter et al. 1996; Westneat et al. 1998; Baker et al. 2001; Gronlund et al. 2002), have typically been explained by males being sexually inept or sperm depleted. However, the pattern of aspermic copulation observed in the fowl suggests that these copulations may have a function since performed by sexually mature males that were not sperm depleted. The effect aspermic copulations had on female polyandry therefore was investigated. In paper III, females were exposed to

15 aspermic copulations, simulated by fitting females temporarily with a harness and copulations occurring under controlled conditions. Females exposed to aspermic copulations responded in the same way as inseminated females, thus with reduced propensity to re-mate compared to non-mated females. The reduction in female re- mating propensity was therefore explained by mounting per se. The observed reduced female re-mating propensity can be explained by mounting manipulating females to perceive that they have received more sperm than they have. Exposure to aspermic copulations may in turn discourage females from re-mating, if copulations are costly to females. Female response to mounting lasted for about two days, long enough to translate into females storing fewer sperm from a new male (paper III, figure 3).

Days Figure 3. Aspermic copulations and sperm competition. Standardised number of hydrolysis points counted on the periviteline layer of eggs laid of females experimentally exposed to natural copulations (open circles), aspermic copulations (filled circles) or no exposure to copulations (diamonds), when later exposed to a new male free-ranging for two days. Fewer sperm reaches the eggs of females that were exposed to aspermic copulations than eggs produced by females exposed to any other experimental treatment, indicating that a reduction in female polyandry was translated into a short-tern reduction in sperm competition intensity (for statistics and further details, see paper III, figure 2). Symbols represent means, bars represent standard error.

Some days reduction in sperm competition (figure 3) may be enough time to improve a male’s chances of fertilisation, at least for an egg or two of a females clutch. In the fowl, no pre-copulatory tactic used by males to prevent female polyandry is known, except from males interrupting each others copulations (Pizzari 2001). Males copulating without semen could be a male strategy to reduce female re-mating propensity temporarily, and at the same time allow males to save sperm for future matings. Under what conditions and to what extent male fowl perform aspermic copulations (but from with described above and shown in figure 2) and also how taxonomically widespread it is, is not known. However, in some birds, males typically mate-guard females to prevent polyandry (e.g. Birkhead 1979), and in species that do not mate guard, high copulation frequencies are often observed (≥20 copulations/clutch/female), suggested to be an alternative strategy to mate guarding

16 (Birkhead et al. 1987). In species with high copulation frequencies and frequently observed ‘failing’ of semen transfer in copulations may therefore be interesting to further study to test this idea. In addition, inhibiting female polyandry by mounting may be cheaper than other observed specialised male copulatory traits and seminal products produced by males to reduce female promiscuity (e.g. Chapman et al. 1995).

Further investigating re-mating rates over time of day showed that males preferentially initiated copulations in evenings, when a copulation is more likely to result in fertilisation, compared to mornings (Moore & Byerly 1942; Parker 1945; Christensen & Johnston 1977). Males were also more rapidly attracted to a soliciting female and closer to females at this time of day (paper I). Timing copulations to this time may therefore increase probability of fertilisation (Birkhead & Møller 1992; e.g. Preston et al. 2003). However, sperm competition is likely to be particularly intense in evenings due increased mating propensity by all males at this time of day (paper I). This in turn may reduce the probability of fertilisation in evenings, selecting males to be able to allocate sperm supplies also according to the sperm competition faced.

Sperm allocation and rivalry High re-mating rates results in female fowl typically being polyandrous and an intense sperm competition (Pizzari et al. 2002). Under these conditions males are predicted to allocate sperm supplies differentially according to the risk and intensity of sperm competition (Parker 1998). In paper II, male perception of female polyandry was altered by housing males alone, with one or with three competitors (paper II). When allowed to copulate until lost sexual interest in a single female, males allocated their sperm supplies differentially both according to experimental treatment, but also according to their own competitive ability (i.e. social status). In the absence of any competitors, males minimised their sperm investment. This is a response predicted theoretically because the chance of fertilisation is higher than when facing sperm competition (Parker et al. 1997; Parker 1998; for review see Wedell et al. 2002). As the number of competitors increased from one to three, dominant males increased their sperm investment, but subdominant males maximised their investment in the presence of just one competitor. Dominant males have greater access to females and control over copulations (Cheng & Burns 1988; Pizzari 2001), and the presence of other males may represent an increased risk of another male inseminating the same female (but not necessarily an increased number of males inseminating this female). Subdominant males cannot prevent females from copulation with other males (Pizzari 2001) and the presence of other males is likely to result in more intense sperm competition, predicting males to reduce their sperm investment when competition increases above one competitor. The reduction in male sperm allocation with increased sperm competition intensity is theoretically predicted (Parker et al. 1996; Parker 1998; e.g. Simmons & Kvarnemo 1997; Pilastro et al. 2002) and may allow males to conserve sperm for future less competitive reproductive opportunities. Cues like number of rivals present and also own social status therefore provides information on the risk and intensity of sperm competition enabling males to allocate sperm strategically.

17 Number of competitors Figure 4. Differential sperm allocation and female polyandry. Dominant males (filled columns) increased relative sperm investment as the number of competitors increased, whereas subordinate males (open columns) decreased their sperm investment when the number of competitors increased above one (for statistics and further details, see paper II, figure 1). Columns represent means, bars represent standard error.

Sperm allocation and female quality With limited sperm supplies and variation in female quality, males should inseminate more sperm to females of higher quality (Parker 1983; 1998; Reinhold et al. 2002; e.g. Gage & Barnard 1996; Byrne & Rice 2006). In paper II it was shown that females with larger combs produced larger eggs with larger yolks, thus that female comb size signal maternal quality. Males used this cue for female quality and preferred females with larger combs both in free-ranging groups and in a two-choice situation. Both socially dominant and subordinate males also allocated more sperm to females with larger combs. However, dominant males biased their sperm allocation in favour of large-combed females more distinct, which in turn may bias paternity of high quality offspring in favour of dominant males.

Further, female quality can vary due to genetic relatedness with the male. This is because mating with relatives on one hand can be beneficial and increase an individual’s inclusive fitness, but on the other hand may reduce offspring fitness due to inbreeding depression (Parker 1979; 2006; Kokko & Ots 2006). When males were presented with females related and unrelated to the male, males’ probability to copulate with either of the females did not differ (paper IV). Males showed a tendency to initiate copulations faster with unrelated females, and also to allocate more sperm to sisters. Differentiation between related and unrelated females suggests that males are able to recognise kin. On the other hand, the effect of genetic relatedness was influenced by social familiarity, in other words whether females had been raised with the male or not, and further studies are needed to disentangle the two effects. However, males did not avoid inbreeding. In species with little paternal care, males are predicted to have higher inbreeding tolerance than females (for female response to

18 inbreeding, see below) and particularly high inbreeding tolerance if access to females is constrained. Both criteria were fulfilled in paper IV since males were only allowed to copulate with one female at the time, showing a predicted male response, also to inbreeding (Parker 1979; 2006; Kokko & Ots 2006).

To summarise, males have high re-mating rates and copulate with females independent of their relatedness with the male. Males also target fecund females and copulation at time of day increasing the potential reproductive value of a copulation. Males show plastic behaviours as a response to the selective pressures created by limited sperm supplies and sperm competition. Male differential sperm allocation is triggered by several cues, thus males allocate sperm reserves in a complex manner. The mechanism of male sperm allocation is still unknown, but it is clear that it regulates sperm numbers in an ejaculate within a short time scale, because from the last copulation with one female and to the first copulation with a novel female only about 10 minutes elapsed (paper II).

19 Female strategies Females’ greater investment in offspring than males’, and the high number of male- initiated copulations females were exposed to, resulted in flexible daily remating patterns to avoid intense sexual harassment, female resistance in copulations when the cumulative number of copulations increased (paper I) and when females were currently not laying (paper III, V). In addition, as predicted from females bearing a greater cost of an inbred offspring, females attempted to avoid inbreeding both before and after copulation (paper IV-V).

Responses to sexual harassment In free-ranging groups of fowl, females had their highest re-mating propensity when sperm storages were empty (i.e. after sexual rest), explaining female solicitation by ensuring sperm for fertilisation (paper I). Consistent with this idea, female re-mating propensity declined when the cumulative number of copulations increased (paper I, III, V, figure 5) and in addition, non-laying females (i.e. females that did not lay any eggs within at least 10 days following behavioural observations) with no need for sperm, solicited fewer copulations than laying females (paper I, III, V). The general pattern of female mating behaviours was that when female solicitation rate declined, female resistance in male-initiated copulations increased (paper I, III, V). Already in female-biased groups (2:8 males to females) were female resistance triggered, concluding that the optimal female mating rate is relatively low and much lower than males’ (paper I, figure 1). In the fowl, male-initiated re-mating rates increased due to increased number of males in a group, and not due to individual male re-mating rates increased. An exposure to higher re-mating rates for females in more male-biased sex ratios is intuitive and often shown (e.g. Holland & Rice 1999; Martin & Hosken 2004; Crudgington et al. 2005; Kraaijeveld et al. 2005; Le Galliard et al. 2005; Wigby & Chapman 2005; Head & Brooks 2006). Nevertheless, this variation in group sex ratio not only triggered female resistance in the fowl, but also shaped female daily re- mating patterns. In the most female-biased groups (2:8 males to females), both sexes initiated more copulations in evenings, the time of day inseminations are most likely to result in fertilisation (Moore & Byerly 1942; Parker 1945; Christensen & Johnston 1977). While males, independent of groups’ sex ratios, continued to initiate copulations mainly in evenings, females on the other hand shifted the time of solicitation from evenings to mornings when sex ratios became more male-biased (paper I, figure 5). This pattern of female daily re-mating can be explained by variation in sexual harassment intensity. In male-biased groups, females are not only exposed to more male-initiated mating, but males were also closer to females and more rapidly attracted to soliciting females, particularly in evenings. By the use of daily variation in male re-mating propensity and distribution patterns of populations, females plastically adjusted the timing of solicitation to the level of sexual harassment of a group. The high rate of male-initiated copulations and also male sexual coercion may constrain female pre-copulatory mate choice in evenings. Prolonged sperm storage may therefore enable females to reduce the number of copulations needed to both ensure enough sperm for fertilisation, but also allow flexibility in when during the day to initiate a copulation. Female mating behaviours may therefore give an

20 explanation for the observed copulation peak also in mornings in the fowl. In addition, this behavioural pattern may be an adaptive response if females retain pre-copulatory control of copulation and/or reduce costs associated with copulation by adopting it.

0,12

0,08

0,04 rate of female soliciitations female of rate

0 2:8 4:6 6:4 group sex ratio Figure 5. Female daily re-mating pattern over time of day and sex ratios. Females solicited more copulations (number of solicitations per female, over minutes observed) in the morning (empty columns) than in the evening (filled columns) in the most female-biased groups (i.e. 2:8 males to females), and reversed this trend in male-biased groups (i.e. 6:4 males to females), while there was no difference in solicitation rate in groups of the intermediate sex ratio (for statistics and further details, see paper I, figure 4). Columns represent means, bars represent standard error.

Inbreeding avoidance Contrary to males, females avoided inbreeding, and did so both before and after copulation (paper IV, V). In groups of one male, his two sisters and two females unrelated to the male, females related to him initiated fewer copulations than unrelated females (paper V). Related females also resisted more copulations from the focal male (paper V). The difference in related and unrelated females’ responses to the focal male confirms the suggestion from paper IV that the fowl discriminate between kin and non-kin, independent of social familiarity (females, paper V, males, H. Løvlie, unpublished data). Kin recognition therefore is based on a genetic cue and not on earlier social familiarity (Holmes & Sherman 1982; Holmes 1986). The evolution of a kin recognition mechanism independent of familiarity makes sense in the light of the promiscuous mating system of the species, where social familiarity, for example among offspring, does not necessarily translate into genetic relatedness (similarly shown in e.g. Hain & Neff 2006). A reliable cue used in kin recognition should be a direct predictor of genetic relatedness, for example individual variation in genes of the major histocompatibility complex (MHC, Klein 1986). The MHC is a highly polymorphic gene complex important in the vertebrate immune system (Hughes and Yeager 1998). Due to the high polymorphism of the MHC, MHC-similarity of two individuals is likely to co-vary with their genetic relatedness and may in turn enable kin recognition (Penn 2002; Zelano and Edwards 2002). Confirming the idea, variation in MHC has been implicated in kin discrimination in several vertebrate species, often mediated by olfactory cues (e.g. Brown and Eklund 1994; Olsen et al 1998; Penn and

21 Potts 1999; Rajakaruna et al. 2006). Studies of variation in MHC affecting mate choice in birds (von Schantz et al. 1996; Freeman-Gallant et al. 2003; Ekblom et al. 2004; Richardson et al. 2004; Bonneaud et al 2006) and also that birds respond to olfactory cues (for reviewed see Zelano and Edwards 2002; domestic fowl, Jones & Roper 1997). Together, this offers the possibility that olfaction may be more important in the fowl than earlier assumed and also suggests a link between olfaction, MHC and kin recognition.

Female red junglefowl also avoided inbreeding after copulation. After copulations in free-ranging groups (paper V), more hydrolysis points were found on eggs laid by females unrelated to the focal male, compared to counts on eggs produced by the male’s sisters (figure 6a). A similar pattern in bias of unrelated males’ sperm was found also in paper IV. However, in paper IV female pre-copulatory behaviours was controlled for (i.e. copulations occurred under controlled conditions). This suggests that the pattern of female sperm utilisation found after free-ranging copulations may not solely be due to female pre-copulatory behaviours despite the intuitive explanation it offers. Replicating the experiment from paper IV confirmed the sperm pattern, concluding that females utilised less sperm from a related male independent of female pre-copulatory behaviours, and neither explained by males’ differential sperm allocation and allocation of more sperm to unrelated females (paper IV), nor by female differential ejaculate ejection (paper V). This suggests another female-driven cryptic mechanism other than differential sperm ejection to occur. The bias in sperm utilisation explained by genetic relatedness was heavily reduced when ejaculates of males were inseminated artificially. This suggests that the mechanism causing the bias is either occurring or triggered early in the female reproductive tract, and/or is triggered by a pre-copulatory cue. After insemination, typically only a small proportion of an ejaculate is stored in SSTs (Brillard & Artoine 1990; Bakst et al. 1994). Potential female-driven mechanisms biasing sperm utilisation could therefore be by affecting sperm transport to SSTs, for example by a vaginal barrier selecting against certain males’ sperm, however could also occur by biasing transport from sperm storages to the infundibulum where fertilisation occurs (Olsen & Neher 1948; Etches 1996).

To summarise, females counteract male reproductive strategies by adopting flexible pre-copulatory behaviours explained by variation in - and avoidance of - intense sexual harassment. Females also avoid inbreeding both before and after copulations, the latter by a cryptic female choice still not known and encouraging further investigation. The prolonged female sperm storage that generates the potential for sperm competition in the species may also be the trait enabling the above described female behaviours. Reducing the number of inseminations needed to ensure fertilisation, in combination with avoidance of solicitation at the daily peak in sexual harassment intensity may make it possible for females to seize considerable more control of mate choice and perhaps reduce costs of high re-mating rates (paper I). In addition, female sperm storage enables a prolonged time for female influence on post-copulatory mechanisms (paper IV-V) likely to reduce risk of inbreeding.

22 60

mean sperm number sperm mean 20

0 0246810

15 C

mean sperm number 5

0 0246810 days Days Figure 6. Patterns of female sperm utilisation. (A) After free-ranging copulation with the focal male did unrelated females (empty points, dashed line) retain more sperm (number of sperm counted on the periviteline layer of eggs laid after insemination) in the first days compared to related females (filled points, solid line). (B) Females retained more sperm after copulations with unrelated (empty points, dashed line) compared to related males (filled points, solid line). (C) Most variation in female sperm utilisation earlier explained by relatedness disappeared after artificial insemination (unrelated: empty points, dashed line; related females: filled points, solid line; for statistics and further details, see paper V, figure 1 and 3a,b). Points represent means, bars represent standard error.

23 Discussion When interests differ Sexual reproduction creates the possibility for one sex to manipulate the other, that in turn triggers counteracting responses and creates a potential for sexual conflict over mating and fertilisation (Parker 1979; 2006). In the fowl the sexes’ skewed investment in offspring results in sex-specific reproductive strategies and reproductive interests that may differ for mating rates (paper I), female sperm limitation (paper I, II, III) and inbreeding (paper IV, V).

Male fowl are selected to re-mate at higher rates than females, and due to sexual dimorphism males are able to force females to copulate (McBride et al. 1969; Collias & Collias 1996; Etches 1996; Pizzari et al. 2002). Already in groups with highly female-biased sex ratios (2:8 males to females) males imposed high enough mating rates on females to trigger female resistance in copulations (paper I). However, female costs of high re-mating rates were not directly measured. Although, female counteracting responses triggered by male behaviours suggest the occurrence of intersexual conflict (Arnqvist & Rowe 2005). Confirming this idea, non-laying females resist more than laying females when exposed to male-initiating copulations. “Non- laying” was here defined as when a female was not laying any eggs in at least 10 days following observations. Non-laying females therefore did not gain any benefits from copulation. The observed resistance is therefore a measure of female unwillingness to engage in copulations, suggesting there to be costs for females associating with copulations, and further a sexual conflict over re-mating.

Few studies have focused on the reproductive value of a copulation changing over time, but rather have focused on sexual conflict over mating rates (but see Parker 1970b). In groups with a male-biased sex ratio (6:4 males to females) females were triggered to re-mate in mornings instead of evenings due to the intense sexual harassment in evenings (paper I, figure 5). Copulations in mornings, compared to evenings, are known to be less likely to result in fertilisation (Moore & Byerly 1942; Parker 1945; Christensen & Johnston 1977), which could create a sexual conflict over female sperm limitation (Wedell et al. 2002). Similarly, male extreme sperm allocation and performance of aspermic copulations could result in female sperm limitation (Wedell et al. 2002). Nevertheless, infertility is not very likely to be a cost females bear from copulating in mornings nor through exposure to aspermic copulations. This is because the long female sperm storage may allow females in male-biased groups (paper I) to solicit copulations in mornings ensuring storage of sperm from the preferred males, and thus reduce the need for involving in copulations in evenings when sexual harassment is higher. For females exposed to aspermic copulations (paper III), the prolonged sperm storage may reduce the risk of sperm depletion for the few days polyandry was inhibited. The cost that triggers female counteracting responses is therefore more likely to be the cost of engaging in copulations per se, or from being sexually harassed and not sperm limitation. Alternatively, sperm limitation has been a cost to females earlier in the arms race between males and females over control of

24 paternity and in turn already triggered polyandry (Pizzari 2002) and prolonged sperm storage as the counteracting responses.

Genetic relatedness between partners also created a potential for conflict. Males copulate indiscriminately according to partner relatedness, while females attempted to avoid inbreeding both before and after copulation (paper IV, V). As a result, males sexually coerce sisters to copulate, triggering females resisted directly (paper V). The occurrence of also a post-copulatory inbreeding avoidance mechanism suggests that female pre-copulatory resistance in copulation is circumvented and males are able to constrain female pre-copulatory mate choice. A cryptic female choice then may enable females to counteract male coercion and reduce the risk of inbreeding. This in turn may select for males inseminating more sperm into sisters to overcome female selection against related males’ sperm, continuing the conflict over fertilisation (paper IV). Counteracting responses may thus continue as an arms race between the sexes, where both sexes is an important part of the selective environment of the other sex (Moore & Pizzari 2005; e.g. paper VI), counteracting responses and sexual conflict therefore needs to considered in studies to fully understand the evolution of reproductive strategies.

Behaviours as a predictor of post-copulatory processes This thesis also points out the fact that copulation can be a poor predictor of fertilisation. Typically (particularly in studies of wild species), studies of variation in paternity observe social pairing, or copulation success, and assign offspring paternity to individuals in the population (e.g. birds, Brooker et al. 1990; Blomqvist et al 2002; Forester et al 2003; Hansson et al. 2004). By adopting such an approach we may get detailed information on the outcome of a reproductive event, but it leaves a gap between the observed social pairing and paternity. In other words, we may gain no or very limited information on the mechanisms affecting paternity. In the fowl, after a successful copulation there is about two weeks of potential competition and choice that can affect paternity (Parker et al. 1942; Etches 1996; Pizzari et al. 2002). But by the use of an experimental approach and the possibility to quantify male ejaculate allocation and female sperm utilisation, it has been possible to study and disentangle pre- and post-copulatory effects and mechanism that in other species are impossible. And as a result this thesis has contributed to filling some of this gap between copulation and paternity. The thesis shows that male sperm allocation in the fowl is triggered by several different cues (e.g. sexual familiarity with a female, female quality, male social status, female polyandry level, paper II; genetic relatedness, paper IV) resulting in complex predictions about male sperm allocation pattern and further of paternity. Similarly complex, female selection against males after mating is also triggered by several cues (e.g. male social status, Pizzari & Birkhead 2000; genetic relatedness and social familiarity, paper V) and by several types of cryptic female choice (paper V). Further, the thesis shows that pre- and post-copulatory behaviours can mirror each other, for example when females avoid inbreeding both before and after copulations (paper IV-V). However, pre- and post-copulatory behaviours may in other cases not coincide, which is true with aspermic copulations where behaviourally

25 successful copulations do not result in semen transfer (paper II-III). The later could lead to misleading predictions of paternity made from an observed mating (Garcia- Gonzalez 2004). Further complicating predictions of paternity is that just because one mechanism is known to occur, there may still be other mechanisms taking place, even within the same individual and triggered by the same cue. This is the case with both pre- and post-copulatory inbreeding avoidance in female fowl, and also the occurense of several cryptic female choice mechanisms (paper V). As a result, behavioural observations therefore may tell little or nothing about the post-copulatory mechanisms affecting paternity, but only shows the pre-copulatory outcome of male and female reproductive strategies. To further increase our understanding of post-copulatory mechanisms more studies, enabling the teasing apart of pre- and post-copulatory male and female-driven strategies, are therefore needed.

Conclusions To conclude, this thesis shows that skewed investment in offspring by the sexes results in sex-specific reproductive strategies in the fowl. Males have higher mating rates than females and also copulate indiscriminately according to partner relatedness. This in turn triggers female counteracting responses and flexible re-mating behaviour which may reduce costs of high mating rates and inbred offspring, the later by adopting both pre- and post-copulatory inbreeding avoidance. Female fowl are polyandrous, creating the potential for sperm competition and cryptic female choice. Males respond to the selection pressure caused by sperm competition and limited sperm supplies by allocating sperm supplies differentially in a complex and plastic manner. The patterns of male differential sperm allocation are predicted to – at least theoretically – improve the reproductive return of an insemination and increase the probability of fertilisation. Females exert post-copulatory strategies likely to enable females to retain control of paternity, by both ejecting ejaculates and biasing sperm utilisation even at a later stage in a reproductive event. Further more, the sexes’ different reproductive strategies create a potential for sexual conflict over re-mating rates and inbreeding. Through the fruitful mix of an experimental approach together with behavioural observations and the possibility to quantify physiological processes, the results of this thesis expose cues and mechanisms affecting pre- and post-copulatory processes and determine whether they are male or female driven. As a result, by combining the possible ways male and female pre- and post-copulatory strategies can affect paternity, it becomes challenging – if at all possible – to predict paternity by observing only behaviours. Nevertheless, the fowl is now one of the most well-studied species regarding pre- and post-copulatory sexual selection, contributing significantly to our general understanding of sexual selection and mechanisms affecting paternity, and the results obtained from this thesis will only enhance that position.

26 References Abplanalp, H., K. Sato, D. Napolitano, & J. Reid. 1992. Reproductive performance of inbred congenic leghorns carrying different haplotypes for the major histocompatibility complex. Poultry Sci. 71:9-17. Adkins, E. K. 1974. Electrical recording of copulation in quail. Physiol. Behav. 13:475-477. Andersson, M. 1994. Sexual selection. Princeton Univ. Press, Princeton. Arnqvist, G. & T. Nilsson. 2000. The evolution of polyandry: multiple mating and female fitness in insects. Anim. Behav. 60:145-160. Arnqvist, G., & L. Rowe. 2005. Sexual conflict. Princeton Univ. Press, Princeton. Baker, R. H., R. I. S. Ashwell, T. A. Richards, K. Fowler, T. Chapman, & A. Pomiankowski. 2001. Effects of multiple mating and male eye span on female reproductive output in the stalk-eyed fly, Cyrtodiopsis dalmanni. Behav. Ecol. 12:732-739. Bakst, M. R., & H. C. Cecil. 1997. Techniques for semen evaluation, semen storage, and fertility determination. 4. Sperm motility and metabolism VII. In vitro sperm-egg interaction assay utilising inner perivitelline layer from laid chicken eggs. Poultry Science Association, Savoy, Illinois. Bakst, M. R., G. J. Wishart, & J. P. Brillard. 1994. Oviducal sperm selection, transport and storage in poultry. Pp. 117-143. Poultry Sci. Rev. Banks, E. M. 1956. Social organisation in red jungle fowl hens (Gallus gallus subsp.). Ecology 37:239-248. Bateman, A. J. 1948. Intra-sexual selection in Drosophila. Heredity 2:349-368. Bateson, P. 1982. Preferences for cousins in Japanese quail. Nature 295:236-237. Bensch, S., D. Hasselquist, & T. von Schantz. 1994. Genetic similarity between parents predicts hatching failure: nonincestuous inbreeding in the great reed warbler? Evolution 48:317-326. Birkhead, T. R. 1979. Mate guarding in the magpie Pica pica. Anim. Behav. 27:866-874. Birkhead, T. R. 1991. Sperm depletion in the Bengalese finch Lonchura striata. Behav. Ecol. 2:267-275. Birkhead, T. R. 1998. Cryptic female choice: criteria for establishing female sperm choice. Evolution 52:1212-1218. Birkhead, T. R, and A. P. Møller. 1992. Sperm Competition in Birds. Evolutionary Causes and Consequences. Academic Press, London. Birkhead, T. R., L. Atkin, & A. P. Møller. 1987. Copulation behaviour in birds. Behaviour 101:101-138. Birkhead, T. R., N. Chaline, J. D. Biggins, T. Burke, & T. Pizzari. 2004. Nontransitivity of paternity in a bird. Evolution 58:416-420. Birkhead, T. R., & A. P. Møller. 1992. Sperm competition in birds: Evolutionary causes and consequences. Academic Press, London. Birkhead, T. R., & A. P. Møller. 1998. Sperm competition and sexual selection. Academic Press, London. Birkhead, T. R., J. E. Pellatt, & F. M. Hunter. 1988. Extra-pair copulation and sperm competition in the zebra finch. Nature 334:60-62. Birkhead, T. R., & T. Pizzari. 2002. Postcopulatory sexual selection. Nature Rev. Gen. 3:262- 273. Blomqvist, D., M. Andersson, C. Küpper, I. C. Cuthill, J. Kis, R. B. Lanctot, B. K. Sandercock, T. Székely, J. Wallander, & B. Kempenaers. 2002. Genetic similarity between males and extra-pair parentage in three species of shorebirds. Nature 419:613-615. Bonneaud, C., O. Chastel, P. Federici, H. Westerdahl, & G. Sorci. 2006. Complex Mhc-based mate choice in wild passerine. Proc R. Soc. Lond. B 273:1111-1116.

27 Bretman, A., N. Wedell, & T. Tregenza. 2004. Molecular evidence of post-copulatory inbreeding avoidance in the field cricket Gryllus bimaculatus. Proc. R. Soc. Lond. B 271:159-164. Brillard, J. 1993. Sperm storage and transport following natural mating and artificial insemination. Poultry Sci. 72:923-928. Brillard, J., & H. Antoine. 1990. Storage of sperm in the uterovaginal junction and it's incidence on the numbers of spermatozoa present in the periviteline layer of hens' eggs. Brit. Poultry. Sci. 31:635-644. Brooker, M. G., I. Rowley, M. Adams, & P. R. Baverstock. 1990. Promiscuity: an inbreeding avoidance mechanism in a socially monogamous species? Behav. Ecol. Sociobiol. 26:191- 199. Brown, J. L., & A. Eklund. 1994. Kin recognition and the major histocompatibility complex: an integrated review. Am. Nat. 143:435-461. Byrne, P. G., & W. R. Rice. 2006. Evidence for adaptive male mate choice in the fruit fly Drosophila melanogaster. Proc. R. Soc. Lond. B 273:917-922. Chapman, T., L. F. Liddle, J. M. Kalb, M. F. Wolfner, & L. Partridge. 1995. Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature 373:241-244. Charlesworth, D., & B. Charlesworth. 1987. Inbreeding depression and its evolutionary consequences. Ann. Rev. Ecol. Syst. 18:237-268. Cheng, K. M., & J. T. Burns. 1988. Dominance relationship and mating behavior of domestic cocks - a model to study mate-guarding and sperm competition in birds. Condor 90:697-704. Cheng, K. M., J. T. Burns, & R. N. Shoffner. 1985. Mating behaviour and fertility in domestic chickens. 1. Inbreeding. Appl. Anim. Behav. Sci. 13:371-381. Christensen, V. L., & N. P. Johnston. 1977. Effect of time of day of insemination and the position of the egg in the oviduct on the fertility of turkeys. Poultry Sci. 56 Clark, G. F., & A. Dell. 2006. Molecular models for murine sperm-egg binding. J. Biol. Chem. 281:13853-13856. Clutton-Brock, T. H., & G. A. Parker. 1995. Sexual coercion in animal societies. Anim. Behav. 49:1345-1365. Collias, N., E., E. Collias, C., D. Hunsaker, & L. Minning. 1966. Locality fixation, mobility and social organization within an unconfined population of red jungle fowl. Anim. Behav. 14:550-559. Collias, N. E. 1987. The vocal repertoire of the red junglefowl: A spectrographic classification and the code of communication. Condor 89:510-524. Collias, N. E., & E. C. Collias. 1996. Social organization of a red junglefowl, Gallus gallus, population related to evolutionary theory. Anim. Behav. 51:1337-1354. Cook, P. A., & N. Wedell. 1999. Non-fertile sperm delay female remating. Nature 397:486. Cornwallis, C. K., & T. R. Birkhead. 2006. Social status and availability of females determine patterns of sperm allocation in the fowl. Evolution 60:1486-1493. Craig, J. V., & R. A. Bahruth. 1965. Inbreeding and social dominance ability in chickens. Anim. Behav. 13:109-113. Craig, J. V., & A. L. Bhagwat. 1974. Agonistic and mating behaviour of adult chickens modified by social and physical environments. Appl. Anim. Ethol. 1:57-65. Crudgington, H. S., A. P. Beckerman, L. Brustle, K. Green, & R. R. Snook. 2005. Experimental removal and elevation of sexual selection: Does sexual selection generate manipulative males and resistant females? Am. Nat. 165: S72-S87. Darwin, C. 1871. The descent of man and selection in relation to sex. John Murray, London. Dewsbury, D. A. 1972. Patterns of copulatory behaviour in male mammals. Quart. Rev. Biol. 47:1-33. Dewsbury, D. A. 1982. Ejaculate cost and male choice. Am. Nat. 119:601-610.

28 Eberhard, W. G. 1996. Female control: sexual selection by cryptic female choice. Princeton Univ. Press, Princeton. Ekblom, R., S. A. Sæther, M. Grahn, P. Fiske, A. Kålås, & J. Höglund. 2004. Major histocompatibility complex variation and mate choice in a lekking bird, the great snipe (Gallinago media). Mol. Ecol. 13:3821-3828. Emlen, S. T., & L. W. Oring. 1977. Ecology, sexual selection and the evolution of mating systems. Science 197:215-223. Etches, R. J. 1996. Reproduction in poultry. CAB International, Oxford. Evans, J. P., L. Zane, S. Francescato, & A. Pilastro. 2003. Directional postcopulatory sexual selection revealed by artificial insemination. Nature 421:360-363. Facon, B., V. Ravigné, J. Goudet. 2006. Experimental evidence of inbreeding avoidance in the hermaphroditic snail Physa acuta. Evol. Ecol. 20:395-406. Fisher, R. A. 1930. The genetical theory of natural selection. Clarendon Press, Oxford. Foerster, K., K. Delhey, A. Johnsen, J. T. Lifjeld, & B. Kempenaers. 2003. Females increase offspring heterozygosity and fitness through extra-pair matings. Nature 425:714-717. Freeman-Gallant, C. R., M. Meguerdichian, N. T. Wheelwright, & S. V. Sollecito. 2003. Social pairing and female mating fidelity predicted by restriction fragment length polymorphism similarity at the major histocompatibility complex in a songbird. Mol. Ecol. 12:3077-3083. Froman, D. P., T. Pizzari, A. J. Feltmann, H. Castillo-Juarez, & T. R. Birkhead. 2002. Sperm mobility: mechanisms of fertilizing efficiency, genetic variation and phenotypic relationship with male status in the domestic fowl, Gallus gallus domesticus. Proc. R. Soc. Lond. B 269:607-612. Fumihito, A., T. Miyake, S. I. Sumi, M. Takada, S. Ohno, & N. Kondo. 1994. One subspecies of the red junglefowl (Gallus gallus gallus) suffices as the matriarchic ancestor of all domestic breeds. Proc. Natl. Acad. Sci. USA 91:12505-12509. Fumihito, A., T. Miyake, M. Takada, R. Shingu, T. Endo, T. Gojobori, N. Kondo, & S. Ohno. 1996. Monophyletic origin and unique dispersal patterns of domestic fowls. Proc. Natl. Acad. Sci. USA 93:6792-6795. Gage, A. R., & C. J. Barnard. 1996. Male crickets increase sperm number in relation to competition and female size. Behav. Ecol. Sociobiol. 38:349-353. Gage, M. J. G. 1992. Removal of rival sperm during copulation in a beetle, Tenebrio molitor. Anim. Behav. 44:587-589. Garcia-Gonzalez, F. 2004. Infertile matings and sperm competition: The effect of “nonsperm representation” on intraspecific variation in sperm precedence patterns. Am. Nat. 164:457- 472. Gems, D., & D. L. Riddle. 1996. Longevity in Caenorhabditis elegans reduced by mating but not gamete production. Nature 379:723-735. Gerlach, G., & N. Lysiak. 2006. Kin recognition and inbreeding avoidance in zebrafish, Danio rereio, is based on phenotype matching. Anim. Behav. 71:1371-1377. Gronlund, C. J., M. D. Deangelis, S. Pruett-Jones, P. S. Ward, & J. A. Coyne. 2002. Mate grasping in Drosophila pegasa. Behaviour 139:545-572. Haig, D. 1999. Asymmetric relations: Internal conflicts and the horror of incest. Evol. Human Behav. 20:83-98. Hain, T. J. A., & B. D. Neff. 2006. Promiscuity drives self-referent kin recognition. Curr. Biol. 16:1807-1811. Hamilton, W. D. 1964. The genetic evolution of social behavior. J. Theor. Biol. 7:1-16 Hamilton, W. D. & M. Zuk. 1982. Heritable true fitness and bright birds: a role for parasites? Science 218:384-387. Hansson, B., D. Hasselquist, & S. Bensch. 2004. Do female great reed warblers seek extra-pair fertilizations to avoid inbreeding? Proc. Roy. Soc. Lond. B 271:S290-S292.

29 Harrisson, B. 1987. Den svenska dvärghönan II. Sven. Rasfjäd. Tidskr. 1:12-14. Head, M. L., & R. Brooks. 2006. Sexual coercion and the opportunity of sexual selection in guppies. Anim. Behav. 71:515-522. Holland, B., & W. R. Rice. 1999. Experimental removal of sexual selection reverses intersexual antagonistic coevolution and removes reproductive load. Proc. Natl. Acad. Sci. USA 96:5083-5088. Holmes, W. G. 1986. Kin recognition by phenotype matching in female belding ground- squirrels. Anim. Behav. 34:38-47. Holmes, W. G., & P. W. Sherman. 1982. The ontogeny of kin recognition in 2 species of ground- squirrels. Am. Zool. 22:491-517. Hughes, A. L., & M. Yeager. 1998. Natural selection at major histocompatibility complex loci of vertebrates. Annu. Rev. Genet. 32:415-435. Hunter, F. M., L. S. Davis, & G. D. Miller. 1996. Sperm transfer in the Adelie penquin. Condor 98:410-413. Jennions, M. D., & M. Petrie. 2000. Why do females mate multiply? A review of the genetic benefits. Biol. Rev. Camb. Philos. Soc. 75:21-64. Johnsgard, P. A. 1999. Pheasants of the world. Biology and natural history. Swan Hill Press, Shrewsbury. Johnson, A. L. 2000. Reproduction in the female. Pp. 569-596 in P. D. Sturkie, ed. Avian Physiology. London, Academic Press. Johnston, N. P., & J. E. Parker. 1970. Effect of time of oviposition in relation to insemination on fertility in chicken hens. Poultry Sci. 49:325-327. Jones, R. B., & T. R. Roper. 1997. Olfaction in the domestic fowl: a critical review. Phys. Behav. 62:1009-1018. Keller, L. F., & D. M. Waller. 2002. Inbreeding effects in wild populations. Trends Ecol. Evol. 17:230-241. Klein, J. 1986. Natural history of the major histocompatibility complex. John Wiley and sons, New York. Kokko, H., R. Brooks, J. M. McNamara, & A. I. Houston. 2002. The sexual selection continuum. Proc. R. Soc. Lond. B 269:1331-1340. Kokko, H., & I. Ots. 2006. When not to avoid inbreeding. Evolution 60:467-475. Kraaijeveld, K., B. I. Katsoyannos, M. Stavrinides, N. A. Kouloussis, & T. Chapman. 2005. Mating in wild females of the Mediterranean fruit fly, Ceratitis capitata. Anim. Behav. 69:771-776. Le Galliard, J-F., P. S. Fitze, R. Ferriere, & J. Clobert. 2005. Sex ratio bias, male aggression, and population collapse in lizards. Proc. Natl. Acad. Sci. USA 102: 18231-18236. Lehmann, L., and N. Perrin. 2002. Altruism, dispersal, and phenotype-matching kin recognition. Am. Nat. 159:451-468. Ligon, J. D., & P. W. Zwartjes. 1995. Ornate plumage of male red jungle fowl does not influence mate choice by females. Anim. Behav. 49:117-125. Lodge, J. R., N. S. Fechheimer, & R. G. Jaap. 1971. The relationship of in vivo sperm storage interval to fertility and embryonic survival in the chicken. Biol. Reprod. 5:252-257. Martin, O. Y., & D. J. Hosken, 2004. Reproductive consequences of population divergence through sexual conflict. Curr. Ciol. 14:906-910. Martin, O. Y., D. J. Hosken, & P. I. Ward. 2004. Post-copulatory sexual selection and female fitness in Scathophaga sterciraria. Proc. R. Soc. Lond. B 271:353-359. Mays, H. L., & G. E. Hill. 2004. Choosing mates: good genes versus genes that are a good fit. Trends Ecol. Evol. 19:554-559. McBride, G., I. P. Parer, & F. Foenander. 1969. The social organisation and behaviour of the feral domestic fowl. Anim. Behav. Mono. 2:125-181.

30 Moore, O. K., & T. C. Byerly. 1942. Relation of time of insemination to percent fertility. Poultry Sci. 21:253-255. Moore, A. J., & T. Pizzari. 2005. Quantitative genetic models of sexual conflict based on interacting phenotypes. Am. Nat. 165:S88-S97. Nakatsuru, K., & D. L. Kramer, 1982. Is sperm cheap? Limited male fertility and female choice in the lemon tetra (Pisces, Characidae). Science 216:753-755. Neff, B. D., & T. E. Pitcher. 2005. Genetic quality and sexual selection: an integrated framework for good genes and compatible genes. Mol. Ecol. 14:19-38. Olsen, M. W., & B. H. Neher. 1948. The site of fertilization in the domestic fowl. J. Exp. Zool. 109:355-366. Olsson, M., R. Shine, T. Madsen, A. Gullberg, & H. Tegelstrom. 1996. Sperm selection by females. Nature 383:585-585. Olsson, M., T. Madsen, & R. Shine. 1997. Is sperm really so cheap? Costs of reproduction in male adders, Vipera berus. Proc. R. Soc. Lond. B 264:455-459. Parker, G. A. 1970a. Sperm competition and its evolutionary consequences in the insects. Biol. Rev. 45:525-567. Parker, G. A. 1970b. The reproductive behaviour and the nature of sexual selection in Scatophaga stercoraria L. (Diptera: Scatophagidae) V. The female’s behaviour at the oviposition site. Behaviour 37:140–168. Parker, G. A. 1979. Sexual selection and sexual conflict. Pp. 123-166 in M. S. Blum & N. A. Blum, eds. Sexual selection and reproductive competition in insects. Academic Press, New York. Parker, G. A. 1982. Why are the so many tiny sperm? Sperm competition and the maintenance of two sexes. J. Theor. Biol. 96:281-294. Parker, G. A. 1983. Mate quality and mating decisions. Pp. 141-166 in P. Bateson, ed. Mate choice. Cambridge Univ. Press, New York. Parker, G. A. 1998. Sperm competition and the evolution of ejaculates: Towards a theory base. Pp. 3-54 in T. R. Birkhead & A. P. Møller, ed. Sperm Competition and Sexual Selection. Academic Press, London. Parker, G. A. 2006. Sexual conflict over mating and fertilization: an overview. Philos. T. Roy. Soc. B. 361:235-259. Parker, G. A., M. A. Ball, P. Stockley, & M. J. G. Gage. 1996. Sperm competition games: individual assessment of sperm competition intensity by group spawners. Proc. R. Soc. Lond. B 263:1291-1297. Parker, G. A., M. A. Ball, P. Stockley, & M. J. G. Gage. 1997. Sperm competition games: a prospective analysis of risk assessment. Proc. Roy. Soc. Lond. B 254:1793-1802. Parker, J. E. 1945. Relation of time of day of artificial insemination to fertility and hatchability of hen's eggs. Poultry Sci. 24:314-317. Parker, J. E., F. F. McKenzie, & H. L. Kempster. 1942. Fertility in the male domestic fowl. Miss. Agric. Exp. Stn. Res. Bull. 347:1-50. Penn, D. J. 2002. The scent of genetic compatibility: Sexual selection and the major histocompatibility complex. Ethology 108:1-21. Penn, D. J., & W. K. Potts. 1999. The evolution of mating preferences and major histocompatibility complex genes. Am. Nat. 153:145-164. Petrie, M., A. Krupa, & T. Burke. 1999. Peacocks lek with relatives even in the absence of social and environmental cues. Nature 401:155-157. Pilastro, A., M. Scaggiante, & M. B. Rasotto. 2002. Individual adjustment of sperm expenditure accords with sperm competition theory. Proc. Natl. Acad. Sci. USA 99:9913-9915. Pizzari, T. 2001. Indirect partner choice through manipulation of male behaviour by female fowl, Gallus gallus domesticus. Proc. R. Soc. Lond. B 268:181-186.

31 Pizzari, T. 2002. Sperm allocation, the Coolidge effect and female polyandry. Trends Ecol. Evol. 17:456-456. Pizzari, T. 2003. Food, vigilance, and sperm: the role of male direct benefits in the evolution of female preference in a polygamous bird. Behav. Ecol. 14:593-601. Pizzari, T., & T. R. Birkhead. 2000. Female feral fowl eject sperm of subdominant males. Nature 405:787-789. Pizzari, T., & T. R. Birkhead. 2001. For whom does the hen cackle? The function of postoviposition cackling. Anim. Behav. 61:601-607. Pizzari, T., D. P. Froman, & T. R. Birkhead. 2002. Pre- and post-insemination episodes of sexual selection in the fowl, Gallus g. domesticus. Heredity 88:112-116. Preston, B. T., I. R. Stevenson, P. J.M., & K. Wilson. 2001. Dominant rams lose out by sperm depletion - A waning success in siring counters a ram’s high score in competition for ewes. Nature 409:681-682. Preston, B. T., I. R. Stevenson, & K. Wilson. 2003. Soay rams target reproductive activity towards promiscuous females’ optimal insemination period. Proc. R. Soc. Lond. B 270:2073- 2078. Rajakaruna, R. S., J. A. Brown, K. H. Kaukinene, & K. M. Miller. 2006. Major histocompatibility complex and kin discrimination in Atlantic salmon and brook trout. Mol. Ecol. 15:4569-4575. Reinhold, K. 2002. Modelling the evolution of female choice strategies under inbreeding conditions. Genetica 116:189-195. Reinhold, K., J. Kurtz, & L. Engqvist. 2002. Cryptic male choice: sperm allocation strategies when female quality varies. J. Evol. Biol.15:201-209. Richardson, D. S. J., J. Komdeur, & T. Burke. 2004. Inbreeding in the Seychelles warbler: Environment-dependent maternal effects. Evolution 58:2037-2048. Schaus, J. M., & S. K. Sakaluk. 2001. Ejaculate expenditures of male crickets in response to varying risk and intensity of sperm competition: not all species play games. Behav. Ecol. 12:740-745. Schütz, K. E., & P. Jensen. 2001. Effects of resource allocation on behavioural strategies: A comparison of red junglefowl (Gallus gallus) and two domesticated breeds of poultry. Ethology 107:753-765. Shine, R., M. M. Olsson, & R. T. Mason. 2000. Chastity belts in gartersnakes: the functional significance of mating plugs. Biol. J. Linnean Soc. 70:377-390. Shoffner, R. N., H. J. Sloan, L. M. Winters, T. H. Canfield, & A. M. Pilkey. 1953. Development and performance of inbred lines of chicken. Uni. Minn. Agric. Exp. Stn. Tech. Bull. 207:1- 34. Simmons, L. W. 2005. The evolution of polyandry: Sperm competition, sperm selection, and offspring viability. Ann. Rev. Ecol. Syst. 36:125-146. Simmons, L. W., M. Beveridge, N. Wedell, & T. Tregenza. 2006. Postcopulatory inbreeding avoidance by female crickets only revealed by molecular markers. Mol. Ecol. 15:3817-3824. Simmons, L. W., & C. Kvarnemo. 1997. Ejaculate expenditure by male bushcrickets decreases with sperm competition intensity. Proc. R. Soc. Lond. B 264:1203-1208. Snow, L. S. E., & M. C. B. Andrade. 2005. Multiple sperm storage organs facilitate female control of paternity. Proc. R. Soc. Lond. B 272:1139-1144. Stutt, A. D., & M. T. Siva-Jothy. 2001. Traumatic insemination and sexual conflict in the bed bug Cimes lectularius. Proc. Natl. Acad. Sci. USA 98:5683-5687. Sullivan, M. S. 1991. Flock structure in red junglefowl. Appl. Anim. Behav. Sci. 30:381-386. Sæther, S. A., P. Fiske, and J. A. Kålås. 2001. Male mate choice, sexual conflict and strategic allocation of copulations in a lekking bird. Proc. R. Soc. Lond. B 268:2097-2102.

32 Tregenza, T., & N. Wedell. 2000. Genetic compatibility, mate choice and patterns of parentage: Invited Review. Mol. Ecol. 9:1013-1027. Tregenza, T., & N. Wedell. 2002. Polyandrous females avoid costs of inbreeding. Nature 415:71- 73. Trivers, R. L. 1972. Parental investment and sexual selection. Pp. 136-179 in B. Campbell, ed. Sexual selection and the descent of man 1871-1971. Aldine-Atherton, Chicago. von Schantz, T., H. Wittzell, G. Göransson, M. Grahn, & K. Persson. 1996. MHC genotype and male ornamentation: genetic evidence for the Hamilton-Zuk model. Proc R. Soc. Lond. B 263:265-271. Wagner, R. H., F. Helfenstein, & E. Danchin. 2004. Female choice of young sperm in a genetically monogamous bird. Proc. R. Soc. Lond. B 271:S134-S137. Warren, D. C., & L. Kilpatrick. 1929. Fertilization in the domestic fowl. Poultry Sci. 8:237-256. Wedell, N., M. J. G. Gage, & G. A. Parker. 2002. Sperm competition, male prudence and sperm- limited females. Trends Ecol. Evol. 17:313-320. Westneat, D. F., L. A. McGraw, J. M. Fraterrigo, T. R. Birkhead, & F. Flectcher. 1998. Patterns of courtship behavior and ejaculate characteristics in male red-winged blackbirds. Behav. Ecol. Sociobiol. 43:161-171. Wigby, S. L., & T. Chapman. 2005. Sex peptide causes mating costs in female Drosophila melanogaster. Curr. Biol. 15:316-321. Wishart, G. J. 1987. Regulation of the length of the fertile period in the domestic fowl by numbers of oviducal spermatozoa, as reflected by those trapped in laid eggs. J. Reprod. Fertil. 80:493-498. Wishart, G. J. 1997. Quantitative aspects of sperm:egg interaction in chickens and turkeys. Anim. Reprod. Sci. 48:81-92. Wolfner, M. F. 2002. The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila. Heredity 88:85-93. Zahavi, A. 1975. Mate selection: a selection for a handicap. J. Theor. Biol. 53:205-214. Zeh, J. A., & D. W. Zeh. 1996. The evolution of polyandry I: intragenomic conflict and genetic incompatibility. Proc. R. Soc. Lond. B 263:1711-1717. Zeh, J. A., & D. W. Zeh. 1997. The evolution of polyandry II: post-copulatory defences against genetic incompatibility. Proc. R. Soc. Lond. B 264:69-75. Zeh, J. A., & D. W. Zeh. 2006. Outbred embryos rescue inbred half-siblings in mixed-paternity broods of live-bearing females. Nature 439:201-203. Zelano, B., & S. V. Edwards. 2002. An Mhc component to kin recognition and mate choice in birds: Predictions, progress, and prospects. Am. Nat. 160:S225-S237.

33 Acknowledgements This thesis is based on the work of Tommaso Pizzari and Tim Birkhead. Before them, behavioural ecology and chickens was an unknown combination.

Supervisors first: Pizzari for taking me on as a master student 6 years ago (not 9 as I think it is according to your calculations). Thank you for all your support and that I have been granted access to your great knowledge of science, chickens and sperm. Sven for tricking me into going to a chicken seminar about 6,5 years ago, for always being positive and supportive at Tovetorp and elsewhere, and also for mastering Swedish grammar and knowing how to do a PhD at Zootis. You two together have been the best complimentary to each other I could possibly have wanted and surely it would have been much harder to get to where I am today without the support of both of you!

At other departments: Tack Lena och resten av HMH (SLU, Skara) for att jag fått använda djur och faciliteter. Alf för din hjälpsamhet och ditt trevliga sällskap i en dammig lada. Pelle för att jag har fått använda djungelhönsen och Götala, totalt avgörande för att studierna om inavel har kunna ske. Ben & EGI for letting me be a part of the inspiring and motivating environment that is your department.

På Tovetorp: Adeline och Nisse för att ni har pysslat om de små liven när de ingått i experiment, men kanske ännu viktigare när de inte gjort det. Thomas för allt tekniskt- praktiskt potentiellt strul som inte har urartat till strul på grund av din hjälp. Kristina, Sonja, och Gunilla för er goda mat, extrafika och annan hjälp på Tovetorp.

The chicken people: Charlie for early on teaching me that nearly anything can be done with our system, it’s just a matter of attitude and patience, and later for help with various things like SAS and haemocytometers. Mark for sharing long days in Skara, thanks for driving and may the PUT R.I.P. Claudia for being the first in the group I could ally against the boys with, pasta forno and of course all the help in the field! Gato nero rincorro pullo? Becky for teaching me the art of non-dark chocolate, bed- linen exchange programmes, for understanding my language, de-upsetifying fikas, help and fun! Thanks also to all the field assistants over the years: Emma Brown, Laura Jääskeläinen, Mervii Jaakola, Frances Tyler and Joseph Fletcher.

På Zootis: Ett generellt tack till er alla som skapar denna trevliga miljö vår institution har. Ulf för datorhjälp, Minna för allt-i-allo och Tommy för ”perfekteri” (inklusive att jag har fått åka på konferenser när stipendiepengarna inte har räckt till). Siw, Anette & Berit för att ni har koll på alla blanketter och papper och kvitton och konton och en massa annat som skulle ta så mycket tid om inte ni hade vetat hur man gör! Bertil för trogen artikelgranskning. Krabb-Anna för lyxkaffe och sällskap långa jobbkvällar, saknat när du är jorden runt. Min ”rumppolare” (inte värre än att våra skrivbord är mot varandra, rumpa-mot-rumpa) och numera, Dr. Maria Lissåker tack för alla frågor och svar under åren. Dan, för allt ditt tjat om deadlines, ägare till terapi-Alfa och en av de få på institutionen som tror på en karriär utanför ’academia’. Johanna, som får mig

34 (utan 4 barn, 3 hundar, 2 hästar & 5 katter) att tänka två gånger innan jag gnäller om att inte ha tid. Samt de blondaste så klart; Alex & Blondie; bara man är snygg så kvittar det, och social intelligens är inte allt (blir nog inte roligare om man har det heller). Samt alla andra som har stöttat mig på ett eller annat sätt över åren. Lycka till allihopa!

Och i vanlig ordning, föräldrar och partner sist: Pappa & Sissel takk for all distansesupport og delt biologiintresse! Anders tack for på-plats-support, inte bara psykologisk (då du är smart nog att fatta vad stress har för påverkan och hur man botar det utan att bli klöst), men också praktiskt med allt från hönsobservationer till rättstavning och mikroskopkirurgi. Att doktorera är inte ett välbetalt jobb från 9-17, utan en livsstil med stor påverkan på också ens närmaste, o d e du tenk!

Och så klart är jag mycket tacksam för det ekonomiska stöd jag har erhållit, som har möjliggjort delar av forskningen, deltagande på konferenser samt att jag har kunnat åka iväg och få handledning: Association for the Study of Animal Behaviour, Ebba & Sven Schwartz stiftelse, Knut & Alice Wallenberg stiftelse, Kungliga Skogs och Lantbruks Akademin, Kungliga Vetenskaps Akademin, Lars Hierta stiftelse, Liljevalch stiftelse, Siléns stiftelse, Stockholms Universitets donationsstipendier, och T. Tullberg stiftelse för zoologisk forskning.

35 Sammanfattning på svenska

När hanar slåss och honor väljer Det evolutionära målet för en individ är att fortplanta sig, helt enkelt för endast generna till de individer som förökar sig kan bli överförda till nästa generation. Det betyder att de individer som producerar många och överlevnadsdugliga avkommor kommer gynnas evolutionärt. Det selektionstryck som orsakas av variation i individers fortplantningsframgång kallas sexuell selektion och är därför ett mycket stark selektionstryck. Sexuell selektion brukar delas in i två huvudprocesser genom vilka individer kan öka sin fortplantningsframgång; han-han-konkurrens (när hanar slåss om tillgången till honor), och honors val av partner (när honor kräset väljer bland hanarna vem som ska få para sig med henne). Att det är hanar som slåss och honor som väljer stämmer bara när honor investerar mer i avkomman än hanar (vilket är det vanligaste bland ryggradsdjur). Hanar kan då ofta öka sin reproduktiva framgång genom att befrukta flera honors ägg, medan honors reproduktiva framgång är begränsad av honans egen äggproduktion. Hanars snabbare fortplantningshastighet leder till att de slåss om tillgången till så många honor som möjligt. För honor blir partnerns kvalité viktigare än antalet partners, vilket leder till honor som noggrant väljer hanar av bra kvalité. Men även om inte honor gynnas av att ha många partners på samma sätt som hanar, vet vi i dagsläget att det är mer regel än undantag i djurvärlden att honor är promiskuösa. Att honor är promiskuösa har en stor påverkan på individers reproduktiva beteenden. Detta är för att om honor erhåller spermier från olika hanar, kan den sexuella selektionen fortsätta också efter parningen. Efter parning är det svårare att se vad som försiggår då allt händer inuti honan (hos djur med inre befruktning iallafall), men då sker han-han-konkurrensen som konkurrens mellan olika hanars spermier -spermiekonkurrens (vilket helt enkelt betyder att spermier från två eller flera hanar befinner sig i en honas reproduktiva system vid samma tidpunkt), och honliga valet kallas då ett kryptiskt honval där valet står mellan olika hanars spermier. Spermiekonkurrens ställer till bekymmer för hanar, då det förlänger konkurrensen om en befruktning och därmed ökar osäkerheten om faderskapet. Ur hanars synvinkel blir spermiekonkurrens ett ”ekonomiskt” problem. Visst, en liten spermie är mycket billigare att producera än ett stort, näringsrikt ägg som hönan producerar, men om man ska ha någon chans att befrukta ett ägg behövs mer än bara ett fåtal spermier. Det är som att köpa lotter i ett lotteri: om man är den enda som köper lotter så är ju vinsten säkrad, fast ju fler som spelar med desto fler lotter behöver man köpa själv för att öka sina chanser att vinna första pris. Och att köpa alla dessa lotter kan bli rätt kostsamt och massor av spermier är inte alls lika billigt att producera längre. Mer alvarligt är att hanars spermieresurser inte är obegränsade. Hanar bör därför – som alla andra när det gäller begränsade resurser – allokera sina resurser på ett ekonomisk sett. Och sätten att vara strategisk på är flera, det kan vara ekonomiskt att ta hänsyn till mängden konkurrens man möter, var man redan har resurser investerade, samt var avkastningen kan bli störst.

36 Hönan å andra sidan har ju potentiellt möjligheten att gynnas av spermie konkurrens genom att flera hanars spermier befinner sig i henne vid samma tid, och då ”besluta” om vem som befruktar ägget då befruktningen sker inuti henne. Detta kan potentiellt ske på en rad olika sätt från att honan gör sig av med ejakulatet direkt efter inseminering (”sperm ejection”), att honan påverkar transporten och lagringen av spermier, eller att ägget i sig påverkar vilken spermie som slutligen släpps igenom och befruktar det. Ett kryptisk honval behöver därför inte vara ett aktivt val direkt styrt av honan, men att det är honan som på något sätt ligger bakom det vissa hanars spermier gynnas före andras.

Att hanar och honor formas av den sexuella selektionen till att ha olika reproduktiva strategier leder till att de reproduktiva intressena för könen ofta inte sammanfaller. Individer av båda könen kan därför försöka att öka sin fortplantningsframgång på bekostnad av individer av det andra könet. Detta kan leda till vad vi kallar en sexuell konflikt. Anpassningar till sexuell selektion hos ena könet kan därför leda till en motrespons hos andra könet i ett försök att återta kontrollen över ett parningstillfälle. Denna konflikt och olika motresponser kan i promiskuösa system fortsätta också efter parningen. Att studera båda könens parningsstrategier, båda före och efter parningen, samt också eventuella motresponser blir därför viktigt för att förstå anpassningar till sexuell selektion och vad som påverkar utfallet av ett parningstillfälle. Jag har i min avhandling studerat hur båda hanar och honors beteenden formas av sexuell selektion och det andra könets motresponser, båda för och efter parning. Och för detta har jag använt djungelhöns och dess domesticerade släkting tamhönan.

Höns och djungelhöns Det röda djungelhönset, Gallus gallus spp., är den vilda anfadern till dagens alla domesticerade höns, en domesticering som skedde för ca 8000 år sedan. Det röda djungelhönset är i dag en utrotningshotad art, medan tamhöns är världens mest talrika fågel. Jag har i min avhandling använt djungelhöns hållna på Götala, en forskningsanläggning tillhörande SLU i Skara (artiklar IV-V), och svenska lanthöns av rasen gammal svensk dvärghöna, hållna på Tovetorp, Stockholm Universitets zoologiska forskningsstation (artiklar I-III). Det röda djungelhönset, men också ursprungliga tamhöns (så som de på Tovetorp), lever i sociala grupper på mellan 2 och 15 individer, med ungefär 1:1-1:4 tuppar per höna. Individer bildar rangordningar, där en hög rang ökar en individs tillgång till resurser. Då djungelhöns och (i alla fall ursprungliga) tamhöns fortfarande är mycket lika, så kallar jag dem båda för ”höns” i fortsättningen.

Lösaktiga höns Hönan tar ensam hand om kycklingarna, vilket leder till klassiska könsroller med tuppar som slåss och hönor som väljer. Höns är promiskuösa mestadels för att tuppar inte låter hönorna vara i fred. Detta i kombination med att hönor sparar spermier i tio- tusentals lagringsorgan i sitt reproduktiva system i upp till två veckor efter en parning (vilket är länge jämfört med däggdjur som ofta sparar spermier under några dagar bara), leder till intensiv spermiekonkurrens och möjligheter till kryptiskt honval utöver ”sperm ejection”.

37 Höns och inavel Hos höns verkar inget av könen sprida sig särskilt långt och både tuppar och hönor stannar ofta i den flock de föds i också efter det att de har blivit könsmogna. Detta är ett potentiellt problem eftersom det ökar risken för inavel, d.v.s. att besläktade individer parar sig med varandra. Inavel är ofta kostsamt då den ökar risken för att recessiva, skadliga alleler kommer till uttryck. Och avkomman kan då bli mindre levnadsduglig. Hos tamhöns vet vi att inavel reducerar tuppars sociala status samt spermiekvalitet och chanserna ökar också att kycklingarna dör tidigt. Individer bör därför undvika inavel. Det finns olika sätt att förhindra inavel. Hos däggdjur och fåglar undviks inavel ofta genom att individer av ena könet lämnar området de föds i när de blir könsmogna. Men som sagt, hönsen har inget sådant spridningsmönster som minskar riskerna för att könsmogna släktingar möts i framtida parningssituationer. Då kan inavel istället förhindras om individer känner igen sina släktingar, och väljer att para sig med obesläktade individer. En släkting till hönsen, vakteln, känner igen och undviker syskon som parningspartners. Så möjligheterna att höns också kan göra så, finns. Vidare kan inavel potentiellt undvikas också efter parning om honan undviker att använda spermier från närbesläktade hanar.

Syftet med avhandlingen Syftet med avhandlingen är att öka vår kunskap och förståelse om hur sexuell selektionen verkar och vad som påverkar individers fortplantingsbeteenden. Jag har använt höns som modellart, och hos dessa studerat båda könens reproduktiva strategier båda före och efter parning. Mera specifikt har jag studerat: • Hanars och honors dagliga parningsmönster och parningsfrekvenser • Hanars förmåga att allokera sina spermieresurser på ett ekonomisk sätt • Det konstiga beteendet att hanar ”fejkar” parningar d.v.s. inte inseminerar spermier vid en parning • Hanars respons mot inavel, före och efter parning • Honors olika sätt att förhindra inavel, både före och efter parning

Sexuella trakasserier (artikel I) Hanar oftare än honor ökar sin fortplantningsframgång genom att ha flera partners. Detta kan leda till att hanar utsätter honor för sexuella trakasserier och -från honans synpunkt- överflödigt många parningsinviter. Detta resulterar ofta i att honan blir mer avtänd och ännu mindre intresserad. Hos hönsen utför tuppar över 90% av alla parningsinviter, och oftast på kvällen efter det att hönorna har lagt sina ägg och då det är större chans att en parning leder till befruktning. I grupper med mestadels hönor utsätts hönorna inte för så intensiva trakasserier, och då blir också hönorna mer intresserade av parningar på kvällen. Men i grupper med många tuppar blir tupparnas ökade intresse för mycket för hönorna, och hönorna väljer istället att initiera parningar på morgonen när tupparna är mindre intresserad och också mer utsprida. Hönorna väljer därför på ett flexibelt sätt sina tillfällen när på dagen de initierar parningar beroende på hur mycket sexuella trakasserier de utsätts för.

38 Tuppars spermieekonomi (artikel II) Tuppar som möter spermiekonkurrens bör vara ekonomiska med sina spermieresurser för att få dem att räcka och därmed öka sannolikheten för framgångsrika befruktningar Genom att låta tuppar para sig med hönor som hade ett litet ”rumpskydd” som förhindrade inseminering och tillät oss att samla in ejakulaten, så kunde tuppars spermieanvändning mätas. När tuppar är ensamma om att para sig med en höna inseminerar de endast en liten mängd spermier, och sparar resten av spermierna till andra parningar. Om tuppar däremot möter konkurrens från andra tuppar beror deras spermieinvestering delvis på vilken rang de har och delvis på hur många andra konkurrenter det finns. Dominanta tuppar ökar mängden spermier inseminerad i en höna om konkurrensen stiger upp till tre andra tuppar. Subdominanta tuppar däremot ökar också sina investeringar, men när det blir för mycket konkurrens (mer än två konkurrenter) så sparar de hellre på resurserna. Detta beror förmodligen på att dominanta tuppar har mer kontroll över vem som har parat sig med ”sina” hönor, och därför möter mindre konkurrens än vad subdominanta tuppar gör även om antalet potentiella konkurrenter är lika för de båda. Subdominanta tuppar däremot ”vet” ju att alla andra tuppar i flocken också har parat sig med en höna innan de själva får chansen. Tuppar tar också andra hänsyn med sina begränsade resurser. De sprider sina investeringar genom att minska antalet spermier de inseminerar i en höna som de redan parat sig med, men kan om de blir presenterade för en ny återigen ejakulera. På det sättet ökar tupparna sina chanser att lyckas befrukta flera hönors ägg. Vidare optimerar tuppar sitt spermieanvändande genom att inseminera mer spermier i hönor som lägger större ägg och därför får mer överlevnadsduglig avkomma. Detta signaleras med kamstorleken på hönan, en stor kam signalerar att hönan lägger stora ägg. Så står valet mellan en höna med liten respektive stor kam, så väljer tuppen hönan med stor kam och investerar fler spermier i henne också.

Fejkade parningar (artikel III) Trots att det evolutionära målet för en individ är att fortplanta sig är det känt i djurvärlden – från insekter till däggdjur – att hanar ibland parar sig med en hona utan att inseminera några spermier. Även fullt könsmogna, erfarna tuppar med fulla spermieresurser gör detta, typiskt efter det att de redan har parat sig och inseminerat spermier i en höna. Genom att utsätta hönor antingen för parningar där tuppar inseminerade sperma, eller ”fejkade” parningar (genom att tillfälligt ha ”rumpskyddet” på dessa hönor i annars naturliga parningar), kunde hönors respons på detta beteende studeras. Och det konstiga är att hönor som utsattes för fejkade parningar var lika ointresserade av att para sig med nya tuppar som de hönor som också mottog sperma i parningarna, båda i motsats till de hönor som inte utsattes för endera av parningarna. Reduktioner i honors promiskuitet efter parning har hittats också hos andra arter, men effekten har då berott på att hanarna har inseminerat stora mängder sperma, inseminerat sofistikerade produkter i sädesvätskan, uppvisat särskilda beteenden eller på annat sätt investerat kostsamt i att minska honans lösaktighet. Fejkade parningar å andra sidan kan vara ett mycket enklare sätt att reducerar hönors promiskuitet. Genom att fejka parningar efter att redan ha inseminerat sperma till en höna kan tuppar kämpa om sitt faderskap, samtidigt som de sparar sperma till andra hönor, och det tillsynes meningslösa beteendet får en mening.

39 Syskonbråk om syskonkärlek (artikel IV) Det röda djungelhönset möter i det vilda båda besläktade och obesläktade individer som potentiella parningspartners. Deras respons till denna risk för inavel var däremot okänd. Genom att låta tuppar få para sig med systrar och obesläktade hönor kunde vi konstatera att tuppar parade sig med både systrar och obesläktade hönor och verkade inte bry sig alls om att förhindra inavel. De till och med inseminerade mer spermier i systrar än i obesläktade hönor (men det berodde också på om de hade växt upp tillsammans eller inte). Hönor däremot använde efter parningen färre spermier från bröder än från obesläktade tuppar att befrukta sina ägg med och minskade därmed risken för inavel (Detta kunde studeras genom att i mikroskop räkna hur många spermier som nådde fram till ägget efter att hönan var parad med en broder, eller en obesläktad tupp). Skillnaden i hur mycket spermier som användes var därmed något hönan styrde över, hur var däremot oklart. Att tuppar hellre investerade fler än färre spermier i systrar kan förklaras med att de försökte komma förbi hönornas selektion mot de egna spermierna, och kan vara en motrespons på hönornas förhindrande av inavel. Men varför blir det så, att hönor bryr sig, men tuppar inte? Hos höns investerar honan mer i avkomman än vad hanen gör. Det betyder att hönan kommer att bära en större kostnad om en avkomma är sjuk, än en tupp. Inavel kan också rent av vara gynnsamt, om man genom att para sig med en släkting kan öka släktingens reproduktiva framgång. Detta gynnas man indirekt av då man har många gener gemensamt med släktingen, och hjälper genom parningen till att sprida dessa gemensamma gener. Av denna anledning bör toleransen mot inavel vara högre i djurvärlden än det hittills har observerats. Både hönor och tuppar beter sig därför optimalt enligt teorin...

Hur hönor förhindrar inavel (artikel V) Det var fortfarande oklart efter den första studien om respons mot inavel, hur hönor påverkade sitt spermieanvändande till fördel för obesläktade tuppars spermier. En rad experiment utfördes därför för att försöka förstå hönors respons till inavelsrisk. I frigående grupper initierade hönor färre parningar med bröder än med obesläktade tuppar och de försökte också hårdare att undvika bröders parningsförsök än om dessa kom från obesläktade tuppar. Hönor känner alltså igen släktingar, och gör så även när individer inte träffas förut, och använder detta för att förhindra inavel. Men hönor försökte också förhindra inavel genom ett kryptiskt honval. Men det var inte genom ”sperm ejection”, vilket kunde konstateras genom att filma alla parningar med fokus på hönans kloak. Inte heller verkar det vara genom en mer ”passiv” mekanism där spermier och ägg från syskon till exempel var för lika och därför inte kompatibla med varandra. Detta för att när ejakulat inseminerades artificiellt, gjorde hönan inte längre något spermieval och således kunde inte heller inavel förhindras. Det är fortfarande oklart hur hönan gör sitt kryptiska spermieval, men antingen sitter mekanismen för att bedöma om ett ejakulat tillhör en broder eller en obesläktad tupp tidigt i hennes reproduktiva system, precis så att mekanismen kringgås när man inseminerar hönor artificiellt. Eller så är det helt enkelt så att hönan behöver får se tuppen för att kunde bedöma hur nära släktingar de är och bestämma vad hon vill göra med spermierna han har levererat. Oavsätt visar det att hönor har flera kryptiska sätt att göra ett val på efter en parning.

40 Konklusion Jag har i denna avhandling visat att könen har olika sätt att försöka öka sin fortplantningsframgång på. Tuppar försöker att öka antalet honor de parar sig och befruktar äggen till. De gör detta genom att para sig ofta och också med systrar. För att reducera risken för att möta konkurrens från andra hanar efter parning utsätter de hönorna för fejkade parningar, vilket leder till att hönorna minska sitt intresse för parningar ett par dagar, och tuppens spermier därför möter mindre konkurrens. Efter parning allokerar tupparna sina resurser på ett sätt som sannolikt ökar chanserna att lyckas i spermiekonkurrensen, får spermierna att räcka till flera honor, samt också investera mer i hönor som lägger större ägg. Hönor å andra sidan försöker undvika kostnaderna associerad med reproduktion. Det gör de genom att undvika för många parningar, och också förhindra produktionen av inavlad avkomma genom att initiera parningar med obesläktade tuppar, men också genom att använda mindre spermier från bröder. Könens olika reproduktiva strategier sammanfaller därför ofta inte, vilket leder till motresponser. Hönor svarar på tuppars parningsiver delvis genom att kämpa emot när tuppar försöker para sig, men också genom att initiera parningar när tupparna är lite mindre sugna för att minska de sexuella trakasserierna de utsätts för. Hönor visar också motresponser till bröders parningar genom att selektera emot deras spermier. Tuppar å andra sidan svarar med att inseminera mer spermier till systrar, kanske som en vidare motrespons till hönornas kryptiska val. Avhandlingen visar därför att vad ena könet gör påverkar det andra och måste inkluderas för att förstå könens fortplantningsbeteenden. Vidare visar avhandlingen att bara för att en parning har skett behöver den inte avgöra utfallet av ett fortplantningstillfälle d.v.s. att den sexuella selektionen sker både före och efter parning. De arbeten som ingår i den här avhandlingen har gett helt nya kunskaper om hanars förmåga att allokera sina spermieresurser på ett komplext sätt och varför de fejkade parningar, samt att honor har flera olika kryptiska sätt att göra val på efter en parning. De var också okänt hur hönsen reagerade på risken för inavel, att de kunde känna igen släktingar, även utan att tidigare ha träffats. Med denna avhandling blir därför höns en av de arter vi idag vet mest om vad gäller sexuell selektion, båda före och efter parning.