BEHAVIOUR, 2008, 75, 763e770 doi:10.1016/j.anbehav.2006.12.029

Available online at www.sciencedirect.com

Male egg carrying in laciniata is favoured by natural not sexual selection

M.GOMENDIO,F.GARCI´A-GONZA´ LEZ, P. REGUERA & A. RIVERO Reproductive Ecology and Biology Group, Department of Evolutionary Ecology, Museo Nacional de Ciencias Naturales (CSIC)

(Received 9 May 2006; initial acceptance 16 June 2006; final acceptance 12 December 2006; published online 31 December 2007; MS. number: 8954)

Two hypotheses could explain the evolution of paternal care: caring males are more attractive to females and mate more often (sexual selection); males care when the benefits in terms of offspring survival exceed the costs (natural selection). To test these hypotheses we used : females can choose whether to lay eggs on plants or on conspecifics, and the extent to which males carry eggs varies between populations. Our results do not support the sexual selection hypothesis: females did not choose to mate with egg-carrying males in either natural populations or experimental contexts. We compared two popu- lations that differ in the extent of male egg carrying and we show that in the population where male egg carrying was more prevalent, parasitism pressure was higher. Field experiments revealed that, in the pop- ulation with high parasitism rate, egg mortality as a result of parasitoid attack was up to 10 times higher on plants than on conspecifics. Egg carrying is thus an effective strategy that protects eggs against parasitoids. We conclude that the main benefit derived by males from egg carrying is an increase in offspring survival, and that males are sensitive to interpopulation differences in egg mortality risks. Male care in this system has evolved despite intermediate levels of paternity certainty because the impact on offspring survival is high, and the costs in terms of loss of mating opportunities low. Thus, our findings support the natural selection hypothesis, although additional work on more populations is needed to verify this.

Ó 2007 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Keywords: golden egg bug; natural selection; parasitism; parasitoid; parental care; paternal care; Phyllomorpha laciniata; sexual selection

Theory predicts that care for offspring will evolve through the costs involved, each sex would be better off if the natural selection when the benefits in terms of offspring other sex provides the care, so a conflict of interests be- survival are greater than the costs to the parents’ residual tween males and females is always present (Parker 1979; reproductive value, such as reduced survival, fecundity Westneat & Sargent 1996; Chapman et al. 2003). This im- and mating opportunities (Williams 1966; Trivers 1972). plies that the behaviour of each sex will be influenced by When offspring survival is substantially improved by the behaviour of the other, so the patterns that we observe parental care, it can be provided by females, by males, or in natural populations must be understood as the resolu- by both sexes. Which sex ends up caring for offspring tion of a game with three players: the male, the female depends partly on the balance of benefits and costs for and the offspring (reviewed in Parker et al. 2002). each sex, and partly on historical constraints. Because of Parental care in is rare, possibly because in most species parents can do little to protect or nurture the offspring (Zeh & Smith 1985; Tallamy & Wood 1986). Exceptions include species in which eggs or young face Correspondence: M. Gomendio, Reproductive Ecology and Biology particularly harsh environments or high predation pres- Group, Department of Evolutionary Ecology, Museo Nacional de Ciencias Naturales (CSIC), Jose´ Gutie´rrez Abascal 2, 28006 Madrid, sures, because in these species parents can improve Spain (email: [email protected]). F. Garcı´a-Gonza´lez is now at offspring survival substantially by keeping environmental the Centre for Evolutionary Biology, Zoology Building (M092), School conditions at an optimal level for the development of the of Animal Biology, The University of Western Australia, Nedlands, offspring (Smith 1976; Tyndale-Biscoe 1984; Wyatt 1986; WA 6009, Australia. Favila 1993; Halffter et al. 1996), protecting offspring 763 0003e3472/08/$32.00/0 Ó 2007 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. 764 ANIMAL BEHAVIOUR, 75,3

against food competitors (Tyndale-Biscoe 1984; Wyatt Whether egg carrying by male P. laciniata constitutes 1986; Halffter et al. 1996), or protecting them against a form of parental care or is merely the consequence of predators (Tallamy & Denno 1981; Nafus & Schreiner intraspecific parasitism remains a point of contention 1988; Kudoˆ et al. 1989, 1995; Scott 1990; Kudoˆ & (see Kaitala et al. 2001; Gomendio & Reguera 2001, for a re- Nakahira 1993; Kudoˆ & Ishibashi 1996) or parasitoids ply). Kaitala et al. (2001) argued that natural selection acts (Ralston 1977). The distribution of parental care among on the ovipositing female, which lays eggs on both male is such that in most species care is provided and female conspecifics whenever the opportunity arises, by the female alone, followed by biparental care and, such as when she encounters a pair engaged in copulation finally, paternal care is the least frequent form of care (Kaitala 1996). However, this hypothesis has not been sup- (Zeh & Smith 1985; Clutton-Brock 1991). Exclusive care ported by data from natural populations (Garcı´a-Gonza´lez by males is uncommon in most taxa because male lifetime & Gomendio 2003b). In contrast, Gomendio & Reguera reproductive success is mainly determined by the number (2001) argued that to understand patterns of care observed of females with whom they mate, and providing care fre- in natural populations it is important to consider not only quently reduces the opportunities for looking for females the benefits to females of laying eggs on conspecifics, but and mating with them (Zeh & Smith 1985; Clutton-Brock also the selective forces operating on the sex that accepts 1991; Queller 1997). to provide care. Thus, while it is clear that females would Recently, another hypothesis has been proposed to benefit from laying eggs on any conspecific (Garcı´a- explain the evolution of care by males in insects, which Gonza´lez & Gomendio 2003a), it remains to be explained suggests that sexual selection is important (Tallamy 2000, why, at the peak of the reproductive season, all (95e 2001). According to this hypothesis, caring males in these 100%) males carry eggs but only 20e40% of females do species benefit from female preferences to mate with so (Gomendio & Reguera 2001). Thus, while some of the males that show their willingness and ability to look after egg carrying by males and females may indeed be the young. Thus, these males would not only avoid the costs result of opportunistic female behaviour, the high levels of reduced mating opportunities, but also benefit from of egg carrying by males (over 77% of all eggs in the their enhanced attractiveness to females. This argument population are carried by males) must partly be explained is similar to the ones developed to explain the evolution by selection acting on the males to accept eggs (Gomendio of paternal care in fish, where territorial males look after & Reguera 2001; Garcı´a-Gonza´lez & Gomendio 2003a, b; the broods of several females simultaneously, and Garcı´a-Gonza´lez et al. 2005), particularly when the females prefer to lay eggs with guarding males (Reynolds evidence available shows that males can easily reject at- et al. 2002, and references therein). tempts by females to lay eggs on them simply by moving Most studies aimed at understanding the evolution of away (Gomendio & Reguera 2001). Females mate with sev- care by males and females have carried out comparative eral males, and several lines of evidence suggest that the analyses to find out how the balance of costs and benefits certainty of paternity is intermediate (Tallamy 2001; for each sex varies between species (Clutton-Brock 1991; Garcı´a-Gonza´lez et al. 2003; Tay et al. 2003). Males at- Thomas 1994; Reynolds et al. 2002). However, even tempt to increase their share of paternity by transferring closely related species often differ in several aspects of more sperm and extending copulation when there are ri- their ecology and life history patterns, making it difficult vals in the vicinity (Garcı´a-Gonza´lez & Gomendio 2004). to control for confounding effects. Perhaps for this reason, Under such conditions, natural selection is expected to most hypotheses are still rather speculative (Balshine et al. favour egg acceptance by males only when the impact in 2002). To test which selective forces favour the appearance terms of offspring survival is very high, and the costs in of male care, one should ideally compare populations of terms of reduced mating opportunities low (Garcı´a- the same species that differ in the extent to which care Gonza´lez et al. 2003, 2005). has spread in the population. Few species show this degree Our aim in this study was to disentangle the potential of intraspecific variability, but the golden egg bug, Phyllo- roles of sexual and natural selection in favouring egg morpha laciniata (Heteroptera, ), is an exception acceptance by males. For this purpose we compared and therefore constitutes an ideal model organism to populations (in central and southern Spain) in which test these hypotheses. egg carrying by males has evolved to a different extent. A We examined which selective forces explain egg carry- higher proportion of males carry eggs in central than in ing by males in P. laciniata. Females of this species can lay southern Spain (81% and 50%, respectively; Reguera eggs both on plants and on the backs of conspecifics 1999). The predictions from the sexual selection and the (males and females). The main sources of egg mortality natural selection hypotheses are easily distinguishable. are predation (mainly by ants) and the parasitoid Gryon (1) The sexual selection hypothesis predicts that egg- bolivari (Hymenoptera: Scelionidae). The survival rates of carrying males should become more attractive to females eggs are low on plants (3%) but are much higher when than noncarrying ones, and that males in the two popula- the eggs are carried by an adult (25%; Reguera & tions should differ in the extent to which they become Gomendio 2002). Given the benefits in terms of egg sur- attractive to females when they carry eggs. (2) The natural vival, females prefer to lay eggs on conspecifics if given selection hypothesis predicts that males in the two popu- the choice (Garcı´a-Gonza´lez & Gomendio 2003a). Egg lations should differ in the extent to which they benefit carrying, however, is costly for the individuals, which be- from offspring survival. Thus, parasitoid pressure, the come more visible (eggs are bright yellow) and less able to main cause of egg mortality on plants (Reguera & escape from predators (Reguera & Gomendio 1999). Gomendio 2002), should be more prevalent in the GOMENDIO ET AL.: NATURAL SELECTION FAVOURS MALE CARE 765 population in which a greater proportion of males carry white eggs would have been laid recently (either just eggs, and male egg carrying should protect eggs from before the current copula or between successive copula- parasitoids. tions). Thus, we distinguished in the analyses white eggs, that is recently laid eggs, from old (yellow) eggs, that is METHODS eggs that individuals were carrying long before the start of the current copula, which includes parasitized eggs. Natural History Hatched eggs were also counted as old eggs. We did not include in the analyses individuals that were recaptured, Phyllomorpha laciniata occurs around the Mediterra- because this could have lead to pseudoreplication. We also nean, where it is typically found in the host plant Parony- excluded from the analyses individuals born during the chia argentea and other Paronychia species (Mineo 1984; reproductive season, because these individuals do not Reguera 1999, and references therein). In the Iberian mate and do not accept eggs until the following year. Peninsula, individuals born in any given year overwinter as sexually immature adults, emerging from their refuges Mate choice experiment around March/April of the following year, once tempera- To find out whether females prefer to mate with egg- tures start to rise. Soon after emergence, individuals start carrying males, we also carried out female choice experi- to copulate, which is followed by oviposition either on ments using individuals from El Espinar (central Spain). plants or on other adults. Females mate with a number Adults were captured during the first week of June 1998 of males during the reproductive period and egg laying and were kept together in constant conditions (25C, takes place throughout the active season (from March/ lights on from 0800 to 2100 hours) in plastic containers April to August). Females lay eggs continually throughout (29 51 cm and 32 cm high) to favour high rates of male the breeding season, producing one egg at a time, and 0e4 egg carrying. eggs daily on average. Eggs change colour as they develop: After a week, all the individuals were isolated for 2 days. eggs are white for some hours after being laid, then they Only males that were carrying eggs were used in the turn to yellow and hatch after about 10e12 days (Kaitala experiment. Males were weighed and 17 females were 1996; Reguera 1999). Hatched eggs remain solidly stuck individually enclosed in a cage (18 18 cm and 10 cm to the backs of the individuals. Eggs parasitized by high) with a pair of haphazardly chosen males that had G. bolivari turn black. Nymphs start an independent life been previously matched by size. This controlled for any as soon as they hatch, and after five instars they become effect of size on female choice, although evidence exists adults. The first adults in any given year can be detected that male size does not influence female choice (Garcı´a- around June in central Spain. These individuals are easily Gonza´lez & Gomendio 2004). Before the female was intro- distinguishable from the sexually mature overwintering duced into the cage, one of the males was haphazardly adults because instead of the dark, heavily sclerotized chosen and the eggs on his back removed (although eggs exoskeleton of the latter, newly emerging adults are pale are firmly glued to the back of males, these are easily re- and soft. These individuals do not copulate during the re- moved with a pair of fine forceps). We then marked each mainder of the breeding season, nor do they carry eggs. male with a dot of blue typists’ correction fluid; one male Individuals cease to be active during August when the was marked on the right side of the pronotum and the host plant becomes dry, and emerge again after the winter other on the left side. (Reguera 1999; Garcı´a-Gonza´lez 2002). Once the experimental units were set up, we monitored the bugs every hour during the day (0800e2100 hours) and Sexual Selection Hypothesis every 2 h under red light at night (2100e0800 hours). Dur- ing this time, we recorded all copulations. Copulations last Field sampling for a long time (in this experiment, X SD ¼ 32:5 6 h), To test whether male egg carrying influences female so the interval between observations was deemed sufficient choice, we conducted field observations in five adjacent to detect all of them. We separated individuals in each localities in central Spain (Villaviciosa de Odo´n, Robledo de enclosure if they did not start mating during the 24 h after Chavela, Colmenar del Arroyo, Valdemorillo and El Espi- we set up the replicates. nar) during 1998 and 1999 throughout the reproductive season. In addition, in 1998 we conducted field observa- tions in southern Spain (Boca de los Frailes, Almerı´a) during Natural Selection Hypothesis 3 days at the peak of the reproductive season in this locality (early April). In those sites that were sampled repeatedly, Field sampling individuals were marked with unique combinations of We sampled populations of P. laciniata in 2004 from two typists’ correction fluid spots to allow for individual locations in Spain: Almerı´a (Pozo de los Frailes, southern identification. We recorded whether individuals were Spain) and Segovia (El Espinar, central Spain). We moni- mating and the number of eggs carried by each individual. tored both populations on a regular basis from the Since no significant differences were found in the patterns beginning of the reproductive season (appearance of first of egg carrying among the central Spain populations, we adults in the spring). Population sampling was carried pooled the data to increase the sample size. out in each population to coincide with the peak of the re- Of particular interest for our predictions was whether productive activity of the bugs in the field, defined as the eggs carried by mating individuals were white, since only period with the highest number of first-generation adults 766 ANIMAL BEHAVIOUR, 75,3

carrying eggs, before the nymphs and second-generation On the day of release (days 3e4), we marked the bugs adults appeared. with coloured correction fluid (Reguera & Gomendio We carried out sampling in an identical way in both 2002). The number of freshly laid eggs and their position populations. For 2 consecutive days (8 and 9 May for on the backs of each individual were recorded before we Almerı´a and 30 and 31 May for Segovia; the peak of released them in the field in two haphazardly chosen reproductive activity differs between these populations P. argentea-containing areas. In each area, we marked three owing to latitudinal and climatic differences) two or three release points, separated by 4e10 m, with wooden sticks people systematically searched an area of ca. 1500 m2 look- nailed to the ground. A total of 95 males and 34 females ing for P. laciniata males. We recorded the number of adult (Almerı´a) and 70 males and 37 females (Segovia) were males, the number of eggs they were carrying and whether released in this way, 12e15 males and 5e6 females per these eggs were unparasitized (yellow or whitish eggs) or release point. parasitized (black eggs). To avoid counting the same male We recaptured marked individuals over 2 consecutive twice, we captured males and temporarily kept them in plas- days (days 10e11). For this purpose the release area was tic boxes (30 20 cm and 8 cm high) with some P. argentea systematically searched within a radius of ca. 20 m from inflorescences. After the sampling was over, males were the release points. We noted the number of eggs that released back into the field. Given the large number of inflo- had been parasitized on the backs of individuals during rescences in the sampling area, and the cryptic nature of the release period. eggs on the inflorescences, finding eggs on plants requires For the plant experiment, 42 (Almerı´a) and 21 (Segovia) a considerable sampling effort and few are usually found females were collected (days 1e2) and kept in plastic (personal observation). For this reason, in this experiment boxes (as above) at ambient temperature. On day 3, each we made no further attempt to find eggs laid on plants. female was individually bagged around a haphazardly selected P. argentea inflorescence (for details see Reguera & Egg manipulation experiments Gomendio 2002). The plant was marked by nailing a small, Two experiments were carried out simultaneously in numbered wooden stick in its proximity. We removed the each population: a mark and recapture experiment (to bags and the females 24 h later (day 4) and recorded the determine the fate of eggs carried by adults in the field) number and position of eggs laid in each inflorescence. and a plant experiment (to determine the fate of eggs laid On day 11, we revisited the plants and recorded the on plants in the field). The experiments took place in both number of eggs that had been parasitized over the 7-day populations to coincide with the period of maximum period. reproductive activity of the bugs (maximum number of e individuals carrying eggs, Almerı´a: 3 13 May; Segovia: 21 Statistical Analyses Junee1 July) in an area adjacent to the sampling area, but separated from it by at least 20 m. The experimental meth- The data were analysed with generalized linear model- odology was identical in both populations. ling techniques available in GLMStat version 6.0 (http:// For the mark and recapture experiment, we collected www.glmstat.com) and Statistica 6.0 (Statsoft, Tulsa, OK, individuals from the experimental area for 2 consecutive U.S.A.) statistical packages. Proportional data (proportion days (days 1e2) and recorded their sex, the number of of egg-carrying adults, proportion of eggs that disappeared eggs they were carrying at the time of collection, and the from, or were parasitized on, the backs of individuals or status of these eggs (unparasitized or parasitized). Eggs plants) were analysed specifying binomial errors and the carried by the bugs were removed with entomological logit link (Crawley 1993). We analysed count data forceps and the bugs were placed in plastic boxes (number of eggs carried per individual) by using Poisson (30 20 cm and 8 cm high) with some P. argentea inflores- errors and the log link. In both cases, the full model was cences. Each box contained approximately 20 males and built by fitting in all potential explanatory variables and 10 females. Individuals were kept in the box for 48 h at their interactions. The significance of each element was ambient temperature to allow females to lay fresh eggs assessed by removing it from the model and analysing on the backs of conspecifics. The purpose of this exercise the resulting change in deviance (Crawley 1993). With was to ensure that all eggs carried by individuals were both binomial and Poisson errors the change in deviance roughly at the same stage of development at the time of attributable to a given factor is distributed as c2. After the release (as parasitoid attack is known to be highly depen- minimal model (the model including only significant dent on the stage of development of the host; Quicke terms and interactions) was obtained, we tested its appro- 1997). We tried to ensure that the mean number of eggs priateness by checking and correcting for overdispersion carried by individuals at the time of release was roughly (Crawley 1993). The significant values given in the text representative of the mean number of eggs they were are for the minimal model while nonsignificant values carrying at the time of collection, as artefacts may arise are those obtained prior to the deletion of the variable when numbers are artificially high (Gomendio & Reguera from the model. 2001). In Almerı´a, the number of eggs on collection Data from the mate choice experiment were analysed (X SE ¼ 2:46 0:16) and release (2.48 0.14) were sim- with a chi-square test between observed frequencies 2 ilar (generalized linear model, GLM: c1 ¼ 0:004, P ¼ 0.94), (number of females mating either egg-carrying or non- but in Segovia the number of eggs on release (3.47 0.20) egg-carrying males) and expected frequencies under the was significantly lower than the number on collection null hypothesis of absence of female choice based on egg 2 (5.59 0.30; c1 ¼ 54:44, P < 0.0001). carrying. GOMENDIO ET AL.: NATURAL SELECTION FAVOURS MALE CARE 767

RESULTS 100 90 (a) Sexual Selection Hypothesis 80 Field sampling 70 60 In populations from central Spain, we analysed egg 50 carrying in 344 single males and 76 mating pairs. If females 40 chose to mate with egg-carrying males we should have found 30 differences in the number of old eggs (yellow eggs) carried by

% Male egg carriers 20 single males and by males in copula, since those are the ones 10 that males were already carrying before the current mating. 0 No differences were found between mating and single males Central Southern either in the proportion of males carrying old eggs (egg carriers: 40/76 copulating males and 152/344 single males; 8 2 (b) c1 ¼ 1:78, P ¼ 0.18) or in the number of old eggs carried 7 (X SE eggs carried: mating males: 3.33 0.4, N ¼ 40; single 2 6 males: 3.27 0.18, N ¼ 152; GLM: c1 ¼ 0:018, P ¼ 0.89). These results remained unaltered regardless of whether we 5 considered hatched eggs as old eggs or not. 4 In southern Spain we found 119 single males and 48 3 mating pairs. The proportion of mating males carrying old Eggs carried eggs did not differ from the proportion of single males 2 carrying old eggs (egg carriers: 21/48 copulating males; 49/ 1 2 119 single males; GLM: c1 ¼ 0:09, P ¼ 0.76), and mating 0 males did not carry more old eggs than single males Central Southern (X SE eggs carried: mating males: 3.67 0.62, N ¼ 21; Figure 1. Comparison between central and southern Spanish popu- 2 single males: 2.96 0.34, N ¼ 49; c1 ¼ 1:13, P ¼ 0.29). lations in (a) the percentage of males carrying old eggs, and (b) the These results remained unaltered regardless of whether mean number of old eggs carried by these males (central Spain: we considered hatched eggs as old eggs or not. N ¼ 83; southern Spain: N ¼ 139). Vertical lines represent SEs. Field sampling data from 2004. Mate choice experiment In 14 of 17 trials the female copulated with one of the two addition, 41 of the 42 marked inflorescences were males. Eight of the females copulated with egg-carrying recovered. In Segovia (central Spain) we recaptured 13 males males and six with males without eggs. The difference and five females, or 16.82% of the total individuals released. between the observed and expected frequencies under the In addition, 18 of the 21 inflorescences were recovered. null hypothesis did not show statistical significance (chi- As expected from the population sampling, in Almerı´a 2 no eggs were parasitized either on the plants or on the square test: c1 ¼ 0:29, P ¼ 0.59). backs of conspecifics. In Segovia, however, parasitism was high: 12.69% of the total eggs carried by recaptured adults Natural Selection Hypothesis (total number of eggs parasitized over total number of eggs Field sampling on release, only for recaptured adults) and 64.70% of the total eggs laid on plants (total number of eggs parasitized In Segovia (central Spain) most of the males carried eggs (81/83 males captured), but in Almerı´a (southern Spain) the proportion of egg-carrying males was significantly lower 2 (91/139 males captured; GLM: c1 ¼ 38:82, P < 0.0001; 35 Fig. 1a). The mean number of eggs carried per male was 30 also significantly higher in Segovia than in Almerı´a (X SE: Segovia: 6.46 0.33; Almerı´a: 2.05 0.19; 25 2 c1 ¼ 262:5, P < 0.0001; Fig. 1b). 20 In Almerı´a none of the eggs carried by males were 15 parasitized. In contrast, in Segovia, 32.53% of the males Percentage carried at least one parasitized egg, and 9.31% of the total 10 eggs carried by males were parasitized (Fig. 2). 5 In both populations the sex ratio was 1:1. We found 262 0 adults in Almerı´a (139 of which were males, 53.05%) and CentralSouthern Central Southern 153 adults in Segovia (83 of which were males, 54.24%); Males carrying parasitized eggs Parasitized eggs thus, densities were higher in Almerı´a. Figure 2. Comparison between central and southern Spanish popu- lations in the percentage of males carrying parasitized eggs and in Egg manipulation experiments the percentage of total eggs in the population that were parasitized In Almerı´a (southern Spain) we recaptured 43 males and (central Spain: N ¼ 83; southern Spain: N ¼ 139). Field sampling 13 females, or 43.41% of the total individuals released. In data from 2004. 768 ANIMAL BEHAVIOUR, 75,3

over total number of eggs laid on plants) were parasitized males should result in a considerable improvement in during the experimental week (Fig. 3). The statistical survival rates. We have focused on the parasitoid wasp G. analysis was done with egg-carrying adult (males plus bolivari because it has a major impact on mortality rates of females) or inflorescence as a replication unit. Eggs suf- eggs on plants (Reguera & Gomendio 2002). Our results fered up to 10 times more parasitism when laid on the in- support both predictions. The number of egg-carrying florescences (X SE proportion of eggs parasitized per males and the mean number of eggs carried per male inflorescence ¼ 66.67 10.69) than when carried on the were significantly higher in the population where parasit- backs of individuals (6.31 3.14 per individual; GLM: oids were most abundant (central Spain). The results of 2 c1 ¼ 28:81, P < 0.0001). the egg manipulation experiment provide an explanation for these results. In central Spain, eggs suffered up to 10 times more parasitism by G. bolivari when laid on the DISCUSSION inflorescences than when carried on the backs of individ- uals. Egg carrying is thus effective in protecting eggs The sexual selection hypothesis suggests that males care against parasitoid attack. Most parasitoids require a long for offspring because they become more attractive to host-handling process (a time during which the parasitoid females and gain increased mating opportunities. Our explores and probes the host to determine whether it is findings from the field sampling do not support this suitable for oviposition; Quicke 1997) before they can hypothesis since mating males in natural populations lay an egg on it. The movement of walking P. laciniata did not carry more old eggs than single males, and this and the vibrations that they typically produce when was the case in populations from both central and they are disturbed (personal observation) are probably suf- southern Spain. Previous work has already shown that ficient to disrupt the egg-laying process, thus protecting mating males do have more recently laid eggs (white eggs) the eggs against parasitoid attack. Thus, males seem to and that these are probably laid by the mating female accept eggs only when the benefits for offspring survival between successive copulations (Garcı´a-Gonza´lez & are substantial, whereas in contexts in which offspring Gomendio 2003b). In the present study we focused on are not at such high risk males are less likely to accept old (yellow) eggs because they were already carried by eggs. We suggest that male acceptance behaviour is impor- the mating male before the beginning of the current cop- tant because males from populations in southern Spain ula, and are therefore the only cue that females could have reject up to 70% of attempts by females to lay eggs, used when choosing mating partners. The results from the whereas rejection rates are lower in populations from mate choice experiment also fail to support the sexual se- other regions (Miettinen & Kaitala 2000). The sensitivity lection hypothesis: when females were given the choice of of males to variation in parasitism pressure thus supports mating with two males, one with and the other without the view that care by males is favoured when offspring eggs, they showed no preferences. These results are in face high mortality risks and the benefits of care thus ex- agreement with an experiment by Kaitala (1998) carried ceed the costs to males. These findings are based on out with females from a different population. Thus, sexual a comparison of two populations; thus, data from more selection does not seem to have been important in the populations are needed to confirm our conclusions. evolution of egg carrying by males. Differences in levels of egg carrying could also result The natural selection hypothesis suggests that males from differences in sex ratio, so that biases in favour of care for offspring because of the benefits in terms of females could result in more eggs being produced and laid offspring survival. This hypothesis has two predictions: (1) on males, or from differences in population densities, males should be more prone to accept eggs in populations since females could lay more eggs on males at high where egg survival on plants is low, and (2) egg carrying by densities because encounter rates are higher (Gomendio & Reguera 2001). The first possibility can be excluded 100 since the sex ratio was 1:1 in both populations. The 90 second possibility can also be excluded because densities 80 were slightly higher in the population in which a smaller 70 proportion of males carried eggs. Finally, differences in 60 female fecundity could also influence the results. Inter- 50 population differences in fecundity are possible because 40 in southern Spain females are heavier than in central 30 Spain, and female body weight is correlated with female fe- 20 % Parasitized eggs cundity (Reguera 1999). However, if differences in fecun- 10 dity were responsible for differences in levels of egg 0 AdultsPlants Adults Plants carrying we would expect higher levels of egg carrying in Central Southern southern Spain, which is the opposite of what we found. Male care is expected to evolve through natural selec- Figure 3. Comparison of the percentage of eggs parasitized in the egg manipulation experiment when carried by individuals ( ) and tion only when a certain degree of paternity has been when laid on P. argentea inflorescences (,) in central and southern achieved, since the benefit is measured in terms of survival Spain (total eggs carried by individuals: central Spain: N ¼ 365; of true genetic offspring. Females mate with several males southern Spain: N ¼ 303; total eggs laid on plants: central Spain: through the season and our own results from previous N ¼ 42; southern Spain: N ¼ 81). studies show that the last male to copulate with a female GOMENDIO ET AL.: NATURAL SELECTION FAVOURS MALE CARE 769 has an intermediate level of paternity (Garcı´a-Gonza´lez and costs to males in terms of loss of mating opportunities et al. 2003). Females tend to lay more eggs after copula- are low. Males are sensitive to variations between popula- tion, so males accepting eggs after they have copulated tions in egg mortality risks, intensifying care when eggs with a female have a 40% chance of carrying their off- are unlikely to survive without it. Males seem willing to spring, and this probability does not change with time provide care under conditions of intermediate certainty of (Garcı´a-Gonza´lez et al. 2003). When levels of paternity paternity when the impact on offspring survival is high. are intermediate, males would be expected to provide care only when the impact on offspring survival is high. Our findings show this to be the case since eggs laid on Acknowledgments plants suffer high mortality rates as a result of parasitoids, For helpful assistance with fieldwork we thank Eva Banda, and egg carrying by males protects them from this cause Jose Antonio Blanco, Francisco Cabrero, Roberto Carbonell, of mortality. Thus, males readily provide care to eggs, Alicia Garcia, Sylvain Gandon, Aurelio Malo and Pablo even if only a proportion of them are their genetic off- Reina. We also thank Professor Virgilio Caleca, University spring, when egg carrying is needed to improve offspring of Palermo, Sicily, for identifying the parasitoid for us. F.G.-G. survival. However, in populations where parasitism is low, was supported by a Ph.D. scholarship from the Ministry of and eggs have better survival prospects on plants, males Education and from the Ministry of Science and Technol- are less likely to carry eggs. ogy. A.R. was funded through a Ramon y Cajal Fellowship Male reproductive success depends to a great extent on from the Spanish Ministry of Education and Science. the number of females with which they mate. When males Further funding was provided by grants from the Asso- care for offspring, one of the main costs is a reduction in the ciation for the Study of Animal Behaviour, the Ministry of time and energy that they can spend looking for other Science and Technology and the Ministry of Education and females and mating with them. This, however, does not Science. We are grateful to Michael Jennions and an seem to be a significant cost for P.laciniata males since, once anonymous referee for constructive comments on the the eggs are glued to their backs, they are free to move and manuscript. they continue to mate with other females. Furthermore, as we showed in this study, egg carrying does not decrease References their mating chances. In natural populations males carry four eggs on average, so back space is not a limiting factor, Balshine, S., Kempenaers, B. & Sze´kely, T. 2002. Conflict and co- as it seems to be in giant water bugs (Heteroptera: Belosto- operation in parental care. Introduction. Philosophical Transactions matidae) (Smith 1997). Similarly, egg carrying does not of the Royal Society of London, Series B, 357, 237e240. seem to decrease foraging efficiency. The fact that egg carry- Chapman, T., Arnqvist, G., Bangham, J. & Rowe, L. 2003. Sexual ing seems to convey a cost only in terms of vulnerability to conflict. Trends in Ecology & Evolution, 18,41e47. predators (Reguera & Gomendio 1999) could also have fav- Clutton-Brock, T. H. 1991. The Evolution of Parental Care. Princeton, oured the evolution of care by males in this species. New Jersey: Princeton University Press. Do our findings from P. laciniata have general implica- Crawley, M. J. 1993. GLIM for Ecologists. Oxford: Blackwell Scientific. tions for the evolution of paternal care in other insects? Favila, M. E. 1993. Some ecological factors affecting the life-style of Taken together, the results from our long-term study Canthon cyanellus cyanellus (Coleoptera Scarabaeidae): an experi- e show that male care is favoured when offspring survival mental approach. Ethology Ecology and Evolution, 5, 319 328. is considerably improved, and costs to males in terms of Garcı´a-Gonza´lez, F. 2002. La evolucio´n del transporte de huevos en loss of mating opportunities are low. This is consistent Phyllomorpha laciniata (Het., Coreidae): una aproximacio´n com- portamental, fisiolo´gica y molecular para explicar su significado with other studies that show that male care is most likely adaptativo en machos y hembras. Ph.D. thesis, Universidad Com- to occur when either males do not suffer a marked loss in plutense de Madrid. mating opportunities or such opportunities are scarce Garcı´a-Gonza´lez, F. & Gomendio, M. 2003a. Oviposition site selec- (Zeh & Smith 1985; Clutton-Brock 1991). In those species tion and oviposition stimulation by conspecifics in the golden egg in which males carry the eggs glued to their body, males bug (Phyllomorpha laciniata): implications on female fitness. can continue to mate with other females while they look af- Behavioral Ecology and Sociobiology, 53, 385e392. ter offspring. In species in which males have to stay at a par- Garcı´a-Gonza´lez, F. & Gomendio, M. 2003b. A field test of the in- ticular site looking after eggs laid on the substrate, the loss traspecific brood parasitism hypothesis in the golden egg bug in mating opportunities may be small when local densities (Phyllomorpha laciniata). Behavioral Ecology and Sociobiology, 53, of receptive females are high, males can mate with several 332e339. females, and males can look after several clutches simulta- Garcı´a-Gonza´lez, F. & Gomendio, M. 2004. Adjustment of copula neously (Reynolds et al. 2002). In both cases, the main ben- duration and ejaculate size according to the risk of sperm compe- efits for males may be in terms of offspring survival. It has tition in the golden egg bug (Phyllomorpha laciniata). Behavioral Ecology, 15,23e30. been suggested that among birds, male care has evolved Garcı´a-Gonza´lez, F., Nu´n˜ez, Y., Ponz, F., Rolda´n, E. R. S. & when extra mating opportunities for males are scarce (re- Gomendio, M. 2003. Sperm competition mechanisms, confidence viewed in Owens 2002). Experimental evidence also shows of paternity and the evolution of paternal care in the golden egg bug that males are more likely to desert offspring when the op- (Phyllomorpha laciniata). Evolution, 57, 1078e1088. portunities for remating are high (Keenleyside 1983; Clut- Garcı´a-Gonza´lez, F., Nu´n˜ez, Y., Ponz, F., Roldan, E. R. S. & ton-Brock 1991; Webb et al. 2002). Gomendio, M. 2005. 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