Timing of oviposition enables dominant female burying beetles to destroy brood-parasitic young

Animal Behaviour 82 (2011) 1227e1233

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Timing of oviposition enables dominant female burying beetles to destroy brood-parasitic young Anne-Katrin Eggert a, b, *, Josef K. Müller b,1 a b

Behavior, Ecology, Evolution, and Systematics (BEES), School of Biological Sciences, Illinois State University Department of Evolutionary Biology and Ecology, Faculty of Biology, University of Freiburg

a r t i c l e i n f o Article history: Received 28 April 2011 Initial acceptance 6 July 2011 Final acceptance 9 August 2011 Available online 29 September 2011 MS. number: A11-00350 Keywords: brood parasitism burying beetle infanticide larvicide Nicrophorus reproductive skew

Infanticide can increase an individual’s reproductive success through reduced competition for its offspring. Here we document the occurrence and effects of infanticidal behaviour by dominant female burying beetles, Nicrophorus vespilloides, in nontolerant breeding associations. Burying beetles feed and guard their young on buried carcasses, and on small carcasses, conspecific females engage in intense aggression. Dominants largely exclude subordinates from the carcass and are the sole caregivers for the larvae. Although subordinates and dominants produce similar egg clutches, only one out of five surviving offspring is the subordinate’s. We hypothesized that much of this skew is due to temporally cued infanticide by dominants. Females oviposit over the course of 24e48 h, and kill larvae they encounter prior to the hatching of their own larvae. Such infanticide can lead to significant skew only if the dominant’s larvae hatch significantly later than the subordinate’s, and if more subordinate than dominant larvae hatch during the infanticidal phase. The subordinate’s share of larvae hatching during the parental phase must be smaller than her share of eggs, and the portion of subordinate larvae killed by the dominant should increase with an increasing difference in oviposition time between the two females. These predictions were met. Dominant females, who had exclusive access to the carcass and larvae, were able to use temporal cues to destroy over half of the subordinates’ first-instar larvae. This is the first study to unambiguously document the primary mechanism underlying reproductive dominance in nontolerant associations of female N. vespilloides. Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Over the last three decades, researchers in animal behaviour have come to accept infanticide as a behavioural strategy used widely by animals to further their own fitness interests. The initially common argument that the killing of conspecific young should be viewed as aberrant rather than adaptive behaviour, resulting from mental disease or inappropriate living conditions in captivity (see Hrdy & Hausfater 1984), has become quite rare. There is now abundant empirical evidence that many infanticidal acts yield selective benefits to the perpetrator, such as increased reproductive opportunities, increased access to limited resources, direct nutritional benefits, or the prevention of misdirected parental care (Hrdy 1979; Elgar & Crespi 1992; Parmigiani & vom Saal 1994; Ebensperger 1998; van Schaik & Janson 2000; Ebensperger & Blumstein 2007). When animals rear their young in groups as communal breeders, or use the same site for reproduction, opportunities for infanticide are numerous, and infanticidal behaviour, both indiscriminate

* Correspondence: A.-K. Eggert, School of Biological Sciences, Illinois State University, Normal, IL 61790-4120, U.S.A. E-mail address: [email protected] (A.-K. Eggert). 1 J. K. Müller is at the Department of Evolutionary Biology and Ecology, Faculty of Biology, University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany.

(Johnstone & Cant 1999) and discriminate (Hager & Johnstone 2004), can be selectively favoured. In a number of animals including many communal breeders, infanticidal individuals rely on temporal cues as indirect indicators of relatedness to distinguish between their own and unrelated young (Elwood 1994). Temporal cues are also used by female burying beetles (genus Nicrophorus) to distinguish their own young from unrelated young (Müller & Eggert 1990). Burying beetles provide parental care in the form of parental defence and regurgitations to the young on small vertebrate carcasses that they inter as a food resource for the developing young (Pukowski 1933). Parental beetles are unable to directly distinguish their own larvae from unrelated conspecific larvae, but females that have oviposited eggs around a carcass kill first-instar larvae that appear on the carcass too soon after oviposition to be the respective female’s own offspring (Müller & Eggert 1990). Females switch from infanticidal to parental behaviour at about the time their own larvae hatch, and subsequently, they accept newly hatched Nicrophorus larvae indiscriminately (Müller & Eggert 1990; Trumbo 1994; Oldekop et al. 2007). Once females have begun to care for larvae, they remain ‘parental’ until the larvae have completed development on the carcass (Müller & Eggert 1990). Breeding females may encounter ‘early’ larvae in a number of different situations. Buried carcasses are sometimes located by

0003-3472/$38.00 Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2011.09.001

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intraspecific or interspecific intruders (Scott 1990; Trumbo 1990a; Robertson 1993), and such intruders, if successful, kill the original residents’ larvae during or after the take-over attempt (Trumbo 1990b), presumably relying on the same time-based mechanism. Even in a situation that superficially appears much more peaceful, time-based infanticide is frequent. When the reproductive resource is relatively large, females tolerate each other’s presence on the carcass and form joint-breeding associations, where females may even feed larvae side by side (Eggert & Müller 1992; Trumbo & Wilson 1993). Despite this apparently cooperative behaviour, both females engage in repeated bouts of infanticide when oviposition is not perfectly synchronous (Eggert & Müller 2000), in apparent attempts to bias the composition of the resulting brood in their own favour. In other species of burying beetles, communally breeding females may destroy each other’s eggs as well (Scott 1997). Here, we focus on a distinctly different type of femaleefemale association, originally described as ‘intraspecific brood parasitism’ (Müller et al. 1990). On smaller carcasses, Nicrophorus females typically engage in violent aggressive interactions whenever they encounter each other, frequently causing serious injuries (Pukowski 1933; Bartlett & Ashworth 1988; Müller et al. 1990; Eggert & Müller 1992). After only a few interactions, they have established a dominance hierarchy, and the subordinate female flees rapidly whenever an encounter is imminent, in an apparent attempt to avoid more physical altercations. From this time on, the subordinate gets minimal access to the carcass, whereas the dominant spends most of her time on or around the carcass (Müller et al. 1990). Nevertheless, in both the field and the laboratory, the subordinate usually oviposits in the soil off the carcass (Eggert et al. 2008) but does not provide parental care, abandoning the carcass prior to the appearance of larvae (Müller et al. 1990, 2007). Thus, only the care-giving dominant female interacts with the larvae and cares for some of the subordinate’s offspring along with her own. Parentage data have shown that such broods contain very few subordinate young, typically one or two, amounting to less than 20% of the surviving offspring in both laboratory and field (Müller et al. 1990, 2007). As to the proximate cause of this pronounced reproductive skew, three candidates have been suggested: higher dominant fecundity, selective ovicide, or selective infanticide committed by dominants (Müller et al. 1990, 2007; Eggert & Müller 1992, 1997; Scott 1997, 1998). Although selective ovicide occurs in communal associations of Nicrophorus tomentosus (Scott 1997), there is no evidence that it occurs in brood-parasitic associations of Nicrophorus vespilloides (Eggert et al. 2008). In N. vespilloides, the greater fecundity of dominant females alone also failed to explain the extreme skew observed among surviving young, leaving selective infanticide as the only untested hypothesis (Eggert et al. 2008). We thus hypothesized that temporally cued larvicide (Müller & Eggert 1990; Eggert & Müller 2000) accounts for the severe reproductive skew in brood-parasitic associations. We assessed the validity of this hypothesis by testing four predictions derived from it. Dominant females can only use temporal cues to selectively cull subordinate larvae if their own larvae begin to hatch significantly later than subordinate larvae. Thus, our first prediction was that the dominant individual’s eggs should begin to hatch significantly later than the subordinate individual’s eggs. Each female lays eggs over 1e2 days, and the larvae hatch over the same time span when temperatures are constant. Second, if the infanticide is to differentially victimize subordinate larvae, then a significantly larger portion of the subordinate’s than the dominant’s larvae must hatch during the dominant’s infanticidal period. Third, if skew is significantly affected by the dominant’s infanticidal activity, then the subordinate’s share of larvae that hatch during the dominant’s parental phase (those that would not fall victim to temporally cued infanticide) must be significantly smaller than her share of all eggs

laid around the carcass. This means that reproductive skew must be significantly greater after the dominant’s infanticidal phase than it is before this phase. Finally, we predicted that a greater differential in the onset of oviposition between subordinate and dominant would result in a larger portion of subordinate (brood-parasitic) larvae being destroyed by infanticide. METHODS General Experimental Procedure and Beetle Maintenance We collected individual N. vespilloides Herbst in baited pitfall traps near Freiburg, Germany, and reared first-generation offspring in the laboratory. Adult offspring were sexed and kept in containers with moist peat in groups of up to six consexuals, in environmental chambers at 20
C on a 16:8 h light:dark cycle. They were fed decapitated mealworms twice a week until we started dye-feeding. For 2 weeks prior to an experiment, females were fed ground beef mixed with a fat-soluble dye twice a week (about 200 mg of Sudan Red 7B, or 400 mg of Sudan Blue II per 20 g of meat). Food dyes result in eggs that are coloured pink or light blue, respectively. The first clutch of eggs produced after dye-feeding can be unequivocally assigned to a ‘red’ or a ‘blue’ female. Experimental beetles were between 20 and 60 days of adult age, but the two females placed on the same carcass differed by less than 7 days in age. We made every effort to avoid systematic differences in age between dominants and subordinates, because age is known to affect female reproductive performance (Lock et al. 2007; Creighton et al. 2009; Trumbo 2009). Females that were placed on the same carcass differed in pronotum width by at least 0.25 mm. Eggs and experimental containers were kept at a constant temperature of 20
C throughout the experiments. Because temperature strongly affects the duration of embryonic development, keeping parent beetles and eggs at identical temperatures is important. Therefore, in all of our experiments, eggs were kept in the centre of an environmental chamber and containers with beetles were rotated around this centre to minimize temperature variation. Timing of Larval Hatching Experimental females were measured, marked individually, and left with a male for 48 h to ensure a sufficient supply of sperm for fertilization of eggs. Three hours before ‘lights off’, two females that had been fed different dyes were placed on a mouse carcass of approximately 15 g (0.3 g) mass in a transparent plastic box (20  20  6 cm) half-filled with compacted moist peat. Flexible thin metal wire was tied around the mouse and its loose end fastened to one corner of the box to keep the beetles from continually moving the carcass about the box and causing significant inadvertent destruction of eggs. The containers were kept under the beetles’ normal photoperiod until the carcass was buried, at which time they were transferred to a dark chamber. All further handling occurred in the dark under red light. The beetles and their carcass were transferred to a new container with peat after 42 h. The original container was searched for eggs, which subsequently were stored on moist filter paper and checked for hatching every 4 h. The beetles and their carcass were transferred again 48 h and 96 h later, if necessary, when a female’s first larva hatched, and once the dominant had begun to accept larvae (see below). Prior to each transfer, and whenever we inspected a carcass for larvae, we recorded the position of both females relative to the carcass in the original container. The peat in used containers was always searched for eggs. At a constant temperature of 20
C, the time from oviposition of eggs to hatching of larvae in N. vespilloides is relatively constant (Müller 1987: mean þ SD ¼ 56 þ 1.5 h, N ¼ 138 eggs; Oldekop et al.

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2007: mean þ SD ¼ 57 þ 1.1 h, N ¼ 25 families). Thus, larval hatching times are closely correlated with time of oviposition, such that hatching time can be used as a surrogate for oviposition time (Müller & Eggert 1990). Although egg size has no effect on time to hatching in N. vespilloides, late eggs within a clutch develop about 3 h faster than early eggs (Smiseth et al. 2006). However, this effect has no bearing on the results presented here, because we only looked at a female’s first larvae.

presence on the carcass was very clear, as subordinates were hardly ever observed on carcasses (1% of all inspections, 4/444) while dominants spent most of their time on the carcass (89%, 394/444). The larger female became dominant in 93% of all pairs (26/28), and the smaller female became dominant in the remaining 7% of pairs (2/28). Injuries occurred regularly; in about one-third of all pairs (9 of 28), one or both females had incurred nontrivial injuries at the time the experiment was terminated.

Definition of Dominance, Injuries, and Testing for Infanticidal Behaviour

Subordinate Contribution to Egg Numbers

The female that was observed on the carcass on more occasions was considered dominant. We noted injuries from aggressive interactions, which typically affect the limbs. Injuries were considered trivial if only one or two tarsal or antennal segments were missing, as occurs even in monogamous breeding pairs or single females. To test the dominant female’s infanticidal or parental disposition, we first started adding three first-instar larvae to the carcass 46 h after placing the females on a carcass. These larvae had been produced by other pairs that we had placed on carcasses 1 day prior to the experimental females. Every 4 h thereafter, we first inspected the carcass visually for any surviving larvae. If none were found, we added three more first-instars to the carcass. If surviving larvae were present, we added another set of three first-instars and terminated the trial 4 h later, at which time we always found more than three surviving larvae on the carcass. We then recorded as the time of transition to parental behaviour the time at which the first survivors had been placed on the carcass. A trial was also terminated if we found more than three larvae during any of our inspections (which happened on three occasions and indicated that we had overlooked larvae in the previous inspection). Whenever more than three larvae were found on a carcass at time x, we assumed that larvae that had been added 4 and 8 h earlier had been accepted, and thus we marked x8 h as the time of transition to parental behaviour. We then searched the peat for eggs one last time, and continued to record hatching times until all larvae had hatched. This protocol effectively assesses the infanticidal versus parental disposition of the tested adult, and similar protocols have been used to this effect in several earlier studies (Müller & Eggert 1990; Eggert & Müller 2000; Oldekop et al. 2007). Once females become parental, they do not reverse to an infanticidal state unless all larvae disappear. Thus, the female’s disposition at a particular time indicates whether or not any larvae that hatched at that time would survive, as females are unable to directly assess the relatedness of larvae.

Similar to an earlier study on the same species (Eggert et al. 2008), dominants laid significantly more eggs than subordinates (paired t test: t27 ¼ 2.226, P ¼ 0.035). We found an average of 33.8  1.8 eggs laid by dominants and an average of 28.1  2.0 eggs laid by subordinates (N ¼ 28 pairs). On average, a subordinate clutch contained just slightly less than half (mean  SE ¼ 44.9  2.3%) of all eggs laid around a carcass. Relative Time of Larval Hatching Consistent with our first prediction, the subordinate female’s larvae began to hatch significantly earlier than the dominant’s. In 22 of 28 pairs, the subordinate’s larvae started hatching before the dominant’s did, in two pairs, both females’ larvae hatched at the same time, and in four pairs, the dominant’s larvae started hatching earlier. Hatching of the subordinate’s first larva occurred, on average, 17 h earlier than hatching of the dominant’s first larva (71.7  1.3 h and 88.4  2.7 h, respectively, after the females’ first contact with the carcass; paired t test: t27 ¼ 5.787, P < 0.0001; see Fig. 1). Based on a larval development time of 56 h (Müller 1987), the subordinate’s first eggs were laid, on average, 16 h after first contact with the carcass, and those of the dominant were laid 32 h after first contact with the carcass. Larvae Affected by Infanticide Dominants killed larvae that arrived on the carcass early. Most dominant females showed infanticidal behaviour until their own first larvae began to hatch, or even until shortly after their own larvae started hatching. Only two (of 28) dominant females accepted larvae that had been added to the carcass before their own larvae hatched (4 h earlier in both cases). On average, dominants

10

Analyses

RESULTS Body Size and Dominance, Presence on the Carcass, and Injuries Although tolerant associations are occasionally observed on carcasses of this size, there were none in this experiment. The distinction between dominant and subordinate on the basis of

8 Number of females

We excluded three trials from all analyses because, in one trial, one of the females did not lay eggs and was killed by the other female on that carcass, and in two trials, the carcass was not buried within 1 day. In three other trials, over 30% of eggs were unfertilized, and those trials were not available for analyses that involved the timing of larval hatching. This left a total of 28 pairs of females to be analysed. We compared a number of reproductive variables in paired t tests, comparing values of dominant and subordinate females from the same trial to each other. Values are given as means  SE.

6

Subordinate Dominant

4 2 0

76 100 124 64 88 112 Onset of larval hatching after carcass detection (h)

Figure 1. Onset of larval hatching in egg clutches produced by dominant and subordinate burying beetles. The X axis shows a series of 4 h intervals after the females first detected the carcass; the Y axis shows the number of clutches in which larvae began to hatch in the respective interval (total N ¼ 28 broods).

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started to accept larvae that had been added to the carcass about 3.7  1.5 h after their own larvae began to hatch and 20.3  0.4 h after the subordinate’s larvae hatched, and thus, the delay between the hatching of a female’s first larva and the time at which larvae were first accepted was significantly greater for the subordinate (paired t test: t27 ¼ 5.86, P < 0.0001). Thus, the dominants’ infanticidal behaviour covered a large part of the period during which subordinate larvae hatched. Consistent with our second prediction, a majority of the larvae that hatched during the infanticidal phase, and would thus have fallen victim to the dominant’s infanticidal behaviour, were the subordinate’s offspring (89.1%, 417 of 468). The percentage of the subordinate’s larvae that hatched during the infanticidal phase (58.1%, 417 of 718) was greater than that of the dominant’s (6.1%, 51 of 839; c21 ¼ 497.62, P < 0.0001; see Fig. 2). In most broods, fewer than 10% of the dominant’s and more than 40% of the subordinate’s larvae hatched in the infanticidal phase and would have been killed and eaten by the dominant female. Larvae Hatching during Parental Phase We will refer to larvae that hatched during the dominant’s parental phase (i.e. after the dominant had ceased killing larvae) as ‘acceptable’ larvae. Roughly a quarter (mean  SE ¼ 27.4  2.7%) of all such acceptable larvae in the brood were the subordinate’s. The subordinate’s contribution to the entire brood after temporally based larvicide was thus significantly smaller than it was at the egg stage (27.4 versus 44.9%; paired t test of portion of eggs and acceptable larvae contributed by the subordinate in the same trial: t27 ¼ 7.432, P < 0.0001), which is consistent with our third prediction. Hatching Differential and Selective Infanticide The earlier a subordinate’s larvae began to hatch relative to the dominant’s larvae, the higher was their mortality due to early infanticide by the dominant female (regression: R2adj ¼ 0.623, F1,26 ¼ 45.63, P < 0.0001). The greater the differential between the subordinate and dominant females’ first-hatched larvae (i.e. the greater the difference in oviposition times), the greater was the percentage of subordinate larvae that hatched during the dominant female’s infanticidal phase, and thus, that would have been killed and eaten (see Fig. 3). This finding is consistent with our fourth prediction.

100 90 80

Infanticidal phase Parental phase

% Larvae

70 60 50

80 60 40 20 0 −20

0

20 40 Hatching differential (h)

60

Figure 3. Percentage of larvae produced by subordinate female burying beetles that were subject to infanticide by the dominant female (hatching in the dominant’s infanticidal phase) as a function of the oviposition differential between the two females (lag in hatching time between the first subordinate and first dominant larva).

DISCUSSION Access to Carcass and Larvae as a Result of Fights Consistent with earlier findings (Müller et al. 1990, 2007; Eggert et al. 2008), the dominant females in our observations had exclusive control of the carcass during larval hatching. Subordinates were hardly ever observed on the carcass before or during parental care, despite the fact that our experimental design forced the subordinate to stay in the vicinity of the carcass. In an earlier study, subordinates were rarely seen on the carcass even during the first day after contact with the carcass (Müller et al. 1990). As subordinates hide in the soil, avoid both the carcass and the dominant female, and flee as soon as they perceive the dominant to be close, fights become increasingly rare over time (J. K. Müller & A.-K. Eggert, personal observation). In the experiment described here, the dominant was usually found on the carcass when we added larvae, while the subordinate was typically buried in the substrate away from the carcass. Subordinates that were visible on the surface were usually attempting to escape by flying or climbing the sides of the container, never attempting to fight the dominant. This is consistent with the observation that subordinates usually abandon the carcass before larvae are found on the carcass (Müller et al.1990, 2007). Monopolizing the carcass thus gives the dominant complete control over the fate of first-instar larvae arriving on the carcass. This situation clearly qualifies as intraspecific brood parasitism insofar as the dominant female (host) is fooled into raising unrelated offspring, diverting resources away from her own young (Roldán & Soler 2011). Timing of Oviposition and Effects of Dominant Infanticide

40 30 20 10 0

100 % Subordinate larvae killed

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N = 718

N = 839

Subordinates’ larvae

Dominants’ larvae

Figure 2. Percentage of larvae produced by subordinate and dominant female burying beetles that hatched during the dominant female’s infanticidal and parental phases, respectively. Total number of larvae produced by subordinates and dominants is indicated below the X axis (N ¼ 28 broods). Larvae that hatched during the infanticidal phase did not survive.

Our study clearly demonstrates that differential larvicide by the dominant female is an important cause of the pronounced reproductive skew observed in brood-parasitic associations of N. vespilloides in the laboratory and the field (Müller et al.1990, 2007). Only the dominant or host female has direct contact with larvae because she excludes the subordinate from the carcass. Our results show that in brood-parasitic associations, the dominant initiates oviposition almost an entire day (17 h) later than the subordinate, such that many of the subordinate’s larvae hatch long before those of the dominant. Using the internal mechanism that causes a behavioural switch around the time that her own larvae begin to hatch (Müller & Eggert 1990; Eggert & Müller 2000), the dominant is then

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able to selectively cull any larvae that hatch too early to be her own offspring. More than half of the subordinate’s larvae, but only 6% of the dominant’s, hatched during the dominant’s infanticidal phase. Clearly, the timing of oviposition is vitally important for reproductive skew in brood-parasitic associations. We can make these statements with certainty although we never actually added any of the two females’ own offspring to the carcass because multiple studies have demonstrated that female burying beetles in general, and N. vespilloides in particular, are unable to discriminate between own and unrelated offspring if temporal cues are not available (Müller & Eggert 1990; Trumbo 1994; Eggert & Müller 2000; Lock et al. 2004; Oldekop et al. 2007). When females are in their infanticidal phase, they invariably destroy any burying beetle larvae they encounter, be they related or not, and they do not revert back to this behaviour once they are caring for larvae unless the entire brood is destroyed (Eggert & Müller 2000). Other Causes of Reproductive Skew In the nontolerant, brood-parasitic associations we focused on here, a small degree of skew is already manifest at the egg stage. Consistent with an earlier study (Eggert et al. 2008), we found subordinate females in the present study to be slightly less fecund than dominants. This component of skew appears to be caused by the subordinate’s limited opportunities to feed on the carcass (Eggert et al. 2008). In the present study, reproductive skew was far more pronounced after temporally cued larvicide than at the egg stage: among the larvae that hatched late enough to be accepted and cared for by dominant females, only 27% were the subordinate’s, whereas among all eggs, 45% were the subordinate’s. However, our experimental subjects were all well fed prior to the experiment, and in the field, limited access to food may well lead to greater fecundity differences between the females. Significant ovicide, which may account for skew in the North American congener N. tomentosus (Scott 1997), does not appear to occur in N. vespilloides (Eggert et al. 2008). Skew after Temporal Infanticide versus Eventual Skew Even after temporally cued larvicide, however, skew was less pronounced than previously observed among surviving offspring, where the subordinate’s offspring contribution was only 16% (in the laboratory: Müller et al. 1990) or 17% (in the field: Müller et al. 2007) of the entire brood. We suggest that the additional skew is generated during brood reduction. Parental beetles in monogamous pairs on small carcasses cull excess offspring to obtain a brood size that the carcass can support, a phenomenon known as brood reduction (Bartlett 1987; Bartlett & Ashworth 1988; Trumbo 1990c; Creighton 2005). Dominants probably show the same behaviour in mixed broods. In our trials, the number of first-instar larvae that hatched after the dominant’s infanticidal phase had ended (37.6  2.0) exceeded the carrying capacity of a 15 g carcass, and some of these larvae would fall victim to brood reduction. We suggest that more of the subordinate’s larvae are killed during this brood reduction. If each dominant female had culled all larvae that hatched after 30 or more larvae were already present on the carcass, subordinates would have contributed an average of 17.5  2.8% of all surviving larvae. However, this calculation assumes that late larvae are the sole victims of brood reduction, which may or may not be the case; at present, no information is available about the criteria that parental beetles use when choosing which larvae to cull. Timing of Oviposition and Associated Costs and Benefits Many researchers working on burying beetle reproduction have assumed that the ephemeral nature of carrion constrains oviposition

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to occur as quickly as possible, with little individual or systematic variation. Female N. vespilloides breeding in a noncompetitive situation (single, or in monogamous pairs), usually act according to this assumption and begin oviposition about 12 h after their first contact with the carcass, such that larvae begin to hatch after about 68 h (Eggert & Müller 2000). However, in tolerant breeding associations, both females oviposit much later than that, without any systematic difference between females (Eggert & Müller 2000). The present study documents a status-associated difference between females in nontolerant associations: the subordinate oviposited at a time that was fairly similar to single females, but dominants began oviposition significantly later. Late oviposition aids dominant females in their selective infanticide of unrelated larvae because it increases the contrast between the hatching times of larvae produced by the subordinate and the dominant female, allowing better discrimination with the same indirect temporal mechanism of recognition. If dominants benefit from late oviposition through improved discrimination, why do subordinates not oviposit later to deny the dominant this benefit? It is possible that subordinates are constrained to oviposit earlier because of nutritional limitations or injury risks. A female’s nutritional condition affects the number of eggs she can lay (Eggert et al. 2008), and may affect how long she can afford to wait before ovipositing, or over how long a period she can continue to lay eggs. Female N. vespilloides in monogamous pairs usually gain significant amounts of weight in their first days on the carcass, despite the fact that they oviposit during that time, indicating significant food intake on the carcass; subordinates would largely be denied this benefit of carcass residency. In our experiment, females were fed vertebrate carrion for 2 weeks prior to encountering a carcass, which might have reduced nutritional effects compared to the field, where many females caught in baited pitfall traps are in very poor nutritional condition. However, the early onset of oviposition in subordinates may be tactical rather than constrained. Larvae that hatch early are at increased risk of death due to temporally cued infanticide, but latehatching larvae may be at greater risk of being culled in the context of brood-reduction behaviour (Bartlett 1987; Trumbo 1990c). If the dominant female has any information about the time at which her competitor begins to oviposit, she should adjust her own oviposition accordingly to minimize the survival of subordinate young. The lack of a significant correlation between the time at which the dominant and the subordinate begin to oviposit (correlation coefficient: r ¼ 0.11), however, suggests that the dominant does not know when her competitor begins to oviposit. If the competitors do not know each other’s oviposition times, the most productive oviposition pattern for a subordinate may involve a bet-hedging tactic in which she oviposits over an extended period to ensure some degree of overlap in hatching with the dominant’s first larvae. Even for the dominant, there must be limits to how late oviposition can still produce young, or we might not see any overlap in the hatching periods of the females’ larvae, or even any reproductive attempts by subordinates. As time passes, the condition of the carcass deteriorates more and more, and the dominant risks losing the entire resource to decomposition. When the first egg clutch is destroyed and females produce a replacement clutch at a later time, the resulting brood is significantly smaller than a first brood because of reduced food quality and quantity (Müller 1987). Decomposition clearly limits the benefits of late oviposition. In the experiment presented here, we used carcasses that were frozen after death and thawed immediately prior to being presented to the beetles. Most carcasses available to beetles in the field are probably 1e2 days old, which could increase the need for the beetles to exploit them quickly. However, the subordinate’s contribution to the brood in

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brood-parasitic associations is similar in the field (16%) and the laboratory (17%), despite the fact that most carcasses in the field are 6e36 h old when they are found by free-flying beetles (Müller et al. 1990, 2007). This suggests that oviposition and infanticide in the field follow a pattern similar to the one described here. A Shift in Acceptance Threshold May Optimize Survival and Condition of Own Offspring A comparison with singly breeding females reveals another difference in temporal discrimination between females in competitive versus noncompetitive breeding situations. Uncontested females begin accepting larvae, unrelated or not, an average of 8 h before their own larvae begin to hatch (Müller & Eggert 1990), whereas dominant females in the present study often killed some of their own larvae, remaining infanticidal until 4 h after their own larvae began to hatch. A similar extension of the infanticidal period also appears to occur in tolerant breeding associations of N. vespilloides females, where both females commit infanticide (Eggert & Müller 2000). By shifting the switch from infanticidal to parental behaviour to a later time, dominants may be able to destroy a greater proportion of unrelated larvae. Destroying a few of their own offspring is relatively inconsequential to the reproductive success of dominants, as the brood will be further reduced in the process of adaptive brood reduction. Factors Affecting the Switch from Infanticidal to Parental Behaviour In our experiment, acceptance of larvae by dominants was closely correlated with the hatching of their own first larvae (Pearson correlation: r26 ¼ 0.896), suggesting that the time of oviposition significantly affects the time of the switch from infanticidal to parental behaviour. This is a point that we have argued in earlier papers (Müller & Eggert 1990; Eggert & Müller 2000), and it requires a physiological mechanism whereby females can determine how much time has passed since they started to lay eggs. Oldekop et al. (2007, page 1998) experimentally disrupted the light cycle experienced by beetles during carcass burial, as well as disturbing reproductive attempts on buried carcasses with 6 h of light exposure after 24 h of darkness, and they concluded that the timing of the behavioural switch is ‘sensitive to photic cues and access to a carcass and is not affected by oviposition’. This statement is not compatible with our current results. In their first two experiments, oviposition and infanticide in the experimental group were both delayed by the same amount after an artificially long light period at the start of carcass burial. In their third experiment, the artificial light period occurred after 24 h of darkness, which the beetles would have probably perceived as conditions on a buried carcass, but it resulted in only a very slightly delayed acceptance time, which may be indicative of a more conservative acceptance behaviour similar to competitive situations, rather than a truly disrupted timing mechanism. If the timing had been truly disrupted, we should have seen a shift in acceptance greater than the 4 h or so that they observed in experiment 3. We are not arguing here that access to a carcass and light do not play a role in this context, but our results strongly suggest that oviposition plays a very significant role as well. Temporal Cues as Indicators of Relatedness in Other Taxonomic Groups Hrdy (1979) identified four types of reproductive benefits that infanticidal individuals could derive from an infanticidal act. The larvicide we describe here is obviously a case of resource competition: by killing unrelated larvae, dominant females secure a larger share of the carcass for their own young. If dominants had no

means of selectively destroying subordinate offspring, more larvae would arrive on the carcass and a greater share of the dominants’ own offspring would die in the course of brood reduction. The use of the time of birth, oviposition or hatching of an offspring as a cue of maternity is not unique to burying beetles. In communal breeders, producing eggs or young too early can frequently be fatal. In communally breeding birds, early eggs are tossed out of the nest or buried by later-laying females (acorn woodpecker, Melanerpes formicivorus: Mumme et al. 1983; Koenig et al. 1995; groove-billed ani, Crotophaga sulcirostris: Vehrencamp 1977; Vehrencamp et al. 1988; smooth-billed ani, Crotophaga ani: Loflin 1983; guira cuckoo, Guira guira: Macedo & Bianchi 1997; Macedo et al. 2001). In mammals, pregnant females are much more likely to kill young, especially in the latter half of pregnancy, than are females after parturition (dwarf mongoose, Helogale parvula: Rasa 1994; house mouse, Mus musculus domesticus: Manning et al. 1995; meerkat, Suricata suricatta: CluttonBrock et al. 1998, 2001; banded mongoose, Mungos mungo: Gilchrist 2006). As females typically use the timing of their own reproductive events (oviposition, parturition) as a reference point for infanticidal behaviour, reproductive synchrony with dominant females can be important in many animals, especially in communal or cooperative breeders (Ebensperger 1998; Poikonen et al. 2008; Saltzman et al. 2008). In all these examples, females change their behaviour at the time of their own oviposition or parturition event; female burying beetles, however, must switch from infanticidal to parental behaviour days after oviposition and thus require a mechanism enabling them to measure time such that the behavioural switch will occur simultaneously with the hatching of their own larvae. Many male mammals are famously infanticidal when they first take over a group of females that already have unrelated young (e.g. lions, Panthera leo: Packer & Pusey 1983; langurs, Presbytes entellus: Hrdy 1974). Many male rodents that switch from infanticidal to parental behaviour when their own young are born appear to use the time of mating and/or cohabitation with a female as a cue to their paternity of offspring (e.g. gerbils, Meriones unguiculatus: Elwood 1977, 1980; house mice, Mus musculus: vom Saal 1985; white-footed mice, Peromyscus leucopus: Cicirello & Wolff 1990; various other rodents: see references in Ebensperger & Blumstein 2007). Male mice make use of the number of days (diurnal lightedark cycles, Perrigo et al. 1990) to estimate the time at which their own young should be born, but in burying beetles, the exact mechanism is not understood (Oldekop et al. 2007). In some communal mammals in which dominant females commit infanticide based on temporal information, infanticide may lead to reproductive restraint by subordinates (banded mongoose, Mungos mungo: Gilchrist 2006; common marmoset, Callithrix jacchus: Saltzman et al. 2008; meerkat, Suricata suricatta: Young et al. 2008). In burying beetles, in contrast, subordinates fail to show any reproductive restraint, apparently laying as many eggs as they possibly can (Eggert et al. 2008). Subordinates act as if the risk of losing many potential offspring to infanticide by dominants at the first-instar stage is not important, since it is required to maximize the odds of producing any surviving offspring on a carcass monopolized by a dominant competitor. This suggests that especially for small individuals, carcasses are too rare and valuable to pass up a chance at current reproduction for potential future opportunities (Eggert & Müller 1997; Scott 1998). Acknowledgments We thank Scott Sakaluk for helpful comments on the manuscript and continued moral and material support. The work was supported by DFG grants to J.K.M. (Mu-1175/2-1 and 3-1).

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