Variation in wild-caught beetles: parental care

In our previous work, we observed that the provision of post-hatching care is highly variable, with either parent leaving the brood at any time, from soon after larval hatching to larval dispersal from the carcass22. Similar observations have been made by other researchers, both in the laboratory23 and in field studies of N. vespilloides24 and in other burying beetle species25. We quantified variation in the supply of care, under standardized conditions, by breeding wild-caught individuals in the laboratory (see 'Methods'). We bred 34 pairs, each in a box with a one-way exit port, through which the adults were free to leave at any time but could not return22, and noted the time of parental departure. While parents are caring for offspring they spend virtually all their time on, or very close to, the carcass. Previous studies have shown that once individuals stop caring in nature, they move away from the carcass and do not return25. Furthermore, even when parents were given the opportunity to return to their brood in laboratory experiments, they did not provide substantial levels of care26. Therefore it is extremely unlikely that actively caring parents accidentally wandered through the exit port in our experiments.

We found considerable continuous variation in the duration of parental attendance at the carcass, in both males and females (Fig. 1). At one extreme, both parents left before the larvae hatched in two of the 34 breeding attempts, whereas in five cases both parents stayed until larvae dispersed away to pupate. We also observed qualitative differences in carcass preparation. In nine cases, we noted that parents had made a feeding incision in the carcass before their larvae hatched (Fig. 1), but in the remaining 25 breeding attempts parents delayed biting a feeding incision until after their larvae had hatched. Parents potentially assist larvae in gaining access to resources on the carcass in two key ways: they bite a feeding incision, presumably so that newly-hatched larvae can more easily penetrate the carcass and feed upon it; and they provision offspring directly. Our experiment found variation in both forms of parental care in wild-caught beetles, bred under standard conditions in the laboratory.

Fig. 1 Variation in the duration of parental care in wild-caught individuals. Variation in the duration of maternal (orange bars) and paternal (purple bars) care, and the timing of biting the feeding incision in the carcass by wild-caught parents under laboratory conditions (green bars, data shown only for 9 pairs that inserted an incision prior to larval hatching). Data are scaled relative to the timing of larval hatching at 0 h. n = 34 pairs. Horizontal bars indicate when we removed parents in the No Care (blue bar) and Control (red bar) treatments. Note that each treatment reduces variation in the extent of parental assistance supplied to larvae, both in biting the feeding incision and caring for offspring after hatching Full size image

Variation in wild-caught beetles: larval mandibles

The high level of variation in the duration of parental attendance at the carcass, and in the extent to which the carcass is prepared prior to larval hatching, means that larvae may receive no parental assistance in gaining access to the resources on the carcass. The larval trait most likely to influence their performance, independently of the parents, is the larval mandibles because they are essential for gaining access to the carrion nest and consuming it. We began by testing whether larvae cope with variable levels of parental care by exhibiting adaptive phenotypic plasticity in the relative size of their mandibles. We paired wild-caught individuals and bred them in the laboratory under standard conditions (see 'Methods'). We exposed larvae to one of two different social environments, lying close to each extreme of the continuous variation in parental assistance that we documented in our first experiment (illustrated on Fig. 1). At one extreme, we created a ‘No Care’ environment by removing parents after carcass preparation was complete but before the larvae had hatched. At the other extreme we made a ‘Control’ environment by keeping parents within the breeding box so that they remained with their young until larval dispersal, and were able to provide care during this time. Although the level of care provided by parents in the Control environment was highly variable, parents still provided substantially more care for their young on average than those in the No Care populations (see below). We predicted that if mandibles exhibited adaptive phenotypic plasticity then they should be relatively larger for a given body size in the No Care environment than in the Control environment, to compensate for the lack of post-hatching parental assistance.

We collected third instar larvae from each treatment and weighed them. Then we dissected out the mandibles, mounted them, and measured their length (see 'Methods and materials', Supplementary Fig. 1). Although selection on the mandible is likely acting on first-instar larvae, we focused on the third instar to maximise precision in measuring both mandible length and body mass. This approach is justified by two observations. First, Nicrophorus beetles show consistency in morphology across instars: a larva with relatively large mandibles in its first instar will also have relatively large mandibles in its third instar27,28. This means that morphological adaptations that facilitate self-feeding in first instar larvae will be apparent across larval development. Second, developmental and genetic studies of other insects indicate that the mechanisms governing mouthpart size do not differ between larval instars29. Thus, we should be able to observe the correlated evolution of the mandibles of the third instar when selection has acted on the developmental mechanisms that dictate mandible size in the first instar.

We found no evidence for adaptive phenotypic plasticity in the mandible size of offspring from wild-caught parents. The scaling relationship between mandible size and body size did not differ significantly between the two different care treatments (ordinary least squares regression, OLS: t 103 = −1.21, P = 0.23; major axis regression, MA: LR = 1.55, P = 0.21, Fig. 2). Furthermore, whether raised in a No Care or the Control treatment, the slope of scaling relationship between mandible size and body size (i.e., the allometric slope, β) did not differ significantly from 0 (OLS regression, Control: t 52 = 1.60, P = 0.12; No Care: t 52 = −0.11, P = 0.91; Combined populations: t 106 = 1.58, P = 0.12. MA regression, Control: r 52 = 0.22, P = 0.12; No Care: r 52 = −0.02, P = 0.91; Combined populations: r 106 = 0.15, P = 0.12, Supplementary Table 1). We found instead a high level of variation in the relationship between mandible size and larval mass. Furthermore, on average, larval mandibles were consistently large, irrespective of larval mass (Fig. 2). One interpretation of this result is that by maintaining relatively large mandibles for self-feeding, larvae are adapted on average to anticipate the worst possible scenario of receiving no parental assistance at all in accessing the resources on the carcass.

Fig. 2 Larval mandible allometry in offspring of wild-caught parents. The allometric relationship between larval mandible length and larval body mass in the offspring of wild-caught parents. Larvae were either raised either in a Control environment (red filled datapoints, red solid line, n = 54) or a No Care environment (blue open datapoints, blue dashed line, n = 54). Ordinary least squares regression lines are shown with 95% confidence intervals Full size image

Experimental evolution

We exploited the high level of natural variation in parental care, and in the relative size of the larval mandibles, to establish populations of burying beetles in the laboratory (n = 4) that we subjected to experimental evolution. We imposed two different but predictable regimes of parental care, applied experimentally at each generation. Two populations experienced the Control treatment, which was identical to the Control treatment in the previous experiment on wild-caught individuals. Here both parents were left with their offspring throughout larval development. Most broods in this treatment received at least 24 h of maternal care (Fig. 1). Parents also cut a feeding incision cut into the carcass for their brood (Fig. 1). The remaining two populations experienced the same No Care treatment as described above. All broods in this treatment predictably experienced no post-hatching care at all (see 'Methods').

Experimentally evolved adaptations in parents

To determine the likelihood that parents in this treatment cut a feeding incision in the carcass for their brood, we assayed the populations after 13 generations of experimental evolution. We found that No Care parents had evolved to be more likely to insert a feeding incision in the carcass before we removed them experimentally (see Supplementary Materials, Supplementary Fig. 2). Specifically, parents from the No Care populations were approximately twice as likely to make an incision into the carcass prior to larval hatching, than individuals from either the Control populations or the Wild populations (binomial GLM: z = 5.28, P < 0.001). Therefore, after 13 generations of experimental evolution, parents predictably cut a feeding incision in the carcass for their broods in the No Care populations.

Next, we investigated whether this feeding incision functioned to promoted larval fitness (see 'Methods'). We allowed pairs of beetles from a stock laboratory population to prepare a carcass and removed the adults before they could make an incision. We cut a small feeding incision in half the carcasses ourselves, keeping control carcasses without an incision. Then we added ten newly-hatched larvae to each type of carcass and let them develop without any parental care. We found that larvae survived better on a carcass when we had cut a small feeding incision in it, than when we had not (binomial GLM: z = 9.07, P < 0.001, Supplementary Fig. 3), thus replicating the results of a previously published experiment30. Therefore, by advancing the time at which they bit a feeding incision in the carcass, No Care parents promoted their offspring’s fitness and in this way adapted to the No Care social environment that we had imposed experimentally. Even though larvae in the No Care populations received no post-hatching care, they did experience some degree of parental assistance in gaining access to resources on the carcass, through the feeding incision bitten by their parents.

Experimentally evolved adaptations in offspring

The level of parental assistance received by larvae in each experimental population was thus more predictable than observed in wild burying beetles, even though the mean level of care supplied differed between the experimental treatments (see Fig. 1, 'Methods'). We expected that consistent exposure to a more predictable environment after hatching should induce an evolutionary change in the relative size of the larval mandibles, according to the level of care supplied. Specifically, selection on larvae to be self-reliant, and maintain relatively large mandibles, should be relaxed in our experimental populations because here larvae could depend on parents for at least some degree of assistance in accessing the resources on the carcass. Our expectation was that in the experimental populations we should see a corresponding evolved change in the scaling relationship between the larval mandibles and larval size: the greater the supply of predictable parental assistance, the more strongly larval mandible size should scale with larval mass.

To test this prediction, we measured the gradient (β) of the scaling relationship between mandible size and larval size in the two experimental populations. Before measuring mandible size, we put each population through a common garden environment to eliminate any potentially confounding environmental effects (see 'Methods'). We found that β was now significantly positive for both experimental populations, unlike the ancestral wild population, such that larval mandible length now increased with larval mass. Furthermore, β differed between experimental populations (Supplementary Table 2, Fig. 3), and in proportion to the level of parental assistance (interaction between experimental care regime and larval mass on larval mandible length, OLS: t 163 = −2.87, P = 0.005; MA: Likelihood ratio = 9.65, P = 0.002, Fig. 3). Larvae from the Control populations could rely on extensive parental assistance in penetrating the carcass, and they had relatively smaller mandibles. Larvae from the No Care populations could depend on less parental help, and they had relatively larger mandibles. Importantly, neither population exhibited the scaling relationship that we found in wild-type larvae, which had even larger mandibles on average for a given body size (Fig. 2). In general, exposure to different levels of predictable parental care drove the evolution of new larval mandible scaling relationships in both the Control populations and the No Care populations. Because some level of parental care was supplied predictably in all the experimentally evolving populations, there was no risk that the larvae in these treatments would ever experience the worst-case scenario of no feeding incision in the carcass and no post-hatching care. By contrast, in wild populations, where the supply of care is far more unpredictable, this is an outcome that some larvae can experience (Fig. 1). Perhaps larvae in wild populations maintain such relatively large mandibles as a conservative bet-hedging strategy, and this explains why they are larger than those seen in the experimentally evolving populations.

Fig. 3 Larval mandible allometry in the experimentally evolving populations. The allometric relationship between larval mandible length and larval body mass in the offspring from experimental populations evolving in a Control environment (red filled datapoints, red solid line, n = 82) and a No Care environment (blue open datapoints, blue dashed line, n = 86). Ordinary least squares regression lines are shown with 95% confidence intervals Full size image

Finally, we investigated whether the new scaling relationships in the Control and No Care populations were adaptive. Specifically, we asked whether smaller larvae from the No Care populations were more likely to survive in a No Care environment than smaller larvae from the Control environment. We focused particularly on smaller larvae because the evolved difference between populations in relative mandible size was most pronounced in this subset of individuals (Fig. 3).

We added broods of ten larvae, drawn either from the Control population or the No Care population, onto a carcass prepared by stock beetles. We measured how well smaller individuals survived when given no assistance in penetrating the carcass, and no post-hatching care. We predicted that smaller larvae from the No Care populations would have a greater chance of survival under these social conditions than smaller larvae from the Control populations because the No Care larval mandibles were relatively larger. Overall, we found that more offspring survived from the No Care populations than the Control populations (binomial GLM: z = 4.02, P < 0.001). This shows that larvae from the No Care populations were better adapted to a No Care environment than larvae from the Control populations. Furthermore, the smallest survivor from each brood was indeed smaller in the No Care populations than in the Control populations (GLM: χ2 = 4.16, P = 0.04, Fig. 4). This result is consistent with the possibility that smaller larvae are more likely to survive without post-hatching care when their mandibles are disproportionately large. Nevertheless, further work is required to determine how much of the increase in larval survival is due to the mandibles alone. The scaling relationship between the larval mandibles and larval size is not the only larval adaptation to have evolved in the No Care populations. In separate work, we have found that other larval adaptations also contribute to larval survival during development in a No Care environment, including more synchronised egg hatching within clutches31.

Fig. 4 The effect of experimental evolution in the Control and No Care environments on the size of the smallest surviving larva. The mass of the smallest surviving larva in the brood, in relation to the social environment experienced by the experimentally evolving populations, when broods of 10 larvae were left on a carcass prepared by stock beetles with no incision and no post-hatching care (Control: n = 12; No Care: n = 27). Means with standard errors are shown Full size image

To sum up, we found that in wild populations of burying beetles larval mandible size could not be predicted by larval size, and the supply of parental care was also highly variable. We suggest that the high level of variation in larval morphology can be explained by the highly variable levels of parental assistance on offer to larvae (Fig. 1, refs. 23,26). When we enforced more predictable levels of care experimentally, we evolved larvae with mandibles that scaled much more predictably with their size (Fig. 3). However, the mechanism that maintains high levels of variation in this scaling relationship within wild populations (Fig. 2) remains to be determined in future work. We cannot tell from our data whether it is due to variation within broods, or among them. Variation within broods could exist as a bet-hedging adaptation for an unpredictable supply of care. Alternatively, variation among broods could arise if parents induce appropriate offspring adaptations for the extent of care they intend to provide (see ref. 32 for an example from birds). Whatever the mechanism at work, the high level of variation in relative mandible size sequestered in natural populations explains in part why we were able to detect evolutionary change so rapidly in our experimental populations.

More generally, our experiments show how cooperative interactions can influence morphological evolution in a social partner. In our experimental populations, we varied the degree to which parents helped their larvae so that they consistently provided either relatively little assistance (only biting a feeding incision in the No Care populations) or substantially more (biting a feeding incision and provisioning for at least 24 h in the Control populations). This, in turn, changed the optimal size of the larval mandibles. The greater the extent of parental assistance, the greater the cost to smaller larvae of maintaining relatively large, relatively redundant mandibles—and the greater the strength of selection to produce smaller mandibles. By predictably contributing more to larval nourishment, across the generations, parents caused an evolved reduction in larval traits for self-feeding. Previous work has shown that antagonistic social interactions can generate escalating evolutionary arms races: when one party evolves to invest more in a social trait it provokes the antagonist to invest an even greater amount (e.g., refs. 7,11,33,34). Our study shows that cooperative interactions can yield the opposite evolutionary outcome. When one party evolves to invest more, the cooperating partner evolves to invest less.