Dynamics of evolutionary succession and coordination between opposite adaptations in cuckoo hosts under antagonistic coevolution

Adaptations are driven by specific natural selection pressures throughout biological evolution. However, these cannot inherently align with future shifts in selection dynamics, thus

manifesting in opposing directions. We performed field experiments on cuckoo hosts to investigate the coexistence and conflict between two evolutionarily successive but opposing behavioral

adaptations—egg retrieval and rejection. Our findings provide key insights. (1) Egg rejection against brood parasites in hosts reshapes egg retrieval to flexible reactions—retrieval,

ignoring, or outright rejection of foreign eggs outside the nest cup, departing from instinctual retrieval. (2) Parasitism pressure and egg mimicry by parasites remarkably alter the

proportions of the three host reactions. Host species with higher parasitism pressure exhibit frequent and rapid rejection of non-mimetic foreign eggs and reduced ignoring or retrieval

responses. Conversely, heightened egg mimicry enhances retrieval behaviors while diminishing ignoring responses. (3) Cuckoos employ consistent mechanisms for rejecting foreign eggs inside or

outside the nest cup. Direct rejection of eggs outside the nest cup shows that rejection precedes retrieval, indicating prioritization of specific adaptation over instinct. (4) Cuckoo hosts

navigate the conflict between the intentions and motivations associated with egg rejection and retrieval by ignoring foreign eggs, a specific outcome of the rejection–retrieval tradeoff.

Biological evolution involves changes in organism population properties that transcend the lifetime of a single individual1. Such changes are adaptations wherein organisms interact with

various factors, including conspecifics, other species, and the environment2,3. Natural selection changes in direction and intensity over time, and therefore, does not always remain

constant4. Consequently, a current adaptation cannot predict or guarantee its adaptability in the future during evolution. For example, the well-known blind spot in the human eye results

from a conflict between optic nerve conduction and the inner lining of the retina5. The blind spot provides evidence that an adaptation cannot predict consequences but evolves based on

previous events, even if they are contradictory. Behavior is the forerunner of evolution, but studying different behavioral adaptations along an evolutionary trajectory is difficult due to

the lack of fossil or anatomical evidence6,7. However, egg retrieval and rejection behaviors in birds may provide a testable case for such studies.

Egg retrieval behavior refers to the act of an incubating bird retrieving eggs that have accidentally rolled out of the nest. This behavior is believed to be a common ancestral behavior

among ground-nesting birds that have not evolved elegant nest knitting8. As egg Retrieval cannot be suspended after stimuli until the bird completes the entire process, the behavior is

regarded as a fixed pattern of instinctive behavior8,9. In contrast, egg rejection is a more recent and specific adaptation that has evolved as a defense against brood parasitism10,11. Egg

rejection is flexible and can be enhanced via learning and social transmission12,13,14. Notably, the motivation and intention for egg rejection oppose those of egg retrieval; the former

refers to recognizing and rejecting eggs that are inside nests, whereas the latter refers to distinguishing and retrieving eggs that are outside nests. Therefore, these two behaviors are

successive adaptations on an evolutionary axis related to different levels of egg discrimination, but the natural selection direction for behavioral intention is the opposite. For egg

retrieval, the birds need to distinguish eggs from non-egg-shaped objects, while for egg rejection, they need to identify parasite eggs from their own eggs. Consequently, common hosts of

brood parasites face and cope with the conflict between egg retrieval and rejection when they possess both behavioral adaptations because when confronted with an egg outside the nest cup,

the former will stimulate the birds to retrieve it whereas the latter will trigger them to reject it. Studies on the relationship between these two behaviors will contribute to revealing the

evolutionary and maintained mechanisms between two successive but opposing adaptations.

Despite evidence of the conflict between egg retrieval and rejection, studies on the topic are limited15. Approximately 100 species of obligate brood parasites exist globally16, including

the famous parasitic cuckoos (Cuculidae spp.) and cowbirds (Icteridae spp.), all of which are altricial birds (except for Heteronetta atricapilla, an obligate parasitic duck). However, only

two studies on egg retrieval behavior have been conducted in altricial birds15,17, of which only one focused on the relationship between egg retrieval and rejection15. As the studied species

have not been exploited by any parasitic species, their relationship with possible brood parasites is unclear15. In this study, we conducted a field experiment18 to study and compare the

behavioral relationship between egg retrieval and rejection in two closely related sympatric breeding species: the Oriental magpie-robin (Copsychus saularis) and white-rumped shama (C.

malabaricus) (Fig. 1A, B). The studied population of magpie-robins was exploited by the common cuckoo (Fig. 2), whereas the shamas were not parasitized by any of the cuckoo species.

(A) Male white-rumped shama; (B) male Oriental magpie-robin; (C) shama nest with a manipulated conspecific egg in ONC treatment; (D) magpie-robin nest with a manipulated conspecific egg in

ONC treatment; (E) shama nest with a manipulated model egg in ONC treatment; (F) magpie-robin nest with a manipulated model egg in ONC treatment. A, B were photographed by Li Cheng with

permission.

A Male magpie-robin feeding cuckoo chick; B female magpie-robin feeding cuckoo chick. The images were photographed by Guoqiang Yu with permission.

This study aimed to determine the effect of two natural selection aspects (cuckoo parasitism and host defense) on two opposite behavioral adaptations (egg retrieval and rejection) and how

hosts cope with the conflict between these adaptations. Furthermore, the pattern of egg rejection and retrieval reactions is expected to change according to the selection intensity of cuckoo

parasitism and host defense. Specifically, magpie-robins are expected to reject more foreign eggs than shamas, whereas both species should reject more non-mimetic eggs than highly mimetic

foreign eggs.

This study was performed in the Nonggang National Nature Reserve, Guangxi Zhuang Autonomous Region, Southwestern China, during the breeding seasons (i.e., April–July of the two study years,

2021–2022). The study area is located in the Sino-Vietnamese border region (22°13′N, 106°42′E), a typical limestone area located in the northern margin of the tropics at an altitude between

150 and 650 m. Mean annual rainfall and temperature in this region are 1150–1550 mm and 20.8–22.4 °C, respectively19. Nest boxes were used to provide nest sites for the two studied species,

the Oriental magpie-robins and white-rumped shamas, which breed in sympatry in the study area, with similar nest and egg phenotypes (Fig. 1). The species are closely related within the genus

Copsychus, according to the taxonomic information presented in “Birds of the World” from the Cornell Lab of Ornithology20,21. Magpie-robins are a common host for the common cuckoo22,

whereas the parasitic host status of shamas is unclear. However, it is assumed that shamas are a potential host for cuckoos21. The studied magpie-robin population was confirmed to be

exploited by the common cuckoo (Fig. 2), unlike the shama population. However, the magpie-robin parasitism rate was unknown, as the hole entrances in the nest boxes used to perform an

experiment may have been too small (6 cm in diameter) for a cuckoo entrance. Although no parasitism was found in the nest boxes, cuckoo fledglings fed by magpie-robins from natural nests

were frequently observed in the images (Fig. 2). Thus, we predicted that magpie-robins experienced a higher degree of selection intensity from cuckoo parasitism than that experienced by

shamas. We conducted artificial parasitism to confirm this prediction by testing the egg recognition capacity of these two species (see the field procedure for the first treatment below for

details). Additionally, the findings of our daily investigation during the egg-laying stage showed no signs of intraspecific parasitism in the magpie-robin or shama nests. Notably, using egg

recognition capacity rather than parasitism rates to represent parasitism pressure was more feasible in this study for several reasons: (1) the egg recognition capacity would reflect the

intensity of interaction between the hosts and parasites23,24; (2) parasitism rates may be underestimated or overestimated owing to different levels of egg rejection in hosts25,26; and (3)

egg rejection rates were expected to differ between the two studied species and, thus, using egg recognition capacity as a representation of parasitism pressure ensured greater comparability

among species.

To investigate the egg recognition capacity of the studied species, we conducted an experiment simulating artificial parasitism during the first study year. The experimental protocol termed

the “inside nest cup (INC)” treatment involved the introduction of a model egg (identical in size to that of the host egg) into the nest cup to replace one of the authentic host eggs. The

model egg was crafted from blue polymer clay to mirror the prevailing phenotype of common cuckoo eggs in China, given that magpie-robins are documented to be parasitized by common cuckoos

using blue eggs22. The treatment was performed the day after the hosts completed their clutches (magpie-robins: n = 39, excluding one predation case; shama: n = 35). Subsequently, the

manipulated clutches were monitored daily for 6 days to confirm the hosts’ responses, which were classified into acceptance (model eggs incubated by the hosts), ejection (model eggs ejected

by the hosts), or desertion (clutches deserted by the hosts). A control trial was used to control for manipulation disturbances (n = 30 for magpie-robins and shamas), in which host clutches

followed the same investigation procedure as the INC treatment without model egg replacement. No desertion or other abnormal phenomena were observed in the control trials; therefore, both

ejection and desertion were regarded as rejections by the hosts. The model egg in this treatment was non-mimetic to the host egg. Thus, the recognition capacity in this study referred to

host recognition based on non-mimetic eggs. Here, we used the egg rejection rate to represent egg recognition capacity. To avoid pseudo-replication, the time between the first and last

experimental nests was limited to approximately 37 days according to the reproductive cycle (i.e., from nest building to fledging) of the hosts20,27.

To investigate the relationship between the reaction patterns associated with egg rejection and retrieval, we carried out the “outside nest cup (ONC)” treatment. This treatment encompassed

two groups of trials, each involving the replacement of either one model or one conspecific egg instead of one of the clutch eggs. At the same time, the substituted egg was positioned on the

nest platform outside the nest cup, at a distance of 2 cm from the rim of the nest cup (Fig. 1C–F). The timing of the manipulation and investigation procedures were the same as those in the

INC treatment. The host responses in ONC treatment were classified into retrieval (the eggs outside the nest cups were retrieved and brought back into the nest cups by the hosts), ejection

(the eggs outside the nest cups were ejected by the hosts), ignoring (the eggs outside the nest cups were neither retrieved nor ejected by the hosts), or desertion (the hosts deserted the

clutches). Some observed nests were randomly selected (n = 27) to monitor the behavioral reaction using a mini camera (WJO3, Hisilicon, Shenzhen, Guangdong, China). Additionally, control

trials (n = 25 for both studied species) were conducted to control for manipulation disturbances, and no desertions or other abnormal phenomena were observed.

Two rejection reactions were identified during the treatment: Reaction A, in which ejection and desertion were combined as rejections (applicable to INC and ONC treatments), and reaction, B

which included the ignoring response as rejection, as this behavior could be construed as a mode of rejection wherein hosts abstain from retrieving the eggs (only applicable for ONC

treatment). We used reactions A or B to analyze the results because ignoring cannot be easily categorized. For the accepting reaction of INC, the hosts would incubate the eggs without

recognition. For the rejecting reaction, the hosts would directly reject the eggs after recognition. The ignoring reaction, however, resembled an undecided process. Using the classification

of reaction A or B to analyze the hosts’ responses to different mimetic levels of parasite eggs would help us reveal the role of the ignoring reaction. Therefore, reactions A and B did not

differ for the INC treatment yet diverged within the context of the ONC treatment (Fig. 3). Notably, conspecific eggs within the conspecific group of the ONC treatment were randomly selected

from the same clutches to mitigate the potential influence of inter-female variation in egg phenotypes. Therefore, the model and conspecific egg groups in ONC treatment represent

non-mimetic and highly mimetic eggs, respectively. Additionally, each experimental nest of ONC treatment received only one egg type (either a model or conspecific egg), while both the

magpie-robin and shama groups received two egg types (magpie-robin: n = 24 for the model group excluding one predation case, n = 24 for the conspecific group; shamas: n = 23 for the model

group, n = 25 for the conspecific group).

The black egg refers to the experimental egg with manipulation. In the INC treatment, the black egg refers to a model egg, while in the ONC treatment, the black egg refers to a model or

conspecific egg. Figure created in PowerPoint.

Using the aforementioned experimental design, we simulated a situation based on two aspects of natural selection: Egg recognition capacity and foreign egg mimicry. Egg recognition capacity

is selected by, and thus reflects the intensity of, cuckoo parasitism (one aspect of natural selection) and was divided into two levels according to the two studied species (magpie-robin and

shama). Although egg recognition capacity may persist owing to past selection, it reflects the intensity of interaction with parasites28,29. However, this will not influence our study

because we did not focus on past or present selection but rather aimed to determine the effect of such intensity changes on host behavior. Foreign egg mimicry reflects the intensity of host

defense (another aspect of natural selection) because stronger host defense will promote the parasites to evolve more optimized egg mimicry. Foreign egg mimicry was divided into two levels:

non-mimetic and highly mimetic. This allowed us to study the relationship between egg retrieval and rejection under two aspects of natural selection that were related yet different. Egg

recognition capacity represents the intensity of encountering parasites whereas egg mimicry reflects the levels of cognition. The egg recognition capacity can be studied under different

levels of egg mimicry whereas the same level of egg mimicry can lead to different capacities of egg recognition between hosts.

Four Markov chain Monte Carlo technique–generalized linear mix models (MCMC-GLMM) were built and used to investigate the effects of experimental manipulation on host reactions. In the first

two models, the response variable was either reaction A or B to the model egg, while the fixed effects were species (magpie-robin or shama), treatment (INC or ONC treatment), clutch size,

and laying date (of the first egg). In the second two models, the response variable was either reaction A or B of ONC treatment, and the fixed effects were group (model or conspecific egg),

species, clutch size, and laying date. Nest identity was a random effect in all models. The MCMC-GLMM calculates the posterior estimate using Bayesian analyses, which provide a posterior

mean and 95% credible interval30. Cox regression models were used to analyze the reaction time associated with egg rejection or retrieval. This analysis was performed by incorporating both

the incidence of a rejection or retrieval event and its latency (i.e., the timing of the daily investigation) into the survival function. As the Cox model assumes a consistent shape for the

survival function, we imposed constrained time intervals of investigation in this study that spanned six days with daily frequency31. In instances where no occurrence transpired during the

six-day window, a latency period of six days without an event was encompassed in the Cox models as a censored value. Furthermore, Kaplan–Meier curves of survival probability were established

to illustrate the significant results derived from the Cox models. MCMC-GLMM, Cox regression, and Kaplan–Meier curves were generated using the packages MCMCglmm, survminer, and survival,

respectively, in R (v. 4.2.2) for Windows (R Foundation for Statistical Computing, Vienna, Austria).

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

The videos revealed that both the magpie-robins and shamas used their bills to eject or retrieve eggs, and all ejections were performed via grasping. Magpie-robins exhibited a rejection rate

of 48.72% in response to the blue model eggs in the INC treatment, comprising 84.21% ejections and 15.79% desertions (Table 1). In comparison, shamas rejected 22.86% of the blue model eggs,

which was less than half the rejection rate of magpie-robins. Ejection and desertion accounted for 75% and 25% of the rejection cases, respectively. The MCMC-GLMM indicated that the model

egg rejection rate of magpie-robins was significantly higher than that of shamas (Species: posterior mean = −0.3, 95% CI = −0.46 to −0.14, P