Evolution and genetic architecture of sex-limited polymorphism in cuckoos.

Sex-limited polymorphism has evolved in many species including our own. Yet, we lack a detailed understanding of the underlying genetic variation and evolutionary processes at work. The brood parasitic common cuckoo (Cuculus canorus) is a prime example of female-limited color polymorphism, where adult males are monochromatic gray and females exhibit either gray or rufous plumage. This polymorphism has been hypothesized to be governed by negative frequency-dependent selection whereby the rarer female morph is protected against harassment by males or from mobbing by parasitized host species. Here, we show that female plumage dichromatism maps to the female-restricted genome. We further demonstrate that, consistent with balancing selection, ancestry of the rufous phenotype is shared with the likewise female dichromatic sister species, the oriental cuckoo (Cuculus optatus). This study shows that sex-specific polymorphism in trait variation can be resolved by genetic variation residing on a sex-limited chromosome and be maintained across species boundaries.

Host learning selects for the coevolution of greater egg mimicry and narrower antiparasitic egg-rejection thresholds.

Egg rejection is an effective and widespread antiparasitic defense to eliminate foreign eggs from the nests of hosts of brood parasitic birds. Several lines of observational and critical experimental evidence support a role for learning by hosts in the recognition of parasitic versus own eggs; specifically, individual hosts that have had prior or current experience with brood parasitism are more likely to reject foreign eggs. Here we confirm experimentally the role of prior experience in altering subsequent egg-rejection decisions in the American robin Turdus migratorius, a free-living host species of an obligate brood parasite, the brown-headed cowbird Molothrus ater. We then model the coevolutionary trajectory of both the extent of mimicry of host eggs by parasitic eggs and the host's egg rejection thresholds in response to an increasing role of learning in egg recognition. Critically, with more learning, we see the evolution of both narrower (more discriminating) rejection thresholds in hosts and greater egg mimicry in parasites. Increasing host clutch size (number of eggs/nest) and increasing parasite load (parasitism rate) also have narrowing effects on the egg-rejection threshold. Together, these results suggest that learning from prior experience with egg rejection may play an important role in the coevolution of egg-mimetic lineages of brood parasites and the refined egg rejection defenses of hosts.

Soft calls do not modulate aggressiveness in male common cuckoos toward conspecific territorial intruders: A sequential playback study.

In many bird species loud broadcast calls serve as aggressive signals with a large effective radius, whereas soft calls may indicate aggression at a closer distance, and are often directed at a nearby conspecific individual. Male common cuckoos (Cuculus canorus) are famous for their simple cu-coo calls, which are long-range broadcast calls, uttered in long sequences during the breeding season. Cuckoos also produce soft calls, including a single gowk (wah), a series of gowks (gowk series), or a harsher single variant, the guo call. To our knowledge, no previous study has attempted to understand the function of cuckoos' soft calls. We conducted a sequential playback experiment by (i) first attracting male cuckoos with "cu-coo" calls, and then (ii) playing heterospecific control calls or one of several types of cuckoo soft calls. While the "cu-coo" call attracted focal males, neither the soft conspecific calls nor the heterospecific control calls elicited an additional response. Finally, the gowk series calls did not alter cuckoos' approach to the speaker when played together with "cu-coo" calls. As cuckoos' soft calls did not increase or decrease aggression, they instead may advertise the cuckoo's presence and position for a nearby conspecific.

Quantitative analysis of vocalisation types in male common cuckoos' "gowk" call complex.

Acoustic communication of animals often contains two types of vocalizations: loud sounds for long-range and soft sounds for short-range signalling. Brood parasitic common cuckoos (Cuculus canorus) are suitable study objects for research on acoustic signalling as they have a simple acoustic repertoire, of which the loud territorial advertisement calls of males, the "cu-coo", is the most famous type. Although a distinct group of soft calls has also been reported in early naturalists' works, no systematic studies compared them acoustically. Even the classification of these soft calls is lacking. Here we surveyed these neglected types of calls, and evaluated them through bioacustic analyses. Specifically, we compared the following soft calls: single gowk, guo, and the series of gowk calls. The advertisement call ("cu-coo") was used as the referent for these comparisons. As the male's gowk series call most typically contains 3-5 elements with decreasing volume (disappearing at the end), we compared the first two elements of this call. Our analyses revealed great acoustic similarities between three call types (gowk series call 1st note, gowk series call 2nd note, and single gowk call), and the distinctiveness of the other call type (guo) from this group. Structurally the gowk cannot be regarded as a separate call from a male's gowk series call, and the only difference is that the series call contains a set of the gowk calls. Our classification of cuckoo calls offers the possibility for further research into their functional importance and communicative role in male-male or female-male social contexts.

Syntax errors do not disrupt acoustic communication in the common cuckoo.

When acoustic communication signals are distorted, receivers may misunderstand the signal, rendering it ineffective. Common cuckoos (Cuculus canorus) are popularly known for the males' simple, two-note advertisement calls, the "cu-coo" used for declaring the male's breeding territories. Cuckoos do not learn their calls (vocal non-learners), so they are expected to have a limited ability to produce different acoustic signals. Nevertheless, male cuckoos appear to make syntax errors (e.g., repeated, reversed, or fragmented elements) even in their simple advertisement calls. We conducted a playback experiment with male cuckoos, broadcasting ten call types, including seven modified calls with errors (e.g. "cu-cu", and "coo-cu") and three natural calls used for comparisons ("cu-coo", "cu-cu-coo", and interspecific control). Male cuckoos responded in a manner suggesting that the presence of the first ("cu") note of the natural 2-note "cu-coo" call in any form or combination yield effective signals. However, through the elevated frequency (by about 200 Hz) and greater speed of the "cu" note, the natural 3-note version "cu-cu-coo" call appears to have gained a novel communicative function in signalling with female cuckoos. Thus, syntax errors in calls with the "cu" element are not responsible for changing the function of the male cuckoos' "cu-coo" call.

Male common cuckoos use a three-note variant of their "cu-coo" call for duetting with conspecific females.

Duetting is a coordinated form of acoustic communication with participants uttering calls or songs simultaneously and/or sequentially. Duetting is often observed in pair-bonded species, with mated females and males both contributing to the communal vocal output. We observed duetting between the sexes in the common cuckoo (Cuculus canorus), an obligate brood parasitic species without known pair formation. Specifically, female cuckoos use their sex-specific bubbling calls for duetting, while male cuckoos use a 3-note variant ("cu-cu-coo") of their typical and well-known 2-note ("cu-coo") territorial advertisement calls. The maximum frequency of the elements in the male's 3-note variants was higher relative to the 2-note calls, while durations of both the elements and the inter-element intervals were shorter. The vast majority (95 %) of the 3-note calling was detected together with the bubbling call, implying an intersexual duetting function, with the female calls preceding these male calls in 67 % of cases. The two call types in duetting followed each other rapidly (mean response time of females was 1.30 ± 0.71 SD s, and 0.76 ± 0.53 SD s in males), and typically overlapped with each other (95 %). Frequently (90 %), the male call was repeated 2-3 times, whereas the female call was repeated less frequently (9%). Our results are consistent with a main function of duetting in intersexual communication and coordination between female and male cuckoos.

Female-female aggression and male responses to the two colour morphs of female common cuckoos.

Female-only colour polymorphism is rare in birds, but occurs in brood parasitic cuckoos (Cuculidae). Obligate brood parasites leave incubation and parental care to other species (hosts), so female-female interactions can play a role in how parasites guard critical resources (host nests) within their laying areas. The plumage of adult female common cuckoos (Cuculus canorus) is either rufous (typically rare) or grey (common), whereas adult male conspecifics are monochromatic (grey). In previous studies, hosts and conspecific males responded with less intensity toward the rare female morph in support of a negative frequency-dependent benefit of female plumage polychromatism. Here, we assessed responses of both conspecific females and males to vocal playbacks of female calls, coupled with one of two 3D models of the different morphs of female cuckoos. At our study population, the rufous female morph was as common as the grey morph; therefore, we predicted similarly high rates of conspecific responses in both treatments. Both female and male cuckoos responded to playbacks acoustically, which demonstrated the primary role of acoustic communication in social interactions amongst cuckoos. Following this, some cuckoos flew closer to the models to inspect them visually. As predicted, no significant differences were detected between the live cuckoos' responses toward the two colour morphs in this population. We conclude that dichromatism in female cuckoos evolved to serve one or more functions other than conspecific signalling.

Host alarm calls attract the unwanted attention of the brood parasitic common cuckoo.

It is well known that avian brood parasites lay their eggs in the nests of other bird species, called hosts. It remains less clear, however, just how parasites are able to recognize their hosts and identify the exact location of the appropriate nests to lay their eggs in. While previous studies attributed high importance to visual signals in finding the hosts' nests (e.g. nest building activity or the distance and direct sight of the nest from vantage points used by the brood parasites), the role of host acoustic signals during the nest searching stage has been largely neglected. We present experimental evidence that both female and male common cuckoos Cuculus canorus pay attention to their host's, the great reed warbler's Acrocephalus arundinaceus alarm calls, relative to the calls of an unparasitized species used as controls. Parallel to this, we found no difference between the visibility of parasitized and unparasitized nests during drone flights, but great reed warblers that alarmed more frequently experienced higher rates of parasitism. We conclude that alarm calls might be advantageous for the hosts when used against enemies or for alerting conspecifics, but can act in a detrimental manner by providing important nest location cues for eavesdropping brood parasites. Our results suggest that host alarm calls may constitute a suitable trait on which cuckoo nestlings can imprint on to recognize their primary host species later in life. Our study contributes to the growing body of knowledge regarding the context-dependency of animal signals, by providing a novel example of a beneficial acoustic trait intercepted by a heterospecific and used against the emitter.

Anti-parasitic egg rejection by great reed warblers (Acrocephalus arundinaceus) tracks differences along an eggshell color gradient.

One of the most effective defenses against avian brood parasitism is the rejection of the foreign egg from the host's nest. Until recently, most studies have tested whether hosts discriminate between own and foreign eggs based on the absolute differences in avian-perceivable eggshell coloration and maculation. However, recent studies suggest that hosts may instead contrast egg appearances across a directional eggshell color gradient. We assessed which discrimination rule best explained egg rejection by great reed warblers Acrocephalus arundinaceus, a frequent host to an egg-mimetic race of common cuckoos Cuculus canorus. We deployed 3D-printed model eggs varying in blue-green to brown coloration and in the presence of maculation. Using visual modeling, we calculated the absolute chromatic and achromatic just-noticeable differences (JNDs), as well as directional JNDs across a blue-green to brown egg color gradient, between host and model eggs. While most model eggs were rejected by great reed warblers, browner eggs were rejected with higher probability than more blue-green eggs, and the rejection probability did not depend on maculation. Directional egg color discrimination shown here and in a suite of recent studies on other host species may shape the cognitive decision rules that hosts use to recognize foreign eggs and affect the course of evolution in parasitic egg mimicry.

Multiple parasitism reduces egg rejection in the host (Acrocephalus arundinaceus) of a mimetic avian brood parasite (Cuculus canorus).

A host that has been targeted by an avian brood parasite can recover most of its potential fitness loss by ejecting the foreign egg(s) from its nest. The propensity for some hosts to engage in egg rejection behavior has put selective pressure on their parasites to evolve mimetic eggshells resembling the host's own shell colors and maculation. In turn, hosts have counterevolved increasingly more sophisticated detection methods such as narrowing visual egg acceptance thresholds or using social cues to recognize parasitism. However, multiple cognitive mechanisms acting simultaneously could theoretically interfere with one another and ultimately decrease egg rejection accuracy, especially if these heuristics yield differing targets for rejection. By painting hosts own eggs, we studied a host species of the common cuckoo Cuculus canorus, the great reed warbler Acrocephalus arundinaceus, and tested its responses to the presence of "foreign" eggs of varying quantity, colors, and uniformity. Using reflectance spectra of egg background coloration and avian perceptual modeling, we then estimated the sensory thresholds triggering egg rejection by this host for each treatment. As previously reported, rejection rates were positively related to the perceptual distance between own and foreign eggs in the nests in all treatments. However, rejection thresholds were more permissive (error prone) both with greater proportions of foreign eggs per clutch and/or when the suite of foreign eggs was perceptually more variable within the nest. These results suggest that parasites, through multiple parasitism, can partially overcome the evolution of hosts' recognition of mimetic parasite eggs. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Are both notes of the common cuckoo's call necessary for familiarity recognition?

Common cuckoos (Cuculus canorus) are best known for their simple two-note calls ("cu-coo"), which are uttered only by males during the breeding season. A previous playback study revealed that territorial males were more tolerant toward playbacks of the calls of familiar, neighbouring individuals than toward unfamiliar, stranger simulated intruders, exhibiting the classical "dear-enemy" phenomenon. Here we experimentally assessed whether the acoustic cues for familiarity recognition are encoded in the first and/or second note of these simple calls. To do so, we played mixed sound files to radio-tagged cuckoos, where the first part of the two-note calls was taken from strangers and the second part from neighbours, or vice versa. As controls, we used behavioural data from two-note neighbour and two-note stranger call playbacks. Cuckoos responded consistently to the two types of mixed sound files. When either the first or second note of the call was taken from a stranger and the other note from a neighbour, they responded to these sound files similarly to two-note playbacks of strangers: they approached the speaker of the playbacks more closely and the calling response-latency to playbacks was longer than to familiar controls. These findings point to the importance of both notes in familiarity recognition. We conclude that familiarity recognition in male common cuckoos needs the complete structure of the two-note cuckoo call, which is characteristic for this species.

Common cuckoos (Cuculus canorus) affect the bacterial diversity of the eggshells of their great reed warbler (Acrocephalus arundinaceus) hosts.

The common cuckoo (Cuculus canorus) is an avian brood parasite, laying its eggs in the nests of other bird species, where these hosts incubate the parasitic eggs, feed and rear the nestlings. The appearance of a cuckoo egg in a host nest may change the bacterial community in the nest. This may have consequences on the hatchability of host eggs, even when hosts reject the parasitic egg, typically within six days after parasitism. The present study revealed the bacterial community of cuckoo eggshells and those of the great reed warbler (Acrocephalus arundinaceus), one of the main hosts of cuckoos. We compared host eggs from non-parasitized clutches, as well as host and cuckoo eggs from parasitized clutches. As incubation may change bacterial assemblages on eggshells, we compared these egg types in two stages: the egg-laying stage, when incubation has not been started, and the mid-incubation stage (ca. on days 5-7 in incubation), where heat from the incubating female dries eggshells. Our results obtained by the 16S rRNA gene sequencing technique showed that fresh host and cuckoo eggs had partially different bacterial communities, but they became more similar during incubation in parasitized nests. Cluster analysis revealed that fresh cuckoo eggs and incubated host eggs in unparasitized nests (where no cuckoo effect could have happened) were the most dissimilar from the other groups of eggs. Cuckoo eggs did not reduce the hatchability of great reed warbler eggs. Our results on the cuckoo-great reed warbler relationship supported the idea that brood parasites may change bacterial microbiota in the host nest. Further studies should reveal how bacterial communities of cuckoo eggshells may vary by host-specific races (gentes) of cuckoos.

Experimental shifts in egg-nest contrasts do not alter egg rejection responses in an avian host-brood parasite system.

Obligate brood parasitic birds exploit their hosts to provide care for unrelated young in the nest. Potential hosts can reduce the cost of parasitism by rejecting foreign eggs from the nest. Observational, comparative, and experimental studies have concluded that most hosts use the coloration and patterning of eggshells to discriminate between own and foreign eggs in the nest. However, an alternative hypothesis is that birds use the colour contrasts between eggshells and the nest lining to identify parasitic eggs (egg-nest contrast hypothesis). In support of this hypothesis, we found that the avian perceivable chromatic contrasts between dyed eggs and unmanipulated nest linings significantly and negatively covaried with the rejection rates of different dyed eggs of the great reed warbler Acrocephalus arundinaceus, a frequently parasitized host of the common cuckoo Cuculus canorus. To experimentally test whether egg-nest contrasts influence rejection, we reciprocally dyed both eggs and the nest lining of this host species with one of two colours: orange and green. Contrary to the egg-nest contrast hypothesis, host rejection patterns in response to dyed eggs were not altered by dyeing nests, relative to unmanipulated control eggs and nests. In turn, experimental egg colour was the only significant predictor of egg rejection rate. Our results demonstrate that egg-nest contrast is a collateral, not a causal factor in egg rejection, and confirm the conclusions of previous studies that hosts can rely on the parasitic egg's appearance itself to recognize the foreign egg in the nest.

Naïve hosts of avian brood parasites accept foreign eggs, whereas older hosts fine-tune foreign egg discrimination during laying.

BACKGROUND: Many potential hosts of social parasites recognize and reject foreign intruders, and reduce or altogether escape the negative impacts of parasitism. The ontogenetic basis of whether and how avian hosts recognize their own and the brood parasitic eggs remains unclear. By repeatedly parasitizing the same hosts with a consistent parasitic egg type, and contrasting the responses of naïve and older breeders, we studied ontogenetic plasticity in the rejection of foreign eggs by the great reed warbler (Acrocephalus arundinaceus), a host species of the common cuckoo (Cuculus canorus). RESULTS: In response to experimental parasitism before the onset of laying, first time breeding hosts showed almost no egg ejection, compared to higher rates of ejection in older breeders. Young birds continued to accept foreign eggs when they were subjected to repeated parasitism, whereas older birds showed even higher ejection rates later in the same laying cycle. CONCLUSIONS: Our results are consistent with the hypotheses that (i) naïve hosts need to see and learn the appearance of their own eggs to discriminate and reject foreign eggs, whereas (ii) experienced breeders possess a recognition template of their own eggs and reject parasitic eggs even without having to see their own eggs. However, we cannot exclude the possibility that other external cues and internal processes, accumulated simply with increasing age, may also modify age-specific patterns in egg rejection (e.g. more sightings of the cuckoo by older breeders). Future research should specifically track the potential role of learning in responses of individual hosts between first and subsequent breeding attempts by testing whether imprinting on a parasitized clutch reduces the rates of rejecting foreign eggs in subsequent parasitized clutches.

Sex allocation in relation to host races in the brood-parasitic common cuckoo (Cuculus canorus).

Sex allocation theory and empirical evidence both suggest that natural selection should favour maternal control of offspring sex ratio in relation to their ability to invest in the offspring. Generalist parasites constitute a particularly interesting group to test this theory as different females commonly utilize different host species showing large variation in provisioning ability. The common cuckoo (Cuculus canorus) is a generalist brood parasite that lays its eggs in the nest of many different passerine birds, but each female tends to specialize on one particular host species giving rise to highly specialized host races. The different host species show large variation in their ability to invest in the parasitic offspring, presenting an opportunity for female cuckoos to bias offspring sex ratio in relation to host species quality. Here, we investigate host-race specific sex allocation controlling for maternal identity in the common cuckoo. We found no evidence of any significant relationship between host race and sex ratio in one sympatric population harbouring three different host races, or in a total of five geographically separated populations. There was also no significant association between host quality, as determined by species-specific female host body mass, and cuckoo sex ratio. Finally, we found no significant relationship between individual cuckoo maternal quality, as determined by her egg volume, and sex ratio within each host race. We conclude that the generalist brood-parasitic common cuckoo show no significant sex-ratio bias in relation to host race and discuss this finding in light of gene flow and host adaptations.

Competition with a host nestling for parental provisioning imposes recoverable costs on parasitic cuckoo chick's growth.

Chicks of the brood parasitic common cuckoo (Cuculus canorus) typically monopolize host parental care by evicting all eggs and nestmates from the nest. To assess the benefits of parasitic eviction behaviour throughout the full nestling period, we generated mixed broods of one cuckoo and one great reed warbler (Acrocephalus arundinaceus) to study how hosts divide care between own and parasitic young. We also recorded parental provisioning behaviour at nests of singleton host nestlings or singleton cuckoo chicks. Host parents fed the three types of broods with similar-sized food items. The mass of the cuckoo chicks was significantly reduced in mixed broods relative to singleton cuckoos. Yet, after the host chick fledged from mixed broods, at about 10-12 days, cuckoo chicks in mixed broods grew faster and appeared to have compensated for the growth costs of prior cohabitation by fledging at similar weights and ages compared to singleton cuckoo chicks. These results are contrary to suggestions that chick competition in mixed broods of cuckoos and hosts causes an irrecoverable cost for the developing brood parasite. Flexibility in cuckoos' growth dynamics may provide a general benefit to ecological uncertainty regarding the realized successes, failures, and costs of nestmate eviction strategies of brood parasites.

A shared chemical basis of avian host-parasite egg colour mimicry.

Avian brood parasites lay their eggs in other birds' nests and impose considerable fitness costs on their hosts. Historically and scientifically, the best studied example of circumventing host defences is the mimicry of host eggshell colour by the common cuckoo (Cuculus canorus). Yet the chemical basis of eggshell colour similarity, which impacts hosts' tolerance towards parasitic eggs, remains unknown. We tested the alternative scenarios that (i) cuckoos replicate host egg pigment chemistry, or (ii) cuckoos use alternative mechanisms to produce a similar perceptual effect to mimic host egg appearance. In parallel with patterns of similarity in avian-perceived colour mimicry, the concentrations of the two key eggshell pigments, biliverdin and protoporphyrin, were most similar between the cuckoo host-races and their respective hosts. Thus, the chemical basis of avian host-parasite egg colour mimicry is evolutionarily conserved, but also intraspecifically flexible. These analyses of pigment composition reveal a novel proximate dimension of coevolutionary interactions between avian brood parasites and hosts, and imply that alternative phenotypes may arise by the modifications of already existing biochemical and physiological mechanisms and pathways.

Alternative mechanisms of increased eggshell hardness of avian brood parasites relative to host species.

Obligate brood parasitic birds lay their eggs in nests of other species and parasite eggs typically have evolved greater structural strength relative to host eggs. Increased mechanical strength of the parasite eggshell is an adaptation that can interfere with puncture ejection behaviours of discriminating hosts. We investigated whether hardness of eggshells is related to differences between physical and chemical traits from three different races of the parasitic common cuckoo Cuculus canorus, and their respective hosts. Using tools developed for materials science, we discovered a novel correlate of increased strength of parasite eggs: the common cuckoo's egg exhibits a greater microhardness, especially in the inner region of the shell matrix, relative to its host and sympatric non-host species. We then tested predictions of four potential mechanisms of shell strength: (i) increased relative thickness overall, (ii) greater proportion of the structurally harder shell layers, (iii) higher concentration of inorganic components in the shell matrix, and (iv) elevated deposition of a high density compound, MgCO(3), in the shell matrix. We confirmed support only for hypothesis (i). Eggshell characteristics did not differ between parasite eggs sampled from different host nests in distant geographical sites, suggesting an evolutionarily shared microstructural mechanism of stronger parasite eggshells across diverse host-races of brood parasitic cuckoos.

Constraints on host choice: why do parasitic birds rarely exploit some common potential hosts?

1. Why are some common and apparently suitable resources avoided by potential users? This interesting ecological and evolutionary conundrum is vividly illustrated by obligate brood parasites. Parasitic birds lay their eggs into nests of a wide range of host species, including many rare ones, but do not parasitize some commonly co-occurring potential hosts. 2. Attempts to explain the absence of parasitism in common potential hosts are limited and typically focused on single-factor explanations while ignoring other potential factors. We tested why thrushes Turdus spp. are extremely rarely parasitized by common cuckoos Cuculus canorus despite breeding commonly in sympatry and building the most conspicuous nests among forest-breeding passerines. 3. No single examined factor explained cuckoo avoidance of thrushes. Life-history traits of all six European thrush species and the 10 most frequently used cuckoo hosts in Europe were similar except body/egg size, nest design and nestling diet. 4. Experiments (n = 1211) in several populations across Europe showed that host defences at egg-laying and incubation stages did not account for the lack of cuckoo parasitism in thrushes. However, cross-fostering experiments disclosed that various factors during the nestling period prevent cuckoos from successfully parasitizing thrushes. Specifically, in some thrush species, the nest cup design forced cuckoo chicks to compete with host chicks with fatal consequences for the parasite. Other species were reluctant to care even for lone cuckoo chicks. 5. Importantly, in an apparently phylogenetically homogenous group of hosts, there were interspecific differences in factors responsible for the absence of cuckoo parasitism. 6. This study highlights the importance of considering multiple potential factors and their interactions for understanding absence of parasitism in potential hosts of parasitic birds. In the present study, comparative and experimental procedures are integrated, which represent a novel approach that should prove useful for the understanding of interspecific ecological relationships in general.

Discordancy or template-based recognition? Dissecting the cognitive basis of the rejection of foreign eggs in hosts of avian brood parasites.

Many avian hosts have evolved antiparasite defence mechanisms, including egg rejection, to reduce the costs of brood parasitism. The two main alternative cognitive mechanisms of egg discrimination are thought to be based on the perceived discordancy of eggs in a clutch or the use of recognition templates by hosts. Our experiments reveal that the great reed warbler (Acrocephalus arundinaceus), a host of the common cuckoo (Cuculus canorus), relies on both mechanisms. In support of the discordancy mechanism, hosts rejected their own eggs (13%) and manipulated ('parasitic') eggs (27%) above control levels in experiments when manipulated eggs were in the majority but when clutches also included a minority of own eggs. Hosts that had the chance to observe the manipulated eggs daily just after laying did not show stronger rejection of manipulated eggs than when the eggs were manipulated at clutch completion. When clutches contained only manipulated eggs, in 33% of the nests hosts showed rejection, also supporting a mechanism of template-based egg discrimination. Rejection using a recognition template might be more advantageous because discordancy-based egg discrimination is increasingly error prone with higher rates of multiple parasitism.