Translocated wild birds are predisposed to learn songs of their ancestral population.

Population differences in socially learned mating signals like oscine birdsong are particularly vulnerable to breakdown through dispersal.1 Despite this challenge, geographic variation in learned signals is ubiquitous.2 A proposed explanation for this pattern is that birds express predispositions to selectively learn and produce population-typical songs.3,4,5 While experimental studies on lab-reared birds have shown the existence of within-species learning predispositions,6,7,8,9,10 it remains unclear whether and how learning predispositions influence song acquisition in the wild. Here, we investigated innate song learning predispositions in wild pied flycatchers (Ficedula hypoleuca) by measuring the songs of individuals translocated as eggs from a Dutch population to a breeding population in Sweden. We compared the songs of the adult males hatched from these translocated eggs with those from the ancestral and receiving populations. Songs of translocated males closely resemble the local Swedish songs to which they were exposed during development, supporting the importance of social learning. However, translocated males selectively learned those local Swedish song elements that sound the most "Dutch-like." As a result, their songs are significantly shifted toward those of the ancestral Dutch population. This suggests that innate learning predispositions track ongoing song evolution in wild populations of pied flycatchers. We propose that as songs continue to diverge over time, this coevolutionary relationship between song and learning predispositions may contribute to the emergence of incipient pre-mating barriers.

Species-specific song responses emerge as a by-product of tuning to the local dialect.

Oscine birds preferentially respond to certain sounds over others from an early age, which focuses subsequent learning onto sexually relevant songs.1,2,3 Songs vary both across species and, due to cultural evolution, among populations of the same species. As a result, early song responses are expected to be shaped by selection both to avoid the fitness costs of cross-species learning4 and to promote learning of population-typical songs.5 These sources of selection are not mutually exclusive but can result in distinct geographic patterns of song responses in juvenile birds: if the risks of interspecific mating are the main driver of early song discrimination, then discrimination should be strongest where closely related species co-occur.4 In contrast, if early discrimination primarily facilitates learning local songs, then it should be tuned to songs typical of the local dialect.5,6,7 Here, we experimentally assess the drivers of song discrimination in nestling pied flycatchers (Ficedula hypoleuca). We first demonstrate that early discrimination against the songs of the closely related collared flycatcher (F. albicollis) is not strongly affected by co-occurrence. Second, across six European populations, we show that nestlings' early song responses are tuned to their local song dialect and that responses to the songs of collared flycatchers are similarly weak as to those of other conspecific dialects. Taken together, these findings provide clear experimental support for the hypothesis that cultural evolution, in conjunction with associated learning predispositions, drives the emergence of pre-mating reproductive barriers.

Tissue-specific patterns of regulatory changes underlying gene expression differences among Ficedula flycatchers and their naturally occurring F1 hybrids.

Changes in interacting cis- and trans-regulatory elements are important candidates for Dobzhansky-Muller hybrid incompatibilities and may contribute to hybrid dysfunction by giving rise to misexpression in hybrids. To gain insight into the molecular mechanisms and determinants of gene expression evolution in natural populations, we analyzed the transcriptome from multiple tissues of two recently diverged Ficedula flycatcher species and their naturally occurring F1 hybrids. Differential gene expression analysis revealed that the extent of differentiation between species and the set of differentially expressed genes varied across tissues. Common to all tissues, a higher proportion of Z-linked genes than autosomal genes showed differential expression, providing evidence for a fast-Z effect. We further found clear signatures of hybrid misexpression in brain, heart, kidney, and liver. However, while testis showed the highest divergence of gene expression among tissues, it showed no clear signature of misexpression in F1 hybrids, even though these hybrids were found to be sterile. It is therefore unlikely that incompatibilities between cis-trans regulatory changes explain the observed sterility. Instead, we found evidence that cis-regulatory changes play a significant role in the evolution of gene expression in testis, which illustrates the tissue-specific nature of cis-regulatory evolution bypassing constraints associated with pleiotropic effects of genes.

Heterospecific mating interactions as an interface between ecology and evolution.

Reproductive interference (costly interspecific sexual interactions) is well-understood to promote divergence in mating-relevant traits (i.e. reproductive character displacement: RCD), but it can also reduce population growth, eventually leading to local extinction of one of the species. The ecological and evolutionary processes driven by reproductive interference can interact with each other. These interactions are likely to influence whether the outcome is coexistence or extinction, but remain little studied. In this paper, we first develop an eco-evolutionary perspective on reproductive interference by integrating ecological and evolutionary processes in a common framework. We also present a simple model to demonstrate the eco-evolutionary dynamics of reproductive interference. We then identify a number of factors that are likely to influence the relative likelihoods of extinction or RCD. We discuss particularly relevant factors by classifying them into four categories: the nature of the traits responding to selection, the mechanisms determining the expression of these traits, mechanisms of reproductive interference and the ecological background. We highlight previously underappreciated ways in which these factors may influence the relative likelihoods of RCD and local extinction. By doing so, we also identify questions and future directions that will increase our holistic understanding of the outcomes of reproductive interference.

Experience modulates an insect's response to anthropogenic noise.

In response to anthropogenic noise, vertebrates express modified acoustic communication signals either through individual plasticity or local population adaptation. In contrast, how insects respond to this stressor is poorly studied. Field crickets Gryllus bimaculatus use acoustic signals to attract and locate mates and are commonly found in noisy roadside environments, offering a powerful system to study the effects of anthropogenic noise on insect communication. Rapid repetition of sexual calls (chirps) is essential to attract females, but calling incurs energetic costs and attracts predators. As a result, males are predicted to reduce calling rates when background noise is high. Here, we combine observations and experimental playbacks to show that the responses of field cricket males to anthropogenic noise also depend on their previous experience with passing cars. First, we show that males living on highway edges decrease their chirp rate in response to passing cars. To assess whether this behavioral response depends on previous exposure to car noise, we then broadcast recordings of car noise to males located at different distances from the road and, therefore, with different previous exposure to car noise. Although all tested individuals responded to broadcasted traffic noise, males closest to the road decreased their chirp rate less than individuals calling further from the road. These results suggest that regular exposure to anthropogenic noise may decrease individuals' sensitivity and behavioral responses to noise, allowing them to maintain effective signaling rates. Behavioral plasticity modulated by experience may thus allow some insect species to cope with human-induced environmental stressors.

The syntax-semantics interface in animal vocal communication.

Syntax (rules for combining words or elements) and semantics (meaning of expressions) are two pivotal features of human language, and interaction between them allows us to generate a limitless number of meaningful expressions. While both features were traditionally thought to be unique to human language, research over the past four decades has revealed intriguing parallels in animal communication systems. Many birds and mammals produce specific calls with distinct meanings, and some species combine multiple meaningful calls into syntactically ordered sequences. However, it remains largely unclear whether, like phrases or sentences in human language, the meaning of these call sequences depends on both the meanings of the component calls and their syntactic order. Here, leveraging recently demonstrated examples of meaningful call combinations, we introduce a framework for exploring the interaction between syntax and semantics (i.e. the syntax-semantic interface) in animal vocal sequences. We outline methods to test the cognitive mechanisms underlying the production and perception of animal vocal sequences and suggest potential evolutionary scenarios for syntactic communication. We hope that this review will stimulate phenomenological studies on animal vocal sequences as well as experimental studies on the cognitive processes, which promise to provide further insights into the evolution of language. This article is part of the theme issue 'What can animal communication teach us about human language?'

Collective Action Promoted by Key Individuals.

Explaining why individuals participate in risky group behaviors has been a long-term challenge. We experimentally studied the formation of groups of birds (mobs) that aggressively confront predators and avian nest parasites and developed a theoretical model to evaluate the conditions under which mobs arise. We presented taxidermied mounts of predators on adult birds (hawks and owls) and of nest threats (crows and cuckoos) at different distances to nests of Phylloscopus warblers. Even when alone, birds are aggressive toward predators of adult birds, both at and away from their nests. By contrast, birds aggressively confront nest threats alone only when they have a nest nearby. However, strong initial responses by nest owners lead individuals without nearby nests to increase their responses, thereby generating a mob. Building on these findings, we derive the conditions in which individuals are incentivized to invest more when joining a high-gain individual compared to when acting alone. Strong responses of high-gain individuals acting alone tend to reduce the investments of other high-gain individuals that subsequently join. However, individuals that benefit sufficiently little from acting alone increase their investments when joining a high-gain individual and can even be sufficiently incentivized to join in when they would otherwise not act alone. Together, these results suggest an important role for key individuals in the generation of some group behaviors.

Call combinations in birds and the evolution of compositional syntax.

Syntax is the set of rules for combining words into phrases, providing the basis for the generative power of linguistic expressions. In human language, the principle of compositionality governs how words are combined into a larger unit, the meaning of which depends on both the meanings of the words and the way in which they are combined. This linguistic capability, i.e., compositional syntax, has long been considered a trait unique to human language. Here, we review recent studies on call combinations in a passerine bird, the Japanese tit (Parus minor), that provide the first firm evidence for compositional syntax in a nonhuman animal. While it has been suggested that the findings of these studies fail to provide evidence for compositionality in Japanese tits, this criticism is based on misunderstanding of experimental design, misrepresentation of the importance of word order in human syntax, and necessitating linguistic capabilities beyond those given by the standard definition of compositionality. We argue that research on avian call combinations has provided the first steps in elucidating how compositional expressions could have emerged in animal communication systems.

Climate-driven build-up of temporal isolation within a recently formed avian hybrid zone.

Divergence in the onset of reproduction can act as an important source of reproductive isolation (i.e., allochronic isolation) between co-occurring young species, but evidence for the evolutionary processes leading to such divergence is often indirect. While advancing spring seasons strongly affect the onset of reproduction in many taxa, it remains largely unexplored whether contemporary spring advancement directly affects allochronic isolation between young species. We examined how increasing spring temperatures affected onset of reproduction and thereby hybridization between pied and collared flycatchers (Ficedula spp.) across habitat types in a young secondary contact zone. We found that both species have advanced their timing of breeding in 14 years. However, selection on pied flycatchers to breed earlier was weaker, resulting in a slower response to advancing springs compared to collared flycatchers and thereby build-up of allochronic isolation between the species. We argue that a preadaptation to a broader niche use (diet) of pied flycatchers explains the slower response to raising spring temperature, but that reduced risk to hybridize may contribute to further divergence in the onset of breeding in the future. Our results show that minor differences in the response to environmental change of co-occurring closely related species can quickly cause allochronic isolation.

Wild Birds Use an Ordering Rule to Decode Novel Call Sequences.

The generative power of human language depends on grammatical rules, such as word ordering, that allow us to produce and comprehend even novel combinations of words [1-3]. Several species of birds and mammals produce sequences of calls [4-6], and, like words in human sentences, their order may influence receiver responses [7]. However, it is unknown whether animals use call ordering to extract meaning from truly novel sequences. Here, we use a novel experimental approach to test this in a wild bird species, the Japanese tit (Parus minor). Japanese tits are attracted to mobbing a predator when they hear conspecific alert and recruitment calls ordered as alert-recruitment sequences [7]. They also approach in response to recruitment calls of heterospecific individuals in mixed-species flocks [8, 9]. Using experimental playbacks, we assess their responses to artificial sequences in which their own alert calls are combined into different orderings with heterospecific recruitment calls. We find that Japanese tits respond similarly to mixed-species alert-recruitment call sequences and to their own alert-recruitment sequences. Importantly, however, tits rarely respond to mixed-species sequences in which the call order is reversed. Thus, Japanese tits extract a compound meaning from novel call sequences using an ordering rule. These results demonstrate a new parallel between animal communication systems and human language, opening new avenues for exploring the evolution of ordering rules and compositionality in animal vocal sequences.

Reproductive character displacement of female, but not male song discrimination in an avian hybrid zone.

Divergence of male sexual signals and female preferences for those signals often maintains reproductive boundaries between closely related, co-occurring species. However, contrasting sources of selection, such as interspecific competition, can lead to weak divergence or even convergence of sexual signals in sympatry. When signals converge, assortative mating can be maintained if the mating preferences of females diverge in sympatry (reproductive character displacement; RCD), but there are few explicit examples. Pied flycatchers (Ficedula hypoleuca) are sympatric with collared flycatchers (F. albicollis) on the Baltic island of Öland, where males from both species compete over nestboxes, their songs converge, and the two species occasionally hybridize. We compare song discrimination of male and female pied flycatchers on Öland and in an allopatric population on the Swedish mainland. Using field choice trials, we show that male pied flycatchers respond similarly to the songs of both species in sympatry and allopatry, while female pied flycatchers express stronger discrimination against heterospecific songs in sympatry than in allopatry. These results are consistent with RCD of song discrimination of female pied flycatchers where they co-occur with collared flycatchers, which should maintain species assortative mating despite convergence of male sexual signals.

Competition-driven build-up of habitat isolation and selection favoring modified dispersal patterns in a young avian hybrid zone.

Competition-driven evolution of habitat isolation is an important mechanism of ecological speciation but empirical support for this process is often indirect. We examined how an on-going displacement of pied flycatchers from their preferred breeding habitat by collared flycatchers in a young secondary contact zone is associated with (a) access to an important food resource (caterpillar larvae), (b) immigration of pied flycatchers in relation to habitat quality, and (c) the risk of hybridization in relation to habitat quality. Over the past 12 years, the estimated access to caterpillar larvae biomass in the habitat surrounding the nests of pied flycatchers has decreased by a fifth due to shifted establishment possibilities, especially for immigrants. However, breeding in the high quality habitat has become associated with such a high risk of hybridization for pied flycatchers that overall selection currently favors pied flycatchers that were forced to immigrate into the poorer habitats (despite lower access to preferred food items). Our results show that competition-driven habitat segregation can lead to fast habitat isolation, which per se caused an opportunity for selection to act in favor of future "voluntarily" altered immigration patterns and possibly strengthened habitat isolation through reinforcement.

Song discrimination by nestling collared flycatchers during early development.

Pre-zygotic isolation is often maintained by species-specific signals and preferences. However, in species where signals are learnt, as in songbirds, learning errors can lead to costly hybridization. Song discrimination expressed during early developmental stages may ensure selective learning later in life but can be difficult to demonstrate before behavioural responses are obvious. Here, we use a novel method, measuring changes in metabolic rate, to detect song perception and discrimination in collared flycatcher embryos and nestlings. We found that nestlings as early as 7 days old respond to song with increased metabolic rate, and, by 9 days old, have increased metabolic rate when listening to conspecific when compared with heterospecific song. This early discrimination between songs probably leads to fewer heterospecific matings, and thus higher fitness of collared flycatchers living in sympatry with closely related species.

Experimental evidence for compositional syntax in bird calls.

Human language can express limitless meanings from a finite set of words based on combinatorial rules (i.e., compositional syntax). Although animal vocalizations may be comprised of different basic elements (notes), it remains unknown whether compositional syntax has also evolved in animals. Here we report the first experimental evidence for compositional syntax in a wild animal species, the Japanese great tit (Parus minor). Tits have over ten different notes in their vocal repertoire and use them either solely or in combination with other notes. Experiments reveal that receivers extract different meanings from 'ABC' (scan for danger) and 'D' notes (approach the caller), and a compound meaning from 'ABC-D' combinations. However, receivers rarely scan and approach when note ordering is artificially reversed ('D-ABC'). Thus, compositional syntax is not unique to human language but may have evolved independently in animals as one of the basic mechanisms of information transmission.

A blueprint for vocal learning: auditory predispositions from brains to genomes.

Memorizing and producing complex strings of sound are requirements for spoken human language. We share these behaviours with likely more than 4000 species of songbirds, making birds our primary model for studying the cognitive basis of vocal learning and, more generally, an important model for how memories are encoded in the brain. In songbirds, as in humans, the sounds that a juvenile learns later in life depend on auditory memories formed early in development. Experiments on a wide variety of songbird species suggest that the formation and lability of these auditory memories, in turn, depend on auditory predispositions that stimulate learning when a juvenile hears relevant, species-typical sounds. We review evidence that variation in key features of these auditory predispositions are determined by variation in genes underlying the development of the auditory system. We argue that increased investigation of the neuronal basis of auditory predispositions expressed early in life in combination with modern comparative genomic approaches may provide insights into the evolution of vocal learning.

Learning and signal copying facilitate communication among bird species.

Signals relevant to different sets of receivers in different contexts create a conflict for signal design. A classic example is vocal alarm signals, often used both during intraspecific and interspecific interactions. How can signals alert individuals from a variety of other species in some contexts, while also maintaining efficient communication among conspecifics? We studied heterospecific responses to avian alarm signals that drive the formation of anti-predator groups but are also used during intraspecific interactions. In three species-rich communities in the western Himalayas, alarm signals vary drastically across species. We show that, independently of differences in their calls, birds respond strongly to the alarm signals of other species with which they co-occur and much more weakly to those of species with which they do not co-occur. These results suggest that previous exposure and learning maintain heterospecific responses in the face of widespread signal divergence. At an area where only two species regularly interact, one species' calls incorporate the call of the other. We demonstrate experimentally that signal copying allows strong responses even without previous exposure and suggest that such hybrid calls may be especially favoured when pairwise interactions between species are strong.

The role of ecological constraint in driving the evolution of avian song frequency across a latitudinal gradient.

Just as features of the physical and biotic environment constrain evolution of ecological and morphological traits, they may also affect evolution of communication systems. Here we analyze constraints on rates of vocal evolution, using a large dataset of New World avian sister taxa. We show that species breeding in tropical forests sing at generally lower frequencies and across narrower bandwidths than species breeding in open habitats, or at high latitudes. We attribute these restrictions on birdsong frequency to the presence of high-frequency insect noise and greater degradation of high-frequency sounds in tropical forests. We fit Ornstein-Uhlenbeck models to show that recent evolution of song frequency has been more greatly constrained in tropical forests than elsewhere, that is, songs have shown less tendency to diverge over time in tropical forests, consistent with inferred acoustic restrictions. In addition, we find that song frequency has evolved more rapidly overall at high latitudes in both forest and open habitats. Besides a larger available sound window, other factors contributing to more rapid divergence at high latitudes may include an overall increased intensity of sexual selection, occupation of more divergent habitats, and the presence of fewer competing species.

Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing peg size on kidney clearance and tumor uptake with improved 64-copper PET imaging.

Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t(1/2) = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope (64)Cu (t(1/2) = 12 h). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site-specific attachment of monodispersed PEGn of increasing molecular size (n = 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake, and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80%ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h observed with diabodies that were pegylated on surface lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.

A latitudinal gradient in rates of evolution of avian syllable diversity and song length.

We ask whether rates of evolution in traits important for reproductive isolation vary across a latitudinal gradient, by quantifying evolutionary rates of two traits important for pre-mating isolation-avian syllable diversity and song length. We analyse over 2500 songs from 116 pairs of closely related New World passerine bird taxa to show that evolutionary rates for the two main groups of passerines-oscines and suboscines-doubled with latitude in both groups for song length. For syllable diversity, oscines (who transmit song culturally) evolved more than 20 times faster at high latitudes than in low latitudes, whereas suboscines (whose songs are innate in most species and who possess very simple song with few syllable types) show no clear latitudinal gradient in rate. Evolutionary rates in oscines and suboscines were similar at tropical latitudes for syllable complexity as well as for song length. These results suggest that evolutionary rates in traits important to reproductive isolation and speciation are influenced by latitude and have been fastest, not in the tropics where species diversity is highest, but towards the poles.

The increased risk of predation enhances cooperation.

Theory predicts that animals in adverse conditions can decrease individual risks and increase long-term benefits by cooperating with neighbours. However, some empirical studies suggest that animals often focus on short-term benefits, which can reduce the likelihood that they will cooperate with others. In this experimental study, we tested between these two alternatives by evaluating whether increased predation risk (as a correlate of environmental adversity) enhances or diminishes the occurrence of cooperation in mobbing, a common anti-predator behaviour, among breeding pied flycatchers Ficedula hypoleuca. We tested whether birds would join their mobbing neighbours more often and harass a stuffed predator placed near their neighbours' nests more intensely in areas with a higher perceived risk of predation. Our results show that birds attended mobs initiated by their neighbours more often, approached the stuffed predator significantly more closely, and mobbed it at a higher intensity in areas where the perceived risk of predation was experimentally increased. In such high-risk areas, birds also were more often involved in between-pair cooperation. This study demonstrates the positive impact of predation risk on cooperation in breeding songbirds, which might help in explaining the emergence and evolution of cooperation.