Regulatory and evolutionary impact of DNA methylation in two songbird species and their naturally occurring F1 hybrids.

BACKGROUND: Regulation of transcription by DNA methylation in 5'-CpG-3' context is a widespread mechanism allowing differential expression of genetically identical cells to persist throughout development. Consequently, differences in DNA methylation can reinforce variation in gene expression among cells, tissues, populations, and species. Despite a surge in studies on DNA methylation, we know little about the importance of DNA methylation in population differentiation and speciation. Here we investigate the regulatory and evolutionary impact of DNA methylation in five tissues of two Ficedula flycatcher species and their naturally occurring F1 hybrids. RESULTS: We show that the density of CpG in the promoters of genes determines the strength of the association between DNA methylation and gene expression. The impact of DNA methylation on gene expression varies among tissues with the brain showing unique patterns. Differentially expressed genes between parental species are predicted by genetic and methylation differentiation in CpG-rich promoters. However, both these factors fail to predict hybrid misexpression suggesting that promoter mismethylation is not a main determinant of hybrid misexpression in Ficedula flycatchers. Using allele-specific methylation estimates in hybrids, we also determine the genome-wide contribution of cis- and trans effects in DNA methylation differentiation. These distinct mechanisms are roughly balanced in all tissues except the brain, where trans differences predominate. CONCLUSIONS: Overall, this study provides insight on the regulatory and evolutionary impact of DNA methylation in songbirds.

Recombination rate variation shapes genomic variability of phylogeographic structure in a widespread North American songbird (Aves: Certhia americana).

The nonrandom distribution of chromosomal characteristics and functional elements-genomic architecture-impacts the relative strengths and impacts of population genetic processes across the genome. Due to this relationship, genomic architecture has the potential to shape variation in population genetic structure across the genome. Population genetic structure has been shown to vary across the genome in a variety of taxa, but this body of work has largely focused on pairwise population genomic comparisons between closely related taxa. Here, we used whole genome sequencing of seven phylogeographically structured populations of a North American songbird, the Brown Creeper (Certhia americana), to determine the impacts of genomic architecture on phylogeographic structure variation across the genome. Using multiple methods to infer phylogeographic structure-ordination, clustering, and phylogenetic methods-we found that recombination rate variation explained a large proportion of phylogeographic structure variation. Genomic regions with low recombination showed phylogeographic structure consistent with the genome-wide pattern. In regions with high recombination, we found strong phylogeographic structure, but with discordant patterns relative to the genome-wide pattern. In regions with high recombination rate, we found that populations with small effective population sizes evolve relatively more rapidly than larger populations, leading to discordant signatures of phylogeographic structure. These results suggest that the interplay between recombination rate variation and effective population sizes shape the relative impacts of selection and genetic drift in different parts of the genome. Overall, the combined interactions of population genetic processes, genomic architecture, and effective population sizes shape patterns of variability in phylogeographic structure across the genome of the Brown Creeper.

Will a changing climate affect hatching success in cavity-nesting birds: A case study with Eastern Bluebirds (Sialia sialis)?

A significant body of evidence indicates that climate change is influencing many aspects of avian ecology. Yet, how climate change is affecting, and is expected to influence some aspects of the breeding ecology of cavity-nesting birds remains uncertain. To explore the potential linkage between timing of first clutch, and the influence of ambient temperature on hatching success, we used Eastern Bluebird (Sialia sialis) nest records over a nine-year period from Alabama, USA. We investigated changes to annual clutch initiation dates, as well as variability in hatching success associated with ambient air temperatures during the incubation period. Using a simple linear model, we observed earlier annual egg laying dates over the nine years of this study with a difference of 24 days between earliest egg-laying date of the season. Daily temperature minima increased 2 °C across the nine-year time frame of this study. These data also indicate that Eastern Bluebird hatching success was the highest when mean ambient air temperature during incubation was between 19 °C and 24 °C (78%, as opposed to 69% and 68% above and below this temperature range, respectively). Our findings of increasing maxima, earlier maxima each year, and the lower minima of temperatures within our study area could expand the breadth of temperatures experienced by nesting Eastern Bluebirds possibly exposing them to temperatures outside of what promotes nesting success. These findings with a cavity-nesting bird highlight an optimal range of ambient temperatures associated with highest hatching success, conditions likely to be affected by climate change.

Vocal convergence and social proximity shape the calls of the most basal Passeriformes, New Zealand Wrens.

Despite extensive research on avian vocal learning, we still lack a general understanding of how and when this ability evolved in birds. As the closest living relatives of the earliest Passeriformes, the New Zealand wrens (Acanthisitti) hold a key phylogenetic position for furthering our understanding of the evolution of vocal learning because they share a common ancestor with two vocal learners: oscines and parrots. However, the vocal learning abilities of New Zealand wrens remain unexplored. Here, we test for the presence of prerequisite behaviors for vocal learning in one of the two extant species of New Zealand wrens, the rifleman (Acanthisitta chloris). We detect the presence of unique individual vocal signatures and show how these signatures are shaped by social proximity, as demonstrated by group vocal signatures and strong acoustic similarities among distantly related individuals in close social proximity. Further, we reveal that rifleman calls share similar phenotypic variance ratios to those previously reported in the learned vocalizations of the zebra finch, Taeniopygia guttata. Together these findings provide strong evidence that riflemen vocally converge, and though the mechanism still remains to be determined, they may also suggest that this vocal convergence is the result of rudimentary vocal learning abilities.

Mixed-species flock sizes and compositions influence flock members' success in three field experiments with novel feeders.

Mixed-species groups and aggregations are quite common and may provide substantial fitness-related benefits to group members. Individuals may benefit from the overall size of the mixed-species group or from the diversity of species present, or both. Here we exposed mixed-species flocks of songbirds (Carolina chickadees, Poecile carolinensis, tufted titmice, Baeolophus bicolor, and the satellite species attracted to these two species) to three different novel feeder experiments to assess the influence of mixed-species flock size and composition on ability to solve the feeder tasks. We also assessed the potential role of habitat density and traffic noise on birds' ability to solve these tasks. We found that likelihood of solving a novel feeder task was associated with mixed-species flock size and composition, though the specific social factor involved depended on the particular species and on the novel feeder. We did not find an influence of habitat density or background traffic noise on likelihood of solving novel feeder tasks. Overall, our results reveal the importance of variation in mixed-species group size and diversity on foraging success in these songbirds.

Supplemental structured surveys and pre-existing detection models improve fine-scale density and population estimation with opportunistic community science data.

Density and population estimates aid in conservation and stakeholder communication. While free and broadly available community science data can effectively inform species distribution models, they often lack the information necessary to estimate imperfect detection and area sampled, thus limiting their use in fine-scale density modeling. We used structured distance-sampling surveys to model detection probability and calculate survey-specific detection offsets in community science models. We estimated density and population for 16 songbird species under three frameworks: (1) a fixed framework that assumes perfect detection within a specified survey radius, (2) an independent framework that calculates offsets from an independent source, and (3) a calibration framework that calculates offsets from supplemental surveys. Within the calibration framework, we examined the effects of calibration dataset size and data pooling. Estimates of density and population size were consistently biased low in the fixed framework. The independent and calibration frameworks produced reliable estimates for some species, but biased estimates for others, indicating discrepancies in detection probability between structured and community science surveys. The calibration framework produced reliable population estimates with as few as 10 calibration surveys with positive detections. Data pooling dramatically decreased bias. This study provides conservationists and managers with a cost-effective method of estimating density and population.

Call combination production is linked to the social environment in Western Australian magpies (Gymnorhina tibicen dorsalis).

It has recently become clear that some language-specific traits previously thought to be unique to humans (such as the capacity to combine sounds) are widespread in the animal kingdom. Despite the increase in studies documenting the presence of call combinations in non-human animals, factors promoting this vocal trait are unclear. One leading hypothesis proposes that communicative complexity co-evolved with social complexity owing to the need to transmit a diversity of information to a wider range of social partners. The Western Australian magpie (Gymnorhina tibicen dorsalis) provides a unique model to investigate this proposed link because it is a group-living, vocal learning species that is capable of multi-level combinatoriality (independently produced calls contain vocal segments and comprise combinations). Here, we compare variations in the production of call combinations across magpie groups ranging in size from 2 to 11 birds. We found that callers in larger groups give call combinations: (i) in greater diversity and (ii) more frequently than callers in smaller groups. Significantly, these observations support the hypothesis that combinatorial complexity may be related to social complexity in an open-ended vocal learner, providing an important step in understanding the role that sociality may have played in the development of vocal combinatorial complexity. This article is part of the theme issue 'The power of sound: unravelling how acoustic communication shapes group dynamics'.

A refined magnetic pulse treatment method for magnetic navigation experiments with adequate sham control: a case study on free-flying songbirds.

Migratory songbirds may navigate by extracting positional information from the geomagnetic field, potentially with a magnetic-particle-based receptor. Previous studies assessed this hypothesis experimentally by exposing birds to a strong but brief magnetic pulse aimed at remagnetizing the particles and evoking an altered behaviour. Critically, such studies were not ideally designed because they lacked an adequate sham treatment controlling for the induced electric field that is fundamentally associated with a magnetic pulse. Consequently, we designed a sham-controlled magnetic-pulse experiment, with sham and treatment pulse producing a similar induced electric field, while limiting the sham magnetic field to a value that is deemed insufficient to remagnetize particles. We tested this novel approach by pulsing more than 250 wild, migrating European robins (Erithacus rubecula) during two autumn seasons. After pulsing them, five traits of free-flight migratory behaviour were observed, but no effect of the pulse could be found. Notably, one of the traits, the migratory motivation of adults, was significantly affected in only one of the two study years. Considering the problem of reproducing experiments with wild animals, we recommend a multi-year approach encompassing large sample size, blinded design and built-in sham control to obtain future insights into the role of magnetic-particle-based magnetoreception in bird navigation.

Removal of older males increases extra-pair siring success of yearling males.

In animals, reproductive performance typically improves over time early in life. Several ultimate and proximate mechanisms may contribute to such an age-related improvement and these mechanisms can act in a relative or in an absolute sense. Low performance of young individuals may be the consequence of a comparison or competition with older individuals (relative), or it may be due to specific traits of young individuals and be unrelated to the presence of older competitors (absolute). Here, we perform a test to disentangle whether the effect of age class (yearling or older) on male extra-pair siring success is relative or absolute. Male age is the most consistent predictor of male extra-pair siring success across bird species, yet the mechanisms underlying this pattern are not well understood. Low extra-pair siring success of yearling males may be a consequence of the presence of older ("adult") males (hypothesis 1), because adult males are more successful in intra- and intersexual interactions or because females prefer to copulate with adult males when available (relative preference). Alternatively, low extra-pair siring success of yearlings may be independent of the presence of adult males (hypothesis 2), for example, if yearling males on average invest less in extra-pair behavior or if females avoid them as extra-pair mates, independent of the availability of older males (absolute preference). To distinguish between these 2 hypotheses, we experimentally manipulated the age structure of a nest-box-breeding population of blue tits (Cyanistes caeruleus) by removing almost all adult males, and compared patterns of extra-pair paternity in the experimental year with those from the preceding 15 "control" years. Removal of adult males resulted in a substantial increase in the extra-pair siring success of yearling males compared to the "control" years, but did not affect the population-level frequency of extra-pair paternity or its spatial patterns. Our results provide clear evidence that extra-pair siring success of yearlings can increase and that it depends on the presence of older males in the population, indicating a relative effect of age on reproductive performance. These results suggest that older males outcompete yearling males in direct or indirect interactions, in sperm competition or as a result of differences in attractiveness to females.

Social restructuring during harsh environmental conditions promotes cooperative behaviour in a songbird.

Cooperation may emerge from intrinsic factors such as social structure and extrinsic factors such as environmental conditions. Although these factors might reinforce or counteract each other, their interaction remains unexplored in animal populations. Studies on multilevel societies suggest a link between social structure, environmental conditions and individual investment in cooperative behaviours. These societies exhibit flexible social configurations, with stable groups that overlap and associate hierarchically. Structure can be seasonal, with upper-level units appearing only during specific seasons, and lower-level units persisting year-round. This offers an opportunity to investigate how cooperation relates to social structure and environmental conditions. Here, we study the seasonal multilevel society of superb fairy-wrens (Malurus cyaneus), observing individual responses to experimental playback of conspecific distress calls. Individuals engaged more in helping behaviour and less in aggressive/territorial song during the harsher non-breeding season compared to the breeding season. The increase in cooperation was greater for breeding group members than for members of the same community, the upper social unit, comprised of distinct breeding groups in association. Results suggest that the interaction between social structure and environmental conditions drives the seasonal switch in cooperation, supporting the hypothesis that multilevel societies can emerge to increase cooperation during harsh environmental conditions.

High prevalence of haemosporidian parasites in Eurasian jays.

Avian haemosporidians are vector-borne parasites, infecting a great variety of birds. The order Passeriformes has the highest average infection probability; nevertheless, some common species of Passeriformes have been rather poorly studied. We investigated haemosporidians in one such species, the Eurasian jay Garrulus glandarius (Corvidae), from a forest population in Hesse, Central Germany. All individuals were infected with at least one haemosporidian genus (overall prevalence: 100%). The most common infection pattern was a mixed Haemoproteus and Leucocytozoon infection, whereas no Plasmodium infection was detected. Results on lineage diversity indicate a rather pronounced host-specificity of Haemoproteus and Leucocytozoon lineages infecting birds of the family Corvidae.

Diet of moulting Swainson's Thrushes (Catharus ustulatus) and Tennessee Warblers (Leiothlypis peregrina) at a stopover site during fall migration measured with fecal DNA metabarcoding.

Moult and migration are energetically demanding and require adequate nutrition. In some species, individuals may interrupt their fall migration to moult at discrete stopover locations outside of their breeding grounds (i.e., moult-migration) leading to competing nutritional demands for moult and migration. Here, we use DNA barcoding of fecal samples to compare the diet of moulting and actively migrating (post-moult) Swainson's Thrushes (Catharus ustulatus) and Tennessee Warblers (Leiothlypis peregrina) during their fall migration stopover at a large urban greenspace in Montreal, Canada. Diet differed according to moult status, species, and seasonality. Swainson's Thrushes had a broad diet with frequent detections of both insects and berry-producing shrubs; while detections in Tennessee Warblers' diets were mainly arthropods. For both species, more actively migrating individuals consumed fleshy-fruiting plants than moulting individuals. A higher proportion of moulting birds consumed arthropods compared to active migrants, due to either arthropod availability or a dietary preference for proteinaceous foods to grow feathers. Both species and moult classes consumed more native plants than non-native plants later in the season. We show the importance of managing urban greenspaces with native plants and diverse food sources that can provide for the different dietary needs of migratory birds.

Body size shapes song in honeyeaters.

Birdsongs are among the most distinctive animal signals. Their evolution is thought to be shaped simultaneously by habitat structure and by the constraints of morphology. Habitat structure affects song transmission and detectability, thus influencing song (the acoustic adaptation hypothesis), while body size and beak size and shape necessarily constrain song characteristics (the morphological constraint hypothesis). Yet, support for the acoustic adaptation and morphological constraint hypotheses remains equivocal, and their simultaneous examination is infrequent. Using a phenotypically diverse Australasian bird clade, the honeyeaters (Aves: Meliphagidae), we compile a dataset consisting of song, environmental, and morphological variables for 163 species and jointly examine predictions of these two hypotheses. Overall, we find that body size constrains song frequency and pace in honeyeaters. Although habitat type and environmental temperature influence aspects of song, that influence is indirect, likely via effects of environmental variation on body size, with some evidence that elevation constrains the evolution of song peak frequency. Our results demonstrate that morphology has an overwhelming influence on birdsong, in support of the morphological constraint hypothesis, with the environment playing a secondary role generally via body size rather than habitat structure. These results suggest that changing body size (a consequence of both global effects such as climate change and local effects such as habitat transformation) will substantially influence the nature of birdsong.

Genome Assembly of Pyrocephalus nanus: A Step Toward the Genetic Conservation of the Endangered Little Vermilion Flycatcher of the Galapagos Islands.

Incredibly powerful whole genome studies of conservation genetics, evolution, and biogeography become possible for non-model organisms when reference genomes are available. Here, we report the sequence and assembly of the whole genome of the little vermilion flycatcher (Pyrocephalus nanus; family Tyrannidae), which is an endemic, endangered, and declining species of the Galapagos Islands. Using PacBio HiFi reads to assemble long contigs and Hi-C reads for scaffolding, we assembled a genome of 1.07 Gb comprising 267 contigs in 152 scaffolds, scaffold N50 74 M, contig N50 17.8 M, with 98.9% assigned to candidate chromosomal sequences and 99.72% of the BUSCO passeriformes 10,844 single-copy orthologs present. In addition, we used the novel HiFiMiTie pipeline to fully assemble and verify all portions of the mitochondrial genome from HiFi reads, obtaining a mitogenome of 17,151 bases, containing 13 protein-coding genes, 22 tRNAs, 2 rRNAs, two control regions, and a unique structure of control region duplication and repeats. These genomes will be a critical tool for much-needed studies of phylogenetics, population genetics, biogeography, and conservation genetics of Pyrocephalus and related genera. This genome and other studies that use it will be able to provide recommendations for conservation management, taxonomic improvement, and to understand the evolution and diversification of this genus within the Galapagos Islands.

The effect of environmental variation on the relationship between survival and risk-taking behaviour in a migratory songbird.

Temporal changes in environmental conditions may play a major role in the year-to-year variation in fitness consequences of behaviours. Identifying environmental drivers of such variation is crucial to understand the evolutionary trajectories of behaviours in natural contexts. However, our understanding of how environmental variation influences behaviours in the wild remains limited. Using data collected over 14 breeding seasons from a collared flycatcher (Ficedula albicollis) population, we examined the effect of environmental variation on the relationship between survival and risk-taking behaviour, a highly variable behavioural trait with great evolutionary and ecological significance. Specifically, using annual recapture probability as a proxy of survival, we evaluated the specific effect of predation pressure, food availability, and mean temperature on the relationship between annual recapture probability and risk-taking behaviour (measured as flight initiation distance [FID]). We found a negative trend, as the relationship between annual recapture probability and FID decreased over the study years and changed from positive to negative. Specifically, in the early years of the study, risk-avoiding individuals exhibited a higher annual recapture probability, whereas in the later years, risk-avoiders had a lower annual recapture probability. However, we did not find evidence that any of the considered environmental factors mediated the variation in the relationship between survival and risk-taking behaviour.

Toxic to the touch: The makings of lethal mantles in pitohui birds and poison dart frogs.

How do chemically defended animals resist their own toxins? This intriguing question on the concept of autotoxicity is at the heart of how species interactions evolve. In this issue of Molecular Ecology (Molecular Ecology, 2024, 33), Bodawatta and colleagues report on how Papua New Guinean birds coopted deadly neurotoxins to create lethal mantles that protect against predators and parasites. Combining chemical screening of the plumage of a diverse collection of passerine birds with genome sequencing, the researchers unlocked a deeper understanding of how some birds sequester deadly batrachotoxin (BTX) from their food without poisoning themselves. They identified that birds impervious to BTX bear amino acid substitutions in the toxin-binding site of the voltage-gated sodium channel Nav1.4, whose function is essential for proper contraction and relaxation of vertebrate muscles. Comparative genetic and molecular docking analyses show that several of the substitutions associated with insensitivity to BTX may have become prevalent among toxic birds through positive selection. Intriguingly, poison dart frogs that also co-opted BTX in their lethal mantles were found to harbour similar toxin insensitivity substitutions in their Nav1.4 channels. Taken together, this sets up a powerful model system for studying the mechanisms behind convergent molecular evolution and how it may drive biological diversity.

Window into the songbird brain reveals superdiffusive migration of adult-born neurons.

In a recent study, Shvedov and colleagues used live two-photon imaging in transgenic zebra finches to reveal migration patterns of neuroblasts through the complex environment of the postembryonic brain. This study highlights the value of ubiquitin C/green fluorescent protein (UBC-GFP) transgenic zebra finches in studying adult neurogenesis and advances our understanding of dispersed long-distance neuronal migration in the adult brain, shedding light on this understudied phenomenon.

Migration increases mitochondrial oxidative capacity without increasing reactive oxygen species emission in a songbird.

Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000 km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation.

Goal-directed and flexible modulation of syllable sequence within birdsong.

Songs constitute a complex system of vocal signals for inter-individual communication in songbirds. Here, we elucidate the flexibility which songbirds exhibit in the organizing and sequencing of syllables within their songs. Utilizing a newly devised song decoder for quasi-real-time annotation, we execute an operant conditioning paradigm, with rewards contingent upon specific syllable syntax. Our analysis reveals that birds possess the capacity to modify the contents of their songs, adjust the repetition length of particular syllables and employing specific motifs. Notably, birds altered their syllable sequence in a goal-directed manner to obtain rewards. We demonstrate that such modulation occurs within a distinct song segment, with adjustments made within 10 minutes after cue presentation. Additionally, we identify the involvement of the parietal-basal ganglia pathway in orchestrating these flexible modulations of syllable sequences. Our findings unveil an unappreciated aspect of songbird communication, drawing parallels with human speech.