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There is considerable evidence for mitochondrial-nuclear co-adaptation as a key evolutionary driver. Hypotheses regarding the roles of sex-linkage have emphasized Z-linked nuclear genes with mitochondrial function (N-mt genes), whereas it remains contentious whether the perfect co-inheritance of W genes with mitogenomes could hinder or facilitate co-adaptation. Young (neo-) sex chromosomes that possess relatively many N-mt genes compared to older chromosomes provide unprecedented hypothesis-testing opportunities. Eastern Yellow Robin (EYR) lineages in coastal and inland habitats with different climates are diverged in mitogenomes, and in a ~ 15.4 Mb nuclear region enriched with N-mt genes, in contrast with otherwise-similar nuclear genomes. This nuclear region maps to passerine chromosome 1A, previously found to be neo-sex in the inland EYR genome. To compare sex-linked Chr1A-derived genes between lineages, we assembled and annotated the coastal EYR genome. We found that: (i) the coastal lineage shares a similar neo-sex system with the inland lineage, (ii) neo-W and neo-Z N-mt genes are not more diverged between lineages than are comparable non-N-mt genes, and showed little evidence for broad positive selection, (iii) however, W-linked N-mt genes are more diverged between lineages than are their Z-linked gametologs. The latter effect was ~7 times stronger for N-mt than non-N-mt genes, suggesting that W-linked N-mt genes might have diverged between lineages under environmental selection through co-evolution with mitogenomes. Finally, we identify a candidate gene driver for divergent selection, NDUFA12. Our data represent a rare example suggesting a possible role for W-associated mitochondrial-nuclear interactions in climate-associated adaptation and lineage differentiation., (© 2024. The Author(s).)

Museum genomics provide an opportunity to investigate population demographics of extinct species, especially valuable when research prior to extinction was minimal. The Bachman's warbler (Vermivora bachmanii) is hypothesized to have gone extinct due to loss of its specialized habitat. However, little is known about other potential contributing factors such as natural rarity or changes to connectivity following habitat fragmentation. We examined mitochondrial DNA (mtDNA) and genome-wide SNPs using specimens collected from breeding and migration sites across the range of the Bachman's warbler. We found no signals of strong population structuring across the breeding range of Bachman's warblers in both mtDNA and genome-wide SNPs. Thus, long-term population isolation did not appear to be a significant contributor to the extinction of the Bachman's warbler. Instead, our findings support the theory that Bachman's warblers underwent a rapid decline likely driven by habitat destruction, which may have been exacerbated by the natural rarity, habitat specificity and low genetic diversity of the species., (© 2024. The Author(s).)

When sex chromosomes evolve recombination suppression, the sex-limited chromosome (Y/W) commonly degenerate by losing functional genes. The rate of Y/W degeneration is believed to slow down over time as the most essential genes are maintained by purifying selection, but supporting data are scarce especially for ZW systems. Here, we study W degeneration in Sylvioidea songbirds where multiple autosomal translocations to the sex chromosomes, and multiple recombination suppression events causing separate evolutionary strata, have occurred during the last ~ 28.1-4.5 million years (Myr). We show that the translocated regions have maintained 68.3-97.7% of their original gene content, compared to only 4.2% on the much older ancestral W chromosome. By mapping W gene losses onto a dated phylogeny, we estimate an average gene loss rate of 1.0% per Myr, with only moderate variation between four independent lineages. Consistent with previous studies, evolutionarily constrained and haploinsufficient genes were preferentially maintained on W. However, the gene loss rate did not show any consistent association with strata age or with the number of W genes at strata formation. Our study provides a unique account on the pace of W gene loss and reinforces the significance of purifying selection in maintaining essential genes on sex chromosomes., (© 2024. The Author(s).)

Breeding responses of organisms to environmental changes may profoundly depend on an individual's age, as an age-environment interaction may be expected to affect the expression of reproductive traits. However, little is known about how this interaction affects short-lived species that experience various environmental conditions in adulthood. Here, we used a 32-year dataset from the collared flycatcher, Ficedula albicollis, population to test whether and how the environment interacts with age to shape female age-specific reproduction. To characterise environmental variation, we applied the remotely sensed normalised difference vegetation index (NDVI), estimating vegetation productivity, and used it as a surrogate for habitat quality. Then, we analysed how within-individual age and NDVI determine patterns in laying date, clutch size, offspring production, and recruitment. We found that young and old females, but not middle-aged females, breeding under low NDVI started to lay eggs later and produced smaller clutches than females of the same age breeding under higher NDVI. No such effects were observed for offspring production or recruitment. Our study provides evidence that both an individual's age and the environmental variation experienced during adulthood may be crucial for shaping reproductive patterns in short-lived avian species, as has been found in long-lived birds., (© 2023. Springer Nature Limited.)

Genetic data can be highly informative for answering questions relevant to practical conservation efforts, but remain one of the most neglected aspects of species recovery plans. Framing genetic questions with reference to practical and tractable conservation objectives can help bypass this limitation of the application of genetics in conservation. Using a single-nucleotide polymorphism dataset from reduced-representation sequencing (DArTSeq), we conducted a genetic assessment of remnant populations of the endangered forty-spotted pardalote (Pardalotus quadragintus), a songbird endemic to Tasmania, Australia. Our objectives were to inform strategies for the conservation of genetic diversity in the species and estimate effective population sizes and patterns of inter-population movement to identify management units relevant to population conservation and habitat restoration. We show population genetic structure and identify two small populations on mainland Tasmania as 'satellites' of larger Bruny Island populations connected by migration. Our data identify management units for conservation objectives relating to genetic diversity and habitat restoration. Although our results do not indicate the immediate need to genetically manage populations, the small effective population sizes we estimated for some populations indicate that they are vulnerable to genetic drift, highlighting the urgent need to implement habitat restoration to increase population size and to conduct genetic monitoring. We discuss how our genetic assessment can be used to inform management interventions for the forty-spotted pardalote and show that by assessing contemporary genetic aspects, valuable information for conservation planning and decision-making can be produced to guide actions that account for genetic diversity and increase chances of recovery in species of conservation concern., (© 2023. The Author(s).)

The impact of climate change on spring phenology poses risks to migratory birds, as migration timing is controlled predominantly by endogenous mechanisms. Despite recent advances in our understanding of the underlying genetic basis of migration timing, the ways that migration timing phenotypes in wild individuals may map to specific genomic regions requires further investigation. We examined the genetic architecture of migration timing in a long-distance migratory songbird (purple martin, Progne subis subis) by integrating genomic data with an extensive dataset of direct migratory tracks. A moderate to large amount of variance in spring migration arrival timing was explained by genomics (proportion of phenotypic variation explained by genomics = 0.74; polygenic score R 2  = 0.24). On chromosome 1, a region that was differentiated between migration timing phenotypes contained genes that could facilitate nocturnal flights and act as epigenetic modifiers. Overall, these results advance our understanding of the genomic underpinnings of migration timing., (© 2023. The Author(s).)

The ability of animals to sync the timing and location of molting (the replacement of hair, skin, exoskeletons or feathers) with peaks in resource availability has important implications for their ecology and evolution. In migratory birds, the timing and location of pre-migratory feather molting, a period when feathers are shed and replaced with newer, more aerodynamic feathers, can vary within and between species. While hypotheses to explain the evolution of intraspecific variation in the timing and location of molt have been proposed, little is known about the genetic basis of this trait or the specific environmental drivers that may result in natural selection for distinct molting phenotypes. Here we take advantage of intraspecific variation in the timing and location of molt in the iconic songbird, the Painted Bunting (Passerina ciris) to investigate the genetic and ecological drivers of distinct molting phenotypes. Specifically, we use genome-wide genetic sequencing in combination with stable isotope analysis to determine population genetic structure and molting phenotype across thirteen breeding sites. We then use genome-wide association analysis (GWAS) to identify a suite of genes associated with molting and pair this with gene-environment association analysis (GEA) to investigate potential environmental drivers of genetic variation in this trait. Associations between genetic variation in molt-linked genes and the environment are further tested via targeted SNP genotyping in 25 additional breeding populations across the range. Together, our integrative analysis suggests that molting is in part regulated by genes linked to feather development and structure (GLI2 and CSPG4) and that genetic variation in these genes is associated with seasonal variation in precipitation and aridity. Overall, this work provides important insights into the genetic basis and potential selective forces behind phenotypic variation in what is arguably one of the most important fitness-linked traits in a migratory bird., (© 2023. The Author(s).)

Comparisons of genomic variation among closely related species often show more differentiation in mitochondrial DNA (mtDNA) and sex chromosomes than in autosomes, a pattern expected due to the differing effective population sizes and evolutionary dynamics of these genomic components. Yet, introgression can cause species pairs to deviate dramatically from general differentiation trends. The yellowhammer (Emberiza citrinella) and pine bunting (E. leucocephalos) are hybridizing avian sister species that differ greatly in appearance and moderately in nuclear DNA, but that show no mtDNA differentiation. This discordance is best explained by adaptive mtDNA introgression-a process that can select for co-introgression at nuclear genes with mitochondrial functions (mitonuclear genes). To better understand these discordant differentiation patterns and characterize nuclear differentiation in this system, we investigated genome-wide differentiation between allopatric yellowhammers and pine buntings and compared it to what was seen previously in mtDNA. We found significant nuclear differentiation that was highly heterogeneous across the genome, with a particularly wide differentiation peak on the sex chromosome Z. We further investigated mitonuclear gene co-introgression between yellowhammers and pine buntings and found support for this process in the direction of pine buntings into yellowhammers. Genomic signals indicative of co-introgression were common in mitonuclear genes coding for subunits of the mitoribosome and electron transport chain complexes. Such introgression of mitochondrial DNA and mitonuclear genes provides a possible explanation for the patterns of high genomic heterogeneity in genomic differentiation seen among some species groups., (© 2022. The Author(s), under exclusive licence to The Genetics Society.)

Elaborate ornamental traits are commonly assumed to be honest signals of individual quality, owing to the presumed costs involved in their production and/or maintenance. Such traits are often highly variable, possibly because of condition-dependence and/or high underlying genetic variation, and it has been suggested that their expression should be more sensitive to condition and/or more heritable than non-ornamental traits. Many bird species display colourful plumage with multiple distinct patches of different developmental origins, forming complex colour phenotypes. Despite this complexity, colourful ornaments are often studied in isolation, without comparison to suitable non-ornamental controls. Based on plumage reflectance data collected over 8 years, we assessed the signalling potential of the multidimensional male colour phenotype in a tropical bird: the purple-crowned fairy-wren Malurus coronatus. Specifically, we tested the predictions that the expression of putative ornamental colours (purple and black - the breeding colours - and blue) is (1) more variable, (2) more heritable and (3) more condition-dependent compared to year-round non-ornamental colours (buff-white and brown). Our results show that ornamental colours exhibit greater levels of variability, and some chromatic components of purple and blue colouration appear slightly heritable (h² = 0.19-0.30). However, contrary to predictions of heightened condition-dependence in ornaments, only brightness of the buff-white and brown colouration increased with male body condition, although brightness of the purple colouration was related to male age as expected. Despite partial support for predictions, the lack of consistent patterns illustrates the complexity of visual signals and highlights the need to study colour phenotypes in their entirety., (© 2021. The Author(s), under exclusive licence to The Genetics Society.)

We investigated time course of photoperiodically driven transcriptional responses in physiologically contrasting seasonal life-history states in migratory blackheaded buntings. Birds exhibiting unstimulated winter phenotype (photosensitive state; responsive to photostimulation) under 6-h short days, and regressed summer phenotype (photorefractory state; unresponsiveness to photostimulation) under 16-h long days, were released into an extended light period up to 22 h of the day. Increased tshβ and dio2, and decreased dio3 mRNA levels in hypothalamus, and low prdx4 and high il1β mRNA levels in blood confirmed photoperiodic induction by hour 18 in photosensitive birds. Further, at hours 10, 14, 18 and 22 of light exposure, the comparison of hypothalamus RNA-Seq results revealed transcriptional differences within and between states. Particularly, we found reduced expression at hour 14 of transthyretin and proopiomelanocortin receptor, and increased expression at hour 18 of apolipoprotein A1 and carbon metabolism related genes in the photosensitive state. Similarly, valine, leucine and isoleucine degradation pathway genes and superoxide dismutase 1 were upregulated, and cocaine- and amphetamine-regulated transcript and gastrin-releasing peptide were downregulated in the photosensitive state. These results show life-history-dependent activation of hypothalamic molecular pathways involved in initiation and maintenance of key biological processes as early as on the first long day.

In today's environmental crisis, conservationists are increasingly confronted with terminally endangered species whose last few surviving populations may be affected by allelic introgression from closely related species. Yet there is a worrying lack of evidence-based recommendations and solutions for this emerging problem. We analyzed genome-wide DNA markers and plumage variability in a critically endangered insular songbird, the Black-winged Myna (BWM, Acridotheres melanopterus). This species is highly threatened by the illegal wildlife trade, with its wild population numbering in the low hundreds, and its continued survival urgently depending on ex-situ breeding. Its three subspecies occur along a geographic gradient of melanism and are variably interpreted as three species. However, our integrative approach revealed that melanism poorly reflects the pattern of limited genomic differentiation across BWM subspecies. We also uncovered allelic introgression into the most melanistic subspecies, tertius, from the all-black congeneric Javan Myna (A. javanicus), which is native to the same islands. Based on our results, we recommend the establishment of three separate breeding programs to maintain subspecific traits that may confer local adaptation, but with the option of occasional cross-breeding between insurance populations in order to boost genetic diversity and increase overall viability prospects of each breeding program. Our results underscore the importance of evidence-based integrative approaches when determining appropriate conservation units. Given the rapid increase of terminally endangered organisms in need of ex-situ conservation, this study provides an important blueprint for similar programs dealing with phenotypically variable species.

The Northern Wheatear (Oenanthe oenanthe, including the nominate and the two subspecies O. o. leucorhoa and O. o. libanotica) and the Seebohm's Wheatear (Oenanthe seebohmi) are today regarded as two distinct species. Before, all four taxa were regarded as four subspecies of the Northern Wheatear. Their classification has exclusively been based on ecological and morphological traits, while their molecular characterization is still missing. With this study, we used next-generation sequencing to assemble 117 complete mitochondrial genomes covering O. o. oenanthe, O. o. leucorhoa and O. seebohmi. We compared the resolution power of each individual mitochondrial marker and concatenated marker sets to reconstruct the phylogeny and estimate speciation times of three taxa. Moreover, we tried to identify the origin of migratory wheatears caught on Helgoland (Germany) and on Crete (Greece). Mitogenome analysis revealed two different ancient lineages that separated around 400,000 years ago. Both lineages consisted of a mix of subspecies and species. The phylogenetic trees, as well as haplotype networks are incongruent with the present morphology-based classification. Mitogenome could not distinguish these presumed species. The genetic panmixia among present populations and taxa might be the consequence of mitochondrial introgression between ancient wheatear populations.

The keen visual systems of birds have been relatively well-studied. The foundations of avian vision rest on their cone and rod photoreceptors. Most birds use four cone photoreceptor types for color vision, a fifth cone for achromatic tasks, and a rod for dim-light vision. The cones, along with their oil droplets, and rods are conserved across birds - with the exception of a few shifts in spectral sensitivity - despite taxonomic, behavioral and ecological differences. Here, however, we describe a novel photoreceptor organelle in a group of New World flycatchers (Empidonax spp.) in which the traditional oil droplet is replaced with a complex of electron-dense megamitochondria surrounded by hundreds of small, orange oil droplets. The photoreceptors with this organelle were unevenly distributed across the retina, being present in the central region (including in the fovea), but absent from the retinal periphery and the area temporalis of these insectivorous birds. Of the many bird species with their photoreceptors characterized, only the two flycatchers described here (E. virescens and E. minimus) possess this unusual retinal structure. We discuss the potential functional significance of this unique sub-cellular structure, which might provide an additional visual channel for these small predatory songbirds.

The X and Z sex chromosomes play a disproportionately large role in intrinsic postzygotic isolation. The underlying mechanisms of this large X/Z effect are, however, still poorly understood. Here we tested whether faster rates of molecular evolution caused by more intense positive selection or genetic drift on the Z chromosome could contribute to the large Z effect in two closely related passerine birds, the Common Nightingale (Luscinia megarhynchos) and the Thrush Nightingale (L. luscinia). We found that the two species differ in patterns of molecular evolution on the Z chromosome. The Z chromosome of L. megarhynchos showed lower levels of within-species polymorphism and an excess of non-synonymous polymorphisms relative to non-synonymous substitutions. This is consistent with increased levels of genetic drift on this chromosome and may be attributed to more intense postcopulatory sexual selection acting on L. megarhynchos males as was indicated by significantly longer sperm and higher between-male variation in sperm length in L. megarhynchos compared to L. luscinia. Interestingly, analysis of interspecific gene flow on the Z chromosome revealed relatively lower levels of introgression from L. megarhynchos to L. luscinia than vice versa, indicating that the Z chromosome of L. megarhynchos accumulated more hybrid incompatibilities. Our results are consistent with the view that postcopulatory sexual selection may reduce the effective population size of the Z chromosome and thus lead to stronger genetic drift on this chromosome in birds. This can result in relatively faster accumulation of hybrid incompatibilities on the Z and thus contribute to the large Z effect.

Songbirds communicate through learned vocalizations, using a forebrain circuit with convergent similarity to vocal-control circuitry in humans. This circuit is incomplete in female zebra finches, hence only males sing. We show that the UTS2B gene, encoding Urotensin-Related Peptide (URP), is uniquely expressed in a key pre-motor vocal nucleus (HVC), and specifically marks the neurons that form a male-specific projection that encodes timing features of learned song. UTS2B-expressing cells appear early in males, prior to projection formation, but are not observed in the female nucleus. We find no expression evidence for canonical receptors within the vocal circuit, suggesting either signalling to other brain regions via diffusion or transduction through other receptor systems. Urotensins have not previously been implicated in vocal control, but we find an annotation in Allen Human Brain Atlas of increased UTS2B expression within portions of human inferior frontal cortex implicated in human speech and singing. Thus UTS2B (URP) is a novel neural marker that may have conserved functions for vocal communication.

Leaf warblers (Aves; Phylloscopidae) are a diverse clade of insectivorous, canopy-dwelling songbirds widespread across the Old World. The taxonomy of Australasian leaf warblers is particularly complex, with multiple species-level divergences between island taxa in the region requiring further scrutiny. We use a combination of morphology, bioacoustics, and analysis of thousands of genome-wide markers to investigate and describe a new species of Phylloscopus leaf warbler from the island of Rote in the Lesser Sundas, Indonesia. We show that this new Rote Leaf Warbler is morphologically and genomically highly distinct from its congenerics, but do not find vocal differentiation between different island taxa. We discuss the behaviour and ecology of this highly distinctive new species, and make recommendations about its conservation status. We believe this constitutes the first description of a novel bird species that is partly based on insights from massive amounts of genome-wide DNA markers.

Although edge-tolerant species are known to benefit from habitat fragmentation, less is known about the population genetic impacts fragmentation may exert on edge-tolerant species. We examined the landscape genomic structure of an edge-tolerant forest-dependent bird species, the Striped Tit-Babbler Mixornis gularis, in the heavily urbanized island of Singapore to determine if two centuries of fragmentation have led to signs of isolation and loss of population-genetic diversity in different parts of the island. We obtained a high-quality complete reference genome with 78x coverage. Using almost 4000 SNPs from double-digest RAD-Sequencing across 46 individuals, we found that the population has likely experienced a recent contraction in effective population size and presently exhibits low population genetic diversity. Using empirical and simulation-based landscape genomic analyses, we also found that the subtle population genetic structure observed in the Striped Tit-Babbler population in Singapore is likely driven by isolation by distance resulting from limited dispersal. Our results demonstrate that population genetic impoverishment and subdivision can accumulate at relatively rapid rates in edge-tolerant bird species such as the Striped Tit-Babbler as a result of fragmentation, and that subtle spatial genetic structure can be detected over fine spatial and temporal scales using relatively few multilocus genomic SNPs.

Knowledge about the underlying genetic architecture of phenotypic traits is needed to understand and predict evolutionary dynamics. The number of causal loci, magnitude of the effects and location in the genome are, however, still largely unknown. Here, we use genome-wide single-nucleotide polymorphism (SNP) data from two large-scale data sets on house sparrows and collared flycatchers to examine the genetic architecture of different morphological traits (tarsus length, wing length, body mass, bill depth, bill length, total and visible badge size and white wing patches). Genomic heritabilities were estimated using relatedness calculated from SNPs. The proportion of variance captured by the SNPs (SNP-based heritability) was lower in house sparrows compared with collared flycatchers, as expected given marker density (6348 SNPs in house sparrows versus 38 689 SNPs in collared flycatchers). Indeed, after downsampling to similar SNP density and sample size, this estimate was no longer markedly different between species. Chromosome-partitioning analyses demonstrated that the proportion of variance explained by each chromosome was significantly positively related to the chromosome size for some traits and, generally, that larger chromosomes tended to explain proportionally more variation than smaller chromosomes. Finally, we found two genome-wide significant associations with very small-effect sizes. One SNP on chromosome 20 was associated with bill length in house sparrows and explained 1.2% of phenotypic variation (V P ), and one SNP on chromosome 4 was associated with tarsus length in collared flycatchers (3% of V P ). Although we cannot exclude the possibility of undetected large-effect loci, our results indicate a polygenic basis for morphological traits.

The relative importance of intrinsic constraints imposed by evolved physiological trade-offs versus the proximate effects of temperature for interspecific variation in embryonic development time remains unclear. Understanding this distinction is important because slow development due to evolved trade-offs can yield phenotypic benefits, whereas slow development from low temperature can yield costs. We experimentally increased embryonic temperature in free-living tropical and north temperate songbird species to test these alternatives. Warmer temperatures consistently shortened development time without costs to embryo mass or metabolism. However, proximate effects of temperature played an increasingly stronger role than intrinsic constraints for development time among species with colder natural incubation temperatures. Long development times of tropical birds have been thought to primarily reflect evolved physiological trade-offs that facilitate their greater longevity. In contrast, our results indicate a much stronger role of temperature in embryonic development time than currently thought.

To obtain aggregate evidence for the molecular basis of musical abilities and the effects of music, we integrated gene-level data from 105 published studies across multiple species including humans, songbirds and several other animals and used a convergent evidence method to prioritize the top candidate genes. Several of the identified top candidate genes like EGR1, FOS, ARC, BDNF and DUSP1 are known to be activity-dependent immediate early genes that respond to sensory and motor stimuli in the brain. Several other top candidate genes like MAPK10, SNCA, ARHGAP24, TET2, UBE2D3, FAM13A and NUDT9 are located on chromosome 4q21-q24, on the candidate genomic region for music abilities in humans. Functional annotation analyses showed the enrichment of genes involved in functions like cognition, learning, memory, neuronal excitation and apoptosis, long-term potentiation and CDK5 signaling pathway. Interestingly, all these biological functions are known to be essential processes underlying learning and memory that are also fundamental for musical abilities including recognition and production of sound. In summary, our study prioritized top candidate genes related to musical traits.

Maternal reproductive investment can critically influence offspring phenotype, and thus these maternal effects are expected to be under strong natural selection. Knowledge on the extent of heritable variation in the physiological mechanisms underlying maternal effects is however limited. In birds, resource allocation to eggs is a key mechanism for mothers to affect their offspring and different components of the egg may or may not be independently adjusted. We studied the heritability of egg components and their genetic and phenotypic covariation in great tits (Parus major), using captive-bred full siblings of wild origin. Egg mass, testosterone (T) and androstenedione (A4) hormone concentrations showed moderate heritability, in agreement with earlier findings. Interestingly, yolk triiodothyronine hormone (T3), but not its precursor, thyroxine hormone (T4), concentration was heritable. An immune factor, albumen lysozyme, showed moderate heritability, but yolk immunoglobulins (IgY) did not. The genetic correlation estimates were moderate but statistically nonsignificant; a trend for a positive genetic correlation was found between A4 and egg mass, T and lysozyme and IgY and lysozyme, respectively. Interestingly, phenotypic correlations were found only between A4 and T, and T4 and T3, respectively. Given that these egg components are associated with fitness-related traits in the offspring (and mother), and that we show that some components are heritable, it opens the possibility that natural selection may shape the rate and direction of phenotypic change via egg composition.

Recent evidence has implicated steroid hormones, specifically estrogens, in the rapid modulation of cognitive processes. Songbirds have been a useful model system in the study of complex cognitive processes including birdsong, a naturally learned vocal behavior regulated by a discrete steroid-sensitive telencephalic circuitry. Singing behavior is known to be regulated by long-term actions of estrogens but rapid steroid modulation of this behavior has never been examined. We investigated if acute actions of estrogens regulate birdsong in canaries (Serinus canaria). In the morning, male canaries sing within minutes after light onset. Birds were injected with fadrozole, a potent aromatase inhibitor, or vehicle within 2-5 minutes after lights on to implement a within-subjects experimental design. This single injection of fadrozole reduced the motivation to sing as well as song acoustic stereotypy, a measure of consistency over song renditions, on the same day. By the next day, however, all song measures that were affected had returned to baseline. This study indicates that estrogens also act in a rapid fashion to regulate two distinct features of song, a learned vocal behavior.

Landscape features influence individual dispersal and as a result can affect both gene flow and genetic variation within and between populations. The landscape of British Columbia, Canada, is already highly heterogeneous because of natural ecological and geological transitions, but disturbance from human-mediated processes has further fragmented continuous habitat, particularly in the central plateau region. In this study, we evaluated the effects of landscape heterogeneity on the genetic structure of a common resident songbird, the black-capped chickadee (Poecile atricapillus). Previous work revealed significant population structuring in British Columbia that could not be explained by physical barriers, so our aim was to assess the pattern of genetic structure at a microgeographic scale and determine the effect of different landscape features on genetic differentiation. A total of 399 individuals from 15 populations were genotyped for fourteen microsatellite loci revealing significant population structuring in this species. Individual- and population-based analyses revealed as many as nine genetic clusters with isolation in the north, the central plateau and the south. Moreover, a mixed modelling approach that accounted for non-independence of pairwise distance values revealed a significant effect of land cover and elevation resistance on genetic differentiation. These results suggest that barriers in the landscape influence dispersal which has led to the unexpectedly high levels of population isolation. Our study demonstrates the importance of incorporating landscape features when interpreting patterns of population differentiation. Despite taking a microgeographic approach, our results have opened up additional questions concerning the processes influencing dispersal and gene flow at the local scale.

With methods for sequencing thousands of loci for many individuals, phylogeographic studies have increased inferential power and the potential for applications to new questions. In songbirds, strong patterns of inter-chromosomal synteny, the published genome of a songbird and the ability to obtain thousands of genetic loci for many individuals permit the investigation of differentiation between and diversity within lineages across chromosomes. Here, we investigate patterns of differentiation and diversity in Certhia americana, a widespread North American songbird, using next-generation sequencing. Additionally, we reassess previous phylogeographic studies within the group. Based on ~30 million sequencing reads and more than 16,000 single-nucleotide polymorphisms in 41 individuals, we identified a strong positive relationship between genetic differentiation and chromosome size, with a negative relationship between genetic diversity and chromosome size. A combination of selection and drift may explain these patterns, although we found no evidence for selection. Because the observed genomic patterns are very similar between widespread, allopatric clades, it is unlikely that selective pressures would be so similar across such different ecological conditions. Alternatively, the accumulation of fixed differences between lineages and loss of genetic variation within lineages due to genetic drift alone may explain the observed patterns. Due to relatively higher recombination rates on smaller chromosomes, larger chromosomes would, on average, accumulate fixed differences between lineages and lose genetic variation within lineages faster, leading to the patterns observed here in C. americana.

Mobile organisms are expected to show population differentiation only over fairly large geographical distances. However, there is growing evidence of discrepancy between dispersal potential and realized gene flow. Here we report an intriguing pattern of differentiation at a very small spatial scale in the forest thrush (Turdus lherminieri), a bird species endemic to the Lesser Antilles. Analysis of 331 individuals from 17 sampling sites distributed over three islands revealed a clear morphological and genetic differentiation between these islands isolated by 40-50 km. More surprisingly, we found that the phenotypic divergence between the two geographic zones of the island of Guadeloupe was associated with a very strong genetic differentiation (Fst from 0.073-0.153), making this pattern a remarkable case in birds given the very small spatial scale considered. Molecular data (mitochondrial control region sequences and microsatellite genotypes) suggest that this strong differentiation could have occurred in situ, although alternative hypotheses cannot be fully discarded. This study suggests that the ongoing habitat fragmentation, especially in tropical forests, may have a deeper impact than previously thought on avian populations.

Ring species provide particularly clear demonstrations of how one species can gradually evolve into two, but are rare in nature. In the greenish warbler (Phylloscopus trochiloides) species complex, a ring of populations wraps around Tibet. Two reproductively isolated forms co-exist in central Siberia, with a gradient of genetic and phenotypic characteristics through the southern chain of populations connecting them. Previous genetic evidence has proven inconclusive, however, regarding whether species divergence took place in the face of continuous gene flow and whether hybridization between the terminal forms of the ring ever occurred. Here we use genome-wide analyses to show that, although spatial patterns of genetic variation are currently mostly as expected of a ring species, historical breaks in gene flow have existed at more than one location around the ring, and the two Siberian forms have occasionally interbred. Substantial periods of geographical isolation occurred not only in the north but also in the western Himalayas, where there is now an extensive hybrid zone between genetically divergent forms. Limited asymmetric introgression has occurred directly between the Siberian forms, although it has not caused a blending of those forms, suggesting selection against introgressed genes in the novel genetic background. Levels of reproductive isolation and genetic introgression are consistent with levels of phenotypic divergence around the ring, with phenotypic similarity and extensive interbreeding across the southwestern contact zone and strong phenotypic divergence and nearly complete reproductive isolation across the northern contact zone. These results cast doubt on the hypothesis that the greenish warbler should be viewed as a rare example of speciation by distance, but demonstrate that the greenish warbler displays a continuum from slightly divergent neighbouring populations to almost fully reproductively isolated species.

Determining the mechanisms responsible for the distribution of genetic diversity in natural populations has occupied a central role in molecular evolution. Our study was motivated by the unprecedented observation that a widespread Eurasian flycatcher, Ficedula albicilla, exhibited no variation at the mitochondrial DNA (mtDNA) ND2 gene in 75 individuals sampled over a 5000-km distance. In contrast, its sister species, F. parva, had low but considerably higher levels of mtDNA variation. We assessed whether natural selection or demographic factors could explain the absence of mtDNA variation in F. albicilla. Eighteen nuclear genes were sequenced to estimate the two species' phylogeographic histories, and for comparison to the mtDNA data. Multilocus coalescence analyses suggested that F. albicilla experienced a population expansion perhaps following a population bottleneck. Simulations based on this demographic history, however, did not replicate the extremely low level of mtDNA variation. Historical range changes based on ecological niche models also failed to explain the observed mtDNA patterns. Neutrality tests (DHEW and ML-HKA) suggested a non-neutral pattern in the mtDNA of F. albicilla. We found a transmembrane-skewed distribution of nonsynonymous substitutions between the two species, three of which caused functional change; the results implied that positive selection could have targeted mtDNA. Several lines of evidence support selection rather than demographic history as the main force influencing the patterns of mtDNA variation. Despite the influence of natural selection, many of the phylogeographic inferences derived from mtDNA were robust, including species limits and a high level of gene flow among populations within species.

Unravelling the genomic landscape of divergence between lineages is key to understanding speciation. The naturally hybridizing collared flycatcher and pied flycatcher are important avian speciation models that show pre- as well as postzygotic isolation. We sequenced and assembled the 1.1-Gb flycatcher genome, physically mapped the assembly to chromosomes using a low-density linkage map and re-sequenced population samples of each species. Here we show that the genomic landscape of species differentiation is highly heterogeneous with approximately 50 'divergence islands' showing up to 50-fold higher sequence divergence than the genomic background. These non-randomly distributed islands, with between one and three regions of elevated divergence per chromosome irrespective of chromosome size, are characterized by reduced levels of nucleotide diversity, skewed allele-frequency spectra, elevated levels of linkage disequilibrium and reduced proportions of shared polymorphisms in both species, indicative of parallel episodes of selection. Proximity of divergence peaks to genomic regions resistant to sequence assembly, potentially including centromeres and telomeres, indicate that complex repeat structures may drive species divergence. A much higher background level of species divergence of the Z chromosome, and a lower proportion of shared polymorphisms, indicate that sex chromosomes and autosomes are at different stages of speciation. This study provides a roadmap to the emerging field of speciation genomics.

Colour polymorphism exemplifies extreme morphological diversity within populations. It is taxonomically widespread but generally rare. Theory suggests that where colour polymorphism does occur, processes generating and maintaining it can promote speciation but the generality of this claim is unclear. Here we confirm, using species-level molecular phylogenies for five families of non-passerine birds, that colour polymorphism is associated with accelerated speciation rates in the three groups in which polymorphism is most prevalent. In all five groups, colour polymorphism is lost at a significantly greater rate than it is gained. Thus, the general rarity and phylogenetic dispersion of colour polymorphism is accounted for by a combination of higher speciation rate and higher transition rate from polymorphism to monomorphism, consistent with theoretical models where speciation is driven by fixation of one or more morphs. This is corroborated by evidence from a species-level molecular phylogeny of passerines, incorporating 4,128 (66.5%) extant species, that polymorphic species tend to be younger than monomorphic species. Our results provide empirical support for the general proposition, dating from classical evolutionary theory, that colour polymorphism can increase speciation rates.

The role of natural selection in shaping adaptive trait differentiation in natural populations has long been recognized. Determining its molecular basis, however, remains a challenge. Here, we search for signals of selection in candidate genes for colour and its perception in a passerine bird. Pied flycatcher plumage varies geographically in both its structural and pigment-based properties. Both characteristics appear to be shaped by selection. A single-locus outlier test revealed 2 of 14 loci to show significantly elevated signals of divergence. The first of these, the follistatin gene, is expressed in the developing feather bud and is found in pathways with genes that determine the structure of feathers and may thus be important in generating variation in structural colouration. The second is a gene potentially underlying the ability to detect this variation: SWS1 opsin. These two loci were most differentiated in two Spanish pied flycatcher populations, which are also among the populations that have the highest UV reflectance. The follistatin and SWS1 opsin genes thus provide strong candidates for future investigations on the molecular basis of adaptively significant traits and their co-evolution.

Neo-sex chromosomes often originate from sex chromosome-autosome fusions and constitute an important basis for the study of gene degeneration and expression in a sex chromosomal context. Neo-sex chromosomes are known from many animal and plant lineages, but have not been reported in birds, a group in which genome organization seems particularly stable. Following indications of sex linkage and unexpected sex-biased gene expression in warblers (Sylvioidea; Passeriformes), we have conducted an extensive marker analysis targeting 31 orthologues of loci on zebra finch chromosome 4a in five species, representative of independent branches of Passerida. We identified a region of sex linkage covering approximately the first half (10 Mb) of chromosome 4a, and associated to both Z and W chromosomes, in three Sylvioidea passerine species. Linkage analysis in an extended pedigree of one species additionally confirmed the association between this part of chromosome 4a and the Z chromosome. Markers located between 10 and 21 Mb of chromosome 4a showed no signs of sex linkage, suggesting that only half of the chromosome was involved in this transition. No sex linkage was observed in non-Sylvioidea passerines, indicating that the neo-sex chromosome arose at the base of the Sylvioidea branch of the avian phylogeny, at 47.4-37.6 millions years ago (MYA), substantially later than the ancestral sex chromosomes (150 MYA). We hypothesize that the gene content of chromosome 4a might be relevant in its transition to a sex chromosome, based on the presence of genes (for example, the androgen receptor) that could offer a selective advantage when associated to Z-linked sex determination loci.

The genetic correlation between the sexes in the expression of secondary sex traits in wild vertebrate populations has attracted very few previous empirical efforts of field researchers. In southern European populations of pied flycatchers, a sexually selected male ornament is also expressed by a proportion of females. Additive genetic variances in ornament size and expression, transmission mechanisms (autosomal vs Z-linkage) and maternal effects are examined by looking at patterns of familial resemblance across three generations. Size of the secondary sex trait has a genetic basis common to both sexes, with estimated heritability being 0.5 under an autosomal model of inheritance. Significant additive genetic variance in males was also confirmed through a cross-fostering experiment. Heritability analyses were only partially consistent with previous molecular genetics evidence, as only two out of the three predictions supported Z-linkage and lack of significant mother-daughter resemblance could be due to small sample sizes caused by limited female trait expression. Therefore, the evidence was mixed as to the contribution of the Z chromosome and autosomal genes to trait size. The threshold heritability of trait expression in females was lower, around 0.3, supporting autosomal-based trait expression in females. Environmental (birth date) and parental effects on ornament size mediated by the mother's condition after accounting for maternal and paternal genetic influences are also highlighted. The genetic correlation between the sexes did not differ from one, indicating that selection on the character on either sex entails a correlated response in the opposite sex.

One of the most debated questions in evolutionary biology is whether female choice of males with exaggerated sexual displays can evolve as a correlated response to selection acting on genes coding for male attractiveness or high overall viability. To date, empirical studies have provided support for parts of this scenario, but evidence for all key genetic components in a natural population is lacking. Here we use animal-model quantitative genetic analysis on data from over 8,500 collared flycatchers (Ficedula albicollis) followed for 24 years to quantify all of the key genetic requirements of both fisherian and 'good-genes' models on sexual selection in the wild. We found significant additive genetic variances of all the main components: male ornament (forehead patch size), female mate choice for this ornament, male fitness and female fitness. However, when the necessary genetic correlations between these components were taken into account, the estimated strength of indirect sexual selection on female mate choice was negligible. Our results show that the combined effect of environmental influences on several components reduces the potential for indirect sexual selection in the wild. This study provides insight into the field of sexual selection by showing that genes coding for mate choice for an ornament probably evolve by their own pathways instead of 'hitchhiking' with genes coding for the ornament.

Evolutionary theory predicts that local population divergence will depend on the balance between the diversifying effect of selection and the homogenizing effect of gene flow. However, spatial variation in the expression of genetic variation will also generate differential evolutionary responses. Furthermore, if dispersal is non-random it may actually reinforce, rather than counteract, evolutionary differentiation. Here we document the evolution of differences in body mass within a population of great tits, Parus major, inhabiting a single continuous woodland, over a 36-year period. We show that genetic variance for nestling body mass is spatially variable, that this generates different potential responses to selection, and that this diversifying effect is reinforced by non-random dispersal. Matching the patterns of variation, selection and evolution with population ecological data, we argue that the small-scale differentiation is driven by density-related differences in habitat quality affecting settlement decisions. Our data show that when gene flow is not homogeneous, evolutionary differentiation can be rapid and can occur over surprisingly small spatial scales. Our findings have important implications for questions of the scale of adaptation and speciation, and challenge the usual treatment of dispersal as a force opposing evolutionary differentiation.

Understanding the capacity of natural populations to adapt to their local environment is a central topic in evolutionary biology. Phenotypic differences between populations may have a genetic basis, but showing that they reflect different adaptive optima requires the quantification of both gene flow and selection. Good empirical data are rare. Using data on a spatially structured island population of great tits (Parus major), we show here that a persistent difference in mean clutch size between two subpopulations only a few kilometres apart has a major genetic component. We also show that immigrants from outside the island carry genes for large clutches. But gene flow into one subpopulation is low, as a result of a low immigration rate together with strong selection against immigrant genes. This has allowed for adaptation to the island environment and the maintenance of small clutches. In the other area, however, higher gene flow prevents local adaptation and maintains larger clutches. We show that the observed small-scale genetic difference in clutch size is not due to divergent selection on the island, but to different levels of gene flow from outside the island. Our findings illustrate the large effect of immigration on the evolution of local adaptations and on genetic population structure.

Ricklefs claims to show that morphological evolution in birds is associated with speciation events--that is, it is punctuational--by inference from data on only species number, clade age and character variance from a range of passerine clades. He suggests that variance increases in proportion with clade age under gradual change, but in proportion to the logarithm of species number if change is punctuational. Here I show that both clade age and the logarithm of species number independently predict variance under both gradual and punctuational change, rendering Ricklefs' results uninformative about his central hypothesis.

The major histocompatibility complex (MHC) has a central role in the specific immune defence of vertebrates. Exon 3 of MHC class I genes encodes the domain that binds and presents peptides from pathogens that trigger immune reactions. Here we develop a fast population screening method for detecting genetic variation in the MHC class I genes of birds. We found evidence of at least 15 exon 3 sequences in the investigated great reed warbler individual. The organisation of the great reed warbler MHC class I genes suggested that a locus-specific screening protocol is impractical due to the high similarity between alleles across loci, including the introns flanking exon 3. Therefore, we used motif-specific PCR to amplify two subsets of alleles (exon 3 sequences) that were separated with by DGGE. The motif-specific primers amplify a substantial proportion of the transcribed class I alleles (2-12 alleles per individual) from as many as six class I loci. Although not exhaustive, this gives a reliable estimate of the class I variation. The method is highly repeatable and more sensitive in detecting genetic variation than the RFLP method. The motif-specific primers also allow us to avoid screening pseudogenes. In our study population of great reed warblers, we found a high level of genetic variation in MHC class I, and no less than 234 DGGE genotypes were detected among 248 screened individuals.

Comprehensive long-term studies of isolated populations provide valuable comparative data that may be used to evaluate different methods for quantifying the relationship between genetic diversity and fitness. Here, we report on data collected from large and well-characterized cohorts of the two numerically dominant species of Darwin's finches on Isla Daphne Major, Galápagos, Ecuador - Geospiza fortis and G. scandens. Multilocus microsatellite (SSR) genetic diversity estimates (heterozygosity and d2) and pedigree-based estimates of the inbreeding coefficient (f) were compared to each other and to two fitness components: lifespan and recruitment. In the larger sample of G. fortis, heterozygosity (H) was correlated with both fitness components, but no relationship was detected in the smaller sample of G. scandens. Analyses of the inbreeding coefficient detected highly significant relationships between f and recruitment, but no relationship between f and overall lifespan. The d2 statistic showed no relationship to either fitness component. When the two SSR-based estimators were compared to f, d2 was correlated with f in G. fortis in the predicted direction, while in G. scandens the relationship was positive. Multilocus heterozygosity was correlated with f in G. fortis but not in the G. scandens sample. A pedigree simulation demonstrated that the variation in true autozygosity can be large among individuals with the same level of inbreeding. This observation may supplement the interpretation of patterns relevant to the local (locus-specific) and general (genome-wide) effects hypotheses, which have been proposed to explain the mechanism responsible for associations between genetic diversity and fitness.

Females in a variety of species commonly mate with multiple males, and there is evidence that they benefit by producing offspring of higher genetic quality; however, the nature of these genetic benefits is debated. Enhanced offspring survival or quality can result from intrinsic effects of paternal genes---'good genes'--or from interactions between the maternal and paternal genomes--'compatible genes'. Evidence for the latter process is accumulating: matings between relatives lead to decreased reproductive success, and the individual level of inbreeding--measured as average heterozygosity--is a strong fitness predictor. Females should thus benefit from mating with genetically dissimilar males. In many birds, social monogamy restricts mate choice, but females may circumvent this by pursuing extra-pair copulations. Here we show that female blue tits, Parus caeruleus, increase the heterozygosity of their progeny through extra-pair matings. Females thereby produce offspring of higher reproductive value, because less inbred individuals have increased survival chances, a more elaborate male secondary sexual trait (crown colour) and higher reproductive success. The cost of inbreeding may therefore be an important factor driving the evolution of female extra-pair mating.

A growing body of empirical and theoretical work supports the plausibility of sympatric speciation, but there remain few examples in which all the essential components of the process are well understood. The African indigobirds Vidua spp. are host-specific brood parasites. Indigobird nestlings are reared along with host young, and mimic the mouth markings of their respective hosts. As adults, male indigobirds mimic host song, whereas females use these songs to choose both their mates and the nests they parasitize. These behavioural mechanisms promote the cohesion of indigobird populations associated with a given host species, and provide a mechanism for reproductive isolation after a new host is colonized. Here we show that all indigobird species are similar genetically, but are significantly differentiated in both mitochondrial haplotype and nuclear allele frequencies. These data support a model of recent sympatric speciation. In contrast to the cuckoo Cuculus canorus, in which only female lineages are faithful to specific hosts, host switches have led to speciation in indigobirds because both males and females imprint on their hosts.

House-sparrow populations have declined sharply in Western Europe in recent decades, but the reasons for this decline have yet to be identified, despite intense public interest in the matter. Here we use a combination of field experimentation, genetic analysis and demographic data to show that a reduction in winter food supply caused by agricultural intensification is probably the principal explanation for the widespread local extinctions of rural house-sparrow populations in southern England. We show that farmland populations exhibit fine-level genetic structuring and that some populations are unable to sustain themselves (sinks), whereas others act as sources.

Cryptically coloured animals commonly occur in several distinct pattern variants. Such phenotypic diversity may be promoted by frequency-dependent predation, in which more abundant variants are attacked disproportionately often, but the hypothesis has never been explicitly tested. Here we report the first controlled experiment on the effects of visual predators on prey crypticity and phenotypic variance, in which blue jays (Cyanocitta cristata) searched for digital moths on computer monitors. Moth phenotypes evolved via a genetic algorithm in which individuals detected by the jays were much less likely to reproduce. Jays often failed to detect atypical cryptic moths, confirming frequency-dependent selection and suggesting the use of searching images, which enhance the detection of common prey. Over successive generations, the moths evolved to become significantly harder to detect, and they showed significantly greater phenotypic variance than non-selected or frequency-independent selected controls.