Mutual mate guarding with limited sexual conflict in a sex-role-reversed shorebird.

Mate guarding is typically considered a male strategy to protect paternity. However, under some circumstances, females might also benefit from guarding their mate. Female mate guarding might be particularly important in socially polyandrous species in which females compete for access to care-giving males. Because males also benefit from being near their partner to avoid paternity loss, pair members may have a mutual interest in mate guarding in polyandrous species. We studied the time spent together and movements that lead to separation, as behavioral measures of mate guarding, in the classically polyandrous red phalarope (Phalaropus fulicarius). We equipped 64 breeding pairs with miniaturized telemetry loggers with GPS to assess variation in mate-guarding intensity in relation to breeding phenology and season, nest attendance, and the occurrence of extrapair paternity. We show that red phalarope pairs were almost continuously together in the days before clutch initiation with no sex bias in separation movements, indicating mutual contribution to mate guarding. Our results suggest that in red phalaropes, both pair members guard their mate, with limited sexual conflict arising through biases in the operational sex ratio and a trade-off with male nest attendance. We found no clear relationship between mate-guarding intensity and the occurrence of extrapair paternity. In this non-territorial socially polyandrous species, mutual mate guarding might be the process underlying the evolution of a brief but strong social pair bond, with no other purpose than producing a clutch for a care-giving male.

Micro Germline-Restricted Chromosome in Blue Tits: Evidence for Meiotic Functions.

The germline-restricted chromosome (GRC) is likely present in all songbird species but differs widely in size and gene content. This extra chromosome has been described as either a microchromosome with only limited basic gene content or a macrochromosome with enriched gene functions related to female gonad and embryo development. Here, we assembled, annotated, and characterized the first micro-GRC in the blue tit (Cyanistes caeruleus) using high-fidelity long-read sequencing data. Although some genes on the blue tit GRC show signals of pseudogenization, others potentially have important functions, either currently or in the past. We highlight the GRC gene paralog BMP15, which is among the highest expressed GRC genes both in blue tits and in zebra finches (Taeniopygia guttata) and is known to play a role in oocyte and follicular maturation in other vertebrates. The GRC genes of the blue tit are further enriched for functions related to the synaptonemal complex. We found a similar functional enrichment when analyzing published data on GRC genes from two nightingale species (Luscinia spp.). We hypothesize that these genes play a role in maintaining standard maternal inheritance or in recombining maternal and paternal GRCs during potential episodes of biparental inheritance.

Weak antagonistic fitness effects can maintain an inversion polymorphism.

The study of chromosomal inversion polymorphisms has received much recent attention, particularly in cases where inversions have drastic effects on phenotypes and fitness (e.g. lethality of homozygotes). Less attention has been paid to the question of the maintenance of inversion polymorphisms that show only weak effects. Here, we study the maintenance of such an inversion polymorphism that links 250 genes on chromosome Tgu11 in the zebra finch (Taeniopygia guttata). Based on data from over 6000 captive birds, we estimated the effects of this inversion on a wide range of fitness-related traits. We found that, compared with the ancestral allele A, the inverted allele D had small additive beneficial effects on male siring success and on female fecundity. These fitness-enhancing effects may explain the initial spread of the derived D allele (allele frequency 53%). However, individuals that were homozygous for D had a slightly lower survival rate, which may explain why the D allele has not spread to fixation. We used individual-based simulations to examine how an inversion polymorphism with such antagonistic fitness effects behaves over time. Our results indicate that polymorphisms become stabilized at an intermediate allele frequency if the inversion links an additively beneficial allele of small effect size to a recessive weakly deleterious mutation, overall resulting in weak net heterosis. Importantly, this conclusion remains valid over a wide range of selection coefficients against the homozygous DD (up to lethality), suggesting that the conditions needed to maintain the polymorphism may frequently be met. However, the simulations also suggest that in our zebra finch populations, the estimated recessive deleterious effect of the D allele (on survival in captivity) is not quite large enough to prevent fixation of the D allele in the long run. Estimates of fitness effects from free-living populations are needed to validate these results.

Mendelian nightmares: the germline-restricted chromosome of songbirds.

Germline-restricted chromosomes (GRCs) are accessory chromosomes that occur only in germ cells. They are eliminated from somatic cells through programmed DNA elimination during embryo development. GRCs have been observed in several unrelated animal taxa and show peculiar modes of non-Mendelian inheritance and within-individual elimination. Recent cytogenetic and phylogenomic evidence suggests that a GRC is present across the species-rich songbirds, but absent in non-passerine birds, implying that over half of all 10,500 bird species have extensive germline/soma genome differences. Here, we review recent insights gained from genomic, transcriptomic, and cytogenetic approaches with regard to the genetic content, phylogenetic distribution, and inheritance of the songbird GRC. While many questions remain unsolved in terms of GRC inheritance, elimination, and function, we discuss plausible scenarios and future directions for understanding this widespread form of programmed DNA elimination.

Machine learning reveals cryptic dialects that explain mate choice in a songbird.

Culturally transmitted communication signals - such as human language or bird song - can change over time through cultural drift, and the resulting dialects may consequently enhance the separation of populations. However, the emergence of song dialects has been considered unlikely when songs are highly individual-specific, as in the zebra finch (Taeniopygia guttata). Here we show that machine learning can nevertheless distinguish the songs from multiple captive zebra finch populations with remarkable precision, and that 'cryptic song dialects' predict strong assortative mating in this species. We examine mating patterns across three consecutive generations using captive populations that have evolved in isolation for about 100 generations. We cross-fostered eggs within and between these populations and used an automated barcode tracking system to quantify social interactions. We find that females preferentially pair with males whose song resembles that of the females' adolescent peers. Our study shows evidence that in zebra finches, a model species for song learning, individuals are sensitive to differences in song that have hitherto remained unnoticed by researchers.

Occasional paternal inheritance of the germline-restricted chromosome in songbirds.

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby outcompeting other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness.

Fitness costs of female choosiness are low in a socially monogamous songbird.

Female mate choice is thought to be responsible for the evolution of many extravagant male ornaments and displays, but the costs of being too selective may hinder the evolution of choosiness. Selection against choosiness may be particularly strong in socially monogamous mating systems, because females may end up without a partner and forego reproduction, especially when many females prefer the same few partners (frequency-dependent selection). Here, we quantify the fitness costs of having mating preferences that are difficult to satisfy, by manipulating the availability of preferred males. We capitalize on the recent discovery that female zebra finches (Taeniopygia guttata) prefer males of familiar song dialect. We measured female fitness in captive breeding colonies in which one-third of females were given ample opportunity to choose a mate of their preferred dialect (two-thirds of all males; "relaxed competition"), while two-thirds of the females had to compete over a limited pool of mates they preferred (one-third of all males; "high competition"). As expected, social pairings were strongly assortative with regard to song dialect. In the high-competition group, 26% of the females remained unpaired, yet they still obtained relatively high fitness by using brood parasitism as an alternative reproductive tactic. Another 31% of high-competition females paired disassortatively for song dialect. These females showed increased levels of extra-pair paternity, mostly with same-dialect males as sires, suggesting that preferences were not abolished after social pairing. However, females that paired disassortatively for song dialect did not have lower reproductive success. Overall, females in the high-competition group reached equal fitness to those that experienced relaxed competition. Our study suggests that alternative reproductive tactics such as egg dumping can help overcome the frequency-dependent costs of being selective in a monogamous mating system, thereby facilitating the evolution of female choosiness.

A sex chromosome inversion is associated with copy number variation of mitochondrial DNA in zebra finch sperm.

The propulsion of sperm cells via movement of the flagellum is of vital importance for successful fertilization. While the exact mechanism of energy production for this movement varies between species, in avian species energy is thought to come predominantly from the mitochondria located in the sperm midpiece. Larger midpieces may contain more mitochondria, which should enhance the energetic capacity and possibly promote mobility. Due to an inversion polymorphism on their sex chromosome TguZ, zebra finches (Taeniopygia guttata castanotis) exhibit large within-species variation in sperm midpiece length, and those sperm with the longest midpieces swim the fastest. Here, we test through quantitative real-time PCR in zebra finch ejaculates whether the inversion genotype has an effect on the copy number of mitochondrial DNA (mtDNA). We find that zebra finches carrying the derived allele (correlated with longer sperm midpieces) have more copies of the mtDNA in their ejaculates than those homozygous for the ancestral allele (shorter midpieces). We suggest downstream effects of mtDNA copy number variation on the rate of adenosine triphosphate production, which in turn may influence sperm swimming speed and fertilization success. Central components of gamete energy metabolism may thus be the proximate cause for a fitness-relevant genetic polymorphism, stabilizing a megabase-scale inversion at an intermediate allele frequency in the wild.

Violating the normality assumption may be the lesser of two evils.

When data are not normally distributed, researchers are often uncertain whether it is legitimate to use tests that assume Gaussian errors, or whether one has to either model a more specific error structure or use randomization techniques. Here we use Monte Carlo simulations to explore the pros and cons of fitting Gaussian models to non-normal data in terms of risk of type I error, power and utility for parameter estimation. We find that Gaussian models are robust to non-normality over a wide range of conditions, meaning that p values remain fairly reliable except for data with influential outliers judged at strict alpha levels. Gaussian models also performed well in terms of power across all simulated scenarios. Parameter estimates were mostly unbiased and precise except if sample sizes were small or the distribution of the predictor was highly skewed. Transformation of data before analysis is often advisable and visual inspection for outliers and heteroscedasticity is important for assessment. In strong contrast, some non-Gaussian models and randomization techniques bear a range of risks that are often insufficiently known. High rates of false-positive conclusions can arise for instance when overdispersion in count data is not controlled appropriately or when randomization procedures ignore existing non-independencies in the data. Hence, newly developed statistical methods not only bring new opportunities, but they can also pose new threats to reliability. We argue that violating the normality assumption bears risks that are limited and manageable, while several more sophisticated approaches are relatively error prone and particularly difficult to check during peer review. Scientists and reviewers who are not fully aware of the risks might benefit from preferentially trusting Gaussian mixed models in which random effects account for non-independencies in the data.

A global analysis of song frequency in passerines provides no support for the acoustic adaptation hypothesis but suggests a role for sexual selection.

Animals use acoustic signals for communication, implying that the properties of these signals can be under strong selection. The acoustic adaptation hypothesis predicts that species in dense habitats emit lower-frequency sounds than those in open areas because low-frequency sounds propagate further in dense vegetation than high-frequency sounds. Signal frequency may also be under sexual selection because it correlates with body size and lower-frequency sounds are perceived as more intimidating. Here, we evaluate these hypotheses by analysing variation in peak song frequency across 5,085 passerine species (Passeriformes). A phylogenetically informed analysis revealed that song frequency decreases with increasing body mass and with male-biased sexual size dimorphism. However, we found no support for the predicted relationship between frequency and habitat. Our results suggest that the global variation in passerine song frequency is mostly driven by natural and sexual selection causing evolutionary shifts in body size rather than by habitat-related selection on sound propagation.

A test for meiotic drive in hybrids between Australian and Timor zebra finches.

Meiotic drivers have been proposed as a potent evolutionary force underlying genetic and phenotypic variation, genome structure, and also speciation. Due to their strong selective advantage, they are expected to rapidly spread through a population despite potentially detrimental effects on organismal fitness. Once fixed, autosomal drivers are cryptic within populations and only become visible in between-population crosses lacking the driver or corresponding suppressor. However, the assumed ubiquity of meiotic drivers has rarely been assessed in crosses between populations or species. Here we test for meiotic drive in hybrid embryos and offspring of Timor and Australian zebra finches-subspecies that have evolved in isolation for about two million years-using 38,541 informative transmissions of 56 markers linked to either centromeres or distal chromosome ends. We did not find evidence for meiotic driver loci on specific chromosomes. However, we observed a weak overall transmission bias toward Timor alleles at centromeres in females (transmission probability of Australian alleles of 47%, nominal p = 6 × 10-5). While this is in line with the centromere drive theory, it goes against the expectation that the subspecies with the larger effective population size (i.e., the Australian zebra finch) should have evolved the more potent meiotic drivers. We thus caution against interpreting our finding as definite evidence for centromeric drive. Yet, weak centromeric meiotic drivers may be more common than generally anticipated and we encourage further studies that are designed to detect also small effect meiotic drivers.

Proximate Causes of Infertility and Embryo Mortality in Captive Zebra Finches.

AbstractSome species show high rates of reproductive failure, which is puzzling because natural selection works against such failure in every generation. Hatching failure is common in both captive and wild zebra finches (Taeniopygia guttata), yet little is known about its proximate causes. Here we analyze data on reproductive performance (the fate of >23,000 eggs) based on up to 14 years of breeding of four captive zebra finch populations. We find that virtually all aspects of reproductive performance are negatively affected by inbreeding (mean r=-0.117); by an early-starting, age-related decline (mean r=-0.132); and by poor early-life nutrition (mean r=-0.058). However, these effects together explain only about 3% of the variance in infertility, offspring mortality, fecundity, and fitness. In contrast, individual repeatability of different fitness components varied between 15% and 50%. As expected, we found relatively low heritability in fitness components (median: 7% of phenotypic variation and 29% of individually repeatable variation). Yet some of the heritable variation in fitness appears to be maintained by antagonistic pleiotropy (negative genetic correlations) between male fitness traits and female and offspring fitness traits. The large amount of unexplained variation suggests a potentially important role of local dominance and epistasis, including the possibility of segregating genetic incompatibilities.

Author Correction: Reproducibility of animal research in light of biological variation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reproducibility of animal research in light of biological variation.

Context-dependent biological variation presents a unique challenge to the reproducibility of results in experimental animal research, because organisms' responses to experimental treatments can vary with both genotype and environmental conditions. In March 2019, experts in animal biology, experimental design and statistics convened in Blonay, Switzerland, to discuss strategies addressing this challenge. In contrast to the current gold standard of rigorous standardization in experimental animal research, we recommend the use of systematic heterogenization of study samples and conditions by actively incorporating biological variation into study design through diversifying study samples and conditions. Here we provide the scientific rationale for this approach in the hope that researchers, regulators, funders and editors can embrace this paradigm shift. We also present a road map towards better practices in view of improving the reproducibility of animal research.

Offspring performance is well buffered against stress experienced by ancestors.

Evolution should render individuals resistant to stress and particularly to stress experienced by ancestors. However, many studies report negative effects of stress experienced by one generation on the performance of subsequent generations. To assess the strength of such transgenerational effects we propose a strategy aimed at overcoming the problem of type I errors when testing multiple proxies of stress in multiple ancestors against multiple offspring performance traits, and we apply it to a large observational dataset on captive zebra finches (Taeniopygia guttata). We combine clear one-tailed hypotheses with steps of validation, meta-analytic summary of mean effect sizes, and independent confirmatory testing. We find that drastic differences in early growth conditions (nestling body mass 8 days after hatching varied sevenfold between 1.7 and 12.4 g) had only moderate direct effects on adult morphology (95% confidence interval [CI]: r = 0.19-0.27) and small direct effects on adult fitness traits (r = 0.02-0.12). In contrast, we found no indirect effects of parental or grandparental condition (r = -0.017 to 0.002; meta-analytic summary of 138 effect sizes), and mixed evidence for small benefits of matching environments between parents and offspring, as the latter was not robust to confirmatory testing in independent datasets. This study shows that evolution has led to a remarkable robustness of zebra finches against undernourishment. Our study suggests that transgenerational effects are absent in this species, because CIs exclude all biologically relevant effect sizes.

The role of genetic constraints and social environment in explaining female extra-pair mating.

Why do females of socially monogamous species engage in extra-pair copulations? This long-standing question remains a puzzle, because the benefits of female promiscuous behavior often do not seem to outweigh the costs. Genetic constraint models offer an answer by proposing that female promiscuity emerges through selection favoring alleles that are either beneficial for male reproductive success (intersexual pleiotropy hypothesis) or beneficial for female fecundity (intrasexual pleiotropy hypothesis). A previous quantitative genetic study on captive zebra finches, Taeniopygia guttata, reported support for the first, but not for the second hypothesis. Here, we re-examine both hypotheses based on data from lines selected for high and low male courtship rate. In contrast to previous conclusions, our new analyses clearly reject the hypothesis that male and female promiscuity are genetically homologous traits. We find some support for a positive genetic correlation between female promiscuity and fecundity. This study also shows that the behavioral outcome of extra-pair courtships primarily depends on individual-specific female preferences and not on the "attractiveness" of the social mate. In contrast, patterns of paternity are strongly influenced by the social partner and the pair bond, presumably reflecting variation in copulation behavior, fertility, or sperm competitiveness.

Programmed DNA elimination of germline development genes in songbirds.

In some eukaryotes, germline and somatic genomes differ dramatically in their composition. Here we characterise a major germline-soma dissimilarity caused by a germline-restricted chromosome (GRC) in songbirds. We show that the zebra finch GRC contains >115 genes paralogous to single-copy genes on 18 autosomes and the Z chromosome, and is enriched in genes involved in female gonad development. Many genes are likely functional, evidenced by expression in testes and ovaries at the RNA and protein level. Using comparative genomics, we show that genes have been added to the GRC over millions of years of evolution, with embryonic development genes bicc1 and trim71 dating to the ancestor of songbirds and dozens of other genes added very recently. The somatic elimination of this evolutionarily dynamic chromosome in songbirds implies a unique mechanism to minimise genetic conflict between germline and soma, relevant to antagonistic pleiotropy, an evolutionary process underlying ageing and sexual traits.

Traffic noise exposure depresses plasma corticosterone and delays offspring growth in breeding zebra finches.

The impact of human activity on the acoustic environment is overwhelming, with anthropogenic noise reaching even remote areas of the planet. The World Health Organization has identified noise pollution as one of the leading environmental health risks in humans, and it has been linked to a myriad of short- and long-term health effects in exposed individuals. However, less is known about the health effects of anthropogenic noise exposure on animals. We investigated long- and short-term effects of traffic noise on zebra finches breeding in small communal aviaries, using a repeated measures design. Birds bred in both noise and no-noise conditions, and we measured baseline plasma glucocorticoid levels before, during and after breeding. In addition, we assayed immune function, measured reproductive success and offspring growth and compared rates of extra-pair paternity of breeding adults. Breeding birds had significantly lower baseline plasma corticosterone levels when exposed to traffic noise than when they were not exposed to noise playback. In addition, the nestlings reared during noise exposure were lighter than nestlings of the same parents when breeding in control conditions. Our results suggest that traffic noise poses a more severe hurdle to birds at more vulnerable stages of their life history, such as during reproductive events and ontogeny. While chronic exposure to traffic noise in our birds did not, by itself, prove to be a sufficient stressor to cause acute effects on health or reproductive success in exposed individuals, it did result in disruptions to normal glucocorticoid profiles and delayed offspring growth. However, animals living in urban habitats are exposed to a multitude of anthropogenic disturbances, and it is likely that even species that appear to be thriving in noisy environments may suffer cumulative effects of these multiple disturbances that may together impact their fitness in urban environments.