Genome of an iconic Australian bird: High‐quality assembly and linkage map of the superb fairy‐wren (Malurus cyaneus)

The superb fairy‐wren, Malurus cyaneus, is one of the most iconic Australian passerine species. This species belongs to an endemic Australasian clade, Meliphagides, which diversified early in the evolution of the oscine passerines. Today, the oscine passerines comprise almost half of all avian species diversity. Despite the rapid increase of available bird genome assemblies, this part of the avian tree has not yet been represented by a high‐quality reference. To rectify that, we present the first high‐quality genome assembly of a Meliphagides representative: the superb fairy‐wren. We combined Illumina shotgun and mate‐pair sequences, PacBio long‐reads, and a genetic linkage map from an intensively sampled pedigree of a wild population to generate this genome assembly. Of the final assembled 1.07‐Gb genome, 975 Mb (90.4%) was anchored onto 25 pseudochromosomes resulting in a final superscaffold N50 of 68.11 Mb. This high‐quality bird genome assembly is one of only a handful which is also accompanied by a genetic map and recombination landscape. In comparison to other pedigree‐based bird genetic maps, we find that the fairy‐wren genetic map more closely resembles those of Taeniopygia guttata and Parus major maps, unlike the Ficedula albicollis map which more closely resembles that of Gallus gallus. Lastly, we also provide a predictive gene and repeat annotation of the genome assembly. This new high‐quality, annotated genome assembly will be an invaluable resource not only regarding the superb fairy‐wren species and relatives but also broadly across the avian tree by providing a novel reference point for comparative genomic analyses.     

Indirect fitness benefits through extra‐pair mating are large for an inbred minority,but cannot explain widespread infidelity among red‐winged fairy‐wrens

Extra‐pair paternity (EPP) has been suggested to improve the genetic quality of offspring, but evidence has been equivocal. Benefits of EPP may be only available to specific individuals or under certain conditions. Red‐winged fairy‐wrens have extremely high levels of EPP, suggesting fitness benefits might be large and available to most individuals. Furthermore, extreme philopatry commonly leads to incestuous social pairings, so inbreeding avoidance may be an important selection pressure. Here, we quantified the fitness benefits of EPP under varying conditions and across life‐stages. Extra‐pair offspring (EPO) did not appear to have higher fitness than within‐pair offspring (WPO), neither in poor years nor in the absence of helpers‐at‐the‐nest. However, EPP was beneficial for closely related social pairs, because inbred WPO suffered an overall 75% reduction in fitness. Inbreeding depression was nonlinear and reduced nestling body condition, first year survival and reproductive success. Our comprehensive study indicates that EPP should be favored for the 17% of females paired incestuously, but cannot explain the widespread infidelity in this species. Furthermore, our finding that fitness benefits of EPP only become apparent for a small part of the population could potentially explain the apparent absence of fitness differences in population wide comparisons of EPO and WPO.     

Piecing together female extra‐pair mate choice: females really do prefer more ornamented males

Evolutionary biologists have long been fascinated by extravagant male traits that abound across the animal kingdom and yet convey no apparent benefits to survival. From isopods to elephants, from armaments to ornaments, researchers have spent decades studying male–male competition and female mate choice in an effort to understand the significance of these secondary sexual characteristics. Among socially monogamous species, a frequently proposed explanation for the existence of male ornaments is that they are indicators of male genetic quality subject to female extra‐pair mate choice. However, despite over two decades of extensive research into extra‐pair paternity (EPP), the evidence that females actually choose more ornamented extra‐pair sires is surprisingly scant. Consequently, whether EPP and female choice have contributed to the evolution of male ornaments in socially monogamous species, and what fitness benefits (if any) they signal to females, remains unclear. Progress in this field has been hampered by the challenge of dissociating clear female choice for ornamentation from confounding factors. In this issue of Molecular Ecology, Whittingham & Dunn (2016) use an experimental approach in a bird species with very high rates of EPP to tease apart these correlative effects. In doing so, they demonstrate clearly that male ornamentation is subject to female extra‐pair mate choice. Their findings further suggest that EPP can be adaptive for females, and represent an important step forward in validating the role of EPP as an evolutionary driver of ornamental elaboration in socially monogamous species.     

Neighbouring‐group composition and within‐group relatedness drive extra‐group paternity rate in the European badger (Meles meles)

Extra‐group paternity (EGP) occurs commonly among group‐living mammals and plays an important role in mating systems and the dynamics of sexual selection; however, socio‐ecological and genetic correlates of EGP have been underexplored. We use 23 years of demographic and genetic data from a high‐density European badger (Meles meles) population, to investigate the relationship between the rate of EGP in litters and mate availability, mate incompatibility and mate quality (heterozygosity). Relatedness between within‐group assigned mothers and candidate fathers had a negative quadratic effect on EGP, whereas the number of neighbouring‐group candidate fathers had a linear positive effect. We detected no effect of mean or maximum heterozygosity of within‐group candidate fathers on EGP. Consequently, EGP was associated primarily with mate availability, subject to within‐group genetic effects, potentially to mitigate mate incompatibility and inbreeding. In badgers, cryptic female choice, facilitated by superfecundation, superfoetation and delayed implantation, prevents males from monopolizing within‐group females. This resonates with a meta‐analysis in group‐living mammals, which proposed that higher rates of EGP occur when within‐group males cannot monopolize within‐group females. In contrast to the positive meta‐analytic association, however, we found that EGP associated negatively with the number of within‐group assigned mothers and the number of within‐group candidate fathers; potentially a strategy to counter within‐group males committing infanticide. The relationship between the rate of EGP and socio‐ecological or genetic factors can therefore be intricate, and the potential for cryptic female choice must be accounted for in comparative studies.     

Ecological opportunities and individual condition as predictors of extra‐pair paternity in a south‐temperate swallow (Tachycineta leucorrhoa)

Ecological and physiological factors such as breeding density, breeding synchrony, and adult body condition can all affect extra‐pair mating behavior, but the relative importance of these factors may vary among species. White‐rumped Swallows (Tachycineta leucorrhoa) nesting in Buenos Aires Province, Argentina, exhibit high rates of extra‐pair paternity, with 77% of nests having extra‐pair young. Our objective was to determine the extent to which extra‐pair paternity in this species is explained by breeding synchrony, breeding density, and adult body condition. Our study of a population of White‐rumped Swallows breeding in nest boxes was conducted during two consecutive breeding seasons (September – early January 2006–2008). We found that neither breeding synchrony nor density of neighbors predicted levels of extra‐pair paternity in our study population. Leaner females were more likely to engage in extra‐pair behavior and fledged more nestlings, but did not differ in structural size from females that did not engage in extra‐pair behavior, suggesting that female mass is an important predictor of mating decisions and fitness for these aerial insectivores. Male body condition was not related to male extra‐pair behavior. The mass of female White‐rumped Swallows may affect their flying ability such that, during their fertile period, they are exposed to more potential extra‐pair mates during longer foraging flights. Being lighter may also improve the ability of females to provision nestlings later in the breeding cycle.     

Mating strategies in dominant meerkats: evidence for extra‐pair paternity in relation to genetic relatedness between pair mates

Rates of extra‐pair paternity (EPP) have frequently been associated with genetic relatedness between social mates in socially monogamous birds. However, evidence is limited in mammals. Here, we investigate whether dominant females use divorce or extra‐pair paternity as a strategy to avoid the negative effects of inbreeding when paired with a related male in meerkats Suricata suricatta, a species where inbreeding depression is evident for several traits. We show that dominant breeding pairs seldom divorce, but that rates of EPP are associated with genetic similarity between mates. Although extra‐pair males are no more distantly related to the female than social males, they are more heterozygous. Nevertheless, extra‐pair pups are not more heterozygous than within‐pair pups. Whether females benefit from EPP in terms of increased fitness of the offspring, such as enhanced survival or growth, requires further investigations.     

Chronic stress in superb fairy‐wrens occupying remnant woodlands: Are noisy miners to blame?

Interactions between competing species may be intensified when they are restricted to small patches of remnant habitat, potentially increasing physiological stress in individuals. The effects of interspecific competition on stress in wildlife remain largely unexplored. In Australia, remnant woodlands are often dominated by aggressive honeyeaters, especially the noisy miner (Manorina melanocephala). Harassment of smaller birds by miners may result in their exclusion from suitable woodland habitat. We tested whether the presence of noisy miners is also associated with elevated stress in a model species of small passerine bird, the superb fairy‐wren (Malurus cyaneus). We sampled wrens from six sites, three remnant woodlands with noisy miners and three larger fragments of reserved habitat without noisy miners. Differential white blood cell counts were used to infer levels of chronic stress. We also assessed variation in body condition and the prevalence of blood parasites (Haemoproteus spp.) to test for associations between stress and parasitemia. The mean heterophil‐to‐lymphocyte (H:L) ratio was 1.8 × higher among superb fairy‐wrens living in miner‐dominated woodlands, suggesting higher levels of chronic stress. Individuals with higher stress appeared to be in poorer condition, as indicated by fat scores and residual body mass. Prevalence of blood parasites was generally high and was highest in reserved habitat (59%) where miners were absent. Birds with blood parasites living in these habitats had higher H:L ratios but the intensity of infection and H:L ratio was inversely related. Our results suggest that birds persisting in the presence of noisy miners might experience chronic stress, but further study is necessary to separate the relative importance of noisy miner aggression from other potential stressors in small patches of degraded woodland. Stress induced by interspecific aggression should be considered in future studies of wildlife living in remnant vegetation.     

Differences in perceived predation risk associated with variation in relative size of extra‐pair and within‐pair offspring

Extra‐pair paternity (EPP) is a widespread phenomenon in birds. Researchers have long hypothesized that EPP must confer a fitness advantage to extra‐pair offspring (EPO), but empirical support for this hypothesis is definitively mixed. This could be because genetic benefits of EPP only exist in a subset of environmental contexts to which a population is exposed. From 2013 to 2015, we manipulated perceived predator density in a population of tree swallows (Tachycineta bicolor) breeding in New York to see whether fitness outcomes of extra‐pair and within‐pair offspring (WPO) varied with predation risk. In nests that had been exposed to predators, EPO were larger, longer‐winged and heavier than WPO. In nonpredator nests, WPO tended to be larger, longer‐winged and heavier than EPO, though the effect was nonsignificant. We found no differences in age, morphology or stress physiology between extra‐pair and within‐pair sires from the same nest, suggesting that additive genetic benefits cannot fully explain the differences in nestling size that we observed. The lack of an effect of predator exposure on survival or glucocorticoid stress physiology of EPO and WPO further suggests that observed size differences do not reflect more general variation in intrinsic genetic quality. Instead, we suggest that size differences may have arisen through differential investment into EPO and WPO by females, perhaps because EPO and WPO represent different reproductive strategies, with each type of nestling conferring a fitness advantage in specific ecological contexts.     

Experimentally altering pre‐breeding sex steroids reduces extra‐pair paternity in female tree swallows

Extra‐pair paternity commonly occurs in many socially monogamous animal species, yet the reasons why females mate with males outside the social pair bond remain poorly understood. Because sex steroids mediate aggressive and sexual behaviors that peak prior to egg laying in female birds, we tested whether they also influence the rate of extra‐pair paternity in nests of female tree swallows (Tachycineta bicolor). In one treatment, we experimentally elevated testosterone (T) using implants containing exogenous T, and in another treatment, we blocked the estrogenic and androgenic actions of T using implants containing 1,4,6‐androstatrien‐3,17‐dione in combination with flutamide (ATD+F). Females with empty implants served as controls. Nests of females treated with T and those with ATD+F were less likely to contain extra‐pair offspring and had a lower proportion of extra‐pair offspring, compared to control females. Treating females with T also delayed clutch initiation dates and disrupted incubation behavior, as found in previous studies, but clutch sizes of T females did not differ from controls. Females treated with ATD+F tended to lay larger clutches than control and T‐treated females, which may be due to their clutch sizes not declining with later initiation dates as found in the T and control treatments; however, after controlling for brood size, ATD+F females produced nestlings that were lighter at day 16 than control females. Although the effect of T on extra‐pair paternity that we observed in tree swallows is consistent with previous studies in female birds, our results demonstrate that blocking the estrogenic and/or androgenic actions of T during pre‐breeding also lowers extra‐pair paternity. Overall, our study suggests that extra‐pair paternity in tree swallows is mediated by sex steroids of females.     

When to start and when to stop: Effects of climate on breeding in a multi‐brooded songbird

Climate warming has been shown to affect the timing of the onset of breeding of many bird species across the world. However, for multi‐brooded species, climate may also affect the timing of the end of the breeding season, and hence also its duration, and these effects may have consequences for fitness. We used 28 years of field data to investigate the links between climate, timing of breeding, and breeding success in a cooperatively breeding passerine, the superb fairy‐wren (Malurus cyaneus). This multi‐brooded species from southeastern Australia has a long breeding season and high variation in phenology between individuals. By applying a “sliding window” approach, we found that higher minimum temperatures in early spring resulted in an earlier start and a longer duration of breeding, whereas less rainfall and more heatwaves (days > 29°C) in late summer resulted in an earlier end and a shorter duration of breeding. Using a hurdle model analysis, we found that earlier start dates did not predict whether or not females produced any young in a season. However, for successful females who produced at least one young, earlier start dates were associated with higher numbers of young produced in a season. Earlier end dates were associated with a higher probability of producing at least one young, presumably because unsuccessful females kept trying when others had ceased. Despite larger scale trends in climate, climate variables in the windows relevant to this species’ phenology did not change across years, and there were no temporal trends in phenology during our study period. Our results illustrate a scenario in which higher temperatures advanced both start and end dates of individuals’ breeding seasons, but did not generate an overall temporal shift in breeding times. They also suggest that the complexity of selection pressures on breeding phenology in multi‐brooded species may have been underestimated.     

Strategic promiscuity helps avoid inbreeding at multiple levels in a cooperative breeder where both sexes are philopatric

In cooperative breeders, the tension between the opposing forces of kin selection and kin competition is at its most severe. Although philopatry facilitates kin selection, it also increases the risk of inbreeding. When dispersal is limited, extra-pair paternity might be an important mechanism to avoid inbreeding, but evidence for this is equivocal. The red-winged fairy-wren is part of a genus of cooperative breeders with extreme levels of promiscuity and male philopatry, but is unique in that females are also strongly philopatric. Here, we test the hypothesis that promiscuity is an important inbreeding avoidance mechanism when both sexes are philopatric. Levels of extra-pair paternity were substantial (70% of broods), but did not arise through females mating with their helpers, but via extra-group mating. Offspring were more likely to be sired by extra-pair males when the social pair was closely related, and these extra-pair males were genetically less similar to the female than the social male and thus, inbreeding is avoided through extra-pair mating. Females were consistent in their choice of the extra-pair sire over time and preferred early moulting males. Despite neighbouring males often being close kin, they sired 37% of extra-pair offspring. However, females that gained paternity from neighbours were typically less related to them than females that gained paternity further away. Our study is the first to suggest that mating with both closely related social partners and neighbours is avoided. Such sophistication in inbreeding avoidance strategies is remarkable, as the extreme levels of promiscuity imply that social context may provide little cue to relatedness.     

Flexible mating patterns in an obligate brood parasite

Polygamous mating is expected to occur in obligate avian brood parasites because the lack of parental care reduces the need for a stable reproductive bond. Unlike nesting species, an absence of the constraints resulting from raising offspring might also favour a flexible mating system that adjusts to changing ecological conditions. Information on brood parasites' mating systems and their spatio‐temporal variation is, however, still scant. Here we analysed the genetic mating patterns of Great Spotted Cuckoos Clamator glandarius in two populations in northern and southern Spain and compared the results with those of previous studies. Parentage analyses showed high levels of polygamy in both populations that contrast with a prevalence of monogamy previously reported in the southern population. We suggest that the differences arise from an increase in population density, which in turn increases the probability of intraspecific encounters and therefore opportunities for mating. We also found that a greater number of mates increased the number of offspring produced, both in males and in females. The increase in offspring production in females might be the result of enhanced fertilization success during the lengthy laying period. Our data, combined with previous reports, demonstrate plasticity in the genetic mating patterns of the Great Spotted Cuckoo that may be associated with large fluctuations in population density.     

A likelihood‐based approach for assessment of extra‐pair paternity and conspecific brood parasitism in natural populations

Genotypes are frequently used to assess alternative reproductive strategies such as extra‐pair paternity and conspecific brood parasitism in wild populations. However, such analyses are vulnerable to genotyping error or molecular artefacts that can bias results. For example, when using multilocus microsatellite data, a mismatch at a single locus, suggesting the offspring was not directly related to its putative parents, can occur quite commonly even when the offspring is truly related. Some recent studies have advocated an ad‐hoc rule that offspring must differ at more than one locus in order to conclude that they are not directly related. While this reduces the frequency with which true offspring are identified as not directly related young, it also introduces bias in the opposite direction, wherein not directly related young are categorized as true offspring. More importantly, it ignores the additional information on allele frequencies which would reduce overall bias. In this study, we present a novel technique for assessing extra‐pair paternity and conspecific brood parasitism using a likelihood‐based approach in a new version of program cervus . We test the suitability of the technique by applying it to a simulated data set and then present an example to demonstrate its influence on the estimation of alternative reproductive strategies.     

Inclusive fitness of 'kissing cousins': new evidence of a role for kin selection in the evolution of extra-pair mating in birds

Social monogamy is nearly ubiquitous across avian taxa,but evidence from a proliferation of studies utilizing molecular paternity analysis suggests that sexual monogamy is the rare exception rather than the rule (Griffith et al. 2002). Efforts to explain the prevalence of extra-pair paternity (EPP) have largely focused on the potential fitness benefits for offspring genetic quality, as females are less likely to benefit directly from seeking extra-pair mates. In particular, there has been considerable interest in the degree to which EPP may represent an adaptive female strategy to avoid inbreeding (or outbreeding)depression when paired with a highly related (or unrelated)social mate (Kempenaers 2007). Others have argued that, because relatives share many genes identical by descent,females might increase their own inclusive fitness by providing additional breeding opportunities to genetically related males (Waser et al. 1986; Kokko & Ots 2006). Thus, in the absence of significant inbreeding depression, pursuing EPP with relatives should be favoured by kin selection, although there exist few unambiguous empirical examples of such preferences in the literature. In this issue of Molecular Ecology, Wang &Lu (2011) present an analysis of mating patterns with respect to genetic relatedness of social and extra-pair partners in the ground tit (Parus humilis), a facultative cooperative breeder in which socially monogamous pairs occasionally form cooperative groups with unpaired helper males (Fig. 1). Consistent with the predictions of the kin-selection hypothesis, females in both bi-parental and cooperative groups preferentially engaged in extra-pair matings with relatives, irrespective of relatedness to their social mates, and while suffering no apparent costs of inbreeding depression in their progeny. These finding shave several exciting implications for our understanding of avian mating system diversity and the evolution of cooperative breeding.     

A molecular analysis of African lion (Panthera leo) mating structure and extra‐group paternity in Etosha National Park

The recent incorporation of molecular methods into analyses of social and mating systems has provided evidence that mating patterns often differ from those predicted by group social organization. Based on field studies and paternity analyses at a limited number of sites, African lions are predicted to exhibit a strict within‐pride mating system. Extra‐group paternity has not been previously reported in African lions; however, observations of extra‐group associations among lions inhabiting Etosha National Park in Namibia suggest deviation from the predicted within‐pride mating pattern. We analysed variation in 14 microsatellite loci in a population of 164 African lions in Etosha National Park. Genetic analysis was coupled with demographic and observational data to examine pride structure, relatedness and extra‐group paternity (EGP). EGP was found to occur in 57% of prides where paternity was analysed (n = 7), and the overall rate of EGP in this population was 41% (n = 34). Group sex ratio had a significant effect on the occurrence of EGP (P < 0.05), indicating that variation in pride‐level social structure may explain intergroup variation in EGP. Prides with a lower male‐to‐female ratio were significantly more likely to experience EGP in this population. The results of this study challenge the current models of African lion mating systems and provide evidence that social structure may not reflect breeding structure in some social mammals.     

Patterns of extra‐pair paternity in mountain chickadees

Extra‐pair paternity (EPP) is common in chickadees and often attributed to the good genes hypothesis. Females generally seek dominant males, who are typically larger, older and sing at higher rates than subordinate males, as extra‐pair sires. In other songbird species, habitat quality and urbanization have been found to influence EPP. Mountain chickadees commonly inhabit suburban habitat, and previous research on our population has shown urbanization may provide benefits to these adaptable songbirds. Here, we ask how individual condition and urbanization influence rates of EPP in mountain chickadees. Over three breeding seasons, we monitored mountain chickadee nests in urban and rural habitat, and determined parentage by genotyping adults and nestlings at six microsatellite loci. Extra‐pair paternity is common in mountain chickadees, with extra‐pair offspring (EPO) in 43.2% of nests and accounting for 17.9% of offspring. We found tenuous support for the good genes hypothesis with females tending to engage in EPCs with older males. However, we did not find an influence of male or female condition on the proportion of EPO in a nest. In addition, we did not find a significant effect of habitat on EPP rates, suggesting the impacts of urbanization on mountain chickadee reproduction may not extend to altering extra‐pair behaviour.     

Extra‐pair mating in a socially monogamous and paternal mouth‐brooding cardinalfish

Many vertebrates form monogamous pairs to mate and care for their offspring. However, genetic tools have increasingly shown that offspring often arise from matings outside of the monogamous pair bond. Social monogamy is relatively common in coral reef fishes, but there have been few studies that have confirmed monogamy or extra‐pair reproduction, either for males or for females. Here, long‐term observations and genetic tools were applied to examine the parentage of embryos in a paternally mouth‐brooding cardinalfish, Sphaeramia nematoptera. Paternal care in fishes, such as mouth‐brooding, is thought to be associated with a high degree of confidence in paternity. Two years of observations confirmed that S. nematoptera form long‐term pair bonds within larger groups. However, genetic parentage revealed extra‐pair mating by both sexes. Of 105 broods analysed from 64 males, 30.1% were mothered by a female that was not the partner and 11.5% of broods included eggs from two females. Despite the high paternal investment associated with mouth‐brooding, 7.6% of broods were fertilized by two males. Extra‐pair matings appeared to be opportunistic encounters with individuals from outside the immediate group. We argue that while pair formation contributes to group cohesion, both males and females can maximize lifetime reproductive success by taking advantage of extra‐pair mating opportunities.     

Levels of extra‐pair paternity are associated with parental care in penduline tits (Remizidae)

In most passerine birds, individuals attempt to maximize their fitness by providing parental care while also mating outside their pair bond. A sex‐specific trade‐off between these two behaviours is predicted to occur, as the fitness benefits of extra‐pair mating differ between the sexes. We use nest observations and parentage analysis to reveal a negative association between male care and the incidence of extra‐pair paternity across three species of penduline tit (Remizidae). This provides evidence of a trade‐off between these two behaviours, possibly due to the devaluing of paternal care by extra‐pair offspring.     

Extra pair paternity in birds: a review of interspecific variation and adaptive function

The application of molecular genetic techniques has revolutionized our view of avian mating systems. Contrary to prior expectations, birds are only very rarely sexually monogamous, with 'extra-pair offspring' found in approximately 90% of species. Even among socially monogamous species, over 11% of offspring are, on average, the result of extra-pair paternity (EPP). Based on over 150 molecular genetic studies of EPP in birds, we review two topical areas: (i) ecological explanations for interspecific variation in the rate of EPP; and (ii) evidence bearing on the adaptive function of EPP. We highlight the remaining challenges of understanding the relative roles of genes and ecology in determining variation between taxa in the rate of extra paternity, and testing for differences between extra-pair offspring and those sired within-pair.