Exposure to per- and polyfluoroalkyl substances (PFAS) impairs avian reproduction and development, yet uptake and elimination pathways in birds remain poorly resolved. To better understand these processes, we quantified PFAS in whole blood (n = 124) from adult and juvenile house sparrows (Passer domesticus) at 16 urban-industrial sites. Dietary pathways were traced using stable isotopes of carbon and nitrogen from feathers (n = 66). At our most contaminated site, we recaptured individuals across eight months to assess seasonal trends in PFAS burdens. We found that exposure to perfluorosulfonic acids (PFSAs) was highest near fire training areas, while perfluorocarboxylic acids (PFCAs) were highest near oil production facilities. PFOS, PFDoA, and PFTeDA were higher in juveniles than adults, and females exhibited lower PFNA, PFUdA, and PFTeDA than males, consistent with maternal transfer of PFAS into eggs. Young fledglings showed tightly clustered, depleted δ13C values (-22.9 ± 0.8‰) compared to adults (-18.9 ± 3.6‰), indicating selective invertebrate provisioning as another source of early life exposure. In recaptured juveniles, ∑PFCAs and ∑PFSAs declined throughout development (0.43-1.25% per day). This enabled estimation of compound-specific elimination half-lives, which ranged from 29 days (PFBS) to 91 days (PFNA). Adults showed slower elimination rates. Our findings indicate pronounced early life vulnerability to PFAS exposure in terrestrial birds.

An emerging question in evolutionary research is the extent to which mitochondrial variation across species and populations drives functional differences in fitness-related traits. The asymmetric introgression and narrow mitochondrial clines seen in long-tailed finch (Poephila acuticauda) subspecies is suggestive of selection but there is no direct evidence of fitness related differences in admixed birds. Asymmetric introgression of the eastern P. a. hecki mito-type into P. a. acuticauda, suggests that the former may possess a selective energetic advantage such as superior escape flight performance. We employed a spatial tracking software, Argus, to quantify the take-off flight performance of 158 wild-derived, captive long-tailed finches as they were released repeatedly, from which we obtained nine flight metrics. Average force and maximum vertical distance travelled were repeatable across all individuals and significantly lower in P. a. hecki compared to P. a. acuticauda males. Hence, contrary to predictions, P. a. hecki did not display escape flight performance superior to P. a. acuticauda suggesting take-off flight performance in wild long-tailed finches is not related to the observed asymmetry in the introgression of mito-type between the subspecies. Furthermore, in intraspecific female hybrids, in which we might theoretically expect a mismatch between mitochondrial haplotype and mitonuclear genes, we also found no evidence of impaired flight take-off performance, relative to those with matched mitochondrial and mitonuclear genes (here: female P. a. acuticauda). Finally, male and female hybrids did not differ in the average force and maximum vertical distance travelled, again suggesting that take-off performance is not significantly worse in the heterogametic sex, as predicted by Haldane's rule. The angle of the take-off flight was a key determinant of our flight performance metrics and consequently, future work could benefit from better directing the flight path of tracked birds.

Ecological risk assessments estimate chemical exposures in wildlife from contaminant concentrations within their surrounding environment. This approach precludes a detailed understanding of interactions between biota and contaminant sources. It also overlooks how the uneven and irregular use of space by animals influences variability in exposure. Here, we conducted a meta-analysis to examine whether site-specific variability in lead exposure was associated with the home range size and movement-related traits of terrestrial bird species. We constructed a series of linear models to test the effect of ecological and environmental predictors on variability in lead exposure, which we quantified using the coefficient of variation for site-specific feather lead concentrations. Accounting for variance across study, site, species, and phylogeny, we show that birds with larger home ranges exhibited more variable levels of lead exposure than those with more restricted movement patterns. This relationship was moderated by foraging niche and was significant in ground foraging birds but non-significant in generalists and arboreal-aerial foragers, which may relate to differences in dietary lead sources and niche complexity. Site-specific variability in feather lead concentrations was further structured by feather type, with contour feathers exhibiting lower variability than wing, tail, or mixed feather samples. Our findings highlight how the use of space at varying scales might influence divergent patterns of chemical exposure in terrestrial birds. We discuss how incorporating movement-related traits into wildlife risk assessments can reduce uncertainties associated with spatial variability in exposure and improve the ecological relevance of monitoring outcomes.

BACKGROUND: Social connections provide individuals with multiple benefits. Individuals, however, are often constrained in how they socially organize due to ecological and environmental factors that affect individual space-use and movement patterns. Weather is one such factor that influences individual movements, and thus social structure. While on longer time scales (i.e., seasonally) the impacts of weather are relatively predictable, on shorter time scales (i.e., sub-daily), the impacts of weather on social organization are less predictable yet are largely overlooked. METHODS: In this study, we examined the influence of short-term weather components, specifically wind and temperature, on the social structure of free-living zebra finches (Taeniopygia castanotis) in the Australian arid zone. Our goal was to characterize if social network structure was impacted by hourly changes in these important weather components. To do so, we used an automated radio telemetry system to concurrently track 128 wild zebra finches for 12 consecutive days in the Australian spring in order to examine the relationships between weather components and social structure. Using Bayesian network analyses to account for the uncertainty in association strengths among individuals, we examined network structure, as measured by density and modularity, in relation to hourly wind speed, temperature, and time of day. Additionally, to assess if weather impacted the synchronization of group-level movements, we calculated proximity ratios between neighbouring individuals, which we related to wind and temperature. RESULTS: We observed that network modularity increased during hours with higher mean wind speed and was highest in the morning and evening hours. In contrast, network density was not related to wind speed. Additionally, neither modularity nor density showed a significant relationship with temperature during the tracking period, however, this period did not cover a large temperature gradient. Group-level movement patterns as measured by proximity ratios between neighbouring individuals showed no relationship with either wind or temperature. CONCLUSIONS: Our results suggest that changes in wind impact social structure in zebra finches. Given the critical role that network modularity plays in social information transfer, increased wind could have important downstream consequences. Stochastic and more frequent changes in weather due to climate change could thus potentially disproportionately impact species, such as the nomadic zebra finch, that rely on locating ephemeral resources.

Most of our understanding of the fundamental processes of mutation and recombination stems from a handful of disparate model organisms and pedigree studies of mammals, with little known about other vertebrates. To gain a broader comparative perspective, we focused on the zebra finch ( Taeniopygia castanotis ), which, like other birds, differs from mammals in its karyotype (which includes many micro-chromosomes), in the mechanism by which recombination is directed to the genome, and in aspects of ontogenesis. We collected genome sequences from three generation pedigrees that provide information about 80 meioses, inferring 202 single-point de novo mutations, 1,088 crossovers, and 275 non-crossovers. On that basis, we estimated a sex-averaged mutation rate of 5.0 × 10 -9 per base pair per generation, on par with mammals that have a similar generation time (~2–3 years). Also as in mammals, we found a paternal germline mutation bias at later stages of gametogenesis (of 1.7:1) but no discernible difference between sexes in early development. Examining recombination patterns, we found that the sex-averaged crossover rate on macro-chromosomes is 0.93 cM/Mb, with a pronounced enrichment of crossovers near telomeres. In contrast, non-crossover rates are more uniformly distributed. On micro-chromosomes, sex-averaged crossover rates are substantially higher (3.96 cM/Mb), in accordance with crossover homeostasis, and both crossover and non-crossover events are more uniformly distributed. At a finer scale, recombination events overlap CpG islands more often than expected by chance, as expected in the absence of PRDM9. Estimates of the degree of GC-biased gene conversion (59%), the mean non-crossover conversion tract length (~32 bp), and the non-crossover-to-crossover ratio (5.4:1) are all comparable to those reported in primates and mice. Therefore, properties of germline mutation and recombination resolutions remain similar over large phylogenetic distances.

Birds and mammals converged upon the same physical mechanism of vocal fold vibration1,2 to produce the wide variety of communicative vocal signals critical for their reproduction, social interactions, survival, and speciation.3,4,5 Recent work reported high-frequency (7-11 kHz) vocalizations in zebra finches,6 termed "heat" or "incubation" calls, that are suggested to have close-range communicative relevance in the context of global warming.6,7 However, their acoustics are poorly described, and by what biophysical mechanism they are produced remains unknown. We recorded heat-associated vocalizations in adult zebra finches in vivo and showed that they are extremely soft, frequency-modulated vocalizations with source levels of 13.9 ± 3.3 dB sound pressure level (SPL) at 1 m and dominant frequencies of 6.8 ± 0.6 kHz. Through in vitro experiments, we establish that these vocalizations are aerodynamic whistles produced inside the avian larynx, not syrinx, during inspiration. Respiratory air flow during whistle production is higher than during regular song and consistent with thermal panting for evaporative cooling.7,8 Laryngeal geometry and dimensional flow analysis suggest that these whistles are laminar-flow whistles that occur when a flow boundary layer is in a transition phase from laminar to turbulent flows.9,10,11 Our data imply that in earlier experiments,6,7 playbacks were conducted at 30 dB above the physiologically relevant playback level (i.e., several magnitudes). Furthermore, heat whistles are at least 35 dB below the behavioral perception limit even of adults12 with more sensitive hearing than juveniles.13,14 Therefore, we show-contra previous works6,7-that heat whistles cannot function as adaptive signals or cues in parent-embryo communication. VIDEO ABSTRACT.

Industrial fluoride emissions negatively impact animal health and remain elevated over large areas surrounding point sources. Here, we examined the geographic and demographic determinants of fluoride exposure in 42 house sparrows (Passer domesticus) from 14 sites around two aluminium smelting operations in Australia. We found that mean bone fluoride concentrations increased with age and were significantly higher in adult (1062 mg/kg) than in juvenile (603 mg/kg) house sparrows. A corresponding increase was observed relative to body size metrics, indicating an association between fluoride accumulation and individual growth. In adults, this was accompanied by changes in bone mineral composition, with a significant decline in bone phosphorus relative to calcium in fluoride exposed birds. Bone fluoride concentrations in 86 % of adult house sparrows exceeded background levels (600 mg/kg) previously reported for passerine birds inhabiting uncontaminated areas of New Zealand and North America. Bone fluoride decreased significantly with distance from aluminium smelting operations and, in adult house sparrows, only fell below background levels (600 mg/kg) at a distance of at least 9.8 km from smelting facilities. The vegetated buffer zones surrounding both smelters were insufficient to prevent elevated exposure to fluoride contamination. Our findings demonstrate the potentially far-reaching ecological impacts of industrial fluoride emissions and their age-related bioaccumulation in a sedentary urban bird.

Spermatozoa exhibit striking morphological variation across the animal kingdom. In passerine birds, sperm exhibit considerable variation in size, yet the basic sperm phenotype is highly conserved; sperm are filiform, the head is corkscrew-shaped, and the midpiece is elongated and twisted around the flagellum. A significant departure from this typical sperm morphology has been reported in the sister species, the Eurasian bullfinch (Pyrrhula pyrrhula) and Azores bullfinch (P. murina). Here, we report a second evolutionary shift in passerine sperm phenotype in the nominate subspecies of the red-browed finch (Neochmia temporalis temporalis); sperm are nonfiliform, with an ellipsoid head and an extremely short midpiece restricted to the nuclear-axoneme junction. Additionally, we show that the sperm phenotype of the red-browed finch is similar to the putatively neotenous sperm described in the two bullfinch species. Using whole-genome data, we found no evidence that the unusual sperm phenotype of the red-browed finch is associated with reduced genetic variation or a population bottleneck. In contrast, using data on relative testes size, we find some support for the hypothesis that relaxed postcopulatory sexual selection, via a lack of sperm competition, may, at least in part, explain the unusual sperm of the red-browed finch. We also discuss the possible roles of mutation, genetic drift, and genetic hitchhiking in the evolutionary origins and maintenance of neotenous sperm phenotypes. Finally, we suggest that these dramatic evolutionary shifts in sperm phenotype warrant further investigation and highlight the need for a greater understanding of the developmental and genomic basis of sperm phenotype.

Long-term social monogamy, a prevalent mating system in avian species, is often associated with increased cooperation and coordination as well as reduced sexual conflict. Although many studies have highlighted the benefits of long-term partnerships for individuals, there remains a lack of insight into how closely partners associate with one another behaviorally. To date, studies investigating pair cohesion in seasonal and long-term partnerships are typically restricted to arrivals at the nest or feeding sites during the breeding season. Using fine-scale automated tracking data on chirruping wedgebills (Psopodes cristatus), a territorial socially monogamous species, we characterized how partners coordinate their movement during and after the breeding season. We used 12 pair-bonded individuals with consistently high localization rates that were tracked for a period between 32 and 69 days, with an average of 260,000 localizations per individual. We demonstrate that pairs (1) had extremely similar home ranges with a similarity index of 0.93 versus 0.18 for non-pairs, (2) maintained consistently closer proximity than expected from movement without paying attention to a partner, and (3) followed each other as they moved, with individuals following their moving partner in 42% of cases during and in 47% of cases after breeding. Our findings show that pair cohesion in socially monogamous territorial species can be very high in both a breeding and non-breeding context, illustrating that strong coordination among partners has important functions beyond reproduction and parental care.

Climate change is altering the frequency and intensity of environmental extremes, and the diurnal rates of environmental change. The daily reaction norms of wild animals show spatial and temporal plasticity to allow appropriate physiological responses to predictable environmental challenges, but these responses have rarely been quantified in wild birds. We addressed this by determining whether physiological stress indices (corticosterone [CORT], glucose [GLU], and circulating heat shock proteins [Hsp]) vary with ambient temperature (Ta) or time of day across a 60 latitudinal gradient (320-380 S). We sampled house sparrows, Passer domesticus, at three locations with varying daily rates of Ta increases in 2015 and in 2019-2020 to determine whether physiological responses to capture and handling stress change diurnally. As predicted, plasma CORT and GLU increased during stress. Baseline CORT and GLU levels did not change during the day, but the amplitude of plasma CORT increase (delta CORT) in response to capture decreased as Ta increased, as was the case for GLU, in birds with initially high baseline CORT. Neither baseline nor stress-related plasma CORT or GLU differed consistently across sampling locations. Heat-shock cognate 70 (Hsc70) and heat-shock protein 90α (Hsp90) showed clear temporal dynamics across the day. Thus, the reaction norms of heat shock proteins are temporally plastic during the day and in response to daily Ta changes, as are the reaction norms of CORT and GLU in response to acute stress. However, the study provides little evidence for such plasticity in reaction norms as a function of average local thermal conditions.

Duetting, a cooperative vocal behaviour performed by mated pairs, is a distinctive vocal behaviour among many species in specifically primates and birds. Yet, the exact features of duets that may make them a stronger territorial signal are still not well understood. One hypothesis is that the precision of duet coordination can indicate the quality of a pair bond or dedication of a pair, and thus the degree of threat posed to a rival pair. To address the implications of duetting precision in a territorial context, we here determined to what extent the antiphonal duetting behaviour in the chirruping wedgebill (Psophodes cristatus), a territorial, socially monogamous passerine, is affected by the precision of fine-level duet coordination. We tested this with playback experiments where we broadcast coordinated and uncoordinated duets at mated pairs, predicting that pairs would exhibit stronger responses to coordinated duets than to uncoordinated ones and sing more coordinated after the simulated intrusion. We found that neither response intensity nor coordination of either sex differed in responses to playback of coordinated and uncoordinated duets. Since chirruping wedgebills did respond consistently to playback, we suggest that either (1) fine-level coordination of duetting does not hold a function in joint resource defence in this species, (2) playback stimuli were too threatening for them to adjust their coordination on a level we could detect or (3) they do not discriminate between our coordinated and uncoordinated playback treatments. We highlight the notion that there may be variety in functions of duetting at play within and across avian species, and that different aspects of duets such as coordination and intensity may hold different functions.

As a result of a warming global climate, understanding how organisms adjust their behaviour to environmental thermal conditions has become an increasingly important question in animal biology. Temperature‐driven adjustments in parental care are potentially important given the repercussions on offspring size, quality and survival. In 2015 and 2016 we monitored parental care for 83 zebra finch Taeniopygia castanotis breeding attempts in the wild with known brood sizes. We recorded the frequency of parental visits to the nest together with mean maximum ambient temperature experienced between day 7 and 14 of the nestling period. We found that for each increase of 1°C in the daytime temperature there was a 0.91% reduction in the hourly rate of parental visits, whilst also accounting for other variables such as nestling age, time of season, and wind speed. Our data suggest that nestlings may receive less food under thermally challenging conditions, which is consistent with recent studies that demonstrate offspring are smaller when reared during periods of high temperature. Understanding the behavioural drivers that may contribute to the production of smaller offspring in extreme heat conditions could prove useful to forecast long‐term consequences for fitness triggered by climate change.

Whilst there is a growing appreciation that mitochondrial divergence across lineages is not selectively neutral, less work has examined the functional differences that may exist in closely divergent taxa. We measured mitochondrial oxygen consumption in the blood of two subspecies of an Australian songbird-the long-tailed finch, Poephila acuticauda-before and after 10 days of heat treatment at 40 °C to explore mitochondrial metabolic plasticity in response to thermal stress. There were significant differences between subspecies in the efficiency of oxidative phosphorylation, with P. a. hecki having higher energy production efficiency than P. a. acuticauda independent of heat treatment. Mitochondrial metabolism increased significantly after the treatment in 4 out of 6 variables in both subspecies, with P. a. hecki showing higher oxygen consumption rates in acclimating to 40 °C. In the same experiment, we also measured circulating levels of corticosterone to assess the effect of the treatment on stress and to explore a possible mechanistic link with mitochondrial metabolism. The heat significantly increased baseline corticosterone, but at an individual level, corticosterone and mitochondrial metabolism were unrelated, indicating that functional plasticity in response to the thermal challenge was not mechanistically determined by corticosterone. Whilst the geographic ranges of the 2 subspecies differ in climate, the extent to which the functional divergence in mitochondrial efficiency reflects selectively neutral or adaptive divergence requires further research. Nonetheless, the reduced metabolic flexibility of P. a. acuticauda after heat suggests that future increases in the frequency and intensity of heatwaves may impose asymmetric effects on the 2 subspecies.

Most of our understanding of the fundamental processes of mutation and recombination stems from a handful of disparate model organisms and pedigree studies of mammals, with little known about other vertebrates. To gain a broader comparative perspective, we focused on the zebra finch (Taeniopygia castanotis), which, like other birds, differs from mammals in its karyotype (which includes many micro-chromosomes), in the mechanism by which recombination is directed to the genome, and in aspects of ontogenesis. We collected genome sequences from three generation pedigrees that provide information about 80 meioses, inferring 202 single-point de novo mutations, 1,174 crossovers, and 275 non-crossovers. On that basis, we estimated a sex-averaged mutation rate of 5.0 × 10-9 per base pair per generation, on par with mammals that have a similar generation time (~2-3 years). Also as in mammals, we found a paternal germline mutation bias at later stages of gametogenesis (of 1.7:1) but no discernible difference between sexes in early development. Examining recombination patterns, we found that the sex-averaged crossover rate on macro-chromosomes (1.05 cM/Mb) is again similar to values observed in mammals, as is the spatial distribution of crossovers, with a pronounced enrichment near telomeres. In contrast, non-crossover rates are more uniformly distributed. On micro-chromosomes, sex-averaged crossover rates are substantially higher (4.21 cM/Mb), as expected from crossover homeostasis, and both crossover and non-crossover events are more uniformly distributed. At a finer scale, recombination events overlap CpG islands more often than expected by chance, as expected in the absence of PRDM9. Despite differences in the mechanism by which recombination events are specified and the presence of many micro-chromosomes, estimates of the degree of GC-biased gene conversion (59%), the mean non-crossover conversion tract length (~32 bp), and the non-crossover-to-crossover ratio (5.4:1) are all comparable to those reported in primates and mice. The similarity of mutation and recombination properties in zebra finch to those in mammals suggest that they are conserved by natural selection.

The zebra finch has been used in laboratory studies to understand the effects of ambient temperature on development and reproduction. We characterize the thermal range in which the species breeds in the wild in Australia. Our data reveal that zebra finches breed over an extremely wide range of ambient temperatures (-5.2 °C to 46.2 °C), and that embryos are not as buffered from ambient temperatures as generally considered, regularly spending time at temperatures between 10 °C and 40 °C. To place the zebra finch in a broader context we examined the thermal breeding range of 327 other Australian terrestrial species. These thermal ranges vary extensively, but there was no indication of a phylogenetic signal for this trait, suggesting it is a relatively labile trait. The range of temperatures in which zebra finch breeds is at the 90th percentile of Australian terrestrial species, indicating that it has a relatively high level of plasticity in coping with thermal conditions. We also found that the zebra finch breeds in relatively high temperatures, with 10 % of observed zebra finch breeding attempts being made in conditions with a 30-day average maximum temperature of 31-38.2 °C. Again, however, around 20 % of Australian bird species were found to breed in higher average maximum temperatures. Nevertheless, the temperatures we have characterized provide insight into the capacity of embryo, nestling, and adult zebra finches to cope across a wide range of ambient temperatures. The zebra finch is a good species for further experimental work in the laboratory to understand tolerance, plasticity, and the effects of temperature on development and physiology. Our findings will help to interpret past and future studies in this important area of research, and provide the appropriate context for future studies to design ecologically relevant manipulations of temperature.

Lead (Pb) is a highly toxic and widespread environmental pollutant and can severely harm body tissues as well as DNA. Pb could potentially damage telomeres, whose length and shortening rate are linked with cellular senescence, physiological state, and mortality. Yet, studies investigating Pb and telomere dynamics in natural systems remain inconclusive. In this study, we used a free-living house sparrow (Passer domesticus) population in Broken Hill, Australia, chronically exposed to varying levels of environmental Pb, to assess the effects of Pb on telomere length and telomere rate of change. Using all data from adults and juveniles, we found that mean blood Pb concentration had a negative relationship with telomere lengths measured at capture sites, such that a standard deviation increase in the concentration of blood Pb was associated with an 8 % decrease in telomere length. In a series of robustness analyses we found that this negative relationship existed at both the individual and the site levels. Although not statistically significant, the relationship between telomere length and soil Pb also appeared to be consistent with that found for blood Pb. Our results demonstrated that while exposure to Pb damages telomeres in free-living house sparrows, the biological effect is relatively weak, and is only identified with a sample size of over 500 individuals. Nevertheless, our data reveal that in this urban setting in Australia, a human commensal bird is suffering from lead-induced damage to telomeres. Given the well-established relationship between telomere shortening and life-span, our study highlights a clear risk of Pb contamination on the biota of the urban area, including humans.

Birds and mammals converged upon the same physical mechanism of vocal fold vibration to produce their broad range of voiced sounds critical to communication. The frequency range of vocal fold vibration is limited per species by biophysical constraints to 3-4 octaves. However, recent work reported vocalizations in zebra finches with apparent fundamental frequencies of 7-11 kHz that far exceed the range of regular calls and song (0.5-1.5 kHz). These "heat" or "incubation" calls are suggested to have close-range communicative relevance in the global temperature rise context, but their acoustics are poorly described and by what biophysical mechanism they are produced remains unknown. We recorded heat calls in adult zebra finches in vivo and show they are extremely soft, frequency-modulated calls with source levels of 13.9 ± 3.3 dB SPL at one meter with dominant frequencies of 6.8 ± 0.6 kHz. Through a series of in vitro experiments, we establish that these calls are aerodynamic whistles produced inside the avian larynx, not syrinx, during inspiration. Respiratory air flow during whistle production is an order of magnitude higher than song and consistent with thermal panting for evaporative cooling. Laryngeal geometry and dimensional flow analysis suggest that these whistles are laminar flow whistles that occur when a flow boundary layer is in a transition phase from laminar to turbulent flows. Birds, like some rodents, are thus able to produce both voiced sounds and aerodynamical whistles in their vocal tract. ### Competing Interest Statement The authors have declared no competing interest.

Spermatozoa exhibit striking morphological variation across the animal kingdom. In passerine birds, sperm exhibit considerable variation in size, yet the basic sperm phenotype is highly conserved; sperm are filiform, the head is corkscrew-shaped, and the midpiece is elongated and twisted around the flagellum. A significant departure from this typical sperm morphology has been reported in the sister species, the Eurasian bullfinch (Pyrrhula pyrrhula) and Azores bullfinch (P. murina). Here, we report a second evolutionary shift in passerine sperm phenotype in the nominate subspecies of the red-browed finch (Neochmia temporalis temporalis); sperm are non-filiform, with an ellipsoid head, and an extremely short midpiece restricted to the nuclear-axoneme junction. Additionally, we show that the sperm phenotype of the red-browed finch is similar to the putatively neotenous sperm described in the two bullfinch species. Using whole-genome data, we found no evidence that the unusual sperm phenotype of the red-browed finch is associated with reduced genetic variation or a population bottleneck. In contrast, we find some support for the hypothesis that relaxed post-copulatory sexual selection may, at least in part, explain the unusual sperm of the red-browed finch. We also discuss the possible roles of mutation, genetic drift, and genetic hitchhiking, in the evolutionary origins and maintenance of neotenous sperm phenotypes. Finally, we suggest that these dramatic evolutionary shifts in sperm phenotype warrant further investigation and highlight the need for a greater understanding of the developmental and genomic basis of sperm phenotype. ### Competing Interest Statement The authors have declared no competing interest.

Carotenoid pigments produce the yellow and red colors of birds and other vertebrates. Despite their importance in social signaling and sexual selection, our understanding of how carotenoid ornamentation evolves in nature remains limited. Here, we examine the long-tailed finch Poephila acuticauda, an Australian songbird with a yellow-billed western subspecies acuticauda and a red-billed eastern subspecies hecki, which hybridize where their ranges overlap. We found that yellow bills can be explained by the loss of C(4)-oxidation, thus preventing yellow dietary carotenoids from being converted to red. Combining linked-read genomic sequencing and reflectance spectrophotometry measurements of bill color collected from wild-sampled finches and laboratory crosses, we identify four loci that together explain 53% of variance in this trait. The two loci of largest effect contain the genes CYP2J19, an essential enzyme for producing red carotenoids, and TTC39B, an enhancer of carotenoid metabolism. A paucity of protein-coding changes and an enrichment of associated upstream variants suggest that the loss of C(4)-oxidation results from cis-regulatory evolution. Evolutionary genealogy reconstruction indicates that the red-billed phenotype is ancestral and that yellow alleles at CYP2J19 and TTC39B first arose and fixed in acuticauda approximately 100 kya. Yellow alleles subsequently introgressed into hecki less than 5 kya. Across all color loci, acuticauda-derived variants show evidence of selective sweeps, implying that yellow bill coloration has been favored by natural selection. Our study illustrates how evolutionary transitions between yellow and red coloration can be achieved by successive selective events acting on regulatory changes at a few interacting genes.

Understanding genetic incompatibilities and genetic introgression between incipient species are major goals in evolutionary biology. Mitochondrial genes evolve rapidly and exist in dense gene networks with coevolved nuclear genes, suggesting that mitochondrial respiration may be particularly susceptible to disruption in hybrid organisms. Mitonuclear interactions have been demonstrated to contribute to hybrid dysfunction between deeply divergent taxa crossed in the laboratory, but there are few empirical examples of mitonuclear interactions between younger lineages that naturally hybridize. Here, we use controlled hybrid crosses and high-resolution respirometry to provide the first experimental evidence in a bird that inter-lineage mitonuclear interactions impact mitochondrial aerobic metabolism. Specifically, respiration capacity of the two mitodiscordant backcrosses (with mismatched mitonuclear combinations) differs from one another, although they do not differ significantly from the parental groups or mitoconcordant backcrosses as we would expect of mitonuclear disruptions. In the wild hybrid zone between these subspecies, the mitochondrial cline centre is shifted west of the nuclear cline centre, which is consistent with the direction of our experimental results. Our results therefore demonstrate asymmetric mitonuclear interactions that impact the capacity of cellular mitochondrial respiration and may help to explain the geographic discordance between mitochondrial and nuclear genomes observed in the wild.