Human-mediated gene flow is increasingly altering the genetic composition of populations, yet conservation assessments typically focus on nuclear introgression alone. We synthesise evidence showing that mitochondrial DNA (mtDNA) introgression can have distinct consequences for performance, adaptation, and the viability of animal populations. Unlike most nuclear loci, mtDNA is uniparentally inherited, nonrecombining, and encodes core components of cellular energy metabolism that must function in tight coordination with nuclear genes. As a result, introgression of nonnative mtDNA can disrupt co-adapted mito-nuclear interactions, sometimes generating sex-specific or environment-dependent costs. Conversely, mtDNA introgression can also be neutral or beneficial in certain circumstances. We argue that ignoring mtDNA risks unintended maladaptation and that mitochondrial and nuclear genetics should be considered jointly in conservation planning.

The environmental conditions organisms experience during early development can have powerful and sustained effects on morphology, physiology, behaviour, and performance. Such developmental effects can influence reproductive success, survival, and life-history strategies and can be transmitted across generations (i.e. trans and intergenerational effects). In this way, developmental effects can be powerful drivers of evolutionary change. Given the developmental environment affects a range of phenotypic traits, it has been proposed that physiological responses to developmental conditions are modulated through cellular mechanisms that are shared across cell and tissue types, such as mitochondrial function. Mitochondrial respiratory function is highly sensitive to environmental conditions and exposure to adverse conditions during development can have sustained effects on different aspects of aerobic respiration in mitochondria. However, it is currently unknown if these effects are widespread across taxonomic groups and which components of mitochondrial respiratory function are most likely to be affected by the environment during development. We compiled data from 86 studies to examine the effects of developmental stressors (nutritional imbalance, glucocorticoid hormone exposure, parental care deprivation, and psychological disturbance) on mitochondrial respiratory function using meta-analysis. We sought to uncover whether there are general effects of developmental stressors on different aspects of mitochondrial respiratory function (antioxidants, metabolic capacity, oxidative damage, oxidative stress, and aerobic respiration). We tested how the type of developmental stressor, together with timing of exposure (prenatal versus postnatal), and sex and taxon of the test subjects influenced the magnitude, direction, and duration of effects on mitochondrial respiratory function. Finally, we tested which aspects of mitochondrial respiratory function were most impacted by developmental stressors. We found that exposure to glucocorticoids, parental care deprivation, and psychological disturbances during development generally decreased mitochondrial respiratory function. Generally, these developmental stressors increased the production of reactive oxygen species and oxidative damage and reduced aerobic respiration, metabolic capacity, and antioxidant levels. Nutritional imbalances during development (including both restricted and excessive nutrition) had a slight negative effect on mitochondrial respiratory function, but this effect may be influenced by publication bias. Overall, our results show that exposure to stressors during development negatively affects mitochondrial respiratory function, suggesting that changes in cellular metabolism may link developmental stressors to variation in whole animal traits and individual fitness.

Sexual dichromatism, characterized by sex-specific differences in coloration, is widespread among birds and often involves carotenoid-based pigmentation. Despite extensive research on the social and ecological environments favoring sexual dichromatism, the molecular mechanisms underlying its development and evolution remain largely unexplored. In this study, we investigated the genetic and molecular processes giving rise to sexual dichromatism in the red ketocarotenoid-based plumage of northern cardinals (Cardinalis cardinalis). We quantified carotenoid concentrations in plasma and feather follicles, confirmed that homologs of CYP2J19, BDH1L, and TTC39B catalyze the production of C-4 ketocarotenoids, and performed gene expression analyses across tissues. Males showed significantly higher plasma and feather ketocarotenoid concentrations, upregulated CYP2J19 and TTC39B expression in liver and feather tissues, and upregulated carotenoid transport gene expression in the gut and feather follicles. Females exhibited a dramatic upregulation of BCO2 in their feather follicles, facilitating carotenoid degradation and attenuating red pigmentation. Additionally, sex-biased expression of hormonal regulators, such as HSD17B4 and ZNF131, in the feather follicle suggests hormonal modulation influences dichromatism. These findings indicate that sex-specific regulation of carotenoid processing genes underpins the vivid red coloration in males and the drab phenotype in females, likely maintained by a balance between natural and sexual selection, with mechanisms of sexual antagonism affecting divergent gene expression. This work advances understanding of the molecular basis of avian sexual dimorphism, highlighting key genetic pathways involved in carotenoid-based coloration and providing a foundation for further research into the evolution of sexually dichromatic traits.

A recent review of honest signalling theory criticised constraint-based (index) explanations for traits that serve as honest signals of individual quality, contending that such explanations offer only proximate mechanisms and fail to explain evolutionary stability or the origin of reliability. A key point in this critique is the contention that ultimate explanations require the possibility of cheating. The authors advocate for the Signaling Trade-off Theory-where trade-offs make cheating evolutionarily unfavourable-as essentially a complete explanation for the evolution of signal honesty. Here, we argue that this critique rests on a false dichotomy between proximate and ultimate explanation. When signal production is mechanistically embedded within vital cellular processes, this shared pathway inherently restricts the evolution of cheating. Using avian ketocarotenoid colouration as a model, we show that condition-dependent signal production is not explained by trade-offs because pigment transformation is coupled to mitochondrial energy metabolism and core cellular performance, making high-signal expression unattainable for low-condition individuals. Deception may therefore be physiologically inaccessible rather than merely costly. Moreover, the enzymatic machinery underlying ketocarotenoid production likely evolved for visual function before being co-opted for social assessment, such that condition-dependent colour expression preceded assessment of colouration in social interactions. Uncheatable honest signals can thus arise as an exaptation of biochemical processes needed to sustain complex life rather than as outcomes of unfavourable trade-offs.

Sex and mitochondria are inextricably linked in the eukaryotic tree of life, a confounding situation given the uniparental inheritance of mitochondria and the biparental inheritance that sexual reproduction entails. Unisexual vertebrate lineages, which arise via hybridization and asexually pass on their genetic material to clonal descendants, provide a unique opportunity to study mitochondrial evolution without potentially confounding effects of sex. Hybridity and clonality set unisexual vertebrates apart from other vertebrates and establish a distinct genetic environment that shapes their evolution, especially dynamics between mitochondrial and nuclear genomes. Here, we provide a perspective on the mitonuclear genomic interactions experienced by unisexual vertebrates and the implications of these interactions on mitochondrial function and integration into organismal performance and fitness. Building upon the hypothesis that sexual reproduction arose to maintain coadaptation between co-functioning nuclear and mitochondrial genes, we propose that unisexual vertebrates may be confined to predominantly 'young' lineages because mitonuclear incompatibilities-arising from either hybridity or clonality-increase the probability of extinction over time (the Mitonuclear Erosion Hypothesis). We provide a multidisciplinary collection of strategies to disentangle the effects of clonality and hybridity and quantify the relative degree to which these characteristics contribute to differences in mitochondrial function, organismal performance and fitness. This article is part of the theme issue 'Evolutionary genetics of mitochondria: on diverse and common evolutionary constraints across eukarya'.

Carotenoid-based coloration is an essential feature of avian diversity and has important roles in communication and mate choice. The red feathers of birds from phylogenetically diverse orders and families are pigmented with C4-ketocarotenoids produced via the successive action of Cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L) on yellow dietary precursors. Yet, the biochemistry of these enzymes remains incompletely understood. Here we present a series of experiments characterizing the substrates, intermediates, and products of CYP2J19 and BDH1L expressed in heterologous cell culture. We confirm that CYP2J19 preferentially hydroxylates the 4 and 4' positions of β-ring substrates, but can also hydroxylate the 3 and 3 positions of C4-ketocarotenoids. We confirm that BDH1L catalyzes the conversion of zeaxanthin to canary xanthophyll B (ε,ε'-carotene-3,3'-dione) a major pigment in plumage of many yellow bird species. These results suggest that the actions of CYP2J19 and/or BDH1L can explain the presence of many metabolically transformed carotenoids in avian tissues.

The carotenoid-based colours of birds are a celebrated example of biological diversity and an important system for the study of evolution. Recently, a two-step mechanism, with the enzymes cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), was described for the biosynthesis of red ketocarotenoids from yellow dietary carotenoids in the retina and plumage of birds. A common assumption has been that all birds with ketocarotenoid-based plumage coloration used this CYP2J19/BDH1L mechanism to produce red feathers. We tested this assumption in house finches (Haemorhous mexicanus) by examining the catalytic function of the house finch homologues of these enzymes and tracking their expression in birds growing new feathers. We found that CYP2J19 and BDH1L did not catalyse the production of 3-hydroxy-echinenone (3-OH-echinenone), the primary red plumage pigment of house finches, when provided with common dietary carotenoid substrates. Moreover, gene expression analyses revealed little to no expression of CYP2J19 in liver tissue or growing feather follicles, the putative sites of pigment metabolism in moulting house finches. Finally, although the hepatic mitochondria of house finches have high concentrations of 3-OH-echinenone, observations using fluorescent markers suggest that both CYP2J19 and BDH1L localise to the endomembrane system rather than the mitochondria. We propose that house finches and other birds that deposit 3-OH-echinenone as their primary red plumage pigment use an alternative enzymatic pathway to produce their characteristic red ketocarotenoid-based coloration.

In many species of birds, red carotenoid coloration serves as an honest signal of individual quality, but the mechanisms that link carotenoid coloration to animal performance remain poorly understood. Most birds that display red carotenoid coloration of feathers, bills, or legs ingest yellow carotenoids and metabolically convert the yellow pigments to red. Here, we review two lines of investigation that have rapidly advanced understanding of the production of red carotenoid coloration in birds, potentially providing an explanation for how red coloration serves as a signal of quality: the identification of the genes that enable birds to be red and the confirmation of links between production of red pigments and core cellular function. CYP2J19 and BDH1L were identified as key enzymes that catalyze the conversion of yellow carotenoids to red carotenoids both in the retinas of birds for enhanced color vision and in the feathers and bills of birds for ornamentation. This CYP2J19 and BDH1L pathway was shown to be the mechanism for production of red coloration in diverse species of birds and turtles. In other studies, it was shown that male House Finches (Haemorhous mexicanus) have high concentrations of red carotenoids within liver mitochondria and that redness is positively associated with mitochondrial function. These observations suggested that the CYP2J19 and BDH1L pathway might be tightly associated with mitochondrial function. However, it was subsequently discovered that male House Finches do not use the CYP2J19 and BDH1L pathway to produce red pigments and that both CYP2J19 and BDH1L localize in the endoplasmic reticulum, not the mitochondria. Thus, we have the most detailed understanding of links between cellular function and redness in a bird species for which the enzymes to convert yellow to red pigments remain unknown, while we have the best understanding of the enzymatic pathways to red in species for which links to cellular function are largely unstudied. Deducing whether and how signals of quality arise from these distinct mechanisms of ornamental coloration is a current challenge for scientists interested in the evolution of honest signaling.

Aerobic respiration in mitochondria is the source for most of the energy that powers complex animals, and maintaining energy flow from mitochondria near the optimum needed for life processes presents challenges for complex animals. Environments of most animals change rapidly. Moreover, individuals pass through developmental stages with different energy demands, and they shift life-history states that require modified production of adenosine triphosphate (ATP). To adjust to changing conditions, all complex animals display some capacity for acclimatization through phenotypic flexibility, whereby key aspects of mitochondrial respiration are reversibly altered. Phenotypic flexibility is a universal feature of the energy-production mechanisms of animals, but all animals face limitations in the range of environments and circumstances to which they can acclimatize. We discuss multiple examples of such phenotypic flexibility in animals, focusing on the different mechanisms employed that acclimatize mitochondrial respiration to exogenous and endogenous challenges. Genotype sets the range of phenotypes related to mitochondrial respiration that is available to an animal. Numerous studies document adaptive evolution of both mitochondrial and nuclear genes that directly affect the range of environments that will support oxidative phosphorylation. Phenotypic flexibility can obscure evolutionary changes in response to changing energy demands, and understanding the interplay of capacity for acclimatization and adaptive evolution of mitochondrial systems presents major challenges for physiological and evolutionary biologists.

Birds exhibit a variety of migration strategies. Because sustained flapping flight requires the production of elevated levels of energy compared to typical daily activities, migratory birds are well-documented to have several physiological adaptations to support the energy demands of migration. However, even though mitochondria are the source of ATP that powers flight, the respiratory performance of the mitochondria is almost unstudied in the context of migration. We hypothesized that migratory species would have higher mitochondrial respiratory performance during migration compared to species that do not migrate. To test this hypothesis, we compared variables related to mitochondrial respiratory function between two confamilial bird species-the migratory Gray Catbird (Dumetella carolinensis) and the non-migratory Northern Mockingbird (Mimus polyglottos). Birds were captured at the same location along the Alabama Gulf Coast, where we assumed that Gray Catbirds were migrants and where resident Northern Mockingbirds live year-round. We found a trend in citrate synthase activity, which suggests that Gray Catbirds have a greater mitochondrial volume in their pectoralis muscle, but we observed no other differences in mitochondrial respiration or complex enzymatic activities between individuals from the migrant vs. the non-migrant species. However, when we assessed the catbirds included in our study using well-established indicators of migratory physiology, birds fell into two groups: a group with physiological parameters indicating a physiology of birds engaged in migration and a group with the physiology of birds not migrating. Thus, our comparison included catbirds that appeared to be outside of migratory condition. When we compared the mitochondrial performance of these three groups, we found that the mitochondrial respiratory capacity of migrating catbirds was very similar to that of Northern Mockingbirds, while the catbirds judged to be not migrating were lowest. One explanation for these observations is these species display very different daily flight behaviors. While the mockingbirds we sampled were not breeding nor migrating, they are highly active birds, living in the open and engaging in flapping flights throughout each day. In contrast, Gray Catbirds live in shrubs and fly infrequently when not migrating. Such differences in baseline energy needs likely confounded our attempt to study adaptations to migration.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are bacterial defences that target bacteriophages and mobile genetic elements. How these defences evolve in novel host environments remains largely unknown. We studied the evolution of the CRISPR-Cas system in Mycoplasma gallisepticum (also named Mycoplasmoides gallisepticum), a bacterial pathogen of poultry that jumped into a passerine host ~30 years ago. Over the decade following the host shift, all isolates displaying a functional CRISPR-Cas system were found not only to harbour completely new sets of spacers, but the DNA protospacer adjacent motif recognized by the main effector M. gallisepticum Cas9 (MgCas9) was also different. These changes in CRISPR-Cas diversity and specificity are consistent with a change in the community of phages and mobile elements infecting M. gallisepticum as it colonized the novel host. In the years following the host shift, we also detected a gradual rise in isolates displaying non-functional MgCas9. After 12 years, all circulating isolates harboured inactive forms only. This loss of CRISPR-Cas function comes at a time when the passerine host is known to have evolved widespread resistance, which in turn drove the evolution of increasing M. gallisepticum virulence through antagonistic coevolution. Such striking concordance in the rise of inactivated forms of CRISPR-Cas and the evolution of host resistance suggests that the inactivation of the CRISPR-Cas system was necessary for enabling adaptive bacterial responses to host-driven selection. We highlight the need to consider both host and pathogen selection pressures on bacteria for understanding the evolution of CRISPR-Cas systems and the key factors driving the emergence of a pathogenic bacterium in a novel host.

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.

The migratory movements undertaken by birds are among the most energetically demanding behaviours observed in nature. Mitochondria are the source of aerobic energy production on which migration depends, but a key component of mitochondrial function, mitochondrial remodelling, has not been investigated in the context of bird migration. We measured markers of mitochondrial remodelling in the skeletal muscles of the Gambel’s (migratory) and Nuttall’s (non-migratory) white-crowned sparrows within and outside migratory periods. Gambel’s were collected in (i) a non-migration period (baseline), (ii) preparation to depart for spring migration (pre-migration) and (iii) active autumn migration (mid-migration). Nuttall’s were collected at timepoints corresponding to baseline and mid-migration in Gambel’s. Across all sampling periods, we found that migratory birds had greater mitochondrial remodelling compared with non-migratory birds. Furthermore, birds from the migratory population also displayed flexibility, increasing several markers of mitochondrial remodelling (e.g. NRF1, OPA1 and Drp1) pre- and during migration. Further, the greater levels of mitochondrial remodelling and its upregulation during migration were specific to the pectoralis muscle used in flapping flight. Our study is the first to show that mitochondrial remodelling supports migration in Gambel’s white-crowned sparrows, indicating a highly specific and efficient phenotype supporting the increased energetic demands of migration.

Carotenoid pigmentation produces the yellow and red coloration of birds and other vertebrates, but our understanding of the genetic architecture of carotenoid ornamentation is largely limited to studies of novel color variants observed in captively bred populations. The complexity of carotenoid-based color evolution in nature remains poorly characterized. Here, we examine the long-tailed finch Poephila acuticauda , an Australian songbird with two hybridizing subspecies that differ in bill coloration: yellow in western subspecies acuticauda and red in eastern subspecies hecki . We characterize the carotenoid composition of each subspecies and find that yellow bills can be explained by the loss of C(4)-oxidation, thus blocking yellow dietary pigments from being metabolized 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 the ketolation via C(4)-oxidation of dietary carotenoids, and TTC39B , an enhancer of ketocarotenoid production. Evolutionary genealogy reconstruction indicates that the red-billed phenotype is ancestral and yellow alleles at both CYP2J19 and TTC39B arose and fixed in acuticauda approximately 100 kya. Yellow alleles then introgressed into hecki less than 5 kya. Across all four loci, acuticauda derived variants show evidence of selective sweeps, implying that yellow bill coloration has been favored by natural selection. Our study suggests that the frequent adaptive evolutionary transitions between red and yellow ornamentation in nature can have a simple genetic basis. Significance We studied variation in carotenoid ornamentation of an Australian songbird with two hybridizing subspecies that differ in bill color: one yellow and the other red. We identified a single metabolic process, C(4)-oxidation, underlying the distinct carotenoid composition of these two bill colors. Genetic association mapping revealed four major effect loci that explained most of the observed variation the trait, including the oxidative ketolation enzyme CYP2J19 and the carotenoid ketolation enhancer gene TTC39B . Evolutionary reconstruction indicates that yellow alleles are derived, ancient (~100 kya), and under positive selection. This has driven their recent (<5 kya) adaptive introgression across the hybrid zone. These findings have important implications for understanding the role of natural selection in phenotypic evolution in natural systems. ### Competing Interest Statement The authors have declared no competing interest.

In birds, the process of speciation is closely associated with transitions in ornamentation, including coloration, plumage pattern, and song. To investigate the origins of these shifts and their connection to genetic changes, we conducted a study on one of the most highly ornamented songbirds, the Painted Bunting (Passerina ciris). The male Painted Buntings exhibits a stunning array of colors, with a red chest, blue head, green back, green coverts, and pink rump. In addition, Painted Buntings show a high level of genetic structure, with eastern and western populations that have fixed genetic differences in both nuclear and mitochondrial genes. Using non-invasive spectrophotometry techniques, we measured the coloration of six plumage patches on 88 museum specimens of male Painted Buntings in definitive plumage from across the range of the species. We predicted that there would be divergence between the genetically distinct eastern and western populations in ornamental coloration that is perceptible to a bunting but imperceptible to a human observer. However, we measured no consistent nor substantial difference in the plumage coloration of males from different populations. The observation of substantial divergence in nuclear and mitochondrial genotype with no change in ornamental coloration between populations of a brightly colored bird has important implications for the role of sexual selection in the process of speciation.

In a hybrid zone between two tropical lekking birds, yellow male plumage of one species has introgressed asymmetrically replacing white plumage of another via sexual selection. Here, we present a detailed analysis of the plumage trait to uncover its physical and genetic bases and trace its evolutionary history. We determine that the carotenoid lutein underlies the yellow phenotype and describe microstructural feather features likely to enhance color appearance. These same features reduce predicted water shedding capacity of feathers, a potential liability in the tropics. Through genome-scale DNA sequencing of hybrids and each species in the genus, we identify BCO2 as the major gene responsible for the color polymorphism. The BCO2 gene tree and genome-wide allele frequency patterns suggest that carotenoid-pigmented collars initially arose in a third species and reached the hybrid zone through historical gene flow. Complex interplay between sexual selection and hybridization has thus shaped phenotypes of these species, where conspicuous sexual traits are key to male reproductive success.

Use of large caliber repeating rifles makes large birds and mammals vulnerable to overharvest or simply mass destruction. This lesson was unscored when unregulated use of firearms caused the local extinction of nearly every species of large mammal and bird in the southeastern United States by the beginning of the twentieth century. During the same period in southern India, restricted access to firearms and a culture of respect for wildlife allowed large mammals and birds to co–exist with a large human population. The survival of megafauna rests on the will of a society to stop uncontrolled killing.