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Diving animals must sustain high muscle activity with finite oxygen (O2) to forage underwater. Studies have shown that some diving mammals exhibit changes in the metabolic phenotype of locomotory muscles compared with non-divers, but the pervasiveness of such changes across diving animals is unclear, particularly among diving birds. Here, we examined whether changes in muscle phenotype and mitochondrial abundance are associated with dive capacity across 17 species of ducks from three distinct evolutionary clades (tribes) in the subfamily Anatinae: the longest diving sea ducks, the mid-tier diving pochards and the non-diving dabblers. In the gastrocnemius (the primary swimming and diving muscle), mitochondrial volume density in both oxidative and glycolytic fiber types was 70% and 30% higher in sea ducks compared with dabblers, respectively. These differences were associated with preferential proliferation of the subsarcolemmal subfraction, the mitochondria adjacent to the cell membrane and nearest to capillaries, relative to the intermyofibrillar subfraction. Capillary density and capillary-to-fiber ratio were positively correlated with mitochondrial volume density, with no variation in the density of oxidative fiber types across tribes. In the pectoralis, sea ducks had greater abundance of oxidative fiber types than dabblers, whereas pochards were intermediate between the two. These data suggest that skeletal muscles of sea ducks have a heightened capacity for aerobic metabolism and an enhanced ability to utilize O2 stores in the blood and muscle while diving., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Evidence from a variety of organisms points to convergent evolution on the mitochondria associated with a physiological response to oxygen deprivation or temperature stress, including mechanisms for high-altitude adaptation. Here, we examine whether demography and/or selection explains standing mitogenome nucleotide diversity in high-altitude adapted populations of three Andean waterfowl species: yellow-billed pintail (Anas georgica), speckled teal (Anas flavirostris), and cinnamon teal (Spatula cyanoptera). We compared a total of 60 mitogenomes from each of these three duck species (n = 20 per species) across low and high altitudes and tested whether part(s) or all of the mitogenome exhibited expected signatures of purifying selection within the high-altitude populations of these species. Historical effective population sizes (Ne) were inferred to be similar between high- and low-altitude populations of each species, suggesting that selection rather than genetic drift best explains the reduced genetic variation found in mitochondrial genes of high-altitude populations compared to low-altitude populations of the same species. Specifically, we provide evidence that establishment of these three Andean waterfowl species in the high-altitude environment, coincided at least in part with a persistent pattern of negative purifying selection acting on oxidative phosphorylation (OXPHOS) function of the mitochondria. Our results further reveal that the extent of gene-specific purifying selection has been greatest in the speckled teal, the species with the longest history of high-altitude occupancy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Graham et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Diving animals must sustain high activity with limited O 2 stores to successfully capture prey. Studies suggest that increasing body O 2 stores supports breath-hold diving, but less is known about metabolic specializations that underlie underwater locomotion. We measured maximal activities of 10 key enzymes in locomotory muscles (gastrocnemius and pectoralis) to identify biochemical changes associated with diving in pathways of oxidative and substrate-level phosphorylation and compared them across three groups of ducks-the longest diving sea ducks (eight spp.), the mid-tier diving pochards (three spp.) and the non-diving dabblers (five spp.). Relative to dabblers, both diving groups had increased activities of succinate dehydrogenase and cytochrome c oxidase, and sea ducks further showed increases in citrate synthase (CS) and hydroxyacyl-CoA dehydrogenase (HOAD). Both diving groups had relative decreases in capacity for anaerobic metabolism (lower ratio of lactate dehydrogenase to CS), with sea ducks also showing a greater capacity for oxidative phosphorylation and lipid oxidation (lower ratio of pyruvate kinase to CS, higher ratio of HOAD to hexokinase). These data suggest that the locomotory muscles of diving ducks are specialized for sustaining high rates of aerobic metabolism, emphasizing the importance of body O 2 stores for dive performance in these species.

The processes leading to divergence and speciation can differ broadly among taxa with different life histories. We examine these processes in a small clade of ducks with historically uncertain relationships and species limits. The green-winged teal (Anas crecca) complex is a Holarctic species of dabbling duck currently categorized as three subspecies (Anas crecca crecca, A. c. nimia, and A. c. carolinensis) with a close relative, the yellow-billed teal (Anas flavirostris) from South America. A. c. crecca and A. c. carolinensis are seasonal migrants, while the other taxa are sedentary. We examined divergence and speciation patterns in this group, determining their phylogenetic relationships and the presence and levels of gene flow among lineages using both mitochondrial and genome-wide nuclear DNA obtained from 1,393 ultraconserved element (UCE) loci. Phylogenetic relationships using nuclear DNA among these taxa showed A. c. crecca, A. c. nimia, and A. c. carolinensis clustering together to form one polytomous clade, with A. flavirostris sister to this clade. This relationship can be summarized as (crecca, nimia, carolinensis)(flavirostris). However, whole mitogenomes revealed a different phylogeny: (crecca, nimia)(carolinensis, flavirostris). The best demographic model for key pairwise comparisons supported divergence with gene flow as the probable speciation mechanism in all three contrasts (crecca-nimia, crecca-carolinensis, and carolinensis-flavirostris). Given prior work, gene flow was expected among the Holarctic taxa, but gene flow between North American carolinensis and South American flavirostris (M ∼0.1-0.4 individuals/generation), albeit low, was not expected. Three geographically oriented modes of divergence are likely involved in the diversification of this complex: heteropatric (crecca-nimia), parapatric (crecca-carolinensis), and (mostly) allopatric (carolinensis-flavirostris). Our study shows that ultraconserved elements are a powerful tool for simultaneously studying systematics and population genomics in systems with historically uncertain relationships and species limits., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Introgression of beneficial alleles has emerged as an important avenue for genetic adaptation in both plant and animal populations. In vertebrates, adaptation to hypoxic high-altitude environments involves the coordination of multiple molecular and cellular mechanisms, including selection on the hypoxia-inducible factor (HIF) pathway and the blood-O 2 transport protein hemoglobin (Hb). In two Andean duck species, a striking DNA sequence similarity reflecting identity by descent is present across the ~20 kb β-globin cluster including both embryonic (HBE) and adult (HBB) paralogs, though it was yet untested whether this is due to independent parallel evolution or adaptive introgression. In this study, we find that identical amino acid substitutions in the β-globin cluster that increase Hb-O 2 affinity have likely resulted from historical interbreeding between high-altitude populations of two different distantly-related species. We examined the direction of introgression and discovered that the species with a deeper mtDNA divergence that colonized high altitude earlier in history (Anas flavirostris) transferred adaptive genetic variation to the species with a shallower divergence (A. georgica) that likely colonized high altitude more recently possibly following a range shift into a novel environment. As a consequence, the species that received these β-globin variants through hybridization might have adapted to hypoxic conditions in the high-altitude environment more quickly through acquiring beneficial alleles from the standing, hybrid-origin variation, leading to faster evolution.

For illustrative purposes, in this article we use "Johansen Plots" as a graphical way of simultaneously visualizing the inter-connected variables that compose the convective steps of the gas transport cascade. These plots are used to reflect on some of the physiological characteristics seen in five species of birds, four of which sojourn to, or are native to, high altitudes (the barnacle goose, bar-headed goose, Andean goose, speckled teal and ruddy duck). These species were chosen to emphasize the diversity of responses to hypoxia that can exist within a single family. This diversity likely arose for many possible reasons, including local adaptation to hypoxia, differences in flight or diving abilities, or as a result of other phylogenetically-based differences across waterfowl in physiology, behaviour, and/or life style., (Copyright © 2020 Elsevier Inc. All rights reserved.)

High-altitude environments require that animals meet the metabolic O 2 demands for locomotion and thermogenesis in O 2 -thin air, but the degree to which convergent metabolic changes have arisen across independent high-altitude lineages or the speed at which such changes arise is unclear. We examined seven high-altitude waterfowl that have inhabited the Andes (3812-4806 m elevation) over varying evolutionary time scales, to elucidate changes in biochemical pathways of energy metabolism in flight muscle relative to low-altitude sister taxa. Convergent changes across high-altitude taxa included increased hydroxyacyl-coA dehydrogenase and succinate dehydrogenase activities, decreased lactate dehydrogenase, pyruvate kinase, creatine kinase, and cytochrome c oxidase activities, and increased myoglobin content. ATP synthase activity increased in only the longest established high-altitude taxa, whereas hexokinase activity increased in only newly established taxa. Therefore, changes in pathways of lipid oxidation, glycolysis, and mitochondrial oxidative phosphorylation are common strategies to cope with high-altitude hypoxia, but some changes require longer evolutionary time to arise., Competing Interests: ND, LA, GN, GS, KM No competing interests declared, (© 2020, Dawson et al.)

We examined the morphology of the lungs of five species of high-altitude resident ducks from Lake Titicaca in the Peruvian Andes (yellow-billed pintail [Anas georgica], cinnamon teal [Anas cyanoptera orinomus], puna teal [Anas puna], speckled teal [Anas flavirostris oxyptera], and ruddy duck [Oxyura jamaicensis ferruginea]) and compared them with those of the high-altitude migratory bar-headed goose (Anser indicus) and the low-altitude migratory barnacle goose (Branta leucopsis). We then determined the relationship between mass-specific lung volume, the volume densities of the component parts of the lung, and previously reported hypoxia-induced increases in pulmonary O 2 extraction. We found that the mass-specific lung volumes and the mass-specific volume of the exchange tissue were larger in the lungs of high-altitude resident birds. The bar-headed goose had a mass-specific lung volume that fell between those of the low-altitude species and the high-altitude residents, but a mass-specific volume of exchange tissue that was not significantly different than that of the high-altitude residents. The data suggest that the mass-specific volume of the lung may increase with evolutionary time spent at altitude. We found an inverse relationship between the percentage increase in pulmonary O 2 extraction and the percentage increase in ventilation across species that was independent of the volume density of the exchange tissue, at least for the resident Andean birds., (© 2020 Anatomical Society.)

The cardiovascular system is critical for delivering O 2 to tissues. Here, we examined the cardiovascular responses to progressive hypoxia in four high-altitude Andean duck species compared with four related low-altitude populations in North America, tested at their native altitude. Ducks were exposed to stepwise decreases in inspired partial pressure of O 2 while we monitored heart rate, O 2 consumption rate, blood O 2 saturation, haematocrit (Hct) and blood haemoglobin (Hb) concentration. We calculated O 2 pulse (the product of stroke volume and the arterial-venous O 2 content difference), blood O 2 concentration and heart rate variability. Regardless of altitude, all eight populations maintained O 2 consumption rate with minimal change in heart rate or O 2 pulse, indicating that O 2 consumption was maintained by either a constant arterial-venous O 2 content difference (an increase in the relative O 2 extracted from arterial blood) or by a combination of changes in stroke volume and the arterial-venous O 2 content difference. Three high-altitude taxa (yellow-billed pintails, cinnamon teal and speckled teal) had higher Hct and Hb concentration, increasing the O 2 content of arterial blood, and potentially providing a greater reserve for enhancing O 2 delivery during hypoxia. Hct and Hb concentration between low- and high-altitude populations of ruddy duck were similar, representing a potential adaptation to diving life. Heart rate variability was generally lower in high-altitude ducks, concurrent with similar or lower heart rates than low-altitude ducks, suggesting a reduction in vagal and sympathetic tone. These unique features of the Andean ducks differ from previous observations in both Andean geese and bar-headed geese, neither of which exhibit significant elevations in Hct or Hb concentration compared with their low-altitude relatives, revealing yet another avian strategy for coping with high altitude., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Along with manipulating habitat, the direct release of domesticated individuals into the wild is a practice used worldwide to augment wildlife populations. We test between possible outcomes of human-mediated secondary contact using genomic techniques at both historical and contemporary timescales for two iconic duck species. First, we sequence several thousand ddRAD-seq loci for contemporary mallards (Anas platyrhynchos) throughout North America and two domestic mallard types (i.e., known game-farm mallards and feral Khaki Campbell's). We show that North American mallards may well be becoming a hybrid swarm due to interbreeding with domesticated game-farm mallards released for hunting. Next, to attain a historical perspective, we applied a bait-capture array targeting thousands of loci in century-old (1842-1915) and contemporary (2009-2010) mallard and American black duck (Anas rubripes) specimens. We conclude that American black ducks and mallards have always been closely related, with a divergence time of ~600,000 years before present, and likely evolved through prolonged isolation followed by limited bouts of gene flow (i.e., secondary contact). They continue to maintain genetic separation, a finding that overturns decades of prior research and speculation suggesting the genetic extinction of the American black duck due to contemporary interbreeding with mallards. Thus, despite having high rates of hybridization, actual gene flow is limited between mallards and American black ducks. Conversely, our historical and contemporary data confirm that the intensive stocking of game-farm mallards during the last ~100 years has fundamentally changed the genetic integrity of North America's wild mallard population, especially in the east. It thus becomes of great interest to ask whether the iconic North American mallard is declining in the wild due to introgression of maladaptive traits from domesticated forms. Moreover, we hypothesize that differential gene flow from domestic game-farm mallards into the wild mallard population may explain the overall temporal increase in differentiation between wild black ducks and mallards, as well as the uncoupling of genetic diversity and effective population size estimates across time in our results. Finally, our findings highlight how genomic methods can recover complex population histories by capturing DNA preserved in traditional museum specimens., (© 2019 John Wiley & Sons Ltd.)

Diet and host phylogeny drive the taxonomic and functional contents of the gut microbiome in mammals, yet it is unknown whether these patterns hold across all vertebrate lineages. Here, we assessed gut microbiomes from ∼900 vertebrate species, including 315 mammals and 491 birds, assessing contributions of diet, phylogeny, and physiology to structuring gut microbiomes. In most nonflying mammals, strong correlations exist between microbial community similarity, host diet, and host phylogenetic distance up to the host order level. In birds, by contrast, gut microbiomes are only very weakly correlated to diet or host phylogeny. Furthermore, while most microbes resident in mammalian guts are present in only a restricted taxonomic range of hosts, most microbes recovered from birds show little evidence of host specificity. Notably, among the mammals, bats host especially bird-like gut microbiomes, with little evidence for correlation to host diet or phylogeny. This suggests that host-gut microbiome phylosymbiosis depends on factors convergently absent in birds and bats, potentially associated with physiological adaptations to flight. Our findings expose major variations in the behavior of these important symbioses in endothermic vertebrates and may signal fundamental evolutionary shifts in the cost/benefit framework of the gut microbiome. IMPORTANCE In this comprehensive survey of microbiomes of >900 species, including 315 mammals and 491 birds, we find a striking convergence of the microbiomes of birds and animals that fly. In nonflying mammals, diet and short-term evolutionary relatedness drive the microbiome, and many microbial species are specific to a particular kind of mammal, but flying mammals and birds break this pattern with many microbes shared across different species, with little correlation either with diet or with relatedness of the hosts. This finding suggests that adaptation to flight breaks long-held relationships between hosts and their microbes., (Copyright © 2020 Song et al.)

The cardiovascular system is critical for delivering O2 to tissues. Here we examine the cardiovascular responses to progressive hypoxia in four high-altitude Andean duck species compared to four related low-altitude populations in North America, tested at their native altitude. Ducks were exposed to stepwise decreases in inspired partial pressure of O2 while we monitored heart rate, O2 consumption rate, blood O2 saturation, haematocrit (Hct), and blood haemoglobin concentration [Hb]. We calculated O2 pulse (the product of stroke volume and the arterial-venous O2 content difference), blood O2 concentration, and heart rate variability. Regardless of altitude, all eight populations maintained O2 consumption rate with minimal change in heart rate or O2 pulse, indicating that O2 consumption was maintained by either a constant arterial-venous O2 content difference (an increase in the relative O2 extracted from arterial blood) or by a combination of changes in stroke volume and the arterial-venous O2 content difference. Three high-altitude taxa (yellow-billed pintails, cinnamon teal, and speckled teal) had higher Hct and [Hb], increasing the O2 content of arterial blood, and potentially providing a greater reserve for enhancing O2 delivery during hypoxia. Hct and [Hb] between low- and high-altitude populations of ruddy duck were similar, representing a potential adaptation to diving life. Heart rate variability was generally lower in high-altitude ducks, concurrent with similar or lower heart rates than low-altitude ducks, suggesting a reduction in vagal and sympathetic tone. These unique features of the Andean ducks differ from previous observations in both Andean geese and bar-headed geese, neither of which exhibit significant elevations in Hct or [Hb] compared to their low-altitude relatives, revealing yet another avian strategy for coping with high altitude., (© 2020. Published by The Company of Biologists Ltd.)

Flightlessness in birds is the product of changes in suites of characters-including increased body size and reduced anterior limbs-that have evolved repeatedly and independently under similar ecological conditions (generally insularity). It remains unknown whether this phenotypic convergence extends to the genomic level, partially because many losses of flight occurred long ago (such as in penguins or ratites), thus complicating the study of the genetic pathways to flightlessness. Here, we use genome sequencing to study the evolution of flightlessness in a group of ducks that are current and dynamic exemplars of this major functional transition. These recently diverged Tachyeres steamer ducks differ in their ability to fly: one species is predominantly flighted and three are mainly flightless. Through a genome-wide association analysis, we identify two narrow candidate genomic regions implicated in the morphological changes that led to flightlessness, and reconstruct the number of times flightlessness has evolved in Tachyeres. The strongest association is with DYRK1A, a gene that when knocked out in mice leads to alterations in growth and bone morphogenesis. These findings, together with phylogenetic and demographic analyses, imply that the genomic changes leading to flightlessness in Tachyeres may have evolved once, and that this trait remains functionally polymorphic in two species., (© 2019 The Author(s). Evolution © 2019 The Society for the Study of Evolution.)

Aim: To investigate the structure and rate of gene flow among populations of habitat-specialized species to understand the ecological and evolutionary processes underpinning their population dynamics and historical demography, including speciation and extinction., Location: Peruvian and Argentine Andes., Taxon: Two subspecies of torrent duck ( Merganetta armata )., Methods: We sampled 156 individuals in Peru ( M. a. leucogenis; Chillón River, n  = 57 and Pachachaca River, n  = 49) and Argentina ( M. a. armata; Arroyo Grande River, n  = 33 and Malargüe River, n  = 17), and sequenced the mitochondrial DNA (mtDNA) control region to conduct coarse and fine-scale demographic analyses of population structure. Additionally, to test for differences between subspecies, and across genetic markers with distinct inheritance patterns, a subset of individuals (Peru, n  = 10 and Argentina, n  = 9) was subjected to partial genome resequencing, obtaining 4,027 autosomal and 189 Z-linked double-digest restriction-associated DNA sequences., Results: Haplotype and nucleotide diversities were higher in Peru than Argentina across all markers. Peruvian and Argentine subspecies showed concordant species-level differences (Φ ST mtDNA = 0.82; Φ ST autosomal = 0.30; Φ ST Z chromosome = 0.45), including no shared mtDNA haplotypes. Demographic parameters estimated for mtDNA using IM and IMa2 analyses, and for autosomal markers using ∂a∂i (isolation-with-migration model), supported an old divergence (mtDNA = 600,000 years before present (ybp), 95% HPD range = 1.2 Mya to 200,000 ybp; and autosomal ∂a∂i  = 782,490 ybp), between the two subspecies, characteristic of deeply diverged lineages. The populations were well-differentiated in Argentina but moderately differentiated in Peru, with low unidirectional gene flow in each country., Main Conclusions: We suggest that the South American Arid Diagonal was preexisting and remains a current phylogeographic barrier between the ranges of the two torrent duck subspecies, and the adult territoriality and breeding site fidelity to the rivers define their population structure., Competing Interests: None declared.

During periods of reduced O 2 supply, the most profound changes in gene expression are mediated by hypoxia-inducible factor (HIF) transcription factors that play a key role in cellular responses to low-O 2 tension. Using target-enrichment sequencing, we tested whether variation in 26 genes in the HIF signaling pathway was associated with high altitude and therefore corresponding O 2 availability in three duck species that colonized the Andes from ancestral low-altitude habitats in South America. We found strong support for convergent evolution in the case of two of the three duck species with the same genes (EGLN1, EPAS1), and even the same exons (exon 12, EPAS1), exhibiting extreme outliers with a high probability of directional selection in the high-altitude populations. These results mirror patterns of adaptation seen in human populations, which showed mutations in EPAS1, and transcriptional regulation differences in EGLN1, causing changes in downstream target transactivation, associated with a blunted hypoxic response.

Recently evolved species typically share genetic variation across their genomes due to incomplete lineage sorting and/or ongoing gene flow. Given only subtle allele frequency differences at most loci and the expectation that divergent selection may affect only a tiny fraction of the genome, distinguishing closely related species based on multi-locus data requires substantial genomic coverage. In this study, we used ddRAD-seq to sample the genomes of five recently diverged, New World "mallards" (Anas spp.), a group of dabbling duck species characterized by diagnosable phenotypic differences but minimal genetic differentiation. With increased genomic sampling, we aimed to characterize population structure within this group and identify genomic regions that may have experienced divergent selection during speciation. We analyzed 3,017 autosomal ddRAD-seq loci and 177 loci from the Z-chromosome. In contrast to previous studies, the ddRAD-seq data were sufficient to assign individuals to their respective species or subspecies and to generate estimates of gene flow in a phylogenetic framework. We find limited evidence of contemporary gene flow between the dichromatic mallard and several monochromatic taxa, but find evidence for historical gene flow between some monochromatic species pairs. We conclude that the overall genetic similarity of these taxa likely reflects retained ancestral polymorphism rather than recent and extensive gene flow. Thus, despite recurring cases of hybridization in this group, our results challenge the current dogma predicting the genetic extinction of the New World monochromatic dabbling ducks via introgressive hybridization with mallards. Moreover, ddRAD-seq data were sufficient to identify previously unknown outlier regions across the Z-chromosome and several autosomal chromosomes that may have been involved in the diversification of species in this recent radiation., (© 2019 John Wiley & Sons Ltd.)

We examined the control of breathing and respiratory gas exchange in six species of high-altitude duck that independently colonized the high Andes. We compared ducks from high-altitude populations in Peru (Lake Titicaca at ∼3800 m above sea level; Chancay River at ∼3000-4100 m) with closely related populations or species from low altitude. Hypoxic ventilatory responses were measured shortly after capture at the native altitude. In general, ducks responded to acute hypoxia with robust increases in total ventilation and pulmonary O 2 extraction. O 2 consumption rates were maintained or increased slightly in acute hypoxia, despite ∼1-2°C reductions in body temperature in most species. Two high-altitude taxa - yellow-billed pintail and torrent duck - exhibited higher total ventilation than their low-altitude counterparts, and yellow-billed pintail exhibited greater increases in pulmonary O 2 extraction in severe hypoxia. In contrast, three other high-altitude taxa - Andean ruddy duck, Andean cinnamon teal and speckled teal - had similar or slightly reduced total ventilation and pulmonary O 2 extraction compared with low-altitude relatives. Arterial O 2 saturation ( S aO 2 ) was elevated in yellow-billed pintails at moderate levels of hypoxia, but there were no differences in S aO 2  in other high-altitude taxa compared with their close relatives. This finding suggests that improvements in S aO 2  in hypoxia can require increases in both breathing and haemoglobin-O 2 affinity, because the yellow-billed pintail was the only high-altitude duck with concurrent increases in both traits compared with its low-altitude relative. Overall, our results suggest that distinct physiological strategies for coping with hypoxia can exist across different high-altitude lineages, even among those inhabiting very similar high-altitude habitats., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Resolving evolutionary relationships and establishing population structure depends on molecular diagnosability that is often limited for closely related taxa. Here, we use 3,200 ddRAD-seq loci across 290 mallards, American black ducks, and putative hybrids to establish population structure and estimate hybridization rates. We test between traditional assignment probability and accumulated recombination events based analyses to assign hybrids to generational classes. For hybrid identification, we report the distribution of recombination events complements ADMIXTURE simulation by extending resolution past F4 hybrid status; however, caution against hybrid assignment based on accumulated recombination events due to an inability to resolve F1 hybrids. Nevertheless, both analyses suggest that there are relatively few backcrossed stages before a lineage's hybrid ancestry is lost and the offspring are effectively parental again. We conclude that despite high rates of observed interspecific hybridization between mallards and black ducks in the middle part of the 20th century, our results do not support the predicted hybrid swarm. Conversely, we report that mallard samples genetically assigned to western and non-western clusters. We indicate that these non-western mallards likely originated from game-farm stock, suggesting landscape level gene flow between domestic and wild conspecifics., Competing Interests: PL, TJ, & KGM have no financial interest to report.

Phylogeographic studies often infer historical demographic processes underlying species distributions based on patterns of neutral genetic variation, but spatial variation in functionally important genes can provide additional insights about biogeographic history allowing for inferences about the potential role of adaptation in geographic range evolution. Integrating data from neutral markers and genes involved in oxygen (O 2 )-transport physiology, we test historical hypotheses about colonization and gene flow across low- and high-altitude regions in the Ruddy Duck ( Oxyura jamaicensis ), a widely distributed species in the New World. Using multilocus analyses that for the first time include populations from the Colombian Andes, we also examined the hypothesis that Ruddy Duck populations from northern South America are of hybrid origin. We found that neutral and functional genes appear to have moved into the Colombian Andes from both North America and southern South America, and that high-altitude Colombian populations do not exhibit evidence of adaptation to hypoxia in hemoglobin genes. Therefore, the biogeographic history of Ruddy Ducks is likely more complex than previously inferred. Our new data raise questions about the hypothesis that adaptation via natural selection to high-altitude conditions through amino acid replacements in the hemoglobin protein allowed Ruddy Ducks to disperse south along the high Andes into southern South America. The existence of shared genetic variation with populations from both North America and southern South America as well as private alleles suggests that the Colombian population of Ruddy Ducks may be of old hybrid origin. This study illustrates the breadth of inferences one can make by combining data from nuclear and functionally important loci in phylogeography, and underscores the importance of complete range-wide sampling to study species history in complex landscapes.

Hypoxia at high altitudes constrains O 2 supply to support metabolism, thermoregulation in the cold, and exercise. High-altitude natives that somehow overcome this challenge-who live, reproduce, and sometimes perform impressive feats of exercise at high altitudes-are a powerful group in which to study the evolution of physiological systems underlying hypoxia resistance. Here, we sought to determine whether a common pulse oximetry system for rodents (MouseOx Plus) can be used reliably in studies of high-altitude birds by examining the hypoxia responses of the Andean goose. We compared concurrent measurements of heart rate obtained using pulse oximetry versus electrocardiography. We also compared our measurements of peripheral arterial O 2 saturation (SaO 2 ) in uncannulated birds with published data collected from blood samples in birds that were surgically implanted arterial cannulae. Responses to acute hypoxia were measured during stepwise reductions in inspired partial pressure of O 2 . Andean geese exhibited very modest breathing and heart rate responses to hypoxia but were nevertheless able to maintain normal O 2 consumption rates during severe hypoxia exposure down to 5 kPa O 2 . There were some minor quantitative differences between uncannulated and cannulated birds, which suggest that surgery, cannulation, and/or other sources of variability between studies had modest effects on the hypoxic ventilatory response, heart rate, blood hemoglobin, and hematocrit. Nevertheless, measurements of heart rate and SaO 2 by pulse oximetry had small standard errors and were generally concordant and well correlated with measurements using other techniques. We conclude that the MouseOx Plus pulse oximetry system can be a valuable tool for studying the cardiorespiratory physiology of waterfowl without the deleterious effects of surgery/cannulation.

High-altitude bar-headed geese ( Anser indicus ) and Andean geese ( Chloephaga melanoptera ) have been shown to preferentially increase tidal volume over breathing frequency when increasing ventilation during exposure to hypoxia. Increasing tidal volume is a more effective breathing strategy but is also thought to be more mechanically and metabolically expensive. We asked whether there might be differences in the mechanics or morphology of the respiratory systems of high-altitude transient bar-headed geese and high-altitude resident Andean geese that could minimize the cost of breathing more deeply. We compared these two species with a low-altitude migratory species, the barnacle goose ( Branta leucopsis ). We ventilated anesthetized birds to measure mechanical properties of the respiratory system and used CT scans to quantify respiratory morphology. We found that the respiratory system of Andean geese was disproportionately larger than that of the other two species, allowing use of a deeper breathing strategy for the same energetic cost. The relative size of the respiratory system, especially the caudal air sacs, of bar-headed geese was also larger than that of barnacle geese. However, when normalized to respiratory system size, the mechanical cost of breathing did not differ significantly among these three species, indicating that deeper breathing is enabled by morphological but not mechanical differences between species. The metabolic cost of breathing was estimated to be <1% of basal metabolic rate at rest in normoxia. Because of differences in the magnitude of the ventilatory response, the cost of breathing was estimated to increase 7- to 10-fold in bar-headed and barnacle geese in severe hypoxia, but less than 1-fold in Andean geese exposed to the same low atmospheric P O 2 ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Local adaptation frequently occurs across populations as a result of migration-selection balance between divergent selective pressures and gene flow associated with life in heterogeneous landscapes. Studying the effects of selection and gene flow on the adaptation process can be achieved in systems that have recently colonized extreme environments. This study utilizes an endemic South American duck species, the speckled teal (Anas flavirostris), which has both high- and low-altitude populations. High-altitude speckled teal (A. f. oxyptera) are locally adapted to the Andean environment and mostly allopatric from low-altitude birds (A. f. flavirostris); however, there is occasional gene flow across altitudinal gradients. In this study, we used next-generation sequencing to explore genetic patterns associated with high-altitude adaptation in speckled teal populations, as well as the extent to which the balance between selection and migration have affected genetic architecture. We identified a set of loci with allele frequencies strongly correlated with altitude, including those involved in the insulin-like signaling pathway, bone morphogenesis, oxidative phosphorylation, responders to hypoxia-induced DNA damage, and feedback loops to the hypoxia-inducible factor pathway. These same outlier loci were found to have depressed gene flow estimates, as well as being highly concentrated on the Z-chromosome. Our results suggest a multifactorial response to life at high altitudes through an array of interconnected pathways that are likely under positive selection and whose genetic components seem to be providing an effective genomic barrier to interbreeding, potentially functioning as an avenue for population divergence and speciation., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Many high-altitude vertebrates have evolved increased capacities in their oxygen transport cascade (ventilation, pulmonary diffusion, circulation and tissue diffusion), enhancing oxygen transfer from the atmosphere to mitochondria. However, the extent of interspecies variation in the control processes that dictate hypoxia responses remains largely unknown. We compared the metabolic, cardiovascular and respiratory responses to progressive decreases in inspired oxygen levels of bar-headed geese ( Anser indicus ), birds that biannually migrate across the Himalayan mountains, with those of Andean geese ( Chloephaga melanoptera ) and crested ducks ( Lophonetta specularioides ), lifelong residents of the high Andes. We show that Andean geese and crested ducks have evolved fundamentally different mechanisms for maintaining oxygen supply during low oxygen (hypoxia) from those of bar-headed geese. Bar-headed geese respond to hypoxia with robust increases in ventilation and heart rate, whereas Andean species increase lung oxygen extraction and cardiac stroke volume. We propose that transient high-altitude performance has favoured the evolution of robust convective oxygen transport recruitment in hypoxia, whereas life-long high-altitude residency has favoured the evolution of structural enhancements to the lungs and heart that increase lung diffusion and stroke volume., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

High altitude flight in rarefied, extremely cold and hypoxic air is a very challenging activity. Only a few species of birds can achieve it. Hitherto, the structure of the lungs of such birds has not been studied. This is because of the rarity of such species and the challenges of preparing well-fixed lung tissue. Here, it was posited that in addition to the now proven physiological adaptations, high altitude flying birds will also have acquired pulmonary structural adaptations that enable them to obtain the large amounts of oxygen (O2) needed for flight at high elevation, an environment where O2 levels are very low. The Andean goose (Chloephaga melanoptera) normally resides at altitudes above 3000 meters and flies to elevations as high as 6000 meters where O2 becomes limiting. In this study, its lung was morphologically- and morphometrically investigated. It was found that structurally the lungs are exceptionally specialized for gas exchange. Atypically, the infundibulae are well-vascularized. The mass-specific volume of the lung (42.8 cm3.kg-1), the mass-specific respiratory surface area of the blood-gas (tissue) barrier (96.5 cm2.g-1) and the mass-specific volume of the pulmonary capillary blood (7.44 cm3.kg-1) were some of the highest values so far reported in birds. The pulmonary structural specializations have generated a mass-specific total (overall) pulmonary morphometric diffusing capacity of the lung for oxygen (DLo2) of 0.119 mlO2.sec-1.mbar-1.kg-1, a value that is among some of the highest ones in birds that have been studied. The adaptations of the lung of the Andean goose possibly produce the high O2 conductance needed to live and fly at high altitude.

The metabolic cost of breathing at rest has never been successfully measured in birds, but has been hypothesized to be higher than in mammals of a similar size because of the rocking motion of the avian sternum being encumbered by the pectoral flight muscles. To measure the cost and work of breathing, and to investigate whether species resident at high altitude exhibit morphological or mechanical changes that alter the work of breathing, we studied 11 species of waterfowl: five from high altitudes (>3000 m) in Perú, and six from low altitudes in Oregon, USA. Birds were anesthetized and mechanically ventilated in sternal recumbency with known tidal volumes and breathing frequencies. The work done by the ventilator was measured, and these values were applied to the combinations of tidal volumes and breathing frequencies used by the birds to breathe at rest. We found the respiratory system of high-altitude species to be of a similar size, but consistently more compliant than that of low-altitude sister taxa, although this did not translate to a significantly reduced work of breathing. The metabolic cost of breathing was estimated to be between 1 and 3% of basal metabolic rate, as low or lower than estimates for other groups of tetrapods., (© 2017. Published by The Company of Biologists Ltd.)

Torrent ducks inhabit fast-flowing rivers in the Andes from sea level to altitudes up to 4500 m. We examined the mitochondrial physiology that facilitates performance over this altitudinal cline by comparing the respiratory capacities of permeabilized fibers, the activities of 16 key metabolic enzymes and the myoglobin content in muscles between high- and low-altitude populations of this species. Mitochondrial respiratory capacities (assessed using substrates of mitochondrial complexes I, II and/or IV) were higher in highland ducks in the gastrocnemius muscle - the primary muscle used to support swimming and diving - but were similar between populations in the pectoralis muscle and the left ventricle. The heightened respiratory capacity in the gastrocnemius of highland ducks was associated with elevated activities of cytochrome oxidase, phosphofructokinase, pyruvate kinase and malate dehydrogenase (MDH). Although respiratory capacities were similar between populations in the other muscles, highland ducks had elevated activities of ATP synthase, lactate dehydrogenase, MDH, hydroxyacyl CoA dehydrogenase and creatine kinase in the left ventricle, and elevated MDH activity and myoglobin content in the pectoralis. Thus, although there was a significant increase in the oxidative capacity of the gastrocnemius in highland ducks, which correlates with improved performance at high altitudes, the variation in metabolic enzyme activities in other muscles not correlated to respiratory capacity, such as the consistent upregulation of MDH activity, may serve other functions that contribute to success at high altitudes., (© 2016. Published by The Company of Biologists Ltd.)

Documenting patterns of host specificity in parasites relies on the adequate definition of parasite species. In many cases, parasites have simplified morphology, making species delimitation based on traditional morphological characters difficult. Molecular data can help in assessing whether widespread parasites harbour cryptic species and, alternatively, in guiding further taxonomic revision in cases in which there is morphological variation. The duck louse genus Anaticola (Phthiraptera: Philopteridae), based on current taxonomy, contains both host-specific and widespread species. Mitochondrial and nuclear DNA sequences of samples from this genus were used to document patterns of host specificity. The comparison of these patterns with morphological variations in Anaticola revealed a general correspondence between the groups identified by DNA sequences and morphology, respectively. These results suggest that a more thorough taxonomic review of this genus is needed. In general, the groups identified on the basis of molecular data were associated with particular groups of waterfowl (e.g. dabbling ducks, sea ducks, geese) or specific biogeographic regions (e.g. North America, South America, Australia, Eurasia)., (© 2016 The Royal Entomological Society.)

Estimating the frequency of hybridization is important to understand its evolutionary consequences and its effects on conservation efforts. In this study, we examined the extent of hybridization in two sister species of ducks that hybridize. We used mitochondrial control region sequences and 3589 double-digest restriction-associated DNA sequences (ddRADseq) to identify admixture between wild lesser scaup (Aythya affinis) and greater scaup (A. marila). Among 111 individuals, we found one introgressed mitochondrial DNA haplotype in lesser scaup and four in greater scaup. Likewise, based on the site-frequency spectrum from autosomal DNA, gene flow was asymmetrical, with higher rates from lesser into greater scaup. However, using ddRADseq nuclear DNA, all individuals were assigned to their respective species with >0.95 posterior assignment probability. To examine the power for detecting admixture, we simulated a breeding experiment in which empirical data were used to create F1 hybrids and nine generations (F2-F10) of backcrossing. F1 hybrids and F2, F3 and most F4 backcrosses were clearly distinguishable from pure individuals, but evidence of admixed histories was effectively lost after the fourth generation. Thus, we conclude that low interspecific assignment probabilities (0.011-0.043) for two lesser and nineteen greater scaup were consistent with admixed histories beyond the F3 generation. These results indicate that the propensity of these species to hybridize in the wild is low and largely asymmetric. When applied to species-specific cases, our approach offers powerful utility for examining concerns of hybridization in conservation efforts, especially for determining the generational time until admixed histories are effectively lost through backcrossing., (© 2015 John Wiley & Sons Ltd.)

A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.