Kevin G. McCracken

Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level

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.

Genomic approaches to understanding population divergence and speciation in birds

ABSTRACT The widespread application of high-throughput sequencing in studying evolutionary processes and patterns of diversification has led to many important discoveries. However, the barriers to utilizing these technologies and interpreting the resulting data can be daunting for first-time users. We provide an overview and a brief primer of relevant methods (e.g., whole-genome sequencing, reduced-representation sequencing, sequence-capture methods, and RNA sequencing), as well as important steps in the analysis pipelines (e.g., loci clustering, variant calling, whole-genome and transcriptome assembly). We also review a number of applications in which researchers have used these technologies to address questions related to avian systems. We highlight how genomic tools are advancing research by discussing their contributions to 3 important facets of avian evolutionary history. We focus on (1) general inferences about biogeography and biogeographic history, (2) patterns of gene flow and isolation upon secondary contact and hybridization, and (3) quantifying levels of genomic divergence between closely related taxa. We find that in many cases, high-throughput sequencing data confirms previous work from traditional molecular markers, although there are examples in which genome-wide genetic markers provide a different biological interpretation. We also discuss how these new data allow researchers to address entirely novel questions, and conclude by outlining a number of intellectual and methodological challenges as the genomics era moves forward.

Mito-nuclear discord in six congeneric lineages of Holarctic ducks (genus Anas).

Many species have Holarctic distributions that extend across Europe, Asia and North America. Most genetics research on these species has examined only mitochondrial (mt) DNA, which has revealed wide variance in divergence between Old World (OW) and New World (NW) populations, ranging from shallow, unstructured genealogies to deeply divergent lineages. In this study, we sequenced 20 nuclear introns to test for concordant patterns of OW–NW differentiation between mtDNA and nuclear (nu) DNA for six lineages of Holarctic ducks (genus Anas). Genetic differentiation for both marker types varied widely among these lineages (idiosyncratic population histories), but mtDNA and nuDNA divergence within lineages was not significantly correlated. Moreover, compared with the association between mtDNA and nuDNA divergence observed among different species, OW–NW nuDNA differentiation was generally lower than mtDNA divergence, at least for lineages with deeply divergent mtDNA. Furthermore, coalescent estimates indicated significantly higher rates of gene flow for nuDNA than mtDNA for four of the six lineages. Thus, Holarctic ducks show prominent mito‐nuclear discord between OW and NW populations, and we reject differences in sorting rates as the sole cause of the within‐species discord. Male‐mediated intercontinental gene flow is likely a leading contributor to this discord, although selection could also cause increased mtDNA divergence relative to weak nuDNA differentiation. The population genetics of these ducks contribute to growing evidence that mtDNA can be an unreliable indicator of stage of speciation and that more holistic approaches are needed for species delimitation.

Becoming pure: identifying generational classes of admixed individuals within lesser and greater scaup populations

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.

Interspecific hybridization contributes to high genetic diversity and apparent effective population size in an endemic population of mottled ducks (Anas fulvigula maculosa)

Under drift-mutation equilibrium, genetic diversity is expected to be correlated with effective population size (Ne). Changes in population size and gene flow are two important processes that can cause populations to deviate from this expected relationship. In this study, we used DNA sequences from six independent loci to examine the influence of these processes on standing genetic diversity in endemic mottled ducks (Anas fulvigula) and geographically widespread mallards (A. platyrhynchos), two species known to hybridize. Mottled ducks have an estimated census size that is about two orders-of-magnitude smaller than that of mallards, yet these two species have similar levels of genetic diversity, especially at nuclear DNA. Coalescent analyses suggest that a population expansion in the mallard at least partly explains this discrepancy, but the mottled duck harbors higher genetic diversity and apparent Ne than expected for its census size even after accounting for a population decline. Incorporating gene flow into the model, however, reduced the estimated Ne of mottled ducks to 33xa0% of the equilibrium Ne and yielded an estimated Ne consistent with census size. We also examined the utility of these loci to distinguish among mallards, mottled ducks, and their hybrids. Most putatively pure individuals were correctly assigned to species, but the power for detecting hybrids was low. Although hybridization with mallards potentially poses a conservation threat to mottled ducks by creating a risk of extinction by hybridization, introgression of mallard alleles has helped maintain high genetic diversity in mottled ducks and might be important for the adaptability and survival of this species.

Mitochondrial physiology in the skeletal and cardiac muscles is altered in torrent ducks, Merganetta armata, from high altitudes in the Andes

ABSTRACT Torrent ducks inhabit fast-flowing rivers in the Andes from sea level to altitudes up to 4500u2005m. 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. Summary: Torrent ducks display markedly different physiological and enzymatic specializations at high altitude, characterized by increased mitochondrial respiratory capacity, increased myoglobin content and reorganized enzymatic capacities.

Respiratory mechanics of eleven avian species resident at high and low altitude

ABSTRACT 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. Highlighted Article: Work and cost of breathing in 11 species of waterfowl are reported to be lower than previously predicted in birds, but without effect of altitudinal habitat despite mechanical differences in highland species.

Divergent respiratory and cardiovascular responses to hypoxia in bar-headed geese and Andean birds

ABSTRACT 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. Highlighted Article: When exposed to progressive hypoxia, bar-headed geese (biannual high-altitude migrators) increase ventilation and heart rate, whereas Andean geese (lifelong high-altitude residents) increase lung oxygen extraction and cardiac stroke volume.

Respiratory mechanics and morphology of Tibetan and Andean high-altitude geese with divergent life histories

ABSTRACT 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 PO2. Summary: Larger, but mechanically similar, respiratory systems enable the slow, deep breathing pattern of geese that reside (Andean geese) or migrate (bar-headed geese) at high altitude compared with low-altitude resident species (barnacle geese).

Validation of a Pulse Oximetry System for High-Altitude Waterfowl by Examining the Hypoxia Responses of the Andean Goose (Chloephaga melanoptera)

Hypoxia at high altitudes constrains O2 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 O2 saturation (SaO2) 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 O2. Andean geese exhibited very modest breathing and heart rate responses to hypoxia but were nevertheless able to maintain normal O2 consumption rates during severe hypoxia exposure down to 5 kPa O2. 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 SaO2 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.