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. The convergent evolution of similar traits in different species could be due to repeated changes at the genetic level or different changes that produce the same phenotypic effect. To investigate the extent to which convergence in phenotype is caused by repeated mutations, we investigated the molecular basis of convergent changes in the oxygenation properties of hemoglobin (Hb) in eight pairs of high- and low-altitude waterfowl taxa from the Andes. The results revealed that convergent increases in Hb-O₂ affinity in highland taxa involved a combination of unique and repeated amino acid replacements. However, convergent changes in Hb function generally did not involve parallel substitutions, indicating that repeatability in the evolution of protein function does not require repeatability at the sequence level.

When populations become locally adapted to contrasting environments, alleles that have high fitness in only one environment may be quickly eliminated in populations adapted to other environments, such that gene flow is partly restricted. The stronger the selection, the more rapidly immigrant alleles of lower fitness will be eliminated from the population. However, gene flow may continue to occur at unlinked loci, and adaptive divergence can proceed in the face of countervailing gene flow if selection is strong relative to migration (s > m). We studied the population genetics of the major hemoglobin genes in yellow-billed pintails (Anas georgica) experiencing contrasting partial pressures of oxygen in the Andes of South America. High gene flow and weak population subdivision were evident at seven putatively neutral loci in different chromosomal linkage groups. In contrast, amino acid replacements (Ser-β13, Ser-β116, and Met-β133) in the βA hemoglobin subunit segregated by elevation between lowland and highland populations with significantly elevated F_ST. Migration rates for the βA subunit alleles were approximately 17–24 times smaller than for five unlinked reference loci, the αA hemoglobin subunit (which lacks amino acid replacements) and the mitochondrial DNA control region. The βA subunit alleles of yellow-billed pintails were half as likely to be transferred downslope, from the highlands to the lowlands, than in the opposite direction upslope. We hypothesize that migration between the lowlands and highlands is restricted by local adaptation, and the βA hemoglobin subunit is a likely target of selection related to high-altitude hypoxia; however, gene flow may be sufficiently high to retard divergence at most unlinked loci. Individuals homozygous for lowland alleles may have relatively little difficulty dispersing to the highlands initially but may experience long-term fitness reduction. Individuals homozygous for highland genotypes, in contrast, would be predicted to have difficulty dispersing to the lowlands if their hemoglobin alleles confer high oxygen affinity, predicted to result in chronic erythrocytosis at low elevation. Heterozygous individuals may have a dispersal advantage if their hemoglobin has a wider range of function due to the presence of multiple protein isoforms with a mixture of different oxygen affinities.

Hypoxia is one of the most important factors affecting survival at high altitude, and the major hemoglobin protein is a likely target of selection. We compared population genetic structure in the αA and βA hemoglobin subunits (HBA2 and HBB) of five paired lowland and highland populations of Andean dabbling ducks to unlinked reference loci. In the hemoglobin genes, parallel amino acid replacements were overrepresented in highland lineages, and one to five derived substitutions occurred at external solvent‐accessible positions on the α and β subunits, at α¹β¹ intersubunit contacts, or in close proximity to inositolpentaphosphate (IPP) binding sites. Coalescent analyses incorporating the stochasticity of drift and mutation indicated that hemoglobin alleles were less likely to be transferred between highland and lowland populations than unlinked alleles at five other loci. Amino acid replacements that were overrepresented in the highlands were rarely found within lowland populations, suggesting that alleles segregating at high frequency in the highlands may be maladaptive in the lowlands and vice versa. Most highland populations are probably nonmigratory and locally adapted to the Altiplano, but gene flow for several species may be sufficiently high to retard divergence at unlinked loci. Heterozygosity was elevated in the αA or βA subunits of highland populations exhibiting high gene flow between the southern lowlands and the highlands and in highland species that disperse seasonally downslope to midelevation environments from the central Andean plateau. However, elevated heterozygosity occurred more frequently in the αA subunit but not simultaneously in both subunits, suggesting that selection may be more constrained by epistasis in the βA subunit. Concordant patterns among multiple species with different evolutionary histories and depths of historical divergence and gene flow suggest that the major hemoglobin genes of these five dabbling duck species have evolved adaptively in response to high‐altitude hypoxia in the Andes.

Philornis downsi (Dodge and Aitken) is a bird-parasitic muscid fly native to mainland South America that recently invaded the Galápagos Islands where it is parasitizing Darwin’s finches and other land birds. This parasite was previously known only from Argentina, Brazil, and Trinidad and Tobago. The first report of P. downsi from mainland Ecuador is provided, supporting the hypothesis that the invasion route of P. downsi from its native range to the Galápagos Islands includes mainland Ecuador. Four different morphologies of pupal exuviae, which belong to different fly species, were uncovered. Dipteran puparia were found in 20% of naturally occurring nests collected in 2013 and in 27% of nests in 2014 at the two sites in western Ecuador. P. downsi accounted for 74% and 40% of the puparia in 2013 and 2014, respectively. Two new bird host species for the genus Philornis were recorded: the streak-headed woodcreeper ( Lepidocolaptes souleyetii ) and the fasciated wren ( Campylorhynchus fasciatus ). The levels of nest parasitism found were much lower than levels reported for the Galápagos Islands. Finally, parasitism of P. downsi by a parasitoid in the genus Brachymeria (Hymenoptera, Chalcididae) is documented. Overall, 9% of fly puparia collected in 2013, and 19% of those collected in 2014, had parasitoid emergence holes.