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.
When multiple species evolve similar changes in phenotype in response to a shared environmental challenge, it suggests that the convergently evolved character state is adaptive under the changed conditions and that it evolved under the influence of directional selection. A key question in evolutionary genetics concerns the extent to which such cases of phenotypic convergence are caused by convergent or parallel substitutions in the underlying genes. This question has important implications for understanding the inherent repeatability of evolution at the molecular level [1–9]. In principle, the convergent evolution of a given phenotype may be attributable to (i) unique substitutions, (ii) parallel substitutions (where identical-by-state alleles with independent mutational origins fix independently in different lineages), or (iii) collateral substitutions (where shared, identical-by-descent alleles fix independently in different lineages) [8]. In the last case, allele-sharing between species may be due to the retention of ancestral polymorphism or a history of introgressive hybridization—either way, the function-altering alleles that contribute to phenotypic convergence do not have independent mutational origins. One especially powerful means of assessing the pervasiveness of repeated evolution at the sequence level is to exploit natural experiments where phylogenetically replicated changes in protein function have evolved in multiple taxa as an adaptive response to a shared environmental challenge. For example, there are good reasons to expect that vertebrate species living at very high altitudes will have convergently evolved hemoglobins (Hbs) with increased O2-binding affinities [10,11]. Under severe hypoxia, an increased blood-O2 affinity can help ensure tissue O2 supply by safeguarding arterial O2 saturation while simultaneously maintaining the pressure gradient that drives O2 diffusion from the peripheral capillaries to the cells of respiring tissues [12–18]. Evolutionary adjustments in blood-O2 affinity often stem directly from structural changes in the tetrameric (α2β2) Hb protein. Genetically based changes in the oxygenation properties of Hb can be brought about by amino acid mutations that increase intrinsic Hb-O2 affinity and/or mutations that suppress the sensitivity of Hb to the inhibitory effects of allosteric co-factors in the red blood cell [19–22] (S1 Fig). Derived increases in Hb-O2 affinity have been documented in some high-altitude birds and mammals [23–30], but other comparative studies have not revealed consistent trends [31–34]. Additional comparisons between conspecific populations and closely related species are needed to assess the validity of empirical generalizations about the relationship between Hb-O2 affinity and native elevation in vertebrates. Previous surveys of sequence variation in the globin genes of Andean waterfowl documented repeated amino acid substitutions in the major Hb isoforms of multiple high-altitude taxa [35,36], but the functional effects of the substitutions were not assessed so it was not known whether the repeated changes contributed to convergent changes in the oxygenation properties of Hb. Here we report a comparative analysis of Hb function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergent changes in biochemical phenotype, and to assess the extent to which convergent changes in phenotype are attributable to repeated amino acid substitutions. We measured the functional properties of native Hb variants in each population and species, and we used protein engineering experiments based on site-directed mutagenesis to measure the functional effects of repeated substitutions that were implicated in convergent increases in Hb-O2 affinity in high-altitude taxa. In six of the eight taxon pairs, the high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique, parallel, and collateral amino acid replacements. In comparisons involving high- and low-altitude populations of three different species, function-altering amino acid polymorphisms emerged as highly significant outliers in genome scans of nucleotide differentiation, with derived, affinity-enhancing mutations present at high frequency in the high-altitude populations. In combination with results of the functional experiments, the population genomic analyses provide an independent line of evidence that the observed changes in Hb function are attributable to positive directional selection.
To characterize the red cell Hb isoform composition of each species, we analyzed blood samples from individual specimens using a combination of isoelectric focusing (IEF) and tandem mass spectrometry (MS/MS). Consistent with data from other anseriform birds [38,39], the waterfowl species that we examined expressed two distinct isoforms, HbA (pI = 8. 0–8. 2) and HbD (pI = 7. 0–7. 2) with the major HbA isoform comprising ~70–80% of total Hb (S1 Table). The major HbA isoform incorporates α-chain products of the αA-globin gene and the minor HbD isoform incorporates products of the tandemly linked αD-globin gene; both isoforms incorporate β-chain products of the same βA-globin gene [38,39]. Since avian HbD has a consistently higher O2-affinity than HbA in all avian taxa examined to date [28,30,32,39], upregulating HbD expression could be expected to provide an efficient means of increasing blood-O2 affinity in response to environmental hypoxia. However, it appears that high-altitude Andean waterfowl do not avail themselves of this option, as we observed no difference in relative isoform abundance between pairs of high- and low-altitude sister taxa (Wilcoxon signed-rank test, W = 12, N = 7 pairwise comparisons, P>0. 05; S1 Table). MS/MS analysis confirmed that subunits of the two adult Hb isoforms represent products of the adult-expressed αA-, αD-, and βA-globin genes; products of the embryonic α- and β-type globin genes were not detected. By combining αD-globin sequences with previously published αA- and βA-globin sequences for the same individual specimens, we identified all amino acid differences that distinguish the HbA and HbD isoforms of each pair of high- and low-altitude taxa (Fig 1). Full alignments of αA-, αD-, and βA-globin amino acid sequences are shown in S2 Fig, and the direction of changes in character state at all substituted sites are shown in S3–S5 Figs. Comparisons of the South American species revealed repeated amino acid replacements at five sites that distinguish the HbA isoforms of high- and low-altitude sister taxa, including repeated replacements at one site in the αA-globin gene (α77Ala→Thr in Andean goose, torrent duck, Puna teal, and speckled teal) and four sites in the βA-globin gene (β13Gly→Ser in ruddy ducks and speckled teal, β94Asp→Glu in crested duck and Puna teal, and both β116Ala→Ser and β133Leu→Met in yellow-billed pintail and speckled teal) (Fig 1). The derived pair of βA-globin amino acid variants ‘116Ser-133Met’ that are shared between sympatric high-altitude populations of yellow-billed pintails and speckled teal are clearly identical-by-descent (S6 Fig). Independent evidence for hybridization between the two species [40,41] suggests that the ‘116Ser-133Met’ βA-globin allele in high-altitude yellow-billed pintails was derived via introgression from high-altitude speckled teals. The same is true for a shared β13 (Gly/Ser) polymorphism, although the derived Ser variant is present at low-frequency in yellow-billed pintails. The repeated amino acid changes at βA-globin sites 13,116 and 133 therefore represent collateral replacements, rather than true parallel replacements, as they do not have independent mutational origins in each species. Three of the eight pairs of high- and low-altitude taxa had structurally distinct HbD isoforms due to 1–2 amino acid substitutions in the αD-globin gene (Fig 1). Repeated substitutions at αD96 occurred in Orinoco goose (Val→Ala) and silver teal (Ala→Val), but the direction of the change in character-state was different in each case (S5 Fig). In both interspecific comparisons (Andean goose vs. Orinoco goose, and Puna teal vs. silver teal), αD96Ala is associated with a higher HbD O2-affinity. However, the individual effects of amino acid replacements at αD96 could not be isolated in either comparison because of potentially confounding replacements in the β-chain (β86Ala→Ser in Andean goose, and β94Asp→Glu in Puna teal) (Fig 1). We measured the O2-binding properties of purified HbA and HbD variants from each taxon and we estimated P50 (the PO2 at which Hb is half-saturated with O2) as an index of Hb-O2 affinity. We focus primarily on measures of Hb-O2 affinity in the presence of Cl- ions and IHP (P50 (KCl+IHP) ), as this is the experimental treatment that is most relevant to in vivo conditions in avian red blood cells. The experiments revealed that O2-affinities of HbD were consistently higher (P50 values were lower; S2 Table) than those of HbA, consistent with data from other birds [28,30,32,39]. Comparisons between high- and low-altitude sister taxa revealed appreciable differences in the O2-affinity of the major HbA isoform in six of eight cases, and in each of these six cases the HbA of the high-altitude taxon exhibited the higher O2-affinity (i. e. , lower P50) (Fig 2A; S2 Table). The only two taxon pairs that did not exhibit appreciable differences in Hb-O2 affinity were those involving conspecific populations of ruddy ducks and torrent ducks (Fig 2A; S2 Table). In contrast to the altitudinal trend for HbA, O2-affinities of the minor HbD isoform were not consistently higher in high-altitude taxa (Fig 2B). However, there were three taxon pairs in which O2-affinities of HbA and HbD were both markedly higher in the high-altitude taxa than in the corresponding low-altitude taxa (crested duck, Puna teal, and speckled teal), a pattern that implicates causative mutations in the β-chain subunit, which is shared by both isoforms. Comparisons involving purified Hb variants from birds with known genotypes provide a means of identifying the specific amino acid mutations that are responsible for evolved changes in Hb-O2 affinity. Below we describe the functional effects of unique and repeated replacements, and we report model-based inferences about the structural mechanisms responsible for the observed changes in Hb-O2 affinity. The fact that high-altitude taxa exhibited higher Hb-O2 affinities than their lowland sister taxa in six of eight pairwise comparisons is an intriguing trend and is suggestive of adaptive convergence, but the overall pattern does not permit conclusive inferences about the adaptive significance of observed changes in Hb function in any particular high-altitude population or species. In principle, genome-wide analyses of nucleotide differentiation between individual pairs of high- and low-altitude populations can provide an independent means of assessing whether altitudinal differences in globin allele frequencies may be attributable to a history of spatially varying selection. Accordingly, we used restriction-site associated DNA sequencing (RAD-Seq) to survey genome-wide patterns of nucleotide differentiation between high- and low-altitude populations of three separate species: cinnamon teal, yellow-billed pintail, and speckled teal. In each of these three pairwise population comparisons, function-altering amino acid polymorphisms in the αA- and/or βA-globin genes emerged as highly significant outliers in the genome-wide distribution of site-specific FST values (Fig 7). Indirect inferences about the adaptive significance of these polymorphisms are corroborated by results of the functional experiments, which demonstrated that the derived variants at these sites contributed to increases in Hb-O2 affinity in high-altitude populations of all three species (αA9 in cinnamon teal and the two-site ‘116–133’ βA-globin haplotypes shared by yellow-billed pintail and speckled teal). Since the βA-globin allele of high-altitude yellow-billed pintail was derived via introgressive hybridization with high-altitude speckled teal, the combined results of our functional experiments and population genomic analyses provide strong evidence for positive selection on introgressed allelic variants. This finding contributes to a growing body of evidence that introgressive hybridization can provide an important source of adaptive genetic variation in animal populations [58–60]. Convergent increases in Hb-O2 affinity in high-altitude waterfowl taxa were caused by a combination of unique amino acid replacements (as in the case of cinnamon teal, where the causative mutation was not shared with other highland taxa), parallel replacements (as in the case of high-altitude crested ducks and Puna teal that shared independently derived β94Asp→Glu mutations), and collateral replacements (as in the case of yellow-billed pintail and speckled teal that shared identical-by-descent β-globin alleles due to a history of introgressive hybridization). Andean goose appears to represent another case where the evolution of a derived increase in Hb-O2 affinity is attributable to one or more unique substitutions, although additional experiments will be required to pinpoint the causative change (s). These results demonstrate that convergent changes in protein function can occur through multiple historical paths involving multiple possible mutations. Among the Andean waterfowl taxa that we examined, we identified only a single case where a convergent increase in Hb-O2 affinity was attributable to a true parallel amino acid substitution (β94Asp→Glu in high-altitude crested ducks and Puna teal). The limited number of function-altering parallel substitutions in the Hbs of Andean waterfowl stands in contrast to patterns of functional evolution in vertebrate opsin proteins, where convergent changes in the wavelengths of maximum absorbance (spectral tuning) are very often attributable to parallel amino acid substitutions [61,62]. In vertebrate opsins, the more pervasive patterns of parallelism may reflect the fact that genetically based changes in spectral tuning can only be achieved via specific mutational replacements at a limited number of key residues in the active site [63]. Our findings are more consistent with results of experimental evolution studies in microbes and yeast where replicated changes in fitness involved little to no parallelism at the underlying sequence level [64,65]. Our comparative survey also identified numerous parallel substitutions that had no effect on the inherent oxygenation properties of Hb, although we cannot rule out the possibility that the derived variants contributed to changes in other structural or functional properties. Our results for waterfowl Hbs provide two important lessons about repeated evolution at the molecular level: (i) most cases of convergence in protein function did not involve true parallel substitutions (indicating that similar phenotypic outcomes can be produced by multiple possible mutations), and (ii) most parallel substitutions produced no change in Hb-O2 affinity (convergent or otherwise). These findings demonstrate that parallel substitutions cannot be interpreted as prima facie evidence for adaptive evolution [66,67], and that the functional significance (and, hence, adaptive significance) of specific substitutions needs to be experimentally tested in order to support conclusions about the molecular basis of phenotypic evolution.
Blood and tissue samples were obtained from Andean waterfowl at high- and low-altitude localities as described previously [35]. Samples from Orinoco geese, Abyssinian blue-winged geese, and Hartlaub’s ducks were obtained from Sylvan Heights Waterfowl Park (Scotland Neck, North Carolina). Animals were handled in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Alaska (certification numbers 02-01-152985 and 05-05-152985). We characterized Hb isoform composition in the mature erythrocytes of 106 wild-caught birds (median sample size = 14 individuals per species) (S1 Table). Native Hb components were separated by means of IEF using precast Phast gels (pH 3–9) (GE Healthcare; 17-0543-01). IEF gel bands were excised and digested with trypsin, and MS/MS was used to identify the resultant peptides, as described previously [26,28,32,68]. Database searches of the resultant MS/MS spectra were performed using Mascot (Matrix Science, v1. 9. 0, London, UK); peptide mass fingerprints were queried against a custom database of avian globin sequences, including the full complement of embryonic and adult α- and β-type globin genes that have been annotated in avian genome assemblies [38,69–73]. We identified all significant protein hits that matched more than one peptide with P<0. 05. After separating the HbA and HbD isoforms by native gel IEF, the relative abundance of the two isoforms was quantified densitometrically using Image J [74]. The αA- and βA-globin genes were amplified and sequenced according to protocols described previously [35,36]. For all specimens used as subjects in the experimental analyses of Hb function, we extracted RNA from whole blood using the RNeasy kit (Qiagen, Valencia, CA), and we amplified full-length cDNAs of the αD-globin gene using a OneStep RT-PCR kit (Qiagen, Valencia, CA). We designed paralog-specific primers using 5’ and 3’ UTR sequences, as described by Opazo et al. [38]. We cloned reverse transcription (RT) -PCR products into pCR4-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA), and we sequenced at least five clones per sample in order to recover both alleles. This enabled us to determine full diploid genotypes for αD-globin in each specimen. The sequences were analyzed using Geneious Pro ver. 5. 4. 3. All new sequences were deposited in GenBank under accessions numbers KT988975-KT988992 and KU160516-KU160529. For each amino acid difference between pairs of high- and low-altitude sister taxa, we identified ancestral and derived states by comparison with orthologous sites in a large number of other waterfowl species (n = 117 sequences for αA-globin, 96 for βA-globin, and 57 for αD-globin). Alignments of variable sites in the αA-, βA-, and αD-globin genes are shown in S3, S4 and S5 Figs, respectively. For each divergent site in each pair of sister taxa, unordered parsimony (using the trace character function in Mesquite [75]) yielded unambiguous inferences of character polarity. One notable case of homoplasy in the βA-globin gene involved sites 116 and 133 in high-altitude yellow-billed pintails and speckled teal (S4 Fig), two species that are known to hybridize in nature [40,41]. To assess whether identical two-site ‘β116Ser-β133Met’ haplotypes from the two species were identical-by-descent, we reconstructed haplotype networks of βA-globin coding sequence using the median-joining algorithm [76], as implemented in the program Network 4. 6 (Fluxus Technology, Suffolk, UK). We conducted the analysis on a sample of 257 βA-globin sequences (116 from yellow-billed pintails and 141 from speckled teal) obtained from sympatric high- and low-altitude populations of both species. Sixty individuals representing three species of Andean ducks (speckled teal, cinnamon teal, and yellow-billed pintail) were selected for genome-wide surveys of nucleotide variation using single-digest RAD-Seq [77]. For each species, ten male specimens were selected from high-altitude (≥3,211 m above sea level), and ten were selected from low-altitude (≤ 914 m). Total genomic DNA was extracted from muscle tissue using a DNeasy Tissue Kit (Qiagen, Valencia, California, USA) and normalized using a Qubit Fluorometer (Invitrogen, Grand Island, New York, USA). DNA samples were submitted to Floragenex (Eugene, Oregon, USA) for single-digest RAD-Seq using SbfI, which recognizes an 8-nucleotide (CCTGCAGG) restriction site. Digested DNAs were ligated to barcodes and sequencing adaptors and then sequenced on the Illumina HiSeq 2000 with single-end 100 bp chemistry. Following Illumina sequencing, sequences were demultiplexed and trimmed to yield RAD sequences of 90 bp. Data analysis and bioinformatics pipelines were provided by Floragenex [77–79]. The Floragenex RAD unitag assembler and BSP pipelines v. 2. 0 were used to create a RAD-Seq ‘unitag’ assembly and Bowtie alignments of SAMtools pileup sequences to the reference assembly. Genotypes at each nucleotide site were determined using the VCF popgen v. 4. 0 pipeline to generate a customized VCF 4. 1 (variant call format) database with parameters set as follows: minimum AF for genotyping = 0. 075, minimum Phred score = 15, minimum depth of sequencing coverage = 10x, and allowing missing genotypes from up to 10% of individuals at each site. To filter out base calls that were not useful due to low quality scores or insufficient coverage, genotypes at each nucleotide site were inferred using the Bayesian maximum likelihood algorithm described by Hohenlohe et al. [79]. This algorithm calculates the likelihood of each possible genotype at each site using a multinomial sampling distribution, which gives the probability of observing a set of read counts (n1, n2, n3, n4) for a particular genotype, where ni is the read count for each of the four possible nucleotides at each site, excluding ambiguous reads with low quality scores. The genotyping algorithm incorporates the site-specific sequencing error rate, and assigns the most likely diploid genotype to each site using a likelihood ratio test and significance level of α = 0. 05. A total of 372 million sequence reads were obtained with an average depth of 7. 6 (±2. 4 SD) million reads per sample for yellow-billed pintail and speckled teal and 3. 3 (±1. 4 SD) million reads per sample for cinnamon teal, corresponding to an average of 140,671 (±27,856) RAD loci. After filtering and genotyping, the RAD-Seq survey yielded 49,670 SNPs associated with 18,998 distinct loci in yellow-billed pintail, 47,731 SNPs associated with 19,433 distinct loci in speckled teal, and 18,145 SNPs associated with 9,300 distinct loci in cinnamon teal, respectively. The mean depth of coverage was 36. 8 (±10. 0 SD) reads per site with an average per site quality score of 166. 2 (±31. 3 SD) for yellow-billed pintail and speckled teal, and 39. 8 (±24. 4 SD) reads per site with an average per site quality score of 177. 6 (±26. 4 SD) for cinnamon teal. Illumina reads were submitted to the European Nucleotide Archive and can be accessed under the short read archive (SRA) accession number PRJEB11624. Sequencing coverage and quality scores were summarized using the software VCFtools v. 0. 1. 11 [80]. Custom perl scripts were first used to filter triploid or tetraploid sites and convert the Floragenex-generated VCF file to a biallelic, VCF v4. 0 compatible format. We then calculated Weir and Cockerham’s [81] estimator of FST for each SNP in comparisons between high- and low-altitude population samples. We purified HbA and HbD variants from hemolysates of 1–4 specimens per species, all of which had known αA-, αD-, and βA-globin genotypes. In the case of ruddy ducks and yellow-billed pintails, previous population surveys of sequence polymorphism in the αA- and βA-globin genes had revealed multiple amino acid haplotypes segregating within high- and/or low-altitude populations [35,36]. In each case we purified HbA and HbD variants from individuals that were homozygous for each of the alternative allelic variants. Hemolysates of each individual specimen were dialyzed overnight against 20 mM Tris buffer (pH 8. 4). The two tetrameric HbA and HbD isoforms were then separated using a HiTrap Q-HP column (GE Healthcare; 1 ml 17-1153-01) and equilibrated with 20 mM Tris buffer (pH 8. 4). HbD was eluted against a linear gradient of 0–200 mM NaCl. The samples were desalted by means of dialysis against 10 mM HEPES buffer (pH 7. 4) at 4°C, and were then concentrated by using a 30 kDa centrifuge filter (Amicon, EMD Millipore). We measured O2-equilibria of purified Hb solutions under standard conditions (37°C, pH 7. 4,0. 3 mM heme) using a modified diffusion chamber where absorption at 436 nm was monitored during stepwise changes in equilibration gas mixtures generated by precision Wösthoff gas-mixing pumps [28,32,39,56,82,83]. In order to characterize intrinsic Hb-O2 affinities and mechanisms of allosteric regulatory control, we measured O2-equilibria in the presence of Cl- ions (0. 1M KCl), in the presence of IHP (IHP/Hb tetramer ratio = 2. 0), in the simultaneous presence of both effectors, and in the absence of both effectors (stripped). Free Cl- concentrations were measured with a model 926S Mark II chloride analyzer (Sherwood Scientific Ltd, Cambridge, UK). We estimated values of P50 and n50 (Hill’s cooperativity coefficient at half-saturation) by fitting the Hill equation Y = PO2n/ (P50n + PO2n) to the experimental O2 saturation data by means of nonlinear regression (Y = fractional O2 saturation; n, cooperativity coefficient). The model-fitting was based on 5–8 equilibration steps between 30% and 70% oxygenation. The αA- and βA-globin sequences of yellow-billed pintail were synthesized by Eurofins MWG Operon (Huntsville, AL, USA) after optimizing the nucleotide sequences in accordance with E. coli codon preferences. The synthesized αA-βA globin gene cassette was cloned into a custom pGM vector system along with the methionine aminopeptidase (MAP) gene, as described by Natarajan et al. [27,84]. We engineered each of the β-chain codon substitutions using the QuikChange II XL Site-Directed Mutagenesis kit from Stratagene (LaJolla, CA, USA). Each engineered codon change was verified by DNA sequencing. Recombinant Hb expression was carried out in the JM109 (DE3) E. coli strain as described in Natarajan et al. [27,84]. To ensure the post-translational cleaving of N-terminal methionines from the nascent globin chains, we co-transformed a plasmid (pCO-MAP) containing an additional copy of the MAP gene. Both pGM and pCO-MAP plasmids were cotransformed and subject to dual selection in an LB agar plate containing ampicillin and kanamycin. The expression of each rHb mutant was carried out in 1. 5 L of TB medium. Bacterial cells were grown in 37°C in an orbital shaker at 200 rpm until absorbance values reached 0. 6–0. 8 at 600 nm. The bacterial cultures were induced by 0. 2 mM IPTG and were then supplemented with hemin (50 μg/ml) and glucose (20 g/L). The bacterial culture conditions and the protocol for preparing cell lysates were described previously [27–29,32,84]. The bacterial cells were resuspended in lysis buffer (50 mM Tris, 1 mM EDTA, 0. 5 mM DTT, pH 7. 6) with lysozyme (1 mg/g wet cells) and were incubated in the ice bath for 30 min. Following sonication of the cells, 0. 5–1. 0% polyethylenimine solution was added, and the crude lysate was then centrifuged at 15000 g for 45 min at 4°C. The rHbs were purified by two-step ion-exchange chromatography. Using high-performance liquid chromatography, the samples were passed through a prepacked anion-exchange column (Q-Sepharose) followed by passage through a cation-exchange column (SP-Sepharose). The clarified supernatant was subjected to overnight dialysis in CAPS buffer (20 mM CAPS with 0. 5mM EDTA, pH 9. 7) at 4°C. The samples were passed through the Q-column and the rHb solutions were eluted against a linear gradient of 0–1. 0 M NaCl. The eluted samples were desalted by overnight dialysis with 20 mM HEPES pH 7. 4 (4°C). Dialyzed samples were then passed through the SP-Sepharose column (HiTrap SPHP, 1 mL, 17-1151-01; GE Healthcare) equilibrated with 20 mM HEPES (pH 7. 4). The rHb samples were eluted with a linear gradient of 20 mM HEPES (pH 9. 2). Samples were concentrated and desalted by overnight dialysis against 10 mM HEPES buffer (pH 7. 4) and were stored at -80°C prior to the measurement of O2-equilibrium curves. The purified rHb samples were analyzed by means of sodium dodecyl sulphate (SDS) -polyacrylamide gel electrophoresis. After preparing rHb samples as oxyHb, deoxyHb, and carbonmonoxy derivatives, we measured absorbance at 450–600 nm to confirm that the absorbance maxima match those of the native HbA samples. Results of isoelectric focusing analyses indicated that each of the purified rHb mutants was present as a tetrameric assembly, and this was further confirmed by cooperativity coefficients (n50) >1. 00 in the O2-equilibrium experiments. In vitro measurements of O2-binding properties were conducted in the same manner for rHbs and native Hb samples. Homology-based structural modeling was performed with Modeller 9. 15 [85] using human Hbs in different ligation states (PDB, 2hhb and 1hho) as templates. Models were evaluated on the SWISS-MODEL server [86]. All models had QMEAN values between 0. 71 and 0. 78. Structural mining was performed using PISA [87], PyMol (Schrödinger, New York, NY), and SPACE [88].
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.
Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level