A key question in evolutionary biology concerns the relative importance of different sources of adaptive genetic variation in natural populations. In addition to new mutations and standing variation, recent studies of natural animal populations have revealed numerous cases in which putative adaptations are attributable to allelic variants that were introduced via introgressive hybridization (Song et al. 2011;Heliconius Genome Consortium 2012;Pardo-Diaz et al. 2012;Hedrick 2013;Huerta-S anchez et al. 2014;Natarajan, Projecto-Garcia, et al. 2015;Racimo et al. 2015;Enciso-Romero et al. 2017;Miao et al. 2017;vonHoldt et al. 2017;Jones et al. 2018). In contrast to de novo mutations and low-frequency variants at mutation-drift equilibrium, alleles derived from other species have already been pretested by selection, albeit on a different genetic background. For this reason, selectively introgressed alleles may be especially likely to comprise coadapted combinations of mutations (Hedrick 2013).
In cases where a given species has colonized a new environment, adaptation to newly encountered challenges may be facilitated and expedited by introgressive hybridization with a closely related resident species that has already evolved refined solutions to those same challenges. In this way, the recipient species capitalizes on adaptive solutions that evolved over a longer period of time in the donor species. Just such a scenario has been described in the case of the Tibetan mastiff, an ancient and phenotypically distinct dog breed that was originally bred as a flock guardian at high altitudes in the Himalayas and Tibetan Plateau (Messerschmidt 1983;Li et al. 2008). Tibetan mastiffs do extraordinarily well at high altitude compared with other dog breeds, and their exceptional hypoxia tolerance appears to stem in part from a legacy of introgressive hybridization with the Tibetan wolf (Canis lupus laniger) (Miao et al. 2017;vonHoldt et al. 2017), a high-altitude subspecies of the gray wolf that can be found at elevations >5,000-m above sea level (Sharma et al. 2004). Population genomic studies of both Tibetan mastiff and Tibetan wolf have identified specific genes that exhibit evidence of positive selection, and therefore represent candidate loci for high-altitude adaptation Article ß The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Open Access (Wang et al. 2013;Gou et al. 2014;Zhang et al. 2014;Fan et al. 2016). In Tibetan mastiffs, one of the strongest signatures of positive selection is observed for the chromosomal region spanning the b-globin gene cluster (Wang et al. 2013;Gou et al. 2014;Fan et al. 2016), a compact set of tandemly duplicated genes that encode the b-type subunits of hemoglobin (Hb), the red blood cell protein responsible for circulatory O 2 transport.
Vertebrate Hb is a heterotetramer that consists of two achain and two b-chain subunits (a 2 b 2 ). Genetically based changes in the oxygenation properties of Hb are known to play a key role in hypoxia adaptation of mammals and other vertebrates (Weber 2007;Storz, Scott, et al. 2010;Storz 2016Storz , 2019)). Under severe hypoxia, an increased Hb-O 2 affinity can improve tissue O 2 delivery if the benefit of safeguarding arterial O 2 saturation more than offsets the cost associated with a diminished diffusion gradient for O 2 unloading in the systemic circulation (Bencowitz et al. 1982;Willford et al. 1982;Storz 2016).
The adult Hb of Tibetan mastiffs is distinguished from that of all other domestic dog breeds by a pair of amino acid replacements at adjacent residue positions in the b-chain subunit (Gou et al. 2014). Due to evidence for a recent selective sweep in the chromosomal region spanning the adultexpressed b-globin gene, Gou et al. (2014) suggested that one or both of the mastiff-specific substitutions are responsible for an adaptive increase in Hb-O 2 affinity. Subsequent genomic analyses revealed that both amino acid replacements-along with the entire b-globin gene cluster-were introgressed from the Tibetan wolf (Miao et al. 2017). Thus, the adult Hbs of Tibetan mastiff and Tibetan wolf are distinguished from those of all other dogs and wolves by the same pair of b-chain amino acid replacements.
The evidence for positive selection on the wolf-derived bglobin variants in Tibetan mastiff suggests that the introgression contributed to hypoxia adaptation at high altitude. However, like most purported examples of adaptive introgression, the phenotypic effects of the introgressed variants have not been experimentally tested (Racimo et al. 2015;Suarez-Gonzalez et al. 2018).
All canids share the same complement of adult Hb genes, including a tandemly linked pair of nearly identical a-type globin genes on Chromosome 6 and a set of triplicated b-type genes on Chromosome 21: d-globin (HBD), b/d-globin (HBB/ D), and b-globin (HBB). The HBB/D gene is a chimeric fusion gene that originated via unequal crossing-over between the tandemly linked HBD and HBB genes (Gaudry et al. 2014) (fig. 1A). The recombination event that produced the chimeric HBB/D fusion gene occurred in the stem lineage of carnivores and is therefore shared by all extant species in the group (Gaudry et al. 2014). The nonchimeric HBB gene at the 3 0 end of the cluster is orthologous to the adult b-globin gene of humans and other mammals (Hoffmann et al. 2008;Opazo et al. 2008aOpazo et al. , 2008bOpazo et al. , 2009;;Janecka et al. 2015). However, in canids and other caniform carnivores (bears, pinnipeds, and members of the superfamily Musteloidea), HBB is a nonexpressed pseudogene (hereafter referred to as HBBps; fig. 1A) (Gaudry et al. 2014;Zald ıvar-L opez et al. 2017). We generated sequence data which revealed that orthologs of the adultexpressed aand b-type globin genes are almost completely invariant at the amino acid level in all domestic dogs and wolves. The only exceptions are the adult-expressed HBB/D genes of Tibetan mastiffs and Tibetan wolves, which are distinguished from those of all other dogs and wolves by two missense substitutions at adjacent sites: G13S and L14M (fig. 1B). In principle, the sharing of the derived Ser-b13 and Met-b14 amino acid states between Tibetan mastiffs and Tibetan wolves could be attributable to the parallel fixation of de novo mutations that arose independently in both lineages or the parallel fixation of pre-existing variants that were inherited from the common ancestor of wolves and domestic dogs. However, genomic analyses clearly demonstrate that HBB/D and the rest of the b-globin gene cluster of Tibetan mastiffs was derived via introgression from the Tibetan wolf (Miao et al. 2017).
Intriguingly, the derived amino acid states in HBB/D that are shared between Tibetan mastiff and Tibetan wolf are present in the tandemly linked HBBps pseudogene of all canids (fig. 1B). This suggests the hypothesis that the pair of missense substitutions in the HBB/D gene were originally derived via gene conversion from the paralogous HBBps pseudogene of Tibetan wolf. To test this hypothesis, we used bioinformatics methods to unravel the history of interparalog gene conversion across the canine b-globin gene cluster. In the b-globin gene clusters of Tibetan mastiff and Tibetan wolf, our results revealed that a recent HBBps ! HBB/D conversion event has overwritten the entire 5 0 end of the HBB/D coding sequence, extending from 65-bp upstream of the start codon to the middle of the first intron (fig. 1C and supplementary table S1, Supplementary Material online). Thus, the HBBps-derived conversion tract spans all of HBB/ D exon 1, which includes the two missense mutations responsible for G13S and S14M.
The conversion event that introduced the two missense mutations into the adult-expressed HBB/D gene occurred initially in the Tibetan wolf lineage (fig. 2). Thus, Ser-b13 and Met-b14 were introduced into the HBB/D gene of Tibetan wolf via one form of intragenomic lateral transfer (ectopic gene conversion from a paralogous pseudogene) and were subsequently introduced into the Tibetan mastiff genome via a second form of lateral transfer (introgression). In both Tibetan wolf and Tibetan mastiff, the paired variants were eventually fixed (and, at least in Tibetan mastiff, population genomic data suggest that the variants were fixed via positive directional selection) (Wang et al. 2013;Gou et al. 2014). However, the variants did not arise as de novo point mutations in the HBB/D genes of either the wolf or mastiff. Ancestral state reconstructions suggest that the canidspecific G13S and L14M substitutions in the HBBps gene occurred after the gene had become transcriptionally inactivated (fig. 3). The joint fixation of the Ser-13 and Met-14 variants in both Tibetan wolf and in Tibetan mastiff, in Signore et al. . doi:10.1093/molbev/msz097 MBE combination with the strong signatures of positive selection in Tibetan mastiff (Wang et al. 2013;Gou et al. 2014;Fan et al. 2016), suggests the hypothesis that the amino acid replacements are responsible for an adaptive increase in Hb-O 2 affinity. To test this hypothesis, we first characterized the oxygenation properties of native canid Hbs to measure the net effect of the two substitutions, and we then conducted site-directed mutagenesis experiments involving recombinantly expressed Hbs to measure their independent and joint effects.
To test the net effects of the b-chain amino acid substitutions G13S and L14M, we measured the oxygenation properties of purified native Hbs from Tibetan wolf, Tibetan mastiff, and multiple breeds of domestic dog (which have Hbs that are structurally identical to that of gray wolf). In the absence of allosteric effectors (stripped), domestic dog Hb has an O 2 affinity (measured as P 50 , the P O2 at which Hb is 50% saturated) of 8.96 6 1.06 Torr. Under the same experimental conditions, O 2 affinities of Tibetan wolf and Tibetan mastiff Hbs are significantly higher (i.e., P 50 's are lower: 5.16 6 1.05 and 4.72 6 1.15 Torr, respectively) (fig. 4A and supplementary table S2, Supplementary Material online). In each of the examined canids, Hb-O 2 affinity was reduced to a similar extent in the presence of Cl À ions and the organic phosphate 2,3diphosphoglycerate (DPG), the two most important allosteric cofactors in mammalian red cells (fig. 4A and supplementary table S2, Supplementary Material online). These results indicate that the increased Hb-O 2 affinity of Tibetan wolf and Tibetan mastiff is attributable to an increase in intrinsic O 2 affinity, not to a suppression of sensitivity to allosteric cofactors.
In the presence of both Cl À and DPG, domestic dog Hb has a significantly lower Bohr effect (i.e., a lower sensitivity to pH) than Tibetan mastiff (ANCOVA, F[1, 2] ¼ 4107, P ¼ 0.0002) and the Tibetan wolf (ANCOVA, F[1, 2] ¼ 2646, P ¼ 0.0004; fig. 4B andsupplementary table S2, Supplementary Material online). The Bohr effect describes the reduction in Hb-O 2 affinity caused by a decline in pH, which allows more complete O 2 unloading to acidic tissues during exercise (Bohr et al. 1904). Since an increased Hb-O 2 affinity can inhibit O 2 unloading in the systemic circulation, the enhanced pH sensitivity of Tibetan canid Hbs should mitigate this potentially negative effect on tissue oxygenation by increasing O 2 unloading to metabolizing cells.
To investigate the structural mechanism responsible for the increased Hb-O 2 affinity conferred by the G13S and L14M Hb Function in High-Altitude Tibetan Canids . doi:10.1093/molbev/msz097 MBE substitutions, we conducted a molecular modeling analysis using the crystal structure of domestic dog Hb (Bhatt et al. 2011) 5B). In the four individual subunits of the Hb tetramer, the heme group is held in place by a coordination bond between the iron atom and the imidazole side chain of the "proximal histidine" (a87, b92) in the F-helix. On the opposite side of the heme plane, the iron-O 2 bond is stabilized by a hydrogen bond with the imidazole side chain of the "distal histidine" (a58, b63) in the E-helix. In the deoxygenated state, the ferrous heme iron, Fe(II), is situated outside the heme plane, away from the imidazole side chain of the distal histidine (Perutz 1970). Upon transition to the oxygenated state, movement of the E-and F-helices shifts Fe(II) into the heme plane, thereby facilitating O 2 binding. Consequently, substitutions that reorient the E-or F-helices may alter heme reactivity by shifting the distance of Fe(II) from the heme plane. Although G13S alone is sufficient to create the hydrogen bond between Trp-15 and Ser-72, L14M is required to produce the necessary reorientation of the Ehelix, which is predicted to tighten up the connection between the A-, E-, and F-helices and shift Fe(II) closer to heme plane. Thus, the model-based prediction is that both G13S and L14M are required to produce the observed increase in O 2 affinity, and that either mutation by itself would be insufficient.
The Hb tetramer undergoes an oxygenation-linked transition in quaternary structure, from the deoxy (tense-or Tstate) to the oxy state (relaxed-or R-state). During this transition the a 1 b 1 and a 2 b 2 dimers slide and rotate in relation to one another, resulting in the breakage and formation of numerous intra-and inter-subunit bonds (Perutz 1970). The change in enthalpy caused by the breakage/formation of ). Thus, amino acid substitutions that affect the T ! R transition are expected to change DH 0 . As the G13S and L14M substitutions in Tibetan canid Hbs tighten up the connection between the A-, E-, and F-helices, inducing a coordinated shift in quaternary structure that moves the Fe(II) closer to heme plane, the T-state is shifted towards a more R-like conformation. This is predicted to alter DH T!R of the protein in addition to increasing Hb-O 2 affinity.
To test predictions of our modeling analysis, we synthesized and experimentally tested four recombinant Hb (rHb) mutants representing each possible two-site combination of ancestral/derived amino acid states at b13 and b14. Consistent with model-based predictions, experimental measurements on the rHb mutants revealed that G13S and L14M only produce a significant increase in Hb-O 2 affinity in combination (fig. 6A). Our model predicts that the joint effect of G13S and L14M (also revealed in the comparison between the native Hbs of domestic dog/gray wolf vs. Tibetan mastiff/ Tibetan wolf) reduce the overall movement of the E-helix during the transition between deoxy-and oxygenated states, thereby altering DH T!R . To test this, we measured O 2 equilibrium curves on domestic dog and Tibetan canid rHbs at two separate temperatures, 25 C and 37 C, to quantify the enthalpy and entropy of oxygenation (DH and DS, respectively). We then calculated the Gibbs free energy of oxygenation (DG) according to the relation DG ¼ DH -TDS. The FIG. 3. Ancestral state estimates reveal that the canid-specific G13S and L14M substitutions in HBB occurred after the gene was pseudogenized in the stem lineage of caniform carnivores. Branches are color coded according to inferred expression levels of Hb isoforms that incorporate the product of HBB relative to isoforms that incorporate the product of HBB/D. For all examined caniform carnivores, % expression of HBB is zero (supplementary table S5, Supplementary Material online). For extant species, amino acid states at b13-14 in the products of HBB/D and HBB are shown to the right of each branch tip, and maximum likelihood estimates of ancestral states are indicated for each internal node in the line of descent of dogs and wolves. Values above the ancestral amino acid estimates represent the posterior probabilities of each two-site combination.
Hb Function in High-Altitude Tibetan Canids . doi:10.1093/molbev/msz097 MBE Hbs of Tibetan mastiff and Tibetan wolf exhibit lower values of both DH and DS (estimated as the slope and Y-intercept of the van't Hoff plot, respectively; fig. 6B and supplementary table S3, Supplementary Material online). The negative slopes displayed in figure 6B reflect the endothermic nature of heme deoxygenation (Roughton et al. 1936;Atha and Ackers 1974), which dictates a negative relationship between Hb-O 2 affinity and temperature (Weber and Campbell 2011). As predicted by our modeling results, the difference in the enthalpy of oxygenation between the alternative rHb mutants (DDH) indicates that the 13Ser-14Met genotype (characteristic of Tibetan canids) has a (numerically) lower DH relative to the 13Gly-14Leu genotype (characteristic of domestic dogs and gray wolves), in both the presence and absence of allosteric effectors. As differential binding of allosteric effectors can alter the overall DH (Weber and Campbell 2011), the observed difference in DH between the rHb mutants in the absence of allosteric effectors (fig. 6B and supplementary table S3, Supplementary Material online) suggests this difference is due to changes in DH T!R .
Numerous statistical approaches have been developed to detect introgression using genomic polymorphism data, and such analyses can be integrated with population genetic tests for evidence of positive selection or associations with phenotype (Racimo et al. 2015). However, conclusive evidence that introgressed alleles have contributed to adaptive phenotypic evolution in the recipient species requires experimental measurements of phenotypic effects. Specifically, experimental evidence is required to document that the introgressed allele increases fitness on the genetic background of the recipient species, or that it contributes to a change in phenotype in the direction that is predicted to be adaptive (Natarajan, Projecto-Garcia, et al. 2015;Suarez-Gonzalez et al. 2018). Our experimental results demonstrate that the derived amino acid changes in Tibetan mastiff and Tibetan wolf reduce Hb P 50 (i.e., increase Hb-O 2 affinity) by $50%, relative to the wildtype dog Hb. This change in Hb-O 2 affinity is in the direction that is expected to enhance arterial O 2 saturation under hypoxic conditions and is therefore consistent with population genetic evidence for positive selection in relation to altitude (Wang et al. 2013;Gou et al. 2014;Fan et al. 2016).
In addition to the genomic evidence for adaptive introgression in Tibetan mastiffs (Miao et al. 2017), our results indicate that the causative G13S and L14M mutations in the donor species, Tibetan wolf, initially arose and were fixed in a nonexpressed pseudogene (HBBps). Missense mutations introduced by gene conversion from paralogous pseudogenes have been documented to have seemingly neutral or deleterious phenotypic effects in other species (Storz, Runck, et al. 2010;Casola et al. 2012;Natarajan, Hoffmann, et al. 2015). The Hb mutations in Tibetan wolf Hb appear to represent a rare case in which such mutations have been favored by selection and have contributed to an adaptive change in phenotype.
The evidence for positive selection on the introgressed missense mutations in Tibetan mastiff (Wang et al. 2013;Gou et al. 2014;Fan et al. 2016) suggests that they conferred an adaptive benefit in the high-altitude environment shared by both Tibetan mastiff (a recent arrival) and Tibetan wolf (the long-term resident). The bG13S and bL14M mutations in HBBps must have been neutral when they first occurred in the common ancestor of canids because all available evidence indicates that this gene was a nonexpressed pseudogene, as it is not transcribed in any extant caniform carnivores (fig. 3) (Gaudry et al. 2014;Zald ıvar-L opez et al. 2017). This represents a possible example of the Dykhuizen-Hartl effect (Kimura 1983) whereby an initially neutral mutation later becomes beneficial upon a change in the external environment and/or genetic background. In the case of the Tibetan wolf, the b13/14 mutations could have experienced two separate changes in fitness effects. With regard to changes in genetic background, the initially neutral b13/14 mutations (Turek, Kreuzer, and Ringnalda 1978;Turek, Kreuzer, Turek-Maischeider, et al. 1978;Bencowitz et al. 1982;Willford et al. 1982;Storz, Scott, et al. 2010;Storz 2016Storz , 2019)). Thus, an affinity-enhancing mutation that is deleterious at low altitude could have beneficial effects on tissue O 2 delivery at extremely high altitudes, a trade-off that has been documented in other mammals (Eaton et al. 1974;Turek, Kreuzer, and Ringnalda 1978;Turek, Kreuzer, Turek-Maischeider, et al. 1978;Chappell and Snyder 1984). Due to elevational differences in optimal blood P 50 , affinity-altering Hb mutations can be expected to have different selection coefficients in highland dogs and wolves than in their lowland conspecifics.
Compared with de novo mutations and segregating allelic variants that contribute to adaptive phenotypic change, introgressed alleles that were previously fixed in a different species have been pretested by selection. For this reason, it has been suggested that selectively introgressed alleles may be especially likely to combine multiple mutations (e.g., epistatic modifiers) that interact to fine-tune a selected phenotypic effect and/or mitigate deleterious pleiotropic effects (Hedrick 2013). This conjecture has not been previously tested because it requires experimental measurements of the phenotypic effects of individual mutations. The epistatic interaction Hb Function in High-Altitude Tibetan Canids . doi:10.1093/molbev/msz097 MBE that we observed between the two b-globin mutations in Tibetan wolf and Tibetan mastiff indicates that neither mutation alone would have conferred an adaptive advantage at high altitude, as the increased Hb-O 2 affinity required both changes in tandem. This suggests that the molecular underpinnings of the putative Hb adaptation in Tibetan mastiff (involving mutations that arose in a nonexpressed gene and which originally fixed in Tibetan wolf) may be qualitatively distinct from functionally similar changes in protein function that could have evolved via the sequential fixation of de novo mutations during the breed's relatively short duration of residency at high altitude.
The b-globin gene cluster of the domestic dog was mined from genome build CanFam3.1 (chr21: 28,060,000-28,239,000). The annotated domestic dog b-globin gene cluster was used as a reference sequence for the assembly of Tibetan mastiff and gray wolf b-globin clusters using previously published SRA files (Tibetan mastiff: SRX445517, SRX445523, and SRX445529; gray wolf: SRX655650, SRX3424047, and SRX3424050). Reads were mapped to the reference sequence using Geneious 7.1.9 (Biomatters) under high stringency (maximum 5% mismatches and gaps allowed). Single nucleotide polymorphism tables built previously for the Tibetan wolf (Zhang et al. 2014) were used to verify single nucleotide polymorphisms in the coding sequences of b-type globin genes.
Discrete genomic regions containing adult-type b-globin genes (HBD, HBB/D, and HBBps; from the 5 0 mRNA cap to the 3 0 poly-A signal) were aligned using MUSCLE (Edgar 2004) and tests of gene conversion were performed using GENECONV (Sawyer 1989) under default parameters.
Ancestral amino acid states were estimated from an alignment of HBB orthologs from 34 mammalian species (supplementary table S4, Supplementary Material online). Using an input phylogeny based on results of previous studies (KoepfLi et al. 2008;Li et al. 2008;Meredith et al. 2011;Fan et al. 2016), reconstructed sequences were estimated using baseml, as implemented in the software package PAML 4.7 (Yang 2007). Reconstructed sequences were estimated under the GTR þ G substitution model, which was determined by MODELTEST (Posada and Crandall 1998) to provide the best fit to the data. The same phylogeny, along with experimental measures of Hb isoform expression (supplementary table S5, Supplementary Material online), was also used to estimate ancestral HBB expression using the Phytools package in R (Revell 2012).
We collected blood from Tibetan wolves at an altitude of 4,300 m in the KeKe XiLi area of the Qinghai-Tibetan Plateau, and from Tibetan mastiffs and domestic dogs at an altitude of 2,300 m in Xining, China. Animals were handled and blood samples were collected in accordance with regulations of the Animal Experimental and Medical Ethics Committee of the Qinghai University Medical College, Qinghai University. RNA was extracted from $200 ll of flash frozen whole blood from domestic dogs (n ¼ 2), Tibetan mastiffs (n ¼ 3), and Tibetan wolves (n ¼ 2) using an RNeasy Universal Plus Mini Kit (Qiagen). cDNA was
Blood samples ($200 ll) were added to a 5Â volume of ice cold water and incubated on ice for 30 min to lyse the red blood cells. Samples were centrifuged at 20,000 Â g for 10 min to remove cell debris. Buffer was added to the supernatants to a final concentration of 0.01-M HEPES/0.2-M NaCl (pH 7.4) and passed through a PD-10 desalting column (GE Healthcare) equilibrated with 25 ml of 0.01-M HEPES (pH 7.4). Hb proteins eluted from the PD-10 column were concentrated using Amicon Ultra-4 Centrifugal Filter Units (Millipore). O 2 -equilibrium curves for Hb solutions (0.1-mM Hb in 0.1-M HEPES/0.05-M ethylenediaminetetraacetic acid buffer) were measured at 37 C using a Blood Oxygen Binding System (Loligo Systems). O 2 -equilibrium curves were measured in the absence (stripped) and presence of chloride ions (0.1-M KCl) and organic phosphates (0.2-mM DPG). Each Hb solution was sequentially equilibrated with three to five different oxygen tensions (P O2 ) at saturation levels between 30% and 70% while the absorbance was continually monitored at 430 nm (deoxy peak) and 421 nm (oxy/deoxy isosbestic point). Hill plots (log[fractional saturation/[1 À fractional saturation]] vs. logP O2 ) constructed from these measurements were used to determine the P O2 at half saturation (P 50 ) and the cooperativity coefficient (n 50 ) from the vintercept and slope of these plots, respectively. P 50 values were measured at three different pH levels, where the pH of working solutions were adjusted with NaOH to as near 7.2, 7.4, or 7.6 as possible, then precisely measured with a pH-1 Micro pH meter and a needle-type pH microsensor (PreSens Precision Sensing GmbH). Individual measurements were pooled according to species/breed and a linear regression was fit to plots of log P 50 versus pH. The resulting equation was used to estimate P 50 values at pH 7.40 (6SE). O 2 -equilibrium curves were measured for rHbs as described above, but at both 37 and 25 C. Van't Hoff plots were created from these measurements (log P 50 vs. 1/temperature) and a linear regression was fit to each plot. The Gibbs free energy of oxygenation (DG) was calculated from these regressions according to the relation DG ¼ DH -TDS, where DH (enthalpy of oxygenation) is derived from the slope of the regression, multiplied by -R (the universal gas constant), and DS (entropy of oxygenation) is derived from the Y-intercept, multiplied by R.
Modeling of canid Hbs was performed using Modeller 9.19 (Webb and Sali 2014). Oxy and deoxy structures of human Hb (PDB ID, 1hho and 2hhb, respectively) were used as templates. The root-mean-square deviations between the template and model for domestic dog and Tibetan wolf Hbs were 0.12 and 0.15 A ˚in both oxy and deoxy formats. Graphics were prepared using the PyMOL Molecular Graphics System, Version v1.7. Vector Construction and Site-Directed Mutagenesis
The canid globin sequences were synthesized by Invitrogen GeneArt Gene Synthesis (Carlsbad, CA) after optimizing the nucleotide sequences in accordance with Escherichia coli codon preferences. The synthesized globin gene cassette was cloned into a custom pGM vector system along with the methionine aminopeptidase (MAP) gene, as described previously (Natarajan et al. 2011(Natarajan et al. , 2013)). We engineered each of the b-chain codon substitutions by whole plasmid amplification using mutagenic primers and Phusion High-Fidelity DNA Polymerase (New England BioLabs, Ipswitch, MA), phosphorylation with T4 Polynucleotide Kinase (New England BioLabs), and circularization with an NEB Quick Ligation Kit (New England BioLabs). Each engineered codon change was verified by DNA sequencing.
Expression and Purification of rHbs rHb expression was carried out in the Escherichia coli JM109 (DE3) strain as described previously (Natarajan et al. 2011(Natarajan et al. , 2013)). Bacterial cell lysates were then loaded onto a HiTrap Q HP anion exchange column (GE Healthcare) equilibrated with 20-mM Tris/0.5-mM ethylenediaminetetraacetic acid (pH 8.3) and eluted with a linear gradient of 0-0.25-M NaCl. Hb-containing fractions were then loaded on to a SP HP cation exchange column (GE Healthcare) and eluted with a linear pH gradient (pH 6.8-8.4). Eluted Hb factions were concentrated using Amicon Ultra-4 Centrifugal Filter Units (Millipore) and oxygenation properties were measured as described above.
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