Divergent and parallel routes of biochemical adaptation in high-altitude from the Qinghai-Tibet Plateau

Xiaojia Zhua,b,1, Yuyan Guana,b,1, Anthony V. Signorec, Chandrasekhar Natarajanc, Shane G. DuBayd,e, Yalin Chenga,b, Naijian Hana, Gang Songa, Yanhua Qua, Hideaki Moriyamac, Federico G. Hoffmannf,g, Angela Fagoh, Fumin Leia,b,2, and Jay F. Storzc,2 aKey Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, 100101 Beijing, China; bUniversity of Chinese Academy of Sciences, 100049 Beijing, China; cSchool of Biological Sciences, University of Nebraska, Lincoln, NE 68588; dCommittee on Evolutionary Biology, University of Chicago, Chicago, IL 60637; eLife Sciences Section, Integrative Research Center, Field Museum of Natural History, Chicago, IL 60605; fDepartment of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS 39762; gInstitute for Genomics, Biocomputing and Biotechnology, Mississippi State University, Mississippi State, MS 39762; and hDepartment of Bioscience, Aarhus University, DK-8000 Aarhus, Denmark

Edited by Cynthia M. Beall, Case Western Reserve University, Cleveland, OH, and approved January 17, 2018 (received for review November 25, 2017)

When different species experience similar selection pressures, the native species have followed similar or different routes of high- probability of evolving similar adaptive solutions may be influenced altitude adaptation (2). The two highest elevation plateaus in the by legacies of evolutionary history, such as lineage-specific changes world, the Andean Altiplano in South America and the Qinghai- in genetic background. Here we test for adaptive convergence in Tibet Plateau in Asia, have very different physiographic and hemoglobin (Hb) function among high-altitude passerine birds that biogeographic histories and are inhabited by members of phy- are native to the Qinghai-Tibet Plateau, and we examine whether logenetically distinct faunas. In the Andes, the passerine birds convergent increases in Hb–O2 affinity have a similar molecular basis that inhabit the highest elevations include representatives of the in different species. We documented that high-altitude parid and globally distributed Passeri clade (oscines) as well as representa- EVOLUTION aegithalid species from the Qinghai-Tibet Plateau have evolved de- tives of the exclusively Neotropical Tyranni clade (suboscines). rived increases in Hb–O2 affinity in comparison with their closest The avifauna of the Qinghai-Tibet Plateau has a very different lowland relatives in East Asia. However, convergent increases in phylogenetic composition, and the passerine birds that inhabit the

Hb–O2 affinity and convergence in underlying functional mecha- highest elevations include a highly disproportionate number of tits nisms were seldom attributable to the same amino acid substitutions in the family Paridae and long-tailed tits in the family in different species. Using ancestral protein resurrection and site- (3–7). Tits are widely distributed throughout the northern hemi- directed mutagenesis, we experimentally confirmed two cases in sphere and tropical Africa, whereas long-tailed tits are mainly which parallel substitutions contributed to convergent increases in restricted to Eurasia; both groups have their center of diversity in

Hb–O2 affinity in codistributed high-altitude species. In one case East Asia (7, 8). involving the ground ( humilis) and gray-crested tit At high altitude, the challenge of matching reduced O2 avail- (Lophophanes dichrous), parallel amino acid replacements with ability with an undiminished cellular O2 demand is especially acute affinity-enhancing effects were attributable to nonsynonymous substitutions at a CpG dinucleotide, suggesting a possible role Significance for mutation bias in promoting recurrent changes at the same site. Overall, most altitude-related changes in Hb function were caused Mountain ranges and highland plateaus in different parts of by divergent amino acid substitutions, and a select few were the world provide an opportunity to investigate the extent to caused by parallel substitutions that produced similar phenotypic which native species have followed similar or different routes effects on the divergent genetic backgrounds of different species. of adaptation to the challenges of life at high altitude. Here we demonstrate that high-altitude songbirds from the Qinghai- hemoglobin | hypoxia | mutation bias | biochemical adaptation | Tibet Plateau independently evolved derived increases in he- convergence moglobin–O2 affinity in comparison with their closest lowland relatives in East Asia. In comparisons that also included more hen different species experience similar selection pres- distantly related high-altitude avian taxa, site-directed muta- Wsures in a shared environment, the probability that they will genesis experiments revealed two cases in which convergent – evolve similar adaptations may be influenced by differences in increases in hemoglobin O2 affinity were caused by identical population size (which determines levels of standing genetic var- amino acid substitutions at the same sites. However, most iation and the rate of input of new mutations) and/or differences adaptive convergence in protein function was attributable to in the duration of residency in that environment (which deter- different amino acid substitutions in different species. mines the time available for new mutations to arise). The proba- Author contributions: F.L. and J.F.S. designed research; X.Z., Y.G., A.V.S., C.N., S.G.D., Y.C., bility of evolving similar adaptive solutions may also be influenced N.H., G.S., Y.Q., H.M., F.G.H., A.F., and F.L. performed research; X.Z., A.V.S., C.N., H.M., by legacies of evolutionary history. Prior genetic changes may F.G.H., A.F., F.L., and J.F.S. analyzed data; and X.Z., F.L., and J.F.S. wrote the paper. preclude or potentiate future changes in a particular trait, in which The authors declare no conflict of interest. case the “happenstance of a realized beginning” (1) may play an This article is a PNAS Direct Submission. outsize role in channeling subsequent pathways of evolutionary Published under the PNAS license. change. Due to lineage-specific changes in genetic background, Data deposition: The sequences reported in this paper have been deposited in GenBank different species may hit upon idiosyncratic solutions to the same (accession nos. MG772099–MG772439). problem simply because they evolved from different ancestral 1X.Z. and Y.G. contributed equally to this work. starting points at the onset of selection. 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. Mountain ranges and highland plateaus in different parts of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. world provide an opportunity to investigate the extent to which 1073/pnas.1720487115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1720487115 PNAS Latest Articles | 1of6 for small, active endotherms like passerine birds that cannot For a subset of six tit species, we experimentally examined rely on metabolic suppression as a general strategy of hypoxia functional properties of native Hbs purified from red blood cells. tolerance. To compensate for the reduced partial pressure of The set of species used in these experiments included extreme high O2 (PO2) in inspired air, physiological adjustments involving alpine specialists such as the (Parus humilis, elevational = – numerous steps in the O2-transport pathway can help sustain O2 range 3,100 5,500 m above sea level), which is endemic to the flux to the tissue mitochondria in support of aerobic ATP syn- Qinghai-Tibet Plateau (6, 7), as well as predominantly highland thesis (9–11). In combination with changes in the cardiorespi- species such as the rufous-vented tit (Periparus rubidiventris, 2,400– ratory system and microcirculation, changes in the oxygenation 4,300 m) and gray-crested tit (Lophophanes dichrous, 2,300– properties of hemoglobin (Hb) can enhance the O2 capacitance 4,600 m), and predominantly lowland species such as the oriental of the blood (the total amount of O unloaded for a given tit (Parus minor, sea level–2,000 m) and the marsh tit (Poecile 2 – arterio-venous difference in O2 tension). Under severe hypoxia, palustris, sea level 2,100 m). We also sampled multiple specimens an increased Hb–O2 affinity safeguards arterial O2 saturation, from high- and low-altitude subspecies of the broadly distributed thereby securing tissue oxygenation, albeit at a lower pressure coal tit [Periparus ater aemodius (2,100–4,600 m) and Periparus ater pekinensis (sea level–1,800 m)]. In addition to analyzing the native gradient for O2 diffusion from the peripheral capillaries to the Hbs of these seven taxa (six distinct species, including geo- cells of respiring tissues (12). Evolutionary increases in Hb–O2 affinity may be caused by amino acid mutations that increase the graphically distinct subspecies of Periparus ater), we also func- tionally tested recombinantly expressed Hbs (rHbs) from an intrinsic O2 affinity of the Hb tetramer and/or mutations that suppress the sensitivity of Hb to the affinity-reducing effects of additional pair of high- and low-altitude sister species in the family − allosteric cofactors (nonheme ligands such as Cl ions and or- Aegithalidae: the black-browed bushtit ( bonvaloti)and ganic phosphates) (12, 13). the sooty bushtit (Aegithalos fulginosus), respectively. We per- In the Andes, birds that are high-altitude natives have generally formed experiments on rHbs for these two species because we did not have adequate quantities of blood from wild-caught birds to evolved derived increases in Hb–O2 affinity in comparison with their closest lowland relatives (14–17). However, convergent in- purify native Hbs. creases in Hb–O2 affinity in different high-altitude species are sel- dom attributable to convergent or parallel changes at the amino Hb Isoform Composition. During adulthood, most species ex- acid level (17). Here we test whether different high-altitude parid press two structurally distinct Hb isoforms that incorporate dif- ferent α-chain subunits but share identical β-chains. The major and aegithalid species from the Qinghai-Tibet Plateau have in- αA βA αA dependently evolved increased Hb–O affinities in comparison with HbA isoform ( 2 2) incorporates products of the -globin 2 gene, and the minor HbD isoform (αD βA ) incorporates products their closest lowland relatives in East Asia, and we examine 2 2 of the αD-globin gene (18–20). We used isoelectric focusing (IEF) whether convergent changes in Hb function have a similar molec- analysis to characterize Hb isoform composition in the red blood ular basis in different phylogenetic lineages. In this context we make cells of each of the focal species. These analyses revealed that the comparisons among different high-altitude parid and aegithalid minor HbD isoform accounted for 20–30% of total Hb (Table S1), species in the Sino-Himalayan region, and, using previously pub- – consistent with data from the majority of other passerine taxa lished data (14 17), we also make comparisons with a phyloge- examined to date (15, 17, 19–21). netically diverse set of high-altitude Andean birds. If lineage- specific changes in genetic background or happenstances of Oxygenation Properties of HbA and HbD Isoforms. We purified HbA biogeographic history predisposed the Andean and Sino-Himalayan and HbD isoforms from red cell lysates of select specimens with to follow different evolutionary paths, then representa- known αA-, αD-, and βA-globin genotypes. We then measured the tives of the two distinct avifaunas may exhibit qualitatively dis- oxygenation properties of purified HbA and HbD solutions in tinct adaptations to hypoxia. If such contingencies influence the the presence and absence of two main allosteric cofactors that adaptive evolution of Hb function, such that highland repre- − regulate Hb–O2 affinity: Cl ions (added as 0.1 M KCl) and sentatives of different lineages hit upon different solutions to the inositol hexaphosphate (IHP, a chemical analog of inositol same problem, this may be reflected in clade-specific or region- pentaphosphate). Experimental measurements on purified Hb specific patterns of molecular parallelism. samples from all species revealed that the HbD isoform exhibi- ted a uniformly higher O2 affinity than HbA, both in the absence Results and Discussion (“stripped”) and presence of allosteric cofactors (Fig. 1 A–C and We conducted a survey of sequence variation in the adult- Table S2). This is indicated by the lower values of P50 (the PO2 at expressed α- and β-type globin genes in a set of 162 bird speci- which heme is 50% saturated) for HbD relative to HbA. This = mens representing 13 tit species in the family Paridae (n 135 consistent isoform differentiation in Hb–O2 affinity suggests that specimens) and 4 long-tailed tit species in Aegithalidae (n = 27 the up-regulation of HbD could provide a ready means of opti- specimens). We collected these specimens from localities span- mizing blood oxygenation properties in response to changes in ning a broad range of elevations on the Qinghai-Tibet Plateau, O2 availability. However, the absence of altitude-related differ- the mountains of Southwest China, and in eastern China. Phy- ences in the HbA/HbD ratio among species (Table S1) indicates logenetic relationships and elevational ranges of focal species are that regulatory adjustments in red cell Hb isoform composition summarized in Fig. S1. do not play an important role in hypoxia adaptation. Both HbA

ABCDFig. 1. O2 affinities of Hbs from high- and low-alti- (H) (H) Periparus rubidiventris (H) (H) tude Asian passerines. (A–C) P values (mean ± SE) for Parus humilis Lophophanes dichrous (H) Aegithalos bonvaloti 50 P. minor (L) Poecile palustris (L) P. ater aemodius A. fulginosus (L) P. ater pekinensis (L) purified HbA and HbD isoforms of Parus humilis and 50 50 ∗ ∗ Parus minor (A), (B) Lophophanes dichrous and Poecile ∗ 40 40 palustris (B), and Periparus rubidiventris, Periparus ater ∗ aemodius,andPeriparus ater pekinensis (C). (D) P50 ∗ 30 ∗ ∗ 30 values for purified recombinant HbA isoforms of

(torr) (torr) Aegithalos bonvaloti and Aegithalos fulginosus. 50 20 50 P P 20 Cross-hatched and solid bars show P50 values in the absence (stripped) and presence of anionic cofactors, 10 10 respectively. Asterisks denote statistically significant 0 0 differences (P < 0.05) in the presence of KCl + IHP. H, HbAHbD HbA HbD HbA HbD HbA high altitude; L, low altitude.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1720487115 Zhu et al. and HbD exhibited cooperative O2 binding, as indicated by Hill that of Periparus ater aemodius; HbD–O2 affinity of Periparus coefficients at half-saturation (n50) >2 in the presence of anions rubidiventris was significantly higher than that of both Periparus (Table S2). ater subspecies (Fig. 1C and Table S2). Similar to each of the other species-level comparisons, experi- Evolved Changes in Hb–O2 Affinity and Its Structural Basis. Experi- ments on recombinantly expressed HbA isoforms from the high- mental results for the nine focal taxa revealed that highland spe- and low-altitude pair of Aegithalos sister species revealed that the – cies generally have higher Hb O2 affinities in comparison with high-altitude Aegithalos bonvaloti had a significantly higher Hb–O2 their close lowland relatives. Within the Parus, the high- affinity relative to the low-altitude Aegithalos fulginosus in the − altitude Parus humilis and the low-altitude Parus minor provide presence of Cl and IHP (Fig. 1D and Table S2). This ∼7 torr A the basis for an especially informative comparison because the two difference in Hb–O2 affinity is attributable to a single α P119A closely related species have completely nonoverlapping elevational substitution in Aegithalos bonvaloti (Fig. 2). This same substitution ranges (Fig. S1). The experiments revealed that the HbA and is responsible for an evolved increase in Hb–O2 affinity in the bar- HbD isoforms of Parus humilis exhibited significantly higher O2 headed goose, Anser indicus (27–29), a high-altitude species re- affinities than the corresponding isoforms of Parus minor,bothin nowned for its trans-Himalayan migratory flights. The αAP119A the absence and presence of allosteric cofactors (Fig. 1A and − substitution eliminates a van der Waals contact between the an- Table S2). In the presence of Cl and IHP, P50 values for HbA and cestral Pro αA119 and Met βA55 on opposing subunits of the same HbD of Parus humilis were lower than those of Parus minor by αβ dimer. The loss of this intradimer contact destabilizes de- factors of 2.0-fold and 1.6-fold, respectively (Table S2). The dif- oxygenated Hb and shifts the allosteric T↔R equilibrium in favor ference in HbA–O2 affinity is attributable to the independent or A A of the high-affinity R state, thereby increasing overall O2 affinity. combined effects of 17 substitutions: 12 in α and 5 in β (Fig. 2). αA – Thus, the parallel P119A substitutions in Aegithalos bonvaloti The qualitatively similar species difference in HbD O2 affinity αA αD and Anser indicus and the parallel A34T substitutions in (Fig. 1A) is associated with eight substitutions: three in in Lophophanes dichrous and Parus humilis are both predicted to addition to the above-mentioned βA substitutions (Fig. 2). increase Hb–O2 affinity by shifting the allosteric equilibrium but Within the Poecile/Lophophanes clade, the high-altitude via opposite mechanisms: destabilization of the T state in the Lophophanes dichrous Poecile palustris and the low-altitude also former case and stabilization of the R state in the latter. have nonoverlapping elevational ranges (Fig. S1). Our experiments Although the comparison between high- and low-altitude revealed that HbA and HbD of Lophophanes dichrous have sig- subspecies of Periparus ater revealed no differentiation in overall nificantly higher O2 affinities than the corresponding isoforms of –

Hb O affinity, all species-level comparisons between close rel- EVOLUTION the low-altitude Poecile palustris (Fig. 1B and Table S2). Species 2 – atives were consistent in that the highland species always had differences in HbA O2 affinity are attributable to the independent – or combined effects of five substitutions (three in αA,twoinβA) higher Hb O2 affinities than the lowland species. In each case, the evolved changes were exclusively attributable to an increase (Fig. 2). A similar difference in HbD–O2 affinity is associated with five substitutions (three in αD in addition to the above-mentioned in intrinsic O2 affinity (as revealed by P50 values for stripped βA substitutions) (Fig. 2). Among the αA substitutions, it is notable Hbs), as there were no appreciable differences in sensitivity to αA allosteric cofactors (Table S2). that Lophophanes dichrous shares the same A34T substitution – α β In principle, altitude-related changes in Hb O2 affinity could be as Parus humilis (Fig. 2). This site is located at an intradimer 1 1/ – α β contact surface. Mutations at such sites are known to affect attributable to derived increases in Hb O2 affinity in high-altitude 2 2 species, derived reductions in low-altitude species, or a combina- Hb–O2 affinity and quaternary structural stability (22–26). Ho- mology modeling analysis suggests that the replacement of Ala tion of both. These alternatives can be distinguished by deciphering with Thr at αA34 introduces four new hydrogen bonds between the character polarity of causative amino acid substitutions. For opposing α-andβ-chain subunits of the oxygenated R conforma- example, if a given high-altitude species has evolved a derived in- crease in Hb–O affinity relative to its low-altitude sister species, tion; the same intersubunit α1β1/α2β2 interface is not altered ap- 2 preciably in the deoxygenated T conformation (Fig. 3 and Table then the evolved change in protein function must be attributable to S3). The allosteric equilibrium between the oxygenated (R) and one or more substitutions where the derived amino acid state is deoxygenated (T) conformations of the Hb tetramer is a primary fixed in the high-altitude species. As described below, we estimated determinant of Hb–O2 affinity. The differential stabilization of globin gene trees to identify the phylogenetic intervals in which oxygenated Hb therefore increases O2 affinity by shifting the T↔R potentially causative amino acid substitutions occurred in the his- allosteric equilibrium in favor of the high-affinity R state. tory of each high-altitude lineage. Within the genus Periparus, experiments revealed no appre- ciable differentiation in overall Hb–O2 affinity between the high- Genealogical Discordance of Globin Genes. To infer the polarity of and low-altitude subspecies of Periparus ater, as HbA–O2 affinity character state change at substituted sites in HbA and HbD, we was slightly higher in the lowland Periparus ater pekinensis, and estimated DNA-based phylogenies of the αA-, αD-, and βA-globin HbD–O2 affinity was higher in the highland Periparus ater genes from all 17 species. Estimated phylogenies revealed high aemodius (Fig. 1C and Table S2). HbA–O2 affinity of the high- levels of genealogical discordance among loci (Fig. S2), likely altitude Periparus rubidiventris was virtually identical to that of reflecting a history of incomplete lineage sorting and/or intro- Periparus ater pekinensis and therefore was slightly higher than gressive hybridization.

Fig. 2. Variable residue positions in a multiple alignment of αA-, αD-, and βA-globin sequences from the set of parid and aegithalid species used in the experimental analysis of Hb function. Subunits of the major HbA isoform are encoded by the αA- and βA- globin genes, whereas those of the minor HbD iso- form are encoded by the αD-andβA-globin genes. High- and low-altitude natives are denoted by a (H) and (L), respectively.

Zhu et al. PNAS Latest Articles | 3of6 A BC119Gly 34Ala 120Lys 111Thr α β 1 1 122Phe 31Arg 31Arg 122Phe

Fig. 3. The αAA34T substitution in Parus humilis and

Lophophanes dichrous increases Hb–O2 affinity by 34Thr 34Ala adding new hydrogen bonds that stabilize the oxy- 117Leu 127Gln 117Leu 131Gln genated R conformation of the Hb tetramer relative 103Gln 30Arg to the deoxygenated T conformation. (A) Structural 30Arg 108Asp model of avian Hb in the liganded R state. (B)A 36Tyr zoomed-in view of the intradimer α1β1 interface of R- state Hb with the ancestral Ala at αA34. Four hy- 126Asp drogen bonds (depicted as dashed lines) are pre- β α 2 dicted at this interface. (C) Replacing Ala with Thr at 2 35Tyr α34 produces a twofold increase in the number of hydrogen bonds at the interface (Table S3), thereby increasing the stability of the R state.

Molecular Convergence and Parallelism. It is critically important to a significant increase in intrinsic Hb–O2 affinity on the AncParidae account for genealogical discordance when testing for evidence background, and this affinity difference persisted in the presence − of molecular parallelism and convergence (30, 31). If amino acid of Cl and IHP (Fig. 4). This result indicates that the parallel substitutions in the αA- and αD-globin genes were mapped onto αAA34T substitutions in Parus humilis and Lophophanes the branches of the species tree (Fig. S1) rather than the ap- dichrous contributed to convergent increases in Hb–O2 affinity propriate gene trees (Fig. S2 A and B), it would create the false in each of the two high-altitude species, but the effect of αAA34T appearance that multiple substitutions had occurred indepen- alone was not sufficient to completely recapitulate the evolved dently in different lineages. change in O2 affinity in either species. Thus, additional amino acid Estimates of gene tree topologies for the αA-, αD-, and βA-globin substitutions in the α-and/orβ-globins of both Parus humilis and genes permit an assessment of the true prevalence of molecular Lophophanes dichrous musthavecontributedtothederivedin- A convergence and parallelism in our set of focal taxa. Some degree creases in Hb–O2 affinity. Interestingly, although α A34T pro- of homoplasy (due to convergence, parallelism, or mutational duced a significant increase in O2 affinity on the AncParidae reversion) is expected due to chance alone. The key question background, reversion of this mutation to the ancestral state A concerns the number of convergent or parallel substitutions that (α T34A) did not produce significant reductions in Hb–O2 af- actually contributed to convergent increases in Hb–O2 affinity in finity on the wild-type backgrounds of either Parus humilis or different high-altitude taxa; this applies to substitutions in HbA Lophophanes dichrous (Fig. 4). Asymmetry in the effects of forward and HbD of both Parus humilis and Lophophanes dichrous,HbD mutations on ancestral backgrounds and reverse mutations on de- of Periparus rubidiventris,andHbAofAegithalos bonvaloti.The rived backgrounds, a phenomenon documented in numerous pro- sequence data revealed that few derived amino acid states are tein-engineering studies, is attributable to epistatic interactions shared between high-altitude taxa to the exclusion of lowland taxa. between the focal mutations and residues at other substituted sites Within the set of high-altitude parid and aegithalid taxa that we that distinguish the two backgrounds (31–36). examined, it appears that there is only one true parallel sub- stitution that is associated with convergent increases in Hb–O2 The Possible Role of Mutation Bias in Promoting Parallel Substitutions. affinity: A34T in the αA-globin orthologs of Parus humilis and Intriguingly, the parallel αAA34T substitutions in both Lopho- Lophophanes dichrous (Fig. 2). phanes dichrous and Parus humilis are attributable to non- synonymous substitutions at a CpG dinucleotide. In both species, Functional Test of Adaptive Parallelism. The αA-globin gene tree replacement of the ancestral Ala for Thr at αA34 was caused by shown in Fig. S2A indicates that the shared, derived Thr αA34 CpG→CpA mutations in the first codon position. Depending on variants in Parus humilis and Lophophanes dichrous must have had the methylation status of the cytosine, transition mutations at CpG independent mutational origins. To test whether the parallel sites are expected to occur at a much higher rate than non-CpG A α A34T substitutions contributed to derived increases in Hb–O2 point mutations at the same nucleotide position (37–40). This affinity in each of these two high-altitude species, we recon- suggests the hypothesis that the αAA34T mutation may be espe- αA βA structed the and sequences of the most recent common cially likely to contribute to evolved increases in Hb–O2 affinity ancestor of the family Paridae, “AncParidae,” which is also the simply because the underlying CpG→CpA transition mutation will most recent common ancestor of Parus and Lophophanes (Fig. S1). occur at a higher rate than nonsynonymous mutations at non-CpG This enabled us to test the effect of the αAA34T mutation on an sites that produce similar affinity-enhancing effects. If adaptation evolutionarily relevant genetic background. Ancestral amino acid is mutation-limited, an increase in the rate of mutation to a ben- states were estimated with a high level of statistical confidence, eficial allele results in a commensurate increase in the allele’s as site-specific posterior probabilities averaged 0.997 for the probability of fixation (41–44). The extent to which evolution is α-chain and 1.000 for the β-chain. The reconstructed ancestral mutation-limited in natural populations is not generally known, Hb was estimated to possess Ala αA34 with a posterior proba- but a growing body of evidence suggests that mutation bias is an bility of 0.988. Overall, the AncParidae Hb differed from the important cause of substitution bias and parallelism in adaptive wild-type Hbs of Parus humilis and Lophophanes dichrous at protein evolution (15, 45, 46). 12and6aminoacidsites,respectively(Fig. S3). An examination of αA-globin nucleotide sequences in a phylo- In vitro functional tests on the purified rHbs confirmed that genetically diverse set of passerines revealed that the CpG di- AncParidae Hb exhibited a significantly lower Hb–O2 affinity nucleotide involving the third position of codon 33 and the first than the wild-type Hbs of both Parus humilis and Lophophanes position of codon 34 is clearly the ancestral state (and, hence, Ala is dichrous, indicating that each of the two high-altitude species the ancestral amino acid at αA34) (Fig. S4). Thus, if the parallel A independently evolved derived increases in Hb–O2 affinity due to α A34T substitutions in Lophophanes dichrous and Parus humilis the effects of one or more lineage-specific substitutions. Site- were partly attributable to mutation bias, it is a bias that is not unique directed mutagenesis experiments revealed that αAA34T produced to parid species. On the basis of mutational accessibility alone, there

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1720487115 Zhu et al. is consistent with comparative data from other high-altitude birds (14–17, 21, 29) and demonstrates that there are multiple possible ways of evolving an increased Hb–O2 affinity without sacrificing allosteric regulatory capacity. In mammals, by contrast, evolutionary changes in Hb–O affinity often involve changes in responsiveness − 2 to Cl and/or organic phosphates (47–52). Despite the pervasive convergence in Hb–O2 affinity (and con- vergence in the underlying functional mechanisms) among high- altitude avian taxa, the limited amount of convergence and paral- lelism at the amino acid level demonstrates that changes in Hb–O2 affinity can be produced by numerous possible amino acid substi- tutions. The analysis of sequence variation in our set of parid and aegithalid species revealed several parallel amino acid substitu- tions, only one of which contributed to convergent increases in Hb– A O2 affinity in different high-altitude tit species: α A34T in Parus humilis and Lophophanes dichrous. Most parallel substitutions were not uniquely associated with convergent increases in Hb–O2 affinity in highland lineages. In studies of genetic convergence and paral- lelism, experimental testing of individual substitutions is necessary to distinguish between signal (replicated substitutions that con- tribute to adaptive convergence in phenotype) and noise (repli- Fig. 4. O2 affinities of recombinantly expressed Hbs representing wild-type genotypes of Parus humilis and Lophophanes dichrous and the reconstructed cated substitutions that have no effect on the selected phenotype). genotype of their most recent common ancestor AncParidae. Site-directed In broader comparisons involving other avian taxa, we docu- mutagenesis experiments revealed that αAA34T produces a significant affinity- mented one additional example in which parallel substitutions enhancing effect on the ancestral background (AncParidae), as indicated by contributed to convergent increases in Hb–O2 affinity in different αA the reduction in P50, but does not completely recapitulate the evolved in- high-altitude species: the P119A substitution in Aegithalos creases in Hb–O2 affinity in Parus humilis or Lophophanes dichrous. Cross- bonvaloti (Aegithalidae: Passeriformes) and the distantly related hatched and solid bars show P50 values in the absence (stripped) and presence bar-headed goose, Anser indicus (Anatidae: Anseriformes). Re- of anionic cofactors, respectively. Mutational reversions (αAA34T) on the wild- sults of site-directed mutagenesis experiments confirm that the EVOLUTION type backgrounds of Parus humilis and Lophophanes dichrous did not produce parallel αAP119A substitutions have significant affinity-enhancing a symmetrical, affinity-reducing effect. The asterisk denotes a statistically sig- effects in the Hbs of both species (Fig. 1D and Table S2) (29). The nificant difference (P < 0.05) in the presence of KCl + IHP. αAP119A mutation also produces a similar affinity-enhancing ef- fect in human Hb (27). All else being equal with respect to effect αA sizes and mutational pleiotropy, allelic variants that have additive is no reason to think that A34T substitutions were more likely to effects across a wide range of genetic backgrounds can be expec- contribute to Hb adaptation in parid species than in other passerines. ted to make more consistent contributions to convergent adapta- tion than those with context-dependent effects. Overall, we did not Broader Patterns of Convergence and Parallelism Involving High- observe any striking clade-specific patterns of parallelism that Altitude Birds from the Andes and the Qinghai-Tibet Plateau. An could be interpreted as evidence that the high-altitude parid or expansion of the comparative analysis to include other high- aegithalid species had hit upon adaptive solutions that were not altitude birds revealed several convergent and parallel substitutions equally accessible to other high-altitude avian taxa. shared with parid and aegithalid species from the Qinghai-Tibet Plateau. The number of such substitutions is reduced significantly Methods if we restrict our attention to shared, derived replacements that are – Sample Collection. We collected a total of 162 bird specimens (representing associated with increases in Hb O2 affinity in high-altitude line- 17 species) from high-altitude localities on the Qinghai-Tibet Plateau and the ages. In the Andean birds, convergent and parallel substitutions mountains of Southwest China and in low-altitude localities throughout were not uncommon, but site-directed mutagenesis experiments eastern China. The collection of all bird specimens was authorized by the revealed that a small fraction of such substitutions (i.e., N/G83S, Forestry Administrations of Qinghai Province and Xizang Autonormous Re- A A86S, D94E, and A116S in β -globin) actually contributed to gion and was in compliance with the National Wildlife Conservation Law of convergent increases in Hb–O2 affinity in two or more high-altitude China. All birds were handled in accordance with regulations of the species (16, 17). These same sites were all invariant in our sample Experimental and Medical Ethics Committee of the Institute of Zoology, Chi- of parid and aegithalid species. nese Academy of Sciences. Details are provided in SI Methods.

Conclusions Hb Isoform Composition and Sequence Variation. To characterize Hb isoform We documented that high-altitude parid and aegithalid species composition in the red blood cells of wild-caught birds, we separated native from the Qinghai-Tibet Plateau have typically evolved in- Hbs by means of IEF and used densitometric measurements to quantify isoform abundance (20). Details regarding cloning and sequencing protocols creased Hb–O2 affinities in comparison with lowland relatives. are provided in SI Methods. All sequences have been deposited in GenBank This pattern is consistent with data from a large and phyloge- – netically diverse set of Andean birds (14–17). The data for (accession nos. MG772099 MG772439). parid and aegithalid birds further bolster a remarkably strong empirical generalization and demonstrate that repeated increases Protein Purification and in Vitro Analysis of Hb Function. Using hemolysates of – bird specimens with known globin genotypes, we isolated and purified the in Hb O2 affinity in high-altitude birds represent one of the most HbA and HbD isoforms by means of anion-exchange FPLC using a HiTrap Q HP striking examples of convergent biochemical adaptation in verte- column (GE Healthcare). Details regarding O2-binding experiments are brates (12). provided in SI Methods. The phylogenetically replicated changes in Hb–O2 affinity provide the opportunity to assess whether such changes consis- Vector Construction, Mutagenesis, and Recombinant Expression. The αA-andβA-globin tently involve the same functional mechanisms and, if so, whether sequences were synthesized in accordance with Escherichia coli codon pref- such changes are attributable to divergent, convergent, or parallel erences. The α-β globin gene cassette was cloned into a custom pGM vector substitutions at the amino acid level. With regard to functional system, and recombinant Hb expression was carried out in the E. coli JM109 mechanisms, the fact that interspecific variation in Hb–O2 affinity (DE3) strain (25, 53). Details regarding mutagenesis and protein purification is exclusively attributable to evolved changes in intrinsic O2 affinity are provided in SI Methods.

Zhu et al. PNAS Latest Articles | 5of6 Phylogenetic Analysis. We used complete exon and intron sequences to esti- ACKNOWLEDGMENTS. We thank N. Zhao, C. Dai, W. Wang, R. Zhang, mate phylogenies of each globin gene. Details regarding alignments, sub- D. Zhang, C. Zhang, B. Gao, Q. Quan, Y. Wu, and Z. Yin for assistance with field stitution models, and ancestral reconstructions are provided in SI Methods. work, E. Petersen for assistance in the laboratory, and P. Alström for help with specimen identification. This study was funded by the Strategic Priority Re- search Program, Chinese Academy of Sciences Grant XDB13020300 (to F.L.), State Structural Modeling. We modeled structures of avian Hbs using MODELER Key Program of the National Science Foundation of China Grant 31630069 ver. 9.19 (54). We used Protein Data Bank (PDB) ID 1hho and 3hhb as (to F.L.), Key Laboratory of Zoological Systematics and Evolution Grant templates for oxygenated and deoxygenated Hb conformations. We O529YX5105 (to F.L.), NIH Grant HL087216 (to J.F.S.), National Science Foun- performed additional calculations using PISA (55) and PyMOL ver. 1.8 dation Grants MCB-1517636 and RII Track-2 FEC-1736249 (to J.F.S.), and Danish (Schrödinger). Council for Independent Research, Natural Sciences Grant 4181-00094 (to A.F.).

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6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1720487115 Zhu et al. Supporting Information

Zhu et al. 10.1073/pnas.1720487115 SI Methods procedure using the BOBS diffusion chamber and gas-mixing Specimen Collection. The collection of all bird specimens was system (Loligo Systems). P50 values were measured at three dif- authorized by the local forestry administrations and was in ferent pH levels (∼7.0, 7.3, and 7.5) using a pH-1 Micro pH meter compliance with the National Wildlife Conservation Law of and a needle-type pH microsensor (PreSens Precision Sensing China. All birds were handled in accordance with regulations of GmbH). A linear regression was fit to plots of logP50 vs. pH, and the Animal Experimental and Medical Ethics Committee of the the resulting equation was used to correct P50 values to pH 7.4. Institute of Zoology, Chinese Academy of Sciences. Voucher specimens were cataloged in the ornithological collection of the Vector Construction and Site-Directed Mutagenesis. The synthesized αA βA National Zoological Museum, Institute of Zoology, Chinese - globin gene cassette was cloned into a custom pGM vector Academy of Sciences. system (Promega) along with the methionine aminopeptidase (MAP) gene, as described previously (25, 53). We engineered each of the Sample Preservation. We collected pectoral muscle from each bird α-andβ-chain codon substitutions via whole-plasmid amplification specimen and preserved subsamples in RNAhold (TransGen) and using mutagenic primers and Phusion High-Fidelity DNA Poly- 100% ethanol as sources of RNA and genomic DNA, re- merase, phosphorylation with T4 Polynucleotide Kinase, and cir- spectively. For a subset of specimens, we also collected 20–100 μL cularization with an NEB Quick Ligation Kit (New England of whole blood from the brachial or ulnar vein using heparinized BioLabs). Each engineered codon change was verified by DNA microcapillary tubes. Red blood cells were separated from the sequencing. plasma fraction by centrifugation, and the packed red cells were subsequently stored at −80 C° before protein purification. Expression and Purification of Recombinant Hbs. Recombinant Hbs were expressed in JM109 (DE3) E. coli host cells. Bacterial cells PCR, Cloning, and Sequencing. For each of the 162 bird specimens, were selected in LB agar with dual antibiotics (ampicillin and including those used as subjects in the experimental analyses of kanamycin) to ensure that transformants received both pGM and Hb function, we extracted DNA from pectoral muscle tissue using pCO-MAP plasmids for expression. The expression of each rHb the DNeasy Tissue kit (Qiagen). We annotated globin genes in mutant was carried out in 1.5 L of TB medium (Research Prod- the genome assembly of Parus humilis (56) and designed paralog- ucts International). Bacterial cells were grown at 37 °C in an or- specific primers using 5′ and 3′ UTR sequences. We then PCR- bital shaker at 200 rpm until absorbance values reached 0.6–0.8 at amplified the αA-, αD-, and βA-globin genes using KD-Plus DNA 600 nm. The bacterial cultures were induced by 0.2 mM isopropyl polymerase (TransGen). To isolate RNA from muscle tissue, we β-D-1-thiogalactopyranoside (IPTG) and were then supplemented used the Mag-BIND Total RNA kit (Omega), and we then with hemin (50 μg/mL) and glucose (20 g/L). The bacterial culture amplified full-length cDNAs of the αA-, αD-, and βA-globin genes conditions and the protocol for preparing cell lysates are described using a One-Step RT-PCR SuperMix kit (TransGen). Following in Natarajan et al. (53). gel purification, we cloned PCR and RT-PCR products using the We purified each rHb sample by means of two-step ion-exchange Blunt Simple Cloning kit (TransGen). For each species, we se- chromatography as described previously (15–17, 21, 25, 26, 29, 53, quenced full-length coding regions or cDNAs of at least three to 60). We then passed the samples through an anion-exchange column five individual specimens, and we sequenced four to six clones per (HiTrap Q-XL, 17–5159-01; GE Healthcare) equilibrated with gene to recover both alleles. This protocol enabled us to de- 20 mM Tris·HCl buffer (0.5 mM EDTA, pH 8.0) and eluted using termine diploid genotypes for each of the three adult-expressed a linear gradient of 0–0.5 M NaCl. We then passed the eluted globin genes in each individual bird. fractions through a cation-exchange column (HiTrap SP-XL, 17– 5161-01; GE Healthcare) equilibrated with 20 mM Hepes buffer Protein Purification and in Vitro Analysis of Hb Function. We purified (0.5 mM EDTA, pH 8.0) followed by elution using a linear gradient HbA and HbD from hemolysates of wild-caught birds by means of of 0–0.5 M NaCl. We desalted the samples by means of dialysis anion-exchange FPLC using an Äkta Pure system and a HiTrap against 10 mM Hepes buffer (pH 7.4) at 4 °C, and we concentrated Q HP column (GE Healthcare). This procedure removes en- the eluted fractions of each rHb sample by means of centrifugal dogenous organic phosphates, yielding stripped Hb samples. filtration. We confirmed the integrity of purified rHb samples using Using purified Hb solutions (0.3 mM heme), we measured O2 SDS-PAGE and IEF. After preparing rHb samples as oxygenated equilibrium curves at 37 °C in 0.1 M Hepes buffer (pH 7.4) in the Hb, deoxygenated Hb, and carbonmonoxy derivatives, we mea- absence (stripped) and presence of 0.1 M KCl and IHP (at sured absorbance at 450–600 nm to confirm that the absorbance twofold molar excess over tetrameric Hb) and in the simulta- maxima matched those of the native HbA samples. neous presence of KCl and IHP. For the analyses of purified native Hbs, we measured O2 equilibria of 3-μL thin-film samples Phylogeny Estimation. We aligned the full globin gene sequences in a modified diffusion chamber where absorption at 436 nm was (exons + introns) using MUSCLE version 3.5 (61) and verified monitored during stepwise changes in the equilibration of N2/O2 that exon length was conserved. In all cases we estimated max- mixtures generated by precision Wösthoff gas-mixing pumps imum likelihood (ML) trees in MEGA version 7.0 (62) using a +Γ (57–59). We estimated values of P50 and n50 by fitting the Hill GTR model of nucleotide substitution with five rate classes, n n n equation Y = PO2 /(P50 + PO2 ) to the experimental O2 satu- and we evaluated support with 1,000 bootstrap pseudoreplicates. ration data by means of nonlinear regression (Y = fractional O2 We estimated Bayesian trees in MrBayes version 3.2 (63) using saturation; n, cooperativity coefficient). The nonlinear fitting default priors, running four simultaneous chains for 2 × 107 − was based on five to eight equilibration steps. Free Cl con- generations and sampling trees every 1,000 generations. We centrations were measured with a model 926S Mark II chloride assessed convergence by measuring the SD of the split frequency analyzer (Sherwood Scientific Ltd). For the analysis of purified among parallel chains, using a split frequency of <0.01 as the rHbs representing AncParidae and the wild-type genotypes of criterion for convergence. We discarded trees collected before Lophophanes dichrous and Parus humilis, we used a similar chains converged.

Zhu et al. www.pnas.org/cgi/content/short/1720487115 1of6 Ancestral Sequence Reconstruction. We reconstructed the αA- and version 4.8 (64). The AncParidae sequences were estimated with βA-globin sequences of the most recent common ancestor of the high levels of statistical confidence. In the reconstructed αA- family Paridae, using Aegithalos as an outgroup. For the full set globin sequence, only one amino acid state was estimated with a αA βA of parid species, orthologs of both - and -globin form posterior probability <0.98 (at αA115 Ser and Ala had posterior monophyletic groups, so it is possible to designate a single an- probabilities of 0.583 and 0.417, respectively), but this site was not cestral node (AncParidae) that is common to both gene trees and the species tree. Using pruned trees with no redundant sequence variable within the set of focal species that were used in the A A βA haplotypes, we estimated α - and β -globin amino acid se- functional experiments. In the reconstructed -globin sequence, quences of AncParidae using the ML approach implemented all amino acid states were estimated with a posterior proba- in Phylogenetic Analysis by Maximum Likelihood (PAML) bility of 1.00.

Elevational Range (m) 0 1000 2000 3000 4000 5000 6000

Periparus ater aemodius

Periparus ater pekinensis

Periparus rubidiventris

Pardaliparus venustulus

Poecile palustris

Poecile davidi

Poecile montanus Paridae Lophophanes dichrous

Parus minor

Parus monticulus

Parus spilonotus

Parus humilis

Cyanistes cyanus

Sylviparus modestus

Aegithalos bonvaloti Aegithalidae Aegithalos fulginosus

Fig. S1. Phylogenetic relationships and elevational distributions (ranges and midpoints) of 14 Asian passerines in the family Paridae and two in the family Aegithalidae. The tree topology is based on data from Johansson et al. (65) and Li et al. (66).

Zhu et al. www.pnas.org/cgi/content/short/1720487115 2of6 (A) αA (B) αD (C) βA Pe. ater + Pe. ater + 84 Pe. rubidiventris Pe. rubidiventris 1.00 Pe. ater + 68 Pa. humilis Pe. rubidiventris Pa. venustulus 0.94 97 Po. montanus 0.95 Pa. humilis Po. palustris

Pa. monticolus Pa. spilonotus Lo. dichrous Pa. minor Pa. spilonotus Po. montanus

Po. montanus 75 94 0.92 Pa. minor + Po. davidi 1.00 Pa. monticolus Po. palustris + Po. davidi Po. palustris Aegithalos 0.99 Po. palustris Po. palustris Pa. humilis Lo. dichrous + Po. davidi Pa. spilonotus Pa. venustulus 99 97 1.00 Cy. cyanus 1.00 0.86 Pa. monticolus 96 Pa. minor Pa. venustulus 81 1.00 1.00 100 Pa. minor 1.00 Cy. cyanus Po. montanus 97 100 Cy. cyanus 1.00 Pa. spilonotus 1.00 93 Lo. dichrous 100 1.00 + Po. davidi 100 1.00 Sy. modestus 1.00 Sy. modestus 100 100 1.00 Sy. modestus 100 0.99 Aegithalos 1.00 Aegithalos 0.01 0.01 0.01

Fig. S2. Phylogenies of αA-globin (A), αD-globin (B), and βA-globin (C) genes exhibit high levels of genealogical discordance. Species names are color-coded according to the four clades depicted in the species tree (Fig. S1).

A) αA-globin

AncParidae Parus humilis Lophophanes dichrous

AncParidae Parus humilis Lophophanes dichrous

B) βA-globin

AncParidae Parus humilis Lophophanes dichrous

AncParidae Parus humilis Lophophanes dichrous

Fig. S3. Wild-type αA-globin (A) and βA-globin (B) sequences of Parus humilis, Lophophanes dichrous, and their reconstructed common ancestor, AncParidae. Posterior probabilities for ancestral amino acid states averaged 0.997 across 141 α-chain sites and 1.000 across 146 β-chain sites.

Zhu et al. www.pnas.org/cgi/content/short/1720487115 3of6 Fig. S4. Phylogeny of parid and aegithalid species (with color-coded species names), along with a diverse set of representative species from other passerine families. The nucleotide states to the right of the branch tips show recurrent nonsynonymous substitutions at the first and second codon positions of codon 34 in the αA-globin gene. Parallel changes at the first codon position are attributable to substitutions at a CpG dinucleotide, CpG→CpA, which results in the replacement of Ala with Thr at αA34. The third position of the codon immediately preceding αA34 is cytosine, C, in all but two species (Catamenia analis and Acanthisitta chloris), clearly indicating that the CpG dinucleotide represents the ancestral state for all oscine and suboscine passerines (i.e., the entire clade sister to Acanthisitta chloris). Thus, in the set of species shown here, the CpG dinucleotide has been eliminated multiple times independently, either by synonymous C→T substitutions at nucleotide site 102 (third position of the codon specifying residue 33) or nonsynonymous G→A substitutions at nucleotide site 103 (first position of the codon specifying residue 34). The high rate of parallelism at these two nucleotide sites is consistent with the high mutability of CpG sites relative to non-CpG sites.

Zhu et al. www.pnas.org/cgi/content/short/1720487115 4of6 Table S1. Relative mean percentage concentration of the minor HbD isoform in the red blood cells of birds from the Qinghai- Tibet Plateau (H) and lowlands of eastern China (L) Species N Mean % HbD (range)

Periparus rubidiventris (H) 2 25.0 (23.4–26.5) Periparus ater aemodius (H) 2 29.5 (26.9–31.9) Periparus ater pekinensis (L) 2 29.7 (27.6–31.6) Lophophanes dichrous (H) 5 28.2 (22.0–30.7) Poecile palustris (L) 3 30.6 (28.6–33.3) Parus humilis (H) 5 23.1 (21.1–26.3) Parus minor (L) 2 24.6 (23.9–25.2)

Table S2. O2 affinities (P50, torr), cooperativity coefficients (n50), and anion sensitivities (Δlog-P50[(KCl + IHP) stripped]) of purified HbA and HbD isoforms from birds from the Qinghai-Tibetan Plateau (H) and lowland regions of eastern China (L) Stripped KCl + IHP

Taxon Hb isoform P50 n50 P50 n50 Δlog-P50[(KCl + IHP) stripped]

Parus humilis (H) HbA 1.73 ± 0.04 1.53 ± 0.06 21.37 ± 0.48 1.98 ± 0.10 1.09 HbD 1.68 ± 0.06 1.57 ± 0.10 16.50 ± 0.38 2.23 ± 0.12 0.99 Parus minor (L) HbA 4.26 ± 0.08 1.96 ± 0.09 42.37 ± 0.40 1.92 ± 0.04 1.00 HbD 3.79 ± 0.06 1.81 ± 0.07 26.14 ± 0.10 3.23 ± 0.04 0.84 Periparus rubidiventris (H) HbA 3.69 ± 0.07 2.03 ± 0.07 34.27 ± 0.84 2.21 ± 0.13 0.97 HbD 2.46 ± 0.02 1.98 ± 0.04 18.03 ± 0.20 2.40 ± 0.07 0.87 Periparus ater aemodius (H) HbA 3.82 ± 0.05 1.95 ± 0.05 37.25 ± 0.92 2.42 ± 0.13 0.99 HbD 2.67 ± 0.03 2.05 ± 0.06 22.56 ± 0.29 3.04 ± 0.12 0.93 Periparus ater pekinensis (L) HbA 3.64 ± 0.06 1.85 ± 0.06 34.64 ± 0.58 1.81 ± 0.06 0.98 HbD 2.67 ± 0.02 1.81 ± 0.03 23.97 ± 0.41 2.38 ± 0.09 0.95 Lophophanes dichrous (H) HbA 4.06 ± 0.05 2.00 ± 0.06 41.08 ± 0.86 2.39 ± 0.13 1.01 HbD 2.38 ± 0.05 1.84 ± 0.09 22.64 ± 0.13 2.52 ± 0.04 0.98 Poecile palustris (L) HbA 3.88 ± 0.03 1.82 ± 0.03 44.72 ± 0.86 2.04 ± 0.09 1.06 HbD 2.71 ± 0.07 1.93 ± 0.11 25.46 ± 0.25 2.04 ± 0.05 0.97 Aegithalos bonvaloti (H) HbA* 4.28 ± 0.04 1.56 ± 0.04 27.66 ± 0.48 1.77 ± 0.05 0.81 Aegithalos fulginosus (L) HbA* 5.15 ± 0.13 1.37 ± 0.06 34.29 ± 0.64 1.64 ± 0.06 0.82

− O2 equilibria were measured in 0.1 mM Hepes buffer at pH 7.4 (± 0.01) and 37 °C in the absence (stripped) and presence of Cl ions (0.1 M KCl]) and IHP (at twofold molar excess over tetrameric Hb). Anion sensitivities are indexed by the difference in log-transformed − values of P50 in the presence and absence of anions (Cl and IHP). The higher the Δlog P50 value, the higher the anion sensitivity of Hb– O2 affinity. *Recombinantly expressed HbA isoforms.

Zhu et al. www.pnas.org/cgi/content/short/1720487115 5of6 Table S3. Predicted hydrogen bonds in the intradimer α1β1/α1β1 interface in deoxygenated (T) and oxygenated (R) states of avian Hb  Conformation αA34 amino acid state Hydrogen bonds Distance, Å

Deoxy (T) Ala Arg 31 [NH1] : Gln 127 [OE1] 2.66 Arg 31 [NH1] : Phe 122 [O] 3.38 Arg 31 [NH2] : Phe 122 [O] 3.36 Gln 103 [OE1] : Gln 131 [NE2] 2.86 Thr 111 [OG1]: Lys 120 [N] 3.52 Leu 117 [O] : Arg 30 [NH2] 2.70 Asp 126 [OD2] : Tyr 35 [OH] 2.86

Thr Gln-103 [NE2] : Asp-108 [OD1] 2.98 Arg-31 [NH2] : Phe-122 [O] 2.66 Thr-34 [OG1] : Pro-124 [O] 3.59 Arg-31 [NE] : Gln-127 [OE1] 2.75 Leu-117 [O] : Arg-30 [NH2] 2.82 Asp-126 [OD2] : Tyr-35 [OH] 3.36 Thr-111 [OG1] : Lys-120 [N] 3.52

Oxy (R) Ala Arg-31 [NH1] : Phe-122 [O] 3.05 Leu-117 [O] : Arg-30 [NH2] 3.71 Asp-126 [OD2] : Tyr-35 [OH] 3.16 Tyr 36 [OH] : Gln 131 [NE2] 3.60

Thr Arg-31 [NE ] : Gln-127 [OE1] 2.66 Arg-31 [NH2] : Phe-122 [O] 2.76 Gln-103 [NE2] : Asp-108 [O] 3.52 Thr-111 [OG1] : Gly-119 [O] 2.66 Gln-103 [OE1] : Gln-127 [NE2] 3.77 Thr-111 [OG1] : Lys-120 [N] 3.69 Leu-117 [O] : Arg-30 [NH1] 3.54 Leu-117 [O] : Arg-30 [NH2] 3.25

Results are shown for two alternative Hb variants, those with the ancestral Ala αA34 and those with the derived Thr αA34, as in Parus humilis and Lophophanes dichrous.

Zhu et al. www.pnas.org/cgi/content/short/1720487115 6of6