Article

Gene Turnover in the Avian Globin Gene Families andEvolutionary Changes in Hemoglobin Isoform ExpressionJuan C Opazoy1 Federico G Hoffmanny23 Chandrasekhar Natarajan4 Christopher C Witt56

Michael Berenbrink7 and Jay F Storz4

1Instituto de Ciencias Ambientales y Evolutivas Facultad de Ciencias Universidad Austral de Chile Valdivia Chile2Department of Biochemistry Molecular Biology Entomology and Plant Pathology Mississippi State University3Institute for Genomics Biocomputing and Biotechnology Mississippi State University4School of Biological Sciences University of Nebraska Lincoln5Department of Biology University of New Mexico6Museum of Southwestern Biology University of New Mexico7Institute of Integrative Biology University of Liverpool Liverpool United KingdomyThese authors contributed equally to this work

Corresponding author E-mail jstorz2unledu

Associate editor James McInerney

Abstract

The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low ratesof gene gain and loss in multigene families To investigate this possibility and to explore the phenotypic consequences ofvariation in gene copy number we examined evolutionary changes in the families of genes that encode the a- and b-typesubunits of hemoglobin (Hb) the tetrameric a2b2 protein responsible for blood-O2 transport A comparative genomicanalysis of 52 bird species revealed that the size and membership composition of the a- and b-globin gene families haveremained remarkably constant during approximately 100 My of avian evolution Most interspecific variation in genecontent is attributable to multiple independent inactivations of the aD-globin gene which encodes the a-chain subunit ofa functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes Due to con-sistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform HbA (whichincorporates products of the aA-globin gene) recurrent losses of aD-globin contribute to among-species variation inblood-O2 affinity Analysis of HbAHbD expression levels in the red blood cells of 122 bird species revealed high variabilityamong lineages and strong phylogenetic signal In comparison with the homologous gene clusters in mammals the lowretention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmentalregulation of Hb synthesis in birds may be more highly conserved with orthologous genes having similar stage-specificexpression profiles and similar functional properties in disparate taxa

Key words avian genomics gene deletion gene duplication gene family evolution hemoglobin protein evolution

IntroductionRelative to the genomes of mammals avian genomes arecharacterized by lower rates of chromosomal evolution andlower levels of structural variation within and among species(Burt et al 1999 Shetty et al 1999 Derjusheva et al 2004Griffin et al 2007 2008 Backstrom et al 2008 Ellegren 20102013 Zhang et al 2014) At the nucleotide sequence levelbirds have lower substitution rates relative to mammals andsquamate reptiles but they have somewhat higher rates thancrocodilians (the sister group to birds representing the onlyother extant lineage of archosaurs) and turtles the sistergroup to archosaurs (Mindell et al 1996 Nam et al 2010Shaffer et al 2013 Green et al 2014) The high level ofconserved synteny among avian genomes suggests thatbirds may have experienced relatively low rates of gene turn-over in multigene families This hypothesis can be tested bycharacterizing rates and patterns of gene turnover in well-characterized multigene familiesmdashsuch as the globin gene

superfamilymdashthat share a high degree of conserved syntenyacross all amniote lineages

The a- and b-globin genes encode subunits of the tetra-meric a2b2 hemoglobin (Hb) protein which is responsible forblood-O2 transport in the red blood cells of jawed vertebrates(gnathostomes) The a- and b-globin genes were tandemlylinked in the last common ancestor of gnathostomesa chromosomal arrangement that has been retained insome lineages of ray-finned fishes lobe-finned fishes andamphibians (Jeffreys et al 1980 Hosbach et al 1983Gillemans et al 2003 Fuchs et al 2006 Opazo et al 2013Schwarze and Burmester 2013) In amniote vertebrates thea- and b-globin genes are located on different chromosomesdue to transposition of the proto b-globin gene(s) to a newchromosomal location after the ancestor of amniotes di-verged from amphibians (Hardison 2008 Patel et al 2008Hoffmann Storz et al 2010 Hoffmann Opazo Storz et al2012)

The Author 2014 Published by Oxford University Press on behalf of the Society for Molecular Biology and EvolutionThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License(httpcreativecommonsorglicensesby-nc40) which permits non-commercial re-use distribution and reproduction in anymedium provided the original work is properly cited For commercial re-use please contact journalspermissionsoupcom Open AccessMol Biol Evol 32(4)871ndash887 doi101093molbevmsu341 Advance Access publication December 9 2014 871

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Because the vertebrate Hb genes comprise one of the mostintensively studied multigene families from a functionalstandpoint (Hardison 2001 2012 Hoffmann Storz et al2010 Storz Opazo et al 2011 Hoffmann Opazo Storz2012 Storz et al 2013 Burmester and Hankeln 2014) theexamination of globin gene family evolution provides an op-portunity to assess the phenotypic consequences of changesin gene content Specifically changes in gene copy numberand divergence between paralogs can promote Hb isoformdifferentiation which can result in physiologically importantmodifications of blood-O2 transport and aerobic energy me-tabolism Changes in the size and membership compositionof the a- and b-globin gene families may produce changes inthe developmental regulation of Hb synthesis and may there-fore constrain or potentiate functional divergence betweenHb isoforms that incorporate the products of paralogousglobin genes (Berenbrink et al 2005 Berenbrink 2007Opazo et al 2008a 2008b Runck et al 2009 HoffmannStorz et al 2010 Storz Hoffmann et al 2011 Storz Opazoet al 2011 Grispo et al 2012 Damsgaard et al 2013 Opazoet al 2013 Storz et al 2013) Indeed over a deeper timescaleof animal evolution the co-option and functional divergenceof more ancient members of the globin gene superfamily haveplayed a well-documented role in evolutionary innovation(Hoffmann Opazo et al 2010 Blank et al 2011 HoffmannOpazo Hoogewijs et al 2012 Hoffmann Opazo Storz 2012Hoogewijs et al 2012 Schwarze and Burmester 2013Schwarze et al 2014)

All gnathostome taxa that have been examined to dateexpress structurally and functionally distinct Hb isoformsduring different stages of prenatal development (Hardison2001) and some tetrapod groups coexpress different Hb iso-forms during postnatal life The majority of birds and non-avian reptiles coexpress two functionally distinct Hb isoformsin definitive erythrocytes HbA (with a-chain subunitsencoded by the aA-globin gene) and HbD (with a-chainsubunits encoded by the aD-globin gene fig 1) In adultbirds HbA and HbD represent major and minor isoformsrespectively where HbD typically accounts for 10ndash40 oftotal Hb (Grispo et al 2012)

Most bird species have retained each of three tandemlylinked a-type globin genes that were present in the lastcommon ancestor of tetrapod vertebrates 50-aE-aD-aA-30

(Hoffmann and Storz 2007 Hoffmann Storz et al 2010Grispo et al 2012) The proto aE- and aA-globin genes orig-inated via tandem duplication of an ancestral proto a-globingene and aD-globin originated subsequently via duplicationof the proto aE-globin gene (Hoffmann and Storz 2007Hoffmann Storz et al 2010) Both duplication events oc-curred in the stem lineage of tetrapods after divergencefrom the ancestor of lobe-finned fishes approximately 370ndash430 Ma (Hoffmann and Storz 2007) In all tetrapods that havebeen examined to date the linkage order of the three a-typeglobin genes reflects their temporal order of expressionduring development aE-globin is exclusively expressed inlarvalembryonic erythroid cells derived from the yolk sacaD-globin is expressed in both primitive and definitive ery-throid cells and aA-globin is expressed in definitive erythroid

cells during later stages of prenatal development and postna-tal life (Cirotto et al 1987 Ikehara et al 1997 Alev et al 20082009 Storz Hoffmann et al 2011)

Most variation in the size of the avian a-globin gene familyis attributable to multiple independent deletions or inactiva-tions of the aD-globin gene (Zhang et al 2014) Due to con-sistent differences in O2-binding properties between the HbAand HbD isoforms (Grispo et al 2012) losses of the aD-globingene likely contribute to among-species variation in blood-O2

affinity which has important physiological consequences Inall bird species that have been examined to date HbD ischaracterized by a higher O2-binding affinity relative toHbA (Grispo et al 2012) and it also exhibits other uniqueproperties such as a propensity to self-associate upon deox-ygenation (Cobb et al 1992 Knapp et al 1999 Rana and Riggs2011) which may enhance the cooperativity of O2 binding(Riggs 1998)

Comparative studies of endothermic vertebrates have doc-umented a positive scaling relationship between body massand blood-O2 affinity (Schmidt-Nielsen and Larimer 1958Lutz et al 1974 Bunn 1980) although the pattern is not asclear in birds as it is in mammals (Baumann and Baumann1977 Lutz 1980) Available data suggest that in general thehigher the mass-specific metabolic rate of an animal thehigher the partial pressure of O2 (PO2) at which the bloodreleases O2 to the cells of respiring tissues This high unloadingtension preserves a steep O2 diffusion gradient between cap-illary blood and perfused tissue thereby enhancing O2 flux tothe tissue mitochondria Given that relative HbAHbD expres-sion levels should be an important determinant of blood-O2

affinity it is of interest to assess whether such expressionlevels are phylogenetically correlated with body mass Oneclear prediction is that avian taxa with especially high mass-specific metabolic rates like hummingbirds and passerines

FIG 1 Postnatally expressed Hb isoforms in avian red blood cells Themajor isoform HbA (aA

2b2) has a-type subunits encoded by the aA-globin gene and the minor isoform HbD (aD

2b2) has a-type subunitsencoded by the aD-globin gene Both isoforms share identical b-typesubunits encoded by the bA-globin gene The remaining members of thea- and b-globin gene families (aE- r- bH- and E-globin) are not ex-pressed at appreciable levels in the definitive erythrocytes of adult birdsWithin each gene cluster the intergenic spacing is not drawn to scale

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have evolved a reduced HbD expression level which (all elsebeing equal) would produce a reduced blood-O2 affinity

The main objectives of the present study were to assess thephenotypic consequences of changes in gene copy numberand to assess relationships between rates of gene retentionexpression levels and functional constraint The specific aimswere 1) to characterize patterns of gene turnover and inter-paralog gene conversion in the a- and b-globin gene clustersof birds 2) to estimate the number of independent inactiva-tionsdeletions of the aD-globin gene during the diversifica-tion of birds 3) to characterize among-species variation in therelative expression of HbA and HbD isoforms 4) to recon-struct evolutionary changes in HbAHbD expression levelsduring the diversification of birds and 5) to characterize var-iation in functional constraint among paralogous globingenes To achieve these aims we conducted a comparativegenomic analysis of the avian a- and b-globin gene clusters in52 species (supplementary table S1 Supplementary Materialonline) building on our previous analysis of high-coveragegenome assemblies for 22 species (Zhang et al 2014) Weconducted molecular evolution analyses based on globin se-quences from a phylogenetically diverse set of 83 bird speciesincluding 50 newly generated sequences from species forwhich full genome sequences were not available Finallyusing experimental measures of protein expression for 122bird species we conducted a phylogenetic comparative anal-ysis to reconstruct evolutionary changes in HbAHbD isoformabundance

Results and Discussion

Genomic Structure of the Avian a- and b-GlobinGene Families

To characterize the genomic structure of the avian globingene clusters we analyzed contigs from a set of 24 bird speciesthat contained the full complement of a- and b-type globingenes Our comparative genomic analysis of these 24 speciesrevealed that the size and membership composition of theglobin gene families have remained remarkably constant asmost bird species have retained an identical complement ofthree tandemly linked a-type globin genes (50-aE-aD-aA-30)and four tandemly linked b-type globin genes (50-r-bH-bA-E-30 fig 2) In eutherian mammals in contrast the a- andb-globin gene clusters have experienced high rates of geneturnover due to lineage-specific duplication and deletionevents resulting in functionally significant variation inglobin gene repertoires among contemporary species(Hoffmann et al 2008a 2008b Opazo et al 2008a Opazoet al 2009 Gaudry et al 2014) In rodents alone thenumber of functional a-type globin genes ranges from 2 to8 (Hoffmann et al 2008b Storz et al 2008) and the number offunctional b-type globin genes ranges from 3 to 7 (Hoffmannet al 2008a) To quantify this apparent difference in thetempo of gene family evolution in birds and mammals weused a stochastic birthndashdeath model to estimate rates of geneturnover () in the globin gene clusters of 24 bird species and22 species of eutherian mammals that span a similar range ofdivergence times Results of this analysis revealed that the rate

of gene turnover in the avian globin gene clusters is roughlytwo times lower than in eutherian mammals (= 00013 vs00023) Available data suggest that the a- and b-globin geneclusters of squamate reptiles have also undergone a high rateof gene turnover relative to homologous gene clusters in birds(Hoffmann Storz et al 2010 Storz Hoffmann et al 2011)

Inference of Orthologous Relationships and Detectionof Interparalog Gene Conversion

To reconstruct phylogenetic relationships and to examinepatterns of molecular evolution we analyzed all available a-and b-type globin sequences from a phylogenetically diverseset of 83 bird species Phylogeny reconstructions based oncoding sequence confirmed that the paralogous a-typeglobin genes grouped into three well-supported clades andclearly identifiable orthologs of each gene are present in otheramniote taxa (fig 3) Similarly the phylogeny reconstructionofb-type genes recovered four clades representing ther-bH-bA- and E-globin paralogs (fig 4) The four avian b-type para-logs are reciprocally monophyletic relative to the b-typeglobins of other amniotes indicating that they representthe products of at least three successive rounds of tandemduplication in the stem lineage of birds The phylogeny re-vealed several cases of interparalog gene conversion betweenthe embryonic r- and E-globin genes as documented previ-ously in analyses of the chicken and zebra finch b-globin geneclusters (Reitman et al 1993 Hoffmann Storz et al 2010)A history of interparalog gene conversion between r- andE-globin is evident in several cases where sequences fromgenes that are positional homologs of r-globin are nestedwithin the clade of E-globin sequences and vice versa(fig 4) For example r- and E-globin coding sequences ofdowny woodpecker (Picoides pubescens) were placed sisterto one another in what is otherwise a clade of r-globinsequences (fig 4) indicating that the E-globin gene of thisspecies has been completely overwritten by rE gene con-version Additional examples of interparalog gene conversionbetween the r- and E-globin genes are evident in phylogenyreconstructions with more extensive taxon sampling (supple-mentary fig S1 Supplementary Material online)

In both chicken and zebra finch the bH-globin gene isexpressed at scarcely detectable levels in definitive erythro-cytes (Alev et al 2008 2009) But whereas the bH-globin geneis quiescent in the primitive erythrocytes of the developingchick embryo (Alev et al 2008) it is highly expressed duringthe analogous stage of embryogenesis in zebra finch (Alevet al 2009) Analyses of chicken b-type genes by Reitmanet al (1993) suggested that the two embryonic genes(r and E) form a reciprocally monophyletic group relativeto bH and bA although recent analyses based on more ex-tensive sequence data recovered the topology (bH(bA(r E)))(Alev et al 2009 Hoffmann Storz et al 2010) Results of theBayesian analysis shown in figure 4 are consistent with thesemore recent analyses although posterior probabilities for keynodes varied markedly depending on which alignments wereused Maximum-likelihood analyses did not provide betterresolution as bootstrap support values for key nodes were

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FIG 2 Genomic structure of the a- and b-globin gene clusters in 24 bird species The phylogeny depicts a time-calibrated supertree (see Materials andMethods for details) The 24 species represented in the figure are those for which we have accounted for the full repertoire of a- and b-type globingenes Truncated genes may have been produced by partial deletion events but in some cases the apparently truncated coding sequences mayrepresent assembly artifacts

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FIG 3 Bayesian phylogeny depicting relationships among avian a-type globin genes based on nucleotide sequence data Sequences of a-type globingenes from other tetrapod vertebrates (axolotl salamander [Ambystoma mexicanum] western clawed frog [Xenopus tropicalis] anole lizard [Anoliscarolinensis] painted turtle [Chrysemys picta] platypus [Ornithorhynchus anatinus] and human [Homo sapiens]) were used as outgroups Bayesianposterior probabilities are shown for the ancestral nodes of each clade of orthologous avian genes

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FIG 4 Bayesian phylogeny depicting relationships among avian b-type globin genes based on nucleotide sequence data Sequences of b-type globingenes from other tetrapod vertebrates (anole lizard [Anolis carolinensis] Chinese softshell turtle [Pelodiscus sinensis] green sea turtle [Chelonia mydas]and painted turtle [Chrysemys picta]) were used as outgroups Bayesian posterior probabilities are shown for the ancestral nodes of each clade oforthologous avian genes and for select nodes uniting clades of paralogous genes Branch-tip labels with rsquo+rsquo symbols denote genes that have beenpartially overwritten by interparalog gene conversion (see text for details)

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consistently less than 50 Likelihood-based topology testscomparing the two best-supported arrangements ((bHbA)(r E)) and (bH (bA (r E))) were not statistically significant(supplementary table S2 Supplementary Material online)Based on available data we regard the relationship amongbH bA and the two embryonic b-type globins (r E) as anunresolved trichotomy

Gene Turnover in the Avian a- and b-Globin GeneFamilies

The comparative genomic analysis revealed that most varia-tion in the size of the avian globin gene families is attributableto deletions or inactivations of individual genes from the an-cestral repertoire of a- and b-type globins We documentedlineage-specific duplications of the bA-globin gene in severalspecies including downy woodpecker crested ibis (Nipponianippon) and emperor penguin (Aptenodytes forsteri) In bothdowny woodpecker and crested ibis the 30-bA gene copies areclearly pseudogenes (fig 2) We also identified putativelypseudogenized copies of duplicated r- and E-globin genesin several species (fig 2)

We found no evidence for lineage-specific gene gains viatandem duplication in the a-globin gene family althoughanalyses of Hb isoform diversity at the protein level suggestthat duplications of postnatally expressed a-type globin geneshave occurred in at least two species of Old World vulturesGriffon vulture Gyps fulvus (Grispo et al 2012) and Reurouppellrsquosgriffon G rueppellii (Hiebl et al 1988) Most variation in genecontent in the avian b-globin gene family is attributable tooccasional lineage-specific deletions or inactivations of thebH- r- and E-globin genes (fig 2)

In the avian a-globin gene family most interspecific vari-ation in gene content is attributable to independent deletionsor inactivations of the aD-globin gene In the sample of24 species for which we have contigs containing completea-globin gene clusters parsimony suggests that there weremultiple independent inactivations of aD-globin (fig 2)Specifically the aD-globin gene appears to have been deletedor otherwise pseudogenized in golden-collared manakin(Manacus vitellinus) little egret (Egretta gazetta) Adeliepenguin (Pygoscelis adeliae) emperor penguin (A forsteri)killdeer (Charadrius vociferus) and hoatzin (Opisthocomushoazin) Partial deletions of the aD-globin gene in both pen-guin species were likely inherited from a common ancestorconsistent with evidence that penguins do not expressHbD during postnatal life (fig 5 supplementary table S3Supplementary Material online) The aD-globin appears tohave been deleted in killdeer and pseudogenized in hoatzinKilldeer (representing Charadriiformes + Gruiformes) andhoatzin (Opisthocomiformes) are sister taxa in our treewhich is based on the total-evidence phylogenomic time-tree of Jarvis et al (2014) Although this suggests that inacti-vation of aD-globin occurred in the common ancestor ofkilldeer and hoatzin these taxa diverged during a rapidpost-KPg radiation and their phylogenetic positions are stillpoorly resolved even with whole-genome data (Jarvis et al2014) Furthermore the period during which killdeer and

hoatzin shared an exclusive common ancestor would havebeen exceedingly brief supporting the hypothesis of indepen-dent inactivation in each lineage In light of this uncertaintyparsimony suggests either four or five independent losses ofthe aD-globin gene in the set of species included in our com-parative analysis

The repeated inactivations of aD-globin in multipleavian lineages are reflective of a broader macroevolutionarytrend among tetrapods Genomic evidence indicates that theaD-globin gene has been independently inactivated or deletedin amphibians and mammals (Cooper et al 2006 Fuchs et al2006 Hoffmann and Storz 2007 Hoffmann et al 2008bHoffmann Storz et al 2010 Hoffmann et al 2011) Somemammalian lineages have retained an intact transcriptionallyactive copy of aD-globin (Goh et al 2005) but there is noevidence that the product of this seemingly defunct gene isassembled into functionally intact Hbs at any stage of devel-opment The HbD isoform is not expressed in the definitiveerythrocytes of crocodilians (Weber and White 1986 1994Grigg et al 1993 Weber et al 2013) suggesting that theaD-globin gene has been inactivated or deleted in theextant sister group of birds Alternatively the crocodilian aD

ortholog (if present) may only be expressed during embryonicdevelopment in which case the HbD isoform may havesimply gone undetected in studies of red blood cells fromadult animals The fact that the aD-globin gene has beendeleted or inactivated multiple times in birds may simplyreflect the fact that it encodes the a-type subunit of aminor Hb isoform it may therefore be subject to less stringentfunctional constraints than the paralogous aE- and aA-globingenes that encode subunits of major (less dispensable) Hbisoforms that are expressed during embryonic and postnataldevelopment respectively

Evolutionary Changes in HbAHbD Isoform Expression

The assembly of a2b2 Hb tetramers is governed by electro-static interactions between a- and b-chain monomers andbetween ab dimers (Bunn and McDonald 1983 Mrabet et al1986 Bunn 1987) For this reason and also because of possibleinterparalog variation in mRNA stability the relative tran-script abundance of coexpressed a- and b-type globingenes in hematopoietic cells may not accurately predict theproportional representation of the encoded subunit isoformsin functionally intact tetrameric Hbs We therefore directlymeasured the relative abundance of HbA and HbD isoformsin red cell lysates rather than relying on measures of aAaD

transcript abundance as proxy measures of proteinabundance

Our survey of HbAHbD expression levels in 122 avianspecies revealed high variability among lineages (supplemen-tary table S3 Supplementary Material online) HbD ac-counted for approximately 20ndash40 of total Hb in themajority of species but this isoform was altogether absentin all or most representatives of five major avian cladesAequornithia (core waterbirds represented by one speciesof stork two herons two pelicans one cormorant and twopenguins) Columbiformes (represented by six species of

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pigeons and doves) Coraciiformes (represented by one spe-cies of roller) Cuculiformes (represented by three species ofcuckoo and one coucal) and Psittaciformes (representedby seven species of parrot from the families Psittacidaeand Psittaculidae) On average HbD expression was also

quite low in swifts and hummingbirds (Apodiformes) notexceeding 25 of total Hb in any species HbD was expressedat very low levels in several species of hummingbirds(mean 1 SD) 34 12 in the wedge-billed hummingbirdSchistes geoffroyi (n = 7 individuals) 32 07 in the

FIG 5 Inferred evolutionary changes in relative expression level of the HbD isoform ( of total Hb) during the diversification of birds Expression dataare based on experimental measures of protein abundance in the definitive red blood cells of 122 bird species (n = 1ndash30 individuals per species 267specimens in total) Terminal branches are color-coded according to the measured HbD expression level of each species and internal branches arecolor-coded according to maximum-likelihood estimates of ancestral character states Branch lengths are proportional to time See Materials andMethods for details regarding the construction of the time-calibrated supertree

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sparkling violetear Colibri coruscans (n = 7) and 16 20 inthe bronzy Inca Coeligena coeligena (n = 7 supplementarytable S3 Supplementary Material online) In contrast HbDseldom accounted for less than 25 of total Hb in 42 exam-ined species of passerines (supplementary table S3Supplementary Material online)

The relative expression level of HbD exhibited strong phy-logenetic inertia (fig 5) Pagelrsquos lamda (Pagel 1999) whichvaries from zero (no phylogenetic inertia) to 10 (strong phy-logenetic inertia) was 0981 for the percentage of HbD acrossthe avian phylogeny Blombergrsquos K was 0861 (significantlygreater than zero Plt 0001) indicating that there was lesstrait similarity among species than expected for a trait evolv-ing under perfect Brownian motion (K = 1) These results in-dicate that measured HbD expression levels exhibit astatistically significant pattern of phylogenetic conservatismalthough the deviation from expectations under Brownianmotion could be partly attributable to measurement erroror sampling error (Blomberg et al 2003 Garland et al 2005)

Body mass exhibited even stronger phylogenetic inertiathan HbD expression in the same set of taxa (Pagelrsquoslambda = 0999 Blombergrsquos K=380) Regression analysisusing a phylogenetic generalized least-squares model revealedno evidence for an association between body mass and HbDexpression (R2 = 001 P = 062) Thus if blood-O2 affinityscales inversely with mass-specific metabolic rate in birds assuggested by Lutz et al (1974) results of our analysis suggestthat the scaling relationship is not attributable to evolution-ary changes in HbAHbD expression levels Instead any suchrelationship must be attributable to evolutionary changes inthe inherent oxygenation properties of Hb isoforms andorchanges in intraerythrocytic concentrations of cellular cofac-tors that allosterically regulate Hb-O2 affinity

Variation in Functional Constraint among Paralogs

To infer relative levels of functional constraint among the dif-ferent globin paralogs we used a codon-based maximum-like-lihood approach to measure variation in the ratio ofnonsynonymous-to-synonymous substitution rates (= dNdS) For the a-type genes the model that provided the best fitto the data permitted four independent-values one for eachavian paralog and one for all avian outgroup branches (table1) If rates of gene retention and relative expression levels arepositively correlated with the degree of functional constraintthen the clear prediction is that the aD-globin clade will becharacterized by a higher -value than the aA-globin cladeConsistent with expectations the estimated -value for theaD-globin gene was appreciably higher (0281 vs 0237 respec-tively table 1) The exclusively embryonic aE-globin gene wascharacterized by the lowest-value (0127 table 1) In the caseof theb-type globin genes the model that provided the best fitto the data permitted three independent -values for 1) bH

and bA 2) r and E and 3) all nonavian branches (table 2)Similar to the pattern observed for the a-type genes the em-bryonic r- and E-globin genes were characterized by lower -values relative to bH- and bA-globin (0084 vs 0100 table 2)Observed patterns for both the a- and b-type globins are

consistent with the idea that genes expressed early in devel-opment are generally subject to more stringent functionalconstraints relative to later-expressed members of the samegene family (Goodman 1963)

Tests of Positive Selection

Likelihood ratio tests (LRTs) revealed significant variationin -values among sites in the alignments of each of theavian a-type paralogs (table 1) and results of Bayes empiricalBayes (BEB) analyses suggested evidence for a history ofpositive selection at several sites as indicated by site-specific-values greater than 10 (table 1) Statistical evidence forpositive selection at aA64 and aA115 is especially intriguingin light of evidence from site-directed mutagenesis experi-ments that charge-changing mutations at both sites producesignificant changes in the O2 affinity of mouse Hb (Natarajanet al 2013) LRTs also revealed significant variation in-valuesamong sites in the alignment of avian bA-globin sequencesand results of the BEB analysis suggested evidence for a historyof positive selection at two sites b4 and b55 (table 2)Statistical evidence for positive selection at both sites is in-triguing in light of evidence from protein-engineering exper-iments Site-directed mutagenesis experiments involvingmammalian Hbs have documented affinity-altering effectsof substitutions at b4 and the adjacent b5 residue posi-tion that perturb the secondary structure of the b-chain A-helix (Fronticelli et al 1995 Tufts et al 2015) Likewise site-directed mutagenesis experiments involving recombinanthuman Hb have documented affinity-enhancing effects ofsubstitutions at b55 that eliminate intradimer (a1b1)atomic contacts (Jessen et al 1991 Weber et al 1993)Protein-engineering experiments involving recombinantavian Hbs are needed to probe the functional effects ofamino acid substitutions at each of these candidate sites forpositive selection

Physiological Implications

Due to the fact that the HbD isoform (which incorporatesproducts of aD-globin) has a consistently higher O2-affinitythan HbA (which incorporates products of aA-globin [Grispoet al 2012]) repeated inactivations of the aD-globin gene canbe expected to contribute to among-species variation inblood-O2 affinity To evaluate the phenotypic consequencesof aD-globin inactivation in definitive erythrocytes we com-piled standardized data on the oxygenation properties of pu-rified HbA and HbD isoforms from 11 bird species (Grispoet al 2012 Projecto-Garcia et al 2013 Cheviron et al 2014table 3) In all 11 species HbD exhibited higher O2-affinity inthe presence of allosteric modulators of Hb-O2 affinity Cl

ions (added as 01 M KCl) and inositol hexaphosphate (IHP[a chemical analog of inositol pentaphosphate that is endog-enously produced in avian red blood cells] present atsaturating or near-saturating concentrations) We compiledin vitro measurements of O2-affinity on purified HbA andHbD isoforms under the same ldquoKCl+IHPrdquo treatment andwe calculated the expected O2-affinity of the compositemixture of HbA and HbD using a weighted average of

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isoform-specific P50 values (the partial pressure of O2 at whichheme is 50 saturated) based on the naturally occurring rel-ative concentrations of the two Hb isoforms in each speciesThe assumption that the avian HbA and HbD isoforms haveadditive effects on blood-O2 affinity is validated by experi-mental studies on the oxygenation properties of isolated iso-forms and composite hemolystes (Weber et al 2004 Grispo

et al 2012) We calculated the expected increase in blood P50

(ie reduction in blood-O2 affinity) caused by the completeloss of HbD expression by comparing the predicted P50 ofthe HbA+HbD composite mixture and the measured P50 ofHbA in isolation Although the P50 of a compositeldquoHbA+HbDrdquo hemolysate is not expected to be identical tothe P50 of whole blood (Berenbrink 2006) the loss of HbD

Table 1 Maximum-Likelihood Analysis of the Ratio of Nonsynonymous-to-Synonymous Substitution Rates x (=dNdS) in the a-Type GlobinGenes of Birds

Models ln L No Parameters Parameter Estimates Candidate Sites for Positive Selection Test

Site modelsmdashaE-globin

M0 3887294 98 x0 = 0154 NA

M1a 3759181 99 x0 = 0047 (p0 = 084) NAx1 = 1000 (p1 = 016)

M2 3758104 101 x0 = 0047 (p0 = 084) 53 A (x = 1901 P = 0894) M2 vs M1ax1 = 1000 (p1 = 015) Not Significantx2 = 2107 (p2 = 001)

M3 3737840 102 x0 = 0016 (p0 = 071) M3 vs M0x1 = 0325 (p1 = 024) P ltlt 103

x2 = 1387 (p2 = 005)

Site modelsmdashaD-globin

M0 5666162 96 x0 = 0236 NA

M1a 5377678 97 x0 = 0080 (p0 = 066) NAx1 = 1000 (p1 = 034)

M2 5358625 99 x0 = 0083 (p0 = 064) 12 P (x = 2393 P = 0576) M2 vs M1ax1 = 1000 (p1 = 030) 17 G (x = 2987 P = 0788) P ltlt 103

x2 = 3253 (p2 = 006) 23 L (x = 3236 P = 0880)29 D (x = 3585 P = 1000)48 H (x = 3580 P = 0998)49 P (x = 3569 P = 0994)63 A (x = 2581 P = 0639)

M3 5320501 100 x0 = 0016 (p0 = 044) M3 vs M0x1 = 0292 (p1 = 040) P ltlt 103

x2 = 1306 (p2 = 016)

Site modelsmdashaA-globin

M0 5166185 112 x0 = 0214 NA

M1a 4867244 113 x0 = 0050 (p0 = 074) NAx1 = 1000 (p1 = 026)

M2 4837546 115 x0 = 0049 (p0 = 073) 5 N (x = 4457 P = 0872) M2 vs M1ax1 = 1000 (p1 = 024) 49 H (x = 5271 P = 0788) P ltlt 103

x2 = 4862 (p2 = 003) 64 A (x = 4911 P = 0880)115 S (x = 5263 P = 1000)

M3 4830789 116 x0 = 0021 (p0 = 065) M3 vs M0x1 = 0374 (p1 = 021) P ltlt 103

x2 = 1216 (p2 = 014)

Branch models

1x 19867046 341 x_all branches = 0200 NA

2x 19861435 342 x_a-type genes = 0214 NA 1x vs 2xx_other branches = 0157 P ltlt 103

4x 19831984 344 x_aE total group = 0127 NA 2x vs 4xx_aD total group = 0281 P ltlt 103

x_aA total group = 0237x_other branches = 0158

7x 19831607 347 x_aE stem = 0097 NA 4x vs 7xx_aE crown = 0128 Not Significantx_aD stem = 0483x_aD crown = 0280x_aA stem = 0152x_aA crown = 0239x_other branches = 0159

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expression should produce a commensurate increase in P50 inboth cases

In hummingbirds which typically have very low HbD ex-pression (table 2 and fig 5 supplementary table S3Supplementary Material online) loss of HbD is predicted toproduce a modest increase in blood P50 ranging from +13 for the violet-throated starfrontlet Coeligena violifer to +86for the giant hummingbird Patagona gigas In contrast inpasserines (which typically have much higher HbD expressionlevels table 2 and fig 5 supplementary table S3Supplementary Material online) the predicted increase inblood P50 was +154 for house wren Troglodytes aedonand +180 for rufous-collared sparrow Zonotrichia capensisThe calculations for passerines provide an indication of themagnitude of change in blood-O2 affinity that must haveaccompanied the quantitative reduction or whole-sale lossof HbD expression in other avian lineages (fig 5) These results

illustrate the potentially dramatic physiological consequencesof inactivating the aD-globin gene Because HbD is also ex-pressed at high levels during prenatal development (Cirottoet al 1987 Alev et al 2008 2009) inactivation or deletion ofaD-globin could affect O2-delivery to the developing embryoIntriguingly representatives of several taxa (families Cuculidae[cuckoos] and Columbidae [pigeons and doves] and the su-perfamily Psittacoidea [parrots]) do not appear to expressHbD during postnatal life (fig 5 supplementary table S3Supplementary Material online) but they have retained aD-globin genes with intact reading frames In such taxa HbDmay still be expressed during embryonic development asdocumented in domestic pigeon (Ikehara et al 1997)

ConclusionsOur results indicate that recurrent deletions and inactivationsof the aD-globin gene entailed significant reductions in blood-

Table 2 Maximum-Likelihood Analysis of the Ratio of Nonsynonymous-to-Synonymous Substitution Rates x (=dNdS) in the b-Type GlobinGenes of Birds

Models ln L No Parameters Parameter Estimates Candidate Sites for Positive Selection Test

Site models

M0 13679145 313 x0 = 0102 NA

M1a 13223916 314 x0 = 0070 (p0 = 0905) NAx1 = 1000 (p1 = 0095)

M2 13221227 316 x0 = 0071 (p0 = 0905) 4 S (x = 189 P = 0927) M2 vs M1x1 = 1000 (p1 = 0079) 43 A (x = 128 P = 0553) Not significantx2 = 169 (p2 = 0016) 55 I (x = 135 P = 0675)

M3 13018598 317 x0 = 0022 (p0 = 0608) M3 vs M0x1 = 0153 (p1 = 0304) P ltlt 103

x2 = 0697 (p2 = 0088)

Branch models

1x 13679145 313 x0 = 0103 NA

3x 13659727 316 x0 = 0192 NA 1x vs 3xx_r= x_E= 0084 P ltlt 0001x_bH = x_bA = 0100

5x 13659190 318 x0 = 0192 3x vs 5xx_r= 0082 Not significantx_E= 0086x_bH = 0106x_bA = 0094

9x 13653580 322 x0 = 0188 5x vs 9xx_r stem = 0244 Plt 005x_E stem = 999x_bH stem = 0575 3x vs 9x

x_bA stem = 361 Not significantx_r crown = 0080x_E crown = 0083x_bH crown = 0093x_bA crown = 0105

Site modelsmdashbA-globin

M0 4248522 123 x0 = 0979

M1 4117445 124 x0 = 0053 (p0 = 0877)x1 = 1000 (p1 = 0123)

M2 4113220 126 x0 = 0055 (p0 = 0876) 4 T (x = 3414 P = 0954) M2 vs M1x1 = 1000 (p1 = 0109) 55 L (x = 3251 P = 0952) Plt 005x2 = 3149 (p2 = 0014)

M3 4072267 127 x0 = 0022 (p0 = 0752)x1 = 0316 (p1 = 0226)x2 = 2080 (p2 = 0021)

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O2 affinity in numerous avian lineagesmdashchanges that mayhave been compensated by functional changes in the remain-ing pre- or postnatally expressed globins These results pro-vide a concrete example of evolutionary changes in aphysiologically important phenotype that were caused byrecurrent gene losses

In mammals evolutionary changes in globin gene con-tent have given rise to variation in the functional diversityof Hb isoforms that are expressed at different stages ofprenatal development (Opazo et al 2008a 2008b) Forexample duplicate copies of different b-type globingenes have been independently co-opted for fetal expres-sion in anthropoid primates and artiodactyls (StorzOpazo et al 2011 Storz et al 2013) which demonstrates

how gene duplication may provide increased scope forevolutionary innovation because redundant gene copiesare able to take on new functions or divide up ancestralfunctions In birds the relative stasis in globin gene familyevolution suggests that the developmental regulation ofHb synthesis may be more highly conserved with ortho-logous genes having similar stage-specific expression pro-files and carrying out similar functions in disparate taxa Incomparison with the globin gene clusters of mammalsand squamate reptiles the general absence of redundantgene duplicates in the avian gene clusters suggests lessopportunity for the divergence of paralogs to facilitatethe acquisition of novel protein functions andor expres-sion patterns

Table 3 O2 Affinities (P50 torr) and Cooperativity Coefficients (n50) of Purified HbA and HbD Isoforms from 11 Bird Species

Species Hb Isoform Abundance () P50(KCl+IHP) (torr) n50

Accipitriformes

Griffon vulture Gyps fulvus HbA 66 2884 198HbD 34 2661 199HbA+HbD 2808 198

Anseriformes

Graylag goose Anser anser HbA 92 4395a 300a

HbD 8 2979a 251a

HbA+HbD 4268 296

Apodiformes

Amazilia hummingbird Amazilia amazilia HbA 88 2984 242HbD 12 2320 240HbA+HbD 2904 242

Green-and-white hummingbird Amazilia viridicauda HbA 89 2424 207HbD 11 2036 229HbA+HbD 2381 209

Violet-throated starfrontlet Coeligena violifer HbA 88 1912 170HbD 12 1701 246HbA+HbD 1887 179

Giant hummingbird Patagona gigas HbA 78 2586 249HbD 22 1656 256HbA+HbD 2381 251

Great-billed hermit Phaethornis malaris HbA 76 2813 204HbD 24 2492 272HbA+HbD 2736 220

Galliformes

Ring-necked pheasant Phasianus colchicus HbA 54 2951 255HbD 46 2424a 246a

HbA+HbD 2709 251

Passeriformes

House wren Troglodytes aedon HbA 64 2587 211HbD 36 1628 236HbA+HbD 2242 220

Rufous-collared sparrow Zonotrichia capensis HbA 67 3813 229HbD 33 2049 240HbA+HbD 3231 232

Struthioformes

Ostrich Struthio camelus HbA 79 3273a 285HbD 21 2290a 244HbA+HbD 3067 276

NOTEmdashO2 equilibria were measured in 01 mM HEPES buffer at pH 74 ( 001) and 37 C in the presence of allosteric effectors ([Cl] 01 M [HEPES] 01 M IHPHb tetramerratio 20 or 420 as indicated [heme] 030ndash100 mM (for details of experimental conditions see Grispo et al [37] Projecto-Garcia et al [80] and Cheviron et al [81]) P50 andn50 values were derived from single O2 equilibrium curves where each value was interpolated from linear Hill plots (correlation coefficient r 4 0995) based on four or moreequilibrium steps between 25 and 75 saturation The reported P50 for the HbA+HbD composite hemolysate is the weighted mean P50 of both isoforms in their naturallyoccurring relative concentrationsaSaturating IHPHb4 ratio (420)

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Materials and Methods

Analysis of Globin Gene Family Evolution

The 52 avian genome assemblies that we analyzed were de-rived from multiple sources (Hillier et al 2004 Dalloul et al2010 Huang et al 2013 Zhan et al 2013 Jarvis et al 2014Zhang et al 2014) Details regarding all examined genomeassemblies and scaffolds are provided in supplementarytable S1 Supplementary Material online We identified con-tigs containinga- andb-globin genes in the genome assemblyof each species by using BLAST (Altschul et al 1990) to makecomparisons with the a- and b-globin genes of chicken Wethen annotated globin genes within the identified gene clus-ters of each species by using GENSCAN (Burge and Karlin1997) in combination with BLAST2 (Tatusova and Madden1999) to make comparisons with known exon sequencesfollowing Hoffmann et al (Hoffmann Storz et al 2010Hoffmann et al 2011) Due to incomplete sequence coveragein the genome assemblies of several species there were somecases where it was not possible to ascertain the full extent ofconserved synteny

To estimate rates of gene turnover in the globin geneclusters of birds and mammals we used a stochastic birthndashdeath model of gene family evolution (Hahn et al 2005)as implemented in the program CAFE v31 (Han et al2013) As input for the program we used ultrametric treesbased on well-resolved phylogenies of birds (Jarvis et al 2014)and eutherian mammals (Meredith et al 2011) The analysiswas based on data for 24 bird species for which we hadcomplete sequence coverage of the a- and b-globin geneclusters For comparison we used genome sequenceassemblies for 22 mammal species representing each of themajor eutherian lineages as reported in Zhang et al (2014)These sets of birds and eutherian mammals span a similarrange of divergence times and therefore provide a goodbasis for comparing lineage-specific rates of gene familyevolution

Sample Collection

We preserved blood and tissue samples from 250 voucherspecimens representing 68 bird species that were collectedfrom numerous localities in South America (supplementarytable S4 Supplementary Material online) In addition to the250 wild-caught museum-vouchered specimens we also ob-tained blood samples from captive individuals representing17 species Our proteomic analysis of Hb isoform compositionwas based on blood samples from a total of 267 specimens(see below)

All wild-caught birds were captured in mist-nets and werethen bled and euthanized in accordance with guidelines ofthe Ornithological Council (Fair and Paul 2010) and protocolsapproved by the University of New Mexico IACUC (Protocolnumber 08UNM033-TR-100117 Animal Welfare Assurancenumber A4023-01) Acquisition and study of Peruvian sam-ples was carried out under permits issued by the managementauthorities of Peru (76-2006-INRENA-IFFS-DCB 087-2007-INRENA-IFFS-DCB 135-2009-AG-DGFFS-DGEFFS 0199-

2012-AG-DGFFS-DGEFFS and 006-2013-MINAGRI-DGFFSDGEFFS) For each individual bird we collected wholeblood from the brachial or ulnar vein using heparinizedmicrocapillary tubes Red blood cells were separated fromthe plasma fraction by centrifugation and the packed redcells were then snap-frozen in liquid nitrogen and werestored at 80 C We collected liver and pectoral musclefrom each specimen as sources of genomic DNA and globinmRNA respectively Muscle samples were snap-frozen or pre-served using RNAlater and were subsequently storedat 80 C prior to RNA isolation Voucher specimens werepreserved along with ancillary data and were deposited inthe collections of the Museum of Southwestern Biologyof the University of New Mexico and the Centro deOrnitologıa y Biodiversidad (CORBIDI) Lima PeruComplete specimen data are available via the ARCTOSonline database (supplementary table S4 SupplementaryMaterial online)

Molecular Cloning and Sequencing

To augment the set of globin sequences derived from thegenome assemblies and other public databases we clonedand sequenced the adult globin genes (aA- aD- andbA-globin) from 34 additional bird species We used theRNeasy Mini Kit (Qiagen Valencia CA) to isolate total RNAfrom blood or pectoral muscle Using a few representativespecies from each order we used 50- and 30-RACE (rapidamplification of cDNA ends [Invitrogen Life TechnologiesCarlsbad CA]) to obtain cDNA sequence for the 50- and 30-untranslated regions (UTRs) of each adult-expressed globingene We then aligned the 50- and 30-UTRs of each sequencedgene and designed paralog-specific primers This strategy en-abled us to obtain complete cDNA sequences for each globinparalog using the OneStep RT-PCR kit (Qiagen Valencia CA)We cloned gel-purified RT-PCR products into pCR4-TOPOvector using the TOPO TA Cloning Kit (Invitrogen LifeTechnologies Grand Island NY) and we then sequencedat least 4ndash6 colonies per individual All sequences weredeposited in GenBank under the accession numbersKM522867ndashKM522916

Phylogenetic Analysis and Assignment of OrthologousRelationships

We used phylogenetic reconstructions to resolve orthologyfor all annotated globin genes including truncated sequencesThe a- and b-type globin sequences were aligned separatelyand when possible amino acid sequence alignments werebased on conceptual translations of nucleotide sequenceAll alignments were performed using the L-INS-i strategyfrom MAFFT v 68 (Katoh and Toh 2008) and phylogenieswere estimated using both maximum-likelihood and Bayesianapproaches We used Treefinder (v March 2011 [Jobb et al2004]) for the maximum-likelihood analyses and MrBayes(Ronquist and Huelsenbeck 2003) for Bayesian estimates ofeach phylogeny In the maximum-likelihood analyses we usedthe ldquopropose modelrdquo routine of Treefinder to select the best-fitting models of nucleotide substitution with an

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independent model for each codon position in analyses basedon nucleotide sequences Model selection was based on theAkaike information criterion with correction for small samplesize In the case of MrBayes we ran six simultaneous chains for2 107 generations sampling every 25 103 generationsusing default priors A given run was considered to havereached convergence once the likelihood scores reached anasymptotic value and the average standard deviation of splitfrequencies remained less than 001 We discarded all treesthat were sampled prior to convergence and we evaluatedsupport for the nodes and parameter estimates from a ma-jority rule consensus of the last 2500 trees

For the b-type globin genes we analyzed four separate setsof sequences 1) an inclusive data set that included all genesapparent pseudogenes and truncated sequences (containingat least one full-length exon) that were derived from thegenome assemblies 2) a data set restricted to genes withfull-length coding sequences and intact reading frames and3) the set of full-length coding sequences plus 34 bA-globincDNA sequences from avian taxa that do not currently havefully sequenced genomes We used each of these data sets toperform likelihood-based topology tests (Shimodaira 2002)

For the b-type globin genes we inferred orthologousrelationships by using an approach that combined theldquoorthology-by-contentrdquo and ldquoorthology-by-contextrdquo criteria(Song et al 2011 2012) We applied the orthology-by-contentcriterion to assign genes to the bH bA or r+E groups (basedon phylogenetic relationships) and we then used theorthology-by-context criterion to assign genes in the lattergroup as r- or E-globin (based on positional homology)

Molecular Evolution Analysis

To measure variation in functional constraint among theavian a- and b-type globin genes and to test for evidenceof positive selection we estimated (=dNdS the ratio of therate of nonsynonymous substitution per nonsynonymous site[dN] to the rate of synonymous substitution per synonymoussite [dS] site) using a maximum-likelihood approach(Goldman and Yang 1994) implemented in the CODEMLprogram v 48 (Yang 2007) In all cases we used LRTs tocompare nested sets of models (Yang 1998) For each align-ment we first compared models that allow to vary amongcodons (M0 vs M3 M1a vs M2a) The latter LRT constitutesa test of positive selection as the alternative model (M2a)allows a subset of sites to have 4 1 in contrast to the nullmodel (M1a) that only includes site classes with lt 1 Theseanalyses revealed statistically significant variation in amongsites in the alignment of bA-globin sequences (table 2) so wethen used the BEB approach (Yang et al 2005) to calculate theposterior probability that a given site belongs to the site classwith 4 1 which suggests a possible history of positiveselection

Characterizing Hb Isoform Composition of Avian Red BloodCellsWe used isoelectric focusing (IEF PhastSystem GE HealthcareBio-Sciences Piscataway NJ) to characterize Hb isoform com-position in red cell lysates from each of the 267 bird

specimens representing 68 species (n = 1ndash30 individuals perspecies) After separating native Hbs using precast IEF gels (pH3ndash9) red (heme-containing) bands were excised from the geland digested with trypsin The resultant peptides were thenidentified by means of tandem mass spectrometry (MSMS)Database searches of the resultant MSMS spectra were per-formed using Mascot (Matrix Science v190 London UK)whereby peptide mass fingerprints were used to querya custom database of avian a- and b-chain sequencesAfter separating the HbA and HbD isoforms by native gelIEF and identifying each of the constituent subunits byMSMS the relative abundance of the different isoforms inthe hemolysates of each individual was quantified densitome-trically using Image J (Abramoff et al 2004)

Supertree Construction

We constructed a time-calibrated supertree of all study spe-cies by starting with a backbone provided by a dated total-evidence phylogeny from Jarvis et al (2014) This phylogenywas based on nucleotide sequence data derived from 48whole avian genomes and 19 fossil calibrations Usingrecent avian taxonomic classifications we were able to un-ambiguously assign each study species to its appropriatebranch in the Jarvis et al (2014) backbone tree Subtrees foreach branch were obtained from the dated supertree of Jetzet al (2012) which was constructed using the Hackett et al(2008) backbone Divergence times in trees from Jetz et altended to be older than the ones from Jarvis et al (2014) weresolved topological or branch-length conflicts by favoringthe Jarvis et al (2014) tree because it was based on farmore sequence data Before grafting time-calibrated subtreesonto the backbone we uniformly rescaled the subtree bran-chlengths so that shared nodes were the same age as theywere in the backbone (following Sibly et al 2012)

Comparative Analysis of HbAHbD Expression Levels

For the comparative analysis of HbAHbD expression levelswe combined our experimental data for 68 species with pub-lished data for an additional 54 species (supplementary tableS3 Supplementary Material online) yielding data for a phy-logenetically diverse set of 122 species in total We used thefinal supertree to conduct comparative analyses under aBrownian motion model of evolution in R v 2151Maximum-likelihood estimates of ancestral states for percentexpression of HbD were generated using APE (Paradis et al2004) Phylogenetic inertia in expression level (percent) ofHbD was quantified using Blombergrsquos K (Blomberg et al2003) and Pagelrsquos Lambda (Pagel 1999) calculated usingGEIGER (Harmon et al 2008) We compiled body massdata for each species from Museum of SouthwesternBiology specimens and Dunning (Dunning 2008) and wetested for an association between body mass and expressionlevel of HbD using a phylogenetic generalized least-squaresmodel as implemented in APE (Paradis et al 2004) We log-transformed body mass and arcsin-squareroot-transformedHbD percent prior to all analyses

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Supplementary MaterialSupplementary figure 1 and tables S1ndashS4 are available atMolecular Biology and Evolution online (httpwwwmbeoxfordjournalsorg)

Acknowledgments

The authors thank T Valqui E Bautista and the students andstaff of the MSB Bird Division and CORBIDI for assistance withfield collections The authors also thank E D Jarvis M T PGilbert and G Zhang for sharing genomic sequence data F BJensen and M F Bertelsen for providing blood samples ofselect species H Moriyama and K Williams for assistancewith the protein expression experiments and J Fjeldsa forproviding the bird illustrations They also thank E D Jarvisand two anonymous reviewers for helpful comments andsuggestions This work was funded by grants from theNational Institutes of HealthNational Heart Lung andBlood Institute to JFS (R01 HL087216 and HL087216-S1)grants from the National Science Foundation to JFS (IOS-0949931) FGH (EPS-0903787) and CCW (DEB-1146491) agrant from the Biotechnology and Biological SciencesResearch Council to MB (BBD0127321) and a grant fromFondo Nacional de Desarrollo Cientifico y Tecnologico toJCO (FONDECYT 1120032)

ReferencesAbramoff MD Magelhaes PJ Ram SJ 2004 Image processing with image

J Biophoton Int 1136ndash42Alev C McIntyre BAS Nagai H Shin M Shinmyozu K Jakt LM Sheng G

2008 bA the major b-globin in definitive red blood cells is presentfrom the onset of primitive erythropoiesis in chicken Dev Dyn 2371193ndash1197

Alev C Shinmyozu K McIntyre BAS Sheng G 2009 Genomic organi-zation of zebra finch a and b globin genes and their expression inprimitive and definitive blood in comparison with globins inchicken Dev Genes Evol 219353ndash360

Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic localalignment search tool J Mol Biol 215403ndash410

Backstrom N Karaiskou N Leder EH Gustafsson L Primmer CRQvarnstrom A Ellegren H 2008 A gene-based genetic linkagemap of the collared flycatcher (Ficedula albicollis) reveals extensivesynteny and gene-order conservation during 100 million years ofavian evolution Genetics 1791479ndash1495

Baumann FH Baumann HR 1977 A comparative study of oxygen trans-port in bird blood Respir Physiol 31333ndash343

Berenbrink M 2006 Evolution of vertebrate haemoglobins histidine sidechains specific buffer value and Bohr effect Respir Physiol Neurobiol154165ndash184

Berenbrink M 2007 Historical reconstructions of evolving physiologicalcomplexity O2 secretion in the eye and swimbladder of fishes J ExpBiol 2101641ndash1652

Berenbrink M Koldkjaer P Kepp O Cossins AR 2005 Evolution ofoxygen secretion in fishes and the emergence of a complex physi-ological system Science 3071752ndash1757

Blank M Kiger L Thielebein A Gerlach F Hankeln T Marden MCBurmester T 2011 Oxygen supply from the birdrsquos eye perspectiveglobin E is a respiratory protein in the chicken retina J Biol Chem28626507ndash26515

Blomberg SP Garland T Ives AR 2003 Testing for phylogenetic signal incomparative data behavioral traits are more labile Evolution 57717ndash745

Bunn HF 1980 Regulation of hemoglobin-function in mammals AmZool 20199ndash211

Bunn HF 1987 Subunit assembly of hemoglobin an important deter-minant of hematologic phenotype Blood 691ndash6

Bunn HF McDonald MJ 1983 Electrostatic interactions in the assemblyof haemoglobin Nature 306498ndash500

Burge C Karlin S 1997 Prediction of complete gene structures inhuman genomic DNA J Mol Biol 26878ndash94

Burmester T Hankeln T 2014 Function and evolution of vertebrateglobins Acta Physiol 211501ndash514

Burt DW Bruley C Dunn IC Jones CT Ramage A Law AS Morrice DRPaton IR Smith J Windsor D et al 1999 The dynamics of chromo-some evolution in birds and mammals Nature 402411ndash413

Cheviron ZA Natarajan C Projecto-Garcia J Eddy DK Jones J CarlingMD Witt CC Moriyama H Weber RE Fago A et al 2014Integrating evolutionary and functional tests of adaptive hypothe-ses a case study of altitudinal differentiation in hemoglobin functionin an Andean sparrow Zonotrichia capensis Mol Biol Evol 312948ndash2962

Cirotto C Panara F Arangi I 1987 The minor hemoglobins of primitiveand definitive erythrocytes of the chicken embryomdashevidence forHemoglobin-L Development 101805ndash813

Cobb JA Manning D Kolatkar PR Cox DJ Riggs AF 1992Deoxygenation-linked association of a tetrameric component ofchicken hemoglobin J Biol Chem 2671183ndash1189

Cooper SJB Wheeler D De Leo A Cheng JF Holland RAB Graves JAMHope RM 2006 The mammalian aD-globin gene lineage and a newmodel for the molecular evolution of a-globin gene clusters at thestem of the mammalian radiation Mol Phylogenet Evol 38439ndash448

Dalloul RA Long JA Zimin AV Aslam L Beal K Blomberg LA BouffardP Burt DW Crasta O Crooijmans RPMA et al 2010 Multi-platformnext-generation sequencing of the domestic turkey (Meleagris gal-lopavo) genome assembly and analysis PLoS Biol 8(9)e1000475

Damsgaard C Storz JF Hoffmann FG Fago A 2013 Hemoglobin isoformdifferentiation and allosteric regulation of oxygen binding in theturtle Trachemys scripta Am J Physiol Regul Integr Comp Physiol305R961ndashR967

Derjusheva S Kurganova A Habermann F Gaginskaya E 2004 Highchromosome conservation detected by comparative chromosomepainting in chicken pigeon and passerine birds Chromosome Res12715ndash723

Dunning JBJ 2008 CRC Handbook of avian body masses Boca Raton(FL) CRC Press

Ellegren H 2013 The evolutionary genomics of birds Ann Rev Ecol EvolSyst 44239ndash259

Ellegren H 2010 Evolutionary stasis the stable chromosomes of birdsTrends Ecol Evol 25283ndash291

Fair JM Paul E 2010 Guidelines to the use of wild birds in researchWashington (DC) Ornithological Council

Fronticelli C Teresa Sana M Perez-Alvarado GC Karavitis M Lu A-LBrinigar WS 1995 Allosteric modulation by tertiary structure inmammalian Hbs J Biol Chem 27030588ndash30592

Fuchs C Burmester T Hankeln T 2006 The amphibian globin generepertoire as revealed by the Xenopus genome Cytogenet GenomeRes 112296ndash306

Garland T Bennett AF Rezende EL 2005 Phylogenetic approaches incomparative physiology J Exp Biol 2083015ndash3035

Gaudry MJ Storz JF Butts GT Campbell KL Hoffman FG 2014Repeated evolution of chimeric fusion genes in the b-globin genefamily of laurasiatherian mammals Genome Biol Evol 61219ndash1233

Gillemans N McMorrow T Tewari R Wai AWK Burgtorf C Drabek DVentress N Langeveld A Higgs D Tan-Un K et al 2003 Functionaland comparative analysis of globin loci in pufferfish and humansBlood 1012842ndash2849

Goh SH Lee YT Bhanu NV Cam MC Desper R Martin BM MoharramR Gherman RB Miller JL 2005 A newly discovered human a-globingene Blood 1061466ndash1472

Goldman N Yang ZH 1994 Codon-based model of nucleotide substi-tution for protein-coding DNA sequences Mol Biol Evol 11725ndash736

Goodman M 1963 Manrsquos place in the phylogeny of the primates asreflected in serum proteins In Washburn S editor Classification

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and human evolution Chicago (IL) Viking Fund Publicationsp 204ndash230

Griffin DK Robertson LB Tempest HG Vignal A Fillon V CrooijmansRPMA Groenen MAM Deryusheva S Gaginskaya E Carre W et al2008 Whole genome comparative studies between chicken andturkey and their implications for avian genome evolution BMCGenomics 9168

Green RE Braun EL Armstrong J Earl D Nguyen N Hickey GVandewege MW St John JA Capella-Gutierrez S Castoe TA et al2014 Three crocodilian genomes reveal ancestral patterns of evo-lution among archosaurs Science 3461335

Griffin DK Robertson LBW Tempest HG Skinner BM 2007 The evo-lution of the avian genome as revealed by comparative molecularcytogenetics Cytogenet Genome Res 11764ndash77

Grigg GC Wells RMG Beard LA 1993 Allosteric control of oxygenbinding by hemoglobin during embryonic development in the croc-odile Crocodylus porosusmdashthe role of red-cell organic phosphatesand carbon dioxide J Exp Biol 17515ndash32

Grispo MT Natarajan C Projecto-Garcia J Moriyama H Weber RE StorzJF 2012 Gene duplication and the evolution of hemoglobin isoformdifferentiation in birds J Biol Chem 28737647ndash37658

Hackett SJ Kimball RT Reddy S Bowie RCK Braun EL Braun MJChojnowski JL Cox WA Han K-L Harshman J et al 2008 A phy-logenomic study of birds reveals their evolutionary history Science3201763ndash1768

Hahn MW De Bie T Stajich JE Nguyen C Cristianini N 2005 Estimatingthe tempo and mode of gene family evolution from comparativegenomic data Genome Res 151153ndash1160

Han MV Thomas GWC Lugo-Martinez J Hahn MW 2013 Estimatinggene gain and loss rates in the presence of genome assembly andannotation error using CAFE 3 Mol Biol Evol 301987ndash1997

Hardison RC 2001 Organization evolution and regulation of the globingenes In Steinberg MH Forget BG Higgs DR Nagel RL editorsDisorders of hemoglobin genetics pathophysiology and clinicalmanagement Cambridge Cambridge University Press p 95ndash115

Hardison RC 2008 Globin genes on the move J Biol 735Hardison RC 2012 Evolution of hemoglobin and its genes Cold Spring

Harb Perspect Med 2a011627Harmon LJ Weir JT Brock CD Glor RE Challenger W 2008

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Hiebl I Weber RE Schneeganss D Kosters J Braunitzer G 1988 High-altitude respiration of birdsmdashstructural adaptations in the majorand minor hemoglobin components of adult Ruppells griffon (Grypsrueppellii)mdasha new molecular pattern for hypoxia tolerance BiolChem Hoppe-Seyler 369217ndash232

Hillier LW Miller W Birney E Warren W Hardison RC Ponting CP BorkP Burt DW Groenen MAM Delany ME et al 2004 Sequence andcomparative analysis of the chicken genome provide unique per-spectives on vertebrate evolution Nature 432695ndash716

Hoffmann FG Opazo JC Hoogewijs D Hankeln T Ebner B VinogradovSN Bailly X Storz JF 2012 Evolution of the globin gene family indeuterostomes lineage-specific patterns of diversification and attri-tion Mol Biol Evol 291735ndash1745

Hoffmann FG Opazo JC Storz JF 2008a New genes originated viamultiple recombinational pathways in the b-globin gene family ofrodents Mol Biol Evol 252589ndash2600

Hoffmann FG Opazo JC Storz JF 2008b Rapid rates of lineage-specificgene duplication and deletion in the a-globin gene family Mol BiolEvol 25591ndash602

Hoffmann FG Opazo JC Storz JF 2010 Gene cooption and convergentevolution of oxygen transport hemoglobins in jawed and jawlessvertebrates Proc Natl Acad Sci U S A 10714274ndash14279

Hoffmann FG Opazo JC Storz JF 2011 Differential loss and retention ofcytoglobin myoglobin and globin-E during the radiation of verte-brates Genome Biol Evol 3588ndash600

Hoffmann FG Opazo JC Storz JF 2012 Whole-genome duplicationsspurred the functional diversification of the globin gene superfamilyin vertebrates Mol Biol Evol 29303ndash312

Hoffmann FG Storz JF 2007 The aD-globin gene originated via dupli-cation of an embryonic a-like globin gene in the ancestor of tetra-pod vertebrates Mol Biol Evol 241982ndash1990

Hoffmann FG Storz JF Gorr TA Opazo JC 2010 Lineage-specific pat-terns of functional diversification in the a- and b-globin gene fam-ilies of tetrapod vertebrates Mol Biol Evol 271126ndash1138

Hoogewijs D Ebner B Germani F Hoffmann FG Fabrizius A Moens LBurmester T Dewilde S Storz JF Vinogradov SN et al 2012Androglobin a chimeric globin in metazoans that is preferentiallyexpressed in mammalian testes Mol Biol Evol 291105ndash1114

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Huang Y Li Y Burt DW Chen H Zhang Y Qian W Kim H Gan S ZhaoY Li J et al 2013 The duck genome and transcriptome provideinsight into an avian influenza virus reservoir species Nature Genet45776ndash783

Ikehara T Eguchi Y Kayo S Takei H 1997 Isolation and sequencing oftwo a-globin genes a(A) and a(D) in pigeon and evidence forembryo-specific expression of the a(D)-globin gene Biochemicaland Biophys Res Commun 234450ndash453

Jarvis ED Mirarab S Aberer AJ Li B Houde P Li C Ho SYW Faircloth BCNabholz B Howard JT et al 2014 Whole genome analyses resolveearly branches in the tree of life of modern birds Science 3461320ndash1331

Jeffreys AJ Wilson V Wood D Simons JP Kay RM Williams JG 1980Linkage of adult a-globin and b-globin genes in X laevis and geneduplication by tetraploidization Cell 21555ndash564

Jessen TH Weber RE Fermi G Tame J Braunitzer G 1991 Adaptation ofbird hemoglobins to high-altitudesmdashdemonstration of molecularmechanism by protein engineering Proc Natl Acad Sci U S A 886519ndash6522

Jetz W Thomas GH Joy JB Hartmann K Mooers AO 2012 The globaldiversity of birds in space and time Nature 491444ndash448

Jobb G von Haeseler A Strimmer K 2004 TREEFINDER a powerfulgraphical analysis environment for molecular phylogenetics BMCEvol Biol 418

Katoh K Toh H 2008 Recent developments in the MAFFT multiplesequence alignment program Brief Bioinform 9286ndash298

Knapp JE Oliveira MA Xie Q Ernst SR Riggs AF Hackert ML 1999 Thestructural and functional analysis of the hemoglobin D componentfrom chicken J Biol Chem 2746411ndash6420

Lutz PL 1980 On the oxygen-affinity of bird blood Am Zool 20187ndash198

Lutz PL Longmuir IS Schmidt-Nielsen K 1974 Oxygen affinity of birdblood Respir Physiol 17269ndash275

Meredith RW Janecka JE Gatesy J Ryder OA Fisher CA Teeling ECGoodbla A Eizirik E Sim~ao TLL Stadler T et al 2011 Impacts of theCretaceous terrestrial revolution and KPg extinction on mammaldiversification Science 334521ndash524

Mindell DP Knight A Baer C Huddleston CJ 1996 Slow rates of mo-lecular evolution in birds and the metabolic rate and body temper-ature hypotheses Mol Biol Evol 13422ndash426

Mrabet NT McDonald MJ Turci S Sarkar R Szabo A Bunn HF 1986Electrostatic attraction governs the dimer assembly of human he-moglobin J Biol Chem 2615222ndash5228

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Natarajan C Inoguchi N Weber RE Fago A Moriyama H Storz JF 2013Epistasis among adaptive mutations in deer mouse hemoglobinScience 3401324ndash1327

Opazo JC Butts GT Nery MF Storz JF Hoffmann FG 2013 Whole-genome duplication and the functional diversification of teleost fishhemoglobins Mol Biol Evol 30140ndash153

Opazo JC Hoffmann FG Storz JF 2008a Differential loss of embryonicglobin genes during the radiation of placental mammals Proc NatlAcad Sci U S A 10512950ndash12955

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Opazo et al doi101093molbevmsu341 MBE at U

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Opazo JC Hoffmann FG Storz JF 2008b Genomic evidence for inde-pendent origins of b-like globin genes in monotremes and therianmammals Proc Natl Acad Sci U S A 1051590ndash1595

Opazo JC Sloan AM Campbell KL Storz JF 2009 Origin and ascendancyof a chimeric fusion gene the b-globin gene of paenungulatemammals Mol Biol Evol 261469ndash1478

Pagel M 1999 Inferring the historical patterns of biological evolutionNature 401877ndash884

Paradis E Claude J Strimmer K 2004 APE analyses of phylogenetics andevolution in R language Bioinformatics 20289ndash290

Patel VS Cooper SJB Deakin JE Fulton B Graves T Warren WC WilsonRK Graves JAM 2008 Platypus globin genes and flanking loci sug-gest a new insertional model for beta-globin evolution in birds andmammals BMC Biol 634

Projecto-Garcia J Natarajan C Moriyama H Weber RE Fago A ChevironZA Dudley R McGuire JA Witt CC Storz JF 2013 Repeated eleva-tional transitions in hemoglobin function during the evolution ofAndean hummingbirds Proc Natl Acad Sci U S A 11020669ndash20674

Rana MS Riggs AF 2011 Indefinite noncooperative self-association ofchicken deoxy hemoglobin D Proteins Struct Funct Bioinform 791499ndash1512

Reitman M Grasso JA Blumenthal R Lewit P 1993 Primary sequenceevolution and repetitive elements of the Gallus gallus (chicken)b-globin cluster Genomics 18616ndash626

Riggs AF 1998 Self-association cooperativity and supercooperativity ofoxygen binding by hemoglobins J Exp Biol 2011073ndash1084

Ronquist F Huelsenbeck JP 2003 MrBayes 3 Bayesian phylogeneticinference under mixed models Bioinformatics 191572ndash1574

Runck AM Moriyama H Storz JF 2009 Evolution of duplicated b-globingenes and the structural basis of hemoglobin isoform differentiationin Mus Mol Biol Evol 262521ndash2532

Schmidt-Nielsen K Larimer JL 1958 Oxygen dissociation curves of mam-malian blood in relation to body size Am J Physiol 195424ndash428

Schwarze K Burmester T 2013 Conservation of globin genes in theldquoliving fossilrdquo Latimeria chalumnae and reconstruction of the evo-lution of the vertebrate globin family Biochim Biophys Acta-ProteinsProteomics 18341801ndash1812

Schwarze K Campbell KL Hankeln T Storz JF Hoffman FG Burmester T2014 The globin gene repertoire of lampreys convergent evolutionof hemoglobin and myoglobin in jawed and jawless vertebrates MolBiol Evol 312708ndash2721

Shaffer HB Minx P Warren DE Shedlock AM Thomson RC ValenzuelaN Abramyan J Amemiya CT Badenhorst D Biggar KK et al 2013The western painted turtle genome a model for the evolution ofextreme physiological adaptations in a slowly evolving lineageGenome Biol 14R28

Shetty S Griffin DK Graves JAM 1999 Comparative painting revealsstrong chromosome homology over 80 million years of bird evolu-tion Chromosome Res 7289ndash295

Shimodaira H 2002 An approximately unbiased test of phylogenetictree selection Syst Biol 51492ndash508

Sibly RM Witt CC Wright NA Venditti C Jetz W Brown JH 2012Energetics lifestyle and reproduction in birds Proc Natl Acad SciU S A 10910937ndash10941

Song G Hsu C-H Riemer C Zhang Y Kim HL Hoffmann F Zhang LHardison RC Green ED Miller W et al 2011 Conversion events ingene clusters BMC Evol Biol 11226

Song G Riemer C Dickins B Kim HL Zhang L Zhang Y Hsu C-HHardison RC Nisc Comparative Sequencing P Green ED et al2012 Revealing mammalian evolutionary relationships by compar-ative analysis of gene clusters Genome Biol Evol 4586ndash601

Storz JF Hoffmann FG Opazo JC Moriyama H 2008 Adaptive func-tional divergence among triplicated a-globin genes in rodentsGenetics 1781623ndash1638

Storz JF Hoffmann FG Opazo JC Sanger TJ Moriyama H 2011Developmental regulation of hemoglobin synthesis in the greenanole lizard Anolis carolinensis J Exp Biol 214575ndash581

Storz JF Opazo JC Hoffmann FG 2011 Phylogenetic diversificationof the globin gene superfamily in chordates IUBMB Life 63313ndash322

Storz JF Opazo JC Hoffmann FG 2013 Gene duplication genome du-plication and the functional diversification of vertebrate globinsMol Phylogenet Evol 66469ndash478

Tatusova TA Madden TL 1999 BLAST 2 SEQUENCES a new tool forcomparing protein and nucleotide sequences FEMS Microbiol Lett174247ndash250

Tufts DM Natarajan C Revsbech IG Projecto-Garcia J HoffmannFG Weber RE Fago A Moriyama H Storz JF 2015 Epistasisconstrains mutational pathways of hemoglobin adaptationin high-altitude pikas Mol Biol Evol 32(2)287ndash298

Weber RE Fago A Malte H Storz JF Gorr TA 2013 Lack of conventionaloxygen-linked proton and anion binding sites does not impair allo-steric regulation of oxygen binding in dwarf caiman hemoglobinAm J Physiol Regul Integr Comp Physiol 305R300ndashR312

Weber RE Jessen TH Malte H Tame J 1993 Mutant hemoglobins(a(119)-Ala and b(55)-Ser)mdashfunctions related to high-altitude res-piration in geese J Appl Physiol 752646ndash2655

Weber RE Voelter W Fago A Echner H Campanella E Low PS 2004Modulation of red cell glycolysis interactions between vertebratehemoglobins and cytoplasmic domains of band 3 red cell mem-brane proteins Am J Physiol Regul Integr Comp Physiol 287R454ndashR464

Weber RE White FN 1986 Oxygen binding in alligator blood related totemperature diving and alkaline tide Am J Physiol 251R901ndashR908

Weber RE White FN 1994 Chloride-dependent organic phosphate sen-sitivity of the oxygenation reaction in crocodilian hemoglobins J ExpBiol 1921ndash11

Yang Z 2007 PAML 4 phylogenetic analysis by maximum likelihoodMol Biol Evol 241586ndash1591

Yang ZH 1998 Likelihood ratio tests for detecting positive selection andapplication to primate lysozyme evolution Mol Biol Evol 15568ndash573

Yang ZH Wong WSW Nielsen R 2005 Bayes empirical Bayes inferenceof amino acid sites under positive selection Mol Biol Evol 221107ndash1118

Zhan X Pan S Wang J Dixon A He J Muller MG Ni P Hu L Liu Y HouH et al 2013 Peregrine and saker falcon genome sequences provideinsights into evolution of a predatory lifestyle Nat Genet 45563ndash566

Zhang G Li C Li Q Li B Larkin DM Lee C Storz JF Antunes AGreenwold MJ Meredith RW et al 2014 Comparative genomicsreveals insights into avian genome evolution and adaptation Science3461311ndash1320

887

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entral on March 31 2015

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