TheGlobinGeneRepertoireofLampreys:ConvergentEvolution ...campbelk/SchwarzeMBE2014.pdf · Thorsten Burmester*,1 1 Institute of Zoology and Zoological Museum ,UniversityofHamburg,Hamburg,Germany

Article

The Globin Gene Repertoire of Lampreys Convergent Evolutionof Hemoglobin and Myoglobin in Jawed and Jawless VertebratesKim Schwarze1 Kevin L Campbell2 Thomas Hankeln3 Jay F Storz4 Federico G Hoffmann56 andThorsten Burmester1

1Institute of Zoology and Zoological Museum University of Hamburg Hamburg Germany2Department of Biological Sciences University of Manitoba Winnipeg MB Canada3Institute of Molecular Genetics Johannes Gutenberg University of Mainz Mainz Germany4School of Biological Sciences University of Nebraska Lincoln5Department of Biochemistry Molecular Biology Entomology and Plant Pathology Mississippi State University6Institute for Genomics Biocomputing and Biotechnology Mississippi State University

Corresponding author E-mail thorstenburmesteruni-hamburgde

Associate editor John True

Abstract

Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomyand physiology Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin andevolution of the globin genes of vertebrates a superfamily that includes the well-known model proteins hemoglobin andmyoglobin Here we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) whichrevealed 23 intact globin genes and two hemoglobin pseudogenes Analyses of the genome of the Arctic lamprey(Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences The majority of theglobin genes in both lamprey species correspond to the known agnathan hemoglobins Both genomes harbor twocopies of globin X an ancient globin gene that has a broad phylogenetic distribution in the animal kingdomSurprisingly we found no evidence for an ortholog of neuroglobin in the lamprey genomes Expression and phylogeneticanalyses identified an ortholog of cytoglobin in the lampreys in fact our results indicate that cytoglobin is the onlyorthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans Notably we alsofound two globins that are highly expressed in the heart of P marinus thus representing functional myoglobins Bothgenes have orthologs in L camtschaticum Phylogenetic analyses indicate that these heart-expressed globins are notorthologous to the myoglobins of jawed vertebrates (Gnathostomata) but originated independently within theagnathans The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of function-ally analogous myoglobins and hemoglobins of gnathostomes indicating that specialized respiratory proteins for O2

transport in the blood and O2 storage in the striated muscles evolved independently in both lineages This dual con-vergence of O2-transport and O2-storage proteins in agnathans and gnathostomes involved the convergent co-option ofdifferent precursor proteins in the ancestral globin repertoire of vertebrates

Key words Agnatha convergent evolution cytoglobin gene duplication hemoglobin myoglobin

IntroductionTransport and storage of molecular O2 in vertebrates is ac-complished by proteins of the globin superfamily Globins aresmall heme-containing proteins that reversibly bind O2 andother gaseous ligands and which are present in essentially allanimal phyla The globin superfamily is a classical modelsystem to study the function and evolution of proteinsgenes and gene families (Hardison 1996 Graur and Li 2000Gillemans et al 2003 Vinogradov et al 2007) In addition totheir role in O2 supply globins may also be instrumental inthe detoxification of reactive O2 and nitrogen species regu-lation of apoptosis and signal transduction (Dickerson andGeis 1983 Weber and Vinogradov 2001 Wittenberg andWittenberg 2003 Burmester and Hankeln 2009)

The vertebrate globins provide a classic example ofhow gene duplication can facilitate the evolution of new

protein functions The globin repertoire of extant vertebratesis the product of successive genome and gene duplicationevents followed by differential gene retention among lineages(Hoffmann et al 2011 Storz et al 2011 2013 HoffmannOpazo and Storz 2012) Eight distinct globin types havebeen identified in the jawed vertebrates (Gnathostomata)Hemoglobin (Hb) which transports O2 in red blood cells(Dickerson and Geis 1983) and myoglobin (Mb) which sup-plies O2 to the mitochondria of cardiac and striated musclecells (Wittenberg and Wittenberg 1989) are the best knownmembers of the globin superfamily and are among the mostintensively investigated proteins in the biomedical sciencesAlthough Mb is a monomer Hb from jawed vertebratesforms a heterotetramer of two - and two -chains(Dickerson and Geis 1983) with each of the latter belongingto distinct globin families that originated through gene

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duplication before the radiation of gnathostomes Different- and -subtypes have evolved independently by gene du-plication in the gnathostome classes (Hoffmann Storz et al2010 Schwarze and Burmester 2013) In many cases the Hbsubtypes are expressed at different stages of developmentMore recently other globins with less well-defined functionshave been discovered These include neuroglobin (Ngb)(Burmester et al 2000) and androglobin (Adgb) (Hoogewijset al 2012) which originated early in the evolution of meta-zoan animals and have orthologs in protostomes and deu-terostomes Adgb and cytoglobin (Cygb) (Kawada et al 2001Burmester et al 2002 Trent and Hargrove 2002) appear to bepresent in all gnathostomes whereas globin E (GbE)(Kugelstadt et al 2004 Blank Kiger et al 2011) globin X(GbX) (Roesner et al 2005 Blank Wollberg et al 2011)and globin Y (GbY) (Fuchs et al 2006) have been secondarilylost in multiple vertebrate lineages independently(Hoffmann et al 2011 Schwarze and Burmester 2013 Storzet al 2013)

Agnatha that is lampreys (Petromyzontiformes) and hag-fishes (Myxiniformes) are jawless vertebrates that divergedfrom the ancestor of gnathostomes (jawed vertebrates) about500 Ma (Kuraku and Kuratani 2006 Smith et al 2013) Extantagnathans are most likely monophyletic and are referred to asCyclostomata (Kuraku et al 1999 Kuratani and Ota 2008)Despite their common name and analogous respiratory func-tions O2-transport Hbs of jawed and jawless vertebrates arestructurally distinct and are not orthologous Previous phylo-genetic analyses suggested that agnathan Hbs share a closeraffinity to gnathostome Cygb than to red blood cell Hbs ingnathostomes that share an analogous function in circulatoryO2 transport (Katoh and Miyata 2002 Hoffmann Opazoet al 2010) Agnathan hemoglobins (aHbs) are usually mono-mers in the oxygenated state and polymerize to dimers ortetramers when deoxygenated (Fago et al 2001) The O2-binding properties of lamprey aHbs resemble those ofgnathostome Hbs with both exhibiting low O2 affinitiesand large Bohr effects (Wald and Riggs 1951 Fago et al2001) As in the case of gnathostome Hbs lamprey aHbsconsist of multiple distinct subunit isoforms which may bedifferentially expressed during ontogeny For exampleLanfranchi et al (1994) demonstrated a switch in aHb expres-sion during development of the Lombardy brook lamprey(Lampetra zanandreai) which can be considered analogousto the switch of embryonic to adult Hb chains ingnathostomes

Lampreys occupy a key phylogenetic position for illumi-nating the origins and evolution of vertebrate globins Therecent availability of the genomic sequences of the sea lam-prey Petromyzon marinus (Smith et al 2013) and the Arcticlamprey Lethenteron camtschaticum (Mehta et al 2013) pro-vides the opportunity to characterize the globin repertoire ofthese species Genome mining and expression analyses showthe presence of functional Hb-like Mb-like and Cygb genes inthese species and suggest that Ngb has been lostComparisons with the globins of jawed vertebrates by phylo-genetic reconstructions and gene synteny analyses furtherallowed us to unravel the evolution of the vertebrate

globin family The results suggest that jawed and jawless ver-tebrates convergently evolved ldquoHbsrdquo and ldquoMbsrdquo with O2

transportstorage functions in blood and striated muscle re-spectively from a common globin ancestor

Results

The Globin Gene Repertoire of the Sea Lamprey andthe Arctic Lamprey

In silico analyses of the genome assembly (Smith et al 2013)and transcriptome data sets from the sea lamprey (P mar-inus) identified 25 globin genes (supplementary table S1Supplementary Material online) At least partial sequencesof these genes were found in the genome data and are dis-tributed on 14 scaffolds Two of the globin genes (aHb ps1 andaHb ps2) which are located on scaffolds GL476413 (739046ndash740131) and GL480013 (24143ndash25251) respectively har-bored premature stop codons and splice site mutations Nocorresponding expressed sequence tags (ESTs) were found forthese genes consistent with the interpretation that they arepseudogenes Two other genes (sea lamprey GbX2 and aHb5csee below for the nomenclature) were only partially repre-sented in the genome and transcriptome assemblies Theopen reading frames of the other globin genes are composedof either three or five exons and range from 426 to 669 bp(141ndash222 amino acids) in length

Analyses of the genome of the Arctic lamprey (Lcamtschaticum) (Mehta et al 2013) revealed 23 globingenes (supplementary table S2 Supplementary Materialonline) which are distributed on seven scaffolds In additionsix contigs were found that include at least fragments ofglobin genes By comparison with the globin repertoire ofthe sea lamprey and with other globin sequences the fulllength open reading frames of 14 globin genes could be de-duced Two globin genes which were not fully represented inthe Arctic lamprey genome assembly have full-length cDNAsequences at EMBLGenBank (aHb1 and aHb7) Thus 16globin genes of Arctic lamprey were full length Ten otherglobin genes were likely incomplete in the current genomeassembly (or represent truncated pseudogenes) and full-length coding sequences could not be determined by com-bining other data

An alignment of the deduced amino acid sequences of allglobins from the sea lamprey (fig 1 and supplementary fig S1Supplementary Material online) and the Arctic lamprey (sup-plementary fig S1 Supplementary Material online) revealedconservation of the typical residues required for heme-coor-dination and ligand stabilization particularly the proximaland distal histidines at positions F8 (eighth amino acid ofhelix F) and E7 respectively and the highly conserved phe-nylalanine at CD1

In both lamprey species partial sequences of putative Adgborthologs (Hoogewijs et al 2012) were detected (P marinusscaffold GL476565 L camtschaticum KE994039) but due tothe fragmentary nature of the Adgb genes they were notincluded in further analyses

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Phylogeny of Vertebrate Globins

To assess orthologous and paralogous relationships of lam-prey globins a phylogenetic analysis was conducted based onan alignment of 136 vertebrate globins (supplementary tableS3 and fig S2 Supplementary Material online) The Bayesiantree showed well-supported monophyletic clades of verte-brate Ngb Cygb GbY Mb GbE and - and -Hb respec-tively which were therefore collapsed (fig 2) The full versionof the tree is given in supplementary figure S3 SupplementaryMaterial online

The lamprey globins fell into three separate clades Onesequence of each species was placed with gnathostome GbX(the GbX2 proteins were not included in the phylogeneticanalyses because parts of the sequences were missing in bothlamprey species [supplementary fig S1 SupplementaryMaterial online]) a second group included putative orthologsof Cygb from lampreys which were placed as sister tognathostome Cygb (posterior probability [PP]frac14 056) andthe third group placed the remaining lamprey globins and

available hagfish globins together with high support(PPfrac14 10) The clade containing the gnathostome-specificglobins (GbY Mb GbE - and -Hbs) was supported by099 PP and was sister to the clade of aHbsagnathan myo-globins (aMbs) (031) Within gnathostome globins there wasstrong support for grouping Mb with GbE and -Hb with-Hb consistent with the results of previous phylogeneticand synteny analyses (Hoffmann et al 2011 HoffmannOpazo and Storz 2012 Hoffmann Opazo Hoogewijs et al2012 Schwarze and Burmester 2013)

Two Paralogous GbX Genes in Lampreys

Sequence comparisons and phylogenetic analyses identifiedtwo paralogous GbX genes (GbX1 and GbX2) in both lampreyspecies In the sea lamprey genome the GbX genes are onscaffolds GL477600 and GL476484 respectively in the Arcticlamprey genome they reside on KE993715 and KE993935 Asmentioned neither of the two GbX2 orthologs had completesequence Like other vertebrate GbX genes (Roesner et al

FIG 1 Alignment of 23 sea lamprey globin proteins with human myoglobin (MB) and -hemoglobin (HbA1) Incomplete sea lamprey sequences arenot shown The -helical structure of sea lamprey aHb5 is shown on top of the alignments Amino acids strictly conserved between the globins areshaded (black 100 conservation dark gray 80 light gray 60) The functionally important phenylalanine (F) at CD1 and the distal and proximalhistidines (H) at E7 and E8 are indicated The globin consensus numbering is given below the sequences

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2005 Blank Wollberg et al 2011 Blank and Burmester 2012)the GbX1 genes display a five exonfour intron structure andinclude the 30- and 50-extensions typical for GbX The availablesequences of the two sea lamprey GbX proteins exhibited54 identity and 69 similarity (Arctic lamprey 4562)Sea lamprey and Arctic lamprey GbX1 proteins share 97of the amino acids Both GbX1 sequences display N-terminalmyristoylation and palmitoylation sites at positions 2 and 3respectively as observed in other vertebrate GbXs (BlankWollberg et al 2011) Gene synteny analyses moreover re-vealed that the lamprey GbX1 genes are flanked by SRP14 andPLEKHG these genes and their orientation are also conserved

in the GbX region of Xenopus tropicalis (supplementary fig S4Supplementary Material online) A PLEKHG paralog is locatedadjacent to GbX2 of L camtschaticum suggesting that theGbX paralogs in lampreys originated through duplication ofthe genomic region

Identification of Lamprey Hbs

The P marinus genome harbors at least 18 intact aHb genesand two aHb pseudogenes as identified by sequence com-parisons and phylogenetic analyses The aHb genes are dis-tributed on nine scaffolds of the genome assembly

FIG 2 Simplified Bayesian phylogenetic tree of agnathan globins The numbers at the nodes are posterior probabilities The bar represents 04 PAMdistance Sea lamprey globins are colored in red Arctic lamprey globins are blue The common names of the species are given See supplementary tableS1 Supplementary Material online for details of the proteins and supplementary figure S3 Supplementary Material online for the full version ofthe tree

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(supplementary fig S5 Supplementary Material online)Scaffold GL478636 includes aHb1 and aHbs11-14 which areall in the same orientation With the exception of aHb1 theseaHbs form a common clade in the phylogenetic tree withaHbs9 and 10 (fig 2)

Nine aHb genes could be assigned to one of the four pre-viously sequenced adult aHb chains of the sea lamprey (Li andRiggs 1970 Hombrados et al 1983 1987 Qiu et al 2000) Thegenes ENSPMAG00000007266 ENSPMAG00000007259 andENSPMAG00000007276 which reside on scaffold GL477137translate into proteins with identical amino acid sequencesthat correspond to the major aHb component of thisspecies PM II (AF248645 (Qiu et al 2000) and were thusnamed aHbs2andashc (supplementary fig S5 SupplementaryMaterial online) The nucleotide sequences of genesENSPMAG00000007266 (aHb5a) and ENSPMAG00000007259(aHb5b) on scaffold GL477423 were identical reflecting eithera recent duplication event or an assembly artifact They cor-respond to the Hb component PM V which is the best stud-ied Hb subunit of the sea lamprey (Li and Riggs 1970Hendrickson et al 1973 Hombrados et al 1983 Honzatkoet al 1985) A gene fragment represented by a 30-exon onscaffold GL480013 (30606-30701 aHb5c) is also identical toPM V Gene ENSPMAG00000005317 on scaffold GL477423closely resembles PM V (99 identity) and was namedaHb5d Genes ENSPMAG00000001587 (aHb1 incorrectly an-notated on scaffold GL478636) and ENSPMAG00000005328(aHb3 on scaffold GL477423) match the protein sequences ofPM I (P09967) and PM III (P09968) respectively (Hombradoset al 1987)

Nine newly identified genes represent previously unchar-acterized aHbs of the sea lamprey They were named accord-ing to the topology of the phylogenetic tree (fig 2) aHb6(scaffold GL479302 428488ndash432549) was not annotated byENSEMBL but has an ortholog in the Arctic lamprey (seebelow) aHb6 is highly represented in ESTs of embryonicP marinus aHb7 is also found among the embryonic ESTs(full sequence in EE278870) though only exon 3 is presentin the genome assembly (GL487383 3904-4026) aHb8(ENSPMAG00000005367) is another embryonic Hb andshares 98 amino acid identity with Hb1 mRNA of Lcamtschaticum (see below) aHb9 (ENSPMAG00000008540)resides on scaffold GL476782 whereas aHbs11ndash14 are onGL478636 aHb11 is coded by ENSPMAG00000001592whereas aHb12ndash14 correspond to a misannotated genewhich is covered by ENSPMAG00000001587 aHb10 is repre-sented by an EST (FD718926) though only the 50-exon islocated on GL478504 Transcripts of all six genes aHb9ndash14were found in the ESTs from P marinus embryos or larvaesuggesting a specific function in early life stages (supplemen-tary table S4 Supplementary Material online)

The genome of the Arctic lamprey L camtschaticumalso revealed 18 aHb genes (supplementary table S2Supplementary Material online) of which 14 full-lengthcoding sequences could be deduced Four additional aHbgenes with partial sequences were identified In cases wherethe orthology of lamprey globins could be inferred the Lcamtschaticum aHb genes were named according to the

putative P marinus ortholog Sequence comparisons andphylogenetic analyses revealed seven aHb genes in theArctic lamprey that appeared to have 11 orthologs in Pmarinus aHb1 aHb6 aHb7 aHb9 aHb10 aHb11 andaHb12 This approach did not allow a reliable assignment oforthology of aHb13 and aHb14 which was thus deduced fromthe positions of the genes in the genome aHb14 appears tohave been duplicated in the Arctic lamprey Three genes onscaffold KE993857 and a gene on contig APJL01123255 differin only 2ndash6 bp and translate into identical amino acid se-quences Three aHb genes on scaffold KE993857 resemblethe aHb2 genes of the sea lamprey which reside on scaffoldGL477137 of that species In addition the two scaffolds shareconserved synteny of the genes AZIN1 and KLHL10 (supple-mentary figs S5ndashS7 Supplementary Material online) theArctic lamprey genes were therefore named aHb2-c accordingto their positions in the genome An additional gene onAPJL01123255 which closely resembles the aHb2-c geneswas named aHb2d BLAST searches showed that L camtscha-ticum aHb2c corresponds to the previously identified Hb1mRNA of this species The aHb2 proteins differ in five to sixamino acids from the major components of the adult Hb of Pmarinus aHb2 whereas aHb3 and 5 are apparently not rep-resented in the L camtschaticum genome Arctic lampreyaHb15 closely resembles aHb2 but no clear ortholog couldbe assigned and we continued the numbering of the aHbgenes Because L camtschaticum aHb16ndash18 are only repre-sented by one or two exons no clear ortholog could beassigned

The globin genes of L camtschaticum were found on fourscaffolds of the current genome assembly (supplementary figS6 Supplementary Material online) In addition three contigsinclude aHb genes Scaffold KE993782 is orthologous withscaffolds GL478636 and GL476782 of P marinus (supplemen-tary fig S7 Supplementary Material online) This large scaffoldincludes the region corresponding to scaffold GL478636 of Pmarinus where aHb1 aHbs11ndash13 and aHb14ab are locatedand the region corresponding to P marinus GL476782 thatharbors aHb9 Scaffold KE993782 also includes aHb7 and thefragmentary genes aHb17 and aHb18 The gene NPRL3 wasfound adjacent to this aHb cluster of both lampreys Syntenyanalyses moreover show the conservation of NPRL3 50 to the-Hb cluster of gnathostome vertebrates in a tail-to-tail ori-entation (fig 3A) Moreover scaffolds GL478636 of P marinusand KE993782 of L camtschaticum also share WDR90 andRAB40 genes which both reside downstream of the gnathos-tome -Hb cluster (fig 3A)

Two Functional Mbs in Lampreys

Romero-Herrera et al (1979) reported the tryptic pattern andthe amino acid composition of a putative Mb from the car-diac muscle of P marinus The translated amino acid se-quence of ENSPMAG00000006056 provided an identicalmatch to this protein and was thus designated as agnathanmyoglobin 1 (aMb1) The phylogenetic analysis revealed aclose affinity between aMb1 and the translated product ofENSPMAG00000008310 (fig 2) which was therefore named

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FIG 3 Synteny analyses of selected lamprey globin genes Orthologous genes are shown in the same color (A) The genes NPRL3 RAB40 and WDR90link the aHb cluster on scaffold KE993782 with the gnathostome Hb cluster (B) FOXK2 and RAB40 paralogs (hatched) link the Arctic lamprey Cygb-scaffold (KE993827) with the gnathostome Cygb locus whereas RNF157 FOXJ1 and EXOC7 link this latter scaffold to aMb1ndashaHb6 cluster (scaffoldKE993736)

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aMb2 Sequence comparison further identified a putativeortholog of P marinus aMb1 on KE993736 of the Arctic lam-prey plus a partial sequence that corresponds to exon 2 oncontigs APJL01135086 and APJL01176948

Quantitative real-time reverse transcription polymerasechain reaction (qRT-PCR) experiments revealed the presenceof mRNAs of both aMb1 and aMb2 in the heart of the sealamprey whereas expression levels in most other tissues in-cluding blood were much lower (fig 4A and B) Moderatelevels of aMb1 and aMb2 mRNA were also found in skeletalmuscle This suggests that both aMb1 and aMb2 act as func-tional Mbs in the sea lamprey aHb5a which was used ascontrol showed the highest mRNA level in the blood (fig4D) mRNA in situ hybridization (ISH) studies showed strongaMb1 antisense signals in the myonucleus of the myofibersand a more diffuse staining in the remaining tissue (fig 5G)Hybridization with sense probes which served as negativecontrols gave no signal (fig 4B D F and H) In heart tissueaMb1 antisense probe showed a diffuse expression patternsimilar to the aHb5a mRNA distribution in heart andskeletal muscle (fig 5) By contrast antisense probes ofaHb5a gave strong ISH signals in the erythrocytes in bloodvessels (fig 5A)

A Putative Cygb in Lampreys

The agnathan Cygbs (from the sea lamprey the Arctic lam-prey and the European brook lamprey) were identified asputative orthologs of gnathostome Cygb on the basis of se-quence comparisons and expression patterns In the Bayesiantree these proteins grouped with the gnathostome Cygbsalbeit with low support (056 PP) (fig 2) qRT-PCR analysesshowed low to moderate expression levels in a variety of sealamprey tissues with brain eyes gills and muscles having thehighest Cygb mRNA levels (fig 4C)

Exon 1 of the sea lamprey Cygb resides on the genomicscaffold GL478089 whereas exons 2 and 3 reside on GL477469(supplementary fig S5 Supplementary Material online) Inthe Arctic lamprey the full length Cygb is on scaffoldKE993827 (supplementary fig S6 Supplementary Materialonline) Synteny analyses showed that WFIKKN2 is presenton the 30-side of the Cygb genes (supplementary fig S7Supplementary Material online) Notably FOXK2 andRAB40 genes reside downstream of both agnathan andgnathostome Cygb (fig 3B) In addition genes RNF157FOXJ1 and EXOC7 are located upstream of sea lampreyaHb6ndashaMb1 whereas homologous genes are positioneddownstream of the gnathostome Cygb (fig 3B)

FIG 4 Quantification of mRNA levels of selected sea lamprey globins in different tissues Using qRT-PCR the mRNA copy numbers of the aMb1 (A) andMb2 (B) the putative Cygb (C) and aHb5a (D) were obtained aMb1 and aMb2 were detected in heart brain gill and skeletal muscle aHb5a was mosthighly expressed in blood whereas Cygb showed a widespread distribution

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Discussion

The Diversity of Lamprey Hbs

We identified 18 functional aHb and two aHb pseudogenes inthe genome of the sea lamprey P marinus Only four Hbchains had been identified previously in protein biochemicalstudies (named here aHb1 aHb2 aHb3 and aHb5) and rep-resent subunit components of the adult aHb (Li and Riggs1970 Hombrados et al 1983 1987 Qiu et al 2000) (supple-mentary table S1 Supplementary Material online) Anotherfive aHb genes closely resemble one of these chains (4 98identity) and most likely represent recent duplicates whoseproducts were not distinguishable from one another in theoriginal protein studies This interpretation is supported by

tandemly linked chromosomal arrangements of aHb2a band c and aHb5a and d respectively

Products of the other nine intact aHb genes were notpreviously identified as subunits in studies on the nativeadult Hb proteins These studies did not examine earlier lifestages and it is likely that these loci are predominantly ex-pressed prior to metamorphosis This interpretation is sup-ported by the expression pattern which was derived from thetranscriptomes and analyzed at Biosample (httpwwwncbinlmnihgovbiosample last accessed July 22 2014) Thesedata show preferential expression of aHb6 aHb7 andaHb12 in the eggs aHb9 aHb10 aHb11 and aHb14 in theembryos and aHb7 aHb9 aHb11 aHb12 aHb13 and aHb14in the larvae (supplementary table S4 Supplementary

FIG 5 ISH of sea lamprey aHb5a (A E) and aMb1 (C G) antisense RNA probes in heart (A C) and muscle (E G) cryosections aHb5a mRNA wasdetected in erythrocytes (A) which reside in the blood vessels but not in the muscle tissue (E) Expression of aMb1 mRNA was detected as diffusestaining in heart sections (C) and in myonucleus of myofibers in muscle (G) Sense probes which were used as negative controls showed no signals (B DF H) Scale barfrac14 100mm

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Material online) This observation confirms a differential ex-pression of aHbs in adults and earlier life stages (Lanfranchiet al 1994) which may reflect functional differentiation of theaHb isoforms that have distinct O2-binding properties (Birdet al 1976) Of note the phylogenetic tree shows that two ofthe aHbs expressed in eggs (aHb6 and aHb7) represent theearliest branching lineages (fig 2)

The aHb repertoire of the Arctic lamprey L camtschaticumincludes at least 18 distinct genes but the aHb protein of thisspecies has not been functionally characterized Neverthelessthree aHb mRNA sequences are available at EMBLGenBankwhich correspond to aHb1 aHb2 and aHb7 of this study It isunknown whether these chains code for components of theadult aHb However such an interpretation is supported bythe fact that the orthologs of aHb1 and aHb2 are also presentin the adult Hb of the sea lamprey Notably both sea lampreyaHb5 and aHb2 and Arctic lamprey aHb2 genes have multi-ple copies in the genomes suggesting a high level of expres-sion and that their encoded products are incorporated asmajor subunit isoforms of adult aHb

Gene Duplication Genome Duplication and theOrigins of Vertebrate-Specific Globins

Phylogenetic analyses indicate that Adgb GbX and Ngb areancient globins that originated prior to the radiation ofProtostomia and Deuterostomia (Roesner et al 2005 Blankand Burmester 2012 Hoffmann Opazo Hoogewijs et al 2012Hoogewijs et al 2012 Storz et al 2013) (fig 6) We identifiedtwo globins that correspond to GbX confirming the earlydivergence of this globin type Putative Adgb genes werefound in the genomes but were not further analyzed becauseof their fragmentary nature Notably the assemblies of the Pmarinus and L camtschaticum genomes do not contain anNgb ortholog and no Ngb-like transcripts were found in theESTs of the agnathans This suggests that Ngb has been de-leted in the Agnathamdasha surprising finding given that thisancient highly conserved globin protein is present in everygnathostome taxon that has been examined to date (with thepossible exception of sharks see Venkatesh et al 2007 2014)

The last common ancestor of Gnathostomata andAgnatha had at least six (fig 6A and B) or five (fig 6C) distinctglobin types In the most parsimonious scenario (fig 6C) thelast common ancestor of Gnathostomata and Agnatha pos-sessed Adgb GbX Ngb Cygb and a globin locus that even-tually gave rise to agnathan aHbs and aMbs gnathostomeHbs and Mbs as well as gnathostome GbE and GbY Thusremarkably Cygb is the only orthologous vertebrate-specificglobin lineage that has been retained in both gnathostomesand agnathans Invertebrate globins including those fromtunicates hemichordates and cephalochordates may wellgroup with Adgb Ngb and GbX but they are not nestedwithin the set of vertebrate-specific globin discussed here(Storz et al 2011 2013 Blank and Burmester 2012Hoffmann Opazo Hoogewijs et al 2012 Hoogewijs et al2012)

Gene synteny may provide important clues regardingthe origins of vertebrate-specific globins Notably the

aHb locus in the lamprey genomes that includes aHb1aHb7 aHbs11ndash14 aHb17 and aHb18 is flanked by the geneNPRL3 upstream and by genes RAB40 and WDR90 down-stream copies of these same genes are located in the samepositions in the -Hb gene cluster of amniote vertebrates(corresponding to the P-terminus of human Chromosome16) (fig 3A) This pattern of conserved synteny reflects aparalogous relationship between the agnathan aHb genesand the gnathostome -Hb genes that likely stems fromone or two rounds of whole-genome duplication (WGD) inthe vertebrate common ancestor The weight of availableevidence suggests that two rounds of WGD occurred

FIG 6 Hypothesized evolution of respiratory function in vertebrateglobins The three possible positions of Cygb are depicted in simplifiedmodels illustrating alternative relationships among the eight primaryvertebrate globin types (AndashC) One bar indicates the origin of O2-storagefunction (and possibly pentacoordination) whereas two bars indicatethe origin of blood O2-tansport function The circle indicates the lastcommon ancestor of the vertebrate-specific globins and the arrow thetime of divergence of Agnatha and Gnathostomata Note that if lastcommon ancestor of the vertebrate-specific globins already had an O2-storage function this function may have also been lost in Cygb

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prior to the split between agnathans and gnathostomes(Kuraku and Kuratani 2006 Kuraku 2008 2010 Kurakuet al 2009 Smith et al 2013) although the genomic organi-zation of Hox clusters in the lamprey L camtschaticum hasbeen interpreted as evidence that WGDs may have occurredindependently in the lampreys and gnathostomes (Mehtaet al 2013) Conserved synteny between the agnathan genecluster that contains aHb1 aHb7 aHbs11ndash14 aHb17 andaHb18 and the gnathostome -Hb gene cluster and the 31pattern of conserved synteny between the agnathan aHb6aMb1 cluster agnathan Cygb and gnathostome Cygb (fig 3)are both consistent with the view that at least one round ofWGD occurred prior to the divergence of agnathans andgnathostomes In combination with the phylogenetic recon-struction (fig 2) patterns of conserved synteny suggest apossible orthologous relationship between the Cygb genesof agnathans and gnathostomes as Cygb is flanked byFOXK2 and RAB40 genes in the genomes of both taxa (fig 3B)

Convergent Evolution of Agnathan and GnathostomeHbs and Mbs

Vertebrate Hb and Mb are famous for their respiratory func-tions Working in tandem they jointly ensure an adequatecellular O2 supply for aerobic energy production (Dickersonand Geis 1983 Weber and Vinogradov 2001 Wittenberg andWittenberg 2003) Although the functional properties ofagnathan aHbs have been well documented we have conclu-sively demonstrated that lampreys also possess two distinctaMbsmdashproteins highly expressed in cardiac muscle that mayhave an O2-storage function analogous to that of gnathos-tome Mb In fact we could assign sea lamprey aMb1 to aprotein previously isolated from the heart of this species(Romero-Herrera et al 1979) and further documented thatthis gene and a second gene (aMb2) are expressed in cardiacmuscle and to lesser degrees in brain gills and skeletal muscle(figs 4 and 5) Putative orthologs of both proteins were iden-tified in the Arctic lamprey

The lamprey aMbs are clearly not orthologous to gnathos-tome Mb (fig 2) rather it appears that the aHb and aMb geneclusters represent products of repeated rounds of tandemduplication that were specific to the agnathan lineageThus ancestral agnathan and gnathostome globins each in-dependently evolved functions related to erythrocyte-basedO2 transport referred to as Hb-function and muscle-specificO2 supply referred to as Mb-function This conclusion doesnot depend on the phylogenetic position of Cygb which re-mains unresolved (fig 2) There are good reasons to supposethat the O2-storage function more closely approximates theancestral state of the MbHb progenitor proteins as an au-thentic O2-transport function requires the prior existence of acirculatory system In principle the evolution of a circulatoryO2-transport function from an ancestral O2-storage functionwould involve several key steps 1) Switching the site of ex-pression from tissue to blood cells 2) a reduction in O2-bind-ing affinity and 3) the evolution of cooperative O2-binding bymeans of oxygenation-linked changes in the quaternary struc-ture of a multimeric subunit assembly (as in the tetrameric

Hb of gnathostomes) or oxygenation-linked changes in poly-merization state (as in the Hbs of agnathans) which are typ-ically monomeric in oxy-state protein and self-associate intodimers or higher-level polymers upon deoxygenation (Waldand Riggs 1951 1998 Fago et al 2001)

Evidence for the convergent evolution of O2-transport Hbsin agnathans and gnathostomes has been documented pre-viously (Hoffmann Opazo et al 2010) The original phyloge-netic analyses indicated that agnathan Hbs are more closelyrelated to Cygb than to the progenitors of the - and -chainHbs of gnathostomes In this study phylogenetic analysis of afar more extensive set of globin sequences (including anagnathan ortholog of Cygb) confirmed the independent or-igins of O2-transport Hbs in agnathans and gnathostomesand also documented that agnathan aHbs are not many-to-one orthologs of gnathostome Cygb (figs 2 and 6) Wealso document evidence that suggests the possibility of con-vergence between muscle-specific Mbs in the two vertebratelineages although an O2-storage function for the lastcommon ancestor cannot be excluded (fig 6) This dual con-vergence of O2-transport Hbs and O2-storage Mbs involvedthe convergent co-option of different precursor proteins inthe ancestral globin repertoire of vertebrates

Notably functional Hbs and Mbs have also been observedin a variety of invertebrates (Weber and Vinogradov 2001)From the phylogenetic trees it appears likely that these pro-teins emerged several times convergently from a globin an-cestor as well (see eg Roesner et al 2005 Blank andBurmester 2012 Hoffmann Opazo Hoogewijs et al 2012)For example the emergence of a functional Hb from amuscle-based Mb analog has been demonstrated in snails(Lieb et al 2006) During the evolution of eukaryotes thefunctional versatility of the globin-based heme structureand its potential for reversible O2-binding appears to havebeen repeatedly recruited for respiratory functions involvingO2-storage and O2-transport

Conclusion

The Emergence of Vertebrate Globin Diversity

Once the ancestors of contemporary vertebrates reached acertain threshold of body size and internal complexity the pas-sive diffusion of O2 became insufficient to meet metabolic de-mands and this presumably favored the evolution of specificrespiratory specializations to sustain sufficient O2 supply tointernal tissues These include respiratory surfaces such asgills a circulatory system and proteins that reversibly bind O2

for transport and storage Vertebrates as well as many inverte-brates have recruited globin proteins to serve respiratory func-tions It is uncertain whether the last common ancestor of allcurrent metazoan globins already had a function in O2 supplyIn fact globin proteins could have evolved reversible O2-bind-ing from an acylated membrane-bound hexacoordinate GbX-likeancestorwithadistinctmembrane-relatedfunctioninlipidprotection or signaling (Blank and Burmester 2012)

Gnathostome Cygbs do not exhibit membrane bindingbut are able to reversibly bind both lipids and O2 (Reederet al 2011) The actual position of Cygb in the vertebrate

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globin tree is not well resolved and three possible scenariosare conceivable (fig 6) Because respiratory functions havebeen conclusively demonstrated for aHbs Hbs Mbs andGbE it is tempting to assume a similar function in the ances-tral proto-globin that gave rise to these vertebrate globintypes (fig 6C) It remains uncertain whether this also appliesto Cygb Regardless we have demonstrated that the physio-logical division of labor between Mb-like and Hb-like respira-tory proteins evolved convergently in Agnatha andGnathostomata most likely with the advent of larger bodysize along with the evolution of active muscles and a closedcirculatory system Finally given the ancient origin of Ngb inMetazoa and its high sequence conservation among verte-bratesmdashwhich suggests a functionally important rolemdashtheapparent loss of this gene in lampreys is surprising It willbe interesting to see whether this nerve-specific globin issimilarly lost in hagfish andor other vertebrate lineagessuch as sharks (Venkatesh et al 2007 2014)

Materials and Methods

Data Collection and Sequence Analyses

Using the BLAST algorithm putative globin genes were iden-tified in the genomic sequences and ESTs of the sea lampreythat are archived in ENSEMBL (httpwwwensemblorg lastaccessed July 22 2014) and GenBank (httpwwwncbinlmnihgov last accessed July 22 2014) The genomic sequences ofthe Arctic lamprey were accessed at httpjlampreygenomeimcba-staredusg (last accessed July 22 2014) (Mehta et al2013) Gene models were built by hand and with the help ofGenScan (httpgenesmiteduGENSCANhtml last accessedJuly 22 2014) These tools were also used to annotate flankinggenes Intronndashexon boundaries were identified with Spidey(httpwwwncbinlmnihgovspidey last accessed July 222014) Preliminary analyses and translation into amino acidswere performed with GeneDoc 27 (Nicholas et al 1997)Myristoylation and palmitoylation sites were predicted byMyristoylator (httpwebexpasyorgmyristoylator lastaccessed July 22 2014) (Bologna et al 2004) and CSS-Palm20 (httpcsspalmbiocuckooorg last accessed July 22 2014)(Ren et al 2008) respectively

Multiple Sequence Alignment and PhylogeneticReconstruction

Sequences of 136 vertebrate globins were collected from thelamprey genomes and from EMBLGenBank (supplementarytable S3 Supplementary Material online) The data set cov-ered 20 globins from P marinus and 14 globins from Lcamtschaticum Incomplete globin sequences were excludedMoreover some closely related globin genes translate intoidentical proteins (supplementary tables S1 and S2Supplementary Material online) and are represented onlyby a single sequence in phylogenetic analyses We furthercollected all available globin sequences of other agnathansfrom the databases the other vertebrate globins were selectedto represent each of the distinct globin types and to cover abroad range of taxa Alternative multiple alignments of theamino acid sequences were generated by MAFFT with the

FFT-NS-i L-INS-i and G-INS-i strategies (Katoh and Toh 2008Katoh et al 2009) MUSCLE (Edgar 2004) PROMALS3D (Peiet al 2008) and T-coffee (Notredame et al 2000) The qualityof each alignment was evaluated with MUMSA (httpmsasbcsuse last accessed July 22 2014) (Lassmann andSonnhammer 2005) The alignment generated by MAFFT L-INS-i received the highest MUMSA score and was used forphylogenetic analyses Tree reconstructions were carried outwith MrBayes 321 (Huelsenbeck and Ronquist 2001 Ayreset al 2012) ProtTest (Abascal et al 2005) was used to selectthe most appropriate model of amino acid evolution (LG Leand Gascuel 2008) applying the Akaike Information CriterionThe LG model was coded with general time reversible as fixedprior with the prset command by specifying the aarevmatprand statefreqpr options A gamma distribution of substitutionrates was assumed and Bayesian trees were constructed Twoindependent runs with one cold and three heated chainswere performed for 5000000 generations Starting treeswere random and the trees were sampled every 1000th gen-eration Posterior probabilities were estimated on the final3000 trees The Ngb and GbX proteins were defined as out-groups because they diverged from the other globins prior tothe separation of Protostomia and Deuterostomia (Roesneret al 2005 Blank and Burmester 2012)

Gene Synteny Analyses

Gene orders and sequences were obtained from the genomeassemblies of Homo sapiens (Annotation Release 104) Gallusgallus (build 31) and X tropicalis (build 11) which are avail-able at NCBI (httpwwwncbinlmnihgovprojectsmap-view last accessed July 22 2014) Syntenic regions wereidentified by comparison with the gene orders in theglobin-containing contigs from the P marinus and Lcamtschaticum genomes

In Silico Analysis of Globin Expression Pattern

The ESTs of P marinus as available at GenBank weresearched with the identified globin sequences employingtBLASTn and BLASTn searches Information regarding thestage-specific expression pattern of each hit was obtainedfrom Biosample (httpwwwncbinlmnihgovbiosample)

RNA Extraction and cDNA Cloning

Two adult sea lampreys (63 cm 7317 g and 58 cm 5353 g)were collected from the Elbe estuary in June 2013 Tissuessamples were harvested immediately placed on dry ice andstored at 80 C Subsamples of skeletal muscle brain eyeliver heart and blood for subsequent RNA extraction wereplaced in RNAlater (Qiagen Hilden Germany) Total RNAwas extracted separately from each of these tissues usingthe Crystal RNA Mini Kit (Biolab Products GeuroodenstorfGermany) Briefly about 1 cm3 of tissue was placed inliquid nitrogen and ground to a fine powder with a mortarand pestle homogenized in 1 ml peqGOLD Trifast (PEQLABErlangen Germany) and 200ml of chloroform added Theaqueous phase was then purified using the filter and silicacolumn method following the manufacturerrsquos instructions

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Samples were treated with DNase (RNase-free DNase Qiagen)and the quality of the RNA was assessed by gel electrophoresisThe RevertAid H Minus First Strand cDNA Synthesis Kit(Thermo Scientific Bonn Germany) was used for reverse tran-scription of 1mg total RNA with oligo-(dT)18 primer in a finalvolume of 20ml For amplification of the selected sea lampreyglobin cDNAs specific oligonucleotides spanning the full-length coding sequences were designed (supplementarytable S5 Supplementary Material online) The PCR productswere cloned into standard cloning vectors (pGEM-T Promegaor pJET 12 Thermo Scientific) and sequenced by a commercialservice (GATC Konstanz Germany)

Quantitative Real-Time Reverse Transcription PCR

Globin mRNA expression levels were estimated by qRT-PCRon an ABI 7500 real-time PCR system using the ABI PowerSYBR Green master mix (Applied Biosystems DarmstadtGermany) RNA samples from muscle brain eye liverheart and blood tissue were examined qRT-PCR amplifica-tion was performed (40 amplification cycles 95 C for 15 s60 C for 15 s 72 C for 30 s) with a final cDNA amountequivalent to 50 ng total RNA 200 nM of each oligonucleo-tide and water to a final volume of 20ml Fluorescence wasmeasured at the end of each amplification cycle To avoidamplification of genomic DNA oligonucleotide primers thatincluded intron-spanning positions were employed (supple-mentary table S5 Supplementary Material online) Each ex-periment was performed in triplicate Negative controls(without cDNA) were run as a single experiment The speci-ficity of the amplification reaction was analyzed by dissocia-tion curve analyses Analysis of qRT-PCR results wasperformed with the ABI 7500 Sequence Detection software206 (Applied Biosystems) Absolute mRNA copy numberswere calculated by means of the standard curve method withdilutions 107ndash102 of the recombinant plasmid The sampleswere normalized according to 1mg total RNA

In Situ Hybridization

Digoxigenin-labeled antisense and sense riboprobes from theannotated lamprey aMb1 and aHb5a genes were constructedusing the DIG RNA Labeling Kit (Roche DiagnosticsMannheim Germany) The plasmids containing the globincDNAs were linearized with NcoI (antisense probe) andNotI (sense probe) and used as templates The labeledprobes were purified by lithium chloride precipitation andtheir integrity was checked by gel electrophoresis The effi-ciency of digoxigenin labeling was determined by dot blots

Frozen heart and muscle samples were equilibrated for 20min at 20 C and cryosectioned at 16mm thickness Thesections were mounted on poly-L-lysine cover slides (FisherScientific Schwerte Germany) fixed for 20 min on ice in 4paraformaldehyde in phosphate-buffered saline (PBS)(140 mM NaCl 27 mM KCl 81 mM Na2HPO4 15 mMKH2PO4 pH 69) and rinsed twice in PBS at room tempera-ture (RT) The sections were acetylated in 05 acetic anhy-dride in 01 M triethanolamine (pH 80) for 10 min washedwith PBS dehydrated in a graded ethanol series (70 9095 100) and dried For hybridization the probe mix

(1000 ngml probe 25 mgml tRNA 50 mM DTT) was dena-tured for 10 min at 65 C and mixed at a ratio 15 with hy-bridization buffer (50 deionized formamide 10 dextransulfate 1 Denhardtrsquos solution 300 mM NaCl 10 mM TrisndashHCl pH 80 1 mM ethylenediaminetetraacetic acid [EDTA]pH 80) Hybridization was carried out at 58 C for 16 h Theslides were rinsed twice in 4 SSC (20 SSC 3 M NaCl 03 Msodium citrate pH 70) for 10 min at RT treated for 30 min at37 C with RNase A (018 Kunitz unitml Roth KarlsruheGermany) in 10 mM Tris pH 80 05 M NaCl 05 mMEDTA followed by additional washing steps (2 5 min atRT in 2 SSC 1 mM DTT for 10 min in 1 SSC 1 mMDTT at RT 10 min in 05 SSC 1 mM DTT at RT and30 min in 01 SSC 1 mM DTT at 60 C)

After equilibration for 5 min in PBS01 Tween-20 and5 min in Buffer B (100 mM TrisndashHCl 150 mM NaCl pH 7505 blocking reagent Roche Diagnostics MannheimGermany) the slides were incubated for 2 h at 37 C with al-kaline-phosphatase-coupled antidigoxigenin antibody (RocheDiagnostics) diluted 15000 in Buffer B Unbound antibodieswere removed by two 15-min washes in 100 mM TrisndashHCl150 mM NaCl pH 75 followed by an 15-min incubation in100 mM TrisndashHCl 100 mM NaCl 50 mM MgCl2 pH 95 Thevisualization of the probes was carried out with the nitro-bluetetrazolium5-bromo-4-chloro-3rsquo-indolyphosphate substratesystem After 16 h the color reaction was stopped by washingin 100 mM TrisndashHCl 1 mM EDTA pH 74 for 15 min Slideswere rinsed for 30 s in 95 ethanol air dried embedded in 1PBSglycerin (19) covered by a coverslip fixed by nail polishand analyzed with an Olympus BX51 research microscope

Supplementary MaterialSupplementary file S1 tables S1ndashS5 and figures S1ndashS7 areavailable at Molecular Biology and Evolution online (httpwwwmbeoxfordjournalsorg)

Acknowledgments

The authors thank Miriam Geurootting Walter Zeeck and ClausZeeck for their help with the collection of lampreys andKatharina Kruszewski and Anthony Signore for their helpwith sequence data This work is supported by a grant ofthe Deutsche Forschungsgemeinschaft to TB (BU 95618)KS was supported by a PhD fellowship from the University ofHamburg JFS acknowledges support from NIH grantHL087216 FGH acknowledges support from NSF grantEPS TH acknowledges funding by the Johannes GutenbergUniversity Centre for Computational Sciences Mainz (SRFN)

ReferencesAbascal F Zardoya R Posada D 2005 ProtTest selection of best-fit

models of protein evolution Bioinformatics 212104ndash2105Ayres DL Darling A Zwickl DJ Beerli P Holder MT Lewis PO

Huelsenbeck JP Ronquist F Swofford DL Cummings MP et al2012 BEAGLE an application programming interface and high-per-formance computing library for statistical phylogenetics Syst Biol61170ndash173

Bird DJ Lutz PL Potter IC 1976 Oxygen dissociation curves of the bloodof larval and adult lampreys (Lampetra fluviatilis) J Exp Biol 65449ndash458

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Blank M Burmester T 2012 Widespread occurrence of N-terminal ac-ylation in animal globins and possible origin of respiratory globinsfrom a membrane-bound ancestor Mol Biol Evol 293553ndash3561

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

Blank M Wollberg J Gerlach F Reimann K Roesner A Hankeln T FagoA Weber RE Burmester T 2011 A membrane-bound vertebrateglobin PLoS One 6e25292

Bologna G Yvon C Duvaud S Veuthey AL 2004 N-Terminal myristoy-lation predictions by ensembles of neural networks Proteomics 41626ndash1632

Burmester T Ebner B Weich B Hankeln T 2002 Cytoglobin a novelglobin type ubiquitously expressed in vertebrate tissues Mol BiolEvol 19416ndash421

Burmester T Hankeln T 2009 What is the function of neuroglobin JExp Biol 2121423ndash1428

Burmester T Weich B Reinhardt S Hankeln T 2000 A vertebrate globinexpressed in the brain Nature 407520ndash523

Dickerson RE Geis I 1983 Hemoglobin structure function evolu-tion and pathology San Francisco (CA) BenjaminCummingsPub Co

Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-racy and high throughput Nucleic Acids Res 321792ndash1797

Fago A Giangiacomo L DrsquoAvino R Carratore V Romano M Boffi AChiancone E 2001 Hagfish hemoglobins structure function andoxygen-linked association J Biol Chem 27627415ndash27423

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

Gillemans N McMorrow T Tewari R Wai AW 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

Graur D Li W-H 2000 Fundamentals of molecular evolution 2nd edSunderland (MA) Sinauer Associates Inc

Hardison RC 1996 A brief history of hemoglobins plant animal protistand bacteria Proc Natl Acad Sci U S A 935675ndash5679

Hendrickson WA Love WE Karle J 1973 Crystal structure analysis of sealamprey hemoglobin at 2 angstrom resolution J Mol Biol 74331ndash361

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

Hombrados I Rodewald K Allard M Neuzil E Braunitzer G 1987Primary structure of the minor haemoglobins from the sea lamprey(Petromyzon marinus Cyclostomata) Biol Chem Hoppe Seyler 368145ndash154

Hombrados I Rodewald K Neuzil E Braunitzer G 1983Haemoglobins LX Primary structure of the major haemoglobin ofthe sea lamprey Petromyzon marinus (var Garonne Loire)Biochimie 65247ndash257

Honzatko RB Hendrickson WA Love WE 1985 Refinement of a mo-lecular model for lamprey hemoglobin from Petromyzon marinus JMol Biol 184147ndash164

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

Huelsenbeck JP Ronquist F 2001 MRBAYES Bayesian inference of phy-logenetic trees Bioinformatics 17754ndash755

Katoh K Asimenos G Toh H 2009 Multiple alignment of DNA se-quences with MAFFT Methods Mol Biol 53739ndash64

Katoh K Miyata T 2002 Cyclostome hemoglobins are possibly para-logous to gnathostome hemoglobins J Exp Biol 55246ndash249

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

Kawada N Kristensen DB Asahina K Nakatani K Minamiyama Y Seki SYoshizato K 2001 Characterization of a stellate cell activation-asso-ciated protein (STAP) with peroxidase activity found in rat hepaticstellate cells J Biol Chem 27625318ndash25323

Kugelstadt D Haberkamp M Hankeln T Burmester T 2004Neuroglobin cytoglobin and a novel eye-specific globin fromchicken Biochem Biophys Res Commun 325719ndash725

Kuraku S 2008 Insights into cyclostome phylogenomics pre-2R or post-2R Zool Sci 25960ndash968

Kuraku S 2010 Palaeophylogenomics of the vertebrate ancestormdashimpact of hidden paralogy on hagfish and lamprey gene phylogenyIntegr Comp Biol 50124ndash129

Kuraku S Hoshiyama D Katoh K Suga H Miyata T 1999 Monophyly oflampreys and hagfishes supported by nuclear DNA-coded genesJ Mol Evol 49729ndash735

Kuraku S Kuratani S 2006 Time scale for cyclostome evolution inferredwith a phylogenetic diagnosis of hagfish and lamprey cDNA se-quences Zool Sci 231053ndash1064

Kuraku S Meyer A Kuratani S 2009 Timing of genome duplicationsrelative to the origin of the vertebrates did cyclostomes divergebefore or after Mol Biol Evol 2647ndash59

Kuratani S Ota KG 2008 Hagfish (Cyclostomata vertebrata) searchingfor the ancestral developmental plan of vertebrates Bioessays 30167ndash172

Lanfranchi G Pallavicini A Laveder P Valle G 1994 Ancestral hemo-globin switching in lampreys Dev Biol 164402ndash408

Lassmann T Sonnhammer EL 2005 Automatic assessment of alignmentquality Nucleic Acids Res 337120ndash7128

Le SQ Gascuel O 2008 An improved general amino acid replacementmatrix Mol Biol Evol 251307ndash1320

Li SL Riggs A 1970 The amino acid sequence of hemoglobinV from the lamprey Petromyzon marinus J Biol Chem 2456149ndash6169

Lieb B Dimitrova K Kang HS Braun S Gebauer W Martin A Hanelt BSaenz SA Adema CM Markl J 2006 Red blood with blue-bloodancestry intriguing structure of a snail hemoglobin Proc Natl AcadSci U S A 10312011ndash12016

Mehta TK Ravi V Yamasaki S et al 2013 Evidence for at least six Hoxclusters in the Japanese lamprey (Lethenteron japonicum) Proc NatlAcad Sci U S A 11016044ndash16049

Nicholas KB Nicholas HB Jr Deerfield DWI 1997 GeneDoc analysis andvisualization of genetic variation EMBNEWNEWS 414

Notredame C Higgins DG Heringa J 2000 T-Coffee a novel method forfast and accurate multiple sequence alignment J Mol Biol 302205ndash217

Pei J Kim BH Grishin NV 2008 PROMALS3D a tool for multiple pro-tein sequence and structure alignments Nucleic Acids Res 362295ndash2300

Qiu Y Maillett DH Knapp J Olson JS Riggs AF 2000 Lamprey hemo-globin Structural basis of the Bohr effect J Biol Chem 27513517ndash13528

Reeder BJ Svistunenko DA Wilson MT 2011 Lipid binding to cytoglo-bin leads to a change in haem co-ordination a role for cytoglobin inlipid signalling of oxidative stress Biochem J 434483ndash492

Ren J Wen L Gao X Jin C Xue Y Yao X 2008 CSS-Palm 20 an updatedsoftware for palmitoylation sites prediction Protein Eng Des Sel 21639ndash644

2720

Schwarze et al doi101093molbevmsu216 MBE at U

niversity of Nebraka-L

incoln Libraries on Septem

ber 18 2014httpm

beoxfordjournalsorgD

ownloaded from

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

Roesner A Fuchs C Hankeln T Burmester T 2005 A globin gene ofancient evolutionary origin in lower vertebrates evidence for twodistinct globin families in animals Mol Biol Evol 2212ndash20

Romero-Herrera AE Lieska N Nasser S 1979 Characterization of themyoglobin of the lamprey Petromyzon marinus J Mol Evol 14259ndash266

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 18341801ndash1812

Smith JJ Kuraku S Holt C Sauka-Spengler T Jiang N Campbell MSYandell MD Manousaki T Meyer A Bloom OE et al 2013Sequencing of the sea lamprey (Petromyzon marinus) genome pro-vides insights into vertebrate evolution Nat Genet 45415ndash421

Storz JF Opazo JC Hoffmann FG 2011 Phylogenetic diversification ofthe 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

Trent JT Hargrove MS 2002 A ubiquitously expressed human hexa-coordinate hemoglobin J Biol Chem 27719538ndash19545

Venkatesh B Kirkness EF Loh YH Halpern AL Lee AP Johnson JDandona N Viswanathan LD Tay A Venter JC et al 2007 Surveysequencing and comparative analysis of the elephant shark(Callorhinchus milii) genome PLoS Biol 5e101

Venkatesh B Lee AP Ravi V Maurya AK Lian MM Swann JB Ohta YFlajnik MF Sutoh Y Kasahara M et al 2014 Elephant shark genomeprovides unique insights into gnathostome evolution Nature 505174ndash179

Vinogradov SN Hoogewijs D Bailly X Mizuguchi K Dewilde S Moens LVanfleteren JR 2007 A model of globin evolution Gene 398132ndash142

Wald G Riggs A 1951 The hemoglobin of the sea lamprey Petromyzonmarinus J Gen Physiol 3545ndash53

Weber RE Vinogradov SN 2001 Nonvertebrate hemoglobins functionsand molecular adaptations Physiol Rev 81569ndash628

Wittenberg BA Wittenberg JB 1989 Transport of oxygen in muscleAnnu Rev Physiol 51857ndash878

Wittenberg JB Wittenberg BA 2003 Myoglobin function reassessedJ Exp Biol 2062011ndash2020

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