BACKGROUND
metabolism in metazoans requires oxidation of organic molecules.
thus natural selection has presumably favored proteins that can reversibly bind and transport oxygen  <cit> .
such oxygen-binding proteins likely originated from enzymes whose primary function was to protect the body from oxygen toxicity, and, secondarily, these enzymes acquired the ability to carry oxygen molecules  <cit> .
several different classes of oxygen-carrying proteins, or respiratory pigments, are found across animal life.
although these molecules can reversibly bind oxygen, their binding affinities and evolutionary origins differ.
in animals, oxygen-binding proteins are usually divided into two main groups: proteins that use iron to bind oxygen, including hemoglobins and hemerythrins, and hemocyanins that use copper  <cit> .
although hemoglobins and hemocyanins have been extensively investigated , knowledge on the evolutionary history of hemerythrins is limited  <cit> .
interestingly, medical sciences have increasingly been taking advantage of oxygen-binding proteins as blood substitutes  <cit>  or as carrier proteins for synthetic vaccines,  making further study of oxygen binding protein diversity and evolution appealing.
hemerythrins  are a non-heme oligomeric protein family within the ‘four-helical up-and-down bundle’ fold and ‘all alpha proteins’ class according to the structural classification of proteins database   <cit> .
oxygen-binding hr proteins contain approximately  <dig> amino acid residues in a single domain and transport oxygen with the aid of two iron ions that bind directly to the polypeptide chain.
residues involved in iron binding include histidines  in positions  <dig>  <dig>   <dig>   <dig>  and  <dig>  glutamic acid residue  in position  <dig> and aspartic acid residue  in position  <dig> .
presence of these signature residues indicates putative respiratory function for hrs  <cit> .
functional hr subunits usually form a homooctamer, although dimeric, trimeric, or tetrameric hrs have been observed in some sipunculid species, including phascolosoma arcuatum, p. agasizii, and siphonosoma funafuti .
the crystal structure of hrs consists of a bundle of four antiparallel α-helices  formed by polypeptides: an a α-helix formed by  <dig> amino acid residues from position  <dig> to  <dig>  b α-helix with  <dig> amino acids residues from position  <dig> to  <dig>  c α-helix formed by  <dig> amino acids residues from position  <dig> to  <dig>  and d α-helix formed by  <dig> amino acids residues from position  <dig> to  <dig>  using t. zostericola as the reference sequence  <cit> .
the core of active sites contains two iron atoms bridged by two carboxylate groups from aspartate and glutamate residues and an oxygen-containing ligand  <cit> .
binding of oxygen apparently requires other currently unknown cellular factors since purified hr, by itself, usually does not bind oxygen  <cit> .
observed oxygen binding capacity is about 25% greater in hrs than heme-based proteins, including hemoglobins  <cit> .
although hr-like proteins have also been reported in prokaryotes  oxygen binding hrs have only been reported from marine invertebrates belonging to annelida  brachiopoda, priapulida, bryozoa, and a single species of both cnidaria  and arthropoda   <cit> .
given this phylogenetic breadth of animals, whether all metazoan hrs share a common origin is debated  <cit> .
overall, hr proteins exhibit variation in their quaternary structure, and four groups have been reported based mainly on their primary structure and location within animal bodies  <cit> .
specifically, hemerythrins found in vascular tissue, referred to here as circulating hemerythrins , and muscle-specific myohemerythrins , have been better characterized compared to the other two hemerythrin groups, ovohemerythrins  and neurohemerythrins   <cit> .
chrs are polymeric intracellular proteins that occur inside nucleated cells, hemerythrocytes or pink blood cells located in coelomic fluid or vascular systems of hr-bearing organisms  <cit> .
in contrast, myohrs are monomeric cytoplasmic proteins present in muscle cells of annelids  <cit> .
the main difference between these groups is the presence of a five-codon insertion found in myohr immediately before the d α-helix.
expression of myohrs seems not to be restricted to chrs-bearing organisms, considering that some annelid species possess both myohrs and hemoglobins  <cit> .
the other two groups of hrs, ovohemerythrin  and neurohemerythrin , are also intracellular and non-circulating.
ovohr was identified in oocytes of the leech theromyzon tessulatum and its presence during oogenesis possibly suggests a complex function in iron storage and detoxification  <cit> .
on the other hand, nhr was recently discovered in neural and non-neuronal tissues from the body wall of the leech hirudo medicinalis, and it exhibits upregulation in response to septic injury  <cit> .
nevertheless, vanin et al.
suggested that nhr of leech may in fact be a myohr  <cit> .
such diversification in hr function may have involved gene duplications resulting in new proteins via neo- or subfunctionalization  <cit> .
moreover, ovohrs and nhrs have only been reported in the literature a few times, and more studies are required to understand their function and evolution.
annelids have the greatest diversity of oxygen-binding proteins among metazoans  <cit>  and it is the only phylum from which all subtypes of hr proteins have been documented  <cit> .
while hrs of annelids have been studied since the middle of the 20th century, until the 1990s, hrs were recorded only from sipunculids and from a single polychaete family, magelonidae  <cit> .
later, vanin et al.
<cit>  found hrs in a nereid and a leech and bailly et al.
<cit>  discovered hrs genes in seven annelid species, suggesting hrs are broadly distributed in annelids.
given the diversity of lifestyles among annelids known to have hrs  <cit> , and the lack of information about hrs in general  <cit> , the occurrence and diversity of these molecules may be higher than currently recognized.
thus, to examine a wide diversity of annelid taxa for hrs and to further understand how different forms of hrs are evolutionarily related to each other, we employed approaches to survey hrs from a diverse array of annelid transcriptomes in silico.
we identified hrs in  <dig> taxa and further describe the molecular diversity and evolution of hrs in the light of annelid phylogeny  <cit> .
along with this, we assess whether described hr subtypes consist of evolutionary lineages or result of independent adaptations to different organismal tissues.
RESULTS
our bioinformatic analyses  recovered a total of  <dig> unique nucleotide sequences of hemerythrin-like genes.
following translation pfam domain evaluation and manual removal of sequences with less than  <dig> amino acid residues,  <dig> putative novel hr genes were retained from all taxa examined in this study, representing  <dig> annelid species in  <dig> different families .
novel hr genes accession numbers for each species is available in additional file  <dig>  the number of expressed hrs in a given species ranged from one in alciopa sp., cossura longocirrata, enchytraeus albidus, schizobranchia insignis, and syllis cf.
hyaline to  <dig> in magelona berkeleyi and phascolosoma agassizii.fig.
<dig> alignment of nucleotide dataset of few species.
showing the five-codon insertion  between c α-helix  and d α-helix
following trimming alignment of translated transcripts possessed  <dig> residue positions, with nearly all sequences, the exception being aphrodita japonica, starting with a methionine residue.
we decided to keep the apparently incomplete sequence from a. japonica due to its high similarity with the remaining sequences.
all sequences in the alignment contained signature residues involved in iron binding, indicating putative respiratory function for these putative hrs  <cit> .
for the  <dig> sequences,  <dig> were unique and  <dig> identical for at least two species at the amino acid level.
sequences were assembled into a final dataset containing  <dig> sequences being  <dig> new, two hr sequences from lingula reevii, a brachiopod, and nine annelid sequences previous used as “queries”, with  <dig> aligned nucleotides positions.
of these,  <dig> sequences contained a five-codon insertion between the c and d α-helices described for myohr, but not chrs   <cit> .
datasets supporting conclusions of this article are available in the figshare repository, under dois: https://doi.org/ <dig> /m <dig> figshare.
<dig> v <dig> and https://doi.org/ <dig> /m <dig> figshare.
<dig> v <dig>
every species analyzed possessed at least one copy of myohr gene.
bayesian inference rooted using two brachiopod hr sequences revealed two major clades with one corresponding to chrs clade  and the other supported monophyly for a myohr clade .
the leech nhr sequence included in the dataset was found inside the myohr clade .
within the myohr clade low nodal support values and polytomies were found.
however, such results are not uncommon for single protein or protein family trees  <cit> .fig.
<dig> bayesian tree using mrbayes  <dig> .
<dig>  <cit>  rooted with two hr sequences from lingula reevii, a brachiopod.
the blue clade represents the chrs othologs sequences with a prediction of the consensus chr sequence quaternary structure using i-tasser  <dig>   <cit> .
black, purple, and green sequences represent the myohr sequences and orange sequence is the only nhr from hirudo medicinalis.
the green clade shows the paralogs sequences from c. variopedatus and m. taylori and the purple clade represents the identical orthologs myohr sequences.
the number after the name of each sequence indicates the genbank accession numbers for each hr gene and it is indicated in additional file 2: table s1
our analysis found  <dig> putative chr sequences lacking the characteristic five-codon insertion before the d α-helix that define myohrs, distributed across nine different families.
besides well-known records of chrs in sipunculids, such as themiste pyroides and phascolosoma agassizii, we discovered chrs in the sipunculid thysanocardia nigra as well as six annelid families; amphinomidae aproditidae, capitellidae, oweniidae, sabellidae, and spionidae .
the topology of the hr gene tree did not mirror recent phylogenies of annelida based on phylogenomic datasets  <cit> .
for example, we found  <dig> hr sequences identical at the nucleotide level  belonging to distant annelid families indicating a strong conservation among those orthologs.
several of these sequences were prepared and sequenced at different times, making cross contamination unlikely.
those  <dig> identical sequences differed  <dig> %  from the consensus of all others myohr sequences and the majority of nonsynonymous substitutions are concentrated in a and b α-helices.
regarding paralogs multiple copies of hr genes were found for several species, including two paralogs from both chaetopterus variopedatus and mesochaetopterus taylori, with these paralogs forming a monophyletic clade .
both species were from chaetopteridae suggesting a recent paralogous duplication  <cit> .
given the presence of multiple hrs, apparently early annelids already contained several copies of hr genes, with some paralogs arising later .
differences in evolutionary rates between chrs and myohrs sequences was accessed and relative rates of change in different positions were calculated using two different approaches.
sites with high variation were found not to have significantly different rates among inter-helices sites using diverge  <cit> .
for helix regions a and c α-helices had one site each with high evolutionary rate, while d α-helix did not have any sites with a high evolutionary rate, indicating a highly conservative region.
at the same time, b α-helix had five sites, suggesting that this helix is evolving faster than others .
relax  <cit>  was also used to assess differences in selection on chrs relative to myohrs while accounting for lineage-specific rate differences.
similar to the diverge analyses, no significant differences  were found between the two sets of genes.
thus, the gene duplication event leading to chrs versus myohrs does not seem to have been accompanied by a significant change in substitution rate or selection differences on the two gene lineages.
alternatively, any evidence of such a rate or selection difference could have been lost during the 500+ my since these genes diverged.fig.
<dig> hr showing differences in evolutionary rates between chrs and myohrs sequences calculated with diverge  <cit>  using a  <dig>  cutoff.
a and c α-helices presented one site each above the cutoff value, d α-helix did not present any sites, and b α-helix presented five sites.
colored dots represent sites above cutoff value, and different colors are only to aid illustration
discussion
as demonstrated here expression of hr genes among annelids is much more common than previously reported, revealing unrecognized diversity of these genes in this phylum.
all  <dig> of the examined species possessed actively transcribed myohrs, while chrs were less frequently recovered from these transcriptomes.
although this diversity and wide distribution of hrs in annelids could, in part, be explained by the need to carry oxygen  secondary functional specializations could also be important for driving diversification.
for example, hrs participate in iron storage, metal detoxification, and immunity in some annelids   <cit> .
our findings build on recent publications demonstrating that sequence diversity among hr-bearing species was larger than traditionally suspected  <cit> .
however, those studies were based predominately on genomic data.
in this case, use of transcriptomes shows that hrs genes were present and expressed.
bayesian phylogenetic reconstruction recovered monophyletic clades for annelid chrs sequences as well as myohrs sequences with strong support corroborating vanin et al.’s  <cit>  previous findings.
the presence of multiple copies of myohr genes across the annelid phylogeny implies these proteins have undergone several instances of gene duplication during their evolution, as previously reported for other bacterial, archeal, and eukaryotic taxa  <cit> .
moreover, the unexpected diversity of myohrs could be associated with functional diversification of this gene, as observed for myoglobins  <cit>  and also for hrs involved in heavy metal detoxification and aspects of innate immunity  <cit> .
classification of specific hr subtypes  was not validated by the gene genealogy.
although our analyses used whole organisms , our results failed to recover hr proteins that corresponded to ovohrs or nhrs.
these categories, however, were described based on limited differences in amino acid sequence and do not reflect distinct monophyletic subgroups within the hr gene family.
given this, recognizing only two primary types of hrs, circulating hrs  and non-circulating hrs , is perhaps more appropriate.
although tentative, this reinterpretation deserves further consideration.
incongruence between our gene genealogy relative to current knowledge of annelid evolutionary history indicates that hrs have a complex history which possibly involved events of gene losses and duplications, paralogs replacements and lateral gene transfer  <cit> .
although previous work supported the idea of a monomeric protein as an ancestral myohr within annelids  <cit> , the presence of chrs in paramphinome jeffreysii , galathowenia oculata , phascolosoma agassizii, themiste pyroides, and thysanocardia nigra , all members of lineages near the base of the annelid tree  <cit>  , indicates that both chrs and myohrs were likely present in the ancestor of annelids, which date back to the cambrian  <cit> .
interestingly, hrs from multiple species, representing hundreds of millions of years of evolution, possessed identical amino acid, and in some cases nucleotide, sequences , suggesting a level of conservation and selection orders of magnitude greater than most of the genome.
additional studies across metazoan together with studies of the gene structure of hr proteins and physiological aspects of organisms are the next important steps toward a better understanding of the evolutionary patterns involved in this family of oxygen carrying proteins.fig.
<dig> lophotrochozoa and annelid relationships based on current knowledge  <cit> .
underlined phyla represent the hr-bearing representants.
annelid taxa in blue possess both chr and myohr genes and taxa in red possess just myohr genes
CONCLUSIONS
our findings demonstrate that sequence diversity among hr-bearing annelid species is much greater than traditionally suspected and that many of these hrs are actively expressed.
there are two primary types of hrs circulating hrs  and non-circulating hrs , instead of the four subtypes reported in the literature.
incongruence between our gene genealogy relative to current knowledge of annelid evolutionary history indicates that hrs have a complex history.
our findings indicate that both chrs and myohrs were likely present in the ancestor of annelids, as both subtypes occur in all lineages near the base of the annelid tree.

 metabolism in metazoans requires oxidation of organic molecules. thus natural selection has presumably favored proteins that can reversibly bind and transport oxygen. such oxygen-binding proteins likely originated from enzymes whose primary function was to protect the body from oxygen toxicity, and, secondarily, these enzymes acquired the ability to carry oxygen molecules. several different classes of oxygen-carrying proteins, or respiratory pigments, are found across animal life. although these molecules can reversibly bind oxygen, their binding affinities and evolutionary origins differ. in animals, oxygen-binding proteins are usually divided into two main groups: proteins that use iron to bind oxygen, including hemoglobins and hemerythrins, and hemocyanins that use copper. although hemoglobins and hemocyanins have been extensively investigated, knowledge on the evolutionary history of hemerythrins is limited. interestingly, medical sciences have increasingly been taking advantage of oxygen-binding proteins as blood substitutes or as carrier proteins for synthetic vaccines, making further study of oxygen binding protein diversity and evolution appealing. hemerythrins are a non-heme oligomeric protein family within the ‘four-helical up-and-down bundle’ fold and ‘all alpha proteins’ class according to the structural classification of proteins database. oxygen-binding hr proteins contain approximately amino acid residues in a single domain and transport oxygen with the aid of two iron ions that bind directly to the polypeptide chain. residues involved in iron binding include histidines in positions and glutamic acid residue in position and aspartic acid residue in position. presence of these signature residues indicates putative respiratory function for hrs. functional hr subunits usually form a homooctamer, although dimeric, trimeric, or tetrameric hrs have been observed in some sipunculid species, including phascolosoma arcuatum, p. agasizii, and siphonosoma funafuti. the crystal structure of hrs consists of a bundle of four antiparallel α-helices formed by polypeptides: an a α-helix formed by amino acid residues from position to b α-helix with amino acids residues from position to c α-helix formed by amino acids residues from position to and d α-helix formed by amino acids residues from position to using t. zostericola as the reference sequence. the core of active sites contains two iron atoms bridged by two carboxylate groups from aspartate and glutamate residues and an oxygen-containing ligand. binding of oxygen apparently requires other currently unknown cellular factors since purified hr, by itself, usually does not bind oxygen. observed oxygen binding capacity is about 25% greater in hrs than heme-based proteins, including hemoglobins. although hr-like proteins have also been reported in prokaryotes oxygen binding hrs have only been reported from marine invertebrates belonging to annelida brachiopoda, priapulida, bryozoa, and a single species of both cnidaria and arthropoda. given this phylogenetic breadth of animals, whether all metazoan hrs share a common origin is debated. overall, hr proteins exhibit variation in their quaternary structure, and four groups have been reported based mainly on their primary structure and location within animal bodies. specifically, hemerythrins found in vascular tissue, referred to here as circulating hemerythrins, and muscle-specific myohemerythrins, have been better characterized compared to the other two hemerythrin groups, ovohemerythrins and neurohemerythrins. chrs are polymeric intracellular proteins that occur inside nucleated cells, hemerythrocytes or pink blood cells located in coelomic fluid or vascular systems of hr-bearing organisms. in contrast, myohrs are monomeric cytoplasmic proteins present in muscle cells of annelids. the main difference between these groups is the presence of a five-codon insertion found in myohr immediately before the d α-helix. expression of myohrs seems not to be restricted to chrs-bearing organisms, considering that some annelid species possess both myohrs and hemoglobins. the other two groups of hrs, ovohemerythrin and neurohemerythrin, are also intracellular and non-circulating. ovohr was identified in oocytes of the leech theromyzon tessulatum and its presence during oogenesis possibly suggests a complex function in iron storage and detoxification. on the other hand, nhr was recently discovered in neural and non-neuronal tissues from the body wall of the leech hirudo medicinalis, and it exhibits upregulation in response to septic injury. nevertheless, vanin et al. suggested that nhr of leech may in fact be a myohr. such diversification in hr function may have involved gene duplications resulting in new proteins via neo- or subfunctionalization. moreover, ovohrs and nhrs have only been reported in the literature a few times, and more studies are required to understand their function and evolution. annelids have the greatest diversity of oxygen-binding proteins among metazoans and it is the only phylum from which all subtypes of hr proteins have been documented. while hrs of annelids have been studied since the middle of the 20th century, until the 1990s, hrs were recorded only from sipunculids and from a single polychaete family, magelonidae. later, vanin et al. found hrs in a nereid and a leech and bailly et al. discovered hrs genes in seven annelid species, suggesting hrs are broadly distributed in annelids. given the diversity of lifestyles among annelids known to have hrs, and the lack of information about hrs in general, the occurrence and diversity of these molecules may be higher than currently recognized. thus, to examine a wide diversity of annelid taxa for hrs and to further understand how different forms of hrs are evolutionarily related to each other, we employed approaches to survey hrs from a diverse array of annelid transcriptomes in silico. we identified hrs in taxa and further describe the molecular diversity and evolution of hrs in the light of annelid phylogeny. along with this, we assess whether described hr subtypes consist of evolutionary lineages or result of independent adaptations to different organismal tissues.  our bioinformatic analyses recovered a total of unique nucleotide sequences of hemerythrin-like genes. following translation pfam domain evaluation and manual removal of sequences with less than amino acid residues, putative novel hr genes were retained from all taxa examined in this study, representing annelid species in different families. novel hr genes accession numbers for each species is available in additional file the number of expressed hrs in a given species ranged from one in alciopa sp., cossura longocirrata, enchytraeus albidus, schizobranchia insignis, and syllis cf. hyaline to in magelona berkeleyi and phascolosoma agassizii.fig. alignment of nucleotide dataset of few species. showing the five-codon insertion between c α-helix and d α-helix following trimming alignment of translated transcripts possessed residue positions, with nearly all sequences, the exception being aphrodita japonica, starting with a methionine residue. we decided to keep the apparently incomplete sequence from a. japonica due to its high similarity with the remaining sequences. all sequences in the alignment contained signature residues involved in iron binding, indicating putative respiratory function for these putative hrs. for the sequences, were unique and identical for at least two species at the amino acid level. sequences were assembled into a final dataset containing sequences being new, two hr sequences from lingula reevii, a brachiopod, and nine annelid sequences previous used as “queries”, with aligned nucleotides positions. of these, sequences contained a five-codon insertion between the c and d α-helices described for myohr, but not chrs. datasets supporting conclusions of this article are available in the figshare repository, under dois: https://doi.org/ /m figshare. v and https://doi.org/ /m figshare. v every species analyzed possessed at least one copy of myohr gene. bayesian inference rooted using two brachiopod hr sequences revealed two major clades with one corresponding to chrs clade and the other supported monophyly for a myohr clade. the leech nhr sequence included in the dataset was found inside the myohr clade. within the myohr clade low nodal support values and polytomies were found. however, such results are not uncommon for single protein or protein family trees.fig. bayesian tree using mrbayes. rooted with two hr sequences from lingula reevii, a brachiopod. the blue clade represents the chrs othologs sequences with a prediction of the consensus chr sequence quaternary structure using i-tasser. black, purple, and green sequences represent the myohr sequences and orange sequence is the only nhr from hirudo medicinalis. the green clade shows the paralogs sequences from c. variopedatus and m. taylori and the purple clade represents the identical orthologs myohr sequences. the number after the name of each sequence indicates the genbank accession numbers for each hr gene and it is indicated in additional file 2: table s1 our analysis found putative chr sequences lacking the characteristic five-codon insertion before the d α-helix that define myohrs, distributed across nine different families. besides well-known records of chrs in sipunculids, such as themiste pyroides and phascolosoma agassizii, we discovered chrs in the sipunculid thysanocardia nigra as well as six annelid families; amphinomidae aproditidae, capitellidae, oweniidae, sabellidae, and spionidae. the topology of the hr gene tree did not mirror recent phylogenies of annelida based on phylogenomic datasets. for example, we found hr sequences identical at the nucleotide level belonging to distant annelid families indicating a strong conservation among those orthologs. several of these sequences were prepared and sequenced at different times, making cross contamination unlikely. those identical sequences differed % from the consensus of all others myohr sequences and the majority of nonsynonymous substitutions are concentrated in a and b α-helices. regarding paralogs multiple copies of hr genes were found for several species, including two paralogs from both chaetopterus variopedatus and mesochaetopterus taylori, with these paralogs forming a monophyletic clade. both species were from chaetopteridae suggesting a recent paralogous duplication. given the presence of multiple hrs, apparently early annelids already contained several copies of hr genes, with some paralogs arising later. differences in evolutionary rates between chrs and myohrs sequences was accessed and relative rates of change in different positions were calculated using two different approaches. sites with high variation were found not to have significantly different rates among inter-helices sites using diverge. for helix regions a and c α-helices had one site each with high evolutionary rate, while d α-helix did not have any sites with a high evolutionary rate, indicating a highly conservative region. at the same time, b α-helix had five sites, suggesting that this helix is evolving faster than others. relax was also used to assess differences in selection on chrs relative to myohrs while accounting for lineage-specific rate differences. similar to the diverge analyses, no significant differences were found between the two sets of genes. thus, the gene duplication event leading to chrs versus myohrs does not seem to have been accompanied by a significant change in substitution rate or selection differences on the two gene lineages. alternatively, any evidence of such a rate or selection difference could have been lost during the 500+ my since these genes diverged.fig. hr showing differences in evolutionary rates between chrs and myohrs sequences calculated with diverge using a cutoff. a and c α-helices presented one site each above the cutoff value, d α-helix did not present any sites, and b α-helix presented five sites. colored dots represent sites above cutoff value, and different colors are only to aid illustration discussion as demonstrated here expression of hr genes among annelids is much more common than previously reported, revealing unrecognized diversity of these genes in this phylum. all of the examined species possessed actively transcribed myohrs, while chrs were less frequently recovered from these transcriptomes. although this diversity and wide distribution of hrs in annelids could, in part, be explained by the need to carry oxygen secondary functional specializations could also be important for driving diversification. for example, hrs participate in iron storage, metal detoxification, and immunity in some annelids. our findings build on recent publications demonstrating that sequence diversity among hr-bearing species was larger than traditionally suspected. however, those studies were based predominately on genomic data. in this case, use of transcriptomes shows that hrs genes were present and expressed. bayesian phylogenetic reconstruction recovered monophyletic clades for annelid chrs sequences as well as myohrs sequences with strong support corroborating vanin et al.’s previous findings. the presence of multiple copies of myohr genes across the annelid phylogeny implies these proteins have undergone several instances of gene duplication during their evolution, as previously reported for other bacterial, archeal, and eukaryotic taxa. moreover, the unexpected diversity of myohrs could be associated with functional diversification of this gene, as observed for myoglobins and also for hrs involved in heavy metal detoxification and aspects of innate immunity. classification of specific hr subtypes was not validated by the gene genealogy. although our analyses used whole organisms, our results failed to recover hr proteins that corresponded to ovohrs or nhrs. these categories, however, were described based on limited differences in amino acid sequence and do not reflect distinct monophyletic subgroups within the hr gene family. given this, recognizing only two primary types of hrs, circulating hrs and non-circulating hrs, is perhaps more appropriate. although tentative, this reinterpretation deserves further consideration. incongruence between our gene genealogy relative to current knowledge of annelid evolutionary history indicates that hrs have a complex history which possibly involved events of gene losses and duplications, paralogs replacements and lateral gene transfer. although previous work supported the idea of a monomeric protein as an ancestral myohr within annelids, the presence of chrs in paramphinome jeffreysii, galathowenia oculata, phascolosoma agassizii, themiste pyroides, and thysanocardia nigra, all members of lineages near the base of the annelid tree, indicates that both chrs and myohrs were likely present in the ancestor of annelids, which date back to the cambrian. interestingly, hrs from multiple species, representing hundreds of millions of years of evolution, possessed identical amino acid, and in some cases nucleotide, sequences, suggesting a level of conservation and selection orders of magnitude greater than most of the genome. additional studies across metazoan together with studies of the gene structure of hr proteins and physiological aspects of organisms are the next important steps toward a better understanding of the evolutionary patterns involved in this family of oxygen carrying proteins.fig. lophotrochozoa and annelid relationships based on current knowledge. underlined phyla represent the hr-bearing representants. annelid taxa in blue possess both chr and myohr genes and taxa in red possess just myohr genes  our findings demonstrate that sequence diversity among hr-bearing annelid species is much greater than traditionally suspected and that many of these hrs are actively expressed. there are two primary types of hrs circulating hrs and non-circulating hrs, instead of the four subtypes reported in the literature. incongruence between our gene genealogy relative to current knowledge of annelid evolutionary history indicates that hrs have a complex history. our findings indicate that both chrs and myohrs were likely present in the ancestor of annelids, as both subtypes occur in all lineages near the base of the annelid tree. 
gene tree topology supported monophyly of chrs and a myohr clade that included nhrs sequences, indicating these designations are functional rather than evolutionary. notably, the topology of the hr gene tree did not mirror the phylogeny of annelida as presently understood, and we found evidence of extensive hr gene duplication and loss in annelids. despite extensive study on hemoglobins and hemocyanins, little is known about hemerythrin evolutionary history. sequences of putative hr genes were identified from annelid species in different families and bayesian inference revealed two major clades with strong statistical support.
 despite extensive study on hemoglobins and hemocyanins, little is known about hemerythrin evolutionary history. four subgroups of hrs have been documented, including: circulating hr, myohemerythrin, ovohemerythrin, and neurohemerythrin. annelids have the greatest diversity of oxygen carrying proteins among animals and are the only phylum in which all hr subgroups have been documented. to examine hr diversity in annelids and to further understand evolution of hrs, we employed approaches to survey annelid transcriptomes in silico.  sequences of putative hr genes were identified from annelid species in different families and bayesian inference revealed two major clades with strong statistical support. notably, the topology of the hr gene tree did not mirror the phylogeny of annelida as presently understood, and we found evidence of extensive hr gene duplication and loss in annelids. gene tree topology supported monophyly of chrs and a myohr clade that included nhrs sequences, indicating these designations are functional rather than evolutionary.  the presence of several chrs in early branching taxa suggests that a variety of hrs were present in the common ancestor of extant annelids. although our analysis was limited to expressed-coding regions, our findings demonstrate a greater diversity of hrs among annelids than previously reported. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords blood pigmentsrespiratory proteinstranscriptomeannelidahttp://dx.doi.org/ /100000155division of environmental biologydeb-1036537halanych kenneth m. http://dx.doi.org/ /100000141division of ocean sciences1155188halanych kenneth m. issue-copyright-statement© the author 2017 