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. 2017 Sep 6;9(10):2580–2591. doi: 10.1093/gbe/evx181

Broad Phylogenetic Occurrence of the Oxygen-Binding Hemerythrins in Bilaterians

Elisa M Costa-Paiva 1,2,*, Carlos G Schrago 1, Kenneth M Halanych 2
PMCID: PMC5629950  PMID: 29016798

Abstract

Animal tissues need to be properly oxygenated for carrying out catabolic respiration and, as such, natural selection has presumably favored special molecules that can reversibly bind and transport oxygen. Hemoglobins, hemocyanins, and hemerythrins (Hrs) fulfill this role, with Hrs being the least studied. Knowledge of oxygen-binding proteins is crucial for understanding animal physiology. Hr genes are present in the three domains of life, Archaea, Bacteria, and Eukaryota; however, within Animalia, Hrs has been reported only in marine species in six phyla (Annelida, Brachiopoda, Priapulida, Bryozoa, Cnidaria, and Arthropoda). Given this observed Hr distribution, whether all metazoan Hrs share a common origin is circumspect. We investigated Hr diversity and evolution in metazoans, by employing in silico approaches to survey for Hrs from of 120 metazoan transcriptomes and genomes. We found 58 candidate Hr genes actively transcribed in 36 species distributed in 11 animal phyla, with new records in Echinodermata, Hemichordata, Mollusca, Nemertea, Phoronida, and Platyhelminthes. Moreover, we found that “Hrs” reported from Cnidaria and Arthropoda were not consistent with that of other metazoan Hrs. Contrary to previous suggestions that Hr genes were absent in deuterostomes, we find Hr genes present in deuterostomes and were likely present in early bilaterians, but not in nonbilaterian animal lineages. As expected, the Hr gene tree did not mirror metazoan phylogeny, suggesting that Hrs evolutionary history was complex and besides the oxygen carrying capacity, the drivers of Hr evolution may also consist of secondary functional specializations of the proteins, like immunological functions.

Keywords: metazoa, transcriptome, evolutionay history, oxygen-binding protein

Introduction

Oxygen-binding proteins are ancient molecules that probably evolved from enzymes that protected the organism against the toxic oxygen (Terwilliger 1998). Considering that metabolism in metazoans requires oxidation of organic molecules, natural selection has likely favored proteins that can reversibly bind and transport oxygen to body tissues (Schmidt-Rhaesa 2007). In metazoans, four families of oxygen-binding proteins are known, usually divided into two main groups: proteins that are use iron to bind oxygen, including hemoglobins and hemerythrins (Hr), and two nonhomologous families of hemocyanins that are use copper (Terwilliger etal. 1976; Burmester 2002). Although these molecules can reversibly bind oxygen, their binding affinities and evolutionary origins differ and the diversity of blood pigments in animals is clearly underestimated (Martín-Durán etal. 2013; Koch etal. 2016; Costa-Paiva etal. 2017).

The evolution of both hemoglobins and hemocyanins has been extensively studied (Burmester 2002, 2015; Lecomte etal. 2005; Vinogradov etal. 2006; Decker etal. 2007), however knowledge of Hr genes is still limited (Vanin etal. 2006). Hemerythrin is an ancient protein family present in all three domains of life (Fukami-Kobayashi etal. 2007; Bailly etal. 2008; Alvarez-Carreño etal. 2016). However, in animals, Hr records are restricted to marine invertebrates within Annelida (which include sipunculids; Struck etal. 2007; Weigert etal. 2014), Brachiopoda, Priapulida, Bryozoa, and a single species of both Cnidaria (Nematostella vectensis) and Arthropoda (Calanus finmarchicus) (Klippenstein 1980; Vanin etal. 2006; Bailly etal. 2008; Martín-Durán etal. 2013; Costa-Paiva etal. 2017). Bailly etal. (2008) suggested that the Hr gene was lost in the ancestor of deuterostomes and conserved only in a few protostomes, leading to questions of Hr homology across metazoans (Bailly etal. 2008; Martín-Durán etal. 2013). A complex evolutionary history of lateral gene transfer, duplications, and gene loses appear to have play an important role in Hr evolution in animals (Alvarez-Carreño etal. 2016).

Hr sequences were originally characterized from sipunculids (Sanders-Loehr and Loehr 1979) for which three Hr sequences were recorded, two from coelomic hemerythrocytes or circulating Hr (cHrs) from Phascolopsis gouldii and Themiste dyscritum and one myohemerythrin (myoHr) from retractor muscle of Themiste zostericola. The difference between cHrs and myoHr sequences is a five-residue insertion in the myoHr sequence between residues 90 and 91 flanked by the C and D helices (Sanders-Loehr and Loehr 1979; Kurtz 1992). Previous workers reported that there were four distinct subtypes of Hrs: polymeric cHrs and monomeric myoHrs, ovohemerythrins (ovoHr), and neurohemerythrins (nHr) (Baert etal. 1992; Coutte etal. 2001; Vergote etal. 2004). However more recent work (Vanin etal. 2006; Costa-Paiva etal. 2017) has confirmed that there are only two types of Hrs (myoHr and cHr). Recent studies show that occurrence, diversity, and expression of Hrs in animals is much greater than currently recorded (Martín-Durán etal. 2013; Costa-Paiva etal. 2017). We employed a stringent approach to scan for Hrs in a diverse array of metazoan transcriptomes and genomes. We examine Hr evolutionary history in the light of animal phylogeny (Whelan etal. 2015; Halanych 2016; Kocot etal. 2017).

Materials and Methods

Sample Collection

Information on species employed herein is provided in table 1. Transcriptomes of these species were collected as part of the WormNet II project to resolve annelid phylogeny with a variety of techniques, including intertidal sampling, dredge and box cores. All samples collected were preserved in RNALater or frozen at −80 °C.

Table 1.

List of All Taxa Analyzed, Including Total Number of Contigs after Assembly, and Number of Putative Hr Genes (for Undelined Taxa)a

Taxon Total Contigs Number Hr Genes Number Accession Number
CHOANOFLAGELATA — —
Acanthoeca spectabilis W.Ellis, 1930 198,922
Salpingoeca pyxidium Kent, 1881 202,399
METAZOA
Ctenophora
Beroe abyssicola Mortensen, 1927 83,798 — —
Coeloplana astericola Mortensen, 1927 222,614 — —
Dryodora glandiformis (Mertens, 1833) 101,598 — —
Euplokamis dunlapae Mills, 1987 321,550 — —
Mnemiopsis leidyi A. Agassiz, 1865 385,798 —
Pleurobrachia bachei A. Agassiz, 1860 38,856 —
*Pleurobrachia bachei A. Agassiz, 1860 – — —
Vallicula multiformis Rankin, 1956 339,814 — —
Porifera
*Amphimedon queenslandica Hooper and van Soest, 2006 — — —
Hyalonema populiferum Schulze, 1899 58,839 — —
Kirkpatrickia variolosa (Kirkpatrick, 1907) 100,231
Latrunculia apicalis Ridley and Dendy, 1886 76,210
Rossella fibulata Schulze and Kirkpatrick, 1910 40,103 — —
Sympagella nux Schmidt, 1870 85,237 — —
Placozoa
*Trichoplax adhaerens Schulze, 1883 — — —
Cnidaria
*Acropora digitifera (Dana, 1846) — — —
Gersemia antarctica (Kukenthal, 1902) 20,023 — —
*Hydra vulgaris Pallas, 1766 — — —
* Nematostella vectensis Stephenson, 1935 — — —
Hit with NW_001833871.1, Pfam domain not confirmed
Hit with NW_001834356.1, Pfam domain not confirmed
*Orbicella faveolata (Ellis and Solander, 1786) — — —
Periphylla periphylla (Peron and Lesueur, 1810) 212,658 — —
*Pseudodiploria strigosa (Dana, 1846) — — —
Acoela (reads) — —
Childia submaculatum SRX1534054 (29,856,889) — —
Convolutriloba macropyga SRX1343815 (210,917,52) — —
Diopisthoporus gymnopharyngeus SRX1534055 (33,284,316) — —
Diopisthoporus longitubus SRX1534056 (44,491,819) — —
Eumecynostomum macrobursalium SRX1534057 (47,195,086) — —
Isodiametra pulchra SRX1343817 (268,267,139) — —
Echinodermata
Apostichopus californicus (Stimpson, 1857) 134,640 1 KY929257
Astrotoma agassizii Lyman, 1875 156,062 — —
Labidiaster annulatus Sladen, 1889 108,871 — —
Labidiaster sp. 168,720 1 KY929242
Leptosynapta clarki Heding, 1928 242,126 1 KY929245
Hemichordata
Balanoglossus aurantiaca Girard, 1853 143,815 3 KY929217-9
Cephalodiscus gracilis Harmer, 1905 57,139 4 KY929226-9
Cephalodiscus hodgsoni Ridewood, 1907 200,052 1 KY929230
Cephalodiscus nigrescens Lankester, 1905 11,565 1 KY929231
Harrimaniidae gen sp. (from Iceland) 230,054 — —
Harrimaniidae gen sp. (from Norway) 274,434 — —
Ptychodera bahamensis Spengel, 1893 115,310 — —
Rhabdopleura sp. 4,790 — —
Saccoglossus mereschkowskii Wagner, 1885 145,937 — —
Schizocardium brasiliense Spengel, 1893 101,493 — —
Stereobalanus canadensis Spengel, 1893 12,741 6 KY929266-71
Torquaratoridae gen. sp. 102,971
Staurozoa gen. sp. 45,023 — —
Chordata
*Oikopleura dioica Fol, 1872 — — —
*Homo sapiens Linnaeus, 1758 — — —
Annelida
Arenicola loveni Kinberg, 1866 27,028 — —
Arhynchite pugettensis Fisher, 1949 20,724 1 KY929214
Aulodrilus japonicus Yamaguchi, 1953 109,361 2 KY929215-6
Capilloventer sp. 221,627 5 KY929220-4
Chloeia pinnata Moore, 1911 130,037 1 KY929232
Dichogaster saliens (Beddard 1893) 98,665 1 KY929233
Diopatra cuprea (Bosc, 1802) 138,779 1 KY929234
Dodecaceria pulchra Day, 1955 229,501 1 KY929235
Eunice norvegica (Linnaeus, 1767) 122,784 1 KY929236
Hermodice carunculata (Pallas, 1766) 110,813 — —
Lumbrineris crassicephala Hartman, 1965 196,426 2 KY929246-7
Marphysa sanguinea (Montagu, 1813) 110,924 2 KY929248-9
Ophiodromus pugettensis (Johnson, 1901) 92,341 — —
Ophryotrocha globopalpata Blake and Hilbig, 1990 129,450 1 KY929253
Palola sp. 211,279 1 KY929254
Sphaerodorum papillifer Moore, 1909 52,411
Brachiopoda
Glottidia pyramidata (Stimpson, 1860) 131,562 1 KY929237
Hemithiris psittacea (Gmelin, 1791) 103,581 2 KY929239-40
Laqueus californicus (Koch, 1848) 133,086 1 KY929243
Macandrevia cranium (O. F. Müller, 1776) 9,695 — —
Phoronida
Phoronis psammophila Cori, 1889 193,702 1 KY929259
Phoronopsis harmeri Pixell, 1912 283,821
Novocrania anomala (O. F. Müller, 1776) 117,369 1 KY929251
Mollusca
Alexandromenia crassa Odhner, 1920 111,729 — —
Amphimeniidae gen. sp. 130,196 — —
Aplacophora gen. sp. 109,736 — —
Cavibelonia sp. 144,105 — —
Entonomenia tricarinata (Salvini-Plawen, 1978) 147,128 — —
Epimenia babai Salvini-Plawen, 1997 71,819 — —
Falcidens caudatus (Heath, 1918) 132,816 — —
Graptacme eborea (Conrad, 1846) 144,601 1 KY929238
Helluoherpia aegiri Handl and Buchinger, 1996 95,935 — —
Hypomenia sp. 93,699 1 KY929241
Kruppomenia borealis Odhner, 1920 142,815 — —
Leptochiton rugatus (Carpenter in Pilsbry, 1892) 115,512 1 KY929244
Macellomenia sp. 107,525 — —
Meiomenia swedmarki Morse, 1979 118,867 — —
Micromenia fodiens (Schwabl, 1955) 230,891 1 KY929250
Neomenia carinata Tullberg, 1875 172,727 — —
Nuculana pernula (O. F. Müller, 1779) 34,274 1 KY929252
Phyllomenia sp. 170,739 — —
Prochaetoderma californicum Schwabl, 1963 293,209 — —
Proneomeniidae gen. sp. 99,165 2 KY929262-3
Scutopus ventrolineatus Salvini-Plawen, 1968 221,900
Simrothiella margaritacea (Koren and Danielssen, 1877) 99,722 — —
Spathoderma clenchi Scheltma, 1985 111,974 — —
Nemertea
Malacobdella grossa (Müller, 1779) 79,313
Paranemertes peregrina Coe, 1901 99,203 2 KY929255-6
Parborlasia corrugatus (McIntosh, 1876) 911,662
Tubulanus polymorphus Renier, 1804 109,120
Bryozoa
Pectinatella magnifica (Leidy, 1851) 191,465 1 KY929258
Cycliophora
Symbion americanus Obst, Funch and Kristensen, 2006 135,725
Entoprocta
Barentsia gracilis M. Sars, 1835 146,310
Loxosoma pectinaricola Franzen, 1962 144,339
Platyhelminthes
Acipensericola petersoni Bullard, Snyder, Jensen and Overstreet, 2008 152,140
Cardicola currani Bullard and Overstreet, 2004 86,962 1 KY929225
Cardicola palmeri Bullard and Overstreet, 2004 52,837
Elaphrobates euzeti Bullard and Overstreet, 2003 118,013
Elopicola sp. 64,384
Hapalorhynchus sp. 42,863
Myliobaticola richardheardi Bullard and Jensen, 2008 15,147
Myliobaticola sp. 73,883
Psettarium anthicum Bullard and Overstreet, 2006 39,616
Sanguinicola sp. 145,041
Selachohemecus olsoni Short, 1954 135,169 2 KY929264-5
Orthonectida
Orthonectida gen. sp. 231,032
Arthropoda
Calanus finmarchicus (Gunnerus, 1770) –
Hit with ES3871551, Pfam domain not confirmed
Colossendeis megalonyxHoek, 1881 114,203
*Limulus polyphemus (Linnaeus, 1758) –
Priapulida
Priapulus sp. 50,034 2 KY929260-1
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a

GenBank accession numbers are also provided here and detailed in supplementary file 1, Supplementary Material online. Genomes are marked with asterisks, all others are transcriptomes. Transcriptomes of acoels presented total reads numbers, instead of contigs.

Data Collection and Sequence Assembly

RNA extraction, cDNA preparation and high-throughput sequencing generally followed Kocot etal. (2011) and Whelan etal. (2015). Total RNA was extracted from either whole animals (for small specimens) or the body wall and coelomic region (for larger specimens). RNAs were purified after extraction using TRIzol (Invitrogen) or the RNeasy kit (Qiagen) with on-column DNase digestion, respectively. In order to reverse transcribe single stranded RNA template, we used the SMART cDNA Library Construction Kit (Clonetech) and double stranded cDNA synthesis was completed with The Advantage 2 PCR system (Clontech). Libraries were barcoded and sequenced with Illumina technology by The Genomic Services Lab at the Hudson Alpha Institute (Huntsville, Alabama, USA). Because sequencing was performed from 2012 to 2015, Paired End (PE) runs were of 100 or 125 bp lengths, utilizing either v3 or v4 chemistry on Illumina HiSeq 2000 or 2500 platforms (San Diego, California). To facilitate sequence assembly, paired-end transcriptome data were digitally normalized to an average k-mer coverage of 30 using normalize-by-median.py (Brown etal. 2012) and assembled using Trinity r2013-02-25 with default settings (Grabherr etal. 2011).

Data Mining and Gene Identification

Methods employed were similar to those in Costa-Paiva etal. (2017). Two complementary approaches were utilized to mine transcriptomic data from 100 metazoan species and two choanoflagellate species for putative Hr genes in silico (table 1). Additionally, we surveyed genomes, a transcriptome, and ESTs from Genbank for 20 species (table 1) including chordates, cnidarians, ctenophores, acoels, placozoan, and arthropods in order to search for Hr similarity.

The first approach employed BLASTX (Altschul etal. 1990) with e-value cutoff of 10−6 in order to compare each assembled transcriptome contig (“queries”) to a protein database composed of 19 Hrs sequences from the National Center for Biotechnology (NCBI) database (supplementary file 2, Supplementary Material online) of at least 110 amino acid residues and previously identified as Hrs (n = 7), myoHrs (n = 10), or “nHr” (n = 2). The BLASTX approach assured that any transcriptome contig with a significant “hit” to an Hr would be further evaluated in the pipeline. Initial contigs recovered from BLAST searches were then utilized in BLASTX searches against the NCBI protein database (minimum e-value of 10−10) and only top hits longer than 300 nucleotides were retained and considered putative Hr genes.

A second approach processed the transcriptomic data from the same species (table 1) through the Trinotate annotation pipeline (http://trinotate.github.io/) (Grabherr etal. 2011), which utilizes a BLAST-based approach to provide, among others, GO annotation (The Gene Ontology Consortium 2004). Transcripts annotated as Hrs, using the 10−6e-value cutoff obtained by using BLASTX, were also considered putative Hr-like gene orthologs.

Contigs putatively identified as Hr genes by both approaches were subsequently translated into amino acids using TransDecoder with default settings (Haas etal. 2013). Since TransDecoder can produce multiple open reading frames (ORFs), all translations were additionally subject to a Pfam domain evaluation using the EMBL-EBI database with an e-value cutoff of 10−5. Translations returning an Hr Pfam domain and that were longer than 100 amino acids residues were retained for subsequent analyses. Moreover, we manually evaluated the presence of residues involved in iron binding, which are: histidine residues (His) in positions 26, 56, 75, 79, and 108; glutamic acid residue (Glu) in position 60; and aspartic acid residue (Asp) in position 113, numbered by reference sequence T. zostericola. Presence of these signature residues indicates putative respiratory function for Hrs. Transcripts passing the criteria described above were considered Hr genes (table 1).

For additional genomes, transcriptome, and ESTs from Genbank, we employed BLASTP, tBLASTn, or BLASTN (Altschul etal. 1990) depending on the type of data available for each species (table 1), at an e-value cutoff of 10−6. We compared the database with the query composed of 19 Hrs sequences from NCBI database (supplementary file 2, Supplementary Material online) as above. Sequences with a significant “hit” to an Hr were additionally subject to a Pfam domain evaluation using the EMBL-EBI database with an e-value cutoff of 10−5.

Sequence Alignment

The protein data set consisted of 77 sequences, including 19 Hr sequences previous used as “queries” (supplementary file 2, Supplementary Material online), and a remaining 58 sequences from translated transcripts (supplementary file 1, Supplementary Material online). All sequences were initially aligned with MAFFT using the “accurate E-INS-i” algorithm (Katoh and Standley 2013), followed by visual inspection and manual curation in order to remove spuriously aligned sequences based on similarity to the protein alignment as a whole. Subsequently, ends of aligned sequences were manually trimmed in Geneious 9.1.3 (Kearse etal. 2012) to exclude 5′ residues leading to the putative start codon and 3′ residues following the first two amino acids subsequent to the end of the D α-helix. The resulting alignment was used for all subsequent analyses (supplementary file 3, Supplementary Material online).

Phylogenetic Analysis

ProtTest3.4 was applied to carry out statistical selection of best-fit models of protein evolution for the data set using the Akaike and Bayesian Information Criteria (AIC and BIC, respectively) methods (Darriba etal. 2011). Bayesian phylogenetic inference was performed with MrBayes 3.2.1 (Ronquist and Huelsenbeck 2003) with two independent runs with four Metropolis-coupled chains were run for 107 generations, sampling the posterior distribution every 500 generations. In order to confirm if chains achieved stationary and determine an appropriate burn-in, we evaluated trace plots of all MrBayes parameter output in Tracer v1.6 (Rambaut etal. 2014). The first 25% of samples were discarded as burn-in and a majority rule consensus tree generated using MrBayes. Bayesian posterior probabilities were used for assessing statistical support of each bipartition.

Evolutionary Rate Analyses

The protein alignment (supplementary file 3, Supplementary Material online) was also used in DIVERGE (Gu etal. 2013) to examine site-specific shifted evolutionary rates and assesses whether there has been a significant change in evolutionary rate after duplication or speciation events by calculating the coefficient of divergence (θD) and determining if the null hypothesis of no functional divergence between Hrs with the five residue indel between C and D α-helices could be statistically rejected. We employed a cutoff of 0.8 for detection of site-specific shifted evolutionary rates (supplementary file 4, Supplementary Material online).

Results

Our in silico analyses (fig. 1) recovered 238 unique nucleotide sequences of hemerythrin-like genes from 108 transcriptomes and 11 genome gene models encompassing 20 metazoan phyla and two choanoflagellate species (table 1). Following translation, Pfam domain evaluation (presence of Hr domain), and removal of sequences with <100 amino acid residues, 58 putative novel Hr genes were retained from all taxa examined in this study, representing 36 metazoan species distributed in 11 different phyla (table 1, supplementary file 1, Supplementary Material online). Hrs had been reported previously in four out of these 11 phyla, namely, Annelida, Brachiopoda, Priapulida, and Bryozoa (Bailly etal. 2008; Martín-Durán etal. 2013; Costa-Paiva etal. 2017). However, we report Hrs in Echinodermata, Hemichordata, Mollusca, Nemertea, Phoronida, and Platyhelminthes. Tertiary structure of Hrs was inferred using I-TASSER (Yang etal. 2015) and putative respiratory function and high similarity among their tertiary structure was confirmed for representative Hr genes in each newly recorded phylum (fig. 2). We did not find any Hr genes in either choanoflagellate species, Acoela, Arthropoda, Chordata, Cnidaria, Ctenophora, Cycliophora, Entoprocta, Placozoa, Porifera, and Orthonectida (table 1).

Fig. 1.—

Fig. 1.—

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Flow chart of bioinformatics pipeline. Rounded blue rectangles represent input/output files, yellow ovals represent software or scripts, and the green hexagon represents a step which involving manual evaluation. Nineteen metazoan Hrs sequences previous used as query sequences from Genbank (supplementary file 2, Supplementary Material online) were also included in the data set.

Fig. 2.—

Fig. 2.—

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Tertiary structure of Hrs from a representative of each newly recorded phylum was inferred using ITASSER (Yang etal. 2015) and confirmed that all sequences have a putative respiratory function and also showed the high similarity among their tertiary structure. Each figure on the right indicated the position of amino acids related to iron binding. (A) Echinodermata—Leptosynapta clarki; (B) Hemichordata—Balanoglossus aurantiaca; (C) Mollusca—Nuculana pernula; (D) Nemertea—Paranemertes peregrina; (E) Phoronida—Phoronis psammophila; (F) Platyhelminthes—Selachomecus olsoni.

Alignment of translated transcripts included 122 residue positions. All sequences started with a methionine residue and contained signature residues involved in iron binding, indicating putative respiratory function (Thompson etal. 2012). For the 58 putatively novel Hr sequences, 34 were unique and 24 were identical for at least two species at the amino acid level. New sequences were combined with 19 publically available Hrs to produce a final data set of 77 Hr sequences (supplementary files 2 and 3, Supplementary Material online; Figshare file DOI: 10.6084/m9.figshare.4715092).

Bayesian inference analysis (fig. 3) recovered several strongly supported clades, as well as less resolved regions (which are often observed in gene genealogies; DeSalle 2015). We found Hr orthologs in 12 additional annelid species (table 1), augmenting previous counts (Bailly etal. 2008; Costa-Paiva etal. 2017). All 58 novel Hr sequences included the five-residue insertion before the D α-helix, consistent with myoHrs (Costa-Pavia etal. 2017). Those sequences were distributed throughout the gene tree in clades with representatives from other phyla (fig. 3, orange and gray clades). As expected (Costa-Paiva etal. 2017), leech “nHr” was strongly supported (P = 1) as sister lineage to a myoHr sequence from the same species. Similarly, the priapulid “nHr” was a strongly supported as sister lineage to a myoHr sequence from the same priapulid species (fig. 3, yellow clades, P ≥ 0.99).

Fig. 3.—

Fig. 3.—

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Bayesian tree using MrBayes 3.2.1 (Ronquist and Huelsenbeck 2003) midpointed rooted. The blue clade represents cHrs with the five residue deletion between C and D α-helices; gray clades represent clades with protostome myoHr sequences; orange clades represent clades with protostomes and deuterostomes myoHr sequences; green clades represent clades with only deuterostomes myoHr sequences and yellow clades represents sequences of myoHr and “nHr” from a leech and a priapulid. The number after the name of each sequence indicates the GenBank accession numbers for each Hr gene and it is indicated in supplementary file 1, Supplementary Material online.

All new 58 sequences possessed the five-residue insertion before the D α-helix characteristic of myoHrs (Bailly etal. 2008; Costa-Paiva etal. 2017) (fig. 3, except blue clade). We used DIVERGE software (Gu etal. 2013) to look for differences in evolutionary rates between annelid cHrs and other sequences, as well as relative rates of change in different positions were calculated and for helix regions. A and B α-helices each had five sites with an elevated evolutionary rate, whereas C and D α-helices had eight and seven sites, respectively, with elevated rates indicating that later helices are likely evolving faster than the others.

The topology of the Hr gene tree, as expected, did not mirror recent phylogenies of Metazoa based on phylogenomic data sets (Whelan etal. 2015; Halanych 2016; Kocot etal. 2017). We found two clades with exclusively protostome composition. One clade (fig. 3, gray clade, P < 0.8) included representatives of Annelida, Mollusca, Platyhelminthes, Brachiopoda, Nemertea, and Priapulida, and the second clade, the cHr clade (fig. 3, blue clade) included only annelids. Moreover, we found Hrs in eight species of Echinodermata and Hemichordata and observed strongly supported clades (fig. 3, green clades, P = 1) exclusively comprised of deuterostome Hr sequences. However, we also found supported clades (fig. 3, orange clades, P > 0.9) where deuterostomes Hr sequences were clustered with Hr sequences from protostomes as annelids, brachiopods, and mollusk. Because most of our data are from transcriptomes, we must be cautious about comments on the absence of genes as they may be in the genome but were not expressed in the sampled tissue at time of collection. Nonetheless, we did not find any Hr genes in transcriptomes of choanoflagellates, arthropods, cnidarians, ctenophores, cycliophorans, entoprocts, orthonectids, sponges, and acoels (table 1). However, we also screened the available genomes of arthropods, cnidarians, a ctenophore, chordates, a placozoan, and a sponge for Hr genes and obtained negative results, including previous Hr records from Nematostella vectensis and Calanus finmarchicus (Martín-Durán etal. 2013). Sequences from N. vectensis (XP_001634535.1 and XP_001622541.1) did not match any Pfam domain and when a BLASTp search was performed, neither sequence was similar to Hr sequences. Sequence from C. finmarchicus (ES387155), also available in Genbank and assigned as a myoHr, did not match any Pfam domain and overall similarity with other Hr sequences.

Discussion

Distribution of Hr genes spans the breadth of Bilateria, but the absence in nonbilaterians contradicts earlier reports. Previously, the distribution of Hrs was thought to be limited to protostomes, in a single ecdysozan phyla (Priapulida) and three lophotrochozoan phyla (Annelida, Brachiopoda, and Bryozoa) (Vanin etal. 2006; Bailly etal. 2008). Here, we discovered actively transcribed Hrs in 36 species from 11 phyla, including the first ever record in deuterostomes. A previous study (Bailly etal. 2008) suggested that deuterostomes lost Hr genes and there was limited conservation of Hrs in protostome lineages after the protostome–deuterostome split. Nonetheless, since we found expressed Hrs in three species of Echinodermata and five species of Hemichordata, we suggest that, at least the ancestor of Deuterostomes and the ancestor of Ambulacraria had at least one copy of an Hr gene (fig. 4). Moreover, we do not observe Hrs in animal lineages that branched off from other metazoans prior to bilaterians. Note, the prior report of Hrs in a cnidarian (Martín-Durán etal. 2013) that is not homologous to the bilaterian Hrs. Both sequences from N. vectensis previously attributed to Hrs did not match the Hr Pfam domain structure and, also, did not present a significant similarity with Hr sequences when a BLAST strategy was employed. The homology of the previously reported crustacean C. finmarchicus Hr (Martín-Durán etal. 2013) is also not supported based on sequence data, although our analysis of ecdysozoans were limited.

Fig. 4.—

Fig. 4.—

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Hypothesized relationships among metazoan phyla derived from recent phylogenomic studies (Whelan etal. 2015; Halanych 2016; Cannon etal. 2016; Kocot etal. 2017). Pink rectangles represent new Hr records, blue rectangles represent previous records confirmed by our results, and purple rectangle represents exclusively annelid cHrs. MA is metazoan ancestor, BA is bilaterian ancestor, and NA is nephrozoan ancestor.

Martín-Durán etal. (2013) suggested that the metazoan ancestor already had a respiratory functional Hr gene followed by frequent gene losses in various lineages. Our findings suggested a scenario with an Hr-bearing nephrozoan ancestor and not the last common bilaterian ancestor, since we did not find any evidence of the presence of Hrs in nonbilaterian metazoans or in the earliest branching bilaterian lineage, Acoela (fig. 4). Bayesian reconstruction of the Hr gene tree was incongruent to current knowledge of metazoan phylogeny (Whelan etal. 2015; Halanych 2016; Kocot etal. 2017), which is not surprising as this gene family is presumably under heavy selection to supply different demands to carry oxygen in various animal lineages. A similar evolutionary pattern is observed across the three domains of life (for Alvarez-Carreño etal. 2016), or even within annelids (Costa-Paiva etal. 2017). The fact that the gene tree includes subclades with disparate taxa (e.g., echinoderms and annelids, orange clades on fig. 3) suggested that Hrs have a complex history, supporting notions of gene loss and duplication, and possibly lateral gene transfer (Martín-Durán etal. 2013; Alvarez-Carreño etal. 2016).

Traditional classification of specific Hr subtypes in cHrs, myoHrs, ovoHrs, and nHrs (Baert etal. 1992; Coutte etal. 2001; Vergote etal. 2004) was not validated by the gene genealogy. Although many of our transcriptomes used whole organisms (including reproductive and nerve tissues), our results failed to recover Hr proteins that corresponded to ovoHrs or nHrs, corroborating Costa-Paiva etal.’s (2017) previous findings. Classification of myoHrs and cHrs had traditionally relied on differences regarding the monomeric or polimeric form, respectively, and the presence or absence of a five-amino-acid indel between the C and D α-helices (Sanders-Loehr and Loehr 1979; Kurtz 1992; Vanin etal. 2006; Costa-Paiva etal. 2017). The cHr subtype of Hrs, which lacks the five residues between C and D α-helices, is present exclusively in Annelida (fig. 3, blue clade) and represent a novelty in bilaterian Hr evolution. Although rare, there are few records of Hrs present in the vascular systems of priapulids (Weber etal. 1979; Weber and Fange 1980) and brachiopods (Richardson etal. 1987), however they possess five-residues between C and D α-helices. Our findings were able in recognize only two primary types of Hrs, based on molecular differences, myoHrs and exclusive annelid cHrs (Costa-Paiva etal. 2017). Furthermore, our results from DIVERGE, concerning differences in evolutionary rates between annelid cHrs and other sequences, as well as relative rates of change in different positions showed that Hr molecules presented differences in an evolutionary rate, with the C and D α-helices (C and D) evolving faster than the A and B α-helices.

Although the distribution of Hrs in animals is likely tied to the need to deliver oxygen to tissues, as corroborated by our results from modeling the tertiary structure of observed Hr genes (fig. 2), many of metazoan lineages we examined also possess hemoglobins to carry oxygen (Mangum 1992; Coutte etal. 2001). We suggest that although the observed pattern could be explained by the need to carry oxygen, secondary functional specializations could also be important for driving diversification (Coates and Decker 2016). Additional 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.

Supplementary Material

Supplementary data are available at Genome Biology and Evolution online.

Competing Interests

The authors declare that they have no competing interests.

Supplementary Material

Supplementary Data
Click here for additional data file. (46.8KB, zip)

Acknowledgements

We thank Fernando Avila Queiroz for valuable help with the figures. A Fellowship for to E.M.C-P. was provided by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil). Use of SkyNet computational resources at Auburn University is acknowledged. This work was funded by National Science Foundation grant DEB-1036537 to K. M. Halanych and Scott R. Santos and OCE-1155188 to K. M. Halanych. This is Molette Biology Laboratory contribution 69 and Auburn University Marine Biology Program contribution 165.

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