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Discovery of Novel Hemocyanin-Like Genes in Metazoans.

Introduction

Four families of oxygen-binding and/or transporting proteins have been characterized into two major groups: iron based, including hemoglobins (Hbs) and hemerythrins (Hrs), and copper based, including two non-homologous families of hemocyanins (Hcs) (Terwilliger et al., 1976; Burmester, 2002; Coates and Decker, 2017). These proteins are inextricably linked to metazoan evolution because they are constrained by oxygen requirements of tissues; and, therefore, selection has presumably favored proteins that can reversibly bind and transport oxygen (Terwilliger, 1998; Schmidt-Rhaesa, 2007). Although these molecules can reversibly bind oxygen, their binding affinities and evolutionary origins differ, and it is generally regarded that the diversity of blood pigments in animals has been underestimated (Martih-Duran et al., 2013; Koch et al., 2016; Costa-Paiva et al., 2017a, b).

Hemocyanins are extracellular, oligomeric proteins that bind molecular oxygen reversibly between two copper ions ([Cu.sub.A] and [Cu.sub.B]; Markl and Decker, 1992) in a side-on bridging arrangement. These megamolecules, which can be similar in size to viruses or ribosomes (up to 13 MDa), are found predominantly in the hemolymph of gastropod molluscs and numerous arthropods (Burmester, 2002; Coates and Decker, 2017). They are also characterized by their affinity for oxygen, which can vary from low to moderate; and they are regulated by a variety of allosteric factors (e.g., urea, calcium) related to specific ecophysiological adaptations (Decker et al., 2007). Arthropod and mollusc Hcs, despite being given the same name due to the conserved dicupric active sites, are distinct in their sequence and structural compositions (Terwilliger, 1998; van Holde et al., 2001). Hcs present in Arthropoda (HcAs) are organized into hexamers, where each subunit has three separate domains: (I) five or six a-helices, (II) four a-helices grouped together, which contain the dicopper active site, and (III) seven antiparallel strands forming a [beta]-barrel (Magnus et al., 1994). HcAs are members of a protein superfamily that includes (a) insect and crustacean phenoloxidases whose functions include sclerotization of the cuticle, wound healing, and humoral immune defense (Whitten and Coates, 2017); (b) hexamerins (HEX), metal-free proteins present in insects that do not bind oxygen but that are considered storage proteins associated with molting cycles or nutritional conditions (Burmester, 1999a); (c) copper-free pseudo-hemocyanins or cryptocyanins (pHc or CRY) that are similar to hemocyanin but appear to act as storage proteins in the hemolymph (Burmester, 1999b); and (d) hexamerin receptors (reHEX) that are present in dipterans and are related to their own ligands (Burmester and Scheller, 1996). Although these proteins form a functionally diverse superfamily, primary structures have highly conserved core elements that allow their evolutionary history to be traced (Burmester, 2001).

Mollusc Hcs (HcMs) consist of paralogous functional units derived from successive gene duplication events (Markl, 2013). They are large proteins (up to 13 MDa) with numerous polypeptide channels consisting of 10-12 or more peptides and 7 or 8 functional units connected to each other by bridging peptides (Van Holde et al., 2001). Each functional unit consists of two domains: the tyrosinase or a-helical core domain where the active site is located, and the [beta]-sandwich domain (Cuff et al., 1998). Functionally, the [alpha] domain in HcMs corresponds to domain II in HcAs, and the [beta] domain corresponds to domain HI (Decker et al., 2007). There is high similarity between HcAs and HcMs near the ligands of the [Cu.sub.B] site, comprising a segment of about 50 residues, or 10% of the polypeptide chain length (Drexel at al., 1987;Preaux et al., 1988). HcAs and HcMs are structurally heterogeneous and represent two different classes of proteins; however, the similar segment around [CU.SUB.B] indicates that both Hcs have evolved independently from a common ancestral mononuclear copper protein (Markl and Decker, 1992).

Despite extensive studies on Hcs over the years (Burmester 2002, 2015; Coates and Nairn, 2014), knowledge remains limited for animals, except molluscs and arthropods, where Hcs are broadly distributed (Burmester, 2001; Lieb et al., 2001; Markl, 2013; Kato et al., 2018). Isolated records of Hc in single species of Porifera (Amphimedon queenslandica), Hemichordata (Saccoglossus kowalevski), and Ctenophora (Mnemiopsis leidyi) (Martfn-Duran et al., 2013) have been reported. The presence of He-like proteins in the tunicate Ciona intestinalis, with putative phenoloxidase-like activity, suggested that respiratory Hcs evolved from a phenoloxidase (Immesberger and Burmester, 2004), in addition to the well-characterized inducible phenoloxidase activity of HcAs and HcMs (reviewed by Coates and Nairn, 2014). Thus, given the absence of functional data for non-bilaterian animals (i.e., ctenophores and sponges), and given recent evidence concerning greater Hc distribution in animals, we revive the discussion about whether all metazoan Hcs shared a common origin with frequent losses (Martfn-Duran et al., 2013), or whether Hcs originated separately after the Lophotrochozoa and the Ecdysozoa diverged (van Holde et al., 2001). In addition to metazoan records, similarities of HcAs with sequences in fungi (Aspergilus niger) have also been reported, suggesting that the origin of Hcs (or type III copper proteins, more generally) occurred in Opistho-konta, followed by multiple independent loss events (Martin-Duran et al., 2013). However, evidence is scant to establish what the function of this ancestral protein might have been (Burmester, 2015), and large gaps in sampling exist across Metazoa. In order to understand the range of natural variation in Hcs, we searched for Hcs in a diverse array of metazoan transcriptomes by using a phylogenetic approach to examine Hc evolutionary history in the context of animal phylogeny.

Materials and Methods

Sample collection

Information on species employed herein is provided in Tables 1 and A1. Transcriptomes of these species were collected using a variety of techniques, which included intertidal sampling, dredge, and box cores. Samples collected were preserved in RNALater (ThermoFisher Scientific, Waltham, MA) or frozen at-80 [degrees]C.

Data collection and sequence assembly

Methods for RNA extraction, cDNA preparation, and high-throughput sequencing generally followed Kocot et al. (2011) and Whelan et al. (2015). Total RNA was extracted either from whole animals (for small specimens) or from the body wall and coelomic region (for larger specimens). RNA purifications were performed after extraction using TRIzol (Invitrogen, Carlsbad, CA) or the RNeasy kit (Qiagen, Hilden, Germany) with on-column DNase digestion (Qiagen). Reverse transcription used a single-stranded RNA template and the SMART cDNA Library Construction Kit (Clontech, Mountain View, CA) with double-stranded cDNA synthesis employing the Advantage 2 PCR (polymerase chain reaction) system (Clontech). The cDNA libraries were barcoded and sequenced by using Illumina (San Diego, C A) technology by the Genomic Services Lab at the Hudson Alpha Institute (Hunts-ville, AL). 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. Cd-hit (Li and Godzik, 2006) was employed to look for redundant sequences. In order to facilitate sequence assembly, PE transcriptome data were digitally normalized to an average k-mer coverage of 30 using normalize-by-median. py (Brown et al., 2012; McDonald and Brown, 2013), and data were assembled using Trinity r2013-02-25 with default settings (Grabherr et al., 2011).

Data mining and gene identification

Methods employed here were similar to those described in Costa-Paiva et al. (2017a). Two approaches were utilized to mine transcriptomic data from 179 metazoan species and 2 choanoflagellate species for putative Hc genes in silico (Table A1).

The first approach employed BLASTX (Altschul et al., 1990) at an e-value cutoff of [10.sup.-6] in order to compare each assembled transcriptome contig ("queries") to a protein database composed of 22 Hc sequences of at least 500 amino acid residues from the National Center for Biotechnology Information (NCBI) database (Table A2). The BLASTX approach assured that any transcriptome contig with a significant "hit" to an Hc would be further evaluated in the pipeline. Then, initial contigs recovered from these BLASTX searches were utilized in a second set of BLASTX searches against the NCBI protein database (minimum e-value of [10.sup.-10]), and only top hits longer than 600 nucleotides were retained and considered as putative Hc genes.

A second approach processed the transcriptomic data from the same species (Table Al) through the Trinotate annotation pipeline (Grabherr et al., 2011), which utilizes a BLAST-based approach to provide, among others, GO annotation (Gene Ontology Consortium, 2004). Transcripts annotated as Hcs, using the [10.sup.-5] e-value cutoff obtained by using BLASTX. were also considered putative He-like gene orthologs.

Contigs identified as putative Hc genes using the two approaches described above were subsequently translated into amino acids using TransDecoder with default settings (Haas et al., 2013). All translations were additionally subject to a Pfam domain evaluation using the EMBL-EBI database with an e-value cutoff of [10.sup.-5]. Translations that returned Hc domains N, M, or C or Hc beta associated with tyrosinases in Pfam and that were longer than 200 amino acid residues were retained for subsequent analyses. Transcripts passing the criteria described above were considered Hc genes (Tables 1, A1).

Sequence alignment

Because Hcs have been treated as two distinct proteins (Terwilliger, 1998), two protein data sets were formed based on the Pfam domain evaluation results: (a) an HcM data set, and (b) an HcA data set.

HcM data set sequence alignment. The HcM data set included eight mollusc sequences previously used as "queries" (Table A2) and five new sequences from translated transcripts. Because HcM consists of a series of functional units that contain [alpha] and [beta] domains, we opted to use partial sequences consisting of two functional units (2[[alpha] domain + [beta] domain]) for each sequence, with the exception of Meso-chaetopterus sequences, which presented just one single functional unit each. The data set was aligned with MAFFT using the default "FFT-NS-2" 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 et al., 2012) to exclude 5' residues of the putative start of a tyrosinase (tyr) domain and 3' residues following the amino acid subsequent to the end of the second Hc domain. The resulting alignment was used for all subsequent analyses (Supplemental File 1, available online).

HcA data set sequence alignment. The HcA data set was formed using 40 HcA superfamily sequences (Burmester, 2001; Aguilera et al., 2013; Martin-Duran et al., 2013; Table A3) and a remaining 16 sequences from translated transcripts presenting at least 2 of the 3 HcA domains I, II, and in. The data set was aligned with MAFFT, using the default "FFT-NS-2" 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, aligned sequences were trimmed using trimAl (Capella-Gutierrez et al., 2009) with a 90% gap threshold in order to eliminate poorly aligned regions. The resulting alignment was used for all subsequent analyses (Supplemental File 2, available online).

Phylogenetic analysis

For each data set, ProtTest3.4 was applied to carry out statistical selection of best-fit models of protein evolution for each data set separately, using the Akaike and the Bayesian Information Criteria methods (Darriba et ai, 2011).

Bayesian phylogenetic inference was performed with MrBayes 3.2.1 (Ronquist and Huelsenbeck, 2003) for each database separately, employing two independent Markov chain Monte Carlo runs. In each run, four Metropolis-coupled chains were sampled every 500th cycle for 10 (7) generations. In order to confirm whether chains achieved stationarity and to determine an appropriate burn-in, we evaluated trace plots of all MrBayes parameters output in Tracer v1.6 (Rambaut et al., 2014). The first 25% of samples were discarded as burn-in, and a majority-rule consensus tree was generated using MrBayes. Bayesian posterior probabilities were used for assessing statistical support of each bipartition.

Contamination screening

We undertook procedures to reduce the chance of false positives in our analysis, meaning genes that are not homologous to Hcs or genes obtained from contamination. In most samples included in our work, the total RNA was extracted from the body wall and coelomic region, which excluded any possible contamination from food residues. Moreover, all the species included in our analyses in which Hcs were found were prepared and sequenced separately from any mollusc or arthropod species, which makes cross contamination highly unlikely.

Furthermore, for in silico analyses, we opted for a more conservative approach and employed a very stringent e-value cutoff (<[10.sup.-5]). After translation, proteins from contigs identified as putative Hc genes were subsequently subject to a Pfam domain evaluation as described above and a BLASTP (Altschul et ai, 1990) search against the NCBI protein database (minimum e-value of [10.sup.-10]). The top hits were Hc sequences from either molluscs (for new HcM sequences) or arthropods (for new HcA sequences), and this could be easily explained because sequences of Hcs from other metazoan species are still rare in NCBI.

Therefore, because we opted for a very conservative approach, we decided not to include two sequences identified as HcAs in the data set: one from an annelid, Cossura lon-gocirrata, and one from a cycliophoran, Symbion americanus. Although these sequences had an Hc Pfam domain and presented the six conservative histidine residues, they also showed high identity values (>80%) to crustacean Hcs when submitted to BLASTP and MEGABLAST searches.

Results

Mollusc hemocyanin

Our bioinformatic pipeline (Fig. 1) recovered a total of 18 unique protein sequences of mollusc Hc-like genes from 181 transcriptomes representing 15 metazoan phyla and 2 cho-anoflagellate species (Table AI). Following translation, Pfam domain evaluation, and removal of sequences with fewer than 200 amino acid residues, 5 putative novel HcM genes were retained from all taxa examined here, representing 2 annelid species from Chaetopteridae (Table 1; Supplemental File 1, available online). For both choanoflagellate species and all other metazoan phyla, we did not find any HcM gene (Table AI). Alignment of translated transcripts possessed 956 residue positions, and partial sequences consisted of 2 functional units (2[[alpha] domain + [beta] domain]) for each sequence, with the exception of Mesochaetopterus sequences, which had 1 functional unit each. New sequences were combined with 8 publicly available HcMs previously used as "queries" (Table A2), to produce a final data set of 13 HcM sequences (Fig. 2; Supplemental File 1, available online; Costa-Paiva, 2018b).

The Bayesian inference analysis (Fig. 3) revealed two highly supported clades (Fig. 3, posterior probability [p.p.] = 0.99), even though other internal nodes were less resolved, as is often observed in gene genealogies (DeSalle, 2015). Aside from previous mollusc records (Markl, 2013), we found novel HcM genes in two annelid species (Table 1), both within Chaeto-pteridae. All five sequences included canonical functional units composed of [alpha] and [beta] domains. The topology of the HcM gene tree did not mirror recent phylogenies of Mollusca (Kocot et al., 2011). We found a strongly supported subgroup (Fig. 3, pink clade B, p.p. = 0.99) with two Phyllochaetopterus Hcs and HcM sequences from caudofoveats, cephalopods, and bivalves. However, the remaining Phyllochaetopterus sequence formed a clade with other Mesochaetopterus sequences (Fig. 3, blue clade A, p.p. = 0.76), which is in a clade with other polyplacophorans, gastropods, and cephalopods.

Our data were based on available transcriptomes, and the absence of a certain gene when searching in transcriptomes does not necessarily mean that it is absent from the genome. Thus, we carefully addressed the fact that we did not find any HcM genes in any other metazoan and choanoflagellates, as expected (Table A1).

Arthropod hemocyanin

Our bioinformatic analyses for HcA (Fig. 1) recovered a total of 137 unique protein sequences of arthropod Hc-like genes from 181 transcriptomes from 15 metazoan phyla and 2 choanoflagellate species (Table 1). After nucleic acid translation, Pfam domain evaluation, and removal of sequences with fewer than 200 amino acid residues, 16 putative novel genes representing at least 2 of the 3 HcA domains were retained, representing 16 individual species distributed across 4 phyla (Table 1). Of these 16 new putative HcA genes, just one possessed all three canonical arthropod domains (I, II, and III): the annelid Streblosoma hartmanae (Table 1). The remaining 15 sequences contained 2 domains (I and II or II and III). Domain II, the location of the dicopper active site, with six histidine residues, was found in each species; therefore, we included these records as putative HcA genes (Fig. 4). In order to understand the relationship of these putative HcA genes with other members of the Hc superfamily, the alignment of translated transcripts possessing 359 residue positions was performed fora data set containing these 16 putative HcA genes and 40 publicly available sequences from HcA superfamily representatives (Burmester, 2001; Aguilera et at, 2013; Martin-Duran et al., 2013) (Supplemental File 2, available online; Costa-Paiva, 2018a).

The Bayesian inference analysis of the HcA superfamily revealed 5 supported clades: (A) a green clade (Fig. 5, p.p. = 1) formed by hexapod hexamerin sequences; (B) a blue clade (Fig. 5, p.p. = 1) formed exclusively by crustacean HcAs, cryptocyanins, and pseudo-hemocyanins; (C) an orange clade (Fig. 5, p.p. = 1) formed by myriapod and chelicerate HcAs; (D) a pink clade (Fig. 5, p.p. = 1) formed by hexapod and crustacean prophenoloxidase sequences; and (E) a yellow clade (Fig. 5, p.p. = 1) formed by non-arthropod HcA sequences, including the 16 novel sequences from annelids, hemichor-dates, sponges, and ctenophores. The circled clade inside the yellow clade was formed exclusively by ctenophore HcA sequences.

Discussion

Herein, we confirm that the distribution of Hc genes is more widespread than previously reported. Our results describe actively transcribed HcM genes in 2 chaetopterid annelid species and HcA genes in 16 species distributed across 4 metazoan phyla (Table 1; Fig. 6). Of the four phyla, HcAs in Ctenophora, Porifera, and Hemichordata were reported previously (Aguilera et al., 2013; Martin-Duran et al., 2013). Importantly, the presence of HcAs in Annelida represents new records. Our work is contrary to the traditional view that Hcs are restricted to molluscs and arthropods (Burmester, 2001; Markl, 2013), corroborating the recent findings of Martin-Duran et al (2013) that the presence of Hcs, as well as other oxygen carrier molecules such as Hrs (Costa-Paiva et al., 2017a) and globins (Koch et al., 2016), is underestimated in animals. Although our results cannot empirically prove that these newly discovered genes effectively transport oxygen, we present evidence here that this function is entirely possible (e.g., orthology to Hcs, Pfam structure, and GO). Additionally, these genes were identified based on sequence similarity to previously well-characterized Hcs, under the assumption that sequence similarity is generally indicative of function (Gabal-don and Huynen, 2004).

Previous studies (e.g., Martin-Duran et al., 2013) have demonstrated that the a domain (tyr domain) has a wide distribution across metazoan lineages, with the exception of arthropods, which can be explained by the expansion and diversification of Hc domain II in this group of animals. However, the tyr domain itself can play several roles in addition to respiratory function, such as melanin biosynthesis (Sugumaran. 2002). Although respiratory function requires the presence of both domains [alpha] and [beta], as we found in molluscs, the presence of few functional units in chaetopterids could indicate another function besides oxygen transport for these molecules, in agreement with recent studies on the functional versatility of Hcs. Examples include antimicrobial peptide production, host-symbiont dynamics, and Hc-derived phenoloxidase activity (Coates and Nairn, 2014; Kremer et al., 2014; Zhuang et al., 2015).

In relation to HcAs, our data corroborate previous findings of HcAs in hemichordates, sponges, and ctenophores (Martin-Duran et al., 2013). Moreover, not all of our newly discovered genes possessed the three domains, but all of them were considered to be an HcA because they possessed domain II, where the active site is located (Decker et al., 2007). The presence of domain II spread across metazoan lineages and the presence of the same domain in amebozoans and in the filamentous fungus Aspergilus niger (Martin-Duran et al., 2013) are likely to be a unikont synapomorphy, as suggested before. Despite previous suggestions that domain I (N domain) of HcA can be used as a specific molecular signature of the

Panarthropoda (Martin-Duran et al., 2013), our findings concerning the presence of domain I in annelids and ctenophores contradict this idea (Fig. 6).

Interestingly, Annelida is the lineage that presents the greatest repertoire of oxygen transport proteins reported to date, possessing all the main superfamily proteins: Hrs, Hbs, Hc As, and HcMs. This fact could be explained by their ancient origin and the early radiation of this group and also by the great diversification of life forms and habitats, leading to a great variety of oxygen absorption and transport strategies inside the body (Schumway, 1979; Rouse and Pleijel, 2001). Furthermore, the high diversity of oxygen-binding proteins can be found even in the same lineage within annelids, as observed in chaetopterids. Based on our data, a single individual of Mesochaetopterus alipes can actively transcribe Hrs (Costa-Paiva et al., 2017b) and mollusc-like Hcs. Other annelids also presented more than one family of oxygen-binding protein: for example, some species of Terebellidae, Opheliidae, and Sipuncula (Bailly et al., 2008; Liu et al., 2013). Such organisms may simultaneously express more than one protein or may have different protein expression in different parts of the body (Bailly et aL, 2008).

Although this unexpected large repertoire of oxygen-binding proteins in an annelid could be partially explained by the need to transport oxygen molecules, secondary functional specializations could also have been an important trigger for the diversification of these molecules throughout their evolutionary history in this group. There are records of Hr molecules involved in other functions besides oxygen loading, such as in the storage of iron atoms, detoxification of heavy metals, and innate immunity pathways of some species of annelids (e.g., Theromyzon tessulatum, Hirudo medicinalis, and Neanthes diversicolor; Baert et al., 1992; Demuynck et al., 1993; Vergote et aL, 2004; reviewed by Coates and Decker, 2017). Moreover, Hcs are known for contributing in many ways to immune defenses, such as the inhibition of viral replication, the production of precursors of antimicrobial peptides, and the conformational switch to a phenoloxidase-like enzyme (Coates and Nairn, 2014; Coates and Talbot, 2018).

Our results support the idea of the presence of Hcs being widespread across metazoans (Fig. 6) with an evolutionary history characterized by frequent losses (Aguilera et al., 2013; Martin-Duran et aL, 2013). Such losses are observed within several lineages of molluscs and arthropods--for example, the gastropod family Planorbidae, which lacks HcM in its he-molymph and utilizes extracellular Hb for oxygen transport (Ochiai et al., 1989; Arndt and Santoro, 1998), probably due to Hb's higher affinity for oxygen than the ancestral Hc (Lieb et al., 2001). The same can be found in crustacean lineages, as branchiopods, ostracods, copepods, cirripeds, and decapods that lost HcA and presented Hb for handling oxygen (Ter-williger and Ryan, 2001).

The revised distribution in expression of HcM and HcA genes across metazoans could be explained by the differences in physiochemical properties of the oxygen-binding domains and the life histories of disparate animal lineages. Obtaining functional data on these newly discovered Hc genes is needed to evaluate the significance of their widespread occurrence in metazoans and oxygen-binding and/or transport proteins in general.

Acknowledgments

We thank Fernando Avila Queiroz for valuable help with the figures. EMC-P was supported by CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior, Brazil). The use of SkyNet computational resources at Auburn University is acknowledged. This work was funded by National Science Foundation grants DEB-1036537 to KMH and Scott R. Santos and OCE-1155188 to KMH. This is Molette Biology Laboratory contribution 86 and Auburn University Marine Biology Program contribution 183. We are grateful to the three anonymous reviewers and the editorial board for their candor.

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Appendix

Table Al

List of all taxa analyzed and total number of contigs after assembly
Table A1
List of all taxa analyzed and total number of contigs after assembly

Taxon                                                   Total contig
                                                        number

Choanoflagelata
Acanthoeca spectabilis W.Ellis, 1930                    198,922
Salpingoeca pyxidium Kent                               202,399
Metazoa
Porifera
Hyalonema papilliferum Schulze, 1899                     58,839
Kirkpatrickia variolosa (Kirkpatrick, 1907)             100,231
Latrunculia apicalis Ridley & Dendy, 1886                76.210
Rossella fibulata Schulze & Kirkpatrick. 1910            40,103
Sympagella mix Schmidt, 1870                             85,237
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

Vallicula multiformis Rankin, 1956                      339,814
Cnidaria
Gersemia antarctica (Kukenthal, 1902)                    20,023
Periphylla periphylla (Peron & Lesueur, 1810)           212,658
Staurozoa gen. sp.                                       45.023
Echinodermata
Apostichopus californicus (Stimpson, 1857)              134,640
Astrotomma agassizii Lyman, 1875                        156,062
Labidiaster annulatus Sladen, 1889                      108,871
Labidiaster sp.                                         168,720
Leptosynapta clarki Heding. 1928                        242,126
Hemichordata
Balanoglossus aurantiaca Girard, 1853                   143,815
Cephalodisctts gracilis Harmer, 1905                     57,139
Cephalodiscus hodgsoni Ridewood, 1907                   200,052
Cephalodiscus nigrescens Lankester, 1905                 11.565
Harrimaniidae gen sp. (from Iceland)                    230,054
Harrimaniidae gen sp. (from Norway)                     274.434
Ptychodera bahamensis Spengel, 1893                     115,310
Rhabdopleura sp.                                           4790
Saccoglossus mereschkowskii Wagner, 1885                145,937
Schizocardium brasiliense Spengel. 1893                 101,493
Stereobalanus canadensis Spengel, 1893                   12,741
Torquaratoridae gen. sp.                                102,971
Annelida
Abarenicola pacifica Healy & Wells. 1959                 94,376
Aeolosoma sp.                                           190,647
Aglaophamus verrilli (Mcintosh, 1885)                   118,343
Alciopa sp.                                             233,051
Amphisamyrha galapagensis Zottoli. 1983                  14,313
Ancistrosyllis groenlandica Mcintosh, 1878               94,924
Andiorrhinus sp.                                        139,858
Ankyrodrilus legaeus Holt, 1965                          54,246
Antarctodrilus proboscidea (Brinkhurst & Fulton, 1979)   49,656
Aphelochaeta sp.                                        165,566
Aphrodita japonica Marenzeller, 1879                    120,025
Arabella sp.                                            217,183
Areco reco Righi, Ayres & Bittencourt, 1978             170,510
Arenkola loveni Kinberg, 1866                            27,028
Arhynchite pugettensis Fisher, 1949                      20,724
Arichlidon gathofi Watson Russell, 2000                 140,980
Aricidea quadrilobata Webster & Benedict. 1887           81,139
Armandia sp.                                            137,440
Aspidosiphon laevis Quatrefages, 1865                   168,072
Auchenoplax crinita Ehlers, 1887                        144,974
Aulodrilus japonicus Yamaguchi, 1953                    109,361
Aulolytus tuberculatus (Schmarda, 1861)                 137,934
Axiothella rubrocincta (Johnson. 1901)                  107,215
Bathydrilus rohdei (Jamieson, 1977)                     226,538
Bdellodrilus illuminatus (Moore, 1894)                   67,562
Bhawania goodei Webster, 1884                            70,615
Boccardia proboscidea Hartman, 1940                     117,570
Cambarincola hold Hoffman, 1963                          46,015
Capilloventer sp.                                       221,627
Chaetogaster diaphanus (Gruithuisen, 1828)              128,034
Chaetopterus variopedatus (Renter, 1804)                147,132
Chaetacanthus magnificus (Grube, 1876)                   95,443
Chaetozone sp.                                          143,597
Chloeia pinnata Moore, 1911                             130,037
Chone sp.                                               106,577
Cirrandus spectabilis (Kinberg, 1866)                   120,244
Clymenella torquala (Leidy, 1855)                       111.567
Cossura longocirrata Webster & Benedict, 1887            75,079
Crucigera zygophora (Johnson, 1901)                     116,092
Dichogaster saliens (Beddard 1893)                       98,665
Diopatra cuprea (Bosc. 1802)                            138,779
Dodecaceria pulchra Day, 1955                           229,501
Dorvdrilus michaelseni Piguet, 1913                     136,096
Eteone sp.                                               41,912
Enchytraeus crypticus Westheide & Graefe, 1992          161,842
Eulalia myriacyclum (Schmarda, 1861)                    110,762
Eunice norvegica (Linnaeus, 1767)                       122,784
Euphrosine capensis Kinberg. 1857                        72,220
Eupolymnia nebulosa (Montagu, 1819)                     139,021
Galathowenia oculata (Zachs, 1923)                      179,612
Galeolaria caespitosa Lamarck, 1818                     143,655
Gatesona chaetophora (Bouche, 1972)                     104,334
Glossodrilus sp.                                        122,993
Glycera americana Leidy, 1855                           126,229
Glycera dibranchiata Ehlers, 1868                       101,455
Glycinde armigera Moore. 1911                            79,528
Glyplonolobdella antarctica (Sawyer & White, 1969)       64,208
Goniada brunnea Treadwell, 1906                          89,398
Harmothoe oculinarum (Storm, 1879)                       94,991
Hemipodia simplex (Gmbe, 1857)                           55,653
Hermenia verruculosu Grube, 1856                        111,026
Hermodice canmculuta (Pallas, 1766)                     110,813
Heteromastus filiformis (Claparede, 1864)               148,196
Histriobdella homari Beneden. 1858                      143,130
Idanihursus sp.                                         201,049
Laetmonice producta Grube, 1876                          73,530
Leanira sp.                                             115,908
Lumbrineris crassicephala Hartman, 1965                 196,426
Lumbrineris perkinsi Carrera-Parra, 2001                144,648
Lysilla sp.                                             104,324
Magelona berkeleyi Jones, 1971                           50,123
Marphysa sanguined (Montagu, 1813)                      110,924
Melinna maculata Webster, 1879                          135,712
Mesochaetopterus alipes Monro, 1928                      83,209

Microphthalmus similis Bobretzky, 1870                  169,427
Myxicola infundibulum (Montagu, 1808)                   217,996
Naineris laevigata (Grube, 1855)                        218,272
Neosabellaria cementarium (Moore, 1906)                  82,479
Nephasoma flagriferum (Selenka, 1885)                   170,216
Nephtys incisa Malmgren, 1865                           188,338
Nicolea macrobranchia (Schmarda. 1861)                   53,572
Nicomache venticola Blake & Hilbig, 1990                124,708
Notomaslus tenuis Moore, 1909                           129,745
Odontosyllis gibba Claparede, 1863                      131,487
Oenone fulgida (Savigny in Lamarck, 1818)               144,726
Ophelina acuminata Orsted. 1843                          81,846
Ophiodromus pugettensis (Johnson. 1901)                  92,341
Ophryotrocha globopalpata Blake & Hilbig, 1990          129,450
Owenia fusifonnis Delle Chiaje, 1844                    106,476
Palola sp.                                              211,279
Paralvinella palmiformis Desbruyeres & Laubier. 1986     85,363
Paramphinome jeffreysii (Mcintosh. 1868)                165.337
Pectinaria gouldii (Verrill. 1874)                       92,091
Phascolosoma agassizii Keferstein. 1866                  87,403
Pherecardia striata (Kinberg, 1857)                     216,722
Phyllochaetopterus prolifica Potts, 1914                193,836
Pista macrolobata Hessle, 1917                          126,764
Prionospio dubia Day. 1961                              119,949
Sabaco elongatus (Verrill, 1873)                         84,082
Schizobranchia insignis Bush, 1905                      102,002
Sclerolinum brattstromi Webb, 1964                      149,694
Scolelepis squamata (Miiller, 1806)                     147.343
Serpula vermicularis Linnaeus, 1767                     151,097
Siboglinum ekmani Jagersten. 1956                       270,658
Siboglinum fiordicum Webb, 1963                          75,226
Sphaerodorum papillifer Moore, 1909                      52,411
Spirobranchus kraussii (Baird, 1865)                    167,761
Sternaspis scutata (Ranzani. 1817)                       10,634
Stemaspis sp.                                            10,878
Streblosoma hartmanae Kritzler. 1971                    108,080
Styiodrilus heringianus Claparede, 1862                 239,935
Stygocapitelia subterranea Knollner, 1934                74,556
Syllis cf. Hyalina Grube. 1863                          106.283
Terebellides stroemii Sars. 1835                        169,760
Tharyx kirkegaardi Blake. 1991                          114,157
Thelepus crispus Johnson. 1901                           67,478
Themiste pyroides (Chamberlin, 1919)                     88,157
Travisia brevis Moore, 1923                              69.827
Trypanosyilis sp.                                       167,501
Brachiopoda
Glottidia pyramidata (Stimpson, 1860)                   131,562
Hemithiris psittacea (Gmelin, 1791)                     103,581
Laqueus californicus (Koch, 1848)                       133,086
Macandrevia cranium (O. F. Miiller, 1776)                  9695
Novocrania anomala (O. F. Miiller, 1776)                117,369
Phoronida
Phoronis psammophila Cori. 1889                         193,702
Phoronopsis harmeri Pixell, 1912                        283,821
Nemertea
Malacobdella grossa (Miiller. 1779)                      79,313
Paranemertes peregrina Coe. 1901                         99,203
Parborlasia corrugatus (Mcintosh. 1876)                 911.662
Tubidaniis polymorphic Renier. 1804                     109,120
Bryozoa
Pectinatella magnified (Leidy. 1851)                    191,465
Cycliophora
Symbion americanus Obst. Funch & Kristensen, 2006       135,725
Entoprocta
Barentsia gracilis M. Sars, 1835                        146,310
Loxosoma pectinaricola Franzen, 1962                    144,339
Platyhelminthes
Acipensericola petersoni Bullard, Snyder, Jensen &
Overstreet, 2008                                        152,140
Cardicola currani Bullard & Overstreet, 2004             86.962
Cardicola palmeri Bullard & Overstreet, 2004             52,837
Elaphrobates euzeti Bullard & Overstreet, 2003          118,013
Elopicola sp.                                            64,384
Hapalorhynchus sp.                                       42,863
Myliobaticola richardheardi Bullard & Jensen. 2008       15,147
Myliobaticola sp.                                        73,883
Psettarium anthicum Bullard & Overstreet, 2006           39,616
Sanguinicola sp.                                        145,041
Selachohemecus olsoni Short. 1954                       135,169
Orthonectida
Orthonectida gen. sp.                                   231,032
Priapulida
Priapulus sp.                                            50.034

Taxon                                           Number and type of
                                                putative Hc genes

Choanoflagelata
Acanthoeca spectabilis W.Ellis, 1930
Salpingoeca pyxidium Kent
Metazoa
Porifera
Hyalonema papilliferum Schulze, 1899
Kirkpatrickia variolosa (Kirkpatrick, 1907)     1 partial HcA, domains
                                                II + III
Latrunculia apicalis Ridley & Dendy, 1886       1 partial HcA. domains
                                                II + III
Rossella fibulata Schulze & Kirkpatrick. 1910
Sympagella mix Schmidt, 1870
Ctenophora
Beroe abyssicola Mortensen, 1927
Coeloplana astericola Mortensen. 1927           1 partial HcA, domains
                                                I + II
Dryodora glandiformis (Mertens. 1833)
Euplokamis dunlapae Mills, 1987
Mnemiopsis leidyi A. Agassiz, 1865              1 partial HcA, domains
                                                II + III
Pleurobrachia bachei A. Agassiz. 1860           2 partial HcAs, domains
                                                II + III

Vallicula multiformis Rankin, 1956
Cnidaria
Gersemia antarctica (Kukenthal, 1902)
Periphylla periphylla
(Peron & Lesueur, 1810)
Staurozoa gen. sp.
Echinodermata
Apostichopus californicus (Stimpson, 1857)
Astrotomma agassizii Lyman, 1875
Labidiaster annulatus Sladen, 1889
Labidiaster sp.
Leptosynapta clarki Heding. 1928
Hemichordata
Balanoglossus aurantiaca Girard, 1853
Cephalodisctts gracilis Harmer, 1905
Cephalodiscus hodgsoni Ridewood, 1907
Cephalodiscus nigrescens Lankester, 1905
Harrimaniidae gen sp. (from Iceland)            1 partial HcA. domains
                                                II + III
Harrimaniidae gen sp. (from Norway)
Ptychodera bahamensis Spengel, 1893
Rhabdopleura sp.
Saccoglossus mereschkowskii Wagner, 1885
Schizocardium brasiliense Spengel. 1893
Stereobalanus canadensis Spengel, 1893
Torquaratoridae gen. sp.
Annelida
Abarenicola pacifica Healy & Wells. 1959
Aeolosoma sp.
Aglaophamus verrilli (Mcintosh, 1885)
Alciopa sp.
Amphisamyrha galapagensis Zottoli. 1983
Ancistrosyllis groenlandica Mcintosh, 1878
Andiorrhinus sp.
Ankyrodrilus legaeus Holt, 1965
Antarctodrilus proboscidea
(Brinkhurst & Fulton, 1979)
Aphelochaeta sp.
Aphrodita japonica Marenzeller, 1879
Arabella sp.
Areco reco Righi, Ayres & Bittencourt, 1978
Arenkola loveni Kinberg, 1866
Arhynchite pugettensis Fisher, 1949
Arichlidon gathofi Watson Russell, 2000
Aricidea quadrilobata Webster & Benedict. 1887
Armandia sp.
Aspidosiphon laevis Quatrefages, 1865
Auchenoplax crinita Ehlers, 1887
Aulodrilus japonicus Yamaguchi, 1953
Aulolytus tuberculatus (Schmarda, 1861)
Axiothella rubrocincta (Johnson. 1901)
Bathydrilus rohdei (Jamieson, 1977)
Bdellodrilus illuminatus (Moore, 1894)
Bhawania goodei Webster, 1884
Boccardia proboscidea Hartman, 1940
Cambarincola hold Hoffman, 1963
Capilloventer sp.
Chaetogaster diaphanus (Gruithuisen, 1828)
Chaetopterus variopedatus (Renter, 1804)
Chaetacanthus magnificus (Grube, 1876)
Chaetozone sp.
Chloeia pinnata Moore, 1911
Chone sp.
Cirrandus spectabilis (Kinberg, 1866)
Clymenella torquala (Leidy, 1855)
Cossura longocirrata
Webster & Benedict, 1887
Crucigera zygophora (Johnson, 1901)
Dichogaster saliens (Beddard 1893)
Diopatra cuprea (Bosc. 1802)
Dodecaceria pulchra Day, 1955
Dorvdrilus michaelseni Piguet, 1913             1 partial HcA, domains
                                                II + III
Eteone sp.
Enchytraeus crypticus
Westheide & Graefe, 1992
Eulalia myriacyclum (Schmarda, 1861)
Eunice norvegica (Linnaeus, 1767)
Euphrosine capensis Kinberg. 1857
Eupolymnia nebulosa (Montagu, 1819)             1 partial HcA. domains
                                                II + HI
Galathowenia oculata (Zachs, 1923)
Galeolaria caespitosa Lamarck, 1818
Gatesona chaetophora (Bouche, 1972)
Glossodrilus sp.
Glycera americana Leidy, 1855
Glycera dibranchiata Ehlers, 1868
Glycinde armigera Moore. 1911
Glyplonolobdella antarctica
(Sawyer & White, 1969)
Goniada brunnea Treadwell, 1906
Harmothoe oculinarum (Storm, 1879)
Hemipodia simplex (Gmbe, 1857)
Hermenia verruculosu Grube, 1856
Hermodice canmculuta (Pallas, 1766)
Heteromastus filiformis (Claparede, 1864)
Histriobdella homari Beneden. 1858
Idanihursus sp.
Laetmonice producta Grube, 1876
Leanira sp.
Lumbrineris crassicephala Hartman, 1965
Lumbrineris perkinsi Carrera-Parra, 2001        1 partial HcA. domains
                                                11 + III
Lysilla sp.
Magelona berkeleyi Jones, 1971
Marphysa sanguined (Montagu, 1813)
Melinna maculata Webster, 1879
Mesochaetopterus alipes Monro, 1928             2HcMs

Microphthalmus similis Bobretzky, 1870
Myxicola infundibulum (Montagu, 1808)
Naineris laevigata (Grube, 1855)
Neosabellaria cementarium (Moore, 1906)
Nephasoma flagriferum (Selenka, 1885)
Nephtys incisa Malmgren, 1865
Nicolea macrobranchia (Schmarda. 1861)
Nicomache venticola Blake & Hilbig, 1990
Notomaslus tenuis Moore, 1909
Odontosyllis gibba Claparede, 1863
Oenone fulgida (Savigny in Lamarck, 1818)
Ophelina acuminata Orsted. 1843
Ophiodromus pugettensis (Johnson. 1901)
Ophryotrocha globopalpata
Blake & Hilbig, 1990
Owenia fusifonnis Delle Chiaje, 1844
Palola sp.
Paralvinella palmiformis
Desbruyeres & Laubier. 1986
Paramphinome jeffreysii (Mcintosh. 1868)        1 partial HcA, domains
                                                n + III
Pectinaria gouldii (Verrill. 1874)
Phascolosoma agassizii Keferstein. 1866
Pherecardia striata (Kinberg, 1857)
Phyllochaetopterus prolifica Potts, 1914        3HcMs
Pista macrolobata Hessle, 1917                  1 partial HcA. domains
                                                II + III
Prionospio dubia Day. 1961
Sabaco elongatus (Verrill, 1873)
Schizobranchia insignis Bush, 1905
Sclerolinum brattstromi Webb, 1964
Scolelepis squamata (Miiller, 1806)
Serpula vermicularis Linnaeus, 1767
Siboglinum ekmani Jagersten. 1956
Siboglinum fiordicum Webb, 1963
Sphaerodorum papillifer Moore, 1909
Spirobranchus kraussii (Baird, 1865)
Sternaspis scutata (Ranzani. 1817)
Stemaspis sp.
Streblosoma hartmanae Kritzler. 1971            1 HcA, domains I + II
                                                + III
Styiodrilus heringianus Claparede, 1862         1 partial HcA. domains
                                                II + III
Stygocapitelia subterranea Knollner, 1934
Syllis cf. Hyalina Grube. 1863
Terebellides stroemii Sars. 1835                1 partial HcA, domains
                                                II + III
Tharyx kirkegaardi Blake. 1991
Thelepus crispus Johnson. 1901                  1 partial HcA, domains
                                                II + III
Themiste pyroides (Chamberlin, 1919)
Travisia brevis Moore, 1923
Trypanosyilis sp.
Brachiopoda
Glottidia pyramidata (Stimpson, 1860)
Hemithiris psittacea (Gmelin, 1791)
Laqueus californicus (Koch, 1848)
Macandrevia cranium (O. F. Miiller, 1776)
Novocrania anomala (O. F. Miiller, 1776)
Phoronida
Phoronis psammophila Cori. 1889
Phoronopsis harmeri Pixell, 1912
Nemertea
Malacobdella grossa (Miiller. 1779)
Paranemertes peregrina Coe. 1901
Parborlasia corrugatus (Mcintosh. 1876)
Tubidaniis polymorphic Renier. 1804
Bryozoa
Pectinatella magnified (Leidy. 1851)
Cycliophora
Symbion americanus Obst.
Funch & Kristensen, 2006
Entoprocta
Barentsia gracilis M. Sars, 1835
Loxosoma pectinaricola Franzen, 1962
Platyhelminthes
Acipensericola petersoni
Bullard, Snyder, Jensen &
Overstreet, 2008
Cardicola currani Bullard & Overstreet, 2004
Cardicola palmeri Bullard & Overstreet, 2004
Elaphrobates euzeti Bullard & Overstreet, 2003
Elopicola sp.
Hapalorhynchus sp.
Myliobaticola richardheardi
Bullard & Jensen. 2008
Myliobaticola sp.
Psettarium anthicum
Bullard & Overstreet, 2006
Sanguinicola sp.
Selachohemecus olsoni Short. 1954
Orthonectida
Orthonectida gen. sp.
Priapulida
Priapulus sp.

                                                GenBank accession
Taxon                                           number

Choanoflagelata
Acanthoeca spectabilis W.Ellis, 1930
Salpingoeca pyxidium Kent
Metazoa
Porifera
Hyalonema papilliferum Schulze, 1899
Kirkpatrickia variolosa (Kirkpatrick, 1907)     MF998096
Latrunculia apicalis Ridley & Dendy, 1886       MF998097
Rossella fibulata Schulze & Kirkpatrick. 1910
Sympagella mix Schmidt, 1870
Ctenophora
Beroe abyssicola Mortensen, 1927
Coeloplana astericola Mortensen. 1927           MF998091
Dryodora glandiformis (Mertens. 1833)
Euplokamis dunlapae Mills, 1987
Mnemiopsis leidyi A. Agassiz, 1865              MF99810I
Pleurobrachia bachei A. Agassiz. 1860           MF998107.
                                                MF998I08
Vallicula multiformis Rankin, 1956
Cnidaria
Gersemia antarctica (Kukenthal, 1902)
Periphylla periphylla (Peron & Lesueur, 1810)
Staurozoa gen. sp.
Echinodermata
Apostichopus californicus (Stimpson, 1857)
Astrotomma agassizii Lyman, 1875
Labidiaster annulatus Sladen, 1889
Labidiaster sp.
Leptosynapta clarki Heding. 1928
Hemichordata
Balanoglossus aurantiaca Girard, 1853
Cephalodisctts gracilis Harmer, 1905
Cephalodiscus hodgsoni Ridewood, 1907
Cephalodiscus nigrescens Lankester, 1905
Harrimaniidae gen sp. (from Iceland)            MF998095
Harrimaniidae gen sp. (from Norway)
Ptychodera bahamensis Spengel, 1893
Rhabdopleura sp.
Saccoglossus mereschkowskii Wagner, 1885
Schizocardium brasiliense Spengel. 1893
Stereobalanus canadensis Spengel, 1893
Torquaratoridae gen. sp.
Annelida
Abarenicola pacifica Healy & Wells. 1959
Aeolosoma sp.
Aglaophamus verrilli (Mcintosh, 1885)
Alciopa sp.
Amphisamyrha galapagensis Zottoli. 1983
Ancistrosyllis groenlandica Mcintosh, 1878
Andiorrhinus sp.
Ankyrodrilus legaeus Holt, 1965
Antarctodrilus proboscidea
(Brinkhurst & Fulton, 1979)
Aphelochaeta sp.
Aphrodita japonica Marenzeller, 1879
Arabella sp.
Areco reco Righi, Ayres & Bittencourt, 1978
Arenkola loveni Kinberg, 1866
Arhynchite pugettensis Fisher, 1949
Arichlidon gathofi Watson Russell, 2000
Aricidea quadrilobata Webster & Benedict. 1887
Armandia sp.
Aspidosiphon laevis Quatrefages, 1865
Auchenoplax crinita Ehlers, 1887
Aulodrilus japonicus Yamaguchi, 1953
Aulolytus tuberculatus (Schmarda, 1861)
Axiothella rubrocincta (Johnson. 1901)
Bathydrilus rohdei (Jamieson, 1977)
Bdellodrilus illuminatus (Moore, 1894)
Bhawania goodei Webster, 1884
Boccardia proboscidea Hartman, 1940
Cambarincola hold Hoffman, 1963
Capilloventer sp.
Chaetogaster diaphanus (Gruithuisen, 1828)
Chaetopterus variopedatus (Renter, 1804)
Chaetacanthus magnificus (Grube, 1876)
Chaetozone sp.
Chloeia pinnata Moore, 1911
Chone sp.
Cirrandus spectabilis (Kinberg, 1866)
Clymenella torquala (Leidy, 1855)
Cossura longocirrata Webster & Benedict, 1887
Crucigera zygophora (Johnson, 1901)
Dichogaster saliens (Beddard 1893)
Diopatra cuprea (Bosc. 1802)
Dodecaceria pulchra Day, 1955
Dorvdrilus michaelseni Piguet, 1913             MF998093
Eteone sp.
Enchytraeus crypticus Westheide & Graefe, 1992
Eulalia myriacyclum (Schmarda, 1861)
Eunice norvegica (Linnaeus, 1767)
Euphrosine capensis Kinberg. 1857
Eupolymnia nebulosa (Montagu, 1819)             MF998094
Galathowenia oculata (Zachs, 1923)
Galeolaria caespitosa Lamarck, 1818
Gatesona chaetophora (Bouche, 1972)
Glossodrilus sp.
Glycera americana Leidy, 1855
Glycera dibranchiata Ehlers, 1868
Glycinde armigera Moore. 1911
Glyplonolobdella antarctica
(Sawyer & White, 1969)
Goniada brunnea Treadwell, 1906
Harmothoe oculinarum (Storm, 1879)
Hemipodia simplex (Gmbe, 1857)
Hermenia verruculosu Grube, 1856
Hermodice canmculuta (Pallas, 1766)
Heteromastus filiformis (Claparede, 1864)
Histriobdella homari Beneden. 1858
Idanihursus sp.
Laetmonice producta Grube, 1876
Leanira sp.
Lumbrineris crassicephala Hartman, 1965
Lumbrineris perkinsi Carrera-Parra, 2001        MF998098
Lysilla sp.
Magelona berkeleyi Jones, 1971
Marphysa sanguined (Montagu, 1813)
Melinna maculata Webster, 1879
Mesochaetopterus alipes Monro, 1928             MF998099,
                                                MF998100
Microphthalmus similis Bobretzky, 1870
Myxicola infundibulum (Montagu, 1808)
Naineris laevigata (Grube, 1855)
Neosabellaria cementarium (Moore, 1906)
Nephasoma flagriferum (Selenka, 1885)
Nephtys incisa Malmgren, 1865
Nicolea macrobranchia (Schmarda. 1861)
Nicomache venticola Blake & Hilbig, 1990
Notomaslus tenuis Moore, 1909
Odontosyllis gibba Claparede, 1863
Oenone fulgida (Savigny in Lamarck, 1818)
Ophelina acuminata Orsted. 1843
Ophiodromus pugettensis (Johnson. 1901)
Ophryotrocha globopalpata Blake & Hilbig, 1990
Owenia fusifonnis Delle Chiaje, 1844
Palola sp.
Paralvinella palmiformis
Desbruyeres & Laubier. 1986
Paramphinome jeffreysii (Mcintosh. 1868)        MF998102
Pectinaria gouldii (Verrill. 1874)
Phascolosoma agassizii Keferstein. 1866
Pherecardia striata (Kinberg, 1857)
Phyllochaetopterus prolifica Potts, 1914        MF998103,
                                                MF998104,
                                                MF998105
Pista macrolobata Hessle, 1917                  MF998106
Prionospio dubia Day. 1961
Sabaco elongatus (Verrill, 1873)
Schizobranchia insignis Bush, 1905
Sclerolinum brattstromi Webb, 1964
Scolelepis squamata (Miiller, 1806)
Serpula vermicularis Linnaeus, 1767
Siboglinum ekmani Jagersten. 1956
Siboglinum fiordicum Webb, 1963
Sphaerodorum papillifer Moore, 1909
Spirobranchus kraussii (Baird, 1865)
Sternaspis scutata (Ranzani. 1817)
Stemaspis sp.
Streblosoma hartmanae Kritzler. 1971            MF998109
Styiodrilus heringianus Claparede, 1862         MF9981I0
Stygocapitelia subterranea Knollner, 1934
Syllis cf. Hyalina Grube. 1863
Terebellides stroemii Sars. 1835                MF998112
Tharyx kirkegaardi Blake. 1991
Thelepus crispus Johnson. 1901                  MF998113
Themiste pyroides (Chamberlin, 1919)
Travisia brevis Moore, 1923
Trypanosyilis sp.
Brachiopoda
Glottidia pyramidata (Stimpson, 1860)
Hemithiris psittacea (Gmelin, 1791)
Laqueus californicus (Koch, 1848)
Macandrevia cranium (O. F. Miiller, 1776)
Novocrania anomala (O. F. Miiller, 1776)
Phoronida
Phoronis psammophila Cori. 1889
Phoronopsis harmeri Pixell, 1912
Nemertea
Malacobdella grossa (Miiller. 1779)
Paranemertes peregrina Coe. 1901
Parborlasia corrugatus (Mcintosh. 1876)
Tubidaniis polymorphic Renier. 1804
Bryozoa
Pectinatella magnified (Leidy. 1851)
Cycliophora
Symbion americanus Obst.
Funch & Kristensen, 2006
Entoprocta
Barentsia gracilis M. Sars, 1835
Loxosoma pectinaricola Franzen, 1962
Platyhelminthes
Acipensericola petersoni
Bullard, Snyder, Jensen &
Overstreet, 2008
Cardicola currani Bullard
& Overstreet, 2004
Cardicola palmeri Bullard
& Overstreet, 2004
Elaphrobates euzeti Bullard
& Overstreet, 2003
Elopicola sp.
Hapalorhynchus sp.
Myliobaticola richardheardi
Bullard & Jensen. 2008
Myliobaticola sp.
Psettarium anthicum Bullard
& Overstreet, 2006
Sanguinicola sp.
Selachohemecus olsoni Short. 1954
Orthonectida
Orthonectida gen. sp.
Priapulida
Priapulus sp.


For underlined taxa, number and type of putative hemocyanin (He) genes and accession numbers are also provided. HcA. arthropod hemocyanin: HcM. mollusc hemocyanin.
Table A2
Queries used to search the assembled translated transcriptomes

Taxon                     Protein

Arthropoda
Archispirosrepnts gigas   Hc subunit type I
Cherax quadricarinatus    Hc
Cupiennius salei          Hc subunit 1
Cyamus scammoni           Hc
Limulus polyphemus        Hc II
Macrobrachium nipponense  Hc
Nebalia kensleyi          Hc
Penaeus monodon           Hc
Periplaneta americana     Hc subunit 1 precursor
Scutigera coleoptrata     Hc subunit A
Zootermopsis nevadensis   Hc
Mollusca
Aplysia California!       Hc
Falcidens crossotus       Hc fgh, partial
Haliotis rubra            Hc type 1
Lepidochitona cinerea     Hc, partial
Nucula nucleus            Hc isoform 1
Octopus bimaculoides      Hc units G and H-like, partial
Sepiella maindroni        Hc
Spirilla spirilla         Hc, partial
Porifera
Amphimedon queenslandica  Phenoloxidase subunit 2-like
Ctenophora
Mnemiopsis leidyi         Hc

Hemichordata
Saccoglossus kowalevski   He-like, partial

Taxon                     GenBank accession number

Arthropoda
Archispirosrepnts gigas   CCC55877.1
Cherax quadricarinatus    AFP23115.1
Cupiennius salei          CAC44749.1
Cyamus scammoni           ABB59715.1
Limulus polyphemus        NP_001301072.1
Macrobrachium nipponense  AHJ90473.1
Nebalia kensleyi          ACV33306.1
Penaeus monodon           AEB77775.1
Periplaneta americana     CAR85701.1
Scutigera coleoptrata     CAC69246.1
Zootermopsis nevadensis   KD[R.sup.2]1641.1
Mollusca
Aplysia California!       CAD88977.1
Falcidens crossotus       CAQ30425.1
Haliotis rubra            ANE23704.1
Lepidochitona cinerea     CBW46988.1
Nucula nucleus            CAH 10286.1
Octopus bimaculoides      XP_014789967
Sepiella maindroni        AGV74427.1
Spirilla spirilla         ADT91164.1
Porifera
Amphimedon queenslandica  XP_003390261.1
Ctenophora
Mnemiopsis leidyi         Contig ML0910 (see supplementary data
                          in Martfn-Duran et at, 2013)
Hemichordata
Saccoglossus kowalevski   ACY92544

All mollusc hemocyanin sequences were also included in the data set
previous to the alignment. He, hemocyanin.


Table A3
Table A3
Arthropod hemocyanin superfamily protein sequences used in Burmester
(2001), Aguilera et al. (2013), and Martin-Durdn et al. (2013). with
gene accession numbers for each species

Protein and taxon         GenBank accession number

Prophenoloxidase
Penaeus monodon           AAD45201
Pacifastacus leniusculus    X83494
Tenebrio molitor          AB020738
Bombvx mori               BBA08368
Manduca sexta             AAC05796
Drosophila melanogaster   NP476812
Neobellieria bullata      AAD45526
Galleria mellonella       AAK64363
Anopheles gambiae         AF004915
Hemocyanin
Arthropoda
Eurypelma californicum    AJ290429
Limulus polyphemus          P04253
Callinectes sapidus       AAF64305
Cupiennius salei          CAC44749
Penaeus semisulcatus      AAM77690
Pacifastacus leniusculus  AAM81357
Panaeus vannamei          CAA57880
Panulirus interruptus       P04254
Homarus americanus        AJ272095
Palinurus vulgaris        CAC69243
Scutigera coleptrata      CAC69246
Non-Arthropoda
Amphimedon quenslandica   XP003390261
Mnemiopsis leidyi         Contig ML0910 (see supplementary data in
                          Martin-Duran et al., 2013)
Mnemiopsis leidyi         Contig ML0463 (see supplementary data in
                          Martin-Duran et al., 2013)
Mnemiopsis leidyi         Contig ML0447 (see supplementary data in
                          Martin-Duran et al., 2013)
Saccoglossus kowalevskii  ACY92544
Cryptocyanin
Cancer magister           AF091261
Pseudo-hemocyanin
Homarus americanus        CAB38042
Homarus americanus        CAB38043
Metacarcinus magister     AAD09762
Hexamerin
Locusta migratoria          U74469
Drosophila melanogaster   NP476624
Periplanela americana     AAB09629
Blaberus discoidalis      AAA74579
Spodoptera litura         CAB55603
Camponotus festinatus     AJ251271
Apriona germari           AAM44045
Plodia interpunctella     AAK71136
Bracon hebetor              125974
Anopheles gambiae         AF020870
Anopheles merits          AF020875


ELISA M. COSTA-PAIVA (1,2,*), CARLOS G. SCHRAGO (1,*), CHRISTOPHER J. COATES (3), AND KENNETH M. HALANYCH (2)

(1) Departamento de Genetica, Laboratorio de Blologia Evolutiva Tedrica e Aplicada, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ CEP 21941-901, Brazil; (2) Department of Biological Sciences, Molette Biology Laboratory for Environmental and Climate Change Studies, Auburn University, Auburn, Alabama 36849; and 'Department of Biosciences, College of Science, Swansea University, Singleton Park, Swansea, Wales SA2 8PP, United Kingdom

Received 11 March 2018; Accepted 14 August 2018: Published online 5 November 2018.

(*) To whom correspondence should be addressed. E-mail: elisapolychaeta@gmail.com (EMC-P), carlos.schrago@gmail.com (CGS).

Abbreviations: GO, Gene Ontology; Hbs. hemoglobins: He, hemocyanin; HcA, arthropod hemocyanin; HcM, mollusc hemocyanin; Hrs. hemerythrins; PCR, polymerase chain reaction; PE, paired end; p.p.. posterior probability; tyr, tyrosinase domain.

Online enhancements: data files.
Table 1
List of taxa analyzed with novel genes and total number of contigs
after assembly

Taxon                                        Total contig number

Metazoa
Porifera
Kirkpatrickia variolosa (Kirkpatrick, 1907)  100,231
Latrunculia apicalis Ridley & Dendy, 1886     76,210
Ctenophora
Coeloplana astericola Mortensen, 1927        222,614
Mnemiopsis leidyi A. Agassiz. 1865           385,798
Pleurobrachia bachei A. Agassiz. 1860         38,856
Hemichordata
Harrimaniidae gen sp. (from Iceland)         230,054
Annelida
Dorydrilus michaelseni Piguet. 1913          136,096
Eupolymnia nebulosa (Montagu, 1819)          139,021
Lumbrineris perkinsi Carrera-Parra, 2001     144,648
Mesochaetopterus alipes Monro, 1928           83,209
Paramphinome jeffreysii (Mcintosh. 1868)     165,337
Phyllochaelopterus prolifica Potts. 1914     193,836

Pista macrolobata Hessle, 1917               126,764
Streblosoma hartmanae Kritzler. 1971         108,080
Stylodrilus heringiamts Claparede. 1862      239,935
Terebellides stroemii Sars, 1835             169,760
Thelepus crispus Johnson, 1901                67,478

Taxon                                  Number and type of putative Hc
                                       genes
Metazoa
Porifera
Kirkpatrickia variolosa
(Kirkpatrick, 1907)                    1 partial HcA. domains II + III
Latrunculia apicalis
Ridley & Dendy, 1886                   1 partial HcA. domains II + III
Ctenophora
Coeloplana astericola Mortensen, 1927  1 partial HcA, domains I + II
Mnemiopsis leidyi A. Agassiz. 1865     1 partial HcA, domains II + III
Pleurobrachia bachei A. Agassiz. 1860  2 partial HcAs. domains II + III
Hemichordata
Harrimaniidae gen sp. (from Iceland)   1 partial HcA. domains II + HI
Annelida
Dorydrilus michaelseni Piguet. 1913    1 partial HcA, domains II + III
Eupolymnia nebulosa (Montagu, 1819)    1 partial HcA. domains II + III
Lumbrineris perkinsi
Carrera-Parra, 2001                    1 partial HcA, domains II + III
Mesochaetopterus alipes
Monro, 1928                            2 HcMs
Paramphinome jeffreysii
(Mcintosh. 1868)                       1 partial HcA. domains II + III
Phyllochaelopterus
prolifica Potts. 1914                  3HcMs

Pista macrolobata Hessle, 1917         1 partial HcA, domains II + III
Streblosoma hartmanae Kritzler. 1971   1 HcA, domains I + II + HI
Stylodrilus heringiamts
Claparede. 1862                        1 partial HcA, domains II + III
Terebellides stroemii Sars, 1835       1 partial HcA. domains II + HI
Thelepus crispus Johnson, 1901         I partial HcA, domains II + III

Taxon                                        GenBank accession number

Metazoa
Porifera
Kirkpatrickia variolosa (Kirkpatrick, 1907)  MF998096
Latrunculia apicalis Ridley & Dendy, 1886    MF998097
Ctenophora
Coeloplana astericola Mortensen, 1927        MF998091
Mnemiopsis leidyi A. Agassiz. 1865           MF998101
Pleurobrachia bachei A. Agassiz. 1860        MF998107. MF998108
Hemichordata
Harrimaniidae gen sp. (from Iceland)         MF998095
Annelida
Dorydrilus michaelseni Piguet. 1913          MF998093
Eupolymnia nebulosa (Montagu, 1819)          MF998094
Lumbrineris perkinsi Carrera-Parra, 2001     MF998098
Mesochaetopterus alipes Monro, 1928          MF998099, MF998100
Paramphinome jeffreysii (Mcintosh. 1868)     MF998102
Phyllochaelopterus prolifica Potts. 1914     MF998I03, MF998104.
                                             MF998105
Pista macrolobata Hessle, 1917               MF998I06
Streblosoma hartmanae Kritzler. 1971         MF998109
Stylodrilus heringiamts Claparede. 1862      MF998110
Terebellides stroemii Sars, 1835             MF998112
Thelepus crispus Johnson, 1901               MF998113

Number and type of putative hemocyanin (Hc) genes and accession
numbers are also provided. HcA, arthropod hemocyanin; HcM, mollusc
hemocyanin.
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Author:Costa-Paiva, Elisa M.; Schrago, Carlos G.; Coates, Christopher J.; Halanych, Kenneth M.
Publication:The Biological Bulletin
Article Type:Report
Date:Dec 1, 2018
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