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Hox, Parahox, EHGbox, and NK genes in bivalve molluscs: evolutionary implications.

ABSTRACT Molluscs belong to one of the two protostome superphyla, the Lophotrochozoa. Among molluscs, bivalves show a characteristic shell morphology separated bilaterally into two plates. The connection between the modification of the development and evolution of the molluscan body plan can be study identifying and obtaining information on the genes that regulate the development of bivalves. Homeobox genes are involved in body plan formation and in the regulation of many developmental processes in bilateria. For this reason, the evolutionary history of Hox, ParaFlox, EHGbox, and NK homeobox gene families could be crucial to understand the evolution of molluscan body plans and phylogeny. The aim of this work was to compare these genes from different Bivalvia families. In this study, 22 homeobox gene fragments from five bivalve +species were identified, and then these homeodomain sequences were compared with those available from other bivalves. The Hox cluster in bivalve molluscs has 11 genes. Regarding the ParaHox cluster, current data suggest that the ParaHox genes are also conserved in bivalves. Two EHGbox genes are well conserved in bivalves (en and Gbx); however, there are no data about the presence of the third one (Mnx). The NK cluster has not been examined in depth in Bivalvia; however, the conservation of Tlx and NK2 genes in different species can be confirmed. The study of the genes reported here might contribute to the understanding of their evolutionary history within the phylum and the involvement of these genes in the body plan of bivalves.

KEY WORDS: bivalve, EHGbox, Hox, mollusc, NK genes, ParaHox


Molluscs belong to the superphylum Lophotrochozoa that also includes annelids, platyhelminths, and a range of other phyla members (Graham 2000). They show a typical developmental process that commences as spiral cleavage and develops into trochophore larva (Cragg 2006). Modifications of early development in molluscs lead directly to the innovation of the body plan. Among molluscs, bivalves show a distinct shell morphology; the shell plate is separated bilaterally into two plates. Bivalve molluscs are included in the monophyletic clade Conchifera together with the Monoplacophora, Cephalopoda, Scaphopoda, and Gastropoda classes (Kocot et al. 2011, Smith et al. 2011). Recently, a phylogenetic backbone for Bivalvia has been proposed by Gonzalez et al. (2016). To get a better understanding of the connection between the modification of the development and evolution of the molluscan body plan, it is important to obtain information on the genes and their genetic activities that regulate the development of bivalves.

A set of genes that have been used to gain insight into phylogenetic relationships is homeobox genes. Homeobox genes are involved in body plan formation and in the regulation of many developmental processes in Bilateria. These genes are believed to have diversified before the last common bilaterian ancestor (Brooke et al. 1998, de Rosa et al. 1999, Kourakis & Martindale 2000, Balavoine et al. 2002). Homeobox genes are characterized by a particular DNA sequence, the homeobox, which encodes a helix-loop-helix DNA-binding domain called the homeodomain. Many homeodomain proteins have a role as transcription factors with important roles in embryonic development and cell differentiation (Gehring 1987). The homeobox superfamily can be subdivided into 11 different classes: ANTP, PRD, LIM, POU, HNF, SINE, TALE, CUT, PROS, ZF, and CERS (Holland et al. 2007). The ANTP superclass is composed of four different subclasses: Hox, ParaHox, EHGbox, and NK.

Hox genes are clustered within the genome of many animals and play essential roles in patterning the body plan of metazoans. Development depends on Hox genes, indicating a close link between Hox cluster evolution and evolutionary changes in the anterior-posterior body axis, the evolution of segment diversity (Tessmar-Raible & Arendt 2003), and furthermore, there is evidence that changes in Hox expression patterns and protein functions contributed to a variety of small morphological changes during animal evolution (Carroll et al. 2005). Members of this gene family have been reported in bilaterians, cnidarians (Larroux et al. 2007), placozoans (Jakob et al. 2004), and ctenophores (Ryan et al. 2010), which suggests that Hox genes emerged prior to the divergence between bilaterians and those phyla. The Hox clusters of all bilateria derive from an ancestral cluster that possessed at least eight Hox class genes (de Rosa et al. 1999, Garcia-Fernandez 2005). The number of Hox genes within a cluster varies among the Bilateria, and in some instances, these variations could be due to the emergence of new genes as a result of gene duplication events (Balavoine et al. 2002, Garcia-Fernandez 2005). Hox genes are grouped into four classes: anterior, paralog group (PG) 3, central, and posterior. Siniakov et al. (2013) inferred that the last common mollus-cannelid ancestor had a single 11-gene Hox cluster: 9 anterior, paralog, and central class genes, plus 2 posterior class genes. The Ecdysozoa are characterized by the presence of Hox genes of the Ubx and AbdB types (Greiner et al. 1997), whereas the Lophotrochozoa are characterized by the presence of Hox genes of the Lox5, Lox2, Lox4, Post-1, and Post-2 types that have not been described in nonlophotrochozoan phyla, de Rosa et al. (1999) found that the Lox5, Lox2, and Lox4 genes were also presented in molluscs. Furthermore, they found that both brachiopods and annelids have two distinct Hox genes, called Post-1 and Post-2. Thomas-Chollier et al. (2010) identified 11 Hox genes in the gastropod Lottia gigantea (Sowerby, 1834) genome and found that these genes are clustered on a single scaffold and display the same orientation except for one of them, the last posterior gene. Takeuchi et al. (2012) and Morino et al. (2013) also identified 10 genes in the oyster Pinctada fucata (Hox1, Hox2, Hox3, Hox4, Hox5, Lox5, Lox4, Lox3, Post1, and Post2); however, they were not able to clearly assign another fragment (LoxZ) as ortholog of Antp/Hox7. Zhang et al. (2012) identified the same 10 Hox genes in Crassostrea gigas but not the Antp ortholog. In the same report, Zhang et al. found that the Crassostrea Hox cluster was disrupted.

ParaHox genes are closely related to Hox genes. The three ParaHox genes (Gsx, Xlox, and Cdx) were first described as a gene cluster in the amphioxus Branchiostoma floridae (Hubbs, 1922) by Brooke et al. (1998). It is widely believed that Hox and ParaHox are sister clusters that arose through the duplication of a single and ancestral hypothetical ProtoHox cluster (Brooke et al. 1998, Thomas-Chollier et al. 2010, for review see Ferrier 2010). The expression of these genes suggests that ParaHox genes are involved in the anterior-posterior development of the digestive system and in the patterning of the nervous system (Fritsch et al. 2015, Holland 2001, Samadi & Steiner 2010a).

The EHGbox cluster includes engrailed (en), motor neuron restricted (Mnx), and gastrulation brain homeobox (Gbx). These genes are considered part of the "Extended Hox group" (Pollard & Holland 2000). In cephalopods, the en gene has a role in delimitating the molluscan shell compartment and may be involved in early stages of neurogenesis (Baratte et al. 2007). This gene has also been studied in gastropods (Degnan et al. 1997, Moshel et al. 1998, Nederbragt et al. 2002). The Mnx gene is involved in neuron differentiation (Ferrier et al. 2001). Gbx genes are related to the Drosophila melanogaster (Meigen, 1830) unplugged (unp) gene, whose function is required for the formation of the tracheal branches that penetrate the central nervous system (Chiang et al. 1995). The expression patterns of Gbx family members implicate these genes in the anterior-posterior patterning and in the development of the nervous system (Rhinn et al. 2004, Tour et al. 2001).

The NK homeobox gene cluster (NK cluster) is probably the most ancient homeobox gene duster, dating at least from the base of the Bilateria. The earliest metazoans, like sponges, possessed multiple AK-like genes, but no Hox, ParaHox, or EHGbox genes have been detected in their genome (Larroux et al. 2006, Larroux et al. 2007, Peterson & Sperling 2007, Gazave et al. 2008, Mendivil Ramos et al. 2012) except for the Cdx gene reported recently in two calcareous sponges by Fortunato et al. (2014). The NK cluster in the last common ancestor to protostomes and deuterostomes probably contained a cluster of nine NK genes: Msx, NKl/slouch, NK3/bagpipe, NK4/tinman, Tlx/cl5, NK7, NK6/hgtx, NK5/Hmx, and Lbxl ladybird genes (Holland 2001, Garcia-Fernandez 2005, Larroux et al. 2007, Wotton et al. 2009). In the fly Drosophila melanogaster, the NK homeobox gene cluster retained an ancestral organization (Kim & Nirenberg 1989), but in the chordate lineage, it underwent breakage and dispersal. NK genes are used at all levels of the bilaterian developmental program and play a primary role in the mesoderm patterning and specification of distinct mesodermal derivatives (Garcia-Fernandez 2005, Saudemont et al. 2008).

Identification of homeobox genes would help in the construction of the history of this conserved gene family and their evolutionary relationships. The number and the type of homeobox genes in a particular animal can be indicative of its phylogenetic relationships and may help assign an animal to one bilaterian clade or another. The aim of this work was to compare Hox, ParaHox, EHG, and NK genes from different Bivalvia families. In the present study, 22 homeobox gene fragments from five bivalve species belonging to five different Bivalvia families were identified, and then these homeodomain sequences were compared with those available from other bivalve molluscs. The presence of Hox, ParaHox, EHG, and NK genes in bivalve molluscs and their evolutionary history is discussed in the Results and Discussion sections.



Bivalve molluscs were collected from Noia, Galicia (Spain). Specimens of five species were used in the study, namely the black scallop Mimachlamys varia (Pectinidae), the Mediterranean mussel Mytilus galloprovincialis (Mytilidae), the flat oyster Ostrea edulis (Ostreidae), the razor clam Solen marginatus (Solenidae), and the clam Venerupis pullastra (Veneridae). A total of 40 adults corresponding to the five species were investigated. Tissue samples were either freshly used or frozen.

Genomic DNA Extraction and Polymerase Chain Reaction Conditions

Genomic DNA isolation from the muscular mass of adult bivalves was performed in liquid nitrogen as described by Ashburner (1989) with the modifications specified by Mesias-Gansbiller et al. (2012). Genomic DNA was stored in aliquots at -20[degrees]C. For the polymerase chain reaction (PCR), a previously described degenerate primer pair was used (Carpintero et al. 2004), which correspond to highly conserved homeodomain

regions from helix 1 and 3: 5'-ATGCGGATCCAGACSYTG GARYTGGARAA RGA RTTYCW Y-3 ' and 5'-ATG CAAGCTTCATSCKNCGRTTYTGRAACCARATYTT NAY-3' (Thermo Fisher Scientific, Inc., MA). Polymerase chain reaction was performed in 50-[micro]l reactions containing IX PCR buffer, 3.5 mM Mg[Cl.sub.2], 0.2 mM dNTPs (Roche Molecular Biochemicals, Mannheim, Germany), 0.5 [micro]M of each primer, 1.5 U Platinum Taq DNA Polymerase (Invitrogen, Breda, The Netherlands), and 200 ng of genomic DNA. The PCR cycle involved an initial denaturation step at 95[degrees]C for 5 min, then 39 cycles of denaturation at 95[degrees]C, 1 min, 40[degrees]C, 1 min, and 72[degrees]C, 30 sec followed by 10 min at 72[degrees]C. Amplified PCR products were visualized in a 2% MS-8 agarose (Pronadisa, Spain) gel and were purified according to the manufacturer's instructions for the MiniElute Gel Extraction Kit (Qiagen, Hilden, Germany).

Cloning and Sequencing of Homeobox PCR Products

Purified PCR products were cloned into the pGem-T Easy Vector System II (Promega, Fitchburg, WI), transformed in Escherichia coli JM109, and the DNA of individual clones was recovered using GenElute Plasmid Miniprep Kit (Sigma-Aldrich, St. Louis, MO). Sequencing of the clones was performed using an ABI Prism dRhodamine Terminator Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA). The 22 new sequences were deposited in the EMBL-EBI gene bank and are listed and marked in bold in Figures 1 and 2. Classification of these gene fragments into paralogy groups was determined by comparison of their derived amino acid sequence, using diagnostic amino acids, to the homeodomain of known Hox and ParaHox genes. These included all available Hox, ParaHox, EHG, and NK genes from bivalve molluscs and Drosophila melanogaster, and Lox5 and Xlox from the cephalopod Euprynma scolopes (Berry, 1913). Names were assigned to the new sequences beginning with three letters indicating the abbreviated species name (the first letter of the genus and the first two letters of the specific epithet), followed by the name of the gene.

Data Collection

Amino acid sequences encoded by the Hox, ParaHox, EHG, and NK genes of 15 bivalve molluscs were retrieved: Crassostrea gigas (Thunberg, 1793), Crassostrea virginica (Grnelin, 1791), Ensis ensis (Linnaeus, 1758), Mimachlamys varia (Linnaeus, 1758), Mytilus galloprovincialis (Lamarck, 1819), Ostrea edulis (Linnaeus, 1758), Patinopecten yessoensis (Jay, 1857), Pecten maximus (Linnaeus, 1758), Pinctada fucata (Gould, 1850), Placopecten magellanicus (Grnelin, 1791), Ruditapes philippinarum (Adams & Reeve, 1850), Solen marginatus (Pulteney, 1799), Transennella tantilla (Gould. 1853), Venerupispullastra (Montagu, 1803), and Yoldia eightsi (Couthouy in Jay, 1839). Sequences and accession numbers are listed in Figures 1 and 2. Species names and references are shown in Table 1.

Phylogenetic Analyses

The Hox, ParaHox, EHG, and NK gene sequences isolated in this study, minus the PCR primers, were aligned to the related known homeodomains of Bivalvia by Clustal Omega (Sievers et al. 2011) set at default parameters. The identity between sequences was calculated as a percentage of identity (100 X number of matches/total number of amino acids).

For the phylogenetic analyses of bivalve homeobox sequences, one distance method, neighbor-joining (NJ) (Felsenstein 1996, Saitou & Nei 1987) and two character methods, maximum parsimony (MP) (Felsenstein 1996) and maximum likelihood (ML) (Jones et al. 1992), were used. Distance and character methods were performed using the MEGA5 package (Tarnura et al. 2011). The evolutionary distances were computed using the JTT matrix-based model of amino acid substitution. A discrete gamma distribution was used to model evolutionary rate differences among sites (eight categories). The MP tree was obtained using the close-neighbor-interchange algorithm. A BioNJ tree was used as the input tree to generate the ML tree. The statistical robustness of the nodes was evaluated by bootstrapping (2,000 replicates), and it is shown next to the tree branches.


Identification o/Hox and ParaHox Genes in Bivalves

Twenty-two homeoboxes from Mimachlamys varia, Mytilus galloprovincialis, Ostrea edulis, Solen marginatus, and Venerupis pullastra were recovered. After PCR amplification of DNA, homologues from eight distinct Hox classes were identified: labial (lab) and PG-2 genes from the anterior class, PG-3 class genes, and PG-4, PG-5, Lox5, Hox7, and Lox4 genes from the central class. Cdx genes from the ParaHox family were also isolated from four bivalve species. The corresponding homeodomains were aligned to Drosophila or mollusc orthologues together with all know sequences previously published of bivalves (Figs. 1 and 2). Preliminary searches against public databases revealed a high similarity of the recovered sequences to homeodomains of the ANTP class. The derived amino acid sequences were designated Mga lab (33 aa), Oed lab (23 aa), Sma lab (23 aa), Vpu lab (23 aa), Sma PG-2 (23 aa), Sma PG-3 (23 aa), Vpu PG-3 (23 aa), Mga PG-4 (23 aa), Sma PG-4 (23 aa), Mga PG-5A (23 aa), Mga PG-5B (23 aa), Oed PG-5 (35 aa), Cva Lox5 (23 aa), Sma Lox5 (23 aa), Vpu Lox 5 (23 aa), Vpu Hox7 (39 aa), Sma Lox4 (23 aa), Cva Cad (23 aa), Mga Cdx (23 aa), Oed Cdx (45 aa), Vpu CdxA (23 aa), and Vpu CdxB (23 aa). The derived amino acid sequences included positions 22-44 of the homeodomain or longer. In all these fragments, residues 39-44 of the third a-helix of the homeodomain, which is part of the putative nucleotide sequence recognition site of homeobox proteins, are well conserved.

The orthological assignments of these new Hox genes were determined by phylogenetic analyses by the ML (Fig. 3), neighbor-joining, and MP methods (Appendix Figs. Al and A2) and/or the presence of key diagnostic amino acid residues, which results in the assignment to specific paralog groups (PG).

In the anterior class of the Hox complex, lab and pb orthologues were isolated. The four new laboratory sequences (Mga lab, Oed lab, Sma lab, and Vpu lab) presented three residues that are characteristic of PG-1 proteins in deuterostomes and protostomes, Ala29, Asn41, and Thr43 (Fig. 1). These labial homeodomains have significant amino acid sequence identities with other bivalve members of the PG-1 class (95% 100% identity). The orthology assignment of these lab fragments was also confirmed by phylogenetic analyses (Fig. 3 and Appendix Figs. Al and A2). Mga lab, Oed Lab, Sma lab, and Vpu lab are grouped into a single clade with other bivalve lab genes with moderate/high bootstrap values (ML bootstrap values 62%; MP, 56%; and NJ, 87%).

The PG-2 fragment from Solen marginatus conserved the three amino acid residues that are considered indicative of the pb class, Lys, Cys, and Asp at positions 24, 27, and 39, respectively (Fig. 1). The Solen fragment is identical to the PG-2 fragments from Crassostrea, Mytilus, Pecten, and Patinopecten. Sma PG-2 is placed into the same clade with the bivalve pb sequences with strong support (bootstrap values NJ, 94%; ML, 82%; and MP, 74%; Fig. 3 and Appendix Figs. A1 and A2).

Two new fragments belonged to the PG-3 class, Sma PG-3 and Vpu PG-3. They contain a Leu at position 37 that is characteristic of Ecdysozoa and Lophotrochozoa (Fig. 1). The Arg at position 24 is found in all bilaterians, except in some insects. These fragments have also a significant amino acid sequence identity with other members of the PG-3 class (96%-100%). Phylogenetic analyses confirm the orthology of these genes with high support (NJ, 85%; ML, 78%; and MP, 75%; Fig. 3 and Appendix Figs. Al and A2).

In the central class, members of the Dfd, src, Lox5, Antp, Lox 2, and Lox4 families were isolated (Figs. 1 and 2). The PG-4 fragments from Mytilus and Solen have a Ser at position 41, which is considered indicative of the Dfd class. Mga PG-4 and Sma PG-4 have a significant amino acid sequence identity with other PG-4 proteins (91%-96%). Maximum likelihood phylogenetic analysis groups Mga PG-4 and Sma PG-4 together with other Dfd genes but with low support (Fig. 3), whereas NJ and MP analyses fail to assign these two fragments in the same group with other members of the Dfd family, although they are not included in a different clade either, perhaps due to the short homeodomain sequence that was obtained (Appendix Figs. A1 and A2).

Three fragments from Mytilus and Ostrea showed to be orthologues of the PG-5 group (Fig. 1). First, they conserved a Lys at position 24 and especially the Asn at position 39, which is characteristic of the PG-5 proteins. In the PG-5 group, the central part of the homeodomain is well conserved; the sequences are almost identical within the Mollusca in all the bivalve sequences. All bivalve sequences show a Tyr and a Lys at positions 22 and 24, respectively, whereas other bilaterians exhibit a Phe and an Arg, as reported by Pernice et al. (2006). Second, the homeodomain of the Oed and Mga PG-5 gene product has significant amino acid sequence identity with other members of the PG-5 family from bivalves and Drosophila (91 % 100% identity). Third, phylogenetic analyses confirm that Mga PG-5 A and PG-5B and Oed PG-5 are orthologues of the PG-5 genes with moderate support (NJ, 56%; ML, 49%; and MP 45%; Fig. 3 and Appendix Figs. Al and A2).

The Lox5 sequences (Cva Lox5, Sma Lox5, and Vpu Lox5) present a key signature residue, which is characteristic of Lox proteins in lophotrochozoans, Gly39 (Fig. 1). These Lox homeodomains have significant amino acid sequence identities with other bivalve members of the PG-5 class (91 % 96% identity). The assignment of these Lox5 fragments was also confirmed by phylogenetic analyses (Fig. 3 and Appendix Figs. Al and A2). Cva Lox5, Sma Lox5, and Vpu Lox5 are grouped into a single clade with other bivalve Lox5 genes with moderate/low bootstrap values.

A single gene Antp ortholog was isolated in Venerupis pullastra (Vpu Hox7). The deduced amino acid sequence conserved key residues of the Antp class, Ala37 and Cys39. Arg29 is also well conserved (Fig. 1). A high amino acid identity with other bivalve orthologues was also found. Phylogenetic analyses by NJ. MP, and ML group Vpu Hox7 together with Dme Antp and Yei Antp, although with low support (Fig. 3 and Appendix Figs. Al and A2). The Ruditapes philippinarum Lox4 gene (Tph Lox4) is also grouped in the same clade (Fig. 2) raising some doubts about its correct assignment as an Uhx ortholog (Barucca et al. 2003). Morino et al. (2013) and Zhang et al. (2012) were not able to identify an ortholog of Antp/Hox7 gene in the oysters Pinctada fucata and Crassostrea gigas, respectively. A Hox7 gene was not isolated in the oyster Ostrea edulis either, perhaps due to a not very exhaustive search or alternatively the gene may have been lost in the Ostreidae family.

A Lox4 gene was isolated in Solen marginatus. The signature residues of this class, His24 and Lys29, are present in the Solen fragment (Fig. 2). These residues are clearly primitive and are present in other lophotrochozoans (Pernice et al. 2006). The derived amino acid sequence of Sma Lox4 is identical to other Lox4 fragments from bivalves. The percentage of identity with the Ruditapes Lox4 fragment is lower (86%); moreover, this latest sequence fails to present the signature residues of this class. In fact, TphLox4 clusters with other genes from Antp group (NJ 82%) and presents the signature residues of the Hox7 group. Phylogenetic analyses assign Sma Lox4 to the same group as other bivalve fragments. In this case, the bootstrap values are over 82% (94% NJ; 93% ML; and 82% MP; Fig. 3 and Appendix Figs. A1 and A2).

In the ParaPlox complex, Cdx/Cad homologues were isolated in Mimachlamys, Mytilus, Ostrea, and Venerupis (Fig. 2). Cad sequences present two characteristic residues of the Cdx class, an lie at position 28 and a Lys at position 31, except for a Thr substitution for lie at position 28 in Vpu CdxA. These Cdx homeodomains have significant amino acid sequence identities with other bivalve Cad sequences (87%-96% identity). The assignment of these Cdx fragments was also confirmed by phylogenetic analyses. The fragments Cva Cad, Mga Cdx, Oed Cdx, Vpu CdxA, and Vpu CdxB group into a single clade with other Cad homeodomains with high support (95% NJ; 88% MP; and 78% ML; Fig. 3 and Appendix Figs. A1 and A2).

Diversity of Hox, ParaHox, EHGbox, and NK Genes in Bivalve Molluses

The evolutionary history of homeobox gene families is crucial to understand the evolution of bilaterian body plans and phylogeny. Hox, ParaHox, EHGbox, and NK genes and their distribution in Bivalvia have been studied in several species. According to de Rosa et al. (1999), the number of Hox genes increased from eight genes in the protostome ancestor to at least 10 Hox genes in the lophotrochozoan ancestor (Fig. 4). Different studies are consistent with the presence of a single Hox cluster in Mollusca (de Rosa et al. 1999, Callaerts et al. 2002, Pernice et al. 2006, Biscotti et al. 2007) and particularly in Bivalvia (Barucca et al. 2003, Carpintero et al. 2004, Canapa et al. 2005, Perez-Paralle et al. 2005, Iijima et al. 2006, Zhang et al. 2012, Morino et al. 2013, Lozano et al. 2014).

Adding the new sequences to the sequences already known in bivalves, all Hox genes identified in other lophotrochozoans are also present in bivalve molluscs. On the basis of the predicted amino acid sequences (Figs. 1 and 2) and phylogenetic analyses of the homeodomain (Fig. 3 and Appendix Fig. Al and A2), bivalves possess orthologues to two anterior genes (Hox1 and Hox2), a PG-3 gene, six central class genes (PG-4, PG-5, Lox5, Hox7, Lox4, and Lox2), and two posterior class genes (Fig. 4). According to the phylogenetic analyses of this study and the presence of signature residues, there are several genes that do not seem to be correctly assigned, Pye-Hox5b and TpliLox4 should be included in the Hox7 group and CgiHoxB7 and PyeHox7 in the Lox5 and Lox2 groups, respectively. Thus, the Hox cluster in bivalve molluscs has probably 11 genes (Fig. 4). This is in agreement with the results obtained in the gastropod molluscs Lottia gigantea (Thomas-Chollier et al. 2010) and Gibbula varia (Linnaeus, 1758) (Samadi & Steiner 2009, 2010b) and the bivalves Pecten maximus (Canapa et al. 2005) and Pinctadafucata (Takcuchi et al. 2012, Morino et al. 2013). In the gastropod Patella vulgata (Linnaeus, 1758), only six Hox genes (Hox3, HB1, HB2, HB3, Lox4, and Lox2) were described by de Rosaet al. (1999). In the bivalves Crassostreagigas (Zhang et al. 2012) and Patinopecten yessoensis (Iijima et al. 2006), only 9-10 Hox genes have been identified.

In different species of Cephalopoda, all Hox genes presented in other lophotrochozoans are described with the exception of Hox2/proboscipedia (Callaerts et al. 2002; Pernice et al. 2006) except in Nautilus pompilius (Linnaeus, 1758), where Hox2 has been detected (Iijima et al. 2006). Homologues of this gene have been reported in bivalves (Barucca et al. 2003, Carpintero et al. 2004, This paper) and in gastropods (Degnan & Morse 1993). Then, the search for pb may not have been exhaustive or the gene may have been lost in several cephalopods. In the cephalopod Octopus bimaculoides (Pickford & McConnaughey, 1949), not only pb but also Hox3/zen and Hox4/Dfd could be lost (Albertin et al. 2015). Lophotrochozoans, and among them molluscs, are characterized by the presence of Lox2, Lox4, Lox5, Post-1, and Post-2. These genes are a clue to help assign an organism to one particular bilaterian clade. All these genes have been identified in bivalves (Barucca et al. 2003, Canapa et al. 2005, Iijima et al. 2006, Zhang et al. 2012, This paper). The genes Lox2 and Lox4 are thought to derive by duplication from a single ancestor (Balavoine et al. 2002). Their presence in Cephalopoda, Gastropoda, and Bivalvia, among the Mollusca, and in brachiopods (de Rosa et al. 1999, Canapa et al. 2005, Iijima et al. 2006, Pernice et al. 2006) could indicate that this duplication preceded the divergence between the Lophotrochozoa and the other protostomes. Finally, the post homeodomains of bivalve molluscs show a Ser at position 23 and a Leu or a Lys at position 37, which may be considered mollusc-specific residues (Fig. 2).

Genes belonging to the three ParaHox classes (Gsx, Xlox, and Cdx) are present in Mollusca (Barucca et al. 2003, Barucca et al. 2006, Iijima et al. 2006, Pernice et al. 2006, Samadi & Steiner 2010a); however, in Bivalvia the three ParaHox genes have only been identified in the oysters Pinctada fucata (Takeuchi et al. 2012, Morino et al. 2013) and Crassostrea gigas (Zhang et al. 2012, Paps et al. 2015). In some gastropods (Degnan & Morse 1993, Iijima et al. 2006), cephalopods (Callaerts et al. 2002, Iijima et al. 2006), and some bivalves species (Barucca et al. 2003, Canapa et al. 2005. Iijima et al. 2006, This paper), only Xlox and/or Cdx genes have been identified. Xlox and Cdx genes from bivalves cluster in two different clades with high bootstrap support (Fig. 3 and Appendix). The presence of the Gsx gene cannot be confirmed with the present data. Hox genes can be lost in evolution, and a similar phenomenon could occur with ParaHox genes. This loss could precede the divergence of the lineages or be independently in each lineage. The presence of the three ParaHox genes in deuterostomes and cnidarians (Brooke et al. 1998, Finnerty & Martindale 1999) indicates that the origin of Hox and ParaHox genes occurred prior to the evolutionary split between the Cnidaria and the Bilateria (Salo et al. 2001). The situation in protostomes is more complex because the distribution of ParaHox is more sporadic. In protostomes, all three ParaHox genes have been identified in Sipuncula (Ferrier & Holland 2001) and in the polyplacophora Nuttallochiton mirandus (Thiele, 1906) (Barucca et al. 2006). A Gsx cognate was also identified in the polyplacophora Acanthopleura japonica (Lischke, 1873) (Iijima et al. 2006). In other lophotrochozoan and in ecdysozoan, the studied species possess just one or two ParaHox genes (Cdx and Xlox), also including the acoela Nemertoderma westbladi (Westblad, 1937) (Jimenez-Guri et al. 2006). It has been proposed by Barucca et al. (2006) that the preservation of all three genes in Mollusca, particularly Gsx could be related to the fact that some molluscs exhibit a clear anterior-posterior axis, whereas the presence of two or a single ParaHox gene could be due to secondary losses related to the modifications in body structure that took place in the more evolved molluscan classes. A Gsx gene was detected in the gastropod Gibbula varia, where this gene was involved in the patterning of mouth, foregut, and nervous system (Samadi & Steiner, 2010a). Current data in bivalves suggest that, with the exception of gene losses, the ParaHox genes are as widely conserved as the Hox genes (Fig. 4).

The EHGbox cluster includes engrailed (en), motor neuron restricted (Mnx), and gastrulation brain homeobox (Gbx) (Fig. 4). The en gene was first identified in Bivalvia (the clam Transennella tantilla, the pectinid Placopecten magellanicus, and the oyster Crassostrea virginica) by Wray et al. (1995). The engrailed gene has been also identified in the oysters Pinctadafucata (Zhou et al. 2008) and Crassostrea gigas, where a duplication has been reported (Zhang et al. 2012). All these genes were included in the same clade with moderate/low support (Fig. 3 and Appendix). Paralogues of engrailed have been found in different species of cephalopods (Wray et al. 1995, Baratte et al. 2007), gastropods (Degnan et al. 1997, Nederbragt et al. 2002, Iijima et al. 2008), polyplacophora (Jacobs et al. 2000), and scaphopods (Wanninger & Haszprunar 2001) where the en gene has a conserved role in molluscan shell formation. In relation to the Gbx genes, they have been previously identified in three cephalopods, two gastropods, one solenogaster, and six bivalves. The first Gbx gene identified in molluscs was isolated by Degnan and Morse (1993) in the gastropod Haliotis rufescens (Swainson, 1822). A Gbx gene has also been identified in the cephalopod Euprymna scolopes by Callaerts et al. (2002). Iijima et al. (2006) isolated two Gbx genes in Solenogastres and the cephalopod Nautilus pompilius. In bivalve molluscs, a Gbx gene was isolated from Pecten maximus (Barucca et al. 2003) and Gbx genes were identified in five bivalve species: Solen marginatus, Mimachlamys varia, Venerupis pullastra, Ostrea edulis, and Mytilus galloprovincialis (MeslasGansbiller et al. 2012). Phylogenetic analyses included all Gbx genes in the same group with strong support (Fig. 3 and Appendix Figs. Al and A2). These data make it possible to confirm that the homeodomain of the Gbx family was highly conserved among five distinct families of bivalve molluscs (Solenidae, Pectinidae, Veneridae, Ostreidae, and Mytilidae), and the presence of Gbx genes in Mollusca, Annelida, Echiura, Echinodermata, and Arthropoda supports the idea that Gbx genes were already present in the last common ancestor of the deuterostomes and protostomes. There are no data available about Mnx genes in bivalves.

Regarding NK homeobox genes, six or seven NK genes probably existed in the last common ancestor of sponges and eumetazoans. The NK cluster in the last common ancestor to protostomes and deuterostomes probably contained a cluster of nine NK genes: Msx, NK1/slouch, NK3/bagpipe, NK4/tinman, Tlx/t-15, NK7, NK6/hgtx, NK5/Hmx, and Lbx j ladybird (Holland 2001, Garcia-Fernandez 2005, Larroux et al. 2007, Wotton et al. 2009). The NK gene cluster has not been examined in depth in Mollusca. One Msx gene was isolated from the gastropod Lymnaea stagnalis (Linnaeus, 1758) (Iijima et al. 2006), and an NK4 gene was shown to be involved in muscle development in the cephalopod Sepia officinalis (Linnaeus, 1758) (Navet et al. 2008). Related to Bivalvia, in Crassostrea gigas, six NK genes (NK3, Tlx, NK6, Hmx, Lbx, and NK2) have been recently identified by Zhang et al. (2012). The presence of nine NK genes in the oyster Pinctada fucata has been reported, however data are not available, except for Tlx and NK2 genes (Morino et al. 2013). Our research group has previously isolated two Tlx genes from Venerupis pullastra and Ostrea edulis and one NK2 gene from O. edulis (Mesias-Gansbiller et al. 2013). Genes of the NK2 class have a distinctive homeobox and encode proteins that have a tyrosine at homeodomain position 54, and the Tlx family of homeobox genes is distinguished by a Thr in position 47 of the homeodomain. The assignment of these genes was confirmed by phylogenetic analyses with strong bootstrap support (Fig. 3 and Appendix Figs. Al and A2). The presence of the Tlx and NK2 genes in oysters and clams suggests that these genes could be conserved in bivalves in general, although its presence has not been yet determined in basal bivalves as Yoldia (Barucca et al. 2003). It is plausible that the absence of other NK genes reflects the limited amount of research for this class of genes in Mollusca.

The list of the genes reported here may contribute to the understanding of their evolutionary history within the phylum. Further investigations of their genetics activities will facilitate the elucidation of the involvement of these genes in the establishment of the body plan of bivalves.


We would like to thank Arturo Silva for providing the samples and John Souto for helpful comments on the English version of the manuscript. C. Mesias-Gansbiller was financially supported by the AECID del Ministerio de Asuntos Exteriores de Espana.


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Laboratorio de Biolog'ia Molecular y del Desarrollo, Departamento de Bioquimica y Biologia Molecular, Instituto de Acuicultura, Universidad de Santiago de Compostela, Constantino Candeira sjn, 15782 Santiago de Compostela, Galicia, Spain

* Corresponding author. E-mail:

DOI: 10.2983/035.035.0119

List of species analyzed in this article and
abbreviations used in figures and references.

 Common name          Species name            Family     Abbreviation

Akoya pearl     Pinctada fucata             Pteriidae        Pfu
oyster          (Gould, 1850)

Antarctic       Yoldia eightsi              Yoldiidae        Yei
clam            (Jay, 1839)

Black scallop   Mimachlamys varia           Pectinidae       Cva
                (Linnaeus, 1758)

Blue mussel     Mytilus galloprovincialis   Mytilidae        Mga
                (Lamarck, 1819)

European        Solen marginatus            Solenidae        Sma
razor clam      (Pulteney, 1799)

Flat oyster     Ostrea edulis (Linnaeus,    Ostreidae        Oed

Giant scallop   Placopecten magellanicus    Pectinidae       Pmag
                (Gmelin, 1791)

Great scallop   Pecten maximus (Linnaeus,   Pectinidae       Pma

Japanese        Patinopecten yessoensis     Pectinidae       Pye
scallop         (Jay, 1857)

Manila clam     Ruditapes philippinarum     Veneridae        Tph
                (Adams & Reeve, 1850)

Pacific         Crassostrea gigas           Ostreidae        Cgi
oyster          (Thunberg, 1793)

Pullet          Venerupis pullastra         Veneridae        Vpu
carpet shell    (Montagu, 1803)

Purple          Transennella tantilla       Veneridae        Tta
transennella    (Gould, 1853)

Sword razor     Ensis ensis (Linnaeus,       Pharidae        Een

Virginia        Crassostrea virginica       Ostreidae        Cvi
oyster          (Gmelin, 1791)

 Common name                      References

Akoya pearl     Zhou et al. 2008, Takeuchi et al, 2012,
oyster          Morino et al. 2013

Antarctic       Barucca et al. 2003

Black scallop   This paper, Mesias-Gansbiller et al. 2012

Blue mussel     This paper, Barucca et al. 2003, Perez-Paralle
                et al. 2005, Mesias-Gansbiller et al. 2012

European        This paper, Mesias-Gansbiller et al. 2012
razor clam

Flat oyster     This paper, Mesias-Gansbiller et al. 2012,
                Mesias-Gansbiller et al. 2013

Giant scallop   Wray et al. 1995

Great scallop   Barucca et al. 2003, Carpintero et al. 2004,
                Canapa et al. 2005

Japanese        Iijima et al. 2006

Manila clam     Barucca et al. 2003

Pacific         Zhang et al. 2012

Pullet          This paper, Mesias-Gansbiller et al. 2012,
carpet shell    Mesias-Gansbiller et al. 2013

Purple          Wray et al. 1995, Jacobs et al. 2000

Sword razor     Barucca et al. 2003

Virginia        Wray et al. 1995
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Article Details
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Author:Perez-Paralle, M. Luz; Pazos, Antonio J.; Mesias-Gansbiller, Crimgilt; Sanchez, Jose L.
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:4EUSP
Date:Apr 1, 2016
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