Integrative and Comparative Biology Advance Access originally published online on August 2, 2007
Integrative and Comparative Biology 2007 47(6):854-864; doi:10.1093/icb/icm077
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The Hox gene complement of a pelagic chaetognath, Flaccisagitta enflata
*Kewalo Marine Laboratory, University of Hawaii, 41 Ahui Street, Honolulu, HI 96813, USA; Biological Sciences, Auburn University, 101 Rouse, Auburn, AL 36830, USA
Correspondence: 1E-mail: matus{at}hawaii.edu
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Chaetognaths are transparent marine animals that are ubiquitous and abundant members of oceanic zooplanktonic communities. Their phylogenetic position within the Metazoa, however, has remained obscure since their discovery. Morphology and embryology have traditionally allied chaetognaths with deuterostomes, but molecular evidence suggests otherwise. Two recent multigene expressed sequence tag (EST) molecular phylogenomic studies suggest that chaetognaths are either sister to the Lophotrochozoa (Matus et al. 2006) or to all protostomes (Marlétaz et al. 2006). We have isolated eight Hox genes, one Parahox gene, and Mox, a related homeodomain gene, from the pelagic chaetognath, Flaccisagitta enflata. Although chaetognath central class Hox genes lack the Lox5 or "spiralian" parapeptide, a diagnostic amino-acid motif that has been utilized previously to assign lophotrochozoan affinity, they do possess a central class Hox gene that has a partial "Ubd-A peptide" found in both ecdysozoan and lophotrochozoan Ubx/Abd-A/Lox2/Lox4 genes. Additionally, we report the presence of two distinct chaetognath posterior Hox genes that possess both ecdysozoan and lophotrochozoan signature amino-acid motifs. The phylogenetic position of chaetognaths, as well as the evolution of the Hox cluster, is discussed in light of these data.
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Introduction |
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Chaetognaths are active predators possessing a torpedo-like, coelomate body with lateral and caudal fins (Fig. 1A). They are hermaphroditic with direct development, lacking both a trochophore-like (i.e., lophotrochozoan) and dipleurula-like (i.e., deuterostome) larval stage. Several features (Fig. 1B) have allied them with deuterostomes (Willmer 1990
), including a tripartite adult body plan, postanal tail, and developmental features such as radial cleavage, enterocoely, and posterior fate of the blastopore (deuterostomy). Molecular data (Telford and Holland 1993; Wada and Satoh 1994; Halanych 1996; Erber et al. 1998; Mallatt and Winchell 2002), "total evidence" (Zrzavy et al. 1998; Giribet et al. 2000; Peterson and Eernisse 2001) and mitochondrial genomic studies (Helfenbein et al. 2004; Papillon et al. 2004) have failed to support the inclusion of the chaetognaths within Deuterostomia. These, and other studies have been unable to discriminate whether chaetognaths are sister to protostomes (Giribet et al. 2000; Helfenbein et al. 2004) (Fig. 1C), within Ecdysozoa (Halanych 1996; Peterson and Eernisse 2001) (Fig. 1D) or within Lophotrochozoa (Erber et al. 1998; Haase et al. 2001; Shimotori and Goto 2001) (Fig. 1E). The most recent attempts to place the Chaetognatha have utilized expressed sequence tag (EST) data, both from the benthic chaetognath Spadella cephaloptera (Marlétaz et al. 2006) and from the pelagic chaetognath, Flaccisagitta enflata (Matus et al. 2006). While taxonomic sampling and gene number varied between these two studies, both definitively showed that chaetognaths are protostomes, although their suggested affinity within the protostomes differed. Based on 79 ribosomal genes, S. cephaloptera was shown to be sister to all protostomes (Marlétaz et al. 2006) (Fig. 1B), while analyses based on 72 genes by Phillippe et al. (2005), as well as increased taxonomic sampling using the single nuclear gene, tropomyosin, suggested that chaetognaths were sister to lophotrochozoans (Matus et al. 2006) (Fig. 1E).
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In both of these recent EST analyses (Marlétaz et al. 2006
; Matus et al. 2006) taxonomic sampling, particularly within the protostomes is sparse. As with many recent EST phylogenies (Philippe et al. 2005; Bourlat et al. 2006), Lophotrochozoa is only represented by Mollusca, Annelida, and Platyhelminthes, while Ecdysozoa is represented by Nematoda, Tardigrada, and Arthropoda. This represents only six of the 
26 phyla that make up these clades. Increased taxonomic sampling from within these groups might result in better resolution of the chaetognath phylogenetic position.
Another set of genes that have been used to gain insight into phylogenetic relationships is Hox genes. Hox genes are developmental regulatory genes involved in body-plan formation that are believed to have diversified before the last common bilaterian ancestor (Brooke et al. 1998; de Rosa et al. 1999; Kourakis and Martindale 2000; Balavoine et al. 2002). Their highly conserved nature has made them useful for revealing relationships of several enigmatic metazoan groups, e.g., brachiopods and priapulids (de Rosa et al. 1999) and dicyemids (Kobayashi et al. 1999). Orthological assignments of certain Hox genes can be determined by phylogenetic analysis and/or the presence of key diagnostic amino-acid residues (Sharkey et al. 1997; Anderson et al. 1998; de Rosa et al. 1999; Kobayashi et al. 1999; Kourakis and Martindale 2000; Telford 2000; Balavoine et al. 2002), which results in assignment to specific paralog groups (PG). While studying chaetognath development, we used degenerate PCR to sample the genome of the pelagic chaetognath, F. enflata for Hox genes to elucidate phylogenetic placement of arrow worms within the Metazoa. Chaetognaths possess definitive members from all four primordial bilaterian Hox paralogy classes (anterior, PG 3, central, and posterior), consistent with an ancestral bilaterian cluster of 8–10 genes (Fig. 2). Although genomic linkage data are not yet available, there is no evidence of cluster duplication in F. enflata.
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Adult chaetognaths (F. enflata,) were obtained from Kaneohe Bay, Kaneohe, Hawaii, and kindly identified by Erik V. Thuesen (The Evergreen State College, Washington, USA). Hox and Parahox genes were initially isolated by PCR from genomic DNA using degenerate primers (see, Supplementary Materials). Flanking sequence was obtained with gene-specific primers by nested 5' and 3' RACE PCR using embryonic and adult cDNA as a template. Sequences have been reported to GenBank under accession numbers EF490810–EF490820. Putative orthological assignments were made via BLASTX searches of the GenBank. An amino-acid alignment of the 60 amino-acid homeodomain (HD) and the 12 amino acids directly flanking the 3' end of the homeodomain was made using representative sequences from acoel (Symsagittifera roscoffensis), ecdysozoan (Tribolium castaneum: beetle), lophotrochozoan (Nereis virens and Capitella capitata sp. I: annelids), and deuterostome (Branchiostoma floridae: cephalochordate) representatives (see, Supplementary Materials). A Bayesian phylogenetic analysis was conducted utilizing the MPI version of MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001
) on the University of Hawaii Dell computing cluster, using a mixed amino-acid model with gamma, which selected RtRev with a 100% posterior probability with 10,000,000 generations sampled every 100 generations with four chains over four independent runs. A summary tree was produced from the last 3,96,000 trees representing 39,600,000 stationary generations. Additionally, Neighbor Joining (NJ) (using mean AA distances) was conducted using phylogenetic analysis using parsimony (*and other methods; PAUP*) v4.0b10 (Swofford 2000). ProtTest (Abascal et al. 2005) also selected the rtrev + G model, which was used for maximum likelihood (ML) analyses conducted using RAXML v2.2.1 (Stamatakis 2006). An initial search was conducted in RAXML v2.2.1 (Stamatakis 2006) using 500 searches using the rtrev + G model. A consensus of this search is reported. ML bootstrap analysis was also conducted with RAXML v2.2.1 with 500 iterations and 15 independent searches, bootstrapped with SeqBoot (Felsenstein 1989), for each iteration. Additional details of phylogenetic analyses, GenBank accession numbers, alignments, and primer sequences are available in Supplementary Materials as well as being available upon request. |
Results and discussion |
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Previous work on the benthic chaetognath, S. cephaloptera, identified a chaetognath Hox cluster of at least six genes (Papillon et al. 2003
), although for many of the genes only partial HDs were recovered. Due to the strong sequence conservation of the HD throughout metazoan evolution, however, obtaining flanking-sequence information is necessary to make more definitive orthological and paralogy assignments. Our initial PCR survey on F. enflata genomic DNA, utilizing primers specific to Hox-class HD genes, resulted in the identification of nine unique Hox gene fragments as well as the identification of the posterior ParaHox gene, caudal, and a HD gene related to the Hox cluster, mox (Minguillon and Garcia-Fernandez 2003). Using RACE PCR from adult and embryonic cDNA templates we isolated additional sequence information from eight of the nine Hox genes isolated in the initial PCR surveys from F. enflata genomic DNA. Based upon both predicted amino-acid sequences (Fig. 3) and a phylogenetic analysis of the HD and 3' flanking sequence (Fig. 4), F. enflata possesses orthologs to anterior (Fen Hox1), PG3 (Fen Hox3), four central class genes (Fen Hox4, Hox6, Hox7, and Hox8), two posterior class genes (Fen PostA and Fen PostB), and one gene (Fen MedPost) that appears to be the ortholog of the benthic chaetognath, S. cephaloptera MedPost gene. Failure to recover PG2 (Hox2 and proboscipedia) genes from the initial genomic DNA survey might not be surprising due to the presence of an intron in the HD of PG2 genes in many taxa (Kmita-Cunisse et al. 1998; Callaerts et al. 2002). Additionally, while flanking sequence for the PG3 gene, FenHox3, was unable to be recovered by RACE PCR, it shares 100% amino-acid identity and 85% nucleotide identity (12 changes, all third codon) with the ortholog isolated from the benthic chaetognath S. cephaloptera (Papillon et al. 2003) as well as identity with other bilaterian PG3 genes suggesting that it is correctly classified as a PG3 gene (SF1).
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The phylogenetic analyses suggest orthology for all of the chaetognath Hox genes isolated in this study, with 100% posterior probability (pp) for Fen Mox, Fen Hox1, Fen MedPost, Fen Cdx, and the two posterior genes, Fen PostA and Fen PostB with known bilaterian PG and 95% pp for Fen Hox6 with a polychaete Lox5 gene (Fig. 4). While the remaining chaetognath Hox genes are not supported by >95% pp, their suggested orthology seems likely, as their position is supported by other methods of phylogenetic analysis, including ML and NJ. The Fen Hox4 clusters with other PG4 genes with 88% pp. The Fen Hox7 clusters with other protostome PG7 genes (Tribolium antp and Nereis Hox7 with 81% pp) and together is sister to deuterostome Branchiostoma Hox6 and Hox7 genes (83% pp). The Fen Hox8 is sister group to protostome and deuterostome PG8 genes (Ubx, Abd-A, Lox2, Lox4, and Branchiostoma Hox8; 79% pp). Finally, the two chaetognath MedPost genes, from F. enflata and S. cephaloptera cluster together with 100% pp, forming the sister group to posterior Hox and Parahox genes (81% pp) (Fig. 4).
PG4 genes (Dfd and Hox4) are characterized by the presence of a diagnostic amino-acid motif found immediately 3' to the HD (LPNTK). The chaetognath gene (Fen Hox4) possesses this motif, making this a definitive PG4 gene (Fig. 3A). Since this amino-acid motif is present in all metazoan PG4 genes surveyed to date (protostomes and deuterostomes), this motif has not been useful in assessing phylogenetic relatedness relative to other lineages. As it has already been established that the chaetognaths are protostomes (Helfenbein et al. 2004; Papillon et al. 2004; Marlétaz et al. 2006; Matus et al. 2006), of particular interest are those Hox genes that have been used to assign taxa to either Lophotrochozoa or Ecdysozoa, namely Lox5, Ubx, AbdA, Lox2, and Lox4 (de Rosa et al. 1999; Telford 2000; Balavoine et al. 2002).
The five central class Hox genes recovered from F. enflata, as well as those isolated from the benthic S. cephaloptera (Papillon et al. 2003), lack the "Lox5" or "spiralian" (de Rosa et al. 1999) peptide motif found in the 3' flanking region of all lophotrochozoan Lox5 genes, a central class gene considered to be orthologous to either ecdysozoan Fushi-tarazu (PG6) genes (Telford 2000) or to Antennapedia (PG7) genes (de Rosa et al. 1999; Balavoine et al. 2002) (Fig. 3B).
Another diagnostic amino-acid motif, the Ubd-A parapeptide, is found 3' to the HD in ecdysozoan Ubx and AbdA genes as well as in lophotrochozoan lox2 and lox4 genes (PG8) (de Rosa et al. 1999; Balavoine et al. 2002). One chaetognath central class Hox gene, Fen Hox 8, possesses four of the seven amino acids (QxIxEMx) found in the Ubd-A parapeptide (Figs. 3C and 5), which suggests that it may be an ortholog of protostome ubx/abdA/lox2/lox4 genes.
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Previous work on the benthic chaetognath, S. cephaloptera, identified at least six chaetognath Hox genes, including a PG3 gene, four central class genes of unknown orthology/paralogy, and a gene which the authors believed to possess both central and posterior class diagnostic residues (Spadella MedPost), but no definitive posterior class Hox genes (Papillon et al. 2003
). When the amino-acid sequences of these six S. cephaloptera Hox gene fragments are compared to those recovered from F. enflata in this study (SF1 and Fig. 3) it appears that SceMed1 is an ortholog of FenHox7, SceMed2 is an ortholog of FenHox6, and SceMed3 is an ortholog of FenHox8. An ortholog of SceMed4, which has been suggested to be related to PG5 (sex combs reduced) genes (Papillon et al. 2005) does not appear to have been identified in F. enflata. This is consistent with phylogenetic analyses, which failed to identify a PG5 gene as well (Fig. 4).
Based upon predicted amino-acid sequence (Fig. 3D) and phylogenetic analyses (Fig. 4), the S. cephaloptera MedPost gene appears to be orthologous to the F. enflata MedPost gene and may be a chaetognath-specific Hox gene. Both MedPost genes share the central class diagnostic amino acids: QT (positions 6–7 in the HD), LTR(R/K)RRI (positions 26–32) and a glutamic acid (E) at position 59 (Papillon et al. 2003) (Fig. 3C and D). 5' of the HD, Fen MedPost has a definitive hexapeptide (SIYPWM), an amino-acid motif important functionally for interaction with extradenticle/pbx/homothorax proteins (Berthelsen et al. 1999; Ryoo et al. 1999), but missing from all posterior class genes. However, both chaetognath MedPost genes also possess diagnostic posterior class residues (K3, A14, R18, Y20, V21, and Q36), which are thought to be exclusive to posterior class genes with the exception of the Q36 in the priapulid Ubx gene (Fig. 3C and D).
Bayesian, ML, and NJ phylogenetic analyses place these chaetognath MedPost genes as sister to posterior class and cdx genes (Fig. 4), although with differing statistical support. While Bayesian posterior probability for this grouping is 81%, below the accepted 95% confidence level, it is still five times more likely than any other grouping for the chaeotognath MedPost genes. ML bootstrap support for this node was only 30% (see, Supplementary Materials). However, in the initial RAXML v 2.1.2 (Stamatakis 2006) searches, 99% of the 500 replicates performed identified a ML tree that placed MedPost genes as the sister group to posterior class Hox genes (Fig. 4 and Supplementary Materials). When those amino acids that are diagnostic of central class Hox genes were removed, both ML and Bayesian analyses place the MedPost genes within the posterior class (see, Supplementary Material). When posterior class diagnostic amino acids were removed, the MedPost genes remained as the sister group to the posterior genes (see, Supplementary Materials). Together, the results of the phylogenetic analyses suggest that chaetognath MedPost genes are definitive central class genes, but are consistently placed as the sister group to the posterior class Hox genes.
The MedPost gene has been suggested to retain features of an ancestral central/posterior class gene (Papillon et al. 2003). This was based largely on the absence of definitive posterior class genes from chaetognaths. We have isolated two posterior class genes using degenerate primers specific to posterior Hox genes. The two chaetognath posterior Hox genes (Fen PostA and Fen PostB) cluster with posterior genes with 100% pp in Bayesian phylogenetic analyses (Fig. 4). Additionally, they possess amino acids diagnostic of posterior class Hox genes, including K3 in PostA, and Y20 in PostA and PostB. Interestingly, both chaetognath posterior genes share a threonine at position 24, which is found in lophotrochozoan posterior1 and in the acoel, Symsagitiffera posterior Hox gene (Fig. 3D). The chaetognath PostA gene shares an arginine at position 36 that has been suggested to be ecdysozoan-specific (AbdominalB genes) (de Rosa et al. 1999; Balavoine et al. 2002; Cook et al. 2004), but further taxonomic sampling has shown it to be found in an acoel (Symsagitiffera Post) and two hemichordate posterior genes (Ptychodera Hox9/10 and Hox11/13a) but is absent from lophotrochozoan posterior class genes (Fig. 3D). Thus, the two chaetognath posterior Hox genes share plesiomorphic characters with lophotrochozoans, ecdysozoans, and deuterostomes making phylogenetic interpretations difficult. The presence of definitive posterior class genes in chaetognaths, however, suggests that the MedPost genes are more likely to be a chaetognath-specific innovation rather than evolutionary antecedents of central and posterior class genes, as previously suggested by others (Papillon et al. 2003). Additionally, the identification of posterior class Hox genes in both cnidarians (Ryan et al. 2007) and acoels (Cook et al. 2004), organisms believed to have diverged prior to chaetognaths (Collins et al. 2005; Jondelius et al. 2002; Ruiz-Trillo et al. 1999, 2002) during metazoan evolution, further bolsters the conclusion that chaetognath MedPost genes are not directly descended from the Hox posterior-central class gene ancestor.
The absence of a definitive Lox5 motif and the presence of a partial UbdA parapeptide in a chaetognath have implications for both the evolution of the bilaterian Hox cluster and the phylogenetic position of the chaetognaths and can be interpreted in two ways. First, the chaetognaths may have diverged prior to the lophotrochozoan/ecdysozoan split (Fig. 6A). Alternatively, because the plesiomorphic condition of both the Lox5 and the UbdA peptides is unclear, we cannot rule out that both the lack of diagnostic residues (Lox5) and divergence (UbdA) could be due to secondary changes unique to the chaetognath lineage (Fig. 6B). In such a case, Chaetognatha may be sister to Lophotrochozoa but the phylogenetic signal within their Hox genes has been erased. Additionally, due to the nature of PCR-biased sampling, one cannot rule out that a second gene containing a UbdA parapeptide exists in chaetognaths. However, the presence of one gene that only possesses a partial UbdA parapeptide suggests that the protostome PG8 genes, Ubx and AbdA (ecdysozoan) and Lox2 and Lox4 (lophotrochozoan) duplicated after their divergence from the chaetognaths (Fig. 6A).
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Given that recent phylogenetic EST analyses places chaetognaths as either sister to protostomes (Marlétaz et al. 2006
) or sister to lophotrochozoans (Matus et al. 2006), the question arises as to whether the data from chaetognath Hox genes sheds any light on these alternative hypotheses. Chaetognaths clearly possess a near complete set of Hox genes, with members from all four classes (anterior, PG3, central, and posterior) as well as a potentially unique class, the MedPost gene, which supports previous theories of an ancestral bilaterian Hox cluster of 8–10 genes (de Rosa et al. 1999; Kourakis and Martindale 2000; Balavoine et al. 2002). One explanation for the data presented here is a sister-group relationship between chaetognaths and all other protostomes. Cnidarians have been shown to possess both anterior class (PG1 and PG2) and posterior class genes (Ryan 2007: 2025), suggesting that the cnidarian–bilaterian ancestor possessed at least three Hox genes (two anterior and one posterior) (Fig. 6). Acoels are the earliest branching metazoan that possesses definitive central class Hox genes (Cook et al. 2004). The protostome–deuterostome ancestor would have then undergone expansion of central and anterior class Hox genes, including the innovation of a definitive Hox4/dfd paralog with the LPTNK motif found 3' of all Hox4 genes (but absent from the acoel central class gene) (Fig. 3A). The chaetognath–protostome ancestor would then have possessed a Hox cluster of at least eight genes including a central class gene possessing an Ubd-A parapeptide. The gene containing this motif would then have duplicated into Ubx/AbdA and Lox2/Lox4 following the split from the chaetognaths. The Lox5 peptide would have evolved in lophotrochozoans following the split from ecdysozoans. Chaetognaths likely evolved a MedPost gene specific to their lineage since a clear ortholog of this gene has not been recovered from any other metazoan (Fig. 6A).
If chaetognaths are actually the sister group to lophotrochozoans as suggested by EST analysis, mitochondrial genomic analyses, and tropomyosin phylogeny (Matus et al. 2006), this would suggest the scenario presented in Fig. 6B. A central Hox gene containing the "Ubd-A parapeptide" would have evolved at the base of the protostomes, and genes containing this motif would have diverged subsequently in ecdysozoans (Ubx and AbdA) and the remainder of the lophotrochozoans (Lox2 and Lox4), to the exclusion of chaetognaths. The Lox5 parapeptide would have evolved after the chaetognath–lophotrochozoan split (Fig. 6B). Unfortunately, due to the nature of degenerate PCR, it is possible that we did not isolate all of the Hox genes in the chaetognath genome, which could have profound impacts on the interpretation of these results. Total genome sequencing of a chaetognath will greatly aid in the characterization of the chaetognath Hox cluster and perhaps shed further light on the evolution of Hox genes in the protostomes.
Placement as either sister to protostomes or sister to Lophotrochozoa raises interesting issues about several key morphological and embryological innovations. The unique set of chaetognath morphological and developmental characters, such as radial cleavage, enterocoely, fate of the blastopore, brachyury staining (Takada et al. 2002), direct development, a tripartite body plan, and protostome-like central nervous system (Hyman 1959; Willmer 1990), may provide insight into the protostomian or lophotrochozoan ancestor. If these features are bilaterian plesiomorphies, the conflict between chaetognaths possessing "deuterostome-like" morphology and embryology can be reconciled with modern molecular phylogenetic approaches. The observation that chaetognaths may possess a single complete complement of Hox genes and that adults and embryos are readily available provides an opportunity to better understand morphological, genomic, and developmental evolution at a key node in the Metazoa.
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Supplementary data |
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Supplementary data are available at ICB online.
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Acknowledgments |
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We wish to thank Allison Sweeney and Sonke Johnson for the opportunity to participate in this symposium. We would also like to thank Deirdre Killebrew and the University of Hawaii Dell Cluster for computational resources, and Casey Dunn and Joe Ryan for help with phylogenetic analyses. This work was supported by NSF support to M.Q.M. (IBN-0236565) and K.H.M. (IBN-0333843). This work is AU Marine Biology Program contribution 26.
Conflict of interest: None declared.
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From the symposium "Integrative Biology of Pelagic Invertebrates" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2007, at Phoenix, Arizona.
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