INTRODUCTION

Acoels and nemertodermatids are marine, soft-bodied, flattened acoelomates (Brusca et al., 2016) that have a unique epidermis with pulsatile activity. The mouth is located ventrally with an incomplete gut and no anus. These organisms lack discrete organs, such as a circulatory system and gonads. According to the traditional view prior to 2010, acoel flatworms and nemertodermatid flatworms (recently called acoelomorphs) were categorized as early-diverged extant orders of the class Turbellaria of the phylum Platyhelminthes, which included planarians, parasitic flukes, and tapeworms (Hyman, 1951; Brusca and Brusca, 2003). Planarians, parasitic flukes, and tapeworms are regarded as representatives of the phylum and are considered primitive bilaterians, but few studies have focused on acoels and nemertodermatids.

Xenoturbella bocki is found at depths of approximately 200 m in cold Baltic seawater and was first described only about 70 years ago (Westblad, 1949). Since then, several species of Xenoturbella have been recorded (Rouse et al., 2016; Nakano et al., 2017). Although morphological and anatomical similarities between Xenoturbella and acoelomorphs have been discussed, X. bocki has been considered an enigmatic animal, and its phylogenetic position has been obscure (Telford, 2008).

Molecular phylogeny is a powerful tool for inferring the phylogenetic relationships of metazoans that differ in morphology and embryology. Nucleotide substitutions in DNA likely occur neutrally, such that the substitution rate can be used to deduce the relationship without prejudicing the interpretation, which would be the case if it were based on specific morphological traits or embryological modes and features. X. bocki has been extensively used for molecular phylogenetic studies. A first round of molecular phylogenetic reports discusses the phylogenetic position of Xenoturbella (Norén et al., 1997; Bourlat et al., 2003; Bourlat et al., 2006) in relation to their enigmatic anatomy and life cycle (Lundin, 1998; Nakano et al., 2013; Perea-Atienza et al., 2015).

There has been considerable debate about the placement of Xenoturbella relative to acoel and nemertodermatid flatworms. Repeated analyses of molecular phylogeny have nearly reached a consensus that acoels, nemertodermatids, and xenoturbellids form a clade called Xenacoelomorpha (Phillipe et al., 2011). However, it remains a matter of debate whether Xenacoelomorpha is monophyletic or whether these organisms are sisters to all bilaterians or to ambulacrarians (echinoderms + hemichordates) (Ruiz-Trillo et al., 1999; Telford, 2008; Hejnol et al., 2009; Mwinyi et al., 2010; Ruiz-Trillo et al., 2014; Simakov et al., 2015; Cannon et al., 2016; Dittmann et al., 2018).

Most molecular phylogenetic studies are based on comparisons of quantitative molecular changes. Comparative studies of qualitative molecular traits may provide additional or more reliable support for those derived from quantitative analyses. An interesting example is encountered in a diagnostic peptide motif known as the “spiralian peptide” (Bayascas et al., 1998) or the “Lox5 peptide” (de Rosa et al., 1999). This motif is composed of KLTGP pentapeptides and is present immediately carboxyterminal to the homeodomains (the functionally important centers of these molecules) that are encoded by the central Hox genes. These genes include Lox5 and Hox6 in lophotrochozoans, but not in ecdysozoans or deuterostomes (see Fig. 2).

This motif is also shared by Lox5 in Dicyema japonicus (Kobayashi et al., 1999), which supports the inclusion of dicyemids in the lophotrochozoan group (Lu et al., 2017). Recently, Fröbius and Funch (2017) reported the presence of another peptide motif, KSLND, in Lox5 (Hox6/7) of rotifers (see Fig. 2). Because this motif is shared with chaetognaths, the authors suggested a close relationship between rotifers and chaetognaths, which are sometimes called Gnathifera. A recent molecular phylogenetic analysis of chaetognaths also supports this rotifer/chaetognath clustering (Marlétaz et al., 2019).

Brauchle et al. (2018) recently surveyed all 11 classes of homeobox-containing genes in xenacoelomorphs and showed the presence of anterior, central, and posterior Hox genes, which are each in the ATNP-type homeodomain family. However, no studies have examined the presence of diagnostic peptides in xenacoelomorphs thus far. Recently, we decoded the genome of the acoel, Praesagittifera naikaiensis (Arimoto et al., 2019), which is a marine acoel worm (Yamasu, 1982) that is easily found along the seashores of the Seto Inland Sea and may be widely used for studies of acoel biology (Hikosaka-Katayama and Hikosaka, 2015). In the present study, we examined the presence or absence of spiralian peptide-like sequences in the Hox genes of acoels, nemertodermatids, and xenoturbellids.

MATERIALS AND METHODS

Genomic and transcriptomic sequence reads of the acoel, Praesagittifera naikaiensis, have been deposited in the sequence read archive of the DNA Data Bank of Japan (DDBJ) under accession No. PRJDB7329 (Arimoto et al., 2019). All data are also available from the GigaScience GigaDB repository ( http://dx.doi.org/10.5524/100564) and at  http://marinegenomics.oist.jp/p_naikaiensis/viewer/info?project_id=71. Gene models have been annotated using BLAST searches (Camacho et al., 2009) against the NCBI RefSeq protein database release 88, and the results have been integrated into the database (Arimoto et al., 2019).

Table 1.

Homeobox genes in xenacoelomorphs.

Fig. 1.

Hox genes in acoels, a nemertodermatid, and Xenoturbella. (A) Alignments of amino acid sequences of the homeodomain encoded by anterior Hox1-related genes (top), central Hox4/5/6-related genes (upper middle), and posterior Hox9/11/13-related genes (lower middle). An alignment of the acoel Evx homeodomain is shown as a control (bottom). Sequence identity and similarity among homeodomains are indicated by * and:, respectively. Conserved amino acid residues are colored according to the properties of their side chains using the Clustal X color scheme ( http://www.jalview.org/help/html/colorSchemes/clustal.html ). Abbreviations of species names: (acoels) Pn, Praesagittifera naikaiensis; Sr, Symsagittifera roscoffensis; Hm, Hofstenia miamia; Is, Isodiametra pulchra; (a nemertodermatid) Nw, Nemertoderma westbladi; and (a xenoturbellid) Xb, Xenoturbella bocki. (B) Molecular phylogeny showing relationships among homeobox genes in xenacoelomorphs. A maximum-likelihood tree was constructed using the sequences of residues 1–60. Acoel Evx was used as an outgroup. Numbers at nodes show bootstrap support (1000 iterations) for the clustering. Clustering of anterior Hox-1-related, central Hox4/5/6-related, and posterior Hox9/11/13-related genes is evident.

Among the 53 predicted homeobox genes in the database, three genes were identified as ANTP-type homeodomain proteins and used in this study (Table 1). Homeodomain amino-acid sequences of xenacoelomorphs were retrieved from the NCBI data bank (Table 1). Data sources for other metazoans are shown in  Supplementary Table 1 (10.2108.zsj.36.395.s1.pdf). Amino-acid alignments were generated using MAFFT version 7.305 (Katoh et al., 2002), and a molecular phylogenetic tree was constructed using the maximum likelihood method and IQ-TREE version 1.6.10 (Nguyen et al., 2015). All homeodomain sequences were compared, and the LG+G4 substitution model was selected as the optimal one for inferring a phylogenetic tree. The nodes of the tree were evaluated using 1000 bootstrap replications. Spiralian-peptide-like sequences were identified by manually aligning amino-acid sequences at positions 50–60 of the homeodomain and its carboxyl-terminal flanking region. The identities and similarities of amino-acid sequences were checked by visual inspection.

RESULTS

Praesagittifera naikaiensis contains three Hox genes

Among the 53 predicted homeobox genes in the P. naikaiensis genome database, three genes were identified as ANTP-type homeodomain proteins (Table 1; Fig. 1A). The three genes correspond to an anterior Hox1-like gene (tentatively called PnHox1), a central Hox4/5/6-like gene (PnHox4/5/6), and a posterior Hox9/11/13-like gene (PnHox9/11/13) (Table 1; Fig. 1A). The presence of one representative for the anterior Hox-cluster gene (tentatively called Hox1), the central one (tentatively called Hox4/5/6), and the posterior one (tentatively called Hox9/11/13) has been reported in the acoels Symsagittifera roscoffensis (Moreno et al., 2009), Hofstenia miamia, and Isodiametra pulchra (Cook et al., 2004) (Table 1).

A nemertodermatid, Nemertoderma westbladi, contains one anterior, one central, and two posterior Hox genes (Jimenez-Guri et al., 2006; Brauchle et al., 2018) (Table 1). Since the reported nemertodermatid central Hox4/5/6 ortholog was partial (Jimenez-Guri et al., 2006), we could not include it in further analyses. In addition, NwHox9/11/13-2 was only used in this analysis because NwHox9/11/13-2 shows more similarity to others than NwHox9/11/13-1. On the other hand, X. bocki contains five copies, one anterior, three central (XbHox4/5/6-1, -2, and -3), and one posterior gene (Brauchle et al., 2018) (Table 1).

Molecular phylogeny was carried out for xenacoelomorph Hox genes using 60 amino-acid residues of the homeodomain. Tree topologies with high bootstrap support were not obtained in the first several trials using a dataset that included all three XbHox4/5/6s of X. bocki due to a long-branch attraction, which was mainly caused by XbHox4/5/6-1. We selected XbHox4/5/6-3 since this homeodomain sequence exhibited greater similarity to those of other acoelomorphs (Fig. 1A). In addition, we found that using Evx (another member of the ANTP family) as an outgroup resulted in the most reliable tree topology (Fig. 1B). The tree demonstrated clustering of xenacoelomorph members Hox1, Hox4/5/6 and Hox9/11/13, which had 77, 69, and 90% bootstrap support, respectively.

The tree topology differed among the three Hox genes but generally displayed grouping of acoel homeodomains, especially as seen in Hox4/5/6, as well as a close relationship between P. naikaiensis and S. roscoffensis in all three clusters. The topology also indicates that Xenoturbella homeodomains diverged first in the Hox1 and Hox4/5/6 clusters (Fig. 1B). These results paved the way for the subsequent examination of the presence of spiralian Hox-peptide-like sequences in xenacoelomorph Hox genes.

Fig. 2.

Alignments of amino acid sequences at positions 50–60 of the homeodomain and the adjacent carboxyl flanking region of central Hox4/5/6 genes. Shared peptide sequences are boxed by magenta in acoels, blue in lophotrochozoans, and green in gnathiferans. No comparable sequence of nemertodermatids has been reported yet. Ac, acoels; Xe, Xenoturbella bocki. For abbreviations of metazoan species,  see Supplementary Table 1 (10.2108.zsj.36.395.s1.pdf). A broad relationship of bilaterians is drawn based on Simakov et al. (2015) and Giribet (2016).

Fig. 3.

Alignments of amino acid sequences at positions 50–60 of the homeodomain and following the carboxyl-flanking region of anterior Hox1 genes. Peptide sequences shared by all three clades of Xenacoelomorph are boxed in magenta. Ac, acoel; Ne, nemertodermatid; Xe, Xenoturbella bocki. For abbreviations of metazoan species,  see Supplementary Table S1 (10.2108.zsj.36.395.s1.pdf).

DISCUSSION

We have reported on the presence of specific Hox-peptide sequences in xenacoelomorphs: (a) a Hox4/5/6-specific peptide sequence shared by acoels but not by Xenoturbella, although the status of nemertodermatids remains unclear; (b) a peptide sequence in Hox1 shared by all three xenacoelomorph members; and (c) a peptide sequence in Hox9/11/13 shared by the two acoels, S. roscoffensis and P. naikaiensis. Together with previous reports on clustering of the three xenacoelomorphs, the present results suggest that (1) Xenacoelomorpha is a discrete taxon or phylum that unites acoels, nemertodermatids, and xenoturbellids; (2) Acoela is a discrete clade in that phylum; and (3) the acoels S. roscoffensis and P. naikaiensis are closely related in comparison with other acoel species. The third notion is also supported by molecular phylogeny results based on a comparison of the whole mitochondrial genome sequences (Arimoto et al., 2019).

One possible argument against the present analysis is whether so few amino acids genuinely constitute a diagnostic peptide motif. Nevertheless, we identified this motif as constituting at least five consecutive identical amino acids that correspond to those in lophotrochozoan-specific Lox5 peptide (De Rosa et al., 1999) and rotifer/chaetognath Hox6 peptide (Fröbius and Funch, 2017). In particular, the acoel-specific Hox4/5/6 peptide consists of seven identical amino acids (Fig. 2), while the xenacoelomorph-specific Hox1 peptide consists of five (Fig. 3), as does the Hox9/11/13 peptide shared by P. naikaiensis and S. roscoffensis (Fig. 4). The homeodomain is the functionally important center of these proteins, so it is expected for the amino acids adjoining both the N- and C-terminal regions to manifest sequence similarity among phylogenetically closely related species. For example, lophotrochozoans share an -RAK- sequence at the C-terminus of Post2 (Fig. 4). The presence of such peptides also supports the clustering of lophotrochozoan members (Fig. 4).

Fig. 4.

Alignments of amino acid sequences at positions 50–60 of the homeodomain and adjacent carboxyl-flanking region of posterior Hox9/11/13 genes. A shared peptide sequence was found in both the acoels Praesagittifera naikaiensis and S. roscoffensis. Three peptides shared by lophotrochozoans are boxed. Ac, acoels; Ne, nemertodermatid; Xe, X. bocki. For abbreviations of metazoan species,  see Supplementary Table S1 (10.2108.zsj.36.395.s1.pdf).

The present results provide further qualitative support of the clustering of Acoela, Nemertodermatida, and Xenoturbellida into the phylum Xenacoelomorpha. Molecular phylogenetic studies (e.g., Phillipe et al., 2011; Simakov et al., 2015) indicate that Acoela and Nemertodermatida comprise a discrete sister clade to Xenoturbellida. However, due to a lack of nemertodermatid Hox4/5/6-peptide data, the present study could not provide a support for the sister relationship between Acoela and Nemertodermatida.

On the other hand, the present results shed little light on the question of whether Xenacoelomorpha is a sister to all bilaterians or to deuterostomes. We carefully compared amino acid sequences of the C-terminal of the homeodomains and the adjacent carboxyl-flanking region between xenacoelomorphs and ambulacrarians (deuterostomes) or protostomes, but we did not find any clues about their relationship. In other words, specific Hox peptides are likely conserved among some clades of metazoans like xenacoelomorphs, lophotrochozoans, or rotifers/chaetognaths. Because Hox peptides of xenacoelomorphs were not shared with deuterostomes (Figs. 2–4), it is impossible to deduce whether there is a close relationship between Xenacoelomorpha and Ambulacraria. This interesting issue should be investigated in future studies.