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Chromosome-level genome assemblies of 2 hemichordates provide new insights into deuterostome origin and chromosome evolution [1]

['Che-Yi Lin', 'Institute Of Cellular', 'Organismic Biology', 'Academia Sinica', 'Taipei', 'Ferdinand Marlétaz', 'Center For Life S Origins', 'Evolution', 'Department Of Genetics', 'Environment']

Date: 2024-06 

Deuterostomes are a monophyletic group of animals that includes Hemichordata, Echinodermata (together called Ambulacraria), and Chordata. The diversity of deuterostome body plans has made it challenging to reconstruct their ancestral condition and to decipher the genetic changes that drove the diversification of deuterostome lineages. Here, we generate chromosome-level genome assemblies of 2 hemichordate species, Ptychodera flava and Schizocardium californicum, and use comparative genomic approaches to infer the chromosomal architecture of the deuterostome common ancestor and delineate lineage-specific chromosomal modifications. We show that hemichordate chromosomes (1N = 23) exhibit remarkable chromosome-scale macrosynteny when compared to other deuterostomes and can be derived from 24 deuterostome ancestral linkage groups (ALGs). These deuterostome ALGs in turn match previously inferred bilaterian ALGs, consistent with a relatively short transition from the last common bilaterian ancestor to the origin of deuterostomes. Based on this deuterostome ALG complement, we deduced chromosomal rearrangement events that occurred in different lineages. For example, a fusion-with-mixing event produced an Ambulacraria-specific ALG that subsequently split into 2 chromosomes in extant hemichordates, while this homologous ALG further fused with another chromosome in sea urchins. Orthologous genes distributed in these rearranged chromosomes are enriched for functions in various developmental processes. We found that the deeply conserved Hox clusters are located in highly rearranged chromosomes and that maintenance of the clusters are likely due to lower densities of transposable elements within the clusters. We also provide evidence that the deuterostome-specific pharyngeal gene cluster was established via the combination of 3 pre-assembled microsyntenic blocks. We suggest that since chromosomal rearrangement events and formation of new gene clusters may change the regulatory controls of developmental genes, these events may have contributed to the evolution of diverse body plans among deuterostomes.

Competing interests: We have read the journal’s policy and one of the authors of this manuscript has the following competing interests: D.S.R. is the paid consultant and shareholder of Dovetail Genomics. The other authors have declared that no competing interests exist.

Funding: This work was supported by grants 112-2326-B-001-004 (Y.H.S.) and 110-2621-B-001-001-MY3 (J.K.Y.) from the National Science and Technology Council, Taiwan ( https://www.nstc.gov.tw/?l=en ), grant AS-GC-111-L01 from Academia Sinica, Taiwan ( https://www.sinica.edu.tw/en/ ) (Y.H.S. and J.K.Y.), and grant PID2019-103921GB-I00 from Ministerio de Economía y Competitividad, Spain ( https://portal.mineco.gob.es/en-us/Pages/index.aspx ) (J.J.T.). P.M.M.G. was funded by a postdoctoral fellowship from Junta de Andalucía ( https://www.juntadeandalucia.es/ ) (DOC_00397). F.M. is supported by the Royal Society Fellowship ( https://royalsociety.org/ ) URF\R1\191161 and the BBSRC grant BB/V01109X/1 ( https://www.ukri.org/councils/bbsrc/ ). D.S.R. was supported by the Molecular Genetics Unit at the Okinawa Institute for Science and Technology ( https://www.oist.jp/ ), and is grateful for support from the Marthella Foskett Brown Chair in Biological Sciences at UC Berkeley ( https://www.berkeley.edu/ ). D.S.R. and C.J.L. were supported by the Chan Zuckerberg BioHub ( https://www.czbiohub.org/ ). The sponsors or funders play no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

( a ) A simplified phylogenetic tree of major branches in Planulozoa (Cnidaria+Bilateria). ( b ) Macrosynteny conservation among deuterostome species including BFL, SCA, PFL, and SPU. Horizontal bars with numbers above represent chromosomes of each species. The conserved synteny blocks between 2 species are connected by curve lines (minimum of 4 gene pairs within a maximum distance of 75 genes between 2 matches). The data underlying this figure can be found in S1 Data . BFL, Branchiostoma floridae; PFL, Ptychodera flava; SCA, Schizocardium californicum; SPU, Strongylocentrotus purpuratus.

Hemichordates comprise 2 groups, the solitary enteropneusts and the colonial pterobranchs. In this study, we generated chromosome-level genome assemblies for 2 enteropneusts, the ptychoderid Ptychodera flava and spengelid Schizocardium californicum. Phylogenomic data showed that Ptychoderidae and Spengelidae are sister groups, together with Harrimaniidae constituting Enteropneusta [ 7 , 24 ]. Our comparative genomic analysis showed remarkable macro-syntenic conservation among deuterostome species. Based on the principle of parsimony and comparative analyses with outgroups, we deduced that the last common ancestor of deuterostomes possessed 24 ancestral linkage group (ALGs) that match the BALGs as previously proposed [ 19 ]. We also discovered lineage-specific rearrangements that reflect the temporal progression towards the chromosomal architectures of extant deuterostomes. While our phylogenetic analysis using synteny-based characters supports a monophyletic deuterostome grouping, we did not identify shared derived macrochromosomal rearrangements that distinguish deuterostomes from other bilaterians. Our results confirm that the genomic architectures of deuterostomes retain more ancestral traits than those of protostomes, consistent with a very short evolutionary distance from the last common ancestor of bilaterians to the origin of deuterostomes. Our study thus provides a roadmap for understanding chromosomal evolution and contributes to deciphering the possible developmental genetic changes underlying the emergence of diverse body plans in deuterostomes.

Among deuterostomes, vertebrates show extensive genomic duplications [ 20 ], but comparisons of sea urchin with other bilaterians [ 19 ], and analysis of sub-chromosomal assemblies of hemichordates [ 15 ] (1) implied that the chromosomes of the deuterostome ancestor retained the 24 bilaterian ancestral linkage groups (BALGs); and (2) identified subsequent rearrangement in the sea urchin and chordate lineages [ 19 ]. Assembling a complete picture of deuterostome genome evolution, however, requires comparisons including chromosome-scale assemblies of hemichordates. Analyses of karyotype evolution including all deuterostome phylum-level lineages could yield important insights into deuterostome ancestry and the evolution of their diverse body plans.

Comparison of diverse metazoan genomes has revealed extensive conservation of chromosome-scale linkage (i.e., “macrosynteny”) across animals [ 13 – 17 ] and enabled the reconstruction of ancestral chromosome-scale units (chromosomes or chromosome arms) [ 18 – 21 ]. These reconstructions have been used to identify shared and derived synteny patterns that can help to resolve long-standing evolutionary questions, infer lineage-specific chromosomal rearrangements, and clarify animal phylogenetic relationships that have been difficult to resolve using conventional phylogenetic approaches [ 18 – 23 ]. For example, identifications of synapomorphic traits of chromosomal fusion-with-mixing events among sponge, cnidarian, and bilaterian genomes provide strong evidence to support the hypothesis that ctenophores are the sister group to all other animals [ 18 ].

The evolutionary events that gave rise to the diverse body plans of deuterostomes remains one of the major mysteries in biology. It is widely accepted that the Deuterostomia includes Echinodermata, Hemichordata, and Chordata, as these animals are characterized by several unique developmental and morphological features, including radial cleavage, deuterostomy, enterocoely formation of the mesoderm, mesoderm-derived skeletal tissues, and pharyngeal openings/slits [ 1 – 3 ]. Despite these common characters, the different deuterostome lineages have evolved distinct body plans. Chordates are defined by their dorsal tubular central nervous system, notochord, and segmented somites [ 4 ], while echinoderms evolved a pentaradially symmetrical adult body, calcitic endoskeleton, and a water vascular system [ 5 ]; and hemichordates are characterized by a tripartite body organization, which includes a proboscis, collar, and trunk [ 6 ]. Molecular phylogenetic analyses have supported a sister group relationship between Echinodermata and Hemichordata, forming a clade called Ambulacraria [ 3 , 7 , 8 ] ( Fig 1A ). While subsequent phylogenomic studies have reinforced support for the ambulacrarian clade, some have suggested a sister group relationship between Ambulacraria and Xenacoelomorpha (a group of marine worms lacking definitive coeloms) and even questioned the monophyletic grouping of the Deuterostomia [ 9 – 12 ]. Due to the long evolutionary history of deuterostome lineages and the difficulties in assigning definitive stem fossils during the early diversification of the group, it remains challenging to postulate the ancestral condition of their common ancestor, let alone to decipher the genomic basis underlying the origins of diverse body plans and phylogenetic affiliations. To address these issues, it is helpful to reconstruct the ancestral genome architectures at major nodes of the animal tree using species that occupy key phylogenetic positions, and trace the subsequent evolutionary trajectories along each lineage.

Results and discussion

Chromosome-level genome assemblies of 2 hemichordates Deuterostomes are composed of 3 major phyla, including hemichordates, echinoderms, and chordates, with the former 2 constituting a group called Ambulacraria (Fig 1A). Previous short read-based genome sequencing of 2 hemichordate species, Saccoglossus kowalevskii and Ptychodera flava, provided a cornerstone for studies on deuterostome evolution [15]. The fragmented nature of these genome assemblies, however, limits our understanding of chromosome evolution among deuterostome lineages. To address this issue, we employed PacBio long-read and Hi-C technologies to sequence genomes of 2 enteropneust hemichordates P. flava (PFL) and Schizocardium californicum (SCA) (S1 Fig). The long read-based genome assemblies of PFL and SCA consist of 1.16 Gbp and 0.93 Gbp, respectively (S1 Fig). After consideration of HiC contacts (S2 Fig), 23 chromosome-scale scaffolds were obtained for both genomes, which matches the 2N = 46 karyotype of PFL [15]. Protein-coding genes were annotated in the 2 genome assemblies using transcriptome data and ab initio prediction approaches, resulting in 35,856 (PFL) and 27,463 (SCA) annotated genes with high BUSCO scores (S1 Fig). Therefore, these 2 hemichordate genome assemblies reached chromosome level with high completeness in gene annotation. The 23 chromosomes of the 2 hemichordate species generally exhibit a one-to-one correspondence based on pairwise comparisons of the positions of orthologous genes (Figs 1B and S3A). This correspondence further supports the chromosomal-scale accuracy of the independently conducted genome assemblies, since conserved syntnies are unlikely to be generated spuriously by assembly errors. Extending this analysis to sea urchin (Strongylocentrotus purpuratus, SPU) and amphioxus (Branchiostoma floridae, BFL), which are representative echinoderm and chordate species, we confirmed chromosome-scale syntenic conservation (macrosynteny) among deuterostomes (Figs 1B and S3B). Given that macro-syntenic conservation has been used to reconstruct ancestral genome architectures and identify lineage-specific chromosomal rearrangement events [19,20], we broadened the synteny analysis by including additional species within and outside the deuterostome superphylum. This approach allowed us to confirm the genomic architecture of the last common ancestor (LCA) of deuterostomes and explore how it evolved among deuterostome lineages.

Stepwise changes in chromosomal architectures within the sea urchin lineage We expect that chromosomal fusion-with-mixing events would occur in a stepwise process as evolution proceeds. As such, 2 distinct chromosomes (at t 0 ) would fuse (at t 1 ), either by end-end fusion or centric insertion, and this event would be followed by rounds of intrachromosomal inversions and translocations (at t 2 ) until the fused chromosome became scrambled (at t s ) (as illustrated in S10 Fig). We therefore postulate that comparing chromosome architectures between species with a relatively short divergence time should allow us to identify the evolutionary state of individual chromosomes during this stepwise process. We thus analyzed 2 additional sea urchin species, L. variegatus (LVA) and L. pictus (LPI), for which chromosomal-level genome assemblies are available for syntenic comparison [16,27]. LVA and LPI are within the genus Lytechinus, which share a common ancestor with S. purpuratus 50 million years ago (mya) [28]. By analyzing syntenic conservation of these 3 sea urchin species (S11 Fig), we inferred that their LCA (tentatively assumed to be sea urchin LCA) possessed 21 ALGs (SALGs) due to 2 shared chromosomal fusion events, J1↘B3 and E⊗(B2⊗C2) (node S in S10 Fig). These 2 fusions were also observed in the recently decoded sea urchin P. lividus genome [26], indicating a common genomic trait of currently available sea urchin genomes. We also deduced 20 ALGs (LALGs) in the Lytechinus LCA, owing to a Lytechinus-specific chromosomal fusion event (G●D) (node L in S10 Fig). Descending from the Lytechinus LCA, L. variegatus and L. pictus each underwent a distinct chromosomal fusion event, F●(J1⊗B3) into L. variegatus LVA1 and F●C1 into L. pictus LPI5), independently resulting in 1N = 19 chromosomes for both species. Based on the phylogenetic relationships and deduced chromosomal architectures (S10 Fig), we construct a putative history of several chromosomal fusion events. For example, 2 echinoderm ALGs (J1 and B3 at t 0 ) fused via centric insertion after which a translocation event resulted in the sea urchin ALG J1↘B3 (at t 2 ). This chromosome then underwent extensive recombinations to become the Lytechinus ALG J1⊗B3 (at t s ). In the lineage leading to L. variegatus, but not L. pictus, end-end fusion of Lytechinus ALGs F and J1⊗B3 resulted in the extant LVA1 chromosome (at t 1 ). Within the LVA1 chromosome, we observed no obvious translocation between regions descended from LALGs J1⊗B3 and F, suggesting that the end-end fusion likely occurred recently in the lineage leading to L. variegatus. In L. pictus, chromosome LPI5 was derived from end-end fusion of LALGs F and C1 followed by a translocation event. Intriguingly, the independent, species-specific fusion event of the 2 Lytechinus species involved the same chromosome (LALG F). Such recent chromosomal fusions may alter recombination rate and cause reproductive isolation, as observed during nematode speciation [29]. Together, the fusion events in sea urchins clearly illustrate how stepwise changes may occur in chromosomal architectures. In several fusion-with-mixing cases, we did not observe transitional states (e.g., SALG E⊗(B2⊗C2) resulted from EALGs E and B2⊗C2, S10 Fig), implying that these fusion events occurred at a relatively ancient time. Assuming that intrachromosomal rearrangements occurred at a constant rate, we postulate the order of fusion events based on synteny patterns. For example, in comparison with the centric insertion pattern of SALG J1↘B3, SALG E⊗(B2⊗C2) exhibits fusion-with-mixing, suggesting that the fusion of EALGs E and B2⊗C2 occurred earlier than that of EALGs J1 and B3. Therefore, from the echinoderm LCA that possessed 23 ALGs to the sea urchin LCA (or more specifically, the LCA of the 3 sea urchin species under investigation) that contained 21 ALGs, there may have been a transitional state with 1N = 22 chromosomes, when EALGs E and B2⊗C2 were already fused but J1 and B3 remained separated. Intriguingly, it has been reported that the haploid genomes of Cidaris cidaris and Arbacia punctulata, which respectively belong to an early branching sea urchin group and an euechinoid outgroup of Lytechinus and S. purpuratus, each contain 22 chromosomes [30,31], suggesting that only 1 fusion event occurred in early branching sea urchins. Thus, we hypothesize that EALGs E and B2⊗C2 fused before the divergence of the sister subclasses of sea urchins, cidaroids, and euechinoids, at least 268 mya [32]. The second fusion event, involving EALGs J1 and B3, possibly occurred later, after the emergence of Arbacia and before the divergence of Lytechinus and S. purpuratus (i.e., between ~185 and 50 mya) [33]. If that is the case, the LCA of all living sea urchins would have possessed 1N = 22 chromosomes, instead of the presumed 21 ancestral chromosomes illustrated in S10 Fig. Future synteny analyses and chromosomal architecture reconstructions using genomes of early branching sea urchins will help to resolve this question.

Hox clusters in rearranged chromosomes Hox genes are typically arranged in clusters and specify bilaterian body regions along the anteroposterior axis [43]. Contrary to their structural and functional conservation, we find that Hox clusters are located in chromosomes that underwent fusion with extensive mixing among the 10 bilaterian species we examined, with the sole exception of amphioxus BFL16 (S16 Fig). In the LCA of bilaterians, the Hox cluster was inferred to be positioned in BALG B2. The descendant of this ALG (DALG B2) contributed to the ambulacrarian-specific fusion with DALG C2 to form AALG B2⊗C2. Subsequently, its descendant in echinoderms further underwent an additional fusion-with-mixing with ALG E to give rise to a chromosome resembling SPU1 in sea urchins. Meanwhile, in hemichordates, AALG B2⊗C2 split into HALGs B2⊗C2-a and B2⊗C2-b (represented by the extant PFL18 and PFL23, S16 Fig). Intriguingly, this splitting event in the hemichordate ancestor separated the Hox cluster and the distalless gene, which are commonly linked in vertebrate genomes [44]. This genetic feature appears to be unique to hemichordates, as the Hox cluster and distalless gene are located in the same chromosome in all other deuterostome species we examined (i.e., amphioxus BFL16, sea star POC9, and sea urchin SPU1). Nevertheless, it remains unclear whether the separation of the Hox cluster and distalless gene during the hemichordate-specific chromosomal split would have resulted in functional consequences related to the origin of the hemichordate body plan. BALG B2 is also involved in different fusion-with-mixing events in the 5 spiralian species, with the spiralian Hox clusters located on the highly rearranged RPH14, SCO9, PYE1, PEC4, and SBE9 (S16 Fig). It is tempting to speculate that these chromosome rearrangement events may have changed the regulatory landscape of Hox genes and contributed to the evolution of lineage-specific body plans. Further studies would certainly be required to test this hypothesis. While intrachromosomal rearrangement events are highly associated with the accumulation of transposable elements (TEs) [45,46], Hox clusters are known to be largely devoid of TEs in chordates [47,48]. The exclusion of TEs from Hox clusters is thought to be chordate-specific, as this trend was not detected in 5 protostome species that have been analyzed (including 4 insects and the nematode Caenorhabditis elegans) [48]. The observation that most Hox clusters are situated in chromosomes that underwent fusion-with-mixing prompted us to analyze TE densities in the Hox-bearing chromosomes. We observed a clear drop-off of TE densities (including DNA transposons (DNA), long terminal repeats (LTR), long interspersed nuclear elements (LINE), and short interspersed nuclear elements (SINE)) within Hox clusters compared with the non-Hox regions of the same chromosomes; this trend was observed in all 9 bilaterian species we examined (Figs 5A and S20–S22). The overall TE densities in Hox-bearing chromosomes were similar to the densities observed across entire genomes (S23 Fig). The exclusion of TEs in Hox clusters is particularly apparent in amphioxus BFL and hemichordate PFL (approximately 77% less than the density of non-Hox regions) in which the Hox clusters are relatively intact (Fig 4). Therefore, the trend of lower TE densities in Hox clusters is broadly observed across bilaterians and is not limited to chordates. The mechanism that suppresses TE invasion (either by selection against insertions or inhibition of such mutations) remains in effect even when Hox clusters are situated in otherwise highly rearranged chromosomes. We also noticed that many genes neighboring Hox clusters, except for the evx genes, are highly rearranged and their orthologous genes are commonly found in different chromosomes (S24 and S25 Figs). This result is consistent with the observation that TEs exist at higher densities outside of Hox clusters, where they can promote intrachromosomal rearrangements. Further characterizations of TE distributions within Hox clusters revealed a higher density of TEs around the posterior Hox genes (between Hox9 and Hox15) within the amphioxus BFL Hox cluster. This higher density is consistent with a previous observation of repeat islands between the amphioxus posterior Hox genes that may contribute to the highly derived posterior region of the amphioxus Hox cluster [47,49,50]. Despite the generally low TE density across the Hox cluster of hemichordate PFL, we noticed that the inversion of Hox13b and Hox13c coincides with the presence of more TEs near the posterior end of the Hox cluster (Fig 5B, PFL). Similarly, the numbers, positions, and orientations of Hox genes between Hox5 and Hox11/13 in the 3 sea urchin species (SPU, LVA, and LPI) have undergone notable changes, which is in line with the higher densities of TEs detected in these regions (Fig 5B). Taken together, these results indicate that exclusion of TEs from Hox clusters appears to be a conserved feature in bilaterians. Nevertheless, TE invasions sometimes occur in the posterior regions of deuterostome Hox clusters, and these invasions have likely contributed to local rearrangements of Hox genes. Our observations are reminiscent of the proposed “deuterostome posterior flexibility” model, which explains how the posterior Hox genes evolved faster in deuterostomes than in protostomes [50,51]. In conclusion, the distributions of TEs both outside and within certain regions of Hox clusters coincide with intrachromosomal gene rearrangements, which may modify TAD structures of Hox clusters and alter the transcriptional regulation of Hox genes.

Evolutionary history of the pharyngeal gene cluster The pharyngeal gene cluster contains 4 transcription factor genes (in the order of nkx2.1, nkx2.2, pax1/9, and foxa) and 2 non-transcription factor genes (slc25a21a and mipol1), and their expression in the pharyngeal slits and surrounding endoderm is considered to be a deuterostome-specific feature [15]. Three additional genes, msx, cnga, and egln3, which respectively encode a homeobox transcription factor, a subunit of cyclic nucleotide-gated channels and Egl-9 family hypoxia inducible factor 3, are also linked to the cluster in some deuterostome species [9,15,52]. The complete pharyngeal gene cluster has so far only been found in deuterostomes, but some of the genes are also linked in protostomes [9]. It has thus been proposed that rather than being a deuterostome-specific trait, an intact cluster may have already been present in the LCA of bilaterians and was later dispersed in protostome lineages [9]. To gain insight into the evolutionary history of the pharyngeal cluster, we analyzed gene complements of the cluster in several bilaterian and non-bilaterian genomes (Fig 6A and 6B). In all the deuterostome genomes we analyzed, we found that xrn2, which encodes a 5′ to 3′ exoribonuclease, is associated with the aforementioned pharyngeal genes and usually located upstream and adjacent to nkx2.1. Based on the gene repertoire and linkage relationships in the deuterostome genomes, we deduced that the complete complement of the pharyngeal cluster in the LCA of deuterostomes included 10 genes. The complement began with xrn2, followed by 3 transcription factor genes (nkx2.1, nkx2.2, and msx), then cnga, pax1/9, slc25a21, mipol1, and foxa, and finally egln3. Several lineage-specific changes then took place within the pharyngeal clusters of deuterostomes (Figs 6B and S26). In the hemichordate PFL, cnga was duplicated, and ghrA genes invaded the pharyngeal cluster between the cnga and pax1/9 genes. In the sea urchin SPU, the pharyngeal cluster is broken into 3 parts, although the 3 parts are still located on the same chromosome (SPU5), and the second part (including msx, cnga, pax1/9, and slc25a21) is inverted. PPT PowerPoint slide

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TIFF original image Download: Fig 6. A possible evolutionary history of the pharyngeal gene cluster. The pharyngeal gene architectures are shown for presumed last common ancestors at key phylogenetic nodes (a) and selected living metazoan species (b) (see S26 Fig for the complete dataset). Genes that are commonly linked together are shown in the same color; homeobox-containing genes, including nkx2.1, nkx2.2 and msx, are in green, mipol1 and foxa genes are in blue, and pax1/9 and slc25a21 are in red. The gray circles indicate genes that are located within the pharyngeal gene cluster. Double slashes are introduced when more than 3 genes are located in between 2 pharyngeal genes. Because pax genes of cnidarians and sponges do not show one-to-one correspondence with those of bilaterians, we surveyed the locations of all potential pax genes and found that none is linked with the other pharyngeal-related genes in cnidarians and sponges. https://doi.org/10.1371/journal.pbio.3002661.g006 In all 6 spiralian genomes we analyzed, orthologs of xrn2 were found to be adjacent to nkx2.1, and mipol1 and foxa genes were also linked (Figs 6B and S26). In a previous study [9], paired gene linkages of nkx2.1 and nkx2.2, pax1/9 and slc25a21, and mipol1 and foxa were also identified in various protostomes. These results support the existence of 3 microsyntenic blocks, including (1) xrn2 and nkx2 genes; (2) pax1/9 and slc25a21; and (3) mipol1 and foxa, as conserved features of bilaterians. Intriguingly, most of the orthologous genes of the pharyngeal cluster are located on the same chromosome, regardless of whether the microsyntenic relationships are maintained. Based on these observations, we considered 2 scenarios for the evolution of the pharyngeal gene cluster: (1) the LCA of bilaterians (similar to the LCA of deuterostomes) possessed a complete pharyngeal gene cluster that later broke up into 3 microsyntenic blocks in protostomes; (2) the LCA of bilaterians (similar to the LCA of protostomes) had the pharyngeal genes arranged in 3 microsyntenic blocks in the same chromosome that became closely linked to form a compact cluster in deuterostomes. To find evidence supporting or excluding these scenarios, we analyzed the genomic positions of the orthologous genes in outgroups to the bilaterians, including several cnidarians and sponges (S26 Fig). In the coral AMI, we observed a syntenic block containing xrn2, nkx2, msx-related, and cgna genes. Other cnidarian species either had preserved parts of this syntenic block (e.g., xrn2 and nkx2 are adjacent in the coral XSP; msx-related and cgna are linked in the sea anemone SCAL) or they lacked the syntenic relationships (S26 Fig). Additionally, slc25a21 was absent in all 6 cnidarian genomes we analyzed. This gene was likely lost in cnidarians, because an ortholog of slc25a21 was identified in the sponge genomes. Moreover, except for the pax genes, orthologs of the other pharyngeal genes are located on the same chromosome of most cnidarian genomes we analyzed. In the 2 sponge genomes, orthologs of the pharyngeal genes are mostly located on different chromosomes or scaffolds, and no microsyntenic blocks were identified. We can therefore infer using the parsimony principle that one microsyntenic block (composed of xrn2, nkx2, msx-related, and cgna genes) was already present in the LCA of bilaterians and cnidarians, and the other pharyngeal genes were located on the same chromosomes but had not yet formed microsyntenic blocks. The 2 additional microsyntenic pairs (pax1/9-slc25a21 and mipol1-foxa) were established in the bilaterian LCA and persist in extant protostomes and deuterostomes. In the lineage leading to the examined spiralian species, the more ancient syntenic block was likely partially disrupted, with only xrn2 and nkx2 genes remaining tightly associated. During the evolution of deuterostomes, the 3 microsyntenic blocks became linked and the egln gene was added at the end, forming the complete pharyngeal gene cluster. Our data therefore support a scenario in which the compact pharyngeal gene cluster of deuterostomes was gradually established from preexisting bilaterian microsyntenic blocks on the deuterostome stem. We cannot, however, rule out the scenario in which individual genes or small blocks distributed along an ancestral chromosome assembled into an ordered cluster in the bilaterian ancestor before breaking into 3 microsyntenic blocks in protostomes. Assembly of the 3 microsyntenic blocks into the deuterostome pharyngeal gene cluster plausibly contributes to the co-regulation of the genes. Indeed, similar temporal expression profiles of the pharyngeal cluster genes are observed among deuterostomes, while orthologs of these genes in protostome and non-bilaterian species display more divergent expression profiles [42]. These results support the idea that clustering of the pharyngeal genes in deuterostomes likely contributes to their co-regulation.

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