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Article Morphological Stasis and Proteome Innovation in

László Bányai, Krisztina Kerekes, Mária Trexler and László Patthy *

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary; [email protected] (L.B.); [email protected] (K.K.); [email protected] (M.T.) * Correspondence: [email protected]; Tel.: +36-1382-6751

 Received: 24 May 2018; Accepted: 11 July 2018; Published: 16 July 2018 

Abstract: , extant representatives of , are prototypic examples of evolutionary stasis; they preserved a morphology and body-plan most similar to the fossil chordates from the early Cambrian. Such a low level of morphological is in harmony with a low rate of amino acid substitution; proteins were shown to evolve slower than those of the slowest evolving , the elephant shark. Surprisingly, a study comparing the predicted proteomes of Chinese amphioxus, belcheri and the Florida amphioxus, Branchiostoma floridae has led to the conclusion that the rate of creation of novel combinations is orders of magnitude greater in lancelets than in any other Metazoa, a finding that contradicts the notion that high rates of protein innovation are usually associated with major evolutionary innovations. Our earlier studies on a representative sample of proteins have provided evidence suggesting that the differences in the domain architectures of predicted proteins of these two species reflect annotation errors, rather than true innovations. In the present work, we have extended these studies to include a larger sample of genes and two additional lancelet species, Asymmetron lucayanum and Branchiostoma lanceolatum. These analyses have confirmed that the domain architecture differences of orthologous proteins of the four lancelet species are because of errors of prediction, the error rate in the given species being inversely related to the quality of the transcriptome dataset that was used to aid gene prediction.

Keywords: amphioxus; Asymmetron; Branchiostoma; domain architecture; gene prediction; annotation; lancelet; proteome; stasis; transcriptome

1. Introduction Cephalochordates (lancelets), the basal group of extant chordates, diverged from other chordates (urochordates and ) about 550 Mya [1,2]. Lancelets are frequently referred to as living fossils because they have preserved a morphology and body-plan most similar to the fossil chordates from the early Cambrian and middle Cambrian period [3–6]. Lancelets, consisting of three genera (Branchiostoma, Epigonichthys and Asymmetron), are widely distributed in tropical and temperate shallow seas inhabiting inshore waters protected from wave action. Phylogenetic analyses based on amino acid sequences of protein-coding genes of amphioxus species have revealed that the Asymmetron clade diverged first from the common ancestor of extant lancelets, followed by the Epigonichthys and Branchiostoma clades [7]. The divergence time estimates of the various amphioxus species were shown to fit well with the time estimates for the closure of seaways by continental drift, providing a plausible explanation for the differences in the geographical distribution of the diverging species. These explanations implicitly assume that the common ancestor of the extant lancelets also existed in an epibenthic state, inhabiting sandy seafloors of inshore waters [7].

Genes 2018, 9, 353; doi:10.3390/genes9070353 www.mdpi.com/journal/genes Genes 2018, 9, 353 2 of 13

The striking conservation of the morphology in cephalochordates may be illustrated by the fact that although the Floridian Branchiostoma floridae and the Mediterranean Branchiostoma lanceolatum diverged ~100 Mya, they are very similar. The most plausible explanation for such morphological conservation is that their developmental program is unchanged. In harmony with this expectation, Somorjai and coworkers have shown that the expression patterns of key developmental genes are practically identical in B. lanceolatum and B. floridae [8]. The recent observation of Holland et al. [9] that of B. floridae, when fertilized with sperm of Asymmetron lucayanum (or vice versa), develop through embryonic and larval stages, has provided the most extreme example of hybridization that has ever been demonstrated among multicellular as it occurs between species that diverged ~120–160 million years ago. Wilson and coworkers were the first to point out that the chief molecular barriers to interspecific hybridization are the regulatory system differences between the maternal and paternal , which must function in concert if an interspecific zygote is to develop [10,11]. As a corollary, it was suggested that higher rates of regulatory evolution create a greater probability of developmental incompatibility between related animals [10,11]. were shown to lose their potential for interspecific hybridization much slower than because the rate of regulatory evolution is significantly higher in mammals than in birds [10–12]. Accordingly, the finding that B. floridae and A. lucayanum can give rise to interspecific hybrids also supports the notion that the rate of regulatory evolution is extremely low in the cephalochordate lineage. Cephalochordates are thus prototypic examples of phenotypic stasis; practically no morphological change over very long evolutionary periods. The underlying process causing phenotypic stasis is that populations well adapted to their local environment are subject to stabilizing selection as long as the local environment remains essentially unchanged, that is, the populations occupy relatively stable niches [13–15]. For the phenotypic stasis of lancelets, this explanation would imply that their common ancestor was well adapted to seafloors of inshore waters and that such environments (albeit on different continents) were relatively stable in the last ~100–160 million years. Studies on the proteomes of lancelets have provided evidence compatible with the notion that they are subject primarily to stabilizing selection; analyses of the sequences of protein-coding genes of B. floridae and A. lucayanum have shown that strong purifying selection holds for the majority of genes and that these proteins are evolving more slowly than in the case of the slowest-evolving vertebrates [16,17]. Surprisingly, a study comparing the proteomes of the Chinese lancelet, B. belcheri, and the Florida lancelet, B. floridae, has concluded that the rate of creation of novel protein domain combinations is orders of magnitude greater in lancelets than in any other Metazoa [18]. In this study, the authors have compared the presence–absence status of protein domain combinations in proteins of various species and concluded that lancelets acquired three-fold more domain combinations than any vertebrate. According to their estimates, lancelets gained new domain pairs at a rate of >10 per Myr, a rate 10–100-fold higher than that observed in other metazoan lineages. The authors have assumed that such an unusually high rate of creation of novel protein domain combinations may be a result of continued activity of transposable elements in the lancelet lineage [18]. Interestingly, the unusually high rate of domain architecture differences observed when the proteomes of B. belcheri and B. floridae were compared is not accompanied by a high amino acid substitution rate. In agreement with the data of Delsuc et al. [2] and Putnam et al. [16], the authors have also found that the rate of amino-acid substitutions is much lower than in urochordates or vertebrates. Thus, whereas the low rate of amino acid substitution appears to be in harmony with phenotypic stasis and stabilizing selection in lancelets, the unusually high rate of creation of novel proteins (with novel domain combinations) is surprising as it would contradict not only the dominance of stabilizing selection, but also the general observation that high rates of creation of novel proteins are usually associated with major evolutionary innovations, such as those that accompanied the appearance of Metazoa and vertebrates [19,20]. Genes 2018, 9, 353 3 of 13

In an earlier work, we have suggested a possible explanation for these contradictions: the differences in domain architecture of orthologous proteins of B. belcheri and B. floridae might reflect gene prediction errors rather than true innovations [21]. The plausibility of this assumption is supported by the fact that genome-annotation of intron-rich genomes is still difficult; the exact genomic structure of protein-coding genes of higher eukaryotes is correctly predicted for only about 60% of the genes [22]. Furthermore, as we have pointed out earlier, when predicted proteomes are compared, the contribution of gene prediction errors to domain architecture differences of orthologs may be far greater than those due to true gene rearrangements [23]. In the present work, we have extended our studies to include the genomes and transcriptomes of two additional lancelet species, Asymmetron lucayanum [17,24] and B. lanceolatum [25]. Furthermore, in addition to the sample of one hundred randomly selected genes analyzed in our earlier work [21], we have also performed in-depth analysis of key developmental genes that are critical for determining anteroposterior and dorvosventral patterning and left–right asymmetric development of amphioxus. The results of our analyses indicate that the domain architecture differences of orthologous proteins of the four lancelet species are due to errors of gene prediction, and that the rate of gene prediction error is inversely related to the quality of the transcriptome dataset used to aid gene prediction. The proportion of correctly predicted proteins was found to increase in the order B. lanceolatum << B. floridae < B. belcheri < A. lucayanum, reflecting differences in the information-content of their transcriptome datasets.

2. Materials and Methods We have randomly selected one hundred proteins of B. belcheri from the dataset Branchiostoma.belcheri HapV2_proteins.fa deposited on the website of the genome data of Chinese lancelet [26] as described previously [21]. In the present work, we have also selected key developmental genes that are known to be critical for determining anteroposterior (AP) and dorvosventral (DV) patterning, and left-right (LR) asymmetric development of amphioxus [8,27–30]. Sequences of the latter group of B. belcheri proteins were downloaded from the Chinese Lancelet Genome website [26]. The genes selected represent constituents of several signaling pathways (the Wnt/β-catenin, Nodal, and Bmp pathways) that pattern the dorsoventral axis; constituents of the Nodal and Bmp pathways that regulate development of left-right asymmetry; and HOX, OTX, KROX, and PBX genes that are crucial for determining anteroposterior patterning. We have used the reciprocal best-hit method to identify orthologs of the selected proteins, using the Branchiostoma.belcheri HapV2_proteins.fa dataset for B. belcheri, the National Center for Biotechnology Information’s (NCBI) non-redundant database of B. floridae proteins, the transcriptomes of A. lucayanum and B. lanceolatum, as well as the human section of the high quality, manually curated UniProtKB/Swiss-Prot database. As the mean identity of orthologous proteins of the Chinese and Florida lancelets was calculated to be 81.2% [18], we have used a cut off-value of 60% amino acid sequence identity as the minimum requirement for lancelet orthologs. The domain architectures of proteins (defined as the linear sequence of Pfam-A domains) were determined using Pfam [31]. Multiple sequence alignments were obtained using Clustal Omega [32]. In cases where orthologous proteins of B. belcheri, B. floridae, B. lanceolatum, and A. lucayanum differed in domain architecture, we assumed that domain architecture deviation is more likely to reflect errors in gene prediction than true changes in gene structure [23]. To distinguish the correct and erroneous members of the ortholog pair, we have compared their domain architectures with those of their orthologs present in the UniProtKB/Swiss-Prot database; the proteins with the same domain architecture as those of orthologous Swiss-Prot entries were judged to be correctly predicted. In the case of some lancelet proteins where human orthologs could not be identified unambiguously in the Swiss-Prot database, the proteins were analyzed with the MisPred tools [33,34] to check whether they are erroneous (incomplete or mispredicted). Genes 2018, 9, 353 4 of 13

To decide whether the domain architecture deviation of the erroneous member(s) of the ortholog groups reflects error in gene prediction or true change in gene structure, we have re-annotated these genes using the FixPred protocol [35]. The FixPred pipeline attempts to correct mispredicted sequences by findingGenes 2018 experimental, 9, x FOR PEER REVIEW evidence for the correct sequence version in complementary DNA4 (cDNA),of 13 expressed sequence tag (EST), and transcriptome databases and by re-analyses of genomic sequences. (cDNA), expressed sequence tag (EST), and transcriptome databases and by re‐analyses of genomic 3. Resultssequences.

3.1. Absence3. Results of Orthologs in Some Lancelet Species May Reflect Errors of Gene Prediction Out of the randomly selected proteins of B. belcheri, 76% had orthologs in the human section of 3.1. Absence of Orthologs in Some Lancelet Species May Reflect Errors of Gene Prediction the UniProtKB/Swiss-Prot database (Supplementary Table S1). In this dataset, 93% of the B. belcheri proteins hadOut orthologs of the randomly in A. lucayanumselected proteins, 92% inof B. belcheri floridae,, 76% but had only orthologs 79% had in matches the human in B.section lanceolatum of . the UniProtKB/Swiss‐Prot database (Supplementary Table S1). In this dataset, 93% of the B. belcheri If a B. belcheri protein had no ortholog in another lancelet species (although orthologs are present in proteins had orthologs in A. lucayanum, 92% in B. floridae, but only 79% had matches in B. lanceolatum. the Swiss-Prot database), we assumed that this reflects an error of genome annotation rather than true If a B. belcheri protein had no ortholog in another lancelet species (although orthologs are present in loss ofthe the Swiss gene.‐Prot In database), such cases, we we assumed subjected that genomic this reflects and/or an error transcriptomic of genome annotation databases rather to analysis than by FixPredtrue tools loss of to the find gene. evidence In such forcases, the we presence subjected of genomic the ‘missing’ and/or transcriptomic gene. Here, we databases illustrate to analysis this of analysisby FixPred with Ryk tools receptor to find tyrosineevidence for kinase. the presence of the ‘missing’ gene. Here, we illustrate this type of analysis with Ryk receptor tyrosine kinase. Ryk Receptor Tyrosine Kinase Ryk Receptor Tyrosine Kinase Protein 247370_PRF0 of B. belcheri has a domain organization typical of Ryk receptor tyrosine kinases; theyProtein have 247370_PRF0 a Wnt-inhinitory of B. belcheri factor has (WIF) a domain domain organization that serves typical to bind of Ryk Wnts receptor [36]. Ryk tyrosine proteins are presentkinases; in they all have groups a Wnt of‐inhinitory , factor as well (WIF) as indomain that serves [37], therefore,to bind Wnts our [36]. observation Ryk proteins that are Ryk present in all groups of Bilateria, as well as in Cnidaria [37], therefore, our observation that Ryk proteins proteins are missing from the predicted proteome of B. floridae was surprising (Supplementary Table S1). are missing from the predicted proteome of B. floridae was surprising (Supplementary Table S1). Our Our earlier analysis of the B. floridae genome and transcriptome, however, provided evidence for a RYK earlier analysis of the B. floridae genome and transcriptome, however, provided evidence for a RYK gene gene that encodes a a protein protein with with the the same same domain domain organization organization as that as that of B. of belcheriB. belcheri [21].[ 21Analysis]. Analysis of the of the transcriptometranscriptome of A. of lucayanum A. lucayanumidentified identified full-length full‐length transcripts that that confirm confirm the the existence existence and andactivity activity of a RYKof a geneRYK gene in this in this lancelet lancelet species. species. The The transcriptome transcriptome of B.B. lanceolatum lanceolatum alsoalso contained contained transcript transcript fragmentsfragments derived derived from from its RYKits RYKgene; gene; although although the the full-lengthfull‐length protein protein could could not not be reconstructed be reconstructed from from thesethese fragments, fragments, they they confirm confirm the the existence existence ofof an active active RYKRYK genegene in B. in lanceolatumB. lanceolatum (Figure(Figure 1). 1).

FigureFigure 1. Ryk1. Ryk receptor receptor tyrosine tyrosine kinase genes genes are are present present in lancelet in lancelet species. species. (A) Although (A) Although the Branchiostoma belcheri genome encodes a full‐length Ryk receptor tyrosine kinase (247370_PRF0), no the Branchiostoma belcheri genome encodes a full-length Ryk receptor tyrosine kinase (247370_PRF0), no ortholog is found in the predicted proteome of . The transcriptome of Asymmetron lucayanum contains transcripts encoding full‐length Ryk receptor tyrosine kinase (GETC01129892.1), but in the case of B. lanceolatum, only fragments of the RYK gene are represented

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ortholog is found in the predicted proteome of Branchiostoma floridae. The transcriptome of Asymmetron lucayanum contains transcripts encoding full-length Ryk receptor tyrosine kinase (GETC01129892.1), but in the case of B. lanceolatum, only fragments of the RYK gene are represented in its transcriptome; (B) Analysis of the B. floridae genome and transcriptome with FixPred provided evidence for a RYK gene that encodes a protein (RYK_BRAFL) with the same domain architecture as those of B. belcheri (RYK_BRABE) and A. lucayanum (RYK_ASYLU). The figure illustrates the Pfam domain architectures of the proteins and the position of protein fragments of the Ryk receptor tyrosine kinases. Color code for Pfam-A domains: Wnt-inhinitory factor (WIF)—green; Pkinase_Tyr—red.

3.2. Domain Architecture Differences of Orthologs of Lancelet Species May Reflect Errors of Gene Prediction As we have outlined in the Materials and Methods, the rationale of our approach was the assumption that lancelet proteins with the same domain organization as those of orthologous Swiss-Prot entries are correctly predicted. In the dataset of randomly selected proteins that have orthologs in the human Swiss-Prot database (Supplementary Table S1), 69%, 58%, 51%, and 7% of the proteins of A. lucayanum, B. belcheri, B. floridae, and B. lanceolatum, respectively, had the same domain architecture as their human ortholog. The extremely low proportion of B. lanceolatum proteins with the correct domain architecture probably reflects the fact that its transcriptome represents a relatively low coverage of its gene set. The cDNA library of B. lanceolatum, constructed from around 1.4 million reads, could be assembled into contigs with an average length of ~490 bp [25], and such short contigs are likely to encode only fragments of proteins [25]. Conversely, our observation that A. lucayanum has the highest proportion of the proteins with the correct domain architecture is explained by the fact that its transcriptome [17] is a rich source of experimental evidence for genes expressed in various tissues of lancelets. The authors have obtained ~146 million and ~177 million reads for the adult and 20h larvae libraries and assembled them into contigs with weighted median lengths of 1886 bp and 1635 bp, respectively, thus the transcriptome of A. lucayanum is more likely to contain full-length transcripts of the proteins. Because a gene mispredicted in one of the lancelet species may be correctly predicted in another species, the percentage of lancelet proteins with correct domain architecture is higher than in any of the individual species; 76% of the ortholog groups contained at least one amphioxus protein with the correct domain architecture. In the dataset of proteins involved in developmental regulation (Supplementary Table S2), all the lancelet proteins had orthologs in the human UniProtKB/Swiss-Prot dataset and 92% of the ortholog groups contained at least one amphioxus protein with the correct domain architecture; 92%, 82%, and 91% of the proteins of A. lucayanum, B. belcheri, and B. floridae, respectively, had the same domain architecture as their human ortholog, but only a lower proportion (69%) of the orthologs had full-length transcripts in the B. lanceolatum transcriptome. We have shown that deviations in the domain organization of proteins of B. belcheri, B. floridae, or A. lucayanum from those of their human orthologs reflect errors of gene prediction; in these cases, FixPred found genomic and/or transcriptomic evidence for the conservation of the domain architectures. In the case of B. lanceolatum, however, full-length versions of the protein fragments could not be reconstructed. In summary, our data suggest that the percentage of correct primary protein predictions tends to increase in the order B. lanceolatum << B. floridae < B. belcheri < A. lucayanum, and that re-annotation with FixPred can verify the conservation of domain architectures, provided that genomic sequences are available. We can illustrate these conclusions with examples selected from Supplementary Tables S1 and S2. Genes 2018, 9, 353 6 of 13

3.2.1. WAP, Follistatin, Immunoglobulin, Kunitz and NTR Domain-Containing (WFIKKN) Protein Proteins 328450_PRF0 and 328460 PRF0 of B. belcheri are orthologous with the C-terminal and N-terminal parts of XP_002607180.1 of B. floridae, respectively [21]. Comparison of the domain structures of 328450_PRF0, 328460_PRF0, and XP_002607180.1 with those of their human orthologs has revealed that XP_002607180.1 of B. floridae has the domain organization typical of WFIKKN proteins of vertebrates (Figure2). The ‘fragmentation’ of the WFIKKN protein of B. belcheri was shown to be the result of an error of the prediction of the B. belcheri proteome that could be corrected by re-annotation of the B. belcheri WFIKKN gene [21]. The transcriptome of B. lanceolatum had no transcript Genes 2018, 9, x FOR PEER REVIEW 6 of 13 derived from its WFIKKN gene. On the other hand, the transcriptome of A. lucayanum contains three transcriptstranscript (GESY01032163.1, derived from its GESY01032162.1, WFIKKN gene. On the and other GESY01012772.1) hand, the transcriptome of the ofWFIKKN A. lucayanumgene and the genome assemblycontains three contained transcripts a single(GESY01032163.1, scaffold containing GESY01032162.1, the 5 and0 part GESY01012772.1) half of the WFIKKN of the WFIKKNgene (Sequence ID: LZCU01164105.1),gene and the genome permitting assembly contained the reconstruction a single scaffold of containing the full-length the 5′ part sequence half of the usingWFIKKN the FixPred gene (Sequence ID: LZCU01164105.1), permitting the reconstruction of the full‐length sequence using protocol.the Analysis FixPred ofprotocol. the genomes Analysis andof the transcriptomes genomes and transcriptomes of B. belcheri of ,B.B. belcheri floridae, B., floridae and A., and lucayanum A. thus confirm thatlucayanum the domain thus confirm architectures that the domain of WFIKKN architectures proteins of WFIKKN are proteins conserved are conserved in lancelets. in lancelets.

Figure 2. WAP, Follistatin, Immunoglobulin, Kunitz and NTR Domain‐Containing (WFIKKN) genes Figure 2. WAP, Follistatin, Immunoglobulin, Kunitz and NTR Domain-Containing (WFIKKN) genes encoding proteins with domain architectures typical of vertebrate WFIKKN proteins are present in encodingthe proteins genomes with of B. belcheri domain, B. floridae architectures and A. lucayanum typical. (A of) Proteins vertebrate 328450_PRF0 WFIKKN and 328460_PRF0 proteins are of present in the genomesB. belcheri of B. correspond belcheri, B. to floridaedifferent andregionsA. of lucayanum the WFIKKN.(A protein) Proteins of B. 328450_PRF0belcheri and the WFIKKN and 328460_PRF0 of B. belcheriproteincorrespond of B. floridae to (XP_002607185.1). different regions The transcriptome of the WFIKKN of A. lucayanum protein contains of B. belcheri three transcriptsand the WFIKKN protein of(GESY01012772.1,B. floridae (XP_002607185.1). GESY01032162.1, and The GESY01032163.1) transcriptome of of theA. WFIKKN lucayanum genecontains and the genome three transcripts assembly of A. lucayanum contains a single scaffold encoding the 5′ part half of the WFIKKN gene (GESY01012772.1,(LZCU01164105.1); GESY01032162.1, (B) Analysis of andthe transcriptome GESY01032163.1) and genome of of the A. lucayanumWFIKKN withgene FixPred and has the genome 0 assemblyprovided of A. lucayanum evidence thatcontains its WFIKKN a gene single encodes scaffold a protein encoding (WFIKKN_ASYLU) the 5 part with half the same of the domainWFIKKN gene (LZCU01164105.1);architecture as (B those) Analysis of B. belcheri of the (WFIKKN_BRABE) transcriptome and and B. genomefloridae (WFIKKN_BRAFL). of A. lucayanum Thewith figure FixPred has providedillustrates evidence the that Pfam its domainWFIKKN organizationsgene encodes of the aproteins protein and (WFIKKN_ASYLU) the position of protein with fragments the same of domain WFIKKN proteins. Color code for Pfam‐A domains: WAP—green; Kazal—red; Immunoglobulin— architecture as those of B. belcheri (WFIKKN_BRABE) and B. floridae (WFIKKN_BRAFL). The figure blue; Kunitz—yellow; netrin (NTR)—purple. illustrates the Pfam domain organizations of the proteins and the position of protein fragments of WFIKKN3.2.2. proteins.Calcium‐Binding Color code Mitochondrial for Pfam-A Carrier domains: Protein WAP—green; Kazal—red; Immunoglobulin—blue; Kunitz—yellow;The 330210_PRF0 netrin (NTR)—purple. protein of B. belcheri has the domain architecture typical of calcium‐binding mitochondrial carrier proteins, containing an EF‐hand domain and three Mito_carr domains 3.2.2. Calcium-Binding(Supplementary Table Mitochondrial S1). The predicted Carrier B. floridae Protein orthologue (XP_002602990.1) differs in domain architecture in that it has a C‐terminal extension with a Peptidase_M2 domain. Because the The 330210_PRF0XP_002602990.1 protein protein sequence of B. belcheriviolated basichas dogmas the domain that MisPred architecture uses to detect typical gene ofprediction calcium-binding mitochondrialerrors [33,34], carrier we proteins,suspected that containing the B. floridae an protein EF-hand is mispredicted. domain Analyses and three of EST Mito_carr databases domains have confirmed this suspicion; ESTs originating from the putative ‘fusion’ region were found to align

Genes 2018, 9, 353 7 of 13

(Supplementary Table S1). The predicted B. floridae orthologue (XP_002602990.1) differs in domain architecture in that it has a C-terminal extension with a Peptidase_M2 domain. Because the XP_002602990.1 protein sequence violated basic dogmas that MisPred uses to detect gene prediction errors [33,34], we suspected that the B. floridae protein is mispredicted. Analyses of EST databases have confirmed this suspicion; ESTs originating from the putative ‘fusion’ region were found to align eitherGenes with 2018, 9, thex FOR N-terminal PEER REVIEW or the C-terminal fusion parts, but not both parts of7 of the13 chimeric XP_002602990.1 protein, permitting the correction of the sequence [21]. Our conclusion that lancelets either with the N‐terminal or the C‐terminal fusion parts, but not both parts of the chimeric contain classicalXP_002602990.1 calcium-binding protein, permitting mitochondrial the correction carrier of the sequence proteins [21]. is Our also conclusion supported that lancelets by the analysis of the transcriptomecontain classical of A. calcium lucayanum‐binding; itmitochondrial contains full-length carrier proteins transcripts is also supported (e.g., by GETC01125846.1) the analysis of of the calcium-bindingthe transcriptome mitochondrial of A. lucayanum carrier; it contains protein full gene‐length (Figure transcripts3). The (e.g., transcriptome GETC01125846.1) of of B.the lanceolatum had severalcalcium transcript‐binding fragments mitochondrial derived carrier protein from its gene calcium-binding (Figure 3). The transcriptome mitochondrial of B. lanceolatum carrier protein gene. had several transcript fragments derived from its calcium‐binding mitochondrial carrier protein Althoughgene. these Although fragments these fragments did not permitdid not permit the reconstruction the reconstruction of of the the full-lengthfull‐length protein, protein, one of one of them matchingthem the C-terminalmatching the part C‐terminal of the part protein of the (JT887996.1) protein (JT887996.1) supports supports the conclusion the conclusion that that the the protein does not containprotein a Peptidase_M2 does not contain domain a Peptidase_M2 as it has domain a stop as it codon has a stop in the codon same in the region same asregion its orthologs as its in the other lanceletorthologs species. in the other Analysis lancelet of species. the genomes Analysis andof the transcriptomes genomes and transcriptomes of lancelets of lancelets thus confirms thus that the confirms that the domain organizations of calcium‐binding mitochondrial carrier proteins are domain organizationsconserved in lancelets. of calcium-binding mitochondrial carrier proteins are conserved in lancelets.

Figure 3. Genes encoding proteins with domain architectures typical of vertebrate calcium‐binding Figure 3. Genes encoding proteins with domain architectures typical of vertebrate calcium-binding mitochondrial carrier proteins are present in the genomes of B. belcheri, B. floridae, B. lanceolatum, and A. mitochondriallucayanum carrier. (A) The proteins B. floridae are protein present XP_002602990.1 in the genomes is an oforthologB. belcheri of the, B. calcium floridae‐binding, B. lanceolatum , and A. lucayanummitochondrial.( Acarrier) The proteinB. floridae of B. belcheriprotein (330210_PRF0), XP_002602990.1 but it contains is an a C ortholog‐terminal extension of the calcium-binding with a mitochondrialPeptidase_M2 carrier domain. protein The of transcriptomeB. belcheri (330210_PRF0), of A. lucayanum contains but it full contains‐length transcripts a C-terminal of the extension gene with a Peptidase_M2(GESY01078193.1) domain. Theencoding transcriptome a protein with of aA. domain lucayanum architecturecontains characteristic full-length of calcium transcripts‐binding of the gene mitochondrial carrier proteins. The transcriptome of B. lanceolatum contains several transcripts of the (GESY01078193.1)calcium‐binding encoding mitochondrial a protein carrier with protein a gene domain and one architecture of them, matching characteristic the C‐terminal of calcium-bindingpart of mitochondrialthe protein carrier (JT887996.1), proteins. supports The transcriptome the conclusion that of B.it does lanceolatum not have containsa C‐terminal several Peptidase_M2 transcripts of the calcium-bindingdomain; (B mitochondrial) The corrected sequence carrier (CMC1_BRAFL) protein gene of and the one calcium of them,‐binding matching mitochondrial the carrier C-terminal part of the proteinprotein (JT887996.1), of B. floridae [21] supportshas the same the domain conclusion architecture that as its it doesorthologs not of have B. belcheri a C-terminal (CMC1_BRABE) Peptidase_M2 and A. lucayanum (CMC1_ASYLU). The figure illustrates the Pfam domain architectures of the proteins B domain;and ( ) the The position corrected of protein sequence fragments (CMC1_BRAFL) of calcium‐binding mitochondrial of the calcium-binding carrier proteins. Color mitochondrial code for carrier protein ofPfamB. floridae‐A domains:[21] EF has‐hand—green; the same Mito_carr—red; domain architecture Peptidase_M2—blue. as its orthologs of B. belcheri (CMC1_BRABE) and A. lucayanum (CMC1_ASYLU). The figure illustrates the Pfam domain architectures of the proteins andthe position of protein fragments of calcium-binding mitochondrial carrier proteins. Color code

for Pfam-A domains: EF-hand—green; Mito_carr—red; Peptidase_M2—blue. Genes 2018, 9, 353 8 of 13

3.2.3. Histone H2A Deubiquitinase

HistoneGenes 2018 H2A, 9, x deubiquitinases FOR PEER REVIEW are conserved proteins involved in the regulation8 of of 13 nucleosome dissociation and transcription activation. The domain architecture of the human protein Q5VVJ2 (MYSM1_HUMAN)3.2.3. Histone consistsH2A Deubiquitinase of a Myb_DNA-binding, a SWIRM, and a JAB domain. The architectures of the predictedHistoneB. belcheri H2A deubiquitinasesand B. floridae are conservedproteins, proteins however, involved differed in the from regulation that of nucleosome the human ortholog in as muchdissociation as one of and the transcriptionB. belcheri activation.orthologs The fragments domain architecture (256530_PRF0) of the human lacks theprotein JAB Q5VVJ2 domain, another (MYSM1_HUMAN) consists of a Myb_DNA‐binding, a SWIRM, and a JAB domain. The architectures orthologof fragment the predicted (256520_PRF0) B. belcheri and B. lacks floridae the proteins, Myb_DNA-binding however, differed from and that SWIRM of the human domains, ortholog whereas the B. floridaeinprotein as much (B6MUN4.1) as one of the B. belcheri lacks orthologs the Myb_DNA-binding fragments (256530_PRF0) domain. lacks the Our JAB analyses domain, another have confirmed that theseortholog deviations fragment in domain (256520_PRF0) architecture lacks the Myb_DNA reflect errors‐binding in geneand SWIRM prediction domains, that whereas could the be corrected with FixPredB. floridae (Figure protein4). (B6MUN4.1) The conclusion lacks the that Myb_DNA histone‐binding H2A domain. deubiquitinases Our analyses of have lancelets confirmed retained the that these deviations in domain architecture reflect errors in gene prediction that could be corrected domain architecturewith FixPred (Figure characteristic 4). The conclusion of Myb that Like, histone SWIRM H2A deubiquitinases And MPN Domainsof lancelets 1retained (MYSM1 the ) genes is also supporteddomain by architecture the analysis characteristic of the of transcriptome Myb Like, SWIRM of A.And lucayanum MPN Domains; it contains1 (MYSM1) full-length genes is also transcripts (e.g., GETC01084111.1)supported by the ofanalysis the MYSM1 of the transcriptomegene. The of transcriptome A. lucayanum; it contains of B. lanceolatum full‐length transcriptshad only (e.g., one transcript fragmentGETC01084111.1) derived from its of theMYSM1 MYSM1gene. gene. AnalysisThe transcriptome of the genomesof B. lanceolatum and transcriptomeshad only one transcript of B. belcheri, B. fragment derived from its MYSM1 gene. Analysis of the genomes and transcriptomes of B. belcheri, floridae, andB. floridaeA. lucayanum, and A. lucayanumthus confirm thus confirm that the that domain the domain architectures architectures of MYSM1of MYSM1 proteins proteins areare conserved in lancelets.conserved in lancelets.

Figure 4. Myb Like, SWIRM And MPN Domains 1 (MYSM1) genes encoding proteins with domain Figure 4. architecturesMyb Like, typical SWIRM of histone And H2A MPN deubiquitinases Domains 1are (MYSM1) present in the genes genomes encoding of B. belcheri proteins, B. floridae with, domain architecturesand A. typical lucayanum of histone. (A) The architectures H2A deubiquitinases of the predicted are histone present H2A in deubiquitinases the genomes of ofB. belcheriB. belcheri and , B. floridae, and A. lucayanumB. floridae deviate.(A) from The the architectures conserved domain of the architecture predicted of these histone proteins H2A in that deubiquitinases one of the fragments of B. belcheri of the B. belcheri protein (256530_PRF0) lacks the JAB domain, another fragment of the B. belcheri ortholog and B. floridae deviate from the conserved domain architecture of these proteins in that one of the (256520_PRF0) lacks the Myb_DNA‐binding and SWIRM domains, whereas the B. floridae protein fragments(B6MUN4.1) of the B. lacks belcheri the Myb_DNAprotein‐ (256530_PRF0)binding domain. The lacks transcriptome the JAB of domain, A. lucayanum another contains fragment full‐ of the B. belcherilengthortholog transcripts (256520_PRF0) of the MYSM1 lacksgene, but the the Myb_DNA-binding transcriptome of B. lanceolatum and SWIRM had only domains,one transcript whereas the B. floridae fragmentprotein (B6MUN4.1)derived from its lacks MYSM1 the gene; Myb_DNA-binding (B) Re‐annotation of domain.the MYSM1 The genes transcriptome of B. belcheri and of A.B. lucayanum floridae with the FixPred protocol has shown that these deviations are due errors in gene prediction, contains full-length transcripts of the MYSM1 gene, but the transcriptome of B. lanceolatum had only permitting the correction of these sequences (MYSM1_BRABE, MYSM1_BRAFL). The figure illustrates one transcriptthe Pfam fragment domain architectures derived fromof the itsproteins MYSM1 and position gene; of (B protein) Re-annotation fragments of MYSM1 of the proteins.MYSM1 genes of B. belcheriColorand B.code floridae for Pfamwith‐A domains: the FixPred Myb_DNA protocol‐binding—green; has shown SWIRM—red; that these deviationsJAB—blue. are due errors in gene prediction, permitting the correction of these sequences (MYSM1_BRABE, MYSM1_BRAFL). The figure

illustrates the Pfam domain architectures of the proteins and position of protein fragments of MYSM1 proteins. Color code for Pfam-A domains: Myb_DNA-binding—green; SWIRM—red; JAB—blue.

3.2.4. Chordin Chordin is a key developmental protein that dorsalizes early embryonic tissues by binding to ventralizing TGF-β family bone morphogenetic proteins and sequestering them in latent Genes 2018, 9, 353 9 of 13

Genes 2018, 9, x FOR PEER REVIEW 9 of 13 complexes.3.2.4. The Chordin domain architecture of the human protein is conserved in B. floridae (Q0Q581_BRAFL), the domain architectureChordin is a ofkey the developmental predicted protein sequence that ofdorsalizes the B. belcheriearly chordateortholog embryonic (Bb_056190F), tissues by however, is markedlybinding different; to ventralizing it lacks TGF two‐β family of the bone chordin morphogenetic (CHRD) proteins domains, and sequestering the C-terminal them in von latent Willebrand factor typecomplexes. C domain The (VWC) domain domain architecture is missing, of the the human penultimate protein VWCis conserved domain in is B. truncated, floridae and the (Q0Q581_BRAFL), the domain architecture of the predicted sequence of the B. belcheri ortholog protein has a long C-terminal extension containing a polycystic disease (PKD)_channel domain (Bb_056190F), however, is markedly different; it lacks two of the chordin (CHRD) domains, the C‐ (Figure5).terminal Because von the Willebrand protein architecture factor type C ofdomain Bb_056190F (VWC) domain violates is missing, basic dogmas the penultimate of MisPred, VWC we have suspecteddomain that the is differencetruncated, and between the protein the has domain a long C organizations‐terminal extension of the containingB. belcheri a polycysticand B. kidney floridae proteins reflects andisease error (PKD)_channel of the prediction domain of (Figure the B. 5). belcheri Becauseprotein the protein and architecture not true innovation.of Bb_056190F Re-annotationviolates of basic dogmas of MisPred, we have suspected that the difference between the domain organizations the CHRD gene of B. belcheri with the FixPred protocol has shown that this deviation is due to an error of the B. belcheri and B. floridae proteins reflects an error of the prediction of the B. belcheri protein and in gene prediction,not true innovation. permitting Re‐annotation the correction of the CHRD of this gene sequence. of B. belcheri Our with conclusion the FixPred that protocol lancelets has contain ‘classical’ chordinsshown that is this also deviation supported is due by to thean error analysis in gene of prediction, the transcriptome permitting the of A.correction lucayanum of this; it contains full-lengthsequence. transcripts Our conclusion (e.g., GETC01124733.1) that lancelets contain of the‘classical’CHRD chordinsgene. is The also supported transcriptome by the analysis of B. lanceolatum of the transcriptome of A. lucayanum; it contains full‐length transcripts (e.g., GETC01124733.1) of the had several transcript fragments derived from its CHRD gene. Although these fragments did not CHRD gene. The transcriptome of B. lanceolatum had several transcript fragments derived from its permit theCHRD reconstruction gene. Although of these the fragments full-length did protein,not permit an the ESTreconstruction (JT869941.1) of the full matching‐length protein, the C-terminal part of thean protein EST (JT869941.1) supports matching the conclusion the C‐terminal that part the proteinof the protein does supports not contain the conclusion a PKD_channel that the domain; it has a stopprotein codon does in not the contain same a regionPKD_channel as its domain; orthologs it has in a the stop other codon lancelet in the same species. region Analysis as its of the orthologs in the other lancelet species. Analysis of the genomes and transcriptomes of B. belcheri, B. genomes and transcriptomes of B. belcheri, B. floridae, and A. lucayanum thus confirm that the domain floridae, and A. lucayanum thus confirm that the domain architectures of chordins are conserved in architectureslancelets. of chordins are conserved in lancelets.

Figure 5. Chordin (CHRD) genes encoding proteins with domain architectures typical of vertebrate Figure 5. Chordinchordins are (CHRD present) in genes the genomes encoding of B. belcheri proteins, B. floridae with, domainand A. lucayanum architectures. (A) The characteristic typical of vertebrate chordins aredomain present architecture in the of genomes vertebrate of chordinsB. belcheri is conserved, B. floridae in B., floridae and A. (Q0Q581), lucayanum but .(theA structure) The characteristic of domain architecturethe B. belcheri ortholog of vertebrate (Bb_056190F) chordins is markedly is conserved different in in thatB. it floridae lacks some(Q0Q581), of the domains but and the structure of the B. belcherihas a longortholog C‐terminal (Bb_056190F) extension containing is markedly a polycystic different kidney disease in that (PKD)_channel it lacks some domain. of The the domains transcriptome of A. lucayanum contains full‐length transcripts of the CHRD gene (GETC01124733.1). and has aThe long transcriptome C-terminal of extension B. lanceolatum containing had several a polycystictranscripts of kidney the CHRD disease gene and (PKD)_channel one of them domain. The transcriptomematching the of CA.‐terminal lucayanum part ofcontains the protein full-length (JT869941.1) transcripts supports the of conclusion the CHRD that genethe protein (GETC01124733.1). does

The transcriptome of B. lanceolatum had several transcripts of the CHRD gene and one of them matching the C-terminal part of the protein (JT869941.1) supports the conclusion that the protein does not have a PKD_channel domain; (B) Re-annotation of the B. belcheri CHRD gene with the MisPred/FixPred protocol has shown that these deviations are due to errors in gene prediction, permitting the correction of this sequence (CHRD_BRABE). The figure illustrates the Pfam domain architectures of the proteins and position of protein fragments of chordins. Color code for Pfam-A domains: von Willebrand factor type C domain (VWC)—green; chordin (CHRD)—red; PKD_channel—blue. Genes 2018, 9, 353 10 of 13

3.3. Lancelet Proteins with Domain Architectures Apparently Unique to Cephalochordates Twenty-four percent of the randomly selected proteins of B. belcheri orthologs had domain architectures with no clear equivalents in the human section of the UniProtKB/Swiss-Prot database (Supplementary Table S3). These proteins belong to two categories: they are variants of vertebrate-specific proteins (e.g., the plasminogen-related protein 040970_PFF0 and its orthologs) or they are epaktologs, proteins only related through shared domains [38] rather than orthologs of human proteins. There are several proteins in Supplementary Table S3 that appear to be correctly predicted as suggested by the observation that they pass the quality control of MisPred and that the orthologs from three lancelet species have identical domain architectures. It must be emphasized, however, that the identity of domain architectures of lancelet orthologs, per se, does not guarantee that they are valid lancelet-specific innovations. Because of the similarity of lancelet genomes and the similarity of gene-prediction protocols, genome annotation may yield similarly erroneous predictions. This danger has been illustrated with the case of protein 018300_PFF0 of B. belcheri and its B. floridae ortholog, XP_002588697.1 that contains a Lectin_C domain and five tandem fibronectin type II (fn2) domains [21].

4. Discussion The ENCODE Genome Annotation Assessment Project [39] has shown that—although none of the gene prediction strategies produce perfect predictions—evidence-based prediction methods exploiting experimental data (protein, messenger RNA, ESTs, etc.) were the most accurate, suggesting that the reliability of gene predictions increases as a function of the information-content of the experimental datasets. The original gene models of B. floridae and B. belcheri analyzed in the present work have been predicted by integrating the results obtained by de novo, -based, and transcriptome-based gene prediction methods [16,18]. In the case of B. belcheri, prediction was aided by ~300 million ESTs [18], whereas in the case of B. floridae gene, models were based on only about 480,000 ESTs [16]. Oulion and coworkers [25] have also pointed out the relatively low coverage of lancelet genomes by the B. floridae transcriptome; whereas 83% of the predicted genes of the B. floridae complete genome sequence are represented in the B. lanceolatum transcriptome, only 41% of the genes were found to match the much smaller dataset from the B. floridae transcriptome [40]. Accordingly, it is likely that as the B. floridae gene models were based on a significantly smaller dataset of ESTs than gene models of B. belcheri, misprediction may be more severe in the case of B. floridae genes. In harmony with this expectation, we have found that a higher proportion of B. belcheri proteins have the same domain architecture as their human Swiss-Prot orthologs than in the case B. floridae proteins (see Section 3.2). The uncertainty in gene predictions is also reflected by the fact that, whereas 30,392 protein-coding genes were predicted for the Chinese lancelet genome [18], the Florida lancelet genome was predicted to contain only 21,900 protein-coding genes [16]. It must be emphasized that both gene number estimates were based on draft genomes, thus genes are likely to be fragmented onto several contigs, which is known to cause significant errors in gene prediction [41]. In a recent study, Francis and Wörheide have also pointed out major problems with the original genome assembly and genome annotation of the B. floridae genome [42]. These authors have shown that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals. Whereas correctly assembled, properly annotated genomes of model organisms have intron to intergenic sequence ratios close to 1:1, this does not hold for the genome of the lancelet B. floridae. The original gene models [16] had annotated almost 90% of the genome as genes, assigning the majority (85%) of the genome to introns. Through re-annotation of the B. floridae scaffolds using the reads from a paired-end RNA sequencing (RNAseq) library, these authors have shown that more of Genes 2018, 9, 353 11 of 13 the genome is intergenic than intronic, suggesting that the original genome annotation has erroneously fused or missed many genes. The transcriptome of A. lucayanum [17] provided so far the richest source of data for evidence-based estimation of the number of genes in lancelet genomes. Based on the transcriptome of A. lucayanum, Yue et al. have identified 23,245 coding sequences (CDSs) (adult library) and 22,941 CDSs (20-h larvae library) of A. lucayanum, respectively [17,24]. The authors have also re-annotated the genome assembly of B. floridae, leading them to conclude that this lancelet genome contains 28,586 genes [17], a number significantly higher than the number (~22,000) originally predicted [16]. The results of our analyses confirm that the domain architecture differences of orthologous proteins of various lancelet species reflect errors of gene prediction; the proportion of correctly predicted proteins increases in the order B. lanceolatum << B. floridae < B. belcheri < A. lucayanum, reflecting differences in the information-content of their transcriptomes.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/9/7/353/s1, Table S1: Comparison of the domain architectures of randomly selected lancelet proteins with those of their human Swiss-Prot orthologs, Table S2: Comparison of the domain architectures of key developmental proteins of lancelets with those of their human Swiss-Prot orthologs, Table S3: Comparison of the domain architectures of randomly selected lancelet proteins with novel domain architectures. Author Contributions: L.P. and L.B. conceived and designed the experiments; K.K., M.T., L.B., and L.P. performed the experiments; K.K., M.T., L.B., and L.P. analyzed the data; L.P. wrote the paper. Funding: This research and the APC were funded by Grant GINOP-2.3.2-15-2016-00001 of the National Research, Development and Innovation Office of Hungary. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

References

1. Blair, J.E.; Hedges, S.B. Molecular phylogeny and divergence times of animals. Mol. Biol. Evol. 2005, 22, 2275–2284. [CrossRef][PubMed] 2. Delsuc, F.; Brinkmann, H.; Chourrout, D.; Philippe, H. and not cephalochordates are the closest living relatives of vertebrates. Nature 2006, 439, 965–968. [CrossRef][PubMed] 3. Chen, J.-Y.; Dzik, J.; Edgecombe, G.D.; Ramskold, L.; Zhou, G.-Q. A possible Early Cambrian chordate. Nature 1995, 377, 720–722. [CrossRef] 4. Chen, J.-Y.; Huang, D.-Y.; Li, C.-W. An early Cambrian -like chordate. Nature 1999, 402, 518–522. [CrossRef] 5. Lacalli, T. The Middle Cambrian fossil and the evolution of chordate swimming. EvoDevo 2012, 3, 12. [CrossRef][PubMed] 6. Morris, S.C.; Whittington, H.B. The animals of the . Sci. Am. 1979, 241, 122–135. [CrossRef] 7. Igawa, T.; Nozawa, M.; Suzuki, D.G.; Reimer, J.D.; Morov, A.R.; Wang, Y.; Henmi, Y.; Yasui, K. Evolutionary history of the extant amphioxus lineage with shallow-branching diversification. Sci. Rep. 2017, 7, 1157. [CrossRef][PubMed] 8. Somorjai, I.; Bertrand, S.; Camasses, A.; Haguenauer, A.; Escriva, H. Evidence for stasis and not genetic piracy in developmental expression patterns of Branchiostoma lanceolatum and Branchiostoma floridae, two amphioxus species that have evolved independently over the course of 200 Myr. Dev. Genes Evol. 2008, 218, 703–713. [CrossRef][PubMed] 9. Holland, N.D.; Holland, L.Z.; Heimberg, A. Hybrids between the Florida amphioxus (Branchiostoma floridae) and the Bahamas lancelet (Asymmetron lucayanum): Developmental morphology and chromosome counts. Biol. Bull. 2015, 228, 13–24. [CrossRef][PubMed] 10. Wilson, A.C.; Maxson, L.R.; Sarich, V.M. Two types of molecular evolution. Evidence from studies of interspecific hybridization. Proc. Natl. Acad. Sci. USA 1974, 71, 2843–2847. [CrossRef][PubMed] 11. Prager, E.M.; Wilson, A.C. Slow evolutionary loss of the potential for interspecific hybridization in birds: A manifestation of slow regulatory evolution. Proc. Natl. Acad. Sci. USA 1975, 72, 200–204. [CrossRef] [PubMed] Genes 2018, 9, 353 12 of 13

12. Fitzpatrick, B.M. Rates of evolution of inviability in birds and mammals. Evolution 2004, 58, 1865–1870. [CrossRef][PubMed] 13. Estes, S.; Arnold, S.J. Resolving the paradox of stasis: Models with stabilizing selection explain evolutionary divergence on all timescales. Am. Nat. 2007, 169, 227–244. [CrossRef][PubMed] 14. Hendry, A. Evolutionary biology: The Elvis paradox. Nature 2007, 446, 147–149. [CrossRef][PubMed] 15. Haller, B.C.; Hendry, A.P. Solving the paradox of stasis: Squashed stabilizing selection and the limits of detection. Evolution 2014, 68, 483–500. [CrossRef][PubMed] 16. Putnam, N.H.; Butts, T.; Ferrier, D.E.; Furlong, R.F.; Hellsten, U.; Kawashima, T.; Robinson-Rechavi, M.; Shoguchi, E.; Terry, A.; Yu, J.K.; et al. The amphioxus genome and the evolution of the chordate karyotype. Nature 2008, 453, 1064–1071. [CrossRef][PubMed] 17. Yue, J.X.; Yu, J.K.; Putnam, N.H.; Holland, L.Z. The transcriptome of an amphioxus, Asymmetron lucayanum, from the Bahamas: A window into chordate evolution. Genome Biol. Evol. 2014, 6, 2681–2696. [CrossRef] [PubMed] 18. Huang, S.; Chen, Z.; Yan, X.; Yu, T.; Huang, G.; Yan, Q.; Pontarotti, P.A.; Zhao, H.; Li, J.; Yang, P.; et al. Decelerated genome evolution in modern vertebrates revealed by analysis of multiple lancelet genomes. Nat. Commun. 2014, 5, 5896. [CrossRef][PubMed] 19. Patthy, L. Modular assembly of genes and the evolution of new functions. Genetica 2003, 118, 217–231. [CrossRef][PubMed] 20. Tordai, H.; Nagy, A.; Farkas, K.; Bányai, L.; Patthy, L. Modules, multidomain proteins and organismic complexity. FEBS J. 2005, 272, 5064–5078. [CrossRef][PubMed] 21. Bányai, L.; Patthy, L. Putative extremely high rate of proteome innovation in lancelets might be explained by high rate of gene prediction errors. Sci. Rep. 2016, 6, 30700. [CrossRef][PubMed] 22. Harrow, J.; Nagy, A.; Reymond, A.; Alioto, T.; Patthy, L.; Antonarakis, S.E.; Guigó, R. Identifying protein-coding genes in genomic sequences. Genome Biol. 2009, 10, 201. [CrossRef][PubMed] 23. Nagy, A.; Szláma, G.; Szarka, E.; Trexler, M.; Bányai, L.; Patthy, L. Reassessing domain architecture evolution of metazoan proteins: Major impact of gene prediction errors. Genes 2011, 2, 449–501. [CrossRef][PubMed] 24. Yue, J.X.; Kozmikova, I.; Ono, H.; Nossa, C.W.; Kozmik, Z.; Putnam, N.H.; Yu, J.K.; Holland, L.Z. Conserved noncoding elements in the most distant genera of Cephalochordates: The goldilocks principle. Genome Biol. Evol. 2016, 8, 2387–2405. [CrossRef][PubMed] 25. Oulion, S.; Bertrand, S.; Belgacem, M.R.; Le Petillon, Y.; Escriva, H. Sequencing and analysis of the Mediterranean amphioxus (Branchiostoma lanceolatum) transcriptome. PLoS ONE 2012, 7, e36554. [CrossRef] [PubMed] 26. The Lancelet (Branchiostoma belcheri) Genome Sequencing and Annotation Project Database. Available online: http://genome.bucm.edu.cn/lancelet/ (accessed on 1 March 2018). 27. Schilling, T.F.; Knight, R.D. Origins of anteroposterior patterning and regulation during chordate evolution. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2001, 356, 1599–1613. [CrossRef][PubMed] 28. Kozmikova, I.; Yu, J.K. Dorsal-ventral patterning in amphioxus: Current understanding, unresolved issues, and future directions. Int. J. Dev. Biol. 2017, 61, 601–610. [CrossRef][PubMed] 29. Soukup, V.; Kozmik, Z. The Bmp signaling pathway regulates development of left-right asymmetry in amphioxus. Dev. Biol. 2018, 434, 164–174. [CrossRef][PubMed] 30. Soukup, V.; Yong, L.W.; Lu, T.M.; Huang, S.W.; Kozmik, Z.; Yu, J.K. The controls left-right asymmetric development in amphioxus. EvoDevo 2015, 6, 5. [CrossRef][PubMed] 31. Finn, R.D.; Coggill, P.; Eberhardt, R.Y.; Eddy, S.R.; Mistry, J.; Mitchell, A.L.; Potter, S.C.; Punta, M.; Qureshi, M.; Sangrador-Vegas, A.; et al. The Pfam protein families database: Towards a more sustainable future. Nucleic Acids Res. 2016, 44, D279–D285. [CrossRef][PubMed] 32. Sievers, F.; Wilm, A.; Dineen, D.G.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Söding, J.; et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 2011, 7, 539. [CrossRef][PubMed] 33. Nagy, A.; Hegyi, H.; Farkas, K.; Tordai, H.; Kozma, E.; Bányai, L.; Patthy, L. Identification and correction of abnormal, incomplete and mispredicted proteins in public databases. BMC Bioinform. 2008, 9, 353. [CrossRef] [PubMed] 34. Nagy, A.; Patthy, L. MisPred: A resource for identification of erroneous protein sequences in public databases. Database (Oxf.) 2013, 2013, bat053. [CrossRef][PubMed] Genes 2018, 9, 353 13 of 13

35. Nagy, A.; Patthy, L. FixPred: A resource for correction of erroneous protein sequences. Database 2014, 2014, bau032. [CrossRef][PubMed] 36. Patthy, L. The WIF module. Trends Biochem. Sci. 2000, 25, 12–13. [CrossRef] 37. Putnam, N.H.; Srivastava, M.; Hellsten, U.; Dirks, B.; Chapman, J.; Salamov, A.; Terry, A.; Shapiro, H.; Lindquist, E.; Kapitonov, V.V.; et al. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 2007, 317, 86–94. [CrossRef][PubMed] 38. Nagy, A.; Bányai, L.; Patthy, L. Reassessing domain architecture evolution of metazoan proteins: Major impact of errors caused by confusing paralogs and epaktologs. Genes 2011, 2, 516–561. [CrossRef][PubMed] 39. Guigó, R.; Flicek, P.; Abril, J.F.; Reymond, A.; Lagarde, J.; Denoeud, F.; Antonarakis, S.; Ashburner, M.; Bajic, V.B.; Birney, E.; et al. EGASP: The human ENCODE Genome Annotation Assessment Project. Genome Biol. 2006, 7, S2. [CrossRef][PubMed] 40. Yu, J.K.; Wang, M.C.; Shin, T.; Kohara, Y.; Holland, L.Z.; Satoh, N.; Satou, Y. A cDNA resource for the cephalochordate amphioxus Branchiostoma floridae. Dev. Genes Evol. 2008, 218, 723–727. [CrossRef][PubMed] 41. Denton, J.F.; Lugo-Martinez, J.; Tucker, A.E.; Schrider, D.R.; Warren, W.C.; Hahn, M.W. Extensive error in the number of genes inferred from draft genome assemblies. PLoS Comput. Biol. 2014, 10, e1003998. [CrossRef] [PubMed] 42. Francis, W.R.; Wörheide, G. similar ratios of introns to intergenic sequence across genomes. Genome Biol. Evol. 2017, 9, 1582–1598. [CrossRef][PubMed]

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