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ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

Coelacanths are a critically valuable species to explore the gene changes that took place in the transition from aquatic to terrestrial life. One interesting and biologically relevant feature of the genus Latimeria is ureotelism. However not all urea is excreted from the body; in fact high concentrations are retained in plasma and seem to be involved in osmoregulation. The purine catabolic pathway, which leads to urea production in Latimeria, has progressively lost some steps, reflecting an enzyme loss during diversification of terrestrial species. We report the results of analyses of the liver and testis transcriptomes of the Indonesian coelacanth Latimeria menadoensis and of the genome of Latimeria chalumnae, which has recently been fully sequenced in the framework of the coelacanth genome project. We describe five genes, uricase, 5-hydroxyisourate hydrolase, parahox neighbor B, allantoinase, and allantoicase, each coding for one of the five enzymes involved in urate degradation to urea, and report the identification of a putative second form of 5-hydroxyisourate hydrolase that is characteristic of the genus Latimeria. The present data also highlight the activity of the complete purine pathway in the coelacanth liver and suggest its involvement in the maintenance of high plasma urea concentrations. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 334–341, 2014. © 2013 Wiley Periodicals, Inc.

Urea, one of the most common excreted products of vertebrates together with urate and ammonia, is produced by two pathways: the urea cycle and purine catabolism. Purine catabolism begins with adenine and guanine degradation to xanthine, which is converted to urate, the central metabolite in all vertebrates (Fig. 1). Urate is oxidized by urate oxidase (uricase, UOX) (E.C. 1.7.3.3) to 5-hydroxyisourate, which in turn is transformed by 5-hydroxyisourate hydrolase (HIUase) (E.C. 3.5.2.17) to 5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylate. This molecule is converted to allantoin through catalysis by parahox neighbor B (PRHOXNB, also known as 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, E.C.4.1.1.-). The latter two steps have recently been discovered through phylogenetic comparison of whole genomes (Ramazzina et al., 2006). Allantoin is degraded by allantoinase (ALN) (E.C. 3.5.2.5) to allantoate, which is hydrolyzed to urea and ureidoglycolate by allantoicase (ALC) (E.C. 3.5.3.4). Finally urea is converted by urease (E.C. 3.5.1.5) to ammonia and carbon dioxide (Fig. 1).

image

Figure 1. The purine catabolism pathway. The terminal purine catabolism step is not identical in all taxonomic groups: the end product of this pathway in reptiles, birds, hominoid primates, humans, and some platyrrhini is urate, allantoin in placental mammals (other than primates) and catarrhini, and allantoate in some teleosts. In contrast, amphioxus, elasmobranchs, other teleosts, dipnoans, amphibians, monotremes, marsupials, and coelacanths display a “complete” pathway that produces urea. Vertebrate taxonomic groups are reported on the left of their end product (Keilin, 1959; Urich, 1994; Hayashi et al., 2000; Keebaugh and Thomas, 2009). Enzymes are reported on the right between their substrate and product.

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Some of these enzymes have been lost during vertebrate evolution, with a truncation of the pathway in higher animals and a consequent change in the excreted end product (Fig. 1). Loss of the final steps of the pathway may be considered to be an adaptation to terrestrial life, since high plasma ammonia can lead to toxemia when water is scarce (Keilin, 1959; Shambaugh, 1977); in such conditions urate excretion is more convenient. The study of Latimeria, a lobe-finned fish and one of the nearest extant relatives to the tetrapod ancestor, could provide insights into the evolution of uricolysis. Furthermore, the high plasma urea levels described in Latimeria (Schmidt-Nielsen, 1964) seem to be involved in osmoregulation (Brown and Brown, 1967), thus providing an intriguing example of the evolutionary convergence with elasmobranchs (Wood et al., 1995). Investigation of the carbamoyl phosphate synthetase (CPS I)–arginase (ARG2) system, the other source of urea, in the African coelacanth Latimeria chalumnae has uncovered potentially adaptive novelties in the hepatic urea cycle that arose during the evolution of terrestrial vertebrates (Amemiya et al., 2013).

The Latimeria gene inventory of purine catabolism enzymes reported in this study documents a complete pathway including five enzymes. An additional transcript of HIUase in testis identified in this work suggests neofunctionalization. Phylogenetic analyses and microsynteny conservation of the five enzymes indicate a closer relationship of coelacanths to tetrapods than to teleosts. This suggested to us that the enzyme pattern found in Latimeria is probably an ancestral condition and that its characterization may be a fundamental starting point to understand the evolutionary changes involved in purine catabolism during the radiation of terrestrial vertebrates.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

Latimeria menadoensis sampling is described in Makapedua et al. (2011), liver and testis transcriptome sequencing and assembly are described in the work (Pallavicini et al., 2013). L. chalumnae sampling, genome sequencing, and assembly are described in Amemiya et al. (2013).

Nucleotide sequences of UOX, PRHOXNB, ALN, ALC, HIUase A, HIUase B, and transthyretin (TTR) (accession numbers HF678434–HF678440) were obtained from high-quality transcripts generated by RNA-sequencing analysis of L. menadoensis liver and testis (Canapa et al., 2012; Pallavicini et al., 2013). The same set of genes was investigated in the genome of L. chalumnae (Amemiya et al., 2013). Each retrieved sequence was validated using tBLASTn on the NCBI database, in order to allow annotation of the putative protein, and analyzed using ProtParam (Gasteiger et al., 2005, http://web.expasy.org/protparam/) for the biophysical parameters: molecular weight, isoelectric point, and estimated half-life (based on the “N-end rule”). The genetic distance between respective nucleotide sequences of the two Latimeria species was calculated as p-distance percentage with PAUP (Swofford, 2002). Latimeria coding sequences (CDS) were aligned with CDS from zebrafish, puffer fish, Xenopus, platypus, and mouse as in Keebaugh and Thomas (2009). The ω rate (non-synonymous/synonymous mutations) was obtained with KaKs_calculator (Zhang et al., 2006) according to Goldman and Yang (1994). The expression levels of purine catabolism pathway enzymes and of the housekeeping gene phosphoglycerate kinase (PGK) were computed in L. menadoensis liver as Fragments Per Kilobase of exons per Million sequenced fragments (FPKM) using CLC Genomics Workbench 4.5.1. Expression levels for testis were also calculated for comparison (data not shown). The sequences of other vertebrate species used in phylogenetic analyses and multiple alignments are listed in Table S1.

Vertebrate UOX, HIUase-TTR, PRHOXNB, ALN, and ALC amino acid alignments were performed with CLUSTALW2 using default parameters (Larkin et al., 2007). HIUase-TTR phylogenetic analysis was performed using Bayesian inference (BI) with MrBayes program (Huelsenbeck et al., 2001) by applying the WAG amino acid model (Whelan and Goldman, 2001). Analyses were run for 2,000,000 generations with sampling every 100 generations; the first 5,000 were discarded as burn-in, and obtainment of stationarity was considered when the average standard deviation of split frequencies reached a value < 0.007. A maximum parsimony (MP) analysis was performed with PAUP using tree bisection–reconnection (TBR) branch swapping and random stepwise additions with 100 replications. Only minimal trees were retained. Bootstrap values refer to 10,000 replications. Parsimony informative sites: 104.

UOX, PRHOXNB, ALN, and ALC phylogenetic trees were obtained with MrBayes (1,000,000 generations, sampling every 100 generations, burn-in 2,500 for each analysis). The amino acid models applied are annotated in each tree legend (S-Figs. 1–4).

Conservation of microsynteny was analyzed for UOX, HIUase A, HIUase B, PRHOXNB, ALN, and ALC by comparing Ensembl gene annotations (release 69—Oct. 2012) for L. chalumnae, Anolis carolinensis, Bos taurus, Callithrix jacchus, Danio rerio, Gallus gallus, Gasterosteus aculeatus, Homo sapiens, Macaca mulatta, Mus musculus, Oreochromis niloticus, Ornithorhynchus anatinus, Oryzias latipes, Petromyzon marinus, Tetraodon nigroviridis, and Xenopus tropicalis.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

Transcripts for the genes of the five enzymes involved in the degradation of urate to urea were identified from the analysis of the L. menadoensis liver transcriptome. The genetic distance between L. menadoensis and L. chalumnae nucleotide sequences ranges from 0.053% to 0.182% (Table 1) and is in line with data reported for other genes (Amemiya et al., 2013; Forconi et al., 2013; Pallavicini et al., 2013). Such low genetic distance allows combining the sequence information from the two coelacanths. Alignment of L. menadoensis transcripts to L. chalumnae genomic scaffolds permitted better definition of transcript features and genomic annotation.

Table 1. L. menadoensis sequence annotation
 Accession numberLengthp-DistanceaLengthpIbMWcHalf-lifed
  1. a

    Percentage p-distance between L. menadoensis and L. chalumnae nucleotide sequences.

  2. b

    PI: inferred protein isoelectric point.

  3. c

    MW: inferred protein molecular weight.

  4. d

    Half-life: estimated half-life calculated on the “N-ter rule” through ProtParam (Gasteiger et al., 2005) on the inferred proteins.

UOXHF6784344,180 bp0.141306 aa6.9035.3330 hr
HIUase AHF6784382,976 bp0.067141 aa9.4815.7530 hr
PRHOXNBHF6784352,858 bp0.182175 aa6.2219.9830 hr
ALNHF6784361,888 bp0.053464 aa6.9051.2030 hr
ALCHF6784371,410 bp0.146404 aa6.5845.3330 hr

A second putative HIUase transcript, that we designated HIUase B, was identified in addition to the sequences coding for the 5 enzymes making up the pathway. HIUase B appears to be composed by a partial cadherin-like sequence at the 5′ terminus (eight inferred exons, 1,215 nt) and three HIUase exons (493 nt). The alignment of the two HIUases shows that two exons, transcribing the 5′ UTR and the 5′ end of CDS in HIUase A, are missing in the B form. The 19% divergence calculated between the two HIUase forms in their comparable portion indicates the existence of two different genes. Moreover, analysis of microsynteny conservation in the flanking regions of the two forms suggests that the localization of the A form in the genome of L. chalumnae, where it is linked downstream to a region containing genes GAS8 and DNDBB1, is similar to the tetrapod pattern (Fig. 2). The synteny of genes downstream of HIUase A is not conserved in piscine species (data not shown). A sequence similar to cadherin 1 (CDH1-like) was identified about 30 kb upstream the coelacanth HIUase A gene. O. anatinus, X. tropicalis, some cetartiodactyla, and carnivora also have a similar CDH1-like region upstream of the HIUase gene (Fig. 2). The B form of the HIUase gene is linked, upstream of its CDS, to a region containing CDH1 and TMCO7, which are arranged in the opposite direction. CDH3, the gene commonly found upstream of CDH1 and TMCO7 in tetrapods, is not annotated in L. chalumnae, nor in G. gallus. However chicken CDH1 is found close to the single HIUase gene on chromosome 11, whereas in coelacanths it is more than 100 kb away from HIUase B. Moreover it was not possible to infer the relative proximity of the HIUase A and HIUase B loci as the genes were found on two separate scaffolds (Fig. 2). TTR sequences, that is, the other members of the TTR/HIUase family, were included in the phylogenetic analyses together with several representative vertebrate HIUases, to clarify the relationships between the HIUase forms of the two coelacanths. Phylogenetic trees show two main clades corresponding to HIUase and TTR proteins. In both MP and BI trees, which share a similar topology, the two putative HIUase forms of Latimeria are nested together in the same node in a sister clade to the tetrapod HIUases (Fig. 3). The expression pattern of the A form in the Indonesian coelacanth indicates higher transcription in liver (14.10 FPKM) than testis (2.66 FPKM), whereas the B form is more expressed in testis than liver (3.59 FPKM vs. 0.18 FPKM) (Fig. 4A).

image

Figure 2. HIUase microsyntenies in L. chalumnae and comparison with orthologous loci for other vertebrates. Coelacanth HIUase A and HIUase B loci are found on two separate scaffolds. CDH3, a gene commonly found in the region of CDH1 and TMCO7 genes in tetrapods, is not annotated in L. chalumnae.

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image

Figure 3. Phylogenetic trees of the TTR/HIUase family. Left: Bayesian inference (midpoint rooting, posterior probabilities >95); right: maximum parsimony (midpoint rooting, bootstrap majority consensus values >50; length tree = 772). The trees show two main clades corresponding to HIUase and TTR proteins, respectively. HIUase gene duplication in coelacanth appears to be lineage-specific.

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image

Figure 4. Expression levels of genes of purine catabolism enzymes. A: Expression levels of the HIUase A and HIUase B genes in L. menadoensis liver and testis. The different expression profiles of the two forms suggest a newly acquired function for HIUase B. B: Expression levels of purine catabolism enzyme transcripts in L. menadoensis liver. Values are expressed as FPKM. The expression of the housekeeping gene phosphoglycerate kinase (PGK) in liver is also reported for comparison.

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With regard to the genes of other enzymes, allantoinase was the most highly expressed (95.31 FPKM) while PRHOXNB showed the lowest expression (4.96 FPKM) (Fig. 4B). UOX, PRHOXNB, ALN, and ALC expression in testis was below the 1.00 FPKM threshold (data not shown).

Molecular phylogenetic analyses (Fig. 3, S-Figs. 1–4) reveal a number of different relationships among the coelacanth proteins: in the PRHOXNB, ALN, and ALC trees the Latimeria sequences form a sister clade to the tetrapod sequences, whereas in the UOX tree they cluster with teleost sequences.

Microsyntenies are reported in Supplementary Figures 5–8. The flanking gene arrangement suggests that coelacanth PRHOXNB and ALC adhere to a tetrapod pattern: the CDX2 gene, which in sarcopterygian genomes is found downstream PRHOXNB, is missing in teleosts; COLEC11 and RPS7 synteny upstream of ALC appears to be a feature of the tetrapod lineage.

On the other hand the microsynteny relationships of UOX and ALN appear to be conserved across all the vertebrates analyzed, even though the latter locus displays a gene loss in amniota with the exception of A. carolinensis. In this representative of reptiles the ALN sequence is retained in the genome under purifying selection (Keebaugh and Thomas, 2010) even though purine catabolism ends with production of urate in this taxon.

A putative peroxisomal translocation signal 1 (PTS1) characterized by the sequence SKL or by conservative variants (S/A/C)(K/R/H)(L/M), identified at the UOX C-terminus (Fig. 5), may indicate a subcellular localization of the enzyme (Rachubinski and Subramani, 1995); no translocation signals were identified in the two HIUases, PRHOXNB, ALN, or ALC.

image

Figure 5. Multiple alignment of UOX C-termini in representative vertebrate species. The conserved PTS1 sequences are in bold (S/A/C)(K/R/H)(L/M). Coelacanths display an intact PTS1 suggesting a peroxisomal location for the UOX enzyme.

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The ω rates (non-synonymous/synonymous substitutions) of L. menadoensis and other representative vertebrates (zebrafish, puffer fish, Xenopus, platypus, and mouse) are reported for each gene in Table 2. All enzymes appear to be under purifying selection (Ka/Ks < 1).

Table 2. ω Ratio between L. menadoensis and other vertebrate sequences
 UOXHIUasePRHOXNBALNALC
  1. a

    ALC in mouse is non-functional.

Zebrafish0.050.100.090.07
Puffer fish0.050.280.110.080.08
Xenopus0.070.130.080.180.06
Platypus0.070.300.100.310.07
Mouse0.060.110.100.08a

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

Among the vertebrate adaptations associated with the passage from aquatic to terrestrial life, the evolution of purine catabolism is particularly intriguing: comparison of data from the various phylogenetic lineages shows a complex picture due to several gene losses and pseudogene conversion events that led to shortening of the pathway in “higher vertebrates” (Remy et al., 1951; Keilin, 1959; Yeldandi et al., 1991; Wu et al., 1992; Urich, 1994; Oda et al., 2002). One such instance is the loss of UOX activity in hominoids, birds, and reptiles (Remy et al., 1951; Keilin, 1959; Urich, 1994). Over the years, several hypotheses have been advanced to explain the adaptive role of high serum uric acid resulting from UOX inactivity in hominoids (Haldane, 1955; Orowan, 1955; Ames et al., 1981; Watanabe et al., 2002).

In this scenario Latimeria, which has seemingly ancient morphological and physiological characteristics and represents one of the nearest relatives to the tetrapod ancestor, is a particularly interesting species for exploring some pre-adaptations that facilitated the transition to land. Urate oxidase catalyzes the first step in the uricolytic pathway and its presence in prokaryotes and eukaryotes suggests an ancient origin (Oda et al., 2002). Surprisingly, while in hominoids UOX has been classified as a pseudogene, in reptiles and birds a defective gene has been reported to evolve under purifying selection, suggesting that in these taxa the gene, no longer involved in purine catabolism, may have acquired new functions (Keebaugh and Thomas, 2010). The phylogenetic analysis indicates that the UOX sequences of the two coelacanths and of Protopterus annectens (lungfish) are more closely related to UOX sequences of the teleost clade than to those of other sarcopterygian clades (S-Fig. 1). The ω ratio values show strong purifying selection (Table 2).

The C-terminal sequence of the Latimeria UOX enzyme (Fig. 5) encodes a peroxisome translocation signal (PTS1, Gould et al., 1989; Miura et al., 1992) that suggests translocation to the organelle as shown for all homologous functional vertebrate enzymes (Hayashi et al., 2000). PTS1 is absent in the reptile A. carolinensis and in the chicken G. gallus, which encode a slightly larger defective protein (Wu et al., 1989; Yeldandi et al., 1990, 1991; Oda et al., 2002). Despite the purifying selection reported by Keebaugh and Thomas (2010) in such lineages, the long bird/reptile branches shown in Supplementary Figure 1 could be explained by the presence of small regions under positive selection hidden by larger regions under purifying selection, which thus go undetected (Keebaugh and Thomas, 2010).

The second step of the uric acid degradation pathway is catalyzed by HIUase. This step and PRHOXNB activity have been described recently (Ramazzina et al., 2006). The HIUase enzymes show high sequence similarity with TTR, which arose by gene duplication in vertebrates (both belong to the TTR-like family). TTRs are a group of plasma proteins that carry thyroid hormones to the brain; they are highly conserved in vertebrates, whereas HIUases are found in a wider range of organisms including bacteria, plants, fungi, and vertebrates (Eneqvist et al., 2003). In TTRs the conserved “TAVV” domain at the C-terminus is replaced by “YRGS” (Eneqvist et al., 2003; Lee et al., 2005).

HIUase-TTR phylogenetic analysis highlighted two genes closely related to HIUases in the two coelacanths. Their expression in liver and microsynteny conservation indicate that HIUase A may have maintained its original function. The assembled transcript of HIUase B is characterized by a portion encoding a cadherin-like sequence at the 5′ end and by three exons of HIUase in frame. A similar association with a cadherin-like sequence has been found in the single HIUase of Mustela putorius furo (AES10673). In addition the potential activity of HIUase B may be unaffected by the absence of the HIUase first coding exon; indeed Power et al. (2009) have described alternative splicing isoforms of HIUase devoid of the first coding exon in some fish species. However, despite the current lack of evidence of the translation of this second form, HIUase B expression in testis, its extremely low expression in liver, and the presence of a partial cadherin-like sequence capable of binding Ca2+ suggest a possible different function.

The presence of two HIUase forms also in teleosts seems to be due to the teleost-specific whole genome duplication (TGD, 3R hypothesis). Tree topology and the phylogenetic distance between the two forms suggest that the gene duplication in coelacanths was due to a lineage-specific event; the second form may have arisen due to recombination; indeed analysis of HIUase B microsynteny placed the gene in a region that in tetrapods contains CDH3 (not annotated in the contig of L. chalumnae), CDH1, and TMCO7 genes.

PRHOXNB, the third enzyme in the pathway, is found neither in reptiles nor in birds (S-Fig. 6) and appears to be under relaxed selection in humans and hominoids (Keebaugh and Thomas, 2009, 2010). The low transcript expression and half-life predictions inferred in silico (Table 1) may indicate a role for the protein as a rate-limiting step in the pathway. Alternatively the low amount of PRHOXNB transcripts could be offset by a faster translation rate or by higher catalytic activity.

For ALN and ALC, the lack of PTS1 at C-terminus in Latimeria might preclude a subcellular localization in peroxisomes; in contrast, ALN and ALC show a bona fide PTS1 sequence in several teleost species (Hayashi et al., 2000). The coelacanth proteins might be localized in the mitochondria as suggested for Xenopus (Usuda et al., 1994).

In conclusion, the significance of the present work lies in the identification of the components of the purine catabolism enzyme pathway in the basal-most living sarcopterygian and in the clarification of their evolutionary history and ancestral state in sarcopterygians.

Identification of the complete functional purine pathway in Latimeria indicates that the loss of function of some genes in the lineage that ultimately led to tetrapods occurred after the transition from aquatic to terrestrial life, presumably in relation to adaptation to new environments. The conservation of an ancestral form of the pathway in Latimeria may also be related to the need for high urea production, which would be required to maintain high plasma osmolarity.

In addition, microsynteny and phylogenetic analysis of the five purine catabolism genes also evinced a clear difference between teleost and tetrapod clades. However, their situation in Latimeria (i.e., possessing features very similar to those of tetrapods) suggests that the divergence is not the result of adaptation to terrestrial life, since such features are shared by the whole sarcopterygian lineage.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

The authors belonging to Dipartimento di Scienze della Vita e dell'Ambiente of Università Politecnica delle Marche are affiliated with Istituto Nazionale Biosistemi e Biostrutture (INBB). M.S. is supported by Deutsche Forschungsgemeinschaft. We are grateful to A.C. Keebaugh and J.W. Thomas for sharing the sequence alignment for Ka/Ks estimation, to anonymous reviewers and to C.T. Amemiya for their editorial suggestions.

LITERATURE CITED

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

Supporting Information

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

Additional supporting information may be found in the online version of this article at the publisher's web-site.

FilenameFormatSizeDescription
jezb22515-sm-0001-SupFigS1.tif25481K

Figure S1. Phylogenetic analysis of UOX. Bayesian inference of UOX amino acid sequences. Numbers on branches indicate posterior probability values (>95). Amino acid model: mixed (Jones posterior probability: 0.892; WAG posterior probability: 0.108). The Branchiostoma floridae sequence was the out-group.

jezb22515-sm-0002-SupFigS2.tif25481K

Figure S2. Phylogenetic analysis of PRHOXNB. Bayesian inference of PRHOXNB amino acid sequences. Midpoint rooting. Numbers on branches indicate posterior probability values (>95). Amino acid model: Jones.

jezb22515-sm-0003-SupFigS3.tif25481K

Figure S3. Phylogenetic analysis of ALN. Bayesian inference of ALN amino acid sequences. Numbers on branches indicate posterior probability values (>95). Amino acid model: WAG. The Strongylocentrotus purpuratus sequence was the out-group.

jezb22515-sm-0004-SupFigS4.tif25481K

Figure S4. Phylogenetic analysis of ALC. Bayesian inference of ALC amino acid sequences. Numbers on branches indicate posterior probability values (>95). Amino acid model: mixed (WAG posterior probability: 0.999; Jones posterior probability: 0.001). The Branchiostoma belcheri tsingtauense sequence was the out-group.

jezb22515-sm-0005-SupFigS5.tif25481K

Figure S5. Microsyntenies of UOX. Arrows indicate the transcriptional orientation, brackets indicate the arrangement of distant loci on the same chromosome.

jezb22515-sm-0006-SupFigS6.tif25481K

Figure S6. Microsyntenies of PRHOXNB. Arrows indicate the transcriptional orientation.

jezb22515-sm-0007-SupFigS7.tif25481K

Figure S7. Microsyntenies of ALN. Arrows indicate the transcriptional orientation, brackets indicate the arrangement of distant loci on the same chromosome. ALN of Anolis carolinensis (not annotated in Ensembl) was identified by homology and is delineated by the coordinates reported in the figure.

jezb22515-sm-0008-SupFigS8.tif25481K

Figure S8. Microsyntenies of ALC. Arrows indicate the gene orientation, brackets indicate the arrangement of distant loci on the same chromosome.

jezb22515-sm-0009-SupTabS1.docx22K

Table S1. Accession number list of the sequences used in the phylogenetic analyses and for UOX multiple sequence alignment.

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