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1 Genomics 93 (2009) Contents lists available at ScienceDirect Genomics journal homepage: Neuropeptide Y-family peptides and receptors in the elephant shark, Callorhinchus milii confirm gene duplications before the gnathostome radiation Tomas A. Larsson a, Boon-Hui Tay b, Görel Sundström a, Robert Fredriksson a, Sydney Brenner b, Dan Larhammar a,, Byrappa Venkatesh b a Department of Neuroscience Uppsala University Box Uppsala, Sweden b Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Biopolis, Singapore, article info abstract Article history: Received 27 May 2008 Accepted 9 October 2008 Available online 4 December 2008 Keywords: Evolution Genome duplication NPY Elephant shark We describe here the repertoire of neuropeptide Y (NPY) peptides and receptors in the elephant shark Callorhinchus milii, belonging to the chondrichthyans that diverged from the rest of the gnathostome (jawed vertebrate) lineage about 450 million years ago and the first chondrichthyan with a genome project. We have identified two peptide genes that are orthologous to NPY and PYY (peptide YY) in other vertebrates, and seven receptor genes orthologous to the Y1, Y2, Y4, Y5, Y6, Y7 and Y8 subtypes found in tetrapods and teleost fishes. The repertoire of peptides and receptors seems to reflect the ancestral configuration in the predecessor of all gnathostomes, whereas other lineages such as mammals and teleosts have lost one or more receptor genes or have acquired 1 2 additional peptide genes. Both the peptides and receptors showed broad and overlapping mrna expressionwhich may explainwhy some receptor gene losses could take place in some lineages, but leaves open the question why all the known ancestral receptors have been retained in the elephant shark Elsevier Inc. All rights reserved. Introduction The formation of additional genes by tetraploidization has been found to be common in many lineages. In vertebrates, large-scale duplication events have been proposed to have taken place twice before the origin of gnathostomes referred to as the one-to-four theory [1] or 2R theory (for two rounds of tetraploidization) [2] and again in the actinopterygian (ray-finned fish) lineage called 3R [3 12] and later independently in several other vertebrate lineages [13 17]. The recent accumulation of genome sequences from several vertebrate species has facilitated the identification of ancestrally duplicated genomic regions, although this is still a difficult task due to loss of genes after duplications and lineage specific duplication and translocation events. Early indications pointing towards tetraploidization as an important factor in the evolution of vertebrate gene families came from the study of rather limited chromosome regions where paralogous chromosome segments were evident [18]. The Hox gene Abbreviations: NPY, Neuropeptide Y; PYY, Peptide YY. Sequence data from this article have been deposited with the Genbank accession nos. EU637845, EU637846, EU637847, EU637848, EU637849, EU637850, EU637851, EU637852, EU and EU Corresponding author. addresses: Tomas.Larsson@neuro.uu.se (T.A. Larsson), mcblab46@imcb.a-star.edu.sg (B.-H. Tay), Gorel.Sundstrom@neuro.uu.se (G. Sundström), robert.fredriksson@neuro.uu.se (R. Fredriksson), sydney_brenner@a-star.edu.sg (S. Brenner), Dan.Larhammar@neuro.uu.se (D. Larhammar), mcbbv@imcb.a-star.edu.sg (B. Venkatesh). clusters comprise one such widely cited example, with four similar chromosomal regions in sarcopterygians (located on human chromosomes 2, 7, 12 and 17) as compared to one region in the cephalochordate amphioxus [19]. In teleost species, the extra tetraploidization has been responsible for generating even more paralogs with an ancestral repertoire of eight Hox clusters in this lineage. For a review of the extensive literature regarding the evolution of Hox clusters and its implications for the understanding of vertebrate evolution, see Hoegg and Meyer [20]. In order to fully understand the mechanism of expansion of many vertebrate gene families, the comparison of lineages with divergence times bracketing any particular duplication event is crucial. Until recently, no genome sequence was available for any cartilaginous fish species. This lack of sequence information has been remedied by the survey-sequencing (with 1.4 coverage) of the small (about 910 Mb) genome of the elephant shark Callorhinchus milii [21,22]. This chondrichthyan belongs to the chimaeras, a lineage that diverged from the elasmobranchs (sharks and rays) approximately 374 million years ago (Mya) [23] and was chosen for sequencing in order to better understand the early evolution of vertebrate genomes. The initial analysis of around 15,000 C. milii gene fragments suggested that it may contain four Hox clusters corresponding to the four that are present in sarcopterygians [22]. This supports the idea that the large-scale duplication events occurred before the split of chondrichthyes and osteichthyes (bony vertebrates) approximately 450 Mya. Furthermore, the C. milii genome was found to have a higher sequence similarity [24] and conservation of synteny with humans as compared to teleosts [22] /$ see front matter 2008 Elsevier Inc. All rights reserved. doi: /j.ygeno

2 T.A. Larsson et al. / Genomics 93 (2009) The increased evolutionary rate and disruption of conserved syntenic blocks in teleosts is most probably a result of the extra genome duplication in this lineage [4,8,10,11]. It has been shown that duplication events lead to increase in evolutionary rates of paralogs [11,25 29] and also to an increase in the frequency of rearrangements [30]. A recent study of the single protocadherin cluster in C. milii showed that this gene cluster was more stable compared to sarcopterygian protocadherin clusters and the duplicated clusters found in teleosts due to lower levels of gene conversion and rearrangements. The conservation of synteny of protocadherin genes was higher between C. milii and human than between C. milii and fugu [31]. The crucial position of C. milii in vertebrate phylogeny and the fact that the C. milii genome seems to be very stable strengthens the usefulness of this genome for comparison with other vertebrate genomes. The neuropeptide Y (NPY) system is an old neuroendocrine system of receptors and peptides with gene family members in vertebrates as well as in some invertebrates. The effects of NPY in mammals include stimulation of appetite, anxiolysis, vasoconstriction, and effects on circadian rhythms, pain signaling and pituitary hormone release [32]. Peptide YY (PYY) acts in mammals as a hypothalamic feedback hormone to reduce appetite [33,34] and also inhibits gut motility and secretion [35]. The function of the NPY system in chondrichthyans has not been studied in detail. A few reports have investigated the vascular responses of NPY peptides in elasmobranchs and found that NPY and PYY cause vasoconstriction and raise the blood pressure [36 38]. The evolutionary history and repertoire of both the peptide and the receptor gene families have been studied thoroughly in tetrapods and bony fishes but information about the repertoire in cartilaginous fishes has been incomplete so far. Earlier studies of the evolution of this system indicated that whole genome duplications have been involved in the expansion of both the peptide gene and receptor gene families. The NPY family of peptides is located close to the Hox clusters in both tetrapods and teleosts [39,40]. Several other gene families in these regions were found to have a similar evolutionary history, thereby strengthening this duplication scenario [40,41]. The presence of NPY and its duplicate PYY in both agnathans and tetrapods indicate that this duplication took place before the origin of gnathostomes [42]. In a similar way, the receptor genes are located on chromosomal segments proposed to have been duplicated by large-scale events as evident from the comparison between several vertebrate species [43 45]. The hypothetical ancestral gnathostome gene repertoire is thought to have consisted of seven receptor genes and two peptide genes inferred from the repertoire found in the genomes of several sequenced vertebrates [40,45]. So far, only two receptor genes have been found in Lampetra fluviatilis, consistent with the proposition that the two proposed large-scale duplication events occurred on each side of the divergence of cyclostomes. The NPY receptors are rhodopsin-like G protein-coupled receptors and can be divided into three subfamilies (Y1, Y2 and Y5) with only about 30% identity. Included in the Y1 subfamily are Y1, Y4, Y6 and Y8. These receptors are approximately 50% identical to each other. The Y2 and Y7 receptors are members of the Y2 subfamily and show approximately the same degree of identity as seen when comparing Y1 subfamily members. Y5 is the single member of the Y5 subfamily. No Y3 receptor has been found to be encoded by a separate gene. In mammals the Y1, Y2, Y4, Y5 and Y6 receptors are present whereas a rather different repertoire is present in teleosts. So far, Y1 has only been found in Danio rerio whereas Y2, Y4, Y7, Y8a and Y8b have been found in several teleosts [45,46]. Three full-length receptors belonging to the Y1 subfamily (Y1, Y4, Y6) have previously been cloned in the chondrichthyan spiny dogfish, Squalus acanthias, and a partial sequence of a fourth receptor (Y5) is also available [46,47]. Peptides belonging to the NPY family have been isolated or cloned from a few cartilaginous fishes; NPY has been cloned in the spotted torpedo ray, Torpedo marmorata [48] and both NPY and PYY have been isolated from the small-spotted catshark, Scyliorhinus canicula [49,50]. PYY has been isolated from both the spiny dogfish, S. acanthias [51] and the longnose skate, Raja rhina [52]. In this paper we report the repertoire of both NPY peptides and receptors as found in the genome of the elephant shark, C. milii as well as the expression patterns in thirteen tissues. Results Identification of peptide sequences The two C. milii peptide sequences were compared with sequences from other species. This revealed that the NPY prepropeptide (Genbank accession nr. EU637845) from elephant shark displays 85% identity to the prepropeptide cloned from the spotted torpedo. Mature NPY display 100% identity to both spotted torpedo and small-spotted catshark. There are only three amino acid differences between the mature NPY peptide in human and elephant shark (Fig. 1). PYY (Genbank accession nr. EU637846) is less conserved, the mature peptide is identical with the peptide from the spiny dogfish and the small-spotted catshark and has two amino acid differences compared to the peptide from longnose skate and nine amino acid differences compared to the mature human PYY peptide (Fig. 1). The introns in the two peptide genes are located at the same positions as seen in other vertebrate species. Identification and phylogenetic analysis of NPY receptor sequences The phylogenetic analyses of C. milii receptor sequences revealed that the NPY receptor repertoire consists of Y1, Y2, Y4, Y5, Y6, Y7 and Fig. 1. Alignment of the Callorhinchus milii NPY peptide sequence together with NPY sequences from other chondrichthyans and human (panel A) and Callorhinchus milii PYY peptide sequence together with PYY sequences from other chondrichthyans and human (panel B). The portion corresponding to the mature peptide is marked with a grey box in the alignment. Gaps are denoted with and identical positions are depicted with.. Species abbreviations are: Cmi Callorhinchus milii, Sca Scyliorhinus canicula, Tma Torpedo marmorata, Hsa Homo sapiens, Rrh Raja rhina and Sac Squalus acanthias.

3 256 T.A. Larsson et al. / Genomics 93 (2009) Y8 (Genbank accession nos. EU637847, EU637848, EU637849, EU637850, EU637851, EU and EU637853). All seven receptor sequences clustered in clades with high bootstrap or quartet-puzzling support values (i.e. in the Y1, Y2 or Y5 subfamily) (see Fig. 3 and Supplementary file 1). The two L. fluviatilis receptor sequences with similarity to the Y1 subfamily [53] and the Y2 subfamily (Genbank accession nos. AAL66410 and EU743622, respectively) cluster in the Y1 and Y2 clades as expected but with low bootstrap support and have therefore been referred to as Y1-like and Y2 Y7-like in Fig. 3 and Supplementary file 1. The C. milii Y5 sequence obtained with degenerate PCR and RACE (see Material and methods) cluster together with other Y5 sequences (Fig. 3). When analyzed together with the previously reported partial S. acanthias Y5 sequence [46] these two chondrichthyan Y5 sequences clustered together as expected (see panel B Supplementary file 1) despite the short alignment (Supplementary file 4) in this analysis (182 aa, see Material and methods section for details). The inclusion of the rapidly evolving zebrafish Y4 sequence with different amino acid composition compared to the other sequences (described in detail in [47]) did not influence the position of the C. milii Y4 sequence that clustered together with S. acanthias Y4 as expected. Expression of NPY peptide and receptor genes RT-PCR revealed that both peptides were expressed in the brain and eye. NPY was expressed in all tissues investigated except pancreas and spleen while PYY had a narrower expression pattern (Fig. 2A). RT-PCR for the receptor genes showed large overlaps in expression patterns and at least one receptor gene expressed in every tissue examined (albeit a very faint band for Y5 in pancreas) (Fig. 2B). Fig. 2. Agarose gel images showing the results of RT-PCR in thirteen Callorhinchus milii tissues using primers for the NPY and PYY genes (panel A) and primers for the seven NPY receptor genes (panel B). Actin was used as control to verify quality of samples. All receptor genes except Y4 were expressed in the brain and eye as well as the kidney. Discussion We describe here the repertoire of NPY peptides and receptors in the elephant shark C. milii. This cartilaginous fish species has two peptide genes coding for NPY and PYY, respectively, and seven receptor genes coding for receptor subtypes Y1, Y2, Y4, Y5, Y6, Y7 and Y8. Comparisons of the two peptide sequences with previously reported sequences [42, 48 52] made it possible to assign their identity as NPY and PYY, respectively, with great certainty despite the short sequence length (Fig. 1). The observed expression patterns in the thirteen tissues investigated indicate broader expression in C. milii than previously seen in other species [42,45] (Fig. 2A). The repertoire of peptides and receptors reported here is the same as that proposed to have been present in the ancestor of gnathostomes [40,45]. Earlier studies have shown differential loss of paralogs in vertebrate lineages, while the present study indicates that the chondrichthyan lineage has retained all gene family members present in the gnathostome ancestor. The expansion of many gene families, including the NPY peptide and receptor genes, has been attributed to large-scale duplication events in vertebrate evolution. The present study confirms our hypothesis [40,43] and firmly establishes that the origin of NPY and PYY as well as the seven receptor subtypes predates the origin of gnathostomes. A schematic picture showing the proposed evolutionary history for the receptor gene family in early vertebrates and the repertoire in four extant gnathostomes are shown in Fig. 4. As Y8 seems to be missing in both chicken and mammals it may have been lost in the amniote ancestor. The Y7 gene was recently reported to exist in the platypus (Ornithorhynchus anatinus) genome [54] while it seems to have been lost in other mammals. Furthermore, Y6 is a pseudogene in several mammalian lineages [55]. Teleost fishes seem to have lost Y5 and Y6. Also Y1 is missing in four sequenced fish genomes [45] but is present in zebrafish. To further clarify the order of the local receptor duplications that generated the clusters of the Y1, Y2 and Y5 subfamilies, thorough analysis of data from cyclostomes and early chordates is required. Also included in this analysis are two full length lamprey receptor sequences, the first of which have been reported previously [53] and the second showing highest identity to gnathostome Y2/Y7 and is in the process of being characterized functionally. The phylogenetic positions of the two L. fluviatilis sequences analyzed in this study are consistent with at least one duplication before the divergence of cyclostomes from the chordate tree (see Fig. 3). In the genome database of the sea lamprey (Petromyzon marinus) several genes with similarity to NPY receptor genes are present (data not shown). One of these sequences is the full-length ortholog of the L. fluviatilis Y1-like receptor gene [53], and two short non-overlapping fragments seem to be orthologous to the L. fluviatilis Y2/Y7 presented here. A few additional partial sequences were found that may correspond to another Y1 subfamily receptor and one Y5-like receptor (albeit with a frameshift) but further studies of sequences and chromosomal locations are required to confirm their identity. The low sequence identity among the members of the NPY receptor Y1 subfamily (approximately 50%) and the differential loss of paralogs in different vertebrate lineages have made the relationships of these receptors somewhat hard to resolve. As seen in the present study the resolution of the phylogenetic trees is not clear in all instances, namely the two river lamprey (L. fluviatilis) sequences and the positions of some Y4 and Y8 sequences (see Fig. 3). Previous studies in our lab have used both phylogenetic analyses and synteny information to deduce the relationships among these receptors [44,45]. Despite the lack of positional information for the C. milii genes it was possible to assign subfamily membership for all of its receptors. Furthermore, the C. milii sequences always clustered together with the corresponding S. acanthias sequence in all four

4 T.A. Larsson et al. / Genomics 93 (2009) bp) intron seen in mammalian Y1 sequences. An extended intron is present also in the Y1 sequences of S. acanthias [47], sturgeon and zebrafish [46]. With the exception of the Y1 receptor and the recently characterized teleost receptor Y8a, NPY receptors usually lack introns in the coding region. We speculate that the most parsimonious explanation is that the Y1 intron was inserted after the duplication events and that the Y8a intron is an even later intron insertion specific to some teleost species. All of the shark receptor sequences have the expected GPCR-features including consensus sequences for N-linked glycosylation in the aminoterminal domain and conserved cysteines assumed to serve as attachment sites for palmitate in the carboxyterminal cytoplasmic tail. All Y2 and Y7 sequences including the two shark sequences have an unusual histidine residue at position six after the well conserved motif after TM3 with the consensus sequence D/E-R-H/Y [56] suggesting that this substitution occurred soon after the local triplication. Likewise, all Y5 sequences share a rare valine at position one, thus deviating from the consensus sequence. This may imply differences in activation and/or inactivation for the three subfamilies of Y receptors. The RT-PCR of receptor genes in C. milii showed surprisingly broad and overlapping mrna expression patterns (Fig. 2B). Similar expression as seen for the three receptor genes in S. acanthias [47] could be detected for Y1, Y4 and Y6 in the kidney, Y4 in eye and muscle and Y6 in eye and intestine. However, there are also considerable differences in expression patterns between C. milii and S. acanthias [47], possibly reflecting the large evolutionary distance (about 374 My since the two lineages separated [22]) between these two species. Notable is the lack of expression of Y4 in C. milii brain given that Y4 was the only gene found to be expressed in the brain of S. acanthias among the three so far investigated in this species [47]. However, because RT-PCR is semiquantitative (at best), quantitative studies need to be performed before more definitive conclusions can be drawn regarding subfunctionalization. The relatively low coverage (1.4 ) of the current C. milii genome assembly leaves open the possibility that more NPY peptide and receptor genes are present in this species that remain to be found. It would be very interesting if some of the duplicates predicted to have existed in the Y2 and Y5 subfamilies as well as duplicates of NPY and PYY resulting from the ancestral tetraploidizations would be found to still exist in the elephant shark genome. With more information on chromosomal linkage of the shark genes it will be possible to deduce the ancestral genomic regions of the NPY peptide and receptor genes. Material and methods Database searches Fig. 3. Phylogenetic neighbor-joining tree constructed in MEGA 4.0 [57] depicting the relationships of NPY receptor genes. The genes described in this study are shown in grey. The Callorhinchus milii sequences are shaded in the figure. Brackets to the right in the figure indicate orthologous clusters. Species abbreviations are: Hsa Homo sapiens, Cfa Canis familiaris, Ssc Sus scrofa, Cpo Cavia porcellus, Rno Rattus norvegicus, Mmu Mus musculus, Gga Gallus gallus, Cmi Callorhinchus milii, Sac Squalus acanthias, Dre Danio rerio, Ocu Oryctolagus cuniculus, Lch Latimeria chalumnae, Tru Takifugu rubripes and Lfl Lampetra fluviatilis. The two Lampetra fluviatilis sequences are underlined in the figure. For a comparison of the topology of trees obtained with the neighbor-joining method and the quartet puzzling method [58] see Supplementary file 1. cases where sequences from this species were available (Fig. 3 and Supplementary file 1). The unique intron in Y1 sequences has earlier been used to distinguish this gene from other members of the NPY receptor family [47]. In the C. milii Y1 sequence the Y1 intron was found in the same position as seen in other species. Furthermore, the intron seems to be larger (N3000 bp) as compared to the short (around Recently a low-coverage ( 1.4 ) sequence of the elephant shark genome was generated [22]. This assembly is comprised of 608,273 sequences (scaffolds, contigs and singletons) with a combined length of 793 Mb. This is estimated to represent 75% of the genome. BLAST searches were performed against the C. milii genome sequences ( with human Y1, Y2, Y4 and Y5 (Genbank accession nos. AAA59920, AAA93170, AAH99637 and NP_006165), chicken Y6 and Y7 (Genbank accession nos. NP_ and NP_ ), Xenopus tropicalis Y8 (Genbank accession nr. NP_ ) and D. rerio Y1, Y2, Y4, Y7, Y8a and Y8b (Genbank accession nos. EU046342, XP_ , AF037400, AY585098, NM_ and AF030245) amino acid sequences as query sequences. The T. marmorata NPY and S. acanthias PYY (Genbank accession nos. P28674 and A respectively) were used as queries to search for peptide genes. Our search identified eight scaffolds of which two showed high similarity to neuropeptides and the rest to receptors. Since some of the gene sequences were incomplete, we completed their coding sequences by doing 5 RACE and/or 3 RACE using cdna from appropriate tissues (see below).

5 258 T.A. Larsson et al. / Genomics 93 (2009) Fig. 4. Proposed evolutionary history of the NPY receptor family by local and large-scale duplication events followed by loss of different paralogs in different lineages. The receptor genes found in the genomes of four sequenced vertebrates (including Callorhinchus milii) are shown in the lower part of the figure. Note that the linkage of genes in Callorhinchus milii is proposed based on the position of orthologous genes in other species. The for Homo sapiens Y6 indicates that this is a pseudogene. Degenerate PCR No sequence corresponding to the Y5 sequence was found in the database searches. To find the Y5 sequence, a degenerate PCR was carried out using the following primers and conditions: forward primer 5 -GAR GAR AAY GAR ATG ATH AAY YTI AC-3 and reverse primer 5 -AAI CCR TAN ARD ATI GGR TT-3. The PCR cycles comprised of a denaturation step at 95 C for 2 min, 35 cycles of 95 C for 30 s, 45 C for 2 min, 72 C for 1 min followed by a final elongation step at 72 C for 5 min. A 540 bp fragment representing the elephant shark Y5 sequence was obtained and thereafter extended using RACE (see below). RACE and RT-PCR Total RNA was isolated from the elephant shark tissues using TRIzol reagent (Invitrogen, USA) according to manufacturer's protocol. Total RNA was treated with RNase-free Cloned DNAse I (TaKaRa Bio Inc., Shiga, Japan) to eliminate any genomic DNA in the RNA preparation, and the absence of genomic DNA was confirmed by amplifying a fragment of an actin gene that encodes an intron. One microgram purified total RNA from each tissue was reverse transcribed and single-strand 5 RACE-ready and 3 RACE-ready cdna were prepared using SMART RACE cdna Amplification Kit (Clontech, USA). 5 RACE and 3 RACE were carried out according to manufacturer's protocol in a

6 T.A. Larsson et al. / Genomics 93 (2009) nested PCR using two gene-specific primers and Universal Primer and Nested Universal Primer supplied by the manufacturer. The genespecific primers used for RACE PCR are given in Table 1 of Appendix A. The following is a typical PCR condition used for amplifying RACE products: a denaturation step at 95 C for 2 min, 35 cycles of 95 C for 30 s, 60 C for 1 min, 72 C for 1 min followed by a final elongation step at 72 C for 5 min. Identity of PCR products was confirmed by sequencing using standard BigDye Terminator v3.1 chemistry on an ABI 3730xl DNA analyzer. The expression patterns of the genes were analyzed by RT-PCR using the 5 RACE-ready cdna as template. The primers used for the RT-PCR together with the primers for the actin (internal control for the quality of cdna) are listed in Table 2 of Appendix A. Representative RT- PCR products were sequenced to confirm their identity. Alignments and phylogenetic analyses The C. milii receptor sequences were aligned with other vertebrate NPY receptor sequences (see Supplementary file 2 for full list of accession numbers) and human somatostatin receptor 1 (SSTR1, to be used as outgroup in subsequent phylogenetic analyses) using MEGA 4.0 [57] with the following settings: pairwise alignment gap opening penalty 35, pairwise alignment gap extension penalty 0.75, multiple alignment gap opening penalty 15, multiple alignment gap extension penalty 0.3 using the Gonnet protein weight matrix. The alignment was cut to remove poorly aligned N-terminal and C-terminal parts so that the final alignment spanned the start of TM1 to the end of TM7 (including the large Y5-specific insertion, see Supplementary file 3). An additional alignment was made including the partial S. acanthias Y5 sequence described earlier (Genbank ID EU046362). This alignment was made using the same settings as the full alignment and was cut based on the ends of the S. acanthias Y5 sequence. The two alignments used in the analyses are available as Supplementary files 3 and 4. Phylogenetic analyses were carried out using the neighbor-joining method as implemented in MEGA 4.0 [57] and the Quartet-puzzling method in Treepuzzle 5.2 [58]. The neighbor-joining analyses were made with Poisson corrected distances, complete deletion of gaps and 1000 bootstrap replicates. The quartet-puzzling analyses were made with 25,000 puzzling steps using the JTT matrix, with eight gamma rates and the alpha parameter estimated from the dataset. Parameters were estimated using the exact and quartet sampling+nj tree options. Amino acid frequencies were estimated from the dataset. All trees were visualized with tree explorer in MEGA 4.0 and rooted with the human somatostatin receptor 1. Acknowledgments We thank Janine Danks, Justin Bell and Terry Walker for their help in collecting elephant shark tissues. The work in DL's lab was supported by grants from the Swedish Research Council and Carl Trygger's Foundation, Sweden and the work in BV's lab was supported by the Biomedical Research Council of the A STAR, Singapore. The authors would like to thank the anonymous reviewers for comments improving the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.ygeno References [1] S. Ohno, Gene duplication and the uniqueness of vertebrate genomes circa , Semin. Cell Dev. Biol. 10 (1999) [2] A.L. Hughes, Phylogenies of developmentally important proteins do not support the hypothesis of two rounds of genome duplication early in vertebrate history, J. Mol. Evol. 48 (1999) [3] A. Meyer, M. 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