Neuropeptide Y (NPY) and its related peptides named peptide YY (PYY) and pancreatic polypeptide (PP) comprise a system of neuronal and endocrine peptides that act on several G-protein-coupled receptors in vertebrates. They are involved in the regulation of a broad range of functions including appetite/satiety, gut motility, cardiovascular activity, pituitary release of hormones, circadian rhythm, and many more (see Pedrazzini et al., 2003; Brumovsky et al., 2007; Mercer et al., 2011; Zhang et al., 2011 for reviews). NPY and PYY arose by duplication of a common ancestral peptide gene before the vertebrate radiation (Larhammar, 1996). In mammals NPY is almost exclusively neuronal whereas PYY is primarily expressed in endocrine cells in the gastrointestinal tract. In ray finned fishes, PYY too is expressed in the nervous system (Cerdá-Reverter et al., 2000; Söderberg et al., 2000). PP is a local duplicate of PYY (Hort et al., 1995) previously found only in tetrapods (Larhammar, 1996; Cerdá-Reverter and Larhammar, 2000; Conlon, 2002; Sundstrom et al., 2008) and is expressed in pancreatic islets.

Mammals generally possess four receptors for the NPY-family peptides, namely subtypes Y1, Y2, Y4, and Y5 (Larhammar and Salaneck, 2004). Subtype Y3 was postulated from pharmacological experiments but does not exist as a separate gene (Herzog et al., 1993; Jazin et al., 1993). A fifth receptor gene, Y6, is expressed in a few mammals such as mouse and rabbit but is a pseudogene in many others including primates (Matsumoto et al., 1996), pig (Wraith et al., 2000), and guinea-pig (Starbäck et al., 2000). With its three peptides and four receptors, the NPY system in humans and most other mammals displays a degree of complexity resembling many other vertebrate peptide-receptor systems for neuronal and endocrine peptides, for instance the melanocortin system (Dores and Baron, 2011; Liang et al., 2013), the opioid system (Sundstrom et al., 2010), the oxytocin-vasopressin system (Ocampo Daza et al., 2011; Yamaguchi et al., 2012), and the somatostatin-cortistatin system (Tostivint et al., 2008; Ocampo Daza et al., 2012). However, previous evolutionary studies of the NPY receptor family have shown that a larger number of receptors existed in the early stages of vertebrate evolution before the emergence of jawed vertebrates, Gnathostomata: by sequence-based phylogenetic analyses and comparison of gene locations on chromosomes, we were able to deduce an ancestral vertebrate set of no less than seven NPY-family receptors (Larhammar and Salaneck, 2004; Larsson et al., 2008), more than for any other known peptide-receptor family. Subsequently, this repertoire was confirmed by our identification of all seven receptor subtypes in a cartilaginous fish (Chondrichthyes), the holocephalan elephant shark, or ghost shark, Callorhinchus milii (Larsson et al., 2009). Thus, the following evolutionary scenario was corroborated: an ancestral pre-vertebrate chromosome carried the genes for a Y1-like, a Y2-like, and a Y5-like receptor subtype. The two basal vertebrate tetraploidizations (Nakatani et al., 2007; Putnam et al., 2008) quadrupled this chromosome, thereby generating four similar chromosomal regions that probably had as many as 12 (4 × 3) family members, unless some were lost already after the first tetraploidization. In extant vertebrate lineages a total of seven family members have been found to remain: four Y1-subfamily genes with one on each of the four chromosomes resulting from the tetraploidizations (Y1, Y4, Y6, and Y8), two Y2-like genes (Y2 linked to Y1, Y7 linked to Y6), and a single surviving Y5 gene (linked to Y1 and Y2) (Larsson et al., 2008, 2009).

Of the vertebrates investigated to date only the elephant shark has maintained all seven of these ancestral vertebrate receptor genes (Larsson et al., 2009). All of the other gnathostome lineages seem to have suffered losses, although some of the genome databases may be incomplete. Among amphibians, the western clawed frog Silurana (Xenopus) tropicalis seems to have lost Y6 which appears to be a pseudogene in the frog Pelophylax esculentus (previously called Rana esculenta; Sundstrom et al., 2012). Amniotes have lost Y8, or possibly this gene was lost independently in birds and mammals (Larsson et al., 2009). The mammalian lineage subsequently lost also Y7. The Y6 gene is a pseudogene in several mammals as mentioned above. In the large and heterogeneous group of rayfinned fishes (Actinopterygii), the teleosts are the most carefully studied. In the true teleost species (Euteleostei) with sequenced genomes, the receptors Y1, Y2, Y4, Y7, and Y8, as well as a duplicate of Y8 called Y8b, have been identified, albeit only the zebrafish genome contains all of these (Larsson et al., 2008; Salaneck et al., 2008). Thus, the Y8 duplicate seems to be the only surviving copy resulting from the teleost-specific third tetraploidization, 3R (Jaillon et al., 2004). Also, this suggests that Y5 and Y6 are missing in Euteleostei. A local duplicate of Y2, named Y2-2, has been found in zebrafish and medaka (Fallmar et al., 2011). In addition, Y5 and Y6 have been identified in more basally diverging rayfinned fish species, namely a sturgeon and a bichir, as well as an early diverging teleost, the silver arowana, Osteoglossum bicirrhosum (Salaneck et al., 2008). Also, we have previously cloned the genes for Y5 and Y6 from the coelacanth Latimeria chalumnae (Lch; Larsson et al., 2007). The receptor gene duplication scenario is further supported by findings in lampreys of members of the Y1, Y2, and Y5 lineages (Salaneck et al., 2001; Larsson et al., 2009; Xu et al., 2012). In summary, of all gnathostome species that have been previously investigated only the elephant shark seems to have retained the complete ancestral repertoire of seven NPY receptor genes.

The West Indian Ocean coelacanth, Lch, is an important species for studies of vertebrate evolution as it is one of only two extant and closely related coelacanth species representing a very early Actinistia branch among the lobe finned fishes, Sarcopterygii. The relationship of coelacanths to lungfishes (Dipnoi) and tetrapods has been difficult to resolve. Recent data suggest that the coelacanths are slightly more closely related to the lungfishes than either of these groups is to tetrapods (Shan and Gras, 2011). The Actinopterygii-Sarcopterygii divergence took place approximately 424 million years ago (Mya) according to a recent estimate based on molecular data (Chen et al., 2012). The coelacanth and tetrapod lineages may have parted only a few tens of millions of years later, well before the split of the amphibian and amniote lineages approximately 350 Mya. Thus, it is of great interest to see which genetic and phenotypic Sarcopterygian characters had already arisen before the coelacanth-tetrapod divergence. In September 2011, a new genome assembly became available for the coelacanth. We present here a genomic analysis of the NPY system in Lch, identifying not only the receptor repertoire but also the genes for the three peptide ligands that bind to these receptors.

The PreEnsembl sequence database (ENSEMBL LatCha1 released September 2011, database version 69.1) for the West Indian Ocean coelacanth Lch was searched with Blast/Blat using as query sequences the human NPY-family receptors Y1, Y2, and Y4, and the zebrafish receptors Y7 and 8b. Five new receptors were identified, all but Y4 are annotated in Ensembl68. Amino acid sequences for NPY receptors from the following species were used for alignments: human (Homo sapiens), chicken (Gallus gallus), zebrafish (Danio rerio), elephant shark (C. milii), mouse (Mus musculus), and coelacanth (L. chalumnae). A list of genes with corresponding accession numbers is provided in Table 1 with the coelacanth NPY receptor sequences in bold.

Amino acid alignments were made in Jalview 2.8 version 14.0 (Waterhouse et al., 2009) using the MUSCLE and Clustal W web tool with standard settings. Phylogenetic Neighbor-Joining (NJ; Saitou and Nei, 1987) trees were made by using Clustal X version 2.0.12 (Larkin et al., 2007), standard settings and 1000 bootstrap replicates were applied. The tree shown in Figure 2 was rooted with the human somatostatin receptor sequence SSTR1. Phylogenetic trees were also made with the Maximum Likelihood (ML) method (Guindon et al., 2010).

The seven NPY-family receptor genes identified in the coelacanth genome database display orthology to the subtypes previously deduced to have arisen in the ancestor of the jawed vertebrates, namely subtypes Y1, Y2, Y4, Y5, Y6, Y7, and Y8 (Figure 3). Thus, the coelacanth has retained the complete ancestral repertoire like the elephant shark, but in contrast to mammals, chicken, and teleost fishes, all of which has lost one or more of the ancestral receptors. Our findings in the coelacanth corroborate our previous gene duplication scenario for the family of NPY receptors (Figure 6), with an ancestral local gene triplication, defining the three subfamilies of Y1, Y2, and Y5, followed by the chromosome quadruplication resulting from the two basal vertebrate tetraploidizations (Larhammar and Salaneck, 2004). Recent studies of other receptor families for neuroendocrine peptide-receptors have also revealed surprising ancestral vertebrate complexity, with six ancient somatostatin receptors (Ocampo Daza et al., 2012), six ancestral oxytocin-vasopressin receptors (Ocampo Daza et al., 2011; Yamaguchi et al., 2012; Ocampo Daza, 2013), and four ancestral GnRH (gonadotropin-releasing hormone) receptors (Kim et al., 2011), followed by losses of genes in mammals as well as other vertebrate lineages.

The clades in the NJ tree shown in Figure 2 corresponding to the seven receptor subtypes have high bootstrap support, except for the Y4–Y8 clade which often has been found to display poor resolution (Larsson et al., 2008, 2009) due to faster evolution of the Y4 sequences (Lundell et al., 2002) and the deviating amino acid composition of the zebrafish Y4 receptor, initially named Ya (Starbäck et al., 1999; see Salaneck et al., 2003). Analyses with the ML method give similar topology with this repertoire of species (not shown). Also when additional species are included for NJ and ML, these receptor subtypes fail to form completely separate clades with high bootstrap values, but the orthology relationships between tetrapods and teleosts have been confirmed by conserved synteny for Y4 and Y8 (Larsson et al., 2008). The seven coelacanth receptors can clearly be identified as orthologs of the previously identified seven ancestral vertebrate receptors, as shown by the tree in Figure 2.

Within each clade in Figure 2, some deviations from the established evolutionary relationships between species can be seen except Y7 which does indeed conform to the conventional species topology. As has been observed for several other gene or protein families, the zebrafish branch is more basal than the elephant shark, namely for Y1 and Y2. This is probably due to a higher evolutionary rate in the teleost fish lineage as compared to cartilaginous fishes and sarcopterygians, as has been observed for conserved non-coding elements (Lee et al., 2011). Also the mouse Y6 branching can probably be accounted for by its higher evolutionary rate. Analyses using the ML method displays similar minor deviations from the species relationships (not shown).

The coelacanth genome project has insufficient coverage for assembly into large scaffolds and therefore does not allow analyses of conserved synteny. Nevertheless, the Y1 and Y5 genes were found to be located close together in a head-to-head orientation like in mammals and chicken, with a similar distance as in human, supporting coregulation of the genes. Y1 and Y5 are known to be coexpressed in brain regions of rat (Parker and Herzog, 1998, 1999; Wolak et al., 2003) and mouse (Naveilhan et al., 1998; Oberto et al., 2007). In the paraventricular region of the hypothalamus, both Y1 and Y5 are known to contribute to the appetite-stimulating effect of NPY as reviewed in Mercer et al. (2011), probably in slightly different ways (Lecklin et al., 2002, 2003). However, the most important promoters and regulatory elements remain to be functionally identified. Indeed, comparison of the pig promoter regions (Wraith, 1999) with those in human (Herzog et al., 1997) suggested that multiple alternative start sites and/or 5′UTR exons exist that may differ even between these mammals, indicating complicated regulation of the two genes.

Based upon previous studies of the highly conserved peptides NPY and PYY, the identification of these in the coelacanth was expected and both of the mature peptides were found to be highly conserved. Indeed, the coelacanth PYY sequence appears to be identical to that of the deduced vertebrate ancestral PYY sequence (Larhammar, 1996), whereas mammalian PYY has undergone a few amino acid replacements. Possibly, the strong conservation of PYY in the coelacanth indicates a broader expression, like in teleost fishes and lamprey where PYY is expressed in both endocrine cells and neurons (Söderberg et al., 1994, 2000; Cerdá-Reverter et al., 2000; Kurokawa and Suzuki, 2002; Montpetit et al., 2005; Murashita et al., 2006, 2009; Wall and Volkoff, 2013), whereas mammalian PYY is almost exclusively expressed in endocrine cells. Broad distribution and/or usage has been found to correlate with higher conservation (Jordan et al., 2005; Khaitovich et al., 2005).

Our finding of PP in the coelacanth genome was unexpected because this peptide has previously only been identified in tetrapods (Larhammar, 1996; Cerdá-Reverter and Larhammar, 2000; Conlon, 2002; Sundstrom et al., 2008). The discovery of PP in the coelacanth could push back its origin by approximately 50 Myr, from a minimum of 340 Myr to a minimum of 390 Myr (Blair and Hedges, 2005). This may even raise the possibility that PP arose before the divergence of lobefinned fishes and rayfinned fishes, and that the PP gene has been lost in the rayfinned fish lineage. The NPY-family peptides so far described in teleosts have been assigned as NPYa, NPYb, PYYa, and PYYb (previously called PY), with the a and b duplicates having resulted from the teleost tetraploidization 3R (Sundstrom et al., 2008). PP in mammals binds to Y4 with higher affinity than either NPY or PYY, showing that partner preference has evolved between PP and Y4 in this lineage (Lundell et al., 1995, 1996; Berglund et al., 2001). In chicken, in contrast, all three peptides bind to Y4 with similar affinities (Lundell et al., 2002). Interestingly, Lch PP does not have the change from the ancestral residue Gln-34 to Pro-34 that is found in all PP sequences, except a few bird sequences that have a histidine at this position and the caecilian Typhlonectes natans (an amphibian) which has a serine (Conlon et al., 1998; Conlon, 2002). The coelacanth has retained the ancestral Gln-34 found also in all NPY and PYY sequences. This may indicate that Lch PP has a binding profile that differs from PP in tetrapods with regard to receptor subtype preferences.

Detailed studies of peptide-receptor interactions will require cloning and functional expression of the coelacanth receptors and synthesis of the peptides, especially Lch PP. Hopefully future crystallization of NPY-family receptors will provide good templates for structural modeling of the coelacanth receptors and thereby help explain how this peptide-receptor system has evolved in the vertebrates. Unfortunately, studies of the tissue distribution of the mRNA for the NPY-family peptides and receptors cannot be easily performed with this endangered species.

The large repertoire of NPY-family receptors in the coelacanth may either mean that it has retained the ancestral functions for the seven vertebrate receptors or evolved novel functions for some of them. If ancestral functions are maintained, this might give clues to what functions may have disappeared in mammals with the loss of receptors Y7 and Y8, as well as Y6 in several mammalian lineages. Another possibility is that any such functions are partially or completely mediated by the four to five receptors that still exist in mammals. Information on such scenarios may be obtained from studies of species that represent additional basally diverging lineages, such as lungfishes (which unfortunately have very large genomes, thereby complicating bioinformatic analyses) and basal actinopterygians like the spotted gar whose genome was recently assembled (Amores et al., 2011). This species is especially interesting as it diverged before the third tetraploidization took place in the teleost lineage. Also basal lineages among the teleosts have recently added valuable information on gene family evolution, especially the recently assembled genome for the European eel (Henkel et al., 2012). Clearly considerable genetic complexity arose at an early stage in vertebrate evolution that can be deduced by comparing distantly related vertebrate lineages. An inevitable conclusion is that for several gene families, mammals must be considered to have degenerated by gene loss.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.