Author Correction: The mid-developmental transition and the evolution of animal body plans.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The mid-developmental transition and the evolution of animal body plans.

Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development--known as the phylotypic period--is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

The round goby genome provides insights into mechanisms that may facilitate biological invasions.

BACKGROUND: The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success. RESULTS: We report a highly contiguous long-read-based genome and analyze gene families that we hypothesize to relate to the ability of these fish to deal with novel environments. The analyses provide novel insights from the large evolutionary scale to the small species-specific scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate immune system, an ancient duplication in red light vision accompanied by red skin fluorescence, evolutionary patterns of epigenetic regulators, and the presence of osmoregulatory genes that may have contributed to the round goby's capacity to invade cold and salty waters. A recurring theme across all analyzed gene families is gene expansions. CONCLUSIONS: The expanded innate immune system of round goby may potentially contribute to its ability to colonize novel areas. Since other gene families also feature copy number expansions in the round goby, and since other Gobiidae also feature fascinating environmental adaptations and are excellent colonizers, further long-read genome approaches across the goby family may reveal whether gene copy number expansions are more generally related to the ability to conquer new habitats in Gobiidae or in fish.

Multiple chromosomal rearrangements in a hybrid zone between Littorina saxatilis ecotypes.

Both classical and recent studies suggest that chromosomal inversion polymorphisms are important in adaptation and speciation. However, biases in discovery and reporting of inversions make it difficult to assess their prevalence and biological importance. Here, we use an approach based on linkage disequilibrium among markers genotyped for samples collected across a transect between contrasting habitats to detect chromosomal rearrangements de novo. We report 17 polymorphic rearrangements in a single locality for the coastal marine snail, Littorina saxatilis. Patterns of diversity in the field and of recombination in controlled crosses provide strong evidence that at least the majority of these rearrangements are inversions. Most show clinal changes in frequency between habitats, suggestive of divergent selection, but only one appears to be fixed for different arrangements in the two habitats. Consistent with widespread evidence for balancing selection on inversion polymorphisms, we argue that a combination of heterosis and divergent selection can explain the observed patterns and should be considered in other systems spanning environmental gradients.

Insights into bilaterian evolution from three spiralian genomes.

Current genomic perspectives on animal diversity neglect two prominent phyla, the molluscs and annelids, that together account for nearly one-third of known marine species and are important both ecologically and as experimental systems in classical embryology. Here we describe the draft genomes of the owl limpet (Lottia gigantea), a marine polychaete (Capitella teleta) and a freshwater leech (Helobdella robusta), and compare them with other animal genomes to investigate the origin and diversification of bilaterians from a genomic perspective. We find that the genome organization, gene structure and functional content of these species are more similar to those of some invertebrate deuterostome genomes (for example, amphioxus and sea urchin) than those of other protostomes that have been sequenced to date (flies, nematodes and flatworms). The conservation of these genomic features enables us to expand the inventory of genes present in the last common bilaterian ancestor, establish the tripartite diversification of bilaterians using multiple genomic characteristics and identify ancient conserved long- and short-range genetic linkages across metazoans. Superimposed on this broadly conserved pan-bilaterian background we find examples of lineage-specific genome evolution, including varying rates of rearrangement, intron gain and loss, expansions and contractions of gene families, and the evolution of clade-specific genes that produce the unique content of each genome.

Larval body patterning and apical organs are conserved in animal evolution.

BACKGROUND: Planktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data. RESULTS: To compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3ß antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the 'apical plate'. We also unraveled a molecular signature of the apical organ--devoid of six3 but expressing foxj, irx, nkx3 and hox--that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla. CONCLUSIONS: The similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ--comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells--was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae.

The brittle star genome illuminates the genetic basis of animal appendage regeneration.

Species within nearly all extant animal lineages are capable of regenerating body parts. However, it remains unclear whether the gene expression programme controlling regeneration is evolutionarily conserved. Brittle stars are a species-rich class of echinoderms with outstanding regenerative abilities, but investigations into the genetic bases of regeneration in this group have been hindered by the limited genomic resources. Here we report a chromosome-scale genome assembly for the brittle star Amphiura filiformis. We show that the brittle star genome is the most rearranged among echinoderms sequenced so far, featuring a reorganized Hox cluster reminiscent of the rearrangements observed in sea urchins. In addition, we performed an extensive profiling of gene expression during brittle star adult arm regeneration and identified sequential waves of gene expression governing wound healing, proliferation and differentiation. We conducted comparative transcriptomic analyses with other invertebrate and vertebrate models for appendage regeneration and uncovered hundreds of genes with conserved expression dynamics, particularly during the proliferative phase of regeneration. Our findings emphasize the crucial importance of echinoderms to detect long-range expression conservation between vertebrates and classical invertebrate regeneration model systems.

Development and Validation of an Automated Classifier to Diagnose Acute Otitis Media in Children.

Importance: Acute otitis media (AOM) is a frequently diagnosed illness in children, yet the accuracy of diagnosis has been consistently low. Multiple neural networks have been developed to recognize the presence of AOM with limited clinical application. Objective: To develop and internally validate an artificial intelligence decision-support tool to interpret videos of the tympanic membrane and enhance accuracy in the diagnosis of AOM. Design, Setting, and Participants: This diagnostic study analyzed otoscopic videos of the tympanic membrane captured using a smartphone during outpatient clinic visits at 2 sites in Pennsylvania between 2018 and 2023. Eligible participants included children who presented for sick visits or wellness visits. Exposure: Otoscopic examination. Main Outcomes and Measures: Using the otoscopic videos that were annotated by validated otoscopists, a deep residual-recurrent neural network was trained to predict both features of the tympanic membrane and the diagnosis of AOM vs no AOM. The accuracy of this network was compared with a second network trained using a decision tree approach. A noise quality filter was also trained to prompt users that the video segment acquired may not be adequate for diagnostic purposes. Results: Using 1151 videos from 635 children (majority younger than 3 years of age), the deep residual-recurrent neural network had almost identical diagnostic accuracy as the decision tree network. The finalized deep residual-recurrent neural network algorithm classified tympanic membrane videos into AOM vs no AOM categories with a sensitivity of 93.8% (95% CI, 92.6%-95.0%) and specificity of 93.5% (95% CI, 92.8%-94.3%) and the decision tree model had a sensitivity of 93.7% (95% CI, 92.4%-94.9%) and specificity of 93.3% (92.5%-94.1%). Of the tympanic membrane features outputted, bulging of the TM most closely aligned with the predicted diagnosis; bulging was present in 230 of 230 cases (100%) in which the diagnosis was predicted to be AOM in the test set. Conclusions and Relevance: These findings suggest that given its high accuracy, the algorithm and medical-grade application that facilitates image acquisition and quality filtering could reasonably be used in primary care or acute care settings to aid with automated diagnosis of AOM and decisions regarding treatment.

A genome resource for the marine annelid Platynereis dumerilii.

The marine annelid Platynereis dumerilii is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of P. dumerilii and of the closely related P. massiliensis and P. megalops, to facilitate comparative genomic approaches and help explore Platynereis biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of ~1.47 Gbp for P. dumerilii, of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 580 miRNA loci. We further explore the high genetic variation (~3% heterozygosity) within the Platynereis species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of Platynereis genomic resources.

Orthology prediction methods: a quality assessment using curated protein families.

The increasing number of sequenced genomes has prompted the development of several automated orthology prediction methods. Tests to evaluate the accuracy of predictions and to explore biases caused by biological and technical factors are therefore required. We used 70 manually curated families to analyze the performance of five public methods in Metazoa. We analyzed the strengths and weaknesses of the methods and quantified the impact of biological and technical challenges. From the latter part of the analysis, genome annotation emerged as the largest single influencer, affecting up to 30% of the performance. Generally, most methods did well in assigning orthologous group but they failed to assign the exact number of genes for half of the groups. The publicly available benchmark set (http://eggnog.embl.de/orthobench/) should facilitate the improvement of current orthology assignment protocols, which is of utmost importance for many fields of biology and should be tackled by a broad scientific community.

Evolution of vertebrate opioid receptors.

The opioid peptides and receptors have prominent roles in pain transmission and reward mechanisms in mammals. The evolution of the opioid receptors has so far been little studied, with only a few reports on species other than tetrapods. We have investigated species representing a broader range of vertebrates and found that the four opioid receptor types (delta, kappa, mu, and NOP) are present in most of the species. The gene relationships were deduced by using both phylogenetic analyses and chromosomal location relative to 20 neighboring gene families in databases of assembled genomes. The combined results show that the vertebrate opioid receptor gene family arose by quadruplication of a large chromosomal block containing at least 14 other gene families. The quadruplication seems to coincide with, and, therefore, probably resulted from, the two proposed genome duplications in early vertebrate evolution. We conclude that the quartet of opioid receptors was already present at the origin of jawed vertebrates approximately 450 million years ago. A few additional opioid receptor gene duplications have occurred in bony fishes. Interestingly, the ancestral receptor gene duplications coincide with the origin of the four opioid peptide precursor genes. Thus, the complete vertebrate opioid system was already established in the first jawed vertebrates.

Neuropeptide Y-family peptides and receptors in the elephant shark, Callorhinchus milii confirm gene duplications before the gnathostome radiation.

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 expression which may explain why 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.

Sensory receptor repertoire in cyprid antennules of the barnacle Balanus improvisus.

Barnacle settlement involves sensing of a variety of exogenous cues. A pair of antennules is the main sensory organ that the cyprid larva uses to explore the surface. Antennules are equipped with a number of setae that have both chemo- and mechanosensing function. The current study explores the repertoire of sensory receptors in Balanus improvisus cyprid antennules with the goal to better understand sensory systems involved in the settling behavior of this species. We carried out transcriptome sequencing of dissected B. improvisus cyprid antennules. The generated transcriptome assembly was used to search for sensory receptors using HMM models. Among potential chemosensory genes, we identified the ionotropic receptors IR25a, IR8a and IR93a, and several divergent IR candidates to be expressed in the cyprid antennules. We found one gustatory-like receptor but no odorant receptors, chemosensory or odorant-binding proteins. Apart from chemosensory receptors, we also identified 13 potential mechanosensory genes represented by several transient receptor potential channels (TRP) subfamilies. Furthermore, we analyzed changes in expression profiles of IRs and TRPs during the B. improvisus settling process. Several of the sensory genes were differentially expressed during the course of larval settlement. This study gives expanded knowledge about the sensory systems present in barnacles, a taxonomic group for which only limited information about receptors is currently available. It furthermore serves as a starting point for more in depth studies of how sensory signaling affects settling behavior in barnacles with implications for preventing biofouling.

Genomic architecture of parallel ecological divergence: Beyond a single environmental contrast.

The study of parallel ecological divergence provides important clues to the operation of natural selection. Parallel divergence often occurs in heterogeneous environments with different kinds of environmental gradients in different locations, but the genomic basis underlying this process is unknown. We investigated the genomics of rapid parallel adaptation in the marine snail Littorina saxatilis in response to two independent environmental axes (crab-predation versus wave-action and low-shore versus high-shore). Using pooled whole-genome resequencing, we show that sharing of genomic regions of high differentiation between environments is generally low but increases at smaller spatial scales. We identify different shared genomic regions of divergence for each environmental axis and show that most of these regions overlap with candidate chromosomal inversions. Several inversion regions are divergent and polymorphic across many localities. We argue that chromosomal inversions could store shared variation that fuels rapid parallel adaptation to heterogeneous environments, possibly as balanced polymorphism shared by adaptive gene flow.

Phylogenetic and chromosomal analyses of multiple gene families syntenic with vertebrate Hox clusters.

BACKGROUND: Ever since the theory about two rounds of genome duplication (2R) in the vertebrate lineage was proposed, the Hox gene clusters have served as the prime example of quadruplicate paralogy in mammalian genomes. In teleost fishes, the observation of additional Hox clusters absent in other vertebrate lineages suggested a third tetraploidization (3R). Because the Hox clusters occupy a quite limited part of each chromosome, and are special in having position-dependent regulation within the multi-gene cluster, studies of syntenic gene families are needed to determine the extent of the duplicated chromosome segments. We have analyzed in detail 14 gene families that are syntenic with the Hox clusters to see if their phylogenies are compatible with the Hox duplications and the 2R/3R scenario. Our starting point was the gene family for the NPY family of peptides located near the Hox clusters in the pufferfish Takifugu rubripes, the zebrafish Danio rerio, and human. RESULTS: Seven of the gene families have members on at least three of the human Hox chromosomes and two families are present on all four. Using both neighbor-joining and quartet-puzzling maximum likelihood methods we found that 13 families have a phylogeny that supports duplications coinciding with the Hox cluster duplications. One additional family also has a topology consistent with 2R but due to lack of urochordate or cephalochordate sequences the time window when these duplications could have occurred is wider. All but two gene families also show teleost-specific duplicates. CONCLUSION: Based on this analysis we conclude that the Hox cluster duplications involved a large number of adjacent gene families, supporting expansion of these families in the 2R, as well as in the teleost 3R tetraploidization. The gene duplicates presumably provided raw material in early vertebrate evolution for neofunctionalization and subfunctionalization.

Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions.

BACKGROUND: One of the many gene families that expanded in early vertebrate evolution is the neuropeptide (NPY) receptor family of G-protein coupled receptors. Earlier work by our lab suggested that several of the NPY receptor genes found in extant vertebrates resulted from two genome duplications before the origin of jawed vertebrates (gnathostomes) and one additional genome duplication in the actinopterygian lineage, based on their location on chromosomes sharing several gene families. In this study we have investigated, in five vertebrate genomes, 45 gene families with members close to the NPY receptor genes in the compact genomes of the teleost fishes Tetraodon nigroviridis and Takifugu rubripes. These correspond to Homo sapiens chromosomes 4, 5, 8 and 10. RESULTS: Chromosome regions with conserved synteny were identified and confirmed by phylogenetic analyses in H. sapiens, M. musculus, D. rerio, T. rubripes and T. nigroviridis. 26 gene families, including the NPY receptor genes, (plus 3 described recently by other labs) showed a tree topology consistent with duplications in early vertebrate evolution and in the actinopterygian lineage, thereby supporting expansion through block duplications. Eight gene families had complications that precluded analysis (such as short sequence length or variable number of repeated domains) and another eight families did not support block duplications (because the paralogs in these families seem to have originated in another time window than the proposed genome duplication events). RT-PCR carried out with several tissues in T. rubripes revealed that all five NPY receptors were expressed in the brain and subtypes Y2, Y4 and Y8 were also expressed in peripheral organs. CONCLUSION: We conclude that the phylogenetic analyses and chromosomal locations of these gene families support duplications of large blocks of genes or even entire chromosomes. Thus, these results are consistent with two early vertebrate tetraploidizations forming a paralogon comprising human chromosomes 4, 5, 8 and 10 and one teleost tetraploidization. The combination of positional and phylogenetic data further strengthens the identification of orthologs and paralogs in the NPY receptor family.

Clines on the seashore: The genomic architecture underlying rapid divergence in the face of gene flow.

Adaptive divergence and speciation may happen despite opposition by gene flow. Identifying the genomic basis underlying divergence with gene flow is a major task in evolutionary genomics. Most approaches (e.g., outlier scans) focus on genomic regions of high differentiation. However, not all genomic architectures potentially underlying divergence are expected to show extreme differentiation. Here, we develop an approach that combines hybrid zone analysis (i.e., focuses on spatial patterns of allele frequency change) with system-specific simulations to identify loci inconsistent with neutral evolution. We apply this to a genome-wide SNP set from an ideally suited study organism, the intertidal snail Littorina saxatilis, which shows primary divergence between ecotypes associated with different shore habitats. We detect many SNPs with clinal patterns, most of which are consistent with neutrality. Among non-neutral SNPs, most are located within three large putative inversions differentiating ecotypes. Many non-neutral SNPs show relatively low levels of differentiation. We discuss potential reasons for this pattern, including loose linkage to selected variants, polygenic adaptation and a component of balancing selection within populations (which may be expected for inversions). Our work is in line with theory predicting a role for inversions in divergence, and emphasizes that genomic regions contributing to divergence may not always be accessible with methods purely based on allele frequency differences. These conclusions call for approaches that take spatial patterns of allele frequency change into account in other systems.

Comparative mitogenomic analysis of three species of periwinkles: Littorina fabalis, L. obtusata and L. saxatilis.

The flat periwinkles, Littorina fabalis and L. obtusata, offer an interesting system for local adaptation and ecological speciation studies. In order to provide genomic resources for these species, we sequenced their mitogenomes together with that of the rough periwinkle L. saxatilis by means of next-generation sequencing technologies. The three mitogenomes present the typical repertoire of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a putative control region. Although the latter could not be fully recovered in flat periwinkles using short-reads due to a highly repetitive fragment, in L. saxatilis this problem was overcome with additional long-reads and we were able to assemble the complete mitogenome. Both gene order and nucleotide composition are similar between the three species as well as compared to other Littorinimorpha. A large variance in divergence was observed across mitochondrial regions, with six- to ten-fold difference between the highest and the lowest divergence rates. Based on nucleotide changes on the whole molecule and assuming a molecular clock, L. fabalis and L. obtusata started to diverge around 0.8 Mya (0.4-1.1 Mya). The evolution of the mitochondrial protein-coding genes in the three Littorina species appears mainly influenced by purifying selection as revealed by phylogenetic tests based on dN/dS ratios that did not detect any evidence for positive selection, although some caution is required given the limited power of the dataset and the implemented approaches.

Neuropeptide Y-family receptors Y6 and Y7 in chicken. Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region.

The peptides of the neuropeptide Y (NPY) family exert their functions, including regulation of appetite and circadian rhythm, by binding to G-protein coupled receptors. Mammals have five subtypes, named Y1, Y2, Y4, Y5 and Y6, and recently Y7 has been discovered in fish and amphibians. In chicken we have previously characterized the first four subtypes and here we describe Y6 and Y7. The genes for Y6 and Y7 are located 1 megabase apart on chromosome 13, which displays conserved synteny with human chromosome 5 that harbours the Y6 gene. The porcine PYY radioligand bound the chicken Y6 receptor with a K(d) of 0.80 +/- 0.36 nm. No functional coupling was demonstrated. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue. Porcine PYY bound chicken Y7 with a K(d) of 0.14 +/- 0.01 nm (mean +/- SEM), whereas chicken PYY surprisingly had a much lower affinity, with a Ki of 41 nm, perhaps as a result of its additional amino acid at the N terminus. Truncated peptide fragments had greatly reduced affinity for Y7, in agreement with its closest relative, Y2, in chicken and fish, but in contrast to Y2 in mammals. This suggests that in mammals Y2 has only recently acquired the ability to bind truncated PYY. Chicken Y7 has a much more restricted tissue distribution than other subtypes and was only detected in adrenal gland. Y7 seems to have been lost in mammals. The physiological roles of Y6 and Y7 remain to be identified, but our phylogenetic and chromosomal analyses support the ancient origin of these Y receptor genes by chromosome duplications in an early (pregnathostome) vertebrate ancestor.

Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss.

We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13-36 and NPY 18-36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.