Comparative screening identifies lineage-associated divergence in DRD4 in cetaceans

Comparative screening identifies lineage-associated divergence in DRD4 in cetaceans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative screening identifies lineage-associated divergence in DRD4 in cetaceans Yumi Shimozato This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8992414/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neuromodulatory pathways, particularly dopaminergic signaling, are thought to play important roles in regulating motivation, learning, and exploratory behavior across vertebrates. Major ecological transitions, such as the shift from terrestrial to fully aquatic environments in cetaceans, may be associated with lineage-specific molecular variation in these systems. However, identifying candidate loci involved in such divergence remains challenging due to the large number of genes contributing to neural function. Here, we conducted a comparative screening of eight genes associated with dopaminergic signaling across selected mammalian lineages, including cetaceans and a phylogenetically distinct terrestrial reference lineage. This exploratory analysis revealed that DRD4 uniquely exhibited patterns consistent with episodic lineage-associated divergence in cetaceans relative to other examined genes. Given the limited taxonomic sampling, these findings do not constitute evidence of adaptive evolution. Instead, they highlight DRD4 as a candidate locus for expanded phylogenetic and functional investigation. This hypothesis-generating study provides a foundation for future analyses incorporating broader taxonomic representation and complementary methodological approaches to evaluate the evolutionary significance of DRD4 variation. Evolutionary Biology Marine and Freshwater Biology Keywords: DRD4 dopaminergic signaling cetaceans comparative genomics molecular evolution lineage-associated divergence Figures Figure 1 Figure 2 Introduction Understanding how conserved neuromodulatory systems evolve across divergent mammalian lineages remains a central question in evolutionary neurobiology. Dopaminergic signaling plays a broadly conserved role in motivation, reward processing, and behavioral regulation across vertebrates (Savitz et al., 2006 ). At the molecular level, dopamine receptors belong to the G-protein-coupled receptor (GPCR) superfamily, one of the largest and evolutionarily dynamic gene families in vertebrates (Fredriksson et al., 2003 ). Variation within these systems has been associated with functional diversity across taxa while maintaining conserved structural frameworks. Comparative studies suggest that lineage-specific ecological pressures can shape components of conserved neural systems (Donaldson & Young, 2008 ). In particular, transitions to novel environments may be accompanied by modifications in neuromodulatory pathways rather than wholesale restructuring of neural architectures (O’Connell & Hofmann, 2011 ). Cetaceans represent one such lineage that underwent a profound ecological transition from terrestrial to fully aquatic environments, accompanied by extensive sensory and behavioral adaptations (Hof et al., 2005 ). At the same time, primates provide a relevant comparative context due to well-documented variation in dopaminergic genes across lineages (Shimada et al., 2004 ). One approach to addressing how conserved signaling systems evolve is to perform hypothesis-driven comparative screening of candidate genes within relevant pathways. Rather than attempting genome-wide inference, such screening may identify loci exhibiting lineage-associated divergence patterns that warrant further investigation. In this study, we conducted a comparative analysis of eight dopamine signaling-related genes across representative mammalian lineages, including cetaceans and primates. The inclusion of primates was not intended to represent a unique behavioral contrast, but rather to provide a phylogenetically informative terrestrial comparison for evaluating lineage-associated patterns of molecular variation. Among the genes examined, DRD4 emerged as the only locus showing patterns consistent with episodic lineage-associated divergence in cetaceans. These findings do not demonstrate adaptive evolution but instead highlight DRD4 as a candidate gene for further comparative investigation into potential lineage-associated molecular divergence within conserved neuromodulatory systems. Materials and Methods Sequence data and orthology assignment Coding sequences for eight candidate genes involved in dopaminergic signaling, plasticity, and social behavior (DRD1, DRD2, DRD4, COMT, SLC6A4, OXTR, BDNF, and GRIN2B) were retrieved for Tursiops truncatus , Delphinapterus leucas , Pan troglodytes , and Homo sapiens . Orthologous protein-coding sequences were obtained from Ensembl (release 110) and NCBI RefSeq using gene annotations verified by reciprocal BLAST searches. Sequence alignment Protein sequences were aligned using MAFFT v7.525 with the automatic strategy selection option (--auto). Alignment quality was inspected and regions of low confidence were excluded prior to downstream analyses. Codon-based nucleotide alignments were subsequently generated using PAL2NAL, preserving reading frames and removing gaps introduced by protein alignment. Phylogenetic framework and branch specification Species relationships were specified according to established mammalian phylogeny. Branches corresponding to Tursiops truncatus , Delphinapterus leucas , and Pan troglodytes were individually designated as foreground branches in branch-site analyses. All remaining branches were treated as background lineages. Tests for lineage-associated divergence Patterns consistent with episodic lineage-associated divergence were explored using the adaptive Branch-Site Random Effects Likelihood (aBSREL) model implemented in HyPhy v2.5. Branch-specific likelihood ratio tests were conducted for each candidate gene, testing foreground branches independently. Multiple testing correction across all tested branches was performed using the Benjamini–Hochberg false discovery rate (FDR), with significance assessed at q < 0.05. Given the limited taxonomic sampling, branch-site estimates were interpreted cautiously, as sparse phylogenetic representation may inflate ω estimates. Reproducibility All sequence alignments, phylogenetic trees, and HyPhy output files are provided as Supplementary Materials. Results Overview of branch-site analyses We examined eight candidate genes related to dopaminergic signaling, reward processing, synaptic plasticity, and social behavior (DRD1, DRD2, DRD4, COMT, SLC6A4, OXTR, BDNF, and GRIN2B) for patterns consistent with episodic lineage-associated divergence using branch-site models. Branches corresponding to Tursiops truncatus , Delphinapterus leucas , and Pan troglodytes were examined individually as foreground lineages. Across most genes and branches, likelihood ratio tests did not detect significant deviations from neutral or purifying selection after correction for multiple testing. Conservation across candidate genes Six of the eight candidate genes (DRD1, DRD2, SLC6A4, OXTR, BDNF, and GRIN2B) showed strong conservation across all examined lineages. Branch-site tests yielded non-significant results for all tested foreground branches (q = 1.0 in all cases), with estimated dN/dS ratios remaining well below 1. These results are consistent with pervasive purifying selection acting on the coding sequences of these genes across cetaceans and primates. Lineage-associated divergence patterns in DRD4 In contrast to the other candidate genes, DRD4 exhibited patterns consistent with episodic lineage-associated divergence in cetacean lineages. For the Tursiops truncatus branch, aBSREL detected a significant signal following multiple testing correction (q < 0.00001), with a maximum estimated ω value of approximately 701.9 and 28 codon sites contributing strong empirical support (EBF ≥ 100). Similarly, the Delphinapterus leucas branch showed a significant signal (q = 0.00378), with a maximum ω of approximately 989.1 and 14 supported codon sites (Table 1; Fig. 1 ). Given the limited taxonomic sampling, these estimates should be interpreted cautiously, as sparse phylogenetic representation can inflate ω values. No significant branch-level signals were detected for DRD4 in the Pan troglodytes or Homo sapiens lineages. Site-level analyses of DRD4 To further characterize the observed patterns in cetacean lineages, we conducted site-level analyses using MEME. Multiple codon sites showed strong statistical support for episodic divergence (EBF ≥ 100) in both dolphin and beluga branches. Mapping of these sites onto the predicted protein structure revealed that several supported codons were located within transmembrane domains of the DRD4 receptor (Fig. 2 ; Supplementary Table S1). Other candidate genes A nominal signal was detected for COMT on the Tursiops truncatus branch (p = 0.0428); however, this signal did not remain significant after false discovery rate correction (q = 0.128). No other candidate genes showed comparable lineage-associated divergence patterns on any tested branch. Discussion The present exploratory screening identified DRD4 as the only gene among the examined dopaminergic signaling-related loci showing patterns consistent with episodic lineage-associated divergence in cetaceans. While such patterns may reflect lineage-associated molecular variation, limited taxonomic sampling prevents robust inference of adaptive evolution. Sparse phylogenetic representation can inflate ω estimates in branch-site models, and therefore the observed signals should be interpreted cautiously (Ding et al., 2002 ). Importantly, the present analysis detects codon substitution patterns rather than functional changes. Consequently, any linkage between sequence divergence and behavioral or motivational traits remains speculative and requires further empirical validation. DRD4 is a well-characterized neuromodulatory receptor involved in dopaminergic signaling pathways (Ebstein et al., 1996 ; Savitz et al., 2006 ) and belongs to the G-protein-coupled receptor family, an evolutionarily diverse class of signaling molecules (Fredriksson et al., 2003 ). Molecular variation in this gene has been associated with neural functional diversity across taxa (Shimada et al., 2004 ). However, whether the divergence observed in cetacean lineages reflects adaptive modification or neutral evolutionary processes remains to be determined. Given the exploratory nature of this screening and the limited number of genes examined, the findings should not be interpreted as evidence for widespread modification of motivational pathways. Instead, they highlight DRD4 as a candidate locus for future comparative investigation. Neuromodulatory systems are known to undergo lineage-associated modifications in response to ecological transitions (Donaldson & Young, 2008 ), and molecular divergence within conserved neural components may represent one pathway through which evolutionary pressures shape lineage-specific trajectories. Future studies incorporating broader taxonomic sampling and complementary analytical approaches will be necessary to evaluate the evolutionary significance of DRD4 variation. Declarations Competing Interests The author declares that there are no financial or non-financial competing interests related to this work. Ethics Approval No ethical approval was required for this study because all data were obtained from publicly available genomic databases. Funding No funding was received for conducting this study. Author Contributions Conceptualization, data collection, analysis, and manuscript preparation were performed by the author. Data Availability All sequence data used in this study were obtained from publicly accessible databases (Ensembl and NCBI RefSeq). Alignment files and analysis outputs are provided as Supplementary Materials. References Ebstein RP, Novick O, Umansky R et al (1996) Dopamine D4 receptor (DRD4) exon III polymorphism associated with the human personality trait of novelty seeking. Nat Genet 12:78–80. https://doi.org/10.1038/ng0196-78 Ding YC, Chi HC, Grady DL et al (2002) Evidence of positive selection acting at the human dopamine receptor D4 gene locus. Proc Natl Acad Sci U S A 99:309–314. https://doi.org/10.1073/pnas.012464099 Donaldson ZR, Young LJ (2008) Oxytocin, vasopressin, and the neurogenetics of. sociality Sci 322:900–904. https://doi.org/10.1126/science.1158668 Fredriksson R, Lagerström MC, Lundin LG, Schiöth HB (2003) The G-protein-coupled receptors in the human genome form five main families. Mol Pharmacol 63:1256–1272. https://doi.org/10.1124/mol.63.6.1256 Hof PR, Chanis R, Marino L (2005) Cortical complexity in cetacean brains Anat Rec A Discov Mol. Cell Evol Biol 287:1142–1152. https://doi.org/10.1002/ar.a.20258 O’Connell LA, Hofmann HA (2011) The vertebrate mesolimbic reward system and social behavior network: a comparative synthesis. J Comp Neurol 519:3599–3639. https://doi.org/10.1002/cne.22735 Savitz J, Solms M, Ramesar R (2006) The molecular genetics of cognition: dopamine, COMT and BDNF. Genes Brain Behav 5:311–328. https://doi.org/10.1111/j.1601-183X.2005.00163.x Shimada MK, Inoue-Murayama M, Ueda Y et al (2004) Polymorphism in the second intron of dopamine receptor D4 gene in humans and apes Biochem. Biophys Res Commun 316:1186–1190. https://doi.org/10.1016/j.bbrc.2004.03.006 Tables Table 1. Summary of aBSREL tests for dolphin branches across candidate genes. Gene Branch p-value q-value Max ω Sites EBF≥100 DRD2 Tursiops truncatus 1 1 0.04 – DRD4 Tursiops truncatus 0 0 701.85 28 DRD4 Delphinapterus leucas 0.00084 0.0038 989.11 14 COMT Tursiops truncatus 0.0428 0.128 932.22 5 SLC6A4 Tursiops truncatus 1 1 0.59 – DRD1 Tursiops truncatus 1 1 0.06 – BDNF Tursiops truncatus 1 1 0.04 – GRIN2B Tursiops truncatus 1 1 0.04 – OXTR Tursiops truncatus 1 1 0.59 – Summary of branch-site analysis results for DRD4. Foreground branches tested using aBSREL are shown with corresponding likelihood ratio statistics, corrected q-values, and estimated ω values (dN/dS). Significant episodic divergence signals were detected only in cetacean branches after multiple testing correction. Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryTableS1DRD4MEMEsites.pdf Supplementary Table S1 Codon sites in DRD4 identified by MEME as showing statistical support for episodic divergence in cetacean lineages. Sites are listed with corresponding empirical Bayes factors (EBF) and inferred location within predicted receptor domains. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8992414","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598424518,"identity":"b196a959-36d6-432d-95ff-2f8c34064fe2","order_by":0,"name":"Yumi Shimozato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYFACxgYgkoByKoCYmbmBaC1A1hmQFkZCWiBqIRRjGxIXFzC4driB8ecOi2h+/sPPH3ycVxvN3w7U8qNiG24ttxMbmHnPSOTObDhm2Dhz2/HcGYeBtvWcuY1fC2ObRO6Ggw2GzbzbjuU2ALUARfBrYfwJ0nKY/WMz75xjufOJ0cLAC9JyjAdoS0MNUC8BLZJALYdBWmb28BTOnHHsQO5GoJaD+PzCdzv94cOfbXW5/fzHN3z4UFOXO+/84YMPflTg1gICB5DYhzFECII6UhSPglEwCkbBCAEADCRiOG+/lxwAAAAASUVORK5CYII=","orcid":"","institution":"Ritsumeikan University","correspondingAuthor":true,"prefix":"","firstName":"Yumi","middleName":"","lastName":"Shimozato","suffix":""}],"badges":[],"createdAt":"2026-02-28 06:20:10","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8992414/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8992414/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103810850,"identity":"b8907495-cb18-4f33-928d-8bd7d7eaf294","added_by":"auto","created_at":"2026-03-03 08:14:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116859,"visible":true,"origin":"","legend":"\u003cp\u003eBranch-site screening results for DRD4 across examined mammalian lineages.\u003cbr\u003e\nBar plots indicate likelihood ratio test support for episodic lineage-associated divergence in foreground branches tested using aBSREL. Significant signals were detected in the \u003cem\u003eTursiops truncatus\u003c/em\u003e and \u003cem\u003eDelphinapterus leucas\u003c/em\u003e branches after multiple testing correction, whereas other examined genes and lineages showed no significant deviations.\u003cbr\u003e\nThese results reflect codon substitution patterns and do not constitute evidence of adaptive evolution.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8992414/v1/d849a1407abdfd4c5ba93244.png"},{"id":103810851,"identity":"5001e7db-85b3-4d9c-a3b0-a8eaf0617bdd","added_by":"auto","created_at":"2026-03-03 08:14:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6873,"visible":true,"origin":"","legend":"\u003cp\u003eMapping of codon sites showing statistical support for episodic divergence in DRD4 identified by MEME.\u003cbr\u003e\nSupported sites (EBF ≥ 100) are shown in relation to predicted transmembrane domains of the receptor protein. Several supported codons were located within transmembrane regions in cetacean lineages.\u003cbr\u003e\nSite-level analyses indicate patterns consistent with lineage-associated divergence but do not imply functional modification.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8992414/v1/ff4709c775c08689be08445f.png"},{"id":104400322,"identity":"bafc8777-4ae7-4574-8d0e-ce6d278ebdeb","added_by":"auto","created_at":"2026-03-11 12:09:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":708265,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8992414/v1/4ed78742-3b22-4bba-9924-efd79ec9de06.pdf"},{"id":103810852,"identity":"bf9e7c2c-887a-4727-8e71-3d1a393996de","added_by":"auto","created_at":"2026-03-03 08:14:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table S1\u003c/strong\u003e\u003cbr\u003e\nCodon sites in DRD4 identified by MEME as showing statistical support for episodic divergence in cetacean lineages.\u003cbr\u003e\nSites are listed with corresponding empirical Bayes factors (EBF) and inferred location within predicted receptor domains.\u003c/p\u003e","description":"","filename":"SupplementaryTableS1DRD4MEMEsites.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8992414/v1/3bf57be57ec63c1e2f369a59.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eComparative screening identifies lineage-associated divergence in DRD4 in cetaceans\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUnderstanding how conserved neuromodulatory systems evolve across divergent mammalian lineages remains a central question in evolutionary neurobiology. Dopaminergic signaling plays a broadly conserved role in motivation, reward processing, and behavioral regulation across vertebrates (Savitz et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). At the molecular level, dopamine receptors belong to the G-protein-coupled receptor (GPCR) superfamily, one of the largest and evolutionarily dynamic gene families in vertebrates (Fredriksson et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Variation within these systems has been associated with functional diversity across taxa while maintaining conserved structural frameworks.\u003c/p\u003e \u003cp\u003eComparative studies suggest that lineage-specific ecological pressures can shape components of conserved neural systems (Donaldson \u0026amp; Young, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In particular, transitions to novel environments may be accompanied by modifications in neuromodulatory pathways rather than wholesale restructuring of neural architectures (O\u0026rsquo;Connell \u0026amp; Hofmann, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Cetaceans represent one such lineage that underwent a profound ecological transition from terrestrial to fully aquatic environments, accompanied by extensive sensory and behavioral adaptations (Hof et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). At the same time, primates provide a relevant comparative context due to well-documented variation in dopaminergic genes across lineages (Shimada et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne approach to addressing how conserved signaling systems evolve is to perform hypothesis-driven comparative screening of candidate genes within relevant pathways. Rather than attempting genome-wide inference, such screening may identify loci exhibiting lineage-associated divergence patterns that warrant further investigation.\u003c/p\u003e \u003cp\u003eIn this study, we conducted a comparative analysis of eight dopamine signaling-related genes across representative mammalian lineages, including cetaceans and primates. The inclusion of primates was not intended to represent a unique behavioral contrast, but rather to provide a phylogenetically informative terrestrial comparison for evaluating lineage-associated patterns of molecular variation. Among the genes examined, DRD4 emerged as the only locus showing patterns consistent with episodic lineage-associated divergence in cetaceans.\u003c/p\u003e \u003cp\u003eThese findings do not demonstrate adaptive evolution but instead highlight DRD4 as a candidate gene for further comparative investigation into potential lineage-associated molecular divergence within conserved neuromodulatory systems.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSequence data and orthology assignment\u003c/h2\u003e \u003cp\u003eCoding sequences for eight candidate genes involved in dopaminergic signaling, plasticity, and social behavior (DRD1, DRD2, DRD4, COMT, SLC6A4, OXTR, BDNF, and GRIN2B) were retrieved for \u003cem\u003eTursiops truncatus\u003c/em\u003e, \u003cem\u003eDelphinapterus leucas\u003c/em\u003e, \u003cem\u003ePan troglodytes\u003c/em\u003e, and \u003cem\u003eHomo sapiens\u003c/em\u003e. Orthologous protein-coding sequences were obtained from Ensembl (release 110) and NCBI RefSeq using gene annotations verified by reciprocal BLAST searches.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSequence alignment\u003c/h3\u003e\n\u003cp\u003eProtein sequences were aligned using MAFFT v7.525 with the automatic strategy selection option (--auto). Alignment quality was inspected and regions of low confidence were excluded prior to downstream analyses. Codon-based nucleotide alignments were subsequently generated using PAL2NAL, preserving reading frames and removing gaps introduced by protein alignment.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic framework and branch specification\u003c/h3\u003e\n\u003cp\u003eSpecies relationships were specified according to established mammalian phylogeny. Branches corresponding to \u003cem\u003eTursiops truncatus\u003c/em\u003e, \u003cem\u003eDelphinapterus leucas\u003c/em\u003e, and \u003cem\u003ePan troglodytes\u003c/em\u003e were individually designated as foreground branches in branch-site analyses. All remaining branches were treated as background lineages.\u003c/p\u003e\n\u003ch3\u003eTests for lineage-associated divergence\u003c/h3\u003e\n\u003cp\u003ePatterns consistent with episodic lineage-associated divergence were explored using the adaptive Branch-Site Random Effects Likelihood (aBSREL) model implemented in HyPhy v2.5. Branch-specific likelihood ratio tests were conducted for each candidate gene, testing foreground branches independently. Multiple testing correction across all tested branches was performed using the Benjamini\u0026ndash;Hochberg false discovery rate (FDR), with significance assessed at q\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eGiven the limited taxonomic sampling, branch-site estimates were interpreted cautiously, as sparse phylogenetic representation may inflate ω estimates.\u003c/p\u003e\n\u003ch3\u003eReproducibility\u003c/h3\u003e\n\u003cp\u003eAll sequence alignments, phylogenetic trees, and HyPhy output files are provided as Supplementary Materials.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eOverview of branch-site analyses\u003c/h2\u003e \u003cp\u003eWe examined eight candidate genes related to dopaminergic signaling, reward processing, synaptic plasticity, and social behavior (DRD1, DRD2, DRD4, COMT, SLC6A4, OXTR, BDNF, and GRIN2B) for patterns consistent with episodic lineage-associated divergence using branch-site models. Branches corresponding to \u003cem\u003eTursiops truncatus\u003c/em\u003e, \u003cem\u003eDelphinapterus leucas\u003c/em\u003e, and \u003cem\u003ePan troglodytes\u003c/em\u003e were examined individually as foreground lineages. Across most genes and branches, likelihood ratio tests did not detect significant deviations from neutral or purifying selection after correction for multiple testing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConservation across candidate genes\u003c/h3\u003e\n\u003cp\u003eSix of the eight candidate genes (DRD1, DRD2, SLC6A4, OXTR, BDNF, and GRIN2B) showed strong conservation across all examined lineages. Branch-site tests yielded non-significant results for all tested foreground branches (q\u0026thinsp;=\u0026thinsp;1.0 in all cases), with estimated dN/dS ratios remaining well below 1. These results are consistent with pervasive purifying selection acting on the coding sequences of these genes across cetaceans and primates.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLineage-associated divergence patterns in DRD4\u003c/h2\u003e \u003cp\u003eIn contrast to the other candidate genes, DRD4 exhibited patterns consistent with episodic lineage-associated divergence in cetacean lineages. For the \u003cem\u003eTursiops truncatus\u003c/em\u003e branch, aBSREL detected a significant signal following multiple testing correction (q\u0026thinsp;\u0026lt;\u0026thinsp;0.00001), with a maximum estimated ω value of approximately 701.9 and 28 codon sites contributing strong empirical support (EBF\u0026thinsp;\u0026ge;\u0026thinsp;100). Similarly, the \u003cem\u003eDelphinapterus leucas\u003c/em\u003e branch showed a significant signal (q\u0026thinsp;=\u0026thinsp;0.00378), with a maximum ω of approximately 989.1 and 14 supported codon sites (Table\u0026nbsp;1; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the limited taxonomic sampling, these estimates should be interpreted cautiously, as sparse phylogenetic representation can inflate ω values.\u003c/p\u003e \u003cp\u003eNo significant branch-level signals were detected for DRD4 in the \u003cem\u003ePan troglodytes\u003c/em\u003e or \u003cem\u003eHomo sapiens\u003c/em\u003e lineages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSite-level analyses of DRD4\u003c/h2\u003e \u003cp\u003eTo further characterize the observed patterns in cetacean lineages, we conducted site-level analyses using MEME. Multiple codon sites showed strong statistical support for episodic divergence (EBF\u0026thinsp;\u0026ge;\u0026thinsp;100) in both dolphin and beluga branches. Mapping of these sites onto the predicted protein structure revealed that several supported codons were located within transmembrane domains of the DRD4 receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Supplementary Table S1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOther candidate genes\u003c/h2\u003e \u003cp\u003eA nominal signal was detected for COMT on the \u003cem\u003eTursiops truncatus\u003c/em\u003e branch (p\u0026thinsp;=\u0026thinsp;0.0428); however, this signal did not remain significant after false discovery rate correction (q\u0026thinsp;=\u0026thinsp;0.128). No other candidate genes showed comparable lineage-associated divergence patterns on any tested branch.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present exploratory screening identified DRD4 as the only gene among the examined dopaminergic signaling-related loci showing patterns consistent with episodic lineage-associated divergence in cetaceans. While such patterns may reflect lineage-associated molecular variation, limited taxonomic sampling prevents robust inference of adaptive evolution. Sparse phylogenetic representation can inflate ω estimates in branch-site models, and therefore the observed signals should be interpreted cautiously (Ding et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImportantly, the present analysis detects codon substitution patterns rather than functional changes. Consequently, any linkage between sequence divergence and behavioral or motivational traits remains speculative and requires further empirical validation. DRD4 is a well-characterized neuromodulatory receptor involved in dopaminergic signaling pathways (Ebstein et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Savitz et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and belongs to the G-protein-coupled receptor family, an evolutionarily diverse class of signaling molecules (Fredriksson et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Molecular variation in this gene has been associated with neural functional diversity across taxa (Shimada et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). However, whether the divergence observed in cetacean lineages reflects adaptive modification or neutral evolutionary processes remains to be determined.\u003c/p\u003e \u003cp\u003eGiven the exploratory nature of this screening and the limited number of genes examined, the findings should not be interpreted as evidence for widespread modification of motivational pathways. Instead, they highlight DRD4 as a candidate locus for future comparative investigation. Neuromodulatory systems are known to undergo lineage-associated modifications in response to ecological transitions (Donaldson \u0026amp; Young, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and molecular divergence within conserved neural components may represent one pathway through which evolutionary pressures shape lineage-specific trajectories.\u003c/p\u003e \u003cp\u003eFuture studies incorporating broader taxonomic sampling and complementary analytical approaches will be necessary to evaluate the evolutionary significance of DRD4 variation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe author declares that there are no financial or non-financial competing interests related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethical approval was required for this study because all data were obtained from publicly available genomic databases.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNo funding was received for conducting this study.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eConceptualization, data collection, analysis, and manuscript preparation were performed by the author.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll sequence data used in this study were obtained from publicly accessible databases (Ensembl and NCBI RefSeq).\u003c/p\u003e\n\u003cp\u003eAlignment files and analysis outputs are provided as Supplementary Materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEbstein RP, Novick O, Umansky R et al (1996) Dopamine D4 receptor (DRD4) exon III polymorphism associated with the human personality trait of novelty seeking. Nat Genet 12:78\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ng0196-78\u003c/span\u003e\u003cspan address=\"10.1038/ng0196-78\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing YC, Chi HC, Grady DL et al (2002) Evidence of positive selection acting at the human dopamine receptor D4 gene locus. Proc Natl Acad Sci U S A 99:309\u0026ndash;314. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.012464099\u003c/span\u003e\u003cspan address=\"10.1073/pnas.012464099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonaldson ZR, Young LJ (2008) Oxytocin, vasopressin, and the neurogenetics of. sociality Sci 322:900\u0026ndash;904. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1158668\u003c/span\u003e\u003cspan address=\"10.1126/science.1158668\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFredriksson R, Lagerstr\u0026ouml;m MC, Lundin LG, Schi\u0026ouml;th HB (2003) The G-protein-coupled receptors in the human genome form five main families. Mol Pharmacol 63:1256\u0026ndash;1272. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1124/mol.63.6.1256\u003c/span\u003e\u003cspan address=\"10.1124/mol.63.6.1256\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHof PR, Chanis R, Marino L (2005) Cortical complexity in cetacean brains Anat Rec A Discov Mol. Cell Evol Biol 287:1142\u0026ndash;1152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ar.a.20258\u003c/span\u003e\u003cspan address=\"10.1002/ar.a.20258\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Connell LA, Hofmann HA (2011) The vertebrate mesolimbic reward system and social behavior network: a comparative synthesis. J Comp Neurol 519:3599\u0026ndash;3639. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cne.22735\u003c/span\u003e\u003cspan address=\"10.1002/cne.22735\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavitz J, Solms M, Ramesar R (2006) The molecular genetics of cognition: dopamine, COMT and BDNF. Genes Brain Behav 5:311\u0026ndash;328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1601-183X.2005.00163.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1601-183X.2005.00163.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimada MK, Inoue-Murayama M, Ueda Y et al (2004) Polymorphism in the second intron of dopamine receptor D4 gene in humans and apes Biochem. Biophys Res Commun 316:1186\u0026ndash;1190. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2004.03.006\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2004.03.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Summary of aBSREL tests for dolphin branches across candidate genes.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"587\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBranch\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eq-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMax \u0026omega;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSites EBF\u0026ge;100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eDRD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003eTursiops truncatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDRD4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTursiops truncatus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e701.85\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDRD4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDelphinapterus leucas\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.00084\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0038\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e989.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eCOMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003eTursiops truncatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0428\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e932.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eSLC6A4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003eTursiops truncatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eDRD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003eTursiops truncatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eBDNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003eTursiops truncatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eGRIN2B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003eTursiops truncatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eOXTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003eTursiops truncatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSummary of branch-site analysis results for DRD4.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Foreground branches tested using aBSREL are shown with corresponding likelihood ratio statistics, corrected q-values, and estimated ω values (dN/dS).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Significant episodic divergence signals were detected only in cetacean branches after multiple testing correction.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Ritsumeikan University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Keywords: DRD4, dopaminergic signaling, cetaceans, comparative genomics, molecular evolution, lineage-associated divergence","lastPublishedDoi":"10.21203/rs.3.rs-8992414/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8992414/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeuromodulatory pathways, particularly dopaminergic signaling, are thought to play important roles in regulating motivation, learning, and exploratory behavior across vertebrates. Major ecological transitions, such as the shift from terrestrial to fully aquatic environments in cetaceans, may be associated with lineage-specific molecular variation in these systems. However, identifying candidate loci involved in such divergence remains challenging due to the large number of genes contributing to neural function.\u003c/p\u003e \u003cp\u003eHere, we conducted a comparative screening of eight genes associated with dopaminergic signaling across selected mammalian lineages, including cetaceans and a phylogenetically distinct terrestrial reference lineage. This exploratory analysis revealed that DRD4 uniquely exhibited patterns consistent with episodic lineage-associated divergence in cetaceans relative to other examined genes.\u003c/p\u003e \u003cp\u003eGiven the limited taxonomic sampling, these findings do not constitute evidence of adaptive evolution. Instead, they highlight DRD4 as a candidate locus for expanded phylogenetic and functional investigation. This hypothesis-generating study provides a foundation for future analyses incorporating broader taxonomic representation and complementary methodological approaches to evaluate the evolutionary significance of DRD4 variation.\u003c/p\u003e","manuscriptTitle":"Comparative screening identifies lineage-associated divergence in DRD4 in cetaceans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 08:14:10","doi":"10.21203/rs.3.rs-8992414/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c17f2bd-61b0-4aec-9936-9167b98da52a","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63685213,"name":"Evolutionary Biology"},{"id":63685214,"name":"Marine and Freshwater Biology"}],"tags":[],"updatedAt":"2026-03-03T08:14:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 08:14:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8992414","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8992414","identity":"rs-8992414","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}