Evolutionary dynamics of the vertebrate Wnt gene repertoire

Evolutionary dynamics of the vertebrate Wnt gene repertoire | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Evolutionary dynamics of the vertebrate Wnt gene repertoire View ORCID Profile Lily G. Fogg , View ORCID Profile Maxime Policarpo , View ORCID Profile Walter Salzburger doi: https://doi.org/10.1101/2025.07.04.663182 Lily G. Fogg 1 Zoological Institute, Department of Environment Sciences, University of Basel , Basel, 4051, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lily G. Fogg Maxime Policarpo 1 Zoological Institute, Department of Environment Sciences, University of Basel , Basel, 4051, Switzerland 2 Evolution of Sensory Systems Research Group, Max Planck Institute for Biological Intelligence , Seewiesen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maxime Policarpo Walter Salzburger 1 Zoological Institute, Department of Environment Sciences, University of Basel , Basel, 4051, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Walter Salzburger For correspondence: lily.fogg{at}unibas.ch maxime.policarpo{at}bi.mpg.de Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The Wnt gene family plays a central role in vertebrate development, yet its evolutionary dynamics across lineages remain largely unexplored. Here, we present the most comprehensive analysis of Wnt gene evolution in vertebrates to date, leveraging a large-scale comparative genomics approach of 38,886 Wnt gene sequences mined from 1,961 species. We first investigate overall patterns of gene retention, duplication, and loss, and then focus specifically on the impact of whole-genome duplications (WGDs). We uncover striking variation in Wnt gene repertoire sizes, with ray-finned fishes (Actinopterygii) exhibiting the largest repertoires – even after excluding taxa with recent WGDs. Notably, we identify extreme expansions in polyploid cyprinids, including an octoploid hybrid harbouring 99 Wnt genes, the highest number observed. Unexpectedly elevated Wnt copy numbers in diploid species, such as the Antarctic lanternfish ( Electrona antarctica ) and the brook lamprey ( Lethenteron reissneri ), point to lineage-specific expansions with potential adaptive significance. Evolutionary rate analyses reveal that certain Wnt clades – especially Wnt8 and Wnt16 – exhibit elevated dN/dS ratios and high birth–death rates, indicative of repeated episodes of relaxed constraint or adaptive diversification. Contrary to our expectations, there was no relationship between developmental expression timing and evolutionary rates, suggesting pleiotropic regulation and functional redundancy of Wnt genes. Altogether, our findings reveal pervasive, lineage-specific remodelling of Wnt gene repertoires, shaped by both genome duplication history and divergent evolutionary trajectories. This work provides a high-resolution framework for understanding the molecular evolution of a key developmental toolkit and highlights candidate genes for future studies of vertebrate eco-morphological diversity. Introduction The development and maintenance of animal form and function are regulated by signalling pathways that govern key processes, such as cell fate determination, tissue patterning, and organogenesis. Among these, the Wnt signalling pathway plays a pivotal role in development, influencing a wide array of biological processes including embryogenesis, stem cell maintenance, and the regulation of cell proliferation and differentiation ( 1 - 3 ). Given its central importance, dysregulation of the Wnt pathway is implicated in a variety of diseases, including cancer and developmental disorders ( 4 , 5 ). The WNT ligands, key components of the Wnt signalling pathway, are secreted signalling proteins encoded by a family of genes that have been found in species across eukaryotes, from sponges to humans ( 6 , 7 ). In vertebrates, the Wnt gene family includes a large repertoire of genes that can be classified into at least 12 subfamilies based on their functional properties and sequence homology ( 7 ). Each gene clade is expressed at different points during development and plays distinct roles in developmental processes. Due to its critical role in development and cellular regulation, the Wnt signalling pathway is thought to be highly conserved across vertebrates ( 8 ). Furthermore, because morphological divergence increases over ontogeny, genes expressed at early and middle stages of development are thought to evolve under stronger purifying selective pressure than genes expressed late in development ( 9 ). However, these conclusions were drawn from studies focusing on a limited number of species or specific lineages. Despite the critical role of the Wnt genes in vertebrate biology, the evolutionary history and dynamics of the Wnt gene family and their link to expression patterns remain poorly understood beyond specific knowledge generated from a handful of model organisms. Here, we conduct a comprehensive analysis of the Wnt gene family across a diversity of vertebrate species, utilizing a large-scale genomic approach to explore the evolutionary dynamics of these genes. By mining 1,961 vertebrate genomes, we characterized the diversification, gene gain and loss patterns, and lineage-specific expansions of the Wnt gene repertoire across all major vertebrate clades (ray-finned fishes, amphibians, reptiles, birds, and mammals). Furthermore, we investigated the potential associations between the Wnt gene repertoire and the developmental timing of gene expression and lineage-specific morphologies. Through this study, we provide a deeper understanding of the evolutionary history and functional diversification of the Wnt gene family across vertebrates and its implications for development and disease. Results and Discussion Vertebrate Wnt gene repertoire variation and expansion after whole-genome duplication By extracting Wnt genes from 1,961 vertebrate species, we found that the number of Wnt genes per genome varied substantially across species and clades ( Fig. 1 ; Fig. S1), reflecting both ancestral genome duplication events and lineage-specific evolutionary processes. Notably, while the loss of Wnt genes in a given species can arise from genome sequencing or assembly artifacts, our dense species sampling leads to an overall estimation of the number of Wnt copies across species reflecting true biological variations. This is supported by a strong phylogenetic signal (λ = 0.9, p < 2e-16) in copy number across the vertebrate phylogeny, i.e, closely related species tend to have similar numbers of Wnt genes, consistent with a pattern shaped by shared evolutionary history. Download figure Open in new tab Fig. 1. The Wnt gene repertoire in vertebrates. Phylogeny with 1898 vertebrate species, for which a genome assembly with more than 90% complete BUSCO genes was available. The branches are coloured by (sub)class. The number of Wnt genes per species are shown as bars, coloured as indicated in the lower left panel. Species with expanded Wnt gene repertoires are highlighted (indicated by black silhouettes). For the most part, these species have undergone additional whole-genome duplications, except for Electrona antarctica and Lethenteron reissneri . Inset plot is a visualisation of the size of the Wnt gene repertoire per gene clade per species class. Branches in phylogeny at the top are coloured by (sub)class. The minimum, mean and maximum copy numbers are represented by dark blue, light blue and red circles, respectively. Note that if there is no dark blue circle visible then the minimum is equal to the maximum. A phylogeny with full species names is available on the Figshare repository. Animal silhouettes were obtained from PhyloPic.org. All Wnt subfamilies were retrieved in all vertebrate classes, with the exception of Wnt16 , which could not be retrieved from the Dipnoi genome investigated here ( Neoceratodus forsteri ). Notably, Actinopterygii (ray-finned fishes) exhibited the largest Wnt gene repertoires, with a median of 24.6 Wnt genes per genome compared to 16.6 – 22 genes in the other vertebrate classes (Agnatha: 21.8; Chondrichthyes: 19.3; Coelacanth: 19; Dipnoi: 17; Amphibia: 20.9; Mammalia: 17.5; Lepidosauria: 16.9; Aves/Crocodilia: 16.7; Testudines: 19.6). The higher number of Wnt genes in ray-finned fish genomes is probably the result of a high retention of members of this gene family subsequent to the teleost whole genome duplication (WGD), and potentially linked to the extremely diverse morphologies in this class ( 10 ). Notably, this pattern persisted even after excluding non-diploid species, which have undergone more recent lineage-specific WGDs. The polyploid species, which had the highest Wnt copy numbers observed, included several linages in the Salmonidae, Cyprinidae, Catostomidae, and Acipenseridae families ( Fig. 1 ). Particularly striking was the hybrid octoploid species Carassius auratus × Cyprinus carpio , which had 99 Wnt genes, followed by its tetraploid parental species, C. auratus and C. carpio, with 55 and 51 genes, respectively. By leveraging these recent and independent WGD events in teleosts, we assessed whether certain Wnt subfamilies were preferentially retained. The average ratio of Wnt gene numbers in polyploid species compared to closely related diploid species ranged from 1.53 for Wnt6 to 2.18 for Wnt16 (Fig. S5). However, these differences were not statistically significant, suggesting either that there is no preferential retention of particular Wnt subfamilies following WGDs, or that these WGDs are too recent for sufficient post-duplication gene loss to reveal clear patterns of differential gene retention. Furthermore, while, as expected, we could observe significantly higher evolutionary rates of Wnt coding sequences in polyploid species ( p = 4.699e-05, Fig. S5), we could not decipher if these accelerations were mainly driven by positive or relaxed selection, i.e., driven by neo-functionalisation or non-functionalisation, also likely due to the relatively recent nature of these WGD events. Wnt gene repertoire expansion without additional whole-genome duplication Among species without additional WGD events, elevated Wnt gene numbers were found in the Antarctic lanternfish Electrona antarctica (36 genes), the Asiatic brook lamprey Lethenteron reissneri (45 genes) and most species in the Eloposteoglossocephala ( 11 ) (range: 24 – 31 genes; mean: 28 genes) ( Fig. 1 ). This finding suggests either localized gene family expansion or possible assembly artifacts ( 12 ). To investigate these unusual cases further, we examined the genomic characteristics of Wnt genes in E. antarctica and L. reissneri . In E. antarctica , the expansion was driven predominantly by remarkable duplications of Wnt8a.1 , with 14 copies – more than in any other species examined (Fig. S2). Genomic coverage levels for these paralogs were similar to other Wnt genes and to BUSCO reference genes, indicating that these duplicates are unlikely to be assembly artifacts (Fig. S2). However, experimental validation ( e.g., via Sanger sequencing) would be required to confirm their authenticity. Evolutionarily, the Wnt8a.1 genes in E. antarctica exhibited elevated dN/dS ratios relative to both Wnt8a.1 orthologs in other species and to other Wnt genes, suggesting a history of relaxed purifying selection or possible positive selection (Fig. S2). The relatively compact size of Wnt8a in fish (in E. antarctica , Wnt8a is approximately 1.5 kb compared to, for example, Wnt4a at >20 kb), together with its genomic architecture and known bicistronic structure ( 13 ), may facilitate tandem duplication, potentially contributing to its lineage-specific expansion. In L. reissneri , the elevated Wnt copy number was due to expansions in Wnt5a and Wnt7bb , with 9 and 13 copies respectively – again the highest copy numbers in these gene clades among all species examined (Fig. S3). Genomic coverage for these paralogs was lower than BUSCO genes but consistent with other Wnt genes in this genome (Fig. S3), leaving their authenticity inconclusive. These patterns may reflect true expansions or potential assembly or annotation issues, which are not uncommon in lamprey genomes due to their high repeat content and fragmented assemblies ( 14 ). Nonetheless, if genuine, these expansions point to ongoing Wnt diversification even in early-diverging vertebrate lineages such as Agnatha. In Eloposteoglossocephala, which includes Osteoglossomorpha ( e.g., Arapaima and elephantfishes) and Elopomorpha (eels and tarpons), the increased size of the Wnt gene repertoire was due to elevated copy number across several Wnt clades, specifically Wnt1, Wnt2, Wnt4, Wnt6, Wnt10, Wnt11, and Wnt16 . These slightly more elevated copy numbers were consistently observed across species in the clade ( Fig. 1 ). The reasons for this pattern may arise from higher retention of duplicates from the teleost WGD compared to Clupeocephalans. Additionally, ecological or morphological factors unique to Eloposteoglossocephala may have favoured the maintenance of extra Wnt copies. Indeed, species in this clade exhibit a wide range of life history strategies, such as long-distance catadromous migrations, air-breathing adaptations, and reproductive strategies involving complex parental care ( 15 - 17 ). Thus, the elevated Wnt copy numbers in this clade may reflect adaptive pressures related to the group’s distinctive evolutionary and ecological contexts, independent of whole-genome duplication. Selective pressures and birth-death dynamics of Wnt genes To assess the evolutionary dynamics of Wnt genes, we first analysed codon-level substitution rates (dN/dS) across gene clades and across vertebrate classes and then used gene tree – species tree reconciliation methods to compute duplication and loss rates (per gene per million years) for each subfamily and each vertebrate class. While the overall distributions of Wnt dN/dS values were similar between vertebrate classes ( Fig. 2 ; Fig. S4), there were striking differences in terms of duplication rates. The most dynamic Wnt gene repertoires were found in Actinopterygii and Mammalia, with significantly higher birth rates than in the other classes ( Fig. 3a ), as well as higher death rates than in most classes ( Fig. 3b ). In fishes, the expanded and highly dynamic Wnt repertoires might have contributed to the exceptionally diverse morphological features of this group, along with their diversification and adaptation to diverse aquatic niches. In both ray-finned fishes and mammals, gene clades with the highest birth rates included Wnt8 , Wnt10 , and Wnt16 ( Fig. 3c and 3d ). Notably, this pattern persisted even when polyploid fish clades (which inflate birth rate estimates) were excluded. Furthermore, in all vertebrate classes except in Agnatha, we observed that, with a mean dN/dS of 0.237, Wnt16 genes were evolving faster than the other subfamilies, which had mean dN/dS ranging from 0.03 for Wnt1 to 0.16 for Wnt10 ( Fig. 3e ). Download figure Open in new tab Fig. 2. dN/dS of Wnt genes across vertebrate classes. For each vertebrate (sub)class (coloured as in Fig. 1 ), the distributions of the dN/dS values of the different Wnt genes are shown as boxplots (first quartile −1.5 interquartile range; first quartile; mean; third quartile; third quartile +1.5 interquartile range; dots represent outliers) for all vertebrate species for which a genome assembly with more than 90% complete BUSCO genes was available. Samples sizes for each vertebrate (sub)class can be retrieved from Supplementary Table 1. Download figure Open in new tab Fig. 3. Birth and death rates of Wnt genes across vertebrate classes. Birth (a) and death (b) rates of all Wnt genes per vertebrate class (coloured as in Fig. 1 ) shown as boxplots (first quartile −1.5 interquartile range; first quartile; mean; third quartile; third quartile +1.5 interquartile range; dots represent outliers). Note that the classes containing representatives with additional whole-genome duplications (WGDs) have been split into species with (Actinopterygii and Amphibia) and without (Actinopterygii_woWGD and Amphibia_woWGD) these additional WGDs. (c-d) Mean birth vs. death rates per Wnt clade for the classes with the highest birth rates: Actinopterygii (c) and Mammalia (d). (e) Relationship between mean dN/dS and mean birth rate per Wnt clade. Black line shows a linear regression. Note that the colour legend for the different Wnt clades represented in panels c-e is located in the bottom right of the figure. The elevated duplication rates in these Wnt gene clades suggest that the biological functions associated with Wnt8 , Wnt10 , and Wnt16 may be subject to increased lineage-specific diversification. For example, the dynamics observed in Wnt8 could reflect its central role in embryonic development – particularly axis formation, neural patterning, and somitogenesis ( 13 , 18 ) – where shifts in expression timing or regulatory architecture may promote developmental innovation. Similarly, the high turnover of Wnt10 may relate to its roles in epithelial-mesenchymal interactions, skin appendage and hair follicle development ( 19 , 20 ), and bone morphogenesis ( 21 ), processes that are often tightly linked to lineage-specific morphological traits. Finally, Wnt16 is implicated in diverse biological processes, including bone homeostasis in mammals ( 22 ), as well as hematopoietic stem cell development in zebrafish ( 23 ), indicating its functional versatility across vertebrates. In contrast, certain Wnt subfamilies exhibited remarkably low duplication rates across vertebrate classes, suggesting strong evolutionary constraint. Wnt1, Wnt3, Wnt4, and Wnt7 were the least dynamic gene clades, with low mean dN/dS values and birth rates ( Fig. 2 ; Fig. 3e ). This evolutionary conservation suggests that the functions of these genes are highly constrained and likely important across diverse lineages. Indeed, these gene subfamilies are involved in critical and conserved developmental processes. For instance, Wnt1 , Wnt3 and Wnt7 all play central roles in the development of the nervous system ( 24 - 31 ), while Wnt4 is crucial for gonad development and sexual maturation ( 32 , 33 ). The evolutionary stability of these genes implies that functional indispensability may limit the tolerance for duplication and mutation, leading to strong purifying selection that maintains their structure and function across vertebrates. Finally, we observed a positive correlation between the mean birth rate of Wnt subfamilies and their mean evolutionary rate (linear model: R 2 = 0.54; p = 0.007; Fig. 3e ), showing that when genes are being duplicated, the evolutionary rates of their coding sequences increase, consistent with their neo-, sub- or non-functionalisation. Notably, the mean birth rate of Wnt16 was also the highest (0.004), followed by Wnt8 (0.0034) and Wnt10 (0.003). Pleiotropy, developmental constraint, and lineage-specific selection in Wnt gene evolution To investigate whether developmental expression patterns influence the evolutionary dynamics of Wnt genes, we analyzed three well-characterized model organisms representing major vertebrate clades: Danio rerio (Actinopterygii), Xenopus laevis (Amphibia), and Mus musculus (Mammalia). Specifically, we tested the hypothesis that Wnt genes expressed earlier in development would exhibit lower evolutionary rates, as early-expressed genes are often subject to stronger purifying selection due to their involvement in foundational developmental processes ( 34 - 36 ). Contrary to expectations, no significant correlation was detected between the timing of developmental expression and dN/dS values across species (Fig. S7-9). This lack of association may reflect the complex and often stage-specific roles of Wnt genes during development. Wnt signalling is known to act repeatedly across embryogenesis and later life stages, with many genes playing roles in multiple tissues and developmental contexts. As a result, even genes with early embryonic expression may be subject to divergent evolutionary pressures across other stages or tissues. Additionally, the high degree of pleiotropy observed in Wnt genes may buffer their evolutionary rates against simple temporal constraints. In such cases, compensatory mechanisms, redundancy within the Wnt gene family, or dosage sensitivity may dilute the relationship between expression timing and sequence constraint. Moreover, available developmental expression datasets may not fully capture the spatial-temporal complexity of Wnt gene regulation, especially for genes with dynamic or tissue-specific expression. While global patterns of evolutionary constraint in the Wnt gene family did not correlate with developmental timing, we tested if we could detect signatures of shifts in selective pressure during specific evolutionary transitions. Given the central role of Wnt signalling in appendage development, we explored the relationship between Wnt gene evolution and the loss of limbs, which happened several times independently in amphibian and lepidosaurians (six independent times when considering species included in this study; Fig. 4a ). Download figure Open in new tab Fig. 4. The dynamics of Wnt genes in lineages with limb loss. (a) Phylogeny of diploid amphibian and lepidosaurian species. Species with limbs are displayed in blue while species which have undergone limb loss are displayed orange. (b) Relationship between the dN/dS values of Wnt10b and Wnt4 and the presence or absence of limbs. pGLS R 2 and adjusted p values (Bonferroni correction) are indicated. Strikingly, we found that Wnt10b and Wnt4 exhibited significantly elevated dN/dS ratios in lineages that have undergone limb loss (pGLS R² values of 0.20 and 0.13, with corresponding adjusted p values of 3.13e-05 and 0.13, respectively; Fig. 4b ; Supplementary Table 1), suggesting a relaxation of purifying selection or the onset of positive selection following the loss of limbs. These findings are consistent with the known roles of both genes in appendage development. Wnt4 has been implicated in early limb bud formation and mesenchymal cell proliferation ( 37 , 38 ), while Wnt10b is involved in bone development and outgrowth and patterning of the limb ( 21 , 39 , 40 ). The elevated dN/dS values in limbless taxa indicate that these genes may have experienced reduced functional constraints or adaptive shifts in regulatory or developmental roles following the loss of limbs. Conclusion In this study, we have assembled a comprehensive Wnt gene atlas from nearly 2000 vertebrate genomes, providing a valuable resource for future comparative and functional studies in non-model organisms. This study reveals widespread variation in vertebrate Wnt genes, shaped by both whole-genome duplications and lineage-specific expansions. Teleosts, particularly polyploid species, show the most extensive Wnt gene repertoires, but we also identify unexpected gene expansions in species without recent WGDs, indicating localized duplication events. Gene duplication and evolutionary rate analyses highlight Wnt8, Wnt10, and Wnt16 as particularly dynamic subfamilies, potentially contributing to lineage-specific morphological innovation. In contrast, the strong conservation of Wnt1, Wnt3, Wnt4, and Wnt7 underscores their essential developmental roles. The positive link between duplication rate and sequence evolution supports models of post-duplication divergence. Although developmental expression timing did not predict sequence constraint, shifts in selective pressure following limb loss in amphibians and lepidosaurs, especially in Wnt4 and Wnt10b , indicate that changes in the evolutionary rates of genes can accompany major morphological transitions. Together, our findings highlight how genome architecture, developmental function, and ecological context interact to shape the evolution of a key signalling pathway in vertebrates. Materials and Methods Genome Data All genome assemblies available on the National Center for Biotechnology Information (NCBI) database ( 41 ) on 6 th November 2023 were downloaded using genome_updater and the following options: -T “7742” (restricts the genome search to vertebrate species), -d “refseq,genbank” (searches both the RefSeq and Genbank databases), and -A 1 (retains only one genome assembly per species). A total of 3533 vertebrate genomes were initially downloaded, of which 169 were excluded as the assemblies were described as “partial”, leaving a total of 3360 vertebrate genomes (Agnatha: 8; Chondrichthyes: 26; Actinopterygii: 1325; Dipnoi: 2; Coelacanth: 1; Amphibia: 85; Mammalia: 776; Lepidosauria: 122; Testudines: 36; Crocodilia: 5; Aves: 974). Alternative scaffolds were removed from the Homo sapiens ( GCA_000001405.40 ) and Danio rerio ( GCA_000002035.6 ) genomes. Genome completeness assessment The completeness of the vertebrate genomes used for this study was assessed using a reimplementation of BUSCO, known as compleasm v0.2.4 ( 12 , 42 ), using the vertebrata odb10 database, except for three extremely large genomes (Dipnoi: Protopterus annectens and Neoceratodus forsteri ; Amphibia: Ambystoma mexicanum) , for which BUSCO results were retrieved from previous studies ( 43 - 45 ). Since it is expected that genomes with a large proportion of missing BUSCO genes will produce biased estimates for the number of Wnt genes, we only selected high-quality genome assemblies on the basis of a high BUSCO score threshold: 90% complete BUSCO genes. In jawed vertebrates, 1955 genome assemblies contained at least 90% complete BUSCO genes. Consistent with previous studies, and likely due to the lack of a suitable BUSCO gene dataset for agnathan species, the eight agnathan genome assemblies had low BUSCO scores (between 17.1 and 76.4%) ( 46 ). Thus, the five highest agnathan genomes (with BUSCO scores above 60%) were retained in our analyses. Similarly, there were only two genome assemblies available for Dipnoi ( Neoceratodus forsteri and Protopterus annectens ), and the best of these two was retained ( N. forsteri , 83.1% complete) to represent this class. Finally, one lepidosaur species ( Sceloporus occidentalis ) was removed from the analyses, as this genome is contaminated with amphibian DNA ( 46 ). Vertebrate species tree The phylogenetic context of the species retained after BUSCO filtering was estimated by reconstructing a vertebrate species tree as described in ( 46 ). All subspecies were discarded to avoid redundancy in the species representation, leaving 1898 species for the phylogeny. Firstly, single-copy orthologs were identified by running BUSCO v5.7.1 ( 12 ) on each genome assembly using the closest available database ( Actinopterygii odb10 database : Actinopterygii; Aves odb10 : Aves; Mammalia odb10 : Mammalia; Tetrapoda odb10 : Amphibia, Crocodylia, Lepidosauria, and Testudines; Vertebrata odb10 : Agnatha, Chondrichthyes, Coelacanth, and Dipnoi). The genome assemblies of two species had to be split prior to running BUSCO due to their large size ( Rana muscosa and Ambystoma mexicanum ). Single-copy orthologs present in at least half of the species were aligned using MUSCLE v5.1 ( 47 ) and alignments trimmed using trimAl v1.4.1 ( 48 ). Maximum likelihood gene trees were constructed for each ortholog using IQ-TREE v2.0 using automated model selection by ModelFinder ( 49 ). We then inferred an unrooted species tree using ASTRAL v5.7.8 ( 50 ) and the resulting tree was rooted and dated using the least squared dating method ( 51 ) implemented in IQ-TREE v2.0 (using calibration dates, extracted from TimeTree.org ( 52 ), available in Supplementary Table 1). A vertebrate species tree was generated by merging these species tree, using the R package “ape” ( 53 ) and the function “bind.tree”. Wnt gene mining First, we built a Wnt database by extracting annotated Wnt genes (GenBank and RefSeq databases) from 28 vertebrate species. Genes annotated as partial or as pseudogenes were discarded, and we verified that retained genes were best matching to Wnt sequences by performing a BLASTP against the UniProt database. For each genome included in this study, we first performed a TBLASTN using this Wnt database as query, the genome as target and an e-value of 1e-3. We then extracted nonoverlapping tblastn hit regions using SAMtools, and these regions were extended by 100,000 bp upstream and downstream. Again, after this extension, overlapping regions were merged. Potential Wnt genes were predicted on these regions using miniprot, using the Wnt database as query. We discarded miniprot predictions for which the number of exons was lower than the number of exons inferred from tblastn results ( i.e., number of non-overlapping tblastn hit in this gene region), as this was indicative that miniprot likely merged two nearby Wnt genes. Miniprot results were further filtered to keep only predictions which lengths were higher than 95% of the query ( i.e., higher than 95% of a Wnt genes from our Wnt database). If two or more predictions on the same genomic region met these criteria, we kept the one that spanned the shortest genomic region. We repeated this filtering operation by decreasing the threshold length, until no miniprot prediction was found any more. Each miniprot predictions were translated to a protein sequence using EMBOSS transeq and this protein was used as query in a blastp against the UniProt database. Only predictions best-matching to a Wnt gene were retained. If a prediction contained one or more frameshifts, these were discarded before the translation. Retained miniprot predicted sequences were then classified as complete genes if they had a length higher or equal to 75% of their best blastp mach against the Wnt database, as pseudogenes if they contained one or more loss-of-function mutations (premature stop codon or frameshift), or as incompletes if no loss-of-function mutation was retrieved, and if their length was shorter than 75% of their best blastp mach against the Wnt database. We also classified predictions containing stretches of ambiguous (“N”) nucleotides as incomplete genes. Assessment of gene classification bias and species filtering criteria To ensure that genes were not disproportionately assigned to the incomplete or pseudogene categories, correlations were assessed between the number of complete genes and the number of incomplete genes or pseudogenes. These analyses were conducted both across all species and within a subset of species which had a lower number of complete Wnt genes, i.e., fewer than 12 (Fig. S6). A very weak negative correlation was identified between the number of complete genes and pseudogenes, but not between complete and incomplete genes, when all species were considered. However, this correlation was not observed when analyses were restricted to species with low numbers of complete Wnt genes. These findings suggest that contracted Wnt repertoires in these species are unlikely to result solely from pseudogenisation or misclassification by the gene mining pipeline. Nevertheless, to minimize potential bias arising from genome assembly or annotation quality, species with exceptionally low numbers of complete Wnt genes (fewer than six) were excluded from downstream analyses. Wnt gene trees and gene verification Gene trees were reconstructed using similar methods for each of the following groups: 1) all Wnt sequences mined, 2) per Wnt gene subfamily, and 3) per Wnt gene subfamily and species class. Firstly, given the large number of gene sequences, a template alignment was first generated by aligning 30 random sequences from the dataset using MUSCLE v5.1 ( 47 ) and the final alignment was generated by adding all other sequences to the template using the option “-add” in MAFFT v7.467 ( 54 ). For the per-gene clade trees, 10 randomly selected sequences from another gene clade were also added as outgroups. The final alignment was trimmed using trimAl v1.4.1 ( 48 ) and maximum likelihood gene trees were generated using IQ-TREE v2.0 ( 49 ) with the optimal model determined by ModelFinder ( 55 ). Trees were rooted in iTOL ( 56 ) and visualised using Taxonium ( 57 ) and the R package ggtree ( 58 ). Following initial gene tree construction, the Wnt gene sequences were manually verified. Firstly, visual inspection was used to identify sequences : 1) causing disparity between the topology of the gene tree and the vertebrate species tree, and 2) sequences with long branches which could represent erroneous predictions and/or distort the tree topology ( 59 ). All questionable sequences were manually re-predicted using a slower but more accurate exon prediction program, known as EXONERATE v2.2.0 ( 60 ). If the EXONERATE prediction disagreed with the prediction originally generated by miniprot, the former was used and the gene was accordingly (re-)classified as “complete”, “pseudogene”, or “incomplete”. This process was repeated iteratively, resulting in the manual verification of 995 genes. The final dataset comprised 38,886 complete Wnt gene sequences. We computed the phylogenetic signal of the total number of Wnt genes using the phylosig function implemented in phytools ( 61 ), with the lambda method. Sequence evolution analyses Branch rates of sequence evolution ( i.e. , ω or dN/dS ratio) were estimated from per-gene clade, per-species class alignments and phylogenies generated as described in the “ Wnt gene trees and gene verification” section. To obtain maximum likelihood ω ratios per branch, we fitted the Muse Gaut (+ GTR) model of codon substitution ( 62 ), implemented in HyPhy ( 63 ). Branches with a lack (dS 1) were discarded. Birth-death rate calculations The per-gene clade, per-species class phylogenies were used to reconstruct the evolutionary dynamics of the Wnt gene family. Firstly, nodes with low bootstrap values (<95%) were collapsed into polytomies using the R package ape ( 53 ). TreeRecs ( 64 ) was used to find the best root per gene tree, and NOTUNG v2.9.1.5 ( 65 ) was used with the phylogenomics option to reconcile the rooted gene tree with the species tree for each gene clade and species class. From the reconciliation results, we extracted the number of duplications, losses, and gene copy number for each internal node. We then computed birth (λ) and death rates (μ) per gene per million years, from duplication and losses numbers, following the approach of Niimura et al. (66) . Branches smaller than two million years were excluded ( 67 ). Rates were calculated for each gene clade within each species class, and mean birth and death rates were computed by averaging birth and death rates across all branches. Developmental timing of Wnt gene expression and correlation with evolutionary rates To investigate the relationship between developmental timing of Wnt gene expression and rates of molecular evolution, transcriptomic and sequence data were analyzed for three model species: Danio rerio, Xenopus laevis, and Mus musculus . For each species, a developmental time-course of gene expression was used to determine the timing of Wnt gene activity across embryogenesis. Expression data for D. rerio were obtained from the EMBL-EBI Expression Atlas ( https://www.ebi.ac.uk/gxa/experiments/E-ERAD-475 ) and were based on an RNA-seq dataset published by White et al. ( 68 ). The X. laevis data were downloaded from Xenbase ( https://www.xenbase.org ) and were based on an RNA-seq dataset published by Session et al. (69) . The data for M. musculus were obtained from the MGI-Mouse Gene Expression Database (GXD; https://www.informatics.jax.org/expression.shtml ). For each gene, TPM values were used to define two key developmental time points: (i) peak expression, the stage with the highest TPM value; and (ii) earliest expression, the first stage with TPM > 0. Developmental stages were filtered to retain similar stages across species using on morphological criteria broadly conserved across vertebrates ( e.g., zygote, gastrulation, somitogenesis, and pharyngula stages). Each stage was assigned a numerical rank to permit regression-based analysis of expression timing as a continuous variable. For each species, expression data were integrated with a curated set of Wnt gene sequences mined in this study. Publicly annotated transcripts from Ensembl ( 70 ) were matched to mined transcripts using phylogenetic inference, ensuring orthology and accurate mapping between expression profiles and evolutionary rate data. Only Wnt genes with both reliable expression classification and dN/dS estimates were included (see section on Sequence evolution analyses for details of dN/dS filtering). To assess whether the timing of gene expression is predictive of evolutionary constraint, dN/dS values were regressed against the numerical ranks of peak and earliest expression stages using generalized least squares (GLS) models in R. This allowed for species-specific tests of the hypothesis that genes expressed earlier in development are subject to stronger purifying selection. Retention of Wnt genes following whole-genome duplications in teleosts Across teleost species included in our study, there were six independent recent whole genome duplications: one in Salmoniformes, four in Cyprinidae, and one in Catostomidae. For each of these polyploid lineages, we computed the mean number of genes per across polyploid species, for each Wnt subfamily. In the same way, we computed the mean number of gene per species and per Wnt subfamily for close diploid species (species included available in Supplementary Table 1). Then, for each WGD lineage, we could compute the ratio between the with these mean number of Wnt genes and the mean number of Wnt genes in close diploid species. We tested if the computed ratios were significantly different between Wnt subfamilies using pairwise Wilcoxon tests (Supplementary Table 1). The p values were adjusted with the Bonferonni correction. For the same set of species, we then used RELAX, implemented in HyPhy, to test if branches corresponding to Wnt genes of polyploid species were evolving under intensified or relaxed selection. We also tested, as control, branches corresponding to Wnt genes of close diploid species. A branch was considered to evolve under relaxed selection if the RELAX p value was < 0.05 and the K parameter <= 1 or under intensified selection if the p value was 1. Correlation between Wnt selective pressure and loss of limbs For each Wnt gene retrieved in lepidosaurian and amphibian, we performed a pGLS using the R package “caper” ( 71 ) with, as response, the dN/dS and, as predictor, the presence or absence of limbs. Only Wnt genes retrieved in at-least three independent clades with limb losses were retained. Resulting p values were corrected using the Bonferroni method. Funding This work was funded by a Swiss National Science Foundation (SNSF) grant awarded to W.S. 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Fogg , Maxime Policarpo , Walter Salzburger bioRxiv 2025.07.04.663182; doi: https://doi.org/10.1101/2025.07.04.663182 Share This Article: Copy Citation Tools Evolutionary dynamics of the vertebrate Wnt gene repertoire Lily G. Fogg , Maxime Policarpo , Walter Salzburger bioRxiv 2025.07.04.663182; doi: https://doi.org/10.1101/2025.07.04.663182 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Evolutionary Biology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)