Fish as Model Systems to Study Epigenetic Drivers in Human Self-Domestication and Neurodevelopmental Cognitive Disorders

Citation: Anastasiadi, D.; Piferrer, F.; Wellenreuther, M.; Benítez Burraco, A. Fish as Model Systems to Study Epigenetic Drivers in Human Self-Domestication and Neurodevelopmental Cognitive Disorders. Genes 2022,13, 987. https://doi.org/10.3390/ genes13060987 Academic Editor: Toshifumi Yokota Received: 25 February 2022 Accepted: 11 May 2022 Published: 31 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). genes G C A T T A C G G C A T Article Fish as Model Systems to Study Epigenetic Drivers in Human Self-Domestication and Neurodevelopmental Cognitive Disorders Dafni Anastasiadi 1,* , Francesc Piferrer 2, Maren Wellenreuther 1,3 and Antonio Benítez Burraco 4 1Seafood Technologies, The New Zealand Institute for Plant and Food Research, Nelson 7010, New Zealand; maren.wellenr[email protected] 2Institut de Ciències del Mar, Spanish National Research Council (CSIC), 08003 Barcelona, Spain; [email protected] 3School of Biological Sciences, The University of Auckland, Auckland 1010, New Zealand 4Department of Spanish, Linguistics, and Theory of Literature (Linguistics), Faculty of Philology, University of Seville, 41004 Seville, Spain; [email protected] *Correspondence: [email protected] Abstract: Modern humans exhibit phenotypic traits and molecular events shared with other domesticates that are thought to be by-products of selection for reduced aggression. This is the human self-domestication hypothesis. As one of the first types of responses to a novel environment, epigenetic changes may have also facilitated early self-domestication in humans. Here, we argue that fish species, which have been recently domesticated, can provide model systems to study epigenetic drivers in human self-domestication. To test this, we used in silico approaches to compare genes with epigenetic changes in early domesticates of European sea bass with genes exhibiting methylation changes in anatomically modern humans (comparison 1), and neurodevelopmental cognitive disorders considered to exhibit abnormal self-domestication traits, i.e., schizophrenia, Williams syndrome, and autism spectrum disorders (comparison 2). Overlapping genes in comparison 1 were involved in processes like limb morphogenesis and phenotypes like abnormal jaw morphology and hypopigmentation. Overlapping genes in comparison 2 affected paralogue genes involved in processes such as neural crest differentiation and ectoderm differentiation. These findings pave the way for future studies using fish species as models to investigate epigenetic changes as drivers of human self-domestication and as triggers of cognitive disorders. Keywords: domestication; epigenetics; vertebrates; cognitive disorders; human evolution; DNA methylation; domestication syndrome; self-domestication; neural crest; fish 1. Introduction Domestication is defined as an “evolutionary process that substantially reshapes the genetic, physiological and behavioral profile of a species to adapt to a human-made environment” [ 1 ]. Historically and contemporarily, this process has affected the evolutionary trajectories of several economically and culturally important vertebrate species. Domestication usually involves selection of less aggressive and more human-tolerant individuals, plus some other specific features of interest such as meat, wool, and milk production. Nonetheless, new additional phenotypic traits emerge repeatedly in independent vertebrate domestication events, even at the early stages of living in a human-made environment prior to deliberate selection; a phenomenon first noticed by Darwin himself and currently known as the “domestication syndrome” [ 2 ]. The domestication syndrome has been predominantly described in mammals, likely due to the large number of mammalian domesticates with a long domestication history, sometimes dating back millennia (e.g., dogs). Phenotypic traits of the domestication syndrome, which are not necessarily Genes 2022,13, 987. https://doi.org/10.3390/genes13060987 https://www.mdpi.com/journal/genes Genes 2022,13, 987 2 of 20 present in all domesticates, include a decreased size of the brain, heart and teeth, vertebrae variability, caudal vertebrae changes, shorter muzzle, more frequent estrous cycles, floppy ears, curly tail and hair, and depigmentation [ 3 , 4 ]. The morphological and physiological traits have all been considered to have arisen as by-products of selection for increased tameness. Since these traits are associated with the final sites of migration of neural crest cells, mild developmental deficits affecting their development, migration or differentiation have been suggested by some authors as the underlying mechanisms of the appearance of the domestication syndrome, termed the “neural crest cell hypothesis” (NCCH) [2,5]. Modern humans, compared to extant apes and extinct hominins, exhibit phenotypic traits similar to those of other domesticated vertebrates, suggesting these may have also been produced as a by-product of selection for reduced aggression and increased sociality [ 6 – 8 ]. This is called the human self-domestication hypothesis [ 6 – 8 ] (HSD). Domestication syndrome-like morphological traits in anatomically modern humans (AMH) include a decreased brain and teeth size, facial robusticity, and sexual dimorphism, as well as neoteny [ 6 , 7 , 9 ]. Behavioral traits include reduced aggression, increased sociability, prolonged playing behavior, and overall more flexible social skills [ 6 , 7 , 9 ]. To understand and evaluate the HSD hypothesis, we need to distinguish between deliberate selection for improved traits, as occurred in, e.g., agricultural animals, and non-deliberate selection for prosociality arising from adaptation to novel environments, as expected for species hypothesized to have gone through a self-domestication process, like bonobos [ 6 ]. The latter should be seen through the lens of domestication being a multi-stage process, where non-deliberate selection arises in response to the new selective environment, e.g., often involving a lack of predators and an increase in food availability [ 7 ], changed environmental conditions [ 10 ], or the colonization of new environments [ 11 ], which are all known factors promoting prosocial behavior. Empirical support for the HSD hypothesis is challenging to obtain, nevertheless, comparative genomics have provided tentative support for it [ 12 ]. Recent results of an elegant study by Zanella et al. [ 13 ] using a molecular genetics approach are consistent with both the NCCH and the process of human self-domestication, specifically with regard to changes in the skull and the face [ 13 , 14 ]. This paper shows how in silico analyses of paleogenetic data and neurodevelopmental studies of human-altered phenotypes (e.g., Williams syndrome) can be integrated with experimental in vitro and in vivo research for empirically validating aspects of the HSD hypothesis. Still, the HSD hypothesis is not universally accepted, since the hypothesis presents some challenges, especially in relation to testing it [ 7 ], but also to the target of selection, which has been suggested to be socially-mediated emotional control and plasticity rather than selection against aggression [15]. Neurodevelopmental disorders in humans characterized by social and cognitive impairments may present traits of the domestication syndrome in an altered manner and thus may be linked to altered self-domestication. This is consistent with the view of selfdomestication as a variable phenotype in the human species, e.g., [ 16 ], with this variability depending on genetic and environmental factors. More generally, this is consistent with the view that cognitive diseases mostly result from the alteration of recently evolved brain mechanisms, as these mechanisms lack compensatory mechanisms to respond to ontogenetic damage. People with autism spectrum disorders (ASD) and schizophrenia (SZ) exhibit abnormal aggressive behavior, abnormal responses to social cues, as well as tooth, ear, and facial anomalies [ 17 , 18 ]. In ASD, increased head and brain size, and generalized overgrowth are also present, while in SZ, decreased brain volume and reproductive dysfunctions occur [ 19 , 20 ]. Accordingly, these two cognitive disorders can be regarded as “less self-domesticated” and “more self-domesticated” phenotypes, respectively [ 19 , 20 ]. Moreover, Williams syndrome (WS), caused by the hemideletion of 28 genes in chromosome 7, is a clear example of a “more self-domesticated” phenotype [ 9 , 13 ]. People with WS show hypersociability, decreased aggression, reduced head and brain size, pointy ears, small teeth and jaws, depigmentation and accelerated sexual maturity [ 9 ]. Zanella et al. [ 13 ] used cell lines derived from WS subjects to establish the molecular links of morphological and Genes 2022,13, 987 3 of 20 behavioral domesticated traits in humans with neural crest development and migration processes. Therefore, cognitive disorders and the gene networks associated with them may be used as models for further testing the HSD hypothesis. Domestication is a process of adaptation to a new selective environment and has been considered likely to involve epigenetic changes [ 21 – 25 ]. Epigenetic mechanisms offer a way for novel phenotypes to emerge rapidly in response to environmental changes and to prime the offspring, when inherited, to face environments based on the parental experience [ 26 – 28 ]. In the first stages of domestication, which coincides with the emergence of domestication syndrome traits, epigenetic changes established during early development might regulate gene expression in the neural crest, and be maintained throughout adulthood and inherited by the offspring. Multigenerational epigenetic inheritance is nearly ubiquitous in diverse animal species (see [ 29 ] for review). The persistence of the domestication environment, together with the stability and small effect of epigenetic changes in mild developmental deficits of the neural crest, could be expected to accelerate the adaptation [ 30 ]. After several generations, epigenetic changes could be genetically assimilated as genetic variants [25,31,32], hardwiring these changes. Partial evidence for this process comes from studies on mammals (dogs-wolves [ 23 ]), birds (red jungle fowl-modern chickens [ 33 ]), and fish species. The same process could be hypothesized to account for the first steps of HSD, as most differences between extinct hominins and AMHs are epigenetic by nature, having impacted features that are related to the domestication syndrome, particularly the morphology of the face [ 34 ]. To date, DNA methylation, but also DNA hydroxymethylation, have been the epigenetic mechanisms of focus, even though other epigenetic mechanisms like histone modifications and variants or non-coding RNAs may also play a role in domestication. Unfortunately, the latter cannot be studied in ancient DNA remains. Domestication of fish species has a distinct history from terrestrial vertebrates [ 35 ], although it is scientifically considered to represent a similar process [ 36 ]. Until the 20th century, the majority of seafood relied on wild animal captures, with a few exceptions like the common carp (Cyprinus cyprio) in China ~8000 years ago or Nile tilapia (Oreochromis nilocitus) in Egypt ~3500 years ago [ 36 , 37 ]. In the last century, the domestication of aquatic species has expanded rapidly, with an estimated number of 368 vertebrates that have been domesticated for aquaculture, including teleost fish, frogs and reptiles [ 38 ]. Nevertheless, the majority of species are at the early stages of domestication, without closed life cycles in captivity and in the absence of deliberate selection for specific traits [ 38 ]. Nonetheless, in parallel with the domestication process, phenotypic traits involving the domestication syndrome, with changes in growth, reproduction, morphology, pigmentation and behaviour, have become manifested in domesticated fish [ 39 – 41 ]. Furthermore, sequencing of fish genomes has revolutionized vertebrate comparative genomics and has greatly contributed to our understanding of selection targets, evolutionary changes and speciation. Subsequently, fish have been suggested to serve as suitable models for human biomedical research [ 42 , 43 ]. Recently, epigenetic patterns emerging during the first stages of domestication, in the absence of genetic differences, have been studied in salmonids [ 44 , 45 ], European sea bass (Dicentrarchus labrax) [ 39 ], Nile tilapia (Oreochromis niloticus) [ 46 , 47 ] and grass carp (Ctenopharyngodon idellus) [ 48 ]. These epigenetic patterns of domestication are present in the sperm of several species, i.e., salmonids [ 45 , 49 – 51 ], showing the potential of intergenerational transfer, while in the European sea bass ~20% are found in early embryos, showcasing the importance of developmental aspects during early domestication [ 39 ]. Taken together, (1) the recent domestication events in fish, (2) the high degree of parallelism between fish and human domestication, particularly, the absence of deliberate selection in both domestication events, (3) the increasing availability of fish methylomes and (4) the use of fish as animal models in biomedical research, render fish promising candidate models to identify the epigenetic mechanisms that lead to the emergence of HSD, including their abnormal manifestation in neurodevelopmental disorders. Comparative epigenomic studies between domesticated animals and humans are expected to demonstrate parallel or contrasting processes operating in addition to traditional Genes 2022,13, 987 4 of 20 genetic aspects [ 15 ]. Here, we argue that fish hold great advantages as models to study epigenetic drivers in HSD. To test our argument, we use comparative approaches to epigenomic patterns, exemplified by the best-studied modification, DNA methylation, between humans and the European sea bass. The European sea bass was chosen because (1) 25 years of selective breeding resulted in selective sweeps in genes similar to those found under positive selection in all domesticates tested, e.g., glutamate receptors [ 52 , 53 ], (2) it presents traits of the domestication syndrome shared with those found in terrestrial vertebrates, e.g., depigmentation and cranial changes [ 39 ], and (3) epigenetic patterns of domestication have been assessed in four tissue types representative of all three embryonic layers, thus reducing bias due to tissue-specificity [ 39 ]. In the present study, we compare epigenetic patterns of domesticated sea bass with epigenetic patterns of (1) AMH as opposed to archaic hominins (Neanderthals and Denisovans), and (2) neurodevelopmental cognitive disorders with an abnormal presentation of traits parallel to the domestication syndrome (SZ, WS and ASD; Figure S1). The goal of these comparisons was to investigate whether genes or pathways were consistently altered during the steps of early domestication in European sea bass and humans, with a potential impact on our species-specific distinctive cognition and behavior. 2. Materials and Methods 2.1. Data Collection Comparative epigenomic analyses were divided into two major groups including early domesticates of the European sea bass vs. (1) AMH and (2) neurodevelopmental cognitive disorders. For this, we compiled five lists of genes identified as differentially methylated in the literature (Figure S1). 2.1.1. European Sea Bass Early Domesticates In European sea bass, we previously conducted work to generate genome-wide DNA methylation patterns (Reduced Representation Bisulfite Sequencing, RRBS) in fish captured in the wild vs. offspring of wild fish reared in hatchery [ 39 ]. DNA methylation data from the brain, muscle, liver and testis can be accessed through the NCBI Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/, accessed on 30 May 2022) with accession codes GSE104366 and GSE125124. Since these data were published, the European sea bass genome has been included in the Ensembl database (https://www.ensembl.org/, accessed on 30 May 2022). The genome assembly v1.0 in Ensembl is the same used for data analysis by Anastasiadi and Piferrer [ 39 ], however, gene annotation has since been updated according to the Ensembl Gene Annotation pipelines. To facilitate comparative epigenomic analysis with humans, we converted the list of genes with differentially methylated regions (DMRs) to the Ensembl genebuild released version from April 2020. To do this, the genomic coordinates (chromosome, start, end position) of DMRs and surrounding 5000 bp regions were intersected with the genebuild Dicentrarchus_labrax.seabass_V1.0.101.gtf. Chromosome names were as in the primary assembly. A total of 1181 unique genes with DMRs were identified in early domesticates. 2.1.2. Anatomically Modern Humans (AMH) A detailed map of the evolutionary dynamics of DNA methylation in human groups was recently published [ 34 ]. DMRs specific to the AMH-lineage as compared to other hominin lineages, i.e., Denisovan and Neanderthal, were identified using a conservative approach to minimize false positives and variability due to factors such as sex or age, as well as DNA methylation data from chimpanzee samples. AMH-lineage DMRs are a set of 873 DMRs that overlap with the gene body or the promoter up to 5000 bp upstream of 588 genes (Supplementary Dataset S2 of Gokhman et al. [ 34 ]; Figure S1). The list of genes with DMRs was supplied by [ 34 ] with UCSC (University of California Santa Cruz) identifiers (IDs) and we used the https://biotools.fr/human/ucsc_id_converter (accessed on 30 May 2022) tool to convert them to Ensembl IDs to facilitate comparative epigenomics with the European sea bass. Genes 2022,13, 987 5 of 20 2.1.3. Neurodevelopmental Cognitive Disorders WS has a clear genetic origin with the hemideletion of 28 genes at 7q11.23. Some of these genes, e.g., BAZ1B, are involved in epigenetic regulation, such as chromatin remodeling, providing a link to the impact on epigenomic patterns in WS [ 13 ]. Differential DNA methylation between patients with WS and healthy individuals as controls has been reported in at least two cases in the literature. DMRs identified using the Infinium ® HumanMethylation450 BeadChip array (Illumina, San Diego, CA, USA) in the blood of 20 WS patients vs. 15 healthy controls found DMRs intersecting 551 unique genes [ 54 ]. Differentially methylated cytosines (DMCs) were detected more recently in the blood of a larger sample of 90 WS patients vs. 34 healthy controls using the same array and these intersected with 143 unique genes [ 55 ]. The two gene lists were combined for further analysis as genes differentially methylated (DM) in WS, with a total of 624 different genes. SZ is a complex psychiatric disorder and epigenome-wide association studies (EWAS) have been carried out to explore the role of DNA methylation in SZ pathophysiology, with discordant results. Recently, a meta-analysis of five EWAS datasets was published, including samples taken from different parts of the brain (frontal cortex, cerebellum, hippocampus, and prefrontal cortex), between 3 and 47 samples per study and using either the Infinium HumanMethylation450 BeadChip or HumanMethylation27 BeadChip (Illumina, San Diego, CA, USA) [ 56 ]. A total of 513 genes were commonly DM in combinations of 4–5 EWAS and these were used here for further analysis as the DM genes in SZ. ASD refers to a group of complex neurodevelopmental disorders with heterogeneous symptoms and underlying etiology. ASD heritability is complex and genetic variants involved are diverse with their number ranging between 1000 and 3000 genes reflecting ASD heterogeneity [ 57 ]. Other molecular aspects to better understand ASD include epigenetic variants and several studies were published in the last years. This allowed us to apply more stringent criteria for inclusion in this study, mainly a minimum number of 15 samples and identification of DMRs, which are considered more robust than DMCs only. Genes from four studies published in the last 4 years, thus, included: (a) 31 genes with DM that were at the same time differentially expressed and common in three independent studies based on blood samples [ 58 ], (b) 181 core genes with DMRs detected using all three approaches in blood cells [ 57 ], (c) 145 unique differentially expressed genes with DMRs in blood cells from three ASD subphenotypes (severe, intermediate, mild) and a group of combined cases [ 59 ], and (d) 58 genes with DMRs detected in postmortem brain samples [ 60 ]. The four datasets combined led to a list of 411 unique ASD genes. 2.2. Comparative Analyses The BioMart data mining tool from Ensembl was used to identify orthologues of human genes from the genome assembly GRCh38.p13 in the European sea bass genome. Duplicate entries were eliminated for further analysis. Thus, we identified unique orthologues as follows: 589 for AMH, 506 for WS, 532 for SZ, and 367 for ASD (Figure S1). The BioMart tool was used to identify paralogues of the human genes involved in neurodevelopmental cognitive disorders in the human genome (GRCh38.p13), in turn used to identify orthologues in the European sea bass genome. Duplicate entries from the combined list of original orthologues and orthologues of human paralogues were eliminated and the number of homologs finally available for comparative analyses was as follows: 3460 for WS, 4000 for SZ and 2994 for ASD. Pairwise comparisons were performed with the fish early domesticates (FED) as a reference and one human group as its pair. Thus, four pairwise comparisons occurred every time: (1) FED vs. AMH, (2) FED vs. WS, (3) FED vs. SZ, and (4) FED vs. ASD. Overlaps between gene lists were identified and visualized using the InteractiVenn tool [ 61 ]. The significance of overlap was tested using Fisher’s exact test for testing the independence of two variables represented by a contingency table. As the genomic background for gene overlap testing, the total number of 23,382 genes in the European sea bass genome (Ensembl genebuild released April 2020) was set. Furthermore, we performed Monte Carlo Genes 2022,13, 987 6 of 20 permutations to test whether overlaps were higher than expected by chance. Random samples of genes were drawn without replacement from the 23,882 total gene list according to the specific gene list each time, e.g., to test the overlap of orthologues FED vs. AMH, 1181 genes for FED vs. 589 genes for AMH were randomly drawn in each iteration. The process was repeated 10,000 times and each time the length of the intersection or overlap between the two genes lists was counted. The standard score of permutation was calculated as observed-mean(permuted)/sd(permuted) and the p-value as: times permuted overlap is higher than observed overlap divided by the number of permutations (10,000). Fisher’s tests and permutations were performed using R (v. 4.0.0) [ 62 ] and Rstudio (v. 1.4.1717) [ 63 ]. The Enrichr tool was used for enrichment analyses and knowledge discovery of gene sets [ 64 – 66 ]. Enrichment analyses were performed for the initial lists of genes (FED, AMH, WS, SZ and ASD). Enriched pathways from the databases BioPlanet, Wikipathway, Mammalian Phenotype and GO-terms Biological Process were kept for further comparisons, which included overlap testing as previously carried out, with background on the total number of terms found in each library on Erichr. Reduction and visualization of GO-terms were aided by REViGO [ 67 ]. IDs of pathways were entered in InteractiVenn to detect overlaps and Fisher’s exact tests were run to detect the statistical significance of the overlap. Enrichment analyses were also performed for the genes that overlapped in a pairwise manner between FED genes and homologs (combined lists of orthologues and orthologues of paralogues). 3. Results 3.1. Differentially Methylated Genes during Early Domestication in European Sea Bass and in Humans Are Shared The early stages of domestication are expected to be associated with DNA methylation changes. To compare DNA methylation changes associated with the early stages of domestication between European sea bass and human, two gene lists were retrieved. In FED 1181 genes with DMRs were detected as compared to wild fish. For humans, based on limited availability and accessibility to early AMH domesticate samples, DNA methylation patterns of present-day AMHs compared to other hominins and primates were considered the most relevant proxy. A total of 589 genes with DMRs were detected as orthologues of AMH. We detected an overlap of 45 genes between FED and AMH and this was significant (Fisher’s test, odds ratio = 1.577, p= 0.004; Figure 1a). Furthermore, we found 1.7 times more genes in common between the two gene lists than expected by chance alone (z-score = 13.62, p= 3 ×10−4; Figure 1b). Genes 2022, 13, x FOR PEER REVIEW 7 of 22 Figure 1. Overlap of genes with epigenetic changes in fish early domesticates (FED) and anatomically modern humans (AMH). The overlap was tested using Fisher’s exact test for count data (a) and permutations (b). The results of permutations are represented as the distribution of the number of overlaps (shaded grey area) with the mean number of permuted overlaps (black vertical line) and significance threshold set to 0.05 (red line). The observed number of overlaps is shown by the green line and the distance of observed vs. expected (random) overlaps is shown with the black arrow. The z-score and the p-value indicate the significance of the overlap. Among the genes with DMRs in both groups (Table 1), we detected several genes that were repeatedly found to be involved in domestication in several species. For example, ADAM metallopeptidases with thrombospondin type 1 motifs, ephrin (eph) receptors, members of the integrin family (alpha or beta), or fibroblast growth factor receptors have been detected in other domesticates (see Dataset 1 from [39] Anastasiadi and Piferrer for overview and Montague et al., Pendleton et al., Zhang et al., Kukekova et al., Carneiro et al. [68–72] for each species). One of these genes is nuclear factor I X (NFIX in humans and nfxib in fish) which was found to be in the top 10 genes with DMRs in AMHs showing a strong correlation between methylation and expression [34]. Several lines of evidence suggest that hypermethylation of NFIX associates with its downregulation in the AMH lineage [34]. In FEDs, nfixb was hypermethylated in the testis (+29.98%) but hypomethylated in the muscle tissue (−35.88%). In other tissues, other nuclear factor 1 isoforms contained DMRs: in muscle tissue, nuclear factor 1 a-type contained 2 DMRs with opposite methylation patterns (+20.69% and −42.30%), and in brain tissue, nuclear factor 1 a-type contained 2 hypomethylated DMRs (−27.36% and −34.02%) and nuclear factor 1 b-type isoform X2 contained a hypomethylated DMR (−30.14%). Table 1. Common genes differentially methylated in European sea bass early domesticates and anatomically modern humans. Gene Name Gene Description Ensembl Gene Stable ID ADAM metallopeptidase with thrombospondin type 1 motif, 17 ENSDLAG00005007818 ArfGAP with GTPase domain, ankyrin repeat and PH domain 1 ENSDLAG00005018378 ATPase copper transporting beta ENSDLAG00005026064 Figure 1. Overlap of genes with epigenetic changes in fish early domesticates (FED) and anatomically modern humans (AMH). The overlap was tested using Fisher’s exact test for count data ( a ) and permutations ( b ). The results of permutations are represented as the distribution of the number of overlaps (shaded grey area) with the mean number of permuted overlaps (black vertical line) and significance threshold set to 0.05 (red line). The observed number of overlaps is shown by the green line and the distance of observed vs. expected (random) overlaps is shown with the black arrow. The z-score and the p-value indicate the significance of the overlap. Genes 2022,13, 987 7 of 20 Among the genes with DMRs in both groups (Table 1), we detected several genes that were repeatedly found to be involved in domestication in several species. For example, ADAM metallopeptidases with thrombospondin type 1 motifs, ephrin (eph) receptors, members of the integrin family (alpha or beta), or fibroblast growth factor receptors have been detected in other domesticates (see Dataset 1 from [ 39 ] Anastasiadi and Piferrer for overview and Montague et al., Pendleton et al., Zhang et al., Kukekova et al., Carneiro et al. [68–72] for each species). One of these genes is nuclear factor I X (NFIX in humans and nfxib in fish) which was found to be in the top 10 genes with DMRs in AMHs showing a strong correlation between methylation and expression [ 34 ]. Several lines of evidence suggest that hypermethylation of NFIX associates with its downregulation in the AMH lineage [ 34 ]. In FEDs, nfixb was hypermethylated in the testis (+29.98%) but hypomethylated in the muscle tissue ( − 35.88%). In other tissues, other nuclear factor 1 isoforms contained DMRs: in muscle tissue, nuclear factor 1 a-type contained 2 DMRs with opposite methylation patterns (+20.69% and − 42.30%), and in brain tissue, nuclear factor 1 a-type contained 2 hypomethylated DMRs ( − 27.36% and − 34.02%) and nuclear factor 1 b-type isoform X2 contained a hypomethylated DMR (−30.14%). We performed enrichment analyses to get insight into the functional roles of the overlapping genes. GO Biological Process enrichment analysis highlighted processes such as limb morphogenesis (GO:0035108, p= 0.045), histone modifications (GO:0016570, p= 0.024), T cell apoptotic processes (GO:0070231, p= 0.014), or granulocyte activation (GO:0036230, p= 0.021) as common (Figure 2a; for full list Table S1). Analysis of MGI Mammalian Phenotypes showed enrichment in traits typical of the domestication syndrome, such as abnormal snout morphology (MP:0000443, p-adjusted = 0.031) or hypopigmentation (MP:0005408, p-adjusted = 0.034; Figure 2b; for full list Table S2). Enrichment of WikiPathways showed that affected pathways include endochondral ossification with skeletal dysplasia (WP4808, p= 0.008), endochondral ossification (WP474, p= 0.008), or androgen receptor signaling pathway (WP138, p= 0.015; for full list Table S3). Genes 2022, 13, x FOR PEER REVIEW 10 of 22 Figure 2. Enrichment analysis of overlapping genes with epigenetic changes in fish early domesticates and anatomically modern humans. (a) GO Biological Process terms enrichment where for each GO-term the color indicates the log10-transformed p-value of enrichment. The semantic space x (yaxis) and the semantic space y (x-axis) are the result of multidimensional scaling done by REViGO and represent semantic similarities between GO-terms. (b) Enrichment of mammalian phenotypes (MGI Mammalian Phenotype 2014). Each phenotype term (y-axis) is attributed significance values after enrichment tests (performed by Enrichr) which include the −log10-transformed p-value of enrichment (x-axis and length of grey lines) and the combined score estimated by Enrichr (color of the bubble and legend). To facilitate visualization according to the significance of enrichment based on p-values, terms are ranked in descending order from top to bottom. 3.2. Early Domestication in European Sea Bass and Neurodevelopmental Cognitive Disorders Affect Paralogue Genes Genes exhibiting DNA methylation changes in patients with neurodevelopmental cognitive disorders with traits parallel to the domestication syndrome such as SZ, WS and ASD were obtained from the literature. A total of 532 genes with DM were orthologues to SZ patients, 506 genes with DM in WS patients and 367 genes with DM in ASD patients. These gene lists of orthologues were compared to the genes of FED to evaluate whether DNA methylation in common genes was affected by these conditions. The pairwise overlaps were not significant in all cases, with 28 genes overlapping in SZ (odds ratio = 1.04, p = 0.439; Figure S2a), 31 overlapping in WS (odds ratio = 1.233, p = 0.88; Figure S2b) and 23 genes overlapping in ASD (odds ratio = 1.262, p = 0.169; Figure S2c). Permutation testing for the pairwise comparisons showed that the number of overlaps was within the range expected by chance in the case of SZ (z-score = −0.59, p = 0.216; Figure S2d) and ASD (zscore = 2.61, p = 0.062; Figure S2f), and only marginally significant in the case of WS (zscore = 3.75, p = 0.049; Figure S2e). In an attempt to overcome the constraints of the conservative approach applied here for orthologues and since key candidate genes of domestication were present in all pairwise comparisons, e.g., protocadherins, ADAM metallopeptidases, collagens and glutamate receptors, we then focused on comparisons of functional properties. Orthologue genes were submitted for enrichment analyses and pairwise comparisons were performed at the pathway level following the reasoning that similar processes may be affected by different genes. We considered four libraries targeted by Enrichr as the most informative in our case: Bioplanet, WikiPathways, GO-terms Biological Process, and MGI Mammalian Phenotype. Terms in all four libraries were examined for enrichment according to the gene lists we provided (FED, SZ, WS, and ASD) and pairwise comparisons of terms were performed as follows: (1) FED vs. SZ, (2) FED vs. WS, and (3) FED vs. ASD (Figure S3). In Figure 2. Enrichment analysis of overlapping genes with epigenetic changes in fish early domesticates and anatomically modern humans. ( a ) GO Biological Process terms enrichment where for each GO-term the color indicates the log10-transformed p-value of enrichment. The semantic space x (y-axis) and the semantic space y (x-axis) are the result of multidimensional scaling done by REViGO and represent semantic similarities between GO-terms. ( b ) Enrichment of mammalian phenotypes (MGI Mammalian Phenotype 2014). Each phenotype term (y-axis) is attributed significance values after enrichment tests (performed by Enrichr) which include the − log10-transformed p-value of enrichment (x-axis and length of grey lines) and the combined score estimated by Enrichr (color of the bubble and legend). To facilitate visualization according to the significance of enrichment based on p-values, terms are ranked in descending order from top to bottom. Genes 2022,13, 987 8 of 20 Table 1. Common genes differentially methylated in European sea bass early domesticates and anatomically modern humans. Gene Name Gene Description Ensembl Gene Stable ID adamts17 agap1 atp7b ADAM metallopeptidase with thrombospondin type 1 motif, 17 ENSDLAG00005007818 ArfGAP with GTPase domain, ankyrin repeat and PH domain 1 ENSDLAG00005018378 ATPase copper transporting beta ENSDLAG00005026064 bcr carm1 BCR activator of RhoGEF and GTPase ENSDLAG00005004082 coactivator-associated arginine methyltransferase 1 ENSDLAG00005025319 celsr1a cemip coro7 dab2ipb cadherin EGF LAG seven-pass G-type receptor 1a ENSDLAG00005009488 cell migration inducing hyaluronidase 1 ENSDLAG00005002105 coronin 7 ENSDLAG00005014078 DAB2 interacting protein b ENSDLAG00005020932 DIP2C ephb3a disco-interacting protein 2 homolog Ca ENSDLAG00005023732 eph receptor B3a ENSDLAG00005000091 eps8l2 EPS8 like 2 ENSDLAG00005011013 EYA2 EYA transcriptional coactivator and phosphatase 2 ENSDLAG00005013401 fbrsl1 fibrosin-like 1 ENSDLAG00005019385 fgfrl1a fibroblast growth factor receptor like 1a ENSDLAG00005002545 galnt18a UDP-N-acetylalphaDgalactosamine:polypeptideN-acety- lgalactosaminyltransferase 18a ENSDLAG00005020537 gli3 GLI family zinc finger 3 ENSDLAG00005018034 itga11b integrin, alpha 11b ENSDLAG00005013142 kaznb kazrin, periplakin interacting protein b ENSDLAG00005001674 lasp1 LIM and SH3 protein 1 ENSDLAG00005018795 lhpp phospholysine phosphohistidine inorganic pyrophosphate phosphatase ENSDLAG00005006011 lmx1bb LIM homeobox transcription factor 1, beta b ENSDLAG00005025877 magi1b membrane associated guanylate kinase, WW and PDZ domain containing 1b ENSDLAG00005022108 mast2 microtubule associated serine/threonine kinase 2 ENSDLAG00005007444 meis2a Meis homeobox 2a ENSDLAG00005007335 msmo1 methylsterol monooxygenase 1 ENSDLAG00005023171 ncor2 nuclear receptor corepressor 2 ENSDLAG00005024501 neurl1aa neuralized E3 ubiquitin protein ligase 1Aa ENSDLAG00005018019 nfixb nuclear factor I X ENSDLAG00005016844 pacs2 phosphofurin acidic cluster sorting protein 2 ENSDLAG00005000298 parvb parvin, beta ENSDLAG00005021030 phactr3b phosphatase and actin regulator 3b ENSDLAG00005012177 prex1 phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 ENSDLAG00005024474 rab3il1 RAB3A interacting protein (rabin3)-like 1 ENSDLAG00005012050 runx3 RUNX family transcription factor 3 ENSDLAG00005000657 sh3pxd2aa SH3 and PX domains 2Aa ENSDLAG00005018046 si:ch211–243o19.4 si:ch211–243o19.4 ENSDLAG00005011826 smoc1 SPARC related modular calcium binding 1 ENSDLAG00005010838 sorcs2 sortilin-related VPS10 domain containing receptor 2 ENSDLAG00005020908 tbc1d22a TBC1 domain family, member 22a ENSDLAG00005010430 tgfbr2b transforming growth factor beta receptor 2b ENSDLAG00005010792 ZNF423 zinc finger protein 423 ENSDLAG00005008914 ENSDLAG00005005197 ENSDLAG00005012304 Genes 2022,13, 987 9 of 20 3.2. Early Domestication in European Sea Bass and Neurodevelopmental Cognitive Disorders Affect Paralogue Genes Genes exhibiting DNA methylation changes in patients with neurodevelopmental cognitive disorders with traits parallel to the domestication syndrome such as SZ, WS and ASD were obtained from the literature. A total of 532 genes with DM were orthologues to SZ patients, 506 genes with DM in WS patients and 367 genes with DM in ASD patients. These gene lists of orthologues were compared to the genes of FED to evaluate whether DNA methylation in common genes was affected by these conditions. The pairwise overlaps were not significant in all cases, with 28 genes overlapping in SZ (odds ratio = 1.04, p= 0.439; Figure S2a), 31 overlapping in WS (odds ratio = 1.233, p= 0.88; Figure S2b) and 23 genes overlapping in ASD (odds ratio = 1.262, p= 0.169; Figure S2c). Permutation testing for the pairwise comparisons showed that the number of overlaps was within the range expected by chance in the case of SZ (z-score = − 0.59, p= 0.216; Figure S2d) and ASD (z-score = 2.61, p= 0.062; Figure S2f), and only marginally significant in the case of WS (z-score = 3.75, p= 0.049; Figure S2e). In an attempt to overcome the constraints of the conservative approach applied here for orthologues and since key candidate genes of domestication were present in all pairwise comparisons, e.g., protocadherins, ADAM metallopeptidases, collagens and glutamate receptors, we then focused on comparisons of functional properties. Orthologue genes were submitted for enrichment analyses and pairwise comparisons were performed at the pathway level following the reasoning that similar processes may be affected by different genes. We considered four libraries targeted by Enrichr as the most informative in our case: Bioplanet, WikiPathways, GO-terms Biological Process, and MGI Mammalian Phenotype. Terms in all four libraries were examined for enrichment according to the gene lists we provided (FED, SZ, WS, and ASD) and pairwise comparisons of terms were performed as follows: (1) FED vs. SZ, (2) FED vs. WS, and (3) FED vs. ASD (Figure S3). In 42% of the comparisons, there was no overlap of terms, while in three cases there were between 1 and 4 terms overlapping. The overlaps of terms were significant only in cases of SZ for WikiPathways (odds ratio = 4.477, p= 0.003; Figure S3d) and GO Biological Process (odds ratio = 2.442, p= 0.002; Figure S3g). WikiPathways included endochondral ossification with skeletal dysplasia (WP4808) and endochondral ossification (WP474) like in the enrichment of orthologue genes overlapping in AMH, but also neural crest differentiation (WP2064). GO Biological Process enriched included development of the renal system (GO:0072001), kidney (GO:0001822), or ureteric bud (GO:0001657), as well as regulation of immune cells such as T-helper 17 and alpha-beta T (GO:2000317, GO:0046639, or GO:2000320). Taken together these results indicate that further comparative analyses could reveal more additional similarities. To investigate the role of gene families, we compared gene lists containing not only the orthologues but also the paralogues of genes. The FED gene list was maintained in the original format and served as the control in the pairwise comparisons completed as above. For the other three gene lists (SZ, WS, and ASD), paralogues in the human genome were obtained by Biomart, merged with the original genes and then orthologues in the European sea bass genome were identified, resulting in lists containing unique homologues (orthologues and paralogues). The gene lists contained 4000 homologues for SZ, 3460 homologues for WS, and 2994 homologues for ASD. Overlap between all pairwise comparisons was significant with 241 genes common in SZ (odds ratio = 1.258, p= 0.001; Figure 3a, Dataset S1), 236 in WS (odds ratio = 1.470, p= 4.422 × 10 −7 ; Figure 3b, Dataset S2), and 178 overlapping in ASD (odds ratio = 1.222, p= 0.011; Figure 3c, Dataset S3). Since these gene lists contain ~8 times more genes than previously, the significance of the overlaps could be attributed to larger numbers. To test whether the number of overlaps could be expected by chance due to large number of genes, we performed Monte Carlo permutations using random sampling of genes from the whole genome as previously. We found that overlaps between gene lists were higher than expected by chance in all cases, including SZ (z-score = 49.03, p= 0; Figure 3d), WS (z-score = 33.01, p= 0; Figure 3e), and ASD Genes 2022,13, 987 16 of 20 have been associated with the domestication phenotype, including HSD. These findings contribute to our understanding of the initial molecular changes happening during early (self-)domestication and pave the way for future studies using fish as models to investigate epigenetic changes as drivers of HSD, but also as etiological factors of human-specific cognitive diseases. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13060987/s1, Figure S1: Conceptual design of the study; Figure S2: Overlap of orthologue genes differentially methylated in fish early domesticates (FED) and cognitive disorders; Figure S3: Overlap of pathways enriched associated with orthologue genes differentially methylated in fish early domesticates (FED) and cognitive disorders; Table S1: Enrichment of Gene Ontology (GO) Biological Process (BP) terms associated with genes shared between early fish domesticates and anatomically modern humans; Table S2: Enrichment of Mammalian Phenotype (2014) terms associated with genes shared between early fish domesticates and anatomically modern humans; Table S3: Enrichment of Wikipathways associated with genes shared between early fish domesticates and anatomically modern humans; Table S4: Enrichment of Wikipathways associated with genes shared between early fish domesticates and human groups with schizophrenia; Table S5: Enrichment of Wikipathways associated with genes shared between early fish domesticates and human groups with Williams syndrome; Table S6: Enrichment of Wikipathways associated with genes shared between early fish domesticates and human groups with autism spectrum disorders; Table S7: Enrichment of Gene Ontology (GO) Biological Process (BP) terms associated with genes shared between early fish domesticates and human groups with schizophrenia; Table S8: Enrichment of Gene Ontology (GO) Biological Process (BP) terms associated with genes shared between early fish domesticates and human groups with Williams syndrome; Table S9: Enrichment of Gene Ontology (GO) Biological Process (BP) terms associated with genes shared between early fish domesticates and human groups with autism spectrum disorders; Dataset S1: homologue genes with epigenetic changes in human groups with schrizophrenia; Dataset S2: homologue genes with epigenetic changes in human groups with Williams syndrome; Dataset S3: homologue genes with epigenetic changes in human groups with autism spectrum disorders. Author Contributions: Conceptualization, D.A., F.P., M.W., A.B.B.; methodology, D.A.; formal analysis, D.A.; investigation, D.A.; data curation, D.A.; writing—original draft preparation, D.A.; writing—review and editing, F.P., M.W., A.B.B.; visualization, D.A.; supervision, F.P., M.W., A.B.B. All authors have read and agreed to the published version of the manuscript. Funding: D.A. was supported by a Marsden grant Te P ¯ utea Rangahau managed by the Royal Society of New Zealand Te Ap ¯ arangi to M.W. (MFP-PAF-1801). This research was supported in part by grant PID2020-114516GB-I00 funded by MCIN/AEI/ 10.13039/501100011033 to A.B.B., and AEI grant PID2019-108888RB-I00 to F.P. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data used in this study have been previously published and the details are included in the Materials and Methods section. 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