domain: shs sdv

text:
Thomas Cucchi

H Harbers

D Neaux

Marie Balasse

L Garbé

D Fiorillo

H Bocherens

D Drucker

Clément Zanolli

R Cornette

of morphological diversification in Western Europe wild boars (Sus scrofa) and the consequences of the Neolithic transition

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Over the last few centuries, the human niche construction [START_REF] Odling-Smee | Niche construction: the neglected process in evolution[END_REF] has become "the world's greatest evolutionary force" [START_REF] Palumbi | Humans as the world's greatest evolutionary force[END_REF], generating a change in the ecological and evolutionary dynamics of species on a global scale [START_REF] Hendry | Human influences on evolution, and the ecological and societal consequences[END_REF]. The human engineering of ecosystems with habitat fragmentation [START_REF] Cheptou | Adaptation to fragmentation: evolutionary dynamics driven by human influences[END_REF], urbanisation [START_REF] Alberti | Global urban signatures of phenotypic change in animal and plant populations[END_REF][START_REF] Mcdonnell | Adaptation and Adaptedness of Organisms to Urban Environments[END_REF] and the disruption of natural dynamics [START_REF] Colautti | Invasions and extinctions through the looking glass of evolutionary ecology[END_REF] has induced rapid microevolutionary changes through strong directional selection, resulting in rapid diversification [START_REF] Hendry | Human influences on evolution, and the ecological and societal consequences[END_REF][START_REF] Hendry | Human influences on rates of phenotypic change in wild animal populations[END_REF][START_REF] Hendry | The speed of ecological speciation[END_REF]. With the emergence of agriculture and, more generally speaking, the Neolithic transition, the human niche construction caused a major ecological disturbance, with the opening and fragmentation of the landscape, the emergence and spread of domesticated animals and plants from several independent centres across Eurasia, the protection of livestock from predators and competitors, and the dispersal of invasive species and pathogens [START_REF] Ellis | People have shaped most of terrestrial nature for at least 12,000 years[END_REF][START_REF] Sullivan | Human behaviour as a long-term ecological driver of nonhuman evolution[END_REF]. To date, the influence of the Neolithic niche construction has been mainly explored through human-induced species distribution [START_REF] Boivin | Ecological consequences of human niche construction: Examining long-term anthropogenic shaping of global species distributions[END_REF][START_REF] Cucchi | Tracking the Near Eastern origins and European dispersal of the western house mouse[END_REF][START_REF] Van Der Merwe | The large «true» Mediterranean Island as a model for the Holocene human impact on the European vertebrate fauna? Recent data and new reflections. The Holocene history of the European vertebrate fauna. Modern aspects of research[END_REF], and over the temporal depth of the direct human influence over species during the process of animal domestication [START_REF] Ameen | Specialized sledge dogs accompanied Inuit dispersal across the North American Arctic[END_REF][START_REF] Brassard | Unexpected morphological diversity in ancient dogs compared to modern relatives[END_REF][START_REF] Clutton-Brock | The process of domestication[END_REF][START_REF] Cucchi | Bones geometric morphometrics illustrate 10th millennium cal. BP domestication of autochthonous Cypriot wild boar (Sus scrofa circeus nov. ssp)[END_REF]Fages et al., 2019;[START_REF] Frantz | Animal domestication in the era of ancient genomics[END_REF][START_REF] Larson | The Evolution of Animal Domestication[END_REF][START_REF] Zeder | Core questions in domestication research[END_REF]. Yet, its consequences on non-domestic species evolution remain to be fully documented and understood [START_REF] Erlandson | Human niche construction and evolutionary theory[END_REF][START_REF] Sarrazin | Evolution in the Anthropocene[END_REF].

To contribute to this research avenue, this study explores the influence of the Neolithic transition in Western Europe on the phenotypic diversification of an indigenous species: the wild boar (Sus scrofa). Emerging in the Near East around 9500 BCE, the Neolithic dispersal reached central-western Europe by 5850 BCE through demic and cultural diffusion via the Danube River and the northern Mediterranean coasts, with the adaptation of technoeconomic strategies to different ecological conditions along the way [START_REF] Manen | The Neolithic Transition in the Western Mediterranean: a Complex and Non-Linear Diffusion Process-The Radiocarbon Record Revisited[END_REF][START_REF] Manen | The neolithisation of the Western Mediterranean: New debates about an old issue[END_REF]. Sus s. scrofa was present in North Western Europe since the Middle Pleistocene, and was evidenced in MIS 7 by 250 ka BCE [START_REF] Auguste | Évolution des peuplements mammaliens en Europe du Nord-Ouest durant le Pléistocène moyen et supérieur. Le cas de la France septentrionale[END_REF]. These animals were common and widespread from France to Belarus from the early Holocene onwards, varying in body size according to Bergman's rule, with northern individuals from north-eastern Germany being larger than southern ones from Spain and Italy (Albarella et al., 2009a). Despite the recent intensification or reintroduction and introgression with domestic pigs on the genetic make-up of wild boar populations in Europe [START_REF] Goedbloed | Reintroductions and genetic introgression from domestic pigs have shaped the genetic population structure of Northwest European wild boar[END_REF], their deep genetic structure across Europe remains mainly determined by the post glacial dispersal from southern refuges located in Spain, Italy for Western Europe, and the Balkans for Central Europe [START_REF] De Jong | Spatial genetic structure of European wild boar, with inferences on late-Pleistocene and Holocene demographic history[END_REF][START_REF] Niedziałkowska | Clear phylogeographic pattern and genetic structure of wild boar Sus scrofa population in Central and Eastern Europe[END_REF], with a loss of genetic diversity towards the northern range of the species [START_REF] Vilaça | Mitochondrial phylogeography of the European wild boar: the effect of climate on genetic diversity and spatial lineage sorting across Europe[END_REF].

The earliest anthropogenic influence over the genetic diversity of European Sus scrofa has been documented by paleogenetics with the introduction of domestic pigs of Near East ancestry by Neolithic stock-herders along the two dispersal waves mentioned previously [START_REF] Larson | Ancient DNA, pig domestication, and the spread of the Neolithic into Europe[END_REF]. These pigs stemmed from the long domestication process of Near Eastern Sus scrofa populations between 9500 and 8000 BCE [START_REF] Price | The Archaeology of Pig Domestication in Eurasia[END_REF], from intensive hunting to first evidence of body size reduction compared to their wild relatives by 8000 BCE [START_REF] Price | Long-term morphological changes and evolving human-pig relations in the northern Fertile Crescent from 11,000 to 2000 cal. bc[END_REF]. These small pigs, entered Europe by 6500 BCE, arriving in Central Western Europe around 5400 BCE [START_REF] Evin | Unravelling the complexity of domestication: a case study using morphometrics and ancient DNA analyses of archaeological pigs from Romania[END_REF][START_REF] Larson | Ancient DNA, pig domestication, and the spread of the Neolithic into Europe[END_REF][START_REF] Ottoni | Pig Domestication and Human-Mediated Dispersal in Western Eurasia Revealed through Ancient DNA and Geometric Morphometrics[END_REF]. By 5000 BCE, the Near Eastern lineage no longer existed in the European pig genome, which, after this date, had 96% of its genetic variants coming from European wild boars. The extent of this genomic replacement is considered to have stemmed from introgressive hybridization facilitated by the small population size of Near Eastern pigs and a lack of barrier between them and the local wild boar populations [START_REF] Ameen | Specialized sledge dogs accompanied Inuit dispersal across the North American Arctic[END_REF].

The introduction of domestic pigs into Europe by successive waves of newcomers from the Near East induced the co-occurence of several lineages of Sus scrofa during part of the Neolithic period : the indigenous wild boar populations in their natural habitat, the domestic pig populations of Near Eastern origin reared by Neolithic people and their feralized populations.

Indeed, thanks to paleogenetics, we know that hybridization and gene flow between wild, domestic and feral Sus scrofa populations, occurred all along the Neolithic [START_REF] Ameen | Specialized sledge dogs accompanied Inuit dispersal across the North American Arctic[END_REF].

This gene flow can be highlighted by traditional pig husbandry still undertaken in Papua New Guinea, where interbreeding is part of the rearing practices, with sows being left to fray with wild male boars [START_REF] Dwyer | Boars, barrows, and breeders: The reproductive status of domestic pig populations in mainland New Guinea[END_REF], or in Mediterranean Europe [START_REF] Albarella | A week on the plateau: Pig husbandry, mobility and resource exploitation in central Sardinia[END_REF][START_REF] Hadjikoumis | Traditional pig herding practices in southwest Iberia: Questions of scale and zooarchaeological implications[END_REF][START_REF] Halstead | A pig fed by hand is worth two in the bush: Ethnoarchaeology of pig husbandry in Greece and its archaeological implications[END_REF]. This does not exclude likely presence of commensal wild boars for which there is no genetic evidence. This co-habitation of several lineages of Sus scrofa produced new hybrid communities, with different ranges of interactions between suids and humans societies in Neolithic Europe [START_REF] Albarella | A week on the plateau: Pig husbandry, mobility and resource exploitation in central Sardinia[END_REF][START_REF] Balasse | Wild game or farm animal? Tracking human-pig relationships in ancient times through stable isotope analysis[END_REF], where boundaries between wild and domestic populations were not as tight as those currently engraved in the industrial and zootechnical views on animal domestication [START_REF] Bogaard | Reconsidering domestication from a process archaeology perspective[END_REF]Stépanoff and Vigne, 2018).

Considering the new ecological and evolutionary forces introduced into Europe by the Neolithic transition, we wanted to explore the extent of its influence on the morphological diversification of Sus s. scrofa in Western Europe, over 4500 years or archaeological record. The remains are dated from the Middle Mesolithic, by 7500 BCE, i.e. before the spread of farmers and their domestic pigs, until the Late Neolithic 3000 BCE, when the new social and economic organization of the Bronze Age emerged. During this timeframe, we attempt to (1) understand whether the Neolithic transition influenced the phenotypic evolution of the autochtonous wild boars, (2) define the phenotypic diversity of the several lineages of Sus scrofa present in Europe at that period and (3) determine to what extent Neolithic societies of Europe have intentionally altered the phenotypic traits of domestic Sus scrofa. To track these intraspecific changes in bone morphology, we had to comprehensively capture enough organismal complexity using high resolution phenotypic data with three dimensional coordinate points analyzed via geometric morphometrics [START_REF] Bardua | A practical guide to sliding and surface semilandmarks in morphometric analyses[END_REF][START_REF] Goswami | High-Density Morphometric Analysis of Shape and Integration: The Good, the Bad, and the Not-Really-a-Problem[END_REF] and morphometric mapping (Bondioli et al., 2010a;[START_REF] Morimoto | Exploring femoral diaphyseal shape variation in wild and captive chimpanzees by means of morphometric mapping: a test of Wolff's law[END_REF]. To track the morphological evolution across the archaeological record, we relied on three skeleton bones (calcaneus, humerus and mandible) chosen for their good preservation in the archaeological record, following an experimental research program on the effects of captivity over skeletal development in European wild boars (http://anr-domexp.cnrs.fr/). This experimental research showed that functional changes due to mobility reduction and changes due to directional selection induced different morphological trajectories from the wild norm of variation, in both the calcaneus form (Harbers et al., 2020a) and the humerus shaft microanatomy (Harbers et al., 2020b). Likewise, the change in feeding behaviour with captivity induced functional responses in the skull and mandible, different from morphological changes induced by the last 200 years of selective breeding [START_REF] Neaux | The effect of captivity on craniomandibular and calcaneal ontogenetic trajectories in wild boar[END_REF][START_REF] Neaux | Constraints associated with captivity alter craniomandibular integration in wild boar[END_REF][START_REF] Neaux | How Changes in Functional Demands Associated with Captivity Affect the Skull Shape of a Wild Boar (Sus scrofa)[END_REF]. All this evidence supports the use of these phenotypic markers to disentangle, functional changes due to a cultural control of the individual's environment [START_REF] Hecker | Domestication revisited: its implications for faunal analysis[END_REF], from selective breeding at a population scale, during the Neolithic transition. Such approach has already proven useful to identify the local domestication of the feral populations of wild boars by PPNB Neolithic people of Cyprus [START_REF] Cucchi | Bones geometric morphometrics illustrate 10th millennium cal. BP domestication of autochthonous Cypriot wild boar (Sus scrofa circeus nov. ssp)[END_REF].

For this study, Sus scrofa from Mesolithic contexts will provide the baseline (T0) for the variation norm of Sus scrofa prior to the Neolithic transition in Europe. The Sus scrofa bones from Neolithic contexts were not divided beforehand into a wild or domestic category using a bone size criterion, as is usually done in zooarchaeological and paleogenetic studies, for three main reasons. First, as previously said, the wild and domestic boundaries might not be a relevant concept to approach these interactions during the Neolithic. Then, these preliminary categories based on bone size differences are too subjective and might introduce interpretation biases.

Third, size changes are not easily related to any clear biotic or abiotic factors. We have therefore chosen to assess the extent of Neolithic anthropogenic influence over Sus scrofa morphological diversity by confronting the archaeological specimen to a gradient of phenotypic variation representing (1) the reaction norms of Sus scrofa populations (Mesolithic and current) living in their natural habitat or (2) in contexts of controlled locomotion and feeding behaviours and (3) the phenotypic divergence induced by selective breeding over the last centuries. The relatedness of the Neolithic specimens to this anthropogenic gradient of phenotypic diversification in Sus scrofa was assessed using predictive and machine learning approaches. We also investigated the relationship between Neolithic suids and humans using stable carbon (δ 13 C) and nitrogen (δ 15 N) isotope ratios in bone collagen, as a greater proximity to humans may have induced changes in the feeding environment and diet of suids [START_REF] Balasse | Wild game or farm animal? Tracking human-pig relationships in ancient times through stable isotope analysis[END_REF][START_REF] Balasse | Wild, domestic and feral? Investigating the status of suids in the Romanian Gumelniţa (5th mil. cal BC) with biogeochemistry and geometric morphometrics[END_REF][START_REF] Brusgaard | Hunting before herding: A zooarchaeological and stable isotopic study of suids (Sus sp.) at Hardinxveld-Giessendam, the Netherlands (5450-4250 cal BC)[END_REF][START_REF] Cucchi | Social Complexification and Pig ( Sus scrofa ) Husbandry in Ancient China: A Combined Geometric Morphometric and Isotopic Approach[END_REF][START_REF] Maring | Possible wild boar management during the Ertebølle period. A carbon and nitrogen isotope analysis of Mesolithic wild boar from Fannerup F, Denmark[END_REF].

MATERIAL

From 101 archaeological Sus scrofa bones collected, we could analyze 73: calcanei (41), humeri (19) and mandibles (13) from adults and sub-adults (Supplementary Table S1). We analysed calcanea with epiphysis known to fuse around 2 years in wild boars [START_REF] Bridault | Wild boar-age at death estimates: the relevance of new modern data for archaeological skeletal material. 1[END_REF]. The humeri all had a complete distal shaft with a fused distal epiphysis, occurring in wild boar by 19-23 months [START_REF] Bull | Tooth eruption and epiphysial fusion in pigs and wild boar[END_REF]. The mandibles belonged to specimens between 12 and 60 months old, according to both the tooth eruption [START_REF] Legge | Practice with science: Molar tooth eruption ages in domestic, feral and wild pigs (Sus scrofa)[END_REF] and wear stages (Horard-Herbin, 1997) of the premolars and molars. These bones were collected from 13 archaeological sites in France, Switzerland and Germany (Figure 1). Within the Sus scrofa specimens, 28 came from five hunter-gatherer contexts dated from the Middle and Late Mesolithic between 8700 and 5000 BCE, and 45 specimens from eight Neolithic settlements between 5500 and 3000 BCE (Supplementary Table S1 andText S2). The latter includes 30 specimens of Sus scrofa (25 calcanei and 5 humeri) from the Neolithic site of Chalain, on which stable isotope analysis was performed and compared with the stable isotope analyses on large wild and domestic herbivores found at the same site, including 14 deer (Cervus elaphus), 5 aurochs (Bos primigenius) and 9 cattle (Bos taurus) (Supplementary Table S2). All Mesolithic specimens were considered as hunted wild boars, representative of the phenotypic variation of wild boars before the Neolithic transition. To assess the amount of anthropogenic diversification in Neolithic Sus scrofa, we used a comparative approach from a collection of Sus scrofa covering the phenotypic continuum from wild boars hunted in their natural habitat to highly selected domestic breeds, whose environment and reproduction was fully controlled by humans (SI3). The hunted wild boars included a sample of 28 Mesolithic specimens from Western Europe contexts (Figure 1) and a sample of 32 specimens from current populations hunted in France and Switzerland. We have also included 24 captive wild boars from an experiment undertaken at a zoological reserve of the Natural History Museum of Paris (see details of the experiment in (Harbers et al., 2020a)).

They were collected from a control population of wild boars (sampled by 8 specimens) and raised in captivity from the age of 6 months to 2 years. These 24 specimens were randomly split into two groups with the same male to female sex ratio. One group was raised in a 3000 m 2 pen and the second in a 100 m 2 stall where males and females were separated. Modern pig samples included 23 specimens from traditional European landraces including 5 free-ranging Corsican landrace pigs and 9 specimens of industrial breeds. A sample of five hybrids (crossed between wild boars and pigs of different breeds) is also included. The topography of the shaft cortical thickness is rendered by a standardized morphometric map with chromatic scale from thinnest (dark blue) to thickest (red). Red areas represent area of muscular insertions (see Harbers et al. 2020b for further methodological details).

METHODS

Geometric Morphometrics

The morphometric study associate the 3D surface semilandmarks geometric morphometrics (3D GM) of the calcaneus and mandible [START_REF] Bardua | A practical guide to sliding and surface semilandmarks in morphometric analyses[END_REF][START_REF] Mitteroecker | A brief review of shape, form, and allometry in geometric morphometrics, with applications to human facial morphology[END_REF] and the 3D morphometric mapping of the humerus shaft cortical thickness (Bondioli et al., 2010a(Bondioli et al., , 2010b)). The 3D models of the calcaneus were reconstructed from medical CT scans and photogrammetry after segmentation in Avizo v.8.0 or reconstruction using Agisoft Photoscan 1.2.6.2834. Mandible 3D models were reconstructed from medical CT and laser scanning using an ARTEC Space Spider. All three acquisitions methods were compared using a common 3D GM framework without statistical biases [START_REF] Waltenberger | Three-dimensional surface scanning methods in osteology: A topographical and geometric morphometric comparison[END_REF]. The humeri were scanned using a medical CT and reconstructed to generate the external (periosteum) and internal (endosteum) 3D surfaces of the bone used to compute cortical thickness.

3D GM analyses were performed on 3D models using landmark and semilandmark protocols on curves and surfaces previously detailed for the calcaneus (Harbers et al., 2020a) and the mandible [START_REF] Neaux | How Changes in Functional Demands Associated with Captivity Affect the Skull Shape of a Wild Boar (Sus scrofa)[END_REF]. For the mandibles, the protocol was adapted to the symphysis and ramus, which are the most common anatomical parts of the mandibles in the archaeological record. The acquisition and standardisation protocol for 3D morphometric mapping of the humeral shaft cortical thickness follows (Harbers et al., 2020b). We adjusted the protocol to ensure its applicability to archaeological humeri missing the proximal end of the diaphysis by mapping only the distal half of the shaft (3-50%). Despite this reduction, all the entheses distinguishing free-ranging wild boars, captive wild boars and pigs are observable (Figure 1, Harbers et al., 2020b). We used the cross-sectional area (CSA) at the distal end of the diaphysis (Figure 1c) as a proxy of the humerus size. The Pearson correlation coefficient showed that this variable was almost perfectly correlated (ρ = 0.965) with the bone volume. Since the latter is also strongly correlated to body mass [START_REF] Campione | The accuracy and precision of body mass estimation in non-avian dinosaurs[END_REF]Harbers et al., 2020b), we used the cross-sectional area as a body mass proxy of archaeological Sus scrofa. Size variation differences between the nine subgroups of wild boars and domestic pigs, in the humeral CSA and calcaneus and mandible centroid sizes, were tested using a factorial ANOVA and visualised with boxplots. To assess the structuration of size variation and identify the occurrence of several populations within Neolithic Sus scrofa, we performed model-based Gaussian mixture analyses using density estimation on size variables.

The differences in humeral cortical thickness topography and calcaneus shape variation between the nine subgroups of wild boars and domestic pigs were tested with factorial MANOVAs. Patterns of morphological differentiation (in a morphospace) among wild boars and pig samples were visualised using canonical variate analyses (CVA). CVA morphospaces were performed after principal component analysis (PCA) computed on Procrustes coordinates in Mesolithic and modern Sus scrofa, retaining 95% of variance for data dimensionality reduction to prevent artificial group discrimination [START_REF] Mitteroecker | Linear discrimination, ordination, and the visualization of selection gradients in modern morphometrics[END_REF].

Neolithic Sus scrofa are projected into this canonical morphospace, using a generic function (predict) which will get fitted values according to the multiple linear model computed for the Mesolithic and modern data. To assess the interaction mode (hunting, control or reproductive isolation) with human Neolithic societies for each Neolithic Sus scrofa, we used a k-nearest neighbor (kNN) classification in the two first canonical axes of the morphospace via a training model of three groups representing each of these interaction modes: (1) modern and Mesolithic wild-caught wild boars, (2) modern captive wild boars, and (3) modern pigs including industrial breeds and landraces. The value of "k" was chosen as the square root of the number of specimen's "n" in the comparative material following [START_REF] Lall | A Nearest Neighbor Bootstrap For Resampling Hydrologic Time Series[END_REF] ; therefore, k = 9 for the humerus dataset (n = 86), k = 10 for the calcaneus dataset (n = 95) and k = 7 for the mandible dataset (n = 43). To check for the reliability of the kNN classification we assessed its congruence with the prediction obtained by fitting their values with the multiple linear discriminant model (CVA) performed for the three groups described previously.

To visualise calcaneus and mandible shape deformations along the canonical axes, we calculated the theoretical minimum and maximum shapes for each axis, then associated them, using a heatmap, on the landmarks corresponding to the distance between the minimum and maximum values along the first two canonical axes. To visualise variations in humerus cortical thickness topography along the canonical axes, we calculated the theoretical minimum and maximum morphometric map for each axis.

All the statistics were performed using R (R Core Team, 2017). ANOVA and PCA were performed using the R package "stat" (R Core Team, 2017). Model based Gaussian mixture analyses of size univariates were performed with "Mclust 5" [START_REF] Scrucca | mclust 5: clustering, classification and density estimation using Gaussian finite mixture models[END_REF]. LDA prediction using the package "MASS" (Venables et al., 2002) and visualisations were performed using "geomorph" (Adams and Otárola-Castillo, 2013) and "rgl" (Adler and Murdoch, 2017).

The kNN algorithm was performed using the package 'class' [START_REF] Torgo | Data mining with R: learning with case studies[END_REF].

Stable Isotope study

A piece of bone was cut from the diaphysis of the Sus scrofa calcaneus or humerus. Bone surfaces were cleaned by abrasion and reduced to powder. A fraction comprised between 355 and 710 µm was used for collagen extraction. The extraction used approximately 150 to 250 mg of powder and followed the procedure described in [START_REF] Bocherens | Isotopic biogeochemistry (13C,15N) of fossil vertebrate collagen: application to the study of a past food web including Neandertal man[END_REF]. Coupled measurements of δ 15 N and δ 13 C were conducted on 300 to 400 μg of collagen on an elemental analyser Thermo Flash 2000 interfaced with a Thermo DeltaVAdvantage IRMS. The analytical precision, estimated from the repeated analysis (N = 8 to 12 within each run) of our laboratory standard (alanine normalised to the IAEA-600 caffeine international standard), was 0.1‰ for δ 13 C and δ 15 N values. Over the course of these analyses, the alanine standard gave mean values of 0.58‰ for δ 15 N (expected value: 0.60‰), -22.08‰ for δ 13 C (expected value: -22.17‰), 40,4 % for C content (expected value: 40.4%) and 15.6% for N content (expected value: 15.7%).

The aurochs, deer and cattle bone collagen were extracted within the framework of a previous study, following the same procedure. The δ 13 C were published [START_REF] Drucker | Carbon stable isotopes of mammal bones as tracers of canopy development and habitat use in temperate and boreal contexts[END_REF] but the δ 15 N values remain unpublished.

RESULTS

Bone size variation in modern and ancient Sus scrofa

All four phenotypic markers displayed a common pattern of size variation (Figure 2), with overall significant differences among the samples of wild, captive and domestic Sus scrofa for the humerus cross section (ANOVA= F:10.81, p<0.0001), the centroid sizes of calcaneus (ANOVA= F:9.302, p<0.0001), the mandible symphysis (ANOVA= F:2.635, p=0.0463) and the ramus (ANOVA= F:3.428, p=0.0251). All four markers displayed (1) an important size decrease between Mesolithic and extant wild boar populations of Western Europe (-16% for the calcaneus) and (2) an increase in bone size (+15 % for the calcaneus) in traditional and industrial pig breeds compared to modern hunted or captive wild boars, or even Mesolithic wild boars.

Captivity in wild boars does not induce any significant size changes in calcaneum and mandible but it does show an increase in the CSA of the humerus compared to free ranging wild boars (Figure 2), suggesting that captivity induce an increase in wild boar body mass but not of their body size.

Size variation in Neolithic

External and structural bone variation in modern and ancient Sus scrofa

The variation in bone shape and humeral cortical thickness in Mesolithic and modern wild and domestic Sus scrofa displayed congruent patterns of diversification in the CVA morphospace, albeit with slight discrepancies (Figure 3). This variation is mainly influenced by human-driven selection between modern wild boars and modern pig breeds (CV1), while a plastic response to the human-driven control of mobility in wild boars is aligned with the second canonical axis (CV2), showing the prevalence of artificial selection over anthropogenic behavioural control in the phenotypic diversification of this species. It is worth noting that hybrids between wild boars and pigs (which were only available for the calcaneus and humerus) show a different diversification pattern: closer to hunted wild boars for the calcaneal shape variation (Figure 3a) and closer to landrace and industrial pig breeds for the topography humeral shaft cortical thickness, showing the greater influence of heredity in the former (Figure 3b). Finally, one can also note that Mesolithic and extant wild boars display similar humeral cortical topography (Figure 3b) but clear phenotypic divergence in their calcaneal shape variation (Figure 3a). For the mandibular-ramus (Figure 3c) and symphysis (Figure 3d), the divergence due to selection and plasticity is found along both the CV1 and CV2, but also resembles the pattern observed for post-cranial bones (even though the morphospace is rotated).

When projecting the variation of Neolithic Sus scrofa over the pattern of phenotypic diversification described previously, we observed that it was centred, albeit greater, within the phenotypic variation of Mesolithic and modern wild boars in their natural habitat; however, patterns differed according to the markers considered. Variability in humeral cortical topography stretches towards the directional selection trajectory of pig breeds and overlaps with the variation of landraces pigs (Figure 3b). On the other hand, the calcaneus shape variation spreads towards captive wild boars without overlapping (Figure 3a), the mandible-ramus displays a variation intermediate between hunted and captive wild boars (Figure 3c), while the symphysis reaches the range of captive wild boars (Figure 3d).

Neolithisation and phenotypic diversification in Sus scrofa

CVA prediction and kNN Classification of the Neolithic Sus scrofa according to the hunted, captive or domestic phenotypic comparative models computed separately for each morphospace (Figure 4a, SI4) shows that 92% of the Neolithic Sus scrofa have a phenotypic variation aligned with the variability of wild boar living in a natural habitat, suggesting that they behaved like wild boar, in an environment without human ecological disturbance, apart from harvesting. The projection of these morphometric inferences along the timeline from Mesolithic to Late Neolithic (Figure 4c) shows that by 5200 BCE, in Early Neolithic contexts of Southern France, and by 4000 BCE in Middle Neolithic France (Bercy) and Germany (Vaihingen), a new morphotype of small Sus scrofa can be observed. This small morphotype is on average 27% smaller than the large morphotype present since the Mesolithic and up to 29% smaller when compared with the large Neolithic Sus scrofa populations. Moreover, between the Mesolithic and the Late Neolithic, the large hunted Sus scrofa displays a 4% size increase.

CVA predictions and kNN algorithm identify only four Neolithic Sus scrofa with the phenotypic signature, for the humerus and mandible, of adult wild boars raised in captivity (Figure 4a). These captive specimens are observed around 5200 BCE in Early Neolithic contexts of Fontbrégoua, circa 4750 BCE at Gazel (context of transition between Early and Middle Neolithic) and later, by 4000 BCE, in the Middle Neolithic contexts of Bercy in Northern France.

Finally, the first phenotype that could be considered under anthropogenic reproductive selection was observed in the middle Neolithic site of Noyen-sur-Seine around 3800 BCE and in the late Neolithic site of Chalain around 3000 BCE. These five domesticated phenotypes were only detected via the morphometric mapping of the humeral cortical thickness, in both small and large morphotypes (Figure 4a).

Results from stable isotope analysis at Chalain

The collagen extraction yield averaged 136 mg/g (24-199 mg/g) and the collagen carbon and nitrogen contents averaged 42.3% (39.5-44.2%) and 15.2% (14.0-16.2%). The C:N atomic ratios varied from 3.24 to 3.31 in Sus scrofa and 3.17 to 3,25 in the remaining fauna (SI 2).

These criteria testify to the high preservation of bone collagen at this site and support the suitability of all stable isotope data for interpretation.

The δ 13 C values measured in Sus scrofa vary between -22.3‰ and -19.6‰ (Figure 4b). These values are higher than those measured in aurochs (-24.6‰ to -23.6‰) and red deer (-24.3‰ to -22.6‰) and partly overlap with those measured in domestic cattle (-23.5‰ to -22.0‰) at the same site (Figure 4). In Sus scrofa, the δ 15 N values vary from 4.2‰ to 8.0‰ and are on average higher (6.4‰,) than those measured in aurochs, red deer and cattle when considered together (mean = 5.8‰, from 4.9‰ to 6.8‰) (Supplementary Table S2 and Figure 4). The δ13C and δ15N values of the four suids with a "wild caught" phenotype and a small size do not differ from the large size wild caught Sus. The three suids classified in the "domestic pig" phenotypic category have intermediate δ 13 C values (-21.3‰ to -21.1‰), two of which have the lowest δ 15 N values of this dataset for Sus scrofa (4.2‰ and 4.4‰).

DISCUSSION

Using wild boars from the Mesolithic, as with the T0 for Sus Scrofa evolution during the Holocene, we first observed that current wild populations of Sus scrofa in the area under study have lost 16% of their body size compared to wild populations living in Western Europe some 2500 years before the Neolithic transition. This supports our current understanding of a global body mass reduction of large mammals by the end of the Pleistocene [START_REF] Smith | Body size downgrading of mammals over the late Quaternary[END_REF].

Considering that body size in wild boars follows Bergman's rule (Albarella et al., 2009b), it has been argued that their body size reduction over the last 10 000 years was mainly due to adaptations to the global temperature increase during the Holocene [52,53]. However, human disturbance as the driver of the reduction in the body size of large mammals since the Late Pleistocene is also proposed [START_REF] Pelletier | Will human influences on evolutionary dynamics in the wild pervade the Anthropocene?[END_REF][START_REF] Pigeon | Fluctuating effects of genetic and plastic changes in body mass on population dynamics in a large herbivore[END_REF][START_REF] Smith | Body size downgrading of mammals over the late Quaternary[END_REF][START_REF] Sullivan | Human behaviour as a long-term ecological driver of nonhuman evolution[END_REF], with the unbalanced human harvesting of large prey as a key driver for body size reduction in large mammals [START_REF] Darimont | The unique ecology of human predators[END_REF][START_REF] Darimont | Human predators outpace other agents of trait change in the wild[END_REF]. Interestingly, we found a slight increase of wild boar body size during the Neolithic, in agreement with what has been found for wild boar populations during the Neolithic and Bronze Age in the Iberian Peninsula [START_REF] Navarrete Belda | Size changes in wild and domestic pig populations between 10,000 and 800 cal. BC in the Iberian Peninsula: Evaluation of natural versus social impacts in animal populations during the first domestication stages[END_REF]. This suggests that the harvesting behaviour inducing size-selective harvesting of wild Sus scrofa populations occurred later, probably over the last two centuries, when harvesting pressure accelerated drastically [START_REF] Coltman | Undesirable evolutionary consequences of trophy hunting[END_REF][START_REF] Pigeon | Fluctuating effects of genetic and plastic changes in body mass on population dynamics in a large herbivore[END_REF].

With the Neolithic transition in Western Europe by the 6 th millennium BCE, we can observe the co-existence of a large and a small morphotype of Sus scrofa, with an almost 30% size difference during the whole Neolithic. The occurrence of small-sized Sus scrofa from the early Neolithic onward in Europe would be congruent with the well-documented arrival in Western Europe of small populations of Sus scrofa domesticated from a Near Eastern population [START_REF] Evin | Unravelling the complexity of domestication: a case study using morphometrics and ancient DNA analyses of archaeological pigs from Romania[END_REF][START_REF] Krause-Kyora | Use of domesticated pigs by Mesolithic hunter-gatherers in northwestern Europe[END_REF][START_REF] Ottoni | Pig Domestication and Human-Mediated Dispersal in Western Eurasia Revealed through Ancient DNA and Geometric Morphometrics[END_REF]. The factors explaining this size difference compared to endemic European wild boar have not been fully investigated yet, but they might be inherited from both the wild ancestral populations in the Near East, considered as a smaller sub-species (Sus scrofa lybicus) [START_REF] Groves | Current views on taxonomy and zoogeography of the genus Sus, in: Pigs and Humans: 10,000 Years of Interaction[END_REF], with their body size reduction being the process of domestication by 8000 BCE in this area [START_REF] Price | Long-term morphological changes and evolving human-pig relations in the northern Fertile Crescent from 11,000 to 2000 cal. bc[END_REF]. Both genetic legacies have left a strong phenotypic imprint on this population which then dispersed throughout Europe [START_REF] Evin | Unravelling the complexity of domestication: a case study using morphometrics and ancient DNA analyses of archaeological pigs from Romania[END_REF][START_REF] Ottoni | Pig Domestication and Human-Mediated Dispersal in Western Eurasia Revealed through Ancient DNA and Geometric Morphometrics[END_REF]. Despite their size difference, however, both the large and small Neolithic Sus scrofa in our dataset displayed a shape variability within the norm of reaction of wild boars behaving in their natural habitat, without any mobility control.

The wild behaviour suggested by their bone morphology is not contradicted by their feeding behaviour, assessed through the stable isotopic study of the 30 large and small Sus scrofa from the Late Neolithic site of Chalain (Figure 4b). Here, the δ 13 C values measured in aurochs and red deer reflect a forested habitat, where the canopy effect results in lower δ 13 C values in plants [START_REF] Van Der Merwe | The large «true» Mediterranean Island as a model for the Holocene human impact on the European vertebrate fauna? Recent data and new reflections. The Holocene history of the European vertebrate fauna. Modern aspects of research[END_REF] and in the herbivores feeding on them (bone collagen δ 13 C values in modern deer from deciduous forests average -23.8‰ and are mostly ≤ -22.6‰; [START_REF] Drucker | Can carbon-13 in large herbivores reflect the canopy effect in temperate and boreal ecosystems? Evidence from modern and ancient ungulates[END_REF], values corrected for the fossil fuel effect). The higher δ 13 C values measured in Sus scrofa may not be directly comparable to those measured in herbivores because their diet may have included a significant proportion of fruits [START_REF] Ballari | A review of wild boar S us scrofa diet and factors affecting food selection in native and introduced ranges[END_REF][START_REF] Schley | Diet of wild boar Sus scrofa in Western Europe, with particular reference to consumption of agricultural crops[END_REF]. Fruits have higher δ 13 C values (by 1‰) compared to leaves in understory vegetation [START_REF] Roberts | Stable carbon, oxygen, and nitrogen, isotope analysis of plants from a South Asian tropical forest: Implications for primatology[END_REF], and this difference is inherited in frugivorous species compared to folivorous species (+1-1.5‰ [START_REF] Krigbaum | Stable isotope canopy effects for sympatric monkeys at Taï Forest, Côte d'Ivoire[END_REF]. Taking this into account, the Sus scrofa found at Chalain would have occupied a wide range of habitats from forests to open environments, while their δ 15 N values suggest a low to moderate contribution of animal protein in their diet. Overall, these results are in agreement with the opportunistic omnivorous feeding behaviour and wide foraging habitat observed in modern wild boars [START_REF] Ballari | A review of wild boar S us scrofa diet and factors affecting food selection in native and introduced ranges[END_REF], as well as in Mesolithic European wild boars which were alternatively found foraging in woodlands on a very low animal protein diet (in Denmark [START_REF] Maring | Possible wild boar management during the Ertebølle period. A carbon and nitrogen isotope analysis of Mesolithic wild boar from Fannerup F, Denmark[END_REF]), in a wider range of habitats with varying degree of openness (in the Netherlands; [START_REF] Brusgaard | Hunting before herding: A zooarchaeological and stable isotopic study of suids (Sus sp.) at Hardinxveld-Giessendam, the Netherlands (5450-4250 cal BC)[END_REF] or in an open environment and with a greater animal protein component (in Belgium; [START_REF] Bocherens | Isotopic biogeochemistry (13C,15N) of fossil vertebrate collagen: application to the study of a past food web including Neandertal man[END_REF]).

Both morphometric and isotopic signatures suggest that the evolutionary interaction between both large and small Neolithic Sus scrofa populations and the Neolithic societies of Western Europe was mainly driven by harvesting (hunting). This hypothesis is supported by the recent integrated study of Late Mesolithic-Neolithic human-suid interaction in Rhine-Meuse, which found no evidence of small suid management during the Neolithic occupation [START_REF] Brusgaard | Hunting before herding: A zooarchaeological and stable isotopic study of suids (Sus sp.) at Hardinxveld-Giessendam, the Netherlands (5450-4250 cal BC)[END_REF]. However, previous studies from Eastern Europe in Late Neolithic Romania have considered small Sus scrofa as domestic livestock living in close proximity to the Neolithic settlements [START_REF] Balasse | Wild game or farm animal? Tracking human-pig relationships in ancient times through stable isotope analysis[END_REF][START_REF] Balasse | Wild, domestic and feral? Investigating the status of suids in the Romanian Gumelniţa (5th mil. cal BC) with biogeochemistry and geometric morphometrics[END_REF][START_REF] Evin | Unravelling the complexity of domestication: a case study using morphometrics and ancient DNA analyses of archaeological pigs from Romania[END_REF], suggesting that suid interactions with Neolithic societies/villagers has differed at both ends of the Neolithic dispersal in Europe.

Nevertheless, the co-occurrence of large and small wild morphotypes of Sus scrofa in Neolithic Western Europe raises several questions. Were these small Sus scrofa feral populations, stemming from the domestic Sus scrofa introduced by early Neolithic farmers, roaming free and independently from the care of Neolithic people and hunted throughout the period? If so, can we consider that events of admixture and gene flow between the small and large populations were limited in time and space, preventing the morphological homogenisation of the two lineages? Were these small Sus scrofa under the care of Neolithic people and therefore "domestic" [START_REF] Dwyer | Boars, barrows, and breeders: The reproductive status of domestic pig populations in mainland New Guinea[END_REF], albeit under an extensive management system as seen in the preindustrial diversity of pig production in New Guinea (see synthesis and references in [START_REF] Price | The Archaeology of Pig Domestication in Eurasia[END_REF]) where they are considered as livestock but behave freely in a natural habitat, with occasional proximity to the settlement? In these extensive management systems, females are captured (along with the occasional male) and penned for several months for impregnating and then left free to roam in order to be harvested in the wild for pork [START_REF] Dwyer | Boars, barrows, and breeders: The reproductive status of domestic pig populations in mainland New Guinea[END_REF]. Their short period of captive life would not have left a strong enough imprint in their bone variation for our morphometric approach to detect. Whatever the answers, the emergence of this small, wild type of Sus scrofa has produced a new phenotypic and genetic diversity in the Sus scrofa populations of Western Europe.

Beyond the body-sized diversity of Sus scrofa, fostered by the translocation of domesticated pigs of Near Eastern ancestry, few anthropogenic morphological changes beyond the phenotypic variation of Sus scrofa living freely in its natural habitat can be observed during the Neolithic niche construction in Europe. The first phenotypic change observed during the Early and Middle Neolithic is a plastic response of tarsal and skull bones similar to the variation induced by a life in captivity (Harbers et al., 2020a;[START_REF] Neaux | Constraints associated with captivity alter craniomandibular integration in wild boar[END_REF][START_REF] Neaux | How Changes in Functional Demands Associated with Captivity Affect the Skull Shape of a Wild Boar (Sus scrofa)[END_REF]. The rare occurrence of this plastic response to captivity in the Neolithic Sus scrofa suggests that captivity was a rare practice in Neolithic Europe and that wild capture management [START_REF] Dwyer | Person, place or pig: Animal attachments and human transactions in New Guinea[END_REF], as described above, would have been the main interaction at that time.

According to this management system, only a few females and occasionally castrated males were kept in captivity, which would explain why so few occurrences are observable in the archaeological record. The limitations of our study, aside from the finite number of individuals to cover such a long time period over such a large geographic area, are therefore double. First, our methodological approach relies on adult and sub-adult specimens, while husbandry practices could necessitate the culling of most males before the age of two. This means that the phenotypic variation of a large proportion of the population may not have been sampled in this study, reducing the possibility to capture phenotypic adaptations to Neolithic habitat transformations at a greater scale. Further age-free phenotypic markers, therefore, need to be developed. The second limitation is the reductionist character of a comparative approach relying on a modern referential, even with experimental conditions such as the one presented here. The complexity of the potential configuration encompassing the interaction between people and suids during the Neolithic can never be fully covered or deciphered.

Around 6000 years ago, by the Middle and especially the Late Neolithic, the 3D topography of the humeral shaft cortical thickness can pick rare but significant phenotypic changes along the selective trajectory towards modern pig breeds (Figure 3b). To understand the human intention behind these changes, one need to consider first that differences in cortical topography between hunted wild boars and current pig breeds are due to functional changes driven by selection for fast growth and muscle production, altering the gait of the animal (Harbers et al., 2020b). Then, that humeral shaft topography in wild boars is mainly influenced by body mass variation (Harbers et al., 2020b). Accordingly, the similarity in the shaft cortical topography of some Middle and Late Neolithic Sus scrofa with current pig breeds suggests that they might have reached extreme body masses leading to a functional change in their humeral shaft anatomy.

Was their diet controlled for extreme weight gain? Are we witnessing the early evidence of selection for fast body growth or greater fat production? It is impossible to answer this question yet; although, in the pre-industrial pig husbandry systems of New Guinea, there are ethnographic examples of captured wild boars fattened to the point that they cannot stand on their feet [START_REF] Dwyer | Boars, barrows, and breeders: The reproductive status of domestic pig populations in mainland New Guinea[END_REF]. The isotopic signature of Late Neolithic specimens of Chalain, where "domestic", "wild" and "captive" phenotypes are present, did not provide any evidence that these "domestic" pigs had a diet richer in animal protein compared to the others (Figure 4b). However, if these special pigs had been fattened with a grain-rich diet, we would not have been able to observe it with these isotopic proxies, unless the grains were grown on manured fields [START_REF] Price | The Archaeology of Pig Domestication in Eurasia[END_REF].

CONCLUSION

This study has provided strong confirmation that the archaeological record can help track the impact of a millennia of human niche construction on the evolution of animals [START_REF] Laland | Cultural niche construction: An introduction[END_REF] and contribute to the documentation of their evolutionary trajectory [START_REF] Sarrazin | Evolution in the Anthropocene[END_REF]. We found that the Neolithic Niche Construction had inceptive but limited impact on the phenotypic evolution of Sus scrofa in Western Europe. In the 4500-year timeframe of our study we did not find any evidence of size-selective harvesting, though a strong size decline between Pre-Neolithic and modern wild boar populations was observed, suggesting that harvesting pressures on size in Sus scrofa populations did not happen during the Neolithic transition. The main impact was the translocation of allochthonous domestic populations of a smaller size, which increased the local phenotypic diversity of Sus scrofa in Western Europe. The feralisation of these alien populations did not seem to induce a novel selective pressure on local populations or a phenotypic homogenization among the local and feral populations. Nevertheless, the greater phenotypic diversity in Western Europe Sus scrofa produced by this introduction could have fostered the local domestication in Europe. A loose rearing model, which would have induced no observable traits for most of the Neolithic Sus scrofa in our dataset, might have been the most common husbandry practice for these animals during this period; nonetheless, rare evidence of phenotypic divergence from the variation seen in boars in a natural habitat was documented. The first is observed from the Early Neolithic onwards and is most likely due to major mobility constraints. Later in the Neolithic, phenotypic changes suggest new selective pressures on Sus scrofa populations cared for by Neolithic people, maybe as early evidence of selective breeding for fast growth or fat production. Further research is still required, therefore, including paleogenomic and metabolomic studies on relevant and well-dated samples from Mesolithic to modern times in order to document the rapid evolutionary pace of cultural processes.

Figure 1 :

 1 Figure 1: a. Geographical location of the modern wild boars (grey filled circles) and archaeological Sus crofa (red filled circles) samples in western Europe: Ranchot (RAN), Arconciel (ARC), Gazel (GAZ), Chalain (CHA), Fontbrégoua (FON), Roquemissou (ROQ), Grotte de l'Aigle (AIG), Camprafaud (CAM), Roucadour (ROU) Téviec (TEV), Noyen (NOY), Vaihingen (VAI) and Bercy (BER). b. Chronological and cultural contexts of the Sus scrofa archaeological samples. c. Position of the phenotypic markers selected for the study of the Sus scrofa skeleton with their respective protocol of morphometric digitization: 3D surface and curve landmarks and semilandmarks respectively of mandible's symphysis and ramus (see Neaux et al. 2020 for methodological details), 3D surface semilandmarks of the calcaneus (see Harbers et al. 2020a for methodological details) and the 3D morphometric mapping and Cross Section Area (CSA) of the humeral shaft (See Harbers et al. 2020b for methodological details).

  Sus scrofa calcaneus covers the whole size range from the smallest wild boars to some of the largest industrial pig breeds. Regarding the body mass proxy (humeral shaft cross section area), Neolithic Sus scrofa are within the range of Mesolithic and modern wild boars. According to model-based density estimations, calcaneus size variation is clustered into two groups: a small one, similar in size to current wild boars, and a large one, similar in size to current pigs and Mesolithic wild boars. Current pigs, from landraces to industrial breeds have a larger CSA than extant and Mesolithic wild boars. Density estimations found three components of variation in the humeral CSA of Neolithic Sus scrofa: (1) a small one, similar to small extant wild boars, (2) a medium one, similar to large extant wild boars, and (3) a large one, similar to current-day pigs or Mesolithic wild boars.

Figure 2 :

 2 Figure 2: Size variation in modern and archaeological Sus scrofa of Western Europe: (a) humerus shaft cross-section area (CSA), centroid size of (b) calcaneus (c) mandible's ramus, (d) mandible's symphysis. Modern samples are presented with notched boxplots. Model-based Gaussian mixture of size variation in archaeological Sus scrofa were performed for humerus shaft CSA (a) and calcaneus centroid size (b) with model-based clustering results displayed by different symbols and named accordingly. Abbreviations: WB=wild boars, WB exp=experimental wild boars, DP=domestic pigs.

Figure 3 :

 3 Figure 3: Phenotypic variation of Neolithic Sus scrofa compared to Mesolithic, modern wild captive and domestic Sus scrofa from Western Europe. Morphospaces and shape deformations from first and second CVA axes were computed from the shape variation of the calcaneus (a), the morphometric mapping of the humeral shaft cortical thickness with the four muscle attachment displayed (b), the mandible-ramus (c) and the mandible's symphysis (d).

Figure 4 :

 4 Figure 4: a. Pie charts representing the classification of Neolithic specimens according to the kNN algorithms in the canonical morphospace of each bone marker (humerus, calcaneus, mandibular ramus and symphysis). The pie charts are spread horizontally along the chronology of the archaeological contexts and vertically according to their latitudinal positions. b. Diagram displaying the distribution of the d 13 C and d 15 N values obtained from the bone collagen of Sus scrofa specimens from Chalain 3, 4 and 19. Also included are the isotopic values for aurochs, red deer and cattle (Drucker and Bocherens, 2009). Sus scrofa individuals are displayed according to their kNN identification and their model-based size clustering (Small=small dot, Large=large dot) c. Phenotypic diversification of Sus scrofa from Mesolithic to Late Neolithic displayed by size variation and kNN identification of each bone (CSA=Cross Section Area). The large and small size of the bone pictograms correspond to their size cluster identified by model-based Gaussian Density estimations.

ACKNOWLEDGMENTS

We Are most grateful to Jacqueline Studer for granting us access to the wild boar collections of the Natural History museum of Geneva.

We are most grateful to Jill Cucchi for copyediting this manuscript.

FUNDINGS

This research has been funded by the ANR, through the Domexp project (ANR-13-JSH3-0003-01); LabEx ANR-10-LABX-0003-BCDiv, in the 'Investissements d'avenir' programme ANR-11-IDEX-0004-02 and project Emergence SU-19-3-EMRG-02.

"For the purpose of Open Access, a CC-BY public copyright licence has been applied by the authors to the present document and will be applied to all subsequent versions up to the

DATA AVAILABILITY

The morphometric dataset for this study can be found in the Dryad repository following this link: https://datadryad.org/stash/share/bIB4h5ellfpy2nafCVwhNm3Im1xOIRIX9lCR6Bu08DE.

CONFLICT OF INTERESTS

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

Author Accepted Manuscript arising from this submission." S1. Details for each archaeological Sus scrofa bones studied with information on their archaeological stratigraphic contexts, anatomical position, lateralisation, ageing and sexing.

SUPPLEMENTARIES Table

Text S2: Description and references for the archaeological contexts from which the phenotypic markers of the study have been collected.

Table S3: Description of the modern corpus of Sus scrofa specimens used for the study. Table S5. Stable isotope extraction yields from Sus scrofa remains collected at the archaeological site of Chalain (France).


Christel Vaché
email: christel.vache@inserm.fr

Nicolas Cubedo3

Luke Mansard1

Jérôme Sarniguet3

David Baux1

Valérie Faugère1

Corinne Baudoin1

Melody Moclyn1

Renaud Touraine4

Geneviève Lina- Granade5

Mireille Cossée1

Anne Bergougnoux1

Vasiliki Kalatzis

Mireille Rossel

Anne-Françoise Roux1

Anne-Françoise Roux

Identification and in vivo functional investigation of a HOMER2 nonstop variant causing hearing loss

DFNA68 is a rare subtype of autosomal dominant nonsyndromic hearing impairment caused by heterozygous alterations in the HOMER2 gene. To date, only 5 pathogenic or likely pathogenic coding variants, including two missense substitutions (c.188 C > T and c.587 G > C), a single base pair duplication (c.840dupC) and two short deletions (c.592_597delACCACA and c.832_836delCCTCA) have been described in 5 families. In this study, we report a novel HOMER2 variation, identified by massively parallel sequencing, in a Sicilian family suffering from progressive dominant hearing loss over 3 generations. This novel alteration is a nonstop substitution (c.1064 A > G) that converts the translational termination codon (TAG) of the gene into a tryptophan codon (TGG) and is predicted to extend the HOMER2 protein by 10 amino acids. RNA analyses from the proband suggested that HOMER2 transcripts carrying the nonstop variant escaped the non-stop decay pathway.

Finally, in vivo studies using a zebrafish animal model and behavioral tests clearly established the deleterious impact of this novel HOMER2 alteration on hearing function. This study identifies the fourth causal variation responsible for DFNA68 and describes a simple in vivo approach to assess the pathogenicity of candidate HOMER2 variants.

INTRODUCTION

Approximately 20-30% of hereditary nonsyndromic hearing loss (NSHL) cases diagnosed in infancy and early childhood present an autosomal dominant inheritance pattern (ADNSHL) [START_REF] Morton | Genetic epidemiology of hearing impairment[END_REF]. For the vast majority of these cases, the HL is described as postlingual, bilateral and progressive. To date at least 51 causal genes have been identified (Hereditary Hearing Loss Homepage; https://hereditaryhearingloss.org/; accessed in May, 2022) and one of them is HOMER2 (OMIM*604799).

The HOMER2 gene is located on chromosome 15, spans 103.7 kb (UCSC Genome browser; http://genome.ucsc.edu/; accessed in May, 2022), and encodes several isoforms of the HOMER homolog 2 protein, including two long isoforms (HOMER2a: 343 aa, NP_004830.2 and HOMER2b: 354 aa, NP_955362.1) due to alternative splicing (NM_004839.4 and NM_199330.2, respectively). These HOMER2 isoforms belongs to the HOMER family of scaffolded proteins (encoded as short and long isoforms by alternative splicing of the HOMER1, HOMER2 and HOMER3 genes), which are involved in Ca2+ homeostasis, cytoskeletal organization and synaptic plasticity (for review see [START_REF] Shiraishi-Yamaguchi | The Homer family proteins[END_REF]). The long HOMER proteins are all characterized by the presence of two main domains: (i) a conserved Nterminal Enabled Vasodilator-stimulated phosphoprotein Homology 1 (EVH1) domain that interacts with proline-rich sequences, (ii) a C-terminal domain, comprising a coiled-coiled region with a CDC42-binding domain and two leucine zipper motifs, responsible for multimerization between HOMER family members and interaction with the CDC42 small GTPase [START_REF] Shiraishi-Yamaguchi | Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines[END_REF].

Azaiez et al. [START_REF] Azaiez | HOMER2, a stereociliary scaffolding protein, is essential for normal hearing in humans and mice[END_REF] were the first to establish the causality of a heterozygous missense HOMER2 variation, NM_199330.2:c.587 G > C, p.(Arg196Pro) (published as NM_004839.4:c.554 G > C, p.(Arg185Pro)) in a dominantly inherited form of NSHL (DFNA68). Thereafter, three other HOMER2 pathogenic heterozygous variants were identified in three families suffering from ADNSHL: the NM_199330.2:c.840dupC, p.(Met281HisfsTer9) variant (published as c.840_841insC) in a large Chinese family [START_REF] Lu | Whole exome sequencing identified a second pathogenic variant in HOMER2 for autosomal dominant non-syndromic deafness[END_REF], the NM_199330.2:c.592_597delACCACA, p.(Thr198_Thr199del) variant in an Italian family [START_REF] Morgan | Lights and Shadows in the Genetics of Syndromic and Non-Syndromic Hearing Loss in the Italian Population[END_REF], and the NM_199330.2:c.832_836delCCTCA, p.(Pro278AlafsTer10) variant in a Spanish family [START_REF] Lachgar | A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family[END_REF]. Another variant (NM_199330.2:c.188 C > T, p.(Pro63Leu)) reported in an Iranian family with dominant deafness was also described [START_REF] Noorian | Whole-Exome Sequencing in Idiopathic Short Stature: Rare Mutations Affecting Growth[END_REF] and assumed as likely pathogenic by the authors although the variation was of uncertain significance when applying the ACMG criteria.

In this article, we have identified a new HOMER2 variant segregating in a large Sicilian family suffering from ADNSHL. This alteration, which was not reported in databases, consists of a nonstop substitution, a very rare variant class. RNA analysis from the proband's peripheral blood sample demonstrated that it results in the production of abnormal RNA transcripts with a 3′ coding sequence extension. Finally, its deleterious effect on hearing was established using zebrafish behavioral tests. With this study we expanded the genotype spectrum associated with DFNA68 and developed a simple zebrafish experiment to test future candidate pathogenic HOMER2 variants.

MATERIALS AND METHODS

Patients

A forty-eight-year-old female (index case) and her fifteen-year-old daughter, both suffering from progressive bilateral nonsyndromic hearing loss, were referred to our laboratory for genetic analyses. For each individual, pure tone audiometry, in a frequency range of 250-8000 Hz, was performed to evaluate the severity of their hearing loss in accordance with the recommendations of the International Bureau for Audiophonology (https://www.biap.org/en/recommandations/recommendations/tc-02-classification/213-rec-02-1-en-audiometric-classification-of-hearingimpairments/file). In addition, a family history questionnaire was completed by the clinicians. Informed consent for genetic analyses was obtained from all participants or their legal representatives. Approval was obtained by the Institutional Review Board (IRB) of CHU de Montpellier: (2018_IRB-MTP_05-05 obtained on the 15th June 2018).

Sequencing and validation

DNA from each participant was isolated from peripheral blood samples using standard procedures. Massively parallel sequencing (MPS) was performed with a hearing loss gene panel on an Illumina® MiniSeq sequencer (Illumina,San Diego, CA, USA). The workflow used to identify candidate pathogenic variations has been already described [START_REF] Baux | Combined genetic approaches yield a 48% diagnostic rate in a large cohort of French hearing-impaired patients[END_REF] and the 84 screened genes are listed in Supplementary Table 1. This approach included a copy-number variant analysis using the MobiCNV algorithm (https://github.com/mobidic/MobiCNV) and a direct visualization of the sequenced reads with the Integrative Genomics Viewer (IGV) software (v2.7.2) [START_REF] Thorvaldsdóttir | Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration[END_REF]. Whole exome sequencing (WES) libraries were prepared using the KAPA® HyperPlus kit (Roche, Madison, WI, USA) according to the KAPA®HyperCap v3.0 workflow. Whole exome enrichment was then performed using the KAPA® HyperExome probes (~43 Mb capture target size) and an Illumina® NextSeq 500 instrument (Illumina) was used for sequencing. Samples were analyzed using an in-house pipeline (MobiDL, https://github.com/mobidic/MobiDL), which performs the secondary and tertiary analyses, and uses BWA [START_REF] Li | Fast and accurate long-read alignment with Burrows-Wheeler transform[END_REF] as aligner and both GATK HaplotypeCaller [START_REF] Auwera G Van Der | Genomics in the cloud: using Docker, GATK, and WDL in Terra[END_REF] and Google DeepVariant [START_REF] Poplin | A universal SNP and small-indel variant caller using deep neural networks[END_REF] as variant callers. Furthermore, copy-number variation (CNV) was assessed using the GATK4 CNV calling module and a pipeline (https://gitlab.univ-nantes.fr/benjamin.co/svexome/-/tree/add_vcf_support) based on the GATK recommendations. Variant filtering was based on: (i) a dominant mode of inheritance, (ii) a genotype/phenotype correlation and (iii) a minor allele frequency (MAF) threshold of 0.1%. Sanger sequencing or MPS were performed in order to validate identified variants and build a comprehensive familial genetic profile. Primer sequences used for PCR-Sanger sequencing are listed in Supplementary Table 2.

In silico analysis and data availability

The reference sequences used in this study were NM_199330.2,NM_153676.3 and NM_194248.2. for the HOMER2, USH1C and OTOF genes, respectively. Functional consequences of variants of interest were evaluated using the online variant interpretation platform Mobidetails [START_REF] Baux | MobiDetails: online DNA variants interpretation[END_REF] (https://mobidetails-iurc-montp-inserm.fr/MD/; accessed in March, 2023). The Human Gene Mutation Database (HGMD® Professional 2020.3; https://portal.biobase-international.com) and the Deafness Variation Database (DVD) (https://deafnessvariationdatabase.org/) were also consulted (accessed in March, 2023). The Human Genome Variation Society recommendations [START_REF] Den Dunnen | HGVS Recommendations for the Description of Sequence Variants: 2016 Update[END_REF] v20.05 (http://varnomen.hgvs.org/) were followed for the description of the variants.

The newly identified variant was classified using the Intervar web server (http://wintervar.wglab.org/) in accordance with the adapted ACMG/AMP guidelines for variant interpretation in the context of hearing loss [START_REF] Oza | Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss[END_REF] and deposited in the Leiden Open Variation Database Global Variome Shared Instance (LOVD GVShared, individuals #00411547 to #00411552).

cDNA analysis of HOMER2

Total RNA from the proband and a control was isolated from peripheral blood samples collected in PAXgeneTM Blood RNA Tubes using the Nucleo Spin® RNA II isolation kit (Macherey-Nagel, Düren, Germany). RNA quality and concentration were estimated with a 4200 TapeStation system (Agilent Technologies, Santa Clara, USA). RNA samples were used as templates for cDNA synthesis using the SuperScriptTM III Reverse Transcriptase (Invitrogen, Cergy-Pontoise, France) and PCR amplifications were performed with HOMER2-specific primers (Supplementary Table 2). PCR products were size-separated on agarose gel and Sanger sequenced.

Zebrafish husbandry

This study was conducted in accordance with the recommendations of INSERM, Montpellier University and the European Convention for the Protection of Animals used for Experimental and Scientific Purposes. The zebrafish Tg(pou4f3:gap43-GFP)s356t transgenic line, redefined later as Brn3c:mGFP [START_REF] Decarvalho | Developmental expression of the POU domain transcription factor Brn-3b (Pou4f2) in the lateral line and visual system of zebrafish[END_REF], was maintained according to recommended Federation of European Laboratory Animal Science Associations (FELASA) procedures on a 14:10 h light/dark photoperiod at 28 °C [START_REF] Aleström | Zebrafish: Housing and husbandry recommendations[END_REF]. Embryos were obtained from natural spawning and incubated in a water tank at 28 °C. Embryos and larvae were staged by days post-fertilization (dpf).

mRNA synthesis and injection

The pShuttleTM Gateway® PLUS ORF clone for HOMER2 (GC-H5096) and its mutated version (HOMER2, c.1064 A > G, 30 bp 5′ GGCTGGCCGAGGCCCAGGCCCCGCCCGTGA-3′ extension) were purchased from GeneCopoeiaTM (GeneCopoeia, Rockville, MD, USA). The ORF sequences were subcloned into the expression vector pCS2 + (gift from Marc Kirschner (Addgene plasmid # 17095; http://n2t.net/addgene:17095; RRID: Addgene_17095)) using the GatewayTM LR ClonaseTM II Enzyme Mix (Invitrogen, Carlsbad, CA, USA). The pCS2+ mCherry expression vector [START_REF] Hirsinger | Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes[END_REF] (gift from Georges Lutfalla; DIMNP, CNRS, University of Montpellier, Montpellier, France) was also used.

Transcription was carried out using the mMESSAGE mMACHINETM T7 or SP6 transcription kit (Invitrogen) for HOMER2 wild type, HOMER2 mutated or mCherry RNA synthesis, respectively. RNA quality and concentration were assessed by electrophoresis and spectrophotometry (NanoDrop One/OneC, Thermo Fisher Scientific, Waltham, MA, USA). A dilution range was performed to determine the optimal mRNA quantity that allowed a good ratio of toxicity/phenotype analysis. Therefore, 80 pg of HOMER2 RNA (wild type sequence: A1064-RNA or mutated sequence: G1064-RNA) were coinjected with 50 pg of mCherry RNA, 1 nL at the one-to two-cell stage. Twenty-four hours after microinjection, mCherry fluorescence was used as a quality control of injection and translation; all negative embryos were discarded.

Acoustic startle response (ASR) test

At 5 dpf, zebrafish larvae with normal morphology were transferred into a 96-well plate with 300 μl of water per well and placed into a Zebrabox®(ViewPoint, Lissieu, France). In the device, larvae in the plate were isolated from environmental surrounding noise. After a 30 min period of adaptation, larvae were submitted to 3 noise stimulations (70 dB, 400 Hz, 1 s per stimulus, 5 min apart). The study was conducted in light condition and zebrafish activity was quantified using the quantization mode of Zebralab® software (ViewPoint), as previously described [START_REF] Crouzier | Morphological, behavioral and cellular analyses revealed different phenotypes in Wolfram syndrome wfs1a and wfs1b zebrafish mutant lines[END_REF]. The quantity of movements during the entire experiment was measured for each larva. Baseline activity levels were subtracted from the activity levels during the sound stimulations (10 s pre-stimulus period before each stimulation) in order to normalize the values. The activity for the 3 stimuli were pooled into 1 s time to assess ASR.

Visual motor response (VMR) assay

Following the ASR test, each 96-well plate was then used to assess the locomotor activity of the larvae in response to visual stimuli. This test quantifies the activity of zebrafish larvae in response to light changes using infrared tracking system. The VMR protocol consisted of a 30min dark-adaptation period followed by two periods, each composed of 10 min of brightness (100% of light intensity) and 10min of darkness, the duration of the whole protocol was 70min. Zebrafish activity was quantified with the Zebralab® software and data were analyzed using 1 min time bins to assess the VMR.

RESULTS

Clinical findings

The proband of this study (III.5) was a 48-year-old female suffering from progressive sensorineural nonsyndromic hearing loss (NSHL). The clinical diagnosis of its deafness was made at the age of 7 years, but an earlier onset was presumed by the patient in the light of her educational difficulties. At the age of 28 years, audiometric assessment revealed a bilateral downward-sloping severe 1st degree HL according to the BIAP recommendations (Fig. 1a). When she was 47 years old, she was fitted with hearing aids. Her daughter (IV.5) was diagnosed at the age of 3 years and an air conduction test performed at age 13 years showed a bilateral downward-sloping mild 1st degree HL (Fig. 1a). The family anamnesis revealed 6 additional affected members over 4 generations (I.1, II.3, II.4, III.2, III.6 and IV), consistent with an autosomal dominant inheritance pattern of the pathology (Fig. 1b).

DNA and RNA analysis

Following our laboratory diagnostic approach, massive parallel sequencing (MPS) analysis using a panel of 84 deafness-causing genes was firstly performed in the proband (III.5) to identify candidate pathogenic variants. This screening strategy highlighted the presence of a HOMER2 A > G transition (c.1064 A > G) in the heterozygous state (Fig. 2). This variant occurred within the translational termination (stop) codon (TAG) of the gene and changed it to a tryptophan codon (TGG). According to the HOMER2 human reference genome sequence, a downstream inframe stop codon was detected, leading to a predicted 10 amino acids C-terminal extension of the protein (Fig. 2). This substitution was absent from all the interrogated databases and was considered as a variant of uncertain significance according to the ACMG/AMP hearing loss guidelines. Sanger sequencing validated the genotype of the proband and revealed that her affected daughter was also a heterozygous carrier (Fig. 1b). In addition, MPS data analysis highlighted two other variants, both in the heterozygous state, in two genes implicated in hearing loss (Supplementary Table S3). The first one was a previously described pathogenic variation (c.778 G > T, p.(Glu260Ter)) in USH1C [START_REF] Abdi | Diversity of the Genes Implicated in Algerian Patients Affected by Usher Syndrome[END_REF], a gene responsible for both nonsyndromic (OMIM#602092) and syndromic (OMIM#276904) recessive deafness. The second one was an intronic transversion (c.327+5 G > T) in OTOF, a gene associated with recessive NSHL (OMIM#601071). This last substitution was present in the Genome Aggregation Database (gnomAD v2.1.1) with a minor allele frequency (MAF) of 0.13% in European (Finnish) and was referred in ClinVar (Variation ID: 335462) as a variant of uncertain significance or a likely benign variation. Splicing predictions obtained with the MaxEntScan and SPiP tools were in favor of a deleterious effect of the variant via an alteration of the donor consensus splice site of exon 4. By contrast, the deep neural network SpliceAI did not predict this adverse event but indicated a moderate increase in the probability that a cryptic donor splice site, located 48-bp upstream of the natural donor splice site, could be used. These two USH1C and OTOF variants were validated by Sanger sequencing in the proband. The OTOF variation was also present in the proband's daughter (Fig. 1b).

In order to exclude the implication of a non-explored DFNA gene in the familial hearing loss, whole exome sequencing (WES) was then carried out with DNA samples from four affected (II.4, III.5, IV.1 and IV.5) and one unaffected (III.4) individuals. After filtering for MAF, genotype/phenotype correlation and a dominant mode of inheritance, the only selected variant was the HOMER2 nonstop substitution. Further segregation of this variant in the family showed it was only detected in the affected members (Fig. 1b).

As the USH1C and OTOF variants identified in the proband could be important for the other family members in a context of genetic counselling, a segregation analysis was additionally performed with Sanger sequencing or MPS. Unexpectedly, this analysis highlighted the presence of another well-known USH1C pathogenic variant (c.496 + 1 G > A; ClinVar variation ID: 371732) in the unaffected IV.2 and IV.3 individuals, presumably inherited from their father III.1 (Fig. 1b and Supplementary Table S3).

Finally, the consequence of the c.1064 A > G HOMER2 variation at the RNA level was assessed by RT-PCR study on total RNA extracted from proband's blood sample. Sanger sequencing revealed that HOMER2 transcripts carrying the c.1064 A > G were present and not subject to a major degradation by the non-stop decay pathway (Fig. 3).

Zebrafish behavioral assays

In order to determine the pathogenicity of the HOMER2 nonstop variant identified in this study we performed in vivo assays using a zebrafish model expressing human HOMER 2 protein when injected with synthetized HOMER2 wildtype RNA (A1064-RNA) or mutated HOMER2 RNA (G1064-RNA) (Fig. 4a and Supplementary Fig. 1a).

A behavioral test, based on recording the acoustic startle response (ASR) was firstly conducted to assess a potential hearing defect in 5 dpf larvae injected with G1064-RNA. The larval locomotor activity in response to noise stimuli above noise background was tracked and differences in the ratio responsive to non-responsive larvae were estimated between the 3 groups: noninjected controls (NI), injected with the wildtype A1064-RNA (A1064) and injected with the mutant G1064-RNA (G1064). ASR data analyses, from three independent tests, highlighted a significant decrease in the number of responsive larvae exposed to noise (70 db, 400 HZ) in the G1064 group compared to the A1064 group (p < 0.0001), whereas no significance difference was observed between the A1064 and NI groups (Fig. 4b).

To exclude a locomotion impairment due to the G1064-RNA injection, a visual motor response (VMR) assay was systematically performed after each ASR experimentation (Fig. 4c). The locomotor reflex was induced by flash of light, independently to acoustic stimulation. Statistical analyses of data obtained from all VMR tests (Fig. 4c) indicated that the locomotor activity of larvae from the G1064 group was not impacted by the overexpression of the mutated HOMER2 RNA in both ON phases and OFF phases, in comparison with the A1064 group.

As a complement to these behavioral tests, β-actin normalized HOMER2 RNA levels and ear diameters from injected 3 dpf larvae (Supplementary Fig. 1b,c) were also studied. For these two analyses, no significant difference was observed between the A1064 and G1064 groups.

Altogether, these results obtained from zebrafish experimentations characterized the c.1064 A > G HOMER2 substitution as a causal variant responsible for hearing loss.

DISCUSSION

Single-nucleotide pathogenic substitutions that change stop codons into sense codons, termed nonstop variants (also known as no-stop, stop-loss or readthrough variations), are an extremely rare type of alteration (387 nonstop variants among 278,993 single pathogenic nucleotide substitutions reported in the HGMD database (accessed in March, 2023)). In this study we identified a HOMER2 nonstop variation segregating with the hearing loss (HL) phenotype described in a large Sicilian family. This result was obtained by massively parallel sequencing of a HL gene panel, confirmed by a whole exome sequencing analysis and validated by Sanger sequencing.

To date only four pathogenic HOMER2 variants have been clearly associated with DFNA68, a missense and a two small deletions in three European families [START_REF] Azaiez | HOMER2, a stereociliary scaffolding protein, is essential for normal hearing in humans and mice[END_REF][START_REF] Morgan | Lights and Shadows in the Genetics of Syndromic and Non-Syndromic Hearing Loss in the Italian Population[END_REF][START_REF] Lachgar | A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family[END_REF] and a single nucleotide duplication in a Chinese family [START_REF] Lu | Whole exome sequencing identified a second pathogenic variant in HOMER2 for autosomal dominant non-syndromic deafness[END_REF]. In these families, all affected individuals suffering from bilateral progressive nonsyndromic hearing loss (NSHL) affecting preferentially the high frequencies with an onset (when indicated) in the first decade of life (7-9 years old), which is consistent with the clinical data collected from the family described herein. It was reported that the severity of the hearing impairment could be variant dependent [START_REF] Lu | Whole exome sequencing identified a second pathogenic variant in HOMER2 for autosomal dominant non-syndromic deafness[END_REF][START_REF] Lachgar | A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family[END_REF], but due to the limited number of cases a fine genotype/phenotype correlation remains difficult to establish. Concerning the fifth reported variant, c.188 C > T, no family history or clinical data was reported [START_REF] Noorian | Whole-Exome Sequencing in Idiopathic Short Stature: Rare Mutations Affecting Growth[END_REF].

Nonstop pathogenic alterations can lead to: (i) mRNA degradation due to activation of the non-stop decay (NSD) pathway in response to the presence of stalled or collided ribosomes [START_REF] Powers | New insights into no-go, non-stop and nonsense-mediated mRNA decay complexes[END_REF], (ii) degradation of the mutated protein by protein quality control mechanisms [START_REF] Torres-Torronteras | A novel nonstop mutation in TYMP does not induce nonstop mRNA decay in a MNGIE patient with severe neuropathy[END_REF][START_REF] Pang | A novel nonstop mutation in the stop codon and a novel missense mutation in the type II 3betahydroxysteroid dehydrogenase (3beta-HSD) gene causing, respectively, nonclassic and classic 3beta-HSD deficiency congenital adrenal hyperplasia[END_REF] or (iii) functional alteration of the mutated protein [START_REF] Sun | Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2[END_REF][START_REF] Bock | A nonstop variant in REEP1 causes peripheral neuropathy by unmasking a 3'UTR-encoded, aggregationinducing motif[END_REF]. As results obtained from studies on animal models [START_REF] Azaiez | HOMER2, a stereociliary scaffolding protein, is essential for normal hearing in humans and mice[END_REF] strongly suggest that the pathogenicity of HOMER2 variants implicated in DFNA68 is not due to haplo-insufficiency, a massive HOMER2 mRNA degradation by NSD linked to the nonstop variant appeared unlikely. To test this hypothesis, RTPCR analyses were performed and confirmed that transcripts carrying the HOMER2 nonstop variation were not a major target of the NSD system. This result was in accordance with data obtained from a meta-analysis [START_REF] Hamby | A meta-analysis of single base-pair substitutions in translational termination codons ("nonstop" mutations) that cause human inherited disease[END_REF], which suggested that alternative stop codons in close proximity to the mutated stop codons (0-49 nucleotides downstream) were not subject to NSD.

In order to validate the pathogenicity of the c.1064 A > G HOMER2 substitution, we then performed in vivo behavioral tests on zebrafish larvae injected with wild type or mutated HOMER2 RNA. The zebrafish model has been already used by Azaiez et al. [START_REF] Azaiez | HOMER2, a stereociliary scaffolding protein, is essential for normal hearing in humans and mice[END_REF] to study the impact of the c.587 G > C HOMER2 variant. In this previous publication, the effect of the variant was determined by measurements of the otic capsule area in 3 dpf larvae while in vivo behavioral tests were conducted in mice. In our study, we performed the behavioral assays on zebrafish larvae at 5 dpf, using the ASR and VMR conventional tests, which is a much more rapid and straightforward model as we were able to directly compare wild type and variant phenotypes. The ASR test revealed a clear decrease in larval activity when injected with the mutated HOMER2 RNA, whereas the VMR assay did not revealed any change in their locomotor capacity, highlighting a hearing impairment. This result was also validated by a western blot experiment detecting human HOMER2 proteins in injected embryos and a qRT-PCR study of the HOMER2 RNA that confirmed the integrity of the injected RNAs in larvae at 3dpf. In order to compare our results to those obtained by Azaiez et al. [START_REF] Azaiez | HOMER2, a stereociliary scaffolding protein, is essential for normal hearing in humans and mice[END_REF], we also measured the ear diameters of 3 dpf larvae injected with wild type or mutated HOMER2 RNAs, and showed that contrary to the c.587 G > C variant, the nonstop alteration did not impair the morphology of the ears.

Taking into consideration all these results, and according to ACMG/AMP guidelines, we classified the c.1064 A > G HOMER2 transition as a class V pathogenic variant.

The exact molecular mechanism involved in the pathogenicity of the HOMER2 nonstop variant has not been determined. A dominant-negative effect on the wild type protein or a gain-of function have been discussed for the previously identified pathogenic alterations. In the case of the nonstop variant, the 10 amino-acids protein extension could inhibit the multimerization process, which occurs via the C-terminal domains of the proteins or could compete for other partner proteins resulting in a dominant-negative effect. In summary, in this article, we identified a new HOMER2 variation associated with progressive dominant NSHL. This variant consisting of a nonstop substitution, a very rare type of pathogenic variation, expands the mutational spectrum of the HOMER2 gene. Identification of this pathogenic variant and 3 other alterations in 2 deafness causing genes will have important consequences for the proband's family concerning medical care and genetic counseling. Furthermore, this study highlights the major importance of developing functional tests to unambiguously characterize the pathogenicity of a variants of uncertain significance. Finally, we believe that using zebrafish animal model and simple behavioral assays, such as ASR and VMR, should be considered and encouraged in a molecular diagnostic strategy when necessary. Percentage values of responsive and non-responsive larvae from the non-injected group (NI), A1064-RNA injected group (A1064) and G1064-RNA injected group (G1064) are displayed in a stacked column chart. The number of larvae in each group is indicated (n). ****p value < 0.0001; ns: not significant. c Visual motor response (VMR) test. Left chart: profiles of larvae from the A1064 group (black dots) and the G1064 group (gray dots) focused on the two ON/OFF sessions of the VMR test. Average of the total distance traveled every 1 min bin is plotted for each group. Right chart: average distances swam by each group during the two pooled ON or OFF sessions. Differences between the two groups is not significant (ns).

Fig. 1

 1 Fig. 1 Audiograms of the proband and her daughter, pedigree chart of the family and segregation of the identified variants. a Superimposed audiograms of the patient at the age of 28 years old (solid line) and her daughter at the age of 13 years old (dotted line) showing bilateral downward-sloping severe and mild 1st degree hearing loss, respectively. b Pedigree of the proband's family with segregation of the heterozygous variants when present: HOMER2, c.1064 A > G (M1 ±); USH1C, c.778 G > T (M2 ±); OTOF, c.327 + 5 G > T (M3 ±) and USH1C, c.496 + 1 G > A (M4 ±). The proband (III.5) is indicated by a black arrow, filled symbols denote affected individuals, brackets indicate a person not involved in the study.

Fig. 2

 2 Fig. 2 Identification of the HOMER2 variant. On the left, Sanger sequencing electropherogram of the heterozygous c.1064 A > G HOMER2 variant segregating with the autosomal dominant nonsyndromic hearing loss in the proband's family. On the right, consequences of the variant at the DNA and protein (expected) levels. The native termination (stop) codon is indicated with bold black font and black star whereas the subsequent in-frame one is indicated with bold gray font and gray star. The 10 amino-acid residues extension of the expected protein is also shown.

Fig. 3

 3 Fig. 3 Consequence of the c.1064 A > G HOMER2 variant on the RNA. a Schematic representation of the study and agarose gel electrophoresis of the RT-PCR products from the proband (III.5) and a control. Primers (forward: F, reverse: R) were indicated with black arrows (b) Sanger sequencing electropherograms of the HOMER2 transcripts from the proband and a control.

Fig. 4

 4 Fig.4Behavioral responses of larvae at 5 dpf. a Schematic representation of the 3′ end of the 2 full length in vitro synthesized RNAs (A1064-RNA and G1064-RNA). b Acoustic startle response (ASR) assay. Left chart: profiles of control non-injected larvae with normal response for each acoustic stimulation. Right chart: percentage values of responsive and non-responsive larvae from non-injected control. Percentage values of responsive and non-responsive larvae from the non-injected group (NI), A1064-RNA injected group (A1064) and G1064-RNA injected group (G1064) are displayed in a stacked column chart. The number of larvae in each group is indicated (n). ****p value < 0.0001; ns: not significant. c Visual motor response (VMR) test. Left chart: profiles of larvae from the A1064 group (black dots) and the G1064 group (gray dots) focused on the two ON/OFF sessions of the VMR test. Average of the total distance traveled every 1 min bin is plotted for each group. Right chart: average distances swam by each group during the two pooled ON or OFF sessions. Differences between the two groups is not significant (ns).

ACKNOWLEDGEMENTS

We are grateful to the family members who contributed to this study. We thank Dr Yoan Arribat, University of Lausanne (Switzerland), for his help with Gateway cloning and Dr Benjamin Cogné, CHU of Nantes (France), for the GATK pipeline.

FUNDING

This work was partially funded by the association SOS Rétinite France and the French Agency of BioMedicine (AOR AMP 2017).

DATA AVAILABILITY

The data that support this study are available from the corresponding author upon reasonable request. The variants, individual and phenotype described in this paper is available to the LOVD GVShared, Individual ID number #00411547 to #00411552 (e.g., https://databases.lovd.nl/shared/individuals/00411547).

AUTHOR CONTRIBUTIONS

Conceptualization, CV, MR, and A-FR; Human molecular genetic investigations, CV, LM, VF, CB, and MM; Clinical investigations, RT and GL-N; Zebrafish investigations, CV, NC, JS, and MR; Software, DB, Writing-original draft preparation, CV and MR; Writingreview and editing, A-FR, VK, MC, and AB. Supervision, A-FR and MR. All authors have read and agreed to the published version of the paper.

COMPETING INTERESTS

The authors declare no competing interests.

ETHICS APPROVAL

The institutional review board (IRB) of CHU de Montpellier approved the experimental protocol (2018_IRB-MTP_05-05 obtained on the 15th June 2018). The zebrafish study was conducted in accordance with the recommendations of INSERM, Montpellier University and the European Convention for the Protection of Animals used for Experimental and Scientific Purposes.