Alexandra Houssaye
email: houssaye@mnhn.fr

Florian Martin

Jean-Renaud Boisserie

Fabrice Lihoreau

Paleoecological inferences from long bone microanatomical specializations in Hippopotamoidea (Mammalia, Artiodactyla)

Keywords: Anthracotheriinae, bone microanatomy, Bothriodontinae, Hippopotamoidea, Microbunodontinae, semi-aquatic lifestyle, weight-bearing

Hippopotamoids are herbivorous mammals that originated in the late middle Eocene. This taxon includes animals with a great variety of sizes and body proportions, from small and gracile forms with slender limbs to heavy massive ones. Many hippopotamoids have previously been considered semi-aquatic but recent studies have highlighted a diversity of ecologies. This study focuses on bone microanatomy, one of the various proxies that enable inferring the ecology of extinct taxa. The comparative analysis of the inner structure of the stylopod bones in various hippopotamoids, based on both transverse and longitudinal virtual sections, highlights a diversity of patterns and clarifies previously proposed hypotheses about the ecology of the sampled hippopotamoids. The filling of the medullary area by spongious deposits in the pygmy hippopotamus, Choeropsis liberiensis, appears associated with frequent incursions into the water by an animal that essentially forages in forests. The common hippopotamus, Hippopotamus amphibious, which spends most of the day submerged in water, shows a greater filling of the medullary area by spongious bone and a thicker cortex. These observations coupled with comparisons with diverse terrestrial and semi-aquatic mammals of various sizes confirm that semi-aquatic lifestyle and heavy weight-bearing are associated with similar microanatomical specializations causing an increase in bone mass. However, for a given mass, comparisons enable determining if an additional increase in bone compactness occurs, as in Hippopotamus amphibious, in which case a semi-aquatic lifestyle could be inferred. Accordingly, this study suggests an essentially terrestrial lifestyle for Microbunodon minimum, Bothriodon velaunus, Elomeryx borbonicus, Merycopotamus medioximus, Paenanthracotherium bergeri, and probably also Saotherium cf. S. mingoz, a slight degree of water dependence in Brachyodus onoideus, and a stronger one in Libycosaurus bahri and Hexaprotodon garyam, though less intense than in Hippopotamus amphibius. Comparisons with other large terrestrial and semi-aquatic taxa, and based on a large part of the diaphysis, are required to better decipher the microanatomical changes associated with a semi-aquatic lifestyle from those linked to loading in heavy quadrupedal mammals.

Introduction

Hippopotamoids, including hippopotamids and anthracotheres, are herbivorous mammals that emerged in the late middle Eocene [START_REF] Soe | New remains of Siamotherium pondaungensis (Cetartiodactyla, Hippopotamoidea) from the Eocene of Pondaung, Myanmar: paleoecologic and phylogenetic implications[END_REF]. This clade is assumed to correspond to the sister group of Cetacea based on both molecular (Irwin and Arnason 1994;[START_REF] Gatesy | Evidence from milk casein genes that cetaceans are close relatives of hippopotamid artiodactyls[END_REF][START_REF] Gatesy | More DNA support for a Cetacea/Hippopotamidae clade: the blood-clotting protein gene gamma-fibrinogen[END_REF][START_REF] Montgelard | Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences[END_REF]; [START_REF] Ursing | Analyses of mitochondrial genomes strongly support a hippopotamus-whale clade[END_REF][START_REF] Nikaido | Phylogenetic relationships among cetartiodactyls based on insertions of short and long interpersed elements: hippopotamuses are the closest extant relatives of whales[END_REF][START_REF] Arnason | Mammalian mitogenomic relationships and the root of the eutherian tree[END_REF][START_REF] Zhou | Phylogenomic analyses and improved resolution of Cetartiodactyla[END_REF]) and morphological [START_REF] Geisler | Morphological support for a close relationship between hippos and whales[END_REF]Boisserie et al. 2005a;[START_REF] Geisler | Phylogenetic relationships of cetaceans to terrestrial artiodactyls[END_REF][START_REF] Gatesy | A phylogenetic blueprint for a modern whale[END_REF] studies. Its abundant fossil record illustrates a wide spatio-temporal distribution. Among hippopotamoids, anthracotheres lived from the late middle Eocene to the early Pleistocene in Eurasia, northern America, and Africa [START_REF] Kron | Anthracotheriidae[END_REF]Dennel 2005;[START_REF] Lihoreau | Family Anthracotheriidae[END_REF][START_REF] Holroyd | Anthracotheriidae. In Cenozoic Mammals of Africa[END_REF][START_REF] Tsubamoto | Recognition of Microbunodon (Artiodactyla, Anthracotheriidae) from the Eocene of China[END_REF][START_REF] Rincon | First Central American record of Anthracotheriidae (Mammalia, Bothriodontinae) from the early Miocene of Panama[END_REF][START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF][START_REF] Grandi | Prominatherium dalmatinum from the late Eocene of Grancona (Vicenza, NE Italy). The oldest terrestrial mammal of the Italian peninsula[END_REF][START_REF] Soe | New remains of Siamotherium pondaungensis (Cetartiodactyla, Hippopotamoidea) from the Eocene of Pondaung, Myanmar: paleoecologic and phylogenetic implications[END_REF]Scherler et al. 2019;[START_REF] Lihoreau | The Libycosaurus (Hippopotamoidea, Artiodactyla) intercontinental dispersal event at the early late Miocene revealed by new fossil remains from Kasserine area, Tunisia[END_REF][START_REF] Grossman | First record of Sivameryx (Cetartiodactyla, Anthracotheriidae) from the lower Miocene of Israel highlights the importance of the Levantine Corridor as a dispersal route between Eurasia and Africa[END_REF]. Anthracotheres are assumed to correspond to stem Hippopotamidae (Boisserie et al. 2005a[START_REF] Boisserie | Morphology and phylogenetic relationships of the earliest known hippopotamids (Cetartiodactyla, Hippopotamidae, Kenyapotaminae)[END_REF][START_REF] Boisserie | Emergence of Hippopotamidae: new scenarios[END_REF][START_REF] Orliac | Early Miocene hippopotamids (Cetartiodactyla) constrain the phylogenetic and spatiotemporal settings of hippopotamid origin[END_REF][START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF]. Currently 27 genera are recognized and distributed in three subfamilies: the Anthracotheriinae, the Microbunodontinae, and the Bothriodontinae [START_REF] Lihoreau | Family Anthracotheriidae[END_REF], including animals with a great variety of sizes, from small (< 25 kg) to medium and large (> 1,000 kg) forms. Anthracotheriinae and Bothriodontinae were medium to very large-sized animals, whereas Microbunodontinae were exclusively small-sized anthracotheres with slender limbs. Anthracotheres have been previously considered as essentially semi-aquatic hippo-like forms (e.g., [START_REF] Falconer | Note of the fossil camel on the Sivalik Hills[END_REF][START_REF] Rütimeyer | Über Anthracotherium magnum und hippoideum[END_REF]. More recent studies have highlighted their great diversity of habitats and diets [START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF] and that anthracotheres can be used as ecological markers to reconstruct the paleoenvironmental history of the landmasses they occupied [START_REF] Lihoreau | Family Anthracotheriidae[END_REF]. Hippopotamids are first known from the early Miocene in Africa, and then spread to Asia and Europe [START_REF] Boisserie | Family Hippopotamidae[END_REF][START_REF] Orliac | Early Miocene hippopotamids (Cetartiodactyla) constrain the phylogenetic and spatiotemporal settings of hippopotamid origin[END_REF]). These small-sized (~30 kg for Morotochoerus; [START_REF] Orliac | Early Miocene hippopotamids (Cetartiodactyla) constrain the phylogenetic and spatiotemporal settings of hippopotamid origin[END_REF] to very large (mean weight of 1500 kg for the extant genus Hippopotamus; [START_REF] Klingel | Hippopotamus amphibius -common hippopotamus[END_REF] animals are assumed to have adopted semi-aquatic habits in the latest Miocene [START_REF] Boisserie | Family Hippopotamidae[END_REF]Boisserie et al. 2011).

Semi-aquatic forms occurred independently in various hippopotamoid lineages, so that the question of the habitat of numerous fossil taxa, and notably their degree of water dependence, remains unresolved [START_REF] Lihoreau | Family Anthracotheriidae[END_REF]Boisserie et al. 2011;[START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF][START_REF] Grandi | Prominatherium dalmatinum from the late Eocene of Grancona (Vicenza, NE Italy). The oldest terrestrial mammal of the Italian peninsula[END_REF] and cannot be answered by sedimentological and morphological data only. For this purpose, various approaches can be added, such as studies on sensorial abilities (e.g., [START_REF] Orliac | Osteological connections of the petrosal bone of the extant Hippopotamidae Hippopotamus amphibius and Choeropsis liberiensis[END_REF], on dental microwear and mesowear (e.g., [START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF][START_REF] Merceron | Can dental microwear textures record inter-individual dietary variations?[END_REF][START_REF] Boisserie | Correlating the success of Hippopotaminae with the C4 grass expansion in Africa: relationship and diet of early Pliocene hippopotamids from Langebaanweg, South Africa[END_REF][START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF], and isotopic investigations (e.g., Boisserie et al. 2005a;[START_REF] Nelson | Isotopic reconstructions of habitat change surrounding the extinction of Sivapithecus, a Miocene hominoid, in the Siwalik Group of Pakistan[END_REF][START_REF] Clementz | Identifying aquatic habits of herbivorous mammals through stable isotope analysis[END_REF][START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF][START_REF] Tütken | Late Oligocene ambient temperatures reconstructed by stable isotope analysis of terrestrial and aquatic vertebrate fossils of Enspel, Germany[END_REF]. Morphofunctional studies on the postcranial skeleton are also required. However, to date, such data relative to hippopotamoids are extremely limited. They consist of limited studies on the skeleton of Hippopotamus amphibius (Gratiolet 1867) and fossil taxa (Kowalesky 1873;Scott and Jepsen 1940;[START_REF] Dineur | Le genre Brachyodus, Anthracotheriidae (Artiodactyla, Mammalia) du Miocène inférieur d'Europe et d'Afrique[END_REF]Pickford 2008), and on detailed descriptions of the forelimb and hind limb myology of Choeropsis liberiensis and H. amphibius, respectively [START_REF] Fisher | Forelimb myology of the pygmy hippopotamus (Choeropsis liberiensis)[END_REF][START_REF] Tsubamoto | Recognition of Microbunodon (Artiodactyla, Anthracotheriidae) from the Eocene of China[END_REF]. In addition, there are data on swimming and aquatic bottom-walking abilities in H. amphibius [START_REF] Coughlin | Hippopotamus underwater locomotion: reduced-gravity movements for a massive mammal[END_REF][START_REF] Endo | Three-dimensional CT examination of the forefoot and hindfoot of the hippopotamus and tapir during a semiaquatic walking[END_REF].

Beyond studies on bone shape and muscle insertions, the analysis of bone microanatomical adaptations can also be very useful for paleobiological inferences. Indeed, bone inner structure reflects the functional constraints imposed on the skeleton and thus the functional requirements of the organism (e.g., Ruff 1992;[START_REF] Ruimerman | A theoretical framework for strain-related trabecular bone maintenance and adaptation[END_REF][START_REF] Volpato | Textural characteristics of the iliac-femoral trabecular pattern in a bipedally trained Japanese macaque[END_REF][START_REF] Mielke | Trabecular architecture in the sciuromorph femoral head: allometry and functional adaptation[END_REF]. The form-function relationships of bone microanatomy have been widely used in the context of adaptation to an aquatic lifestyle, focusing on both aquatic and semi-aquatic forms (e.g., [START_REF] Canoville | Evolution of humeral microanatomy and lifestyle in amniotes, and some comments on palaeobiological inferences[END_REF][START_REF] Dumont | Inner architecture of vertebral centra in terrestrial and aquatic mammals: a two-dimensional comparative study[END_REF][START_REF] Amson | Gradual adaptation of bone structure to aquatic lifestyle in extinct sloths from Peru[END_REF]Diaz-Berenguer et al. 2019;[START_REF] Klein | Microstructure of vertebrae, ribs, and gastralia of Triassic sauropterygians-new insights into the microanatomical processes involved in aquatic adaptations of marine reptiles[END_REF]). Yet, hippopotamoids also include large quadrupedal taxa. Adaptations to heavy weight bearing are also observed in the bone microanatomy of large quadrupeds [START_REF] Wall | The correlation between high limb-bone density and aquatic habits in recent mammals[END_REF]Houssaye et al. 2016a). However, they can be hard to decipher from those linked to a semi-aquatic lifestyle, as both are characterized by an increase in bone compactness [START_REF] Cooper | Aquatic habits of cetacean ancestors: integrating bone microanatomy and stable isotopes[END_REF]Houssaye et al. 2016b). Indeed, both semi-aquatic taxa swimming or walking on the bottom at shallow depths and heavy terrestrial quadrupeds generally exhibit an increased compactness characterized by a more or less extensive thickening of the cortex and filling of the medullary cavity by cancellous bone [START_REF] Nakajima | Comparative humeral microanatomy of terrestrial, semiaquatic, and aquatic carnivorans using micro-focus CT scan[END_REF]Houssaye et al. 2016a,c;[START_REF] Houssaye | From land to water: evolutionary changes in long bone microanatomy of otters (Mammalia: Mustelidae)[END_REF]. The increased bone mass in semi-aquatic taxa is assumed to hydrostatically counter buoyancy to dive and stay at shallow depths but also to counter waves to improve stability in rough waters [START_REF] Wall | The correlation between high limb-bone density and aquatic habits in recent mammals[END_REF][START_REF] Taylor | Functional significance of bone ballast in the evolution of buoyancy control strategies by aquatic tetrapods[END_REF]. Conversely, in heavy animals, such a structure is believed to increase bone strength and improve the absorption of impact energy [START_REF] Oxnard | From giant ground sloths to human osteoporosis: an essay on the architecture and biomechanics of bone[END_REF][START_REF] Augat | The role of cortical bone and its microstructure in bone strength[END_REF]. Investigations on phylogenetically close taxa showing light and massive terrestrial and semi-aquatic forms thus constitute a great opportunity to better characterize these microanatomical specializations that have convergently evolved multiple times in heavy and/or semi-aquatic (and aquatic) amniotes (Houssaye et al. 2016a,c). Only limited microanatomical data are available for hippopotamoids beyond the mid-diaphyseal sections of modern hippopotamids [START_REF] Cooper | Aquatic habits of cetacean ancestors: integrating bone microanatomy and stable isotopes[END_REF]Houssaye et al. 2016a). [START_REF] Cooper | Aquatic habits of cetacean ancestors: integrating bone microanatomy and stable isotopes[END_REF] investigated the microanatomical features of three extinct hippopotamoid species from two genera, the microbunodontine Microbunodon silistrense and the bothriodontines Merycopotamus dissimilis and Merycopotamus medioximus, in order to infer their paleoecology, suggesting a terrestrial lifestyle for Mi. silistrense and a semi-aquatic one for the two Merycopotamus species.

The objectives of the present study are to investigate the microanatomical adaptive features of hippopotamoid bones by analyzing the inner structure of limb long bones of diverse hippopotamoids supposed to illustrate various degrees of water dependency, taking into account their body proportions, and to discuss inferences related to the paleoecology of the sampled fossil forms.

It is predicted that 1) the bones of terrestrial forms will show a tubular organization, i.e., a compact cortex surrounding an empty medullary cavity, typical of terrestrial amniotes [START_REF] Laurin | Bone microanatomy and lifestyle: a descriptive approach[END_REF]Houssaye et al. 2018), 2) the bones will be more compact in the largest terrestrial species, with a thicker cortex and some trabeculae in the medullary area, 3) the species most dependent on the aquatic environment will also show more compact bones, and 4) the species that are both large and highly dependent on the aquatic environment will be much more compact.

Material and Methods

Material

The material examined consists of the humeri and femora of various hippopotamoids.

Stylopod bones are assumed to bear a strong morphofunctional signal in their microanatomy (e.g., Quemeneur et al. 2013). They were therefore selected in order to investigate the long bone microanatomical adaptations related to the posture and lifestyle of these extinct taxa.

Moreover, most of the comparative data for ungulates are available only for these bones.

Fossil material includes the bones of four hippopotamoid subfamilies (Tables1, 2). (Cuvier 1822) is a microbunodontine from the late Oligocene of Europe. The sampled specimens are from the collections from La Milloque, France (Lihoreau et al. 2004). This small-sized species (around 20 kg) is lightly built with long and slender limbs, the hind limb being longer and more robust than the forelimb [START_REF] Cabard | Mammalia, Artiodactyla, Anthracotheriidae) de l'Oligocène supérieur d'Europe de l'Ouest[END_REF][START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF]Lihoreau et al. 2004;[START_REF] Lihoreau | Family Anthracotheriidae[END_REF]. Microbunodon is assumed to be terrestrial and to have lived in closed habitats (dense forest) based on its morphology (e.g., low orbits, no specialization for subaquatic hearing, autopod showing a digitigrade stance, weakly elongated metapodials ;[START_REF] Cabard | Mammalia, Artiodactyla, Anthracotheriidae) de l'Oligocène supérieur d'Europe de l'Ouest[END_REF][START_REF] Vignaud | Geology and palaeontology of the Upper Miocene Toros-Menalla hominid locality, Chad[END_REF][START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF][START_REF] Lihoreau | Family Anthracotheriidae[END_REF], which is corroborated at the generic level by micro-and mesowear data, and isotopic studies [START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF][START_REF] Lihoreau | Family Anthracotheriidae[END_REF][START_REF] Nelson | Isotopic reconstructions of habitat change surrounding the extinction of Sivapithecus, a Miocene hominoid, in the Siwalik Group of Pakistan[END_REF].

Microbunodon minimum

The sampled anthracotheriine is Paenanthracotherium bergeri, from the Oligocene of La Bénissons-Dieu, France. It is large (< 950 kg; estimation based on talus measurement following Martinez and Sudre [1995]) and heavily-built with short and robust limb bones (Roman and Boucher 1936) and may have lived in swamps as was historically supposed for the closely related Anthracotherium (Cuvier 1822). However, the low position of its orbits on the skull (see plates in Roman and Boucher 1936;Scherler et al. 2019), though not incompatible with a semi-aquatic lifestyle, does not indicate a hippo-like lifestyle at the interface between air and water.

Within Bothriodontinae, Bothriodon velaunus, from the early Oligocene of Ronzon, France [START_REF] Lihoreau | Family Anthracotheriidae[END_REF]) is a medium sized animal (< 150 kg; estimation based on talus measurement following Martinez and Sudre [1995]) displaying a lightly-built skeleton with gracile legs [START_REF] Filhol | Etude des mammifères fossiles de Saint-Gerand le Puy (Allier)[END_REF][START_REF] Lihoreau | Family Anthracotheriidae[END_REF]. Though little is known about its ecology, its diet is supposed to have been predominantly folivorous, based on cusp morphology [START_REF] Lihoreau | Family Anthracotheriidae[END_REF]. It is similar to Elomeryx borbonicus (<130 kg; estimated from Martinez and Sudre [1995]), from the late Oligocene of Saint-Henri/Saint-André, Marseille, France [START_REF] Lihoreau | Family Anthracotheriidae[END_REF]. The hind limbs of Elomeryx borbonicus are longer and more robust than its forelimbs [START_REF] Geais | Le Brachyodus borbonicus des argiles de St-Henri (près Marseille)[END_REF]. If the proposed occurrence of webbed feet [START_REF] Geais | Le Brachyodus borbonicus des argiles de St-Henri (près Marseille)[END_REF]) suggests a semi-aquatic lifestyle, supposedly in marshy habitats, the position of the superior border of the orbit and the morphology of the tympanic bulla and of the auditory tube are typical of terrestrial animals [START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF].

Libycosaurus bahri, from the late Miocene of Toros-Ménalla, Chad [START_REF] Vignaud | Geology and palaeontology of the Upper Miocene Toros-Menalla hominid locality, Chad[END_REF], displays a heavy (< 1,600 kg) hippo-like morphology with short and stocky limbs, although being more elongated and slender than those of the common hippopotamus [START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF]. This sexually body-mass-dimorphic taxon appears to be semi-aquatic, based among others on its highly elevated orbits and external nares, the morphology of the tympanic bulla, and on isotopic studies [START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF][START_REF] Boisserie | Emergence of Hippopotamidae: new scenarios[END_REF]. Finally, Brachyodus onoideus, from the early Miocene of Nancray and Neuville, near Orléans, France [START_REF] Lihoreau | Family Anthracotheriidae[END_REF], is much larger (< 2,350 kg; estimate based on talus measurement following Martinez and Sudre [1995]). This large-sized species displays strong limb bones albeit being more elongated and slender than those of the common hippo [START_REF] Dineur | Le genre Brachyodus, Anthracotheriidae (Artiodactyla, Mammalia) du Miocène inférieur d'Europe et d'Afrique[END_REF]. A terrestrial mode of life has been suggested for this species based on the conformation of its limbs [START_REF] Dineur | Le genre Brachyodus, Anthracotheriidae (Artiodactyla, Mammalia) du Miocène inférieur d'Europe et d'Afrique[END_REF] but the amphibious position of the sensory organs on the head along with the morphology of the tympanic bulla potentially enabling underwater directional hearing rather suggest hydrophilic and water immersive habits (Orliac et al. 2013). Trackways from the lower Miocene of Spain possibly attributable to this species seem to indicate a gregarious behavior close to that of hippos with trails in palustrine environments (Diaz Martinez et al. 2020).

Within hippopotamines, Saotherium cf. S. mingoz [START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF], from the early Pliocene of Kossom Bougoudi, Chad [START_REF] Boisserie | Family Hippopotamidae[END_REF][START_REF] Boisserie | Morphology and phylogenetic relationships of the earliest known hippopotamids (Cetartiodactyla, Hippopotamidae, Kenyapotaminae)[END_REF], is of intermediate size (<1,000 kg; estimated from Martinez and Sudre [1995]) between the extant C. liberiensis (< 275 kg) and H. amphibius (< 2,000 kg). It does not show adaptations to a semi-aquatic lifestyle [START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF]Boisserie 2005[START_REF] Boisserie | Family Hippopotamidae[END_REF]; Weston and Boisserie 2010) but isotopic data suggest water dependence [START_REF] Zazzo | Herbivore paleodiet and paleoenvironmental changes in Chad during the Pliocene using stable isotope ratios of tooth enamel carbonate[END_REF]. As for Hexaprotodon garyam, from the late Miocene of Toros-Ménalla, Chad [START_REF] Boisserie | Family Hippopotamidae[END_REF][START_REF] Vignaud | Geology and palaeontology of the Upper Miocene Toros-Menalla hominid locality, Chad[END_REF] Weston and Boisserie 2010), weighting up to 2,200 kg [START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF]estimated from Martinez and Sudre [1995]), it displays a set of morphological [START_REF] Vignaud | Geology and palaeontology of the Upper Miocene Toros-Menalla hominid locality, Chad[END_REF] and isotopic features [START_REF] Jacques | Les préférences écologiques (paléorégimes alimentaires, paléohabitats) des grands mammifères herbivores des sites à hominidés du Miocène supérieur du Nord Tchad. Reconstitution au moyen de l'analyse isotopique en carbone et oxygène du carbonate de l'émail dentaire[END_REF]) that might indicate a semi-aquatic lifestyle [START_REF] Vignaud | Geology and palaeontology of the Upper Miocene Toros-Menalla hominid locality, Chad[END_REF]Boisserie et al. 2005b;[START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF]. The extant hippopotamine, C. liberiensis, about six times lighter than H. amphibius, is more gracile with slender elongated limbs (Eltringham 1999;Nowak and Paradiso 1983;Robinson, 2013), whereas H. amphibius displays a massive barrelshaped body supported by a heavily-built skeleton with short and stocky limbs (Eltringham 1999;[START_REF] Klingel | Hippopotamus amphibius -common hippopotamus[END_REF]Nowak and Paradiso 1983). Common hippopotamuses inhabit wetlands with permanent water bodies deep enough to enable immersion, such as lakes, rivers, pools, swamps, marshes, and ponds, in the vicinity of reed beds and grasslands (Eltringham 1999;[START_REF] Klingel | Hippopotamus amphibius -common hippopotamus[END_REF]Nowak and Paradiso 1983). Choeropsis inhabits wetter, closer, and denser environments than the common hippopotamus, which is consistent with its less webbed toes and its nocturnal habits. It lives close to streams, rivers, or swamps, and can dive, thus showing a lifestyle close to that of extant forest tapirs (Eltringham 1999;Nowak and Paradiso 1983;Robinson 2013), despite additional adaptations for a semi-aquatic lifestyle, such as the ability to occlude ears and nostrils when diving.

The comparative material includes terrestrial and semi-aquatic ungulates covering and extending the possible ecologies as well as the size range of the hippopotamoid sample (see Table 1; Fig. 1). It includes the basal cetancodont Indohyus indirae and archaeocetes belonging to the Pakicetidae, Remingtonocetidae, and Protocetidae, in order to illustrate various ecological steps in the progressive adaptation to an aquatic lifestyle in the cetacean lineage. It also includes small to medium-sized even-toed ungulates with long and slender legs (e.g., Hyemoschus aquaticus, Potamochoerus porcus), but also medium-to large-sized even-toed and odd-toed ungulates, from gracile (e.g., Giraffa Camelopardalis, Alces alces) to heavily-built (e.g., Syncerus caffer, Rhinoceros unicornis) animals (Table 1).

Methods

Data acquisition

Bones were scanned using high-resolution computed tomography at: 1) the IC2MP, University of Poitiers (UMR 7285; EasyTom XL duo, RX Solutions), 2) the ISEM, University of Montpellier (UMR 5554; EasyTom 150, RX Solutions) 3) the Steinmann-Institut, University of Bonn (Germany; GE phoenix|X-ray v|tome|xs 240), 4) the AST-RX platform of the Muséum National d'Histoire Naturelle, Paris (UMS 2700; GE phoenix|X-ray v|tome|xs 240), and 5) the Equine Diagnostic Imaging Centre at the Royal Veterinary College, London (GE Lightspeed), with reconstructions performed using X-Act (RX Solutions), DATOX/RES, phoenix datos|x or MEDVIEW software (MedImage). Voxel size naturally varies pending on specimen size (Table 1).

Transverse virtual thin-sections were made for each bone at the plane assumed to cross the growth center (where the cortex is the thickest in the case of a non-uniform thickness, see Houssaye et al. 2018, and at the intersection of the nutrient arteries the central axis of the medullary cavity, see Houssaye and Prévoteau 2020). Sagittal and coronal virtual sections were made at the planes crossing the core of the transverse section. Image visualization and virtual sections were performed using VGSTUDIOMAX, versions 2.2 (Volume Graphics Inc.).

Data analysis

Quantitative parameters were used to describe the transverse sections, except those of extinct taxa showing an insufficient contrast between bone and the infilling sediment (see Table 1).

The choice of parameters follows Houssaye et al. (2018), using the BoneJ plugin (Doube et al.

2010) of ImageJ (Wayne Rasband National Institutes of Health, USA) and the software Bone

Profiler [START_REF] Girondot | Bone profiler: a tool to quantify, model, and statistically compare bone-section compactness profiles[END_REF]: A) CSAb (Cross-Sectional Area of bone), representing the surface occupied by bony tissue; B) CSAt (total Cross-Sectional Area), as the surface of the whole transverse section; C) C (Compactness), as the ratio of CSAb over CSAt; D) R, as the radius of the section approximated as a circle calculated based on the perimeter/2π, and used as a proxy of size; E) CSS (Cross-Sectional Shape), as the ratio between the maximal and minimal second moments of area (Imax/Imin); F) Zpol (Polar Section Modulus) representing the resistance of a section to torsion and bending (see [START_REF] Ruff | Long bone articular and diaphyseal structure in Old World monkeys and apes. I: locomotor effects[END_REF][START_REF] Ksepka | Bone histology in extant and fossil penguins (Aves: Sphenisciformes)[END_REF]; G) RMeanT, as the relative mean thickness of cortical bone (after separation by segmentation of the cortical and medullary areas), calculated as the absolute mean thickness of cortical bone divided by R; H) RSDT, as the relative standard deviation of cortical bone thickness; I) P: the extent of the medullary cavity as measured by the relative distance from the center of the section to the point where the most abrupt change in compactness occurs; and J) S: the width of the transitional zone between the compact cortex and the medullary cavity as measured by the reciprocal of the slope of the compactness profile at the inflection point.

We tested the influence of size on all parameters by performing linear regressions of each parameter to R (lm function). We comparatively analyzed the values of each parameter for the hippopotamoids separately, all rhinos, the (dominantly) terrestrial and the (dominantly) aquatic ungulates (Table 1;Figs S2

; S4).
We tested the phylogenetic signal for each parameter on the raw data. For that we averaged the values obtained for each species when several specimens from the same species were available. Then we calculated the K-statistic following [START_REF] Blomberg | Testing for phylogenetic signal in comparative data: behavioral traits are more labile[END_REF] for each parameter and performed randomization tests. The K-statistic compares the observed phylogenetic signal in a trait with the signal under a Brownian motion model of trait evolution. A K-value > 1 implies more similarity between relatives than expected under Brownian motion; K < 1 highlights convergences.

For this testing, we used two different consensus trees (Fig. 1) based on phylogenies from [START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF][START_REF] Lihoreau | The Libycosaurus (Hippopotamoidea, Artiodactyla) intercontinental dispersal event at the early late Miocene revealed by new fossil remains from Kasserine area, Tunisia[END_REF], Boisserie et al. (2011[START_REF] Boisserie | Basal hippopotamines from the upper Miocene of Chorora, Ethiopia[END_REF], and Gomes [START_REF] Rodrigues | Unexpected evolutionary patterns of dental ontogenetic traits in cetartiodactyl mammals[END_REF] for the relationships within Hippopotamoidea, the proposition of [START_REF] Thewissen | Whales originated from aquatic artiodactyls in the Eocene epoch of India[END_REF] and [START_REF] Vautrin | From limb to fin: an Eocene protocetid forelimb from Senegal sheds new light on the early locomotor evolution of cetaceans[END_REF] for the position of Indohyus and Cetacea, and recent phylogenetic hypotheses based on molecular data for extant representatives of modern clades of laurasiatherian mammals [START_REF] Gilbert | Mitochondrial and nuclear phylogenies of Cervidae (Mammalia, Ruminantia): systematics, morphology, and biogeography[END_REF][START_REF] Bibi | A multi-calibrated mitochondrial phylogeny of extant Bovidae (Artiodactyla, Ruminantia) and the importance of the fossil record to systematics[END_REF][START_REF] Foley | Mammal madness: is the mammal tree of life not yet resolved?[END_REF][START_REF] Soe | New remains of Siamotherium pondaungensis (Cetartiodactyla, Hippopotamoidea) from the Eocene of Pondaung, Myanmar: paleoecologic and phylogenetic implications[END_REF]). These phylogenies differ by the relationships observed within Hippopotamoidea. The first hypothesis links Hippopotamidae to archaic bothriodontines, i.e., the African Paleogene clade including Bothriogenys and Brachyodus. The second hypothesis proposes the Hippopotamidae as sister-group of the whole Bothriodontinae.

In order to analyze quantitatively the distribution of the different specimens in the microanatomical morphospace, but also for the purpose of evaluating how the different microanatomical and cross-sectional parameters explain the variations observable in our sample, we conducted normalized PCAs (David and Jacobs 2014).

All statistical analyses were performed using the statistical software R (R Core Team 2014).

Results

Humerus

Qualitative description

Humeral sections of Choeropsis do not show a particular thickening of the cortex (Figs. 2A-B, 3A), as compared to most quadrupedal mammals (see Houssaye et al. 2018;[START_REF] Canoville | Evolution of humeral microanatomy and lifestyle in amniotes, and some comments on palaeobiological inferences[END_REF]; Fig. S1). However, whereas these taxa show a generally tubular diaphysis with a compact cortex surrounding an empty medullary cavity, the humerus medullary space of Choeropsis is filled with a spongious tissue. The thickness of the cortex increases distal to the deltoid tuberosity but is then rather homogeneous in the rest of the diaphysis. The organization appears rather similar in Saotherium (Figs. 2C, 3B), Libycosaurus (Figs. 2J,3H) and in one specimen of Hexaprotodon (TM 55-XX-05; Figs. 2F,3D), but also in Brachyodus (Figs. 2H,3F), though its spongiosa is poorly preserved. The spongiosa is also poorly preserved in Bothriodon whose cortex is thinner than in the other taxa (Figs. 2I,3G). In Hippopotamus the thickness of the cortex varies along the diaphysis distal to the deltoid tuberosity, with a pronounced thickening around the growth center (Figs. 2D-E, 3C). As in Choeropsis a spongiosa fills the medullary area. Such a thickening of the cortex is also observed in one specimen of Hexaprotodon garyam (TM 258-01-27; Figs. 2G,3E) and, to a lesser extent, in Paenanthracotherium (Figs. 2M,3K). The spongiosa in the medullary cavity of the later is not preserved or was absent. As for Elomeryx and Microbunodon, they show a tubular organization (Figs. 2K-L, 3I-J). As for Merycopotamus, sections of Me. medioximus (Lihoreau et al. 2004) from [START_REF] Cooper | Aquatic habits of cetacean ancestors: integrating bone microanatomy and stable isotopes[END_REF] show an organization close to that of Elomeryx and Microbunodon (Fig. S1A) but that of Me. dissimilis [START_REF] Falconer | Note of the fossil camel on the Sivalik Hills[END_REF] is filled with some trabeculae (Fig. S1B).

Quantitative comparisons

Compactness (C) is the highest in Hippopotamus. It is higher than in rhinos and in Hexaprotodon (TM 258-01-27), which show higher values than the other taxa (even than most aquatic ones; Fig. S2A). Choeropsis globally displays a higher compactness than the other terrestrial ungulates, but it is much lower than in rhinos. The other hippopotamoids show compactness values slightly (especially Brachyodus) lower than those of Choeropsis.

The cross-sectional shape (CSS) is strongly higher in aquatic taxa (Fig. S2B); as for the others, it is maximal in the comparative terrestrial taxa and globally much higher in rhinos, Hippopotamus, Microbunodon, and Bothriodon than in Choeropsis, Elomeryx, Brachyodus, and Hexaprotodon, which thus present more rounded sections (Fig. S2C). The relative mean cortical thickness (RMeanT) is the highest in Hippopotamus and Hexaprotodon (TM 258-01-27). It varies strongly within rhinos where it is generally higher than in other terrestrial ungulates. RMeanT is much higher in Brachyodus than in Choeropsis, Elomeryx, Bothriodon, Microbunodon, and the comparative terrestrial and aquatic taxa (Fig. S2D). The cortical thickness is rather homogeneous in most terrestrial and in aquatic taxa, which show the lowest RSDT values (Fig. S2E). This is also the case for Microbunodon, Elomeryx, and Bothriodon.

However it is slightly higher in Choeropsis and much higher in Brachyodus and even more in Hippopotamus and Hexaprotodon garyam . RSDT varies strongly among rhinos, from values close to those of Brachyodus to values higher to those of Hippopotamus.

The extent of the medullary cavity (P) is the highest in Bothriodon, which shows values similar to most terrestrial forms (Fig. S2F). P is rather similar in Microbunodon, Elomeryx, Choeropsis, and Brachyodus, being slightly lower than in most terrestrial taxa. It is lower in rhinos and Hexaprotodon, and clearly low in Hippopotamus. Values vary strongly in aquatic taxa. S, the transition zone (between the compact cortex and the medullary cavity), is rather similar in Choeropsis, Microbunodon, Elomeryx, and Hexaprotodon compared to most other terrestrial ungulates (Fig. S2G). It is higher in rhinos and much higher in Hippopotamus, and intermediate between Choeropsis and rhinos for Bothriodon and Brachyodus. It varies a lot among aquatic taxa. As for the polar section modulus (Zpol), it varies a lot within rhinos where it is the highest but is extremely constant in Hippopotamus and Choeropsis. It is high in Hippopotamus, Brachyodus, and Hexaprotodon and very high in rhinos (Fig. S2H).

Quantitative analyses

All parameters, except Zpol (in mm 3 ), are dimensionless ratios. All parameters except CSS are correlated with size (Table 3). This shows, beyond a size effect, a strong allometry in the data, except for the shape of the diaphyseal sections (CSS).

All parameters show a significant phylogenetic signal (at 5%; Table 3), whatever the chosen phylogeny (Fig. 1), with K values however always below 1. This signal, as significant in R, could be partly associated with the allometry, but not only, as CSS is not significantly correlated with size.

A first PCA was performed on the humerus data but as the specimen of Maiacetus was driving too much the variation along PC2, another one was conducted without this specimen.

The two first axes of the PCA on humerus data represent 78.30% of the variance with 64.55 for the first axis and 13.75% for the second one. They are both correlated with size (Table 3) and commented hereafter. The first axis rather clearly discriminates most terrestrial ungulates (negative values) from semi-aquatic and heavy ones (Fig. 4). Specimens of Hippopotamus group together on the positive part. Specimens of Choeropsis are rather intermediary between most terrestrial ungulates and the heavy and semi-aquatic ones. Both specimens of Microbunodon and the specimen of Bothriodon clearly group with most terrestrial ungulates; it is the same for both specimens of Elomeryx, which are close to Choeropsis'. Conversely, the two specimens of Brachyodus are between Choeropsis and rhinos. The specimen of Hexaprotodon TM 258-01-27 is very close to Hippopotamus. The first axis is essentially driven by RSDT, P, C, and Zpol (RMeanT and S to a slightly lower extent), which all show allometry. Zpol and S covary, and P varies antagonistically with C. This axis shows that larger humeri show a higher compactness (C), a reduced medullary area (P, RMeanT), resulting in a higher resistance to bending and torsion (Zpol), but also a wider transition zone between the compact cortex and the medullary cavity (S), associated with the filling of the medullary cavity by a spongiosa, and also a higher variation of the cortical thickness in the transverse section (RSDT), which means a more heterogeneous growth in width of the bone.

The second axis poorly discriminates groups. This axis is essentially driven by the crosssectional geometry (CSS), and to a lesser extent by the transition zone (S) and Zpol.

Femur

Qualitative analyses

Choeropsis shows a femoral inner organization similar to that of the humerus, except that the trabecular network appears much looser in most of the diaphysis (Fig. 5A-B). In Hippopotamus, the cortex is thicker than in Choeropsis but its thickness is rather homogeneous along the diaphysis (Fig. 5C-D). Both taxa show a medullary space partially filled by a loose spongiosa. Conversely, in all the other hippopotamoids sampled, the medullary space appears empty (Figs. 5E-J, 6C-H) so that their organization is tubular. Only the thickness of the cortical layer varies, being the lowest in Elomeryx, whose section is similar to those of Me. medioximus and Mi. silistrense (Cooper et al. 2006).

Quantitative comparisons

Femoral compactness in transverse sections of Elomeryx is rather similar to that of most terrestrial ungulates (Fig. S3). However, it is progressively higher in Choeropsis, Hexaprotodon, rhinos, Brachyodus, Libycosaurus, and Hippopotamus that show the highest compactness values, with aquatic ungulates (Fig. S4). CSS values are much higher in rhinos than in all other taxa; hippopotamoids show values similar to (e.g., Elomeryx) or lower (especially Choeropsis and Hippopotamus) than those of the terrestrial ungulates. Elomeryx, like rhinos and most terrestrial ungulates, has rather low values of relative cortical thickness (RMeanT), which are minimal in the aquatic ungulates. Values are higher in Choeropsis and Libycosaurus, and much higher in Hippopotamus, Brachyodus, and Hexaprotodon. RSDT is rather low in Elomeryx, most terrestrial and aquatic ungulates. It is slightly higher in Choeropsis and progressively much higher in Hexaprotodon, Libycosaurus, and rhinos, Hippopotamus, and Brachyodus. The size of the medullary cavity in Elomeryx is like in most terrestrial ungulates, whereas it is smaller in rhinos and Choeropsis, and much smaller in Brachyodus, Hexaprotodon, Libycosaurus, and Hippopotamus. It is minimal in aquatic taxa.

The transition zone (S) is much wider in rhinos than in all other taxa and minimal for most terrestrial ungulates. As for the polar section modulus (Zpol), it is similar as in most terrestrial ungulates in Choeropsis, Elomeryx, and aquatic ungulates, but higher in Hippopotamus and Libycosaurus, much higher in rhinos and Hexaprotodon, and very high in Brachyodus.

Quantitatives analyses

In the femora, all variables, except CSS, are significantly correlated with size (Table 4).

All parameters show a significant phylogenetic signal, for both phylogenies (Table 4), with high K values for C and, to a lesser extent, P. Again, the signal could be partly associated with the allometry.

The first two axes represent 72.93% (54.07, and 18.86%, respectively) of the variance for the femur data. Only PC1 is correlated with size (Table 4). Again, C and RMeanT vary antagonistically to P. Zpol covaries with RSDT. As for the humerus, PC1 distinguishes most terrestrial ungulates from rhinos and massive hippopotamoids (Fig. 7). Choeropsis specimens, like those of Tapirus terrestris and some large terrestrial ungulates (e.g., Alces americanus, Bubalus bubalis, Syncerus caffer), are intermediate between most terrestrial ungulates and rhinos and large hippopotamoids. Elomeryx groups with most terrestrial ungulates, whereas Libycosaurus and Hexaprotodon are between Choeropsis and Hippopotamus, and Brachyodus gathers with Hippopotamus. The first axis essentially discriminates based on RSDT, C, P, Zpol, and RMeanT. Bones on the positive part of the axis show a higher compactness, a reduced medullary cavity, and a wider transition zone, with a cortical thickness being more heterogeneous along the transverse section. As it is correlated with size (but not only), this trend can be generalized to femora when they become larger in our sample. Along the second axis, rhinos are clearly distinct from hippopotamoids and from most terrestrial ungulates.

They group with tapirs and the aquatic Remingtonecetus. The second axis is essentially driven by CSS and S and, to a lesser extent, by RMeanT.

Discussion

Hippopotamoid microanatomical features

Humerus

This study reveals a high variability in humerus microanatomy within hippopotamoids. It clearly shows for the first time, thanks to longitudinal sections, that the medullary area of Choeropsis liberiensis is completely filled by a spongious tissue. This is also the case for Hippopotamus amphibius, as previously shown in [START_REF] Wall | The correlation between high limb-bone density and aquatic habits in recent mammals[END_REF] and in other hippopotamoids.

It is clear in Saotherium cf. S. mingoz, Hexaprotodon garyam, and Libycosaurus bahri, likely in Brachyodus onoideus, but it is unclear for Bothriodon velaunus and Paenanthracotherium bergeri. A spongious medullary area occurs in only a few terrestrial mammals with most of them showing a tubular organization [START_REF] Canoville | Evolution of humeral microanatomy and lifestyle in amniotes, and some comments on palaeobiological inferences[END_REF][START_REF] Laurin | Bone microanatomy and lifestyle: a descriptive approach[END_REF]Houssaye et al. 2018; Fig. S1). The spongiosa is tighter and more extended along the whole diaphysis in these hippopotamoids than in some sloths, anteaters and armadillos (Montanez-Rivera et al. 2018) as well as some otters [START_REF] Houssaye | From land to water: evolutionary changes in long bone microanatomy of otters (Mammalia: Mustelidae)[END_REF], but rather close to what is observable in tapirs (AH, pers. obs.), the giant anteater, large ground sloths [START_REF] Amson | The postcranial musculoskeletal system of xenarthrans: insights from over two centuries of research and future directions[END_REF], and large otters [START_REF] Houssaye | From land to water: evolutionary changes in long bone microanatomy of otters (Mammalia: Mustelidae)[END_REF], whereas it is tighter in rhinos (AH, pers. obs.). The thickening of the cortex near the growth center in the humerus of Hippopotamus and one specimen of Hexaprotodon garyam is similar to what is observed in the sea otter [START_REF] Houssaye | From land to water: evolutionary changes in long bone microanatomy of otters (Mammalia: Mustelidae)[END_REF], some rhinos (Fig. S1X; AH, pers. obs.), and maybe also in the ground sloth Hapalops [START_REF] Amson | The postcranial musculoskeletal system of xenarthrans: insights from over two centuries of research and future directions[END_REF]. It evokes what is observed in the astrapothere Parastrapotherium sp., though the cortex of the latter is less thick -approximating the condition in Libycosaurus bahri and Brachyodus onoideus -and in the notungulate Nesodon imbricatus, which nevertheless shows an empty medullary cavity and is maybe closer to Paenanthracotherium bergeri (Houssaye et al. 2016b). The organization in Choeropsis appears close to that of Tapirus terrestris (Fig. S1U). Elomeryx borbonicus and Mi. minimum, like most specimens of Merycopotamus (except that of Me. dissimilis; [START_REF] Cooper | Aquatic habits of cetacean ancestors: integrating bone microanatomy and stable isotopes[END_REF]; Fig. S1A-B), display a tubular inner structure rather close to that of large terrestrial mammals, like Alces and Bubalus bubalus, but also of the small semiaquatic Hyemoschus aquaticus and Indohyus indirae.

A high compactness, as compared to most terrestrial ungulates, is observed in all hippopotamoids except Elomeryx, Microbunodon, and Me. medioximus, and it is maximal in Hippopotamus and Hexaprotodon, which show a much thicker cortex around the growth center. The more massive taxa (Hippopotamus, Hexaprotodon, Brachyodus, and rhinos) show the most heterogeneous distribution of cortical bone along the transverse section, and thus a heterogeneous bone growth in diameter. Their bone structure suggests a high resistance to torsion and bending (though less than for rhinos) as indicated by their high polar section modulus values.

Femur

A spongious organization is also observed in the femur of Choeropsis and Hippopotamus. In Choeropsis, it evokes what is observed in some large otters (Lontra) and Tapirus terrestris with an empty core of the medullary area (Houssaye and Botton-Divet 2018; Fig. S3T). The spongiosa is also less tight than in Hippopotamus, where it is less tight than in rhinos (Fig. S3V-Y). Hippopotamus does not show a thickening of the cortex around the growth center as in the humerus though its cortex is thicker than in Choeropsis. All other hippopotamoids sampled display a medullary area with as much spongious deposits as in Choeropsis, or less.

There is less variability in microanatomical features among hippopotamoid femora. Most, except Elomeryx borbonicus, show a cortex thicker than in the biggest terrestrial ungulates, like Syncerus caffer, or than the putative semi-aquatic Indohyus indirae (Fig. S3B,J), and rather evoking Pteronura brasiliensis [START_REF] Houssaye | From land to water: evolutionary changes in long bone microanatomy of otters (Mammalia: Mustelidae)[END_REF]. Elomeryx We generally observe the same interspecific variations in compactness in the femur than in the humerus. All hippopotamoids except Elomeryx show a high compactness as compared to terrestrial ungulates, especially Hippopotamus, though to a much lesser extent than in the humerus (like for one specimen of Hexaprotodon). The cortex is proportionally thicker in Hippopotamus, Brachyodus, and Hexaprotodon. As for the humerus, the more massive taxa show the most heterogeneous organization along the section. Contrary to what is observed for the humerus, the cross-sectional shape (as indicated by CSS) differs significantly between hippos (whose femoral diaphysis is more cylindrical) and rhinos, which also show a larger transition zone linked to their tighter spongiosa. The bone structure of Brachyodus and Hexaprotodon (and of rhinos) suggests a higher resistance to torsion and bending than in the other taxa.

Microanatomical features linked to size

There is a phylogenetic signal in all parameters analyzed. However, as size also follows the phylogeny (whatever the phylogeny chosen), and as all parameters (except CSS, for both bones) are correlated with size, it is difficult to distinguish the phylogenetic signal from the allometry. In the quantitative analyses, the impact of size appears to be the main driver of variation. The analyses have shown that in both humeri and femora an increase in bone absolute width is associated with a relative increase in compactness, a more heterogeneous growth in width of the bone, a wider transition zone between the compact cortex and the open medullary cavity, a more reduced medullary area, and the filling of the medullary cavity by a spongiosa. This confers to the bone a higher resistance to bending and torsion. This is consistent with previous studies including heavy taxa (Houssaye et al. 2016a,b). The intensity of the observed microanatomical specializations -increase in cortical thickness and spongiosa in the medullary area -are more intense in humeri than in femora in many hippopotamoids (see below).

If the size parameter (radius of the section approximated as a circle), which reflects animal weight because long bone diameter is known to be correlated with body mass [START_REF] Anderson | Long-bone circumference and weight in mammals, birds and dinosaurs[END_REF], appears as the main driver distinguishing hippopotamoids and rhinos from other ungulates in the quantitative analyses, the qualitative observations allow to better distinguish and characterize the microanatomical specializations of the various taxa analyzed.

Indeed, qualitative observations are also based on longitudinal sections and not only transverse ones and thus cover a much larger proportion of the bone. Moreover, the quantitative parameters do not include enough information about the trabecular bone that would allow to better decipher between taxa, notably between rhinos and hippos in our case.

Ecological inferences

In Choeropsis, humeri and femora display rather similar microanatomical organizations, characteristically differing from most terrestrial ungulates by showing a filling of the medullary area by spongious bone. Both bones show microanatomical features very similar to those of Tapirus terrestris (despite a slightly thinner cortex in the latter). Both animals are of approximately the same weight and forage in similar habitats, i.e., lowland forests close to streams, rivers, or swamps [START_REF] Medici | Order Perissodactyla, Family Tapiridae (Tapirs)[END_REF]. The filling of the medullary area is also observed in other semi-aquatic mammals (e.g., large otters [START_REF] Houssaye | From land to water: evolutionary changes in long bone microanatomy of otters (Mammalia: Mustelidae)[END_REF]) and is probably associated with their frequent forays into the water.

Hippopotamus displays a thickening of the cortex -stronger in the humerus than in the femur, especially near the growth center -and the filling of the medullary area by spongious bone. This is consistent with Hippopotamus being more specialized than Choeropsis for an aquatic lifestyle as it spends most of the day submerged in water. Its nevertheless still active locomotion on land, impeding more drastic functional aquatic adaptations [START_REF] Fisher | Forelimb myology of the pygmy hippopotamus (Choeropsis liberiensis)[END_REF][START_REF] Tsubamoto | Recognition of Microbunodon (Artiodactyla, Anthracotheriidae) from the Eocene of China[END_REF], is consistent with the absence of osteosclerosis (strong increase in whole bone compactness; see [START_REF] Houssaye | Pachyostosis" in aquatic amniotes: a review[END_REF].

Both extant hippopotamids show muscular adaptations associated with body weight support and increased resistance against water, especially in the forelimb to propel the trunk forward and in the autopod loading for stability on wet ground [START_REF] Fisher | Forelimb myology of the pygmy hippopotamus (Choeropsis liberiensis)[END_REF].

Modifications of the pectoral muscles might explain the stronger compactness observed in hippo humeri than femora.

The humeral sections of Hippopotamus evoke what is observed in some rhinos.

However, Hippopotamus femora differ from those of rhinos, whose diaphyses show a heterogeneous (proximo-distal) organization along the diaphysis [START_REF] Wall | The correlation between high limb-bone density and aquatic habits in recent mammals[END_REF]AH pers. obs.) whereas it is homogeneous in Hippopotamus. Longitudinal sections of the long bones of diverse extant rhino species would be required in order to better analyze the variation observed among rhino transverse sections (Fig. S1W-A') considering the strong changes occurring along the diaphysis [START_REF] Wall | The correlation between high limb-bone density and aquatic habits in recent mammals[END_REF]AH, pers. obs.). This would also allow to determine more precisely to which rhinos Hippopotamus is the closest, some rhinos being exclusively terrestrial whereas others spend most of their time in shallow waters [START_REF] Dinerstein | Family Rhinocerotidae (Rhinoceroses)[END_REF], and to link more precisely the microanatomical features observed with the habitat and mass.

Saotherium cf. S. mingoz has a humerus inner structure very close to that of Choeropsis, despite being much larger, so that this filling could be only related to mass and not necessarily to a semi-aquatic lifestyle. Femur data were unfortunately not available to verify this hypothesis.

Humeri in Mi. minimum and humeri and femora in Elomeryx borbonicus, as for Me. medioximus [START_REF] Cooper | Aquatic habits of cetacean ancestors: integrating bone microanatomy and stable isotopes[END_REF]; Fig. S1A), have a tubular inner structure close to that of large terrestrial ungulates, like Alces and Bubalus but also to the small Hyemoschus, and Indohyus for the humerus. The cortical layer is slightly thicker than in ungulates of similar sizes. This suggests a lifestyle possibly similar to that of Hyemoschus, which is essentially terrestrial, living in closed habitats, but foraging along the banks of rivers at night, which is in agreement with previous ecological reconstructions [START_REF] Lihoreau | Systématique et Paléoécologie Des Anthracotheriidae [Artiodactyla ; Suiformes] Du Mio-Pliocène de l'Ancien Monde : Implications Paléobiogéographiques[END_REF][START_REF] Lihoreau | Family Anthracotheriidae[END_REF]; Table 2). No femur is available for Microbunodon. The higher morphological robustness of the hind limb in Elomeryx borbonicus is not reflected in the microanatomy of its stylopod bone.

The humerus of Bothriodon velaunus is close to those of Microbudondon and

Elomeryx, though it shows a thinner cortex and a spongious transition zone between a compact cortex and a probably empty core (at least) of the medullary area, with a transverse section close to that of Tapirus terrestris. This taxon, very close in size (< 150 kg) and morphology to Elomeryx borbonicus, probably also shared a similar lifestyle (Table 2).

The humerus of Paenanthracotherium bergeri shows a rather thick cortex, slightly thicker than in Bubalus bubalis for a similar diameter, and the medullary area appears essentially void of trabeculae (if it is not a preservation artefact), except a few at the transition zone. This organization is rather similar to that of the notungulate Nesodon imbricatus (< 500 kg; [START_REF] Cassini | Body mass estimation in early Miocene native South American ungulates: a predictive equation based on 3D landmarks[END_REF][START_REF] Forasiepi | New toxodontid (Notoungulata) from the early Miocene of Mendoza, Argentina[END_REF]Houssaye et al. 2016b), which suggests an essentially terrestrial lifestyle for this rather massive (< 950 kg) and heavily built animal, in accordance with Scherler et al. (2019;Table 2). This could be compatible with walking, but not underwater swimming or bottom walking in swamps. Unfortunately, no femur was available to further explore this comparison.

The humerus of Brachyodus onoideus resembles that of Choeropsis. The bad preservation of the core of the bone prevents us from knowing to which degree the medullary cavity is filled with a spongiosa and thus to state if it was more similar to Choeropsis, to some rhinos, or to the astrapothere Parastrapotherium (Houssaye et al. 2016b). Its femur seems clearly void of trabeculae in the medullary area and thus differs from Choeropsis, Hippopotamus, and rhinos. It also differs from Parastrapotherium whose cortex is thinner and the transition zone large (Houssaye et al. 2016b). In Brachyodus onoideus, humerus and femur, like in Choeropsis, present similar microanatomical organizations, which might suggest comparable relative use of the forelimbs and hind limbs in these two taxa. More specimens would naturally be required to confirm this hypothesis. The relatively thick cortex is consistent with its heavy weight (< 2,350 kg), the spongious tissue in the medullary area of the humerus with a semi-aquatic lifestyle, but animals with aquatic affinities of similar size, i.e., Hippopotamus and the Asiatic rhinos combine a thick cortex and spongious tissue in the medullary cavity. Brachyodus onoideus, if semi-aquatic (Orliac et al. 2013), would probably have had reduced immersive habits as compared to these species.

The humerus of Libycosaurus bahri is close to those of Choeropsis and Brachyodus onoideus. As opposed to in Brachyodus onoideus, the spongiosa is clearly well developed and extended in the medullary area. As for the femur, it is also void of trabeculae but with a spongious transition zone. Libycosaurus bahri is a heavy (< 1,600 kg) animal with a hippolike morphology. Its microanatomical features are consistent with a semi-aquatic lifestyle as suggested by previous works [START_REF] Boisserie | Emergence of Hippopotamidae: new scenarios[END_REF][START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF]; Table 2).

The specimens of Hexaprotodon garyam differ in their microanatomical specialization, despite a similar size. One humerus (TM 115-06-01) is close to that of Saotherium cf. S. mingoz, whereas the other (TM 258-01-31) has a much thicker cortex, especially near the growth center. This difference in cortical thickness, and thus of compactness, is also observed, though to a lesser extent, between the two femora. Both humeri show spongious bone extending in the medullary area. Like for Brachyodus onoideus and Libycosaurus bahri, the femora show a relatively thick cortex but an open medullary cavity. Both microanatomical patterns are compatible with a semi-aquatic lifestyle for this heavy taxon (<2,200 kg), as suggested by previous studies [START_REF] Vignaud | Geology and palaeontology of the Upper Miocene Toros-Menalla hominid locality, Chad[END_REF]Boisserie et al. 2005b;[START_REF] Lihoreau | Description and palaeobiology of a new species of Libycosaurus (Cetartiodactyla, Anthracotheriidae) from the late Miocene of Toros-Menalla, northern Chad[END_REF]; Table 2). However, if one specimen (TM 115-06-01) is more compatible with the lifestyle of Choeropsis, the other (TM 258-01-31) is more similar to Hippopotamus.

Hexaprotodon garyam and Libycosaurus bahri, like Hippopotamus amphibius and contrary to Choeropsis and Brachyodus onoideus, display a stronger compactness in the humerus than in the femur. Libycosaurus bahri and the most compact Hexaprotodon garyam specimens evoke what is observed in Parastrapotherium. This taxon, supposedly with some species as heavy as Hippopotamus, was assumed to be a megaherbiore frequently foraging in aquatic environments (Avilla and Vizcaino, 2005;[START_REF] Flynn | Cenozoic Andean faunas: shedding new light on South American mammal evolution, biogeography, environments, and tectonics[END_REF]. Parastrapotherium nevertheless shows a lower increase in compactness and extension of the spongiosa than Hippopotamus. Hippopotamus is so far the hippopotamoid displaying the strongest increase in bone compactness. Hippopotamus is among the heaviest hippopotamoids but not strongly heavier than some others (e.g., Hexaprotodon and Brachyodus). This taxon might thus illustrate the strongest degree of adaptation to a semi-aquatic lifestyle among hippopotamoids.

Only Pyrotherium romeroi (of about 3,500 kg; [START_REF] Shockey | Pyrotherium macfaddeni, sp. nov. (late Oligocene, Bolivia) and the pedal morphology of pyrotheres[END_REF], considered showing extreme microanatomical adaptation to heavy-weight support, exhibits more compact stylopod bones than Hippopotamus, at least near the growth center (no longitudinal section is available for Pyrotherium romeroi). This extreme increase in bone mass, if indeed not associated with a semi-aquatic lifestyle, remains poorly misunderstood and requires further investigation. Indeed, heavier terrestrial taxa do not show such a high inner compactness (Houssaye et al. 2016a). Only the sea otter shows a similarly high compactness near the growth center [START_REF] Houssaye | From land to water: evolutionary changes in long bone microanatomy of otters (Mammalia: Mustelidae)[END_REF] but this specialization is linked to its almost exclusively aquatic lifestyle and not to loading. Comparisons with other large terrestrial and semi-aquatic taxa are required to better decipher the microanatomical changes associated with a semi-aquatic lifestyle from those linked to loading in heavy quadrupedal mammals.

Conclusion

This microanatomical study of the stylopod bones of various hippopotamoids and comparisons with diverse terrestrial and semi-aquatic quadrupedal ungulates highlight converging specializations for a semi-aquatic lifestyle and loading. This consists of an increase in bone compactness by filling the medullary area with cancellous bone and a thickening of the cortex. However, comparisons between animals of similar weight suggest, in the case of excessive compactness, a semi-aquatic lifestyle. In this context, the microstructure of Choeropsis liberiensis appears to be consistent with an animal that is mainly foraging in forests but frequently invades the water, whereas that of Hippopotamus amphibius shows a stronger compactness consistent with an animal that spends a large part of its time standing in the water or walking/swimming on the bottom. Accordingly, on the ground of microanatomical features, the extinct taxa Microbunodon minimum, Bothriodon velaunus, Elomeryx borbonicus, Merycopotamus medioximus, Paenanthracotherium bergeri, and probably also Saotherium cf. S. mingoz are inferred as essentially terrestrial animals, Brachyodus onoideus as slightly water-dependent, and Libycosaurus bahri and Hexaprotodon garyam as clearly semi-aquatic, although less specialized for this ecology than Hippopotamus amphibius. In order to better decipher the microanatomical changes associated with a semiaquatic lifestyle in relation to those associated with loading in heavy quadrupedal mammals, microanatomical investigations, preferentially including a large part of the diaphysis, and functional analyses on limb bones of other large terrestrial and semi-aquatic taxa are strongly needed. 1. Color code as in Fig. 4

  borbonicus sections are similar to those of Hyemoschus, Bubalus, and Alces. As for Tapirus terrestris, Parastrapotherium sp., and Nesodon imbricatus, they display a relatively thinner cortex with a spongious transition zone between the compact cortex and the medullary cavity and an open medullary cavity (Fig. S3T; Houssaye et al. 2016b), which thus differs from the patterns observed in the sampled hippopotamoids, though only slightly to Choeropsis'. The organizations observed in most hippopotamoids sampled (i.e., except Choeropsis, Hippopotamus, and Elomeryx) appear thus only similar to what is observed in various otter femora (Houssaye and Botton-Divet 2018) and Syncerus caffer, based on the comparative material available to date.

CFig. 2

 2 Fig. 1 Two consensus phylogenetic trees including the extant and extinct taxa sampled. A from Boisserie et al. (2011) for the hippopotamines, Lihoreau et al. (2015; SI fig. 17) and Boisserie et al. (2017) for the other hippopotamoids; B from Lihoreau et al. (2015, 2019) and Gomes Rodrigues et al. (2019) for the hippopotamoids, and A,B from Thewissen et al. (2007)

Fig. 3

 3 Fig. 3 Humerus transverse sections near the growth center. A Choeropsis liberiensis MNHN 1978-104; B Saotherium cf. S. mingoz KB 03-97-170; C Hippopotamus amphibius MNHN 1971-308; D Hexaprotodon garyam TM 55-XX-05; E Hexaprotodon garyam TM 258-01-27; F Brachyodus onoideus MNHN Neu 75; G Bothriodon velaunus 2004-6-1792-RON; H Libycosaurus bahri TM 98-99-01; I Elomeryx borbonicus UCBL-FSL 8572; J Microbunodon minimum UP-L.M. 1968 MA 377; K Paenanthracotherium bergeri UCBL-FSL 213779. Scale bars equal: A,G,I,J-5 mm; F,H-10 mm; B,C,D,E,K-15 mm. Partial sedimentary filling of the bone in B, D,E,(F), G, H, and K.

Fig. 4

 4 Fig. 4 Distribution of the specimens in the morphospace along the two first axes of the humerus PCA with the contribution of the different parameters according to the principal component (PC)1 and PC2 axes. Abbreviations as listed in Table 1

Fig. 7

 7 Fig. 7 Distribution of the specimens in the morphospace along the two first axes of the femur PCA with the contribution of the different parameters according to the principal component (PC)1 and PC2 axes. Abbreviations as listed in Table1. Color code as in Fig.4

Table 2 . Supposed lifestyle of the hippopotamoids sampled based

 2

	1
	(sub)Family	Species	. Abb	number Mass Inventory	e Bon	Resol
	Equidae		Ec	380-	MNHN	H,F 78,90(Pa)
		Equus caballus		600	ZM AC
					1880-29
	Tapiridae		Tp	150-	MNHN	H,F 80,80(M)
		Tapirus pinchaque		200	ZM AC
					1982-34
			Tt	180-	MNHN	H,F 55,55(P)
		Tapirus terrestris		300	ZM MO
					1939-225
					ZFMK 462 H,F 48,82(B)
	Rhinocerotidae	Ceratotherium	Cs	1,350-	MNHN	H,F 78,68(Pa)
		simum		3,500	ZM MO
					2005-297
					JRHRVC	H,F 684,771(L
					uncat.		)
		Dicerorhinus	Ds	600-	MNHN	H,F 58,65(Pa)
		sumatrensis		775	ZM AC
				kg	1903-300
		Diceros bicornis	Db	800-	UMZC	H	518(L)
				1,300	H.6481
					NHMUK	H	561(L)
					M92402
		Rhinoceros	Rs	1,200-	MNHN	H,F 76,76(Pa)
		sondaicus		1,500	ZM AC
					A7970
		Rhinoceros	Ru	<	MNHN	H,F 76,76(Pa)
		unicornis		2,000	ZM AC
				kg	1960-59
	Tayassuidae		Pt	15-42 MNHN	H,F 38,38(P)
		Pecari tajacu			ZM MO
					1917-263
					MNHN	H,F 40,40(P)
					ZM MO
					2000-352
	Camelidae	Lama guanicoe	Lg	140 90-	M7388 STIPB	H,F 55,55(B)
	Suidae	porcus Potamochoerus	Pp	115 50-	1944-234 ZM MO MNHN	H,F 53,53(P)
					MNHN	H,F 61,61(P)
					ZM MO
					C.G. 1971-
					34
		Sus scrofa	Ss	44-320	STIPB M56	H,F 173,206(B )

on literature and on this study. H: humerus; F: femur.

Table 3 Values obtained for the tests of a size effect and phylogenetic signal in the various

 3

Acknowledgments

We warmly thank J. Lesur, A. Verguin, C. Argot and G. Billet (MNHN, Paris, France); F. Saragoza, A. Bonnet and E. Magne (Musée Crozatier, Le Puy-en-Velay, France); D. Berthet and F. Vigouroux (Musée des Confluences, Lyon, France); E. Robert (Université Claude Bernard Lyon I, Lyon, France); L. Costeur and F. Dammeyer (Naturhistorisches Museum Basel, Basel, Switzerland); G. Garcia and M. Brunet (PALEVOPRIM, Université de Poitiers, Poitiers, France); C. Nekoulnang Djetounako and M. Adoum, (Centre National de Recherche pour le Dévelopement, N'Djamena, Chad) with support of L. Andossa and M. Hassane Taïsso (Université de N'djaména, Chad) for the loan of the hippopotamoid specimens. We thank A. Mazurier (IC2MP, UMR 7285 CNRS-UP, Poitiers, France), R. Lebrun (ISEM, plateforme MRI, Montpellier, France), M. Garcia Sanz (AST-RX platform, UMS 2700, MNHN), and the Steinmann-Institut (University of Bonn, Germany), for providing beamtime and support, and 1 2

Declarations

Funding We acknowledge financial support from the ANR SPLASH (ANR-15-CE32-0010).

AH also acknowledges financial support from the ERC-2016-STG-715300.

Conflicts of interest/Competing interests None

Ethics approval Not applicable

Consent to participate Not applicable

Consent for publication Not applicable

Availability of data and material Microtomographic scans will be available under request to the authors. 3D image data (.tif format) obtained by micro-tomography of MNHN specimens will be permanently saved by the MNHN DSI and identified by the inventory number of the specimen. They will be visible as work carried out on the interface https://3dtheque.mnhn.fr/ and made available via the interface http://colhelper.mnhn.fr/. All 3D image data obtained by micro-tomography at the IC2MP (University of Poitiers) are permanently saved by PALEVOPRIM under the responsibility of the director of the research unit (currently: JRB).

Inventory will be communicated upon request, and data will be made available depending on the policy of the original specimen repository institution.

Code availability Not applicable


MD, MSPH Flore Salviat

PhD Cécile Charles

MD, PhD Catherine Uzan
email: catherine.uzan@aphp.fr

Shall patients' anxiety influence surgical decisions for atypical breast lesions? A substudy of the prospective NOMAT trial

Atypical breast lesions (ABL) identified on image-guided biopsies are associated with both a 10-25% probability of concomitant cancer at surgery, and a long-term increased risk of breast cancer. Many such ABL may be considered as part of the "overdiagnosis" of current breast cancer screening strategies. Proposing a systematic surgery, as has long been done, leads to procedures with no cancer associated on definitive specimen in 75 to 90% of women which can be considered as unnecessary. Current guidelines (from NCCN and (1)) recommend to attempt avoiding surgery of lesions at low risk of upgrading (based on pathological classification or models), with close follow up.

However, some patients may be less tolerant to uncertainty and have a lower acceptance of followup strategies. NOMAT (clinicaltrials.gov NCT02523612; French Ethics Committee approval 2015-A00045-44) is a national multicenter prospective trial (2) whose primary objective was to validate a prediction model for upgrade at surgery, among women with atypical breast lesions. A secondary objective was to describe the evolution of state anxiety and identify factors associated with this evolution.

Women aged 18 or older were eligible for the study if diagnosed with an ABL (including flat epithelial atypia [FEA], atypical ductal [ADH] or lobular hyperplasia [ALH] and lobular carcinoma in situ [LCIS] of classic type) on image-guided biopsy for breast microcalcifications were included in this trial between July 7, 2015 and May 29, 2018, in 18 French centers [START_REF] Uzan | Prospective Multicenter Study Validate a Prediction Model for Surgery Uptake Among Women with Atypical Breast Lesions[END_REF]. The final patients' diagnoses were classified as "cancer" if either an invasive adenocarcinoma, or a ductal carcinoma in situ (DCIS) or a pleomorphic lobular carcinoma in situ (LCIS) was retrieved on the surgical specimen.

Psychological self-reported outcomes were collected at baseline, including anxiety (State-Trait Anxiety Inventory, STAI) and intolerance of uncertainty (Intolerance of Uncertainty Scale). "Before surgery" was at the end of the surgical consultation meaning after the announcement of ABL on biopsy, the risk of cancer associated and explanation on surgical procedure. Patients completed the state-anxiety scale again post-surgery (between day 21 and day 30).

We compared the state-anxiety scores after versus before surgery using a paired Wilcoxon-Mann-Whitney test. The association between candidate variables and changes in anxiety score levels before versus after surgery were assessed using a Wilcoxon-Mann-Whitney test. Statistical analyses were performed on R version 3.6.1.

  Two hundred forty one (80.3%) and 210 (70.0%) women completed the STAI-state inventory questionnaire before and after surgery respectively. The mean state-anxiety score of respondents was significantly lower after surgery in the global population (42.70 [SD 13.07] vs 35.37 [SD 11.68], p<0.001) as well as in patients who did not get a final diagnosis of cancer (42.63 [SD 13.06] vs 33.59 [SD 10.76], p<0.001).

Figure 1

 1 Figure 1 Evolution of state anxiety before and after surgery in patients who filled both questionnaires

All outside the submitted work: Roche (Personnal fees, non-financial support), MSD (Personnal fees), Astra Zeneca (Non-financial support) Flore salviat and Cecile Charles declare that there are no competing interests.

One hundred ninety-nine patients (66.3%) answered both questionnaires. Among them, 118 patients (59.3%) had stable, 65 (32.7%) decreased and 16 (8.0%) increased anxiety levels after surgery (Figure 1). Patients with increased anxiety were younger than patients with decreased or stable levels (n=180) (mean: 50.07 years [SD: 8.06], vs 55.52 years [SD: 9.37], p=0.026). Patients with decreased anxiety had a higher baseline STAI-Trait score than patients with increased or stable levels (n=118) (mean: 46.10 [SD: 8.81] vs 38.88 [SD: 8.84], p<0.001) and a higher baseline level of intolerance of uncertainty (mean: 62.29 [SD: 19.16] vs 53.32 [SD: 16.24], p=0.002). Of note, state anxiety and intolerance to uncertainty are highly correlated (Kruskall-Wallis p= 4.10 -7 ).

Among 162 women who did have cancer at surgery, 56 (34.6%) had a decreased, 98 (60.5%) a stable and 8 (4.9%) an increased STAI-state anxiety score. There again, the total score of intolerance to uncertainty at baseline was higher in patients with decreased anxiety as compared to the others (mean: 63.36 [SD: 18.74] vs 52.00 [SD: 14.85], p=0.0002). The form of ABL significantly affected the score of STAI-ETAT before surgery (p=0.004) and after surgery (p=0.01), patients with ADH were more anxious than patients with no ADH.

Overall, surgery allowed a decreased level of anxiety in one third of the women, whether they had a final diagnosis of cancer or not. These women had consistent higher anxiety and intolerance to certainty levels at baseline. The present design raises, but cannot entirely answer the question whether surgical abstention would preclude such anxiety relief. In addition to clinical and pathological features (i.e age, initial radiological microcalcifications, focus size and complete radiological target excision after biopsy) [START_REF] Uzan | Prospective Multicenter Study Validate a Prediction Model for Surgery Uptake Among Women with Atypical Breast Lesions[END_REF][START_REF] Rageth | Atypical ductal hyperplasia and the risk of underestimation: tissue sampling method, multifocality, and associated calcification significantly influence the diagnostic upgrade rate based on subsequent surgical specimens[END_REF], breast surgeons should take into account psychological aspects, including anxiety associated with the situation, tolerance to uncertainty, and patients preferences overall, in the surgery decision balance. A well-conducted shared decision process appears of critical importance in this situation.