Alexandre Assemat

Mickaël Mourlam
email: mickael.mourlam@umontpellier.fr

Romain Weppe

Jacob Maugoust

Maeva Pierre-Olivier Antoine

Judith Orliac

The ossicular chain of Cainotheriidae (Mammalia, Artiodactyla)

Keywords: Morphometry, Malleus, Incus, Stapes, Paleogene, Quercy

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Introduction

The middle ear ossicles -the malleus, the incus, and the stapesform a bony chain contained within the air-filled middle ear cavity. Their presence is a hallmark of Mammalia (e.g., [START_REF] Simpson | Mesozoic Mammals and the Polyphyletic Origin of Mammals[END_REF][START_REF] Luo | A new mammaliaform from the early Jurassic and evolution of mammalian characteristics[END_REF]. The three ossicles are the smallest bones of the mammalian skeleton and they play a fundamental role in hearing process. Indeed, the ossicular chain transmits the airborne sound waves from the tympanic membrane (at the interface between the outer and middle ear) to the fluid-filled cochlea of the inner ear, while performing an anatomical impedance match between this two media (e.g., [START_REF] Wever | Physiological Acoustics[END_REF][START_REF] Dallos | The Auditory Periphery: Biophysics and Physiology[END_REF][START_REF] Schubert | History of research on hearing[END_REF][START_REF] Killion | Impedance matching by the combined effects of the outer and middle ear[END_REF][START_REF] Peake | Impedance matching, optimum velocity, and ideal middle ears[END_REF][START_REF] Hemilä | What middle ear parameters tell about impedance matching and high-frequency hearing[END_REF][START_REF] Nummela | The Physics of Sound in Air and Water[END_REF][START_REF] Mason | Structure and function of the mammalian middle ear. II: Inferring function from structure[END_REF]. Morphology of the ossicular chain and its specificity within different mammalian groups has been intensely studied for systematic purposes (e.g., [START_REF] Doran | Morphology of the mammalian ossicula auditus[END_REF][START_REF] Fleischer | Studien am Skelett des Gehörorgans der Säugetiere, einschließlich des Menschen[END_REF][START_REF] Schmelzle | Phylogenetic transformations of the ear ossicles in marsupial mammals, with special reference to diprotodontians: a character analysis[END_REF][START_REF] Wible | A reexamination of the Carnivora malleus (Mammalia, Placentalia)[END_REF][START_REF] Mason | Of mice, moles and guinea-pigs: functional morphology of the middle ear in living mammals[END_REF][START_REF] Stoessel | Comparative anatomy of the middle ear ossicles of extant hominids-Introducing a geometric morphometric protocol[END_REF]Maier & Ruf, 2016a, 2016b;[START_REF] Kerber | Morphology of the Middle Ear Ossicles in the Rodent Perimys (Neoepiblemidae) and a Comprehensive Anatomical and Morphometric Study of the Phylogenetic Transformations of these Structures in Caviomorphs[END_REF][START_REF] Loza | Evolutionary transformations of the malleus in pinnipeds, with emphasis on Southern Hemisphere taxa[END_REF], or functional aspects (e.g., [START_REF] Fleischer | Evolutionary principles of the mammalian middle ear[END_REF][START_REF] Nummela | Scaling of the mammalian middle ear[END_REF][START_REF] Nummela | Scaling of the marsupial middle ear and its functional significance[END_REF][START_REF] Puria | Tympanic-membrane and malleus-incus-complex co-adaptations for high-frequency hearing in mammals[END_REF]. Most of these works deal with extant taxa, and, because of their fragility and small size, ossicles are rarely preserved -or at least retrieved -in the fossil records. Yet, when retrieved, they bring useful observations for the systematics or ecology of taxa, or both [e.g., systematic position of Pakicetidae [START_REF] Thewissen | Origin of underwater hearing in whales[END_REF] and hearing mechanisms in early cetaceans [START_REF] Nummela | Eocene evolution of whale hearing[END_REF][START_REF] Nummela | Sound transmission in archaic and modern whales: anatomical adaptations for underwater hearing[END_REF] among Artiodactyla]. Still, fossil ossicles are rarely preserved all three together, and almost never found in anatomical connection.

Here, we describe a broad sample of isolated ossicles of Cainotheriidae from Paleogene karstic infillings from Quercy (Tarn-et-Garonne, France). Cainotheriids are an extinct family of small artiodactyls (even-toed ungulates) documented in the fossil record between the late Eocene and the middle Miocene in Western Europe [START_REF] Blondel | New data on the Cainotheriidae (Mammalia, Artiodactyla) from the early Oligocene of south-western France[END_REF]. Because of their unique dental morphology, the phylogenetic position of Cainotheriidae within artiodactyls is still debated. They have been related to different European endemic families [START_REF] Romer | Vertebrate paleontology[END_REF][START_REF] Webb | The phylogeny of hornless ruminants and a description of the cranium of Archaeomeryx[END_REF][START_REF] Gentry | The phylogeny of the Artiodactyla[END_REF] without reaching a consensus, or to modern groups of artiodactyls such as ruminants [START_REF] Geisler | Phylogenetic relationships of extinct cetartiodactyls: results of simultaneous analyses of molecular, morphological, and stratigraphic data[END_REF][START_REF] O'leary | Impact of increased character sampling on the phylogeny of Cetartiodactyla (Mammalia): combined analysis including fossils[END_REF][START_REF] Lihoreau | Hippos stem from the longest sequence of terrestrial cetartiodactyl evolution in Africa[END_REF] or tylopods [START_REF] Geisler | Morphological support for a close relationship between hippos and whales[END_REF][START_REF] Geisler | Phylogenetic Relationships of Cetaceans to Terrestrial Artiodactyls[END_REF][START_REF] Thewissen | Whales originated from aquatic artiodactyls in the Eocene epoch of India[END_REF]).

The recent phylogenetic study of [START_REF] Weppe | Cainotheriidae (Mammalia, Artiodactyla) from Dams (Quercy, SW France): phylogenetic relationships and evolution around the Eocene-Oligocene transition (MP19-MP21)[END_REF] retrieved Cainotheriidae closely related to the European endemic families Mixtotheriidae, Anoplotheriidae and Robiacinidae.

Cainotheriidae are particularly abundant in karstic localities from Quercy, southwestern France. This family, which includes at least five genera within two sub-families ranges from rabbit-sized species to size of a small ruminant [START_REF] Erfurt | Endemic European Paleogene Artiodactyls: Cebochoeridae, Choeropotamidae, Mixtotheriidae, Cainotheriidae, Anoplotheriidae, Xiphodontidae, and Amphimerycidae[END_REF][START_REF] Theodor | Micro-Computed Tomographic Scanning of the Ear Region of Cainotherium: Character Analysis and Implications[END_REF].

Contrary to many European endemic ungulates which went extinct at the end of the Eocene [START_REF] Sudre | Ungulates from Paleogene of Western Europe: relationships between their evolution and environmental changes during that period[END_REF][START_REF] Blondel | The Eocene-Oligocene ungulates from Western Europe and their environment[END_REF], cainotheriids made it through the Eocene/Oligocene transition and they are one of the very few artiodactyl groups to diversify during Oligocene times [START_REF] Blondel | New data on the Cainotheriidae (Mammalia, Artiodactyla) from the early Oligocene of south-western France[END_REF]. The ossicles we describe here originate from two loci from the karstic network of Dams, discovered in 2016. This karstic network was emptied during the extensive phosphate exploitation that took place in Quercy during the late 19 th century. The network, however, still houses a great quantity of clay infillings including two channels that yielded a great quantity of cainotheriid remains, namely DAM1 and DAM3.

These two infillings within Dams network bracket the Eocene-Oligocene transition [START_REF] Weppe | Cainotheriidae (Mammalia, Artiodactyla) from Dams (Quercy, SW France): phylogenetic relationships and evolution around the Eocene-Oligocene transition (MP19-MP21)[END_REF]) and they document a period that corresponds to a major faunal turnover in Western Europe linked to climatic, geographic and oceanic circulation changes [START_REF] Legendre | Les communautés de mammifères d'Europe occidentale de I'Eocène supérieur et Oligocène: structures et milieux[END_REF][START_REF] Berggren | Eocene-Oligocene climatic and biotic evolution: an overview[END_REF]. Based on the unprecedented sample from Dams, including a total of 18 mallei, 28 incudes and three stapedes, and on an in-situ ossicular chain from Pech Desse (Quercy, France, late Oligocene), we discuss the intra-and interspecific variability of ossicle morphology within Cainotheriidae and describe for the first time a reconstructed ossicular chain for a Paleogene terrestrial artiodactyl species.

Material and methods

Material

Most of the specimens included in this analysis come from the Dams karstic network located near Caylus (Tarn-et-Garonne) in Quercy (SW France). All specimens studied are curated at the University of Montpellier (UM). The Dams material was collected after screenwashing of 40kg from DAM1 and 30kg from DAM3 in 2016. Raw fossil material was first concentrated by wet screening of the red clays collected in Dams locality, (0.7-mm mesh size), and then picked up with smooth tweezers, under a stereomicroscope. The material consists of 16 mallei, 16 incudes, and two stapedes from DAM1 (late Eocene, Mammalian Paleogene reference level 19 [MP19]; [START_REF] Weppe | Cainotheriidés (Mammalia, Artiodactyla) et Grande Coupure : nouveau matériel des phosphorites du Quercy[END_REF] and 12 incudes (among which two are in connection with the malleus head), and one stapes from DAM3 (early Oligocene, MP22; [START_REF] Weppe | Cainotheriidés (Mammalia, Artiodactyla) et Grande Coupure : nouveau matériel des phosphorites du Quercy[END_REF]. The taxonomic identification of the isolated ossicles from Dams relies on a strong corpus of evidences: 1) the relative abundance of mammalian fossil remains; 90% of the remains (cranial, dental and postcranial) from DAM1 belong to the small cainotheriid artiodactyl Paroxacron valdense. Therefore, all the mallei and 16 incudes upon 18, that can be referred to the same morph, likely correspond, based on the relative abundance criterion, to Paroxacron valdense; 2) Artiodactyla hallmark; the incus presents a processus longum slightly smaller than the processus brevis which is a characteristic of Artiodactyla [START_REF] Doran | Morphology of the mammalian ossicula auditus[END_REF][START_REF] Wilkie | The Auditory Organ of the Ox (Bos taurus )[END_REF][START_REF] Thewissen | Origin of underwater hearing in whales[END_REF][START_REF] Thewissen | Phylogenetic aspects of Cetacean origins: A morphological perspective[END_REF][START_REF] Milinkovitch | Evolutionary biology. Even-toed fingerprints on whale ancestry[END_REF]; 3) incudo-mallear joint association; the association between the malleus and the incus, besides general size compatibility and match between the articular surfaces, is based on fused incudo-mallear complexes found in DAM3.

Based on these criterions, the ossicles from DAM1 are all assigned to Paroxacron valdense, the only cainotheriid species retrieved in this channel [START_REF] Weppe | Cainotheriidés (Mammalia, Artiodactyla) et Grande Coupure : nouveau matériel des phosphorites du Quercy[END_REF]. In contrast, five different cainotheriid species co-occur in DAM3, making specific attribution of the isolated ossicles impossible (in the current state of our knowledge). A list of the included material is provided in supplementary information Table S1. Other incudes of similar size have been found in both levels (DAM1 and DAM3), but they are not included in this study due to their markedly different morphology that would point to rodents or chiropterans instead of artiodactyls. In addition to the ossicles from Dams localities, we reconstruct in this work the in-situ location of the cainotheriid ossicular chain based on a basicranium (UM PDS 3353) from the late Oligocene locality of Pech Desse (MP28, Quercy; [START_REF] Hugueney | Biochronologie mammalienne dans le Paléogène et le Miocène inférieur du Centre de la France : synthèse réactualisée[END_REF] that preserves the ossicles trapped in the middle ear cavity. Two cainotheriine species are retrieved in Pech Desse, namely Plesiomeryx cf. cadurcensis and Caenomeryx cf. procommunis [START_REF] Remy | Biochronologie des phosphorites du Quercy: mise à jour des listes fauniques et nouveaux gisements de mammifères fossiles[END_REF]. Based on the overall larger dimensions of the specimen, it is here referred to as Caenomeryx cf. procommunis.

Micro CT scanning and virtual reconstruction

The ossicles were scanned, using the high-resolution micro CT-scanner EasyTom of the technical facility of the Montpellier Rio Imaging platform, at a high voltage (150 kV) using a copper filter and small sample holders (2 and 4 cm diameters), allowing to be close to the Xray source and therefore, to reach a voxel size of 11.89 µm for isolated specimens, and 23.81 µm for the partial cranium UM PDS 3353. They were reconstructed virtually in 3D using the threshold tool of Avizo 9.5 (VSG-FEI) software. The specimens partly encrusted with matrix were cleaned using the manual segmentation tool of the same software. The virtual reconstruction of the in-situ location of the ossicular chain of cainotheriid from Pech Desse was realized using the freeware MorphoDig [START_REF] Lebrun | MorphoDig, an open-source 3D freeware ded-MorphoDig, an open-source 3D freeware ded-icated to biology[END_REF]. Anatomical terminology used in this study mainly follows that of [START_REF] Wible | A reexamination of the Carnivora malleus (Mammalia, Placentalia)[END_REF]; orientations are based on the reconstruction of the in-situ ossicle chain of the cranium UM PDS 3353.

Geometric morphometric analysis

Nine mallei and nine incudes from DAM1 and five incudes from DAM3 are included in the geometric morphometric analyses, other specimens being discarded due to their fragmentary condition. In addition, the incus and malleus from the Pech Desse specimen were also added to the analyses. Only three stapedes were unearthed in Dams localities, which does not allow a proper discussion of the morphometrical variability of this ossicle. To quantify the malleus and incus shape variations, we digitized a set of 3D landmarks using MorphoDig software (MorphoDig 0.8) (Fig. 1). Nine landmarks were placed on the malleus including five on the articular area. The first one was placed at the highest point of the curve along the medial margin of the articular area. The second one occupies the same position on the distal margin of the articular area. The third landmark is located at the center of the ridge of the articular area. The fourth and the fifth landmarks were placed on the deepest points along the dorsal and ventral borders of the articular facet, respectively. The sixth landmark was placed at the extremity of the muscular process and the seventh one is located on the notch between the muscular process and the manubrium. The eighth is positioned at the deepest point of the angulation formed by the neck of the malleus with the manubrium. The last one was placed at the extremity of the anterior process. On the incus, we positioned nine landmarks, including five on the articular area. The first one was placed on the deepest part on the lateral border of the articular area and the second one at an equivalent location on the medial border.

Landmarks three and four were placed on the most salient points of the articular surface i.e., on the dorsal and ventral edge, respectively. The fifth landmark is located at the central position of the bulge formed by the intersection of the two articular facets. Landmarks six and seven are positioned on the incudal body. The sixth one is at the middle of the swelling along the upper border of the incudal body and the seventh was placed at the middle of the curve joining the short and the long process posteroventrally. The eighth landmark was placed internally to the long process where it becomes thinner, while the ninth one was positioned at the extremity of the short process. The treatment of the raw dataset was performed using R software version 3.4.4 (R Core Team, 2018). All 10 replicates were scaled to unit centroid size (i.e. "the square root of the sum of squared distances from each landmark to the centroid of the configuration" Claude, 2008:139), translated, rotated, and superimposed through the Generalized partial Procrustes Analysis (pGPA) following [START_REF] Claude | Morphometrics with R[END_REF][START_REF] Claude | Log-shape ratios, Procrustes superimposition, elliptic Fourier analysis: three worked examples in R. Hystrix It[END_REF]; see also [START_REF] Bookstein | Introduction to methods for landmark data[END_REF][START_REF] Rohlf | Rotational fit (Procrustes) Methods[END_REF][START_REF] Dryden | Statistical Shape Analysis[END_REF]. Then, to apprehend the shape variability of the malleus and incus, we performed a Principal Component Analysis (PCA;

Pearson type) on the Procrustes coordinates (resulting from the pGPA). Error measurement follows [START_REF] Yezerinac | Measurement error and morphometric studies: statistical power and observer experience[END_REF] see also [START_REF] Claude | A geometric morphometric assessment of the effects of environment and cladogenesis on the evolution of the turtle shell[END_REF][START_REF] Claude | Morphometrics with R[END_REF]Claude, : 65-66, 2013)). Data and script are available in supplementary material dataset 1.

Data availability statement

All specimens studied are curated at the University of Montpellier (UM) and can be freely consulted upon request. The 3D models of the middle ear reconstruction of Caenomeryx cf.

procommunis is available in open access on MorphoMuseuM (https://morphomuseum.com/ ; Assemat, in press). The scripts used to perform the morphometric analyses are provided in supplementary information.

Description Malleus (Figs. 2-3)

The description of the cainotheriid malleus (Fig. 2) primarily relies on material from the DAM1 locality as DAM3 only yielded malleus articular surfaces fossilized in anatomical connection with the incus. The malleus is the most lateral part of the ossicular chain. In life, it contacts the tympanic membrane by a long, flat-shaped manubrium and a short and angular lateral process. The malleo-incudal complex is unfused for all documented specimens from DAM1. The globose head of the malleus bears the articular surface for the incus. The latter is divided into two facets separate by a deep and wide groove; the superior articular facet for the incus lies in the dorsal aspect, while the inferior articular facet, of about the same size, lies at ca. 35° angle to it (Fig. 2A). The general shape of the articular surface is saddle-like and is asymmetrical related to the fact that the groove is wider and shallower on the dorsomedial part. On the anterior surface, the head comes to a small, rounded point that we identify as a blunt capitular spine [START_REF] Wible | A reexamination of the Carnivora malleus (Mammalia, Placentalia)[END_REF]. A thin and sharp bony crest joins the basis of this spine to the anterior process, the outer lamella [START_REF] Henson | Some morphological and functional aspects of certain structures of the middle ear in bats and insectivores[END_REF]. Lateral to the capitular spine, the surface of the head is carved by a small pit. The neck of the malleus is relatively straight and it forms with the head an obtuse angle which confers to the malleus a general sigmoidal shape. It is relatively broad and lines the osseous lamina on the posteromedial edge.

The osseous lamina consists of a particularly thin portion of bone bearing a depression on the lateroventral aspect of the malleus (Fig. 2D); there is no clear demarcation with the basis of the manubrium. The specimens of our sample seem to display a short blunt anterior process, also known as processus gracilis, or prearticular, and mentioned in living artiodactyls [START_REF] Wible | A reexamination of the Carnivora malleus (Mammalia, Placentalia)[END_REF]Maier & Ruf, 2016b). It might have displayed a much thinner terminal part but it would have been broken away during the fossilization process and left no trace. As illustrated for carnivorans by [START_REF] Wible | A reexamination of the Carnivora malleus (Mammalia, Placentalia)[END_REF], the base of the manubrium corresponds to the confluence of the neck, lateral process, and ventral margin of the osseous lamina. The manubrium is long with a flat and thin tympanic surface. The latter is much wider than the lateral edge which bears a ridge becoming narrower at the manubrium's extremity.

Unfortunately, the manubrium is partly broken on all isolated specimens. It displays a welldeveloped lateral process at the posterior margin of its base. The medial margin of the bone bears a strong and conical muscular process forming a 40° angle with the manubrium. The course of the chorda tympani nerve is marked on the ventral aspect of the muscular process.

Comparison. The small and flat head of the malleus of the cainotheriids from Dams is closer in proportion and shape to Ruminantia as illustrated in Capreolus and Giraffa by [START_REF] Fleischer | Studien am Skelett des Gehörorgans der Säugetiere, einschließlich des Menschen[END_REF] than to Sus, Hippopotamus, and Camelus. Like ruminants, they also display a long neck and a wide osseous lamina. In suids, hippos, and camelids, the neck is shorter and the osseous lamina remains smaller and closely appressed to the head. Compared to hippos and suids, the cainotheriid malleus also displays a shorter but stockier muscular process which is closer to the manubrium. The muscular process global shape in cainotheriids is relatively similar to that of some living ruminants (e.g. Bos taurus, Ovis aries, and Cervus elaphus).

Inter-and intraspecific variation. The morphology of the 16 mallei from DAM1 shows a noticeable variability mainly affecting the length of the malleus neck, the angulation of the lateral process, the shape, depth and orientation of the articular surface, and also the location, length and shape of the muscular process (Fig. 2G-H; Fig. S1A-H). In addition, in comparison with the specimens from DAM1, the malleus from Pech Desse displays a different morphology of its articular surface. In order to quantify this inter-and intraspecific shape variation of the malleus, we performed two pGPAs, one with all available mallei (interspecific analysis; see Material and methods) and one without the Pech Desse specimen (intraspecific analysis). Due to small breakages on the latter specimen, interspecific shape variation has been quantified by taking into account only the first seven landmarks. For the interspecific analysis, measurement error is 6.92 % for centroid size and 16.88 % for shape. Inter-specimen size and shape variations are stronger than the intra-specimen ones (specimen factor is significant in both ANOVA (F = 135.4; p < 0.001) and Procrustes ANOVA (F = 50.23; p = 0.001); see also [START_REF] Claude | Log-shape ratios, Procrustes superimposition, elliptic Fourier analysis: three worked examples in R. Hystrix It[END_REF]. The Fig. 3A presents the projection of the specimens on the first factorial plane. PC1 (52.96 % of the variance) clearly separates the Pech Desse specimen from the other mallei and highlights deep morphological difference in term of shape and orientation of the articular facets of the two species. The mallei from DAM1 present intraspecific variation mainly on PC2 (19.72 % of the variance) that corresponds with small size variation of the articular surface and slight variations of orientation of the body of the malleus in regards to its head. More precisely, projection of individuals on the first factorial plane (48.82 % of the variance) of the intraspecific analysis (Fig. 3D; where measurement error for centroid size is 5.08 % with specimen factor significant (F = 187.7; p < 0.001) and measurement error for shape is 23.41% with specimen factor significant (F = 33.72; p = 0.001)), underlines these variations. Indeed specimens from DAM1 are distributed more or less randomly along the first two PCs that both stand for size variation of the articular surface.

Furthermore, PC1 also presents variation of orientation of the anterior process and PC2 highlights variation of orientation of the muscular process and of the main body in regards to the malleus head. This disparity of orientation of the malleus body can also be observed directly by superimposing all the mallei while keeping the same position for the articular surface (Figs. 2G-H, 3E-F).

Incus (Figs. 4-5)

The incus is the middle ossicle joining the malleus and the stapes. It is conical and stocky in shape (Fig. 4 A-B). The wide articular area of the incudomallear joint is composed of two asymmetrical facets. It displays the same asymmetry as previously described for the malleus. A salient edge, of different size depending on the specimen, separates the articular surface from the incudal body (Fig. 4D). The dorsal aspect of the incus is variably convex, from nearly flat to strongly domed. The cainotheriid incus displays two processes of similar length, set apart by a wide angle; the processus brevis and the processus longum.

The processus brevis prolongs posteriorly the incudal body. The processus longum, which connects the stapes by the lenticular apophysis, is located on the ventral edge of the incus, posterior to the articular surface. The distal extremity of the processus longum of all incudes of our sample is broken so that no lenticular apophysis is documented. A groove starts at the base of the processus longum and runs all along of it. The separation between the two processes is strengthened by the length of the body of the incus. The processus brevis of the cainotheriid incus displays a thinning at its extremity while the processus longum is thicker.

The extremity of the processus longum ends in a small spike that corresponds to the attachment of the broken lenticular apophysis.

Comparison. The cainotheriid incus is morphologically similar to that of other artiodactyls with two processes of about the same length, whereas in most other mammals, the processus longum is longer than the processus brevis [START_REF] Doran | Morphology of the mammalian ossicula auditus[END_REF]. Cainotheriid incudes from Dams differ from those of Camelus in having further apart processes, separated by a wider angle.

The articular area of cainotheriid incudes appears to be shallower than in camels, but deeper than in bovines (Doran, 1878, Pl. 61). The body of studied incudes is close in shape to that of Sus scrofa. It is more massive in the hippos and the llama which also display a more cylindrical shape. Ruminants display a wide range of incudal body morphologies [START_REF] Doran | Morphology of the mammalian ossicula auditus[END_REF][START_REF] Wilkie | The Ossicula Auditûs of the Sheep (Ovis aries)[END_REF][START_REF] Wilkie | The Auditory Organ of the Ox (Bos taurus )[END_REF]. The scarcity of illustrations available in the literature prevents a broader comparison among artiodactyls.

Inter-and intraspecific variation. The incudes from DAM1 and DAM3 show a noticeable variability of the depth of the articular facet and of the angulation between the processus brevis and the processus longum. They also display a variation of their dorsal part bulge (Fig. 4F, Fig. 5A-B, Fig. S1I-M). In order to quantify inter-and intraspecific shape variations, we performed a pGPA on a dataset of nine three-dimensional landmarks (Fig. 1B; see Material and methods section, Geometric morphometric analysis) for a sample of 15 incudes from DAM1, DAM3 and Pech Desse. For this analysis, measurement error is 3.14 % for centroid size, 18.82 % for shape and inter-specimen size (F = 309.4; p < 0.001) and shape (F = 44.13; p = 0.001) variations are significantly stronger than the intra-specimen ones. The two first PCs explain 56.32 % of the variance (Fig. 5A). The main shape variation contributions of PC1 (39.12 % of the variance) correspond with an elongation of the processus brevis, correlated with a smoother and thinner dorsal bulge toward the positive values (Fig. 5 B). PC1 also displays shape and orientation variations of the articular facets following the same trends observed for the interspecific variation of the malleus (see above). PC2 (17.20 % of the variance) mainly highlights the variation of angulation between the two processes (Fig. 5 C).

The projection of the specimens on the first factorial plane results in two slightly overlapping groups corresponding to DAM1 and DAM3 localities. DAM3's morphospace, with a surface that covers 41 % of the whole sample's morphospace, is nearly five times more expanded than that of DAM1 (that covers 8.6 %). Only one specimen from DAM3 lies within DAM1's morphospace. Incudes from DAM1 present intraspecific variability along PC1 and PC2, highlighting mostly variation of the angulation between the two processes and also, small size variation of the articular surface. PC1 also clearly isolates the Pech Desse specimen from the incudes retrieved in Dams. Indeed, in terms of Mahalanobis distance on the first five PCs, the specimens from DAM3 are always retrieved at least twice as far from the Pech Desse specimen as from the centroid of DAM1 (see Tab. S2).

Stapes (Fig. 6)

The stapes is the most proximal element of the ossicular chain. Located medioposteriorly relative to the other ossicles, it is composed of a head, two crura (anterior and posterior), and a footplate. Regarding its connectivity, in life, the head articulates with the lenticular apophysis of the incus while the footplate sits on the fenestra vestibuli, retained by the annular ligament. The isolated nature of the stapes and the high resolution of µCT-scan acquisition performed here permit us to describe the structure of this ossicle in cainotheriids in more detail than was possible for [START_REF] Orliac | Fallen in a dead ear: intralabyrinthine preservation of stapes in fossil artiodactyls[END_REF].

The stapedes from DAM1 present a global trapezoidal shape with two long crura separated by a wide foramen intercrurale (Fig. 6 A-B). The latter has the same size on the medial and lateral surface and it extends from the head to the large footplate. The two DAM1 stapedes are very similar in terms of size and shape of the foramen intercrurale. Their footplate are roughly oval in shape and concavo-convex, with a prominent umbo. In the specimen from DAM3, the footplate is bean-shaped (Fig. 6 C vs M). In the DAM1 stapes, the rim is thick along its whole length, unlike that of DAM3, the rim of which is thicker posteriorly and much thinner anteriorly. On the stapes head, the articular facet for the lenticular apophysis of the processus longum is narrow and oval, slightly convex anteroposteriorly and slightly concave mediolaterally (Fig. 6 A,D). The processus muscularis stapedis, located on the top of the posterior crus, appears to be larger in the DAM1 stapedes than in that from DAM3. The general shape of the bone is asymmetrical in specimens from both DAM1 and DAM3, with the anterior crus longer and slenderer than the posterior one.

The posterior crus is slightly straighter in DAM1 stapedes.

Comparisons. The morphology of the cainotheriid stapedes from Dams differs from those of the cow and llama which present a more rectangular general shape in lateral view, due to more symmetrical crura and smaller footplates [START_REF] Doran | Morphology of the mammalian ossicula auditus[END_REF]Fleisher, 1973;[START_REF] Costeur | Middle ear bones of a mid-gestation ruminant foetus extracted from X-ray computed tomography[END_REF]. The cainotheriid stapedes from Dams, like in hippopotamids, display a wide foramen intercrurale extending to footplate; hippopotamids however differ in having a smaller head and a more elliptic (i.e. not asymmetrical) footplate (Fleisher, 1973, fig. 41;[START_REF] Orliac | Fallen in a dead ear: intralabyrinthine preservation of stapes in fossil artiodactyls[END_REF], fig. 2C-D). Asymmetrical crura as observed in cainotheriids are also observed in camelids (Camelus bactrianus; [START_REF] Bai | A functional anatomy of the external and middle ear of the bactrian camel (Camelus bactrianus)[END_REF], suoids (Tayassu tajacu and Microstonyx erymanthius; [START_REF] Orliac | Fallen in a dead ear: intralabyrinthine preservation of stapes in fossil artiodactyls[END_REF] and ruminants (e.g. Giraffa camelopardalis; [START_REF] Doran | Morphology of the mammalian ossicula auditus[END_REF].

Reconstruction of the ossicular chain of the cainotheriin Caenomeryx filholi

The marked intra-specific variability of ossicle shape makes it difficult to reconstruct an articulated ossicular chain based on composite material. We therefore performed a reconstruction of the ossicular chain based on in-situ ossicles preserved in the middle ear cavity of the basicranium UM PDS 3353 from Pech Desse (MP 28, Quercy). The malleus and the stapes were preserved within the left bullar space, while the right side preserved the malleus and the incus. Due to postmortem soft-tissue decay, the ossicles were no longer in connection. A complete middle ear was therefore virtually reconstructed using the left bulla, petrosal and incus and mirror-3D models of the right malleus and stapes. Based on the relative position of the tympanic ring of the bulla and of the fenestra vestibuli of the petrosal, we propose a reconstruction of the three-dimensional orientation of the ossicles within the middle ear cavity (Fig. 7A-D). The malleus is positioned so that it closes anterodorsally the tympanic ring with its anterior process, and that the manubrium contacts the tympanic membrane with its flat part oriented ventrally. Unfortunately, the anterior process of the malleus is broken away on both sides, and the connectivity with the bulla could not be fully assessed. The incus contacts the malleus via the articular area, while it connects the petrosal by soft tissues fixed on the processus brevis. The contact with the stapes is realized by the lenticular apophysis, a very fragile structure that is not preserved here. Nevertheless, according to the location of the fenestra vestibuli (determining the position of the stapes within the middle ear cavity), this process seems to have been orthogonal to the processus longum of the incus. The 3D model of the middle ear reconstruction is available on MorphoMuseuM (Assemat, in press).

Compared to the mallei from DAM1, the articular surface of the malleus from Pech Desse presents a wider angle between the superior and inferior articular facets (nearly 90° for Pech Desse). In addition, the shape of their articular surfaces are clearly distinct; while the articular surface of the mallei of DAM1 (in posterior view) clearly disrupt from the neck of the malleus with a subrectangular shape that extends ventrodorsally, the head of the malleus of Pech Desse is much slender ventrodorsally and somehow extends continuously the neck of the malleus medially (Fig. 2A-B vs Fig. 7I; see also Fig. 3A-C). The orientation of the muscular process is also more medial. The malleus is generally more gracile in the specimen from Pech Desse. It preserves the delicate structure of the manubrium's extremity which is spatulated (Fig. 7C-D), for the DAM1 material, it is difficult to assess if the manubrium is not spatulated or if this feature was broken away on specimens. The shape of the incus from Pech Dess reflects the differences observed at the level of the malleus articular surface. The stapes from Pech Desse (Fig. 7M-P) also exhibits a quite different morphology from that of DAM1, with a general profile slenderer anteroposteriorly, a smaller head (might be due to in situ partial preservation), slight differences in crura width and orientation, and footplate outlines that are oval instead of being bean-shaped. However, the morphology of the stapedial footplate is very similar, being elongated, concavo-convex in dorsal view, and bearing a wide stapedial footplate rim (Fig. 7M-P). The observation of the contact between the petrosal and the bulla (Fig. 7Q) using µCT-scan data have not permitted us to confirm the presence of the processus internus praearticularis (pipa; Maier & Ruf, 2016b), not visible in intracranial view between the tegmen tympani and the basisphenoid bone because of tight contact between the two structures.

Discussion

Inter-and intraspecific variability of the shape of the ossicles has been described for a few group of mammals such as the African mole-rats (Bathyergidae, Lange et al. 2007), the hominoid primates [START_REF] Stoessel | Comparative anatomy of the middle ear ossicles of extant hominids-Introducing a geometric morphometric protocol[END_REF]) and the golden moles (Chrysochloridae, Mason et al. 2018). The sample from DAM1 composed of 16 mallei and 16 incudes, all referred to Paroxacron valdense, allows the consideration of intraspecific variability of these ossicles in this small extinct artiodactyl. The malleus shows a wide shape range; variations mainly affect the size of the articular facet and the rotation of malleus head relative to the body (Fig. 2 G-H). This considerable variability of the shape might result in non-negligible variations in the orientation of the ossicle chain in the auditory area or, in turn, in a similar variation range at the incus level. Indeed, just as for the malleus, the incudes from DAM1, also all referred to Paroxacron valdense, show a remarkable variability of shape (Fig. 4E-F; Fig. 5), mostly concerning the length of the processus brevis, the angle between the two processes, the dorsal bulging of the body, and the width of the articular facet.

Despite relatively significant shape variation, the morphospace of the incudes specimens from DAM1 -representing one single species -is smaller than that of those from DAM3, and rather well separated from it, except for one DAM3 specimen that lies within shown by the wide array of morphologies observed within and between higher rank mammal groups (e.g., [START_REF] Doran | Morphology of the mammalian ossicula auditus[END_REF][START_REF] Fleischer | Studien am Skelett des Gehörorgans der Säugetiere, einschließlich des Menschen[END_REF][START_REF] Nummela | Scaling of the mammalian middle ear[END_REF][START_REF] Schmelzle | Phylogenetic transformations of the ear ossicles in marsupial mammals, with special reference to diprotodontians: a character analysis[END_REF][START_REF] Mason | Of mice, moles and guinea-pigs: functional morphology of the middle ear in living mammals[END_REF][START_REF] Solntseva | Adaptive features of the middle ear of mammal in ontogeny[END_REF]. The morphology of the stapes has, for example, been proposed as a hallmark for major divisions among Placentalia [START_REF] Novacek | Origin and transformation of the mammalian stapes[END_REF]. The variation of shape observed among cainotheriid incudes between DAM1 and DAM3 supports the potential interest of ossicles at the generic level. Yet, establishing a morphotype reference based on ossicles for each cainotheriid genus seems most unlikely given the usual scarcity of these smallest bones among the fossil material collected in localities. At a wider scale, ossicles are likely to provide interesting signal at the Artiodactyla level and documentation of the morphology of the cainotheriid ossicle chain could be of interest to address the phylogenetic relationships of this extinct family. Actually, cainotheriids have been proposed to be closely related to tylopods (e.g., [START_REF] Gentry | The phylogeny of the Artiodactyla[END_REF][START_REF] Thewissen | Whales originated from aquatic artiodactyls in the Eocene epoch of India[END_REF], or closer to ruminants (e.g., [START_REF] Geisler | Phylogenetic relationships of extinct cetartiodactyls: results of simultaneous analyses of molecular, morphological, and stratigraphic data[END_REF][START_REF] O'leary | Impact of increased character sampling on the phylogeny of Cetartiodactyla (Mammalia): combined analysis including fossils[END_REF]. Recent results based on dental evidence placed them together with Anoplotheriidae and Mixtotheriidae, close to Ruminantia [START_REF] Weppe | Cainotheriidés (Mammalia, Artiodactyla) et Grande Coupure : nouveau matériel des phosphorites du Quercy[END_REF]. One stapes has been described for Anoplotheriidae (Diplobune minor; Orliac et al. 2017, fig. 3); it is morphologically very close to that described here for Paroxacron valdense, with asymmetrical crura, a large slender head, a wide foramen intercrurale, and a somewhat convex footplate. However, the general morphological signal carried by the ossicular chain is difficult to interpret at the Artiodactyla level yet, because modern groups also display a wide range of morphologies and specializations. The morphology of the malleus of Dams cainotheriids is close in proportion and shape to that of Ruminantia with a long neck, a wide osseous lamina, and a similar global shape of the muscular process. Compared to camelids and hippos, the manubrium of Caenomeryx is much more spatulated and shows a morphology close to rodents (e.g., Rattus Microtus, Fleischer 1973:fig.29, 31), carnivorans (e.g. Mustela, Fleischer 1973:fig.53) or primates (e.g., Galago, Macaca, Fleischer 1973:fig.18-19); the spatulated aspect strongly recalls that of golden moles [START_REF] Willi | Ossicular differentiation of airborne and seismic stimuli in the Cape golden mole (Chrysochloris asiatica)[END_REF]. The meaning of spatulated manubrium in terms of sound transmission remains unclear as it is found in a wide array of mammalian species.

The cainotheriid incus brings little information and is morphologically similar to that of other artiodactyls with two processes of about the same length [START_REF] Doran | Morphology of the mammalian ossicula auditus[END_REF]. Finally, the stapedial morphology seems to be closer to that of Anoplotheriidae than to any modern representatives of Artiodactyla illustrated in the literature. However, knowledge of early members of Ruminantia and Camelidae is necessary before a proper discussion is engaged on morphological proximity of Cainotheriidae with one modern group or the other.

The present cainotheriid sample provides a very first glimpse into Paleogene artiodactyl ossicle evolution and variability. Compared to the ossicles from DAM1 (dated at ca. 35 Ma), and DAM3 (ca. 32 Ma), the in-situ ossicular chain from Pech Desse (ca. 25 Ma) exhibits a quite different morphology, with more gracile elements. These morphological changes could echo a shift in cainotheriid ecological habits between the late Eocene and the late Oligocene. This shift may relate with deep environmental changes, such as the opening of the vegetation cover that occurred after the Grande Coupure in Western Europe [START_REF] Collinson | Vegetational and floristic changes around the Eocene/Oligocene boundary in Western and Central Europe[END_REF][START_REF] Cavagnetto | Preliminary palynological data on floristic and climatic changes during the Middle Eocene-Early Oligocene of the eastern Ebro Basin, northeast Spain[END_REF]. However, documentation of the ossicular morphology of other Oligocene-Miocene cainotheriid genera (i.e., Plesiomeryx and Cainotherium) is necessary to properly address this question in the light of the phylogenetical signal, as revealed by craniodental morphology.

Conclusions

The unprecedented fossil ossicles sample from the karstic network of Dams, including a total of 18 mallei, 28 incudes and three stapedes, allows the documentation of intra-and interspecific variability of auditory ossicle morphology within Cainotheriidae. This descriptive work constitutes the first description of a reconstructed ossicular chain of a terrestrial Paleogene artiodactyl species. Despite considerable intraspecific variability, the malleus, the incus, and the stapes appear to be taxonomically informative at the Cainotheriidae scale. This highlights the interest of picking these tiny bones (~1-2 mm long) when sorting out the sediments. Internal investigation of fossils by µCT-scan imaging will certainly also allow for completing our knowledge of Paleogene artiodactyl ossicles, thereby widening our observations and conclusions, notably from a phylogenetic perspective. andS2) at putative location of the processus internus prearticularis (pipa). Abbreviations: eam, external auditory meatus; fc, fenestra cochleae; fv, fenestra vestibuli; hm, head of malleus; ib, incus bulge; lp, lateral process of malleus; mn, manubrium; mp, muscular process of malleus; pb, processus brevis of the incus; pl, processus longum of the incus; pop, paroccipital process; pt, petrosal bone; sf, stapedial footplate; sh, stapes head; tb, tympanic bulla. Orientations: A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bars, A-B = 5 mm; C-P = 1 mm.

Figure captions

  DAM1 morphospace. This individual from DAM3 could be a representative of the genus Paroxacron and could either document the species Paroxacron bergeri or Paroxacron sp. retrieved in DAM3 (Weppe, 2018). Indeed, the genus Paroxacron crosses the Eocene/Oligocene transition and is found both in DAM1 (with the single oxacronine cainotheriid species Paroxacron valdense) and DAM3 fossiliferous levels. The larger morphospace covered by the specimens from DAM3 compared to DAM1 could be explained by the co-occurrence in DAM3 of three different cainotheriid genera (Oxacroninae: Paroxacron; Cainotheriinae: Plesiomeryx and Caenomeryx), comprising five different species. The morphology of the younger ossicles from Pech Desse, assigned to Caenomeryx cf. procommunis, is also markedly different from those described from DAM1 and DAM3, confirming the potential systematic and phylogenetic interest of ossicular morphology, as

Figure 1 .

 1 Figure 1. Position of landmarks used in the Generalized partial Procrustes analysis (pGPA) and principal component analysis (PCA) on the malleus (A) and the incus (B) of late Eocene and early Oligocene Cainotheriidae from Dams localities (DAM1 and DAM3, Phosphorites of Quercy, SW France).

Figure 2 .

 2 Figure 2. Left cainotheriid malleus from Dams DAM1 (DAM1 330) in (A) posterior, (B) medial, (C) anterior, (D) lateral, (E) medioventral, and (F) dorsolateral views. G-H, Illustration of morphological variability as observed in (G) posterior and (H) dorsomedial views of a right malleus). Abbreviations: ap, anterior process; as, articular surface; cs, capitular spine; hm, head of malleus; iaf, inferior articular facet; lp, lateral process; mn, manubrium; mp, muscular process; nm, neck of malleus; ol, osseous lamina; ola, outer lamella; saf, superior articular facet. Orientations: A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bar = 1 mm.

Figure 3 .

 3 Figure 3. PCAs on Procrustes coordinates illustrating morphological variability of the cainotheriid malleus from DAM1 (late Eocene, Phosphorites of Quercy, SW France). (A) Inter-and intraspecific variation of the shape of the malleus based on 7 landmarks; projection of specimens from DAM1 (in black) and Pech Desse (in orange) on the first factorial plane; (B-C; E-F) Patterns of variation along PCs with maximal values in red and minimal in blue; (B) shape variation on PC1 of (A); (C) shape variation on PC2 of (A); (D) Intraspecific variation of the shape of the malleus based on 9 landmarks; projection of specimens from DAM1 on the first two PCs; (E) shape variation on PC1 of (D); (F) shape variation on PC2 of (D). See text for more details.

Figure 4 .

 4 Figure 4. Left cainotheriid incus from Dams DAM1 (DAM 1 307) in (A) dorsomedial, (B) lateral, (C) posterior, (D) anterior, views. E-F, Illustration of morphological variability range as observed in (E) lateral and (F) dorsal views. Abbreviations: iaf, inferior articular facet; ib, incus bulge; pb, processus brevis; pl, processus longum; plg, processus longum groove; saf, superior articular facet. Orientations: A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bar = 0.5 mm.

Figure 5 .

 5 Figure 5. PCA on Procrustes coordinates illustrating morphological variability of the cainotheriid incus from the localities of DAM1 (grey surface; late Eocene), DAM3 (pink surface; early Oligocene) and Pech Desse (orange square; late Oligocene). (A) Projection of individuals on the first factorial plane; (B-C) Patterns of variation along PC1 (B) and PC2 (C) with maximal values in red and minimal in blue.

Figure 6 .

 6 Figure 6. Right stapes of Paleogene Cainotheriidae from the Phosphorites of Quercy, SW France. (A-I) Paroxacron valdense from DAM1, late Eocene (A-E, DAM 1 316,) (F-I. DAM 1 317) -A, ventral view; B, dorsal view; C, lateral view; D, posterior view; E, antero dorsal view. DAM 1 317 (F-I) -F, ventral view; G, posterior view; H, dorsal view; I, lateral view. (J-M) Cainotheriidae indet. from DAM3, early Oligocene (DAM 3 13) J, ventral view; K, posterior view; L, dorsal view; M, lateral view; Abbreviations: ac, anterior crus; fi, foramen intercrurale; pc, posterior crus; pm, processus muscularis; sfr, stapedial footplate rim; sh, stapes head; um, umbo. Orientations: A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bars = 0.5 mm.

Figure 7 .

 7 Figure 7. 3D reconstruction of the middle ear of Caenomeryx cf. procommunis from Pech Desse, late Oligocene, Phosphorites of Quercy, SW France (UM PDS 3353). A-B: Left middle ear with in-situ ossicles; C-D: virtually reassembled composite left ossicle chain in ventral (C) and dorsal (D) views; E-H: incus; I-L, malleus; M-P, stapes; Q, Petro-tympanic complex of UM PDS 3353 showing orthogonal slices (S1 and S2) at putative location of the

Acknowledgements

We are especially grateful to Thierry Pélissié and Gilles Escarguel who organized the field campaign in Dams (since 2016) and who are in charge of field excavation and prospection in the Phosphorites of Quercy; we are also thankful to all the team of the Cloup d'Aural and to the Quercy research team (C. Blondel, PALEVOPRIM, Poitiers; M. Godinot, MNHN, Paris; S. Couette, EPHE, Dijon; Margot Bernardi, EPHE, Dijon; M. Vianey-Liaud, ISEM, Montpellier; Christian Bousquet, Cloup d'Aural) for their work in the field. Many thanks to M. Longuet for her help with sorting out the ossicles of DAM1. Finally, we are very grateful to M.J. Mason, S. Nummela, and L. Costeur for their fruitful comments and suggestion that helped us improving substantially this manuscript. This work was financially supported by the ANR program DEADENDER (ANR-18-CE02-0003-01) -PI M.J. Orliac. This is ISEM publication n° 2020-024.

Author contributions

A. Assemat performed the morphological study, the statistical analyses, and wrote the paper.


Craig Baker-Austin

Carla Pruzzo

James D Oliver

Delphine Destoumieux-Garzon

Vibrios -from genes to ecosystems

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++++++++++++

This special edition of Environmental Microbiology "Vibrios -from Genes to Ecosystems" brings together and captures the sheer breadth of research across this fascinating genus of microorganisms, through a variety of research, opinion and review articles. Perhaps one of the most captivating aspects related to vibrios is the duality that they represent in microbiology. On one hand they are important autochthonous constituents of marine and coastal waters throughout the world, playing critical ecological roles through a myriad of phylogenetically and functionally diverse representatives; on the other, unfortunately, vibrios constitute a uniquely potent group of human and animal pathogens capable of causing an array of diseases such as gastroenteritis, sepsis, and wound infections. Through the most famous (or infamous) member of this genus -V. cholerae, cholera has remained a global scourge, and alongside the seafood pathogen V. parahaemolyticus, the only pathogens that have emerged from aquatic and marine sources to cause bona fide global pandemics.

Certainly, the research studies presented in this special edition draw on many aspects of this duality and provide critical insights regarding the growing importance of these bacteria, perhaps in the process highlighting that vibrios constitute a separate field of study in and of itself.

Several review articles in this special edition highlight why these bacteria are a critically important topic to study in microbiology. [START_REF] Vezzulli | Global emergence of environmental non-O1/O139 Vibrio cholerae infections linked with climate change: a neglected research field?[END_REF] outlines the growing This article is protected by copyright. All rights reserved.

Editorial

importance of non-cholera V. cholerae (non O1/O139) using a variety of epidemiological, microbiological and public health data, and asks the pertinent question if this member of the Vibrionaceae family deserves greater research emphasis, particularly given the impact of climate change on this important human pathogen. Similarly, [START_REF] Froelich | In hot water: effects of climate change on Vibriohuman interactions[END_REF] outline how and why a warming climatic system may place humans in greater contact -and consequently risk, of pathogenic vibrios. The risk of contamination is also explored by [START_REF] Canty | A comparison between farmed oysters using floating cages and oysters grown on-bottom reveals more potentially human pathogenic Vibrio in the on-bottom oysters[END_REF] who study the effect of farming practices on the prevalence of the human pathogen V. vulnificus in oysters. Moreover, the review article by Hernández-Cabanyero and Amaro (2020) provides insights into the evolution and life strategies of this pathogen in response to selected environmental parameters. This article also contributes to fill an important gap in the existing knowledge on the biology of V. vulnificus and stresses its importance as a zoonotic pathogen linked to fish farms and climate change.

Several aspects related to the fascinating biology and genomics of vibrios are described here too. Sozhamman and Waldminghaus (2020) outline the importance of dual chromosomes in vibrios -focussing on the pathogen V. cholerae. Single-chromosome V. cholerae have been previously identified, and these particular strains are essential to study fundamental questions on chromosome biology in general, and DNA replication in particular. Furthering our understanding of the biology of these organisms, [START_REF] Crisan | The Vibrio cholerae type VI secretion system: toxins, regulators and consequences[END_REF] Using a variety of methodologies, including molecular microbiological approaches, sequencing and comparative bioinformatics they demonstrate both the integral role of the competence regulon as well as conserved nature of this system in pathogenic V. cholerae, further outlining the role of chitin and HGT in this context.

Two articles show that the multiple host and environmental stressors acting on the selection of Vibrio virulence factors may lead to cases of conflicting or coincidental selection. In V. splendidus, [START_REF] Oyanedel | Vibrio splendidus Oantigen structure: a trade-off between virulence to oysters and resistance to grazers[END_REF] show that the structure of the O-antigen is detrimental for virulence but essential for resistance to amoeba grazing, which likely explains it hypervariability in environmental isolates. In contrast, the article by [START_REF] Robino | Resistance of the oyster pathogen Vibrio tasmaniensis LGP32 against grazing by Vannella sp. marine amoeba involves Vsm and CopA virulence factors[END_REF] shows that resistance to copper and toxic factors (metalloproteases) in V. tasmaniensis are beneficial in the interaction with both oysters and marine amoeba. How the interactions of vibrios with environmental predators and metazoan hosts may be beneficial or costly for Vibrio virulence is discussed in [START_REF] Destoumieux-Garzón | Vibrio-bivalve interactions in health and disease[END_REF], who review the current knowledge on Vibrio-bivalve interactions. Critically important to understand how and why these bacteria can cause infections in humans and animals is the need for reliable, tractable and repeatable model systems, and especially those that constitute and reflect real exposure hazards. [START_REF] Hubert | A universal oyster infection model demonstrates that Vibrio vulnificus Type 6 secretion systems have antibacterial activity in vivo[END_REF] outline an approach to generate 'artificial' marine snow, This article is protected by copyright. All rights reserved.

Editorial

into which bacteria such as V. vulnificus can be incorporated to facilitate extensive uptake by oysters. This represents a highly promising and adaptable model for bacterial study within filter-feeding shellfish -the major route through which many of these species cause disease in humans. This study also outlines the growing importance of the T6SS in terms of potentially modulating numbers of these pathogens in the environment, critically important information to more fully understand the ecology of V. vulnificus.

Other articles of this special edition tackle the vast complexity of Vibrio biology and ecology highlighting how science is moving on fast in this fascinating research field. The opinion paper by [START_REF] Baker-Austin | The new tools revolutionizing Vibrio science[END_REF] outlines some of the most exciting, innovative and translational scientific advances that are currently being applied to these ecologically, environmentally and clinically important bacteria. From this paper, as well as from the other contributions of the special edition, it is evident that the coordinated use of different experimental approaches across various disciplines, not just microbiological, is the most fruitful strategy to decipher structures and mechanisms underlying the duality of life style of Vibrio bacteria, and to clarify the still obscure aspects of their interactions with both the environment and their host. We feel that the enormous breadth of research captured in this special edition -encompassing microbiology, molecular biology, genetics, and bioinformatics, among others -outline the true interdisciplinary nature of vibrios, and underline why they continue to represent such a fascinating and rewarding group of microorganisms to study.

This article is protected by copyright. All rights reserved.

  provide a cogent overview of the importance of the type 6 secretions system (T6SS) as proteinaceous weapons used by a variety of Gram-negative bacteria. Utilizing V. cholerae as an exemplar model organism for T6SS they reveal the broad yet growing importance of these secretion This article is protected by copyright. All rights reserved.

  Editorialsystems as intrinsic mechanisms to evade phagocytic eukaryotes, cause intestinal inflammation, and compete against other bacteria with toxins that disrupt lipid membranes, cell walls and actin cytoskeletons.Recently, Vibrio bacteria have also garnered attention as host for molecular biology and biotechnology applications. As outlined in the review article by[START_REF] Hoff | Vibrio natriegens: an ultrafast-growing marine bacterium as emerging synthetic biology chassis[END_REF], this is the case with V. natriegens, the fastest-growing non-pathogenic bacterium known to date. Spanning all the way from its initial isolation to its latest applications, the article focuses on V. natriegens successful applications in synthetic biology and biotechnology in recent years, and examines missing methods and tools which need to be established in order to exploit the full potential of this fascinating bacterium. Identifying the mechanisms underpinning virulence traits and understanding the drivers of their selection is key for the prevention of human and animal diseases caused by vibrios. Here are a series of articles that study virulence determinants in environmental and clinical strains. The article by Piel et al. (2019) reveals the importance of a plasmid highly prevalent in oyster farms, which encodes a T6SS involved in virulence in the oyster pathogen, V. crassostreae. While the T6SS contributes to the dampen the immune defenses of the oyster, Drebes Dörr and Blokesch (2020) show that T6SSs in environmental isolates of V. cholerae are major players of interbacterial competition while others, which harbor an actin cross-linking domain on the VgrG protein, and confer resistance to amoeba grazing. Seibt et al. (2020) explore the role of the σ54-dependent transcriptional regulator VasH in the expression of such secretion This article is protected by copyright. All rights reserved. system in pandemic cholera causing El Tor strain A1552, which requires specialized growth signals of low temperature and high osmolarity for T6SS expression. A study presented here by Stutzmann et al. (2020) compared the chitin-induced competence and transformation behavior in a variety of pandemic O1 El Tor V. cholerae isolates as well as an O139 isolate.

This article is protected by copyright. All rights reserved.