Article https://doi.org/10.1038/s41467-022-34656-0 Ruminant inner ear shape records 35 million years of neutral evolution Received: 11 November 2021 Bastien Mennecart 1 , Laura Dziomber 2,3, Manuela Aiglstorfer 4, Faysal Bibi 5, Daniel DeMiguel 6,7,8, Masaki Fujita9, Mugino O. Kubo 10, Accepted: 2 November 2022 Flavie Laurens11, Jin Meng 12, Grégoire Métais13, Bert Müller 14, María Ríos15, Gertrud E. Rössner16,17, Israel M. Sánchez 8, Georg Schulz 14,18, Shiqi Wang 19 & Loïc Costeur 1 Check for updates Extrinsic and intrinsic factors impact diversity. On deep-time scales, the 1234567890():,; 1234567890():,; extrinsic impact of climate and geology are crucial, but poorly understood. Here, we use the inner ear morphology of ruminant artiodactyls to test for a deep-time correlation between a low adaptive anatomical structure and both extrinsic and intrinsic variables. We apply geometric morphometric analyses in a phylogenetic frame to X-ray computed tomographic data from 191 ruminant species. Contrasting results across ruminant clades show that neutral evolu- tionary processes over time may strongly influence the evolution of inner ear morphology. Extant, ecologically diversified clades increase their evolutionary rate with decreasing Cenozoic global temperatures. Evolutionary rate peaks with the colonization of new continents. Simultaneously, ecologically restric- ted clades show declining or unchanged rates. These results suggest that both climate and paleogeography produced heterogeneous environments, which likely facilitated Cervidae and Bovidae diversification and exemplifies the effect of extrinsic and intrinsic factors on evolution in ruminants. The Earth has undergone a general declining trend in global tem- Taxonomic diversity and body mass are major parameters used peratures for the last 45 million years1. This has led to the estab- for inferring evolutionary rates and diversification episodes in mam- lishment of permanent polar ice caps, which have strengthened the mals in deep time (e.g., ref. 8,). However, these proxies have significant climatic belts leading to widespread continental seasonality and drawbacks that prevent them from being effective, especially for aridification. Climate is a major factor influencing distribution and determining reliable evolutionary rates. Body mass is a parameter diversity of life on the planet and it plays a cardinal role in specia- known to be strongly correlated with ecological factors. Extinct taxa tion and extinction, best recognized on macro-evolutionary or are often represented by fragmentary remains only, with a generally geological time scales2–4. Moreover, climate variability, in particular discontinuous record leading to difficulties in tracing the entire evo- in periods of declining global temperatures, is known to induce lutionary history of a clade. Indeed, teeth are mostly well-preserved environmental heterogeneity and is hereby a strong selection fac- and widely distributed in the fossil record and represent one of the tor. Changing climatic conditions and related tectonic activity (i.e., only possibilities to establish correlation between diversification and changes in local/regional to continental scale paleogeography) extrinsic factors on large scales and for whole clades9. However, they involve environmental shifts, modification in resource availability, are also directly linked to feeding habits, making them prone to eco- and habitat fragmentation, paving paths for innovations to arise5 logical plasticity. Investigating the entire evolution of a clade requires and offer potential for diversification6. An example of this influence focusing on a structure that evolves neutrally through time. The bony can be observed throughout the last 5 million years of human labyrinth is an anatomical structure that has received attention evolution7. recently in evolutionary research and preliminarily proven to A full list of affiliations appears at the end of the paper. e-mail: mennecartbastien@gmail.com Nature Communications | (2022)13:7222 1 Article https://doi.org/10.1038/s41467-022-34656-0 accurately reflect the kinship (i.e., phylogeny) between species —and scenarios. Both the increase of maximum size, according to the hence their evolution over time10,11. The bony labyrinth is preserved as Depéret-Cope’s rule, and an increase of the evolutionary rate of size, a hollow structure inside the petrosal bone, which is the densest hard are demonstrated in the younger taxa of almost all clades. The tissue of the mammalian skeleto-dental system after teeth. It thus morphological evolutionary rates of the bony labyrinth through preserves extraordinarily well in the fossil record. Since the advent of time vary depending on the considered clade. Bony labyrinth CT-scanning techniques, allowing an assembling of a virtual endocast, morphological evolutionary rates clearly react to intrinsic factors the petrosal bone and the bony labyrinth have helped to clarify various responding to their respective ecological optimum combined with aspects of mammalian evolution. Examples are found in the early steps environmental and geographical conditions. of mammalian evolution in the Mesozoic12, the mammalian phylogeny reconstructed using the wealth of morphological characters they can Results/Discussion yield10,13,14, or the evolution and dispersal of modern humans from Principal component analysis Africa11. In addition, inner ear functions for hearing and balance are The PCA polymorphospace possesses a strong phylogenetic signal critical in the interaction between and within animals and their envir- (permutation test p < 0.001). The average amount of shape change onments. Yet, data show that bony labyrinth morphology may only along the branches of the phylogenetic tree is indeed very small, in partly depend on ecological and physiological parameters such as comparison to the null hypothesis of no phylogenetic signal. The habitat type15,16, or locomotion capabilities17,18. Predominantly, the dataset displays similar covariances among species to the covariances morphological signal recorded in the bony labyrinth is strongly related expected under Brownian motion (Pagel’s λ 0.83, p < 0.001). However, to phylogeny10,19 and is considered to reflect neutral evolution20. The the variance is mostly within the clade, not between clades (Bloom- low selection pressure on this morphological structure may be due to berg’s K 0.36). its important developmental constraints10.The bony labyrinth may 21.99% of bony labyrinth shape disparity is present along the thus be an appropriate proxy to investigate deep-time neutral evolu- PC1 axis (Supplementary Data 1). The relative size of the anterior tion in vertebrates. and posterior semicircular canals in comparison to that of the Ruminants are one of the most diverse groups of large mam- cochlea is smaller in the most negative values of PC1 in comparison mals living today. They have evolved under the climatic fluctuations to the most positive ones. The anterior and posterior semicircular of the last 45 Ma from tiny forms without cranial appendages to the canals are more rounded in the most negative PC1 values than the modern group with a diversity in body sizes, locomotor capacities, positive ones where they are dorsally extended. The lateral semi- and cranial appendages (i.e., pronghorns, ossicones, antlers, or circular canal in the most negative values of PC1 branches dorsally horns). Ruminants are abundant in the fossil record and very well above the posterior ampulla, in the vestibule between the latter and represented in paleontological and neontological museum’s col- the base of the common crus (Supplementary Data 1). In the most lections. These collections document their survival through the positive values of PC1, the lateral semicircular canal directly bran- worldwide Eocene-Oligocene cooling and aridification event 34 ches in the posterior ampulla. In the most negative values, the million years ago and show how they successfully adapted to new vestibular aqueduct runs parallel to the midline of the common crus habitats. Thereafter, they highly diversified during the Miocene along its course and extends above the end of the common crus Climatic Optimum ca. 18–15 Ma when cranial appendages first dorsally. In the most positive values, the vestibular aqueduct is occurred in all modern groups21. They have distributed and diver- curved and shorter than the common crus (Supplementary Data 1). sified in South America at the onset of the ice age ca. 3 Ma during the The PC2 axis represents 10.60% of the data variation. The bony Great American Biotic Interchange (GABI) and, finally, became clo- labyrinth located in the most positive values of PC2 are displaying a sely linked to human evolution. Ruminants have thus not only bigger cochlea than in the most negative PC2 values, especially accompanied and survived the major climatic and geological events considering the first turn of the cochlea (Supplementary Data 1). In of the Cenozoic, but highly benefited in species diversity. Through the most negative values of PC2, the ovoid lateral semicircular canal these radiations, they became one of the taxonomically richest directly branches in the posterior ampulla. In the most positive components of the terrestrial vertebrate fauna. Yet, little is known values of PC2, the rounded lateral semicircular canal branches about how they precisely reacted to environmental changes and anteriorly to the posterior ampulla in the vestibule. The fenestra how their diversification and radiations were impacted by intrinsic vestibuli is narrower in the most negative values of PC2 than in the and extrinsic factors. Due to intrinsic factors related to ecological positive ones. The vestibular aqueduct is extremely short and optimum (e.g., metabolism), the evolutionary rates of the bony curved in the most negative values of PC2, while it is straight and labyrinth morphology may differ from one clade to the other longer than the common crus in the most positive values. depending on Cenozoic climatic events (extrinsic factors), high- The most negative values along PC1 are occupied by tragulids, lighting the current picture of the ruminant differences observed in while members of Pecora have higher values (Supplementary Data 1). their diversity and success. For all of these reasons, we use the No clear trend can be observed within Pecora along the PC1 axis. Along ruminant bony labyrinth shape as a proxy to explore how the evo- the PC2 axis, the negative values are mostly occupied by bovids within lution of the different ruminant lineages have been impacted by the Pecora. The positive values of PC2 are occupied by all members of intrinsic and extrinsic factors. Pecora and Stem Ruminantia without clear distinction. The Bovidae In this study, we investigate if the bony labyrinth is a good represents the largest morphospace within the dataset, having the proxy to analyze the phylogeny and evolution of a whole mamma- maximum and minimum PC1 and PC2 values (Supplementary Data 1). lian clade, directly including fossil data into the analysis. Since bony When PC1 vs PC2 scores are further inspected through time, we find labyrinth morphology may partly evolve neutrally and its size evo- that the Early-Middle Miocene Dorcatherium specimens have the lution may be more strongly related to ecological factors, a com- highest values along the PC1 axis within the Tragulidae, marking a parison between both parameters is likely to clarify different transition between the Stem Ruminantia and the modern Tragulidae aspects of ruminant evolution. In addition, we test if bony labyrinth (Supplementary Fig. 1). Interestingly, until the transition between the morphological evolutionary rates help understand the relationships Middle and Late Miocene, Pecora display a similar shape disparity between intrinsic and extrinsic factors shaping the evolutionary along the PC1 and the PC2 axes of Stem Ruminantia (Supplementary processes. Our results show that bony labyrinth shape morphology Fig. 1). The morphological increase in disparity of the Bovidae also took compares well to molecular-based phylogenetic hypotheses. place during this period and diverges taxonomically in the fossil Bony labyrinth morphology and size display different evolutionary record. Nature Communications | (2022)13:7222 2 Article https://doi.org/10.1038/s41467-022-34656-0 Between-group PCA branching in the vestibule between the posterior ampulla and the base The overall classification accuracy when considering the bg-PCA is 58.5 of the common crus. Along the CV2 axis (16.9%), the negative values % (Supplementary Data). Stem Ruminantia, Tragulidae, Antilocapridae, are mostly occupied by Bovidae, while Stem Ruminantia occupy the Dromomerycidae, and Cervidae are above 60% of classification accu- highest values (Supplementary Data 1). A trend can be observed with racy with the Tragulidae culminating at 100%. In contrast, Giraffidae, the Giraffomorpha and the Cervidae having the lower CV2 positive Moschidae, and Stem Pecora are below 50% of classification accuracy scores, the Moschidae and Antilocapridae having slightly higher with Moschidae having the lowest cross-validated classification result CV2 scores, and finally the Stem Pecora having just lower CV2 scores (36.4%). Stem Pecora are largely reattributed as stem ruminants than the Stem Ruminantia. The Stem Ruminantia and Stem Pecora (27.3%), while Giraffidae are reattributed as Dromomerycidae (12.5%). possess an enlarged cochlea, longer in Stem Ruminantia than Stem The bg-PC1 axis represents 70.92% of the variance (Supplemen- Pecora, and a large rounded fenestra vestibule. The base of the very tary Data 1). A clear distinction between the Tragulidae and all the elongated vestibular aqueduct is anterior to the axis of the common remaining ruminant groups can be observed. The Tragulidae bony crus, and the lateral semicircular canal branches high in the posterior labyrinth differs from that of the other ruminants by having a longer ampulla. In Stem Ruminantia, the posterior semicircular canal is cochlea, a longer common crus than the vestibular aqueduct, and a remarkably higher than the anterior semicircular canal, while in the very oblique lateral semicircular canal branching in the vestibule Stem Pecora they are of similar height. The Antilocapridae, Gir- between the posterior ampulla and the base of the common crus. affomorpha, and Cervidae are relatively close morphologically to the Along the bg-PC2 axis (12.85%), the negative values are mostly occu- Stem Pecora. Nevertheless, Antilocapridae differ markedly from the pied by Bovidae, while Stem Ruminantia and Stem Pecora mostly other Crown Pecora by retaining a large rounded fenestra vestibule, occupy the highest values along bg-PC2 (Supplementary Data 1). All while Giraffomorpha, Cervidae, Bovidae, and Moschidae possess an other ruminant groups overlap along bg-PC2 scores. The stem rumi- ovoid fenestra vestibule. Members of the Bovidae are generally char- nants possess a long cochlea, a large fenestra vestibuli, the base of the acterized by a short and oblique vestibular aqueduct and a very obli- vestibular aqueduct is anterior to the axis of the common crus, and the que lateral semicircular canal that branches, similarly to members of posterior semicircular canal is remarkably higher than the anterior the Moschidae, in the vestibule anterior to the posterior ampulla. semicircular canal. In the Stem Pecora, the cochlea is shorter, the Posterior and anterior semicircular canals are relatively of the same posterior and anterior semicircular canals are of similar height, and the height in Bovidae, while the anterior one is higher than the posterior lateral semicircular canal branches high in the posterior ampulla. The one in Moschidae. Their vestibular aqueduct is longer than in Bovidae. Antilocapridae, Giraffidae, and Cervidae are relatively close morpho- logically to the Stem Pecora. Members of the Bovidae are generally Inner ear shape reflects phylogeny characterized by a short and slightly oblique vestibular aqueduct and a As expected according to previous analyses of the bony labyrinth in very oblique lateral semicircular canal that branches, similarly to vertebrates10,15,22–24, the impact of the phylogeny on the shape of the members of the Moschidae, in the vestibule anterior to the posterior bony labyrinth is significant in ruminants. The bg-PCA shows that ampulla. Pecora, except Bovidae younger than 10-9 Ma, have retained an overall When observing bg-PC1 vs bg-PC2 through time, the Early-Middle bony labyrinth morphology similar to that of the most ancestral stem Miocene Dorcatherium specimens have the lowest values along bg-PC1 ruminants studied (Supplementary Fig. 2). The speciose extant bovids axis within the Tragulidae, making a transition between the Stem (more than 139 living species25) started their long-lasting diversifica- Ruminantia and the modern Tragulidae (Supplementary Fig. 2). The tion in the Late Miocene, expressed in taxonomy and reflected in Stem Pecora and the Stem Ruminantia display a relatively similar mean disparity of their bony labyrinth morphology. They colonized very shape along the bg-PC1 and the bg-PC2 axes (Supplementary Fig. 2). A diverse environments and habitats, hereby exemplifying the largest first shape shift occurs for the Crown Ruminantia during the Early Miocene. Considering the Bovidae, similarly to their disparity of shape (PCA), a shift in the characterizing shape of the family can be traced back to the transition between the Middle and Late Miocene. Canonical variates analysis The overall classification accuracy when considering the CVA is 80.4 % (Supplementary Data 1). Stem Pecora, Tragulidae, Antilocapridae, Cervidae, and Bovidae are above 60% of classification accuracy with the Tragulidae culminating at 96.6%. Stem Ruminantia, Giraffidae, and Dromomerycidae, however, are below 50% of classification accuracy. Dromomerycidae, wich have a null cross-validated classification result, could equally be confused with Stem Pecora, Giraffidae, and Cervidae (33.3%). Stem Ruminantia can be confused with Stem Pecora (30.0%), while Giraffidae may be confused with Bovidae (37.5%). The CV1 axis represents 45.2% of the total variance (Fig. 1). The Tragulidae are isolated among the highest CV1 values (15 to 20). All other ruminants are located under the CV1 value of 5. We can observe a trend towards the lower values in the repartition of the different clades starting from the Stem Ruminantia, Stem Pecora, Moschidae, Antilo- Fig. 1 | Evolution of ruminant bony labyrinth morphology through time. Shape capridae, Giraffidae, Cervidae, and Bovidae. This trend is even clearer changes are observed along CV axes (CV1 45.23% & CV2 16.84%). Groups are Stem considering CV1 vs CV2 through time (Fig. 1). Along the CV2 axis Ruminantia (light yellow), Tragulidae (dark blue), Stem Pecora (light dark blue), (16.9%), we can observe a similar trend in the repartition of the data. Antilocapridae (dark red), Dromomerycidae (orange), Giraffomorpha (dark light The position of the Tragulidae is just intermediate to the Cervidae and blue), Cervidae (light blue), Moschidae (yellow), and Bovidae (red). Bivariate plot of Bovidae. The Tragulidae bony labyrinth differs from that of the other CV1 vs CV2 and animated gif of the Fig. 2 can be found in Supplementary Data 1. ruminants by having a longer cochlea, a longer common crus than the Thickened circles are the bony labyrinth specimens from Supplementary Fig. 3. vestibular aqueduct, and a very oblique lateral semicircular canal Silhouettes of the families modified from ref. 107. Nature Communications | (2022)13:7222 3 Article https://doi.org/10.1038/s41467-022-34656-0 ecological diversity among Ruminantia26. The bony labyrinth of tra- see accelerating rates of morphological evolution of the general gulids (the only living non-pecoran ruminants) was morphologically pecoran bony labyrinth shape (Supplementary Fig. 4). These cold derived early in its evolutionary history and has remained thus con- pulses and periods are known to have triggered the development of served thereafter10 (Fig. 1). Maximizing the separation of the between- more arid and more heterogeneous environments and habitats, group means in relation to the variation within the groups’ ratio especially at mid-latitudes44–46. Such a fragmentation increases the according to specified selected clades, we clearly observe a shape diversity of potential habitats, stimulating clades to diversify ecologi- continuum through time from the basalmost and oldest stem rumi- cally and taxonomically47. Comparing different ruminant clades offers nants to different pecoran families (Fig. 1). The bony labyrinth a more complex picture. The evolutionary rates for Tragulidae are morphologies characterizing the Pronghorn are the closest to those of constantly low and not supported by the climatic model (β = 0). This the stem pecorans, while those of bovids are more derived, confirming result indicates that they may have been more stenopotent and unable the consensus reached by molecular phylogenetic hypotheses27–29. to expand into more arid or colder environments. Low bony labyrinth evolutionary rates may here express a certain phenotypic stasis, which Rates of evolution show radiation events is otherwise known in groups qualified as “living fossils”, where mor- Ruminants went through several episodes of radiation in relation to phology and the ecological niche have barely changed over millions of environmental changes10,21,28. The evolutionary rates of the bony years. In contrast to this, we find a positive correlation in gir- labyrinth morphology reveal different scenarios for the different affomorphs (β > 0). Giraffomorphs have a very low diversity today with clades of ruminants. The Early Miocene emerges as a period when only two living genera restricted to African intertropical savannas and evolutionary rates show accelerations at the base of crown pecoran limited to subtropical forests. Despite a strong increase in global clades, especially in bovids and giraffomorphs (Fig. 2). This has already species diversity within crown Giraffidae during the Middle and Late been supposed based on molecular data28. The rapid morphological Miocene, their rates of evolution decline after this period (Fig. 3). This diversification at the base of these pecoran clades may represent the concurs with a decrease of global temperatures and the extinction of effect of an ecological opportunity30 since only these pecoran clades stem giraffomorphs (palaeomerycoids and non-giraffid giraffoids), entered Africa at this precise period, while few other ruminants are which testifies to a drop in morphological disparity due to the known prior to that time from Africa31–33. Other more significant evo- shrinking of their habitats. Biological factors must be postulated here. lutionary accelerations occur at terminal nodes in clades with recent The less efficient longer gestation periods in giraffes may have accel- radiative events (Supplementary Data 1). These include duikers erated the disappearance of these large ruminants25 within the context (Cephalophini, of which 19 species arose in the last million years in the of global cooling. Moreover, diversification is known to slow down forests of central Africa25,34) or Odocoileini in cervids. These examples when a large part of a clade disappears48, accentuating the effect of confirm that rates of speciation and morphological evolution are evolutionary rate decrease. Both cervids and bovids benefited from positively correlated on macroevolutionary time-scales35. For example, the global temperature decline related to permanent polar glaciations insular taxa are known to strongly diverge morphologically from their through adaptation to more heterogeneous habitats and seasonal cli- mainland ancestors (e.g., Köhler and Moyà-Solà36). Indeed, clades mates, which is reflected in the evolutionary rates of the bony labyrinth including specimens and species that are insular since the Pleistocene (β < 0; Fig. 3). In particular, increasing aridification related to the global (e.g., Rusa timorensis, Cervus astylodon, and Cervus nippon from Japan) cooling in Africa from the early Late Miocene onwards led to habitat reveal acceleration of rates in the evolution of the bony labyrinth. heterogeneity, thus promoting an ecological opportunity for taxa Bovids and cervids diversify greatly from the Middle Miocene onwards through adaptive radiation and, ultimately, to the diversification of and start colonizing a large range of biomes. As an example, many floras and faunas49. However, global temperatures alone cannot caprine bovids are typical mountain dwellers. They probably origi- explain the huge increase in the evolutionary rate found in Cervidae nated on the rising Middle Miocene Tibetan Plateau and diversified in during the Plio-Pleistocene (Δ AIC < 4 without any effect of smoothing response to this new ecological opportunity29 highlighted by an parameters; Supplementary Data 1). Indeed, the last 3 million years increase in their evolutionary rate (Fig. 2). Warm climate open envir- experienced an explosion of cervid species diversity, especially in onment dwellers such as the very speciose antelope clades and their South America. During this time at least 19 extant species evolved most recent common ancestors are recorded in the latest Middle to regionally and which currently represent a third of total cervid species Late Miocene of Africa37,38. Other clades display significantly lower diversity. The interplay between a new ecological opportunity for evolutionary rates throughout their history, such as the Tragulidae North American deer, entering South America during the Great (Supplementary Data 1), which are arising in the Eocene39,40. Based on American Biotic Interchange50, and environmental heterogeneity fos- the bony labyrinth, this clade remains a less morphologically diversi- tered by the progressive strengthening of seasonality51, coincides with fied group throughout the Cenozoic, reflected by its general body the accelerating rates of the bony labyrinth morphological evolution in bauplan. This is probably related to a narrower ecological plasticity cervids and their increase in taxonomic and morphological diversity and adaptive disadvantage in digestive physiology displayed by a documented over this short period. Bovids do also show an increase of more restricted distribution across climate zones than other their bony labyrinth evolutionary rates but to a lesser extent than ruminants25,41,42. Moreover, the skull morphology of this lineage shows cervids despite a higher taxonomical diversity today. Extrinsic factors paedomorphosis43, which may explain the observed significant involving the colonization of South America and an adaptive radiation decrease in evolutionary rate. may have been preponderant for cervids. North American pronghorns (Antilocapridae) also show a rapid Morphological evolution and the correlations to climate and acceleration of their evolutionary rates during the last 3 million years. geological events However, in contrast to cervids and bovids, they remained ecologically Morphological traits, like hypsodonty or appendage characters, have poorly diversified and inhabited open habitats throughout their entire been shown to correlate to environmental changes21. This is not sys- history52. This may explain the relatively low evolutionary rates of the tematically the case considering the morphological evolution of the clade during most of its history. The drastic decrease in species ruminant bony labyrinth. The phenotypic evolution of the bony diversity (with only one species being recorded today) and a possible labyrinth morphology was tested against global temperature using the evolutionary bottleneck53 may explain a derived bony labyrinth mor- EnvExp model implemented in RPANDA (see Material and Methods phology for Antilocapra in comparison to those of fossil antilocaprids and8). Colder periods, such as at the Eocene-Oligocene boundary, the (Fig. 4G, H, e.g., shape of the vestibular and cochlear aqueducts). This late Oligocene glaciations, or the early Late Miocene to the present, all may have driven the acceleration in rates of morphological evolution. Nature Communications | (2022)13:7222 4 Article https://doi.org/10.1038/s41467-022-34656-0 original data of the inner ear region representing 16% of the all known ruminant diversity including 16.5% of the known Tragulidae and ca. 22% of the known Bovidae and Cervidae. The following ruminant clades are included in the analysis, and for each pecoran lineage one of the earliest representatives is included (Supplementary Data 2): Antilocapridae, Bovidae, Cervidae, Dromomerycidae, Girafformorpha sensu55, Moschidae, as well as Middle Miocene Tragulidae and several members of Stem Pecora and Stem Ruminantia (Fig. 4 and Supple- mentary Fig. 3). More specific information concerning the specimens (taxonomical group, inventory number, host institution, age, and locality) is given in Supplementary Data 2. Petrosal bones were scanned using high resolution hard X-ray computed tomography from the following institutions: Biomaterials Science Center of the University of Basel (CH), nanotom® m (phoenix| x-ray, GE Sensing & Inspection Technologies); Department of Anthropology of the University of Zurich (CH), Nikon XTH 225 ST; Department of Geosciences of the University of Fribourg (CH), Bruker Skyscan 2211; Plateforme d’Accès Scientifique à la Tomographie à Rayon X (AST-RX) of the Muséum national d’Histoire naturelle in Paris (FR), GE Sensing and Inspection Technologies phoenix X-ray v|tome|x L240-180; Plateforme Montpellier Ressources Imagerie (MRI) of the University of Montpellier 2 (FR), Skyscan 1076 in vivo; Microscopy and Imaging Facility (MIF) of the American Museum of Natural History Fig. 2 | Evolutionary rates of the ruminant bony labyrinth morphology based (USA), GE Phoenix Vtome x L240; Nanoscale Research Facility of the on PC scores through the phylogenetic tree. Significant decrease in the evolu- University of Florida (USA), Phoenix v|tome|x M (GE’s Measurement & tionary rates (blue circle) is observed within the tragulid evolution, while a sig- Control business); Staatliche Naturwissenschaftliche Sammlungen nificant increase of the evolutionary rates (red circle) is observed at the base of the Bayerns (G), nanotom® m (phoenix|x-ray, GE Sensing & Inspection Giraffomorpha during the Early Miocene and within the South American deer, Technologies); Staatliches Museum für Naturkunde Stuttgart (G), insular cervini, and the duikers during the Pleistocene (Supplementary Data 1). Bruker Skyscan 1272; The Natural History Museum London of United Methodology and statistical results produced by the R packages RRphylo91 and Kingdom (UK), Nikon Metrology HMX-ST 225; Institute of Vertebrate Phytools88 are provided in Material and Methods and Supplementary Data 1. Sil- houettes of the families modified from ref. 107. Same color code as in Fig. 1 for ages. Paleontology and Paleoanthropology, Chinese Academy of Sciences (CHINA), GE v|tome|x m300&180 (GE Measurement & Control, Wuntsdorf, Germany); Museo Nacional de Ciencias Naturales-CSIC Population size reduction, such as in bottleneck situations, is a major (ES), NIKON CT-SCAN- XT H-160; University Museum, University of factor enhancing the influence of genetic drift and highlights the Tokyo (JA), TX225-ACTIS (TESCO Corporation) and ScanXmate- impact of neutral evolution on bony labyrinth morphology20. B100TSS110 (Comscantecno Co. Ltd.). Pixel resolution mostly varies The evolution of bony labyrinth morphology in extant and extinct between 15 and 60 μm. During petrosal bone acquisition, 1440 ruminants confirm that extrinsic factors (e.g., climate and tectonics), equiangular radiographs were taken over 360° using an adjustable as well as intrinsic factors (biological traits combined with ecological range of accelerating voltage of 90 kV and a beam current of 200 µA opportunities) over long time scales have contributed in triggering the for recent material to 180 kV with a beam current of 30 µA for fossils. global morphological diversification and diversity of this group. The Segmentation was performed with AVIZO® 9.0 Lite software (FEI bony labyrinth shape can reflect functional requirements under strong Visualization Sciences Group, Houston). selection pressure, best observed in extreme cases (e.g., dolphin18). Digitization of the bony labyrinth was performed using Landmark However, a growing body of evidence shows that its shape is strongly Editor 3.6 software56. The landmarks data are available in Supple- correlated to phylogeny10,19. Neutral evolutionary processes seem to be mentary Data 1. The landmarking protocol has been improved from driving its morphological evolution, visible at the microevolutionary Mennecart et al10. (see Supplementary Fig. 5). The primary semiland- level. This demonstrates that fine scale bony labyrinth shape differ- mark curves were resampled in R v4.1.357 to produce an equidistant ences in populations of closely related species are significant22,54. Our repartition of 307 points along the curves58 using the “digit.curves” contrasting results on evolutionary rate shifts in various groups show function of the R package geomorph v4.0.359,60. The semi-landmarks striking similarities to molecular evolutionary rates. They confirm that were then slid along the curves using the bending energy61. bony labyrinth shape is a powerful tool for investigating the complex links between morphology and evolution, and their forcing factors Phylogeny over geological timescales. These new results complement those from A phylogenetic tree was obtained using Mesquite 3.04 software62 com- the analysis of molecular data derived from extant taxa. Combining bining specific phylogenetic hypotheses of the 191 species into a com- both results is likely to yield a clear picture on the evolutionary history bined tree (Parabos63, Leptobos64, Antilocapridae52, Dromomerycidae65, of mammals and, specifically, on the forcing factors shaping their Reduncini66, Myotragus67, duikers68, extant Ruminantia28, Stem Rumi- diversity and evolution. nantia and Stem Pecora69,70, Giraffomorpha55, Moschidae71, Cervidae72, Giraffidae73). We retain the familial topology where Antilocapridae is the Methods sister clade of all other extant pecorans, Giraffomorpha is the sister Sample composition, data acquisition and digitization clade of Cervidae and Bovoidea, and Bovoidea is composed of Our research complies with all relevant ethical regulations. We selec- Moschidae and Bovidae28. Giraffomorpha contains Giraffidae, Palaeo- ted and scanned petrosal bones, which house the bony labyrinth, from merycidae, and Climacoceratidae55. The giraffid from Kohfidisch (Tur- 306 specimens representing 191 ruminant species from the early Oli- olian; MN11) is currently described as Giraffidae gen. indet74. However, gocene (ca. 33 Ma) to the present (Supplementary Data 2). The the morphology of the bony labyrinth shows typical structures that resulting dataset encompasses the most extensive study to date using recall those observed in the Birgerbohlinia schaubi specimens of similar Nature Communications | (2022)13:7222 5 Article https://doi.org/10.1038/s41467-022-34656-0 order to assess our expectation that there should be a strong phylo- genetic signal in our data, we performed a permutation test (rando- mized rounds: 10.00084) based on the phylogenetic tree. Klingenberg and Gidaszewski84 defined that “The empirical p-value for the test is the proportion of permuted data sets in which the sum of squared changes is shorter or equal to the value obtained for the original data.” Marriott85 and Edgington86 suggested that 1.000 permutations are a reasonable minimum for a test at 5% level of significance, while 5.000 are a reasonable minimum at the 1% level87. Moreover, the Pagel’s λ and Bloomberg’s K phylogenetic signal values have been calculated using the function “phylosig” in the R package phytools v1.0.388. All sup- porting data are given in the Supplementary Data 1. To characterize the morphological similarities within the different clades, a between- groups PCA (bg-PCA) and a Canonical Variates Analysis (CVA) were performed. bg-PCA and CVA provide complementary information89,90. A bg-PCA observes the variance between groups (here defined as Stem Ruminantia, Tragulidae, Stem Pecora, Antilocapridae, Giraffomorpha, Cervidae, Moschidae, and Bovidae) without standardizing the within- groups variance89. A standardization is performed when using the CVA. The CVA maximizes the separation of the between-groups means Fig. 3 | Evolutionary rates of the different ruminant families’ bony labyrinth relative to the variation within the groups’ ratio according to the morphology based on PC scores compared with the global temperature curve1 specified chosen grouping variable89. The bg-PCA was computed using (gray dots). The Middle Miocene Climatic Optimum (mMCO) and the Great the function “groupPCA”and the CVA using the function “CVA”, both in American Biotic Interchange (GABI) are highlighted since they are important fac- the R package Morpho v2.983. To test the performance of the classifi- tors in the ruminant evolution. Methodology and statistical results produced by the cation model, both analyses were cross-validated using leaving-one- R package RPanda93 are provided in Material and Methods and Supplementary out cross-validation for the bg-PCA and Jackknife Cross-validation for Data 1. Groups are Tragulina (dark blue; Stem Ruminantia + Tragulidae), Antiloca- pridae (dark red), Giraffomorpha (dark light blue), Cervidae (light blue), Moschidae the CVA. All supporting data of the bg-PCA and the CVA are given in (yellow), Bovidae (red). Silhouettes of the families modified from107. Supplementary Data 1 (script) and in Supplementary Data 1 (support- ing information). age from the Crevillente-2 locality (Turolian; MN11; 8.5 Ma75). Never- Evolutionary rate. The evolutionary rate of the bony labyrinth mor- theless, based on several morphological differences (the thickness of phology within the phylogeny was calculated using the function the first cochlear turn, orientation of the vestibular aqueduct, and the “RRphylo” in the R package RRphylo v2.6.091. This function performs a anterior canal shape), we cannot confirm that Giraffidae gen. indet. from phylogenetic ridge regression92. The significance of the evolutionary Kohfidisch is Birgerbohlinia schaubi. We attribute this specimen to Bir- rate changes (acceleration and deceleration) were tested using the gerbohlinia sp., sister taxon of Birgerbohlinia schaubi. Dromomerycidae function “search.shift” in the R package RRphylo v2.6.091 to highlight has been considered to be closely related to Cervidae55. However, we the significant changes in the rates within the phylogenetic tree. We retain the phylogenetic hypothesis of a close relation to Antilocapridae76 plot the calculated evolutionary rate values on the phylogenetic tree based on morphological similarities of the bony labyrinth. Hoplitomeryx using the function “contMap” in the R package phytools v1.0.388. All is a vigorously discussed taxon from a phylogenetic viewpoint for which supporting data concerning RRphylo are given in Supplemen- a new family has been proposed77. Studies of the horncores of Hoplito- tary Data 1. meryx show bovid affinity for this taxon76,78. We keep this phylogenetic We tested whether the bony labyrinth morphology evolu- hypothesis here, because the morphology of the bony labyrinth, espe- tionary rate is correlated, anticorrelated, or not correlated (β) cially the lateral semicircular canal orientation, is typical for bovids. with an environmental function through time using the function The nodes of the main clades are calibrated using the fossil “fit_t_env“ in the R package RPANDA v2.093. The environmental record. The oldest known ruminant is Archaeomeryx from the middle function is the isotopic data of the δ18O used as a proxy for the Eocene of Asia, ca. 44 Ma79. The origin of the crown Pecora is older than temperature curve provided by Zachos et al1. Because each family 37 Ma39. The giraffomorph Bedenomeryx is known from 24 Ma80. The has a potentially different ecological optimum and reacts differ- oldest moschid is ca. 18 Ma81, while the oldest bovid ca. 18.75 Ma. Node ently to the environmental changes, we separately analyzed each calibration within families is estimated based on several sources in the pecoran clade and the Tragulina in a first analysis. We then ana- literature10,37,55. The phylogenetic tree can be consulted in Supple- lyzed the Pecora as a whole and the Tragulina in a second ana- mentary Fig. 6 and the nexus file is given in the Supplementary Data 1. lysis. The significance of the results was tested against different levels of smoothing of the temperature curve, compared to the Statistical analysis resulting Aikake values. This implies that if these correlations are Shape variation and phylogenetic signal. Shape variation in bony always significant in a clade, regardless of the smoothing para- labyrinth morphology (disparity and similarity) was studied using a meter, temperature is not the only parameter influencing evolu- geometric morphometrics approach implemented in MorphoJ82 and R tionary rates. All supporting data concerning RPANDA are given in v4.1.357. When several specimens of a species were available, we cre- Supplementary Data 1. ated a mean shape for the species using the function “mshape” of the To compare the impact of the topology of the phylogenetic package geomorph v.4.0.359,60 for the analyses that are using only one tree on the evolutionary results, similar analyses have also been specimen per species in a phylogenetic framework (dataset with performed using the size of the specimens based on the bony 191 species). We performed a Principal Component Analysis (PCA) labyrinth centroid size. Bony labyrinth size has been proven to be computed using the function “procSym” of the R package Morpho a good proxy for body mass estimation in ruminants94 and has v2.983 to study the shape variation within the dataset in its natural been used here to explore size evolution and diversification in scale, including intraspecific variation (dataset with 306 specimens). In ruminants. Bony labyrinth morphology is constrained by Nature Communications | (2022)13:7222 6 Article https://doi.org/10.1038/s41467-022-34656-0 Evolutionary rates of the bony labyrinth are more heterogenous and explained in detail in the text. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability The datasets generated during and analyzed during the current study (Landmark data) are available in the Supplementary Data 1. All mate- rials from which shape data are generated are housed in museum collections. Details on the location of these collections can be found in Supplementary Data 2. 3D reconstructed models of the bony labyr- inths are either published and open acces in MorphoMuseuM102–106 (https://morphomuseum.com/) or will be. The not yet published models are available from the corresponding authors upon reasonable request. 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We are also grateful to all the persons and institutions 98. du Toit, J. T. & Owen-Smith, N. Body size, population metabolism, who scanned for us. Tandra Fairbanks (NMB) is thanked for her help and habitat specialization among large African herbivores. Am. with the English. B.M. and L.C. are grateful to the Swiss National Nat. 133, 736–740 (1989). Science Foundation for supporting this research through the projects 99. Mennecart B., Becker D., & Berger J. -P. Mandible shape of rumi- 200021_178853 and 200021_159854/1 on the ear region evolution in nants: between phylogeny and feeding habits. In: Ruminants: ruminants. B.M. P300P2_161065 and P3P3P2_161066 on the evolution Anatomy, behavior, and diseases, (ed. Mendes R. E.) 205–226 of the early ruminants. F.B. acknowledges support from a Gerstner (Nova Science Publishers, 2012). Scholarship at the American Museum of Natural History. G.R. thanks 100. Bokma, F. et al. Testing for Depéret’s rule (body size increase) in the German Research Foundation project RO 1197/3-1. G.M., B.M., and mammals using combined extinct and extant data. Syst. Biol. 65, L.C. want to thank the Museum National d’Histoire Naturelle de Paris 98–108 (2016). for financing the Ast-RX-2013-051 Project. D.D.M. acknowledges R+D 101. Besiou, E., Choupa, M. N., Lyras, G. & van der Geer, A. Body mass +I project ref. PID2020-116220GB-I00 from the Ministerio de Ciencia e divergence in sympatric deer species of Pleistocene Crete Innovación/Agencia Estatal de Investigación/10.13039/ (Greece). Palaeontol. Electron. 25, a23 (2022). 501100011033/. I.S. acknowledges support by the Spanish Ministry of 102. Mennecart B., Métais G., Tissier J., Rössner G. E., & Costeur L. 3D Science and Innovation (projects ref. PID2020-117289GB-I00 and models related to the publication: Reassessment of the enigmatic PID2020-116220GB-I00), and the Generalitat de Catalunya (CERCA ruminant Miocene genus Amphimoschus Bourgeois, 1873 (Mam- Programme). M.K. is funded by Japan Society for the Promotion of malia, Artiodactyla, Ruminantia, Pecora). MorphoMuseuM 7, Science (KAKENHI Grant No. 19K04060). M.R. is supported by FCT- e131 (2021). CEEC postdoctoral funding (CEECIND/02199/2018). M.R. acknowl- 103. Mennecart, B., Perthuis de, A. D. & Costeur, L. 3D models related to edges MINECO project CGL2011-25754 for providing funding for the the publication: The first French tragulid skull (Mammalia, Rumi- analysis. S.W. thanks the Strategic Priority Research Program of nantia, Tragulidae) and associated tragulid remains from the Chinese Academy of Sciences (XDB26000000) and the National Middle Miocene of Contres (Loir-et-Cher, France). MorphoMu- Natural Science Foundation of China (41872001). G.S. and B.Mü. seuM 3, e4 (2018). acknowledge financial support from the Swiss National Science 104. Aiglstorfer, M., Costeur, L., Mennecart, B. & Heizmann, E. P. J. Foundation in the frame of the R’equip initiative (316030_133802). Micromeryx? eiselei - a new moschid species from Steinheim am Albuch, Germany, and the first comprehensive description of Author contributions moschid cranial material from the Miocene of Central Europe. All the authors have approved the submitted version (and any sub- MorphoMuseuM 3, e4 (2107). stantially modified version that involves the author’s contribution to the 105. Costeur, L. & Mennecart, B. 3D models related to the publication: study); AND to have agreed both to be personally accountable for the Prenatal growth stages show the development of the ruminant author’s own contributions and to ensure that questions related to the bony labyrinth and petrosal bone. MorphoMuseuM 2, e3 accuracy or integrity of any part of the work, even ones in which the (2016). author was not personally involved, are appropriately investigated, 106. Mennecart, B. & Costeur, L. 3D models related to the publication: a resolved, and the resolution documented in the literature. Conception Dorcatherium (Mammalia, Ruminantia, Middle Miocene) petrosal or design of the work: B.M., L.D., L.C.; the acquisition: B.M., L.D., M.A., bone and the tragulid ear region. MorphoMuseuM 2, e2 F.B., D.D.M., M.F., M.K., F.L., J.M., G.M., B.Mü., M.R., G.R., I.S., G.S., S.W., (2016). L.C.; the analysis: B.M., L.D.; the interpretation of data: B.M., L.D., L.C.; 107. Mennecart, B. et al. Allometric and phylogenetic aspects of stapes have drafted the work: B.M., L.D., L.C.; substantively revised it: B.M., L.D., morphology in ruminantia (Mammalia, Artiodactyla). Front. Earth M.A., F.B., D.D.M., M.F., M.K., F.L., J.M., G.M., B.Mü., M.R., G.R., I.S., Sci. 8, 176 (2020). G.S., S.W., L.C. Acknowledgements Competing interests We would like to thank all the curators, collection manager, and The authors declare no competing interests. scientists who helped us allowed us to study and provided access to material: C. Argot and G. Billet (Muséum national d’Histoire naturelle, Additional information Paris), D. Geraads (Museum national d’Histoire Naturelle) for provid- Supplementary information The online version contains ing the Sivatherium specimen, E. Robert (Université Claude Bernard, supplementary material available at Lyon 1), D. Berthet (Musée des Confluences Lyon), M. Orliac (Uni- https://doi.org/10.1038/s41467-022-34656-0. versité Montpellier 2) for providing the bony labyrinth of Bachither- ium, F. Duranthon and Y. Laurent (Muséum d’histoire naturelle, Correspondence and requests for materials should be addressed to Toulouse), S. Legal, P. Coster, C. Balm, O. Maridet, O. Lapauze and J. Bastien Mennecart. Tissier (Parc Naturel Régional du Luberon and excavation team Murs Project), A. de Perthuie for accessing his private collection, Christiane Peer review information Nature Communications thanks Marcus Clauss, Zeitler, R. Ziegler and E. Heizmann (Staatliches Museum für Nat- Alessandro Urciuoli and Vera Weisbecker for their contribution to the urkunde Stuttgart), P. Brewer, A. Garbout, and F. Ahmed (Natural peer review of this work. Peer reviewer reports are available. History Museum, London), U. Göhlich and G. Daxner-Höck (Nat- urhistorisches Museum Wien), A. Van der Geer (Netherland Centre for Reprints and permissions information is available at Biodiversity Leiden), R. C. Hulbert Jr. (University of Florida, Gaines- http://www.nature.com/reprints ville), A. M. García Forner and P. Montoya (Museu de Geologia de la Universitat de València, Burjassot) for providing the petrosals of Publisher’s note Springer Nature remains neutral with regard to jur- Birgerbohlinia, M. Pina (University of Manchester) for scanning isdictional claims in published maps and institutional affiliations. Nature Communications | (2022)13:7222 10 Article https://doi.org/10.1038/s41467-022-34656-0 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2022 1 Naturhistorisches Museum Basel, Augustinergasse 2, 4001 Basel, Switzerland. 2Institute of Plant Sciences, University of Bern, 3013 Bern, Switzerland. 3 Oeschger Centre for Climate Change Research, University of Bern, 3012 Bern, Switzerland. 4Naturhistorisches Museum Mainz / Landessammlung für Naturkunde Rheinland-Pfalz, Reichklarastraße 10, 55116 Mainz, Germany. 5Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Berlin 10115, Germany. 6Fundación ARAID, Zaragoza, Spain. 7Departamento de Ciencias de la Tierra, Área de Paleontología / Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA). Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain. 8Institut Català de Palaeontologia Miquel Crusafont (ICP), Edifici Z, c/de les columnes s/n, Universitat Autònoma de Barcelona, 08193Cerdanyola del Vallès, Barcelona, Spain. 9 National Museum of Nature and Science, Tsukuba, Japan. 10Department of Natural Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan. 11Swiss National Data and Service Center for the Humanities, 4123 Allschwil, Switzerland. 12American Museum of Natural History, 10024 New York; Earth and Environmental Sciences, Graduate Center, City University of New York, New York, NY 10016, USA. 13CR2P - Centre de Recherche en Paléontologie - Paris, UMR 7207, CNRS, MNHN, Sorbonne Université. Muséum national d’Histoire naturelle, CP38, 8 rue Buffon, 75005 Paris, France. 14Biomaterials Science Center, Department of Biomedical Engineering, University of Basel, Gewerbestrasse 14, 4123 Allschwil, Switzerland. 15 Department of Earth Sciences, GeoBioTec, Nova School of Science and Technology, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal. 16Staatliche Naturwissenschaftliche Sammlungen Bayerns - Bayerische Staatssammlung für Paläontologie und Geologie, Richard- Wagner-Strasse 10, 80333 Munich, Germany. 17Department für Geo- und Umweltwissenschaften, Paläontologie & Geobiologie, Ludwig-Maximilians- Universität München, Richard-Wagner-Strasse 10, 80333 Munich, Germany. 18Micro- and Nanotomography Core Facility, Department of Biomedical Engi- neering, University of Basel Gewerbestrasse 14, 4123 Allschwil, Switzerland. 19Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, 142 Xizhimenwai Street, Beijing 100044, China. e-mail: mennecartbastien@gmail.com Nature Communications | (2022)13:7222 11