TYPE Original Research PUBLISHED 19 May 2023 DOI 10.3389/fevo.2023.1173341 First digital study of the frontal OPEN ACCESS sinus of stem-Canini (Canidae, Carnivora): evolutionary and EDITED BY Stergios D. Zarkogiannis, University of Oxford, United Kingdom REVIEWED BY George Lyras, ecological insights throughout National and Kapodistrian University of Athens, Greece Emmanuel Paul Gilissen, advanced diagnostic in Royal Museum for Central Africa, Belgium Alejandro Pérez Ramos, Malaga University, Spain paleobiology *CORRESPONDENCE Saverio Bartolini-Lucenti Samuele Frosali 1, Saverio Bartolini-Lucenti 1,2*, saverio.bartolinilucenti@unifi.it Joan Madurell-Malapeira 1,3, Alessandro Urciuoli 2,4,5, Lorenzo Rook lorenzo.rook@unifi.it Loïc Costeur 6 and Lorenzo Rook 1,7* RECEIVED 24 February 2023 1 Department of Earth Science, University of Florence, Florence, Italy, 2 Institut Català de Paleontologia ACCEPTED 24 April 2023 Miquel Crusafont, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain, 3 Department of PUBLISHED 19 May 2023 Geology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain, 4 Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain, 5 Division of Palaeoanthropology, Senckenberg Research CITATION Institute and Natural History Museum Frankfurt, Frankfurt am Main, Germany, 6 Naturhistorisches Frosali S, Bartolini-Lucenti S, Museum Basel, Basel, Switzerland, 7 Changes Foundation, Sapienza Università di Roma, Roma, Italy Madurell-Malapeira J, Urciuoli A, Costeur L and Rook L (2023) First digital study of the frontal sinus of stem-Canini (Canidae, Carnivora): evolutionary and ecological insights Introduction: The phylogenetic and ecological importance of paranasal sinuses throughout advanced diagnostic in in carnivorans was highlighted by several previous authors, mostly in extant paleobiology. species. Nevertheless, no specific study on this feature on extant canids, and no Front. Ecol. Evol. 11:1173341. doi: 10.3389/fevo.2023.1173341 one on fossil representatives of the family, has been published up to now. Here, COPYRIGHT we analyze for the first time the paranasal sinus of extant and fossil canids through © 2023 Frosali, Bartolini-Lucenti, Madurell- computed tomographic techniques to characterize them morphologically and Malapeira, Urciuoli, Costeur and Rook. This is morphometrically, making ecological inferences. an open-access article distributed under the terms of the Creative Commons Attribution Methods: To do so, we applied for the first time an innovative deformation-based License (CC BY). The use, distribution or morphometric approach. reproduction in other forums is permitted, provided the original author(s) and the Results: The results obtained for extant species highlight a remarkable correlation copyright owner(s) are credited and that the between morphology and ecomorphotypes previously defined by some original publication in this journal is cited, in accordance with accepted academic practice. scholars (namely hypercarnivorous group-hunters; small-prey hypercarnivores, No use, distribution or reproduction is mesocarnivores, hypocarnivores). Our results thus support the direct relationship permitted which does not comply with these between diet preferences and the development of frontal sinus in canids. terms. Regarding fossil specimens, we reconstructed for the first time the frontal sinus of three Eucyon species and compared it to those of living forms. Discussion: The best-preserved specimen, the only known cranium of Eucyon adoxus dated to the Late Pliocene of Saint-Estève (France), displayed similarities with hypercarnivorous group-hunter canids by the large sinus prominences. Given that the overall craniodental morphology of E. adoxus suggests that it acted as a small prey hypercarnivore—similar to extant Canis simensis—the aforementioned affinities might have evolved independently, in relation to high stresses during feeding. Overall, our study demonstrates that morphological inspection and deformation- based geometric morphometrics complement each other and allow a thorough investigation of sinus shape variability, thus enabling the study of sinus morphology in other fossil carnivorans with the ultimate goal of inferring their ecological preferences. KEYWORDS innovative morphometrics, sustainable cultural heritage, Canidae, paranasal sinuses, paleoecology, Eucyon Frontiers in Ecology and Evolution 01 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 1. Introduction paranasal cavity in paleontological and paleoanthropological research, for its potential at different levels, e.g., evolutionary, biomechanical, 1.1. Dispersal of the tribe Canini in Eurasia ecological one (Balzeau et al., 2022). In carnivorans, the first introduction of the frontal sinus as a Currently, the members of the family Canidae are widely taxonomic-useful character was proposed by Huxley (1880) and is distributed across Eurasia and Africa, but geologically speaking this is today widely recognized by the scientific community as a just a recent achievement. Throughout most of their history, the diagnostic feature in the phylogenetic analysis of Canidae. The distribution of canids was restricted to North America (Wang and basal state (lack of the sinus) is manifested externally by a shallow Tedford, 2008; Tedford et al., 2009; Sotnikova and Rook, 2010). The depression or a groove, creasing the dorsal surface of the major biotic events related to the dispersal of the subfamily Caninae postorbital process (Tedford et al., 1995): this condition is present (to which all extant canids belong to) and their evolution in Eurasia in almost all the living species of the tribe Vulpini and it is indeed coincided with faunal turnovers that, in these continents, occurred known as the “vulpine crease.” In members of the tribe Canini, this between 5.5 and 0.5 Ma. One of the two major events of the Canini cavity inflates and expands its size: the most derived condition is dispersal in Eurasia was the “Eucyon event” (sensu Sotnikova and shown by Canis, where the sinus penetrates the postorbital process Rook, 2010) that took place at the end of the Miocene (the other main to its tip and expands posteriorly along the rostrodorsal surface of event was the so-called “Canis event” at the beginning of the Late the braincase, ultimately reaching the frontoparietal suture Pliocene; Sotnikova and Rook, 2010). Species of the genus Eucyon (Tedford et al., 1995). This enlargement of the frontal region (Tedford and Qiu, 1996) indeed appeared in Central Asia, Europe, and produces a deeply convex morphology of the dorsal surface of the Africa by the latest Miocene (Figure 1). For instance, the remarkable cranium above the orbits and an enlargement, in width, of the Late Miocene occurrences of Eucyon monticinensis (Rook, 1992) in postorbital constriction. Europe, Eucyon davisi (Merriam, 1911) in Asia, and Eucyon intrepidus Among other cranial features, like dentition morphology or snout (Morales et al., 2005) in Africa, were roughly contemporaneous. The length, the development of the frontal sinus is linked to the type of diet genus then experienced multiple radiations in Eurasia and Africa, the species evolved. Particularly, large canids with hypercarnivorous reaching a relatively high diversity in the Pliocene of Eurasia (Rook, diets tend to have more developed sinuses and enlarged, domed frontals. 2009; Figure 1). The diversification of the Canini in Asia peaked at the In durophagous carnivorans, both in Hyaenidae and Borophaginae, the beginning of the Early Pliocene, as evidenced by the appearance of frontal sinus extends backward toward the posterior tip of the skull and, Nurocyon chonokhariensis (Sotnikova, 2006) and Eucyon zhoui as a result, there is a large air space between the braincase and the (Tedford and Qiu, 1996), and by the increase in number of E. davisi parietal bones above it (Paulli, 1900; Joeckel, 1998; Vinuesa et al., 2015). finds (Sotnikova and Rook, 2010). The European diversity peaked Werdelin (1989) and Joeckel (1998) proposed that such a domed head later with the arrival of “Canis” michauxi (Martin, 1973), Eucyon in bone-cracking hyaenids and canids serves to transmit the great adoxus (Martin, 1973), and Eucyon odessanus (Odintzov, 1967) in the stresses from the premolars to the back of the skull, thereby reducing Late Ruscinian (Late Pliocene; Sotnikova and Rook, 2010) (Figure 1). the bending stress of the rostrum. The main hypothesis to explain the In the Early Pleistocene, after the “Canis event,” there was a species presence of the frontal sinus in carnivorans is indeed that these turnover on the continent and the species of the genus Eucyon went structures are useful for the even dissipation of stresses linked to feeding almost completely extinct, being limited to the Chinese Eucyon minor mechanics. Some studies (among others Curtis and Van Valkenburgh, (Teilhard de Chardin and Piveteau, 1930) and “Canis” kuruksaensis 2014) also pointed out a relationship between the intraspecific variations (Sotnikova, 1989), while Canis sensu lato underwent a wide of the morphology and size of the frontal sinus and the diet of the diversification. This turnover included Canis teilhardi (Qiu et al., individual (the softer the food eaten, the smaller the frontal sinus; Curtis 2004), Canis longdanensis (Qiu et al., 2004), Canis chihliensis (Teilhard et al., 2018; Pérez-Ramos et al., 2020; Ruiz et al., 2023). Other hypotheses de Chardin and Piveteau, 1930), Canis (Xenocyon) brevicephalus (Qiu have been suggested to explain the function of these structures, among et al., 2004), and Canis (Xenocyon) dubius (Teilhard de Chardin, 1940) others the idea that frontal sinuses are useful for brain cooling, olfaction, in China, and the western forms like the renown Canis etruscus potential sensory enhancement ability, or for the maximization of (Forsyth-Major, 1877), Canis arnensis (Del Campana, 1913), Canis muscle attachment areas, or even hibernation (Joeckel, 1998; McGreevy borjgali (Bartolini-Lucenti et al., 2020), Canis (Xenocyon) falconeri et al., 2004; Vinuesa et al., 2015, 2016; Vinuesa, 2018; Pérez-Ramos et al., (Forsyth-Major, 1877), and Canis accitanus (Garrido and Arribas, 2020). However, none of these explanations is entirely satisfactory and 2008; Sotnikova and Rook, 2010). The diversity of the Canis s.l. group one hypothesis does not necessarily exclude another. The present paper decreased in the second half of the Early Pleistocene of Eurasia, offers the first in-depth study of the frontal sinus of extant and fossil becoming mostly limited to the large hypercarnivorous form Canis canids using computed tomography and innovative 3D methodologies (Xenocyon) lycaonoides (Kretzoi, 1938) and small wolf Canis (never applied to these structures) to characterize and analyze them mosbachensis (Soergel, 1925) (and its possible zoogeographic extreme and, eventually, draw ecological inferences on the considered fossil taxa. Canis variabilis Pei, 1934) (Rook et al., 2023). 2. Materials and methods 1.2. Brief overview of biomechanics of the frontal sinus in carnivorans 2.1. Studied and comparative material The frontal sinus is an often-overlooked anatomical structure. This study reports the first digital analysis of the frontal sinuses Nevertheless, recent studies unveil the renovated interest for this of three stem-Canini of the genus Eucyon: the only known cranium Frontiers in Ecology and Evolution 02 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 FIGURE 1 Stratigraphic, phylogenetic, and geographical scheme of Eucyon spp. in the framework of extant canids evolution and zoogeography from Miocene to modern times. The phylogenetic relationships are compiled using widely accepted and recent molecular and total evidence analyses (e.g., Zrzavý et al., 2018; Perri et al., 2021). Solid lines represent undisputed patterns of affinities; divergence between solid-lines groups are time-calibrated using average time of divergence expressed in these works (e.g., Perri et al., 2021). Dashed lines account for disputed topologies of trees (e.g., paraphyly of Lupulella; relationship among Vulpini; cf. Wayne and Ostrander, 2007 and Zrzavý et al., 2018). Time of the nodes in the dashed-lined groups should not be taken as significative. Considering the disputed nature of several Eucyon spp. (as expressed in the text) we reported only their chronological and geographic distribution. Data are taken from: Tedford and Qiu (1996), Sotnikova (2006), Spassov and Rook (2006), De Bonis et al. (2007), Montoya et al. (2009), Rook (2009), Tedford et al. (2009), Werdelin et al. (2015), Bartolini-Lucenti and Rook (2021), Valenciano et al. (2022). ‘Eucyon event’ is taken from Sotnikova and Rook (2010). Colors of the chronological ranges represents clades. Ranges of other canids are taken from previously cited literature and also from: Stiner et al. (2001), Boudadi-Maligne (2010), Werdelin and Sanders (2010), Bartolini-Lucenti et al. (2018), Marciszak et al. (2021), Tamvakis et al. (2022), Martínez-Navarro et al. (2023). Color continents maps next to specific names represent presence of the species in one/more than one continent(s), following the color code of the boxes in the figure. of the species Eucyon adoxus, from the Late Pliocene of Saint-Estève (MZUF), the Giacomo Doria Natural History Museum of Genoa (France; Rook, 2009) and stored at the Natural History Museum of (CE) and the Earth Science Department of the University of Florence Basel (NMB Rss.45); a cranium (F:AM 97057) of Eucyon davisi from (DST). Additional specimens were downloaded from the digital the Early-Late Pliocene of Xiakou, Yushe Basin (China; Tedford and repository MorphoSource.1 The considered comparative sample Qiu, 1996), currently stored at the American Museum of Natural includes (Supplementary Table S1) four specimens of Canis lupus, History of New York (U.S.A.); and, the only known cranial specimen Linnaeus (1758), four of Canis lupaster (Hemprich and Ehrenberg, of Eucyon monticinensis, from the latest Late Miocene of Cava 1828/1834), three of Canis simensis Rüppell (1835/1840), two of Monticino (Italy, Bartolini-Lucenti et al., 2022) and stored in the Canis aureus (Linnaeus, 1758), one of Canis latrans Civic Museum of Natural Sciences of Faenza. The objective of the study is the comparison of the sinus of these fossil species to that of selected extant Canidae. The extant comparative specimens used here are held at the “La Specola” Zoology Museum of Florence 1 www.morphosource.org Frontiers in Ecology and Evolution 03 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 Say (1823), one of Lupulella adusta (Sundevall, 1847), four of (prosthion-occipital condyles); LmaxS, maximum rostrocaudal length Lupulella mesomelas (Schreber, 1775), two of Lycaon pictus of the frontal sinus; TL, total length of the cranium (measured from (Temminck, 1820), and one of Vulpes lagopus (Linnaeus, 1758). To prosthion-akrokranion); Vol, volume of the frontal sinuses. compare morphologically and morphometrically the specimens we used the digital analysis of images derived from CT scans via the 2.2.3. Morphometric variables and software Amira (ver. 5.4.5, Thermo Fisher). Tomographic scans of ecomorphogroups the material from Florence and Faenza were acquired at the Medical We used Autodesk Meshmixer (ver. 3.5.474, Autodesk) and Artec Radiology ward (“SOS Radiologia”) of the San Giovanni di Dio Studio 15 Professional (ver. 15.0.3.425, Artec) software to ameliorate Hospital (Florence, Italy) using a Siemens Somatom Definition AS and regularize the mesh structure, as well as to measure the sinus scanner. Eucyon adoxus was scanned at the Biomaterials Science volume (Vol), sinus maximum rostrocaudal length (LmaxS), the Center of the University of Basel, Switzerland using a Phoenix cranial length (TL) and the condylobasal length (CBL). The latter was Nanotom (GE). The specimen of Eucyon davisi was scanned at the used to estimate the body mass of each specimen, using the regression University of Texas high-resolution X-ray Computed Tomography formula proposed by Van Valkenburgh (1990). Van Valkenburgh and Facility (Austin, Texas, U.S.A.). For the specimens downloaded from Koepfli (1993), studying extant canids dietary preferences, established Morphosource, we report the info included in the metadata and four distinct ecological groups. The differed for their morphological accompanying files of the raw data: m-81.001, Canis simensis, features and the proportional amount of vertebrate prey in their diet: originally held at the AMNH Mammal Collection, was scanned by Group 1 and 2 have >70% of vertebrate meat in their diet, thus are the University of Texas high-resolution X-ray Computed considered hypercarnivore canids. They are distinguished by the fact Tomography Facility (Austin, Texas, U.S.A.). 80-50-290, Canis that Group 1 canids cooperatively hunt large prey, whereas Group 2 latrans, kept at the University of Arkansas Museum and was scanned are hypercarnivorous specialized in small prey. Group 3 canids are by the MicroCT Imaging Consortium for Research and Outreach mesocarnivorous, with an income of 70–50% of vertebrate meat in (Fayetteville, Arkansas, U.S.A.). 368.443, Lycaon pictus was scanned their diet. Group 4 represent the hypocarnivorous dogs with <50% of by the University of Texas high-resolution X-ray Computed meat. We used these well attested groups to investigate the ecological Tomography Facility (Austin, Texas, U.S.A.), while the original implication of the development of the frontal sinus. sample comes from NMNH – Division of Mammals. The data To assess the presence of correlation among the variables obtained from these images made the reconstruction of the frontal considered in the analysis, we performed ordinary least squares (OLS) sinuses of all the specimens possible. We decided to use the left sinus regressions on natural log-transformed linear variables (ln TL, ln only to ease the alignment of the surfaces and, consequently, their LmaxS, in mm), natural log-transformed cube root of the left sinus analysis with deformation-based geometric morphometric methods. volume (ln Vol, in mm3), and on the natural log-transformed body mass (ln BM, in kg). The regressions were performed for the whole Canidae sample, as well as for two subsets based on ecological 2.2. Nomenclature and abbreviations preferences (group hunters vs. non-group hunters) due to the difference noted in one of the ecological groups. We also checked the 2.2.1. Proposed nomenclature of the frontal homogeneity of the slopes and intercepts between group hunters and sinuses the rest of the sample via analysis of the covariance (ANCOVA). All We propose for the first time a nomenclature to describe the frontal regressions were performed in R Studio (v. 2023.03.0 + 386 “Cherry sinus in canids and carnivorans (Figure 2). The sinus is divided into three Blossom” release 3c53477afb13ab959aeb5b34df1f10c237b256c3, regions: rostral, caudal, and ventral ones. The rostral and caudal regions 2023-03-0936; RStudio Team, 2021) for R (v. 4.1.2; R Core Team, are characterized by specific features on their dorsal, medial, lateral, and 2021) using the lm() function of the base package ‘stats’, whereas ventral surfaces. The ventral region is generally described for its medial we used the anova() function of the ‘car’ v. 3.0–11 (Fox and Weisberg, and lateral sides and its rostral and caudal margins. The features, 2019) to perform the ANCOVA. although variable, can be recognized on the outer surface. Here we report the names and abbreviations for the analyzed specimens. Abbreviations 2.2.4. Diffeomorphic surface matching analyses and their definition, in alphabetical order: ci, caudal incision (if more To further inspect the shape variation in the canid sinus, than one: medial ci, lateral ci, etc.); dcll, dorsocaudal lateral lobe; dcml, we employed a diffeomorphic surface matching (DSM) approach, dorsocaudal medial lobe; ds, dorsal sulcus (if the sulcus is simple it may which is a recently-developed landmark-free geometric be named “longitudinal ds”; if it branches, it can have “a transverse part morphometrics method that allows the direct comparison between of the ds”; etc.); l, lobe (a large inflated portion of the sinus), lb, lobule (a continuous surfaces by relying on their geometrical correspondence smaller and less prominent lobe); p, prominence (general term to (Glaunès and Joshi, 2006; Durrleman et al., 2014). This method takes describe an inflation of the surface of the sinus); pol, postorbital lobe; ri, into account all data points at the same time and does not require rostral incision (generally divides the rostral region of the sinus in two point-to-point correspondence, thus allowing the use of surfaces sides: the rostrolateral lobe and the rostromedial one); rll, rostrolateral composed by a different number of faces (Durrleman et al., 2012). lobe; rml, rostromedial lobe; vl, ventral lobe (the tip of the ventral From an operational viewpoint, DSM first estimates the average shape region); vlp, ventrolateral prominence. for the considered sample and identifies its most variable portions, attaching a number of initial control points to them. The deformations 2.2.2. Other abbreviations used in the text from the average to each of the analyzed specimens are then modeled List of abbreviations used in the paper, in alphabetical order: BM, as smooth and invertible functions (that are, diffeomorphisms) and body mass; CBL, condylobasal length of the cranium compiled to build an atlas for the entire sample. Compared with Frontiers in Ecology and Evolution 04 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 FIGURE 2 Nomenclature of the main parts of the frontal sinus of Canidae. Eucyon adoxus (NMB Rss.45, A–D) and C. lupaster (MZUF-2714, E,F) were taken as models because their sinuses represent, respectively, a quite complex and one of the simplest versions of this paranasal structure (not to scale). The color map (A,B) represents the principal areas of the sinus: yellow, rostral region; blue, caudal region; red, ventral region. ci, caudal incision; dcll, dorsocaudal lateral lobe; dcml, dorsocaudal medial lobe; ds, dorsal sulcus; l, lobe; lb., lobule; p: prominence; pol, postorbital lobe; ri, rostral incision; rll, rostrolateral lobe; rml, rostromedial lobe; vl, ventral lobe; vlp, ventrolateral prominence. landmarks, correspondence between surfaces is not strictly TABLE 1 List of landmarks (LM) used in the analysis and their number and definition. homologous, which is particularly convenient for structures with a very complex 3D morphology that does not allow to identify a large LM number Definition number of homologous points, as in the case of frontal sinuses and of 1 Rostralmost (Anteriormost) point on the rostromedial lobe. other craniodental structures (e.g., semicircular canals and enamel/ Caudalmost (Posteriormost) point of the incision between dentine junction; Urciuoli et al., 2020; Zanolli et al., 2022). 2 rostromedial and lateral lobe. Prior to performing DSM analyses, the surfaces required a number of preparatory steps. First the 3D models of the left sinus of each 3 Lateralmost point of the lateral lobe. specimen were decimated to 40,000 triangles (±100 triangles) in 4 Medialmost point on the medial side of the sinus. Amira and saved in PLY format. Given the complexity and variability 5 Caudalmost (Posteriormost) point of the sinus. of the sinus, the alignment methods used for preprocessing the 6 Ventralmost point of the frontal sinus. surfaces in other DSM analyses (Beaudet et al., 2016; Urciuoli et al., 2020) did not yield satisfactory results. To circumvent this issue, we defined a number of landmarks (see Table 1) that allowed us to The obtained deformation fields (i.e., the output of the DSM generally describe its morphology and orientation. The aforementioned analyses) were then inspected with different multivariate methods to landmarks were placed on each individual in Amira software and used identify the possible presence of patterns of shape variation in the to perform a General Procrustes alignment in R using the procSym() data. First, we computed a principal component analysis (PCA), function of the ‘Morpho’ v. 4.1.3 (Schlager, 2017) package. The which allows to reduce the high dimensionality of the data and to obtained aligned configurations were then used to extract the concentrate the variance in the first axes (components). The resulting rotational matrix and scaling factor for each individual, which were components were then used to perform a canonical variate analysis then applied to the corresponding surface using two functions, (CVA)—with the CVA() function of the ‘Morpho’ package—using computeTransform() and applyTransform(), of the ‘Morpho’ package. hunting and dietary preferences (group hunters, hypercarnivory of The aligned surfaces were then converted into legacy VTK files using small prey, and mesocarnivory) as grouping factor, thus enabling us the open-source software Paraview v. 5.6.0.2 The VTK files were then to assess the presence of a group structure within the data. We included analyzed with Deformetrica ver. 4.3 (Bône et al., 2018) using the only the first seven components (accounting for the 71.9% of total ‘DeterministicAtlas’ analysis with Kernel size = 0.10, resulting in the variance) as these returned the best classification results, a practice identification of 7,800 control points. The V. lagopus individual was not commonly used in high-dimensional shape data (e.g., Zanolli et al., included in the analysis due to its very reduced sinus morphology. R 2022). The fossil specimens (E. adoxus NMB Rss.45, E. davisi F:AM scripts and data used here for the analyses are available at the following 97057) and the hypocarnivore Lupulella adusta (MZUF-8496) were link http://dx.doi.org/10.5281/zenodo.7836886. plotted a posteriori onto the shape space. For these specimens, we also computed cross-validated posterior probabilities and typicality probabilities using their CVA scores. Posterior probabilities assume that an individual must belong to one of the a priori defined groups 2 www.paraview.org and are expressed as a percentage. In contrast, the interpretation of Frontiers in Ecology and Evolution 05 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 typicality probabilities is less intuitive, as they test the null hypothesis of this paranasal cavity. Eucyon adoxus possesses instead a well- of group membership for a given specimen and return a p-value, developed frontal sinus with a marked rostral incision that identifies two which allows to reject the null hypothesis (i.e., the specimen is an prominent rostral lobes. On the dorsal surface, the sinus of E. adoxus outlier for the considered group) at p < 0.05. Finally—as for the shows evident prominences along the main axis of the sinus, marked by morphometric variables—we assessed the possible presence of a dorsal sulcus. The ventral lobe is elongated ventrally, giving the frontal allometric trends in the shape data by regressing each of the obtained sinus a triangular-like outline, in lateral view. The postorbital lobe is PCs and CVs against the log-transformed body mass (in kg) in R. developed, resembling in shape the morphology of the process of the frontal bone: indeed, the sinus visibly invades the whole length of the process (Figure 3). This species shows also a trigonal dorsocaudal lobe 3. Results and a marked ventrolateral prominence jutting caudally. The sinus of Eucyon davisi is rostrocaudally elongated but with fairly inflated 3.1. Description of the sinuses of Eucyon generalized structures (Figure 3). The rostrocaudal elongation is remarkable: even if they do not reach the frontoparietal suture, the sinus The three species of the genus Eucyon considered in this study show covers a fair portion of the endocranial cavity. There is no rostromedial quite different morphologies of the frontal sinuses. The cranium of lobe, whereas the rostrolateral one is prominent, in dorsal and lateral E. monticinensis MSF 466, at present the only cranium of the species ever views (Figure 3). In lateral view, the ventral lobe is slender compared to recovered and described, is extremely deformed due to taphonomic the inflated and rounded dorsocaudal lobe. The postorbital lobe is processes. The deformation affecting the fossil involved a dorsoventral reduced and sharp, jutting pointily on the lateral plane. compression, with a left lateral component, so that the cranium seems to lie on the left side. The left sinus, the only one preserved in MSF 466, is thus heavily deformed (Figure 3). Despite this problematic, the 3D model 3.2. Morphological and morphometric shows that this species presents quite a small sinus, with reduced lobes. comparison of the frontal sinuses in Further comments on its development are hindered by the deformation: Canidae it is indeed hard to understand the dorsoventral extension of the sinus, as well as the development of its ventral lobe. The other species of the Figure 4 shows the final reconstruction of the frontal sinuses of a genus, also shown in Figure 3, show instead a very different morphology single specimen for each fossil and extant species, considered in FIGURE 3 Crania of the three species of Eucyon here analyzed. (A–A’,D–D′) Eucyon adoxus (NMB Rss.45) from St. Estève (Late Pliocene, MN 15; southern France), in dorsal (A–A’) and in left lateral (D–D’) views. (B–B’,E-E’) Eucyon monticinensis (MSF 466) from Cava Monticino (latest Miocene, 5.61– 5.33 Ma; Emilia-Romagna, Italy), in dorsal (B–B’) and left lateral (D–D’) views. (C–C’,F-F’) Eucyon davisi (F:AM 97057) from near Xiakou (late Early-Late Pliocene, ~4.0–3.0 Ma; Yushe Basin, Shanxi, China), in dorsal (C–C’) and left lateral (F–F’) views. (A’–F’) Highlight the position of the frontal sinuses within the crania. Frontiers in Ecology and Evolution 06 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 FIGURE 4 Dorsal and left lateral views of the crania of fossil and extant Canidae analyzed here, with the frontal sinuses in their natural position. (A–A’) NMB Rss.45, E. adoxus. (B–B’) MSF 466, E. monticinensis. (C–C’) F:AM 97057, E. davisi. (D–D’) MZUF-1474, V. lagopus. (E–E’) MZUF-1715, L. mesomelas. (F–F’) MZUF-11878, C. aureus. (G–G’) MZUF-1851, C. lupaster. (H-H’) MZUF-8496, L. adusta. (I–I’) 88-50-290, C. latrans. (J–J’) MZUF-13781, C. simensis. (K–K′) 368.443, L. pictus. (L–L’) MZUF-11874, C. lupus. relationship to the cranium. Figure 5 shows the morphology of the left rostromedial and rostrolateral lobes, while C. latrans and L. mesomelas, sinus of a single specimen for each species considered. As it is visible despite still having two fairly small rostral lobes, show a deeper rostral from Figures 4, 5, the frontal sinus is a fairly variable structure, incision (which in the case of L. mesomelas is also wider). The showing a wide range of dimensions and morphologies in the postorbital lobe in C. aureus, C. lupaster and L. mesomelas is reduced members of the family Canidae. The analysis shows the almost total and barely diversified on the lateral side of the rostrolateral lobe. It also lack of sinus in V. lagopus, which are not well defined nor possess has comparable position in rostrocaudal length of the sinus. The proper lobes, rather many small chambers barely connected to each similarity between L. mesomelas and C. aureus and C. lupaster also other. On the contrary, all the other specimens analyzed possessed extends in lateral view: a three-sided outline, with no or modestly well-formed sinus, distinguished from one another for their degree of developed ventrolateral prominence. Canis latrans has a deeper and external complexity. Despite these differences all species shared a more developed sinus, in both dorsal and lateral views. Canis simensis rounded three-sided outline in lateral view and the presence of at least has a deep, wide, and rounded rostral incision, with two well-formed one rostral lobe. In all the species of Canis, two rostral lobes separated rostral lobes, especially the rostrolateral one. In general terms, the by the rostral incision are visible, despite their different degree of sinus of the Ethiopian wolf is characterized by a relatively poor development in the various species. Canis aureus and C. lupaster show rostrocaudal elongation but a proportionally dorsoventrally deep very similar frontal sinuses with a shallow rostral incision and a small extension. Indeed, in lateral view the sinus shows an angulated and Frontiers in Ecology and Evolution 07 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 FIGURE 5 Dorsal and lateral views of the left frontal sinus of the examined species. Each model has been reported to same width (and true scale, i.e., 2cm, is reported per each specimen between dorsal and lateral view). (A–A’) NMB Rss.45, E. adoxus. (B–B’) MSF 466, E. monticinensis. (C–C’) F:AM 97057, E. davisi. (D–D′) MZUF-11880, C. aureus. (E–E’) 88-50-290, C. latrans. (F–F′) MZUF-2714, C. lupaster. (G–G’) MZUF-2032, C. lupus. (H–H′) MZUF-13781, C. simensis. (I–I′) MZUF-1474, V. lagopus. (J–J’) MZUF-3293, L. mesomelas. (K–K′) MZUF-8496, L. adusta. (L–L’) 368.443, L. pictus. wide ventral lobe. Moreover, it displays a series of caudal incisions prominences of the dorsal surface is similar to that of the wolf. which denote multiple caudal lobes. Canis lupus possesses the most Caudally, the sinus of L. pictus is rounded and subdivided into complex sinus in the genus Canis, rivaled only by that of L. pictus. In different smaller lobules by evident incisions, similarly but more than addition to a deep rostral incision, C. lupus shows two well-developed in C. lupus. In L. adusta, the sinus is particularly proportionally and domed rostral lobes and a dorsal sulcus that branches medially elongated rostrocaudally and marked by the absence of the and laterally, shaping different prominences. The African hunting dog, rostromedial lobe. Furthermore, it possesses an enlarged rostrolateral L. pictus, have a complex and lobose sinus, marked by the developed lobe. Despite the breadth of this part, the postorbital lobe is not so rostrolateral lobe and a reduced rostromedial one. In this species, the expanded, yet invading the zygomatic process of the frontal. The postorbital lobe is rostrally advanced and further pneumatized in two postorbital lobe is parted in two lobules in the considered specimen. separated lobules. The degree of complexity and of additional Despite the generic attribution, the sinuses of L. adusta and Frontiers in Ecology and Evolution 08 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 L. mesomelas differ greatly from one another. This is evident in the of the cranium, body mass) (Table 2). The results are reported in dorsal shape of the sinuses (slender in L. adusta, in dorsal view, and Table 3 and Figures 6, 7. The sinus of E. monticinensis was not characterized by prominences visible, in lateral view), the morphology considered because it was too deformed for this kind of analysis. of the rostral region and in the development of the caudal lobe and of The bivariate regressions for the considered morphometric the ventrocaudal promincence of L. adusta (the latter almost in as variables show that they are highly correlated with one another and proper lobe). The morphology of the sinus of E. davisi and with the body mass (Figures 6, 7; Table 3). Both ln LmaxS and ln Vol E. monticinensis resembles that of L. adusta for the absence of a show a strong correlation with ln BM, which explains a similarly rostromedial lobe and a developed rostolateral one. Nevertheless, the large amount of the variance of the two variables (ca. 70–75%) and sinus of E. monticinensis is rostrocaudally shorter compared to the one follows a negative allometry relationship (i.e., the body mass grows of L. adusta. The reduction in rostrocaudal length is similar to slower than either volume or the maximum sinus length) C. aureus, C. lupaster, and L. mesomelas. Nevertheless, the taphonomic (Figures 6A,B). The correlation between ln LmaxS and ln Vol with deformation of the cranium (thus of the sinus) hinders proper ln TL is similarly strong, explaining approximately 70% of total comparison with the other extant canids. Eucyon davisi instead shows variance, yet shows a positive allometry relationships (i.e., the skull a proportionally reduced rostrolateral lobe when compared to the length increases faster than both volume and maximum sinus large and inflated one of L. adusta. Furthermore, the sinus of the fossil length) (Figures 6C,D). In all plots of Figure 6, V. lagopus and species is more inflated with almost no subdivisions, whereas L. adusta C. lupus lie at the two extremes of variability, as the former has an shows numerous lobules individualized by sulci and incisions. Despite extremely reduced sinus, whereas the latter has the largest and most these differences the sinus of L. adusta is closest in terms of general developed sinus of all the considered species. Between these two resemblance to that of E. davisi. The sinus of E. adoxus differs from extremes lie all the other extant species. Eucyon adoxus is located that of medium-sized canids (C. aureus, C. lupaster, C. latrans, close to the variability of C. simensis, for its similar values of ln Vol C. simensis, and L. mesomelas) for its rostrocaudal elongated and and ln LmaxS, and to the lower ranges of Groups 1 hypercarnivores. pointed shape, in dorsal view. In this regard, it resembles C. lupus, Eucyon davisi lies a bit apart from the ecomorphological groups. The though proportionally smaller. Even in lateral view, E. adoxus has a comparable values of ln LmaxS and ln TL, E. davisi is often close to proportionally deep sinus, unlike previous taxa. The dorsal surface L. adusta (as in Figures 6A,C,D). Considering the relationship shows some prominences identified by a branched sulcus resembling between ln LmaxS and ln Vol (Figure 7) we have seen an interesting those of C. latrans and, to minor extent, C. lupus. The presence of a pattern of distribution. Group-hunters canids (Group 1 of Van bulging and inflated rostromedial sulcus marks a sharp difference with Valkenburgh and Koepfli, 1993) are separated from the other canids, L. adusta. especially mesocarnivores (Group 3) and small-prey hypercarnivores Another feature used to describe the morphology and (Group 2). This is highlighted by the regression lines with different development of these paranasal cavities is their caudal extension, in R2 plotted in the graph (Figure 7; Table 3). The distinction in the particular relative to the frontoparietal suture. As visible in Figure 4, lines suggests the presence of an allometric grade shift between only in C. lupus and L. pictus the frontal sinus reaches the group-hunter taxa and the rest of Canidae. This is further confirmed frontoparietal suture, whereas in the other species, despite their by the ANCOVA results, showing the homogeneity of the allometric difference, it never extends further caudally. Other species that have slopes (F = 0.005, p = 0.947) and the presence of significant well-developed frontal sinuses with a considerable caudal extension, differences between the intercepts (F = 136.03, p = <0.001) of the two i.e., close to the frontoparietal suture, are C. latrans, C. simensis, and groups. Hence, group hunters appear to possess a more voluminous L. adusta. If the cranium is observed in lateral view (Figure 4), it is sinus for a similar sinus length when compared to non-group hunter relevant to note the relationship between the frontal sinus and the canid taxa. Apparently, L. adusta lies together with the large-prey endocranium. The frontal sinus in V. lagopus is evidently reduced. In hypercarnivores, unlike E. davisi or E. adoxus, which clustered close, C. lupus the frontal sinus covers a good portion of the cranial cavity, almost overlapped, to C. simensis. whereas in the other species it covers different portions of the braincase. In C. aureus the overlap between the frontal sinus and the brain is the smallest (Figure 4). The second is C. lupaster, and then 3.3. Diffeomorphic analyses C. simensis and C. latrans which have similar parts of the braincase covered by the frontal sinus. Lycaon pictus has a large portion of the The principal component analysis (PCA) performed on the braincase topped by the frontal sinus even if the overlapping region is deformation fields obtained from the DSM analyses performed on not as large as in C. lupus. In Lupulella spp., L. adusta seems to possess the left sinus (Figure 8; Table 3; see Supplementary Material) allows a longer region of the braincase covered by the frontal sinus compared discerning the four different ecological groups included in the to L. mesomelas. As mentioned above, in the fossil species the sinus study, particularly when the first three principal components (PCs; expands caudally into a modest portion of the endocranial cavity. This 46.2% of total variance) are considered at the same time. The extension is comparable in both E. adoxus and E. davisi. The bivariate regressions between the PCs and log-transformed BM preservation of E. monticinensis specimen (MSF 466) does not allow identify the presence of a significant allometric relationship only for any comparison. PC2 (Table 3), where the body mass explains half (R2 = 0.429, As far as the morphometric analyses are concerned, the study p < 0.001) of its total variance, thus suggesting that BM differences considered in particular the relationship between different parameters play a limited role in explaining the separation observed between extrapolated by the tridimensional renderings of the frontal sinus the analyzed taxa. The PC1 accounts for the 20.7% of the variability. (e.g., sinus volume, maximum rostrocaudal length of the sinus) and Along this axis the groups fail to separate properly, with the characteristics of the considered specimens/species (e.g., total length majority of the specimens located on the positive side of the axis Frontiers in Ecology and Evolution 09 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 TABLE 2 Results of the measurements of the specimens examined. Species Cat. Num. Vol (mm3) LmaxS (mm) TL (mm) BM (kg) Eucyon adoxus NMB Rss.45 2692.9 28.8 182 15.5 Eucyon davisi F:AM 97057 3014.6 30.6 167.3 11.4 Eucyon monticinensis MSF 466 985.3* 22.1* - 12.0+ MZUF-11880 1640.2 24.1 154.2 7.4 Canis aureus MZUF-11878 1517.8 20.3 151.5 9.2 Canis latrans 88-50-250 3469.3 29.5 197.0 19.6 MZUF-1851 1683.4 21.6 156.4 9.6 MZUF-2140 675.2 15.5 156.3 8.7 Canis lupaster MZUF-2110 712.8 18.4 150.5 8.5 MZUF-2714 1549.1 20.4 146.5 8.5 MZUF-2032 11406.5 50.7 220.1 26.0 MZUF-11874 8772.2 46.5 223.2 26.5 Canis lupus MZUF-16534 9342.2 44.8 231.5 29.2 DST-N01 4437.5 39.6 212.6 22.3 m-81.001 2579.7 28.4 208.4 22.3 Canis simensis CE818 1789.9 24.3 187.2 16.3 MZUF-13781 2935.8 28.8 203.4 21.7 Lupulella adusta MZUF-8496 1920.9 31.4 166.5 13.2 MZUF-1843 1000.1 19.3 - - MZUF-1898 1755.5 24.0 150.1 9.4 Lupulella. mesomelas MZUF-1128 942.2 19.7 153.6 9.8 MZUF-1715 2382.4 26.8 147.1 - MZUF-3293 1293.6 22.9 132.3 6.5 368.443 5913.1 44.1 204.0 21.4 Lycaon pictus MZUF-1127 3323.3 36.0 192.9 20.0 Vulpes lagopus MZUF-1474 29.5 9.5 113.1 4.4 *these measures must be taken with caution; +estimation taken from Bartolini-Lucenti and Rook (2021), based on the regression formula by Van Valkenburgh (1990) based on length of the lower carnassial. TABLE 3 Results of ordinary least-squares (OLS) regressions between natural log-transformed metric variables, natural log-transformed body mass, principal components (PCs), and canonical variates (CVs) used to assess the correlation between the used variables. R2 p Slope SE 95% CI Intercept SE 95% CI Canidae (n = 25) ln LmaxS vs. ln Vol 0.887 <0.001 0.936 0.071 0.788 1.084 0.904 0.181 0.527 1.281 ln Vol vs. ln BM 0.699 <0.001 0.638 0.088 0.454 0.821 0.883 0.233 0.398 1.368 ln LmaxS vs. ln BM 0.738 <0.001 0.652 0.082 0.481 0.823 1.6 0.217 1.154 2.056 ln Vol vs. ln TL 0.695 <0.001 1.767 0.242 1.266 2.269 −6.564 1.247 −9.149 −3.979 ln LmaxS vs. ln TL 0.718 <0.001 1.788 0.232 1.307 2.268 −5.912 1.195 −8.390 −3.434 PC1 vs. ln BM 0.122 0.062 −0.402 0.204 −0.827 0.022 1.07 0.546 −0.069 2.209 PC2 vs. ln BM 0.429 <0.001 0.554 0.135 0.272 0.837 −1.426 0.363 −2.183 −0.668 PC3 vs. ln BM 0.016 0.567 0.096 0.165 −0.249 0.441 −0.264 0.443 −1.188 0.661 CV1 vs. ln BM 0.317 0.004 −4.742 1.448 −7.763 −1.722 11.853 3.883 3.753 19.952 CV2 vs. ln BM 0.281 0.007 −2.409 0.793 −4.063 −0.754 6.492 2.128 2.054 10.931 Group hunters (n = 6) ln LmaxS vs. ln Vol 0.091 0.002 0.731 0.104 0.443 1.0187 1.628 0.304 0.783 2.472 Non-group hunters (n = 19) ln LmaxS vs. ln Vol 0.86 <0.001 0.75 0.075 0.59 0.91 1.304 0.181 0.919 1.689 Significant correlations are in bold. Frontiers in Ecology and Evolution 10 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 FIGURE 6 Bivariate regressions between (A) the natural log-transformed body mass (ln BM) vs. the log-transformed maximum rostrocaudal length of sinus (ln LmaxS). (B) The log-transformed body mass (ln BM) vs. the log-transformed of cubic root of volume of the sinus (ln Vol). (C) The natural log- transformed total length of the cranium (ln TL) vs. the log-transformed maximum rostrocaudal length of sinus (ln LmaxS). (D) the natural log- transformed total length of the cranium (ln TL) vs. the log-transformed of cubic root of volume of the sinus (ln Vol). Lines represent OLS (ordinary least squares regressions) best-fit lines for the whole canid sample. There is a significant correlation between all the variables (see Table 3 for further details). and very few on negative values. Canis lupus and L. pictus (Group 1) the negative values of the axis. Most of the specimens of Group 1 show a wide-range variability along PC1. Apart from one specimen canids have high positive values of PC2. The fossil species have of L. mesomelas, both Canis simensis (Group 2) and canids of small positive values of PC2. The PC3 accounts for the 10.8% of the Group 3 placed in the positive side of PC1. This position variability. Along this axis the groups are almost all clustered corresponds to proportionally inflated (especially laterolaterally and together toward positive values (between −0.25 and 0.5). Eucyon dorsoventrally) but rostrocaudally shortened sinuses – see spp. lie within this cluster of extant species. Figure 8C. This pattern is confirmed by their morphology (see The canonical variate analysis (CVA; Figure 9) performed on the Figure 5). Eucyon adoxus lies close to C. simensis, with a slightly low first four principal components achieves good classification results after negative value of PC1. Eucyon davisi falls within the variability of cross-validation, with 95.2% of correctly classified individuals. A visual Groups 1 and 3. PC2 accounts for 14.7% of the morphological inspection of the bivariate plot of CV1 and CV2 confirms this result variability. Along this axis the ecological groups are almost and shows a very good separation between the a priori identified separated, with Groups 2 placed on the positive side of PC2, ecological groups, with no overlap among them (Figure 9) along CV1 whereas the majority of Group 3 and the specimen of L. adusta have (80.5% of total variance). Notably, CV1 and CV2 show a strong and Frontiers in Ecology and Evolution 11 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 4. Discussion 4.1. Frontal sinus: phylogenetic signal and ecological relevance Among carnivorans, the frontal sinus as anatomical structure has been deeply studied in Hyaenidae (Vinuesa et al., 2016) and recently in Ursidae (Pérez-Ramos et al., 2020). Frontal sinus in fossil hyaenids were firstly described by Joeckel (1998), evidencing that bone-cracking forms like Adcrocuta possessed very enlarged frontal sinus overlapping the whole brain cavity and other jackal-like, wolf-like and transitional bone-cracker forms with lesser enlarged sinus which only overlap from 70 to 80% of the brain cavity like Hyaenotherium, Palinhyaena (Qiu et al., 1979) or Ictitherium (Joeckel, 1998; see also Vinuesa et al., 2015, 2016; Vinuesa, 2018, for further discussion). Despite the interesting convergent evolution between hyaenas and canids (see Supplementary Material), even in terms of frontal sinus development, that of fossil canids was never studied in detail, except for a few cases. FIGURE 7 For instance, some scholars hypothesized a posteriorly enlarged Bivariate regressions between the natural log-transformed of cubic frontal sinus in some hypercarnivorous and durophagous American root of volume of the sinus (ln Vol) and log-transformed maximum fossil canids like Borophagus (Cope, 1892) and Epicyon (Leidy, 1858) rostrocaudal length of sinus (ln LmaxS). Lines represent OLS (ordinary least squares regressions) best-fit lines for the whole canid sample (see Werdelin, 1989; Tseng and Wang, 2010). (black) and hypercarnivorous group-hunting canids (red). There is a In Caninae, i.e., extant canids, the features of the frontal sinus significant correlation between volume and the length of the sinus were described for the first time by Huxley (1880), who used them as (see Table 3 for further details). one of the main characteristics to differentiate between “Alopecoid” and “Thooid” canids. In his view, the division is justified by the lack of the frontal sinus in the former and their retention in the latter. Some significant correlation with the body mass (Table 3), which explains less scholars discarded this interpretation relating it to an increasing size than half (CV1: R2 = 0.317, p = 0.004; CV2: R2 = 0.281, p = 0.007) of its (e.g., Matthew, 1924), whereas others maintained the unrelatedness variance. Thus implying that BM plays a role in the shape differences between frontal sinus development and body size (we will discuss this captured by these axes, while not being the main driver of shape further on). The latter interpretation favored the validity of frontal variation. This is also partially reflected by the distribution of the sinus features for phylogenetic interpretations such as those of Berta groups along the CV1, all clearly separated from one another. (1988) or Tedford et al., (1995, 2009). The studies of the late Specimens of Groups 1, 2 and 3 cluster fairly well and lie at the opposite R.H. Tedford, together with X. Wang and B.E. Taylor, are among the sides of the axis, respectively in its negative (Group 1) and positive most relevant analyses of the frontal sinus in extant and fossil Caninae. (Groups 2–3) portion. The two extremes are related to completely In their papers, the authors studied the absence or presence of the different shapes of the sinus: the lowest values of the CV1 coincide with sinus, and its development in terms of expansion toward the rostrocaudally elongated and narrow sinus, with strong rostrolateral postorbital process of the frontal, in lateral sense, and the frontoparietal lobes; on the contrary, the high values of CV1 correspond to suture, in caudal sense. Anatomical and paleontological evidence laterolaterally inflated and short sinus, see Figure 9B. In between them supports the interpretation of a phylogenetic information in the lies the single specimen of L. adusta. The fossil species are located in expansion of the frontal sinus: the early forms of Caninae (e.g., the negative side of the axis: E. adoxus is enclosed in the variance of Leptocyon Matthew, 1918) lack a developed sinus (Tedford et al., Group 1 canids, whereas the E. davisi has the lowest value of CV1. On 2009), as does most of the extant and fossil representatives of the the CV2 (19.5% of variance) the specimens do not separate greatly and genus Vulpes and Urocyon (Baird, 1858). In juxtaposition to this most of them are rather comprised between −2 and 2. Only C. simensis condition, Canis spp. have inflated sinuses that always invade the lies far, with peculiarly negative low values. Along this axis, E. adoxus postorbital process and, in some taxa, extends toward the confirms its position close to Group 1 canids. Eucyon davisi has very frontoparietal suture. The similarities between Leptocyon, Urocyon and high values, lying far from any group. This corresponds to fairly Vulpes in terms of morphological features and the timing of their rostrocaudal short sinuses with developed rostral lobes. The posterior paleontological record (the genus Leptocyon is the oldest of the probabilities computed for E. adoxus, E. davisi and L. adusta on the subfamily Caninae known since the Oligocene, whereas Vulpes and basis of CVA scores classify these taxa as group-hunters, with a Urocyon are the oldest of extant canids to appear in the fossil record; posterior probability of 100% for both fossil species and 94% for Tedford et al., 2009) suggest the primitive nature of the absence/ L. adusta (Table 4). In contrast, the typicality probabilities—which do reduction of the frontal sinus in Caninae (confirmed also in Canidae not imply that each specimen must belong to at least one of the groups by other observations, e.g., Wang, 1994; Wang et al., 1999). And at the as posterior probabilities do— show that L. adusta and E. davisi same time the development in Canis apparently marks the derivate represent outliers for all the considered ecological groups (p < 0.05). In state of these taxa. This observation is corroborated by empirical turn, E. adoxus matches the variability of group-hunters (p = 0.30; evidence and was often used also with a systematic purpose. Tedford Table 4) confirming the result of posterior probabilities. and Qiu (1996) included the lateral inflation of the sinus into the Frontiers in Ecology and Evolution 12 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 FIGURE 8 Bivariate scatterplots of PCA results performed on the deformation fields obtained from DSM analysis of sinus shape (variance for each component included within parentheses): (A) PC2 vs. PC1; (B) PC3 vs. PC1. (C) Maximum (below) and minimum (above) extreme conformations of shape variation are shown in dorsal (left), lateral (middle), and medial (right) views for each PC: PC1, PC2, and PC3. Cumulative deformations from mean shape are mapped on the surface by means of a pseudocolor scale ranging from dark blue (no displacement) to dark red (0.11mm). Black arrows correspond to the vectors identifying the direction and amount of displacement. Symbols and convex hulls are colored group-wise as follows: dark red – hypercarnivorous group hunters; grey – mesocarnivores; blue – hypercarnivores on small prey; green – hypocarnivores. FIGURE 9 (A) Bivariate scatterplots of CVA results performed on the deformation fields obtained from DSM analysis of sinus shape (variance for each canonical variate included within parentheses). (B) Maximum (below) and minimum (above) extreme conformations of shape variation are shown in dorsal (left), lateral (middle), and medial (right) views for each CV: CV1 and CV2. Cumulative deformations from mean shape are mapped on the surface by means of a pseudocolor scale ranging from dark blue (no displacement) to dark red (0.11mm). Black arrows correspond to the vectors identifying the direction and amount of displacement. Symbols and convex hulls are colored group-wise as follows: dark red – hypercarnivorous group hunters; grey – mesocarnivores; blue – hypercarnivores on small prey; green – hypocarnivores. Frontiers in Ecology and Evolution 13 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 TABLE 4 Posterior and typicality probabilities computed for E. adoxus, E. davisi, and L. adusta based on canonical variate analysis (CVA) scores obtained using hunting strategy as a grouping factor. Group hunters Hypercarnivores Mesocarnivores Posterior probabilities E. adoxus NMB Rss.45 100% 0% 0% E. davisi F:AM 97057 100% 0% 0% L. adusta MZUF-8496 94% 0% 6% Typicality probabilities E. adoxus NMB Rss.45 0.38 <0.001 <0.001 E. davisi F:AM 97057 <0.001 <0.001 <0.001 L. adusta MZUF-8496 0.01 <0.001 <0.001 Note that while posterior probabilities represent the likelihood of belonging to a group (expressed as a percentage), typicality probabilities are given as p-values for the null hypothesis of group membership so that for p < 0.05, the specimen can be considered an outlier for a given group. Highest posterior probabilities and significant typicality probabilities are in bold. postorbital process as one of the diagnostic features of the genus 4.2. Frontal sinus and inferred diet Eucyon, a synapomorphy shared with Canis. Sotnikova (2006) preferences described a peculiar Canini from the Pliocene of Mongolia as Nurocyon chonokhariensis thanks to a combination of primitive and Despite the variability displayed by this structure, the comparison derived features, among which sharing a large frontal sinus that of different morphologies of the frontal sinus possessed by the invades the postorbital process with Canis and Eucyon. different canids has shown some patterns (Figure 5). Most of the Nevertheless, the information from the structure and features discussed above seem to correspond to an ecological affinity development of the frontal sinus has also led to some phylogenetic rather than a phylogenetic relationship between species with similar reconstructions that contrast with molecular evidence. One of the sinus morphologies. Indeed, in some cases, phylogenetically related most striking probably is relationship between the crab-eating fox taxa differ more than with distantly related ones. For instance, the (Cerdocyon Smith, 1839) and the raccoon dog (Nyctereutes): some species identified as mesocarnivorous (C. aureus, C. latrans, morphological analyses (Tedford et al., 2009) include the two taxa C. lupaster and L. mesomelas; n.b., all members of Group 3) have within the Cerdocyonina (subtribe erected by Tedford et al., 2009 to comparable general shapes of the sinuses. They show, e.g., shallower include the clade of South American Caninae) whereas the molecular rostral incisions and modest and simple rostrolateral lobes, less and total-evidence phylogenies show the distance between them (see developed compared to the hypercarnivorous ones, like C. lupus also Figure 1). While the position of Cerdocyon is confirmed, (Group 1) and C. simensis (Group 2). Moreover, mesocarnivorous Nyctereutes is deeply rooted within the tribe Vulpini, well separated species share fairly smooth dorsal surface of the sinus, without marked from Canini (among others, Lindblad-Toh et al., 2005; Wayne and sulci on it. The only exception to this smooth pattern is C. latrans, Ostrander, 2007; Chen and Zhang, 2012; Zrzavý et al., 2018; Figure 1). which shows a particularly evident caudal prominence, identified by This incongruence can be explained by observing the numerous a transverse dorsal sulcus (Figure 5). As far as hypercarnivorous similarities that the crab-eating fox and the raccoon dog show in their species are concerned, it should be noted that Groups 1 and 2 show a cranial and dentognathic features. Among these comparable features great difference between them: the former has simpler lobes in the there are the prominent subangular lobe, the expanded angular rostral region whereas the latter has more developed and more process, the reduced canines, and the development of the frontal complex rostral lobes. Furthermore, the sinus of C. simensis has low sinus, clearly visible from a direct study of cranial specimens (Tedford prominences on its dorsal side, although some sulci are still visible on et al., 1995). These characteristics are linked to diet and ecological its surface, and, in general terms, are deeper than those of preferences and thus most probably they represent convergent mesocarnivorous canids. On the contrary, the sinus of C. lupus is features evolved in two distantly related groups, in different context considerably more complex and marked by deep sulci and evident (Eurasia for Nyctereutes and South America for Cerdocyon) prominences (Figure 5). A great complexity and pneumatization is (Bartolini-Lucenti et al., 2018). Indeed, the development of sinuses evident also in L. pictus. Lupulella adusta, here represented by a single in Nyctereutes has been related by some authors to the possibility of specimen, has its own peculiarities. Unlike the mesocarnivorous producing relatively large bite forces while feeding (Curtis, 2014; species, the sinus is developed, elongated rostrocaudally and possesses Curtis and Van Valkenburgh, 2014), just like in larger canids (Slater relatively high prominences on its dorsal surface. The grouping of taxa et al., 2009; Tseng and Wang, 2010). Similar ecological interpretation, according to ecological affinity is testified also by the morphometric rather than phylogenetic one, was applied to the Late Miocene taxon analyses here carried out (Figures 6–9). Independently from the Eucyon ferox (Miller and Carranza-Castañeda, 1998). Once methodology and the application (i.e., log-transformed measures or considered the first member of the genus Canis, also due to the 3D meshes), the results coherently point out two distinct groups, development of its frontal sinus, a recent reassessment of its general separated from one another. All this evidence fits with the inferences features suggests a more plausible interpretation of the taxon to the of other scholars (e.g., Curtis and Van Valkenburgh, 2014) on the genus Eucyon and with a hypercarnivorous diet (Bartolini-Lucenti degree of pneumatization and the development of the frontal sinus and Rook, 2021). As in the case of Nyctereutes, the development of according to feeding ecology. The grade shift between group hunters the sinus of E. ferox is thus plausibly related to its diet. (Group 1) and the others, despite their diet (namely Group 2–3) add Frontiers in Ecology and Evolution 14 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 further support to this interpretation (Figure 7). Interestingly, the species, which lies close to L. adusta in the linear models (Figure 6) position of L. adusta matches the hypothesis: larger stresses during but does not follow the allometric grade shift between natural feeding (whether from food items or prey hunting) seem to result in log-transformed volume and maximum length (Figure 7) and clearly comparably larger and convoluted sinuses in group hunters and differs from all other groups when total shape is considered hypocarnivorous canids. If such inference can be easily drawn from (Figures 8, 9). Group 1 species, in the case of Group 4 ones (though here represented Different is the case of E. adoxus. The development of the by a single specimen), at the very least, the results corroborate the rostromedial lobe, or the prominent convoluted dorsal morphology observation by Curtis and Van Valkenburgh (2014) regarding does not support a similarity to mesocarnivorous species (Group 3). N. procyonoides (another typical hypocarnivorous, i.e., Group 4, taxon, This interpretation can be disregarded also considering the results of following Van Valkenburgh and Koepfli, 1993). the morphometric analyses (Figures 6–9). It also differs from the Therefore, our analyses confirm how relevant frontal sinus is as an proportionally wider and shallower sinus of C. simensis. anatomical structure and yet the numerous parameters that influence Morphologically and morphometrically, our results suggest that its development. Surely, like Matthew (1924) hypothesized, body mass E. adoxus was subject to a relevant amount of stress, comparable, has an influence on the development of the sinus. Nevertheless, the proportionally, to that experienced by Group 1 canids. Such a influence of phylogeny pointed out by several scholars (e.g., Berta, similarity contrasts with its cranial and dentognathic features, which 1988; Tedford et al., 2009; see above) is undeniable. Generally, do not support an ascription to this ecomorphogroup (see also members of the tribe Vulpini do not have frontal sinus, whereas, on Bartolini-Lucenti and Rook, 2021). It could be argued that even the the contrary, Canini always do possess the sinus, with different single specimen of L. adusta falls close to hypercarnivorous group- development (Figures 4, 5). When comparing the morphology of the hunters in some analyses (Figures 7, 8; Table 4) and yet the sinus within Canini, some phylogenetically controlled features seem morphological features of this canid are clearly hypocarnivorous. Even to be present (e.g., an expanded rostromedial lobe in Canis s.s. unlike in this case, an ascription of E. adoxus to hypocarnivorous group can Lupulella). Nevertheless, other smaller scale differences or peculiar be easily excluded for its dentognathic features. The elongated similarities between the species here considered cannot be easily cranium, the diastemata between premolars, with their pointy cusps, explained with phylogeny. The third parameter with a relevant role in and sharp molars are more definitely close to those of C. simensis. shaping the frontal sinus of canids is their ecology, in terms of dietary Nevertheless, as reminded above, the distinction in terms of frontal habits. Indeed, this structure is linked to a complex and interrelated sinus development between E. adoxus and C. simensis is significant. set of characteristics which must be simultaneously taken into Studying the frontal sinus of Group 2 species is difficult as three out consideration when studying fossil species. Despite the deformation of four species ascribed at this ecomorphogroup are of the genus that hinders morphometric analyses, the sinus of E. monticinensis is Vulpes, with virtually no sinus [V. lagopus, see the ‘protosinus’ in modestly developed and characterized by a similarity with the genus Figure 5; Vulpes macrotis (Merriam, 1888) and Vulpes corsac Lupulella, rather than Canis. This similarity is shared also by E. davisi. (Linnaeus, 1768)]. The only representant that possesses a sinus is In both fossil taxa, the developed rostrolateral lobe marks this C. simensis, and even in this species it is not particularly expanded nor similarity, especially with L. adusta. Despite the mild deformations, so convoluted (see Figure 5). Almost resembling the condition of the similarity of the sinus of E. monticinensis with the species of the Group 3 canids (e.g., C. aureus or C. lupaster). The hypercarnivorous African Lupulella is interesting for its timing. Eucyon monticinensis diet of this canid, based primarily on rodents like the big-headed represents one of the oldest undisputed records of Canidae in western African mole-rat (Tachyoryctes macrocephalus Rüppell, 1842) Eurasia (age of the type locality: ca 5.6–5.3 Ma; Rook, 1992; Bartolini- (Kingdon, 1989), does not imply elevated stresses for their masticatory Lucenti et al., 2022; Figure 1), part of the ‘Eucyon event’ of the Late apparatus. This explains the morphology and modest development of Miocene (Sotnikova and Rook, 2010). The time of divergence of the C. simensis sinus, despite the degree of carnivory especially in genus Lupulella from the Canis-clade following recent molecular comparison with mesocarnivorous (Group 3). Much evidence analyses (e.g., Perri et al., 2021) is estimated around 5.1 Ma. There are supports this: for instance, Van Valkenburgh and Koepfli (1993) Late Miocene records of Eucyon in Africa (e.g., E. intrepidus from showed that, indeed, mesocarnivorous species have more robust Kenya), which nevertheless were recently disputed by some authors mandibles compared to Group 2 species. The cranial and dental for the scanty nature and the inconsistency of their features (see morphology of E. adoxus fit with the interpretation of this taxon as Werdelin et al., 2015 for a deeper discussion on the issue). Taxa typical small-prey hypercarnivorous (Group 2). The more convoluted sinus of ‘Eucyon event’, e.g., E. monticinensis and E. davisi from Asia of E. adoxus cannot be explained suggesting that it was capable of (Sotnikova and Rook, 2010), could be at the base of the radiation of hunting large prey, like some Group 3 taxa, as neither its linear this jackal-like canids in Eurasia but also in Africa. Lupulella might morphometric parameters (Bartolini-Lucenti and Rook, 2021) nor the be the extant descendant of such dispersal and radiation. The tridimensional morphology of the sinus support it. More plausibly the interpretation does not contrast with present phylogenetic hypotheses preferred prey of this canid was larger than those of C. simensis. (e.g., Zrzavý et al., 2018) and finds support in the patterns of Bartolini-Lucenti and Rook (2021) used the least regression equation morphological and morphometric affinities obtained here. Regarding of Van Valkenburgh et al. (2003) to estimate typical prey size of E. davisi, linear morphometric parameters were not conclusive in E. adoxus, obtaining a prey weight of 5 kg. This estimation greatly classifying the diet of this fossil taxa with any of extant dietary groups, exceeds the average size of the larger typical prey of C. simensis if not for a similarity with Group 3 species (Bartolini-Lucenti and (T. macrocephalus: ~400–900 g, Yalden, 1985) and it is close to extant Rook, 2021). Dentognathic features do not hint at any peculiar dietary leporid lagomorphs like hares (genus Lepus Linnaeus, 1758). A specialization to hypo- or hypercarnivory (Rook, 2009). Our results connection between elongated cranium and mandible, characterized show certain degree of peculiarity of E. davisi compared to other by diastemata, and a lagomorph-based diet was proposed for other Frontiers in Ecology and Evolution 15 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 fossil canids, e.g., Cynotherium sardous (Studiati, 1857) from Sardinia 1989). Although the latter site is younger than type locality of (see Lyras et al., 2006; Lyras and Van der Geer, 2006; Madurell- E. adoxus, St. Estève (Roussillon Basin; France; Nomade et al., 2014), Malapeira et al., 2015). Lagomorphs experienced a moment of great the former site is one of the few other occurrences of E. adoxus in radiation at the beginning of the Pliocene (Lopez-Martinez, 2008) and Europe (Rook, 2009). The Pliocene leporine lagomorph Hypolagus dispersal events characterizing different regions and bioprovinces of (extinct) and Oryctolagus were surely smaller compared to the larger Eurasia (Maridet et al., 2007). Among the large rodents and rodents before mentioned, but still robust forms compared to the lagomorphs of the Early-Late Pliocene of western Europe, several taxa extant species, like Oryctolagus laynensis (López Martínez, 1977) (see were present, e.g., Castor (Linnaeus, 1758); Trogontherium (von López-Martínez, 1989; Lopez-Martinez, 2008). They were recorded in Waldheim, 1809); Hystrix (Linnaeus, 1758); Hypolagus (Dice, 1917); the late Early and Late Pliocene of western Europe, also in the and Oryctolagus (Lilljeborg, 1874) (see Hugueney, 2004; Lopez- Roussillon basin (Mein and Aymar, 1984) together with Trischizolagus Martinez, 2008). Of these, the former three are generally of large size (Radulesco and Samson, 1967) (another leporine lagomorph). Eucyon (Hugueney, 2004), despite the presence of a small-sized Trogontherium davisi apparently fed on smaller prey, since the estimated prey size is minus (Newton, 1890) that was found in Red Crag (MN15, UK, around 2 kg (Bartolini-Lucenti and Rook, 2021). Interestingly even for Fostowicz-Frelik, 2008) and Perrier-Les Etouaries (Hugueney et al., E. monticinensis the estimated prey size is around 5 kg FIGURE 10 Restoration of the three Eucyon species studied here with their fossil crania highlighting the position of the frontal sinuses, in red. Top: Eucyon davisi and the cranium F:AM 97057, on the right. Centrum: Eucyon adoxus and the specimen NMB Rss.45, on the left. Bottom: Eucyon monticinensis with fragmented cranium MSF 466. Artwork by Cecilia Loddi. Frontiers in Ecology and Evolution 16 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 (Bartolini-Lucenti and Rook, 2021). Nevertheless, neither the latter Funding two Eucyon spp. possess a specialized dentition nor mandibular features allowing us to narrow down a preferred diet. The morphology SB-L is supported by the Tuscany Region funds for young of their sinuses seems non-conclusive either. researchers “Giovanisì” (www.giovanisi.it): “POR FSC 2014–2020” funds for “Progetti di alta formazione attraverso l’attivazone di Assegni di Ricerca.” LR acknowledges the support of CHANGES Foundation 5. Concluding remarks to the University of Florence (Earth Sciences Department), funded by the Italian Ministry of University and Research, PNRR, Missione 4 Despite the known relevance of the frontal sinus for the phylogeny Componente 2, “Dalla ricerca all’impresa,” Investimento 1.3, and ecology of Carnivora, no previous work has ever focused Project PE_0000020. specifically on the morphology of this structure in extant and fossil canids nor described it in detail. This study represents the first attempt to characterize the frontal sinus of Canidae, using CT and micro-CT Acknowledgments scans to assess its 3D shape via an innovative 3D geometric morphometric method (i.e., diffeomorphic surface matching analysis), The authors are thankful to the kindness of the curators who which allows the direct comparison between continuous surfaces in a allowed us to scan the specimens in their collections or to access landmark-free fashion. them online: Paolo Agnelli, curator of the Zoological Museum “La The results for extant Caninae show a great correspondence Specola” (Museum of Natural History of the University of between morphology and renowned ecomorphotype (see Van Florence, Italy); Enzo Bagnaresi and Marco Sami of the Civic Valkenburgh and Koepfli, 1993) supporting previous interpretations Museum of Natural Sciences of Faenza (Italy); Laurel Lamb of the on the direct influence of diet and feeding behavior on the relative University of Arkansas Museum (Fayetteville, Arkansas, U.S.A.); development of frontal sinus in the different canid species. Regarding Neil Duncan, Collections Manager of the American Museum of the fossil species, we were able to reconstruct for the first time the Natural History (New York, U.S.A.); Jessie Maisano, Department whole sinuses of three Eucyon spp. (Figure 10) and compare them with of Geological Sciences, Jackson School of Geosciences, University the extant species. The deformation of the specimen of E. monticinensis of Texas at Austin (U.S.A.). They are indebted to the staff of the from Cava Monticino does not allow a proper study of the sinus. On San Giovanni di Dio Hospital (Florence, Italy), especially to the contrary, E. adoxus, one of the most enigmatic canids of the Elisabetta Peruzzi and Giovanni Dedola and all the technicians of Pliocene of the western Eurasia, show a sinus with developed Medical Radiology ward (“SOS Radiologia”) for granting us access prominences testifying to a relevant stress during feeding, to their CT-scanner and support while processing raw data. proportionally like hypercarnivorous canids, if not even group-hunters. We thank greatly Georgios Lyras for kindly providing us the scan The dentognathic features of E. adoxus do not support an interpretation of E. davisi. They thank Georg Schulz and Bert Müller for access of this early Canini as a hunter of large prey but more of small prey to the CT-scanner at the Biomaterials Science Center of the specialist, like C. simensis. The resemblance between these two species University of Basel. We thank Cecilia Loddi for the gorgeous can also be retraced to the cranial and mandibular characteristics. restoration of our Eucyon species. The novel combination of morphological, linear and 3D morphometric analyses has allowed the first rigorous characterization of the frontal sinus of fossil canids of the genus Eucyon (Figure 10), Conflict of interest revealing the importance of such anatomical structure and its deep connection with size, phylogeny, and ecology of the considered The authors declare that the research was conducted in the species. The methodology and the analyses here carried out for the absence of any commercial or financial relationships that could first time could be useful tools to help characterizing the frontal sinus be construed as a potential conflict of interest. and investigating the dietary preferences of fossil carnivorans. Publisher’s note Data availability statement All claims expressed in this article are solely those of the authors Publicly available datasets were analyzed in this study. This data and do not necessarily represent those of their affiliated can be found at: http://dx.doi.org/10.5281/zenodo.7836886. organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or Author contributions endorsed by the publisher. SF segmented the tomographic data and prepared a preliminary draft of the text with the help of SB-L and LR. SB-L prepared the linear Supplementary material and 3D data and performed statistical analyses. AU carried out the geometric morphometric analysis. SB-L, JM-M, and AU prepared the The Supplementary material for this article can be found online manuscript with the contribution of SF, LR, and LC. All author revised at: https://www.frontiersin.org/articles/10.3389/fevo.2023.1173341/ and approved the final version of the text. full#supplementary-material Frontiers in Ecology and Evolution 17 frontiersin.org Frosali et al. 10.3389/fevo.2023.1173341 References Baird, S.F. (1858). Mammals: Upon the zoology of the several Pacific railroad routes. Glaunès, J. A., and Joshi, S. (2006). “Template estimation form unlabeled point set data Reports, explorations and surveys for railroad route from Mississippi River to Pacific Ocean and surfaces for computational anatomy” in MICCAI 2006 workshop proceedings. 8: xix–xlviii, 1–757. Washington, DC: Tucker. MFCA’06 workshop. Mathematical foundations of computational anatomy: Geometrical and statistical methods for modelling biological shape variability. eds. X. Pennec and S. Balzeau, A., Albessard-Ball, L., Kubicka, A. M., Filippo, A., Beaudet, A., Santos, E., Joshi (Copenhagen: INRIA/MICCAI), 29–39. et al. (2022). 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