Turbinal bones are still one of the last frontiers of the tetrapod skull: hypotheses, challenges and perspectives.

Turbinals are bony or cartilaginous structures that are present in the nasal cavity of most tetrapods. They are involved in key functions such as olfaction, heat, and moisture conservation, as well as protection of the respiratory tract. Despite recent studies that challenged long-standing hypotheses about their physiological and genomic correlation, turbinals remain largely unexplored, particularly for non-mammalian species. Herein, we review and synthesise the current knowledge of turbinals using an integrative approach that includes comparative anatomy, physiology, histology and genomics. In addition, we provide synonyms and correspondences of tetrapod turbinals from about 80 publications. This work represents a first step towards drawing hypotheses of homology for the whole clade, and provides a strong basis to develop new research avenues.

Mammalian maxilloturbinal evolution does not reflect thermal biology.

The evolution of endothermy in vertebrates is a major research topic in recent decades that has been tackled by a myriad of research disciplines including paleontology, anatomy, physiology, evolutionary and developmental biology. The ability of most mammals to maintain a relatively constant and high body temperature is considered a key adaptation, enabling them to successfully colonize new habitats and harsh environments. It has been proposed that in mammals the anterior nasal cavity, which houses the maxilloturbinal, plays a pivotal role in body temperature maintenance, via a bony system supporting an epithelium involved in heat and moisture conservation. The presence and the relative size of the maxilloturbinal has been proposed to reflect the endothermic conditions and basal metabolic rate in extinct vertebrates. We show that there is no evidence to relate the origin of endothermy and the development of some turbinal bones by using a comprehensive dataset of µCT-derived maxilloturbinals spanning most mammalian orders. Indeed, we demonstrate that neither corrected basal metabolic rate nor body temperature significantly correlate with the relative surface area of the maxilloturbinal. Instead, we identify important variations in the relative surface area, morpho-anatomy, and complexity of the maxilloturbinal across the mammalian phylogeny and species ecology.

Craniogenetic studies in Sus scrofa: With emphasis on the 'orbitosphenoid' problem.

The orbitosphenoid is a skeletal element of the endocranium of extant mammals. However, it has also been described in many of their fossil ancestors. Craniogenetic studies show that it is composed of two types of bone: first, the cartilaginous ala orbitalis and parts of the trabecular plate are transformed by endochondral ossification; second, so-called 'appositional bone' ('Zuwachsknochen') arises directly from the perichondrium of the two optic pilae and spreads in all directions and overlays the remaining cartilage and the endochondral ossifications. For some time, both bone types can be distinguished microscopically, but later in craniogenesis they fuse completely to become the presphenoid sensu lato of the osteocranium. We interpret the 'appositional bone' as neomorphic mode to reinforce the endocranial bone structures, which are the ossification of the delicate cartilaginous template of the chondrocranium. We studied the ossifications of the presphenoidal skull region in a series of ontogenetic stages of the pig Sus scrofa. We applied conventional histology as well as stained and unstained µCT scans. We can show the above-mentioned modes of ossification, and we can demonstrate the contribution of 'appositional bone' well into neonatal and infantile stages. The ossifications of the presphenoid (including the orbitosphenoid) are very slender in therapsids and early mammaliaforms as previously described by other authors. In mammaliaforms, they tend to become thicker and closely connected with the frontal bone, which may be due to the contribution of neomorphic appositional bone. We assume that thereby the presphenoid sensu lato becomes an enforcement of the orbital pillars.

Auditory region circulation in Lagomorpha: the internal carotid artery pattern revisited.

The internal carotid artery (ICA) is one of the major vessels in the cranial circulation. Characters concerning the ICA, such as its course in the auditory region, have been employed frequently in phylogenetic analyses of mammals, including extinct taxa. In lagomorphs, however, our knowledge on vascular features of the auditory region has been based predominantly on living species, mostly on the European rabbit. We present the first survey on 11 out of 12 extant genera and key fossil taxa such as stem lagomorphs and early crown representatives (Archaeolagus and Prolagus). The ICA pattern shows a modified transpromontorial course in stem taxa (Litolagus, Megalagus and Palaeolagus) and Archaeolagus, which we propose as the ancestral character state for Lagomorpha, similar to that for the earliest rodents, plesiadapids and scandentians. The ICA pattern in leporids is perbullar, but shows structural similarities to stem taxa, whereas the extrabullar ICA course in Ochotona is apparently a highly derived condition. Prolagus shows a mixed character state between leporids and Ochotona in its ICA route. The persistence of the transpromontorial ICA course and similarities in the carotid canal structure among stem taxa and crown leporids support morphological conservatism in Lagomorpha, in contrast to their sister clade Rodentia. This article is part of the theme issue 'The mammalian skull: development, structure and function'.

Ontogenetic transformation of the cartilaginous nasal capsule in mammals, a review with new observations on bats.

The nasal capsule, as the most rostral part of the chondrocranium, is a critical point of connection with the facial skeleton. Its fate may influence facial form, and the varied fates of cartilage may be a vehicle contributing to morphological diversity. Here, we review ontogenetic changes in the cartilaginous nasal capsule of mammals, and make new observations on perinatal specimens of two chiropteran species of different suborders. Our observations reveal some commonalities between Rousettus leschenaultii and Desmodus rotundus, such as perinatal ossification of the first ethmoturbinal. However, in Rousettus, ossification of turbinals is demonstrated as either perichondrial or endochondral. In Desmodus, perichondrial and endochondral ossification of the posterior nasal cupula is observed at birth, a part of the nasal capsule previously shown to persist as cartilage into infancy in Rousettus. Combined with prior findings on cranial cartilages we identify several diverse transformational mechanisms by which cartilage as a tissue type may contribute to morphological diversity of the cranium. First, cartilage differentiates in an iterative fashion to increase nasal complexity, but still retains the capacity for later elaboration via de novo bone emanating outward before or after cartilage ossifies. Second, cartilage acts as a driver of growth at growth centers, or via interstitial growth (e.g., septal cartilage). Finally, cartilage as a tissue may influence the timing of ossification and union of the facial and basicranial skeleton. In particular, cartilage at certain points of ontogeny may "model" via selective resorption, showing some similarity to bone.

Reconstruction of evolutionary changes in fat and toxin consumption reveals associations with gene losses in mammals: A case study for the lipase inhibitor PNLIPRP1 and the xenobiotic receptor NR1I3.

The inactivation of ancestral protein-coding genes (gene loss) can be associated with phenotypic modifications. Within placental mammals, repeated losses of PNLIPRP1 (gene inhibiting fat digestion) occurred preferentially in strictly herbivorous species, whereas repeated NR1I3 losses (gene involved in detoxification) occurred preferentially in strictly carnivorous species. It was hypothesized that lower fat contents of herbivorous diets and lower toxin contents of carnivorous diets cause relaxed selection pressure on these genes, resulting in the accumulation of mutations and ultimately to convergent gene losses. However, because herbivorous and carnivorous diets differ vastly in their composition, a fine-grained analysis is required for hypothesis testing. We generated a trait matrix recording diet and semi-quantitative estimates of fat and toxin consumption for 52 placental species. By including data from 31 fossil taxa, we reconstructed the ancestral diets in major lineages (grundplan reconstruction). We found support that PNLIPRP1 loss is primarily associated with low levels of fat intake and not simply with herbivory/carnivory. In particular, PNLIPRP1 loss also occurred in carnivorous lineages feeding on a fat-poor diet, suggesting that the loss of this gene may be beneficial for occupying ecological niches characterized by fat-poor food resources. Similarly, we demonstrated that carnivorous species are indeed less exposed to diet-related toxins, suggesting that the loss of NR1I3 and related genes (NR1I2 and UGT1A6) resulted from relaxed selection pressure. This study illustrates the need of detailed phenotype studies to obtain a deeper understanding of factors underlying gene losses and to progress in understanding genomic causes of phenotypic variation in mammals.

The squirrel is in the detail: Anatomy and morphometry of the tail in Sciuromorpha (Rodentia, Mammalia).

In mammals, the caudal vertebrae are certainly among the least studied elements of their skeleton. However, the tail plays an important role in locomotion (e.g., balance, prehensility) and behavior (e.g., signaling). Previous studies largely focused on prehensile tails in Primates and Carnivora, in which certain osteological features were selected and used to define tail regions (proximal, transitional, distal). Interestingly, the distribution pattern of these anatomical characters and the relative proportions of the tail regions were similar in both orders. In order to test if such tail regionalization can be applied to Rodentia, we investigated the caudal vertebrae of 20 Sciuridae and six Gliridae species. Furthermore, we examined relationships between tail anatomy/morphometry and locomotion. The position of selected characters along the tail was recorded and their distribution was compared statistically using Spearman rank correlation. Vertebral body length (VBL) was measured to calculate the proportions of each tail region and to perform procrustes analysis on the shape of relative vertebral body length (rVBL) progressions. Our results show that tail regionalization, as defined for Primates and Carnivora, can be applied to almost all investigated squirrels, regardless of their locomotor category. Moreover, major locomotor categories can be distinguished by rVBL progression and tail region proportions. In particular, the small flying squirrels Glaucomys volans and Hylopetes sagitta show an extremely short transitional region. Likewise, several semifossorial taxa can be distinguished by their short distal region. Moreover, among flying squirrels, Petaurista petaurista shows differences with the small flying squirrels, mirroring previous observations on locomotory adaptations based on their inner ear morphometry. Our results show furthermore that the tail region proportions of P. petaurista, phylogenetically more basal than the small flying squirrels, are similar to those of bauplan-conservative arboreal squirrels.

Two Functional Epithelial Sodium Channel Isoforms Are Present in Rodents despite Pronounced Evolutionary Pseudogenization and Exon Fusion.

The epithelial sodium channel (ENaC) plays a key role in salt and water homeostasis in tetrapod vertebrates. There are four ENaC subunits (α, ß, γ, δ), forming heterotrimeric αßγ- or δßγ-ENaCs. Although the physiology of αßγ-ENaC is well understood, for decades the field has stalled with respect to δßγ-ENaC due to the lack of mammalian model organisms. The SCNN1D gene coding for δ-ENaC was previously believed to be absent in rodents, hindering studies using standard laboratory animals. We analyzed all currently available rodent genomes and discovered that SCNN1D is present in rodents but was independently lost in five rodent lineages, including the Muridae (mice and rats). The independent loss of SCNN1D in rodent lineages may be constrained by phylogeny and taxon-specific adaptation to dry habitats, however habitat aridity does not provide a selection pressure for maintenance of SCNN1D across Rodentia. A fusion of two exons coding for a structurally flexible region in the extracellular domain of δ-ENaC appeared in the Hystricognathi (a group that includes guinea pigs). This conserved pattern evolved at least 41 Ma and represents a new autapomorphic feature for this clade. Exon fusion does not impair functionality of guinea pig (Cavia porcellus) δßγ-ENaC expressed in Xenopus oocytes. Electrophysiological characterization at the whole-cell and single-channel level revealed conserved biophysical features and mechanisms controlling guinea pig αßγ- and δßγ-ENaC function as compared with human orthologs. Guinea pigs therefore represent commercially available mammalian model animals that will help shed light on the physiological function of δ-ENaC.

"Forever young"-Postnatal growth inhibition of the turbinal skeleton in brachycephalic dog breeds (Canis lupus familiaris).

In short snouted (brachycephalic) dogs (Canis lupus familiaris), several genetic mutations cause postnatal growth inhibition of the viscerocranium. Thus, for example, the pug keeps a snub nose like that observed in neonate dogs in general. However, little is known how far intranasal structures like the turbinal skeleton are also affected. In the present study, we provide the first detailed morphological and morphometric analyses on the turbinal skeleton of pug, Japanese chin, pekingese, King Charles spaniel, and Cavalier. In order to elucidate how a shortened snout affects turbinal shape, size, and density, our sample covers different degrees of brachycephaly. Macerated skulls of 1 juvenile and 17 adult individuals were investigated by µCT and virtual 3D reconstructions. In addition, histological serial sections of two prenatal and one neonate whippet were taken into account. All investigated postnatal stages show three frontoturbinals and three ethmoturbinals similar to longer snouted breeds, whereas the number of interturbinals is reduced. The shape of the entire turbinal skeleton simplifies with decreasing snout length, that is, within a minimized nasal cavity the turbinals decrease proportionally in surface area and surface density due to a looser arrangement. We interpret these apparent reductions as a result of spatial constraint which affects postnatal appositional bone growth and the position of the turbinals inside the nasal cavity. The turbinal skeleton of brachycephalic dogs arrests at an early ontogenetic stage, corresponding with previous studies on the dermal bones. Hence, we assume an association between the growth of intranasal structures and facial elongation.

Oldest known multituberculate stapes suggests an asymmetric bicrural pattern as ancestral for Multituberculata.

Middle ear ossicles (malleus, incus, stapes) are known for few multituberculate taxa, and three different stapedial morphotypes have been suggested: (i) slender, columelliform and microperforate, (ii) robust and rod-like, and (iii) bicrural. Reinvestigation of Upper Jurassic (Kimmeridgian) mammalian petrosals from the Guimarota coal mine in central Portugal (Western Europe) revealed an asymmetric bicrural stapes (ABS) in the paulchoffatiid Pseudobolodon oreas The middle ear ossicles displaced inside the osseous vestibule were detected by a µCT analysis. The Kimmeridgian age of the Guimarota stapes exceeds the stapes from the Early Cretaceous (Barremian) of Asia (about 122-124 Ma) by approximately 30 Myr, and is only slightly younger than the stapes of the recently described Oxfordian euharamiyidan Arboroharamiya allinhopsoni The Guimarota stapes indicates that the stapes of Lambdopsalis, described as columelliform and microperforate (small stapedial foramen), does not represent a general condition for multituberculates. The stapes of Pseudobolodon is bicrural, the anterior crus sits centrally on the oval footplate, and the stapedial head is simple and smaller than the footplate. We hypothesize that the ABS evolved from the symmetric bicrural stapes (SBS) of non-mammaliaform cynodonts. The ABS appears to be the ancestral morphotype of the mammalian SBS, and the mammalian columelliform imperforate stapes.

The anterior process of the malleus in extant Lagomorpha (Mammalia).

The anterior anchoring of the malleus of 30 extant species of Lagomorpha (rabbits, hares, pikas) has been studied on the basis of histological serial sections and µCT-scans. It is shown that former studies of Oryctolagus, Lepus, and Ochotona are incomplete, because the rostral part of the processus anterior of the malleus is always lacking due to damage of this extremely delicate structure. Our study shows that in perinatal stages of Leporidae the praearticulare develops a prominent processus internus that fits into a groove at the ventral side of the tegmen tympani; this "tongue and groove"-arrangement may act as a hinge. In adult stages, the rostral end of the praearticulare fuses synostotically with the medial process of the ectotympanic. Torsional strain produced by rotation around the axis of the middle ear ossicles at sound transmission must, therefore, be experienced by the extremely thin but highly elastic bony pedicle of the processus internus praearticularis. The free ending processus anterior of a late fetal Ochotona shows a short processus internus praearticularis, which does not articulate with the tegmen tympani. During postnatal development the middle ear of Ochotona becomes considerably remodelled: not only does excessive pneumatization of the tegmen tympani and tympanic cavity wall occur, but the short processus anterior is fused synostotically to a bone trabecula of the tegmen tympani meshwork. The thin and elastic bone bridges are not equivalent in Leporidae and Ochotonidae, that is, they must have evolved convergently. Fleischer's classification with Oryctolagus possessing a "freely mobile type" of middle ear ossicles cannot be supported by our observations. The same holds true for Ochotona, which does not represent a "freely mobile type" either. Thus, we suggest for the lagomorph middle ear ossicles a new category: the "bone elasticity type."

Evolution of the mammalian middle ear: a historical review.

Here we present a brief, historical review of research into the mammalian middle ear structures. Most of their essential homologies were established by embryologists, notably including Reichert, during the 19th century. The evolutionary dimension was confirmed by finds of fossil synapsids, mainly from the Karroo of South Africa. In 1913, Ernst Gaupp was the first to present a synthesis of the available embryological and paleontological data, but a number of morphological details remained to be solved, such as the origin of the tympanic membrane. Gaupp favoured an independent origin of the eardrum in anurans, sauropsids, and mammals; we support most of his ideas. The present review emphasizes the problem of how the mammalian middle ear structures that developed at the angle of the lower jaw were transferred to the basicranium; the ontogenesis of extant marsupials provides important information on this question.

The anterior process of the malleus in Cetartiodactyla.

The anterior fixation of the anterior process of the malleus has been studied in a number of Cetartiodactyla. This anterior process, also known as processus gracilis, is provided by the prearticular (gonial), a dermal bone, whereas the cartilage of Meckel becomes resorbed in perinatal ontogenetic stages. Posteriorly, the prearticular fuses with the cartilaginous caput of the malleus; rostrally, the prearticular (= processus gracilis) is always fixed to the anterior crus of the ectotympanic by an extremely thin splint (thickness < 50 µm). From the rostral part of the processus gracilis all studied cetartiodactyls develop a processus internus of considerable size. This process was known as 'ossiculum accessorium mallei' in former times, and its homology has been disputed; from our microscopic-anatomical study we can definitely state that it is always a 'processus internus praearticularis'. This process contacts the fascia of the tensor tympani muscle. In non-ruminants, it articulates dorsally with the tegmen tympani and dorsolaterally with the ectotympanic; whereas the processus gracilis becomes more and more slender, the processus internus may grow to considerable size and normally is fused to the adjacent bones already in juveniles. However, in ruminants, the tegmen tympani tends to disappear and the processus internus praearticularis appears as relatively small bony knob at the floor of the medial cranial cavity, and it seems not to be fused to the surrounding bones; in later age stages, it may become secondarily overgrown by the petrosal. This dorsally exposed processus internus praearticularis seems to be a synapomorphy of the Ruminantia. The functional meaning of this internal process of the prearticular, which is also developed to a minor degree in Carnivora, remains unclear at the moment - but we present some speculations about this.

Bony labyrinth morphometry indicates locomotor adaptations in the squirrel-related clade (Rodentia, Mammalia).

The semicircular canals (SCs) of the inner ear detect angular acceleration and are located in the bony labyrinth of the petrosal bone. Based on high-resolution computed tomography, we created a size-independent database of the bony labyrinth of 50 mammalian species especially rodents of the squirrel-related clade comprising taxa with fossorial, arboreal and gliding adaptations. Our sampling also includes gliding marsupials, actively flying bats, the arboreal tree shrew and subterranean species. The morphometric anatomy of the SCs was correlated to the locomotion mode. Even if the phylogenetic signal cannot entirely be excluded, the main significance for functional morphological studies has been found in the diameter of the SCs, whereas the radius of curvature is of minor interest. Additionally, we found clear differences in the bias angle of the canals between subterranean and gliding taxa, but also between sciurids and glirids. The sensitivity of the inner ear correlates with the locomotion mode, with a higher sensitivity of the SCs in fossorial species than in flying taxa. We conclude that the inner ear of flying and gliding mammals is less sensitive due to the large information flow into this sense organ during locomotion.

Morphology of the nasal capsule of primates--with special reference to Daubentonia and Homo.

Primitive mammals are considered macrosmatic. They have very large and complicated nasal capsules, nasal cavities with extensive olfactory epithelia, and relatively large olfactory bulbs. The complicated structures of the nasal capsule follow a relatively conservative "bauplan," which is normally easy to see in earlier fetal stages; especially in altricial taxa it differentiates well into postnatal life. As anteriormost part of the chondrocranium, the nasal capsule is at first cartilaginous. Most of it ossifies endochondrally, but "appositional bone" ("Zuwachsknochen") is also common. Many fetal structures become resorbed. Together, all surviving bone structures form the ethmoid bone, but cartilages of the external nose and of the vomeronasal complex can persist throughout life. We describe in detail the anatomy of Daubentonia madagascariensis based on a fetal stage (41 mm HL) and an adult skull was analyzed by µCT. We found that the nasal capsule of this species is by far the most complicated one of all extant Primates. We also describe older fetuses of Homo sapiens (35 and 63 mm HL) as representative of a derived primate. The most significant feature of man--and probably of all anthropoids--is the complete loss of the recessus frontoturbinalis and its associated structures. It can be demonstrated that the evolutionary reductions within the primate nasal capsule mainly affect those structures associated with olfaction, whereas cartilages that are important for the biomechanics of the facial skull of the fetus persist.

Nasal anatomy of the non-mammaliaform cynodont Brasilitherium riograndensis (Eucynodontia, Therapsida) reveals new insight into mammalian evolution.

The mammalian nasal cavity is characterized by a unique anatomy with complex internal features. The evolution of turbinals was correlated with endothermic and macrosmatic adaptations in therapsids and in early mammals, which is still apparent in their twofold function (warming and moistening of air, olfaction). Fossil evidence for the transformation from the nonmammalian to the mammalian nasal cavity pattern has been poor and inadequate. Ossification of the cartilaginous nasal capsule and turbinals seems to be a feature that occurred only very late in synapsid evolution but delicate ethmoidal bones are rarely preserved. Here we provide the first µCT investigation of the nasal cavity of the advanced non-mammaliaform cynodont Brasilitherium riograndensis from the Late Triassic of Southern Brazil, a member of the sister-group of mammaliaforms, in order to elucidate a critical anatomical transition in early mammalian evolution. Brasilitherium riograndensis already had at least partially ossified turbinals as remnants of the nasoturbinal and the first ethmoturbinal are preserved. The posterior nasal septum is partly ossified and contributes to a mesethmoid. The nasal cavity is posteriorly expanded and forms a distinctive pars posterior (ethmoidal recess) that is ventrally separated from the nasopharyngeal duct by a distinct lamina terminalis. Thus, our observations clearly demonstrate that principal features of the mammalian nasal cavity were already present in the sister-group of mammaliaforms.

Comparative anatomy and systematic implications of the turbinal skeleton in Lagomorpha (Mammalia).

In order to elucidate the systematic relevance of the turbinal skeleton in Lagomorpha the ethmoidal regions of 6 ochotonid, 21 leporid, and 2 outgroup species (Sciurus vulgaris, Tupaia sp.) species were investigated by high-resolution computed tomography (µCT). Number and shape of turbinals correspond to major clades and to several genera. All Lagomorpha under study have a deeply excavated nasoturbinal that is continuous with the lamina semicircularis; a feature likely to be an autapomorphy of lagomorphs. In particular, the olfactory turbinals (frontoturbinals, ethmoturbinals, and interturbinals) provide new systematic information. The plesiomorphic lagomorph pattern comprises two frontoturbinals, three ethmoturbinals, and one interturbinal between ethmoturbinal I and II. Ochotonidae are derived from the lagomorph goundplan by loss of ethmoturbinal III; an interturbinal between the two frontoturbinals is an autapomorphy of Leporidae. Pronolagus is apomorphic in having a very slender first ethmoturbinal, but shows a puzzling pattern in decreasing the number of turbinals. Pronolagus rupestris and Romerolagus diazi have independently reduced their turbinals to just two fronto- and two ethmoturbinals, which is the lowest number among the sampled lagomorphs. In contrast, the more derived leporid genera under study (Oryctolagus, Caprolagus, Sylvilagus, and Lepus) show a tendency to increase the number of turbinals, either by developing an ethmoturbinal IV (Caprolagus hispidus, Lepus arcticus) or by additional interturbinals. Intraspecific variation was investigated in Ochotona alpina, Oryctolagus cuniculus, and Lepus europaeus and is restricted to additional interturbinals in the frontoturbinal recess of the two leporids.

High morphological variation of vestibular system accompanies slow and infrequent locomotion in three-toed sloths.

The semicircular canals (SCs), part of the vestibular apparatus of the inner ear, are directly involved in the detection of angular motion of the head for maintaining balance, and exhibit adaptive patterns for locomotor behaviour. Consequently, they are generally believed to show low levels of intraspecific morphological variation, but few studies have investigated this assumption. On the basis of high-resolution computed tomography, we present here, to our knowledge, the first comprehensive study of the pattern of variation of the inner ear with a focus on Xenarthra. Our study demonstrates that extant three-toed sloths show a high level of morphological variation of the bony labyrinth of the inner ear. Especially, the variation in shape, relative size and angles of their SCs greatly differ from those of other, faster-moving taxa within Xenarthra and Placentalia in general. The unique pattern of variation in three-toed sloths suggests that a release of selection and/or constraints on their organ of balance is associated with the observed wide range of phenotypes. This release is coincident with their slow and infrequent locomotion and may be related, among other possible factors, to a reduced functional demand for a precise sensitivity to movement.