An X-radiographic and SEM study of the osseous inner ear of multituberculates and monotremes (Mammalia): implications for mammalian phylogeny and evolution of hearing

Multituberculate petrosals with well-preserved, three-dimensional internal anatomy from the Late Cretaceous/early Paleocene Bug Creek Anthills, Montana, U.S.A., are described from X-radiographic and SEM images, as well as from conventional visual observations, and are compared with the anatomy of the osseous inner ear in monotremes and in primitive non-therian and therian mammals. Results of this study indicate that: (1) the cochlea of at least some multituberculates retained a lagena, previously known only in monotremes among mammals; (2) an enlarged vestibule evolved in several lineages of multituberculates independently, and hence is not a synapomorphy of the order; (3) the cochlear canal lacks osseous laminae in support of the short, wide basilar membrane, which was probably inefficient in responding to high-frequency airborne vibrations; and (4) consequently, bone-conducted hearing in some multituberculate species may have been important in interpretation of their surroundings. Comparisons with the inner ear of monotremes and primitive therians indicate that curvature of the cochlea and cribriform plates for passage of vestibulocochlear nerve branches through the petrosal are unlikely homologues between monotremes and therians. From non-therian to therian mammals, there is a distinct morphological gap in the inner ear transition, characterized by acquisition of a number of neomorphs in the therian inner ear; an intermediate stage has yet to be discovered.     

Mammalian Faunal Succession in the Cretaceous of the Kyzylkum Desert

Both metatherians and eutherians are known from the Early Cretaceous (Barremian, 125 mya; million years ago) of China, while eutherian-dominated mammalian faunas appeared in Asia at least by the earliest Late Cretaceous (Cenomanian, 95 mya). The approximately 99–93 my old (Cenomanian) Sheikhdzheili l.f. from western Uzbekistan is a small sample of only eutherians, including three zhelestids and a possible zalambdalestoid. The much better-known 90 my old (Turonian) Bissekty l.f. at Dzharakuduk in the central Uzbekistan includes 15 named and unnamed species, based on ongoing analyses. Of these, 12 are eutherians represented by at least the three groups—asioryctitheres, zalambdalestids, and zhelestids—plus an eutherian of uncertain position—Paranyctoides. Zalambdalestids and zhelestids have been argued to be related to the origin of the placental gliriforms (Euarchontoglires) and ferungulates (Laurasiatheria), respectively. Although there are four previously recognized metatherians, we believe three are referable to the deltatheroid Sulestes karakshi and the fourth, Sailestes quadrans, may belong to Paranyctoides. There is one multituberculate and one symmetrodont in the Bissekty l.f. While comparably aged (Turonian) localities in North America have somewhat similar non-therians, they have more metatherians and no eutherians. The next younger localities (early Campanian, ∼80 mya) in North America have both a zhelestid and Paranyctoides, suggesting dispersal of eutherians from Asia. At Dzharakuduk, the approximately 85 my old (late Turonian/Coniacian) Aitym l.f. is much less well known than the Bissekty l.f., but yields nearly identical taxa, with two non-therians, one metatherian, and six eutherians.     

Brain gangliosides in monotremes, marsupials and placentals: phylogenetic and thermoregulatory aspects

The concentration and composition of brain gangliosides of 17 mammalian species belonging to the subclasses of Prototheria (monotremes), Metatheria (marsupials), and Eutheria (placentals) were investigated. The mean concentration of brain gangliosides ranges from 525 to 610 micrograms NeuAc/g wet wt in monotremes, 445-900 micrograms in marsupials and from 630 to 1130 micrograms in the placentals. In the phylogenetic series of mammals, a decrease in the complexity of brain ganglioside composition becomes obvious: a drastic reduction in the number of individual ganglioside fractions particularly those of the c-pathway of biosynthesis, took place from the level of monotremes to that of the marsupials and placentals. In monotremes, marsupials and "lower" placentals (insectivores) the percentage of alkali-labile gangliosides is relatively low (between traces and 5%), whereas in the higher evolved mammals it amounts to about 20% of all gangliosides. The ratio of the contents of the two major mammalian ganglioside fractions GD1a and GT1b is generally in the range of 1.0 and even higher; in the heterothermic platypus from the monotremes and in hibernators among the placental mammals, however, it is much lower (about 0.8). These data support the hypothesis that the brain ganglioside composition not only depends on the phylogenetic level of nervous organization (cephalization) but is additionally correlated with the state of thermal adaptation.     

Relationships of the dog-like marsupials, deltatheroidans and early tribosphenic mammals

A cladistic analysis of the major groups of early metatherian mammals shows that the Deltatheroida and South American dog-like Borhyaenoidea are closely related and are placed in a new supercohort Deltatheralia. while all other metatherians are placed in the supercohort Marsupialia. A reassessment of Early Cretaceous mammals with tribosphenic molars shows that metatherians and eutherians apparently evolved independently from a peramuran-like ancestor, and an entoconid. distinctly basined talonid and large protocone were acquired separately in each group. Consequently. the Tribosphenida ( sensu McKenna) is apparently paraphyletic. Aegialodontia are regarded as structurally ancestral to Metatheria but not Eutheria. The known fossil record supports the view that differentation of the stocks which gave rise to metatherians and eutherians apparently occurred in Late Jurassic or Early Cretaceous time.     

The phylogenetic relationships of argyrolagid marsupials

New material of the oldest known argyrolagid marsupial Proargyrolagus bolivianus from the late Oligocene of Bolivia is described. The new specimen preserves previously unknown aspects of the anterior dentition that solve the long-standing homology problem concerning the identity (i2) of the procumbent lower incisors in argyrolagids. This new anatomical information is incorporated into a morphology-based phylogenetic analysis of all extant marsupial families and Argyrolagidae, with the aim of testing the monophyly of Paucituberculata and evaluating the relationships among extant marsupial families. Eleven features support the monophyly of Paucituberculata, the following three unique among Marsupialia: small size of the paraconid, procumbent second lower incisor, and supraoccipital without distinct lambdoid crest resulting in globular form of braincase. Paucituberculata is the sister group of an Australian clade of marsupials that includes Dromiciops, but these results are not robust, as shown by sensitivity analyses. The foramen ovale surrounded completely by the alisphenoid supports the association of Dromiciops with diprotodontians.     

Expression and function of pannexins in the inner ear and hearing

Pannexin (Panx) is a gene family encoding gap junction proteins in vertebrates. So far, three isoforms (Panx1, 2 and 3) have been identified. All of three Panx isoforms express in the cochlea with distinct expression patterns. Panx1 expresses in the cochlea extensively, including the spiral limbus, the organ of Corti, and the cochlear lateral wall, whereas Panx2 and Panx3 restrict to the basal cells of the stria vascularis in the lateral wall and the cochlear bony structure, respectively. However, there is no pannexin expression in auditory sensory hair cells. Recent studies demonstrated that like connexin gap junction gene, Panx1 deficiency causes hearing loss. Panx1 channels dominate ATP release in the cochlea. Deletion of Panx1 abolishes ATP release in the cochlea and reduces endocochlear potential (EP), auditory receptor current/potential, and active cochlear amplification. Panx1 deficiency in the cochlea also activates caspase-3 cell apoptotic pathway leading to cell degeneration. These new findings suggest that pannexins have a critical role in the cochlea in regard to hearing. However, detailed information about pannexin function in the cochlea and Panx mutation induced hearing loss still remain largely undetermined. Further studies are required.     

Mouse models to study inner ear development and hereditary hearing loss

Hereditary sensorineural hearing loss, derived from inner ear defects, is the most common hereditary disability with a prevalence of 1 in 1,000 children, although it can be present in up to 15% of births in isolated communities. The mouse serves as an ideal animal model to identify new deafness-related genes and to study their roles in vivo. This review describes mouse models for genes that have been linked with hearing impairment (HI) in humans. Mutations in several groups of genes have been linked with HI in both mice and humans. Mutant mice have been instrumental in elucidating the function and mechanisms of the inner ear. For example, the roles of collagens and tectorins in the tectorial membrane, as well as the necessity of intact links between the hair cell projections, stereocilia and kinocilia, have been discovered in mice. Accurate endolymph composition and the proteins which participate in its production were found to be crucial for inner ear function, as well as several motor proteins such as prestin and myosins. Two systematic projects, KOMP and EUCOMM, which are currently being carried out to create knock-out and conditional mutants for every gene in the mouse genome, promise that many additional deafness-related genes will be identified in the next years, providing models for all forms of human deafness.     

Ontogenetic change of bone microstructures and its ethological implication in Champsosaurus (Diapsida,Choristodera)

Compact cortex in a Champsosaurus (Diapsida, Choristodera) femur is ontogenetically replaced with extensively developed cancellous bone. This histological shift, together with retention of calcified cartilage to late ontogenetic stage, was previously considered to show that adult champsosaurs were more adapted to aquatic environments than juveniles. However, the new histological examination reveals the nearly amedullar condition of a juvenile femur consisting of thick periosteal cortex and less cancellous bone tissue and the amedullar but more porous condition of adult femora. This likely demonstrates that the femoral inner structure of the juvenile is denser than those of the adults, and therefore, juveniles were more aquatic. It is suggested that morphological variations between two sympatric species of Champsosaurus reflect sexual dimorphism in a single species and limb bones with more robust morphology, showing better terrestrial adaptation for nesting on land, belong to females. The similarity of gross limb bone morphology between juveniles and inferred adult males indicates aquatic habitats for juveniles, coincident with the new interpretation of bone microstructures. No differences are, however, recognised in femoral microstructure between inferred sexes in adults. The possibly lowered density of femur in adults is considered as an adaptation to increase the mobility in water.     

Water channel proteins in the inner ear and their link to hearing impairment and deafness

The inner ear is a fluid-filled sensory organ that transforms mechanical stimuli into the senses of hearing and balance. These neurosensory functions depend on the strict regulation of the volume of the two major extracellular fluid domains of the inner ear, the perilymph and the endolymph. Water channel proteins, or aquaporins (AQPs), are molecular candidates for the precise regulation of perilymph and endolymph volume. Eight AQP subtypes have been identified in the membranous labyrinth of the inner ear. Similar AQP subtypes are also expressed in the kidney, where they function in whole-body water regulation. In the inner ear, AQP subtypes are ubiquitously expressed in distinct cell types, suggesting that AQPs have an important physiological role in the volume regulation of perilymph and endolymph. Furthermore, disturbed AQP function may have pathophysiological relevance and may turn AQPs into therapeutic targets for the treatment of inner ear diseases. In this review, we present the currently available knowledge regarding the expression and function of AQPs in the inner ear. We give special consideration to AQP subtypes AQP2, AQP4 and AQP5, which have been studied most extensively. The potential functions of AQP2 and AQP5 in the resorption and secretion of endolymph and of AQP4 in the equilibration of cell volume are described. The pathophysiological implications of these AQP subtypes for inner ear diseases, that appear to involve impaired fluid regulation, such as Menière's disease and Sj?gren's syndrome, are discussed.     

Emx2 and early hair cell development in the mouse inner ear

Emx2 is a homeodomain protein that plays a critical role in inner ear development. Homozygous null mice die at birth with a range of defects in the CNS, renal system and skeleton. The cochlea is shorter than normal with about 60% fewer auditory hair cells. It appears to lack outer hair cells and some supporting cells are either absent or fail to differentiate. Many of the hair cells differentiate in pairs and although their hair bundles develop normally their planar cell polarity is compromised. Measurements of cell polarity suggest that classic planar cell polarity molecules are not directly influenced by Emx2 and that polarity is compromised by developmental defects in the sensory precursor population or by defects in epithelial cues for cell alignment. Planar cell polarity is normal in the vestibular epithelia although polarity reversal across the striola is absent in both the utricular and saccular maculae. In contrast, cochlear hair cell polarity is disorganized. The expression domain for Bmp4 is expanded and Fgfr1 and Prox1 are expressed in fewer cells in the cochlear sensory epithelium of Emx2 null mice. We conclude that Emx2 regulates early developmental events that balance cell proliferation and differentiation in the sensory precursor population.     

Sexual dimorphism in Champsosaurus (Diapsida, Choristodera)

Two nearly complete specimens of Champsosaurus (Diapsida, Choristodera) with distinctive morphologies, from the Tullock Formation (Early Paleocene) of northeastern Montana, USA, were described as different species. The limb bones of C. ambulator are more robust than those of C. laramiensis, indicating that C. ambulator was more adapted for walking than C. laramiensis. The phylogenetic significance of these limb bone morphologies, however, appears questionable because similar dimorphic variations occur in a closely related genus and champsosaurs from other geologic ages and locations. Female champsosaurs may have been better adapted to a terrestrial life than males due to nesting behavior on land, resulting in variable limb bone morphologies between sexes. The observed morphologic variations are, hence, hypothesized to reflect sexual dimorphism rather than sympatry of species. The C. ambulator-shaped humeri and femora, demonstrating a terrestrial adaptation, are suggested to belong to females and C. laramiensis-shaped limb bones to males. No significant variations of humeral and femoral morphologies occur in small champsosaur specimens, suggesting an aquatic niche for juveniles like adult males.     

Inositol phospho-oligosaccharide stimulates cell proliferation in the early developing inner ear

The ability of an inositol phospho-oligosaccharide (POS) to mimic the mitogenic effects of nerve growth factor (NGF) and insulin on the early development of the inner ear was investigated. POS (10 microM) stimulated the incorporation of [3H]thymidine into the cochleovestibular ganglion by 3.9-fold. NGF (50 ng/ml) stimulation was 4.7-fold. POS and NGF showed no additivity. Cells induced to proliferate by POS overlapped with those expressing NGF receptors. POS, like insulin, potentiated the mitogenic effect of bombesin on the otic vesicle epithelium. DNA synthesis in the presence of bombesin (100 nM) plus POS (10 microM) was increased by 6.4-fold. POS stimulation was not additive with insulin. The results suggest that POS may play a role in growth factor regulation of cell proliferation during embryonic development.     

Functional-adaptive analysis of the hindlimb anatomy of extant marsupials and the paleobiology of the Paleocene marsupials Mayulestes ferox and Pucadelphys andinus

This article analyzes the adaptations of the hindlimb of two Early Paleocene marsupials, Mayulestes ferox and Pucadelphys andinus. This analysis is based on detailed comparisons with various extant marsupials, both South American and Australian. In the case of the South American opossums, original myological data were collected and osteological-myological associations were related to their locomotor behavior. The use of Australian genera helped to improve the appraisal of the locomotory habits of the fossil taxa. Several features are indicative of the ability of Mayulestes to climb or walk on uneven surfaces (e.g., very mobile hip joint, astragalocalcaneal joint pattern), and some other features emphasize a relative agility (e.g., strongly everted iliac blades, morphology of the distal epiphysis of the femur, medially stabilized cruroastragalar joint). Pucadelphys exhibits a hindlimb relatively similar morphologically to that of Mayulestes, but with features indicating slightly increased agility and a terrestrial component that is more emphasized than in Mayulestes. The Tiupampa fossils were therefore more agile than most living didelphids and resembled the condition observed in living dasyurids more. These conclusions complement a previous study performed on the forelimb of these fossils.     

Tissue-specific requirements for FGF8 during early inner ear development

Several members of the FGF gene family have been shown to intervene from various tissue sources to direct otic placode induction and otic vesicle formation. In this study we define the roles of FGF8, found in different expression domains during this process, in mice and chickens. By conditional inactivation of Fgf8 in distinct tissue compartments we demonstrate that Fgf8 is required in the mesoderm and endoderm during early inner ear development. In the chicken embryo, overexpression of Fgf8 from various tissue sources during otic specification leads to a loss of otic tissue. In contrast ectopic overexpression of Fgf10, a major player during murine otic induction, does not influence otic vesicle formation in chicken embryos but results in the formation of ectopic structures with a non-otic character. This study underlines the crucial role of a defined Fgf8 expression pattern controlling inner ear formation in vertebrates.     

Functional-adaptive anatomy of the axial skeleton of some extant marsupials and the paleobiology of the paleocene marsupials Mayulestes ferox and Pucadelphys andinus

In this study, the axial skeletons of two Early Paleocene marsupials, Mayulestes ferox and Pucadelphys andinus, were analyzed functionally and compared to that of six South American and three Australian species of extant marsupials. In the case of the South American opossums, myological data of the epaxial musculature were collected and analyzed and osteological-myological associations were related to locomotor behavior. Various features of the vertebral column that relate to diet or to locomotor or postural patterns were pointed out. These features include: the craniocaudal development of the neural process of the axis; the position of the anticlinal vertebra; the morphology of the neural processes of the thoracolumbar vertebrae (orientation, length, and craniocaudal width); the length, orientation, and curvature of the transverse processes of the lumbar vertebrae; and the length and robustness of the caudal vertebrae. In both fossil forms the vertebral column is mobile and allows a great range of flexion and extension of the spine, more so than in most of the living didelphids. It is emphasized here that the analysis of the axial skeleton complements and improves the conclusions provided by the forelimb and hindlimb analyses. It is proposed that Mayulestes and Pucadelphys represent an ancestral morphotype suggesting that the generalized type of locomotion of Paleocene marsupials was partly terrestrial with some climbing ability.     

Dispersal,vicariance, and the Late Cretaceous to early tertiary land mammal biogeography from South America to Australia

A review of paleontological, phyletic, geophysical, and climatic evidence leads to a new scenario of land mammal dispersal among South America, Antarctica, and Australia in the Late Cretaceous to early Tertiary epochs. New fossil land vertebrate material has been recovered from all three continents in recent years. As regards Gondwana, the present evidence suggests that monotreme mammals and ratite birds are of Mesozoic origin, based on both geochronological and phyletic grounds. The occurrence of monotremes in the early Paleocene (ca. 62 Ma) faunas of Patagonia and of ratites in late Eocene (ca. 41-37 m.y.) faunas of Seymour Island (Antarctic Peninsula) probably is an artifact of a much older and widespread Gondwana distribution prior to the Late Cretaceous Epoch. Except for South American microbiotheres being australidelphians, marsupial faunas of South America and Australia still are fundamentally disjunct. New material from Seymour Island (Microbiotheriidae) indicates the presence there of a derived taxon that resides in a group that is the sister taxon of most Australian marsupials. There is no compelling evidence that dispersal between Antarctica and Australia was as recent as ca. 41 Ma or later. In fact, the derived marsupial and placental land mammal fauna of Seymour Island shows its greatest affinity with Patagonian forms of Casamayoran age (ca. 51–54 m.y.). This suggests an earlier dispersal of more plesiomorphic marsupials from Patagonia to Australia via Antarctica, and vicariant disjunction subsequently. This is consistent with geophysical evidence that the South Tasman Rise was submerged by 64 Ma and with geological evidence that a shallow water marine barrier was present from then onward. The scenario above is consistent with molecular evidence suggesting that australidelphian bandicoots, dasyurids, and diprotodontians were distinct and present in Australia at least as early as the 63-Ma-old australidelphian microbiotheres and the ancient but not basal australidelphian,Andinodelphys, in the Tiupampa Fauna of Bolivia. Land mammal dispersal to Australia typically has been considered to be at a low level of probability (e.g., by sweepstakes dispersal). This study suggests that the marsupial colonizers of Australia included already recognizable members of the Peramelina, Dasyuromorphia, and Diprotodontia, at least, and entered via a filter route rather than by a sweepstakes dispersal.To whom correspondence should be addressed.