Ontogenetic and phylogenetic transformations of the ear ossicles in marsupial mammals

This study is based on the examination of histological sections of specimens of different ages and of adult ossicles from macerated skulls representing a wide range of taxa and aims at addressing several issues concerning the evolution of the ear ossicles in marsupials. Three-dimensional reconstructions of the ear ossicles based on histological series were done for one or more stages of Monodelphis domestica, Caluromys philander, Sminthopsis virginiae, Trichosurus vulpecula, and Macropus rufogriseus. Several common trends were found. Portions of the ossicles that are phylogenetically older develop earlier than portions representing more recent evolutionary inventions (manubrium of the malleus, crus longum of the incus). The onset of endochondral ossification in the taxa in which this was examined followed the sequence; first malleus, then incus, and finally stapes. In M. domestica and C. philander at birth the yet precartilaginous ossicles form a supportive strut between the lower jaw and the braincase. The cartilage of Paauw develops relatively late in comparison with the ear ossicles and in close association to the tendon of the stapedial muscle. A feeble artery traverses the stapedial foramen of the stapes in the youngest stages of M. domestica, C. philander, and Sminthopsis virginiae examined. Presence of a large stapedial foramen is reconstructed in the groundplan of the Didelphidae and of Marsupialia. The stapedial foramen is absent in all adult caenolestids, dasyurids, Myrmecobius, Notoryctes, peramelids, vombatids, and phascolarctids. Pouch young of Perameles sp. and Dasyurus viverrinus show a bicrurate stapes with a sizeable stapedial foramen. Some didelphids examined to date show a double insertion of the Tensor tympani muscle. Some differences exist between M. domestica and C. philander in adult ossicle form, including the relative length of the incudal crus breve and of the stapes. Several differences exist between the malleus of didelphids and that of some phalangeriforms, the latter showing a short neck, absence of the lamina, and a ventrally directed manubrium. Hearing starts in M. domestica at an age in which the external auditory meatus has not yet fully developed, the ossicles are not fully ossified, and the middle ear space is partially filled with loose mesenchyme. The ontogenetic changes in hearing abilities in M. domestica between postnatal days 30 and 40 may be at least partially related to changes in middle ear structures.     

Thermogenic capabilities of the opossum Monodelphis domestica when warm and cold acclimated: similarities between American and Australian marsupials

1. Monodelphis domestica is a small marsupial mammal from South America. Its thermogenic abilities in the cold were determined when the opossums were both warm (WA) and cold (CA) acclimated. Maximum heat production of M. domestica was obtained at low temperatures in helium-oxygen. 2. Basal metabolic rate (BMR) in the WA animals was 3.2 W/kg and mean body temperature was 32.6 degrees C at 30 degrees C. These values were lower than those generally reported for marsupials. Nevertheless, these M. domestica showed considerable metabolic expansibility in response to cold. Sustained (summit) metabolism was 8-9 times BMR, while peak metabolism was 11-13 times BMR. These maximum values were equal to, or above, those expected in small placentals. 3. Cold acclimation altered the thermal responses of M. domestica, particularly in warm TaS. However, summit metabolism was not significantly increased; nor did M. domestica show a significant thermogenic response to noradrenaline, which in many small placentals elicits non-shivering thermogenesis. The thermoregulatory responses of this American marsupial were, in most aspects, similar to those of Australian marsupials. This suggests that the considerable thermoregulatory abilities of marsupials are of some antiquity.     

Ontogenetic and phylogenetic transformations of the vomeronasal complex and nasal floor elements in marsupial mammals

Histological sections and three-dimensional reconstructions of section-series were used to document the anatomy of the vomeronasal complex and other aspects of the ethmoidal region in representatives of 13 families and six orders of marsupial mammals, including for the first time Microbiotheria. The changes during growth of several features were examined in ontogenetic series. Marsupials are very conservative in comparison with eutherians regarding the vomeronasal complex. All have a vomeronasal organ and a nasopalatine duct, have no nasopalatine duct cartilage, have no (or just an incipient) palatine cartilage, and the overall construction of the nasal floor is uniform across species. Most features examined show a high degree of homoplasy (e.g. presence of glandular ridges, isolated dorsal process of the paraseptal cartilage), and their systematic value is confined to low taxonomic levels. Significant ontogenetic changes occur in features usually discussed in the systematic/taxonomic literature. Amongst the didelphids examined, Caluromys philander shows several autapomorphies. It is hypothesized that the opening of the VNO into the upper end of the nasopalatine duct was present in the marsupial groundplan. Most marsupials have a large and horizontal anterior transverse lamina, the plesiomorphic condition, which becomes oblique in diprotodontians. Some features are autapomorphies of well-supported monophyletic groups of marsupials, e.g. the conspicuous internasal communication of perameliformes and the 'tube-like' or ring-shaped paraseptal cartilage of vombatiformes. An outer bar joining the middle (and not the dorsal-most portion) of the paraseptal cartilage characterizes Australasian marsupials and Dromiciops, with the exclusion of perameliformes, and evolved independently in Caluromys philander.     

OSSIFICATION HETEROCHRONY IN THE THERIAN POSTCRANIAL SKELETON AND THE MARSUPIAL–PLACENTAL DICHOTOMY

Postcranial ossification sequences in 24 therian mammals and three outgroup taxa were obtained using clear staining and computed tomography to test the hypothesis that the marsupial forelimb is developmentally accelerated, and to assess patterns of therian postcranial ossification. Sequence rank variation of individual bones, phylogenetic analysis, and algorithm-based heterochrony optimization using event pairs were employed. Phylogenetic analysis only recovers Marsupialia, Australidelphia, and Eulipotyphla. Little heterochrony is found within marsupials and placentals. However, heterochrony was observed between marsupials and placentals, relating to late ossification in hind limb long bones and early ossification of the anterior axial skeleton. Also, ossification rank position of marsupial forelimb and shoulder girdle elements is more conservative than that of placentals; in placentals the hind limb area is more conservative. The differing ossification patterns in marsupials can be explained with a combination of muscular strain and energy allocation constraints, both resulting from the requirement of active movement of the altricial marsupial neonates toward the teat. Peramelemorphs, which are comparatively passive at birth and include species with relatively derived forelimbs, differ little from other marsupials in ossification sequence. This suggests that ossification heterochrony in marsupials is not directly related to diversity constraints on the marsupial forelimb and shoulder girdle.     

Mammary glands in male marsupials: I. Primordia in neonatal opossums Didelphis virginiana and Monodelphis domestica

Neonates of the American didelphid marsupials Didelphis virginiana and Monodelphis domestica were sexed by karyotype and histologically examined on the day of birth. Mammary anlagen were found in both sexes of both species, but the neonatal males had less than one-third of the full female complement of mammary glands. Male neonates of both species also had paired scrotal bulges anterior to the genital tubercle but these were never present in females, once again raising the question of whether the pouch and scrotum are homologous structures. Mammary anlagen are not found in male neonates of the Australian marsupial species so far studied, which suggests a dichotomy in the control of some aspects of sexual differentiation in the two marsupial lineages.     

Ossification heterochrony in the therian postcranial skeleton and the marsupial-placental dichotomy.

Postcranial ossification sequences in 24 therian mammals and three outgroup taxa were obtained using clear staining and computed tomography to test the hypothesis that the marsupial forelimb is developmentally accelerated, and to assess patterns of therian postcranial ossification. Sequence rank variation of individual bones, phylogenetic analysis, and algorithm-based heterochrony optimization using event pairs were employed. Phylogenetic analysis only recovers Marsupialia, Australidelphia, and Eulipotyphla. Little heterochrony is found within marsupials and placentals. However, heterochrony was observed between marsupials and placentals, relating to late ossification in hind limb long bones and early ossification of the anterior axial skeleton. Also, ossification rank position of marsupial forelimb and shoulder girdle elements is more conservative than that of placentals; in placentals the hind limb area is more conservative. The differing ossification patterns in marsupials can be explained with a combination of muscular strain and energy allocation constraints, both resulting from the requirement of active movement of the altricial marsupial neonates toward the teat. Peramelemorphs, which are comparatively passive at birth and include species with relatively derived forelimbs, differ little from other marsupials in ossification sequence. This suggests that ossification heterochrony in marsupials is not directly related to diversity constraints on the marsupial forelimb and shoulder girdle.     

Unusually similar patterns of antibody V segment diversity in distantly related marsupials

A pattern of coevolution between the V gene segments of Ig H and L chains has been noted previously by several investigators. Species with restricted germline V(H) diversity tend to have limited germline V(L) diversity, whereas species with high levels of germline V(H) diversity have more diverse V(L) gene segments. Evidence for a limited pool of V(H) but diverse V(L) gene segments in a South American opossum, Monodelphis domestica, is consistent with this marsupial being an exception to the pattern. To determine whether M. domestica is unique or the norm for marsupials, the V(H) and V(L) of an Australian possum, Trichosurus vulpecula, were characterized. The Ig repertoire in T. vulpecula is also derived from a restricted V(H) pool but a diverse V(L) pool. The V(L) gene segments of T. vulpecula are highly complex and contain lineages that predate the separation of marsupials and placental mammals. Thus, neither marsupial follows a pattern of coevolution of V(H) and V(L) gene segments observed in other mammals. Rather, marsupial V(H) and V(L) complexity appears to be evolving divergently, retaining diversity in V(L) perhaps to compensate for limited V(H) diversity. There is a high degree of similarity between the V(H) and V(L) in M. domestica and T. vulpecula, with the majority of V(L) families being shared between both species. All marsupial V(H) sequences isolated so far form a common clade of closely related sequences, and in contrast to the V(L) genes, the V(H) likely underwent a major loss of diversity early in marsupial evolution.     

Autopodial development in the sea turtles Chelonia mydas and Caretta caretta

The manus and pes were studied using whole-mount and histological preparations of ontogenetic series of Chelonia mydas and Caretta caretta. Patterns of connectivity and sequences of chondrification events are similar to those reported for other turtle species, with respect to both the primary axis and the digital arch. There is no evidence of anterior condensations in the region distal to the radius and the tibia, supporting the hypothesis that the radiale and tibiale are absent in turtles. The three middle metacarpals are the first elements to start ossification in the manus of C. mydas, while ossification has not started in the pes. In the hatchling of C. mydas, most carpals have started ossification, whereas tarsals are mostly still cartilaginous. In C. caretta, the first carpals to ossify are the ulnare and intermedium, followed by the pisiform. Among metatarsals, the fifth hooked metatarsal is the last one to start ossification. The fibulare and intermedium fuse early in chondrogenesis, later becoming the astragalocalcaneum. Ossification in the carpals of C. caretta starts while tarsals are still cartilaginous. The derived autopodial proportions in each autopodium of adults are laid out at the condensation stage, and features that were present in basal turtles are absent at all stages examined (developmental penetrance). In contrast to this, conservatism is expressed in the presence of similar patterns of connectivity during early chondrogenesis, and in the development of overall proportions of the manus versus pes. As in adult anatomy, the development of the autopodium of marine turtles is a mosaic of derived and plesiomorphic features.     

Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart)

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.     

Oxygen consumption and thermoregulatory responses in three species of South American marsupials

Oxygen consumption (VO(2)), body temperature (T(b)) and wet thermal conductance (C(wet)), under resting conditions, exposure to low ambient temperature (T(a)) and during sustained exercise (treadmill running) were measured in three phylogenetic related (same family; Didelphidae) South American marsupials possessing similar body masses: Caluromys philander (arboreal/fruit and insect eating), Philander opossum (terrestrial and arboreal/omnivore), and Metachirus nudicaudatus (terrestrial/omnivore). Our measurements of VO(2) and C(wet) under resting conditions agree with those previously reported for other marsupials. We expected that C. philander would show a lower maximal sustained VO(2), compared to the other two species, based on its more reduced skeletal muscle mass. However, the values obtained for C. philander were not statistically different (ANOVA) from those obtained for the other two species. When exposed to low ambient temperature (12 degrees C), differences among the three species were detected, i.e., M. nudicaudatus did not survive, while the other two species were able to reduce their T(b) under such conditions. C. philander gradually decreases its T(b) when cold exposed, and P. opossum shows a more pronounced T(b) drop only when exposure to low ambient temperatures occurs for a more prolonged period of time.     

Milk composition in an American marsupial (Monodelphis domestica).

1. Twenty one sequential milk samples from an American marsupial (Monodelphis domestica) were analyzed. 2. The solids fraction was relatively low initially (10% w/w) and then increased to about 30% at mid-lactation where it remained until lactation ceased. 3. During the first 50 days of lactation the relative proportions of protein, fat and carbohydrate were 36, 28 and 34%, respectively, of the solids fraction. 4. Sodium and magnesium concentrations were constant; however levels of potassium and magnesium varied substantially. 5. The milk of M. domestica exhibits a similar pattern of quantitative and qualitative changes to that seen in Australian marsupials.     

Physical mapping of the IGF2 locus in the South American opossum Monodelphis domestica

The South American opossum Monodelphis domestica has been a model organism for marsupials for many years and has recently been the subject of a large-scale genome sequencing effort that will provide the foundation for comparative studies of gene function and regulation. Genomic imprinting is one mechanism of gene regulation that has received increasing attention due to the impact of imprinting defects on development and disease. We have mapped the imprinted insulin-like growth factor II (IGF2) gene of M. domestica as a first step in understanding the regulatory mechanisms involved in genomic imprinting in this marsupial.     

Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.     

Ontogenesis of the scapula in marsupial mammals, with special emphasis on perinatal stages of didelphids and remarks on the origin of the therian scapula

The development of the scapula was studied in embryonic and postnatal specimens of Monodelphis domestica and perinatal specimens of Philander opossum, Caluromys philander, and Sminthopsis virginiae using histological sections and 3D reconstructions. Additionally, macerated skeletons of postnatal M. domestica were examined. This study focused on the detachment of the scapulocoracoid from the sternum and on the acquisition of a supraspinous fossa, a supraspinatus muscle, and a scapular spine, all these events associated with the origin of the therian shoulder girdle. In none of the specimens is there a continuity of the cartilaginous scapulocoracoid with the sternum, even though the structures are in close proximity, especially in S. virginiae. At birth, the first rib laterally presents a pronounced boss that probably contacts the humerus during certain movements. Only the acromial portion of the scapular spine, which originates from the anterior margin of the scapular blade, is preformed in cartilage. The other portion is formed by appositional bone ("Zuwachsknochen"), which expands from the perichondral ossification of the scapula into an intermuscular aponeurosis between the supra- and infraspinous muscles. This intermuscular aponeurosis inserts more or less in the middle of the lateral surface of the developing scapula. Thus, the floor of the supraspinous fossa is present from the beginning of scapular development, simultaneously with the infraspinous fossa. The homology of the therian spine with the anterior border of the sauropsid and monotreme scapula is questioned. We consider the dorsal portion (as opposed to the ventral or acromial portion) of the scapular spine a neomorphic structure of therian mammals.     

The mole's thumb -- evolution of the hand skeleton in talpids (Mammalia)

Osteological specimens representing 15 out of the 16 currently recognized talpid genera were examined and scored for seven discrete morphological characters of the hand. The phylogenetic distribution of these characters was studied in the context of alternative hypotheses of talpid relationships, using three species of shrews and a hedgehog as outgroups. All talpids show a similar number and arrangement of carpal bones. The most obvious differences concern the presence of additional sesamoid bones, the relative size of the os falciforme when present, and the degree of fusion of the scaphoid and lunate in the proximal carpal row. Marked differences in the relative length and proportions of the metacarpals also exist. The development of the carpals in Talpa europaea was studied through examination of histological sections of the hand of an embryo and a neonate. Whereas carpal anatomy in the neonate mirrors the arrangement and proportions of the adult, in the embryo the scaphoid and lunate are still separate, there are no signs of the os falciforme, and the size proportions of metacarpals to carpals are obviously different to those of the adult. A prehallux or tibial sesamoid, serial homologue to the os falciforme or prepollex (a radial sesamoid), does not have an obvious functional role, and its presence might be the result of a common epigenetic control in the hand and the foot resulting in a non-adaptive structure in the latter.     

Monotreme ossification sequences and the riddle of mammalian skeletal development

The developmental differences between marsupials, placentals, and monotremes are thought to be reflected in differing patterns of postcranial development and diversity. However, developmental polarities remain obscured by the rarity of monotreme data. Here, I present the first postcranial ossification sequences of the monotreme echidna and platypus, and compare these with published data from other mammals and amniotes. Strikingly, monotreme stylopodia (humerus, femur) ossify after the more distal zeugopodia (radius/ulna, tibia/fibula), resembling only the European mole among all amniotes assessed. European moles also share extreme humeral adaptations to rotation digging and/or swimming with monotremes, suggesting a causal relationship between adaptation and ossification heterochrony. Late femoral ossification with respect to tibia/fibula in monotremes and moles points toward developmental integration of the serially homologous fore- and hindlimb bones. Monotreme cervical ribs and coracoids ossify later than in most amniotes but are similarly timed as homologous ossifications in therians, where they are lost as independent bones. This loss may have been facilitated by a developmental delay of coracoids and cervical ribs at the base of mammals. The monotreme sequence, although highly derived, resembles placentals more than marsupials. Thus, marsupial postcranial development, and potentially related diversity constraints, may not represent the ancestral mammalian condition.     

Shape, variance and integration during craniogenesis: contrasting marsupial and placental mammals

Studies of morphological integration can provide insight into developmental patterns, even in extinct taxa known only from skeletal remains, thus making them an important tool for studies of evolutionary development. However, interpreting patterns of integration and assessing their significance for organismal evolution requires detailed understanding of the developmental interactions that shape integration and how those interactions change through ontogeny. Thus far, relatively little comparative data have been produced for this important topic, and the data that do exist are overwhelmingly from humans and their close relatives or from laboratory models such as mice. Here, we compare data on shape, variance and integration through postnatal ontogeny for a placental mammal, the least shrew, Cryptotis parva, and a marsupial mammal, the gray short-tailed opossum, Monodelphis domestica. Cranial variance decreased dramatically from early to late ontogeny in Cryptotis, but remained stable through ontogeny in Monodelphis, potentially reflecting functional constraints related to the short gestation and early ossification of oral bones in marsupials. Both Cryptotis and Monodelphis showed significant changes in cranial integration through ontogeny, with a mixture of increased, decreased and stable levels of integration in different cranial regions. Of particular note is that Monodelphis showed an unambiguous decrease in integration of the oral region through ontogeny, potentially relating to their early ossification. Selection at different stages of development may have markedly different effects if patterns of integration change substantially through ontogeny. Our results suggest that high integration of the oral region combined with functional constraints for suckling during early postnatal ontogeny may drive the stagnant variance observed in Monodelphis and potentially other marsupials.     

Carpal bone kinematics and ligament lengthening studied for the full range of joint movement

Present data on carpal kinematics and carpal ligament behaviour are limited to flexion and deviation movements of the hand. These motions do not represent all the wrist-joint motions which are important for the activities of daily living. The goal of this project was to obtain insight into carpal kinematics and carpal ligament behaviour during motions of the hand covering the full range of motion of the wrist joint.

The carpals and the ligaments of four wrist-joint specimens were provided with radiopaque markers. These joints were subjected to Röntgenstereophotogrammetric experimentation in a large number of hand positions to determine carpal positions and ligament lengths. The movements of the carpal bones were described by means of finite helical axes (FHA).

It was found that the movements of the carpals in the distal row closely resemble those of the hand. Conversely, the motions of the carpals of the proximal row appeared not to be directly proportional to the hand motions and exhibited clear out-of-plane movements. Furthermore, it could be shown that movements of the hand into the ulnodorsal quadrant of the full range of hand motion corresponds to larger helical rotations and translations for most of the carpals than when the hand was moved into any other quadrant. The maximal ligament length changes determined did not exceed the length changes reported for pure flexion and pure deviation movements of the hand.     

Constraints on the morphological evolution of marsupial shoulder girdles

Throughout their evolutionary histories, marsupial mammals have been taxonomically and morphologically less diverse than their sister taxa the placentals. Because of this, it has been proposed that the evolution of marsupials has been constrained by the functional requirements of their mode of reproduction. Marsupials give birth after short gestation times to immature neonates that immediately crawl, under the power of their precociously developed shoulder girdles, to the teat where they attach and complete their early development. Using a novel approach incorporating adult and embryological morphological data, this study is the first to both: (1) statistically support adult patterns of morphological divergence consistent with the constraint hypothesis, and (2) identify ontogenetic patterns of morphological change that demonstrate that the constraint was responsible, at least in part, for their formation. As predicted by the marsupial constraint, the shoulder girdles of adult marsupials are less diverse than those of adult placentals, and adult marsupial scapulae are less morphologically diverse than adult marsupial pelves. Furthermore, marsupials that complete an extensive crawl to the teat are restricted to a common pattern of ontogenetic scapular shape change, strongly supporting the hypothesis that the morphological development of the marsupial scapula has been limited evolutionarily by its obligate role in the crawl to the teat. Because this study establishes that ontogenetic and evolutionary morphological change is correlated within mammalian scapulae, it is probable that the marsupial constraint also restricted the morphological divergence of the scapula over evolutionary time by limiting ontogenetic change in the scapula. These findings, coupled with the importance of the shoulder girdle in mammalian locomotor specialization, support the conclusion that the low morphological diversity of marsupial forms over evolutionary time could be directly due to the constraint on marsupial morphological evolution caused by the functional requirements of the crawl to the teat.