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.     

The relationships of mammals

A cladistic analysis generates alternative hypotheses regarding both the origin and the interrelationships of mammals to those most widely accepted at the present time. It is proposed that the tritylodontids are more closely related to mammals than is Probainognathus ; that the non-therian mammals do not constitute a monophyletic group; and that the monotremes are related to the modern therians, the ear ossicles among other characters having evolved only once. The multituberculates may be related to the monotremes.
It is argued that the current views are variously based on an overemphasis of superficial dental similarities, misinterpretation of the structure of the mammalianbraincaseand too readyacceptance of parallel evolution amongstthe groups concerned. The hypotheses proposed here are apparently much more parsimonious.     

Review of the monotreme fossil record and comparison of palaeontological and molecular data

Monotremes have traditionally been considered a remnant group of mammals descended from archaic Mesozoic stock, surviving to the present day on the relatively isolated Australian continent. Challenges to this orthodoxy have been spurred by discoveries of 'advanced' Cretaceous monotremes (Steropodon galmani, Archer, M., et al., 1985. First Mesozoic mammal from Australia-an Early Cretaceous monotreme, Nature. 318, 363-366) as well as by results from molecular data linking monotremes to therian mammals (specifically to marsupials in some studies). This paper reviews the monotreme fossil record and briefly discusses significant new information from additional Cretaceous Australian material. Mesozoic monotremes (including S. galmani) were a diverse group as evidenced by new material from the Early Cretaceous of New South Wales and Victoria currently under study. Although most of these new finds are edentulous jaws (limiting dental comparisons and determination of dietary niches), a range of sizes and forms has been determined. Some of these Cretaceous jaws exhibit archaic features-in particular evidence for the presence of a splenial bone in S. galmani-not seen in therian mammals or in post-Mesozoic (Tertiary and Quaternary) monotreme taxa. Tertiary monotremes were either archaic ornithorhynchids (toothed platypuses in the genera Monotrematum and Obdurodon) or tachyglossids (large echidnas in the genera Megalibgwilia and Zaglossus). Quaternary ornithorhynchid material is referable to the sole living platypus species Ornithorhynchus anatinus. Quaternary echidnas, however, were moderately diverse and several forms are known (Megalibgwilia species; 'Zaglossus' hacketti; Zaglossus species and Tachyglossus aculeatus).     

Biophysical stimuli induced by passive movements compensate for lack of skeletal muscle during embryonic skeletogenesis

In genetically modified mice with abnormal skeletal muscle development, bones and joints are differentially affected by the lack of skeletal muscle. We hypothesise that unequal levels of biophysical stimuli in the developing humerus and femur can explain the differential effects on these rudiments when muscle is absent. We find that the expression patterns of four mechanosensitive genes important for endochondral ossification are differentially affected in muscleless limb mutants, with more extreme changes in the expression in the humerus than in the femur. Using finite element analysis, we show that the biophysical stimuli induced by muscle forces are similar in the humerus and femur, implying that the removal of muscle contractile forces should, in theory, affect the rudiments equally. However, simulations in which a displacement was applied to the end of the limb, such as could be caused in muscleless mice by movements of the mother or normal littermates, predicted higher biophysical stimuli in the femur than in the humerus. Stimuli induced by limb movement were much higher than those induced by the direct application of muscle forces, and we propose that movements of limbs caused by muscle contractions, rather than the direct application of muscle forces, provide the main mechanical stimuli for normal skeletal development. In muscleless mice, passive movement induces unequal biophysical stimuli in the humerus and femur, providing an explanation for the differential effects seen in these mice. The significance of these results is that forces originating external to the embryo may contribute to the initiation and progression of skeletal development when muscle development is abnormal.     

The septomaxilla of fossil and recent synapsids and the problem of the septomaxilla of monotremes and armadillos

The septomaxilla is a paired intramembranous ossification in the external nares that occurs in Lepidosauria among Recent Sauropsida and is purported to be present in Monotremata and Dasypodidae (armadillos) among Recent Mammalia. A review of neontological and palaeontological evidence regarding this element in mammals supports the following conclusions: (1) monotremes have a true septomaxilla resembling that known for non-mammalian therapsids and some Mesozoic mammals; (2) the element in dasypodids is a neomorph; it neither resembles the septomaxilla of other synapsids nor does it exhibit the same relationship to the developing nasal-floor cartilage as the septomaxilla of lepidosaurs and monotremes; (3) a septomaxilla is lacking in all Recent therians, and there is no evidence that this bone is fused to the premaxilla in Recent therians, as has been suggested by previous authors.     

Evolution of the Monotremes: Phylogenetic Relationship to Marsupials and Eutherians, and Estimation of Divergence Dates Based on α-Lactalbumin Amino Acid Sequences

The amino acid sequences of the -lactalbumins of the echidna, Tachyglossus aculeatus, and the platypus, Ornithorhynchus anatinus, were compared with each other and with those of 13 eutherian and 3 marsupial species. Phylogenetic parsimony analyses, in which selected mammalian lysozymes were used as outgroups, yielded trees whose consensus indicated that the two monotremes are sister taxa to marsupials and eutherians and that the latter two clades are each other's closest relatives. The data do not support the notion of a Marsupionta (monotreme–marsupial) clade. Pairwise comparison between the -lactalbumins yielded maximum-likelihood distances from which divergence dates were estimated on the basis of three calibration points. The distance data support the view that the echidna and platypus lineages diverged from their last common ancestor at least 50 to 57 Ma (million years ago) and that monotremes diverged from marsupials and eutherian mammals about 163 to 186 Ma.     

Testes weight, body weight and mating systems in marsupials and monotremes

Relationships between testes weight, body weight and mating systems were examined in 40 marsupial species and in the extant monotremes. Relationships between relative testes weight and mating systems in marsupials resemble those previously described for primates. Thus relative testes weights are greatest in those marsupials where females mate with multiple males during the fertile period, i.e. polyandrous species (e.g. Antechinus ftavipes, Isoodon obesulus, Perameles nasuta, Potorous tridactylus, Macropus eugenii and M. agilis) and smallest in monandrous forms (e.g. Petauroides volans and Petaurus breviceps ) where females usually mate with a single male. These findings are consistent with effects of sperm competition upon the evolution of relative testes sizes in marsupials. Where field studies on marsupial mating systems are lacking, we make predictions based upon examination of their relative testes weights. Tarsipes rostratus, Acrobates pygmaeus, Macropus rufogriseus and Sarcophilus harrisii are predicted to engage in multiple matings and sperm competition. Conversely, Lasiorhinus latifrons, Cercatetus concinnus and Pseudoantechinus macdonnellensis are predicted to be monandrous in their mating behaviour. The monotremes ( Ornithorhynchus anatinus, Tachyglossus aculeatus and Zaglossus bruijnii ) are characterized by possession of very large testes; monotremes are shown to have significantly greater relative testes weights than marsupials, primates or avian species. This taxonomic difference is unlikely to be related lo the occurrence of oviparity or to the abdominal position of the testes in the Monotremata. Their mating systems are not known in detail, but some evidence for multiple matings (and hence for sperm competition) exists for Tachyglossus aculeatus so that their large testes may be adaptive in this context.     

The hind limb step cycle of Iguana and primitive reptiles

The hind limb step cycle of Iguana is described and compared with other lizards. In generalized lizards with well developed limbs, the femur retracts through a large arc and rotates as it retracts, flexion of the crus on the metatarsus occurs during the initial part of the propulsive phase and extension of the knee and ankle during the final part, the crus rotates to face laterally, the metatarsus is laterally directed at the time of pedal plantarflexion, and pedal plantarflexion involves an initial rotation of the metatarsus around its long axis followed by an extension of the ankle joint. The evolutionary significance of the differences in the hind limb step cycle of lizards and salamanders is considered and it is shown that in many, but not all, features, primitive reptiles would have been like salamanders. The primitive step cycle differs from the mammalian pattern in the large amount of rotation of the limb segments, the large amount of femoral retraction, and the reduced amount of lateral movement of the hip relative to the knee that occurs during femoral retraction.     

The septomaxilla of fossil and recent synapsids and the problem of the septomaxilla of monotremes and armadillos

The septomaxilla is a paired intramembranous ossification in the external nares that occurs in Lepidosauria among Recent Sauropsida and is purported to be present in Monotremata and Dasypodidae (armadillos) among Recent Mammalia. A review of neontological and palaeontological evidence regarding this element in mammals supports the following conclusions: (1) monotremes have a true septomaxilla resembling that known for non-mammalian therapsids and some Mesozoic mammals; (2) the element in dasypodids is a neomorph; it neither resembles the septomaxilla of other synapsids nor does it exhibit the same relationship to the developing nasal-floor cartilage as the septomaxilla of lepidosaurs and monotremes; (3) a septomaxilla is lacking in all Recent therians, and there is no evidence that this bone is fused to the premaxilla in Recent therians, as has been suggested by previous authors.     

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.     

A Tachyglossid-Like Humerus from the Early Cretaceous of South-Eastern Australia

A partial right humerus has been recovered from the Early Cretaceous (Albian) Eumeralla Formation at Dinosaur Cove in south-eastern Australia. General morphology, size and the presence of a single epicondylar foramen (the entepicondylar) suggest that the bone is from a mammal or an advanced therapsid reptile. The humerus is similar in size, shape and torsion to the equivalent bone of extant and late Neogene echidnas (Tachyglossidae) but, contrary to the situation in extant monotremes, in which the ulna and radius articulate with a single, largely bulbous condyle, it bears a shallow, pulley-shaped (i.e. trochlear-form) ulnar articulation that is confluent ventro-laterally with the bulbous radial condyle. This form of ulnar articulation distinguishes this bone from the humeri of most advanced therapsids and members of several major groups of Mesozoic mammals, which have a condylar ulnar articulation, but parallels the situation found in therian mammals and in some other lineages of Mesozoic mammals. As in extant monotremes the distal humerus is greatly expanded transversely and humeral torsion is strong. Transverse expansion of the distal humerus is evident in the humeri of the fossorial docodont Haldanodon, highly-fossorial talpids and some clearly fossorial dicynodont therapsids, but the fossil shows greatest overall similarity to extant monotremes and it is possible that the peculiar elbow joint of extant monotremes evolved from a condition approximating that of the fossil. On the basis of comparisons with Mesozoic and Cainozoic mammalian taxa in which humeral morphology is known, the Dinosaur Cove humerus is tentatively attributed to a monotreme. However, several apparently primitive features of the bone exclude the animal concerned from the extant families Tachyglossidae and Ornithorhynchidae and suggest that, if it is a monotreme, it is a stem-group monotreme. Whatever, the animal's true affinity, the gross morphology of its humerus indicates considerable capacity for rotation-thrust digging.     

Forelimb Kinematics of Rats Using XROMM,with Implications for Small Eutherians and Their Fossil Relatives

The earliest eutherian mammals were small-bodied locomotor generalists with a forelimb morphology that strongly resembles that of extant rats. Understanding the kinematics of the humerus, radius, and ulna of extant rats can inform and constrain hypotheses concerning typical posture and mobility in early eutherian forelimbs. The locomotion of Rattus norvegicus has been extensively studied, but the three-dimensional kinematics of the bones themselves remains under-explored. Here, for the first time, we use markerless XROMM (Scientific Rotoscoping) to explore the three-dimensional long bone movements in Rattus norvegicus during a normal, symmetrical gait (walking). Our data show a basic kinematic profile that agrees with previous studies on rats and other small therians: rats maintain a crouched forelimb posture throughout the step cycle, and the ulna is confined to flexion/extension in a parasagittal plane. However, our three-dimensional data illuminate long-axis rotation (LAR) movements for both the humerus and the radius for the first time. Medial LAR of the humerus throughout stance maintains an adducted elbow with a caudally-facing olecranon process, which in turn maintains a cranially-directed manus orientation (pronation). The radius also shows significant LAR correlated with manus pronation and supination. Moreover, we report that elbow flexion and manus orientation are correlated in R. norvegicus: as the elbow angle becomes more acute, manus supination increases. Our data also suggest that manus pronation and orientation in R. norvegicus rely on a divided system of labor between the ulna and radius. Given that the radius follows the flexion and extension trajectory of the ulna, it must rotate at the elbow (on the capitulum) so that during the stance phase its distal end lies medial to ulna, ensuring that the manus remains pronated while the forelimb is supporting the body. We suggest that forelimb posture and kinematics in Juramaia, Eomaia, and other basal eutherians were grossly similar to those of rats, and that humerus and radius LAR may have always played a significant role in forelimb and manus posture in small eutherian mammals.     

In vivo strains in the femur of the nine‐banded armadillo (Dasypus novemcinctus)

The capacity of limb bones to resist the locomotor loads they encounter depends on both the pattern of those loads and the material properties of the skeletal elements. Among mammals, understanding of the interplay between these two factors has been based primarily on evidence from locomotor behaviors in upright placentals, which show limb bones that are loaded predominantly in anteroposterior bending with minimal amounts of torsion. However, loading patterns from the femora of opossums, marsupials using crouched limb posture, show appreciable torsion while the bone experiences mediolateral (ML) bending. These data indicated greater loading diversity in mammals than was previously recognized, and suggested the possibility that ancestral loading patterns found in sprawling lineages (e.g., reptilian sauropsids) might have been retained among basal mammals. To further test this hypothesis, we recorded in vivo locomotor strains from the femur of the nine‐banded armadillo (Dasypus novemcinctus), a member of the basal xenarthran clade of placental mammals that also uses crouched limb posture. Orientations of principal strains and magnitudes of shear strains indicate that armadillo femora are exposed to only limited torsion; however, bending is essentially ML, placing the medial aspect of the femur in compression and the lateral aspect in tension. This orientation of bending is similar to that found in opossums, but planar strain analyses indicate much more of the armadillo femur experiences tension during bending, potentially due to muscles pulling on the large, laterally positioned third trochanter. Limb bone safety factors were estimated between 3.3 and 4.3 in bending, similar to other placental mammals, but lower than opossums and most sprawling taxa. Thus, femoral loading patterns in armadillos show a mixture of similarities to both opossums (ML bending) and other placentals (limited torsion and low safety factors), along with unique features (high axial tension) that likely relate to their distinctive hindlimb anatomy. J. Morphol. 26:889–899, 2015. © 2015 Wiley Periodicals, Inc.     

Postcranial anatomy of Haldanodon exspectatus (Mammalia, Docodonta) from the Late Jurassic (Kimmeridgian) of Portugal and its bearing for mammalian evolution

The postcranium of the Late Jurassic docodont Haldanodon exspectatus Kühne & Krusat, 1972 is represented by a partial skeleton and isolated bones of other individuals from the Late Jurassic (Kimmeridgian) of the Guimarota coal mine in Portugal. Haldanodon exhibits adaptations for a fossorial lifestyle such as stout and short limb bones and humeri with greatly expanded distal joints and strong deltopectoral crests. Short first and second phalanges and moderately curved and laterally compressed terminal phalanges with lateral grooves suggest that Haldanodon was a scratch-digger. The dorso-ventrally elongated, triangular scapula has a convex transverse profile with strongly laterally reflected anterior and posterior scapula margins, enclosing a deep trough-like 'infraspinous fossa'. A supraspinous fossa is not developed. The saddle-shaped glenoid facet is mainly formed by the coracoid and orientated antero-ventrally indicating a sprawling gait. No epiphyses were detected and the wide size range of humerus and femur possibly indicate a lifelong growth. Haldanodon is more derived than Morganucodon by complete reduction of the procoracoid, absence of the procoracoid foramen, and a peg-like coracoid. It shares with monotremes a postscapular fossa that is absent in Morganucodon . A PAUP analysis based on 280 cranio-dental and postcranial characters corroborated the position of Haldanodon above morganucodontids and below Hadrocodium .  © 2005 The Linnean Society of London, Zoological Journal of the Linnean Society , 2005, 145 , 219–248.     

The genomes of the South American opossum (Monodelphis domestica) and platypus (Ornithorhynchus anatinus) encode a more complete purine catabolic pathway than placental mammals

The end product of purine catabolism varies amongst vertebrates and is a consequence of independent gene inactivation events that have truncated the purine catabolic pathway. Mammals have traditionally been grouped into two classes based on their end product of purine catabolism: most mammals, whose end product is allantoin due to an ancient loss of allantoinase (ALLN), and the hominoids, whose end product is uric acid due to recent inactivations of urate oxidase (UOX). However little is known about purine catabolism in marsupials and monotremes. Here we report the results of a comparative genomics study designed to characterize the purine catabolic pathway in a marsupial, the South American opossum (Monodelphis domestica), and a monotreme, the platypus (Ornithorhynchus anatinus). We found that both genomes encode a more complete set of genes for purine catabolism than do eutherians and conclude that a near complete purine catabolic pathway was present in the common ancestor of all mammals, and that the loss of ALLN is specific to placental mammals. Our results therefore provide a revised history for gene loss in the purine catabolic pathway and suggest that marsupials and monotremes represent a third class of mammals with respect to their end products of purine catabolism.     

Sprawling locomotion in the lizard Sceloporus clarkii: the effects of speed on gait, hindlimb kinematics, and axial bending during walking

Although the hindlimb is widely considered to provide the propulsive force in lizard locomotion, no study to date has analysed kinematic patterns of hindlimb movements for more than one stride for a single individual and no study has considered limb and axial kinematics together. In this study, kinematic data from several individuals of the Sceloporus clarkii are used to describe the movement patterns of the axial skeleton and hindlimb at different speeds, to analyse how kinematics change with speed, and to compare and contrast these findings with the inferred effects of speed cited in the literature. Angular limb movements and axial bending patterns (standing wave with nodes on the girdles) did not change with speed. Only the relative speed of retracting the femur and flexing the knee during limb retraction changes with speed. Based on these data and similar results from a recent study of salamanders, it appears that, over a range of speeds involving a walking trot, sprawling vertebrates increase speed by simply retracting the femur relatively faster, thus this simple functional adjustment may be a general mechanism to increase speed in tetrapods. The demonstration that femoral retraction alone is the major speed effector in Sceloporus clarkii lends strong functional support to ecomorphological implications of limb length (and especially femur length and caudifemoralis size) in locomotory ecology and performance in phrynosomatid lizards. It also lends support to inferences about the caudifemoralis muscle as a preadaptation to terrestrial locomotion and as a key innovation in the evolution of bipedalism.