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.     

DIFFERENCES IN THE TIMING OF PRECHONDROGENIC LIMB DEVELOPMENT IN MAMMALS: THE MARSUPIAL–PLACENTAL DICHOTOMY RESOLVED

In contrast to placentals, marsupials are born with forelimbs that are greatly developmentally advanced relative to their hind limbs. Despite significant interest, we still do not know why this is the case, or how this difference is achieved developmentally. Studies of prechondrogenic and chondrogenic limbs have supported the traditional hypothesis that marsupial forelimb development is accelerated in response to the functional requirements of the newborn's crawl to the teat. However, limb ossification studies have concluded that, rather than the forelimb being accelerated, hind limb development is delayed. By increasing the taxonomic coverage and number of prechondrogenic events relative to previous studies, and combining traditional phylogenetic analyses of event sequences with novel analyses of relative developmental rates, this study demonstrates that the timing of limb development in marsupials is more complex than commonly thought. The marsupial phenotype was derived through two independent evolutionary changes in developmental rate: (1) an acceleration of the forelimb's first appearance and (2) a delay of hind limb development from the bud stage onward. Surprisingly, this study also provides some support for an evolutionary acceleration of the marsupial hind limb's first appearance. Further study is needed on the developmental and genetic mechanisms driving these major evolutionary transitions.     

Heterochrony and post‐natal growth in mammals – an examination of growth plates in limbs

Mammals display a broad spectrum of limb specializations coupled with different locomotor strategies and habitat occupation. This anatomical diversity reflects different patterns of development and growth, including the timing of epiphyseal growth plate closure in the long bones of the skeleton. We investigated the sequence of union in 15 growth plates in the limbs of about 400 specimens, representing 58 mammalian species: 34 placentals, 23 marsupials and one monotreme. We found a common general pattern of growth plate closure sequence, but one that is universal neither between species nor in higher‐order taxa. Locomotor habitat has no detectable correlation with the growth plate closure sequence, but observed patterns indicate that growth plate closure sequence is determined more strongly through phylogenetic factors. For example, the girdle elements (acetabulum and coracoid process) always ossify first in marsupials, whereas the distal humerus is fused before the girdle elements in some placentals. We also found that heterochronic shifts (changes in timing) in the growth plate closure sequence of marsupials occur with a higher rate than in placentals. This presents a contrast with the more limited variation in timing and morphospace occupation typical for marsupial development. Moreover, unlike placentals, marsupials maintain many epiphyses separated throughout life. However, as complete union of all epiphyseal growth plates is recorded in monotremes, the marsupial condition might represent the derived state.     

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.     

Integration of the mammalian shoulder girdle within populations and over evolutionary time

Morphological integration has the potential to link morphological variation within populations with morphological evolution among species. This study begins to investigate this link by comparing integration among shoulder girdle elements (e.g. scapular blade, glenoid, coracoid, etc.) during the origin and evolution of therian mammals, and within modern bat, opossum and mouse populations. In this study, correlations among skeletal elements and patterns of allometry are used as proxies for integration. Results suggest that shoulder girdle elements tended to vary and evolve independently during the origin of mammals and subsequent radiation of placentals, consistent with the elements’ distinct developmental and evolutionary origins. This finding suggests that skeletal element correlations, and therefore integration, can be conserved over large taxonomic and temporal scales. However, marsupials display a different pattern in which shoulder girdle elements tend to be more integrated, with the exception of the coracoid. This finding is consistent with a shift in the pattern of skeletal element integration coincident with the appearance of the marsupial mode of reproduction. This finding provides further evidence that development can play a significant role in the establishment of patterns of skeletal element correlation and that patterns of skeletal element correlation can themselves evolve when faced with sufficient selective pressures.     

Conserved relative timing of cranial ossification patterns in early mammalian evolution

We analyzed a comprehensive data set of ossification sequences including seven marsupial, 13 placental and seven sauropsid species. Data are provided for the first time for two major mammalian clades, Chiroptera and Soricidae, and for two rodent species; the published sequences of three species were improved with additional sampling. The relative timing of the onset of ossification in 17 cranial elements was recorded, resulting in 136 event pairs, which were treated as characters for each species. Half of these characters are constant across all taxa, 30% are variable but phylogenetically uninformative, and 19% potentially deliver diagnostic features for clades of two or more taxa. Using the conservative estimate of heterochronic changes provided by the program Parsimov, only a few heterochronies were found to diagnose mammals, marsupials, or placentals. A later onset of ossification of the pterygoid with respect to six other cranial bones characterizes therian mammals. This result may relate to the relatively small size of this bone in this clade. One change in relative onset of ossification is hypothesized as a potential human autapomorphy in the context of the sampling made: the earlier onset of the ossification of the periotic with respect to the lacrimal and to three basicranial bones. Using the standard error of scaled ranks across all species as a measure of each element's lability in developmental timing, we found that ossification of early, middle, and late events are similarly labile, with basicranial traits the most labile in timing of onset of ossification. Despite marsupials and placental mammals diverging at least 130 Ma, few heterochronic shifts in cranial ossification diagnose these clades.     

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.     

Skeletal development in the African elephant and ossification timing in placental mammals

We provide here unique data on elephant skeletal ontogeny. We focus on the sequence of cranial and post-cranial ossification events during growth in the African elephant (Loxodonta africana). Previous analyses on ossification sequences in mammals have focused on monotremes, marsupials, boreoeutherian and xenarthran placentals. Here, we add data on ossification sequences in an afrotherian. We use two different methods to quantify sequence heterochrony: the sequence method and event-paring/Parsimov. Compared with other placentals, elephants show late ossifications of the basicranium, manual and pedal phalanges, and early ossifications of the ischium and metacarpals. Moreover, ossification in elephants starts very early and progresses rapidly. Specifically, the elephant exhibits the same percentage of bones showing an ossification centre at the end of the first third of its gestation period as the mouse and hamster have close to birth. Elephants show a number of features of their ossification patterns that differ from those of other placental mammals. The pattern of the initiation of the ossification evident in the African elephant underscores a possible correlation between the timing of ossification onset and gestation time throughout mammals.     

Skeletal ossification and sequence heterochrony in xenarthran evolution

Previous analyses of how mammals vary in their ossification sequences have focused on monotremes, marsupials, and boreoeutherian placentals. Here, we focus on the sequence of cranial and postcranial ossification events during growth in the xenarthran skull and skeleton, including armadillos, anteaters, and sloths. We use two different methods to quantify sequence heterochrony: sequence analysis of variance (ANOVA) and event‐paring/Parsimov. Our results indicate that Parsimov is conservative and does not detect clear heterochronic shifts between xenarthran and boreoeutherian placentals. Sequence‐ANOVA performs better, but both methods exhibit sensitivity to the artifactual accumulation of ties. By controlling for ties and taking into account results that the methods have in common, our analysis suggests that xenarthrans significantly differ from other placentals by a late ossification of the sternum and an early ossification of the phalanges and pubis. We interpret these differences as showing that heterochrony plays a role in the skeletal development of xenarthrans, a change from previous studies that have emphasized the developmental homogeneity of the skeleton across placental mammals.     

Postcranial Skeleton of the Cretaceous Mammal Akidolestes cifellii and Its Locomotor Adaptations

Spalacotheroid “symmetrodontans” are a group of extinct Mesozoic mammals. They are basal taxa in the trechnotherian clade that includes modern marsupials and placentals. Therefore, fossils of spalacotheroids can provide information on the ancestral condition from which marsupials and placentals likely have evolved. Here, we describe the postcranial skeleton of Akidolestes cifellii, a spalacotheroid species from the Lower Cretaceous Yixian Formation of northeastern China. Our comparison of the skeletal features of Akidolestes and the closely related Zhangheotherium and Maotherium indicates some major morphological and functional differences in the postcranium among these spalacotheroid mammals. Akidolestes shows characters for terrestrial habitat preference. Overall it appears to be a generalized terrestrial mammal. Akidolestes differs from Zhangheotherium and Maotherium in some characteristics of the scapula, the pelvis, and the hind limb, some of which can be directly correlated with different locomotor capabilities, and possibly also habitat preferences. This suggests that a greater ecomorphological differentiation occurred in these stem therian mammals than previously thought and that ecological differentiation is a major pattern in early therian mammal evolution.     

Why are There Fewer Marsupials than Placentals? On the Relevance of Geography and Physiology to Evolutionary Patterns of Mammalian Diversity and Disparity

Placental mammals occupy a larger morphospace and are taxonomically more diverse than marsupials by an order of magnitude, as shown by quantitative and phylogenetic studies of several character complexes and clades. Many have suggested that life history acts as a constraint on the evolution of marsupial morphology. However, the frequent circumvention of constraints suggests that the pattern of morphospace occupation in marsupials is more a reflection of lack of ecological opportunity than one of biases in the production of variants during development. Features of marsupial physiology are a potential source of biases in the evolution of the group; these could be coupled with past macroevolutionary patterns that followed conditions imposed by global temperature changes. This is evident at the K/Pg boundary and at the Eocene/Oligocene boundary. The geographic pattern of taxonomic and morphological diversity in placental clades mirrors that of extant placentals as a whole versus marsupials: placentals of northern origin are more diverse those of southern one and include the clades that are outliers in taxonomic (rodents and bats) and ecomorphological (whales and bats) richness.     

Three-dimensional kinematic analysis of the pectoral girdle during upside-down locomotion of two-toed sloths (Choloepus didactylus, Linné 1758)

Background

Theria (marsupials and placental mammals) are characterized by a highly mobile pectoral girdle in which the scapula has been shown to be an important propulsive element during locomotion. Shoulder function and kinematics are highly conservative during locomotion within quadrupedal therian mammals. In order to gain insight into the functional morphology and evolution of the pectoral girdle of the two-toed sloth we here analyze the anatomy and the three-dimensional (3D) pattern of shoulder kinematics during quadrupedal suspensory ('upside-down') locomotion.

Methods

We use scientific rotoscoping, a new, non-invasive, markerless approach for x-ray reconstruction of moving morphology (XROMM), to quantify in vivo the 3D movements of all constituent skeletal elements of the shoulder girdle. Additionally we use histologic staining to analyze the configuration of the sterno-clavicular articulation (SCA).

Results

Despite the inverse orientation of the body towards gravity, sloths display a 3D kinematic pattern and an orientation of the scapula relative to the thorax similar to pronograde claviculate mammalian species that differs from that of aclaviculate as well as brachiating mammals. Reduction of the relative length of the scapula alters its displacing effect on limb excursions. The configuration of the SCA maximizes mobility at this joint and demonstrates a tensile loading regime between thorax and limbs.

Conclusions

The morphological characteristics of the scapula and the SCA allow maximal mobility of the forelimb to facilitate effective locomotion within a discontinuous habitat. These evolutionary changes associated with the adoption of the suspensory posture emphasized humeral influence on forelimb motion, but allowed the retention of the plesiomorphic 3D kinematic pattern.     

Osseous inner ear structures and hearing in early marsupials and placentals

Based on the internal anatomy of petrosal bones as shown in radiographs and scanning electron microscopy, the inner ear structures of Late Cretaceous marsupials and placentals (about 65 Myr ago) from the Bug Creek Anthills locality of Montana, USA, are described. The inner ears of Late Cretaceous marsupials and placentals are similar to each other in having the following tribosphenic therian synapomorphies: a fully coiled cochlea, primary and secondary osseous spiral laminae, the perilymphatic recess merging with the scala tympani of the cochlea, an aqueductus cochleae, a true fenestra cochleae, a radial pattern of the cochlear nerve and an elongate basilar membrane extending to the region between the fenestra vestibuli and fenestra cochleae. The inner ear structures of living therians differ from those of their Late Cretaceous relatives mainly in having a greater number of spiral turns of the cochlea and a longer basilar membrane. Functionally, a coiled cochlea not only permits the development of an elongate basilar membrane within a restricted space in the skull but also allows a centralized nerve system to innervate the elongate basilar membrane. Qualitative and quantitative analyses show that, with a typical therian inner ear, Late Cretaceous marsupials and placentals were probably capable of high-frequency hearing.     

The root of the mammalian tree inferred from whole mitochondrial genomes

Morphological and molecular data are currently contradictory over the position of monotremes with respect to marsupial and placental mammals. As part of a re-evaluation of both forms of data we examine complete mitochondrial genomes in more detail. There is a particularly large discrepancy in the frequencies of thymine and cytosine (T-C) between mitochondrial genomes that appears to affect some deep divergences in the mammalian tree. We report that recoding nucleotides to RY-characters, and partitioning maximum-likelihood analyses among subsets of data reduces such biases, and improves the fit of models to the data, respectively. RY-coding also increases the signal on the internal branches relative to external, and thus increases the phylogenetic signal. In contrast to previous analyses of mitochondrial data, our analyses favor Theria (marsupials plus placentals) over Marsupionta (monotremes plus marsupials). However, a short therian stem lineage is inferred, which is at variance with the traditionally deep placement of monotremes on morphological data.     

Heterochronic shifts in the ossification sequences of surface- and subsurface-dwelling skinks are correlated with the degree of limb reduction

Scincid lizards exhibit a variety of limb anatomies which reflect the functional requirements of different modes of life. Besides surface dwellers which show neither body elongation nor limb reduction, there are numerous examples that can be arranged as increasingly serpentiform taxa moving in sand, humus or leaf litter. We explored the question of whether limb reduction and body elongation in skinks are linked to heterochronic shifts in the ossification sequences. The study material comprises skinks showing four different morphotypes: Liopholis whitii, Lerista bougainvillii, Hemiergis peronii and Saiphos equalis. Results showed that (i) scincid lizards with limb reductions exhibit an earlier onset of ossification in the cervical vertebrae, and (ii) ossification starts earlier in the pectoral girdle (scapula and coracoid) and pelvic girdle (ilium, ischium and pubis) relative to the timing of the onset in elements of the forelimbs and hind limbs. Furthermore, they show (iii) an earlier strengthening of the premaxilla, which first completes the anterior part of the dorsal cranial roof, and (iv) an earlier onset of ossification in the forelimb elements than in the equivalent elements of the hind limbs. The species showing the least limb reduction (L. bougainvillii) had the greatest developmental similarity to the normally proportioned surface-dwelling species (L. whitii). S. equalis, as the morphotype with the greatest deviation from the normally proportioned, pentadactyle form, varies the most from L. whitii. The heterochronic shifts in the ossification sequences are linked to a shift in the emphasis from limbed locomotion to trunk locomotion in the species with body elongation and/or limb reduction.     

Cranial modularity shifts during mammalian evolution

The mammalian skull has been studied as several separate functional components for decades, but the study of modularity is a more recent, integrative approach toward quantitative examination of independent subsets of highly correlated traits, or modules. Although most studies of modularity focus on developmental and genetic systems, phenotypic modules have been noted in many diverse morphological structures. However, few studies have provided empirical data for comparing modules across higher taxonomic levels, limiting the ability to assess the broader evolutionary significance of modularity. This study uses 18-32 three-dimensional cranial landmarks to analyze phenotypic modularity in 106 mammalian species and demonstrates that cranial modularity is generally conserved in the evolution of therian mammals (marsupials and placentals) but differs between therians and monotremes, the two extant subclasses of Mammalia. Within therians, cluster analyses identify six distinct modules, but only three modules display significant integration in all species. Monotremes display only two highly integrated modules. Specific hypotheses of functional and developmental influences on cranial bones were tested. Theoretical correlation matrices for bones were constructed on the basis of shared function, tissue origin, or mode of ossification, and all three of these models are significantly correlated with observed correlation matrices for the mammalian cranium.     

Cranial modularity and sequence heterochrony in mammals

Heterochrony, the temporal shifting of developmental events relative to each other, requires a degree of autonomy among those processes or structures. Modularity, the division of larger structures or processes into autonomous sets of internally integrated units, is often discussed in relation to the concept of heterochrony. However, the relationship between the developmental modules derived from studies of heterochrony and evolutionary modules, which should be of adaptive importance and relate to the genotype-phenotype map, has not been explicitly studied. I analyzed a series of sectioned and whole cleared-and-stained embryological and neonatal specimens, supplemented with published ontogenetic data, to test the hypothesis that bones within the same phenotypic modules, as determined by morphometric analysis, are developmentally integrated and will display coordinated heterochronic shifts across taxa. Modularity was analyzed in cranial bone ossification sequences of 12 therian mammals. A dataset of 12-18 developmental events was used to assess if modularity in developmental sequences corresponds to six phenotypic modules, derived from a recent morphometric analysis of cranial modularity in mammals. Kendall's tau was used to measure rank correlations, with randomization tests for significance. If modularity in developmental sequences corresponds to observed phenotypic modules, bones within a single phenotypic module should show integration of developmental timing, maintaining the same timing of ossification relative to each other, despite differences in overall ossification sequences across taxa. Analyses did not find any significant conservation of developmental timing within the six phenotypic modules, meaning that bones that are highly integrated in adult morphology are not significantly integrated in developmental timing.     

Rearrangements of mitochondrial transfer RNA genes in marsupials

Summary The nucleotide sequences of the mitochondrial origin of light-strand replication and the five tRNA genes surrounding it were determined for three marsupials. The region was found to be rearranged, leaving only the tRNA^Tyr gene at the same position as in placental mammals andXenopus. Distribution of the same rearranged genotype among two marsupial families indicates that the events causing the rearrangements took place in an early marsupial ancestor. The putative mitochondrial light-strand origin of replication in marsupials contains a hairpin structure similar to other vertebrate origins and, in addition, extensive flanking sequences that are not found in other vertebrates. Sequence comparisons among the marsupials as well as placentals indicate that the tRNA^Tyr gene has been evolving under more constraints than the other tRNA genes.Deceased July 21, 1991     

Relationships Among the Families and Orders of Marsupials and the Major Mammalian Lineages Based on Recombination Activating Gene-1

Controversies remain over the relationships among several of the marsupial families and between the three major extant lineages of mammals: Eutheria (placentals), Metatheria (marsupials), and Prototheria (monotremes). Two opposing hypotheses place the marsupials as either sister to the placental mammals (Theria hypothesis) or sister to the monotremes (Palimpsest or Marsupionta hypothesis). A nuclear gene that has proved useful for analyzing phylogenies of vertebrates is the recombination activation gene-1 (RAG1). RAG1 is a highly conserved gene in vertebrates and likely entered the genome by horizontal transfer early in the evolution of jawed vertebrates. Phylogenetic analyses were performed on RAG1 sequences from seven placentals, 28 marsupials, and all three living monotreme species. Phylogenetic analyses of RAG1 sequences support many of the traditional relationships among the marsupials and suggest a relationship between bandicoots (order Peramelina) and the marsupial mole (order Notoryctemorphia), two lineages whose position in the phylogenetic tree has been enigmatic. A sister relationship between South American shrew opossums (order Paucituberculata) and all other living marsupial orders is also suggested by RAG1. The relationship between the three major groups of mammals is consistent with the Theria hypothesis, with the monotremes as the sister group to a clade containing marsupials and placentals.     

The developmental reduction of the marsupial coracoid: A case study in Monodelphis domestica

During their embryogenesis, marsupials develop a unique structure, the shoulder arch, which provides the structural and muscle‐attachment support necessary for the newborn's crawl to the teat. One of the most pronounced and important aspects of the shoulder arch is an enlarged coracoid. After marsupial newborns reach the teat, the shoulder arch is remodeled and the coracoid is reduced to a small process on the scapula. Although an understanding of marsupial coracoid reduction has the potential to provide insights into both, marsupial evolution and the origin of mammals, little is known about the morphological and cellular processes controlling this process. To remedy this situation, this study examined the morphological and cellular mechanisms behind coracoid reduction in the gray short‐tailed opossum, Monodelphis domestica. A quantitative, morphometric study of shoulder girdle development revealed that the coracoid is reduced in size relative to other aspects of the shoulder girdle by growing at a slower rate. Using a series of molecular assays for cell death, no evidence was found for programmed cell death playing a role in the reduction of coracoid size in marsupials (in contrast to hypotheses of previous researchers). Although it is likely the case that coracoid growth is reduced through a relatively lower rate of cellular proliferation, differences in proliferative rates in the coracoid and scapula were not great enough to be quantified using standard molecular assays. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.