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Phenotypic variation among species is a product of evolutionary changes to developmental programs 1 , 2 . However, how these changes generate novel morphological traits remains largely unclear. Here we studied the genomic and developmental basis of the mammalian gliding membrane, or patagium—an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species. Through comparative genomic analysis of 15 marsupial genomes, both from gliding and non-gliding species, we find that the Emx2 locus experienced lineage-specific patterns of accelerated cis -regulatory evolution in gliding species. By combining epigenomics, transcriptomics and in-pouch marsupial transgenics, we show that Emx2 is a critical upstream regulator of patagium development. Moreover, we identify different cis -regulatory elements that may be responsible for driving increased Emx2 expression levels in gliding species. Lastly, using mouse functional experiments, we find evidence that Emx2 expression patterns in gliders may have been modified from a pre-existing program found in all mammals. Together, our results suggest that patagia repeatedly originated through a process of convergent genomic evolution, whereby regulation of Emx2 was altered by distinct cis -regulatory elements in independently evolved species. Thus, different regulatory elements targeting the same key developmental gene may constitute an effective strategy by which natural selection has harnessed regulatory evolution in marsupial genomes to generate phenotypic novelty. Patagia—the mammalian gliding membrane—repeatedly originated through a process of convergent genomic evolution, whereby the regulation of Emx2 was altered by distinct cis -regulatory elements in independently evolved species.

Marsupials and placental mammals exhibit significant differences in reproductive and life history strategies. Marsupials are born highly underdeveloped after an extremely short period of gestation, leading to prioritized development of structures critical for post-birth survival in the pouch. Critically, they must undergo accelerated development of the orofacial region compared to placentals. Previously, we described the accelerated development of the orofacial region in the carnivorous Australian marsupial, the fat-tailed dunnart Sminthopsis crassicaudata , that has one of the shortest gestations of any mammal. By combining genome comparisons of the mouse and dunnart with functional data for the enhancer-associated chromatin modifications, H3K4me3 and H3K27ac, we investigated divergence of craniofacial regulatory landscapes between these species. This is the first description of genome-wide face regulatory elements in a marsupial, with 60,626 putative enhancers and 12,295 putative promoters described. We also generated craniofacial RNA-seq data for the dunnart to investigate expression dynamics of genes near predicted active regulatory elements. While genes involved in regulating facial development were largely conserved in mouse and dunnart, the regulatory landscape varied significantly. Additionally, a subset of dunnart-specific enhancers was associated with genes highly expressed only in dunnart relating to cranial neural crest proliferation, embryonic myogenesis, and epidermis development. Comparative RNA-seq analyses of facial tissue revealed dunnart-specific expression of genes involved in the development of the mechanosensory system. Accelerated development of the dunnart sensory system likely relates to the sensory cues received by the nasal–oral region during the postnatal journey to the pouch. Together, these data suggest that accelerated face development in the dunnart may be driven by dunnart-specific enhancer activity. Our study highlights the power of marsupial–placental comparative genomics for understanding the role of enhancers in driving temporal shifts in development. Marsupials are a distinctive group of mammals best known for their defining trait: a pouch. Unlike monotremes, which lay eggs, and placental mammals, in which young develop fully in the womb, marsupials give birth to highly premature offspring. These young complete most of their development within the mother’s pouch. Because of this strategy, marsupial newborns possess traits critical for immediate survival outside the womb. These include well-developed forelimbs for climbing into the pouch and a mature oral region for attaching to the teat and suckling. Evolution has shaped the development of the head to match the diverse environments vertebrates inhabit. Marsupials diverged from placental mammals over 160 million years ago, and their requirement for early limb and oral development makes them a powerful system for investigating the genetic mechanisms underlying development. The fat-tailed dunnart (Sminthopsis crassicaudata), often called a marsupial mouse, is emerging as an important model organism. With its short gestation period and extremely undeveloped state at birth, the dunnart provides an excellent comparison to the laboratory mouse – a well-established placental model – for studying evolutionary and developmental differences. Cook et al. investigated the genes and regulatory elements driving early orofacial development in the dunnart, comparing their findings with existing craniofacial expression and epigenomic data in mice. Their results showed that although the genes involved in craniofacial development are highly conserved between the two species, the regulatory elements controlling those genes differ markedly. In dunnarts, regulatory elements linked to skin, nervous system, and muscle development were highly active, whereas in mice they were inactive or only weakly expressed. An important feature of marsupial development is the postnatal retention of the periderm (a transient outer cell layer in embryonic skin). This may support gas exchange in prematurely born young. In addition, elevated activity of genes regulating sensory and muscle development suggests early maturation of mechanosensory and olfactory systems, which are essential for the newborn’s journey to the pouch. Notably, pharyngeal and facial muscles develop before the skeletal system, contrasting with the developmental sequence in placental mammals. In summary, marsupials exhibit a unique reproductive strategy, in which young are born in an extremely underdeveloped state and survival depends on accelerated development of specific traits after birth. By comparing gene regulation in marsupials and placentals, researchers gain insights into how evolution shaped developmental pathways. Expanding genomic resources for the dunnart, such as genome editing tools and transgenic models, will further enhance its role as a powerful comparative system for evolutionary developmental biology.

Developmental constraints can have significant influence on the magnitude and direction of evolutionary change, and many studies have demonstrated that these effects are manifested on macroevolutionary scales. Phenotypic integration, or the strong interactions among traits, has been similarly invoked as a major influence on morphological variation, and many studies have demonstrated that trait integration changes through ontogeny, in many cases decreasing with age. Here, we unify these perspectives in a case study of the ontogeny of the mammalian cranium, focusing on a comparison between marsupials and placentals. Marsupials are born at an extremely altricial state, requiring, in most cases, the use of the forelimbs to climb to the pouch, and, in all cases, an extended period of continuous suckling, during which most of their development occurs. Previous work has shown that marsupials are less disparate in adult cranial form than are placentals, particularly in the oral apparatus, and in forelimb ontogeny and adult morphology, presumably due to functional selection pressures on these two systems during early postnatal development. Using phenotypic trajectory analysis to quantify prenatal and early postnatal cranial ontogeny in 10 species of therian mammals, we demonstrate that this pattern of limited variation is also apparent in the development of the oral apparatus of marsupials, relative to placentals, but not in the skull more generally. Combined with the observation that marsupials show extremely high integration of the oral apparatus in early postnatal ontogeny, while other cranial regions show similar levels of integration to that observed in placentals, we suggest that high integration may compound the effects of the functional constraints for continuous suckling to ultimately limit the ontogenetic and adult disparity of the marsupial oral apparatus throughout their evolutionary history.

Most of our understanding of forebrain development comes from research of eutherian mammals, such as rodents, primates, and carnivores. However, as the cerebral cortex forms largely prenatally, observation and manipulation of its development has required invasive and/or ex vivo procedures. Marsupials, on the other hand, are born at comparatively earlier stages of development and most events of forebrain formation occur once attached to the teat, thereby permitting continuous and non-invasive experimental access. Here, we take advantage of this aspect of marsupial biology to establish and characterise a resourceful laboratory model of forebrain development: the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-sized carnivorous Australian marsupial. We present an anatomical description of the postnatal development of the body, head and brain in dunnarts, and provide a staging system compatible with human and mouse developmental stages. As compared to eutherians, the orofacial region develops earlier in dunnarts, while forebrain development is largely protracted, extending for more than 40 days versus ca. 15 days in mice. We discuss the benefits of fat-tailed dunnarts as laboratory animals in studies of developmental biology, with an emphasis on how their accessibility in the pouch can help address new experimental questions, especially regarding mechanisms of brain development and evolution.

Early pregnancy is a critical time for successful reproduction; up to half of human pregnancies fail before the development of the definitive chorioallantoic placenta. Unlike the situation in eutherian mammals, marsupial pregnancy is characterised by a long pre-implantation period prior to the development of the short-lived placenta, making them ideal models for study of the uterine environment promoting embryonic survival pre-implantation. Here we present a transcriptomic study of pre-implantation marsupial pregnancy, and identify differentially expressed genes in the Sminthopsis crassicaudata uterus involved in metabolism and biosynthesis, transport, immunity, tissue remodelling, and uterine receptivity. Interestingly, almost one quarter of the top 50 genes that are differentially upregulated in early pregnancy are putatively involved in histotrophy, highlighting the importance of nutrient transport to the conceptus prior to the development of the placenta. This work furthers our understanding of the mechanisms underlying survival of pre-implantation embryos in the earliest live bearing ancestors of mammals.

Changes in life history are expected when new sources of extrinsic mortality impact on natural populations. We report a new disease, devil facial tumor disease, causing an abrupt transition from iteroparity toward single breeding in the largest extant carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), in which males can weigh as much as 14 kg and females 9 kg. This change in life history is associated with almost complete mortality of individuals from this infectious cancer past their first year of adult life. Devils have shown their capacity to respond to this disease-induced increased adult mortality with a 16-fold increase in the proportion of individuals exhibiting precocious sexual maturity. These patterns are documented in five populations where there are data from before and after disease arrival and subsequent population impacts. To our knowledge, this is the first known case of infectious disease leading to increased early reproduction in a mammal. The persistence of both this disease and the associated life-history changes pose questions about longer-term evolutionary responses and conservation prospects for this iconic species.

The ectotympanic, malleus and incus of the developing mammalian middle ear (ME) are initially attached to the dentary via Meckel's cartilage, betraying their origins from the primary jaw joint of land vertebrates. This recapitulation has prompted mostly unquantified suggestions that several suspected—but similarly unquantified—key evolutionary transformations leading to the mammalian ME are recapitulated in development, through negative allometry and posterior/medial displacement of ME bones relative to the jaw joint. Here we show, using µCT reconstructions, that neither allometric nor topological change is quantifiable in the pre-detachment ME development of six marsupials and two monotremes. Also, differential ME positioning in the two monotreme species is not recapitulated. This challenges the developmental prerequisites of widely cited evolutionary scenarios of definitive mammalian middle ear (DMME) evolution, highlighting the requirement for further fossil evidence to test these hypotheses. Possible association between rear molar eruption, full ME ossification and ME detachment in marsupials suggests functional divergence between dentary and ME as a trigger for developmental, and possibly also evolutionary, ME detachment. The stable positioning of the dentary and ME supports suggestions that a ‘partial mammalian middle ear’ as found in many mammaliaforms—probably with a cartilaginous Meckel's cartilage—represents the only developmentally plausible evolutionary DMME precursor.

Background In contrast to placental neonates, in which all cranial bones are ossified, marsupial young have only the bones of the oral region and the exoccipital ossified at birth, in order to facilitate suckling at an early stage of development. In this study, we investigated whether this heterochronic shift in the timing of cranial ossification constrains cranial disparity in marsupials relative to placentals. Methods We collected three-dimensional (3D) landmark data about the crania of a wide range of extant placentals and marsupials, and from six fossil metatherians (the clade including extant marsupials and their stem relatives), using a laser scanner and a 3D digitizer. Principal components analysis and delta variance tests were used to investigate the distribution and disparity of cranial morphology between different landmark sets (optimizing either number of landmarks or number of taxa) of the whole skull and of individual developmental or functional regions (neurocranium, viscerocranium, oral region) for extant placentals and marsupials. Marsupial and placental data was also compared based on shared ecological aspects including diet, habitat, and time of peak activity. Results We found that the extant marsupial taxa investigated here occupy a much smaller area of morphospace than the placental taxa, with a significantly ( P <0.01) smaller overall variance. Inclusion of fossil taxa did not significantly increase the variance of metatherian cranial shape. Fossil forms generally plotted close to or within the realm of their extant marsupial relatives. When the disparities of cranial regions were investigated separately, significant differences between placentals and marsupials were seen for the viscerocranial and oral regions, but not for the neurocranial region. Conclusion These results support the hypothesis of developmental constraint limiting the evolution of the marsupial skull, and further suggest that the marsupial viscerocranium as a whole, rather than just the early-ossifying oral region, is developmentally constrained.

We tested if growth rates of recent taxa are unequivocally separated between endotherms and ectotherms, and compared these to dinosaurian growth rates. We therefore performed linear regression analyses on the log-transformed maximum growth rate against log-transformed body mass at maximum growth for extant altricial birds, precocial birds, eutherians, marsupials, reptiles, fishes and dinosaurs. Regression models of precocial birds (and fishes) strongly differed from Case's study (1978), which is often used to compare dinosaurian growth rates to those of extant vertebrates. For all taxonomic groups, the slope of 0.75 expected from the Metabolic Theory of Ecology was statistically supported. To compare growth rates between taxonomic groups we therefore used regressions with this fixed slope and group-specific intercepts. On average, maximum growth rates of ectotherms were about 10 (reptiles) to 20 (fishes) times (in comparison to mammals) or even 45 (reptiles) to 100 (fishes) times (in comparison to birds) lower than in endotherms. While on average all taxa were clearly separated from each other, individual growth rates overlapped between several taxa and even between endotherms and ectotherms. Dinosaurs had growth rates intermediate between similar sized/scaled-up reptiles and mammals, but a much lower rate than scaled-up birds. All dinosaurian growth rates were within the range of extant reptiles and mammals, and were lower than those of birds. Under the assumption that growth rate and metabolic rate are indeed linked, our results suggest two alternative interpretations. Compared to other sauropsids, the growth rates of studied dinosaurs clearly indicate that they had an ectothermic rather than an endothermic metabolic rate. Compared to other vertebrate growth rates, the overall high variability in growth rates of extant groups and the high overlap between individual growth rates of endothermic and ectothermic extant species make it impossible to rule out either of the two thermoregulation strategies for studied dinosaurs.

Marsupials have a slow rate of development and this allows a detailed examination of thermoregulatory developmental changes and stages. We quantified the cooling rates of marsupial dunnarts ( Sminthopsis crassicaudata ) at 40–56 days (d) old, and torpor and basking behaviour in animals given the option to bask in four age groups from 60 to 150 d. The development of thermoregulation was a continuum, but was characterised by three major thermoregulatory stages: (1) at 40 d, animals were unable to maintain a constant high body temperature during short-term cold exposure; (2) at 60 d, animals could maintain a high T b for the first part of the night at an ambient temperature of 15.0 ± 0.7 °C; later in the night, they entered an apparent torpor bout but could only rewarm passively when basking under a heat lamp; (3) from ~90 d, they expressed prolonged torpor bouts and were able to rewarm endogenously. Young newly weaned 60 d animals were able to avoid hypothermia by basking. In this case, basking was not an optional behavioural method of reducing the cost of rewarming from torpor, but was essential for thermoregulation independent of the nest temperature. Results from our study suggest that basking is a crucial behavioural trait that permits young marsupials and perhaps other juvenile altricial mammals to overcome the developmental stage between poikilothermy early in development and full endothermy later in life.