First monotreme from the Late Cretaceous of South America.

Monotremata is a clade of egg-lying mammals, represented by the living platypus and echidnas, which is endemic to Australia, and adjacent islands. Occurrence of basal monotremes in the Early Cretaceous of Australia has led to the consensus that this clade originated on that continent, arriving later to South America. Here we report on the discovery of a Late Cretaceous monotreme from southern Argentina, demonstrating that monotremes were present in circumpolar regions by the end of the Mesozoic, and that their distinctive anatomical features were probably present in these ancient forms as well.

Investigating the utility of using fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas (Tachyglossus aculeatus).

This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesterone metabolite concentrations were elevated above baseline values (448.0 ± 156.3 ng/g) during pregnancy and the luteal phase. However, compared to plasma progesterone the rise in fecal progesterone metabolite concentrations after copulation was delayed (3.3 ± 0.4 versus 8.3 ± 0.6 days, respectively), such that pregnancy was more reliably detected in its latter half when using fecal samples. Mating and oviposition were observed for 14 of the 19 pregnancies resulting in an estimated gestation of 16.7 ± 0.2 days (range 16.0-18.1 d). The estrogen enzyme-immunoassays tested (n = 3) in this study were not suitable for the fecal samples of the echidna. Fecal progesterone metabolites are an effective tool for confirming the timing and occurrence of estrous cycles in captive echidna colonies and can assist zookeepers in identifying possible causes of sub-optimal reproductive success without the unnecessary stress of repeated capture and anaesthesia for blood collection.

Validation of a non-invasive assessment technique for quantifying faecal glucocorticoid metabolite concentrations in the short-beaked echidna (Tachyglossus aculeatus).

The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic-pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response.

Fluorescence and UV-visible reflectance in the fur of several Rodentia genera.

Mammals are generally brown in colour, but recent publications are showing that they may not be as uniform as once assumed. Monotremes, marsupials, and a handful of eutherians reflect various colours when lit with UV light, mostly purple. Because of these still scarce records, we aimed to explore UV reflectance among rodent genera, the most diverse mammalian group, and the group of eutherians with the most common records of biofluorescence. Here we report structures like nails and quills reflected green, but for most genera, it was faded. However, Hystrix, Erethizon, and Ctenomys showed intense and contrasting green glow, while Chaetomys presented a vivid orange anogenital. The main available explanation of fluorescence in mammals relies on porphyrin. This explanation applies to the cases like Chaetomys, where specimens showed anogenital orange biofluorescence, but does not apply to the green biofluorescence we observed. In our sample, because the structures that reflected green were all keratinized, we have reasons to believe that biofluorescence results from keratinization and is a structurally-based colouration. However, not all spines/quills equally biofluoresced, so we cannot rule out other explanations. Since Rodentia is the most common mammalian group with reports on biofluorescence, this trait likely serves various functions that match the species diversity of this group.

Evolution of the Short Form of DNMT3A, DNMT3A2, Occurred in the Common Ancestor of Mammals.

Genomic imprinting is found in marsupial and eutherian mammals, but not in monotremes. While the primary regulator of genomic imprinting in eutherians is differential DNA methylation between parental alleles, conserved imprinted genes in marsupials tend to lack DNA methylation at their promoters. DNA methylation at eutherian imprinted genes is mainly catalyzed by a DNA methyltransferase (DNMT) enzyme, DNMT3A. There are two isoforms of eutherian DNMT3A: DNMT3A and DNMT3A2. DNMT3A2 is the primary isoform for establishing DNA methylation at eutherian imprinted genes and is essential for eutherian genomic imprinting. In this study, we investigated whether DNMT3A2 is also present in the two other mammalian lineages, marsupials and monotremes. We identified DNMT3A2 in both marsupials and monotremes, although imprinting has not been identified in monotremes. By analyzing genomic sequences and transcriptome data across vertebrates, we concluded that the evolution of DNMT3A2 occurred in the common ancestor of mammals. In addition, DNMT3A/3A2 gene and protein expression during gametogenesis showed distinct sexual dimorphisms in a marsupial, the tammar wallaby, and this pattern coincided with the sex-specific DNA methylation reprogramming in this species as it does in mice. Our results show that DNMT3A2 is present in all mammalian groups and suggests that the basic DNMT3A/3A2-based DNA methylation mechanism is conserved at least in therian mammals.

Musculoskeletal anatomy and nomenclature of the mammalian epipubic bones.

Despite the well-established anatomy nomenclature for the marsupial skeleton, there are no names for the epipubic bone structures. Epipubic bones are paired bones articulating with the pubis and projecting cranially in the ventral body wall, present on the pelvic girdle of cynodonts, monotremes and marsupials. These bones were commonly thought to be related to pouch support in marsupials and more recently associated with locomotion. The parts of the epipubic bones have not been named and this has impeded proper morphological analysis. We analyzed the epipubic bones of 302 skeletons comprising American and Australian marsupials, as well as 27 monotreme skeletons, and dissected 10 marsupials for myological attachments analysis. We suggest the following nomenclature for the epipubic bone structures: crest for the cranial end, shaft for the body of the bone, lateral tubercle and the medial articular process. Some markings on the epipubic bone include the oblique line, pertaining to the attachment of external abdominal oblique muscle from the opposite side. The pyramidalis line is the suggested nomenclature for the pyramidalis muscle attachment and the inguinal ligament line for the inguinal ligament attachment. Regarding myology and attachments, based on dissections and review of the literature, the muscles pyramidalis, pectineus, external and internal abdominal oblique, transversus abdominis and rectus abdominis and the structures linea alba, linea semilunaris and the inguinal ligament are connected to the epipubic bone. As has been previously noted, anatomically, epipubic bones are so named due to their position (epi-above, pubic-pubis), and the same applies to structures such as the "epipubic process" or "epipubic cartilage" in amphibians and reptiles. While testing epipubic bone homology in vertebrates is beyond the scope of this work, we believe that using "epipubic bones" or epipubic cartilage/process as standardized terms for the structures found in the most cranial part of the superior ramus of the pubis would facilitate better anatomical communication. This should be valid for other similar terms, such as "epipubes" or "prepubis", that might occur in the literature in relation to this same physiographic position, and it should also be named as epipubic. We believe that this nomenclature will help in future morphologic studies.

T. Thomson Flynn and the monotreme egg from oocyte maturation to germ layer formation.

Knowledge of oocyte development and the early differentiation of the germ layers in monotremes stems largely from two articles by J. P. Hill and T. Thomson Flynn. The completeness of their account was due to the large series of echidna ovaries and eggs collected on Tasmania by Flynn, an Australian biologist of whom a brief account is given. A striking finding in the oocyte and early embryo of monotremes was the presence of a latebra connected to the yolk bed beneath the germinal disc as described in birds and several reptiles. A further resemblance was the presence early in vitellogenesis of a striate zone beneath the zona pellucida. Cleavage resulted first in a lens-shaped blastodisc encircled by cells called vitellocytes that later fused to form a germ-ring. The blastodisc gave rise to a blastoderm that eventually became unilaminar but comprised two cell types identified as ectoderm and primitive endoderm. Eventually these segregated into two layers and formed a blastula. This sequence resembled that in marsupials except there was no obvious distinction between future embryonic and extra-embryonic regions. As the blastoderm extended over the surface of the yolk, it was preceded by the germ-ring that eventually played a role in forming the yolk navel. This was a unique feature of monotreme development. It is shown that Flynn played an important role in analysis of the material as well as in its collection.

A Survey of the Nematode Parasites of the Short-beaked Echidna Tachyglossus aculeatus (Monotremata: Tachyglossidae) from Southwestern Australia with the Description of a New Subfamily, Genus and Species of the Subuluridae Travassos, 1914 (Nematoda: Ascaridida).

INTRODUCTION: Nematodes were found in the digestive tracts of 15 short-beaked echidnas Tachyglossus aculeatus (Shaw, 1792) collected from southwestern Australia between August 1964 and March 2020. METHODS: Specimens were prepared for microscopic examination as temporary wet mounts, measurements were made, figures prepared using a drawing tube and light micrographs taken. All nematodes were identified to at least genus level and a bootstrap analysis of the helminth community was carried out. RESULTS: Two previously described species, one species identified to genus and one new nematode species were recovered, including Nicollina ridei (Nicollinidae), Parapharyngodon anomalus (Pharyngodonidae), Parastrongyloides sp. (Strongyloididae) and Echidnonema coronorum n. g., n. sp. (Subuluridae, Echidnonematinae n. sf.). Echidnonema coronorum n. sp. can be distinguished from all other subulurids in having a mouth encircled by six leaf-like elements separated by six small pointed elements. The buccal complex is also unique to this species and is composed of three parts, including an anterior buccal capsule and a posterior pharyngeal portion divided into two; the proximal part contains lobes which are ornamented with numerous irregular teeth. A bootstrap estimate of species richness of 99.9% indicated that there were few, if any, additional species to be discovered in the southwestern Australian population of short-beaked echidnas. CONCLUSION: Morphological analysis supports the status of E. coronorum as a new species and the erection of the Echidnonematinae as a new subfamily to accommodate it. The helminth community of the population of short-beaked echidnas studied appears to be depauperate and contains species unique to the region.

Quantitative analysis of cerebellar morphology in monotreme, metatherian and eutherian mammals.

To superficial inspection, the mammalian cerebellum appears to be a stereotypical structure that varies little in morphology across mammals. In the present study, the volumes of components of the corpus cerebelli, foliation of the cerebellar cortex and the volumes of the pontine and deep cerebellar nuclei have been measured and compared in three species of monotreme, 90 species of marsupial and 57 species of eutherian mammal. In all three mammalian groups, the volume of the corpus cerebelli scales isometrically with brain volume, and pontine nuclear volume also scales isometrically with cerebellar volume. The ratio of hemisphere to vermal cerebellar cortex is comparable in all mammals at small cerebellar volume, but elaboration of cerebellar hemispheres is largely confined to large cerebella of eutherian mammals. At small cerebellar volumes, diprotodontid metatherians have proportionally large cerebellar hemispheres compared to non-diprotodontid metatherians, and metatherian cerebella in general have a high volume of central white matter for a given cerebellar cortex volume compared to eutherians. The degree of foliation of the cerebellum scales similarly in therian mammals, but is relatively low in the monotremes for the volume of their corpus cerebelli. Among metatherians, cerebellar foliation is stronger among diprotodontid as compared to non-diprotodontids. Although the cerebellum has a similar structure in all mammals, there are subtle differences in structure between different mammal groups with possible functional implications.

Quantitative Analysis of the Timing of Development of the Cerebellum and Precerebellar Nuclei in Monotremes, Metatherians, Rodents, and Humans.

We have used a quantitative statistical approach to compare the pace of development in the cerebellum and precerebellar systems relative to body size in monotremes and metatherians with that in eutherians (rodents and humans). Embryos, fetuses, and early postnatal mammals were scored on whether key structural events had been reached in the development of the cerebellum itself (CC-corpus cerebelli; 10 milestones), or the pontine and inferior olivary precerebellar nuclear groups (PC; 4 milestones). We found that many early cerebellar and precerebellar milestones (e.g., formation of Purkinje cell layer and deep cerebellar nuclei) were reached at a smaller absolute body length in both metatherians and eutherians together, compared to monotremes. Some later milestones (e.g., formation of the external granular layer and primary fissuration) were reached at a smaller body length in metatherians than eutherians. When the analysis was performed with proportional body length expressed as a natural log-transformed ratio of length at birth, milestones were reached at a much smaller proportional body length in rodents and humans than in the metatherians or monotremes. The findings are consistent with the slower pace of metabolic activity and embryonic development in monotremes. They also indicate slightly advanced maturation of some early features of the cerebellum in some metatherians (i.e., early cerebellar development in dasyurids relative to body size), but do not support the notion of an accelerated development of the cerebellum to cope with the demands of early birth. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1998-2013, 2020. © 2019 American Association for Anatomy.

Deciphering Ancestral Sex Chromosome Turnovers Based on Analysis of Male Mutation Bias.

The age of sex chromosomes is commonly obtained by comparing the substitution rates of XY gametologs. Coupled with phylogenetic reconstructions, one can refine the origin of a sex chromosome system relative to specific speciation events. However, these approaches are insufficient to determine the presence and duration of ancestral sex chromosome systems that were lost in some species. In this study, we worked with genomic and transcriptomic data from mammals and squamates and analyzed the effect of male mutation bias on X-linked sequences in these groups. We searched for signatures indicating whether monotremes shared the same sex chromosomes with placental mammals or whether pleurodonts and acrodonts had a common ancestral sex chromosome system. Our analyses indicate that platypus did not share the XY chromosomes with placental mammals, in agreement with previous work. In contrast, analyses of agamids showed that this lineage maintained the pleurodont XY chromosomes for several million years. We performed multiple simulations using different strengths of male mutation bias to confirm the results. Overall, our work shows that variations in substitution rates due to male mutation bias could be applied to uncover signatures of ancestral sex chromosome systems.

Quantitative analysis of forebrain pallial morphology in monotremes and comparison with that in therians.

We have made quantitative volumetric analyses of cerebral cortical (pallial) structures in the brains of three species of monotreme (Ornithorhynchus anatinus, Tachyglossus aculeatus, Zaglossus bruijni) and compared the findings with similar measurements in a range of therian mammals (6 marsupials and 50 placentals). We have found that although the iso- and periallocortical grey matter volume of the monotremes is about what would be expected for their brain size, the proportion of iso- and periallocortical white matter in monotremes is substantially lower than that in the forebrains of therians. This suggests that the forebrains of the three monotremes have fewer association, commissural and/or projection connections than those of similarly sized forebrains of therian mammals. We also found that the iso- and periallocortex of the platypus is relatively smooth-surfaced compared to similarly sized brains of therian mammals, with a distinct caudal shift in the positioning of cortical white matter in the forebrain, consistent with expansion of the posterior thalamic radiation. Central laminated olfactory structures (anterior olfactory nucleus and piriform cortex) are large in the tachyglossid monotremes (Tachyglossus aculeatus and Zaglossus bruijni) and large in xenarthran placental mammals, suggesting convergence of the forebrain structure of monotreme formivores with that of similarly specialized therians like the xenarthrans Myrmecophaga tridactyla and Dasypus novemcinctus.

A Functional Perspective on the Evolution of the Cochlea.

This review summarizes paleontological data as well as studies on the morphology, function, and molecular evolution of the cochlea of living mammals (monotremes, marsupials, and placentals). The most parsimonious scenario is an early evolution of the characteristic organ of Corti, with inner and outer hair cells and nascent electromotility. Most remaining unique features, such as loss of the lagenar macula, coiling of the cochlea, and bony laminae supporting the basilar membrane, arose later, after the separation of the monotreme lineage, but before marsupial and placental mammals diverged. The question of when hearing sensitivity first extended into the ultrasonic range (defined here as >20 kHz) remains speculative, not least because of the late appearance of the definitive mammalian middle ear. The last significant change was optimizing the operating voltage range of prestin, and thus the efficiency of the outer hair cells' amplifying action, in the placental lineage only.

Conceptus Coats of Marsupials and Monotremes.

Mammals evolved from oviparous reptiles that laid eggs in a dry, terrestrial environment, thus requiring large amounts of yolk to support development and tough, outer coats to protect them. Eutherian mammals such as humans and mice exhibit an "extreme" form of viviparity in which yolk and conceptus coats have become largely redundant. However, the "other" mammals-monotremes and marsupials-have retained and modified some features of reptilian development that provide valuable insights into the evolution of viviparity in mammals. Most striking of these are the conceptus coats, which include the zona pellucida, the mucoid coat, and the shell coat. We discuss current knowledge of these coats in monotremes and marsupials, their possible roles, and recently identified components such as the zona pellucida protein ZPAX, conceptus coat mucin (CCM), and nephronectin (NPNT).

Evolutionary origins and diversification of testis-specific short histone H2A variants in mammals.

Eukaryotic genomes must accomplish both compact packaging for genome stability and inheritance, as well as accessibility for gene expression. They do so using post-translational modifications of four ancient canonical histone proteins (H2A, H2B, H3, and H4) and by deploying histone variants with specialized chromatin functions. Some histone variants are conserved across all eukaryotes, whereas others are lineage-specific. Here, we performed detailed phylogenomic analyses of "short H2A histone" variants found in mammalian genomes. We discovered a previously undescribed typically-sized H2A variant in monotremes and marsupials, H2A.R, which may represent the common ancestor of the short H2As. We also discovered a novel class of short H2A histone variants in eutherian mammals, H2A.Q We show that short H2A variants arose on the X Chromosome in the common ancestor of all eutherian mammals and diverged into four evolutionarily distinct clades: H2A.B, H2A.L, H2A.P, and H2A.Q However, the repertoires of short histone H2A variants vary extensively among eutherian mammals due to lineage-specific gains and losses. Finally, we show that all four short H2As are subject to accelerated rates of protein evolution relative to both canonical and other variant H2A proteins including H2A.R. Our analyses reveal that short H2As are a unique class of testis-restricted histone variants displaying an unprecedented evolutionary dynamism. Based on their X-Chromosomal localization, genetic turnover, and testis-specific expression, we hypothesize that short H2A variants may participate in genetic conflicts involving sex chromosomes during reproduction.

Weird Animals, Sex, and Genome Evolution.

Making my career in Australia exposed me to the tyranny of distance, but it gave me opportunities to study our unique native fauna. Distantly related animal species present genetic variation that we can use to explore the most fundamental biological structures and processes. I have compared chromosomes and genomes of kangaroos and platypus, tiger snakes and emus, devils (Tasmanian) and dragons (lizards). I particularly love the challenges posed by sex chromosomes, which, apart from determining sex, provide stunning examples of epigenetic control and break all the evolutionary rules that we currently understand. Here I describe some of those amazing animals and the insights on genome structure, function, and evolution they have afforded us. I also describe my sometimes-random walk in science and the factors and people who influenced my direction. Being a woman in science is still not easy, and I hope others will find encouragement and empathy in my story.

Comparative anatomy of neonates of the three major mammalian groups (monotremes, marsupials, placentals) and implications for the ancestral mammalian neonate morphotype.

The existing different modes of reproduction in monotremes, marsupials and placentals are the main source for our current understanding of the origin and evolution of the mammalian reproduction. The reproductive strategies and, in particular, the maturity states of the neonates differ remarkably between the three groups. Monotremes, for example, are the only extant mammals that lay eggs and incubate them for the last third of their embryonic development. In contrast, marsupials and placentals are viviparous and rely on intra-uterine development of the neonates via choriovitelline (mainly marsupials) and chorioallantoic (mainly placentals) placentae. The maturity of a newborn is closely linked to the parental care strategy once the neonate is born. The varying developmental degrees of neonates are the main focus of this study. Monotremes and marsupials produce highly altricial and nearly embryonic offspring. Placental mammals always give birth to more developed newborns with the widest range from altricial to precocial. The ability of a newborn to survive and grow in the environment it was born in depends highly on the degree of maturation of vital organs at the time of birth. Here, the anatomy of four neonates of the three major extant mammalian groups is compared. The basis for this study is histological and ultrastructural serial sections of a hatchling of Ornithorhynchus anatinus (Monotremata), and neonates of Monodelphis domestica (Marsupialia), Mesocricetus auratus (altricial Placentalia) and Macroscelides proboscideus (precocial Placentalia). Special attention was given to the developmental stages of the organs skin, lung, liver and kidney, which are considered crucial for the maintenance of vital functions. The state of the organs of newborn monotremes and marsupials are found to be able to support a minimum of vital functions outside the uterus. They are sufficient to survive, but without capacities for additional energetic challenges. The organs of the altricial placental neonate are further developed, able to support the maintenance of vital functions and short-term metabolic increase. The precocial placental newborn shows the most advanced state of organ development, to allow the maintenance of vital functions, stable thermoregulation and high energetic performance. The ancestral condition of a mammalian neonate is interpreted to be similar to the state of organ development found in the newborns of marsupials and monotremes. In comparison, the newborns of altricial and precocial placentals are derived from the ancestral state to a more mature developmental degree associated with advanced organ systems.

Marsupial and monotreme cathelicidins display antimicrobial activity, including against methicillin-resistant Staphylococcus aureus.

With the growing demand for new antibiotics to combat increasing multi-drug resistance, a family of antimicrobial peptides known as cathelicidins has emerged as potential candidates. Expansions in cathelicidin-encoding genes in marsupials and monotremes are of specific interest as the peptides they encode have evolved to protect immunologically naive young in the harsh conditions of the pouch and burrow. Our previous work demonstrated that some marsupial and monotreme cathelicidins have broad-spectrum antibacterial activity and kill resistant bacteria, but the activity of many cathelicidins is unknown. To investigate associations between peptide antimicrobial activity and physiochemical properties, we tested 15 cathelicidin mature peptides from tammar wallaby, grey short-tailed opossum, platypus and echidna for antimicrobial activity against a range of bacterial and fungal clinical isolates. One opossum cathelicidin ModoCath4, tammar wallaby MaeuCath7 and echidna Taac-CATH1 had broad-spectrum antibacterial activity and killed methicillin-resistant Staphylococcus aureus. However, antimicrobial activity was reduced in the presence of serum or whole blood, and non-specific toxicity was observed at high concentrations. The active peptides were highly charged, potentially increasing binding to microbial surfaces, and contained amphipathic helical structures, which may facilitate membrane permeabilisation. Peptide sequence homology, net charge, amphipathicity and alpha helical content did not correlate with antimicrobial activity. However active peptides contained a significantly higher percentage of cationic residues than inactive ones, which may be used to predict active peptides in future work. Along with previous studies, our results indicate that marsupial and monotreme cathelicidins show potential for development as novel therapeutics to combat increasing antimicrobial resistance.

Immune-endocrine interactions in marsupials and monotremes.

Interactions between the immune and endocrine systems are not well studied in marsupials and monotremes. One exception to this is the phenomenon of semelparity, which is well covered in the literature as this is an unusual reproductive strategy amongst mammals and is only observed in some dasyurid and didelphid marsupials. Thymus involution provides a direct link between the endocrine and immune systems and warrants further study in marsupials and monotremes. The thymus is a primary immune tissue which is essential for overall immune function. Whilst the organ is large in juvenile animals, it begins to involute around puberty due to the suppressive effects of sex steroids. Thymus involution has a significant effect on the immune system, as it signals the onset of immune aging and decline in function. The output of naïve T lymphocytes by the thymus decreases, increasing susceptibility of aged individuals to infection and cancers. Understanding the links between the immune and endocrine system in marsupials and monotremes may shed light on diseases such as devil facial tumour disease (DFTD) which threatens the future of the Tasmanian devil. We hypothesise that changes in sex hormones around puberty may drive changes in the immune system, such as thymus involution, which may make devils more susceptible to DFTD as they age. In addition, the Schwann cell origin of DFTD may enable tumours to respond to sex hormones, as occurs in similar cancers in humans.

Inner ear labyrinth anatomy of monotremes and implications for mammalian inner ear evolution.

The monophyletic clade Monotremata branches early from the rest of the mammalian crown group in the Jurassic and members of this clade retain many ancestral mammalian traits. Thus, accurate and detailed anatomical descriptions of this group can offer unique insight into the early evolutionary history of Mammalia. In this study, we examine the inner ear anatomy of two extant monotremes, Ornithorhynchus anatinus and Tachyglossus aculeatus, with the primary goals of elucidating the ancestral mammalian ear morphology and resolving inconsistencies found within previous descriptive literature. We use histological serial sections and high-resolution microcomputed tomography (µCT) for correlating soft tissue features of the vestibule and cochlea to the osseous labyrinth endocast. We found that in both monotremes the scala tympani coils to a lesser degree than scala vestibuli and scala media, although all three scalae show an apical coil inside the osseous cochlear tube. The helicotrema (conduit between scala tympani and scala vestibuli) is in subapical position, and the cochlear and lagenar ganglia and their associated nerve fibers are not enclosed by bone. In comparison, in extant therian mammals (i.e., marsupials and placentals) the helicotrema is located at the apex of the osseous cochlear canal, the three scalae coil to the same degree and the cochlear ganglion is enclosed by the primary bony lamina. Whether the lagenar ganglion is lost in therian mammals or integrated into the cochlear ganglion is still debated. The presence of a sensory lagenar macula at the apex of the membranous cochlear duct, innervated by a separate lagenar nerve and ganglion is a plesiomorphic condition of amniotes that monotremes share. A separate osseous lagenar canaliculus for the lagenar nerve, and the coiling of the distended lagenar sac at the end of the cochlear duct are autapomorphies of monotremes. Based on our findings we hypothesize that the ancestral inner ear of stem mammaliaforms is characterized by a straight or slightly curved osseous cochlear canal, a lagenar macula, lagenar nerve fibers separated from a larger bundle of cochlear nerve fibers, the presence of an organ of Corti and an intra-otic cochlear ganglion suspended by membranous connective tissue. Among the major Mesozoic clades of crown mammals, cladotherians and gondwanatherians most likely acquired a fully functioning organ of Corti but lost the sensory lagenar macula, like extant therians. However, Mesozoic spalacotherioids, multituberculates and eutriconodonts likely retained the mammaliaform condition. J. Morphol. 278:236-263, 2017. © 2016 Wiley Periodicals,Inc.