Eutherian Species Research Articles

The role of stem cells in uterine involution.

Uterine remodeling during pregnancy and repair postpartum are fundamental to the successful propagation of eutherian species. The most drastic remodeling occurs in species with invasively implanting embryos, including humans and mice. During embryo implantation, embryonic trophoblasts breach the epithelium, penetrating into the stroma. Stromal cell decidualization, which is critical for the establishment and maintenance of early pregnancy, occurs throughout the implantation site. Trophoblasts further invade into and remodel uterine spiral arteries, which is necessary for placental formation. The uterus increases in size up to 24-fold, which is largely attributed to myometrial expansion. Uterine changes that occur during pregnancy must then be resolved postpartum. Following parturition, the uterus repairs the remodeled tissue in the process of uterine involution. During involution, the majority of the endometrium is regenerated to replace the tissue that is shed postpartum. The myometrium returns to the pre-gravid state which is thought to occur through apoptosis and autophagy of smooth muscle cells. Although we understand the general process of postpartum uterine involution, the detailed mechanisms, particularly the role of putative stem cells, are poorly understood. This review discusses the evidence for the existence of epithelial, stromal and myometrial stem cells and their role in uterine involution. Gaps in knowledge and areas for future research are also considered. Studies of both postpartum and menstrual uterine repair, which likely involve similar mechanisms, are described under the broad definition of uterine involution. Although the primary focus of this review is human, mouse models are discussed to provide additional information.

Parasite infestation influences life history but not boldness behavior in placental live-bearing fish

Parasites can negatively affect the reproductive success of hosts. Placental species may be particularly susceptible, because parasite-induced stress during pregnancy could potentially influence embryo development. Here, we examine the consequences of a trematode infestation (black spot disease, BSD) for fetal development and adult behavior in 19 natural populations of the placental live-bearing fish species Poeciliopsis retropinna (Poeciliidae) in Costa Rica. First, we observed substantial variation in parasite infestation among populations which correlated with a number of local environmental conditions (elevation, river width, depth, and flow velocity). Furthermore, we observed substantial variation in parasite infestation among females within populations associated with maternal age and size. We found that the infestation rate significantly influenced embryonic development, with more heavily parasitized females producing smaller and worse-conditioned offspring at birth, possibly, because a costly immune response during pregnancy limits, either directly or indirectly, nourishment to developing embryos. Finally, a behavioral experiment in the field showed that the infestation rate did not affect an individual’s boldness. Our study indicates that in placental live-bearing fish parasite infestation leads to reduced embryo provisioning during pregnancy, resulting in a smaller offspring size and quality at birth potentially with negative implications for offspring fitness.

Single nuclei RNA-seq of mouse placental labyrinth development.

The placenta is the interface between mother and fetus in all eutherian species. However, our understanding of this essential organ remains incomplete. A substantial challenge has been the syncytial cells of the placenta, which have made dissociation and independent evaluation of the different cell types of this organ difficult. Here, we address questions concerning the ontogeny, specification, and function of the cell types of a representative hemochorial placenta by performing single nuclei RNA sequencing (snRNA-seq) at multiple stages of mouse embryonic development focusing on the exchange interface, the labyrinth. Timepoints extended from progenitor-driven expansion through terminal differentiation. Analysis by snRNA-seq identified transcript profiles and inferred functions, cell trajectories, signaling interactions, and transcriptional drivers of all but the most highly polyploid cell types of the placenta. These data profile placental development at an unprecedented resolution, provide insights into differentiation and function across time, and provide a resource for future study.

A single-cell transcriptome atlas of marsupial embryogenesis and Xinactivation.

Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of Xdosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160million years ago. They exhibit distinctive developmental features, including late implantation1 and imprinted Xchromosome inactivation2, which is associated with expression of the XIST-like noncoding RNA RSX3. Here we perform a single-cell RNA-sequencing analysis of embryogenesis and Xchromosome inactivation in a marsupial, the grey short-tailed opossum (Monodelphis domestica). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian-marsupial divergence. RSX coating and inactivation of the Xchromosome occurs early and rapidly. This observation supports the hypothesis that-in organisms with early Xchromosome inactivation-imprinted Xchromosome inactivation prevents biallelic Xsilencing. We identify XSR, an RSXantisense transcript expressed from the active Xchromosome, as a candidate for the regulator of imprinted Xchromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and Xdosage compensation.

Historical biogeography of smoothhound sharks (genus Mustelus) of Southern Africa reveals multiple dispersal events from the Northern Hemisphere

Members of the smoothhound shark genus Mustelus display a widespread distribution pattern across ocean basins with a high degree of sub-regional endemism. The patterns and processes that resulted in smoothhound biodiversity and present-day distribution remain largely unknown. We infer the phylogenetic relationships of the genus Mustelus, based on sequence data (3474 bp) from three mitochondrial genes (CR, NADH-2 and 12S-16SrRNA) and a nuclear gene (KBTBD2) from seven species of Mustelus distributed across the eastern Atlantic- and Indo-Pacific oceans. Using the CR and KBTBD2 dataset, we infer the phylogeographic placement of Old World Mustelus, with particular reference to species from southern Africa. Using a near-complete phylogeny of the genus including Old World and New World species of Mustelus and publicly available sequences of the NADH-2 gene, we found supporting evidence indicating a major cladogenic event separating placental and aplacental species. Biogeographical analyses further revealed that the radiation of Mustelus in the southern African region was driven primarily by long-distance dispersal during the upper Miocene to lower Pleistocene. The placement of the placental blackspotted smoothhound Mustelus punctulatus at the base of the placental non-spotted clade suggests the secondary loss of black spots in the genus, and this was also supported by the ancestral state reconstruction. The results furthermore suggest that the Southern Hemisphere species of the genus arose from multiple separate dispersal events from the Northern Hemisphere which is in line with the earliest record of Mustelus in the Northern Hemisphere.

Secretion and Function of Pituitary Prolactin in Evolutionary Perspective.

The hypothalamo-pituitary system developed in early vertebrates. Prolactin is an ancient vertebrate hormone released from the pituitary that exerts particularly diverse functions. The purpose of the review is to take a comparative approach in the description of prolactin, its secretion from pituitary lactotrophs, and hormonal functions. Since the reproductive and osmoregulatory roles of prolactin are best established in a variety of species, these functions are the primary subjects of discussion. Different types of prolactin and prolactin receptors developed during vertebrate evolution, which will be described in this review. The signal transduction of prolactin receptors is well conserved among vertebrates enabling us to describe the whole subphylum. Then, the review focuses on the regulation of prolactin release in mammals as we have the most knowledge on this class of vertebrates. Prolactin secretion in response to different reproductive stimuli, such as estrogen-induced release, mating, pregnancy and suckling is detailed. Reproduction in birds is different from that in mammals in several aspects. Prolactin is released during incubation in avian species whose regulation and functional significance are discussed. Little information is available on prolactin in reptiles and amphibians; therefore, they are mentioned only in specific cases to explain certain evolutionary aspects. In turn, the osmoregulatory function of prolactin is well established in fish. The different types of pituitary prolactin in fish play particularly important roles in the adaptation of eutherian species to fresh water environments. To achieve this function, prolactin is released from lactotrophs in hyposmolarity, as they are directly osmosensitive in fish. In turn, the released prolactin acts on branchial epithelia, especially ionocytes of the gill to retain salt and excrete water. This review will highlight the points where comparative data give new ideas or suggest new approaches for investigation in other taxa.

An irregular hourglass pattern describes the tempo of phenotypic development in placental mammal evolution

Organismal development is defined by progressive transformations that ultimately give rise to distinct tissues and organs. Thus, temporal shifts in ontogeny often reflect key phenotypic differences in phylogeny. Classical theory predicts that interspecific morphological divergence originates towards the end of embryonic or fetal life stages, i.e. the early conservation model. By contrast, the hourglass model predicts interspecific variation early and late in prenatal ontogeny, though with a phylogenetically similar mid-developmental period. This phylotypic period, however, remains challenging to define within large clades such as mammals. Thus, molecular and morphological tests on a mammalian hourglass have not been entirely congruent. Here, we report an hourglass-like pattern for mammalian developmental evolution. By comparing published data on the timing of 74 homologous characters across 51 placental species, we demonstrated that variation in the timing of development decreased late in embryogenesis––when organ formation is highly active. Evolutionary rates of characters related to this timeframe were lowest, coinciding with a phylotypic period that persisted well beyond the pharyngula ‘stage’. The trajectory culminated with elevated variation in a handful of fetal and perinatal characters, yielding an irregular hourglass pattern. Our study invites further quantification of ontogeny across diverse amniotes and thus challenges current ideas on the universality of developmental patterns.

Computational identification of cell-specific variable regions in ChIP-seq data.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is used to identify genome-wide DNA regions bound by proteins. Given one ChIP-seq experiment with replicates, binding sites not observed in all the replicates will usually be interpreted as noise and discarded. However, the recent discovery of high-occupancy target (HOT) regions suggests that there are regions where binding of multiple transcription factors can be identified. To investigate ChIP-seq variability, we developed a reproducibility score and a method that identifies cell-specific variable regions in ChIP-seq data by integrating replicated ChIP-seq experiments for multiple protein targets on a particular cell type. Using our method, we found variable regions in human cell lines K562, GM12878, HepG2, MCF-7 and in mouse embryonic stem cells (mESCs). These variable-occupancy target regions (VOTs) are CG dinucleotide rich, and show enrichment at promoters and R-loops. They overlap significantly with HOT regions, but are not blacklisted regions producing non-specific binding ChIP-seq peaks. Furthermore, in mESCs, VOTs are conserved among placental species suggesting that they could have a function important for this taxon. Our method can be useful to point to such regions along the genome in a given cell type of interest, to improve the downstream interpretative analysis before follow-up experiments.

ACE2 diversity in placental mammals reveals the evolutionary strategy of SARS-CoV-2.

The recent emergence of SARS-CoV-2 is responsible for the current pandemic of COVID-19, which uses the human membrane protein ACE2 as a gateway to host-cell infection. We performed a comparative genomic analysis of 70 ACE2 placental mammal orthologues to identify variations and contribute to the understanding of evolutionary dynamics behind this successful adaptation to infect humans. Our results reveal that 4% of the ACE2 sites are under positive selection, all located in the catalytic domain, suggesting possibly taxon-specific adaptations related to the ACE2 function, such as cardiovascular physiology. Considering all variable sites, we selected 30 of them located at the critical ACE2 binding sites to the SARS-CoV-like viruses for analysis in more detail. Our results reveal a relatively high diversity of ACE2 between placental mammal species, while showing no polymorphism within human populations, at least considering the 30 inter-species variable sites. A perfect scenario for natural selection favored this opportunistic new coronavirus in its trajectory of infecting humans. We suggest that SARS-CoV-2 became a specialist coronavirus for human hosts. Differences in the rate of infection and mortality could be related to the innate immune responses, other unknown genetic factors, as well as non-biological factors.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells from the Tasmanian Devil (Sarcophilus harrisii) Express Immunomodulatory Factors and a Tropism Toward Devil Facial Tumor Cells.

Marsupials have long attracted scientific interest because of their unique biological features and their position in mammalian evolution. Mesenchymal stem cells (MSCs) are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory, and regenerative properties. MSCs have been harvested from various tissues in numerous eutherian species; however, there are no descriptions of MSCs derived from a marsupial. In this study, we have generated Tasmanian devil (Sarcophilus harrisii) MSCs from devil induced pluripotent stem cells (iPSCs), thus providing an unlimited source of devil MSCs and circumventing the need to harvest tissues from live animals. Devil iPSCs were differentiated into MSCs (iMSCs) through both embryoid body formation assays (EB-iMSCs) and through inhibition of the transforming growth factor beta/activin signaling pathway (SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly proliferative and express the MSC-specific surface proteins CD73, CD90, and CD105, in addition to the pluripotency transcription factors OCT4/POU5F1, SOX2, and NANOG. Expression of the marsupial pluripotency factor POU5F3, a paralogue of OCT4/POU5F1, is significantly reduced in association with the transition from pluripotency to multipotency. Devil iMSCs readily differentiate along the adipogenic, osteogenic, and chondrogenic pathways in vitro, confirming their trilineage differentiation potential. Importantly, in vitro teratoma assays confirmed their multipotency, rather than pluripotency, since the iMSCs only formed derivatives of the mesodermal germ layer. Devil iMSCs show a tropism toward medium conditioned by devil facial tumor cells and express a range of immunomodulatory and anti-inflammatory factors. Therefore, devil iMSCs will be a valuable tool for further studies on marsupial biology and may facilitate the development of an MSC-based treatment strategy against Devil Facial Tumor Disease.

Homeobox genes in endometrium: from development to decidualization.

The eutherian species evolved an elaborate uterus to allow viviparity. For successful pregnancy, the uterus must not only be differentiated, but must also function optimally and any defects in uterus differentiation and/or function can lead to infertility. The homoebox gene HOXA10 has emerged to be a key player in both uterine development and its optimal functioning in adulthood. Within the Abd-B family, the posterior Hoxa genes play a dominant role in anterio-posterior segmentation of the Müllerian ducts in mammals, with Hoxa10 having a central role in uterine segmentation. In the adult endometrium, HOXA10 is expressed by endometrial cells and is regulated in a cyclic manner under the influence of ovarian steroids. During embryo implantation, expression of HOXA10 is increased in endometrial stromal cells by signals from the embryo to govern stromal cell transformation to decidual cells. Once decidualization is initiated, HOXA10 is rapidly downregulated to activate expression of pro-invasive factors to promote trophoblast invasion. We propose that HOXA10 governs embryo implantation in a three-step process: 1) acquisition of endometrial receptivity, 2) responding to signals from the blastocyst to modify receptive endometrium for decidualization 3) making the decidua conductive for trophoblast invasion and placentation. There is currently ample evidence that expression of HOXA10 is deregulated in a variety of "endometriopathies" such as endometriosis and endometrial cancers. Overall, HOXA10 appears to be the master regulator of endometrial health and a central determinant of fertility in mammals.

Abstract 017: Cytolytic Natural Killer Cells Play a Direct Role in Causing Hypertension and Intrauterine Growth Restriction in Pregnant Rats

The R educed U terine P erfusion P ressure (RUPP) rat model of placental ischemia mirrors many of the hallmark features of preeclampsia (PE) such as hypertension, intrauterine growth restriction (IUGR), and immune activation, which includes increased cytolytic natural killer cells (cNKs). We have previously demonstrated that depletion of NK cells in RUPP rats improves PE pathophysiology. In this study we tested the hypothesis that RUPP-stimulated NKs have increased cytotoxicity and play a direct role in inflammation, oxidative stress, hypertension, and intrauterine growth restriction in pregnant rats. NK cells were isolated from the placentas of normal pregnant (NP) and RUPP rats on gestation day (GD) 19 and assessed for cytotoxic activity. RUPP NK cells demonstrated a 5-fold increase in cytotoxic activity vs NP NK cells. On GD 12, 5x10 6 NK cells from RUPP rats were infused into a subset of NP rats. On GD 19, circulating and placental total NK cells and cNKs were quantified via flow cytometry in NP, RUPP, and NP+RUPP NK rats. MAP, fetal and placental weights, placental reactive oxygen species (ROS), and placental cytokines were also measured. Placental cNK cells (%gated) were significantly increased in RUPP (18.5±3.6%) and NP+RUPP NK (16.1±3.6%) compared to NP (4.1±0.8%) controls. Circulating cNKs followed a similar trend. MAP increased from 100 mmHg in NP to 126 mmHg in RUPP, and 119 mmHg in NP+RUPP NK (p<0.05 vs NP). Fetal weight decreased from 2.4±0.03 g in NP to 2.0±0.04 g in RUPP and to 2.1±0.02 g in NP+RUPP NK (p<0.05 vs NP). Placental weights followed a similar trend. Placental cNK cytokines were significantly increased in RUPP and in NP recipients of RUPP NK cells: TNF-α: NP- 23±3.1, RUPP- 62.3±7.8 pg/mg, NP+RUPP NK-43.9±5.7 pg/mg (p<0.05 vs NP); IFNγ: NP- 3.3±0.7, RUPP- 7.6±1.2 pg/mg, NP+RUPP NK-6.6±1.9 pg/mg (p<0.05 vs NP). Placental VEGF decreased from 6.5±1.3 pg/mg in NP to 2±0.8pg/mg in RUPP and 2.9±0.6 pg/mg in NP+RUPP NK. Circulating levels followed a similar trend. Placental ROS significantly increased from 27.3±4.6 RLUs/min/mg in NP to 63.1±5.7 RLUs/min/mg in RUPP and 84±17.6 RLUs/min/mg in NP+RUPP NK. These data demonstrate a direct role for cNKs in causing PE pathophysiology and identifies cNKs as a novel therapeutic target for treatment of PE.

Odontogenic ameloblast-associated (ODAM) is inactivated in toothless/enamelless placental mammals and toothed whales

BackgroundThe gene for odontogenic ameloblast-associated (ODAM) is a member of the secretory calcium-binding phosphoprotein gene family. ODAM is primarily expressed in dental tissues including the enamel organ and the junctional epithelium, and may also have pleiotropic functions that are unrelated to teeth. Here, we leverage the power of natural selection to test competing hypotheses that ODAM is tooth-specific versus pleiotropic. Specifically, we compiled and screened complete protein-coding sequences, plus sequences for flanking intronic regions, for ODAM in 165 placental mammals to determine if this gene contains inactivating mutations in lineages that either lack teeth (baleen whales, pangolins, anteaters) or lack enamel on their teeth (aardvarks, sloths, armadillos), as would be expected if the only essential functions of ODAM are related to tooth development and the adhesion of the gingival junctional epithelium to the enamel tooth surface.ResultsWe discovered inactivating mutations in all species of placental mammals that either lack teeth or lack enamel on their teeth. A surprising result is that ODAM is also inactivated in a few additional lineages including all toothed whales that were examined. We hypothesize that ODAM inactivation is related to the simplified outer enamel surface of toothed whales. An alternate hypothesis is that ODAM inactivation in toothed whales may be related to altered antimicrobial functions of the junctional epithelium in aquatic habitats. Selection analyses on ODAM sequences revealed that the composite dN/dS value for pseudogenic branches is close to 1.0 as expected for a neutrally evolving pseudogene. DN/dS values on transitional branches were used to estimate ODAM inactivation times. In the case of pangolins, ODAM was inactivated ~ 65 million years ago, which is older than the oldest pangolin fossil (Eomanis, 47 Ma) and suggests an even more ancient loss or simplification of teeth in this lineage.ConclusionOur results validate the hypothesis that the only essential functions of ODAM that are maintained by natural selection are related to tooth development and/or the maintenance of a healthy junctional epithelium that attaches to the enamel surface of teeth.

Morphological adaptation of the eutherian gastrointestinal tract to diet

Although adaptations of the gastrointestinal tract to diet have been widely documented, statistical evaluations of corresponding correlations between diet and morphology are scarce. We use a dataset that scores the complexity of the stomach, caecum and colon in 599 Eutherian species, with a higher complexity score indicating a higher degree of macroanatomical differentiation, and link these data to information on body mass and the estimated fibre content of the natural diet, controlling for phylogeny. The results indicate that dietary niche differentiation occurs along, rather than within, major Eutherian lineages, and that stomach and large intestine complexity are often positively correlated. Large intestine complexity represents a convergent adaptation to high-fibre diets across lineages (although not all species ingesting high-fibre diets have complex large intestines). By contrast, stomach complexity cannot be linked to diet, but represents a taxon-specific signal in the sense of homoplasies. In particular, the functions of stomach complexity in the smallest and the largest Eutheria with complex stomachs (the Myomorpha and the Cetacea) remain to be elucidated.

Morphological adaptation of the eutherian gastrointestinaltract to diet

Although adaptations of the gastrointestinal tract to diet have been widely documented, statistical evaluations of corresponding correlations between diet and morphology are scarce. We use a dataset that scores the complexity of the stomach, caecum and colon in 599 Eutherian species, with a higher complexity score indicating a higher degree of macroanatomical differentiation, and link these data to information on body mass and the estimated fibre content of the natural diet, controlling for phylogeny. The results indicate that dietary niche differentiation occurs along, rather than within, major Eutherian lineages, and that stomach and large intestine complexity are often positively correlated. Large intestine complexity represents a convergent adaptation to high-fibre diets across lineages (although not all species ingesting high-fibre diets have complex large intestines). By contrast, stomach complexity cannot be linked to diet, but represents a taxon-specific signal in the sense of homoplasies. In particular, the functions of stomach complexity in the smallest and the largest Eutheria with complex stomachs (the Myomorpha and the Cetacea) remain to be elucidated .

Label-Retaining, Putative Mesenchymal Stem Cells Contribute to Murine Myometrial Repair During Uterine Involution.

Uterine remodeling during pregnancy is a fundamental, dynamic process required for successful propagation of eutherian species. The uterus can increase in size up to 40-fold during pregnancy, which is largely attributed to expansion of the myometrium by hyperplasia and hypertrophy. After pregnancy, the uterus repairs the remodeled or "damaged" tissue during uterine involution (INV). Little is known about this repair process, particularly the role of mesenchymal stem/progenitor cells. The objective of this study was to identify and characterize putative mesenchymal stem/progenitor cells in the murine myometrium using a combination of label retention and mesenchymal stem cell (MSC) marker expression and a pregnancy and uterine INV model. Tet-off transgenic mice with the Cre-lox system were used to specifically label mesenchymal cells (ie, myometrial and endometrial stromal cells) within the uterus while avoiding other cell types (eg, epithelial, immune, and endothelial cells) to identify slowly dividing cells and assess their stem cell qualities. We identified myometrial label-retaining cells (LRCs) that persisted for at least 3 months, expressed CD146 and CD140b (MSC markers), and proliferated at a higher rate during uterine INV compared with nonlabeled cells. The LRCs did not appear to express either estrogen receptor alpha or progesterone receptor, nor did the number of LRCs change at different estrous stages or in response to exogenous estradiol or progesterone administration, suggesting that LRCs were not involved in normal estrous cycling. The results from this study provide important insight into putative stem/progenitor cells in the myometrium and their possible role in uterine physiology.

Single cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development.

ABSTRACTThe mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis.

Serial Homology and Correlated Characters in Morphological Phylogenetics: Modeling the Evolution of Dental Crests in Placentals.

Accurate modeling of the complexity of morphological evolution is crucial for morphological phylogenetics and for performing tests on a wide variety of evolutionary scenarios. In this context, morphological integration and the problem of correlated categorical characters represent a major challenge. In particular, the magnitude and implications of correlations among serially homologous structures such as teeth have been much debated but were never tested statistically within a broad phylogenetic context. Here, we present a large-scale empirical study analyzing the serial variation of cingular crests on successive molars (M1, M2, and M3) of 274 placental species in a phylogenetic context. Both likelihood analyses and analysis of phylogenetic co-distributions demonstrated highly correlated evolution in the entire sample and thus the non-independence of these serial features at a macroevolutionary scale. Likelihood analyses show that their serial variation should be better scored within a single composite character model with constrained paths for transitions enabling simultaneous changes on all three molars, which suggests a strong developmental or genetic integration. These results are congruent with current genetic and developmental knowledge related to dental morphological variation and call into question the frequent use of separate characters scored on serially homologous structures of the dentition in phylogenetic analyses. Overall, they provide long-overdue and clear empirical evidence that in-depth studies of patterns of integration constitute an essential step toward more realistic character construction and modeling. This approach is critical for more accurate morphological phylogenetics and, more generally, for testing macroevolutionary scenarios on groups of correlated characters.

A pan-mammalian map of interhemispheric brain connections predates the evolution of the corpus callosum

The brain of mammals differs from that of all other vertebrates, in having a six-layered neocortex that is extensively interconnected within and between hemispheres. Interhemispheric connections are conveyed through the anterior commissure in egg-laying monotremes and marsupials, whereas eutherians evolved a separate commissural tract, the corpus callosum. Although the pattern of interhemispheric connectivity via the corpus callosum is broadly shared across eutherian species, it is not known whether this pattern arose as a consequence of callosal evolution or instead corresponds to a more ancient feature of mammalian brain organization. Here we show that, despite cortical axons using an ancestral commissural route, monotremes and marsupials share features of interhemispheric connectivity with eutherians that likely predate the origin of the corpus callosum. Based on ex vivo magnetic resonance imaging and tractography, we found that connections through the anterior commissure in both fat-tailed dunnarts (Marsupialia) and duck-billed platypus (Monotremata) are spatially segregated according to cortical area topography. Moreover, cell-resolution retrograde and anterograde interhemispheric circuit mapping in dunnarts revealed several features shared with callosal circuits of eutherians. These include the layered organization of commissural neurons and terminals, a broad map of connections between similar (homotopic) regions of each hemisphere, and regions connected to different areas (heterotopic), including hyperconnected hubs along the medial and lateral borders of the cortex, such as the cingulate/motor cortex and claustrum/insula. We therefore propose that an interhemispheric connectome originated in early mammalian ancestors, predating the evolution of the corpus callosum. Because these features have been conserved throughout mammalian evolution, they likely represent key aspects of neocortical organization.

Characterization of the complete mitochondrial genome of the common smoothhound shark, Mustelus mustelus (Carcharhiniformes: Triakidae)

We present the complete mitochondrial genome of the common smoothhound , Mustelus mustelus,which is 16,755 bp long, contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and non-coding control region. All protein-coding genes begin with the ATG codon, except for the COI gene, which begins with GTG. Six protein-coding genes terminated with the TAA codon, and six with incomplete codons, T or TA. The phylogenetic reconstruction places M. mustelus within the genus Mustelus, with the closest relationship to the placental species, M. griseus. This mitogenome provides valuable information to further unravel the evolution of alternate reproductive modes within the genus.