Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Enhancer evolution across 20 mammalian species.

The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution.

Fossils document evolutionary changes of jaw joint to mammalian middle ear.

The dual jaw joint of Morganucodon1,2 consists of the dentary-squamosal joint laterally and the articular-quadrate one medially. The articular-quadrate joint and its associated post-dentary bones constitute the precursor of the mammalian middle ear. Fossils documenting the transition from such a precursor to the mammalian middle ear are poor, resulting in inconsistent interpretations of this hallmark apparatus in the earliest stage of mammaliaform evolution1-5. Here we report mandibular middle ears from two Jurassic mammaliaforms: a new morganucodontan-like species and a pseudotribosphenic shuotheriid species6. The morganucodontan-like species shows many previously unknown post-dentary bone morphologies1,2 and exhibits features that suggest a loss of load-bearing function in its articular-quadrate joint. The middle ear of the shuotheriid approaches the mammalian condition in that it has features that are suitable for an exclusively auditory function, although the post-dentary bones are still attached to the dentary. With size reduction of the jaw-joint bones, the quadrate shifts medially at different degrees in relation to the articular in the two mammaliaforms. These changes provide evidence of a gradual loss of load-bearing function in the articular-quadrate jaw joint-a prerequisite for the detachment of the post-dentary bones from the dentary7-12 and the eventual breakdown of the Meckel's cartilage13-15 during the evolution of mammaliaforms.

Jurassic shuotheriids show earliest dental diversification of mammaliaforms.

Shuotheriids are Jurassic mammaliaforms that possess pseudotribosphenic teeth in which a pseudotalonid is anterior to the trigonid in the lower molar, contrasting with the tribosphenic pattern of therian mammals (placentals, marsupials and kin) in which the talonid is posterior to the trigonid1-4. The origin of the pseudotribosphenic teeth remains unclear, obscuring our perception of shuotheriid affinities and the early evolution of mammaliaforms1,5-9. Here we report a new Jurassic shuotheriid represented by two skeletal specimens. Their complete pseudotribosphenic dentitions allow reidentification of dental structures using serial homology and the tooth occlusal relationship. Contrary to the conventional view1,2,6,10,11, our findings show that dental structures of shuotheriids can be homologized to those of docodontans and partly support homologous statements for some dental structures between docodontans and other mammaliaforms6,12. The phylogenetic analysis based on new evidence removes shuotheriids from the tribosphenic ausktribosphenids (including monotremes) and clusters them with docodontans to form a new clade, Docodontiformes, that is characterized by pseudotribosphenic features. In the phylogeny, docodontiforms and 'holotherians' (Kuehneotherium, monotremes and therians)13 evolve independently from a Morganucodon-like ancestor with triconodont molars by labio-lingual widening their posterior teeth for more efficient food processing. The pseudotribosphenic pattern passed a cusp semitriangulation stage9, whereas the tribosphenic pattern and its precursor went through a stage of cusp triangulation. The two different processes resulted in complex tooth structures and occlusal patterns that elucidate the earliest diversification of mammaliaforms.

Identification of constrained sequence elements across 239 primate genomes.

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

FSC-certified forest management benefits large mammals compared to non-FSC.

More than a quarter of the world's tropical forests are exploited for timber1. Logging impacts biodiversity in these ecosystems, primarily through the creation of forest roads that facilitate hunting for wildlife over extensive areas. Forest management certification schemes such as the Forest Stewardship Council (FSC) are expected to mitigate impacts on biodiversity, but so far very little is known about the effectiveness of FSC certification because of research design challenges, predominantly limited sample sizes2,3. Here we provide this evidence by using 1.3 million camera-trap photos of 55 mammal species in 14 logging concessions in western equatorial Africa. We observed higher mammal encounter rates in FSC-certified than in non-FSC logging concessions. The effect was most pronounced for species weighing more than 10 kg and for species of high conservation priority such as the critically endangered forest elephant and western lowland gorilla. Across the whole mammal community, non-FSC concessions contained proportionally more rodents and other small species than did FSC-certified concessions. The first priority for species protection should be to maintain unlogged forests with effective law enforcement, but for logged forests our findings provide convincing data that FSC-certified forest management is less damaging to the mammal community than is non-FSC forest management. This study provides strong evidence that FSC-certified forest management or equivalently stringent requirements and controlling mechanisms should become the norm for timber extraction to avoid half-empty forests dominated by rodents and other small species.

A species-level timeline of mammal evolution integrating phylogenomic data.

High-throughput sequencing projects generate genome-scale sequence data for species-level phylogenies1-3. However, state-of-the-art Bayesian methods for inferring timetrees are computationally limited to small datasets and cannot exploit the growing number of available genomes4. In the case of mammals, molecular-clock analyses of limited datasets have produced conflicting estimates of clade ages with large uncertainties5,6, and thus the timescale of placental mammal evolution remains contentious7-10. Here we develop a Bayesian molecular-clock dating approach to estimate a timetree of 4,705 mammal species integrating information from 72 mammal genomes. We show that increasingly larger phylogenomic datasets produce diversification time estimates with progressively smaller uncertainties, facilitating precise tests of macroevolutionary hypotheses. For example, we confidently reject an explosive model of placental mammal origination in the Palaeogene8 and show that crown Placentalia originated in the Late Cretaceous with unambiguous ordinal diversification in the Palaeocene/Eocene. Our Bayesian methodology facilitates analysis of complete genomes and thousands of species within an integrated framework, making it possible to address hitherto intractable research questions on species diversifications. This approach can be used to address other contentious cases of animal and plant diversifications that require analysis of species-level phylogenomic datasets.

Climate change increases cross-species viral transmission risk.

At least 10,000 virus species have the ability to infect humans but, at present, the vast majority are circulating silently in wild mammals1,2. However, changes in climate and land use will lead to opportunities for viral sharing among previously geographically isolated species of wildlife3,4. In some cases, this will facilitate zoonotic spillover-a mechanistic link between global environmental change and disease emergence. Here we simulate potential hotspots of future viral sharing, using a phylogeographical model of the mammal-virus network, and projections of geographical range shifts for 3,139 mammal species under climate-change and land-use scenarios for the year 2070. We predict that species will aggregate in new combinations at high elevations, in biodiversity hotspots, and in areas of high human population density in Asia and Africa, causing the cross-species transmission of their associated viruses an estimated 4,000 times. Owing to their unique dispersal ability, bats account for the majority of novel viral sharing and are likely to share viruses along evolutionary pathways that will facilitate future emergence in humans. Notably, we find that this ecological transition may already be underway, and holding warming under 2 °C within the twenty-first century will not reduce future viral sharing. Our findings highlight an urgent need to pair viral surveillance and discovery efforts with biodiversity surveys tracking the range shifts of species, especially in tropical regions that contain the most zoonoses and are experiencing rapid warming.

Cancer risk across mammals.

Cancer is a ubiquitous disease of metazoans, predicted to disproportionately affect larger, long-lived organisms owing to their greater number of cell divisions, and thus increased probability of somatic mutations1,2. While elevated cancer risk with larger body size and/or longevity has been documented within species3-5, Peto's paradox indicates the apparent lack of such an association among taxa6. Yet, unequivocal empirical evidence for Peto's paradox is lacking, stemming from the difficulty of estimating cancer risk in non-model species. Here we build and analyse a database on cancer-related mortality using data on adult zoo mammals (110,148 individuals, 191 species) and map age-controlled cancer mortality to the mammalian tree of life. We demonstrate the universality and high frequency of oncogenic phenomena in mammals and reveal substantial differences in cancer mortality across major mammalian orders. We show that the phylogenetic distribution of cancer mortality is associated with diet, with carnivorous mammals (especially mammal-consuming ones) facing the highest cancer-related mortality. Moreover, we provide unequivocal evidence for the body size and longevity components of Peto's paradox by showing that cancer mortality risk is largely independent of both body mass and adult life expectancy across species. These results highlight the key role of life-history evolution in shaping cancer resistance and provide major advancements in the quest for natural anticancer defences.

Large-scale across species transcriptomic analysis identifies genetic selection signatures associated with longevity in mammals.

Lifespan varies significantly among mammals, with more than 100-fold difference between the shortest and longest living species. This natural difference may uncover the evolutionary forces and molecular features that define longevity. To understand the relationship between gene expression variation and longevity, we conducted a comparative transcriptomics analysis of liver, kidney, and brain tissues of 103 mammalian species. We found that few genes exhibit common expression patterns with longevity in the three organs analyzed. However, pathways related to translation fidelity, such as nonsense-mediated decay and eukaryotic translation elongation, correlated with longevity across mammals. Analyses of selection pressure found that selection intensity related to the direction of longevity-correlated genes is inconsistent across organs. Furthermore, expression of methionine restriction-related genes correlated with longevity and was under strong selection in long-lived mammals, suggesting that a common strategy is utilized by natural selection and artificial intervention to control lifespan. Our results indicate that lifespan regulation via gene expression is driven through polygenic and indirect natural selection.

Fossoriality and evolutionary development in two Cretaceous mammaliamorphs.

Mammaliamorpha comprises the last common ancestor of Tritylodontidae and Mammalia plus all its descendants1. Tritylodontids are nonmammaliaform herbivorous cynodonts that originated in the Late Triassic epoch, diversified in the Jurassic period2-5 and survived into the Early Cretaceous epoch6,7. Eutriconodontans have generally been considered to be an extinct mammalian group, although different views exist8. Here we report a newly discovered tritylodontid and eutriconodontan from the Early Cretaceous Jehol Biota of China. Eutriconodontans are common in this biota9, but it was not previously known to contain tritylodontids. The two distantly related species show convergent features that are adapted for fossorial life, and are the first 'scratch-diggers' known from this biota. Both species also show an increased number of presacral vertebrae, relative to the ancestral state in synapsids or mammals10,11, that display meristic and homeotic changes. These fossils shed light on the evolutionary development of the axial skeleton in mammaliamorphs, which has been the focus of numerous studies in vertebrate evolution12-17 and developmental biology18-28. The phenotypes recorded by these fossils indicate that developmental plasticity in somitogenesis and HOX gene expression in the axial skeleton-similar to that observed in extant mammals-was already in place in stem mammaliamorphs. The interaction of these developmental mechanisms with natural selection may have underpinned the diverse phenotypes of body plan that evolved independently in various clades of mammaliamorph.

The timing and effect of the earliest human arrivals in North America.

The peopling of the Americas marks a major expansion of humans across the planet. However, questions regarding the timing and mechanisms of this dispersal remain, and the previously accepted model (termed 'Clovis-first')-suggesting that the first inhabitants of the Americas were linked with the Clovis tradition, a complex marked by distinctive fluted lithic points1-has been effectively refuted. Here we analyse chronometric data from 42 North American and Beringian archaeological sites using a Bayesian age modelling approach, and use the resulting chronological framework to elucidate spatiotemporal patterns of human dispersal. We then integrate these patterns with the available genetic and climatic evidence. The data obtained show that humans were probably present before, during and immediately after the Last Glacial Maximum (about 26.5-19 thousand years ago)2,3 but that more widespread occupation began during a period of abrupt warming, Greenland Interstadial 1 (about 14.7-12.9 thousand years before AD 2000)4. We also identify the near-synchronous commencement of Beringian, Clovis and Western Stemmed cultural traditions, and an overlap of each with the last dates for the appearance of 18 now-extinct faunal genera. Our analysis suggests that the widespread expansion of humans through North America was a key factor in the extinction of large terrestrial mammals.

Skeleton of a Cretaceous mammal from Madagascar reflects long-term insularity.

The fossil record of mammaliaforms (mammals and their closest relatives) of the Mesozoic era from the southern supercontinent Gondwana is far less extensive than that from its northern counterpart, Laurasia1,2. Among Mesozoic mammaliaforms, Gondwanatheria is one of the most poorly known clades, previously represented by only a single cranium and isolated jaws and teeth1-5. As a result, the anatomy, palaeobiology and phylogenetic relationships of gondwanatherians remain unclear. Here we report the discovery of an articulated and very well-preserved skeleton of a gondwanatherian of the latest age (72.1-66 million years ago) of the Cretaceous period from Madagascar that we assign to a new genus and species, Adalatherium hui. To our knowledge, the specimen is the most complete skeleton of a Gondwanan Mesozoic mammaliaform that has been found, and includes the only postcranial material and ascending ramus of the dentary known for any gondwanatherian. A phylogenetic analysis including the new taxon recovers Gondwanatheria as the sister group to Multituberculata. The skeleton, which represents one of the largest of the Gondwanan Mesozoic mammaliaforms, is particularly notable for exhibiting many unique features in combination with features that are convergent on those of therian mammals. This uniqueness is consistent with a lineage history for A. hui of isolation on Madagascar for more than 20 million years.

OrthoMaM v12: a database of curated single-copy ortholog alignments and trees to study mammalian evolutionary genomics.

To date, the databases built to gather information on gene orthology do not provide end-users with descriptors of the molecular evolution information and phylogenetic pattern of these orthologues. In this context, we developed OrthoMaM, a database of ORTHOlogous MAmmalian Markers describing the evolutionary dynamics of coding sequences in mammalian genomes. OrthoMaM version 12 includes 15,868 alignments of orthologous coding sequences (CDS) from the 190 complete mammalian genomes currently available. All annotations and 1-to-1 orthology assignments are based on NCBI. Orthologous CDS can be mined for potential informative markers at the different taxonomic levels of the mammalian tree. To this end, several evolutionary descriptors of DNA sequences are provided for querying purposes (e.g. base composition and relative substitution rate). The graphical web interface allows the user to easily browse and sort the results of combined queries. The corresponding multiple sequence alignments and ML trees, inferred using state-of-the art approaches, are available for download both at the nucleotide and amino acid levels. OrthoMaM v12 can be used by researchers interested either in reconstructing the phylogenetic relationships of mammalian taxa or in understanding the evolutionary dynamics of coding sequences in their genomes. OrthoMaM is available for browsing, querying and complete or filtered download at https://orthomam.mbb.cnrs.fr/.

The decoupled nature of basal metabolic rate and body temperature in endotherm evolution.

The origins of endothermy in birds and mammals are important events in vertebrate evolution. Endotherms can maintain their body temperature (Tb) over a wide range of ambient temperatures primarily using the heat that is generated continuously by their high basal metabolic rate (BMR)1. There is also an important positive feedback loop as Tb influences BMR1-3. Owing to this interplay between BMRs and Tb, many ecologists and evolutionary physiologists posit that the evolution of BMR and Tb must have been coupled during the radiation of endotherms3-5, changing with similar trends6-8. However, colder historical environments might have imposed strong selective pressures on BMR to compensate for increased rates of heat loss and to keep Tb constant9-12. Thus, adaptation to cold ambient temperatures through increases in BMR could have decoupled BMR from Tb and caused different evolutionary routes to the modern diversity in these traits. Here we show that BMR and Tb were decoupled in approximately 90% of mammalian phylogenetic branches and 36% of avian phylogenetic branches. Mammalian BMRs evolved with rapid bursts but without a long-term directional trend, whereas Tb evolved mostly at a constant rate and towards colder bodies from a warmer-bodied common ancestor. Avian BMRs evolved predominantly at a constant rate and without a long-term directional trend, whereas Tb evolved with much greater rate heterogeneity and with adaptive evolution towards colder bodies. Furthermore, rapid shifts that lead to both increases and decreases in BMRs were linked to abrupt changes towards colder ambient temperatures-although only in mammals. Our results suggest that natural selection effectively exploited the diversity in mammalian BMRs under diverse, often-adverse historical thermal environments.

The generality of cryptic dietary niche differences in diverse large-herbivore assemblages.

Ecological niche differences are necessary for stable species coexistence but are often difficult to discern. Models of dietary niche differentiation in large mammalian herbivores invoke the quality, quantity, and spatiotemporal distribution of plant tissues and growth forms but are agnostic toward food plant species identity. Empirical support for these models is variable, suggesting that additional mechanisms of resource partitioning may be important in sustaining large-herbivore diversity in African savannas. We used DNA metabarcoding to conduct a taxonomically explicit analysis of large-herbivore diets across southeastern Africa, analyzing ∼4,000 fecal samples of 30 species from 10 sites in seven countries over 6 y. We detected 893 food plant taxa from 124 families, but just two families-grasses and legumes-accounted for the majority of herbivore diets. Nonetheless, herbivore species almost invariably partitioned food plant taxa; diet composition differed significantly in 97% of pairwise comparisons between sympatric species, and dissimilarity was pronounced even between the strictest grazers (grass eaters), strictest browsers (nongrass eaters), and closest relatives at each site. Niche differentiation was weakest in an ecosystem recovering from catastrophic defaunation, indicating that food plant partitioning is driven by species interactions, and was stronger at low rainfall, as expected if interspecific competition is a predominant driver. Diets differed more between browsers than grazers, which predictably shaped community organization: Grazer-dominated trophic networks had higher nestedness and lower modularity. That dietary differentiation is structured along taxonomic lines complements prior work on how herbivores partition plant parts and patches and suggests that common mechanisms govern herbivore coexistence and community assembly in savannas.

Global assessment of effective population sizes: Consistent taxonomic differences in meeting the 50/500 rule.

Effective population size (Ne) is a particularly useful metric for conservation as it affects genetic drift, inbreeding and adaptive potential within populations. Current guidelines recommend a minimum Ne of 50 and 500 to avoid short-term inbreeding and to preserve long-term adaptive potential respectively. However, the extent to which wild populations reach these thresholds globally has not been investigated, nor has the relationship between Ne and human activities. Through a quantitative review, we generated a dataset with 4610 georeferenced Ne estimates from 3829 populations, extracted from 723 articles. These data show that certain taxonomic groups are less likely to meet 50/500 thresholds and are disproportionately impacted by human activities; plant, mammal and amphibian populations had a <54% probability of reaching N ̂ e = 50 and a <9% probability of reaching N ̂ e = 500. Populations listed as being of conservation concern according to the IUCN Red List had a smaller median N ̂ e than unlisted populations, and this was consistent across all taxonomic groups. N ̂ e was reduced in areas with a greater Global Human Footprint, especially for amphibians, birds and mammals, however relationships varied between taxa. We also highlight several considerations for future works, including the role that gene flow and subpopulation structure plays in the estimation of N ̂ e in wild populations, and the need for finer-scale taxonomic analyses. Our findings provide guidance for more specific thresholds based on Ne and help prioritise assessment of populations from taxa most at risk of failing to meet conservation thresholds.

Late-surviving stem mammal links the lowermost Cretaceous of North America and Gondwana.

Haramiyida was a successful clade of mammaliaforms, spanning the Late Triassic period to at least the Late Jurassic period, but their fossils are scant outside Eurasia and Cretaceous records are controversial1-4. Here we report, to our knowledge, the first cranium of a large haramiyidan from the basal Cretaceous of North America. This cranium possesses an amalgam of stem mammaliaform plesiomorphies and crown mammalian apomorphies. Moreover, it shows dental traits that are diagnostic of isolated teeth of supposed multituberculate affinities from the Cretaceous of Morocco, which have been assigned to the enigmatic 'Hahnodontidae'. Exceptional preservation of this specimen also provides insights into the evolution of the ancestral mammalian brain. We demonstrate the haramiyidan affinities of Gondwanan hahnodontid teeth, removing them from multituberculates, and suggest that hahnodontid mammaliaforms had a much wider, possibly Pangaean distribution during the Jurassic-Cretaceous transition.

Phylogenomic conflict coincides with rapid morphological innovation.

Evolutionary biologists have long been fascinated with the episodes of rapid phenotypic innovation that underlie the emergence of major lineages. Although our understanding of the environmental and ecological contexts of such episodes has steadily increased, it has remained unclear how population processes contribute to emergent macroevolutionary patterns. One insight gleaned from phylogenomics is that gene-tree conflict, frequently caused by population-level processes, is often rampant during the origin of major lineages. With the understanding that phylogenomic conflict is often driven by complex population processes, we hypothesized that there may be a direct correspondence between instances of high conflict and elevated rates of phenotypic innovation if both patterns result from the same processes. We evaluated this hypothesis in six clades spanning vertebrates and plants. We found that the most conflict-rich regions of these six clades also tended to experience the highest rates of phenotypic innovation, suggesting that population processes shaping both phenotypic and genomic evolution may leave signatures at deep timescales. Closer examination of the biological significance of phylogenomic conflict may yield improved connections between micro- and macroevolution and increase our understanding of the processes that shape the origin of major lineages across the Tree of Life.

Molecular Clocks without Rocks: New Solutions for Old Problems.

Molecular data have been used to date species divergences ever since they were described as documents of evolutionary history in the 1960s. Yet, an inadequate fossil record and discordance between gene trees and species trees are persistently problematic. We examine how, by accommodating gene tree discordance and by scaling branch lengths to absolute time using mutation rate and generation time, multispecies coalescent (MSC) methods can potentially overcome these challenges. We find that time estimates can differ - in some cases, substantially - depending on whether MSC methods or traditional phylogenetic methods that apply concatenation are used, and whether the tree is calibrated with pedigree-based mutation rates or with fossils. We discuss the advantages and shortcomings of both approaches and provide practical guidance for data analysis when using these methods.