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.

A comparative genomics multitool for scientific discovery and conservation.

The Zoonomia Project is investigating the genomics of shared and specialized traits in eutherian mammals. Here we provide genome assemblies for 131 species, of which all but 9 are previously uncharacterized, and describe a whole-genome alignment of 240 species of considerable phylogenetic diversity, comprising representatives from more than 80% of mammalian families. We find that regions of reduced genetic diversity are more abundant in species at a high risk of extinction, discern signals of evolutionary selection at high resolution and provide insights from individual reference genomes. By prioritizing phylogenetic diversity and making data available quickly and without restriction, the Zoonomia Project aims to support biological discovery, medical research and the conservation of biodiversity.

Divergent evolution of developmental timing in the neocortex revealed by marsupial and eutherian transcriptomes.

Only mammals evolved a neocortex, which integrates sensory-motor and cognitive functions. Significant diversifications in the cellular composition and connectivity of the neocortex occurred between the two main therian groups: marsupials and eutherians. However, the developmental mechanisms underlying these diversifications are largely unknown. Here, we compared the neocortical transcriptomes of Sminthopsis crassicaudata, a mouse-sized marsupial, with those of eutherian mice at two developmentally equivalent time points corresponding to deeper and upper layer neuron generation. Enrichment analyses revealed more mature gene networks in marsupials at the early stage, which reverted at the later stage, suggesting a more precocious but protracted neuronal maturation program relative to birth timing of cortical layers. We ranked genes expressed in different species and identified important differences in gene expression rankings between species. For example, genes known to be enriched in upper-layer cortical projection neuron subtypes, such as Cux1, Lhx2 and Satb2, likely relate to corpus callosum emergence in eutherians. These results show molecular heterochronies of neocortical development in Theria, and highlight changes in gene expression and cell type composition that may underlie neocortical evolution and diversification. This article has an associated 'The people behind the papers' interview.

An Early Cretaceous eutherian and the placental-marsupial dichotomy.

Molecular estimates of the divergence of placental and marsupial mammals and their broader clades (Eutheria and Metatheria, respectively) fall primarily in the Jurassic period. Supporting these estimates, Juramaia-the oldest purported eutherian-is from the early Late Jurassic (160 million years ago) of northeastern China. Sinodelphys-the oldest purported metatherian-is from the same geographic area but is 35 million years younger, from the Jehol biota. Here we report a new Jehol eutherian, Ambolestes zhoui, with a nearly complete skeleton that preserves anatomical details that are unknown from contemporaneous mammals, including the ectotympanic and hyoid apparatus. This new fossil demonstrates that Sinodelphys is a eutherian, and that postcranial differences between Sinodelphys and the Jehol eutherian Eomaia-previously thought to indicate separate invasions of a scansorial niche by eutherians and metatherians-are instead variations among the early members of the placental lineage. The oldest known metatherians are now not from eastern Asia but are 110 million years old from western North America, which produces a 50-million-year ghost lineage for Metatheria.

Solenodon genome reveals convergent evolution of venom in eulipotyphlan mammals.

Venom systems are key adaptations that have evolved throughout the tree of life and typically facilitate predation or defense. Despite venoms being model systems for studying a variety of evolutionary and physiological processes, many taxonomic groups remain understudied, including venomous mammals. Within the order Eulipotyphla, multiple shrew species and solenodons have oral venom systems. Despite morphological variation of their delivery systems, it remains unclear whether venom represents the ancestral state in this group or is the result of multiple independent origins. We investigated the origin and evolution of venom in eulipotyphlans by characterizing the venom system of the endangered Hispaniolan solenodon (Solenodon paradoxus). We constructed a genome to underpin proteomic identifications of solenodon venom toxins, before undertaking evolutionary analyses of those constituents, and functional assessments of the secreted venom. Our findings show that solenodon venom consists of multiple paralogous kallikrein 1 (KLK1) serine proteases, which cause hypotensive effects in vivo, and seem likely to have evolved to facilitate vertebrate prey capture. Comparative analyses provide convincing evidence that the oral venom systems of solenodons and shrews have evolved convergently, with the 4 independent origins of venom in eulipotyphlans outnumbering all other venom origins in mammals. We find that KLK1s have been independently coopted into the venom of shrews and solenodons following their divergence during the late Cretaceous, suggesting that evolutionary constraints may be acting on these genes. Consequently, our findings represent a striking example of convergent molecular evolution and demonstrate that distinct structural backgrounds can yield equivalent functions.

Diverse phylogenomic datasets uncover a concordant scenario of laurasiatherian interordinal relationships.

Resolving the interordinal relationships in the mammalian superorder Laurasiatheria has been among the most intractable problems in higher-level mammalian systematics, with many conflicting hypotheses having been proposed. The present study collected three different sources of genome-scale data with comprehensive taxon sampling of laurasiatherian species, including two protein-coding datasets (4,186 protein-coding genes for an amino acid dataset comprising 2,761,247 amino acid residues and a nucleotide dataset comprising 5,516,340 nucleotides from 1st and 2nd codon positions), an intronic dataset (1,210 introns comprising 1,162,723 nucleotides) and an ultraconserved elements (UCEs) dataset (1,246 UCEs comprising 1,946,472 nucleotides) from 40 species representing all six laurasiatherian orders and 7 non-laurasiatherian outgroups. Remarkably, phylogenetic trees reconstructed with the four datasets using different tree-building methods (RAxML, FastTree, ASTRAL and MP-EST) all supported the relationship (Eulipotyphla, (Chiroptera, ((Carnivora, Pholidota), (Cetartiodactyla, Perissodactyla)))). We find a resolution of interordinal relationships of Laurasiatheria among all types of markers used in the present study, and the likelihood ratio tests for tree comparisons confirmed that the present tree topology is the optimal hypothesis compared to other examined hypotheses. Jackknifing subsampling analyses demonstrate that the results of laurasiatherian tree reconstruction varied with the number of loci and ordinal representatives used, which are likely the two main contributors to phylogenetic disagreements of Laurasiatheria seen in previous studies. Our study provides significant insight into laurasiatherian evolution, and moreover, an important methodological strategy and reference for resolving phylogenies of adaptive radiation, which have been a long-standing challenge in the field of phylogenetics.

Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals.

Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These "molecular vestiges" provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

Beyond Brownian Motion and the Ornstein-Uhlenbeck Process: Stochastic Diffusion Models for the Evolution of Quantitative Characters.

Gaussian processes, such as Brownian motion and the Ornstein-Uhlenbeck process, have been popular models for the evolution of quantitative traits and are widely used in phylogenetic comparative methods. However, they have drawbacks that limit their utility. Here we describe new, non-Gaussian stochastic differential equation (diffusion) models of quantitative trait evolution. We present general methods for deriving new diffusion models and develop new software for fitting non-Gaussian evolutionary models to trait data. The theory of stochastic processes provides a mathematical framework for understanding the properties of current and future phylogenetic comparative methods. Attention to the mathematical details of models of trait evolution and diversification may help avoid some pitfalls when using stochastic processes to model macroevolution.

Speciation and gene flow in two sympatric small mammals from Madagascar, Microgale fotsifotsy and M. soricoides (Mammalia: Tenrecidae).

Madagascar's shrew tenrecs (Mammalia: Tenrecidae; Microgale, Nesogale) represent an excellent system for studying speciation. Most species are endemic to the island's eastern humid forests, a region renowned for high levels of biodiversity and a high rate of in situ diversification. We set out to understand the speciation dynamics in a clade of recently described taxa: Microgale fotsifotsy and M. soricoides, which have nearly identical distributions in the moist evergreen forest, and M. nasoloi, which occurs in the western dry deciduous forest. A phylogenetic analysis using mitochondrial DNA data recovered two distinct clades of M. fotsifotsy: a south clade that is sister to, and broadly sympatric with, M. soricoides, and a north clade that is sister to the dry-forest and distantly allopatric species M. nasoloi. To better understand this result, we analysed cranioskeletal measurements and performed demographic analyses using nuclear sequence data from ultraconserved elements. Nuclear data did not support a sister relationship between M. soricoides and the south clade of M. fotsifotsy but did demonstrate introgression between these clades, which probably explains the discordance between nuclear and mitochondrial phylogenies. Demographic analyses also revealed the absence of gene flow between the north and south clades of M. fotsifotsy. Morphometric data revealed several major differences between M. soricoides and M. fotsifotsy, as well as more subtle differences between the two clades of M. fotsifotsy. In light of these results, we treat the south clade of M. fotsifotsy as a new candidate species. Our findings demonstrate the utility of integrating multiple data types to understand complex speciation histories, and contribute to a growing body of evidence that species diversity on Madagascar is underestimated.

New insights from RADseq data on differentiation in the Hottentot golden mole species complex from South Africa.

Golden moles (Family Chrysochloridae) are small subterranean mammals, endemic to sub-Saharan Africa, and many of the 21 species are listed as threatened on the IUCN Red List. Most species have highly restricted ranges; however two species, the Hottentot golden mole (Amblysomus hottentotus) and the Cape golden mole (Chrysochloris asiatica) have relatively wide ranges. We recently uncovered cryptic diversity within A. hottentotus, through a phylogeographic analysis of this taxon using two mitochondrial gene regions and a nuclear intron. To further investigate this cryptic diversity, we generated nuclear SNP data from across the genome of A. hottentotus, by means of double-digest restriction-site associated DNA sequencing (ddRADSeq), and mapped reads to the Cape golden mole genome. We conducted a phylogenetic analysis and investigated population differentiation. Our results support the distinctiveness of A. h. meesteri. Furthermore, we provide evidence from nuclear SNPs in support of our previous finding that Central coastal samples represent a unique cryptic lineage that is highly divergent from A. h. pondoliae farther south. Although mtDNA suggests that Umtata may represent a unique lineage sister to A. h. longiceps, mito-nuclear discordance from our RADseq data indicate that these samples may instead be closer to A. h. pondoliae, and therefore may not represent a distinct lineage. We stress the importance of recognizing that understudied populations, such as that of Umtata, may represent populations or ESUs under threat and in need of conservation attention. We present a high-quality filtered SNP dataset, comprising thousands of SNPs, which may serve as a useful resource for future golden mole studies. We have thus added to the growing body of research demonstrating the power and utility of RADseq to investigate population differentiation.

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.

Ontogenetic and life history trait changes associated with convergent ecological specializations in extinct ungulate mammals.

Investigating life history traits in mammals is crucial to understand their survival in changing environments. However, these parameters are hard to estimate in a macroevolutionary context. Here we show that the use of dental ontogenetic parameters can provide clues to better understand the adaptive nature of phenotypic traits in extinct species such as South American notoungulates. This recently extinct order of mammals evolved in a context of important geological, climatic, and environmental variations. Interestingly, notoungulates were mostly herbivorous and acquired high-crowned teeth very early in their evolutionary history. We focused on the variations in crown height, dental eruption pattern, and associated body mass of 69 notoungulate taxa, placed in their phylogenetic and geological contexts. We showed that notoungulates evolved higher crowns several times between 45 and 20 Ma, independently of the variation in body mass. Interestingly, the independent acquisitions of ever-growing teeth were systematically accompanied by eruption of molars faster than permanent premolars. These repeated associations of dental innovations have never been documented for other mammals and raise questions on their significance and causal relationships. We suggest that these correlated changes could originate from ontogenetic adjustments favored by structural constraints, and may indicate accelerated life histories. Complementarily, these more durable and efficient dentitions could be selected to cope with important ingestions of abrasive particles in the context of intensified volcanism and increasing aridity. This study demonstrates that assessing both life history and ecological traits allows a better knowledge of the specializations of extinct mammals that evolved under strong environmental constraints.

Rapid morphological evolution in placental mammals post-dates the origin of the crown group.

Resolving the timing and pattern of early placental mammal evolution has been confounded by conflict among divergence date estimates from interpretation of the fossil record and from molecular-clock dating studies. Despite both fossil occurrences and molecular sequences favouring a Cretaceous origin for Placentalia, no unambiguous Cretaceous placental mammal has been discovered. Investigating the differing patterns of evolution in morphological and molecular data reveals a possible explanation for this conflict. Here, we quantified the relationship between morphological and molecular rates of evolution. We show that, independent of divergence dates, morphological rates of evolution were slow relative to molecular evolution during the initial divergence of Placentalia, but substantially increased during the origination of the extant orders. The rapid radiation of placentals into a highly morphologically disparate Cenozoic fauna is thus not associated with the origin of Placentalia, but post-dates superordinal origins. These findings predict that early members of major placental groups may not be easily distinguishable from one another or from stem eutherians on the basis of skeleto-dental morphology. This result supports a Late Cretaceous origin of crown placentals with an ordinal-level adaptive radiation in the early Paleocene, with the high relative rate permitting rapid anatomical change without requiring unreasonably fast molecular evolutionary rates. The lack of definitive Cretaceous placental mammals may be a result of morphological similarity among stem and early crown eutherians, providing an avenue for reconciling the fossil record with molecular divergence estimates for Placentalia.

Karyotype Analysis of the New Talpa Species Talpa aquitania (Talpidae; Insectivora) from Northern Spain.

Karyotypes of 3 male Talpa specimens from northern Spain were analyzed. The mesostyles of upper molars and cytochrome b sequence analysis identified these specimens as belonging to Talpa aquitania, a new Talpa species recently described from northern Spain and southern France. We describe here for the first time the karyotype of Talpa aquitania. Its diploid number is 2n = 34 and NFa = 64, and all chromosomes including the sex chromosomes are biarmed, either metacentric or submetacentric. G-banding demonstrated that the karyotypes of T. aquitania and T. occidentalis (the most closely related species) are almost identical. However, the karyotype of T. aquitania differs from the karyotypes of both T. europaea and T. occidentalis in that it has a medium-sized biarmed Y chromosome rather than a dot-like chromosome and that chromosome 16 is submetacentric in T. aquitania but has a small p-arm in both T. europaea and T. occidentalis. Pericentromeric C-bands were scarce and only clearly visible in a few chromosomal pairs. In addition, C-banding demonstrated that half of the 14p, the 16p, and the Y chromosome are all heterochromatic. rDNA genes were located at the secondary constriction in autosomal pair 3, a common feature in the karyotypes of all Talpa species. Hybridization signals for telomeric repeats were found on the telomeres and the pericentric regions of some chromosomes and co-localized in the secondary constriction of pair 3 with the rDNA genes. In conclusion, the karyotype of T. aquitania from northern Spain is very similar to the karyotype of other species belonging to the genus Talpa.

Molecular evolution of type II MAGE genes from ancestral MAGED2 gene and their phylogenetic resolution of basal mammalian clades.

Type II melanoma-associated antigens (MAGE) are a subgroup of about a dozen proteins found in various locations in the genome and expressed in normal tissues, thus are not related to cancer as the type I MAGE genes. This gene family exists as a single copy in non-mammals and monotremata, but found as two copies in metatherians and occur as a diverse group in all eutherians. Our studies suggest MAGED2 as the ancestor of this subfamily and the most likely evolutionary history of eutherian type II MAGE genes is hereby proposed based on synteny conservation, phylogenetic relations, genome location, homology conservation, and the protein and gene structures. Type II genes can be divided into two: those with 13 exons (MAGED1, MAGED2, TRO, and MAGED4) and those with only one exon (MAGEE1, MAGEE2, MAGEF1, NSMCE3, MAGEH1, MAGEL2, and NDN) with different evolutionary patterns. Our results suggest a need to change the gene nomenclature to MAGE1 (the ancestral gene), currently designated as LOC103095671 and LOC100935086, in opossum and Tasmanian devil, respectively, and MAGE2 (the duplicated one), currently designated as LOC100617402 and NDNL2, respectively, to avoid confusion. We reconstructed the phylogenetic relationships among 23 mammalian species using the combined sequences of MAGED1, MAGED2, MAGEL2, and NDN, because of their high divergence, and found high levels of support, being able to resolve the phylogenetic relationships among Euarchontoglires, Laurasiatheria, Afrotheria, and Xenarthra, as an example that small, but phylogenetically informative sequences, can be very useful for resolving basal mammalian clades.

[DNA molecular identification of Manis pentadactyla].

The study was aimed to establish a stable, accurate site specific PCR identification system to identify Manis pentadactyla and its adulterants using DNA molecular identification. The genomic DNA was extracted from experimental samples using the DNA extraction kit. The Cytb and CO Ⅰ genes were amplified using PCR and sequenced bi-directionally. Obtained sequences were assembled using the BioEdit software. The neighbor-joining tree was constructed by MEGA 6.0. Specific identification primers were designed according to the specific allelets, and PCR reaction system was optimized. The results indicated that the Cytb and CO Ⅰ sequence both were able to be used to identify M. pentadactyla and its adulterants. With the specific primers CO Ⅰ-S10/A5, the M. pentadactyla could be amplified a 400 bp DNA band when the annealing temperature ranged from 55 to 60 ℃ and the amount of DNA template ranged from 3 to 100 ng within 35 PCR cycles. However, other adulterants displayed no relevant bands. So that primers CO Ⅰ- S10 / A5 can be used to identify the M. pentadactyla with the adulterants.

Eutherians experienced elevated evolutionary rates in the immediate aftermath of the Cretaceous-Palaeogene mass extinction.

The effect of the Cretaceous-Palaeogene (K-Pg) mass extinction on the evolution of many groups, including placental mammals, has been hotly debated. The fossil record suggests a sudden adaptive radiation of placentals immediately after the event, but several recent quantitative analyses have reconstructed no significant increase in either clade origination rates or rates of character evolution in the Palaeocene. Here we use stochastic methods to date a recent phylogenetic analysis of Cretaceous and Palaeocene mammals and show that Placentalia likely originated in the Late Cretaceous, but that most intraordinal diversification occurred during the earliest Palaeocene. This analysis reconstructs fewer than 10 placental mammal lineages crossing the K-Pg boundary. Moreover, we show that rates of morphological evolution in the 5 Myr interval immediately after the K-Pg mass extinction are three times higher than background rates during the Cretaceous. These results suggest that the K-Pg mass extinction had a marked impact on placental mammal diversification, supporting the view that an evolutionary radiation occurred as placental lineages invaded new ecological niches during the Early Palaeocene.

A genomic timescale for placental mammal evolution.

The precise pattern and timing of speciation events that gave rise to all living placental mammals remain controversial. We provide a comprehensive phylogenetic analysis of genetic variation across an alignment of 241 placental mammal genome assemblies, addressing prior concerns regarding limited genomic sampling across species. We compared neutral genome-wide phylogenomic signals using concatenation and coalescent-based approaches, interrogated phylogenetic variation across chromosomes, and analyzed extensive catalogs of structural variants. Interordinal relationships exhibit relatively low rates of phylogenomic conflict across diverse datasets and analytical methods. Conversely, X-chromosome versus autosome conflicts characterize multiple independent clades that radiated during the Cenozoic. Genomic time trees reveal an accumulation of cladogenic events before and immediately after the Cretaceous-Paleogene (K-Pg) boundary, implying important roles for Cretaceous continental vicariance and the K-Pg extinction in the placental radiation.

Brawn before brains in placental mammals after the end-Cretaceous extinction.

Mammals are the most encephalized vertebrates, with the largest brains relative to body size. Placental mammals have particularly enlarged brains, with expanded neocortices for sensory integration, the origins of which are unclear. We used computed tomography scans of newly discovered Paleocene fossils to show that contrary to the convention that mammal brains have steadily enlarged over time, early placentals initially decreased their relative brain sizes because body mass increased at a faster rate. Later in the Eocene, multiple crown lineages independently acquired highly encephalized brains through marked growth in sensory regions. We argue that the placental radiation initially emphasized increases in body size as extinction survivors filled vacant niches. Brains eventually became larger as ecosystems saturated and competition intensified.

Phylogenomics and the Genetic Architecture of the Placental Mammal Radiation.

The genomes of placental mammals are being sequenced at an unprecedented rate. Alignments of hundreds, and one day thousands, of genomes spanning the rich living and extinct diversity of species offer unparalleled power to resolve phylogenetic controversies, identify genomic innovations of adaptation, and dissect the genetic architecture of reproductive isolation. We highlight outstanding questions about the earliest phases of placental mammal diversification and the promise of newer methods, as well as remaining challenges, toward using whole genome data to resolve placental mammal phylogeny. The next phase of mammalian comparative genomics will see the completion and application of finished-quality, gapless genome assemblies from many ordinal lineages and closely related species. Interspecific comparisons between the most hypervariable genomic loci will likely reveal large, but heretofore mostly underappreciated, effects on population divergence, morphological innovation, and the origin of new species.