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.

The developmental basis for scaling of mammalian tooth size.

When evolution leads to differences in body size, organs generally scale along. A well-known example of the tight relationship between organ and body size is the scaling of mammalian molar teeth. To investigate how teeth scale during development and evolution, we compared molar development from initiation through final size in the mouse and the rat. Whereas the linear dimensions of the rat molars are twice that of the mouse molars, their shapes are largely the same. Here, we focus on the first lower molars that are considered the most reliable dental proxy for size-related patterns due to their low within-species variability. We found that scaling of the molars starts early, and that the rat molar is patterned equally as fast but in a larger size than the mouse molar. Using transcriptomics, we discovered that a known regulator of body size, insulin-like growth factor 1 (Igf1), is more highly expressed in the rat molars compared to the mouse molars. Ex vivo and in vivo mouse models demonstrated that modulation of the IGF pathway reproduces several aspects of the observed scaling process. Furthermore, analysis of IGF1-treated mouse molars and computational modeling indicate that IGF signaling scales teeth by simultaneously enhancing growth and by inhibiting the cusp-patterning program, thereby providing a relatively simple mechanism for scaling teeth during development and evolution. Finally, comparative data from shrews to elephants suggest that this scaling mechanism regulates the minimum tooth size possible, as well as the patterning potential of large teeth.

From teeth to pad: tooth loss and development of keratinous structures in sirenians.

Sirenians are a well-known example of morphological adaptation to a shallow-water grazing diet characterized by a modified feeding apparatus and orofacial morphology. Such adaptations were accompanied by an anterior tooth reduction associated with the development of keratinized pads, the evolution of which remains elusive. Among sirenians, the recently extinct Steller's sea cow represents a special case for being completely toothless. Here, we used µ-CT scans of sirenian crania to understand how motor-sensor systems associated with tooth innervation responded to innovations such as keratinized pads and continuous dental replacement. In addition, we surveyed nine genes associated with dental reduction for signatures of loss of function. Our results reveal how patterns of innervation changed with modifications of the dental formula, especially continuous replacement in manatees. Both our morphological and genomic data show that dental development was not completely lost in the edentulous Steller's sea cows. By tracing the phylogenetic history of tooth innervation, we illustrate the role of development in promoting the innervation of keratinized pads, similar to the secondary use of dental canals for innervating neomorphic keratinized structures in other tetrapod groups.

Phylogenetic Signal and Bias in Paleontology.

An unprecedented amount of evidence now illuminates the phylogeny of living mammals and birds on the Tree of Life. We use this tree to measure the phylogenetic value of data typically used in paleontology (bones and teeth) from six data sets derived from five published studies. We ask three interrelated questions: 1) Can these data adequately reconstruct known parts of the Tree of Life? 2) Is accuracy generally similar for studies using morphology, or do some morphological data sets perform better than others? 3) Does the loss of non-fossilizable data cause taxa to occur in misleadingly basal positions? Adding morphology to DNA data sets usually increases congruence of resulting topologies to the well-corroborated tree, but this varies among morphological data sets. Extant taxa with a high proportion of missing morphological characters can greatly reduce phylogenetic resolution when analyzed together with fossils. Attempts to ameliorate this by deleting extant taxa missing morphology are prone to decreased accuracy due to long-branch artifacts. We find no evidence that fossilization causes extinct taxa to incorrectly appear at or near topologically basal branches. Morphology comprises the evidence held in common by living taxa and fossils, and phylogenetic analysis of fossils greatly benefits from inclusion of molecular and morphological data sampled for living taxa, whatever methods are used for phylogeny estimation. [Concatenation; fossilization; morphology; parsimony; systematics; taphonomy; total-evidence.].

Developmental origins and homologies of the hyracoid dentition.

Understanding the origins of morphological specializations in mammals is a key goal in evolutionary biology. It can be accomplished by studying dental homology, which is at the core of most evolutionary and developmental studies. Here, we focused on the evolution and development of the specialized dentition of hyraxes for which dental homologies have long been debated, and could have implications on early placental evolution. Specifically, we analysed dental mineralization sequences of the three living genera of hyraxes and 17 fossil species using X-ray computed microtomography. Our results point out the labile position of vestigial upper teeth on jaw bones in extant species, associated with the frequently unusual premolar shape of deciduous canines over 50 Ma of hyracoid evolution. We proposed two evolutionary and developmental hypotheses to explain these original hyracoid dental characteristics. (a) The presence of a vestigial teeth on the maxilla in front of a complex deciduous canine could be interpreted as extra-teeth reminiscent of early placental evolution or sirenians, an order phylogenetically close to hyracoids and showing five premolars. (b) These vestigial teeth could also correspond to third incisors with a position unusually shifted on the maxilla, which could be explained by the dual developmental origin of these most posterior incisors and their degenerated condition. This integrative study allows discussion on the current evolutionary and developmental paradigms associated with the mammalian dentition. It also highlights the importance of nonmodel species to understand dental homologies.

Open data and digital morphology.

Over the past two decades, the development of methods for visualizing and analysing specimens digitally, in three and even four dimensions, has transformed the study of living and fossil organisms. However, the initial promise that the widespread application of such methods would facilitate access to the underlying digital data has not been fully achieved. The underlying datasets for many published studies are not readily or freely available, introducing a barrier to verification and reproducibility, and the reuse of data. There is no current agreement or policy on the amount and type of data that should be made available alongside studies that use, and in some cases are wholly reliant on, digital morphology. Here, we propose a set of recommendations for minimum standards and additional best practice for three-dimensional digital data publication, and review the issues around data storage, management and accessibility.

Inner ear development in cetaceans.

Cetaceans face the challenge of maintaining equilibrium underwater and obtaining sensory input within a dense, low-visibility medium. The cetacean ear represents a key innovation that marked their evolution from terrestrial artiodactyls to among the most fully aquatic mammals in existence. Using micro-CT and histological data, we document shape and size changes in the cetacean inner ear during ontogeny, and demonstrate that, as a proportion of gestation time, the cetacean inner ear is precocial in its growth compared with that of suid artiodactyls. Cetacean inner ears begin ossifying and reach near-adult shape as early as at 32% of the gestation period, and near-adult dimensions as early as at 27% newborn total length. Our earliest embryos with measurable inner ears (13% newborn length) exhibit a flattened cochlea (i.e. smaller distance from cochlear apex to round window) compared with later and adult stages. Inner ears of Sus scrofa have neither begun ossifying nor reached near-adult dimensions at 55% of the gestation period, but have an adult-like ratio of cochlear diameters to each other, suggesting an adult-like shape. The precocial development of the cetacean inner ear complements previous work demonstrating precocial development of other cetacean anatomical features such as the locomotor muscles to facilitate swimming at the moment of birth.

Phylogeny, paleontology, and primates: do incomplete fossils bias the tree of life?

Paleontological systematics relies heavily on morphological data that have undergone decay and fossilization. Here, we apply a heuristic means to assess how a fossil's incompleteness detracts from inferring its phylogenetic relationships. We compiled a phylogenetic matrix for primates and simulated the extinction of living species by deleting an extant taxon's molecular data and keeping only those morphological characters present in actual fossils. The choice of characters present in a given living taxon (the subject) was defined by those present in a given fossil (the template). By measuring congruence between a well-corroborated phylogeny to those incorporating artificial fossils, and by comparing real vs. random character distributions and states, we tested the information content of paleontological datasets and determined if extinction of a living species leads to bias in phylogeny reconstruction. We found a positive correlation between fossil completeness and topological congruence. Real fossil templates sampled for 36 or more of the 360 available morphological characters (including dental) performed significantly better than similarly complete templates with random states. Templates dominated by only one partition performed worse than templates with randomly sampled characters across partitions. The template based on the Eocene primate Darwinius masillae performs better than most other templates with a similar number of sampled characters, likely due to preservation of data across multiple partitions. Our results support the interpretation that Darwinius is strepsirhine, not haplorhine, and suggest that paleontological datasets are reliable in primate phylogeny reconstruction.

Ossification sequence and genetic patterning in the mouse axial skeleton.

We provide novel data on vertebral ontogeny in the mouse, the mammalian model-of-choice for developmental studies. Most previous studies on ossification sequences in mice have focused on pooled elements of the spine (cervicals, thoracics, lumbars, sacrals, and caudals). Here, we contribute data on ossification sequences in the neural arches and centra to provide a comparative basis upon which to evaluate mammalian diversity of the axial skeleton. In attempt to explain the ossification pattern observed, we compared our observations with the phenotype of Cdx over-expresser mice. We use high-resolution X-ray microtomography and clearing and staining techniques to quantify the precise sequential ossification pattern of the mouse spine. We show that micro-CT scans perform better in all cases whereas clearing and staining exhibit sensitivity to the presence of semi-opaque tissue. We observe that the centra of wild-type mice always ossify after neural arches and that the ossification of the neural arches proceeds from two loci. The ossification of the centra appears more complex, especially in the neck where ossification is delayed and does not just follow the order of the vertebrae along the anterior-posterior axis. Our findings also suggest that Cdx genes' expression levels may be involved in the delayed ossification in the neck centra.

Development and embryonic staging in non-model organisms: the case of an afrotherian mammal.

Studies of evolutionary developmental biology commonly use 'model organisms' such as fruit flies or mice, and questions are often functional or epigenetic. Phylogenetic investigations, in contrast, typically use species that are less common and mostly deal with broad scale analyses in the tree of life. However, important evolutionary transformations have taken place at all taxonomic levels, resulting in such diverse forms as elephants and shrews. To understand the mechanisms underlying morphological diversification, broader sampling and comparative approaches are paramount. Using a simple, standardized protocol, we describe for the first time the development of soft tissues and some parts of the skeleton, using µCT-imaging of developmental series of Echinops telfairi and Tenrec ecaudatus, two tenrecid afrotherian mammals. The developmental timing of soft tissue and skeletal characters described for the tenrecids is briefly compared with that of other mammals, including mouse, echidna, and the opossum. We found relatively few heterochronic differences in development in the armadillo vs. tenrec, consistent with a close relationship of Xenarthra and Afrotheria. Ossification in T. ecaudatus continues well into the second half of overall gestation, resembling the pattern seen in other small mammals and differing markedly from the advanced state of ossification evident early in the gestation of elephants, sheep, and humans.

Skeletal development in sloths and the evolution of mammalian vertebral patterning.

Mammals show a very low level of variation in vertebral count, particularly in the neck. Phenotypes exhibited at various stages during the development of the axial skeleton may play a key role in testing mechanisms recently proposed to explain this conservatism. Here, we provide osteogenetic data that identify developmental criteria with which to recognize cervical vs. noncervical vertebrae in mammals. Except for sloths, all mammals show the late ossification of the caudal-most centra in the neck after other centra and neural arches. In sloths with 8-10 ribless neck vertebrae, the caudal-most neck centra ossify early, matching the pattern observed in cranial thoracic vertebrae of other mammals. Accordingly, we interpret the ribless neck vertebrae of three-toed sloths caudal to V7 as thoracic based on our developmental criterion. Applied to the unusual vertebral phenotype of long-necked sloths, these data support the interpretation that elements of the axial skeleton with origins from distinct mesodermal tissues have repatterned over the course of evolution.

RUNX2 tandem repeats and the evolution of facial length in placental mammals.

BACKGROUND: When simple sequence repeats are integrated into functional genes, they can potentially act as evolutionary 'tuning knobs', supplying abundant genetic variation with minimal risk of pleiotropic deleterious effects. The genetic basis of variation in facial shape and length represents a possible example of this phenomenon. Runt-related transcription factor 2 (RUNX2), which is involved in osteoblast differentiation, contains a functionally-important tandem repeat of glutamine and alanine amino acids. The ratio of glutamines to alanines (the QA ratio) in this protein seemingly influences the regulation of bone development. Notably, in domestic breeds of dog, and in carnivorans in general, the ratio of glutamines to alanines is strongly correlated with facial length. RESULTS: In this study we examine whether this correlation holds true across placental mammals, particularly those mammals for which facial length is highly variable and related to adaptive behavior and lifestyle (e.g., primates, afrotherians, xenarthrans). We obtained relative facial length measurements and RUNX2 sequences for 41 mammalian species representing 12 orders. Using both a phylogenetic generalized least squares model and a recently-developed Bayesian comparative method, we tested for a correlation between genetic and morphometric data while controlling for phylogeny, evolutionary rates, and divergence times. Non-carnivoran taxa generally had substantially lower glutamine-alanine ratios than carnivorans (primates and xenarthrans with means of 1.34 and 1.25, respectively, compared to a mean of 3.1 for carnivorans), and we found no correlation between RUNX2 sequence and face length across placental mammals. CONCLUSIONS: Results of our diverse comparative phylogenetic analyses indicate that QA ratio does not consistently correlate with face length across the 41 mammalian taxa considered. Thus, although RUNX2 might function as a 'tuning knob' modifying face length in carnivorans, this relationship is not conserved across mammals in general.

Skeletal development in the African elephant and ossification timing in placental mammals.

We provide here unique data on elephant skeletal ontogeny. We focus on the sequence of cranial and post-cranial ossification events during growth in the African elephant (Loxodonta africana). Previous analyses on ossification sequences in mammals have focused on monotremes, marsupials, boreoeutherian and xenarthran placentals. Here, we add data on ossification sequences in an afrotherian. We use two different methods to quantify sequence heterochrony: the sequence method and event-paring/Parsimov. Compared with other placentals, elephants show late ossifications of the basicranium, manual and pedal phalanges, and early ossifications of the ischium and metacarpals. Moreover, ossification in elephants starts very early and progresses rapidly. Specifically, the elephant exhibits the same percentage of bones showing an ossification centre at the end of the first third of its gestation period as the mouse and hamster have close to birth. Elephants show a number of features of their ossification patterns that differ from those of other placental mammals. The pattern of the initiation of the ossification evident in the African elephant underscores a possible correlation between the timing of ossification onset and gestation time throughout mammals.

Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny.

The fossil record suggests a rapid radiation of placental mammals following the Cretaceous-Paleogene (K-Pg) mass extinction 65 million years ago (Ma); nevertheless, molecular time estimates, while highly variable, are generally much older. Early molecular studies suffer from inadequate dating methods, reliance on the molecular clock, and simplistic and over-confident interpretations of the fossil record. More recent studies have used Bayesian dating methods that circumvent those issues, but the use of limited data has led to large estimation uncertainties, precluding a decisive conclusion on the timing of mammalian diversifications. Here we use a powerful Bayesian method to analyse 36 nuclear genomes and 274 mitochondrial genomes (20.6 million base pairs), combined with robust but flexible fossil calibrations. Our posterior time estimates suggest that marsupials diverged from eutherians 168-178 Ma, and crown Marsupialia diverged 64-84 Ma. Placentalia diverged 88-90 Ma, and present-day placental orders (except Primates and Xenarthra) originated in a ∼20 Myr window (45-65 Ma) after the K-Pg extinction. Therefore we reject a pre K-Pg model of placental ordinal diversification. We suggest other infamous instances of mismatch between molecular and palaeontological divergence time estimates will be resolved with this same approach.

High morphological variation of vestibular system accompanies slow and infrequent locomotion in three-toed sloths.

The semicircular canals (SCs), part of the vestibular apparatus of the inner ear, are directly involved in the detection of angular motion of the head for maintaining balance, and exhibit adaptive patterns for locomotor behaviour. Consequently, they are generally believed to show low levels of intraspecific morphological variation, but few studies have investigated this assumption. On the basis of high-resolution computed tomography, we present here, to our knowledge, the first comprehensive study of the pattern of variation of the inner ear with a focus on Xenarthra. Our study demonstrates that extant three-toed sloths show a high level of morphological variation of the bony labyrinth of the inner ear. Especially, the variation in shape, relative size and angles of their SCs greatly differ from those of other, faster-moving taxa within Xenarthra and Placentalia in general. The unique pattern of variation in three-toed sloths suggests that a release of selection and/or constraints on their organ of balance is associated with the observed wide range of phenotypes. This release is coincident with their slow and infrequent locomotion and may be related, among other possible factors, to a reduced functional demand for a precise sensitivity to movement.

Skeletal ossification and sequence heterochrony in xenarthran evolution.

Previous analyses of how mammals vary in their ossification sequences have focused on monotremes, marsupials, and boreoeutherian placentals. Here, we focus on the sequence of cranial and postcranial ossification events during growth in the xenarthran skull and skeleton, including armadillos, anteaters, and sloths. We use two different methods to quantify sequence heterochrony: sequence analysis of variance (ANOVA) and event-paring/Parsimov. Our results indicate that Parsimov is conservative and does not detect clear heterochronic shifts between xenarthran and boreoeutherian placentals. Sequence-ANOVA performs better, but both methods exhibit sensitivity to the artifactual accumulation of ties. By controlling for ties and taking into account results that the methods have in common, our analysis suggests that xenarthrans significantly differ from other placentals by a late ossification of the sternum and an early ossification of the phalanges and pubis. We interpret these differences as showing that heterochrony plays a role in the skeletal development of xenarthrans, a change from previous studies that have emphasized the developmental homogeneity of the skeleton across placental mammals.

The new framework for understanding placental mammal evolution.

An unprecedented level of confidence has recently crystallized around a new hypothesis of how living placental mammals share a pattern of common descent. The major groups are afrotheres (e.g., aardvarks, elephants), xenarthrans (e.g., anteaters, sloths), laurasiatheres (e.g., horses, shrews), and euarchontoglires (e.g., humans, rodents). Compared with previous hypotheses this tree is remarkably stable; however, some uncertainty persists about the location of the placental root, and (for example) the position of bats within laurasiatheres, of sea cows and aardvarks within afrotheres, and of dermopterans within euarchontoglires. A variety of names for sub-clades within the new placental mammal tree have been proposed, not all of which follow conventions regarding priority and stability. More importantly, the new phylogenetic framework enables the formulation of new hypotheses and testing thereof, for example regarding the possible developmental dichotomy that seems to distinguish members of the newly identified southern and northern radiations of living placental mammals.

Nomenclature and placental mammal phylogeny.

An issue arising from recent progress in establishing the placental mammal Tree of Life concerns the nomenclature of high-level clades. Fortunately, there are now several well-supported clades among extant mammals that require unambiguous, stable names. Although the International Code of Zoological Nomenclature does not apply above the Linnean rank of family, and while consensus on the adoption of competing systems of nomenclature does not yet exist, there is a clear, historical basis upon which to arbitrate among competing names for high-level mammalian clades. Here, we recommend application of the principles of priority and stability, as laid down by G.G. Simpson in 1945, to discriminate among proposed names for high-level taxa. We apply these principles to specific cases among placental mammals with broad relevance for taxonomy, and close with particular emphasis on the Afrotherian family Tenrecidae. We conclude that no matter how reconstructions of the Tree of Life change in years to come, systematists should apply new names reluctantly, deferring to those already published and maximizing consistency with existing nomenclature.

A phylogenetic estimate for golden moles (Mammalia, Afrotheria, Chrysochloridae).

BACKGROUND: Golden moles (Chrysochloridae) are small, subterranean, afrotherian mammals from South Africa and neighboring regions. Of the 21 species now recognized, some (e.g., Chrysochloris asiatica, Amblysomus hottentotus) are relatively common, whereas others (e.g., species of Chrysospalax, Cryptochloris, Neamblysomus) are rare and endangered. Here, we use a combined analysis of partial sequences of the nuclear GHR gene and morphological characters to derive a phylogeny of species in the family Chrysochloridae. RESULTS: Although not all nodes of the combined analysis have high support values, the overall pattern of relationships obtained from different methods of phylogeny reconstruction allow us to make several recommendations regarding the current taxonomy of golden moles. We elevate Huetia to generic status to include the species leucorhinus and confirm the use of the Linnean binomial Carpitalpa arendsi, which belongs within Amblysominae along with Amblysomus and Neamblysomus. A second group, Chrysochlorinae, includes Chrysochloris, Cryptochloris, Huetia, Eremitalpa, Chrysospalax, and Calcochloris. Bayesian methods make chrysochlorines paraphyletic by placing the root within them, coinciding with root positions favored by a majority of randomly-generated outgroup taxa. Maximum Parsimony (MP) places the root either between chrysochlorines and amblysomines (with Chlorotalpa as sister taxon to amblysomines), or at Chlorotalpa, with the former two groups reconstructed as monophyletic in all optimal MP trees. CONCLUSIONS: The inclusion of additional genetic loci for this clade is important to confirm our taxonomic results and resolve the chrysochlorid root. Nevertheless, our optimal topologies support a division of chrysochlorids into amblysomines and chrysochlorines, with Chlorotalpa intermediate between the two. Furthermore, evolution of the chrysochlorid malleus exhibits homoplasy. The elongate malleus has evolved just once in the Cryptochloris-Chrysochloris group; other changes in shape have occurred at multiple nodes, regardless of how the root is resolved.

Dental eruption in afrotherian mammals.

BACKGROUND: Afrotheria comprises a newly recognized clade of mammals with strong molecular evidence for its monophyly. In contrast, morphological data uniting its diverse constituents, including elephants, sea cows, hyraxes, aardvarks, sengis, tenrecs and golden moles, have been difficult to identify. Here, we suggest relatively late eruption of the permanent dentition as a shared characteristic of afrotherian mammals. This characteristic and other features (such as vertebral anomalies and testicondy) recall the phenotype of a human genetic pathology (cleidocranial dysplasia), correlations with which have not been explored previously in the context of character evolution within the recently established phylogeny of living mammalian clades. RESULTS: Although data on the absolute timing of eruption in sengis, golden moles and tenrecs are still unknown, craniometric comparisons for ontogenetic series of these taxa show that considerable skull growth takes place prior to the complete eruption of the permanent cheek teeth. Specimens showing less than half (sengis, golden moles) or two-thirds (tenrecs, hyraxes) of their permanent cheek teeth reach or exceed the median jaw length of conspecifics with a complete dentition. With few exceptions, afrotherians are closer to median adult jaw length with fewer erupted, permanent cheek teeth than comparable stages of non-afrotherians. Manatees (but not dugongs), elephants and hyraxes with known age data show eruption of permanent teeth late in ontogeny relative to other mammals. While the occurrence of delayed eruption, vertebral anomalies and other potential afrotherian synapomorphies resemble some symptoms of a human genetic pathology, these characteristics do not appear to covary significantly among mammalian clades. CONCLUSION: Morphological characteristics shared by such physically disparate animals such as elephants and golden moles are not easy to recognize, but are now known to include late eruption of permanent teeth, in addition to vertebral anomalies, testicondy and other features. Awareness of their possible genetic correlates promises insight into the developmental basis of shared morphological features of afrotherians and other vertebrates.