Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

Phylogenetic congruence, conflict and consilience between molecular and morphological data.

Morphology and molecules are important data sources for estimating evolutionary relationships. Modern studies often utilise morphological and molecular partitions alongside each other in combined analyses. However, the effect of combining phenomic and genomic partitions is unclear. This is exacerbated by their size imbalance, and conflict over the efficacy of different inference methods when using morphological characters. To systematically address the effect of topological incongruence, size imbalance, and tree inference methods, we conduct a meta-analysis of 32 combined (molecular + morphology) datasets across metazoa. Our results reveal that morphological-molecular topological incongruence is pervasive: these data partitions yield very different trees, irrespective of which method is used for morphology inference. Analysis of the combined data often yields unique trees that are not sampled by either partition individually, even with the inclusion of relatively small quantities of morphological characters. Differences between morphology inference methods in terms of resolution and congruence largely relate to consensus methods. Furthermore, stepping stone Bayes factor analyses reveal that morphological and molecular partitions are not consistently combinable, i.e. data partitions are not always best explained under a single evolutionary process. In light of these results, we advise that the congruence between morphological and molecular data partitions needs to be considered in combined analyses. Nonetheless, our results reveal that, for most datasets, morphology and molecules can, and should, be combined in order to best estimate evolutionary history and reveal hidden support for novel relationships. Studies that analyse only phenomic or genomic data in isolation are unlikely to provide the full evolutionary picture.

Extended embryo retention and viviparity in the first amniotes.

The amniotic egg with its complex fetal membranes was a key innovation in vertebrate evolution that enabled the great diversification of reptiles, birds and mammals. It is debated whether these fetal membranes evolved in eggs on land as an adaptation to the terrestrial environment or to control antagonistic fetal-maternal interaction in association with extended embryo retention (EER). Here we report an oviparous choristodere from the Lower Cretaceous period of northeast China. The ossification sequence of the embryo confirms that choristoderes are basal archosauromorphs. The discovery of oviparity in this assumed viviparous extinct clade, together with existing evidence, suggests that EER was the primitive reproductive mode in basal archosauromorphs. Phylogenetic comparative analyses on extant and extinct amniotes suggest that the first amniote displayed EER (including viviparity).

Galeaspid anatomy and the origin of vertebrate paired appendages.

Paired fins are a major innovation1,2 that evolved in the jawed vertebrate lineage after divergence from living jawless vertebrates3. Extinct jawless armoured stem gnathostomes show a diversity of paired body-wall extensions, ranging from skeletal processes to simple flaps4. By contrast, osteostracans (a sister group to jawed vertebrates) are interpreted to have the first true paired appendages in a pectoral position, with pelvic appendages evolving later in association with jaws5. Here we show, on the basis of articulated remains of Tujiaaspis vividus from the Silurian period of China, that galeaspids (a sister group to both osteostracans and jawed vertebrates) possessed three unpaired dorsal fins, an approximately symmetrical hypochordal tail and a pair of continuous, branchial-to-caudal ventrolateral fins. The ventrolateral fins are similar to paired fin flaps in other stem gnathostomes, and specifically to the ventrolateral ridges of cephalaspid osteostracans that also possess differentiated pectoral fins. The ventrolateral fins are compatible with aspects of the fin-fold hypothesis for the origin of vertebrate paired appendages6-10. Galeaspids have a precursor condition to osteostracans and jawed vertebrates in which paired fins arose initially as continuous pectoral-pelvic lateral fins that our computed fluid-dynamics experiments show passively generated lift. Only later in the stem lineage to osteostracans and jawed vertebrates did pectoral fins differentiate anteriorly. This later differentiation was followed by restriction of the remaining field of fin competence to a pelvic position, facilitating active propulsion and steering.

Phylogenetic analyses of ray-finned fishes (Actinopterygii) using collagen type I protein sequences.

Ray-finned fishes (Actinopterygii) are the largest and most diverse group of vertebrates, comprising over half of all living vertebrate species. Phylogenetic relationships between ray-finned fishes have historically pivoted on the study of morphology, which has notoriously failed to resolve higher order relationships, such as within the percomorphs. More recently, comprehensive genomic analyses have provided further resolution of actinopterygian phylogeny, including higher order relationships. Such analyses are rightfully regarded as the 'gold standard' for phylogenetics. However, DNA retrieval requires modern or well-preserved tissue and is less likely to be preserved in archaeological or fossil specimens. By contrast, some proteins, such as collagen, are phylogenetically informative and can survive into deep time. Here, we test the utility of collagen type I amino acid sequences for phylogenetic estimation of ray-finned fishes. We estimate topology using Bayesian approaches and compare the congruence of our estimated trees with published genomic phylogenies. Furthermore, we apply a Bayesian molecular clock approach and compare estimated divergence dates with previously published genomic clock analyses. Our collagen-derived trees exhibit 77% of node positions as congruent with recent genomic-derived trees, with the majority of discrepancies occurring in higher order node positions, almost exclusively within the Percomorpha. Our molecular clock trees present divergence times that are fairly comparable with genomic-based phylogenetic analyses. We estimate the mean node age of Actinopteri at ∼293 million years (Ma), the base of Teleostei at ∼211 Ma and the radiation of percomorphs beginning at ∼141 Ma (∼350 Ma, ∼250-283 Ma and ∼120-133 Ma in genomic trees, respectively). Finally, we show that the average rate of collagen (I) sequence evolution is 0.9 amino acid substitutions for every million years of divergence, with the α3 (I) sequence evolving the fastest, followed by the α2 (I) chain. This is the quickest rate known for any vertebrate group. We demonstrate that phylogenetic analyses using collagen type I amino acid sequences generate tangible signals for actinopterygians that are highly congruent with recent genomic-level studies. However, there is limited congruence within percomorphs, perhaps due to clade-specific functional constraints acting upon collagen sequences. Our results provide important insights for future phylogenetic analyses incorporating extinct actinopterygian species via collagen (I) sequencing.

Coevolution of enamel, ganoin, enameloid, and their matrix SCPP genes in osteichthyans.

We resolve debate over the evolution of vertebrate hypermineralized tissues through analyses of matrix protein-encoding secretory calcium-binding phosphoprotein (SCPP) genes and phylogenetic inference of hypermineralized tissues. Among these genes, AMBN and ENAM are found in both sarcopterygians and actinopterygians, whereas AMEL and SCPP5 are found only in sarcopterygians and actinopterygians, respectively. Actinopterygian AMBN, ENAM, and SCPP5 are expressed during the formation of hypermineralized tissues on scales and teeth: ganoin, acrodin, and collar enamel in gar, and acrodin and collar enameloid in zebrafish. Our phylogenetic analyses indicate the emergence of an ancestral enamel in stem-osteichthyans, whereas ganoin emerged in stem-actinopterygians and true enamel in stem-sarcopterygians. Thus, AMBN and ENAM originated in concert with ancestral enamel, SCPP5 evolved in association with ganoin, and AMEL evolved with true enamel. Shifts in gene expression domain and timing explain the evolution of different hypermineralized tissues. We propose that hypermineralized tissues in osteichthyans coevolved with matrix SCPP genes.

Collagen Sequence Analysis Reveals Evolutionary History of Extinct West Indies Nesophontes (Island-Shrews).

Ancient biomolecule analyses are proving increasingly useful in the study of evolutionary patterns, including extinct organisms. Proteomic sequencing techniques complement genomic approaches, having the potential to examine lineages further back in time than achievable using ancient DNA, given the less stringent preservation requirements. In this study, we demonstrate the ability to use collagen sequence analyses via proteomics to assist species delimitation as a foundation for informing evolutionary patterns. We uncover biogeographic information of an enigmatic and recently extinct lineage of Nesophontes across their range on the Caribbean islands. First, evolutionary relationships reconstructed from collagen sequences reaffirm the affinity of Nesophontes and Solenodon as sister taxa within Solenodonota. This relationship helps lay the foundation for testing geographical isolation hypotheses across islands within the Greater Antilles, including movement from Cuba toward Hispaniola. Second, our results are consistent with Cuba having just two species of Nesophontes (N. micrus and N. major) that exhibit intrapopulation morphological variation. Finally, analysis of the recently described species from the Cayman Islands (N. hemicingulus) indicates that it is a closer relative to N. major rather than N. micrus as previously speculated. This proteomic sequencing improves our understanding of the origin, evolution, and distribution of this extinct mammal lineage, particularly with respect to the approximate timing of speciation. Such knowledge is vital for this biodiversity hotspot, where the magnitude of recent extinctions may obscure true estimates of species richness in the past.

Morphological Phylogenetics Evaluated Using Novel Evolutionary Simulations.

Evolutionary inferences require reliable phylogenies. Morphological data have traditionally been analyzed using maximum parsimony, but recent simulation studies have suggested that Bayesian analyses yield more accurate trees. This debate is ongoing, in part, because of ambiguity over modes of morphological evolution and a lack of appropriate models. Here, we investigate phylogenetic methods using two novel simulation models-one in which morphological characters evolve stochastically along lineages and another in which individuals undergo selection. Both models generate character data and lineage splitting simultaneously: the resulting trees are an emergent property, rather than a fixed parameter. Standard consensus methods for Bayesian searches (Mki) yield fewer incorrect nodes and quartets than the standard consensus trees recovered using equal weighting and implied weighting parsimony searches. Distances between the pool of derived trees (most parsimonious or posterior distribution) and the true trees-measured using Robinson-Foulds (RF), subtree prune and regraft (SPR), and tree bisection reconnection (TBR) metrics-demonstrate that this is related to the search strategy and consensus method of each technique. The amount and structure of homoplasy in character data differ between models. Morphological coherence, which has previously not been considered in this context, proves to be a more important factor for phylogenetic accuracy than homoplasy. Selection-based models exhibit relatively lower homoplasy, lower morphological coherence, and higher inaccuracy in inferred trees. Selection is a dominant driver of morphological evolution, but we demonstrate that it has a confounding effect on numerous character properties which are fundamental to phylogenetic inference. We suggest that the current debate should move beyond considerations of parsimony versus Bayesian, toward identifying modes of morphological evolution and using these to build models for probabilistic search methods. [Bayesian; evolution; morphology; parsimony; phylogenetics; selection; simulation.].

The dermal skeleton of the jawless vertebrate Tremataspis mammillata (Osteostraci, stem-Gnathostomata).

Osteostracans are the closest jawless relatives of jawed vertebrates, informing the gradual assembly of the vertebrate mineralised skeleton. Conflicting interpretations of their dermal skeletal histology arise from failure to account for topological variation, obscuring their significance in elucidating vertebrate skeletal evolution. To resolve this, we characterize the cranial and trunk dermal skeleton of a single individual of Tremataspis mammillata (Osteostraci, Thyestiida) at submicron resolution using synchrotron- and computed- tomography. Our results show that the architecture of the Tremataspis dermal skeleton is, for the most part, conserved over the skeleton and is broadly consistent with previous histological hypotheses based on 2D thin section study. We resolve debate over the homology of the basal layer, identifying it as osteogenic acellular isopedin rather than odontogenic elasmodine or metaplastic ossification of the stratum compactum of the dermis. We find topological variation between all dermal skeletal elements studied, and particularly between the cranial and postcranial dermal skeleton. This variation can be largely explained by reduction in differentiation due to geometric constraints imposed within smaller skeletal elements, such as scales. Our description of the dermal skeleton of Tremataspis mammillata provides a foundation for interpreting data from cursory topological samples of dermal skeletal diversity obtained in other osteostracans. This reveals general aspects of histological structure that must be ancestral for osteostracans and, likely, ancestral jawed vertebrates. Finally, we draw the distinction between hypotheses and descriptions in palaeohistology.

The nature of aspidin and the evolutionary origin of bone.

Bone is the key innovation underpinning the evolution of the vertebrate skeleton, yet its origin is mired by debate over interpretation of the most primitive bone-like tissue, aspidin. This has variously been interpreted as cellular bone, acellular bone, dentine or an intermediate of dentine and bone. The crux of the controversy is the nature of unmineralized spaces pervading the aspidin matrix, which have alternatively been interpreted as having housed cells, cell processes or Sharpey's fibres. Discriminating between these hypotheses has been hindered by the limits of traditional histological methods. Here, we use synchrotron X-ray tomographic microscopy to reveal the nature of aspidin. We show that the spaces exhibit a linear morphology incompatible with interpretations that they represent voids left by cells or cell processes. Instead, these spaces represent intrinsic collagen fibre bundles that form a scaffold about which mineral was deposited. Aspidin is thus acellular dermal bone. We reject hypotheses that it is a type of dentine, cellular bone or transitional tissue. Our study suggests that the full repertoire of skeletal tissue types was established before the divergence of the earliest known skeletonizing vertebrates, indicating that the corresponding cell types evolved rapidly following the divergence of cyclostomes and gnathostomes.

Parsimony, not Bayesian analysis, recovers more stratigraphically congruent phylogenetic trees.

Reconstructing evolutionary histories requires accurate phylogenetic trees. Recent simulation studies suggest that probabilistic phylogenetic analyses of morphological data are more accurate than traditional parsimony techniques. Here, we use empirical data to compare Bayesian and parsimony phylogenies in terms of their congruence with the distribution of age ranges of the component taxa. Analysis of 167 independent morphological data matrices of fossil tetrapods finds that Bayesian trees exhibit significantly lower stratigraphic congruence than the equivalent parsimony trees. As such, taking stratigraphic data as an independent benchmark indicates that parsimony analyses are more accurate for phylogenetic reconstruction of morphological data. The discrepancy between simulated and empirical studies may result from historic data peaking practices or some complexities of empirical data as yet unaccounted for.

Histology and affinity of anaspids, and the early evolution of the vertebrate dermal skeleton.

The assembly of the gnathostome bodyplan constitutes a formative episode in vertebrate evolutionary history, an interval in which the mineralized skeleton and its canonical suite of cell and tissue types originated. Fossil jawless fishes, assigned to the gnathostome stem-lineage, provide an unparalleled insight into the origin and evolution of the skeleton, hindered only by uncertainty over the phylogenetic position and evolutionary significance of key clades. Chief among these are the jawless anaspids, whose skeletal composition, a rich source of phylogenetic information, is poorly characterized. Here we survey the histology of representatives spanning anaspid diversity and infer their generalized skeletal architecture. The anaspid dermal skeleton is composed of odontodes comprising spheritic dentine and enameloid, overlying a basal layer of acellular parallel fibre bone containing an extensive shallow canal network. A recoded and revised phylogenetic analysis using equal and implied weights parsimony resolves anaspids as monophyletic, nested among stem-gnathostomes. Our results suggest the anaspid dermal skeleton is a degenerate derivative of a histologically more complex ancestral vertebrate skeleton, rather than reflecting primitive simplicity. Hypotheses that anaspids are ancestral skeletonizing lampreys, or a derived lineage of jawless vertebrates with paired fins, are rejected.

Histology of the heterostracan dermal skeleton: Insight into the origin of the vertebrate mineralised skeleton.

Living vertebrates are divided into those that possess a fully formed and fully mineralised skeleton (gnathostomes) versus those that possess only unmineralised cartilaginous rudiments (cyclostomes). As such, extinct phylogenetic intermediates of these living lineages afford unique insights into the evolutionary assembly of the vertebrate mineralised skeleton and its canonical tissue types. Extinct jawless and jawed fishes assigned to the gnathostome stem evidence the piecemeal assembly of skeletal systems, revealing that the dermal skeleton is the earliest manifestation of a homologous mineralised skeleton. Yet the nature of the primitive dermal skeleton, itself, is poorly understood. This is principally because previous histological studies of early vertebrates lacked a phylogenetic framework required to derive evolutionary hypotheses. Nowhere is this more apparent than within Heterostraci, a diverse clade of primitive jawless vertebrates. To this end, we surveyed the dermal skeletal histology of heterostracans, inferred the plesiomorphic heterostracan skeleton and, through histological comparison to other skeletonising vertebrate clades, deduced the ancestral nature of the vertebrate dermal skeleton. Heterostracans primitively possess a four-layered skeleton, comprising a superficial layer of odontodes composed of dentine and enameloid; a compact layer of acellular parallel-fibred bone containing a network of vascular canals that supply the pulp canals (L1); a trabecular layer consisting of intersecting radial walls composed of acellular parallel-fibred bone, showing osteon-like development (L2); and a basal layer of isopedin (L3). A three layered skeleton, equivalent to the superficial layer L2 and L3 and composed of enameloid, dentine and acellular bone, is possessed by the ancestor of heterostracans + jawed vertebrates. We conclude that an osteogenic component is plesiomorphic with respect to the vertebrate dermal skeleton. Consequently, we interpret the dermal skeleton of denticles in chondrichthyans and jawless thelodonts as independently and secondarily simplified. J. Morphol. 276:657-680, 2015. © 2015 The Authors Journal of Morphology Published by Wiley Periodicals, Inc.