Thomas J. Near

Resolution of ray-finned fish phylogeny and timing of diversification

Ray-finned fishes make up half of all living vertebrate species. Nearly all ray-finned fishes are teleosts, which include most commercially important fish species, several model organisms for genomics and developmental biology, and the dominant component of marine and freshwater vertebrate faunas. Despite the economic and scientific importance of ray-finned fishes, the lack of a single comprehensive phylogeny with corresponding divergence-time estimates has limited our understanding of the evolution and diversification of this radiation. Our analyses, which use multiple nuclear gene sequences in conjunction with 36 fossil age constraints, result in a well-supported phylogeny of all major ray-finned fish lineages and molecular age estimates that are generally consistent with the fossil record. This phylogeny informs three long-standing problems: specifically identifying elopomorphs (eels and tarpons) as the sister lineage of all other teleosts, providing a unique hypothesis on the radiation of early euteleosts, and offering a promising strategy for resolution of the “bush at the top of the tree” that includes percomorphs and other spiny-finned teleosts. Contrasting our divergence time estimates with studies using a single nuclear gene or whole mitochondrial genomes, we find that the former underestimates ages of the oldest ray-finned fish divergences, but the latter dramatically overestimates ages for derived teleost lineages. Our time-calibrated phylogeny reveals that much of the diversification leading to extant groups of teleosts occurred between the late Mesozoic and early Cenozoic, identifying this period as the “Second Age of Fishes.”

Early bursts of body size and shape evolution are rare in comparative data.

George Gaylord Simpson famously postulated that much of lifes diversity originated as adaptive radiations—more or less simultaneous divergences of numerous lines from a single ancestral adaptive type. However, identifying adaptive radiations has proven difficult due to a lack of broad‐scale comparative datasets. Here, we use phylogenetic comparative data on body size and shape in a diversity of animal clades to test a key model of adaptive radiation, in which initially rapid morphological evolution is followed by relative stasis. We compared the fit of this model to both single selective peak and random walk models. We found little support for the early‐burst model of adaptive radiation, whereas both other models, particularly that of selective peaks, were commonly supported. In addition, we found that the net rate of morphological evolution varied inversely with clade age. The youngest clades appear to evolve most rapidly because long‐term change typically does not attain the amount of divergence predicted from rates measured over short time scales. Across our entire analysis, the dominant pattern was one of constraints shaping evolution continually through time rather than rapid evolution followed by stasis. We suggest that the classical model of adaptive radiation, where morphological evolution is initially rapid and slows through time, may be rare in comparative data.

Phylogeny and tempo of diversification in the superradiation of spiny-rayed fishes

Spiny-rayed fishes, or acanthomorphs, comprise nearly one-third of all living vertebrates. Despite their dominant role in aquatic ecosystems, the evolutionary history and tempo of acanthomorph diversification is poorly understood. We investigate the pattern of lineage diversification in acanthomorphs by using a well-resolved time-calibrated phylogeny inferred from a nuclear gene supermatrix that includes 520 acanthomorph species and 37 fossil age constraints. This phylogeny provides resolution for what has been classically referred to as the “bush at the top” of the teleost tree, and indicates acanthomorphs originated in the Early Cretaceous. Paleontological evidence suggests acanthomorphs exhibit a pulse of morphological diversification following the end Cretaceous mass extinction; however, the role of this event on the accumulation of living acanthomorph diversity remains unclear. Lineage diversification rates through time exhibit no shifts associated with the end Cretaceous mass extinction, but there is a global decrease in lineage diversification rates 50 Ma that occurs during a period when morphological disparity among fossil acanthomorphs increases sharply. Analysis of clade-specific shifts in diversification rates reveal that the hyperdiversity of living acanthomorphs is highlighted by several rapidly radiating lineages including tunas, gobies, blennies, snailfishes, and Afro-American cichlids. These lineages with high diversification rates are not associated with a single habitat type, such as coral reefs, indicating there is no single explanation for the success of acanthomorphs, as exceptional bouts of diversification have occurred across a wide array of marine and freshwater habitats.

Assessing Concordance of Fossil Calibration Points in Molecular Clock Studies: An Example Using Turtles

Although still controversial, estimation of divergence times using molecular data has emerged as a powerful tool to examine the tempo and mode of evolutionary change. Two primary obstacles in improving the accuracy of molecular dating are heterogeneity in DNA substitution rates and accuracy of the fossil record as calibration points. Recent methodological advances have provided powerful methods that estimate relative divergence times in the face of heterogeneity of nucleotide substitution rates among lineages. However, relatively little attention has focused on the accuracy of fossil calibration points that allow one to translate relative divergence times into absolute time. We present a new cross‐validation method that identifies inconsistent fossils when multiple fossil calibrations are available for a clade and apply our method to a molecular phylogeny of living turtles with fossil calibration times for 17 of the 22 internal nodes in the tree. Our cross‐validation procedure identified seven inconsistent fossils. Using the consistent fossils as calibration points, we found that despite their overall antiquity as a lineage, the most species‐rich clades of turtles diversified well within the Cenozoic. Many of the truly ancient lineages of turtles are currently represented by a few, often endangered species that deserve high priority as conservation targets.

TEMPO OF HYBRID INVIABILITY IN CENTRARCHID FISHES (TELEOSTEI: CENTRARCHIDAE)

Abstract Hybrid viability decreases with divergence time, a pattern consistent with a so‐called speciation clock. However, the actual rate at which this clock ticks is poorly known. Most speciation‐clock studies have used genetic divergence as a proxy for time, adopting a molecular clock and often far‐distant calibration points to convert genetic distances into age. Because molecular clock assumptions are violated for most genetic datasets and distant calibrations are of questionable utility, the actual rate at which reproductive isolation evolves may be substantially different than current estimates suggest. We provide a robust measure of the tempo at which hybrid viability declines with divergence time in a clade of freshwater fishes (Centrarchidae). This incompatibility clock is distinct from a speciation clock because speciation events in centrarchids appear to be driven largely by prezygotic isolation. Our analyses used divergence times estimated with penalized likelihood applied to a phylogeny derived from seven gene regions and calibrated with six centrarchid fossils. We found that hybrid embryo viability declined at mean rate of 3.13% per million years, slower than in most other taxa investigated to date. Despite measurement error in both molecular estimated ages and hatching success of hybrid crosses, divergence time explained between 73% and 90% of the variation in hybrid viability among nodes. This high correlation is consistent with the gradual accumulation of many genetic incompatibilities of small effect. Hybrid viability declined with the square of time, consistent with an increasing rate of accumulation of incompatibilities between divergent genomes (the snowball effect). However, the quadratic slope is due to a lag phase resulting from heterosis among young species pairs, a phenomenon rarely considered in predictions of hybrid fitness. Finally, we found that reciprocal crosses often show asymmetrical hybrid viabilities. We discuss several alternative explanations for this result including possible deleterious cytonuclear interactions. Speciation‐clock studies have been a small cottage industry recently, but there are still novel insights to be gained from analyses of more taxonomic groups. However, between‐group comparisons require more careful molecular‐clock calibration than has been the norm.

Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes

The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6–5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity.

FOSSIL CALIBRATIONS AND MOLECULAR DIVERGENCE TIME ESTIMATES IN CENTRARCHID FISHES (TELEOSTEI: CENTRARCHIDAE)

Abstract Molecular clock methods allow biologists to estimate divergence times, which in turn play an important role in comparative studies of many evolutionary processes. It is well known that molecular age estimates can be biased by heterogeneity in rates of molecular evolution, but less attention has been paid to the issue of potentially erroneous fossil calibrations. In this study we estimate the timing of diversification in Centrarchidae, an endemic major lineage of the diverse North American freshwater fish fauna, through a new approach to fossil calibration and molecular evolutionary model selection. Given a completely resolved multi‐gene molecular phylogeny and a set of multiple fossil‐inferred age estimates, we tested for potentially erroneous fossil calibrations using a recently developed fossil cross‐validation. We also used fossil information to guide the selection of the optimal molecular evolutionary model with a new fossil jackknife method in a fossil‐based model cross‐validation. The centrarchid phylogeny resulted from a mixed‐model Bayesian strategy that included 14 separate data partitions sampled from three mtDNA and four nuclear genes. Ten of the 31 interspecific nodes in the centrarchid phylogeny were assigned a minimal age estimate from the centrarchid fossil record. Our analyses identified four fossil dates that were inconsistent with the other fossils, and we removed them from the molecular dating analysis. Using fossil‐based model cross‐validation to determine the optimal smoothing value in penalized likelihood analysis, and six mutually consistent fossil calibrations, the age of the most recent common ancestor of Centrarchidae was 33.59 million years ago (mya). Penalized likelihood analyses of individual data partitions all converged on a very similar age estimate for this node, indicating that rate heterogeneity among data partitions is not confounding our analyses. These results place the origin of the centrarchid radiation at a time of major faunal turnover as the fossil record indicates that the most diverse lineages of the North American freshwater fish fauna originated at the Eocene‐Oligocene boundary, approximately 34 mya. This time coincided with major global climate change from warm to cool temperatures and a signature of elevated lineage extinction and origination in the fossil record across the tree of life. Our analyses demonstrate the utility of fossil cross‐validation to critically assess individual fossil calibration points, providing the ability to discriminate between consistent and inconsistent fossil age estimates that are used for calibrating molecular phylogenies.

Estimating divergence times of notothenioid fishes using a fossil- calibrated molecular clock

Hypotheses concerning the diversification of notothenioid fishes have relied extensively on estimates of divergence times using molecular clock methods. The timing of diversification of the cold adapted antifreeze glycoprotein (AFGP)-bearing Antarctic notothenioid clade in the middle to late Miocene has been correlated with the onset of polar climatic conditions along the Antarctic Continental Shelf. Critical examination of the previous molecular clock analyses of notothenioids reveals several problems associated with heterogeneity of nucleotide substitution rates among lineages, the application of potentially inappropriate nucleotide substitution rates, and the lack of confidence intervals for divergence time estimates. In this study, the notothenioid partial gene mtDNA 12S-16S rRNA (PG-rRNA) molecular clock was reanalysed using a tree-based maximum likelihood strategy that attempts to account for rate heterogeneity of nucleotide substitution rates among lineages using the penalized likelihood method, and bootstrap resampling to estimate confidence intervals of divergence time estimates. The molecular clock was calibrated using the notothenioid fossil Proeleginops grandeastmanorum. Divergence time estimates for all nodes in the PG-rRNA maximum likelihood tree were substantially older than previous estimates. In particular, the estimated age of the AFGP-bearing Antarctic notothenioid clade predates the onset of extensive sea ice and development of polar conditions by at least 10 million years. Despite caveats involving the fossil calibration and limitations of the PG-rRNA dataset, these divergence time estimates provide initial observations for the development of a novel model of the diversification of cold adapted Antarctic notothenioid fishes.

Molecular and fossil evidence place the origin of cichlid fishes long after Gondwanan rifting

Cichlid fishes are a key model system in the study of adaptive radiation, speciation and evolutionary developmental biology. More than 1600 cichlid species inhabit freshwater and marginal marine environments across several southern landmasses. This distributional pattern, combined with parallels between cichlid phylogeny and sequences of Mesozoic continental rifting, has led to the widely accepted hypothesis that cichlids are an ancient group whose major biogeographic patterns arose from Gondwanan vicariance. Although the Early Cretaceous (ca 135 Ma) divergence of living cichlids demanded by the vicariance model now represents a key calibration for teleost molecular clocks, this putative split pre-dates the oldest cichlid fossils by nearly 90 Myr. Here, we provide independent palaeontological and relaxed-molecular-clock estimates for the time of cichlid origin that collectively reject the antiquity of the group required by the Gondwanan vicariance scenario. The distribution of cichlid fossil horizons, the age of stratigraphically consistent outgroup lineages to cichlids and relaxed-clock analysis of a DNA sequence dataset consisting of 10 nuclear genes all deliver overlapping estimates for crown cichlid origin centred on the Palaeocene (ca 65–57 Ma), substantially post-dating the tectonic fragmentation of Gondwana. Our results provide a revised macroevolutionary time scale for cichlids, imply a role for dispersal in generating the observed geographical distribution of this important model clade and add to a growing debate that questions the dominance of the vicariance paradigm of historical biogeography.

Molecular Evidence for Acanthocephala as a Subtaxon of Rotifera

Rotifers are free-living animals usually smaller than 1 mm that possess a characteristic wheel organ. Acanthocephalans (thorny-headed worms) are larger endoparasitic animals that use vertebrates and arthropods to complete their life cycle. The taxa Acanthocephala and Rotifera are considered separate phyla, often within the taxon Aschelminthes. We have reexamined the relationship between Rotifera and Acanthocephala using 18S rRNA gene sequences. Our results conclusively show that Acanthocephala is the sister group of the rotifer class Bdelloidea. Rotifera was nonmonophyletic in all molecular analyses, which supports the hypothesis that the Acanthocephala represent a taxon within the phylum Rotifera and not a separate phylum. These results agree with a previous cladistic study of morphological characters.