Bayesian Inference Under the Multispecies Coalescent with Ancient DNA Sequences.

Ancient DNA (aDNA) is increasingly being used to investigate questions such as the phylogenetic relationships and divergence times of extant and extinct species. If aDNA samples are sufficiently old, expected branch lengths (in units of nucleotide substitutions) are reduced relative to contemporary samples. This can be accounted for by incorporating sample ages into phylogenetic analyses. Existing methods that use tip (sample) dates infer gene trees rather than species trees, which can lead to incorrect or biased inferences of the species tree. Methods using a multispecies coalescent (MSC) model overcome these issues. We developed an MSC model with tip dates and implemented it in the program bpp. The method performed well for a range of biologically realistic scenarios, estimating calibrated divergence times and mutation rates precisely. Simulations suggest that estimation precision can be best improved by prioritizing sampling of many loci and more ancient samples. Incorrectly treating ancient samples as contemporary in analyzing simulated data, mimicking a common practice of empirical analyses, led to large systematic biases in model parameters, including divergence times. Two genomic datasets of mammoths and elephants were analyzed, demonstrating the method's empirical utility.

Tip dating and Bayes factors provide insight into the divergences of crown bird clades across the end-Cretaceous mass extinction.

The origin of crown birds (Neornithes) remains contentious owing to conflicting divergence time hypotheses obtained from alternative sources of data. The fossil record suggests limited diversification of Neornithes in the Late Mesozoic and a substantial radiation in the aftermath of the Cretaceous-Palaeogene (K-Pg) mass extinction, approximately 66 Ma. Molecular clock studies, however, have yielded estimates for neornithine origins ranging from the Early Cretaceous (130 Ma) to less than 10 Myr before the K-Pg. We use Bayes factors to compare the fit of node ages from different molecular clock studies to an independent morphological dataset. Our results allow us to reject scenarios of crown bird origins deep in the Early Cretaceous, as well as an origin of crown birds within the last 10 Myr of the Cretaceous. The scenario best supported by our analyses is one where Neornithes originated between the Early and Late Cretaceous (ca 100 Ma), while numerous divergences within major neoavian clades either span or postdate the K-Pg. This study affirms the importance of the K-Pg on the diversification of modern birds, and the potential of combined-evidence tip-dating analyses to illuminate recalcitrant 'rocks versus clocks' debates.

MEGA11: Molecular Evolutionary Genetics Analysis Version 11.

The Molecular Evolutionary Genetics Analysis (MEGA) software has matured to contain a large collection of methods and tools of computational molecular evolution. Here, we describe new additions that make MEGA a more comprehensive tool for building timetrees of species, pathogens, and gene families using rapid relaxed-clock methods. Methods for estimating divergence times and confidence intervals are implemented to use probability densities for calibration constraints for node-dating and sequence sampling dates for tip-dating analyses. They are supported by new options for tagging sequences with spatiotemporal sampling information, an expanded interactive Node Calibrations Editor, and an extended Tree Explorer to display timetrees. Also added is a Bayesian method for estimating neutral evolutionary probabilities of alleles in a species using multispecies sequence alignments and a machine learning method to test for the autocorrelation of evolutionary rates in phylogenies. The computer memory requirements for the maximum likelihood analysis are reduced significantly through reprogramming, and the graphical user interface has been made more responsive and interactive for very big data sets. These enhancements will improve the user experience, quality of results, and the pace of biological discovery. Natively compiled graphical user interface and command-line versions of MEGA11 are available for Microsoft Windows, Linux, and macOS from www.megasoftware.net.

Trilobite evolutionary rates constrain the duration of the Cambrian explosion.

Trilobites are often considered exemplary for understanding the Cambrian explosion of animal life, due to their unsurpassed diversity and abundance. These biomineralized arthropods appear abruptly in the fossil record with an established diversity, phylogenetic disparity, and provincialism at the beginning of Cambrian Series 2 (∼521 Ma), suggesting a protracted but cryptic earlier history that possibly extends into the Precambrian. However, recent analyses indicate elevated rates of phenotypic and genomic evolution for arthropods during the early Cambrian, thereby shortening the phylogenetic fuse. Furthermore, comparatively little research has been devoted to understanding the duration of the Cambrian explosion, after which normal Phanerozoic evolutionary rates were established. We test these hypotheses by applying Bayesian tip-dating methods to a comprehensive dataset of Cambrian trilobites. We show that trilobites have a Cambrian origin, as supported by the trace fossil record and molecular clocks. Surprisingly, they exhibit constant evolutionary rates across the entire Cambrian, for all aspects of the preserved phenotype: discrete, meristic, and continuous morphological traits. Our data therefore provide robust, quantitative evidence that by the time the typical Cambrian fossil record begins (∼521 Ma), the Cambrian explosion had already largely concluded. This suggests that a modern-style marine biosphere had rapidly emerged during the latest Ediacaran and earliest Cambrian (∼20 million years), followed by broad-scale evolutionary stasis throughout the remainder of the Cambrian.

Fossils improve phylogenetic analyses of morphological characters.

Fossils provide our only direct window into evolutionary events in the distant past. Incorporating them into phylogenetic hypotheses of living clades can help time-calibrate divergences, as well as elucidate macroevolutionary dynamics. However, the effect fossils have on phylogenetic reconstruction from morphology remains controversial. The consequences of explicitly incorporating the stratigraphic ages of fossils using tip-dated inference are also unclear. Here, we use simulations to evaluate the performance of inference methods across different levels of fossil sampling and missing data. Our results show that fossil taxa improve phylogenetic analysis of morphological datasets, even when highly fragmentary. Irrespective of inference method, fossils improve the accuracy of phylogenies and increase the number of resolved nodes. They also induce the collapse of ancient and highly uncertain relationships that tend to be incorrectly resolved when sampling only extant taxa. Furthermore, tip-dated analyses under the fossilized birth-death process outperform undated methods of inference, demonstrating that the stratigraphic ages of fossils contain vital phylogenetic information. Fossils help to extract true phylogenetic signals from morphology, an effect that is mediated by both their distinctive morphology and their temporal information, and their incorporation in total-evidence phylogenetics is necessary to faithfully reconstruct evolutionary history.

Phylogeny and time scale of diversification in the fossil-rich sunfishes and black basses (Teleostei: Percomorpha: Centrarchidae).

Species of the North American freshwater fish lineage Centrarchidae are apex predators in their habitats and are among the world's most popular sport fishes. Centrarchids boast a rich fossil record that extends from the latest Eocene to the Pleistocene. To investigate the phylogeny and timing of diversification of Centrarchidae, we deploy a dataset of DNA sequences of 16 nuclear genes sampled from nearly all of the recognized and undescribed species. We also utilize previously published morphological datasets to assess the phylogenetic placement of one of the oldest known centrarchid fossils, †Plioplarchus whitei. A Bayesian multispecies coalescent species tree analysis provides insight on relationships that evaded resolution in earlier studies, such as the relationships of Acantharchus pomotis, the resolution of a clade consisting of species previously synonymized under the Spotted Bass, Micropterus punctulatus, and a clade of recently described species previously considered populations of the Redeye Bass, Micropterus coosae. This new molecular phylogeny and the inclusion of †P. whitei and other centrarchid fossils in the tip-dated fossilized birth-death analysis results in a new hypothesis of the timing of diversification in Centrarchidae that contextualizes the ages of centrarchid fossils to the timing of speciation among the extant species. In addition to providing new temporal perspectives on the diversification of freshwater fishes in North America, this study may close of the chapter of centrarchid phylogeny inferred using Sanger-sequenced genes, as the use of genomic-scale datasets becomes mainstream in the phylogenetics of fishes.

A Simulation-Based Evaluation of Tip-Dating Under the Fossilized Birth-Death Process.

Bayesian molecular dating is widely used to study evolutionary timescales. This procedure usually involves phylogenetic analysis of nucleotide sequence data, with fossil-based calibrations applied as age constraints on internal nodes of the tree. An alternative approach is tip-dating, which explicitly includes fossil data in the analysis. This can be done, for example, through the joint analysis of molecular data from present-day taxa and morphological data from both extant and fossil taxa. In the context of tip-dating, an important development has been the fossilized birth-death process, which allows non-contemporaneous tips and sampled ancestors while providing a model of lineage diversification for the prior on the tree topology and internal node times. However, tip-dating with fossils faces a number of considerable challenges, especially, those associated with fossil sampling and evolutionary models for morphological characters. We conducted a simulation study to evaluate the performance of tip-dating using the fossilized birth-death model. We simulated fossil occurrences and the evolution of nucleotide sequences and morphological characters under a wide range of conditions. Our analyses of these data show that the number and the maximum age of fossil occurrences have a greater influence than the degree of among-lineage rate variation or the number of morphological characters on estimates of node times and the tree topology. Tip-dating with the fossilized birth-death model generally performs well in recovering the relationships among extant taxa but has difficulties in correctly placing fossil taxa in the tree and identifying the number of sampled ancestors. The method yields accurate estimates of the ages of the root and crown group, although the precision of these estimates varies with the probability of fossil occurrence. The exclusion of morphological characters results in a slight overestimation of node times, whereas the exclusion of nucleotide sequences has a negative impact on inference of the tree topology. Our results provide an overview of the performance of tip-dating using the fossilized birth-death model, which will inform further development of the method and its application to key questions in evolutionary biology.

Olson's Gap or Olson's Extinction? A Bayesian tip-dating approach to resolving stratigraphic uncertainty.

Adaptive radiations and mass extinctions are of critical importance in structuring terrestrial ecosystems. However, the causes and progress of these transitions often remain controversial, in part because of debates surrounding the completeness of the fossil record and biostratigraphy of the relevant fossil-bearing formations. The early-middle Permian, when a substantial faunal turnover in tetrapods coincided with a restructuring of the trophic structure of ecosystems, is such a time. Some have suggested the transition is obscured by a gap in the tetrapod fossil record (Olson's Gap), while others suggest a correlation between North American and Russian tetrapod-bearing formations allows the interval to be documented in detail. The latter biostratigraphic scheme has been used to support a mass extinction at this time (Olson's Extinction). Bayesian tip-dating methods used frequently in phylogenetics are employed to resolve this debate. Bayes factors are used to compare the results of analyses incorporating tip age priors based on different stratigraphic hypotheses, to show which stratigraphic scheme best fits the morphological data and phylogeny. Olson's Gap is rejected, and the veracity of Olson's Extinction is given further support. Tip-dating approaches have great potential to resolve debates surrounding the stratigraphic ages of critical formations where appropriate morphological data is available.

Tip-dating and the origin of Telluraves.

Despite a relatively vast accumulation of molecular data, the timing of diversification of modern bird lineages remains elusive. Accurate dating of the origination of Telluraves-a clade of birds defined by their arboreality-is of particular interest, as it contains the most species-rich avian group, the passerines. Historically, neontological studies have estimated a Cretaceous origin for the group, but more recent studies have recovered Cenozoic dates, closer to the oldest known fossils for the group. We employ total-evidence dating to estimate divergence times that are expected to be both less sensitive to prior assumptions and more accurate. Specifically, we use a large collection of morphological character data from arboreal bird fossils, along with combined molecular sequence and morphological character data from extant taxa. Our analyses recover a Late Cretaceous origin for crown Telluraves, with a few lineages crossing the K-Pg boundary. Following the K-Pg boundary, our results show the group underwent rapid diversification, likely benefiting from increased ecological opportunities in the aftermath of the extinction event. We find very little confidence for the precise topological placement of many extinct taxa, possibly due to rapid diversification, paucity of character data, and rapid morphological differentiation during the early history of the group.

Armed conflict and population displacement as drivers of the evolution and dispersal of Mycobacterium tuberculosis.

The "Beijing" Mycobacterium tuberculosis (Mtb) lineage 2 (L2) is spreading globally and has been associated with accelerated disease progression and increased antibiotic resistance. Here we performed a phylodynamic reconstruction of one of the L2 sublineages, the central Asian clade (CAC), which has recently spread to western Europe. We find that recent historical events have contributed to the evolution and dispersal of the CAC. Our timing estimates indicate that the clade was likely introduced to Afghanistan during the 1979-1989 Soviet-Afghan war and spread further after population displacement in the wake of the American invasion in 2001. We also find that drug resistance mutations accumulated on a massive scale in Mtb isolates from former Soviet republics after the fall of the Soviet Union, a pattern that was not observed in CAC isolates from Afghanistan. Our results underscore the detrimental effects of political instability and population displacement on tuberculosis control and demonstrate the power of phylodynamic methods in exploring bacterial evolution in space and time.

A comparison of methods for estimating substitution rates from ancient DNA sequence data.

BACKGROUND: Phylogenetic analysis of DNA from modern and ancient samples allows the reconstruction of important demographic and evolutionary processes. A critical component of these analyses is the estimation of evolutionary rates, which can be calibrated using information about the ages of the samples. However, the reliability of these rate estimates can be negatively affected by among-lineage rate variation and non-random sampling. Using a simulation study, we compared the performance of three phylogenetic methods for inferring evolutionary rates from time-structured data sets: regression of root-to-tip distances, least-squares dating, and Bayesian inference. We also applied these three methods to time-structured mitogenomic data sets from six vertebrate species. RESULTS: Our results from 12 simulation scenarios show that the three methods produce reliable estimates when the substitution rate is high, rate variation is low, and samples of similar ages are not all grouped together in the tree (i.e., low phylo-temporal clustering). The interaction of these factors is particularly important for least-squares dating and Bayesian estimation of evolutionary rates. The three estimation methods produced consistent estimates of rates across most of the six mitogenomic data sets, with sequence data from horses being an exception. CONCLUSIONS: We recommend that phylogenetic studies of ancient DNA sequences should use multiple methods of inference and test for the presence of temporal signal, among-lineage rate variation, and phylo-temporal clustering in the data.

Tip-dating and homoplasy: reconciling the shallow molecular divergences of modern gharials with their long fossil record.

Simultaneously analysing morphological, molecular and stratigraphic data suggests a potential resolution to a major remaining inconsistency in crocodylian evolution. The ancient, long-snouted thoracosaurs have always been placed near the Indian gharial Gavialis, but their antiquity (ca 72 Ma) is highly incongruous with genomic evidence for the young age of the Gavialis lineage (ca 40 Ma). We reconcile this contradiction with an updated morphological dataset and novel analysis, and demonstrate that thoracosaurs are an ancient iteration of long-snouted stem crocodylians unrelated to modern gharials. The extensive similarities between thoracosaurs and Gavialis are shown to be an almost 'perfect storm' of homoplasy, combining convergent adaptions to fish-eating, as well resemblances between genuinely primitive traits (thoracosaurs) and atavisms (Gavialis). Phylogenetic methods that ignore stratigraphy (parsimony and undated Bayesian methods) are unable to tease apart these similarities and invariably unite thoracosaurs and Gavialis. However, tip-dated Bayesian approaches additionally consider the large temporal gap separating ancient (thoracosaurs) and modern (Gavialis) iterations of similar long-snouted crocodyliforms. These analyses robustly favour a phylogeny which places thoracosaurs basal to crocodylians, far removed from modern gharials, which accordingly are a very young radiation. This phylogenetic uncoupling of ancient and modern gharial-like crocs is more consistent with molecular clock divergence estimates, and also the bulk of the crocodylian fossil record (e.g. all unequivocal gharial fossils are very young). Provided that the priors and models attribute appropriate relative weights to the morphological and stratigraphic signals-an issue that requires investigation-tip-dating approaches are potentially better able to detect homoplasy and improve inferences about phylogenetic relationships, character evolution and divergence dates.

Bayesian Morphological Clock Methods Resurrect Placoderm Monophyly and Reveal Rapid Early Evolution in Jawed Vertebrates.

The phylogeny of early gnathostomes provides an important framework for understanding one of the most significant evolutionary events, the origin and diversification of jawed vertebrates. A series of recent cladistic analyses have suggested that the placoderms, an extinct group of armoured fish, form a paraphyletic group basal to all other jawed vertebrates. We revised and expanded this morphological data set, most notably by sampling autapomorphies in a similar way to parsimony-informative traits, thus ensuring this data (unlike most existing morphological data sets) satisfied an important assumption of Bayesian tip-dated morphological clock approaches. We also found problems with characters supporting placoderm paraphyly, including character correlation and incorrect codings. Analysis of this data set reveals that paraphyly and monophyly of core placoderms (excluding maxillate forms) are essentially equally parsimonious. The two alternative topologies have different root positions for the jawed vertebrates but are otherwise similar. However, analysis using tip-dated clock methods reveals strong support for placoderm monophyly, due to this analysis favoring trees with more balanced rates of evolution. Furthermore, enforcing placoderm paraphyly results in higher levels and unusual patterns of rate heterogeneity among branches, similar to that generated from simulated trees reconstructed with incorrect root positions. These simulations also show that Bayesian tip-dated clock methods outperform parsimony when the outgroup is largely uninformative (e.g., due to inapplicable characters), as might be the case here. The analysis also reveals that gnathostomes underwent a rapid burst of evolution during the Silurian period which declined during the Early Devonian. This rapid evolution during a period with few articulated fossils might partly explain the difficulty in ascertaining the root position of jawed vertebrates.

Inferences from tip-calibrated phylogenies: a review and a practical guide.

Molecular dating of phylogenetic trees is a growing discipline using sequence data to co-estimate the timing of evolutionary events and rates of molecular evolution. All molecular-dating methods require converting genetic divergence between sequences into absolute time. Historically, this could only be achieved by associating externally derived dates obtained from fossil or biogeographical evidence to internal nodes of the tree. In some cases, notably for fast-evolving genomes such as viruses and some bacteria, the time span over which samples were collected may cover a significant proportion of the time since they last shared a common ancestor. This situation allows phylogenetic trees to be calibrated by associating sampling dates directly to the sequences representing the tips (terminal nodes) of the tree. The increasing availability of genomic data from ancient DNA extends the applicability of such tip-based calibration to a variety of taxa including humans, extinct megafauna and various microorganisms which typically have a scarce fossil record. The development of statistical models accounting for heterogeneity in different aspects of the evolutionary process while accommodating very large data sets (e.g. whole genomes) has allowed using tip-dating methods to reach inferences on divergence times, substitution rates, past demography or the age of specific mutations on a variety of spatiotemporal scales. In this review, we summarize the current state of the art of tip dating, discuss some recent applications, highlight common pitfalls and provide a 'how to' guide to thoroughly perform such analyses.

Topology, divergence dates, and macroevolutionary inferences vary between different tip-dating approaches applied to fossil theropods (Dinosauria).

Dated phylogenies of fossil taxa allow palaeobiologists to estimate the timing of major divergences and placement of extinct lineages, and to test macroevolutionary hypotheses. Recently developed Bayesian 'tip-dating' methods simultaneously infer and date the branching relationships among fossil taxa, and infer putative ancestral relationships. Using a previously published dataset for extinct theropod dinosaurs, we contrast the dated relationships inferred by several tip-dating approaches and evaluate potential downstream effects on phylogenetic comparative methods. We also compare tip-dating analyses to maximum-parsimony trees time-scaled via alternative a posteriori approaches including via the probabilistic cal3 method. Among tip-dating analyses, we find opposing but strongly supported relationships, despite similarity in inferred ancestors. Overall, tip-dating methods infer divergence dates often millions (or tens of millions) of years older than the earliest stratigraphic appearance of that clade. Model-comparison analyses of the pattern of body-size evolution found that the support for evolutionary mode can vary across and between tree samples from cal3 and tip-dating approaches. These differences suggest that model and software choice in dating analyses can have a substantial impact on the dated phylogenies obtained and broader evolutionary inferences.

Multiple morphological clocks and total-evidence tip-dating in mammals.

Morphological integration predicts that correlated characters will coevolve; thus, each distinct suite of correlated characters might be expected to evolve according to a separate clock or 'pacemaker'. Characters in a large morphological dataset for mammals were found to be evolving according to seven separate clocks, each distinct from the molecular clock. Total-evidence tip-dating using these multiple clocks inflated divergence time estimates, but potentially improved topological inference. In particular, single-clock analyses placed several meridiungulates and condylarths in a heterodox position as stem placentals, but multi-clock analyses retrieved a more plausible and orthodox position within crown placentals. Several shortcomings (including uneven character sampling) currently impact upon the accuracy of total-evidence dating, but this study suggests that when sufficiently large and appropriately constructed phenotypic datasets become more commonplace, multi-clock approaches are feasible and can affect both divergence dates and phylogenetic relationships.

Inferring node dates from tip dates in fossil Canidae: the importance of tree priors.

Tip-dating methods are becoming popular alternatives to traditional node calibration approaches for building time-scaled phylogenetic trees, but questions remain about their application to empirical datasets. We compared the performance of the most popular methods against a dated tree of fossil Canidae derived from previously published monographs. Using a canid morphology dataset, we performed tip-dating using BEAST v. 2.1.3 and MrBayes v. 3.2.5. We find that for key nodes (Canis, approx. 3.2 Ma, Caninae approx. 11.7 Ma) a non-mechanistic model using a uniform tree prior produces estimates that are unrealistically old (27.5, 38.9 Ma). Mechanistic models (incorporating lineage birth, death and sampling rates) estimate ages that are closely in line with prior research. We provide a discussion of these two families of models (mechanistic versus non-mechanistic) and their applicability to fossil datasets.

Phylogenetic analysis of molecular and morphological data highlights uncertainty in the relationships of fossil and living species of Elopomorpha (Actinopterygii: Teleostei).

Elopomorpha is one of the three main clades of living teleost fishes and includes a range of disparate lineages including eels, tarpons, bonefishes, and halosaurs. Elopomorphs were among the first groups of fishes investigated using Hennigian phylogenetic methods and continue to be the object of intense phylogenetic scrutiny due to their economic significance, diversity, and crucial evolutionary status as the sister group of all other teleosts. While portions of the phylogenetic backbone for Elopomorpha are consistent between studies, the relationships among Albula, Pterothrissus, Notacanthiformes, and Anguilliformes remain contentious and difficult to evaluate. This lack of phylogenetic resolution is problematic as fossil lineages are often described and placed taxonomically based on an assumed sister group relationship between Albula and Pterothrissus. In addition, phylogenetic studies using morphological data that sample elopomorph fossil lineages often do not include notacanthiform or anguilliform lineages, potentially introducing a bias toward interpreting fossils as members of the common stem of Pterothrissus and Albula. Here we provide a phylogenetic analysis of DNA sequences sampled from multiple nuclear genes that include representative taxa from Albula, Pterothrissus, Notacanthiformes and Anguilliformes. We integrate our molecular dataset with a morphological character matrix that spans both living and fossil elopomorph lineages. Our results reveal substantial uncertainty in the placement of Pterothrissus as well as all sampled fossil lineages, questioning the stability of the taxonomy of fossil Elopomorpha. However, despite topological uncertainty, our integration of fossil lineages into a Bayesian time calibrated framework provides divergence time estimates for the clade that are consistent with previously published age estimates based on the elopomorph fossil record and molecular estimates resulting from traditional node-dating methods.

An evaluation of fossil tip-dating versus node-age calibrations in tetraodontiform fishes (Teleostei: Percomorphaceae).

Time-calibrated phylogenies based on molecular data provide a framework for comparative studies. Calibration methods to combine fossil information with molecular phylogenies are, however, under active development, often generating disagreement about the best way to incorporate paleontological data into these analyses. This study provides an empirical comparison of the most widely used approach based on node-dating priors for relaxed clocks implemented in the programs BEAST and MrBayes, with two recently proposed improvements: one using a new fossilized birth-death process model for node dating (implemented in the program DPPDiv), and the other using a total-evidence or tip-dating method (implemented in MrBayes and BEAST). These methods are applied herein to tetraodontiform fishes, a diverse group of living and extinct taxa that features one of the most extensive fossil records among teleosts. Previous estimates of time-calibrated phylogenies of tetraodontiforms using node-dating methods reported disparate estimates for their age of origin, ranging from the late Jurassic to the early Paleocene (ca. 150-59Ma). We analyzed a comprehensive dataset with 16 loci and 210 morphological characters, including 131 taxa (95 extant and 36 fossil species) representing all families of fossil and extant tetraodontiforms, under different molecular clock calibration approaches. Results from node-dating methods produced consistently younger ages than the tip-dating approaches. The older ages inferred by tip dating imply an unlikely early-late Jurassic (ca. 185-119Ma) origin for this order and the existence of extended ghost lineages in their fossil record. Node-based methods, by contrast, produce time estimates that are more consistent with the stratigraphic record, suggesting a late Cretaceous (ca. 86-96Ma) origin. We show that the precision of clade age estimates using tip dating increases with the number of fossils analyzed and with the proximity of fossil taxa to the node under assessment. This study suggests that current implementations of tip dating may overestimate ages of divergence in calibrated phylogenies. It also provides a comprehensive phylogenetic framework for tetraodontiform systematics and future comparative studies.

Phylogenetic relationships and timing of diversification in gonorynchiform fishes inferred using nuclear gene DNA sequences (Teleostei: Ostariophysi).

The Gonorynchiformes are the sister lineage of the species-rich Otophysi and provide important insights into the diversification of ostariophysan fishes. Phylogenies of gonorynchiforms inferred using morphological characters and mtDNA gene sequences provide differing resolutions with regard to the sister lineage of all other gonorynchiforms (Chanos vs. Gonorynchus) and support for monophyly of the two miniaturized lineages Cromeria and Grasseichthys. In this study the phylogeny and divergence times of gonorynchiforms are investigated with DNA sequences sampled from nine nuclear genes and a published morphological character matrix. Bayesian phylogenetic analyses reveal substantial congruence among individual gene trees with inferences from eight genes placing Gonorynchus as the sister lineage to all other gonorynchiforms. Seven gene trees resolve Cromeria and Grasseichthys as a clade, supporting previous inferences using morphological characters. Phylogenies resulting from either concatenating the nuclear genes, performing a multispecies coalescent species tree analysis, or combining the morphological and nuclear gene DNA sequences resolve Gonorynchus as the living sister lineage of all other gonorynchiforms, strongly support the monophyly of Cromeria and Grasseichthys, and resolve a clade containing Parakneria, Cromeria, and Grasseichthys. The morphological dataset, which includes 13 gonorynchiform fossil taxa that range in age from Early Cretaceous to Eocene, was analyzed in combination with DNA sequences from the nine nuclear genes and a relaxed molecular clock to estimate times of evolutionary divergence. This "tip dating" strategy accommodates uncertainty in the phylogenetic resolution of fossil taxa that provide calibration information in the relaxed molecular clock analysis. The estimated age of the most recent common ancestor (MRCA) of living gonorynchiforms is slightly older than estimates from previous node dating efforts, but the molecular tip dating estimated ages of Kneriinae (Kneria, Parakneria, Cromeria, and Grasseichthys) and the two paedomorphic lineages, Cromeria and Grasseichthys, are considerably younger.