Conservation of Affinity Rather Than Sequence Underlies a Dynamic Evolution of the Motif-Mediated p53/MDM2 Interaction in Ray-Finned Fishes.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 µM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of "functional affinity" in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Phylogenomics, Lineage Diversification Rates, and the Evolution of Diadromy in Clupeiformes (Anchovies, Herrings, Sardines, and Relatives).

Migration independently evolved numerous times in animals, with a myriad of ecological and evolutionary implications. In fishes, perhaps the most extreme form of migration is diadromy, the migration between marine and freshwater environments. Key and longstanding questions are: how many times has diadromy evolved in fishes, how frequently do diadromous clades give rise to non-diadromous species, and does diadromy influence lineage diversification rates? Many diadromous fishes have large geographic ranges with constituent populations that use isolated freshwater habitats. This may limit gene flow among some populations, increasing the likelihood of speciation in diadromous lineages relative to non-diadromous lineages. Alternatively, diadromy may reduce lineage diversification rates if migration is associated with enhanced dispersal capacity that facilitates gene flow within and between populations. Clupeiformes (herrings, sardines, shads and anchovies) is a model clade for testing hypotheses about the evolution of diadromy because it includes an exceptionally high proportion of diadromous species and several independent evolutionary origins of diadromy. However, relationships among major clupeiform lineages remain unresolved and existing phylogenies sparsely sampled diadromous species, limiting the resolution of phylogenetically-informed statistical analyses. We assembled a phylogenomic dataset and used multi-species coalescent and concatenation-based approaches to generate the most comprehensive, highly-resolved clupeiform phylogeny to date, clarifying associations among several major clades and identifying recalcitrant relationships needing further examination. We determined that variation in rates of sequence evolution (heterotachy) and base-composition (non-stationarity) had little impact on our results. Using this phylogeny, we characterized evolutionary patterns of diadromy and tested for differences in lineage diversification rates between diadromous, marine, and freshwater lineages. We identified thirteen transitions to diadromy, all during the Cenozoic Era (ten origins of anadromy, two origins of catadromy, and one origin of amphidromy), and seven losses of diadromy. Two diadromous lineages rapidly generated non-diadromous species, demonstrating that diadromy is not an evolutionary dead-end. We discovered considerably faster transition rates out of diadromy than to diadromy. The largest lineage diversification rate increase in Clupeiformes was associated with a transition to diadromy, but we uncovered little statistical support for categorically faster lineage diversification rates in diadromous versus non-diadromous fishes. We propose that diadromy may increase the potential for accelerated lineage diversification, particularly in species that migrate long distances. However, this potential may only be realized in certain biogeographic contexts, such as when diadromy allows access to ecosystems in which there is limited competition from incumbent species.

The impact of paleoclimatic changes on body size evolution in marine fishes.

Body size is an important species trait, correlating with life span, fecundity, and other ecological factors. Over Earth's geological history, climate shifts have occurred, potentially shaping body size evolution in many clades. General rules attempting to summarize body size evolution include Bergmann's rule, which states that species reach larger sizes in cooler environments and smaller sizes in warmer environments, and Cope's rule, which poses that lineages tend to increase in size over evolutionary time. Tetraodontiform fishes (including pufferfishes, boxfishes, and ocean sunfishes) provide an extraordinary clade to test these rules in ectotherms owing to their exemplary fossil record and the great disparity in body size observed among extant and fossil species. We examined Bergmann's and Cope's rules in this group by combining phylogenomic data (1,103 exon loci from 185 extant species) with 210 anatomical characters coded from both fossil and extant species. We aggregated data layers on paleoclimate and body size from the species examined, and inferred a set of time-calibrated phylogenies using tip-dating approaches for downstream comparative analyses of body size evolution by implementing models that incorporate paleoclimatic information. We found strong support for a temperature-driven model in which increasing body size over time is correlated with decreasing oceanic temperatures. On average, extant tetraodontiforms are two to three times larger than their fossil counterparts, which otherwise evolved during periods of warmer ocean temperatures. These results provide strong support for both Bergmann's and Cope's rules, trends that are less studied in marine fishes compared to terrestrial vertebrates and marine invertebrates.

Phylogenomics of Bony-Tongue Fishes (Osteoglossomorpha) Shed Light on the Craniofacial Evolution and Biogeography of the Weakly Electric Clade (Mormyridae).

Bonytongues (Osteoglossomorpha) constitute an ancient clade of teleost fishes distributed in freshwater habitats throughout the world. The group includes well-known species such as arowanas, featherbacks, pirarucus, and the weakly electric fishes in the family Mormyridae. Their disjunct distribution, extreme morphologies, and electrolocating capabilities (Gymnarchidae and Mormyridae) have attracted much scientific interest, but a comprehensive phylogenetic framework for comparative analysis is missing, especially for the species-rich family Mormyridae. Of particular interest are disparate craniofacial morphologies among mormyrids which might constitute an exceptional model system to study convergent evolution. We present a phylogenomic analysis based on 546 exons of 179 species (out of 260), 28 out of 29 genera, and all six families of extant bonytongues. Based on a recent reassessment of the fossil record of osteoglossomorphs, we inferred dates of divergence among transcontinental clades and the major groups. The estimated ages of divergence among extant taxa (e.g., Osteoglossomorpha, Osteoglossiformes, and Mormyroidea) are older than previous reports, but most of the divergence dates obtained for clades on separate continents are too young to be explained by simple vicariance hypotheses. Biogeographic analysis of mormyrids indicates that their high species diversity in the Congo Basin is a consequence of range reductions of previously widespread ancestors and that the highest diversity of craniofacial morphologies among mormyrids originated in this basin. Special emphasis on a taxon-rich representation for mormyrids revealed pervasive misalignment between our phylogenomic results and mormyrid taxonomy due to repeated instances of convergence for extreme craniofacial morphologies. Estimation of ancestral phenotypes revealed contingent evolution of snout elongation and unique projections from the lower jaw to form the distinctive Schnauzenorgan. Synthesis of comparative analyses suggests that the remarkable craniofacial morphologies of mormyrids evolved convergently due to niche partitioning, likely enabled by interactions between their exclusive morphological and electrosensory adaptations. [Africa; ancestral state estimation; diversity; exon capture; freshwater fishes; Phylogenomics.].

Evolutionary determinism and convergence associated with water-column transitions in marine fishes.

Repeatable, convergent outcomes are prima facie evidence for determinism in evolutionary processes. Among fishes, well-known examples include microevolutionary habitat transitions into the water column, where freshwater populations (e.g., sticklebacks, cichlids, and whitefishes) recurrently diverge toward slender-bodied pelagic forms and deep-bodied benthic forms. However, the consequences of such processes at deeper macroevolutionary scales in the marine environment are less clear. We applied a phylogenomics-based integrative, comparative approach to test hypotheses about the scope and strength of convergence in a marine fish clade with a worldwide distribution (snappers and fusiliers, family Lutjanidae) featuring multiple water-column transitions over the past 45 million years. We collected genome-wide exon data for 110 (∼80%) species in the group and aggregated data layers for body shape, habitat occupancy, geographic distribution, and paleontological and geological information. We also implemented approaches using genomic subsets to account for phylogenetic uncertainty in comparative analyses. Our results show independent incursions into the water column by ancestral benthic lineages in all major oceanic basins. These evolutionary transitions are persistently associated with convergent phenotypes, where deep-bodied benthic forms with truncate caudal fins repeatedly evolve into slender midwater species with furcate caudal fins. Lineage diversification and transition dynamics vary asymmetrically between habitats, with benthic lineages diversifying faster and colonizing midwater habitats more often than the reverse. Convergent ecological and functional phenotypes along the benthic-pelagic axis are pervasive among different lineages and across vastly different evolutionary scales, achieving predictable high-fitness solutions for similar environmental challenges, ultimately demonstrating strong determinism in fish body-shape evolution.

Exon-capture data and locus screening provide new insights into the phylogeny of flatfishes (Pleuronectoidei).

There is an extensive collection of literature on the taxonomy and phylogenetics of flatfishes (Pleuronectiformes) that extends over two centuries, but consensus on many of their evolutionary relationships remains elusive. Phylogenetic uncertainty stems from highly divergent results derived from morphological and genetic characters, and between various molecular datasets. Deciphering relationships is complicated by rapid diversification early in the Pleuronectiformes tree and an abundance of studies that incompletely and inconsistently sample taxa and genetic markers. We present phylogenies based on a genome-wide dataset (4,434 nuclear markers via exon-capture) and wide taxon sampling (86 species spanning 12 of 16 families) of the largest flatfish suborder (Pleuronectoidei). Nine different subsets of the data and two tree construction approaches (eighteen phylogenies in total) are remarkably consistent with other recent molecular phylogenies, and show strong support for the monophyly of all families included except Pleuronectidae. Analyses resolved a novel phylogenetic hypothesis for the family Rhombosoleidae as being within the Pleuronectoidea rather than the Soleoidea, and failed to support the subfamily Hippoglossinae as a monophyletic group. Our results were corroborated with evidence from previous phylogenetic studies to outline regions of persistent phylogenetic uncertainty and identify groups in need of further phylogenetic inference.

Testing the Utility of Alternative Metrics of Branch Support to Address the Ancient Evolutionary Radiation of Tunas, Stromateoids, and Allies (Teleostei: Pelagiaria).

The use of high-throughput sequencing technologies to produce genome-scale data sets was expected to settle some long-standing controversies across the Tree of Life, particularly in areas where short branches occur at deep timescales. Instead, these data sets have often yielded many well-supported but conflicting topologies, and highly variable gene-tree distributions. A variety of branch-support metrics beyond the nonparametric bootstrap are now available to assess how robust a phylogenetic hypothesis may be, as well as new methods to quantify gene-tree discordance. We applied multiple branch-support metrics to a study of an ancient group of marine fishes (Teleostei: Pelagiaria) whose interfamilial relationships have proven difficult to resolve due to a rapid accumulation of lineages very early in its history. We analyzed hundreds of loci including published ultraconserved elements and newly generated exonic data along with their flanking regions to represent all 16 extant families for more than 150 out of 284 valid species in the group. Branch support was typically lower at inter- than intra-familial relationships regardless of the type of marker used. Several nodes that were highly supported with bootstrap had a very low site and gene-tree concordance, revealing underlying conflict. Despite this conflict, we were able to identify four consistent interfamilial clades, each comprised of two or three families. Combining exons with their flanking regions also produced increased branch lengths at the deep branches of the pelagiarian tree. Our results demonstrate the limitations of employing current metrics of branch support and species-tree estimation when assessing the confidence of ancient evolutionary radiations and emphasize the necessity to embrace alternative measurements to explore phylogenetic uncertainty and discordance in phylogenomic data sets.[Concatenation; exons; introns; phylogenomics; species-tree methods; target capture.].

Comprehensive phylogeny of ray-finned fishes (Actinopterygii) based on transcriptomic and genomic data.

Our understanding of phylogenetic relationships among bony fishes has been transformed by analysis of a small number of genes, but uncertainty remains around critical nodes. Genome-scale inferences so far have sampled a limited number of taxa and genes. Here we leveraged 144 genomes and 159 transcriptomes to investigate fish evolution with an unparalleled scale of data: >0.5 Mb from 1,105 orthologous exon sequences from 303 species, representing 66 out of 72 ray-finned fish orders. We apply phylogenetic tests designed to trace the effect of whole-genome duplication events on gene trees and find paralogy-free loci using a bioinformatics approach. Genome-wide data support the structure of the fish phylogeny, and hypothesis-testing procedures appropriate for phylogenomic datasets using explicit gene genealogy interrogation settle some long-standing uncertainties, such as the branching order at the base of the teleosts and among early euteleosts, and the sister lineage to the acanthomorph and percomorph radiations. Comprehensive fossil calibrations date the origin of all major fish lineages before the end of the Cretaceous.

Biogeography, habitat transitions and hybridization in a radiation of South American silverside fishes revealed by mitochondrial and genomic RAD data.

Rivers and lake systems in the southern cone of South America have been widely influenced by historical glaciations, carrying important implications for the evolution of aquatic organisms, including prompting transitions between marine and freshwater habitats and by triggering hybridization among incipient species via waterway connectivity and stream capture events. Silverside fishes (Odontesthes) in the region comprise a radiation of 19 marine and freshwater species that have been hypothesized on the basis of morphological or mitochondrial DNA data to have either transitioned repeatedly into continental waters from the sea or colonized marine habitats following freshwater diversification. New double digest restriction-site associated DNA data presented here provide a robust framework to investigate the biogeographical history of and habitat transitions in Odontesthes. We show that Odontesthes silversides originally diversified in the Pacific but independently colonized the Atlantic three times, producing three independent marine-to-freshwater transitions. Our results also indicate recent introgression of marine mitochondrial haplotypes into two freshwater clades, with more recurring instances of hybridization among Atlantic- versus Pacific-slope species. In Pacific freshwater drainages, hybridization with a marine species appears to be geographically isolated and may be related to glaciation events. Substantial structural differences of estuarine gradients between these two geographical areas may have influenced the frequency, intensity and evolutionary effects of hybridization events.

New insights about species delimitation in red snappers (Lutjanus purpureus and L. campechanus) using multilocus data.

Lutjanus campechanus and Lutjanus purpureus are two commercially important lutjanid fishes (snappers) with non-sympatric distribution throughout Western Atlantic. Even though both taxa have traditionally been regarded as valid species, their taxonomic status remains under debate. In the present study, we used phylogeographic approaches and molecular methods of species delimitation to elucidate the taxonomic issues between both species, based on 1478 base pairs from four genomic regions. We found haplotypes shared between the two species, particularly in relation to nuclear DNA (nuDNA) sequences. The molecular delimitation of species supported the discrimination of L. purpureus and L. campechanus as distinct evolutionary units. Nonetheless, a unidirectional gene flow was found from L. campechanus towards L. purpureus. Therefore, it seems plausible to infer that L. campechanus and L. purpureus are two evolutionary units in which the apparent sharing of haplotypes should be driven by introgression.

On trends and patterns in macroevolution: Williston's law and the branchiostegal series of extant and extinct osteichthyans.

BACKGROUND: The branchiostegal series consists of an alignment of bony elements in the posterior portion of the skull of osteichthyan vertebrates. We trace the evolution of the number of elements in a comprehensive survey that includes 440 extant and 66 extinct species. Using a newly updated actinopterygian tree in combination with phylogenetic comparative analyses, we test whether osteichthyan branchiostegals follow an evolutionary trend under 'Williston's law', which postulates that osteichthyan lineages experienced a reduction of bony elements over time. RESULTS: We detected no overall macroevolutionary trend in branchiostegal numbers, providing no support for 'Williston's law'. This result is robust to the subsampling of palaeontological data, but the estimation of the model parameters is much more ambiguous. CONCLUSIONS: We find substantial evidence for a macroevolutionary dynamic favouring an 'early burst' of trait evolution over alternative models. Our study highlights the challenges of accurately reconstructing macroevolutionary dynamics even with large amounts of data about extant and extinct taxa.

Genomics overrules mitochondrial DNA, siding with morphology on a controversial case of species delimitation.

Species delimitation is a major quest in biology and is essential for adequate management of the organismal diversity. A challenging example comprises the fish species of red snappers in the Western Atlantic. Red snappers have been traditionally recognized as two separate species based on morphology: Lutjanus campechanus (northern red snapper) and L. purpureus (southern red snapper). Recent genetic studies using mitochondrial markers, however, failed to delineate these nominal species, leading to the current lumping of the northern and southern populations into a single species ( L. campechanus). This decision carries broad implications for conservation and management as red snappers have been commercially over-exploited across the Western Atlantic and are currently listed as vulnerable. To address this conflict, we examine genome-wide data collected throughout the range of the two species. Population genomics, phylogenetic and coalescent analyses favour the existence of two independent evolutionary lineages, a result that confirms the morphology-based delimitation scenario in agreement with conventional taxonomy. Despite finding evidence of introgression in geographically neighbouring populations in northern South America, our genomic analyses strongly support isolation and differentiation of these species, suggesting that the northern and southern red snappers should be treated as distinct taxonomic entities.

Comparative phylogeography of trans-Andean freshwater fishes based on genome-wide nuclear and mitochondrial markers.

The Neotropical region represents one of the greatest biodiversity hot spots on earth. Despite its unparalleled biodiversity, regional comparative phylogeographic studies are still scarce, with most focusing on model clades (e.g. birds) and typically examining a handful of loci. Here, we apply a genome-wide comparative phylogeographic approach to test hypotheses of codiversification of freshwater fishes in the trans-Andean region. Using target capture methods, we examined exon data for over 1,000 loci combined with complete mitochondrial genomes to study the phylogeographic history of five primary fish species (>150 individuals) collected from eight major river basins in Northwestern South America and Lower Central America. To assess their patterns of genetic structure, we inferred genealogical concordance taking into account all major aspects of phylogeography (within loci, across multiple genes, across species and among biogeographic provinces). Based on phylogeographic concordance factors, we tested four a priori biogeographic hypotheses, finding support for three of them and uncovering a novel, unexpected pattern of codiversification. The four emerging inter-riverine patterns are as follows: (a) Tuira + Atrato, (b) Ranchería + Catatumbo, (c) Magdalena system and (d) Sinú + Atrato. These patterns are interpreted as shared responses to the complex uplifting and orogenic processes that modified or sundered watersheds, allowing codiversification and speciation over geological time. We also find evidence of cryptic speciation in one of the species examined and instances of mitochondrial introgression in others. These results help further our knowledge of the historical geographic factors shaping the outstanding biodiversity of the Neotropics.

Post-Cretaceous bursts of evolution along the benthic-pelagic axis in marine fishes.

Ecological opportunity arising in the aftermath of mass extinction events is thought to be a powerful driver of evolutionary radiations. Here, we assessed how the wake of the Cretaceous-Palaeogene (K-Pg) mass extinction shaped diversification dynamics in a clade of mostly marine fishes (Carangaria), which comprises a disparate array of benthic and pelagic dwellers including some of the most astonishing fish forms (e.g. flatfishes, billfishes, remoras, archerfishes). Analyses of lineage diversification show time-heterogeneous rates of lineage diversification in carangarians, with highest rates reached during the Palaeocene. Likewise, a remarkable proportion of Carangaria's morphological variation originated early in the history of the group and in tandem with a marked incidence of habitat shifts. Taken together, these results suggest that all major lineages and body plans in Carangaria originated in an early burst shortly after the K-Pg mass extinction, which ultimately allowed the occupation of newly released niches along the benthic-pelagic habitat axis.

Phylogenetic classification of bony fishes.

BACKGROUND: Fish classifications, as those of most other taxonomic groups, are being transformed drastically as new molecular phylogenies provide support for natural groups that were unanticipated by previous studies. A brief review of the main criteria used by ichthyologists to define their classifications during the last 50 years, however, reveals slow progress towards using an explicit phylogenetic framework. Instead, the trend has been to rely, in varying degrees, on deep-rooted anatomical concepts and authority, often mixing taxa with explicit phylogenetic support with arbitrary groupings. Two leading sources in ichthyology frequently used for fish classifications (JS Nelson's volumes of Fishes of the World and W. Eschmeyer's Catalog of Fishes) fail to adopt a global phylogenetic framework despite much recent progress made towards the resolution of the fish Tree of Life. The first explicit phylogenetic classification of bony fishes was published in 2013, based on a comprehensive molecular phylogeny ( www.deepfin.org ). We here update the first version of that classification by incorporating the most recent phylogenetic results. RESULTS: The updated classification presented here is based on phylogenies inferred using molecular and genomic data for nearly 2000 fishes. A total of 72 orders (and 79 suborders) are recognized in this version, compared with 66 orders in version 1. The phylogeny resolves placement of 410 families, or ~80% of the total of 514 families of bony fishes currently recognized. The ordinal status of 30 percomorph families included in this study, however, remains uncertain (incertae sedis in the series Carangaria, Ovalentaria, or Eupercaria). Comments to support taxonomic decisions and comparisons with conflicting taxonomic groups proposed by others are presented. We also highlight cases were morphological support exist for the groups being classified. CONCLUSIONS: This version of the phylogenetic classification of bony fishes is substantially improved, providing resolution for more taxa than previous versions, based on more densely sampled phylogenetic trees. The classification presented in this study represents, unlike any other, the most up-to-date hypothesis of the Tree of Life of fishes.

Widespread ecomorphological convergence in multiple fish families spanning the marine-freshwater interface.

The theoretical definition and quantification of convergence is an increasingly topical focus in evolutionary research, with particular growing interest on study scales spanning deep phylogenetic divergences and broad geographical areas. While much progress has recently been made in understanding the role of convergence in driving terrestrial (e.g. anole lizards) and aquatic (e.g. cichlids) radiations, little is known about its macroevolutionary effects across environmental gradients. This study uses a suite of recently developed comparative approaches integrating diverse aspects of morphology, dietary data, habitat affiliation and phylogeny to assess convergence across several well-known tropical-temperate fish families in the percomorph suborder Terapontoidei, a clade with considerable phenotypic and ecological diversity radiating in both marine and freshwater environments. We demonstrate significant widespread convergence across many lineages occupying equivalent trophic niches, particularly feeding habits such as herbivory and biting of attached prey off hard substrates. These include several examples of convergent morphotypes evolving independently in marine and freshwater clades, separated by deep evolutionary divergences (tens of millions of years). The Terapontoidei present a new example of the macroevolutionary dynamics of morphological and ecological coevolution in relation to habitat and trophic preferences, at a greater phylogenetic and habitat scale than most well-studied adaptive radiations.

Herbivory Promotes Dental Disparification and Macroevolutionary Dynamics in Grunters (Teleostei: Terapontidae), a Freshwater Adaptive Radiation.

Trophic shifts into new adaptive zones have played major (although often conflicting) roles in reshaping the evolutionary trajectories of many lineages. We analyze data on diet, tooth, and oral morphology and relate these traits to phenotypic disparification and lineage diversification rates across the ecologically diverse Terapontidae, a family of Australasian fishes. In contrast to carnivores and most omnivores, which have retained relatively simple, ancestral caniniform tooth shapes, herbivorous terapontids appear to have evolved a variety of novel tooth shapes at significantly faster rates to meet the demands of plant-based diets. The evolution of herbivory prompted major disparification, significantly expanding the terapontid adaptive phenotypic continuum into an entirely novel functional morphospace. There was minimal support for our hypothesis of faster overall rates of integrated tooth shape, spacing, and jaw biomechanical evolution in herbivorous terapontids in their entirety, compared with other trophic strategies. There was, however, considerable support for accelerated disparification within a diverse freshwater clade containing a range of specialized freshwater herbivores. While the evolutionary transition to herbivorous diets has played a central role in terapontid phenotypic diversification by pushing herbivores toward novel fitness peaks, there was little support for herbivory driving significantly higher lineage diversification compared with background rates across the family.

Phylogenetic placement of enigmatic percomorph families (Teleostei: Percomorphaceae).

Percomorphs are a large and diverse group of spiny-finned fishes that have come to be known as the "bush at the top" due to their persistent lack of phylogenetic resolution. Recently, the broader Euteleost Tree of Life project (EToL) inferred a well-supported phylogenetic hypothesis that groups the diversity of percomorphs into nine well-supported series (supraordinal groups): Ophidiaria, Batrachoidaria, Gobiaria, Syngnatharia, Pelagiaria, Anabantaria, Carangaria, Ovalentaria, and Eupercaria. The EToL also provided, for the first time, a monophyletic definition of Perciformes - the largest order of vertebrates. Despite significant progress made in accommodating the diversity of percomorph taxa into major clades, some 62 families (most previously placed in "Perciformes", as traditionally defined) were not examined by the EToL. Here, we provide evidence for the phylogenetic affinities of 10 of those 62 families, seven of which have largely remained enigmatic. This expanded taxonomic sampling also provides further support for the nine EToL supraordinal series. We examined sequences from 21 genes previously used by the EToL and added two fast-evolving mitochondrial markers in an attempt to increase resolution within the rapid percomorph radiations. We restricted the taxonomic sampling to 1229 percomorph species, including expanded sampling from recent studies. Results of maximum likelihood analysis revealed that bathyclupeids (Bathyclupeidae), galjoen fishes (Dichistiidae), kelpfishes (Chironemidae), marblefishes (Aplodactylidae), trumpeters (Latridae), barbeled grunters (Hapalogenyidae), slopefishes (Symphysanodontidae), and picarel porgies (formerly Centracanthidae) are members of the series Eupercaria ("new bush at the top"). The picarel porgies and porgies (Sparidae) are now placed in the same family (Sparidae). Our analyses suggest a close affinity between the orders Spariformes (including Lethrinidae, Nemipteridae and Sparidae) and Lobotiformes (including the tripletails or Lobotidae, the barbeled grunters, and tigerperches or Datnioididae), albeit support for this group is low. None of the newly examined families belong in the order Perciformes, as recently defined. Finally, we confirm results from other recent studies that place the Australasian salmons (Arripidae) within Pelagiaria, and the false trevallies (Lactariidae) close to flatfishes, jacks, and trevallies, within Carangaria.

Fossil-based comparative analyses reveal ancient marine ancestry erased by extinction in ray-finned fishes.

The marine-freshwater boundary is a major biodiversity gradient and few groups have colonised both systems successfully. Fishes have transitioned between habitats repeatedly, diversifying in rivers, lakes and oceans over evolutionary time. However, their history of habitat colonisation and diversification is unclear based on available fossil and phylogenetic data. We estimate ancestral habitats and diversification and transition rates using a large-scale phylogeny of extant fish taxa and one containing a massive number of extinct species. Extant-only phylogenetic analyses indicate freshwater ancestry, but inclusion of fossils reveal strong evidence of marine ancestry in lineages now restricted to freshwaters. Diversification and colonisation dynamics vary asymmetrically between habitats, as marine lineages colonise and flourish in rivers more frequently than the reverse. Our study highlights the importance of including fossils in comparative analyses, showing that freshwaters have played a role as refuges for ancient fish lineages, a signal erased by extinction in extant-only phylogenies.

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.