Benjamin P. Keck

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.

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.

Phylogeny and Temporal Diversification of Darters (Percidae: Etheostomatinae)

Discussions aimed at resolution of the Tree of Life are most often focused on the interrelationships of major organismal lineages. In this study, we focus on the resolution of some of the most apical branches in the Tree of Life through exploration of the phylogenetic relationships of darters, a species-rich clade of North American freshwater fishes. With a near-complete taxon sampling of close to 250 species, we aim to investigate strategies for efficient multilocus data sampling and the estimation of divergence times using relaxed-clock methods when a clade lacks a fossil record. Our phylogenetic data set comprises a single mitochondrial DNA (mtDNA) gene and two nuclear genes sampled from 245 of the 248 darter species. This dense sampling allows us to determine if a modest amount of nuclear DNA sequence data can resolve relationships among closely related animal species. Darters lack a fossil record to provide age calibration priors in relaxed-clock analyses. Therefore, we use a near-complete species-sampled phylogeny of the perciform clade Centrarchidae, which has a rich fossil record, to assess two distinct strategies of external calibration in relaxed-clock divergence time estimates of darters: using ages inferred from the fossil record and molecular evolutionary rate estimates. Comparison of Bayesian phylogenies inferred from mtDNA and nuclear genes reveals that heterospecific mtDNA is present in approximately 12.5% of all darter species. We identify three patterns of mtDNA introgression in darters: proximal mtDNA transfer, which involves the transfer of mtDNA among extant and sympatric darter species, indeterminate introgression, which involves the transfer of mtDNA from a lineage that cannot be confidently identified because the introgressed haplotypes are not clearly referable to mtDNA haplotypes in any recognized species, and deep introgression, which is characterized by species diversification within a recipient clade subsequent to the transfer of heterospecific mtDNA. The results of our analyses indicate that DNA sequences sampled from single-copy nuclear genes can provide appreciable phylogenetic resolution for closely related animal species. A well-resolved near-complete species-sampled phylogeny of darters was estimated with Bayesian methods using a concatenated mtDNA and nuclear gene data set with all identified heterospecific mtDNA haplotypes treated as missing data. The relaxed-clock analyses resulted in very similar posterior age estimates across the three sampled genes and methods of calibration and therefore offer a viable strategy for estimating divergence times for clades that lack a fossil record. In addition, an informative rank-free clade-based classification of darters that preserves the rich history of nomenclature in the group and provides formal taxonomic communication of darter clades was constructed using the mtDNA and nuclear gene phylogeny. On the whole, the appeal of mtDNA for phylogeny inference among closely related animal species is diminished by the observations of extensive mtDNA introgression and by finding appreciable phylogenetic signal in a modest sampling of nuclear genes in our phylogenetic analyses of darters.

Dispersal, vicariance, and timing of diversification in Nothonotus darters

The species diversity of North American freshwater fishes is unparalleled among temperate regions of the planet. This diversity is concentrated in the Central Highlands of eastern North America and this distribution pattern has inspired different models involving either dispersal or vicariance to explain the high species diversity of North American fishes. The most popular of these models is the Central Highlands vicariance hypothesis (CHVH), which proposes an ancient and diverse widespread fauna that existed across a previously continuous highland landscape that is much different from today. The mechanisms of isolation in the CHVH involve specific instances of vicariance that affected several diverse lineages of Central Highlands fishes. We tested predictions of the CHVH and alternative models using a cytochrome b‐inferred phylogeny of the darter clade Nothonotus. A Bayesian mixed‐model method was used for phylogenetic analysis. The phylogenetic data set included all 20 recognized Nothonotus species, and most species were represented with multiple sequences. We were able to convert genetic branch lengths to absolute age using external fossil calibrations in the freshwater perciform fish clade Centrarchidae. Using a well‐resolved Nothonotus phylogeny and divergence time estimates, we identify equal numbers of instances of both vicariance and dispersal among disjunct regions of the Central Highlands, biogeographic pseudocongruence, rather recent speciation in Nothonotus, and a surprisingly large amount of speciation within highland areas. With regard to Nothonotus, previous Central Highlands biogeographic models offer little in the way of providing possible mechanisms responsible for diversification in the clade. Patterns of speciation in Nothonotus are similar to those discovered in recent efforts that have included speciation as a parameter into classic models of island biogeography.

GEOGRAPHIC AND TEMPORAL ASPECTS OF MITOCHONDRIAL REPLACEMENT IN NOTHONOTUS DARTERS (TELEOSTEI: PERCIDAE: ETHEOSTOMATINAE).

A growing number of molecular studies have identified mitochondrial replacement among closely related animal species, but there has been limited investigation into the phylogenetic, geographic, and temporal patterns, especially in more inclusive clades. We present a phylogenetic analysis of DNA sequences collected from mitochondrial and nuclear genes sampled from all 20 species of the darter clade Nothonotus and reveal extensive mtDNA replacement in N. rufilineatus. Using phylogenetic trees, haplotype networks, analysis of molecular variance (AMOVAs), and distributions of minimum pairwise genetic distances, we discovered that the mtDNA of N. rufilineatus has been replaced by that of different sympatric species of Nothonotus in different river drainages. In the Cumberland River, N. rufilineatus populations were fixed for N. camurus mtDNA. In the upper Tennessee River, N. rufilineatus contained N. chlorobranchius and N. camurus mtDNA. Most surprising, our analyses indicated that N. rufilineatus has acted as a “conduit species,” facilitating the introgression of N. chlorobranchius mtDNA into N. camurus in the upper Tennessee River. We identified several potential mechanisms for the observed pattern of introgression, and suggest experiments to assess their relative contributions. Comparisons among darter subclades indicated that the mitochondrial lineage of the clade is most influential in determining if the lineage is a mitochondrial donor or recipient.

Patterns of Natural Hybridization in Darters (Percidae: Etheostomatinae)

Abstract Hybridization is an evolutionarily important process with varied outcomes that depend on interacting factors of time since common ancestry, behavioral differences, and environmental conditions. Hybridization is relatively common in teleost fishes, and patterns from naturally occurring hybrids and experimental interspecific crosses provide insight into the evolution of reproductive barriers that lead to speciation. It has been several decades since records of hybrid darter specimens (Percidae: Etheostomatinae) have been collected and analyzed. We assembled a dataset of 252 reported natural hybrids from museum collections and literature reports that comprise 67 unique hybrid combinations involving 63 darter species. Frequencies of unique hybrid combinations decrease with phylogenetic distance and are lower between species with different egg deposition behaviors. Large range size and egg burying reproductive behavior were characteristics of the species most frequently identified as a parental species of hybrid specimens. Evidence of genetic introgression was not consistently predicted by the frequency of records for a particular hybrid combination. This dataset likely underestimates the amount of hybridization that has occurred among darter species, because of the relatively narrow evolutionary time frame during which specimens have been collected and identified.

Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related darter lineages (Teleostei: Percidae: Etheostomatinae).

Investigations into the phylogenetics of closely related animal species are dominated by the use of mitochondrial DNA (mtDNA) sequence data. However, the near-ubiquitous use of mtDNA to infer phylogeny among closely related animal lineages is tempered by an increasing number of studies that document high rates of transfer of mtDNA genomes among closely related species through hybridization, leading to substantial discordance between phylogenies inferred from mtDNA and nuclear gene sequences. In addition, the recent development of methods that simultaneously infer a species phylogeny and estimate divergence times, while accounting for incongruence among individual gene trees, has ushered in a new era in the investigation of phylogeny among closely related species. In this study we assess if DNA sequence data sampled from a modest number of nuclear genes can resolve relationships of a species-rich clade of North American freshwater teleost fishes, the darters. We articulate and expand on a recently introduced method to infer a time-calibrated multi-species coalescent phylogeny using the computer program (*)BEAST. Our analyses result in well-resolved and strongly supported time-calibrated darter species tree. Contrary to the expectation that mtDNA will provide greater phylogenetic resolution than nuclear gene data; the darter species tree inferred exclusively from nuclear genes exhibits a higher frequency of strongly supported nodes than the mtDNA time-calibrated gene tree.

Fish functional traits correlated with environmental variables in a temperate biodiversity hotspot.

The global biodiversity crisis has invigorated the search for generalized patterns in most disciplines within the natural sciences. Studies based on organismal functional traits attempt to broaden implications of results by identifying the response of functional traits, instead of taxonomic units, to environmental variables. Determining the functional trait responses enables more direct comparisons with, or predictions for, communities of different taxonomic composition. The North American freshwater fish fauna is both diverse and increasingly imperiled through human mediated disturbances, including climate change. The Tennessee River, USA, contains one of the most diverse assemblages of freshwater fish in North America and has more imperiled species than other rivers, but there has been no trait-based study of community structure in the system. We identified 211 localities in the upper Tennessee River that were sampled by the Tennessee Valley Authority between 2009 and 2011 and compiled fish functional traits for the observed species and environmental variables for each locality. Using fourth corner analysis, we identified significant correlations between many fish functional traits and environmental variables. Functional traits associated with an opportunistic life history strategy were correlated with localities subject to greater land use disturbance and less flow regulation, while functional traits associated with a periodic life history strategy were correlated with localities subject to regular disturbance and regulated flow. These are patterns observed at the continental scale, highlighting the generalizability of trait-based methods. Contrary to studies that found no community structure differences when considering riparian buffer zones, we found that fish functional traits were correlated with different environmental variables between analyses with buffer zones vs. entire catchment area land cover proportions. Using existing databases and fourth corner analysis, our results support the broad application potential for trait-based methods and indicate trait-based methods can detect environmental filtering by riparian zone land cover.

Mitochondrial genome primers for Lake Malawi cichlids

Resolving the evolutionary history of rapidly diversifying lineages like the Lake Malawi Cichlid Flock demands powerful phylogenetic tools. Although this clade of over 500 species of fish likely diversified in less than two million years, the availability of extensive sequence data sets, such as complete mitochondrial genomes, could help resolve evolutionary patterns in this group. Using a large number of newly developed primers, we generated whole mitochondrial genome sequences for 14 Lake Malawi cichlids. We compared sequence divergence across protein‐coding regions of the mitochondrial genome and also compared divergence in the mitochondrial loci to divergence at two nuclear protein‐coding loci, Mitfb and Dlx2. Despite the widespread sharing of haplotypes of identical sequences at individual loci, the combined use of all protein‐coding mitochondrial loci provided a bifurcating phylogenetic hypothesis for the exemplars of major lineages within the Lake Malawi cichlid radiation. The primers presented here could have substantial utility for evolutionary analyses of mitochondrial evolution and hybridization within this diverse clade.

A New Species of Nothonotus Darter (Teleostei: Percidae) from the Caney Fork in Tennessee, USA

ABSTRACT We describe a new species of Nothonotus from the Caney Fork River drainage, Tennessee, USA, and assess morphological variation in N. microlepidus and N. sanguifluus with specimens sampled from all known populations. We used Linear Discriminant Analysis (LDA) on a meristic dataset from 831 individuals to delimit species of the N. maculatus species group endemic to the Cumberland River drainage. We assigned populations to groups in the LDA on the basis of phylogenetic lineages identified in previous studies of Nothonotus darters using mitochondrial and nuclear gene DNA sequences. The discriminant functions were able to reasonably differentiate among N. microlepidus, N. sanguifluus and the new species of Nothonotus. The discriminant functions did not reliably differentiate among N. sanguifluus populations sampled from different tributaries in the Cumberland River drainage. Two populations of N. sanguifluus were not included in the LDA because of limited material and thus could not be assigned to a group; these are tentatively assigned to N. sanguifluus partially on the basis of predictions using the model from the LDA and the estimated phylogeny. Phylogenetic and multivariate analyses of morphological characters allow differentiation of the new species from its closest relatives, N. microlepidus and N. sanguifluus. Additionally, the new species can be differentiated from other species in the N. maculatus species group with a combination of characters, including having red in all median fins of breeding-condition males, the presence of a suborbital bar, extensive red in the pectoral fins, and lower counts of lateral line scales as compared with N. sanguifluus. With the description of this new species of Nothonotus there are now five described endemic darters occurring in the Caney Fork above Great Falls.