Breeding behaviour predicts patterns of natural hybridization in North American minnows (Cyprinidae).

Premating barriers such as variation in reproductive behaviour can evolve quickly, but because gametic and postzygotic incompatibilities often evolve more slowly, circumstances that bring gametes into contact can breach the boundaries of premating isolation. In aquatic environments, the gametes of organisms with external fertilization are released into a constantly moving environment and may come into contact with heterospecific gametes. In fishes, nest association (spawning in another species' nest) is a behaviour that brings gametes from different species into close spatiotemporal proximity. These interactions might increase chances of hybridization, especially when multiple species associate with a single nest builder. This study addresses these interactions in the largest clade of North American freshwater fishes, the minnows (Cyprinidae). We compiled a list of over 17,000 hybrid specimens in conjunction with species distribution data, breeding behaviours, and an inferred phylogeny to test if breeding behaviour, in addition to evolutionary history, is an important predictor of hybridization. We find that breeding behaviour is a significant predictor of hybridization, even when phylogenetic relatedness and divergence time are accounted for. Specifically, nest associates are more likely to hybridize with other nest associates whereas non-nesting species had relatively low rates of hybridization.

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.

Continental monophyly of cichlid fishes and the phylogenetic position of Heterochromis multidens.

The incredibly species-rich cichlid fish faunas of both the Neotropics and Africa are generally thought to be reciprocally monophyletic. However, the phylogenetic affinity of the African cichlid Heterochromis multidens is ambiguous, and this distinct lineage could make African cichlids paraphyletic. In past studies, Heterochromis has been variously suggested to be one of the earliest diverging lineages within either the Neotropical or the African cichlid radiations, and it has even been hypothesized to be the sister lineage to a clade containing all Neotropical and African cichlids. We examined the phylogenetic relationships among a representative sample of cichlids with a dataset of 29 nuclear loci to assess the support for the different hypotheses of the phylogenetic position of Heterochromis. Although individual gene trees in some instances supported alternative relationships, a majority of gene trees, integration of genes into species trees, and hypothesis testing of putative topologies all supported Heterochromis as belonging to the clade of African cichlids.

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.

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.

Pelagic larval duration predicts extinction risk in a freshwater fish clade.

Pelagic larval duration (PLD) can influence evolutionary processes ranging from dispersal to extinction in aquatic organisms. Using estimates of PLD obtained from species of North American darters (Percidae: Etheostomatinae), we demonstrate that this freshwater fish clade exhibits surprising variation in PLD. Comparative analyses provide some evidence that higher stream gradients favour the evolution of shorter PLD. Additionally, similar to patterns in the marine fossil record in which lower PLD is associated with greater extinction probability, we found a reduced PLD in darter lineages was evolutionarily associated with extinction risk. Understanding the causes and consequences of PLD length could lead to better management and conservation of organisms in our increasingly imperiled aquatic environments.

Erosion of heterogeneous rock drives diversification of Appalachian fishes.

The high levels of biodiversity supported by mountains suggest a possible link between geologic processes and biological evolution. Freshwater biodiversity is high not only in tectonically active settings but also in tectonically quiescent montane regions such as the Appalachian Mountains. We show that erosion through different rock types drove allopatric divergence between lineages of the Greenfin Darter (Nothonotus chlorobranchius), a fish species endemic to rivers draining metamorphic rocks in the Tennessee River basin in the United States. In the past, metamorphic rock preferred by N. chlorobranchius was more widespread, but as erosion exposed other rock types, lineages of this species were progressively isolated in tributaries farther upstream, where metamorphic rock remained. Our results suggest a geologic mechanism for initiating allopatric diversification in mountains long after tectonic activity ceases.

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.

Species tree estimation and the historical biogeography of heroine cichlids.

Heroine cichlids are major components of the fish faunas in both Central America and the Caribbean. To examine the evolutionary patterns of how cichlids colonized both of these regions, we reconstructed the phylogenetic relationships among 23 cichlid lineages. We used three phylogenetically novel nuclear markers (Dystropin b, Myomesin1, and Wnt7b) in combination with sequence data from seven other gene regions (Nd2, Rag1, Enc1, Sreb2, Ptr, Plagl2, and Zic1) to elucidate the species tree of these cichlids. The species examined represent major heroine lineages in South America, Central America, and the Greater Antilles. The individual gene trees of these groups were topologically quite discordant. Therefore, we combined the genetic partitions and inferred the species tree using both concatenation and a coalescent-based Bayesian method. The two resulting phylogenetic topologies were largely concordant but differed in two fundamental ways. First, more nodes in the concatenated tree were supported with substantial or 100% Bayesian posterior support than in the coalescent-based tree. Second, there was a minor, but biogeographically critical, topological difference between the concatenated and coalescent-based trees. Nevertheless, both analyses recovered topologies consistent with the Greater Antillean heroines being phylogenetically nested within the largely Central American heroine radiation. This study suggests that reconstructions of cichlid phylogeny and historical biogeography should account for the vagaries of individual gene histories.

A young clade repeating an old pattern: diversity in Nothonotus darters (Teleostei: Percidae) endemic to the Cumberland River.

Hypotheses of diversification in eastern North American freshwater fishes have focused primarily on allopatric distributions of species between disjunct highland areas and major river systems. However, these hypotheses do not fully explain the rich diversity of species within highland regions and river systems. Relatively old diversification events at small geographic scales have been observed in the Barcheek Darter subclade that occurs in the Cumberland River drainage (CRD) in Kentucky and Tennessee, United States of America, but it is unknown if this pattern is consistent in other darter subclades. We explored phylogeographic diversity in two species of Nothonotus darters, N. microlepidus and N. sanguifluus, endemic to the CRD to compare phylogenetic patterns between Barcheek Darters and species of Nothonotus. We collected sequence data for a mitochondrial gene (cytb) and three nuclear genes (MLL, S7 and RAG1) from 19 N. microlepidus and 35 N. sanguifluus specimens. Gene trees were estimated using maximum likelihood and Bayesian methods, and a 'species tree' was inferred using a Bayesian method. These trees indicate that species diversity in Nothonotus is underestimated. Five distinct lineages were evident, despite retained ancestral polymorphism and unsampled extirpated populations. Comparison of chronograms for Barcheek Darters and Nothonotus revealed that microendemism resulting from species diversification at small geographic scales in the CRD is a consistent pattern in both old and young darter subclades. Our analyses reveal that geographic isolating mechanisms that result in similar phylogeographic patterns in the CRD are persistent through long expanses of evolutionary time.

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.

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.

Assessing phylogenetic resolution among mitochondrial, nuclear, and morphological datasets in Nothonotus darters (Teleostei: Percidae).

External morphological characters are the basis of our understanding of diversity and species relationships in many darter clades. The past decade has seen the publication of many studies utilizing mtDNA sequence data to investigate darter phylogenetics, but only recently have nuclear genes been used to investigate darter relationships. Despite a long tradition of use in darter systematics few studies have examined the phylogenetic utility of external morphological characters in estimating relationships among species in darter clades. We present DNA sequence data from the mitochondrial cytochrome b (cytb) gene, the nuclear encoded S7 intron 1, and discretely coded external morphological characters for all 20 species in the darter clade Nothonotus. Bayesian phylogenetic analyses result in phylogenies that are in broad agreement with previous studies. The cytb gene tree is well resolved, while the nuclear S7 gene tree lacks phylogenetic resolution, node support, and is characterized by a lack of reciprocal monophyly for many of the Nothonotus species. The phylogenies resulting from analysis of the morphological dataset lack resolution, but nodes present are found in the cytb and S7 gene trees. The highest resolution and node support is found in the Bayesian combined data phylogeny. Based on our results we propose continued exploration of the phylogenetic utility of external morphological characters in other darter clades. Given the extensive lack of reciprocal monophyly of species observed in the S7 gene tree we predict that nuclear gene sequences may have limited utility in intraspecific phylogeographic studies of Nothonotus 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.