Richard E. Broughton

The Tree of Life and a New Classification of Bony Fishes

The tree of life of fishes is in a state of flux because we still lack a comprehensive phylogeny that includes all major groups. The situation is most critical for a large clade of spiny-finned fishes, traditionally referred to as percomorphs, whose uncertain relationships have plagued ichthyologists for over a century. Most of what we know about the higher-level relationships among fish lineages has been based on morphology, but rapid influx of molecular studies is changing many established systematic concepts. We report a comprehensive molecular phylogeny for bony fishes that includes representatives of all major lineages. DNA sequence data for 21 molecular markers (one mitochondrial and 20 nuclear genes) were collected for 1410 bony fish taxa, plus four tetrapod species and two chondrichthyan outgroups (total 1416 terminals). Bony fish diversity is represented by 1093 genera, 369 families, and all traditionally recognized orders. The maximum likelihood tree provides unprecedented resolution and high bootstrap support for most backbone nodes, defining for the first time a global phylogeny of fishes. The general structure of the tree is in agreement with expectations from previous morphological and molecular studies, but significant new clades arise. Most interestingly, the high degree of uncertainty among percomorphs is now resolved into nine well-supported supraordinal groups. The order Perciformes, considered by many a polyphyletic taxonomic waste basket, is defined for the first time as a monophyletic group in the global phylogeny. A new classification that reflects our phylogenetic hypothesis is proposed to facilitate communication about the newly found structure of the tree of life of fishes. Finally, the molecular phylogeny is calibrated using 60 fossil constraints to produce a comprehensive time tree. The new time-calibrated phylogeny will provide the basis for and stimulate new comparative studies to better understand the evolution of the amazing diversity of fishes.

Multi-locus phylogenetic analysis reveals the pattern and tempo of bony fish evolution

Over half of all vertebrates are “fishes”, which exhibit enormous diversity in morphology, physiology, behavior, reproductive biology, and ecology. Investigation of fundamental areas of vertebrate biology depend critically on a robust phylogeny of fishes, yet evolutionary relationships among the major actinopterygian and sarcopterygian lineages have not been conclusively resolved. Although a consensus phylogeny of teleosts has been emerging recently, it has been based on analyses of various subsets of actinopterygian taxa, but not on a full sample of all bony fishes. Here we conducted a comprehensive phylogenetic study on a broad taxonomic sample of 61 actinopterygian and sarcopterygian lineages (with a chondrichthyan outgroup) using a molecular data set of 21 independent loci. These data yielded a resolved phylogenetic hypothesis for extant Osteichthyes, including 1) reciprocally monophyletic Sarcopterygii and Actinopterygii, as currently understood, with polypteriforms as the first diverging lineage within Actinopterygii; 2) a monophyletic group containing gars and bowfin (= Holostei) as sister group to teleosts; and 3) the earliest diverging lineage among teleosts being Elopomorpha, rather than Osteoglossomorpha. Relaxed-clock dating analysis employing a set of 24 newly applied fossil calibrations reveals divergence times that are more consistent with paleontological estimates than previous studies. Establishing a new phylogenetic pattern with accurate divergence dates for bony fishes illustrates several areas where the fossil record is incomplete and provides critical new insights on diversification of this important vertebrate group.

Variable microsatellite markers amplify across divergent lineages of cyprinid fishes (subfamily Leusicinae)

Thomas F. Turner1,∗, Thomas E. Dowling2, Richard E. Broughton3 & John R. Gold4 1Department of Biology and Museum of Southwestern Biology, University of New Mexico, Albuquerque, NM 87131; 2Department of Biology, Arizona State University, Tempe, AZ 85287; 3Department of Zoology, University of Oklahoma, Norman, OK 73019; 4Center for Biosystematics and Biodiversity, Texas A&M University, College Station, TX 77843 (∗Corresponding author: E-mail: turnert@unm.edu)

Quantification of Homoplasy for Nucleotide Transitions and Transversions and a Reexamination of Assumptions in Weighted Phylogenetic Analysis

Nucleotide transitions are frequently down-weighted relative to transversions in phylogenetic analysis. This is based on the assumption that transitions, by virtue of their greater evolutionary rate, exhibit relatively more homoplasy and are therefore less reliable phylogenetic characters. Relative amounts of homoplastic and consistent transition and transversion changes in mitochondrial protein coding genes were determined from character-state reconstructions on a highly corroborated phylogeny of mammals. We found that although homoplasy was related to evolutionary rates and was greater for transitions, the absolute number of consistent transitions greatly exceeded the number of consistent transversions. Consequently, transitions provided substantially more useful phylogenetic information than transversions. These results suggest that down-weighting transitions may be unwarranted in many cases. This conclusion was supported by the fact that a range of transition: transversion weighting schemes applied to various mitochondrial genes and genomic partitions rarely provided improvement in phylogenetic estimates relative to equal weighting, and in some cases weighting transitions more heavily than transversions was most effective.

Diversification on an ecologically constrained adaptive landscape

We used phylogenetic analysis of body‐size ecomorphs in a crustacean species complex to gain insight into how spatial complexity of ecological processes generates and maintains biological diversity. Studies of geographically widespread species of Hyalella amphipods show that phenotypic evolution is tightly constrained in a manner consistent with adaptive responses to alternative predation regimes. A molecular phylogeny indicates that evolution of Hyalella ecomorphs is characterized by parallel evolution and by phenotypic stasis despite substantial levels of underlying molecular change. The phylogeny suggests that species diversification sometimes occurs by niche shifts, and sometimes occurs without a change in niche. Moreover, diversification in the Hyalella ecomorphs has involved the repeated evolution of similar phenotypic forms that exist in similar ecological settings, a hallmark of adaptive evolution. The evolutionary stasis observed in clades separated by substantial genetic divergence, but existing in similar habitats, is also suggestive of stabilizing natural selection acting to constrain phenotypic evolution within narrow bounds. We interpret the observed decoupling of genetic and phenotypic diversification in terms of adaptive radiation on an ecologically constrained adaptive landscape, and suggest that ecological constraints, perhaps acting together with genetic and functional constraints, may explain the parallel evolution and evolutionary stasis inferred by the phylogeny.

First Sequenced Mitochondrial Genome from the Phylum Acanthocephala(Leptorhynchoides thecatus) and Its Phylogenetic Position Within Metazoa

The complete sequence of the mitochondrial genome of Leptorhynchoides thecatus (Acanthocephala) was determined, and a phylogenetic analysis was carried out to determine its placement within Metazoa. The genome is circular, 13,888 bp, and contains at least 36 of the 37 genes typically found in animal mitochondrial genomes. The genes for the large and small ribosomal RNA subunits are shorter than those of most metazoans, and the structures of most of the tRNA genes are atypical. There are two significant noncoding regions (377 and 294 bp), which are the best candidates for a control region; however, these regions do not appear similar to any of the control regions of other animals studied to date. The amino acid and nucleotide sequences of the protein coding genes of L. thecatus and 25 other metazoan taxa were used in both maximum likelihood and maximum parsimony phylogenetic analyses. Results indicate that among taxa with available mitochondrial genome sequences, Platyhelminthes is the closest relative to L. thecatus, which together are the sister taxon of Nematoda; however, long branches and/or base composition bias could be responsible for this result. The monophyly of Ecdysozoa, molting organisms, was not supported by any of the analyses. This study represents the first mitochondrial genome of an acanthocephalan to be sequenced and will allow further studies of systematics, population genetics, and genome evolution.

Phylogenetic Relationships in the North American Cyprinid Genus Cyprinella (Actinopterygii: Cyprinidae) Based on Sequences of the Mitochondrial ND2 and ND4L Genes

Abstract Shiners of the cyprinid genus Cyprinella are abundant and broadly distributed in eastern and central North America. Thirty species are currently placed in the genus: these include six species restricted to Mexico and three barbeled forms formerly placed in different cyprinid genera (primarily Hybopsis). We conducted a molecular phylogenetic analysis of all species of Cyprinella found in the United States, using complete nucleotide sequences of the mitochondrial, protein-coding genes ND2 and ND4L. Maximum-parsimony analysis recovered a single most-parsimonious tree for Cyprinella. Among historically recognized, nonbarbeled Cyprinella, the mitochondrial (mt) DNA tree indicated that basal lineages in Cyprinella are comprised largely of species with linear breeding tubercles and that are endemic to Atlantic and/or Gulf slope drainages, whereas derived lineages are comprised of species broadly distributed in the Mississippi basin and the American Southwest. The Alabama Shiner, C. callistia, was basal in the mtDNA tree, although a monophyletic Cyprinella that included C. callistia was not supported in more than 50% of bootstrap replicates. There was strong bootstrap support (89%) for a clade that included all species of nonbarbeled Cyprinella (except C. callistia) and two barbeled species, C. labrosa and C. zanema. The third barbeled species, C. monacha, fell outside of Cyprinella sister to a species of Hybopsis. Within Cyprinella were a series of well-supported species groups, although in some cases bootstrap support for relationships among groups was below 50%. A derived clade consisting of C. spiloptera, C. whipplei, C. venusta, and the southwestern C. lutrensis group was strongly supported. The species C. lutrensis and C. lepida were not monophyletic, suggesting further study and revision within this group are warranted. In general, the most-parsimonious mtDNA tree was similar in terms of relationships among species to those proposed more than 40 years ago by R. H. Gibbs.

Mitochondrial-Nuclear Interactions: Compensatory Evolution or Variable Functional Constraint among Vertebrate Oxidative Phosphorylation Genes?

Oxidative phosphorylation (OXPHOS), the major energy-producing pathway in aerobic organisms, includes protein subunits encoded by both mitochondrial (mt) and nuclear (nu) genomes. How these independent genomes have coevolved is a long-standing question in evolutionary biology. Although mt genes evolve faster than most nu genes, maintenance of OXPHOS structural stability and functional efficiency may involve correlated evolution of mt and nu OXPHOS genes. The nu OXPHOS genes might be predicted to exhibit accelerated evolutionary rates to accommodate the elevated substitution rates of mt OXPHOS subunits with which they interact. Evolutionary rates of nu OXPHOS genes should, therefore, be higher than that of nu genes that are not involved in OXPHOS (nu non-OXPHOS). We tested the compensatory evolution hypothesis by comparing the evolutionary rates (synonymous substitution rate dS and nonsynonymous substitution rate dN) among 13 mt OXPHOS genes, 60 nu OXPHOS genes, and 77 nu non-OXPHOS genes in vertebrates (7 fish and 40 mammal species). The results from a combined analysis of all OXPHOS subunits fit the predictions of the hypothesis. However, results from two OXPHOS complexes did not fit this pattern when analyzed separately. We found that the dN of nu OXPHOS genes for “core” subunits (those involved in the major catalytic activity) was lower than that of “noncore” subunits, whereas there was no significant difference in dN between genes for nu non-OXPHOS and core subunits. This latter finding suggests that compensatory changes play a minor role in the evolution of OXPHOS genes and that the observed accelerated nu substitution rates are due largely to reduced functional constraint on noncore subunits.

SIGNIFICANT ROLE FOR HISTORICAL EFFECTS IN THE EVOLUTION OF REPRODUCTIVE ISOLATION: EVIDENCE FROM PATTERNS OF INTROGRESSION BETWEEN THE CYPRINID FISHES, LUXILUS CORNUTUS AND LUXILUS CHRYSOCEPHALUS

Samples of Luxilus cornutus, Luxilus chrysocephalus, and their hybrids were collected along hypothesized routes of dispersal from Pleistocene refugia to examine the significance of geographic variation in patterns of introgression between these species. Patterns of allozyme and mitochondrial DNA (mtDNA) variation were generally consistent with those from previous studies. Tests of Hardy‐Weinberg equilibrium revealed significant deficiencies of heterozygotes in all samples, indicating some form of reproductive isolation. Mitochondrial DNAs of each species were not equally represented in F1 hybrids; however, this bias was eliminated when the two largest samples were excluded from the analysis. Backcross hybrids exhibited biased mtDNA introgression, as samples from Lake Erie (eastern) and Lake Michigan (western) drainages showed significant excesses of mtDNAs from L. chrysocephalus and L. cornutus, respectively, relative to frequencies of diagnostic allozyme markers. The extent and direction of allozyme and mtDNA introgression was quantified by calculating isolation index values from morphologically “pure” individuals of each species from each locality. Analysis of variance of these measures identified limited introgression of allozyme variants with no geographic pattern, but significant differences in direction of mtDNA introgression between drainages (i.e., postglacial dispersal route). Association between patterns of mtDNA introgression and dispersal route across the latitudinal width of the contact zone is best explained by genetic divergence during past isolation of ancestral populations from these drainages. These results identify a significant role for historical effects in the evolution of reproductive isolation and the process of speciation.

Genetic consequences of population reduction and geographic isolation in the critically endangered frog, Rana sevosa.

Abstract Anthropogenic habitat fragmentation and reduction are major causes of population declines and extinction. As these processes intensify, our ability to rescue imperiled taxa is critically dependent on an understanding of historical, demographic, and genetic parameters of diminishing populations. We assessed the effects of recent geographic isolation and population reduction on genetic variability for endangered Dusky Gopher Frogs, Rana sevosa. Only two populations of R. sevosa exist, each is geographically isolated and restricted to a single breeding pond, and one of them may have gone locally extinct. Therefore, we studied the largest and perhaps only population of R. sevosa. The only option for comparison to non-isolated populations was of its ecologically similar sister species (other Gopher Frogs, R. capito) and of the sister species to R. sevosa and R. capito (Crawfish Frogs, R. areolata). Variation in seven microsatellite DNA loci was assessed for each population to determine the effects of isolation and population bottleneck on R. sevosa. In comparison to the average non-isolated population, R. sevosa had significantly lower genetic variation and a strong signature of population bottleneck. In fact, R. sevosa had HO that was 72%, HE that was 81%, and A that was 61% of the average non-isolated population. Results indicated a severe, negative genetic consequence of recent population reduction and geographic isolation via lack of gene flow, enhanced effects of drift, and inbreeding. Extensive demographic data have been collected for R. sevosa beginning when the species was rediscovered in 1987 and continuing through our study. These previously collected demographic data aid in interpretation of our genetic data and discussion of implications for conservation and management.