Rachel Lockridge Mueller

A new time-scale for ray-finned fish evolution

The Actinopterygii (ray-finned fishes) is the largest and most diverse vertebrate group, but little is agreed about the timing of its early evolution. Estimates using mitochondrial genomic data suggest that the major actinopterygian clades are much older than divergence dates implied by fossils. Here, the timing of the evolutionary origins of these clades is reinvestigated using morphological, and nuclear and mitochondrial genetic data. Results indicate that existing fossil-based estimates of the age of the crown-group Neopterygii, including the teleosts, Lepisosteus (gar) and Amia (bowfin), are at least 40 Myr too young. We present new palaeontological evidence that the neopterygian crown radiation is a Palaeozoic event, and demonstrate that conflicts between molecular and morphological data for the age of the Neopterygii result, in part, from missing fossil data. Although our molecular data also provide an older age estimate for the teleost crown, this range extension remains unsupported by the fossil evidence. Nuclear data from all relevant clades are used to demonstrate that the actinopterygian whole-genome duplication event is teleost-specific. While the date estimate of this event overlaps the probable range of the teleost stem group, a correlation between the genome duplication and the large-scale pattern of actinopterygian phylogeny remains elusive.

Evolutionary Rates, Divergence Dates, and the Performance of Mitochondrial Genes in Bayesian Phylogenetic Analysis

The mitochondrial genome is one of the most frequently used loci in phylogenetic and phylogeographic analyses, and it is becoming increasingly possible to sequence and analyze this genome in its entirety from diverse taxa. However, sequencing the entire genome is not always desirable or feasible. Which genes should be selected to best infer the evolutionary history of the mitochondria within a group of organisms, and what properties of a gene determine its phylogenetic performance? The current study addresses these questions in a Bayesian phylogenetic framework with reference to a phylogeny of plethodontid and related salamanders derived from 27 complete mitochondrial genomes; this topology is corroborated by nuclear DNA and morphological data. Evolutionary rates for each mitochondrial gene and divergence dates for all nodes in the plethodontid mitochondrial genome phylogeny were estimated in both Bayesian and maximum likelihood frameworks using multiple fossil calibrations, multiple data partitions, and a clock-independent approach. Bayesian analyses of individual genes were performed, and the resulting trees compared against the reference topology. Ordinal logistic regression analysis of molecular evolution rate, gene length, and the G-shape parameter a demonstrated that slower rate of evolution and longer gene length both increased the probability that a gene would perform well phylogenetically. Estimated rates of molecular evolution vary 84-fold among different mitochondrial genes and different salamander lineages, and mean rates among genes vary 15-fold. Despite having conserved amino acid sequences, cox1, cox2, cox3, and cob have the fastest mean rates of nucleotide substitution, and the greatest variation in rates, whereas rrnS and rrnL have the slowest rates. Reasons underlying this rate variation are discussed, as is the extensive rate variation in cox1 in light of its proposed role in DNA barcoding.

LTR Retrotransposons Contribute to Genomic Gigantism in Plethodontid Salamanders

Among vertebrates, most of the largest genomes are found within the salamanders, a clade of amphibians that includes 613 species. Salamander genome sizes range from ∼14 to ∼120 Gb. Because genome size is correlated with nucleus and cell sizes, as well as other traits, morphological evolution in salamanders has been profoundly affected by genomic gigantism. However, the molecular mechanisms driving genomic expansion in this clade remain largely unknown. Here, we present the first comparative analysis of transposable element (TE) content in salamanders. Using high-throughput sequencing, we generated genomic shotgun data for six species from the Plethodontidae, the largest family of salamanders. We then developed a pipeline to mine TE sequences from shotgun data in taxa with limited genomic resources, such as salamanders. Our summaries of overall TE abundance and diversity for each species demonstrate that TEs make up a substantial portion of salamander genomes, and that all of the major known types of TEs are represented in salamanders. The most abundant TE superfamilies found in the genomes of our six focal species are similar, despite substantial variation in genome size. However, our results demonstrate a major difference between salamanders and other vertebrates: salamander genomes contain much larger amounts of long terminal repeat (LTR) retrotransposons, primarily Ty3/gypsy elements. Thus, the extreme increase in genome size that occurred in salamanders was likely accompanied by a shift in TE landscape. These results suggest that increased proliferation of LTR retrotransposons was a major molecular mechanism contributing to genomic expansion in salamanders.

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution

Significance Aedes albopictus is a highly adaptive species that thrives worldwide in tropical and temperate zones. From its origin in Asia, it has established itself on every continent except Antarctica. This expansion, coupled with its ability to vector the epidemic human diseases dengue and Chikungunya fevers, make it a significant global public health threat. A complete genome sequence and transcriptome data were obtained for the Ae. albopictus Foshan strain, a colony derived from mosquitoes from its historical origin. The large genome (1,967 Mb) comprises an abundance of repetitive DNA classes and expansions of the numbers of gene family members involved in insecticide resistance, diapause, sex determination, immunity, and olfaction. This large genome repertory and plasticity may contribute to its success as an invasive species. The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

Locomotor performance at low temperature and the evolution of nocturnality in geckos

Nocturnal geckos are active at body temperatures 10–35°C below the thermal optima for maximum rate of aerobic metabolism (V.O2max) of diurnal lizards. Therefore, given ancestral (diurnal) lizard physiology, nocturnality causes a substantial thermal handicap in locomotor performance. In prior studies, we hypothesized that a low minimum cost of locomotion (Cmin) in geckos was an adaptation that increased locomotor endurance capacity at low, nocturnal temperatures. However, Cmin is only part of an integrated system that, in conjunction with the maximum rate of oxygen consumption, sets the maximum speed that can be sustained aerobically (termed the maximum aerobic speed or MAS). We conducted the first phylogenetic analysis of MAS and V.O2max lizards and found that the greatest changes in MAS, Cmin and V.O2max (at activity temperatures) in the evolutionary history of lizards all coincided with the evolution of nocturnality in geckos. Geckos active at 15–25°C did not become optimized for nocturnal temperatures, or fully offset the thermal effects of nocturnality by evolving maximal rates of oxygen consumption comparable to diurnal lizards active at 35°C. Geckos did evolve MAS twice that of diurnal lizards running at low temperatures by evolving a remarkably low Cmin. Allometric analysis and phylogenetically independent contrasts of V.O2max , Cmin, and MAS indicate a 72% evolutionary decrease in V.O2max , (at activity temperatures) and a 50% evolutionary decrease in Cmin concordant with the evolution of nocturnality in geckos. Experimental measurements show that decreased Cmin in six species of gecko increased MAS by 50–120% compared to diurnal lizards at low temperatures. Thus, geckos sufficiently overcame the near paralyzing effects of nocturnal temperatures, but only offset about 50% of the decrease in MAS resulting from the low maximum rate of oxygen consumption. Although the nocturnal environment remains severely suboptimal, the evolution of a low cost of locomotion in the ancestor of geckos was highly adaptive for nocturnality. We also present a generalized approach to ecophysiological evolution that integrates phylogeny with the causal relationships among environment, physiology, and performance capacity. With respect to a clade, two hypotheses are central to our integrative approach: (1) a change of an environmental variable (e.g., temperature) causes a performance handicap; and (2) evolution of a physiological variable (e.g., minimum cost of locomotion [Cmin]) increases performance in the derived environment. To test the hypothesis that evolution of a physiological variable is adaptive in nature, we suggest determining if individuals in nature perform at levels exceeding the performance capacity of their hypothetical ancestors and if this additional performance capacity is due to the evolution of the physiological variable in question.

Branch length estimation and divergence dating: estimates of error in Bayesian and maximum likelihood frameworks

BackgroundEstimates of divergence dates between species improve our understanding of processes ranging from nucleotide substitution to speciation. Such estimates are frequently based on molecular genetic differences between species; therefore, they rely on accurate estimates of the number of such differences (i.e. substitutions per site, measured as branch length on phylogenies). We used simulations to determine the effects of dataset size, branch length heterogeneity, branch depth, and analytical framework on branch length estimation across a range of branch lengths. We then reanalyzed an empirical dataset for plethodontid salamanders to determine how inaccurate branch length estimation can affect estimates of divergence dates.ResultsThe accuracy of branch length estimation varied with branch length, dataset size (both number of taxa and sites), branch length heterogeneity, branch depth, dataset complexity, and analytical framework. For simple phylogenies analyzed in a Bayesian framework, branches were increasingly underestimated as branch length increased; in a maximum likelihood framework, longer branch lengths were somewhat overestimated. Longer datasets improved estimates in both frameworks; however, when the number of taxa was increased, estimation accuracy for deeper branches was less than for tip branches. Increasing the complexity of the dataset produced more misestimated branches in a Bayesian framework; however, in an ML framework, more branches were estimated more accurately. Using ML branch length estimates to re-estimate plethodontid salamander divergence dates generally resulted in an increase in the estimated age of older nodes and a decrease in the estimated age of younger nodes.ConclusionsBranch lengths are misestimated in both statistical frameworks for simulations of simple datasets. However, for complex datasets, length estimates are quite accurate in ML (even for short datasets), whereas few branches are estimated accurately in a Bayesian framework. Our reanalysis of empirical data demonstrates the magnitude of effects of Bayesian branch length misestimation on divergence date estimates. Because the length of branches for empirical datasets can be estimated most reliably in an ML framework when branches are <1 substitution/site and datasets are ≥1 kb, we suggest that divergence date estimates using datasets, branch lengths, and/or analytical techniques that fall outside of these parameters should be interpreted with caution.

Slow DNA Loss in the Gigantic Genomes of Salamanders

Evolutionary changes in genome size result from the combined effects of mutation, natural selection, and genetic drift. Insertion and deletion mutations (indels) directly impact genome size by adding or removing sequences. Most species lose more DNA through small indels (i.e., ∼1–30 bp) than they gain, which can result in genome reduction over time. Because this rate of DNA loss varies across species, small indel dynamics have been suggested to contribute to genome size evolution. Species with extremely large genomes provide interesting test cases for exploring the link between small indels and genome size; however, most large genomes remain relatively unexplored. Here, we examine rates of DNA loss in the tetrapods with the largest genomes—the salamanders. We used low-coverage genomic shotgun sequence data from four salamander species to examine patterns of insertion, deletion, and substitution in neutrally evolving non-long terminal repeat (LTR) retrotransposon sequences. For comparison, we estimated genome-wide DNA loss rates in non-LTR retrotransposon sequences from five other vertebrate genomes: Anolis carolinensis, Danio rerio, Gallus gallus, Homo sapiens, and Xenopus tropicalis. Our results show that salamanders have significantly lower rates of DNA loss than do other vertebrates. More specifically, salamanders experience lower numbers of deletions relative to insertions, and both deletions and insertions are skewed toward smaller sizes. On the basis of these patterns, we conclude that slow DNA loss contributes to genomic gigantism in salamanders. We also identify candidate molecular mechanisms underlying these differences and suggest that natural variation in indel dynamics provides a unique opportunity to study the basis of genome stability.

Genome size, cell size, and the evolution of enucleated erythrocytes in attenuate salamanders

Within the salamander family Plethodontidae, five different clades have evolved high levels of enucleated red blood cells, which are extremely unusual among non-mammalian vertebrates. In each of these five clades, the salamanders have large genomes and miniaturized or attenuated body forms. Such a correlation suggests that the loss of nuclei in red blood cells may be related, in part, to the interaction between large genome size and small body size, which has been shown to have profound morphological consequences for the nervous and visual systems in plethodontids. Previous work has demonstrated that variation in both the level of enucleated cells and the size of the nuclear genome exists among species of the monophyletic plethodontid genus Batrachoseps. Here, we report extensive intraspecific variation in levels of enucleated red blood cells in 15 species and provide measurements of red blood cell size, nucleus size, and genome size for 13 species of Batrachoseps. We present a new phylogenetic hypothesis for the genus based on 6150bp of mitochondrial DNA sequence data from nine exemplar taxa and use it to examine the relationship between genome size and enucleated red blood cell morphology in a phylogenetic framework. Our analyses demonstrate positive direct correlations between genome size, nucleus size, and both nucleated and enucleated cell sizes within Batrachoseps, although only the relationship between genome size and nucleus size is significant when phylogenetically independent contrasts are used. In light of our results and broader studies of comparative hematology, we propose that high levels of enucleated, variably sized red blood cells in Batrachoseps may have evolved in response to rheological problems associated with the circulation of large red blood cells containing large, bulky nuclei in an attenuate organism.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development

BackgroundChromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution — some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 – 75 Gb, 12–74 Gb of which are lost from pre-somatic cell lineages at germline – soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown.ResultsHere, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome.ConclusionsOur results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

DNA transposons have colonized the genome of the giant virus Pandoravirus salinus

BackgroundTransposable elements are mobile DNA sequences that are widely distributed in prokaryotic and eukaryotic genomes, where they represent a major force in genome evolution. However, transposable elements have rarely been documented in viruses, and their contribution to viral genome evolution remains largely unexplored. Pandoraviruses are recently described DNA viruses with genome sizes that exceed those of some prokaryotes, rivaling parasitic eukaryotes. These large genomes appear to include substantial noncoding intergenic spaces, which provide potential locations for transposable element insertions. However, no mobile genetic elements have yet been reported in pandoravirus genomes.ResultsHere, we report a family of miniature inverted-repeat transposable elements (MITEs) in the Pandoravirus salinus genome, representing the first description of a virus populated with a canonical transposable element family that proliferated by transposition within the viral genome. The MITE family, which we name Submariner, includes 30 copies with all the hallmarks of MITEs: short length, terminal inverted repeats, TA target site duplication, and no coding capacity. Submariner elements show signs of transposition and are undetectable in the genome of Pandoravirus dulcis, the closest known relative Pandoravirus salinus. We identified a DNA transposon related to Submariner in the genome of Acanthamoeba castellanii, a species thought to host pandoraviruses, which contains remnants of coding sequence for a Tc1/mariner transposase. These observations suggest that the Submariner MITEs of P. salinus belong to the widespread Tc1/mariner superfamily and may have been mobilized by an amoebozoan host. Ten of the 30 MITEs in the P. salinus genome are located within coding regions of predicted genes, while others are close to genes, suggesting that these transposons may have contributed to viral genetic novelty.ConclusionsOur discovery highlights the remarkable ability of DNA transposons to colonize and shape genomes from all domains of life, as well as giant viruses. Our findings continue to blur the division between viral and cellular genomes, adhering to the emerging view that the content, dynamics, and evolution of the genomes of giant viruses do not substantially differ from those of cellular organisms.