Homology-Aware Phylogenomics at Gigabase Scales.

Obstacles to inferring species trees from whole genome data sets range from algorithmic and data management challenges to the wholesale discordance in evolutionary history found in different parts of a genome. Recent work that builds trees directly from genomes by parsing them into sets of small $k$-mer strings holds promise to streamline and simplify these efforts, but existing approaches do not account well for gene tree discordance. We describe a "seed and extend" protocol that finds nearly exact matching sets of orthologous $k$-mers and extends them to construct data sets that can properly account for genomic heterogeneity. Exploiting an efficient suffix array data structure, sets of whole genomes can be parsed and converted into phylogenetic data matrices rapidly, with contiguous blocks of $k$-mers from the same chromosome, gene, or scaffold concatenated as needed. Phylogenetic trees constructed from highly curated rice genome data and a diverse set of six other eukaryotic whole genome, transcriptome, and organellar genome data sets recovered trees nearly identical to published phylogenomic analyses, in a small fraction of the time, and requiring many fewer parameter choices. Our method's ability to retain local homology information was demonstrated by using it to characterize gene tree discordance across the rice genome, and by its robustness to the high rate of interchromosomal gene transfer found in several rice species.

A biomechanical model of agonist-initiated contraction in the asthmatic airway.

This paper presents a modelling framework in which the local stress environment of airway smooth muscle (ASM) cells may be predicted and cellular responses to local stress may be investigated. We consider an elastic axisymmetric model of a layer of connective tissue and circumferential ASM fibres embedded in parenchymal tissue and model the active contractile force generated by ASM via a stress acting along the fibres. A constitutive law is proposed that accounts for active and passive material properties as well as the proportion of muscle to connective tissue. The model predicts significantly different contractile responses depending on the proportion of muscle to connective tissue in the remodelled airway. We find that radial and hoop-stress distributions in remodelled muscle layers are highly heterogenous with distinct regions of compression and tension. Such patterns of stress are likely to have important implications, from a mechano-transduction perspective, on contractility, short-term cytoskeletal adaptation and long-term airway remodelling in asthma.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

A method for determining the dependence of calcium oscillations on inositol trisphosphate oscillations.

In some cell types, oscillations in the concentration of free intracellular calcium ([Ca2+]) are accompanied by oscillations in the concentration of inositol 1,4,5-trisphosphate ([IP3]). However, in most cell types it is still an open question as to whether oscillations in [IP3] are necessary for Ca2+ oscillations in vivo, or whether they merely follow passively. Using a wide range of models, we show that the response to an artificially applied pulse of IP3 can be used to distinguish between these two cases. Hence, we show that muscarinic receptor-mediated, long-period Ca2+ oscillations in pancreatic acinar cells depend on [IP3] oscillations, whereas short-period Ca2+ oscillations in airway smooth muscle do not.

A simple and practical method to acquire geometrically correct images with resonant scanning-based line scanning in a custom-built video-rate laser scanning microscope.

Most currently available confocal or two-photon laser scanning microscopes (LSMs) allow acquisition rates of the order of 1-5 images s(-1), which is too slow to fully resolve dynamic changes in intracellular messenger concentration in living cells or tissues. Several technologies exist to obtain faster imaging rates, either in the video-rate range (30 images s(-1)) or beyond, but the most versatile technology available today is based on resonant scanners for horizontal line scanning. These scanning devices have several advantages over designs based on acousto-optical deflectors or Nipkow discs, but a drawback is that the scanning pattern is not a linear but rather a sinusoidal function of time. This puts additional constraints on the hardware necessary to read-in the image data flow, one of which is the generation of a pixel clock that varies in frequency with the position of the pixel on the scanned line. We describe a practical solution to obtain a variable pixel clock add-on that is easy to build and is easy to integrate into a custom-built LSM based on resonant scanning technology. In addition, we discuss some important hardware and software design aspects that simplify the construction of a resonant scanning-based LSM for high-speed, high-resolution imaging. Finally, we demonstrate that the microscope can be used to resolve calcium puffs triggered by photolytically increasing the intracellular concentration of inositol trisphosphate.

Isolation and characterization of the laure olfactory behavioral mutant in the zebrafish, Danio rerio.

To initiate a genetic analysis of olfactory development and function in the zebrafish, Danio rerio, we developed a behavioral genetic screen for mutations affecting the olfactory sensory system. First, we characterized olfactory responses of wild-type zebrafish to various odors. We found that 3-day-old juvenile zebrafish reacted to the amino acid L-cysteine with an aversive behavioral response. We isolated one mutant, laure (lre), which showed no aversive behavioral response to L-cysteine at 3 days of development, and carried out a preliminary characterization of this mutant's defects. We found that lre mutant fish were also defective in their response to L-serine and L-alanine, but not to taurocholic acid, as young adults. In addition, lre mutant fish had significantly fewer primary olfactory sensory neurons than normal, and the axons of these neurons did not form the characteristic axon termination pattern in the developing olfactory bulb. Nevertheless, the olfactory epithelium of lre mutant fish showed normal or near normal electrophysiological responses to several odorants. Our data suggest that the behavioral defects observed in the lre mutant result from the disruption of the developing olfactory sensory neurons and their axonal connections within the olfactory bulb. The isolation of the lre mutant shows that our behavior-based screen represents a viable approach for carrying out a genetic dissection of olfactory behaviors in this vertebrate model system.

Evidence of a novel transduction pathway mediating detection of polyamines by the zebrafish olfactory system.

To better understand the full extent of the odorant detection capabilities of fish, we investigated the olfactory sensitivity of zebrafish to a monoamine and several polyamines using electrophysiological and activity-dependent labeling techniques. Electro-olfactogram (EOG) recording methods established the relative stimulatory effectiveness of these odorants as: spermine >> spermidine approximately agmatine > glutamine > putrescine >or= cadaverine >or= histamine > artificial freshwater. The detection threshold for the potent polyamines was approximately 1 micromol l(-1). Cross-adaptation experiments suggested that multiple receptors are involved in polyamine detection. Three observations indicated that polyamine signaling may involve a transduction cascade distinct from those used by either amino acids or bile salts. Like bile salts and the adenylate cyclase activator forskolin, but unlike amino acid odorants, polyamines failed to stimulate activity-dependent labeling of olfactory sensory neurons with the cation channel permeant probe agmatine, suggesting a signaling pathway different from that used by amino acid stimuli. Also supporting distinct amino acid and polyamine signaling pathways is the finding that altering phospholipase C activity with the inhibitor U-73122 significantly reduced amino acid-evoked responses, but had little effect on polyamine- (or bile salt-) evoked responses. Altering cyclic nucleotide-mediated signaling by adenylate cyclase activation with forskolin, which significantly reduced responses to bile salts, failed to attenuate polyamine responses, suggesting that polyamines and bile salts do not share a common transduction cascade. Collectively, these findings suggest that polyamines are a new class of olfactory stimuli transduced by a receptor-mediated, second messenger signaling pathway that is distinct from those used by amino acids or bile salts.

Absolute diversification rates in angiosperm clades.

The extraordinary contemporary species richness and ecological predominance of flowering plants (angiosperms) are even more remarkable when considering the relatively recent onset of their evolutionary diversification. We examine the evolutionary diversification of angiosperms and the observed differential distribution of species in angiosperm clades by estimating the rate of diversification for angiosperms as a whole and for a large set of angiosperm clades. We also identify angiosperm clades with a standing diversity that is either much higher or lower than expected, given the estimated background diversification rate. Recognition of angiosperm clades, the phylogenetic relationships among them, and their taxonomic composition are based on an empirical compilation of primary phylogenetic studies. By making an integrative and critical use of the paleobotanical record, we obtain reasonably secure approximations for the age of a large set of angiosperm clades. Diversification was modeled as a stochastic, time-homogeneous birth-and-death process that depends on the diversification rate (r) and the relative extinction rate (epsilon). A statistical analysis of the birth and death process was then used to obtain 95% confidence intervals for the expected number of species through time in a clade that diversifies at a rate equal to that of angiosperms as a whole. Confidence intervals were obtained for stem group and for crown group ages in the absence of extinction (e = 0.0) and under a high relative extinction rate (epsilon = 0.9). The standing diversity of angiosperm clades was then compared to expected species diversity according to the background rate of diversification, and, depending on their placement with respect to the calculated confidence intervals, exceedingly species-rich or exceedingly species-poor clades were identified. The rate of diversification for angiosperms as a whole ranges from 0.077 (epsilon = 0.9) to 0.089 (epsilon = 0.0) net speciation events per million years. Ten clades fall above the confidence intervals of expected species diversity, and 13 clades were found to be unexpectedly species poor. The phylogenetic distribution of clades with an exceedingly high number of species suggests that traits that confer high rates of diversification evolved independently in different instances and do not characterize the angiosperms as a whole.

Scaling of accuracy in extremely large phylogenetic trees.

The accuracy of phylogenetic inference was examined in simulated data sets up to nearly 10,000 taxa, the size of the largest set of homologous genes in existing molecular sequence databases. Even with a simple search algorithm (maximum parsimony without branch swapping), the number of characters needed to estimate 80% of a tree correctly can scale remarkably well at optimal substitution rates (on the order of log N, where N is the number of taxa). In other words, the number of taxa in an analysis can be doubled and only an arithmetic increase in the number of characters is required to maintain the same level of accuracy. Even substitution rates that are much higher than normally used in phylogenetic studies did not affect the scaling too adversely. However, scaling is usually worse than log N for more stringent levels of accuracy. Moreover, errors are not distributed randomly throughout the tree. Shallow nodes are remarkably easy to reconstruct and display favourable log-linear scaling. The deepest nodes are extremely difficult to reconstruct accurately, even with branch swapping, and the scaling is poor. Therefore, the strategy of sequencing large numbers of homologous genes may not always provide global solutions to extreme phylogenetic problems and alternative strategies may be required.

Propagation of intercellular calcium waves in C6 glioma cells transfected with connexins 43 or 32.

In this study, gap junction-deficient C6 glioma cells, transfected with either connexin 43 (Cx43) or 32 (Cx32), have been used to evaluate the ability of these connexins to pass intercellular Ca2+ waves. Ca2+ waves, observed with fluorescence imaging using fura-2 or fluo-3, were initiated by mechanical stimulation in the presence of a supra-perfusion of the extracellular fluid or by the non-contact technique of flash photolysis of intracellular caged-IP3. Following manual mechanical stimulation, the parental C6 glioma cells and cells expressing Cx43 and Cx32 gap junctions all propagated intercellular Ca2+ waves. Ca2+ waves in cells expressing Cx43 traveled approximately twice the distance as compared to waves in cells expressing Cx32 or parental cells. The cells expressing Cx43 were also about twice as sensitive to ATP as cells expressing Cx32. In the presence of a supra-perfusion of extracellular fluid, the Ca2+ waves in parental cells were almost abolished while the mechanically induced Ca2+ waves in the cells expressing Cx43 and Cx32 propagate similar but limited distances of several cells in a direction opposite to the fluid flow. The photolytic release of IP3, but not Ca2+, in cells expressing Cx43 or Cx32 resulted in the propagation of Ca2+ waves that traveled distances similar to those observed in the presence of supra-perfusion. Parental C6 glioma cells did not initiate intercellular Ca2+ waves when stimulated by photolysis. From these studies we conclude that (1) both Cx43 and Cx32 based gap junctions are permeable to IP3 and can serve to communicate Ca2+ waves, (2) that Ca2+ wave propagation via gap junctions was dependent on the diffusion of IP3 but not Ca2+, (3) that an extracellular messenger capable of communicating waves is released from only the stimulated cell, and (4) that simultaneous intracellular and extracellular signaling can occur to enhance the propagation of intercellular Ca2+ waves.

Astrocyte-endothelial cell calcium signals conveyed by two signalling pathways.

Astrocytes and endothelial cells are in close contact with each other at the blood-brain barrier, where important molecular transports take place. Despite these key morphological and functional properties, little is known regarding the dynamic signalling processes that occur between these two cell types. We investigated astrocyte-endothelial cell calcium signalling mechanisms in a coculture model prepared from primary rat cortical astrocytes and ECV304 cells. We used flash photolysis of caged inositol-trisphosphate (IP3) and gentle mechanical stimulation to trigger astrocyte-endothelial cell calcium signals and to investigate the underlying propagation mechanisms. Photolytically releasing IP3 in a single cell triggered increases in cytoplasmic calcium concentration that propagated between astrocytes and endothelial cells in either direction. These propagating calcium signals did not cross cell-free zones and were not affected by fast superfusion or by the purinergic inhibitors apyrase and suramin, indicating that they are communicated through an intracellular pathway in conjunction with gap junctions. Electrophysiological experiments confirmed a low degree of astrocyte-endothelial cell electrical cell-to-cell coupling. Mechanical stimulation of a single cell also triggered astrocyte-endothelial cell calcium signals but, in contrast to the former triggering mode, these signals crossed cell-free zones and were significantly inhibited by apyrase, thus indicating the involvement of an extracellular and purinergic messenger. Astrocyte-endothelial cell calcium signalling also occurred in cocultures prepared with astrocytes and primary rat brain capillary endothelial cells. We conclude that astrocytes and endothelial cells can exchange fast-acting calcium signals (time scale of seconds) that can be communicated through an intracellular/gap junctional pathway and an extracellular purinergic pathway.

Sources of error and confidence intervals in estimating the age of angiosperms from rbcL and 18S rDNA data.

Molecular estimates of the age of angiosperms have varied widely, and many greatly predate the Early Cretaceous appearance of angiosperms in the fossil record, but there have been few attempts to assess confidence limits on ages. Experiments with rbcL and 18S data using maximum likelihood suggest that previous angiosperm age estimates were too old because they assumed equal rates across sites-use of a gamma distribution of rates to correct for site-to-site variation gives 10-30 my (million years) younger ages-and relied on herbaceous angiosperm taxa with high rates of molecular evolution. Ages based on first and second codon positions of rbcL are markedly older than those based on third positions, which conflict with the fossil record in being too young, but all examined data partitions of rbcL and 18S depart substantially from a molecular clock. Age estimates are surprisingly insensitive to different views on seed-plant relationships. Randomization schemes were used to quantify confidence intervals due to phylogenetic uncertainty, substitutional noise, and lineage effects (deviations from a molecular clock). Estimates of the age of crown-group angiosperms range from 68 to 281 mya (million years ago), depending on data, tree, and assumptions, with most ∼140-190 mya (Early Jurassic-earliest Cretaceous). Approximate 95% confidence intervals on ages are wider for rbcL than 18S, ranging up to 160 my for phylogenetic uncertainty, 90 my for substitutional noise, and 70 my for lineage effects. These intervals overlap the oldest occurrences of angiosperms in the fossil record, as well as some estimates from previous molecular studies.

Assessment of the accuracy of matrix representation with parsimony analysis supertree construction.

Despite the growing popularity of supertree construction for combining phylogenetic information to produce more inclusive phylogenies, large-scale performance testing of this method has not been done. Through simulation, we tested the accuracy of the most widely used supertree method, matrix representation with parsimony analysis (MRP), with respect to a (maximum parsimony) total evidence solution and a known model tree. When source trees overlap completely, MRP provided a reasonable approximation of the total evidence tree; agreement was usually > 85%. Performance improved slightly when using smaller, more numerous, or more congruent source trees, and especially when elements were weighted in proportion to the bootstrap frequencies of the nodes they represented on each source tree ("weighted MRP"). Although total evidence always estimated the model tree slightly better than nonweighted MRP methods, weighted MRP in turn usually out-performed total evidence slightly. When source studies were even moderately nonoverlapping (i.e., sharing only three-quarters of the taxa), the high proportion of missing data caused a loss in resolution that severely degraded the performance for all methods, including total evidence. In such cases, even combining more trees, which had positive effects elsewhere, did not improve accuracy. Instead, "seeding" the supertree or total evidence analyses with a single largely complete study improved performance substantially. This finding could be an important strategy for any studies that seek to combine phylogenetic information. Overall, our results suggest that MRP supertree construction provides a reasonable approximation of a total evidence solution and that weighted MRP should be used whenever possible.

High-speed digital microscopy.

High-speed imaging is an ideal technique to accurately resolve the temporal and spatial characteristics of rapid events at either the molecular or cellular level. In this article, the digital imaging techniques used to simultaneously acquire transillumination phase-contrast images, at 240 images s(-1) (high-speed), to characterize ciliary beat frequency, and fluorescence images, at 30 images s(-1) (fast), to measure intracellular calcium concentration ([Ca2+]i), are described. With this technique, a precise correlation between the changes in ciliary beat frequency with changes in [Ca2+]i can be made. Simultaneous imaging is achieved by using different wavelengths of light to form the phase-contrast and fluorescent images and selectively directing these light wavelengths to different cameras with dichroic mirrors and bandpass filters. High-speed images compatible with standard video recording equipment are obtained by prematurely resetting the raster scan of a CCD camera with additional vertical synchronization pulses. The fast [Ca2+]i images are determined using the ratiometric dye fura-2 and a recording technique that monitors rapid changes in fluorescence at a single wavelength and uses intermittent reference images for calibration.

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26.

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

Error, bias, and long-branch attraction in data for two chloroplast photosystem genes in seed plants.

Sequences of two chloroplast photosystem genes, psaA and psbB, together comprising about 3,500 bp, were obtained for all five major groups of extant seed plants and several outgroups among other vascular plants. Strongly supported, but significantly conflicting, phylogenetic signals were obtained in parsimony analyses from partitions of the data into first and second codon positions versus third positions. In the former, both genes agreed on a monophyletic gymnosperms, with Gnetales closely related to certain conifers. In the latter, Gnetales are inferred to be the sister group of all other seed plants, with gymnosperms paraphyletic. None of the data supported the modern "anthophyte hypothesis," which places Gnetales as the sister group of flowering plants. A series of simulation studies were undertaken to examine the error rate for parsimony inference. Three kinds of errors were examined: random error, systematic bias (both properties of finite data sets), and statistical inconsistency owing to long-branch attraction (an asymptotic property). Parsimony reconstructions were extremely biased for third-position data for psbB. Regardless of the true underlying tree, a tree in which Gnetales are sister to all other seed plants was likely to be reconstructed for these data. None of the combinations of genes or partitions permits the anthophyte tree to be reconstructed with high probability. Simulations of progressively larger data sets indicate the existence of long-branch attraction (statistical inconsistency) for third-position psbB data if either the anthophyte tree or the gymnosperm tree is correct. This is also true for the anthophyte tree using either psaA third positions or psbB first and second positions. A factor contributing to bias and inconsistency is extremely short branches at the base of the seed plant radiation, coupled with extremely high rates in Gnetales and nonseed plant outgroups.

Phylogenetic systematics of the tribe Millettieae (Leguminosae) based on chloroplast trnK/matK sequences and its implications for evolutionary patterns in Papilionoideae.

Phylogenetic relationships in the tribe Millettieae and allies in the subfamily Papilionoideae (Leguminosae) were reconstructed from chloroplast trnK/matK sequences. Sixty-two accessions representing 57 traditionally recognized genera of Papilionoideae were sampled, including 27 samples from Millettieae. Phylogenies were constructed using maximum parsimony and are well resolved and supported by high bootstrap values. A well-supported "core Millettieae" clade is recognized, comprising the four large genera Millettia, Lonchocarpus, Derris, and Tephrosia. Several other small genera of Millettieae are not in the core Millettieae clade. Platycyamus is grouped with Phaseoleae (in part). Ostryocarpus, Austrosteenisia, and Dalbergiella are neither in the core Millettieae or Phaseoleae clade. These taxa, along with core Millettieae and Phaseoleae, form a monophyletic sister group to Indigofereae. Cyclolobium and Poecilanthe are close to Brongniartieae. Callerya and Wisteria belong to a large clade that includes all the legumes that lack the inverted repeat in their chloroplast genome, which confirms previous rbcL and phytochrome gene family phylogenies. The evolutionary history of four characters was examined in Millettieae and allies: the presence of canavanine, inflorescence types, the dehiscence of pods, and the presence of winged pods. trnK/matK sequence analysis suggests that the presence of a pseudoraceme or pseudopanicle and the accumulation of nonprotein amino acids are phylogenetically informative for Millettieae and allies with only a few exceptions.

Improved bootstrap confidence limits in large-scale phylogenies, with an example from Neo-Astragalus (Leguminosae).

Phylogenetic analyses of large data sets pose special challenges, including the apparent tendency for the bootstrap support for a clade to decline with increased taxon sampling of that clade. We document this decline in data sets with increasing numbers of taxa in Astragalus, the most species-rich angiosperm genus. Support for one subclade, Neo-Astragalus, declined monotonically with increased sampling of taxa inside Neo-Astragalus, irrespective of whether parsimony or neighbor-joining methods were used or of which particular heuristic search algorithm was used (although more stringent algorithms tended to yield higher support). Three possible explanations for this decline were examined, including (1) mistaken assignment of the most recent common ancestor of the taxon sample (and its bootstrap support) with the most recent common ancestor of the clade from which it was sampled; (2) computational limitations of heuristic search strategies; and (3) statistical bias in bootstrap proportions, especially that from random homoplasy distributed among taxa. The best explanation appears to be (3), although computational shortcomings (2) may explain some of the problem. The bootstrap proportion, as currently used in phylogenetic analysis, does not accurately capture the classical notion of confidence assessments on the null hypothesis of nonmonophyly, especially in large data sets. More accurate assessments of confidence as type I error levels (relying on iterated bootstrap methods) remove most of the monotonic decline in confidence with increasing numbers of taxa.