Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia.

BACKGROUND: Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes. RESULTS: Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30% of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome. CONCLUSIONS: Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20%.

An angiosperm-wide analysis of the gynodioecy-dioecy pathway.

BACKGROUND AND AIMS: About 6 % of an estimated total of 240 000 species of angiosperms are dioecious. The main precursors of this sexual system are thought to be monoecy and gynodioecy. A previous angiosperm-wide study revealed that many dioecious species have evolved through the monoecy pathway; some case studies and a large body of theoretical research also provide evidence in support of the gynodioecy pathway. If plants have evolved through the gynodioecy pathway, gynodioecious and dioecious species should co-occur in the same genera. However, to date, no large-scale analysis has been conducted to determine the prevalence of the gynodioecy pathway in angiosperms. In this study, this gap in knowledge was addressed by performing an angiosperm-wide survey in order to test for co-occurrence as evidence of the gynodioecy pathway. METHODS: Data from different sources were compiled to obtain (to our knowledge) the largest dataset on gynodioecy available, with 275 genera that include at least one gynodioecious species. This dataset was combined with a dioecy dataset from the literature, and a study was made of how often dioecious and gynodioecious species could be found in the same genera using a contingency table framework. KEY RESULTS: It was found that, overall, angiosperm genera with both gynodioecious and dioecious species occur more frequently than expected, in agreement with the gynodioecy pathway. Importantly, this trend holds when studying different classes separately (or sub-classes, orders and families), suggesting that the gynodioecy pathway is not restricted to a few taxa but may instead be widespread in angiosperms. CONCLUSIONS: This work complements that previously carried out on the monoecy pathway and suggests that gynodioecy is also a common pathway in angiosperms. The results also identify angiosperm families where some (or all) dioecious species may have evolved from gynodioecious precursors. These families could be the targets of future small-scale studies on transitions to dioecy taking phylogeny explicitly into account.

Dioecy is associated with higher diversification rates in flowering plants.

In angiosperms, dioecious clades tend to have fewer species than their nondioecious sister clades. This departure from the expected equal species richness in the standard sister clade test has been interpreted as implying that dioecious clades diversify less and has initiated a series of studies suggesting that dioecy might be an 'evolutionary dead end'. However, two of us recently showed that the 'equal species richness' null hypothesis is not valid in the case of derived char acters, such as dioecy, and proposed a new test for sister clade comparisons; preliminary results, using a data set available in the litterature, indicated that dioecious clades migth diversify more than expected. However, it is crucial for this new test to distinguish between ancestral and derived cases of dioecy, a criterion that was not taken into account in the available data set. Here, we present a new data set that was obtained by searching the phylogenetic literature on more than 600 completely dioecious angiosperm genera and identifying 115 sister clade pairs for which dioecy is likely to be derived (including > 50% of the dioecious species). Applying the new sister clade test to this new dataset, we confirm the preliminary result that dioecy is associated with an increased diversification rate, a result that does not support the idea that dioecy is an evolutionary dead end in angiosperms. The traits usually associated with dioecy, that is, an arborescent growth form, abiotic pollination, fleshy fruits or a tropical distribution, do not influence the diversification rate. Rather than a low diversification rate, the observed species richness patterns of dioecious clades seem to be better explained by a low transition rate to dioecy and frequent losses.

Confronting the constraints of morphological taxonomy in the Botryosphaeriales.

Identification of fungi and the International Code of Nomenclature underpinning this process, rests strongly on the characterisation of morphological structures. Yet, the value of these characters to define species in many groups has become questionable or even superfluous. This has emerged as DNA-based techniques have increasingly revealed cryptic species and species complexes. This problem is vividly illustrated in the present study where 105 isolates of the Botryosphaeriales were recovered from both healthy and diseased woody tissues of native Acacia spp. in Namibia and South Africa. Thirteen phylogenetically distinct groups were identified based on Internal Transcribed Spacer (ITS) rDNA PCR-RFLP and translation elongation factor 1-α (TEF1-α) sequence data, two loci that are known to be reliable markers to distinguish species in the Botryosphaeriales. Four of these groups could be linked reliably to sequence data for formerly described species, including Botryosphaeria dothidea, Dothiorella dulcispinae, Lasiodiplodia pseudotheobromae and Spencermartinsia viticola. Nine groups, however, could not be linked to any other species known from culture and for which sequence data are available. These groups are, therefore, described as Aplosporella africana, A. papillata, Botryosphaeria auasmontanum, Dothiorella capri-amissi, Do. oblonga, Lasiodiplodia pyriformis, Spencermartinsia rosulata, Sphaeropsis variabilis and an undescribed Neofusicoccum sp. The species described here could not be reliably compared with the thousands of taxa described in these genera from other hosts and regions, where only morphological data are available. Such comparison would be possible only if all previously described taxa are epitypified, which is not a viable objective for the two families, Botryosphaeriaceae and Aplosporellaceae, in the Botryosphaeriales identified here. The extent of diversity of the Botryosphaeriales revealed in this and other recent studies is expected to reflect that of other undersampled regions and hosts, and illustrates the urgency to find more effective ways to describe species in this, and indeed other, groups of fungi.

Patterns of molecular evolution in dioecious and non-dioecious Silene.

Dioecy (i.e. having separate sexes) is a rather rare breeding system in flowering plants. Such rareness may result from a high probability of extinction in dioecious species because of less efficient dispersal and the costs of sexual selection, which are expected to harm dioecious species' survival on the long term. These handicaps should decrease the effective population size (Ne) of dioecious species, which in turn should reduce the efficacy of selection. Moreover, sexual selection in dioecious species is expected to specifically affect some genes, which will evolve under positive selection. The relative contribution of these effects is currently unknown and we tried to disentangle them by comparing sequence evolution between dioecious and non-dioecious species in the Silene genus (Caryophyllaceae), where dioecy has evolved at least twice. For the dioecious species in the section Melandrium, where dioecy is the oldest, we found a global reduction of purifying selection, while on some, male-biased genes, positive selection was found. For section Otites, where dioecy evolved more recently, we found no significant differences between dioecious and non-dioecious species. Our results are consistent with the view that dioecy is an evolutionary dead end in flowering plants, although other scenarios for explaining reduced Ne cannot be ruled out. Our results also show that contrasting forces act on the genomes of dioecious plants, and suggest that some time is required before the genome of such plants bears the footprints of dioecy.

Salbutamol: beware of the paradox!

A 7-year-old known asthmatic presented with an acute severe asthma attack to the Accident and Emergency department. Following a poor response to salbutamol and ipratropium nebulisers, he was given intravenous salbutamol and aminophylline. Over the course of the following 3 h, there was improvement in his bronchospasm with decreasing oxygen requirement, however, his respiratory rate showed an upward trend. Serial blood gas estimations showed a worsening metabolic acidosis unresponsive to two fluid boluses of 20 ml/kg of normal saline. Lactate levels were subsequently measured and found to be high, accounting for the metabolic acidosis. High lactate levels were attributed to intravenous salbutamol. His blood gases and lactate level returned to normal within 3 h after stopping intravenous salbutamol. He was recommended on salbutamol nebulisers while still continuing on intravenous aminophylline. He continued to improve and was discharged home after 4 days.

Silene as a model system in ecology and evolution.

The genus Silene, studied by Darwin, Mendel and other early scientists, is re-emerging as a system for studying interrelated questions in ecology, evolution and developmental biology. These questions include sex chromosome evolution, epigenetic control of sex expression, genomic conflict and speciation. Its well-studied interactions with the pathogen Microbotryum has made Silene a model for the evolution and dynamics of disease in natural systems, and its interactions with herbivores have increased our understanding of multi-trophic ecological processes and the evolution of invasiveness. Molecular tools are now providing new approaches to many of these classical yet unresolved problems, and new progress is being made through combining phylogenetic, genomic and molecular evolutionary studies with ecological and phenotypic data.

Some fungi, zearalenone and other mycotoxins in chicken rations, stock feedstuffs, lucerne and pasture grasses in the communal farming area of Rhenosterkop in South Africa.

Mycotoxins may be present in feeds without any visible signs of mould contamination. There is a need for rapid and accurate measurement of mycotoxins for purposes of continual monitoring and identification of high risk commodities. Samples from commercial chicken feed (maize kernels), cattle feed (lucerne, grass and hay) and milk were analysed for the presence of certain mycotoxins and cultured for fungi. Results of fungal profiles showed that most samples were contaminated by moulds belonging to the genera, Alternaria, Aspergillus, Fusarium, Penicillium, Mucor, Phoma and Rhizopus. All the chicken feed samples tested contained mycotoxins either below the recommended safe levels for poultry or below the detection limits of the ELISA tests. However, samples of grass contained levels of zearalenone greater than the allowable concentration for dairy and beef cattle (250 ppm), which may be linked to the presence of Fusarium scirpi identified in the fungal profiles. The levels of AFMI were below the detection limits of the ELISA tests, which may be attributed to the low levels of aflatoxins found in the feed (grass) samples. The presence of the fungus in samples analysed is not evidence for the presence of mycotoxins.

Extension and use of a physical map of the Thinopyrum-derived Lr19 translocation.

Twenty-nine deletion mutant lines were used to extend a physical map of the Lr19 translocated chromosome segment. One hundred and forty-four Sse8387I/MseI and 32 EcoRI/MseI primer combinations were used to obtain 95 Thinopyrum-specific AFLP markers. The physical map confirmed that terminal deletions had mostly occurred, however, it appears that intercalary deletions and primer or restriction site mutations were also induced. The markers allowed for grouping of the deletion mutant lines into 19 clusters, with 7 AFLP markers mapping in the same marker bin as Lr19. Primary and secondary Lr19 allosyndetic recombinants were subsequently physically mapped employing AFLP, RFLP, SCAR and microsatellite markers and the data integrated with the deletion map. A further shortened, tertiary Lr19 recombinant was derived following homologous recombination between the proximally shortest secondary recombinant, Lr19-149-299, and distally shortest recombinant, Lr19-149-478. The tertiary recombinant could be confirmed employing the mapped markers and it was possible to identify new markers on this recombinant that can be used to reduce the translocation still further.

Steps in the evolution of heteromorphic sex chromosomes.

We review some recently published results on sex chromosomes in a diversity of species. We focus on several fish and some plants whose sex chromosomes appear to be 'young', as only parts of the chromosome are nonrecombining, while the rest is pseudoautosomal. However, the age of these systems is not yet very clear. Even without knowing what proportions of their genes are genetically degenerate, these cases are of great interest, as they may offer opportunities to study in detail how sex chromosomes evolve. In particular, we review evidence that recombination suppression occurs progressively in evolutionarily independent cases, suggesting that selection drives loss of recombination over increasingly large regions. We discuss how selection during the period when a chromosome is adapting to its role as a Y chromosome might drive such a process.

Recombination and base composition: the case of the highly self-fertilizing plant Arabidopsis thaliana.

BACKGROUND: Rates of recombination can vary among genomic regions in eukaryotes, and this is believed to have major effects on their genome organization in terms of base composition, DNA repeat density, intron size, evolutionary rates and gene order. In highly self-fertilizing species such as Arabidopsis thaliana, however, heterozygosity is expected to be strongly reduced and recombination will be much less effective, so that its influence on genome organization should be greatly reduced. RESULTS: Here we investigated theoretically the joint effects of recombination and self-fertilization on base composition, and tested the predictions with genomic data from the complete A. thaliana genome. We show that, in this species, both codon-usage bias and GC content do not correlate with the local rates of crossing over, in agreement with our theoretical results. CONCLUSIONS: We conclude that levels of inbreeding modulate the effect of recombination on base composition, and possibly other genomic features (for example, transposable element dynamics). We argue that inbreeding should be considered when interpreting patterns of molecular evolution.

Effects of spontaneously chosen crank rate variations on electromyographic responses in sub-maximal arm exercise in inexperienced subjects.

The aim of the present study was to compare electromyographic responses during arm exercises with a crank rate chosen spontaneously ( T(S)) or set at 20% below or above ( T(-20), T(+20)) the spontaneously chosen crank rate (SCCR). Ten male physical education students performed arm exercises with intensities ranging from 20% to 80% of maximal power. Muscular activity levels were analysed for the biceps brachii and the triceps brachii muscles using integrated rectified surface electromyography (iEMG). All values were presented as the mean and standard deviation. During T(S), the sum of iEMG for the two muscles studied was significantly ( P<0.05) lower than during T(+20) for each power output. No significant differences were observed in iEMG values between T(S) and T(-20). The hypothesis that SCCR relates to a minimisation of muscle activation during an upper body exercise was not confirmed. Variations superior or inferior to a 20% increase of the iEMG responses do not influence it. Moreover, the selection of crank rates depends on the power output and the SCCR increased significantly ( P<0.05) with increasing power output.

Plasma lactate recovery from maximal exercise with correction for variations in plasma volume.

BACKGROUND: To compare plasma lactate concentrations and plasma lactate kinetics during recovery, for measured and corrected values for changes in plasma volume, after a maximal aerobic exercise. METHODS: Sixteen male subjects performed an incremental and maximal exercise in order to reach maximal aerobic power. Prior to the exercise, at the end and during recovery (2, 5, 12 and 30 min), blood samples were collected through an antecubital catheter. Samples were analysed for lactate, hematocrit and hemoglobin in order to calculate changes in plasma volume. Plasma lactate concentrations ([La]p) were corrected for changes in plasma volume. Plasma lactate kinetics was estimated through the ratio between [La]p after 5 min recovery minus [La]p after 30 min to time (25 min) and expressed in percentage per minute. RESULTS: Maximal changes in plasma volume (-19.7 +/- 3.8%) were correlated to maximal measured [La]p (r=0.66, p<0.01). Maximal measured [La]p values (14.9 +/- 2.6 mmol x l-1) were 17.3% higher (p<0.001) than corrected values (12.7 +/-2.0 mmol x l-1). The kinetics of [La]p decrease was significantly higher (p<0.001) for measured values (2.38 +/- 0.29 % x min-1) than for corrected values (2.22 +/- 0.33 % x min-1). CONCLUSIONS: These results suggested that changes in plasma volume must be taken into account when peak postexercise plasma lactate concentration or lactate recovery curves are analysed.

Spontaneously chosen crank rate variations in submaximal arm exercise with inexperienced subjects. Effects on cardiorespiratory and efficiency parameters.

The aim of the present study was to compare the physiological responses during arm exercises when the crank rate was chosen spontaneously (TS) or set at +/- 20 % (T-20, T+ 20) of the spontaneously chosen crank rate (SCCR). Eight physical education male students, aged 22 +/- 3.2 years, performed an upper body exercise in which intensities ranged from unload to 80 % of maximal power. No significant difference was observed in oxygen uptake, ventilation, gross and net efficiency values between TS and T+ 20 or T-20. Nevertheless, oxygen uptake and ventilation were significantly (p < 0.05) lower and gross and net efficiencies higher (p < 0.05) during T-20 than T+ 20. No significant difference was noticed for heart rate, delta and work efficiency between TS, T-20 and T+ 20. The hypothesis that SCCR is the most economical one according to the efficiency parameters was not quite verified. However, crank rates lower than SCCR could be interesting because they increase gross efficiency compared to higher crank rates. Moreover, the selection of crank rates depends on power output. Indeed, SCCR increased significantly (p < 0.05) with power output. In the physical reconditioning of injured or handicapped subjects, the latter are very sensitive to the power output, and the crank rate could be another constraint.

Synonymous codon usage, accuracy of translation, and gene length in Caenorhabditis elegans.

In many unicellular organisms, invertebrates, and plants, synonymous codon usage biases result from a coadaptation between codon usage and tRNAs abundance to optimize the efficiency of protein synthesis. However, it remains unclear whether natural selection acts at the level of the speed or the accuracy of mRNAs translation. Here we show that codon usage can improve the fidelity of protein synthesis in multicellular species. As predicted by the model of selection for translational accuracy, we find that the frequency of codons optimal for translation is significantly higher at codons encoding for conserved amino acids than at codons encoding for nonconserved amino acids in 548 genes compared between Caenorhabditis elegans and Homo sapiens. Although this model predicts that codon bias correlates positively with gene length, a negative correlation between codon bias and gene length has been observed in eukaryotes. This suggests that selection for fidelity of protein synthesis is not the main factor responsible for codon biases. The relationship between codon bias and gene length remains unexplained. Exploring the differences in gene expression process in eukaryotes and prokaryotes should provide new insights to understand this key question of codon usage.

RPE responses during arm and leg exercises: effect of variations in spontaneously chosen crank rate.

The aim of this study was two-fold. First, the rating of perceived exertion (RPE) was compared between two different upper and lower body exercises. Subjects (n = 12) performed with spontaneously chosen crank or pedal rates: (i) incremental maximum power tests (Test 1), with an initial work rate of 50% of maximal power followed by increases of 10% at each 120-sec. work stage and (ii) tests (Test 2) with exercise bouts set at 20, 40, 60, and 80% of maximal power separated by passive recovery periods. Second, the effects of variations in spontaneously chosen crank rate on RPE was analysed using the second test performed only with upper body. Subjects performed Test 2 three times with crank rates spontaneously chosen by the subjects, set at plus or minus 20% of spontaneously chosen crank rate. During both Tests 1 and 2 for upper or lower body, RPE increased linearly (p<.01) with power output. No significant difference was noticed between upper and lower body tests; however, RPE was significantly different (p<.05) between Test 1 results for upper and lower body at 70, 80, 90, and 100% of maximal power. The greater RPE at high power output could be linked to the important effect of fatigue during upper body exercise. Among the three crank-rate conditions, no significant difference in RPE was noticed. The choice of crank rate does not seem to influence the perception of exertion in upper body cycling exercise.

Does recombination improve selection on codon usage? Lessons from nematode and fly complete genomes.

Understanding the factors responsible for variations in mutation patterns and selection efficacy along chromosomes is a prerequisite for deciphering genome sequences. Population genetics models predict a positive correlation between the efficacy of selection at a given locus and the local rate of recombination because of Hill-Robertson effects. Codon usage is considered one of the most striking examples that support this prediction at the molecular level. In a wide range of species including Caenorhabditis elegans and Drosophila melanogaster, codon usage is essentially shaped by selection acting for translational efficiency. Codon usage bias correlates positively with recombination rate in Drosophila, apparently supporting the hypothesis that selection on codon usage is improved by recombination. Here we present an exhaustive analysis of codon usage in C. elegans and D. melanogaster complete genomes. We show that in both genomes there is a positive correlation between recombination rate and the frequency of optimal codons. However, we demonstrate that in both species, this effect is due to a mutational bias toward G and C bases in regions of high recombination rate, possibly as a direct consequence of the recombination process. The correlation between codon usage bias and recombination rate in these species appears to be essentially determined by recombination-dependent mutational patterns, rather than selective effects. This result highlights that it is necessary to take into account the mutagenic effect of recombination to understand the evolutionary role and impact of recombination.