Diversification and the evolution of dispersal ability in the tribe Brassiceae (Brassicaceae).

BACKGROUND AND AIMS: Dispersal and establishment ability can influence evolutionary processes such as geographic isolation, adaptive divergence and extinction probability. Through these population-level dynamics, dispersal ability may also influence macro-evolutionary processes such as species distributions and diversification. This study examined patterns of evolution of dispersal-related fruit traits, and how the evolution of these traits is correlated with shifts in geographic range size, habitat and diversification rates in the tribe Brassiceae (Brassicaceae). METHODS: The phylogenetic analysis included 72 taxa sampled from across the Brassiceae and included both nuclear and chloroplast markers. Dispersal-related fruit characters were scored and climate information for each taxon was retrieved from a database. Correlations between fruit traits, seed characters, habitat, range and climate were determined, together with trait-dependent diversification rates. KEY RESULTS: It was found that the evolution of traits associated with limited dispersal evolved only in association with compensatory traits that increase dispersal ability. The evolution of increased dispersal ability occurred in multiple ways through the correlated evolution of different combinations of fruit traits. The evolution of traits that increase dispersal ability was in turn associated with larger seed size, increased geographic range size and higher diversification rates. CONCLUSIONS: This study provides evidence that the evolution of increased dispersal ability and larger seed size, which may increase establishment ability, can also influence macro-evolutionary processes, possibly by increasing the propensity for long-distance dispersal. In particular, it may increase speciation and consequent diversification rates by increasing the likelihood of geographic and thereby reproductive isolation.

The mating of a fly.

Courtship in Drosophila is influenced by a wide variety of genes, in that many different kinds of pleiotropic mutations lead to defective courtship. This may seem to be a truism, but the broad temporal and spatial expression of most of the fly's "neuro genes" makes it difficult to exclude elements of such genes' actions as materially underlying reproductive behavior. "Courtship genes" that seem to play more particular roles were originally identified as sensory, learning, or rhythm mutations; their reproductive abnormalities have been especially informative for revealing components of male or female actions that might otherwise have gone unnoticed. Further behavioral mutations seemed originally to be courtship-specific, turned out not to have that property, and have led to a broadened perspective on the nature and action of Drosophila's sex-determination genes.

Interspecific genetic control of courtship song production and reception in Drosophila.

The genetic control of courtship song differences between Drosophila melanogaster and Drosophila simulans males was investigated by producing hybrids from reciprocal crosses. The song rhythm difference between the parental species appears to be due to sex-linked genes, whereas the basic interpulse-interval difference is autosomally inherited. Hybrid females show selective preferences for artificially generated songs carrying intermediate "hybrid" characteristics.

Independent photoreceptive circadian clocks throughout Drosophila.

Transgenic Drosophila that expressed either luciferase or green fluorescent protein driven from the promoter of the clock gene period were used to monitor the circadian clock in explanted head, thorax, and abdominal tissues. The tissues (including sensory bristles in the leg and wing) showed rhythmic bioluminescence, and the rhythms could be reset by light. The photoreceptive properties of the explanted tissues indicate that unidentified photoreceptors are likely to contribute to photic signal transduction to the clock. These results show that autonomous circadian oscillators are present throughout the body, and they suggest that individual cells in Drosophila are capable of supporting their own independent clocks.

Molecular transfer of a species-specific behavior from Drosophila simulans to Drosophila melanogaster.

Drosophila males modulate the interpulse intervals produced during their courtship songs. These song cycles, which are altered by mutations in the clock gene period, exhibit a species-specific variation that facilitates mating. We have used chimeric period gene constructs from Drosophila melanogaster and Drosophila simulans in germline transformation experiments to map the genetic control of their song rhythm difference to a small segment of the amino acid encoding information within this gene.

Assessment of exposure to secondhand smoke at outdoor bars and family restaurants in Athens, Georgia, using salivary cotinine.

Exposure to secondhand smoke (SHS) in outdoor settings is a growing public health concern due to recent indoor smoking bans. The objective of this study was to measure salivary cotinine, a metabolite of nicotine, in subjects aged 21-30 exposed to SHS outside bars and restaurants in Athens, Georgia. Nonsmokers participated during 6-hr periods in outdoor standing or seating areas of bars and restaurants where indoor smoking was banned, as well as a control outdoor location with no smokers over six weekends during the summer and early fall of 2007. Pre- and post-exposure saliva samples (N = 25 person-days at the bar site, N = 28 person-days at the restaurant site, and N = 11 person-days at the control) were collected and analyzed for cotinine. The mean change in the response, (ln(post) - ln(pre)) salivary cotinine levels, was significantly impacted by the type of site (bar, restaurant, control) (F = 5.09; d.f. = 2, 6.7; p = 0.0455). The median percent increase in salivary cotinine from pre-test to post-test was estimated to be 162%, 102%, and 16% at the bar, restaurant, and control sites, respectively, values that were significant increases at bars (t = 4.63; d.f. = 9.24; p = 0.0011) and restaurants (t = 4.33; d.f. = 4.47; p = 0.0097) but not at the control sites. On average, these pre-test to post-test increases in salivary cotinine were significantly higher at bar sites than control sites (t = 3.05; d.f. = 9.85; p = 0.0176) and at restaurant sites compared with control sites (t = 2.35; d.f. = 5.09; p = 0.0461). Nonsmokers outside restaurants and bars in Athens, Georgia, have significantly elevated salivary cotinine levels indicative of secondhand smoke exposure.

Evidence that the TIM light response is relevant to light-induced phase shifts in Drosophila melanogaster.

Light is a major environmental signal for the entrainment of circadian rhythms. In Drosophila melanogaster, recent experiments suggest that photic information is transduced to the clock through the timeless gene product, TIM. We provide genetic and spectral evidence supporting the relevance of TIM light responses to clock resetting. A missense mutant TIM, TIM-SL, exhibits greater sensitivity to light in both TIM protein disappearance and locomotor activity phase shifting assays. We show that the wavelength dependence of light-induced decreases in TIM levels and that of light-mediated phase shifting are virtually identical. Analysis of dose response of TIM disappearance in a variety of mutant genotypes suggests cell-autonomous light responses that are largely independent of the canonical visual transduction pathway.

Antibodies to the period gene product of Drosophila reveal diverse tissue distribution and rhythmic changes in the visual system.

Polyclonal antibodies were prepared against the period gene product, which influences biological rhythms in D. melanogaster, by using small synthetic peptides from the per sequence as immunogens. The peptide that elicited the best antibody reagent was a small domain near the site of the pers (short period) mutation. Specific immunohistochemical staining was detected in a variety of tissue types: the embryonic CNS; a few cell bodies in the central brain of pupae; these and other cells in the central brain of adults, as well as imaginal cells in the eyes, optic lobes, and the gut. The intensity of per-specific staining in the visual system was found to oscillate, defining a free-running circadian rhythm with a peak in the middle of the night.

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla.

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.

The circadian clock of fruit flies is blind after elimination of all known photoreceptors.

Circadian rhythms are entrained by light to follow the daily solar cycle. We show that Drosophila uses at least three light input pathways for this entrainment: (1) cryptochrome, acting in the pacemaker cells themselves, (2) the compound eyes, and (3) extraocular photoreception, possibly involving an internal structure known as the Hofbauer-Buchner eyelet, which is located underneath the compound eye and projects to the pacemaker center in the brain. Although influencing the circadian system in different ways, each input pathway appears capable of entraining circadian rhythms at the molecular and behavioral level. This entrainment is completely abolished in glass(60j) cry(b) double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome.

A promoterless period gene mediates behavioral rhythmicity and cyclical per expression in a restricted subset of the Drosophila nervous system.

Transgenic flies carrying a 7.2 kb piece of DNA from the period (per) gene were analyzed for the presence of circadian locomotor activity rhythms and fluctuations of per-encoded mRNA and protein. The 5' end of this genomic fragment is within the first intron, which precedes the coding region. This promotorless fragment could rescue circadian behavioral rhythms and mediate spatial expression of PER in a subset of wild-type per cells within the CNS and PNS. In one behaviorally rhythmic line, PER protein was found in only "per lateral neurons." In the rhythmic transgenics, per mRNA and protein levels undergo circadian cycling, as previously described for wild type. Cycling of PER in brain cells of flies carrying the same 7.2 kb piece of per DNA under the control of a heat shock promoter corroborated the hypothesis that per's molecular cyclings and behavioral rhythmicity are causally related.

The strength and periodicity of D. melanogaster circadian rhythms are differentially affected by alterations in period gene expression.

The per gene of D. melanogaster influences or participates in the generation of biological rhythms. Previous experiments have identified the head as the location from which per exerts its effect on circadian rhythms. To localize further this region and to examine the effects of altered levels and altered spatial expression patterns of the per gene on circadian rhythms of locomotor activity, we have characterized transformed lines containing per gene constructs missing substantial cis-acting regulatory information. The data suggest that wild-type levels of per gene expression are necessary in only a small fraction of the nervous system for near wild-type periods, whereas a larger fraction of per-expressing cells in the brain contributes to the strength of the circadian rhythms.

The timSL mutant of the Drosophila rhythm gene timeless manifests allele-specific interactions with period gene mutants.

To identify new components of the Drosophila circadian clock, we screened chemically mutagenized flies for suppressors or enhancers of the long periods characteristic of the period (per) mutant allele perL. We isolated a novel mutant that maps to the rhythm gene timeless (tim). This novel allele, timSL, alters the temporal pattern of perL protein nuclear localization and restores temperature compensation to perL flies. timSL more generally manifests specific interactions with different per alleles. The identification of this first period-altering tim allele provides further evidence that TIM is a major component of the clock, and the allele-specific interactions with PER provide evidence that the PER/TIM heterodimer is a unit of circadian function. Although timSL fails to restore PER-L/TIM temperature insensitivity in yeast, it alters the TIM phosphorylation pattern during the late night. The effects on phosphorylation suggest that timSL functions as a partial bypass suppressor of perL and provide evidence that the TIM phosphorylation program contributes to the circadian timekeeping mechanism.

Drosophila CRY is a deep brain circadian photoreceptor.

cry (cryptochrome) is an important clock gene, and recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cry(b), inhibits circadian photoresponses. Restricting CRY expression to specific fly tissues shows that CRY expression is needed in a cell-autonomous fashion for oscillators present in different locations. CRY overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted CRY expression also rescues all circadian defects of cry(b) behavior. As wild-type pacemaker neurons express CRY, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pace-maker and a behavioral output system.

Novel features of drosophila period Transcription revealed by real-time luciferase reporting.

The rapid turnover of luciferase and the sensitive, non-invasive nature of its assay make this reporter gene uniquely situated for temporal gene expression studies. To determine the in vivo regulatory pattern of the Drosophila clock gene period (per), we generated transgenic strains carrying a luciferase cDNA fused to the promoter region of the per gene. This has allowed us to monitor circadian rhythms of bioluminescence from pacemaker cells within the head for several days in individual living adults. These high time-resolution experiments permitted neuronal per transcription and opens the door to vastly simplified experiments in general chronobiology and studies of temporally regulated transcription in a wide range of experimental systems.

Transplanted Drosophila excretory tubules maintain circadian clock cycling out of phase with the host.

Circadian rhythms in behaviors and physiological processes are driven by conserved molecular mechanisms involving the rhythmic expression of clock genes in the brains of animals [1]. The persistence of similar molecular rhythms in peripheral tissues in vitro [2] [3] suggests that these tissues contain self-sustained circadian clocks that may be linked to rhythmic physiological functions. It is not known how brain and peripheral clocks are organized into a synchronized timing system; however, it has been assumed that peripheral clocks submit to a master clock in the brain. To address this matter we examined the expression of two clock genes, period (per) and timeless (tim), in host and transplanted abdominal organs of Drosophila. We found that excretory organs in tissue culture display free-running, light-sensitive oscillations in per and tim gene activity indicating that they house self-sustained circadian clocks. To test for humoral factors, we monitored cycling of the TIM protein in excretory tubules transplanted into host flies entrained to an opposite light-dark cycle. We show that the clock protein in the donor tubules cycled out of phase with that in the host tubules, indicating that different organs may cycle independently, despite sharing the same hormonal milieu. We suggest that one way to achieve circadian coordination of physiological sub-systems in higher animals may be through the direct entrainment of light-sensitive clocks by environmental signals.

Tripping along the trail to the molecular mechanisms of biological clocks.

Solving the mechanism of circadian clocks has become an important goal, in part because daily rhythms are running in such a wide variety of organisms, and contribute to many aspects of their well being. Systematic genetic approaches to studying 'the clock' were initiated in fruitflies more than 20 years ago as a novel means by which neural-pacemaking mysteries might be solved. Such chronogenetic investigations gained momentum when they spread to other species, and became molecular. However, the molecular studies were misleading, that is, until some elementary neuro-anatomical observations, involving the expression of a 'clock gene' in Drosophila, gave the experiments in this molecular-neurogenetic area of chronobiology a new direction. The initially neuro-descriptive studies led to the current investigations that involve negatively acting transcription factors and other clock molecules that are presumed to interact with them. In addition, new mutants and clones have been isolated in a timely manner. These mutations and molecules should permit chronogeneticists, working on a wide variety of organisms, to unravel further details of how the clock works, how environmental information finds its way to it, and how it sends information out into the organism's physiology, biochemistry and behavior.

Cryptochromes: sensory reception, transduction, and clock functions subserving circadian systems.

Cryptochromes (CRYs) are blue-light-absorbing proteins involved in a variety of biological phenomena. In animals, CRYs exhibit a certain versatility with regard to these organisms' circadian rhythms, as has been revealed by the effects of mutations and molecular manipulations. The rhythm system of Drosophila uses one gene's worth of CRY protein to transmit light into a circadian clock within the brain, which controls the fly's sleep-wake cycles. In fact, the relevant pacemaking neurons are themselves circadian photoreceptive structures. In peripheral tissues and others located posterior to the brain, Drosophila CRY may be a photoreceptive molecule and also part of the pacemaker mechanism. Mice have two CRY-encoding genes. They are expressed in many tissues, including the retina and a clock structure within the brain. In the former location, mouse CRY may play a circadian-photoreceptive role, along with that mediated by rhodopsins found elsewhere in the retina. In the latter tissue, the hypothalamic suprachiasmatic nucleus, mouse CRYs are closely connected to the multimolecule murine clock mechanism.

Abnormalities of male-specific FRU protein and serotonin expression in the CNS of fruitless mutants in Drosophila.

The fruitless gene in Drosophila produces male-specific protein (FRU(M)) involved in the control of courtship. FRU(M) spatial and temporal patterns were examined in fru mutants that exhibit aberrant male courtship. Chromosome breakpoints at the locus eliminated FRU(M). Homozygous viable mutants exhibited an intriguing array of defects. In fru(1) males, there were absences of FRU(M)-expressing neuronal clusters or stained cells within certain clusters, reductions of signal intensities in others, and ectopic FRU(M) expression in novel cells. fru(2) males exhibited an overall decrement of FRU(M) expression in all neurons normally expressing the gene. fru(4) and fru(sat) mutants only produced FRU(M) in small numbers of neurons at extremely low levels, and no FRU(M) signals were detected in fru(3) males. This array of abnormalities was inferred to correlate with the varying behavioral defects exhibited by these mutants. Such abnormalities include courtship among males, which has been hypothesized to involve anomalies of serotonin (5-HT) function in the brain. However, double-labeling uncovered no coexpression of FRU(M) and 5-HT in brain neurons. Yet, a newly identified set of sexually dimorphic FRU(M)/5-HT-positive neurons was identified in the abdominal ganglion of adult males. These sexually dimorphic neurons (s-Abg) project toward regions of the abdomen involved in male reproduction. The s-Abg neurons and the proximal extents of their axons were unstained or absent in wild-type females and exhibited subnormal or no 5-HT immunoreactivity in certain fru-mutant males, indicating that fruitless controls the formation of these cells or 5-HT production in them.

Two novel doubletime mutants alter circadian properties and eliminate the delay between RNA and protein in Drosophila.

Phosphorylation is an important feature of pacemaker organization in Drosophila. Genetic and biochemical evidence suggests involvement of the casein kinase I homolog doubletime (dbt) in the Drosophila circadian pacemaker. We have characterized two novel dbt mutants. Both cause a lengthening of behavioral period and profoundly alter period (per) and timeless (tim) transcript and protein profiles. The PER profile shows a major difference from the wild-type program only during the morning hours, consistent with a prominent role for DBT during the PER monomer degradation phase. The transcript profiles are delayed, but there is little effect on the protein accumulation profiles, resulting in the elimination of the characteristic lag between the mRNA and protein profiles. These results and others indicate that light and post-transcriptional regulation play major roles in defining the temporal properties of the protein curves and suggest that this lag is unnecessary for the feedback regulation of per and tim protein on per and tim transcription.