Taxon-specific, phased siRNAs underlie a speciation locus in monkeyflowers.

Taxon-specific small RNA loci are widespread in eukaryotic genomes, yet their role in lineage-specific adaptation, phenotypic diversification, and speciation is poorly understood. Here, we report that a speciation locus in monkeyflowers (Mimulus), YELLOW UPPER (YUP), contains an inverted repeat region that produces small interfering RNAs (siRNAs) in a phased pattern. Although the inverted repeat is derived from a partial duplication of a protein-coding gene that is not involved in flower pigmentation, one of the siRNAs targets and represses a master regulator of floral carotenoid pigmentation. YUP emerged with two protein-coding genes that control other aspects of flower coloration as a "superlocus" in a subclade of Mimulus and has contributed to subsequent phenotypic diversification and pollinator-mediated speciation in the descendant species.

Two MYB Proteins in a Self-Organizing Activator-Inhibitor System Produce Spotted Pigmentation Patterns.

Many organisms exhibit visually striking spotted or striped pigmentation patterns. Developmental models predict that such spatial patterns can form when a local autocatalytic feedback loop and a long-range inhibitory feedback loop interact. At its simplest, this self-organizing network only requires one self-activating activator that also activates a repressor, which inhibits the activator and diffuses to neighboring cells. However, the molecular activators and inhibitors fully fitting this versatile model remain elusive in pigmentation systems. Here, we characterize an R2R3-MYB activator and an R3-MYB repressor in monkeyflowers (Mimulus). Through experimental perturbation and mathematical modeling, we demonstrate that the properties of these two proteins correspond to an activator-inhibitor pair in a two-component, reaction-diffusion system, explaining the formation of dispersed anthocyanin spots in monkeyflower petals. Notably, disrupting this pattern impacts pollinator visitation. Thus, subtle changes in simple activator-inhibitor systems are likely essential contributors to the evolution of the remarkable diversity of pigmentation patterns in flowers.

Morphospace exploration reveals divergent fitness optima between plants and pollinators.

The obligate mutualism and exquisite specificity of many plant-pollinator interactions lead to the expectation that flower phenotypes (e.g., corolla tube length) and corresponding pollinator traits (e.g., hawkmoth proboscis length) are congruent as a result of coevolution by natural selection. However, the effect of variation in flower morphology on the fitness of plants and their pollinators has not been quantified systematically. In this study, we employed the theoretical morphospace paradigm using a combination of 3D printing, electronic sensing, and machine vision technologies to determine the influence of two flower morphological features (corolla curvature and nectary diameter) on the fitness of both parties: the artificial flower and its hawkmoth pollinator. Contrary to the expectation that the same flower morphology maximizes the fitness of both plant and pollinator, we found that the two parties have divergent optima for corolla curvature, with non-overlapping fitness peaks in flower morphospace. The divergent fitness optima between plants and pollinators could lead to evolutionary diversification in both groups.

Shape matters: corolla curvature improves nectar discovery in the hawkmoth Manduca sexta.

1. We measured the effects of variation in corolla curvature and nectary aperture radius on pollinator foraging ability using the hawkmoth Manduca sexta and 3D-printed artificial flowers whose shapes were mathematically specified. 2. In dimorphic arrays containing trumpet-shaped flowers and flat-disk flowers, hawkmoths were able to empty the nectaries of significantly more trumpet-shaped flowers regardless of nectary aperture size. Interestingly, trumpet-shaped flowers needed to deviate only slightly from the flat-disk morphotype in order to significantly increase hawkmoth foraging ability. 3. Whole-flower three-dimensional shape, particularly corolla curvature, has the potential to act as a mechanical guide for Manduca sexta, further implicating direct flower-proboscis contact as an important contributor to foraging success during flower handling in hawkmoths.

Three floral volatiles contribute to differential pollinator attraction in monkeyflowers (Mimulus).

Flowering plants employ a wide variety of signals, including scent, to attract the attention of pollinators. In this study we investigated the role of floral scent in mediating differential attraction between two species of monkeyflowers (Mimulus) reproductively isolated by pollinator preference. The emission rate and chemical identity of floral volatiles differ between the bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated M. cardinalis. Mimulus lewisii flowers produce an array of volatiles dominated by d-limonene, ß-myrcene and E-ß-ocimene. Of these three monoterpenes, M. cardinalis flowers produce only d-limonene, released at just 0.9% the rate of M. lewisii flowers. Using the Bombus vosnesenskii bumblebee, an important pollinator of M. lewisii, we conducted simultaneous gas chromatography with extracellular recordings in the bumblebee antennal lobe. Results from these experiments revealed that these three monoterpenes evoke significant neural responses, and that a synthetic mixture of the three volatiles evokes the same responses as the natural scent. Furthermore, the neural population shows enhanced responses to the M. lewisii scent over the scent of M. cardinalis. This neural response is reflected in behavior; in two-choice assays, bumblebees investigate artificial flowers scented with M. lewisii more frequently than ones scented with M. cardinalis, and in synthetic mixtures the three monoterpenes are necessary and sufficient to recapitulate responses to the natural scent of M. lewisii. In this system, floral scent alone is sufficient to elicit differential visitation by bumblebees, implying a strong role of scent in the maintenance of reproductive isolation between M. lewisii and M. cardinalis.

The genetic control of flower-pollinator specificity.

The ca. 275,000 species of flowering plants are the result of a recent adaptive radiation driven largely by the coevolution between plants and their animal pollinators. Identification of genes and mutations responsible for floral trait variation underlying pollinator specificity is crucial to understanding how pollinator shifts occur between closely related species. Petunia, Mimulus, and Antirrhinum have provided a high standard of experimental evidence to establish causal links from genes to floral traits to pollinator responses. In all three systems, MYB transcription factors seem to play a prominent role in the diversification of pollinator-associated floral traits.

Bulk segregant analysis of an induced floral mutant identifies a MIXTA-like R2R3 MYB controlling nectar guide formation in Mimulus lewisii.

The genetic and developmental basis of many ecologically important floral traits (e.g., carotenoid pigmentation, corolla tube structure, nectar volume, pistil and stamen length) remains poorly understood. Here we analyze a chemically induced floral mutant of Mimulus lewisii through bulk segregant analysis and transgenic experiments and identify a MIXTA-like R2R3 MYB gene that controls nectar guide formation in M. lewisii flowers, which involves epidermal cell development and carotenoid pigmentation.

Using experimental evolution to investigate geographic range limits in monkeyflowers.

Every species occupies a restricted geographic distribution, but it is unclear why natural selection at the range margin fails to increase tolerance to limiting environmental variables and thereby allow continual range expansion. Models indicate that the interplay of demographic asymmetries, dispersal, divergent natural selection, and adaptive trade-offs across spatially varying environments can give rise to stable range limits. Here we examine sister species of the monkeyflowers Mimulus cardinalis and M. lewisii to identify traits that might contribute to the evolution of the species' ranges and to ask whether adaptive trade-offs between environments can limit their geographic distribution. In the Sierra Nevada Mountains of California, M. cardinalis is found from low to mid elevation and M. lewisii is found from mid to high elevation. We transplanted segregating populations of interspecific hybrids to low and high elevation and cross-pollinated those that survived to flowering to create selected populations that evolved at low or high elevation. When grown in a common environment, the progeny of hybrids selected at high elevation flowered earlier compared to a greenhouse control population, whereas hybrids selected at low elevation displayed increased warm-temperature photosynthetic capacity. If adaptation to one environment entails a cost to adaptation in other environments, then selected hybrid populations should display reduced fitness, relative to an unselected control population, when grown in an environment in which they were not selected. Two such trade-offs were observed in this study, where hybrids selected at high elevation displayed reduced biomass when grown in temperatures characteristic of low elevation and hybrids selected at low elevation showed reduced resistance to freezing. These results identify traits under selection for range expansion and suggest that adaptive trade-offs can contribute to limiting the geographic distribution of species.

Criterion validity of the BBUSQ-22: a questionnaire assessing bowel and urinary tract symptoms in women.

To evaluate the criterion validity of the Birmingham Bowel and Urinary Symptoms Questionnaire (BBUSQ-22). Forty-four women presenting with urinary or bowel symptoms to a urogynaecology/functional bowel clinic, completed three disease-specific questionnaires (Birmingham Bowel and Urinary Symptoms Questionnaire, Sheffield Prolapse Symptoms Questionnaire and King's Health Questionnaire) and two generic questionnaires (SF-36 and EuroQol). Wilcoxon rank sum tests, Spearman's rank correlations and multiple regression analyses were undertaken. All clinically hypothesised correlations between the questionnaires' domains were found significant, and all domains hypothesised to be predictors of the BBUSQ-22 domains were proven to be prognostic, without explaining too much variability to doubt their uniqueness. The similarity among the measures has been proved without such relationships being too strong to call into question the distinctiveness of the concepts being measured. Overall, criterion validity is demonstrated, and the clinical usefulness of the addition this questionnaire makes to the area is highlighted.

Development and psychometric testing of a symptom index for pelvic organ prolapse.

A wide range of symptoms are commonly ascribed to pelvic organ prolapse including pain, awareness of lump, bowel, bladder and sexual dysfunction. The aim of this work was to develop and validate an instrument to quantify symptoms related to pelvic organ prolapse. Consultation with symptomatic women and specialists in coloproctology, urology, gynaecology and sexual health resulted in a questionnaire with 25 questions. In total, 203 women participated in a psychometric testing of this instrument, 152 cases with prolapse and 51 controls without. The content validity, criterion validity, reliability and responsiveness of the questionnaire were evaluated. The questionnaire proved a reliable and valid instrument for the assessment of symptoms related to uterovaginal prolapse. It is also sensitive to change.

Mutations in CAX1 produce phenotypes characteristic of plants tolerant to serpentine soils.

Plant tolerance of serpentine soils is potentially an excellent model for studying the genetics of adaptive variation in natural populations. A large-scale viability screen of Arabidopsis thaliana mutants on a defined nutrient solution with a low Ca(2+) : Mg(2+) ratio (1 : 24 mol : mol), typical of serpentine soils, yielded survivors with null alleles of the tonoplast calcium-proton antiporter CAX1. cax1 mutants have most of the phenotypes associated with tolerance to serpentine soils, including survival in solutions with a low Ca(2+) : Mg(2+) ratio; requirement for a high concentration of Mg(2+) for maximum growth; reduced leaf tissue concentration of Mg(2+); and poor growth performance on 'normal' levels of Ca(2+) and Mg(2+). A physiological model is proposed to explain how loss-of-function cax1 mutations could produce all these phenotypes characteristic of plants adapted to serpentine soils, why 'normal' plants are unable to survive on serpentine soil, and why serpentine-adapted plants are unable to compete on 'normal' soils.

Allele substitution at a flower colour locus produces a pollinator shift in monkeyflowers.

The role of major mutations in adaptive evolution has been debated for more than a century. The classical view is that adaptive mutations are nearly infinite in number with infinitesimally small phenotypic effect, but recent theory suggests otherwise. To provide empirical estimates of the magnitude of adaptive mutations in wild plants, we conducted field studies to determine the adaptive value of alternative alleles at a single locus, YELLOW UPPER (YUP). YUP controls the presence or absence of yellow carotenoid pigments in the petals of pink-flowered Mimulus lewisii, which is pollinated by bumblebees, and its red-flowered sister species M. cardinalis, which is pollinated by hummingbirds. We bred near-isogenic lines (NILs) in which the YUP allele from each species was substituted into the other. M. cardinalis NILs with the M. lewisii YUP allele had dark pink flowers and received 74-fold more bee visits than the wild type, whereas M. lewisii NILs with the M. cardinalis yup allele had yellow-orange flowers and received 68-fold more hummingbird visits than the wild type. These results indicate that an adaptive shift in pollinator preference may be initiated by a single major mutation.

Components of reproductive isolation between the monkeyflowers Mimulus lewisii and M. cardinalis (Phrymaceae).

Evolutionists have long recognized the role of reproductive isolation in speciation, but the relative contributions of different reproductive barriers are poorly understood. We examined the nature of isolation between Mimulus lewisii and M. cardinalis, sister species of monkeyflowers. Studied reproductive barriers include: ecogeographic isolation; pollinator isolation (pollinator fidelity in a natural mixed population); pollen competition (seed set and hybrid production from experimental interspecific, intraspecific, and mixed pollinations in the greenhouse); and relative hybrid fitness (germination, survivorship, percent flowering, biomass, pollen viability, and seed mass in the greenhouse). Additionally, the rate of hybridization in nature was estimated from seed collections in a sympatric population. We found substantial reproductive barriers at multiple stages in the life history of M. lewisii and M. cardinalis. Using range maps constructed from herbarium collections, we estimated that the different ecogeographic distributions of the species result in 58.7% reproductive isolation. Mimulus lewisii and M. cardinalis are visited by different pollinators, and in a region of sympatry 97.6% of pollinator foraging bouts were specific to one species or the other. In the greenhouse, interspecific pollinations generated nearly 50% fewer seeds than intraspecific controls. Mixed pollinations of M. cardinalis flowers yielded >75% parentals even when only one-quarter of the pollen treatment consisted of M. cardinalis pollen. In contrast, both species had similar siring success on M. lewisii flowers. The observed 99.915% occurrence of parental M. lewisii and M. cardinalis in seeds collected from a sympatric population is nearly identical to that expected, based upon our field observations of pollinator behavior and our laboratory experiments of pollen competition. F1 hybrids exhibited reduced germination rates, high survivorship and reproduction, and low pollen and ovule fertility. In aggregate, the studied reproductive barriers prevent, on average, 99.87% of gene flow, with most reproductive isolation occurring prior to hybrid formation. Our results suggest that ecological factors resulting from adaptive divergence are the primary isolating barriers in this system. Additional studies of taxa at varying degrees of evolutionary divergence are needed to identify the relative importance of pre- and postzygotic isolating mechanisms in speciation.

The acute effect of magnetic stimulation of the pelvic floor on involuntary detrusor activity during natural filling and overactive bladder symptoms.

OBJECTIVE: To evaluate the effect of magnetic stimulation of the pelvic floor (MSPF) on involuntary detrusor activity observed during natural filling, and on the overactive bladder symptom complex. PATIENTS AND METHODS: Eighteen women with detrusor overactivity on conventional cystometry underwent ambulatory urodynamic monitoring over two filling cycles. Fluid intake was standardized, provocative manoeuvres applied at regular intervals and symptoms documented contemporaneously. During the second filling cycle MSPF was delivered whenever the detrusor pressure increased by > 5 cmH2O. The women were subsequently treated with MSPF for 6 weeks; their lower urinary tract symptoms were assessed before and after treatment. RESULTS: Comparing the second (stimulated) cycle with the first (unstimulated) cycle, cystometric capacity was higher (373 vs 224 mL, P < 0.03). and involuntary detrusor activity of shorter duration (370 vs 427 s, P < 0.82) and lower amplitude (53 vs 63 cmH2O, P < or = 0.05). All women tolerated the procedure comfortably, but nine found it too time-consuming and withdrew. In the nine women who completed treatment there was no consistent change in overactive bladder symptoms. CONCLUSIONS: In this pilot study, MSPF during natural filling was associated with a decrease in the amplitude of involuntary detrusor contractions and a significant increase in cystometric capacity. However, MSPF had a variable effect on sensations of urgency, both acutely and after treatment, and currently there is no evidence to suggest that MSPF has an enduring effect on symptoms of the overactive bladder.

Leaf stomatal and epidermal cell development: identification of putative quantitative trait loci in relation to elevated carbon dioxide concentration in poplar.

Genetic variation in stomatal initiation and density, and epidermal cell size and number were examined in a hybrid pedigree of Populus trichocarpa T. & G. and P. deltoides Marsh in both ambient ([aCO2]) and elevated ([eCO2]) concentrations of CO2. We aimed to link anatomical traits with the underlying genetic map of F2 Family 331, composed of 350 markers across 19 linkage groups. Leaf stomatal and epidermal cell traits showed pronounced differences between the original parents. We considered the following traits in the F2 population: stomatal density (SD), stomatal index (SI), epidermal cell area (ECA) and the number of epidermal cells per leaf (ECN). In [eCO2], adaxial SD and SI were reduced in the F2 population, whereas ECA increased and ECN remained unchanged. In [aCO2], four putative quantitative trait loci (QTL) with logarithm of the odds ratio (LOD) scores greater than 2.9 were found for stomatal traits on linkage group B: adaxial SI (LOD scores of 5.4 and 5.2); abaxial SI (LOD score of 3.3); and SD (LOD score of 3.2). These results imply that QTL for SI and SD share linkage group B and are under genetic control. More moderate LOD scores (LOD scores >/= 2.5) suggest QTL for SI on linkage groups A and B and for SD on linkage groups B, D and X with a probable co-locating quantitative trait locus for SI and SD on linkage group D (position 46.3 cM). The QTL in both [aCO2] and [eCO2] for adaxial SD were co-located on linkage group X (LOD scores of 3.5 and 2.6, respectively) indicating a similar response across both treatments. Putative QTL were located on linkage group A (position 89.2 cM) for both leaf size and ECN in [aCO2] and for ECA at almost the same position. The data provide preliminary evidence that leaf stomatal and cell traits are amenable to QTL analysis.

A scoring system for the assessment of bowel and lower urinary tract symptoms in women.

OBJECTIVE: To develop a simple scoring system for a validated 22-item questionnaire used to assess bowel and urinary dysfunction in women. SETTING: A urogynaecology clinic, a functional bowel clinic, a district general hospital and a general practice. POPULATION: One hundred and one women referred with functional bowel and/or urinary symptoms and 131 asymptomatic controls. METHODS: A user manual has been prepared. Individual responses to questions are categorised into normal and abnormal and odds ratio tests applied to reflect their sensitivity. RESULTS: Scoring methods have been detailed, and appropriate and sensitive cutoff points defined. CONCLUSIONS: The use of this validated questionnaire is now aided by a user manual, facilitating health care evaluation research into the effects of pelvic surgery on pelvic floor symptomatology. A simple scoring system is provided, making the questionnaire a valuable and accessible research tool.

Review of current practice to establish success after vasectomy.

BACKGROUND: This study aimed to examine the criteria used by surgeons in a district general hospital to confirm success following vasectomy, to establish the proportion of men undergoing vasectomy in whom the procedure was unsuccessful according to those criteria, and to evaluate their subsequent management. METHODS: All 15 surgeons performing vasectomy indicated that they required two consecutive azoospermic postvasectomy semen specimens before they advised couples that the vasectomy was successful. Results of postvasectomy semen analysis (PVSA) for all 240 primary vasectomies performed over a 12-month interval were analysed. Minimum follow-up was 30 (range 30-42; median 37) months. RESULTS: At follow-up 72 men (30 per cent) had not returned postvasectomy samples that fulfilled the criteria, including 18 who were azoospermic on the first PVSA 3 months after vasectomy but who failed to produce a second specimen. In 24 men (10 per cent) who failed to comply with the PVSA protocol, there was no documentation of any further action being taken. No pregnancies were reported in the partners of the study group during this interval and only one patient underwent repeat vasectomy. CONCLUSION: The results suggest that the strict requirement of two consecutive azoospermic postvasectomy semen specimens may be unjustified, leads to a high level of non-compliance and causes unnecessary delay in confirming success of the procedure.

Abundant Pathogenic Variation in the New Hybrid Rust Melampsora xcolumbiana on Hybrid Poplar.

ABSTRACT The recently described rust hybrid Melampsora xcolumbiana was discovered as a result of its novel pathogenic variation on Populus trichocarpa x P. deltoides (TxD) hybrid poplar. To characterize this pathogenic variation, 10 commercial TxD clones, all F(1) clones, were chosen as host differentials. Fourteen mononuredinial isolates of Pacific Northwestern field collections of M. xcolumbiana, from 1996 to 1998 inclusive, were determined to be 13 distinct pathotypes. In contrast, four Southeastern isolates of M. medusae could not be distinguished on the same TxD host differentials, although they can be distinguished as pathotypes using P. deltoides differentials. The first three pathotypes of M. xcolumbiana (Mxc1, Mxc2, and Mxc3) and a Mississippi isolate of M. medusae were inoculated onto a three-generation TxD pedigree, formerly used to characterize the Mmd1 gene for resistance to M. medusae. Resistance to the Mxc3 pathotype mapped to the same linkage group (group Q) as the Mmd1 gene. In contrast, linked genes for resistance to Mxc1 and to Mxc2 were located on linkage group O, and were unlike Mmd1 and Mxc3 in that they were inherited from P. deltoides. The latter two genes resembled Mmd1 and Mxc3 in that infection type was correlated with quantitative traits such as uredinial density and latent period. Pathogenic variation in M. xcolumbiana matches resistance genes from both P. trichocarpa and P. deltoides and reveals the vulnerability to hybrid rust of commercial TxD hybrid poplar clones.

Quantitative trait loci and candidate gene mapping of bud set and bud flush in populus.

The genetic control of bud phenology in hybrid poplar was studied by mapping quantitative trait loci (QTL) affecting the timing of autumn bud set and spring bud flush. The founders of the mapping pedigree were collected from widely separated latitudes to maximize segregating variation for dormancy-related traits in the F(2) generation-the female Populus trichocarpa parent is from Washington State (48 degrees N) and the male P. deltoides parent is from Texas (31 degrees N). Bud set and bud flush timing were measured on the F(2) generation in a replicated clonal field trial. Using a linkage map constructed of AFLP and microsatellite markers, three QTL controlling bud set and six QTL controlling bud flush were detected. Additionally, five candidate genes believed to be involved in perception of photoperiod (PHYB1, PHYB2) or transduction of abscisic acid response signals (ABI1B, ABI1D, and ABI3) were placed on the QTL map. PHYB2 and ABI1B were found to be coincident with QTL affecting bud set and bud flush.