Evolvability and trait function predict phenotypic divergence of plant populations.

Understanding the causes and limits of population divergence in phenotypic traits is a fundamental aim of evolutionary biology, with the potential to yield predictions of adaptation to environmental change. Reciprocal transplant experiments and the evaluation of optimality models suggest that local adaptation is common but not universal, and some studies suggest that trait divergence is highly constrained by genetic variances and covariances of complex phenotypes. We analyze a large database of population divergence in plants and evaluate whether evolutionary divergence scales positively with standing genetic variation within populations (evolvability), as expected if genetic constraints are evolutionarily important. We further evaluate differences in divergence and evolvability-divergence relationships between reproductive and vegetative traits and between selfing, mixed-mating, and outcrossing species, as these factors are expected to influence both patterns of selection and evolutionary potentials. Evolutionary divergence scaled positively with evolvability. Furthermore, trait divergence was greater for vegetative traits than for floral (reproductive) traits, but largely independent of the mating system. Jointly, these factors explained ~40% of the variance in evolutionary divergence. The consistency of the evolvability-divergence relationships across diverse species suggests substantial predictability of trait divergence. The results are also consistent with genetic constraints playing a role in evolutionary divergence.

Redefining hyperviscosity in acute leukemia: Potential implications for red cell transfusions in the microvasculature.

Hyperleukocytosis is an emergency of acute leukemia leading to blood hyperviscosity, potentially resulting in life-threatening microvascular obstruction, or leukostasis. Due to the high number of red cells in the circulation, hematocrit/hemoglobin levels (Hct/Hgb) are major drivers of blood viscosity, but how Hct/Hgb mediates hyperviscosity in acute leukemia remains unknown. In vivo hemorheological studies are difficult to conduct and interpret due to issues related to visualizing and manipulating the microvasculature. To that end, a multi-vessel microfluidic device recapitulating the size-scale and geometry of the microvasculature was designed to investigate how Hct/Hgb interacts with acute leukemia to induce "in vitro" leukostasis. Using patient samples and cell lines, the degree of leukostasis was different among leukemia immunophenotypes with respect to white blood cell (WBC) count and Hct/Hgb. Among lymphoid immunophenotypes, severe anemia is protective against in vitro leukostasis and Hct/Hgb thresholds became apparent above which in vitro leukostasis significantly increased, to a greater extent with B-cell acute lymphoblastic leukemia (ALL) versus T-cell ALL. In vitro leukostasis in acute myeloid leukemia was primarily driven by WBC with little interaction with Hct/Hgb. This sets the stage for prospective clinical studies assessing how red cell transfusion may affect leukostasis risk in immunophenotypically different acute leukemia patients.

Simulated pollinator decline has similar effects on seed production of female and hermaphrodite Lobelia siphilitica, but different effects on selection on floral traits.

PREMISE: Pollinator decline, by reducing seed production, is predicted to strengthen natural selection on floral traits. However, the effect of pollinator decline on gender dimorphic species (such as gynodioecious species, where plants produce female or hermaphrodite flowers) may differ between the sex morphs: if pollinator decline reduces the seed production of females more than hermaphrodites, then it should also have a larger effect on selection on floral traits in females than in hermaphrodites. METHODS: To simulate pollinator decline, we experimentally reduced pollinator access to female and hermaphrodite Lobelia siphilitica plants. We compared the seed production of plants in the reduced pollination treatment to plants that were exposed to ambient pollination conditions. Within each treatment, we also measured directional selection on four floral traits of females and hermaphrodites. RESULTS: Experimentally reducing pollination decreased seed production of both females and hermaphrodites by ~21%. Reducing pollination also strengthened selection on floral traits, but this effect was not larger in females than in hermaphrodites. Instead, reducing pollination intensified selection for taller inflorescences in hermaphrodites, but did not intensify selection on any floral trait in females. CONCLUSIONS: Our results suggest that pollinator decline will not have a larger effect on either seed production or selection on floral traits of female plants. As such, any effect of pollinator decline on seed production may be similar for gender dimorphic and monomorphic species. However, the potential for floral traits of females (and thus of gender dimorphic species) to evolve in response to pollinator decline may be limited.

Label-free hematology analysis using deep-ultraviolet microscopy.

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.

Global gene flow releases invasive plants from environmental constraints on genetic diversity.

When plants establish outside their native range, their ability to adapt to the new environment is influenced by both demography and dispersal. However, the relative importance of these two factors is poorly understood. To quantify the influence of demography and dispersal on patterns of genetic diversity underlying adaptation, we used data from a globally distributed demographic research network comprising 35 native and 18 nonnative populations of Plantago lanceolata Species-specific simulation experiments showed that dispersal would dilute demographic influences on genetic diversity at local scales. Populations in the native European range had strong spatial genetic structure associated with geographic distance and precipitation seasonality. In contrast, nonnative populations had weaker spatial genetic structure that was not associated with environmental gradients but with higher within-population genetic diversity. Our findings show that dispersal caused by repeated, long-distance, human-mediated introductions has allowed invasive plant populations to overcome environmental constraints on genetic diversity, even without strong demographic changes. The impact of invasive plants may, therefore, increase with repeated introductions, highlighting the need to constrain future introductions of species even if they already exist in an area.

Plasticity in floral longevity and sex-phase duration of Lobelia siphilitica in response to simulated pollinator declines.

PREMISE: Pollinator declines can reduce the quantity and quality of pollination services, resulting in less pollen deposited on flowers and lower seed production by plants. In response to these reductions, plant species that cannot autonomously self-pollinate and thus are dependent on pollinators to set seed could plastically adjust their floral traits. Such plasticity could increase the opportunity for outcross pollination directly, as well as indirectly by affecting inflorescence traits. METHODS: To test whether plants can respond to pollinator declines by plastically adjusting their floral traits, we simulated declines by experimentally reducing pollinator access to Lobelia siphilitica plants and measuring two traits of early- and late-season flowers: (1) floral longevity; and (2) sex-phase duration. To test whether plasticity in these floral traits affected inflorescence traits, we measured daily display size and phenotypic gender. RESULTS: We found that experimentally reducing pollination did not affect female-phase duration, but did extend the male-phase duration of early-season flowers by 13% and the longevity of late-season flowers by 12.8%. However, plants with an extended male phase did not have a more male-biased phenotypic gender, and plants with an extended floral longevity did not have a larger daily display. CONCLUSIONS: Our results suggest that plants can respond to pollinator declines by plastically adjusting both the longevity and sex-phase duration of their flowers. If this plasticity increases the opportunity for outcross pollination, then it could be one mechanism by which pollinator-dependent plant species maintain seed production as pollinators decline.

Phenotypic plasticity masks range-wide genetic differentiation for vegetative but not reproductive traits in a short-lived plant.

Genetic differentiation and phenotypic plasticity jointly shape intraspecific trait variation, but their roles differ among traits. In short-lived plants, reproductive traits may be more genetically determined due to their impact on fitness, whereas vegetative traits may show higher plasticity to buffer short-term perturbations. Combining a multi-treatment greenhouse experiment with observational field data throughout the range of a widespread short-lived herb, Plantago lanceolata, we (1) disentangled genetic and plastic responses of functional traits to a set of environmental drivers and (2) assessed how genetic differentiation and plasticity shape observational trait-environment relationships. Reproductive traits showed distinct genetic differentiation that largely determined observational patterns, but only when correcting traits for differences in biomass. Vegetative traits showed higher plasticity and opposite genetic and plastic responses, masking the genetic component underlying field-observed trait variation. Our study suggests that genetic differentiation may be inferred from observational data only for the traits most closely related to fitness.

Point-of-Care Diagnostic Assays and Novel Preclinical Technologies for Hemostasis and Thrombosis.

Hemostasis is a complex wound-healing process involving numerous mechanical and biochemical mechanisms and influenced by many factors including platelets, coagulation factors, and endothelial components. Slight alterations in these mechanisms can lead to either prothrombotic or bleeding consequences, and such hemostatic imbalances can lead to significant clinical consequences with resultant morbidity and mortality. An ideal hemostasis assay would not only address all the unique processes involved in clot formation and resolution but also take place under flow conditions to account for endothelial involvement. Global assays do exist; however, these assays are not flow based. Flow-based assays have been limited secondary to their large blood volume requirements and low throughput, limiting potential clinical applications. Microfluidic-based assays address the aforementioned limitations of both global and flow-based assays by utilizing standardized devices that require low blood volumes, offer reproducible analysis, and have functionality under a range of shear stresses and flow conditions. While still largely confined to the preclinical space, here we aim to discuss these novel technologies and potential clinical implications, particularly in comparison to the current, commercially available point-of-care assays.

Defence by duplication: The relation between phenotypic glyphosate resistance and EPSPS gene copy number variation in Amaranthus palmeri.

Gene copy number variation (CNV) has been increasingly associated with organismal responses to environmental stress, but we know little about the quantitative relation between CNV and phenotypic variation. In this study we quantify the relation between variation in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) copy number using digital drop PCR and variation in phenotypic glyphosate resistance in 22 populations of Amaranthus palmeri (Palmer Amaranth), a range-expanding agricultural weed. Overall, we detected a significant positive relation between population mean copy number and resistance. The majority of populations exhibited high glyphosate resistance yet maintained low-resistance individuals, resulting in bimodality in many populations. We also investigated threshold models for the relation between copy number and resistance, and found evidence for a threshold of ~15 EPSPS copies: there was a steep increase in resistance below the threshold, followed by a much shallower increase. Across 924 individuals, as copy number increased the range of variation in resistance decreased, yielding an increasing frequency of high phenotypic resistance individuals. Among populations we detected a decline in variation (s.d.) as mean phenotypic resistance increased from moderate to high, consistent with the prediction that as phenotypic resistance increases in populations, stabilizing selection decreases variation in the trait. Our study demonstrates that populations of A. palmeri can harbour wide variation in EPSPS copy number and phenotypic glyphosate resistance, reflecting the history of, and template for future, resistance evolution.

Simulated pollinator declines intensify selection on floral traits that facilitate selfing and outcrossing in Impatiens capensis.

PREMISE: Anthropogenic environmental change is causing pollinator populations to decline. These declines should intensify selection for floral traits that facilitate outcrossing by making plants more attractive to pollinators and/or for floral traits that facilitate selfing in the absence of pollinators. However, the effect of pollinator declines on selection on floral traits could be modified by other environmental factors such as herbivores. METHODS: We studied the effect of simulated pollinator declines on selection on floral traits of Impatiens capensis, a mixed-mating species that produces both obligately selfing cleistogamous flowers and primarily outcrossing chasmogamous flowers. We measured directional selection differentials via seeds per plant on two traits that facilitate outcrossing (chasmogamous flower size and number) and one trait that facilitates selfing (cleistogamous flower number) in ambient, reduced pollinator access, and reduced pollinator access combined with increased foliar herbivory treatments. RESULTS: Reduced pollinator access intensified selection for larger chasmogamous flowers and more cleistogamous flowers. In contrast, increased herbivory did not affect selection on any floral trait. CONCLUSIONS: Reduced pollinator access intensified selection for a trait that facilitates outcrossing, suggesting that even species such as I. capensis that can autonomously self-pollinate have the potential to respond to pollinator declines by evolving floral traits that reinforce interactions between plants and pollinators. However, reduced pollinator access also intensified selection for a trait that facilitates selfing, suggesting that I. capensis could adapt to pollinator declines by evolving floral traits that maintain the production of both selfed and outcrossed seeds.

Beyond balancing selection: frequent mitochondrial recombination contributes to high-female frequencies in gynodioecious Lobelia siphilitica (Campanulaceae).

Gynodioecy is a sexual system in which females and hermaphrodites co-occur. In most gynodioecious angiosperms, sex is determined by an interaction between mitochondrial male-sterility genes (CMS) that arise via recombination and nuclear restorer alleles that evolve to suppress them. In theory, gynodioecy occurs when multiple CMS types are maintained at equilibrium frequencies by balancing selection. However, some gynodioecious populations contain very high frequencies of females. High female frequencies are not expected under balancing selection, but could be explained by the repeated introduction of novel CMS types. To test for balancing selection and/or the repeated introduction of novel CMS, we characterised cytoplasmic haplotypes from 61 populations of Lobelia siphilitica that vary widely in female frequency. We confirmed that mitotype diversity and female frequency were positively correlated across populations, consistent with balancing selection. However, while low-female populations hosted mostly common mitotypes, high-female populations and female plants hosted mostly rare, recombinant mitotypes likely to carry novel CMS types. Our results suggest that balancing selection maintains established CMS types across this species, but extreme female frequencies result from frequent invasion by novel CMS types. We conclude that balancing selection alone cannot account for extreme population sex-ratio variation within a gynodioecious species.

Integrated automated particle tracking microfluidic enables high-throughput cell deformability cytometry for red cell disorders.

Investigating individual red blood cells (RBCs) is critical to understanding hematologic diseases, as pathology often originates at the single-cell level. Many RBC disorders manifest in altered biophysical properties, such as deformability of RBCs. Due to limitations in current biophysical assays, there exists a need for high-throughput analysis of RBC deformability with single-cell resolution. To that end, we present a method that pairs a simple in vitro artificial microvasculature network system with an innovative MATLAB-based automated particle tracking program, allowing for high-throughput, single-cell deformability index (sDI) measurements of entire RBC populations. We apply our technology to quantify the sDI of RBCs from healthy volunteers, Sickle cell disease (SCD) patients, a transfusion-dependent beta thalassemia major patient, and in stored packed RBCs (pRBCs) that undergo storage lesion over 4 weeks. Moreover, our system can also measure cell size for each RBC, thereby enabling 2D analysis of cell deformability vs cell size with single cell resolution akin to flow cytometry. Our results demonstrate the clear existence of distinct biophysical RBC subpopulations with high interpatient variability in SCD as indicated by large magnitude skewness and kurtosis values of distribution, the "shifting" of sDI vs RBC size curves over transfusion cycles in beta thalassemia, and the appearance of low sDI RBC subpopulations within 4 days of pRBC storage. Overall, our system offers an inexpensive, convenient, and high-throughput method to gauge single RBC deformability and size for any RBC population and has the potential to aid in disease monitoring and transfusion guidelines for various RBC disorders.

Two days of calorie deprivation impairs high level cognitive processes, mood, and self-reported exertion during aerobic exercise: A randomized double-blind, placebo-controlled study.

Military personnel and emergency responders perform cognitively-demanding tasks during periods of sustained physical exertion and limited caloric intake. Cognitive function is preserved during short-term caloric restriction, but it is unclear if preservation extends to combined caloric restriction and physical exertion. According to the "reticular-activating hypofrontality" model, vigorous exertion impairs prefrontal cortex activity and associated functions. This double-blind, placebo-controlled, crossover study examined cognitive function during sustained exertion while volunteers were calorically-deprived. Twenty-three volunteers were calorie-depleted for two days on one occasion and fully-fed on another. They completed intermittent bouts of exercise at 40-65% VO2peak while prefrontal cortex-dependent tasks of cognitive control, mood, and perceived exertion were assessed. Calorie deprivation impaired accuracy on the task-switching task of set-shifting (p < .01) and decreased sensitivity on the go/no-go task of response inhibition (p < .05). Calorie deprivation did not affect risk taking on the Rogers risk task. During exercise, calorie deprivation, particularly on day 2, increased perceived exertion (p < .05) and impaired mood states of tension, depression, anger, vigor, fatigue, and confusion (all p < .01). Physical exertion during severe calorie deprivation impairs cognitive control, mood, and self-rated exertion. Reallocation of cerebral metabolic resources from the prefrontal cortex to structures supporting movement may explain these deficits.

What Are the Environmental Determinants of Phenotypic Selection? A Meta-analysis of Experimental Studies.

Although many selection estimates have been published, the environmental factors that cause selection to vary in space and time have rarely been identified. One way to identify these factors is by experimentally manipulating the environment and measuring selection in each treatment. We compiled and analyzed selection estimates from experimental studies. First, we tested whether the effect of manipulating the environment on selection gradients depends on taxon, trait type, or fitness component. We found that the effect of manipulating the environment was larger when selection was measured on life-history traits or via survival. Second, we tested two predictions about the environmental factors that cause variation in selection. We found support for the prediction that variation in selection is more likely to be caused by environmental factors that have a large effect on mean fitness but not for the prediction that variation is more likely to be caused by biotic factors. Third, we compared selection gradients from experimental and observational studies. We found that selection varied more among treatments in experimental studies than among spatial and temporal replicates in observational studies, suggesting that experimental studies can detect relationships between environmental factors and selection that would not be apparent in observational studies.

Physiological effects of temperature do not explain prevalence of females in populations of gynodioecious Lobelia siphilitica growing in warmer climates.

PREMISE OF THE STUDY: Gynodioecy is a sexual polymorphism whereby female and hermaphroditic plants co-occur within populations. In many gynodioecious species, stressful abiotic environments are associated with higher frequencies of females. This association suggests that abiotic stress affects the relative fitness of females and hermaphrodites and, thus, the maintenance of gynodioecy. METHODS: To test whether abiotic stress affects the fitness of females and hermaphrodites, we grew open-pollinated Lobelia siphilitica families in temperature regimes characteristic of the southern portion of the species' range (where females are common) and the northern portion of the range (where females are rare). We measured physiological and phenological traits that are indicative of heat stress, and fitness components of females and hermaphrodites that could affect the maintenance of gynodioecy. KEY RESULTS: Contrary to expectations if growth at high temperatures is stressful, we found that the hot treatment increased leaf chlorophyll content, decreased the percentage of plants that delayed flowering initiation, and did not affect the quantum efficiency of photosystem II. Growth at high temperatures did not affect the magnitude of the difference in rosette size (a correlate of flower number) between females and hermaphrodites, or the variance in pollen viability among hermaphrodites. CONCLUSIONS: We found that growing-season temperatures typical of high female L. siphilitica populations were not stressful and did not affect either the fitness of females compared to hermaphrodites or variation in fitness among hermaphrodites. Consequently, further research is necessary to explain correlations between abiotic environmental factors and the frequency of females in this and other gynodioecious species.

Why is gynodioecy a rare but widely distributed sexual system? Lessons from the Lamiaceae.

Gynodioecy, a sexual system where females and hermaphrodites co-occur, is found in << 1% of angiosperm species. To understand why gynodioecy is rare, we need to understand why females are maintained in some lineages, but not in others. We modelled the evolution of gynodioecy in the Lamiaceae, and investigated whether transition rates between gynodioecious and nongynodioecious states varied across the family. We also investigated whether the evolution of gynodioecy was correlated with the evolution of a herbaceous growth form and temperate distribution. Transition rates differed between Lamiaceae subfamilies. In the Nepetoideae, there were many transitions towards gynodioecy (n = 11), but also many reversions to nongynodioecy (n = 29). In addition, a herbaceous growth form, but not a temperate distribution, affected the rate of transitions both towards and away from gynodioecy; transitions towards gynodioecy occurred ˜16 times more frequently and transitions away from gynodioecy occurred ˜11 times more frequently in herbaceous lineages than in woody lineages. Within the Lamiaceae, lineages in which gynodioecy has frequently evolved also have a high rate of reversions to the nongynodioecious state. Consequently, to understand why gynodioecy is rare, we need to understand why sexual systems are more evolutionarily labile in some lineages than in others.

Long-term effectiveness and generality of practical functional assessment and skill-based treatment.

There are several considerations to address when conducting functional communication training for challenging behavior in a school setting, such as the need for schedule thinning and maintenance across staff and the need to establish a variety of appropriate classroom skills. There are several strategies for conducting schedule thinning following functional communication training and for transferring effects across people or settings. However, there are few examples of these processes in natural settings with relevant caregivers and with long-term maintenance of effects. We implemented a functional assessment and skill-based treatment process with six children with autism in a specialized school setting and extended treatment until challenging behavior was reduced to near-zero levels across multiple staff and settings. Follow-up data indicate that effects were still observed 1 year posttreatment and the use of crisis procedures (e.g., physical restraint) was eliminated for all participants.

The effect of experimental pollinator decline on pollinator-mediated selection on floral traits.

Human-mediated environmental change, by reducing mean fitness, is hypothesized to strengthen selection on traits that mediate interactions among species. For example, human-mediated declines in pollinator populations are hypothesized to reduce mean seed production by increasing the magnitude of pollen limitation and thus strengthen pollinator-mediated selection on floral traits that increase pollinator attraction or pollen transfer efficiency. To test this hypothesis, we measured two female fitness components and six floral traits of Lobelia siphilitica plants exposed to supplemental hand-pollination, ambient open-pollination, or reduced open-pollination treatments. The reduced treatment simulated pollinator decline, while the supplemental treatment was used to estimate pollen limitation and pollinator-mediated selection. We found that plants in the reduced pollination treatment were significantly pollen limited, resulting in pollinator-mediated selection for taller inflorescences and more vibrant petals, both traits that could increase pollinator attraction. This contrasts with plants in the ambient pollination treatment, where reproduction was not pollen limited and there was not significant pollinator-mediated selection on any floral trait. Our results support the hypothesis that human-mediated environmental change can strengthen selection on traits of interacting species and suggest that these traits have the potential to evolve in response to changing environments.