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Background & Aims: Many liver diseases are driven by inflammation, but imaging to non-invasively diagnose and quantify liver inflammation has been underdeveloped. The inflammatory liver microenvironment is aberrantly oxidising owing in part to reactive oxygen species generated by myeloid leucocytes. We hypothesised that magnetic resonance imaging using the oxidatively activated probe Fe-PyC3A will provide a non-invasive biomarker of liver inflammation., Methods: A mouse model of drug-induced liver injury was generated through intraperitoneal injection of a hepatoxic dose of acetaminophen. A mouse model of steatohepatitis was generated via a choline-deficient, l-amino acid defined high-fat diet (CDAHFD). Images were acquired dynamically before and after intravenous injection of Fe-PyC3A. The contrast agent gadoterate meglumine was used as a non-oxidatively activated negative control probe in mice fed CDAHFD. The (post-pre) Fe-PyC3A injection change in liver vs. muscle contrast-to-noise ratio (ΔCNR) recorded 2 min post-injection was correlated with liver function test values, histologic scoring assigned using the NASH Clinical Research Network criteria, and intrahepatic myeloid leucocyte composition determined by flow cytometry., Results: For mice receiving i.p. injections of acetaminophen, intrahepatic neutrophil composition correlated poorly with liver test values but positively and significantly with ΔCNR (r = 0.64, p <0.0001). For mice fed CDAHFD, ΔCNR generated by Fe-PyC3A in the left lobe was significantly greater in mice meeting histologic criteria strongly associated with a diagnosis NASH compared to mice where histology was consistent with likely non-NASH ( p  = 0.0001), whereas no differential effect was observed using gadoterate meglumine. In mice fed CDAHFD, ΔCNR did not correlate strongly with fractional composition of any specific myeloid cell subpopulation as determined by flow cytometry., Conclusions: Magnetic resonance imaging using Fe-PyC3A merits further evaluation as a non-invasive biomarker for liver inflammation., Impact and Implications: Non-invasive tests to diagnose and measure liver inflammation are underdeveloped. Inflammatory cells such as neutrophils release reactive oxygen species which creates an inflammatory liver microenvironment that can drive chemical oxidation. We recently invented a new class of magnetic resonance imaging probe that is made visible to the scanner only after chemical oxidation. Here, we demonstrate how this imaging technology could be applied as a non-invasive biomarker for liver inflammation., Competing Interests: EMG holds equity in, and receives consulting income from Reveal Pharmaceuticals, and receives consulting income from Collagen Medical, LLC. PC holds equity in, and receives consulting income from Collagen Medical LLC, holds equity in Reveal Pharmaceuticals, and has research funding from Takeda, Pliant Therapeutics, and Janssen. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Authors.)

Background: Health care accreditation is a widely accepted mechanism for improving the quality of care and promoting patient safety. An integral dimension of health care quality is the patient experience of care. However, the influence of accreditation on the patient experience is unclear. The Home Health Consumer Assessment of Healthcare Providers and Systems (HHCAHPS) survey is the standard for collecting patient care experience data in the home health setting. The aim of this study was to examine the association of Joint Commission accreditation on patients' experience of care by comparing HHCAHPS ratings from Joint Commission-accredited and non-Joint Commission-accredited home health agencies (HHAs)., Methods: This multiyear observational study used 2015-2019 HHCAHPS data obtained from the Centers for Medicare & Medicaid Services (CMS) website and Joint Commission databases. The data set included 1,454 (23.8%) Joint Commission-accredited and 4,643 (76.2%) non-Joint Commission-accredited HHAs. Dependent variables included three composite measures of care (Care of Patients, Provider-Patient Communications, and Specific Care Issues) and two global rating measures. Data were analyzed using a series of longitudinal random effects logistic regression models., Results: This study found no association between Joint Commission accreditation and the two global HHCAHPS measures, modest significant increases for Joint Commission-accredited HHAs in measure rates for the Care of Patients and Communication composite measures (p < 0.05), and a more significant increase for the Specific Care Issues composite measure related to medication safety and home safety (p < 0.001)., Conclusions: These findings suggest that Joint Commission accreditation may be positively associated with some patient experience of care outcomes. This relationship was most pronounced when there was significant overlap between the focus of the accreditation standards and focus of the HHCAHPS items., (Copyright © 2023 The Joint Commission. Published by Elsevier Inc. All rights reserved.)

SNIO-CBP, a single-nanometer iron oxide (SNIO) nanoparticle functionalized with a type I collagen-binding peptide (CBP), was developed as a T 1 -weighted MRI contrast agent with only endogenous elements for fast and noninvasive detection of liver fibrosis. SNIO-CBP exhibits 6.7-fold higher relaxivity compared to a molecular gadolinium-based collagen-binding contrast agent CM-101 on a per CBP basis at 4.7 T. Unlike most iron oxide nanoparticles, SNIO-CBP exhibits fast elimination from the bloodstream with a 5.7 min half-life, high renal clearance, and low, transient liver enhancement in healthy mice. We show that a dose of SNIO-CBP that is 2.5-fold lower than that for CM-101 has comparable imaging efficacy in rapid (within 15 min following intravenous injection) detection of hepatotoxin-induced liver fibrosis using T 1 -weighted MRI in a carbon tetrachloride-induced mouse liver injury model. We further demonstrate the applicability of SNIO-CBP in detecting liver fibrosis in choline-deficient L -amino acid-defined high-fat diet mouse model of nonalcoholic steatohepatitis. These results provide a platform with potential for the development of high relaxivity, gadolinium-free molecular MRI probes for characterizing chronic liver disease.

Liver fibrosis plays a critical role in the evolution of most chronic liver diseases and is characterized by a buildup of extracellular matrix, which can progress to cirrhosis, hepatocellular carcinoma, liver failure, or death. Now, there are no noninvasive methods available to accurately assess disease activity (fibrogenesis) to sensitively detect early onset of fibrosis or to detect early response to treatment. Here, we hypothesized that extracellular allysine aldehyde (Lys Ald ) pairs formed by collagen oxidation during active fibrosis could be a target for assessing fibrogenesis with a molecular probe. We showed that molecular magnetic resonance imaging (MRI) using an extracellular probe targeting these Lys Ald pairs acts as a noninvasive biomarker of fibrogenesis and demonstrated its high sensitivity and specificity in detecting fibrogenesis in toxin- and dietary-induced mouse models, a cholestasis rat model of liver fibrogenesis, and in human fibrotic liver tissues. Quantitative molecular MRI was highly correlated with fibrogenesis markers and enabled noninvasive detection of early onset fibrosis and response to antifibrotic treatment, showing high potential for clinical translation.

Liver fibrogenesis is accompanied by upregulation of lysyl oxidase enzymes, which catalyze oxidation of lysine ε-amino groups on the extracellular matrix proteins to form the aldehyde containing amino acid allysine (Lys Ald ). Here, we describe the design and synthesis of novel manganese-based MRI probes with high signal amplification for imaging liver fibrogenesis. Rational design of a series of stable hydrazine-equipped manganese MRI probes gives Mn-2CHyd with the highest affinity and turn-on relaxivity (4-fold) upon reaction with Lys Ald . A dynamic PET-MRI study using [ 52 Mn]Mn-2CHyd showed low liver uptake of the probe in healthy mice. The ability of the probe to detect liver fibrogenesis was then demonstrated in vivo in CCl 4 -injured mice. This study enables further development and application of manganese-based hydrazine-equipped probes for imaging liver fibrogenesis.

Altered host-microbe interactions and increased intestinal permeability have been implicated in disease pathogenesis. However, the mechanisms by which intestinal microbes affect epithelial barrier integrity remain unclear. Here, we investigate the impact of bacterial metabolism of host-produced bile acid (BA) metabolites on epithelial barrier integrity. We observe that rats fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) exhibit reduced intestinal abundance of host-produced conjugated BAs at early time points, coinciding with increased gut permeability. We show that in vitro, conjugated BAs protect gut epithelial monolayers from damage caused by bacterially produced unconjugated BAs through micelle formation. We then demonstrate that inhibition of bacterial BA deconjugation with a small-molecule inhibitor prevents the development of pathologic intestinal permeability and hepatic inflammation in CDAHFD-fed rats. Our study identifies a signaling-independent, physicochemical mechanism for conjugated BA-mediated protection of epithelial barrier function and suggests that rational manipulation of microbial BA metabolism could be leveraged to regulate gut barrier integrity.

Orthotopic liver transplantation (OLT) is a lifesaving therapy for patients with irreversible liver damage caused by autoimmune liver diseases (AutoD) including autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). Currently, it is unclear how access to transplantation differs among patients with various etiologies of liver disease. Our aim is to evaluate the likelihood of transplant and the long-term patient and graft survival after OLT for each etiology for transplantation from 2000 to 2021. We conducted a large retrospective study of United Network for Organ Sharing (UNOS) liver transplant patients in five 4-year eras with five cohorts: AutoD (PBC, PSC, AIH cirrhosis), alcohol-related liver disease (ALD), hepatocellular carcinoma (HCC), viral hepatitis, and nonalcoholic steatohepatitis (NASH). We conducted a multivariate analysis for probability of transplant. Intent-to-treat (ITT) analysis was performed to assess the 10-year survival differences for each listing diagnosis while accounting for both waitlist and post-transplant survival. Across all eras, autoimmune conditions had a lower adjusted probability of transplant of 0.92 (0.92, 0.93) compared to ALD 0.97 (0.97, 0.97), HCC 1.08 (1.07, 1.08), viral hepatitis 0.99 (0.99, 0.99), and NASH 0.99 (0.99, 1.00). Patients with AutoD had significantly better post-transplant patient and graft survival than ALD, HCC, viral hepatitis, and NASH in each and across all eras (p-values all < 0.001). Patients with AutoD had superior ITT survival (p-value < 0.001, log rank test). In addition, the waitlist survival for patients with AutoD compared to other listing diagnoses was improved with the exception of ALD, which showed no significant difference (p-value = 0.1056, log rank test). Despite a superior 10-year graft and patient survival in patients transplanted for AutoD, patients with AutoD have a significantly lower probability of receiving a liver transplant compared to those transplanted for HCC, ALD, viral hepatitis, and NASH. Patients with AutoD may benefit from improved liver allocation while maintaining superior waitlist and post-transplant survival. Decreased access in spite of appropriate outcomes for patients poses a significant risk for increased morbidity for patients with AutoD.

Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis-induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.

Despite advancements in diabetic care, diabetic kidney transplant recipients have significantly worse outcomes than non-diabetics., Aim: Our study aims to demonstrate the impact of diabetes, types I and II, on American young adults (18-40 years old) requiring kidney transplantation., Methods: Using the United Network for Organ Sharing database, we conducted a population cohort study that included all first-time, kidney-only transplant recipients during 2002-2019, ages 18-40 years old. Patients were grouped according to indication for transplant. Primary outcomes were cumulative all-cause mortality and death-censored graft failure. Death-censored graft failure and patient survival at 1, 5, and 10 years were calculated via the Kaplan-Meier method. Multivariate Cox regression was used to assess for potential confounders., Results: Of 42 466 transplant recipients, 3418 (8.1%) had end-stage kidney disease associated with diabetes. At each time-point, cumulative mortality was higher in diabetics compared to patients with non-diabetic causes of renal failure. Conversely, cumulative graft failure was similar between the groups. Adjusted hazard ratios for all-cause mortality and graft failure in diabetics were 2.99 (95% CI 2.67-3.35; p < .01) and 0.98 (95% CI 0.92-1.05, p < .01), respectively., Conclusion: Diabetes mellitus in young adult kidney transplant recipients is associated with a nearly three-fold increase in mortality, reflecting a relatively vulnerable patient population. Identifying the underlying causes of poor outcomes in this population should be a priority for future study., (© 2022 Asian Pacific Society of Nephrology.)

Widely acknowledged is the disproportionate number of COVID-19 cases among nursing home residents. This observational study examined the relationship between accreditation status and COVID-19 case rates in states where the numbers and proportions of Joint Commission accredited facilities made such comparisons possible (Illinois (IL), Florida (FL), and Massachusetts (MA)). COVID-19 data were accessed from the Centers for Medicare & Medicaid Services (CMS) Nursing Home Compare Public Use File, which included retrospective COVID-19 data submitted by nursing homes to the Centers for Disease Control and Prevention (CDC) National Healthcare Safety Network. The outcome variable was the total number of nursing home-identified COVID-19 cases from June 2020 to January 2021. Joint Commission accreditation status was the independent variable. Mediating factors included state, and county-level case rates. Increases in the county rate had a significant association with higher nursing home COVID-19 case rates ( p  < .001). After adjusting for county case rates, no differences were observed in the mean group case rates for accredited and nonaccredited nursing homes. However, comparing predicted case rates to actual case rates revealed that accredited nursing homes were more closely aligned with their predicted rates. Performance of the nonaccredited nursing homes was more variable and had proportionally more outliers compared to accredited nursing homes. Community prevalence of COVID-19 is the strongest predictor of nursing home cases. While accreditation status did not have an impact on overall mean group performance, nonaccredited nursing homes had greater variation in performance and a higher proportion of negative outliers. Accreditation was associated with more consistent performance during the COVID-19 pandemic, despite being located in counties with a higher prevalence of COVID-19.

Background & Aims: During liver fibrosis, tissue repair mechanisms replace necrotic tissue with highly stabilized extracellular matrix proteins. Extracellular matrix stabilization influences the speed of tissue recovery. Here, we studied the expression and function of peroxidasin (PXDN), a peroxidase that uses hydrogen peroxide to cross-link collagen IV during liver fibrosis progression and regression., Methods: Mouse models of liver fibrosis and cirrhosis patients were analyzed for the expression of PXDN in liver and serum. Pxdn -/- and Pxdn +/+ mice were either treated with carbon tetrachloride for 6 weeks to generate toxin-induced fibrosis or fed with a choline-deficient L-amino acid-defined high-fat diet for 16 weeks to create nonalcoholic fatty liver disease fibrosis. Liver histology, quantitative real-time polymerase chain reaction, collagen content, flowcytometry and immunostaining of immune cells, RNA-sequencing, and liver function tests were analyzed. In vivo imaging of liver reactive oxygen species (ROS) was performed using a redox-active iron complex, Fe-PyC3A., Results: In human and mouse cirrhotic tissue, PXDN is expressed by stellate cells and is secreted into fibrotic areas. In patients with nonalcoholic fatty liver disease, serum levels of PXDN increased significantly. In both mouse models of liver fibrosis, PXDN deficiency resulted in elevated monocyte and pro-fibrolysis macrophage recruitment into fibrotic bands and caused decreased accumulation of cross-linked collagens. In Pxdn -/- mice, collagen fibers were loosely organized, an atypical phenotype that is reversible upon macrophage depletion. Elevated ROS in Pxdn -/- livers was observed, which can result in activation of hypoxic signaling cascades and may affect signaling pathways involved in macrophage polarization such as TNF-a via NF-kB. Fibrosis resolution in Pxdn -/- mice was associated with significant decrease in collagen content and improved liver function., Conclusion: PXDN deficiency is associated with increased ROS levels and a hypoxic liver microenvironment that can regulate recruitment and programming of pro-resolution macrophages. Our data implicate the importance of the liver microenvironment in macrophage programming during liver fibrosis and suggest a novel pathway that is involved in the resolution of scar tissue., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Dysnatremias are a rare but significant event in liver transplantation. While recipient pre-transplant hypernatremia has been demonstrated to increase post-transplant mortality, the degree of hypernatremia and the impact of its resolution have been less well characterized. Here, we used multivariate Cox regression with a comprehensive list of donor and recipient factors in order to conduct a robust multivariate retrospective database study of 54,311 United Network for Organ Sharing (UNOS) liver transplant patients to analyze the effect of pre-transplant serum sodium on post-transplant mortality, post-transplant length of hospitalization, and post-transplant graft survival. Mortality and graft failure increased in a stepwise fashion with increasing pre-transplant hypernatremia: 145 -150 mEq/L (HR = 1.118 and HR = 1.113), 150-155 mEq/L (HR = 1.324 and HR = 1.306), and > 155 mEq/L (HR = 1.623 and HR = 1.661). Pre-transplant hypo- and hypernatremia also increased length of post-transplant hospitalization: < 125 mEq/L (HR = 1.098), 125-130 mEq/L (HR = 1.060), 145 -150 mEq/L (HR = 1.140), and 150-155 mEq/L (HR = 1.358). Resolution of hypernatremia showed no significant difference in mortality compared with normonatremia, while unresolved hypernatremia significantly increased mortality (HR = 1.254), including a durable long-term increased mortality risk for patients with creatinine < 2 mg/dL and MELD < 25. Pre-transplant hypernatremia serves as a morbid prognostic indicator for post-transplant morbidity and mortality., (© 2021 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

Abstract: A 33-year-old man presented with acute painless loss of vision in his right eye after hand-foot-mouth disease (HFMD). Examination confirmed a right optic neuropathy. Neuroimaging and routine evaluations for alternative causes for an optic neuropathy were negative. He was treated with high dose corticosteroids and made an almost complete visual recovery. Postinfectious optic neuritis has been reported after a vast array of infections including: varicella zoster virus, influenza virus, herpes simplex virus, Epstein-Barr Virus, Lyme disease, and many others. Although Coxsackie virus infections are a known cause of HFMD and have been reported to cause maculopathy, to the best of our knowledge, this is the first reported case of optic neuritis after HFMD in the English language ophthalmic literature., Competing Interests: The authors report no conflicts of interest., (Copyright © 2020 by North American Neuro-Ophthalmology Society.)

Background: Aggressive acceptance of liver allografts has driven utilization of marginal allografts. Our aim was to assess the impact of the aggressive phenotype on transplant center outcomes over time., Methods: We used a cohort of 148 361 candidates from the Organ Procurement and Transplantation Network for liver transplantation between 2002 and 2016 in 134 centers. Using the Discard Risk Index, we designated high probability discard allografts by the top 10th percentile for likelihood of discard. Aggressive phenotype was defined by usage of high probability discard (HPD) allografts (top 10th percentile). Our analysis of survival on waitlist and graft survival after transplantation included a comprehensive list of center level covariates across three equal time periods (2002-2006, 2007-2011, and 2012-2016)., Results: After adjusting for recipient and center-level factors, aggressive centers had improving graft survival over time. Aggressive vs non-aggressive centers: 2002-2006 HR 1.12 (1.05-1.19), 2007-2011 HR 1.13 (1.05-1.22), 2012-2016 HR 0.99 (0.89-1.10). Aggressive centers had improved waitlist survival compared with non-aggressive centers after adjusting for allograft disparity., Conclusions: Aggressive phenotype had a positive impact on waitlist survival, and graft survival in aggressive centers have improved to benchmark levels over time. These findings serve as justification for aggressive utilization of allografts., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 μm non-motile cocci, S. aureus deforms smaller than 0.5 μm in the sub-micron channels of the OLCN. Here we present the μSiM-CA (Microfluidic - Silicon Membrane - Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The μSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the μSiM-CA. However the μSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Risk adjustment is critical in the comparison of quality of care and health care outcomes for providers. Electronic health records (EHRs) have the potential to eliminate the need for costly and time-consuming manual data abstraction of patient outcomes and risk factors necessary for risk adjustment., Methods: Leading EHR vendors and hospital focus groups were asked to review risk factors in the New York State (NYS) coronary artery bypass graft (CABG) surgery statistical models for mortality and readmission and assess feasibility of EHR data capture. Risk models based only on registry data elements that can be captured by EHRs (one for easily obtained data and one for data obtained with more difficulty) were developed and compared with the NYS models for different years., Results: Only 6 data elements could be extracted from the EHR, and outlier hospitals differed substantially for readmission but not for mortality. At the patient level, measures of fit and predictive ability indicated that the EHR models are inferior to the NYS CABG surgery risk model [eg, c-statistics of 0.76 vs. 0.71 (P<0.001) and 0.76 vs. 0.74 (P=0.009) for mortality in 2010], although the correlation of the predicted probabilities between the NYS and EHR models was high, ranging from 0.96 to 0.98., Conclusions: A simplified risk model using EHR data elements could not capture most of the risk factors in the NYS CABG surgery risk models, many outlier hospitals were different for readmissions, and patient-level measures of fit were inferior.

Objectives: This study examined the relationship between resident race and immunization status in long-term care facilities (LTCFs). Race was captured at the resident and the facility racial composition level., Design: Thirty-six long-term care facilities varying in racial composition and size were selected for site visits., Setting: LTCFs were urban and rural, CMS certified, and non-hospital administered., Measurements: Chart abstraction was used to determine race, immunization, and refusal status for the 2010-2011 flu season (influenza 1), the 2011-2012 flu season (influenza 2), and the pneumococcal pneumonia vaccine for all residents over 65 years old., Results: Thirty-five LTCFs submitted sufficient data for inclusion, and 2570 resident records were reviewed. Overall immunization rates were 70.5% for influenza 1, 74.1% for influenza 2, and 65.6% for pneumococcal pneumonia. Random effects logistic regression indicated that as the percent of Black residents increased, the immunization rate significantly decreased (immunization 1, p < 0.018, immunization 2, p < 0.002, pneumococcal pneumonia, p = 0.0059), independent of the effect of resident race which had less of an impact on rates., Conclusions: This study found considerable LTCF variation and racial disparities in immunization rates. Compared to Blacks, Whites were vaccinated at higher rates regardless of the LTCF racial composition. Facilities with a greater proportion of Black residents had lower immunization rates than those with primarily White residents. Facility racial mix is a stronger predictor of influenza immunization than resident race. Black residents had significantly higher vaccination refusal rates than White residents for immunization 2. Further studies examining LTCF-level factors that affect racial disparities in immunizations in LTCFs are needed.

The breakdown of heterostyly to homostyly is a classic system for the investigation of evolutionary transitions from outcrossing to selfing. Loss of sexual polymorphism is characterized by changes to population morph structure and floral morphology. Here, we used molecular phylogeography to investigate the geographical context for the breakdown process in Primula chungensis, a species with distylous and homostylous populations. We genotyped plants from 20 populations throughout the entire range in south-west China using the chloroplast intergenic spacer (trnL-trnF), nuclear internal transcribed spacer (ITS) and 10 nuclear microsatellite loci, and determined the genetic relationships among populations and the variation in floral traits associated with homostyle evolution. The marker data identified two multi-population lineages (Tibet and Sichuan) and one single-population lineage (Yunnan), a pattern consistent with at least two independent origins of homostyly. Evidence from flower and pollen size variation is consistent with the hypothesis that transitions to selfing have arisen by the same genetic mechanism involving recombination and/or mutation at the distyly linkage group. Nevertheless, flowers of homostylous lineages have followed divergent evolutionary trajectories following their origin, resulting in populations with both approach and reverse herkogamy. Our study illustrates a rare example of the near-complete replacement of sexual polymorphism by floral monomorphism in a heterostylous species., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

The floral polymorphism tristyly involves three style morphs with a reciprocal arrangement of stigma and anther heights governed by two diallelic loci (S and M). Tristyly functions to promote cross-pollination, but modifications to stamen position commonly cause transitions to selfing. Here, we integrate whole-genome sequencing and genetic mapping to investigate the genetic architecture of the M locus and the genetic basis of independent transitions to selfing in tristylous Eichhornia paniculata. We crossed independently derived semi-homostylous selfing variants of the long- and mid-styled morph fixed for alternate alleles at the M locus (ssmm and ssMM, respectively), and backcrossed the F 1 to the parental ssmm genotype. We phenotyped and genotyped 462 backcross progeny using 1450 genotyping-by-sequencing (GBS) markers and performed composite interval mapping to identify quantitative trait loci (QTL) governing style-length and anther-height variation. A QTL associated with the primary style-morph differences (style length and anther height) mapped to linkage group 5 and spanned ~13-27.5 Mbp of assembled sequence. Bulk segregant analysis identified 334 genes containing SNPs potentially linked to the M locus. The stamen modifications characterizing each selfing variant were governed by loci on different linkage groups. Our results provide an important step towards identifying the M locus and demonstrate that transitions to selfing have originated by independent sets of mating-system modifier genes unlinked to the M locus, a pattern inconsistent with a recombinational origin of selfing variants at a putative supergene., (© 2016 Her Majesty the Queen in Right of Canada Molecular Ecology © 2016 John Wiley & Sons Ltd.)

Premise of the Study: Populations of heterostylous species are characterized by two or three floral morphs with reciprocal positioning of stigmas and anthers. Theoretical models predict equal morph frequencies (isoplethy) when disassortative mating is prevalent in populations, but biased morph ratios may occur when variation in the expression of heterostyly causes deviations from intermorph mating., Methods: We explore the role of sex-organ deployment in governing morph ratios in two closely related genera of Boraginaceae, exhibiting striking variation in floral traits associated with the heterostylous syndrome. We sampled 66 populations of six species of Glandora and 39 populations of three species of Lithodora across their distributional range in the Mediterranean. In each population we estimated morph ratios and measured several floral traits. We used phylogenetically corrected and noncorrected regressions to test the hypothesis that differences in sex-organ reciprocity and herkogamy are associated with deviations from isoplethy., Key Results: Biased morph ratios occurred in 24% of populations, particularly in Lithodora. Populations biased for the long-styled morph (L-morph) were more frequent than the short-styled morph (S-morph). Distylous species were less likely to exhibit biased ratios than species with stigma-height dimorphism. In Lithodora fruticosa, a species lacking reciprocity, decreased herkogamy in the S-morph was associated with increasing L-morph bias, perhaps resulting from self-interference., Conclusion: Striking variation in the expression of heterostyly in Glandora and Lithodora is associated with biased morph ratios, which probably result from pollinator-mediated mating asymmetries within populations., (© 2017 Botanical Society of America.)

Most heterostylous plants possess a reciprocal arrangement of stigmas and anthers (reciprocal herkogamy), heteromorphic self-incompatibility, and ancillary polymorphisms of pollen and stigmas. The topographical complementarity hypothesis proposes that ancillary polymorphisms function in the rejection of incompatible pollen thus promoting disassortative pollination. Here, we test this hypothesis by investigating patterns of pollen transfer and capture in populations of dimorphic Armeria maritima and A. pubigera and distylous Limonium vulgare (Plumbaginaceae), and by studying pollen adherence and germination patterns in A. maritima following controlled hand-pollinations. Armeria lacks reciprocal herkogamy allowing the evaluation of the extent to which ancillary polymorphisms affect the composition of pollen loads. We compared the amounts of compatible and incompatible pollen on stigmas in natural populations and calculated the proficiencies of pollen transfer for each mating type. We detected disassortative pollination in each species, and mating types did not differ in compatible pollen capture, although cob stigmas captured more incompatible pollen. Controlled hand-pollinations revealed the failure of incompatible pollen to adhere and germinate on stigmas. Our results provided evidence that, while structural in nature, pollen-stigma dimorphisms are tightly associated with heteromorphic incompatibility and likely function to promote disassortative pollination, especially in the absence of reciprocal herkogamy., (© 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.)

Mating patterns and natural selection play important roles in determining whether genetic polymorphisms are maintained or lost. Here, we document an atypical population of Lapeirousia anceps (Iridaceae) with a bimodal distribution of floral-tube length and investigate the reproductive mechanisms associated with this pattern of variation. Flowers were visited exclusively by the long-proboscid fly Moegistorhynchus longirostris (Nemestrinidae), which exhibited a unimodal distribution of proboscis length and displayed a preference for long-tubed phenotypes. Despite being visited by a single pollinator species, allozyme markers revealed significant genetic differentiation between open-pollinated progeny of long- and short-tubed phenotypes suggesting mating barriers between them. We obtained direct evidence for mating barriers between the floral-tube phenotypes through observations of pollinator foraging, controlled hand pollinations and measurements of pollen competition and seed set. Intermediate tube-length phenotypes produced fewer seeds in the field than either long- or short-tubed phenotypes. Although floral-tube length bimodality may not be a stable state over long timescales, reproductive barriers to mating and low 'hybrid' fitness have the potential to contribute to the maintenance of this state in the short term., (© 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.)

The evolution of predominant self-fertilization from cross-fertilization in plants is accompanied by diverse changes to morphology, ecology and genetics, some of which likely result from regulatory changes in gene expression. We examined changes in gene expression during early stages in the transition to selfing in populations of animal-pollinated Eichhornia paniculata with contrasting mating patterns. We crossed plants from outcrossing and selfing populations and tested for the presence of allele-specific expression (ASE) in floral buds and leaf tissue of F1 offspring, indicative of cis-regulatory changes. We identified 1365 genes exhibiting ASE in floral buds and leaf tissue. These genes preferentially expressed alleles from outcrossing parents. Moreover, we found evidence that genes exhibiting ASE had a greater nonsynonymous diversity compared to synonymous diversity in the selfing parents. Our results suggest that the transition from outcrossing to high rates of self-fertilization may have the potential to shape the cis-regulatory genomic landscape of angiosperm species, but that the changes in ASE may be moderate, particularly during the early stages of this transition., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

The stable coexistence within populations of females, males, and hermaphrodites (subdioecy) is enigmatic because theoretical models indicate that maintenance of this sexual system involves highly restricted conditions. Subdioecy is more commonly interpreted as a transitory stage along the gynodioecious pathway from hermaphroditism to dioecy. The widespread, North American, aquatic plant Sagittaria latifolia is largely composed of monoecious or dioecious populations; however, subdioecious populations with high frequencies of hermaphrodites (mean frequency = 0.50) characterize the northern range boundary of dioecy in eastern North America. We investigated two hypotheses for the origin of subdioecy in this region. Using polymorphic microsatellite loci, we evaluated whether subdioecy arises through selection on standing genetic variation for male sex inconstancy in dioecious populations, or results from hybridization between monoecious and dioecious populations. We found evidence for both pathways to subdioecy, although hybridization was the more common mechanism, with genetic evidence of admixture in nine of 14 subdioecious populations examined. Hybridization has also played a role in the origin of androdioecious populations in S. latifolia, a mechanism not often considered in the evolution of this rare sexual system. Our study demonstrates how hybridization has the potential to play a role in the diversification of plant sexual systems., (© 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.)

Premise of the Study: Variation in the mating system of hermaphroditic plant populations is determined by interactions between genetic and environmental factors operating via both pre- and postmating processes. Models predicting the maintenance of intermediate outcrossing rates in animal-pollinated plants often assume that the mating system is primarily controlled by floral morphology and pollinator availability, but rarely has the influence of postpollination processes on variation in outcrossing been examined., Methods: We investigated the influence of stylar discrimination between illegitimate and legitimate pollen-tube growth and the pollen-load capacity of stigmas on mating-system variation in the annual, tristylous species Eichhornia paniculata using controlled crosses and genetic markers. This species exhibits an exceptionally broad range of outcrossing rates in natural populations., Key Results: There was significant variation among populations in the pollen-load capacity of stigmas and the ability of styles to discriminate between illegitimate vs. legitimate pollen. There was strong correspondence between stylar-discrimination ability and variation in outcrossing rate among populations and style morphs. The combination of stigmatic pollen-load capacity and stylar discrimination explained more than 80% of the variation in outcrossing rates among populations., Conclusions: The finding that stigmatic pollen-load capacity and stylar-discrimination ability contributed significantly to explaining the wide range of outcrossing rates in E. paniculata suggests that postpollination mechanisms play an important role in governing mating patterns in this species. The difference in levels of stylar discrimination between outcrossing and selfing populations may reflect a trade-off between selection for increased outcrossing and greater reproductive assurance., (© 2016 Botanical Society of America.)

Background and Aims: Flowering plants display considerable variation in mating system, specifically the relative frequency of cross- and self-fertilization. The majority of estimates of outcrossing rate do not account for temporal variation, particularly during the flowering season. Here, we investigated seasonal variation in mating and fertility in Incarvillea sinensis (Bignoniaceae), an annual with showy, insect-pollinated, 'one-day' flowers capable of delayed selfing. We examined the influence of several biotic and abiotic environmental factors on day-to-day variation in fruit set, seed set and patterns of mating., Methods: We recorded daily flower number and pollinator abundance in nine 3 × 3-m patches in a population at Mu Us Sand land, Inner Mongolia, China. From marked flowers we collected data on daily fruit and seed set and estimated outcrossing rate and biparental inbreeding using six microsatellite loci and 172 open-pollinated families throughout the flowering period., Key Results: Flower density increased significantly over most of the 50-d flowering season, but was associated with a decline in levels of pollinator service by bees, particularly on windy days. Fruit and seed set declined over time, especially during the latter third of the flowering period. Multilocus estimates of outcrossing rate were obtained using two methods (the programs MLTR and BORICE) and both indicated high selfing rates of ∼80 %. There was evidence for a significant increase in levels of selfing as the flowering season progressed and pollinator visitation declined. Biparental inbreeding also declined significantly as the flowering season progressed., Conclusions: Temporal variation in outcrossing rates may be a common feature of the mating biology of annual, insect-pollinated plants of harsh environments but our study is the first to examine seasonal mating-system dynamics in this context. Despite having large flowers and showy floral displays, I. sinensis attracted relatively few pollinators. Delayed selfing by corolla dragging largely explains the occurrence of mixed mating in I. sinensis, and this mode of self-fertilization probably functions to promote reproductive assurance when pollinator service is limited by windy environmental conditions and competition from co-occurring flowering plants., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background and Aims: The balance between stochastic forces and negative frequency-dependent selection largely determines style morph frequencies in heterostylous populations. Investigation of morph frequencies at geographical range limits can provide insights into the forces maintaining the floral polymorphism, and the factors causing biased morph ratios. Here, we investigate style morph frequencies in populations at the south-western European range limit of tristylous Lythrum salicaria, to explore the role of demographic and geographical factors influencing morph ratios in its native range., Methods: We measured morph composition and evenness, and the size of 96 populations, along a north to south latitudinal transect from Galicia to Andalucia, Iberian Peninsula, traversing a steep climatic gradient. To examine the potential influence of morph-specific fitness components on morph ratios, we examined reproductive traits in 19 populations., Key Results: Most populations of L. salicaria were trimorphic (94·79 %), the majority exhibiting 1 : 1 : 1 morph ratios (68·75 %). Populations with biased morph ratios had a deficiency of the short-styled morph. Population size and morph evenness were positively associated with latitude, with smaller populations and those with less even morph ratios occurring towards the south. Greater variance in morph evenness was evident at the southern range margin. There were no consistent differences in components of reproductive fitness among style morphs, but southern populations produced less fruit and seed than more northerly populations., Conclusions: Our results demonstrate the influence of finite population size on morph frequencies in L. salicaria. However, they also illustrate the resilience of Iberian populations to the factors causing deviations from isoplethy and morph loss, especially at the southern range limit where populations are smaller. The maintenance of tristyly in small populations of L. salicaria may be aided by the genetic connectivity of populations in agricultural landscapes resulting from gene flow through pollen and seed dispersal., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Sexual dimorphism in resource allocation is expected to change during the life cycle of dioecious plants because of temporal differences between the sexes in reproductive investment. Given the potential for sex-specific differences in reproductive costs, resource availability may contribute to variation in reproductive allocation in females and males. Here, we used Rumex hastatulus, a dioecious, wind-pollinated annual plant, to investigate whether sexual dimorphism varies with life-history stage and nutrient availability, and determine whether allocation patterns differ depending on reproductive commitment. To examine if the costs of reproduction varied between the sexes, reproduction was either allowed or prevented through bud removal, and biomass allocation was measured at maturity. In a second experiment to assess variation in sexual dimorphism across the life cycle, and whether this varied with resource availability, plants were grown in high and low nutrients and allocation to roots, aboveground vegetative growth and reproduction were measured at three developmental stages. Males prevented from reproducing compensated with increased above- and belowground allocation to a much larger degree than females, suggesting that male reproductive costs reduce vegetative growth. The proportional allocation to roots, reproductive structures and aboveground vegetative growth varied between the sexes and among life-cycle stages, but not with nutrient treatment. Females allocated proportionally more resources to roots than males at peak flowering, but this pattern was reversed at reproductive maturity under low-nutrient conditions. Our study illustrates the importance of temporal dynamics in sex-specific resource allocation and provides support for high male reproductive costs in wind-pollinated plants., (© 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Flowering plants possess an unrivaled diversity of mechanisms for achieving sexual and asexual reproduction, often simultaneously. The commonest type of asexual reproduction is clonal growth (vegetative propagation) in which parental genotypes (genets) produce vegetative modules (ramets) that are capable of independent growth, reproduction, and often dispersal. Clonal growth leads to an expansion in the size of genets and increased fitness because large floral displays increase fertility and opportunities for outcrossing. Moreover, the clonal dispersal of vegetative propagules can assist "mate finding," particularly in aquatic plants. However, there are ecological circumstances in which functional antagonism between sexual and asexual reproductive modes can negatively affect the fitness of clonal plants. Populations of heterostylous and dioecious species have a small number of mating groups (two or three), which should occur at equal frequency in equilibrium populations. Extensive clonal growth and vegetative dispersal can disrupt the functioning of these sexual polymorphisms, resulting in biased morph ratios and populations with a single mating group, with consequences for fertility and mating. In populations in which clonal propagation predominates, mutations reducing fertility may lead to sexual dysfunction and even the loss of sex. Recent evidence suggests that somatic mutations can play a significant role in influencing fitness in clonal plants and may also help explain the occurrence of genetic diversity in sterile clonal populations. Highly polymorphic genetic markers offer outstanding opportunities for gaining novel insights into functional interactions between sexual and clonal reproduction in flowering plants.

Mating patterns in heterostylous species with intramorph compatibility have the potential to deviate from symmetrical disassortative mating owing to ecological and reproductive factors influencing pollen dispersal. Here, we investigate potential and realized patterns of mating in distylous Luculia pinceana (Rubiaceae), a species with intramorph compatibility. Our analysis provides an opportunity to test Darwin's hypothesis that reciprocal herkogamy promotes disassortative pollen transfer. We combined measurements of sex-organ reciprocity and pollen production to predict potential pollen transfer and mating patterns in a population from SW China. Marker-based paternity analysis was then used to estimate realized patterns of disassortative and assortative mating at the individual and floral morph levels. Both potential and realized mating patterns indicated a significant component of disassortative mating, satisfying theoretical conditions for the maintenance of floral dimorphism. Levels of assortative mating (37.7%) were significantly lower than disassortative mating (62.3%), but numerous offspring resulting from intramorph mating were detected in the majority of maternal seed families in both floral morphs. Our results provide empirical support for Darwin's cross-promotion hypothesis on the function of reciprocal herkogamy, but indicate that in most heterostylous species strong diallelic incompatibility may be a general requirement for complete disassortative mating., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Invasion genetics is a relatively new discipline that investigates patterns of genetic variation in populations of invasive species and their ecological and evolutionary consequences. Evolutionary biologists have a long-standing interest in colonizing species, owing to their short life cycles and widespread distributions, but not until publication of The Genetics of Colonizing Species (1965), edited by H.G. Baker and G.L. Stebbins, was a synthesis on the genetics and evolution of colonizers available. Here, I make the case that the Baker and Stebbins volume is the foundational document for invasion genetics, and in conjunction with the increased use of genetic markers and development of invasion biology, resulted in the birth of this new field over the past two decades. I consider the historical origins and legacy of the Baker and Stebbins volume and review some of the key issues that were addressed. I provide biographical sketches of the two editors, emphasizing their contrasting backgrounds and personalities. I review examples from my own work on plant invasions that are relevant to issues discussed by contributors to the volume. These include the following: determinants of invasion success, life history trade-offs, generalist vs. specialist strategies, general-purpose genotypes, adaptive phenotypic plasticity, mating systems and the influence of bottlenecks on genetic variation. I conclude by posing several key questions in invasion genetics and argue that one of the main challenges that the area faces is to integrate experimental field studies of the ecology and demography of populations with the largely descriptive approaches that have tended to dominate most research to date., (© 2014 John Wiley & Sons Ltd.)

Genetic diversity in populations of invasive species is influenced by a variety of factors including reproductive systems, ploidy level, stochastic forces associated with colonization and multiple introductions followed by admixture. Here, we compare genetic variation in native and introduced populations of the clonal plant Oxalis pes-caprae to investigate the influence of reproductive mode and ploidy on levels of diversity. This species is a tristylous geophyte native to South Africa. Invasive populations throughout much of the introduced range are composed of a sterile clonal pentaploid short-styled form. We examined morph ratios, ploidy level, reproductive mode and genetic diversity at nuclear microsatellite loci in 10 and 12 populations from South Africa and the Western Mediterranean region, respectively. Flow cytometry confirmed earlier reports of diploids and tetraploids in the native range, with a single population containing pentaploid individuals. Introduced populations were composed mainly of pentaploids, but sexual tetraploids were also found. There was clear genetic differentiation between ploidy levels, but sexual populations from both regions were not significantly different in levels of diversity. Invasive populations of the pentaploid exhibited dramatically reduced levels of diversity but were not genetically uniform. The occurrence of mixed ploidy levels and stylar polymorphism in the introduced range is consistent with multiple introductions to the Western Mediterranean. This inference was supported by variation patterns at microsatellite loci. Our study indicates that some invasive populations of Oxalis pes-caprae are not entirely clonal, as often assumed, and multiple introductions and recombination have the potential to increase genetic variation in the introduced range., (© 2014 John Wiley & Sons Ltd.)

The transition from outcrossing to selfing is predicted to reduce the genome-wide efficacy of selection because of the lower effective population size (Ne) that accompanies this change in mating system. However, strongly recessive deleterious mutations exposed in the homozygous backgrounds of selfers should be under strong purifying selection. Here, we examine estimates of the distribution of fitness effects (DFE) and changes in the magnitude of effective selection coefficients (Nes) acting on mutations during the transition from outcrossing to selfing. Using forward simulations, we investigated the ability of a DFE inference approach to detect the joint influence of mating system and the dominance of deleterious mutations on selection efficacy. We investigated predictions from our simulations in the annual plant Eichhornia paniculata, in which selfing has evolved from outcrossing on multiple occasions. We used range-wide sampling to generate population genomic datasets and identified nonsynonymous and synonymous polymorphisms segregating in outcrossing and selfing populations. We found that the transition to selfing was accompanied by a change in the DFE, with a larger fraction of effectively neutral sites (Nes < 1), a result consistent with the effects of reduced Ne in selfers. Moreover, an increased proportion of sites in selfers were under strong purifying selection (Nes > 100), and simulations suggest that this is due to the exposure of recessive deleterious mutations. We conclude that the transition to selfing has been accompanied by the genome-wide influences of reduced Ne and strong purifying selection against deleterious recessive mutations, an example of purging at the molecular level., (Copyright © 2015 by the Genetics Society of America.)

Background and Aims: The balance between stochastic forces and frequency-dependent mating largely governs style morph frequencies in heterostylous populations. In clonal species, deviations from equal morph ratios often result from founder events and unfavourable conditions for sexual reproduction. The aim of this study was to investigate whether different flooding regimes, because of their influence on sexual vs. clonal reproduction, are associated with regional variation in morph frequencies and floral trait differentiation in populations of the clonal, tristylous, aquatic Eichhornia azurea (Pontederiaceae) in the Pantanal wetlands of Brazil., Methods: Style morph frequencies were sampled from 73 populations distributed across four flooding regimes differing in depth and duration. Measurements of flower size, sex-organ dimension, pollen size and pollen production were made in selected populations, and pollinator assemblages and their functional traits were recorded., Key Results: Most populations of E. azurea were tristylous (78 %), but the majority exhibited uneven morph ratios. The frequency of the mid-styled morph was significantly lower than that of the long- and short-styled morphs. Morph evenness was positively associated with population size but not with flooding regime. There were significant phenotypic differences among flooding regimes for all floral traits, including populations with reduced flower size, sex-organ length and smaller pollen. Pollinator assemblages varied with flood duration., Conclusions: The similar morph structure and evenness of populations, regardless of flooding regime, suggest that sexual reproduction and clonal dispersal are sufficiently common to prevent the signature of founder events from dominating in a region. However, the pervasive occurrence of biased morph ratios in most populations suggests that many are in a non-equilibrium state. The reduced frequency of the mid-styled morph in trimorphic and dimorphic populations may be associated with the weak self-incompatibility of this morph resulting in selfing and inbreeding depression. Clonality in E. azurea and the weak self-incompatibility of the mid-styled morph may make it more vulnerable to geitonogamous selfing., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Willows (Salix: Salicaceae) form a major ecological component of Holarctic floras and consequently are an obvious target for a DNA-based identification system. We surveyed two to seven plastid genome regions (~3.8 kb; ~3% of the genome) from 71 Salix species across all five subgenera, to assess their performance as DNA barcode markers. Although Salix has a relatively high level of interspecific hybridization, this may not sufficiently explain the near complete failure of barcoding that we observed: only one species had a unique barcode. We recovered 39 unique haplotypes, from more than 500 specimens, that could be partitioned into six major haplotype groups. A unique variant of group I (haplotype 1*) was shared by 53 species in three of five Salix subgenera. This unusual pattern of haplotype sharing across infrageneric taxa is suggestive of either a massive nonrandom coalescence failure (incomplete lineage sorting), or of repeated plastid capture events, possibly including a historical selective sweep of haplotype 1* across taxonomic sections. The former is unlikely as molecular dating indicates that haplotype 1* originated recently and is nested in the oldest major haplotype group in the genus. Further, we detected significant non-neutrality in the frequency spectrum of mutations in group I, but not outside group I, and demonstrated a striking absence of geographical (isolation by distance) effects in the haplotype distributions of this group. The most likely explanation for the patterns we observed involves recent repeated plastid capture events, aided by widespread hybridization and long-range seed dispersal, but primarily propelled by one or more trans-species selective sweeps., (© 2014 John Wiley & Sons Ltd.)

Background and Aims: Variation in the relative female and male reproductive success of flowering plants is widespread, despite the fundamental hermaphroditic condition of the majority of species. In many hermaphroditic populations, environmental conditions and their influence on development and size can influence the gender expression of individuals through the formation of hermaphroditic and unisexual flowers. This study investigates the hypothesis that the bulbous, animal-pollinated, perennial Lilium apertum (Liliaceae) exhibits a form of size-dependent gender modification known as gender diphasy, in which the sexual expression of individuals depends on their size, with plants often changing sex between seasons., Methods: Variation in floral traits was examined in relation to their size using marked individuals in natural populations, and also under glasshouse conditions. Measurements were taken of the height, flower number, floral sex expression, flower size, flower biomass and pollen production of individuals over consecutive years between 2009 and 2012 in seven populations in south-west China., Key Results: Flowers of L. apertum are either perfect (hermaphroditic) or staminate (male) and, in any given season, plants exhibit one of three sex phenotypes: only hermaphrodite flowers, a mixture of hermaphroditic and male flowers, or only male flowers. Transitions between each of these sex phenotypes were observed over consecutive years and were commonly size-dependent, particularly transitions from small plants bearing only male flowers to those that were taller with hermaphroditic flowers. Hermaphroditic flowers were significantly larger, heavier and produced more pollen than male flowers., Conclusions: The results for L. apertum are consistent with the 'size advantage hypothesis' developed for animal species with sex change. The theory predicts that when individuals are small they should exhibit the sex for which the costs of reproduction are less, and this usually involves the male phase. L. apertum provides an example of gender diphasy, a rare sexual system in flowering plants., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The evolution of self-fertilization from outcrossing has occurred on numerous occasions in flowering plants. This shift in mating system profoundly influences the morphology, ecology, genetics and evolution of selfing lineages. As a result, there has been sustained interest in understanding the mechanisms driving the evolution of selfing and its environmental context. Recently, patterns of molecular variation have been used to make inferences about the selective mechanisms associated with mating system transitions. However, these inferences can be complicated by the action of linked selection following the transition. Here, using multilocus simulations and comparative molecular data from related selfers and outcrossers, we demonstrate that there is little evidence for strong bottlenecks associated with initial transitions to selfing, and our simulation results cast doubt on whether it is possible to infer the role of bottlenecks associated with reproductive assurance in the evolution of selfing. They indicate that the effects of background selection on the loss of diversity and efficacy of selection occur rapidly following the shift to high selfing. Future comparative studies that integrate explicit ecological and genomic details are necessary for quantifying the independent and joint effects of selection and demography on transitions to selfing and the loss of genetic diversity., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Gender strategies involve three fundamental sex phenotypes - female, male and hermaphrodite. Their frequencies in populations typically define plant sexual systems. Patterns of sex-ratio variation in a geographical context can provide insight into transitions among sexual systems, because environmental gradients differentially influence sex phenotype fitness. Here, we investigate sex-ratio variation in 116 populations of Sagittaria latifolia at the northern range limit in eastern N. America and evaluate mechanisms responsible for the patterns observed. We detected continuous variation in sex phenotype frequencies from monoecy through subdioecy to dioecy. There was a decline in the frequency and flower production of females in northerly populations, whereas hermaphrodite frequencies increased at the range limit, and in small populations. Tests of a model of sex-ratio evolution, using empirical estimates of fitness components, indicated that the relative female and male contribution of males and hermaphrodites to fitness is closer to equilibrium expectations than female frequencies. Plasticity in sex expression and clonality likely contribute to deviations from equilibrium expectations., (© 2014 The Authors. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.)

Clonality is often implicated in models of the evolution of dioecy, but few studies have explicitly compared clonal structure between plant sexual systems, or between the sexes in dioecious populations. Here, we exploit the occurrence of monoecy and dioecy in clonal Sagittaria latifola (Alismataceae) to evaluate two main hypotheses: (i) clone sizes are smaller in monoecious than dioecious populations, because of constraints imposed on clone size by costs associated with geitonogamy; (ii) in dioecious populations, male clones are larger and flower more often than female clones because of sex-differential reproductive costs. Differences in clone size and flowering could result in discordance between ramet- and genet-based sex ratios. We used spatially explicit sampling to address these hypotheses in 10 monoecious and 11 dioecious populations of S. latifolia at the northern range limit in Eastern North America. In contrast to our predictions, monoecious clones were significantly larger than dioecious clones, probably due to their higher rates of vegetative growth and corm production, and in dioecious populations, there was no difference in clone size between females and males; ramet- and genet-based sex ratios were therefore highly correlated. Genotypic diversity declined with latitude for both sexual systems, but monoecious populations exhibited lower genotypic richness. Differences in life history between the sexual systems of S. latifolia appear to be the most important determinants of clonal structure and diversity., (© 2014 John Wiley & Sons Ltd.)

Heteromorphic sex chromosomes have originated independently in many species, and a common feature of their evolution is the degeneration of the Y chromosome, characterized by a loss of gene content and function. Despite being of broad significance to our understanding of sex chromosome evolution, the genetic changes that occur during the early stages of Y-chromosome degeneration are poorly understood, especially in plants. Here, we investigate sex chromosome evolution in the dioecious plant Rumex hastatulus, in which X and Y chromosomes have evolved relatively recently and occur in two distinct systems: an ancestral XX/XY system and a derived XX/XY1Y2 system. This polymorphism provides a unique opportunity to investigate the effect of sex chromosome age on patterns of divergence and gene degeneration within a species. Despite recent suppression of recombination and low X-Y divergence in both systems, we find evidence that Y-linked genes have started to undergo gene loss, causing ∼ 28% and ∼ 8% hemizygosity of the ancestral and derived X chromosomes, respectively. Furthermore, genes remaining on Y chromosomes have accumulated more amino acid replacements, contain more unpreferred changes in codon use, and exhibit significantly reduced gene expression compared with their X-linked alleles, with the magnitude of these effects being greatest for older sex-linked genes. Our results provide evidence for reduced selection efficiency and ongoing Y-chromosome degeneration in a flowering plant, and indicate that Y degeneration can occur soon after recombination suppression between sex chromosomes.

Adaptation to climate, evolving over contemporary time scales, could facilitate rapid range expansion across environmental gradients. Here, we examine local adaptation along a climatic gradient in the North American invasive plant Lythrum salicaria. We show that the evolution of earlier flowering is adaptive at the northern invasion front where it increases fitness as much as, or more than, the effects of enemy release and the evolution of increased competitive ability. However, early flowering decreases investment in vegetative growth, which reduces fitness by a factor of 3 in southern environments where the North American invasion commenced. Our results demonstrate that local adaptation can evolve quickly during range expansion, overcoming environmental constraints on propagule production.

Differentiation of female sexual organs in flowering plants is rare and contrasts with the wide range of male reproductive strategies. An unusual example involves diplostigmaty, the possession of spatially and temporally distinct stigmas in Sebaea (Gentianaceae). Here, the single pistil within a flower has an apical stigma, as occurs in most flowering plants, but also a secondary stigma that occurs midway down the style, which is physically discrete and receptive several days after the apical stigma. We examined the function of diplostigmaty in Sebaea aurea, an insect-pollinated species of the Western Cape of South Africa. Floral manipulations and measurements of fertility and mating patterns provided evidence that basal stigmas function to enable autonomous delayed self-pollination, without limiting opportunities for outcrossing and thus avoiding the costs of seed discounting. We suggest that delayed selfing serves as a mechanism of reproductive assurance in populations with low plant density. The possession of dimorphic stigma function provides a novel example of a flexible mixed-mating strategy in plants that is responsive to changing demographic conditions.

Objectives: To compare obese versus non-obese Jamaican adolescents' risk for type 2 diabetes (T2D) and cardiovascular diseases (CVDs); and to explore a suitable and economical method of screening for these risk factors in the school settings., Design: A descriptive cross-sectional study of adolescents' risk for T2D and CVD. All the participants were examined at their respective schools., Setting: Jamaica, West Indies., Population: 276 Jamaican adolescents aged 14-19 years, randomly selected from grades 9 to 12 from 10 high schools on the island and included both boys and girls. All ethnicities on the island were represented., Main Outcome Measures: High fasting blood glucose, total cholesterol, glycated haemoglobin (HbA1c), blood pressure, body mass index (BMI), waist circumference, waist-to-hip ratio, family history of obesity, T2D and CVDs, low physical activity, and presence of Acanthosis Nigricans. All blood measures were analysed using the finger prick procedure., Results: Waist circumference, waist-to-hip ratio, Acanthosis Nigricans, total cholesterol, family history of T2D and blood pressure were the strongest predictors of BMI (p=0.001). Over one-third of the participants were overweight. Jamaican adolescent females had a significantly higher number of risk factors and were less physically active than males (p<0.05). Over 80% of participants reported ≥3 risk factors for T2D and CVD. Participants with BMI ≥25 reported five or more risk factors. One-third of the overweight participants were classified with metabolic syndrome., Conclusions: Jamaican adolescents are at risk of T2D and CVD. Family history of disease and anthropometric measures identified more participants at risk than did the blood measures. Jamaican adolescent females reported more risk factors for T2D and CVD as compared to males. Collection of this type of data was feasible within the school settings. All data were collected in 1 day per school. Intervention measures are needed to educate Jamaican adolescents to reduce overweight and subsequently the risk factors.

Flowering plants are characterized by striking variation in reproductive systems, and the evolutionary lability of their sexual traits is often considered a major driver of lineage diversification. But, evolutionary transitions in reproductive form and function are never entirely unconstrained and many changes exhibit strong directionality. Here, I consider why this occurs by examining transitions in pollination, mating and sexual systems, some of which have been considered irreversible. Among pollination systems, shifts from bee to hummingbird pollination are rarely reversible, whereas transitions from animal to wind pollination are occasionally reversed. Specialized pollination systems can become destabilized through a loss of pollinator service resulting in a return to generalized pollination, or more commonly a reliance on self-pollination. Homomorphic and heteromorphic self-incompatibility systems have multiple origins but breakdown to self-compatibility occurs much more frequently with little evidence for subsequent gains, at least over short time-spans. Similarly, numerous examples of the shift from outcrossing to predominant self-fertilization are known, but cases of reversal are very limited supporting the view that autogamy usually represents an evolutionary dead-end. The evolution of dioecy from hermaphroditism has also been considered irreversible, although recent evidence indicates that the occurrence of sex inconstancy and hybridization can lead to the origin of derived sexual systems from dioecy. The directionality of many transitions clearly refutes the notion of unconstrained reproductive flexibility, but novel adaptive solutions generally do not retrace earlier patterns of trait evolution.

Frequency-dependent selection should drive dioecious populations toward a 1:1 sex ratio, but biased sex ratios are widespread, especially among plants with sex chromosomes. Here, we develop population genetic models to investigate the relationships between evolutionarily stable sex ratios, haploid selection, and deleterious mutation load. We confirm that when haploid selection acts only on the relative fitness of X- and Y-bearing pollen and the sex ratio is controlled by the maternal genotype, seed sex ratios evolve toward 1:1. When we also consider haploid selection acting on deleterious mutations, however, we find that biased sex ratios can be stably maintained, reflecting a balance between the advantages of purging deleterious mutations via haploid selection, and the disadvantages of haploid selection on the sex ratio. Our results provide a plausible evolutionary explanation for biased sex ratios in dioecious plants, given the extensive gene expression that occurs across plant genomes at the haploid stage., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

Backgrounds and Aims: The spatial separation of stigmas and anthers (herkogamy) in flowering plants functions to reduce self-pollination and avoid interference between pollen dispersal and receipt. Little is known about the evolutionary relationships among the three main forms of herkogamy - approach, reverse and reciprocal herkogamy (distyly) - or about transitions to and from a non-herkogamous condition. This problem was examined in Exochaenium (Gentianaceae), a genus of African herbs that exhibits considerable variation in floral morphology, including the three forms of herkogamy., Methods: Using maximum parsimony and maximum likelihood methods, the evolutionary history of herkogamic and non-herkogamic conditions was reconstructed from a molecular phylogeny of 15 species of Exochaenium and four outgroup taxa, based on three chloroplast regions, the nuclear ribosomal internal transcribed spacer (ITS1 and 2) and the 5·8S gene. Ancestral character states were determined and the reconstructions were used to evaluate competing models for the origin of reciprocal herkogamy., Key Results: Reciprocal herkogamy originated once in Exochaenium from an ancestor with approach herkogamy. Reverse herkogamy and the non-herkogamic condition homostyly were derived from heterostyly. Distylous species possessed pendent, slightly zygomorphic flowers, and the single transition to reverse herkogamy was associated with the hawkmoth pollination syndrome. Reductions in flower size characterized three of four independent transitions from reciprocal herkogamy to homostyly., Conclusions: The results support Lloyd and Webb's model in which distyly originated from an ancestor with approach herkogamy. They also demonstrate the lability of sex organ deployment and implicate pollinators, or their absence, as playing an important role in driving transitions among herkogamic and non-herkogamic conditions.

Background and Aims: Populations of dioecious flowering plants commonly exhibit heterogeneity in sex ratios and deviations from the equilibrium expectation of equal numbers of females and males. Yet the role of ecological and demographic factors in contributing towards biased sex ratios is currently not well understood., Methods: Species-level studies from the literature were analysed to investigate ecological correlates of among-population sex-ratio variation and metapopulation models and empirical data were used to explore the influence of demography and non-equilibrium conditions on flowering sex ratios., Key Results: The survey revealed significant among-population heterogeneity in sex ratios and this was related to the degree of sampling effort. For some species, sex-ratio bias was associated with the proportion of non-reproductive individuals, with greater male bias in populations with a lower proportion of individuals that were flowering. Male-biased ratios were also found at higher altitudes and latitudes, and in more xeric sites. Simulations and empirical data indicated that clonal species exhibited greater heterogeneity in sex ratios than non-clonal species as a result of their slower approach to equilibrium. The simulations also indicated the importance of interactions between reproductive mode and founder effects, with greater departures from equilibrium in clonal populations with fewer founding individuals., Conclusions: The results indicate that sex-based differences in costs of reproduction and non-equilibrium conditions can each play important roles in affecting flowering sex ratios in populations of dioecious plants.