Polyploid goldback and silverback ferns (Pentagramma) occupy a wider, colder, and wetter bioclimatic niche than diploid counterparts.

PREMISE: The western North American fern genus Pentagramma (Pteridaceae) is characterized by complex patterns of ploidy variation, an understanding of which is critical to comprehending both the evolutionary processes within the genus and its current diversity. METHODS: We undertook a cytogeographic study across the range of the genus, using a combination of chromosome counts and flow cytometry to infer ploidy level. Bioclimatic variables and elevation were used to compare niches. RESULTS: We found that diploids and tetraploids are common and widespread, and triploids are rare and sporadic; in contrast with genome size inferences in earlier studies, no hexaploids were found. Diploids and tetraploids show different geographic ranges: only tetraploids were found in the northernmost portion of the range (Washington, Oregon, and British Columbia) and only diploids were found in the Sierra Nevada of California. Diploid, triploid, and tetraploid cytotypes were found to co-occur in relatively few localities: in the southern (San Diego County, California) and desert Southwest (Arizona) parts of the range, and along the Pacific Coast of California. CONCLUSIONS: Tetraploids occupy a wider bioclimatic niche than diploids both within P. triangularis and at the genus-wide scale. It is unknown whether the wider niche of tetraploids is due to their expansion upon the diploid niche, if diploids have contracted their niche due to competition or changing abiotic conditions, or if this wider niche occupancy is due to multiple origins of tetraploids.

Rapid sequential determination of the explosives 2,4,6-trinitrotoluene and cyclotrimethylenetrinitramine in forensic samples employing a graphite sheet sensor and cyclic square-wave stripping voltammetry.

The development of a portable analytical procedure is described for rapid sequential detection and quantification of the explosives 2,4,6-trinitrotoluene (TNT) and cyclotrimethylenetrinitramine (RDX) in forensic samples using a graphite sheet (GS). A single GS platform works as a collector of explosive residues and detector after its assembly into a 3D-printed cell. The detection strategy is based on cyclic square-wave stripping voltammetry. The cathodic scan from + 0.1 to -1.0 V with accumulation at 0.0 V enables the TNT detection (three reduction peaks), and the anodic scan from + 0.2 to + 1.55 V with accumulation at -0.9 V provides the RDX detection (two oxidation processes). Low detection limit values (0.1 µmol L-1 for TNT and 2.4 µmol L-1 for RDX) and wide linear ranges (from 1 to 150 µmol L-1 for TNT and from 20 to 300 µmol L-1 for RDX) were obtained. The sensor did not respond to pentaerythritol tetranitrate (PETN), which was evaluated as a potential interferent, because plastic explosives contain mixtures of TNT, RDX, and PETN. The GS electrode was also evaluated as a collector of TNT and RDX residues spread on different surfaces to simulate forensic scenarios. After swiping over different surfaces (metal, granite, wood, cloths, hands, money bills, and cellphone), the GS electrode was assembled in the 3D-printed cell ready to measure both explosives by the proposed method. In all cases, the presence of TNT and RDX was confirmed, attesting the reliability of the proposed device to act as collector and sensor.

Population size as a major determinant of mating system and population genetic differentiation in a narrow endemic chasmophyte.

BACKGROUND: Mating system is one of the major determinants of intra- and interspecific genetic structure, but may vary within and between plant populations. Our study model included all known populations of Moehringia tommasinii (Caryophyllaceae), a narrow endemic plant inhabiting rock crevices in the northwestern Adriatic, and some populations of co-occurring and widespread M. muscosa, an ecologically divergent relative with an overlapping flowering period. We performed reciprocal crosses within and between taxa and used molecular markers to assess the extent of gene flow within and between populations and taxa. Using coefficient of inbreeding, population size, seed weight, pollen-to-ovule ratio, and flower display size, we also looked for evidence of a selfing syndrome. RESULTS: A surprisingly high variation in mating systems was observed among populations of M. tommasinii. These populations exhibited genetic structuring, with their size positively correlated with both seed weight and pollen production. Although a selfing syndrome could not be confirmed as the majority of selfing resulted from allogamous treatments, the occurrence of selfing was notable. In the presence of M. muscosa, at a site where both species coexist closely, a distinct pattern of fruit production was observed in M. tommasinii following various pollination treatments. Molecular and morphometric data provided evidence of hybridization followed by local extinction at this site. CONCLUSIONS: Population size proved to be the most important factor affecting the mating system in genetically structured populations of M. tommasinii. Lighter seeds and lower pollen production observed in populations with pronounced selfing do not provide enough evidence for the selfing syndrome. Detected gene flow between M. tommasinii and the sympatric M. muscosa suggested weak reproductive barriers between the taxa, which could pose a conservation problems for the former species. Hybridization leading to local extinction may also resulted in floral polymorphism and disruption of mating patterns of M. tommasinii.

Hybridization and introgression are prevalent in Southern European Erysimum (Brassicaceae) species.

BACKGROUND AND AIMS: Hybridization is a common and important force in plant evolution. One of its outcomes is introgression - the transfer of small genomic regions from one taxon to another by hybridization and repeated backcrossing. This process is believed to be common in glacial refugia, where range expansions and contractions can lead to cycles of sympatry and isolation, creating conditions for extensive hybridization and introgression. Polyploidization is another genome-wide process with a major influence on plant evolution. Both hybridization and polyploidization can have complex effects on plant evolution. However, these effects are often difficult to understand in recently evolved species complexes. METHODS: We combined flow cytometry, analyses of transcriptomic sequences and pollen tube growth assays to investigate the consequences of polyploidization, hybridization and introgression on the recent evolution of several Erysimum (Brassicaceae) species from the South of the Iberian Peninsula, a well-known glacial refugium. This species complex differentiated in the last 2 million years, and its evolution has been hypothesized to be determined mainly by polyploidization, interspecific hybridization and introgression. KEY RESULTS: Our results support a scenario of widespread hybridization involving both extant and 'ghost' taxa. Several taxa studied here, most notably those with purple corollas, are polyploids, probably of allopolyploid origin. Moreover, hybridization in this group might be an ongoing phenomenon, as pre-zygotic barriers appeared weak in many cases. CONCLUSIONS: The evolution of Erysimum spp. has been determined by hybridization to a large extent. Species with purple (polyploids) and yellow flowers (mostly diploid) exhibit a strong signature of introgression in their genomes, indicating that hybridization occurred regardless of colour and across ploidy levels. Although the adaptive value of such genomic exchanges remains unclear, our results demonstrate the significance of hybridization for plant diversification, which should be taken into account when studying plant evolution.

Ploidy effects on the relationship between floral phenotype, reproductive investment, and fitness in an autogamous species complex.

PREMISE: The relationships between reproductive investment, phenotype, and fitness have been broadly studied in cross-pollinated plants in contrast to selfing species, which are considered less interesting in this area because they are supposed to be a dead end in any evolutionary pathway. Still, selfing plants are unique systems to study these questions since the position of reproductive structures and traits related to flower size play an important role in female and male pollination success. METHODS: Erysimum incanum s.l. is a selfing species complex that has three levels of ploidy (diploids, tetraploids, and hexaploids) and traits that are typically associated with the selfing syndrome. Here, we used 1609 plants belonging to these three ploidies to characterize the floral phenotype and spatial configuration of reproductive structures, reproductive investment (pollen and ovule production), and plant fitness. Then, we used structural equation modelling to analyze the relationship between all these variables across ploidy levels. RESULTS: An increase in ploidy level leads to bigger flowers with anthers exserted farther and more pollen and ovules. In addition, hexaploid plants had higher absolute values for herkogamy, which is positively correlated with fitness. Ovule production significantly mediated the natural selection acting on different phenotypic traits and pollen production, a pattern that is maintained across ploidies. CONCLUSIONS: The changes in floral phenotypes, reproductive investment, and fitness with ploidy level suggest that genome duplication can be a driver for transitions in reproductive strategy by modifying the investment in pollen and ovules and linking them with plant phenotype and fitness.

The impact of habitat loss on pollination services for a threatened dune endemic plant.

Habitat loss is currently a major threat to biodiversity, affecting species interactions, such as plant-pollinator interactions. This is particularly important in self-incompatible plants relying on pollinators to reproduce and sustain their populations. Here, we evaluated how habitat loss affects the pollination system, plant individual-pollinator species interaction network, and plant reproductive fitness of the self-incompatible Jasione maritima var. sabularia, a threatened taxon from dune systems. This plant is a pollinator generalist, visited by 108 species from distinct taxonomic groups. Results suggest that increasing habitat loss led to a significant decline in pollinator richness, increased pollen limitation, and a decrease in reproductive fitness of J. maritima var. sabularia. Visitation rate per individual did not significantly change with available area, indicating that the quality of pollen differed across populations. The topology of the network between J. maritima var. sabularia individuals and its pollinator species did not change, which may be attributed to the stability in the core of pollinator species. This suggests that the lower fitness of plants with increasing habitat degradation may be explained not only by the lower richness of peripheral pollinators but also by the genetic structure of the plant populations, as there is a possible higher transference of less quality pollen by pollinators, ultimately compromising the persistence of plant populations. Our study highlights the need of future studies to integrate the fine details provided by individual-level networks, which will increase our understanding of the pattern of species interactions and its consequences for the fitness of threatened plant populations.

Flow cytometric analysis of pollen and spores: An overview of applications and methodology.

Pollen grains are the male gametophytes in a seed-plant life cycle. Their small, particulate nature and crucial role in plant reproduction have made them an attractive object of study using flow cytometry (FCM), with a wide range of applications existing in the literature. While methodological considerations for many of these overlap with those for other tissue types (e.g., general considerations for the measurement of nuclear DNA content), the relative complexity of pollen compared to single cells presents some unique challenges. We consider these here in the context of both the identification and isolation of pollen and its subunits, and the types of research applications. While the discussion here mostly concerns pollen, the general principles described here can be extended to apply to spores in ferns, lycophytes, and bryophytes. In addition to recommendations provided in more general studies, some recurring and notable issues related specifically to pollen and spores are highlighted.

Isolation of plant nuclei for estimation of nuclear DNA content: Overview and best practices.

A critical aspect for obtaining accurate, reliable, and high-resolution estimates of nuclear DNA content is the release of nuclei from the cytoplasm in sufficient amounts, while maintaining their integrity throughout the analysis, protecting their DNA from degradation by endonucleases, and enabling stoichiometric DNA staining. In embryophytes, the most common method consists of chopping the plant material with a sharp razor blade to release nuclei into an isolation buffer, filtering the homogenate, and staining the nuclei in buffered suspension with a fluorochrome of choice. Despite the recent description of alternative approaches to isolate nuclei, the chopping procedure remains the most widely adopted method, due to its simplicity, rapidity, and effectiveness. In this review article, we discuss the specifics of nuclei isolation buffers and the distorting effects that secondary metabolites may have in nuclear suspensions and how to test them. We also present alternatives to the chopping procedure, options for filtering and fluorochromes, and discuss the applications of these varied approaches. A summary of the best practices regarding the isolation of plant nuclei for the estimation of nuclear DNA content is also provided.

Genetic similarities versus morphological resemblance: Unraveling a polyploid complex in a Mediterranean biodiversity hotspot.

The Balkan Peninsula is recognized as one of the hotspots of biodiversity in Europe. This area has shown since the Last Glacial Maximum appropriate conditions for species diversification and hybridization, which has led to the existence of numerous taxonomically unresolved entities. Here, we focus on the Western Balkans and explore the genetic structure and relationships among species belonging to the V. austriaca - V. orbiculata diploid-polyploid complex, including populations showing intermediate morphologies. A combination of nuclear markers (microsatellites), plastid DNA regions (trnH-psbA, ycf6-psbM) and ploidy level estimations using flow cytometry are employed to assess the genetic structure and evolutionary dynamics of this polyploid complex. To reconstruct the evolutionary history, an approximate Bayesian computation approach is combined with projections of the species distribution models onto the climatic scenarios of the Mid-Holocene (6 ka BP) and Last Glacial Maximum (22 ka BP). Four main groups were found: one well-established entity within the diploid level, V. dalmatica, a second diploid-tetraploid group which corresponds to V. orbiculata, a hexaploid cluster harboring V. austriaca subsp. jacquinii individuals, and an enigmatic tetraploid group. According to the molecular data obtained, this latter cluster represents an allopolyploid cryptic lineage −with V. orbiculata and V. dalmatica as putative parents− morphologically similar to V. orbiculata, but genetically more related to V. austriaca subsp. jacquinii. Veronica dalmatica and this "uncertain tetraploid" group are involved in the formation of the hexaploid taxon V. austriaca subsp. jacquinii, with the possibility of recent gene flow among different cytotypes. The present study supports a scenario of diversification from a diploid common ancestor leading to two different but interrelated lineages. The first one would correspond with the diploid V. orbiculata plus tetraploid individuals of this species arising through allo- and autopolyploidization, and the second one would involve all ploidy levels with allopolyploidization being prevalent.

Insights into amebiasis using a human 3D-intestinal model.

Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human-parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three-dimensional (3D)-intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria-like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D-intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.

On-Surface Synthesis and Collective Spin Excitations of a Triangulene-Based Nanostar.

Triangulene nanographenes are open-shell molecules with predicted high spin state due to the frustration of their conjugated network. Their long-sought synthesis became recently possible over a metal surface. Here, we present a macrocycle formed by six [3]triangulenes, which was obtained by combining the solution synthesis of a dimethylphenyl-anthracene cyclic hexamer and the on-surface cyclodehydrogenation of this precursor over a gold substrate. The resulting triangulene nanostar exhibits a collective spin state generated by the interaction of its 12 unpaired π-electrons along the conjugated lattice, corresponding to the antiferromagnetic ordering of six S=1 sites (one per triangulene unit). Inelastic electron tunneling spectroscopy resolved three spin excitations connecting the singlet ground state with triplet states. The nanostar behaves close to predictions from the Heisenberg model of an S=1 spin ring, representing a unique system to test collective spin modes in cyclic systems.

Verteporfin targeting YAP1/TAZ-TEAD transcriptional activity inhibits the tumorigenic properties of gastric cancer stem cells.

Gastric carcinomas (GC) are heterogeneous tumors, composed of a subpopulation of cluster of differentiation-44 (CD44)+ tumorigenic and chemoresistant cancer stem cells (CSC). YAP1 and TAZ oncoproteins (Y/T) interact with TEA domain family member 1 (TEAD) transcription factors to promote cell survival and proliferation in multiple tissues. Their activity and role in GC remain unclear. This work aimed to analyze Y/T-TEAD activity and molecular signature in gastric CSC, and to assess the effect of verteporfin, a Food and Drug Administration-approved drug preventing Y/T-TEAD interaction, on gastric CSC tumorigenic properties. Y/T-TEAD molecular signature was investigated using bioinformatical (KmPlot database), transcriptomic and immunostaining analyses in patient-derived GC and cell lines. Verteporfin effects on Y/T-TEAD transcriptional activity, CSC proliferation and tumorigenic properties were evaluated using in vitro tumorsphere assays and mouse models of patient-derived GC xenografts. High expressions of YAP1, TAZ, TEAD1, TEAD4 and their target genes were associated with low overall survival in nonmetastatic human GC patients (n = 444). This Y/T-TEAD molecular signature was enriched in CD44+ patient-derived GC cells and in cells resistant to conventional chemotherapy. Verteporfin treatment inhibited Y/T-TEAD transcriptional activity, cell proliferation and CD44 expression, and decreased the pool of tumorsphere-forming CD44+ /aldehyde dehydrogenase (ALDH)high gastric CSC. Finally, verteporfin treatment inhibited GC tumor growth in vivo; the residual tumor cells exhibited reduced expressions of CD44 and ALDH1, and more importantly, they were unable to initiate new tumorspheres in vitro. All these data demonstrate that Y/T-TEAD activity controls gastric CSC tumorigenic properties. The repositioning of verteporfin targeting YAP1/TAZ-TEAD activity could be a promising CSC-based strategy for the treatment of GC.

The role of multiple reproductive barriers: strong post-pollination interactions govern cytotype isolation in a tetraploid-octoploid contact zone.

BACKGROUND AND AIMS: Polyploidy is an important contributor to sympatric speciation and assortative mating is a key mechanism driving cytotype interactions in contact zones. While strong reproductive barriers can mediate the coexistence of different cytotypes in sympatry, positive frequency-dependent mating disadvantage ultimately drives the transition to single-ploidy populations. However, comprehensive estimates of reproductive isolation among cytotypes and across multiple barriers are rare. We quantify the strength of isolation across multiple reproductive stages in a tetraploid-octoploid contact zone to understand the potential for coexistence. METHODS: Assortative mating due to flowering asynchrony, pollinator behaviour, morphological overlap, self-fertilization and gametic competition between tetraploid and octoploid Gladiolus communis in a contact zone in the Western Iberian Peninsula were assessed in natural and experimental populations to quantify reproductive isolation (RI) between cytotypes. KEY RESULTS: Tetraploids and octoploids have a high degree of overlap in flowering time and similar floral morphology, and are visited by generalist insects without cytotype foraging preferences, resulting in weak pre-pollination RI (from 0.00 to 0.21). In contrast, post-pollination isolation resulting from gametic selection was a strong barrier to inter-cytotype mating, with ploidy composition in stigmatic pollen loads determining the levels of RI (from 0.54 to 1.00). Between-cytotype cross-incompatibility was relatively high (RI from 0.54 to 0.63) as was isolation acquired through self-pollination (RI of 0.59 in tetraploids and 0.39 in octoploids). CONCLUSIONS: Total RI was high for both tetraploids (from 0.90 to 1.00) and octoploids (from 0.78 to 0.98). Such high rates of assortative mating will enable cytotype coexistence in mixed-ploidy populations by weakening the impacts of minority cytotype exclusion. This study reveals the key role of gametic selection in cytotype siring success and highlights the importance of comprehensive estimates across multiple reproductive barriers to understand cytotype interactions at contact zones.

Environmental differences are correlated with the distribution pattern of cytotypes in Veronica subsection Pentasepalae at a broad scale.

BACKGROUND AND AIMS: The distribution of cytotypes and its potential correlation with environmental variables represent a cornerstone to understanding the origin and maintenance of polyploid lineages. Although many studies have addressed this question in single species at a regional scale, only a few have attempted to decipher this enigma in groups of closely related species at a broad intercontinental geographical scale. Here, we consider approx. 20 species of a diploid-polyploid complex (Veronica subsect. Pentasepalae) of recent and rapid diversification represented in Europe and North Africa to study the frequency and distribution of cytotypes and their relationship to environmental variables. METHODS: A total of 680 individuals (207 populations) were sampled. Ploidy levels were determined using flow cytometry. Ecological differentiation among cytotypes was tested using climatic and environmental variables related to temperature, precipitation, vegetation and biogeographical region, among others, and by performing univariate and multivariate (constrained principal coordinates analysis) analyses. KEY RESULTS: Four ploidy levels (2x, 4x, 6x and 8x) were found and genome downsizing was observed to occur within the group. Plants of different ploidy level are ecologically differentiated, with hexaploids and octoploids occurring in wetter and colder habitats with a higher seasonality than diploids. A south to north distribution pattern was found, with diploids occupying southern refugial areas and octoploids being more frequent in northern regions of Europe above the permafrost boundary. CONCLUSIONS: The distribution of cytotypes can be explained by ecological differentiation, the geographical position of refuge areas during the Quaternary climatic oscillations as well as by ice and permafrost retreat patterns. The Balkan Peninsula constitutes the most important contact zone between cytotypes. This work provides the first comprehensive ploidy screening within V. subsect. Pentasepalae at a broad scale and indicates that polyploidy and genome downsizing might have contributed to the colonization of new habitats in a recently diverged polyploid complex.

Is selfing a reproductive assurance promoting polyploid establishment? Reduced fitness, leaky self-incompatibility and lower inbreeding depression in neotetraploids.

PREMISE: Newly formed polyploids face significant obstacles to persistence and population establishment because of fitness costs of intercytotype mating. Selfing provides the opportunity to escape mate limitation, enabling production of new individuals and increasing the likelihood of fixation of new polyploid lineages. Still, association between self-compatibility and polyploidy is not always clear. We compared self-incompatibility and inbreeding depression in neotetraploids and their diploid progenitor to explore the direct effects of whole genome duplications on self-incompatibility and the implications of ploidy-driven changes for polyploid establishment. METHODS: Outcross and self-pollinations were performed in diploids and synthetic neotetraploids of Jasione maritima var. maritima, and reproductive success was measured through fruit and seed production and seed germination. Self- and outcross offspring were grown under controlled conditions, and plant performance was measured through several fitness parameters. RESULTS: Neotetraploids showed an overall lower performance than diploids. Reproductive success was negatively affected by selfing in both cytotypes. However, greater variation in the expression of self-incompatibility was observed in neotetraploids; additionally, developmental and physiological parameters were not affected by selfing on neotetraploids, with an overall similar fitness of outcrossed and selfed individuals, resulting in lower inbreeding depression indexes. CONCLUSIONS: Neotetraploids might have benefited from selfing at initial stages after their formation. Genome duplications resulted in leaky self-incompatibility, enabling the production of offspring under minority cytotype disadvantage with similar fitness as outcrossed offspring. Our results support theoretical assumptions that selfing might be important for neopolyploid establishment, although changes in self-incompatibility might not be abrupt.

Virulence factor genotyping of Helicobacter pylori isolated from Costa Rican dyspeptic patients.

BACKGROUND AND AIMS: Costa Rica is one of the countries with the highest incidence and mortality rates for gastric cancer. Helicobacter pylori infection rates are high in the whole country. We have previously shown that H. pylori CagA+ is significantly associated with atrophic gastritis (AG) of the antrum in a dyspeptic population. The aim of this work is to determine if other H. pylori virulence factors (vacA, dupA, oipA, iceA and babA2) are associated with atrophic gastritis (AG) or duodenal ulcer (DU). METHODS: The presence of virulence genes in Costa Rican H. pylori isolates was analyzed by PCR in 151 cultured strains from patients with dyspeptic symptoms. Endoscopic and histopathological diagnoses were available. Odds-ratio and 95% confidence intervals for AG patients vs. non-atrophic gastritis (NAG) or DU patients vs. no duodenal ulcer (NDU) patients were calculated. RESULTS: Amongst the studied isolates, 82% had the cagA+, 76.2% had the vacA s1m1, 97.0% had the oipA+, 21.0% had the icea1, 79.0% had the iceA2, 44.0% had the babA2+ and 76.0% the dupA+ genotypes. Infection with H pylori cagA+, dupA+, oipA+, iceA, babA2+, and vacA s1m1 genotypes was not associated with AG risk. The frequency of the dupA gene was 78.7 and 60.9% in isolates from patients with NDU and DU, respectively, and its presence was significantly associated with decreased risk of duodenal ulcer [odds-ratio: 0.33, p = 0.024, confidence interval 95% (0.11-0.85)]. CONCLUSION: H. pylori dupA genotype is inversely associated with DU risk in this population.

Catalysis Concepts in Medicinal Inorganic Chemistry.

Catalysis has strongly emerged in the field of medicinal inorganic chemistry as a suitable tool to deliver new drug candidates and to overcome drawbacks associated to metallodrugs. In this Concept article, we discuss representative examples of how catalysis has been applied in combination with metal complexes to deliver new therapy approaches. In particular, we explain key achievements in the design of catalytic metallodrugs that damage biomolecular targets and in the development of metal catalysis schemes for the activation of exogenous organic prodrugs. Moreover, we discuss our recent discoveries on the flavin-mediated bioorthogonal catalytic activation of metal-based prodrugs; a new catalysis strategy in which metal complexes are unconventionally employed as substrates rather than catalysts.

Fitness benefits and costs of floral advertising traits: insights from rayed and rayless phenotypes of Anacyclus (Asteraceae).

PREMISE OF THE STUDY: Ray flowers commonly observed in daisies' flowering heads are a well-known example of advertising structures for enhancing pollinator attraction. Despite this, ray loss has occurred in multiple lineages, which still rely on pollinators, suggesting that rayless phenotypes could also be adaptive for animal-pollination. Here, we investigate the benefits and costs of these specialized floral advertising structures by comparing rayed and rayless phenotypes in two hybridizing closely related species. METHODS: We assessed the advantages and costs of ray production in terms of floral visitor's attraction, pollen limitation, and female reproductive success using the broad natural variation on ray size and number at the contact zone of A. clavatus (rayed) and A. valentinus (rayless). In addition, we experimentally explored the effect of rays under controlled neighborhoods and the effect of ray removal on fruit production. KEY RESULTS: In sympatry, rayed phenotypes attracted significantly more visitors than rayless plants, in which seed production was pollen limited. However, rayed phenotypes did not show higher fruit set or seed production than rayless phenotypes. Fruit set and seed production benefited from denser neighborhood displays and larger individual floral displays, respectively. The removal of ray florets did not appear to enable resource reallocation to fruit production. CONCLUSIONS: Rayless heads compensated their lower visitation rate by means of a higher number of flowers per head achieving similar fecundity levels to rayed plants. The larger size of rayless heads might thus indicate an inflorescence-level trade-off between attraction and fertility.

Waiting for ICU admission may increase the risk of death-A plea for better resource organization.

BACKGROUND: Mortality is high in critically ill patients. In order to study the risk factors associated with mortality in these patients, we conducted an observational retrospective study in the general Intensive Care Unit (ICU) of Faro Hospital. METHODS: All patients discharged from the general ICU in the year 2015 were evaluated for inclusion. Mortality was characterized in the first 48 hours of ICU stay, at the time of discharge from ICU, and at discharge from hospital. Collected variables included demographic variables (age), and ICU variables: type of ICU admission (scheduled surgery, urgent surgery, medical and trauma), Simplified Acute Physiology Score (SAPS II), main diagnosis, hospital length of stay (HLS) before ICU (BICULS), in ICU (ICULS) and after ICU (AICULS). RESULTS: When comparing survivors with non-survivors, we found that age, disease severity expressed by SAPS II and BICULS were significantly higher in non-survivors. After multivariate regression analysis, BICULS was still significantly associated with mortality in the hospital. CONCLUSION: Further studies are needed to characterize whether this longer BICULS is related to non-modifiable prior conditions or whether it is related to delayed ICU admission, which is a modifiable factor.

The challenge of species delimitation in the diploid-polyploid complex Veronica subsection Pentasepalae.

A reliable taxonomic framework and the identification of evolutionary lineages are essential for effective decisions in conservation biodiversity programs. However, phylogenetic reconstruction becomes extremely difficult when polyploidy and hybridization are involved. Veronica subsection Pentasepalae is a diploid-polyploid complex of ca. 20 species with ploidy levels ranging from 2x to 10x. Here, DNA-ploidy level estimations and AFLP fingerprinting were used to determine the evolutionary history, and species boundaries were reviewed in an integrated approach including also previous data (mainly morphology and sequence-based phylogenetic reconstructions). Molecular analyses were performed for 243 individuals from 95 populations, including for the first time all taxa currently recognized within the subsection. Phylogenetic reconstruction identified four main groups corresponding almost completely to the four clusters identified by genetic structure analyses. Multiple autopolyploidization events have occurred in the tetraploid V. satureiifolia giving rise to octoploid entities in central Europe and north of Spain, whereas hybridization is demonstrated to have occurred in several populations from the Balkan Peninsula. Furthermore, our study has established the taxonomic status of taxa, for the most part recovered as monophyletic. Cryptic taxa within the group have been identified, and a new species, Veronica dalmatica, is fully described. This study highlights the implications of polyploidy in species delimitation, and illustrates the importance to conserve polyploid populations as potential sources of diversification due to evolutionary significance of genome duplications in plant evolution.