Polyploidy and hybridization in the Mediterranean: unravelling the evolutionary history of Centaurium (Gentianaceae).

BACKGROUND AND AIMS: Polyploidy is considered one of the main mechanisms of plant evolution and speciation. In the Mediterranean Basin, polyploidy has contributed to making this region a biodiversity hotspot, along with its geological and climatic history and other ecological and biogeographical factors. The Mediterranean genus Centaurium (Gentianaceae) comprises ~25 species, of which 60 % are polyploids, including tetraploids and hexaploids. To date, the evolutionary history of centauries has been studied using Sanger sequencing phylogenies, which have been insufficient to fully understand the phylogenetic relationships in this lineage. The goal of this study is to gain a better understanding of the evolutionary history of Centaurium by exploring the mechanisms that have driven its diversification, specifically hybridization and polyploidy. We aim to identify the parentage of hybrid species, at the species or clade level, as well as assessing whether morphological traits are associated with particular ploidy levels. METHODS: We sequenced RADseq markers from 42 samples of 28 Centaurium taxa, and performed phylogenomic analyses using maximum likelihood, summary coalescent SVDquartets and Neighbor-Net approaches. To identify hybrid taxa, we used PhyloNetworks and the fastSTRUCTURE algorithm. To infer the putative parental species of the allopolyploids, we employed genomic analyses (SNIPloid). The association between different traits and particular ploidy levels was explored with non-metric multidimensional scaling. KEY RESULTS: Our phylogenetic analyses confirmed the long-suspected occurrence of recurrent hybridization. The allopolyploid origin of the tetraploid C. serpentinicola and the hexaploids C. mairei, C. malzacianum and C. centaurioides was also confirmed, unlike that of C. discolor. We inferred additional signatures of hybridization events within the genus and identified morphological traits differentially distributed in different ploidy levels. CONCLUSIONS: This study highlights the important role that hybridization has played in the evolution of a Mediterranean genus such as Centaurium, leading to a polyploid complex, which facilitated its diversification and may exemplify that of other Mediterranean groups.

Relevance of DNA preservation for future botany and ecology.

The use of molecular methods in plant systematics and taxonomy has increased during the last decades; however, the accessibility of curated genetic samples and their metadata is a bottleneck for DNA-based genetic studies in botany. Plant biodiversity DNA banks and DNA-friendly collections could be critical suppliers of curated genetic material for researchers in the current context of plant biodiversity loss. Here, we aimed to understand the potential of plant DNA banks and DNA-friendly collections to enhance the growth and openness of scientific knowledge. The preservation of genetic material should become part of a natural collection's process for the generation of extended specimens enabling the preservation of both the phenotype and genotype and contributing to the generation of data networks which cross-fertilize other fields. These curated collections are advantageous in endangered species research, detecting processes related to extinction, giving a genetic dimension to IUCN assessments or completing the Leipzig Catalogue of Vascular Plants. Therefore, DNA collections are fundamental in producing FAIR data, responsible research and innovation (RRI) and meeting the goals of international conservation programmes. The completion of natural collections is important for current research efforts and furthermore vital to support future research in an era of ongoing plant biodiversity loss.

The role of cyclic nucleotides in bacterial antimicrobial resistance and tolerance.

Nucleotide signalling molecules - mainly cyclic 3',5'-adenosine phosphate (cAMP), bis-(3',5')-cyclic diguanosine monophosphate (c-di-GMP), and bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) - contribute to the regulation of cellular pathways. Numerous recent works have focused on the involvement of these cyclic nucleotide phosphates (cNPs) in bacterial resistance and tolerance to antimicrobial treatment. Indeed, the rise of antimicrobial resistance (AMR) is a rising global threat to human health, while the rise of antimicrobial tolerance underlies the development of AMR and long-term infections, placing an additional burden on this problem. Here, we summarise the current understanding of cNP signalling in bacterial physiology with a focus on our understanding of how cNP signalling affects AMR and antimicrobial tolerance in different bacterial species. We also discuss additional cNP-related drug targets in bacterial pathogens that may have therapeutic potential.

Genetic Diversity and Population Structure Assessed Using Microsatellite (SSR) Markers from Relict Populations of Nuphar pumila (Nymphaeaceae).

The genus Nuphar (Nymphaeaceae) comprises aquatic plant species inhabiting freshwater bodies of the Northern hemisphere temperate regions known as yellow water-lilies. Nuphar lutea and N. pumila are the only representatives in the European continent and present different ecologies: the former is a widespread generalist, while the latter is restricted to northern latitudes or high-altitudes due to its requirements for colder and oligotrophic waters. The Central Europe mountainous areas, the Massif Central (France) and the Cantabrian Mountains (north Iberian Peninsula) harbor relict isolated N. pumila populations endangered by eutrophication and hybridization with N. lutea. We aim to detect hybridization processes in the Massif Central and Cantabrian Mountains populations and compare the genetic diversity of N. pumila in the relict populations of Central Europe by using microsatellite (SSR) markers. No evidence of hybridization was found in the Iberian population, whereas the admixture between N. pumila and N. lutea in the Massif Central populations could be due to hybridization or ancient introgression. Our current knowledge would benefit from genetic diversity studies focusing on both species throughout their distributional range. The Iberian and Massif Central N. pumila populations were genetically distinct, representing two different clusters from other relict populations, with low genetic diversity and a genetic boundary within Central Europe.

What Hides in the Heights? The Case of the Iberian Endemism Bromus picoeuropeanus.

Bromus picoeuropeanus is a recently described species belonging to a complex genus of grasses. It inhabits stony soils at heights ranging from 1600 to 2200 m in Picos de Europa (Cantabrian Mountains, northern Spain). This species is morphologically very similar to B. erectus, partially sharing its presumed distribution range. We aim to determine the relationship between these species and their altitudinal ranges in Picos de Europa and the Cantabrian Mountains by conducting phylogenetic analyses based on nuclear (ETS and ITS) and chloroplastic (trnL) markers. Phylogenetic trees were inferred by Maximum Likelihood and Bayesian Inference. Haplotype networks were estimated based on the plastid marker. Although the ITS topologies could not generate exclusive clades for these species, the ETS analyses generated highly supported B. picoeuropeanus exclusive clades, which included locations outside its altitudinal putative range. The ETS-ITS and ETS-ITS-trnL topologies generated B. picoeuropeanus exclusive clades, whereas the trnL-based trees and haplotype networks were unable to discriminate B. erectus and B. picoeuropeanus. This evidence suggests that B. picoeuropeanus is a separate species with a larger distribution than previously thought, opening new questions regarding the evolution of B. erectus and other similar species in European mountainous systems. However, more information is needed regarding B. picoeuropeanus susceptibility to temperature rises.

Effect of extracellular matrix on adhesion, viability, actin cytoskeleton and K+ currents of cells expressing human ether à go-go channels.

Ether à go-go (EAG) potassium channels possess oncogenic properties and have gained great interest as research tools for cancer detection and therapy. Besides, EAG electrophysiological properties are regulated through the cell cycle and determined by cytoskeletal interactions. Thus, because of the pivotal role of extracellular matrix (ECM) and cytoskeleton in cancer progression, we studied the effect of ECM components on adhesion, viability, actin organization and EAG currents in wild-type CHO cells (CHO-wt) and cells expressing human EAG channels (CHO-hEAG). At short incubation times, adhesion and viability of CHO-hEAG cells grown on collagen, heparin or poly-lysine were lower than CHO-wt cells, however, only CHO-hEAG sustained growing under total serum starvation. CHO-hEAG cells grown on poly-lysine did not organize their cytoskeleton but when grown on collagen or fibronectin displayed lamellipodia and stress fibers, respectively. Interestingly, EAG expressing cells displayed special actin structures suggesting a dynamic actin cytoskeleton, such structures were not exhibited by wild-type cells. EAG current density was significantly lower in cells grown on collagen at short incubation times. Finally, we studied potential associations between hEAG channels and integrins or actin filaments by confocal microscopy. No association between beta1-integrins and hEAG channels was found, however, a very strong co-localization was observed between hEAG channels and actin filaments, supported by immunoblot experiments in which hEAG channels were found in the insoluble fraction (associated to cytoskeleton). Our results suggest ECM components as potential modulators of oncogenic human-EAG expressing cells and emphasize the relationship between potassium channels, cytoskeleton, ECM and cancer.

Role of extracellular matrix-cell interaction and epidermal growth factor (EGF) on EGF-receptors and actin cytoskeleton arrangement in infantile pituitary cells.

Epidermal growth factor (EGF) induces changes in cell morphology, actin cytoskeleton, and adhesion processes in cultured infantile pituitary cells. The extracellular matrix, through integrin engagement, collaborates with growth factors in cell signaling. We have examined the participation of collagen I/III and collagen plus fibronectin in the EGF response of infantile pituitary cells with respect to their cell morphology and actin cytoskeleton. As a comparison, we have used poly-lysine as a substrate. Infantile cells elicit the EGF response when they are associated with extracellular matrix proteins, but no response can be obtained with poly-lysine as the substrate. Cells acquire a flattened shape and organize their actin filaments and vinculin as in focal adhesions. Because the EGF receptor (EGFR) is linked to the actin cytoskeleton in other cells structuring a microdomain in cell signaling, we have investigated this association and substrate adhesion participation in infantile pituitary cells. The proportion of EGFR associated with the actin cytoskeleton is approximately 31%; no difference has been observed between the substrates used. Cells in suspension show actin-associated EGFR, suggesting an association independent of cell adhesion. However, no colocalization of EGFRs with actin fibers has been observed, suggesting an indirect association. Compared with beta(1)-integrin, which is linked to actin fibers through structural proteins, EGFR binds more strongly with the actin cytoskeleton. This study thus shows cell adhesion dependence on the EGF effect in the actin cytoskeleton arrangement; this is probably favored by the actin fiber/EGFR association that facilitates the cell signaling pathways for actin cytoskeleton organization in infantile pituitary cells.

Epidermal growth factor induces an increase in cell adhesion and an arrangement of actin skeleton in stress fibres in pituitary cultured cells from infantile rats but not adult rats.

The rat anterior pituitary gland undergoes changes in its cyto-architecture during the second and third weeks of postnatal life. However, little is known about the factors that regulate these tissue conformational changes. The epidermal growth factor (EGF) is one of the growth factors that are synthesized by the pituitary gland, and almost all of the pituitary cells have EGF receptors (EGFR). In addition to the effects of the EGF on mitosis and differentiation, this growth factor can modulate cell adhesion, cell migration, and cytoskeletal organization. In this study we focussed our attention in examining the effects of EGF on the adhesion of cells to the extracellular matrix and on the actin cytoskeletal arrangement of pituitary cells from infantile and adult rats. Our results show that in infantile cells the EGF induces cell adhesion with increase in cell surface area. The arrangement of actin-F in infantile EGF-treated cells was in stress fibers and vinculin acquired a striped shape at the membrane border, suggesting the assembly of focal adhesion contacts. In contrast, in adult pituitary cells EGF does not induce any change in cell adhesion, and the cells maintain a rounded shape with an arrangement of actin-F in thin cortical bands even though, immuno-localization of the EGFR was observed in adult cells cultured in defined medium. We also looked for the EGFR in membrane preparations from infantile and adult pituitaries, and a marked difference in membrane EGFR was observed between them, the infantile pituitaries showing a significantly higher amount. Our results suggest that in infantile cells EGF induces the assembly of focal adhesion contacts, and that in adult cells the receptor of this growth factor is uncoupled of the signaling pathway by which a rearrangement of actin cytoskeleton occurs.