Mariana Castro

Theoretical study of the static first hyperpolarizability of azo-enaminone compounds

In this work the static electric properties of azo-enaminones, with special emphasis to the vector component of the first hyperpolarizability βvec, are determined at the Hartree–Fock (HF) level with the electron correlation (EC) effects included through the second-order Moller–Plesset perturbation theory (MP2). The ab initio results, in accordance with previous semiempirical calculations, show that appropriate choices of substituents to be incorporated to the molecular structure can have a marked influence on the first hyperpolarizability. An initial study about the changes on the βvec values of these compounds, as a result of the incorporation of different donor groups, indicates that this property increases as function of the donor group strength tending to a saturated value. A comparison between our HF and MP2 results, for all compounds studied here, show that the βvec values are strongly affected by the effects of the electron correlation correction.

Reproductive strategy of the invasive Oxalis pes-caprae: distribution patterns of floral morphs, ploidy levels and sexual reproduction

Oxalis pes-caprae, a tristylous flowering plant native to South Africa, is described in the western Mediterranean basin as an asexual—only 5x short-styled morph (5x S-morph) invasive weed losing all mating partners after introduction. The objective of this study was to reassess the patterns of floral morph and cytotype distribution and the sexual reproduction ability in this invaded range. For that, floral morph and cytotype composition were evaluated in 39 populations of O. pes-caprae in a methodical sampling. The reproductive success of natural populations was assessed as fruit and seed production and seed germination for all floral morphs and cytotypes detected. Self- and morph-incompatibility were also studied with controlled hand pollinations. A remarkable diversity in floral morph and cytotype composition was observed. Furthermore, we observed successful sexual reproduction in several localities across the surveyed area. The S-morph is still dominant in this invaded area, and although it was mostly 5x, an additional cytotype (4x) was also recorded. Records of both a mid-styled morph (M-morph) and an area with trimorphic populations of this species are reported here for the first time in the invasive range of the Mediterranean basin. The long-styled morph appears to occur randomly across the surveyed area, while the M-morph is concentrated mainly in Estremadura province (Portugal), where a breakdown in the incompatibility system was observed. These distribution patterns may result from events of sexual reproduction after incompatibility breakdown and/or from multiple introduction events from the native area. The ability to reproduce sexually, undetected so far, may have important impacts in the population dynamics and major consequences for the adaptation and selection potential of O. pes-caprae in this invaded area.

Hidden diversity in wild Beta taxa from Portugal: Insights from genome size and ploidy level estimations using flow cytometry

Crop wild relatives constitute a broad pool of potentially useful genetic resources for plant breeders. The genus Beta L. (Amaranthaceae) is an important source of crops, primarily for sugar production. Until recently, species within Section Beta were mostly cytogenetically uniform, with diploidy being prevalent. Still, with the discovery of tetraploid individuals of the wild B. macrocarpa in the Canary Islands, a large-scale study was necessary to evaluate the cytogenetic diversity within the wild Beta. For that, genome size and ploidy level of B. vulgaris subsp. maritima and B. macrocarpa from 21 populations across Portugal mainland and islands, including all know populations of the later taxon, were estimated using propidium iodide flow cytometry. This work revealed a cytogenetically diverse scenario. The analyzed populations were mostly diploid, except for one population of B. vulgaris subsp. maritima that presented both diploid and tetraploid individuals, and for two populations of B. macrocarpa where two or three cytotypes (diploids, tetraploids and/or hexaploids) were found. The nuclear DNA content of diploid individuals was estimated as 1.44±0.035 and 1.41±0.027 pg/2C for B. vulgaris subsp. maritima and B. macrocarpa, respectively. Also, leaves of both species presented variable levels of endopolyploidy. The obtained results are discussed within the context of interspecific hybridization and cryptic diversity and constitute significant data for the conservation of these wild Beta crop relatives.

Genome size stability despite high chromosome number variation in Carex gr. laevigata

PREMISE OF THE STUDY In organisms with holocentric chromosomes like Carex species, chromosome number evolution has been hypothesized to be a result of fission, fusion, and/or translocation events. Negative, positive, or the absence of correlations have been found between chromosome number and genome size in Carex. METHODS Using the inferred diploid chromosome number and 80 genome size measurements from 26 individuals and 20 populations of Carex gr. laevigata, we tested the null hypothesis of chromosome number evolution by duplication and deletion of whole chromosomes. KEY RESULTS Our results show a significant positive correlation between genome size and chromosome number, but the slope of such correlation supports the hypothesis of proliferation and removal of repetitive DNA fragments to explain genome size variation rather than duplication and deletion of whole chromosomes. CONCLUSIONS Our results refine the theory of the holokinetic drive: this mechanism is proposed to facilitate repetitive DNA removal (or any segmental deletion) when smaller homologous chromosomes are preferentially inherited, or repetitive DNA proliferation (or any segmental duplication) when larger homologs are preferred. This study sheds light on how karyotype evolution plays an important role in the diversification of the species of the genus Carex.

Genome size variation and incidence of polyploidy in Scrophulariaceae sensu lato from the Iberian Peninsula

In this work, genome size variation and polyploidy incidence in Scrophulariaceae from the Iberian Peninsula were addressed, with the obtained results providing important background information for subsequent studies, with several future perspectives being opened.

How much do we know about the frequency of hybridisation and polyploidy in the Mediterranean region

Natural hybridisation and polyploidy are currently recognised as drivers of biodiversity, despite early scepticism about their importance. The Mediterranean region is a biodiversity hotspot where geological and climatic events have created numerous opportunities for speciation through hybridisation and polyploidy. Still, our knowledge on the frequency of these mechanisms in the region is largely limited, despite both phenomena are frequently cited in studies of Mediterranean plants. We reviewed information available from biodiversity and cytogenetic databases to provide the first estimates of hybridisation and polyploidy frequency in the Mediterranean region. We also inspected the most comprehensive modern Mediterranean Flora (Flora iberica) to survey the frequency and taxonomic distribution of hybrids and polyploids in Iberian Peninsula. We found that <6% of Mediterranean plants were hybrids, although a higher frequency was estimated for the Iberian Peninsula (13%). Hybrids were concentrated in few families and in even fewer genera. The overall frequency of polyploidy (36.5%) was comparable with previous estimates in other regions; however our estimates increased when analysing the Iberian Peninsula (48.8%). A surprisingly high incidence of species harbouring two or more ploidy levels was also observed (21.7%). A review of the available literature also showed that the ecological factors driving emergence and establishment of new entities are still poorly studied in the Mediterranean flora, although geographic barriers seem to play a major role in polyploid complexes. Finally, this study reveals several gaps and limitations in our current knowledge about the frequency of hybridisation and polyploidy in the Mediterranean region. The obtained estimates might change in the future with the increasing number of studies; still, rather than setting the complete reality, we hope that this work triggers future studies on hybridisation and polyploidy in the Mediterranean region.

Invasion Fosters Change: Independent Evolutionary Shifts in Reproductive Traits after Oxalis pes-caprae L. Introduction.

Biological invasions offer optimal scenarios to study evolutionary changes under contemporary timescales. After long-distance dispersal, exotic species have to cope with strong mate limitation, and shifts toward uniparental reproduction have been hypothesized to be selectively advantageous. Oxalis pes-caprae is a clonal tristylous species native to South Africa, and invasive in Mediterranean regions worldwide. It reproduces sexually and asexually but the importance of each strategy differs between ranges. Native populations reproduce mostly sexually while in invasive ones asexual reproduction is the prevailing strategy due to the dominance of pentaploid monomorphic populations. Nevertheless, two contrasting scenarios have been observed after introduction: transition toward clonality, and re-acquisition of sexuality fueled by multiple introductions of compatible mates. Here, we aimed to assess evolutionary changes of reproductive traits in O. pes-caprae invasive populations and evaluate whether these traits could be related with invasion success and prevalence of certain forms in the western Mediterranean basin. Sexual and asexual reproduction traits were quantified under optimal conditions in a common garden experiment including native and invasive sexual, predominately asexual, and obligated asexual individuals. Different reproductive, ecological, and genetic constraints created by long-distance dispersal seem to have generated different selective pressures in sexual and asexual traits, with our results supporting evolutionary changes in invasive populations of O. pes-caprae. Native plants had higher sexual fitness, while a transition toward clonality was clear for invasive forms, supporting clonal reproduction as a major trait driving invasion. Differences were also observed among invasive plants, with sexual forms having increased dispersal potential; thus, they are expected to be in advantage in comparison with predominantly asexual and obligated asexual plants, and may become widespread in the future. Historical processes, like the initial introduction of predominantly asexual forms followed by sexual forms more recently, could be in the origin of current distribution patterns of O. pes-caprae in the western Mediterranean. This study shows that invasion processes are very dynamic and that ecological and genetic constraints determined by the invasion process may originate different reproductive strategies that are likely to determine invasion success.

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.

Cytogeography of the subalpine marsh marigold polyploid complex (Caltha leptosepala s.l., Ranunculaceae)

PREMISE OF THE STUDY Unrecognized variation in ploidy level can lead to an underestimation of species richness and a misleading delineation of geographic range. Caltha leptosepala (Ranunculaceae) comprises a complex of hexaploids (6x), rare nonaploids (9x), and dodecaploids (12x), all with unknown distributions. We delineate the geographic distribution and contact zones of the cytotypes, investigate morphologies of cytotypes and subspecies, and discuss the biogeography and evolutionary history of the polyploid complex. METHODS Using cytologically determined specimens as reference, propidium iodide flow cytometry was performed on silica-dried samples and herbarium specimens from across the range of C. leptosepala s.l. Genome size estimates from flow cytometry were used to infer cytotypes. A key morphological character, leaf length-to-width ratio, was measured to evaluate whether these dimensions are informative for taxon and/or cytotype delimitation. KEY RESULTS Dodecaploids were more northerly in distribution than hexaploids, and a single midlatitude population in the Northern Rockies yielded nonaploids. Genome size estimates were significantly different between all cytotypes and between hexaploid subspecies. Leaf length-to-width ratios were significantly different between subspecies and some cytotypes. CONCLUSIONS Caltha leptosepala presents clear patterns of cytotype distribution at the large scale. Marked differences in morphology, range, and genome size were detected between the hexaploid subspecies, C. leptosepala subsp. howellii in the Cascade-Sierra axis and C. leptosepala subsp. leptosepala in the Rockies. Sympatry between cytotypes in the Cascades and a parapatric distribution in the Northern Rockies suggest unique origins and separate lineages in the respective contact zones.

Pollen flow between flowers of the same morph in invasive populations of Oxalis pes-caprae L. in the western Mediterranean region

Oxalis pes-caprae is a tristylous South African geophyte that is invasive in regions with Mediterranean climate. Given the introduction of the short-styled morph only, vegetative reproduction was, until recently, the reproductive mode known for this invaded area. The detection of new floral morphs, fruit and seed production in natural populations of the invaded range, together with the reported weakening in the heteromorphic incompatibility system, raised the question on whether the short-styled flowers are able to exchange pollen among themselves, and if so, which mechanisms account for it. Thus, flower morphometric analysis and field experiments were conducted to assess pollinator visitation rates and pollen flow in three invasive populations. Flower morphological changes suggesting the evolution from tristyly towards semi-homostyly were observed. Moreover, O. pes-caprae flowers were visited by different functional groups of pollinators, mostly hymenopteran. Dye flow experiments revealed that the short-styled flowers were able to successfully exchange pollen among themselves in natural conditions. All of this, together with the weakening of the incompatibility system, constitutes further data that might help to explain the recently reported occurrence of sexual reproduction in this invaded range, which by itself bears important implications for the invasion process of O. pes-caprae.