Phytochemical and Cytogenetic Study of Two Centaurea Species from Croatia: the Particular Case of Diploid and Tetraploid C. salonitana.

Natural wild populations of C. rupestris and C. salonitana were studied to determine possible relationships between the volatile oil (VO) composition and ploidy level. The chemical composition of the volatile oil was investigated using the GC/MS technique. The predominant components of the VO of diploid and tetraploid C. salonitana were hexadecanoic acid and α-linoleic acids, while in C. rupestris they were germacrene D and ß-caryophyllene in one population and heptacosane and germacrene D, in another. The nuclear DNA amounts (2 C DNA), determined by flow cytometry, were 3.54 pg for C. rupestris, 3.39 pg for the diploid and 6.79 pg for the tetraploid population of C. salonitana. Evidence that the degree of ploidy solely influences the chemical composition of the essential oil of C. salonitana was not found. The results presented are the first data to be reported on the DNA content of the studied Centaurea populations from Croatia, as well as on the chemical composition of C. salonitana volatile oil.

Morphologic, genetic, and biogeographic continua among subspecies hinder the conservation of threatened taxa: the case of Centaurea aspera ssp. scorpiurifolia (Asteraceae).

Subspecies are widely included as conservation units because of their potential to become new species. However, their practical recognition includes variable criteria, such as morphological, genetic, geographic and other differences. Centaurea aspera ssp. scorpiurifolia is a threatened taxon endemic to Andalusia (Spain), which coexists in most of its distribution area with similar taxa. Because of the difficulty to identify it using morphology alone, we aimed to sample all the populations cited as ssp. scorpiurifolia as exhaustively as possible, morphologically characterise them, and analyse their genetic structuring using microsatellites, to better understand difficulties when conserving subspecies. Three different Centaurea species were found which were easily identified. Within C. aspera, two genetic populations and some admixed individuals were observed, one including ssp. scorpiurifolia individuals and the other including individuals identified as subspecies aspera, stenophylla, and scorpiurifolia. A morphological continuum between these two genetic populations and a wide overlapping of their biogeographic distribution were also found. This continuum can affect the conservation of ssp. scorpiurifolia because of potential misidentifications and harmful effects of subspecific hybridization. Misidentifications could be partly overcome by using as many different traits as possible, and conservation priority should be given to populations representative of the ends of this continuum.

Trait variation, trade-offs, and attributes may contribute to colonization and range expansion of a globally distributed weed.

PREMISE: Trait variation, trade-offs, and attributes can facilitate colonization and range expansion. We explored how those trait features compare between ancestral and nonnative populations of the globally distributed weed Centaurea solstitialis. METHODS: We measured traits related to survival, size, reproduction, and dispersal in field sampling following major environmental gradients; that of elevation in Anatolia (ancestral range) and that of precipitation in Argentina (nonnative range). We also estimated abundance. RESULTS: We found that overall variation in traits in ancestral populations was similar to that in nonnative populations. Only one trait-seed mass-displayed greater variation in ancestral than nonnative populations; coincidentally, seed mass has been shown to track global range expansion of C. solstitialis. Traits displayed several associations, among which seed mass and number were positively related in both ranges. Many traits varied with elevation in the ancestral range, whereas none varied with precipitation in the nonnative one. Interestingly, most traits varying with elevation within the ancestral range also displayed differences in attributes between ancestral and nonnative ranges. Unexpectedly, ancestral plants were more fecund than nonnative plants, but density was greater in the nonnative than ancestral range, indicating that C. solstitialis survives at larger proportions in the nonnative than ancestral range. CONCLUSIONS: Our results suggest that maintaining levels of trait variation in nonnative populations comparable to those in ancestral populations, avoiding trait trade-offs, and developing differences in trait attributes between ranges can play a major role in the success of many weeds in novel environments.

CcMYB6-1 and CcbHLH1, two novel transcription factors synergistically involved in regulating anthocyanin biosynthesis in cornflower.

Anthocyanins in cornflower (Centaurea cyanus) is catalysed by a set of biosynthesis genes, however, the potential mechanism of transcriptional regulation remains unclear. In the present study, we traced the dynamic changes of petal colour development from white to violet and finally to blue on the same petal in cornflower. Pigment analysis showed that anthocyanin accumulation dramatically increased with petal colour development. Subsequently, nine libraries from above three colour regions were constructed for RNA-seq and 105,506 unigenes were obtained by de novo assembling. The differentially expressed genes among three colour regions were significantly enriched in the phenylpropanoid biosynthesis and flavonoid biosynthesis pathways, leading to the excavation and analysis of 46 biosynthesis genes involved in this process. Furthermore, four R2R3-CcMYBs clustered into subgroup 4 or subgroup 6 and one CcbHLH1 clustered into IIIf subgroup were screened out by phylogenetic analysis with Arabidopsis homologues. The promoters of flavanone 3-hydroxylase (CcF3H) and dihydroflavonol 4-reductase (CcDFR) were further isolated to investigate upstream regulation mechanism. CcMYB6-1 significantly upregulated the activity of above two promoters and stimulated anthocyanin accumulation by dual luciferase assay and transient expression in tobacco leaves, and its activity was obviously enhanced when co-infiltrated with CcbHLH1. Moreover, both yeast two-hybrid and bimolecular fluorescence complementation assays indicated the protein-protein interaction between these two activators. Based on these obtained results, it reveals that CcMYB6-1 and CcbHLH1 are two novel transcription factors synergistically involved in regulating anthocyanin biosynthesis. This study provides insights into the regulatory mechanism of anthocyanin accumulation in cornflower.

Islands as a crossroad of evolutionary lineages: A case study of Centaurea sect. Centaurea (Compositae) from Sardinia (Mediterranean Basin).

The Mediterranean Basin is a biodiversity hotspot, where islands play a key role because of their high biological diversity, degree of endemicity and human pressure. One of these islands, Sardinia, is a good evolutionary laboratory, especially for the study of complex genera, such as Centaurea. In particular, endemic species of Centaurea sect. Centaurea from Sardinia provides an interesting case study of plant evolution on continental islands. We attempted to clarify the processes leading to the diversification of Centaurea species on Sardinia using bi-parentally inherited nuclear markers and maternally inherited plastid markers. Our plastid results revealed the presence of five lineages of sect. Centaurea on the island. Three of them were defined as three species: C. ferulacea, C. filiformis and C. horrida. The other two lineages highlighted the complex evolutionary history of the two polyploids C. corensis and C. magistrorum. Multiple colonization events from the mainland involving the C. deusta and C. paniculata lineages among others, have led to the diversity of sect. Centaurea on Sardinia. One colonization event likely followed a southern path via the land connection between the mainland, the Calabrian Plate and Sardinia. A second pathway likely followed a northern connection, probably through the Tuscan Archipelago. Implications of these findings on conservation efforts for Centaurea endemics on Sardinia are also discussed.

Expansion history and environmental suitability shape effective population size in a plant invasion.

The margins of an expanding range are predicted to be challenging environments for adaptation. Marginal populations should often experience low effective population sizes (Ne ) where genetic drift is high due to demographic expansion and/or census population size is low due to unfavourable environmental conditions. Nevertheless, invasive species demonstrate increasing evidence of rapid evolution and potential adaptation to novel environments encountered during colonization, calling into question whether significant reductions in Ne are realized during range expansions in nature. Here we report one of the first empirical tests of the joint effects of expansion dynamics and environment on effective population size variation during invasive range expansion. We estimate contemporary values of Ne using rates of linkage disequilibrium among genome-wide markers within introduced populations of the highly invasive plant Centaurea solstitialis (yellow starthistle) in North America (California, USA), and within native Eurasian populations. As predicted, we find that Ne within the invaded range is positively correlated with both expansion history (time since founding) and habitat quality (abiotic climate). History and climate had independent additive effects with similar effect sizes, indicating an important role for both factors in this invasion. These results support theoretical expectations for the population genetics of range expansion, though whether these processes can ultimately arrest the spread of an invasive species remains an unanswered question.

Native and Invading Yellow Starthistle (Centaurea solstitialis) Microbiomes Differ in Composition and Diversity of Bacteria.

Invasive species could benefit from being introduced to locations with more favorable species interactions, including the loss of enemies, the gain of mutualists, or the simplification of complex interaction networks. Microbiomes are an important source of species interactions with strong fitness effects on multicellular organisms, and these interactions are known to vary across regions. The highly invasive plant yellow starthistle (Centaurea solstitialis) has been shown to experience more favorable microbial interactions in its invasions of the Americas, but the microbiome that must contribute to this variation in interactions is unknown. We sequenced amplicons of 16S rRNA genes to characterize bacterial community compositions in the phyllosphere, ectorhizosphere, and endorhizosphere of yellow starthistle plants from seven invading populations in California, USA, and eight native populations in Europe. We tested for the differentiation of microbiomes by geography, plant compartment, and plant genotype. Bacterial communities differed significantly between native and invading plants within plant compartments, with consistently lower diversity in the microbiome of invading plants. The diversity of bacteria in roots was positively correlated with plant genotype diversity within both ranges, but this relationship did not explain microbiome differences between ranges. Our results reveal that these invading plants are experiencing either a simplified microbial environment or simplified microbial interactions as a result of the dominance of a few taxa within their microbiome. Our findings highlight several alternative hypotheses for the sources of variation that we observe in invader microbiomes and the potential for altered bacterial interactions to facilitate invasion success.IMPORTANCE Previous studies have found that introduced plants commonly experience more favorable microbial interactions in their non-native range, suggesting that changes to the microbiome could be an important contributor to invasion success. Little is known about microbiome variation across native and invading populations, however, and the potential sources of more favorable interactions are undescribed. Here, we report one of the first microbiome comparisons of plants from multiple native and invading populations, in the noxious weed yellow starthistle. We identify clear differences in composition and diversity of microbiome bacteria. Our findings raise new questions about the sources of these differences, and we outline the next generation of research that will be required to connect microbiome variation to its potential role in plant invasions.

Potential limits to the benefits of admixture during biological invasion.

Species introductions often bring together genetically divergent source populations, resulting in genetic admixture. This geographic reshuffling of diversity has the potential to generate favourable new genetic combinations, facilitating the establishment and invasive spread of introduced populations. Observational support for the superior performance of admixed introductions has been mixed, however, and the broad importance of admixture to invasion questioned. Under most underlying mechanisms, admixture's benefits should be expected to increase with greater divergence among and lower genetic diversity within source populations, though these effects have not been quantified in invaders. We experimentally crossed source populations differing in divergence in the invasive plant Centaurea solstitialis. Crosses resulted in many positive (heterotic) interactions, but fitness benefits declined and were ultimately negative at high source divergence, with patterns suggesting cytonuclear epistasis. We explored the literature to assess whether such negative epistatic interactions might be impeding admixture at high source population divergence. Admixed introductions reported for plants came from sources with a wide range of genetic variation, but were disproportionately absent where there was high genetic divergence among native populations. We conclude that while admixture is common in species introductions and often happens under conditions expected to be beneficial to invaders, these conditions may be constrained by predictable negative genetic interactions, potentially explaining conflicting evidence for admixture's benefits to invasion.

Sympatric diploid and tetraploid cytotypes of Centaurea stoebe s.l. do not differ in arbuscular mycorrhizal communities and mycorrhizal growth response.

PREMISE OF THE STUDY: Genome duplication is associated with multiple changes at different levels, including interactions with pollinators and herbivores. Yet little is known whether polyploidy may also shape belowground interactions. METHODS: To elucidate potential ploidy-specific interactions with arbuscular mycorrhizal fungi (AMF), we compared mycorrhizal colonization and assembly of AMF communities in roots of diploid and tetraploid Centaurea stoebe s.l. (Asteraceae) co-occurring in a Central European population. In a follow-up greenhouse experiment, we tested inter-cytotype differences in mycorrhizal growth response by combining ploidy, substrate, and inoculation with native AMF in a full-factorial design. KEY RESULTS: All sampled plants were highly colonized by AMF, with the Glomeraceae predominating. AMF-community composition revealed by 454-pyrosequencing reflected the spatial distribution of the hosts, but not their ploidy level or soil characteristics. In the greenhouse experiment, the tetraploids produced more shoot biomass than the diploids did when grown in a more fertile substrate, while no inter-cytotype differences were found in a less fertile substrate. AMF inoculation significantly reduced plant growth and improved P uptake, but its effects did not differ between the cytotypes. CONCLUSIONS: The results do not support our hypotheses that the cytotype structure in a mixed-ploidy population of C. stoebe is mirrored in AMF-community composition and that ploidy-specific fungal communities contribute to cytotype co-existence. Causes and implications of the observed negative growth response to AMF are discussed.

Is population genetic structure of vascular plants shaped more by ecological or geographic factors? A study case on the Mediterranean endemic Centaurea filiformis (Asteraceae).

All known populations of the Sardinian endemic Centaurea filiformis Viv. (Asteraceae) were studied in order to understand the impact of both geographic and ecological factors on the genetic structuring of this species. Fourteen populations and 234 individuals were sampled. The demographic structure of the populations and the reproductive ecology were estimated in 28 plots. Population genetic analyses were based on SSR markers. Genetic structure was investigated by spatial Bayesian methods. Average densities of 0.51 individuals m-2 were detected, with a prevalence of adults. Ten species of pollinators were identified; C. filiformis ability to self-pollinate and myrmecochory were demonstrated experimentally. The populations displayed an average heterozygosity value of He  = 0.576 and high genetic differentiation (overall FST  = 0.218). Bayesian analysis suggests that five is the most probable number of gene pools of origin. A strong correlation between geographic distances and genetic distances among populations was highlighted. The demographic population structure of C. filiformis is dominated by adults, suggesting that it is a stable-regressive or senile species, investing more in local persistence than colonisation ability. Despite the scattered distribution, the populations studied do not present evidence of genetic erosion. The analysis of genetic differentiation reveals very high differentiation levels among populations, thus indicating that effective barriers exist against gene flow. A general conclusion is that population distribution results in a clear genetic structure for the populations studied, and that geography and not ecology is shaping the present distribution of this species.

Local pre-adaptation to disturbance and inbreeding-environment interactions affect colonisation abilities of diploid and tetraploid Centaurea stoebe.

Primary colonisation in invasive ranges most commonly occurs in disturbed habitats, where anthropogenic disturbance may cause physical damage to plants. The tolerance to such damage may differ between cytotypes and among populations as a result of differing population histories (adaptive differentiation between ruderal verus natural habitats). Moreover, founder populations often experience inbreeding depression, the effects of which may increase through physical damage due to inbreeding-environment interactions. We aimed to understand how such colonisation processes differ between diploid and tetraploid Centaurea stoebe populations, with a view to understanding why only tetraploids are invasive. We conducted a clipping experiment (frequency: zero, once or twice in the growing season) on inbred versus outbred offspring originating from 37 C. stoebe populations of varying cytotype, range and habitat type (natural versus ruderal). Aboveground biomass was harvested at the end of the vegetation period, while re-sprouting success was recorded in the following spring. Clipping reduced re-sprouting success and biomass, which was significantly more pronounced in natural than in ruderal populations. Inbreeding depression was not detected under benign conditions, but became increasingly apparent in biomass when plants were clipped. The effects of clipping and inbreeding did not differ between cytotypes. Adaptive differentiation in disturbance tolerance was higher among populations than between cytotypes, which highlights the potential of pre-adaptation in ruderal populations during early colonisation on anthropogenically disturbed sites. While the consequences of inbreeding increased through clipping-mediated stress, they were comparable between cytotypes, and consequently do not contribute to understanding the cytotype shift in the invasive range.

Phytochemical and Cytogenetic Characterization of Centaurea solstitialis L. (Asteraceae) from Croatia.

The cytogenetic characterization of Centaurea solstitialis L. (Asteraceae) showed a chromosome number of 2n = 16. Karyotype is composed by four pairs of metacentric, two pairs of submetacentric and two pairs of subtelocentric chromosomes. Physical mapping of two rDNA probes revealed two loci of 35S and one locus of 5S rRNA genes. Chromomycin fluorochrome banding revealed that all rDNA loci were GC rich. The genome size (2C-value) of 1.95 pg classes this species in the group of very small genomes. Chemical composition of C. solstitialis volatile oil (VO) from Croatia, studied with gas chromatography-mass spectrometry showed dominant components as it follows: hexadecanoic acid, α-linolenic acid, germacrene D and heptacosane. Antioxidant capacity, measured by ferric reducing power assay and 2,2-diphenyl-1-picrylhydrazyl methods, as well as inhibition of acetyl- and butyrylcholinesterase of VO was lower comparing to a standard solutions. Volatile oil tested with disc diffusion method showed good inhibitory potential against Pseudomonas aeruginosa, Escherichia coli and all tested fungi: Candida albicans, Penicillium funiculosum and Aspergillus fumigatus. The microdilution method showed best activity against Chronobacter sakazakii and A. fumigatus.

Population genomic analyses reveal a history of range expansion and trait evolution across the native and invaded range of yellow starthistle (Centaurea solstitialis).

Identifying sources of genetic variation and reconstructing invasion routes for non-native introduced species is central to understanding the circumstances under which they may evolve increased invasiveness. In this study, we used genome-wide single nucleotide polymorphisms to study the colonization history of Centaurea solstitialis in its native range in Eurasia and invasions into the Americas. We leveraged this information to pinpoint key evolutionary shifts in plant size, a focal trait associated with invasiveness in this species. Our analyses revealed clear population genomic structure of potential source populations in Eurasia, including deep differentiation of a lineage found in the southern Apennine and Balkan Peninsulas and divergence among populations in Asia, eastern Europe and western Europe. We found strongest support for an evolutionary scenario in which western European populations were derived from an ancient admixture event between populations from eastern Europe and Asia, and subsequently served as the main genetic 'bridgehead' for introductions to the Americas. Introductions to California appear to be from a single source region, and multiple, independent introductions of divergent genotypes likely occurred into the Pacific Northwest. Plant size has evolved significantly at three points during range expansion, including a large size increase in the lineage responsible for the aggressive invasion of the California interior. These results reveal a long history of colonization, admixture and trait evolution in C. solstitialis, and suggest routes for improving evidence-based management decisions for one of the most ecologically and economically damaging invasive species in the western United States.

Speciation and genetic diversity in Centaurea subsect. Phalolepis in Anatolia.

Mountains of Anatolia are one of the main Mediterranean biodiversity hotspots and their richness in endemic species amounts for 30% of the flora. Two main factors may account for this high diversity: the complex orography and its role as refugia during past glaciations. We have investigated seven narrow endemics of Centaurea subsection Phalolepis from Anatolia by means of microsatellites and ecological niche modelling (ENM), in order to analyse genetic polymorphisms and getting insights into their speciation. Despite being narrow endemics, all the studied species show moderate to high SSR genetic diversity. Populations are genetically isolated, but exchange of genes probably occurred at glacial maxima (likely through the Anatolian mountain arches as suggested by the ENM). The lack of correlation between genetic clusters and (morpho) species is interpreted as a result of allopatric diversification on the basis of a shared gene pool. As suggested in a former study in Greece, post-glacial isolation in mountains would be the main driver of diversification in these plants; mountains of Anatolia would have acted as plant refugia, allowing the maintenance of high genetic diversity. Ancient gene flow between taxa that became sympatric during glaciations may also have contributed to the high levels of genetic diversity.

Neighbour Origin and Ploidy Level Drive Impact of an Alien Invasive Plant Species in a Competitive Environment.

Our understanding of the potential mechanisms driving the spread and naturalization of alien plant species has increased over the past decades, but specific knowledge on the factors contributing to their increased impact in the introduced range is still urgently needed. The native European plant Centaurea stoebe occurs as two cytotypes with different life histories (monocarpic diploids, allo-polycarpic tetraploids). However, only tetraploids have been found in its introduced range in North America, where C. stoebe has become a most prominent plant invader. Here, we focus on the ploidy level of C. stoebe and origin of neighbouring community in explaining the high impact during the invasion of new sites in the introduced range. We conducted a mesocosm experiment under open-field conditions with the diploid (EU2x) and tetraploid (EU4x) cytotype of Centaurea stoebe from its native European (EU) range, and with the invasive tetraploid (NA4x) cytotype from the introduced North American (NA) range in competition with EU (old) or NA (new) neighbouring plant communities. In the presence of competition, the biomass of EU neighbouring community was reduced to a comparable level by all three geo-cytotypes of C. stoebe. In contrast, the biomass of the NA neighbouring community was reduced beyond when competing with tetraploid, but not with diploid C. stoebe. The fact that the biomass of all three geo-cytotypes of C. stoebe was correlated with the biomass of the EU neighbouring community, but not with that of the NA neighbouring community suggests that different mechanisms underlie the competitive interactions between C. stoebe and its old vs. new neighbouring communities, such as competition for the same limiting resources at home vs competition through novel allelo-chemicals or differential resource uptake strategies in the introduced range. We therefore caution to simply use the ecosystem impact assessed at home to predict impact in the introduced range.

Allogamy-Autogamy Switch Enhance Assortative Mating in the Allotetraploid Centaurea seridis L. Coexisting with the Diploid Centaurea aspera L. and Triggers the Asymmetrical Formation of Triploid Hybrids.

Hybridization between tetraploids and their related diploids is generally unsuccessful in Centaurea, hence natural formation of triploid hybrids is rare. In contrast, the diploid Centaurea aspera and the allotetraploid C. seridis coexist in several contact zones where a high frequency of triploid hybrids is found. We analyzed the floral biology of the three taxa to identify reproductive isolation mechanisms that allow their coexistence. Flowering phenology was recorded, and controlled pollinations within and between the three taxa were performed in the field. Ploidy level and germination of progeny were also assessed. There was a 50% flowering overlap which indicated a phenological shift. Diploids were strictly allogamous and did not display mentor effects, while tetraploids were found to be highly autogamous. This breakdown of self-incompatibility by polyploids is first described in Centaurea. The asymmetrical formation of the hybrid was also found: all the triploid intact cypselae came from the diploid mothers pollinated by the pollen of tetraploids. Pollen and eggs from triploids were totally sterile, acting as a strong triploid block. These prezygotic isolation mechanisms ensured higher assortative mating in tetraploids than in diploids, improving their persistence in the contact zones. However these mechanisms can also be the cause of the low genetic diversity and high genetic structure observed in C. seridis.

Mountains and refuges: Genetic structure and evolutionary history in closely related, endemic Centaurea in continental Greece.

Mountains of continental Greece are one of the main Mediterranean biodiversity hotspots, very rich in endemic species. The speciation in this area might have resulted from two main factors: a complex orography and its role as a refugium during past glaciations. We have investigated genetic diversity and population structure for a group of narrow endemics of Centaurea subsect. Phalolepis, with three main goals: to investigate population structure of these narrow endemics, to check whether patterns of genetic variation are in agreement with recognized species boundaries, and to get insights into the process of diversification within this group. Fifteen populations belonging to seven species were genotyped using cpDNA (rpl32-trnL region) sequences and nuclear microsatellites (eight loci). SSR were used to assess genetic variability, to analyse molecular variance, to identify genetic barriers, to estimate recent and historical gene flow, and to carry out a model-based Bayesian clustering. Analysis of cpDNA was used to construct a haplotype network. Despite being narrow endemics, all the studied species show moderate to high SSR genetic diversity. Genetic isolation of populations is very high, with no current gene flow among them. Patterns of genetic structure indicate that there are more genetic clusters than there are currently recognized taxa. Genetic data suggest that isolation in mountain ranges and subsequent allopatric speciation would be the main driver of diversification in the group; the refugial nature of the mountains of continental Greece has allowed the maintenance of high within-population genetic diversity.

Fragment size does not matter when you are well connected: effects of fragmentation on fitness of coexisting gypsophiles.

Most habitat fragmentation studies have focused on the effects of population size on reproductive success of single species, but studies assessing the effects of both fragment size and connectivity, and their interaction, on several coexisting species are rare. In this study, we selected 20 fragments along two continuous gradients of size and degree of isolation in a gypsum landscape in central Spain. In each fragment, we selected 15 individuals of each of three dominant gypsophiles (Centaurea hyssopifolia, Lepidium subulatum and Helianthemum squamatum, 300 plants per species, 900 plants in total) and measured several reproductive traits: inflorescence number, fruit set, seed set and seed mass. We hypothesised that plant fitness would be lower on small and isolated fragments due to an interaction between fragment size and connectivity, and that response patterns would be species-specific. Overall, fragment size had very little effect on reproductive traits compared to that of connectivity. We observed a positive effect of fragment connectivity on C. hyssopifolia fitness, mediated by the increased seed predation in plants from isolated fragments, resulting in fewer viable seeds per capitulum and lower seed set. Furthermore, seed mass was lower in plants from isolated fragments for both C. hyssopifolia and L. subulatum. In contrast, few reproductive traits of H. squamatum were affected by habitat fragmentation. We discuss the implications of species-specific responses to habitat fragmentation for the dynamics and conservation of gypsum plant communities. Our results highlight the complex interplay among plants and their mutualistic and antagonistic visitors, and reinforce the often-neglected role of habitat connectivity as a key component of the fragmentation process.

Contemporary evolution of plant reproductive strategies under global change is revealed by stored seeds.

Global change is expected to impose new selection pressures on natural populations. Phenotypic responses, such as earlier phenology in response to climate warming, have been repeatedly observed in the field. The recent pollinator decline is also expected to change selection on reproductive traits in flowering plants. However, it remains unclear whether short-term adaptation of plant reproductive strategies occurs in response to global change. In this study, we report the evolution of some important reproductive traits of the annual self-incompatible weed Centaurea cyanus. In a common garden experiment, we germinated stored seeds, sampled 18 years apart from the same location, in a region where warmer springs and indices of pollinator decline have been reported. Compared to the ancestral population (1992), our results showed that plants of the descendant population (2010) flowered earlier and also produced larger capitula with longer receptivity and a larger floral display. QST -FST comparisons indicated that natural selection has likely contributed to the evolution of some of the traits investigated. Lower FST within temporal samples than among spatial samples further suggests a limited role of gene flow from neighbouring populations. We therefore propose that trait shifts could partly be due to adaptation to global change.

A rare case of a natural contact zone in Morocco between an autopolyploid and an allopolyploid of Centaurea aspera with sterile tetraploid hybrids.

A new contact zone between Centaurea aspera and Centaurea seridis was found in Morocco. Chromosome counts and flow cytometry showed that both taxa were tetraploid (4x = 44). A literature review and morphometric analysis established that C. aspera corresponds to the autopolyploid C. aspera subsp. gentilii and C. seridis corresponds to the allopolyploid C. seridis var. auriculata. This contact area was compared with the homologous contact zones in Spain formed by the diploid C. aspera subsp. stenophylla and the tetraploid C. seridis subsp. maritima. Natural hybrids between parental species were frequent in both areas. In Spain, hybrids were triploid (from reduced gametes A and gamete AB), highly sterile and exerted a 'triploid block'. In Morocco, cytometry showed that hybrids were tetraploid and, therefore, probably fertile, but all the capitula lacked achenes. It is likely that the resulting genome of the new tetraploid hybrid (AAAB), through the fusion of reduced gametes AA (from subsp. gentilii) and AB (from var. auriculata), could explain irregularities in meiosis through formation of aneuploid gametes and, therefore, infertility of the hybrid. Moroccan sterile tetraploid hybrids develop, but have the identical irregularities to Spanish triploids, probably due to the odd number of homologous chromosomes. The new hybrid is first described as C. x subdecurrens nothosubsp. paucispinus. In addition, distribution and ecological traits are analysed.