Extensive pollen-mediated gene flow across intensively managed landscapes in an insect-pollinated shrub native to semiarid habitats.

Our knowledge of the impact of landscape fragmentation on gene flow patterns is mainly drawn from tropical and temperate ecosystems, where landscape features, such as the distance of a tree to the forest edge, drive connectivity and mating patterns. Yet, the structure of arid and semiarid plant communities - with open canopies and a scattered distribution of trees - differs greatly from those that are well-characterized in the literature. As a result, we ignore whether the documented consequences of landscape fragmentation on plant mating and gene flow patterns also hold for native plant communities in arid and semiarid regions. We investigated the relative contribution of plant traits, pollinator activity, and individual neighbourhood in explaining variation in mating and gene flow patterns of an insect-pollinated semiarid arborescent shrub, Ziziphus lotus, at three sites embedded in highly altered agriculture landscapes. We used 14 SSRs, seed paternity analyses, and individual mixed effect mating models (MEMMi) to estimate the individual mating variables and the pollen dispersal kernel at each site. Individual spatial location, flower density, and floral visitation rate explained most of the variation of mating variables. Unexpectedly, individual correlated paternity was very low and shrubs surrounded by the most degraded matrix exhibited an increased fraction of pollen immigration and a high effective number of pollen donors per mother shrub. Overall, our results reveal that an active pollinator assemblage ensures highly efficient mating, and maintains pollen-mediated gene flow and notable connectivity levels, even in highly altered landscapes, potentially halting genetic isolation within and between distant sites.

Multiple founder events explain the genetic diversity and structure of the model allopolyploid grass Brachypodium hybridum in the Iberian Peninsula hotspot.

BACKGROUND AND AIMS: It is accepted that contemporary allopolyploid species have originated recurrently, but very few cases have been documented using multiple natural formations of the same species. To extend our knowledge, we have investigated the multiple origins, genetic variation and structure of the allotetraploid grass Brachypodium hybridum with respect to its progenitor diploid species B. distachyon (D genome) and B. stacei (S genome). For this, our primary focus is the Iberian Peninsula, an evolutionary hotspot for the genus Brachypodium. METHODS: We analysed 342 B. hybridum individuals from 36 populations using ten nuclear SSR loci and two plastid loci. The B. hybridum genetic profiles were compared with those previously reported for B. stacei and B. distachyon. In addition, phylogenetic analysis of the plastid data was performed for a reduced subset of individuals. KEY RESULTS: The nuclear simple sequence repeat (SSR) genetic analysis detected medium to high genetic diversity, with a strong south-to-north genetic structure cline, and a high selfing rate in B. hybridum. Comparative genetic analysis showed a close relatedness of current B. hybridum D allelic profiles with those of B. distachyon, but a lack of similarity with those of B. stacei, suggesting another B. stacei source for the B. hybridum S alleles. Plastid analysis detected three different bidirectional allopolyploidization events: two involved distinct B. distachyon-like ancestors and one involved a B. stacei-like ancestor. The south-eastern Iberian Peninsula B. hybridum populations were more genetically diverse and could have originated from at least two hybridization events whereas north-eastern/north-western Iberian Peninsula B. hybridum populations were less diverse and may have derived from at least one hybridization event. CONCLUSIONS: The genetic and evolutionary evidence supports the plausible in situ origin of the south-eastern and northern Iberian Peninsula B. hybridum allopolyploids from their respective local B. distachyon and unknown B. stacei ancestors. The untapped multiple origins and genetic variation detected in these B. hybridum populations opens the way to future evolutionary analysis of allopolyploid formation and genomic dominance and expression in the B. hybridum-B. distachyon-B. stacei grass model complex.

Variation in functional responses to water stress and differentiation between natural allopolyploid populations in the Brachypodium distachyon species complex.

Background and Aims: Some polyploid species show enhanced physiological tolerance to drought compared with their progenitors. However, very few studies have examined the consistency of physiological drought response between genetically differentiated natural polyploid populations, which is key to evaluation of the importance of adaptive evolution after polyploidization in those systems where drought exerts a selective pressure. Methods: A comparative functional approach was used to investigate differentiation of drought-tolerance-related traits in the Brachypodium species complex, a model system for grass polyploid adaptive speciation and functional genomics that comprises three closely related annual species: the two diploid parents, B. distachyon and B. stacei, and the allotetraploid derived from them, B. hybridum. Differentiation of drought-tolerance-related traits between ten genetically distinct B. hybridum populations and its ecological correlates was further analysed. Key Results: The functional drought response is overall well differentiated between Brachypodium species. Brachypodium hybridum allotetraploids showed a transgressive expression pattern in leaf phytohormone content in response to drought. In contrast, other B. hybridum physiological traits correlated to B. stacei ones. Particularly, proline and water content were the traits that best discriminated these species from B. distachyon under drought. Conclusions: After polyploid formation and/or colonization, B. hybridum populations have adaptively diverged physiologically and genetically in response to variations in aridity.

Environmental isolation explains Iberian genetic diversity in the highly homozygous model grass Brachypodium distachyon.

BACKGROUND: Brachypodium distachyon (Poaceae), an annual Mediterranean Aluminum (Al)-sensitive grass, is currently being used as a model species to provide new information on cereals and biofuel crops. The plant has a short life cycle and one of the smallest genomes in the grasses being well suited to experimental manipulation. Its genome has been fully sequenced and several genomic resources are being developed to elucidate key traits and gene functions. A reliable germplasm collection that reflects the natural diversity of this species is therefore needed for all these genomic resources. However, despite being a model plant, we still know very little about its genetic diversity. As a first step to overcome this gap, we used nuclear Simple Sequence Repeats (nSSR) to study the patterns of genetic diversity and population structure of B. distachyon in 14 populations sampled across the Iberian Peninsula (Spain), one of its best known areas. RESULTS: We found very low levels of genetic diversity, allelic number and heterozygosity in B. distachyon, congruent with a highly selfing system. Our results indicate the existence of at least three genetic clusters providing additional evidence for the existence of a significant genetic structure in the Iberian Peninsula and supporting this geographical area as an important genetic reservoir. Several hotspots of genetic diversity were detected and populations growing on basic soils were significantly more diverse than those growing in acidic soils. A partial Mantel test confirmed a statistically significant Isolation-By-Distance (IBD) among all studied populations, as well as a statistically significant Isolation-By-Environment (IBE) revealing the presence of environmental-driven isolation as one explanation for the genetic patterns found in the Iberian Peninsula. CONCLUSIONS: The finding of higher genetic diversity in eastern Iberian populations occurring in basic soils suggests that these populations can be better adapted than those occurring in western areas of the Iberian Peninsula where the soils are more acidic and accumulate toxic Al ions. This suggests that the western Iberian acidic soils might prevent the establishment of Al-sensitive B. distachyon populations, potentially causing the existence of more genetically depauperated individuals.

The interplay between aridity and competition determines colonization ability, exclusion and ecological segregation in the heteroploid Brachypodium distachyon species complex.

A higher competitive advantage of polyploid plants compared with their parental diploids is frequently invoked to explain their establishment success, colonization of novel environments and cytotypic ecological segregation, yet there is scarce experimental evidence supporting such hypotheses. Here, we investigated whether differential competitive ability of species of the Brachypodium distachyon (Poaceae) species complex, a model system for genomic, ecological and evolutionary studies of temperate grasses, contributes to explaining their ecological segregation as well as their coexistence in diploid/allotetraploid contact zones. We conducted two field experiments in dry and humid localities to evaluate the tolerance to competition of diploids and allotetraploids in densely occupied environments, and to parameterize models of intra- and intercytotype competition as a mechanism for species exclusion/coexistence. We provide experimental evidence supporting the hypothesis that, under natural field conditions, allotetraploids have superior ecological success compared with one of their parental diploids in terms of both colonizing competitive habitats and intercytotypic competition, with the balance of intra/intercytotype competition favoring polyploid population establishment. These findings, together with previous data on ecogeographic segregation and adaptive response to water stress, suggest that the interplay between aridity and competitive outcome determines the ability to colonize competitive environments, the exclusion of diploids, especially in arid localities, and species geographic segregation.

Facilitation contributes to Mediterranean woody plant diversity but does not shape the diversity-productivity relationship along aridity gradients.

The diversity-productivity relationship (humped-back model (HBM)) and the stress-gradient (SGH) hypotheses may be connected when productivity is limited primarily by aridity. We analytically connect both hypotheses and assess the contribution of facilitation to woody plant richness along the aridity gradient of the Western Mediterranean floristic region. We monitored regeneration niches of woody plants, obtaining rarefied species richness and plant relative interaction indices in 54 forests and scrublands in a 1750-km geographical range across Spain, Morocco and the Canary Islands. We verified the monotonic increase in facilitation with aridity postulated by SGH and the humped-shape pattern of species richness expected from HBM, which became manifest after expanding the aridity gradient or crossing vegetation types. Along the gradient, interaction balance turned into facilitation earlier in forest than in scrublands. The effects of aridity and interaction balance on species diversity were additive rather than interdependent. Facilitation is an important driver of woody species richness at macroecological scales because it added up to diversity in most sites, with enhanced contribution with increased stress. The HBM was not shaped by species interactions. Results suggest that facilitation may act in Mediterranean vegetation buffering against critical transitions between states allowing woody plant communities to cope with the rise in aridity expected with global warming.

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines.

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.

Environmental niche variation and evolutionary diversification of the Brachypodium distachyon grass complex species in their native circum-Mediterranean range.

UNLABELLED: • PREMISE OF THE STUDY: We conducted environmental niche modeling (ENM) of the Brachypodium distachyon s.l. complex, a model group of two diploid annual grasses (B. distachyon, B. stacei) and their derived allotetraploid (B. hybridum), native to the circum-Mediterranean region. We (1) investigated the ENMs of the three species in their native range based on present and past climate data; (2) identified potential overlapping niches of the diploids and their hybrid across four Quaternary windows; (3) tested whether speciation was associated with niche divergence/conservatism in the complex species; and (4) tested for the potential of the polyploid outperforming the diploids in the native range.• METHODS: Geo-referenced data, altitude, and 19 climatic variables were used to construct the ENMs. We used paleoclimate niche models to trace the potential existence of ancestral gene flow among the hybridizing species of the complex.• KEY RESULTS: Brachypodium distachyon grows in higher, cooler, and wetter places, B. stacei in lower, warmer, and drier places, and B. hybridum in places with intermediate climatic features. Brachypodium hybridum had the largest niche overlap with its parent niches, but a similar distribution range and niche breadth.• CONCLUSIONS: Each species had a unique environmental niche though there were multiple niche overlapping areas for the diploids across time, suggesting the potential existence of several hybrid zones during the Pleistocene and the Holocene. No evidence of niche divergence was found, suggesting that species diversification was not driven by ecological speciation but by evolutionary history, though it could be associated to distinct environmental adaptations.

Environmental aridity is associated with cytotype segregation and polyploidy occurrence in Brachypodium distachyon (Poaceae).

• The ecological and adaptive significance of plant polyploidization is not well understood and no clear pattern of association between polyploid frequency and environment has emerged. Climatic factors are expected to predict cytotype distribution. However, the relationship among climate, cytotype distribution and variation of abiotic stress tolerance traits has rarely been examined. • Here, we use flow cytometry and root-tip squashes to examine the cytotype distribution in the temperate annual grass Brachypodium distachyon in 57 natural populations distributed across an aridity gradient in the Iberian Peninsula. We further investigate the link between environmental aridity, ploidy, and variation of drought tolerance and drought avoidance (flowering time) traits. • Distribution of diploids (2n = 10) and allotetraploids (2n = 30) in this species is geographically structured throughout its range in the Iberian Peninsula, and is associated with aridity gradients. Importantly, after controlling for geographic and altitudinal effects, the link between aridity and polyploidization occurrence persisted. Water-use efficiency varied between ploidy levels, with tetraploids being more efficient in the use of water than diploids under water-restricted growing conditions. • Our results indicate that aridity is an important predictor of polyploid occurrence in B. distachyon, suggesting a possible adaptive origin of the cytotype segregation.

Variation and fitness costs for tolerance to different types of herbivore damage in Boechera stricta genotypes with contrasting glucosinolate structures.

• Analyses of plant tolerance in response to different modes of herbivory are essential to an understanding of plant defense evolution, yet are still scarce. Allocation costs and trade-offs between tolerance and plant chemical defenses may influence genetic variation for tolerance. However, variation in defenses also occurs for the presence or absence of discrete chemical structures; yet, the effects of intraspecific polymorphisms on tolerance to multiple herbivores have not been evaluated. • Here, in a glasshouse experiment, we investigated the variation for tolerance to different types of herbivore damage, and direct allocation costs, in 10 genotypes of Boechera stricta (Brassicaceae), a wild relative of Arabidopsis, with contrasting foliar glucosinolate chemical structures (methionine-derived glucosinolates vs glucosinolates derived from branched-chain amino acids). • We found significant genetic variation for tolerance to different types of herbivore. Structural variations in the glucosinolate profile did not influence tolerance to damage, but predicted plant fitness. Levels of constitutive and induced glucosinolates varied between genotypes with different structural profiles, but we did not detect any cost of tolerance explaining the genetic variation in tolerance among genotypes. • Trade-offs between plant tolerance to multiple herbivores may not explain the existence of intermediate levels of tolerance to damage in plants with contrasting chemical defensive profiles.

Assessing ecological specialization of an ant-seed dispersal mutualism through a wide geographic range.

Specialization in species interactions is of central importance for understanding the ecological structure and evolution of plant-animal mutualisms. Most plant-animal mutualisms are facultative and strongly asymmetric. In particular, myrmecochory (seed dispersal by ants) has been regarded as a very generalized interaction. Although some recent studies have suggested that only a few ant species are really important for dispersal, no rigorous measurement of the specialization in ant-seed dispersal mutualisms has been performed. Here, we use individual plants as basic units for replication to investigate the generalization-specialization of the herb Helleborus foetidus on its ant dispersers over a considerable part of its geographical range. We define generalization in terms of diversity components (species richness and evenness) of the ant visitor that realizes dispersal by removing diaspores. We obtain truly comparable values of ant visitor diversity, distinguishing among different functional groups of visitors and identifying incidental visitors and real ant dispersers. Using null model approaches, we test the null hypothesis that ant-mediated dispersal is a generalized mutualism. At least two premises should be confirmed to validate the hypothesis: (1) diaspores are dispersed by multiple ant-visitor species, and (2) diaspore dispersal is significantly equitable. Though up to 37 ant species visited diaspores across 10 populations, only two large formicines, Camponotus cruentatus and Formica lugubris, were responsible for the vast majority of visits resulting in dispersal in most populations and years, which strongly suggests that ant seed dispersal in H. foetidus is ecologically specialized. Interestingly, specialization degree was unrelated to dispersal success across populations. Our study offers new insights into the spatiotemporal dynamics of myrmecochory. We propose the existence of an alternative scenario to extensive generalization. In this new scenario, generalization is replaced by ecological specialization, which is determined by the intrinsic traits of the plant species rather than by the ecological context in which the interaction takes place.

Conflicting selection on diaspore traits limits the evolutionary potential of seed dispersal by ants.

Conflicts of selection on diaspore traits throughout the dispersal cycle can limit the evolutionary consequences of seed dispersal. However, these conflicts have never been investigated in directed dispersal systems. We explored conflicts of selection through life stages of dispersal in the myrmecochorous herb Helleborus foetidus. Seeds are subject to two contrasting partial selective scenarios. Undispersed seeds are subject to positive directional selection on seed size characters, whereas seeds dispersed are subject to stabilizing selection for size. In both scenarios, seedling establishment determined the magnitude and direction of selection. This does not reflect ant preferences for seed size. However, total selection still depends largely on ant activity, as ants control the relative importance of each selective scenario. We advocate the use of analytical approaches combining multiplicative fitness and microenvironment-specific selection to more realistically estimate the realized selection on traits functional during several life stages. This approach may be extended to any organism dispersing offspring to different environments.

Climate change decreases suitable areas for rapeseed cultivation in Europe but provides new opportunities for white mustard as an alternative oilseed for biofuel production.

Oilseed crops, including several mustards, are cultivated as biofuel sources worldwide. However, common mustard crops (e.g., the rapeseed Brassica napus) grow naturally in mesic temperate regions, which are expected to be impaired by global warming and increased aridity. In particular, increased aridity is predicted to reduce the oil concentration and seed yield of rapeseed crops. There is therefore an urgent need to identify alternative bioenergy crops that are preadapted to future climatic conditions. An alternative to conventional Brassica species for biodiesel production is the white mustard Sinapis alba, which is native to the circum-Mediterranean region and has a high seed lipid content. S. alba grows spontaneously in olive groves and other widespread Mediterranean crops; accordingly, it could be easily cultivated by companion planting to improve ecosystem function by decreasing soil loss, controlling microbial disease, and assisting in the maintenance of biodiversity. In this study, using species distribution modeling, we predicted climatically suitable areas for the cultivation of S. alba in Western Europe across the Mediterranean Basin under present climatic conditions and several climate change scenarios. We show that current climatically suitable areas for S. alba cultivation do not overlap with those for B. napus. Unlike B. napus, S. alba could be cultivated throughout most of the circum-Mediterranean region. According to our models, increases in aridity and annual mean temperatures will expand the climatically suitable areas for S. alba in the Mediterranean Basin. However, suitable areas for the cultivation of B. napus will decrease significantly in Western Europe. Our results indicate that S. alba is a strong, environmentally safe candidate for biofuel production throughout the Mediterranean Basin and other Western European countries, especially under climate change scenarios that are expected to impair current oilseed crops.

Neutral molecular markers support common origin of aluminium tolerance in three congeneric grass species growing in acidic soils.

Aluminium (Al) toxicity is the main abiotic stress limiting plant productivity in acidic soils that are widely distributed among arable lands. Plant species differ in the level of Al resistance showing intraspecific and interspecific variation in many crop species. However, the origin of Al-tolerance is not well known. Three annual species, difficult to distinguish phenotypically and that were until recently misinterpreted as a single complex species under Brachypodium distachyon, have been recently separated into three distinct species: the diploids B. distachyon (2n = 10) and B. stacei (2n = 20), and B. hybridum (2n = 30), the allotetraploid derived from the two diploid species. The aims of this work were to know the origin of Al-tolerance in acidic soil conditions within these three Brachypodium species and to develop new DNA markers for species discrimination. Two multiplex SSR-PCRs allowed to genotype a group of 94 accessions for 17 pentanucleotide microsatellite (SSRs) loci. The variability for 139 inter-microsatellite (ISSRs) markers was also examined. The genetic relationships obtained using those neutral molecular markers (SSRs and ISSRs) support that all Al-tolerant allotetraploid accessions of B. hybridum have a common origin that is related with both geographic location and acidic soils. The possibility that the adaptation to acidic soils caused the isolation of the tolerant B. hybridum populations from the others is discussed. We finally describe a new, easy, DNA barcoding method based in the upstream-intron 1 region of the ALMT1 gene, a tool that is 100 % effective to distinguish among these three Brachypodium species.

Development and characterization of microsatellite primers in the endangered Mediterranean shrub Ziziphus lotus (Rhamnaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed to characterize and evaluate patterns of genetic diversity and structure in the endangered Mediterranean shrub Ziziphus lotus (Rhamnaceae). METHODS AND RESULTS: Twenty microsatellite primers were developed for Z. lotus, of which 14 were polymorphic. We evaluated microsatellite polymorphism in 97 specimens from 18 Spanish and seven Moroccan populations. Between two and eight alleles were found per locus, and the average number of alleles was 5.54. Observed heterozygosity and expected heterozygosity ranged from 0.08 to 0.90 and from 0.08 to 0.82, respectively. Nine of these primers also amplified microsatellite loci in Z. jujuba. CONCLUSIONS: The microsatellite markers described here will be useful in studies on genetic variation, population genetic structure, and gene flow in the fragmented habitat of this species. These markers are a valuable resource for designing appropriate conservation measures for the species in the Mediterranean range.

Natural variation, differentiation, and genetic trade-offs of ecophysiological traits in response to water limitation in Brachypodium distachyon and its descendent allotetraploid B. hybridum (Poaceae).

Differences in tolerance to water stress may underlie ecological divergence of closely related ploidy lineages. However, the mechanistic basis of physiological variation governing ecogeographical cytotype segregation is not well understood. Here, using Brachypodium distachyon and its derived allotetraploid B. hybridum as model, we test the hypothesis that, for heteroploid annuals, ecological divergence of polyploids in drier environments is based on trait differentiation enabling drought escape. We demonstrate that under water limitation allotetraploids maintain higher photosynthesis and stomatal conductance and show earlier flowering than diploids, concordant with a drought-escape strategy to cope with water stress. Increased heterozygosity and greater genetic variability and plasticity of polyploids could confer a superior adaptive capability. Consistent with these predictions, we document (1) greater standing within-population genetic variation in water-use efficiency (WUE) and flowering time in allotetraploids, and (2) the existence of (nonlinear) environmental clines in physiology across allotetraploid populations. Increased gas exchange and diminished WUE occurred at the driest end of the gradient, consistent with a drought-escape strategy. Finally, we found that allotetraploids showed weaker genetic correlations than diploids congruous with the expectation of relaxed pleiotropic constraints in polyploids. Our results suggest evolutionary divergence of ecophysiological traits in each ploidy lineage.

Large-scale adaptive divergence in Boechera fecunda, an endangered wild relative of Arabidopsis.

Many biological species are threatened with extinction because of a number of factors such as climate change and habitat loss, and their preservation depends on an accurate understanding of the extent of their genetic variability within and among populations. In this study, we assessed the genetic divergence of five quantitative traits in 10 populations of an endangered cruciferous species, Boechera fecunda, found in only several populations in each of two geographic regions (WEST and EAST) in southwestern Montana. We analyzed variation in quantitative traits, neutral molecular markers, and environmental factors and provided evidence that despite the restricted geographical distribution of this species, it exhibits a high level of genetic variation and regional adaptation. Conservation efforts therefore should be directed to the preservation of populations in each of these two regions without attempting transplantation between regions. Heritabilities and genetic coefficients of variation estimated from nested ANOVAs were generally high for leaf and rosette traits, although lower (and not significantly different from 0) for water-use efficiency. Measures of quantitative genetic differentiation, Q ST, were calculated for each trait from each pair of populations. For three of the five traits, these values were significantly higher between regions compared with those within regions (after adjustment for neutral genetic variation, F ST). This suggested that natural selection has played an important role in producing regional divergence in this species. Our analysis also revealed that the B. fecunda populations appear to be locally adapted due, at least in part, to differences in environmental conditions in the EAST and WEST regions.

Update on the genomics and basic biology of Brachypodium: International Brachypodium Initiative (IBI).

The scientific presentations at the First International Brachypodium Conference (abstracts available at http://www.brachy2013.unimore.it) are evidence of the widespread adoption of Brachypodium distachyon as a model system. Furthermore, the wide range of topics presented (genome evolution, roots, abiotic and biotic stress, comparative genomics, natural diversity, and cell walls) demonstrates that the Brachypodium research community has achieved a critical mass of tools and has transitioned from resource development to addressing biological questions, particularly those unique to grasses.

A gain-of-function polymorphism controlling complex traits and fitness in nature.

Identification of the causal genes that control complex trait variation remains challenging, limiting our appreciation of the evolutionary processes that influence polymorphisms in nature. We cloned a quantitative trait locus that controls plant defensive chemistry, damage by insect herbivores, survival, and reproduction in the natural environments where this polymorphism evolved. These ecological effects are driven by duplications in the BCMA (branched-chain methionine allocation) loci controlling this variation and by two selectively favored amino acid changes in the glucosinolate-biosynthetic cytochrome P450 proteins that they encode. These changes cause a gain of novel enzyme function, modulated by allelic differences in catalytic rate and gene copy number. Ecological interactions in diverse environments likely contribute to the widespread polymorphism of this biochemical function.