Genome-wide methylome stability and parental effects in the worldwide distributed Lombardy poplar.

BACKGROUND: Despite the increasing number of epigenomic studies in plants, little is known about the forces that shape the methylome in long-lived woody perennials. The Lombardy poplar offers an ideal opportunity to investigate the impact of the individual environmental history of trees on the methylome. RESULTS: We present the results of three interconnected experiments on Lombardy poplar. In the first experiment, we investigated methylome variability during a growing season and across vegetatively reproduced generations. We found that ramets collected over Europe and raised in common conditions have stable methylomes in symmetrical CG-contexts. In contrast, seasonal dynamics occurred in methylation patterns in CHH context. In the second experiment, we investigated whether methylome patterns of plants grown in a non-parental environment correlate with the parental climate. We did not observe a biological relevant pattern that significantly correlates with the parental climate. Finally, we investigated whether the parental environment has persistent carry-over effects on the vegetative offspring's phenotype. We combined new bud set observations of three consecutive growing seasons with former published bud set data. Using a linear mixed effects analysis, we found a statistically significant but weak short-term, parental carry-over effect on the timing of bud set. However, this effect was negligible compared to the direct effects of the offspring environment. CONCLUSIONS: Genome-wide cytosine methylation patterns in symmetrical CG-context are stable in Lombardy poplar and appear to be mainly the result of random processes. In this widespread poplar clone, methylation patterns in CG-context can be used as biomarkers to infer a common ancestor and thus to investigate the recent environmental history of a specific Lombardy poplar. The Lombardy poplar shows high phenotypic plasticity in a novel environment which enabled this clonal tree to adapt and survive all over the temperate regions of the world.

High levels of effective long-distance dispersal may blur ecotypic divergence in a rare terrestrial orchid.

BACKGROUND: Gene flow and adaptive divergence are key aspects of metapopulation dynamics and ecological speciation. Long-distance dispersal is hard to detect and few studies estimate dispersal in combination with adaptive divergence. The aim of this study was to investigate effective long-distance dispersal and adaptive divergence in the fen orchid (Liparis loeselii (L.) Rich.). We used amplified fragment length polymorphism (AFLP)-based assignment tests to quantify effective long-distance dispersal at two different regions in Northwest Europe. In addition, genomic divergence between fen orchid populations occupying two distinguishable habitats, wet dune slacks and alkaline fens, was investigated by a genome scan approach at different spatial scales (continental, landscape and regional) and based on 451 AFLP loci. RESULTS: We expected that different habitats would contribute to strong divergence and restricted gene flow resulting in isolation-by-adaptation. Instead, we found remarkably high levels of effective long-distance seed dispersal and low levels of adaptive divergence. At least 15% of the assigned individuals likely originated from among-population dispersal events with dispersal distances up to 220 km. Six (1.3%) 'outlier' loci, potentially reflecting local adaptation to habitat-type, were identified with high statistical support. Of these, only one (0.22%) was a replicated outlier in multiple independent dune-fen population comparisons and thus possibly reflecting truly parallel divergence. Signals of adaptation in response to habitat type were most evident at the scale of individual populations. CONCLUSIONS: The findings of this study suggest that the homogenizing effect of effective long-distance seed dispersal may overwhelm divergent selection associated to habitat type in fen orchids in Northwest Europe.

Genetic structure and seed-mediated dispersal rates of an endangered shrub in a fragmented landscape: a case study for Juniperus communis in northwestern Europe.

BACKGROUND: Population extinction risk in a fragmented landscape is related to the differential ability of the species to spread its genes across the landscape. The impact of landscape fragmentation on plant population dynamics will therefore vary across different spatial scales. We quantified successful seed-mediated dispersal of the dioecious shrub Juniperus communis in a fragmented landscape across northwestern Europe by using amplified fragment length polymorphism (AFLP) markers. Furthermore we investigated the genetic diversity and structure on two spatial scales: across northwestern Europe and across Flanders (northern Belgium). We also studied whether seed viability and populations size were correlated with genetic diversity. RESULTS: Unexpectedly, estimated seed-mediated dispersal rates were quite high and ranged between 3% and 14%. No population differentiation and no spatial genetic structure were detected on the local, Flemish scale. A significant low to moderate genetic differentiation between populations was detected at the regional, northwest European scale (PhiPT = 0.10). In general, geographically nearby populations were also genetically related. High levels of within-population genetic diversity were detected but no correlation was found between any genetic diversity parameter and population size or seed viability. CONCLUSIONS: In northwestern Europe, landscape fragmentation has lead to a weak isolation-by-distance pattern but not to genetic impoverishment of common juniper. Substantial rates of successful migration by seed-mediated gene flow indicate a high dispersal ability which could enable Juniperus communis to naturally colonize suitable habitats. However, it is not clear whether the observed levels of migration will suffice to counterbalance the effects of genetic drift in small populations on the long run.

Occasional long-distance dispersal may not prevent inbreeding in a threatened butterfly.

BACKGROUND: To set up successful conservation measures, detailed knowledge on the dispersal and colonization capacities of the focal species and connectivity between populations is of high relevance. We developed species-specific nuclear microsatellite molecular markers for the grayling (Hipparchia semele), a butterfly endemic to Europe and of growing conservation concern in North-West Europe, and report on its population genetics, in a fragmented, anthropogenic landscape in Belgium. Our study included samples from 23 different locations nested in two regions and additional historical samples from two locations. We assessed contemporary, long-distance dispersal based on genetic assignment tests and investigated the effect of habitat loss and fragmentation on the population genetic structure and genetic variation using data of nine microsatellite loci. RESULTS: Detected dispersal events covered remarkably long distances, which were up to ten times larger than previously reported colonisation distances, with the longest movement recorded in this study even exceeding 100 km. However, observed frequencies of long-distance dispersal were low. Our results point to the consequences of the strong population decline of the last decades, with evidence of inbreeding for several of the recently sampled populations and low estimates of effective population sizes (Ne) (ranging from 20 to 54 individuals). CONCLUSIONS: Our study shows low frequencies of long-distance dispersal, which is unable to prevent inbreeding in most of the local populations. We discuss the significance for species conservation including future translocation events and discuss appropriate conservation strategies to maintain viable grayling (meta) populations in highly fragmented, anthropogenic landscapes.

Genetic structure of recently fragmented suburban populations of European stag beetle.

Habitat loss and fragmentation due to urbanization can negatively affect metapopulation persistence when gene flow among populations is reduced and population sizes decrease. Inference of patterns and processes of population connectivity derived from spatial genetic analysis has proven invaluable for conservation and management. However, a more complete account of population dynamics may be obtained by combining spatial and temporal sampling. We, therefore, performed a genetic study on European stag beetle (Lucanus cervus L.) populations in a suburban context using samples collected in three locations and during the period 2002-2016. The sampling area has seen recent landscape changes which resulted in population declines. Through the use of a suite of F ST, clustering analysis, individual assignment, and relatedness analysis, we assessed fine scale spatiotemporal genetic variation within and among habitat patches using 283 individuals successfully genotyped at 17 microsatellites. Our findings suggested the three locations to hold demographically independent populations, at least over time scales of relevance to conservation, though with higher levels of gene flow in the past. Contrary to expectation from tagging studies, dispersal appeared to be mainly female-biased. Although the life cycle of stag beetle suggests its generations to be discrete, no clear temporal structure was identified, which could be attributed to the varying duration of larval development. Since population bottlenecks were detected and estimates of effective number of breeders were low, conservation actions are eminent which should include the establishment of suitable dead wood for oviposition on both local and regional scales to increase (re)colonization success and connectivity among current populations.

Reintroduced Native Populus nigra in Restored Floodplain Reduces Spread of Exotic Poplar Species.

Exotic Populus taxa pose a threat to the success of riparian forest restoration in floodplain areas. We evaluated the impact of exotic Populus taxa on softwood riparian forest development along the river Common Meuse after introducing native Populus nigra and after the re-establishment of the natural river dynamics. We sampled 154 poplar seedlings that spontaneously colonized restored habitat and assessed their taxonomy based on diagnostic chloroplast and nuclear microsatellite markers. Furthermore, by using a paternity analysis on 72 seedlings resulting from six open pollinated P. nigra females, we investigated natural hybridization between frequently planted cultivated poplars and native P. nigra. The majority of the poplar seedlings from the gravel banks analyzed where identified as P. nigra; only 2% of the sampled seedlings exhibited genes of exotic poplar species. Similarly, the majority of the seedlings from the open pollinated progenies were identified as P. nigra. For three seedlings (4%), paternity was assigned to a cultivar of P. × canadensis. Almost two decades after reintroducing P. nigra, the constitution of the seed and pollen pools changed in the study area in favor of reproduction of the native species and at the expense of the exotic poplar species. This study indicates that, although significant gene flow form exotic poplars is observed in European floodplains, restoration programs of the native P. nigra can vigorously outcompete the exotic gene flows and strongly reduce the impact of exotic Populus taxa on the softwood riparian forest development.

Transgenerational effects in asexually reproduced offspring of Populus.

The response of trees to a changing climate can be affected by transgenerational phenotypic plasticity, i.e. phenotypic variation that is conserved and transferred to the offspring. Transgenerational plasticity that is influenced by epigenetics (heritable changes in gene function that do not result from changes in DNA sequence) during both sexual and asexual reproduction are of major relevance for adaptation of plants to climate change. To understand the transgenerational effects on the responses of vegetatively propagated poplar (Populus deltoides and P. trichocarpa) ramets (cuttings) to a changing environment, we tested whether the temperature and photoperiod experienced by the mother trees (genets) persistently affects the phenology of the cuttings grown in a common environment. We weekly monitored the bud phenology of the cuttings collected from the parent trees that have been growing across Europe along a >2100 km latitudinal gradient for at least 18 years. In addition, we asked whether there was variation in DNA methylation as measured by Methylation Sensitive Amplified Fragment Length Polymorphism (MSAPs) in the clones due to the different environmental conditions experienced by the parent trees. Our results indicate a transgenerational effect on bud phenology in the asexually reproduced offspring (vegetative cuttings). The temperatures experienced by the parent tree clones (from different geographic regions) altered the bud flush of the cuttings in the common garden. However, no significant epigenetic variation was detected in the cuttings of the parent trees within single genotypes growing under different climates. In sum, our results show that trees have the potential to respond to rapid climate change but the mechanism behind these changes needs to be further investigated by more powerful molecular methods like whole-genome bisulphite sequencing techniques.

Variability in DNA Methylation and Generational Plasticity in the Lombardy Poplar, a Single Genotype Worldwide Distributed Since the Eighteenth Century.

In the absence of genetic diversity, plants rely on the capacity of phenotypic plasticity to cope with shifts in environmental conditions. Understanding the mechanisms behind phenotypic plasticity and how local phenotypic adjustments are transferred to clonal offspring, will provide insight into its ecological and evolutionary significance. Epigenetic changes have recently been proposed to play a crucial role in rapid environmental adaptation. While the contribution of epigenetic changes to phenotypic plasticity has been extensively studied in sexual reproducing model organisms, little work has been done on vegetative generations of asexual reproducing plant species. We studied the variability of DNA methylation and bud set phenology of the Lombardy poplar (Populus nigra cv. Italica Duroi), a cultivated tree representing a single genotype worldwide distributed since the eighteenth century. Bud set observations and CpG methyl polymorphisms were studied on vegetative offspring resulting from cuttings grown for one season in a common glasshouse environment. The cuttings were collected from 60 adult Lombardy poplars growing in different environments. The physiological condition of the cuttings was determined by measuring weight and nutrient condition. Methylation sensitive amplified polymorphisms were used to obtain global patterns of DNA methylation. Using logistic regression models, we investigated correlations among epigenotype, bud phenology, and the climate at the home site of the donor trees, while accounting for physiological effects. We found significant epigenetic variation as well as significant variation in bud phenology, in the absence of genetic variation. Remarkably, phenology of bud set observed at the end of the growing season in the common environment was significantly correlated with climate variables at the home site of the mother trees, specifically the average temperature of January and monthly potential evapotranspiration. Although we could not directly detect significant effects of epigenetic variation on phenology, our results suggest that, in the Lombardy poplar, epigenetic marks contribute to the variation of phenotypic response that can be transferred onto asexually reproduced offspring resulting in locally adapted ecotypes. This contributes to the growing evidence that epigenetic-based transgenerational inheritance might be relevant for adaptation and evolution in contrasting or rapidly changing environments.

The contribution of mating system variation to reproductive isolation in two closely related Centaurium species (Gentianaceae) with a generalized flower morphology.

In closely related plant species that display strong similarities in phenology and pollinator communities, differences in breeding system and associated shifts in floral traits may have important effects on the magnitude and direction of heterospecific pollen flow and hybridization. Here, we quantified the strength of several pre- and postzygotic barriers acting between the facultatively outcrossing Centaurium erythraea and the predominantly selfing C. littorale via a suite of experiments, and estimated the frequency of hybridization in the field using molecular markers. The reproductive barriers primarily responsible for preventing hybridization were essentially prezygotic and these acted asymmetrically. Due to differences in floral display, pollen production, and pollen transfer rates, heterospecific pollen flow occurred predominantly from C. erythraea to C. littorale. In C. littorale, on the other hand, close anther-stigma positioning and resulting higher capacity for autonomous selfing functioned as an efficient barrier to counterbalance the higher risk for hybrid mating. In both species the action of all reproductive barriers resulted in a small opportunity for hybrid establishment, which was confirmed by the occurrence of only ∼1% putative hybrids in the field. Our findings confirm that differences in breeding system affect heterospecific pollen transfer patterns and that autonomous selfing may efficiently prevent hybridization.