Genetic response of a perennial grass to warm and wet environments interacts and is associated with trait means as well as plasticity.

Potential for rapid evolution is an important mechanism allowing species to adapt to changing climatic conditions. Although such a potential has been largely studied in various short-lived organisms, to what extent we can observe similar patterns in long-lived plant species, which often dominate natural systems, is largely unexplored. We explored potential for rapid evolution in Festuca rubra, long-lived grass with extensive clonal growth dominating in alpine grasslands. We used field sowing experiment simulating expected climate change in our model region. Specifically, we exposed seeds from 5 independent seed sources to novel climatic conditions by shifting them along a natural climatic grid and explored genetic profiles of established seedling after 3 years. Data on genetic profiles of plants selected under different novel conditions indicate that different climate shifts select significantly different pools of genotypes from common seed pools. Increasing soil moisture was more important selective pressure than increasing temperature or interaction of the two climatic factors. This can indicate negative genetic interaction in response to the combined effects, or that the effects of different climates are interactive rather than additive. The selected alleles were found in genomic regions likely affecting function of specific genes or their expression. Many of these were also linked to morphological traits (mainly to trait plasticity) suggesting these changes may have a consequence for plant performance. Overall, these data indicate that even long-lived plant species may experience strong selection by climate, and their populations thus have the potential to rapidly adapt to these novel conditions.

Evidence of the Ability of Microsatellite Method to Distinguish Cannabis Strains with High Cannabinoid Content.

Introduction: Cannabis is a plant with high potential for use in several sectors of the industry; however, it is also a controversial crop due to its tetrahydrocannabinol (THC) content. Moreover, the plant has a rather unclarified classification. Traditionally, two types of Cannabis have been distinguished, hemp as a source of fiber and low THC content, and marijuana with high THC levels, which is used as a drug. With the increasing use of CBD strains and wide range of commercially used THC strains, it is becoming paramount to be able to develop an easy and reliable method for Cannabis strain differentiation. The use of simple sequence repeat markers, or microsatellites, seems to be an applicable choice. Materials and Methods: In this study, 52 strains of Cannabis with variable cannabinoid content were collected from growers from different geographical regions and analyzed using 17 different microsatellite markers. For more precise differentiation, five strains were selected and a higher number of individuals of each were analyzed. Results: Fragment analysis and cluster analysis showed that when one to three individual plants per strain were analyzed, the method was able to classify these samples into distinguishable groups with similar gene structure. They also revealed that when a larger sample set was used (10 individual plants per strain), highly specific strain clusters could be fully discriminated. Conclusion: Our study involved the highest number of cannabinoid-rich strains up to now and showed that the microsatellite method can be used to reliably differentiate Cannabis strains and show their relationships.

Development of microsatellite markers for the perennial plant Tofieldia calyculata.

PREMISE: Polymorphic microsatellite markers were developed to study genetic diversity and genetic structure of populations of the locally endangered species Tofieldia calyculata (Tofieldiaceae). METHODS AND RESULTS: Nineteen polymorphic microsatellite loci were developed using DNA-enriched libraries sequenced by Ilumina technology and were then used to genotype 101 individuals from five populations from Austria, Slovakia, Poland, and the Czech Republic. Of the markers tested, 68% were polymorphic in four of the five investigated populations, with one marker polymorphic in all populations. The number of alleles per locus in the populations ranged from one to 11. Levels of observed and expected heterozygosity ranged from 0.00 to 0.75 and from 0.00 to 0.84, respectively. Moreover, six of the 19 loci amplified when tested in the congeneric species T. pusilla. CONCLUSIONS: The 19 newly developed microsatellite markers can be used to describe genetic diversity and population structure of populations of T. calyculata.

Evolutionary potential of a widespread clonal grass under changing climate.

Adaptive responses are probably the most effective long-term responses of populations to climate change, but they require sufficient evolutionary potential upon which selection can act. This requires high genetic variance for the traits under selection and low antagonizing genetic covariances between the different traits. Evolutionary potential estimates are still scarce for long-lived, clonal plants, although these species are predicted to dominate the landscape with climate change. We studied the evolutionary potential of a perennial grass, Festuca rubra, in western Norway, in two controlled environments corresponding to extreme environments in natural populations: cold-dry and warm-wet, the latter being consistent with the climatic predictions for the country. We estimated genetic variances, covariances, selection gradients and response to selection for a wide range of growth, resource acquisition and physiological traits, and compared their estimates between the environments. We showed that the evolutionary potential of F. rubra is high in both environments, and genetic covariances define one main direction along which selection can act with relatively few constraints to selection. The observed response to selection at present is not sufficient to produce genotypes adapted to the predicted climate change under a simple, space for time substitution model. However, the current populations contain genotypes which are pre-adapted to the new climate, especially for growth and resource acquisition traits. Overall, these results suggest that the present populations of the long-lived clonal plant may have sufficient evolutionary potential to withstand long-term climate changes through adaptive responses.

Unmasking cryptic biodiversity in polyploids: origin and diversification of Aster amellus aggregate.

Background and Aims: The origin of different cytotypes by autopolyploidy may be an important mechanism in plant diversification. Although cryptic autopolyploids probably comprise the largest fraction of overlooked plant diversity, our knowledge of their origin and evolution is still rather limited. Here we study the presumed autopolyploid aggregate of Aster amellus, which encompasses diploid and hexaploid cytotypes. Although the cytotypes of A. amellus are not morphologically distinguishable, previous studies showed spatial segregation and limited gene flow between them, which could result in different evolutionary trajectories for each cytotype. Methods: We combine macroevolutionary, microevolutionary and niche modelling tools to disentangle the origin and the demographic history of the cytotypes, using chloroplast and nuclear markers in a dense population sampling in central Europe. Key Results: Our results revealed a segregation between diploid and hexaploid cytotypes in the nuclear genome, where each cytotype represents a monophyletic lineage probably homogenized by concerted evolution. In contrast, the chloroplast genome showed intermixed connections between the cytotypes, which may correspond to shared ancestral relationships. Phylogeny, demographic analyses and ecological niche modelling supported an ongoing differentiation of the cytotypes, where the hexaploid cytotype is experiencing a demographic expansion and niche differentiation with respect to its diploid relative. Conclusions: The two cytotypes may be considered as two different lineages at the onset of their evolutionary diversification. Polyploidization led to the occurrence of hexaploids, which expanded and changed their ecological niche.

Microsatellite markers for study of the invasive species Rumex alpinus (Polygonaceae).

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed to study genetic diversity, population structure, and dispersal strategies of the highly invasive species Rumex alpinus (Polygonaceae). METHODS AND RESULTS: Fifteen polymorphic microsatellite loci were developed using a 454 sequencing approach and used to genotype 72 individuals from six populations in Austria and France. All markers were polymorphic in at least one investigated population, and the number of alleles ranged from one to four alleles per locus. Observed and expected heterozygosity ranged from 0.06 to 1.0 and from 0.18 to 0.72, respectively. CONCLUSIONS: These 15 markers will be useful for characterizing dispersal strategies and gene flow assessment between R. alpinus populations and other, often weedy Rumex species.

Adaptive differentiation of Festuca rubra along a climate gradient revealed by molecular markers and quantitative traits.

Species response to climate change is influenced by predictable (selective) and unpredictable (random) evolutionary processes. To understand how climate change will affect present-day species, it is necessary to assess their adaptive potential and distinguish it from the effects of random processes. This will allow predicting how different genotypes will respond to forecasted environmental change. Space for time substitution experiments are an elegant way to test the response of present day populations to climate variation in real time. Here we assess neutral and putatively adaptive variation in 11 populations of Festuca rubra situated along crossed gradients of temperature and moisture using molecular markers and phenotypic measurements, respectively. By comparing population differentiation in putatively neutral molecular markers and phenotypic traits (QST-FST comparisons), we show the existence of adaptive differentiation in phenotypic traits and their plasticity across the climatic gradient. The observed patterns of differentiation are due to the high genotypic and phenotypic differentiation of the populations from the coldest (and wettest) environment. Finally, we observe statistically significant covariation between markers and phenotypic traits, which is likely caused by isolation by adaptation. These results contribute to a better understanding of the current adaptation and evolutionary potential to face climate change of a widespread species. They can also be extrapolated to understand how the studied populations will adjust to upcoming climate change without going through the lengthy process of phenotyping.

Strong fluctuations in aboveground population size do not limit genetic diversity in populations of an endangered biennial species.

Assessing genetic diversity within populations of rare species and understanding its determinants are crucial for effective species protection. While a lot is known about the relationships between genetic diversity, fitness, and current population size, very few studies explored the effects of past population size. Knowledge of past population size may, however, improve our ability to predict future population fates. We studied Gentianella praecox subsp. bohemica, a biennial species with extensive seed bank. We tested the effect of current, past minimal and maximal population size, and harmonic mean of population sizes within the last 15 years on genetic diversity and fitness. Maximum population size over the last 15 years was the best predictor of expected heterozygosity of the populations and was significantly related to current population size and management. Plant fitness was significantly related to current as well as maximum population size and expected heterozygosity. The results suggested that information on past population size may improve our understanding of contemporary genetic diversity across populations. They demonstrated that despite the strong fluctuations in population size, large reductions in population size do not result in immediate loss of genetic diversity and reduction of fitness within the populations. This is likely due to the seed bank of the species serving as reservoir of the genetic diversity of the populations. From a conservation point of view, this suggests that the restoration of small populations of short-lived species with permanent seed bank is possible as these populations may still be genetically diverse.

Development of SSR markers by 454 sequencing in the endemic species Gentianella praecox subsp. bohemica (Gentianaceae).

PREMISE OF THE STUDY: Polymorphic microsatellite loci were developed and used to genotype individuals of Gentianella praecox subsp. bohemica (Gentianaceae), a highly protected taxon in Europe, to study the genetic structure of the remaining populations. METHODS AND RESULTS: Thirty-eight primer pairs were successfully amplified; of these, 12 polymorphic microsatellite loci were developed using a 454 sequencing approach and used to genotype 180 individuals of G. praecox subsp. bohemica from six populations. Allelic richness ranged between one and nine alleles per locus. We detected a high frequency of polyploid individuals (77.8%). The highest average percentage of heterozygous genotypes was identified for samples from the Hroby population (75.5%). All loci can also be amplified in the congeneric species G. praecox subsp. praecox, G. amarella subsp. amarella, and G. obtusifolia subsp. sturmiana. CONCLUSIONS: These markers will provide knowledge on patterns of gene flow and population genetic structure, which is necessary for current protection actions and for effective conservation of this species in the future.

Selection on meiosis genes in diploid and tetraploid Arabidopsis arenosa.

Meiotic chromosome segregation is critical for fertility across eukaryotes, and core meiotic processes are well conserved even between kingdoms. Nevertheless, recent work in animals has shown that at least some meiosis genes are highly diverse or strongly differentiated among populations. What drives this remains largely unknown. We previously showed that autotetraploid Arabidopsis arenosa evolved stable meiosis, likely through reduced crossover rates, and that associated with this there is strong evidence for selection in a subset of meiosis genes known to affect axis formation, synapsis, and crossover frequency. Here, we use genome-wide data to study the molecular evolution of 70 meiosis genes in a much wider sample of A. arenosa. We sample the polyploid lineage, a diploid lineage from the Carpathian Mountains, and a more distantly related diploid lineage from the adjacent, but biogeographically distinct Pannonian Basin. We find that not only did selection act on meiosis genes in the polyploid lineage but also independently on a smaller subset of meiosis genes in Pannonian diploids. Functionally related genes are targeted by selection in these distinct contexts, and in two cases, independent sweeps occurred in the same loci. The tetraploid lineage has sustained selection on more genes, has more amino acid changes in each, and these more often affect conserved or potentially functional sites. We hypothesize that Pannonian diploid and tetraploid A. arenosa experienced selection on structural proteins that mediate sister chromatid cohesion, the formation of meiotic chromosome axes, and synapsis, likely for different underlying reasons.

Efficacy of S-adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)-DHPA, against the growth of Cryptosporidium parvum in vitro.

D-eritadenine and (S)-DHPA are aliphatic adenosine analogues known to target S-adenosylhomocysteine hydrolase (SAHH) and potent antiviral compounds. In the present study, we demonstrate that these two compounds also display efficacy against recombinant SAHH enzyme of the protozoan parasite Cryptosporidium parvum, as well as inhibition of parasite growth in vitro. Our data confirm that SAHH could serve as a rational drug target in cryptosporidial infection and antiviral adenosine analogues are potential candidates for drug development against cryptosporidiosis.