eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account.

BACKGROUND: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management. RESULTS: Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA. CONCLUSIONS: These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents' detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg.

Molecular and quantitative genetic variation within and between populations of the declining grassland species Saxifraga granulata.

Formerly common plant species are expected to be particularly susceptible to recent habitat fragmentation. We studied the population genetics of 19 recently fragmented Saxifraga granulata populations (max. distance 61 km) in Luxembourg and neighboring Germany using RAPD markers and a common garden experiment. We assessed (1) the relationships between plant fitness, quantitative genetic variation, molecular genetic variation, and population size; and (2) the relative importance of genetic drift and selection in shaping genetic variation. Molecular genetic diversity was high but did not correlate with population size, habitat conditions, or plant performance. Genetic differentiation was low (F ST = 0.079 ± 0.135), and there was no isolation by distance. Longevity, clonality, and the long-lived seed bank of S. granulata may have prevented strong genetic erosion and genetic differentiation among populations. However, genetic distinctness increased with decreasing genetic diversity indicating that random genetic drift occurred in the studied populations. Quantitative and molecular genetic variations were correlated, and their differentiation (Q ST vs. F ST) among S. granulata populations was similar, suggesting that mainly random processes have shaped the quantitative genetic differentiation among populations. However, pairwise quantitative genetic distances increased with geographic and climatic distances, even when adjusted for molecular genetic distances, indicating diversifying selection. Our results indicate that long-lived clonal species may be buffered at least temporarily against the negative effects of fragmentation. The relationship between quantitative genetic and geographic distance may be a more sensitive indicator of selection than Q ST-F ST differences.

Getting rid of 'rain' and 'stars': Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg.

ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets ('stars'). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets ('stars') and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve).

Genetic diversity and differentiation of populations of Anthyllis vulneraria along elevational and latitudinal gradients.

The abundant centre model (ACM) predicts that the suitability of environmental conditions for a species decreases from the centre of its distribution toward its range periphery and, consequently, its populations will become scarcer, smaller and more isolated, resulting in lower genetic diversity and increased differentiation. However, little is known about whether genetic diversity shows similar patterns along elevational and latitudinal gradients with similar changes in important environmental conditions. Using microsatellite markers, we studied the genetic diversity and structure of 20 populations each of Anthyllis vulneraria along elevational gradients in the Alps from the valleys to the elevational limit (2500 m) and along a latitudinal gradient (2500 km) from Central Europe to the range margin in northern Scandinavia. Both types of gradients corresponded to an 11.5°C difference in mean annual temperature. Genetic diversity strongly declined and differentiation increased with latitude in line with the predictions of the ACM. However, as population size did not decline with latitude and genetic diversity was not related to population size in A. vulneraria, this pattern is not likely to be due to less favorable conditions in the North, but due to serial founder effects during the post-glacial recolonization process. Genetic diversity was not related to elevation, but we found significant isolation by distance along both gradients, although the elevational gradient was shorter by orders of magnitude. Subarctic populations differed genetically from alpine populations indicating that the northern populations did not originate from high elevational Alpine ones. Our results support the notion that postglacial latitudinal colonization over large distances resulted in a larger loss of genetic diversity than elevational range shifts. The lack of genetic diversity in subarctic populations may threaten their long-term persistence in the face of climate change, whereas alpine populations could benefit from gene flow from low-elevation populations.

eDNA-based detection of the invasive crayfish Pacifastacus leniusculus in streams with a LAMP assay using dependent replicates to gain higher sensitivity.

LAMP assays are becoming increasingly popular in the field of invasive species detection but are still underused in eDNA-based monitoring. Here, we propose a LAMP assay designed to detect the North American crayfish species Pacifastacus leniusculus in water samples from streams. The presence of P. leniusculus was detected through this new LAMP assay in all but one of the nine sites sampled. No correlation was found between ddPCR absolute concentration measurements and the number of LAMP-positive technical replicates. However, we showed that using dependent technical replicates could significantly enhance the detection sensitivity of the LAMP assay. Applied to other assays, it could improve sensitivity and thus allow for a more efficient use of eDNA-based LAMP assays for invasive species detection in aquatic ecosystems.

Altitude and latitude have different effects on population characteristics of the widespread plant Anthyllis vulneraria.

Widespread plants may provide natural models for how population processes change with temperature and other environmental variables and how they may respond to global change. Similar changes in temperature can occur along altitudinal and latitudinal gradients, but hardly any study has compared the effects of the two types of gradients. We studied populations of Anthyllis vulneraria along a latitudinal gradient from Central Europe to the range limit in the North and an altitudinal gradient in the Alps from 500 m to the altitudinal limit at 2500 m, both encompassing a change in annual mean temperature of c. 11.5 °C. Plant size and reproduction decreased, but plant density increased along both gradients, indicating higher recruitment and demographic compensation among vital rates. Our results support the view that demographic compensation may be common in widespread species in contrast to the predictions of the abundant centre model of biogeography. Variation in temperature along the gradients had the strongest effects on most population characteristics, followed by that in precipitation, solar radiation, and soil nutrients. The proportion of plants flowering, seed set and seed mass declined with latitude, while the large variation in these traits along the altitudinal gradient was not related to elevation and covarying environmental variables like annual mean temperature. This suggests that it will be more difficult to draw conclusions about the potential impacts of future climate warming on plant populations in mountains, because of the importance of small-scale variation in environmental conditions.

Ecological niche differences between two polyploid cytotypes of Saxifraga rosacea.

PREMISE: Different cytotypes of a species may differ in their morphology, phenology, physiology, and their tolerance of extreme environments. We studied the ecological niches of two subspecies of Saxifraga rosacea with different ploidy levels: the hexaploid Central European endemic subspecies sponhemica and the more widely distributed octoploid subspecies rosacea. METHODS: For both cytotypes, we recorded local environmental conditions and mean plant trait values in populations across their areas of distribution, analyzed their distributions by niche modeling, studied their performance at two transplant sites with contrasting conditions, and experimentally tested their cold resistance. RESULTS: Mean annual temperature was higher in hexaploid than in octoploid populations and experiments indicated that frost tolerance of the hexaploid is lower than that of the octoploid. Reproduction of octoploids from Central Europe was higher than that of hexaploids at a transplant site in subarctic Iceland, whereas the opposite was true in temperate Luxembourg, indicating adaptation of the octoploids to colder conditions. Temperature variables were also most important in niche models predicting the distribution of the two cytotypes. Genetic differences in survival among populations were larger for the octoploids than for the hexaploids in both field gardens, suggesting that greater genetic variability may contribute to the octoploid's larger distributional range. CONCLUSIONS: Our results support the hypotheses that different cytotypes may have different niches leading to spatial segregation, and that higher ploidy levels can result in a broader ecological niche and greater tolerance of more extreme conditions.

Genetic structure of colline and montane populations of an endangered plant species.

Due to land-use intensification, lowland and colline populations of many plants of nutrient-poor grasslands have been strongly fragmented in the last decades, with potentially negative consequences for their genetic diversity and persistence. Populations in mountains might represent a genetic reservoir for grassland plants, because they have been less affected by land-use changes. We studied the genetic structure and diversity of colline and montane Vosges populations of the threatened perennial plant Arnica montana in western central Europe using AFLP markers. Our results indicate that in contrast to our expectation even strongly fragmented colline populations of A. montana have conserved a considerable amount of genetic diversity. However, mean seed mass increased with the proportion of polymorphic loci, suggesting inbreeding effects in low diversity populations. At a similar small geographical scale there was a clear IBD pattern for the montane Vosges but not for the colline populations. However, there was a strong IBD-pattern for the colline populations at a large geographical scale suggesting that this pattern is a legacy of historical gene flow, as most of the colline populations are today strongly isolated from each other. Genetic differentiation between colline and montane Vosges populations was strong. Moreover, results of a genome scan study indicated differences in loci under selection, suggesting that plants from montane Vosges populations might be maladapted to conditions at colline sites. Our results suggest caution in using material from montane populations of rare plants for the reinforcement of small genetically depauperate lowland populations.

Divergent selection along climatic gradients in a rare central European endemic species, Saxifraga sponhemica.

BACKGROUND AND AIMS: The effects of habitat fragmentation on quantitative genetic variation in plant populations are still poorly known. Saxifraga sponhemica is a rare endemic of Central Europe with a disjunct distribution, and a stable and specialized habitat of treeless screes and cliffs. This study therefore used S. sponhemica as a model species to compare quantitative and molecular variation in order to explore (1) the relative importance of drift and selection in shaping the distribution of quantitative genetic variation along climatic gradients; (2) the relationship between plant fitness, quantitative genetic variation, molecular genetic variation and population size; and (3) the relationship between the differentiation of a trait among populations and its evolvability. METHODS: Genetic variation within and among 22 populations from the whole distribution area of S. sponhemica was studied using RAPD (random amplified polymorphic DNA) markers, and climatic variables were obtained for each site. Seeds were collected from each population and germinated, and seedlings were transplanted into a common garden for determination of variation in plant traits. KEY RESULTS: In contrast to previous results from rare plant species, strong evidence was found for divergent selection. Most population trait means of S. sponhemica were significantly related to climate gradients, indicating adaptation. Quantitative genetic differentiation increased with geographical distance, even when neutral molecular divergence was controlled for, and QST exceeded FST for some traits. The evolvability of traits was negatively correlated with the degree of differentiation among populations (QST), i.e. traits under strong selection showed little genetic variation within populations. The evolutionary potential of a population was not related to its size, the performance of the population or its neutral genetic diversity. However, performance in the common garden was lower for plants from populations with reduced molecular genetic variation, suggesting inbreeding depression due to genetic erosion. CONCLUSIONS: The findings suggest that studies of molecular and quantitative genetic variation may provide complementary insights important for the conservation of rare species. The strong differentiation of quantitative traits among populations shows that selection can be an important force for structuring variation in evolutionarily important traits even for rare endemic species restricted to very specific habitats.

Genomics and the challenging translation into conservation practice.

The global loss of biodiversity continues at an alarming rate. Genomic approaches have been suggested as a promising tool for conservation practice as scaling up to genome-wide data can improve traditional conservation genetic inferences and provide qualitatively novel insights. However, the generation of genomic data and subsequent analyses and interpretations remain challenging and largely confined to academic research in ecology and evolution. This generates a gap between basic research and applicable solutions for conservation managers faced with multifaceted problems. Before the real-world conservation potential of genomic research can be realized, we suggest that current infrastructures need to be modified, methods must mature, analytical pipelines need to be developed, and successful case studies must be disseminated to practitioners.

Effects of inbreeding and interpopulation crosses on performance and plasticity of two generations of offspring of a declining grassland plant.

PREMISE OF THE STUDY: Inbreeding depression is a major evolutionary force and an important topic in conservation genetics because habitat fragmentation leads to increased inbreeding in the populations of many species. Crosses between populations may restore heterozygosity, resulting in increased performance (heterosis), but may also lead to the disruption of coadapted gene complexes and to decreased performance (outbreeding depression). METHODS: We investigated the effects of selfing and of within and between population crosses on reproduction and the performance of two generations of offspring of the declining grassland plant Saxifraga granulata (Saxifragaceae). We also subjected the first generation of offspring to a fertilization and two stress treatments (competition and defoliation) to investigate whether the effects of inbreeding and interpopulation gene flow depend on environmental conditions. KEY RESULTS: Inbreeding depression affected all traits in the F(1) generation (δ = 0.07-0.55), but was stronger for traits expressed late during development and varied among families. The adaptive plasticity of offspring from selfing and from interpopulation crosses in response to nutrient addition was reduced. Outbreeding depression was also observed in response to stress. Multiplicative fitness of the F(2) generation after serial inbreeding was extremely low (δ > 0.99), but there was heterosis after crossing inbred lines. Outbreeding depression was not observed in the F(2). CONCLUSIONS: Continuous inbreeding may drastically reduce the fitness of plants, but effects may be environment-dependent. When assessing the genetic effects of fragmentation and interpopulation crosses, the possible effects on the mean performance of offspring and on its adaptive plasticity should be considered.

Local adaptation in the monocarpic perennial Carlina vulgaris at different spatial scales across Europe.

Spatial variation in environmental conditions can lead to local adaptation of plant populations, particularly if gene flow among populations is low. Many studies have investigated adaptation to contrasting environmental conditions, but little is known about the spatial scale of adaptive evolution. We studied population differentiation and local adaptation at two spatial scales in the monocarpic grassland perennial Carlina vulgaris. We reciprocally transplanted seedlings among five European regions (northwestern Czech Republic, central Germany, Luxembourg, southern Sweden and northwestern Switzerland) and among populations of different sizes within three of the regions. We recorded survival, growth and reproduction over three growing periods. At the regional scale, several performance traits and the individual fitness of C. vulgaris were highest if the plants were grown in their home region and they decreased with increasing transplant distance. The effects are likely due to climatic differences that increased with the geographical distance between regions. At the local scale, there were significant interactions between the effects of the population of origin and the transplant site, but these were not due to an enhanced performance of plants at their home site and they were not related to the geographical or environmental distance between the site of origin and the transplant site. The size of the population of origin did not influence the strength of local adaptation. The results of our study suggest that C. vulgaris consists of regionally adapted genotypes, and that distance is a good predictor of the extent of adaptive differentiation at large scales ( > 200 km) but not at small scales. We conclude that patterns of local adaptation should be taken into account for the efficient preservation of genetic resources, when assessing the status of a plant species and during conservation planning.

Effects of pollen quantity and quality on reproduction and offspring vigor in the rare plant Scorzonera humilis (Asteraceae).

We studied the effects of pollinator exclusion, interparental distance, and supplementary hand pollination on reproduction and progeny vigor in Scorzonera humilis (Asteraceae), a rare plant of fragmented, nutrient-poor grasslands. Caged flowers produced no seeds and selfed flowers only very rarely, indicating that S. humilis is mainly self-incompatible. Seed production, seed mass, and seed germination following between-population crosses were consistently, but not significantly, higher than after within-population crosses. Seed set increased with local density of conspecifics, indicating that the reduced plant density in fragmented populations may reduce plant reproductive success. Seed set was pollen limited in all four populations studied. Supplementary hand-pollination strongly increased the survival of offspring, indicating that either pollinators transferred pollen from related individuals resulting in inbreeding depression in spite of the incompatibility system or that higher pollen loads increased pollen competition and the selectivity among gametes. In one of the populations, adding pollen from a different population strongly increased progeny fitness compared with both natural pollination and pollen supplementation from the same population. The results indicate that S. humilis is sensitive to inbreeding and that pollen limitation can reduce both the number and quality of offspring.