Summary• The specificity of orchids for their fungi can vary substantially, from highly specialist interactions to more generalist interactions, but little is known about the evolutionary history of the mycorrhizal specificity of orchids.• Here, we used a network analysis approach to investigate orchid mycorrhizal associations in 16 species of the genus Orchis sampled across 11 different regions in Europe. We first examined in detail the structure of the network of associations and then tested for a phylogenetic signal in mycorrhizal specificity and identified the fungi with which the orchids associated.• We found 20 different fungal lineages that associated with species of the genus Orchis, most of them being related to members of the Tulasnellaceae (84.33% of all identified associations) and a smaller proportion being related to members of the Ceratobasidiaceae (9.97%). Species associations formed a nested network that is built on asymmetric links among species. Evolution of mycorrhizal specificity in Orchis closely resembles a Brownian motion process, and the interaction between Orchis and Tulasnellaceae fungi is significantly influenced by the phylogenetic relationships between the Orchis species.• Our results provide evidence of the presence of phylogenetic conservatism in mycorrhizal specificity in orchids and demonstrate that evolutionary processes may be an important factor in generating patterns of mycorrhizal associations.

Summary• Seed dispersal and the subsequent recruitment of new individuals into a population are important processes affecting the population dynamics, genetic diversity and spatial genetic structure of plant populations.• Spatial patterns of seedling recruitment were investigated in two populations of the terrestrial orchid Orchis purpurea using both univariate and bivariate point pattern analysis, parentage analysis and seed germination experiments.• Both adults and recruits showed a clustered spatial distribution with cluster radii of c . 4-5 m. The parentage analysis resulted in offspring-dispersal distances that were slightly larger than distances obtained from the point pattern analyses. The suitability of microsites for germination differed among sites, with strong constraints in one site and almost no constraints in the other.• These results provide a clear and coherent picture of recruitment patterns in a tuberous, perennial orchid. Seed dispersal is limited to a few metres from the mother plant, whereas the availability of suitable germination conditions may vary strongly from one site to the next. Because of a time lag of 3-4 yr between seed dispersal and actual recruitment, and irregular flowering and fruiting patterns of adult plants, interpretation of recruitment patterns using point patterns analyses ideally should take into account the demographic properties of orchid populations.

Environmental DNA (eDNA) analysis is a rapid, non-invasive, cost-efficient biodiversity monitoring tool with enormous potential to inform aquatic conservation and management. Development is ongoing, with strong commercial interest, and new uses are continually being discovered. General applications of eDNA and guidelines for best practice in freshwater systems have been established, but habitat-specific assessments are lacking. Ponds are highly diverse, yet understudied systems that could benefit from eDNA monitoring. However, eDNA applications in ponds and methodological constraints specific to these environments remain unaddressed. Following a stakeholder workshop in 2017, researchers combined knowledge and expertise to review these applications and challenges that must be addressed for the future and consistency of eDNA monitoring in ponds. The greatest challenges for pond eDNA surveys are representative sampling, eDNA capture, and potential PCR inhibition. We provide recommendations for sampling, eDNA capture, inhibition testing, and laboratory practice, which should aid new and ongoing eDNA projects in ponds. If implemented, these recommendations will contribute towards an eventual broad standardisation of eDNA research and practice, with room to tailor workflows for optimal analysis and

Forest fragmentation is expected to affect patch occupancy patterns, population size and population viability of plant populations through changes in both patch area and isolation. We tested the hypothesis that patch area has had a significant effect on patch occupancy and population size of Primula elatior, a common forest herbaceous plant species in Flanders, Belgium. The hypothesis that plants from small populations have lower fitness as reflected by several characteristics related to reproduction was also tested. Finally, the probability of P. elatior colonizing presently empty patches was investigated. Patch area proved to be the most important factor explaining population size. Patch area, spatial isolation and within-patch habitat characteristics all contributed significantly to the explanation of the distribution pattern of P. elatior. Plants from small populations had a significantly lower individual fitness than plants from large populations. Small populations produced significantly fewer seeds per fruit and per plant than did large populations. Individual seed mass decreased with increasing population size, but total seed mass increased with increasing population size. Plant-to-plant variability in the proportion of flowers setting fruit, number of seeds per fruit and number of seeds per plant decreased with increasing population size. Skewed pin-thrum ratios and lower pollination intensity may explain the reduced fecundity in small populations. Geographical isolation had a significant effect on the probability of P. elatior colonizing empty patches. The results show that patch area and isolation may influence regional persistence of plant populations through altered colonization probabilities and reduced reproductive success of small populations.