Disentangling phylogenetic relationships proves challenging for groups that have evolved recently, especially if there is ongoing reticulation. Although they are in most cases immediately isolated from diploid relatives, sets of sibling allopolyploids often hybridize with each other, thereby increasing the complexity of an already challenging situation. Dactylorhiza (Orchidaceae: Orchidinae) is a genus much affected by allopolyploid speciation and reticulate phylogenetic relationships. Here, we use genetic variation at tens of thousands of genomic positions to unravel the convoluted evolutionary history of Dactylorhiza. We first investigate circumscription and relationships of diploid species in the genus using coalescent and maximum likelihood methods, and then group 16 allotetraploids by maximum affiliation to their putative parental diploids, implementing a method based on genotype likelihoods. The direction of hybrid crosses is inferred for each allotetraploid using information from maternally inherited plastid RADseq loci. Starting from age estimates of parental taxa, the relative ages of these allotetraploid entities are inferred by quantifying their genetic similarity to the diploids and numbers of private alleles compared with sibling allotetraploids. Whereas northwestern Europe is dominated by young allotetraploids of postglacial origins, comparatively older allotetraploids are distributed further south, where climatic conditions remained relatively stable during the Pleistocene glaciations. Our bioinformatics approach should prove effective for the study of other naturally occurring, nonmodel, polyploid plant complexes.

Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S . pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S . pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25.

The orchid family is the largest in the angiosperms, but little is known about the molecular basis of the significant variation they exhibit. We investigate here the transcriptomic divergence between two European terrestrial orchids, Dactylorhiza incarnata and Dactylorhiza fuchsii, and integrate these results in the context of their distinct ecologies that we also document. Clear signals of lineage‐specific adaptive evolution of protein‐coding sequences are identified, notably targeting elements of biotic defence, including both physical and chemical adaptations in the context of divergent pools of pathogens and herbivores. In turn, a substantial regulatory divergence between the two species appears linked to adaptation/acclimation to abiotic conditions. Several of the pathways affected by differential expression are also targeted by deviating post‐transcriptional regulation via sRNAs. Finally, D. incarnata appears to suffer from insufficient sRNA control over the activity of RNA‐dependent DNA polymerase, resulting in increased activity of class I transposable elements and, over time, in larger genome size than that of D. fuchsii. The extensive molecular divergence between the two species suggests significant genomic and transcriptomic shock in their hybrids and offers insights into the difficulty of coexistence at the homoploid level. Altogether, biological response to selection, accumulated during the history of these orchids, appears governed by their microenvironmental context, in which biotic and abiotic pressures act synergistically to shape transcriptome structure, expression and regulation.

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Animal pollinators mediate gene flow among plant populations, but in contrast to well‐studied topographic and (Pleistocene) environmental isolating barriers, their impact on population genetic differentiation remains largely unexplored. Comparing how these multifarious factors drive microevolutionary histories is, however, crucial for better resolving macroevolutionary patterns of plant diversification. Here we combined genomic analyses with landscape genetics and niche modelling across six related Neotropical plant species (424 individuals across 33 localities) differing in pollination strategy to test the hypothesis that highly mobile (vertebrate) pollinators more effectively link isolated localities than less mobile (bee) pollinators. We found consistently higher genetic differentiation (FST) among localities of bee‐ than vertebrate‐pollinated species with increasing geographical distance, topographic barriers and historical climatic instability. High admixture among montane populations further suggested relative climatic stability of Neotropical montane forests during the Pleistocene. Overall, our results indicate that pollinators may differentially impact the potential for allopatric speciation, thereby critically influencing diversification histories at macroevolutionary scales.

Whole‐genome duplication has shaped the evolution of angiosperms and other organisms, and is important for many crops. Structural reorganization of chromosomes and repatterning of gene expression are frequently observed in allopolyploids, with physiological and ecological consequences. Recurrent origins from different parental populations are widespread among polyploids, resulting in an array of lineages that provide excellent models to uncover mechanisms of adaptation to divergent environments in early phases of polyploid evolution. We integrate here transcriptomic and ecophysiological comparative studies to show that sibling allopolyploid marsh orchid species (Dactylorhiza, Orchidaceae) occur in different habitats (low nutrient fens vs. meadows with mesic soils) and are characterized by a complex suite of intertwined, pronounced ecophysiological differences between them. We uncover distinct features in leaf elemental chemistry, light‐harvesting, photoprotection, nutrient transport and stomata activity of the two sibling allopolyploids, which appear to match their specific ecologies, in particular soil chemistry differences at their native sites. We argue that the phenotypic divergence between the sibling allopolyploids has a clear genetic basis, generating ecological barriers that maintain distinct, independent lineages, despite pervasive interspecific gene flow. This suggests that recurrent origins of polyploids bring about a long‐term potential to trigger and maintain functional and ecological diversity in marsh orchids and other groups.

Whole-genome duplication, in particular allopolyploidy, has shaped the evolution of angiosperms and other organisms. Structural reorganization of chromosomes and repatterning of gene expression is frequently observed in early generation allopolyploids, with potential ecological consequences. The relative contributions of environmental and intrinsic drivers to these changes are unknown. We show here that in marsh orchids (Dactylorhiza, Orchidaceae), recurrently-formed allopolyploids are characterized by distinct eco-physiologies matching their respective environments, providing us with an excellent study system to address this question. Here we integrate eco-physiological and transcriptomic comparative studies to reveal a complex suite of intertwined, pronounced differences between sibling allopolyploids. We show that Dactylorhiza majalis that is distributed in Central and Southern Europe favors meadows with mesic soils. Its sibling allopolyploid D. traunsteineri occurs in fens, characterized by soils depleted by macro- and micronutrients, mainly in previously glaciated European areas. We further uncover distinct features in their nutrient transport, leaf elemental chemistry, light-harvesting, photoprotection, and stomata activity, that appear all linked to their distinct ecologies, in particular soil chemistry differences at their native sites. Recurrent polyploidization hence enriches biodiversity through eco-physiological diversification, providing the opportunity for sibling allopolyploids to evolve as distinct evolutionary units, despite pervasive interspecific gene flow.

Animal pollinators mediate gene flow among plant populations, but, in contrast to well-studied topographic and (Pleistocene) environmental isolating barriers, their impact on population genetic differentiation remains largely unexplored. Comparatively investigating how these multifarious factors drive microevolutionary histories is, however, crucial for better resolving macroevolutionary patterns of plant diversification. We here combined genomic analyses with landscape genetics and niche modelling across six related Neotropical plant species (424 individuals across 33 localities) differing in pollination strategy to test the hypothesis that highly mobile (vertebrate) pollinators more effectively link isolated localities than less mobile (bee) pollinators. We found consistently higher genetic differentiation (FST) among localities of bee- than vertebrate-pollinated species with increasing geographic distance, topographic barriers and historic climatic instability. High admixture among montane populations further suggested relative climatic stability of Neotropical montane forests during the Pleistocene. Overall, our results indicate that pollinators may differentially impact the potential for allopatric speciation, thereby critically influencing diversification histories at macroevolutionary scales.