Repeat Dynamics across Timescales: A Perspective from Sibling Allotetraploid Marsh Orchids (Dactylorhiza majalis s.l.).

To provide insights into the fate of transposable elements (TEs) across timescales in a post-polyploidization context, we comparatively investigate five sibling Dactylorhiza allotetraploids (Orchidaceae) formed independently and sequentially between 500 and 100K generations ago by unidirectional hybridization between diploids D. fuchsii and D. incarnata. Our results first reveal that the paternal D. incarnata genome shows a marked increased content of LTR retrotransposons compared to the maternal species, reflected in its larger genome size and consistent with a previously hypothesized bottleneck. With regard to the allopolyploids, in the youngest D. purpurella both genome size and TE composition appear to be largely additive with respect to parents, whereas for polyploids of intermediate ages we uncover rampant genome expansion on a magnitude of multiple entire genomes of some plants such as Arabidopsis. The oldest allopolyploids in the series are not larger than the intermediate ones. A putative tandem repeat, potentially derived from a non-autonomous miniature inverted-repeat TE (MITE) drives much of the genome dynamics in the allopolyploids. The highly dynamic MITE-like element is found in higher proportions in the maternal diploid, D. fuchsii, but is observed to increase in copy number in both subgenomes of the allopolyploids. Altogether, the fate of repeats appears strongly regulated and therefore predictable across multiple independent allopolyploidization events in this system. Apart from the MITE-like element, we consistently document a mild genomic shock following the allopolyploidizations investigated here, which may be linked to their relatively large genome sizes, possibly associated with strong selection against further genome expansions.

Recurrent allopolyploidizations diversify ecophysiological traits in marsh orchids (Dactylorhiza majalis s.l.).

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.

Analysis of trait-performance-fitness relationships reveals pollinator-mediated selection on orchid pollination traits.

PREMISE: The role of pollinators in evolutionary floral divergence has spurred substantial effort into measuring pollinator-mediated phenotypic selection and its variation in space and time. For such estimates, the fitness consequences of pollination processes must be separated from other factors affecting fitness. METHODS: We built a fitness function linking phenotypic traits of food-deceptive orchids to female reproductive success by including pollinator visitation and pollen deposition as intermediate performance components and used the fitness function to estimate the strength of pollinator-mediated selection through female reproductive success. We also quantified male performance as pollinarium removal and assessed similarity in trait effects on male and female performance. RESULTS: The proportion of plants visited at least once by an effective pollinator was moderate to high, ranging from 53.7% to 85.1%. Tall, many-flowered plants were often more likely to be visited and pollinated. Given effective pollination, pollen deposition onto stigmas tended to be more likely for taller plants. Pollen deposition further depended on traits affecting the physical fit of pollinators to flowers (flower size, spur length), though the exact relationships varied in time and space. Using the fitness function to assess pollinator-mediated selection through female reproductive success acting on multiple traits, we found that selection varied detectably among taxa after accounting for sampling uncertainty. Across taxa, selection on most traits was stronger on average and more variable when pollination was less reliable. CONCLUSIONS: These results support pollination-related trait-performance-fitness relationships and thus pollinator-mediated selection on traits functionally involved in the pollination process.

Asymmetric contributions of seed and pollen to gene dispersal in the marsh orchid Dactylorhiza umbrosa in Asia Minor.

Orchids differ from other plants in their extremely small and partly air-filled seeds that can be transported long distances by wind. Seed dispersal in orchids is expected to contribute strongly to overall gene flow, and orchids generally express low levels of genetic differentiation between populations and low pollen to seed flow ratios. However, studies in orchids distributed in northern Europe have often found a poor geographic structuring of genetic variation. Here, we studied geographic differentiation in the marsh orchid Dactylorhiza umbrosa, which is widely distributed in upland regions from Asia Minor to Central Asia. These areas were less affected by Pleistocene ice ages than northern Europe and the orchid should have been able to survive the last ice age in local refugia. In the plastid genome, which is dispersed by seeds, populations at close distance were clearly divergent, but the differentiation still increased with geographic distance, and a significant phylogeographic structure had developed. In the nuclear genome, which is dispersed by both seeds and pollen, populations showed an even stronger correlation between genetic and geographic distance, but average levels of differentiation were lower than in the plastid genome, and no phylogeographic structure was evident. Combining plastid and nuclear data, we found that the ratio of pollen to seed dispersal (mp/ms) decreases with physical distance. Comparison with orchids that grow in parts of Europe that were glaciated during the last ice suggests that a balanced structure of genetic diversity develops only slowly in many terrestrial orchids, despite efficient seed dispersal.

Phylogenomic Relationships of Diploids and the Origins of Allotetraploids in Dactylorhiza (Orchidaceae).

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.

Restriction-site associated DNA sequencing supports a sister group relationship of Nigritella and Gymnadenia (Orchidaceae).

The orchid genus Nigritella is closely related to Gymnadenia and has from time to time been merged with the latter. Although Nigritella is morphologically distinct, it has been suggested that the separating characters are easily modifiable and subject to rapid evolutionary change. So far, molecular phylogenetic studies have either given support for the inclusion of Nigritella in Gymnadenia, or for their separation as different genera. To resolve this issue, we analysed data obtained from Restriction-site associated DNA sequencing, RADseq, which provides a large number of SNPs distributed across the entire genome. To analyse samples of different ploidies, we take an analytical approach of building a reduced genomic reference based on de novo RADseq loci reconstructed from diploid accessions only, which we further use to map and call variants across both diploid and polyploid accessions. We found that Nigritella is distinct from Gymnadenia forming a well-supported separate clade, and that genetic diversity within Gymnadenia is high. Within Gymnadenia, taxa characterized by an ITS-E ribotype (G. conopsea s.str. (early flowering) and G. odoratissima), are divergent from taxa characterized by ITS-L ribotype (G. frivaldii, G. densiflora and late flowering G. conopsea). Gymnigritella runei is confirmed to have an allopolyploid origin from diploid Gymnadenia conopsea and tetraploid N. nigra ssp. nigra on the basis of RADseq data. Within Nigritella the aggregation of polyploid members into three clear-cut groups as suggested by allozyme and nuclear microsatellite data was further supported.

Orchid colonization: multiple parallel dispersal events and mosaic genetic structure in Dactylorhiza majalis ssp. lapponica on the Baltic island of Gotland.

Background and Aims: The island of Gotland in the Baltic Sea has had no contact with surrounding continental areas since the withdrawal of the Weichselian ice sheet at approx. 17 ka BP. Plants present on Gotland must have arrived by long-distance dispersal, so populations are expected to exhibit reduced levels of genetic diversity compared with populations on surrounding mainlands. However, orchids have very small seeds, which appear well adapted to long-distance dispersal, and they should therefore be less affected than other plant species by colonization bottlenecks. The aim of this study was to analyse the genetic structure of orchids colonizing isolated islands, using the marsh orchid Dactylorhiza majalis ssp. lapponica as a case study. Methods: More than 500 samples from 27 populations were analysed for 15 plastid and eight nuclear marker loci. Population diversity and differentiation patterns were compared for nuclear and plastid marker systems and analysed in relation to geographical location. Key Results: We found high genetic diversity but no clear geographical structure of genetic differentiation between populations on Gotland. However, the between-population differentiation in plastid and nuclear markers were correlated and the greatest diversity was found at sites at comparatively high elevations, which were the first to emerge above the water after the Ice Age. Conclusions: The regional population on Gotland has been established by a minimum of four dispersal events from continental regions. Subsequent gene flow between sites has not yet homogenized the differentiation pattern originating from initial colonization. We conclude that long-distance seed dispersal in orchids has a strong impact on structuring genetic diversity during periods of expansion and colonization, but contributes less to gene flow between populations once a stable population structure has been achieved.

Adaptive sequence evolution is driven by biotic stress in a pair of orchid species (Dactylorhiza) with distinct ecological optima.

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.

Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity.

BACKGROUND AND AIMS: Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes. METHODS: Ploidy and proportions of endoreplicated genome were determined using DAPI (4',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato. KEY RESULTS: Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe. CONCLUSIONS: Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.

Altered gene expression and ecological divergence in sibling allopolyploids of Dactylorhiza (Orchidaceae).

BACKGROUND: Hybridization and polyploidy are potent forces that have regularly stimulated plant evolution and adaptation. Dactylorhiza majalis s.s., D. traunsteineri s.l. and D. ebudensis are three allopolyploid species of a polyploid complex formed through unidirectional (and, in the first two cases, recurrent) hybridization between the widespread diploids D. fuchsii and D. incarnata. Differing considerably in geographical extent and ecological tolerance, the three allopolyploids together provide a useful system to explore genomic responses to allopolyploidization and reveal their role in adaptation to contrasting environments. RESULTS: Analyses of cDNA-AFLPs show a significant increase in the range of gene expression of these allopolyploid lineages, demonstrating higher potential for phenotypic plasticity than is shown by either parent. Moreover, allopolyploid individuals express significantly more gene variants (including novel alleles) than their parents, providing clear evidence of increased biological complexity following allopolyploidization. More genetic mutations seem to have accumulated in the older D. majalis compared with the younger D. traunsteineri since their respective formation. CONCLUSIONS: Multiple origins of the polyploids contribute to differential patterns of gene expression with a distinct geographic structure. However, several transcripts conserved within each allopolyploid taxon differ between taxa, indicating that habitat preferences shape similar expression patterns in these independently formed tetraploids. Statistical signals separate several transcripts - some of them novel in allopolyploids - that appear correlated with adaptive traits and seem to play a role favouring the persistence of individuals in their native environments. In addition to stabilizing the allopolyploid genome, genetic and epigenetic alterations are key determinants of adaptive success of the new polyploid species after recurrent allopolyploidization events, potentially triggering reproductive isolation between the resulting lineages.

Stable epigenetic effects impact adaptation in allopolyploid orchids (Dactylorhiza: Orchidaceae).

Epigenetic information includes heritable signals that modulate gene expression but are not encoded in the primary nucleotide sequence. We have studied natural epigenetic variation in three allotetraploid sibling orchid species (Dactylorhiza majalis s.str, D. traunsteineri s.l., and D. ebudensis) that differ radically in geography/ecology. The epigenetic variation released by genome doubling has been restructured in species-specific patterns that reflect their recent evolutionary history and have an impact on their ecology and evolution, hundreds of generations after their formation. Using two contrasting approaches that yielded largely congruent results, epigenome scans pinpointed epiloci under divergent selection that correlate with eco-environmental variables, mainly related to water availability and temperature. The stable epigenetic divergence in this group is largely responsible for persistent ecological differences, which then set the stage for species-specific genetic patterns to accumulate in response to further selection and/or drift. Our results strongly suggest a need to expand our current evolutionary framework to encompass a complementary epigenetic dimension when seeking to understand population processes that drive phenotypic evolution and adaptation.

Polymorphic populations of Dactylorhiza incarnata s.l. (Orchidaceae) on the Baltic island of Gotland: morphology, habitat preference and genetic differentiation.

BACKGROUND AND AIMS: Organisms may be polymorphic within natural populations, but often the significance and genetic background to such polymorphism is not known. To understand the colour polymorphism expressed in the diploid marsh-orchids Dactylorhiza incarnata, morphological, habitat and genetic differentiation was studied in mixed populations on the island of Gotland, supplemented with genetic marker data from adjacent areas. METHODS: A total of 398 accessions was investigated for plastid haplotype and three nuclear microsatellites. Morphometric data and vegetation data were obtained from a subset of 104 plants. KEY RESULTS: No clear pattern of habitat differentiation was found among the colour morphs. Within sites, the yellow-flowered morph (ochroleuca) was slightly larger than the others in some flower characters, whereas the purple-flowered morph with spotted leaves (cruenta) was on average smaller. However, populations of the same colour morph differed considerably between sites, and there was also considerable overlap between morphs. Morphs were often genetically differentiated but imperfectly separated within sites. Most populations were characterized by significant levels of inbreeding. The ochroleuca morph constitutes a coherent, highly homozygous sublineage, although introgression from purple-flowered morphs occurs at some sites. The cruenta morph was genetically variable, although Gotland populations formed a coherent group. Purple-flowered plants with unspotted leaves (incarnata in the strict sense) were even more variable and spanned the entire genetic diversity seen in the other morphs. CONCLUSIONS: Colour polymorphism in D. incarnata is maintained by inbreeding, but possibly also by other ecological factors. The yellow-flowered morph may best be recognized as a variety of D. incarnata, var. ochroleuca, and the lack of anthocyanins is probably due to a particular recessive allele in homozygous form. Presence of spotted leaves is an uncertain taxonomic character, and genetic differentiation within D. incarnata would be better described by other morphological characters such as leaf shape and stature and size and shape of lip and spur.

ETS and plastid sequence data indicate a spontaneous origin of Scandinavian betony, Betonica officinalis L.

BACKGROUND AND AIMS: Betony (Betonica officinalis L.) is one of the rarest and most spectacular plants in the Scandinavian flora. A long-term question has been whether it is spontaneous or introduced, or whether it comprises both spontaneous and introduced populations. This study aimed to answer this question by analyzing sequence data from the nuclear external transcribed spacer (ETS) region and three regions of the plastid genome, the trnT-trnL intergenic spacer (IGS) region, tRNA-Leu (trnL) intron, and the trnS-trnG IGS. MATERIALS AND METHODS: Altogether 41 samples from 11 European countries were analyzed. A unique duplication in the trnT-trnL IGS was detected in material from Skåne (southern Sweden), the "Skåne-duplication." Populations with this duplication are united on a moderately supported branch in the phylogeny based on plastid sequences. A distinct heath genotype from Yorkshire was discovered in the phylogeny based on plastid sequences and in a comparative cultivation. RESULTS: Phylogeny based on ETS sequences does not support any Scandinavian group, whereas a principal coordinates analysis ordination based on variable ETS positions indicated a spontaneous origin for all Scandinavian populations, which comprise a genetically well-defined subgroup of the species, most closely related to other spontaneous populations from adjacent parts of continental parts of northern Europe. DISCUSSION: Seven possible naturally occurring localities remain in Scandinavia, five in central Skåne, southernmost Sweden, and two on the southwestern part of the Danish island of Lolland.

Polyploid evolution and plastid DNA variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in Scandinavia.

The Dactylorhiza incarnata/maculata complex (Orchidaceae) was used as a model system to understand genetic differentiation processes in a naturally occurring polyploid complex with much of ongoing diversification and wide distribution in recently glaciated areas in northern Europe. Data were obtained for 12 hypervariable regions in the plastid DNA genome. A total of 166 haplotypes were found in a sample of 1099 plants. Allopolyploid taxa have inherited their plastid genomes from D. maculata s.l. Overall haplotype diversity of the combined group of allopolyploid taxa was comparable to that of maternal D. maculata s.l., but populations of allopolyploids were also more strongly differentiated from each other and contained lower numbers of haplotypes than populations of D. maculata s.l. In addition to haplotypes found in extant D. maculata s.l., the allopolyploids also contained several distinct and widespread haplotypes that were not found in any of the parental lineages. Some of these haplotypes were shared between widespread allopolyploids. Divergent allopolyploids with small distributions did not seem to originate from local polyploidization events, but rather as segregates of already existing allopolyploids. Genetic diversification of allopolyploid Dactylorhiza is the result of repeated polyploid formation, secondary hybridization and introgression between already existing polyploids and extant representatives of parental lineages, hybridization between independently derived polyploid lineages, and phyletic diversification in the group of allopolyploids. Although some polyploid taxa must have evolved after the last glaciation, genetic material from the parental lineages has been transferred continuously for longer periods of time. This combination of processes may explain the taxonomic complexity encountered in Dactylorhiza and other polyploid complexes distributed in previously glaciated parts of Europe.

Habitat-driven variation in mycorrhizal communities in the terrestrial orchid genus Dactylorhiza.

Orchid species are critically dependent on mycorrhizal fungi for completion of their life cycle, particularly during the early stages of their development when nutritional resources are scarce. As such, orchid mycorrhizal fungi play an important role in the population dynamics, abundance, and spatial distribution of orchid species. However, less is known about the ecology and distribution of orchid mycorrhizal fungi. In this study, we used 454 amplicon pyrosequencing to investigate ecological and geographic variation in mycorrhizal associations in fourteen species of the orchid genus Dactylorhiza. More specifically, we tested the hypothesis that variation in orchid mycorrhizal communities resulted primarily from differences in habitat conditions where the species were growing. The results showed that all investigated Dactylorhiza species associated with a large number of fungal OTUs, the majority belonging to the Tulasnellaceae, Ceratobasidiaceae and Sebacinales. Mycorrhizal specificity was low, but significant variation in mycorrhizal community composition was observed between species inhabiting different ecological habitats. Although several fungi had a broad geographic distribution, Species Indicator Analysis revealed some fungi that were characteristic for specific habitats. Overall, these results indicate that orchid mycorrhizal fungi may have a broad geographic distribution, but that their occurrence is bounded by specific habitat conditions.

Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data.

Polyploidy is common in higher plants, and speciation in polyploid complexes is usually the result of reticulate evolution. We examined variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable plastid DNA loci to describe speciation patterns and species relationships in the Dactylorhiza incarnata/maculata polyploid complex (marsh orchids; Orchidaceae) in Greece. Several endemic taxa with restricted distribution have been described from this area, and to propose meaningful conservation priorities, detailed relationships need to be known. We identified four independently derived allopolyploid lineages, which is a pattern poorly correlated with prevailing taxonomy. Three lineages were composed of populations restricted to small areas and may be of recent origins from extant parental lineages. One lineage with wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece has high levels of genetic diversity at the polyploid level. This diversity has accumulated over a long time and may include genetic variants originating from now extinct parental populations. Our data also indicate that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.