The origin and speciation of orchids.

Orchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis. We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants. The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica. These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation.

Genomic, spatial and morphometric data for discrimination of four species in the Mediterranean Tamus clade of yams (Dioscorea, Dioscoreaceae).

BACKGROUND AND AIMS: Among the numerous pantropical species of the yam genus, Dioscorea, only a small group occurs in the Mediterranean basin, including two narrow Pyrenean endemics (Borderea clade) and two Mediterranean-wide species (D. communis and D. orientalis, Tamus clade). However, several currently unrecognized species and infraspecific taxa have been described in the Tamus clade due to significant morphological variation associated with D. communis. Our overarching aim was to investigate taxon delimitation in the Tamus clade using an integrative approach combining phylogenomic, spatial and morphological data. METHODS: We analysed 76 herbarium samples using Hyb-Seq genomic capture to sequence 260 low-copy nuclear genes and plastomes, together with morphometric and environmental modelling approaches. KEY RESULTS: Phylogenomic reconstructions confirmed that the two previously accepted species of the Tamus clade, D. communis and D. orientalis, are monophyletic and form sister clades. Three subclades showing distinctive geographic patterns were identified within D. communis. These subclades were also identifiable from morphometric and climatic data, and introgression patterns were inferred between subclades in the eastern part of the distribution of D. communis. CONCLUSIONS: We propose a taxonomy that maintains D. orientalis, endemic to the eastern Mediterranean region, and splits D. communis sensu lato into three species: D. edulis, endemic to Macaronesia (Canary Islands and Madeira); D. cretica, endemic to the eastern Mediterranean region; and D. communis sensu stricto, widespread across western and central Europe. Introgression inferred between D. communis s.s. and D. cretica is likely to be explained by their relatively recent speciation at the end of the Miocene, disjunct isolation in eastern and western Mediterranean glacial refugia and a subsequent westward recolonization of D. communis s.s. Our study shows that the use of integrated genomic, spatial and morphological approaches allows a more robust definition of species boundaries and the identification of species that previous systematic studies failed to uncover.

Rapid Parallel Adaptation to Anthropogenic Heavy Metal Pollution.

The impact of human-mediated environmental change on the evolutionary trajectories of wild organisms is poorly understood. In particular, capacity of species to adapt rapidly (in hundreds of generations or less), reproducibly and predictably to extreme environmental change is unclear. Silene uniflora is predominantly a coastal species, but it has also colonized isolated, disused mines with phytotoxic, zinc-contaminated soils. To test whether rapid, parallel adaptation to anthropogenic pollution has taken place, we used reduced representation sequencing (ddRAD) to reconstruct the evolutionary history of geographically proximate mine and coastal population pairs and found largely independent colonization of mines from different coastal sites. Furthermore, our results show that parallel evolution of zinc tolerance has occurred without gene flow spreading adaptive alleles between mine populations. In genomic regions where signatures of selection were detected across multiple mine-coast pairs, we identified genes with functions linked to physiological differences between the putative ecotypes, although genetic differentiation at specific loci is only partially shared between mine populations. Our results are consistent with a complex, polygenic genetic architecture underpinning rapid adaptation. This shows that even under a scenario of strong selection and rapid adaptation, evolutionary responses to human activities (and other environmental challenges) may be idiosyncratic at the genetic level and, therefore, difficult to predict from genomic data.

Biogeography and genome size evolution of the oldest extant vascular plant genus, Equisetum (Equisetaceae).

BACKGROUND AND AIMS: Extant plant groups with a long fossil history are key elements in understanding vascular plant evolution. Horsetails (Equisetum, Equisetaceae) have a nearly continuous fossil record dating back to the Carboniferous, but their phylogenetic and biogeographic patterns are still poorly understood. We use here the most extensive phylogenetic analysis to date as a framework to evaluate their age, biogeography and genome size evolution. METHODS: DNA sequences of four plastid loci were used to estimate divergence times and investigate the biogeographic history of all extant species of Equisetum. Flow cytometry was used to study genome size evolution against the framework of phylogenetic relationships in Equisetum. KEY RESULTS: On a well-supported phylogenetic tree including all extant Equisetum species, a molecular clock calibrated with multiple fossils places the node at which the outgroup and Equisetum diverged at 343 Mya (Early Carboniferous), with the first major split among extant species occurring 170 Mya (Middle Jurassic). These dates are older than those reported in some other recent molecular clock studies but are largely in agreement with a timeline established by fossil appearance in the geological record. Representatives of evergreen subgenus Hippochaete have much larger genome sizes than those of deciduous subgenus Equisetum, despite their shared conserved chromosome number. Subgenus Paramochaete has an intermediate genome size and maintains the same number of chromosomes. CONCLUSIONS: The first divergences among extant members of the genus coincided with the break-up of Pangaea and the resulting more humid, warmer climate. Subsequent tectonic activity most likely involved vicariance events that led to species divergences combined with some more recent, long-distance dispersal events. We hypothesize that differences in genome size between subgenera may be related to the number of sperm flagellae.

Microsatellites and petal morphology reveal new patterns of admixture in Orchis hybrid zones.

PREMISE: The genetic structure of hybrid zones provides insight into the potential for gene flow to occur between plant taxa. Four closely related European orchid species (Orchis anthropophora, O. militaris, O. purpurea, and O. simia) hybridize when they co-occur. We aimed to characterize patterns of hybridization in O. militaris-O. purpurea, O. purpurea-O. simia, and O. anthropophora-O. simia hybrid zones using molecular and morphological data. METHODS: We used 11 newly isolated nuclear microsatellites to genotype 695 individuals collected from seven hybrid zones and six allopatric parental populations in France. Geometric morphometric analysis was conducted using 15 labellum landmarks to capture the main aspects of petal shape. RESULTS: Backcrossing was asymmetric toward O. militaris in multiple O. militaris-O. purpurea hybrid zones. Hybrids in O. purpurea-O. simia and O. anthropophora-O. simia hybrid zones were largely limited to F1 and F2 generations, but further admixture had occurred. These patterns were reflected in labellum geometric morphometric data, which correlated strongly with nuclear microsatellite data in all three species combinations. CONCLUSIONS: The coexistence of parental and admixed individuals in these Orchis hybrid zones implies they are likely to be tension zones being maintained by a balance between gene flow into the hybrid zone and selection acting against admixed individuals. The pattern of admixture in the three species combinations suggests intrinsic selection acting on the hybrids is weaker in more closely related taxa.

Conservation of the Threatened Species, Pulsatilla vulgaris Mill. (Pasqueflower), is Aided by Reproductive System and Polyploidy.

Population loss due to habitat disturbance is a major concern in biodiversity conservation. Here we investigate the genetic causes of the demographic decline observed in English populations of Pulsatilla vulgaris and the consequences for conservation. Using 10 nuclear microsatellite markers, we compare genetic variation in wild populations with restored and seed-regenerated populations (674 samples). Emergence of genetic structure and loss of allelic variation in natural populations are not as evident as expected from demographic trends. Restored populations show genetic variation comparable to their source populations and, in general, to the wild ones. Genetic homogeneity is observed in regeneration trials, although some alleles not captured in source populations are detected. We infer that polyploidy, longevity, and clonal reproduction have provided P. vulgaris with the standing genetic variation necessary to make the species resilient to the effects of demographic decline, suggesting that the use of multiple sources for reintroduction may be beneficial to mimic natural gene flow and the availability of multiple allele copies typical of polyploid species.

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization.

BACKGROUND: Satellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids (Paphiopedilum subgenus Parvisepalum and representative species in the other subgenera/sections across the genus). A new satellite repeat in Paphiopedilum subgenus Parvisepalum, SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from P. armeniacum (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA. RESULTS: SatA comprises c. 14.5% of the P. armeniacum genome and is specific to subgenus Parvisepalum. It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin-loop motifs. A new karyotype of P. vietnamense (2n = 28) is presented and shows that the chromosome number in subgenus Parvisepalum is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven Paphiopedilum species of subgenus Parvisepalum (2n = 26-28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific. CONCLUSIONS: We conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to Paphiopedilum subgenus Parvisepalum. SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus Parvisepalum and indeed of all Paphiopedilum could be either 2n = 26 or 28, if P. vietnamense is sister to all species in the subgenus as suggested by the ITS data.

Orchid conservation: further links.

BACKGROUND: Due in great part to their often complex interactions with mycorrhizal fungi, pollinators and host trees, Orchidaceae present particular challenges for conservation. Furthermore, orchids, as potentially the largest family of angiosperms with >26000 species, species complexes and frequent hybrid formation, are complex to catalogue. Following a highlight in 2015, a further seven papers focusing on orchids, their interactions with beneficial organisms, pollinators and mycorrhiza, and other factors relating to their conservation, including threats from human utilization and changing land use, are presented here. CONCLUSIONS: The production of an online flora of all known plants and an assessment of the conservation status of all known plant species as far as possible, to guide conservation action are the first two targets of the Global Strategy for Plant Conservation Without knowing how many species there are and how they should be circumscribed, neither of these targets is achievable. Orchids are a fascinating subject for fundamental research with rapid species evolution, specific organ structure and development, but they also suffer from high levels of threat. Effective orchid conservation must take account of the beneficial interactions with fungi and pollinators and the potentially detrimental effects of over-collection and changes in land use.

Marked hybridization and introgression in Ophrys sect. Pseudophrys in the western Iberian Peninsula.

PREMISE OF THE STUDY: Orchids in the genus Ophrys represent extraordinary cases of tight coevolution between plants and their pollinators, and as a result, they present opportunities for studying hybridization, or a lack thereof, during speciation. However, few studies assess the real effect of hybridization in diversification. The three most representative species of section Pseudophrys in the western Iberian Peninsula-O. dyris, O. fusca, and O. lutea-were chosen to study evolutionary relationships and examine speciation. METHODS: Using eight specific nuclear microsatellite loci, 357 individuals from 28 locations were studied; 142 of these samples were also studied with four plastid microsatellite loci. Data were analyzed using Bayesian cluster analysis, a median-joint network, and multivariate analysis. KEY RESULTS: Many O. dyris and O. fusca specimens had three or four alleles and were therefore treated as tetraploid. Ophrys dyris is poorly genetically separated from O. fusca, and pure populations are rare. Ophrys fusca and O. lutea are distinct, but hybrids/introgressed individuals were detected in most of the populations and supported by plastid haplotypes. Ophrys fusca is subdivided into three well-delimited genetic lineages with a strict geographic correspondence confirmed by plastid haplotypes. CONCLUSIONS: Because postzygotic barriers are weak, leakage in this highly specialized orchid-pollinator system contributes to hybridization and introgression. These leakages may have occurred during periods of past climate change, promoting homogenization and the potential for generations of new biodiversity via production of novel genotypes/phenotypes interacting with pollinators.

Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size.

Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low-abundance repeat-derived DNA, supporting a scenario where amplified repeats continually accumulate due to infrequent DNA removal. Our results indicate that a lack of deletion and low turnover of repetitive DNA are major contributors to the evolution of extremely large genomes and show that their size cannot simply be accounted for by the activity of a small number of high-abundance repeat families.

Orchid conservation: making the links.

Orchidaceae, one of the largest families of flowering plants, present particular challenges for conservation, due in great part to their often complex interactions with mycorrhizal fungi, pollinators and host trees. In this Highlight, we present seven papers focusing on orchids and their interactions and other factors relating to their conservation.

Rock outcrop orchids reveal the genetic connectivity and diversity of inselbergs of northeastern Brazil.

BACKGROUND: Because of their fragmented nature, inselberg species are interesting biological models for studying the genetic consequences of disjoint populations. Inselbergs are commonly compared with oceanic islands, as most of them display a marked ecological isolation from the surrounding area. The isolation of these rock outcrops is reflected in the high number of recorded endemic species and the strong floristic differences between individual inselbergs and adjacent habitats. We examined the genetic connectivity of orchids Epidendrum cinnabarinum and E. secundum adapted to Neotropical inselbergs of northeastern Brazil. Our goals were to identify major genetic divergences or disjunctions across the range of the species and to investigate potential demographic and evolutionary mechanisms leading to lineage divergence in Neotropical mountain ecosystems. RESULTS: Based on plastid markers, high genetic differentiation was found for E. cinnabarinum (FST = 0.644) and E. secundum (FST = 0.636). Haplotypes were not geographically structured in either taxon, suggesting that restricted gene flow and genetic drift may be significant factors influencing the diversification of these inselberg populations. Moreover, strong differentiation was found between populations over short spatial scales, indicating substantial periods of isolation among populations. For E. secundum, nuclear markers indicated higher gene flow by pollen than by seeds. CONCLUSIONS: The comparative approach adopted in this study contributed to the elucidation of patterns in both species. Our results confirm the ancient and highly isolated nature of inselberg populations. Both species showed similar patterns of genetic diversity and structure, highlighting the importance of seed-restricted gene flow and genetic drift as drivers of plant diversification in terrestrial islands such as inselbergs.

A universe of dwarfs and giants: genome size and chromosome evolution in the monocot family Melanthiaceae.

• Since the occurrence of giant genomes in angiosperms is restricted to just a few lineages, identifying where shifts towards genome obesity have occurred is essential for understanding the evolutionary mechanisms triggering this process. • Genome sizes were assessed using flow cytometry in 79 species and new chromosome numbers were obtained. Phylogenetically based statistical methods were applied to infer ancestral character reconstructions of chromosome numbers and nuclear DNA contents. • Melanthiaceae are the most diverse family in terms of genome size, with C-values ranging more than 230-fold. Our data confirmed that giant genomes are restricted to tribe Parideae, with most extant species in the family characterized by small genomes. Ancestral genome size reconstruction revealed that the most recent common ancestor (MRCA) for the family had a relatively small genome (1C = 5.37 pg). Chromosome losses and polyploidy are recovered as the main evolutionary mechanisms generating chromosome number change. • Genome evolution in Melanthiaceae has been characterized by a trend towards genome size reduction, with just one episode of dramatic DNA accumulation in Parideae. Such extreme contrasting profiles of genome size evolution illustrate the key role of transposable elements and chromosome rearrangements in driving the evolution of plant genomes.

Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae).

Fritillaria (Liliaceae) is a genus of approximately 140 species of bulbous perennial plants that includes taxa of both horticultural and medicinal importance. As well as being commercially valuable, Fritillaria species have attracted attention because of their exceptionally large genome sizes, with all values recorded to date in excess of 30Gb. Despite such interest in the genus, phylogenetic relationships between the majority of species have remained untested. Here we present the first phylogenetic reconstruction of relationships to encompass most of the currently recognised species diversity in the genus. Three regions of the plastid genome were sequenced in 117 individuals of Fritillaria, representing 92 species (c. 66% of the genus) and in representatives of nine other genera of Liliaceae. Eleven low-copy nuclear gene regions were also screened in selected species for their potential utility. Phylogenetic analysis of a combined plastid dataset using maximum parsimony and Bayesian inference provided support for the monophyly of the majority of currently recognised subgenera. However, subgenus Fritillaria, which is by far the largest of the subgenera and includes the most important species used in traditional Chinese medicine, is found to be polyphyletic. Moreover, several taxa that were represented by multiple individuals show evidence of species non-monophyly. The Japanese endemic subgenus Japonica, which contains the species with the largest recorded genome size for any diploid plant, is resolved as sister to the predominantly Middle Eastern and Central Asian subgenus Rhinopetalum. Whilst relationships between most of the major Fritillaria lineages can now be resolved, our results also highlight the need for data from additional independently evolving loci; an endeavour that may be particularly challenging in light of the huge nuclear genomes found in these plants.

Phylogenetics, ancestral state reconstruction, and a new infrafamilial classification of the pantropical Ochnaceae (Medusagynaceae, Ochnaceae s.str., Quiinaceae) based on five DNA regions.

Ochnaceae s.str. (Malpighiales) are a pantropical family of about 500 species and 27 genera of almost exclusively woody plants. Infrafamilial classification and relationships have been controversial partially due to the lack of a robust phylogenetic framework. Including all genera except Indosinia and Perissocarpa and DNA sequence data for five DNA regions (ITS, matK, ndhF, rbcL, trnL-F), we provide for the first time a nearly complete molecular phylogenetic analysis of Ochnaceae s.l. resolving most of the phylogenetic backbone of the family. Based on this, we present a new classification of Ochnaceae s.l., with Medusagynoideae and Quiinoideae included as subfamilies and the former subfamilies Ochnoideae and Sauvagesioideae recognized at the rank of tribe. Our data support a monophyletic Ochneae, but Sauvagesieae in the traditional circumscription is paraphyletic because Testulea emerges as sister to the rest of Ochnoideae, and the next clade shows Luxemburgia+Philacra as sister group to the remaining Ochnoideae. To avoid paraphyly, we classify Luxemburgieae and Testuleeae as new tribes. The African genus Lophira, which has switched between subfamilies (here tribes) in past classifications, emerges as sister to all other Ochneae. Thus, endosperm-free seeds and ovules with partly to completely united integuments (resulting in an apparently single integument) are characters that unite all members of that tribe. The relationships within its largest clade, Ochnineae (former Ochneae), are poorly resolved, but former Ochninae (Brackenridgea, Ochna) are polyphyletic. Within Sauvagesieae, the genus Sauvagesia in its broad circumscription is polyphyletic as Sauvagesia serrata is sister to a clade of Adenarake, Sauvagesia spp., and three other genera. Within Quiinoideae, in contrast to former phylogenetic hypotheses, Lacunaria and Touroulia form a clade that is sister to Quiina. Bayesian ancestral state reconstructions showed that zygomorphic flowers with adaptations to buzz-pollination (poricidal anthers), a syncarpous gynoecium (a near-apocarpous gynoecium evolved independently in Quiinoideae and Ochninae), numerous ovules, septicidal capsules, and winged seeds with endosperm are the ancestral condition in Ochnoideae. Although in some lineages poricidal anthers were lost secondarily, the evolution of poricidal superstructures secured the maintenance of buzz-pollination in some of these genera, indicating a strong selective pressure on keeping that specialized pollination system.

The genome sequence of the English holly, Ilex aquifolium L. (Aquifoliaceae).

We present a genome assembly from an individual Ilex aquifolium (the English holly; Eudicot; Magnoliopsida; Aquifoliales; Aquifoliaceae). The genome sequence is 800.0 megabases in span. Most of the assembly is scaffolded into 20 chromosomal pseudomolecules. The assembled mitochondrial and plastid genomes have lengths of 538.43 kilobases and 157.52 kilobases in length, respectively.

The genome sequence of rosebay willowherb Chamaenerion angustifolium (L.) Scop., 1771 (syn. Epilobium angustifolium L., 1753) (Onagraceae).

We present a genome assembly from an individual Chamaenerion angustifolium (fireweed; Tracheophyta; Magnoliopsida; Myrtales; Onagraceae). The genome sequence is 655.9 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 495.18 kilobases and 160.41 kilobases in length, respectively.

Upregulation of the Renin-Angiotensin System Is Associated with Patient Survival and the Tumour Microenvironment in Glioblastoma.

Glioblastoma is a highly aggressive disease with poor survival outcomes. An emerging body of literature links the role of the renin-angiotensin system (RAS), well-known for its function in the cardiovascular system, to the progression of cancers. We studied the expression of RAS-related genes (ATP6AP2, AGTR1, AGTR2, ACE, AGT, and REN) in The Cancer Genome Atlas (TCGA) glioblastoma cohort, their relationship to patient survival, and association with tumour microenvironment pathways. The expression of RAS genes was then examined in 12 patient-derived glioblastoma cell lines treated with chemoradiation. In cases of glioblastoma within the TCGA, ATP6AP2, AGTR1, ACE, and AGT had consistent expressions across samples, while AGTR2 and REN were lowly expressed. High expression of AGTR1 was independently associated with lower progression-free survival (PFS) (p = 0.01) and had a non-significant trend for overall survival (OS) after multivariate analysis (p = 0.095). The combined expression of RAS receptors (ATP6AP2, AGTR1, and AGTR2) was positively associated with gene pathways involved in hypoxia, microvasculature, stem cell plasticity, and the molecular characterisation of glioblastoma subtypes. In patient-derived glioblastoma cell lines, ATP6AP2 and AGTR1 were upregulated after chemoradiotherapy and correlated with an increase in HIF1A expression. This data suggests the RAS is correlated with changes in the tumour microenvironment and associated with glioblastoma survival outcomes.

The genome sequence of common fleabane, Pulicaria dysenterica (L.) Bernh. (Asteraceae).

We present a genome assembly from an individual Pulicaria dysenterica (common fleabane; Tracheophyta; Magnoliopsida; Asterales; Asteraceae). The genome sequence is 833.2 megabases in span. Most of the assembly is scaffolded into 9 chromosomal pseudomolecules. The mitochondrial and plastid genomes were assembled and have lengths of 375.47 kilobases and 150.94 kilobases respectively.

Effective population size in a partially clonal plant is not predicted by the number of genetic individuals.

Estimating effective population size (N e) is important for theoretical and practical applications in evolutionary biology and conservation. Nevertheless, estimates of N e in organisms with complex life-history traits remain scarce because of the challenges associated with estimation methods. Partially clonal plants capable of both vegetative (clonal) growth and sexual reproduction are a common group of organisms for which the discrepancy between the apparent number of individuals (ramets) and the number of genetic individuals (genets) can be striking, and it is unclear how this discrepancy relates to N e. In this study, we analysed two populations of the orchid Cypripedium calceolus to understand how the rate of clonal versus sexual reproduction affected N e. We genotyped >1000 ramets at microsatellite and SNP loci, and estimated contemporary N e with the linkage disequilibrium method, starting from the theoretical expectation that variance in reproductive success among individuals caused by clonal reproduction and by constraints on sexual reproduction would lower N e. We considered factors potentially affecting our estimates, including different marker types and sampling strategies, and the influence of pseudoreplication in genomic data sets on N e confidence intervals. The magnitude of N e/N ramets and N e/N genets ratios we provide may be used as reference points for other species with similar life-history traits. Our findings demonstrate that N e in partially clonal plants cannot be predicted based on the number of genets generated by sexual reproduction, because demographic changes over time can strongly influence N e. This is especially relevant in species of conservation concern in which population declines may not be detected by only ascertaining the number of genets.