Alpine plants grow in harsh environments and are thought to face occasional frost during the sensitive reproductive phase. Apomixis (asexual reproduction via seed) can be advantageous when sexual reproduction is disturbed by cold stress. Apomictic polyploids tend to grow in colder climates than their sexual diploid relatives. Whether cold temperatures actually induce apomixis was unknown to date.

Polyploidy in angiosperms is an influential factor to trigger apomixis, the reproduction of asexual seeds. Apomixis is usually facultative, which means that both sexual and apomictic seeds can be formed by the same plant. Environmental abiotic stress, e.g. light stress, can change the frequency of apomixis. Previous work suggested effects of stress treatments on meiosis and megasporogenesis. We hypothesized that polyploidy would alter the stress response and hence reproductive phenotypes of different cytotypes. The main aims of this research were to explore with prolonged photoperiods, whether polyploidy alters proportions of sexual ovule and sexual seed formation under light stress conditions. We used three facultative apomictic, pseudogamous cytotypes of the Ranunculus auricomus complex (diploid, tetraploid, and hexaploid). Stress treatments were applied by extended light periods (16.5 h) and control (10 h) in climate growth chambers. Proportions of apomeiotic vs. meiotic development in the ovule were evaluated with clearing methods, and mode of seed formation was examined by single seed flow cytometric seed screening (ssFCSS). We further studied pollen stainability to understand effects of pollen quality on seed formation. Results revealed that under extended photoperiod, all cytotypes produced significantly more sexual ovules than in the control, with strongest effects on diploids. The stress treatment affected neither the frequency of seed set nor the proportion of sexual seeds nor pollen quality. Successful seed formation appears to be dependent on balanced maternal: paternal genome contributions. Diploid cytotypes had mostly sexual seed formation, while polyploid cytotypes formed predominantly apomictic seeds. Pollen quality was in hexaploids better than in diploids and tetraploids. These findings confirm our hypothesis that megasporogenesis is triggered by light stress treatments. Comparisons of cytotypes support the hypothesis that ovule development in polyploid plants is less sensitive to prolonged photoperiods and responds to a lesser extent with sexual ovule formation. Polyploids may better buffer environmental stress, which releases the potential for aposporous ovule development from somatic cells, and may facilitate the establishment of apomictic seed formation.

Polyploidization and the shift to apomictic reproduction are connected to changes in DNA cytosine-methylation. Cytosine-methylation is further sensitive to environmental conditions. We, therefore, hypothesize that DNA methylation patterns would differentiate within species with geographical parthenogenesis, i.e., when diploid sexual and polyploid apomictic populations exhibit different spatial distributions. On natural populations of the alpine plant Ranunculus kuepferi, we tested differences in methylation patterns across two cytotypes (diploid, tetraploid) and three reproduction modes (sexual, mixed, apomictic), and their correlation to environmental data and geographical distributions. We used methylation-sensitive amplified fragment-length polymorphism (methylation-sensitive AFLPs) and scored three types of epiloci. Methylation patterns differed independently between cytotypes versus modes of reproduction and separated three distinct combined groups (2x sexual + mixed, 4x mixed, and 4x apomictic), with differentiation of 4x apomicts in all epiloci. We found no global spatial autocorrelation, but instead correlations to elevation and temperature gradients in 22 and 36 epiloci, respectively. Results suggest that methylation patterns in R. kuepferi were altered by cold conditions during postglacial recolonization of the Alps, and by the concomitant shift to facultative apomixis, and by polyploidization. Obligate apomictic tetraploids at the highest elevations established a distinct methylation profile. Methylation patterns reflect an ecological gradient rather than the geographical differentiation.

High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic species occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown.

Intensive transformation of lowland rainforest into oil palm and rubber monocultures is the most common land-use practice in Sumatra (Indonesia), accompanied by invasion of weeds. In the Jambi province, Centotheca lappacea is one of the most abundant alien grass species in plantations and in jungle rubber (an extensively used agroforest), but largely missing in natural rainforests. Here, we investigated putative genetic differentiation and signatures for adaptation in the introduced area. We studied reproductive mode and ploidy level as putative factors for invasiveness of the species. We sampled 19 populations in oil palm and rubber monocultures and in jungle rubber in two regions (Bukit Duabelas and Harapan). Amplified fragment length polymorphisms (AFLP) revealed a high diversity of individual genotypes and only a weak differentiation among populations (FST = 0.173) and between the two regions (FST = 0.065). There was no significant genetic differentiation between the three land-use systems. The metapopulation of C. lappacea consists of five genetic partitions with high levels of admixture; all partitions appeared in both regions, but with different proportions. Within the Bukit Duabelas region we observed significant isolation-by-distance. Nine AFLP loci (5.3% of all loci) were under natural diversifying selection. All studied populations of C. lappacea were diploid, outcrossing and self-incompatible, without any hints of apomixis. The estimated residence time of c. 100 years coincides with the onset of rubber and oil palm planting in Sumatra. In the colonization process, the species is already in a phase of establishment, which may be enhanced by efficient selection acting on a highly diverse gene pool. In the land-use systems, seed dispersal might be enhanced by adhesive spikelets. At present, the abundance of established populations in intensively managed land-use systems might provide opportunities for rapid dispersal of C. lappacea across rural landscapes in Sumatra, while the invasion potential in rainforest ecosystems appears to be moderate as long as they remain undisturbed.

Polyploidy is one of the most important evolutionary processes in plants. In natural populations, polyploids usually emerge from unreduced gametes which either fuse with reduced ones, resulting in triploid offspring (triploid bridge), or with other unreduced gametes, resulting in tetraploid embryos. The frequencies of these two pathways, and male versus female gamete contributions, however, are largely unexplored. Ranunculus kuepferi occurs with diploid, triploid and autotetraploid cytotypes in the Alps, whereby diploids are mostly sexual, while tetraploids are facultative apomicts. To test for the occurrence of polyploidization events by triploid bridge, we investigated 551 plants of natural populations via flow cytometric seed screening. We assessed ploidy shifts in the embryo to reconstruct female versus male gamete contributions to polyploid embryo and/or endosperm formation. Seed formation via unreduced egg cells (BIII hybrids) occurred in all three cytotypes, while only in one case both gametes were unreduced. Polyploids further formed seeds with reduced, unfertilized egg cells (polyhaploids and aneuploids). Pollen was highly variable in diameter, but only pollen >27 μm was viable, whereby diploids produced higher proportions of well-developed pollen. Pollen size was not informative for the formation of unreduced pollen. These results suggest that a female triploid bridge via unreduced egg cells is the major pathway toward polyploidization in R. kuepferi, maybe as a consequence of constraints of endosperm development. Triploids resulting from unreduced male gametes were not observed, which explains the lack of obligate sexual tetraploid individuals and populations. Unreduced egg cell formation in diploids represents the first step toward apomixis.

Hybridization and polyploidization play important roles in plant evolution but it is still not fully clarified how these evolutionary forces contribute to the establishment of apomicts. Apomixis, the asexual reproduction via seed formation, comprises several essential alterations in development compared to the sexual pathway. Furthermore, most natural apomicts were found to be polyploids and/or hybrids. The Ranunculus auricomus complex comprises diploid sexual and polyploid apomictic species and represents an excellent model system to gain knowledge on origin and evolution of apomixis in natural plant populations. In this study, the second generation of synthetically produced homoploid (2x) and heteroploid (3x) hybrids derived from sexual R. auricomus species was analyzed for aposporous initial cell formation by DIC microscopy. Complete manifestation of apomixis was determined by measuring single mature seeds by flow cytometric seed screen. Microscopic analysis of the female gametophyte formation indicated spontaneous occurrence of aposporous initial cells and several developmental irregularities. The frequency of apospory was found to depend on dosage effects since a significant increase in apospory was observed, when both F1 parents, rather than just one, were aposporous. Other than in the F1 generation, diploid Ranunculus F2 hybrids formed BIII seeds and fully apomictic seeds. The results indicate that hybridization rather than polyploidization seems to be the functional activator of apomictic reproduction in the synthetic Ranunculus hybrids. In turn, at least two hybrid generations are required to establish apomictic seed formation.

Hybridization of species with porous genomes can eventually lead to introgression via repeated backcrossing. The potential for introgression between species is reflected by the extent of segregation distortion in later generation hybrids. Here we studied a population of hybrids between Salix purpurea and S. helvetica that has emerged within the last 30 years on a glacier forefield in the European Alps due to secondary contact of the parental species. We used 5758 biallelic SNPs produced by RAD sequencing with the aim to ascertain the predominance of backcrosses (F1 hybrid x parent) or F2 hybrids (F1 hybrid x F1 hybrid) among hybrid offspring. Further, the SNPs were used to study segregation distortion in the second hybrid generation.

Apomictic plants expand their geographical distributions more to higher elevations compared to their sexual progenitors. It was so far unclear whether this tendency is related to mode of reproduction itself or represents a side effect of polyploidy. Apomixis is advantageous for range expansions as no mating partners and pollinators are needed (Baker's rule). Polyploidy is thought to infer fitness advantages and a higher vigour that would enable plants to adjust better to more extreme climatic conditions. However, little is known about actual performance of plants at higher elevations. We analyzed 81 populations of Ranunculus kuepferi from the whole distribution area in the European Alps to quantify apomictic versus sexual seed formation via flow cytometric seed screening. Seed set and vegetative growth were measured as fitness parameters. All parameters were correlated to geographical distribution, elevation, temperature and precipitation. Flow cytometric seed screening revealed predominantly obligate sexuality (88.9 %) and facultative apomixis in diploid populations, while tetraploid populations are predominantly facultative (65.4 %) to obligate apomictic. Apomictic seed formation correlated significantly to higher elevations, which explains also the observed niche shift to lower temperatures. However, within the tetraploid range, there is no apparent correlation of degree of facultative apomixis to geographical distance. Apomixis appeared in diploids three times independently in separated, otherwise sexual populations in the southwestern refugial areas of the Alps. Diploid apomixis was not successful in range expansions, and obligate sexual polyploids were not observed. Polyploidy may relate to cold tolerance as an adaptation to conditions at high elevations, where diploid sexuals have no fitness advantage. Instead, facultative apomixis may have aided colonization of higher elevations and range expansions in the Alps without mate and pollinator limitation, but did not necessarily involve long-distance dispersal. A direct influence of low temperatures on unreduced gamete formation cannot be ruled out.

Biological invasions can be associated with shifts of the species' climatic niches but the incidence of such shifts is under debate. The reproductive system might be a key factor controlling such shifts because it influences a species' evolutionary flexibility. However, the link between reproductive systems and niche dynamics in plant invasions has been little studied so far. We compiled global occurrence data sets of 13 congeneric sexual and apomictic species pairs, and used principal components analysis (PCA) and kernel smoothers to compare changes in climatic niche optima, breadths and unfilling/expansion between native and alien ranges. Niche change metrics were compared between sexual and apomictic species. All 26 species showed changes in niche optima and/or breadth and 14 species significantly expanded their climatic niches. However, we found no effect of the reproductive system on niche dynamics. Instead, species with narrower native niches showed higher rates of niche expansion in the alien ranges. Our results suggest that niche shifts are frequent in plant invasions but evolutionary potential may not be of major importance for such shifts. Niche dynamics rather appear to be driven by changes of the realized niche without adaptive change of the fundamental climatic niche.

In the absence of sex and recombination, genomes are expected to accumulate deleterious mutations via an irreversible process known as Muller's ratchet, especially in the case of polyploidy. In contrast, no genome-wide mutation accumulation was detected in a transcriptome of facultative apomictic, hexaploid plants of the Ranunculus auricomus complex. We hypothesize that mutations cannot accumulate in flowering plants with facultative sexuality because sexual and asexual development concurrently occurs within the same generation. We assume a strong effect of purging selection on reduced gametophytes in the sexual developmental pathway because previously masked recessive deleterious mutations would be exposed to selection.

Apomixis, the asexual reproduction via seeds, is associated to polyploidy and hybridization. To identify possible signatures of apomixis, and possible candidate genes underlying the shift from sex to apomixis, microarray-based gene expression patterns of live microdissected ovules at four different developmental stages were compared between apomictic and sexual individuals of the Ranunculus auricomus complex. Following predictions from previous work on mechanisms underlying apomixis penetrance and expressivity in the genus, gene expression patterns were classified into three categories based on their relative expression in apomicts compared to their sexual parental ancestors. We found evidence of misregulation and differential gene expression between apomicts and sexuals, with the highest number of differences detected during meiosis progression and emergence of aposporous initial (AI) cells, a key developmental stage in the ovule of apomicts where a decision between divergent reproductive pathways takes place. While most of the differentially expressed genes (DEGs) could not be annotated, gene expression was classified into transgressive, parent of origin and ploidy effects. Genes related to gametogenesis and meiosis demonstrated patterns reflective of transgressive and genome dosage effects, which support the hypothesis of a dominant factor controlling apomixis in Ranunculus and modulated by secondary modifiers. Three genes with probable functions in sporogenesis and gametogenesis development are identified and characterized for future studies.

Polyploidy is common in the genus Salix. However, little is known about the origin, parentage and genomic composition of polyploid species because of a lack of suitable molecular markers and analysis tools. We established a phylogenomic framework including species of all described sections of Eurasian shrub willows. We analyzed the genomic composition of seven polyploid willow species in comparison to putative diploid parental species to draw conclusions on their origin and the effects of backcrossing and post-origin evolution. We applied recently developed programs like SNAPP, HyDe, and SNiPloid to establish a bioinformatic pipeline for unravelling the complexity of polyploid genomes. RAD sequencing revealed 23,393 loci and 320,010 high quality SNPs for the analysis of relationships of 35 species of Eurasian shrub willows (Salix subg. Chamaetia/Vetrix). Polyploid willow species appear to be predominantly of allopolyploid origin. More ancient allopolyploidization events were observed for two hexaploid and one octoploid species, while our data suggested a more recent allopolyploid origin for the included tetraploids and identified putative parental taxa. SNiPloid analyses disentangled the different genomic signatures resulting from hybrid origin, backcrossing, and secondary post-origin evolution in the polyploid species. Our RAD sequencing data demonstrate that willow genomes are shaped by ancient and recent reticulate evolution, polyploidization, and post-origin divergence of species.

The exposure to environmental stress can trigger epigenetic variation, which may have several evolutionary consequences. Polyploidy seems to affect the DNA methylation profiles. Nevertheless, it abides unclear whether temperature stress can induce methylations changes in different cytotypes and to what extent a treatment shift is translated to an epigenetic response. A suitable model system for studying these questions is Ranunculus kuepferi, an alpine perennial herb. Diploid and autotetraploid individuals of R. kuepferi were exposed to cold (+7°C day/+2°C night; frost treatment -1°C cold shocks for 3 nights per week) and warm (+15° day/+10°C night) conditions in climate growth chambers for two consecutive flowering periods and shifted from one condition to the other after the first flowering period. Methylation-sensitive amplified fragment-length polymorphism markers were applied for both years, to track down possible alterations induced by the stress treatments. Patterns of methylation suggested that cytotypes differed significantly in their profiles, independent from year of treatment. Likewise, the treatment shift had an impact on both cytotypes, resulting in significantly less epiloci, regardless the shift's direction. The AMOVAs revealed higher variation within than among treatments in diploids. In tetraploids, internally-methylated loci had a higher variation among than within treatments, as a response to temperature's change in both directions, and support the hypothesis of temperature stress affecting the epigenetic variation. Results suggest that the temperature-sensitivity of DNA methylation patterns shows a highly dynamic phenotypic plasticity in R. kuepferi, as both cytotypes responded to temperature shifts. Furthermore, ploidy level, even without effects of hybridization, has an important effect on epigenetic background variation, which may be correlated with the DNA methylation dynamics during cold acclimation.

The taxonomic status of many Southern European taxa of the Ranunculus auricomus complex remains uncertain despite this region's proximity to the native ranges of the sexual progenitor species of the complex. We investigated whether additional sexual progenitor species are present in the Mediterranean region. Utilizing target enrichment of 736 single-copy nuclear gene regions and flow cytometry, we analyzed phylogenomic relationships, the ploidy level, and the reproductive mode in representatives of 16 populations in Southern Europe, with additional sequence data from herbarium collections. Additionally, phased sequence assemblies from suspected nothotaxa were mapped to previously described sexual progenitor species in order to determine hybrid ancestry. We found the majority of Mediterranean taxa to be tetraploid, with hybrid populations propagating primarily via apomixis. Phylogenomic analysis revealed that except for the progenitor species, the Mediterranean taxa are often polyphyletic. Most apomictic taxa showed evidence of mixed heritage from progenitor species, with certain progenitor genotypes having mapped more to the populations from adjacent geographical regions. Geographical trends were found in phylogenetic distance, roughly following an east-to-west longitudinal demarcation of the complex, with apomicts extending to the southern margins. Additionally, we observed post-hybridization divergence between the western and eastern populations of nothotaxa in Southern Europe. Our results support a classification of apomictic populations as nothotaxa, as previously suggested for Central Europe.

Emerging polyploids may depend on environmental niche shifts for successful establishment. Using the alpine plant Ranunculus kuepferi as a model system, we explore the niche shift hypothesis at different spatial resolutions and in contrasting parts of the species range.

The reconstruction of reticulate evolutionary histories in plants is still a major methodological challenge. Sequences of the ITS nrDNA are a popular marker to analyze hybrid relationships, but variation of this multicopy spacer region is affected by concerted evolution, high intraindividual polymorphism, and shifts in mode of reproduction. The relevance of changes in secondary structure is still under dispute. We aim to shed light on the extent of polymorphism within and between sexual species and their putative natural as well as synthetic hybrid derivatives in the Ranunculus auricomus complex to test morphology-based hypotheses of hybrid origin and parentage of taxa. We employed direct sequencing of ITS nrDNA from 68 individuals representing three sexuals, their synthetic hybrids and one sympatric natural apomict, as well as cloning of ITS copies in four representative individuals, RNA secondary structure analysis, and landmark geometric morphometric analysis on leaves. Phylogenetic network analyses indicate additivity of parental ITS variants in both synthetic and natural hybrids. The triploid synthetic hybrids are genetically much closer to their maternal progenitors, probably due to ploidy dosage effects, although exhibiting a paternal-like leaf morphology. The natural hybrids are genetically and morphologically closer to the putative paternal progenitor species. Secondary structures of ITS1-5.8S-ITS2 were rather conserved in all taxa. The observed similarities in ITS polymorphisms suggest that the natural apomict R. variabilis is an ancient hybrid of the diploid sexual species R. notabilis and the sexual species R. cassubicifolius. The additivity pattern shared by R. variabilis and the synthetic hybrids supports an evolutionary and biogeographical scenario that R. variabilis originated from ancient hybridization. Concerted evolution of ITS copies in R. variabilis is incomplete, probably due to a shift to asexual reproduction. Under the condition of comprehensive inter- and intraspecific sampling, ITS polymorphisms are powerful for elucidating reticulate evolutionary histories.

The genus Acronychia (Citrus family, Rutaceae) contains 49 species of trees and shrubs that are found mainly in rain forest. The genus has a large distributional range from mainland southern Asia to Australia and New Caledonia, but most species are endemic to either New Guinea or Australia. This study aimed to provide the first detailed molecular phylogeny of Acronychia and use it to test the taxonomic value of fruit morphological characters, and infer the historical biogeography of the genus.

Hybridisation and polyploidy are major forces contributing to plant speciation. Homoploid (2x) and heteroploid (3x) hybrids, however, represent critical stages for evolution due to disturbed meiosis and reduced fertility. Apomixis--asexual reproduction via seeds--can overcome hybrid sterility, but requires several concerted alterations of developmental pathways to result in functional seed formation. Here, we analyse the reproductive behaviours of homo- and heteroploid synthetic hybrids from crosses between sexual diploid and tetraploid Ranunculus auricomus species to test the hypothesis that developmental asynchrony in hybrids triggers the shift to apomictic reproduction. Evaluation of male and female gametophyte development, viability and functionality of gametes shows developmental asynchrony, whereas seed set and germinability indicate reduced fitness in synthetic hybrids compared to sexual parents. We present the first experimental evidence for spontaneous apospory in most hybrids as an alternative pathway to meiosis, and the appearance of functional apomictic seeds in triploids. Bypassing meiosis permits these triploid genotypes to form viable seed and new polyploid progeny. Asynchronous development causes reduced sexual seed set and emergence of apospory in synthetic Ranunculus hybrids. Apomixis is functional in triploids and associated with drastic meiotic abnormalities. Selection acts to stabilise developmental patterns and to tolerate endosperm dosage balance shifts which facilitates successful seed set and establishment of apomictic lineages.

Sex determination systems in plants can involve either female or male heterogamety (ZW or XY, respectively). Here we used Illumina short reads, Oxford Nanopore Technologies (ONT) long reads and Hi-C reads to assemble the first chromosome-scale genome of a female willow tree (Salix dunnii), and to predict genes using transcriptome sequences and available databases. The final genome sequence of 328 Mb in total was assembled in 29 scaffolds, and includes 31,501 predicted genes. Analyses of short-read sequence data that included female and male plants suggested a male heterogametic sex-determining factor on chromosome 7, implying that, unlike the female heterogamety of most species in the genus Salix, male heterogamety evolved in the subgenus Salix. The S. dunnii sex-linked region occupies about 3.21 Mb of chromosome 7 in females (representing its position in the X chromosome), probably within a pericentromeric region. Our data suggest that this region is enriched for transposable element insertions, and about one-third of its 124 protein-coding genes were gained via duplications from other genome regions. We detect purifying selection on the genes that were ancestrally present in the region, though some have been lost. Transcriptome data from female and male individuals show more male- than female-biased genes in catkin and leaf tissues, and indicate enrichment for male-biased genes in the pseudo-autosomal regions. Our study provides valuable genomic resources for further studies of sex-determining regions in the family Salicaceae, and sex chromosome evolution.