Uniparental silencing of 5S rRNA genes in plant allopolyploids - insights from Cardamine (Brassicaceae).

While the phenomenon of uniparental silencing of 35S rDNA in interspecific hybrids and allopolyploids is well documented, there is a notable absence of information regarding whether such silencing extends to the 5S RNA component of ribosomes. To address this gap in knowledge, we analyzed the 5S and 35S rDNA expression in Cardamine (Brassicaceae) allopolyploids, namely C. × insueta (2n = 3x = 24, genome composition RRA), C. flexuosa (2n = 4x = 32, AAHH), and C. scutata (2n = 4x = 32, PPAA) which share a common diploid ancestor (AA). We employed high-throughput sequencing of transcriptomes and genomes and phylogenetic analyses of 5S rRNA variants. The genomic organization of rDNA was further scrutinized through clustering and fluorescence in situ hybridization. In the C. × insueta allotriploid, we observed uniparental dominant expression of 5S and 35S rDNA loci. In the C. flexuosa and C. scutata allotetraploids, the expression pattern differed, with the 35S rDNA being expressed from the A subgenome, whereas the 5S rDNA was expressed from the partner subgenome. Both C. flexuosa and C. scutata but not C. × insueta showed copy and locus number changes. We conclude that in stabilized allopolyploids, transcription of ribosomal RNA components occurs from different subgenomes. This phenomenon appears to result in the formation of chimeric ribosomes comprising rRNA molecules derived from distinct parental origins. We speculate that the interplay of epigenetic silencing and rDNA rearrangements introduces an additional layer of variation in multimolecule ribosomal complexes, potentially contributing to the evolutionary success of allopolyploids.

Achromobacter seleniivolatilans sp. nov. and Buttiauxella selenatireducens sp. nov., isolated from the rhizosphere of selenium hyperaccumulator Cardamine hupingshanesis.

Two Gram-stain-negative bacterial strains, R39T and R73T, were isolated from the rhizosphere soil of the selenium hyperaccumulator Cardamine hupingshanesis in China. Strain R39T transformed selenite into elemental and volatile selenium, whereas strain R73T transformed both selenate and selenite into elemental selenium. Phylogenetic and phylogenomic analyses indicated that strain R39T belonged to the genus Achromobacter, while strain R73T belonged to the genus Buttiauxella. Strain R39T (genome size, 6.68 Mb; G+C content, 61.6 mol%) showed the closest relationship to Achromobacter marplatensis LMG 26219T and Achromobacter kerstersii LMG 3441T, with average nucleotide identity (ANI) values of 83.6 and 83.4 %, respectively. Strain R73T (genome size, 5.22 Mb; G+C content, 50.3 mol%) was most closely related to Buttiauxella ferragutiae ATCC 51602T with an ANI value of 86.4 %. Furthermore, strain A111 from the GenBank database was found to cluster with strain R73T within the genus Buttiauxella through phylogenomic analyses. The ANI and digital DNA-DNA hybridization values between strains R73T and A111 were 97.5 and 80.0% respectively, indicating that they belong to the same species. Phenotypic characteristics also differentiated strain R39T and strain R73T from their closely related species. Based on the polyphasic analyses, strain R39T and strain R73T represent novel species of the genera Achromobacter and Buttiauxella, respectively, for which the names Achromobacter seleniivolatilans sp. nov. (type strain R39T=GDMCC 1.3843T=JCM 36009T) and Buttiauxella selenatireducens sp. nov. (type strain R73T=GDMCC 1.3636T=JCM 35850T) are proposed.

Effects of selenoprotein extracts from Cardamine hupingshanensis on growth, selenium metabolism, antioxidant capacity, immunity and intestinal health in largemouth bass Micropterus salmoides.

This study aimed to assess the impact of dietary selenoprotein extracts from Cardamine hupingshanensis (SePCH) on the growth, hematological parameters, selenium metabolism, immune responses, antioxidant capacities, inflammatory reactions and intestinal barrier functions in juvenile largemouth bass (Micropterus salmoides). The base diet was supplemented with four different concentrations of SePCH: 0.00, 0.30, 0.60 and 1.20 g/Kg (actual selenium contents: 0.37, 0.59, 0.84 and 1.30 mg/kg). These concentrations were used to formulate four isonitrogenous and isoenergetic diets for juvenile largemouth bass during a 60-day culture period. Adequate dietary SePCH (0.60 and 1.20 g/Kg) significantly increased weight gain and daily growth rate compared to the control groups (0.00 g/Kg). Furthermore, 0.60 and 1.20 g/Kg SePCH significantly enhanced amounts of white blood cells, red blood cells, platelets, lymphocytes and monocytes, and levels of hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin in the hemocytes. In addition, 0.60 and 1.20 g/Kg SePCH increased the mRNA expression levels of selenocysteine lyase, selenophosphate synthase 1, 15 kDa selenoprotein, selenoprotein T2, selenoprotein H, selenoprotein P and selenoprotein K in the fish liver and intestine compared to the controls. Adequate SePCH not only significantly elevated the activities of antioxidant enzymes (Total superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase), the levels of total antioxidant capacity and glutathione, while increased mRNA transcription levels of NF-E2-related factor 2, Cu/Zn-superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. However, adequate SePCH significantly decreased levels of malondialdehyde and H2O2 and the mRNA expression levels of kelch-like ECH-associated protein 1a and kelch-like ECH-associated protein 1b in the fish liver and intestine compared to the controls. Meanwhile, adequate SePCH markedly enhanced the levels of immune factors (alkaline phosphatase, acid phosphatase, lysozyme, complement component 3, complement component 4 and immunoglobulin M) and innate immune-related genes (lysozyme, hepcidin, liver-expressed antimicrobial peptide 2, complement component 3 and complement component 4) in the fish liver and intestine compared to the controls. Adequate SePCH reduced the levels of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin 8, interleukin 1ß and interferon γ), while increasing transforming growth factor ß1 levels at both transcriptional and protein levels in the liver and intestine. The mRNA expression levels of mitogen-activated protein kinase 13 (MAPK 13), MAPK14 and nuclear factor kappa B p65 were significantly reduced in the liver and intestine of fish fed with 0.60 and 1.20 g/Kg SePCH compared to the controls. Histological sections also demonstrated that 0.60 and 1.20 g/Kg SePCH significantly increased intestinal villus height and villus width compared to the controls. Furthermore, the mRNA expression levels of tight junction proteins (zonula occludens-1, zonula occludens-3, Claudin-1, Claudin-3, Claudin-5, Claudin-11, Claudin-23 and Claudin-34) and Mucin-17 were significantly upregulated in the intestinal epithelial cells of 0.60 and 1.20 g/Kg SePCH groups compared to the controls. In conclusion, these results found that 0.60 and 1.20 g/Kg dietary SePCH can not only improve growth, hematological parameters, selenium metabolism, antioxidant capacities, enhance immune responses and intestinal functions, but also alleviate inflammatory responses. This information can serve as a useful reference for formulating feeds for largemouth bass.

A novel selenite-tolerant rhizosphere bacterium Wautersiella enshiensis sp. nov., isolated from Chinese selenium hyperaccumulator, Cardamine hupingshanensis.

Selenium (Se) is a dietary essential trace element for humans with various physiological functions and it could also be accumulated by some plant species, like Astragalus bisulcatus, Stanleya pinnata, and Cardamine hupinshanensis. A novel Gram-stain-negative, facultatively anaerobic, selenite-tolerant bacterium, designated strain YLX-1T , was isolated from the rhizosphere of a Se hyperaccumulating plant, Cardamine hupingshanensis in Enshi, China. Phylogenetic analysis based on 16 S rRNA gene sequences indicated that strain YLX-1T is a potential new species in the genus Wautersiella. Strain YLX-1T could grow in the temperature range of 4-37°C (optimally at 28°C) and in the pH range of 5-9 (optimum pH 7), which also could tolerate Se up to 6000 mg Se/L via producing extracellular red nano-Se with 100-300 nm size. However, it could predominantly accumulate selenocystine (SeCys2 ) in the cell under lower Se stress (1.5 mg Se/L). These results would help broaden our knowledge about the Se accumulation and transformation mechanism involved in rhizosphere bacteria like strain YLX-1T in C. hupingshanensis. Based on polyphasic data, we propose the creation of the new species Wautersiella enshiensis sp. nov., strain YLX-1T ( = CCTCC M 2013671) which will be promising to produce nano-Se as fertilizer, food additives or medicine.

Evolution of hygrophytic plant species in the Anatolia-Caucasus region: insights from phylogenomic analyses of Cardamine perennials.

BACKGROUND AND AIMS: Southwestern Asia is a significant centre of biodiversity and a cradle of diversification for many plant groups, especially xerophytic elements. In contrast, little is known about the evolution and diversification of its hygrophytic flora. To fill this gap, we focus on Cardamine (Brassicaceae) species that grow in wetlands over a wide altitudinal range. We aimed to elucidate their evolution, assess the extent of presumed historical gene flow between species, and draw inferences about intraspecific structure. METHODS: We applied the phylogenomic Hyb-Seq approach, ecological niche analyses and multivariate morphometrics to a total of 85 Cardamine populations from the target region of Anatolia-Caucasus, usually treated as four to six species, and supplemented them with close relatives from Europe. KEY RESULTS: Five diploids are recognized in the focus area, three of which occur in regions adjacent to the Black and/or Caspian Sea (C. penzesii, C. tenera, C. lazica), one species widely distributed from the Caucasus to Lebanon and Iran (C. uliginosa), and one western Anatolian entity (provisionally C. cf. uliginosa). Phylogenomic data suggest recent speciation during the Pleistocene, likely driven by both geographic separation (allopatry) and ecological divergence. With the exception of a single hybrid (allotetraploid) speciation event proven for C. wiedemanniana, an endemic of southern Turkey, no significant traces of past or present interspecific gene flow were observed. Genetic variation within the studied species is spatially structured, suggesting reduced gene flow due to geographic and ecological barriers, but also glacial survival in different refugia. CONCLUSIONS: This study highlights the importance of the refugial regions of the Black and Caspian Seas for both harbouring and generating hygrophytic species diversity in Southwestern Asia. It also supports the significance of evolutionary links between Anatolia and the Balkan Peninsula. Reticulation and polyploidization played a minor evolutionary role here in contrast to the European relatives.

The evolutionary history of Cardamine bulbifera shows a successful rapid postglacial Eurasian range expansion in the absence of sexual reproduction.

BACKGROUND AND AIMS: Sexual reproduction is known to drive plant diversification and adaptation. Here we investigate the evolutionary history and spatiotemporal origin of a dodecaploid (2n = 12x = 96) Eurasian deciduous woodland species, Cardamine bulbifera, which reproduces and spreads via vegetative bulb-like structures only. The species has been among the most successful range-expanding understorey woodland plants in Europe, which raises the question of the genetic architecture of its gene pool, since its hexaploid (2n = 6x = 48) but putatively outcrossing closest relative, C. quinquefolia, displays a smaller distribution range in Eastern Europe towards the Caucasus region. Cardamine bulbifera belongs to a small monophyletic clade of four species comprising also C. abchasica (2n = 2x = 16) and C. bipinnata (unknown ploidy) from the Caucasus region. METHODS: We sequenced the genomes of the two polyploids and their two putative ancestors using Illumina short-read sequencing technology (×7-8 coverage). Covering the entire distribution range, genomic data were generated for 67 samples of the two polyploids (51 samples of C. bulbifera, 16 samples of C. quinquefolia) and 6 samples of the putative diploid taxa (4 samples of C. abchasica, 2 samples of C. bipinnata) to unravel the evolutionary origin of the polyploid taxa using phylogenetic reconstructions of biparentally and maternally inherited genetic sequence data. Ploidy levels of C. bulbifera and C. quinquefolia were analysed by comparative chromosome painting. We used genetic assignment analysis (STRUCTURE) and approximate Bayesian computation (ABC) modelling to test whether C. bulbifera represents genetically differentiated lineages and addressed the hypothesis of its hybrid origin. Comparative ecological modelling was applied to unravel possible niche differentiation among the two polyploid species. KEY RESULTS: Cardamine bulbifera was shown to be a non-hybridogenous, auto-dodecaploid taxon of early Pleistocene origin, but with a history of past gene flow with its hexaploid sister species C. quinquefolia, likely during the last glacial maximum in shared refuge areas in Eastern Europe towards Western Turkey and the Crimean Peninsula region. The diploid Caucasian endemic C. abchasica is considered an ancestral species, which also provides evidence for the origin of the species complex in the Caucasus region. Cardamine bulbifera successfully expanded its distribution range postglacially towards Central and Western Europe accompanied by a transition to exclusively vegetative propagation. CONCLUSIONS: A transition to vegetative propagation in C. bulbifera is hypothesized as the major innovation to rapidly expand its distribution range following postglacially progressing woodland vegetation throughout Europe. Preceding and introgressive gene flow from its sister species C. quinquefolia in the joint refuge area is documented. This transition and ecological differentiation may have been triggered by preceding introgressive gene flow from its sister species in the joint East European refuge areas.

Transcriptome, proteome, and metabolome reveal the mechanism of tolerance to selenate toxicity in Cardamine violifolia.

Cardamine violifolia was found here to accumulate selenium (Se) to over 9000 mg kg-1 dry weight. To investigate the mechanism of Se accumulation and tolerance in C. violifolia, metabolome, transcriptome, and proteome technologies were applied to C. violifolia seedlings treated with selenate. Several sulfate transporter (Sultr) genes (Sultr1;1, Sultr1;2, and Sultr2;1) and sulfur assimilatory enzyme genes showed high expression levels in response to selenate. Many calcium protein and cysteine-rich kinase genes of C. violifolia were downregulated, whereas selenium-binding protein 1 (SBP1) and protein sulfur deficiency-induced 2 (SDI2) of C. violifolia were upregulated by selenate. The expression of genes involved in the ribosome and posttranslational modifications and chaperones in C. violifolia were also detected in response to selenate. Based on the results of this study and previous findings, we suggest that the downregulated expression of calcium proteins and cysteine-rich kinases, and the upregulated expression of SBP1 and SDI2, were important contributors to the Se tolerance of C. violifolia. The downregulation of cysteine-rich kinases and calcium proteins would enhance Se tolerance of C. violifolia is a novel proposition that has not been reported on other Se hyperaccumulators. This study provides us novel insights to understand Se accumulation and tolerance in plants.

Integration analysis of PacBio SMRT- and Illumina RNA-seq reveals candidate genes and pathway involved in selenium metabolism in hyperaccumulator Cardamine violifolia.

BACKGROUND: Cardamine violifolia, native to China, is one of the selenium (Se) hyperaccumulators. The mechanism of Se metabolism and tolerance remains unclear, and only limited genetic information is currently available. Therefore, we combined a PacBio single-molecule real-time (SMRT) transcriptome library and the Illumina RNA-seq data of sodium selenate (Na2SeO4)-treated C. violifolia to further reveal the molecular mechanism of Se metabolism. RESULTS: The concentrations of the total, inorganic, and organic Se in C. violifolia seedlings significantly increased as the Na2SeO4 treatment concentration increased. From SMRT full-length transcriptome of C. violifolia, we obtained 26,745 annotated nonredundant transcripts, 14,269 simple sequence repeats, 283 alternative splices, and 3407 transcription factors. Fifty-one genes from 134 transcripts were identified to be involved in Se metabolism, including transporter, assimilatory enzyme, and several specific genes. Analysis of Illumina RNA-Seq data showed that a total of 948 differentially expressed genes (DEGs) were filtered from the four groups with Na2SeO4 treatment, among which 11 DEGs were related to Se metabolism. The enrichment analysis of KEGG pathways of all the DEGs showed that they were significantly enriched in five pathways, such as hormone signal transduction and plant-pathogen interaction pathways. Four genes related to Se metabolism, adenosine triphosphate sulfurase 1, adenosine 5'-phosphosulfate reductase 3, cysteine (Cys) desulfurase 1, and serine acetyltransferase 2, were regulated by lncRNAs. Twenty potential hub genes (e.g., sulfate transporter 1;1, Cys synthase, methionine gamma-lyase, and Se-binding protein 1) were screened and identified to play important roles in Se accumulation and tolerance in C. violifolia as concluded by weighted gene correlation network analysis. Based on combinative analysis of expression profiling and annotation of genes as well as Se speciation and concentration in C. violifolia under the treatments with different Na2SeO4 concentrations, a putative Se metabolism and assimilation pathway in C. violifolia was proposed. CONCLUSION: Our data provide abundant information on putative gene transcriptions and pathway involved in Se metabolism of C. violifolia. The findings present a genetic resource and provide novel insights into the mechanism of Se hyperaccumulation in C. violifolia.

Trait-dependent resemblance of the flowering phenology and floral morphology of the allopolyploid Cardamine flexuosa to those of the parental diploids in natural habitats.

Allopolyploids possess complete sets of genomes derived from different parental species and exhibit a range of variation in various traits. Reproductive traits may play a key role in the reproductive isolation between allopolyploids and their parental species, thus affecting the thriving of allopolyploids. However, empirical data, especially in natural habitats, comparing reproductive trait variation between allopolyploids and their parental species remain rare. Here, we documented the flowering phenology and floral morphology of the allopolyploid wild plant Cardamine flexuosa and its diploid parents C. amara and C. hirsuta in their native range in Switzerland. The flowering of C. flexuosa started at an intermediate time compared with those of the parents and the flowering period of C. flexuosa overlapped with those of the parents. Cardamine flexuosa resembled C. hirsuta in the size of flowers and petals and the length/width ratio of petals, while it resembled C. amara in the length/width ratio of flowers. These results provide empirical evidence of the trait-dependent variation of allopolyploid phenotypes in natural habitats at the local scale. They also suggest that the variation in some reproductive traits in C. flexuosa is associated with self-fertilization. Therefore, it is helpful to consider the mating system in furthering the understanding of the processes that may have shaped trait variation in polyploids in nature.

How diploidization turned a tetraploid into a pseudotriploid.

PREMISE OF THE STUDY: Despite being highly fertile and occupying a large geographic region, the North American heartleaf bittercress (Cardamine cordifolia; Brassicaceae) has a puzzling triploid-like chromosome number (2n = 3x = 24). As most triploids are sterile, we embarked on a detailed analysis of the C. cordifolia genome to elucidate its origin and structure. METHODS: Mitotic and meiotic chromosome complement of C. cordifolia was analyzed by comparative chromosome painting using chromosome-specific BAC contigs of Arabidopsis thaliana. Resulting chromosome patterns were documented by multicolor fluorescence microscopy and compared with known ancestral and extant Brassicaceae genomes. KEY RESULTS: We discovered that C. cordifolia is not a triploid hybrid but a diploidized tetraploid with the prevalence of regular, diploid-like meiotic pairing. The ancestral tetraploid chromosome number (2n = 32) was reduced to a triploid-like number (2n = 24) through four terminal chromosome translocations. CONCLUSIONS: The structure of the pseudotriploid C. cordifolia genome results from a stepwise diploidization process after whole-genome duplication. We showed that translocation-based descending dysploidy (from n = 16 to n = 12) was mediated by the formation of five new chromosomes. The genome of C. cordifolia represents the diploidization process in statu nascendi and provides valuable insights into mechanisms of postpolyploidy rediploidization in land plants. Our data further suggest that chromosome number alone does not need to be a reliable proxy of species' evolutionary past and that the same chromosome number may originate either by polyploidization (hybridization) or due to descending dysploidy.

Cytotype distribution patterns, ecological differentiation, and genetic structure in a diploid-tetraploid contact zone of Cardamine amara.

UNLABELLED: • PREMISE OF THE STUDY: Contact zones between diploids and their autopolyploid descendants represent a unique evolutionary venue for studying polyploid establishment, cytotype coexistence, and interactions. Here, we examine cytotype coexistence in a diploid-tetraploid contact zone of a perennial herb, Cardamine amara, located north of the Alps by assessing cytotype spatial patterns, ecological divergence, and genetic variation and structure.• METHODS: Flow cytometry was applied to screen DNA ploidy levels in 302 populations (3296 individuals) and the genetic variation of a selection of 25 populations was examined using microsatellite and AFLP markers. Environmental (landscape and climatic) data were analyzed to assess ecological differentiation between the cytotypes.• KEY RESULTS: A parapatric distribution of the cytotypes with a relatively wide (over 100 km in some regions) secondary contact zone was identified. Mixed-ploidy populations, documented for the first time in this species, as well as triploid individuals were found along the diploid-tetraploid borderline. Different climatic requirements of the two main cytotypes were revealed, mirrored in their altitudinal separation. The tetraploids were genetically differentiated from both the diploids and the modeled, in silico autotetraploid genotypes, in accordance with the assumed polyploid origin and spread linked to past glaciations, and largely independent evolution in allopatry.• CONCLUSIONS: The observed spatial and genetic patterns likely reflect the evolutionary and colonization history of the two cytotypes and have been maintained by multiple factors such as ecological divergence, limited gene flow between the cytotypes, and the restricted dispersal capacity.

When fathers are instant losers: homogenization of rDNA loci in recently formed Cardamine × schulzii trigenomic allopolyploid.

Recently formed allopolyploids represent an excellent system to study the impacts of hybridization and genomic duplication on genome structure and evolution. Here we explored the 35SrRNA genes (rDNA) in the Cardamine × schulzii allohexaploid that was formed by two subsequent hybridization events within the past c. 150 yr. The rDNA loci were analyzed by cloning, next generation sequencing (NGS), RT-PCR and FISH methods. The primary C. × insueta triploid hybrid derived from C. rivularis (♀) and C. amara (♂) had gene ratios highly skewed towards maternal sequences. Similarly, C. × schulzii, originating from the secondary hybridization event involving C. × insueta (♀) and C. pratensis (♂), showed a reduction in paternal rDNA homeologs despite an excess of chromosomes inherited from C. pratensis. We also identified novel rDNA loci in C. × schulzii, suggesting that lost loci might be slowly reinstalled by translocation (but not recombination) of genes from partner genomes. Prevalent clonal propagation of allopolyploids, C. × insueta and C. × schulzii, indicates that concerted evolution of rDNA may occur in the absence of extensive meiotic cycles. Adoption of NGS in rDNA variant analysis is highly informative for deciphering the evolutionary histories of allopolyploid species with ongoing homogenization processes.

Multiple hybridization events in Cardamine (Brassicaceae) during the last 150 years: revisiting a textbook example of neoallopolyploidy.

BACKGROUND AND AIMS: Recently formed allopolyploid species represent excellent subjects for exploring early stages of polyploid evolution. The hexaploid Cardamine schulzii was regarded as one of the few nascent allopolyploid species formed within the past ∼150 years that presumably arose by autopolyploidization of a triploid hybrid, C. × insueta; however, the most recent investigations have shown that it is a trigenomic hybrid. The aims of this study were to explore the efficiency of progenitor-specific microsatellite markers in detecting the hybrid origins and genome composition of these two allopolyploids, to estimate the frequency of polyploid formation events, and to outline their evolutionary potential for long-term persistence and speciation. METHODS: Flow-cytometric ploidy-level screening and genotyping by progenitor-specific microsatellite markers (20 microsatellite loci) were carried out on samples focused on hybridizing populations at Urnerboden, Switzerland, but also including comparative material of the parental species from other sites in the Alps and more distant areas. KEY RESULTS: It was confirmed that hybridization between the diploids C. amara and C. rivularis auct. gave rise to triploid C. × insueta, and it is inferred that this has occurred repeatedly. Evidence is provided that C. schulzii comprises three parental genomes and supports its origin from hybridization events between C. × insueta and the locally co-occurring hypotetraploid C. pratensis, leading to two cytotypes of C. schulzii: hypopentaploid and hypohexaploid. Each cytotype of C. schulzii is genetically uniform, suggesting their single origins. CONCLUSIONS: Persistence of C. schulzii has presumably been achieved only by perennial growth and clonal reproduction. This contrasts with C. × insueta, in which multiple origins and occasional sexual reproduction have generated sufficient genetic variation for long-term survival and evolutionary success. This study illustrates a complex case of recurrent hybridization and polyploidization events, and highlights the role of triploids that promoted the origin of trigenomic hybrids.

Context-dependent resistance against butterfly herbivory in a polyploid herb.

Spatial variation in biotic interactions and natural selection are fundamental parts of natural systems, and can be driven by differences in both trait distributions and the local environmental context of the interaction. Most studies of plant-animal interactions have been performed only in natural settings, making it difficult to disentangle the effects of traits and context. To assess the relative importance of trait differences and environmental context for among-population variation in plant resistance to herbivory, we compared oviposition by the butterfly Anthocharis cardamines on two ploidy types of the herb Cardamine pratensis under experimentally controlled conditions with oviposition in natural populations. Under controlled conditions, plants from octoploid populations were significantly more preferred than plants from tetraploid populations. This difference was largely mediated by differences in flower size. Among natural populations, there was no difference in oviposition rates between the two ploidy types. Our results suggest that differences in oviposition rates among populations of the two cytotypes in the field are caused mainly by differences in environmental context, and that the higher attractiveness of octoploids to herbivores observed under common environmental conditions is balanced by the fact that they occur in habitats which harbor lower densities of butterflies. This illustrates that spatial variation in biotic interactions is the net result of differences in trait distributions of the interacting organisms and differences in environmental context, and that variation in both traits and context are important in understanding species interactions.

The widespread crucifer species Cardamine flexuosa is an allotetraploid with a conserved subgenomic structure.

The origin of Cardamine flexuosa (Wavy Bittercress) has been a conundrum for more than six decades. Here we identify its parental species, analyse its genome structure in comparison to parental genomes and describe intergenomic structural variations in C. flexuosa. Genomic in situ hybridization (GISH) and comparative chromosome painting (CCP) uncovered the parental genomes and the chromosome composition of C. flexuosa and its presumed diploid progenitors. Cardamine flexuosa is an allotetraploid (2n = 4x = 32), originating from two diploid species, Cardamine amara and Cardamine hirsuta (2n = 2x = 16). The two parental species display almost perfectly conserved chromosomal collinearity for seven out of the eight chromosomes. A 13 Mb pericentric inversion distinguishes chromosome CA1 from CH1. A comparative cytomolecular map was established for C. flexuosa by CCP/GISH. Whereas conserved chromosome collinearity between the C. amara and C. hirsuta subgenomes might have promoted intergenomic rearrangements through homeologous recombination, only one reciprocal translocation between two homeologues has occurred since the origin of C. flexuosa. The genome of C. flexuosa demonstrates that allopolyploids can maintain remarkably stable subgenomes over 10(4) -10(5)  yr throughout a wide distribution range. By contrast, the rRNA genes underwent genome-specific elimination towards a diploid-like number of loci.

The more the merrier: recent hybridization and polyploidy in cardamine.

This article describes the use of cytogenomic and molecular approaches to explore the origin and evolution of Cardamine schulzii, a textbook example of a recent allopolyploid, in its ~110-year history of human-induced hybridization and allopolyploidy in the Swiss Alps. Triploids are typically viewed as bridges between diploids and tetraploids but rarely as parental genomes of high-level hybrids and polyploids. The genome of the triploid semifertile hybrid Cardamine × insueta (2n = 24, RRA) was shown to combine the parental genomes of two diploid (2n = 2x = 16) species, Cardamine amara (AA) and Cardamine rivularis (RR). These parental genomes have remained structurally stable within the triploid genome over the >100 years since its origin. Furthermore, we provide compelling evidence that the alleged recent polyploid C. schulzii is not an autohexaploid derivative of C. × insueta. Instead, at least two hybridization events involving C. × insueta and the hypotetraploid Cardamine pratensis (PPPP, 2n = 4x-2 = 30) have resulted in the origin of the trigenomic hypopentaploid (2n = 5x-2 = 38, PPRRA) and hypohexaploid (2n = 6x-2 = 46, PPPPRA). These data show that the semifertile triploid hybrid can promote a merger of three different genomes and demonstrate how important it is to reexamine the routinely repeated textbook examples using modern techniques.

Novel antagonistic interactions associated with plant polyploidization influence trait selection and habitat preference.

Polyploidization is an important mechanism for sympatric speciation in plants. Still, we know little about whether plant polyploidization leads to insect host shifts, and if novel interactions influence habitat and trait selection in plants. We investigated herbivory by the flower bud gall-forming midge Dasineura cardaminis on tetraploids and octoploids of the herb Cardamine pratensis. Gall midges attacked only octoploid plant populations, and a transplantation experiment confirmed this preference. Attack rates were higher in populations that were shaded, highly connected or occurred along stream margins. Within populations, late-flowering individuals with many flowers were most attacked. Galling reduced seed production and significantly influenced phenotypic selection on flower number. Our results suggest that an increase in ploidy may lead to insect host shifts and that plant ploidy explains insect host use. In newly formed plant polyploids, novel interactions may alter habitat preferences and trait selection, and influence the further evolution of cytotypes.

Cytotype diversity and genome size variation in eastern Asian polyploid Cardamine (Brassicaceae) species.

BACKGROUND AND AIMS: Intraspecific ploidy-level variation is an important aspect of a species' genetic make-up, which may lend insight into its evolutionary history and future potential. The present study explores this phenomenon in a group of eastern Asian Cardamine species. METHODS: Plant material was sampled from 59 localities in Japan and Korea, which were used in karyological (chromosome counting) and flow cytometric analyses. The absolute nuclear DNA content (in pg) was measured using propidium iodide and the relative nuclear DNA content (in arbitrary units) was measured using 4,6-diamidino-2-phenylindole fluorochrome. KEY RESULTS: Substantial cytotype diversity was found, with strikingly different distribution patterns between the species. Two cytotypes were found in C. torrentis sensu lato (4x and 8x, in C. valida and C. torrentis sensu stricto, respectively), which displays a north-south geographical pattern in Japan. Hypotheses regarding their origin and colonization history in the Japanese archipelago are discussed. In Korean C. amaraeiformis, only tetraploids were found, and these populations may in fact belong to C. valida. C. yezoensis was found to harbour as many as six cytotypes in Japan, ranging from hexa- to dodecaploids. Ploidy levels do not show any obvious geographical pattern; populations with mixed ploidy levels, containing two to four cytotypes, are frequently observed throughout the range. C. schinziana, an endemic of Hokkaido, has hexa- and octoploid populations. Previous chromosome records are also revised, showing that they are largely based on misidentified material or misinterpreted names. CONCLUSIONS: Sampling of multiple populations and utilization of the efficient flow cytometric approach allowed the detection of large-scale variation in ploidy levels and genome size variation attributable to aneuploidy. These data will be essential in further phylogenetic and evolutionary studies.

Hybridization between two polyploid Cardamine (Brassicaceae) species in North-western Spain: discordance between morphological and genetic variation patterns.

BACKGROUND AND AIMS: Hybridization is an important evolutionary phenomenon, and therefore a detailed understanding of the dynamics of interspecific gene flow and resulting morphological and genetic patterns is of widespread interest. Here hybridization between the polyploids Cardamine pratensis and C. raphanifolia at four localities is explored. Using different types of data, the aim is to provide simultaneous and direct comparisons between genotype and phenotype variation patterns in the studied hybrid populations. METHODS: Evidence of hybridization has been gathered from morphology, molecular markers (amplified fragment length polymorphism and chloroplast DNA sequences), pollen viability, karyology and nuclear DNA content. KEY RESULTS: All data support extensive gene flow occurring in the hybrid populations. A wide range of morphological and genetic variation is observed, which includes both parental and intermediate types. Unbalanced pollen fertility and several ploidy levels are recorded. CONCLUSIONS: Incongruence reported between genotype and phenotype suggests that parental phenotypes are affected by introgression, and intermediate hybrid phenotypes can be genetically closer to one of the parents. Thus, it is evident that morphology, when used alone, can be misleading for interpreting hybridization, and critical evaluation of other data is needed.