Oligo-barcode illuminates holocentric karyotype evolution in Rhynchospora (Cyperaceae).

Holocentric karyotypes are assumed to rapidly evolve through chromosome fusions and fissions due to the diffuse nature of their centromeres. Here, we took advantage of the recent availability of a chromosome-scale reference genome for Rhynchospora breviuscula, a model species of this holocentric genus, and developed the first set of oligo-based barcode probes for a holocentric plant. These probes were applied to 13 additional species of the genus, aiming to investigate the evolutionary dynamics driving the karyotype evolution in Rhynchospora. The two sets of probes were composed of 27,392 (green) and 23,968 (magenta) oligonucleotides (45-nt long), and generated 15 distinct FISH signals as a unique barcode pattern for the identification of all five chromosome pairs of the R. breviuscula karyotype. Oligo-FISH comparative analyzes revealed different types of rearrangements, such as fusions, fissions, putative inversions and translocations, as well as genomic duplications among the analyzed species. Two rounds of whole genome duplication (WGD) were demonstrated in R. pubera, but both analyzed accessions differed in the complex chain of events that gave rise to its large, structurally diploidized karyotypes with 2n = 10 or 12. Considering the phylogenetic relationships and divergence time of the species, the specificity and synteny of the probes were maintained up to species with a divergence time of ~25 My. However, karyotype divergence in more distant species hindered chromosome mapping and the inference of specific events. This barcoding system is a powerful tool to study chromosomal variations and genomic evolution in holocentric chromosomes of Rhynchospora species.

Epigenetic histone H3 phosphorylation marks discriminate between univalent- and bivalent-forming chromosomes during canina asymmetrical meiosis.

BACKGROUND AND AIMS: Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2n = 5x = 35 chromosomes in somatic cells. They evolved a unique form of asymmetrical meiosis characterized by two types of chromosomes: (1) chromosomes forming bivalents and distributed in the normal sexual way; and (2) chromosomes occurring as univalents and transferred by a female gamete only. In the mature pollen of pentaploid species, seven bivalent-derived chromosomes are transmitted to offspring, and 21 unpaired univalent chromosomes are eliminated during microsporogenesis. To discriminate between bivalent- and univalent-forming chromosomes, we studied histone H3 phosphorylation patterns regulating meiotic chromosome condensation and segregation. METHODS: We analysed histone modification patterns during male canina meiosis in two representative dogrose species, 5x Rosa canina and 5x Rosa rubiginosa, by immunohistochemical and molecular cytogenetics approaches. Immunostaining of meiotic cells included α-tubulin, histone H3 phosphorylation (H3S10p, H3S28p and H3T3p) and methylation (H3K4me3 and H3K27me3) marks. In addition, fluorescent in situ hybridization was carried out with an 18S rDNA probe. KEY RESULTS: In the first meiotic division, univalent chromosomes underwent equational division into chromatids, while homologues in bivalents were segregated as regular dyads. In diakinesis, bivalent chromosomes displayed strong H3 phosphorylation signals in proximal regions, spreading to the rest of the chromosome. In contrast, in univalents, the H3 phosphorylation signals were weaker, occurring mostly outside proximal regions largely overlapping with the H3K4me3 signals. Reduced phosphorylation was associated with relative under-condensation of the univalent chromosomes, particularly at early diakinesis. CONCLUSIONS: We hypothesize that the absence of pairing and/or recombination in univalent chromosomes negatively affects the histone H3 phosphorylation of their chromatin and perhaps the loading of meiotic-specific cohesins. This apparently destabilizes cohesion of sister chromatids, leading to their premature split in the first meiotic division.

Untangling the hedge: Genetic diversity in clonally and sexually transmitted genomes of European wild roses, Rosa L.

While European wild roses are abundant and widely distributed, their morphological taxonomy is complicated and ambiguous. In particular, the polyploid Rosa section Caninae (dogroses) is characterised by its unusual meiosis, causing simultaneous clonal and sexual transmission of sub-genomes. This hemisexual reproduction, which often co-occurs with vegetative reproduction, defies the standard definition of species boundaries. We analysed seven highly polymorphic microsatellite loci, scored for over 2 600 Rosa samples of differing ploidy, collected across Europe within three independent research projects. Based on their morphology, these samples had been identified as belonging to 21 dogrose and five other native rose species. We quantified the degree of clonality within species and at individual sampling sites. We then compared the genetic structure within our data to current rose morpho-systematics and searched for hemisexually co-inherited sets of alleles at individual loci. We found considerably fewer copies of identical multi-locus genotypes in dogroses than in roses with regular meiosis, with some variation recorded among species. While clonality showed no detectable geographic pattern, some genotypes appeared to be more widespread. Microsatellite data confirmed the current classification of subsections, but they did not support most of the generally accepted dogrose microspecies. Under canina meiosis, we found co-inherited sets of alleles as expected, but could not distinguish between sexually and clonally inherited sub-genomes, with only some of the detected allele combinations being lineage-specific.

The Data Portal "Chromosome Numbers of the Flora of Germany": Progress After Five Years, Recent Developments, and Future Strategies.

Chromosome numbers and information on ploidy level are of great importance for better understanding of plant evolution and taxonomy but also for species identification. Technical developments in flow cytometry dramatically improved the measurement of genome size and triggered a renewed interest in chromosome numbers. Web-based portals make these kind of data accessible for a wide audience in both academia and citizen science. The specialised database of German plant chromosome counts and ploidy estimates comprises to date more than 14,000 records covering 52% of the German taxa. The database is accessed about 70 times per month and became an integral part of several more comprehensive initiatives. One example is the Rothmaler Flora of Germany, which now for the first time features chromosome data with verified origin from the region of interest. Another is the German plant information hub FloraWeb currently amended by several new data sources with the chromosome number database as a major component.

Variation in Volatile Organic Compounds in native, synanthropic accessions and cultivars of the musk strawberry (Fragaria moschata Duchesne ex Weston).

Prior to the world-wide dominance of F. ×ananassa in strawberry production, native species had been cultivated in European gardens for centuries. Especially the musk strawberry (F. moschata) had been highly appreciated due to its fruit size and extraordinary aroma. Detailed studies on the diversity of the species' fruit aroma are lacking, although breeding aims to improve strawberry aroma by complex crossings during recent years. Today a few cultivars, abandoned synanthropic occurrences and native populations of this species exist in Germany. Here we characterised aroma profiles of F. moschata accessions by analysing Volatile Organic Compounds. In particular, differences between native and cultivated accessions as well as the diversity in aroma profiles of native populations were investigated. Profiles of Volatile Organic Compounds were analysed by immersion stir bar sorptive extraction-gas chromatography-quadrupol mass spectrometry (imm-SBSE-GC-qMS). These data were compared with a genetic characterisation of samples based on eight microsatellite loci using univariate and multivariate statistical analyses. High amounts of furanones and the key compound methyl anthranilate were characteristic for the aroma profile of F. moschata. We detected a considerable diversity of Volatile Organic Compounds among accessions of F. moschata, particularly among genetically distinct samples from the same population. Native accessions contained more terpenoids but less esters and were moderately differentiated from cultivated samples. The observed patterns of Volatile Organic Compounds indicate that cultivated accessions had been selected for favourable aroma profiles and thus showing traces of domestication. Moreover, native populations harbour a great diversity of Volatile Organic Compounds, which could be also considered for future breeding efforts.

Phylogeography of Artemisia frigida (Anthemideae, Asteraceae) based on genotyping-by-sequencing and plastid DNA data: Migration through Beringia.

Artemisia frigida is a temperate grassland species that has the largest natural range among its genus, with occurrences across the temperate grassland biomes of Eurasia and North America. Despite its wide geographic range, we know little about the species' distribution history. Hence, we conducted a phylogeographical study to test the hypothesis that the species' distribution pattern is related to a potential historical migration over the 'Bering land bridge'. We applied two molecular approaches: genotyping-by-sequencing (GBS) and Sanger sequencing of the plastid intergenic spacer region (rpl32 - trnL) to investigate genetic differentiation and relatedness among 21 populations from North America, Middle Asia, Central Asia and the Russian Far East. Furthermore, we identified the ploidy level of individuals based on GBS data. Our results indicate that A. frigida originated in Asia, spread northwards to the Far East and then to North America across the Bering Strait. We found a pronounced genetic structuring between Middle and Central Asian populations with mixed ploidy levels, tetraploids in the Far East, and nearly exclusively diploids in North America except for one individual. According to phylogenetic analysis, two populations of Kazakhstan (KZ2 and KZ3) represent the most likely ancestral diploids that constitute the basally branching lineages, and subsequent polyploidization has occurred on several occasions independently. Mantel tests revealed weak correlations between genetic distance and geographical distance and climatic conditions, which indicates that paleoclimatic fluctuations may have more profoundly influenced A. frigida's spatial genetic structure and distribution than the current environment.

Population genetics and plant growth experiments as prerequisite for conservation measures of the rare European aquatic plant Luronium natans (Alismataceae).

Information provided by population genetic studies is often necessary to effectively protect endangered species. In general, such data is scarce for aquatic plants and this holds also for Luronium natans, an aquatic macrophyte endemic to northwestern and western Europe. It is threatened across its whole distribution range due to human influences, in particular due to eutrophication and intensive fish farming. In spite of habitat protection populations continue to decline and re-introductions are one possibility to prevent the species' extinction. Therefore, insights in genetic diversity and relatedness of source populations is warranted. Thus, we performed Amplified Fragment-Length Polymorphism (AFLP) on two large populations in Saxony, Germany (Großenhainer Pflege and Niederspree), complemented with numerous additional occurrences from Europe. In addition, we conducted experiments on plant growth to assess optimal conditions for ex-situ cultivation taking water temperature, water level and substrate into account. We revealed considerably high levels of genetic diversity within populations (Shannon Indices ranged from 0.367 to 0.416) implying that populations are not restricted to clonal growth only but reproduce also by open-pollinated flowers. Remarkably, the two geographically close Saxon populations were genetically distant to each other but subpopulations within a locality were completely intermingled. Concerning optimal cultivation conditions, longest roots were obtained at temperatures >14°C and saturated, but not submerging water levels. Thus, our findings advocate for a re-introduction scheme from nearby source populations and provide detailed information on successful ex-situ cultivation.

Power and Weakness of Repetition - Evaluating the Phylogenetic Signal From Repeatomes in the Family Rosaceae With Two Case Studies From Genera Prone to Polyploidy and Hybridization (Rosa and Fragaria).

Plant genomes consist, to a considerable extent, of non-coding repetitive DNA. Several studies showed that phylogenetic signals can be extracted from such repeatome data by using among-species dissimilarities from the RepeatExplorer2 pipeline as distance measures. Here, we advanced this approach by adjusting the read input for comparative clustering indirectly proportional to genome size and by summarizing all clusters into a main distance matrix subjected to Neighbor Joining algorithms and Principal Coordinate Analyses. Thus, our multivariate statistical method works as a "repeatomic fingerprint," and we proved its power and limitations by exemplarily applying it to the family Rosaceae at intrafamilial and, in the genera Fragaria and Rosa, at the intrageneric level. Since both taxa are prone to hybridization events, we wanted to show whether repeatome data are suitable to unravel the origin of natural and synthetic hybrids. In addition, we compared the results based on complete repeatomes with those from ribosomal DNA clusters only, because they represent one of the most widely used barcoding markers. Our results demonstrated that repeatome data contained a clear phylogenetic signal supporting the current subfamilial classification within Rosaceae. Accordingly, the well-accepted major evolutionary lineages within Fragaria were distinguished, and hybrids showed intermediate positions between parental species in data sets retrieved from both complete repeatomes and rDNA clusters. Within the taxonomically more complicated and particularly frequently hybridizing genus Rosa, we detected rather weak phylogenetic signals but surprisingly found a geographic pattern at a population scale. In sum, our method revealed promising results at larger taxonomic scales as well as within taxa with manageable levels of reticulation, but success remained rather taxon specific. Since repeatomes can be technically easy and comparably inexpensively retrieved even from samples of rather poor DNA quality, our phylogenomic method serves as a valuable alternative when high-quality genomes are unavailable, for example, in the case of old museum specimens.

Ancient Origin of Two 5S rDNA Families Dominating in the Genus Rosa and Their Behavior in the Canina-Type Meiosis.

The genus Rosa comprises more than 100 woody species characterized by intensive hybridization, introgression, and an overall complex evolutionary history. Besides many diploid species (2n = 2x = 14) polyploids ranging from 3x to 10x are frequently found. Here we analyzed 5S ribosomal DNA in 19 species covering two subgenera and the major sections within subg. Rosa. In addition to diploids and polyploids with regular meiosis, we focused on 5x dogroses (Rosa sect. Caninae), which exhibit an asymmetric meiosis differentiating between bivalent- and univalent-forming chromosomes. Using genomic resources, we reconstructed 5S rDNA units to reveal their phylogenetic relationships. Additionally, we designed locus-specific probes derived from intergenic spacers (IGSs) and determined the position and number of 5S rDNA families on chromosomes. Two major 5S rDNA families (termed 5S_A and 5S_B, respectively) were found at variable ratios in both diploid and polyploid species including members of the early diverging subgenera, Rosa persica and Rosa minutifolia. Within subg. Rosa species of sect. Rosa amplified the 5S_A variant only, while taxa of other sections contained both variants at variable ratios. The 5S_B family was often co-localized with 35S rDNA at the nucleolar organizer regions (NOR) chromosomes, whereas the co-localization of the 5S_A family with NOR was only exceptionally observed. The allo-pentaploid dogroses showed a distinct distribution of 5S rDNA families between bivalent- and univalent-forming chromosomes. In conclusion, two divergent 5S rDNA families dominate rose genomes. Both gene families apparently arose in the early history of the genus, already 30 myrs ago, and apparently survived numerous speciation events thereafter. These observations are consistent with a relatively slow genome turnover in the Rosa genus.

Asymmetrical canina meiosis is accompanied by the expansion of a pericentromeric satellite in non-recombining univalent chromosomes in the genus Rosa.

BACKGROUND AND AIMS: Despite their abundant odd-ploidy (2n = 5x = 35), dogroses (Rosa sect. Caninae) are capable of sexual reproduction due to their unique meiosis. During canina meiosis, two sets of chromosomes form bivalents and are transmitted by male and female gametes, whereas the remaining chromosomes form univalents and are exclusively transmitted by the egg cells. Thus, the evolution of chromosomes is expected to be driven by their behaviour during meiosis. METHODS: To gain insight into differential chromosome evolution, fluorescence in situ hybridization was conducted for mitotic and meiotic chromosomes in four dogroses (two subsections) using satellite and ribosomal DNA probes. By exploiting high-throughput sequencing data, we determined the abundance and diversity of the satellite repeats in the genus Rosa by analysing 20 pentaploid, tetraploid and diploid species in total. KEY RESULTS: A pericentromeric satellite repeat, CANR4, was found in all members of the genus Rosa, including the basal subgenera Hulthemia and Hesperhodos. The satellite was distributed across multiple chromosomes (5-20 sites per mitotic cell), and its genomic abundance was higher in pentaploid dogroses (2.3 %) than in non-dogrose species (1.3 %). In dogrose meiosis, univalent chromosomes were markedly enriched in CANR4 repeats based on both the number and the intensity of the signals compared to bivalent-forming chromosomes. Single-nucleotide polymorphisms and cluster analysis revealed high intragenomic homogeneity of the satellite in dogrose genomes. CONCLUSIONS: The CANR4 satellite arose early in the evolution of the genus Rosa. Its high content and extraordinary homogeneity in dogrose genomes is explained by its recent amplification in non-recombining chromosomes. We hypothesize that satellite DNA expansion may contribute to the divergence of univalent chromosomes in Rosa species with non-symmetrical meiosis.

The fate of ribosomal RNA genes in spontaneous polyploid dogrose hybrids [Rosa L. sect. Caninae (DC.) Ser.] exhibiting non-symmetrical meiosis.

Dogroses represent an exceptional system for studying the effects of genome doubling and hybridization: their asymmetrical meiosis enables recombination in bi-parentally inherited chromosomes but prevents it in maternally inherited ones. We employed fluorescent in situ hybridization, genome skimming, amplicon sequencing of genomic and cDNA as well as conventional cloning of nuclear ribosomal DNA in two phylogenetically distinct pentaploid (2n = 5x = 35) species, Rosa canina and Rosa inodora, and their naturally occurring reciprocal hybrids, Rosa dumalis (5x) and Rosa agrestis (5x, 6x). Both progenitor species differed in composition, meiotic behaviour and expression of rDNA loci: R. canina (five 18S and 5-8 5S loci) was dominated by the Canina ribotypes, but R. inodora (four 18S loci and 7-8 5S loci) by the Rubiginosa ribotype. The co-localized 5S/18S loci occurred on either bivalent-forming (R. canina) or univalent-forming (R. inodora) chromosomes. Ribosomal DNA loci were additively inherited; however, the Canina ribotypes were dominantly expressed, even in genotypes with relatively low copy number of these genes. Moreover, we observed rDNA homogenization towards the paternally transmitted Canina ribotype in 6x R. agrestis. The here-observed variation in arrangement and composition of rDNA types between R. canina and R. inodora suggests the involvement of different genomes in bivalent formation. This results supports the hypothesis that the asymmetrical meiosis arose at least twice by independent ancient hybridization events.

To be or not to be the odd one out--allele-specific transcription in pentaploid dogroses (Rosa L. sect. Caninae (DC.) Ser).

BACKGROUND: Multiple hybridization events gave rise to pentaploid dogroses which can reproduce sexually despite their uneven ploidy level by the unique canina meiosis. Two homologous chromosome sets are involved in bivalent formation and are transmitted by the haploid pollen grains and the tetraploid egg cells. In addition the egg cells contain three sets of univalent chromosomes which are excluded from recombination. In this study we investigated whether differential behavior of chromosomes as bivalents or univalents is reflected by sequence divergence or transcription intensity between homeologous alleles of two single copy genes (LEAFY, cGAPDH) and one ribosomal DNA locus (nrITS). RESULTS: We detected a maximum number of four different alleles of all investigated loci in pentaploid dogroses and identified the respective allele with two copies, which is presumably located on bivalent forming chromosomes. For the alleles of the ribosomal DNA locus and cGAPDH only slight, if any, differential transcription was determined, whereas the LEAFY alleles with one copy were found to be significantly stronger expressed than the LEAFY allele with two copies. Moreover, we found for the three marker genes that all alleles have been under similar regimes of purifying selection. CONCLUSIONS: Analyses of both molecular sequence evolution and expression patterns did not support the hypothesis that unique alleles probably located on non-recombining chromosomes are less functional than duplicate alleles presumably located on recombining chromosomes.

Microsatellite analyses of artificial and spontaneous dogrose hybrids reveal the hybridogenic origin of Rosa micrantha by the contribution of unreduced gametes.

Dogroses are characterized by a unique meiosis system, the so-called canina meiosis, which facilitates sexual reproduction at odd-number ploidy. The mostly pentaploid somatic level of dogroses is restored by a merger of haploid sperm cells and tetraploid egg cells. We analyzed experimental hybrids between different dogrose species using microsatellites to determine pollen-transmitted alleles. This information was used to reconstruct the putative hybridogenic origin of Rosa micrantha and R. dumalis and to estimate the frequency of spontaneous hybridization in a natural population. We found no evidence for the hybrid origin of R. dumalis, but our data suggest that R. micrantha presumably arose by hybridization between R. rubiginosa and R. canina or R. corymbifera. We observed only hexaploid individuals of R. micrantha, thus the establishment of this hybridogenic species was favored when unreduced gametes contributed to their origin. We demonstrate that spontaneous hybrids originated infrequently from the parental species in a natural population, but hybridization was often associated with the formation of unreduced gametes. We postulate that unreduced gametes play a major role in the evolutionary success of dogrose hybrids because they provide highly homologous chromosomes crucial for bivalent formation during canina meiosis and thus ensuring this unique form of sexual reproduction.

AFLP markers as a tool to reconstruct complex relationships: A case study in Rosa (Rosaceae).

The genus Rosa has a complex evolutionary history caused by several factors, often in conjunction: extensive hybridization, recent radiation, incomplete lineage sorting, and multiple events of polyploidy. We examined the applicability of AFLP markers for reconstructing (species) relationships in Rosa, using UPGMA clustering, Wagner parsimony, and Bayesian inference. All trees were well resolved, but many of the deeper branches were weakly supported. The cluster analysis showed that the rose cultivars can be separated into a European and an Oriental cluster, each being related to different wild species. The phylogenetic analyses showed that (1) two of the four subgenera (Hulthemia and Platyrhodon) do not deserve subgeneric status; (2) section Carolinae should be merged with sect. Cinnamomeae; (3) subsection Rubigineae is a monophyletic group within sect. Caninae, making sect. Caninae paraphyletic; and (4) there is little support for the distinction of the five other subsections within sect. Caninae. Comparison of the trees with morphological classifications and with previous molecular studies showed that all methods yielded reliable trees. Bayesian inference proved to be a useful alternative to parsimony analysis of AFLP data. Because of their genome-wide sampling, AFLPs are the markers of choice to reconstruct (species) relationships in evolutionary complex groups.

The molecular phylogeny of Rebutia (Cactaceae) and its allies demonstrates the influence of paleogeography on the evolution of South American mountain cacti.

The tropical Andes harbor a major part of the world's plant biodiversity. The montane cacti of the tribes Browningieae, Cereeae, and Trichocereeae underwent extensive radiation and thus are well suited as a model group to study the diversification of Andean plants. We reconstructed their phylogeny employing three noncoding chloroplast regions and explained it in the context of the geological history of South America. We found that the clade of cephalia-bearing cacti with naked pericarpels is centered in northeastern Brazil, whereas almost all other clades comprise Andean species. The spatial split between the clades was probably caused by the Andean uplift and the concurrent formation of intracontinental marine basins in the Tertiary. The phylogenetic reconstructions based on parsimony and Bayesian approaches do not reflect the traditional delimitation of the tribes and of the large genera. Our results suggest that Rebutia s.l. and Echinopsis s.l. are not monophyletic and that Sulcorebutia, Weingartia, and Cintia should be united into one genus. Even though this "Weingartia-complex" and the genus Gymnocalycium are similar in size and morphological diversity, Gymnocalycium has a very high molecular divergence suggesting a comparably older radiation.

Different evolutionary histories of two Phragmidium species infecting the same dog rose hosts.

Rust fungi in the genus Phragmidium are frequent pathogens of both wild and cultivated roses. We investigated the occurrence and relationships of rusts on dog roses, Rosa sect. Caninae (Rosa canina, R. corymbifera and R. rubiginosa) in Germany. Two Phragmidium species, P. mucronatum and P. tuberculatum, were able to infect each of the three dog rose species examined. However, the overall infection of R. rubiginosa was significantly lower, which could be important for rose breeding. Despite overlapping host ranges, the evolutionary background of P. tuberculatum and P. mucronatum is quite distinct. Phylogenetic analyses of the D1/D2 region of the LSU rDNA suggest that P. mucronatum shares a common ancestor with other rose rusts, whereas P. tuberculatum evolved from a Rubus-Sanguisorba rust clade and must have undergone a host shift to Rosa spp.