Development and characterization of eleven microsatellite loci for the tropical understory tree Paypayrola blanchetiana Tul. (Violaceae).

Microsatellites markers were developed for Paypayrola blanchetiana (Violaceae), a near-dispersing forest tree forming aggregated populations, to investigate genetic diversity and gene flow among subpopulations in a fragmented environment. Next generation sequencing (Illumina platform) was used to develop ten nuclear microsatellite loci and one plastid microsatellite locus that amplify in P. blanchetiana. Polymorphism was tested in two subpopulations separated by a distance of approximately 11 km. The identified loci contained between two and five alleles per locus. Observed heterozygosity ranged between 0.063 and 0.563 in both subpopulations, while expected heterozygosity ranged from 0.063 to 0.567 in the first, and 0.063-0.627 in the second subpopulation. The microsatellites are among the first in the family Violaceae and will be useful for population genetic studies in this species. Amplification was successful in one further Paypayrola species from Amazonia, which suggest a wider usefulness of the present markers.

Tangled banks: A landscape genomic evaluation of Wallace's Riverine barrier hypothesis for three Amazon plant species.

Wallace's Riverine Barrier hypothesis is one of the earliest biogeographic explanations for Amazon speciation, but it has rarely been tested in plants. In this study, we used three woody Amazonian plant species to evaluate Wallace's Hypothesis using tools of landscape genomics. We generated unlinked single-nucleotide polymorphism (SNP) data from the nuclear genomes of 234 individuals (78 for each plant species) across 13 sampling sites along the Rio Branco, Brazil, for Amphirrhox longifolia (8,075 SNPs), Psychotria lupulina (9,501 SNPs) and Passiflora spinosa (14,536 SNPs). Although significantly different migration rates were estimated between species, the population structure data do not support the hypothesis that the Rio Branco-an allopatric barrier for primates and birds-is a significant genetic barrier for Amphirrhox longifolia, Passiflora spinosa or Psychotria lupulina. Overall, we demonstrated that medium-sized rivers in the Amazon Basin, such as the Rio Branco, are permeable barriers to gene flow for animal-dispersed and animal-pollinated plant species.

Wide but not impermeable: Testing the riverine barrier hypothesis for an Amazonian plant species.

Wallace's riverine barrier hypothesis postulates that large rivers, such as the Amazon and its tributaries, reduce or prevent gene flow between populations on opposite banks, leading to allopatry and areas of species endemism occupying interfluvial regions. Several studies have shown that two major tributaries, Rio Branco and Rio Negro, are important barriers to gene flow for birds, amphibians and primates. No botanical studies have considered the potential role of the Rio Branco as a barrier, while a single botanical study has evaluated the Rio Negro as a barrier. We studied an Amazon shrub, Amphirrhox longifolia (A. St.-Hil.) Spreng (Violaceae), as a model to test the riverine barrier hypothesis. Twenty-six populations of A. longifolia were sampled on both banks of the Rio Branco and Rio Negro in the core Amazon Basin. Double-digest RADseq was used to identify 8,010 unlinked SNP markers from the nuclear genome of 156 individuals. Data relating to population structure support the hypothesis that the Rio Negro acted as a significant genetic barrier for A. longifolia. On the other hand, no genetic differentiation was detected among populations spanning the narrower Rio Branco, which is a tributary of the Rio Negro. This study shows that the strength of riverine barriers for Amazon plants is dependent on the width of the river separating populations and species-specific dispersal traits. Future studies of plants with contrasting life history traits will further improve our understanding of the landscape genetics and allopatric speciation history of Amazon plant diversity.

No evidence of contemporary interploidy gene flow between the closely related European woodland violets Viola reichenbachiana and V. riviniana (sect. Viola, Violaceae).

Viola reichenbachiana (2n = 4x = 20) and V. riviniana (2n = 8x = 40) are closely related species widely distributed in Europe, often sharing the same habitat throughout their overlapping ranges. It has been suggested in numerous studies that their high intraspecific morphological variability and plasticity might have been further increased by interspecific hybridisation in contact zones, given the sympatry of the species and the incomplete sterility of their hybrid. The aims of this study were to: (i) confirm that V. reichenbachiana and V. riviniana have one 4x genome in common, and (ii) determine the impact of hybridisation and introgression on genetic variation of these two species in selected European populations. For our study, we used 31 Viola populations from four European countries, which were analysed using AFLP and sequencing of a variable plastid intergenic spacer, trnH-psbA. Our analysis revealed that V. reichenbachiana exhibited larger haplotype diversity, having three species-specific haplotypes versus one in V. riviniana. The relationships among haplotypes suggest transfer of common haplotypes into V. riviniana from both V. reichenbachiana and hypothetically the other, now extinct, parental species. AFLP analysis showed low overall genetic diversity of both species, with V. riviniana showing higher among-population diversity. None of the morphologically designated hybrid populations had additive AFLP polymorphisms that would have indicated recent hybridisation. Also, kinship coefficients between both species did not indicate gene flow. V. riviniana showed significant population subdivision and significant isolation by distance, in contrast to V. reichenbachiana. The results indicate lack of gene flow between species, high influence of selfing on genetic variability, as well as probably only localised introgression toward V. riviniana.

Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species.

High-throughput DNA sequencing facilitates the analysis of large portions of the genome in nonmodel organisms, ensuring high accuracy of population genetic parameters. However, empirical studies evaluating the appropriate sample size for these kinds of studies are still scarce. In this study, we use double-digest restriction-associated DNA sequencing (ddRADseq) to recover thousands of single nucleotide polymorphisms (SNPs) for two physically isolated populations of Amphirrhox longifolia (Violaceae), a nonmodel plant species for which no reference genome is available. We used resampling techniques to construct simulated populations with a random subset of individuals and SNPs to determine how many individuals and biallelic markers should be sampled for accurate estimates of intra- and interpopulation genetic diversity. We identified 3646 and 4900 polymorphic SNPs for the two populations of A. longifolia, respectively. Our simulations show that, overall, a sample size greater than eight individuals has little impact on estimates of genetic diversity within A. longifolia populations, when 1000 SNPs or higher are used. Our results also show that even at a very small sample size (i.e. two individuals), accurate estimates of FST can be obtained with a large number of SNPs (≥1500). These results highlight the potential of high-throughput genomic sequencing approaches to address questions related to evolutionary biology in nonmodel organisms. Furthermore, our findings also provide insights into the optimization of sampling strategies in the era of population genomics.

Discovery, structure, function, and applications of cyclotides: circular proteins from plants.

Cyclotides are plant-derived cyclic peptides that have a head-to-tail cyclic backbone and three conserved disulphide bonds that form a cyclic cystine knot motif. They occur in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, typically with 10-100 cyclotides in a given plant species, in a wide range of tissues, including flowers, leaves, stems, and roots. Some cyclotides are expressed in large amounts (up to 1g kg(-1) wet plant weight) and their natural function appears to be to protect plants from pests or pathogens. This article provides a brief overview of their discovery, distribution in plants, and applications. In particular, their exceptional stability has led to their use as peptide-based scaffolds in drug design applications. They also have potential as natural 'ecofriendly' insecticides, and as protein engineering frameworks.

Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining.

Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150 000 individual cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies.

Does human-induced habitat transformation modify pollinator-mediated selection? A case study in Viola portalesia (Violaceae).

Pollinator-mediated selection is one of the most important factors driving adaptation in flowering plants. However, as ecological conditions change through habitat loss and fragmentation, the interactions among species may evolve in new and unexpected directions. Human-induced environmental variation is likely to affect selection regimes, but as yet no empirical examples have been reported. In the study reported here, we examined the influence of human-induced habitat transformation on the composition of pollinator assemblages and, hence, pollinator-mediated selection on the flower phenotype of Viola portalesia (Violaceae). Our results indicate that pollinator assemblages differed substantially in terms of species composition and visitation rate between nearby native and transformed habitats. Similarly, the insect species that contributed most to visitation rates differed between plant populations. While the magnitude and sign of pollinator-mediated selection on flower length and width did not differ between sites, selection for flower number lost significance in the transformed habitat, and a significant pattern of disruptive selection for flower shape, undetected in the native habitat, was present in the transformed one. Overall, the results of this study suggest that human-induced habitat change may not only modify the species composition of pollinator assemblages, relaxing the selection process on some flower characters, but they may also create new opportunities for fitness-trait covariation not present in pristine conditions.

Evolution of plant RNA polymerase IV/V genes: evidence of subneofunctionalization of duplicated NRPD2/NRPE2-like paralogs in Viola (Violaceae).

BACKGROUND: DNA-dependent RNA polymerase IV and V (Pol IV and V) are multi-subunit enzymes occurring in plants. The origin of Pol V, specific to angiosperms, from Pol IV, which is present in all land plants, is linked to the duplication of the gene encoding the largest subunit and the subsequent subneofunctionalization of the two paralogs (NRPD1 and NRPE1). Additional duplication of the second-largest subunit, NRPD2/NRPE2, has happened independently in at least some eudicot lineages, but its paralogs are often subject to concerted evolution and gene death and little is known about their evolution nor their affinity with Pol IV and Pol V. RESULTS: We sequenced a approximately 1500 bp NRPD2/E2-like fragment from 18 Viola species, mostly paleopolyploids, and 6 non-Viola Violaceae species. Incongruence between the NRPD2/E2-like gene phylogeny and species phylogeny indicates a first duplication of NRPD2 relatively basally in Violaceae, with subsequent sorting of paralogs in the descendants, followed by a second duplication in the common ancestor of Viola and Allexis. In Viola, the mutation pattern suggested (sub-) neofunctionalization of the two NRPD2/E2-like paralogs, NRPD2/E2-a and NRPD2/E2-b. The dN/dS ratios indicated that a 54 bp region exerted strong positive selection for both paralogs immediately following duplication. This 54 bp region encodes a domain that is involved in the binding of the Nrpd2 subunit with other Pol IV/V subunits, and may be important for correct recognition of subunits specific to Pol IV and Pol V. Across all Viola taxa 73 NRPD2/E2-like sequences were obtained, of which 23 (32%) were putative pseudogenes - all occurring in polyploids. The NRPD2 duplication was conserved in all lineages except the diploid MELVIO clade, in which NRPD2/E2-b was lost, and its allopolyploid derivates from hybridization with the CHAM clade, section Viola and section Melanium, in which NRPD2/E2-a occurred in multiple copies while NRPD2/E2-b paralogs were either absent or pseudogenized. CONCLUSIONS: Following the relatively recent split of Pol IV and Pol V, our data indicate that these two multi-subunit enzymes are still in the process of specialization and each acquiring fully subfunctionalized copies of their subunit genes. Even after specialization, the NRPD2/E2-like paralogs are prone to pseudogenization and gene conversion and NRPD2 and NRPE2 copy number is a highly dynamic process modulated by allopolyploidy and gene death.

Cyclotide proteins and precursors from the genus Gloeospermum: filling a blank spot in the cyclotide map of Violaceae.

Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.

Description of a fertilization-independent obligate apomictic species: Corunastylis apostasioides Fitzg.

The Australian midge orchid Corunastylis apostasioides of the tribe Diurideae has completely eliminated any male contribution in the process of seed formation, which occurs directly from the maternal tissue by a process termed apomixis. Here, we report C. apostasioides to be an obligate apomictic species devoid of any sexuality and compare its development to a close sexual relative C. fimbriata (R. Br.) D.L. Jones & M.A. Clem. Apomictic characteristics in C. apostasioides include production of seed in absence of fertilization, frequently closed flowers, production of immature pollen in non-dehiscent anthers, expansion of ovaries despite the lack of fertilization and the absence of a citronella scent that is found in C. fimbriata produced to attract pollinating vinegar flies (Jones 2006). The nature of apomixis in C. apostasioides was examined by ovule histology and amplified fragment length polymorphism (AFLP) in each case drawing comparison with sexual C. fimbriata. In C. apostasioides the central megaspore mother cell undergoes diplosporic apomixis, while additional embryos are derived from nucellar or integument initials formed by sporophytic apomixis. Typical of apomicts, C. apostasioides is polyploid compared to the sexual C. fimbriata. The divergences of C. apostasioides from sexuality to apomictic development are discussed.

Circular proteins from Melicytus (Violaceae) refine the conserved protein and gene architecture of cyclotides.

Cyclotides are cyclic disulfide rich mini-proteins found in various Rubiaceae (coffee family), Violaceae (violet family) and Cucurbitaceae (squash family) plant species. Within the Violaceae, cyclotides have been found in numerous species of the genus Viola as well as species from two other genera, namely Hybanthus and Leonia. This is the first in-depth report of cyclotides in the genus Melicytus (Violaceae). We present the chromatographic profiles of extracts of eight Melicytus species and one Melicytus hybrid that were found to contain these circular peptides. We isolated and characterised five novel cyclotides (mra1 to mra5) from the aerial parts of a common New Zealand tree, Melicytus ramiflorus. All five peptides show the characteristics of the bracelet subfamily of cyclotides. Furthermore, we isolated 17 non-redundant cDNA clones from the leaves of Melicytus ramiflorus encoding cyclotide prepropeptides. This detailed report on the presence of cyclotides in several species of the genus Melicytus further strengthens our hypothesis that cyclotides are ubiquitous in Violaceae family plants and provides additional insight into the biochemical processing mechanisms that produce the cyclic protein backbone of this unique family of ultra-stable plant proteins.

Molecular phylogenetic analysis of Violaceae (Malpighiales) based on plastid and nuclear DNA sequences.

A phylogenetic analysis of Violaceae is presented using sequences from rbcL, atpB, matK and 18S rDNA from 39 species and 19 genera. The combined analysis of four molecular markers resulted in only one most parsimonious tree, and 33 of all 38 nodes within Violaceae are supported by a bootstrap proportion of more than 50%. Fusispermum is in a basal-most position and Rinorea, Decorsella, Rinoreocarpus and the other Violaceae are successively diverged. The monogeneric subfamily Fusispermoideae is supported, and it shares a number of plesiomorphies with Passifloraceae (a convolute petal aestivation, actinomorphic flowers and connate filaments). The other monogeneric subfamily Leonioideae is sunken within the subfamily Violoideae and is sister to Gloeospermum, sharing some seed morphological characteristics. The present molecular phylogenetic analysis suggests that the convolute, apotact and quincuncial petal aestivation is successively derived within the family. The evolutionary trends of the other morphological characteristics, such as a filament connation, the number of carpels and floral symmetry, are discussed.

GISH and AFLP analyses of novel Brassica napus lines derived from one hybrid between B. napus and Orychophragmus violaceus.

New Brassica napus inbred lines with different petal colors and with canola quality and increased levels of oleic (approximately 70%, 10% higher than that of B. napus parent) and linoleic (28%) acids have been developed in the progenies of one B. napus cv. Oro x Orychophragmus violaceus F5 hybrid plant (2n = 31). Their genetic constituents were analyzed by using the methods of genomic in situ hybridization (GISH) and amplified fragments length polymorphism (AFLP). No intact chromosomes of O. violaceus origin were detected by GISH in their somatic cells of ovaries and root tips (2n = 38) and pollen mother cells (PMCs) with normal chromosome pairing (19 bivalents) and segregation (19:19), though signals of variable sizes and intensities were located mainly at terminal and centromeric parts of some mitotic chromosomes and meiotic bivalents at diakinesis or chromosomes in anaphase I groups and one large patch of chromatin was intensively labeled and separated spatially in some telophase I nuclei and metaphase II PMCs. AFLP analysis revealed that substantial genomic changes have occurred in these lines and O. violaceus-specific bands, deleted bands in 'Oro' and novel bands for two parents were detected. The possible mechanisms for these results were discussed.

Extra divisions and nuclei fusions in microspores from Brassica allohexaploid (AABBCC) x Orychophragmus violaceus hybrids.

Abnormal meiosis and microspore development and related defective mutants have often been reported in plants and wide hybrids. Here extra divisions and nuclei fusions were observed to occur in microspore nuclei of partial hybrids between synthetic Brassica hexaploid (2n = 54, AABBCC) and another crucifer Orychophragmus violaceus (2n = 24). Abnormal spindle were formed and chromosomes were separated into several nuclei of variable sizes after bi-, or multi-polar divisions in the four cells of tetrads. As a consequence, more than eight mini-microspores of different sizes were produced by one tetrad. Genomic in situ hybridization results indicated that no chromosome replication occurred during such divisions. In some tetrads, the four nuclei were fused to form one large cell with increased chromosome number. The extra divisions or fusions appeared only in some flower buds of one plant, some anthers in the same buds, or even in individual cells of tetrads. The possible mechanisms behind these cytological phenomena are discussed.

A continent of plant defense peptide diversity: cyclotides in Australian Hybanthus (Violaceae).

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

Demographic and random amplified polymorphic DNA analyses reveal high levels of genetic diversity in a clonal violet.

We performed demographic and molecular investigations on woodland populations of the clonal herb Viola riviniana in central Germany. We investigated the pattern of seedling recruitment, the amount of genotypic (clonal) variation and the partitioning of genetic variation among and within populations. Our demographic study was carried out in six violet populations of different ages and habitat conditions. It revealed that repeated seedling recruitment takes place in all of these populations, and that clonal propagation is accompanied by high ramet mortality. Our molecular investigations were performed on a subset of three of these six violet populations. Random amplified polymorphic DNA analyses using six primers yielded 45 scorable bands that were used to identify multilocus genotypes, i.e. putative clones. Consistent with our demographic results and independent of population age, we found a large genotypic diversity with a mean proportion of distinguishable genotypes of 0.93 and a mean Simpson's diversity index of 0.99. Using AMOVA we found a strong genetic differentiation among these violet populations with a PhiST value of 0.41. We suggest that a high selfing rate, limited gene flow due to short seed dispersal distances and drift due to founder effects are responsible for this pattern. Although Viola riviniana is a clonal plant, traits associated with sexual reproduction rather than clonality per se are moulding the pattern of genetic variation in this species.