Genotyping-by-sequencing-based high-resolution mapping reveals a single candidate gene for the grapevine veraison locus Ver1.

Veraison marks the transition from berry growth to berry ripening and is a crucial phenological stage in grapevine (Vitis vinifera): the berries become soft and begin to accumulate sugars, aromatic substances, and, in red cultivars, anthocyanins for pigmentation, while the organic acid levels begin to decrease. These changes determine the potential quality of wine. However, rising global temperatures lead to earlier flowering and ripening, which strongly influence wine quality. Here, we combined genotyping-by-sequencing with a bioinformatics pipeline on ∼150 F1 genotypes derived from a cross between the early ripening variety 'Calardis Musqué' and the late-ripening variety 'Villard Blanc'. Starting from 20,410 haplotype-based markers, we generated a high-density genetic map and performed a quantitative trait locus analysis based on phenotypic datasets evaluated over 20 years. Through locus-specific-marker-enrichment and recombinant screening of ∼1000 additional genotypes, we refined the originally postulated 5 Mb veraison locus, Ver1, on chromosome 16 to only 112 kb, allowing us to pinpoint the ethylene response factor (ERF) VviERF027 (VCost.v3 gene ID: Vitvi16g00942, CRIBIv1 gene ID: VIT_16s0100g00400) as veraison candidate gene. Furthermore, the early veraison allele could be traced back to a clonal 'Pinot' variant first mentioned in the 17th century. 'Pinot Precoce Noir' passed this allele over 'Madeleine Royale' to the maternal grandparent 'Bacchus Weiss' and, ultimately, to the maternal parent 'Calardis Musqué'. Our findings are crucial for ripening time control, thereby improving wine quality, and for breeding grapevines adjusted to climate change scenarios that have a major impact on agro-ecosystems in altering crop plant phenology.

A comparative analysis of plastome evolution in autotrophic Piperales.

PREMISE: Many plastomes of autotrophic Piperales have been reported to date, describing a variety of differences. Most studies focused only on a few species or a single genus, and extensive, comparative analyses have not been done. Here, we reviewed publicly available plastome reconstructions for autotrophic Piperales, reanalyzed publicly available raw data, and provided new sequence data for all previously missing genera. Comparative plastome genomics of >100 autotrophic Piperales were performed. METHODS: We performed de novo assemblies to reconstruct the plastomes of newly generated sequence data. We used Sanger sequencing and read mapping to verify the assemblies and to bridge assembly gaps. Furthermore, we reconstructed the phylogenetic relationships as a foundation for comparative plastome genomics. RESULTS: We identified a plethora of assembly and annotation issues in published plastome data, which, if unattended, will lead to an artificial increase of diversity. We were able to detect patterns of missing and incorrect feature annotation and determined that the inverted repeat (IR) boundaries were the major source for erroneous assembly. Accounting for the aforementioned issues, we discovered relatively stable junctions of the IRs and the small single-copy region (SSC), whereas the majority of plastome variations among Piperales stems from fluctuations of the boundaries of the IR and the large single-copy (LSC) region. CONCLUSIONS: This study of all available plastomes of autotrophic Piperales, expanded by new data for previously missing genera, highlights the IR-LSC junctions as a potential marker for discrimination of various taxonomic levels. Our data indicates a pseudogene-like status for cemA and ycf15 in various Piperales. Based on a review of published data, we conclude that incorrect IR-SSC boundary identification is the major source for erroneous plastome assembly. We propose a gold standard for assembly and annotation of high-quality plastomes based on de novo assembly methods and appropriate references for gene annotation.

Extreme plastomes in holoparasitic Balanophoraceae are not the norm.

BACKGROUND: Balanophoraceae plastomes are known for their highly condensed and re-arranged nature alongside the most extreme nucleotide compositional bias known to date, culminating in two independent reconfigurations of their genetic code. Currently, a large portion of the Balanophoraceae diversity remains unexplored, hindering, among others, evolutionary pattern recognition. Here, we explored newly sequenced plastomes of Sarcophyte sanguinea and Thonningia sanguinea. The reconstructed plastomes were analyzed using various methods of comparative genomics based on a representative taxon sampling. RESULTS: Sarcophyte, recovered sister to the other sampled Balanophoraceae s. str., has plastomes up to 50% larger than those currently published. Its gene set contains five genes lost in any other species, including matK. Five cis-spliced introns are maintained. In contrast, the Thonningia plastome is similarly reduced to published Balanophoraceae and retains only a single cis-spliced intron. Its protein-coding genes show a more biased codon usage compared to Sarcophyte, with an accumulation of in-frame TAG stop codons. Structural plastome comparison revealed multiple, previously unknown, structural rearrangements within Balanophoraceae. CONCLUSIONS: For the "minimal plastomes" of Thonningia, we propose a genetic code change identical to sister genus Balanophora. Sarcophyte however differs drastically from our current understanding on Balanophoraceae plastomes. With a less-extreme nucleotide composition, there is no evidence for an altered genetic code. Using comparative genomics, we identified a hotspot for plastome reconfiguration in Balanophoraceae. Based on previously published and newly identified structural reconfigurations, we propose an updated model of evolutionary plastome trajectories for Balanophoraceae, illustrating a much greater plastome diversity than previously known.

The use of Anchored Hybrid Enrichment data to resolve higher-level phylogenetic relationships: A proof-of-concept applied to Asterales (Eudicotyledoneae; Angiosperms).

Anchored Hybrid Enrichment (AHE) is a tool for capturing orthologous regions of the nuclear genome shared in low or single copy across lineages. Despite the increasing number of studies using this method, its usefulness to estimate relationships at deeper taxonomic levels in plants has not been fully explored. Here we present a proof of concept about the performance of nuclear loci obtained with AHE to infer phylogenetic relationships and explore the use of gene sampling schemes to estimate divergence times in Asterales. We recovered low-copy nuclear loci using the AHE method from herbarium material and silica-preserved samples. Maximum likelihood, Bayesian inference, and coalescence approaches were used to reconstruct phylogenomic relationships. Dating analyses were conducted under a multispecies coalescent approach by jointly inferring species tree and divergence times with random gene sampling schemes and multiple calibrations. We recovered 403 low-copy nuclear loci for 63 species representing nine out of eleven families of Asterales. Phylogenetic hypotheses were congruent among the applied methods and previously published results. Analyses with concatenated datasets were strongly supported, but coalescence-based analyses showed low support for the phylogenetic position of families Argophyllaceae and Alseuosmiaceae. Estimated family ages were congruent among gene sampling schemes, with the mean age for Asterales around 130 Myr. Our study documents the usefulness of AHE for resolving phylogenetic relationships at deep phylogenetic levels in Asterales. Observed phylogenetic inconsistencies were possibly due to the non-inclusion of families Phellinceae and Pentaphragmataceae. Random gene sampling schemes produced consistent age estimates with coalescence and species tree relaxed clock approaches.

Evolution of Class II TCP genes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia.

Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.

Rapid identification of inflorescence type markers by genotyping-by-sequencing of diploid and triploid F1 plants of Hydrangea macrophylla.

BACKGROUND: The ornamental crop Hydrangea macrophylla develops highly attractive lacecap (wild type) or mophead inflorescences. The mophead trait, which is mostly favored by consumers, is recessively inherited by the INFLORESCENCE TYPE locus (INF). If lacecap cultivars are crossed with mophead cultivars, then either 50% or all progenies develop lacecap inflorescences, depending on the zygosity at the INF locus. For most cultivars, the zygosity at the INF locus is unknown. Furthermore, the determination of the inflorescence type in offspring populations is time-consuming, because seedlings flower the first time in the 2nd year after sowing. Within this study, we aimed to develop DNA-based markers that allow to determine the zygosity at the INF locus of prospective parental plants and to predict the inflorescence phenotype of seedlings already in the non-flowering stage. RESULTS: By crossing a mophead and a lacecap cultivar of H. macrophylla, we produced a pseudo-backcross F1 population consisting of 422 plants. These plants segregated into 279 lacecap, 73 mophead, 3 intermediate and 67 non-flowering plants, differing significantly from the expected 1:1 segregation ratio. Surprisingly, 75% of these plants were triploid, although both parents were diploid. We found that the lacecap parent produced unreduced pollen, which induced the formation of triploids. 380 randomly selected F1 plants were genotyped by genotyping-by-sequencing (GbS). Using a genome assembly of cultivar 'Sir Joseph Banks', we performed subsequently a bulk sequence analysis with pooled GbS data of diploid versus mophead plants. We identified directly 2 markers tightly linked with the INF locus, each of them explaining 99.7% of the inflorescence phenotype. Using a collection consisting of 56 diploid, triploid or tetraploid H. macrophylla varieties, we detected 6 sequence variants for one of these markers. Two variants were associated with the mophead phenotype. Furthermore, we found by marker analysis a co-segregation between the mophead and the non-flowering trait, which indicates a major flowering time locus next to the INF locus. CONCLUSION: Through bulk sequence analysis of pooled GbS data from diploid and polyploid F1 plants, we identify rapidly tightly linked markers for the inflorescence type, a dominant-recessively inherited trait in the non-model plant species H. macrophylla.

The effects of intervessel pit characteristics on xylem hydraulic efficiency and photosynthesis in hemiepiphytic and non-hemiepiphytic Ficus species.

Xylem vulnerability to cavitation and hydraulic efficiency are directly linked to fine-scale bordered pit features in water-conducting cells of vascular plants. However, it is unclear how pit characteristics influence water transport and carbon economy in tropical species. The primary aim of this study was to evaluate functional implications of changes in pit characteristics for water relations and photosynthetic traits in tropical Ficus species with different growth forms (i.e. hemiepiphytic and non-hemiepiphytic) grown under common conditions. Intervessel pit characteristics were measured using scanning electron microscopy in five hemiepiphytic and five non-hemiepiphytic Ficus species to determine whether these traits were related to hydraulics, leaf photosynthesis, stomatal conductance and wood density. Ficus species varied greatly in intervessel pit structure, hydraulic conductivity and leaf physiology, and clear differences were observed between the two growth forms. The area and diameter of pit aperture were negatively correlated with sapwood-specific hydraulic conductivity, mass-based net assimilation rate, stomatal conductance (gs ), intercellular CO2 concentration (Ci ) and the petiole vessel lumen diameters (Dv ), but positively correlated with wood density. Pit morphology was only negatively correlated with sapwood- and leaf-specific hydraulic conductivity and Dv . Pit density was positively correlated with gs , Ci and Dv , but negatively with intrinsic leaf water-use efficiency. Pit and pit aperture shape were not significantly correlated with any of the physiological traits. These findings indicate a significant role of pit characteristics in xylem water transport, carbon assimilation and ecophysiological adaptation of Ficus species in tropical rain forests.

Multilocus coalescent species delimitation to evaluate traditionally defined morphotypes in Hydrangea sect. Asperae (Hydrangeaceae).

The number of species recognized in section Asperae of the flowering plant genus Hydrangea differs widely between subsequent revisions. This variation is largely centered around the H. aspera species complex, with numbers of recognized species varying from one to nearly a dozen. Despite indications of molecular variation in this complex, no sequence-based species delimitation methods have been employed to evaluate the primarily morphology-based species boundaries. In the present study, a multi-locus coalescent-based approach to species delimitation is employed in order to identify separate evolutionary lines within H. sect. Asperae, using four chloroplast and four nuclear molecular markers. Eight lineages were recovered within the focal group, of which five correspond with named morphotypes. The other three lineages illustrate types of conflict between molecular species delimitation and traditional morphology-based taxonomy. One molecular lineage comprises two named morphotypes, which possibly diverged recently enough to not have developed sufficient molecular divergence. A second conflict is found in H. strigosa. This morphotype is recovered as a separate lineage when occurring in geographic isolation, but when occurring in sympatry with two other morphotypes (H. aspera and H. robusta), the coalescent species delimitation lumps these taxa into a single putative species.

Recalcitrant deep and shallow nodes in Aristolochia (Aristolochiaceae) illuminated using anchored hybrid enrichment.

Recalcitrant relationships are characterized by very short internodes that can be found among shallow and deep phylogenetic scales all over the tree of life. Adding large amounts of presumably informative sequences, while decreasing systematic error, has been suggested as a possible approach to increase phylogenetic resolution. The development of enrichment strategies, coupled with next generation sequencing, resulted in a cost-effective way to facilitate the reconstruction of recalcitrant relationships. By applying the anchored hybrid enrichment (AHE) genome partitioning strategy to Aristolochia using an universal angiosperm probe set, we obtained 231-233 out of 517 single or low copy nuclear loci originally contained in the enrichment kit, resulting in a total alignment length of 154,756bp to 160,150bp. Since Aristolochia (Piperales; magnoliids) is distantly related to any angiosperm species whose genome has been used for the plant AHE probe design (Amborella trichopoda being the closest), it serves as a proof of universality for this probe set. Aristolochia comprises approximately 500 species grouped in several clades (OTUs), whose relationships to each other are partially unknown. Previous phylogenetic studies have shown that these lineages branched deep in time and in quick succession, seen as short-deep internodes. Short-shallow internodes are also characteristic of some Aristolochia lineages such as Aristolochia subsection Pentandrae, a clade of presumably recent diversification. This subsection is here included to test the performance of AHE at species level. Filtering and subsampling loci using the phylogenetic informativeness method resolves several recalcitrant phylogenetic relationships within Aristolochia. By assuming different ploidy levels during bioinformatics processing of raw data, first hints are obtained that polyploidization contributed to the evolution of Aristolochia. Phylogenetic results are discussed in the light of current systematics and morphology.

Next-generation sequencing reveals differentially amplified tandem repeats as a major genome component of Northern Europe's oldest Camellia japonica.

Northern Europe's oldest and largest Camellia japonica growing at the Pillnitz Castle (Germany) for over 200 years is of botanical and cultural importance and is a reference for C. japonica molecular scale analysis. In order to provide a fundament for genome analysis of the genus Camellia, we characterize the C. japonica tandem repeat fraction, constituting 12.5 % of the Pillnitz camellia's genome. A genomic library of the Pillnitz C. japonica was produced and Illumina sequenced to generate 36 Gb of paired-end reads. We performed graph-based read clustering implemented in the RepeatExplorer pipeline to estimate the C. japonica repeat fraction of 73 %. This enabled us to identify and characterize the most prominent satellite DNAs, Camellia japonica satellite 1-4 (CajaSat1-CajaSat4), and the 5S ribosomal DNA (rDNA) by bioinformatics, fluorescent in situ and Southern hybridization. Within the Camellia genus, satellite spreading, array expansion and formation of higher-order structures highlight different modes of repeat evolution. The CajaSat satellites localize at prominent chromosomal sites, including (peri)centromeres and subtelomeres of all chromosomes, thus serving as chromosomal landmarks for their identification. This work provides an insight into the C. japonica chromosome organization and significantly expands the Camellia genomic knowledge, also with respect to the tea plant Camellia sinensis.

LC-MS- and (1)H NMR-Based Metabolomic Analysis and in Vitro Toxicological Assessment of 43 Aristolochia Species.

Species of Aristolochia are used as herbal medicines worldwide. They cause aristolochic acid nephropathy (AAN), a devastating disease associated with kidney failure and renal cancer. Aristolochic acids I and II (1 and 2) are considered to be responsible for these nephrotoxic and carcinogenic effects. A wide range of other aristolochic acid analogues (AAAs) exist, and their implication in AAN may have been overlooked. An LC-MS- and (1)H NMR-based metabolomic analysis was carried out on 43 medicinally used Aristolochia species. The cytotoxicity and genotoxicity of 28 Aristolochia extracts were measured in human kidney (HK-2) cells. Compounds 1 and 2 were found to be the most common AAAs. However, AA IV (3), aristolactam I (4), and aristolactam BI (5) were also widespread. No correlation was found between the amounts of 1 or 2 and extract cytotoxicity against HK-2 cells. The genotoxicity and cytotoxicity of the extracts could be linked to their contents of 5, AA D (8), and AA IIIa (10). These results undermine the assumption that 1 and 2 are exclusively responsible for the toxicity of Aristolochia species. Other analogues are likely to contribute to their toxicity and need to be considered as nephrotoxic agents. These findings facilitate understanding of the nephrotoxic mechanisms of Aristolochia and have significance for the regulation of herbal medicines.

A genome-scale mining strategy for recovering novel rapidly-evolving nuclear single-copy genes for addressing shallow-scale phylogenetics in Hydrangea.

BACKGROUND: Identifying orthologous molecular markers that potentially resolve relationships at and below species level has been a major challenge in molecular phylogenetics over the past decade. Non-coding regions of nuclear low- or single-copy markers are a vast and promising source of data providing information for shallow-scale phylogenetics. Taking advantage of public transcriptome data from the One Thousand Plant Project (1KP), we developed a genome-scale mining strategy for recovering potentially orthologous single-copy markers to address low-scale phylogenetics. Our marker design targeted the amplification of intron-rich nuclear single-copy regions from genomic DNA. As a case study we used Hydrangea section Cornidia, one of the most recently diverged lineages within Hydrangeaceae (Cornales), for comparing the performance of three of these nuclear markers to other "fast" evolving plastid markers. RESULTS: Our data mining and filtering process retrieved 73 putative nuclear single-copy genes which are potentially useful for resolving phylogenetic relationships at a range of divergence depths within Cornales. The three assessed nuclear markers showed considerably more phylogenetic signal for shallow evolutionary depths than conventional plastid markers. Phylogenetic signal in plastid markers increased less markedly towards deeper evolutionary divergences. Potential phylogenetic noise introduced by nuclear markers was lower than their respective phylogenetic signal across all evolutionary depths. In contrast, plastid markers showed higher probabilities for introducing phylogenetic noise than signal at the deepest evolutionary divergences within the tribe Hydrangeeae (Hydrangeaceae). CONCLUSIONS: While nuclear single-copy markers are highly informative for shallow evolutionary depths without introducing phylogenetic noise, plastid markers might be more appropriate for resolving deeper-level divergences such as the backbone relationships of the Hydrangeaceae family and deeper, at which non-coding parts of nuclear markers could potentially introduce noise due to elevated rates of evolution. The herein developed and demonstrated transcriptome based mining strategy has a great potential for the design of novel and highly informative nuclear markers for a range of plant groups and evolutionary scales.

The betrayed thief - the extraordinary strategy of Aristolochia rotunda to deceive its pollinators.

Pollination of several angiosperms is based on deceit. In such systems, the flowers advertise a reward that ultimately is not provided. We report on a previously unknown pollination/mimicry system discovered in deceptive Aristolochia rotunda (Aristolochiaceae). Pollinators were collected in the natural habitat and identified. Flower scent and the volatiles of insects (models) potentially mimicked were analyzed by chemical analytical techniques. Electrophysiological and behavioral tests on the pollinators identified the components that mediate the plant-pollinator interaction and revealed the model of the mimicry system. The main pollinators of A. rotunda were female Chloropidae. They are food thieves that feed on secretions of true bugs (Miridae) while these are eaten by arthropod predators. Freshly killed mirids and Aristolochia flowers released the same scent components that chloropids use to find their food sources. Aristolochia exploits these components to deceive their chloropid pollinators. Aristolochia and other trap flowers were believed to lure saprophilous flies and mimic brood sites of pollinators. We demonstrate for A. rotunda, and hypothesize for other deceptive angiosperms, the evolution of a different, kleptomyiophilous pollination strategy. It involves scent mimicry and the exploitation of kleptoparasitic flies as pollinators. Our findings suggest a reconsideration of plants assumed to show sapromyiophilous pollination.

Intercontinental long-distance dispersal of Canellaceae from the New to the Old World revealed by a nuclear single copy gene and chloroplast loci.

Canellales, a clade consisting of Winteraceae and Canellaceae, represent the smallest order of magnoliid angiosperms. The clade shows a broad distribution throughout the Southern Hemisphere, across a diverse range of dry to wet tropical forests. In contrast to their sister-group, Winteraceae, the phylogenetic relations and biogeography within Canellaceae remain poorly studied. Here we present the phylogenetic relationships of all currently recognized genera of Canellales with a special focus on the Old World Canellaceae using a combined dataset consisting of the chloroplast trnK-matK-trnK-psbA and the nuclear single copy gene mag1 (Maigo 1). Within Canellaceae we found high statistical support for the monophyly of Warburgia and Cinnamosma. However, we also found relationships that differ from previous studies. Cinnamodendron splitted into two clades, a South American clade and a second clade confined to the Antilles and adjacent areas. Cinnamodendron from the Antilles, as well as Capsicodendron, South American Cinnamodendron and Pleodendron were not monophyletic. Consequently, Capsicodendron should be included in the South American Cinnamodendron clade and the genus Pleodendron merged with the Cinnamodendron clade from the Antilles. We also found that Warburgia (restricted to mainland eastern Africa) together with the South American Cinnamodendron and Capsicodendron are sister to the Malagasy genus Cinnamosma. In addition to the unexpected geographical relationships, both biogeographic and molecular clock analyses suggest vicariance, extinction, and at least one intercontinental long-distance-dispersal event. Our dating result contrasts previous work on Winteraceae. Diversification of Winteraceae took place in the Paleocene, predating the Canellaceae diversification by 13 MA in the Eocene. The phylogenetic relationships for Canellaceae supported here offer a solid framework for a future taxonomic revision of the Canellaceae.

Major trends in stem anatomy and growth forms in the perianth-bearing Piperales, with special focus on Aristolochia.

BACKGROUND AND AIMS: The order Piperales has the highest diversity of growth forms among the earliest angiosperm lineages, including trees, shrubs, climbers and herbs. However, within the perianth-bearing Piperales (Asarum, Saruma, Lactoris, Hydnora, Prosopanche, Thottea and Aristolochia), climbing species only occur in the most species-rich genus Aristolochia. This study traces anatomical and morphological traits among these lineages, to detect trends in growth form evolution and developmental processes. METHODS: Transverse stem sections of different developmental stages of representatives of Asarum, Saruma, Lactoris, Hydnora, Thottea and Aristolochia were compared and anatomical traits were linked to growth form evolution. Biomechanical properties of representative climbers were determined in three-point bending tests and are discussed based on the anatomical observations. Growth form evolution of the perianth-bearing Piperales was reconstructed by ancestral character state reconstruction using Mesquite. KEY RESULTS: While species of Asarum and Saruma are exclusively herbaceous, species of the remaining genera show a higher diversity of growth habit and anatomy. This growth form diversity is accompanied by a more complex stem anatomy and appropriate biomechanical properties. The ancestral growth form of the perianth-bearing Piperales is reconstructed with either a shrub-like or herbaceous character state, while the following three backbone nodes in the reconstruction show a shrub-like character state. Accordingly, the climbing habit most probably evolved in the ancestor of Aristolochia. CONCLUSIONS: Since the ancestor of the perianth-bearing Piperales has been reconstructed with a herb- or shrub-like habit, it is proposed that the climbing habit is a derived growth form, which evolved with the diversification of Aristolochia, and might have been a key feature for its diversification. Observed anatomical synapomorphies, such as the perivascular fibres in Lactoris, Thottea and Aristolochia, support the phylogenetic relationship of several lineages within the perianth-bearing Piperales. In addition, the hypothesis that the vegetative organs of the holoparasitic Hydnoraceae are most probably rhizomes is confirmed.

Chemical imitation of yeast fermentation by the drosophilid-pollinated deceptive trap-flower Aristolochia baetica (Aristolochiaceae).

Deceptive flowers, unlike in mutualistic pollination systems, mislead their pollinators by advertising rewards which ultimately are not provided. Although our understanding of deceptive pollination systems increased in recent years, the attractive signals and deceptive strategies in the majority of species remain unknown. This is also true for the genus Aristolochia, famous for its deceptive and fly-pollinated trap flowers. Representatives of this genus were generally assumed to be oviposition-site mimics, imitating vertebrate carrion or mushrooms. However, recent studies found a broader spectrum of strategies, including kleptomyiophily and imitation of invertebrate carrion. A different deceptive strategy is presented here for the western Mediterranean Aristolochia baetica L. We found that this species is mostly pollinated by drosophilid flies (Drosophilidae, mostly Drosophila spp.), which typically feed on fermenting fruit infested by yeasts. The flowers of A. baetica emitted mostly typical yeast volatiles, predominantly the aliphatic compounds acetoin and 2,3-butandiol, and derived acetates, as well as the aromatic compound 2-phenylethanol. Analyses of the absolute configurations of the chiral volatiles revealed weakly (acetoin, 2,3-butanediol) to strongly (mono- and diacetates) biased stereoisomer-ratios. Electrophysiological (GC-EAD) experiments and lab bioassays demonstrated that most of the floral volatiles, although not all stereoisomers of chiral compounds, were physiologically active and attractive in drosophilid pollinators; a synthetic mixture thereof successfully attracted them in field and lab bioassays. We conclude that A. baetica chemically mimics yeast fermentation to deceive its pollinators. This deceptive strategy (scent chemistry, pollinators, trapping function) is also known from more distantly related plants, such as Arum palaestinum Boiss. (Araceae) and Ceropegia spp. (Apocynaceae), suggesting convergent evolution. In contrast to other studies working on floral scents in plants imitating breeding sites, the present study considered the absolute configuration of chiral compounds.

Characterization of the basal angiosperm Aristolochia fimbriata: a potential experimental system for genetic studies.

BACKGROUND: Previous studies in basal angiosperms have provided insight into the diversity within the angiosperm lineage and helped to polarize analyses of flowering plant evolution. However, there is still not an experimental system for genetic studies among basal angiosperms to facilitate comparative studies and functional investigation. It would be desirable to identify a basal angiosperm experimental system that possesses many of the features found in existing plant model systems (e.g., Arabidopsis and Oryza). RESULTS: We have considered all basal angiosperm families for general characteristics important for experimental systems, including availability to the scientific community, growth habit, and membership in a large basal angiosperm group that displays a wide spectrum of phenotypic diversity. Most basal angiosperms are woody or aquatic, thus are not well-suited for large scale cultivation, and were excluded. We further investigated members of Aristolochiaceae for ease of culture, life cycle, genome size, and chromosome number. We demonstrated self-compatibility for Aristolochia elegans and A. fimbriata, and transformation with a GFP reporter construct for Saruma henryi and A. fimbriata. Furthermore, A. fimbriata was easily cultivated with a life cycle of just three months, could be regenerated in a tissue culture system, and had one of the smallest genomes among basal angiosperms. An extensive multi-tissue EST dataset was produced for A. fimbriata that includes over 3.8 million 454 sequence reads. CONCLUSIONS: Aristolochia fimbriata has numerous features that facilitate genetic studies and is suggested as a potential model system for use with a wide variety of technologies. Emerging genetic and genomic tools for A. fimbriata and closely related species can aid the investigation of floral biology, developmental genetics, biochemical pathways important in plant-insect interactions as well as human health, and various other features present in early angiosperms.

Application of the phylogenetic informativeness method to chloroplast markers: a test case of closely related species in tribe Hydrangeeae (Hydrangeaceae).

In evolutionary biology appropriate marker selection for the reconstruction of solid phylogenetic hypotheses is fundamental. One of the most challenging tasks addresses the appropriate choice of genomic regions in studies of closely related species. Robust phylogenetic frameworks are central to studies dealing with questions ranging from evolutionary and conservation biology, biogeography to plant breeding. Phylogenetic informativeness profiles provide a quantitative measure of the phylogenetic signal in markers and therefore a method for locus prioritization. The present work profiles phylogenetic informativeness of mostly non-coding chloroplast regions in an angiosperm lineage of closely related species: the popular ornamental tribe Hydrangeeae (Hydrangeaceae, Cornales, Asterids). A recent phylogenetic study denoted a case of resolution contrast between the two strongly supported clades within tribe Hydrangeeae. We evaluate the phylogenetic signal of 13 highly variable plastid markers for estimating relationships within and among the currently recognized monophyletic groups of this tribe. A selection of combined loci based on their phylogenetic informativeness retrieved more robust phylogenetic hypotheses than simply combining individual markers performing best with respect to resolution, nodal support and accuracy or those presenting the highest number of parsimony informative characters. We propose the rpl32-ndhF intergenic spacer (IGS), trnV-ndhC IGS, trnL-rpl32 IGS, psbT-petB region and ndhA intron as the best candidates for future phylogenetic studies in Hydrangeeae and potentially in other Asterids. We also contrasted the phylogenetic informativeness of coded indels against substitutions concluding that, despite their low phylogenetic informativeness, coded indels provide additional phylogenetic signal that is nearly free of noise. Phylogenetic relationships obtained from our total combined analyses showed improved resolution and nodal support with respect to recently published results.

The Mitochondrial Genome of the Holoparasitic Plant Thonningia sanguinea Provides Insights into the Evolution of the Multichromosomal Structure.

Multichromosomal mitochondrial genome (mitogenome) structures have repeatedly evolved in many lineages of angiosperms. However, the underlying mechanism remains unclear. The mitogenomes of three genera of Balanophoraceae, namely Lophophytum, Ombrophytum, and Rhopalocnemis, have already been sequenced and assembled, all showing a highly multichromosomal structure, albeit with different genome and chromosome sizes. It is expected that characterization of additional lineages of this family may expand the knowledge of mitogenome diversity and provide insights into the evolution of the plant mitogenome structure and size. Here, we assembled and characterized the mitogenome of Thonningia sanguinea, which, together with Balanophora, forms a clade sister to the clade comprising Lophophytum, Ombrophytum, and Rhopalocnemis. The mitogenome of T. sanguinea possesses a multichromosomal structure of 18 circular chromosomes of 8.7-19.2 kb, with a total size of 246,247 bp. There are very limited shared regions and poor chromosomal correspondence between T. sanguinea and other Balanophoraceae species, suggesting frequent rearrangements and rapid sequence turnover. Numerous medium- and small-sized repeats were identified in the T. sanguinea mitogenome; however, no repeat-mediated recombination was detected, which was verified by Illumina reads mapping and PCR experiments. Intraspecific mitogenome variations in T. sanguinea are mostly insertions and deletions, some of which can lead to degradation of perfect repeats in one or two accessions. Based on the mitogenome features of T. sanguinea, we propose a mechanism to explain the evolution of a multichromosomal mitogenome from a master circle, which involves mutation in organellar DNA replication, recombination and repair genes, decrease of recombination, and repeat degradation.

Phylogenetic and comparative analyses of Hydnora abyssinica plastomes provide evidence for hidden diversity within Hydnoraceae.

BACKGROUND: To date, plastid genomes have been published for all but two holoparasitic angiosperm families. However, only a single or a few plastomes represent most of these families. Of the approximately 40 genera of holoparasitic angiosperms, a complete plastid genome sequence is available for only about half. In addition, less than 15 species are currently represented with more than one published plastid genome, most of which belong to the Orobanchaceae. Therefore, a significant portion of the holoparasitic plant plastome diversity remains unexplored. This limited information could hinder potential evolutionary pattern recognition as well as the exploration of inter- and intra-species plastid genome diversity in the most extreme holoparasitic angiosperms. RESULTS: Here, we report the first plastomes of Kenyan Hydnora abyssinica accessions. The plastomes have a typical quadripartite structure and encode 24 unique genes. Phylogenetic tree reconstruction recovers the Kenyan accessions as monophyletic and together in a clade with the Namibian H. abyssinica accession and the recently published H. arabica from Oman. Hydnora abyssinica as a whole however is recovered as non-monophyletic, with H. arabica nested within. This result is supported by distinct structural plastome synapomorphies as well as pairwise distance estimates that reveal hidden diversity within the Hydnora species in Africa. CONCLUSION: We propose to increase efforts to sample widespread holoparasitic species for their plastid genomes, as is the case with H. abyssinica, which is widely distributed in Africa. Morphological reinvestigation and further molecular data are needed to fully investigate the diversity of H. abyssinica along the entire range of distribution, as well as the diversity of currently synonymized taxa.