Rapid evolution of hybrid breakdown following recent divergence with gene flow in Senecio species on Mount Etna, Sicily.

How do nascent species evolve reproductive isolation during speciation with on-going gene flow? How do hybrid lineages become stabilised hybrid species? While commonly used genomic approaches provide an indirect way to identify species incompatibility factors, synthetic hybrids generated from interspecific crosses allow direct pinpointing of phenotypic traits involved in incompatibilities and the traits that are potentially adaptive in hybrid species. Here we report the analysis of phenotypic variation and hybrid breakdown in crosses between closely-related Senecio aethnensis and S. chrysanthemifolius, and their homoploid hybrid species, S. squalidus. The two former species represent a likely case of recent (<200 ky) speciation with gene flow driven by adaptation to contrasting conditions of high- and low-elevations on Mount Etna, Sicily. As these species form viable and fertile hybrids, it remains unclear whether they have started to evolve reproductive incompatibility. Our analysis represents the first study of phenotypic variation and hybrid breakdown involving multiple Senecio hybrid families. It revealed wide range of variation in multiple traits, including the traits previously unrecorded in synthetic hybrids. Leaf shape, highly distinct between S. aethnensis and S. chrysanthemifolius, was extremely variable in F2 hybrids, but more consistent in S. squalidus. Our study demonstrates that interspecific incompatibilities can evolve rapidly despite on-going gene flow between the species. Further work is necessary to understand the genetic bases of these incompatibilities and their role in speciation with gene flow.

Digest: Evolution of plasticity and its potential role in the decline of specialists.

How does plasticity evolve over relatively short timescales? Through a series of common garden and reciprocal transplant experiments, Walter et al. found distinct patterns of variation in the phenotype and gene expression for two closely related Sicilian daisy species of the genus Senecio across an elevational gradient. This suggests that adaptive divergence may produce interspecific differences in both the magnitude and direction of plasticity. The nonadaptive nature of the plasticity found in Senecio aethnensis has important implications for conservation efforts and evolutionary modeling.

MicroRNAs as serum biomarker for Senecio brasiliensis poisoning in cattle.

Senecio spp. is one of the most frequent plant-related poisonings in cattle. Its ingestion generates the disease seneciosis, characterized by hepatic damages. Liver biopsies and serum markers dosage are tools used in diagnosis; however, many breeding cattle are undiagnosed. MicroRNAs are non-coding RNA, stable in biological fluids. Their difference in expression levels may indicate the presence of the poisoning. We analyzed the miRNA profiling to identify potential diagnostic biomarkers for Senecio brasiliensis poisoning. The expression of miR-21, miR-885, miR-122, miR-181b, miR-30a, miR-378, and let-7 f were evaluated in the serum of exposed cattle. At least one histological change was found in liver and lower quantity of albumin and high AST and ALP were also detected. MiRNAs miR-30a, miR-378, miR-21, miR-885, and miR-122 presented significantly higher expression in intoxicated animals than in healthy animals. Furthermore, miR-122, miR-885, and, especially, miR-21 signatures demonstrated high sensitivity and specificity, with potential application for detecting poisoning.

Multi-methodological approach for the Quality assessment of Senecionis scandentis Herba (Qianliguang) in the herbal market.

We set forth to assess the quality of an herbal medicine sold in Hong Kong called Qianliguang by employing a multi-methodological approach. The quality is set by its identity, chemical composition, and bioactivities, among others. Qianliguang (Senecionis scandentis Herba, Senecio scandens Buch.-Ham. ex D.Don) has known antibacterial properties. However, it is poisonous and overconsumption can result in liver damage. Eighteen Qianliguang samples were purchased from herbal shops at various districts in Hong Kong. Samples were first authenticated organoleptically. DNA barcoding at the psbA-trnH, ITS2, and rbcL loci was then conducted to confirm the species. HPLC-UV was performed to screen for the presence of the chemical compounds and to quantify the flavonoid hyperoside. UPLC-MS was used to quantify the amount of the toxic pyrrolizidine alkaloid (PA) adonifoline. Microdilution assay was performed to show the antibacterial effect on Streptococcus aureus and S. pneumoniae. Results showed that five samples were found to be substituted by species belonging to the genus Lespedeza; four samples were mixtures containing not only Qianliguang but also Achyranthes aspera L., Lonicera confusa DC., or Solanum nigrum L. HPLC-UV showed that only ten contained enough hyperoside to meet the standard requirement. In addition, nine samples had adonifoline that exceeded the toxicity standard requirement. In the microdilution assay, samples containing Qianliguang showed inhibition on S. aureus and S. pneumoniae, while among the five Lespedeza sp. samples the antibacterial effects on S. aureus were not detectable; only one sample showed inhibition to S. pneumoniae. Our study illustrated the necessity of using a multi-methodological approach for herbal medicine quality assessment. We also showed that Qianliguang samples in the Hong Kong market were either toxic or adulterated. It is therefore essential to improve the quality control of Qianliguang and probably other herbs in the herbal market.

Adaptive divergence generates distinct plastic responses in two closely related Senecio species.

The evolution of plastic responses to external cues allows species to maintain fitness in response to the environmental variations they regularly experience. However, it remains unclear how plasticity evolves during adaptation. To test whether distinct patterns of plasticity are associated with adaptive divergence, we quantified plasticity for two closely related but ecologically divergent Sicilian daisy species (Senecio, Asteraceae). We sampled 40 representative genotypes of each species from their native range on Mt. Etna and then reciprocally transplanted multiple clones of each genotype into four field sites along an elevational gradient that included the native elevational range of each species, and two intermediate elevations. At each elevation, we quantified survival and measured leaf traits that included investment (specific leaf area), morphology, chlorophyll fluorescence, pigment content, and gene expression. Traits and differentially expressed genes that changed with elevation in one species often showed little changes in the other species, or changed in the opposite direction. As evidence of adaptive divergence, both species performed better at their native site and better than the species from the other habitat. Adaptive divergence is, therefore, associated with the evolution of distinct plastic responses to environmental variation, despite these two species sharing a recent common ancestor.

Adaptive divergence in shoot gravitropism creates hybrid sterility in an Australian wildflower.

Natural selection is responsible for much of the diversity we see in nature. Just as it drives the evolution of new traits, it can also lead to new species. However, it is unclear whether natural selection conferring adaptation to local environments can drive speciation through the evolution of hybrid sterility between populations. Here, we show that adaptive divergence in shoot gravitropism, the ability of a plant's shoot to bend upwards in response to the downward pull of gravity, contributes to the evolution of hybrid sterility in an Australian wildflower, Senecio lautus We find that shoot gravitropism has evolved multiple times in association with plant height between adjacent populations inhabiting contrasting environments, suggesting that these traits have evolved by natural selection. We directly tested this prediction using a hybrid population subjected to eight rounds of recombination and three rounds of selection in the field. Our experiments revealed that shoot gravitropism responds to natural selection in the expected direction of the locally adapted population. Using the advanced hybrid population, we discovered that individuals with extreme differences in gravitropism had more sterile crosses than individuals with similar gravitropic responses, which were largely fertile, indicating that this adaptive trait is genetically correlated with hybrid sterility. Our results suggest that natural selection can drive the evolution of locally adaptive traits that also create hybrid sterility, thus revealing an evolutionary connection between local adaptation and the origin of new species.

ScGST3 and multiple R2R3-MYB transcription factors function in anthocyanin accumulation in Senecio cruentus.

Anthocyanins are important flavonoid pigments involved in the colouring of flowers and fruits. They are synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported into the vacuole for storage. Previous reports have suggested that glutathione S-transferase (GST) is involved in anthocyanin transport. However, due to the limitation of plant materials, most GSTs only participate in the cyanidin or delphinidin transport pathway. Here, an anthocyanin-related GST, ScGST3, was identified from the transcriptome of cineraria. The expression pattern of ScGST3 was highly consistent with anthocyanin accumulation in ray florets. Molecular complementation of Arabidopsis tt19 indicated that the overexpression of ScGST3 restores the anthocyanin-deficient phenotype of the mutant. Virus-induced gene silencing (VIGS) of ScGST3 in carmine and blue cineraria leaves could inhibit anthocyanin accumulation, further confirming the function of ScGST3 in anthocyanin accumulation. In vitro assays showed that ScGST3 increases the water solubility of cyanidin-3-O-glucoside (C3G) and delphinidin-3-O-glucosid (D3G). In addition, we also identified two anthocyanin-related MYB transcription factors, ScMYB3 and ScMYB6. The expression pattern of these two genes was also highly consistent with anthocyanin accumulation. Faded abaxial leaf phenotypes were observed after the silencing of ScMYB3 and ScMYB6, and the expression levels of partial structural genes were repressed. Based on the results from dual-luciferase assays and yeast one-hybrid assays, ScMYB3 can activate the promoter of ScGST3. Collectively, the transcription of ScGST3 is regulated by ScMYB3, which plays an important role in the transport of C3G and D3G in cineraria.

Phenotypic and genotypic parallel evolution in parapatric ecotypes of Senecio.

The independent and repeated adaptation of populations to similar environments often results in the evolution of similar forms. This phenomenon creates a strong correlation between phenotype and environment and is referred to as parallel evolution. However, we are still largely unaware of the dynamics of parallel evolution, as well as the interplay between phenotype and genotype within natural systems. Here, we examined phenotypic and genotypic parallel evolution in multiple parapatric Dune-Headland coastal ecotypes of an Australian wildflower, Senecio lautus. We observed a clear trait-environment association in the system, with all replicate populations having evolved along the same phenotypic evolutionary trajectory. Similar phenotypes have arisen via mutational changes occurring in different genes, although many share the same biological functions. Our results shed light on how replicated adaptation manifests at the phenotypic and genotypic levels within populations, and highlight S. lautus as one of the most striking cases of phenotypic parallel evolution in nature.

Highly Replicated Evolution of Parapatric Ecotypes.

Parallel evolution of ecotypes occurs when selection independently drives the evolution of similar traits across similar environments. The multiple origins of ecotypes are often inferred based on a phylogeny that clusters populations according to geographic location and not by the environment they occupy. However, the use of phylogenies to infer parallel evolution in closely related populations is problematic because gene flow and incomplete lineage sorting can uncouple the genetic structure at neutral markers from the colonization history of populations. Here, we demonstrate multiple origins within ecotypes of an Australian wildflower, Senecio lautus. We observed strong genetic structure as well as phylogenetic clustering by geography and show that this is unlikely due to gene flow between parapatric ecotypes, which was surprisingly low. We further confirm this analytically by demonstrating that phylogenetic distortion due to gene flow often requires higher levels of migration than those observed in S. lautus. Our results imply that selection can repeatedly create similar phenotypes despite the perceived homogenizing effects of gene flow.

Plastid phylogenomics and insights into the inter-mountain dispersal of the Eastern African giant senecios (Dendrosenecio, Asteraceae).

Giant senecios (Dendrosenecio, Asteraceae), endemic to the tropical mountains of Eastern Africa, are one of the most conspicuous alpine plant groups in the world. Although the group has received substantial attention from researchers, its infrageneric relationships are contentious, and the speciation history remains poorly understood. In this study, whole chloroplast genome sequences of 46 individuals were used to reconstruct the phylogeny of giant senecios using Maximum Likelihood and Bayesian Inference methods. The divergence times of this emblematic group were estimated using fossil-based calibrations. Additionally, the ancestral areas were inferred, and ecological niche modeling was used to predict their suitable habitats. Phylogenetic analyses yielded two robustly supported clades. One clade included taxa sampled from Tanzania, while the other clade included species from other regions. Giant senecios likely originated from the North of Tanzania approximately 2.3 million years ago (highest posterior density 95%; 0.77-4.40), then rapidly radiated into the Kenyan and Ugandan mountains within the last one million years. The potential routes of dispersal have been proposed based on the inferred ancestral areas, estimated time, and predicted past suitable niches. Plio-Pleistocene climate oscillations and orogeny instigated early divergence of the genus. Whereas in situ radiation of giant senecios was chiefly driven by multiple long-distance dispersal events followed by episodes of vicariance, and allopatric speciation (geographic and/or altitudinal).

Development of an activity assay for characterizing deoxyhypusine synthase and its diverse reaction products.

Deoxyhypusine synthase transfers an aminobutyl moiety from spermidine to the eukaryotic translation initiation factor 5A (eIF5A) in the first step of eIF5A activation. This exclusive post-translational modification is conserved in all eukaryotes. Activated eIF5A has been shown to be essential for cell proliferation and viability. Recent reports have linked the activation of eIF5A to several human diseases. Deoxyhypusine synthase, which is encoded by a single gene copy in most eukaryotes, was duplicated in several plant lineages during evolution, the copies being repeatedly recruited to pyrrolizidine alkaloid biosynthesis. However, the function of many of these duplicates is unknown. Notably, deoxyhypusine synthase is highly promiscuous and can catalyze various reactions, often of unknown biological relevance. To facilitate in-depth biochemical studies of this enzyme, we report here the development of a simple and robust in vitro enzyme assay. It involves precolumn derivatization of the polyamines taking part in the reaction and avoids the need for the previously used radioactively labeled tracers. The derivatized polyamines are quantified after high-performance liquid chromatography coupled to diode array and fluorescence detectors. By performing kinetic analyses of deoxyhypusine synthase and its paralog from the pyrrolizidine alkaloid-producing plant Senecio vernalis, we demonstrate that the assay unequivocally differentiates the paralogous enzymes. Furthermore, it detects and quantifies, in a single assay, the side reactions that occur in parallel to the main reaction. The presented assay thus provides a detailed biochemical characterization of deoxyhypusine synthase and its paralogs.

Direct formation of the sesquiterpeonid ether liguloxide by a terpene synthase in Senecio scandens.

KEY MESSAGE: SsLOS directly catalyzed formation of the sesquiterpenoid ether liguloxide in the medicinal plant Senecio scandens. Terpene synthases determine the diversity of terpene skeletons and corresponding terpenoid natural products. Oxygenated groups introduced in catalysis of terpene synthases are important for solubility, potential bioactivity and further elaboration of terpenoids. Here we identified one terpene synthase, SsLOS, in the Chinese medicinal plant Senecio scandens. SsLOS acted as the sesquiterpene synthase and utilized (E,E)-farnesyl diphosphate as the substrate to produce a blend of sesquiterpenoids. GC-MS analysis and NMR structure identification demonstrated that SsLOS directly produced the sesquiterpenoid ether, liguloxide, as well as its alcoholic isomer, 6-epi-guaia-2(3)-en-11-ol. Homology modeling and site-directed mutagenesis were combined to explore the catalytic mechanism of SsLOS. A few key residues were identified in the active site and hedycaryol was identified as the neutral intermediate of SsLOS catalysis. The plausible catalytic mechanism was proposed as well. Altogether, SsLOS was identified and characterized as the sesquiterpenoid ether synthase, which is the second terpenoid ether synthase after 1,8-cineol synthase, suggesting some insights for the universal mechanism of terpene synthases using the water molecule in the catalytic cavity.

Diversity and evolution of cytochrome P450s of Jacobaea vulgaris and Jacobaea aquatica.

BACKGROUND: Collectively, plants produce a huge variety of secondary metabolites (SMs) which are involved in the adaptation of plants to biotic and abiotic stresses. The most characteristic feature of SMs is their striking inter- and intraspecific chemical diversity. Cytochrome P450 monooxygenases (CYPs) often play an important role in the biosynthesis of SMs and thus in the evolution of chemical diversity. Here we studied the diversity and evolution of CYPs of two Jacobaea species which contain a characteristic group of SMs namely the pyrrolizidine alkaloids (PAs). RESULTS: We retrieved CYPs from RNA-seq data of J. vulgaris and J. aquatica, resulting in 221 and 157 full-length CYP genes, respectively. The analyses of conserved motifs confirmed that Jacobaea CYP proteins share conserved motifs including the heme-binding signature, the PERF motif, the K-helix and the I-helix. KEGG annotation revealed that the CYPs assigned as being SM metabolic pathway genes were all from the CYP71 clan but no CYPs were assigned as being involved in alkaloid pathways. Phylogenetic analyses of full-length CYPs were conducted for the six largest CYP families of Jacobaea (CYP71, CYP76, CYP706, CYP82, CYP93 and CYP72) and were compared with CYPs of two other members of the Asteraceae, Helianthus annuus and Lactuca sativa, and with Arabidopsis thaliana. The phylogenetic trees showed strong lineage specific diversification of CYPs, implying that the evolution of CYPs has been very fast even within the Asteraceae family. Only in the closely related species J. vulgaris and J. aquatica, CYPs were found often in pairs, confirming a close relationship in the evolutionary history. CONCLUSIONS: This study discovered 378 full-length CYPs in Jacobaea species, which can be used for future exploration of their functions, including possible involvement in PA biosynthesis and PA diversity.

Afro-alpine flagships revisited: Parallel adaptation, intermountain admixture and shallow genetic structuring in the giant senecios (Dendrosenecio).

Distantly related lineages of the enigmatic giant rosette plants of tropical alpine environments provide classical examples of convergent adaptation. For the giant senecios (Dendrosenecio), the endemic landmarks of the East African sky islands, it has also been suggested that parallel adaptation has been important for within-lineage differentiation. To test this hypothesis and to address potential gene flow and hybridization among the isolated sky islands, we organized field expeditions to all major mountains. We sampled all currently accepted species and all but one subspecies and genotyped 460 plants representing 109 populations. We tested whether genetic structuring corresponds to geography, as predicted by a parallel adaptation hypothesis, or to altitudinal belt and habitat rather than mountains, as predicted by a hypothesis of a single origin of adaptations. Bayesian and Neighbor-Net analyses showed that the main genetic structure is shallow and largely corresponds to geography, supporting a hypothesis of recent, rapid radiation via parallel altitude/habitat adaptation on different mountains. We also found evidence for intermountain admixture, suggesting several long-distance dispersals by wind across vast areas of unsuitable habitat. The combination of parallel adaptation, secondary contact, and hybridization may explain the complex patterns of morphological variation and the contradicting taxonomic treatments of these rare enigmatic giants, supporting the use of wide taxonomic concepts. Notably, the within-population genetic diversity was very low and calls for increased conservation efforts.

Senecio as a model system for integrating studies of genotype, phenotype and fitness.

Two major developments have made it possible to use examples of ecological radiations as model systems to understand evolution and ecology. First, the integration of quantitative genetics with ecological experiments allows detailed connections to be made between genotype, phenotype, and fitness in the field. Second, dramatic advances in molecular genetics have created new possibilities for integrating field and laboratory experiments with detailed genetic sequencing. Combining these approaches allows evolutionary biologists to better study the interplay between genotype, phenotype, and fitness to explore a wide range of evolutionary processes. Here, we present the genus Senecio (Asteraceae) as an excellent system to integrate these developments, and to address fundamental questions in ecology and evolution. Senecio is one of the largest and most phenotypically diverse genera of flowering plants, containing species ranging from woody perennials to herbaceous annuals. These Senecio species exhibit many growth habits, life histories, and morphologies, and they occupy a multitude of environments. Common within the genus are species that have hybridized naturally, undergone polyploidization, and colonized diverse environments, often through rapid phenotypic divergence and adaptive radiation. These diverse experimental attributes make Senecio an attractive model system in which to address a broad range of questions in evolution and ecology.

Strong divergent selection at multiple loci in two closely related species of ragworts adapted to high and low elevations on Mount Etna.

Recently diverged species present particularly informative systems for studying speciation and maintenance of genetic divergence in the face of gene flow. We investigated speciation in two closely related Senecio species, S. aethnensis and S. chrysanthemifolius, which grow at high and low elevations, respectively, on Mount Etna, Sicily and form a hybrid zone at intermediate elevations. We used a newly generated genome-wide single nucleotide polymorphism (SNP) dataset from 192 individuals collected over 18 localities along an elevational gradient to reconstruct the likely history of speciation, identify highly differentiated SNPs, and estimate the strength of divergent selection. We found that speciation in this system involved heterogeneous and bidirectional gene flow along the genome, and species experienced marked population size changes in the past. Furthermore, we identified highly-differentiated SNPs between the species, some of which are located in genes potentially involved in ecological differences between species (such as photosynthesis and UV response). We analysed the shape of these SNPs' allele frequency clines along the elevational gradient. These clines show significantly variable coincidence and concordance, indicative of the presence of multifarious selective forces. Selection against hybrids is estimated to be very strong (0.16-0.78) and one of the highest reported in literature. The combination of strong cumulative selection across the genome and previously identified intrinsic incompatibilities probably work together to maintain the genetic and phenotypic differentiation between these species - pointing to the importance of considering both intrinsic and extrinsic factors when studying divergence and speciation.

[Cloning and functional characterization of farnesyl diphosphate synthase in Senecio scandens].

As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize ß-macrocarpene synthase) into BL21 competent cells. The results showed that the content of ß-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.

Cloning and functional characterization of farnesyl diphosphate synthase in Senecio scandens / 中国中药杂志

As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize β-macrocarpene synthase) into BL21 competent cells. The results showed that the content of β-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.

[Identification of stable reference gene by qRT-PCR in Senecio scandens].

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.

Comparative RNA-seq analysis of nickel hyperaccumulating and non-accumulating populations of Senecio coronatus (Asteraceae).

Most metal hyperaccumulating plants accumulate nickel, yet the molecular basis of Ni hyperaccumulation is not well understood. We chose Senecio coronatus to investigate this phenomenon as this species displays marked variation in shoot Ni content across ultramafic outcrops in the Barberton Greenstone Belt (South Africa), thus allowing an intraspecific comparative approach to be employed. No correlation between soil and shoot Ni contents was observed, suggesting that this variation has a genetic rather than environmental basis. This was confirmed by our observation that the accumulation phenotype of plants from two hyperaccumulator and two non-accumulator populations was maintained when the plants were grown on a soil mix from these four sites for 12 months. We analysed the genetic variation among 12 serpentine populations of S. coronatus, and used RNA-seq for de novo transcriptome assembly and analysis of gene expression in hyperaccumulator versus non-accumulator populations. Genetic analysis revealed the presence of hyperaccumulators in two well supported evolutionary lineages, indicating that Ni hyperaccumulation may have evolved more than once in this species. RNA-Seq analysis indicated that putative homologues of transporters associated with root iron uptake in plants are expressed at elevated levels in roots and shoots of hyperaccumulating populations of S. coronatus from both evolutionary lineages. We hypothesise that Ni hyperaccumulation in S. coronatus may have evolved through recruitment of these transporters, which play a role in the iron-deficiency response in other plant species.