Dramatic difference in rate of chromosome number evolution among sundew (Drosera L., Droseraceae) lineages.

Chromosome number change is a driver of speciation in eukaryotic organisms. Carnivorous sundews in the plant genus Drosera L. exhibit single chromosome number variation both among and within species, especially in the Australian Drosera subg. Ergaleium D.C., potentially linked to atypical centromeres that span much of the length of the chromosomes. We critically reviewed the literature on chromosome counts in Drosera, verified the taxonomy and quality of the original counts, and reconstructed dated phylogenies. We used the BiChrom model to test whether rates of single chromosome number increase and decrease, and chromosome number doubling differed between D. subg. Ergaleium and the other subgenera and between self-compatible and self-incompatible lineages. The best model for chromosome evolution among subgenera had equal rates of chromosome number doubling but higher rates of single chromosome number change in D. subg. Ergaleium than in the other subgenera. Contrary to expectation, self-incompatible lineages had a significantly higher rate of single chromosome loss than self-compatible lineages. We found no evidence for an association between differences in single chromosome number changes and diploidization after polyploidy or centromere type. This study presents an exemplar for critically examining published cytological data and rigorously testing factors that may impact the rates of chromosome number evolution.

Expression and function of an S1-type nuclease in the digestive fluid of a sundew, Drosera adelae.

BACKGROUND AND AIMS: Carnivorous plants trap and digest insects and similar-sized animals. Many studies have examined enzymes in the digestive fluids of these plants and have gradually unveiled the origins and gene expression of these enzymes. However, only a few attempts have been made at characterization of nucleases. This study aimed to reveal gene expression and the structural, functional and evolutionary characteristics of an S1-type nuclease (DAN1) in the digestive fluid of an Australian sundew, Drosera adelae, whose trap organ shows unique gene expression and related epigenetic regulation. METHODS: Organ-specificity in Dan1 expression was examined using glandular tentacles, laminas, roots and inflorescences, and real-time PCR. The methylation status of the Dan1 promoter in each organ was clarified by bisulphite sequencing. The structural characteristics of DAN1 were studied by a comparison of primary structures of S1-type nucleases of three carnivorous and seven non-carnivorous plants. DAN1 was prepared using a cell-free protein synthesis system. Requirements for metal ions, optimum pH and temperature, and substrate preference were examined using conventional methods. KEY RESULTS: Dan1 is exclusively expressed in the glandular tentacles and its promoter is almost completely unmethylated in all organs. This is in contrast to the S-like RNase gene da-I of Dr. adelae, which shows similar organ-specific expression, but is controlled by a promoter that is specifically unmethylated in the glandular tentacles. Comparison of amino acid sequences of S1-type nucleases identifies seven and three positions where amino acid residues are conserved only among the carnivorous plants and only among the non-carnivorous plants, respectively. DAN1 prefers a substrate RNA over DNA in the presence of Zn2+, Mn2+ or Ca2+ at an optimum pH of 4.0. CONCLUSIONS: Uptake of phosphates from prey is suggested to be the main function of DAN1, which is very different from the known functions of S1-type nucleases. Evolution has modified the structure and expression of Dan1 to specifically function in the digestive fluid.

A novel approach for reliable qualitative and quantitative prey spectra identification of carnivorous plants combining DNA metabarcoding and macro photography.

Prey spectra (the number and composition of captured arthropods) represent a crucial aspect of carnivorous plant ecology, yet remain poorly studied. Traditional morphology-based approaches for prey identification are time-intensive, require specialists with considerable knowledge of arthropod taxonomy, and are hampered by high numbers of unidentifiable (i.e., heavily digested) prey items. We examined prey spectra of three species of closely-related annual Drosera (Droseraceae, sundews) from tropical northern Australia using a novel DNA metabarcoding approach with in-situ macro photography as a plausibility control and to facilitate prey quantity estimations. This new method facilitated accurate analyses of carnivorous plant prey spectra (even of heavily digested prey lacking characteristic morphological features) at a taxonomic resolution and level of completeness far exceeding morphology-based methods and approaching the 100% mark at arthropod order level. Although the three studied species exhibited significant differences in detected prey spectra, little prey specialisation was observed and habitat or plant population density variations were likely the main drivers of prey spectra dissimilarity.

Organ-specific expression and epigenetic traits of genes encoding digestive enzymes in the lance-leaf sundew (Drosera adelae).

Over the last two decades, extensive studies have been performed at the molecular level to understand the evolution of carnivorous plants. As fruits, the repertoire of protein components in the digestive fluids of several carnivorous plants have gradually become clear. However, the quantitative aspects of these proteins and the expression mechanisms of the genes that encode them are still poorly understood. In this study, using the Australian sundew Drosera adelae, we identified and quantified the digestive fluid proteins. We examined the expression and methylation status of the genes corresponding to major hydrolytic enzymes in various organs; these included thaumatin-like protein, S-like RNase, cysteine protease, class I chitinase, ß-1, 3-glucanase, and hevein-like protein. The genes encoding these proteins were exclusively expressed in the glandular tentacles. Furthermore, the promoters of the ß-1, 3-glucanase and cysteine protease genes were demethylated only in the glandular tentacles, similar to the previously reported case of the S-like RNase gene da-I. This phenomenon correlated with high expression of the DNA demethylase DEMETER in the glandular tentacles, strongly suggesting that it performs glandular tentacle-specific demethylation of the genes. The current study strengthens and generalizes the relevance of epigenetics to trap organ-specific gene expression in D. adelae. We also suggest similarities between the trap organs of carnivorous plants and the roots of non-carnivorous plants.

Biochemical and antifungal characteristics of recombinant class I chitinase from Drosera rotundifolia.

DrChit is class I chitinase involved in the digestion of insect prey of Drosera rotundifolia plants. Herein, we cloned the DrChit-S open reading frame lacking the 5'- sequence coding signal peptide into the pET32a vector and its derivate lacking the thioredoxin tag. After DrChit-S + Trx and DrChit-S-Trx overexpression in Escherichia coli cells and purification on Ni-NTA agarose, both enzymes exhibited maximum activity at pH 6.0 and 38 °C. Surprisingly, the DrChit -S + Trx exerted double enzyme activity and improved all kinetic parameters for FITC-chitin substrate degradation resulting in catalytic efficiency three times higher (46.2 mM-1. s-1) than DrChit-S-Trx (13.63 mM-1. s-1). The 3D-structure of DrChit-S + Trx revealed different spatial arrangement of the three tyrosine residues in chitin-binding site, while their aromatic rings showed better stacking geometry for CH/π interactions with the carbohydrate substrate. In contrast, there were no significant differences between both enzymes when the effect of metal ions and their antifungal potential were tested. Quantitative in vitro assays showed growth suppression of Fusarium poae (40%), Trichoderma viride (43.8%), and Alternaria solani (52.6%) but not Rhizoctonia solani (sp.). Our study indicates that sundew chitinase has potential in biotechnology either for degradation of chitin to oligomers applicable in medicine or for plant defense fortification.

Breeding systems of floral colour forms in the Drosera cistiflora species complex.

Variation in plant breeding systems has implications for pollinator-mediated selection on floral traits and the ecology of populations. Here we evaluate pollinator contribution to seed production, self-compatibility and pollen limitation in different floral colour forms of Drosera cistiflora sensu lato (Droseraceae). These insectivorous perennial plants are endemic to fynbos and renosterveld vegetation in the Cape Floristic Region of South Africa, and the species complex includes five floral colour forms (pink, purple, red, white and yellow), some of which are known to be pollinated by beetles. Controlled hand-pollination experiments were conducted in 15 populations of D. cistiflora s.l. (two to four populations per floral colour form) to test whether the colour forms vary in their degree of self-compatibility and their ability to produce seeds through autonomous self-fertilization. Yellow-flowered forms were highly self-incompatible, while other floral colour forms exhibited partial self-compatibility. Seed set resulting from autonomous selfing was very low, and pollinator dependence indices were high in all populations. Since hand cross-pollination resulted in greater seed set than open pollination in 13 of the 15 populations, we inferred that seed production is generally pollen-limited. Drosera cistiflora s.l. typically exhibits high levels of pollinator dependence and pollen limitation. This is unusual among Drosera species worldwide and suggests that pollinators are likely to mediate strong selection on attractive traits such as floral colour and size in D. cistiflora s.l. These results also suggest that the floral colour forms of D. cistiflora s.l. which are rare and threatened are likely to be vulnerable to local extinction if mutualisms were to collapse indefinitely.

Expression of Drosera rotundifolia Chitinase in Transgenic Tobacco Plants Enhanced Their Antifungal Potential.

In this study, a chitinase gene (DrChit) that plays a role in the carnivorous processes of Drosera rotundifolia L. was isolated from genomic DNA, linked to a double CaMV35S promoter and nos terminator in a pBinPlus plant binary vector, and used for Agrobacterium-mediated transformation of tobacco. RT-qPCR revealed that within 14 transgenic lines analysed in detail, 57% had DrChit transcript abundance comparable to or lower than level of a reference actin gene transcript. In contrast, the transgenic lines 9 and 14 exhibited 72 and 152 times higher expression level than actin. The protein extracts of these two lines exhibited five and eight times higher chitinolytic activity than non-transgenic controls when measured in a fluorimetric assay with FITC-chitin. Finally, the growth of Trichoderma viride was obviously suppressed when the pathogen was exposed to 100 µg of crude protein extract isolated from line 9 and line 14, with the area of mycelium growth reaching only 56.4% and 45.2%, of non-transgenic control, respectively. This is the first time a chitinase from a carnivorous plant with substrate specificity for long chitin polymers was tested in a transgenic plant with the aim of exploring its antifungal potential.

Plastid Genomes of Carnivorous Plants Drosera rotundifolia and Nepenthes × ventrata Reveal Evolutionary Patterns Resembling Those Observed in Parasitic Plants.

Carnivorous plants have the ability to capture and digest small animals as a source of additional nutrients, which allows them to grow in nutrient-poor habitats. Here we report the complete sequences of the plastid genomes of two carnivorous plants of the order Caryophyllales, Drosera rotundifolia and Nepenthes × ventrata. The plastome of D. rotundifolia is repeat-rich and highly rearranged. It lacks NAD(P)H dehydrogenase genes, as well as ycf1 and ycf2 genes, and three essential tRNA genes. Intron losses are observed in some protein-coding and tRNA genes along with a pronounced reduction of RNA editing sites. Only six editing sites were identified by RNA-seq in D. rotundifolia plastid genome and at most conserved editing sites the conserved amino acids are already encoded at the DNA level. In contrast, the N. × ventrata plastome has a typical structure and gene content, except for pseudogenization of the ccsA gene. N. × ventrata and D. rotundifolia could represent different stages of evolution of the plastid genomes of carnivorous plants, resembling events observed in parasitic plants in the course of the switch from autotrophy to a heterotrophic lifestyle.

Structure prediction and network analysis of chitinases from the Cape sundew, Drosera capensis.

BACKGROUND: Carnivorous plants possess diverse sets of enzymes with novel functionalities applicable to biotechnology, proteomics, and bioanalytical research. Chitinases constitute an important class of such enzymes, with future applications including human-safe antifungal agents and pesticides. Here, we compare chitinases from the genome of the carnivorous plant Drosera capensis to those from related carnivorous plants and model organisms. METHODS: Using comparative modeling, in silico maturation, and molecular dynamics simulation, we produce models of the mature enzymes in aqueous solution. We utilize network analytic techniques to identify similarities and differences in chitinase topology. RESULTS: Here, we report molecular models and functional predictions from protein structure networks for eleven new chitinases from D. capensis, including a novel class IV chitinase with two active domains. This architecture has previously been observed in microorganisms but not in plants. We use a combination of comparative and de novo structure prediction followed by molecular dynamics simulation to produce models of the mature forms of these proteins in aqueous solution. Protein structure network analysis of these and other plant chitinases reveal characteristic features of the two major chitinase families. GENERAL SIGNIFICANCE: This work demonstrates how computational techniques can facilitate quickly moving from raw sequence data to refined structural models and comparative analysis, and to select promising candidates for subsequent biochemical characterization. This capability is increasingly important given the large and growing body of data from high-throughput genome sequencing, which makes experimental characterization of every target impractical.

Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V ß-1,3-glucanase.

MAIN CONCLUSION: A gene for ß-1,3-glucanase was isolated from carnivorous sundew. It is active in leaves and roots, but not in digestive glands. Analyses in transgenic tobacco suggest its function in germination. Ancestral plant ß-1,3-glucanases (EC 3.2.1.39) played a role in cell division and cell wall remodelling, but divergent evolution has extended their roles in plant defense against stresses to decomposition of prey in carnivorous plants. As available gene sequences from carnivorous plants are rare, we isolated a glucanase gene from roundleaf sundew (Drosera rotundifolia L.) by a genome walking approach. Computational predictions recognized typical gene features and protein motifs described for other plant ß-1,3-glucanases. Phylogenetic reconstructions suggest strong support for evolutionary relatedness to class V ß-1,3-glucanases, including homologs that are active in the traps of related carnivorous species. The gene is expressed in sundew vegetative tissues but not in flowers and digestive glands, and encodes for a functional enzyme when expressed in transgenic tobacco. Detailed analyses of the supposed promoter both in silico and in transgenic tobacco suggest that this glucanase plays a role in development. Specific spatiotemporal activity was observed during transgenic seed germination. Later during growth, the sundew promoter was active in marginal and sub-marginal areas of apical true leaf meristems of young tobacco plants. These results suggest that the isolated glucanase gene is regulated endogenously, possibly by auxin. This is the first report on a nuclear gene study from sundew.

Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.).

MAIN CONCLUSION: Chitinase gene from the carnivorous plant, Drosera rotundifolia , was cloned and functionally characterised. Plant chitinases are believed to play an important role in the developmental and physiological processes and in responses to biotic and abiotic stress. In addition, there is growing evidence that carnivorous plants can use them to digest insect prey. In this study, a full-length genomic clone consisting of the 1665-bp chitinase gene (gDrChit) and adjacent promoter region of the 698 bp in length were isolated from Drosera rotundifolia L. using degenerate PCR and a genome-walking approach. The corresponding coding sequence of chitinase gene (DrChit) was obtained following RNA isolation from the leaves of aseptically grown in vitro plants, cDNA synthesis with a gene-specific primer and PCR amplification. The open reading frame of cDNA clone consisted of 978 nucleotides and encoded 325 amino acid residues. Sequence analysis indicated that DrChit belongs to the class I group of plant chitinases. Phylogenetic analysis within the Caryophyllales class I chitinases demonstrated a significant evolutionary relatedness of DrChit with clade Ib, which contains the extracellular orthologues that play a role in carnivory. Comparative expression analysis revealed that the DrChit is expressed predominantly in tentacles and is up-regulated by treatment with inducers that mimick insect prey. Enzymatic activity of rDrChit protein expressed in Escherichia coli was confirmed and purified protein exhibited a long oligomer-specific endochitinase activity on glycol-chitin and FITC-chitin. The isolation and expression profile of a chitinase gene from D. rotundifolia has not been reported so far. The obtained results support the role of specific chitinases in digestive processes in carnivorous plant species.

Novel proteases from the genome of the carnivorous plant Drosera capensis: Structural prediction and comparative analysis.

In his 1875 monograph on insectivorous plants, Darwin described the feeding reactions of Drosera flypaper traps and predicted that their secretions contained a "ferment" similar to mammalian pepsin, an aspartic protease. Here we report a high-quality draft genome sequence for the cape sundew, Drosera capensis, the first genome of a carnivorous plant from order Caryophyllales, which also includes the Venus flytrap (Dionaea) and the tropical pitcher plants (Nepenthes). This species was selected in part for its hardiness and ease of cultivation, making it an excellent model organism for further investigations of plant carnivory. Analysis of predicted protein sequences yields genes encoding proteases homologous to those found in other plants, some of which display sequence and structural features that suggest novel functionalities. Because the sequence similarity to proteins of known structure is in most cases too low for traditional homology modeling, 3D structures of representative proteases are predicted using comparative modeling with all-atom refinement. Although the overall folds and active residues for these proteins are conserved, we find structural and sequence differences consistent with a diversity of substrate recognition patterns. Finally, we predict differences in substrate specificities using in silico experiments, providing targets for structure/function studies of novel enzymes with biological and technological significance. Proteins 2016; 84:1517-1533. © 2016 Wiley Periodicals, Inc.

Flavonoids as chemotaxonomic markers in the genus Drosera.

The botanical classification of the huge genus Drosera remains controversial since long. In the present study, the pattern of major phenolic compounds in ten Drosera species belonging to seven different subgenera and/or sections of the genus was investigated for chemotaxonomic allocation. The composition of flavonoids and ellagic acid derivatives in Drosera adelae, Drosera burmannii, Drosera dielsiana, Drosera hilaris, Drosera montana, Drosera petiolaris, and Drosera pygmaea was elucidated for the first time. The scarce data on these compounds in Drosera binata, Drosera aliciae, and Drosera spatulata were complemented significantly. Detailed LC-DAD-MS, LC-NMR, and offline 1D and 2D NMR analyses resulted in the unambiguous identification of around 40 different substances, three of them (8-hydroxy-luteolin-8-O-arabinopyranoside, tricetin-7-O-xylopyranoside and 8-hydroxytricetin-8-O-arabinopyranoside) being natural products described for the first time. The distribution of the compounds characterized underlines their potential to serve as chemotaxonomic markers in this genus.

Population history of the two carnivorous plants Drosera peltata var. nipponica and Drosera rotundifolia (Droseraceae) in Korea.

PREMISE OF THE STUDY: Drosera peltata var. nipponica, an element of the East Asia warm-temperate vegetation, and D. rotundifolia, a widely distributed boreal species, reach one of their northernmost and southernmost limits, respectively, on the Korean Peninsula. Because the Last Glacial Maximum (LGM)-Holocene dynamics of warm-temperate and boreal paleovegetation differed considerably on the Peninsula, the population history of these two sundews is expected to be different, leaving differential imprints in their genetic structure. METHODS: We investigated population genetic structure of D. peltata var. nipponica and D. rotundifolia in South Korea (10 populations of each for 20 allozyme loci) to infer their population history in this region. In addition, we compared the genetic variation harbored in the two sundews to those reported for other carnivorous and wetland plants. KEY RESULTS: Drosera peltata var. nipponica showed no genetic diversity, whereas D. rotundifolia exhibited extremely low within-population variation (He = 0.005) and considerable among-population divergence (FST = 0.817). CONCLUSIONS: Our results suggest that extant populations of D. peltata var. nipponica likely originated from a single ancestral population from southern Japan or southern China through postglacial dispersal. On the contrary, D. rotundifolia probably survived the LGM in situ, with extant populations derived from either one or several small source populations. We argue that separate conservation strategies should be employed, given that the two taxa have different ecological and demographic traits and harbor different levels of genetic diversity.

S-like ribonuclease gene expression in carnivorous plants.

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.

Characteristics of leaf shapes among two parental Drosera species and a hybrid examined by canonical discriminant analysis and a hierarchical Bayesian model.

PREMISE OF THE STUDY: Although nondestructive, convenient species identification is desirable for follow-up research and species conservation, species identification is often confusing, particularly when an interspecific hybrid shows intermediate morphological characteristics between the parental species. • METHODS: Drosera anglica Hudson (2n = 40) and D. rotundifolia L. (20) bear the hybrid Drosera obovata Mert. et Koch (30). The samples were identified based on seed fertility and a cytological investigation (DNA amount) before examination. Then, 13 measured morphological traits-including leaf size, leaf shape, and flowering-were used in a canonical discriminant analysis (CDA). Leaf length and width were used in a hierarchical Bayesian model (HBM). • KEY RESULTS: The majority of the traits of D. obovata were intermediate between the two parental species. However, D. obovata developed larger leaves than the parental species. The identification error of the CDA based on the 13 morphological traits was 4.9%. Errors occurred more often with smaller leaves. When the CDA was used for blade length and width only, the error increased to 6.2%. The HBM, based on the relationships between blade length and width, showed the lowest identification error-4.7%-by improving the identification of small leaves. • CONCLUSIONS: The HBM enabled convenient, nondestructive measurements for species identification by considering nonlinear relationships between morphological traits and measurement error. The HBM is likely to be applicable to various follow-up studies, as well as species conservation.

Is Drosera meristocaulis a pygmy sundew? Evidence of a long-distance dispersal between Western Australia and northern South America.

BACKGROUND AND AIMS: South America and Oceania possess numerous floristic similarities, often confirmed by morphological and molecular data. The carnivorous Drosera meristocaulis (Droseraceae), endemic to the Neblina highlands of northern South America, was known to share morphological characters with the pygmy sundews of Drosera sect. Bryastrum, which are endemic to Australia and New Zealand. The inclusion of D. meristocaulis in a molecular phylogenetic analysis may clarify its systematic position and offer an opportunity to investigate character evolution in Droseraceae and phylogeographic patterns between South America and Oceania. METHODS: Drosera meristocaulis was included in a molecular phylogenetic analysis of Droseraceae, using nuclear internal transcribed spacer (ITS) and plastid rbcL and rps16 sequence data. Pollen of D. meristocaulis was studied using light microscopy and scanning electron microscopy techniques, and the karyotype was inferred from root tip meristem. KEY RESULTS: The phylogenetic inferences (maximum parsimony, maximum likelihood and Bayesian approaches) substantiate with high statistical support the inclusion of sect. Meristocaulis and its single species, D. meristocaulis, within the Australian Drosera clade, sister to a group comprising species of sect. Bryastrum. A chromosome number of 2n = approx. 32-36 supports the phylogenetic position within the Australian clade. The undivided styles, conspicuous large setuous stipules, a cryptocotylar (hypogaeous) germination pattern and pollen tetrads with aperture of intermediate type 7-8 are key morphological traits shared between D. meristocaulis and pygmy sundews of sect. Bryastrum from Australia and New Zealand. CONCLUSIONS: The multidisciplinary approach adopted in this study (using morphological, palynological, cytotaxonomic and molecular phylogenetic data) enabled us to elucidate the relationships of the thus far unplaced taxon D. meristocaulis. Long-distance dispersal between southwestern Oceania and northern South America is the most likely scenario to explain the phylogeographic pattern revealed.

A rapid and efficient method for isolating high quality DNA from leaves of carnivorous plants from the Drosera genus.

Drosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 µg/g of fresh weight to 35.29 µg/g depending of the species of Drosera, with a A260/A280 ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h.

Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated.

Phyloclimatic modeling: combining phylogenetics and bioclimatic modeling.

We investigate the impact of past climates on plant diversification by tracking the "footprint" of climate change on a phylogenetic tree. Diversity within the cosmopolitan carnivorous plant genus Drosera (Droseraceae) is focused within Mediterranean climate regions. We explore whether this diversity is temporally linked to Mediterranean-type climatic shifts of the mid-Miocene and whether climate preferences are conservative over phylogenetic timescales. Phyloclimatic modeling combines environmental niche (bioclimatic) modeling with phylogenetics in order to study evolutionary patterns in relation to climate change. We present the largest and most complete such example to date using Drosera. The bioclimatic models of extant species demonstrate clear phylogenetic patterns; this is particularly evident for the tuberous sundews from southwestern Australia (subgenus Ergaleium). We employ a method for establishing confidence intervals of node ages on a phylogeny using replicates from a Bayesian phylogenetic analysis. This chronogram shows that many clades, including subgenus Ergaleium and section Bryastrum, diversified during the establishment of the Mediterranean-type climate. Ancestral reconstructions of bioclimatic models demonstrate a pattern of preference for this climate type within these groups. Ancestral bioclimatic models are projected into palaeo-climate reconstructions for the time periods indicated by the chronogram. We present two such examples that each generate plausible estimates of ancestral lineage distribution, which are similar to their current distributions. This is the first study to attempt bioclimatic projections on evolutionary time scales. The sundews appear to have diversified in response to local climate development. Some groups are specialized for Mediterranean climates, others show wide-ranging generalism. This demonstrates that Phyloclimatic modeling could be repeated for other plant groups and is fundamental to the understanding of evolutionary responses to climate change.