Nepenthes Research Articles

Genome skimming provides well resolved plastid and nuclear phylogenies, showing patterns of deep reticulate evolution in the tropical carnivorous plant genus Nepenthes (Caryophyllales)

Nepenthes is a genus of carnivorous plants consisting of ~160 species that are distributed in the paleotropics. Molecular systematics has so far not been able to resolve evolutionary relationships of most species because of the limited genetic divergence in previous studies. In the present study, we used a genome-skimming approach to infer phylogenetic relationships on the basis of 81 plastid genes and the highly repetitive rRNA (external transcribed spacer (ETS)–26S) for 39 accessions representing 34 species from eight sections. Maximum-likelihood analysis and Bayesian inference were performed separately for the nuclear and the plastid datasets. Divergence-time estimations were conducted on the basis of a relaxed molecular-clock model, using secondary calibration points. The phylogenetic analyses of the nuclear and plastid datasets yielded well resolved and supported phylogenies. Incongruences between the two datasets were detected, suggesting multiple hybridisation events or incomplete lineage sorting in the deeper and more recent evolutionary history of the genus. The inclusion of several known and suspected hybrids in the phylogenetic analysis provided insights into their parentage. Divergence-time estimations placed the crown diversification of Nepenthes in the early Miocene, c. 20 million years ago. This study showed that genome skimming provides well resolved nuclear and plastid phylogenies that provide valuable insights into the complex evolutionary relationships of Nepenthes.

Plumbagin from a tropical pitcher plant (Nepenthes alata Blanco) induces apoptotic cell death via a p53-dependent pathway in MCF-7 human breast cancer cells

Plumbagin (5-hydroxy-2-methyl-1,4-naphthaquinone) has displayed antitumor activity in vitro and in animal models; however, the underlying molecular mechanisms have not been fully explored. The aim of this study was to investigate the anticancer effects of plumbagin isolated from Nepenthes alata against MCF-7 breast cancer cells. We examined the cytotoxicity, cell cycle regulation, apoptotic cell death, and generation of intracellular reactive oxygen species (ROS) in MCF-7 cells. Plumbagin exhibited potent cytotoxicity in MCF-7 cells (wild-type p53) compared to that in SK-OV-3 (null-type) human epithelial ovarian cancer cells. Specifically, plumbagin upregulated the expression of p21CIP1/WAF1 in MCF-7 cells, causing cell cycle arrest in the G2/M phase through inhibition of cyclin B1 levels. Plumbagin also significantly increased the ratio of Bax/Bcl-2 and release of cytochrome c, resulting in apoptotic cell death in MCF-7 cells. Furthermore, plumbagin dramatically increased the intracellular ROS level, whereas pretreatment with the ROS scavenger N-acetyl cysteine protected against plumbagin-induced cytotoxicity, suggesting that ROS formation plays a pivotal role in antitumor activity in MCF-7 cells. In mice bearing MCF-7 cell xenografts, plumbagin significantly reduced tumor growth and weight without apparent side effects. We therefore concluded that plumbagin exerts anticancer activity against MCF-7 cells through the generation of intracellular ROS, resulting in the induction of apoptosis via a p53-dependent pathway. This study thus identifies a new anticancer mechanism of plumbagin against p53-dependent breast cancer cells and suggests a novel strategy for overcoming of breast cancer therapy.

The diversity of Nepenthes at the post-mining area in Sintang District, West Kalimantan, Indonesia

Setiawan H, Wardhani HAK, Kamaludin, Hutagaol RR, Afriani R. 2018. The diversity of Nepenthes at the post-mining area in Sintang District, West Kalimantan, Indonesia. Biodiversitas 19: 1820-1827. Nepenthe is a tropical pitcher plant found in Indonesian archipelago and lives in nutrient leak habitat. One of Nepenthes nutrient leak habitat commonly found in Kalimantan Island is the post-mining area. This research aimed to know the habitat condition, species composition, species richness and diversity index of Nepenthes in Post-mining area in Sintang District, West Kalimantan Province. Thirty plots, each measuring 5 x 5-meter square, were established to survey the Nepenthes diversity in five locations of the post-mining area in Sintang District. There were 910 individuals of Nepenthes found spreading in two sub-Districts and five areas. Habitat condition in the post-mining area tended to be high in light intensity and temperature but low in humidity. Five Nepenthes species were found, namely N. ampullaria, N. bicalcarata, N. gracilis, N. mirabilis, and N. rafflesiana. Four natural hybrids were also found including N. xhookeriana, N. xkuchingensis, N. xneglecta, and N. xtrichocarpa. The species richness in post-mining area were 1.25 in Danau BTN (Bank Tabungan Negara/State Savings Bank) 0.95 in Dusun Kerangas, 0.58 in Sungai Ana Village, 0.21 in Jerora 1 and 0.20 in Danau Baning. The diversity index in Danau BTN, Dusun Kerangas, Sungai Ana, Jerora 1 and Danau Banning area were of low levels, respectively, 1.66, 1.3, 1.0, 0.64, and 0.68. Cluster analysis of species richness, diversity index, and abiotic factors showed that Danau Baning-Sungai Ana-Jerora 1 was grouped in the same cluster while Danau BTN and Dusun Kerangas were in a separate cluster.

DNA barcoding of the genus Nepenthes (Pitcher plant): a preliminary assessment towards its identification

BackgroundDNA barcoding is impending towards the generation of universal standards for species discrimination with a standard gene region that can be sequenced accurately and within short span of time. In this study, we were successful in developing efficient barcode locus in the Nepenthes genus. A total of 317 accessions were retrieved from GenBank of NCBI which represent 140 different species Nepenthes and evaluated the efficacy of ITS, rbcl and matK barcode candidates using barcode gap, applied distance similarity, and tree-based methods.ResultOur result indicates that single-locus ITS or combined with plastid regions (matK) showed the best species discrimination with distinctive barcoding gaps. Therefore, we tentatively proposed the combination of ITS+matK as a core barcode for Nepenthes genus.ConclusionThis study provides a report on DNA barcoding for unique insectivores’ Nepenthes genus. As the different species of Nepenthes are higly endemic and endangered, it would be a useful study to understand the evolutionary relationship, sketched in emigration, mislabeling and can be a probable assessment for its biodiversity.

Discovery of digestive enzymes in carnivorous plants with focus on proteases.

BackgroundCarnivorous plants have been fascinating researchers with their unique characters and bioinspired applications. These include medicinal trait of some carnivorous plants with potentials for pharmaceutical industry.MethodsThis review will cover recent progress based on current studies on digestive enzymes secreted by different genera of carnivorous plants: Drosera (sundews), Dionaea (Venus flytrap), Nepenthes (tropical pitcher plants), Sarracenia (North American pitcher plants), Cephalotus (Australian pitcher plants), Genlisea (corkscrew plants), and Utricularia (bladderworts).ResultsSince the discovery of secreted protease nepenthesin in Nepenthes pitcher, digestive enzymes from carnivorous plants have been the focus of many studies. Recent genomics approaches have accelerated digestive enzyme discovery. Furthermore, the advancement in recombinant technology and protein purification helped in the identification and characterisation of enzymes in carnivorous plants.DiscussionThese different aspects will be described and discussed in this review with focus on the role of secreted plant proteases and their potential industrial applications.

Regulation of enzyme activities in carnivorous pitcher plants of the genus Nepenthes.

Nepenthes regulates enzyme activities by sensing stimuli from the insect prey. Protein is the best inductor mimicking the presence of an insect prey. Carnivorous plants of the genus Nepenthes have evolved passive pitcher traps for prey capture. In this study, we investigated the ability of chemical signals from a prey (chitin, protein, and ammonium) to induce transcription and synthesis of digestive enzymes in Nepenthes × Mixta. We used real-time PCR and specific antibodies generated against the aspartic proteases nepenthesins, and type III and type IV chitinases to investigate the induction of digestive enzyme synthesis in response to different chemical stimuli from the prey. Transcription of nepenthesins was strongly induced by ammonium, protein and live prey; chitin induced transcription only very slightly. This is in accordance with the amount of released enzyme and proteolytic activity in the digestive fluid. Although transcription of type III chitinase was induced by all investigated stimuli, a significant accumulation of the enzyme in the digestive fluid was found mainly after protein and live prey addition. Protein and live prey were also the best inducers for accumulation of type IV chitinase in the digestive fluid. Although ammonium strongly induced transcription of all investigated genes probably through membrane depolarization, strong acidification of the digestive fluid affected stability and abundance of both chitinases in the digestive fluid. The study showed that the proteins are universal inductors of enzyme activities in carnivorous pitcher plants best mimicking the presence of insect prey. This is not surprising, because proteins are a much valuable source of nitrogen, superior to chitin. Extensive vesicular activity was observed in prey-activated glands.

Keeping an eye on coloration: ecological correlates of the evolution of pitcher traits in the genus Nepenthes (Caryophyllales)

Keeping an eye on coloration: ecological correlates of the evolution of pitcher traits in the genus Nepenthes (Caryophyllales)

Convergent and divergent evolution in carnivorous pitcher plant traps.

Contents Summary 1035 I. Introduction 1035 II. Evolution of the pitcher 1036 III. Convergent evolution 1036 IV. Divergent evolution 1038 V. Adaptive radiation and speciation 1040 VI. Conclusions and perspectives 1040 Acknowledgements 1040 References 1040 SUMMARY: The pitcher trap is a striking example of convergent evolution across unrelated carnivorous plant lineages. Convergent traits that have evolved across pitcher plant lineages are essential for trap function, suggesting that key selective pressures are in action. Recent studies have also revealed patterns of divergent evolution in functional pitcher morphology within genera. Adaptations to differences in local prey assemblages may drive such divergence and, ultimately, speciation. Here, we review recent research on convergent and divergent evolution in pitcher plant traps, with a focus on the genus Nepenthes, which we propose as a new model for research into adaptive radiation and speciation.

ETHNOBOTANICAL, PHYTOCHEMICAL, AND PHARMACOLOGICAL PROPERTIES OF NEPENTHES SPECIES: A REVIEW

The genus Nepenthes (Nepenthaceae) has been utilized in folk medicine for a long time in India and Southeast Asia countries. They are used in the treatment of leprosy, cholera, night blindness, gastrointestinal discomfort, dysentery, stomachache, and bed-wetting among others. This review highlights the ethnobotanical uses, phytochemicals, and pharmacological activities of both crude extracts and pure bioactive compounds of Nepenthes spp. The phytochemical compounds isolated from Nepenthes species include flavonoids, terpenoids, tannins, alkaloids, and steroids among other phytochemicals. A wide range of pharmacological activities was exhibited by the crude extracts and pure bioactive components such as antibacterial, antifungal, antimalarial antioxidant, antidiabetic, antiosteoporotic, anti-inflammatory, cytotoxicity, and hypolipidemic activities. This review revealed that many active compounds are present in Nepenthes spp. However, many pharmacological screenings such as anticancer, antiviral, wound healing, antihelminthic, antidiarrheal properties, among others have not been carried out yet. Therefore, more biological investigations and phytochemical screenings are required to fully explore the genus Nepenthes which may lead to development of new therapeutic agents.

Coprophagous features in carnivorous Nepenthes plants: a task for ureases

Most terrestrial carnivorous plants are specialized on insect prey digestion to obtain additional nutrients. Few species of the genus Nepenthes developed mutualistic relationships with mammals for nitrogen supplementation. Whether dietary changes require certain enzymatic composition to utilize new sources of nutrients has rarely been tested. Here, we investigated the role of urease for Nepenthes hemsleyana that gains nitrogen from the bat Kerivoula hardwickii while it roosts inside the pitchers. We hypothesized that N. hemsleyana is able to use urea from the bats’ excrements. In fact, we demonstrate that 15N-enriched urea provided to Nepenthes pitchers is metabolized and its nitrogen is distributed within the plant. As ureases are necessary to degrade urea, these hydrolytic enzymes should be involved. We proved the presence and enzymatic activity of a urease for Nepenthes plant tissues. The corresponding urease cDNA from N. hemsleyana was isolated and functionally expressed. A comprehensive phylogenetic analysis for eukaryotic ureases, including Nepenthes and five other carnivorous plants’ taxa, identified them as canonical ureases and reflects the plant phylogeny. Hence, this study reveals ureases as an emblematic example for an efficient, low-cost but high adaptive plasticity in plants while developing a further specialized lifestyle from carnivory to coprophagy.

Explorative study of tropical pitcher plants (Nepenthes sp.) types and insects that trapped inside in Sebangau National Park Palangka Raya Central Kalimantan

Pitcher plants (Nepenthes sp.) is a plant with unique shape either of shades of colors, pouch shape and its capability in catching insects. Pitcher plant (Nepenthes sp.) is one of the plants that protected under Law Number. 5 of 1990 about Conservation of Biological Resources and Ecosystem and Government Regulation Number 7/1999 about Preservation of Plants and Animals. Sebangau National Park is one of representative of peat swamp ecosystem and one of some types of pitcher plants (Nepenthes sp.) habitat. This study aimed to determine the types and diversity levels of pitcher plants (Nepenthes sp.) and the trapped insects inside in Sebangau National and to determine the differences of diversity levels of pitcher plants (Nepenthes sp.) and the trapped insects inside in the opened and closed forest in Sebangau National Park. The research type was conducted descriptive qualitative research. The method used survey method with purpossive sampling technique.The result of the study the number of pitcher plants (Nepenthes sp.) obtained in opened forest were three types consist of Nepenthes mirabilis, Nepenthes rafflesiana and Nepenthes gracilis with two types insects trapped inside those were Diptera ordo and Hymenoptera ordo. While the number of pitcher plants (Nepenthes sp.) obtained in closed forest as many as two types consist of Nepenthes ampullaria and Nepenthes rafflesiana with two type insects trapped inside those were Diptera ordo and Hymenoptera ordo. The results of the analysis calculation pitcher plants (Nepenthes sp.) species diversity index in opened and closed forest showed lower category. The diversity in row were 1 and 0,45 with H’ criteria ≤ 1 low diversity. The results of the study of insects trapped inside of pitcher plants (Nepenthes sp.) obtained in opened and closed forest showed low category. The diversity in a row were 0,63 and 0,52 with the criteria of H’ ≤ 1 low diversity.

Nepenthes khasiana Hook. f. (Nepenthaceae) from Assam

Nepenthes khasiana , an endangered species, listed in Appendix I of CITES, is the only species of the genus known to occur in India. It is a rare endemic species hitherto recorded from Meghalaya. It is threatened due to several factors including wanton destruction of its habitat by coal mining and harvesting for medicinal preparations.

Carnivorous Nutrition in Pitcher Plants (Nepenthes spp.) via an Unusual Complement of Endogenous Enzymes.

Plants belonging to the genus Nepenthes are carnivorous, using specialized pitfall traps called "pitchers" that attract, capture, and digest insects as a primary source of nutrients. We have used RNA sequencing to generate a cDNA library from the Nepenthes pitchers and applied it to mass spectrometry-based identification of the enzymes secreted into the pitcher fluid using a nonspecific digestion strategy superior to trypsin in this application. This first complete catalog of the pitcher fluid subproteome includes enzymes across a variety of functional classes. The most abundant proteins present in the secreted fluid are proteases, nucleases, peroxidases, chitinases, a phosphatase, and a glucanase. Nitrogen recovery involves a particularly rich complement of proteases. In addition to the two expected aspartic proteases, we discovered three novel nepenthensins, two prolyl endopeptidases that we name neprosins, and a putative serine carboxypeptidase. Additional proteins identified are relevant to pathogen-defense and secretion mechanisms. The full complement of acid-stable enzymes discovered in this study suggests that carnivory in the genus Nepenthes can be sustained by plant-based mechanisms alone and does not absolutely require bacterial symbiosis.

Nanoporous Monolithic Microsphere Arrays Have Anti-Adhesive Properties Independent of Humidity.

Bioinspired artificial surfaces with tailored adhesive properties have attracted significant interest. While fibrillar adhesive pads mimicking gecko feet are optimized for strong reversible adhesion, monolithic microsphere arrays mimicking the slippery zone of the pitchers of carnivorous plants of the genus Nepenthes show anti-adhesive properties even against tacky counterpart surfaces. In contrast to the influence of topography, the influence of relative humidity (RH) on adhesion has been widely neglected. Some previous works deal with the influence of RH on the adhesive performance of fibrillar adhesive pads. Commonly, humidity-induced softening of the fibrils enhances adhesion. However, little is known on the influence of RH on solid anti-adhesive surfaces. We prepared polymeric nanoporous monolithic microsphere arrays (NMMAs) with microsphere diameters of a few 10 µm to test their anti-adhesive properties at RHs of 2% and 90%. Despite the presence of continuous nanopore systems through which the inner nanopore walls were accessible to humid air, the topography-induced anti-adhesive properties of NMMAs on tacky counterpart surfaces were retained even at RH = 90%. This RH-independent robustness of the anti-adhesive properties of NMMAs significantly contrasts the adhesion enhancement by humidity-induced softening on nanoporous fibrillar adhesive pads made of the same material.

Two New Nepenthes Species from the Philippines and an Emended Description of Nepenthes ramos.

With 50 species of the genus Nepenthes L. currently described from the Philippines, it is without doubt that the country, along with the islands of Sumatra (Indonesia) and Borneo (Indonesia, Malaysia, Brunei), should be considered the center of diversity of the genus. In this work, we describe two new species. One species, N. aenigma sp. nov., is from Ilocos Norte province on Luzon Island and has the—for Nepenthes—unusual ecological preference to grow in dense vegetation in deep shade. The other new species is from Mount Hamiguitan in Davao Oriental province on Mindanao Island. With this new entry, Mount Hamiguitan is now home to four endemic species (N. peltata, N. micramphora, N. hamiguitanensis, N. justinae sp. nov.). Furthermore, we provide an emended description of N. ramos based on field data. Nepenthes kurata is synonymized here with N. ramos.

Proteome analysis of digestive fluids in Nepenthes pitchers.

Carnivorous plants have developed strategies to enable growth in nutrient-poor soils. For the genus Nepenthes, this strategy represents producing pitcher-modified leaves that can trap and digest various prey. These pitchers produce a digestive fluid composed of proteins, including hydrolytic enzymes. The focus of this study was on the identification of these proteins. In order to better characterize and have an overview of these proteins, digestive fluid was sampled from pitchers at different stages of maturity from five species of Nepenthes (N. mirabilis, N. alata, N. sanguinea, N. bicalcarata and N. albomarginata) that vary in their ecological niches and grew under different conditions. Three complementary approaches based on transcriptomic resources, mass spectrometry and in silico analysis were used. This study permitted the identification of 29 proteins excreted in the pitchers. Twenty of these proteins were never reported in Nepenthes previously and included serine carboxypeptidases, α- and β-galactosidases, lipid transfer proteins and esterases/lipases. These 20 proteins display sequence signals allowing their secretion into the pitcher fluid. Nepenthes pitcher plants have evolved an arsenal of enzymes to digest prey caught in their traps. The panel of new proteins identified in this study provides new insights into the digestive process of these carnivorous plants.

Bacterial Diversity and Community Structure in Two Bornean Nepenthes Species with Differences in Nitrogen Acquisition Strategies.

Carnivorous plants of the genus Nepenthes have been studied for over a century, but surprisingly little is known about associations with microorganisms. The two species Nepenthes rafflesiana and Nepenthes hemsleyana differ in their pitcher-mediated nutrient sources, sequestering nitrogen from arthropod prey and arthropods as well as bat faeces, respectively. We expected bacterial communities living in the pitchers to resemble this diet difference. Samples were taken from different parts of the pitchers (leaf, peristome, inside, outside, digestive fluid) of both species. Bacterial communities were determined using culture-independent high-throughput amplicon sequencing. Bacterial richness and community structure were similar in leaves, peristomes, inside and outside walls of both plant species. Regarding digestive fluids, bacterial richness was higher in N. hemsleyana than in N. rafflesiana. Additionally, digestive fluid communities were highly variable in structure, with strain-specific differences in community composition between replicates. Acidophilic taxa were mostly of low abundance, except the genus Acidocella, which strikingly reached extremely high levels in two N. rafflesiana fluids. In N. hemsleyana fluid, some taxa classified as vertebrate gut symbionts as well as saprophytes were enriched compared to N. rafflesiana, with saprophytes constituting potential competitors for nutrients. The high variation in community structure might be caused by a number of biotic and abiotic factors. Nitrogen-fixing bacteria were present in both study species, which might provide essential nutrients to the plant at times of low prey capture and/or rare encounters with bats.

Crystallization of nepenthesin I using a low-pH crystallization screen.

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.

Climate, soils and vicariance - their roles in shaping the diversity and distribution of Nepenthes in Southeast Asia

The Palaeotropical pitcher plant genus Nepenthes (Nepenthaceae) is characterized by specialized nutrient sequestration strategies, narrow endemism, and a patchy distribution in which vicariance is believed to have played a fundamental role. Using recent studies of the effects of climate, soil type and vicariance, we review patterns of diversity and endemism in Nepenthes. First we consider how climate influences the geographical range of the genus and diversity of prey trapping mechanisms. Second, we examine edaphic influences, specifically the relationship between limestone and ultramafic soils and the obligate edaphic endemic Nepenthes that inhabit them. Third, we examine the role of vicariance, with regards to the patchy distribution of suitable habitats throughout Southeast Asia, and the passive dispersal mechanism of Nepenthes seeds. Climate is the principal determinant of variation in pitcher functional traits and in perhumid environments, may drive the evolution of alternative nutrient sequestration strategies. Although little is known about the ecophysiological relationships between soil type and obligate edaphic Nepenthes, ultramafic and limestone substrates may strongly influence vegetation physiognomy, creating a diversity of environmental niches that are exploited by specialized Nepenthes species. Finally, the complex geology and geography of the Malay Archipelago drives diversification through vicariance.

Phylogenetic analysis of niche divergence reveals distinct evolutionary histories and climate change implications for tropical carnivorous pitcher plants

AbstractAimTo analyse the underpinnings of historical drivers of diversity and their contributions to current distributions and future roles in a changing climate, we studied the relationship between ecological niche divergence and phylogenetic signal in tropical carnivorous pitcher plants.LocationSoutheast Asia.MethodsEstimates of realized ecological niches were reconstructed and plotted along a newly created multilocus molecular phylogeny. Phylogenetic signal was analysed by comparisons of calculated phylogenetic relatedness with ecological niche divergence. Current and projected future potentially suitable habitats were mapped for several species of plants with variable evolutionary histories and distributions.ResultsHighland and lowland species had distinct phylogenetic signals. Higher altitude species had significantly lower molecular divergence as compared with the lowland species, yet ecological niches with less overlap. When projected onto a future climate scenario, highland species lose a greater amount of potentially suitable habitat compared to lower altitude species, and the majority of studied higher altitude species will face an overall loss of future suitable habitat.Main conclusionWe conclude that distinct phylogenetic signals not only unravel differing evolutionary histories but also show that the implications of species' tolerances to future changing climate vary. Over the past million years, historical climate change shaped the differing evolution and ecological niches of highland and lowland tropical pitcher plant species. Rapid, recent radiations of the higher altitude species are reflected in limited molecular divergence, which is in sharp contrast with the more gradually evolved and genetically distinct lower altitude species in our study. Our projections for future potentially suitable habitats show that on‐going climate shifts will have detrimental effects on especially the higher altitude species due to a narrower niche tolerance and dramatic loss of potentially suitable habitat.