Carnivorous Plant Genus Research Articles

Micropropagation and genetic transformation of Byblis liniflora

AbstractByblis, a small genus of carnivorous plants predominantly found in Australia, is characterized by its passive trapping mechanism and unique floral features. The chemical composition of Byblis, including identified phenylethanoid glycosides, particularly acteoside, highlights its pharmacological potential with various biological activities. In vitro culture techniques have been established for propagation, with micropropagation protocols developed for different Byblis species. However, information on genetic transformation, vital for trait modification and enhanced pharmacological interest, remains limited. This study focuses on optimizing micropropagation, adventitious regeneration, and genetic transformation methods for Byblis liniflora. Adventitious regeneration rates were highest in medium with reduced Murashige and Skoog salts (MS/10) and sucrose (3 gL−1) concentrations. Zeatin supplementation (1 mgL−1) further improved regeneration rates and bud development with 100% of regenerated root explants and 8.8 shoots per explant. Liquid MB3 medium supplemented with indole-3-acetic acid (IAA) 5 mgL−1 facilitated efficient rooting and acclimatization. The establishment of an efficient Rhizobium-mediated genetic transformation method yielded transgenic plants expressing green fluorescent protein (GFP). Molecular analysis confirmed transgene integration, marking the first successful genetic transformation in the Byblis genus. These advancements pave the way for exploring gene function and enhancing pharmacological properties, thereby broadening our understanding and utilization of carnivorous plants like Byblis.

Fabrication of biomimetic anisotropic crescent-shaped microstructured surfaces by laser shock imprinting

Abstract The crescent-shaped microstructure bionic to the slip zone of the slippery zone of the carnivorous plant genus Nepenthes was fabricated on the surface of copper foil by laser shock imprinting (LSI). The microstructure of crescent-shaped grooves was initially fabricated on the surface of the micro-mold by etching, and then the microstructure was replicated on the surface of copper foil through plastic deformation under laser shock loading. Increasing the laser shock energy or the number of shocks can increase the degree of replication of the crescent-shaped microstructure, the height of the crescent-shaped microstructure, and the contact angle of water droplets on the surface. The wettability of the surface of the crescent microstructure is anisotropic and increases with an increase in offset distance. The anisotropy of the crescent-shaped microstructure causes the solid–liquid contact line in the direction of the bottom of the arc to become a long and approximately straight line. According to the rule that controlling LSI processing parameters can fabricate surfaces with different heights and wettability, a gradient wetting surface consisting of crescent-shaped microstructures was designed to achieve the directional spreading of droplets. By altering the distribution of crescent-shaped microstructures, a type-I flow channel with the ability to limit the spreading range of water droplets was fabricated.

Rampant chloroplast capture in Sarracenia revealed by plastome phylogeny.

Introgression can produce novel genetic variation in organisms that hybridize. Sympatric species pairs in the carnivorous plant genus Sarracenia L. frequently hybridize, and all known hybrids are fertile. Despite being a desirable system for studying the evolutionary consequences of hybridization, the extent to which introgression occurs in the genus is limited to a few species in only two field sites. Previous phylogenomic analysis of Sarracenia estimated a highly resolved species tree from 199 nuclear genes, but revealed a plastid genome that is highly discordant with the species tree. Such cytonuclear discordance could be caused by chloroplast introgression (i.e. chloroplast capture) or incomplete lineage sorting (ILS). To better understand the extent to which introgression is occurring in Sarracenia, the chloroplast capture and ILS hypotheses were formally evaluated. Plastomes were assembled de-novo from sequencing reads generated from 17 individuals in addition to reads obtained from the previous study. Assemblies of 14 whole plastomes were generated and annotated, and the remaining fragmented assemblies were scaffolded to these whole-plastome assemblies. Coding sequence from 79 homologous genes were aligned and concatenated for maximum-likelihood phylogeny estimation. The plastome tree is extremely discordant with the published species tree. Plastome trees were simulated under the coalescent and tree distance from the species tree was calculated to generate a null distribution of discordance that is expected under ILS alone. A t-test rejected the null hypothesis that ILS could cause the level of discordance seen in the plastome tree, suggesting that chloroplast capture must be invoked to explain the discordance. Due to the extreme level of discordance in the plastome tree, it is likely that chloroplast capture has been common in the evolutionary history of Sarracenia.

DIVERSITY, ECOLOGY AND CONSERVATION STATUS OF NEPENTHES IN WEST SUMATRA PROVINCE, INDONESIA

Nepenthes is the largest carnivorous plant genus present in Indonesia. There are 39 species of Nepenthes pitcher plants recorded in Sumatra from lowland to montane forests, and 34 of them are endemic; this represents the greatest species diversity of Nepenthes after Borneo. Field studies were conducted in 2021 and 2022 to increase our knowledge of the diversity, habitats and distributions of Nepenthes in West Sumatra province. Twenty-three species of Nepenthes were recorded from the province, consisting of 15 highland species, 4 mid-elevation species and 4 lowland species. Ecophysiological studies conducted at Bukit Malalak showed clumped distributions of N. bongso, N. dubia, N. eustachya and N. rhombicaulis. Foliar and pitcher fluid nutrient concentrations were found to be similar to those cited in other recent studies although growth rates were slightly more rapid than at Gunung Talang. Bukit Malalak is a new locality for two threatened species, namely N. dubia (CR) and N. rhombicaulis (VU), enlarging their extents of occurrence. In total, nine species from West Sumatra are threatened and conservation actions are urgently needed for these and other Nepenthes species remaining on the island.

Better to risk limb than life: some insects use autotomy to escape passive predation by carnivorous plants

Limb autotomy, the voluntary shedding of body parts as a strategy to escape predation or entrapment, is particularly common in insects and other arthropods that are frequently captured by the carnivorous plant genus Drosera. However, no study has previously examined the effectiveness of autotomy at facilitating escape from these passive, sessile plant predators. Using field observations of numerous Drosera species in Western Australia and Australia’s Northern Territory, we present the first field evidence of limb autotomy being employed by insects and other arthropods to escape capture by carnivorous plants. Most autotomised limbs found on the Drosera traps belonged to large nematoceran flies, probably comprised primarily of Tipulidae (crane flies), which have a characteristically large, slender body with very long limbs that seems likely to allow the effective use of autotomy as a strategy to prevent fatal capture. However, autotomy was overall only rarely observed amongst the Drosera prey as most prey items were small and quickly became completely enveloped by the sticky mucilage, rendering any such escape attempts impossible. Finally, we determined that the likelihood of escape decreased markedly as multiple limbs were lost.

NUTRIENT CONCENTRATIONS IN THREE NEPENTHES SPECIES (NEPENTHACEAE) FROM NORTH SUMATRA

MANSUR, M., SALAMAH, A., MIRMANTO, E. & BREARLEY, F. Q. 2022. Nutrient concentrations in three Nepenthes species (Nepenthaceae) from North Sumatra. Reinwardtia 21(2): 55‒62. — Nepenthes is a genus of carnivorous plants that are unique ornamental plants, but their nutrient concentration relationships have not been studied much, especially in endemic species on the island of Sumatra. So far, the analysis of the nutrient concentration in Nepenthes is mostly limited to leaves. There are few reports of nutrient concentrations in the pitcher fluid and the soil around where it grows. Leaves, pitcher fluid, and soil around the growth sites of each species i.e., Nepenthes sumatrana, N. spectabilis, and N. tobaica, from North Sumatra province were collected for nutrient analyses (N, P, K, Ca, Mg, and Na). The results showed that the nutrient concentrations in the leaves and pitcher fluid in the three Nepenthes species were generally low with those in the leaves greater than in the pitcher fluid. The concentration of nutrients in the leaves of N. sumatrana (lowland species) was least (except for N and Na) when compared to N. spectabilis and N. tobaica (highland species), likely reflecting the poorly fertile soil. In contrast, the nutrient concentration in the pitcher fluid of N. sumatrana was greater than N. spectabilis and N. tobaica. When compared across an extensive data set, we show that leaf N does not change with elevation, whereas P declines and the N:P ratio increases with elevation, suggesting that Nepenthes plants are obtaining sufficient N from prey at higher elevations.

NUTRIENT CONCENTRATIONS IN THREE NEPENTHES SPECIES (NEPENTHACEAE) FROM NORTH SUMATRA

MANSUR, M., SALAMAH, A., MIRMANTO, E. & BREARLEY, F. Q. 2022. Nutrient concentrations in three Nepenthes species (Nepenthaceae) from North Sumatra. Reinwardtia 21(2): 55‒62. — Nepenthes is a genus of carnivorous plants that are unique ornamental plants, but their nutrient concentration relationships have not been studied much, especially in endemic species on the island of Sumatra. So far, the analysis of the nutrient concentration in Nepenthes is mostly limited to leaves. There are few reports of nutrient concentrations in the pitcher fluid and the soil around where it grows. Leaves, pitcher fluid, and soil around the growth sites of each species i.e., Nepenthes sumatrana, N. spectabilis, and N. tobaica, from North Sumatra province were collected for nutrient analyses (N, P, K, Ca, Mg, and Na). The results showed that the nutrient concentrations in the leaves and pitcher fluid in the three Nepenthes species were generally low with those in the leaves greater than in the pitcher fluid. The concentration of nutrients in the leaves of N. sumatrana (lowland species) was least (except for N and Na) when compared to N. spectabilis and N. tobaica (highland species), likely reflecting the poorly fertile soil. In contrast, the nutrient concentration in the pitcher fluid of N. sumatrana was greater than N. spectabilis and N. tobaica. When compared across an extensive data set, we show that leaf N does not change with elevation, whereas P declines and the N:P ratio increases with elevation, suggesting that Nepenthes plants are obtaining sufficient N from prey at higher elevations.

Proofof anthocyanins in the carnivorous plant genus Nepenthes.

Yellow to red colored betalains are a chemotaxonomic feature of Caryophyllales, while in most other plant taxa, anthocyanins are responsible for these colors. The carnivorous plant family Nepenthaceae belongs to Caryophyllales; here, red‐pigmented tissues seem to attract insect prey. Strikingly, the chemical nature of red color in Nepenthes has never been elucidated. Although belonging to Caryophyllales, in Nepenthes, some molecular evidence supports the presence of anthocyanins rather than betalains. However, there was previously no direct chemical proof of this. Using ultra‐high‐performance liquid chromatography‐electrospray ionization‐high‐resolution mass spectrometry, we identified cyanidin glycosides in Nepenthes species and tissues. Further, we reveal the existence of a complete set of constitutively expressed anthocyanin biosynthetic genes in Nepenthes. Thus, here we finally conclude the long‐term open question regarding red pigmentation in Nepenthaceae.

First observations of UV-induced fluorescence in Heliamphora (Sarraceniaceae) and other tepui flora

Seven species of the carnivorous plant genus Heliamphora were studied in the wild across four tepuis of the Venezuelan Guyana. All were found to exhibit UV-induced blue fluorescence in their young and developing pitchers, the fluorescence being largely confined to the downward-pointing trichomes of the pitcher interior, with a small contribution from the nectaries. Subsequent work on cultivated plants confirmed the universality of this trait across all known members of the genus. Fluorescence microscopy localised the blue emissions to the surface of the trichomes and unequivocally showed that it represents true fluorescence. The phenomenon was found to be highly transient, generally being seen only in recently opened pitchers. Whether it has a biological function or is an incidental property remains to be determined. Possible roles in the attraction of prey and pitcher inquilines are discussed.

Metabolomics Analysis Reveals Tissue-Specific Metabolite Compositions in Leaf Blade and Traps of Carnivorous Nepenthes Plants.

Nepenthes is a genus of carnivorous plants that evolved a pitfall trap, the pitcher, to catch and digest insect prey to obtain additional nutrients. Each pitcher is part of the whole leaf, together with a leaf blade. These two completely different parts of the same organ were studied separately in a non-targeted metabolomics approach in Nepenthes x ventrata, a robust natural hybrid. The first aim was the analysis and profiling of small (50–1000 m/z) polar and non-polar molecules to find a characteristic metabolite pattern for the particular tissues. Second, the impact of insect feeding on the metabolome of the pitcher and leaf blade was studied. Using UPLC-ESI-qTOF and cheminformatics, about 2000 features (MS/MS events) were detected in the two tissues. They showed a huge chemical diversity, harboring classes of chemical substances that significantly discriminate these tissues. Among the common constituents of N. x ventrata are phenolics, flavonoids and naphthoquinones, namely plumbagin, a characteristic compound for carnivorous Nepenthales, and many yet-unknown compounds. Upon insect feeding, only in pitchers in the polar compounds fraction, small but significant differences could be detected. By further integrating information with cheminformatics approaches, we provide and discuss evidence that the metabolite composition of the tissues can point to their function.

Agrobacterium tumefaciens mediated transformation of the aquatic carnivorous plant Utricularia gibba

BackgroundThe genus Utricularia belongs to Lentibulariaceae, the largest family of carnivorous plants, which includes terrestrial, epiphytic and aquatic species. The development of specialized structures that evolved for carnivory is a feature of this genus that has been of great interest to biologists since Darwin‘s early studies. Utricularia gibba is itself an aquatic plant with sophisticated bladder traps having one of the most complex suction mechanisms for trapping prey. However, the molecular characterization of the mechanisms that regulate trap development and the biophysical processes involved in prey trapping are still largely unknown due to the lack of a simple and reproducible gene transfer system.ResultsHere, we report the establishment of a simple, fast and reproducible protocol for genetic transformation of U. gibba based on the T-DNA of Agrobacterium tumefaciens. An in vitro selection system using Phosphinotricin as a selective agent was established for U. gibba. Plant transformation was confirmed by histochemical GUS assays and PCR and qRT-PCR analyses. We report on the expression pattern of the 35S promoter and of the promoter of a trap-specific ribonuclease gene in transgenic U. gibba plants.ConclusionsThe genetic transformation protocol reported here is an effective method for studying developmental biology and functional genomics of this genus of carnivorous plants and advances the utility of U. gibba as a model system to study developmental processes involved in trap formation.

Jasmonate-independent regulation of digestive enzyme activity in the carnivorous butterwort Pinguicula × Tina.

Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula × Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion. We identified eight enzymes in the digestive secretion, many of which were previously found in other genera of carnivorous plants. Among them, alpha-amylase is unique in carnivorous plants. Enzymatic activities increased in response to prey capture; however, the tissue content of jasmonic acid and its isoleucine conjugate remained rather low in contrast to the jasmonate response to wounding. Enzyme activities did not increase in response to the exogenous application of jasmonic acid or coronatine. Whereas similar digestive enzymes were co-opted from plant defense mechanisms among carnivorous plants, the mode of their regulation differs. The butterwort has not co-opted jasmonate signaling for the induction of enzyme activities in response to prey capture. Moreover, the presence of alpha-amylase in digestive fluid of P. × Tina, which has not been found in other genera of carnivorous plants, might indicate that non-defense-related genes have also been co-opted for carnivory.

A bio-inspired superhydrophobic surface for fog collection and directional water transport

Simple methods for the collection and transportation of water droplets can solve complex problems, but developing technologies to achieve this is a challenge. To this end, widespread attention in recent years has focused on the preparation and application of superhydrophobic surfaces. Inspired by the structures of trichomes (hairs) of Sarracenia, a genus of carnivorous pitcher plants, we report a bionic high-low rib-like microstructure with superhydrophobic and directional water transport properties. At the same time, we can accelerate fog-collection efficiency by preparing a surface enabling directional slip of liquid. This work provides a good strategy for liquid transportation and fog collection, which has broad application prospects in the collection of rainwater in desert areas and the transmission of microfluidics in mechanical engineering.

Plant systematics and biogeography in the Australasian tropics: part 2

[Extract:] This double issue is the second on the topic of plant systematics and biogeography in the Australasian tropics published in Australian Systematic Botany. The first issue on this topic (vol. 31, 5–6) was published in 2018 and brought together ten papers that contributed original research in a wide range of disciplines, including biogeography, developmental anatomy, evolutionary radiation and taxonomy. This second special issue follows directly on, and showcases 10 additional papers, many across those same disciplines; however, several fascinating papers expand the range of disciplines represented. These include palaeobotanical work that contributes critical new information on the history of horsetails (Equisetum) in Australia (Rozefelds et al. 2019); a detailed account of the taxonomic work on Australian plants of the Wendland family, Court Gardeners at the Royal Gardens of Herrenhausen, Hanover, Germany (Dowe et al. 2019), a little known aspect of Australian botanical history; and lastly a demonstration of the utility of genome skimming approaches for untangling evolutionary relationships in species-rich and morphologically complex tropical Australasian groups, using the carnivorous plant genus Nepenthes as an example (Nauheimer et al. 2019).

Evidence of motile traps in Byblis

Byblis Salisb. is a small genus of carnivorous plants with adhesive traps in the Lamiales family Byblidaceae Domin. There are two perennial species (B. gigantea Lindl. and B. lamellata Conran & Lowrie) with restricted ranges in Western Australia, where they experience a Mediterranean climate. The critically endangered B gigantea is endemic to the Swan River drainage area, now entirely within the Perth metropolitan area, whilst B. lamellata is restricted to the coastal region North of Perth. The genus also contains six currently recognized annual species (B. aquatica Lowrie & Conran, B. filifolia Planch., B. guehoi Lowrie & Conran, B. liniflora Salisb., B. pilbarana Lowrie & Conran, and B. rorida Lowrie & Conran) which inhabit the tropical and semi-arid regions of Northern Australia. The genus also extends to the island of New Guinea (Lowrie 2013; McPherson 2010). All species are found in substrates which are very nutrient-poor (Lowrie 2013; McPherson 2010) and share habitats with representatives of other genera of carnivorous plants (particularly Drosera, but also Utricularia and Nepenthes). Although there are important morphological differences between the Byblis species, all share the same basic structure in that they produce stems from which radiate filiform leaves. Another feature common to all species is the ability to produce fast concentrated growth in response to seasonal rainfall. (Bourke, pers. comm.). It has been observed on many occasions that all Byblis species play host to Miridae bugs from the genus Setocoris (Bourke, pers. comm.). A mutualistic relationship has been proven to exist between another viscid plant genus Roridula and a different genus of the family Miridae, Pameridea (Anderson & Midgley 2003). A similar relationship is strongly suspected in Byblis (Lowrie 2013; Cross et al. 2018).

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.

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.

Evidence for pitcher trait-mediated coexistence between sympatric Nepenthes pitcher plant species across geographical scales

ABSTRACTBackground: According to modern coexistence theory, ecologically similar species can coexist if fitness differences between them are small, or niche differences between them are large. However, these predictions have not been tested extensively in real systems and are difficult to examine in traits-based studies.Aims: The aim of our study was, by using the carnivorous pitcher plant genus Nepenthes as a model system, to examine (1) species growth ranks as a proxy for fitness; (2) modern coexistence theory at a geographical scale; (3) evidence for pitcher trait-mediated resource partitioning between sympatric Nepenthes species.Methods: We used growth ranks, obtained from a survey of experienced Nepenthes nursery owners, as a proxy for fitness. Multivariate pitcher-, vegetative- or combined-trait distances, computed from morphometric-trait data from the literature were used as proxies for niche differences. Data on global Nepenthes species-pair sympatry was modelled against these fitness and niche differences.Results: Niche and growth rate differences were positively and negatively correlated with sympatry, respectively, as expected from theory.Conclusion: Our results agree with theory and suggest that fitness differences can be approximated from average species growth. Prey- and microhabitat-resource partitioning, operating through divergent pitcher and vegetative traits respectively, are likely mechanisms that stabilise the coexistence of sympatric Nepenthes species.

Common Features Between the Proteomes of Floral and Extrafloral Nectar From the Castor Plant (Ricinus Communis) and the Proteomes of Exudates From Carnivorous Plants.

Label-free quantitative proteome analysis of extrafloral (EFN) and floral nectar (FN) from castor (Ricinus communis) plants resulted in the identification of 72 and 37 proteins, respectively. Thirty proteins were differentially accumulated between EFN and FN, and 24 of these were more abundant in the EFN. In addition to proteins involved in maintaining the nectar pathogen free such as chitinases and glucan 1,3-beta-glucosidase, both proteomes share an array of peptidases, lipases, carbohydrases, and nucleases. A total of 39 of the identified proteins, comprising different classes of hydrolases, were found to have biochemical matching partners in the exudates of at least five genera of carnivorous plants, indicating the EFN and FN possess a potential to digest biological material from microbial, animal or plant origin equivalent to the exudates of carnivorous plants.

Molecular phylogeny of bladderworts: A wide approach of Utricularia (Lentibulariaceae) species relationships based on six plastidial and nuclear DNA sequences

The carnivorous plant genus Utricularia L. (bladderwort) comprises about 240 species distributed worldwide and is traditionally classified into two subgenera (Polypompholyx and Utricularia) and 35 sections, based mainly on general and trap morphology. It is one out of the largest carnivorous genera, representing ca. 30% of all carnivorous plant species, and is also the most widely distributed. According to previous phylogenetic studies, most infrageneric sections are monophyletic, but there are several incongruences considering their relationships and also the dissenting position of some species as a result of a too few (mostly one or two) molecular markers analyzed. Thus, here we present a multilocus phylogeny for Utricularia species with a wide taxonomic sampling (78 species and 115 accessions) based on six plastid (rbcL, matK, rpl20-rps12, rps16, trnL-F) and nuclear DNA (ITS region) sequences. The aim is to reconstruct a well-resolved tree to propose evolutionary and biogeographic hypotheses for the radiation of lineages with inferences about the divergence times of clades using a molecular clock approach.