Evolutionary paths to new phenotypes.

Ecological model systems inform on innovative traits in plants and animals.

Convergence in carnivorous pitcher plants reveals a mechanism for composite trait evolution.

Composite traits involve multiple components that, only when combined, gain a new synergistic function. Thus, how they evolve remains a puzzle. We combined field experiments, microscopy, chemical analyses, and laser Doppler vibrometry with comparative phylogenetic analyses to show that two carnivorous Nepenthes pitcher plant species independently evolved similar adaptations in three distinct traits to acquire a new, composite trapping mechanism. Comparative analyses suggest that this new trait arose convergently through "spontaneous coincidence" of the required trait combination, rather than directional selection in the component traits. Our results indicate a plausible mechanism for composite trait evolution and highlight the importance of stochastic phenotypic variation as a facilitator of evolutionary novelty.

As prey and pollinators, insects increase reproduction and allow for outcrossing in the carnivorous plant Dionaea muscipula.

PREMISE: Understanding the factors that limit reproductive success is a key component of plant biology. Carnivorous plants rely on insects as both nutrient sources and pollinators, providing a unique system for studying the effects of both resource and pollen limitation on plant reproduction. METHODS: We conducted a field experiment using wild-growing Dionaea muscipula J. Ellis (Droseraceae) in which we manipulated prey and pollen in a factorial design and measured flower production, number of fruits, and number of seeds. Because understanding reproduction requires knowledge of a plant species' reproductive and pollination biology, we also examined the pollination system, per-visit pollinator effectiveness, and pollen-ovule (P/O) ratio of D. muscipula. RESULTS: Plants that received supplemental prey produced more flowers than control plants. They also had a higher overall fitness estimate (number of flowers × fruit set (total fruits/total flowers) × seeds per fruit), although this benefit was significant only when prey supplementation occurred in the previous growing season. Neither pollen supplementation nor the interaction between pollen and prey supplementation significantly affected overall plant fitness. CONCLUSIONS: This study reinforces the reliance of D. muscipula on adequate prey capture for flower, fruit, and seed production and a mobile pollen vector for reproduction, indicating the importance of considering insects as part of an effective conservation management plan for this species.

Is the co-option of jasmonate signalling for botanical carnivory a universal trait for all carnivorous plants?

The carnivorous plants in the order Caryophyllales co-opted jasmonate signalling from plant defence to botanical carnivory. However, carnivorous plants have at least 11 independent origins, and here we ask whether jasmonate signalling has been co-opted repeatedly in different evolutionary lineages. We experimentally wounded and fed the carnivorous plants Sarracenia purpurea (order Ericales), Cephalotus follicularis (order Oxalidales), Drosophyllum lusitanicum (order Caryophyllales), and measured electrical signals, phytohormone tissue level, and digestive enzymes activity. Coronatine was added exogenously to confirm the role of jasmonates in the induction of digestive process. Immunodetection of aspartic protease and proteomic analysis of digestive fluid was also performed. We found that prey capture induced accumulation of endogenous jasmonates only in D. lusitanicum, in accordance with increased enzyme activity after insect prey or coronatine application. In C. follicularis, the enzyme activity was constitutive while in S. purpurea was regulated by multiple factors. Several classes of digestive enzymes were identified in the digestive fluid of D. lusitanicum. Although carnivorous plants from different evolutionary lineages use the same digestive enzymes, the mechanism of their regulation differs. All investigated genera use jasmonates for their ancient role, defence, but jasmonate signalling has been co-opted for botanical carnivory only in some of them.

Carnivorous Plant Biology: From Gene to Traps.

Carnivorous plants (approximately 850 species) are specific mixotrophic plants which all perform photosynthesis but need mainly nitrogen and phosphorous from animal or protist bodies [...].

Carnivorous plants: Unlocking the secrets of peristome geometry in pitcher plants.

A recent study employs computational models to explore the functional morphology of carnivorous trapping pitchers in Nepenthes. Focusing on the peristome, the study uncovers new dimensions in form-function relationships, offering theoretical insights into the role of complex trap morphology.

Mechanics reveals the role of peristome geometry in prey capture in carnivorous pitcher plants (Nepenthes).

Carnivorous pitcher plants (Nepenthes) are a striking example of a natural pitfall trap. The trap's slippery rim, or peristome, plays a critical role in insect capture via an aquaplaning mechanism that is well documented. While the peristome has received significant research attention, the conspicuous variation in peristome geometry across the genus remains unexplored. We examined the mechanics of prey capture using Nepenthes pitcher plants with divergent peristome geometries. Inspired by living material, we developed a mathematical model that links the peristomes' three-dimensional geometries to the physics of prey capture under the laws of Newtonian mechanics. Linking form and function enables us to test hypotheses related to the function of features such as shape and ornamentation, orientation in a gravitational field, and the presence of "teeth," while analysis of the energetic costs and gains of a given geometry provides a means of inferring potential evolutionary pathways. In a separate modeling approach, we show how prey size may correlate with peristome dimensions for optimal capture. Our modeling framework provides a physical platform to understand how divergence in peristome morphology may have evolved in the genus Nepenthes in response to shifts in prey diversity, availability, and size.

Mutational analysis of mechanosensitive ion channels in the carnivorous Venus flytrap plant.

How the Venus flytrap (Dionaea muscipula) evolved the remarkable ability to sense, capture, and digest animal prey for nutrients has long puzzled the scientific community.1 Recent genome and transcriptome sequencing studies have provided clues to the genes thought to play a role in these tasks.2,3,4,5 However, proving a causal link between these and any aspect of the plant's hunting behavior has been challenging due to the genetic intractability of this non-model organism. Here, we use CRISPR-Cas9 methods to generate targeted modifications in the Venus flytrap genome. The plant detects prey using touch-sensitive trigger hairs located on its bilobed leaves.6 Upon bending, these hairs convert mechanical touch signals into changes in the membrane potential of sensory cells, leading to rapid closure of the leaf lobes to ensnare the animal.7 Here, we generate mutations in trigger-hair-expressed MscS-like (MSL)-family mechanosensitive ion channel genes FLYCATCHER1 (FLYC1) and FLYCATCHER2 (FLYC2)5 and find that double-mutant plants have a reduced leaf-closing response to mechanical ultrasound stimulation. While we cannot exclude off-target effects of the CRISPR-Cas9 system, our genetic analysis is consistent with these and other functionally redundant mechanosensitive ion channels acting together to generate the sensory system necessary for prey detection.

Non-prey biotic interactions in carnivorous plants.

Carnivorous plants often spark broad interest due to their specialized adaptations for trapping and consuming animals. These notable organisms not only fix carbon through photosynthesis, but they also obtain essential nutrients such as nitrogen and phosphate from their captured prey. In typical angiosperms, interactions with animals are usually confined to such processes as pollination and herbivory, but another layer of complexity in these interactions is added for carnivorous plants. Here, we introduce carnivorous plants and their associated organisms - ranging from their prey to their symbionts - and highlight biotic interactions beyond carnivory to discuss how the 'default' interactions typical for flowering plants have changed in the case of the carnivorous plants (Figure 1).

Volatile organic compounds influence prey composition in Sarracenia carnivorous plants.

Sarracenia pitcher plants display interspecific differences in prey, so far only explained by pitcher morphology. We hypothesized that pitcher odours play a role in prey composition. We first compared odour and prey compositions among Sarracenia taxa grown together, forming a kinship gradient from S. purpurea known to capture primarily ants towards S. leucophylla known to capture many flying insects: S. purpurea, S. X mitchelliana, and S. X Juthatip soper & S. X leucophylla horticultural hybrids. We then measured several pitcher traits to disentangle the contributions of morphology and odour to prey variation. The pitcher odours were as diverse as those of generalist-pollinated flowers but with notable differences among taxa, reflecting their relatedness. VOC similarity analyses revealed taxon specificities, that mirrored those revealed by prey similarity analyses. S. X leucophylla stood out by being more specialised in flying insects like bees and moths and by releasing more monoterpenes known to attract flower visitors. S. X Juthatip soper trapped as many bees but fewer moths, sesquiterpenes contributing less to its scent. Ants and Diptera were the main prey of the other two with fatty-acid-derivative-dominated scents. Quantities of the different prey groups can be inferred 98% from quantities of the odour classes and pitcher dimensions. Two syndromes were revealed: ants associated with fatty-acid-derivatives and short pitchers; flying insects associated with monoterpenes, benzenoids and tall pitchers. In S. X leucophylla, emission rate of fatty-acid-derivatives and pitcher length explained most variation in ant captures; monoterpenes and pitcher length explained most variation in bee and moth captures; monoterpenes alone explained most variation in Diptera and wasp captures. Our results suggest that odours are key factors of the diet composition of pitcher plants. They support the hypothesis of perceptual exploitation of insect biases in carnivorous plants and provide new insights into the olfactory preferences of insect groups.

Draft genome and transcriptome of Nepenthes mirabilis, a carnivorous plant in China.

OBJECTIVES: Nepenthes belongs to the monotypic family Nepenthaceae, one of the largest carnivorous plant families. Nepenthes species show impressive adaptive radiation and suffer from being overexploited in nature. Nepenthes mirabilis is the most widely distributed species and the only Nepenthes species that is naturally distributed within China. Herein, we reported the genome and transcriptome assemblies of N. mirabilis. The assemblies will be useful resources for comparative genomics, to understand the adaptation and conservation of carnivorous species. DATA DESCRIPTION: This work produced ~ 139.5 Gb N. mirabilis whole genome sequencing reads using leaf tissues, and ~ 21.7 Gb and ~ 27.9 Gb of raw RNA-seq reads for its leaves and flowers, respectively. Transcriptome assembly obtained 339,802 transcripts, in which 79,758 open reading frames (ORFs) were identified. Function analysis indicated that these ORFs were mainly associated with proteolysis and DNA integration. The assembled genome was 691,409,685 bp with 159,555 contigs/scaffolds and an N50 of 10,307 bp. The BUSCO assessment of the assembled genome and transcriptome indicated 91.1% and 93.7% completeness, respectively. A total of 42,961 genes were predicted in the genome identified, coding for 45,461 proteins. The predicted genes were annotated using multiple databases, facilitating future functional analyses of them. This is the first genome report on the Nepenthaceae family.

Biological Potential of Carnivorous Plants from Nepenthales.

Since Charles Darwin and his book carnivorous plants have aroused interest and heated debate. In addition, there is growing interest in this group of plants as a source of secondary metabolites and in the application of their biological activity. The aim of this study was to trace the recent literature in search of the application of extracts obtained from families Droseraceae, Nepenthaceae, and Drosophyllaceae to show their biological potential. The data collected in the review clearly indicate that the studied Nepenthales species have great biological potential in terms of antibacterial, antifungal, antioxidant, anti-inflammatory, and anticancer use. We proposed that further investigations should include: (i) bioactivity-guided investigations of crude plant extract to connect a particular type of action with a specific compound or a group of metabolites; (ii) a search for new bioactive properties of carnivorous plants; (iii) establishment of molecular mechanisms associated with specific activity. Furthermore, further research should be extended to include less explored species, i.e., Drosophyllum lusitanicum and especially Aldrovanda vesiculosa.

Carnivorous Plants from Nepenthaceae and Droseraceae as a Source of Secondary Metabolites.

Carnivorous plants are able to attract small animals or protozoa and retain them in their specialized traps. Later, the captured organisms are killed and digested. The nutrients contained in the prey bodies are absorbed by the plants to use for growth and reproduction. These plants produce many secondary metabolites involved in the carnivorous syndrome. The main purpose of this review was to provide an overview of the secondary metabolites in the family Nepenthaceae and Droseraceae, which were studied using modern identification techniques, i.e., high-performance liquid chromatography or ultra-high-performance liquid chromatography with mass spectrometry and nuclear magnetic resonance spectroscopy. After literature screening, there is no doubt that tissues of species from the genera Nepenthes, Drosera, and Dionaea are rich sources of secondary metabolites that can be used in pharmacy and for medical purposes. The main types of the identified compounds include phenolic acids and their derivatives (gallic, protocatechuic, chlorogenic, ferulic, p-coumaric acids, gallic, hydroxybenzoic, vanillic, syringic caffeic acids, and vanillin), flavonoids (myricetin, quercetin, and kaempferol derivatives), including anthocyanins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, and cyanidin), naphthoquinones (e.g., plumbagin, droserone, and 5-O-methyl droserone), and volatile organic compounds. Due to the biological activity of most of these substances, the importance of the carnivorous plant as a pharmaceutical crop will increase.

The Localization of Cell Wall Components in the Quadrifids of Whole-Mount Immunolabeled Utricularia dichotoma Traps.

Utricularia (bladderworts) are carnivorous plants. They produce small hollow vesicles, which function as suction traps that work underwater and capture fine organisms. Inside the traps, there are numerous glandular trichomes (quadrifids), which take part in the secretion of digestive enzymes, the resorption of released nutrients, and likely the pumping out of water. Due to the extreme specialization of quadrifids, they are an interesting model for studying the cell walls. This aim of the study was to fill in the gap in the literature concerning the immunocytochemistry of quadrifids in the major cell wall polysaccharides and glycoproteins. To do this, the localization of the cell wall components in the quadrifids was performed using whole-mount immunolabeled Utricularia traps. It was observed that only parts (arms) of the terminal cells had enough discontinuous cuticle to be permeable to antibodies. There were different patterns of the cell wall components in the arms of the terminal cells of the quadrifids. The cell walls of the arms were especially rich in low-methyl-esterified homogalacturonan. Moreover, various arabinogalactan proteins also occurred. Cell walls in glandular cells of quadrifids were rich in low-methyl-esterified homogalacturonan; in contrast, in the aquatic carnivorous plant Aldrovanda vesiculosa, cell walls in the glandular cells of digestive glands were poor in low-methyl-esterified homogalacturonan. Arabinogalactan proteins were found in the cell walls of trap gland cells in all studied carnivorous plants: Utricularia, and members of Droseraceae and Drosophyllaceae.

Cytokinins and auxins in organs of aquatic carnivorous plants: what do they reflect?

BACKGROUND AND AIMS: Aquatic carnivorous plants have typical rootless linear shoots bearing traps and exhibit steep physiological polarity with rapid apical growth. The aim was to analyse auxin and cytokinin metabolites in traps, leaves/shoots and shoot apices in several species of genera Aldrovanda and Utricularia to elucidate how the hormonal profiles reflect the specific organ functions and polarity. METHODS: The main auxin and cytokinin metabolites were analysed in miniature samples (>2 mg dry weight) of different organs of Aldrovanda vesiculosa and six Utricularia species using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. KEY RESULTS: Total contents of biologically active forms (free bases, ribosides) of all four main endogenously occurring cytokinin types were consistently higher in traps than in leaves in four Utricularia species with monomorphic shoots and/or higher than in shoots in two Utricularia species with dimorphic shoots. In Aldrovanda traps, the total content of different cytokinin forms was similar to or lower than that in shoots. In U. australis leaves, feeding on prey increased all cytokinin forms, while no consistent differences occurred in Aldrovanda. In four aquatic Utricularia species with monomorphic shoots, the content of four auxin forms was usually higher in traps than in leaves. Zero IAA content was determined in U. australis leaves from a meso-eutrophic site or when prey-fed. CONCLUSIONS: Different cytokinin and auxin profiles estimated in traps and leaves/shoots of aquatic carnivorous plants indicate an association with different dominant functions of these organs: nutrient uptake by traps versus photosynthetic function of traps. Interplay of cytokinins and auxins regulates apical dominance in these plants possessing strong polarity.

Chemonastic Stalked Glands in the Carnivorous Rainbow Plant Byblis gigantea LINDL. (Byblidaceae, Lamiales).

Carnivorous rainbow plants (Byblis, Byblidaceae, Lamiales) possess sticky flypaper traps for the capture, retention, and digestion of prey (mainly small insects). The trapping system is based on a multitude of millimeter-sized glandular trichomes (also termed stalked glands), which produce adhesive glue drops. For over a century, the trapping system of Byblis was considered passive, meaning that no plant movement is involved. Recently, a remarkable discovery was made: the stalked glands of Byblis are indeed capable of reacting to chemical (protein) stimuli with slow movement responses. This prompted us to investigate this phenomenon further with a series of experiments on the stimulation, kinematics, actuation, and functional morphology of the stalked glands of cultivated Byblis gigantea plants. Measured stalked gland lengths and densities on the trap leaves are similar to the data from the literature. Motion reactions could only be triggered with chemical stimuli, corroborating the prior study on the stalked gland sensitivity. Reaction time (i.e., time from stimulation until the onset of motion) and movement duration are temperature-dependent, which hints towards a tight physiological control of the involved processes. The stalked gland movement, which consist of a sequence of twisting and kinking motions, is rendered possible by the components of the stalk cell wall and is furthermore anatomically and mechanically predetermined by the orientation of cellulose microfibrils in the cell wall. Successive water displacement processes from the stalk cell into the basal cells actuate the movement. The same kinematics could be observed in stalked glands drying in air or submersed in a saturated salt solution. Stimulated and dried stalked glands as well as those from the hypertonic medium were capable of regaining their initial shape by rehydration in water. However, no glue production could be observed afterwards. The long-time overlooked chemonastic movements of stalked glands may help Byblis to retain and digest its prey; however, further research is needed to shed light on the ecological characteristics of the rainbow plant's trapping system.

Selection against early flowering in geothermally heated soils is associated with pollen but not prey availability in a carnivorous plant.

PREMISE: In high-latitude environments, plastic responses of phenology to increasing spring temperatures allow plants to extend growing seasons while avoiding late frosts. However, evolved plasticity might become maladaptive if climatic conditions change and spring temperatures no longer provide reliable cues for conditions important for fitness. Maladaptative phenological responses might be related to both abiotic factors and mismatches with interacting species. When mismatches arise, we expect selection to favor changes in phenology. METHODS: We combined observations along a soil temperature gradient in a geothermally heated area with pollen and prey supplementation experiments and examined how phenotypic selection on flowering time in the carnivorous plant Pinguicula vulgaris depends on soil temperature, and pollen and prey availability. RESULTS: Flowering advanced and fitness decreased with increasing soil temperature. However, in pollen-supplemented plants, fitness instead increased with soil temperature. In heated soils, there was selection favoring later flowering, while earlier flowering was favored in unheated soils. This pattern remained also after artificially increasing pollen and prey availability. CONCLUSIONS: Plant-pollinator mismatches can be an important reason why evolved plastic responses of flowering time to increasing spring temperatures become maladaptive under novel environmental conditions, and why there is selection to delay flowering. In our study, selection for later flowering remained after artificially increasing pollen availability, suggesting that abiotic factors also contribute to the observed selection. Identifying the factors that make evolved phenological responses maladaptive under novel conditions is fundamental for understanding and predicting evolutionary responses to climate warming.

Pitcher geometry facilitates extrinsically powered 'springboard trapping' in carnivorous Nepenthes gracilis pitcher plants.

Carnivorous pitcher plants capture insects in cup-shaped leaves that function as motionless pitfall traps. Nepenthes gracilis evolved a unique 'springboard' trapping mechanism that exploits the impact energy of falling raindrops to actuate a fast pivoting motion of the canopy-like pitcher lid. We superimposed multiple computed micro-tomography images of the same pitcher to reveal distinct deformation patterns in lid-trapping N. gracilis and closely related pitfall-trapping N. rafflesiana. We found prominent differences between downward and upward lid displacement in N. gracilis only. Downward displacement was characterized by bending in two distinct deformation zones whist upward displacement was accomplished by evenly distributed straightening of the entire upper rear section of the pitcher. This suggests an anisotropic impact response, which may help to maximize initial jerk forces for prey capture, as well as the subsequent damping of the oscillation. Our results point to a key role of pitcher geometry for effective 'springboard' trapping in N. gracilis.