Form follows function: morphological diversification and alternative trapping strategies in carnivorous Nepenthes pitcher plants

Carnivorous plants of the genus Nepenthes have evolved a striking diversity of pitcher traps that rely on specialized slippery surfaces for prey capture. With a comparative study of trap morphology, we show that Nepenthes pitcher plants have evolved specific adaptations for the use of either one of two distinct trapping mechanisms: slippery wax crystals on the inner pitcher wall and 'insect aquaplaning' on the wet upper rim (peristome). Species without wax crystals had wider peristomes with a longer inward slope. Ancestral state reconstructions identified wax crystal layers and narrow, symmetrical peristomes as ancestral, indicating that wax crystals have been reduced or lost multiple times independently. Our results complement recent reports of nutrient source specializations in Nepenthes and suggest that these specializations may have driven speciation and rapid diversification in this genus.     

Carnivorous syndrome in Asian pitcher plants of the genus Nepenthes

BACKGROUND AND AIMS: Pitcher plants Nepenthes alata and N. mirabilis are carnivorous species with leaves composed of a photosynthetic part (lamina) and a pitcher trap. This characteristic permitted direct physiological and anatomical comparison between these two distinct parts of the leaves to determine those features involved in the 'carnivorous syndrome', which include low net photosynthetic assimilation rate (A(N)) and low photosynthetic nitrogen use efficiency (PNUE). METHODS: Photosynthetic rate (A(N)) and respiration rate (R(d)) were measured gasometrically, chlorophyll concentration was determined spectrophotometrically and nitrogen concentration was determined using a CHN elemental analyser in lamina and trap separately. Anatomy of N. alata was observed using light, fluorescence and transmission electron microscopy. A(N), foliar nitrogen and chlorophyll concentration were also compared with values for other carnivorous plant species (genera Sarracenia, Drosera) that combine both autotrophic and carnivorous functions into the same physical organ. KEY RESULTS: It was found that the A(N) in Nepenthes lamina was low and PNUE was only slightly higher or similar in comparison with other carnivorous plants. It was not observed that the pitcher had a higher R(d) than the lamina, but A(N) in the pitcher was significantly lower than in the lamina. Nepenthes possesses a cluster of characters that could result in reduced photosynthesis in the pitcher and be responsible for carnivorous function of the leaf: replacement of chlorophyll-containing cells with digestive glands, low chlorophyll and nitrogen concentration, compact mesophyll with a small portion of intercellular spaces, absence of palisade parenchyma and low stomatal density. CONCLUSION: Low photosynthetic capacity, nitrogen efficiency, chlorophyll and nitrogen concentration of Nepenthes pitchers was found, together with a set of features that characterized the carnivorous syndrome. Dual use of leaves for photosynthesis and nutrient gain can decrease photosynthetic efficiency in carnivorous plants in general.     

Feeding enhances photosynthetic efficiency in the carnivorous pitcher plant Nepenthes talangensis

Background and Aims

Cost–benefit models predict that carnivory can increase the rate of photosynthesis (A_N) by leaves of carnivorous plants as a result of increased nitrogen absorption from prey. However, the cost of carnivory includes decreased A_N and increased respiration rates (R_D) of trapping organs. The principal aim of the present study was to assess the costs and benefits of carnivory in the pitcher plant Nepenthes talangensis, leaves of which are composed of a lamina and a pitcher trap, in response to feeding with beetle larvae.

Methods

Pitchers of Nepenthes grown at 200 µmol m⁻² s⁻¹ photosynthetically active radiation (PAR) were fed with insect larvae for 2 months, and the effects on the photosynthetic processes were then assessed by simultaneous measurements of gas exchange and chlorophyll fluorescence of laminae and pitchers, which were correlated with nitrogen, carbon and total chlorophyll concentrations.

Key Results

A_N and maximum (F_v/F_m) and effective quantum yield of photosystem II (Φ_PSII) were greater in the fed than unfed laminae but not in the fed compared with unfed pitchers. Respiration rate was not significantly affected in fed compared with unfed plants. The unfed plants had greater non-photochemical quenching (NPQ) of chlorophyll fluorescence. Higher NPQ in unfed lamina did not compensate for their lower Φ_PSII, resulting in lower photochemical quenching (QP) and thus higher excitation pressure on PSII. Biomass and nitrogen and chlorophyll concentration also increased as a result of feeding. The cost of carnivory was shown by lower A_N and Φ_PSII in pitchers than in laminae, but R_D depended on whether it was expressed on a dry weight or a surface area basis. Correlation between nitrogen and A_N in the pitchers was not found. Cost–benefit analysis showed a large beneficial effect on photosynthesis from feeding as light intensity increased from 200 to 1000 µmol m⁻² s⁻¹ PAR after which it did not increase further. All fed plants began to flower.

Conclusion

Feeding pitchers with insect larvae increases A_N of leaf laminae, due to higher nutrient acquisition, with strong correlation with nitrogen concentration, but A_N of pitchers does not increase, despite increased nitrogen concentration in their tissue. Increased A_N improves growth and reproduction and is likely to increase the competitive advantage of carnivorous over non-carnivorous plants in nutrient-poor habitats.Key words: carnivorous plants, chlorophyll fluorescence, Nepenthes talangensis, nitrogen, pitcher plant, photosynthetic rate, photosystem II, respiration rate     

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.     

A viscoelastic deadly fluid in carnivorous pitcher plants

Background

The carnivorous plants of the genus Nepenthes, widely distributed in the Asian tropics, rely mostly on nutrients derived from arthropods trapped in their pitcher-shaped leaves and digested by their enzymatic fluid. The genus exhibits a great diversity of prey and pitcher forms and its mechanism of trapping has long intrigued scientists. The slippery inner surfaces of the pitchers, which can be waxy or highly wettable, have so far been considered as the key trapping devices. However, the occurrence of species lacking such epidermal specializations but still effective at trapping insects suggests the possible implication of other mechanisms.

Methodology/Principal Findings

Using a combination of insect bioassays, high-speed video and rheological measurements, we show that the digestive fluid of Nepenthes rafflesiana is highly viscoelastic and that this physical property is crucial for the retention of insects in its traps. Trapping efficiency is shown to remain strong even when the fluid is highly diluted by water, as long as the elastic relaxation time of the fluid is higher than the typical time scale of insect movements.

Conclusions/Significance

This finding challenges the common classification of Nepenthes pitchers as simple passive traps and is of great adaptive significance for these tropical plants, which are often submitted to high rainfalls and variations in fluid concentration. The viscoelastic trap constitutes a cryptic but potentially widespread adaptation of Nepenthes species and could be a homologous trait shared through common ancestry with the sundew (Drosera) flypaper plants. Such large production of a highly viscoelastic biopolymer fluid in permanent pools is nevertheless unique in the plant kingdom and suggests novel applications for pest control.     

Degradation of a peptide in pitcher fluid of the carnivorous plant Nepenthes alata Blanco

Carnivorous plants acquire substantial amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes produces trapping organs called pitchers at the tips of tendrils elongated from leaf ends. Acidic fluid is secreted at the bottoms of the pitchers. The pitcher fluid includes several hydrolytic enzymes, and some, such as aspartic proteinase, are thought to be involved in nitrogen acquisition from insect proteins. To understand the nitrogen-acquisition process, it is essential to identify the protein-degradation products in the pitcher fluid. To gain insight into protein degradation in pitcher fluid, we used the oxidized B-chain of bovine insulin as a model substrate, and its degradation by the pitcher fluid of N. alata was investigated using liquid chromatography-mass spectrometry (LC-MS). LC-MS analysis of the degradation products revealed that the oxidized B-chain of bovine insulin was initially cleaved at aromatic amino acids such as phenylalanine and tyrosine. These cleavage sites are similar to those of aspartic proteinases from other plants and animals. The presence of a series of peptide fragments as degradation products suggests that exopeptidase(s) is also present in the pitcher fluid. Amino acid analysis and peptide fragment analysis of the degradation products demonstrated that three amino acids plus small peptides were released from the oxidized B-chain of bovine insulin, suggesting that insect proteins are readily degraded to small peptides and amino acids in the pitcher fluid of N. alata.     

Geographical variation in food web structure in Nepenthes pitcher plants

Abstract. 1. Relative to Nepenthes species in West Malaysia near the evolutionary centre of the genus, outlying species of Nepenthes in the Seychelles, Sri Lanka and Madagascar have fewer species of both prey and predator living in them, fewer and smaller guilds of species, much apparently empty niche space, less complex food webs, and a greater connectance. The ratios of prey to predators, and of connectance (C₁) to the total number of trophic types present remain approximately constant.
2. Differences between the food webs appear to be related in a complex way to the size of the country and its degree of spatial and temporal isolation, the size of the local species pool capable of colonizing the pitchers, and the number of Nepenthes species present. However, the maximal length of food chains in the richest and most complex food webs is probably limited by energetic constraints or environmental predictability.
3. The data may illustrate how food webs change to become more complex, both by the addition of new guilds of species and the addition of species to existing guilds, while at the same time certain properties of the food web are kept approximately constant.     

Fluid physico-chemical properties influence capture and diet in Nepenthes pitcher plants

Background and AimsNepenthes pitcher plants have evolved modified leaves with slippery surfaces and enzymatic fluids that trap and digest prey, faeces and/or plant detritus. Although the fluid’s contribution to insect capture is recognized, the physico-chemical properties involved remain underexplored and may vary among species, influencing their diet type. This study investigates the contributions of acidity and viscoelasticity in the fluid’s capture efficiency of two ant and two fly species in four Nepenthes species with different nutrition strategies.Methods Four Nepenthes species were studied, namely N. rafflesiana, N. gracilis, N. hemsleyana and N. ampullaria. Fluid was collected from pitchers of varying ages from plants growing in the field and immediately transferred to glass vials, and individual ants (tribe Campotini, Fomicinae) and flies (Calliphora vomitoria and Drosophila melanogaster) were dropped in and observed for 5 min. Water-filled vials were used as controls. Survival and lifetime data were analysed using models applied to right-censored observations. Additional laboratory experiments were carried out in which C. vomitoria flies were immersed in pH-controlled aqueous solutions and observed for 5 min.Key Results Pitcher fluid differed among Nepenthes species as regards insect retention capacity and time-to-kill, with differences observed between prey types. Only the fluids of the reputedly insectivorous species were very acidic and/or viscoelastic and retained significantly more insects than the water controls. Viscoelastic fluids were fatal to flies and were able to trap the broadest diversity of insects. Younger viscoelastic fluids showed a better retention ability than older fluids, although with less rapid killing ability, suggesting that a chemical action follows a mechanical one. Insect retention increased exponentially with fluid viscoelasticity, and this happened more abruptly and at a lower threshold for flies compared with ants. Flies were more often retained if they fell into the traps on their backs, thus wetting their wings. Insect retention and death rate increased with fluid acidity, with a lower threshold for ants than for flies, and the time-to-kill decreased with increasing acidity. The laboratory experiments showed that fewer flies escaped from acidic solutions compared with water.Conclusions In addition to viscoelasticity, the pitcher’s fluid acidity and wetting ability influence the fate of insects and hence the diet of Nepenthes. The plants might select the prey that they retain by manipulating the secretion of H⁺ ions and polysaccharides in their pitcher fluid. This in turn might participate in possible adaptive radiation of this genus with regard to nutrient sequestration strategy. These plants might even structurally influence insect fall-orientation and capture-probability, inspiring biomimetic designs for pest control.     

Contribution of pitcher fragrance and fluid viscosity to high prey diversity in a Nepenthes carnivorous plant from borneo

Mechanisms that improve prey richness in carnivorous plants may involve three crucial phases of trapping:attraction, capture and retention.Nepenthes rafflesiana var. typica is an insectivorous pitcher plant that is widespread in northern Borneo.It exhibits ontogenetic pitcher dimorphism with the upper pitchers trapping more flying prey than the lower pitchers.While this difference in prey composition has been ascribed to differences in attraction,the contribution of capture and retention has been overlooked.This study focused on distinguishing between the prey trapping mechanisms, and assessing their relative contribution to prey diversity.Arthropod richness and diversity of both visitors and prey in the two types of pitchers were analysed to quantify the relative contribution of attraction to prey trapping.Rate of insect visits to the different pitcher parts and the presence or absence of a sweet fragrance was recorded to clarify the origin and mechanism of attraction.The mechanism of retention was studied by insect bioassays and measurements of fluid viscosity. Nepenthes rafflesiana was found to trap a broader prey spectrum than that previously described for any Nepenthes species,with the upper pitchers attracting and trapping a greater quantity and diversity of prey items than the lower pitchers.Capture efficiency was low compared with attraction or retention efficiency.Fragrance of the peristome,or nectar rim,accounted mainly for the observed non-specific, better prey attraction by the upper pitchers, while the retentive properties of the viscous fluid in these upper pitchers arguably explains the species richness of their flying prey.The pitchers of N. rafflesiana are therefore more than simple pitfall traps and the digestive fluid plays an important yet unsuspected role in the ecological success of the species.     

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth

Background and Aims

Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated.

Methods

Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma–optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes.

Key Results

The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes.

Conclusions

The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.     

With a flick of the lid: a novel trapping mechanism in Nepenthes gracilis pitcher plants

Carnivorous pitcher plants capture prey with modified leaves (pitchers), using diverse mechanisms such as 'insect aquaplaning' on the wet pitcher rim, slippery wax crystals on the inner pitcher wall, and viscoelastic retentive fluids. Here we describe a new trapping mechanism for Nepenthes gracilis which has evolved a unique, semi-slippery wax crystal surface on the underside of the pitcher lid and utilises the impact of rain drops to 'flick' insects into the trap. Depending on the experimental conditions (simulated 'rain', wet after 'rain', or dry), insects were captured mainly by the lid, the peristome, or the inner pitcher wall, respectively. The application of an anti-slip coating to the lower lid surface reduced prey capture in the field. Compared to sympatric N. rafflesiana, N. gracilis pitchers secreted more nectar under the lid and less on the peristome, thereby directing prey mainly towards the lid. The direct contribution to prey capture represents a novel function of the pitcher lid.     

Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [β-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.     

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata

The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (β-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), β-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes.     

Focus on mast cells in the tumor microenvironment: Current knowledge and future directions

Mast cells (MCs) are crucial cells participating in both innate and adaptive immune processes that play important roles in protecting human health and in the pathophysiology of various diseases, such as allergies, cardiovascular diseases, and autoimmune diseases. In the context of tumors, MCs are a non-negligible population of immune cells in the tumor microenvironment (TME). In most tumor types, MCs accumulate in both the tumor tissue and the surrounding tissue. MCs interact with multiple components of the TME, affecting TME remodeling and the tumor cell fate. However, controversy persists regarding whether MCs contribute to tumor progression or trigger an anti-tumor immune response. This review focuses on the context of the TME to explore the specific properties and functions of MCs and discusses the crosstalk that occurs between MCs and other components of the TME, which affect tumor angiogenesis and lymphangiogenesis, invasion and metastasis, and tumor immunity through different mechanisms. We also anticipate the potential role of MCs in cancer immunotherapy, which might expand upon the success achieved with existing cancer therapies.     

Traps of carnivorous pitcher plants as a habitat: composition of the fluid, biodiversity and mutualistic activities

BACKGROUND: Carnivorous pitcher plants (CPPs) use cone-shaped leaves to trap animals for nutrient supply but are not able to kill all intruders of their traps. Numerous species, ranging from bacteria to vertrebrates, survive and propagate in the otherwise deadly traps. This paper reviews the literature on phytotelmata of CPPs. PITCHER: Fluid as a Habitat The volumes of pitchers range from 0·2 mL to 1·5 L. In Nepenthes and Cephalotus, the fluid is secreted by the trap; the other genera collect rain water. The fluid is usually acidic, rich in O(2) and contains digestive enzymes. In some taxa, toxins or detergents are found, or the fluid is extremely viscous. In Heliamphora or Sarracenia, the fluid differs little from pure water. INQUILINE: Diversity Pitcher inquilines comprise bacteria, protozoa, algae, fungi, rotifers, crustaceans, arachnids, insects and amphibia. The dominant groups are protists and Dipteran larvae. The various species of CPPs host different sets of inquilines. Sarracenia purpurea hosts up to 165 species of inquilines, followed by Nepenthes ampullaria with 59 species, compared with only three species from Brocchinia reducta. Reasons for these differences include size, the life span of the pitcher as well as its fluid. MUTUALISTIC: Activities Inquilines closely interact with their host. Some live as parasites, but the vast majority are mutualists. Beneficial activities include secretion of enzymes, feeding on the plant's prey and successive excretion of inorganic nutrients, mechanical break up of the prey, removal of excessive prey and assimilation of atmospheric N(2). CONCLUSIONS: There is strong evidence that CPPs influence their phytotelm. Two strategies can be distinguished: (1) Nepenthes and Cephalotus produce acidic, toxic or digestive fluids and host a limited diversity of inquilines. (2) Genera without efficient enzymes such as Sarracenia or Heliamphora host diverse organisms and depend to a large extent on their symbionts for prey utilization.     

Adaptive radiation with regard to nutrient sequestration strategies in the carnivorous plants of the genus Nepenthes

Carnivorous pitcher plants of the genus Nepenthes have evolved a great diversity of pitcher morphologies. Selective pressures for maximizing nutrient uptake have driven speciation and diversification of the genus in a process known as adaptive radiation. This leads to the evolution of pitchers adapted to specific and often bizarre source of nutrients, which are not strictly animal-derived. One example is Nepenthes ampullaria with unusual growth pattern and pitcher morphology what enables the plant to capture a leaf litter from the canopy above. We showed that the plant benefits from nitrogen uptake by increased rate of photosynthesis and growth what may provide competitive advantage over others co-habiting plants. A possible impact of such specialization toward hybridization, an important mechanism in speciation, is discussed.     

The nitrogen supply from soils and insects during growth of the pitcher plants Nepenthes mirabilis, Cephalotus follicularis and Darlingtonia californica

This study investigated the nitrogen (N) acquisition from soil and insect capture during the growth of three species of pitcher plants, Nepenthes mirabilis, Cephalotus follicularis and Darlingtonia californica. ¹⁵N/¹⁴N natural abundance ratios (δ¹⁵N) of plants and pitchers of different age, non-carnivorous reference plants, and insect prey were used to estimate proportional contributions of insects to the N content of leaves and whole plants. Young Nepenthes leaves (phyllodes) carrying closed pitchers comprised major sinks for N and developed mainly from insect N captured elsewhere on the plant. Their δ¹⁵N values of up to 7.2‰ were higher than the average δ¹⁵N value of captured insects (mean δ¹⁵N value = 5.3‰). In leaves carrying old pitchers that are acting as a N source, the δ¹⁵N decreased to 3.0‰ indicating either an increasing contribution of soil N to those plant parts which in fact captured the insects or N gain from N₂ fixation by microorganisms which may exist in old pitchers. The δ¹⁵N value of N in water collected from old pitchers was 1.2‰ and contained free amino acids. The fraction of insect N in young and old pitchers and their associated leaves decreased from 1.0 to 0.3 mg g⁻¹. This fraction decreased further with the size of the investigated tiller. Nepenthes contained on average 61.5 ± 7.6% (mean ± SD, range 50–71%) insect N based on the N content of a whole tiller. In the absence of suitable non-carnivorous reference plants for Cephalotus, δ¹⁵N values were assessed across a developmental sequence from young plants lacking pitchers to large adults with up to 38 pitchers. The data indicated dependence on soil N until 4 pitchers had opened. Beyond that stage, plant size increased with the number of catching pitchers but the fraction of soil N remained high. Large Cephalotus plants were estimated to derive 26 ± 5.9% (mean ± SD of the three largest plants; range: 19–30%) of the N from insects. In Cephalotus we observed an increased δ¹⁵N value in sink versus source pitchers of about 1.2‰ on average. Source and sink pitchers of Darlingtonia had a similar δ¹⁵N value, but plant N in this species showed δ¹⁵N signals closer to that of insect N than in either Cephalotus or Nepenthes. Insect N contributed 76.4 ± 8.4% (range 57–90%) to total pitcher N content. The data suggest complex patterns of partitioning of insect and soil-derived N between source and sink regions in pitcher plants and possibly higher dependence on insect N than recorded elsewhere for Drosera species. Received: 14 April 1997 / Accepted: 18 August 1997     

Mutualism between tree shrews and pitcher plants: Perspectives and avenues for future research

Three species of Nepenthes pitcher plants from Borneo engage in a mutualistic interaction with mountain tree shrews, the basis of which is the exchange of nutritional resources. The plants produce modified “toilet pitchers” that produce copious amounts of exudates, the latter serving as a food source for tree shrews. The exudates are only accessible to the tree shrews when they position their hindquarters over the pitcher orifice. Tree shrews mark valuable resources with feces and regularly defecate into the pitchers when they visit them to feed. Feces represent a valuable source of nitrogen for these Nepenthes species, but there are many facets of the mutualism that are yet to be investigated. These include, but are not limited to, seasonal variation in exudate production rates by the plants, behavioral ecology of visiting tree shrews and the mechanism by which the plants signal to tree shrews that their pitchers represent a food source. Further research into this extraordinary animal-plant interaction is required to gain a better understanding of the benefits to the participating species.Key words: Nepenthes, tree shrew, nitrogen sequestration, mutualism, animal-plant interactionsThe pitcher plant genus Nepenthes comprises approximately 120 species, with the centre of diversity lying in the perhumid tropics of Southeast Asia. All species are vines or subscandent shrubs that produce highly modified leaf organs (“pitchers”) which typically attract, trap, retain and digest arthropods for nutritional benefit. The pitchers of almost all Nepenthes species share the same physical components,¹ including the pitcher cup, the peristome and the lid. The pitcher cup usually consists of two main sections: an upper zone which is often covered with wax crystals and anisotropically-oriented semilunate cells^2,3 that assist in the capture and retention of prey; and a lower portion, which contains fluid and is lined with digestive glands.^2,3 The peristome is a ridge of hardened tissue that lines the orifice. Its anisotropic, wettable surface plays a key role in prey capture.^4,5 In most species, the lid is a broad, flat structure which overhangs the orifice and prevents the entry of rainwater which, if unimpeded, can cause the pitchers to overflow, thereby losing digestive enzymes and the products of their activities. The lid is often brightly coloured, has many nectar glands on its surfaces and plays an important role in prey attraction.²The degree of development and/or modification of each pitcher component varies substantially among (and even within) Nepenthes species^2,6,7 and recent research has demonstrated that unique modifications to pitcher structure possessed by several species play important roles in specialized nutrient acquisition strategies.^8–12 One such species, Nepenthes lowii, demonstrates a remarkable nitrogen sequestration strategy, in which mountain tree shrews (Tupaia montana) defecate into its pitchers while feeding on exudates secreted by glands on the inner surface of the pitcher lid. Feces accounts for 57–100% of foliar nitrogen in this species¹³ and N. lowii “toilet pitchers” are ineffective arthropod traps. The large orifices and reflexed, concave lids of N. lowii pitchers induce T. montana to sit astride the pitcher whilst feeding, facilitating fecal deposition.Chin et al.¹⁴ found that two other montane species from Borneo, Nepenthes rajah and Nepenthes macrophylla, also trap tree shrew feces. Detailed analysis of trap geometry revealed that these two species and N. lowii share a unique arrangement of trap characteristics that was not detected by earlier studies on the genus. This involves the production of pitchers with very large orifices, large, concave lids that are reflexed approximately 90° away from the orifice and lid glands that produce copious exudates.¹⁴ The distance from the front of the pitcher orifice to the inner surface of the lid precisely matches the head + body length of T. montana, resulting in the tree shrews'' food source being positioned behind the pitcher orifice and ensuring that the animals'' hindquarters are positioned over the orifice while they feed on the lid gland exudates.Thus, N. lowii, N. macrophylla and N. rajah are all engaged in a mutualism with T. montana, the basis of which is the exchange of nutritional resources that are scarce in these species'' habitats. The interaction with T. montana is facilitated by trap geometry, but all three Nepenthes species produce pitchers that differ substantially in structure, apart from the shared characteristics outlined above.¹⁴ Through a series of modifications to trap structure and geometry—none of which appears to have compromised their ability to trap arthropod prey—N. rajah and N. macrophylla benefit from a highly specialised nitrogen sequestration strategy that is not available to congeners other than N. lowii.Although Clarke et al.¹³ demonstrated that N. lowii derives nutritional benefit from T. montana feces, there are many facets of the association that have yet to be investigated and the discoveries of Chin et al.¹⁴ give rise to a number avenues for further research, several of which are discussed below.The behavioral ecology of T. montana with respect to Nepenthes has not been studied in detail. We do not know whether individual tree shrews defend valuable pitchers against other animals or whether such resources are shared. However, video footage, showing T. montana scent-marking a toilet pitcher of N. lowii after feeding from it, supports the former scenario (Clarke et al.¹³ and Suppl. video). It is not known whether or how, the plants signal to tree shrews that their pitchers provide a nutritional resource (or even how valuable that resource is—the composition and nutritional value of the lid gland exudates has not been determined). When newly-formed pitchers first open, their tissues generally remain soft for several days while they undergo rapid expansion during the final stages of development.¹ During this period, the pitchers are incapable of supporting a tree shrew without suffering significant damage, yet few pitchers of N. lowii, N. macrophylla or N. rajah that we observed exhibited signs of such damage. One possible explanation for this is that the plants signal the tree shrews to indicate whether or not individual pitchers are “open for business.” This might be achieved using variations in color: Tupaia spp. are dichromatic, with sensitivity maxima at ca. 440 and 550–560 nm¹⁵ and the pitchers of all three feces-trapping Nepenthes species utilize combinations of green, red, yellow, orange and purple pigments, which change as individual pitchers age.¹ In N. lowii, the inner surfaces of the feces-trapping pitchers are uniformly dark purple when mature, but when they first open, they are unevenly covered with purple, pink and green patches. The production of copious lid gland exudates in N. lowii appears to commence after the pitchers have hardened and the uniform dark purple color has developed on the inner surfaces.The study by Chin et al.¹⁴ was based on a series of three field trips to northern Borneo that were conducted in March, April and May 2009. The first of these two visits took place during the wet season and heavy rain fell on most days throughout these months. In contrast, May was unusually dry. During this period, many N. rajah plants exhibited signs of stress due to lack of water, including wilting or senescence of developing pitchers and inflorescences. This coincided with an apparent (but unquantified) decline in the number of pitchers that received tree shrew feces: whereas such pitchers were relatively easy to locate during our visits in March and April, they were rare during May. Furthermore, most pitchers that received copious amounts of feces in March and April received none in May. The reasons for this are unknown and may involve changes in the foraging behaviour of T. montana or perhaps a reduction in the quantity and/or quality of the lid gland secretions. Through video recordings, we found that T. montana still visited pitchers of N. rajah during May (Chin et al.¹⁴), but very few fecal pellets were deposited inside them. Tree shrews mark valuable resources using feces,¹⁶ so it is feasible that during periods of decreased nectar production, T. montana alters its foraging behavior to utilize alternative food resources, resulting in decreased rates of defecation into N. rajah pitchers.N. lowii, N. rajah and N. macrophylla are virtually confined to montane habitats above 1,800 m altitude, but the geographical range of T. montana extends well beyond that of the “toilet pitchers” and includes a number of sites that are substantially lower than 1,800 m.¹⁷ Given this, why are the toilet pitchers not found at lower altitudes? Large, fleshy fruits with small seeds (such as figs) comprise a major component of the diet of T. montana,¹⁸ but plants that produce these are relatively scarce in alpine and upper montane equatorial habitats.¹⁹ This could limit the distribution of toilet pitchers in two ways. First, the lack of fleshy fruits at high altitudes might make toilet pitchers a valuable resource for T. montana in upper montane habitats. Furthermore, the density of arthropods at high altitudes is considerably less than in the lowlands.²⁰ This exerts selective pressure on Nepenthes to adopt non-carnivorous nutrient acquisition strategies.¹³ Accordingly, the production of very large, specialized pitchers that receive a steady input of feces may provide a net benefit for the plants, but only at high altitudes. Second, at lower altitudes, fleshy fruits (and arthropods) are more abundant and at these sites the benefits of producing toilet pitchers may be reduced or even negated, hence their absence from smaller mountains.Through their unique pitcher characteristics and trap geometries, a number of Nepenthes species derive supplementary nutrition from a wide variety of arthropod groups, leaf litter and animal feces.^{6,10,13,14,21,22} It is arguable that no other plant family has such a complex and diverse array of interactions with animals. Recent discoveries add to a growing body of evidence to suggest that Nepenthes demonstrate adaptive radiation with regard to nutrient sequestration strategies (see Chin et al.¹⁴ for a more detailed discussion). The findings of Chin et al.¹⁴ provide the strongest support for this hypothesis to date; in addition, they provide the first plausible explanation for the extraordinary size of N. rajah pitchers. This iconic species is the world''s largest carnivorous plant and was first described 150 years ago. The population studied by Chin et al.¹⁴ grows at a site on Mount Kinabalu that has been visited by tourists since 2001 and has been regularly examined by scientists and Sabah Parks staff for more than 30 years. Despite this, the association between N. rajah and T. montana remained undetected until we employed remote survey methods to record pitcher visitors. To date, this technique has been used on just five species of Nepenthes^4,5,13,14 and in each case, remarkable insights into the interactions between animals and Nepenthes have been gained. The potential for further discoveries using this method is therefore high and through new and innovative experimental methodologies now being employed, we anticipate many more exciting discoveries in the near future.