DNA metabarcoding allows non-invasive identification of arthropod prey provisioned to nestling Rufous hummingbirds (Selasphorus rufus).

Hummingbirds consume sugars from nectar, sap and honeydew, and obtain protein, fat and minerals from arthropods. To date, the identity of arthropod taxa in hummingbird diets has been investigated by observation of foraging or examination of alimentary tract contents. Direct examination of nestling provisioning adds the extra complication of disturbance to the young and mother. Here, we show that arthropod food items provisioned to Rufous hummingbird (Selasphorus rufus) nestlings can be identified by a safe and non-invasive protocol using next-generation sequencing (NGS) of DNA from nestling fecal pellets collected post-fledging. We found that females on southern Vancouver Island (British Columbia, Canada) provisioned nestlings with a wide range of arthropod taxa. The samples examined contained three Classes, eight Orders, 48 Families, and 87 Genera, with from one to 15 Families being identified in a single pellet. Soft-bodied Dipterans were found most frequently and had the highest relative abundance; hard-bodied prey items were absent from almost all samples. Substantial differences in taxa were found within season and between years, indicating the importance of multi-year sampling when defining a prey spectrum.

Potential effects of climate change on members of the Palaeotropical pitcher plant family Nepenthaceae.

Anthropogenic climate change is predicted to have profound effects on species distributions over the coming decades. In this paper, we used maximum entropy modelling (Maxent) to estimate the effects of projected changes in climate on extent of climatically-suitable habitat for two Nepenthes pitcher plant species in Borneo. The model results predicted an increase in area of climatically-suitable habitat for the lowland species Nepenthes rafflesiana by 2100; in contrast, the highland species Nepenthes tentaculata was predicted to undergo significant loss of climatically-suitable habitat over the same period. Based on the results of the models, we recommend that research be undertaken into practical mitigation strategies, as approximately two-thirds of Nepenthes are restricted to montane habitats. Highland species with narrow elevational ranges will be at particularly high risk, and investigation into possible mitigation strategies should be focused on them.

Capture mechanism in Palaeotropical pitcher plants (Nepenthaceae) is constrained by climate.

BACKGROUND AND AIMS: Nepenthes (Nepenthaceae, approx. 120 species) are carnivorous pitcher plants with a centre of diversity comprising the Philippines, Borneo, Sumatra and Sulawesi. Nepenthes pitchers use three main mechanisms for capturing prey: epicuticular waxes inside the pitcher; a wettable peristome (a collar-shaped structure around the opening); and viscoelastic fluid. Previous studies have provided evidence suggesting that the first mechanism may be more suited to seasonal climates, whereas the latter two might be more suited to perhumid environments. In this study, this idea was tested using climate envelope modelling. METHODS: A total of 94 species, comprising 1978 populations, were grouped by prey capture mechanism (large peristome, small peristome, waxy, waxless, viscoelastic, non-viscoelastic, 'wet' syndrome and 'dry' syndrome). Nineteen bioclimatic variables were used to model habitat suitability at approx. 1 km resolution for each group, using Maxent, a presence-only species distribution modelling program. KEY RESULTS: Prey capture groups putatively associated with perhumid conditions (large peristome, waxless, viscoelastic and 'wet' syndrome) had more restricted areas of probable habitat suitability than those associated putatively with less humid conditions (small peristome, waxy, non-viscoelastic and'dry' syndrome). Overall, the viscoelastic group showed the most restricted area of modelled suitable habitat. CONCLUSIONS: The current study is the first to demonstrate that the prey capture mechanism in a carnivorous plant is constrained by climate. Nepenthes species employing peristome-based and viscoelastic fluid-based capture are largely restricted to perhumid regions; in contrast, the wax-based mechanism allows successful capture in both perhumid and more seasonal areas. Possible reasons for the maintenance of peristome-based and viscoelastic fluid-based capture mechanisms in Nepenthes are discussed in relation to the costs and benefits associated with a given prey capture strategy.

Tuning of color contrast signals to visual sensitivity maxima of tree shrews by three Bornean highland Nepenthes species.

Three species of Nepenthes pitcher plants (Nepenthes rajah, Nepenthes lowii and Nepenthes macrophylla) specialize in harvesting nutrients from tree shrew excreta in their pitchers. In all three species, nectaries on the underside of the pitcher lid are the focus of the tree shrews' attention. Tree shrews are dichromats, with visual sensitivity in the blue and green wavebands. All three Nepenthes species were shown to produce visual signals, in which the underside of the pitcher lid (the area of highest nectar production) stood out in high contrast to the adjacent area on the pitcher (i.e., was brighter), in the blue and green wavebands visible to the tree shrews. N. rajah showed the tightest degree of "tuning," notably in the green waveband. Conversely, pitchers of Nepenthes burbidgeae, a typical insectivorous species sympatric with N. rajah, did not produce a color pattern tuned to tree shrew sensitivity maxima.

The use of light in prey capture by the tropical pitcher plant Nepenthes aristolochioides.

Nepenthes pitcher plants deploy tube-shaped pitchers to catch invertebrate prey; those of Nepenthes aristolochioides possess an unusual translucent dome. The hypothesis was tested that N. aristolochioides pitchers operate as light traps, by quantifying prey capture under three shade treatments. Flies are red-blind, with visual sensitivity maxima in the UV, blue, and green wavebands. Red celluloid filters were used to reduce the transmission of these wavebands into the interior of the pitchers. Those that were shaded at the rear showed a 3-fold reduction in Drosophila caught, relative to either unshaded control pitchers, or pitchers that were shaded at the front. Thus, light transmitted through the translucent dome is a fundamental component of N. aristolochioides' trapping mechanism.

A carnivorous plant fed by its ant symbiont: a unique multi-faceted nutritional mutualism.

Scarcity of essential nutrients has led plants to evolve alternative nutritional strategies, such as myrmecotrophy (ant-waste-derived nutrition) and carnivory (invertebrate predation). The carnivorous plant Nepenthes bicalcarata grows in the Bornean peatswamp forests and is believed to have a mutualistic relationship with its symbiotic ant Camponotus schmitzi. However, the benefits provided by the ant have not been quantified. We tested the hypothesis of a nutritional mutualism, using foliar isotopic and reflectance analyses and by comparing fitness-related traits between ant-inhabited and uninhabited plants. Plants inhabited by C. schmitzi produced more leaves of greater area and nitrogen content than unoccupied plants. The ants were estimated to provide a 200% increase in foliar nitrogen to adult plants. Inhabited plants also produced more and larger pitchers containing higher prey biomass. C. schmitzi-occupied pitchers differed qualitatively in containing C. schmitzi wastes and captured large ants and flying insects. Pitcher abortion rates were lower in inhabited plants partly because of herbivore deterrence as herbivory-aborted buds decreased with ant occupation rate. Lower abortion was also attributed to ant nutritional service. The ants had higher δ(15)N values than any tested prey, and foliar δ(15)N increased with ant occupation rate, confirming their predatory behaviour and demonstrating their direct contribution to the plant-recycled N. We estimated that N. bicalcarata derives on average 42% of its foliar N from C. schmitzi wastes, (76% in highly-occupied plants). According to the Structure Independent Pigment Index, plants without C. schmitzi were nutrient stressed compared to both occupied plants, and pitcher-lacking plants. This attests to the physiological cost of pitcher production and poor nutrient assimilation in the absence of the symbiont. Hence C. schmitzi contributes crucially to the nutrition of N. bicalcarata, via protection of assimilatory organs, enhancement of prey capture, and myrmecotrophy. This combination of carnivory and myrmecotrophy represents an outstanding strategy of nutrient sequestration.

The carnivorous syndrome in Nepenthes pitcher plants: current state of knowledge and potential future directions.

Nepenthes is the largest genus of pitcher plants, with its centre of diversity in SE Asia. The plants grow in substrates that are deficient in N and offset this deficiency by trapping animal prey, primarily arthropods. Recent research has provided new insights into the function of the pitchers, particularly with regard to prey tapping and retention. Species examined to date use combinations of wettable peristomes, wax layers and viscoelastic fluid to trap and retain prey. In many respects, this has redefined our understanding of the functioning of Nepenthes pitchers. In addition, recent research has shown that several Nepenthes species target specific groups of prey animals, or are even evolving away from a strictly carnivorous mode of operation. Future research into nutrient sequestration strategies and mechanisms of prey attraction would no doubt further enhance our knowledge of the ecology of this remarkable genus.

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 faeces and regularly defecate into the pitchers when they visit them to feed. Faeces 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.

Ion fluxes across the pitcher walls of three Bornean Nepenthes pitcher plant species: flux rates and gland distribution patterns reflect nitrogen sequestration strategies.

Nepenthes pitcher plant species differ in their prey capture strategies, prey capture rates, and pitcher longevity. In this study, it is investigated whether or not interspecific differences in nutrient sequestration strategy are reflected in the physiology and microstructure of the pitchers themselves. Using a non-invasive technique (MIFE), ion fluxes in pitchers of Nepenthes ampullaria Jack, Nepenthes bicalcarata Hook.f., and Nepenthes rafflesiana Jack were measured. Scanning electron microscopy was also used to characterize the distribution of glandular and other structures on the inner pitcher walls. The results demonstrate that nutrient sequestration strategy is indeed mirrored in pitcher physiology and microstructure. Species producing long-lived pitchers with low prey capture rates (N. ampullaria, N. bicalcarata) showed lower rates of NH(4)(+) uptake than N. rafflesiana, a species producing short-lived pitchers with high capture rates. Crucially, species dependent upon aquatic commensals (N. ampullaria, N. bicalcarata) actively manipulated H(+) fluxes to maintain less acid pitcher fluid than found in 'typical' species; in addition, these species lacked the lunate cells and epicuticular waxes characteristic of 'typical' insectivorous congeners. An unexpected finding was that ion fluxes occurred in the wax-covered, non-glandular zones in N. rafflesiana. The only candidates for active transport of aqueous ions in these zones appear to be the epidermal cells lying beneath the lunate cells, as these are the only sites not visibly coated with epicuticular waxes.

Trap geometry in three giant montane pitcher plant species from Borneo is a function of tree shrew body size.

*Three Bornean pitcher plant species, Nepenthes lowii, N. rajah and N. macrophylla, produce modified pitchers that 'capture' tree shrew faeces for nutritional benefit. Tree shrews (Tupaia montana) feed on exudates produced by glands on the inner surfaces of the pitcher lids and defecate into the pitchers. *Here, we tested the hypothesis that pitcher geometry in these species is related to tree shrew body size by comparing the pitcher characteristics with those of five other 'typical' (arthropod-trapping) Nepenthes species. *We found that only pitchers with large orifices and lids that are concave, elongated and oriented approximately at right angles to the orifice capture faeces. The distance from the tree shrews' food source (that is, the lid nectar glands) to the front of the pitcher orifice precisely matches the head plus body length of T. montana in the faeces-trapping species, and is a function of orifice size and the angle of lid reflexion. *Substantial changes to nutrient acquisition strategies in carnivorous plants may occur through simple modifications to trap geometry. This extraordinary plant-animal interaction adds to a growing body of evidence that Nepenthes represents a candidate model for adaptive radiation with regard to nitrogen sequestration strategies.

Tree shrew lavatories: a novel nitrogen sequestration strategy in a tropical pitcher plant.

Nepenthes pitcher plants are typically carnivorous, producing pitchers with varying combinations of epicuticular wax crystals, viscoelastic fluids and slippery peristomes to trap arthropod prey, especially ants. However, ant densities are low in tropical montane habitats, thereby limiting the potential benefits of the carnivorous syndrome. Nepenthes lowii, a montane species from Borneo, produces two types of pitchers that differ greatly in form and function. Pitchers produced by immature plants conform to the 'typical' Nepenthes pattern, catching arthropod prey. However, pitchers produced by mature N. lowii plants lack the features associated with carnivory and are instead visited by tree shrews, which defaecate into them after feeding on exudates that accumulate on the pitcher lid. We tested the hypothesis that tree shrew faeces represent a significant nitrogen (N) source for N. lowii, finding that it accounts for between 57 and 100 per cent of foliar N in mature N. lowii plants. Thus, N. lowii employs a diversified N sequestration strategy, gaining access to a N source that is not available to sympatric congeners. The interaction between N. lowii and tree shrews appears to be a mutualism based on the exchange of food sources that are scarce in their montane habitat.