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This work outlines a simple experimental procedure to quantify behavioral drivers of foraging decisions in free-living animals, temporarily relocating mistletoe plants to novel locations and measuring visitation rates.
Fruiting mistletoes present a model system for understanding decisions made by foraging animals when locating food. Where, when, and how animals find food is central to many ecological questions, relating to the basis of individual foraging decisions and the extent to which these decisions are innate or acquired. Ecologists have paid particular attention to frugivores, quantifying their preference for fruits with specific shapes, colors, or scents, which, over evolutionary time, confer selection for suites of traits in their favored plants whose seeds they disperse.
This work outlines a novel experimental approach to manipulating food plant occurrence and measuring the response of wild, free-living animals, ideally suited to studying the evolutionary origin and ecological maintenance of seed dispersal. This "cut and paste" protocol involves removing an entire fruiting mistletoe plant from its host and either returning it to its original location or moving it to a novel location, affixing it to a 'pseudo-host' of the same or different tree species. By counting visits to the mistletoe and noting the duration, species, and behaviors, a series of comparisons can discern the most important factors affecting foraging decisions and the consequences for both plant and animal. Here, the protocol is illustrated with a case study to determine between-guild differences in mistletoe frugivory.
The experimental approach teases apart the mechanistic basis of search image formation and refinement, spatial learning, interspecific differences in foraging strategies, and how these changes modify seed dispersal effectiveness. Finally, potential modifications are considered with respect to addressing other questions on foraging ecology, plant-animal interactions, and coevolution.
How do animals find food? This is a deceptively simple question, integrating cognition, sensory perception, and metabolic demands with habitat structure, interspecific interactions, and variation in resource availability through space and time. Most of the conceptual advances in the understanding of this topic have come from studying captive animals, where resource quality, quantity, and accessibility can be manipulated1,2. While useful for establishing sensory capabilities, qualitative preferences, and nutritional qualities of food, captive methods do not reveal how animals fulfill these demands in the wild.
Early experimental studies on resource use by free-living animals sought to understand the lower bound of food availability an organism will reach before deciding to feed elsewhere (Charnov's marginal value theorem3). Known as "giving up density," this approach quantifies how much risk an animal is willing to tolerate - e.g., how few acorns per square meter a squirrel is prepared to leave behind when feeding in woodlands of differing densities where predators are variously detectable4. While this framework has been applied to a wide range of food resources and ecosystems, the necessarily constructed basis of the approach limits its application and can confound the interpretation of reported differences5. Further, determinants of giving up density relate more to vigilance, habitat preferences, and competition than foraging ecology (known collectively as the ecology of fear6). This approach is rarely able to capture the attractiveness of a food resource in the wild to a free-living animal. Hence, studies on frugivory are usually based on the observation of wild behavior with implications for both plant and animal being drawn from the resulting behaviors.
Foraging decisions made by frugivores when selecting fruit may hinge on many different traits expressed physically by the plant in terms of abundance, quality, and seasonal availability. How easy the fruits are to locate, consume, and pass through the gut also play a role in the selection by frugivores, making it tricky to separate the potentially learned behavior from the inherited. The current work introduces a new approach to manipulating resource availability and location to measure the response of wild, free-living animals as they forage in their natural habitat. This method is ideally suited to addressing questions regarding the cues different animals use to locate food—in the case showcased here, the energy-rich fruit of hemiparasitic mistletoe plants. The approach involves removing entire mistletoe plants from their host trees and relocating them to other trees of the same or different species.
Note that the case study presented focuses on fruit, frugivores, and the interaction between dietary breadth and the implications for seed dispersal. However, for work on nectarivores or folivores, the same approach can be applied to flowering mistletoes or non-reproductive mistletoes, respectively. Mistletoes are an ideal model to use for this approach, being found in woodlands and forests worldwide and visited by a wide range of animals7. In terms of fruit, although most research has focused on mistletoe fruit specialists that eat little else8, a large range of generalist frugivores and opportunists with a broader diet regularly consume mistletoe fruit9. Finally, their size, growth habit, and physiognomy make them especially amenable to experimental manipulation10.
Research in a semi-arid woodland system demonstrated that foliage density affected the apparency of mistletoes to fruit-eating birds11, but numerous questions remain unanswered. Do birds search for mistletoes or fruiting mistletoes? For those mistletoe populations dependent on a single host species, do birds preferentially search for the mistletoe or for their principal host? Do groups that forage primarily, occasionally, or opportunistically on mistletoe fruit use divergent cues to find mistletoe fruit?
To answer these questions and uncouple the influence of host identity, spatial context, and mistletoe location on bird visitation, a novel relocation protocol was devised; that experiment is used as a case study. The protocol is illustrated with step-by-step instructions to determine how birds locate fruit in a structurally complex woodland. In addition to exploring other questions readily addressed using this technique, consideration is given to how this method could be integrated with other ecological field methods to understand the mechanistic basis of foraging ecology in forest and woodland canopies.
The initial application of this experimental approach was to determine how birds find food in a heterogeneous woodland canopy by relocating entire mistletoe plants. This protocol spans 2 days—selecting the mistletoe on day 1 to manipulation, then affixing, observing, and detaching the mistletoe on day 2. Conduct replicate trials on successive days; select the mistletoe for the next trial on the second day of the first trial. In the illustrative case study, bird visitation to mistletoes was compared among three different host locations, referred to as treatments here.
To do this, a single fruiting Grey Mistletoe (Amyema quandang) plant was cut from its host plant and attached to one of the three locations: 1) its original host tree, 2) a pseudo-host tree, or 3) a novel host tree. The original host treatment maintained both the spatial location and the host identity constant while controlling for the effects of cutting. The psuedo-host treatment involved temporarily affixing the mistletoe to a different individual of the same species as the host (in this case study, Yarran (Acacia homalophylla)) but with few to no existing mistletoes to discern the roles of spatial memory versus host-association. The novel host, an individual of a different tree species that does not host mistletoes (for the case study site, White Cypress Pine (Callitris glaucophylla)) clarified whether the search image used by mistletoe fruit consumers relates to the mistletoe itself or the principal host.
This experimental protocol was developed and experimentally trialled under the provision of and abiding by the Animal Research Authority guidelines of the University of Technology Sydney (UTS ACEC 2013-745). The protocol does not require handling of animals. Native plants were experimentally manipulated under the permission of a National Parks and Wildlife scientific licence (SL101337).
1. Determine suitable site, species, and ethical considerations
2. Identify target individual mistletoe-host pairs
3. Cutting the mistletoe
4. Attaching (pasting) the mistletoe
5. Collect visitation data
6. Collect contextual data on the location of mistletoe plants
7. End-of-observation tasks
Data amounting to a total of 392 h of observation were collected across the 60 replicates, with 26 of the replicate mistletoes receiving visits from 15 species of birds. To determine whether the visiting birds preferred one treatment over another, visitation data were analyzed using generalized linear models (GzLMs)17 with negative binomial distributions (after18,19). Four variables were included as covariates: host height, host canopy cov...
This novel method represents a cost-effective means of understanding the mechanistic basis of foraging differences among species and feeding guilds, revealing the critical role of prior learning and spatial awareness in determining how birds find ripe fruit in structurally complex environments. By uncoupling spatial location from other proximate cues, it was possible to demonstrate that generalist frugivores visit plants in known locations, rather than relying on associations with particular habitats, whereas specialists...
The authors acknowledge that they have no competing financial interests in the pursuit or publication of this research.
The authors recognize John Rawsthorne for initially proposing the cut and paste protocol. Many thanks to the numerous volunteers who dedicated their time to observing the birds. This research was funded by the University of Technology Sydney, Charles Sturt University, Birdlife Australia, and the Ecological Society of Australia as part of a Masters (research) degree.
Name | Company | Catalog Number | Comments |
Alcohol cleansing pads | Forestry Suppliers | 25557 | SmartCompliance First Aid Cabinet Refill |
Ladder | Forestry Suppliers | 90905 | Telesteps 12.5’ Telescopic Ladder |
Motion-triggered camera | Forestry Suppliers | 91269 | Reconyx HF2X HyperFire 2 Camera |
Nylon cable ties | Forestry Suppliers | 17032 | Black is the preferred color |
Pruning Saw | Forestry Suppliers | 81154 | Folding model is preferred to minimize injury, with pole mounted saws advisable if ladders cannot be used to accesss high plants |
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