Temporary Translocation of Entire Mistletoe Plants to Understand the Mechanistic Basis of Animal Foraging Decisions

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 manipulated^1,2. 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

1. Choose the ecosystem type and study location.
   1. Look for a suitable site with abundant mistletoes, one regular mistletoe host, and at least one species of tree/shrub that does not normally host mistletoe.
      NOTE: The case study was carried out in a semi-arid woodland, New South Wales, Australia.
2. Identify the ecologically relevant time of year for the study.
   1. Predetermine when mistletoes are fruiting. Consider that this might be different times of the year, depending on the seasonal profile of a given area.
      NOTE: In this case, mistletoe species fruit for some months over spring-summer.
3. Choose a patch size that satisfies the research question.
   1. If the species being observed is territorial, choose a patch size that reflects that.
   2. If the mistletoe species abundance is patchy, choose several patches and be prepared to reflect the variance in the statistical analysis.
4. Identify the dominant species in the chosen ecosystem and location.
   NOTE: At the case study site, the dominant tree canopy species comprised Callitris glaucophylla (White Cypress Pine), Acacia homalophylla (Yarran), and Casuarina cristata (Belah), with subdominant stands of Allocasuarina luehmannii (Buloke) and Eucalyptus populneus (Bimble box). Amyema quandang (Grey Mistletoe; Loranthaceae) is the principal mistletoe in the area, growing almost exclusively on Acacia homalophylla (Yarran) at the study site.
5. Identify the target mistletoe and become familiar with the animals that forage on it.
   NOTE: For example, in the case study, Grey Mistletoe produces pale-yellow fleshy fruits¹² that are eaten by two mistletoe specialist frugivores (Mistletoebird, Dicaeidae, Dicaeum hirundinaceum, and Painted Honeyeater, Meliphagidae, Grantiella picta) and four generalist frugivores (Silvereye, Zosterops lateralis; Spiny-cheeked Honeyeater, Acanthagenys rufogularis; Singing Honeyeater, Lichenstomus virescens; Striped Honeyeater, Plectorhyncha lanceolata), with numerous other species opportunistically consuming the fruits and occasionally dispersing the seeds^13,12.
6. Choose the ideal number of replicates for the study, considering the total number of days required to complete each 2-day replicate trials. To reduce the number of days that the experiment will run, have an observer observe two replicates simultaneously, with sufficient distance between the two replicates to minimize interference.
   NOTE: In the case study, data for 20 replicates were collected for each of the three relocation treatments (60 individual mistletoes), over the course of 60 days, with 1 day of observation per replicate, randomized across treatments.
7. Conduct a pilot study to prolong the vigor of the mistletoe once cut from the host plant by comparing visitation to mistletoes with and without the cut ends sealed with glue.
   1. If there is no difference in terms of either wilting or bird visitation for the 12 h duration of each trial, consider the mistletoe as retaining sufficient vigor until late afternoon.
   2. If bird visitation is significantly lower to cut mistletoes, select a different mistletoe species and/or a more humid environment where evaporation is slower.
8. Ensure that all the relevant permissions are in place, both to collect native plants and to observe wildlife. Since this protocol involves cutting live mistletoe from the canopy, avoid work in populations of mistletoe of conservation concern. Further, given the reliance of many animals on mistletoe as both a food source and a nesting/roosting location, ensure that the experiment does not cause a lasting disruption to the ecological community under investigation.
   1. Obtain appropriate permissions from the relevant government agency and consult the animal care and ethics committee of the researcher's institution, balancing any short-term impacts with the scientific merit of the proposed study. Note that no wildlife handling is explicitly required for this method.

2. Identify target individual mistletoe-host pairs

1. At least 1 day prior to conducting the experiment, locate suitable mistletoe plants on the appropriate hosts and, if relevant, appropriate pseudo-hosts or novel hosts.
   1. When selecting locations to affix the mistletoe, ensure the branch is sufficiently strong to hold the weight of the mistletoe.
   2. When selecting a target individual mistletoe, consider the thickness of the host branch and whether the selected mistletoe is growing at the terminal end of the branch or midway along.
      1. Do not select host branches above 70 mm diameter or mistletoes growing midway along a host branch that still bears host foliage. Alternatively, prune such branches above the haustorium to avoid difficulty in cutting the host branch or transporting the host foliage along with the mistletoe.
   3. Select the new host location that will allow for affixing the mistletoe in the same orientation as it grew, e.g., if its branches all droop down in a tear drop shape, ensure that they do so at the new location as well (Figure 2).
   4. Inspect each candidate mistletoe closely to ensure that no active nests are located within them or near them.
   5. Choose mistletoe plants that can be safely reached and removed from their host before dawn. If ladders are to be used, ensure that the ground beneath each tree is clear of snakes, animal burrows, and obstructions.
   6. Note the phenology (i.e., presence of ripe fruit or open flowers).
2. Record details of experimental plants. Mark the target plant pair unobtrusively to avoid disturbing animals, e.g., an inconspicuous fabric tag, a stick, or stake in the ground close by or GPS coordinates.

3. Cutting the mistletoe

1. At least 1 h before dawn on the day of observations, remove the mistletoe from its host using a clean pruning saw.
   1. Depending on the branching pattern of the mistletoe, cut either side of the haustorium but cut proximal (i.e., upstream) to the connection with the host and remove the entire mistletoe.
   2. Take care when cutting, undercutting first to minimize damage to the host tree. Remember to be well-positioned and/or have a second person assisting in this process, as the abscised mistletoe may be heavier than anticipated.
      1. For larger mistletoe plants, wind a length of rope around the proximal portion of the host branch (between the trunk and haustorium) before tying it securely to the mistletoe prior to abscission, enabling the plant to be lowered safely to the ground without losing branches that are characteristically brittle.
   3. Thoroughly clean the saw with ethanol after each individual mistletoe removal.

4. Attaching (pasting) the mistletoe

1. Once the mistletoe is removed, affix it to the final location using black cable-ties. Make sure that the mistletoe does not swing unnaturally in the wind or fall off if a larger animal lands on it. Make the cable ties as inconspicuous as possible, cut off long ends, and leave no rubbish behind for curious animals to find.
2. As described in step 2.1.3, ensure that the relocated plant is secured in an orientation similar to its original growth habit.

5. Collect visitation data

1. In addition to noting animal species and the duration of visit, collect behavioral data for distinguishing different kinds of visits, including actively foraging for insects, visiting and probing flowers, taking fruit, agonistic interactions, and loafing. Use timed watches with binoculars or with motion-activated cameras mounted the night before.
   1. If using cameras, conduct initial trials using different sensitivity settings and locations to minimize false triggering.
   2. For timed watches, simultaneously observe multiple mistletoes from the one vantage point. During this period, record every visit to the relocated mistletoes by direct observation from a distance of 5–10 m, noting the identity of each bird and the duration of each visit (as per¹¹). Divide the visiting species into three diet-based functional groups.
      NOTE: This case study used this method, with the observation period of approximately 6.5 h between 7:30 a.m. and 6:30 p.m., in two blocks over the morning and afternoon, avoiding the heat of the day where there was little bird activity while still capturing peak foraging activity^14,15.

6. Collect contextual data on the location of mistletoe plants

1. In addition to noting whether each plant is a control (i.e., cut and returned to its original location) or a relocated plant, record attributes of the host (species, height, diameter), mistletoe (size, foliage density, phenology, height, aspect, number of fruits), and context (distance to nearest mistletoe, distance to other fruiting / flowering plants).
2. Use photo-point monitoring of both mistletoe and pseudo-host as an effective complement to conventional quantitative data collection, with image analysis software readily able to generate estimates of canopy closure and other physiognomic attributes.

7. End-of-observation tasks

1. For seed dispersal studies, estimate the number of fruits removed by counting the total number of ripe fruits before and after the experimental period. Check the ground for fruit caps or fallen fruits before removing the mistletoe.
2. At the end of every data collection day, once visitation data have been collected, remove the relocated mistletoe and collect all the cable ties and any flagging tape or tags.

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 (after^18,19). Four variables were included as covariates: host height, host canopy cover, number of mistletoes on the host, and number of fruits on the treatment mistletoe. When just treatment was included in the model, there was a significant difference in the number of visits by birds to each of the mistletoe relocation treatments. The number of visits to the original host mistletoes was significantly higher than to either mistletoes relocated to pseudo-hosts or novel hosts (Table 1A). When the covariates, host canopy cover, and the number of fruits were included, the difference remained significant, but the habitat characteristics did not influence visitation (Table 1B). Thus, across all birds, spatial cues were more important than resource density, accessibility, or apparency, consistent with the inference that prior experience is more influential than proximate sensory cues in finding ripe fruit.

Table 1: Models exploring the influence of treatment on the number of visits to treatment mistletoes. (A) Generalized linear models; Model (B) includes the host canopy cover and the number of fruits on the treatment mistletoes as covariates. Treatment mistletoes comprised one of the three treatments: In situ (IS), cut from the original host tree and reattached back in the same tree; Same Species (SS), moved to another pseudo-host tree of the same species as the original host; Different Species (DS), moved to a novel host species that never hosts Grey Mistletoe. Significance is shown in bold.

Table 2: Generalized linear models for models comparing the number of visits to treatment mistletoes across dietary guilds, including various covariates. (A) overall dietary guild; (B) Specialist; (C) Generalists; (D) Opportunists. Treatment mistletoes were cut from their original host tree and either replaced in the same tree (In situ, IS), placed in a pseudo-host host of the same species as the original host (Same Species, SS), or placed in a novel host species that does not host mistletoe (Different Species, DS). Models used a Poisson error distribution with a log-linear link, unless otherwise indicated. Significance is shown in bold. * negative binomial with the log link.

To determine whether the dietary breadth of birds influenced their search strategy to locate fruiting mistletoe, dietary functional group (mistletoe specialist, generalist frugivore, and opportunist) were included as a second predictor alongside treatment. A median test was conducted to determine whether the three dietary guilds differed in the number of visits made to the treatment mistletoes. Further analyses were conducted separately for each functional group using a Poisson GzLM with a loglinear link, models created initially including the four selected covariates to find the best model. The resultant guild models were then compared for the overall fit to determine the set of covariates that were most influential for each different dietary group.

The specialist dietary guild included one species: Mistletoebird. The generalist frugivore guild included four species: Spiny-cheeked Honeyeater, Silvereye, Singing Honeyeater, and Striped Honeyeater. The opportunist guild included nine species: Splendid Fairy Wren, Inland Thornbill, Yellow Thornbill, Rufous Whistler, Australian (Mallee) Ringneck, Double-barred Finch, Grey Shrike-thrush, Noisy Miner, and Red-capped Robin. The mistletoe specialist visited treatment mistletoes on 19 occasions: there were 19 visits by generalist frugivores and 34 by opportunists. Visitation by the three guilds differed significantly after accounting for visits to treatment mistletoes, and there was a significant interaction between guild and treatment (Table 2A). The generalists visited the original host mistletoes significantly more than the pseudo-host or novel host mistletoes.

Specialist visitation did not significantly differ among treatments; however, the number of fruits was positively related to visitation frequency (P = 0.001; Table 2B). The number of fruits on treatment mistletoes was not significantly different across treatments (one-way ANOVA: F _{(2, 56)} = 0.266, P = 0.768).

The individual model for generalist frugivores excluded novel host treatment mistletoes as no visits by generalists were recorded at those mistletoes by this dietary group (Table 2B). The best model included host canopy cover and the number of mistletoes on the host tree as covariates. Generalist frugivores visited the original host mistletoes significantly more than they visited the pseudo-host mistletoes (Table 2C). The percentage of host canopy cover and the number of mistletoes on the host tree significantly influenced the visiting generalists as main effects (Table 2C).

The best individual model for opportunists included host height and host canopy cover as covariates. Opportunists visited original host mistletoes significantly more than they visited the pseudo-host or novel host mistletoes and were significantly influenced by the height and canopy cover of the host tree (Table 2D).

Figure 1: Grey Mistletoe (Amyema quandang) in different stages of removal. (A) A mistletoe being removed from its host by cutting below the haustorium with a cleaned pruning saw, and (B) detail of the same mistletoe removed from the host complete with the distal end of the host branch. (C) The three main branches of another, larger mistletoe clump removed from the haustorium; (D) Detail of the three branches showing the cable ties affixing them to one another. Please click here to view a larger version of this figure.

Figure 2: Grey Mistletoe (Amyema quandang) in three different treatments. (A) In situ mistletoe before being removed from its Yarran (Acacia homalophylla) host, and then replaced in its original location. (B,C) Different species treatment mistletoe after being removed from its original host and relocated in this White Cypress tree (Callitris glaucophylla). (D) Same species mistletoe after being removed from its original host and relocated to a new Yarran tree already hosting other mistletoes. Treatment mistletoes circled in red. Please click here to view a larger version of this figure.

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 used more proximate cues of resource availability regardless of the spatial context.

These findings lead to the next question of how and when do foraging birds develop this memory, and how much of a role does it play in the pattern of seed dispersal that drives the spatial pattern of mistletoe occurrence in the landscape? Although this case study used direct observation to collect visitation data, the protocol described herein could be readily applied using motion-triggered cameras^20,21, allowing simultaneous monitoring of multiple sets of mistletoes and yielding new insights into between-species, -guild, -habitat, and -biome differences.

Several refinements are worth considering to maximize data quality, comparability, and ease of application. First, recognize that wild animals can respond both positively and negatively to novelty; hence, be careful to minimize any extraneous changes to their environment both in establishing the experiment and during data collection. By conducting mistletoe relocation predawn, any disturbance to diurnal animals will be minimized. Although logistically more challenging, it appeared that birds were more likely to visit plants if they had been affixed in the dark²², presumably due to disturbance from movement. Second, minor details can be surprisingly important. Trim off the ends of the zip ties, wear dull clothing, and keep all movements to a minimum, especially during data collection. For studies on fruit removal and seed dispersal, as ripe fruits are readily knocked off the peduncle, count fruits after the mistletoes are relocated. Additionally, check the ground after each period of collecting visitation data for fruit caps on the ground, indicative of fruit removal.

Although these representative results and overarching question are related to fruit removal, this protocol could be readily applied to address questions regarding nectarivores and pollination or folivores and arboreal herbivory. In addition to manipulating mistletoe locations (e.g., high vs low to quantify herbivory from ground-based vs arboreal herbivores; in situ vs translocated mistletoes to quantify the influence of resident and transient nectarivores in effecting short- and long-distance pollen transport), resource density can also be manipulated. Thus, by making high- and low-resource density patches by manipulating mistletoe densities and/or flower/fruit/leaf numbers, different resource-use strategies can be discriminated. By integrating these experiments with before and after measurements of the relevant resource, giving up densities can also be estimated, enabling resultant inferences regarding foraging ecology to be contextualized within the broader framework of habitat preferences and predator vigilance.

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.