Using eDNA to experimentally test ungulate browsing preferences
© Nichols et al. 2015
Received: 13 May 2015
Accepted: 28 August 2015
Published: 10 September 2015
Large herbivores may affect ecosystem processes and states, but such effects can be difficult to quantify, especially within multispecies assemblages. To better understand such processes and improve our predictive ability of systems undergoing change, herbivore diets can be studied using controlled feeding trials (or cafeteria tests). With some wildlife, such as large herbivores, it is impractical to empirically verify these findings, because it requires visually observing animals in forested environments, which can disturb them from their natural behaviors. Yet, in field-based cafeteria trials it is nearly impossible to differentiate selection between herbivore species that forage on similar plants and make very similar bite marks. However, during browsing ungulates leave saliva residue which includes some buccal cells and DNA that can be extracted for species identification. Here we used a newly developed eDNA-based method (biteDNA) to test the browsing preferences of four sympatric ungulate species in the wild. Overall, food preferences varied between species, but all species strongly preferred deciduous over coniferous species. Our method allows the study of plant-animal interactions in multispecies assemblages at very fine detail.
In some areas, particularly across Europe and North America, ungulates are currently expanding their population sizes and ranges. These increases can cause dramatic changes to the landscape (Cote et al. 2004). Such changes can be difficult to predict, especially in multi-species assemblages containing several ungulate species with different feeding preferences. Such plant-herbivore relationships are important to understand in the context of trophic interactions, rewilding and species invasions. In forest ecosystems, for example, it is important to understand which trees ungulates prefer and how such preferences may affect plant succession and forestry operations. Dietary studies of such relationships are often done using feeding trials (also called cafeteria tests) where different foods are presented to an animal which then makes a choice as to what it will eat. However, cafeteria tests on wild ungulates are usually only practical where a single species occurs in the area. Ungulate bites tend to look the same and direct observations of the animals’ choices may be difficult or time consuming. Thus, many cafeteria studies are done with ungulates in captivity (Bergvall et al. 2006; Renaud et al. 2003; Shipley and Spalinger 1995). In some field studies, sites are assumed to be visited only by the predominant species, despite other species also being present (Jia et al. 1995; Milligan and Koricheva 2013; van Beest et al. 2010). DNA technology (Taberlet et al. 2012) makes it easier to study cryptic animal behaviors, such as herbivory. Herbivory by browsing herbivores can be cryptic when they are foraging in heavily forested environments with low visibility. We recently developed a new tool to study ungulate browsing based on the fact that when ungulates chew on shrubs or tree branches, they tend to leave behind enough saliva and environmental DNA (eDNA) for species identification (Nichols et al. 2012, 2015; Nichols and Spong 2014). Here, we expanded upon classical cafeteria tests by incorporating our browsed twig environmental DNA (biteDNA) method to differentiate the bites made by four ungulate species on artificial stands composed of three tree species. Thus, this is the first experimental test of foraging preferences using the biteDNA technique.
Our study was performed in an area where moose (Alces alces), roe deer (Capreolus capreolus), fallow deer (Cervus dama), and red deer (Cervus elaphus) co-occur. Moose and roe deer are known to browse extensively whereas red deer and fallow deer are mixed feeders, but fallow deer are known to fall closer to the grazer end of the spectrum (Hofmann 1989). In general, these ungulate species tend to overlap in their use of resources (Latham 1999; Mysterud 2000). During our study period (winter), with an abundance of snow, the animals only available food was browse and supplementary fodder provided by the landowners. In our cafeteria test, we mimicked stands of Scots pine (Pinus sylvestris), European aspen (Populus tremula) and goat willow (Salix caprea) to test the foraging preferences of these forest ungulates. After exposing our stands to the wild ungulates for two weeks, we collected browsed twig samples and used the biteDNA method to identify which ungulate species had browsed each twig.
Results and discussion
144 out of 463 (31 %) browsed twig samples amplified DNA during PCR. All deer species were found to browse at our experimental plots. In our previous biteDNA studies, our amplification success has reached 80 % (Nichols and Spong 2014). We suggest that the freeze–thaw cycles during the study period are a reason behind the low amplification success rate. The samples that failed in our current study were evenly distributed across tree species and height classes. The freeze–thaw cycles may have broken off the buds on the shoots of the willow and aspen making them appear to be bites when they actually were not. Therefore, we caution other users of the biteDNA method to be aware of this. However, this also reveals a strength of the method we used; had we not used biteDNA, we might have overestimated browsing intensity and visitation rates to our cafeteria stations.
Cafeteria tests, like this, provide a way to empirically verify past studies and test ecological theories and this study is the first to experimentally test the browsing preferences of wild ungulates using biteDNA. This method is useful for studying cryptic browsing in particular. When presented with a choice to forage on willow and aspen versus pine (mimicking natural tree stands) all deer species tended to not use pine. In addition, we found that the deer species had different preferences for willow and aspen. Moose and roe deer preferred goat willow whereas red deer showed no clear preference. These preferences have implications in the area of forest management. The knowledge of deer preferences for certain trees may allow forest managers to better predict forest damages and successional stages based on the amount and species’ of trees available to ungulates.
Density estimates according to offtake levels and observations by professional hunters (numbers per 1000 hectares) (Svenska Jägareförbundet, pers comm)
Collection of all browsing samples followed the protocols in Nichols et al. (2012). Briefly, the top 1.5–2 cm of browsed twigs were clipped into two ml centrifuge tubes filled with phosphate-buffered saline solution. We transported these samples at ambient temperatures until they reached our lab (within 1 week) and were then stored at −20 °C until DNA extraction. DNA extraction followed the final (robotic) extraction protocol and all PCR amplifications and fragment analyses followed the protocols found in Nichols et al. (2012).
To test the preferences for tree species we used χ2 tests to see if the distribution in numbers of bites was significantly different from an even distribution. By ‘even distribution’ we mean that if a deer made 50 bites total, an even distribution that indicated no preference between willow and aspen would have been 25 bites on willow and 25 bites on aspen. We used JMP 9.0 for these analyses (SAS Institute Inc., Cary, NC, USA).
RVN and GS designed the experiment and performed the field-work. RVN did the laboratory work and statistical analyses. JPGMC contributed to the conceptual basis and data analysis of the manuscript. All authors contributed to writing the manuscript; RVN took the lead. All authors read and approved the final manuscript.
Funding came from the Swedish Research Council Formas. J.P.G.M.C. was supported by the Swedish thematic research program Wildlife and Forestry and the EU Marie Curie Career Integration Grant ‘HOTSPOT’ (PCIG10-GA-2011-304128). Anita Norman assisted during sample collection and Helena Königsson assisted during laboratory work.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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