vol. 193, no. 2 the american naturalist february 2019 Natural History Note Testing Darwin’s Hypothesis about the Wonderful Venus Flytrap: Marginal Spikes Form a “Horrid q1 Prison” for Moderate-Sized Insect Prey Alexander L. Davis,1 Matthew H. Babb,1 Matthew C. Lowe,1 Adam T. Yeh,1 Brandon T. Lee,1 and Christopher H. Martin1,2,* 1. Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599; 2. Department of Integrative Biology and Museum of Vertebrate Zoology, University of California, Berkeley, California 94720 Submitted May 8, 2018; Accepted September 24, 2018; Electronically published Month XX, 2018 Dryad data: https://dx.doi.org/10.5061/dryad.h8401kn. abstract: Botanical carnivory is a novel feeding strategy associated providing new ecological opportunities (Wainwright et al. with numerous physiological and morphological adaptations. How- 2012; Maia et al. 2013; Martin and Wainwright 2013; Stroud ever, the benefits of these novel carnivorous traits are rarely tested. and Losos 2016). Despite the importance of these traits, our We used field observations, lab experiments, and a seminatural ex- understanding of the adaptive value of novel structures is of- periment to test prey capture function of the marginal spikes on snap ten assumed and rarely directly tested. Frequently, this is be- traps of the Venus flytrap (Dionaea muscipula). Our field and labora- cause it is difficult or impossible to manipulate the trait with- fi tory results suggested inef cient capture success: fewer than one in four out impairing organismal function in an unintended way; prey encounters led to prey capture. Removing the marginal spikes de- creased the rate of prey capture success for moderate-sized cricket prey however, many carnivorous plant traits do not present this by 90%, but this effect disappeared for larger prey. The nonlinear ben- obstacle. efit of spikes suggests that they provide a better cage for capturing more Botanical carnivory is a novel feeding strategy that has abundant insects of moderate and small sizes, but they may also pro- evolved at least nine separate times in over 700 species of vide a foothold for rare large prey to escape. Our observations support angiosperms, typically in areas with severely limited nitrogen Darwin’s hypothesis that the marginal spikes form a “horrid prison” and phosphorus (Roberts and Oosting 1958; Givnish et al. that increases prey capture success for moderate-sized prey, but the de- 1984; Ellison 2006; Król et al. 2012; Givnish 2015). Pitfall creasing benefit for larger prey is unexpected and previously undocu- fi traps evolved independently at least six times and sticky traps mented. Thus, we nd surprising complexity in the adaptive landscape fi for one of the most wonderful evolutionary innovations among all ve times. However, snap traps have most likely evolved only plants. These findings enrich understanding of the evolution and diver- once in the ancestral lineage leading to the aquatic waterwheel sification of novel trap morphology in carnivorous plants. (Aldrovandra vesiculosa)andVenusflytrap (Dionaea musci- pula), which is sister to the sundews (Drosera spp.) and within Keywords: snap trap, carnivorous plant, prey capture performance, novelty, key innovation, exaptation. the Caryophyllales (Cameron 2002; Givnish 2015; Walker et al. 2017). Multiple hypotheses have been proposed for why snap traps evolved, including the ability to capture larger Introduction prey, capture prey more quickly, or more completely digest prey (Darwin 1875; Gibson and Waller 2009). However, The origins of novel structures remain an important and these hypotheses have rarely been tested except for a few field poorly understood problem in evolutionary biology (Mayr studies documenting the size and diversity of arthropod q2 1960; Mozcek 2008). Novel traits are often key innovations prey (Jones 1923; Gibson 1991; Hutchens and Luken 2009; Youngsteadt et al. 2018). * Corresponding author; email: [email protected]. The marginal spikes found in Dionaea are modified tri- ORCIDs: Davis, http://orcid.org/0000-0003-0614-1376; Lowe, http://orcid chomes that extend from the margin of the trap lobes. These .org/0000-0002-8996-3895; Yeh, http://orcid.org/0000-0002-9959-2688; Mar- tin, http://orcid.org/0000-0001-7989-9124. spikes are homologous to the trichomes of sundews but do Am. Nat. 2019. Vol. 193, pp. 000–000. q 2018 by The University of Chicago. not exude any sticky resin and have lost the mucus glands 0003-0147/2019/19302-58452$15.00. All rights reserved. in these spikes (Gibson and Waller 2009). Darwin was the DOI: 10.1086/701433 first to document evidence for carnivory in flytraps and 58452.proof.3d 1 Achorn International 12/11/18 03:12 000 The American Naturalist sundews in a series of careful experiments and proposed that flashlight to illuminate the trap from behind, making any- the marginal spikes of flytraps enhance prey capture success thing inside the trap visible as a silhouette. If the trap contained by providing a cage-like structure around the top of the trap something, it was assigned a value of 1 for “catch,” and if it that contains the prey (Darwin 1875; Gibson and Waller contained nothing, it was assigned a 0 for “miss.” We also 2009). Darwin (1875) also hypothesized that while small noted when a trap was closed on another trap or contained insects will be able to escape between the spikes, a moderately debris inside, such as sticks or grass (these were considered sized insect will be “pushed back again into its horrid prison amiss;n p 7). Both logistic regression and a generalized lin- with closing walls” (p. 312), and large, strong insects will be ear mixed effects model (GLMM; package lme4; Bates et al. able to free themselves. Determining the function of the mar- 2015) in R using RStudio (RStudio Team 2015; R Statistical ginalspikesisimportantforunderstandingtherarityofme- Programming Group 2018) were used to determine whether q3 chanical snap traps. trap length had a significant effect on prey capture rate in Traits that enhance prey capture ability are expected to be the field. strongly selected for given the benefits of additional nutrients and the energetic and opportunity costs associated with a Laboratory Prey Capture Experiments triggered trap missing its intended prey. The marginal spikes provide a novel function that potentially increases prey Plants used in lab experiments were tissue cultured and pur- capture rate and minimizes the costs associated with a failed chased from commercial suppliers (http://bugbitingplants trap-closing event. Nutrients from insect prey increase the .com; http://stores.ebay.com/joelscarnivorousplants). The growth rate of Venus flytraps (Darwin 1878; Roberts and plants were maintained in 40-L terraria under high-output Oosting 1958) at a cost of lower photosynthetic efficiency fluorescent lighting (14L∶10D photocycle) with 8-cm pots q4 of carnivorous plants compared to other plants (Ellison and submerged in 1–4 cm of reverse osmosis water at all times. Gotelli 2009; Pavlovič and Saganová 2015). The traps are Throughout the duration of the experiments, the plants were triggered by an action potential when specialized trigger hairs kept at ambient temperatures under the lights (ranging from are stimulated (Volkov et al. 2008, 2009) and close as quickly 357C during the day to 227C at night) and 50%–90% humid- as 100 ms, forming a cage around the prey item (Poppinga ity, similar to natural conditions in the field during summer et al. 2013). If the trap fails to capture an insect, it takes be- months. Crickets were purchased from PetSmart and kept tween 2 and 3 days for the trap to reopen, during which time in 4-L plastic containers with shelter, water, and a complete it is unable to be used for prey capture. Beyond the energy diet (Fluker’s cricket food). expended to close a trap and the opportunity cost of a miss, To assess the adaptive role of marginal spikes, we set up there is a cost associated with declining trap performance prey capture arenas (fig. 1). Each arena consisted of one plant and trap death. Traps that have closed and reopened have in a petri dish of distilled water, one cricket of known length lower subsequent trap closure speeds and trap gape angle (range: 0.7–2.3 cm) and mass (range: 0.026–0.420 g), cricket (Stuhlman 1948). Additionally, after a few closings, traps rap- food, and a ramp from the dry bottom of the arena to the idly die. plant. The relationship between prey mass and catch rate We measured prey capture efficiency, trap closure time, was plotted to ensure that the relationship was linear and to and the effect of marginal spikes using field observations of account for nonisometric power scaling in cricket hind legs. wild Venus flytraps, laboratory experiments, and a seminatu- Only healthy crickets with all six legs were used for prey ral experiment. By testing the prey capture ability of plants capture trials. Orthopterans make up approximately 10% of with intact spikes and plants with the spikes clipped off, we flytrap prey in the wild (Ellison and Gotelli 2009; C. H. Mar- assessed the novel function of the marginal spike cage for prey tin, personal observation), and this may represent an under- capture. estimate of how often they visit plants in the wild because they may be more likely to escape than less powerful prey, such as Methods ants or small beetles. The crickets used in this study ranged between 7 and 23 mm, which is within the natural distribu- Field Data Collection tion of orthopteran prey sizes in the Green Swamp in which The Green Swamp Preserve, North Carolina, is one of the very large individuals were observed (reaching at least 54 mm; last remaining eastern pine savannah habitats containing en- C. H. Martin, personal observation). All closed traps were demic flytraps.