Southeastern Naturalist 433 J. Annis, J. Coons, C. Helm, and B. Molano-Flores 22001188 SOUTHEASTERN NATURALIST 1V7o(3l.) :1473,3 N–4o3. 73 The Role of Red Leaf Coloration in Prey Capture for Pinguicula planifolia Jenna Annis1, Janice Coons1, Charles Helm2, and Brenda Molano-Flores2,* Abstract - Anthocyanins in the leaves of carnivorous plants are suggested to play a role in prey capture. In this study, we investigated the role of red leaf coloration (an indicator of anthocyanins) on prey capture using Pinguicula planifolia (Chapman’s Butterwort). Overall, red leaves had less prey (i.e., Collembola) than green leaves, suggesting that red coloration does not enhance prey capture for Chapman’s Butterwort. However, the frequent presence of Collembola on leaves suggests that this plant species could be relying on other cues to attract prey (e.g., olfactory cues). Introduction Anthocyanins are prominent in the vegetative tissues of several evolutionarily distinct carnivorous plant families, and an emerging argument suggests that these pigments serve a role in prey capture (Joel 1988, Jürgens et al. 2015, Moran et al. 1999). Schaefer and Ruxton (2008) demonstrated that the markedly red coloration on the pitchers of Nepenthes (tropical pitcher plants) species may enhance prey capture compared to pitchers without red coloration. Ichiishi et al. (1999) suggested that anthocyanins present in the trapping leaves of Dionaea muscipula J. Ellis (Venus Fly-trap) and Drosera spatulata Labill. (Spoon-leaved Sundew) are produced when the plants become nitrogen deficient, and that this pigment production subsequently increases prey attraction in both species, providing a means to regain nutrients lacking in the substrate. Jürgens et al. (2015) proposed that anthocyanins reduce the risk of pollinator–prey conflict in sundew species because the pigments tended to deter pollinating insects while still attracting insect prey, but the red-pigmented leaves also lowered total prey-capture. The debate associated with the involvement of red pigmentation in prey capture is due partly to the widely accepted argument that insects’ color vision is poor in the red spectrum of light (Bennett and Ellison 2009, Chittka et al. 2001). Although it is unlikely that color is the only determining factor in attracting insect prey by carnivorous plants, these studies do present more questions than answers in terms of how these red pigments are beneficial for plant taxa such as Butterwort species that rely on captured prey for nutrients (Ademec and Pavlovič 2018, Legendre 2000). The focus of this study was to determine how red leaf coloration affects prey capture for Chapman’s Butterwort—a species in which leaf color in natural populations ranges from green 1Eastern Illinois University, Biological Sciences Department, 600 Lincoln Avenue, Charleston, IL 61920. 2University of Illinois, Prairie Research Institute, Illinois Natural History Survey, 1816 South Oak Street, Champaign, IL 61820. Corresponding author - molano1@illinois.edu. Manuscript Editor: Jason Cryan Southeastern Naturalist J. Annis, J. Coons, C. Helm, and B. Molano-Flores 2018 Vol. 17, No. 3 434 to red (Gluch 2005). In addition, Annis (2016) verified the presence of anthocyanins associated with red coloration for this species. Materials and Methods We studied a population of Chapman’s Butterwort at the St. Joseph Bay State Buffer Preserve in Gulf County, FL. In mid-March 2015, we marked 20 red plants and 20 green plants by placing bamboo skewers near plants, and measured and recorded the length and width of the sampled leaves. We placed 2 wooden insecttraps (3.5 cm x 5.0 cm x 0.5 cm wood palette) within a 15-cm radius of each marked plant; traps were secured to the ground with metal sod staples. Regardless of the color of the focal plant, 1 trap was painted red, 1 was painted green, and both were smeared with TangleTrap Sticky Coating (The Tangle Company, Grand Rapids, MI). We chose specific paint colors for the green and red traps by using a color palette and matching the closest available paint color to natural leaf colors of Chapman’s Butterwort observed in the field. Although we did our best to match wooden traps and leaf colors, we did not use a spectroradiometer to verify the reflectance and absorption spectra of the traps and leaves (Horner et al. 2018). We set traps for 3 d chosen when no rain was in the forecast and then collected, covered with wax paper, and stored them in plastic bags. We carefully applied white electrical tape to the marked leaf, peeled it off, and placed leaf samples in 70% ethanol on same day as we collected traps. We repeated this prey-capture procedure for the same plants (different leaves) after 2 weeks. We used leaf areas (calculated using dimensions A = πab where A = area, a = width, b = length) to obtain number of prey captured/cm2. We performed a two-way analysis of variance to determine the effects of trap type (wooden or leaf) and trap color (red or green) on prey capture. We identified to the arthropod-order level and determined abundance for each prey order on leaves and wooden traps. Based on this information, we determined taxa percentages caught per trap and per leaf. We conducted all statistical analyses in SPSS Statistics Version 22. Results A 2-factor analysis of variance showed a significant effect of trap color (F1 = 5.3, P ≤ 0.05)] and trap type (F1 = 10.7, P ≤ 0.05) on prey capture, but no significant interaction occurred between trap color and trap type (F1 = 0.0, P > 0.05). Green coloration captured more prey than red coloration, and painted wood blocks captured more prey than leaves (Table 1). All trap colors and types captured a large percentage of Collembola; wooden traps also captured Nematocera followed by Brachycera, whereas leaves captured Arachnida followed by Nematocera and Brachycera (Fig. 1). Discussion In contrast to arguments that certain carnivorous plants utilize anthocyanins to attract prey (Ichiishi et al. 1999, Schaefer and Ruxton 2008), our results do not Southeastern Naturalist 435 J. Annis, J. Coons, C. Helm, and B. Molano-Flores 2018 Vol. 17, No. 3 support that contention; green leaves of Chapman’s Butterwort caught more prey than red leaves, suggesting that red leaf coloration could be a deterrent to prey. Similar results have been found for several sundew species (Foot et al. 2014, Horner et al. 2018, Jurgens et al. 2015). Perhaps differences found for prey preference to color by different species of carnivorous plants relates to differences in the type of Figure 1. Percent of arthropod taxa captured on green wooden traps, red wooden traps, green leaves, and red leaves on or near Chapman’s Butterwort plants. All percent values have been rounded. Table 1. Number of arthropods captured by color (green vs. red) and trap type (wooden blocks vs. leaves) on or near Chapman’s Butterwort plants. Mean ± SE are reported and means within trap color or trap type with different superscript letter differ based on ANOVA (P ≤ 0.05). n = sample size. Variable n Arthropods/cm2 Trap color Green 59 0.6 ± 0.1A Red 60 0.4 ± 0.1B Trap type Artificial 79 0.7 ± 0.0A Leaf 40 0.4 ± 0.1B Southeastern Naturalist J. Annis, J. Coons, C. Helm, and B. Molano-Flores 2018 Vol. 17, No. 3 436 prey captured. The prey capture by Chapman’s Buttwort is similar to other butterwort species in major taxa captured (i.e., Collembola and Arachnida), but different in other taxa (Diptera) and in the percentage of each taxon captured. Zamora (1990) found that the major prey group of the European Pinguicula nevadensis (H. Lindb.) Casper (Grasilla) was Diptera (62.2%), followed by Arachnida (17.1%), and Collembola (7.6%). Pavón et al. (2011) found a similar pattern in Central Mexico for the butterwort Pinguicula moranensis Kunth (53.6% Diptera, 29.1% Collembola, and 13.2% Arachnida). For Chapman’s Butterwort, we found that Collembola was the top prey (Fig. 1). Anthocyanins do not seem to play a role in attracting prey for Chapman’s Butterwort, and Joel et al. (1985) reported that no obvious UV patterns are displayed by butterworts to attract insects; thus, we hypothesize that Chapman’s Butterwort could be relying on nonvisual cues, such as olfactory cues, to attract prey. For example, Lloyd (1942) and Fleischmann (2016) mentioned that butterwort mucilage emits a “delicate fungus-like odor” that may attract insects. The majority of prey captured on red (62%) and green (67%) Chapman’s Butterwort leaves was Collembola. The large majority of most Collembola species’ diet consists of fungal hyphae (Newell 1984), and studies show that these soil arthropods are attracted to the odor of fungi (Bengtsson et al. 1988, Hedlund et al. 1995). Although anthocyanins do not seem to play a role in attracting prey in Chapman’s Butterwort, they could be performing a photoprotection role (Horner et al. 2018). Perspective Future studies should address the: (1) role of olfactory cues to attract prey, possibly including amounts or consistency of mucilage; (2) role of environmental factors (i.e., temperature, humidity, and rainfall) on prey availability; (3) seasonal patterns of prey capture and availability; and (4) the cues that attract different prey as captured by different species of insectivorous plants. These studies could provide a better understanding of prey-capture dynamics associated with this species and other carnivorous plant species. Acknowledgments We thank Samantha Primer, Jean Mengelkoch, David N. Zaya, Mary Ann Feist, Robin Kennedy, Patricia Stampe, and the staff at St. Joseph Bay State Buffer Preserve. Funding was provided by Bok Tower Gardens, US Fish and Wildlife Service, and Eastern Illinois University (The Lewis Hanford Tiffany and Loel Zehner Tiffany Botany Graduate Research Fund, College of Sciences, Graduate School, and Department of Biological Sciences). Literature Cited Ademec, L., and A. Pavlovič. 2018. Mineral nutrition of terrestrial carnivorous plants. Pp. 221-231, In A.M. Ellison and L. Adamec (Eds.). Carnivorous Plants: Physiology, Ecology, and Evolution, Oxford University Press, Oxford, UK. 510 pp. Annis, J.M. 2016. Seeing red in a sea of green: Anthocyanin production in a carnivorous plant, Pinguicula planifolia. M.Sc. Thesis. Eastern Illinois University, Charleston, IL. 93 pp. Southeastern Naturalist 437 J. Annis, J. Coons, C. Helm, and B. Molano-Flores 2018 Vol. 17, No. 3 Bengtsson G., A. Erlandsson., and S. Rundgren, 1988. Fungal odor attracts soil Collembola. Soil Biology and Biochemistry 20:25–30. Bennett, K.F., and A.M. Ellison. 2009. Nectar, not color, may lure insects to their death. Biology Letters 5:469–472. Chittka, L., J. Spaethe, A. Schmidt, and A. Hickelsberger. 2001. Adaptation, constraint, and chance in the evolution of flower color and pollinator color-vision. Pp. 106–126, In L. Chittka and J.D. Thomson (Eds.). Cognitive Ecology of Pollination. Cambridge University Press, Cambridge, UK. 360 pp. Fleischmann, A. 2016. Pinguicula flowers with pollen imitations close at night: Some observations on butterwort flower biology. Carnivorous Plant Newsletter 45:84–92. Foot, G., S.P. Rice, and J. Millett. 2014. Red trap-color of the carnivorous plant does not serve a prey-attraction or camouflage function. Biology Letters 10:20131024. Gluch, O. 2005. Pinguicula species (Lentibulariaceae) from the southeastern United States: Observations of different habitats in Florida. Acta Botanica Gallica 152:197–204. Hedlund, K., A.D.M. Rayner, and S.E. Reynolds. 1995. Fungal odor discrimination in two sympatric species of fungivorous Collembolans. Functional Ecology 9:869–875. Horner, J.D., B.J. Płachno, U. Bauer, and B. Di Giusto. 2018. Attraction of prey. Pp. 157– 166, In A.M. Ellison and L. Adamec (Eds.). Carnivorous Plants: Physiology, Ecology, and Evolution, Oxford University Press, Oxford, UK. 510 pp. Ichiishi, S., T. Nagamitsu, Y. Kondo, T. Iwashina, K. Kondo, and N. Tagashira. 1999. Effects of macro-components and sucrose in the medium on in vitro red-color pigmentation in Dionaea muscipula Ellis and Drosera spatulata Labill. Plant Biotechnology 16:235–238. Joel, D.M. 1988. Mimicry and mutualism in carnivorous pitcher plants (Sarraceniaceae, Nepenthaceae, Cephalotaceae, Bromeliaceae). Biological Journal of the Linnean Society 35:185–197. Joel, D.M., B.E. Juniper, and A. Dafni. 1985. Ultraviolet patterns in the traps of carnivorous plants. New Phytologist 101:585–593. Jürgens, A., T. Witt, A. Sciligo, and A.M. El-Sayed. 2015. The effect of trap color and trap–flower distance on prey and pollinator capture in carnivorous Drosera species. Functional Ecology 29:1026–1037. Legendre, L. 2000. The genus Pinguicula L. (Lentibulariaceae): An overview. Acta Botanica Gallica 147:77–95. Lloyd, F.E. 1942. The Carnivorous Plants, 9th Edition. Ronald Press, New York, NY. 380 pp. Moran, J.A., W.E. Booth, and J.K. Charles. 1999. Aspects of pitcher morphology and spectral characteristics of six Bornean Nepenthes pitcher-plant species: Implications for prey capture. Annals of Botany 83:521–528. Newell, K. 1984. Interactions between two decomposer basidiomycetes and a collembolan under Sitka Spruce: Grazing and its potential effects on fungal distribution and litter decomposition. Soil Biology and Biochemistry 16:235–239. Pavón, N.P., A. Contreras-Ramos, and Y. Islas-Perusquía. 2011. Diversity of arthropods preyed upon by the carnivorous plant Pinguicula moranensis (Lentibulariaceae) in a temperate forest of Central Mexico. The Southwestern Naturalist 56:78–82. Schaefer, H.M., and G.D. Ruxton. 2008. Fatal attraction: Carnivorous plants roll out the red carpet to lure insects. Biology Letters 4:153–155. Zamora, R. 1990. The feeding ecology of a carnivorous plant (Pinguicula nevadense): Prey analysis and capture constraints. Oecologia 84:376–379.