Southeastern Naturalist J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 254 2015 SOUTHEASTERN NATURALIST 14(2):254–266 Distinctions in Pitcher Morphology and Prey Capture of the Okefenokee Variety within the Carnivorous Plant Species Sarracenia minor Jessica D. Stephens1,*, Rebecca L. Godwin2, and Debbie R. Folkerts2 Abstract - Morphological differences in trapping mechanisms of carnivorous plants may be a response to selection pressure to reduce competition among species and even conspecifics. However, few studies have investigated whether variation in plant morphology is related to prey capture. Here, we measured height, peristome width, and outer-trichome density in pitchers of Sarracenia minor (Hooded Pitcher Plant) and its variety, S. minor var. okefenokeensis (S. m. var. okefenokeensis). In addition, we compared arthropod-prey contents of pitchers between the typical short form and the tall Okefenokee variety and across seasons and sampling locations. Similar to findings in previous studies, we found that S. minor was an ant specialist with no influence of season or location. In contrast, S. m. var. okefenokeensis had a more generalist diet, and prey content varied across season and location. Pitchers of S. m. var. okefenokeensis were also significantly larger in height and peristome width with fewer outer trichomes than S. minor. Overall, differences in pitcher morphology appear to be correlated with prey contents. These results have important implications for our understanding of the evolution and diversification of carnivorou s plants. Introduction Scientists have been intrigued with carnivorous adaptations in plants since their discovery in the 1800s. Since then, more than 645 species have been described worldwide resulting from at least 6 independent origins (Albert et al. 1992). Recent strides in our knowledge and understanding of these remarkable plants has led to a clearer definition of phytocarnivory (Juniper et al. 1989), cost-benefit analyses of resource allocation (Givnish et al. 1984, Laakkonen et al. 2006), documentation of genetic and morphological variation (Bauer et al. 2011, Godt and Hamrick 1998, Lloyd 1942), descriptions of various symbiotic plant–arthropod interactions (Folkerts 1999, Stephens and Folkerts 2012), and resolution of phylogenetic relationships (Jobson et al. 2003, Meimberg et al. 2001, Stephens et al. 2015). However, the adaptive significance of trapping morphology and its relationship to prey capture remains poorly known. Examination of prey capture and morphological variation among species plays an important role in our understanding of the evolution of carnivorous plants. The fact that carnivorous plants are highly dependent on prey for nutrients suggests that strong selective pressures have occurred on traits related to prey capture (Ellison 1Department of Plant Biology, University of Georgia, Athens, GA 30602. 2Department of Biological Sciences, Auburn University, Auburn, AL 36849. *Corresponding author - jdstephe@uga.edu. Manuscript Editor: Justin Hart Southeastern Naturalist 255 J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 and Gotelli 2001). Variation in traits such as coloration (Schaefer and Ruxton 2008), trap size (Bhattarai and Horner 2009), nectar (Joel 1986), and volatiles (Jürgens et al. 2009) are all hypothesized to influence amount and type of prey captured. Moreover, large-scale differences in trap types (e.g., sticky traps, pitcher traps, bear traps) suggest prey partitioning (Ellison and Gotelli 2009), and there is some evidence for prey partitioning within sticky-trap species occurring in sympatry (Pinguicula [Karlsson et al. 1987], Drosera [Thum 1986]). Among pitcher plants with leaves modified as passive pitfall traps (i.e., Nepenthes, Cephalotus, Heliamphora, Darlingtonia, and Sarracenia), there is substantial variation in trapping morphology. Specifically, Moran et al. (1999) proposed that the large amount of variation in pitchers among Nepenthes species has most likely led to differences in prey capture. This hypothesis has also been suggested for the genus Sarracenia (Folkerts 1999), a diverse group, which appears to have evolved less than 3 million years ago (Ellison et al. 2012), yet has had numerous divergences in pitcher morphology (Stephens et al. 2015). Sarracenia pitchers have been described in general as having attractive, conductive, glandular, and detentive zones, yet the extent, position, and condition of these leaf areas varies among species (Lloyd 1942). In particular, attractive zones (e.g., upper leaf areas that function to lure prey) appear to vary significantly in nectar glands, shape, color, trichome density, and height (McDaniel 1971, Juniper et al. 1989). In addition, diversity of Sarracenia prey has been documented by a number of authors (Ellison and Gotelli 2009, Folkerts 1992). Previous studies of prey capture and pitcher traits in Sarracenia have primarily focused on variation within S. purpurea L. (Purple Pitcher Plant; Bennet and Ellison 2009, Cresswell 1993, Heard 1998, Wolfe 1981) or S. alata Alph. Wood (Yellow Trumpets; Bhattarai and Horner 2009, Green and Horner 2007). Interestingly, Sarracenia minor Walt. (Hooded Pitcher Plant), which has previously been described as an ant specialist (Fish 1976, Folkerts 1992, Givnish 1989), has a recognized variety, S. minor Walt. var. okefenokeensis Schnell (S. m. var. okefenokeensis) that varies substantially in leaf traits from S. minor. In particular, the variety is known for being taller than the short-form S. minor and, from our observations, has relatively few outer trichomes, which are hypothesized to facilitate the movement of prey toward the mouth of the pitcher (Studnička 2013), yet has the same color and overall appearance as other S. minor. This large variation in leaf traits within the species is an ideal situation for examining the relationship between pitcher morphology and prey capture. The aim of this study was to investigate whether prey and leaf traits vary consistently between S. minor and S. m. var. okefenokeensis. In addition, we examined the differences in these traits and prey types between seasons and sampling locations within and between the species and variety. Leaf traits measured included pitcher height, peristome width, and outer trichome density. We identified prey from the contents of measured leaves in S. minor and S. m. var. okefenokeensis found in adjacent locations in the spring and fall of 2010. We predicted, given apparent phenotypic differences, that S. m. var. okefenokeensis would have a more generalist prey diet than that of the ant-specialist S. minor and that both would have Southeastern Naturalist J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 256 less-diverse prey in the spring as a result of fewer available prey and/or shorter cumulative capture time compared to fall samples. Results from this study highlight the role that leaf traits play in prey capture and allow us to speculate on the evolutionary mechanisms involved in carnivorous-plant diversification . Field-site Description The Okefenokee-Osceola ecosystem is located in southeastern Georgia and extends into northeastern Florida (Fig. 1). This region comprises one of the largest freshwater systems in the US and is protected through the Okefenokee National Wildlife Refuge (ONWR) and Okefenokee Wilderness Area located in Georgia and the Osceola National Forest (ONF) at the Florida-Georgia border. The area protected by ONWR (including the Okefenokee National Wilderness Area) is approximately 177,252 ha of peat-filled wetland comprised of bay swamps, cypress– hardwood swamps, scrub–shrub wetlands, prairies, peat-forming bogs, open water, and wet pine savannahs (Edwards et al. 2013). Although the age and origin of the basin is still debated (see Edwards et al. 2013), the most prevalent hypothesis is Figure 1. Sampling locations of Sarracenia minor (Hooded Pitcher Plant) and S. minor var. okefenokeensis (S. m. var. okefenokeensis). The light-gray shaded region across Florida, Georgia, and South Carolina represents the range of S. minor, while the darker dot is the range of S. m. var. okefenokeensis. Shaded regions in the inset designate the Okefenokee National Wildlife Refuge (ONWR) and Osceola National Forest (ONF) located at the Georgia-Florida border. Major roads are indicated by lines and are labeled accordingly. The gray shaded star within ONWR represents the 2 S. m. var. okefenokeensis collecting sites (boardwalk and Chesser prairie, ~2.24 km apart). Black stars represent the ONF S. minor and GA-121 S. minor sampling locations. Southeastern Naturalist 257 J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 that the area was formed during the Pleistocene/Holocene epoch from a deposit of marine sediments along the eastern side of the swamp (Gibbons 1997). The adjacent depression subsequently filled with tannin-rich, highly acidic freshwater and sandy, low-nutrient soils. This fire-maintained habitat contains the only known populations of Okenfenokee Giant. We collected samples within ONWR at Chesser prairie and along the boardwalk trail (~2.24 km apart; Fig. 1). Areas adjacent to ONWR, including ONF, have similar substrate, hydrology, prairies, and peat formations, but lack the floating tree-islands that characterize the refuge. ONF protects roughly 81,000 ha of swamps, bays, and wet pine flatwoods and is comprised mostly of Taxodium distichum (L.) Rich. (Bald Cypress), Nyssa sylvatica Marsh. (Black Gum), Pinus taeda L. (Loblolly Pine), Pinus ellottii Engelm. (Slash Pine), and Serenoa repens (Bartram) J.K. Small (Saw Palmetto) growing in acidic soils (Avers and Bracy 1975). These areas, including those protected by ONF, contain the only S. minor populations within the Okefenokee- Osceola ecosystem. Our collections of S. minor in ONF were restricted to roadsides due to dense Saw Palmetto in the interior forests; collection sites were located roughly 58.8 km from the ONWR sampling sites. In the fall, we sampled at an additional site along GA highway 121 adjacent to ONWR located approximately 13.8 km from the ONWR and 56.4 km from the ONF sampling site (Fig. 1). Methods Prey composition We collected mature and fully developed S. m. var. okefenokeensis leaves at Chesser prairie in May and November of 2010 (n = 10, 10; respectively) and from the boardwalk trail in November 2010 (n = 10) within the ONWR (Fig. 1). We collected S. minor leaves from ONF during the same time period (n =10, 10; respectively) and from the Highway 121 site in November (n = 10) (Fig. 1). We were careful to ensure that each collected leaf was from a separate individual plant because pitcher plants form clumps along a subterranean rhizome (McPherson 2007). Unfortunately, the species and variety do not occur in syntopy, making it unavoidable to sample them at different sites. Therefore, any habitat differences in prey availability may have influenced prey captured. In order to minimize this effect, we sampled S. minor populations that were as close as possible to S. m. var. okefenokeensis and within the Okefenokee-Osceola ecosystem. We extracted all pitcher contents for each sample and preserved them in 70% ethanol. Most prey items were partially digested and often dismembered, therefore, we counted only arthropod heads and identified all prey to order and in some cas es family level. We estimated prey diversity with Simpson (D') and Shannon (H') diversity indices and made comparisons across seasons and varieties using the vegan package (Oksanen et al. 2010) in R v3.0.2 (R Core Team 2013). We used relative proportion of prey in order to avoid bias due to partial digestion of prey. Examination of preytype differences between seasons, between locations within seasons, and between species and variety within seasons was conducted using a permutation multivariate Southeastern Naturalist J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 258 analysis of variance (perMANOVA) in the vegan package under the “adonis” function at 1000 permutations (Oksanen et al. 2010) in R v3.0.2 (R Core Team 2013). Leaf collection and morphological measurements We measured leaf traits thought to influence prey capture for both the species and variety. Specifically, we measured pitcher height, width of the peristome (pitcher opening), and outer-trichome density in the same leaves used for prey samples. Downward-pointing trichomes within the interior of pitchers are thought to function to keep prey from escaping and are found in all species (Lloyd 1942). Less is known about the function of upward pointing hairs on outer leaf surfaces (Fig. 2), but it is hypothesized that they function to direct crawling prey into the pitcher mouth (Studnička 2013). In addition, these outer trichomes are highly variable among Sarracenia spp. (D.R. Folkerts, pers. observ.). We made quantitative measurements of outer-trichome density using an Olympus SZX10 stereomicroscope with an ocular grid by sampling within a 1.43-mm² patch in 3 sample areas—1 each at the base, middle, and top of each pitcher. We tested correlation between leaf traits using a Spearman’s rank correlation in R v3.0.2 (R Core Team 2013). Suites of traits were then compared between seasons within species and variety, between sampling locations within seasons, and overall between S. m. var. okefenokeensis and S. minor using a permutation multivariate analysis of variance (perMANOVA) conducted in the vegan package under the “adonis” function at 1000 permutations (Oksanen et al. 2010) in R v3.0.2 (R Core Team 2013). We employed a principal components analysis (PCA) with individuals’ scores plotted along the first two axes of variance to visualize leaf-trait variation. Finally, to test correlation between prey types and leaf traits, we conducted a Mantel test (Mantel 1967) comparing a matrix of dissimilarity indices for prey types and leaf traits for all individuals using the vegan package (Oksanen et al. 2010). Figure 2. Upward-pointing trichomes indicated by the arrow found on the outside of the pitcher between S. minor var. okefenokeensis (A.) and S. minor (B.) seen here using a scanning electron microscope after specimens were air dried and sputter coated. Southeastern Naturalist 259 J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 Results In total, we identified 5529 prey items in S. minor and S. m. var. okefenokeensis comprising 8 orders and 9 families. However, approximately 95% of all prey observed were Coleoptera, Diptera, Hymenoptera (mostly Formicidae), or Lepidoptera (Fig. 3). Overall, S. m. var. okefenokeensis had more-diverse prey when compared to S. minor. However, both taxa exhibited lower prey diversity and lower average prey counts per pitcher in the spring versus fall (Table 1). The majority (>95%) of S. minor prey was Formicidae (i.e., ants) in both spring and fall (Fig. 3), with no significant difference in the ONF sampling locations between seasons (F = 1.495, r2 = 0.081, P = 0.08). There was also no difference in prey type between sites in the fall (F = 1.441, r2 = 0.074, P = 0.24). In comparison, S. m. var. okefenokeensis displayed a more generalist diet (Table 1, Fig. 3) and had significantly different prey contents at the Chesser prairie site between seasons (F = 5.998, r2 = 0.250, P < 0.001). S. m. var. okefenokeensis Figure 3. Percent of prey type for S. minor var. okefenokeensis and S. minor across spring and fall sampling. Shadings represent prey type and are labeled accordingly. Other designates all other taxa that were identified but had small contribu tions to prey capture. Southeastern Naturalist J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 260 also differed significantly between sampling locations (i.e., Chesser prairie and the boardwalk) in the fall (F = 4.944, r2 = 0.215, P = 0.002). The difference in prey composition between sampling locations was largely driven by variation in the proportion of Lepidoptera, Coleoptera, and Diptera prey with little difference in the amount of Formicidae captured between Chesser prairie and the boardwalk (25% vs. 33%, respectively). Even with a shift in prey composition for S. m. var. okefenokeensis between seasons and sites, there was still a significant difference in prey compared to S. minor for both the spring (F = 9.357, r2 = 0.355, P < 0.001) and fall (F = 24.892, r2 = 0.396, P < 0.001). All combinations of leaf traits were highly correlated, and therefore were considered as suites of traits in a multivariate analysis. There were no significant differences in leaf traits between seasons for S. m. var. okefenokeensis (F = 1.260, r2 = 0.065, P = 0.303) or for S. minor (F = 0.749, r2 = 0.042, P = 0.45). In addition, there were no significant differences in leaf traits between sampling locations for S. m. var. okefenokeensis (F = 2.745, r2 = 0.13, P = 0.08) or for S. minor (F = 0.813, r2 = 0.043, P = 0.48) during the fall collection. PCA indicated a clustering of leaf traits by species and variety (Fig. 4), and this difference was significant for both the spring (F =32.543, r2 = 0.656, P < 0.001) and fall (F = 27.985, r2 = 0.424, P < 0.001) collections. Taken together, these results suggest that S. minor had a greater density of outer trichomes, a smaller peristome, and was shorter than S. m. var. okefenokeensis throughout the year. Lastly, leaf traits were correlated with prey types in all individuals (r2 = 0.1632, P < 0.001). Discussion Consistent with previous descriptions (Fish 1976, Givnish 1989), our results support S. minor as an ant specialist. Furthermore, the strong affinity for ants was not influenced by season or locality. In contrast, S. m. var. okefenokeensis had a more generalist diet that was significantly different from S. minor and varied in prey composition (with the exception of Formicidae capture) between the 2 sites sampled less than 2.5 km apart within the Okefenokee Swamp. These findings are congruent with predictions about the functional morphology of these pitchers. Specifically, shorter pitchers with dense trichomes on outer surfaces are thought to have an advantage in capturing ground-crawling prey, while taller pitchers with trichomes primarily around the peristome are thought to have an advantage in capture of larger flying prey. Table 1. Shannon (H') and Simpson (D') diversity indices for prey contents and average prey counts per pitcher between Sarracenia minor (Hooded Pitcher Plant) and S. minor var. okefenokeensis (S. m. var. okefenokeensis) between seasons and overall (i.e., seasons combined; n =30) for each taxon. Average prey Shannon (H') Simpson (D') count/pitcher Species Spring Fall Overall Spring Fall Overall Spring Fall S. minor 0.061 0.406 0.299 0.027 0.208 0.152 30 110 S. minor var. okefenokeensis 0.208 1.005 0.739 0.122 0.552 0.408 72 142 Southeastern Naturalist 261 J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 Overall, the S. m. var. okefenokeensis captured a larger proportion of Coleoptera and Diptera prey in the spring, while Lepidoptera adults and Formicidae prey increased in the fall. This seasonal difference in prey type for S. m. var. okefenokeensis, as well as overall lower diversity indices for both plants in the spring, may be partially explained by limited prey availability (Wolda 1988). For example, peaks of Lepidoptera species richness in temperate forest systems tend to be during June and August (Thomas and Thomas 1994). Availability may also explain the higher prey abundance (approximately 70 more individual prey items per pitcher) in the fall for both the S. minor and S. m. var. okefenokeensis. This effect was not likely confounded by pitcher age, because we tried to control for age of pitchers between seasons and between species and variety by consistently selecting mature pitchers that showed no signs of senescence. In addition, previous research on Figure 4. Principal components analysis (PCA) of measured leaf traits for (S. minor; n = 30) and (S. m. var. okefenokeensis; n = 30). Leaf traits represented by the vectors include: Orifice = peristome width, Height, Low = lower trichome density, Mid = middle trichome density, and Up = upper trichome density along the outside of the pitcher. PC1 represents 70% of the variation in traits, and PC2 is 16% of the variation in traits. Southeastern Naturalist J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 262 Sarracenia prey capture has shown that the majority of prey is captured within the first 30 days of a pitcher ’s lifespan (Fish and Hall 1978, Wolfe 1981). Interestingly, S. m. var. okefenokeensis captured more prey per pitcher than S. minor in both the spring and fall (~2.5x and 0.5x more, respectively). This result supports previous research suggesting that larger pitchers capture more prey, possibly due to greater amount of attractants (Bhattarai and Horner 2009). In addition, multiple studies have found that pitcher size tends to positively correlate with prey biomass (Cresswell et al. 1993, Green and Horner 2007, Heard 1998). Although we did not directly sample biomass, the majority of prey captured (e.g., Lepidoptera, Coleoptera, and Diptera) by S. m. var. okefenokeensis tended to be relatively larger than the predominantly Formicidae prey captured by S. minor, suggesting that these plants captured not only a higher number of prey, but also a larger biomass of prey. We cannot conclusively link the differences in pitcher size and amount of prey between S. minor and S. m. var. okefenokeensis because all traits measured were highly correlated. This result could indicate that prey partitioning in Sarracenia may be the result of suites of traits similar to pollination syndromes. The concept of “carnivorous syndromes” may also include traits that we did not measure in this study. For example, it has been suggested that nectar, volatiles, and color may also aid in prey capture (Joel 1986, Juniper et al. 1989). We did not directly measure color, but it should be noted that there is very little difference in overall color or in the white fenestrations between the species and variety. This similarity suggests that color may not be a dominant trait driving differences in prey capture. Bennett and Ellison (2009) arrived at a similar conclusion when examining color cues and sugar availability with “pseudopitchers”, suggesting that nectar is the primary attractant. Nectar glands have been identified along the outer pitcher of S. minor, with denser assemblages towards the upper section of the pitcher (Lloyd 1942), but variation in nectar concentration and extra-floral nectary location across individuals and the varieties has not been established. Another trait that may influence prey capture is pitcher volatiles. It has been speculated that chemical attractants may be partly responsible for the large number of ants comprising the prey of S. minor (Jürgens et al. 2009). Future studies incorporating measures of these traits and manipulation of traits may help us tease apart their relative importance in attracting prey. The overall differences in prey types and leaf traits between S. minor and S. m. var. okefenokeensis may be the result of local adaptation or phenotypic plasticity. Local adaptation (ecotypic distinction) may result from differences in selection pressures between habitats in the swamp and in upland areas, specifically differences in the abundance of crawling prey as well as in hydrology and surrounding vegetation. However, numerous ant species have been documented in the Okefenokee Swamp (Wheeler 1913) as well as in surrounding areas, and both varieties captured a large proportion of ants. The abundance of flying prey (i.e., Lepidoptera and Diptera), however, would less likely be influenced by hydrology and is therefore more likely to be similar in S. minor and S. m. var. okefenokeensis habitats. Despite this, S. m. var. okefenokeensis captured far more flying insects Southeastern Naturalist 263 J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 than S. minor. In contrast, unique environmental conditions within the swamp could be driving a phenotypic plasticity response (ecophenic distinction) causing the dramatically larger size in the Okefenokee variety. It should also be noted that there is a “giant” form of S. psittacina Michx. (Parrot Pitcher Plant) found within the swamp. Environmental conditions have been observed to have effects on pitcher size and color in many Sarracenia species, but transplants of S. m. var. okefenokeensis with native soil into greenhouse conditions maintain their larger size at least for a while (R.O. Determann, Atlanta Botanical Garden, Atlanta, GA; pers. comm.). Ours is the first study, however, in which a difference in trichome density has been noted within S. minor. Future studies using a combination of genetic analyses and reciprocal transplants of S. minor, Parrot Pitcher Plant, and their varieties could aid in understanding the causes of this interesting phenotype. At this point, we can only speculate about the evolutionary sequence of divergence in S. minor. The species may have expanded from an original population in Okefenokee Swamp outward into habitats where other carnivorous plants were already present and experienced ecological character displacement through specializing on ant prey. It is also possible that ant-specialist S. minor expanded into swamp habitat and experienced competitive release in a habitat where other pitcher plants do not occur and ants are somewhat less abundant. Overall, the significant differences between prey types and leaf traits suggest a tight correlation between function and morphology of S. minor pitchers and has possible implications at the genus level. Specifically, the recent divergence of this group at less than 3 million years ago and the morphological divergence in sister taxa (Ellison et al. 2012, Stephens et al. 2015) could indicate that ecological mechanisms, like niche divergence, are responsible for rapid diversification in Sarracenia. More research examining pitcher morphology and prey dynamics could help elucidate the evolutionary history of carnivory and diversification in this genus. Acknowledgments We would like to thank Nathan Hall for field and lab assistance and Chelsea Cunard for discussion on statistical analyses. This research was conducted under an award from the Estuarine Reserves Division, Office of Ocean and Coastal Resource Management, National Ocean Service, National Oceanic and Atmospheric Administration (Award # NA10NOS4200038) and under permit # 41590-10-033 granted by Okefenokee National Wildlife Refuge. Literature Cited Albert, V.A., S.E. Williams, and M.W. Chase. 1992. Carnivorous plants: Phylogeny and structural evolution. Science 257:1491–1495. Avers, P.E., and K.C. Bracy. 1975. Soils and physiography of the Osceola National Forest. US Department of Agriculture, Forest Service, Atlanta, GA. Bauer, U., C.J. Clemente, T. Renner, and W. Federle. 2011. Form follows function: Morphological diversification and alternative trapping strategies in carnivorous Nepenthes pitcher plants. Journal of Evolutionary Biology 25(1):90–102. Southeastern Naturalist J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 264 Bennett, K.F., and A.M. Ellison. 2009. Nectar, not color, may lure insects to their death. Biology Letters 5(4):469–472. Bhattarai, G.P., and J.D. Horner. 2009. The importance of pitcher size in prey capture in the carnivorous plant, Sarracenia alata Wood (Sarraceniaceae). American Midland Naturalist 161:264–272. Cresswell, J.E. 1993. The morphological correlates of prey capture and resource parasitism in pitchers of the carnivorous plant Sarracenia purpurea. American Midland Naturalist. 129:35–41. Edwards, L., J. Ambrose, and L.K. Kirkman. 2013. Okefenokee Swamp. Pp. 494–509, In The Natural Communities of Georgia. The University of Georgia Press, Athens, GA. 704 pp. Ellison, A.M., and N.J. Gotelli. 2001. Evolutionary ecology of carnivorous plants. TREE 16:623–629. Ellison, A.M., and N.J. Gotelli. 2009. Energetics and the evolution of carnivorous plants: Darwin’s “most wonderful plants in the world”. Journal of Experimental Botany 60:19–42. Ellison, A.M., E.D. Butler, E.J. Hicks, R.F.C. Naczi, P.J. Calie, C.D. Bell, and C.C. Davis. 2012. Phylogeny and biogeography of the carnivorous plant family Sarraceniaceae. PLoS ONE 7(6):e39291. Fish, D. 1976. Insect–plant relationships of the insectivorous pitcher plant Sarracenia minor. Florida Entomologist 59:199–203. Fish, D., and D.W. Hall. 1978. Succession and stratification of aquatic insects inhabiting the leaves of the insectivorous pitcher plant, Sarracenia purpurea. American Midland Naturalist 99:172–183. Folkerts, D.R. 1992. Interactions of pitcher plants (Sarracenia: Sarraceniaceae) with their arthropod prey in the southeastern United States. Ph.D. Dissertation. University of Georgia, Athens, GA. Folkerts, D.R. 1999. Pitcher plant wetlands of the southeastern United States: Arthropod associates. Pp. 247–275, In D.P. Batzer, R.B. Rader, and S.A. Wissinger (Eds.). Invertebrates in Freshwater Wetlands of North America: Ecology and Management. John Wiley and Sons, New York, NY. 1120 pp. Gibbons, W. 1997. The natural history of the Okefenokee Swamp. Georgia Wildlife 6(1):4–16. Givnish, T.J. 1989. Ecology and evolution in carnivorous plants. Pp. 243–290, In W.G. Abrahamson (Ed.). Plant–Animal Interactions. McGraw-Hill, New York, NY. 480 pp. Givnish, T.J., E.L. Burkhardt, R.E. Happel, and J.W. Weintraub. 1984. Carnivory in the bromeliad Brocchinia reducta, with a cost/benefit model for the general restriction of carnivorous plants to sunny, moist, nutrient-poor habitats. American Naturalist 124:479–497. Godt, M.J.W., and J.L. Hamrick. 1998. Allozyme diversity in the endangered pitcher plant Sarracenia rubra ssp. alabamensis (Sarraceniaceae) and its close relative S. rubra ssp. rubra. American Journal of Botany 85:802–810. Green, M.L., and J.D. Horner. 2007. The relationship between prey capture and characteristics of the carnivorous pitcher plant, Sarracenia alata Wood. American Midland Naturalist. 158:424–431. Heard, S.B. 1998. Capture rates of invertebrate prey by the pitcher plant, Sarracenia purpurea L. American Midland Naturalist 139:79–89. Southeastern Naturalist 265 J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 Jobson, R.W., J. Playford, K.M.Cameron, and V.A. Albert. 2003. Molecular phylogenetics of Lentibulariaceae inferred from plastid rps16 intron and trnL-F DNA sequences: Implications for character evolution and biogeography. Systematic Botany 28:157–171. Joel, D.M. 1986. Glandular structure in carnivorous plants: Their role in mutual exploitation of insects. Pp. 219–234, In B.E. Juniper and T.R.E. Southwood (Eds.). Insects and the Plant Surface. Edward Arnold, London, UK. Juniper, B.E., R.J. Robins, and D.M. Joel. 1989. The Carnivorous Plants. Academic Press, New York, NY. 353 pp. Jürgens, A., A.M. El-Sayed, and D.M. Suckling. 2009. Do carnivorous plants use volatiles for attracting prey insects? Functional Ecology 23:875–887. Karlsson, P.S., K.O. Nordell, S. Eirefelt, and A. Svensson. 1987. Trapping efficiency of three carnivorous Pinguicula species. Oecologia 73:518–521. Laakkonen, L., R.W. Jobson, and V.A. Albert. 2006. A new model for the evolution of carnivory in the bladderwort plant (Utricularia): Adaptive changes in cytochrome c oxidase (COX) provide respiratory power. Plant Biology 8:758–764. Lloyd, F.E. 1942. The Carnivorous Plants. Ronald Press, New York, NY. 723 pp. Mantel, N. 1967. The detection of disease clustering and a generalized regression approach. Cancer Research 27:209–220. McDaniel, S. 1971. The genus Sarracenia (Sarraceniaceae). Bulletin No. 9 of the Tall Timbers Research Station,Tallahassee, FL. McPherson, S. 2007. Pitcher Plants of the Americas. The McDonald and Woodward Publishing Company, Blacksburg, VA. 183 pp. Meimberg, H., A. Wistuba, P. Dittrich, and G.R.Heubl. 2001. Molecular phylogeny of Nepenthaceae based on cladistic analysis of plastid trnK intron sequences. Plant Biology 3:164–175. 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. Oksanen, J., F. Blanchet, R. Kindt, P. Legendre, R.B. O’Hare, et al. 2010. Vegan: Community ecology package, version 2.0-10, http://vegan.r-forge.r-project.org. Accessed 17 May 2014. R Core Team. 2013. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org. Accessed 17 May 2014. 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. Stephens, J.D., and D.R. Folkerts. 2012. Life-history aspects of Exyra semicrocea (Pitcher Plant Moth) (Lepidoptera: Noctuidae). Southeastern Naturalist 11:111–126. Stephens, J.D., W.L. Rogers, K. Heyduk, J.M. Cruse-Sanders, R.O. Determann, T.C. Glenn, and R.L. Malmberg. 2015. Resolving phylogenetic relationships of the recently radiated carnivorous plant genus Sarracenia using target enrichment. Molecular Phylogenetics and Evolution 85:76–87. Studnička, M. 2013. Different wing in pitchers of the myrmecohagous species Sarracenia minor and S. rubra. Journal of the International Carnivorous Plant Society 42:19–23. Thomas, A.W., and G.M. Thomas. 1994. Sampling strategies for estimating moth-species diversity using a light trap in a northeastern softwood forest. Journal of Lepidopterist’s Society 48:85–105. Southeastern Naturalist J.D. Stephens, R.L. Godwin, and D.R. Folkerts 2015 Vol. 14, No. 2 266 Thum, M. 1986. Segregation of habitat and prey in two sympatric carnivorous plant species, Drosera rotundifolia and Drosera intermedia. Oecologia 70:601–605. Wheeler, W.M. 1913. Ant collected in Georgia by Dr. J.C. Bradley and Mr. W.T. Davis. Psyche: 112–117. Wolda, H. 1988. Insect seasonality: Why? Annual Review of Ecology and Systematics 19:1–18. Wolfe, L.M. 1981. Feeding behavior of a plant: Differential prey capture in old and new leaves of the pitcher plant (Sarracenia purpurea). American Midland Naturalist 106:352–359.