661 A.P. Abbate and J.W. Campbell 22001133 SOUSToHuEthAeSaTstEeRrnN N NaAtuTrUaRlisAtLIST 12V(o3l). :1626,1 N–6o6. 53 Parasitic Beechdrops (Epifagus virginiana): A Possible Ant-Pollinated Plant Anthony P. Abbate1,* and Joshua W. Campbell1 Abstract - Epifagus virginiana (Beechdrop) is an annual flowering plant that parasitizes Fagus grandifolia (American Beech) roots. The pollination biology of the chasmogamous flowers of E. virginiana is unknown. In September 2011–2012, we observed insect visitors to E. virginiana flowers. Insect visitors included Bombus impatiens, Crematogaster spp., and Prenolepis imparis, but were dominated by P. imparis (over 96% of insect visits). In September 2012, we captured ants (Crematogaster and P. imparis) on E. virginiana flowers in three different areas found in the Coastal Plain and Piedmont physiographic provinces. With the use of 2,5-diphenyl tetrazolium bromide (MTT), we tested E. virginiana pollen from flowers and ant integuments for viability. We observed no significant difference (P > 0.05) between the viability of pollen taken directly from E. virginiana flowers and pollen removed from the ants. Our data suggests that ants can be playing an important role in the pollination of E. virginiana. Introduction Epifagus virginiana Bartram (Beechdrops) is an annual flowering plant in the family Orobanchaceae that parasitizes Fagus grandifolia Ehrhart. (American Beech) roots. Due to E. virginiana’s parasitic nature and complete lack of chlorophyll, E. virginiana’s range (Thieret 1969) and density (Tsai and Manos 2010) are completely dependent on F. grandifolia. Epifagus virginiana is a small plant that grows up to 30 cm tall and produces two types of flowers during late summer/ early fall: cleistogamous and chasmogamous flowers (Musselman 1982). Chasmogamous flowers (potentially cross pollinated) are located towards the tips of the stems and are elongated, while cleistogamous flowers (self-fertile) are located closer to the base of the plant and are bud-like in shape. Though the cleistogamous flowers remain closed, the chasmogamous flowers open and produce a nectary that half encircles the ovary to presumably attract potential pollinators (Thieret 1969). Chasmogamous flowers are tubular in shape and up to 1.2 cm in length (Musselman 1982) with pink/purple petals. Many plants lack chasmogamous flowers, and plants that do have chasmogamous flowers produce many more cleistogamous flowers (Musselman 1982). Despite many of the chasmogamous flowers being sterile, some can be fertile and produce fruit and seeds (Thieret 1969). Thieret (1969) suggested that cross-pollination by insects may be required for seed production within the chasmogamous flowers. Despite some limited observations (e.g., Musselman 1982, Thieret 1969), the pollination biology of the chasmogamous flowers of E. virginiana is unknown, and no insect visitors have ever been documented. Cooke and Schively (1904) suggest that chasmogamous flowers may be a left-over relict from an insect-po llinated past. 1High Point University, Biology Department, 833 Montlieu Avenue, High Point, NC 27262. *Corresponding author - abbata08@gmail.com. A.P. Abbate and J.W. Campbell 2013 Southeastern Naturalist Vol. 12, No. 3 662 Methods In September 2011, we conducted a pilot study at the Piedmont Environmental Center (PEC; High Point, NC: 36.015897°N, 79.943835°W) in which we visually observed and filmed E. virginiana flowers for insect visitors using digital cameras (Sony HD Handy Cam) fixed to tripods for a total of 20 hours during morning, midday, afternoon, and night (5 hours per time period). For our observations within PEC, we used two populations of E. virginiana that consisted of approximately 25 plants each. At least two cameras were focused on individual plants during a given observation time within an E. virginiana population. Using the same camera procedures, in September 2012 we expanded our study to the Coastal Plain (Beaver Dam Park, VA: 37.450258°N, 76.526342°W), two Piedmont areas (Uwharrie National Forest, NC: 35.461362°N, 79.968474°W; and PEC), and the Cumberland Plateau in Georgia (34.349288°N, 85.347577°W). Our observations indicated that ants were the primary visitors to the E. virginiana flowers. Many flowering plants are visited by ants but are rarely pollinated by them (e.g., Peakall et al. 1991; Schürch et al. 2000). It has been suggested that ants are poor pollinators because they may secrete chemical substances that inhibit pollen viability (Beattie et al. 1984). Pollen has been shown to lose viability after contact with an ant’s integument in less than 30 minutes (Beattie et al. 1984, Hull and Beattie 1988). The chemicals secreted by ants, which act as an antiseptic to kill bacteria and other microorganisms, originate from the metapleural gland (also called metasternal or metathoricic gland; Hölldobler and Engel-Seigel 1984). Metapleural glands are ubiquitous and considered a shared derived characteristic within Formicidae (Yek and Mueller 2010). We tested our hypothesis that ants are possible pollinators of E. virginiana by “dusting” captured ants with pollen and testing the viability of pollen after removal from the ants. Using 2,5-diphenyl tetrazolium bromide (MTT) in a 40% sucrose solution, we tested the viability of the pollen found on E. virginiana flowers and ants. This chemical stains viable pollen a dark purple indicating the presence of dehydrogenase, which is critical for viable pollen development (Wang et al. 2005). This pollen viability test is considered a good test for viable pollen as it does not stain dead or aborted pollen (Rodriquez-Riano and Dafni 2000). We followed the preparation methods for MTT explained by Hecker (1963). To place pollen onto ants, live ants were affixed onto slides with a thin coating of Elmer’s glue. Anthers containing pollen were moved over each ant’s body several times. We “dusted” a total of 27 ants (25 P. imparis and 2 Crematogaster sp.). After 30 minutes, ants were removed from the glue and MTT was dripped over the specimens onto a slide to remove and test the pollen viability. Ants were then observed by a dissecting scope to ensure that all pollen had been removed. We counted viable and non-viable pollen found on the slides and compared it to pollen taken directly from a flower . 663 A.P. Abbate and J.W. Campbell 2013 Southeastern Naturalist Vol. 12, No. 3 Results During the 20 observation hours in 2011, we observed only two insect visitors: Bombus impatiens Cresson (Eastern Bumble Bee) and Prenolepis imparis Say (Winter or Beech Ant). However, P. imparis accounted for 92.5% of all observed insect visitors (n = 40). Most insect visitors were observed during morning hours, and no visitations were documented during midday or night. We collected both P. imparis and B. impatiens specimens from E. virginiana chasmogamous flowers and photographed them with a scanning electron microscope (Jeol JSM-35CF). The photos revealed E. virginiana pollen adhered to both bee and ant bodies. At all locations in 2012, we visually observed ants rapidly moving from flower to flower presumably seeking nectar produced by chasmogamous flowers. Ant visitation occurred during the morning and afternoon but not during midday or night. Ants were also observed moving from plant to plant in areas where Epifagus plants overlapped. We found two ant species (P. imparis and Crematogaster sp.) in the Piedmont and one ant species (P. imparis) in the Coastal Plain visiting open Epifagus flowers. Although we observed ants in the Cumberland Plateau visiting open flowers, we were unable to capture any. Table 1 details our field observations. Overall, we found high percentages of viable pollen from flowers (81–100%) and pollen removed from ant bodies (65–91%) in all three Epifagus populations that we were able to capture ants. A t-test showed no significant difference (P > 0.05) for pollen viability between pollen taken directly from Epifagus flowers and pollen removed from ants’ integument. Discussion Although ant pollination is rare and potentially overlooked, there are a few documented ant-pollinated plants in North America: Polygonum cascadense Baker (Cascade Knotweed; Hickman 1974), Diamorpha smallii Britton (Elf Orpine; Wyatt 1981), and Paronychia pulvinata Gray (Rocky Mountain Nailwort; Puterbaugh 1998). Cytinus hypocistis (L.) L., a root holoparasite found in the Mediterranean area, is the only parasitic plant that has been documented to Table 1. Percentage of flower visitors observed at each field observation site with the actual number of insects observed in parentheses. Percentages for PEC includes data gathered during 2011 and 2012 field seasons, whereas all other locations are for 2012 onl y. LocationA % ants observed % Bombus observed # hours of observation PEC 93.9 (46) 6.1 (3) 25 UW 100.0 (15) 0.0 5 BD 100.0 (13) 0.0 8 CP 100.0 (4) 0.0 3 Total 96.3 (78) 3.7 (3) 41 APEC = Piedmont Environmental Center, UW = Uwharrie National Forest, BD = Beaver Dam Park, CP = Cumberland Plateau. A.P. Abbate and J.W. Campbell 2013 Southeastern Naturalist Vol. 12, No. 3 664 be cross-pollinated by ants (De Vega et al. 2009). However, Kawakita and Kato (2002) discovered that Balanophora kuroiwai Makino, another root holoparasite, was pollinated within an inflorescence (geitonogamy) by ants. Epifagus virginiana fits the criteria for ant pollination, including having a high density of very small plants overlapping at a uniform height (Wyatt 1981), small inconspicuous flowers, and flowers attached close to the stem. Our results suggest that E. virginiana potentially depends on ants as vectors for pollen transfer from plant to plant, or geitonogamy. During our field observations, P. imparis (Winter or Beech Ant) was the dominant species observed visiting E. virginiana flowers. Bombus impatiens and Crematogaster sp. were infrequent visitors and rarely observed. Prenolepis imparis is tolerant of low temperatures (Talbot 1943). This ant species is frequently active at temperatures below 10 °C and avoids temperatures in excess of 26 °C (Lynch et al. 1980), which explains why P. imparis was observed during the E. virginiana flowering season (September) and not observed during midday. Prenolepis imparis are generally found in oak/beech-dominated forests where they nest in soil and have a generalist diet. Their overall range overlaps with F. grandifolia and E. virginiana. Our data suggests that P. imparis may be a potential pollinator for the chasmogamous flowers of E. virginiana. During our observations, ants were observed moving between plants that were overlapping, which potentially allowed cross pollination. Although E. virginiana populations are not entirely dependent on cross pollination to survive, the chasmogamous flowers and general characteristics of the plant appear to have evolved traits to attract insect visitors (e.g., ants) that are abundant within their inconspicuous habitats that rarely receive more common pollinators. Acknowledgments We thank Dick Thomas for giving us permission to use the Piedmont Environmental Center and Victor Abercrombie for use of his land. We also thank Eleanor Russell for introducing us to this unique plant. The authors also gratefully acknowledge the Undergraduate Research and Creative Works office at High Point University for funding. Literature Cited Beattie, A.J., C. Turnbull, R.B Knox, and E.G. Williams. 1984. Ant inhibition of pollen function: A possible reason why ant pollination is rare. American Journal of Botany 71:421–426. Cooke, E., and A.F. Schively. 1904. Observations on the structure and development of Epiphegus virginiana. 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