D IFFERENTIAL POLLINATOR EFFECTIVENESS AND IMPORTANCE

American Journal of Botany 99(3): 448 458. 2012. D IFFERENTIAL POLLINATOR EFFECTIVENESS AND IMPORTANCE IN A MILKWEED ( A SCLEPIAS, APOCYNACEAE) HYBRID ZONE 1 T ERESA M. STOEPLER 2,7, A NDREA E DGE 3, A NNA S TEEL 4, R OBIN L. O QUINN 5, AND M ARK F ISHBEIN 6 2 The George Washington University, Department of Biological Sciences, 2023 G St. NW, Suite 340, Washington, District of Columbia 20052-0086 USA; 3 University of Louisiana at Lafayette, Department of Biology, P.O. Box 42451, 300 East St. Mary Blvd., Lafayette, Louisiana 70504 USA; 4 Graduate Group in Ecology, University of California, Davis, 1005 Wickson Hall, One Shields Avenue, Davis, California 95616 USA; 5 Department of Biology, Eastern Washington University, 234-C Science Building Cheney, Washington 99004-2440 USA; and 6 Oklahoma State University, Department of Botany, 301 Physical Science, Stillwater, Oklahoma 74078-3013 USA Premise of the study: Exceptions to the ideal of complete reproductive isolation between species are commonly encountered in diverse plant, animal, and fungal groups, but often the causative ecological processes are poorly understood. In flowering plants, the outcome of hybridization depends in part on the effectiveness of pollinators in interspecific pollen transport. In the Asclepias exaltata and A. syriaca (Apocynaceae) hybrid zone in Shenandoah National Park, Virginia, extensive introgression has been documented. The objectives of this study were to (1) determine the extent of pollinator overlap among A. exaltata, A. syriaca, and their hybrids and (2) identify the insect taxa responsible for hybridization and introgression. Methods: We observed focal plants of parental species and hybrids to measure visitation rate, visit duration, and per-visit pollinia removal and deposition, and we calculated pollinator effectiveness and importance. Key results: Visitation rates varied significantly between the 2 yr of the study. Overall, Apis mellifera, Bombus sp., and Epargyreus clarus were the most important pollinators. However, Bombus sp. was the only visitor that was observed to both remove and insert pollinia for both parent species as well as hybrids. Conclusions: We conclude that Bombus may be a key agent of hybridization and introgression in these sympatric milkweed populations, and hybrids are neither preferred nor selected against by pollinators. Thus, we have identified a potential mechanism for how hybrids act as bridges to gene flow between A. exaltata and A. syriaca. These results provide insights into the breakdown of prezygotic isolating mechanisms. Key words: Apocynaceae; Asclepias exaltata; Asclepias syriaca ; hybridization; introgression; pollen deposition; pollen removal; pollination effectiveness; pollinator importance; pollinia. Exceptions to the ideal of complete reproductive isolation between species are commonly encountered in diverse plant, animal, and fungal groups, but often the ecological processes that result in incomplete reproductive barriers are poorly understood ( Helbig et al., 2001 ; Sloan et al., 2008 ; reviewed in Rundle and Nosil, 2005 ). Hybridization is an important evolutionary process that can increase intraspecific diversity, blur species boundaries through the incorporation of alleles from one species into another, or lead to speciation ( Anderson and Hubricht, 1938 ; Arnold, 1997 ; Rieseberg, 1997 ). Hybridization has occurred extensively throughout the evolutionary history of 1 Manuscript received 14 June 2011; revision accepted 3 January 2012. The authors thank S. Broyles for sharing his insights about the Shenandoah National Park hybrid zone, J. Vick for field assistance and insect identifications, the DC Plant-Insect Group, J. Steets, S. Kephart, S. Weller, G. Aldridge, and one anonymous reviewer for providing helpful comments on earlier drafts, and J. T. Lill, S. M. Murphy, and M. Palmer for assistance with statistical analyses. They are grateful to the USA National Park Service for permission to conduct the study and the assistance of W. Cass. This research was funded by a Research Experiences for Undergraduates site at Mississippi State University (NSF Grant #DBI- 0244113 to G. Thibaudeau) and USDA grant #2003-34506-13768 to M.F. 7 Author for correspondence (e-mail: teresa.stoepler@gmail.com); tel.: (202) 994-4408; fax: (202) 994-6100 doi:10.3732/ajb.1100272 flowering plants, despite selection for strong pre- and postzygotic isolating mechanisms (reviewed in Rieseberg, 1997 ; Soltis and Soltis, 2009 ). Hybrids may act as either a barrier or a bridge to gene exchange between parental species. In biotically pollinated plants, if pollinators do not discriminate between parents and hybrids, hybrids are more likely to act as a bridge for gene flow (Grant, 1993 ; Emms and Arnold, 2000 ; Wesselingh and Arnold, 2000 ; Broyles, 2002 ; Campbell, 2003 ). Hybrid bridges are predicted to result in a higher frequency of backcrossing and introgression and potentially lead to decreased reproductive isolation of the parent species ( Grant, 1993 ; Leebens-Mack and Milligan, 1998 ). In contrast, parental pollinators may not be attracted to novel hybrid phenotypes, resulting in hybrid individuals that are reproductively isolated ( Vereecken et al., 2010 ). If novel hybrid phenotypes are unattractive to all pollinators, they potentially could act as a genetic dead end. Under a third scenario, substantial pollen transfer among hybrids may occur if new pollinators are attracted to their phenotypes ( Arnold, 1992 ; Vereecken et al., 2010 ). Depending on the fitness of hybrids, this eventually could lead to hybrid speciation ( Arnold, 1997 ). Despite the importance of pollinator behavior in mediating hybridization, the extent of pollinator fidelity to hybrid vs. parental floral cues and the patterns of pollen movement in natural hybrid zones are not well understood ( Campbell et al., 2002 ). American Journal of Botany 99(3): 448 458, 2012; http://www.amjbot.org/ 2012 Botanical Society of America 448

March 2012] STOEPLER ET AL. POLLINATOR EFFECTIVENESS IN A MILKWEED HYBRID ZONE 449 A key factor determining the outcome of hybridization is the effectiveness of pollinators in pollen transfer between parental species and hybrids. Pollinator effectiveness has been variously defined and measured but refers generally to the relative contribution of a flower-visiting taxon to the fitness of a focal plant species ( Herrera, 1987 ; Ivey et al., 2003 ; reviewed by Ne eman et al., 2010 ). Pollinator effectiveness can be broken down into three main components: the rate of visitation, the rate of pollen removal, and the efficiency of pollen deposition ( Herrera, 1987 ; Inouye et al., 1994 ; Fishbein and Venable, 1996a ). To clarify the distinction between per-visit measures of effectiveness (i.e., removal and deposition rates) and composite measures that include visitation rates, hereafter we will refer to composite measures as pollinator importance and restrict the use of pollinator effectiveness to per-visit measures. Pollination studies have traditionally employed incomplete measures of pollinator importance such as visitation rate alone or inspection of captured insects for pollen grains ( Beattie, 1972 ; Ehrenfeld, 1979 ; Sugden, 1986 ; Young, 1988 ). Although these studies have provided useful estimates of pollinator importance, they cannot detect when visitors are effective at one component of pollination but ineffective at others, rendering visitors unimportant overall ( Thomson and Thomson, 1992 ; Fishbein and Venable, 1996a ; Fenster et al., 2004 ). For example, skipper butterflies (Hesperiidae) accounted for 21% of the total visits to the tropical herb Calathea ovandensis Matuda, yet these visits resulted in only 1% of total fruit set ( Schemske and Horvitz, 1984 ). Comprehensive studies of pollinator effectiveness have become more common but are still the exception rather than the rule (e.g., Mayfield et al., 2001 ; Ivey et al., 2003 ; Fumero-Cab á n and Melend é z-ackerman, 2007 ; Sahli and Conner, 2007 ; Theiss et al., 2007 ). Milkweeds (Asclepias spp., Apocynaceae) are an ideal system in which to study how variation in pollination effectiveness influences hybridization dynamics. In milkweeds, pollen is transferred in discrete masses called pollinia, allowing pollen transfer to be accurately quantified in the field ( Wyatt and Broyles, 1994 ; Fishbein and Venable, 1996a ). Milkweeds are diverse in North America, with over 120 species, most of which occur sympatrically with 1 10 of their congeners ( Woodson, 1954 ; M. Fishbein, unpublished data). Several species pairs are known to form viable F l hybrids and backcrosses in natural populations ( Woodson, 1954 ; Kephart et al., 1988 ; Broyles et al., 1996 ; Broyles, 2002 ; Klips and Culley, 2004 ). Asclepias syriaca L., common milkweed, and A. exaltata L., poke milkweed, are sympatric across eastern North America (Woodson, 1954 ). Asclepias exaltata is mostly found in forest understories, whereas A. syriaca grows in open fields and along roadsides ( Broyles et al., 1996 ). Despite distinct habitat preferences, human disturbance in the form of roads and power-line cuts has brought the two species into closer contact and has increased the opportunity for hybridization ( Wyatt et al., 1993 ; Wyatt, 1996 ). The two species co-occur commonly in forest openings at Shenandoah National Park (SNP), Virginia, USA, where they exhibit partially overlapping flowering phenologies and cross to form viable hybrid offspring ( Broyles, 2002 ). These species exhibit distinct floral morphologies ( Fig. 1 ), including differences in flower color, shape, scent, and the number of flowers per umbel (inflorescence unit), which may provide a basis for pollinator discrimination ( Kephart et al., 1988 ; Broyles et al., 1996 ; M. Fishbein, unpublished data). Nonetheless, hybridization and bidirectional allozyme introgression between the two species occur, though hybrid formation appears to be rare ( Broyles et al., 1996 ; Broyles, 2002 ). Generalist hymenopteran and lepidopteran pollinators of these species at SNP ( Broyles et al., 1996 ) are typical of these and other common and widespread milkweeds ( Robertson, 1891, 1928 ; Betz et al., 1994 ; Wyatt and Broyles, 1994 ; Fishbein and Venable, 1996a ; Theiss et al., 2007 ). Because the SNP A. syriaca A. exaltata hybrid zone has been the subject of intensive genetic study, it provides an excellent system by which to investigate the role of pollinators in mediating hybridization and gene flow. Although introgression between A. exaltata and A. syriaca has been documented and pollen-carrying insects identified, the relative importance of each of the pollinators remains unknown. The current study was designed to provide a mechanistic explanation based on pollinator behavior for the documented pattern of introgression. Specifically, we aimed (1) to determine which insect taxa visit the sympatric milkweeds A. exaltata, A. syriaca, and their hybrids and (2) to test the effectiveness of these insect visitors as pollinators by quantifying the number of pollinia removed and inserted per flower per visit by observing pollinators on focal plants in the field. Removal and insertion measures provide separate estimates of both the effectiveness and importance of floral visitors to each milkweed s male and female fitness components ( Fishbein and Venable, 1996b ). MATERIALS AND METHODS Study site and species We conducted this study at SNP from June 27 to July 6, 2004, and from June 22 to July 6, 2005. This 793 000-km 2 park is located in the Blue Ridge Mountains in the southern Appalachians and is dominated by deciduous forests with many natural and manmade clearings. The Blue Ridge Parkway (Skyline Drive) runs the length of the park along the mountain crest and is characterized by abundant edge and meadow habitat. We established study sites at scattered locations throughout SNP that differed in milkweed composition (i.e., mixtures of A. exaltata, A. syriaca, or hybrid; Appendix 1). In the two years of the study, we used overlapping but non-coincident study sites (Appendix 1). Asclepias exaltata grows primarily in the understory of eastern deciduous forest communities, whereas A. syriaca occurs in a variety of open habitats, including prairies, glades, old fields, roadsides, and other disturbed sites ( Woodson, 1954 ; M. Fishbein, personal observation). At SNP the species exhibit predicted habitat preferences, but they commonly co-occur along roadsides and in clearings, especially along Skyline Drive ( Broyles, 2002 ). Asclepias exaltata and A. syriaca differ in peak flowering period but have overlapping phenologies that permit hybridization. Across their ranges, A. exaltata begins to flower earlier than A. syriaca, and at SNP the overlap in flowering lasts for several weeks ( Broyles et al., 1996 ; Broyles, 2002 ). Because of weather differences between the 2 yr of sampling, we performed observations from peak to late flowering for A. exaltata and early to peak flowering for A. syriaca in 2004 and from early to peak flowering for A. exaltata and early flowering for A. syriaca in 2005. We identified hybrids as plants expressing intermediate states of a suite of quantitative floral and vegetative characters diagnostic of the two parent species ( Table 1, Fig. 1 ). On the basis of the distinct intermediacy of these traits, we are confident that the individuals we identified as hybrids are not genetically pure examples of either parent species and are most likely F 1 s (cf. Broyles, 2002 ); however, it is possible that some of the sampled individuals may be later-generation hybrids or backcrosses. With the use of allozyme markers, Broyles (2002) confirmed that morphologically intermediate plants that appear to be F 1 s have a high probability of being so. Such individuals are rarely encountered at SNP ( Broyles, 2002 ; M. Fishbein, personal observation); however, we intentionally sought out and sampled these putative F 1 individuals. We excluded individuals that appeared to represent backcrosses that were morphologically closer to one of the parents. For convenience, each of Asclepias syriaca, A. exaltata, and their hybrid will be referred to hereafter as a milkweed morphotype. Pollinator observations on focal plants We marked and bagged a single umbel on each focal plant of A. exaltata, A. syriaca, and hybrids ( N = 35, 23, and 26, respectively, in 2004 and N = 66, 54, and 23, respectively, in 2005)

450 AMERICAN JOURNAL OF BOTANY [Vol. 99 Fig. 1. Flowers of Asclepias syriaca, A. exaltata, and their hybrid at Shenandoah National Park, Virginia, showing intermediacy of hybrid morphology. (A) A. syriaca. (B) F 1 hybrid. (C) A. exaltata. before anthesis to exclude visitors before observation periods. White bridal veil bags were secured with twist ties; these bags have been shown to minimally affect ambient environmental conditions around milkweed flowers and to have little effect on nectar volume and sucrose concentration ( Wyatt et al., 1992 ). We acknowledge that through bagging we may have created increased nectar volumes compared with unbagged umbels and that this may have biased visit duration. However, bagging mimics the natural situation when flowers first open or when their nectar has been recharged ( Wyatt and Shannon, 1986 ). A single observer conducted all observations on a single focal plant. The observer conducted measurements of insect visitation rate and pollinator effectiveness on separate plant individuals. In all cases, the focal plant contained only a single umbel that was in flower at the time of observation (the focal umbel). The observer removed the pollination bag from this umbel, counted the number of open flowers, and immediately monitored the umbel from a chair at a distance of circa 10 m using binoculars to avoid affecting visitor behavior. Observers counted only open flowers; however, no umbels contained senescent flowers, and few contained unopened buds. To control for microsite variation, T ABLE 1. Diagnostic morphological characters of Asclepias exaltata, A. syriaca, and their F 1 hybrid. Numbers in parentheses are one standard deviation from the mean. From Kephart et al. (1988), except for number of flowers/umbel and pedicel length (M. Fishbein, unpublished data). Milkweed morphotype Character A. exaltata Hybrid A. syriaca Mean number of flowers/ 23 (7) 38 (15) 67 (20) umbel Mean pedicel length (cm) 3.6 (0.6) 3.4 (0.6) 2.4 (0.5) Corolla color Greenish Green lobes, pink base Light rose to deep pink Corolla lobe posture Strongly reflexed Reflexed with spreading tips Reflexed with spreading tips Corona color White Light pink Rose to pink Corona segment shape Truncate Intermediate Tapered Lower leaf pubescence None Moderately tomentose Densely tomentose Leaf base Cuneate Obtuse to cuneate Rounded to obtuse we paired each plant used for observation of visitation rate with an adjacent plant (within 5 m, almost always within 2 m) of the same morphotype to be used for measurement of pollinator effectiveness during a separate observation period. We staggered observations throughout the day when insect visitors were most active (between 0800 and 1600 h), under a variety of weather conditions ranging from hot, clear, and still to cool, overcast, and windy. We did not conduct any nocturnal observations because visitation during the night was uncommon (M. Fishbein, personal observation; however, on the evening of July 5, 2004, abundant visitation by a small moth was observed on both parental species as well as hybrids; see also Bertin and Wilson, 1980 ; Jennersten and Morse, 1991 ). We stopped observations for moderate to heavy rain, since visitation ceased during these periods. In a few cases, storms or wilted flowers made one of the pairs unsuitable for observation, resulting in a few unpaired samples. In both years, voucher specimens of common visitors were deposited in the Mississippi State University Entomological Museum, Starkville, Mississippi. Insect visitation rate To measure insect visitation rate, we observed each focal umbel for a 30-min period during which we recorded and identified to the lowest taxonomic level possible every visitor to the inflorescence and measured the duration of the visit using a stopwatch. We defined a visit as an insect physically contacting the focal umbel for at least 1 s during an observation period. Because it was impossible to collect visiting insects without disrupting observation periods, we used binoculars to identify all visitors while they were on the umbel; thus, we often were only able to identify visitors to genus or higher taxonomic rank. In 2004, three observers completed 24.5 h of total observation time on A. exaltata ( N = 20), A. syriaca ( N = 13), and hybrid ( N = 15) individuals (total = 48 plants). In 2005, four observers completed 33.5 h of total observation time on A. exaltata (N = 29), A. syriaca (N = 28), and hybrid ( N = 11) individuals (total = 68 plants). Pollinator effectiveness To measure pollinator effectiveness, we recorded the number of pollinaria (pair of pollinia, typically five per flower; for clarity we will refer to these as pollinia hereafter) removed and inserted and the visit duration by each of the first five insect visitors to the focal umbel. We made these measurements on paired plants during observation periods separate from those described previously to measure visitation rates. Before the arrival of each visitor, the observer counted the total number of pollinia in the focal umbel with a hand lens. Immediately after an individual insect s visit, the observer recounted the total number of pollinia in the inflorescence, which enabled the calculation of the number of pollinia that had been removed in that single visit.

March 2012] STOEPLER ET AL. POLLINATOR EFFECTIVENESS IN A MILKWEED HYBRID ZONE 451 The insertion of a pollinium was often visible externally because the other member of the pollinarium or the broken translator ( arm attaching pollinia pairs) projected outward from the stigmatic opening. This method provides a conservative estimate of the number of pollinia inserted; in the closely related species A. tuberosa L., the actual number of insertions is 2.3 times higher than estimates made using this method, and the true value is highly correlated ( Fishbein and Venable, 1996a ). The observer repeated this process for the next four visitors, for which we assume that the small number of pollinia removed and inserted during previous visits had a negligible effect on the probability of pollinia transfer. If more than 30 min lapsed between successive visitors, we terminated the observation period. This occurred for one A. exaltata individual in 2004 and for 20 individuals in 2005 ( A. exaltata : N = 12, A. syriaca : N = 4, and hybrid: N = 4). In 2004, we performed pollinator effectiveness observations on 15 A. exaltata, nine A. syriaca, and 11 hybrid individuals (total = 35 plants). In 2005, we performed pollinator effectiveness observations on 38 A. exaltata, 28 A. syriaca, and 12 hybrid individuals (total = 78 plants). To estimate the overall pollinator importance of each visitor group (see Data analysis ), we multiplied the mean number of visits per 30-min observation period per umbel by the mean number of pollinia removed per flower per visit (male function), and by the mean number of pollinia inserted per flower per visit (female function) for each milkweed morphotype for each year of the study ( Fishbein and Venable, 1996a ; Larsson, 2005 ; Fumero-Cab á n and Mel é ndez-ackerman, 2007 ; Sahli and Conner, 2007 ). To calculate the pollen transfer efficiency (PTE) of each visitor group, we used an equation following Larsson (2005) : PTE pollinia inserted per flower per visit pollinia removed per flower per visit The advantage of measuring pollinator importance is that it identifies the most important pollinator over time; the advantage of measuring pollinator efficiency is that it measures the optimality of a particular pollinator in a single visit. For example, a pollinator may be unimportant but efficient if it has a high ratio of pollinia insertions/removals but is an infrequent visitor. Conversely, an important pollinator may be somewhat inefficient in single visits but can have a significant effect through frequent flower visits. Data analysis We grouped floral visitors (unless noted otherwise) into four main categories corresponding to each of the three most frequent taxa and a fourth category containing all others: Apis ( Apis mellifera Linnaeus, Hymenoptera: Apidae, honeybee), Bombus ( Bombus sp. Latreille, Hymenoptera: Apidae, bumblebee), Epargyreus ( Epargyreus clarus Cramer, Lepidoptera: Hesperiidae, silver-spotted skipper), and Other (Appendix 2). Multiple species of Bombus may be present at SNP, but we were unable to determine species identities from observation posts. Because pollination systems are commonly inferred to be specialized for particular insect families or orders (e.g., bee flowers or butterfly flowers, Faegri and van der Pijl, 1979 ; but see Fishbein and Venable, 1996a ; Waser et al., 1996 ), we conducted tests to compare the most important higher taxa of visitors, Hymenoptera, Lepidoptera, and Diptera, by pooling visitors into these groups. We analyzed data from 2004 and 2005 separately in most cases because of differences in the composition of the pollinator fauna and the overlapping but non-coincident study sites. Despite these limitations, between-year comparisons of pooled pollinator groups were used to gauge the degree of interannual variability; these analyses should be interpreted with appropriate caution. Because only two of the same sites were used in both years of the study (Big Meadows and Naked Creek Overlook; Appendix 1), we did not analyze site differences in insect visitation or pollinator effectiveness. However, in comparing patterns of visitation and effectiveness in the whole data set to the subset of the Big Meadows and Naked Creek sites, we found that the patterns were qualitatively similar (data not shown). For many analyses, normality assumptions were not met, even under a variety of scale transformations. In these cases, nonparametric tests were used. Before all analyses, we examined the residuals to ensure the assumption of homogeneity of variance was met. Because the three milkweed morphotypes differed in the number of flowers per umbel ( Table 1 ), and this trait is known to affect pollinator effectiveness in milkweeds ( Fishbein and Venable, 1996b ), we scaled effectiveness components by the number of flowers per umbel (e.g., pollinia removal was analyzed as the number of pollinia removed per observation period per fl ower ). However, we did not scale visitation rates by the number of flowers because the number of visits per observation period was independent of the number of flowers per umbel ( R 2 = 0.004, df =114, P = 0.521). We tested for variation across milkweed morphotypes in visitation rate, pooled among all insect taxa, using ANOVA. For each morphotype, we evaluated differences between years in visitation rate, pooled among all insect taxa, with Kruskal-Wallis tests. We tested for main effects of insect taxa, milkweed morphotype, year, and their interactions using ANOVA on rank-transformed visitation rates ( Conover and Iman, 1981 ; SAS Institute, 2008 ). We also evaluated variation across milkweed morphotypes in visitation rate for each of the four main visitor groups and for pooled Hymenoptera, Lepidoptera, and Diptera with ANOVA. We used logistic regression to test whether visit duration affected whether pollinia were removed or inserted (tested separately) during one visit. We used G tests to evaluate variation across milkweed morphotypes in the frequency in which any pollinia were removed or inserted (tested separately) in a single visit for each of the main insect groups. We evaluated variation across milkweed morphotypes in the number of pollinia removed per flower per visit ( removal rate hereafter) for each of the main insect groups with two-way ANOVA on rank-transformed pollinia removal rate. All significant Kruskal-Wallis tests were followed by Mann-Whitney U post hoc tests ( Dytham, 2003 ) using the Bonferroni correction to control for type I error inflation (Holm, 1979 ), and all significant ANOVAs were followed by Tukey honestly significant difference (HSD) post hoc tests. Sample sizes were too small to test for differences among morphotypes or insect groups in pollinia insertions. We conducted all statistical analyses with JMP v. 8.0.2 (SAS Institute, Cary, North Carolina, USA) or SAS v. 9.1.3 (SAS Institute). RESULTS Insect visitation rate Flower-visiting insects included diverse members of Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera (Appendix 2). The most common visitors were Apis mellifera, Bombus sp., and Epargyreus clarus (Appendix 2). These three primary visitors hereafter will be referred to as Apis, Bombus, and Epargyreus. We observed a total of 522 visits to focal umbels in 2004 (178 to A. exaltata, 115 to hybrids, and 229 to A. syriaca ) and 657 in 2005 (362 to A. exaltata, 60 to hybrids, and 235 to A. syriaca ) ( Fig. 2 ). Differences in the mean number of visits per umbel per 30-min observation period (hereafter visitation rate ) among the three morphotypes were significant in both years ( Fig. 2 ; 2004: F 2, 44 = 5.26, P = 0.0089; 2005: F 2, 62 = 6.84, P = 0.0021). In 2004, Asclepias syriaca had a visitation rate that was twice that of the hybrids (Tukey HSD: P = 0.013) and 1.77 times higher than that of A. exaltata (P = 0.036). However, in 2005 A. exaltata had a visitation rate 2.2 times higher than did hybrids (Tukey HSD: P = 0.0020). The visitation rate to focal umbels by all visitors combined was not significantly different between years for A. exaltata or for hybrids ( H = 1.86, df = 1, P = 0.17 and H = 0.93, df = 1, P = 0.33, respectively), but A. syriaca received twice as many visits in 2004 than in 2005 ( H = 4.10, df = 1, P = 0.043; Fig. 2 ). The four main visitor groups visited focal umbels at significantly different rates depending on morphotype ( Fig. 3 ). In a three-way ANOVA on ranked visitation rates, variation was not significant between years, which were pooled for further analysis (Appendix S1, see Supplemental Data with the online version of this article). In a two-way ANOVA, the interaction of visitor group and morphotype and the main effect of visitor group were significant, but the main effect of morphotype was not (Appendix S1, see Supplemental Data with the online version of this article). To clarify the pattern of variation across visitor groups, we conducted one-way analyses for each morphotype. In 2004, differences in visitation rate to A. exaltata (H = 27.00, df = 3, P < 0.0001) and to A. syriaca ( H = 28.48, df = 3, P < 0.0001) were significant but not to hybrids ( H = 2.63, df = 3, P = 0.45; Fig. 3A ). In 2005, insect groups differed significantly in visitation rate for all three morphotypes: A. exaltata

452 AMERICAN JOURNAL OF BOTANY [Vol. 99 Fig. 2. Mean number of visits per 30-min observation period per umbel by all insect visitors combined to Asclepias exaltata, hybrids, and A. syriaca in 2004 and 2005. Error bars represent ± 1 SE. Letters above bars represent significant differences among milkweed morphotype means within year according to Tukey honestly significant difference tests ( α = 0.01). ( H = 44.46, df = 3, P < 0.0001), A. syriaca (H = 12.65, df = 3, P = 0.0055), and hybrids ( H = 12.17, df = 3, P = 0.0007; Fig. 3B ). Individual insect groups exhibited significant variation in visitation rates between years. Whereas Bombus visitation rates across all morphotypes were on average over 3 times higher in 2005 compared with 2004, the overall rate of visitation by Epargyreus was over 3 times higher in 2004 compared with 2005 (compare Fig. 3A and 3B ). These taxa also exhibited differential patterns of visitation to morphotypes: Bombus visited A. exaltata and A. syriaca at rates over 2.5 higher in 2005 than in 2004, but Epargyreus visited hybrids and A. syriaca at 2.3 and 5 times higher rates, respectively, in 2004 than in 2005 (compare Fig. 3A and 3B ). Also of note were the especially low visitation rates of Apis on A. exaltata compared with other visitors and the absence of Bombus visitation to A. syriaca in 2004 ( Fig. 3A ). Visitation rates to the three morphotypes also varied when insects were grouped by order, with higher overall visitation rates by Hymenoptera (especially in 2005) and preferences for parental over hybrid morphotypes in 2005 that were much stronger for Lepidoptera than Hymenoptera (Appendix S2). Pollinator effectiveness Roughly one-third (167 of 525, or 31.81%) of the total insect visits to pollinator effectiveness focal plants resulted in the removal of at least one pollinium. Pollinia insertions were rarely observed, with 4.76% (25 of 525) of visits resulting in at least one insertion. For those visitors observed to remove pollinia from at least one milkweed morphotype, longer visits were significantly more likely to result in successful pollinia removal ( χ 2 = 13.18, df = 1, P = 0.0003). On average, visits resulting in successful pollinia removal were 1.6 times longer than unsuccessful visits. This pattern was especially evident for Apis (χ 2 = 24.07, df = 1, P < 0.0001) and Bombus (χ 2 = 16.17, df = 1, P < 0.0001). Similarly, longer visits were significantly more likely to result in successful pollinia insertion ( χ 2 = 8.16, df = 1, P = 0.0043), and visits resulting in successful pollinia insertion averaged twice as long as unsuccessful visits. None of the three primary visitors varied significantly in the frequency of successful visits (i.e., those in which at least one Fig. 3. Mean number of visits per 30-min observation period per umbel by the four main insect visitor groups to Asclepias exaltata, hybrids, and A. syriaca in (A) 2004 and (B) 2005. Error bars represent ±1 SE. Letters above bars represent significant differences among insect group medians within each milkweed morphotype according to Bonferroni-corrected Mann Whitney U tests ( α = 0.008). pollinium was removed) across morphotypes, pooled across years ( Apis : G = 3.94, df = 2, P = 0.14; Bombus : G = 2.94, df = 2, P = 0.23; Epargyreus : G = 1.88, df = 2, P = 0.39). Similarly, the frequency of successful visits pooled across insect visitors within years did not vary across morphotypes in either 2004 ( G = 1.79, df = 2, P = 0.41) or in 2005 ( G = 4.90, df = 2, P = 0.086). However, Bombus removed pollinia significantly more frequently than Epargyreus from hybrids in 2004, and Epargyreus removed pollinia significantly more frequently than Bombus from A. exaltata in 2005, when differences among visitors were tested separately for each morphotype (Appendix S3). Removal rate, or the number of pollinia removed per flower per visit, also differed by morphotype and insect visitor ( Fig. 4A, B ). For example, on hybrids in 2004, Bombus removed pollinia at a rate that was 11 times higher than that of Epargyreus, a significant difference ( Fig. 4A; Appendix S3). In 2005, there was a trend such that Epargyreus removed pollinia at a rate 1.4 times higher than that of Bombus on A. syriaca ( Fig. 4B; Appendix S3). We made too few effectiveness observations for A. syriaca in 2004 to assess differences in removal rates; however, Bombus was never observed to visit A. syriaca in that year, and only three Apis visits (one resulting in pollinia removal) were observed.

March 2012] STOEPLER ET AL. POLLINATOR EFFECTIVENESS IN A MILKWEED HYBRID ZONE 453 Fig. 4. Mean number of pollinia removed per flower per visit in (A) 2004 and (B) 2005 and mean number of pollinia inserted per flower per visit in (C) 2004 and (D) 2005 from the three milkweed morphotypes by Apis mellifera (Apis ), Bombus sp. ( Bombus ), and Epargyreus clarus ( Epargyreus ). In panels A and B, Other includes all other visitors listed in Appendix 2. Error bars represent ±1 SE. Bombus and Epargyreus were the only two visitor groups to successfully insert pollinia in more than one milkweed morphotype in the same year ( Fig. 4C, D ); however, the number of insertions per visit was too low to analyze statistically. There was a trend toward a greater effectiveness of Bombus across morphotypes in that Bombus was the only visitor observed to insert pollinia in hybrid flowers in 2004 ( Fig. 4C ) and was the only visitor that ever inserted pollinia in all three morphotypes ( Fig. 4D ). Pollinator importance Patterns of overall pollinator importance (visitation rate pollination rate) differed between male (pollinia removal) and female (pollinia insertion) components. In both 2004 and 2005, Bombus was the most important pollinator of A. exaltata and hybrids through male function ( Fig. 5A, B ). We did not observe Bombus to visit A. syriaca in 2004, though it was one of the most important pollinators of this species in 2005. Apis was the most important pollinator through male function for A. syriaca in 2004, and it was among the most important pollinators for this species in 2005. Apis also contributed to hybrid fitness through male function in both years though much less than did Bombus. However, Apis was unimportant for A. exaltata in both 2004 and 2005 ( Fig. 5A, B ). Epargyreus was moderately important for pollinia removal for A. exaltata in both years; however, it was unimportant for A. syriaca and hybrids in both years. Other taxa were mostly unimportant except on A. syriaca in 2005, which had pollinia removals by megachilid bees (2 observations), small solitary bees (2 observations), a single fly, and six lepidopterans, including one unidentified hesperiid, one Papilio glaucus Linnaeus, one Danaus plexippus (Linnaeus), and three individuals of Speyeria sp. In terms of female function, Bombus was the only important pollinator for hybrids in 2004 and was the most important pollinator for A. exaltata in 2005 ( Fig. 5C, D ). Bombus was also one of the most important pollinators of A. syriaca in 2005. Apis was the most important for pollinia insertion for A. syriaca in 2004 and for hybrids in 2005. Epargyreus was one of the most important pollinators of A. syriaca in 2005 and was also moderately important for A. syriaca in 2004 and for A. exaltata in 2005. None of the other visitors were observed to insert pollinia in either year and were thus unimportant overall. Asclepias exaltata had no important pollinators through female function in 2004 because no insertions were observed in this species in that year ( Fig. 5C ). Pollen transfer efficiencies differed among visitor groups, across milkweed morphotypes, and between years ( Table 2 ). Bombus had the highest PTE on hybrids in 2004, whereas Apis had the highest PTE on hybrids in 2005. Bombus PTE values across morphotypes in 2005 were roughly equal. Epargyreus had the highest PTE on A. syriaca and on A. exaltata in 2005, whereas Apis had the highest PTE on A. syriaca in 2004. DISCUSSION To our knowledge, we have conducted one of the first studies of pollination effectiveness in a natural, interspecific hybrid zone. Our results demonstrate that a suite of overlapping, generalist bees and butterflies pollinate the milkweeds Asclepias exaltata and A. syriaca and their hybrid, with strong interannual variation in visitation, pollinator effectiveness, and pollinator importance. Apis mellifera, Bombus sp., and Epargyreus clarus were the only visitors observed to remove pollinia from all three of the milkweed morphotypes, whereas Bombus was the only visitor that also inserted pollinia successfully in both parent species and their hybrid ( Fig. 4 ). These data suggest that Bombus is likely a key pollinator for each species individually

454 AMERICAN JOURNAL OF BOTANY [Vol. 99 Fig. 5. Overall pollinator importance (mean number of visits per observation period mean number of pollinia removed or inserted per flower per visit) of the four insect visitor groups. Overall pollinator importance through male function (pollinia removals) shown in (A) 2004 and (B) 2005 and through female function (pollinia insertions) in (C) 2004 and (D) 2005. and has the potential to be an important agent of introgression between A. syriaca and A. exaltata. Visitation rates to hybrids were not significantly different from those to A. exaltata in 2004 and to A. syriaca in 2005 ( Fig. 3 ). No differences existed in the rates of pollinia removal or insertion by the primary pollinators across the two parent species and hybrid, and in some cases, more pollinia were removed and inserted in hybrids than in the parents ( Fig. 4 ). Taken together, these data suggest that pollinators neither prefer nor select against hybrids in the Shenandoah hybrid zone, consistent with the introgression documented between A. syriaca and A. exaltata at SNP ( Broyles, 2002 ). Because our data are limited to a single geographic area studied in consecutive years, the roles of specific pollinators in this system should not be over-generalized. These findings reinforce patterns of pollinator and visitor overlap to parents T ABLE 2. Mean pollen transfer efficiency (PTE, calculated as the mean number of pollinia inserted per flower per visit/the mean number of pollinia removed per flower per visit) of the three principle insect visitor taxa in 2004 and 2005. PTE of all other insect groups is zero for all milkweed morphotypes in both years. Flower visitor Year Milkweed morphotype Apis Bombus Epargyreus 2004 A. exaltata 0 0 0 A. syriaca 0.042 0 0.033 Hybrid 0 0.129 0 2005 A. exaltata 0 0.025 0.042 A. syriaca 0 0.032 0.083 Hybrid 0.125 0.024 0 and hybrids shown in other hybrid systems such as Ipomopsis ( Campbell et al., 1998 ; Wolf et al., 2001 ) and Penstemon (Kimball, 2008 ), and they support the role of hybrids as genetic bridges in milkweeds ( Broyles, 2002 ). The relative importance of Bombus as an agent of milkweed hybridization emerges from a context of variation in components of pollination effectiveness across morphotype visitor combinations and between years. For example, although less important overall ( Fig. 5 ), Epargyreus appeared to be a more effective visitor to A. exaltata than Bombus through pollinia removal in both years and through pollinia insertion in 2004 ( Fig. 4 ). Similarly, the high pollen-transfer efficiencies of Epargyreus in 2005 ( Table 2 ) are obscured by the relatively modest visitation rates of these insects in that year ( Fig. 3 ), which tempered their overall importance. Such variation underscores the advantage of measuring pollination importance as completely as practicable, rather than relying on proxies such as visitation rates, pollinator pollen loads, or per-visit transfer rates in isolation (e.g., Fumero-Cabán and Melendéz-Ackerman, 2007 ). The species-rich pollinating fauna and substantial temporal variation in pollination importance documented in the SNP hybrid zone are characteristic of the widespread and abundant milkweed species that have been best studied with regard to pollination (reviewed by Fishbein and Venable, 1996a ). However, few previous studies have documented insect visitation in areas where both A. syriaca and A. exaltata occur (but see Broyles et al., 1996 ), and none has measured pollinator effectiveness. The importance of Apis and Bombus corroborates what has been found in previous pollination studies of these milkweeds. Either Apis mellifera ( Willson and Bertin, 1979 ; Pleasants, 1991 ; Betz et al., 1994 ; Shore 1993 ), Bombus spp.

March 2012] STOEPLER ET AL. POLLINATOR EFFECTIVENESS IN A MILKWEED HYBRID ZONE 455 ( Morse, 1982 ; Jennersten and Morse, 1991 ), or both taxa ( Kephart and Theiss, 2004 ; Theiss et al., 2007 ) have been shown to be the most common visitors or the visitors bearing the most pollinia of A. syriaca. Similarly, Betz et al. (1994), found that both Apis mellifera and Bombus pennsylvanicus carried more pollinia than any other visitors to A. exaltata. In the only other study of both milkweeds at a single site, Broyles et al. (1996) found Apis to be the most common visitor to both species, also at SNP. Ours is the first study, to our knowledge, to show that, at least in some years, Epargyreus also may be a comparably important pollinator, especially for A. syriaca. Several plant and pollinator factors could have contributed to the observed high level of interannual variation. Variation in visitation rates between years could be due to fluctuations in pollinator populations, which could in turn alter competition among pollinators for floral resources (nectar in the case of Asclepias flowers). Although Bombus did not visit A. syriaca in 2004, total visitation rates by hymenopterans were roughly the same in 2004 and 2005 (Appendix S2, see Supplemental Data with the online version of this article), suggesting that solitary bees and wasps took the place of Bombus in 2004. Smaller bees and wasps may avoid Bombus by only visiting umbels where Bombus is not present or by foraging in areas where Bombus is not abundant ( Hingston and McQuillan, 1999 ). In addition, although Bombus visitation may vary considerably between years, clonal perennials such as A. syriaca, A. exaltata, and their hybrids are likely to be less sensitive to fluctuations in pollination as many annual, nonclonal species would be, and these characteristics may buffer these milkweed populations against interannual pollinator variability. The high variability in Bombus visitation in the 2 years of the study suggests that Bombus may not represent a consistent means of interspecific pollen transfer. In 2004, Bombus was not observed to visit A. syriaca, and it visited A. exaltata at a rate nearly one-half that in 2005. In 2005, Bombus was one of the most common visitors to A. syriaca, and visitation to A. exaltata was comparatively high. Because Bombus is a key pollinator in this system, opportunities for hybridization between A. exaltata and A. syriaca appear to have been more limited in 2004 compared with 2005. If years like 2004 are common, when Bombus visitation was much less frequent, this may explain why hybrid formation is rare ( Broyles, 2002 ). Epargyreus also appears to be an important pollinator, especially for A. syriaca. Epargyreus was one of the most consistent visitors, especially in 2004 when it contributed over 30% of all visits to the three morphotypes (Appendix 2). Apis appears to be important for pollination of A. syriaca and hybrids, as well as backcrosses involving these morphotypes, but not for F 1 hybrid formation nor for backcrosses to A. exaltata. Importantly, introgression has been shown to be asymmetric (toward A. syriaca ) at SNP ( Broyles, 2002 ), consistent with the direction of backcrossing that would be facilitated by Apis. Future work should explore whether Apis is primarily responsible for the observed asymmetry of introgression. We conclude that there are no pollinator-mediated limitations to hybrids acting as bidirectional bridges for gene flow between A. exaltata and A. syriaca at SNP. Despite high levels of variation between years in floral visitation and pollen transfer rates, the three parental and hybrid morphotypes share the same set of overlapping generalist pollinators. On the basis of consistency in overall importance, Bombus may be a key player in the processes of hybrid formation and introgression within the SNP hybrid zone. For the A. exaltata A. syriaca hybrid zone, we have demonstrated patterns of interannual variation in pollinator importance that help to explain why hybrid formation may be episodic and introgression asymmetric. Similar patterns of temporal variation and asymmetry have been found to be driven by analogous prezygotic processes in a variety of plant and animal hybrid zones ( Green and Parent, 2003 ; Tagane et al., 2008 ; Gomes et al., 2009 ; Arnold et al., 2010 ; Kokudai et al., 2011 ). LITERATURE CITED A NDERSON, E., AND L. H UBRICHT. 1938. Hybridization in Tradescantia. III. The evidence for introgressive hybridization. American Journal of Botany 25 : 396 402. A RNOLD, M. L. 1992. Natural hybridization as an evolutionary process. Annual Review of Ecology and Systematics 24 : 521 524. A RNOLD, M. L. 1997. Natural hybridization and evolution. Oxford University Press, Oxford, UK. A RNOLD, M. L., S. T ANG, S. J. KNAPP, AND N. H. M ARTIN. 2010. Asymmetric introgression hybridization among Louisiana iris species. Genes 1 : 9 22. B EATTIE, A. J. 1972. The pollination ecology of Viola, 2. Pollen loads of insect visitors. Watsonia 9 : 13 25. BERTIN, R. I., AND M. F. WILSON. 1980. Effectiveness of diurnal and nocturnal pollination of two milkweeds. Canadian Journal of Botany 58 : 1744 1746. B ETZ, R. F., R. D. STRUVEN, J. E. WALL, AND F. B. H EITLER. 1994. Insect pollinators of 12 milkweed ( Asclepias ) species. In R. G. Wickett, P. D. Lewis, A. Woodliffe, and P. Pratt [eds.], Proceedings of the Thirteenth North American Prairie Conference: Spirit of the land, our prairie legacy, 45 60. Corporation of the City of Windsor, Windsor, Ontario, Canada. B ROYLES, S. B. 2002. Hybrid bridges to gene flow: A case study in milkweeds ( Asclepias ). Evolution; International Journal of Organic Evolution 56 : 1943 1953. B ROYLES, S. B., C. V AIL, AND S. L. S HERMAN-BROYLES. 1996. Pollination genetics of hybridization in sympatric populations of Asclepias exaltata and A. syriaca (Asclepiadaceae). American Journal of Botany 83 : 1580 1584. C AMPBELL, D. R. 2003. Natural selection in Ipomopsis hybrid zones: Implications for ecological speciation. The New Phytologist 161 : 83 90. CAMPBELL, D. R., N. M. WASER, AND G. T. PEDERSON. 2002. Predicting patterns of mating and potential hybridization from pollinator behavior. American Naturalist 159 : 438 450. C AMPBELL, D. R., N. M. WASER, AND P. G. WOLF. 1998. Pollen transfer by natural hybrids and parental species in an Ipomopsis hybrid zone. Evolution; International Journal of Organic Evolution 52 : 1602 1611. CONOVER, W. J., AND R. L. IMAN. 1981. Rank transformations as a bridge between parametric and nonparametric statistics. The American Statistician 35 : 124 129. D YTHAM, C. 2003. Choosing and using statistics: A biologist s guide. Blackwell, Berlin. EHRENFELD, J. G. 1979. Pollination of three species of Euphorbia subgenus Chamaesyce, with special reference to bees. American Midland Naturalist 101 : 87 98. E MMS, S. K., AND M. K. ARNOLD. 2000. Site-to-site differences in pollinator visitation patterns in a Louisiana iris hybrid zone. Oikos 91 : 568 578. F AEGRI, K., AND L. VAN DER PIJL. 1979. The principles of pollination ecology. Pergamon Press, Oxford, UK. F ENSTER, C. B., W. S. ARMBRUSTER, P. WILSON, M. R. DUDASH, AND J. D. T HOMSON. 2004. Pollination syndromes and floral specialization. Annual Review of Ecology Evolution and Systematics 35 : 375 403. F ISHBEIN, M., AND D. L. VENABLE. 1996a. Diversity and temporal change in the effective pollinators of Asclepias tuberosa. Ecology 77 : 1061 1073. FISHBEIN, M., AND D. L. VENABLE. 1996b. Evolution of inflorescence design: Theory and data. Evolution; International Journal of Organic Evolution 50 : 2165 2177.

456 AMERICAN JOURNAL OF BOTANY [Vol. 99 F UMERO-CAB Á N, J. J., AND E. J. MEL É NDEZ-ACKERMAN. 2007. Relative pollination effectiveness of floral visitors of Pitcairnia angustifolia (Bromeliaceae). American Journal of Botany 94 : 419 424. G OMES, B., C. A. SOUSA, M. T. NOVO, F. B. FREITAS, R. ALVES, A. R. CÔ RTE- REAL, P. S ALGUEIRO, ET AL. 2009. Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal. BMC Evolutionary Biology 9 : 262. G RANT, V. 1993. Effects of hybridization and selection on floral isolation. Proceedings of the National Academy of Sciences, USA 90 : 990 993. GREEN, D. M., AND C. PARENT. 2003. Variable and asymmetric introgression in a hybrid zone in the toads, Bufo americanus and Bufo fowleri. Copeia 2003 : 34 43. H ELBIG, A. J., M. SALOMON, S. B ENSCH, AND I. S EIBOLD. 2001. Male-biased gene flow across an avian hybrid zone: Evidence from mitochondrial and microsatellite DNA. Journal of Evolutionary Biology 14 : 277 287. H ERRERA, C. M. 1987. Components of pollinator quality Comparative analysis of a diverse insect assemblage. Oikos 50 : 79 90. H INGSTON, A. B., AND P. B. M CQUILLAN. 1999. Displacement of Tasmanian native megachilid bees by the recently introduced bumblebee Bombus terrestris (Linnaeus, 1758) (Hymenoptera: Apidae). Australian Journal of Zoology 47 : 59 65. H OLM, S., 1979. A simple sequentially rejective multiple test procedure. Scandinavian Journal of Statistics 6 : 65 70. I NOUYE, D. W., D. E. GILL, M. R. DUDASH, AND C. B. FENSTER. 1994. A model and lexicon for pollen fate. American Journal of Botany 81 : 1517 1530. I VEY, C. T., P. MARTINEZ, AND R. W YATT. 2003. Variation in pollinator effectiveness in swamp milkweed, Asclepias incarnata (Apocynaceae). American Journal of Botany 90 : 214 225. J ENNERSTEN, O., AND D. H. MORSE. 1991. The quality of pollination by diurnal and nocturnal insects visiting common milkweed, Asclepias syriaca. American Midland Naturalist 125 : 18 28. KEPHART, S., AND K. THEISS. 2004. Pollinator-mediated isolation in sympatric milkweeds ( Asclepias ): Do floral morphology and insect behavior influence species boundaries? The New Phytologist 161 : 265 277. K EPHART, S. R., R. WYATT, AND D. P ARRELLA. 1988. Hybridization in North American Asclepias.1. Morphological evidence. Systematic Botany 13 : 456 473. K IMBALL, S. 2008. Links between floral morphology and floral visitors along an elevational gradient in a Penstemon hybrid zone. Oikos 117 : 1064 1074. K LIPS, R. A., AND T. M. CULLEY. 2004. Natural hybridization between prairie milkweeds, Asclepias sullivantii and Asclepias syriaca: Morphological, isozyme and hand-pollination evidence. International Journal of Plant Sciences 165 : 1027 1037. K OKUDAI, C. B. S., F. M. SENE, AND M. H. MANFRIN. 2011. Sympatry and asymmetric introgression between the cactophilic species Drosophila serido and Drosophila antonietae (Diptera: Drosophilidae). Annals of the Entomological Society of America 104 : 434 442. LARSSON, M. 2005. Higher pollinator effectiveness by specialist than generalist flower-visitors of unspecialized Knautia arvensis (Dipsacaceae). Oecologia 146 : 394 403. L EEBENS-MACK, J., AND B. G. MILLIGAN. 1998. Pollination biology in hybridizing Baptisia (Fabaceae) populations. American Journal of Botany 85 : 500 507. M AYFIELD, M. M., N. M. WASER, AND M. V. P RICE. 2001. Exploring the most effective pollinator principle with complex flowers: Bumblebees and Ipomopsis aggregata. Annals of Botany 88 : 591 596. M ORSE, D. H. 1982. The turnover of milkweed pollinia on bumblebees and implications for outcrossing. Oecologia 53 : 187 196. N E EMAN, G., A. J Ü RGENS, L. N EWSTROM-LLOYD, S. G. POTTS, AND A. D AFNI. 2010. A framework for comparing pollinator performance: Effectiveness and efficiency. Biological Reviews of the Cambridge Philosophical Society 85 : 435 451. P LEASANTS, J. M. 1991. Evidence for short-distance dispersal of pollinia in Asclepias syriaca L. Functional Ecology 5 : 75 82. R IESEBERG, L. 1997. Hybrid origins of plants. Annual Review of Ecology and Systematics 28 : 359 389. R OBERTSON, C. 1891. Flowers and insects, Asclepiadaceae to Scrophulariaceae. Transactions of the Academy of Science of St. Louis 5: 569 598. R OBERTSON, C. 1928. Flowers and insects. Lists of visitors of four hundred and fifty-three flowers. Published by the author, Carlinville, Illinois, USA. R UNDLE, H. D., AND P. N OSIL. 2005. Ecological speciation. Ecology Letters 8 : 336 352. SAHLI, H. F., AND J. K. CONNER. 2007. Visitation, effectiveness, and efficiency of 15 genera of visitors to wild radish, Raphanus raphanistrum (Brassicaceae). American Journal of Botany 94 : 203 209. SAS INSTITUTE INC. 2008. SAS/STAT 9.2 user s guide. SAS Institute Inc., Cary, North Carolina, USA. SCHEMSKE, D. W., AND C. C. HORVITZ. 1984. Variation among floral visitors in pollination ability: A precondition for mutualism specialization. Science 225 : 519 521. S HORE, J. S. 1993. Pollination genetics of the common milkweed, Asclepias syriaca L. Heredity 70 : 101 108. S LOAN, D. B., T. G IRAUD, AND M. E. HOOD. 2008. Maximized virulence in a sterilizing pathogen: The anther-smut fungus and its co-evolved hosts. Journal of Evolutionary Biology 21 : 1544 1554. S OLTIS, P. S., AND D. E. SOLTIS. 2009. The role of hybridization in plant speciation. Annual Review of Plant Biology 60 : 561 588. SUGDEN, E. A. 1986. Anthecology and pollinator efficacy of Styrax officinale subsp. redivivum (Styracaceae). American Journal of Botany 73 : 919 930. T AGANE, S., M. H IRAMATSU, AND H. O KUBO. 2008. Hybridization and asymmetric introgression between Rhododendron eriocarpum and R. indicum on Yakushima Island, southwest Japan. Journal of Plant Research 121 : 387 395. T HEISS, K., S. R. KEPHART, AND C. T. I VEY. 2007. Pollinator effectiveness on co-occurring milkweeds ( Asclepias ; Apocynaceae, Asclepiadoideae). Annals of the Missouri Botanical Garden 94 : 505 516. THOMSON, J. D., AND B. A. THOMSON. 1992. Pollen presentation and viability schedules in animal-pollinated plants: Consequences for reproductive success. In R. Wyatt [ed.], Ecology and evolution of plant reproduction, 1 24. Chapman and Hall, New York, New York, USA. VEREECKEN, N. J., S. COZZOLINO, AND F. P. SCHIESTL. 2010. Hybrid floral scent novelty drives pollinator shift in sexually deceptive orchids. BMC Evolutionary Biology 10 : 103. W ASER, N. M., L. CHITTKA, M. V. PRICE, N. M. WILLIAMS, AND J. O LLERTON. 1996. Generalization in pollination systems and why it matters. Ecology 77 : 1043 1060. W ESSELINGH, R. A., AND M. L. A RNOLD. 2000. Pollinator behaviour and the evolution of Louisiana iris hybrid zones. Journal of Evolutionary Biology 13 : 171 180. WILLSON, R. A., AND M. L. BERTIN. 1979. Flower-visitors, nectar production, and inflorescence size of Asclepias syriaca. Canadian Journal of Botany 57 : 1380 1388. W OLF, P. G., D. R. CAMPBELL, N. M. WASER, S. D. S IPES, T. R. TOLER, AND J. K. ARCHIBALD. 2001. Tests of pre- and postpollination barriers to hybridization between sympatric species of Ipomopsis (Polemoniaceae). American Journal of Botany 88 : 213 219. W OODSON, R. E. JR. 1954. The North American species of Asclepias L. Annals of the Missouri Botanical Garden 41 : 1 211. W YATT, R. 1996. More on the southward spread of common milkweed, Asclepias syriaca L. Bulletin of the Torrey Botanical Club 123 : 68 69. W YATT, R., AND S. B. BROYLES. 1994. Ecology and evolution of reproduction in milkweeds. Annual Review of Ecology and Systematics 25 : 423 441. WYATT, R., S. B. BROYLES, AND G. S. DERDA. 1992. Environmental influences on nectar production in milkweeds ( Asclepias syriaca and A. exaltata ). American Journal of Botany 79 : 636 642. W YATT, R., AND T. R. SHANNON. 1986. Nectar production and pollination of Asclepias exaltata. Systematic Botany 11 : 326 334. W YATT, R., A. S TONEBURNER, S. B. BROYLES, AND J. R. ALLISON. 1993. Range extension southward in common milkweed, Asclepias syriaca L. Bulletin of the Torrey Botanical Club 120 : 177 179. Y OUNG, H. J. 1988. Differential importance of beetle species pollinating Dieffenbachia longispatha (Araceae). Ecology 69 : 832 844.