Pollinator recognition by a keystone tropical plant Matthew G. Bettsa,1, Adam S. Hadleya, and W. John Kressb a
Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR 97331; and bDepartment of Botany, National Museum of Natural History, Smithsonian Institution, Washington, DC 20013-7012
Understanding the mechanisms enabling coevolution in complex mutualistic networks remains a central challenge in evolutionary biology. We show for the first time, to our knowledge, that a tropical plant species has the capacity to discriminate among floral visitors, investing in reproduction differentially across the pollinator community. After we standardized pollen quality in 223 aviary experiments, successful pollination of Heliconia tortuosa (measured as pollen tube abundance) occurred frequently when plants were visited by long-distance traplining hummingbird species with specialized bills (x pollen tubes = 1.21 ± 0.12 SE) but was reduced 5.7 times when visited by straight-billed territorial birds (x pollen tubes = 0.20 ± 0.074 SE) or insects. Our subsequent experiments revealed that plants use the nectar extraction capacity of tropical hummingbirds, a positive function of bill length, as a cue to turn on reproductively. Furthermore, we show that hummingbirds with long bills and high nectar extraction efficiency engaged in daily movements at broad spatial scales (∼1 km), but that territorial species moved only short distances (10 individuals (25, 31). This high concentration of nectar resources often results in defense of a single clonal clump by territorial hummingbirds, which strongly constrains pollen flow (28). However, the large body size and specialized morphology of traplining hummingbirds require them to move further to acquire necessary resources (21), thus potentially facilitating gene flow and enhancing plant fitness (32). To test this hypothesis, we assembled movement data on all seven pollinator species (SI Materials and Methods). Pollen tube abundance was positively correlated with the median movement distance that each species moved within 1 d (Fig. 3A) (F = 48.98, R2 = 0.87, P = 0.0004). We also found a strong effect of movement behavioral strategy on the number of pollen tubes; traplining species, which regularly move long distances across landscapes to Betts et al.
acquire nectar resources (32, 33), elicited greater pollen tube abundance than territorial species (6) (Fig. 3B) (GLMM: Z = 4.52, P < 0.0001). Second, the capacity to recognize specialized pollinators could also function to maximize the diversity of conspecific pollen received. Because of the placement of the anthers in H. tortuosa and the foraging position of green hermit and violet sabrewing (Movie S1), these species are more likely, on average, to carry high pollen loads than nonspecialized species. Our data on hummingbird pollen loads support this hypothesis; traplining species carried significantly higher individual loads of H. tortuosa pollen (^x = 154.47; 95% CI = 129.02–183.09) than territorial species (^x = 28.78; 95% CI = 14.15–58.55; GLM: t = 4.63, P < 0.0001). Although H. tortuosa ultimately requires only three grains to fertilize all seeds in the ovary, evidence from other plant species suggests that high pollen abundance on the stigma increases pollen tube competition (or potentially, allows greater opportunity for female choice), thereby increasing the quality of pollen reaching the ovary (34). Indeed, an increase in the number of pollen donors has been shown to be positively correlated with seed weight (34). Third, recognition of morphologically specialized pollinators might reduce the risk of pollen allelopathy or other types of pollen interference, whereby nonspecific pollen is deposited on the stigma by generalist pollinators and interferes with pollen receipt (35). In our study, both trapliners and territorial hummingbird species carried substantial non-Heliconia pollen, and we found no statistical differences between these groups (GLM: t = 1.55, P = 0.121). However, on average, trapliners carried lower loads of non-Heliconia pollen grains (^x = 12.55; 95% CI = 9.58– 16.44) vs. territorial hummingbird species (^x = 29.96; 95% CI = 9.87–89.12). Together, our results suggest that pollinator recognition by H. tortuosa facilitates mate selection and is most likely to do so by (i) increased outcrossing caused by receipt of longer-distance nonself-pollen or (ii) enhanced conspecific pollen diversity. Species in the genus Heliconia lack two mechanisms that promote outcrossing in many flowering plants, namely physiological self-incompatibility and spatial/temporal separation of sexual function (25). However, the often long and highly curved flowers of Heliconia species, including H. tortuosa, are a striking floral mechanism to screen out certain visitors. In many birdpollinated plant species, flower length has been proposed as an adaptation to allow plants to exclude pollinators that are unlikely to be carrying high-quality pollen (29). In H. tortuosa, the long, curved perianth reduces but does not preclude visitation by territorial hummingbirds, which still receive some nectar rewards (29). Repeated deposition of pollen by such locally foraging species of hummingbirds would clearly increase the levels of inbreeding in the plants as well as cause a reduction in the diversity and abundance of conspecific pollen. Because of the energetic costs of fruit and seed development (36), a mechanism that enables a plant to distinguish low- from high-quality pollinators before investing in seed production would confer a considerable adaptive advantage. Plant recognition of pollinators may occur in other plant taxa, particularly in relatively stable tropical systems with high pollinator diversity. One hypothesis is that the fitness gains afforded by pollinator recognition in terms of increasing mutualist specialization and possibly, outcrossing rates may have provided an initial mechanism for corolla lengthening in other plant families. Minor microevolutionary increases in corolla length should carry no fitness benefits until they accumulate sufficiently to induce switches by poor-quality pollinators to alternative flower species (30). However, if plants use minor differences in nectar extraction as a cue to indicate visitation by a high-quality pollinator, corolla lengthening in tandem with pollinator recognition would have immediate benefits. An alternative hypothesis is that pollinator recognition may have evolved to more effectively prevent PNAS Early Edition | 3 of 6
ECOLOGY
A
A
B
C
dropped from 53.7% to 19.9%. This hidden specialization has two important implications. First, pollination and subsequent plant demography could be more vulnerable to population declines or disruptions to movement of specialized pollinator species than would be expected given observations of floral visitors. Indeed, our previous work indicates that interpatch movement of traplining species is disrupted by tropical forest fragmentation (33, 38), with consequent negative impacts on seed set (23). Second, plant–pollinator mutualisms in this system may not be as generalized as superficially apparent, which may result in greater potential for rapid reciprocal selection and therefore, coevolutionary change in H. tortuosa and a few key specialized pollinators. It is now well-known that the high cognitive capacity of many vertebrate pollinators allows them to recognize and specialize on particular flower species (30). However, a growing body of research indicates that plants may also exhibit complex decisionmaking behavior (39, 40). Here, we show for the first time, to our knowledge, that a plant has evolved an effective behavior that allows the recognition of specific hummingbird pollinators and thereby, regulates reproduction accordingly. Materials and Methods We conducted the study in the landscape surrounding the Organization for Tropical Studies Las Cruces Biological Station in southern Costa Rica (8° 47’ 7’’ N, 82° 57’ 32’’ W).
Fig. 2. (A) Boxplot showing how nectar extraction efficiency in H. tortuosa varies across pollinator species. Boxes show first and third quartiles of data. The bill shapes/sizes of the species are to scale in relative terms. Green represents successful pollen tube growth, and red represents limited growth. (B) Relationship between nectar remaining after pollinator visitation and the number of pollen tubes per style for each species (species abbreviations are the same as in Fig. 1). Nectar extraction capacity of hummingbirds strongly influenced the abundance of pollen tubes (r = −0.73). (C) Effect of hand pollination only vs. hand pollination combined with nectar extraction on pollen tube abundance. Pollen tubes were 3.5 times more common when nectar was experimentally extracted than when flowers were hand-pollinated only. Error bars in B and C are ± SE.
pollination by poor pollinators in plants that have already evolved long corollas. Regardless of the initial evolutionary mechanism, candidates to examine for such pollinator recognition are those with long corollas combined with high nectar rewards (e.g., Acanthaceae, Rubiaceae, and Malvaceae) that would result in strong pollinator motivation and a subsequent reduction in morphological filters. Research over the past decade has emphasized the importance of understanding plant–pollinator interactions as networks of species rather than tight, parallel specializations (16). Such networks are highly nested, which means that the most specialized plant species are visited by generalist pollinators and vice versa. This structure has been hypothesized to buffer networks against loss of individual species (37). Our results indicate that, in this tropical system, plant–pollinator relationships are more specialized than assumed given the observational data used to quantify most pollination networks (16). Although we have frequently observed all six species of hummingbirds carrying pollen and visiting the flowers of H. tortuosa (Fig. 4A), pollination and successful fertilization are dominated locally by only two traplining species (i.e., violet sabrewing and green hermit). Indeed, when we compensated for this functional pollination effect, 80.1% of the reproductive contribution to Heliconia was provided by only two species (Fig. 4B); the contribution of all other species combined 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1419522112
Hand-Pollination Experiments. To conduct our initial test of pollen limitation in H. tortuosa, we randomly attributed hand-pollination treatments to 159 flowers during the dry season (January to March) from 2011 to 2014. H. tortuosa flowers open for only a single day before abscising. Treatments constituted covering all inflorescences with fine-mesh bags the night before pollination. We then collected pollen from conspecific flowers 30–100 m away (to ensure no self-fertilization) and applied it immediately (41) to flower stigmas using a toothpick. Stigmas were cleaned of self-pollen using a cotton swab before pollination. All flowers were then rebagged, and styles were collected the next day. We compared treated flowers with 746 flowers that were allowed full pollinator access and collected from the same study area and time period. We applied a similar or greater amount to what we typically observe on captured green hermits and violet sabrewings. To test whether under- or overabundance of pollen during hand supplementations negatively affected pollen tube growth, we conducted an additional experiment, in which we hand-pollinated flowers using the methods above but left them open to pollinators. Our pollinator-accessible hand
A
B
Fig. 3. (A) Relationship between median daily distance moved by pollinators and pollen tube abundance (± SE; R2 = 0.87). Species moving longer distances stimulated significantly more pollen tubes than those tending to make short-distance movements. (B) Individual flowers experimentally exposed to traplining species (GREH, VISA, and STHR) grew significantly more pollen tubes than ones visited by territorial species (GCBR, RTAH, and SBRH). Species abbreviations are the same as in Fig. 1.
Betts et al.
B
Fig. 4. Pollination network showing interactions between the six hummingbird pollinators that we examined (top row of black squares) and plants (bottom row of black squares). Width of connections represents strength of interactions. (A) Pollen-load data collected from the bills, nares, and heads of individual hummingbirds show that H. tortuosa seems to be a pollinator generalist, with contributions to its reproduction spread across all six hummingbird species. However, after taking the differential rates of plant investment after pollinator recognition into account (B), violet sabrewings and green hermits are likely responsible for 80% of successful reproduction. Pollinator recognition, therefore, promotes high levels of functional specialization within this complex network. GCBR, green-crowned brilliant (H. jacula); GREH, green hermit (Phaethornis guy); RTAH, rufous-tailed hummingbird; SBHU, scaly-breasted hummingbird; STHE stripe-throated hummingbird; VISA, violet sabrewing.
pollinations (n = 24, x = 0.85 ± 0.29 SE) did not alter pollination success in relation to unmanipulated open controls (n = 21, x = 0.95 ± 0.30 SE; GLM: t = 0.36, P = 0.72). Aviary Experiment. We captured 128 individual hummingbirds representing six species using a combination of 12-m nets and hall traps (23, 33). Individuals were fitted with unique aluminum bands to allow identification of recaptures and hence, nonindependent samples. We randomly assigned birds to individual flowers within aviaries and randomly assigned treatment to flowers. In this experiment, treatments comprised (i) hand-pollinated flowers with pollen outcrossed from a conspecific flower collected from within 30– 100 m and with pollinators excluded (n = 92) and (ii) hand-pollinated flowers using the same methods but with bird visitation (n = 202). To minimize the risk of pollen degradation (41), pollen was applied immediately after removal from pollen donors. Again, we cleaned stigmas with a cotton swab before pollination; although this process could have negatively affected reproduction, it does not constitute a bias, because it was conducted in all treatments. Floral emasculation is conducted in many hand-pollination studies to remove a potential confounding effect of self-pollen (42). However, in H. tortuosa, emasculation alters the positioning of the style and damages the flower, and both of which could result in estimates of reproductive success that are biased. Finally, floral emasculation risks altering pollinator behavior (42, 43). In bird visitation treatments, we took extreme care to ensure that birds were completely clean of pollen (SI Materials and Methods). We removed pollen using fuchsin jelly (for later use in pollen samples), a photographer’s brush (Fig. S1), and disposable lens-cleaning cloths. Bills, nares, heads, and throats were inspected with a hand lens after cleaning to ensure that no pollen grains remained. Birds were then released into the aviary and permitted to visit experimental flowers (Movie S1). Only one specific treatment flower was available to each bird within an experiment. We used three portable aviaries, each of which was assembled to cover individual plants. Aviaries were 2 × 2 × 3 m and covered in shade cloth mesh. Birds typically visited flowers in 1) in all models that assumed a Poisson distribution by calculating the sum of squared Pearson residuals, the ratio of residuals to rdf (residual degrees of freedom), and the P value based on the appropriate χ2 distribution (48) (Table S2). We detected significant but minor overdispersion in one of three models (Table S2), and therefore, we modeled pollen tube count data in this case using the quasi-Poisson family (49). As another test of whether our results were driven by poor match to the data distribution, we also modeled the presence/absence of pollen tubes in addition to pollen tube abundance. Results did not differ substantially from those assuming a Poisson distribution (SI Materials and Methods and Tables S3–S6). We tested for differences in nectar consumption capacity across species using GLMs with a Gaussian distribution. Green hermit males and females were included separately in analysis of nectar extraction, because they show strong sexual dimorphism in bill length, which is likely to influence nectar extraction efficiency (50). Sexes of this species have different movement behavior, with male home ranges larger than female home ranges (33). We tested assumptions of regressions by inspecting regression residuals for normality against a q-q plot. We tested the effect of experimental nectar extraction on pollen tube abundance using GLMMs with a Poisson distribution. The individual plant was specified as a random effect to account for occasional repeat sampling of
PNAS Early Edition | 5 of 6
ECOLOGY
A
the same plant. These data were analyzed two ways. First, we predicted pollen tube abundance as a function of the amount of nectar removed. Second, we tested the effect of nectar extraction treatments vs. hand pollination-only treatments. Finally, we tested whether territorial species carried more non-Heliconia pollen than traplining species using GLMs with a Gaussian distribution. Data were log(x + 1)-transformed to meet model assumptions. We used the same modeling approach to test whether traplining species carried more Heliconia pollen than trapliners.
ACKNOWLEDGMENTS. C. Argo, A. C. Betts, A. M. Betts, and M. Betts, C. Birkett, K. DeWolfe, C. Dourhard, S. Frey, J. Greer, A. Jack, T. McFadden, K. O’Connell, M. Paniagua, and E. Sandi assisted with data collection. E. Jackson and N. Volpe provided a portion of the hummingbird movement data. We thank M. Harmon, D. Hibbs, F. Jones, U. Kormann, A. Moldenke, and L. Reid for comments on the manuscript and A. Muldoon for statistical advice. The manuscript benefitted from comments from J. Thomson, N. Waser, and an anonymous reviewer. We thank the staff of Las Cruces Biological Station for facilitating collection of field data. This work was funded by National Science Foundation Grants DEB1050954 (to M.G.B. and W.J.K.) and DEB-1457837 (to M.G.B., A.S.H., and W.J.K.).
1. Kondrashov AS (1988) Deleterious mutations and the evolution of sexual reproduction. Nature 336(6198):435–440. 2. Fischer RA (1930) The Genetical Theory of Natural Selection (Clarendon, Oxford). 3. Darwin C (1877) The Various Contrivances by Which Orchids Are Fertilised by Insects (John Murray, London). 4. Dodd ME, Silvertown J, Chase MW (1999) Phylogenetic analysis of trait evolution and species diversity variation among angiosperm families. Evolution 53(3):732–744. 5. Aizen MA, Harder LD (2007) Expanding the limits of the pollen-limitation concept: Effects of pollen quantity and quality. Ecology 88(2):271–281. 6. Stiles FG, Wolf LL (1970) Hummingbird territoriality at a tropical flowering tree. Auk 87(3):467–491. 7. Temeles EJ, Koulouris CR, Sander SE, Kress WJ (2009) Effect of flower shape and size on foraging performance and trade-offs in a tropical hummingbird. Ecology 90(5):1147–1161. 8. Baker HG, Hurd PD (1968) Intrafloral ecology. Annu Rev Entomol 13(1):385–414. 9. Stanton ML, Snow AA, Handel SN (1986) Floral evolution: Attractiveness to pollinators increases male fitness. Science 232(4758):1625–1627. 10. Thompson JN (2005) The Geographic Mosaic of Coevolution (University of Chicago Press, Chicago). 11. Thompson JN (1999) The evolution of species interactions. Science 284(5423):2116–2118. 12. Guimarães PR, Jr, Jordano P, Thompson JN (2011) Evolution and coevolution in mutualistic networks. Ecol Lett 14(9):877–885. 13. Newman E, Manning J, Anderson B (2014) Matching floral and pollinator traits through guild convergence and pollinator ecotype formation. Ann Bot (Lond) 113(2):373–384. 14. Fenster CB, Armbruster WS, Wilson P, Dudash MR, Thomson JD (2004) Pollination syndromes and floral specialization. Annu Rev Ecol Evol Syst 35(1):375–403. 15. Thomson JD, Wilson P (2008) Explaining evolutionary shifts between bee and hummingbird pollination: Convergence, divergence, and directionality. Int J Plant Sci 169(1):23–38. 16. Bascompte J, Jordano P, Olesen JM (2006) Asymmetric coevolutionary networks facilitate biodiversity maintenance. Science 312(5772):431–433. 17. Waser NM, Chittka L, Price MV, Williams NM, Ollerton J (1996) Generalization in pollination systems, and why it matters. Ecology 77(4):1043–1060. 18. Fenster CB (1991) Selection on floral morphology by hummingbirds. Biotropica 23(1): 98–101. 19. Nilsson LA, Johnsson L, Ralison L, Randrianjohany E (1987) Angraecoid orchids and hawkmoths in central Madagascar: Specialized pollination systems and generalist foragers. Biotropica 19(4):310–318. 20. Nuismer SL, Jordano P, Bascompte J (2013) Coevolution and the architecture of mutualistic networks. Evolution 67(2):338–354. 21. Stiles FG (1975) Ecology, flowering phenology, and hummingbird pollination of some Costa Rican Heliconia species. Ecology 56(2):285–301. 22. Feinsinger P (1976) Organization of a tropical guild of nectarivorous birds. Ecol Monogr 46(3):257–291. 23. Hadley AS, Frey SJK, Robinson WD, Kress WJ, Betts MG (2014) Tropical forest fragmentation limits pollination of a keystone understory herb. Ecology 95(8):2202–2212. 24. Knight TM, et al. (2005) Pollen limitation of plant reproduction: Pattern and Process. Annu Rev Ecol Evol Syst 36(1):467–497. 25. Kress WJ (1983) Self-incompatibility in Central American Heliconia. Evolution 37(4): 735–744.
26. Abrahamczyk S, Souto-Vilarós D, Renner SS (2014) Escape from extreme specialization: Passionflowers, bats and the sword-billed hummingbird. Proc Biol Sci 281(1795): 20140888. 27. Maglianesi MA, Blüthgen N, Böhning-Gaese K, Schleuning M (2014) Morphological traits determine specialization and resource use in plant-hummingbird networks in the Neotropics. Ecology 95(12):3325–3334. 28. Linhart YB, et al. (1987) Disturbance and predictability of flowering pattens in birdpollinated cloud forest plants. Ecology 68(6):1696–1710. 29. Wolf LL, Hainsworth FR, Stiles FG (1972) Energetics of foraging: Rate and efficiency of nectar extraction by hummingbirds. Science 176(4041):1351–1352. 30. Heinrich B, Raven PH (1972) Energetics and pollination ecology. Science 176(4035): 597–602. 31. Linhart YB (1973) Ecological and behavioral determinants of pollen dispersal in hummingbird-pollinated Heliconia. Am Nat 107(956):511–523. 32. Stiles FG (1978) Ecological and evolutionary implications of bird pollination. Am Zool 18(4):715–727. 33. Volpe NL, Hadley AS, Robinson WD, Betts MG (2014) Functional connectivity experiments reflect routine movement behavior of a tropical hummingbird species. Ecol Appl 24(8):2122–2131. 34. Murdy WH, Carter MEB (1987) Regulation of the timing of pollen germination by the pistil in Talinum-mengesii (Portulacaceae). Am J Bot 74(12):1888–1892. 35. Loughnan D, Thomson JD, Ogilvie JE, Gilbert B (2014) Taraxacum officinale pollen depresses seed set of montane wildflowers through pollen allelopathy. J Poll Ecol 13(15):146–150. 36. Haig D, Westoby M (1988) On limits to seed production. Am Nat 131(5):757–759. 37. Memmott J, Waser NM, Price MV (2004) Tolerance of pollination networks to species extinctions. Proc Biol Sci 271(1557):2605–2611. 38. Hadley AS, Betts MG (2009) Tropical deforestation alters hummingbird movement patterns. Biol Lett 5(2):207–210. 39. Allmann S, Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles. Science 329(5995):1075–1078. 40. Trewavas A (2002) Mindless mastery. Nature 415(6874):841. 41. Stone JL, Thomson JD, Dent-Acosta SJ (1995) Assessment of pollen viability in handpollination experiments: A review. Am J Bot 82(9):1186–1197. 42. Eckert CG, et al. (2010) Plant mating systems in a changing world. Trends Ecol Evol 25(1):35–43. 43. Kearns CA, Inouye DW (1993) Techniques for Pollination Biologists (University of Colorado Press, Niwot, CO). 44. Dormann CF, Gruber B, Frund J (2008) Introducing the Bipartite Package: Analysing ecological networks. R-News 8(2):8–11. 45. Bates D, et al. (2014) lme4: Linear Mixed-Effects Models Using Eigen and S4 (R Core Team). 46. Pike N (2011) Using false discovery rates for multiple comparisons in ecology and evolution. Methods Ecol Evol 2(3):278–282. 47. Richards SA (2008) Dealing with overdispersed count data in applied ecology. J Appl Ecol 45(1):218–227. 48. Venables WN, Ripley BD (2002) Modern Applied Statistics with S (Springer, Berlin), 4th Ed. 49. Zeileis A, Kleiber C, Jackman S (2008) Regression models for count data in R. J Stat Softw 27(8):1–25. 50. Temeles EJ, Miller JS, Rifkin JL (2010) Evolution of sexual dimorphism in bill size and shape of hermit hummingbirds (Phaethornithinae): A role for ecological causation. Philos Trans R Soc Lond B Biol Sci 365(1543):1053–1063.
6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1419522112
Betts et al.
Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR 97331; and bDepartment of Botany, National Museum of Natural History, Smithsonian Institution, Washington, DC 20013-7012
Understanding the mechanisms enabling coevolution in complex mutualistic networks remains a central challenge in evolutionary biology. We show for the first time, to our knowledge, that a tropical plant species has the capacity to discriminate among floral visitors, investing in reproduction differentially across the pollinator community. After we standardized pollen quality in 223 aviary experiments, successful pollination of Heliconia tortuosa (measured as pollen tube abundance) occurred frequently when plants were visited by long-distance traplining hummingbird species with specialized bills (x pollen tubes = 1.21 ± 0.12 SE) but was reduced 5.7 times when visited by straight-billed territorial birds (x pollen tubes = 0.20 ± 0.074 SE) or insects. Our subsequent experiments revealed that plants use the nectar extraction capacity of tropical hummingbirds, a positive function of bill length, as a cue to turn on reproductively. Furthermore, we show that hummingbirds with long bills and high nectar extraction efficiency engaged in daily movements at broad spatial scales (∼1 km), but that territorial species moved only short distances (10 individuals (25, 31). This high concentration of nectar resources often results in defense of a single clonal clump by territorial hummingbirds, which strongly constrains pollen flow (28). However, the large body size and specialized morphology of traplining hummingbirds require them to move further to acquire necessary resources (21), thus potentially facilitating gene flow and enhancing plant fitness (32). To test this hypothesis, we assembled movement data on all seven pollinator species (SI Materials and Methods). Pollen tube abundance was positively correlated with the median movement distance that each species moved within 1 d (Fig. 3A) (F = 48.98, R2 = 0.87, P = 0.0004). We also found a strong effect of movement behavioral strategy on the number of pollen tubes; traplining species, which regularly move long distances across landscapes to Betts et al.
acquire nectar resources (32, 33), elicited greater pollen tube abundance than territorial species (6) (Fig. 3B) (GLMM: Z = 4.52, P < 0.0001). Second, the capacity to recognize specialized pollinators could also function to maximize the diversity of conspecific pollen received. Because of the placement of the anthers in H. tortuosa and the foraging position of green hermit and violet sabrewing (Movie S1), these species are more likely, on average, to carry high pollen loads than nonspecialized species. Our data on hummingbird pollen loads support this hypothesis; traplining species carried significantly higher individual loads of H. tortuosa pollen (^x = 154.47; 95% CI = 129.02–183.09) than territorial species (^x = 28.78; 95% CI = 14.15–58.55; GLM: t = 4.63, P < 0.0001). Although H. tortuosa ultimately requires only three grains to fertilize all seeds in the ovary, evidence from other plant species suggests that high pollen abundance on the stigma increases pollen tube competition (or potentially, allows greater opportunity for female choice), thereby increasing the quality of pollen reaching the ovary (34). Indeed, an increase in the number of pollen donors has been shown to be positively correlated with seed weight (34). Third, recognition of morphologically specialized pollinators might reduce the risk of pollen allelopathy or other types of pollen interference, whereby nonspecific pollen is deposited on the stigma by generalist pollinators and interferes with pollen receipt (35). In our study, both trapliners and territorial hummingbird species carried substantial non-Heliconia pollen, and we found no statistical differences between these groups (GLM: t = 1.55, P = 0.121). However, on average, trapliners carried lower loads of non-Heliconia pollen grains (^x = 12.55; 95% CI = 9.58– 16.44) vs. territorial hummingbird species (^x = 29.96; 95% CI = 9.87–89.12). Together, our results suggest that pollinator recognition by H. tortuosa facilitates mate selection and is most likely to do so by (i) increased outcrossing caused by receipt of longer-distance nonself-pollen or (ii) enhanced conspecific pollen diversity. Species in the genus Heliconia lack two mechanisms that promote outcrossing in many flowering plants, namely physiological self-incompatibility and spatial/temporal separation of sexual function (25). However, the often long and highly curved flowers of Heliconia species, including H. tortuosa, are a striking floral mechanism to screen out certain visitors. In many birdpollinated plant species, flower length has been proposed as an adaptation to allow plants to exclude pollinators that are unlikely to be carrying high-quality pollen (29). In H. tortuosa, the long, curved perianth reduces but does not preclude visitation by territorial hummingbirds, which still receive some nectar rewards (29). Repeated deposition of pollen by such locally foraging species of hummingbirds would clearly increase the levels of inbreeding in the plants as well as cause a reduction in the diversity and abundance of conspecific pollen. Because of the energetic costs of fruit and seed development (36), a mechanism that enables a plant to distinguish low- from high-quality pollinators before investing in seed production would confer a considerable adaptive advantage. Plant recognition of pollinators may occur in other plant taxa, particularly in relatively stable tropical systems with high pollinator diversity. One hypothesis is that the fitness gains afforded by pollinator recognition in terms of increasing mutualist specialization and possibly, outcrossing rates may have provided an initial mechanism for corolla lengthening in other plant families. Minor microevolutionary increases in corolla length should carry no fitness benefits until they accumulate sufficiently to induce switches by poor-quality pollinators to alternative flower species (30). However, if plants use minor differences in nectar extraction as a cue to indicate visitation by a high-quality pollinator, corolla lengthening in tandem with pollinator recognition would have immediate benefits. An alternative hypothesis is that pollinator recognition may have evolved to more effectively prevent PNAS Early Edition | 3 of 6
ECOLOGY
A
A
B
C
dropped from 53.7% to 19.9%. This hidden specialization has two important implications. First, pollination and subsequent plant demography could be more vulnerable to population declines or disruptions to movement of specialized pollinator species than would be expected given observations of floral visitors. Indeed, our previous work indicates that interpatch movement of traplining species is disrupted by tropical forest fragmentation (33, 38), with consequent negative impacts on seed set (23). Second, plant–pollinator mutualisms in this system may not be as generalized as superficially apparent, which may result in greater potential for rapid reciprocal selection and therefore, coevolutionary change in H. tortuosa and a few key specialized pollinators. It is now well-known that the high cognitive capacity of many vertebrate pollinators allows them to recognize and specialize on particular flower species (30). However, a growing body of research indicates that plants may also exhibit complex decisionmaking behavior (39, 40). Here, we show for the first time, to our knowledge, that a plant has evolved an effective behavior that allows the recognition of specific hummingbird pollinators and thereby, regulates reproduction accordingly. Materials and Methods We conducted the study in the landscape surrounding the Organization for Tropical Studies Las Cruces Biological Station in southern Costa Rica (8° 47’ 7’’ N, 82° 57’ 32’’ W).
Fig. 2. (A) Boxplot showing how nectar extraction efficiency in H. tortuosa varies across pollinator species. Boxes show first and third quartiles of data. The bill shapes/sizes of the species are to scale in relative terms. Green represents successful pollen tube growth, and red represents limited growth. (B) Relationship between nectar remaining after pollinator visitation and the number of pollen tubes per style for each species (species abbreviations are the same as in Fig. 1). Nectar extraction capacity of hummingbirds strongly influenced the abundance of pollen tubes (r = −0.73). (C) Effect of hand pollination only vs. hand pollination combined with nectar extraction on pollen tube abundance. Pollen tubes were 3.5 times more common when nectar was experimentally extracted than when flowers were hand-pollinated only. Error bars in B and C are ± SE.
pollination by poor pollinators in plants that have already evolved long corollas. Regardless of the initial evolutionary mechanism, candidates to examine for such pollinator recognition are those with long corollas combined with high nectar rewards (e.g., Acanthaceae, Rubiaceae, and Malvaceae) that would result in strong pollinator motivation and a subsequent reduction in morphological filters. Research over the past decade has emphasized the importance of understanding plant–pollinator interactions as networks of species rather than tight, parallel specializations (16). Such networks are highly nested, which means that the most specialized plant species are visited by generalist pollinators and vice versa. This structure has been hypothesized to buffer networks against loss of individual species (37). Our results indicate that, in this tropical system, plant–pollinator relationships are more specialized than assumed given the observational data used to quantify most pollination networks (16). Although we have frequently observed all six species of hummingbirds carrying pollen and visiting the flowers of H. tortuosa (Fig. 4A), pollination and successful fertilization are dominated locally by only two traplining species (i.e., violet sabrewing and green hermit). Indeed, when we compensated for this functional pollination effect, 80.1% of the reproductive contribution to Heliconia was provided by only two species (Fig. 4B); the contribution of all other species combined 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1419522112
Hand-Pollination Experiments. To conduct our initial test of pollen limitation in H. tortuosa, we randomly attributed hand-pollination treatments to 159 flowers during the dry season (January to March) from 2011 to 2014. H. tortuosa flowers open for only a single day before abscising. Treatments constituted covering all inflorescences with fine-mesh bags the night before pollination. We then collected pollen from conspecific flowers 30–100 m away (to ensure no self-fertilization) and applied it immediately (41) to flower stigmas using a toothpick. Stigmas were cleaned of self-pollen using a cotton swab before pollination. All flowers were then rebagged, and styles were collected the next day. We compared treated flowers with 746 flowers that were allowed full pollinator access and collected from the same study area and time period. We applied a similar or greater amount to what we typically observe on captured green hermits and violet sabrewings. To test whether under- or overabundance of pollen during hand supplementations negatively affected pollen tube growth, we conducted an additional experiment, in which we hand-pollinated flowers using the methods above but left them open to pollinators. Our pollinator-accessible hand
A
B
Fig. 3. (A) Relationship between median daily distance moved by pollinators and pollen tube abundance (± SE; R2 = 0.87). Species moving longer distances stimulated significantly more pollen tubes than those tending to make short-distance movements. (B) Individual flowers experimentally exposed to traplining species (GREH, VISA, and STHR) grew significantly more pollen tubes than ones visited by territorial species (GCBR, RTAH, and SBRH). Species abbreviations are the same as in Fig. 1.
Betts et al.
B
Fig. 4. Pollination network showing interactions between the six hummingbird pollinators that we examined (top row of black squares) and plants (bottom row of black squares). Width of connections represents strength of interactions. (A) Pollen-load data collected from the bills, nares, and heads of individual hummingbirds show that H. tortuosa seems to be a pollinator generalist, with contributions to its reproduction spread across all six hummingbird species. However, after taking the differential rates of plant investment after pollinator recognition into account (B), violet sabrewings and green hermits are likely responsible for 80% of successful reproduction. Pollinator recognition, therefore, promotes high levels of functional specialization within this complex network. GCBR, green-crowned brilliant (H. jacula); GREH, green hermit (Phaethornis guy); RTAH, rufous-tailed hummingbird; SBHU, scaly-breasted hummingbird; STHE stripe-throated hummingbird; VISA, violet sabrewing.
pollinations (n = 24, x = 0.85 ± 0.29 SE) did not alter pollination success in relation to unmanipulated open controls (n = 21, x = 0.95 ± 0.30 SE; GLM: t = 0.36, P = 0.72). Aviary Experiment. We captured 128 individual hummingbirds representing six species using a combination of 12-m nets and hall traps (23, 33). Individuals were fitted with unique aluminum bands to allow identification of recaptures and hence, nonindependent samples. We randomly assigned birds to individual flowers within aviaries and randomly assigned treatment to flowers. In this experiment, treatments comprised (i) hand-pollinated flowers with pollen outcrossed from a conspecific flower collected from within 30– 100 m and with pollinators excluded (n = 92) and (ii) hand-pollinated flowers using the same methods but with bird visitation (n = 202). To minimize the risk of pollen degradation (41), pollen was applied immediately after removal from pollen donors. Again, we cleaned stigmas with a cotton swab before pollination; although this process could have negatively affected reproduction, it does not constitute a bias, because it was conducted in all treatments. Floral emasculation is conducted in many hand-pollination studies to remove a potential confounding effect of self-pollen (42). However, in H. tortuosa, emasculation alters the positioning of the style and damages the flower, and both of which could result in estimates of reproductive success that are biased. Finally, floral emasculation risks altering pollinator behavior (42, 43). In bird visitation treatments, we took extreme care to ensure that birds were completely clean of pollen (SI Materials and Methods). We removed pollen using fuchsin jelly (for later use in pollen samples), a photographer’s brush (Fig. S1), and disposable lens-cleaning cloths. Bills, nares, heads, and throats were inspected with a hand lens after cleaning to ensure that no pollen grains remained. Birds were then released into the aviary and permitted to visit experimental flowers (Movie S1). Only one specific treatment flower was available to each bird within an experiment. We used three portable aviaries, each of which was assembled to cover individual plants. Aviaries were 2 × 2 × 3 m and covered in shade cloth mesh. Birds typically visited flowers in 1) in all models that assumed a Poisson distribution by calculating the sum of squared Pearson residuals, the ratio of residuals to rdf (residual degrees of freedom), and the P value based on the appropriate χ2 distribution (48) (Table S2). We detected significant but minor overdispersion in one of three models (Table S2), and therefore, we modeled pollen tube count data in this case using the quasi-Poisson family (49). As another test of whether our results were driven by poor match to the data distribution, we also modeled the presence/absence of pollen tubes in addition to pollen tube abundance. Results did not differ substantially from those assuming a Poisson distribution (SI Materials and Methods and Tables S3–S6). We tested for differences in nectar consumption capacity across species using GLMs with a Gaussian distribution. Green hermit males and females were included separately in analysis of nectar extraction, because they show strong sexual dimorphism in bill length, which is likely to influence nectar extraction efficiency (50). Sexes of this species have different movement behavior, with male home ranges larger than female home ranges (33). We tested assumptions of regressions by inspecting regression residuals for normality against a q-q plot. We tested the effect of experimental nectar extraction on pollen tube abundance using GLMMs with a Poisson distribution. The individual plant was specified as a random effect to account for occasional repeat sampling of
PNAS Early Edition | 5 of 6
ECOLOGY
A
the same plant. These data were analyzed two ways. First, we predicted pollen tube abundance as a function of the amount of nectar removed. Second, we tested the effect of nectar extraction treatments vs. hand pollination-only treatments. Finally, we tested whether territorial species carried more non-Heliconia pollen than traplining species using GLMs with a Gaussian distribution. Data were log(x + 1)-transformed to meet model assumptions. We used the same modeling approach to test whether traplining species carried more Heliconia pollen than trapliners.
ACKNOWLEDGMENTS. C. Argo, A. C. Betts, A. M. Betts, and M. Betts, C. Birkett, K. DeWolfe, C. Dourhard, S. Frey, J. Greer, A. Jack, T. McFadden, K. O’Connell, M. Paniagua, and E. Sandi assisted with data collection. E. Jackson and N. Volpe provided a portion of the hummingbird movement data. We thank M. Harmon, D. Hibbs, F. Jones, U. Kormann, A. Moldenke, and L. Reid for comments on the manuscript and A. Muldoon for statistical advice. The manuscript benefitted from comments from J. Thomson, N. Waser, and an anonymous reviewer. We thank the staff of Las Cruces Biological Station for facilitating collection of field data. This work was funded by National Science Foundation Grants DEB1050954 (to M.G.B. and W.J.K.) and DEB-1457837 (to M.G.B., A.S.H., and W.J.K.).
1. Kondrashov AS (1988) Deleterious mutations and the evolution of sexual reproduction. Nature 336(6198):435–440. 2. Fischer RA (1930) The Genetical Theory of Natural Selection (Clarendon, Oxford). 3. Darwin C (1877) The Various Contrivances by Which Orchids Are Fertilised by Insects (John Murray, London). 4. Dodd ME, Silvertown J, Chase MW (1999) Phylogenetic analysis of trait evolution and species diversity variation among angiosperm families. Evolution 53(3):732–744. 5. Aizen MA, Harder LD (2007) Expanding the limits of the pollen-limitation concept: Effects of pollen quantity and quality. Ecology 88(2):271–281. 6. Stiles FG, Wolf LL (1970) Hummingbird territoriality at a tropical flowering tree. Auk 87(3):467–491. 7. Temeles EJ, Koulouris CR, Sander SE, Kress WJ (2009) Effect of flower shape and size on foraging performance and trade-offs in a tropical hummingbird. Ecology 90(5):1147–1161. 8. Baker HG, Hurd PD (1968) Intrafloral ecology. Annu Rev Entomol 13(1):385–414. 9. Stanton ML, Snow AA, Handel SN (1986) Floral evolution: Attractiveness to pollinators increases male fitness. Science 232(4758):1625–1627. 10. Thompson JN (2005) The Geographic Mosaic of Coevolution (University of Chicago Press, Chicago). 11. Thompson JN (1999) The evolution of species interactions. Science 284(5423):2116–2118. 12. Guimarães PR, Jr, Jordano P, Thompson JN (2011) Evolution and coevolution in mutualistic networks. Ecol Lett 14(9):877–885. 13. Newman E, Manning J, Anderson B (2014) Matching floral and pollinator traits through guild convergence and pollinator ecotype formation. Ann Bot (Lond) 113(2):373–384. 14. Fenster CB, Armbruster WS, Wilson P, Dudash MR, Thomson JD (2004) Pollination syndromes and floral specialization. Annu Rev Ecol Evol Syst 35(1):375–403. 15. Thomson JD, Wilson P (2008) Explaining evolutionary shifts between bee and hummingbird pollination: Convergence, divergence, and directionality. Int J Plant Sci 169(1):23–38. 16. Bascompte J, Jordano P, Olesen JM (2006) Asymmetric coevolutionary networks facilitate biodiversity maintenance. Science 312(5772):431–433. 17. Waser NM, Chittka L, Price MV, Williams NM, Ollerton J (1996) Generalization in pollination systems, and why it matters. Ecology 77(4):1043–1060. 18. Fenster CB (1991) Selection on floral morphology by hummingbirds. Biotropica 23(1): 98–101. 19. Nilsson LA, Johnsson L, Ralison L, Randrianjohany E (1987) Angraecoid orchids and hawkmoths in central Madagascar: Specialized pollination systems and generalist foragers. Biotropica 19(4):310–318. 20. Nuismer SL, Jordano P, Bascompte J (2013) Coevolution and the architecture of mutualistic networks. Evolution 67(2):338–354. 21. Stiles FG (1975) Ecology, flowering phenology, and hummingbird pollination of some Costa Rican Heliconia species. Ecology 56(2):285–301. 22. Feinsinger P (1976) Organization of a tropical guild of nectarivorous birds. Ecol Monogr 46(3):257–291. 23. Hadley AS, Frey SJK, Robinson WD, Kress WJ, Betts MG (2014) Tropical forest fragmentation limits pollination of a keystone understory herb. Ecology 95(8):2202–2212. 24. Knight TM, et al. (2005) Pollen limitation of plant reproduction: Pattern and Process. Annu Rev Ecol Evol Syst 36(1):467–497. 25. Kress WJ (1983) Self-incompatibility in Central American Heliconia. Evolution 37(4): 735–744.
26. Abrahamczyk S, Souto-Vilarós D, Renner SS (2014) Escape from extreme specialization: Passionflowers, bats and the sword-billed hummingbird. Proc Biol Sci 281(1795): 20140888. 27. Maglianesi MA, Blüthgen N, Böhning-Gaese K, Schleuning M (2014) Morphological traits determine specialization and resource use in plant-hummingbird networks in the Neotropics. Ecology 95(12):3325–3334. 28. Linhart YB, et al. (1987) Disturbance and predictability of flowering pattens in birdpollinated cloud forest plants. Ecology 68(6):1696–1710. 29. Wolf LL, Hainsworth FR, Stiles FG (1972) Energetics of foraging: Rate and efficiency of nectar extraction by hummingbirds. Science 176(4041):1351–1352. 30. Heinrich B, Raven PH (1972) Energetics and pollination ecology. Science 176(4035): 597–602. 31. Linhart YB (1973) Ecological and behavioral determinants of pollen dispersal in hummingbird-pollinated Heliconia. Am Nat 107(956):511–523. 32. Stiles FG (1978) Ecological and evolutionary implications of bird pollination. Am Zool 18(4):715–727. 33. Volpe NL, Hadley AS, Robinson WD, Betts MG (2014) Functional connectivity experiments reflect routine movement behavior of a tropical hummingbird species. Ecol Appl 24(8):2122–2131. 34. Murdy WH, Carter MEB (1987) Regulation of the timing of pollen germination by the pistil in Talinum-mengesii (Portulacaceae). Am J Bot 74(12):1888–1892. 35. Loughnan D, Thomson JD, Ogilvie JE, Gilbert B (2014) Taraxacum officinale pollen depresses seed set of montane wildflowers through pollen allelopathy. J Poll Ecol 13(15):146–150. 36. Haig D, Westoby M (1988) On limits to seed production. Am Nat 131(5):757–759. 37. Memmott J, Waser NM, Price MV (2004) Tolerance of pollination networks to species extinctions. Proc Biol Sci 271(1557):2605–2611. 38. Hadley AS, Betts MG (2009) Tropical deforestation alters hummingbird movement patterns. Biol Lett 5(2):207–210. 39. Allmann S, Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles. Science 329(5995):1075–1078. 40. Trewavas A (2002) Mindless mastery. Nature 415(6874):841. 41. Stone JL, Thomson JD, Dent-Acosta SJ (1995) Assessment of pollen viability in handpollination experiments: A review. Am J Bot 82(9):1186–1197. 42. Eckert CG, et al. (2010) Plant mating systems in a changing world. Trends Ecol Evol 25(1):35–43. 43. Kearns CA, Inouye DW (1993) Techniques for Pollination Biologists (University of Colorado Press, Niwot, CO). 44. Dormann CF, Gruber B, Frund J (2008) Introducing the Bipartite Package: Analysing ecological networks. R-News 8(2):8–11. 45. Bates D, et al. (2014) lme4: Linear Mixed-Effects Models Using Eigen and S4 (R Core Team). 46. Pike N (2011) Using false discovery rates for multiple comparisons in ecology and evolution. Methods Ecol Evol 2(3):278–282. 47. Richards SA (2008) Dealing with overdispersed count data in applied ecology. J Appl Ecol 45(1):218–227. 48. Venables WN, Ripley BD (2002) Modern Applied Statistics with S (Springer, Berlin), 4th Ed. 49. Zeileis A, Kleiber C, Jackman S (2008) Regression models for count data in R. J Stat Softw 27(8):1–25. 50. Temeles EJ, Miller JS, Rifkin JL (2010) Evolution of sexual dimorphism in bill size and shape of hermit hummingbirds (Phaethornithinae): A role for ecological causation. Philos Trans R Soc Lond B Biol Sci 365(1543):1053–1063.
6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1419522112
Betts et al.
