- Email: [email protected]
Pollinator-induced twisting of flowers sidesteps floral architecture constraints
Magazine R793
trout would aid in determining whether the shared abilities of the former two species are due to common ancestry or convergence. Convergence has been suggested as the reason for other superficially similar ape and human abilities [10] and is most likely the reason why trout have superficially similar partner-choice abilities to humans and chimpanzees. Supplemental Information Supplemental Information including experimental procedures can be found with this article online at http://dx.doi.org/10.1016/j. cub.2014.07.033. Acknowledgments This research was funded by the Gates Cambridge Trust (A.L.V.), Swiss National Science Foundation (R.B.), and Musgrave Fund (A.M.). We thank all those who assisted with the fieldwork, particularly the staff of the Lizard Island Research Station. Madeleine Emms conducted the reliability coding and Nicola Clayton provided comments that improved the manuscript. References 1. Barclay, P. (2013). Strategies for cooperation in biological markets, especially for humans. Evol. Hum. Behav. 34, 164–175. 2. Melis, A.P., Hare, B., and Tomasello, M. (2006). Chimpanzees recruit the best collaborators. Science 311, 1297–1300. 3. Bshary, R., Hohner, A., Ait-el-Djoudi, K., and Fricke, H. (2006). Interspecific communicative and coordinated hunting between groupers and giant moray eels in the Red Sea. PLoS Biol. 4, e431. 4. Shettleworth, S.J. (2010). Cognition, Evolution, and Behavior, 2nd edition (Oxford University Press). 5. Plotnik, J.M., Lair, R., Suphachoksahakun, W., and de Waal, F.B.M. (2011). Elephants know when they need a helping trunk in a cooperative task. Proc. Natl. Acad. Sci. USA. 108, 5116–5121. 6. Seed, A.M., Clayton, N.S., and Emery, N.J. (2008). Cooperative problem solving in rooks (Corvus frugilegus). Proc. R. Soc. Lond. B Biol. Sci. 275, 1421–1429. 7. Vail, A.L., Manica, A., and Bshary, R. (2013). Referential gestures in fish collaborative hunting. Nat. Commun. 4, 1765. 8. Salwiczek, L.H., Pretot, L., Demarta, L., Proctor, D., Essler, J., Pinto, A.I., Wismer, S., Stoinski, T., Brosnan, S.F., and Bshary, R. (2012). Adult cleaner wrasse outperform capuchin monkeys, chimpanzees and orangutans in a complex foraging task derived from cleaner – client reef fish cooperation. PLoS One 7, e49068. 9. Epstein, R., Kirshnit, C.E., Lanza, R.P., and Rubin, L.C. (1984). “Insight” in the pigeon: antecedents and determinants of intelligent performance. Nature 308, 61–62. 10. Greenberg, J.R., Hamann, K., Warneken, F., and Tomasello, M. (2010). Chimpanzee helping in collaborative and noncollaborative contexts. Anim. Behav. 80, 873–880. 1Department
of Zoology, University of Cambridge, Downing Street, Cambridge, CB23EJ, UK. 2Institute of Biology, University of Neuchatel, Rue Emile-Argand 11, CH-2000 Neuchatel, Switzerland. *E-mail: [email protected]
Pollinator-induced twisting of flowers sidesteps floral architecture constraints Michael Bartoš1 and Šteˇpán Janecˇek1,2 Specific pollen placement by zygomorphic flowers on pollinators is one of the key innovations of angiosperm evolution [1]. In most phylogenetic lineages that have evolved zygomorphic flowers, reproductive organs are positioned either in the lower or upper part of the flower. Although these specific positions largely enhance pollen economy, they also represent architectural constraints such that flowers are able to place pollen only on the dorsal or ventral part of pollinators’ bodies [2]. Such constraints can lead to interspecific pollen placement in situations where phylogenetically related species with the same floral architecture share pollinators [3]. Here, we present a simple but ingenious adaptation of Impatiens frithii, a bird-pollinated plant that shares its main pollinator with four other Impatiens species on Mt. Cameroon. In contrast to other species of the genus, the nectar spur of I. frithii is not curved downwards, but slightly upwards. This apparently small modification significantly affects how pollen is placed on birds’ bodies. When a bird forages on nectar, the flower twists as the spur conforms to the shape of the bird’s bill. As a consequence, pollen is placed in an unusual location on the bird’s body — the ventral surface of its bill or head. Our observations demonstrate that a minute change in floral morphology can effectively overcome constraints resulting from the basal floral architecture early in the group’s evolution. We assume that such adaptations can not only help the plants avoid interspecific competition, but as the adaptations create strong reproductive barriers, they may also contribute to plant speciation. The origin of pre-pollination reproductive barriers has been one
of the central topics of evolutionary biology since Darwin’s time, and remains rather mysterious in situations where phylogenetically related species grow in sympatry and share the same pollinators. In these situations, one way to separate gene flow is to place pollen on different parts of pollinators’ bodies [4]. Extremely precise placement that leads to reproductive isolation can be found particularly in orchids [5], which achieve this precision by producing pollinia. Plants with free granular pollen, however, cannot in this way achieve mechanical isolation, as has been well documented, for example, in members of the genera Pedicularis and Stylidium [6,7]. In these cases, the only possible way of creating a sufficiently strong reproductive barrier is to place and pick up pollen sufficiently far apart [6,7]. The evolutionary process that can lead to this situation is nevertheless almost always related to gradual changes in traits (e.g. gradual changes in the reward–stigma distance and/or the reward– anther distance), which result in overlapping pollen placement on pollinators’ bodies by different plant species [8]. Such processes, therefore, cannot lead to effective reproductive isolation [6]. In some cases, architectural constraints can be overcome if a pollinator alters its foraging position, as has been demonstrated in insects collecting pollen on Pedicularis [6] or in perching sunbirds foraging for nectar on Aloe [9]. The endemic species Impatiens frithii has only relatively recently been described from the Bakossi Mountains and Mt. Etinde in western Cameroon [10]. Its floral properties correspond to the birdpollination syndrome. The epiphytic I. frithii grows on smaller trees or lower tree branches, and its longpeduncled red flowers protrude out of the foliage. We observed I. frithii flowering on the slopes of Mt. Cameroon at elevations of 879–1340 m above sea level during the wet season of 2013 (July 24–September 2). To identify its main pollinators we observed seven individuals of I. frithii using remote video systems (163.3 hours of observations). We recorded eighteen arrivals of Cyanomitra oritis, and this sunbird
Current Biology Vol 24 No 17 R794
Figure 1. Two types of pollen placement by Impatiens species. (A) All known Impatiens species place pollen grains on the dorsal part of the head; this panel shows a sunbird Cyanomitra oritis during nectar foraging on Impatiens sakeriana. (B) Flower of I. frithii with atypical spur curvature. (C) Sunbird C. oritis inserting its bill into an I. frithii flower that is still in a normal position (snapshot from a video recording). (D) Nectar spur of I. frithii in an inverted position, fitting the beak shape of C. oritis (snapshot from a video recording).
was also the most frequently observed legitimate visitor on all other co-flowering bird-pollinated Impatiens species that grow on Mt. Cameroon (unpublished data). The typical floral architecture of the genus Impatiens with reproductive
organs in the dorsal part of the flower inevitably leads to pollen deposition on and pick-up from the dorsal part of pollinators’ heads (Figure 1A). In the case of I. frithii, however, the situation is quite different. Thanks to atypical nectar
spur curvature, where the spur of I. frithii is not curved downwards, as is usual, but slightly upwards (Figure 1B), the flower twists during bird foraging on nectar as the spur conforms to the shape of the bird’s bill. Pollen is consequently placed on the ventral surface of the bird’s bill or head (Figure 1C, 1D; Supplemental Movie S1). Changes in pollen placement on sunbirds by closely related sympatric species have been described only in relation to changes in pollinator foraging position. Succulent tree aloes, such as Aloe pluridens and Aloe lineata var. muirii, place their pollen on the underside of the mandible and chin of sunbirds foraging in the head-up position. Aloe africana, by contrast, due to its strongly curved perianth tube, places pollen on the crown of the heads of sunbirds that forage in the upside-down position [9]. The upside-down feeding position requires support for perching, which is provided by the plants’ robust inflorescence. The sunbird C. oritis, which forages nectar from flowers of I. frithii while hovering, cannot forage in the upside-down position, and the unusual pollen placement by I. frithii is facilitated by extraordinary rotation of the flower. This adaptation seems to be very important, considering the fact that several bird-pollinated Impatiens species occurring on Mt. Cameroon share a common pollinator, and that all flower at the same time during the wet season (unpublished data). Change in the placement of pollen is a perfect barrier against pollen transfer among sympatric occurring Impatiens species. The biggest remaining question is whether this adaptation separating pollination systems could be the result of sympatric speciation or whether it evolved in allopatry, and secondary sympatry caused character displacement to reduce pollen competition [8]. Follow-up experimental studies are needed to resolve this question, and the next studies should also determine how tongue and bill movements contribute to the flower twist. Our observation provides evidence for the first known pollination system where plants have overcome floral architecture constraints on
Magazine R795
pollen placement by twisting their nectar spurs and illustrates how evolution sometimes comes up with unexpected solutions. Supplemental Information Supplemental Information includes one supplemental movie and can be found with this article online at http://dx.doi. org/10.1016/j.cub.2014.07.056. Acknowledgements We are grateful for the logistical support of D. Horˇák and O. Sedlácˇek and wish to thank L.F. Ewome, J. Esembe and M.G. Mbonde for their assistance in the field. We would like to thank F. Rooks for English proofreading. This study was supported by the Czech Science Foundation (project No. P505/11/1617), the National Geographic Foundation (project No. 923012) and the long-term research development project RVO 67985939. References 1. Endress, P.K. (1999). Symmetry in flowers: diversity and evolution. Int. J. Plant Sci. 160, S3–S23. 2. Westerkamp, C., and Claßen-Bockhoff, R. (2007). Bilabiate flowers: the ultimate response to bees? Ann. Bot. 100, 361–374. 3. Armbruster, W. S., and Herzig, A. L. (1984). Partitioning and sharing of pollinators by four sympatric species of Dalechampia (Euphorbiaceae) in Panama. Ann. Mo. Bot. Gard. 71, 1–6. 4. Grant, V. (1994). Modes and origins of mechanical and ethological isolation in angiosperms. Proc. Natl. Acad. Sci. USA 91, 3–10. 5. Pauw, A. (2006). Floral syndromes accurately predict pollination by a specialized oil-collecting bee (Rediviva peringueyi, Melittidae) in a guild of South African orchids (Coryciinae). Am. J. Bot. 93, 917–926. 6. Armbruster, W. S., Shi, X. Q., and Huang, S. Q. (2014). Do specialized flowers promote reproductive isolation? Realized pollination accuracy of three sympatric Pedicularis species. Ann. Bot. 113, 331–340. 7. Armbruster, W. S., Edwards, M. E., and Debevec, E. M. (1994). Floral character displacement generates assemblage structure of Western Australian triggerplants (Stylidium). Ecology 75, 315–329. 8. Armbruster, W. S., and Muchhala, N. (2009). Associations between floral specialization and species diversity: cause, effect, or correlation? Evol. Ecol. 23, 159–179. 9. Botes, C., Johnson, S. D., and Cowling, R. M. (2008). Coexistence of succulent tree aloes: partitioning of bird pollinators by floral traits and flowering phenology. Oikos 117, 875–882. 10 Cheek, M., and Csiba, L. (2002). A new epiphytic species of Impatiens (Balsaminaceae) from western Cameroon. Kew Bulletin 57, 669–674. 1Institute
of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, CZ-379 82 Trˇebonˇ, Czech Republic. 2Department of Ecology, Faculty of Science, Charles University in Prague, Vinicˇná 7, CZ-128 44 Praha 2, Czech Republic. E-mail: [email protected]
Reply to Cordi et al. Christian Cajochen1,*, Songül Altanay-Ekici1, Mirjam Münch2, Sylvia Frey1, Vera Knoblauch3, and Anna Wirz-Justice1 In their paper on the influence of the moon on sleep, Cordi et al. [1] have analyzed a large number of subjects and found no significant effects, as opposed to our positive study findings with a smaller cohort [2]. More is not necessarily better. There are two main reasons why we think the comparison of these two data sets is not just comparing a small with a big sample size, since increasing the number of study volunteers in a sleep study does not automatically increase data quality. Several factors and processes influence the quality and structure of human sleep, primarily duration of prior wakefulness, circadian phase and the environmental light– dark cycle (for a review, see [3]). To thoroughly investigate and quantify the contribution of each of these influences on sleep, studies need to be carefully designed with the aim of controlling for each factor. Thus, to investigate a potential rhythmic influence on sleep retrospectively, it is essential that the examined cohort (study volunteers) was studied under very controlled conditions. For instance, light affects our circadian rhythms and in turn our sleep more powerfully than any drug. Consequently, synchronizing study volunteers to the 24-hour light–dark cycle according to their own preferred sleep–wake timing is a requirement for any sleep study which aims at quantifying sleep measures. Chronobiologists call this ‘enforcing circadian entrainment’, which is a sine qua non for proper quantification of the influence of the circadian process and prior wakefulness on sleep. We also consider this a necessary prerequisite when carrying out post-hoc analyses of the potential impact of rhythmic phenomena (such as the lunar cycle) on sleep. To explain this phenomenon with a more allegorical approach, imagine an orchestra with a certain number of musicians. You are trying to recognize a rhythmic characteristic of the music
played by the musicians, but you can only listen to them every 10 minutes for a very short time retrospectively. If the orchestra was not precisely synchronized to the conductor, you will not recognize the melody, even if the number of musicians is massively increased, which does not augment signal quality but instead leads to cacophony. However, if the players (regardless of their number) play tuned synchronously to the conductor (i.e., circadian entrainment), the chance of recognising a melody or a superimposed weaker melody is much greater, even if you sample only every 10 minutes. In chronobiological experiments, therefore, we synchronize study volunteers to the 24-hour light–dark cycle with respect to their own natural sleep timing, to unmask as well as possible the influence of circadian rhythmicity on any variable of interest. If this is ignored, one may probably miss very different rhythmic influences, such as that of the moon, because the signal to noise ratio becomes very weak. That could be the main reason that, although part of folklore, it has up until now been difficult to detect the rather weak influence of the moon on human sleep, since it cannot be revealed just by pooling non-synchronized sleep data. For future studies, we therefore suggest to carefully control the following variables, and to perform a power analysis to estimate the sample size for a targeted statistical effect: circadian phase, light–dark cycle, circadian entrainment, prior duration of wakefulness, menstrual cycle and age. References 1. Cordi, M., Ackermann, S., Bes, F.W., Hartmann, F., Konrad, B.N., Genzel, L., Pawlowski, M., Steiger, A., Schulz, H., Rasch, B., and Dresler, M. (2014). Lunar cycle effects on sleep and the file drawer problem. Curr. Biol. 24, R549–R550. 2. Cajochen, C., Altanay-Ekici, S., Munch, M., Frey, S., Knoblauch, V., and Wirz-Justice, A. (2013). Evidence that the lunar cycle influences human sleep. Curr. Biol. 23, 1485–1488. 3. Cajochen, C., Chellappa, S., and Schmidt, C. (2010). What keeps us awake? The role of clocks and hourglasses, light, and melatonin. Int. Rev. Neurobiol. 93, 57–90. 1Centre
for Chronobiology, Psychiatric Hospital of the University of Basel, 4012 Basel, Switzerland. 2Solar Energy and Building Physics Laboratory, Swiss Federal Institute of Technology Lausanne, 1015 Lausanne, Switzerland. 3Centre for Sleep Medicine, Hirslanden Clinic, 8702 Zollikon, Switzerland. *E-mail: [email protected]
trout would aid in determining whether the shared abilities of the former two species are due to common ancestry or convergence. Convergence has been suggested as the reason for other superficially similar ape and human abilities [10] and is most likely the reason why trout have superficially similar partner-choice abilities to humans and chimpanzees. Supplemental Information Supplemental Information including experimental procedures can be found with this article online at http://dx.doi.org/10.1016/j. cub.2014.07.033. Acknowledgments This research was funded by the Gates Cambridge Trust (A.L.V.), Swiss National Science Foundation (R.B.), and Musgrave Fund (A.M.). We thank all those who assisted with the fieldwork, particularly the staff of the Lizard Island Research Station. Madeleine Emms conducted the reliability coding and Nicola Clayton provided comments that improved the manuscript. References 1. Barclay, P. (2013). Strategies for cooperation in biological markets, especially for humans. Evol. Hum. Behav. 34, 164–175. 2. Melis, A.P., Hare, B., and Tomasello, M. (2006). Chimpanzees recruit the best collaborators. Science 311, 1297–1300. 3. Bshary, R., Hohner, A., Ait-el-Djoudi, K., and Fricke, H. (2006). Interspecific communicative and coordinated hunting between groupers and giant moray eels in the Red Sea. PLoS Biol. 4, e431. 4. Shettleworth, S.J. (2010). Cognition, Evolution, and Behavior, 2nd edition (Oxford University Press). 5. Plotnik, J.M., Lair, R., Suphachoksahakun, W., and de Waal, F.B.M. (2011). Elephants know when they need a helping trunk in a cooperative task. Proc. Natl. Acad. Sci. USA. 108, 5116–5121. 6. Seed, A.M., Clayton, N.S., and Emery, N.J. (2008). Cooperative problem solving in rooks (Corvus frugilegus). Proc. R. Soc. Lond. B Biol. Sci. 275, 1421–1429. 7. Vail, A.L., Manica, A., and Bshary, R. (2013). Referential gestures in fish collaborative hunting. Nat. Commun. 4, 1765. 8. Salwiczek, L.H., Pretot, L., Demarta, L., Proctor, D., Essler, J., Pinto, A.I., Wismer, S., Stoinski, T., Brosnan, S.F., and Bshary, R. (2012). Adult cleaner wrasse outperform capuchin monkeys, chimpanzees and orangutans in a complex foraging task derived from cleaner – client reef fish cooperation. PLoS One 7, e49068. 9. Epstein, R., Kirshnit, C.E., Lanza, R.P., and Rubin, L.C. (1984). “Insight” in the pigeon: antecedents and determinants of intelligent performance. Nature 308, 61–62. 10. Greenberg, J.R., Hamann, K., Warneken, F., and Tomasello, M. (2010). Chimpanzee helping in collaborative and noncollaborative contexts. Anim. Behav. 80, 873–880. 1Department
of Zoology, University of Cambridge, Downing Street, Cambridge, CB23EJ, UK. 2Institute of Biology, University of Neuchatel, Rue Emile-Argand 11, CH-2000 Neuchatel, Switzerland. *E-mail: [email protected]
Pollinator-induced twisting of flowers sidesteps floral architecture constraints Michael Bartoš1 and Šteˇpán Janecˇek1,2 Specific pollen placement by zygomorphic flowers on pollinators is one of the key innovations of angiosperm evolution [1]. In most phylogenetic lineages that have evolved zygomorphic flowers, reproductive organs are positioned either in the lower or upper part of the flower. Although these specific positions largely enhance pollen economy, they also represent architectural constraints such that flowers are able to place pollen only on the dorsal or ventral part of pollinators’ bodies [2]. Such constraints can lead to interspecific pollen placement in situations where phylogenetically related species with the same floral architecture share pollinators [3]. Here, we present a simple but ingenious adaptation of Impatiens frithii, a bird-pollinated plant that shares its main pollinator with four other Impatiens species on Mt. Cameroon. In contrast to other species of the genus, the nectar spur of I. frithii is not curved downwards, but slightly upwards. This apparently small modification significantly affects how pollen is placed on birds’ bodies. When a bird forages on nectar, the flower twists as the spur conforms to the shape of the bird’s bill. As a consequence, pollen is placed in an unusual location on the bird’s body — the ventral surface of its bill or head. Our observations demonstrate that a minute change in floral morphology can effectively overcome constraints resulting from the basal floral architecture early in the group’s evolution. We assume that such adaptations can not only help the plants avoid interspecific competition, but as the adaptations create strong reproductive barriers, they may also contribute to plant speciation. The origin of pre-pollination reproductive barriers has been one
of the central topics of evolutionary biology since Darwin’s time, and remains rather mysterious in situations where phylogenetically related species grow in sympatry and share the same pollinators. In these situations, one way to separate gene flow is to place pollen on different parts of pollinators’ bodies [4]. Extremely precise placement that leads to reproductive isolation can be found particularly in orchids [5], which achieve this precision by producing pollinia. Plants with free granular pollen, however, cannot in this way achieve mechanical isolation, as has been well documented, for example, in members of the genera Pedicularis and Stylidium [6,7]. In these cases, the only possible way of creating a sufficiently strong reproductive barrier is to place and pick up pollen sufficiently far apart [6,7]. The evolutionary process that can lead to this situation is nevertheless almost always related to gradual changes in traits (e.g. gradual changes in the reward–stigma distance and/or the reward– anther distance), which result in overlapping pollen placement on pollinators’ bodies by different plant species [8]. Such processes, therefore, cannot lead to effective reproductive isolation [6]. In some cases, architectural constraints can be overcome if a pollinator alters its foraging position, as has been demonstrated in insects collecting pollen on Pedicularis [6] or in perching sunbirds foraging for nectar on Aloe [9]. The endemic species Impatiens frithii has only relatively recently been described from the Bakossi Mountains and Mt. Etinde in western Cameroon [10]. Its floral properties correspond to the birdpollination syndrome. The epiphytic I. frithii grows on smaller trees or lower tree branches, and its longpeduncled red flowers protrude out of the foliage. We observed I. frithii flowering on the slopes of Mt. Cameroon at elevations of 879–1340 m above sea level during the wet season of 2013 (July 24–September 2). To identify its main pollinators we observed seven individuals of I. frithii using remote video systems (163.3 hours of observations). We recorded eighteen arrivals of Cyanomitra oritis, and this sunbird
Current Biology Vol 24 No 17 R794
Figure 1. Two types of pollen placement by Impatiens species. (A) All known Impatiens species place pollen grains on the dorsal part of the head; this panel shows a sunbird Cyanomitra oritis during nectar foraging on Impatiens sakeriana. (B) Flower of I. frithii with atypical spur curvature. (C) Sunbird C. oritis inserting its bill into an I. frithii flower that is still in a normal position (snapshot from a video recording). (D) Nectar spur of I. frithii in an inverted position, fitting the beak shape of C. oritis (snapshot from a video recording).
was also the most frequently observed legitimate visitor on all other co-flowering bird-pollinated Impatiens species that grow on Mt. Cameroon (unpublished data). The typical floral architecture of the genus Impatiens with reproductive
organs in the dorsal part of the flower inevitably leads to pollen deposition on and pick-up from the dorsal part of pollinators’ heads (Figure 1A). In the case of I. frithii, however, the situation is quite different. Thanks to atypical nectar
spur curvature, where the spur of I. frithii is not curved downwards, as is usual, but slightly upwards (Figure 1B), the flower twists during bird foraging on nectar as the spur conforms to the shape of the bird’s bill. Pollen is consequently placed on the ventral surface of the bird’s bill or head (Figure 1C, 1D; Supplemental Movie S1). Changes in pollen placement on sunbirds by closely related sympatric species have been described only in relation to changes in pollinator foraging position. Succulent tree aloes, such as Aloe pluridens and Aloe lineata var. muirii, place their pollen on the underside of the mandible and chin of sunbirds foraging in the head-up position. Aloe africana, by contrast, due to its strongly curved perianth tube, places pollen on the crown of the heads of sunbirds that forage in the upside-down position [9]. The upside-down feeding position requires support for perching, which is provided by the plants’ robust inflorescence. The sunbird C. oritis, which forages nectar from flowers of I. frithii while hovering, cannot forage in the upside-down position, and the unusual pollen placement by I. frithii is facilitated by extraordinary rotation of the flower. This adaptation seems to be very important, considering the fact that several bird-pollinated Impatiens species occurring on Mt. Cameroon share a common pollinator, and that all flower at the same time during the wet season (unpublished data). Change in the placement of pollen is a perfect barrier against pollen transfer among sympatric occurring Impatiens species. The biggest remaining question is whether this adaptation separating pollination systems could be the result of sympatric speciation or whether it evolved in allopatry, and secondary sympatry caused character displacement to reduce pollen competition [8]. Follow-up experimental studies are needed to resolve this question, and the next studies should also determine how tongue and bill movements contribute to the flower twist. Our observation provides evidence for the first known pollination system where plants have overcome floral architecture constraints on
Magazine R795
pollen placement by twisting their nectar spurs and illustrates how evolution sometimes comes up with unexpected solutions. Supplemental Information Supplemental Information includes one supplemental movie and can be found with this article online at http://dx.doi. org/10.1016/j.cub.2014.07.056. Acknowledgements We are grateful for the logistical support of D. Horˇák and O. Sedlácˇek and wish to thank L.F. Ewome, J. Esembe and M.G. Mbonde for their assistance in the field. We would like to thank F. Rooks for English proofreading. This study was supported by the Czech Science Foundation (project No. P505/11/1617), the National Geographic Foundation (project No. 923012) and the long-term research development project RVO 67985939. References 1. Endress, P.K. (1999). Symmetry in flowers: diversity and evolution. Int. J. Plant Sci. 160, S3–S23. 2. Westerkamp, C., and Claßen-Bockhoff, R. (2007). Bilabiate flowers: the ultimate response to bees? Ann. Bot. 100, 361–374. 3. Armbruster, W. S., and Herzig, A. L. (1984). Partitioning and sharing of pollinators by four sympatric species of Dalechampia (Euphorbiaceae) in Panama. Ann. Mo. Bot. Gard. 71, 1–6. 4. Grant, V. (1994). Modes and origins of mechanical and ethological isolation in angiosperms. Proc. Natl. Acad. Sci. USA 91, 3–10. 5. Pauw, A. (2006). Floral syndromes accurately predict pollination by a specialized oil-collecting bee (Rediviva peringueyi, Melittidae) in a guild of South African orchids (Coryciinae). Am. J. Bot. 93, 917–926. 6. Armbruster, W. S., Shi, X. Q., and Huang, S. Q. (2014). Do specialized flowers promote reproductive isolation? Realized pollination accuracy of three sympatric Pedicularis species. Ann. Bot. 113, 331–340. 7. Armbruster, W. S., Edwards, M. E., and Debevec, E. M. (1994). Floral character displacement generates assemblage structure of Western Australian triggerplants (Stylidium). Ecology 75, 315–329. 8. Armbruster, W. S., and Muchhala, N. (2009). Associations between floral specialization and species diversity: cause, effect, or correlation? Evol. Ecol. 23, 159–179. 9. Botes, C., Johnson, S. D., and Cowling, R. M. (2008). Coexistence of succulent tree aloes: partitioning of bird pollinators by floral traits and flowering phenology. Oikos 117, 875–882. 10 Cheek, M., and Csiba, L. (2002). A new epiphytic species of Impatiens (Balsaminaceae) from western Cameroon. Kew Bulletin 57, 669–674. 1Institute
of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, CZ-379 82 Trˇebonˇ, Czech Republic. 2Department of Ecology, Faculty of Science, Charles University in Prague, Vinicˇná 7, CZ-128 44 Praha 2, Czech Republic. E-mail: [email protected]
Reply to Cordi et al. Christian Cajochen1,*, Songül Altanay-Ekici1, Mirjam Münch2, Sylvia Frey1, Vera Knoblauch3, and Anna Wirz-Justice1 In their paper on the influence of the moon on sleep, Cordi et al. [1] have analyzed a large number of subjects and found no significant effects, as opposed to our positive study findings with a smaller cohort [2]. More is not necessarily better. There are two main reasons why we think the comparison of these two data sets is not just comparing a small with a big sample size, since increasing the number of study volunteers in a sleep study does not automatically increase data quality. Several factors and processes influence the quality and structure of human sleep, primarily duration of prior wakefulness, circadian phase and the environmental light– dark cycle (for a review, see [3]). To thoroughly investigate and quantify the contribution of each of these influences on sleep, studies need to be carefully designed with the aim of controlling for each factor. Thus, to investigate a potential rhythmic influence on sleep retrospectively, it is essential that the examined cohort (study volunteers) was studied under very controlled conditions. For instance, light affects our circadian rhythms and in turn our sleep more powerfully than any drug. Consequently, synchronizing study volunteers to the 24-hour light–dark cycle according to their own preferred sleep–wake timing is a requirement for any sleep study which aims at quantifying sleep measures. Chronobiologists call this ‘enforcing circadian entrainment’, which is a sine qua non for proper quantification of the influence of the circadian process and prior wakefulness on sleep. We also consider this a necessary prerequisite when carrying out post-hoc analyses of the potential impact of rhythmic phenomena (such as the lunar cycle) on sleep. To explain this phenomenon with a more allegorical approach, imagine an orchestra with a certain number of musicians. You are trying to recognize a rhythmic characteristic of the music
played by the musicians, but you can only listen to them every 10 minutes for a very short time retrospectively. If the orchestra was not precisely synchronized to the conductor, you will not recognize the melody, even if the number of musicians is massively increased, which does not augment signal quality but instead leads to cacophony. However, if the players (regardless of their number) play tuned synchronously to the conductor (i.e., circadian entrainment), the chance of recognising a melody or a superimposed weaker melody is much greater, even if you sample only every 10 minutes. In chronobiological experiments, therefore, we synchronize study volunteers to the 24-hour light–dark cycle with respect to their own natural sleep timing, to unmask as well as possible the influence of circadian rhythmicity on any variable of interest. If this is ignored, one may probably miss very different rhythmic influences, such as that of the moon, because the signal to noise ratio becomes very weak. That could be the main reason that, although part of folklore, it has up until now been difficult to detect the rather weak influence of the moon on human sleep, since it cannot be revealed just by pooling non-synchronized sleep data. For future studies, we therefore suggest to carefully control the following variables, and to perform a power analysis to estimate the sample size for a targeted statistical effect: circadian phase, light–dark cycle, circadian entrainment, prior duration of wakefulness, menstrual cycle and age. References 1. Cordi, M., Ackermann, S., Bes, F.W., Hartmann, F., Konrad, B.N., Genzel, L., Pawlowski, M., Steiger, A., Schulz, H., Rasch, B., and Dresler, M. (2014). Lunar cycle effects on sleep and the file drawer problem. Curr. Biol. 24, R549–R550. 2. Cajochen, C., Altanay-Ekici, S., Munch, M., Frey, S., Knoblauch, V., and Wirz-Justice, A. (2013). Evidence that the lunar cycle influences human sleep. Curr. Biol. 23, 1485–1488. 3. Cajochen, C., Chellappa, S., and Schmidt, C. (2010). What keeps us awake? The role of clocks and hourglasses, light, and melatonin. Int. Rev. Neurobiol. 93, 57–90. 1Centre
for Chronobiology, Psychiatric Hospital of the University of Basel, 4012 Basel, Switzerland. 2Solar Energy and Building Physics Laboratory, Swiss Federal Institute of Technology Lausanne, 1015 Lausanne, Switzerland. 3Centre for Sleep Medicine, Hirslanden Clinic, 8702 Zollikon, Switzerland. *E-mail: [email protected]