1. No pollinator is an island – the role of context and previous experience in floral reproductive isolation Bischoff M 1,2,3, Campbell DR 2,3, Jϋrgens A 4 and Raguso RA 1 1 Department of Neurobiology & Behavior, Cornell University, Ithaca, NY 14853 USA 2 Department of Ecology & Evolutionary Biology, University of California, Irvine, CA 92697 USA 3 Rocky Mountain Biological Laboratory, Crested Butte, CO 81224 USA 4 School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg 3209, South Africa

2. Introduction – the evolution of floral diversity Photo: David Inouye Pollinator behaviour is a key factor mediating reproductive isolation and speciation in flowering plants Pollinators respond to floral features (e.g. morphology, colour and scent) Pollinators can learn to handle different floral phenotypes with experience

3. Ipomopsis and their pollinators I. aggregata and Selasphorus platycercus I. tenuituba and Hyles lineata

4. Geographic variation in reproductive isolation of Ipomopsis Grizzly Ridge, CO, USA low elevation (2375 m), warm nights no hybrids, mosaic structure hawkmoths abundant and foraging at night Hyles lineata visit I. tenuituba at night, hummingbirds visit I. aggregata during the day => Complete ethological isolation (Aldridge & Campbell 2007, Evolution 61: 99-110) Poverty Gulch, CO, USA high elevation (3000m), cool nights clinal Ipomopsis hybrid zone Hyles presence sparse, occasionally abundant, foraging during the day Hummingbirds and Hyles visit both species during the day => Formation of Ipomopsis hybrid swarm Poverty Gulch, CO

5. Floral scent of Ipomopsis parental species and hybrids Bischoff et al. 2014, Annals of Botany 113: 533-544. 53 compounds total Bouquets of parental Ipomopsis species and hybrids with large quantitative overlap Indole present only in I. tenuituba at night

6. From floral scent chemistry to behaviour Is the nocturnal indole signal in I. tenuituba influencing wild hawkmoth pollinators in the field? Are there interactions between flower colour and scent?

7. Scent augmentation +1 ng indole Colour manipulation with acrylic paints Score approaches and visits by wild Hyles to single-species or mixed-species arrays of I. tenuituba and I. aggregata Control = paraffin only Field manipulation of floral scent and colour at Grizzly Ridge, CO 1 m

8. In the field – Responses of wild Hyles lineata to trait-swapped Ipomopsis flowers 1. Manipulate scent but not colour in mixed-species arrays: Addition of indole increases Hyles approaches (but not visits) to I. aggregata I. tenuituba always emits indole, addition of extra indole does not alter high visitation rate

9. In the field – Responses of wild Hyles lineata to trait-swapped Ipomopsis flowers 2. Manipulation of scent and colour of I. aggregata only Low overall turnout with few Hyles entering the arrays White colour increased visits indole + white not synergistic

10. In the field – Responses of wild Hyles lineata to trait-swapped Ipomopsis flowers (all flowers emit natural indole) 3. Manipulation of scent and colour for I. tenuituba only High overall turnout with many Hyles entering the arrays Indole increased visits to red White flowers visited more than red flowers Indole + white not synergistic

11. In the field – Responses of wild Hyles lineata to trait-swapped Ipomopsis flowers 4. Manipulate scent with indole and swap natural colour in mixed-species arrays: Large indole plume over entire array All colour x species combinations visited equally Under a concentrated indole plume and in context with I. tenuituba, Hyles will visit I. aggregata!

12. From the field to the lab How does prior experience influence Hyles lineata foraging decisions?

13. In the flight cage – Responses of flower-naïve Hyles lineata to Ipomopsis flowers No-choice assay: record flight of 3-day old moths for 5 min at 15 lux Naïve Hyles lineata show an innate preference for I. tenuituba over I. aggregata Number of hawkmoths

14. In the flight cage – Responses of flower-naïve Hyles lineata to Ipomopsis flowers No-choice assay: record flight of 3-day old moths for 5 min at 15 lux Augmentation of paper discs attached to I. aggregata with 1 ul indole (100ng/ul) A A with indole Indole increases visits from naïve Hyles to I. aggregata

15. In the flight cage – Responses of flower-naïve Hyles lineata to Ipomopsis flowers Indifference to I. aggregata when experience with I. aggregata only Exposure to I. aggregata has not effect on responses to I. tenuituba Exposure to I. tenuituba enhances responses to I. tenuituba and I. aggregata

16. Conclusions Lessons from the field: Indole attracts wild hawkmoths to Ipomopsis flowers but has no effect on visit rate. White flower colour alone does not suffice to attract wild hawkmoths, yet more of the attracted moths visit flowers if white. Thus, both scent (indole) and high visual contrast (white colour), in that order, are required for H. lineata to find and feed from I. tenuituba flowers at dusk. However, no synergism of colour and scent. Lessons from the flight cage: Lab experiments with naïve hawkmoths reveal that preference for indole- scented flowers is innate. Prior experience with I. tenuituba flowers may overcome initial rejection of I. aggregata flowers in the lab. Prior foraging experience and floral neighbourhood context influence the foraging decisions of Hyles lineata.

17. Acknowledgements For assistance in the field and lab, we thank B. Castro, G. Clarke, M. Forster, M. Gallagher, J. Goyret, K. Haynes, S. Klumpers, J. Ogilvie, R. Schaeffer, M. Stang, E. Stone, H. Summers and S. van de Velde. Funding was provided by National Science Foundation Rocky Mountain Biological Laboratory University of California Faculty Research and Travel grant