Pollinator Adaptation and the Evolution of Floral Nectar Sugar Composition

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Year: 2017

Pollinator adaptation and the evolution of floral nectar sugar composition

Abrahamczyk, Stefan ; Kessler, Michael ; Hanley, Daniel ; Karger, Dirk Nikolaus ; Müller, Matthias P. J ; Knauer, Anina C ; Keller, Felix ; Schwerdtfeger, Michael ; Humphreys, Aelys M

DOI: https://doi.org/10.1111/jeb.12991

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-127539 Journal Article Accepted Version

Originally published at: Abrahamczyk, Stefan; Kessler, Michael; Hanley, Daniel; Karger, Dirk Nikolaus; Müller, Matthias P. J; Knauer, Anina C; Keller, Felix; Schwerdtfeger, Michael; Humphreys, Aelys M (2017). Pollinator adaptation and the evolution of floral nectar sugar composition. Journal of Evolutionary Biology, 30(1):112-127. DOI: https://doi.org/10.1111/jeb.12991 C. Knauer 5 This article isprotected Allrights bycopyright. reserved. 10.1111/jeb.12991 version andtheVersi this between differences through the copyediting, typesetting, pagination forpublication accepted This articlehasbeen Abrahamczyk Stefan Pollinator adaptation andthe evolution of floral nectar sugar composition Article type : Research Papers 12-Oct-2016 : Date Accepted :08-Oct-2016 Date Revised :20-Jun-2016 Date Received 4 11548-1300, USA 3 ² Institute of Systematic UniversityBotany, of Germany 7 1 6 Goettingen, Germany Stockholm, Sweden Department of of Department Biology, IslandUniversity, Long 720 Boulevard, Brookville,Northern New York Institute of Plant Science, University of Zuri Science, of University Plant of Institute Nees Institute for Plant Biodiversity, University Department of Life Sciences, Imperial College London, Silwood Park Campus, SL5 Campus, U.K. Berkshire, 7PY Park Silwood London, Sciences, College Life Imperial of Department 2,37073 Untere Karspuele Goettingen, University of Science, of Plant Institute Albrecht-v.-Haller AcceptedDepartment of Ecology, Environment and Plant Sciences, University of Stoc Article 2 , Felix Keller

1 , MichaelKessler 4 , Michael Schwerdtfeger 2 , Daniel Hanley , Daniel ch, Zollikerstr. 107, 8008 Zurich,Switzerland Zurich, Zollikerstr. 107, 8008 Zurich, Switzerland 5 of Bonn, Meckenheimer Allee 170, 53115 Bonn, Bonn, 53115 170, Allee Meckenheimer Bonn, of , Aelys Humphreys M. and undergone full peer review but has notbeen review undergone fullpeer and on of Record. Please cite of Record.Please on and proofreading process, which may lead to 3 , Dirk N. Karger 2 6,7 Matthias P.J.Müller

kholm, 106 91 this article this as doi:

2 , Anina

This article isprotected Allrights bycopyright. reserved. constraint, sucrose phylogenetic AcceptedKey words: 12:409). (CTS Stiftelse Tryggers Carl and by PBZHP3-133420) (grant Researchers a for by AMH Swiss Prospective was funded NF Fellowship CZ.1.07/2.3.00/30.0041). no. Czech Republic (project of the state budget Fundandthe Social bythe European funded was DH der Pflanzenkenntnis. zurFörderung Stiftung andthe the Swiss Nationalfond Stiftung, Adenauer by SA Konrad- funded the on drafts. earlier was for commenting reviewers anonymous several for University and collection, L. Palacký Ackerly, space and Bunnefeld duringliterature D. help S.W. during andA.Egert for E.Benetti Peters help and B.Keller, labwork, advice, for B.Seitz Lüdi collection, P. Endress, J.de R.Vos, Carter, G. help duringdata for collection, for help M. andWeist duringnectar Tompkins A.Schmidt-Lebuhn H. andJ.-M. Piedra, E.A.Tripp, We J. are H.Martinez Hentrich to grateful nectar. Kluge, B. Steudel, Zurichprovided collect to permission Bogor andCibodasthe “Sukkulentensammlung” Singapore, Basel,Osnabrück, Potsdam, St. Bern, Zu Gallen, Article gardens botanical of Freiburg,Berlin, Gö The Bonn, Acknowledgments 771 163 +46(0)8 Phone: [email protected] Humphreys M. Aelys +49Tel. 228 734649 [email protected] Abrahamczyk Stefan authors Corresponding asterids, fructose, glucose,pollination syndrome, phylogenetic conservatism, :

Thomas and P. Kevan for helpful suggestions and for helpful suggestions Thomas Kevan and P. rich, Leiden, Kew, Graz, Graz, Kew, Leiden, rich, ttingen, Halle,Heidelberg, Jena, Munich, Münster, Cochabamba, Santa Cruz, Cochabamba, Santa nectar hasbeen foundnectar to be invariable relatively within any clades, plant with interspecific morphology (Baker & Baker, 1975, 1982, 1983a). On the other hand,the sugar composition floralof preferennutritional constraints the or to adaptation and disaccharide (Baker glucose andthe sucrose Baker, fructose monosaccharides & isas1982), an primarily the comprising solution of nectar, anaqueous chemical composition the that Onthe hand,it remains floral nectar poor. suggested one of thehas been evolution of understanding ABSTRACT angiosperm evolution and floral dive andfloral evolution angiosperm plants and their animal pollinators being thought to be one of the key mechanisms responsible for Acceptedangiosperms (70-80%, extracted from Heywood interactions.pollinator Nectar isthe floral main plantis the of evolution floral rewards understanding central the of evolution to Understanding Introduction unhelpful advancing field. be might this for constraint by conclude discussing why and we maintaining pollinator deception.of Thus, broad study our provid nectarivorous bird-pollinated plants nectar sucrose-poor specialized insome unexpectedly document by Finally, we the plants. presented Article adapttheirpollinators foraging dietaryor behavior requirements to the nectar sugar composition necessarily has to adaptits nectar composition correlated – ina dictatedmanner by pollinator beha abiotic environment into the analyt and andsize floral shape the andwe studies suggest incorporate thatnectar future might evolution fully to needed explain Additional aretherefore for howfactors nectar evolves. explanation partial However, the inferred adaptive process is weak, suggesting that adaptation to PG only provides a two optimal highNSP values, forspecialist-pollin toward rejected: NSP evolves be adaptation hypothesis (PGs),and cannot that the of groups show pollinators.to We analyze ~2,100 species asteof modeling approach to evaluate the hypothesis that nectar sucrose proportion (NSP) isan adaptation pollinator dietary requirements whetheror it is nectar whether as an to sugar concerns debate evolves adaptation composition A longstanding

ical framework. Further, we show that NSP and PG evolution are evolution ical show NSPand Further, we framework. that PG from the Old World, which migh rsification (Stebbins, 1970; Harrison (Stebbins,rsification 1970; to ensure butrather pollination suggests that ‘phylogenetically constrained’. Here we use a reward the provided by vast majority of modern the conceptual dichotomy between adaptation and et al., ated NSP forand low generalist-pollinated plants. rids, spanning several plantfamilies andpollinator ces of aplant’s pollinator(s) as well asto flower vior. This contrasts with vior. es several new insightsinto how nectar evolves 2007) but despite coadaptation between between coadaptation despite but 2007) t represent an overlooked form et al., et the view that a plant that a theview 1999),our climate (Nicolson sucrose nectars, suggested beto the result of se is high- regions tubular In apredominance flowers with water. of there Mediterranean require more with a high proportion of hexose (Nicolson sucrose solutions and this is thought to explain osmola higher have solutions Hexose 1983a). Baker, flowersopen rungreater risk of nectar evaporatio the en to attributed has This been correlation 1961). Percival, proportion; hexose floral andNSP, suchthat correlation between shape is there a long-recognized Inaddition, NSP (high have to flowers highNSPandshallow low have tend tend to flowers or tubular deep Heynemann, 1983; Martínez del Rio Rio del Martínez 1983; Heynemann, sucrose proportion (NSP), reflect adaptations to that interspecific differences innectar sugarcomp Nicolson, 2008), nectar-feeding bats prefer a nectar-feeding proportion sucrose (Baker low prefer 2008), Nicolson, bats a nectar (Nicolson prefer proportion with highsucrose hummingbirds, fructose and glucose, to total sugar content). While of sucrose (relative (insolution) hexo and the composition concentration two contribution or requirementsnutritional differ fromthose nectof adaptations to feed nectar on butare still know (Martínez del Rio, Rio(Martínez del 1990; Martínez sucrose-rich nectar cannot be digested as effici Hansen Hansen nectar occasionally(referre on feed to areknown beetles) flies andsomegroups of kinkajous, short-tongued geckos, mice, Muchhala, 2008). Inaddition, asurp also act (referred as regularly, to Fleming as flowers often visiting pollinators and, by specialists; are feeders nectar bats) andobligate phyllostomid pteropodid sunbirds,honeyeaters, hummingbirds, groups andsome butterflies, wasps, (bees, moths of among nectar of plants sugarcomposition inthe convergence documented studies Early by the pollinated same pollinator group (PG; Baker floral rewardsof evolution floral nectar.and, inparticular, the what drives of understanding questioncall into our perspectives contradictory seemingly 1998; Nicolson & van Wyk, 1998; Galetto & Bernadello, 2003; Rodríguez This article isprotected Allrights bycopyright. reserved. Accepted Article differences beingindependent of et al., Johnson2006; &Nicolson, 2008). These lackanimals special morphological et al., 2007; Petanidou, 2005). This correlation reflects the physical constraints of the physical constraints reflects This 2005). correlation Petanidou, 2007; d to as generalists; e.g. e.g. as generalists; d to the plants’ pollinators (e.g. main rising variety of unspecializedve of rising variety et al., et et al., et 1992; Baker Baker 1992; et al., ently by, or is even toxic to, some generalists 1992). Therefore, ithas frequently been proposed the correlation andnectars between shallow flowers n to be effective pollinators. However, their the dietary requirements of different PGs(e.g. of different the requirements dietary lection against high-hexose nectars inawarm, dry high-hexose against lection 2007). However, high-hexose nectars also tend to to tend also nectars high-hexose However, 2007). ar-feeding specialists with respect to sugar n, rendering them useless pollinatorsto (Baker & osition andconcentration, especially innectar van Tets & Nicolson, 2000; & Baker, 1982, 1983a). Various insects groups of most specialist PGs, suchas moths, bees or rity, and therefore lower evaporation rates, than flies) andseveral groups of vertebrates (e.g. et al., 1998). van Wyk,1993; rtebrates and insects (songbirds, rtebratesinsects (songbirds, and et al., et al., et et al., et 2007; Johnson & & Johnson 2007; 2014). These Nicolson, 2002; 2002; Nicolson, vironment, because because vironment, 1998). Similarly, Galetto et al., et ses, ses, & &

into understanding of how floral nectar evolves. In nectar how evolves. floral of understanding into progress hampered have may and conservatism adaptation between dichotomy perceived the on that and being focus proposed believe constraint the biological generating anyexplicit, mechanism of Crisp, 2012). 2012). Crisp, Rodríguez-Riaño Rodríguez-Riaño phylogenetic constraints (e.g. Schmidt-Lebuhn (e.g.Schmidt-Lebuhn constraints phylogenetic beengenerally centered on aperceived dich has what causes interspecific paidto question attention of differences been the Considerable andsucrose date, To innectar. the has fructose, glucose proportions debate relative of in the 2007). &Thornburg, (Nicolson themselves different plant clades rather thandueto any inna between plantspollinated andpass hummingbirds by

( Riaño

related species to retain their ancestral state over time (e.g. Wiens & Graham, 2005; Cooper is often invoked to explain the lack of variation among close relatives (Wiens & Graham, 2005; Ackerly, 2009; Cooper Cooper 2009; Ackerly, 2005; Graham, & (Wiens even refer to the process of failing time, ancestral orand over of retention traits. We evolutionary describeto apattern of interchangeably 1 sugar solutions themselves butitis among genera, irrespective of irrespective of genera, PG (e.g. among has andinaloes be not to NSP Scrophulariaceae), but and relatives conserved found within been recorded for closely related species in the same plant family (e.g. in Gesneriaceae, Proteaceae and 1998; Nicolson & van Wyk, 1998; Galetto & Bernadello, 2003). Instead, similar NSP has been failed to find NSP findNSP to characteristicvalues failed the individual PGs of (e.g. Given the adaptiveobvious advantage of a itispollinators, surprisingthat more recent studi sufficiently dilute, accessi easily sufficiently because a plantwill only be regularly visited sic Accepted are used inertia here and conservatism phylogenetic phylogenetic constraint, Phylogenetic Article ) on theadaptation to of pollinators NSP (Galetto & Thornburg, Bernadello, 2007; 2003; et al., et 2014). These findings have led to the suggestion that there is a “phylogenetic constraint” constraint” isa led that there “phylogenetic findings the to suggestion have These 2014). et al., 2014). For example, it has been suggested that the differences inNSP differences the that has suggested it been For example, 2014). ble nectar are fulfilled (Nicolson

to to change, adapt tosome also thought to cons to thought also van Wyk otomy betweenotomy adaptation and to pollinators by efficient pollinators, whose requirements of et al., et et al.,

et al., es of plant species pollinated by different PGs have different PGs by pollinatedspecies plant es of te differences inthe requirements of thepollinators are aware that use varies among and authors may general, phylogenetic conservatism constraint or conservatism phylogenetic general, good fitgood between floral traits andanimal 2006; Nicolson Nicolson 2006; 2010;Wiens stasis, lack of variation among closerelatives, erine birds bebecausemight they belong to 1993; Nicolson & Rodríguez- Nicolson & 2007; Thornburg, 1993; titute evidence for ad factor habitator occupy some regionor et al., van Wyk, 1993; et al., et al., or the tendency of closely 2007). 2007). 2010; Losos, 2011; Cook & Cook 2011; Losos, 2010; 2007). We are not aware aware not are We 2007). aptation to pollinators Galetto et al., et al.,

1 , stochastic with respect to be to if found inNSPwere change befavored hypothesis might anon-adaptive Alternatively, angiosperm clade of about 80,000 species ( nectar sugar composition, andphylogeneti pollinator pollinator preferences. We adatasetcompile that to that hypothesis NSP, the isanadaptation nectarsugarcomposition, inparticular evaluate (Hansen, to approach use Butler this framework we King, Here, 2004). & 1997; model-comparison inference and conservatism require donot can be of explanations mechanistic Thus, in a be to evaluated hypotheses uptestable bysetting methods, usingcomparative distinguished physical or constraints the and size the environment.of according the constraints allometric drifting of found floral be shape to ifit to e.g., were adaptation; pollinated by the samePG; Leroiby pollinated the trait in relation to the environment (i.e., convergence of nectar sugar composition for plants then test for a correlation between the trait and the environment, such that there isconvergence of nectar We andPG trait asthe of asthe sugar the define composition environment. We and mints. an adaptation to something else, say, corolla shape and size (Nicolson, 2002; Witt sugar was nectar not composition but be that PG might to that preclude rejected, would adaptation instance, if a correlation between NSP and PG were rejected, the hypothesis that NSP evolves asan reliable evidence of syndromes pollination (Baker et al. 1998). Based on this, we analyzed the Pollinators are thought mainly to besensitive to sucrose (NSP), while the proportion fructose (NFP) and glucose (NGP) are though theor environment changes first King, 2004). We also explore the nature of the hy trait isassociated with a shift an adaptation to something (Westoby pattern of stasis and retention of the ancestral state does not mean that the trait in question isnot processes; e.g.,stabilizingand non-adaptive sele 2002; Ackerly, 2003, 2004; Crisp &Cook, 2012). Both stasis and change can result from both adapti nor does it constitute evidence for or against adaptation (Leroi plant species, irrespective theirof pollinators, butpr among related limited insugar variation closely suggests conservatism composition phylogenetic of (Westoby close be among not relatives, for the might variation pattern what the causal explanation observed This article isprotected Allrights bycopyright. reserved. Accepted Article 2010; Wiens et al., et al., 1995; Blomberg Garland, caseof nectar, documentation Inthe & Blomberg 2011). Losos, 2002; 1995; 2010; Crisp &Cook, 2012). However, this conveys thatonly there islimited trait any in function (i.e.,a newsyndrome; Hansen, pollination Butler 1997; & environmental variable to which to variable environmental et al., (Pagel, 1994; Ackerly, 2004)? 2004)? Ackerly, 1994; (Pagel, et al., 1994; Ackerly, 2004) and such that an adaptive shift in the Bremer, 2009 1995; Ackerly, 2004; Losos, 2011; Hansen, 2014). For For 2014). Hansen, 2011; Losos, 2004; Ackerly, 1995; ction ction anadaptive provides explanation for the pothesized adaptation by asking whether the trait is unprecedented inscope for this purpose, with ovides noovides insightinto how floral nectar evolves, c databroadly sampled for the asterids, a major theproportion totalof sugarconstituted by ), carrots, includingthe heathers daisies, et al., it were hypothesized to be an 1994; Blomberg & Garland, & Blomberg 1994; t not to provide et al., 2013). 2013). ve precedes or accompanies change (the inNSP trait). NSP and is between accurate and ifchange PG correl and PGcannot be rejected, ifthere is evidence Nectar sucrose isan adaptation environment are slow and achieved by st and the environment (PG) is rejected or if there is ev adaptations to pollinators. This would besupported Overall sugar concentration (%) and composition (relative proportions of fructose, glucose and and offructose, glucose proportions (%) sugarconcentration Overall (relative composition stored itinsilica gelfor up ato few to months per species was 1 µl (Morrant collected nectar per volume species.of minimum plant The flowers from young at least three nectar Southeast AsiaandSouth America field inEurope, inthe 2007 2011 September and August nectar between We collected 2007). bacteriaof impact which andyeasts, able are transfer to sucrose (Nicolson to hexoses & Thornburg, the minimize to flowers from by only young nectar collecting sugarcomposition innectar variation Sutherland,Janza 1994; proportional content of each sugar in turn to evaluate support for the following hypotheses. hypotheses. for each content the to following sugarinturn of evaluate support proportional Accepted in plants growing the wild inbotanior in or between populations the wild between differences dueto degree asmall to andonly individuals of intraspecific variationlevel that differences to is documented due hasbeen among mainly 2007) & Thornburg, Nicolson 1998; Galetto, & Torres 1996; Schwerdtfeger, 1983a; microclimate, nectar sugar iscomposition relatively volume and total sugar concentration, which ar nectar to sugar sugarcontent”. In contrast and composition” terms “nectar usingthe “nectar Scholar for available this clade, accessed by searching for rangegeographical Furthermore, andpollinators. a andsucrose) glucose asterids andchose in diversity the (fructose, because structure,of their floral samples for this study. We focused on the proportional content of the three main nectar sugars the nectar Old andanalyzed (e.g. taxa neglected from collected new tropics), World previously Article data compiled sugar for nectar asteridsWe records composition usingpublished and, focusing on data Nectar Material and Methods

et al

et al., ., 1995; Gijbels to pollinators. This would besupported if a correlation between NSP and inbotanicalgardens. Weused glass capillaries to collect 2009). We placed the nectar on filter paper, air dried it paper, airdried nectar filter and on We placed the 2009). cal gardens (e.g. Freeman & Wilken, 1987; ochastic, rather thanadaptive, change. prevent microbial decomposition ofthesugars. that adaptation to the environment israpid and e heavily influencedby water availability and et al., et publications in ISI Web of Knowledge and Google and Google in publications ISIWeb of Knowledge H2 constant within plant constant within species Baker,(Baker & wealth publisheddata of ecology pollination is on if a correlation between the trait NGP) (NFP or idence that shifts in the trait in relation to the ated, such that change in 2014 : Nectar fructose and glucose are not arenot Nectar fructose andglucose : ). We further reduced thepotential for PG (the environment) environment) (the PG Vickery & . The relatively low low relatively The . H1 : Pyke, 1980;Stiles, 1981; andlorikeets Hopper (Psittacidae; Burbridge, 1979; & (Meliphagidae) of genera honeyeaters bodied (Trochilidae) andOld World sunbirds refertobird speciesweIn contrast,1992). the generalist insect-pollinated. we Similarly, recogn a rangeof insects referred wasps,for are smallwide flies as to bees andbutterflies, (e.g. andbeetles) small, flowers, providing and2) to access relatively nectar referred open the to are as bee-pollinated categories developed by Schwerdtfeger (1996): 1) Also, the details of the pollinators of these plants are largely unknown. Instead, we adopted the two Baker & Baker, 1975, 1982, 1983a, 1983b) can be difficult to detect in nature (Schwerdtfeger, 1996). Several concepts for insect syst pollination by short-tonguedpollinated beesandbutterflies sh separate PGs. food plants. Therefore, we treated specialized, nectar-feeding birds from the Old and New Worlds as birdsinthe Old andNew areassociatedWorlds withnectar-feeding characteristics different their of nectarfrom andare able to digest sucrose effici to digest sucrose but not as efficiently as they are as able as Rio (Martínez digest to they hexoses not del efficiently but sucrose digest to the ability to digest sucrose (Martínez delRio, 1990), while others (waxwings; Bombycilidae) are able Some birds starlings, and of these (thrushes, 2008). This article isprotected Allrights bycopyright. reserved. flowerpeckers [Dicaeidae]; Stiles,Amadon, 1950; 1981; Johnson &Nicolson, delHoyo2008; and [Drepaniidae] Hawaiian honeycreepers [Zosteropidae], white-eyes [Pycnonotidae], amounts fruits,of seeds or insects, are referred to Generalist feed passerinePGs. different andnon-passerine birds that nectar, on as well as larger on Johnson we Nicolson, treate 2008), (specialists; & andphysiology withmorphology plantsthat are birds(generalists) contrast are nectar-feeding intheir pollinated unspecialized that by plants mainly Due to the large variety of concepts for defining PGs, these were considered carefully. Because groups ofpollinator Definition was normalized logit by transformation prior to analysis. sucrose) were determined using standard protocols (SI text). The proportional (Schwerdtfeger, 1996). Accepted distinguishingthe species mainly pollingroup, Article This contrasts with the various plantspollinated groups of different by groups Tjørve

et al., (Nectariniidae), sugarbirds ( (Nectariniidae), sugarbirds 2005; Johnson Nicolson,& 2008), take most of their energy ated during theday and night,respectively as specialized, such as New World hummingbirds as suchasNew specialized, hummingbirds World ently. Behavioral differences between specialized mainly pollinated by pollinated by mainly obligate nectar-feeding birds ized a butterfly-pollinated d specialized andunspecialized as birdspecies typical bee-pollinated, flowers zygomorphic often asunspecialized (e.g. [Oriolidae], bulbuls orioles ems exist. The distinction between plants ort-tongued beesort-tongued long-tonguedor bees(e.g. mockingbirds; familiesmockingbirds; inthe Muscicapoidea) lack Promeropidae), and a moth-pollinated and content of each sugar

and some small- and some et al., et et al., et

to absolute time using 110 published age constraints using (Britton absolute constraints S1) time (table inpathd8 age 110 published to height scaling allbranch lengths tree andthen S1),was (table 1.0 setting the to by studies obtained results numerous independent the of of branchlengths, representing distribution realistic The most (figure S1). explored approaches were for providing information branchlengths three branch-length in founded distances (e.g. allmodels rely phylogenetic analyses on trait evolution Many Brownian motion; e.g. Pagel, 1999; Thomas &Frec available. data were and PG for nectar which species, both finalset2063 comprised of The trees within genera). (e.g. uncertainty randomly resolved 100 times to generate aset of 100 trees that reflect some, shallower phylogenetic species place using 2.74 manually to Mesquite and the phylomatic ( phylomatic webpage and the to generate asummary phylogeny. At the high for all studies asterid sampled species information frompublished phylogenetic we used Therefore, genes incomplete. for individual highly rendering data used amongcoverage differ markers studies, in datasetfor available DNA species most nectar the phylogenies DNA-based are but the Published, information Phylogenetic butterfly-pollinated. as with corolla colors were scored long, alandingplatform, narrow andbright Flowers tubes wereplatform scored as pollinated pending corolla withand stamens tubes, stigmas wide long, arelatively without flowers landing (Fenster because this isknown to be areliable method for id based coloration floral categories on 1978) Pijl, used &van andscent der (Faegri morphology, Rothmaler, 2011) or our own field observations. For species where thepollinator isunknownwe generated data (~30% species)of we usedpollinator We informationobtained oneachasterid spec newly the For sugarcomposition. nectar data on published the to used extract publications same World bats, New unspecialized birds, moths, groups: gene defined ninepollinator of butterflies recognized by some authors (Baker & Baker, 1975, 1982, 1983a, 1983b). In the end, we Accepted Abrahamczyk S1; data, genes (supplementary based on multiple phylogenies published Article et al., 2004;Rosas-Guerrero

Webb by specialized by Old birds World in or, the Americas, hummingbirds. ralist insects, bees andwasps, et al., et al., 2014). For example, brightly colored, often red, scentless scentless red, colored, brightly often For 2014). example, 2009 specialized Old World birds and hummingbirds. birdsandhummingbirds. specialized OldWorld er taxonomic level we referred to Smith (Maddison & Maddison, 2009). Polytomies were were Polytomies 2009). &Maddison, (Maddison ). For resolution among and within genera we used among used andwithin we genera ). Forresolution kleton, 2012) and since the assembled tree lacked lacked tree assembled the since and 2012) kleton, entifying the most effective, main PGs of aplant of effective, PGs the most main entifying observations from theliterature (e.g. Jäger& ies’ to affiliation pollinatorone group the from specialized flies, butterflies, et al., et al. et 2007). et al (2011) ., 2016) 2016) ., All models Allmodels were fitted using maximum likelihood (ML) inR( strength of the pull toward those stochastic rates toward of change those also differoptima may (OU-2V) and in the one which inwhich (OU-2), one andgeneralist specialist PGs differ between was allowed to optimum mean theon results of the OU-PG model (see Results) were based simpler hypotheses, three our models further test to Instead, not reported. andare unreliable suboptimal model overall. Results for these more complex models are therefore considered in Thisa resulted estimated. parameters bereliably that could or one more not findings indicated S8), (table and points data preliminary by aremany represented optima allputative not large, putative optima such models soon become highly parameter rich. Although the current dataset is pull or stochastic rate may vary among optima (Beaulieu bypollinated different PGs (OU-PG; putative9 optima putative optima; see Results) and the third allowed the mean optimum to differ among species clades uniform rate definedwith Clades; ashaving allowed second species differ clades the to the (OU-1), optimum among (OU- mean indifferent a value single directional toward optimum change allowed first model The the optimum. toward rate dictated both by the strength of the pull (rate of adaptation) and the rate of (stochastic) change a & Butler (OU) of was (Hansen, Ornstein-Uhlenbeck King, series 1997; Next 2004) models at a values, in or sugar more allow toward change directional, one optimal nectar be These to fitted. clademinimum size for detecting shifts was set to 100. identified; Thomas & Freckleton, 2012). For practi incrementally until no better models were found (i.e., all shifts supported by supported found (i.e., allshifts were better models no until incrementally The number rate of PG. was increased of independently rates’), shifts clades (‘differential and among et al., selective regime, were determined O’Meara, Ancestral determine distribution each2015). the which putativ phylogenetic states, of motmot (Thomas & Freckleton, 2012) and the OU rates’ 2002).Anderson, The BMand‘differential mo 2014 This article isprotected Allrights bycopyright. reserved. Accepted Article Schluter BM; is indirection change (Brownianthat random of constant motion, nullexpectation the from We devised aseries of models (table 1) that allow change in nectar sugar to depart in various ways models adaptive andnon-adaptive in framework: fitting motion aBrownian Model ) and fit compared usingsample-size AIC was model corrected (AICc; values & Burnham 2004) for PG plant andmanually for clade. et al., 1997). The first departure allowed the rate of change in nectar sugar to vary over time sugar changeinnectar rate vary to over of allowed the The first departure 1997). optima alsomay differ(OU-2A). using inthe ‘ace’the (Paradis function ofmodel ape equal-rates models were fitted using fitted & (Beaulieu OUwie were models cality and to avoid inferring spurious shifts the shifts inferring the cality avoid andto spurious s change in the differen dels were fitted using‘transformPhylo.ML’dels were in devised for NSP only (table 1): one inwhich the ). More complex models in which theadaptive et al., 2012) were explored, but with 9 or 16 9or with but explored, were 2012) R Development Core Team Team Core R Development tial rates analysis; 16 Δ AICc

≥ 6were e e for thefor remaining 636 species were generated for birds)andNSP as unspecialized ‘high’ (>0.45) ‘low’or ( andhummingbi moths birds,butterflies, specialized asbinary, as each with PGcoded variable treated ‘s (see Results). To further explore this we tested for correlated change between the NSP and PG. We The results the of above analyses analyses correlation framework: inMarkov fitting acontinuous-time Model Accepted data, Abrahamczyk (see S2; from the taken were literature supplementary species) representing genera,660 55 families, and13 th of comprised sugarcomposition nectar dataset andsubspecies species The of2,116 asterids, composition innectarsugar Variation in topollinator group relation Results whole to those for a set of less inclusive clades). Deta simulated data) and 3) phylogenetic scale (by compar trees), 2) phylogeny alone (by comparing results theof differentialrates analysis to those for thephylogeneticcomparing signal uncertaint phylogenetic test to the effect of 1) performed were sets of analyses Three and simulated phylogeneticto andcompared data to scale ofresults uncertainty Robustness likelihood ratio (LR) test with 4degrees offreedom (d.f.) on each tree. large trees; available from www.evolution.rdg.ac.u each of the 100 trees using the Discrete functions in BayesTraits V2.0 (Quad Precision version for Article for iterations ML fitted10 with were Models 2). (table model independent inthe andfour model on whether the second trait isin state 0 or 1). There are eight possible transitions in the dependent (i.e., depending trait and 0-> in differ ->1 trait may 0transitions 1 one on the other dependent are whether the second trait is in state 0 or 1). The second model states that rates of change in one trait -> (i.e., trait of the -> 1 other 0 and 1 independent mode first The 2006). & Meade, Pagel 1994; (Pagel, models for discrete traits to test whether change in one traitdepends on the state of the second trait fordeviation]) allgeneralist-pollinated plants; tabl suggested that NSP (but not NFP or NGP) isan adaptation to PG of eachnectar data sugarandpollinator across theset of 100 e 16 orders. Roughly two thirds of these data(1480 this study (577 species sampled from Botanical sampled species from (577 this study e S4). Next we used two continuous-time Markov 0 transitions occur at the same rateirrespective of k/BayesTraits.html) and fit was compared using a pecialist’ (bats, specialized flies, bees andwasps, l states that rates of change in one trait are rds) or ‘generalist’ (generalist insects and ils of these analyses are provided in the SI text. ing best-fitting models across the asterids asa ≤ 0.45, the 84

th percentile [mean [mean + standard percentile 1

et al y (by y ., 2016). Data Data 2016). .,

354; tables S3). half Half asfor NSPandthe other the1, same atoccur tables node of these exactly 354; occur Barraclough, 2014). Fifteen rate shifts were al rate shiftswere 2014).Barraclough, Fifteen high ( 3). The stationary variance, ameasure of the rate of drift relative to the strength of selection, is very However, the parameter estimates from this model suggest that the adaptive process is weak (table model isa much better fit to the data ( unspecialized insects andhigh for plantspollinat NSP independent optima were inferred for was best-fitting OU-PGmodel andforThe NSP NFP rates andNGP differential (table 1). Two NSP, NFPandNGPmodels for evolutionary Best-fitting a nectar (figurewith low have sugarconcentration S3). overall and named clades that we are only beginning to be able be detect to to (Smith beginning clades that we only are and named sample analyzed here or, alternatively, suggests correspondence between evolutionary processes named to, almost an clades. major to, or This be sparse relatively of the correspond artifact could Lamiales, + rates generallyGentianales where decrea inEricales, increased generally shifts rates and were found where clades, in most nested shallower or rates rate inwhich were differential 15 forshifts was the model, best NSP model second The shifts were several speedups; and shifts were slowdowns S3). eight Seven (figure 3, table identified 10% of the age theof asterids (tableS2). adaptive process in which species are closer to th specialist-pollinated plants phylogeneticand yield ha unexpectedly sucrose-poor nectar among andmuch (figure birdsandbutterflies, overlap them 1 World degree of variation inthe NSP of species pollinated by plants show askew with respect to the two hexoses, being shifted toward fructose. There isa high lower NSP lower NSP than plantspollinated specialists by (figure 1 nectar sugar composition along the sucrose axis, wi 1), confirm1), that generalist-pollinated plants ar (OU-2,simpler models OU-2V andOU-2A), although a (third fit data the to worse table best overall; distance from the ancestral state to the trait optimum, isextremely long (ln[2]/ This article isprotected Allrights bycopyright. reserved. Accepted Article Abrahamczyk data, S2; [supplementary groups Gardens, 59 from the wild, with samples from th σ 2 /2 α = 2.5x10 5 ) and the phylogenetic half-life, defined as the time needed to evolve half defined as timethe needed half-life, evolve the to ) and phylogenetic the for some species pollinated by Ol for NSP: lowfor NSP NSP: for plantspollinated by generalist birdsand Δ AICc ≥ so found under the overall best model for NFP ( best for model the overall so found under e evolving toward alo 783) than any theof otherBM-based models. et al e wild spanningarangeof families andpollinator eir optimum and that isachievable within about th plants pollinated generalists by beingfound at ., 2016]). A ternary plot shows separation of lf-life estimates of ~10 Ma, i.e., suggestingan ed all by other PGs studiedhere (figure 2). This sed in shallower clades. Most clades identified most PGs, especially by specialized flies, Old specializedby especially PGs, most A ,

figure S2). Only hummingbird-pollinated

d World specialized birdsthat wer NSP optimum than B ). Inparticular, we documented et al., 2011; Humphreys & Humphreys 2011; α = 4.0x10 = 5 Ma). The Δ AICc ≥

into alow-NSP,into specialist-pollinated plant than from a specialist to generalist PG are more likely in a low inalow likely ( sugar background aspecialist PG aremore generalist to from 98% of trees; table 2). This suggests that nectar shif and shifts from high to low high inageneralist ( likely from are background to more and shifts NSP PG reveals the nature of this dependency: a high-NSP, and berejected not between NSP PG could evolution dependent formally, of the model More for any of the 100 trees (P < 0.0001, LR tests with likely,more respectively). areflowers likelymore beto a pollinated by spec high-NSP both and low-NSP round: hold not the associationother This does way flowers). NSP times likely more flowersto pollinate high-NSP and NSP and generalist low very PGshigh than few are 0.0001, Fisher’s Exact test, two-tailed; table S4). Sp and NSP PGWhen are coded asbinaryvariables, analyses: nectarCorrelation sucrose andpollinator proportion group NGP was OU-Clades. both NFP and best for in model second The in andspeedups Ericales. andLamiales occur Gentianales to tended sugars: decreases slowdowns was pattern for the allthree same The of and speedups as shifts. nodes rate occur same to for tended NFPor for 11 both. the at Again, shifts NSP, support strong effect For of scale. phylogenetic NSP, thestrong asterids findings for the confirmed but, overall were a analysis less revealed inclusive any trait. Finally, recovered clades of be with can apparently they because caution should interpreted S3) andthese be with table data (SItext; andsimulated empirical differently toexpectations aneutralfor trait. Howe rate andNSP increases isnot an is andof pattern phylogeny decreases the evolving artifact of rates analysis differed for data empirical differential and simulated (figureS4). Thus, overall, the S5). results the Thus, table some uncertainty. to The (SI text, robust ourof phylogenetic findingsare Estimates of phylogenetic signal were constant across to datasimulated andscale andcompared phylogenetic to uncertainty Robustness trees). Rate estimates for the reverse Accepted Article for NGP ( model best overall Under the away. a nodes few or one only transitions were indistinguishable. indistinguishable. weretransitions into a generalist-pollinated plant( high-NSP there is significant dependency in the data there data (P < issignificantin the dependency ialist than ageneralist pollinator (68 and4 times 4 d.f.).Acomparison rate parameterof estimates associated with (specialists highNSP are three ecialist PGsare likelymore to be associated with specialist-pollinated plantis ver, some shift positions were recovered for both ts first from the ancestral state. In addition, shifts generalists six times more likely to pollinate low- the set of 100 trees for all three sugars and PG Δ AICc q ≥ 34 > > 34 122), there was q likelymore tochange 24 > > 24 q 12;trees) 94% of q 13; 96% of q 13 > q 12; both within and outside the asterids (e.g. with. Similar deception strategies probably occur in deceptive to the pollinators because of the compar Itisthought. possible that this represents anal be innature than may andsuggests that widespread previously thismore (2010) phenomenon explanation. Nectarivorousexplanation. birdsareknown to prefer plants pollinated specialized by OldWorld nect different optimal NSPvalues indifferent plantcl requirements for different different 2002), in preferences &Erhardt, different 1997, andfemales (Rusterholz males because of specialized Old birdsWorld (figure1). The variab species pollinated by most notablyPGs, most by converged theon optimum for that NSP PG. Instea Based theresultson of Baker andBaker (1982), we Unexpected variation innectar sugar 20%) that is extremely sucrose poor (NSP < 0.20 by Old pollinated nectarivorousWorld birdsproduce than sucrose solutions (Nicolson &Fleming, 2003). Our finding, that about athird of the species osmolarity have solutions small to a higher nectar inand hexose changes of the sensitive osmolarity This article isprotected Allrights bycopyright. reserved. Discussion these two sugars (SI text, tables S6, S7,figure S5). were largely mirrored andNGP,thuscontra for NFP was much cladesheight, stronger analyzedfor separately for than findings These asterids overall. relative half-lives tree Evidence short overall to asindicatedfor S5). by adaptation, e.g. phylogenetic differencesin addition, specialistamong asPGs well as clades among were found (table 3, S6, figure overall sugarconcentrationoverall of the nectar is low(5%; Brown composed nectar of hexose they shown prefer have that experiments but concentration Accepted study). Further studies are needed to test this hypothesis. Article

(Petanidou, subgroups pollinator 2005; Goldblatt & Manning,or 1999) composition for groups many pollinator van Wyk van ternative, energy-saving pollination strategy, which is arivorous birds probably requiresarivorous birdsprobably a different ; figureS3), corroborates the results of Brown ades (explored further below). The for variability ility for these insect-pollinated plants may be specialized flies,bees and wasps, butterflies and d, inwe found highvariability theNSPof plant expected plants pollinated by one PG tohave atively energy-poor nectar arethey rewarded many plantspecies many belonging several genera to et al., dicting resultsacross the asterids asa whole for nectar with low sugaroverall concentration (< sucrose-rich nectars wi 1993; et al., et Nicolson & van Wyk, 1998; this this 1998; Wyk, & van Nicolson 2010). This is because the birdsare is the This because 2010). th a th sugar high overall s if the et al. body shape evolution in three-spined stickleback (Voje andsize, inturn be shape Such an which has invoked forcorrelated with could PG. explanation been allometric bounded NSP interpretation isdrifting, the isthat constraints floral by possible third of (Labra interpretation, therefore, is that NSP is evolving possible 3). (table unrealistic rangebut not Another limitsare of the NSPapproachoccupied the for ranges, perhaps themselves therefore representing unreachable adaptive peaks. The optima inferred plants. However, the optima inferred from these models tend to lie outside currently occupied movement toward lowNSPingeneralist-pollinated 2013), perhapsas afunction previous studies ( previous specialist PGs, while being high,aregenerally i bypollinated most obligate nectarfeeders imp generalists and one for specialists plantspollinated by (figure2). Lack differencesof among plants pollinated by forplants one independent values, infactoptimal on two only converge they toward different to NSP isallowed isevolve where overall for NSP evolution best The model optima for each of the nine PGs studied here (table 1). Inspection of the inferred optima reveal that our findings constitute evidence for NSP being an adaptation to PG? NSP between the and But deta what do to andfor extent PG. PGs evolution correlated different statistical strong rejected: we found support for among different NSP convergence on optima lizards (Slater in andhabitat niches incetaceans monitor dietary inrelation to sizeevolution for body invoked ancestral state (Hansen, 1997, 2012; T.F. Hansen, landscape inthe but sense strict increasing from rather divergence an adaptive the trend of adaptive optima. One interpretation ofsuch a model isthat itdoes not represent adaptation to peaks in the two the by explained is not model the of residual variance the of alargeamount andthat movement bystochastic is overwhelmed NSPoptima toward the any evolution adaptive that suggesting it T.F.reach (Hansen, pers. Hansen, 1997; comm.) life isunrealistically suggesting long, thatspecie process a reveals The (table phylogene adaptive weak 3). model best-fitting of the inspection Bernadello, 2003) have failed tode Accepted Articleet al., that NS hypothesis the tested We sucroseNectar proportion as 1992; Baker Baker 1992; et al., et 2009; Hansen, 2012), for example corolla example for 2012), Hansen, 2009; et al., et van Wyk, et al., et 2010; Collar 1998). At first glance, 1998). results our be this cannot suggest that hypothesis 1993; Nicolson & van Wyk, 1998; Galetto of the abiotic environment (Petanidou, 2005; Nicolson an adaptation to pollinator group an adaptationtopollinator et al., et P is P an adaptation to PG (e.g. tect significant differences among inNSP (specialist) FurtherPGs. 2011). For nectar evolution this would suggest slow suggest slow this would For nectar evolution 2011). ndistinguishable. This could bendistinguishable. could reason This why one lies that theNSPrequirements the of individual s are far from their optimum and unlikely ever to as an adaptation to other, unmeasuredvariables other, to an adaptation as . The stochastic variance isalso very high, pers. Suchanin comm.). plants inspecialist-pollinated andhighNSP shape and size (Nicolson, 2002; Witt (Nicolson, 2002; andsize shape et al., 2013). Without formally incorporating Heynemann, 1983; Martínez del Rio Rio del Martínez 1983; Heynemann, et al., terpretation hasbeen 1998;Galetto & et al., 2007). A 2007). et al., tic half- ils of

pollinators (Nicolson (Nicolson pollinators the view that a plant must adapt itsnectar composition to ensure regular visitation by efficient their NSP to thelocal pollinator guild in theliterature (and thisstudy; Nicolson interaction pollinator might better be viewed from asthe andPG environment in the trait – treated This article isprotected Allrights bycopyright. reserved. shift from the ancestral state likely to pollinate alow-NSP flower. Thisasymmetry is specialized nectar feeder is likely more to pollinat likely to bepollinated by a specia is a that suggests as variables flower more data coded binary in NSPandPG the dependency The Is nectar sucrose proportion the trait or the environment? needed to fully explain how NSP evolves. – here not act are ashypothesized directly other factors does it of NSP but evolution component explains less of the residual variance. Together, these results suggest that PG isan important complex The story. isafarmore but superiormodel the to entire data that one fit nevert However, because the simpler models area much worse fit to the data, they do not capture the than rather drift. adaptation that of of interpretation the supports PGs.This specialist versus insight: these models suggest a much stronger adaptive process, at the coarse level of generalist PGs but resultsfrom the simpler OU (OU2,models OU2A and OU2V; table S2)do offer some additional distinguished be alternatives cannot these framework, hypothesis-testing the into variables these Acceptedanimals “capitalize” on the NSP presented by the pl dictated by behavior pollinator rather than vice first findingthat isprimarily nature NSPchanges correlation of suggeststhatThe the the 1980). under(Janzen coevolution strict as thatchange, requires simultaneous expected would in away be andPGNSP iscorrelated in away that means that highto lowshifts from NSPare likelymore inge shifts from a specialist to generalist PG are more in change asimultaneous inNSP preceding occur PG. Once has without or variation been established, Article et al., 2007). A is NSP of be this should perhaps conceptual that consequence 2007). of highNSP/specialist of PG ( list pollinator, irrespective its of NSP (table S4). In contrast, a (cf. “diffuse coevolution” Janzen(1980)).of This contrasts with et al., therein). and2007 references versa. In other words, it suggests that pollinating neralist-pollinated lineages. Thus, theevolution of order to be fully understood, the floral rewards- fullyunderstood, be to order likely inlineages with low-NSP nectar andfurther e a flower andageneralist high-NSP feeder is more certain shiftsare more likely than others, butnot the opposite perspective to that often presented ant andthatthe plants explained by the is findingthatNSP thefirst to q 13 > > 13 q 12; table 2); i.e., changes in NSP may do not necessarily adapt adapt necessarily not do heless heless

shed furtherlight theseon alternatives. different processes govern the relative proportions if even inNFP and/or NGP, of that the beindependent signal cannot that inNSP proportions, Finally, these findings hold true not only for NSP but for NFPandNGPas (tables for NSPbut S7, not S6, well only findingshold true these Finally, figure S5).This contrasts with asterids findingsfor pollinatorplant butby behavi evolve and that the relationship between NSP and PG isperhaps not governed by adaptations of the differences inNSP interspecific that conclusions above both PG how alone cannot explain support governed by floral shape and size, rather than the main PGs of each clade. These results further small, flowers,open suggesting thatthe different have and Asteraceae flowers tubular tend S8). to Ericaceae table and hummingbirds; butterflies Asteraceae are pollinated by bothspecialist and ge PGsspecialist (bees andwasps, birds specialized OW sampled and here), while hummingbirds (Ericaceae) anda low optimum for thedaisy family different clades, irrespective of the main PGs. in revealed optima different models single-optimum Incontrast, upon elaborated above. deception hypothesis for support the birds,providing of further forspecialized nectarivorous inferred optimum is low-NSP the PGs.Oneinteresting specialist example found differences among also but and for generalist PGs(figureS5) specialist optima independent corroborate to the finding of tended evidence for adaptation to PG in analyzed defined PGs broadly the to is adaptation that, at the broadest scale, several factors areneed from these for (table This asterids inferred from t supports models than the 3). overall model for 12 rejected S6)out of clades anda process13 (table rapid adaptive much andprecise was more be for individual clades for anadaptive asterids as not than could model stronger awhole: much Analysis lessof inclusiveclades revealed severa processes indifferent Different the two hexoses in nectar ( Accepteda these sugars (table1) twofor Article contra or anddietary requirements. clades andfordifferent clades sugars? nd suggests either that pollinators some clades andnone atall inot Baker et al., 1998) or, because our analyses were based based were on our analyses or, because 1998) A was highoptimum inferred for the heather family l important insights. Evidence for adaptation was optima inferred for these two clades could be here. Certain clades, however, revealed strong as a whole, where an ad neralist PGs(generalist insects, bees and wasps, (Asteraceae). Ericaceae are entirelypollinated by ed to explain nectar evolution, only one of which of each sugar. Future (experimental) work may her clades. her models Multiple-optima are sensitive to the proportion of aptive model was rejected he idea he This article isprotected Allrights bycopyright. reserved. clades (Baker & Baker, 1975; Nicolson canaccumulate that andamon within of variation amount the limiting constraint", “phylogenetic theon dichotomy between adaptation dietary topollinator requirements andthere beinga has of nectar sugarcomposition centered evolution surrounding the debate historical of the Much andbeyond evolution innectar andconservatism Adaptation adaptation adaptation pollinatorsto is a not sufficient explanation its on own, Rodríguez-Riaño 2007; Thornburg, 2003; Bernadello, diversity,pollinator arelikely to underlie some earl clades areof and clade-specificthat NSPoptima independent of indifferent operating processes maintaining evolutionary process.maintaining the ancestral is retention state of Thus, apattern2014). nothing says its that or of generating something they or havewould succumbed to sele an adaptation. The counter isthat argument re This 2014). a(Hansen, means that interpreted as retained ancestral be cannot (plesiomorphy) state recently (e.g. Revell from anyunderl signalis disconnected phylogenetic Bl (e.g. asprocesses andinterpreted phylogenies read it from be patterns haslong been simply while cannot clear that However, asprocess. 2012), unexplained (Ackerly, 2004; Cattin for adaptation, but where the specific hypothesis being tested leaves some observed variance have invoked both adaptation and conservatism in cases such as ours, where there issome evidence studies in anything empirical Many 2011). andcannot or Losos, explain constrain itself (e.g. patterns patterns remains. inferring phylogenetic processpractice of from 2004; Losos, 2011), why does it persist? We suggest that there are several reasons. of the inadequacy of nectar the literature, the far clarity, longstanding beyond the Given adaptation/conservatism dichotomy (e.g. Leroi the nectar literature. is inlieu full invoked as of conservatism the just mechanistic appears theexplanation, be to case in words, tested. Inother being hypothesis the to related directly a historical describe strong signal not these studies, phylogenetic conservatism isnot invoked asan alternative to adaptation but to Accepted Cooper signal(Losos, 2008; asphylogenetic measured often divergence, Article ii i ) Asaresult of (naïve) interpretation of phylogenetic patterns of trait similarity and ) Because of the cladistic tradition of reco et al., et 2008;Boucher et al., et al., 2004; Escuerdo Escuerdo 2004; et al., 2007). Indeed, results such as those above, of different tained plesiomorphies must be adaptations for beadaptations must tained plesiomorphies 2014;Münkemüller et al., omberg & Garland, 2002), the extent which to extent the Garland, 2002), & omberg ction (Losos, pressures to change Hansen, 2011; gnizing only as autapomorphies adaptations ier claims of phylogeneticier of claims conservatism(Galetto& ying process has only become clear relatively clear relatively become has ying process only 1994; Blomberg & Garland, 2002; Ackerly, et al., et al., 2014). However, although we found that that found we although However, 2014). 2012; Hansen, 2014). However, in phylogeny et al., et al., et 2015). Therefore the is merely a is depiction of merely 2010;CrispCook, &

g Abrahamczyk S., Kessler M., Hanley M., S.,Kessler D., Hanley Karger Abrahamczyk phylogenetic constraints or conservatism. phylogenetic with consideration how of the flor divisions of PGs and incorporating additional factors into the hypothesis-t interactions floralvia rewards evolve by focu whole story. Future studies may increase me We present evidence that NSP isan adaptation to PG in asterids but, importantly, that this isnot the Conclusion 2009). less attention than each phenomenon has separately (but see Ackerly 2003, 2004; Labra, Labra, Ackerly 2004; (but see 2003, hasseparately than phenomenon attention each less reconciliation howof adaptation andconservatism 2014). 2014). The trait may stillbe anadap (Hansen, under study is rejected is at specific that hypothesis the anadaptation all, only not question the mean in trait necessarily not that does inference over “adaptation” but of “inertia” confusing References inertia models”;e.g. Pienaar

the samethe models ( 1991; Wiens & Graham, 2005; Crisp & Cook, 2012), was born. Increasingly, these fields make use of et al., ininferringchallenges involved conservatism as largely different fields. This is mo interpretation dichotomous. isnot necessarily or “inertia”, “adaptation” either species (Labra that variable unmeasured another, to or manner AcceptedBeaulieu 2004; Butler & King, [Hansen, 1997; adaptation (one with inparallel is concerned developing Article 1994; Hansen, 1997), another, dedicated to detecting (niche) conservatism (Harvey & Pagel, &Pagel, (niche) detecting to (Harvey conservatism dedicated Hansen, 1997), another, 1994; Schwerdtfeger M., Humphreys A.M. 2016. Data from: adaptationPollinator andthe iii iv et al., ) Due to the development of the conceptual frameworks for studying adaptation and adaptation for studying frameworks conceptual of the development the to ) Due ) Because of the terminology used in the OUframework of adaptive models (“adaptation-

et al., 2012], the other with a “failure to adapt” [Wiens cf. 2009; Hansen, 2012). Thus, although model inferences described may as inferences be model Thus, although 2012). Hansen, 2009; e.g. Kozake.g.Wiens, & 2010; Münkemüller et al., adaptation inaadaptation comparative framework (Baum & Larsson 1991; Leroi al nectar-pollinator interaction ar use This 2013). be as certainly perceived may conceptually tation to an environment that it chanistic howplant-pollinator understanding of D.N., Müller M.P.J., Knau sing on dense samplingsing on of narrower finer clades, shows strong similarity among closely related st likely historical: as one field was realizingthe can beinterpreted together has received much et al., et al., ises, without the need toinvoke 2015) but because they are because but they 2015) self is evolving inadrift-like er A.C., Keller F., 2010; Kozak & Wiens, 2010]), 2010]), & Wiens, Kozak 2010; esting framework, along et al.,

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24 : 2098-2100. : 2098-2100. . Israel and phylogeny. Caryophylloideae (Caryophyllaceae) inrelati Accepted Article J. Evol.Biol.

26 : 2244-2259. on to floweron length,pollination biology OU-2 OU-2 OU-PG OU-PG OU-Clades OU-Clades Model Hypothesis rates rates Differential BM Table 1. OU-1 OU-1 Accepted Article (NGP). nectar in founded compared for Models proportionmotion Brownian sucrose fructos nectar (NSP), differ between generalist andspecialist PGs As OU-PG is butdirectional toward change two which may optima, on PG may differ among PGs; thus nectar sugar evolution is dependent which several optima, istoward change directional but As OU-1 change in the differential rates analysis; Table S3) S3) Table analysis; rates differential the in change may differ as clades (defined having among theirof own rate which several optima, istoward change directional but As OU-1 lineages lineages As BMbutthe rate ofmay change vary over time andamong constant over time andamong lineages direction, with amean of change zero andarate of thatis change Nectar sugar evolves independently of PG andis random in over time andamong lineages and rate of (stochastic)change toward the optimum are constant toward aglobaloptimum state; the strength of the pulltoward Nectar sugar evolves independently of PG butis directional, optima) optima) k optima) optima) k optima) optima) k of rate shifts and k change) k trait optimum) of pull[=adaptive 1 change] and k of rates) Free = 4 (as OU-1 and2 putative =11 (as OU-1 and9 putative =18 (as OU-1 and 16 putative 16putative and =18(asOU-1 = 3(ratestrength of change, = 2+ = 2(rootstate andrate of parameters n (rootstate, n +1 n

number number e proportion (NFP) and nectar gl (NFP) andnectar proportion e 10096.3 – – – – 10096.3 10134.5 8357.8 8357.8 10134.5 9342.8 10539.4 8820.2 8820.2 10539.4 9901.5 10160.5 8417.5 8417.5 10160.5 9398.0 9149.1 9931.7 (NSP) (NSP) AICc AICc 8004.3 8395.8 8395.8 9344.6 (NFP) AICc AICc

9220.2 9220.2 (NGP) AICc AICc

ucose proportion U2 As OU-2 butthe strength of may pull differ between optima OU-2A AcceptedThis article isprotected Allrights bycopyright. reserved. Table 2. Article OU = Uhlenbeck Ornstein = BM Brownian motion; denoteAICc values inbold the best-fitting model. As OU-2 butthe rate of change may differ between optima OU-2V Definition rateof parameters incompared the correlation analyses.

q q q q Parameter Evolutionary transition 34 24 12 13 Shift from specialist specialist to generalist from in Shift Shift from high to low sucrose in generalist background 0.45 (0.015 0.45 highto low in from sucrose Shift background generalist specialist from to generalist in Shift Shift from high to low sucrose in specialist background 0.018 (0.017 0.018 high to low Shiftfrom sucrose in specialist background Reverse shifts (1 -> 0) Forward (0-> shifts 1) Forward 1 low sucrose background 0.0044 (0.0033 0.0044 background sucrose low high sucrose background 0.00077 (0.00048 0.00077 background high sucrose [=pull]) [=pull]) rates of adaptive change k rates of stochastic change) change) stochastic of rates k = 5 (as OU-2 and2 putative = 5 (as OU-2 and2 putative (median [95% CI]) Estimate 10096.9 – – – – 10096.9 10098.2 – – – – 10098.2 2 – 3.1) – 0.019) – 0.0058) –

0.010) 2 1 table. this directly from the independent model has rate parameters. four model independent the independent model, transition rates in trait transition are model, the sa one independent Rate estimates shown are summaries across the 100 trees; comparison Accepted Article in rates trait transition one because dependent are possiblemodel the under transitions Eight q q q q 31 43 42 21 Shift from low to high sucrose in Shift from generalist to specialist in low sucrose background 0.030 (0.022 0.030 (0.0083 0.045 sucrose inlow generalist specialist background to from Shift low in highsucrose from to background Shift ageneralist (0.00 0.00 background inhighsucrose generalist specialist to from Shift me irrespective of the state of the second trait. Thus aseils akrud0.054 (0.050 aspecialist background s reported in the text were performed across each tree indiv tree each across performed were text the in reported s may vary depending on state state on of t depending may vary q 12= q 34, – 1.51) – – q 0.043) 0.058) – 21= 0.48) q 43, he second trait. In the q idually and cannot be readidually be and cannot 13= q 24 and 24 q 31= q 42, and 42, Core Solanaceae OU-1 3.38 0.15 0.15 3.38 OU-1 Core Solanaceae Core Asteraceae OU-1 1.12 0.062 0.062 1.12 OU-1 Core Asteraceae Gelsemiaceae + Gentianaceae + Apocynaceae OU-1 1.80 0.057 0.057 1.80 OU-1 +Apocynaceae Gentianaceae + Gelsemiaceae Ajugoideae + Lamioideae Core Ericaceae OU-1 5.65 0.11 0.11 5.65 OU-1 0.087 0.19 Core Ericaceae OU-1 Fouqueriaceae+ Polemoniaceae + Primulaceae Asterids Rubiaceae OU-1 OU-1 Rubiaceae Core Acanthaceae OU-1 0.67 0.053 0.053 0.67 OU-1 Core Acanthaceae Gesnerioideae Gesnerioideae Core Plantaginaceae Plantaginaceae Core model could not be rejected (see Table S6). rejected could be not (see Table model AcceptedThis article isprotected Allrights bycopyright. reserved. ArticleTable 3. Parameter

Clade 1 estimates for the best-fitting OU model for asterids overall a OU-PG OU-PG OU-PG OU-PG OU-PG OU-PG OU-PG OU-PG

2 Model 1.37 1.37 81.9 9.74 9.74 81.9 0.87 <0.001 <0.001 0.87 23.4 9.98 9.98 23.4 0.69 0.068 0.068 0.69 2 σ

0.12 0.12 α <0.001 <0.001

23.6 23.6 θ – generalist – – – – – – – – insects

θ – bats 34.7 34.7 34.7 – – 34.7 34.7 34.7 82.4 82.4 82.4 – – 82.4 82.4 82.4 45.3 78.1 99.9 53.3 53.3 99.9 78.1 45.3 36.2 88.5 – – – – 88.5 36.2 36– – – – 23.6 – 85.7 – 85.7 85.7 – 85.7 – 05– – – – 60.5 – 82.4 – 28.8 28.8 – 82.4 – 37– – – – 53.7 – 78.2 78.2 – – 78.2 78– – – – 57.8

nd foreach of the lessinclusive clades analyzed separately, wh θ – bees and wasps

θ – specialized flies

θ – specialized Old World birds

<0.001 θ – unspecialized – – – – – – – – – – birds 23.6 – – 23.6 11.1 23.6 473. 47 4.53 34.7 34.7 34.7 60.5 – – 60.5 13.0 60.5 74.2 – – 74.2 91.8 0.071 53.7 – – 53.7 7.98 53.7 15.6 100 100 15.6 99.8 4.0 248. 24 12.1 82.4 82.4 82.4 827. 82 5.78 78.2 78.2 78.2 – – – – 85.7 6.37 – – – – 74.8 10.2 – – – – 77.8 0.070 θ – butterflies

θ – moths

θ – hummingbirds ere such a x 10 t (th)

5 1/2 (39) 8.98 8.98 (39) (34) 6.26 6.26 (34) (28) 11.0 11.0 (28) (49) (49) 25.9 (67) 1.11 1.11 (67) (62) 15.7 15.7 (62) (56) 5.72 5.72 (56) (46) (46) 5.07 (1) 2.5 (114) (34) 4.20 4.20 (34) (31) (31) 1.17

x vy 10 5

Boraginales BM BM Boraginales Campanulaceae Nepetoideae controlled by controlled adaptation) inadaptation) Ma 2

1 optima differs clades. among move from the ancestral state to the new optimum) and OU-PG isaOU-PG model inwhich the mean optimum differ amongmay pollinator groups (PGs) σ 2 Accepted Article drift stochastic under rate or (e.g. of inis the change factor), anunmeasured to relation σ ). Optimum values are). as shown Optimum back-transformedvalues NSP; estimates S5formean (% figure see variat -1 , θ isthe mean trait optimum, t

OU-PG OU-PG OU-PG 0.35 0.35 0.057 1/2 0.39 0.39 15.5 5.59 5.59 15.5 is thephylogenetic half-life is in Ma with total v y isthe stationary variance ( NA NA 8.16 8.16 – – 6.30 23.3 – – – – 23.3 6.30 A– – – – NA – 70.0 70.0 – – 83.9 σ 2 /2 3 . Not all PGs are present in all clades (see Table S8) so the number of α α ; the balance between the action controlled by by controlled action the between balance ;the is the strength of the “pull” toward the selective optima (rate of tree height (th) in height in (th) (ln(2)/ Ma tree brackets 1.15 1.15 – – ion around the mean). NA – – – – – – 77.7 12.2 – – – – 78.1 0.12

α ; the time needed to α and that andthat 5) NA (57) (56) (56) 3.09 (38) (38) 1.38 NSP (table S3). analysis for nectar glucose andnectar(NGP) fructos (SS) is1– Shift SS support = 3438, (node 0.92). andGesnerioideae SS= (node 0.85) 2276, Plantaginaceae core (node core SS= Solanaceae 2389, with caution: shifts treated node,should be three each at of findingashift from likelihood calculated the S3. support) a Based (shift on measure table support Escalloniales + Bruniales. Clades at which rate shifts occur arenumbered; details are provided in (n named linenot named. *The are orders sampled rates (yellow through intermediate green), comparedthen (blue), background to (black). rates Major sucrose proportion (NSP). Branch figure S2for details). NW = New Word; OW = Old toward lower nectar sucrose proportion (NSP; main plot: all data, n=2116; inset: (PG).Allgroups separategroup along the sucros relative contributions fructose,of glucoseandsucrose to the nectar sugarutilized each by pollinator Figure 1. Figure and lower quartiles, circles quartiles, (defined and lower aslying outliers = 1.5 beyond among ofDistribution NSP plants pollinated diffe by Figure 3: 3: Figure species pollinated by Seeother PGs. table for3 details. Colors andsymbols as infigure 1. by generalistpollinated insects andunspecialized in(PG) andspecialist differences plants generalist asterids. The between are PGs, with main found Accepted Article 2. Figure cultivated plants. Outliers represent several plant families andar Composition of nectar sugars andvariation groups. pollinator among Selective inferredoptima for nectar sucrose f Phylogenetic position rateof shifts inferred exp , where

f exp is the expected frequency of shifts at that node. The differential rates frequency differential of that shifts The node. at isthe expected colors show rates as exceptionally high (red) to exceptionally low low high(red)colors rates as exceptionally to show exceptionally e based data on sampled frombothwildand e axis with generalist pollinator tending groups birds tendingtoward lower NSP than optima World. See textmain for definitions of PGs. ext to Dipsacales +Aquifoliales) isApiales + e proportion (NFP) revealed similar results as for rent Horizontal PGs. line = =median, boxes upper in thedifferential rates analysis for nectar content (NSP)inrelation group to pollinator x the interquartile range[Q3-Q1]). A : Ternary plot of the group means; see 0.53), 0.53), B :

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Accepted Article