Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch

Year: 2014

Floral volatile alleles can contribute to pollinator-mediated reproductive isolation in monkeyflowers (Mimulus)

Byers, Kelsey J R P ; Vela, James P ; Peng, Foen ; Riffell, Jeffrey A ; Bradshaw, HD

Abstract: Pollinator-mediated reproductive isolation is a major factor in driving the diversification of flowering plants. Studies of floral traits involved in reproductive isolation have focused nearly exclusively on visual signals, such as flower color. The role of less obvious signals, such as floral scent, hasbeen studied only recently. In particular, the genetics of floral volatiles involved in mediating differential pollinator visitation remains unknown. The bumblebee-pollinated Mimulus lewisii and hummingbird- pollinated M. cardinalis are a model system for studying reproductive isolation via pollinator preference. We have shown that these two species differ in three floral terpenoid volatiles - D-limonene, ฀-myrcene, and E-฀-ocimene - that are attractive to bumblebee pollinators. By genetic mapping and in vitro enzyme activity analysis we demonstrate that these interspecific differences are consistent with allelic variation at two loci – LIMONENE-MYRCENE SYNTHASE (LMS) and OCIMENE SYNTHASE (OS). M. lewisii LMS (MlLMS) and OS (MlOS) are expressed most strongly in floral tissue in the last stages of floral development. M. cardinalis LMS (McLMS) is weakly expressed and has a nonsense mutation in exon 3. M. cardinalis OS (McOS) is expressed similarly to MlOS, but the encoded McOS enzyme produces no E-฀-ocimene. Recapitulating the M. cardinalis phenotype by reducing the expression of MlLMS by RNAi in transgenic M. lewisii produces no behavioral difference in pollinating bumblebees; however, reducing MlOS expression produces a 6% decrease in visitation. Allelic variation at the OCIMENE SYNTHASE locus likely contributes to differential pollinator visitation, and thus promotes reproductive isolation between M. lewisii and M. cardinalis. OCIMENE SYNTHASE joins a growing list of “speciation genes” (“barrier genes”) in flowering plants.

DOI: https://doi.org/10.1111/tpj.12702

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-100188 Journal Article Accepted Version

Originally published at: Byers, Kelsey J R P; Vela, James P; Peng, Foen; Riffell, Jeffrey A; Bradshaw, H D (2014). Floral volatile alleles can contribute to pollinator-mediated reproductive isolation in monkeyflowers (Mimulus). The Plant Journal, 80(6):1031-1042. DOI: https://doi.org/10.1111/tpj.12702 Accepted Article KF857265, sequences: for numbers Accession

Kelsey J.R.P. Byers Terpene synthase, floral volatiles, speciation, pollination, pollination, speciation, volatiles, floral synthase, Terpene Keywords: 1 ( monkeyflowers in isolation reproductive to pollinator-mediated contribute can alleles volatile Floral Article type : Original Article 06-Oct-2014 : Date Accepted Revised: 28-Sep-2014 Date : 17-Apr-2014 Date Received This article This protectedis by Allcopyright. rights reserved. an 'Accepted Article', doi: 10.1111/tpj.12702 lead to differences betweenversionthis and the Versionof Record. Please cite throughthebeen typesetting,copyediting, pagination and process proofreading which may This article hasbeen accepted for publicationand undergone fullpeer review but has not Running title: Floral scent and reproductive isolation in in isolation reproductive and scent Floral title: Running [email protected] Riffell: A. Jeffrey [email protected] Peng: Foen [email protected] Vela: P. James Kelsey J.R.P. [email protected] Byers: KF857264, KM659024, AHI50308, KF857262, KF857263, AHI50306, AHI50307, corresponding author corresponding M. lewisii M. lewisii M. cardinalis M. lewisii M. lewisii M. lewisii OCIMENE SYNTHASE lewisiiM. OCIMENE M. cardinalisM. SYNTHASEOCIMENE M. cardinalis b Present address: University of Zürich, Institute of Systematic Botany Botany Systematic of Institute of Zürich, University address: Present Mimulus a,b , James P. Vela P. , James LIMONENE-MYRCENE SYNTHASE protein product

OCIMENE SYNTHASE protein product product protein SYNTHASE OCIMENE LIMONENE-MYRCENE SYNTHASE LIMONENE-MYRCENE LIMONENE-MYRCENE SYNTHASE LIMONENE-MYRCENE : [email protected], (206)616-1796 (phone), (206)616-2011 (fax) OCIMENE SYNTHASE protein product product protein SYNTHASE OCIMENE

LIMONENE-MYRCENE SYNTHASE a ) University of Washington, Department of Biology ofBiology Department Washington, of University a , Foen Peng 8008 Zürich, Switzerland Seattle, WA98195-1800 mRNA, complete cds complete mRNA, mRNA, complete cds cds mRNA, complete a , Jeffrey A. Riffell A. , Jeffrey

gene, complete cds cds complete gene, mRNA, cds complete Mimulus Mimulus lewisii mRNA, exon3

a , and H.D. Bradshaw, Jr. Bradshaw, H.D. , and , Mimulus cardinalis this article as a,1

Accepted Article difference in pollinating bumblebees; however, reducing reducing however, bumblebees; pollinating in difference visitation. Allelic variation at the pollinator visitation, and thus promotes reproductive isolation between between isolation reproductive promotes thus and visitation, pollinator by reducing the expression of theexpression by reducing their associated pollinators; the association between pollinator type and suites of signals signals of suites and type pollinator between theassociation pollinators; associated their Flowering1970). plantsvariety of use a signals toadvertise the presence (or illusion) o attributed to their specialized association with different animal pollinators (Grant, 1949; Stebbins, The rapid diversification the ofworld’s estimated 275,000 species of flowering plants has of Introduction SYNTHASEOCIMENE the encoded McOS enzyme produces no no produces enzyme McOS encoded the and has a nonsense mutation in exon 3. floral tissue in the last stages of floral development. development. floral of stages thelast in tissue floral and and By genetic mapping and and mapping By genetic differences are consistent with with areconsistent differences hummingbird-pollinated hummingbird-pollinated pollinator visitation remains unknown. The bumblebee-pollinated bumblebee-pollinated The unknown. remains visitation pollinator of thegenetics particular, In recently. only studied visual signals, such flower as color.of role The lessobvious signals, such as floral scent e nearly focused have isolation reproductive in involved traits of floral Studies plants. Pollinator-mediated reproductive isolation is major a factordriving in d the Summary This article This protectedis by Allcopyright. rights reserved. volatiles - D-limonene, D-limonene, - volatiles these have shown that pollinator We preference. OCIMENE SYNTHASEOCIMENE joins a growing list of “speciation genes” (“barrier genes”) in flowering plan flowering in genes”) (“barrier genes” “speciation of list a growing joins β in vitro ( -myrcene, and and -myrcene, M. cardinalis M. OS ). MlLMS M. lewisii allelicvariation at two loci – enzyme activity analysis we demonstrate that these interspecific interspecific thatthese demonstrate we analysis activity enzyme OCIMENE SYNTHASEOCIMENE by RNAi by transgenicin are a model system for studying reproductive isolation isolation reproductive studying for system model a are E M. cardinalis

E - LMS LMS β - β -ocimene - that are attractive to bumblebee pollinators. pollinators. bumblebee to attractive - thatare -ocimene -ocimene. Recapitulating the Recapitulating-ocimene. the ( MlLMS two species differ in three floral terpenoid terpenoid threefloral two in species differ floral volatiles involved in mediatinginvolved floral volatiles differential in M. cardinalis

) and ) and locus OS ( MlOS McOS M. lewisii OS

LIMONENE-MYRCENE SYNTHASE LIMONENE-MYRCENE likely contributes to differential to differential contributes likely ( expression produces a 6% decrease in in decrease a6% produces expression MlOS ) isexpressed similarly to

LMS Mimulus lewisii produces no behavioral behavioral no produces ) are expressed most strongly in in strongly most expressed are ) ( McLMS M. lewisiiM. M. cardinalis iversification of flowering ) is weakly expressed expressed weakly ) is and and and xclusively on on xclusively f a toreward a f M. cardinalis , has, been phenotype phenotype gives rise rise gives MlOS ten beenten ( via LMS , but ts. ts.

) . Accepted Article al. et al. et genetics (Raguso, 2008a; 2009;Parachnowitsch andPeakall, genetics Raguso, 2008a; 2008b;Whitehead (Raguso, pollination affect that volatiles well-characterized with systems Those lacking. been has evolution and ecology, genetics, neurobiology, biochemistry, synt a identified, occasionally are volatiles of floral production the for responsible flor although However, area. this in progress levels, processing higher-order and receptor activity neural pollinator of analysis scent, floral of analysis chemical including scent, attracting pollinators (Raguso, recent development2008a). The of techniques for study in role strong a play may it that understanding long the despite signal, understudied generally a – is ratios,relative identi Floralamount, and scent –the and Sargent, 2009;Yuan Kay Johnson, 2009; systems of (Harder and variety a somein extent investigated to havebeen shape (Rausher, 2008). Othervisual signals, texture, such as pattern, orientation, anthesis time, siz of avariety in investigated been has which color, floral is signal studied easily notably the sexually deceptive orchids in the genera genera the in orchids deceptive thesexually notably specialized systemsscent where crucial is toplant-pollinator interactions (Raguso, 2008b Muchof work discussing sp the al. as such systems non-orchid to this expanding begun to the concept of pollination syndromes (Fenster of concept syndromesto pollination the (Fenster This article This protectedis by Allcopyright. rights reserved. demonstrating the importanceof volatile floral , 2012), as well as the non-deceptive genus genus non-deceptive the as well as , 2012), , 2008), and , 2008), , 2010) and, 2010) Petunia Ophrys (Klahre (Klahre (Schiestl and Ayasse, 2002; Mant Mant 2002; andAyasse, (Schiestl et al. , 2013a). et al. eciation involving floral volatiles has been done in extremely done in volatileseciation involving floral has been , 2011). studies agrowing Whilethereare of number and genetic and genomic tools, has allowed some some allowed has tools, genomic and genetic and al scent is frequently characterized, and genes and genes characterized, frequently is scent al Gymnadenia s in mediating these largely specialized theselargely plant- s in mediating et al., et al., are separate from those with well-characterizedwith from those separate are ties of volatile compounds emitted by theflowercompounds by volatileties of emitted Chiloglottis Silene 2004). Perhaps the most well-known themost and 2004). Perhaps et al. (Waelti (Waelti (Huber (Huber , 2005; Vereecken (Schiestl and Peakall, Peakall (Schiestl2005; and et al. et et al. , Recent 2005). work has , 2008), , 2008), pollination syndromes syndromes pollination Linanthus et al. hesis of floral scent scent offloral hesis at both the both at , 2010;Xu ing floral et al. et ), most (Chess e, ande, , 2012). et et Accepted Article systems (Gang, 2005), including of angio diverse range a in known are production volatile underlying genes the contrast, In unknown. controllgenetic pollinator pathways the interactions, This article This protectedis by Allcopyright. rights reserved. al. Petunia described. completely not been the underlying genetic mechanisms mediating species-specific volatile emission thisin systemhave le can gene single bya mediated production volatile to near isogenic withlineshigh levels of methyl differentially expressed in the two species. The hawkmoth Thehawkmoth species. two the in expressed differentially is that factor transcription MYB a encodes VII, chromosome on locus the underlie to hypothesized were identified in the the in identifiedwere chromosome VII substantially contribut substantially VII chromosome on thelocus and production methyl benzoate for required absolutely being II chromosome on locus hawkmoth pollinators of pollinators hawkmoth axillaris Arabidopsis differential attraction of pollinators between sister species (Klahre (Klahre species sister between pollinators of attraction differential Petunia molecular genetic with and basis those for effects. the volatiles floral linking research of paucity is a there systems, of these aresult as grown has emission and production of volatile underpinnings thegenetic of knowledge our Although , 1998; Wang and Pichersky, 1998), (Koeduka (Koeduka is the isthe well-developedonly model demonstrating that role the specific volatile a plays in and and (Bohlmann (Bohlmann P. exserta P. et al. Petunia Petunia differ in their production of methyl benzoate, a volatile benzoate,methyl attractive a tothe of production in their differ , 2006;Orlova et al. et P. axillaris , 2000; ,Chen 2000; genome on chromosomesgenome on with the IIVII, and Clarkia . Through QTL mapping, two regions underlying this difference difference this underlying regions two mapping, QTL Through . et al. Antirrhinum (Pichersky ing to methyl benzoate quantity. quantity. benzoate methyl to ing et al. , 2006; Dexter , 2003), and many species of agricultural importance. benzoate production, suggesting that a change in change in a that suggesting production, benzoate (Dudareva (Dudareva et al. ad to differential pollinator attraction; however, however, attraction; pollinator differential to ad ing volatile production in these systems remain productioning in volatile these , 1995;Dudareva et al. Manduca sexta Manduca et al. , 2007), , 2000;Dudareva et al. Silene , 2011). The sister species et al. ODO1 is attracted more strongly strongly more attracted is P. axillaris (Gupta , 1996; Dudareva Dudareva , 1996; , oneof thegenes and plant speciation speciation plant and et al. et al. allele at the , 2003), , 2012), sperm sperm et P. P.

Accepted Article The sisterThe species This article This protectedis by Allcopyright. rights reserved. mediated reproductive isolation for several decades (Hiesey (Hiesey decades several for isolation reproductive mediated pollinator attraction betwee attraction pollinator dif impacting loci multiple of discovery the to led has resources genetic and ecological Yuan Yuan important for the attraction of bumblebee pollinators, including including pollinators, bumblebee of theattraction for important isolation between between isolation Bradshaw, Within 1999). of areas sympatry, pollinator fidelityis responsible98% for of reproductive cardinalis pollinator of floral volatiles three produced by M. cardinalis speciation link. and well-studied ecology, is an obviouschoice for filling in missingthis piece cardinalis codingor regulatory genes? ar 2010), Blackman, and (Rieseberg plants in genes” “specia of discussions other with keeping In isolation? reproductive this, through and visitation, protein Whatfunction? any, role, do these if differencesscent play diffin between volatiles floral of emission thedifferential of underpinnings genetic the Therefore,we ask some global questions, which we begin to address in this manu 2014). et al. ? How many genes are responsible, and how do how and responsible, are genes many How ? , lower-elevationis a hummingbird-pollinated (Hiesey species , 2013c). M. lewisii produces only D-limonene, released at just 0.9% the rate of of rate the 0.9% just at released D-limonene, only produces M. lewisii M. lewisii M. Mimulus lewisii in the central Sierra Nevada mountains of California. Of the three volatiles, volatiles, three the Of California. of mountains Nevada Sierra thecentral in and and n the two species (Bradshaw and Schemske, 2003; Yuan Yuan 2003; Schemske, and (Bradshaw species n thetwo Mimulus M. cardinalis M. cardinalis M. lewisii is a bumblebee-pollinated alpine species, while its sister, sister, its while species, alpine bumblebee-pollinated a is , with its known attractive volatiles, genetic and genomic tools, tools, genomic and genetic volatiles, attractive known its , with – D-limonene, – have served as a model system for studying pollinator- studying for system model a as haveserved (Ramsey e these genes of large effect or effect, small genes effect large of e these et al. the species differ in gene expression and β -myrcene, and and -myrcene, , 2003). Previous workhas shown that et al. Bombus vosnesenskii Bombus , 1971), and thecombination of et al. et M. lewisii erential pollinatorerential E - β of floral the scent- , 1971; ,Schemske 1971; and -ocimene – are are – -ocimene M. lewisii script: What are (Byers et al. , the native , the ferential ferential , 2013b; et al. and and M. , M. tion tion

Accepted Article model for the difference in these three compound these three in model thedifference for (r = 0.474 versus(r =0.474 D-limonene, 0.574versus = r between between th control might loci Mendelian at alleles that correlated (r = 0.975), but neither wa neither but 0.975), (r= correlated limonene from emissionthe areinherited volatiles (0.52:0.48 (0.52:0.48 volatiles similar for ng/flower/hr; F ng/flower/hr; ng/flower/hr; mean =2.8 with other traits that differ between the species (Bradshaw (Bradshaw thespecies between differ that traits other with =27). Complete or partial dominance of the to that of the of to that Whitney 0.864, p and U = =15) much higher that of thethan ocimene. emission The of D-limonenerate for the F one-tailed 0.005, For p U = =27). When a backcross (F backcross a When (mean = 0.5 ng/flower/hr, ng/flower/hr, 0.5 = (mean cardinalis absence of emission of of emission of absence Construction of an F volatiles in floral differences species-specific of mapping Genetic Results This article This protectedis by Allcopyright. rights reserved. revealed revealed patterns of controllinginheritance of loci emission the of D-limonene, M. lewisii : mean =0.0ng/flower/hr; F β -myrcene (F M. lewisii 1 vs. vs. and and β M. lewisii 1 1 -myrcene present:absent; 0.38:0.62 0.38:0.62 present:absent; -myrcene crossbetween x parental inbred line (mean = 55.1 ng/flower/hr, ng/flower/hr, =55.1 (mean line inbred parental β M. cardinalis M. cardinalis 1 -myrcene and and -myrcene : mean = 2.6 ng/flower/hr, ng/flower/hr, 2.6 mean = : M. lewisii N = 9; = 9; one-tailed 0.005,p = U suggesting27), = that highD- levelsof two-tailed 0.036, p= U =25; F E - : mean = 7.6 ng/flower/hr; 7.6 ng/flower/hr; = mean : β ) population) of 100plants was scored forpresence the or . D-limonene and D-limonene and . M. lewisii -ocimene, the the -ocimene, s particularly highlycorrelated with 1 vs. vs. E - β M. lewisiiM. -ocimene, it segregated segregated approximately -ocimene, 1:1 for it both M. lewisii M. lewisii M. e difference in emission of these monoterpenes emission thesemonoterpenes of in e difference inbred line LF10 and and LF10 line inbred β -myrcene). Therefore, we considered a two-locus s between the two speciescontrolling the locus –one between s M. lewisii parent in a dominant manner. The pattern was was Thepattern manner. dominant a in parent M. lewisii two-tailed p=0.600, U=17; F 1 (mean = 47.2 ng/flower/hr; ng/flower/hr; 47.2 (mean= alleles for these floral volatiles is consistent consistent is volatiles floral these for alleles β -myrcene emission rates were veryhighly rates emission -myrcene E - β 1 et al. vs. -ocimene present:absent), suggesting suggesting present:absent), -ocimene M. cardinalisM. allele appears to be semidominant (F semidominant be to appears allele : mean = 3.3 ng/flower/hr, ng/flower/hr, 3.3 = mean : M. cardinalis M. cardinalis , 1998).

M. cardinalis cardinalis M. N E parental inbred line line inbred parental = 9; two-tailed Mann- two-tailed = 9; - : mean = 0.0 0.0 = mean : β -ocimene emission -ocimene rate one-tailed p =0.005, U β -myrcene, and -myrcene, inbred line CE10 line CE10 inbred N 1 vs. vs. = 3) was= 3) similar M. cardinalis M. E - β 1 - :

Accepted Article (N,D)Dxx(S,T,G)xxxE (NSE/DTE) motifs required for Mg for motifsrequired (NSE/DTE) (N,D)Dxx(S,T,G)xxxE population. D-limonene and and D-limonene between markers M02_510K and M02_1500K (Table S4), position 321kbp (mgv1a003660m) is transcribed in M02_1500 accounted for 92% of the difference between between thedifference of 92% for accounted M02_1500 Identification and characterizationof a bifunctional LIMONENE-MYRCE construc was =768) (N population backcross A larger limonene and 98% of the difference in difference the limonene 98% and of Indel markers developed for two of the of two for developed markers Indel synthases/cyclases –excellent candidatescontrolling for D-limonene and terpene three of cluster a is kbp there 226 and kbp 206 positions between 89 Onscaffold kbp. formation (Dudareva (Dudareva formation bin/gbrowse/mimulus/), theortholog of M02_1500K maps to process (Nieuwenhuizen (Nieuwenhuizen process assembled and annotated and annotated assembled The locus associated with D-limonene and and D-limonene with associated locus The lewisii limonene and made cluster the within synthases terpene candidate putative putative between plants) backcross 768 (in norecombinations revealed S5) (Table cluster the production of D-limonene and and D-limonene of production the This article This protectedis by Allcopyright. rights reserved. only the the only flowers M. lewisii LIMONENE-MYRCENE SYNTHASE

The predicted AHI50308 gene product contains the conserved DDxx(D/E) and and DDxx(D/E) conserved the contains product gene AHI50308 predicted The β -myrcene synthases by recombination. RT-P byrecombination. synthases -myrcene β -myrcene production. No transgressive se transgressive No production. -myrcene ortholog (KF857265) of the ofthe (KF857265) ortholog et al. et et al. et M. guttatus , 2003). ,well 2013), as RRx the as β -myrcene, and another, unlinked, locus controlling controlling locus unlinked, another, and -myrcene, genome v1.1 as a reference (http://www.phytozome.net/cgi- areference as v1.1 genome β M. lewisii/cardinalis -myrcene emissions, consistent with a single-locus model for for model asingle-locus with consistent emissions, -myrcene (see Methods). The very tight Methods). among (see linkage The the β -myrcene -myrcene emission 15cM to interval a mapped was M. guttatus M. M. lewisii ted to map the two lociprecision. map the greater with to ted it impractical to resolve the identity of the D- the of identity the resolve to impractical it 2+ binding during the terpene synthesis synthesis terpene the during binding 8 W motif required for cyclic terpene cyclic terpene for W motif required M. lewisii ca terpene synthase gene on scaffold 89 at scaffold on 89at gene synthase terpene candidate genes in the terpene synthase synthase terpene the in genes candidate . 5.3cM from. M02_1500K. Using the CR showed that, of the three candidates, three the of CR showed that, gregationbackcrosswas observed the in flowers. The marker genotype at at genotype marker The flowers. M. guttatusM. and and M. cardinalis M. NE SYNTHASE (LMS) in β scaffold 89 at position 201 -myrcene emission. themselves or the or the themselves emissions of D- E - β -ocimene. -ocimene. M. M. Accepted Article same proportions as observed in the authentic headspace collection from from collection headspace theauthentic in observed as proportions same MYRCENE SYNTHASE (LMS) encoded aby single LIMONE bifunctional a of effect pleiotropic the to due is population mapping backcross the with the frequent occurrence of multi-product terpene synthases (Dudareva (Dudareva synthases terpene multi-product of occurrence thefrequent with The The S1, Fig. 2 for the difference in D-limonene and D-limonene in difference the for supplied with geranyl pyrophosphate (GPP) as a substrate, yielding D-limonene and and D-limonene yielding substrate, a as (GPP) pyrophosphate geranyl with supplied Of note, this is not adefinitive demonstration that McLMS protein (G201X, using MlLMS AHI50308 as the reference allele). transversion mutation in exon 3 of of 3 exon in mutation transversion The The This article This protectedis by Allcopyright. rights reserved. of the difference between between of difference the markers sc4_2325K and M13_2620 (Table S4). markerThe genotype at sc4_2325 accounted 98% for overexpressed in overexpressed in backcross population. The orthologous region of the the of region orthologous The population. backcross complementation of the nonfunctional nonfunctional the of complementation formally possible that a different, tightly-linkedgene instead m necessary toshowconclusively that The locus associated with with associated locus The Identification characterization and of in SYNTHASE OCIMENE (OS) single-locus model for for model single-locus M. cardinalis LMS M. lewisii A ). This thehighsuggests). This that cDNA (KF857264) orthologous to mgv1a003660m, designated designated mgv1a003660m, to orthologous (KF857264) cDNA E. coli E. coli ( E McLMS - (as in Bohlmann β -ocimene production. Noproduction. transgressive segregation-ocimene was observed in the E M. lewisii - β -ocimene emission was mapped to a 7.5 cM -ocimene between midway toa7.5 interval emission was mapped ) coding sequence was not expressed not expressed was sequence ) coding McLMS and LMS β -myrcene emission between between emission -myrcene correlation between D-limonene and M. cardinalis is the causal gene for the phenotypic difference. difference. phenotypic forthe gene causal the is et al. M. cardinalis (KM659024) that results in a nonsense mutation in the , 2000).A crude lysate from the LMS LMS gene in

LMS M. guttatus emissionsof is the isthe gene underlying the locus responsible allele by the allele by the M. lewisii ight be responsible. Transgenic genomea lies 484kbp interval in M. lewisiiM. E M. lewisii M. in vitro - β ( M. lewisii -ocimene, consistent with a a with consistent -ocimene, MlLMS M. lewisii because there is a G66T G66T is a there because TS321K and and et al. flowers β E. coli ). This isconsistent). -myrcene emission-myrcene in allele would be M.cardinalis , 2004). flowers (Table flowers (Table β , was , was culture was was culture -myrcene in the the in -myrcene NE- ; it is Accepted Article flowers. flowers. flower (Fig. 2 (Fig. flower open the in as well as development, floral of and 20mm) (15mm anthesis to daysprior three last to open flower (see Yuan Yuan (see flower to open 5mm) anthesis, to prior (8d bud early from tissue offlowering stages different six with RT-PCR Using LMS between between As with As with S1). (Fig. substitutions acid amino nonsynonymous 19 the by unaltered are which indel marker developed for the An 1). (Fig. cyclases sesquiterpene as annotated are genes otherfour the while synthase, a terpene as (mgv1a020487m) atposition isannotated gene 2538kbp The kbp. kbp 2577 – 2538 position 4 at kbp(2325 –2809kbp) on scaffoldclus isa 4. There This article This protectedis by Allcopyright. rights reserved. that that When overexpressed in overexpressed When SYNTHASE putative the with plants) backcross 768 (in recombinations no revealed S5), (Table contain the same DDxx(D/E) and NSE/DTE Mg andNSE/DTE DDxx(D/E) same the contain in McOS and deletion of an arginine residue at position 308in McOS (Fig. S1). Both sequences differs from atMlOS 19 amino acid residues, incl enzyme (McOS, AHI50307) capable of synthesizing any monoterpene that could we detect. Mc However, under the same conditionsHowever, thesame the under (KF857262) encodes afunctional SYNTHASE OCIMENE (MlOS, AHI50306) (Fig. 2 and and MlOS LMS McOS OS M. lewisii is the gene underlying the locus responsible for the difference in in difference the for responsible thelocus underlying gene the is (seeMethods). expression expression , in the absence of a transgenic complementat transgenic a of theabsence , in B ). isexpressed similarly to McLMS and and M. cardinalis E. coli in vivo is weakly expressed in late-stage floral buds but not expressed in open open in expressed but not buds floral late-stage in expressed weakly is et al. and supplied with GPP as a substrate, the substrate, a as GPP with supplied and , 2013c), we found that both

M. lewisii/cardinalis M. . MlOS M. cardinalis . . 2+ binding motifs, as well as the RRx the well as as motifs, binding uding insertionof aleucine residue at position 238 ter fiveter terpeneof biosynthesis genes on scaffold ortholog of mgv1a003660m, designated designated mgv1a003660m, of ortholog

TS2538 ion test strict is ion demonstration this test a not MlLMS cDNA (KF857263) does not encode an and MlOS M. lewisii E - are expressed in in the expressed are β -ocimene emission-ocimene A , Table S1). S1). , Table 8 W cyclase motif, motif, cyclase W

TS2538 OCIMENE OCIMENE cDNA cDNA TS2538 TS2538 OS Accepted Article most T range =1.2%-56.1%,= 10.2%; mean constructs into emission rates of D-limonene and D-limonene of rates emission Using RNA interference Using RNA parent. Notably, LMS321K-8 had an increase in in increase hadan LMS321K-8 Notably, parent. pollinator studies (for data on three other T other on three data (for studies pollinator T A total ofA total 71T relative towild-type the (range parent =190.4-493.9%, mean 383.4%). = The original T original The plants homozygous for the for homozygous plants pollinator behavioral experiment. The 24 individuals selected for the experiment produced much much produced experiment the for selected individuals 24 The experiment. behavioral pollinator headspacecollection of floral volatiles to MlLMS assayed each transgenic plant in triplicate for floral volatile production. All T All production. volatile floral for triplicate in plant transgenic each assayed ofD-limonene 2.8% production and 9.1% Construction of RNAi knockdowns of knockdowns RNAi of Construction This article This protectedis by Allcopyright. rights reserved. lewisii in compound minor additional an synthesizing for be responsible may MlLMS that indicating verify We recovered 24 We recovered 24 specific floral volatiles on pollinating bumblebees. 0 parent (Table 1, Fig. 3). All other T All other 3). Fig. 1, (Table parent . . One of these T LMS 1 and and plants showed a decrease in emission of terpinolene (range = 0.0%-132.6%, mean = 18.0%), 18.0%), mean= =0.0%-132.6%, (range of terpinolene emission in decrease a showed plants and and MlOS 1 2 LMS321K-8 had very low emission rates of D-limonene and and D-limonene of rates emission low very had LMS321K-8 plants from the self-pollinated progeny of T progeny self-pollinated the from plants OS M. lewisii toproducemuch lower floral volatile levels,comparable t to M. lewisii gene function 1 transgenics (LMS321K-8) was selfed as the parent of T of theparent as selfed was (LMS321K-8) transgenics via (Yuan (Yuan

(inbred line LF10) T LF10) line (inbred M. cardinalis Agrobacterium et al. et in vivo in β -myrcene relative to the wild-type to the -myrcene relative β , wewere2013c), able knock to down the expression of both 1 -myrcene: range= 4.1-50.0%, mean = 12.8%). Interestingly, plants had a similar increase in in increase asimilar had plants MlLMS and to determine the effect of decreased emission of of emission decreased of theeffect determine to and alleles at select the greenhouse population for the bumblebee bumblebee the for population greenhouse the select β -mediated -myrcene production relativetothe -myrcene 2 lines from independent T independent from lines and 1 plants carrying the the carrying plants E - β MlOS LMS -ocimene of 452.8% compared with the in planta in and in stably transformed transformed stably in 1 OS OS LMS321K-8 were assayed using transformation of hairpin RNAi RNAi hairpin of transformation (Table S2). This allowed us both to to both us allowed This S2). (Table MlLMS E 1 M. lewisii - transgenics, see Table S3). S3). Table see transgenics, β -ocimene production production -ocimene β -RNAi transgene, and -myrcene, with a mean mean a with -myrcene, 1 plants had lower lower had plants 2 hose producedin plants usedfor M. lewisii M. lewisii LF10 (D-limonene:

LF10 T M. lewisiiM. M. 0

Accepted Article Waser, 1986)were measured. see of preference, exclusive choices, random from todeviate bumblebee individual of an tendency the as (expressed constancy type)and flower each to visits total of theproportion as (expressed impatiens ( bumblebee on production monoterpene of reduced impact the assay to performed Two one experiments, for the Only two T Only two 34.8%). T 1.8%; 39.6%-58.9%,D-limonene: range = mean 49.2%; = Effects of of Effects were produced from TS2538-1, and these produced similar amounts of D-lim as the the as myrcene levels were similar to found those in 0.9% ofwild-type had a much lower emission rate of of rate emission lower much had a ocimene knockdown phenotype relative to to relative phenotype knockdown ocimene substantially TS2538-1, later so than T emission rates of D-limonene (39.6%) and and (39.6%) D-limonene of rates emission = 57.3-144.5%, mean =88.7%),but much less M. lewisii 0.1-2.4%,mean =1.9%; less D-limonene and This article This protectedis by Allcopyright. rights reserved. M. lewisii wild-type ancestor (range = 93.0-510.5%, mean = 247.2%). The D-limonene and =247.2%). 93.0-510.5%, TheD-limonene mean (rangewild-type ancestor = ) visitation in a captive greenhouse setting. During each experiment, both preference preference experiment, both each During setting. greenhouse captive a in visitation ) 1 MlLMS plant TS2538-1,which was self-pollinatedcreate a to T 1 plants carrying the carrying plants LF10 LF10 ancestor (D-limonene: range =61.3-127.4%, mean =83.6%; and M. lewisii β MlOS -myrcene compared to the wild-type wild-type the to compared -myrcene β -myrcene: range =0.0-4.8%, 0.6%),mean = and more ; knockdowns on bumblebee pollinator behavior behavior pollinator bumblebee on knockdowns β -myrcene is absent from from absent is -myrcene MlLMS MlOS E - -RNAi transgene were recovered, but both had the desired desired the had but both recovered, were transgene -RNAi β -RNAi transgenics and one for the 2 -ocimene (0.8%) relativeto -ocimene (0.8%) lines were not created from this plant. A totalof 80T A this from plant. created were not lines β M. lewisii -myrcene (28.3%)(Fig. T 3). (28.3%)(Fig. -myrcene E M. cardinalisM. - β -ocimene (range = 0.9-3.9%, mean = 1.9%).-ocimene 0.9-3.9%,(range = mean = LF10 ( M. cardinalis β -myrcene: range = 28.3%-41.2%, mean = = mean 28.3%-41.2%, = range -myrcene: (D-limonene: range = 0.03-2.8%, (D-limonene: = = range mean E M. lewisii - β -ocimene: range = 0.8%-2.9%, mean = = mean 0.8%-2.9%, = range -ocimene: 2 M. lewisii population for pollinator studies, studies, pollinator for population ). ). ancestor (D-limonene: range= ancestor (D-limonene: 1 MlOS plantTS2538-2 flowered LF10, as well as lower lower as well as LF10, -RNAi transgenics, were transgenics, -RNAi onene and E - β β -ocimene than the -ocimene than -myrcene: range range -myrcene: Bombus Bombus β -myrcene β - 2 plants plants E - β - Accepted Article foraging bouts,with an average Bateman’s index of -0.1142 ( these using repeated was above described thesimulation 0.0149; of index Bateman’s average the in absent also no significant difference ( difference no significant flowers in this experiment nor for either flower type in the ocimenemay operate asnear-field a olfactory cue, but this behavi wild-type w (49.52%) 833 visits, 1682 Of pollination. effect to required as fully, flower the entered were defined as observable contact with the sexual organs of the flower – – theflower of organs sexual the with contact observable as defined were and contact the flower with their antennae prior to prior antennae their with flower the contact and 4). Bumblebees approaching the approaching Bumblebees 4). constancy when presented with ( flowers with these constancy presented when actual than the values index Bateman’s divergent resulting in average an Bateman’s index of -0.1141. totalof simulations A 96,648 had foraging boutswith wereused 100,000 simulated runs of randomly permuted plant locations, differ significantly was constancy this whether random visitation complete patterns; +1indicates indexof-0.0114 (-1 indicates complete inconstancyregular switching – betweentypes; 0 Bateman’s average an with constancy, for assayed were bouts foraging bumblebee of 39 A total eitherflower type. towards difference behavioral qualitative 1166 (52.95%) were to wild-type wild-type to were (52.95%) 1166 A total of visitsA total 1682 were This article This protectedis by Allcopyright. rights reserved. plants, showing a significant preference for the wild-type wild-type the for preference significant a showing plants, For the MlOS M. lewisii -RNAi knockdown2202 of total experiment, a MlOS and 849(50.48%) were to the -RNAi experiment, with a total of 46 bumblebee foraging bouts showing an an showing bouts foraging bumblebee of 46 total a with experiment, -RNAi Χ observed toflowers the in 2 =0.15, M. lewisii MlOS p = 0.70, Fig. 4). Bumblebees appeared to show no overall overall no toshow appeared Bumblebees 4). Fig. =0.70, -RNAi flowers were noted to fr notedto were flowers -RNAi and 1036 and(47.05%) 1036 towere the ent from random visitation, ent from random bumblebee same the p = 0.97). = data, showing that bumblebees demonstrated no no demonstrated bumblebees that showing data, M. lewisiiM. MlLMS constancy, alwayswithin types). determineTo aborting a potential visit, suggesting that that suggesting visit, potential a aborting MlLMS M. lewisii MlLMS visits thesevisits, Of observed. were -RNAi knockdownexperiment. Visits p =0.95). -RNAi experiment. Constancy was was Constancy experiment. -RNAi orwas not noted for the wild-type flowers (X -RNAi transgenic plants, showing equently wave their antennae equently i.e., M. lewisiiM. MlOS 2 the bumblebee bumblebee the =7.67, more more p =0.0056, Fig. ere tothe ere indicates -RNAi -RNAi E

- β - Accepted Article the allelic variants producing differences in floral volatile emissions between between emissions volatile floral in differences producing variants allelic the limonene, alleles of RNAi knockouts show that the loss-of-function cardinalis that eliminate its abilityproduce to allele due tononsense mutation a in whileexon 3, emission from OCIMENE SYNTHASEOCIMENE LMS limonene, volatile emission phenotypes, and that functional copies of both genes a only D-limonene much lower at levels (0.9%of in floral emission of D-limonene, D-limonene, of emission floral in containing the the containing McLMS circ strong present we loci, these underlying genes weexperiments. Although thesedefinitive performed effects on bumblebees – D-limonene, D-limonene, bumblebees on – effects Mimulus lewisii Discussion This article This protectedis by Allcopyright. rights reserved. likely tomutationsdue in genes: two and and the lack of product from McOS activity OS LMS are not in β β accounts for 92-98% of the phenotypic difference between between difference thephenotypic of 92-98% for accounts -myrcene, and and -myrcene, -myrcene, and and -myrcene, and M. cardinalis M. lewisii produces three floral volatiles with significant neurophysiological and behavioral behavioral and neurophysiological significant with volatiles floral three produces OS LMS ( are sufficient (since we have shown that they are necessary) to produce D- MlOS alleles. However, or or E E , OS - - flowers can be explained at the molecular genetic level; genetic molecular the at explained be can flowers McOS β β -ocimene -ocimene -ocimene, we would have to transform , but in genes very tightly linked to them. To show that the that the Toshow them. to linked tightly very genes in , but β ). In quantitative genetic terms, a terms, genetic quantitative In ). -myrcene, and and -myrcene, E - LIMONENE-MYRCENE SYNTHASE β β -ocimene. -ocimene. -myrcene, and in vivo in M. cardinalis . However, there remains. the formal possibility that LMS M. lewisii E - umstantial evidence (the nonsense mutation in in mutation (thenonsense evidence umstantial McOS β and in vitro -ocimene. very low The level of volatile lackconclusive are the these evidence that E - is very difficult to transform, so we have not have we so transform, to difficult very is OS β ) (Byers ) (Byers has multiple coding sequence differences differences sequence coding multiple has -ocimene, while ) that is consistent with this, and RNAi RNAi and this, with consistent is that ) alleles can recapitulate the can recapitulate alleles et al. llelic variation at loci containing containing atloci variation llelic M. cardinalis , differences 2014). These are ( MlLMS M. lewisii re re necessary to produce D- M. cardinalis M. lewisii , McLMS and and with constructs McLMS M. cardinalis M. and and ) and ) and M. cardinalis produces produces M. lewisii M. is a is null

Accepted Article size molecularin genetic terms, evolut in the probability of fixation of the allele is 2 is allele ofthe fixation of probability the visitationas in difference a detect to experiments visitati pollinator our designed we so 1997), Clark, and (Hartl populations natural in very quickly total visi /2202 visitsgreater wild-type to plant the pollinated pollinated individuals would allow selection to exceed drift as an evolutionary force at this locus locus this at force evolutionary an as drift exceed to selection allow would individuals T Why does the loss of thelossD-limonene of Why does and 4.17). RNAi transgenic plants, plants, transgenic RNAi alleles of results in modesta but significant(6%) decrease in bumblebee visitation, suggesting that alterna analogous (but weaker) representation of the phen weaker)representation (but analogous bumblebee visitation in the greenhouse. In contrast, knocking down emission of of emission down knocking contrast, In greenhouse. the in visitation bumblebee on effect significant no produces compounds two these of emission down knocking substantially knockouts show that the loss-of-function loss-of-function the that show knockouts This article This protectedis by Allcopyright. rights reserved. Surprisingly, despite the high level of production of D-limonene and and D-limonene of production of level thehigh despite Surprisingly, pollinatorvisitation.volatile differential testfor necessaryemission phenotypes to metabolic pathways (Gang, 2005). Given the much higher emission of of emission higher much the Given 2005). (Gang, pathways metabolic synthesis flexible isa but complex process, and buildups of precursorscan be utilized b Terpene (GPP). pyrophosphate geranyl precursor shared the of pool common of a to rerouting due 2 plants used in the greenhouse experiment had surprisingly high levels of of levels high had surprisingly experiment greenhouse the in used plants OCIMENE SYNTHASE OCIMENE M. lewisii and the hummingbird-pollinated hummingbird-pollinated the and M. lewisiiM. can contribute to reproductive isolation between bumblebee- the between isolation reproductive to can contribute may be prone to this effect. As the RNAi technique used here is an an is here used technique theRNAi As effect. this to prone be may β -myrcene have no effect on bumblebee visitation? First, the the First, visitation? bumblebee on effect no have -myrcene ionary genetic terms a selection coefficient ( coefficient selection a terms genetic ionary LMS s , or 12%; an effective population size greater than five five than greater size population effective an ,or 12%; and and small as 5%. Assuming an infinite population size, size, population infinite an Assuming 5%. as small OS ts) would sweep the beneficial allele to fixation tofixation allele beneficial the sweep would ts) otypes resulting from loss-of-functiona mutation alleles can recapitulate the canrecapitulate alleles M. cardinalis . Although 6% is a modest effect effect is amodest 6% Although . β -myrcene in in -myrcene E - β -ocimene inthe -ocimene E - β -ocimene, perhaps -ocimene, perhaps E M. cardinalis M. lewisii - β -ocimene -ocimene s (N ) of 0.06 (130 of) 0.06(130 y alternate y e =1/(4 MlLMS flowers, flowers,

s on ) = ) = tive - Accepted Article infestation in wild populations of pathogen or florivory, herbivory, Data on pollinators. bumblebee of attraction the in role secondary similar olfactory responsesin both bee species. Moreover, multiple roles volatiles these may be playing in to speculate possibilities. on these field Future experiments increasewill our understanding of th bumblebee-flowerinvolvingincluding systems, other those interactions in lie elsewhere (Kessler visitation was seenwith theloss of Simi plant. the within process metabolic other some of byproduct the merely or context, environmental syndrome, pollination some previous of remnant vosnesenskii wild with found be would that those from differ effects these that possible is it Finally, Roels and Kelly, 2011), thus we feel that feelthat 2011),we Roels andKelly, thus It is also possible that the high production of D-limonene and D-limonene of production high the that possible also is It setting. natural a in occur might that effects the and physiology in awild population, fluctuations in volatile production as found h This article This protectedis by Allcopyright. rights reserved. navigation (Bogdany and Taber, 1979). The high production of D-limonene and and D-limonene of production high The 1979). Taber, and (Bogdany navigation ( honeybee for important be to shown been has attraction long-distance flower; patch long-distance attraction to the mediate at Although threevolatiles simi have the (Kessler defens as such plant, the within function another et al. . However, both species are generalist floral visitors, and the the and visitors, floral aregeneralist species both However, . , 2013), as these volatiles are known anti-herbivory compounds (Levin, 1976). 1976). (Levin, compounds anti-herbivory known are volatiles these as 2013), , et al. , 2013), for example in herbivory defense (Arimura M. lewisii E - β lar physical properties, D-limonene and and D-limoneneproperties, physical lar -ocimene, ispossibleit that mainof the volatile role this may B. impatiens B. and and M. lewisii M. cardinalis level rather than at the level of the individual theindividual of level atthe than rather level e against herbivores, nectar robbers, or disease disease or robbers, nectar herbivores, against e larly, although a significant effect on bumblebee bumblebee on effect significant a although larly, is an excellent model for these experiments model these isanexcellent for and B. impatiens are currently are lacking, limiting our ability M. cardinalis M. β - myrcene in myrcene - ere are reflective of the system’s has been used as a model for for model a as used been has M. lewisii . B. vosnesenskii B. M. lewisii β et al. -myrcene may -myrcene serve β Apis mellifera -myrcene may be a be may -myrcene scent elicits , 2004) with, 2004) a serves Bombus Bombus (Bodbyl (Bodbyl ) e e Accepted Article the the language Castellanos in hummingbird-pollinated flower such as as such flower hummingbird-pollinated scent informationvery poorly (GoldsmithGoldsmith, and sothe1982), loss of scent ina retain and 1989), Papi, and (Ioalé ofsmell sense limited very a have Hummingbirds hummingbirds? experiments were similar to those found in wild populations of of populations wild in found tothose similar were experiments in densities Additionally, decisions. landing final in role asignificant may play such as scent, floral by be influenced may decision landing final the point, Atthat flower. individual an to approach visually-guided by a followed signals, al. densely-flowered greenhouse experiments, signals such as the presence or absence of absence or presence the as such signals experiments, greenhouse densely-flowered How might a loss-of-function alleleof the potential effect of asingle change in scent in wilda population. of indicator better a are conditions greenhouse thedense so clusters, large in streambeds montane honeybees (Butler, 1951;Galen and Kevan, 1980; as such pollinators diurnal by decisions landing in role strong a scent plays that M system? this within interaction pollinator in play scent does role What allowing them tointegrate multiple floral cues. realistic more a offer experiments greenhouse the which all three monoterpenes were required monoterpenes which allthree in scents, floral extracted and artificial with experiments behavioral previous in found we those deta in differ results these Although explanation). full a for and Methods Materials SI (see This article This protectedis by Allcopyright. rights reserved. , 2009; Dötterl and Vereecken, –the initial 2010) approach guidedbe patch-level may by visual M. lewisii (Dötterl (Dötterl and Vereecken, Parachnowitsch 2010; et al. , 2004) would likely have no fitness cost, would, 2004) nofitness might likely have and even increase OS M. cardinalisM. promote a pollinator switch from bumblebees to for maximum bumblebee response (Byers response bumblebee maximum for Galen and Kevan, 1983; Lunau, 1992; Majetic especially in relatively weakly scented flowers scented flowers weakly relatively in especially assay for the effect of scent on pollinators by by pollinators on scent of theeffect for assay (an “anti-bee” but not “pro-bird” shift, to use use to shift, “pro-bird” not but “anti-bee” (an M. lewisii et al. et

, 2012). Therefore, even in the , whichgrows along any studies have shown haveshown studies any the greenhouse greenhouse the bumblebees and and bumblebees E et al. - β -ocimene -ocimene il from il from , 2014),

et Accepted Article reinforce visitation behavior. Whether these change these Whether behavior. visitation reinforce access found between between found access signa visual in difference with the combination In pollen. or nectar away carrying fitness by discouraging bumblebee visitors from transferring hetero This article This protectedis by Allcopyright. rights reserved. orthologous terpene synthase genes of other species in and profiles volatile the investigating unclear; is contact secondary during reinforcement The factthat the some insight into this question. it is easily missed, in as where systems in isolation reproductive in factor bea also can scent nose, human by the detectable a that scents strong relatively have volatiles floral of study in used the systems many Although to contribute to genes structural for process. this about drawn being from conclusions genes withknown effects in prezygotic reproductive isolation shoul of 2010). nearly andBlackman, thelimitednumber genes (Rieseberg always regulatoryHowever, referred to as “speciation genes” (Coyne, or 1992) involv genes that thinking current the contradicts reported. Some authors have commented that the role the that commented have authors reported. Some hav pollinators generalist with species sister in isolation reproductive pollinator and genetics scent floral integrating examples no and unknown, largely is bumblebees as such pollinators daytime involving isolation reproductive pollinator-mediated in scent floral in changes of role nighttime pollinatorsa from distance iswell documented (Raguso and Willis, 2003) OS polymorphism between between polymorphism M. lewisii Mimulus . of role strong . The emissions of volatilesfloral in attracting and and this process should not be ignored. beignored. not should process this M. cardinalis M. lewisii ed in prezygotic reproductive isolation – often often – isolation reproductive prezygotic ed in , such a loss of We would suggest, however, that the potential thepotential that however, suggest, would We “barrier genes” (Noor and Feder, 2006) – are –are 2006) Feder, and (Noor genes” “barrier s in florals in volatiles evol and of floral scent in reproductive isolation is is isolation reproductive in scent floral of Mimulus M. cardinalis section E - β d d preclude anygeneral specific pollen to the stigma and -ocimene might serveto -ocimene might is in a structural gene gene structural a in is Erythranthe ved in allopatry or as or allopatry as ved in ls and mechanical mechanical ls and . The potential may provide provide may generalist, generalist, e been e re re Accepted Article Volatiles were first triplicate assayed in F an in mapping fine and QTL Procedures Experimental integrative study possible. is questions, but our work with but ourwork questions, of these subset a answered only have speciation in volatiles floral of role the of studies previous Nearly settings. field or greenhouse relevant ecologically in changes genetic of these results the assa finally, and, transgenics), lines, (near-isogenic materials genetic high-resolution creating differences phenotypic of these basis genetic the determining by proceed can studies Then, 2014). each; seeByers inbred line x and(LF10 CE10 comparedCE10) with M. cardinalis complex mixture complex – one can by begin species-specific looking at differences, identifyingcritic easily-measured visual signals such floral as color and pattern. Here, sisterthe species attractive an as considered be should scent Floral 2002). andAyasse, (Schiestl profiles scent floral tomultiple attracted are channe “private as less serve thus scents floral as cases, generalist in questionable This article This protectedis by Allcopyright. rights reserved. limonene, rate emission for assayed and below) methods and Methods and Materials SI also (see 2014 was then constructed. Headspacevolatiles collected were the describedin manner in B and LF10 between cross a of individuals of 100 β -myrcene, and and -myrcene, can be used as a model for the study of reproductive isolation involving floral volatiles floral involving isolation reproductive of study the for a model as be used can et al. via electrophysiological and behavioral assays (Riffell (Riffell assays behavioral and electrophysiological , 2014) using a Mann-Whitney Mann-Whitney a using 2014) , E - β Mimulus -ocimene. Pearson correlation coefficientswere calculated , an emerging model system, shows that a comprehensive, a comprehensive, that shows system, model emerging , an 1

cross of CE10, backcrossed to CE10 [(LF10 x CE10) x CE10],x backcrossed[(LF10 CE10,x CE10) CE10 to factor even in generalist systems, in withfactor along more even previous results forparent lines the (9samples U test. A coarse mapping population consisting consisting test. population mapping A coarse M. lewisii inbred line LF10 and and LF10 line inbred et al. al volatiles withinal a , 2013; Byers ls” and pollinators pollinators and ls” M. cardinalis cardinalis M. M. lewisii yers et al. pairwise s of D- s of et al. ying of ying , all and and , , ,

Accepted Article those backcross plants with informative recombinations between markers flanking flanking markers between recombinations informative with plants backcross those vol for flowers score to required effort the reduce To backcross. the in markers flanking molecular markers and the markers and flanking molecular the to relative position map their upon based identified were genes candidate and examined, was The The Information. collection headspace time-intensive and more labor- OS were developedfrom SYNTHASEOCIMENE limonene and putative putative the flanking markers with and screened constructed was =768) (N population backcross A larger traits. the terpene synthases/cyclaseson LC250K and TS306K (see Table S5 for all primers). No recombination events were observedwere No recombination among events Table (see primers). all for LC250K TS306K S5 and limonene/ ge the of inspection from clear was it However, map. (QTL) locus trait quantitative low-resolution a creating of intent with the S4) (Table genome the limonene/ with the two most divergent phenotypes (high D-limonene/ (high phenotypes divergent most thetwo with plants backcross of 24 A subset pleiotropy. or linkage potential toinvestigate threescents the for This article This protectedis by Allcopyright. rights reserved. (N = 52) were phenotyped for scent, using a direct extraction assay from flowers rather than than extraction the flowersrather direct scent,assay for using from a were phenotyped 52) = (N Mimulus guttatus LIMONENE-MYRCENE SYNTHASE β β -myrcene and and -myrcene -myrcene and high high and -myrcene β -myrcene emission, primers were designed (sc4_2325K and (TableM13_2620) Markers S4). used in the mapping process M. lewisii genomic region corresponding to the corresponding genomic region E - β -ocimene were, to afirst approximation, segregating as Mendelian E and - β -ocimene) were screened at 34 indel markers evenly spaced across across spaced evenly markers indel at34 screened were -ocimene) M. guttatus M. guttatus M. cardinalis and (M02_510 putative M02_1500), andflanking the scaffold 89. The candidate genes were designated designated were genes candidate The 89. scaffold notypic and phenotypic data that the emission of D- of theemission data that phenotypic and notypic annotation. For the genome sequences, and amplify codominant codominant amplify and sequences, genome method. For further details, see Supplemental Supplemental see details, further For method. to amplify indel polymorphisms in two of of two in polymorphisms indel amplify to β M. lewisii -myrcenelow and LMS region containing containing region locuscontrolling D- atile production, only only production, atile E - β -ocimene; low D- low -ocimene; LMS (N = 107) or =107) (N LMS or or OS

Accepted Article and equivalent stages in in stages equivalent and recombination events were observed between TS2538 and TS2538 between observed were events recombination polymorphism in a terpene synthase designated onTS2538 Terpene synthase expression expression synthase Terpene For details, seeSupplementa vitro In 0.1 than cM. Total RNA was extracted from flower buds collected at 5, 8, 10, 15, and 20mm stages in in stages 20mm and 15, 10, 8, 5, at collected buds flower from extracted was RNA Total (Invitrogen). RT-PCR for both loci for both species was performed with these cDNA, using using cDNA, these with performed was species both for loci both for RT-PCR (Invitrogen). System Synthesis First-Strand III SuperScript the using extracts RNA total from prepared was cDNA the the the two candidate genes and the putative putative the and genes candidate two the This article This protectedis by Allcopyright. rights reserved. a control for background expression levels as described in Yuan in described as levels expression background for a control the following:the et al. et targeted to knocktargeted expression down the of ancestral in thisstate clade (Beardsley for RNAi senseforward/reverse Transgenesis was done in the Transgenesis the wasin done Construction of of Construction OS , 2004; Arabidopsis Biological Resource Center, CD3-447) as described in (Yuan (Yuan in described as CD3-447) Center, Resource Biological Arabidopsis , 2004; locus controlling locus assay for terpene synthase activitysynthase terpene assay for MlLMS MlLMS RNAi forward/reverse for for RNAiforward/reverse and and E M. cardinalisM. - β -ocimene emission, primers were designed to amplify an indel an indel toamplify weredesigned primers emission, -ocimene MlOS l Information and Fäldt Fäldt and l Information M. lewisii in vivo in MlOS RNAi transgenic RNAi transgenic

and . Total RNA was extracted from open flowers of both species. both of RNAwasextracted open flowers from . Total et al. et background, as insect pollination is inferred to be the the to be inferred is pollination insect as background, MlLMS LMS McOS , 2003). Hairpin RNA interference (RNAi) transgenes (RNAi) interference Hairpin RNA 2003). , , defining a candidate region of less than 0.1 cM. For For cM. than0.1 less of region candidate a , defining ; and and ; MlLMS, McLMS or or M. lewisii lewisii M. et al. et MlOS MlUBC (2003). (2003). were constructed in pFGC5941 (Kerschen pFGC5941 in were constructed OS M. guttatus foward/reverse for , defining a candidate region of less less of region candidate a , defining et al. et forward/reverse for for forward/reverse (2013c). Primers usedwere Primers (2013c). scaffold 4. No No 4. scaffold MlUBC et al. McLMS . M. lewisii , 2013c). In MlUBC ; MlOS as

Accepted Article eachcase, targetspecificity theRNAifr of This article This protectedis by Allcopyright. rights reserved. LF10 genome sequence. For For sequence. genome LF10 directionallycloned pFGC5941 into the amplified and directionally cloned into the into cloned directionally and amplified MlOS pFGC5941. A180bp antisense fragment (entirely within the 289bp constancy,were visits “collapsed” level tothe plant – For constancy. and visits) total of (proportion preference for analyzed and transcribed Data were day. each type on each of flower given a of encountering chance equal had an bumblebees that ensure to transgenic) between equalized and counted were flowers opened newly and were removed flowers old activity, bumblebee first tothe prior day of each twoobservers using voice recorders, each following recorded were behavior bumblebee of Observations S2. Figure in depicted is positions, plant randomized including experiments, Supplementa design, see For experimental details of experiments Greenhouse planta then electroporated separately into were reduced to a single visit, since flower numbers were unequal between plants and flowers were flowers and plants between unequal were numbers flower since visit, single toa reduced were , 289bp sensea fragment was amplified and directionallycloned into the transformation of of transformation M. lewisii MlLMS LF10 LF10 following (Yuan , a106 bp fragment of Agrobacterium tumefaciens tumefaciens Agrobacterium Nco agment was assured by BLAST search against the against search BLAST by assured was agment Bam I/ Asc the two types of plants (wild-type and RNAi and RNAi (wild-type of plants types the two HI/ I (sense) and and (sense) I Xba one or two bumblebees at a time. At the start thestart At time. ata bumblebees two or one l Information. The layout of the greenhouse greenhouse the of layout l The Information. for the first six hours of thefirst three days by i.e. I site. Constructs were verified by sequencing, sequencing, by verified were Constructs site. I et al. ,

multiplevisits toone plant in sequence M. lewisii Bam , 2013c). strain GV3101 and used for for andused GV3101 strain HI/ cDNA was amplified and and amplified was cDNA Nco Xba I/ I (antisense) sites. For Asc I amplicon)I was Nco I/ Asc I site ofI site M. lewisii in

Accepted Article greenhouse experiments. experiments. greenhouse scoring, and collection extraction volatile Description Methods: and Materials Supplemental Information Supporting for Legends Fellowship. Denton Writing of College AchievementMelinda Rewards the for Scientists the Foundation and Chapter Seattle Fellowshipthe from Foundation 0718124).ARCS KJRPB byan (DGE is supported also Researc Graduate NSF a and 1209340), (IOS and KJRPB 1121692), DBI 0822709; (5R01GM088805),grants Science Foundation National HDB to grant of Health Institutes National a by supported was work This reviewers. was improved greatly by helpful and thorough critiques fromD. Tholl and two an manuscript The experiments. greenhouse in assistance for M. Sargent care, and plant greenhouse Beeman,E. D. Ewing,We thank P. Acknowledgments the toestimate used was randomness) (complete zero from deviation observed times; th 100,000 repeated identities, plant shuffled with test permutation a in used were data foraging thesame expectation, null the from different met by this required as type, each to access equal equalizin preference; of independent is which 1986), Waser, in (described method Bateman’s using calculated was Constancy bout. foraging a in plants visited constancy ten they or m if analysis for were tightly often Bumblebees only clustered. used This article This protectedis by Allcopyright. rights reserved. Forbush, J. Milne, N. Kurashig J. Milne, Forbush,

in vitro assays for terpene synthase activity, and ric. To determine if observed constancy was was constancy ric. observed To determine if of volatile headspace collection and analysis, analysis, and collection headspace volatile of e fraction of the simulations with a greater than than with e fractionsimulations the greater of a g flowers at the start of the day gave pollinators pollinators gave day of the start atthe flowers g to HDB (FIBR 0328636;1209340),(IOS JAR IOS (FIBR HDB to e, M.Kovic, B.and Watson for h Fellowship to KJRPB KJRPB to h Fellowship p onymous -value. -value. ore

Accepted Article guttatus Bodbyl Roels, S.A. and Kelly, Rapid J.K. (2011) evolution caused bypollinator in loss and the evolution of hummingbird pollination. pollination. hummingbird of evolution and the Beardsley, P.M., Yen, A.,and Olmstead, R.G. AFLP(2003) phylogeny of 1976-1983. and transcript accumulation of (E)- of accumulation and transcript Table S4: Molecular markers used during QTL and fine mapping. fine and QTL during used markers Molecular S4: Table of knockdown RNAi from recovered parents T1 separate four from plants T2 in production Volatile S3: Table experiments. RNAi from recovered plants T1 best the2-4 in Volatile production S2: Table Figure S2: highlighted. differences with McOS and MlOS of sequences Protein S1: Figure used. primers oligonucleotide of Sequences S5: Table the of Products S1: Table This article This protectedis by Allcopyright. rights reserved. Apidologie acros orienting bees for of odor significance The (1979) III. S. Taber, and F.J. Bogdany, defense response in amodel legume. Two-spotted spider mites induce emissionof (E)- Herbivore-ind (2004) J. Bohlmann, and J., Takabayashi, S., Kugimiya, R., Ozawa, G., Arimura, References . Evolution

10(1), 55-62. 10(1), Schematic of the greenhouse experimental setup. setup. experimental greenhouse the of Schematic

MlLMS. 65(9), 2541-2552. 65(9),

in vitro terpene synthaseassays. terpene β -ocimene synthase in in synthase -ocimene Evolution 57(6), 1397-1410. 57(6), Lotus japonicas Lotus

Mimulus . Plant Physiol. Plant section Mimulus s a canyon. canyon. a s β Erythranthe 135(4), -ocimene uced uced

Accepted Article trait loci affecting differences in floral morphology between two species monkeyflower of between ( morphology infloral trait loci differences affecting Quantitative (1998) D.W. Schemske, J.K., McKay, and Frewen, B.E., K.G., Otto, Jr., Bradshaw, H.D., differential pollinator in monkeyflowersdifferential ( attraction Byers, K.J.R.P., Bradshaw,H.D.,Jr., and Riffell, β in Gershe and N.J., Oldham, D., Martin, J., Bohlmann, This article This protectedis by Allcopyright. rights reserved. from volatiles terpenoid of emission Chen, Tholl, F., D., D’Auria,J.C., Farooq,Pichersky, A., E., and Gershenzon, J in pollination hummingbird of evolution duri changes ‘pro-bird’ and ‘Anti-bee’ (2004) J.D. Thomson, and P., Wilson, M.C., Castellanos, pollinator monkeyflowers. in shift Allele (2003) D.W. Schemske, and Jr. H.D., Bradshaw, Genetics Linanthus dichotomus Linanthus in scent and morphology floral in divergence G. (2008)Geographic LeBuhn, Raguso, and R.A., ( discovery worker byhoneybee the of in the food Theimportance Butler,of C.G. (1951) perfume Coyne, Genetics J.A. (1992) and speciation. Apis mellifera -ocimene synthase. -ocimene synthase. Arabidopsis thaliana Arabidopsis 149, 367-382. L.) L.) Proc. R. Soc. Lond. Sci.Lond. B Biol. R.Soc.Proc. Arch. Biochem. Biophys. (Polemoniaceae). : cDNA cloning, characterization, and functional expression of a myrcene/(E)- a of expression functional and characterization, cloning, cDNA : Nature Arabidopsis Penstemon Am. J. Bot. J. Am. 426, 176-178. Nature Nature 375(2), 261-269.375(2), J.A. (2014)Three floralvolatiles contribute to 138, 403-413. 138, flowers. flowers. nzon, J. (2000) Terpenoid secondary metabolism metabolism secondary Terpenoid (2000) J. nzon, 355, 511-515. 95(12), 1652-1659. Mimulus flowers. flowers. substitution at a flower colour locus produces a a produces locus colour flower ata substitution Plant Cell Plant ). J. Evol. Biol. Evol. J. J. Exp. Biol. Exp. J. 15, 481-494.Chess, 15, S.K.R., 17(4), 876-885. 17(4), 217(4), 614-623. 217(4), . (2003)Biosynthesis. and

Mimulus ng the ng ). ). Accepted Article

snapdragon flowers. flowers. snapdragon emission biosynthesis and in benzoate Wood, K.(2000)Developmental methyl regulation of Gorenste L.M., Murfitt, C.J., Mann, N., Dudareva, Cell synt terpene expressionfunction three and of J. (2003) ( J. (2003) J. Fäldt, N., Gorenstein, N., Kolosova, C.M., Kish, D., Martin, N, Dudareva. breweri in production scent floral in involved -anenzyme acetyltransferase CoA:benzylalcohol Acetyl- (1998) E. Pichersky, and R.A., Raguso, K.H., Nam, J.C., D’Auria, N., Dudareva, 1148. the the floral volatile isoeugenol. of thebiosynthesis in involved acetyltransferase petunia a of Characterization (2007) D. Clark, and E., Koeduk C.J., Ma, C.M., Kish, A., Qualley, Dexter, R., This article This protectedis by Allcopyright. rights reserved. novel patterns of S-linalool synthase gene expression in the in expression gene synthase S-linalool of patterns novel a review and perspectives. perspectives. and a review Dötterl, chemical N.J. S. The Vereecken, (2010) and ecology evolutionand of bee-flower interactions: Dudareva, N., Cseke, L., Blanc, V.M., and Pichersky, E. (1996) Evolution of floral scent i scent floral of Evolution (1996) E. Pichersky, and V.M., Blanc, L., Cseke, N., Dudareva, 15(5), 1227-1241. . Plant J. E )- β -Ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: snapdragon: in biosynthesis scent of floral genes myrcenesynthase and -Ocimene 14(3), 297-304. 14(3), Plant Cell Plant Can. J. Zool. J. Can. Plant J. Plant 12(6), 949-961. 49(2), 265-275. 88,668-697. hase genes of a new terpene synthase subfamily. subfamily. synthase terpene new ofa genes hase in, N., Kolosova, N., Kish, C.M., Bohman, C., and N.,Bohman, and in, Kolosova, C., Kish, N., C.M., a, T., Nagegowda, D.A., Dudareva, N., Pichersky, Pichersky, N., Dudareva, D.A., Nagegowda, T., a, C. breweri flower. , Miller, B., and Bohlmann, Bohlmann, and B., , Miller, Plant Cell Cell Plant n Clarkia Clarkia Clarkia 8, 1137- : Plant Plant Accepted Article spruce ( cloning and functional characterization of (+)-3-carene synthase. synthase. (+)-3-carene of characterization functional and cloning syndromes and floral specialization. J.D. (2004) M.R., and Thomson, Dudash, P., W.S., Wilson, C.B., Armbruster, Fenster, Fäldt, J.,Martin,D., Miller, B.,Rawat, S.and Bohl 135, 1893-1902. Dudareva, N., Pichersky, E., and Gershenzon, E., and N., Pichersky, Dudareva, This article This protectedis by Allcopyright. rights reserved. Grant, Pollination V.(1949) systems isolating as mechanisms in angiosperms. Condor Hummi Black-chinned the in smell of Sense (1982) T.H. Goldsmith, and K.M. Goldsmith, Gang, D.R. (2005) Evolution flavorsof and scents. Polemonium viscosum pollinat and polymorphisms floral color, and Scent (1980) P.G. Kevan, and C. Galen,

1207-1213. Galen, C. and Kevan, P.G. (1983) Bumblebee foraging and floral scent dimorphism: dimorphism: scent floral and foraging Bumblebee (1983) P.G. Kevan, and C. Galen, Curtis (Hymenoptera:Apidae) and (Hymenoptera:Apidae) Curtis 84,237-238. Picea abies Picea ): Methyl jasmonate-induced terpene synthase gene expression, and cDNA cDNA and expression, gene synthase terpene jasmonate-induced Methyl ): Nutt. Nutt. The American Midland Naturalist Midland The American Polemonium viscosum Annu. Rev. Ecol. Evol. Syst. Syst. Evol. Ecol. Rev. Annu. J. (2004) Biochemistry (2004) volatiles.J. plant of mann, J. (2003) Traumatic Traumatic (2003) J. mann, Annu. Rev. Plant Biol. Plant Rev. Annu. Nutt. (Polemoniaceae). Nutt. 104(2),281-289. 35, 375-403. Plant Mol. Biol. Mol. Plant 56,301-325. resin defense in Norway Norway in defense resin Evolution 51, 119-133. Can. J. Zool. J. Can. Bombus kirbyellus ion biology in in ion biology Pollination Pollination

Plant Physiol. Plant 3,82-97. ngbird. ngbird. 61, 61,

Accepted Article genetics. and geography, ecology, speciation: plant in pollination animal of role The (2009) R.D. Sargent, and K.M. Kay, hummingbirds. hummingbirds. ins magnetic and discrimination odor size, bulb Olfactory (1989) F. Papi, and P. Ioalé, attraction in two species of of two species in attraction emissi scent Floral (2005) F.P. Schiestl, and W., Sauter, R., Kaiser, F.K., Huber, Inst. WashingtonPubl. 1-213.628, Hartl, D.L. and Clark, A.G. (1997) (1997) A.G. Clark, and D.L. Hartl, evidence for floral adaptation. adaptation. floral for evidence Harder,L.D. and Johnson, S.D. Darwin's (2009) be key volatile for pollinator attraction. Biosystematics, genetics, and physiological ecology of the the of ecology physiological and genetics, Biosystematics, Hiesey, W.M., Nobs,and M.A., Björkman, O. (1971) MA. Sunderland, campion ( campion Gupta, A.K., Akhtar, T., Widmer, Pichersky, A., E.,Schiestl, and IdentificatF.P. (2012) This article This protectedis by Allcopyright. rights reserved. Silene latifolia Physiol. Behav. ) guaiacol guaiacol ) Gymnadenia 45, 995-999. 45, New Phytol. Principles of Population Genetics Population of Principles O Ann. Rev. Ecol. Evol. Syst. Evol. Ecol. Rev. Ann. -methyltransferase involvedbiosynthesis the in of veratrole, a BMC Plant Biol. BMC Plant (Orchidaceae). (Orchidaceae). 183(3), 530-545. autiful contrivances: evolutionary and functional functional and evolutionary contrivances: autiful Experimental Studies on the Nature of Species. V. Species. of Nature the on Studies Experimental 12, 158. 12, Oecologia Oecologia Erythranthe 40,637-656. (4 142(4), 564-575. th ed.) Sinaur Associates Inc., Inc., Associates Sinaur ed.) section of of section on and pollinator pollinator and on Mimulus integrating integrating ion whiteof ensitivity in in ensitivity . Carnegie Carnegie

Accepted Article defence/apparency dilemma of pollinator attractiondeploying by complex f Kessler, D.,Diezel, C., D.G., Clark, Colquhoun, T.A., and Baldwin, (2013)Petunia I.T. flowers solve the in transgenic plants. plants. transgenic in Kerschen, A., Napoli, C.A., Jorgensen, R.A., and Müller, A.E. (2004) Effec This article This protectedis by Allcopyright. rights reserved. (2011) Pollinator choice in in choice Pollinator (2011) M. Saxenhofer, K., Hermann, A., Gurba, Klahre, U., 16(3), 299-306. ester. alcohol coniferyl a of reduction via biosynthesized are ofspices, constituents aromatic characteristic Lewis, N. Dudareva, T.J., Noel, J.P., Baiga, N.G., Koeduka, T., Fridman, E., Gang, D.R., Vassão, D.G.,Jackson, B.L., Kish, C.M., Orlova, I., Spa Curr. Biol. pollinator attraction and seed fitness in fitness seed and attraction pollinator Majetic, C.J., Raguso, R.A.,and Ashman, T-L. (2009) stamen signals. to visual antennae of bees: orientation by bumble flowers of recognition Innate K. (1992) Lunau, Syst. herbivores. and pathogens to ofplants defenses chemical The (1976) D.A. Levin, 7, 121-159. Proc. Natl. Acad. Sci. U.S.A. Sci. Acad. Natl. Proc. 21, 730-739. Can. J. Zool. J. Can. FEBS Letters Petunia 70, 2139-2144. 566, 223-228. 566, 103(26), 10128-10133. 103(26), depends on two major genetic loci for floral scent production. production. scent floral for loci genetic major two on depends Hesperis matronalisHesperis , and Pichersky, E. (2006) Eugenol and isoeugenol, and isoeugenol, (2006) Eugenol Pichersky, E. , and , Bossolini, E., Guerin, P.M., and Kuhlemeier, C. Kuhlemeier, P.M., and Guerin, E., , Bossolini, The sweet smell of success: floral scent affects . Funct. Ecol. tiveness of RNA interference 23, 480-487. loral blends. Annu. Rev. Ecol. Ecol. Rev. Annu. Ecol. Letters ssova, S.M.,

Accepted Article Chiloglottis Pollinator specificity, odour chemistry floral phylogeny of the and Australian sexua Peakall, R.,Ebert,D., Poldy, J., Barrow, R.A., Francke,W., Bower, C.C.,and Schiestl, F.P.(2010) 667-675. scent emission, but not flower size or colour in bee-pollinated bee-pollinated scent in emission, sizeorcolour flower butnot Parachnowitsch, A.(2012)Phenotypic Raguso, andKessler, R.A., A.L., selection floral increase to transport. auxin synthesis enhancement petunia in benzoic revealsmultipleflowers acid and pathways to in among populations and species of the sexually deceptive orchid genus genus orchid deceptive thesexually of species and populations among Does (2005) F.P. Schiestl, and R., Peakall, J., Mant, This article This protectedis by Allcopyright. rights reserved. W.A., Murphy, A.S., Rhodes,D., Pichersky, E., and Dudareva, (20N. Orlova, I., Marshall-Colón,A., Schnepp, J., Wood, B., Varbanova, M., Fr 851-861. production apple. in volatilefor terpene R.G. (2013)Functional genomics reveals thatco a M.Y Wang, A.J., Matich, E.J.D., Bailleul, X., Chen, S.A., Green, N.J., Nieuwenhuizen, 1449-1463. Noor, M.A.F. Feder, (2006) and SpeciationJ.L. genetics: evolving approaches. Plant Cell orchids: implications for pollinator driven speciation. speciation. driven pollinator for implications orchids: 18(12), 3458-3475. 18(12), Plant Physiol, Physiol, Plant mpact terpene synthase gene family can account selection on floral odor promote differentiation differentiation promote odor floral on selection 161, 787-804. Penstemon digitalis New Phytol. New 06) idman, E., Blakeslee, J.J.,Peer,

Ophrys Reduction of benzenoid benzenoid of Reduction 188(2), 437-450. ? Nat. Rev. Genet. Rev. Nat. Evolution . ., and Atkinson, Atkinson, ., and lly deceptive New Phytol. 59(7), 59(7), 7, 195, Accepted Article Raguso, R.A. (2008b) Wake up and smell the roses: the ecology and evolutionof responses to floral traits. In In traits. floral to responses Raguso, R.A. and Willis, M.A. (2003) Hawkmoth pollination Arizona’s in SoronanDesert: Behavioral Rev. Ecol. Evol. Syst. synthase, an enzyme involved in the production of floral scent in in scent of floral production the in involved enzyme an synthase, characterizat and Purification (1995) R. Croteau, and E., Lewinsohn, E., Pichersky, This article This protectedis by Allcopyright. rights reserved. ecology. ecology. Raguso, StartR.A. (2008a) makingchallenge scents: the of integratingchemistry into pollin Biophys. between the the monkeyflowersbetween is reproductive of Components (2003) D.W. Schemske, and Jr., H.D., Bradshaw, J., Ramsey, Boggs, C.L., Rausher, M.D. (2008) Evolutionary transitions in floralcolor. 1534.

Rieseberg, L.H. and Blackman, B.K. (2010) Speciation genes in plants. plants. in genes Speciation (2010) B.K. Blackman, and L.H. Rieseberg, 316(2), 803-807. Entomol. Exp. Appl. Appl. Exp. Entomol. et al .), pp. 43-65. University Chicagoof Press, Chicago. 39, 549-569. 39, Evolution and ecology taking flight, Butterflies as model systems model as Butterflies flight, taking ecology and Evolution Mimulus lewisii 128(1), 196-207. and M. cardinalis M. Int. J. Plant. Sci. Sci. Plant. J. Int. (Phrymaceae). Clarkia breweri Clarkia Ann. Bot. Ann. 169(1), 7-21. Evolution 106(3), 439-455. floral scent. . ion of S-linalool of S-linalool ion Arch. Biochem.Arch. 57(7), 1520- olation olation ation (ed. (ed. Annu. Annu. Accepted Article 349(1), 153-160. in monkeyflowers ( Pollinat (1999) Jr. H.D., Bradshaw, and D.W. Schemske, odour compound: ecological and evolutionary implications. implications. evolutionary and ecological compound: odour Schiestl, F.P. Peakall, and R. Two(2005) orchids differentattract pollinatorsthe sa with olfactory specialization and learning in in learning and specialization olfactory Riffell, J.A.,Lei, H., Abrell, L. and Hildebrand, J.G. (2013)Neural basis of pollina a This article This protectedis by Allcopyright. rights reserved. orchids? orchids? speciation cause odor floral in changes Do (2002) M. Ayasse, and F.P. Schiestl, methyltransferase involved in floral scent production in in production scent floral in involved methyltransferase O- ofS-adenosyl-l-methionine:(iso)eugenol Characterization (1998) E. Pichersky, and J. Wang, shift in sexually deceptive orchids. (2 F.P. Schiestl, and S., Cozzolino, N.J., Vereecken, mechanisms. Adapt Stebbins,(1970) G.L. isolation in two species ofisolation species two in Waelti, M.O., Muhlemann,J.K., Widmer, andA., Schi Plant Syst. Evol. Evol. Syst. Plant Annu. Rev. Ecol. Syst. Syst. Ecol. Rev. Annu. Mimulus 234, 111-119. Silene ive radiation of reproductive characteristics in angiosperms, I, Pollination characteristics in I, angiosperms, reproductive of ive radiation ). Proc. Natl. Acad. Sci. U.S.A. U.S.A. Sci. Acad. Natl. Proc. . J. Evol. Biol. Evol. J. BMC Evol. Biol. Evol. BMC 1, 307-326.1, Manduca sexta Manduca 21(1), 111-121. 21(1), 010) Hybrid floral scent novelty drives pollinator pollinator drives novelty scent floral Hybrid 010) 10(1), 103. estl, (2008)Floral F.P. . or preference and the evolution of floral traits traits of floral theevolution and preference or Science Clarkia breweri 96(21), 11910-11915. Funct. Ecol. Funct. 339(6116), 200-204. 339(6116), . 19(4), 674-680. Arch. Biochem.Arch. Biophys. odourreproductive and in sexually deceptive deceptive sexually in tor’s buffet: me floral

Accepted Article

induced floral mutant identifies a identifies mutant floral induced Yuan, Y-W., Sagawa, J.M.,Di Stilio, V.S., and Bradshaw, H.D., Jr. (2013b) Bulk segreg specificity. flower-polli of control genetic The (2013a) Jr. H.D., and Bradshaw, K.J.R.P., Byers, Y-W., Yuan, adaptation in a sexually deceptive orchid. orchid. deceptive asexually in adaptation Xu, S., Schlüter,P.M.,Grossniklaus, U., and Schiestl, F.P. geneticThe (2012) genetics. Whitehead, M.R. and Peakall, R.Integrating (2009) floral scent,pollination ecol 603. Waser, (1986) N.M. Flower constancy: definition,cause, and measurement. This article This protectedis by Allcopyright. rights reserved. between sister species of species sister between of isolat reproductive pollinator-mediated to contributing QTL anthocyanin major a of dissection Yuan, Y-W., Sagawa, J.M., Young, R.C., Christensen, B.J., and Bradshaw, H.D., Jr. ( Mimulus lewisii Funct. Ecol. Curr. Opin. Plant. Biol. Plant. Opin. Curr. . Genetics 23, 863-874. 194(2), 523-528. Mimulus 16(4), 422-428. MIXTA . Genetics -like R2R3 PLoS Genet. PLoS 194(1), 255-263. MYB 8(8), e1002889. 8(8), controlling guidenectar formation in basisof pollinator Am. Nat. ogypopulation and 2013c)Genetic ant analysisof an 127(5), 593- nator nator ion ion Accepted Article Positions on the lower half of each are from the the from are each of half lower the on Positions queries of the of the queries lewisii complete data on all volatiles produced by samples. Emission values inparen cardinalis

wild-type Table 1 Tables: This article This protectedis by Allcopyright. rights reserved. Figure 1 Figures: limonene limonene D- E β ocimene - -myrcene Volatile Volatile β - limonene-myrcene synthase homologous is a to terpene synthase at 319,982 bpon : Volatile production in transgenic (T transgenic in production Volatile : : . (B) Genetic of maps . (B) Mimulus lewisii M. lewisii M. guttatus M. 50.708) (35.539 (ng/hr) (4.320, (2.056, 43.228 7.049) 3.793) 5.563 2.837 LF10 ,

and and M. cardinalis and (ng/hr) transcripts Putative with BLASTx. Absent Absent (0.649, (0.649, (0.000, (0.000, (0.000, (0.000, 1.419) 0.000) 0.000) 1.024 CE10 M. cardinalis MlLMS theses represent the 90% confidence interval. See Table S2 for S2for Table the See represent confidence 90%theses interval. (20.591, MlLMS 30.120) (ng/hr) (0.765, (0.194, 25.125 . Values are an average of 2-3 independent headspace headspace independent of2-3 average an are Values . 1.757) 0.339) 1.216 0.257 and RNAi RNAi MlOS M. lewisii and their terpene synthases. (A) theirterpene and - 1 parent plants of greenhouse experiment lines) and and lines) experiment greenhouse of plants parent and the homologous regions in M. guttatus (ng/hr) (23.285, 24.354) MlOS (1.047, (1.047, (0.042, (0.042, 23.820 1.185) RNAi RNAi 0.074) 1.116 0.054 . - (% LF10) MlLMS genome scaffolds; annotations are from arefrom annotations scaffolds; genome terpene synthases are highlighted. highlighted. are synthases terpene 451.64 RNAi RNAi .1 118.75 2.81% .6 / 39.34% n/a n/a 9.06% % - MlLMS CE10) RNAi RNAi n/a 0.97% n/a 0.97% n/a (% % Mimulus lewisii - M. guttatus (% LF10) 55.10% 2326.17 MlOS RNAi RNAi

- M. M. . and and MlOS CE10) RNAi RNAi (% M. % M. - Accepted Article preferentially visit preferentially wild-type knockdowns, knockdowns, visitation response to to response visitation Figure 4 lines. (A) emission D-limonene levels of (b)/ according to bud size of size bud to according stages, despite producing no terpenoid volatile that we could detect. Develop ocimene but (c), Figure 3 guttatus This article This protectedis by Allcopyright. rights reserved. common monoterpene precursor), the MlLMS enzyme produces D-limonene (b) and (b)and D-limonene enzyme produces MlLMS common precursor), monoterpene the bacterial overexpression system and and system overexpression bacterial Figure 2 2,538,727 bp on scaffold 4. McLMS flowers. MlLMS in the same relative proportionin thesamerelative as is expressed just prior to flowering and in open flowers, but flowers, open in and flowering to prior just expressed is , : : Stable RNAiknockdowns of : Terpene synthase activity activity synthase : Terpene scaffold 89, and and 89, scaffold McOS

MlOS

Image of bumblebeea visit.typical (B) Greenhouse experiments with experiments Greenhouse MlOS , and asimilarshows expression pattern to M. cardinalis RNAi knockdowns, and the wild-type parent. Bumblebees show the same same the show Bumblebees parent. wild-type the and knockdowns, RNAi McUBC MlLMS M. lewisiiM. M. lewisii . M. lewisii knockdown McOS does not. (B) Temporal expression of terpene synthases synthases of terpene expression (B)Temporal not. does McOS , and the corresponding stageof β in floral the volatile emission. ocimene synthase is homologous to a terpene synthase at synthase terpene toa homologous is synthase ocimene in vitro -myrcene (a)and in vitro MlLMS over over Bombus impatiens Bombus

transgenics as to wild-type to transgenics as and MlOS enzyme assay. Using geranyl pyrophosphate (GPP, the the (GPP, pyrophosphate geranyl Using assay. enzyme and and in vivo in

knockdown transgenics. Response of bumblebees to bumblebees of Response MlOS MlLMS E . (A) Products(A) terpene synthases. of using a - in in β -ocimene respectively. (c), M. lewisii and and , and , and M. lewisii M. lewisii McOS M. cardinalis produce plants with low McLMS M. lewisii is expressed at thesame at isexpressed wild-type and transgenic transgenic and wild-type MlOS produces is not expressed open in expressed not is MlLMS mental mental staging is . Bumblebees Bumblebees . is pictured for for pictured is RNAi RNAi β -myrcene (a) (a) -myrcene E - β in vivo. - Accepted Article A A

This article This protectedis by Allcopyright. rights reserved. Abundance (% of sample) Mimulus cardinalis 100 100 100 100 50 50 50 50 Mimulus lewisii 0 0 0 0 . . 0011.0 10.0 9.0 8.0 M. lewisii MlLMS MlOS McOS a Time (min) a b b c c Sca guttatus Mimulus 02_1500 lewisii Mimulus 201 B ff old 89 proteinase asp 4 257 249 cyclase limonene 251 B .criai B M.cardinalisUBC M.cardinalisOS M. cardinalisUBC M. cardinalisLMS M. lewisiiUBC M. lewisii M. lewisii M. lewisiiLMS Sca 13_3500 guttatus Mimulus 1.365 lewisii Mimulus like dynamin- 5 ff 5 old 4 protein binding acid nucleic 261 01 0open 20 15 10 8 8 .4 .3 .4 2.557 2.549 2.538 2.442 ROI hydrolase isochorismatase equivalentbudstage(mm) equivalentbudstage(mm) 6 7 8 293 287 273 264 like dynamin- 10 protein binding acid nucleic 6 8 9 0 326 306 292 280 268 Limonene-myrcene synthase( 2.534 15 synthase terpene like dynamin- Ocimene synthase( protein tegument large 0open 20 2.542 like dynamin- cyclase (STC) sesquiterpene unknown (DYT1) tapetum 1 dysfunctional 2.551 CENP phosphatase MgH T T STC STC STC Y1DT DYT1 DYT1 DYT1 2.559 MlOS M. lewisii M. lewisii M. lewisiiUBC M. lewisiiOS 5 5 MlLMS ) synthase terpene 01 0open 20 15 10 8 2.568 8 equivalentbudstage(mm) equivalentbudstage(mm) ) 10 2.573 2.577 15 2.579 synthase terpene protein expressed 2 335 321 2.583 0open 20 synthase terpene peptidase cystine

Accepted Article

This article This protectedis by Allcopyright. rights reserved. Abundance (% of wild−type M. lewisii) 100 100 100 100 50 50 50 50 0 0 0 0 . . 0011.0 10.0 9.0 8.0 M. cardinalis MlOS M. lewisii MlLMS Time (min) (a) − a a RNAi − RNAi b b (b) b (c) c c

Accepted Article A

This article This protectedis by Allcopyright. rights reserved. Total visits (%) B 100 20 40 60 80 0 MlLMS wild

wt n =1682visits − − p =0.70 type: 49.52% RNAi: 50.48%

MlLMS− RNAi MlOS wild

wt n =2202visits p =0.0056 − − type: 52.95% RNAi: 47.05%

MlOS− RNAi