This article isThis article by protected copyright. All rightsreserved. 10.1111/plb.12924 doi: Accepted differences to lead between the of thisversion c and Version Record.Please copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review buthasnot title Running Ithaca, USA. Cornell University, andBehavior, Neurobiology of Department 4. Chile. Mar, del Viña Botánico Nacional, Jardín 3. Argentina. Córdoba, Biología Ve de Multidisciplinario Instituto 2. México. de México, Ciudad Autónoma México, de Nacional Universidad de Instituto Ecología, 1. ArticleBenitez Pisano Aimé Rubini pollen fl asymmetric and fragrances in boundaries species of Breakdown :Z Editor Paper :Research Article type BENITEZ DR. SANTIAGO - Vieyra : 2* Mandevilla . -
X Ren X 1,2
, Marcela Moré
- VIEYRA (Orcid ID :0000 ID (Orcid VIEYRA
hybrids: morphology, fragrances morphology, hybrids:
ow 2 Mandevilla , Mauricio A. Cisternas A. , Mauricio .
getal (CONICET getal (CONICET - 0003 : floral morphological intermediacy, novel novel intermediacy, : floral morphological
- 4116
- and pollen flow pollen and 7969) 2, 2, 3
-
, Robert A. Raguso , Robert Universidad Nacional de Córdoba), Córdoba), de Nacional Universidad
ite thisarticle as 4 , Santiago , Santiago
This article isThis article by protected copyright. All rightsreserved. Summary ARGENTINA. (CONICET * Mandevilla Keywords
Accepted Article author Corresponding
compounds not emitted by not emittedby compounds with additional phenotype, atransgressive hybridindividualsshowed putative from volatiles backcrossing ratesof asymmetrical hybridizat extensive suggested movement analogue pollen and individuals intermediate morphologically of presence The analogues. pollen movements using foraging their tracked and le an unsupervised relevant traitsand taxonomically through individual grouping assessing and morphometrics geometric through shape floral characterizing fragrances, floral of composition phenotypi We examined occurs. isolation floral of the how breakdown and interbreeding, to ( signals olfactory and visual of roles inter with individuals of Thepresence respectively. nocturnal hawkmoths, Hymenoptera and guilds:diurnal pollinator andassociated phenotype floral in differences clear with species aresister (Apocynaceae) plant hybrid discovering clues to main providedthe and has origin, hybrid putative intermediacyisof indicator an Phenotypic
, pollination. : Apocynaceae, floral fragrances, geometric morphometry, phenotypic intermediacy, intermediacy, phenotypic morphometry, geometric fragrances, floral Apocynaceae, : -
Universidad Nacional de Córdoba), CC 495, CP 5000, Ciudad de Córdoba, Córdoba, Córdoba, de Ciudad Córdoba, CP 5000, CC 495, de Córdoba), Nacional Universidad
: [email protected] arning algorithm. We quantified the visitation frequencies of floral visitors visitors floral of frequencies the visitation quantified algorithm. We arning mediate phenotypes in a wild population raises questions about the the about raises questions population a wild in phenotypes mediate c variation in a in c variationmixed either parental species parental either ion between ion s in nature. s in
between these putative hybrids and hybrids putative between these i.e. corolla morphology and floral fragrances) as barriers asbarriers floral fragrances) and morphology corolla M. laxa
Mandevilla pentlandiana Mandevilla Instituto Multidisciplinario Multidisciplinario de Instituto Mandevilla .
and and
M. pentlandiana
population
analysing chemicalthe analysing and M. laxa , along with , along
M. laxa
Biología Vegetal Biología . Floral
This article isThis article by protected copyright. All rightsreserved. Bischoff (Waelti studied least well remains scent the in plants, floral isolation reproductive species ( parental than al guilds pollinator attract novel species ifthey parental either to crossing preven may traits floral traits.floral Hybrid on some extent to depends individuals parental al. parental two spec the of comparisons al. ( groups functional bydifferent pollinated despite some being hybridize, species often related of populations that revealed studieshave Many 1997). (Arnold their and species parental thesegene between hybrids rateof flow andthe genotypes o anddistribution frequency the choices determine can foraging because their hybridization ofplant drivers major Pollinators are 1994). & Hodges Arnold 1994; (Grant isolation) ethological traits( specific floral transfer ( pollen interspecific reduces prevents or length) spur pre particular, relat barriersbetween reproductive of breakdown the about raises questions phenotypes, Introduction . 2016; Marques . 2016;
Accepted Article 2015). 2015). Martin 2007; &Campbell Aldridge 2004;
introgression. continued putative betweenvisits occur, oncethey but M. l sympatric swarm between ahybrid presence the of results suggest Our et al A full understanding of the role of floral traits in reproductive isolation requires requires isolation traits reproductive floral in the of of role understanding A full intermediate by characterized often in individuals nature, hybrid of presence The Whether or not hybrids survive and produce fertile descendants by interbreeding with with interbreeding by fertiledescendants survive produce and hybrids Whether not or axa . 2014). Previous studies suggest that floral scent in hybrids canshow atleastthre hybrids floral scent in that suggest studies Previous . 2014). - zygotic barriers to hybridization occur when floral morphology ( morphology floral when occur barriers hybridization to zygotic
and indicate indicate and
et al. e.g. i.e
2016) . temporal or ph or . temporal colour or or colour that in that , promote inappropriate pollen placement, or flower at different times atdifferent flower placement, or pollen , promoteinappropriate
scent) affect pollinator attraction and visitation behaviour ( visitation behaviour and attraction scent) affect pollinator itial hybridization events between these parental species are rare, are species parental these between events itial hybridization ies and natural hybrids with respect to floral traits (Campbell traits(Campbell respect floral with to hybrids natural and ies enological isolation). Of all the traits known to affect to known traits Of allthe isolation). enological e.g.
et al. et al. Hodges & Arnold 1994; Sérsic Sérsic 1994; &Arnold Hodges 2008; Campbell 2008; hybrids a hybrids i.e. nd
M. l mechanical isolation) or when or mechanical isolation) et al. axa (Vereecken (Vereecken
are common and facilitate are commonand 2016). et al. M. pentlandi e.g.
et al.
2001; Ippolito Ippolito 2001; et al. posture ornectar posture
2008; ed species. In In ed species.
2010; Ma 2010; Ma t back f hybrid ana i.e.
e a
- nd et et et
This article isThis article by protected copyright. All rightsreserved. of mixed population anew discovered wehave Recently, origin. ahybrid has it totestwhether it difficult (now making extinct), locality single from a are known er and to issimilar species rareendemic tothis Ezcurra, According (2005). species, athird that possibility the hybridsbetween putative sympatry, of Moré 1998; (Torres &Galetto hawkmoths, respectively nocturnal and Hymenoptera diurnal groups, pollinator distinct by where arepollinated they S1), (Fig. Argentina (Simões pentlandiana Mandevilla f dynamics swarm is hybrid (2007). &Campbell by Aldridge suggested species that related parental between geneflow enhance might swarm ahybrid scentin floral so, plants. If hybrid intermediate chemically and/or morphologically the of presence by canbe facilitated herbivores & by Floate proposed bridge” hypothesis “hybrid mirrors the question hybrid This the swarm. enveloping aplume scent to due floral of introgression) ( parental species both tovisit more likely were cases) both in (hawkmoths vectors (Campbell of hybrids ca and systems, in hybrid attraction can mediatepollinator specificcompounds that volatile shown have studies species(Vereecken parental either species ( ( profiles scent intermediate additive, chemically parental species: 1) to relation in patterns different Accepted Article Cortis ect corolla lobes, features of features of lobes, ect corolla et al. Bischoff Bischoff et al A favourable system in which to study the role of floral scent in reproductive isolation and and isolation reproductive scentin floral of therole study to which system in A favourable Ipomopsis et al. et are reproductively isolated under normal conditions, as a hybrid bridgeas pollinators, for as ahybrid normalconditions, under isolated reproductively are
. 2006) that occur in sympatry throughout Andean Bolivia and extreme north and Bolivia Andean throughout occur sympatry in that . 2006) 2009; n elicit feeding behaviour in conjunction with flower colour and posture among among and posture flowerwith colour conjunction in behaviour elicit feeding n
2016), respectively. One question emerging from these studies is w from is emerging thesestudies Onequestion respectively. 2016), et al. Campbell
in North America (Bischoff America(Bischoff North in
2015
(DC.) Woodson and and (DC.) Woodson ound in the the genus in ound ) or 3) the the ) or presence3) et al. et al. M. grata et al. M. pentlandiana. 2016), 2) the absence of certain compounds present in parental parental present in compounds certain absenceof the 2) 2016), M. pentlandiana M.
2010, 2010, , may actually be of hybrid was suggested by Ezcurra wassuggested by of hybrid be actually , may
Mandevilla M. laxa Marquez of novel ( novel of Whitham (1993), in that plant host shifts by by hostshifts plant inthat (1993), Whitham et al.
However, only two type specimens type of However,two only
(Ruiz & Pav.) Woodson are sister species are sisterspecies Woodson &Pav.) (Ruiz
2015) and and 2015) and et al.
(Apocynaceae) in southern South America. South southern (Apocynaceae) in transgressive) compounds not found in in not found compounds transgressive)
M. laxa
2016 M. laxa M. laxa Zaluzianskia et al ).
have not recorded been not have M. laxa
Manipulative experimental experimental Manipulative . 2007). Across this large zone zone Acrosslarge this . 2007). and and
but smaller flowers it has M. pentlandiana
in South Africa South in promoting hether pollen pollen hether - western M. grata ,
although although
at the at the
This article isThis article by protected copyright. All rightsreserved. Fie S1). (Fig. Accepted Bolivia and Argentina northern incentraland sympatry in mayoccur and environments, woodland distribution. and species system, description Study a Materials vectors. aspollen visitors floral of themovement evaluate experimentally to pollen analogues hybrids, using putative among speciesand flow parental pollen of role address the (Baylac recognition pattern of from field the procedures statistical with data morphometric combining geometric by information anyprior without groupings morphological an firstis the algorithm. While learning an unsupervised through traits relevant and taxonomically through grouping individual Weassessed population. ourstudy in plants flowering floral all ofscent of chemical composition species between morphology Article origin. to determine us their allow would that evidence genetic of population origin ahybrid without traits, extreme having phenotypic but parental the species, of one pentlandiana between speciesboundaries the of represents ararebreach it 1)that population; areThere atleast individuals These thickness. floral traits, and intermediate vegetative showing individuals in north extreme distributions their southern of . Thus, Thus, Our main goal is to main isto goal Our Mandevilla laxa Mandevilla , resulting in a recent hybrid swarm, 2) that intermediate individuals actually belong to to belong actually individuals intermediate swarm, that ain 2) , recenthybrid resulting ld observations ld Mandevilla We characterized the distribution of standing variation in flower shape and the shape the flower and variationin standing of distribution the We characterized nd methods nd three
pollinators in the origin of these hybrid individuals thesehybrid origin of the in pollinators M. laxa M. laxa
. For ther . For potential explanations for the phenotypic variation observed in this mixedthis observed in variation the phenotypic for explanations potential
plants as putative hybrids, with the understanding that we do not yet have that have yet not we understanding do the with hybrids, plants asputative
and and
cannot be clearly assigned to any to assigned clearly be cannot were made during the flowering season flowering the during made were find out whether these individuals showing intermediate floral intermediate floral showing these individuals outwhether find and and M. pentlandiana
a priori a emainder of this paper, we refer to this morphologically intermediate morphologically this to paper, werefer this emainder of M. pentlandiana M. pentlandiana
classification, the s the classification, -
central Argentina (Fig. S1), along with many many alongwith S1), (Fig. central Argentina (Apocynaceae) are perennial vines that grow in grow in that vines (Apocynaceae) areperennial
actually represent hybrid individuals of of these individuals two hybrid represent actually
econd is an unbiased attempt to identify identify attempt to unbiased isan econd such as flower size, shape and leaf and shape size, asflower such
Mandevilla
or 3) the presence of a third species, athird presence of or the 3) , from November 2013 to , from2013 November
we assessed the possibility of of possibility the we assessed et al
species, even to to even species, . 2003). . 2003). M . laxa Moreover, to to Moreover,
and and M. grata M. . This article isThis article by protected copyright. All rightsreserved. Acceptedthe style beneath the stigmatic on concavity deposited this isfirst pollen, carrying wasIf theproboscis already slit. eachcone along it passes when theproboscis attached onto by it is caught withdrawn, interany of thefive through tube the corolla into areinserted proboscis when pollinators’ is achieved chambers.Pollination f anthers, the slitsbetween the with alternating grooves five style The receptive part. Thick slits. five longitudinal by arerepresented anthers adjacent between boundaries The gynostegium. thewith style united are anthers mechanism. The pollination Moré Galetto 1999; strictherkogamy, to due hum by receives visits occasionally contrast, In Article (Moré honeybees and hummingbirds daytime by visitedduring isalso and Sphingidae), laxa Mandevilla for open species remain Flowers both of 1998). &Galetto Torres mowith contrast, In Galetto 1998). sucrose melonand produce concentrated ripe of that resembling M. pentlandiana laxa racemes,dense whereas small and produces species both S1). of distribution (Fig. southernmost the representing Argentina, Province, Córdoba LaQuebrada", Natural Parque "ReservaHídrica the in 2014 February
has salverform flowers with white corollas markedly larger than the tubular greenish flowers of flowers tubular thanthe greenish larger markedly corollas white with flowers has salverform ened stigmatic regions, called“style stigmatic regions, ened re variable sugar concentration than that of of than that concentration sugar re variable Although both species are self speciesare both Although M. pentlandiana
is pollinated in the evening by nocturnal long bynocturnal evening the in is pollinated (Fig. 1, upper panel). Theflowe panel). (Fig. 1, upper et al. - i.e. head is completely enclosed within the lower part of the anther the lower of the part enclosed within is completely head
2007). the base of the corresponding anther corresponding the the baseof M. laxa
-
flowers depend upon the visit of pollinators to set fruits (Torres & (Torres set fruits pollinatorsto of the visit upon depend flowers staminal spaces between the fused anthers. When the proboscis is the proboscis When anthers. fused the staminal between spaces is pollinated primarily by honeybees and bumblebees during daytime and bumblebeesduring and byhoneybees primarily is pollinated
Mandevilla
flowers emit an intense jasmine emit intense flowers an mingbirds (Torres & Galetto 1998). &Galetto 1998). (Torres mingbirds - compatible, autogamy does not occur in unvisited flowers unvisited occur in not does autogamy compatible, - heads”, consist of an upper secretory part and a lower alower partand secretory upper an of consist heads”, M. laxa species show an extremely precise and complex precise and extremely an species show rs of rs of M. pentlandiana
exhibits large and sparse racemes. sparse large racemes. and exhibits M. pentlandiana orming five longitudinal pollination pollination longitudinal five orming - head, forming a complex organ, the organ, head, forminga complex -
- tongued h cone slit. During withdrawal pollen is pollen withdrawal During slit. cone - rich nectar (44.7% w/w; Torres & w/w;Torres nectar (44.7% rich 3 - - like fragrance and produce nectar produce and fragrance like 4
Mandevilla pe Mandevilla (27.7% w/w to 44.2% w/w; 44.2% w/wto (27.7%
days. Elsewhere in Argentina, Argentina, Elsewhere in days.
emit a weak fruity fragrance fragrance emit fruity aweak
- awkmoths (Lepidoptera: (Lepidoptera: awkmoths head (Moré head ntlandiana et al. Mandevilla Mandevilla - et al. cone, with with cone,
2007)
2007). 2007).
.
This article isThis article by protected copyright. All rightsreserved. samethe individual from of flowers Landmarkconfigurations coordinates. matrix Procrustes data of Accepted landmarks ( homologous minimizeto configurations oflandmark the rotation (3) size; and same the centroid they have that so configuration landmark ofthe scaling (2) same centroid; involves analysis This 2011). (Klingenberg program the MorphoJ using was performed of flowers symmetry the bilateral considering thecoordinates on landmark analysis Procrustes Ageneralized 2010). (Rohlf eachindividu of configurations Landmark (13). theanther base of 7), sepals(6, the of tip maximum curvature: minimum of or points landmarkseight were 12). remaining The (11, lobes coro the and tube corolla the between point theinflection and 5) (4, nectarchamber the to entrance the of constriction ovary(3), the into style the of insertion (1, 2), into thepetiole the of calyx insertion we “landmarks” A total13 of lowerpanel). 1, (Fig. visible petalswere five the of three that such mmscale, reference a50 with along flowers of sections individual. per averaged Datawere flower to senescence. due Only first 70% ethanol. in preserved and were sampled Article Zelditch all in homologous arearguably that anatomical loci of dataarecoordinates because the the data, in iscontained morphometrics landmark In 2012). (Gómez populations ( populations levels: within different because study field of growing M. pentlandiana laxa, morphology Flower
et al. three steps: (1) translation of the landmark configuration of all objects so that they share they the sothat objects all of the configuration of landmark three translation steps:(1) One to five five One to among variation shape flower the characterize to morphometrics geometric We used
2012). -
based geometric morphometrics, the spatial information missing from traditional from missing traditional spatialinformation morphometrics, the geometric based et al.
flowers (mean = 2.08) from all flowering individuals (n=63) in the community community the (n=63)in individuals from allflowering = 2.08) (mean flowers and their putative hybrids. andtheirhybrids. putative Zelditch Zelditch
2008) or among species (van der Niet der species (van among or 2008) it enables the interpretation and visualization of variation patterns at patterns variation and of visualization interpretation the it enables et al. - cone (8, 9), tip of the 9), (8, anther cone e.g.
2012
Gómez ). In this way, only shape information is retained in the the in is retained shape only information In ). this way, al were obtained using the program TpsDig ver. 2.16 program 2.16 ver. the using al wereobtained TpsDig et al. Geometric morphometrics of floral shape isa shape floral of Geometricmorphometrics re selected. Five landmarks were anatomical: anatomical: were landmarksFive selected. re individuals in the analysis (Bookstein 1997; 1997; (Bookstein theindividuals in analysis
2006; Benitez 2006; -
day flowers were sampled to avoid difference avoid to flowers were sampled day squared Euclidean distances between between distances Euclidean squared
Digital photos were taken of sagittal sagittal were of taken Digital photos
- cone (10) and tip of the central petal centralpetal the tipof and (10) cone et al. et - Vieyra Vieyra
2010; Kaczorowsk 2010; landmarks et al.
2009), among2009), : discrete : discrete i et al. M.
lla s This article isThis article by protected copyright. All rightsreserved. differing models fitting with Acceptedby estimated model canbe ‘best’ matrix. A covariance the by are the determined of clusters orientation) volume, (shape, features mean.Geometric nearer the points with means, atthe or clusters centred groups by characterized are densities multivariate normal by mixtures of Datagenerated Gaussian distribution. a(multivariate) following et al (Baylac individuals of the identity onthe prior information any without groupings morphological toidentify used method a data, themorphometric to geometric analysis mm). <13 lobes mm corolla <23 or length (flower pentlandiana be assigned to large to flowers too its when were hybrid putative an“intermediate” or be to was cons individual An Ezcurra (2005). following length) corollalobe and length traits (flower fragrances. taking floral and samples for groups different priori An studied population. the in discontinuities to us detectmorphological algorithm, learning allowing putati the of taxonomicdescriptions the Article following the one first groups, into =63) (n individuals flowering classify to two approaches followed We hybridization. of result werethe they whether or species, parental ofputative the phenotypes these individuals test whether to makes difficult it individuals intermediate presence of obs.) pers. hairiness, and Classification 2011). program (Klingenberg from MorphoJ grids transformation the using visualized were changes Morphological coordinates. Procrustes the after were averaged . 2008). Thi . 2008).
classification was needed for purely operative reasons, in order to examine pollen flow pollen examine reasons, order to in purelyoperative for wasneeded classification
First, we divided the individuals into three groups using two taxonomically relevant floral floral relevant taxonomically two using three into individuals groups the wedivided First, though Even
(flower length > 15 mm or corolla lobes > 3mm) or too small to be assigned to to assigned be small to too >3mm)or lobes mm corolla >15 or length (flower
s method assumes that observations are the result of mixed components, each one each one mixedof components, are result the observations s method that assumes superimposition. A Principal Component Analysis (PCA) was then performed on on performed (PCA)wasthen Analysis Component APrincipal superimposition. M. pentlandi
and floral traits andfloral ana ve parental species, and the second using an unsupervised anunsupervised using second the and ve species, parental
and (corolla size and corolla lobe shape; Ezcurra 2005) Ezcurra lobeshape; corolla and size (corolla M. laxa
S
differ in many vegetative ( many in differ eco nd, we applied a Gaussian finite mixture mixture finite a Gaussian weapplied nd,
et al
increased density for increased density . 2003; Cordeiro . 2003; leaf shape, thickness thickness leaf shape,
were extreme M. , the - M. laxa
Estrela between idered idered
a This article isThis article by protected copyright. All rightsreserved. fit.a betterWe stress of indicating values lower with matrix, wasdetermined, distance observed the 2 obtained the how illustrates which dissimilar). Stress, are completely (Dötterl low a in coordinates sample depicts matrix, methodthat on adissimilarity based ordination an (NMDS), Non using wascompared floral of fragrances chemical composition Kruskal using werecompared nocturnal) vs.(diurnal periods andbetween putative hybrids the species and parental ratesamong emission d above) pentlandiana Thirtee Material). Supplementary (GC mass spectrometry and chromatography gas with combined 1998) & Pellmyr (Raguso (Goodrich (SPME) fibers microextraction phase solid methods, fragrance of flower composition of chemical the Characterization coordinates. Procrustes the components of principal firsttwo the of space reduced the in performed was analysis mixture (BIC). Criterion its Bayesian Information on model based best the choose within the of parametrizations 2018 the using analysis this performed modelWe selection. for astatistical criterion then applying and maximumlikelihood, data by shape, (clustermean, parametrizations Acceptedaytime. Article - dimensional geometric space. T geometric dimensional ). We tested models with increasing number of clusters (from 1 to 9) and different 9)to and 1 (from clusters numberof increasing ).modelswith Wetested . For dynamic headspace, 24 samples were obtained, 11 of them at night and 13 during them 13 of and 11 atnight wereobtained, samples 24 dynamic headspace, . For
et al.
Fragrance emission rates were obtained from dynamic headspace samples. headspace samples. from dynamic obtained rateswere emission Fragrance s was fragrance flower of composition Chemical
and four from putative from putative four and 2009). This index generates values between 0 (all compounds are shared) and 100 (they (they 100 and shared) are compounds (all 0 between values indexgenerates This 2009).
- group covariance matrix (for details see Fraley & Raftery 2007), and and 2007), Raftery & see details Fraley (for matrix covariance group n SPME samples were collected: three from three collected: were samples SPME n mclust he Bray hybrids, according to the the to hybrids, according volume and orientation) and/or numbers of components to the the to components numbersof and/or orientation) volume and
- ( package Wallis or Wilcoxon rank su Wilcoxon Wallis or - Curtis index was used to build a dissimilarity matrix a dissimilarity build index to Curtis wasused Scrucca
tudied by means of two complementary complementary two meansof by tudied et al a prioria
. 2016) of R software (R Core Team Rsoftware of (RCore . 2016) et al
- . 2006) and dynamic. 2006) headspace Metric Multidimensional Scaling Scaling Metric Multidimensional m test, respectively. The The mrespectively. test,
- taxonomic D configuration dep D configuration
The Gaussian finite Gaussianfinite The M. laxa M.
classification (see classification Differences in Differences , six from, six - MS; see MS; arts from M. This article isThis article by protected copyright. All rightsreserved. Accepted their and shared visitors (Smith assemblages visitor flower of indices (PS) similarity proportional the we calculated them, was among flow possible pollen Wallis tests. Kruskal using periodsexamined were observation among and seeabove) taxonomic classification, hour flower · asvisits wasfrequency calculated Visitation to ifnot. order and possible, whenever species to wereidentified visitors temperatures. Animal high due to time presumably this interval, during st preliminary hoursbecause 14:00 and between 11:30 were omitted in flowers (215 866 morning ( chosen. was plant focal period,adifferent eachobservation In in period Article visitors Flower Team(R Core 2018). R software using wereperformed analyses statistical of distribution obtainanempirical to vector grouping the of permutations (Dötterl group different anysample froma to than eachother to aremoresimilar groups sampleswithin that indicates groups within and between separation measure of a teststatistic matrix. It yields operates asimilarity on directly that procedure amongsamples. ANO scentcomposition in differences quantitative qualitativeand evaluate to (ANOSIM) similarities of an analysis also performed - 1 . Diffe
M. laxa To analyse if analyse To each periods, fiftyobservation of A total 09:00 to 11:30 hrs.), afternoon (14:00 to 18:30) and evening (19:00 to 21:00) (19:00 to21:00) andevening to18:30) (14:00 afternoon hrs.), to 11:30 09:00 rences in visitation rates among parental species and putative hybrids (following the the hybrids(following putative speciesand parental rates among visitation rences in
, . An . An M. pentlandiana M. laxa, M. laxa, R M. laxa, M. pentlandiana M. laxa,
et al.
visitation frequency. PS is calculated as 1 as1 PS is calculated frequency. visitation value of “0” indicates a completely random grouping, whereas a value of “1” “1” of avalue randomwhereas grouping, acompletely value “0”indicates of et al. 304 in in 304
2009). Statistical of significance 2009). a priori a
2008).
and putative hybrids between November 2013 and February 2014. 2014. February and 2013 hybrids andbetween November putative M. pentlandiana M. pentlandiana
defined groups, based on differences of mean ranks betweenmean ranks of differences on based defined groups, This measurement takes into account both the number of the both account into takes measurement This
and putative hybrids and putative
one of 30 min, were done throughout the flowering theflowering throughout weredone 30min, of one and 347 in the hybrids). in 347 putative and SIM is a commonly used multivariate isacommonly SIM
Flower visits were recorded during during recorded were visits Flower R
was assess was -
½ [(Σ(P udies indicated no floral visitation no indicated udies
share pollinators and, thus, if thus, and, share pollinators R
ed by 10,000 random10,000 ed by ai under the null model. All null the under
– R
P that is a relative isarelative that bi )], P where
Observations Observations in a total of of in atotal ai
and P and - - 1 bi ·
This article isThis article by protected copyright. All rightsreserved. Accepted for (n=335 were collected openflowers the of 2/3 remaining the days, After three site. study anther the surrounding corolla, the to according for asfollows:blue colour agiven of powder fluorescent aspecific received 1993). Individuals (Kearns &Inouye population movement of estimate tothe analogues aspollen powders fluorescent used we a straightforwardmanner. Thus, in individuals different of flowers transfer between assess pollen visitors flower of Movement (see Kislev preparations M. pentlandiana hawkmoths the to attached loads (MZUNC). Pollen de Córdoba Nacional at the Universidad Zoology Museum of of the collection from Lepidoptera Argentina) of Conservation and Research for GICLA (Group Article pollen were carrying they whether season of colleagues (Beccacece by our werecaptured that b hawkmothindividuals of proboscides weinspectedthe addition, In 1988). Nilsson &Rabakonandrianina, by (asdescribed microscope abinocular mothscales using presence of of flowers of atleast pollinators are important hawkmoths that indicated had records visitors. higher indica with values 1, from to range 0 PSvalues respectively. the by made rate total visitation up the of are theproportions
Because moth pollination is difficult to detect by direct observation at night, and previous andprevious at night, observation direct to detectby isdifficult pollination moth Because This compThis laxa M.
M. laxa M. ’ proboscises were compared with reference pollen samples taken from flowers of of fromsamples flowers taken pollen reference with were compared proboscises ’ a priori priori a and their putative hybrids and mounted in glycerine jelly semi jelly glycerine mountedin hybridsand and their putative ,
M. pentlandiana ( n=75 lex pollination of mechanism pollination lex et al. et taxonomic c ) , M. laxa M. pentlandiana M. pentlandiana
1972). - cone using a fine bristle brush in about 1/3 of the open flowers atthe flowers theof 1/3open about in bristlea fine brush using cone , yellow for for , yellow from any of them. Hawkmoth individuals are deposited at the at the aredeposited them.individuals fromof Hawkmoth any
and putative hybrids, using a binocular microscope to confirm to microscope abinocular hybrids,and using putative
lassification. Fluorescent powders were placed within the within were placed powders Fluorescent lassification. ( n=75 M. pentlandiana et al. ) Mandevilla
and putative hybrids ( hybrids and putative
2011) at the study site during the flowering the siteduring study atthe 2011) i
th species (see above) makes it difficult to makes difficult it above) species (see
flower visitor to to flower visitor and red for the putative hybrids, hybrids, putative and the redfor ting greater overlap in flower in greater overlap ting n=75 M. laxa M. laxa
flower visitors in the in visitors flower elonging to speciesfour elonging ) were inspected for the the for ) wereinspected - permanent permanent a th (Moré (Moré
and and b th et al.
taxa, M. M. laxa, M. laxa,
2007), 2007), This article isThis article by protected copyright. All rightsreserved. in both maximumMoreover, the tube in the of ones. apex the towards located but minimum values an 4 (landmarks to nectar chamberentrance the constriction the of anther the position by represented was mainly loadings the PC2 in Variation lobes. corolla reduced and corolla tube ashort scores, showing loading PC1 star developed, a well and tube corolla elongated bothan scores, showing loading PC1 in maximum values (la lobesshape corolla the andin and5) 4 2, (landmarks1, tube theof elongation corolla the changesin by represented mainly with intermingled hybridsappeared putative morpholo maximum of minimumextremes the and species represented parental putative thetwo where axes, the first two by morphospace determined the in were arranged t morphology Flower Results totals. frommarginal inaχ theusing population hybrids abundanceof the on based expectations fromdeparted null analogues pollen received or thatdelivered flowers of number the Wetestedwhether pollination. ofpollen analogues presencethe of Weconsidered colour. pollenfrom of sameadifferent or the received analogues that flowers of percentage wedetermined the Thus, powders). red and yellow remaining the for (blue tomaximize colours visualization different filters of using 250) Leica DC adigitalcamera with equipped DMLB (Leica microscope an epifluorescence pentlandiana Accepted byPC2). 9.62% PC1 and by axes (85.72% PCA two he first Article More than 95% of total variance in the configuration of floral landmarks was explained by by explained landmarks was floral of configuration the in variance total 95% of than More
, 190 for putative , 190 - shaped limb. Meanwhile, Meanwhile, limb. shaped in a flower as an indicator of a floral visit but not necessarily of a successful asuccessful of not necessarily but visit afloral of an indicator in aflower as
ndmarks 11, 12 and 13; Figs. 1, 2). Individuals from 2).Individuals Figs. 1, and 13; 12 ndmarks 11, hybrid individuals hybrid 2
independence test. Expected numbers of flowers were calculated calculated were flowers numbers of Expected test. independence M. pentlandiana M. laxa
and 44 for for and 44
individuals. individuals.
filter for the blue powder and yellow filter filter and yellow powder blue filter for the individuals showed the minimum in individuals the values showed M. laxa, M. pentlan M. laxa, d 5; Figs. 1, 2), which is retracted in the the in 2),which5; Figs.retracted 1, is d M. laxa Individuals of the studied community studiedcommunity the of Individuals - cone (landmarks 8, 9 and 10) and the andthe and 10) 9 8, (landmarks cone
Variation in PC1 loadings was loadings PC1 in Variation ). Flowers were inspected under under were inspected Flowers ). gical variability (Fig. 2). Many (Fig.2). Many variability gical M. laxa diana
and and putative
showed the the showed -
This article isThis article by protected copyright. All rightsreserved. Accepted with and 1,8 linalool monoterpenoids, scent of The salicylate. methyl than amounts compo of scent the monoterpenes dominated whereas scent floral of inthe compounds representative suggested (classes sesquiterpene unidentified an monoterpene and unidentified an analysis: dynamic headspace using detected were not SPME analysis Qualitative fragrance of flower composition of chemical the Characterization laxa resp intermediate individuals, eigth and nine involved clusters third secondand The intermediate or individual. hybrid putative one Article to the all belonging individuals included (19 & Raftery empiricalthe Fraley of following criteria significant as considered be may which a points, difference atleasttwo by BIClower had values solutions Alternative taxa laxa Classification ( entrance tube to corollathe relation in 7) varied and 6 of the sepals (landmarks relativeposition PC2the and PC1
(Fig. S4) (Fig. and six intermediate individuals (Fig. 2). (Fig. intermediate six and individuals 21to and M. laxa - und. Benzenoid esters also were present in presentin were also esters Benzenoid und. GC Nine volatile organic compounds (VOCs)were identi compounds organic Nine volatile based Taxonomic classification - MS (Table S2). Two VOCs found in low amounts were exclusive to to amountsexclusive low in were TwoVOCsfound S2). (Table MS , whereas no volatiles were shared with with shared volatiles were , whereasno .
landmarks 11 and 12; Fig. 12; and 1). 11 landmarks M. pentlandiana T he Gaussian mixture analysis indicated that the best model included four groups. four best the model included indicatedthat mixture analysis he Gaussian
ectively. The fourth one included all the individuals belonging to the all belonging individuals one included Thefourth ectively. . The 24 remaining individuals were not assignable to either of of these either assignable to not were individuals remaining . The24
- by mass spectral ion fragments). Sesquiterpenes were the most werethe Sesquiterpenes fragments). mass spectral ion by cineole, and shared the presence of lina of presence the shared cineole, and M. pentlandiana on
two relevant floral traits assigned 17 individuals to individuals to 17 traitsassigned floral relevant two intermediate individuals
M. laxa M. pentlandiana M. laxa M. pentlandiana M. pentlandiana , with linalool being the being most abundant linalool , with
, with methyl benzoate present in greater present in methyl benzoate , with following the previous classification, and and previous classification, following the
fied from floral headspace using using headspace fromfied floral , dominated by ( by , dominated
(Table S2).(Table was dominated by two two by dominated was lool and methyl salicylate salicylate methyl and lool E,E M. pentlandiana 98). The first cluster The cluster first 98).
) - α - farnesene, farnesene, M. M.
and This article isThis article by protected copyright. All rightsreserved. W =2; d.f. 3.375, (Kruskal hybrids putative the species and betweenparental significantly differ not with shared those whereas heptadecene, with shared Compounds daytime). α sabinen hybrids: putative the specific to were VOCs heptadecene.Four and farnesene, farnesol daytime: linalool, during emitted only Final S3). (Table salicylate to exclusive were benzoate benzyl benzoateand methyl and andafarnesolisomer at night exclusively (1,8 traceamounts in themof two only detected, trans DMNT and pentlandiana benzoate) and benzyl alcohol, benzyl and tridecatetraene) and terpinolene (sabinene, monoterpenoids including here, werefibers identified us VOCs detected not additional 11 of 3).A total Fig. S3, (Table well ashydrocarbons as above, asdescribed benzenoids and sesquiterpenes monoterpenes, included TheseVOCs methods. be markedly varied rates and emission analysis Quantitative - = 4, = 4, Accepted and alcohol benzyl and andnight) day (emitted during terpinolene Article - DMNT DMNT P
= 0.343; putative hybrids hybrids putative = 0.343; Total scent emission rates, calculated per g dry floral mass per inflorescence per hour, did did hour, per floral inflorescence dry mass per g per calculated rates, scentemission Total species chemicalcomplexity, The trans , eight volatiles were recorded, both in daytime night inand both , volatileswererecorded, eight and pentadecane were exclusive to to exclusive were pentadecane and P α
- - = 0 TMTT were detected at night but were present only in traces during daytime. The daytime. The tracesduring in were but only night present at weredetected TMTT terpineol), irregular terpenoids ( terpenoids irregular terpineol), tween taxa. Eighteen different VOCs were detected using dynamic headspace dynamic headspace using detected were VOCs different Eighteen tween taxa. .185) or between diurnal and nocturnal samples (Wilcoxon test, samples(Wilcoxon nocturnal and diurnal between.185) or trans - DMNT (( ly, the headspace of putative headspace putative the ly, of W M. pentlandiana = 9, = 9, E
the hydrocarbon pentadecane (see Table S3). In In S3). Table (see pentadecane hydrocarbon the ) α - 4,8 -
M. laxa terpineol, methyl salicylate, benzyl alcohol, ( alcohol, benzyl salicylate, methyl terpineol, P
- = 0.786; = 0.786; - dimethylnona specificity and temporal patterns of scent composition scentcomposition of patterns andtemporal specificity trans
- were M. pentlandiana cineole and trans
- were 1,8 M. laxa, M. laxa, TMTT only during daytime. Benzaldehyde, daytime. Benzaldehyde, during TMTT only β - - hybrids contained contained hybrids M. laxa TMTT (( myrcene - 1,3,7 - W α cineole, ( cineole,
- = 8, = 8, - terpineol), with with terpineol), , whereas all samples emitted methyl methyl emitted , whereasallsamples triene), benzenoids (benzaldehyde, (benzaldehyde, triene),benzenoids , linalool and and , linalool β E,E . In - myrcene, ( P α )
M. laxa - = 1.0). However, the dry mass dry However,the = 1.0). - - 4,8,12 E,E terpineol (emitted only during during (emittedterpineol only time samples, whereas time whereas samples, ) 13 VOCs, with seven seven VOCs,with 13 - α - , - trimethyl E
farnesene and farnesene and 11 compounds were 11 compounds α ) β - - - terpineol (Table S3). (Table terpineol β myrcene emitted myrcene emitted - - Wallis test, Wallis test, ocimene, M. pentlandiana E,E M. ing SPME ing - 1,3,7,11 ) - α - α - trans H e and e and -
= -
, This article isThis article by protected copyright. All rightsreserved. proboscis( their to of examined flowers sparganura Sappho periods. observation le mellifera pentlandiana observation the outside this study of periods Rachiplusia ( flies), hummingbirds ( visita visitors and Floral from 0.807, laxa among clustered individual, which hybrid aputative adiurnal of sample from exception single to space, corresponding phenotypic each than fragrance of pentlandiana of flower one of Apis mellifera Apis Accepted the its flowersoutside visiting was observed hawkmoth an unidentified scales, and pidopteran Article
samples. Theseclusters M. laxa M. laxa P
= 0.0001; Fig. 3). Six VOCs, mainly monoterpenoids, clearly separated putative putative separated clearly monoterpenoids, VOCs, mainly 3). Fig. Six = 0.0001; and wasps. and Flowers of Flowers the presence of suggested headspace data analysis NMDS The sp.). Hawkmoths (Sphingidae) were not directly observed during the formal observation the formal observation during observed directly sp.).Hawkmoths(Sphingidae) werenot
. and and (0.0253 ± 0.0082 g; ± 0.0082 (0.0253 Putative hybrid flowers were hybridflowers Putative ) and other Hymenoptera ( other Hymenoptera ) and M. laxa M. pentlandiana M. pentlandiana Manduca rustica Manduca ) and Diptera (hoverflies and flesh and Diptera(hoverflies ) and
Finally, flowers of flowers Finally, although an although M. laxa A total of 2.7% (n = 75) of examined flowers of putative hybrids had hybrids putative of examinedflowers of =75) 2.7% (n of A total M. pentlandiana M. Chlorostilbon aureoventris Chlorostilbon
periods. No lepidopteran scales were recorded in the flowers the of in scalesrecorded lepidopteran were No periods.
tion rates (0.1043 ± 0.0229 g; g; ±0.0229 (0.1043
. Four out of ten trapped hawkmoths carried pollen fromcarried pollen hawkmoths trapped outoften . Four differed significantly in fragrance chemical composition (ANOSIM, (ANOSIM, fragrance chemical composition in significantly differed Agrius cingulata Agrius n
flowers, due to scaling of floral of scaling mass. flowers, to due (compounds 1 to 6; Fig 3). 3). 1to 6; (compounds Fig
, = 201). Thus, each = 201). Thus, n
were visited by a wide array of animals including honeybees animals array of awide including by were visited = 2; M. laxa M. laxa, M. pentlandiana M. pentlandiana M. laxa, Bombus opifex Bombus
Manduca diffissa, n Manduca diffissa, visited by by visited n
= 53) is, on average, five average, is, on = 53)
were visited by hummingbirds ( by hummingbirds visited were ; Trochilidae
individual (Sphingidae) was seen visiting flowers visiting was(Sphingidae) seen individual - C. aureoventris flies, Tab M. laxa and wasps), Diptera (hoverflies and flesh and (hoverflies Diptera wasps), and ) and noctuid moths (Lepidoptera: noctuid ) and
flower produces much larger amounts amounts muchflower larger produces = 1; and = 1;and le 1). le 1). and their putative hybrids, with the the hybrids, with putative and their
No scales were recorded in the the in scaleswereNo recorded hummingbirds, flies, flies, hummingbirds, -
Manduca sextaManduca of clusters in the fragrance fragrance the in clusters of fold
larger than that of larger that than C. aureoventris M. laxa M. , n hybrids
= 1). Apis Apis
attached M.
M. and and
R -
= This article isThis article by protected copyright. All rightsreserved. from morphologically be distinguished cannot which some individuals andthe of presence hybrids, sug hybrids. This putative Discussion a,b). 4 (Fig. expected than waslower individuals other of flowers of greater abundance the despite them.contrast, between In moving arefreely visitors flower between analogues Ho (Fig. 4 b). is occurring visitation flower assortative of degree expectedin than higher re on based from expectations null significantly movements departed visitor Observed analogues. as pollen M. laxa visitors flower of Movement pentlandiana pair othertwo the in assemblage visitor between the of differentiation degree a high wasThere ( index similarity to proportional according assemblage, 0.42). = d.f. 2, = 2.68, ( putative hybrids the and species within parental or evening) afternoon, (morning, obse atdifferent recorded were differences significant from No eachother. significantly = 2, d.f.
AcceptedM. pentlandiana Article lative abundance (χ abundance lative Mandevilla laxa Mandevilla
and 115 of 190 for flow of 190 hybrid 115 and P Our findings suggest incomplete reproductive isolation among the parental species and the the and species among parental the isolation reproductive incomplete suggest findings Our for of 335 flowers (189 analysed the of 569 one third Roughly of Flowers
= 0.007, Table 1) than those of those = 1)than Table 0.007, - putative putative
P
= 0.268; putative hybrids, putative = 0.268;
M. laxa in t in M. pentla M. hybrids, PS = 0.08; =0.08; hybrids, PS
he community, movement of flower visitors between visitors flower of movement he community, and the put and the M. laxa, M. pentlandiana M. pentlandiana M. laxa, 2
= 221.98, df = 4, P < 0.0001; Fig. 4 a, b). Intraspecific movement was movement was Intraspecific 4a,b). < 0.0001; df=Fig. P 4, = 221.98, gestion is supported by the morphological intermediacy of most putative most of putative the intermediacy morphological by is supported gestion
and putative hybrids did not differ from expected values, indicating that that indicating values, expected from notdiffer did hybrids putative and
ndiana ative
were visited more frequently (Kruskal more frequently were visited ers) received fluorescent powders delivered by flower flower visitors by delivered powders ers) received fluorescent hybrids M. laxa M. pentlandiana H = 2.31, d.f. = 2, =2, d.f. = 2.31,
showed the highest overlap in their in visitor highest overlap showed the
and their putative hybrid, indicating that some that indicating andhybrid, putative their and the putative putative and the
M. laxa - M. laxa
P -
putative hybrids, PS = 0.74, Table 1). =0.74, PS hybrids, putative wever, observed flow of pollen pollen of flow wever, observed = 0.32; hybrids , PS = 0.10 (Table 1). =0.10 , PS M. laxa M. pentlandiana , which did not differ not did , which - M. pentlandiana Wallis test, Wallis test, , H M. pentlandiana = 1.75, df = 2, P = = P df 2, = 1.75, rvation timesrvation H ; 24 of for of ; 44 24
= 9.80, = 9.80,
and s: M. , H This article isThis article by protected copyright. All rightsreserved. Accepted any on rely not does which analysis priori a intermed nature of ahybrid with our results areconsistent Thus, 2006). Aldridge Campbell& 1999; (Ellis & Johnson wild populations in origins hybrid assess putative to the indicator sha the closer to being pentlandiana pentlandiana M. laxa contact areas two between foundin wereonly individuals Instead, intermediate populations. allopatric spe third hypothetical because this is unlikely scenario This site. the study presentin be origin may speciesahybrid athird without Second, ranges. geographic combined acrosstheir before recorded been not have variants extremeyet phenotypic laxa Mandevilla traits.However, extreme having phenotypic but parentalto the species, of belong one may individuals study. inour individuals Article intermediate nature of hybrid confirm the to needed data havenot genetic the wedo are awarethat plastid system this for available P be not confirmed. could actaspollinators effectively they whether accor and, visitors and hybrids putative addition, hybrids. In putative from flowers pollinate could they that suggest scales, presence of observation time, from circumstantial night evidence during visiting observe hawkmoths not we did though Even parental species. of range phenotypic fragrances, floral M. laxa. trn
and and taxonomic classification, based on two relevant floral traits, and a finite Gaussian mixture Gaussian afinite and traits, floral relevant on two based taxonomic classification,
Remarkably, we detected a transgressive segregation pattern in the chemical the c pattern in segregation a transgressive wedetected Remarkably, Putative hybrid individuals showed an interm an individuals showed hybrid Putative L - M. pentlandiana M. pentlandiana trn , a corolla tube less developed than in in than , tube lessdeveloped acorolla , and a corolla lobe shape which varies in a gradient between the two parental species, parental two the between agradient varies in which shape lobe , acorolla and F
and and was invariable, and the nuclear markers only varied at very few positions. Thus, w fewpositions. very at varied markers only the and nuclear was invariable, i.e. dingly, the movement of pollen analogues was stronger between them. However, between them. However, analogues wasstronger thepollen movementof dingly,
M. pentlandiana the presence of novel or extreme traits in hybrid organisms beyond the beyond organisms hybrid traits in extreme or novel presencethe of pe of Alternative (Supplementary Methods and Results, Table S1, Fig S2 Fig S1, Table and Results, Methods (Supplementary M. laxa (Ezcurra, 2005; this study). this 2005; (Ezcurra, . a priori hypotheses cannot be completely disregarded: First, intermediateFirst, disregarded: completely be cannot hypotheses Phenotypic intermediacy has been traditionally used as an asan used traditionally been intermediacyhas Phenotypic
are common species, well represented in botanical col botanical in represented well are common species,
information, revealed revealed information, cies has not been collected, up to the present, in thein to present, collected, up been not cies has M. laxa M. laxa s, pollen loads carried on probosces, and the the and probosces, carried loads s, on pollen ediate shape in relation to to relation in shape ediate
shared hummingbirds as main as flower hummingbirds shared
but more developed than in in than but more developed
M. laxa three and four groups, respectively groups, four and three reliminary molecular data data molecular reliminary , M. pentlandiana - iate individuals. iate individuals. S3) M. laxa
a re inconclusive: re inconclusive: omposition of of omposition M.
and
lections, lections, or M. The The e . This article isThis article by protected copyright. All rightsreserved. that data show Our experiments. additional behavioural without ascertain to isdifficult hybrids officinalis (Fähnrich products asminor terpineol) ( suitehydrocarbons monoterpene the of and major product multi asingle of actions the confirm that America (Raguso South Nicotiana 1,8 to leading flux pathway or activity increased enzymatic of artefacts pleiotropic represent actually compounds 1,8 of thes that these is for patterns explanation The most parsimonious S3). S2, terpinolene) emitted ex (Rieseberg segregation transgressive include species. Instead, additive, achemically notproduce do individuals hybrid al as au growing ill of investigations preliminary in applied been m knowledge, Lexer 2005; Bateman & in plants (Shipunov hybrids their and species parental among discriminate to applied has been with together clustered six remaining 1 assigned analysis either to assignable taxonomically not c alternative these However,
Accepted Article . (2003). - cineole (eucalyptol) than either parental species, and that the additional/unique monoterpenoid monoterpenoid the that additional/unique and species, parental either cineolethan (eucalyptol) In contrast with floral shape, floral with contrast In
clusive VOCs VOCs clusive (sect. (Lamiaceae; Wise
-
as well as increased amounts of the monoterpenes β the of amounts increased as well as - odern pattern odern cineole em cineole seful tool in the identification of animal cryptic species after the proposal of Baylac of speciesaftertheproposal animal of cryptic in identification the seful tool Alatae the fragrance patterns among patterns fragrance the 7 to two “morphologically intermediate” groups, one to to one intermediate” groups, totwo “morphologically 7 et al. ; Solanaceae) pollinated by the same hawkmoth guild in the same region of of inthe region guild same the same hawkmoth by pollinated ; Solanaceae) et al. et al. - ission (Table S2). This hypothesis derives from chemical studies of studies from chemical derives hypothesis This S2). (Table ission benzyl alcohol and two structurally related monoterpenes (sabinene, α monoterpenes (sabinene, related structurally two alcoholand benzyl
- 2009; Peñaloza 2009; recognition approaches like finite Gaussian mixture analysis have not yet havenot yet mixture analysis Gaussian finite like approaches recognition lassifications were not exactly the same: from 24 individuals which were same:individuals the from 24 exactly werenot lassifications et al. 2003 & 2006). Enzymatic and genetic studies with these species with studies genetic Enzymatic &2006).and 2003
1998). 1998). M. laxa M. M. laxa NMDS ordination of floral fragrance revealed that the putative putative the that floralof revealed fragrance ordination NMDS et al. et al. - product enzyme are sufficient to account for 1,8 accountfor to enzyme sufficient are product The role of novel/transgressive volatiles among putative putative among of volatiles role novel/transgressive The - Ramírez 2012), as et al. et al.
individuals
M. laxa, M. pentlandiana M. pentlandiana M. laxa, or or - defined plant species. However, this approach is approach this species. However, defined plant M. pentlandiana 1999; Seehausen 2004). Putative hybrid individuals individuals Putative hybrid Seehausen 2004). 1999; et al.
was determined previously for for previously was determined intermediate scent between the two parental parental two the between intermediate scent
(Fig. 2). 2). (Fig.
2010; McCarthy McCarthy 2010; e.g.
β - - pinene, sabinene, β sabinene, pinene, , the finite Gaussian mixture Gaussian finite , the myrcene and α myrcene and Geometric morphometric analysis morphometric analysis Geometric and their putative hybrids and hybrids their putative e plants emit larger amounts emit amounts e plants larger M. pentlandian et al . 2016). To our To our . 2016). - terpineol (Tables (Tables terpineol - Salvia myrcene, α - cineole asa a
and the andthe
- - et This article isThis article by protected copyright. All rightsreserved. between uncle between between hybridization ph acrossfloral specificity visitor reduce D bee presentare in which widely in proportions, lower terpenoids plants(Riffell pollinated turn, In 2006). in recorded been have methyl farnesol, of amounts also emits low bee common many to farnesene, acompound pentlandiana instance, For pollinators. of guilds different of combineattractants bouquets asfloral isolation, than thehybrids in amounts lesser greater or significantly in wereemitted (benzyl or acetate) hybrids the in absent wereeither phenotype atransgressive showed th In westernAmerica. of North Mountains theRocky in pollinated) aggregata Africa between hybrids natural the et al. parentalof phenotypes. versions intermediate or additive simply are not that volatile compounds novel by influenced or with correlated be may patterns asymmetrical ö
Accepted may mixed bouquets volatile features,these withmorphological Along 2010). tterl &Vereecken Article
ar why similar populations have not been documented from the extensive zone of sympatry sympatry of zone extensive from the documented been not have similar populations ar why (2016) reported both, intermediate flower morphology and volatile compound emission rates of emission ratesof volatileand compound morphology both, reportedintermediate flower (2016) . In constrast, constrast, . In M. laxa M. laxa M. laxa In our study system, olfactory signals may have contributed to the breakdown of ethological of ethological the contributedto breakdown have may signals system, study olfactory our In been have studies fewfield Relatively
(Polemoniaceae; primarily hummingbird pollinated) and and pollinated) hummingbird (Polemoniaceae; primarily flowers are characterized by high e high by characterized areflowers M. laxa M. laxa and hybrids putative the and Bischoff Bischoff
M. pentlandiana
of pollen movement/gene flow among parental species and putative hybrids hybrids putative and species parental among flow movement/gene pollen of moth M. laxa and putative hybrids emit compounds which are characteristic of hawkmoth of are which characteristic hybridsemit and putative compounds et al. - Zaluzianskya natalensis natalensis Zaluzianskya in the taxa the parental in pollinated plants (Kaiser 1993; Knudsen & Tollsten 1993; Raguso Raguso 1993; Knudsen &Tollsten 1993; plants (Kaiser pollinated et al.
2013; Raguso 2013; and and ;
(2014) described a similar system to our own, with with own, our asimilarto described system (2014) volatiles M. pentlandiana , extending northwards from Jujuy, Argentina into much of into Argentina Jujuy, , from northwards extending enotypes, and thus may have facilitated the initial initial may have facilitatedthe and thus enotypes, ,
allowing the breakdown of ethological barriers. It remains It remains barriers. ethological of the breakdown allowing - salicylate, salicylate, present in one or both both oftheparental or one present in et al. -
pollinated plants (D plants pollinated
(p conducted on floral scent in hybrid plants. hybrid floral scentin on conducted mission rates of the sesquiterpenoid ( themissionrates of sesquiterpenoid entan
2006; Klahre 2006; Klahre
and the subsequent unidirectional interbreeding interbreeding unidirectional subsequent and the and trans - 1 Z. microsiphon Z. - ol, lavender lactone, lactone, lavender ol, - DMNT and DMNT and
- et al. pollinated plants (Dobson 2006; 2006; (Dobson plants pollinated ö tterl & Vereecken 2010), but it butit 2010), tterl &Vereecken I. tenuituba I. eir study, eir study,
2011), bu 2011),
trans (Orobanchaceae) in South South in (Orobanchaceae) - hybrid hybrid TMTT, all of of TMTT,which all α
t they also produce alsoproduce t they (primarily hawkmoth(primarily - pinene). pinene). Ipomopsis Ipomopsis Ipomopsis E,E Ipomopsis Ipomopsis )
- Campbell α
- taxa M. et al. -
This article isThis article by protected copyright. All rightsreserved. Accepted of pollen point. setcou fruit later for and checked visit afterahummingbird bagged immediately flowers virgin using studies Future pollinatesome flowers. may eventually hummingbirds that be mismatch the pentlandiana (Moré flowers pollinate effectively that species, itwasfound hawkmoth spectrum of precise trait mechanismof pollination the revealed that study Aprevious plants. fertilize these visitor for most floral thepotential system diminishes that reproductive featuresacomplex family isolation, of ethological breakdown Rhodes Article 2006; & Sweet agents (Brunet outcrossing and vectors distance pollen as long advantages their Miller 2007; & Campbell Aldridge 1996; Búrquez (Willmott & (Moré flowersto of visitation hawkmoth poll driving units in floral of density the than responsible more traitsare system, in this floral that suggesting interchange, analogue pentlandiana them between (Fig. was stronger analogues pollen of themovement accordingly, not simply oroccur. do haveoverlooked, been populations introgressive determine to needed will be herbarium studies and field Extensive Bolivia. In relation to dipteran floral visitors, their their visitors, floral to dipteran Inrelation et al. et al Mandevilla Even though though Even laxa Mandevilla -
. 2017). . 2017). hawkmoth of in abundance isahallmark Spatiotemporal 2007). variation matching with the proboscis width of hawkmoth pollinators. Despite being visited by a by visited being Despite hawkmothpollinators. of width proboscis the with matching
flowers (pers. obs.). We suspect that hummingbirds would be nectar because be of thieves would hummingbirds We suspect that obs.). (pers. flowers lowest pollen the showed abundance, its bybeesdespite pollinated and, mainly was tween beak width and anther cone slit width. slitwidth. cone andanther width tween beak
flowers in our study. flowersour in olfactory signals an olfactory
and putative and en transfer dynamics. Previous studies led us to expect higher levels of levels of expectto higher studies us led Previous dynamics. en transfer M. laxa et al. from the point of view of mechanical isolation, the Apocynaceae the isolation, mechanical of view from of point the hybrids hybrids
2007). The same mechanism seems to operate in in same operate The to mechanism seems 2007). , which they effectively pollinate elsewhere in Argentina elsewhere Argentina in pollinate effectively they , which
d morphological intermediacy may have contributed to to the contributed have intermediacy may morphological d shared hummingbirds as main flower visitors and, and, visitors flower asmain hummingbirds shared proboscides are too short to reach either the nectar or nectaror reachthe to either short are too proboscides
only those with proboscises narrower than 0.7 mm 0.7 than narrower proboscises with only those However, we do not completely discard discard completely not However,we do
et al.
2014), which counterbalances which counterbalances 2014),
whether additional additional whether ld shed light on this this light shed ld on
4 b). Incontrast, b). 4 M. laxa pollination pollination M.
requires a requires s to s to M. This article isThis article by protected copyright. All rightsreserved. zones. Thehybrid dynamics of (1979) N.H. Barton Accepted pp. UK: 215 (1 M.L. Arnold Introgressive hybridization. (1953) E. Anderson rate. hybridization in differ sites that two preferencebetween Variationin pollinator (2007) Campbell G., D.R. Aldridge References DEB grant UnitedStates NSF by supported RAR were by Contributions CONICET. of are researchers SBV and MM Quebrada”. La Parque Natural Hídrica Secretaría about discussion CeciliaEzcurrafor thank We assistance. molecular laboratory for Laura Marquez Cabrera and Gerardo Salazar, Lidia andto work, field Federic Pisano, Graciela Pisano, Rubini Aluhé Rubini Pisano, Anouk Caviglia, LucreciaEstigarribia, Agustín Izquierdo, Juliana Bianca Bonaparte, Article Acknowledgements zone hybrid natural in consequences, behavioural their traits, and of segregating interpretation meaningful ( floral introgression of studies morphol and floralscent how assessing template for balancedof This kind generation. eachhybrid for scentchemistry and morphology floral baseline characterize to parents, the andbackcrossesto F2 hybrids and F1 crosses produce to controlled s.
Thanks to Marcela Palacio and Pablo Cortina (IMBIV) for assistance in GC in assistance for Cortina (IMBIV) Pablo Marcela and to Palacio Thanks Future studies on this systemthis studies on will Future
de Ambiente from Córdoba Province for allowing us to carry out this study in “Reserva in the this to out study us allowing carry for Province Córdoba de Ambientefrom
997)
Natural hybridization and evolution. and Natural hybridization
e.g.
Ippolito Evolution Evolution et al. evaluate the cytogenetic and genetic aspects, and thecytogenetic evaluate o Sazatornil and Ana Clara Ibañez for assistance during during assistance for Ana and Clara Ibañez Sazatornil o 61
Biological Reviews Reviews Biological 2004; Martin 2004; Martin , 99 Heredity ogy segregate is lacking from most relevant from most relevant islacking segregate ogy – - 110. 1342792. 1342792.
Oxford University Press, Oxford, Oxford University Mandevilla
43,
et al.
341
28, 2008), and should allow a more allowamore should and 2008), –
359. taxonomy. We also thank the the Wealsothank taxonomy.
280
– 307.
Ipomopsis - MS analysis, to to analysis, MS
along with with along
contact contact This article isThis article by protected copyright. All rightsreserved. foraging choices. on hawkmoth orientation flower volatilesand of species: theinfluence S.D Johnson A., Jürgens Campbell D.R., of flowers andevolution Ecology G. (2006) Aldridge Campbell D.R., rate. outc size display and on floral group insectpollinator Impactof (2006) Sweet H.R. J., Brunet pp. UK: 435 Press, Cambridge, University F.L.(1997) Bookstein Context (2015) floraland scent mediatedby isolation D.R. Campbell A., Jürgens R.A., Raguso M., Bischoff species. parental their and (Polemoniaceae) CampbellD.R. A., Jürgens M., Bischoff 2354 of shape Benitez Argentina Argentina). (Córdoba, sus alrededores LaQuebrada" y "Parque Natural Hídrica laReserva de Serrano enelBosque Sphingidae) (Lepidoptera: deesfíngidos Riqueza N Villafañe A.I., Zapata Beccacece H.M., 89 species of investigation for complexes. the recognition G. (2003) Simbolotti VillemantC., M., Baylac Systematics zones. Analysishybrid of (1985) HewittG.M. N.H., Barton
Accepted Article– 98. Evolution Evolution – 2362.
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16,
137 60,
113 –
234 140. – 148.
Morphometric tools for landmark data: geometry and biology geometry and data: landmark for tools Morphometric –
246.
, a sexually deceptive orchid. orchid. deceptive , asexually .
Oxford University Press, University Oxford
Floral biology of hybrid zones. In: Harder L.D., Barret S.C. (Eds) (Eds) BarretS.C. L.D., Harder In: zones. ofFloral hybrid biology
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105, – nguage and environment for statistical computing statistical for andenvironment nguage 372. Manduca sexta Manduca - tongued hawkmothtongued
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– This article isThis article by protected copyright. All rightsreserved. pp. 478 Amsterdam: Press, Elsevier/Academic aprimer. biologists: for Geometricmorphometrics (2012) Sheets H.D. D.L., M.L., Swiderski Zelditch 273, 1,8 synthase, ( Katahira E., T.J., Savage Wise M.L., mothsbe trusted? of Thepollination (1996) A. Búrquez Willmott A.P., two in isolation and reproductive Floral odour (2008) F.P. Schiestl A., Widmer J.K., Muhlemann Waelti M.O., deceptive orchids. sexually in Cozzolin N.J., Vereecken 423 variation. floral shape studying morphometrics for geometric dimensional (2010 Linder H.P. Johnson S.D., LeónM.S.P., de C.P., Zollikofer NietT., der van Journal of Botanical fruit setin Factorsconstraining (1999) L. Galetto C., Torres Acceptedofficinalis Salvia Article –
14891 426.
– 14899. - cineole synthase, and (+) synthase,cineole and species of species of ) cDNA isolation, characterization, and functional expression of (+) expression functional and characterization, isolation, ) cDNA American Journal of Botany Journal American
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1050 205. Hybrid floral scent novelty drives pollinator shift shift drives pollinator scentnovelty floral Hybrid Merremia palmeri Merremia
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This article isThis article by protected copyright. All rightsreserved. shape. theto consensus relation arein PC2 indicated and from PC1 values a finite circles, and individuals hybrid squares, putative the first the two by explained variation hybrids. Shape putative 2. Fig. cm. 1 to equal Scalebars morphometric analysis. geometric h 1. Fig. legends Figure Acceptedybrid Article a priori a . (c) . (c)
Flower shape variation within andbetween within Flower shapevariation (a) population. studied forms the flower Diversity in of Gaussian mixture analysis, and unsupervised learning algorithm. Maximum and minimum and algorithm. learning Maximum andunsupervised mixture analysis, Gaussian Mandevilla laxa. Mandevilla
classification, based on two relevant taxonomic floral traits: triangles, triangles, traits: floral taxonomic relevant two classification, basedon
Lower panels show the the panelsLower show M. laxa Mandevilla laxa, M. pentlandiana laxa,M.pentlandiana Mandevilla position of the 13 landmarks used for the for used landmarks the of 13 position . Grey polygons delimits groups according to according delimits groups polygons . Grey axes of a PCA (n = 62). Symbols indicate Symbols indicate =62). (n aPCA axes of
Mandevilla pentlandiana.
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This article isThis article by protected copyright. All rightsreserved. study. S1. Table fragrance. flower of composition and Method Material Supporting Information Supporting for andblack 190) for size:white sample the circles represents ar the thickness of The visitors. flower χ to contribution signed Their powders. thatreceivedfluorescent flowers of number diagonal) (below expected and diagonal) (above observed Each c table. (a) Contingency pollen analogues. using hybrids, asvisualized putative 4. Fig. determined. been that Note analysis. SIMPER determined by space, as inMDS regions of other those and these loci to nearest samples the between differences in explaining compounds these of importance the in indicate NMDS) diamonds (small black t for loci The structures. associatedchemical and their compounds night symbols represent filled samples, daytime laxa circles, Symbolsindicate: analysis. dynamic headspace through collected scentthe compounds Bray on based al. Knudsen to according classes differentchemical into wereVOCs grouped bouquets. scent floral the to groups compound of different relativecontribution the diagrams Uppershowing Pie panel: hybrids. 3. Fig.
Accepted Article (2006). Medium panel: Non Medium (2006). panel: ; trian
Floral scent variation within and between Floral within scent variation Movement of flower visitors within and and between within Movement visitors flower of
gles, GenBank accession numbers and variable sites for all sitesfor variable numbersand accession GenBank - Curtis dissimilarities, which were calculated from the total standardized peak areas peak of standardized total from the were calculated Curtis dissimilarities, which M. pentlandiana M. pentlandiana
M. laxa M. laxa
(n= 44). (n= - metric multidimensio metric and squares, putative hybrid individuals. Open symbols represent Open individuals. symbols hybrid squares,and putative
s. Molecular analyses and characterization of the chemical ofthe characterization and analyses Molecular rows represents the observed number of visits. The size of the the sizeof The number visits. of observed the represents rows 2
(Pearson's residuals) is indicated. (b) Observed movement of movement of Observed (b) indicated. is residuals) (Pearson's
the position of the double bond in compound 9 has 9 compoundnot in the bond of double position the M. pentlandiana - time samples. Bottom panel: Identified floral floral volatile timesamples. Identified Bottom panel: Mandevilla laxa, M. pentlandiana M. laxa, pentlandiana Mandevilla nal scaling (NMDS) of the floral scent profile profile scent floral the of (NMDS) scaling nal Mandevilla laxa, M. pentlandiana M. pentlandiana laxa, Mandevilla
(n= 335); grey for putative hybrids (n = forhybrids(n grey 335); putative (n=
Mandevilla Mandevilla hese compounds in scentspacehese in compounds sequences used in this this sequences in used and their and their putat ell shows the the ell shows and their andtheir M. ive ive et This article isThis article by protected copyright. All rightsreserved. Accepted Ezcurra (2005). of measures typical the indicates typical dots Black taxa. any to assignable not individuals show dots S4. Figure S1. Table in shown sitesare polymorphic and numbers accession of below) bp S3. Figure Article S1. Table in are shown laxa Mandevilla S2. Figure triangle. awhite with isshown intermediatethe individuals, morphological we found population,where study The dots). S1. Figure system. study the fragrances in S3. Table system. study the fragrances in S2. Table Mandevilla laxa Volatile organic compounds (VOCs) detected using dynamic headspace using (VOCs) detected compounds Volatile organic S using (VOCs) detected compounds Volatile organic
Geographical distribution of distribution Geographical Linear measurements of two key floral traits (flower length and corolla lobe length). Open corollaOpen length). and lobe length floral traits(flower key measurements two Linear of and900 720 above, bp and and 660 alignment490 (between waxy Fragments GBSSI of of bp) 210 and 50 alignment(between Fragment ITS of Mandevilla pentlandiana, Mandevilla laxa Mandevilla pentlandiana, Mandevilla
and and putative hybrid individuals. Genbank accession numbers and polymorphic sites numbers and accession Genbank individuals. putative hybrid
measures (blue box) or in or in box) (blue measures
M. grata Mandevilla laxa Mandevilla , a third species supposed to be a hybrid according to ahybridaccording be speciesto , athird supposed M. pentlandiana
and putati and (blue dots) an (blue
show individuals that fit either in the the in that either individuals fit show
PME
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measures (orange box). Grey box Greybox box). measures (orange Mandevilla pentlandiana, pentlandiana, Mandevilla - GC d M. pentlandiana M. pentlandiana - MS analyses of floral of analyses MS - GC - MS of floral floral of MS (orange This article isThis article by protected copyright. All rightsreserved. Accepted Article
This article isThis article by protected copyright. All rightsreserved. Accepted Article
This article isThis article by protected copyright. All rightsreserved. Accepted Article
This article isThis article by protected copyright. All rightsreserved. Accepted Article