the online version of this article ( doi:10.1007/s10886 - 010 - 9821 - 3 ) contains supplementary material , which is available to authorized users . most insects rely on chemical compounds ( pheromones ) to identify and attract conspecific mates ( wyatt , 2003 ) . the chemical composition of mating pheromones may range from single molecules to diverse , complex mixtures ( symonds and elgar , 2004 , 2008 ; billeter et al . , 2009 ) . because mating pheromones usually consist of species - specific blends , they often mediate prezygotic reproductive isolation among closely related lineages ( card et al . , 1977 ; roelofs et al . , 2002 ) . in insects , mating pheromones are synthesized de novo or from sequestered precursors . in either case , both biotic and abiotic factors influence pheromonal chemical composition ( stennett and etges , 1997 ; groot et al . , 2009 ) . hence , an important question is how insect populations that colonize novel environments respond to environmental changes that influence mating pheromones . in addition , to understand the underlying mechanisms of pheromone evolution in insects , we must examine intraspecific phenotypic variation in pheromone chemistry across habitats , geographic regions , and genetic gradients . orchid ( or euglossine ) bees constitute a diverse lineage of neotropical insect pollinators ( ramrez , 2009 ; ramrez et al . , 2010 ) that are best known for their fragrance - collecting behavior ( darwin , 1862 ; dressler , 1968 ; dodson et al . , 1969 ) . male bees collect volatile compounds from multiple floral and non - floral sources that they use subsequently during courtship display ( dressler , 1982 ; eltz et al . , 2005b ) . males of all species of orchid bees ( 200 ) have enlarged hind - tibial organs in which they deposit perfumes that consist primarily of terpenoids and aromatic compounds ( williams and whitten , 1983 ; eltz et al . , 1999 ; male orchid bees land on the surface of fragrance sources and secrete large quantities of long - chain lipids from cephalic labial glands that dissolve the volatile compounds to be collected ( whitten et al . although it has not been confirmed directly via behavioral experiments , it is likely that perfume chemistry enables females to choose mates and/or discriminate non - specific males ( eltz et al . , 1999 , 2003 , 2005a ; bemb , 2004 ; zimmermann et al . , 2006 ) ( 2009a ) analyzed the chemical composition of the fragrances acquired by male orchid bees from 15 closely related sympatric species in the genus euglossa from central panama . they showed that related lineages of bees accumulate species - specific fragrance blends that have differentiated rapidly during lineage diversification , potentially in response to strong selection against hybridization or mating interference between species . previous studies also have shown that perfume variation tends to be lower within lineages than between lineages ( eltz et al . , 1999 , 2005a , 2008 ; zimmermann et al . , 2006 ) , even when individuals co - occur in the same habitat ( eltz et al . however , the extent of intraspecific perfume variation across geographic regions and genetic gradients has not yet been examined . male orchid bees may accidentally accumulate a considerable diversity of inactive ( noisy ) compounds during the process of perfume collection ( eltz et al . , 2005a ) , as they often collect volatiles from multiple sources , even at local scales ( pemberton and wheeler , 2006 ) . in addition , variation in the availability and abundance of specific chemicals among habitats may result in disparate fragrance phenotypes ( eltz et al . thus , orchid bees must deal with environmentally induced variability while maintaining a reliable chemical communication system . do conspecific male orchid bees collect similar compounds across disparate habitats ? can different chemical compounds serve homologous functions in distant populations ? we designed a comprehensive analysis of fragrance variation within and between interbreeding populations of a single species , euglossa aff . in particular , we focused on elucidating phenotypic change in the perfumes of e. aff . the green orchid bee euglossa viridissima is distributed throughout lowland dry forests in northern mesoamerica ( hinojosa - diaz et al . , 2009 ) , ranging in distribution from southern costa rica to northern mexico ( fig .  1 ) . previous taxonomic treatments recognized e. viridissima as a single distinct species ( e.g. , roubik and hanson , 2004 ) . however , a recent study of populations from the yucatan peninsula ( mexico ) showed instead that this lineage consists of two cryptic sister species ( eltz et al . , 2008 ) . the most prominent morphological difference between the two lineages is in the number of teeth on the mandible of males : two or three dentitions . viridissima ) collect large quantities of the compound 2-hydroxy-6-nona-1,3-dienyl - benzaldehyde ( abbreviated hndb ) , males with two dentitions ( e. viridissima ) lack hndb in their perfumes ( eltz et al . , 2008 ) . a detailed systematic analysis of these lineages based on morphology , chemistry , and genetic data is in preparation ( eltz et al . , native range populations in mesoamerica were el chote , monte pio , el remate , xmatkuil and acg . naturalized populations in the united states were gumbo - limbo and fern forest map of mesoamerica and southern united states indicating sampling localities where male euglossa aff . native range populations in mesoamerica were el chote , monte pio , el remate , xmatkuil and acg . naturalized populations in the united states were gumbo - limbo and fern forest as early as 2003 , bees of e. aff . viridissima were introduced accidentally and naturalized in southeastern united states ( skov and wiley , 2005 ) . since then , vigorous populations have become established throughout most of the southern coastal peninsula of florida ( pemberton and liu , 2008 ; liu and pemberton , 2009 ; pemberton and ramrez , personal observation ) . a chemical analysis of the perfumes of naturalized bees from florida showed that male bees collect multiple fragrance compounds from several plant species , including native , naturalized , and horticultural plants ( pemberton and wheeler , 2006 ) . however , the composition of these fragrances has not been compared with that of native e. aff . viridissima across its native range in mesoamerica and its introduced range in florida ( u.s . ) . we estimated the magnitude of intraspecific geographical variation in fragrance composition and identified volatile compounds that vary within and between bee populations . viridissima to the united states may have resulted in both a genetic bottleneck and a habitat shift , we asked whether changes in perfume chemistry have accompanied the naturalization of this lineage of bees . viridissima lineage were collected from five localities across the native range in costa rica and mexico , and two localities in the introduced range in the u.s . ( florida ) in 2008 and 2009 ( fig .  1 ; supplementary material appendix  1 ) . male bees were lured with synthetic chemical baits consisting of squares of blotter paper impregnated with p - dimethoxybenzene , methyl cinnamate , or eugenol ( sigma - aldrich , st . the impregnated paper squares were suspended 1.5  m aboveground and covered with screen mesh to prevent male bees from accessing the bait . males were captured with hand nets and kept in the shade inside screen cages until subsequent dissection , typically conducted the same day.florida populations were sampled at two natural area preserves . fern forest ( broward county ) is primarily a tropical hardwood forest grading into freshwater cypress swamp and upland pine habitats . gumbo - limbo nature center ( palm beach county ) is located on a barrier island with a tropical hardwood forest and mangroves . mesoamerican populations were sampled from various habitats , including secondary seasonally dry tropical forest ( acg ) , secondary seasonally dry scrubland ( xmatkuil ) , farmland surrounded by stretches of tropical perennial forest ( el chote ) , settlement adjacent to tropical perennial forest ( monte pio ) , and coastal mangroves ( el remate ) . fragrance extraction and chemical analysis male bees were cold - anaesthetized either on ice or inside a freezer ( 20c ) for 5  min immediately prior to dissection . right hind tibiae were removed with clean dissecting scissors and deposited in 2  ml screw - cap autosampler vials ( agilent technologies , santa clara , ca , usa ) , which contained 500  l of hexane to extract the fragrances . to distinguish between exogenous volatiles and endogenous male - produced lipids , acetates , and straight - chain hydrocarbon compounds , labial glands were dissected , extracted , and analyzed from a subset of individuals ( n  =  43 ) using the same protocol described above . all compounds present in both leg extracts and labial glands were excluded from the analysis ( fig .  2 ) . voucher specimens of all samples were either pinned or deposited in 200 proof ethanol . 2overlaid total ion current chromatograms corresponding to hind - leg ( black ) and labial gland ( grey ) extracts from the same individual bee . compounds present in both hind legs and labial glands were considered endogenous in origin and thus were excluded from the analysis . the y axis indicates ion abundancegas chromatography / mass spectrometry ( gc / ms ) was conducted at the department of environmental sciences , policy and management at the university of california berkeley , using a 7890a agilent gc coupled with a 5975c agilent mass selective detector ( agilent technologies , santa clara , ca , usa ) . the gc was fitted with a 30  m long , 0.25  mm internal diam , non - polar agilent hp-5ms capillary column ( cat . # sample aliquots of 1  l were injected by using an agilent 7683b automatic injector operated in splitless mode . the oven temperature was programmed from 60 to 300c at 3c / min , using helium as carrier gas with a constant flow rate set to 0.7  ml / min . mass selective detector ( msd ) scanning parameters ranged from 50 to 550  amu , with a sampling rate of 2.91 scans / sec and a threshold detection of 150 counts.chemical analyses revealed the presence of triclopyr 2-butoxyethyl ester ( hereafter triclopyr bee ) , in florida populations only . viridissima were actively attracted to this compound , we performed attraction bioassays with both captive and wild bees . captive bees were nest - trapped in florida ( august 2009 ) and reared at uc berkeley in an insectary facility maintained at 25c , 4060% humidity , and kept under a 12/12  h light / dark cycle . male bees were not exposed to any fragrances prior to the bioassay , in which we presented male bees with hind leg extracts that contained large quantities of triclopyr bee and as little as possible of other volatile compounds . we applied 100  l of the hexane extract to clean blotter paper squares placed inside a flight cage where the bees were reared . as an experimental control , we presented male bees with labial gland extracts from the same individuals for which fragrance extracts were selected in the experimental treatment . the number of bees displaying fragrance gathering behavior within the first 2  min was recorded.in addition , to test whether male e. aff . viridissima from native - range populations also were attracted to triclopyr bee , we purified this compound from leg extracts using a preparative gc system similar to that of nojima et al . a megabore db-5 capillary column ( 30  m , 0.53  mm i d , 0.5  um film thickness ) was used to separate compounds in an hp 5890 ii gas chromatograph ( 50 to 300c at 10/min ) . injection was on column , with a 30  m retention gap preceding the actual analytical column . the peak of triclopyr bee was captured by using a 40  cm piece column trap ( hp-1 , 0.53 i d ) that was connected to the preparative outlet at the time of elution . the substance retained in the trap repeated runs yielded approximately 75  g of purified triclopyr bee diluted in 1  ml of n - hexane . the bioassay was conducted in a disturbed dry forest near xmatkuil ( merida , mexico ) during the morning of 14 march , 2010 . we applied 200  l of isolated triclopyr bee in n - hexane ( 15  g ) to filter paper ( whatman 1 , 2  cm ) pinned to a small tree at breast height . in the vicinity ( > 4  m away ) , we also exposed two p - dimethoxybenzene baits , which were visited by dozens of male e. aff . bees that landed on the triclopyr bee filter paper were observed briefly to verify volatile collection . overlaid total ion current chromatograms corresponding to hind - leg ( black ) and labial gland ( grey ) extracts from the same individual bee . compounds present in both hind legs and labial glands were considered endogenous in origin and thus were excluded from the analysis . the y axis indicates ion abundance compound characterization we used the software chemstation ve.02.00 ( agilent technologies ) to register chromatogram peaks and save their corresponding spectra in user - built mass - spectral libraries ( ramrez and eltz , unpublished ) which we use to cross - reference additional chromatograms . we further characterized individual compounds by comparing spectra against several published libraries ( adams , 2001 ; pal600k , palisade corporation , usa ) . louis , mo , usa ) , injected and analyzed under the same conditions and instrument described above ( uc berkeley ) , were also used to corroborate compound identities ( table  1 ) . uncharacterized compounds were named based on retention times and ei - mass spectrum ions ( table  1 ) . automatic peak integration was conducted using the rte integrator in the software chemstation ve.02.00 ( agilent technologies ) set to a minimum - area detection threshold equivalent to 0.5% of the area of the largest peak . viridissima ranked by their incidence ( % ) across populationscompound namecompound classretention time ( min)entry # incidence ( % ) contribution to dissimilarity ( % ) usa vs. mesoamericaeugenolphenylpropanoid23.679164798.542-hydroxy-6-nona-1,3-dienyl - benzaldehyde 4 ( hndb4)aromate50.698547419.04caryophyllenesesquiterpene26.288173682.60isolemecin , trans - na35.38861502.25ocimene , beta - monoterpene10.547199443.44benzyl benzoatearomate39.306218423.002-hydroxy-6-nona-1,3-dienyl - benzaldehyde 1 ( hndb1)aromate45.62851411.65m / z:55,69,81,95,107,119,135,147,161,175,189,207,218,426triterpene80.67559401.252-hydroxy-6-nona-1,3-dienyl - benzaldehyde 3 ( hndb3)aromate48.89053382.27m / z:53,65,74,92,120,155unknown16.572373361.17humulene , alpha - sesquiterpene27.673165361.22cadinene , delta - sesquiterpene30.465110350.07benzyl cinnamatena49.863208343.48m / z:55,68,81,93,107,121,133,147,161,175,189,204sesquiterpene25.149245321.26pinene , alpha - monoterpene6.639174280.61m / z:55,69,81,95,107,119,135,147,161,175,189,203,218unknown79.337127260.253-cyclohexane-1ol , 4-methyl-1 m ethylethylmonoterpene15.926180250.232-hydroxy-6-nona-1,3-dienyl - benzaldehyde 2 ( hndb2)aromate47.23152251.18triclopyr 2-butoxyethyl esteraromate53.553762517.37germacrene , dsesquiterpene28.794359250.69similar to elemicinaromate31.781413250.48similar to amorphene , alpha - sesquiterpene30.10545230.29similar to elemicinunknown31.75349230.33similar to amyrin , alpha - triterpene79.800424230.551,4-dimethoxybenzene , aromate15.308179190.14cubene , beta - sesquiterpene28.789194191.03epizonarenesesquiterpene29.50443180.45m / z:55,115,127,173,183,201,215,228,244,269,283,293,311,326,344unknown64.722333180.28cineole , 1,8-monoterpene9.906255180.23copaene , alpha - sesquiterpene24.486182180.20methyl esterna36.067235181.79myrcene , beta - monoterpene8.424254170.25bisabolene , beta - sesquiterpene29.922378160.82pinene , beta - monoterpene7.989175150.30barbatene , beta - sesquiterpene27.227188150.17m / z:55,67,77,81,91,105,119,133,161,204sesquiterpene28.068193150.13m / z:65,77,91,115,129,147,175,244,260unknown51.76672150.26m / z:55,71,99,115,128,141,157,171,186,200,213,228,245,301,326unknown62.691311150.10m / z:55,69,83,95,108,115,127,145,159,173,183,201,215,225,244,253,285,299,324,342unknown66.279334150.16similar to amorphene , alpha - sesquiterpene28.611161140.15similarity percentage ( simper ) was used to calculate the relative contribution of each compound to the observed dissimilarity between us and mesoamerican populations . 1% are indicated in bold most common exogenous fragrance compounds collected by male euglossa aff . viridissima ranked by their incidence ( % ) across populations similarity percentage ( simper ) was used to calculate the relative contribution of each compound to the observed dissimilarity between us and mesoamerican populations . 1% are indicated in bold statistical methods we built a square matrix containing the absolute quantities ( total ion currents ) for each compound , containing all samples from all localities . we compared individual chemical profiles within and between populations via non - metric multidimensional scaling ( mds ) , an ordination technique where a predetermined number of axes of variation are chosen , and non - metric distances are fitted to those dimensions . because ordination via mds makes few assumptions about the nature of the data , any distance measure can be applied . we calculated a triangular distance matrix between samples ( individuals ) using the bray - curtis index of dissimilarity , which has the advantage of being insensitive to compounds jointly absent in sample pairs ( i.e. , pairwise dissimilarities are fixed ) . we computed 2- and 3-dimensional mds plots ( 50 iterations per run ) using the software package ecodist v1.2.2 ( written in r ) . to assess statistically whether orchid bee fragrances exhibit greater dissimilarity between populations than within populations , we conducted an analysis of similarity ( anosim ) test , implemented in the software package vegan v1.15 - 4 ( written in r ) . additional descriptive statistics , tests , and plots were produced using r basic packages , freely available at www.cran.r-project.org . we estimated the relative contribution of individual compounds to the observed ordinal dissimilarities using the similarity percentage ( simper ) method , implemented in the software package primer v6 ( clarke and gorley , 2006 ) . we registered a total of 333 compounds in hind leg extracts of 114 males sampled from seven populations ( fig .  1 ) . by comparing the chemical profiles of hind leg extracts against those from labial glands ( fig . 2 ) we determined that 41 compounds were produced in the bees labial glands ( endogenous origin ) , and included straight chain hydrocarbons , acetates , diacetates , and alcohols ( supplementary material appendix  2 ) . the remaining 292 compounds are thus exogenous in origin , and included monoterpenes , sequiterpenes , bicyclic sesquiterpenes , triterpenes , and other compounds ( table  1 ) . the per - capita number of exogenous compounds across all populations ranged from 1 to 65 chemicals ( average 21.50    12.66 ) with marginally similar means in all seven populations ( anova f  =  3.2118 , p  =  0.075 ) . on average , male bees from fern forest ( u.s . ) exhibited the lowest number of compounds per capita ( 15.83    10.08 ) , whereas el remate ( mexico ) exhibited the highest number ( 29.23    6.24 ; fig . populations were combined , they had significantly fewer compounds per individual ( 17.46    10.78 ) than all mesoamerican populations combined ( 24.14    13.17 ; anova f  =  8.0419 , p  =  0.005 ) . 3boxplots of the number of volatile exogenous compounds per capita in fragrances of male euglossa aff . viridissima in native and introduced populations boxplots of the number of volatile exogenous compounds per capita in fragrances of male euglossa aff . viridissima in native and introduced populations our non - metric multidimensional scaling ( mds ) analysis included exogenous compounds only . when all volatile compounds in the data matrix were included ( coded by relative abundances ) , a strong clustering by population was found ( fig . this observation was corroborated by the anosim results , which indicated that fragrance dissimilarity within populations was lower than among populations ( r  =  0.433 , p  <  0.001 ) . we found a similar pattern when we excluded the two compounds with the highest contribution to the dissimilarity between u.s . and mesoamerica ( fig . 4b ; see simper analysis below ) , namely hndb4 ( one of four stereoisomers ) and triclopyr bee . likewise , mds ordinations based only on the 50 compounds with the highest incidence ranks across all populations ( fig . 4c ) , and the entire dataset transformed into discrete binary ( presence / absence ) characters ( fig . in addition , regardless of which data partition we used , we found that introduced ( u.s . ) and native ( mesoamerica ) populations were strongly differentiated ( fig . 4 ; anosim populations not pooled : r  =  0.483 , p  <  0.001 ) . to explore the possibility that per - capita compound diversity ( number of compounds ) affected population differences , we overlaid a bubble - plot on an mds ordination ( based on all fragrance data ) with circle diameters proportional to the number of compounds per capita ( fig .  5 ) . 3 ) , populations did not appear to cluster based on fragrance diversity alone ( fig . 4non - metric multidimensional scaling ( nmds ) plots based on the chemical composition of exogenous compounds present in hind legs of male euglossa aff . ordination plots were computed based on a all volatile compounds , b all volatile compounds except the two most dissimilar between u.s . and mesoamerican populations ( i.e. , hndb4 , triclopyr bee ) , c the 50 compounds with highest incidence across all populations , and d all volatile compounds coded as binary characters ( presence / absence ) . filled circles and triangles correspond to individuals from naturalized populationsfig . circle diameters correspond to the number of volatile exogenous compounds present in the fragrances of each individual . filled circles and triangles correspond to individuals from naturalized populations non - metric multidimensional scaling ( nmds ) plots based on the chemical composition of exogenous compounds present in hind legs of male euglossa aff . ordination plots were computed based on a all volatile compounds , b all volatile compounds except the two most dissimilar between u.s . and mesoamerican populations ( i.e. , hndb4 , triclopyr bee ) , c the 50 compounds with highest incidence across all populations , and d all volatile compounds coded as binary characters ( presence / absence ) . filled circles and triangles correspond to individuals from naturalized populations bubble plot overlaid on an mds ordination based on all exogenous fragrance compounds . circle diameters correspond to the number of volatile exogenous compounds present in the fragrances of each individual . analyses were performed using the software primer ( clarke and gorley 2006 ) . filled circles and triangles correspond to individuals from naturalized populations our simper analysis revealed that five compounds jointly contributed to > 50% of the observed chemical dissimilarity between populations from u.s . and mesoamerica ( table  1 ) . these included hndb4 ( 19.04% ) , triclopyr bee ( 17.37% ) , eugenol ( 8.54% ) , benzyl cinnamate ( 3.48% ) , and beta - ocimene ( 3.41% ) . with the exception of triclopyr bee , which was present only in u.s . populations , these compounds exhibited high incidence values across all populations ( table  1 ) . the simper analysis also revealed that of the ten most dissimilar compounds between u.s . and mesoamerica , eight were present in both population sets ( table  1 ; fig . 6 except panels f , j ) . of these , six were present in more individuals and had greater relative quantities in mesoamerican than in u.s . the two remaining compounds ( eugenol and caryophyllene ) were present in more individuals and had greater relative amounts in u.s . triclopyr bee and benzyl benzoate were present only in u.s . and mesoamerica , respectively ( fig . bees , and it was detected in 63% of individuals ( n  =  43 ) . benzyl benzoate contributed up to 17.84% of the fragrance composition in individual bees , and it was detected in 18% of mesoamerican individuals 6boxplots of per - capita relative concentration ( relative amount of each compound over all compounds ) of ten chemicals detected in bees sampled from native ( mesoamerica ) and naturalized ( u.s . ) these ten compounds had the greatest contributions to dissimilarity between native and naturalized populations boxplots of per - capita relative concentration ( relative amount of each compound over all compounds ) of ten chemicals detected in bees sampled from native ( mesoamerica ) and naturalized ( u.s . ) populations . these ten compounds had the greatest contributions to dissimilarity between native and naturalized populations we also conducted a simper analysis among all seven populations . in 15 of the 21 possible pairwise comparisons , five compounds jointly explained > 50% of the observed fragrance dissimilarity ( supplementary material appendix  3 ) . in all but two pairwise comparisons , el remate ( mexico ) v. acg ( costa rica ) and el remate v. monte pio ( mexico ) , the compounds hndb4 and eugenol ranked among the top five compounds with highest dissimilarity between populations ( supplementary material appendix  3 ) . sesquiterpene compounds , followed by monoterpenes , contributed the most to the observed dissimilarity between populations ( table  1 ; online appendix  3 ) . viridissima ( eltz et al . , 2008 ) , four stereoisomers of the compound hndb were found in male perfumes ( supplementary material fig .  1 ) . the compound hndb4 was by far the most abundant stereoisomer in the majority of populations , except in el remate ( mexico ) . the fragrances of all individuals in el remate had greater relative amounts of the stereoisomers hndb1 , hndb2 , and hndb3 than all other populations . in some cases , other stereoisomers were more abundant than hndb4 itself ( supplmentary material fig .  1 ) . because our spectral searches against published libraries did not return any significant matches for triclopyr bee , the identification of this compound was conducted by comparison against analytical standards provided by dow agrosciences ( indianapolis , in , us ) . both the retention time and ei - spectrum of the analytical standard of triclopyr 2-butoxyethyl ester ( triclopyr bee ) perfectly matched peaks in our samples ( fig . viridissima were observed displaying fragrance collecting behavior on areas where the herbicide garlon had been applied in fern forest ( pemberton , pers . triclopyr bee is one of three available active ingredients present in commercially available herbicides ( including garlon ) used for broadleaf weed control . the other active ingredients are triclopyr acid and triclopyr triethylamine salt ( tea ) . 
 fig . 7mass spectrum of triclopyr 2-butoxyethyl ester , a prevalent compound found in leg extracts of males from naturalized populations of euglossa aff . viridissima in florida ( u.s . ) and absent in native ( mesoamerican ) populations mass spectrum of triclopyr 2-butoxyethyl ester , a prevalent compound found in leg extracts of males from naturalized populations of euglossa aff . viridissima in florida ( u.s . ) and absent in native ( mesoamerican ) populations in our laboratory bioassay , we presented 20 male bees with three hexane leg extracts containing large amounts of triclopyr bee and trace amounts of other volatile compounds . within the first two minutes of exposure , six , five , and six individual bees exhibited obvious collecting behavior in the three trials , respectively . controls ( labial lipid extracts ) did not elicit fragrance - collecting behavior in any of the trials . our bioassay using purified aliquots of triclopyr bee with native bee populations in the field revealed that male e. aff . viridissima approached the filter paper with triclopyr bee , landed on it , and performed typical collection behavior . euglossine bees acquire species - specific fragrance bouquets from a great diversity of floral and non - floral sources ( williams , 1982 ; whitten et al . , 1993 ; ramrez et al . , 2002 ; pemberton and wheeler , 2006 ; zimmermann et al individual perfume phenotypes may not be attributable to single sources , but instead , emerge from multiple visits to numerous hosts ( eltz et al . although sympatric species of orchid bees , and particularly closely related lineages ( eltz et al . , 1999 ; zimmermann et al . , 2006 , 2009a ) , exhibit divergent perfume phenotypes , it has been unclear to what extent fragrances vary across geographic regions within lineages . viridissima maintain most of the individual compounds of their fragrance phenotypes across distant populations in disparate habitats , but a few major ( abundant ) compounds can be present or absent from perfume bouquets . viridissima in florida have been stable for at least seven years , our data suggest that the observed phenotypic changes have negligible effects on population viability . this observation is congruent with the previous report that male euglossa tridentata and e. erythrochlora sampled in isla del cao , an island 17  km off the coast of costa rica , collect substantially fewer compounds per capita than in mainland populations ( eltz et al . the remarkable qualitative consistency we found in fragrance phenotypes is at odds with the considerable habitat variability among the seven sites sampled , but supports the hypothesis that orchid bee perfume preferences are under strong selection ( zimmermann et al . , 2009a ) . the differences in perfume composition that we observed between native and naturalized populations could be attributed to environmental factors , to differences in chemical preference between populations , or to both . first , species turnover ( beta diversity ) of fragrance hosts across habitats may force phenotypic changes via chemical abundance and availability . in fact , a recent survey of volatile compounds present in resins of amazonian trees revealed not only a vast diversity of monoterpenes and sesquiterpenes , but also pronounced chemical turnover across tree species from different habitats ( courtois et al . , 2009 ) . second , strong female preference for certain fragrance phenotypes may restrict the amount of perfume phenotypic plasticity . although direct evidence for female choice of fragrance phenotypes is still missing for orchid bees , perfume phenotypes are likely to be under strong sexual selection ( zimmermann et al . , 2009a , b ) . hence , it is also conceivable that , at evolutionary timescales , new male fragrance phenotypes may emerge through novel female preferences for perfumes that indicate mate genotypic quality or simply exploit a sensory bias ( andersson , 1994 ) . unpublished data ) , and therefore it is possible that population differences in perfume phenotypes have an underlying genetic basis . rapid evolutionary change in pheromone phenotypes has been demonstrated in several species of insects ( lofstedt , 1993 ; takanashi et al . , 2005 ; groot et al . , viridissima is broadly sympatric with its sister taxon ( e. viridissima ) throughout most of mesoamerica ( eltz et al . the main difference in the perfumes of these two lineages is the presence of hndb in e. aff . viridissima and its complete absence in e. viridissima ( eltz et al . , 2008 , unpublished data ) . our data corroborated the presence of hndb as a major fragrance compound in both native and naturalized populations of e. aff . viridissima and , interestingly , showed that the four known structural stereoisomers of hndb ( eltz et al . , 2008 ) highly variable ratios of hndb stereoisomers were found only in one population ( el remate , mexico ) . although the source of hndb remains unknown , this either suggests that bees acquire hndb from the same host in both native and introduced populations , but from a different source in el remate , or that environmental differences between el remate and the other sites induced changes in the production of hndb stereoisomers by the same host . the chemical structure of hndb is unique , but resembles compounds produced by phytopathological fungi ( suzuki et al . , the presence of large quantities of triclopyr bee in leg extracts of naturalized bee populations , together with our field and laboratory bioassays , demonstrates that male bees are attracted to and actively collect this herbicide active ingredient . indeed , previous studies have shown that males of the orchid bee eufriesea purpurata from brazil collect large quantities of the synthetic pesticide ddt ( roberts et al . , 1982 ; walter and roberts , 2007 ) . although we did not detect triclopyr bee in leg extracts from native populations , male bees from mesoamerica clearly were attracted to this compound . this result suggests that a pre - existing sensory bias , rather than rapid adaptation among introduced populations , may explain the collection of organohalogen synthetic chemicals by male orchid bees . both behavioral and neurophysiological approaches are needed to elucidate the sensory basis of this unusual behavior . viridissima collect a broad diversity of molecules in native and introduced populations . as shown previously for orchid bees ( e.g. , williams and whitten , 1983 ; eltz et al . , 1999 , 2005a ; we found that perfume compounds that differed the most in abundance among populations also tended to be common to all populations . for example , whereas eugenol and hndb were present in > 73% of the sampled individuals and contributed significantly to population dissimilarity , most rare compounds contributed little to population dissimilarity . despite contributing little to overall population differences , rare compounds were diverse in our dataset . viridissima , but most of them were present in few individuals and in low concentrations . because triterpenes exhibit high molecular weights ( > 400 ) , and thus may not volatilize at ambient temperature , they may constitute poor airborne signaling molecules . we speculate that orchid bees actually collect triterpenes accidentally since they are common components of tree resins ( e.g. , burseraceae ) , which are known to contain numerous volatile compounds ( de la cruz - caizares et al . , 2005 ; courtois et al . , 2009 ) . hence , most of the compounds that were abundant in our dataset , such as sesquiterpenes , monoterpenes , and aromates , contributed the most to population differences , and also are likely to constitute the main courtship signaling molecules in orchid bees . in summary , the preference for most fragrance compounds acquired by orchid bees appears to be under strong selection . evidence for this comes from the fact that male bees managed to collect similar compounds across disparate habitats ranging from tropical rain forests to tropical dry forests , to mangroves , to disturbed habitats . however , we also found evidence for pronounced quantitative and qualitative changes across geographic ( and possibly genetic ) gradients . previous studies have suggested that saltational changes in fragrance preferences may constitute an important mechanism of evolution of perfume phenotypes as well as speciation in orchid bees ( eltz et al . , 2008 ; zimmermann et al . , 2009a ) . because mutations that affect the olfactory system of orchid bees can induce the acquisition or loss of specific chemicals , perfume phenotypes have the potential to evolve fast enough to facilitate lineage differentiation ( eltz et al . , 2008 our analysis highlights the opportunistic capacity that orchid bees have to incorporate novel compounds in their perfumes , as illustrated by the gain and loss of several compounds in naturalized populations . whether population - level changes in perfume phenotypes have underlying genetic components , and are thus subject to selection , should be a fruitful area of future research .