An olfactory receptor gene underlies reproductive isolation in perfume-collecting orchid bees

Speciation is facilitated by the evolution of reproductive barriers that prevent or reduce hybridization among diverging lineages. However, the genetic mechanisms that control the evolution of reproductive barriers remain elusive, particularly in natural populations. We identify a gene associated with divergence in chemical courtship signaling in a pair of nascent orchid bee lineages. Male orchid bees collect perfume compounds from flowers and other sources to subsequently expose during courtship display, thereby conveying information on species identity. We show that these two lineages exhibit differentiated perfume blends and that this change is associated with the rapid evolution of a single odorant receptor gene. Our study suggests that reproductive isolation evolved through divergence of a major barrier gene involved in chemically mediated pre-mating isolation via genetic coupling.


Speciation is facilitated by the evolution of reproductive barriers that prevent or reduce 22
hybridization among diverging lineages. However, the genetic mechanisms that control the 23 evolution of reproductive barriers remain elusive, particularly in natural populations. We 24 identify a gene associated with divergence in chemical courtship signaling in a pair of 25 nascent orchid bee lineages. Male orchid bees collect perfume compounds from flowers and 26 other sources to subsequently expose during courtship display, thereby conveying 27 information on species identity. We show that these two lineages exhibit differentiated 28 perfume blends and that this change is associated with the rapid evolution of a single 29 odorant receptor gene. Our study suggests that reproductive isolation evolved through 30 divergence of a major barrier gene involved in chemically mediated pre-mating isolation 31 via genetic coupling. 32 33 34 35 36 Speciation, the formation of new species from a single ancestral species, is facilitated by the 38 emergence of reproductive barriers between lineages and is considered the most fundamental 39 process in the generation of biological diversity (1, 2). While a growing number of studies have 40 revealed that recently formed species often exhibit marked divergence across multiple genomic 41 regions (3), the role of these genomic 'islands of divergence' in reproductive isolation remains 42 controversial (4-7). Even when specific genomic regions can be associated with reproductive 43 isolation, they usually encompass hundreds of genes of unknown function. As a result, few 44 studies have successfully linked specific genetic loci to reproductive barrier traits or determined 45 how they contribute to the speciation process (7-9). Here we combine a large-scale population 46 level approach with high-resolution genome-wide diversification analyses to identify the genetic 47 basis of a phenotypic trait that likely controls reproductive isolation in a pair of orchid bee 48 species. 49 50 Male orchid bees actively collect volatile chemical substances from floral and non-floral sources 51 to concoct highly species-specific perfume blends (10-12), which they subsequently expose 52 during ritualized courtship displays ( Fig. 1) (13, 14). The exact type of information conveyed 53 remains unknown, but perfumes are clearly involved in mating behavior and species recognition 54 (15). Because orchid bees acquire volatile chemicals directly from the environment, their 55 olfactory system is critical for both perfume concoction by males and perfume detection by 56 females, which effectively creates a strong linkage between male trait and female preference. 57 Thus, changes in genes underlying olfactory perception can simultaneously alter the male 58 perfume signal and the female perfume preference through pleiotropic effects (16), a scenario 59 that could lead to the rapid evolution of assortative mating (17, 18). We hypothesize that the 60 differentiation of chemosensory genes drives rapid shifts in chemical perfume composition 61 during the speciation process, facilitated by genetic coupling of male trait and the associated 62 female preference in orchid bees. 63

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To test whether perfume composition evolves rapidly during species formation, we conducted a 70 population-level analysis of perfume chemistry in a pair of orchid bee lineages (Euglossa 71 dilemma and E. viridissima) that diverged ~150,000 years ago (19). We collected male bees 72 across the entire geographical range of each lineage throughout Central America (Fig. 1, Table  73 S1-S2) and analyzed the perfume chemistry of 384 individuals via gas chromatography-mass 74 spectrometry (Table S2). A non-metric multidimensional scaling analysis revealed strong 75 differentiation of perfumes into two distinct lineage-specific chemical phenotypes independent of 76 geography (Fig. 2a, ANOSIM R=0.8, p=0.001). This pattern was driven by both quantitative and 77 qualitative differences of perfume chemistry and held true when using either the entire set of 78 compounds or the 40 most prevalent compounds (Fig. S1, Supplementary Text). This 79 observation supports the hypothesis that those compounds collected by a high number of 80 individuals play a critical role in perfume specificity and private signaling in orchid bees. 81 82 We found that the most striking difference in perfume chemistry between E. dilemma and E. 83 viridissima was the presence of two lineage-specific compounds that are highly prevalent and 84 present in high relative abundance. The compound HNDB (2-hydroxy-6-nona-1,3-dienyl-85 benzaldehyde, (16)) was only present in perfume blends of E. dilemma, and the compound L97 86 (lactone-derivative of linoleic acid, (20)) was only present in perfume blends of E. viridissima 87 ( Fig. S2-S3, Table S3). These two molecules accounted for the highest average proportion of 88 overall perfume content per species (relative abundance HNDB: 55%, L97: 37%, Table S4) and 89 together contributed to 46.3% of the chemical differentiation between E. dilemma and E. 90 viridissima (SIMPER analysis, Table S5). 91 Remarkably, perfume chemistry is the only trait that allows reliable identification and separation 93 of these lineages. Close examination of morphological traits revealed that the number of teeth on 94 the male mandible differs between the two lineages, with most but not all individuals segregating 95 into two groups (19). Males of E. dilemma always have three mandibular teeth, whereas males of 96 E. viridissima are polymorphic for the number of teeth with 89.4% of males exhibiting two teeth 97 and the remaining fraction (10.6%) exhibiting three teeth that are hardly distinguishable from E. 98 dilemma (Fig. S4, Supplementary Text). Together, these results demonstrate that species-99 specificity in perfume chemistry evolved rapidly through changes of few major compounds. 100 These observations are consistent with the hypothesis that perfume chemistry is a mating 101 recognition signal that functions as a pre-mating reproductive barrier among orchid bee lineages. To contrast the divergence we observed in perfume chemistry with genetic differentiation 114 between E. dilemma and E. viridissima, we genotyped 232 males sampled from across their 115 geographic ranges (Fig. 1, Table S2). A principal components analysis of genetic variance (PCA) 116 based on 16,369 single nucleotide polymorphisms (SNPs) revealed that these lineages are 117 genetically distinct in both allopatric and sympatric populations (Fig. 2b, Fig. S5). E. dilemma 118 and E. viridissima were not separated over a single PC axis (Fig. 2b, Fig. S6, Supplementary  119 Text), which is consistent with a scenario of incomplete genome-wide separation between 120 species. This observation was further supported by a genetic clustering analysis that first 121 separated geographically distinct populations within E. dilemma before it separated species 122 (ADMIXTURE, Fig. 2d, Fig. S7 (Fig. 3c), two of which exhibited a highly 152 skewed differential in nucleotide diversity towards E. dilemma (∆π, Fig. 3c), suggesting strong 153 unilateral positive selection in this lineage. We identified signatures of an E. dilemma-specific 154 selective sweep in one of these windows based on allele frequency spectra and haplotypes of the 155 three distinct genetic lineages (Fig. 4a). We did not identify any additional species-specific 156 sweeps (Fig. S10), highlighting that this 50 kb window contains a unique, locally restricted 157 signature of positive selection in E. dilemma. This observation is congruent with strong and 158 recent selective forces driving the divergence between E. dilemma and E. viridissima and 159 suggests that the identified genomic region harbors loci mediating reproductive isolation 160 between these nascent bee lineages.

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Close inspection of the selective sweep region revealed the presence of 14 genes (Fig. 4a) OR41, that we previously identified as divergent between Ed north and Ev (28) (Fig. 4a). This 181 suggests that OR41 evolved under strong positive selection in the common ancestor of E. 182 dilemma after or during the split between E. dilemma and E. viridissima. Re-sequencing of OR41 183 confirmed these results (N=47, Fig. 4b, Table S9-S10, Supplementary Text), and revealed that 184 the protein coding sequences were fixed for 19 substitutions between species, 17 of which were 185 non-synonymous leading to changes in the amino acid sequence of the resulting protein (Fig. 4c).

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A comparison with distantly related Euglossa species demonstrated that all fixed substitutions 187 were derived (Fig. S11)

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Our results show that a simple major phenotypic difference in a reproductive barrier trait 205 between two lineages in the early stages of speciation is maintained despite low genetic 206 differentiation and ongoing gene flow. Only strong selection can counteract such equalizing 207 mechanisms, highlighting the adaptive value of the species-specific major perfume compounds 208 in E. dilemma and E. viridissima. While genome-wide analyses often lack resolution to identify 209 the genes that control barrier traits (3, 6-9, 29), we were able to identify a single genetic locus of 210 adaptive interspecific divergence, leading to a unique opportunity to understand the genomic 211 landscape of speciation on a fine genetic scale in a non-model system. Our findings provide a 212 link between a discrete shift in perfume composition with a single olfactory receptor gene that 213 evolved under strong positive selection, linking a chemosensory barrier trait with an olfactory 214 gene. Perfume composition in orchid bees is intricately connected to the sense of smell (16, 30, 215 31). In fact, E. dilemma and E. viridissima are known to differ in the behavioral preference 216 towards and the sensory detection of HNDB (16) the major perfume compound in E. dilemma. 217 This observation lends support to the hypothesis that the 17 non-synonymous substitutions 218 present in OR41 in the E. dilemma lineage underlie functional changes in sensory perception 219 between species. Accordingly, the data presented here are consistent with the genetic coupling of 220 a reproductive trait and trait preference (17, 18, 32) that evolved through rapid divergent 221 selection in a single gene leading to speciation. 222 223