DOI: 10.5061/dryad.698s5
title: Data from: Amino acid change in an orchid desaturase enables mimicry of the pollinator’s sex pheromone

description: Mimicry illustrates the power of selection to produce phenotypic
 convergence in biology [ 1 ]. A striking example is the imitation of
 female insects by plants that are pollinated by sexual deception of males
 of the same insect species [ 2–4 ]. This involves mimicry of visual,
 tactile, and chemical signals of females [ 2–7 ], especially their sex
 pheromones [ 8–11 ]. The Mediterranean orchid Ophrys exaltata employs
 chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in
 an insect sex pheromone to attract and elicit mating behavior in its
 pollinators, males of the cellophane bee Colletes cunicularius [ 11–13 ].
 A difference in alkene double-bond positions is responsible for
 reproductive isolation between O. exaltata and closely related species,
 such as O. sphegodes [ 13–16 ]. We show that these 7-alkenes are likely
 determined by the action of the stearoyl-acyl-carrier-protein desaturase
 (SAD) homolog SAD5. After gene duplication, changes in subcellular
 localization relative to the ancestral housekeeping desaturase may have
 allowed proto-SAD5’s reaction products to undergo further biosynthesis to
 both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes
 may have led to pollinator-mediated deleterious pleiotropy. Despite
 possible evolutionary intermediates with reduced activity, amino acid
 changes at the bottom of the substrate-binding cavity have conferred
 enzyme specificity for 7-alkene biosynthesis by preventing the binding of
 longer-chained fatty acid (FA) precursors by the enzyme. This change in
 desaturase function enabled the orchid to perfect its chemical mimicry of
 pollinator sex pheromones by escape from deleterious pleiotropy,
 supporting a role of pleiotropy in determining the possible trajectories
 of adaptive evolution.
description: RNA in situ
 hybridization of SAD2-clade desaturases in Ophrys
 flowers.Additional data for
 Figure S1: RNA in situ hybridization of SAD2-clade desaturases (SAD1 and
 SAD2) in Ophrys flowers. (A) Overview illustration showing O. exaltata and
 O. sphegodes flowers from top and front along with the layout and
 direction of view of thin sections (starting at the base of the labellum
 on top toward the apex at the bottom). (B) Two thin sections of O.
 exaltata labellum, at 3 and 5 mm from the base (top) of the labellum. (C)
 From top to bottom, O. sphegodes labellum thin sections at 1, 3, 5, 7 and
 10 mm from the base (top) of the labellum. The antisense panels show
 blackish signal in the epidermal layer of O. sphegodes that is clearly
 stronger than the brownish background signal observed in the sense
 controls. However, no such difference was observed between sense and
 antisense signals in O. exaltata epidermis. The difference between O.
 sphegodes and O. exaltata is consistent with previous gene expression
 analyses [1, 2] that show SAD2-clade genes to be highly expressed in
 mature O. sphegodes labellum, whereas expression in O. exaltata labellum
 was low. The higher level of SAD2 expression as compared to SAD1
 expression in O. sphegodes [1, 2] suggests that ISH signal in panel (C) is
 primarily due to SAD2. References: 1. Xu et al. (2012). PLoS Genet. 8,
 e1002889. 2. Schlüter et al. (2011). Proc. Natl. Acad. Sci. USA 108,
 5696-5701.ISH-data-1_Sph-SAD2+Exa-SAD2.pdfRNA in situ hybridization of SAD3 in Ophrys flowers.Additional data for Figure S1: RNA in situ hybridization of SAD3 (the only SAD3-clade member) in Ophrys flowers. The probes were hybridized (A) with four thin sections of O. exaltata at 1, 3, 5 and 7 mm and (B) with two sections of O. sphegodes at 5 and 7 mm from the base of the lip. Antisense signal was detected in the epidermal layer of both species as compared to the sense control, although this is much clearer in panel (A). Previous gene expression analyses showed SAD3 to be ubiquitously expressed in different plant tissues and developmental stages, suggesting it to encode a housekeeping desaturase [1, 2], and these expression data are consistent with ISH despite low signal intensity in panel (B). It should be noted that qualitative absence of clear antisense signal (e.g. in non-epidermal layers) cannot be taken as evidence of absence of SAD3 expression there. References: 1. Xu et al. (2012). PLoS Genet. 8, e1002889. 2. Schlüter et al. (2011). Proc. Natl. Acad. Sci. USA 108, 5696-5701.ISH-data-2_Sph-SAD3+Exa-SAD3.pdfRNA in situ hybridization of SAD5-clade desaturases in Ophrys flowers.Additional data for Figure S1: RNA in situ hybridization of SAD5-clade desaturases (SAD5 and SAD6) in Ophrys flowers. Panel (A) shows thin sections of two individuals of O. exaltata and (B) one O. sphegodes individual; for each individual, two sections at 5 and 7 mm from the base (top) of the labellum were analysed. SAD5 antisense probe and sense control are shown for O. sphegodes. The antisense panels show black signal in the epidermal layer of O. exaltata (panel A), but not in O. sphegodes (panel B); O. exaltata antisense signal (panel A) is clearly stronger than the brownish background seen with the sense and antisense probes in O. sphegodes (panel B). This is consistent with the expression pattern reported for SAD5 [1], which is highly expressed in O. exaltata but not in O. sphegodes. References: 1. Xu et al. (2012). PLoS Genet. 8, e1002889.ISH-data-3_Exa-SAD5+Sph-SAD5.pdf