Jim Mant

DOES SELECTION ON FLORAL ODOR PROMOTE DIFFERENTIATION AMONG POPULATIONS AND SPECIES OF THE SEXUALLY DECEPTIVE ORCHID GENUS OPHRYS

Abstract —Sexually deceptive orchids from the genus Ophrys attract their pollinators primarily through the chemical mimicry of female hymenopteran sex pheromones, thereby deceiving males into attempted matings with the orchid labellum. Floral odor traits are crucial for the reproductive success of these pollinator‐limited orchids, as well as for maintaining reproductive isolation through the attraction of specific pollinators. We tested for the signature of pollinator‐mediated selection on floral odor by comparing intra and interspecific differentiation in odor compounds with that found at microsatellite markers among natural populations. Three regions from southern Italy were sampled. We found strong floral odor differentiation among allopatric populations within species, among allopatric species and among sympatric species. Population differences in odor were also reflected in significant variation in the attractivity of floral extracts to the pollinator, Colletes cunicularius. Odor compounds that are electrophysiologically active in C. cunicularius males, especially alkenes, were more strongly differentiated among conspecific populations than nonactive compounds in the floral odor. In marked contrast to these odor patterns, there was limited population or species level differentiation in microsatellites (FST range 0.005 to 0.127, mean FST 0.075). We propose that the strong odor differentiation and lack of genetic differentiation among sympatric taxa indicates selection imposed by the distinct odor preferences of different pollinating species. Within species, low FST values are suggestive of large effective population sizes and indicate that divergent selection rather than genetic drift accounts for the strong population differentiation in odor. The higher differentiation in active versus non‐active odor compounds suggests that divergent selection among orchid populations may be driven by local pollinator preferences for those particular compounds critical for pollinator attraction.

Cuticular Hydrocarbons as Sex Pheromone of the Bee Colletes cunicularius and the Key to its Mimicry by the Sexually Deceptive Orchid, Ophrys exaltata

Male Colletes cunicularius bees pollinate the orchid, Ophrys exaltata, after being sexually deceived by the orchid’s odor-mimicry of the female bee’s sex pheromone. We detected biologically active volatiles of C. cunicularius by using gas chromatographic–electroantennographic detection (GC-EAD) with simultaneous flame ionization detection. After identification of the target compounds by coupled gas chromatography–mass spectrometry (GC-MS), we performed behavioral tests using synthetic blends of the active components. We detected 22 EAD active compounds in cuticular extracts of C. cunicularius females. Blends of straight chain, odd-numbered alkanes and (Z)-7-alkenes with 21–29 carbon atoms constituted the major biologically active compounds. Alkenes were the key compounds releasing mating behavior, especially those with (Z)-7 unsaturation. Comparison of patterns of bee volatiles with those of O. exaltata subsp. archipelagi revealed that all EAD-active compounds were also found in extracts of orchid labella. Previous studies of the mating behavior in C. cunicularius showed linalool to be an important attractant for patrolling males. We confirmed this with synthetic linalool but found that it rarely elicited copulatory behavior, in accordance with previous studies. A blend of active cuticular compounds with linalool elicited both attraction and copulation behavior in patrolling males. Thus, linalool appears to function as a long-range attractant, whereas cuticular hydrocarbons are necessary for inducing short-range mating behavior.

A PHYLOGENETIC STUDY OF POLLINATOR CONSERVATISM AMONG SEXUALLY DECEPTIVE ORCHIDS

Abstract Orchids of the genus Chiloglottis are pollinated through the sexual deception of male wasps mainly from the genus Neozeleboria (Tiphiidae: Thynninae). The orchids mimic both the appearance and sex pheromones of wingless female thynnines but provide no reward to the deceived males. Despite the asymmetry of this interaction, strong pollinator specificity is typical. Such plant‐pollinator interactions would seem to be relatively flexible in the plants adaptive response to variation in the local pollinator resource. However, we present DNA sequence data on both orchids and wasps that demonstrate a pattern of pollinator conservatism operating at a range of taxonomic levels. Sequence data from the wasps indicate 15 of 16 Chiloglottis pollinators are closely related members of one clade of Thynninae. A pattern of congruence between orchid and wasp phylogenies is also demonstrated below the generic level, such that related orchids tend to use related thynnine wasps as specific pollinators. Comparative physiological data on the wasp responses to the floral scents of two Chiloglottis species and one outgroup, Arthrochilus, indicate similar attractive volatile chemicals are used by related orchid taxa. By extension, we infer a similarity of sex pheromone signals among related thynnines. Thus, the conservative pattern of pollinator change in sexually deceptive orchids may reflect phylogenetic patterns in the sex pheromones of their pollinators.

Population differentiation in female sex pheromone and male preferences in a solitary bee

Population differentiation in female mating signals and associated male preferences can drive reproductive isolation among segregated populations. We tested this assumption by investigating intraspecific variation in female sex pheromone and associated male odour preferences among distant populations in the solitary bee Colletes cunicularius (L.) by using quantitative gas chromatography and by performing field bioassays with synthetic blends of key sex pheromone compounds. We found significant differences in sex pheromone blends among the bee populations, and the divergence in odour blends correlated positively with geographic distance, suggesting that genetic divergence among distant populations can affect sex pheromone chemistry. Our behavioural experiments, however, demonstrate that synthetic copies of allopatric female sex pheromones were cross-attractive to patrolling males from distant populations, making reproductive isolation by non-recognition of mating signals among populations unlikely. Our data also show that patrolling male bees from different populations preferred odour types from allopatric populations at the two sites of bioassays. These male preferences are not expected to select for changes in the female sex pheromone, but may influence the evolution of floral odour in sexually deceptive orchids of the genus Ophrys that are pollinated by C. cunicularius males.

Specific pollinator attraction and the diversification of sexually deceptive Chiloglottis (Orchidaceae)

Abstract.Evidence indicates that sexually deceptive Chiloglottis R.Br. (Orchidaceae) taxa specifically attract their thynnine wasp (Tiphiidae) pollinators through the floral odour mimicry of female wasp sex pheromones. We use amplified fragment length polymorphisms (AFLPs) to reconstruct the species-level phylogeny of Chiloglottis, make a preliminary evaluation of genetic distinctions between species, and compare the historical association among orchids and their pollinators using wasp sequence data from a previous study. AFLPs show large differences between three sub-generic clades relative to that found among species within each clade. Interspecific genetic barriers are indicated by AFLP discontinuities among species unlike in previously reported DNA sequence data. However, such barriers are demonstrated clearly in only one of the two pairs of sympatric species sampled more intensively. We interpret these patterns as indicating either (i) a rapid and recent radiation of species within each clade following histories of stasis or extinction, or (ii) alternating cycles of divergence and gene flow acting to homogenize genetic differences among species within each of the three clades.

Phylogeography of pollinator-specific sexually deceptive Chiloglottis taxa (Orchidaceae): evidence for sympatric divergence?

Divergence in sexually deceptive orchids is thought to occur through shifts in the attraction of specific pollinators, a process that is mediated by changes in the floral odours that lure sexually excited male insects. We investigated the origin of reproductive isolation in a sexually deceptive species complex of Chiloglottis R.Br. (Orchidaceae: Diurideae). Two geographically separated montane regions in eastern Australia were sampled, each containing sympatric pairs of orchid taxa presently found under the name, Chiloglottis pluricallata. Behavioural tests confirmed at least three distinct orchid taxa that specifically attract different pollinators. An artificial crossing experiment among two taxa from one region demonstrated their interfertility, and confirmed isolation to be a function of pollinator attraction. A phylogeographic analysis using amplified fragment length polymorphisms (AFLPs) indicated that samples from each geographical region are most closely related, a pattern consistent with in situ or sympatric divergence. However, an extensive population genetic study on two taxa from one region failed to entirely reject the possibility of intertaxon gene flow. Although clear genetic differentiation of the taxa is evident in two out of three sites where both grow in direct sympatry, overall, the two taxa are not strongly distinguished by AFLP markers. The reconstruction of a simple bifurcating pattern of divergence may be confounded by a combination of contemporary population‐level processes operating within each taxon, the retention of ancestral polymorphism or intertaxon gene flow.

Key innovation or adaptive change? A test of leaf traits using Triodiinae in Australia

The evolution of novel traits (“key innovations”) allows some lineages to move into new environments or adapt to changing climates, whereas other lineages may track suitable habitat or go extinct. We test whether, and how, trait shifts are linked to environmental change using Triodiinae, C4 grasses that form the dominant understory over about 30% of Australia. Using phylogenetic and relaxed molecular clock estimates, we assess the Australian biogeographic origins of Triodiinae and reconstruct the evolution of stomatal and vascular bundle positioning. Triodiinae diversified from the mid-Miocene, coincident with the aridification of Australia. Subsequent niche shifts have been mostly from the Eremaean biome to the savannah, coincident with the expansion of the latter. Biome shifts are correlated with changes in leaf anatomy and radiations within Triodiinae are largely regional. Symplectrodia and Monodia are nested within Triodia. Rather than enabling biome shifts, convergent changes in leaf anatomy have probably occurred after taxa moved into the savannah biome—they are likely to have been subsequent adaptions rather than key innovations. Our study highlights the importance of testing the timing and origin of traits assumed to be phenotypic innovations that enabled ecological shifts.