Floral volatile alleles can contribute to pollinator-mediated reproductive isolation in monkeyflowers (Mimulus)

Abstract

Pollinator-mediated reproductive isolation is a major factor in driving the diversification of flowering plants. Studies of floral traits involved in reproductive isolation have focused nearly exclusively on visual signals, such as flower color. The role of less obvious signals, such as floral scent, has been studied only recently. In particular, the genetics of floral volatiles involved in mediating differential pollinator visitation remains unknown. The bumblebee-pollinated Mimulus lewisii and hummingbird-pollinated Mimulus cardinalis are a model system for studying reproductive isolation via pollinator preference. We have shown that these two species differ in three floral terpenoid volatiles - d-limonene, β-myrcene, and E-β-ocimene - that are attractive to bumblebee pollinators. By genetic mapping and in vitro analysis of enzyme activity we demonstrate that these interspecific differences are consistent with allelic variation at two loci, LIMONENE-MYRCENE SYNTHASE (LMS) and OCIMENE SYNTHASE (OS). Mimulus lewisii LMS (MlLMS) and OS (MlOS) are expressed most strongly in floral tissue in the last stages of floral development. Mimulus cardinalis LMS (McLMS) is weakly expressed and has a nonsense mutation in exon 3. Mimulus cardinalis OS (McOS) is expressed similarly to MlOS, but the encoded McOS enzyme produces no E-β-ocimene. Recapitulating the M. cardinalis phenotype by reducing the expression of MlLMS by RNA interference in transgenic M. lewisii produces no behavioral difference in pollinating bumblebees; however, reducing MlOS expression produces a 6% decrease in visitation. Allelic variation at the OCIMENE SYNTHASE locus is likely to contribute to differential pollinator visitation, and thus promote reproductive isolation between M. lewisii and M. cardinalis. OCIMENE SYNTHASE joins a growing list of 'speciation genes' ('barrier genes') in flowering plants.

Keywords: Mimulus cardinalis; Mimulus lewisii; floral volatiles; pollination; speciation; terpene synthase.

Figure 1

Mimulus lewisii and M. cardinalis and their terpene synthases. (A) Mimulus lewisii and M. cardinalis. (B) Genetic maps of MlLMS and MlOS and the homologous regions in M. guttatus. Positions on the lower half of each are from the M. guttatus genome scaffolds; annotations are from queries of the M. guttatus transcripts with BLASTx. Putative terpene synthases are highlighted. M. lewisii limonene-myrcene synthase is homologous to a terpene synthase at 319,982 bp on M. guttatus scaffold 89, and M. lewisii ocimene synthase is homologous to a terpene synthase at 2,538,727 bp on scaffold 4.

Figure 2

Terpene synthase activity in vitro and in vivo. (A) Products of terpene synthases using a bacterial overexpression system and in vitro enzyme assay. Using geranyl pyrophosphate (GPP, the common monoterpene precursor), the MlLMS enzyme produces D-limonene (b) and β-myrcene (a) in the same relative proportion as in the floral volatile emission. M. lewisii MlOS produces E-β-ocimene (c), but M. cardinalis McOS does not. (B) Temporal expression of terpene synthases in vivo. MlLMS is expressed just prior to flowering and in open flowers, but McLMS is not expressed in open flowers. MlOS shows a similar expression pattern to MlLMS, and McOS is expressed at the same stages, despite producing no terpenoid volatile that we could detect. Developmental staging is according to bud size of M. lewisii, and the corresponding stage of M. cardinalis is pictured for McLMS, McOS, and McUBC.

Figure 3

Stable RNAi knockdowns of MlLMS and MlOS in M. lewisii produce plants with low emission levels of D-limonene (b)/β-myrcene (a) and E-β-ocimene (c), respectively.

Figure 4

Greenhouse experiments with Bombus impatiens and M. lewisii wild-type and transgenic lines. (A) Image of a typical bumblebee visit. (B) Response of bumblebees to MlLMS RNAi knockdowns, MlOS RNAi knockdowns, and the wild-type parent. Bumblebees show the same visitation response to MlLMS knockdown transgenics as to wild-type M. lewisii. Bumblebees preferentially visit wild-type M. lewisii over MlOS knockdown transgenics.