Giovanni Scopece

Patterns of reproductive isolation in Mediterranean deceptive orchids.

Abstract The evolution of reproductive isolation is of central interest in evolutionary biology. In plants, this is typically achieved by a combination of pre- and postpollination mechanisms that prevent, or limit, the amount of interspecific gene flow. Here, we investigated and compared two ecologically defined groups of Mediterranean orchids that differ in pollination biology and pollinator specificity: sexually deceptive orchids versus food-deceptive orchids. We used experimental crosses to assess the strength of postmating prezygotic, and postzygotic reproductive isolation, and a phylogenetic framework to determine their relative rates of evolution. We found quantitative and qualitative differences between the two groups. Food-deceptive orchids have weak premating isolation but strong postmating isolation, whereas the converse situation characterizes sexually deceptive orchids. Only postzygotic reproductive isolation among food-deceptive orchids was found to evolve in a clock-like manner. Comparison of evolutionary rates, within a common interval of genetic distance, showed that the contribution of postmating barriers was more relevant in the food-deceptive species than in the sexually deceptive species. Asymmetry in prezygotic isolation was found among food-deceptive species. Our results indicate that postmating barriers are most important for reproductive isolation in food-deceptive orchids, whereas premating barriers are most important in sexually deceptive orchids. The different rate of evolution of reproductive isolation and the relative strength of pre- and postmating barriers may have implication for speciation processes in the two orchid groups.

Evolution of Postzygotic Reproductive Isolation in a Guild of Deceptive Orchids

The evolution of reproductive barriers is of central importance for speciation. Here, we investigated three components of postzygotic isolation—embryo mortality, hybrid inviability, and hybrid sterility—in a group of food‐deceptive Mediterranean orchids from the genera Anacamptis, Neotinea, and Orchis. In these orchids, pollinator‐mediated isolation is weak, which suggests that postpollination barriers exist. Based on crossing experiments and a literature survey, we found that embryo mortality caused complete reproductive isolation among 36.3% of the species pairs, and hybrid inviability affected 55.6% of the potentially hybridizing species pairs. Hybrid sterility was assessed experimentally for seven species pairs. A strong reduction of fertility in all investigated hybrids was found, together with clear differences between male and female components of hybrid sterility. Postzygotic isolation was found to evolve gradually with genetic divergence, and late postzygotic isolation (i.e., hybrid inviability and sterility) evolved faster than embryo mortality, which is an earlier postzygotic isolation stage. These results reveal that intrinsic postzygotic isolation strongly contributes to maintaining species boundaries among Mediterranean food‐deceptive orchids while establishing a prominent role for these reproductive barriers in the early stage of species isolation.

Floral isolation is the main reproductive barrier among closely related sexually deceptive orchids.

Floral isolation is an important component of pollinator‐driven speciation. However, up to now, only a few studies have quantified its strength and relative contribution to total reproductive isolation. In this study, we quantified floral isolation among three closely related, sympatric orchid species of the genus Ophrys by directly tracking pollen flow. Ophrys orchids mimic their pollinators’ mating signals, and are pollinated by male insects during mating attempts. This pollination system, called sexual deception, is usually highly specific. However, whether pollinator specialization also conveys floral isolation is currently under debate. In this study, we found strong floral isolation: among 46 tracked pollen transfers in two flowering seasons, all occurred within species. Accounting for observation error rate, we estimated a floral isolation index ≥0.98 among each pair of species. Hand pollination experiments suggested that postpollination barriers were effectively absent among our study species. Genetic analysis based on AFLP markers showed a clear species clustering and very few F1 hybrids in natural populations, providing independent evidence that strong floral isolation prevents significant interspecies gene flow. Our results provide the first direct evidence that floral isolation acts as the main reproductive barrier among closely related plant species with specialized pollination.

Pollination Efficiency and the Evolution of Specialized Deceptive Pollination Systems

The ultimate causes of evolution of highly specialized pollination systems are little understood. We investigated the relationship between specialization and pollination efficiency, defined as the proportion of pollinated flowers relative to those that experienced pollen removal, using orchids with different pollination strategies as a model system. Rewarding orchids showed the highest pollination efficiency. Sexually deceptive orchids had comparably high pollination efficiency, but food‐deceptive orchids had significantly lower efficiency. Values for pollinator sharing (a measure of the degree of generalization in pollination systems) showed the reverse pattern, in that groups with high pollination efficiency had low values of pollinator sharing. Low pollinator sharing may thus be the basis for efficient pollination. Population genetic data indicated that both food‐ and sexually deceptive species have higher degrees of among‐population gene flow than do rewarding orchids. Thus, the shift from food to sexual deception may be driven by selection for more efficient pollination, without compromising the high levels of gene flow that are characteristic of deceptive species.

Specificity in pollination and consequences for postmating reproductive isolation in deceptive Mediterranean orchids

The type of reproductive isolation prevalent in the initial stages of species divergence can affect the nature and rate of emergence of additional reproductive barriers that subsequently strengthen isolation between species. Different groups of Mediterranean deceptive orchids are characterized by different levels of pollinator specificity. Whereas food-deceptive orchid species show weak pollinator specificity, the sexually deceptive Ophrys species display a more specialized pollination strategy. Comparative analyses reveal that orchids with high pollinator specificity mostly rely on premating reproductive barriers and have very little postmating isolation. In this group, a shift to a novel pollinator achieved by modifying the odour bouquet may represent the main isolation mechanism involved in speciation. By contrast, orchids with weak premating isolation, such as generalized food-deceptive orchids, show strong evidence for intrinsic postmating reproductive barriers, particularly for late-acting postzygotic barriers such as hybrid sterility. In such species, chromosomal differences may have played a key role in species isolation, although strong postmating–prezygotic isolation has also evolved in these orchids. Molecular analyses of hybrid zones indicate that the types and strength of reproductive barriers in deceptive orchids with contrasting premating isolation mechanisms directly affect the rate and evolutionary consequences of hybridization and the nature of species differentiation.

Ecology and genetic diversity of the dense-flowered orchid, Neotinea maculata, at the centre and edge of its range

BACKGROUND AND AIMS Species may occur over a wide geographical range within which populations can display large variation in reproductive success and genetic diversity. Neotinea maculata is a rare orchid of conservation concern at the edge of its range in Ireland, where it occurs in small populations. However, it is relatively common throughout the Mediterranean region. Here, factors that affect rarity of N. maculata in Ireland are investigated by comparing Irish populations with those found in Italy, where it is more common. METHODS Vegetation communities, breeding system and genetic diversity were compared using three amplified fragment length polymorphism (AFLP) primer pairs in populations in Ireland and Italy. Vegetation was quantified using quadrats taken along transects in study populations, and hand pollination experiments were performed to assess reliance of N. maculata on pollinators in both Irish and Italian populations. KEY RESULTS Neotinea maculata occupies different vegetation communities in Italian and Irish populations. Breeding system experiments show that N. maculata is 100 % autogamous, and there are no differences in fruit and seed production in selfed, outcrossed and unmanipulated plants. AFLP markers revealed that Irish and Italian populations have similar genetic diversity and are distinct from each other. CONCLUSIONS Neotinea maculata does not suffer any negative effects of autogamous reproduction; it self-pollinates and sets seed readily in the absence of pollinators. It occupies a variety of habitats in both Ireland and Italy; however, Irish populations are small and rare and should be conserved. This could be due to climatic factors and the absence of suitable soil mycorrhizas to allow recruitment from seed.

COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN ORCHIS MASCULA AND ORCHIS PAUCIFLORA

Studies of the strength and nature of reproductive isolation (RI) between species can greatly contribute to our understanding of speciation. Although the role of RI in speciation is well recognized, there is a dearth of information on the contributions of different barriers between related plant species. Here, we estimated multiple components of RI between two Mediterranean orchid sister species (Orchis mascula and Orchis pauciflora), disentangling the strength and absolute contributions of seven different isolating mechanisms. Our survey includes one prepollination, two postpollination prezygotic (pollen–stigma incompatibility, conspecific pollen precedence), two intrinsic postzygotic (embryo mortality and hybrid sterility) and two extrinsic postzygotic (hybrid habitat differentiation and hybrid pollination) isolating mechanisms. We found strong RI between the investigated species, although none of the barriers were able to completely impede gene flow. Five isolating mechanisms contributed positively to the maintenance of species boundaries. Contrary to most surveys of isolating mechanisms, our data speak against a clear predominance of prepollination or of prezygotic barriers but confirm the emerging pattern of multiple barriers contributing to the maintenance of species integrity. These findings suggest an allopatric condition during early phases of species divergence. We discuss our data in the wider context of previous studies carried out in this orchid group by using a comparative approach.

Genic rather than genome-wide differences between sexually deceptive Ophrys orchids with different pollinators

High pollinator specificity and the potential for simple genetic changes to affect pollinator attraction make sexually deceptive orchids an ideal system for the study of ecological speciation, in which change of flower odour is likely important. This study surveys reproductive barriers and differences in floral phenotypes in a group of four closely related, coflowering sympatric Ophrys species and uses a genotyping‐by‐sequencing (GBS) approach to obtain information on the proportion of the genome that is differentiated between species. Ophrys species were found to effectively lack postpollination barriers, but are strongly isolated by their different pollinators (floral isolation) and, to a smaller extent, by shifts in flowering time (temporal isolation). Although flower morphology and perhaps labellum coloration may contribute to floral isolation, reproductive barriers may largely be due to differences in flower odour chemistry. GBS revealed shared polymorphism throughout the Ophrys genome, with very little population structure between species. Genome scans for FST outliers identified few markers that are highly differentiated between species and repeatable in several populations. These genome scans also revealed highly differentiated polymorphisms in genes with putative involvement in floral odour production, including a previously identified candidate gene thought to be involved in the biosynthesis of pseudo‐pheromones by the orchid flowers. Taken together, these data suggest that ecological speciation associated with different pollinators in sexually deceptive orchids has a genic rather than a genomic basis, placing these species at an early phase of genomic divergence within the ‘speciation continuum’.

Transitions between self-compatibility and self-incompatibility and the evolution of reproductive isolation in the large and diverse tropical genus Dendrobium (Orchidaceae)

BACKGROUND AND AIMS The evolution of interspecific reproductive barriers is crucial to understanding species evolution. This study examines the contribution of transitions between self-compatibility (SC) and self-incompatibility (SI) and genetic divergence in the evolution of reproductive barriers in Dendrobium, one of the largest orchid genera. Specifically, it investigates the evolution of pre- and postzygotic isolation and the effects of transitions between compatibility states on interspecific reproductive isolation within the genus. METHODS The role of SC and SI changes in reproductive compatibility among species was examined using fruit set and seed viability data available in the literature from 86 species and ∼2500 hand pollinations. The evolution of SC and SI in Dendrobium species was investigated within a phylogenetic framework using internal transcribed spacer sequences available in GenBank. KEY RESULTS Based on data from crossing experiments, estimations of genetic distance and the results of a literature survey, it was found that changes in SC and SI significantly influenced the compatibility between species in interspecific crosses. The number of fruits produced was significantly higher in crosses in which self-incompatible species acted as pollen donor for self-compatible species, following the SI × SC rule. Maximum likelihood and Bayesian tests did not reject transitions from SI to SC and from SC to SI across the Dendrobium phylogeny. In addition, postzygotic isolation (embryo mortality) was found to evolve gradually with genetic divergence, in agreement with previous results observed for other plant species, including orchids. CONCLUSIONS Transitions between SC and SI and the gradual accumulation of genetic incompatibilities affecting postzygotic isolation are important mechanisms preventing gene flow among Dendrobium species, and may constitute important evolutionary processes contributing to the high levels of species diversity in this tropical orchid group.

Pollinator shifts between Ophrys sphegodes populations: might adaptation to different pollinators drive population divergence?

Local adaptation to different pollinators is considered one of the possible initial stages of ecological speciation as reproductive isolation is a by‐product of the divergence in pollination systems. However, pollinator‐mediated divergent selection will not necessarily result in complete reproductive isolation, because incipient speciation is often overcome by gene flow. We investigated the potential of pollinator shift in the sexually deceptive orchids Ophrys sphegodes and Ophrys exaltata and compared the levels of floral isolation vs. genetic distance among populations with contrasting predominant pollinators. We analysed floral hydrocarbons as a proxy for floral divergence between populations. Floral adoption of pollinators and their fidelity was tested using pollinator choice experiments. Interpopulation gene flow and population differentiation levels were estimated using AFLP markers. The Tyrrhenian O. sphegodes population preferentially attracted the pollinator bee Andrena bimaculata, whereas the Adriatic O. sphegodes population exclusively attracted A. nigroaenea. Significant differences in scent component proportions were identified in O. sphegodes populations that attracted different preferred pollinators. High interpopulation gene flow was detected, but populations were genetically structured at species level. The high interpopulation gene flow levels independent of preferred pollinators suggest that local adaptation to different pollinators has not (yet) generated detectable genome‐wide separation. Alternatively, despite extensive gene flow, few genes underlying floral isolation remain differentiated as a consequence of divergent selection. Different pollination ecotypes in O. sphegodes might represent a local selective response imposed by temporal variation in a geographical mosaic of pollinators as a consequence of the frequent disturbance regimes typical of Ophrys habitats.