Variable post‐zygotic isolation in Drosophila melanogaster/D. simulans hybrids

The study of hybrid inviability reveals cryptic divergence between the genetic interactions that maintain stable phenotypes in the pure species . We characterized the effects of natural variation on the penetrance of hybrid inviability phenotypes in crosses between Drosophila melanogaster and two species of the D. simulans subcomplex, D. simulans and D. sechellia. Using a panel of wild‐caught lines, we studied the levels of genetic variance present in D. simulans and D. sechellia affecting prezygotic and post‐zygotic isolation in hybridizations with D. melanogaster females. We observed extensive variability in the viability of hybrid individuals, dependent on the genotype of the parents, suggesting that intraspecific natural variation manifests directly in hybrid phenotypes. Furthermore, we found that genetic background significantly affects the penetrance of a well‐studied determinant of hybrid inviability: the interaction between Hmr^mel–Lhr^sim. Our results suggest that hybrid inviability – and reproductive isolation generally – can be modified by polymorphisms at multiple loci segregating within the parental species. Just as the penetrance of most mutant phenotypes can be modified by the genetic background within the pure species, the penetrance of hybrid inviability phenotypes is highly influenced by the parental genotypes.     

Evaluation of pre‐ and post‐zygotic mating barriers,hybrid fitness and phylogenetic relationship between Cyprinodon variegatus variegatus and Cyprinodon variegatus hubbsi (Cyprinodontiformes,Teleostei)

The euryhaline fish Cyprinodon variegatus variegatus (Cvv) is capable of tolerating ambient salinities ranging from 0.3 to 167 g l^?1, but incapable of long‐term survival in freshwater (< 2 mM Na⁺). However, a population of this species, now designated as a subspecies (Cyprinodon variegatus hubbsi; Cvh), has been isolated in several freshwater (0.4–1 mM Na⁺) lakes in central Florida for the past ~150 ky. We previously demonstrated that Cvh has a significantly higher affinity for Na⁺ uptake suggesting that it has adapted to its dilute freshwater environment. We here evaluate whether Cvh should be considered a separate species by characterizing pre‐ and post‐zygotic isolation, Na⁺ transport characteristics of the two populations and their hybrids, and developing a molecular phylogeny of Cvv and Cvh populations in Florida using mtDNA sequence data. We found evidence of partial prezygotic isolation with Cvv females mating almost exclusively (89%) with con‐specific males in choice mating experiments. Partial post‐zygotic isolation was also observed with significant (59–89%) reductions in hatching success of hybrid embryos compared with con‐specific embryos. Na⁺ uptake kinetics in hybrids (both Cvv x Cvh and Cvh x Cvv) bred and raised under common garden conditions were intermediate to Cvh (high affinity) and Cvv (low affinity) indicating that observed differences are genetically based. Similar observations were made with respect to short‐term (96 h) survival of juveniles acutely transferred from 7 mM Na⁺ to a range of more dilute (0.1–2 mM Na⁺) freshwater. Finally, although phylogenetic analysis of Cvv and Cvh populations using mtDNA sequence for ND2 were unable to fully resolve a polytomy between Cvh and Cvv populations from northeastern Florida, these data do not falsify the hypothesis that Cvh is of monophyletic origin. Overall, the available data suggest that Cvh should be considered a separate species or at a minimum an evolutionarily significant unit.     

Ongoing speciation within the Anastrepha fraterculus cryptic species complex: the case of the Andean morphotype

The Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) cryptic species complex is currently composed of seven taxonomically recognized morphotypes. Both, pre‐ and post‐zygotic isolation has been documented among four of these morphotypes, revealing that in fact they appear to be distinct biological entities. In order to progress in the full delimitation of species within the complex, we examined reproductive isolation between a Colombian population of the Andean morphotype and populations belonging to four other morphotypes spanning from Mexico to Argentina. Flies from the Andean morphotype exhibited strong pre‐zygotic mating isolation through temporal partitioning of mating activity. Post‐zygotic isolation was observed for crosses of males of all morphotypes and Andean morphotype females, yet most of the F1 hybrid ♂ × F1 hybrid ♀ self‐crosses showed normal levels of fertility, a finding suggesting a nuclear–cytoplasmic interaction according to previous studies. Overall, the Andean morphotype within the complex also appears to be a distinct biological entity. We discuss the implications of these findings for the understanding of speciation mechanisms in the Neotropical genus Anastrepha.     

The effectiveness of pre‐ and post‐zygotic barriers in avoiding hybridization between two snapdragons (Antirrhinum L.: Plantaginaceae)

Reproductive barriers play an important role in the maintenance of species boundaries. However, to date, few studies have provided a detailed analysis of reproductive isolation barriers between species or examined their importance in maintaining species identity. This is the first detailed study into pre‐ and post‐zygotic reproductive isolation barriers in Antirrhinum, based on a mixed population with two species that rarely co‐occur. The study revealed that pollinator constancy and preference and poor hybrid seed viability were the most important reproductive isolating mechanisms. Reproductive isolation was practically complete by both pre‐ and post‐zygotic barriers. Average pre‐zygotic isolation was greater than post‐zygotic isolation, in accordance with the trend observed in flowering plants in which reproductive isolation is principally caused by pre‐zygotic mechanisms. However, average post‐zygotic isolation was also high, in contrast to what was expected among Antirrhinum spp. This case highlights the importance of quantifying the reproductive isolation barriers thoroughly to understand how and why species boundaries are maintained. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 159–172.     

Geographic contrasts between pre‐ and postzygotic barriers are consistent with reinforcement in Heliconius butterflies

Identifying the traits causing reproductive isolation and the order in which they evolve is fundamental to understanding speciation. Here, we quantify prezygotic and intrinsic postzygotic isolation among allopatric, parapatric, and sympatric populations of the butterflies Heliconius elevatus and Heliconius pardalinus. Sympatric populations from the Amazon (H. elevatus and H. p. butleri) exhibit strong prezygotic isolation and rarely mate in captivity; however, hybrids are fertile. Allopatric populations from the Amazon (H. p. butleri) and Andes (H. p. sergestus) mate freely when brought together in captivity, but the female F1 hybrids are sterile. Parapatric populations (H. elevatus and H. p. sergestus) exhibit both assortative mating and sterility of female F1s. Assortative mating in sympatric populations is consistent with reinforcement in the face of gene flow, where the driving force, selection against hybrids, is due to disruption of mimicry and other ecological traits rather than hybrid sterility. In contrast, the lack of assortative mating and hybrid sterility observed in allopatric populations suggests that geographic isolation enables the evolution of intrinsic postzygotic reproductive isolation. Our results show how the types of reproductive barriers that evolve between species may depend on geography.     

Evaluation of reproductive barriers and realisation of interspecific hybridisations depending on genetic distances between species in the genus Helleborus

The genus Helleborus comprises 22 species, which are allocated to six sections. H. x hybridus and H. niger, which belong to different Helleborus sections, are economically important ornamentals. Several other species with minor impact exhibit interesting features, e.g. flower size, flower colour, foliage, scent and disease resistance, which should be introgressed into H. x hybridus or H. niger through interspecific hybridisation. The aims of this study were to investigate whether and which kind of hybridisation barriers occur in crosses between Helleborus species and if they differ in their manifestations, depending on the genetic distance of the respective partners. In order to obtain interspecific hybrids despite crossing barriers, a method to overcome these barriers should be developed. Crossing barriers in Helleborus were localised as predominantly post‐zygotic according to in situ pollen tube staining with aniline blue. For certain crosses, pre‐zygotic barriers could also be assumed, but pollen tube growth was not totally inhibited. Therefore, embryo rescue techniques via ovule culture were established to overcome the post‐zygotic barriers. Ovules were isolated from maternal plants 5–7 weeks after pollination in most cases and then cultured in vitro. Overall, 219 hybrids were successfully obtained, of which 16 were derived from inter‐sectional crosses. Hybrids were verified by flow cytometry and/or by molecular DNA markers.     

Post‐zygotic isolation in cactophilic Drosophila: larval viability and adult life‐history traits of D. mojavensis/D. arizonae hybrids

Drosophila mojavensis and Drosophila arizonae are cactophilic flies that have been used extensively in speciation studies. Incomplete premating isolation, evidence of reinforcement, and a lack of recent introgression between these species point to a potentially important role for post‐zygotic isolating barriers in this system. Other than hybrid male sterility, however, post‐zygotic isolation between D. mojavensis and D. arizonae has received little attention. In this study, we examined viability and life‐history traits of D. mojavensis/D. arizonae F₁ hybrids from sympatric crosses. Specifically, we reared hybrids and purebreds on the natural host cacti of each parental species and compared viability, development time, thorax length, and desiccation resistance between hybrids and purebreds. Interestingly, hybrid females from both crosses performed similarly or even better than purebred females. In contrast, hybrid sons of D. arizonae mothers, in addition to being sterile, had shorter average thorax length than males of both parental species, and hybrid males from both crosses had substantially lower desiccation resistance than D. mojavensis males. The probable cost to hybridization for D. mojavensis females resulting from reduced desiccation resistance of hybrid sons may have been an important selective factor in the history of reinforcement for crosses involving these females.     

Airborne‐pollen pool and mating pattern in a hybrid zone between Pinus pumila and P. parviflora var. pentaphylla

The reproductive isolation barriers and the mating patterns among Pinus pumila, P. parviflora var. pentaphylla and their hybrids were examined by flowering phenology and genetic assays of three life stages: airborne‐pollen grains, adults and seeds, in a hybrid zone on Mount Apoi, Hokkaido, Japan. Chloroplast DNA composition of the airborne‐pollen was determined by single‐pollen polymerase chain reaction. Mating patterns were analysed by estimating the molecular hybrid index of the seed parent, their seed embryos and pollen parents. The observation of flowering phenology showed that the flowering of P. pumila precedes that of P. parviflora var. pentaphylla by about 6 to 10 days within the same altitudinal ranges. Although this prezygotic isolation barrier is effective, the genetic assay of airborne‐pollen showed that the two pine species, particularly P. pumila, still have chances to form F₁ hybrid seeds. Both parental species showed a strong assortative mating pattern; F₁ seeds were found in only 1.4% of seeds from P. pumila mother trees and not at all in P. parviflora var. pentaphylla. The assortative mating was concluded as the combined result of flowering time differentiation and cross‐incompatibility. In contrast to the parental species, hybrids were fertilized evenly by the two parental species and themselves. The breakdown of prezygotic barriers (intermediate flowering phenology) and cross‐incompatibility may account for the unselective mating. It is suggested that introgression is ongoing on Mount Apoi through backcrossing between hybrids and parental species, despite strong isolation barriers between the parental species.     

Asymmetric Assortative Mating Behaviour Reflects Incomplete Pre‐zygotic Isolation in the Nasonia Species Complex

Preference of con‐ over heterospecific mates leading to assortative mating can substantially contribute to pre‐zygotic reproductive isolation and prevent fitness losses if post‐zygotic hybridization barriers already exist. The jewel wasp genus Nasonia displays quite strong and well‐studied post‐zygotic reproductive isolation due to a ubiquitous Wolbachia infection causing cytoplasmic incompatibility between different species. Pre‐zygotic isolation, however, has received far less research attention in this model organism, especially concerning the mechanisms and criteria of mate choice. In the present study, we analysed mate rejection and mate acceptance rates in cross‐comparisons between all four Nasonia species. We put emphasis on observing which sex is more likely to interrupt interspecific matings and how discriminatory behaviour varies across the different species in all possible combinations. We found an asymmetric distribution of assortative mating among the four Nasonia species that appears to be highly influenced by the respective combinations of sex and species. Females appeared to be the main discriminators against heterospecific mating partners, but interestingly, we could also detect mate discrimination and rejection behaviour in males, a widely neglected factor in research on mating behaviour in general and on Nasonia in particular. Moreover, the asymmetry in the assortative mating behaviour was partially reflective of sym‐ or allopatric distributions of natural Nasonia populations.     

Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex

Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.     

Molecular analyses identify hybridization‐mediated nuclear evolution in newly discovered fungal hybrids

Hybridization may be a major driver in the evolution of plant pathogens. In a high elevation Alpine larch stand in Montana, a novel hybrid fungal pathogen of trees originating from the mating of Heterobasidion irregulare with H. occidentale has been recently discovered. In this study, sequence analyses of one mitochondrial and four nuclear loci from 11 Heterobasidion genotypes collected in the same Alpine larch stand indicated that hybridization has increased allelic diversity by generating novel polymorphisms unreported in either parental species. Sequence data and ploidy analysis through flow cytometry confirmed that heterokaryotic (n + n) genotypes were not first‐generation hybrids, but were the result of multiple backcrosses, indicating hybrids are fertile. Additionally, all admixed genotypes possessed the H. occidentale mitochondrion, indicating that the hybrid progeny may have been backcrossing mostly with H. occidentale. Based on reticulate phylogenetic network analysis by PhyloNet, Bayesian assignment, and ordination tests, alleles can be defined as H. irregulare‐like or H. occidentale‐like. H. irregulare‐like alleles are clearly distinct from all known H. irregulare alleles and are derived from the admixing of both Heterobasidion species. Instead, all but one H. occidentale alleles found in hybrids, although novel, were not clearly distinct from alleles found in the parental H. occidentale population. This discovery demonstrates that Alpine larch can be a universal host favouring the interspecific hybridization between H. irregulare and H. occidentale and the hybridization‐mediated evolution of a nucleus, derived from H. irregulare parental species but clearly distinct from it.     

Species‐specific habitat preferences do not shape the structure of a crested newt hybrid zone (Triturus cristatus x T. carnifex)

Reproductive isolation barriers maintain the integrity of species by preventing interspecific gene flow. They involve temporal, habitat or behavioral isolation acting before fertilization, and postzygotic isolation manifested as hybrid mortality or sterility. One of the approaches of how to study reproductive isolation barriers is through the analysis of hybrid zones. In this paper, we describe the structure of a hybrid zone between two crested newt species (Triturus cristatus and T. carnifex) in the southern part of the Czech Republic using morphological, microsatellite, and mitochondrial (mtDNA) markers. Specifically, we tested the hypothesis that the structure of the hybrid zone is maintained by species‐specific habitat preferences. Comparing the genetic structure of populations with geographical and ecological parameters, we found that the hybrid zone was structured primarily geographically, with T. cristatus‐like populations occurring in the northeast and T. carnifex‐like populations in the southwest. Despite T. cristatus tending to occur in deeper ponds and T. carnifex on localities with more shading, the effect of both ecological parameters on the structure of the zone was minimal. Next, we corroborated that T. carnifex individuals and some hybrids possess mtDNA of T. dobrogicus, whose nuclear background was not detected in the studied hybrid zone. Hybridization between T. carnifex and T. dobrogicus (resulting in unidirectional mtDNA introgression) had to predate subsequent formation of the hybrid zone between T. cristatus and T. carnifex. Populations of crested newts in the southern part of the Czech Republic thus represent a genetic mosaic of nuclear and mitochondrial genomes of three species.     

Selection on hybrids of ecologically divergent ecotypes of a marine snail: the relative importance of exogenous and endogenous barriers

Unravelling the form of selection acting on hybrids of ecotypes undergoing ecological speciation is essential to understand the mechanisms behind the evolution of reproductive isolation in the face of gene flow. Shell phenotype is known to be affected by natural selection and is involved in the fitness of the marine snail Littorina saxatilis. Here, we studied the association between shell traits and fitness in hybrids in order to determine the relative role of exogenous and endogenous selection in this hybrid zone of L. saxatilis. We show that directional selection is the predominant mode of selection among hybrids. We also show its heterogeneity, affecting different shell traits, within populations at the level of the microhabitat. Therefore, endogenous selection mechanisms are most probably lacking in this hybrid zone and exogenous barriers (pre‐ and post‐zygotic) are possibly one of the main forces behind the evolution of barriers to gene flow between these ecologically divergent ecotypes. This study shows how this barrier might represent an important type of reproductive isolation within ecological speciation, and this should be taken into account in future studies of speciation in hybrid zones. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 391–400.     

Pre‐zygotic reproductive isolation between two synchronopatric Opuntia (Cactaceae) species in the Brazilian Chaco

• Opuntia (Cactaceae) is known for high rates of hybridization and ploidisation, resulting in the formation of new species. The occurrence of two sympatric and closely related species of Opuntia, O. elata and O. retrorsa, in Brazilian Chaco enabled us to test the hypothesis that pre‐zygotic reproductive isolation mechanisms operate in both species.
• We monitored the flowering period, as well as floral biology, and compared the morphological variation of floral structures through measurements, performed intra‐ and interspecific cross‐pollination tests, and recorded the guild of floral visitors and pollinators.
• Flowering was seasonal and highly synchronous. Floral biology exhibits similar strategies, and although floral morphology differs significantly in many of the compared structures, such morphological variation does not result in the selection of exclusive pollinators. Floral visitors and pollinators are oligolectic bees shared by both species. Opuntia elata and O. retrorsa are self‐compatible. While interspecific cross‐pollination (bidirectional) resulted in germination, the pollen tube did not penetrate the stigma.
• Opuntia elata and O. retrorsa are closely related; however, they are isolated and do not hybridise in Brazilian Chaco. We found that both have weak pre‐pollination barriers, but that they are strongly isolated by pollen–pistil incompatibility, i.e. post‐pollination barrier.

     

Hybridization increases mitochondrial production of reactive oxygen species in sunfish

Mitochondrial dysfunction and oxidative stress have been suggested to be possible mechanisms underlying hybrid breakdown, as a result of mito‐nuclear incompatibilities in respiratory complexes of the electron transport system. However, it remains unclear whether hybridization increases the production of reactive oxygen species (ROS) by mitochondria. We used high‐resolution respirometry and fluorometry on isolated liver mitochondria to examine mitochondrial physiology and ROS emission in naturally occurring hybrids of pumpkinseed (Lepomis gibbosus) and bluegill (L. macrochirus). ROS emission was greater in hybrids than in both parent species when respiration was supported by complex I (but not complex II) substrates, and was associated with increases in lipid peroxidation. However, respiratory capacities for oxidative phosphorylation, phosphorylation efficiency, and O₂ kinetics in hybrids were intermediate between those in parental species. Flux control ratios of capacities for electron transport (measured in uncoupled mitochondria) relative to oxidative phosphorylation suggested that the limiting influence of the phosphorylation system is reduced in hybrids. This likely helped offset impairments in electron transport capacity and complex III activity, but contributed to augmenting ROS production. Therefore, hybridization can increase mitochondrial ROS production, in support of previous suggestions that mitochondrial dysfunction can induce oxidative stress and thus contribute to hybrid breakdown.     

Disruption of recruitment in two endemic palms on Lord Howe Island by invasive rats

Invasive species may have negative impacts on many narrow range endemics and species restricted to oceanic islands. Predicting recent impacts of invasive species on long-lived trees is difficult because the presence of adult plants may mask population changes. We examined the impact of introduced black rats (Rattus rattus) on two palm species restricted to cloud forests and endemic to Lord Howe Island, a small oceanic island in the southern Pacific. We combined estimates of the standing size distribution of these palms with the proximal impacts of rats on fruit survival in areas baited to control rats and in unbaited areas. The size distribution of palms with trunks was comparable across baited and unbaited sites. Small juvenile palms lacking a trunk (<50 cm tall) were abundant in baited areas, but rare in unbaited sites for Lepidorrhachis mooreana, and rare or absent in 3 out of 4 unbaited Hedyscepe canterburyana sites. All ripe fruits were lost to rats in the small fruited L. mooreana. Fruit removal was widespread but less (20–54%) in H. canterburyana. Both palms showed evidence of a reduced capacity to maintain a juvenile bank of palms through regular recruitment as a consequence of over 90 years of rat impact. This will limit the ability of these species to take advantage of episodic canopy gaps. Baiting for rat control reduced fruit losses and resulted in the re-establishment of a juvenile palm bank. Conservation of both endemic palms necessitates control (or eradication) of rat populations on the unique cloud forest summits of the island.     

Is embryo abortion a post‐zygotic barrier to gene flow between Littorina ecotypes?

Genetic incompatibilities contribute to reproductive isolation between many diverging populations, but it is still unclear to what extent they play a role if divergence happens with gene flow. In contact zones between the "Crab" and "Wave" ecotypes of the snail Littorina saxatilis, divergent selection forms strong barriers to gene flow, while the role of post‐zygotic barriers due to selection against hybrids remains unclear. High embryo abortion rates in this species could indicate the presence of such barriers. Post‐zygotic barriers might include genetic incompatibilities (e.g. Dobzhansky–Muller incompatibilities) but also maladaptation, both expected to be most pronounced in contact zones. In addition, embryo abortion might reflect physiological stress on females and embryos independent of any genetic stress. We examined all embryos of >500 females sampled outside and inside contact zones of three populations in Sweden. Females' clutch size ranged from 0 to 1,011 embryos (mean 130 ± 123), and abortion rates varied between 0% and 100% (mean 12%). We described female genotypes by using a hybrid index based on hundreds of SNPs differentiated between ecotypes with which we characterized female genotypes. We also calculated female SNP heterozygosity and inversion karyotype. Clutch size did not vary with female hybrid index, and abortion rates were only weakly related to hybrid index in two sites but not at all in a third site. No additional variation in abortion rate was explained by female SNP heterozygosity, but increased female inversion heterozygosity added slightly to increased abortion. Our results show only weak and probably biologically insignificant post‐zygotic barriers contributing to ecotype divergence, and the high and variable abortion rates were marginally, if at all, explained by hybrid index of females.     

Population structure of a vector‐borne plant parasite

Parasites are among the most diverse groups of life on Earth, yet complex natural histories often preclude studies of their speciation processes. The biology of parasitic plants facilitates in situ collection of data on both genetic structure and the mechanisms responsible for that structure. Here, we studied the role of mating, dispersal and establishment in host race formation of a parasitic plant. We investigated the population genetics of a vector‐borne desert mistletoe (Phoradendron californicum) across two legume host tree species (Senegalia greggii and Prosopis velutina) in the Sonoran desert using microsatellites. Consistent with host race formation, we found strong host‐associated genetic structure in sympatry, little genetic variation due to geographic site and weak isolation by distance. We hypothesize that genetic differentiation results from differences in the timing of mistletoe flowering by host species, as we found initial flowering date of individual mistletoes correlated with genetic ancestry. Hybrids with intermediate ancestry were detected genetically. Individuals likely resulting from recent, successful establishment events following dispersal between the host species were detected at frequencies similar to hybrids between host races. Therefore, barriers to gene flow between the host races may have been stronger at mating than at dispersal. We also found higher inbreeding and within‐host individual relatedness values for mistletoes on the more rare and isolated host species (S. greggii). Our study spanned spatial scales to address how interactions with both vectors and hosts influence parasitic plant structure with implications for parasite virulence evolution and speciation.     

Massive floral display affects insect visits but not pollinator‐mediated pollen transfer in Rhododendron ferrugineum

Fragmentation of natural vegetation creates one of the largest threats to plant–pollinator interactions. Although fragmentation impacts on plant populations have been explored in many, mainly herbaceous, species, the response of wild mass‐flowering species is poorly known. Here, we studied 28 heathland patches dominated by the mass‐flowering shrub Rhododendron ferrugineum, each presenting different R. ferrugineum floral display sizes (total inflorescence number per patch) and patch isolation (median distance to the three nearest patches). We assessed the impacts of these two factors on (i) heathland patch visitor assemblage (considering R. ferrugineum versus surrounding community) and (ii) R. ferrugineum flower visitation rate and pollen transfer limitation (comparing seed set from emasculated to pollen‐supplemented flowers). We found that diversity and abundance of bees visiting R. ferrugineum in heathland patches significantly decreased with decreasing R. ferrugineum floral display, while overall visitor density per patch and flower visitation rate increased. Moreover, a decrease in massive floral display and increase in patch isolation resulted in reduced visitor density in the surrounding community. Even in patches with few individuals, we found disproportionate visitor abundance in R. ferrugineum compared to the surrounding community. Finally, pollen transfer limitation in R. ferrugineum was neither affected by visitation rate nor by patch attributes. By disproportionally attracting pollinators from co‐flowering species, and probably promoting geitonogamous pollen transfer, the mass‐flowering trait appears adequate to compensate, in terms of conspecific pollen transfer, for the decrease in visitor diversity and abundance and in mate availability, which usually result from population fragmentation.     

Habitat preference and flowering‐time variation contribute to reproductive isolation between diploid and autotetraploid Anacamptis pyramidalis

Tetraploid lineages are typically reproductively isolated from their diploid ancestors by post‐zygotic isolation via triploid sterility. Nevertheless, polyploids often also exhibit ecological divergence that could contribute to reproductive isolation from diploid ancestors. In this study, we disentangled the contribution of different forms of reproductive isolation between sympatric diploid and autotetraploid individuals of the food‐deceptive orchid Anacamptis pyramidalis by quantifying the strength of seven reproductive barriers: three prepollination, one post‐pollination prezygotic and three post‐zygotic. The overall reproductive isolation between the two cytotypes was found very high, with a preponderant contribution of two prepollination barriers, that is phenological and microhabitat differences. Although the contribution of post‐zygotic isolation (triploid sterility) is confirmed in our study, these results highlight that prepollination isolation, not necessarily involving pollinator preference, can represent a strong component of reproductive isolation between different cytotypes. Thus, in the context of polyploidy as quantum speciation, that generates reproductive isolation via triploid sterility, ecological divergence can strengthen the reproductive isolation between cytotypes, reducing the waste of gametes in low fitness interploidy crosses and thus favouring the initial establishment of the polyploid lineage. Under this light, speciation by polyploidy involves ecological processes and should not be strictly considered as a nonecological form of speciation.