Outcrossing rates in two self‐compatible,hybridising Rhinanthus species: implications for hybrid formation

The congeners Rhinanthus angustifolius and Rhinanthus minor, two annual hemiparasites pollinated by bumblebees, are known to hybridise in the wild. Both species are self‐compatible, but the capacity for autonomous selfing is higher in R. minor. This suggests a difference in realized outcrossing rates, which have not been determined before in these species. Using microsatellites, both species turned out to have mixed mating systems, but with a much lower multilocus outcrossing rate (0.13) for R. minor compared to R. angustifolius (0.76). We hypothesised that a higher outcrossing rate should lead to a higher chance of heterospecific pollination, and we therefore determined the rate of hybrid formation on each species in an artificial mixed population. Hybrid seeds were produced at low frequency (4.5%), and no significant difference was found between the species. It is therefore likely that post‐pollination processes influence hybrid seed formation to counteract the expected difference in heterospecific pollen deposition. We checked fruit set, seed set and the rate of autonomous selfing in controlled crosses in the greenhouse in 2 years, and found that fruit set (2003) or seed set (2010) were lower in R. angustifolius × R. minor crosses relative to the reciprocal cross. Hybrid seeds produced on R. angustifolius also had a much lower germination rate, so most of the established F₁ hybrid plants have the R. minor cytoplasm. The formation of advanced hybrids depends on pollinator preference, which is biased towards R. angustifolius if present in sufficient numbers, because it offers more rewards.     

Hybridization in Annual Plants: Patterns and DynamicsDuring a Four-Year Study in Mixed Rhinanthus Populations

Hybridization in annual plants is rare, but their short life cycle provides an excellent opportunity to study the dynamics of hybridization. Hybridization occurs between the annual hemiparasites Rhinanthus minor and Rhinanthus angustifolius (Orobanchaceae). Using flower morphology, Kwak (1980) found a prevalence of hybrids close to R. angustifolius in a single population. We aim to find whether this pattern is also found using genetic markers, whether it is generally occurring in mixed populations, and whether these populations are stable over time. We used species-specific genetic markers to determine the number of individuals in a range of hybrid classes in three mixed populations of different ages during four consecutive years. In the young population, F₁ hybrids were found in the first year and mostly hybrids between R. minor and these F₁s in the second year, but in the years after that, hybrids close to R. angustifolius became more abundant. We also found this in the two older populations, where hybrids close to R. angustifolius always occurred in higher frequencies than hybrids close to R. minor. Over time, R. angustifolius strongly increased in frequency in two populations. Patterns of marker presence and absence suggested that advanced-generation hybrids are mainly formed by backcrossing with one of the parents, predominantly R. angustifolius whenever its frequency in the population is higher than 15%. The dynamics of mixed populations depend on the ecological conditions that regulate the presence of the two parental species, and introgression into R. angustifolius seems prevalent.     

HYBRID FITNESS IN THE LOUISIANA IRISES: ANALYSIS OF PARENTAL AND F1 PERFORMANCE

The assumption of hybrid inferiority is central to the two models most widely applied to the prediction of hybrid zone evolution. Both the tension zone and mosaic models assume that natural selection acts against hybrids regardless of the environment in which they occur. To test this assumption, we investigated components of fitness in Iris fulva, I. hexagona and their reciprocal F₁ hybrids under greenhouse conditions. The four cross types were compared on the basis of seed germination, vegetative and clonal growth, and sexual reproduction. In all cases, the hybrids performed as well as, or significantly better than, both of their parents. These results suggest that F₁ hybrids between I. fulva and I. hexagona are at least as fit as their parents. The results of this study are therefore inconsistent with the assumptions of both the tension zone and mosaic models of hybrid zone evolution.     

Fitness of natural willow hybrids in a pioneer mosaic hybrid zone

Hybrid fitness is an important parameter to predict the evolutionary consequences of a hybridization event and to characterize hybrid zones. We studied fitness parameters of F₁ and later‐generation hybrids between the lowland species Salix purpurea and the alpine S. helvetica that have recently emerged during colonization of an alpine glacier forefield. Fruit production (number of capsules per catkin and fruit set) did not differ between hybrids and parents, but the number of seeds per capsule of F₁ hybrids was slightly lower than that of later‐generation hybrids and of the parents. Germination rates and seedling growth were tested on three substrates (pH 4.5, 7.0, and 8.0). Germination rates of seeds collected from F₁ hybrids were lower on acid and neutral substrates, but equal at pH 8.0 compared to all other groups, while the seeds from later‐generation hybrids performed as well as the parents on all three substrates. In seedling growth, the colonizer S. purpurea performed better than all other taxa on all three substrates, while hybrids resembled the subalpine species S. helvetica. Results suggest that endogenous selection acts against F₁ hybrids, but favors fitter genotypes in later‐generation hybrids. Exogenous selection via soil pH appears to be weak during seedling establishment. The pioneer vegetation on the glacier forefield may offer sufficient niche space for hybrid seedlings. Owing to the relatively high fitness of the hybrids and the scattered distribution of hybrids and parental individuals on the glacier forefield, this hybrid zone can be assigned to a mosaic model, probably facilitating gene flow and introgression between the parental species. As establishment of the hybrid zone appears to be linked to a colonization process, we propose to call it a pioneer mosaic hybrid zone.     

Germination characteristics of Rhinanthus minor influence field emergence,competitiveness and potential use in restoration projects

• The facultative root hemi‐parasite Rhinanthus minor is often used in grassland habitat restoration projects to regulate ecosystem structure and function. Its impact on community productivity and diversity as a function of resource supply, sward composition and management has been widely investigated. However, there is a lack of information about the possible influence of seed quality on the efficacy of the hemi‐parasite.
• Ten seed lots from commercial sources were sown in the field and their germination characteristics investigated in the laboratory. Seeds from four lots were also germinated and sown in pots alongside plants of two host species, Lotus corniculatus and Holcus lanatus. Plant establishment, height and flowering density were evaluated for the hemi‐parasite, while plant biomass was measured for both R. minor and its host.
• Two aspects of seed quality influenced the field emergence of seed lots of R. minor, the radicle emergence (%) and the length of the lag period from the beginning of imbibition to germination (mean germination time), which indicates seed vigour. A longer lag period (lower vigour) was associated with higher levels of seedling mortality and lower plant vigour, in terms of plant height and biomass accumulation and was also reflected in the parasitic impact of the seed lots.
• Seed quality, specifically germination and vigour, can influence the establishment, survival, subsequent plant productivity and parasitic impact of R. minor in vegetation restoration projects. Seed quality is discussed as a key factor to consider when predicting the impact of the hemi‐parasite on community productivity and diversity.

     

Reversing expansion of Calamagrostis epigejos in a grassland biodiversity hotspot: Hemiparasitic Rhinanthus major does a better job than increased mowing intensity

Questions

Can hemiparasitic Rhinanthus major originating from a local population suppress the competitive clonal grass Calamagrostis epigejos and reverse its expansion in species‐rich semi‐natural grasslands? Does sowing seeds of R. major facilitate restoration of target meadow vegetation? Is R. major more beneficial for biodiversity restoration/conservation than increased mowing intensity, a conventional measure to suppress C. epigejos?

Location

?ertoryje National Nature Reserve, Bílé Karpaty (White Carpathians) Protected Landscape Area, Czech Republic.

Methods

We conducted a before‐after‐control‐impact experiment in meadow patches heavily infested by C. epigejos: eight blocks, each containing four plots with four treatment combinations: (1) traditional management, i.e. mowing once in summer, (2) mowing in summer and autumn (3) mowing in summer and seed sowing of R. major, (4) mowing in summer and autumn and seed sowing of R. major. Above‐ground biomass of C. epigejos and vegetation composition of each of the plots were monitored every year from 2013 to 2016. To assess the effects of treatments, we analysed biomass production of C. epigejos, herb layer cover and vegetation composition.

Results

Both sowing R. major and an additional autumn meadow cut significantly suppressed C. epigejos. Their effects were additive and of comparable size. Both treatments also had significant but markedly different effects on community composition. Rhinanthus major facilitated directional community composition change towards the regional Brachypodio‐Molinetum meadows. In contrast, increased mowing intensity significantly decreased frequency of threatened species, which however may have also been influenced by R. major.

Conclusions

Sowing of autochthonous R. major seeds was demonstrated as an efficient tool to suppress C. epigejos and facilitate community restoration. It can be combined with an additional meadow cut to further accelerate decline of the grass. The additional cut should however be used as a short‐term practice (1–2 years) only to minimize potential negative effects of its long‐term application on some threatened plant species. The effects of R. major are comparable to those of Rhinanthus alectorolophus reported previously. As a species occurring naturally in species‐rich dry grasslands, R. major has a broader and longer‐term application potential than R. alectorolophus in ecological restoration and conservation of these communities.     

Exogenous selection rather than cytonuclear incompatibilities shapes asymmetrical fitness of reciprocal Arabidopsis hybrids

Reciprocal crosses between species often display an asymmetry in the fitness of F₁ hybrids. This pattern, referred to as isolation asymmetry or Darwin's corollary to Haldane's rule, is a general feature of reproductive isolation in plants, yet factors determining its magnitude and direction remain unclear. We evaluated reciprocal species crosses between two naturally hybridizing diploid species of Arabidopsis to assess the degree of isolation asymmetry at different postmating life stages. We found that pollen from Arabidopsis arenosa will usually fertilize ovules from Arabidopsis lyrata; the reverse receptivity being less complete. Maternal A. lyrata parents set more F₁ hybrid seed, but germinate at lower frequency, reversing the asymmetry. As predicted by theory, A. lyrata (the maternal parent with lower seed viability in crosses) exhibited accelerated chloroplast evolution, indicating that cytonuclear incompatibilities may play a role in reproductive isolation. However, this direction of asymmetrical reproductive isolation is not replicated in natural suture zones, where delayed hybrid breakdown of fertility at later developmental stages, or later‐acting selection against A. arenosa maternal hybrids (unrelated to hybrid fertility, e.g., substrate adaptation) may be responsible for an excess of A. lyrata maternal hybrids. Exogenous selection rather than cytonuclear incompatibilities thus shapes the asymmetrical postmating isolation in nature.     

LOCAL ADAPTATION,PHENOTYPIC DIFFERENTIATION,AND HYBRID FITNESS IN DIVERGED NATURAL POPULATIONS OF ARABIDOPSIS LYRATA

Selection for local adaptation results in genetic differentiation in ecologically important traits. In a perennial, outcrossing model plant Arabidopsis lyrata, several differentiated phenotypic traits contribute to local adaptation, as demonstrated by fitness advantage of the local population at each site in reciprocal transplant experiments. Here we compared fitness components, hierarchical total fitness and differentiation in putatively ecologically important traits of plants from two diverged parental populations from different continents in the native climate conditions of the populations in Norway and in North Carolina (NC, U.S.A.). Survival and number of fruits per inflorescence indicated local advantage at both sites and aster life‐history models provided additional evidence for local adaptation also at the level of hierarchical total fitness. Populations were also differentiated in flowering start date and floral display. We also included reciprocal experimental F₁ and F₂ hybrids to examine the genetic basis of adaptation. Surprisingly, the F₂ hybrids showed heterosis at the study site in Norway, likely because of a combination of beneficial dominance effects from different traits. At the NC site, hybrid fitness was mostly intermediate relative to the parental populations. Local cytoplasmic origin was associated with higher fitness, indicating that cytoplasmic genomes also may contribute to the evolution of local adaptation.     

Performance of two Rhinanthus species under different hydric conditions

Rhinanthus angustifolius and Rhinanthus minor are annual hemiparasitic herbs found in open vegetations. They are closely related, hybridize frequently, and appear to have largely overlapping niches, although some floras report a preference of R. angustifolius for more humid conditions compared to R. minor. We analysed the relative fitness of both species by following their fate from germination to seed production in three different hydric treatments (wet, moist and dry) in a garden experiment. We found that R. angustifolius was either as fit as or fitter than R. minor in all conditions and for all studied parameters, with a slightly higher flower production in R. angustifolius in the wet treatment. Wet conditions had a negative effect on germination and flowering rate, but they increased flower and seed production. These observations show that environmental conditions will influence the composition of mixed Rhinanthus populations.     

QTL affecting fitness of hybrids between wild and cultivated soybeans in experimental fields

The objective of this study was to identify quantitative trait loci (QTL) affecting fitness of hybrids between wild soybean (Glycine soja) and cultivated soybean (Glycine max). Seed dormancy and seed number, both of which are important for fitness, were evaluated by testing artificial hybrids of G. soja × G. max in a multiple‐site field trial. Generally, the fitness of the F₁ hybrids and hybrid derivatives from self‐pollination was lower than that of G. soja due to loss of seed dormancy, whereas the fitness of hybrid derivatives with higher proportions of G. soja genetic background was comparable with that of G. soja. These differences were genetically dissected into QTL for each population. Three QTLs for seed dormancy and one QTL for total seed number were detected in the F₂ progenies of two diverse cross combinations. At those four QTLs, the G. max alleles reduced seed number and severely reduced seed survival during the winter, suggesting that major genes acquired during soybean adaptation to cultivation have a selective disadvantage in natural habitats. In progenies with a higher proportion of G. soja genetic background, the genetic effects of the G. max alleles were not expressed as phenotypes because the G. soja alleles were dominant over the G. max alleles. Considering the highly inbreeding nature of these species, most hybrid derivatives would disappear quickly in early self‐pollinating generations in natural habitats because of the low fitness of plants carrying G. max alleles.     

Isoproturon Resistance in Phalaris minor: Study of Physiobiochemical Parameters in Isoproturon Susceptible and Resistant Biotypes and Their Hybrids

An experiment was conducted to study physio-biochemical parameters to delineate the molecular basis of resistance against isoproturon in Phalaris minor. The reciprocal crosses of isoproturon resistant and susceptible biotypes of P. minor were made during the first year of an experiment and during the next year, these F₁ hybrids with their respective parents were evaluated for physio-biochemical and growth parameters after spraying the weed @ 0.0, 0.5, 1.0 and 1.5 kg ha^?1 with isoproturon at 22^nd and 30^th DAS. The F₁s showed heterosis and the order of resistance against isoproturon in the four biotypes was R×S > R > S×R > S. This indicated that the resistance against isoproturon in P. minor is predominantly governed by cytoplasmic inheritance, but the nuclear inheritance also has some contribution.     

Population genetic diversity and species relationships in the genus Rhinanthus L. based on microsatellite markers

The genus Rhinanthus L. is complex, containing many taxonomically unresolved taxa. In this paper we studied genetic variation and species relationships in 15 populations of six Rhinanthus species from three sections. For this purpose, we developed new microsatellite primers for R. osiliensis and used them to investigate genetic variation in two narrow endemics (R. osiliensis, R. javorkae) and in four widespread species (R. rumelicus R. wagneri, R. angustifolius and R. minor). Species‐specific private alleles were found in all species except R. osiliensis and R. angustifolius. The Bulgarian endemic R. javorkae showed the lowest genetic variation, followed by widespread R. minor and Estonian endemic R. osiliensis. Rhinanthus javorkae and R. minor were genetically most differentiated. Section Cleistolemus is weakly structured genetically, indicating close affinity between R. osiliensis, R. rumelicus, R. wagneri and R. angustifolius.     

Hybrid dwarfness in crosses between wheat (Triticum aestivum L.) and rye (Secale cereale L.): a new look at an old phenomenon

The existence of hybrid dwarfs from intraspecific crosses in wheat (Triticum aestivum) was described 100 years ago, and the genetics underlying hybrid dwarfness are well understood. In this study, we report a dwarf phenotype in interspecific hybrids between wheat and rye (Secale cereale). We identified two rye lines that produce hybrid dwarfs with wheat and have none of the hitherto known hybrid dwarfing genes. Genetic analyses revealed that both rye lines carry a single allelic gene responsible for the dwarf phenotype. This gene was designated Hdw‐R1 (Hybrid dwarf‐R1). Application of gibberellic acid (GA₃) to both intraspecific (wheat–wheat) and interspecific (wheat–rye) hybrids showed that hybrid dwarfness cannot be overcome by treatment with this phytohormone. Histological analysis of shoot apices showed that wheat–rye hybrids with the dwarf phenotype at 21 and 45 days after germination failed to develop further. Shoot apices of dwarf plants did not elongate, did not form new primordia and had a dome‐shaped appearance in the seed. The possible relationship between hybrid dwarfness and the genes responsible for the transition from vegetative to generative growth stage is discussed.     

Detecting hybridization in mixed populations ofRhinanthus minor andRhinanthus angustifolius

Rhinanthus minor andRhinanthus angustifolius are known to hybridize in mixed populations in nature. These hybridization events can have important evolutionary consequences. The development and use of species-specific RAPD and ISSR markers allowed the detection of hybrid individuals not always distinguishable with morphological characters. Two mixed populations of different ages were studied. In a young mixed 2-year-old population, both individuals of the two parental species and F₁ hybrids were found using genetic analysis, showing that hybridization occurred rapidly. Flower morphology of F₁ hybrids was too variable to distinguish all these hybrids from the parental species. This morphological variability of F₁ hybrids was also confirmed in artificial crosses in the greenhouse. In an old and no longer mixed 30-year-old population, onlyR. angustifolius plants and a few genetically introgressed individuals close toR. angustifolius were present. Genetic markers showed traces of past hybridization and introgression. Unidirectional introgression ofR. minor intoR. angustifolius with the complete disappearance ofR. minor from this population was observed.     

Role of seed germination in adaptation and reproductive isolation in Arabidopsis lyrata

Seed germination is an important developmental and life history stage. Yet, the evolutionary impact of germination has mainly been studied in the context of dormancy, or for its role in reproductive isolation between species. Here, we aim to examine multiple consequences of genetic divergence on germination traits between two Arabidopsis lyrata subspecies: ssp. petraea (Eurasia) and ssp. lyrata (North America). Postdormancy germination time, a potentially adaptive trait, showed differentiation between the populations, and quantitative trait loci (QTL) mapping revealed that the trait variation is mainly controlled by two antagonistic loci. These QTL areas contain several candidate genes with known function in postdormancy germination in A. thaliana. The sequence variation of three genes was consistent with differential selection, and they also included fixed nonsynonymous substitutions with potential to account for the phenotypic differentiation. We further show that the divergence between the subspecies has led to a slight but significant reduction in hybrid germination proportions, indicating incipient reproductive isolation. Comparison of reciprocal F₁ and F₂ progenies suggests that Bateson–Dobzhansky–Muller incompatibilities likely act through uniparentally inherited factors. Examination of genomewide transmission ratio distortion further revealed that cytonuclear interactions cause substantial pregermination inviability in the hybrids. These results confirm that seed germination has adaptive potential beyond the dormancy stage and that hybrid seed inviability can be one of the first reproductive barriers to arise during divergence.     

HYBRIDIZATION IN LEOPARD FROGS (RANA PIPIENS COMPLEX): LARVAL FITNESS COMPONENTS IN SINGLE-GENOTYPE POPULATIONS AND MIXTURES

Recognizing the predominant mode of selection in hybrid systems is important in predicting the evolutionary fate of recombinant genotypes. Natural selection is endogenous if hybrid genotypes are at a disadvantage relative to parental species independent of environment. Alternatively, relative fitness can vary in response to environmental variation (exogenous selection), and hybrid genotypes can possess fitness values equal to or greater than that of parental species. I investigated the nature of natural selection in a leopard frog hybrid system by rearing larvae of hybrid and parental genotypes between Rana blairi and R. sphenocephala in 1000-L outdoor experimental ponds. Three hybrid (F₁, backcrossj [B₁], backcross₂ [B₂]) and two parental (R. blairi [BB] and R. sphenocephala [SS]) larval genotypes were produced by artificial fertilzations using adult frogs from a natural population in central Missouri. Resultant larvae were reared in single-genotype populations and two-way mixtures at equal total numbers from hatching to metamorphosis. In single-genotype ponds, F₁ hybrid larvae had highest survival and BB were largest at metamorphosis. When F₁ and SS larvae were mixed together, F₁ hybrids had reduced survival and both F₁ and SS larvae metamorphosed at larger body masses than when reared separately. When mixed, both B₁ and SS larvae had shorter larval period lengths than when reared alone. Higher proportion of B₁ metamorphs were produced when larvae were mixed with either parental species than when reared alone. Larval fitness components as measured by survival, body mass at metamorphosis, proportion of survivors metamorphosing, and larval period length for B₂ hybrid and BB larvae were similar in single-genotype populations and mixtures. Comparison of composite fitness component estimates indicated hybrid genotypes possess equivalent or higher larval fitness relative to both parental species for the life-history fitness components measured. Despite reduced survival of F₁ hybrids in mixtures, backcross-generation hybrid genotypes demonstrated high levels of larval growth, survival, and metamorphosis in mixtures with parental species. Consequently, this study suggests natural hybridization and subsequent backcrossing between R. blairi and R. sphenocephala can produce novel and relatively fit hybrid genotypes capable of successful existence with parental species larvae. Thus, the evolutionary fate of hybrid and parental genotypes in this system may be influenced by exogenous selection mediated by genotypic composition of larval assemblages.     

Multigenerational outbreeding effects in Chinook salmon (Oncorhynchus tshawytscha)

Outbreeding, mating between genetically divergent individuals, may result in negative fitness consequences for offspring via outbreeding depression. Outbreeding effects are of notable concern in salmonid research as outbreeding can have major implications for salmon aquaculture and conservation management. We therefore quantified outbreeding effects in two generations (F₁ hybrids and F₂ backcrossed hybrids) of Chinook salmon (Oncorhynchus tshawytscha) derived from captively-reared purebred lines that had been selectively bred for differential performance based on disease resistance and growth rate. Parental lines were crossed in 2009 to create purebred and reciprocal hybrid crosses (n = 53 families), and in 2010 parental and hybrid crosses were crossed to create purebred and backcrossed hybrid crosses (n = 66 families). Although we found significant genetic divergence between the parental lines (F_ST = 0.130), reciprocal F₁ hybrids showed no evidence of outbreeding depression (hybrid breakdown) or favorable heterosis for weight, length, condition or survival. The F₂ backcrossed hybrids showed no outbreeding depression for a suite of fitness related traits measured from egg to sexually mature adult life stages. Our study contributes to the current knowledge of outbreeding effects in salmonids and supports the need for more research to better comprehend the mechanisms driving outbreeding depression.     

A metabolome‐based core hybridisation strategy for the prediction of rice grain weight across environments

Marker‐based prediction holds great promise for improving current plant and animal breeding efficiencies. However, the predictabilities of complex traits are always severely affected by negative factors, including distant relatedness, environmental discrepancies, unknown population structures, and indeterminate numbers of predictive variables. In this study, we utilised two independent F₁ hybrid populations in the years 2012 and 2015 to predict rice thousand grain weight (TGW) using parental untargeted metabolite profiles with a partial least squares regression method. A stable predictive model for TGW was built based on hybrids from the population in 2012 (r = 0.75) but failed to properly predict TGW for hybrids from the population in 2015 (r = 0.27). After integrating hybrids from both populations into the training set, the TGW of hybrids could be predicted but was largely dependent on population structures. Then, core hybrids from each population were determined by principal component analysis and the TGW of hybrids in both environments were successfully predicted (r > 0.60). Moreover, adjusting the population structures and numbers of predictive analytes increased TGW predictability for hybrids in 2015 (r = 0.72). Our study demonstrates that the TGW of F₁ hybrids across environments can be accurately predicted based on parental untargeted metabolite profiles with a core hybridisation strategy in rice. Metabolic biomarkers identified from early developmental stage tissues, which are grown under experimental conditions, may represent a workable approach towards the robust prediction of major agronomic traits for climate‐adaptive varieties.