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.     

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.     

Fitness of reciprocal F1 hybrids between Rhinanthus minor and Rhinanthus major under controlled conditions and in the field

The performance of first‐generation hybrids determines to a large extent the long‐term outcome of hybridization in natural populations. F₁ hybrids can facilitate further gene flow between the two parental species, especially in animal‐pollinated flowering plants. We studied the performance of reciprocal F₁ hybrids between Rhinanthus minor and R. major, two hemiparasitic, annual, self‐compatible plant species, from seed germination to seed production under controlled conditions and in the field. We sowed seeds with known ancestry outdoors before winter and followed the complete life cycle until plant death in July the following season. Germination under laboratory conditions was much lower for the F₁ hybrid formed on R. major compared with the reciprocal hybrid formed on R. minor, and this confirmed previous results from similar experiments. However, this difference was not found under field conditions, which seems to indicate that the experimental conditions used for germination in the laboratory are not representative for the germination behaviour of the hybrids under more natural conditions. The earlier interpretation that F₁ hybrid seeds formed on R. major face intrinsic genetic incompatibilities therefore appears to be incorrect. Both F₁ hybrids performed at least as well as and sometimes better than R. minor, which had a higher fitness than R. major in one of the two years in the greenhouse and in the field transplant experiment. The high fitness of the F₁ hybrids confirms findings from naturally mixed populations, where F₁ hybrids appear in the first year after the two species meet, which leads to extensive advanced‐hybrid formation and introgression in subsequent generations.     

Rhinanthus minor population genetic structure and subspecies: Potential seed sources of a keystone species in grassland restoration projects

In the last few decades unimproved semi-natural grasslands have been affected by intensification of land use and habitat fragmentation. Because of their biodiversity these species-rich grasslands are of high conservation importance and efforts are under way to restore such habitats. Detailed knowledge of within species diversity will aid deciding on the optimal seed source for such restoration projects, e.g. local genotypes or ecotypes. Rhinanthus minor is a species that is typically found in semi-natural grasslands and is commonly used in grassland restoration projects. This is because R. minor is a hemiparasitic plant that takes minerals and nutrients from its host, which in turn decreases the host's biomass and leads to opportunities for less competitive species in the vegetation. Here, we investigate genetic diversity within and between R. minor populations. This allowed us to test whether the six different subspecies of R. minor that have been described in the UK, based on their morphology, flowering time, and habitat, can be differentiated using molecular markers. We identified moderate levels of genetic differentiation between R. minor populations within the UK. In addition, R. minor individuals from the UK appear to be distinct from R. minor and Rhinanthus angustifolius individuals from other European countries based on microsatellite genotyping and DNA sequencing of cpDNA and rDNA ITS. The molecular markers used in the current study did not separate populations of R. minor based on either their subspecies or habitat. The implication for the use of R. minor in grassland restoration projects seems to be that it is not necessary to use local seeds or seeds from the same subspecies.     

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.     

Shared pollinators and pollen transfer dynamics in two hybridizing species, Rhinanthus minor and R. angustifolius

Gene flow between hybridizing plant species depends strongly on pollinator behaviour, which affects pollen transfer among floral types and reproductive isolation. We examined bumblebee behaviour and pollen transfer between two hybridizing Rhinanthus species that are very similar in ecology and floral traits. The two species, Rhinanthus minor and R. angustifolius, shared similar pollinator guilds and assemblages, but pollinator recruitment and flower visitation rates were higher in R. angustifolius sites, probably because of its higher reward levels and better visibility. When presented with Rhinanthus flowers, bumblebees that previously foraged on R. angustifolius were less prone to visit R. minor inflorescences, while R. minor foragers accepted both species in similar proportions. Although Rhinanthus has been cited as a case of mechanical isolation resulting from interactions between bee behaviour and differences in stigma and anther placement, we found no support for efficient mechanical reproductive isolation. Bumblebees that foraged on R. minor flowers carried more pollen, but pollen placement on their bodies was similar to that of bees that visited R. angustifolius, and cross-specific stigmatic pollen deposition was similar in both directions. However, the asymmetry in pollinator handling time between the two species, due to dissimilar pollen rewards, may have lowered relative heterospecific pollen receipt on R. angustifolius, suggesting that net gene flow resulting from pollen transfer dynamics is more likely towards R. minor, although this effect remains weak and will be most likely counterbalanced by context-based labile pollinator preference.     

Rhinanthus plants found in calcareous fens on Gotland (Sweden): Are they related to Rhinanthus osiliensis from Saaremaa (Estonia)?

Rhinanthus osiliensis is a narrow endemic growing in calcareous spring fens on the island Saaremaa, Estonia. Plants very similar to R. osiliensis have been found in Sweden on the island of Gotland. Fifteen microsatellite markers were used to characterize genetic variation within R. osiliensis and the unknown taxon from Gotland. Results were compared with common Rhinanthus minor and Rhinanthus angstifolius. Our results showed that genetic diversity was remarkably lower in the Rhinanthus sp. from Gotland compared to Estonian endemic R. osiliensis. The microsatellite loci we used distinguished four studied species. In total, 46 species-specific alleles were found. The Neighbor-Joining dendrogram based on Nei's genetic distances, the Bayesian clustering analysis and the principle coordinate analysis showed that R. osiliensis and Rhinanthus sp. from Gotland are genetically differentiated and could not be the same species. In addition, endemic R. osiliensis was closer to widespread R. angustifolius. Further genetic comparison between unknown Rhinanthus sp. and local R. angustifolius and R. minor from Gotland is necessary for determine the species status and checking the possible alternative hypothesis about the origin of the unclear taxon.     

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.     

Genetic diversity and differentiation in six species of the genus Rhinanthus (Orobanchaceae)

The intricate genus Rhinanthus L. contains many taxonomically unresolved taxa. The assessment of genetic diversity may clarify the differentiation and taxonomic relationships between species and help to determine their mating system, which is important for understanding the future prospects of populations and species. Isozymes were used to assess the mating system, genetic diversity, and differentiation in R. rumelicus, R. osiliensis, R. wagneri, R. alectorolophus, and R. angustifolius from sect. Cleistolemus and R. minor from sect. Rhinanthus. Genetic diversity at species level was found to be higher in widespread R. angustifolius and R. rumelicus compared with insular endemic R. osiliensis. Positive inbreeding coefficient revealed dominant inbreeding in endemic R. osiliensis and R. wagneri. We also detected a significant departure from Hardy–Weinberg equilibrium in some populations of R. rumelicus and R. angustifolius. The common R. alectorolophus and R. minor and several populations of R. rumelicus and R. angustifolius displayed a mixed mating mode. Six studied species divided into three distinct clusters. Rhinanthus alectorolophus was the most differentiated taxon among the investigated species. The species of sect. Cleistolemus formed two distinct groups. The first cluster consisted only of R. alectorolophus while another comprised R. rumelicus, R. osiliensis, R. wagneri, and R. angustifolius, indicating their close affinity. Thus, our isozyme results call into question the monophyly of section Cleistolemus and the taxonomic position of R. alectorolophus.     

Hybridization and introgression between the exotic Siberian elm, Ulmus pumila, and the native Field elm, U. minor, in Italy

In response to the first Dutch elm disease (DED) pandemic, Siberian elm, Ulmus pumila, was planted to replace the native elm, U. minor, in Italy. The potential for hybridization between these two species is high and repeated hybridization could result in the genetic swamping of the native species and facilitate the evolution of invasiveness in the introduced species. We used genetic markers to examine the extent of hybridization between these two species and to determine the pattern of introgression. We quantified and compared the level of genetic diversity between the hybrids and the two parental species. Hybrids between U. pumila and U. minor were common. The pattern of introgression was not as strongly biased towards U. pumila as was previously observed for hybrids between U. rubra and U. pumila in the United States. The levels of heterozygosity were similar between U. minor and the hybrids and both groups had higher levels of heterozygosity relative to U. pumila. The programs Structure and NewHybrids indicated the presence of first- (F₁) and second- generation (F₂) hybrids and of backcrosses in the hybrid population. The presence of healthy DED resistant U. minor individuals combined with the self-compatibility of U. minor could help explain the presence of F₂ individuals in Italy. The presence of F₂ individuals, where most of the variability present in the hybrids will be released, could facilitate rapid evolution and the potential evolution of invasiveness of U. pumila in Italy.     

PARENTAL FREQUENCIES AND SPATIAL CONFIGURATION SHAPE BUMBLEBEE BEHAVIOR AND FLORAL ISOLATION IN HYBRIDIZING RHINANTHUS

To shed light on the role played by pollinators in the diversification of angiosperms, focus is needed on how floral isolation varies locally in the early stages of plant divergence. The few studies performed so far have often used species pairs with distinct pollination syndromes and contrasting floral displays. Here, we focus on a hybridizing pair (Rhinanthus minor and Rhinanthus angustifolius) with strong similarities in flower morphology and pollinators (bumblebees). We examined how ethological isolation changes locally in relation to relative Rhinanthus frequencies, spatial configurations, and pollinator assemblages. Interestingly, floral divergence based on adaptation to different pollinators is unlikely in Rhinanthus: no relationship was found between floral isolation and the local pollinator assemblage. In contrast, species frequency and spatial arrangement strongly influenced bumblebee behavior, ethological isolation, and thus potentially hybrid formation. When both Rhinanthus were present in equal proportions, bees generally preferred the more rewarding and conspicuous species. However, when the Rhinanthus frequencies were unbalanced, the more abundant species was preferred, although this was less pronounced when the less rewarding R. minor predominated. Ethological isolation is highly sensitive to site characteristics, and can be as high as in species with contrasting floral displays and pollinator suites, even though flowers are similar.     

Local adaptation, intrinsic coadaptation and the effects of environmental stress on interpopulation hybrids in the copepod Tigriopus californicus

Hybridization between divergent populations may cause a reduction in fitness due either to disruption in local adaptation or disruption in intrinsic coadaptation. We tested for both effects in the tidepool copepod Tigriopus californicus. Fitness surrogates were measured in pure populations and interpopulation hybrids, with broods split between three environmental treatments: (1) Standard: 15 °C/100% seawater; (2) High temperature: 25 °C/100% seawater; and (3) Low salinity: 15 °C/50% seawater. Effects of these treatments were independent of population, providing no evidence for local adaptation. Comparison of mean fitness in pure populations, F₁ hybrids and F₂ hybrids showed that hybridization caused beneficial interactions between alleles at the same locus and detrimental interactions between loci (i.e., disruption of intrinsic coadaptation). The effects of hybridization were environmentally dependent as exposure to the most stressful treatment (high temperature) resulted in the maintenance of F₁ heterosis and a substantial reduction in F₂ breakdown.     

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.     

Impact of hemiparasitic Rhinanthus angustifolius and R. minor on nitrogen availability in grasslands

Root hemiparasites like Rhinanthus angustifolius C.C. Gmel and R. minor L. have a potential to accelerate the restoration of semi-natural grasslands because they may decrease above-ground biomass of the vegetation. This, in turn, may be beneficial for species diversity. It is known that hemiparasites often accumulate high nutrient concentrations in their above-ground parts, resulting in high quality litter. Because of the short life cycle of many parasitic plants, litter is released early in the season and the main part is not removed from the grassland by hay-making. This has been shown to yield an increased nutrient availability locally. We performed an introduction experiment with R. angustifolius and R. minor in three semi-natural grasslands in Flanders (Belgium). In the second year after sowing, the above-ground nitrogen (N) content of the grasses and of the potential host vegetation (excluding the hemiparasite), was increased in the parasitized plots. The reduction of grass (and legume) above-ground biomass in parasitized plots resulted in a decrease in the total above-ground N uptake of grasses, host and total vegetation (ex- and including the parasite, respectively) of the parasitized plots compared to the control. Furthermore, with a tracer experiment (¹⁵N), we demonstrated that the N from the added tracer was relatively less available in parasitized plots, suggesting larger soil N pools in these treatments. This is probably the consequence of increased mineralization, resulting from the high-quality, parasitic litter. Further experiments should be conducted to investigate the impact of hemiparasitic Rhinanthus spp., e.g. on the availability of other nutrients such as phosphorus.     

Interactions of the Hemiparasitic Species Rhinanthus minor with its Host Plant Community at Two Nutrient Levels

For root hemiparasites, host plants are both the source of water and nutrients below-ground, but competitors for light above-ground. Hemiparasites can reduce host biomass, and in this way considerably affect the whole plant community. To investigate these effects, we carried out two experiments in an oligotrophic meadow with a native population of Rhinanthus minor. In the first experiment, removal of R. minor was combined with fertilization in a factorial design, and in the second one, we manipulated R. minor density by thinning. The presence of R. minor decreased the biomass of its host community, mostly by suppressing grasses. In this way, the species was able to counterbalance the effect of fertilization, which increased community biomass and in particular that of grasses. Neither the presence of R. minor nor fertilization affected the total number of species or the Shannon-Wiener diversity index (H’) of the host community. However, H’ of grasses was higher and H’ of forbs (non-leguminous dicots) was lower in the presence of R. minor. Reduction of grasses by R. minor favored mainly the dominant forb Plantago lanceolata, which partly acquired the role of a competitive dominant. Effects of R. minor on community diversity seem to be highly dependent on the relative sensitivities of dominant and subordinate species. Fertilization increased the mortality of seedlings, resulting in a lower number of flowering plants. However, surviving individuals on average produced more flowers. Thinning resulted in lower mortality of R. minor plants. This indicates that intraspecific competition in R. minor populations results in negative density dependence.     

Outbreeding Depression in Hybrids Between Spatially Separated Pink Salmon, Oncorhynchus gorbuscha, Populations: Marine Survival, Homing ability, and Variability in Family Size

Hybridization between distinct populations and introgression of nonnative genes can erode fitness of native populations through outbreeding depression, either by producing a phenotype intermediate to that of both contributing genomes (and maladapted in either population's environment) or by disrupting distinct coadapted complexes of epistatic genes. In salmon, fitness-related traits such as homing ability or family-size distribution may be eroded. We investigated geographically separated pink salmon populations in repeated trials in independent broodyears (odd and even). Hybrids were made between female Auke Creek (Southeast Alaska) pink salmon and Pillar Creek (Kodiak Island, ～1 000 km away) males; hybrids and their offspring were compared to offspring of control crosses of the same females with Auke Creek males. Parentage assignment from microsatellite analysis was used to improve estimates of survival and straying and to examine variation of family size. Hybridization reduced return rates of adults (a proxy for survival at sea) in the F₁ generation in the odd-year broodline (p < 0.0001) but not in the even-year broodline (p = 0.678). Hybridization reduced survival in both the odd- and even-broodyear F₂ (p < 0.005 and p < 0.0001). Hybridization did not appear to impair homing ability; weekly surveys revealed similar straying rates (～2%) by both hybrid and control fish into nearby (～1 km) Waydelich Creek in both generations in both trials. Hybridization did not increase the index of variability (σ²/μ) in family size. Decreased survival in the hybrid F₂ generation supports an epistatic model of outbreeding depression; nonepistatic effects may have contributed to reduced survival in the odd-broodyear F₁ hybrid fish. Outbreeding depression in hybrids of geographically separated populations demonstrates that introgression of nonnative fish can erode fitness, and should be recognized as a potential detriment of both aquaculture and management practices.     

Aneuploidy and isolation in two Hypochoeris species

The annual species Hypochoeris glabra (2n = 10) and the perennial H. radicata (2n = 8) hybridise readily in nature and in experiment. During meiosis in F₁ hybrids the maximum association is a chain of seven and a bivalent indicating that at least three interchanges differentiate the two genomes. The nucleolar chromosomes in the two species are homologous and form a ring bivalent. They are, however, differentiated since in the F₁ hybrid only one nucleolar-organiser region is expressed. Although chromosomal differentiation reduces the egg fertility of F₁ hybrids to about 1%, viable backcross hybrids to H. radicata as pollen parent have been experimentally produced and occur in natural populations. Backcrosses with 8, 9 and rarely 13 chromosomes are found and those with 2n = 8 are fully interfertile with H. radicata. Gene flow may therefore take place in natural populations across an aneuploid barrier. The direction of gene flow in Hypochoeris is probably unidirectional from the annual to the perennial.     

Population genetics of the common long-armed octopus Octopus minor (Sasaki, 1920) (Cephalopoda: Octopoda) in Chinese waters based on microsatellite analysis

Octopus minor (Sasaki, 1920) is a commercially important cephalopod in Chinese waters. To provide a theoretical basis for resource protection and sustainable management, we investigated genetic structure of ten O. minor populations in Chinese waters using microsatellite DNA markers. Eight microsatellite loci revealed high allelic diversity with 11–26 alleles per locus. Observed and expected heterozygosity varied from 0.412 to 0.900 and from 0.337 to 0.845, respectively. The overall F_ST value was 0.198, indicating great genetic differentiation among populations. The F_ST value between Yilan and other populations reached more than 0.3 that may be indicative of subspecies rank. Mantel test showed significant correlations between genetic and geographic distance (R = 0.383, P = 0.004) indicating that genetic differentiation of O. minor conformed to a pattern of isolation-by-distance. Using the Neighbor-joining method, cluster analysis divided nine populations into three groups and divided ten populations into two groups wherein Yilan was distinguished from the other populations. Analysis based on F_ST, D_c values and clustering highlighted the heterogeneity of Yilan and the relative homogeneity between Yilan and Ganyu. The significant population genetic structure of O. minor is related to the combined effects of geographical barriers, current features and life history characteristics.     

Genetic introgression of hybrid Rhododendron x intermedium Tausch is habitat mediated: Evidences from south-eastern Alps (Italy)

Hybridization between Rhododendron ferrugineum L. and R. hirsutum L. in south-eastern Alps was examined in order to (i) evaluate the breeding direction and the extent of backcrossing between hybrids and the parental species, (ii) define which processes facilitate speciation and maintain species identities and (iii) clarify the role of rock geochemistry in hybridization events. Individuals of three hybrid populations were analysed by morphological and molecular markers. The internal transcribed spacer and trnH–psbA distinguished the parental species and F₁ hybrids while only the simple sequence repeat markers recognized genotype classes: F, H, F₁, F₂, BxF (backcross to R. ferrugineum) and BxH (backcross to R. hirsutum). Combining morphological and molecular data, we found that the tested populations had complex genetic structure: the F₁ individuals produce F₂ hybrids and backcross to parental species. Due to R. hirsutum phenology, most backcrossing events were with this parental species (asymmetric hybridization). Geochemical analyses indicate that alkaline soil conditions linked to calcareous dolomitic rocks promoted the genetic assimilation of R. hirsutum. In addition, R. x intermedium shows a higher edaphic adaptation than R. hirsutum as it can be found on a wide range of calcareous-dolomitic rocks as well as on weakly acidic soils of natural or anthropogenic origin.     

Reproductive phenology and physiological traits in the red mangrove hybrid complex (Rhizophora mangle and R. racemosa) across a natural gradient of nutrients and salinity

Factors modulating introgressive hybridization between the red mangrove species Rhizophora mangle and R. racemosa in spatially defined sites are poorly understood. To investigate this, we evaluated the reproductive phenology and the nutrient and physiological traits in those two species and their F₁ hybrids genotyped with microsatellite data across a natural hybrid zone from the Pacific coast of Panama. We found no evidence that reproductive phenology represents a barrier to gene flow, because R. mangle and the F₁ hybrids produced flowers and propagules throughout the annual cycle, while R. racemosa flowered only in the dry season. Soil nutrient concentrations decreased landward, while soil salinity varied only slightly. Foliar nutrients and δ¹⁵N signatures varied according to the soil nutrient gradient, but only foliar phosphorus and carbon varied among species. In contrast, two structural variables (height and trunk diameter) and leaf variables related to salinity tolerance (Na, Cl:Na, K:Na, cation:anion) and water-use efficiency (i.e., δ¹³C) differed among species, suggesting higher salinity tolerance for R. mangle and F₁ hybrids compared with R. racemosa. We conclude that parental species and F₁ hybrids differ in salinity tolerance and water-use efficiency, which could be associated with adaptive evolution of the red mangrove hybrid complex.