A European phylogeography of Rhinanthus minor compared to Rhinanthus angustifolius: unexpected splits and signs of hybridization

Rhinanthus minor and Rhinanthus angustifolius (Orobanchaceae) are annual hemiparasites, which occur sympatrically in Europe and are known to hybridize. We studied chloroplast and nuclear (amplified fragment length polymorphism [AFLP]) diversity in R. minor and compared genetic structuring in this species with R. angustifolius by analyzing the AFLP data for both species simultaneously. The AFLP data revealed that populations in Italy, Greece, and southeast Russia initially identified as R. minor were so distant from the other R. minor populations that they probably belong to another, yet unidentified taxon, and we refer to them as Rhinanthus sp. R. minor s.s. showed a clear geographic genetic structure in both the chloroplast DNA (cpDNA) and nuclear genome. The simultaneous analysis of both species shed new light on the previously published findings for R. angustifolius, because some populations now turned out to belong to R. minor. The admixture analysis revealed very few individuals of mixed R. minor-R.angustifolius ancestry in the natural populations in the west of Europe, while admixture levels were higher in the east. The combined haplotype network showed that haplotype H1 was shared among all species and is likely to be ancestral. H2 was more abundant in R. angustifolius and H3 in R. minor, and the latter probably arose from H1 in this species in the east of Europe. The occurrence of H3 in R. angustifolius may be explained by introgression from R. minor, but without interspecific admixture, these are likely to have been old hybridization events. Our study underlines the importance of including related species in phylogeographic studies.     

Quantification of Leuconostoc populations in mixed dairy starter cultures using fluorescence in situ hybridization

AIMS: Development of a rapid method to identify and quantify Leuconostoc populations in mesophilic starter cultures. METHODS AND RESULTS: 16S rRNA-targeted oligonucleotide probes were used in a whole cell in situ hybridization assay for the identification of the genus Leuconostoc and an undescribed Leuconostoc ribospecies. The probes were fluorescently labelled and used to quantify the Leuconostoc populations in five different mixed starter cultures. CONCLUSIONS: There was a good correlation between the results obtained using fluorescence in situ hybridization (FISH) with that of standard plate counting methods. SIGNIFICANCE AND IMPACT OF THE STUDY: To develop a FISH method capable of identifying and quantifying the Leuconostoc population in starter cultures within 1 day.     

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.     

Detecting linkage disequilibrium in bacterial populations.

The distribution of the number of pairwise differences calculated from comparisons between n haploid genomes has frequently been used as a starting point for testing the hypothesis of linkage equilibrium. For this purpose the variance of the pairwise differences, VD, is used as a test statistic to evaluate the null hypothesis that all loci are in linkage equilibrium. The problem is to determine the critical value of the distribution of VD. This critical value can be estimated either by Monte Carlo simulation or by assuming that VD is distributed normally and calculating a one-tailed 95% critical value for VD, L, L = EVD + 1.645 sqrt(VarVD), where E(VD) is the expectation of VD, and Var(VD) is the variance of VD. If VD (observed) > L, the null hypothesis of linkage equilibrium is rejected. Using Monte Carlo simulation we show that the formula currently available for Var(VD) is incorrect, especially for genetically highly diverse data. This has implications for hypothesis testing in bacterial populations, which are often genetically highly diverse. For this reason we derive a new, exact formula for Var(VD). The distribution of VD is examined and shown to approach normality as the sample size increases. This makes the new formula a useful tool in the investigation of large data sets, where testing for linkage using Monte Carlo simulation can be very time consuming. Application of the new formula, in conjunction with Monte Carlo simulation, to populations of Bradyrhizobium japonicum, Rhizobium leguminosarum, and Bacillus subtilis reveals linkage disequilibrium where linkage equilibrium has previously been reported.     

Mutation-selection balance in mixed mating populations

An approximation to the average number of deleterious mutations per gamete, Q, is derived from a model allowing selection on both zygotes and male gametes. Progeny are produced by either outcrossing or self-fertilization with fixed probabilities. The genetic model is a standard in evolutionary biology: mutations occur at unlinked loci, have equivalent effects, and combine multiplicatively to determine fitness. The approximation developed here treats individual mutation counts with a generalized Poisson model conditioned on the distribution of selfing histories in the population. The approximation is accurate across the range of parameter sets considered and provides both analytical insights and greatly increased computational speed. Model predictions are discussed in relation to several outstanding problems, including the estimation of the genomic deleterious mutation rates (U), the generality of "selective interference" among loci, and the consequences of gametic selection for the joint distribution of inbreeding depression and mating system across species. Finally, conflicting results from previous analytical treatments of mutation-selection balance are resolved to assumptions about the life-cycle and the initial fate of mutations.     

Detecting particular genotypes in populations under nonrandom mating

We investigate the time to formation of particular genotypes in populations with nonrandom mating systems. We employ two main techniques. The first is a study of branching processes with “killing”; these are models which behave just like a standard Galton-Watson branching process with the added possibility of being terminated by the occurrence of a special event in the process. In our case, this special event corresponds to formation or detection of a group of individuals carrying a specific genotype. We then use these results and some natural approximation methods to analyze and interpret the gene formation problem in a simple way.     

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.     

Inter-specific hybridization underlies phenotypic variability in Daphnia populations

Summary In the glacial lakes of the Palaearctic three species of Cladocera commonly coexist: Daphnia hyalina, D. galeata, and D. cucullata. Frequently these populations contain not only animals which are morphologically typical for the species but also individuals of an intermediate phenotype. Electrophoretic investigations of allozyme-patterns in morphologically typical individuals reveal that each species is fixed for a different allele at the GOT locus. Morphologically intermediate animals are heterozygous for the alleles of the two species which they resemble. The allelic pattern at other loci is also consistent with the assumption that morphological intermediates are formed via interspecific hybridization. Very few backcrosses between galeata-hyalina hybrids and their parent species are found, and there is no indication of gene flow between D. cucullata and the other species.     

Transposable element number in mixed mating populations

Theoretical population genetic studies of transposable elements focus almost exclusively on random mating species, whereas many plants reproduce through partial or substantial self-fertilization. Here I develop computer simulation and analytic approximations of simplified element dynamics (transposition balanced by selective elimination) in partially self-fertilizing populations, using Ty1-copia elements for biological inspiration. Under the most plausible models and parameter values, element numbers decrease with self-fertilization when element insertions are deleterious, but may increase when ectopic exchange regulates element number. Conclusions for models of ectopic exchange depend in part on parameters for which little firm empirical evidence is available. Small changes in selfing rate can lead to abrupt changes in element number when homozygous and heterozygous elements have markedly different fitness effects. Equilibrium element numbers can be sensitive to population size, especially at high selfing rates. Elements are frequently lost in small highly selfing populations under the deleterious insertion model. In contrast, small highly selfing populations can accumulate very large numbers of elements under ectopic exchange. Empirical data on element number and localization in plants with different mating systems suggests that deleterious insertion, rather than ectopic exchange, may regulate element number. Limitations to available empirical data, especially the lack of comparison between closely related species differing in mating system, mean that this conclusion is tentative.     

Size hierarchy and mineral status of Rhinanthus angustifolius populations under different grassland management regimes

The size hierarchy and mineral concentrations of adult plants in flower and their seed crop have been investigated in three populations of Rhinanthus angustifolius under a grassland management regime consisting of haying without fertilization. The effect of this type of management on the nutrient budgets of soil or Rhinanthus plants was shown to be small initially, i.e. up to the twelfth year of changed management. Although the mean biomass of plants significantly diminished by 30% in this period, their nutrient concentration was not strongly affected. Small plants consistently had lower concentrations of N and Na than large plants. Prolongation of this management regime beyond twelve years let to a general reduction in the phosphorus concentration of the plants. An observed increase in the concentrations of K, Mn, and Cu, especially in small plants, is tentatively ascribed to an imbalance in mineral nutrition.Nomenclature of vascular plants follows Van der Meijden et al. (1983). Flora van Nederland.     

混播草地不同种群再生性的研究

在不同刈割频率和时间尺度下 ,对混播草地多年生黑麦草 (Lolium perenne)分蘖数和叶片生长、白三叶 (Trifoliumrepens)分枝数和匍匐茎生长及不同种群年产量和组分进行了连续 3年的监测研究 .结果表明 ,刈割能刺激黑麦草叶片、白三叶匍匐茎生长和分枝数发生 ,保持混播草地黑麦草和白三叶的适宜比和稳定共存 ,提高草地年生产力 ,但不同刈割频率和刈割时间对其影响差异不显著 (P >0 .0 5 ) .黑麦草叶片生长对 6月刈割效果比 8月明显 ,而白三叶匍匐茎生长则与之相反 ,黑麦草产量主要取决于叶片生长 ,白三叶产量主要取决于匍匐茎分枝数 .刈割的黑麦草、白三叶产量组分比分别为 5 0 %、15 % ,比试验前约低 10 %、5 % ,而CK为 39%、6 % .     

Detecting hybridization between wild species and their domesticated relatives

The widespread occurrence of free-ranging domestic or feral carnivores (dogs, cats) or ungulates (pigs, goats), and massive releases of captive-reproduced game stocks (galliforms, waterfowl) is raising fear that introgressive hybridization with wild populations might disrupt local adaptations, leading to population decline and loss of biodiversity. Detecting introgression through hybridization is problematic if the parental populations cannot be sampled (unlike in classical stable hybrid zones), or if hybridization is sporadic. However, the use of hypervariable DNA markers (microsatellites) and new statistical methods (Bayesian models), have dramatically improved the assessment of cryptic population structure, admixture analyses and individual assignment testing. In this paper, I summarize results of projects aimed to identify occurrence and extent of introgressive hybridization in European populations of wolves (Canis lupus), wildcats (Felis silvestris), rock partridges and red-legged partridges (Alectoris graeca and Alectoris rufa), using genetic methods. Results indicate that introgressive hybridization can be locally pervasive, and that conservation plans should be implemented to preserve the integrity of the gene pools of wild populations. Population genetic methods can be fruitfully used to identify introgressed individuals and hybridizing populations, providing data which allow evaluating risks of outbreeding depression. The diffusion in the wild of invasive feral animals, and massive restocking with captive-reproduced game species, should be carefully controlled to avoid loss of genetic diversity and disruption of local adaptations.     

Deconvolving sequence variation in mixed DNA populations.

We present an original approach to identifying sequence variants in a mixed DNA population from sequence trace data. The heart of the method is based on parsimony: given a wildtype DNA sequence, a set of observed variations at each position collected from sequencing data, and a complete catalog of all possible mutations, determine the smallest set of mutations from the catalog that could fully explain the observed variations. The algorithmic complexity of the problem is analyzed for several classes of mutations, including block substitutions, single-range deletions, and single-range insertions. The reconstruction problem is shown to be NP-complete for single-range insertions and deletions, while for block substitutions, single character insertion, and single character deletion mutations, polynomial time algorithms are provided. Once a minimum set of mutations compatible with the observed sequence is found, the relative frequency of those mutations is recovered by solving a system of linear equations. Simulation results show the algorithm successfully deconvolving mutations in p53 known to cause cancer. An extension of the algorithm is proposed as a new method of high throughput screening for single nucleotide polymorphisms by multiplexing DNA.     

Detection of minor drug-resistant populations by parallel allele-specific sequencing

We developed a highly sensitive parallel allele-specific sequencing (PASS) assay to simultaneously analyze a large number of viral genomes and detect minor drug-resistant populations at approximately 0.1-0.01% levels. Using this assay on samples from individuals infected with human immunodeficiency viruses (HIV), we successfully detected and quantified minor populations of drug-resistant viruses and performed linkage analysis of multiple-drug resistance mutations. This assay may serve as a useful tool to study drug resistance in HIV and other infectious agents.     

Avian hybridization, mixed pairing and female choice

A review of behavioural studies of hybridization and mixed pairing in birds led to the following conclusions. (1) Hybridization is more common where one of two hybridizing species is rare. (2) There is no tendency in mixed pairs or in interspecific copulations for females to belong to a rarer species than males, as would be predicted by theory based on female choice. (3) There is no evidence that females prefer males of a larger species when hybridizing, in sympatric or in parapatric species. I propose three hypotheses to account for these findings. (1) Scarcity of conspecifics facilitates hybridization in general, no matter which sex is the rare one. (2) Mistakes in mate recognition cause hybridization, especially when females choose wrongly. (3) Supernormal stimuli may sometimes cause hybridization. It is not possible to judge which of these hypotheses is the best supported, and hybridization is likely to have multiple causes.     

Detecting action potentials in neuronal populations with calcium imaging.

The study of neural circuits requires methods for simultaneously recording the activity of populations of neurons. Here, using calcium imaging of neocortical brain slices we take advantage of the ubiquitous distribution of calcium channels in neurons to develop a method to reconstruct the action potentials occurring in a population of neurons. Combining calcium imaging with whole-cell or perforated patch recordings from neurons loaded with acetoxymethyl ester or potassium salt forms of calcium indicators, we demonstrate that each action potential produces a stereotyped calcium transient in the somata of pyramidal neurons. These signals are detectable without averaging, and the signal-to-noise is sufficient to carry out a reconstruction of the spiking pattern of hundreds of neurons, up to relatively high firing frequencies. This technique could in principle be applied systematically to follow the activity of neuronal populations in vitro and in vivo.     

Detecting de novo insulin synthesis in embryonic stem cell-derived populations

Several studies in recent years have described protocols, both genetic- and culture-based, that induce the differentiation of embryonic stem (ES) cells towards a pancreatic beta-cell type. The success of previous protocols in generating insulin-producing beta-cells has been questioned due in part to uncertainty regarding cell lineage but also due to the controversy regarding the source of any insulin detected in these cells. In an attempt to address the latter, we designed a novel assay that can identify de novo insulin synthesis. The method is based on metabolic labeling combined with a modified radio-immunoassay and will routinely detect less than 5 pg/microl of de novo insulin synthesis in lysates from the insulinoma cell line MIN6. This assay failed to detect any newly translated insulin in an ES cell-derived population generated using an adapted version of a previously published, 5-stage differentiation protocol. In combination with other techniques, including immunofluorescent staining and western blot analysis to detect and quantify C-peptide, we conclude that the majority of the insulin found in these differentiated ES cell cultures is medium-derived.