The analysis of pyrrolizidine alkaloids in Jacobaea vulgaris; a comparison of extraction and detection methods

Introduction – Pyrrolizidine alkaloids (PAs) serve an important function in plant defence. Objective – To compare different extraction methods and detection techniques, namely gas chromatography with nitrogen phosphorus detection (GC‐NPD) and liquid chromatography tandem mass spectrometry (LC‐MS/MS) with quadrupole analysers for analysing PAs in Jacobaea vulgaris. Methodology – Both formic acid and sulfuric acid were tested for PA extraction from dry plant material. For GC‐NPD, reduction is required to transform PA N‐oxides into tertiary amines. Zinc and sodium metabisulfite were compared as reducing agents. Results – The lowest PA concentration measured with GC‐NPD was approximately 0.03 mg/g and with LC‐MS/MS 0.002 mg/g. The detection of major PAs by both techniques was comparable but a number of minor PAs were not detected by GC‐NPD. With the LC‐MS/MS procedure higher concentrations were found in plant extracts, indicating that losses may have occurred during the sample preparation for the GC‐NPD method. Zinc proved a more effective reducing agent than sodium metabisulfite. The sample preparation for LC‐MS/MS analysis using formic acid extraction without any reduction and purification steps is far less complex and less time consuming compared to GC‐NPD analysis with sulfuric acid extraction and PA N‐oxide reduction with zinc and purification. Conclusions – In terms of sensitivity and discrimination, formic acid extraction in combination with LC‐MS/MS detection is the method of choice for analysing PAs (both free and N‐oxides forms) in plant material. Copyright © 2009 John Wiley & Sons, Ltd.     

Genetic rescue persists beyond first-generation outbreeding in small populations of a rare plant

Habitat fragmentation commonly causes genetic problems and reduced fitness when populations become small. Stocking small populations with individuals from other populations may enrich genetic variation and alleviate inbreeding, but such artificial gene flow is not commonly used in conservation owing to potential outbreeding depression. We addressed the role of long-term population size, genetic distance between populations and test environment for the performance of two generations of offspring from between-population crosses of the locally rare plant Ranunculus reptans L. Interpopulation outbreeding positively affected an aggregate measure of fitness, and the fitness superiority of interpopulation hybrids was maintained in the second offspring (F2) generation. Small populations benefited more strongly from interpopulation outbreeding. Genetic distance between crossed populations in neutral markers or quantitative characters was not important. These results were consistent under near-natural competition-free and competitive conditions. We conclude that the benefits of interpopulation outbreeding are likely to outweigh potential drawbacks, especially for populations that suffer from inbreeding.     

Among-sibling differences in the phenotypes of juvenile fish depend on their location within the egg mass and maternal dominance rank

We investigated whether among-sibling differences in the phenotypes of juvenile fish were systematically related to the position in the egg mass where each individual developed during oogenesis. We sampled eggs from the front, middle and rear thirds of the egg mass in female brown trout of known dominance rank. In the resulting juveniles, we then measured traits that are related to individual fitness: body size, social status and standard metabolic rate (SMR). When controlling for differences among females in mean egg size, siblings from dominant mothers were initially larger (and had a lower mass-corrected SMR) if they developed from eggs at the rear of the egg mass. However, heterogeneity in the size of siblings from different positions in the egg mass diminished in lower-ranking females. Location of the egg within the egg mass also affected the social dominance of the resulting juvenile fish, although the direction of this effect varied with developmental age. This study provides the first evidence of a systematic basis for among-sibling differences in the phenotypes of offspring in a highly fecund organism.     

Testing hybridization hypotheses and evaluating the evolutionary potential of hybrids in mangrove plant species

Natural hybridization is of marked importance from global to local biological diversity. In mangroves, species ranges overlap extensively with one another and species share a long overlap of flowering time. Although hybridization has been suggested, patterns of hybridization and the evolutionary potential of hybrids are not yet fully understood. This study provides molecular evidence for the parental origins and status of hybrids in the dominant mangrove genus Rhizophora based on comparisons of chloroplast and nuclear phylogenies and estimations of genetic relatedness and structure from inter‐simple sequence repeat (ISSR) markers. Phylogenetic analyses indicate that almost all species can act as maternal parents to hybrids and that hybridization can be bidirectional. Bayesian analyses indicate that hybrids are simple F₁s, and no trace of backcrossing was detected within populations. Hybridization, for the most part, occurs almost only locally and dispersal of hybrid individuals is limited beyond the hybrid sites.     

The quantitative genetic basis of adaptive divergence in the moor frog (Rana arvalis) and its implications for gene flow

Knowledge on the relative contribution of direct genetic, maternal and environmental effects to adaptive divergence is important for understanding the drivers of biological diversification. The moor frog (Rana arvalis) shows adaptive divergence in embryonic and larval fitness traits along an acidification gradient in south-western Sweden. To understand the quantitative genetic basis of this divergence, we performed reciprocal crosses between three divergent population pairs and reared embryos and larvae at acid and neutral pH in the laboratory. Divergence in embryonic acid tolerance (survival) was mainly determined by maternal effects, whereas the relative contributions of maternal, additive and nonadditive genetic effects in larval life-history traits differed between traits, population pairs and rearing environments. These results emphasize the need to investigate the quantitative genetic basis of adaptive divergence in multiple populations and traits, as well as different environments. We discuss the implications of our findings for maintenance of local adaptation in the context of migrant and hybrid fitness.     

Identification of somatic hybrids in plant protoplast fusions with monoclonal antibodies to plasma-membrane antigens

Monoclonal antibodies generated by immunization with a plasma-membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. were used in combination with fluoresceinor rhodamine-labeled goat anti-mouse immunoglobulins to identify heterokaryons in protoplast fusion procedures. Antibody labeling did not inhibit callus formation nor plantlet regeneration. The antibodies are non-invasive and surface labeling provides clear optical discrimination of true heterokaryons from unfused aggregates as well as from parental protoplasts and homokaryons. Labeling is stable throughout fusion and hence by pre-labeling parental protoplast populations the strategy is both versatile and of general applicability.     

Simulating the extinction of parental lineages from introgressive hybridization: the effects of fitness,initial proportions of parental taxa,and mate choice

Genomic introgression among divergent taxa following human-mediated secondary contact is a growing concern for the management and conservation of aquatic biodiversity. We simulated the composition of taxa following admixture and hybridization by independently altering three variables: (1) initial proportion of parental taxa following secondary contact; (2) fitness gradients among parental and introgressant taxa; and, (3) strength of assortative mating among these taxa. Ultimately, we established that parental taxa will trend toward extinction as introgression proceeds in spite of even a heavy fitness penalty for the hybrids. Also, the number of generations required (rate) to reach an arbitrarily determined threshold of extinction (< 5.0%) was inversely related to the strength of the relative fitness gradients among parental and derivative hybridized lineages. Moreover, the rates of extinction for parental taxa depended on the initial relative proportions in the admixture with rare taxa going extinct more rapidly than abundant taxa. Finally, the strength of assortative mating (as an evolved or reinforced mechanism of pre-mating isolation) will affect the rate of extinction. Introgressive hybridization, therefore, emerges as an important risk to structural biodiversity wherever divergent, yet reproductively compatible, taxa come together naturally or are brought together through human activities.     

Selection on domestication traits and quantitative trait loci in crop–wild sunflower hybrids

The strength and extent of gene flow from crops into wild populations depends, in part, on the fitness of the crop alleles, as well as that of alleles at linked loci. Interest in crop–wild gene flow has increased with the advent of transgenic plants, but nontransgenic crop–wild hybrids can provide case studies to understand the factors influencing introgression, provided that the genetic architecture and the fitness effects of loci are known. This study used recombinant inbred lines (RILs) generated from a cross between crop and wild sunflowers to assess selection on domestication traits and quantitative trait loci (QTL) in two contrasting environments, in Indiana and Nebraska, USA. Only a small fraction of plants (9%) produced seed in Nebraska, due to adverse weather conditions, while the majority of plants (79%) in Indiana reproduced. Phenotypic selection analysis found that a mixture of crop and wild traits were favoured in Indiana (i.e. had significant selection gradients), including larger leaves, increased floral longevity, larger disk diameter, reduced ray flower size and smaller achene (seed) mass. Selection favouring early flowering was detected in Nebraska. QTLs for fitness were found at the end of linkage groups six (LG6) and nine (LG9) in both field sites, each explaining 11–12% of the total variation. Crop alleles were favoured on LG9, but wild alleles were favoured on LG6. QTLs for numerous domestication traits overlapped with the fitness QTLs, including flowering date, achene mass, head number, and disk diameter. It remains to be seen if these QTL clusters are the product of multiple linked genes, or individual genes with pleiotropic effects. These results indicate that crop trait values and alleles may sometimes be favoured in a noncrop environment and across broad geographical regions.     

Maternal effects and additive genetic inheritance in the collared lemming Dicrostonyx groenlandicus

We examined patterns of inheritance of size, growth and behavioural traits of collared lemmings (Dicrostonyx groenlandicus). Work was conducted on field-caught parents from the Canadian Arctic and their lab-born progeny. We partitioned inherited variance in traits into additive genetic and maternal effects components by using a half-sib breeding experiment in which each sire was mated to two dams. We found no evidence of statistically significant amounts of additive genetic variance in any of the traits measured. However, significant maternal effects were detected for several size- and growth-related traits. Three behavioural traits involving aggression, dispersal and activity showed no statistically significant inheritance of any kind. The presence of maternal effects may have consequences for population dynamics by causing lags resulting in inappropriate phenotypes being produced under regimes of fluctuating selection pressure. We recommend that maternal effects should be investigated as a potential general cause of population cycles in small mammals.     

Maternal allocation of androgens and antagonistic effects of yolk androgens on sons and daughters

Mothers can influence the phenotype of their offspring by adjustingthe quality of their eggs in relation to sex and reproductivevalue of the progeny. Maternal androgens in the eggs of vertebratesmay mediate such adaptive early maternal effects. However, theevolution of early maternal effects mediated by egg androgensmay be constrained by the inability of mothers to differentiallyallocate androgens to eggs with a male or a female if androgenshave different effects on sons and daughters. In this study,we increased the concentration of androgens in the eggs of barnswallows (Hirundo rustica) within the physiological range ofvariation and analyzed the effect on nestling growth and beggingbehavior. Egg androgens increased body size and mass of sonsbut reduced these characters in daughters when compared to twocontrol groups in a repeated-measures analysis of variance ofdata collected at different ages. However, the differentialeffect of androgen on the two sexes was no longer significantwhen the analysis was restricted to the age of 12 days, whenfinal body size is attained. In a second experiment, we testedwhether mothers differentially allocated androgens to eggs withsons rather than daughters while manipulating a paternal secondarysexual character. Androgen concentration did not vary in relationto paternal ornamentation or embryo sex. Hence, antagonisticeffects of egg androgens on sons and daughters may exist inthe very early posthatching life and may constrain the evolutionof adaptive maternal effects because mothers do not differentiallyallocate androgens in relation to embryo sex.     

Cascading effects of induced terrestrial plant defences on aquatic and terrestrial ecosystem function

Herbivores induce plants to undergo diverse processes that minimize costs to the plant, such as producing defences to deter herbivory or reallocating limited resources to inaccessible portions of the plant. Yet most plant tissue is consumed by decomposers, not herbivores, and these defensive processes aimed to deter herbivores may alter plant tissue even after detachment from the plant. All consumers value nutrients, but plants also require these nutrients for primary functions and defensive processes. We experimentally simulated herbivory with and without nutrient additions on red alder (Alnus rubra), which supplies the majority of leaf litter for many rivers in western North America. Simulated herbivory induced a defence response with cascading effects: terrestrial herbivores and aquatic decomposers fed less on leaves from stressed trees. This effect was context dependent: leaves from fertilized-only trees decomposed most rapidly while leaves from fertilized trees receiving the herbivory treatment decomposed least, suggesting plants funnelled a nutritionally valuable resource into enhanced defence. One component of the defence response was a decrease in leaf nitrogen leading to elevated carbon : nitrogen. Aquatic decomposers prefer leaves naturally low in C : N and this altered nutrient profile largely explains the lower rate of aquatic decomposition. Furthermore, terrestrial soil decomposers were unaffected by either treatment but did show a preference for local and nitrogen-rich leaves. Our study illustrates the ecological implications of terrestrial herbivory and these findings demonstrate that the effects of selection caused by terrestrial herbivory in one ecosystem can indirectly shape the structure of other ecosystems through ecological fluxes across boundaries.     

Maternal immune factors and the evolution of secondary sexual characters

Secondary sexual characters have been hypothesized to revealthe ability of males to resist debilitating parasites. Althoughsuch reliable signaling of parasite resistance may be maintainedby parasite–host coevolution, maternal effects potentiallyprovide a previously neglected factor that could affect thelevel of genetic variation in resistance to parasites. Thatcould be the case because maternal effects have an entirelyenvironmental basis, or because they can maintain considerableamounts of genetic variation through epistatic effects, evenin the presence of strong directional selection. Maternal effectshave been shown to occur as maternal allocation of immune factorsto offspring, and such allocation may depend on the mating prospectsof sons, causing mothers to differentially allocate maternaleffects to eggs in species subject to intense sexual selection.Here we show that a maternal effect through innate antibacterialimmune defense, lysozyme, which is transferred from the motherto the egg in birds, is positively associated with the evolutionof secondary sexual characters. Previous studies have shownthat females differentially allocate lysozyme to their eggswhen mated to attractive males, and elevated levels of lysozymeare associated with reduced hatching failure and superior healthamong neonates and adults. In this study, comparative analysesof lysozyme from eggs of 85 species of birds showed a strongpositive relationship between brightness of male plumage andegg lysozyme, even when controlling for potentially confoundingvariables. These findings suggest that maternal immune factorsmay play a role in the evolution of secondary sexual characters.     

Statistical convenience vs biological insight: consequences of data transformation for the analysis of fitness variation in heterogeneous environments

In plants, more favourable environmental conditions can lead to dramatic increases in both mean fitness and variance in fitness. This results in data that violate the equality-of-variance assumption of anova, a problem that most empiricists would address by log-transforming fitness values. Using heuristic data sets and simple simulations, we show that anova on log-transformed fitness consistently fails to match the outcome of selection in a heterogeneous environment or its sensitivity to environmental frequency. Only anova based on relative fitness within environments accurately predicts the sensitivity of genotype selection to the frequency of alternative environments. Parallel analyses of variance based on absolute fitness and relative fitness can bracket the expected success of alternative genotypes under hard and soft selection, respectively. For example, for Sinapis arvensis growing in full sun and partial shade treatments, families achieving high fitness in the best environment are favoured under hard selection, whereas soft selection favours different families that achieve consistently good performance across environments. Based on these findings, we recommend that log-transformation of fitness should no longer be standard practice in ecological genetics studies. Weighted anova is a preferable method for dealing with unequal variances, and investigators should also make greater use of techniques such as quantile regression or resampling to describe and evaluate fitness variation across heterogeneous environments.