Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

Evaluation of a polymerase chain reaction-restriction fragment length polymorphism assay for dermatophyte and nondermatophyte identification in onychomycosis.

BACKGROUND: Dermatophytes are the main cause of onychomycoses, but various nondermatophyte filamentous fungi are often isolated from abnormal nails. The correct identification of the aetiological agent of nail infections is necessary in order to recommend appropriate treatment. OBJECTIVE: To evaluate a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA for fungal identification in nails on a large number of samples in comparison with cultures. METHODS: Infectious fungi were analysed using PCR-RFLP in 410 nail samples in which fungal elements were observed in situ by direct mycological examination (positive samples). The results were compared with those previously obtained by culture of fungi on Sabouraud agar from the same nail samples. RESULTS: PCR-RFLP identification of fungi in nails allowed validation of the results obtained in culture when Trichophyton spp. grew from infected samples. In addition, nondermatophyte filamentous fungi could be identified with certainty as the infectious agents in onychomycosis, and discriminated from dermatophytes as well as from transient contaminants. The specificity of the culture results relative to PCR-RFLP appeared to be 81%, 71%, 52% and 63% when Fusarium spp., Scopulariopsis brevicaulis, Aspergillus spp. and Candida spp., respectively, grew on Sabouraud agar. It was also possible to identify the infectious agent when direct nail mycological examination showed fungal elements, but negative results were obtained from fungal culture. CONCLUSIONS: Improved sensitivity for the detection of fungi in nails was obtained using the PCR-RFLP assay. Rapid and reliable molecular identification of the infectious fungus can be used routinely and presents several important advantages compared with culture in expediting the choice of appropriate antifungal therapy.

Morphological, ecological and genetic aspects associated with endemism in the Fly Orchid group.

The European genus Ophrys (Orchidaceae) is famous for its insect-like floral morphology, an adaptation for a pseudocopulatory pollination strategy involving Hymenoptera males. A large number of endemic Ophrys species have recently been described, especially within the Mediterranean Basin, which is one of the major species diversity hotspots. Subtle morphological variation and specific pollinator dependence are the two main perceptible criteria for describing numerous endemic taxa. However, the degree to which endemics differ genetically remains a challenging question. Additionally, knowledge regarding the factors underlying the emergence of such endemic entities is limited. To achieve new insights regarding speciation processes in Ophrys, we have investigated species boundaries in the Fly Orchid group (Ophrys insectifera sensu lato) by examining morphological, ecological and genetic evidence. Classically, authors have recognized one widespread taxon (O. insectifera) and two endemics (O. aymoninii from France and O. subinsectifera from Spain). Our research has identified clear morphological and ecological factors segregating among these taxa; however, genetic differences were more ambiguous. Insights from cpDNA sequencing and amplified fragment length polymorphisms genotyping indicated a recent diversification in the three extant Fly Orchid species, which may have been further obscured by active migration and admixture across the European continent. Our genetic results still indicate weak but noticeable phylogeographic clustering that partially correlates with the described species. Particularly, we report several isolated haplotypes and genetic clusters in central and southeastern Europe. With regard to the morphological, ecological and genetic aspects, we discuss the endemism status within the Fly Orchid group from evolutionary, taxonomical and conservation perspectives.

Characterization of microsatellite loci and reliable genotyping in a polyploid plant, Mercurialis perennis (Euphorbiaceae).

For many applications in population genetics, codominant simple sequence repeats (SSRs) may have substantial advantages over dominant anonymous markers such as amplified fragment length polymorphisms (AFLPs). In high polyploids, however, allele dosage of SSRs cannot easily be determined and alleles are not easily attributable to potentially diploidized loci. Here, we argue that SSRs may nonetheless be better than AFLPs for polyploid taxa if they are analyzed as effectively dominant markers because they are more reliable and more precise. We describe the transfer of SSRs developed for diploid Mercurialis huetii to the clonal dioecious M. perennis. Primers were tested on a set of 54 male and female plants from natural decaploid populations. Eight of 65 tested loci produced polymorphic fragments. Binary profiles from 4 different scoring routines were used to define multilocus lineages (MLLs). Allowing for fragment differences within 1 MLL, all analyses revealed the same 14 MLLs without conflicting with merigenet, sex, or plot assignment. For semiautomatic scoring, a combination of as few as 2 of the 4 most polymorphic loci resulted in unambiguous discrimination of clones. Our study demonstrates that microsatellite fingerprinting of polyploid plants is a cost efficient and reliable alternative to AFLPs, not least because fewer loci are required than for diploids.

Fine-scale genetic structure and marginal processes in an expanding population of Biscutella laevigata L. (Brassicaceae).

Evolutionary processes acting at the expanding margins of a species' range are still poorly understood. Genetic drift is considered prevalent in marginal populations, and the maintenance of genetic diversity during recolonization might seem puzzling. To investigate such processes, a fine-scale investigation of 219 individuals was performed within a population of Biscutella laevigata (Brassicaceae), located at the leading edge of its range. The survey used amplified fragment length polymorphisms (AFLPs). As commonly reported across the whole species distribution range, individual density and genetic diversity decreased along the local axis of recolonization of this expanding population, highlighting the enduring effect of the historical colonization on present-day diversity. The self-incompatibility system of the plant may have prevented local inbreeding in newly found patches and sustained genetic diversity by ensuring gene flow from established populations. Within the more continuously populated region, spatial analysis of genetic structure revealed restricted gene flow among individuals. The distribution of genotypes formed a mosaic of relatively homogenous patches within the continuous population. This pattern could be explained by a history of expansion by long-distance dispersal followed by fine-scale diffusion (that is, a stratified dispersal combination). The secondary contact among expanding patches apparently led to admixture among differentiated genotypes where they met (that is, a reshuffling effect). This type of dynamics could explain the maintenance of genetic diversity during recolonization.

Combining palaeodistribution modelling and phylogeographical approaches for identifying glacial refugia in Alpine Primula

Aim We investigated the late Quaternary history of two closely related and partly sympatric species of Primula from the south-western European Alps, P. latifolia Lapeyr. and P. marginata Curtis, by combining phylogeographical and palaeodistribution modelling approaches. In particular, we were interested in whether the two approaches were congruent and identified the same glacial refugia. Location South-western European Alps. Methods For the phylogeographical analysis we included 353 individuals from 28 populations of P. marginata and 172 individuals from 15 populations of P. latifolia and used amplified fragment length polymorphisms (AFLPs). For palaeodistribution modelling, species distribution models (SDMs) were based on extant species occurrences and then projected to climate models (CCSM, MIROC) of the Last Glacial Maximum (LGM), approximately 21 ka. Results The locations of the modelled LGM refugia were confirmed by various indices of genetic variation. The refugia of the two species were largely geographically isolated, overlapping only 6% to 11% of the species' total LGM distribution. This overlap decreased when the position of the glacial ice sheet and the differential elevational and edaphic distributions of the two species were considered. Main conclusions The combination of phylogeography and palaeodistribution modelling proved useful in locating putative glacial refugia of two alpine species of Primula. The phylogeographical data allowed us to identify those parts of the modelled LGM refugial area that were likely source areas for recolonization. The use of SDMs predicted LGM refugial areas substantially larger and geographically more divergent than could have been predicted by phylogeographical data alone

Broad-scale adaptive genetic variation in alpine plants is driven by temperature and precipitation.

Identifying adaptive genetic variation is a challenging task, in particular in non-model species for which genomic information is still limited or absent. Here, we studied distribution patterns of amplified fragment length polymorphisms (AFLPs) in response to environmental variation, in 13 alpine plant species consistently sampled across the entire European Alps. Multiple linear regressions were performed between AFLP allele frequencies per site as dependent variables and two categories of independent variables, namely Moran's eigenvector map MEM variables (to account for spatial and unaccounted environmental variation, and historical demographic processes) and environmental variables. These associations allowed the identification of 153 loci of ecological relevance. Univariate regressions between allele frequency and each environmental factor further showed that loci of ecological relevance were mainly correlated with MEM variables. We found that precipitation and temperature were the best environmental predictors, whereas topographic factors were rarely involved in environmental associations. Climatic factors, subject to rapid variation as a result of the current global warming, are known to strongly influence the fate of alpine plants. Our study shows, for the first time for a large number of species, that the same environmental variables are drivers of plant adaptation at the scale of a whole biome, here the European Alps.

Automated scoring of AFLPs using RawGeno v 2.0, a free R CRAN library.

Amplified Fragment Length Polymorphisms (AFLPs) are a cheap and efficient protocol for generating large sets of genetic markers. This technique has become increasingly used during the last decade in various fields of biology, including population genomics, phylogeography, and genome mapping. Here, we present RawGeno, an R library dedicated to the automated scoring of AFLPs (i.e., the coding of electropherogram signals into ready-to-use datasets). Our program includes a complete suite of tools for binning, editing, visualizing, and exporting results obtained from AFLP experiments. RawGeno can either be used with command lines and program analysis routines or through a user-friendly graphical user interface. We describe the whole RawGeno pipeline along with recommendations for (a) setting the analysis of electropherograms in combination with PeakScanner, a program freely distributed by Applied Biosystems; (b) performing quality checks; (c) defining bins and proceeding to scoring; (d) filtering nonoptimal bins; and (e) exporting results in different formats.

Predicting present and future intra-specific genetic structure through niche hindcasting across 24 millennia.

Paleoclimatic reconstructions coupled with species distribution models and identification of extant spatial genetic structure have the potential to provide insights into the demographic events that shape the distribution of intra-specific genetic variation across time. Using the globeflower Trollius europaeus as a case-study, we combined (1) Amplified Fragment Length Polymorphisms, (2) suites of 1000-years stepwise hindcasted species distributions and (3) a model of diffusion through time over the last 24,000 years, to trace the spatial dynamics that most likely fits the species' current genetic structure. We show that the globeflower comprises four gene pools in Europe which, from the dry period preceding the Last Glacial Maximum, dispersed while tracking the conditions fitting its climatic niche. Among these four gene pools, two are predicted to experience drastic range retraction in the near future. Our interdisciplinary approach, applicable to virtually any taxon, is an advance in inferring how climate change impacts species' genetic structures.

Integrating species distribution models (SDMs) and phylogeography for two species of Alpine Primula.

The major intention of the present study was to investigate whether an approach combining the use of niche-based palaeodistribution modeling and phylo-geography would support or modify hypotheses about the Quaternary distributional history derived from phylogeographic methods alone. Our study system comprised two closely related species of Alpine Primula. We used species distribution models based on the extant distribution of the species and last glacial maximum (LGM) climate models to predict the distribution of the two species during the LGM. Phylogeographic data were generated using amplified fragment length polymorphisms (AFLPs). In Primula hirsuta, models of past distribution and phylogeographic data are partly congruent and support the hypothesis of widespread nunatak survival in the Central Alps. Species distribution models (SDMs) allowed us to differentiate between alpine regions that harbor potential nunatak areas and regions that have been colonized from other areas. SDMs revealed that diversity is a good indicator for nunataks, while rarity is a good indicator for peripheral relict populations that were not source for the recolonization of the inner Alps. In P. daonensis, palaeo-distribution models and phylogeographic data are incongruent. Besides the uncertainty inherent to this type of modeling approach (e.g., relatively coarse 1-km grain size), disagreement of models and data may partly be caused by shifts of ecological niche in both species. Nevertheless, we demonstrate that the combination of palaeo-distribution modeling with phylogeographical approaches provides a more differentiated picture of the distributional history of species and partly supports (P. hirsuta) and partly modifies (P. daonensis and P. hirsuta) hypotheses of Quaternary distributional history. Some of the refugial area indicated by palaeodistribution models could not have been identified with phylogeographic data.

Ecology and life history affect different aspects of the population structure of 27 high-alpine plants.

A plant species' genetic population structure is the result of a complex combination of its life history, ecological preferences, position in the ecosystem and historical factors. As a result, many different statistical methods exist that measure different aspects of species' genetic structure. However, little is known about how these methods are interrelated and how they are related to a species' ecology and life history. In this study, we used the IntraBioDiv amplified fragment length polymorphisms data set from 27 high-alpine species to calculate eight genetic summary statistics that we jointly correlate to a set of six ecological and life-history traits. We found that there is a large amount of redundancy among the calculated summary statistics and that there is a significant association with the matrix of species traits. In a multivariate analysis, two main aspects of population structure were visible among the 27 species. The first aspect is related to the species' dispersal capacities and the second is most likely related to the species' postglacial recolonization of the Alps. Furthermore, we found that some summary statistics, most importantly Mantel's r and Jost's D, show different behaviour than expected based on theory. We therefore advise caution in drawing too strong conclusions from these statistics.

Combining niche modelling and landscape genetics to study local adaptation: A novel approach illustrated using alpine plants

Understanding the factors that shape adaptive genetic variation across species niches has become of paramount importance in evolutionary ecology, especially to understand how adaptation to changing climate affects the geographic range of species. The distribution of adaptive alleles in the ecological niche is determined by the emergence of novel mutations, their fitness consequences and gene flow that connects populations across species niches. Striking demographical differences and source sink dynamics of populations between the centre and the margin of the niche can play a major role in the emergence and spread of adaptive alleles. Although some theoretical predictions have long been proposed, the origin and distribution of adaptive alleles within species niches remain untested. In this paper, we propose and discuss a novel empirical approach that combines landscape genetics with species niche modelling, to test whether alleles that confer local adaptation are more likely to occur in either marginal or central populations of species niches. We illustrate this new approach by using a published data set of 21 alpine plant species genotyped with a total of 2483 amplified fragment length polymorphisms (AFLP), distributed over more than 1733 sampling sites across the Alps. Based on the assumption that alleles that were statistically associated with environmental variables were adaptive, we found that adaptive alleles in the margin of a species niche were also present in the niche centre, which suggests that adaptation originates in the niche centre. These findings corroborate models of species range evolution, in which the centre of the niche contributes to the emergence of novel adaptive alleles, which diffuse towards niche margins and facilitate niche and range expansion through subsequent local adaptation. Although these results need to be confirmed via fitness measurements in natural populations and functionally characterised genetic sequences, this study provides a first step towards understanding how adaptive genetic variation emerges and shapes species niches and geographic ranges along environmental gradients.

Paraoxonase2 polymorphisms are associated with nephropathy in Type II diabetes.

AIMS/HYPOTHESIS: Paraoxonase is a member of a multigene family of three genes. Paraoxonase2 gene polymorphisms have been associated with coronary heart disease in non-diabetic patients and with an increased fasting glycaemia in patients with Type II (non-insulin-dependent) diabetes mellitus. We tested the hypothesis of whether paraoxonase1 and paraoxonase2 polymorphisms were associated with diabetic nephropathy. METHODS: Our case-control study of 299 Swiss patients with Type II diabetes included 147 patients with confirmed diabetic nephropathy. RESULTS: In univariate analyses the two paraoxonase2 polymorphisms were associated with diabetic nephropathy. When subjected to multivariate analyses, both paraoxonase2 polymorphisms remained statistically associated with diabetic nephropathy independent of traditional risk factors (paraoxonase2-148: OR = 2.53, p = 0.003; paraoxonase2-311: OR = 2.67, p = 0.002). In addition, BMI interacted with paraoxonase2 polymorphisms as a risk factor of nephropathy. CONCLUSIONS/INTERPRETATION: The paraoxonase2 gene polymorphisms were significantly associated with diabetic nephropathy independent of traditional risk factors in Type II diabetic patients. The susceptibility to diabetic nephropathy was intensified by the degree of obesity. Pathophysiological pathways should be investigated and could be involved in insulin resistance or lipids metabolism or both.

Association between bipolar disorder and monoamine oxidase A gene polymorphisms: results of a multicenter study

OBJECTIVE: Although genetic factors have been implicated in the etiology of bipolar disorder, no specific gene has been conclusively identified. Given the link between abnormalities in serotonergic neurotransmission and bipolar disorder, a candidate gene association approach was applied to study the involvement of the monoamine oxidase A (MAOA) gene, which codes for a catabolic enzyme of serotonin, in the susceptibility to bipolar disorder. METHOD: In France and Switzerland, 272 patients with bipolar disorder and 122 healthy subjects were typed for three polymorphic markers of the MAOA gene: the MAOA-CA repeat, the MAOA restriction fragment length polymorphism (RFLP), and a repeat directly adjacent to the variable number of tandem repeats (VNTR) locus. RESULTS: A significant difference in the distribution of the alleles for the MAOA-CA repeat was observed between the female bipolar patients and comparison group. CONCLUSIONS: The results obtained in the French and Swiss population confirm findings from two studies conducted in the United Kingdom.

Signature polymorphisms in the internal transcribed spacer region relevant for the differentiation of zoophilic and anthropophilic strains of Trichophyton interdigitale and other species of T. mentagrophytes sensu lato.

Summary Background Dermatophytes are the main cause of superficial mycoses in humans and animals. Molecular research has given useful insights into the phylogeny and taxonomy of the dermatophytes to overcome the difficulties with conventional diagnostics. Objectives The Trichophyton mentagrophytes complex consists of anthropophilic as well as zoophilic species. Although several molecular markers have been developed for the differentiation of strains belonging to T. mentagrophytes sensu lato, correct identification still remains problematic, especially concerning the delineation of anthropophilic and zoophilic strains of T. interdigitale. This differentiation is not academic but is essential for selection of the correct antimycotic therapy to treat infected patients. Methods One hundred and thirty isolates identified by morphological characteristics as T. mentagrophytes sensu lato were investigated using restriction fragment length polymorphism (RFLP) and sequence analysis of the polymerase chain reaction-amplified internal transcribed spacer (ITS) region of the rDNA. Results Species of this complex produced individual RFLP patterns obtained by the restriction enzyme MvaI. Subsequent sequence analysis of the ITS1, 5.8S and ITS2 region of all strains, but of T. interdigitale in particular, revealed single unique polymorphisms in anthropophilic and zoophilic strains. Conclusions Signature polymorphisms were observed to be useful for the differentiation of these strains and epidemiological data showed a host specificity among zoophilic strains of T. interdigitale/Arthroderma vanbreuseghemii compared with A. benhamiae as well as characteristic clinical pictures in humans when caused by zoophilic or anthropophilic strains. The delineation is relevant because it helps in determining the correct treatment and provides clues regarding the source of the infection.