Innate, High Tolerance to Zinc and Lead in Violets Confirmed at the Suspended Cell Level.

Many species of the Viola L. genus (violets) colonize areas with high concentrations of trace elements in the soil, e.g., nickel, cadmium, zinc, and lead. Although tolerance to heavy metals is a common phenomenon in violets, it is not clear whether this is the result of gradual microevolutionary processes as a part of the adaptation to the specific conditions, or whether the tolerance was inherited from the ancestor(s). We developed cell suspension cultures of five plant species: two non-metallophytes-Arabidopsis thaliana (Col-0) and Viola · wittrockiana, and three metallophytes-V. philippica, V. tricolor, and Silene vulgaris subsp. humilis for tolerance tests. The aim of the study was to measure the level of tolerance of violets in comparison with species from the other genera to verify the hypothesis of the high, innate tolerance of the former. We measured cell viability, non-enzymatic antioxidant content, and the accumulation of heavy metals after cell treatment with Zn or Pb. The results indicate they are innate and independent on the ecological status (metallophyte vs. non-metallophyte) and high in comparison with other species tolerance to Zn and Pb in violets. Viability of the cells after Zn and Pb (1000 µM) exposure for 72 h was the highest in violets. Antioxidant content, after heavy metal treatment, increased significantly, particularly in metallophyte violets, indicating their high responsivity to metals. In all species, lead was detected in the protoplasm of the cells, not in the vacuole or cell wall. All violets were characterized by the accumulation capacity of lead. Here, we clearly show that the physiological and biochemical studies conducted with the use of heavy metals on plant cells translate into the heavy metal tolerance of the species.

Environmental impact on the temporal production of chasmogamous and cleistogamous flowers in the mixed breeding system of Viola pubescens.

Viola pubescens is a perennial, mixed breeding herb that produces both chasmogamous and cleistogamous flowers at different times of the season. Once bud type is specified, it does not convert from one form to the other. While temporal production of the two flowers is known to be influenced by environmental factors, the specific environmental cues that signal emergence of each flower type have not been empirically studied. To investigate the environmental parameters driving seasonal development of chasmogamous versus cleistogamous flowers, a native V. pubescens population was examined during the spring and summer of 2016 and 2017. Measurements of light quantity, canopy cover, photoperiod, temperature, soil moisture, soil pH, and the number of chasmogamous and cleistogamous buds were collected on either a weekly or biweekly basis. Independent zero-inflated negative binomial (ZINB) regressions were used to model the odds of bud production (0 versus 1 bud) and bud counts (≥ 1 bud) as a function of the environmental variables. Results of the ZINB models highlight key differences between the environmental variables that influence chasmogamous versus cleistogamous bud development and counts. In addition to the ZINB regressions, individual logistic regressions were fit to the bud data. The logistic models support results of the ZINB models and, more crucially, identify specific environmental thresholds at which each bud type is probable. Collectively, this work offers novel insight into how environmental variables shape temporal development of chasmogamous and cleistogamous flowers, suggests distinct threshold values that may aid in selectively inducing each flower type, and provides insight into how climatic change may impact mixed breeding species.

De novo transcriptome analysis of Viola ×wittrockiana exposed to high temperature stress.

Around the world, pansies are one of the most popular garden flowers, but they are generally sensitive to high temperatures, and this limits the practicality of planting them during the warmest days of the year. However, a few pansy germplasms with improved heat tolerance have been discovered or bred, but the mechanisms of their heat resistance are not understood. In this study, we investigated the transcript profiles of a heat-tolerant pansy inbred line, DFM16, in response to high temperatures using RNAseq. Approximately 55.48 Gb of nucleotide data were obtained and assembled into 167,576 unigenes with an average length of 959 bp, of which, 5,708 genes were found to be differentially expressed after heat treatments. Real-time qPCR was performed to validate the expression profiles of the selected genes. Nine metabolic pathways were found to be significantly enriched, in the analysis of the differentially expressed genes. Several potentially interesting genes that encoded putative transcription regulators or key components involving heat shock protein (HSP), heat shock transcription factors (HSF), and antioxidants biosynthesis, were identified. These genes were highlighted to indicate their significance in response to heat stress and will be used as candidate genes to improve pansy heat-tolerance in the future.

Alpine species in dynamic insular ecosystems through time: conservation genetics and niche shift estimates of the endemic and vulnerable Viola cheiranthifolia.

BACKGROUND AND AIMS: Alpine oceanic ecosystems are considered amongst the most ephemeral and restricted habitats, with a biota highly vulnerable to climate changes and disturbances. As an example of an alpine insular endemic, the past and future population genetic structure and diversity, and the future distribution of Viola cheiranthifolia (Violaceae), endemic to Tenerife (Canary Islands), were estimated. The main goals were to predict distribution changes of this alpine oceanic plant under climate change, and to assist in actions for its conservation. METHODS: To perform population genetic analysis, 14 specific microsatellite markers and algorithms which considered the polyploid condition of V. cheiranthifolia were employed. The niche modelling approach incorporated temperature gradients, topography and snow cover maps. Models were projected into climate change scenarios to assess the extent of the altitudinal shifts of environmental suitability. Finally, simulations were performed to predict whether the environmental suitability loss will affect the genetic diversity of populations. KEY RESULTS: Viola cheiranthifolia presents short dispersal capacity, moderate levels of genetic diversity and a clear population genetic structure divided into two main groups (Teide and Las Cañadas Wall), showing signs of recolonization dynamics after volcanic eruptions. Future estimates of the distribution of the study populations also showed that, despite being extremely vulnerable to climate change, the species will not lose all its potential area in the next decades. The simulations to estimate genetic diversity loss show that it is correlated to suitability loss, especially in Las Cañadas Wall. CONCLUSIONS: The low dispersal capacity of V. cheiranthifolia, coupled with herbivory pressure, mainly from rabbits, will make its adaptation to future climate conditions in this fragile alpine ecosystem difficult. Conservation actions should be focused on herbivore control, population reinforcement and surveillance of niche shifts, especially in Guajara, which represents the oldest isolated population and a genetic reservoir for the species.

Suspended cells of metallicolous and nonmetallicolous Viola species tolerate, accumulate and detoxify zinc and lead.

We studied the zinc and lead accumulation and tolerance level of suspended cells of four Viola species with different metallophyte statuses: Viola lutea ssp. westfalica (obligate metallophyte), V. tricolor (facultative metallophyte), V. arvensis (accidental metallophyte) and V. uliginosa (nonmetallophyte), in order to determine the correlation between cell and plant tolerance. Cells of all studied species/genotypes were tolerant to metal concentrations applied to the medium for 24, 48 and 72 h, more for zinc than for lead, as estimated by cell viability using the alamarBlue assay. Viable cells of each analyzed species/genotype accumulated zinc and particularly lead in very high amounts after treatment with 2000 µM for 72 h (1500-4500 mg kg-1, 24 000-32 000 mg kg-1, respectively), determined by atomic absorption spectrometry. The bioaccumulation factor values confirmed the cells' hyperaccumulation strategy. The cell-activated detoxification mechanism, consisting in deposition of metals in the cell wall and vacuoles, as shown by transmission electron microscopy with X-ray microanalysis, allows the cells to survive despite the high level of metal accumulation. These results indicate innate high tolerance to zinc and lead in violets with different metallophyte statuses and also in the nonmetallophyte, suggesting that evolutionarily developed hypertolerance may occurs in this group as a whole.

Lack of correlation between pollen aperture number and environmental factors in pansies (Viola L., sect. Melanium Ging.) - pollen heteromorphism re-examined.

The development of different pollen morphs by one specimen - pollen heteromorphism - occurs in ca. one-third species of the genus Viola. Melanium section species (pansies) stand out in producing the widest range of apertures among Viola species. Aperture number decreases with elevation increase, and faster germination of five-aperturate pollen as compared with three-aperturate has previously been postulated. We re-examined pollen heteromorphism in the context of its viability, and made correlations with elevation (>1500 m a.s.l. versus <1500 m a.s.l.), soil type (metalliferous versus non-metalliferous; MET versus NMET) and chromosome number based on selected study criteria of ca. 20% karyologically and morphologically strongly differentiated but genetically closely related pansies. A total of 79% of analysed species were heteromorphic, forming three- to six-colp or ate pollen per individual flower. Mean aperture number and pollen viability were not affected by soil type (MET versus NMET). Mean aperture number was also not influenced by elevation or species chromosome number. Positive correlations were established between aperture number and pollen viability, negative between pollen viability and elevation (increasing altitude of 100 m decreased pollen viability by 0.4%) and lack of correlation between chromosome number and pollen viability. The varied frequencies of different pollen morphs among species are not under the general pressure of ecological conditions, as previously postulated for the species of Melanium section. Rather, this trait in pansies, similar to other floral characters (e.g. long, curved nectar spur, 'landing platform', posterior petals with nectar guides), is adaptive but dependent on the breeding system (inbreeding versus outbreeding) of the individual species.

Expression of B-class MADS-box genes in response to variations in photoperiod is associated with chasmogamous and cleistogamous flower development in Viola philippica.

BACKGROUND: Some plants develop a breeding system that produces both chasmogamous (CH) and cleistogamous (CL) flowers. However, the underlying molecular mechanism remains elusive. RESULTS: In the present study, we observed that Viola philippica develops CH flowers with short daylight, whereas an extended photoperiod induces the formation of intermediate CL and CL flowers. In response to long daylight, the respective number and size of petals and stamens was lower and smaller than those of normally developed CH flowers, and a minimum of 14-h light induced complete CL flowers that had no petals but developed two stamens of reduced fertility. The floral ABC model indicates that B-class MADS-box genes largely influence the development of the affected two-whorl floral organs; therefore, we focused on characterizing these genes in V. philippica to understand this particular developmental transition. Three such genes were isolated and respectively designated as VpTM6-1, VpTM6-2, and VpPI. These were differentially expressed during floral development (particularly in petals and stamens) and the highest level of expression was observed in CH flowers; significantly low levels were detected in intermediate CL flowers, and the lowest level in CL flowers. The observed variations in the levels of expression after floral induction and organogenesis apparently occurred in response to variations in photoperiod. CONCLUSIONS: Therefore, inhibition of the development of petals and stamens might be due to the downregulation of B-class MADS-box gene expression by long daylight, thereby inducing the generation of CL flowers. Our work contributes to the understanding of the adaptive evolutionary formation of dimorphic flowers in plants.

From facilitation to competition: temperature-driven shift in dominant plant interactions affects population dynamics in seminatural grasslands.

Biotic interactions are often ignored in assessments of climate change impacts. However, climate-related changes in species interactions, often mediated through increased dominance of certain species or functional groups, may have important implications for how species respond to climate warming and altered precipitation patterns. We examined how a dominant plant functional group affected the population dynamics of four co-occurring forb species by experimentally removing graminoids in seminatural grasslands. Specifically, we explored how the interaction between dominants and subordinates varied with climate by replicating the removal experiment across a climate grid consisting of 12 field sites spanning broad-scale temperature and precipitation gradients in southern Norway. Biotic interactions affected population growth rates of all study species, and the net outcome of interactions between dominants and subordinates switched from facilitation to competition with increasing temperature along the temperature gradient. The impacts of competitive interactions on subordinates in the warmer sites could primarily be attributed to reduced plant survival. Whereas the response to dominant removal varied with temperature, there was no overall effect of precipitation on the balance between competition and facilitation. Our findings suggest that global warming may increase the relative importance of competitive interactions in seminatural grasslands across a wide range of precipitation levels, thereby favouring highly competitive dominant species over subordinate species. As a result, seminatural grasslands may become increasingly dependent on disturbance (i.e. traditional management such as grazing and mowing) to maintain viable populations of subordinate species and thereby biodiversity under future climates. Our study highlights the importance of population-level studies replicated under different climatic conditions for understanding the underlying mechanisms of climate change impacts on plants.

Steep Decline and Cessation in Seed Dispersal by Myrmica rubra Ants.

Myrmecochorous diaspores bear a nutrient-rich appendage, the elaiosome, attractive to ant workers that retrieve them into the nest, detach the elaiosome and reject the seed intact. While this interaction is beneficial for the plant partner by ensuring its seed dispersal, elaiosome consumption has various effects -positive, negative or none - on ants' demography and survival, depending on both the ant/plant species involved. In this context, the contribution of ants to seed dispersal strongly varies according to the ant/plant pairs considered. In this paper, we investigate whether the dynamics of myrmecochory also vary on a temporal scale, for a given pair of partners: Myrmica rubra ants and Viola odorata seeds. During their first encounter with seeds, ants collect all the diaspores and eat the majority of elaiosomes. Both the harvesting effort and the elaiosome consumption decline when seeds are offered on the next week and completely cease for the following weeks. This is related to a decrease in the number of foragers reaching the food source, as well as to a reduced probability for an ant contacting a seed to retrieve it. Seed retrieval is not reactivated after seven weeks without any encounter with V. odorata seeds. By contrast, naive ant colonies only fed with fruit flies do not show a decline of prey harvesting of which the speed of retrieval even increases over the successive weeks. Myrmecochory may thus be labile at the scale of a fruiting season due to the ability of ants to steeply tune and cease for several months the harvesting of these seemingly poorly rewarding items and to maintain cessation of seed exploitation. The present study emphasizes the importance of a long-lasting follow up of the myrmecochory process, to assess the stability of this ant-plant partnership and to identify mechanisms of adaptive harvesting in ants.

Parasites in bloom: flowers aid dispersal and transmission of pollinator parasites within and between bee species.

The dispersal of parasites is critical for epidemiology, and the interspecific vectoring of parasites when species share resources may play an underappreciated role in parasite dispersal. One of the best examples of such a situation is the shared use of flowers by pollinators, but the importance of flowers and interspecific vectoring in the dispersal of pollinator parasites is poorly understood and frequently overlooked. Here, we use an experimental approach to show that during even short foraging periods of 3 h, three bumblebee parasites and two honeybee parasites were dispersed effectively onto flowers by their hosts, and then vectored readily between flowers by non-host pollinator species. The results suggest that flowers are likely to be hotspots for the transmission of pollinator parasites and that considering potential vector, as well as host, species will be of general importance for understanding the distribution and transmission of parasites in the environment and between pollinators.

Albanian violets of the section Melanium, their morphological variability, genetic similarity and their adaptations to serpentine or chalk soils.

Violets of the section Melanium from Albanian serpentine and chalk soils were examined for their taxonomic affiliations, their ability to accumulate heavy metals and their colonization by arbuscular mycorrhizal fungi (AMF). The sequence analysis of the ITS1-5.8S rDNA-ITS2 region showed that all the sampled six Albanian violets grouped between Viola lutea and Viola arvensis, but not with Viola tricolor. The fine resolution of the ITS sequences was not sufficient for a further delimitation of the Albanian violets within the V. lutea-V. arvensis clade. Therefore, the Albanian violets were classified by a set of morphological characters. Viola albanica, Viola dukadjinica and Viola raunsiensis from serpentine soils as well as Viola aetolica from a chalk meadow were unambiguously identified, whereas the samples of Viola macedonica showed high morphological variability. All the violets, in both roots and shoots contained less than or similar levels of heavy metals as their harboring soils, indicating that they were heavy metal excluders. All the violets were strongly colonized by AMF with the remarkable exception of V. albanica. This violet lived as a scree creeper in shallow serpentine soil where the concentration of heavy metals was high but those of P, K and N were scarce.

Reproductive system of a mixed-mating plant responds to climate perturbation by increased selfing.

How plants respond to climatic perturbations, which are forecasted to increase in frequency and intensity, is difficult to predict because of the buffering effects of plasticity. Compensatory adjustments may maintain fecundity and recruitment, or delay negative changes that are inevitable but not immediately evident. We imposed a climate perturbation of warming and drought on a mixed-mating perennial violet, testing for adjustments in growth, reproduction and mortality. We observed several plasticity-based buffering responses, such that the climatic perturbation did not alter population structure. The most substantial reproductive adjustments, however, involved selfing, with a 45% increase in self-pollination by chasmogamous flowers, a 61% increase in the number of cleistogamous flowers that produced at least one fruit and an overall 15% increase in fruit production from selfed cleistogamous flowers. Reproductive assurance thus compensated for environmental change, including low pollinator visitation that occurred independently of our climate treatment. There was also no immediate evidence for inbreeding depression. Our work indicates that plants with vegetative and reproductive flexibility may not be immediately and negatively affected by a climatic perturbation. The stabilizing effects of these reproductive responses in the long term, however, may depend on the implications of significantly elevated levels of selfing.

Metallophyte status of violets of the section Melanium.

Violets from metal-enriched soils have controversially been described as both heavy-metal accumulators and excluders in the literature. The present study solves the issue for violets of the section Melanium (zinc violets, Viola lutea ssp. calaminaria and V. lutea ssp. westfalica; hartsease or wild pansy, Viola tricolor; and mountain pansy, V. lutea). The aims were to determine the concentrations of heavy metals in the soil and in the roots and shoots of field-collected plants, to evaluate the potential impact of colonisation by arbuscular mycorrhizal fungi on heavy-metal concentrations in the plant tissues, and to quantitatively define the localisation of the elements in root cross-sections. When these violets grow in low-metal soils, higher concentrations of the heavy metals were found in the roots and shoots than in the soil, whereas the opposite was seen in samples from high-metal soils. Under all field conditions examined, the roots of all of these species were colonised by arbuscular mycorrhizal fungi. However, V. tricolor was marginally colonised when the concentrations of Zn and P were higher in the soil. Determination of the spatial distribution of the elements in root cross-sections of these violets indicates tissue-specific deposition of elements within the vascular tissue, the cortex, and the rhizodermis. These data indicate that violets of the section Melanium are heavy-metal excluders.

Reconfiguration and the reduction of vortex-induced vibrations in broad leaves.

Flexible plants, fungi and sessile animals reconfigure in wind and water to reduce the drag acting upon them. In strong winds and flood waters, for example, leaves roll up into cone shapes that reduce drag compared with rigid objects of similar surface area. Less understood is how a leaf attached to a flexible leaf stalk will roll up stably in an unsteady flow. Previous mathematical and physical models have only considered the case of a flexible sheet attached to a rigid tether in steady flow. In this paper, the dynamics of the flow around the leaf of the wild ginger Hexastylis arifolia and the wild violet Viola papilionacea are described using particle image velocimetry. The flows around the leaves are compared with those of simplified physical and numerical models of flexible sheets attached to both rigid and flexible beams. In the actual leaf, a stable recirculation zone is formed within the wake of the reconfigured cone. In the physical model, a similar recirculation zone is observed within sheets constructed to roll up into cones with both rigid and flexible tethers. Numerical simulations and experiments show that flexible rectangular sheets that reconfigure into U-shapes, however, are less stable when attached to flexible tethers. In these cases, larger forces and oscillations due to strong vortex shedding are measured. These results suggest that the three-dimensional cone structure in addition to flexibility is significant to both the reduction of vortex-induced vibrations and the forces experienced by the leaf.

Increased genetic diversity of Viola tricolor L. (Violaceae) in metal-polluted environments.

Changes in DNA sequences affecting cryptic intraspecific variability are very important mechanisms of plant microevolutionary processes, initiating species diversification. In polluted environments, intra- and interpopulation changes at the molecular level proceed rapidly and lead to the formation of new ecotypes in a relatively short time. We used ISSR PCR fingerprinting data to analyze the genetic diversity and genetic structure of seven populations of Viola tricolor: four growing on soil contaminated with heavy metals (Zn, Pb, Cd; waste heaps) and three from control soil. The populations from the polluted sites showed higher genetic polymorphism (%(poly)=84%) and gene diversity (H(T)=0.1709) than the control populations (%(poly)=75% and H(T)=0.1448). The number of private markers we detected within metallicolous (MET) populations was more than double that found within non-metallicolous (NON) populations (15 vs. 7). The STRUCTURE and UPGMA analyses showed clear genetic differences between the NON and MET populations. Based on broad analyses of the genetic parameters, we conclude that the effect of these polluted environments on the genetic diversity of the MET populations, separating them from the NON populations, is evidence of microevolutionary processes at species level, leading to species divergence and the emergence of local ecotypes better adapted to their different environments.

Fitness and genetic variation of Viola calaminaria, an endemic metallophyte: implications of population structure and history.

We investigated variations in genetic diversity and plant fitness in a rare endemic metallophyte of calamine soils, Viola calaminaria, in relation to population size, population connectivity and population history in order to evaluate and discuss potential conservation strategies for the species. Mean population genetic diversity (H(s) = 0.25) of V. calaminaria was similar to endemic non-metallophyte taxa. Twenty-one per cent of the genetic variation was partitioned among populations and a low (9%) but significant differentiation was found among geographical regions. Our results did not support the hypothesis that the acquisition of metal tolerance may result in reduced genetic diversity, and suggested that strict metallophytes do not exhibit higher inter-population differentiation resulting from scattered habitats. There were no relationships between population genetic diversity and population size. Significant correlations were found between plant fitness and (i) population size and (ii) connectivity index. Recently-founded populations exhibited the same level of genetic diversity as ancient populations and also possessed higher plant fitness. There was no indication of strong founder effects in recently-established populations. The results suggest that the creation of habitats through human activities could provide new opportunities for conservation of this species.

Intraspecific variation in Viola suavis in Europe: parallel evolution of white-flowered morphotypes.

BACKGROUND AND AIMS: Viola species are commonly grown for their ornamental flowers, but their evolutionary history and taxonomy are often complicated and have been poorly explored so far. This is a study of the polymorphic, typically blue-flowered species Viola suavis, concentrating on the white-flowered populations of uncertain taxonomic assignment that occur in Spain and central and south-eastern Europe. The aim was to resolve their origin and taxonomic status and to study the intraspecific structure and (post)glacial history of this species. METHODS: Viola suavis and five close relatives were sampled from multiple locations and subjected to molecular (AFLP, sequencing of nrDNA ITS) and morphometric analyses. Data on ploidy level and pollen fertility were also obtained, to address an assumed hybrid origin of the white-flowered populations. KEY RESULTS: In V. suavis a strong intraspecific genetic split into two groups was observed, indicating that there has been a long-term isolation and survival in distinct glacial refugia. The white-flowered populations could be placed within the variation range of this species, and it is clear that they evolved independently in two distant areas. Their parallel evolution is supported by both morphological and genetic differentiation. The strongly reduced genetic variation and absence of unique AFLP fragments suggest their derived status and origin from the typical, blue-flowered populations. CONCLUSIONS: These results suggest that intraspecific variation in V. suavis has been largely shaped by population isolations during the last glaciation and subsequent recolonizations, although cultivation and vegetative spread by humans have affected the present picture as well.

Metalliferous and non-metalliferous populations of Viola tricolor represent similar mode of antioxidative response.

Heavy metal-contaminated sites are excellent areas to examine the antioxidative machinery responsible for physiological adaptations of many plant species. Superoxide dismutase (SOD), guaiacol peroxide (GPX), ascorbate peroxide (APX), catalase (CAT) activity and hydrogen peroxide (H(2)O(2)) content were analyzed in leaves and roots of Viola tricolor (Viola) from contaminated soils ('Bukowno', 'Saturn', 'Warpie' heaps), and non-contaminated soil ('Zakopane meadow') to examine the level of oxidative stress and antioxidative response. In leaves, six isoforms of SOD were recognized. Roots possessed two additional bands, named manganese superoxide dismutase (MnSOD)-like form (MnSODI) and Cu/ZnSOD-like form (Cu/ZnSODIV). The H(2)O(2) content in leaves ranged from 554 to 5 098 micromol H(2)O(2)/gf.w. and was negatively correlated with CAT activity. The non-contaminated population was characterized by the lowest CAT activity combined with the highest H(2)O(2) concentration. Two isoforms of CAT, CAT-1 and CAT-2, were recognized in leaves of plants from non-contaminated and contaminated sites, respectively. In roots of individuals from two heaps ('Warpie' and 'Saturn'), two distinct bands for each CAT isoform were observed. A slower migrating band may be an aggregate, exhibiting CAT and MnSODs activities. Both peroxidases (APX and GPX) presented the same pattern of activity, depending on the organ, indicating that in leaves and roots APX and GPX were regulated in parallel. Differences in enzyme activities and H(2)O(2) content between plants from different contaminated sites were statistically significant, but were tightly maintained at a very similar level. Prolonged and permanent heavy metal stress evoked a very similar mode of antioxidative response in specimens of analyzed metalliferous populations not causing measurable oxidative stress. Thus, our results clearly indicate that V. tricolor is a taxon well adapted to heavy metal-contaminated soils, and that differences in enzyme activities and H(2)O(2) content result from adjustment of plants to a variety of conditions.

Genetic structure among and within peripheral and central populations of three endangered floodplain violets.

Understanding the partitioning of genetic variance in peripheral and central populations may shed more light on the effects of genetic drift and gene flow on population genetic structure and, thereby, improve attempts to conserve genetic diversity. We analysed genetic structure of peripheral and central populations of three insect-pollinated violets (Viola elatior, Viola pumila, Viola stagnina) to evaluate to what extent these patterns can be explained by gene flow and genetic drift. Amplified fragment length polymorphism was used to analyse 930 individuals of 50 populations. Consistent with theoretical predictions, peripheral populations were smaller and more isolated, differentiation was stronger, and genetic diversity and gene flow lower in peripheral populations of V. pumila and V. stagnina. In V. elatior, probably historic fragmentation effects linked to its specific habitat type were superimposed on the plant geographic (peripheral-central) patterns, resulting in lower relative importance of gene flow in central populations. Genetic variation between regions (3-6%), among (30-37%) and within populations (60-64%) was significant. Peripheral populations lacked markers that were rare and localized in central populations. Loss of widespread markers in peripheral V. stagnina populations indicated genetic erosion. Autocorrelation within populations was statistically significant up to a distance of 10-20 m. Higher average genetic similarity in peripheral populations than in central ones indicated higher local gene flow, probably owing to management practices. Peripheral populations contributed significantly to genetic variation and contained unique markers, which made them valuable for the conservation of genetic diversity.