Inferences on pathogenic fungus population structures from microsatellite data: new insights from spatial genetics approaches

Landscape genetics, which combines population genetics, landscape ecology and spatial statistics, has emerged recently as a new discipline that can be used to assess how landscape features or environmental variables can influence gene flow and spatial genetic variation. We applied this approach to the invasive plant pathogenic fungus Mycosphaerella fijiensis, which causes black leaf streak disease of banana. Around 880 isolates were sampled within a 50 × 50 km area located in a fragmented banana production zone in Cameroon that includes several potential physical barriers to gene flow. Two clustering algorithms and a new F(ST) -based procedure were applied to define the number of genetic entities and their spatial domain without a priori assumptions. Two populations were clearly delineated, and the genetic discontinuity appeared sharp but asymmetric. Interestingly, no landscape features matched this genetic discontinuity, and no isolation by distance (IBD) was found within populations. Our results suggest that the genetic structure observed in this production area reflects the recent history of M. fijiensis expansion in Cameroon rather than resulting from contemporary gene flow. Finally, we discuss the influence of the suspected high effective population size for such an organism on (i) the absence of an IBD signal, (ii) the characterization of contemporary gene-flow events through assignation methods of analysis and (iii) the evolution of the genetic discontinuity detected in this study.     

In vitro evaluation of Colombian plant extracts against Black Sigatoka (Mycosphaerella fijiensis Morelet)

In this work, 90 dichloromethane and methanol extracts obtained from 45 plants collected at the Natural Reserve Bremen – La Popa (Colombia) and at the Natural Regional Park Ucumarí (NRPU, Colombia) belonging to five botanical families were evaluated at 1000 mg/l, for their in vitro fungicide activity through the ascospore germ tube elongation and the measurement of the mycelial radial growth of Mycosphaerella fijiensis assays. The methanol extracts from the species Lycianthes acutifolia (Solanaceae) and Piper pesaresanum (Piperaceae); as well as, the dichloromethane extracts from P. pesaresanum and those from the Lauraceae family named Nectandra acutifolia and Ocoteca paulii, all inhibited M. fijiensis ascospore germination in 100% in the germinative tube elongation assay. With regards to the effects of the plant extracts on mycelial radial growth, the methanol extracts from P. pesaresanum and the dichloromethane one from N. acutifolia both showed 100% inhibition in this bioassay. Additionally, from the phytochemical screening on the dichloromethane and methanol extracts it was found that compounds such as alkaloids, phenols and terpenes were present in most of the extracts evaluated and they might be the cause of the antifungal activities reported.     

Recent range expansion and agricultural landscape heterogeneity have only minimal effect on the spatial genetic structure of the plant pathogenic fungus Mycosphaerella fijiensis

Understanding how geographical and environmental features affect genetic variation atboth the population and individual levels is crucial in biology, especially in the case ofpathogens. However, distinguishing between these factors and the effects of historicalrange expansion on spatial genetic structure remains challenging. In the present study, weinvestigated the case of Mycosphaerella fijiensis—a plant pathogenic fungusthat has recently colonized an agricultural landscape characterized by the presence ofpotential barriers to gene flow, including several commercial plantations in which diseasecontrol practises such as the use of fungicides are applied frequently, and low hostdensity areas. We first genotyped 300 isolates sampled at a global scale on untreatedplants in two dimensions over a 50 × 80-km area. Using two different clusteringalgorithms, no genetic structure was detected in the studied area, suggesting expansion oflarge populations and/or no influence of potential barriers. Second, we investigatedthe potential effect of disease control practises on M. fijiensis diversity bycomparing populations sampled in commercial vs food-crop plantations. At this local scale,we detected significantly higher allelic richness inside commercial plantations comparedwith the surrounding food-crop plantation populations. Analysis of molecular varianceindicated that 99% of the total genetic variance occurred within populations. Wediscuss the suggestion that high population size and/or high migration rate betweenpopulations might be responsible for the absence of any effect of disease controlpractises on genetic diversity and differentiation.     

Forecasting the airborne spread of Mycosphaerella fijiensis, a cause of black Sigatoka disease on banana: estimations of numbers of perithecia and ascospores

Banana leaves showing different levels of black Sigatoka disease were collected from an unsprayed plantation in Costa Rica during two separate periods representing the wet to dry season transition (October 1993 – February 1994) and the dry to wet season transition (April – September 1995). Laboratory studies were used to investigate the relationship between the release of Mycosphaerella fijiensis ascospores and the amount of inoculum on banana plants showing different levels of infection, as assessed by leaf necrotic area. The number of perithecia present in the necrotic area was used as an indication of potential ascospore loads and was investigated as a series of regression equations. A series of rewetting and incubation regimes was used to investigate spore release under field conditions (21°C and 100% relative humidity in the early morning and 28°C, 60% relative humidity on days when it rained in mid-afternoon). Results suggest that rainfall, combined with a high temperature, may lead to peaks of ascospore release but without necessarily increasing overall numbers released over periods of up to 4 days and that a high level of spore release was less sensitive to changes in temperature once it had been initiated. The exact role of temperature in spore release is still unclear, however, as leaf samples kept at atypically low temperatures also released non-germinating ascospores. An average of 4.5 ascospores was released per perithecium. This does not resolve ambiguities in the literature regarding the number of ascospores present in each perithecium. A linear model relating the average ascospore numbers and necrotic area, using quick estimates of the amounts of necrotic area on the leaves of a random sample of plants across a plantation, is proposed, to give an indication of the relative amount of airborne inoculum potentially available between different plantations.     

The genetic structure of the plant pathogenic fungus Melampsora larici-populina on its wild host is extensively impacted by host domestication

Wild and cultivated plants represent very different habitats for pathogens, especially when cultivated plants bear qualitative resistance genes. Here, we investigated to what extent the population genetic structure of a plant pathogenic fungus collected on its wild host can be impacted by the deployment of resistant cultivars. We studied one of the main poplar diseases, poplar rust, caused by the fungus Melampsora larici‐populina. A thousand and fifty individuals sampled from several locations in France were phenotyped for their virulence profile (ability to infect or not the most deployed resistant cultivar ‘Beaupré’), and a subset of these was genotyped using 25 microsatellite markers. Bayesian assignment tests on genetic data clustered the 476 genotyped individuals into three genetic groups. Group 1 gathered most virulent individuals and displayed evidence for selection and drastic demographic changes resulting from breakdown of the poplar cultivar ‘Beaupré’. Group 2 comprised individuals corresponding to ancestral populations of M. larici‐populina naturally occurring in the native range. Group 3 displayed the hallmarks of strict asexual reproduction, which has never previously been demonstrated in this species. We discuss how poplar cultivation has influenced the spatial and genetic structure of this plant pathogenic fungus, and has led to the spread of virulence alleles (gene swamping) in M. larici‐populina populations evolving on the wild host.     

Hidden founder effects: small‐scale spatial genetic structure in recently established populations of the grassland specialist plant Anthyllis vulneraria

The long‐term establishment success of founder plant populations has been commonly assessed based on the measures of population genetic diversity and among population genetic differentiation, with founder populations expected to carry sufficient genetic diversity when population establishment is the result of many colonists from multiple source populations (the ‘migrant pool’ colonization model). Theory, however, predicts that, after initial colonization, rapid population expansion may result in a fast increase in the extent of spatial genetic structure (SGS), independent of extant genetic diversity. This SGS can reduce long‐term population viability by increasing inbreeding. Using 12 microsatellite markers, we inferred colonization patterns in four recent populations of the grassland specialist plant Anthyllis vulneraria and compared the extent of SGS between recently established and old populations. Assignment analyses of the individuals of recent population based on the genetic composition of nine adjacent putative source populations suggested the occurrence of the ‘migrant pool’ colonization model, further confirmed by high genetic diversity within and low genetic differentiation among recent populations. Population establishment, however, resulted in the build‐up of strong SGS, most likely as a result of spatially restricted recruitment of the progeny of initial colonists. Although reduced, significant SGS was nonetheless observed to persist in old populations. The presence of SGS was in all populations associated with elevated inbreeding coefficients, potentially affecting the long‐term viability of these populations. In conclusion, this study illustrates the importance of studying SGS next to population genetic diversity and differentiation to adequately infer colonization patterns and long‐term establishment success of plant species.     

Genetic variation and structure in native populations of the fire ant Solenopsis invicta: evolutionary and demographic implications

We studied population genetic variation and structure in the fire ant Solenopsis invicta using nuclear genotypic and mitochondrial DNA (mtDNA) sequence data obtained from samples collected throughout its native range. Geographic populations are strongly differentiated at both genomes, with such structure more pronounced in Brazil than in Argentina. Higher-level regional structure is evident from the occurrence of isolation-by-distance patterns among populations, the recognition of clusters of genetically similar, geographically adjacent populations by ordination analysis, and the detection of an mtDNA discontinuity between Argentina and Brazil coinciding with a previously identified landform of biogeographical relevance. Multiple lines of evidence from both genomes suggest that the ancestors of the ants we studied resembled extant northern Argentine S. invicta , and that existing Brazilian populations were established more recently by serial long-distance colonizations and/or range expansions. The most compelling evidence for this is the corresponding increase in F _K (a measure of divergence from a hypothetical ancestor) and decrease in genetic diversity with distance from the Corrientes population in northern Argentina. Relatively deep sequence divergence among several mtDNA clades, coupled with geographical partitioning of many of them, suggests prolonged occupation of South America by S. invicta in more-or-less isolated regional populations. Such populations appear, in some cases, to have come into secondary contact without regaining the capacity to freely interbreed. We conclude that nominal S. invicta in its native range comprises multiple entities that are sufficiently genetically isolated and diverged to have embarked on independent evolutionary paths.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 541–560.     

Genetic Diversity and Spatial Genetic Structure of an Epiphytic Bromeliad in Costa Rican Montane Secondary Forest Patches

Information on genetic variation and its distribution in tropical plant populations relies mainly on studies of ground‐rooted species, while genetic information of epiphytic plants is still limited. Particularly, the effect of forest successional condition on genetic diversity and structure of epiphytes is scanty in the literature. We evaluated the genetic variation and spatial genetic structure of the epiphytic bromeliad Guzmania monostachia (Bromeliaceae, Tillandsioideae) in montane secondary forest patches in Costa Rica. The sampling design included plants on the same trees (i.e., populations), populations within forest patches and patches within secondary forest at two different successional stages (early vs. mid‐succession). Six microsatellites revealed low levels of population genetic variation (A = 2.06, A_E = 1.61, H_E = 0.348), a marked deficiency of heterozygotes (H_O = 0.031) and high inbreeding (f = 0.908). Genetic differentiation was negligible among populations within the same forest patch, but moderate (G_ST = 0.123 ± 0.043) among forest patches. Genetic relatedness between individuals was significantly higher for plants located within the same forest patch and separated by <60 m and decreased as distance between plants increased, becoming significantly negative at distances >400 m. An analysis of molecular variance (AMOVA) showed significant genetic variation between forest patches, but non‐significant variation between successional stages. The selfing breeding system and limited seed dispersal capabilities in G. monostachia could explain the observed levels and partitioning of genetic diversity at this geographic scale. However, these results also suggest that forest fragmentation is likely to influence the degree of local genetic structuring of epiphytic plants by limiting gene flow.     

Isolation by distance and sharp discontinuities in gene frequencies: implications for the phylogeography of an alpine insect species, Carabus solieri

Analysis of genetic isolation by distance (IBD) is of prime importance for the study of processes responsible for spatial population genetic structure and is thus frequently used in case studies. However, the identification of a significant IBD pattern does not necessarily imply the absence of sharp discontinuities in gene frequencies. Therefore, identifying barriers to gene flow and/or secondary contact between differentiated entities remains a major challenge in population biology. Geographical genetic structure of 41 populations (1080 individuals) of an alpine insect species, Carabus solieri, was studied using 10 microsatellite loci. All populations were significantly differentiated and spatially structured according to IBD over the entire range. However, clustering analyses clearly identified three main clusters of populations, which correspond to geographical entities. Whereas IBD also occurs within each cluster, population structure was different according to which group of populations was considered. The southernmost cluster corresponds to the most fragmented part of the range. Consistently, it was characterized by relatively high levels of differentiation associated with low genetic diversity, and the slope of the regression of genetic differentiation against geographical distances was threefold those of the two other clusters. Comparisons of within-cluster and between-cluster IBD patterns revealed barriers to gene flow. A comparison of the two approaches, IBD and clustering analyses, provided us with valuable information with which to infer the phylogeography of the species, and in particular to propose postglacial colonization routes from two potential refugia located in Italy and in southeastern France. Our study highlights strongly the possible confounding contribution of barriers to gene flow to IBD pattern and emphasizes the utility of the model-based clustering analysis to identify such barriers.     

Understanding the recent colonization history of a plant pathogenic fungus using population genetic tools and Approximate Bayesian Computation

Understanding the processes by which new diseases are introduced in previously healthy areas is of major interest in elaborating prevention and management policies, as well as in understanding the dynamics of pathogen diversity at large spatial scale. In this study, we aimed to decipher the dispersal processes that have led to the emergence of the plant pathogenic fungus Microcyclus ulei, which is responsible for the South American Leaf Blight (SALB). This fungus has devastated rubber tree plantations across Latin America since the beginning of the twentieth century. As only imprecise historical information is available, the study of population evolutionary history based on population genetics appeared most appropriate. The distribution of genetic diversity in a continental sampling of four countries (Brazil, Ecuador, Guatemala and French Guiana) was studied using a set of 16 microsatellite markers developed specifically for this purpose. A very strong genetic structure was found (F_st=0.70), demonstrating that there has been no regular gene flow between Latin American M. ulei populations. Strong bottlenecks probably occurred at the foundation of each population. The most likely scenario of colonization identified by the Approximate Bayesian Computation (ABC) method implemented in 𝒟ℐ𝒴𝒜ℬ𝒞 suggested two independent sources from the Amazonian endemic area. The Brazilian, Ecuadorian and Guatemalan populations might stem from serial introductions through human-mediated movement of infected plant material from an unsampled source population, whereas the French Guiana population seems to have arisen from an independent colonization event through spore dispersal.     

Genetic variation in rural and urban populations of Epipactis helleborine (L.) Crantz. (Orchidaceae) in Britain

Genetic variation in Epipactis helleborine in the British Isles was assessed using starch gel electrophoresis of isozymes; 273 individuals were sampled from 13 populations and examined for genetic variation using eight enzyme systems encoded for by 13 loci. Overall, 46% of the loci examined were polymorphic, with an average of 1.69 alleles per locus. Within populations, a mean of 33% of the loci were polymorphic, with a mean number of 1.46 alleles per locus. Levels of genetic variation were compared between urban and well established rural populations to assess the genetic consequences of colonization of the urban sites. The average levels of genetic variation detected in urban populations were lower than that found in rural populations, although there was a much greater range of variation among the urban populations. Large urban populations actually have patterns of variation similar to rural populations and show evidence of multiple founders. This indicates that the high dispersibility of Epipactis seeds can in some cases overcome the predicted loss of genetic variation associated with founder effects during colonization. Small urban populations, however, show significantly lower levels of genetic variation compared with these large urban populations and the rural populations, and it seems likely that this is attributable to single founding events and/or genetic drift.     

Genetic structure and gene flow in a metapopulation of an endangered plant species,Silene tatarica

We investigated the distribution of genetic variation within and between seven subpopulations in a riparian population of Silene tatarica in northern Finland by using amplified fragment length polymorphism (AFLP) markers. A Bayesian approach-based clustering program indicated that the marker data contained not only one panmictic population, but consisted of seven clusters, and that each original sample site seems to consist of a distinct subpopulation. A coalescent-based simulation approach shows recurrent gene flow between subpopulations. Relative high FST values indicated a clear subpopulation differentiation. However, amova analysis and UPGMA-dendrogram did not suggest any hierarchical regional structuring among the subpopulations. There was no correlation between geographical and genetic distances among the subpopulations, nor any correlation between the subpopulation census size and amount of genetic variation. Estimates of gene flow suggested a low level of gene flow between the subpopulations, and the assignment tests proposed a few long-distance bidirectional dispersal events between the subpopulations. No apparent difference was found in within-subpopulation genetic diversity among upper, middle and lower regions along the river. Relative high amounts of linkage disequilibrium at subpopulation level indicated recent population bottlenecks or admixture, and at metapopulation levels a high subpopulation turnover rate. The overall pattern of genetic variation within and between subpopulations also suggested a 'classical' metapopulation structure of the species suggested by the ecological surveys.     

Genetic structure in Red Junglefowl (Gallus gallus) populations: Strong spatial patterns in the wild ancestors of domestic chickens in a core distribution range

Red Junglefowl (Gallus gallus) are among the few remaining ancestors of an extant domesticated livestock species, the domestic chicken, that still occur in the wild. Little is known about genetic diversity, population structure, and demography of wild Red Junglefowl in their natural habitats. Extinction threats from habitat loss or genetic alteration from domestic introgression exacerbate further the conservation status of this progenitor species. In a previous study, we reported extraordinary adaptive genetic variation in the MHC B‐locus in wild Red Junglefowl and no evidence of allelic introgression between wild and domestic chickens was observed. In this study, we characterized spatial genetic variation and population structure in naturally occurring populations of Red Junglefowl in their core distribution range in South Central Vietnam. A sample of 212 Red Junglefowl was obtained from geographically and ecologically diverse habitats across an area of 250 × 350 km. We used amplified fragment‐length polymorphism markers obtained from 431 loci to determine whether genetic diversity and population structure varies. We found that Red Junglefowl are widely distributed but form small and isolated populations. Strong spatial genetic patterns occur at both local and regional scales. At local scale, population stratification can be identified to approximately 5 km. At regional scale, we identified distinct populations of Red Junglefowl in the southern lowlands, northern highlands, and eastern coastal portions of the study area. Both local and long‐distance genetic patterns observed in wild Red Junglefowl may reflect the species’ ground‐dwelling and territorial characteristics, including dispersal barriers imposed by the Annamite Mountain Range. Spatially explicit analyses with neutral genetic markers can be highly informative and here elevates the conservation profile of the wild ancestors of domesticated chickens.     

Genetic structure in large, continuous mammal populations: the example of brown bears in northwestern Eurasia

Knowledge of population structure and genetic diversity and the spatio-temporal demographic processes affecting populations is crucial for effective wildlife preservation, yet these factors are still poorly understood for organisms with large continuous ranges. Available population genetic data reveal that widespread mammals have for the most part only been carefully studied at the local population scale, which is insufficient for understanding population processes at larger scales. Here, we provide data on population structure, genetic diversity and gene flow in a brown bear population inhabiting the large territory of northwestern Eurasia. Analysis of 17 microsatellite loci indicated significant population substructure, consisting of four genetic groups. While three genetic clusters were confined to small geographical areas-located in Estonia, southern Finland and Leningrad oblast, Russia-the fourth cluster spanned a very large area broadly falling between northern Finland and the Arkhangelsk and Kirov oblasts of Russia. Thus, the data indicate a complex pattern where a fraction of the population exhibits large-scale gene flow that is unparalleled by other wild mammals studied to date, while the remainder of the population appears to have been structured by a combination of demographic history and landscape barriers. These results based on nuclear data are generally in good agreement with evidence previously derived using mitochondrial markers, and taken together, these markers provide complementary information about female-specific and population-level processes. Moreover, this study conveys information about spatial processes occurring over multiple generations that cannot be readily gained using other approaches, e.g. telemetry.     

Genetic structure and population differentiation of the Mediterranean pioneer spiny broom Calicotome villosa across the Strait of Gibraltar

The region around the Strait of Gibraltar is considered to be one of the most relevant 'hot spots' of biodiversity in the Mediterranean Basin due to its historical, biogeographical, and ecological features. Prominent among these is its role as a land bridge for the migration and differentiation of species during the Pleistocene, as a consequence of the lowering of sea level and climate changes associated with the Ice Ages. In the present study, we report a multilevel hierarchical investigation of the genetic diversity of Calicotome villosa , a common pioneer legume shrub, at the regional scale. The results of genetic analysis of progeny arrays are consistent with a predominantly outcrossing mating system in all the populations analysed. Geographically, a pattern of population isolation by distance was found, but the Strait accounted for only approximately 2% of the among-population genetic differentiation. Consequently, extensive historical gene flow appears to be the rule for this species in this area. According to the natural history traits of C. villosa (pollination, dispersal, and colonization ability), we hypothesize that gene flow must be strongly influenced by seed dispersal because pollen flow is very limited. Based on the history of trade and land use, cattle and human movements across the Strait must have strongly favoured seed dispersal. We review and discuss these results and compare them with those of other reported studies of genetic and phylogenetic differentiation across the Strait of Gibraltar. It is stressed that colonization ability, which depends upon seed dispersal and life form, can be a more critical factor in gene flow than pollination.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 39–51.     

Genetic and ecological data provide incongruent interpretations of population structure and dispersal in naturally subdivided populations of white-tailed ptarmigan (Lagopus leucura)

The dispersal of individuals among populations affects the demographic and adaptive trajectories of animal populations and is fundamental to understanding population dynamics. White-tailed ptarmigan (Lagopus leucura) are a high elevation grouse species that live year-round in patchily distributed alpine areas in western North America. We investigated the patterns of dispersal and identified barriers to gene flow for a threatened subspecies (L. l. saxatilis) endemic to Vancouver Island, Canada. Connectivity among seven sites was examined using nine microsatellite loci (n = 133 individuals, H(O) = 0.62, mean number of alleles = 10) and direct movement observations using radio-telemetry (n = 118 individuals). Average movement distances of individuals measured by radio-telemetry were 0.63-3.23 km and considerably less than the shortest distance between sampling sites (18 km). Furthermore, despite extensive radio-telemetry data, movement was never observed between any of the seven sampling sites. In contrast, genetic results (STRUCTURE, TESS) showed connectivity among most of the seven sampling sites and suggested that genetic variation is best explained by two clusters of individuals which separated the South sampling site from all other areas of Vancouver Island. Analysis of molecular data also showed a generally consistent pattern of isolation by distance (Mantel test r = 0.11, P < 0.01) with large areas of unsuitable low elevation habitat possibly acting as barriers to gene flow. Despite the naturally subdivided distribution of populations, white-tailed ptarmigan do not fit well into any common definition of a metapopulation. We conclude the incongruities between the genetic and radio-telemetry data are best explained by episodic dispersal patterns. In this study, we demonstrated the importance of combining genetic and ecological data in understanding patterns of dispersal and population structure.     

Joint estimation of contemporary seed and pollen dispersal rates among plant populations

There are few statistical methods for estimating contemporary dispersal among plant populations. A maximum-likelihood procedure is introduced here that uses pre- and post-dispersal population samples of biparentally inherited genetic markers to jointly estimate contemporary seed and pollen immigration rates from a set of discrete external sources into a target population. Monte Carlo simulations indicate that accurate estimates and reliable confidence intervals can be obtained using this method for both pollen and seed migration rates at modest sample sizes (100 parents/population and 100 offspring) when population differentiation is moderate (F(ST) ≥ 0.1), or by increasing pre-dispersal samples (to about 500 parents/population) when genetic divergence is weak (F(ST) = 0.01). The method exhibited low sensitivity to the number of source populations and achieved good accuracy at affordable genetic resolution (10 loci with 10 equifrequent alleles each). Unsampled source populations introduced positive biases in migration rate estimates from sampled sources, although they were minor when the proportion of immigration from the latter was comparatively low. A practical application of the method to a metapopulation of the Australian resprouter shrub Banksia attenuata revealed comparable levels of directional seed and pollen migration among dune groups, and the estimate of seed dispersal was higher than a previous estimate based on conservative assignment tests. The method should be of interest to researchers and managers assessing broad-scale nonequilibrium seed and pollen gene flow dynamics in plants.     

Clonal dispersal and spatial mixing in populations of the plant pathogenic fungus, Sclerotinia sclerotiorum

Two thousand seven hundred and forty-seven isolates of Sclerotinia sclerotiorum were sampled from four field populations of canola in western Canada. Each field was sampled in a grid of 128 50-m 50-m quadrats plus four intensive quadrats each sampled in a diagonal transect. Sampling was done at two phases of the disease cycle: (1) from ascospore inoculum on petals and (2) from disease lesions in stems. A total of 594 unique genotypes was identified by DNA fingerprinting. In each field, a small group of clones represented the majority of the sample, with a large group of clones or genotypes sampled once or twice. Clone frequencies were compared by χ² tests. The difference in profiles of clone frequencies for the two fields sampled in 1991 was not significant, but in 1992 the difference in profiles was marginally significant, indicating some local population substructure. The difference in profiles of clone frequencies for petals and lesions was not significant in each of the two fields sampled in 1991. In each of the two fields sampled in 1992, however, the difference was highly significant, consistent either with selection for some clones or with waves of immigration during the disease cycle. Nine of the 30 most frequently sampled clones from this study were previously recovered in a macrogeographical sample from western Canada in 1990. For spatial analyses, randomization tests indicated no significant spatial aggregation of either clones on petals or clones from lesions. Also, isolates of a clone on petals were not closer to isolates of the same clone from lesions than could be predicted by chance. Both observations suggest spatial mixing of ascospore inoculum from resident or immigrant sources.     

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.