Individual spatial aggregation correlates with between-population variation in fine-scale genetic structure of Silene ciliata (Caryophyllaceae).

Fine-scale genetic structure (FSGS) can vary among populations within species depending on multiple demographic and environmental factors. Theoretical models predict that FSGS should decrease in high-density populations and increase in populations where individuals are spatially aggregated. However, few empirical studies have compared FSGS between populations with different degrees of individual spatial aggregation and microhabitat heterogeneity. In this work, we studied the relationship between spatial and genetic structure in five populations of alpine specialist Silene ciliata Poiret (Caryophyllaceae). We mapped all individuals in each population and genotyped 96 of them using 10 microsatellite markers. We found significant FSGS consistent with an isolation-by-distance process in three of the five populations. The intensity of FSGS was positively associated with individual spatial aggregation. However, no association was found between FSGS and global population density or microhabitat heterogeneity. Overall, our results support theoretical studies indicating that stronger spatial aggregation tends to increase the magnitude of FSGS. They also highlight the relevance of characterizing local plant distribution and microhabitat to better understand the mechanisms that generate intraspecific variation in FSGS across landscapes.

Estimating exotic gene flow into native pine stands: zygotic vs. gametic components.

Monitoring contemporary gene flow from widespread exotic plantations is becoming an important problem in forest conservation genetics. In plants, where both seed and pollen disperse, three components of exotic gene flow with potentially unequal consequences should be, but have not been, explicitly distinguished: zygotic, male gametic and female gametic. Building on a previous model for estimating contemporary rates of zygotic and male gametic gene flow among plant populations, we present here an approach that additionally estimates the third (female gametic) gene flow component, based on a combination of uni- and biparentally inherited markers. Using this method and a combined set of chloroplast and nuclear microsatellites, we estimate gene flow rates from exotic plantations into two Iberian relict stands of maritime pine (Pinus pinaster) and Scots pine (Pinus sylvestris). Results show neither zygotic nor female gametic gene flow but moderate (6-8%) male gametic introgression for both species, implying significant dispersal of pollen, but not of seeds, from exotic plantations into native stands shortly after introduced trees reached reproductive maturity. Numerical simulation results suggest that the model yields reasonably accurate estimates for our empirical data sets, especially for larger samples. We discuss conservation management implications of observed levels of exposure to nonlocal genes and identify research needs to determine potentially associated hazards. Our approach should be useful for plant ecologists and ecosystem managers interested in the vectors of contemporary genetic connectivity among discrete plant populations.

Patterns of pollen dispersal in a small population of the Canarian endemic palm (Phoenix canariensis).

The genetic diversity of small populations is greatly influenced by local dispersal patterns and genetic connectivity among populations, with pollen dispersal being the major component of gene flow in many plants species. Patterns of pollen dispersal, mating system parameters and spatial genetic structure were investigated in a small isolated population of the emblematic palm Phoenix canariensis in Gran Canaria island (Canary Islands). All adult palms present in the study population (n=182), as well as 616 seeds collected from 22 female palms, were mapped and genotyped at 8 microsatellite loci. Mating system analysis revealed an average of 5.8 effective pollen donors (Nep) per female. There was strong variation in correlated paternity rates across maternal progenies (ranging from null to 0.9) that could not be explained by the location and density of local males around focal females. Paternity analysis revealed a mean effective pollen dispersal distance of ∼71 m, with ∼70% of effective pollen originating from a distance of <75 m, and 90% from <200 m. A spatially explicit mating model indicated a leptokurtic pollen dispersal kernel, significant pollen immigration (12%) from external palm groves and a directional pollen dispersal pattern that seems consistent with local altitudinal air movement. No evidence of inbreeding or genetic diversity erosion was found, but spatial genetic structure was detected in the small palm population. Overall, the results suggest substantial pollen dispersal over the studied population, genetic connectivity among different palm groves and some resilience to neutral genetic erosion and subsequently to fragmentation.

PSA: software for parental structure analysis of seed or seedling patches.

Parental structure analysis (PSA) is a computer program to analyse separate contributions of paternal and maternal parents to postdispersal plant offspring. The program provides joint estimates of maternal, paternal and cross-parental correlations within and among a set of predefined groups of seeds or seedlings, as well as derivative estimates of effective parental numbers. PSA utilizes data sets that distinguish between maternal and paternal contributions to the genotype of each offspring in the sample, but does not require parental samples per se. The approach requires assay of codominant diploid markers from both seed coat (maternally inherited) and seedling/embryo (biparentally inherited) tissues for each offspring. A simulation analysis of PSA's performance shows that it provides fairly accurate parental correlation estimates from affordable sampling effort. PSA should be of interest to plant biologists studying the interplay between dispersal, demography and genetics, as well as plant-animal interactions.

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.

Effects of forest plantations on the genetic composition of conspecific native Aleppo pine populations.

Afforestation is a common and widespread management practice throughout the world, yet its implications for the genetic diversity of native populations are still poorly understood. We examined the effect of Aleppo pine (Pinus halepensis) plantations on the genetic composition of nearby conspecific native populations. We focused on two native populations in Israel with different levels of isolation from the surrounding plantations and compared the genetic diversity of naturally established young trees within the native populations with that of local native adults, using nine nuclear microsatellite markers. We found that the genetic composition of the recruits was significantly different from that of local adults in both populations, with allelic frequency changes between generations that could not be ascribed to random drift, but rather to substantial gene flow from the surrounding planted Aleppo pine populations. The more isolated population experienced a lower gene-flow level (22%) than the less isolated population (49%). The genetic divergence between native populations at the adult-tree stage (F(st) = 0.32) was more than twice as high as that of the young trees naturally established around native adults (F(st) = 0.15). Our findings provide evidence for a rapid genetic homogenization process of native populations following the massive planting efforts in the last decades. These findings have important implications for forest management and nature conservation and constitute a warning sign for the risk of translocation of biota for local biodiversity.

Climatic niche and neutral genetic diversity of the six Iberian pine species: a retrospective and prospective view.

Quaternary climatic fluctuations have left contrasting historical footprints on the neutral genetic diversity patterns of existing populations of different tree species. We should expect the demography, and consequently the neutral genetic structure, of taxa less tolerant to particular climatic extremes to be more sensitive to long-term climate fluctuations. We explore this hypothesis here by sampling all six pine species found in the Iberian Peninsula (2464 individuals, 105 populations), using a common set of chloroplast microsatellite markers, and by looking at the association between neutral genetic diversity and species-specific climatic requirements. We found large variation in neutral genetic diversity and structure among Iberian pines, with cold-enduring mountain species (Pinus uncinata, P. sylvestris and P. nigra) showing substantially greater diversity than thermophilous taxa (P. pinea and P. halepensis). Within species, we observed a significant positive correlation between population genetic diversity and summer precipitation for some of the mountain pines. The observed pattern is consistent with the hypotheses that: (i) more thermophilous species have been subjected to stronger demographic fluctuations in the past, as a consequence of their maladaptation to recurrent glacial cold stages; and (ii) altitudinal migrations have allowed the maintenance of large effective population sizes and genetic variation in cold-tolerant species, especially in more humid regions. In the light of these results and hypotheses, we discuss some potential genetic consequences of impending climate change.

Isolation by distance in a continuous population under stochastic demographic fluctuations.

The local density of individuals is seldom uniform in space and time within natural populations. Yet, formal approaches to the process of isolation by distance in continuous populations have encountered analytical difficulties in describing genetic structuring with demographic heterogeneities, usually disregarding local correlations in the movement and reproduction of genes. We formulate exact recursions for probabilities of identity in continuous populations, from which we deduce definitions of effective dispersal () and effective density (D(e)) that generalize results relating spatial genetic structure, dispersal and density in lattice models. The latter claim is checked in simulations where estimates of effective parameters obtained from demographic information are compared with estimates derived from spatial genetic patterns in a plant population evolving in a heterogeneous and dynamic habitat. The simulations further suggest that increasing spatio-temporal correlations in local density reduce and generally decrease the product , with dispersal kurtosis influencing their sensitivity to density fluctuations. As in the lattice model, the expected relationship between the product and the genetic structure statistic a(r) holds under fluctuating density, irrespective of dispersal kurtosis. The product D sigma(2) between observed census density and the observed dispersal rate over one generation will generally be an upwardly biased (up to 400% in simulations) estimator of in populations distributed in spatially aggregated habitats.

Estimating gametic introgression rates in a risk assessment context: a case study with Scots pine relicts.

The estimation of recent gene immigration is fundamental to a wide range of evolutionary and conservation studies. In a risk assessment context, gene flow estimation procedures are needed that are both accurate and readily amenable to formal evaluation of statistical uncertainty. However, genetic methods for estimating recent migration rates that are specific and have been thoroughly evaluated are scarce. Here we use an original and straightforward maximum-likelihood method to estimate recent uniparental gametic immigration from non-local plantations into an endangered population of the Iberian relict pine variety Pinus sylvestris var. nevadensis D. H. Christ. Our approach is not intended to ascertain population membership of individuals, but rather to obtain accurate immigration rate estimates with reliable confidence limits. We found very high (approximately 40%) pollen introgression at the seed-crop level into the Scots pine relict, and substantial (10-15%) male gametic introgression among naturally regenerated recruits. Using numerical simulation, we show that our method yields uniparental gametic immigration estimates that are expected to be virtually unbiased and usually accurate under our sampling conditions. Among four tested methods to estimate the confidence intervals for immigration estimates, the profile-likelihood method was the best, as it outperformed bootstrapping procedures and yielded coverage close to nominal limits under different sample sizes and migration rates. This study presents a method by which researchers can facilitate decision making within a gene flow risk assessment context.

Mating system and pollen gene flow in Mediterranean maritime pine.

Mating systems define the mode of gene transmission across generations, helping to determine the amount and distribution of genetic variation within and among populations of plant species. A hierarchical analysis of Mediterranean maritime pine mating system (61 mother trees from 24 plots, clustered in three populations) was used to identify factors affecting mating patterns and to fit pollen dispersal kernels. Levels of ovule and seed abortion, multi- and single-locus outcrossing rates and correlated paternity were estimated from progeny arrays and correlated with ecological stand variables and biometric tree measures. Pollen dispersal kernels were fitted using TwoGener and KinDist indirect methods and simulations were carried out to identify relevant factors affecting correlated paternity. Maritime pine showed high outcrossing rates (t(m) and t(s) approximately 0.96) and relatively low levels of correlated paternity [an r(p) of 0.018 (Ritland's estimate) or 0.048 (Hardy's estimate)], although higher than in other anemophilous tree species. Mating system parameters had high variation at the single-tree level (99-100%) but no stand or population effect was detected. At the single-tree level, outcrossing rates were correlated with tree (diameter and height) and crown size. In addition, correlated paternity showed a significant negative correlation with tree height, height to crown base and height to the largest crown width, probably reflecting the importance of the trees' 'ecological neighborhoods'. Indirectly estimated pollen dispersal kernels were very leptokurtic (exponential-power distributions with beta<<0.5), with mean dispersal distances from 78.4 to 174.4 m. Fitted dispersal kernels will be useful in building explicit simulation models that include dispersal functions, and which will contribute to current conservation and management programs for maritime pine. Nevertheless, the numerical simulations showed that restricted dispersal, male fertility and phenological overlap could only partially explain the observed levels of correlated paternity; so other factors may also be relevant for the management of this valuable forest tree species.

Measuring the genetic structure of the pollen pool as the probability of paternal identity.

Contemporary pollen flow in forest plant species is measured by the probability of paternal identity (PPI) for two randomly sampled offspring, drawn from a single female, and contrasting that with PPI for two random offspring, drawn from different females. Two different estimation strategies have emerged: (a) an indirect approach, using the 'genetic structure' of the pollen received by different mothers and (b) a direct approach, based on parentage analysis. The indirect strategy is somewhat limited by the assumptions, but is widely useful. The direct approach is most appropriate where a large majority of the true fathers can be identified exactly, which is sometimes possible with high-resolution SSR markers. Using the parentage approach, we develop estimates of PPI, showing that the obvious estimates are severely biased, and providing an unbiased alternative. We then illustrate the methods with SSR data from a 36-tree isolated population of Pinus sylvestris from the Meseta region of Spain, for which categorical paternity assignment was available for over 95% of offspring. For all the females combined, we estimate that PPI=0.0425, indicating uneven male reproductive contributions. Different (but overlapping) arrays of males pollinate different females, and for the average female, PPI=0.317, indicating substantial 'pollen structure' for the population. We also relate the direct measures of PPI to those available from indirect approaches, and show that they are generally comparable.

Patterns of pollen dispersal in a small population of Pinus sylvestris L. revealed by total-exclusion paternity analysis.

Patterns of pollen dispersal were investigated in a small, isolated, relict population of Pinus sylvestris L., consisting of 36 trees. A total-exclusion battery comprising four chloroplast and two nuclear microsatellites (theoretical paternity exclusion probability EP=0.996) was used to assign paternity to 813 seeds, collected from 34 trees in the stand. Long-distance pollen immigration accounted for 4.3% of observed matings. Self-fertilization rate was very high (0.25), compared with typical values in more widespread populations of the species. The average effective pollen dispersal distance within the stand was 48 m (or 83 m excluding selfs). Half of effective pollen was dispersed within 11 m, and 7% beyond 200 m. A strong correlation was found between the distance to the closest tree and the mean mating-distance calculated for single-tree progenies. The effective pollen dispersal distribution showed a leptokurtic shape, with a large and significant departure from that expected under uniform dispersal. A maximum-likelihood procedure was used to fit an individual pollen dispersal distance probability density function (dispersal kernel). The estimated kernel indicated fairly leptokurtic dispersal (shape parameter b=0.67), with an average pollen dispersal distance of 135 m, and 50% of pollen dispersed beyond 30 m. A marked directionality pattern of pollen dispersal was found, mainly caused by the uneven distribution of trees, coupled with restricted dispersal and unequal male success. Overall, results show that the number and distribution of potential pollen donors in small populations may strongly influence the patterns of effective pollen dispersal.

Increased selfing and correlated paternity in a small population of a predominantly outcrossing conifer, Pinus sylvestris.

Outcrossing rate, the rates of ovule and seed abortion, and levels of correlated paternity were estimated in a small population of Pinus sylvestris, a predominantly outcrossing conifer, and were compared with estimates from two widely dispersed woodlands of the same species, showing a range of densities. On average, seed trees of the small population showed an eight-fold higher selfing rate (25 vs. 3%) and a 100-fold greater incidence of correlated paternity (19.6 vs. 0.2%) than did trees from the large populations. No evidence was found of pollen limitation within the remnant stand, as suggested by ovule abortion rates. Investigation of the mating patterns in the small population, based on the unambiguous genealogy of 778 open-pollinated seeds, showed a large departure from random mating. Only 8% of the possible mating pairs within the stand were observed. Correlated paternity rate within a maternal sibship was negatively associated (rs = -0.398, P < 0.050) with the distance to the nearest neighbour, and shared paternity among maternal sibships was negatively correlated (rs = -0.704, P < 0.001) with the distance between seed trees. Numerical simulations, based on the estimated individual pollen dispersal kernel, suggest that restricted dispersal might have been the key factor affecting mating patterns in the small population and, together with low population density, may account for the observed mating system variation between the small and the large populations. The results of this study show that a severe size reduction may substantially affect the mating system of a wind-pollinated, typically outcrossed plant species.

Cross-amplification and sequence variation of microsatellite loci in Eurasian hard pines.

Microsatellite transfer across coniferous species is a valued methodology because de novo development for each species is costly and there are many species with only a limited commodity value. Cross-species amplification of orthologous microsatellite regions provides valuable information on mutational and evolutionary processes affecting these loci. We tested 19 nuclear microsatellite markers from Pinus taeda L. (subsection Australes) and three from P. sylvestris L. (subsection Pinus) on seven Eurasian hard pine species ( P. uncinata Ram., P. sylvestris L., P. nigra Arn., P. pinaster Ait., P. halepensis Mill., P. pinea L. and P. canariensis Sm.). Transfer rates to species in subsection Pinus (36-59%) were slightly higher than those to subsections Pineae and Pinaster (32-45%). Half of the trans-specific microsatellites were found to be polymorphic over evolutionary times of approximately 100 million years (ten million generations). Sequencing of three trans-specific microsatellites showed conserved repeat and flanking regions. Both a decrease in the number of perfect repeats in the non-focal species and a polarity for mutation, the latter defined as a higher substitution rate in the flanking sequence regions close to the repeat motifs, were observed in the trans-specific microsatellites. The transfer of microsatellites among hard pine species proved to be useful for obtaining highly polymorphic markers in a wide range of species, thereby providing new tools for population and quantitative genetic studies.