Large-scale forest fragmentation can increase interpopulation genetic differentiation and erode the genetic variability Distance-weighted fragmentation asexual reproduction remnant plant populations. In this study, we analyze the extent of clonality and the genetic variability and structure within a holm oak Quercus ilex population from Central Spain at 3 patches showing different degrees of fragmentation.
For this purpose, we have typed individuals adults and 86 saplings at 9 microsatellite loci.
We have also found evidence that fragmentation has contributed to reduce genetic variability and increase genetic differentiation in holm oak saplings, indicating that the younger cohorts are suffering some negative genetic consequences of long-term population fragmentation. Finally, analyses of fine spatial genetic structure have revealed significant kinship structures up to 20—50 m that were particularly patent in the 2 less fragmented patches.
Overall, our findings point to long-term genetic shifts in population structure of holm oaks in fragmented landscapes; however, further research is required on pollen dispersal and gene flow in this species. Human activities are responsible for large-scale forest fragmentation and destruction in several ecosystems worldwide Noss and Csuti ; Lindenmayer and Fischer This has often reduced continuous habitats into several smaller spatially isolated remnants, threatening the maintenance of biodiversity in such landscapes Saunders et al.
One of the consequences of fragmentation is the loss of connectivity between patches with its potential-associated genetic effects. In the case of wind-pollinated plants, reduction in fragment size and decreased pollen interchange Distance-weighted fragmentation asexual reproduction increase genetic differentiation between patches and erode local genetic variability Young "Distance-weighted fragmentation asexual reproduction" al.
The latter can reduce both individual fitness and the population's ability to respond to changes in selection pressures that can ultimately compromise its long-term viability Young et al. Moreover, intensive forest management and environmental perturbations can also favor asexual over sexual reproduction strategies, which can strongly reduce population genetic diversity if only some clones dominate and colonize large areas e.
Current empirical evidence on the genetic consequences of forest fragmentation is unclear Lowe et al.
These contrasting results may reflect interspecific differences in mating patterns, generation time, and longevity Young et al. On the other hand, some studies have suggested that the open landscape after fragmentation facilitates airborne pollen dispersal that can compensate for the expected detrimental genetic effects of fragmentation Young et al.
The holm oak Quercus ilexL. In Spain, it is the most prevalent tree species and extends over a surface of approximately 5. The holm oak plays an extremely important role for the functioning of Mediterranean forests, and it is considered a keystone species in these ecosystems Blanco et al.
This species is also of economic importance and has been extensively used by humans for centuries Blanco et al. In addition, large areas of holm oak forests have been cleared for large-scale farming and former woodlands have undergone extensive fragmentation for centuries Blondel and Aronson This long-term human management and alteration has resulted in different levels of forest fragmentation ranging from continuous forest to woodland islands of variable size, savannah-like dehesas, or even extremely isolated trees within farmland areas Blondel Aronson ; Vicente and Ales These human-induced changes in population structure could have reduced the genetic variability of small isolated populations, although long-distance pollen dispersal and the species long lifespan several centuries could have counterbalanced the expected loss of genetic diversity over time Petit et al.
However, in spite of the key role of the holm oak in Mediterranean ecosystems, no study has specifically analyzed the genetic consequences of fragmentation. In addition, the holm oak is known to be a vigorous resprouter after disturbance, with very effective asexual reproduction through root shooting Espelta et al. If clones are not properly discriminated, levels of local genetic diversity and the number of different individuals present in a population may be overestimated Mayes et al.
We have used 9 microsatellite markers to type individuals and discriminate clones, obtain accurate estimates of genetic variability, and analyze genetic structure under such contrasting scenarios of population fragmentation.
In particular, we tested the following predictions: In this study, we considered 3 patches differing in the degree of fragmentation and tree spatial isolation: The crops are mainly barley Hordeum vulgare and wheat Triticum spp. It should be noted that there was an absence of saplings and recruits in Distance-weighted fragmentation asexual reproduction A due to recurrent ploughing and destruction of new recruits. Adults and saplings are evenly distributed in the studied patches, although the latter is more frequent in areas with higher density of adult reproductive trees.
Spatial location Universal Transverse Mercator co-ordinates for each sampled tree was recorded using a Global Positioning System. Rather, we sampled the most Distance-weighted fragmentation asexual reproduction ramets from these clustered formations to cover the entire area of the suspected clone. Map of the study area showing the spatial distribution of the studied holm oak individuals. Only adult reproductive trees are showed. Ellipses indicate study patches: A an extensively cultivated area with highly isolated holm oaks growing within the culture matrix; B a noncultivated area covered with pastures and scattered holm oaks; and C a continuous holm oak Distance-weighted fragmentation asexual reproduction. We used 9 polymorphic microsatellite markers previously "Distance-weighted fragmentation asexual reproduction" for other Quercus species to genotype holm oaks in the study area: We also used Arlequin 3.
Genetic diversity was evaluated calculating the total number of alleles Aallelic richness standardized to the smallest sample size A Runbiased effective of alleles A eobserved heterozygosity H Oexpected heterozygosity H Eand inbreeding coefficient F IS.
All these analyses were performed on the basis of Distance-weighted fragmentation asexual reproduction genotypes, that is, multiple clonal genotypes were removed.
Each study patch and cohort was tested for heterozygosity excess in order to detect recent population bottlenecks using the program Bottleneck 1. We ran Bottleneck under the two-phase model TPM that is supposed to fit microsatellite evolution better than other methods Di Rienzo et al. Statistical significance was assessed with a two-tailed Wilcoxon signed-rank test, the most powerful and robust statistics when using less than 20 polymorphic loci Piry et al. We also investigated the distribution of the allele frequencies using the mode shift test also implemented in the program Bottleneck Piry et al.
For each locus, patch, and cohort, we calculated the M -ratio, defined as the ratio of the number of alleles to the range of allele sizes, a statistic that can detect reductions in population sizes Garza and Williamson M -ratios were calculated as follows: We then calculated mean M -ratios across all loci for each patch.