Drivers of tree fecundity in pedunculate oak (Quercus robur) refugial populations at the species' southwestern range margin.

The current low latitudinal range margins of many extra-tropical plant species consist of small and scattered populations that persist locally in microrefugia. It remains poorly understood how their refugial distribution affects mating patterns and reproductive success. Here we examine flower and acorn production and their determinants in refugial populations of the widespread European forest tree pedunculate oak (Quercus robur). We monitored male flower, female flower and acorn production in 159 adult trees from 12 oak stands over 2 years. We related these and derived parameters to a series of ecological and genetic predictor variables extrinsic (stand size, density and isolation as well as elevation, topography and forest cover) or intrinsic (size, phenology and several genotypic measures) to the target tree. Tree fertility was unrelated to extrinsic factors but determined by tree size, although we detected size-independent variation in reproductive investment. Female flower number accurately predicted acorn crop size. Fruit set differed between years, evidencing the existence of pollen limitation at the landscape but not at the local scale. Fruit set also tended to increase with the number of mates of the target tree. We detected no other evidence for genetic constraints on mating. Reproduction was triggered by a combination of small-scale and landscape-scale drivers. Although short-distance mating prevailed, limited pollen flow did not appear to significantly constrain reproductive success. The high intrinsic ability of populations to maintain their reproductive capacity may help explain their successful long-term persistence in an adverse broader environment.

Unusually limited pollen dispersal and connectivity of Pedunculate oak (Quercus robur) refugial populations at the species' southern range margin.

Low-latitudinal range margins of temperate and boreal plant species typically consist of scattered populations that persist locally in microrefugia. It remains poorly understood how their refugial habitats affect patterns of gene flow and connectivity, key components for their long-term viability and evolution. We examine landscape-scale patterns of historical and contemporary gene flow in refugial populations of the widespread European forest tree Pedunculate oak (Quercus robur) at the species' southwestern range margin. We sampled all adult trees (n = 135) growing in a 20 km long valley and genotyped 724 acorns from 72 mother trees at 17 microsatellite loci. The ten oak stands that we identified were highly differentiated and formed four distinct genetic clusters, despite sporadic historical dispersal being detectable. By far most contemporary pollination occurred within stands, either between local mates (85.6%) or through selfing (6.8%). Pollen exchange between stands (2.6%) was remarkably rare given their relative proximity and was complemented by long-distance pollen immigration (4.4%) and hybridization with the locally abundant Quercus pyrenaica (0.6%). The frequency of between-stand mating events decreased with increasing size and spatial isolation of stands. Overall, our results reveal outstandingly little long-distance gene flow for a wind-pollinated tree species. We argue that the distinct landscape characteristics of oaks' refugial habitats, with a combination of a rugged topography, dense vegetation and humid microclimate, are likely to increase plant survival but to hamper effective long-distance pollen dispersal. Moreover, local mating might be favoured by high tree compatibility resulting from genetic purging in these long-term relict populations.

Colour, design and reward: phenotypic integration of fleshy fruit displays.

The functional or structural linkage among traits [phenotypic integration (PI)] within complex structures can constrain the evolutionary response of individual traits. To analyse whether frugivores with distinct sensory ecology have shaped the patterns of fruit diversification differently, we compared PI values of fleshy fruits that are consumed by birds and mammals. We used phylogenetic comparative analyses of PI among 13 morphological, nutritional and visual fruit traits from 111 Mediterranean plant species. Results showed that morphological traits had higher PI values than nutritional and colour traits. Visual and nutritional traits show positive phylogenetic covariance, while negative covariation occurs between fruits size and nutrients. Importantly, fruits consumed by birds were relatively more integrated than fruits consumed partly or solely by mammals. Hence, we show that major groups of mutualistic frugivores can shape the covariance among some fruit traits differently and thereby influence fruit diversification.

Isolation and characterization of 16 polymorphic microsatellite loci for Frangula alnus (Rhamnaceae).

We report the first 16 polymorphic nuclear microsatellite markers developed for Frangula alnus (Rhamnaceae). Markers were tested on all three subspecies as well as on three local populations, including analyses of both leaf and seed endocarps. A total of 87 alleles were found (mean number of alleles per locus was 5.44) for 72 individuals genotyped. Observed and expected heterozygosities ranged from 0.097 to 0.792 and from 0.093 to 0.794, respectively. The levels of polymorphism and exclusionary power of the developed markers render them applicable for parentage analyses and measurements of seed dispersal through direct comparison of endocarps and adult tree genotypes.

Isolation and characterization of 12 microsatellite loci for Rhamnus alaternus (Rhamnaceae).

Twelve polymorphic microsatellite loci were developed for Rhamnus alaternus using an enriched-library approach. We detected 69 alleles in 49 individuals genotyped (mean number of alleles per locus was 4.79) in two different populations. The values of observed and expected heterozygosities ranged from 0.045 to 0.963 and 0.089 to 0.873 respectively. Levels of polymorphism and the exclusionary power of the developed markers render them readily applicable for studies of contemporary pollen and seed gene flow through parentage analyses.

Contemporary pollen and seed dispersal in a Prunus mahaleb population: patterns in distance and direction.

Pollination and seed dispersal determine the spatial pattern of gene flow in plant populations and, for those species relying on pollinators and frugivores as dispersal vectors, animal activity plays a key role in determining this spatial pattern. For these plant species, reported dispersal patterns are dominated by short-distance movements with a significant amount of immigration. However, the contribution of seed and pollen to the overall contemporary gene immigration is still poorly documented for most plant populations. In this study we investigated pollination and seed dispersal at two spatial scales in a local population of Prunus mahaleb (L.), a species pollinated by insects and dispersed by frugivorous vertebrates. First, we dissected the relative contribution of pollen and seed dispersal to gene immigration from other parts of the metapopulation. We found high levels of gene immigration (18.50%), due to frequent long distance seed dispersal events. Second, we assessed the distance and directionality for pollen and seed dispersal events within the local population. Pollen and seed movement patterns were non-random, with skewed distance distributions: pollen tended moved up to 548 m along an axis approaching the N-S direction, and seeds were dispersed up to 990 m, frequently along the SW and SE axes. Animal-mediated dispersal contributed significantly towards gene immigration into the local population and had a markedly nonrandom pattern within the local population. Our data suggest that animals can impose distinct spatial signatures in contemporary gene flow, with the potential to induce significant genetic structure at a local level.

Differential contribution of frugivores to complex seed dispersal patterns.

Frugivores are highly variable in their contribution to fruit removal in plant populations. However, data are lacking on species-specific variation in two central aspects of seed dispersal, distance of dispersal and probability of dispersal among populations through long-distance transport. We used DNA-based genotyping techniques on Prunus mahaleb seeds dispersed by birds (small- and medium-sized passerines) and carnivorous mammals to infer each seed's source tree, dispersal distance, and the probability of having originated from outside the study population. Small passerines dispersed most seeds short distances (50% dispersed <51 m from source trees) and into covered microhabitats. Mammals and medium-sized birds dispersed seeds long distances (50% of mammals dispersed seeds >495 m, and 50% of medium-sized birds dispersed seeds to >110 m) and mostly into open microhabitats. Thus, dispersal distance and microhabitat of seed deposition were linked through the contrasting behaviors of different frugivores. When the quantitative contribution to fruit removal was accounted for, mammals were responsible for introducing two-thirds of the immigrant seeds into the population, whereas birds accounted for one-third. Our results demonstrate that frugivores differ widely in their effects on seed-mediated gene flow. Despite highly diverse coteries of mutualistic frugivores dispersing seeds, critical long-distance dispersal events might rely on a small subset of large species. Population declines of these key frugivore species may seriously impair seed-mediated gene flow in fragmented landscapes by truncating the long-distance events and collapsing seed arrival to a restricted subset of available microsites.

Mating patterns, pollen dispersal, and the ecological maternal neighbourhood in a Prunus mahaleb L. population.

Gender polymorphism, plant-animal interactions, and environmental heterogeneity are the three important sources of variation in mating system and pollen dispersal patterns. We used progeny arrays and paternity analysis to assess the effects of gender type and density level on variation in mating patterns within a highly isolated population of Prunus mahaleb, a gynodioecious species. All the adult trees in the population were sampled and located. The direct estimate of long-distance insect-mediated pollination events was low (< 10%). Gender expression deeply influenced the mating system, decreasing the outcrossing rates (t(m)) and the pollen pool diversity in hermaphrodite trees. Long intermate distances (> 250 m) were significantly more frequent among female mother trees. Variation in local tree density also affected pollen pool diversity and intermate distance, with a higher effective number of fathers (k(e)) and longer intermate distances for female trees in low-density patches. A canonical correlation analysis showed significant correlations between mating variables and the maternal ecological neighbourhood. Only the first canonical variable was significant and explained 78% of variation. Outcrossing rates tended to decrease, and the relatedness among the fathers tended to increase, when mother trees grew in dense patches with high cover of other woody species and taller vegetation away from the pine forest edge. We highlight the relevance of considering maternal ecological neighbourhood effects on mating system and gene flow studies as maternal trees act simultaneously as receptors of pollen and as sources of the seeds to be dispersed.

Rangewide phylogeography of a bird-dispersed Eurasian shrub: contrasting Mediterranean and temperate glacial refugia.

We studied the phylogeography of alder buckthorn (Frangula alnus), a bird-dispersed shrub or small tree distributed over most of Europe and West Asia and present in three of the four main refugia of West Palaearctic temperate woody plants: the Iberian Peninsula, the Balkans and Anatolia. A total of 78 populations from 21 countries were analysed for chloroplast DNA variation using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and 21 different haplotypes were distinguished. We found a very strong overall population differentiation (GST = 0.81) and phylogeographical structure, and a sharp contrast between the haplotype-rich refugia and the almost completely uniform area of postglacial colonization. The haplotype network comprises three lineages made up of haplotypes from the Iberian Peninsula, Anatolia with the Caucasus, and temperate Europe. The Iberian and the Anatolian branches represent parts of a major lineage that spans over the whole northern Mediterranean Basin and some neighbouring areas and probably dates back to the Tertiary. Many haplotypes of this lineage are distributed locally and most populations are fixed for a single haplotype; these populations have apparently been very stable since their establishment, experiencing negligible gene flow and few mutations. The temperate European lineage consists of one very widespread and abundant plus six locally distributed haplotypes. Four of them are located in Southeast Europe, the putative refugium of all extant temperate European populations. Contrary to populations from Iberia and Anatolia, F. alnus populations from the southeastern European refugium have most genetic variation within populations. Bird-mediated seed dispersal has apparently allowed not only a very rapid postglacial expansion of F. alnus but also subsequent regular seed exchanges between populations of the largely continuous species range in temperate Europe. In contrast, the disjunct F. alnus populations persisting in Mediterranean mountain ranges seem to have experienced little gene flow and have therefore accumulated a high degree of differentiation, even at short distances. Populations from the southern parts of the glacial refugia have contributed little to the postglacial recolonization of Europe, but their long-term historical continuity has allowed them to maintain a unique store of genetic variation.

Seed dispersal by animals: exact identification of source trees with endocarp DNA microsatellites.

A long-standing challenge in studies of seed dispersal by animal frugivores has been the characterization of the spatial relationships between dispersed seeds and the maternal plants, i.e. the seed shadow. The difficulties to track unambiguously the origin of frugivore-dispersed seeds in natural communities has been considered an unavoidable limitation of the research field and precluded a robust analysis of the direct consequences of zoochory. Here we report that the multilocus genotype at simple sequence repeat (SSR; microsatellite) loci of the woody endocarp, a tissue of maternal origin, provides an unequivocal genetic fingerprint of the source tree. By comparing the endocarp genotype against the complete set of genotypes of reproductive trees in the population, we could unambiguously identify the source tree for 82.1% of the seeds collected in seed traps and hypothesize that the remaining 17.9% of sampled seeds come from other populations. Identification of the source tree for Prunus mahaleb seeds dispersed by frugivores revealed a marked heterogeneity in the genetic composition of the seed rain in different microhabitats, with a range of 1-5 distinct maternal trees contributing seeds to a particular landscape patch. Within-population dispersal distances ranged between 0 and 316 m, with up to 62% of the seeds delivered within 15 m of the source trees. Long distance dispersal events, detected by the exclusion of all reproductive trees in the population, accounted for up to 17.9% of the seeds sampled. Our results indicate strong distance limitation of seed delivery combined with infrequent long-distance dispersal events, extreme heterogeneity in the landscape pattern of genetic makeup, and a marked mosaic of multiple parentage for the seeds delivered to a particular patch.

RAPD variation and population genetic structure in Prunus mahaleb (Rosaceae), an animal-dispersed tree.

We examined the patterns of random amplified polymorphic DNA (RAPD) variation among seven Prunus mahaleb (Rosaceae) populations extending over approximately 100 km2 to examine local differentiation in relation to spatial isolation due to both geographical distance and differences in elevation. No less than 51. 4% of the RAPD loci were polymorphic, but very few were fixed and among-population variation accounted for 16.46% of variation in RAPD patterns. Mean gene diversity was 0.1441, with mean Nei's genetic diversity for individual populations ranging between 0.089 and 0.149. Mean GST value across loci was 0.1935 (range, 0.0162-0.4685), giving an average estimate for Nm of 1.191. These results suggest extensive gene flow among populations, but higher GST and lower Nm values relative to other outcrossing, woody species with endozoochorous dispersal, also suggest a process of isolation by distance. The combined effect of both geographical and elevation distances and nonoverlapping flowering and fruiting phenophases on the GST matrix was partially significant, revealing only marginal isolation of the P. mahaleb populations. The matrix correlation between estimated Nm values among populations and the geographical + elevation distance matrices (r = -0.4623, P = 0.07), suggests a marginal trend for more isolated populations to exchange less immigrants. Long-distance seed dispersal by efficient medium-sized frugivorous birds and mammals is most likely associated to the high levels of within-population genetic diversity. However, vicariance factors and demographic bottlenecks (high postdispersal seed and seedling mortality) explain comparatively high levels of local differentiation.

Annual variability in seed production by woody plants and the masting concept: reassessment of principles and relationship to pollination and seed dispersal.

By analyzing 296 published and unpublished data sets describing annual variation in seed output by 144 species of woody plants, this article addresses the following questions. Do plant species naturally fall into distinct groups corresponding to masting and nonmasting habits? Do plant populations generally exhibit significant bimodality in annual seed output? Are there significant relationships between annual variability in seed production and pollination and seed dispersal modes, as predicted from economy of scale considerations? We failed to identify distinct groups of species with contrasting levels of annual variability in seed output but did find evidence that most polycarpic woody plants seem to adhere to alternating supra-annual schedules consisting of either high or low reproduction years. Seed production was weakly more variable among wind-pollinated taxa than animal-pollinated ones. Plants dispersed by mutualistic frugivores were less variable than those dispersed by either inanimate means or animals that predominantly behave as seed predators. We conclude that there are no objective reasons to perpetuate the concept of mast fruiting in the ecological literature as a shorthand to designate a distinct biological phenomenon. Associations between supra-annual variability in seed output and pollination and seed dispersal methods suggest the existence of important reproductive correlates that demand further investigation.