Seed-mediated Gene Flow Research Articles

Origins of a morphological cline between Eucalyptus melanophloia and Eucalyptus whitei

Most theories to explain the origin and maintenance of clines in Eucalyptus are based on a morphological classification system. The true relationships between putative species along clines require detailed investigation of phylogenetic relationships. A cline between Eucalyptus melanophloia and E. whitei was examined using morphological and molecular analyses to determine whether genetic structuring in nuclear and chloroplast DNA along the cline could be explained by secondary contact between independent evolutionary lineages, or whether the cline represents a single species that has undergone primary differentiation. Morphological analysis showed phenotypic variation distributed continuously across the cline and that seedlings bred true to parental type. Microsatellite analysis indicated that there was little genetic structuring across the cline, and low levels of population differentiation. This result was further reinforced by analysis of the cpDNA. The phylogeographic distribution of cpDNA haplotypes is likely to have resulted from restricted seed-mediated gene flow with isolation by distance. A cogent explanation for the cline is that it has arisen by selection on leaf types promoted by a gradient in precipitation with the short-broad, subsessile leaves of E. melanophloia favoured under higher rainfall and the long, narrow, petiolate leaves of E. whitei favoured in arid environments.

Molecular genetic variation in a widespread forest tree species Eucalyptus obliqua (Myrtaceae) on the island of Tasmania

Eucalyptus obliqua L’Hér. is widespread across south-eastern Australia. On the island of Tasmania it has a more-or-less continuous distribution across its range and it dominates much of the wet sclerophyll forest managed for forestry purposes. To understand better the distribution of genetic variation in these native forests we examined nuclear microsatellite diversity in 432 mature individuals from 20 populations of E. obliqua across Tasmania, including populations from each end of three locally steep environmental gradients. In addition, chloroplast microsatellite loci were assessed in 297 individuals across 31 populations. Nuclear microsatellite diversity values in E. obliqua were high (average HE = 0.80) and inbreeding coefficients low (average F = 0.02) within these populations. The degree of differentiation between populations was very low (FST = 0.015). No significant microsatellite differentiation could be found across three locally steep environmental gradients, even though there is significant genetic differentiation in quantitative traits. This suggests that the observed quantitative variation is maintained by natural selection. Population differentiation based on chloroplast haplotypes was high (GST = 0.69) compared with that based on nuclear microsatellites, suggesting that pollen-mediated gene flow is >150 times the level of seed-mediated gene flow in this animal-pollinated species; hence, pollen is likely to be the main mode of gene flow countering selection along local environmental gradients. Implications of these results for silvicultural practices are discussed.

Pollen- and Seed-Mediated Transgene Flow in Commercial Cotton Seed Production Fields

BackgroundCharacterizing the spatial patterns of gene flow from transgenic crops is challenging, making it difficult to design containment strategies for markets that regulate the adventitious presence of transgenes. Insecticidal Bacillus thuringiensis (Bt) cotton is planted on millions of hectares annually and is a potential source of transgene flow.Methodology/Principal FindingsHere we monitored 15 non-Bt cotton (Gossypium hirsutum, L.) seed production fields (some transgenic for herbicide resistance, some not) for gene flow of the Bt cotton cry1Ac transgene. We investigated seed-mediated gene flow, which yields adventitious Bt cotton plants, and pollen-mediated gene flow, which generates outcrossed seeds. A spatially-explicit statistical analysis was used to quantify the effects of nearby Bt and non-Bt cotton fields at various spatial scales, along with the effects of pollinator abundance and adventitious Bt plants in fields, on pollen-mediated gene flow. Adventitious Bt cotton plants, resulting from seed bags and planting error, comprised over 15% of plants sampled from the edges of three seed production fields. In contrast, pollen-mediated gene flow affected less than 1% of the seed sampled from field edges. Variation in outcrossing was better explained by the area of Bt cotton fields within 750 m of the seed production fields than by the area of Bt cotton within larger or smaller spatial scales. Variation in outcrossing was also positively associated with the abundance of honey bees.Conclusions/SignificanceA comparison of statistical methods showed that our spatially-explicit analysis was more powerful for understanding the effects of surrounding fields than customary models based on distance. Given the low rates of pollen-mediated gene flow observed in this study, we conclude that careful planting and screening of seeds could be more important than field spacing for limiting gene flow.

Does flax have the answer to the GM mix-up?

Does flax have the answer to the GM mix-up?

From glacial refugia to modern populations: new assemblages of organelle genomes generated by differential cytoplasmic gene flow in transcontinental black spruce

Assessing species' range-wide cytoplasmic diversity provides valuable insights regarding their dispersal and adaptive potential in a changing environment. Transcontinental chloroplast (cpDNA) and mitochondrial DNA (mtDNA) population structures were compared to identify putative ancestral and new cytoplasmic genome assemblages in black spruce (Picea mariana), a North American boreal conifer. Mean within-population diversity and allelic richness for cpSSR markers were 0.80 and 4.21, respectively, and diminished westward. Population differentiation based on G(ST) was lower for cpDNA than for mtDNA (G(ST) =0.104 and 0.645, respectively) but appeared comparable when estimated using Jost differentiation index (D=0.459 and 0.537, respectively). Further analyses resulted in the delineation of at least three genetically distinct cpDNA lineages partially congruent with those inferred from mtDNA data, which roughly corresponded to western, central and eastern Canada. Additionally, the patterns of variation in Alaska for both cpDNA and mtDNA markers suggested that black spruce survived the last glacial maximum in this northern region. The range-wide comparison of the geographic extent of cytoplasmic DNA lineages revealed that extensive pollen gene flow between ancestral lineages occurred preferentially from west to east during the postglacial expansion of the species, while seed-mediated gene flow remained geographically restricted. This differential gene flow promoted intraspecific cytoplasmic capture that generated new assemblages of cpDNA and mtDNA genomes during the Holocene. Hence, black spruce postglacial colonization unexpectedly resulted in an increase in genetic diversity with possible adaptive consequences.

Geographical patterns of phenotypic diversity and structure of Kenyan wild sorghum populations (Sorghum spp.) as an aid to germplasm collection and conservation strategy

Kenya lies within sorghum centre of diversity. However, information on the relative extent of diversity patterns within and among genetically defined groups of distinct ecosystems is lacking. The objective was to assess the structure and phenotypic diversity of wild sorghum populations across a range of geographical and ecological conditions in the country. Sixty-two wild sorghum populations (30 individuals per population) sampled from four distinct sorghum growing regions of Kenya and covering different agroecologies were characterized for ten qualitative traits. Plant height, number of tillers, panicle sizes and flag leaf dimensions were also recorded. Frequencies of the phenotypic classes of each character were calculated. The Shannon diversity index (H′) was used to estimate the magnitude of diversity. Principal component analysis was used to differentiate populations within and between regions. Wild sorghum is widely distributed in Kenya, occurring in sympatric ranges with cultivated sorghum, and both have overlapping flowering windows. All characters considered displayed great phenotypic diversity. Pooled over characters within regions, the mean H′ ranged between 0.60 and 0.93 in Western and Coast regions, respectively. Wild sorghum was found to show a weak regional differentiation, probably reflecting the importance of seed-mediated gene flow in shaping the wild sorghum population structure. Trait distribution was variable among regions, but there was no conspicuous distribution of the traits studied in any given region. Spontaneous hybridization and introgression of genes from cultivated to wild sorghum seems to be likely, and may already have occurred for a long time, although undocumented. Implications for in situ and ex situ genetic resources conservation are discussed.

Realized gene flow within mixed stands of Quercus robur L. and Q. petraea (Matt.) L. revealed at the stage of naturally established seedling

The estimates of contemporary gene flow assessed based on naturally established seedlings provide information much needed for understanding the abilities of forest tree populations to persist under global changes through migration and/or adaptation facilitated by gene exchange among populations. Here, we investigated pollen- and seed-mediated gene flow in two mixed-oak forest stands (consisting of Quercus robur L. and Q. petraea [Matt.] Liebl.). The gene flow parameters were estimated based on microsatellite multilocus genotypes of seedlings and adults and their spatial locations within the sample plots using models that attempt to reconstruct the genealogy of the seedling cohorts. Pollen and seed dispersal were modelled using the standard seedling neighbourhood model and a modification--the 2-component seedling neighbourhood model, with the later allowing separation of the dispersal process into local and long-distance components. The 2-component model fitted the data substantially better than the standard model and provided estimates of mean seed and pollen dispersal distances accounting for long-distance propagule dispersal. The mean distance of effective pollen dispersal was found to be 298 and 463 m, depending on the stand, while the mean distance of effective seed dispersal was only 8.8 and 15.6 m, which is consistent with wind pollination and primarily seed dispersal by gravity in Quercus. Some differences observed between the two stands could be attributed to the differences in the stand structure of the adult populations and the existing understory vegetation. Such a mixture of relatively limited seed dispersal with occasional long distance gene flow seems to be an efficient strategy for colonizing new habitats with subsequent local adaptation, while maintaining genetic diversity within populations.

Multiple northern refugia for Asian sacred lotus, an aquatic plant with characteristics of ice-age endurance

The effect of palaeoclimate on the distribution of aquatic plants is little known, although these plants typically have much broader geographical distributions than their terrestrial counterparts. We investigated the structure of genetic variation of chloroplast DNA in the Asian sacred lotus (Nelumbo nucifera Gaertn (Nelumbonaceae)) in 15 wild populations across China as well as in four populations from India, Japan and Thailand, to infer the refugia of this ancient plant during Quaternary climatic oscillations. We obtained 37 cpDNA haplotypes in 417 individuals from 19 populations. A moderate to high level of chloroplast genetic differentiation (GST = 0.547, NST = 0.691) and significant phylogeographic structure (NST > GST) were observed, suggesting a low level of recurrent seed-mediated gene flow among the populations. The results of AMOVA analysis also showed that more variation was partitioned among (71%) than within populations (29%). The phylogenetic relationships for the recovered haplotypes showed that haplotypes of wild lotus in north-eastern China significantly diverged from those distributed in central and southern regions of China. A high level of haplotype diversity, rather than reduced genetic diversity, in north-eastern China indicated multiple refugia in northern China during the Quaternary glaciations. Lotus plants have the following two characteristics that facilitated survival through Quaternary glaciations: seeds buried in mud remain viable for thousands of years, and self-heating flowers can reproduce in cold temperatures.

Rapeseed cytoplasm gives advantage in wild relatives and complicates genetically modified crop biocontainment

Biocontainment methods for genetically modified crops closest to commercial reality (chloroplast transformation, male sterility) would be compromised (in absolute terms) by seed-mediated gene flow leading to chloroplast capture. Even in these circumstances, however, it can be argued that biocontainment still represses transgene movement, with the efficacy depending on the relative frequency of seed- and pollen-mediated gene flow. In this study, we screened for crop-specific chloroplast markers from rapeseed (Brassica napus) amongst sympatric and allopatric populations of wild B. oleracea in natural cliff-top populations and B. rapa in riverside and weedy populations. We found only modest crop chloroplast presence in wild B. oleracea and in weedy B. rapa, but a surprisingly high incidence in sympatric (but not in allopatric) riverside B. rapa populations. Chloroplast inheritance models indicate that elevated crop chloroplast acquisition is best explained if crop cytoplasm confers selective advantage in riverside B. rapa populations. Our results therefore imply that chloroplast transformation may slow transgene recruitment in two settings, but actually accelerate transgene spread in a third. This finding suggests that the appropriateness of chloroplast transformation for biocontainment policy depends on both context and geographical location.

Short-distance gene flow in Populus nigra L. accounts for small-scale spatial genetic structures: implications for in situ conservation measures

The European black poplar (Populus nigra L.) is a major species of riparian softwood forests. Due to human influences, it is one of the most threatened tree species in Europe. For restoration purposes, remaining stands may act as source populations. We analysed a natural population of P. nigra for genetic diversity and spatial genetic structure using seven microsatellite markers. For the first time, paternity analysis of seedlings as well as juveniles from a restricted area of natural regeneration was used to quantify pollen- and seed-mediated gene flow, respectively. In both cases, cultivated P. x canadensis trees in vicinity could act as potential parents. Spatial genetic patterns of the adult tree population suggest small-scale isolation by distance due to short-distance gene flow, the major part of which (i.e. 70%) takes place within distances of less than 1 km. This helps to explain the reduced diversity in the juveniles. It has implications for the spatial management of natural regeneration areas within in situ conservation measures.

Response to Bagavathiannan and Van Acker's “Transgenes and national boundaries – The need for international regulations”: Biotechnology developers and regulators already consider transgene movement across national boundaries and the environmental risks posed by adventitious presence of unapproved events are overstated

Bagavathiannan and Van Acker propose greater international cooperation and information sharing in risk assessment for biotechnology-derived crops because pollen- and seed-mediated gene flow across political boundaries may lead to the adventitious presence of unapproved transgenes at sites along the borders of neighboring countries. However, they fail to convince us that something is wrong with the current situation and provide no details of how it could be improved.

Gene Flow, Invasiveness, and Ecological Impact of Genetically Modified Crops

The main environmental concerns about genetically modified (GM) crops are the potential weediness or invasiveness in the crop itself or in its wild or weedy relatives as a result of transgene movement. Here we briefly review evidence for pollen- and seed-mediated gene flow from GM crops to non-GM or other GM crops and to wild relatives. The report focuses on the effect of abiotic and biotic stress-tolerance traits on plant fitness and their potential to increase weedy or invasive tendencies. An evaluation of weediness and invasive traits that contribute to the success of agricultural weeds and invasive plants was of limited value in predicting the effect of biotic and abiotic stress-tolerance GM traits, suggesting context-specific evaluation rather than generalizations. Fitness data on herbicide, insect, and disease resistance, as well as cold-, drought-, and salinity-tolerance traits, are reviewed. We describe useful ecological models predicting the effects of gene flow and altered fitness in GM crops and wild/weedy relatives, as well as suitable mitigation measures. A better understanding of factors controlling population size, dynamics, and range limits in weedy volunteer GM crop and related host or target weed populations is necessary before the effect of biotic and abiotic stress-tolerance GM traits can be fully assessed.

Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms.

Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns. Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden. The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden. Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchids.

Population genetic structure inMyrtus communisL. in a chronically fragmented landscape in the Mediterranean: can gene flow counteract habitat perturbation?

Ancient managed landscapes provide ideal opportunities to assess the consequences of habitat fragmentation on the patterns of genetic diversity and gene flow in long-lived plant species. Using amplified fragment length polymorphism (AFLP) and allozyme markers, we quantified seed-mediated gene flow and population genetic diversity and structure in 14 populations of Myrtus communis (myrtle), a common endozoochorous shrub species of forest patches in lowland agricultural Mediterranean areas. Overall, allozyme diversity for myrtle was low (P(95) = 25%; A = 1.411; H(e) = 0.085) compared to other known populations, and a significant portion of populations (57%) had lower levels of allelic diversity and/or heterozygosity than expected at random, as shown by simulated resampling of the whole diversity of the landscape. We found significant correlations between allozyme variability and population size and patch isolation, but no significant inbreeding in any population. Genetic differentiation among populations for both allozyme and AFLP markers was significant (Phi(ST) = 0.144 and Phi(ST) = 0.142, respectively) but an isolation-by-distance pattern was not detected. Assignment tests on AFLP data indicated a high immigration rate in the populations (ca. 20-22%), likely through effective seed dispersal across the landscape by birds and mammals. Our results suggest that genetic isolation is not the automatic outcome of habitat destruction since substantial levels of seed-mediated gene flow are currently detectable. However, even moderate rates of gene flow seem insufficient in this long-lived species to counteract the genetic erosion and differentiation imposed by chronic habitat destruction.

In-field frequencies and characteristics of oilseed rape with double herbicide resistance

When growing different transgenic herbicide-resistant oilseed rape cultivars side by side, seeds with multiple herbicide resistance can arise, possibly causing problems for the management of volunteer plants. Large-scale field experiments were performed in the years 1999/2000 and 2000/2001 in order to investigate the frequencies and the consequences of the transfer of herbicide resistance genes from transgenic oilseed rape to cultivars grown on neighboring agricultural fields. Transgenic oilseed rape with resistance to glufosinate-ammonium (LibertyLink, LL) and with glyphosate resistance (RoundupReady, RR), respectively, was sown in adjacent 0.5 ha plots, surrounded by about 8 ha non-transgenic oilseed rape. The plots and the field were either in direct contact (0.5 m gap width) or they were separated by 10 m of fallow land. Seed samples taken during harvest in the transgenic plots at different distances were investigated for progeny with resistance to the respective other herbicide. It was found that outcrossing frequencies were reduced to different extents by a 10 m isolation distance. In addition to pollen-mediated transgene flow as a result of outcrossing, we found considerable seed-mediated gene flow by adventitious dispersal of transgenic seeds through the harvesting machine. Volunteer plants with double herbicide resistance emerging in the transgenic plots after harvest were selected by suitable applications of the complementary herbicides Basta and Roundup Ultra. In both years, double-resistant volunteers were largely restricted to the inner edges of the plots. Expression analysis under controlled laboratory conditions of double-resistant plants generated by manual crosses revealed stability of transgene expression even at elevated temperatures. Greenhouse tests with double-resistant oilseed rape plants gave no indication that the sensitivity to a range of different herbicides is changed as compared to non-transgenic oilseed rape.

Seed-Mediated Gene Flow in Wheat: Seed Bank Longevity in Western Canada

Development of genetically modified (GM) wheat has raised concerns about the movement and persistence of transgenes in agroecosystems and the ability of growers to segregate GM from conventional wheat. Wheat as a crop has been studied extensively but the population biology of volunteer wheat is not well characterized. Artificial seed bank studies were conducted in western Canada to provide baseline data on volunteer wheat seed persistence. Seed from two cultivars of Canadian western red spring wheat, ‘AC Splendor’ and ‘AC Superb’, were buried in mesh bags at three depths (0, 2, and 15 cm) in two different environments in the fall of 2003 and 2004. In addition, in 2004, ‘AC Superb’ seed were separated into small and large seed lots and buried with a medium seed lot to examine the influence of seed size on seed bank persistence. Seeds were withdrawn at intervals to assess seed germination and viability and regression analysis conducted on the viable seed at each sample period, after burial. Seed viability was variable within years and sites, and declined exponentially over time. In the spring, approximately 6 mo after initiation, viable seed ranged from 1 to 43%. With the exception of a single site and year, seeds on the soil surface persisted significantly longer than buried seeds and increasing burial depth accelerated loss of viability. The maximum viability of wheat seeds at 0, 2, and 15 cm depth in the spring following planting was 43, 7, and 2%, respectively. The extinction of viability for 99% (EX99) of the seed was estimated from regression analysis. The EX99values of seeds buried at 0, 2, and 15 cm ranged from 493 to 1,114, 319 to 654, and 175 to 352 d after planting (DAP), respectively, with the exception of one site in 2003 where burial depths were not different and all had an EX99value of 456 DAP. Seed size and cultivar did not significantly affect persistence, with the exception of one site in 2003 where the difference in EX99values was 20 DAP. The rapid loss of seed viability limits temporal gene flow via volunteers in years following a wheat crop. Results provide data on spring wheat biology to aid in Canadian environmental biosafety assessments of GM wheat and will be incorporated into a mechanistic model to predict wheat gene flow on the Canadian prairies.

Potential for seed-mediated gene flow in agroecosystems from transgenic safflower (Carthamus tinctorius L.) intended for plant molecular farming

Safflower has been transformed for field scale molecular farming of high-value proteins including several pharmaceuticals. Viable safflower seed remaining in the soil seed bank after harvest could facilitate seed and pollen-mediated gene flow. Seeds may germinate in subsequent years and volunteer plants may flower and potentially outcross with commodity safflower and/or produce seed. Seeds from volunteers could become admixed with conventional crops at harvest, and/or replenish the seed bank. Seed in following crops could be transported locally and internationally and facilitate gene flow in locations where regulatory thresholds and public acceptance differ from Canada. Seed-mediated gene flow was examined in three studies. Safflower seed loss and viability following harvest of commercial fields of a non-transgenic cultivar were determined. We assessed seed longevity of transgenic and non-transgenic safflower, on the soil surface and buried at two depths. Finally, we surveyed commercial safflower fields at different sites and measured density and growth stage of safflower volunteers, in other crops the following year and documented volunteer survival and viable seed production. Total seed loss at harvest in commercial fields, ranged from 231 to 1,069 seeds m(-2) and the number of viable seeds ranged from 81 to 518 seeds m(-2). Safflower has a relatively short longevity in the seed bank and no viable seeds were found after 2 years. Based on the seed burial studies it is predicted that winter conditions would reduce safflower seed viability on the soil surface by >50%, leaving between 40 and 260 viable seeds m(-2). The density of safflower volunteers emerging in the early spring of the following year ranged from 3 to 11 seedlings m(-2). Safflower volunteers did not survive in fields under chemical fallow, but in some cereal fields small numbers of volunteers did survive and generate viable seed. Results will be used to make recommendations for best management practices to reduce seed-mediated gene flow from commercial production of plant molecular farming with safflower.

Creation and analysis of a novel chimeric promoter for the complete containment of pollen- and seed-mediated gene flow

Effective containment of gene flow in transgenic plants requires a promoter that is highly specific for male and female gametes or tissues. Here, we report the creation of a novel pollen-, stigma- and carpel-specific (PSC) promoter through the fusion of the pollen-specific LAT52 and carpel-specific AGL5 enhancers to a stigma-specific SLG promoter. Gene expression analysis showed that fusion of the LAT52 enhancer to the SLG promoter enables the latter to gain pollen-specific activity while the acquirement of carpel-specific activity requires the correct orientation of the inserted AGL5 enhancer in the PSC promoter, and only a forward- but not a reverse-oriented one is functional. The resulting fPSC promoter, when fused to DT-A, generated at least three aberrant gynoecium phenotypes. Type I plants exhibited shortened stigmatic tissues, resembling plants containing the DT-A gene controlled by the SLG promoter. However, type II and III plants displayed partial or complete ablation of gynoecia, and were unable to support the reproductive process. Type II and III plants also produced severely perturbed anthers and pollen in comparison to type I or SLG::DT-A plants, and transgenic pollen grains were unable, when out-crossed with control plants, to pass the transgene to the next generation in all plants examined, indicating that they are selectively eliminated. This tissue-specific ablation or perturbation is highly specific, and does not compromise vegetative growth. Evidently, the fPSC promoter faithfully acquires tissue specificity from the incorporated enhancers and promoter, and should have a practical application for transgene containment in non-fruit and -grain producing plant crops.

Spatial Scales of Pollen and Seed-Mediated Gene Flow in Tropical Rain Forest Trees

Gene flow via seed and pollen is a primary determinant of genetic and species diversity in plant communities at different spatial scales. This paper reviews studies of gene flow and population genetic structure in tropical rain forest trees and places them in ecological and biogeographic context. Although much pollination is among nearest neighbors, an increasing number of genetic studies report pollination ranging from 0.5-14 km for canopy tree species, resulting in extensive breeding areas in disturbed and undisturbed rain forest. Direct genetic measures of seed dispersal are still rare; however, studies of fine scale spatial genetic structure (SGS) indicate that the bulk of effective seed dispersal occurs at local scales, and we found no difference in SGS (Sp statistic) between temperate (N=24 species) and tropical forest trees (N=15). Our analysis did find significantly higher genetic differen- tiation in tropical trees (FST=0.177; N=42) than in temperate forest trees (FST=0.116; N=82). This may be due to the fact that tropical trees experience low but significant rates of self-fertilization and bi-parental inbreed- ing, whereas half of the temperate tree species in our survey are wind pollinated and are more strictly allogamous. Genetic drift may also be more pronounced in tropical trees due to the low population densities of most species.

Strong Genetic Differentiation of Primula sikkimensis in the East Himalaya–Hengduan Mountains

The East Himalaya-Hengduan Mountains region is the center of diversity of the genus Primula, and P. sikkimensis is one of the most common members of the genus in the region. In this study, the genetic diversity and structure of P. sikkimensis populations in China were assessed using inter-simple sequence repeat (ISSR) and chloroplast microsatellite markers. The 254 individuals analyzed represented 13 populations. High levels of genetic diversity were revealed by ISSR markers. At the species level, the expected heterozygosity and Shannon's index were 0.4032 and 0.5576, respectively. AMOVA analysis showed that 50.3% of the total genetic diversity was partitioned among populations. Three pairs of chloroplast microsatellite primers tested yielded a total of 12 size variants and 15 chloroplast haplotypes. Strong cpDNA genetic differentiation (G (ST) = 0.697) and evidence for phylogeographic structure were detected (N (ST )= 0.788, significantly higher than G (ST)). Estimated rates of pollen-mediated gene flow are approximately 27% greater than estimated rates of seed-mediated gene flow in P. sikkimensis. Both seed and pollen dispersal, however, are limited, and gene flow among populations appears to be hindered by the patchiness of the species' habitats and their geographic isolation. These features may have played important roles in shaping the genetic structure of P. sikkimensis. A minimum-spanning tree of chloroplast DNA haplotypes was constructed, and possible glacial refugia of P. sikkimensis were identified.