Outcrossing Rate Research Articles

Estimating Out-Crossing Rate of Bg 379-2 Using Morphological Markers and Confirmation by Molecular Markers

Rice is largely self-fertilized and accordingly a field population of rice is completely composed of near homozygotes. Due to the emergence of off-types, homozygosity will be affected. With the time, this will cause the reduction of genetic purity in some rice varieties. One of the reasons has been suspected to be the high out-crossing frequencies of such varieties. Studies were conducted at the Rice Research and Development Institute, Batalagoda, Sri Lanka to estimate the out-crossing rate of Bg 379-2, a variety having the problem of maintaining genetic purity. Bg 379-2 was allowed to out-cross with Bg 450 and the number of out-crossed plants were counted using dominant morphological markers such as short-round grain and purple culm of pollen donor. A molecular confirmation of out-crossing was also performed using sequence tagged site (STS) molecular marker pTA248. The variety Bg 379-2 showed a potential out-crossing rate of 3.41% and an average out-crossing rate of 1.29% using dominant morphological markers. Polymorphism was clearly detected between parents and out-crossed plants as well as selfed plants of Bg 379-2 using their banding patterns. A similar study can be performed to determine the out-crossing rates of other varieties which show high percentage of off-types in the population for the better understanding of the breeding behavior of the varieties.

Using relational decision trees to model out-crossing rates in a multi-field setting

Nearly three-quarters of the genetically modified maize (the insect resistant type MON 810, also called Bt maize) produced in the EU are cultivated in Spain, where the share of Bt maize cultivation in some regions (Catalonia) is very high (above 70%). In order to ensure coexistence with the production of conventional maize and satisfy the 0.9% EU threshold for adventitious presence of authorized genetically modified (GM) material in conventional (non-GM) maize crops, a set of preventive coexistence measures must be applied. These measures usually include the setup of large and fixed isolation distances, pollen barriers, flowering coincidence, crop rotation and other measures, which are very hard to fulfill in a multi-field setting. Basic empirical and modeling studies that explore the feasibility of coexistence between GM and non-GM crops focus on pair-based interactions between fields while multi-field studies build upon them, attempting to consider the complexity of gene flow under crop management practices.In this study, we use the methodology of relational data mining (which can take into account several coexistence measures at the same time) to predict gene flow from GM to non-GM maize fields under multi-field crop management practices at a local scale. The approach extends the pair-based assessments of out-crossing rate by considering all neighboring fields within the entire study area, along with the farming practices applied to them. The estimation of the out-crossing rates is performed by using a PostgreSQL relational database that is analyzed with the algorithm TILDE for building relational classification trees. In building the trees, TILDE explores the relations describing spatial aspects, maize flowering and crop management practices for the 400ha maize oriented production area Pla de Foixà in Catalonia, Spain, in the period 2004–2006.Our approach proposes a new methodology to predict the level of adventitious presence on a multi-field setting, where the influence of more than one GM field is considered at the same time. The structure of the obtained models can be used in the design of coexistence measures of the second generation, which should not be used individually but treated as synergetic coexistence measures, offering different alternatives to achieve a particular coexistence threshold (e.g., 0.9%, 0.45%, or 0.1%). The possibility to consider multiple measures simultaneously makes farmers more flexible in their management decisions as compared to the rigid use of isolation distance only, which is currently the most commonly recommended coexistence measure.

Recent and Projected Increases in Atmospheric CO2 Concentration Can Enhance Gene Flow between Wild and Genetically Altered Rice (Oryza sativa)

Although recent and projected increases in atmospheric carbon dioxide can alter plant phenological development, these changes have not been quantified in terms of floral outcrossing rates or gene transfer. Could differential phenological development in response to rising CO2 between genetically modified crops and wild, weedy relatives increase the spread of novel genes, potentially altering evolutionary fitness? Here we show that increasing CO2 from an early 20th century concentration (300 µmol mol−1) to current (400 µmol mol−1) and projected, mid-21st century (600 µmol mol−1) values, enhanced the flow of genes from wild, weedy rice to the genetically altered, herbicide resistant, cultivated population, with outcrossing increasing from 0.22% to 0.71% from 300 to 600 µmol mol−1. The increase in outcrossing and gene transfer was associated with differential increases in plant height, as well as greater tiller and panicle production in the wild, relative to the cultivated population. In addition, increasing CO2 also resulted in a greater synchronicity in flowering times between the two populations. The observed changes reported here resulted in a subsequent increase in rice dedomestication and a greater number of weedy, herbicide-resistant hybrid progeny. Overall, these data suggest that differential phenological responses to rising atmospheric CO2 could result in enhanced flow of novel genes and greater success of feral plant species in agroecosystems.

Pollen-mediated gene flow in wheat (Triticum aestivum L.) in a semiarid field environment in Spain

Transgenic wheat (Triticum aestivum L.) varieties are being developed and field-tested in various countries. Concerns regarding gene flow from genetically modified (GM) crops to non-GM crops have stimulated research to estimate outcrossing in wheat prior to the release and commercialization of any transgenic cultivars. The aim is to ensure that coexistence of all types of wheat with GM wheat is feasible in accordance with current regulations. The present study describes the result of a field experiment under the semi-arid climate conditions of Madrid, Spain, at two locations ("La Canaleja" and "El Encin" experimental stations) in Madrid over a 3-year period, from 2005 to 2007. The experimental design consisted of a 50 × 50 m wheat pollen source sown with wheat cultivars resistant to the herbicide chlortoluron ('Deganit' and 'Castan' respectively) and three susceptible receptor cultivars ('Abental', 'Altria' and 'Recital') sown in replicated 1 × 1 m plots at different distances (0, 1, 3, 5, 10, 20, 40, 80 and 100 m) and four directions. Outcrossing rates were measured as a percentage of herbicide-resistant hybrids using an herbicide-screening assay. Outcrossing was greatest near the pollen source, averaging 0.029% at 0 m distance at "La Canaleja" and 0.337% at "El Encin", both below the 0.9% European Union regulated threshold, although a maximum outcrossing rate of 3.5% was detected in one recipient plot. These percentages declined rapidly as the distance increased, but hybrids were detected at different rates at distances of up to 100 m, the maximum distance of the experiment. Environmental conditions, as drought in 2004-2005 and 2005-2006, may have influenced the extent of outcrossing. These assays carried out in wheat under semi-arid conditions in Europe provide a more complete assessment of pollen-mediated gene flow in this crop.

Microevolutionary Distribution of Isogenicity in a Self-fertilizing Fish (Kryptolebias marmoratus) in the Florida Keys

The mangrove rivulus Kryptolebias marmoratus and a closely related species are the world's only vertebrates that routinely self-fertilize. Such uniqueness presents a model for understanding why this reproductive mode, common in plants and invertebrates, is so rare in vertebrates. A survey of 32 highly polymorphic loci in >200 specimens of mangrove rivulus from multiple locales in the Florida Keys, USA, revealed extensive population-genetic structure on microspatial and micro-temporal scales. Observed heterozygosities were severely constrained, as expected for a hermaphroditic species with a mixed-mating system and low rates of outcrossing. Despite the pronounced population structure and the implied restrictions on effective gene flow, isogenicity (genetic identity across individuals) within and among local inbred populations was surprisingly low even after factoring out probable de novo mutations. Results indicate that neither frequent bottlenecks nor directional genetic adaptation to local environmental conditions were the primary driving forces impacting multilocus population-genetic architecture in this self-fertilizing vertebrate species. On the other hand, a high diversity of isogenic lineages within relatively small and isolated local populations is consistent with the action of diversifying selection driven by the extreme spatio-temporal environmental variability that is characteristic of mangrove habitats.

Evolution of outcrossing in experimental populations of Caenorhabditis elegans.

Caenorhabditis elegans can reproduce exclusively by self-fertilization. Yet, males can be maintained in laboratory populations, a phenomenon that continues to puzzle biologists. In this study we evaluated the role of males in facilitating adaptation to novel environments. For this, we contrasted the evolution of a fitness component exclusive to outcrossing in experimental populations of different mating systems. We introgressed a modifier of outcrossing into a hybrid population derived from several wild isolates to transform the wild-type androdioecious mating system into a dioecious mating system. By genotyping 375 single-nucleotide polymorphisms we show that the two populations had similar standing genetic diversity available for adaptation, despite the occurrence of selection during their derivation. We then performed replicated experimental evolution under the two mating systems from starting conditions of either high or low levels of diversity, under defined environmental conditions of discrete non-overlapping generations, constant density at high population sizes (N = 104), no obvious spatial structure and abundant food resources. During 100 generations measurements of sex ratios and male competitive performance showed: 1) adaptation to the novel environment; 2) directional selection on male frequency under androdioecy; 3) optimal outcrossing rates of 0.5 under androdioecy; 4) the existence of initial inbreeding depression; and finally 5) that the strength of directional selection on male competitive performance does not depend on male frequencies. Taken together, these results suggest that androdioecious males are maintained at intermediate frequencies because outcrossing is adaptive.

Two's company, three's a crowd: experimental evaluation of the evolutionary maintenance of trioecy in Mercurialis annua (Euphorbiaceae).

Trioecy is an uncommon sexual system in which males, females, and hermaphrodites co-occur as three clearly different gender classes. The evolutionary stability of trioecy is unclear, but would depend on factors such as hermaphroditic sex allocation and rates of outcrossing vs. selfing. Here, trioecious populations of Mercurialis annua are described for the first time. We examined the frequencies of females, males and hermaphrodites across ten natural populations and evaluated the association between the frequency of females and plant densities. Previous studies have shown that selfing rates in this species are density-dependent and are reduced in the presence of males, which produce substantially more pollen than hermaphrodites. Accordingly, we examined the evolutionary stability of trioecy using an experiment in which we (a) indirectly manipulated selfing rates by altering plant densities and the frequency of males in a fully factorial manner across 20 experimental plots and (b) examined the effect of these manipulations on the frequency of the three sex phenotypes in the next generation of plants. In the parental generation, we measured the seed and pollen allocations of hermaphrodites and compared them with allocations by unisexual plants. In natural populations, females occurred at higher frequencies in denser patches, a finding consistent with our expectations. Under our experimental conditions, however, no combination of plant densities and male frequencies was associated with increased frequencies of females. Our results suggest that the factors that regulate female frequencies in trioecious populations of M. annua are independent of those regulating male frequencies (density), and that the stable co-existence of all three sex phenotypes within populations is unlikely.

Flowering asynchrony and mating system effects on reproductive assurance and mutualism persistence in fragmented fig–fig wasp populations

Plants and animals may experience reproductive Allee effects in fragmented populations, and obligate pollination mutualisms may be especially sensitive to extinction risk via this density-dependent process. In this study we examine how a shift from within-crown reproductive synchrony to asynchrony influences reproductive assurance through contributions to selfing and outcrossing in small, spatially isolated populations of Ficus. The research focuses on the monoecious fig F. petiolaris and consists of phenological analyses and genetic assessments of selfing and outcrossing for populations located in Baja California's Sonoran Desert. Phenological censuses of eight populations revealed within-crown asynchrony in 44% of reproducing trees, with 16% having sufficient overlap of male and female flowering phases to permit selfing via the cycling of pollinating fig wasps within natal trees. In mating system analyses of two of these populations, however, multilocus outcrossing rates (t(m)) were indistinguishable from 1. This result, combined with low levels of inbreeding, indicates selfing to be absent or at best a minor contributor to reproductive assurance. The results indicate that the fitness benefits of within-crown asynchrony lie not with selfing, as commonly asserted, but with increased opportunities for outcross pollen transmission and receipt, changing our understanding of the mechanisms by which reproduction is facilitated and extinction risk minimized in naturally fragmented Ficus populations. Given the role of fig fruit as a keystone food resource in many tropical environments, trait variation leading to reproductive assurance in figs, such as within-crown asynchrony, has broader ecosystem-level implications.

Herkogamy and mate diversity in the wild daffodilNarcissus longispathus: beyond the selfing–outcrossing paradigm in the evolution of mixed mating

Spatial separation of male and female reproductive structures (herkogamy) is a widespread floral trait that has traditionally been viewed as an adaptation that reduces the likelihood of self-pollination. Here we propose that increased herkogamy may also influence another important aspect of plant mating: the diversity of pollen donors siring seeds within fruits. We test this hypothesis in Narcissus longispathus, a wild daffodil species with extensive variation in anther-stigma separation. To study the morphological basis of variation in herkogamy, floral measurements were undertaken in 16 populations of N.longispathus. We then quantified multilocus outcrossing rates and the correlation of outcrossed paternity in three of these populations sampled over several years. Mating system estimates were calculated for each population and year, and also separately for groups of plants that differed markedly in herkogamy within each population and year. In N. longispathus herkogamy was much more variable than other floral traits, and was more closely related to style length than to anther position. Averaged across populations and years, plants with high herkogamy had similar outcrossing rates (0.683) to plants with intermediate (0.648) or low herkogamy (0.590). However, a significant linear trend was found for correlation of outcrossed paternity, which increased monotonically from high herkogamy (0.221), through intermediate herkogamy (0.303) to low herkogamy (0.463) plants. The diversity of pollen donors siring seeds of high herkogamy Narcissus flowers was thus consistently greater than the diversity of pollen donors siring seeds of low herkogamy flowers. Results of this study contribute to the emerging consensus that floral traits can simultaneously influence several aspects of plant mating system in complex ways, thus extending the traditional focus centred exclusively on patterns and relative importance of self- and cross-fertilisation.

Mating System Analysis of Alnus maritima (Seaside Alder), a Rare Riparian Tree

Abstract Effective conservation requires an understanding of the genetic interactions among populations and individuals of a species, particularly those with fragmented, isolated distributions. Alnus maritima (seaside alder) is a rare tree species with an extremely fragmented distribution of highly isolated populations in the Delmarva Peninsula, Georgia, and Oklahoma. We conducted a mating system study to estimate the outcrossing rate, inbreeding coefficient, biparental inbreeding rate, and correlation of paternity in progeny from a Georgia and an Oklahoma population to investigate the effects of isolation on the A. maritima mating system. Data from nine microsatellite loci showed similarly high multilocus outcrossing rates in both populations (tm = 0.94). Individual tree outcrossing rates were also high (tm = 0.873–1.047). There was no significant biparental inbreeding in either population, but there was significantly higher correlated paternity in the Oklahoma population. Results showed the high out...

DNA 표지를 이용한 채종원내 소나무의 교배양식 분석

To assess parameters of mating system in seed orchard, such as outcrossing rates, number of potential pollen contributors, and degree of pollen contamination, seeds, produced in '77 plot of the Japanese red pine (Pinus densiflora S et Z) seed orchard at Anmyeon island, were collected in 2007 and analysed by nSSR and cpSSR markers. Estimates of outcrossing rates ranged from 91.2 to 100% (mean 97.7%) on the basis of the analysis of cpSSR haplotypes and from 81.6 to 100% (mean 95.3%) on the basis of the analysis of nSSR genotypes. By cross checking of both DNA markers, seeds, presumed to be products of self pollination on the basis of single marker, were confirmed as outcrossed seeds, which resulted in cumulative outcrossing rates of 98.9%. On the basis of pooled cpSSR haplotype of each seed, the number of pollen contributors and paternal contribution rates were estimated as 14.8 and 0.512, respectively. In conclusion, considering pretty high level of outcrossing rates observed in a seed orchard, good genetic potential of the seeds, produced in '77 plot of the seed orchard of Japanese red pines at Anmyeon island, may be guaranteed. Investigated results from the analysis of mating system of Japanese red pines in a '77 plot of the seed orchard may also be expected to provide useful information for the management and establishment of the seed orchard of the progressive generation.

Studying comparative suitability of CMS lines

Parental lines constitute the first and foremost step in a hybrid breeding program. In this context, developing a commercially viable cytoplasmic male sterile (CMS) line is considered to be a highly cumbersome process. In fact, the belated success of hybrid rice technology in India was basically due to the nonavailability of a CMS line suited to the tropics. Over the years, a large number of male sterile lines with different cytosterility sources have been developed in India and elsewhere. Only a few-such as IR58025A and IR62829A-are now used commercially in India. They possess all the essential traits-complete and stable male sterility, high outcrossing rate, better grain quality, easy restorability, good combining ability, and adaptability-of a commercially viable CMS line. Some 64 CMS lines from India, China, Malaysia, and IRRI were evaluated along with IR58025A, IR62829A, and standard checks in the wet season of 1995 and 1996 and dry season of 1994 and 1995 to assess their comparative suitability for commercial use.

No Evolutionary Shift in the Mating System of North American Ambrosia artemisiifolia (Asteraceae) Following Its Introduction to China

The mating system plays a key role during the process of plant invasion. Contemporary evolution of uniparental reproduction (selfing or asexuality) can relieve the challenges of mate limitation in colonizing populations by providing reproductive assurance. Here we examined aspects of the genetics of colonization in Ambrosia artemisiifolia, a North American native that is invasive in China. This species has been found to possess a strong self-incompatibility system and have high outcrossing rates in North America and we examined whether there has been an evolutionary shift towards the dependence on selfing in the introduced range. Specifically, we estimated outcrossing rates in one native and five invasive populations and compared levels of genetic diversity between North America and China. Based on six microsatellite loci we found that, like the native North American population, all five Chinese populations possessed a completely outcrossing mating system. The estimates of paternity correlations were low, ranging from 0.028–0.122, which suggests that populations possessed ∼8–36 pollen donor parents contributing to each maternal plant in the invasive populations. High levels of genetic diversity for both native and invasive populations were found with the unbiased estimate of gene diversity ranging from 0.262–0.289 for both geographic ranges based on AFLP markers. Our results demonstrate that there has been no evolutionary shift from outcrossing to selfing during A. artemisiifolia's invasion of China. Furthermore, high levels of genetic variation in North America and China indicate that there has been no erosion of genetic variance due to a bottleneck during the introduction process. We suggest that the successful invasion of A. artemisiifolia into Asia was facilitated by repeated introductions from multiple source populations in the native range creating a diverse gene pool within Chinese populations.

Fluxo gênico recíproco entre cultivares de soja convencional e geneticamente modificada

O objetivo deste trabalho foi determinar o fluxo gênico recíproco entre duas cultivares de soja, uma tolerante e outra sensível ao glifosato, além de aplicar estimadores para determinar a taxa de fecundação cruzada na população e o número de sementes híbridas na progênie. O experimento compôs-se de quatro blocos com 40 fileiras de soja, com 20 fileiras de cada cultivar (CD217 e CD219RR). No estádio R8, cinco fileiras, distantes 0, 5, 1, 2, 4 e 8 m da cultivar adjacente, foram colhidas, trilhadas e analisadas quanto à ocorrência de fluxo gênico. Como características marcadoras, foram utilizadas as cores da flor, hipocótilo e pubescência, e a tolerância ao glifosato. As cultivares contrastam quanto às características analisadas, cada uma condicionada por um gene com dois alelos, em interação de dominância completa. Na progênie da cultivar tolerante, a maior taxa de híbridos encontrada foi 0, 27% e, na progênie da cultivar sensível, identificou-se 0, 83%; pela hipótese do efeito diluição, as taxas de hibridação natural populacional seriam 0, 104 e 0, 388%, respectivamente. O fluxo gênico recíproco entre as cultivares CD217 e CD219RR não é o mesmo em ambas as direções. Os estimadores propostos são úteis para determinar a taxa de híbridos em amostras de sementes.

Short communication. Estimation of cross-fertilization rate in saff lower (Carthamus tinctorius L.)

Safflower is a promising oilseed crop for the production of specialty oils in the Mediterranean area. Oil quality traits such as high oleic acid content, high linoleic acid content, high saturated fatty acid content, or high gamma-tocopherol content have been developed in this crop. The traits are controlled by the genotype of the developing embryo and therefore they are influenced by the presence of foreign pollen. The objective of this research was to study the rate of cross-fertilization in safflower using the high oleic acid trait as a biochemical marker. An experiment in which each high oleic plant was surrounded by 24 low oleic acid plants was conducted over three environments in the same location at Córdoba, Spain. The average rate of cross-fertilization in the three environments was 5.7, 12.1, and 13.2%, though higher frequencies up to 35.9% were detected at the single-plant level and up to 58.3% at the single-head level. The low average outcrossing frequencies identified in this research indicate no need for large isolation distances between conventional cultivars and cultivars with special oil characteristics. However, the occurrence of a significant outcrossing rate should be taken into consideration if transgenic safflower is to be cultivated close to conventional safflower or in areas of distribution of wild Carthamus species.

Genetic characterization and gene flow in different geographical-distance neighbouring natural populations of wild soybean (Glycine soja Sieb. & Zucc.) and implications for protection from GM soybeans

To design appropriate strategies for ex situ and in situ conservation of wild soybean and safeguard the biosafety of the wild soybean gene pool when genetically modified soybeans are grown, it is important to understand its genetic characteristics, and to quantify gene flow and kinship within and between neighboring populations. We analyzed 9 pairs of neighboring populations of wild soybeans using 20 pairs of nuclear SSR markers. Results showed that Chinese wild soybean natural populations had outcrossing rates of 0–3.5% and that most populations contained many kinship families. The kinship families could be attributed to the accumulation of outcrossed offspring within populations during the history of population colonization. Wild soybean is very sensitive to environmental selection, which results in genetic differentiation of populations, and the emergence of specific alleles. We used an index τ to explain why genetic differences would exist between the pairwise populations; the interpopulation genetic differentiation chiefly consisted in the differences of allele frequencies over the genome. We found long-distance dispersal (1.5 km) of wild soybean seeds in a land ecosystem. There was close correlation between genetic and geographical distance among natural populations of Chinese wild soybean. Within a distance of 50 km, there was greater gene flow when the distance between populations was shorter. These findings have implications for ex situ and in situ conservation in an ecogeographical region, and also for protection of the gene pool from contamination by GM soybeans in wild soybean species.

Disentangling the effects of breakdown of self‐incompatibility and transition to selfing in North American Arabidopsis lyrata

AbstractA breakdown of self‐incompatibility (SI) followed by a shift to selfing is commonly observed in the evolution of flowering plants. Both are expected to reduce the levels of heterozygosity and genetic diversity. However, breakdown of SI should most strongly affect the region of the SI locus (S‐locus) because of the relaxation of balancing selection that operates on a functional S‐locus, and a potential selective sweep. In contrast, a transition to selfing should affect the whole genome. We set out to disentangle the effects of breakdown of SI and transition to selfing on the level and distribution of genetic diversity in North American populations of Arabidopsis lyrata. Specifically, we compared sequence diversity of loci linked and unlinked to the S‐locus for populations ranging from complete selfing to fully outcrossing. Regardless of linkage to the S‐locus, heterozygosity and genetic diversity increased with population outcrossing rate. High heterozygosity of self‐compatible individuals in outcrossing populations suggests that SI is not the only factor preventing the evolution of self‐fertilization in those populations. There was a strong loss of diversity in selfing populations, which was more pronounced at the S‐locus. In addition, selfing populations showed an accumulation of derived mutations at the S‐locus. Our results provide evidence that beyond the genome‐wide consequences of the population bottleneck associated with the shift to selfing, the S‐locus of A. lyrata shows a specific signal either reflecting the relaxation of balancing selection or positive selection.

Does cross-pollination occur during seed regeneration at the International Rice Genebank?

Preserving genetic integrity of accessions through cycles of conservation and regeneration is a major objective of the International Rice Genebank (IRG). The careful management of seed lots at all steps of the conservation- regeneration process contributes to this objective. Although cultivated rice is a self-pollinated crop, cross-pollination between accessions can potentially occur during seed regeneration when numerous accessions usually flower at the same time. To estimate the outcrossing rate in Oryza sativa and determine whether the plot design currently used at IRG for seed regeneration permits outcrossing between accessions, an experiment was conducted at IRRI during the 1996 dry season.

The genetic structure and mating system of Acrocomia aculeata (Arecaceae)

Acrocomia aculeata is a perennial, fruit-producing palm tree, native to tropical forests. Its fruits have spurred interest because of their significant potential for use in the cosmetic industry and as feedstock for biofuel. In the present study, the genetic structure and mating system in Acrocomia aculeata were analyzed, using eight nuclear micro-satellite loci and samples from São Paulo and Minas Gerais states, Brazil. By means of Bayesian analysis, these populations were clustered into two or three groups. A high multilocus outcrossing rate suggests that outcrosses were predominant, although a certain degree of biparental inbreeding also occurred. Thus, although monoecious and self-compatible, there is every indication that A. aculeata bears a mixed reproductive system, with a predominance of outcrossing. Given the genetic structure revealed hereby, future conservation strategies and germplasm collecting should be focussed on sampling and preserving individuals from different clusters.

Potential Inbreeding in a Small Population of a Mass Flowering Species, <i>Xanthorrhoea johnsonii</i> (Xanthorrhoeaceae): Is Your Mother My Father?

Xanthorrhoea johnsonii is a long lived slow growing perennial understorey species, that produces a large quantity of passively dispersed seed every 3 - 5 years. Reproductive maturity is not reached until 20 - 30 years of age. The temporal asynchrony of the flowering event in this population was analogous to geographic isolation through fragmentation. A small population of plants flowering in isolation provided the opportunity to examine outcrossing rates, genetic diversity and the paternity of progeny at a small spatial scale (0.2 ha). The geographic location and physical characteristics of the adult plants were recorded, and both adults and their seed were sampled for genetic analysis. Four microsatellite loci were screened for genetic diversity and spatial structure analysis. A population outcrossing rate was estimated, as well as the number of paternal parents required to resolve the progeny multilocus genotypes. High genetic diversity was found in both adults and progeny with an estimated 97% outcrossing rate. All maternal lines required several paternal contributors, with no evidence of dominant paternal genotypes. Pollen transfer occurred between both geographically close and distant plants.