Using genetic markers to directly estimate gene flow and reproductive success parameters in plants on the basis of naturally regenerated seedlings

Estimating seed and pollen gene flow in plants on the basis of samples of naturally regenerated seedlings can provide much needed information about "realized gene flow," but seems to be one of the greatest challenges in plant population biology. Traditional parentage methods, because of their inability to discriminate between male and female parentage of seedlings, unless supported by uniparentally inherited markers, are not capable of precisely describing seed and pollen aspects of gene flow realized in seedlings. Here, we describe a maximum-likelihood method for modeling female and male parentage in a local plant population on the basis of genotypic data from naturally established seedlings and when the location and genotypes of all potential parents within the population are known. The method models female and male reproductive success of individuals as a function of factors likely to influence reproductive success (e.g., distance of seed dispersal, distance between mates, and relative fecundity--i.e., female and male selection gradients). The method is designed to account for levels of seed and pollen gene flow into the local population from unsampled adults; therefore, it is well suited to isolated, but also wide-spread natural populations, where extensive seed and pollen dispersal complicates traditional parentage analyses. Computer simulations were performed to evaluate the utility and robustness of the model and estimation procedure and to assess how the exclusion power of genetic markers (isozymes or microsatellites) affects the accuracy of the parameter estimation. In addition, the method was applied to genotypic data collected in Scots pine (isozymes) and oak (microsatellites) populations to obtain preliminary estimates of long-distance seed and pollen gene flow and the patterns of local seed and pollen dispersal in these species.     

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.     

Fine-Scale Spatial Genetic Structure in Emmer Wheat and the Role of Population Range Position

The extent of spatial genetic structure (SGS) within plant populations depends on seed and pollen dispersal distance, breeding type, level of self-fertilization and effective plant density. Self-fertilizing species with gravity-dispersed seeds are expected to have both small effective population sizes and low pollen movement leading to high genetic structure. Higher SGS can be expected in more patchy and peripheral populations because of lower plant density and population sizes, and lower intensity of gene flow. We tested these predictions analyzing SGS in two core and two peripheral populations of predominantly self-fertilizing emmer wheat. Analysis of SGS with 11 nuclear microsatellites revealed (1) a negative linear relationship between kinship coefficients, calculated for pairs of individuals, and the logarithm of geographical distance between members of the pairs, in all studied populations; and (2) a significant autocorrelation for a distance up to 5 m (core populations) or 20 m (peripheral populations). Pollen flow, estimated from comparison of nuclear and chloroplast variation, was spatially limited, as was seed dispersal. Our results support a hypothesized relationship between SGS intensity and breeding system, the mode of seed dispersal and the population range position (core vs. periphery).     

Pleistocene megafaunal extinctions and the functional loss of long‐distance seed‐dispersal services

Pleistocene extinctions affected mainly large‐bodied animals, determining the loss or changes in numerous ecological functions. Evidence points to a central role of many extinct megafauna herbivores as seed dispersers. An important step in understanding the legacy of extinct mutualistic interactions is to evaluate the roles and effectiveness of megafauna herbivores in seed dispersal. Here we use morphological and ecophysiological allometries to estimate both quantitative and qualitative aspects of seed‐dispersal services likely provided by extinct megafauna. We developed a mechanistic model that encompasses four stages of seed dispersal – seed ingestion, gut retention, animal movement, and seed deposition. We estimate seed‐dispersal kernels through simulations to infer the role of Pleistocene megafauna in promoting long‐distance dispersal and examine how seed dispersal was affected by extinctions. Simulations suggest extinct large‐bodied frugivores would frequently disperse large seeds over a thousand meters, whereas smaller‐bodied frugivores are more likely to deposit the seeds over a few hundred meters. Moreover, events of long‐distance seed dispersal by the extinct megafauna would be up to ten times longer than long‐distance dispersal by smaller‐sized extant mammals. By estimating the combined distribution of seed dispersal distances considering all large‐bodied mammalian frugivores in specific South American Pleistocene assemblages we found that long‐distance dispersal contracted by at least two thirds after the megafauna died out. The disruption of long‐distance dispersal is expected to have consequences for recruitment, spatial and genetic structure of plant populations, population persistence and community composition. Promoting long‐distance seed dispersal was one among other salient features of extinct Pleistocene megafauna that reveal their influence on natural ecosystems. Modeling the consequences of megafaunal extinctions can offer quantitative predictions on the consequences of ongoing defaunation to plant populations and ecological communities.     

Changes of effective gene dispersal distances by pollen and seeds across successive life stages in a tropical tree

Pollen and seed dispersal are the two key processes in which plant genes move in space, mostly mediated by animal dispersal vectors in tropical forests. Due to the movement patterns of pollinators and seed dispersers and subsequent complex spatial patterns in the mortality of offspring, we have little knowledge of how pollinators and seed dispersers affect effective gene dispersal distances across successive recruitment stages. Using six highly polymorphic microsatellite loci and parentage analyses, we quantified pollen dispersal, seed dispersal, and effective paternal and maternal gene dispersal distances from pollen‐ and seed‐donors to offspring across four recruitment stages within a population of the monoecious tropical tree Prunus africana in western Kenya. In general, pollen‐dispersal and paternal gene dispersal distances were much longer than seed‐dispersal and maternal gene dispersal distances, with the long‐distance within‐population gene dispersal in P. africana being mostly mediated by pollinators. Seed dispersal, paternal and maternal gene dispersal distances increased significantly across recruitment stages, suggesting strong density‐ and distance‐dependent mortality near the parent trees. Pollen dispersal distances also varied significantly, but inconsistently across recruitment stages. The mean dispersal distance was initially much (23‐fold) farther for pollen than for seeds, yet the pollen‐to‐seed dispersal distance ratio diminished by an order of magnitude at later stages as maternal gene dispersal distances disproportionately increased. Our study elucidates the relative changes in the contribution of the two processes, pollen and seed dispersal, to effective gene dispersal across recruitment. Overall, complex sequential processes during recruitment contribute to the genetic make‐up of tree populations. This highlights the importance of a multistage perspective for a comprehensive understanding of the impact of animal‐mediated pollen and seed dispersal on small‐scale spatial genetic patterns of long‐lived tree species.     

Powers of discerning: challenges to understanding dispersal processes in natural populations

In this issue of Molecular Ecology, authors Robledo-Arnuncio & Garcia present a compelling approach for quantifying seed dispersal in plant populations. Building upon methods previously used for quantification of pollen dispersal, the authors not only examine the behaviour of the model with respect to sample sizes, dispersal distance, and the kurtosis of the dispersal function but also provide an empirical example using Prunus mahaleb.     

Effects of seed dispersal, adult tree and seedling density on the spatial genetic structure of regeneration at fine temporal and spatial scales

Several demographic factors can produce family structured patches within natural plant populations, particularly limited seed and pollen dispersal and small effective density. In this paper, we used computer simulations to examine how seed dispersal, density, and spatial distribution of adult trees and seedlings can explain the spatial genetic structure (SGS) of natural regeneration after a single reproductive event in a small population. We then illustrated the results of our simulations using genetic (isozymes and chloroplast microsatellites) and demographic experimental data from an Abies alba (silver fir) intensive study plot located in the Southern French Alps (Mont Ventoux). Simulations showed that the structuring effect of limited dispersal on seedling SGS can largely be counterbalanced by high effective density or a clumped spatial distribution of adult trees. In addition, the clumping of natural regeneration far from adult trees, which is common in temperate forest communities where gap dynamics are predominant, further decreases SGS intensity. Contrary to our simulation results, low adult tree density, aggregated spatial distribution of seedlings, and limited seed dispersal did not generate a significant SGS in our A. alba experimental plot. Although some level of long distance pollen and seed flow could explain this lack of SGS, our experimental data confirm the role of spatial aggregation (both in adult trees and in seedlings far from adult trees) in reducing SGS in natural populations.     

VARIATION IN POLLEN FLOW WITHIN AND AMONG POPULATIONS OF IPOMOPSIS AGGREGATA

Pollen dispersal is a major component of gene flow in plant populations. It can influence microevolution within and among populations as well as the evolution of floral characters that affect dispersal. Most previous studies have relied on point estimates to characterize dispersal distances, even though there is likely to be substantial intrapopulational and interpopulational variation. We measured variation in pollen dispersal for the hummingbird-pollinated herb Ipomopsis aggregata (Polemoniaceae), using powdered fluorescent dyes to estimate pollen movement. Analysis of 5–6 natural populations in each of three years indicated that mean and mean squared distances of pollen dispersal, measured over the reproductive lifespan of individual plants, varied more than threefold among populations and years. Dispersal distances also shifted over the season within a given population. Unlike the variation among populations, these seasonal changes were associated in part with changes in flower density. The mean distance of pollen dispersal from an individual plant was unrelated to the date of first flowering, but did reflect two floral characters. Plants with higher variance in stamen length across flowers delivered pollen farther on average, as predicted by computer simulations of pollen carryover. Plants with lower mean stamen lengths also delivered pollen farther. Such effects of plant characters on pollen dispersal are a critical prerequisite for dispersal to evolve in response to its effects on fitness.     

Migration load in plants: role of pollen and seed dispersal in heterogeneous landscapes

Evolution of local adaptation depends critically on the level of gene flow, which, in plants, can be due to either pollen or seed dispersal. Using analytical predictions and individual-centred simulations, we investigate the specific influence of seed and pollen dispersal on local adaptation in plant populations growing in patchy heterogeneous landscapes. We study the evolution of a polygenic trait subject to stabilizing selection within populations, but divergent selection between populations. Deviations from linkage equilibrium and Hardy-Weinberg equilibrium make different contributions to genotypic variance depending on the dispersal mode. Local genotypic variance, differentiation between populations and genetic load vary with the rate of gene flow but are similar for seed and pollen dispersal, unless the landscape is very heterogeneous. In this case, genetic load is higher in the case of pollen dispersal, which appears to be due to differences in the distribution of genotypic values before selection.     

Evaluating the influence of different aspects of habitat fragmentation on mating patterns and pollen dispersal in the bird-pollinated Banksia sphaerocarpa var. caesia

Habitat fragmentation can significantly affect mating and pollen dispersal patterns in plant populations, although the differential effects of the various aspects of fragmentation are poorly understood. In this study, we used eight microsatellite loci to investigate the effect of fragmentation on the mating system and pollen dispersal within one large and eight small population remnants of Banksia sphaerocarpa var. caesia, a bird-pollinated shrub in the southern agricultural region of Western Australia. The large population had a much larger neighbourhood size and lower selfing rate, maternal pollen pool differentiation and within-plot mean pollen dispersal distance than the small populations. Outcrossing was consistently high and ranged from 85.7% ± 2.6 to 98.5% ± 0.9, and mating patterns suggested nearest-neighbour pollination. Pollen immigration into small populations ranged from 2.8% ± 1.8 to 16.5% ± 3.2. Using the small populations, we tested for correlations between various fragmentation variables and mating system and pollen dispersal parameters. We found significant negative linear relationships between population isolation and outcrossing rate; population shape and neighbourhood size; and conspecific density and mean pollen dispersal distance. There were significant positive linear relationships between population shape and pollen pool differentiation and between population size and number of different fathers per seed crop. Our results suggest that birds may use a series of fragmented populations as a vegetation corridor while foraging across the landscape and that population connectivity is a critical determinant of pollinator visitation. Our results also suggest that the effect of a linear population shape on the mating system and pollen dispersal is routinely underestimated.     

Genetic rescue by distant trees mitigates qualitative pollen limitation imposed by fine‐scale spatial genetic structure

Restricted seed dispersal frequently leads to fine‐scale spatial genetic structure (i.e., FSGS) within plant populations. Depending on its spatial extent and the mobility of pollinators, this inflated kinship at the immediate neighbourhood can critically impoverish pollen quality. Despite the common occurrence of positive FSGS within plant populations, our knowledge regarding the role of long‐distance pollination preventing reproductive failure is still limited. Using microsatellite markers, we examined the existence of positive FSGS in two low‐density populations of the tree Pyrus bourgaeana. We also designed controlled crosses among trees differing in their kinship to investigate the effects of increased local kinship on plant reproduction. We used six pollination treatments and fully monitored fruit production, fruit and seed weight, proportion of mature seeds per fruit, and seed germination. Our results revealed positive FSGS in both study populations and lower fruit initiation in flowers pollinated with pollen from highly‐genetically related individuals within the neighbourhood, with this trend intensifying as the fruit development progressed. Besides, open‐pollinated flowers exhibited lower performance compared to those pollinated by distant pollen donors, suggesting intense qualitative pollen limitation in natural populations. We found positive fine‐scale spatial genetic structure is translated into impoverished pollen quality from nearby pollen donors which negatively impacts the reproductive success of trees in low‐density populations. Under this scenario of intrapopulation genetic rescue by distant pollen donors, the relevance of highly‐mobile pollinators for connecting spatially and genetically distant patches of trees may be crucial to safeguarding population recruitment.     

A model for genetic relationship in areally continous plant populations.

Representations are based on plant populations, continuously distributed over their habitats according to specified density patterns. Migration of genetic material takes place via pollen and seed dispersal. Monoecious plants with arbitrary rates of self-fertilization and dioecious plants are considered. The model was constructed with the intention of determining coefficients of inbreeding and kinship for all locations within the seed population after its dispersal over the habitat, assuming the respective genetic relationships of the parental generation to be known. To display the consequences of single components hidden in the general result, the following specifications have been treated: finite population size combined with random dispersal of seed, equilibrium states for hypothetically infinite population size with “limited” dispersal of pollen and seed, random dispersal of pollen, and random dispersal of seed.     

The Limits of Mean-Field Heterozygosity Estimates under Spatial Extension in Simulated Plant Populations

Computational models of evolutionary processes are increasingly required to incorporate multiple and diverse sources of data. A popular feature to include in population genetics models is spatial extension, which reflects more accurately natural populations than does a mean field approach. However, such models necessarily violate the mean field assumptions of classical population genetics, as do natural populations in the real world. Recently, it has been questioned whether classical approaches are truly applicable to the real world. Individual based models (IBM) are a powerful and versatile approach to achieve integration in models. In this study an IBM was used to examine how populations of plants deviate from classical expectations under spatial extension. Populations of plants that used three different mating strategies were placed in a range of arena sizes giving crowded to sparse occupation densities. Using a measure of population density, the pollen communication distance (P(cd)), the deviation exhibited by outbreeding populations differed from classical mean field expectations by less than 5% when P(cd) was less than 1, and over this threshold value the deviation significantly increased. Populations with an intermediate mating strategy did not have such a threshold and deviated directly with increasing isolation between individuals. Populations with a selfing strategy were influenced more by the mating strategy than by increased isolation. In all cases pollen dispersal was more influential than seed dispersal. The IBM model showed that mean field calculations can be reasonably applied to natural outbreeding plant populations that occur at a density in which individuals are less than the average pollen dispersal distance from their neighbors.     

Effective Long-Distance Pollen Dispersal in Centaurea jacea

Background

Agri-environment schemes play an increasingly important role for the conservation of rare plants in intensively managed agricultural landscapes. However, little is known about their effects on gene flow via pollen dispersal between populations of these species.

Methodology/Principal Findings

In a 2-year experiment, we observed effective pollen dispersal from source populations of Centaurea jacea in restored meadows, the most widespread Swiss agri-environment scheme, to potted plants in adjacent intensively managed meadows without other individuals of this species. Potted plants were put in replicated source populations at 25, 50, 100 m and where possible 200 m distance from these source populations. Pollen transfer among isolated plants was prevented by temporary bagging, such that only one isolated plant was accessible for flower visitors at any one time. Because C. jacea is self-incompatible, seed set in single-plant isolates indicated insect mediated effective pollen dispersal from the source population. Seed set was higher in source populations (35.7±4.4) than in isolates (4.8±1.0). Seed set declined from 18.9% of that in source populations at a distance of 25 m to 7.4% at 200 m. At a distance of 200 m seed set was still significantly higher in selfed plants, indicating long-distance effective pollen dispersal up to 200 m. Analyses of covariance suggested that bees contributed more than flies to this long-distance pollen dispersal. We found evidence that pollen dispersal to single-plant isolates was positively affected by the diversity and flower abundance of neighboring plant species in the intensively managed meadow. Furthermore, the decline of the dispersal was less steep when the source population of C. jacea was large.

Conclusions

We conclude that insect pollinators can effectively transfer pollen from source populations of C. jacea over at least 200 m, even when “recipient populations” consisted of single-plant isolates, suggesting that gene flow by pollen over this distance is very likely. Source population size and flowering environment surrounding recipient plants appear to be important factors affecting pollen dispersal in C. jacea. It is conceivable that most insect-pollinated plants in a network of restored sites within intensively managed grassland can form metapopulations, if distances between sites are of similar magnitude as tested here.     

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.     

A model for genetic relationship in areally continuous plant populations

Representations are based on plant populations, continuously distributed over their habitats according to specified density patterns. Migration of genetic material takes place via pollen and seed dispersal. Monoecious plants with arbitrary rates of self-fertilization and dioecious plants are considered. The model was constructed with the intention of determining coefficients of inbreeding and kinship for all locations within the seed population after its dispersal over the habitat, assuming the respective genetic relationships of the parental generation to be known. To display the consequences of single components hidden in the general result, the following specifications have been treated: finite population size combined with random dispersal of seed, equilibrium states for hypothetically infinite population size with “limited” dispersal of pollen and seed, random dispersal of pollen, and random dispersal of seed.     

Fine-scale spatial genetic structure and gene dispersal in Silene latifolia

Plants are sessile organisms, often characterized by limited dispersal. Seeds and pollen are the critical stages for gene flow. Here we investigate spatial genetic structure, gene dispersal and the relative contribution of pollen vs seed in the movement of genes in a stable metapopulation of the white campion Silene latifolia within its native range. This short-lived perennial plant is dioecious, has gravity-dispersed seeds and moth-mediated pollination. Direct measures of pollen dispersal suggested that large populations receive more pollen than small isolated populations and that most gene flow occurs within tens of meters. However, these studies were performed in the newly colonized range (North America) where the specialist pollinator is absent. In the native range (Europe), gene dispersal could fall on a different spatial scale. We genotyped 258 individuals from large and small (15) subpopulations along a 60 km, elongated metapopulation in Europe using six highly variable microsatellite markers, two X-linked and four autosomal. We found substantial genetic differentiation among subpopulations (global F_ST=0.11) and a general pattern of isolation by distance over the whole sampled area. Spatial autocorrelation revealed high relatedness among neighboring individuals over hundreds of meters. Estimates of gene dispersal revealed gene flow at the scale of tens of meters (5–30 m), similar to the newly colonized range. Contrary to expectations, estimates of dispersal based on X and autosomal markers showed very similar ranges, suggesting similar levels of pollen and seed dispersal. This may be explained by stochastic events of extensive seed dispersal in this area and limited pollen dispersal.     

Joint estimation of contemporary seed and pollen dispersal rates among plant populations

There are few statistical methods for estimating contemporary dispersal among plant populations. A maximum-likelihood procedure is introduced here that uses pre- and post-dispersal population samples of biparentally inherited genetic markers to jointly estimate contemporary seed and pollen immigration rates from a set of discrete external sources into a target population. Monte Carlo simulations indicate that accurate estimates and reliable confidence intervals can be obtained using this method for both pollen and seed migration rates at modest sample sizes (100 parents/population and 100 offspring) when population differentiation is moderate (F(ST) ≥ 0.1), or by increasing pre-dispersal samples (to about 500 parents/population) when genetic divergence is weak (F(ST) = 0.01). The method exhibited low sensitivity to the number of source populations and achieved good accuracy at affordable genetic resolution (10 loci with 10 equifrequent alleles each). Unsampled source populations introduced positive biases in migration rate estimates from sampled sources, although they were minor when the proportion of immigration from the latter was comparatively low. A practical application of the method to a metapopulation of the Australian resprouter shrub Banksia attenuata revealed comparable levels of directional seed and pollen migration among dune groups, and the estimate of seed dispersal was higher than a previous estimate based on conservative assignment tests. The method should be of interest to researchers and managers assessing broad-scale nonequilibrium seed and pollen gene flow dynamics in plants.     

Contributions of long‐distance dispersal to population growth in colonising Pinus ponderosa populations

Long‐distance dispersal is an integral part of plant species migration and population development. We aged and genotyped 1125 individuals in four disjunct populations of Pinus ponderosa that were initially established by long‐distance dispersal in the 16th and 17th centuries. Parentage analysis was used to determine if individuals were the product of local reproductive events (two parents present), long‐distance pollen dispersal (one parent present) or long‐distance seed dispersal (no parents present). All individuals established in the first century at each site were the result of long‐distance dispersal. Individuals reproduced at younger ages with increasing age of the overall population. These results suggest Allee effects, where populations were initially unable to expand on their own, and were dependent on long‐distance dispersal to overcome a minimum‐size threshold. Our results demonstrate that long‐distance dispersal was not only necessary for initial colonisation but also to sustain subsequent population growth during early phases of expansion.     

Isolation affects reproductive success in low-density but not high-density populations of two wind-pollinated <Emphasis Type="Italic">Thalictrum</Emphasis> species

Since pollen usually travels limited distances in wind-pollinated plant species, plants growing at low density may become pollen limited. We examined how local pollen availability and population density affect reproductive success in two wind-pollinated, dioecious species, Thalictrum fendleri and Thalictrum dioicum. Distance to the nearest flowering male, the number of flowering males within 2 m, and flower number on those males served as measures of local pollen availability. Increased distance from pollen donors reduced seed set in the lowest-density population of each species, but seed set in high-density populations was not correlated with local pollen availability. For plants in high- and low-density populations at similar distances from pollen donors, this distance only affected seed set in low-density populations. To ensure that differences in resource availability were not causing spurious correlations between seed set and plant density, we constructed low-density artificial arrays in populations of T. dioicum. In these, seed set decreased rapidly with increases in distance from pollen donors. Despite these effects, the density of males in a population was not correlated with average seed set in T. dioicum, and hand pollination in the T. dioicum populations also failed to increase seed set over natural levels. These results suggest that pollen receipt only limits seed set on isolated plants within low- density populations of T. dioicum and T. fendleri.