A question of origin: Where and how to collect seed for ecological restoration

Native plant species are routinely planted or sown in ecological restoration projects, but successful establishment and survival depend on where and how seeds are collected. Research suggests that it is important to use locally adapted seeds. Local populations often show a home-site advantage and non-local genotypes may be maladapted to local environmental conditions. Furthermore, intraspecific hybridisation of local and non-local genotypes may have a negative impact on the genetic structure of local populations via mechanisms such as outbreeding depression. Many species show a strong small-scale genetic differentiation between different habitats so that matching habitats of the restoration and donor site can be more important than minimizing geographical separation. It is a challenge to identify appropriate seed sources because strong small-scale population differentiation makes it difficult to delineate geographically defined seed zones to which seed exchange should be limited. Moreover, it is important to consider the genetic diversity of introduced material because it may be crucial to avoid genetic bottlenecks, inbreeding depression and poor establishment of plant populations. Repeated propagation in stock, which is often required to obtain a sufficient amount of seeds, can further reduce genetic diversity and may select for particular genotypes. Negative impacts of improper seed choice for nursery planting stock may become detectable only after many years, especially in long-lived and slow growing plants. Although scientific information on many species remains limited, the increasing demand for translocation of seed means that mandatory regulations are necessary. Guidelines should prescribe a specification of seed provenance, a record of genetic diversity of wild collections and rules for subsequent processing such as direct transfer and propagation of stock or seed orchards. We use a literature review to evaluate current legislation and to develop recommendations for herbaceous and woody species.     

Genetic differentiation of three endangered wild roses in northeastern Germany: Rosa inodora Fries, Rosa sherardii Davies and Rosa subcollina (H. Christ) Keller

Because of increased interest in the use of local provenances for restoration or landscaping projects, information about the genetic differentiation of plant species is required to delineate provenances for seed collection. To obtain information about population distinctiveness of endangered Rosa species occurring in Brandenburg (northeast Germany), we investigated the genetic differentiation of Rosa inodora, R. sherardii and R. subcollina using RAPD markers. All three species were uncommon in our study region. Φ-statistics, estimated by amova, revealed a low interpopulation differentiation for R. inodora (Φ(PT) = 0.19, P < 0.0001) and higher values for R. sherardii and R. subcollina (Φ(PT) = 0.29 and 0.30, P < 0.0001). UPGMA dendrograms and NMDS showed clear spatial differentiation for all species and a correlation between geographic and genetic distances. Due to predominantly high values of genetic differentiation and spatial patterns of ordination, we suggest small provenance regions for endangered Rosa species for seed collection.     

Seed provenance matters — Effects on germination of four plant species used for ecological restoration

The use of local seed provenances is often recommended in restoration and habitat creation because they are thought to be better adapted to local habitat conditions. However, spatial scales and the degree of population differentiation are not well known and germination is often not included in comparisons between provenances. We analysed germination as a key trait of plant development in five provenances of four species used for ecological restoration on arable land (wildflower strips). Germination was tested under different conditions in growth chambers (early vs. late spring) and in the field (non-competition vs. competition). We also examined the contribution of non-genetic (maternal) effects to population differentiation.Large differences in germination traits were found between the provenances in growth chambers and in the field. The ranking was species-specific, but largely consistent across all tested environments. Local provenances did not generally exhibit higher germination percentages in the field relative to non-local provenances. Due to the high stability of germination traits across various environments, growth chamber tests provided a reliable prediction for provenance differences in the field. The differences among provenances seemed to be largely genetically determined as the inclusion of seed mass in the analysis to control for maternal effects did not decrease the degree differences between-provenance differences. In one species, however, non-genetic contributions to population differentiation were found by comparing F1 seeds grown under homogeneous conditions and original seed material. We conclude that potentially large between-provenance differences in germination traits need to be considered in ecological restoration projects, particularly in non-permanent systems where they may determine vegetation development.     

Does restored plant diversity play a role in the reproductive functionality of Banksia populations?

Vegetation structure and plant species diversity of restoration sites are predicted to directly affect pollinator attraction, with potential impacts on gene flow, reproduction, genetic diversity of future generations, and ultimately restoration success. We compared Banksia attenuata R.Br. (Proteaceae) in a low species diversity restoration site and an adjacent natural remnant. We assessed fecundity genetic diversity in adult plants and their offspring, mating system parameters and pollen dispersal using paternity assignment. Results were compared to an earlier study of reproductive functionality within a high species diversity restoration site that was restored in a similar manner, enabling us to investigate any association between plant species diversity and fecundity. Seed set data indicated no significant differences between restored and adjacent natural sites; however, seed set data between restoration sites was significantly different (2.08 ± 0.39 and 6.89 ± 1.12, respectively). The mean number of fruits (follicles) per inflorescence was not significantly different between restoration sites. Genetic diversity of adult plants and their offspring were comparable in all sites. Higher allelic richness and genetic differentiation in one restored site reflected sourcing beyond local provenance. Low correlated paternity indicated high levels of multiple siring of seeds and paternity assignment demonstrated strong genetic connectivity between sites. Reproductive functionality, as measured by fecundity and genetic diversity in the offspring of B. attenuata, is resilient to low species diversity within a restored plant community. We consider our results in the context of establishing seed production areas (SPAs) that maximize the quantity and genetic quality of Banksia seeds for restoration.     

Rapid genetic identification of local provenance seed collection zones for ecological restoration and biodiversity conservation

The ecological restoration of native plant communities requires the collection of large amounts of seed. Use of non-local provenance seed can have detrimental consequences for the success of restoration if there is a home-site advantage, and for nature conservation through the erosion of natural patterns of population genetic structuring and/or genetic swamping (and extirpation) of locally significant genotypes. As part of an ongoing project to genetically delineate local provenance seed collection zones for species within a large urban bushland remnant of high conservation value, we assessed population genetic differentiation in two widespread coastal leguminous species, Acacia rostellifera and A. cochlearis (Fabaceae), commonly used in restoration programmes in SW Australia. Using amplified fragment length polymorphism (AFLP), we found very high levels of genetic differentiation among populations, with an analysis of molecular variance (AMOVA) showing more than 50% of the total genetic variance to be partitioned among populations (Φ_ST=0.58 and 0.51 for A. rostellifera and A. cochlearis, respectively), and marked non-overlap of almost all potential seed source populations from the local population in ordinations. Our results suggest extremely restricted natural dispersal among populations, possibly due to a combination of low seed set, seed dispersal by ants, clonality, a linear distribution of naturally fragmented populations and possibly low outcrossing rates. We suggest a narrow seed collection zone should be applied to these species for the conservation of genetic diversity and natural patterns of population genetic structure, and we highlight the value and importance of provenance evaluation to ecological restoration.     

The role of seed provenance in the early development of Arbutus unedo seedlings under contrasting watering conditions

In the last decades, several studies have reported the increase of land degradation and desertification in the Mediterranean Basin. Depending on degradation severity, ecological restoration might be needed in order to promote ecosystem recovery. The ecology of the selected species and intra specific variability should be considered in order to improve restoration options, especially facing climate change.The present study tested the hypothesis that seedlings from drier provenances would be better adapted to low water content conditions. Seeds were germinated under controlled temperature after which seedlings were grown in a phytotron under two contrasting watering regimes. Seedling performance was analysed using morphological and physiological parameters.Low water content had a clear negative effect on the seedlings’ aboveground biomass (total dry weight, root collar diameter, leaf dry weight and leaf weight ratio) and a positive effect on belowground biomass (root weight and root:shoot ratio). This response was not unequivocal, since provenances differed in morphological adaptations to low water content. Seedlings from the wettest provenance revealed a higher relative growth rate under high water content but a poor adaptation to limited water availability when compared to the other two provenances. This was observed by the absence of a significant belowground investment in this provenance. Seedlings from the wettest provenance also presented a significant reduction of total leaf area that was not observed in the other two provenances. This can however be hardly considered as a successful adaptation to cope with drought since this provenance produced less sclerophyllous leaves, less belowground biomass and also lower sapwood to leaf area ratio independently from the water content conditions. By contrast, seedlings from the dry provenance with the hottest summer had similar root collar diameter, leaf dry weight and physiological performance under both watering regimes.The observed adaptations to water regimes seem to be related with the climate of the seed source and highlighted the importance of seed provenance in ecological restoration actions using Mediterranean species. This knowledge could improve early establishment success predictions for different plant populations, allowing more reliable and cost-effective management decisions under climate change scenarios.     

Assessing the effect of genetic diversity on the early establishment of the threatened seagrass Posidonia australis using a reciprocal‐transplant experiment

Two common goals for restoration are rapid plant establishment and long‐term plant persistence. The success of transplanted populations may be jeopardized if the donor transplants are not genetically diverse, and/or poorly matched to their new environment. Here, we test the effects of local adaptation and plot‐level genetic diversity on the early establishment phase of a threatened seagrass species, Posidonia australis, by performing a reciprocal transplant experiment across two genetically and geographically distinct populations in southeastern Australia. Posidonia australis is a long‐lived, slow‐growing species that has no seed bank, and the successful transplantation of live shoots and seedlings is the only available restoration method. Our results show a strong effect of local adaptation and genetic diversity on P. australis survivorship and performance over the first 6 months following transplantation. High‐genetic diversity plots displayed higher survival rates and exhibited reduced productivity and increased carbohydrate reserves within the rhizome. This suggests that high‐diversity plots included shoots that were conserving energy stores by actively reducing growth rates during the early stages of transplantation. The lowest diversity plots exhibited high leaf and root productivity and corresponding low carbohydrate reserves. This may be a sign of stress in the low‐diversity transplants, potentially explaining the very low survival rate. We suggest that future restoration efforts source donor transplants from multiple local sources to ensure both local adaptation and sufficient genetic diversity to increase the likelihood of early establishment success.     

Hidden founder effects: small‐scale spatial genetic structure in recently established populations of the grassland specialist plant Anthyllis vulneraria

The long‐term establishment success of founder plant populations has been commonly assessed based on the measures of population genetic diversity and among population genetic differentiation, with founder populations expected to carry sufficient genetic diversity when population establishment is the result of many colonists from multiple source populations (the ‘migrant pool’ colonization model). Theory, however, predicts that, after initial colonization, rapid population expansion may result in a fast increase in the extent of spatial genetic structure (SGS), independent of extant genetic diversity. This SGS can reduce long‐term population viability by increasing inbreeding. Using 12 microsatellite markers, we inferred colonization patterns in four recent populations of the grassland specialist plant Anthyllis vulneraria and compared the extent of SGS between recently established and old populations. Assignment analyses of the individuals of recent population based on the genetic composition of nine adjacent putative source populations suggested the occurrence of the ‘migrant pool’ colonization model, further confirmed by high genetic diversity within and low genetic differentiation among recent populations. Population establishment, however, resulted in the build‐up of strong SGS, most likely as a result of spatially restricted recruitment of the progeny of initial colonists. Although reduced, significant SGS was nonetheless observed to persist in old populations. The presence of SGS was in all populations associated with elevated inbreeding coefficients, potentially affecting the long‐term viability of these populations. In conclusion, this study illustrates the importance of studying SGS next to population genetic diversity and differentiation to adequately infer colonization patterns and long‐term establishment success of plant species.     

Trading populations—can biodiversity offsets effectively compensate for population losses?

Biodiversity offsetting promotes the protection or restoration of biodiversity at one site to compensate for the loss of biodiversity due to development at another site. Thus populations of species at a development site may be extirpated in the belief that offsetting elsewhere will compensate for the loss of biodiversity. In this study we tested the replaceability of roadside populations of the orchid Diuris platichila threatened by development (populations 1–5, n = 50–541 plants) with a potential offset population occurring in nearby natural vegetation (population 6, n = 143 plants). We measured differences in habitat among the populations and associated differences in flowering and fruiting. We also measured genotypic diversity within and among the populations, and the capacity of soil from each population to promote the symbiotic germination of outcrossed seed from the two largest populations (populations 1–2). An evaluation of the performance of the relevant offset policy was also undertaken which was informed by these studies. Compared to the roadside populations, the potential offset site had limited flowering (except after fire) and was genotypically less diverse. Soil from the potential offset site, and populations 2, 3 and 5, supported significantly less seed germination than soil from population 1. Translocating individuals from the most genotypically diverse populations into the offset population could help to buffer against the loss of genetic diversity if offsetting was required; however, the limited reproduction and recruitment opportunities at the offset site could result in the eventual erosion of any initial increase in diversity. The offset policy failed to secure a suitable offset for a hypothesized loss of plants. More generally we conclude that offsetting approaches which do not assess genotypic diversity and recruitment capacity may fail in their objective of protecting species and that maintaining populations in safe sites may be required.     

Co-occurring patterns of endophyte infection and genetic structure in the alpine grass, <Emphasis Type="Italic">Festuca eskia</Emphasis>: implications for seed sourcing in ecological restoration

Choosing the provenance of seed used in ecological restoration could entail its success. An alternative approach to examine local adaptation in seed sourcing is the assessment of genetic structure and diversity based on molecular markers. These types of analyses focus on the genetics of the target plant itself and eliminate the genetic influence of associated organisms, such as Epichloë/Neotyphodium endophytes in grasses. By impacting the fitness of their host, such symbionts may influence population genetic structure and diversity. Therefore, seed sourcing for grasses must consider the influence of their endophytes to increase seed translocation success and minimize the risks associated with this practice. To delineate seed zones for restoration of the alpine fescue Festuca eskia Ramond ex. DC. (Poaceae), we assessed population genetic differentiation and diversity patterns in the species including endophyte occurrence along altitudinal and longitudinal gradients in the Pyrenees Mountains. Twenty-three populations were analysed for endophyte status, and three STS and one SSR marker were used to examine genetic differentiation and diversity patterns. Results showed that F. eskia hosts an asexual form of Epichloë and infection frequency within populations decreased from East to West (100 vs. 8–25%). Molecular markers separated F. eskia into two East and West groups, and endophyte infection and genetic patterns were congruent with molecular data. Little evidence for genetic differentiation or difference in endophyte occurrence associated with altitude was detected. Little variation was found in within population diversity, regardless of provenance altitude and site, and/or endophyte infection frequency. The results of this study suggested the establishment of two distinct management units for F. eskia seed sourcing restoration.     

Differentiation and adaptation in <Emphasis Type="Italic">Brassica nigra</Emphasis> populations: interactions with related herbivores

Local adaptation and population differentiation of plants are well documented, but studies on interactions with natural enemies are rare. In particular, evidence for plant adaptation to the local biotic environment, such as herbivores remains poor. We used the black mustard Brassica nigra, an annual species of river valley and coastal habitats to (1) analyse population differentiation in plant traits and herbivory in a common garden experiment, (2) examine home versus away differences in a reciprocal transplant experiment and (3) test whether plants are adapted to local herbivores or vice versa under standard greenhouse conditions. In the common garden experiment, we found significant differentiation in plant traits, leaf damage and herbivore number among seven populations of B. nigra from France and Germany (distance 15–1,000 km). Differences were particularly strong among coastal and river valley populations and did not necessarily increase with geographical distance. A herbivore removal treatment did not change population differentiation when compared with the control allowing natural colonisation. The reciprocal transplant experiment at a scale of 15–30 km did not reveal local plant adaptation, whilst one dominant herbivore species (Meligethes aeneus) occurred in significantly higher numbers on local plants. A greenhouse experiment combining three aphid (Brevicoryne brassicae) and plant populations of the same provenance indicated herbivore adaptation to their local plants rather than plant adaptation, but overall contrasts between local and non-local combinations were not significant. The results suggest that herbivores may counteract local plant adaptation to other environmental factors. Our study has important implications for plant translocations in ecological restoration projects.     

Stability of species and provenance performance when translocated into different community assemblages

The stability of species and provenance performance across diverse environments is a major issue in restoration, particularly for assisted migration and climate‐adjusted provenancing strategies. This study examines how differences in species and provenance performance are affected by plant community composition in a dry sclerophyll forest restoration experiment. Five indices were measured over 6 years post‐establishment to evaluate the relative performance of community composition using 10 provenances of two focal eucalypts (Eucalyptus pauciflora and Eucalyptus tenuiramis) under six community treatments for E. pauciflora and five for E. tenuiramis. Community treatments varied according to the species planted as the immediate neighbor to the focal species, and included same species, same genus, or one of three different genera. Significant species and provenance differences were observed for all measured performance indices, with no evidence of interaction effects with community treatments. E. tenuiramis was more susceptible to insects and frost, and had poorer establishment but greater growth of the survivors than E. pauciflora. Generally, nonlocal provenances were more susceptible to insect herbivory and frost damage and had higher mortality than local provenances. At this early life‐stage there was no evidence that co‐planted species affected the relative performance of focal species or provenances, arguing transfer functions are likely stable across different planted communities. While species and provenance performance was not affected by community context, focal species differed in their response to upslope migration and any climate‐adjusted provenancing may require staged transfers to avoid maladaptation under contemporary growing conditions.     

Genetic Delineation of Local Provenance in Persoonia longifolia: Implications for Seed Sourcing for Ecological Restoration

Restoration of diverse native plant communities typically requires the collection of large amounts of seed. Thus, practitioners often struggle to find adequate supplies near project sites and need to know from how far they can collect without compromising restoration success—how far does local provenance extend? We addressed this issue by assessing genetic variation within, and differentiation among, 12 potential seed source populations of Persoonia longifolia, a key component of the jarrah forest of Western Australia. An analysis of molecular variance of 66 polymorphic amplified fragment length polymorphism markers partitioned 92% of the total genetic variation within populations and 8% among populations, indicating relatively weak but statistically significant population genetic differentiation. Ordination of these genetic data showed marked west/east and north/south gradients. Pairwise population genetic dissimilarity was correlated with both geographic distance and environmental distance derived from five climate variables. However, partial Mantel tests showed that the relationship between genetic and geographic distance was not independent of environmental distance, suggesting a non‐neutral signature in these markers. Bayesian outlier analysis identified two markers, and spatial analysis method tests identified highly significant associations between these two markers and three environmental variables. Frequency differences at these markers across populations suggested the possibility of climatically adapted provenances. The global significance value from analyses of similarities for these two markers correlated to a general provenance distance of 47 km, in contrast to a threshold of 60 km for the complete dataset. Guidelines for seed sourcing that consider these population genetic data should lead to more effective ecological restoration with this species.     

Reproductive strategy, clonal structure and genetic diversity in populations of the aquatic macrophyte Sparganium emersum in river systems

Many aquatic and riparian plant species are characterized by the ability to reproduce both sexually and asexually. Yet, little is known about how spatial variation in sexual and asexual reproduction affects the genotypic diversity within populations of aquatic and riparian plants. We used six polymorphic microsatellites to examine the genetic diversity within and differentiation among 17 populations (606 individuals) of Sparganium emersum, in two Dutch-German rivers. Our study revealed a striking difference between rivers in the mode of reproduction (sexual vs. asexual) within S. emersum populations. The mode of reproduction was strongly related to locally reigning hydrodynamic conditions. Sexually reproducing populations exhibited a greater number of multilocus genotypes compared to asexual populations. The regional population structure suggested higher levels of gene flow among sexually reproducing populations compared to clonal populations. Gene flow was mainly mediated via hydrochoric dispersal of generative propagules (seeds), impeding genetic differentiation among populations even over river distances up to 50 km. Although evidence for hydrochoric dispersal of vegetative propagules (clonal plant fragments) was found, this mechanism appeared to be relatively less important. Bayesian-based assignment procedures revealed a number of immigrants, originating from outside our study area, suggesting intercatchment plant dispersal, possibly the result of waterfowl-mediated seed dispersal. This study demonstrates how variation in local environmental conditions in river systems, resulting in shifting balances of sexual vs. asexual reproduction within populations, will affect the genotypic diversity within populations. This study furthermore cautions against generalizations about dispersal of riparian plant species in river systems.     

Turning up the heat on the provenance debate: Testing the ‘local is best’ paradigm under heatwave conditions

A key aspect of successful restoration projects is the sourcing of propagation material suited to the environmental and biotic conditions of the proposed planting site. Traditionally, the use of propagules collected locally has been advocated for revegetation on the assumption that this material is better adapted to local conditions. A rapidly changing climate, however, is challenging the assumption that the use of local genetic stock will provide the best restoration outcome in the long term. We tested the ‘local is best’ paradigm using open top chambers to simulate the predicted summer temperatures for 2050 in western Sydney, Australia. We compared the establishment success of Eucalyptus tereticornis and Themeda australis, dominant species in Cumberland Plain Woodland, grown from local versus non‐local seed. All plants survived an exceptional summer heatwave and few differences between temperature treatments were found. No evidence of local superiority was found for survival or growth of non‐reproductive tissues of either species. However, local provenance plants of E. tereticornis suffered significantly greater herbivory in the ambient temperature treatment than one non‐local provenance, and local provenance plants of T. australis demonstrated significant superiority to most non‐local provenances in all categories of reproductive growth. For both species, the provenances from warmer climates demonstrated comparable, and often better, growth performance than the local provenance plants.     

Prevailing negative soil biota effect and no evidence for local adaptation in a widespread Eurasian grass

Background

Soil biota effects are increasingly accepted as an important driver of the abundance and distribution of plants. While biogeographical studies on alien invasive plant species have indicated coevolution with soil biota in their native distribution range, it is unknown whether adaptation to soil biota varies among populations within the native distribution range. The question of local adaptation between plants and their soil biota has important implications for conservation of biodiversity and may justify the use of seed material from local provenances in restoration campaigns.

Methodology/Principal Findings

We studied soil biota effects in ten populations of the steppe grass Stipa capillata from two distinct regions, Europe and Asia. We tested for local adaptation at two different scales, both within (ca. 10–80 km) and between (ca. 3300 km) regions, using a reciprocal inoculation experiment in the greenhouse for nine months. Generally, negative soil biota effects were consistent. However, we did not find evidence for local adaptation: both within and between regions, growth of plants in their ‘home soil’ was not significantly larger relative to that in soil from other, more distant, populations.

Conclusions/Significance

Our study suggests that negative soil biota effects can prevail in different parts of a plant species'' range. Absence of local adaptation points to the possibility of similar rhizosphere biota composition across populations and regions, sufficient gene flow to prevent coevolution, selection in favor of plasticity, or functional redundancy among different soil biota. From the point of view of plant - soil biota interactions, our findings indicate that the current practice of using seeds exclusively from local provenances in ecosystem restoration campaigns may not be justified.     

Testing the “Local Provenance” Paradigm: A Common Garden Experiment in Cumberland Plain Woodland,Sydney, Australia

Seed for restoration projects has traditionally been sourced locally to “preserve” the genetic integrity of the replanted site. Plants grown from locally sourced seeds are perceived to have the advantage of being adapted to local conditions, and the use of local provenance is a requirement of many restoration projects. However, the processes of climate change and habitat fragmentation, with the subsequent development of novel environments, are forcing us to reconsider this basic tenet of restoration ecology. We tested the “local provenance is best” paradigm, by comparing the performance of plants grown from local with non‐local seed sources within a common garden experiment. We selected six species representing a range of growth forms (Acacia falcata, Bursaria spinosa ssp. spinosa, Eucalyptus crebra, E. tereticornis, Hardenbergia violacea and Themeda australis) from an assemblage known as the Cumberland Plain Woodland, a threatened community in western Sydney. Multiple provenances were collected from within the range of each species and grown at two field sites on the Cumberland Plain. Growing time varied between species and ranged from 7 months to 2 years. With the exception of B. spinosa, and to a lesser extent T. australis, we found little evidence that local provenance plants were superior to distant provenances in terms of survival and establishment.     

Patterns of sex ratio variation and genetic diversity in the dioecious forest perennial <Emphasis Type="Italic">Mercurialis perennis</Emphasis>

In small populations of plant species with separate sexes, it can be expected that besides the local environment also stochastic events influence population sex ratios. Biased sex ratios may in turn negatively affect genetic diversity due to increased genetic drift and, in clonal plants, due to reduced sexual reproductive output. Empirical evidence for these processes is scarce, however. We investigated the pattern of sex ratio variation and the distribution of genetic variation of the dioecious clonal forest herb Mercurialis perennis using AFLP markers. Analysis of molecular variance indicated a pronounced genetic structure. Overall within-population genetic diversity was moderate and local sex ratios were slightly male biased. The proportion of male to female plants in large populations slightly increased with increasing light penetration to the herb layer. Small populations, on the contrary, displayed high variability in sex ratios, unrelated to the local light environment. Genotypic diversity decreased with more male-biased sex ratios. We conclude that stochastic events related to small population size and the local forest environment, related to canopy closure, affect the proportion of female plants and indirectly influence local genotypic diversity, likely through the degree of sexual reproduction. This is one of the first studies to report a clear association between gender proportions and genetic diversity of a dioecious plant species in a fairly large survey.     

The effect of herbivores on genotypic diversity in a clonal aquatic plant

In clonal plants, vegetative parts may outcompete seeds in the absence of disturbance, limiting the build‐up of genotypic diversity through repeated seedling recruitment (RSR). Herbivory may provide disturbance and trigger establishment of strong colonizers (seeds) at the expense of strong competitors (clonal propagules). In the clonal aquatic fennel pondweed Potamogeton pectinatus, two distinct herbivore guilds may modify the dynamics of propagation. In winter, Bewick's swans may deplete patches of tubers, promoting seedling establishment in spring. In summer, seed consumption by waterfowl can reduce the density of viable seeds but grazing may also reduce tuber production and hence facilitate seedling establishment. This study is among the first to experimentally test herbivore impact on plant genotypic diversity. We assess the separate and combined effects of both herbivore guilds on genotypic diversity and structure of fennel pondweed beds. Using microsatellites, we genotyped P. pectinatus from an exclosure experiment and assessed the contribution of herbivory, dispersal and sexual reproduction to the population genetic structure. Despite the predominance of clonal propagation in P. pectinatus, we found considerable genotypic diversity. Within the experimental blocks, kinship among genets decreased with geographic distance, clearly identifying a role for RSR in the maintenance of genotypic diversity within the fennel pondweed beds. However, over a period of five years, none of the herbivory treatments affected genotypic diversity. Hence, sexual reproduction on a local scale is important in this putatively clonal plant and possibly sufficient to ensure a relatively high genotypic diversity even in the absence of herbivores. Although we cannot preclude a role of herbivory in shaping genotypic diversity of a clonal plant, after five years of exclusion of the two investigated herbivore guilds no measurable effect on genotypic diversity was detected.