Sex determination and the evolution of dioecy from monoecy in Sagittaria latifolia (Alismataceae)

The role of mutations of small versus large effect in adaptive evolution is of considerable interest to evolutionary biologists. The major evolutionary pathways for the origin of dioecy in plants (the gynodioecy and monoecy-paradioecy pathways) are often distinguished by the number of mutations involved and the magnitude of their effects. Here, we investigate the genetic and environmental determinants of sex in Sagittaria latifolia, a species with both monoecious and dioecious populations, and evaluate evidence for the evolution of dioecy via gynodioecy or monoecy-paradioecy. We crossed plants of the two sexual systems to generate F1, F2 and backcross progeny, and grew clones from dioecious populations in low-and high-fertilizer conditions to examine sex inconstancy in females and males. Several lines of evidence implicate two-locus control of the sex phenotypes. In dioecious populations sex is determined by Mendelian segregation of alleles, with males heterozygous at both the male- and female-sterility loci. In monoecious populations, plants are homozygous for alleles dominant to male sterility in females and recessive to female sterility in males. Experimental manipulation of resources revealed sex inconstancy in males but not females. These results are consistent with predictions for the evolution of dioecy via gynodioecy, rather than the expected monoecy-paradioecy pathway, given the ancestral monoecious condition.     

Tests of a mechanistic model of one hormone regulating both sexes in Buchloe dactyloides (Poaceae)

A mechanistic model of one hormone regulating both sexes in flowering plants was tested in buffalograss (Buchloe dactyloides). This model assumes that one hormone has male and female cell receptors to inhibit one sex and induce the other independently. Three components—the normal range of hormone level in the plant and the sensitivity levels of the two receptors—interact to regulate sex expression. The study organism, buffalograss, is usually considered dioecious, but natural populations consist of varying proportions of male, female, and monoecious plants. Prior research with growth regulators had shown that only gibberellin (GA) had consistent and significant effects on sex expression in this species. To test the model assumption of a hormone with a dual function, GA and a GA inhibitor (paclobutrazol, PAC) were applied to three monoecious genotypes; in two of the genotypes the GA treatment yielded a significantly higher proportion of male inflorescences, and this transition involved both inducing male and inhibiting female. PAC treatment produced exclusively female inflorescences, illustrating the dual effects of GA. To test the predictability of the model, GA was applied to two dwarf female genotypes. These plants were transformed into neuter and near-neuter plants with normal height and vegetative growth, as predicted by our model for genotypes with a physiologically wide overlapping of male and female sterile regions. The model also predicts that male or female plants would be induced to produce inflorescences of the other sex if the hormone level could be shifted from one side of the overlapping sterile regions to the other. This was verified by applying high levels of GA to a normal female genotype that resulted in the production of male inflorescences. However, this is the only normal female that has responded to GA application by producing male inflorescences, and males lose vigor and/or die without producing female inflorescences at high levels of PAC. The model suggests that the constancy of these males and females is due to the relative location of the sensitivity levels in relation to each other and to the hormone range. We conclude that the one-hormone model can facilitate both applied and basic research.     

Environmental conditions affect sex expression in monoecious, but not in male and female plants of Urtica dioica

Urtica dioica is a sub-dioecious plant species, i.e. males and females coexist with monoecious individuals. Under standard conditions, seed sex ratio (SSR, fraction of males) was found to vary significantly among seed samples collected from female plants originating from the same population (0.05–0.76). As a first step, we investigated the extent to which SSR and sex expression of male, female, and monoecious individuals is influenced by external factors. We performed experiments to analyse: (1) whether the environment of a parental plant affects the sex ratio (SR) of its offspring, (2) whether SSR can be affected by environmental conditions before flowering, and (3) whether sex expression of male, female and monoecious plants that have already flowered can be modified by environmental conditions or by application of phyto-hormones. Within the range of our experimental design, SSR was not influenced by external factors, and gender in male and female plants was stable. However, sex expression in monoecious plants was found to be labile: flower sex ratio (FSR, fraction of male flowers) differed considerably between clones from the same individual within treatments, and increased toward 100% maleness under benign conditions. These results provide strong evidence that monoecious individuals are inconstant males, which alter FSR according to environmental circumstances. In contrast, we consider sex expression in male and female individuals to be solely genetically based. The observed variation in SSR between maternal parents cannot be explained by sex-by-environment interactions.     

Complex sex determination in the stinging nettle Urtica dioica

The dioecious species Urtica dioica harbours wide variation in sex ratio of seeds. We conducted a series of crosses to analyse the genetic basis of sex determination in this species. Dutch populations of U. dioica contain low proportions of monoecious individuals beside male and female plants. Self-pollination of monoecious plants always yielded female, male and monoecious plants, generally in a ratio of one female to three male/monoecious individuals. This motivated us to write down a simple model in which gender is determined by one major sex-determination locus with four alleles. In the model males and monoecious plants have distinct genotypes but are both heterozygous at the sex-determination locus. We first made crosses among progeny obtained after self-pollination of monoecious plants. These crosses showed that the monoecious trait generally showed Mendelian inheritance and was passed on to the next generation via both pollen and seeds. Further crosses between monoecious plants and plants from dioecious system indicated that alleles from the dioecious system are often dominant. However, many exceptions to our genetic model are observed which suggest that dominance is incomplete and/or that more genes are involved in sex determination. We discuss to what extent sex determination genes explain the strongly biased seed sex ratios and argue that additional genes, for instance genes for female choice, must also be involved.     

The role of hybridization in the evolution of sexual system diversity in a clonal,aquatic plant

The stable coexistence within populations of females, males, and hermaphrodites (subdioecy) is enigmatic because theoretical models indicate that maintenance of this sexual system involves highly restricted conditions. Subdioecy is more commonly interpreted as a transitory stage along the gynodioecious pathway from hermaphroditism to dioecy. The widespread, North American, aquatic plant Sagittaria latifolia is largely composed of monoecious or dioecious populations; however, subdioecious populations with high frequencies of hermaphrodites (mean frequency = 0.50) characterize the northern range boundary of dioecy in eastern North America. We investigated two hypotheses for the origin of subdioecy in this region. Using polymorphic microsatellite loci, we evaluated whether subdioecy arises through selection on standing genetic variation for male sex inconstancy in dioecious populations, or results from hybridization between monoecious and dioecious populations. We found evidence for both pathways to subdioecy, although hybridization was the more common mechanism, with genetic evidence of admixture in nine of 14 subdioecious populations examined. Hybridization has also played a role in the origin of androdioecious populations in S. latifolia, a mechanism not often considered in the evolution of this rare sexual system. Our study demonstrates how hybridization has the potential to play a role in the diversification of plant sexual systems.     

The transmission of genes via pollen and ovules in gynodioecious angiosperms

Gynodioecy is defined as the class of sexual dimorphism in which females are constant (functionally unisexual) and males are inconstant (bisexual). Quantitative measures describe the sexual performance of males in gynodioecious populations. The relative (male/female) ovule contribution compares the ovule contributions of the sexes to the next generation. Male constancy and inconstancy, the proportions of male genes transmitted via pollen and ovules, are direct measure of maleness and femaleness. Equations permitting the values of the sex parameters to be estimated are derived. The values obtained for twelve species show that gynodioecy is not a single breeding system, but merges into hermaphroditism and dioecy at opposite extremes. The distribution of inconstancies among individual males is described for six species. In most species, seed counts overestimate the ovule contributions of males.     

Clonal genetic structure and diversity in populations of an aquatic plant with combined vs. separate sexes

Clonality is often implicated in models of the evolution of dioecy, but few studies have explicitly compared clonal structure between plant sexual systems, or between the sexes in dioecious populations. Here, we exploit the occurrence of monoecy and dioecy in clonal Sagittaria latifola (Alismataceae) to evaluate two main hypotheses: (i) clone sizes are smaller in monoecious than dioecious populations, because of constraints imposed on clone size by costs associated with geitonogamy; (ii) in dioecious populations, male clones are larger and flower more often than female clones because of sex‐differential reproductive costs. Differences in clone size and flowering could result in discordance between ramet‐ and genet‐based sex ratios. We used spatially explicit sampling to address these hypotheses in 10 monoecious and 11 dioecious populations of S. latifolia at the northern range limit in Eastern North America. In contrast to our predictions, monoecious clones were significantly larger than dioecious clones, probably due to their higher rates of vegetative growth and corm production, and in dioecious populations, there was no difference in clone size between females and males; ramet‐ and genet‐based sex ratios were therefore highly correlated. Genotypic diversity declined with latitude for both sexual systems, but monoecious populations exhibited lower genotypic richness. Differences in life history between the sexual systems of S. latifolia appear to be the most important determinants of clonal structure and diversity.     

SECONDARY SEX CHARACTERISTICS IN SPINACIA OLERACEA L.: QUANTITATIVE EVIDENCE FOR THE EXISTENCE OF AT LEAST THREE SEXUAL MORPHS

Spinacia oleracea L. was grown hydroponically across an experimental salinity gradient with treatments of 0.0, 1.5, 3.0, 4.5, and 6.0 grams of NaCl/liter of nutrient medium. Plant height, root biomass, vegetative biomass, fruit morphology, average fruit weight, and fruit mass: volume ratio were examined in each treatment to determine if differences other than primary sex characteristics exist between male, female, and monoecious plants. Average fruit weight was the only parameter examined that was not useful as a secondary sex characteristic. Orthogonal contrasts examining plant height, vegetative biomass, and root biomass of plants were distinct from one another across the salinity gradient. Similarly, female and monoecious plants differed significantly from one another in terms of root biomass and the average mass: volume ratio of the fruit each sexual morph produced, as well as the proportion of smooth to wrinkled fruit produced. The data indicate that male, female, and monoecious plants are three distinct sexual morphs.     

Impact of monoecy in the genetic structure of a predominately dioecious conifer species, Araucaria angustifolia (Bert.) O. Kuntze

Understanding the mating system of a tree species is important in genetic conservation and tree breeding strategies because it affects the inbreeding and genetic diversity of the descendant populations. Araucaria angustifolia (Bert.) O. Kuntze is a mainly dioecious species that reproduces through outcrossing. However, some monoecious trees have been identified and they may reproduce through self-fertilization. The objective of this study was to confirm the expected relatedness of full-sibs in outcrossed hand-pollinated progenies of female seed trees, self-sibs of hand self-pollinated monoecious seed trees, and to investigate the mating system of open-pollinated progenies of female and monoecious A. angustifolia trees. To do this, eight microsatellite loci were used to genotype hand- and open-pollinated progenies. Our results show that the relatedness of outcrossed hand-pollinated progenies are true full-sibs and progenies from a selfed monoecious seed tree are self-sibs, which confirms the hand-pollination method used. Open-pollinated female seed trees reproduced only by outcrossing, generating progenies with a mixture of full- and half-sibs. Monoecious seed trees reproduced mainly by xenogamy, generating progenies with mixtures of self-sibs, full-sibs, half-sibs and self-half-sibs. We also found that an increase in the effective number of pollen donors ( $ N_{\text{ep}} $ ) would lead to an increase in the total number of alleles ( $ K $ ) within progenies. Our results also suggest that monecious trees have limited potential to modify the genetic structure through selfed seed production due to the very low estimated selfing rate in these trees and the rare occurrence of these trees in natural populations.     

GENETICS OF SEX RATIO VARIATION AMONG NATURAL POPULATIONS OF A DIOECIOUS PLANT

The female-biased sex ratio in the genus Silene is the most widely documented example of sex ratio bias in plants. It has recently been shown that the genetic basis of sex ratio in this species involves a system of sex ratio distorters and restorers, but it is not known if these genetic elements are of fundamental importance in natural populations. I crossed plants from natural populations with known testers to examine the frequencies of sex ratio distorters and restorers in eight populations of S. alba, and to determine the extent that these elements are responsible for the sex ratio variation in nature. The genetic basis of sex ratio varied from one population to another, suggesting that sex ratio evolution occurs over a relatively small spatial scale and is asynchronous among local demes. The resulting variation in the frequency of sex ratio alleles among populations explained most of the sex ratio variation seen in nature, which suggests that the ecology and evolution of this trait in natural populations is governed primarily by underlying dynamics of selfish genetic elements. The possible causes and the evolutionary consequences of population structure at genes controlling sex ratio are discussed, as are the implications of these results for the overall importance of selfish genetic elements in natural populations.     

GEOGRAPHIC VARIATION IN REPRODUCTIVE BEHAVIOR AND SIZE STRUCTURE OF THE AUSTRALIAN CYCAD MACROZAMIA COMMUNIS (ZAMIACEAE)

Macrozamia communis is a common cycad that occurs in eastern New South Wales, Australia, where it forms an understory in various forest or woodland communities. Leaf number of reproductive individuals as well as of nonreproductive ones, taken as an indication of size class, varies clinally from north to south, with reproductive individuals in southern populations having mean leaf numbers about 12 times greater than those at the northern end of the range of this species. Female cone size and mature ovule weight appear to vary also along this gradient. Using leaf number as a predictor of sexual maturity, from 37 to 100% of the individuals present in a population were judged to be mature. The percent of mature individuals participating in the 1987 coning episode ranged from none in three southern populations to 68% elsewhere. In 9 of 10 samples more males than females participated in the 1987 coning episode, with a collective sex ratio slightly male biased for all populations sampled. In three population samples average cone numbers for male and female plants were equal. In two samples males produced more cones per plant than females. Leaf numbers of males and females in individual populations were equal. Few individuals producing cones in 1987 showed evidence of recent prior cone production, suggesting that successive cone production by both sexes is separated by an interval of several years. The periodicity with which fire is used as a fire suppression method may affect the frequency of cone production in some populations, perhaps preventing it entirely. The apparent absence of recruitment in some populations may result from long-term seed collection by humans for horticultural purposes as well as the very poor seed dispersal that appears to characterize this cycad throughout its range. The weevil Tranes internatus was observed occupying the internal tissues of female cones; this insect may be involved in the pollination of M. communis.     

Germinable soil seed banks in abandoned grasslands in central and western Norway and their significance for restoration

Questions: Could the seed bank increase biodiversity during restoration of abandoned, species‐poor, formerly cultivated vegetation? Is it possible to identify how climate, soil and former and present management and vegetation affected the seed bank? Location: The study sites were eight abandoned grasslands, four in Orkdal, central Norway and four in Gaular, western Norway. Methods: 144 seed bank samples were collected from three depths. Each sample was sown and placed in a greenhouse. After three months, the trays were dried and stored at 4°C in a dry place for two months. This was repeated twice. Results: There was a separation of the two regions along the first DCA axis in both the seed bank and in the vegetation analysis and also a clear separation of the seed bank from the vegetation along the second axis. These results are caused by differences in former management as well as temperature, precipitation and soil type between Gaular and Orkdal. We found more annuals, short‐lived species and species demanding light open conditions in the seed bank than in the vegetation probably because these species have the capacity for producing persistent seeds. Most of the species found only in the seed bank were found in very few samples and with few individuals. Conclusion: These results suggest that it may be difficult to increase vegetation biodiversity through restoration of grasslands such as those investigated if the natural soil seed bank is the main seed source.     

Gender variation and the evolution of dioecy in Wurmbea dioica (Liliaceae)

A major obstacle for empirical tests of hypotheses concerning the evolution of dioecy in flowering plants is the limited number of species that possess both cosexual and dioecious populations. Wurmbea dioica (Liliaceae) is a diminutive, fly-pollinated geophyte native to temperate Australia. Marked geographical variation of floral traits is evident, particularly with respect to sex expression. A survey of phenotypic gender in 45 populations from Western Australia (WA), South Australia (SA), Victoria (Vic) and the Australian Capital Territory (ACT) revealed two contrasting patterns. Populations in SA, Vic, and ACT were uniformly dimorphic for gender, containing female and male plants, whereas populations in WA were either monomorphic or dimorphic. In most dimorphic populations varying numbers of male plants produced hermaphrodite flowers (male inconstancy). There was a significant negative relationship between female frequency and the proportion of inconstant male plants. Depending on region and population, male plants produced more flowers of larger size than females. In WA monomorphic populations often occurred on rich, moist soils at high density, whereas dimorphic populations were more commonly found at lower density on shallow soils in drier areas. In an area of sympatry, plants with contrasting sexual systems flowered at different times and were ecologically differentiated. The patterns of gender variation in W. dioica indicate that dioecy has evolved via the gynodioecious pathway. The spread of females in monomorphic populations may be favoured where ecological conditions result in increased selfing and inbreeding depression in hermaphrodites.     

Sex expression,female frequency,and reproductive output in a gynodioecious clonal herb, <Emphasis Type="Italic">Glechoma longituba</Emphasis> (Lamiaceae)

We investigated the sex morphs and frequency of females in 24 natural populations of the clonal, gynodioecious species Glechoma longituba. In addition to the presence of hermaphrodites and females, there were some individuals with intermediate sex expression that could not be categorized as either morph. The majority of populations contained a predominance of hermaphrodites, although female frequency varied from 0 to 100%. The sexual expression of individual ramets did not change over three consecutive years or when ramets were transplanted to different populations, but it did change over the course of a flowering season. Seeds from females weighed more than those from hermaphrodites. The seed set of hermaphrodites was relatively constant across populations; the seed set of females was higher than that of hermaphrodites in most populations, but lower in populations in which females occurred at a very high frequency because of pollen limitation. This led to a negative correlation being detected between the seed set of females and the frequency of females in a population. We postulated that the adjustment of sex expression within seasons could be an adaptive mechanism of reproductive assurance in this system.     

GENDER VARIATION AND SEXUAL DIFFERENCES IN REPRODUCTIVE CHARACTERS AND SEED PRODUCTION IN GYNODIOECIOUS GERANIUM MACULATUM

Variation in sex expression, flowering pattern, and seed production was studied in the self-compatible perennial herb Geranium maculatum in Illinois and Indiana. In a survey of eight populations, female (male-sterile) plants were found in seven (frequencies ranging from 0.5% to 24.3% [median 4.2%]), and intermediate plants (with partly reduced male function) were found in all populations. Gender variation and sexual differences in reproductive characters were studied in detail in two populations. One population consisted of 5% female, 27% intermediate, and 68% hermaphrodite plants; the other consisted of 1% female, 20% intermediate, and 79% hermaphrodite plants. Females produced smaller flowers and began flowering earlier than hermaphrodites. Intermediates produced flowers of an intermediate size and began flowering as early as females. Females and hermaphrodites did not differ in flower number, vegetative size, flowering frequency, survival, or seed size. However, females produced 1.6 times more seeds than hermaphrodites. Intermediates produced 1.3–1.6 times more seeds than hermaphrodites. Some between-year variation in sex expression was observed. Hand-pollination with outcross pollen produced two to four times as many seeds as hand-pollination with self-pollen. A lower outcrossing rate in hermaphrodites than in females may at least partly explain the lower seed set in hermaphrodites. The higher seed production of females, and possibly the high fecundity of the intermediates, should contribute to the maintenance of this sexual polymorphism.     

LIFE-HISTORY STRATEGIES AND SEX RATIOS FOR A CULTIVAR AND A WILD POPULATION OF BUCHLOE DACTYLOIDES (GRAMINEAE)

Buchloe dactyloides (Nutt.) Engelm. is a perennial, sod-forming shortgrass of the grasslands of central North America from Montana to northern Mexico. The objectives of this research were to compare a cultivar (Sharps Improved) and a wild population from the Oklahoma panhandle, both in the field and in the greenhouse over several growing seasons, in regard to life-history strategies, sex ratios, and constancy of sex expression. Sex ratios of offspring from individual burs (usually 1–5 seeds per bur) and for all surviving plants germinated from seeds in the greenhouse were markedly different for the two populations. The wild population exhibited a 1: 1 male/female ratio with no monoecious plants and complete stability in sex expression over 3 yr of study in the greenhouse. In contrast, 13% of the cultivar plants were monoecious, and its unisexual plants showed a female bias. Burs of the two populations, in flats exposed to three separated month-long periods of favorable moisture with intervening “drought” periods, showed differential germinability of seeds within burs. The cultivar had less dormancy and produced a greater number of seedlings in the first and second periods; wild burs significantly exceeded cultivar burs in the number of seedlings during the third period and in the number of burs giving rise to seedlings in all three periods. Field and greenhouse measurements of vegetative (VRE) and sexual reproductive effort (SRE) per plant indicated that SRE was minimal (ca. 2% of aboveground biomass) in relation to asexual ramet propagation by rapidly growing stolons (VRE = ca. 80%). Mean SRE and VRE were not only similar for the populations, but also for the males and females within a population, although the values for individual plants were highly variable. It is concluded that Buchloe dactyloides is largely dioecious, with at least some wild populations showing a 1: 1 male/female ratio and constancy of sex expression, and that the bur as a dispersal unit containing 1–5 seeds is particularly appropriate for the breeding system and habitat ecology of Buchloe dactyloides.     

The genetics of sex determination in stinging nettle (<Emphasis Type="Italic">Urtica dioica</Emphasis>)

Urtica dioica (“stinging nettle”) includes both dioecious and monoecious forms. In most sexually dimorphic angiosperm species, the genetic mechanisms of sex determination are completely unknown. The few species that include both monoecious and dioecious forms provide an unusual opportunity to examine the genetic mechanisms that underlie the separation of sexual functions, through crossing experiments and analysis of progeny segregation. Our focus is on the genetic mechanisms distinguishing monoecious and dioecious forms of U. dioica. A complicated picture of sex determination in this species has resulted from crosses between dioecious and monoecious subspecies, as well as between dioecious and monoecious forms of the same subspecies. Most significant is evidence for a maternal influence on sex determination and for the possibility of gynodioecy as an intermediate stage in the evolutionary pathway to dioecy. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

EFFECTS OF PARENTAGE AND SIZE OF THE POLLEN LOAD ON PROGENY PERFORMANCE IN CAMPANULA AMERICANA

To examine the effects of maternal and paternal parentage and the size of the pollen load on seed size and weight and on progeny performance we conducted a controlled crossing experiment using a natural population of Campanula americana. We found that seed size was positively correlated with early seedling performance for all but one of traits we measured (days to emergence), but was not significantly correlated with any of the later vegetative measures or reproductive output. We detected significant effects due to the maternal parent for the vegetative traits days to emergence, days to first leaf, and final plant height, as well as total seed weight, and mean seed weight per fruit. Significant paternal effects were found for all of the seedling traits except number of leaves after vernalization. The progeny from fruits receiving high pollen loads significantly outperformed the progeny from fruits receiving low pollen loads for the traits days to first and second leaf, numbers of leaves after vernalization, and days to first flower. These results not only demonstrate the importance of parentage and seed weight on progeny performance, but also indicate that variations in the size of the pollen load may be important in seedling establishment in natural populations.     

On the evolution of anisogamy from isogamous monoecy and on the origin of sex

A new hypothesis is established for the evolution of anisogamy in isogamous monoecious haploid unicells. Contrary to present concepts, it is assumed that in such isogamous ancestors the genetically identical gametes are bipolarly different as (+) and (?). The gametic differentiation of a monoecious taxon may be perceived as a phenocopy of that of a related dioecious form. The two gametic phenotypes result from alternative pathways of sexual differentiation. The alternative must be controlled by a sensible switch mechanism that responds to minor differences between vegetative cells when gametogenesis is triggered. Cell-size dependent factors are assumed to be able to influence the switch mechanism causing bigger cells to be of one sex, smaller cells of the other. Fertilization then occurs selectively between big and small gametes because of their sexual difference. Size-different gametes within one species may arise from different types of gametogenesis depending on how many gametes one vegetative cell produces. These assumptions, i.e. the bipolarity of isogametes, in monoecious taxa, size-dependent sex determination, and different types of gametogenesis within one taxon, are justifiable since they are realized independently from each other in various species. It is postulated that mutational fixation of the size-dependent sex determination in such monoecious species creates cell lineages producing sex-different macro- and microgametes and establishes constitutional anisogamous dioecy. The proposed hypothesis clearly separates the evolution of anisogamy as a problem of sexual reproduction from the evolution of sex per se, sex being defined as a bipolarity effecting fertilization.     

The effects of three years of fencing enclosure on soil seed banks and the relationship with above-ground vegetation of degraded alpine grasslands of the Tibetan plateau

Background and Aims

Fenced enclosures have become an important method for re-establishing degraded grassland on the Tibetan plateau, and examination of soil seed banks may provide useful insights to understanding the effects and mechanisms of fencing enclosure on the restoration.

Methods

An investigation was conducted into the effects of enclosure for 3 years on the soil seed banks of degraded natural and sown grasslands at eight study sites. Species composition, soil seed bank density and the relationships with above-ground vegetation were analysed based on 4800 soil core samples and counting of seeds extracted from soil samples.

Results

After 3 years of fencing enclosure, soil seed banks differed between the different communities across the study sites. Species numbers and seed density in soil seed banks decreased from natural grassland to sown grassland, with most seeds occurring in the upper 5 cm soil layer. In these alpine grasslands, relatively few species produced high numbers of seeds, although their occurrence across the eight study sites was variable. Total vegetation cover increased with enclosure due to the colonization capacity of the vegetation rather than soil seed banks.

Conclusions

This study provided evidence that soil seed banks do not play an important role in the restoration of degraded alpine grassland when using fencing enclosures. Further studies conducted over longer periods are needed to address this subject.