Phylogenetic species delimitation in ectomycorrhizal fungi and implications for barcoding: the case of the Tricholoma scalpturatum complex (Basidiomycota)

Population studies have revealed that the fungal ectomycorrhizal morphospecies Tricholoma scalpturatum consists of at least two genetically distinct groups that occur sympatrically in several geographical areas. This discovery prompted us to examine species boundaries and relationships between members formerly assigned to T. scalpturatum and allied taxa using phylogenetic analyses. Sequence data were obtained from three nuclear DNA regions [internal transcribed spacer (ITS), gpd and tef], from 101 carpophores collected over a large geographical range in Western Europe, and some reference sequences from public databases. The ITS was also tested for its applicability as DNA barcode for species delimitation. Four highly supported phylogenetic clades were detected. The two previously detected genetic groups of T. scalpturatum were assigned to the phylospecies Tricholoma argyraceum and T. scalpturatum. The two remaining clades were referred to as Tricholoma cingulatum and Tricholoma inocybeoides. Unexpectedly, T. cingulatum showed an accelerated rate of evolution that we attributed to narrow host specialization. This study also reveals recombinant ITS sequences in T. inocybeoides, suggesting a hybrid origin. The ITS was a useful tool for the determination of species boundaries: the mean value of intraspecific genetic distances in the entire ITS region (including 5.8S rDNA) was <0.2%, whereas interspecific divergence estimates ranged from 1.78% to 4.22%. Apart from giving insights into the evolution of the T. scalpturatum complex, this study contributes to the establishment of a library of taxonomically verified voucher specimens, an a posteriori correlation between phenotype and genotype, and DNA barcoding of ectomycorrhizal fungi.     

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules

The ectomycorrhizal fungus Tricholoma populinum is host-specific with Populus species. T. populinum has wind-dispersed progagules and may be capable of long-distance dispersal. In this study, we tested the hypothesis of a panmictic population between Scandinavia and North America. DNA sequences from five nuclear loci were used to assess phylogeographic structure and nucleotide divergence between continents. Tricholoma populinum was composed of Scandinavian and North American lineages with complete absence of shared haplotypes and only one shared nucleotide mutation. Divergence of these lineages was estimated at approx. 1.7-1.0 million yr ago (Ma), which occurred after the estimated divergence of host species Populus tremula and Populus balsamifera/Populus trichocarpa at 5 Ma. Phylogeographic structure was not observed within Scandinavian or North American lineages of T. populinum. Intercontinental divergence appears to have resulted from either allopatric isolation; a recent, rare long-distance dispersal founding event followed by genetic drift; or the response in an obligate mycorrhizal fungus with a narrow host range to contractions and expansion of host distribution during glacial and interglacial episodes within continents. Understanding present genetic variation in populations is important for predicting how obligate symbiotic fungi will adapt to present and future changing climatic conditions.     

High sexual reproduction and limited contemporary dispersal in the ectomycorrhizal fungus Tricholoma scalpturatum: new insights from population genetics and spatial autocorrelation analysis

Dispersal and establishment are fundamental processes influencing the response of species to environmental changes, and the long-term persistence of populations. A previous study on the symbiotic ectomycorrhizal fungus Tricholoma scalpturatum revealed strong genetic differentiations between populations in Western Europe, suggesting restricted dispersal for this wind-dispersed cosmopolitan fungus. Two distinct genetic groups (genetic groups 1 and 2), co-occurring in some locations, were also identified and could correspond to cryptic species. In the present work, we examine the reproductive strategy and dispersal biology of the two T. scalpturatum's genetic groups. Variable molecular markers (intersimple sequence repeats and intergenic spacer 2-restriction fragment length polymorphisms) and spatial autocorrelation analyses were used to examine fine-scale patterns (< 140 m) of genetic structure, in an effort to determine the physical scale at which genetic structure exists. A total of 473 fruit bodies were mapped and collected over 3 years from two plots located in the south of France, including 219 and 254 samples from group 1 and group 2, respectively. High genetic diversity and the presence of numerous small genets were observed in both groups. Autocorrelation analyses revealed significant positive spatial genetic structures of genets at close distances (up to few metres for both groups). Mantel tests confirmed this isolation-by-distance pattern. These results clearly demonstrate high sexual reproduction and spatial structuring of genets at very small geographical scales in this wind-dispersed ectomycorrhizal fungal species, a pattern consistent with restricted contemporary dispersal of spores.     

Population Evidence of Cryptic Species and Geographical Structure in the Cosmopolitan Ectomycorrhizal Fungus, Tricholoma scalpturatum

Tricholoma scalpturatum is an ectomycorrhizal fungus that forms symbioses with roots of diverse trees and shrubs. It is commonly encountered in a wide range of habitats, across temperate ecosystems. A previous study has revealed a high genetic diversity at a local scale, and ruderal abilities. To examine genetic structure at a large geographical scale, a total of 164 basidiocarps were collected from 30 populations located in Western Europe, from Spain to Scandinavia. These samples were analyzed by three molecular methods with different levels of resolution: inter-simple sequence repeats (ISSRs), restriction fragment length polymorphisms (RFLPs) in the rDNA internal transcribed spacer (ITS), and ITS sequence analysis. Considerable genetic variation was found, and the morphospecies was separated into two genetic groups that were distinct from each other. The ISSR data and the relatively low percentage value (96%) of shared sequence polymorphisms in the ITS between isolates from the two groups, strongly suggest cryptic species and long-lasting separation. No geographical exclusion was detected for these two widely distributed taxa. However, high estimates of population differentiation were observed in each group, including between populations less than a few kilometers apart. This result provides evidence for limited gene flow and/or founding effects. It also indicates that T. scalpturatum does not constitute a random mating population, and the hypothesis of endemism cannot be excluded for this cosmopolitan wind-dispersed fungus.     

Intraspecific competition and light effect on reproduction of Ligularia virgaurea,an invasive native alpine grassland clonal herb

The relationship between sexual reproduction and clonal growth in clonal plants often shows up at the ramet level. However, only a few studies focus on the relationship at the genet level, which could finally account for evolution. The sexual reproduction and clonal growth of Ligularia virgaurea, a perennial herb widely distributed in the alpine grasslands of the Qinghai‐Tibetan Plateau of China, were studied under different competition intensities and light conditions at the genet level through a potted experiment. The results showed that: (1) sexual reproduction did not depend on density or light, and increasing clonal growth with decreasing density and increasing light intensity indicated that intraspecific competition and light intensity may affect the clonal life history of L. virgaurea; (2) both sexual reproduction and clonal growth show a positive linear relationship with genet size under different densities and light conditions; (3) a threshold size is required for sexual reproduction and no evidence of a threshold size for clonal growth under different densities and light conditions; (4) light level affected the allocation of total biomass to clonal and sexual structures, with less allocation to clonal structures and more allocation to sexual structures in full sunlight than in shade; (5) light determined the onset of sexual reproduction, and the genets in the shade required a smaller threshold size for sexual reproduction to occur than the plants in full sunlight; and (6) no evidence was found of trade‐offs between clonal growth and sexual reproduction under different densities and light conditions at the genet level, and the positive correlation between two reproductive modes indicated that these are two integrated processes. Clonal growth in this species may be viewed as a growth strategy that tends to maximize genet fitness.     

大白口蘑分离菌株的DNA鉴定

以松口蘑Tricholoma matsutake子实体为外类群,对大白口蘑T.giganteum野生子实体及其组织分离菌丝进行ITS序列测序,通过DNAStar软件进行比较分析。结果表明大白口蘑ITS序列长度为589bp,松口蘑ITS序列长度为601bp,ITS1和ITS2呈现不同程度的种间多态性;ITS序列测定证实了大白口蘑野生子实体及其组织分离菌丝的同质性,并且ITS区序列在大白口蘑种内不同菌株间的变异程度很小,表明使用通用引物ITS4和ITS5,通过PCR扩增测序即可用于大白口蘑的种质鉴定。     

大白口蘑分离菌株的DNA鉴定

以松口蘑Tricholoma matsutake子实体为外类群,对大白口蘑T. giganteum 野生子实体及其组织分离菌丝进行ITS序列测序,通过DNAStar软件进行比较分析。结果表明大白口蘑ITS序列长度为589bp,松口蘑ITS序列长度为601bp,ITS1和ITS2呈现不同程度的种间多态性;ITS序列测定证实了大白口蘑野生子实体及其组织分离菌丝的同质性,并且ITS区序列在大白口蘑种内不同菌株间的变异程度很小,表明使用通用引物ITS4和ITS5,通过PCR扩增测序即可用于大白口蘑的种质鉴定。     

Re-evaluation of the phylogenetic relationship among species of the genus Tricholoma

The internal transcribed spacer sequences spanning the regions between the 17S and 25S rRNAs (ITS1 and ITS2) and including the complete sequence of the 5.8S rRNA were used for phylogenetic analyses. This approach to define phylogenetic relationships within the genus Tricholoma was tested using different isolates of T. terreum. Fruitbodies identified in nature were analysed in order to allow use of morphology for taxonomy. The isolates from different locations were closely related as could be expected for one species. Thus, the method could be applied to different Tricholoma species. Three clusters within the genus Tricholoma can be distinguished with four additional species not included in any of these clusters. Molecular analyses of two Cortinarius species confirm a phylogenetically distinct genus.     

中国口蘑属种类名录(英文)

通过系统的文献收集 ,汇总了口蘑属 (Tricholoma)在中国的 91个名称记录 ,并对其研究现状和分布进行了简述。文献调查结果表明 ,口蘑属种类广泛分布于中国的 2 7个省区 ,其中以黑龙江、吉林、辽宁、内蒙古、贵州、四川、云南、西藏 ,青海、甘肃、河北、山西和陕西等地的报道较多 ,其他地区如台湾、香港、安徽、福建、广东、广西、海南、河南、湖南、湖北、江苏、山东、新疆 ,浙江也有一些报道。根据《国际植物命名法规》和《真菌、地衣汉语学名命名法规》 ,作者订正了中文文献中的一些拉丁和汉语学名 ,同时对形态描述和鉴定等方面的问题也进行了简要的讨论。在已报道的口蘑属名称记录中 ,能够得到承认的本属名称有 43个种和 3个变种 ,属内其他种类名称的异名有 8个 ,而已经转移到其他属的有 3 0个 ,在命名法上模糊不清名称有 3个 ,错拼名称有 2个 ,另有未定名种 2个。目前能够承认的名称是酸涩口蘑 (T .acerbum)、白棕口蘑 (T .albobrunneum)、白口蘑(T .album)、银盖口蘑 (T .argyraceum)、黑鳞口蘑 (T .atrosquamosum)、橙柄口蘑 (T.aurantiipes)、金黄褶口蘑 (T .auratum)、傻松口蘑 (T .bakamatsutake)、欧洲口蘑 (T .caligatum)、灰环口蘑 (T.cingulatum)、银白口蘑 (T .colum betta)、油口蘑 (T .     

Comparison of clonal diversity in mountain and Piedmont populations of Trillium cuneatum (Melanthiaceae-Trilliaceae), a forest understory species

The balance between clonal and sexual reproduction can vary widely among plant populations, and the extent of clonality may be influenced by the combined effects of historical land use and variation in environmental conditions. We investigated patterns of clonal spread in five Trillium cuneatum populations, two in the Appalachian Mountains characterized by mesic, cooler conditions, and three at lower elevations experiencing warmer, drier conditions and greater disturbance. Using a new measure of the genet effective number and innovative orthogonal contrast methods, we quantified genet structure, contrasting clonal growth in the mountains with that in the Piedmont. Asexual propagation was more common in the Piedmont, where 25% of the sampled ramets were clonally derived, but was much less frequent in the mountains (7% clonal replicates). Hierarchical partitioning of variation in genet diversity showed that the majority (75.8%) of the variation resulted from more vegetative replication in the Piedmont. Most of the remaining variation (21.6%) was attributable to differences between urban and rural Piedmont populations, and a small, statistically nonsignificant fraction of the variation (2.6%) was due to interpopulation differences within the mountains. Higher frequency of cloning may enhance both genetic and demographic population viability in fragmented Piedmont habitats.     

Fine‐scale genetic structure and flowering output of the seagrass Enhalus acoroides undergoing disturbance

Seagrass are under great stress in the tropical coast of Asia, where Enhalus acoroides is frequently the dominant species with a large food web. Here, we investigate the question of the fine‐scale genetic structure of this ecologically important foundation species, subject to severe anthropogenic disturbance in China. The genetic structure will illuminate potential mechanisms for population dynamics and sustainability, which are critical for preservation of biodiversity and for decision‐making in management and restoration. We evaluated the fine‐scale spatial genetic structure (SGS) and flowering output of E. acoroides, and indirectly estimated the relative importance of sexual versus asexual reproduction for population persistence using spatial autocorrelation analysis. Results reveal high clonal diversity for this species, as predicted from its high sexual reproduction output. The stronger Sp statistic at the ramet‐level compared with genet‐level indicates that clonality increases the SGS pattern for E. acoroides. Significant SGS at the genet‐level may be explained by the aggregated dispersal of seed/pollen cohorts. The estimated gene dispersal variance suggests that dispersal mediated by sexual reproduction is more important than clonal growth in this study area. The ongoing anthropogenic disturbance will negatively affect the mating pattern and the SGS patterns in the future due to massive death of shoots, and less frequency of sexual reproduction.     

Clonal structure of Elytrigia atherica along different successional stages of a salt marsh

Elytrigia atherica is a tall clonal grass species typical of higher salt marshes, but is gradually invading to the lower marshes. At young successional stages of a salt marsh, E. atherica is found sparsely dispersed in small groups of ramets. These patches increase in size and ramet density over time, eventually forming extensive swards as succession proceeds. This study investigates the change in the clonal diversity of E. atherica stands during colonization as a result of its reproductive strategy. Clonal diversities of differently sized patches of E. atherica were investigated on two lower salt-marsh sites of different age, 25 years and 35 years, respectively. Microsatellite fingerprint patterns were used to determine genet identities and to estimate relatedness and genetic differentiation between the sites, between patches within sites and within patches. The majority of the patches on both sites contained more than one genet. On the older site, the clonal diversity was higher than on the younger site. However, the clonal diversity tended to decrease with increasing patch size. Low genetic differentiation was found between the two sites, indicating habitat differentiation, whereas differentiation between patches within sites was high. It is reasoned that different environmental conditions could have resulted in different clonal structures: On an older marsh, the increase of successful seedling recruitment, due to more suitable environmental conditions, leads to an increase in clonal diversity. Over time, with increasing ramet density, intraspecific competition is likely to increase, resulting in a decrease of clonal diversity.     

Sexual reproduction and clonal growth in Reinhardtia gracilis (Palmae), an understory tropical palm

Patterns of sexual reproduction and clonal growth were investigated in the understory palm Reinhardtia gracilis var. gracilior over a 3-yr period. R. gracilis is a very abundant clonal palm in the tropical rain forest of Los Tuxtlas, Veracruz, México. Because ramets form clumps, genets are easily identified in the field. Genets were monitored in a 0.5-ha area, and classified by size according to the number of ramets they possessed. In contrast to clonal growth, sexual reproduction was highly dependent on genet size. The probability of reproduction, the number of inflorescences, and the number of fruits produced were positively correlated with genet size. However, neither the probability of producing a ramet, nor the number of ramets produced per genet were correlated with genet size. Over the 3 yr of study, 55% of the genet population had at least one ramet with reproductive structures, while <1% (a single genet in one year) had six ramets with flowers. Thirty-two percent of the mature genets reproduced during each of three consecutive years. In contrast, 58% of the genets produced no new ramets during these 3 yr. No evidence was found of a trade-off between clonal growth and sexual reproduction. Ramet production increases genet size and this in turn increases genet reproductive performance. Clonal growth in this species may be viewed as a growth strategy that tends to maximize genet fitness.     

Development of microsatellite markers from an ectomycorrhizal fungus, <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>, by an ISSR-suppression-PCR method

An inter-simple sequence repeat (ISSR)-suppression-PCR technique established to develop microsatellite markers of plant species was applied to an ectomycorrhizal fungus, Tricholoma matsutake. Six polymorphic SSR markers were developed. All six polymorphic SSR markers were single-locused and co-dominant. Alleles produced by these six single-locused markers ranged from two to nine per locus and the expected heterozygosities were calculated as values from 0.098 to 0.803. The results indicated that the ISSR-suppression-PCR technique was effective and applicable to the development of microsatellite markers from ectomycorrhizal fungi. Furthermore, the six microsatellite loci did not amplify DNA from any other ectomycorrhizal species investigated, except for Tricholoma nauseosum (Swedish matsutake) and Tricholoma fulvocastaneum, suggesting that population genetics and reproduction of T. matsutake could be investigated by the SSR markers developed in the present study.     

Local forest environment largely affects below-ground growth, clonal diversity and fine-scale spatial genetic structure in the temperate deciduous forest herb Paris quadrifolia

Paris quadrifolia (herb Paris) is a long-lived, clonal woodland herb that shows strong differences in local population size and shoot density along an environmental gradient of soil and light conditions. This environmentally based structuring may be mediated by differences in clonal growth and seedling recruitment through sexual reproduction. To study the interrelationship between environmental conditions and spatial patterns of clonal growth, the spatial genetic structure of four P. quadrifolia populations growing in strongly contrasting sites was determined. In the first place, plant excavations were performed in order to (i) determine differences in below-ground growth of genets, (ii) investigate connectedness of ramets and (iii) determine total genet size. Although no differences in internode length were found among sites, clones in moist sites were much smaller (genets usually consisted of 1-3 interconnected shoots, most of them flowering) than genets in dry sites, which consisted of up to 15 interconnected shoots, the majority of which were vegetative. Further, amplified fragment length polymorphism (AFLP) markers were used. Clonal diversity was higher in populations located in moist and productive ash-poplar forests compared to those found in drier and less productive mixed forest sites (G/N: 0.27 and 0.14 and Simpson's D: 0.84 and 0.75, respectively). Patterns of spatial population genetic structure under dry conditions revealed several large clones dominating the entire population, whereas in moist sites many small genets were observed. Nevertheless, strong spatial genetic structure of the genet population was observed. Our results clearly demonstrate that patterns of clonal diversity and growth form of P. quadrifolia differ among environments. Limited seedling recruitment and large clone sizes due to higher connectedness of ramets explain the low clonal diversity in dry sites. In moist sites, higher levels of clonal diversity and small clone sizes indicate repeated seedling recruitment, whereas strong spatial genetic structure suggests limited seed dispersal within populations.     

Genet age in marginal populations of two clonal Carex species in the Siberian Arctic

During a Swedish-Russian expedition to northern Siberia 1994, we sampled two marginal populations of two Carex species at two high arctic sites ( C, stans Drej. on Faddeyevsky Island and C. ensifolia V. Krecz ssp. arctisibirica Jurtz. at north-eastern Taymyr Peninsula), both north of previously documented localities in that areas for the two species. These populations were composed of a few distinct patches of ramet colonies, some of them shaped like fairy rings with dead centres. We measured the size of all colonies and collected samples for detailed morphometric analyses of rhizome growth. By using RAPD (random amplified polymorphic DNA) analysis we established that the largest colony at each site consisted of a single genet, based on 41 polymorphic bands amplified with three primers. Pooled samples from each of two additional colonies of C. stans on Faddeyevsky Island were analysed and showed that clones of the same species at the same site were relatively dissimilar (Dice's similarity index 0.26 0,43), We then assumed that each ramet colony represented a single genet. Based on the morphometric data, we developed a deterministic growth model that simulates the clonal growth of these species and enabled estimates of the time since establishment of the genets. The estimated age of the five C. Stans clones varied from 17 to 154 yr and the age of the two C. ensifolia ssp, arctisibirica clones was well over 3000 yr.     

Longevity of clonal plants: why it matters and how to measure it

Background

Species'' life-history and population dynamics are strongly shaped by the longevity of individuals, but life span is one of the least accessible demographic traits, particularly in clonal plants. Continuous vegetative reproduction of genets enables persistence despite low or no sexual reproduction, affecting genet turnover rates and population stability. Therefore, the longevity of clonal plants is of considerable biological interest, but remains relatively poorly known.

Scope

Here, we critically review the present knowledge on the longevity of clonal plants and discuss its importance for population persistence. Direct life-span measurements such as growth-ring analysis in woody plants are relatively easy to take, although, for many clonal plants, these methods are not adequate due to the variable growth pattern of ramets and difficult genet identification. Recently, indirect methods have been introduced in which genet size and annual shoot increments are used to estimate genet age. These methods, often based on molecular techniques, allow the investigation of genet size and age structure of whole populations, a crucial issue for understanding their viability and persistence. However, indirect estimates of clonal longevity are impeded because the process of ageing in clonal plants is still poorly understood and because their size and age are not always well correlated. Alternative estimators for genet life span such as somatic mutations have recently been suggested.

Conclusions

Empirical knowledge on the longevity of clonal species has increased considerably in the last few years. Maximum age estimates are an indicator of population persistence, but are not sufficient to evaluate turnover rates and the ability of long-lived clonal plants to enhance community stability and ecosystem resilience. In order to understand the dynamics of populations it will be necessary to measure genet size and age structure, not only life spans of single individuals, and to use such data for modelling of genet dynamics.     

Dynamics of distribution and performance of ramets constructing genets: a demographic�Cgenetic study in a clonal plant, Convallaria keiskei

Background and Aims

In clonal plants producing vegetative offspring, performance at the genet level as well as at the ramet level should be investigated in order to understand the entire picture of the population dynamics and the life history characteristics. In this study, demography, including reproduction and survival, the growth patterns and the spatial distributions of ramets within genets of the clonal herb Convallaria keiskei were explored.

Methods

Vegetative growth, flowering and survival of shoots whose genets were identified using microsatellite markers were monitored in four study plots for 3 years (2003–2005). The size structures of ramets in genets and their temporal shifts were then analysed. Their spatial distributions were also examined.

Key Results

During the census, 274 and 149 ramets were mapped in two 1 × 2 m plots, and 83 and 94 ramets in two 2 × 2 m quadrats. Thirty-eight genotypes were identified from 580 samples. Each plot included 5–18 genets, and most ramets belonged to the predominant genet(s) in each plot. Shoots foliated yearly for several years, but flowering ramets did not have an inflorescence the next year. A considerable number of new clonal offspring persistently appeared, forming a bell-shaped curve of the size structure of ramets in each genet. Comparing the structures modelled by the normal distributions suggested variation among ramets belonging to a single genet and variation among genets. Furthermore, spatial analyses revealed clumped and distant distributions of ramet pairs in a genet, in which the distant patterns corresponded to the linearly elongating clonal growth pattern of this species.

Conclusion

Characteristics of ramet performances such as flowering and recruitment of clonal offspring, in addition to growth, played a large part in the regulation of genet dynamics and distribution, which were different among the studied genets. These might be characteristics particularly relevant to clonal life histories.Key words: Clonal plant, Convallaria keiskei, demography, genet, genetic identification, growth pattern, life history, ramet, spatial distribution     

Reproductive demography of ramets and genets in a rhizomatous clonal plant <Emphasis Type="Italic">Convallaria keiskei</Emphasis>

Clonal growth occurring below the ground makes it difficult to identify individuals and demonstrate the demographic features of a focal plant species. In this study, genotypically identified ramets of a rhizomatous clonal herb, Convallaria keiskei Miq., were monitored for their growth, survival, and reproduction from 2003 to 2006. After the monitoring period, their subterranean organs were excavated to explore the underground connections of established ramets and the direction of clonal growth. We then combined data on the fate of the monitored ramets with the information of rhizome connections, clarifying reproductive demography at both the ramet and genet levels. Although each ramet initiated both sexual reproduction (via flowering) and clonal growth, clonal growth tended to precede sexual reproduction. In a surveyed genet, 51.0% of ramets produced flowers and 29.6% generated clonal offspring during the study period. Consequently, we clarified the reproductive demography of C. keiskei: clonal growth tended to precede flowering in a ramet, and a genet can keep reproducing every season at the genet level, despite a ramet not having inflorescence every year.     

Within-population spatial genetic structure, neighbourhood size and clonal subrange in the seagrass Cymodocea nodosa

Abstract The extent of clonality within populations strongly influences their spatial genetic structure (SGS), yet this is hardly ever thoroughly analysed. We employed spatial autocorrelation analysis to study effects of sexual and clonal reproduction on dispersal of the dioecious seagrass Cymodocea nodosa. Analyses were performed both at genet level (i.e. excluding clonal repeats) and at ramet level. Clonal structure was characterized by the clonal subrange, a spatial measure of the linear limits where clonality still affects SGS. We show that the clonal subrange is equivalent to the distance where the probability of clonal identity approaches zero. This combined approach was applied to two meadows with different levels of disturbance, Cadiz (stable) and Alfacs (disturbed). Genotypic richness, the proportion of the sample representing distinct genotypes, was moderate (0.38 Cadiz, 0.46 Alfacs) mostly due to dominance of a few clones. Expected heterozygosities were comparable to those found in other clonal plants. SGS analyses at the genet level revealed extremely restricted gene dispersal in Cadiz (Sp = 0.052, a statistic reflecting the decrease of pairwise kinship with distance), the strongest SGS found for seagrass species, comparable only to values for selfing herbaceous land plants. At Cadiz the clonal subrange extended across shorter distances (20-25 m) than in Alfacs (30-35 m). Comparisons of sexual and vegetative components of gene dispersal suggest that, as a dispersal vector within meadows, clonal spread is at least as important as sexual reproduction. The restricted dispersal and SGS pattern in both meadows indicates that the species follows a repeated seedling recruitment strategy.