American Journal of Botany 90(3): 364–369. 2003.

RANDOM

AMPLIFIED POLYMORPHIC

DNA

ASSESSMENT

MEDITERRANEAN SEAGRASS POSIDONIA OCEANICA1

OF DIVERSITY IN WESTERN POPULATIONS OF THE

MARIA ANGELES JOVER,2 LUCAS DEL CASTILLO-AGUDO,3 MANUEL GARCIA-CARRASCOSA,2 AND JUAN SEGURA4,5 Laboratorio de Biologı´a Marina, Departamento de Biologı´a Animal, Universidad de Valencia, Valencia, Spain; 3Departamento de Microbiologı´a y Ecologı´a, Universidad de Valencia, Valencia, Spain; and 4Departamento de Biologı´a Vegetal, Universidad de Valencia, Valencia, Spain

2

Posidonia oceanica is an endemic Mediterranean seagrass species that has often been assumed to contain low levels of genetic diversity. Random amplified polymorfic DNA (RAPD) markers were used to assess genetic diversity among five populations from three geographical regions (north, central, and south) of the western Mediterranean Sea. Stranded germinating seeds from one of the central populations were also included in the analysis. Forty-one putative genets were identified among 76 ramets based on 28 RAPD markers. Genotypic diversity strongly depended on the spatial structure, age, and maturity of the meadows. The lowest clonal diversity was found in the less structured and youngest prairies. Conversely, a high genotypic diversity was found in the highly structured meadows. The genotypic diversity in these meadows was at the same level as in P. australis and higher than previously reported data for P. oceanica populations in the Tyrrhenian Sea near the coast of Italy. Key words:

clonal analysis; genotypic and genetic diversity; population structure; Posidonia oceanica; RAPDs; seagrass.

Posidonia oceanica (L.) Delile is an endemic Mediterranean seagrass with a dominant role in sublittoral ecosystem dynamics supporting a highly productive and very diversified ecosystem. It is considered to be the climax community on sublittoral soft bottoms (Pe`res and Picard, 1964). Thus, accurate information about the extent, distribution, and nature of the genetic variability in this species is required. Because of the high degree of regression of P. oceanica meadows, this information will contribute to designing the most appropriate conservation strategies for the species. Random amplified polymorphic DNA (RAPD) and microsatellite markers have contributed significantly to our knowledge of the population genetics of seagrasses, showing that considerable genetic and clonal diversity is usually present and that genetic differentiation among populations seems to be the rule for most of the species studied (Reusch, 2001). Published studies about P. oceanica, however, partially contradict these considerations. The RAPD analyses carried out in a P. oceanica population at Island of Ischia (Gulf of Naples, Italy) showed an almost complete clonality (Procaccini and Mazzella, 1996) and a very low genetic distance from other disjunct population located at Blanes, Spain (Procaccini et al., 1996). More recently, Procaccini et al. (2001), using microsatellites, observed the existence of clear patterns of genetic structure in P. oceanica meadows from 17 localities in the Tyrrhenian Sea, but these meadows were largely uniclonal. In this paper we demonstrate that RAPD analysis is a useful approach to identify multilocus genotypes in P. oceanica populations from a wide geographic range along western Mediterranean coasts. Although RAPD markers must be carefully interpreted because of their dominance (Lynch and Milligan, 1994), they are well suited for resolving genets in natural and cultivated clonal

plants (Liu and Furnier, 1993; Gabrielsen and Brochmann, 1997; Bush and Mulcahy, 1999; Fischer et al., 2000; Verburg et al., 2000; Auge et al., 2001), including seagrasses (Waycott, 1998). These molecular markers, consistent within a genet, are very unlikely to be affected by somatic mutations within a genet (Van de Ven and McNicol, 1995). The genetic structure of plant populations reflects the interactions of different processes including long-term evolutionary history of the species (shifts in distribution, habitat fragmentation, and population isolation), mutation, genetic drift, mating system, gene flow, and selection (Slatkin, 1987; Schaal et al., 1998). Patterns of genetic diversity in seagrasses are also influenced by other factors such as the spatial structure, age, and maturity of the meadows, which affect the recruitment potential of seedlings, pollen, and vegetative propagules. Although some of these factors are well known in other seagrass species such as Zostera (Reusch et al., 2000), Thalassia (Kirsten et al., 1998; Schlueter and Guttman, 1998), and P. australis (Waycott and Sampson, 1997), the information for P. oceanica is scarce. Because of this, we present the results of an RAPD survey of different meadow types of P. oceanica distributed along Mediterranean coasts of Europe, the Iberian Peninsula, Balearic Islands, and Morocco. MATERIAL AND METHODS Sample collection—Posidonia oceanica shoots were collected from five locations along the western Mediterranean coasts: one population in France, three in Spain, and one along the north African coast at Morocco (Fig. 1, Table 1). Leaves from individual shoots, always collected at least 5 m apart, were stored in 96% ethanol until processing. For comparative purposes, germinating seeds were also collected (described later). The main characteristics of the P. oceanica sampled meadows are given next.

Manuscript received 11 June 2002; revision accepted 24 October 2002. Author for reprint requests: J. Segura, Departamento de Biologı´a Vegetal, Facultad de Farmacia, Universidad de Valencia, Avda. Vicent A. Estelle´s s/n. Burjassot 46100, Valencia, Spain (phone: 134 963 864 922; FAX: 134 963 864 926; e-mail: [email protected]). 1

Banyuls, France (Plage de les Elmes)—This meadow is constituted by a discontinuous and superficial belt, with a low density of fascicles per square meter and a thin rhizome stratum. The rhizomes present plagiogravitropic growth, covering the substrate surface but without forming compact meadows.

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from San Antonio’s Cape to Santa Pola’s Cape-Island of Tabarca, forming a submerged coastal fringe of ;100 km in length, practically without discontinuity.

Fig. 1. Map of location of the Posidonia oceanica meadows included in the study (see Table 1 for population abbreviations).

Formentera Island, Balearic Archipelago, Spain (Punta Pedrera)—This very developed meadow has both high fascicle density and substrate coverage. The plants extend from sea level, with leaves spreading on the water surface, to their lowest depth of 30 m. The meadows grow on a substrate with a very heterogeneous topography including a lot of channels and cuvettes. From the surface to 15–18 m deep, the Posidonia coverage is always homogeneous. Below 18 m, Posidonia grows in isolated patches on a mixed substrate of blocks and boulders. At the lower limit, plagiogravitropic rhizomes prevail. The samples (F1 to F15) were collected in the homogeneous part of the meadow at 10–11 m. On the other hand, stranded germinating seeds and seedlings (samples S1 to S9) were picked up from the beach and preserved in 96% ethanol. The seed origin is unknown, but they must come from one of the extensive prairies off the Formentera Island or even off neighboring Ibiza Island. Tabarca Island, Spain (La Cantera beach)—This is a Posidonia reef barrier where the superficial meadow shows an important development, reaching several meters high from the bottom level, from the strong vertical growth of the orthogravitropic rhizomes. The samples were collected from the inner margin of the reef barrier at 0.5 m depth (samples T1 to T10) and belong to the ‘‘superficial dwarfism’’ morphotype, described by by Sa´nchez-Lizaso (1993). Leaves have reduced width and length as an adaptation to wave action.

The meadow borders are formed by plagiogravitropic rhizomes that expand to colonize the surrounding coarse, sandy sediment. Posidonia prairies from Plage de les Elmes belong to a very discontinuous belt of meadows located between Cap Be´ar and Cap Cerbe`re. It is not clear if these meadows were well developed several decades ago because historical data are not accurate enough to measure the evolution of the prairies (Pergent et al., 1985). Recent data show that current bottom area is occupied by continuous, relatively limited and localized beds and that areas of dead meadows may indicate a more extensive bottom cover of Posidonia in the past (Ballesta et at., 2000). The Plage de les Elmes is located in an area exposed to wave action and shows an advanced state of regression as a result of several factors, including harbor construction and other man-made changes in the coastline. The meadows of the inner harbor of Banyuls-sur-mer and other places near Plage des Elmes, nevertheless seem well stabilized and present a high level of vitality (Pergent et al., 1991), probably from the protection of wave action and other factors. A faunistic and hydrological study of these Posidonia meadows was carried out by Kerneis (1960). Samples were collected from a series of superficial shoots growing on a rocky substrate at a depth of 1–1.5 m (B11 and B12 samples) and from a patch at a depth of 2.5–4.5 m (B1 to B10 samples).

Chafarinas Archipelago (Southeast Alboran Sea, Moroccan coast)—These Posidonia prairies can be considered as the southwestern limit of the species in the Mediterranean Sea. They form young, expansive but less structured meadows off the sheltered coast of the three small islands, which constitute the archipelago. The P. oceanica meadows of Chafarinas archipelago are geographically isolated. The nearest Posidonia meadows are located to the east, in Beni-Saf (near Oran) off the Algerian coast, and toward the north, off the coasts of Almeria and Granada (Spain). In both cases, the meadows are more than 100 km apart. Samples were collected in a meadow located along the southern coast of Isabel II Island. The meadow is not as extensive and grows between 8 and 10 m deep on a mixed bottom of sand, pebbles, and boulders (Ch1 to Ch15 samples).

Morayra, Spain (Punta de la Estrella)—This highly consolidated meadow grows at a depth of 0.5–25 m on mixed substrates of irregular topography. The upper limit of the prairie is constituted by isolated patches among blocks and boulders. Their upright growth and expansion are limited by hydrodynamic effects. The meadow has a high density of fascicles (350–400 shoots/ m2) with coverage superior to 90%, and a high stratum of orthogravitropic rhizomes with steps more than 1 m in height. There are also many cuvettes up to 2 m deep filled with coarse sediments. The lower limit of the meadow is constituted by isolated shoots growing at a 25-m depth on a muddy sand substrate. The samples were collected from 15 different plants in the upper (samples M1 to M7) and lower (samples M8 to M15) limits of the meadow. The Morayra prairies belong to a group of Posidonia meadows extending

DNA extraction and amplification—The extractions and Polymerase chain reaction (PCR) amplifications of DNA were carried out as described in Del Castillo-Agudo et al. (1995), except that NaOCl treatment was omitted. Epiphytes were scraped from leaves submerged in 70% ethanol, then leaves were washed with distilled sterilized water. NaOCl treatment was omitted because it inhibited DNA amplification. Fragments generated by amplification were separated by size on 1% agarose (SeaKem LE, FMC Bioproducts, Rockland, Maine, USA) gel run in TBE buffer (89 mmol/L Tris base, 89 mmol/L boric acid, and 2 mmol/L EDTA, pH 8), stained with ethidium bromide, and visualized by illumination with UV light (Nebauer et al., 2000). To aid interpretation of band identity between gels, each contained Gene Ruler DNA ladder mix (Fermentas AB, Vilnius, Lithuania).

TABLE 1.

Sampled populations of Posidonia oceanica with abbreviations and geographic location. Sampling station

Latitude

Banyuls-sur-mer (Plage des Elmes)

428319 N

38129 E

Longitude

Depth (m)

Samples

0889 E

4.5 2.5 2–3

388449 N

18249 E

8 10–11

388099 N 358409 N

08279 W 28259 W

0.5 8–10

B1–B10: homogeneous meadow B11–B12: upper limit of the meadow M1–M7: homogeneous meadow M8–M15: lower limit of the meadow R1–R10: different fascicles from a single rhizome F1–F15: homogeneous meadow S1–S9: stranded seedlings T1–T10: upper limit of a Posidonia barrier-reef Ch1–Ch15: homogeneous meadow

Morayra (Punta de la Estrella)

388419 N

Formentera Island (Punta Pedrera) Tabarca (Playa de la Cantera) Chafarinas Islands (Isabel II Island)

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Reproducibility and repeatability of amplification profiles were tested for each primer. Control samples containing all reaction material except DNA were used to test that no self-amplification or DNA contamination occurred. Only those bands that were clear and consistently reproduced in different analysis were considered. Poor amplifications occurred systematically with individuals from different populations; these were excluded from the analysis and they mainly account for the different sample sizes of this study. At least two replicates per sample were amplified and DNA from all the individuals was extracted twice. The bands with the same molecular mass and mobility were treated as identical fragments. Amplified fragments, named by the primer used and the molecular mass in base pairs (bp), were scored as the presence (1) or absence (0) of homologous bands, and a matrix of the different RAPD phenotypes was assembled. Because RAPD markers are dominant, we assumed that each band represented the phenotype at a single biallelic locus (Williams et al., 1990). The heterogeneity of band frequencies across all populations was tested using G2 tests in POPGENE software (Yeh et al., 1997). Clonal diversity analysis—All ramets with an identical band pattern were considered as one genet. Three measures of genotypic (clonal) diversity were calculated according to Ellstrand and Roose (1987): (1) PD, proportion of distinguishable genets (G/N), where G is the number of genets (RAPD phenotypes) detected and N is the number of ramets analyzed; (2) Simpson’s unbiased diversity index, given by D 5 1 2 {[Sni(ni 2 1)]/[N(N 2 1)]}, where ni equals the number of ramets of genet i; and (3) genotypic evenness (Fager, 1972), calculated as E 5 (Dobserved 2 Dminimum)/(Dmaximum 2 Dminimum), where Dminimum 5 [(G 2 1)(2N 2 G)]/[N(N 2 1)] and Dmaximum 5 [N(G 2 1)]/[G(N 2 1)]. D ranges from 0 in a population composed of a single genotype to 1 in a population in which every sampled individual represents a different genotype. E ranges from 0 in a population in which all individuals represent different genotypes or where one genotype dominates and the other genotypes are represented by a single individual to 1 in a population in which all clones are represented by the same number of ramets. Additionally, and for comparative purposes, Shannon’s information index was also calculated to provide a relative estimate of the degree of genetic variation within each population using POPGENE software. Shannon’s analysis has general applications in ecology and is relatively insensitive to the skewing effects caused by the inability to detect heterozygous loci (Dawson et al., 1995). Relationships among genets—A pairwise distance matrix was computed based on Nei’s coefficient of similarity (Nei and Li, 1979), using the RAPDPLOT program of Black (1998). Dendrograms were then created with UPGMA (unweighted pair group method with arithmetic averaging) and neighbor-joining cluster analysis as implemented using the NEIGHBOR program from the Phylip 3.57c package (Felsenstein, 1993). To give a measure of the variability in the data, the original matrix was bootstrapped 500 times via RAPDPLOT and a consensus tree was generated using the NEIGHBOR and CONSENSE programs in PHYLIP. Permutation test probability (PTP) analysis was also performed, using RAPDistance (Armstrong et al., 1996) software, to test whether the resulting tree reflects an actual tree-like signal in the data or merely an artefact of the algorithm (Faith and Cranston, 1991). A cophenetic value matrix was also produced from the tree matrix using the COPH program from NTSYS-pc (Rohlf, 1997). This matrix was then employed to check the goodness of fit of cluster analysis by comparing it to the original pairwise distance matrix using a Mantel test (MXCOMP program from NTSYS-pc). Significance was determined using 1000 permutations. All dendrograms were displayed and printed using TREEVIEW software (Page, 1996). An analysis of molecular variance (AMOVA: Excofier et al., 1992) was used to study the relationships between the P. oceanica genets collected in Morayra in the upper and lower limits of the meadow. The resulting variance components were used as estimates of the genetic divergence among the samples collected in the two bathymetric levels. AMOVA analysis was performed using the WINAMOVA 1.5 program (available from L. Excoffier, Genetics and Biometry Laboratory, University of Geneva, Switzerland).

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TABLE 2. Genotypic and genetic diversity for RAPD data from six populations of Posidonia oceanica. Populations

Banyuls Chafarinas Formentera Morayra Seeds Tabarca Mean

N

G

PD

D

E

I

12 15 15 15 9 10

3 2 9 14 7 6 6.8

0.25 0.13 0.60 0.93 0.78 0.60 0.54

0.44 0.25 0.89 0.99 0.94 0.84 0.73

0.30 0.29 0.6 0.00 0.57 0.43 0.31

0.041 0.022 0.262 0.251 0.313 0.102 0.165

Abbreviations: N, number of samples; G, number of genets (RAPD phenotypes); PD, proportion of distinguishable genets (G/N); D, Simpson index of diversity; E, genotypic evenness; I, Shannon index of diversity.

RESULTS Primer selection and RAPD profiling—Individual Posidonia DNA samples were diluted and bulked by populations to screen 40 primers (Series A and C, Operon Technologies, Alameda, California, USA). On the basis of high reproducibility of patterns, signal intensity, and adequate number of bands (between 6 and 15), two primers (OPA3 and OPA4) were used to screen the 76 samples. The reproducibility of band pattern obtained with OPA3 and OPA4 was confirmed by amplification of DNA of seven leaves from fascicles of a single rhizome (data not shown). Under our conditions, series C primers, previously used by Procaccini and Mazzella (1996) and Procaccini et al. (1996) in P. oceanica, did not meet the criteria cited earlier. The two selected primers generated 28 consistently wellamplified bands, ranging in size from 200 to 2200 bp. Twentysix of these bands were polymorphic among the six populations. The populations differed widely in their polymorphism for the whole set of bands scored. The lowest level of polymorphism (3.6%) was observed in the Chafarinas population, followed by the Banyuls (7.1%), Tabarca (21.4%), Morayra, and Formentera (46.4% each) populations. A high percentage of polymorphic bands (53.6%) was observed in the germinating seeds and seedlings sampled in Morayra. Results of the G2 tests showed statistical heterogeneity of band frequency for 21 of the 28 assayed RAPD loci. Genotypic (clonal) diversity—A total of 41 putative multilocus genotypes were identified out of 76 ramets analyzed. Identical genotypes in different populations were not identified. The proportion of distinguishable genets (PD) and values for Simpson’s diversity index (D) and genotypic evennes (E) are listed in Table 2. The proportion of distinguishable genets varied considerably between the most diverse population, Morayra (0.93), and the least diverse population, Chafarinas (0.13). The lower D values (0.25 and 0.44) were obtained in Chafarinas and Banyuls, respectively. In the remaining four populations, D values were higher than 0.8, indicating that a significant proportion of ramets belonged to different genotypes. The lower E value (0) was obtained in Morayra, a population in which only two samples belonged to the same clone. In Formentera and Tabarca populations, the identified genotypes also tended to be rather evenly distributed (E 5 0.60 and 0.43, respectively) but one of the two multisample clones detected in each population consisted of five and six samples, respectively. Banyuls and Chafarinas had comparable E values (0.29–0.30), and both populations seem to be dominated by a

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large clone consisting of nine samples in Banyuls and 13 in Chafarinas. The mean genetic diversity within populations estimated by Shannon’s indices ranged from 0.022 to 0.317 (Table 2). In general, these values and the measures of genotypic diversity were correlated. Note, however, that the Tabarca population had a lower Shannon index than the populations with comparable values of genotypic diversity (Morayra, Formentera, and seedlings). Relationships among genets—The UPGMA or neighborjoining dendrograms generated from the Nei’s distance matrix revealed similar relationships among the 41 genets; therefore, only the neighbor-joining dendrogram is presented. The unrooted neighbor-joining dendrogram clustered the genets from Moraira, Tabarca, and Formentera within distinct groups according to their geographical origin. Interestingly, the dendogram formed two distinctive subclusters with the Morayra genets sampled at two different bathymetric levels corresponding to upper and lower limits of the meadow. For Banyuls, Chafarinas, and seedling populations, a lack of concordance between genets and their geographic origin was observed. In the three cases, RAPD phenotypes were intermingled in different parts of the dendrogram. So, the consensus tree from the 500 bootstrapped samples indicated a relatively low level of support for the distinctiveness of these populations (Fig. 2). Corroborating this, the PTT test gave a Z value of 10.3. The Mantel test comparing Nei’s distance and cophenetic matrices had a poor but significant correlation (r 5 0.383, P 5 0.002). The AMOVA procedure was finally implemented to study the relationships between the two subset of Morayra data (upper and lower limits of the meadow). The AMOVA showed significant differences between both groups of genets (FST 5 0.50, P , 0.001), confirming the grouping of individuals in the dendogram (Fig. 2). DISCUSSION Results herein represent the first successful use of RAPD markers to characterize genotypic and genetic variation in Posidonia oceanica, a Mediterranean endemic seagrass that provides important functions in shallow sublittoral coastal ecosystems. Previous studies using these molecular markers showed a complete absence of genetic diversity within a P. oceanica meadow and a very low genetic distance from other disjunct populations (Procaccini and Mazzella, 1996; Procaccini et al., 1996). Our RAPD survey of the six western Mediterranean populations of P. oceanica demonstrates that this seagrass displays a wide range of clonal diversity. Thus, whereas some populations showed near clonality, others had maximal genotypic diversity. The level of clonal diversity in P. oceanica seems to be related to the spatial structure, age, and maturity of the meadows. We found low clonal diversity as a trend in the less structured and youngest prairies, such as Banyuls and Chafarinas, respectively, the former because of its pioneer character colonizing the upper beach limits and the latter because it can be dated precisely as being absent in the archipelago in a cartographic survey in 1926. Conversely, a high genotypic diversity was found in highly structured meadows such as Tabarca, Morayra, and Formentera. The high genotypic diversity of these latter long-lived meadows may be related to (Eriksson, 1993; McFadden, 1997; Schla¨pfer and Fischer, 1998) (1)

Fig. 2. Bootstrapped neighbor-joining tree from the Nei’s distance matrix using the 41 genets of Posidonia oceanica. Numbers at the nodes indicate the number of trees with that node (omitted if less than 40% of trees).

association of high genet longevity with high diversity at population foundation and no competitive exclusion of genets and (2) low but continuous seed recruitment over time. The proportion of distinguishable genotypes was 0.54 across all the six sampled Posidonia populations, 0.34 for the less structured meadows (Banyuls and Chafarinas), and 0.71 for the highly structured ones. The D values (for the average and the two meadows types) were, respectively, 0.73, 0.34, and 0.91. All these estimates are higher than the clonal plant average (PD 5 0.17; D 5 0.62), but within the range for predominantly clonal plants (Ellstrand and Roose, 1987). In contrast, the mean genotypic evenness was lower (0.31 vs. 0.68) than that reported by Ellstrand and Roose (1987). Note, however, that the results of Ellstrand and Roose (1987) are based on allozymes, and these markers always detect a lower number of genets than RAPDs (Waycott, 1998; Esselman et al., 1999). Our values for genotypic diversity in the highly structured meadows (Tabarca, Morayra, and Formentera) and seedlings are comparable to those calculated in P. australis on RAPD (Waycott, 1998) and higher than those based on microsatellite data in P. oceanica populations collected in 17 locations along the coasts of the Tyrrhenian Sea in Italy (Proccacini et al., 2001). Thus, the total sample G/N value for the Italian popu-

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lations was 0.116, whereas in our case this value was 0.539. When the highly structured and mature meadows are considered alone, this value is even higher (0.644). The hierarchical organization of clonal plants leads to two different measures of genetic diversity: genotypic variation as the number and frequency of genets and genetic variation as the number and frequency of alleles either calculated across genets or across ramets (McLellan et al., 1997). These two measures are not necessarily correlated; although a large number of genets is likely to comprise a large number of alleles, a population can also consists of only a few, highly heterozygous genets (McLellan et al., 1997). Except for Tabarca populations, where the Shannon and Simpson indices did not correspond, there was a good correlation between the measures of genotypic and genetic variation. The AMOVA revealed the existence of genetic substructuring in Morayra, a population sampled at two different depths, which was in agreement with published work on several seagrasses (see Reusch, 2001, for review) including P. oceanica (Procaccini et al., 2001, and references therein) and P. australis (Waycot and Sampson, 1997; Waycott et al., 1997). All seagrasses reproduce vegetatively by branching of rhizomes and the formation of new leaf shoots and sexually through seeds. The relative importance of sexual reproduction and clonal growth is not well established in P. oceanica, but several studies suggest that sexual reproduction might be sporadic and that seedling establishment is unsuccessful (see Procaccini and Mazzella, 1998). Caye and Meinesz (1984) reported, however, that flowering and fruiting are more frequent in southern latitudes, suggesting a gradient of genetic variability of the meadows from north to south. Procaccini et al. (2001) concluded that, according to the level of variability detected by microsatellite markers, almost 90% of the millions of Posidonia shoots along the western coasts of Italy, including part of the eastern coasts of Corsica and Sardinia, are genetically identical, and therefore clonality can be considered as the primary reproductive mode of the species. Our RAPD data demonstrate, however, strong differences in genotypic diversity between the youngest and less structured prairies, in which only two or three genets were found, to the aged and highly structured one, in which a high number of ramets represented different genets. Waycott (1998) suggests that clonality in P. australis may be primarily a function of meadow formation (i.e., anchorage). The high clonality observed in the youngest P. oceanica meadows sampled (Chafarinas) suggests that this mechanism can also be operative in the Mediterranean congener of P. australis. Given the long lifespan of the genets, both sexual reproduction and immigration of divergent clones will maintain the genetic diversity within the highly structured populations (Moraira, Formentera, and Tabarca). Supporting this idea, Balestri et al. (1998) observed that P. oceanica seedlings might persist for many years if recruited on meadow substrates, indicating the importance of sexual reproduction in meadow maintenance and that sites suitable for recruitment can be separated from sites where flowering plants occur. In summary, and although RAPD analysis must be interpreted with caution, our results demonstrate that P. oceanica meadows could be clearly multiclonal. The apparent contradiction between our results and those of Procaccini et al. (2001) could be either related to a highly different flowering frequency among the two regions studied, or to the different variability signals detected by the different markers used, RAPDs and microsatellites, as suggested by Mariette et al.

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