386
L. Orsini et al.
al growth or sexual reproduction. while seedlings
can only originate by sexual reproduction. By
assessing gene flow among different localities we
established the dispersal range of seedlings
through the relative genetic contribution of the
different adult meadows to the newly formed
populations.
Materials and Methods
Study area and sampling strategy
Posidonia oceanica (1.) Dellie individual shoots
were sampled in three localities (Vada, Meloria
and Gulf of Follonica) along the coasts of
Tuscany (Italy), both in well established populations (Vada A, Meloda A and Follonica A) and in
extensive patches of seedlings adjacent to the
main meadow (Vada 5, Meloria Sand Follonica
S).Al1 seedling populations were distributed at 58 m depth. while the adult populations were sampled both at 7 and 15 m depth. Almost 40 shoots
were collected for each population and DNA was
extracted in CTAB buffer (Procaccini et al. 1996;
Procaccini and Mazzella 1998).
Microsatellite analysis
Six polymorphic loci, one chloroplastic (Poc-trn)
and five nuclear (Poc-5, Poc-26. Poc-35, Poc-42,
Poc-45), previously identified from a Posidonia
oceanica genomic library (Procaccini and
Waycott 1998). were amplified in the 219 individual shoots sampled (for detailed technique see
Procaccini and Waycott 1998).
Statistical analysis
Genetic parameters were calculated considering
only one ramet of each genotype per population
to avoid an artificial increase in N (Procaccini
and Mazzella 1998). Weir and Cockerham's
(1984) estimators of the level of inbreeding,f and
P, were obtained using FSTAT (version 1.2,
Goudet 1995) computer package. Standard deviations were calculated jackknifing over loci and
over populations. Observed and expected heterozygosity levels, allele frequency, number of
polymorphic loci per population were calculated
using GENEPOP 1.2 (Raymond and Rousset
1995) computer package. The Poc-trn locus of
the chloroplastic DNA was not included in analysis of the heterozygosity and related statistics,
since the chloroplast is maternally inherited in
angiosperms. Genotypic diversity in different
populations was calculated by the GIN ratio,
where G is the number of distinct genotypes and
N is the total number of samples. In order to estimate levels of gene flow among populations, we
calculated two statistical indices: i} (Weir and
Cockerham 1984) and Rho (Goodman 1997). The
significance of tJ and Rho were assessed by a
permutation test and over 1000 bootstrap replicates, respectively. The number of migrants per
generation was then calculated for ij(Nm"} and
for Rho (Nm Rho ). OJl2 was applied to calculate
genetic distances (Goldstein et al. 1995). i} was
calculated with FSTAT (Goudet 1995); Rho and
o/.P were calculated with RST Calc package
(Goodman 1997). The UPGMA analysis was performed on OJl2 genetic distance.
Results
Five of the six micro satellite loci screened for
this study were polymorphic in the six populations analysed. The monomorphic locus (Poc-5)
was considered in the analysis to allow comparison with published data. Sixteen alleles were present in total (Table 1). The distribution of allele
frequencies were similar in all populations, with
high frequency alleles characterizing the electrophoretic proflle of each locus. Five private
alleles, three in seedling populations and two in
adult populations, were present in total (Table 1).
Private alleles. when present, were detected in a
maximum of two individuals over the total number of samples screened for each population.
In the three localities, expected heterozygosity values were higher in the seedling populations (average value= 0.245) in respect to the
mature ones (average value= 0.290) (Table 2).
Values of f and F, calculated jackknifing over
populations were positive and significantly different from zero, indicating high homozygosity
both within the single populations and in the
whole set of samples (Table 3). Among the three
more polymorphic loci, Poc-35 gave the higher
values of homozygosity if= 0.741,SD= 0.118, F=
0.731, SD= 0.121).
Of the total 219 ramets sampled in the six
populations, less than 50% had distinct geno-
L. Orsini et al.
al growth or sexual reproduction. while seedlings
can only originate by sexual reproduction. By
assessing gene flow among different localities we
established the dispersal range of seedlings
through the relative genetic contribution of the
different adult meadows to the newly formed
populations.
Materials and Methods
Study area and sampling strategy
Posidonia oceanica (1.) Dellie individual shoots
were sampled in three localities (Vada, Meloria
and Gulf of Follonica) along the coasts of
Tuscany (Italy), both in well established populations (Vada A, Meloda A and Follonica A) and in
extensive patches of seedlings adjacent to the
main meadow (Vada 5, Meloria Sand Follonica
S).Al1 seedling populations were distributed at 58 m depth. while the adult populations were sampled both at 7 and 15 m depth. Almost 40 shoots
were collected for each population and DNA was
extracted in CTAB buffer (Procaccini et al. 1996;
Procaccini and Mazzella 1998).
Microsatellite analysis
Six polymorphic loci, one chloroplastic (Poc-trn)
and five nuclear (Poc-5, Poc-26. Poc-35, Poc-42,
Poc-45), previously identified from a Posidonia
oceanica genomic library (Procaccini and
Waycott 1998). were amplified in the 219 individual shoots sampled (for detailed technique see
Procaccini and Waycott 1998).
Statistical analysis
Genetic parameters were calculated considering
only one ramet of each genotype per population
to avoid an artificial increase in N (Procaccini
and Mazzella 1998). Weir and Cockerham's
(1984) estimators of the level of inbreeding,f and
P, were obtained using FSTAT (version 1.2,
Goudet 1995) computer package. Standard deviations were calculated jackknifing over loci and
over populations. Observed and expected heterozygosity levels, allele frequency, number of
polymorphic loci per population were calculated
using GENEPOP 1.2 (Raymond and Rousset
1995) computer package. The Poc-trn locus of
the chloroplastic DNA was not included in analysis of the heterozygosity and related statistics,
since the chloroplast is maternally inherited in
angiosperms. Genotypic diversity in different
populations was calculated by the GIN ratio,
where G is the number of distinct genotypes and
N is the total number of samples. In order to estimate levels of gene flow among populations, we
calculated two statistical indices: i} (Weir and
Cockerham 1984) and Rho (Goodman 1997). The
significance of tJ and Rho were assessed by a
permutation test and over 1000 bootstrap replicates, respectively. The number of migrants per
generation was then calculated for ij(Nm"} and
for Rho (Nm Rho ). OJl2 was applied to calculate
genetic distances (Goldstein et al. 1995). i} was
calculated with FSTAT (Goudet 1995); Rho and
o/.P were calculated with RST Calc package
(Goodman 1997). The UPGMA analysis was performed on OJl2 genetic distance.
Results
Five of the six micro satellite loci screened for
this study were polymorphic in the six populations analysed. The monomorphic locus (Poc-5)
was considered in the analysis to allow comparison with published data. Sixteen alleles were present in total (Table 1). The distribution of allele
frequencies were similar in all populations, with
high frequency alleles characterizing the electrophoretic proflle of each locus. Five private
alleles, three in seedling populations and two in
adult populations, were present in total (Table 1).
Private alleles. when present, were detected in a
maximum of two individuals over the total number of samples screened for each population.
In the three localities, expected heterozygosity values were higher in the seedling populations (average value= 0.245) in respect to the
mature ones (average value= 0.290) (Table 2).
Values of f and F, calculated jackknifing over
populations were positive and significantly different from zero, indicating high homozygosity
both within the single populations and in the
whole set of samples (Table 3). Among the three
more polymorphic loci, Poc-35 gave the higher
values of homozygosity if= 0.741,SD= 0.118, F=
0.731, SD= 0.121).
Of the total 219 ramets sampled in the six
populations, less than 50% had distinct geno-