Supplementary Material
Detailed methodology for clonal discrimination
Eelgrass is a partially clonal species, combining sexual and vegetative reproductions. The first step of
the genetic analyses is clonal discrimination. This method is based on the multilocus genotype (MLG)
of sampling units, and addresses two questions (Arnaud-Haond et al, 2007a): 1. Do all the replicates
of the same MLG really belong to the same genet (i.e., are they all issued from the same sexual
reproduction event)? 2. Does each distinct MLG really belong to a distinct genet (i.e., are they truly
issued from distinct reproductive events, or are the differences of MLG due to somatic mutations or
scoring errors)? Briefly, the first step consists of the estimation of allelic frequencies, following the
round-robin method (Parks and Werth, 1993). The probability Psex, that two identical MLG derived
from two distinct sexual reproductions (null hypothesis) is then estimated, taking departures from
Hardy-Weinberg equilibrium into account (Arnaud-Haond et al, 2007a). When Psex falls below a
threshold value fixed at 0.01, the null hypothesis is rejected, and the two identical MLGs are
considered as belonging to the same clonal lineage. Once this first step is achieved, slightly different
MLGS belonging to the same genet may, nevertheless, still occur in the dataset, either due to
somatic mutation or scoring errors (Douhovnikoff and Dodd, 2003). If ignored, such differences
would lead to an overestimation of the number of clones in the sample. To test whether these
slightly different MLG (distinct for one locus) belong to distinct genets, a very similar method to that
used with identical MLGs is applied. First, the locus differing among the MLG considered slightly
different is removed , with the result that the MLGs now share an identical reduced genotype. Psex is
used on the set of identical loci in order to estimate the probability that these MLG could actually be
derived from distinct reproductive events. As previously, when psex is lower than 0.01 (null hypothesis
rejected), the slightly different MLG can be considered to belong to the same genet or multilocus
lineage (MLL, Arnaud-Haond et al, 2007a) and the MLG dataset is corrected.
Supplementary Table S1 Estimation of the percentage of Multilocus Genotypes covered by the
sampling of 30 ramets. N is the number of sampling units, standardized to 30 in all quadrats, G the
number of distinct MLGs or MLLs discriminated in each quadrat, R the estimate of genotypic richness
(R=(G-1)/(N-1)). Density corresponds to the estimated number of ramets for 1m². Ntot is the derived
estimate of the number of ramets occurring within the 600m²-quadrat, and Gtot corresponds to the
expected number of MLGs occurring within the quadrat (Gtot=R * Ntot). %Gsampled is the ratio of G and
Gtot in percent, indicating the percentage of MLG covered by the sampling, and Gshared is the number
of MLG shared between 2009 and 2012.
Location
Quadrat
Molène
/
Saint Malo
1
Saint-Malo
2
Roscanvel
2
Arradon
2
Year
N
G
R
2009
2012
2009
2012
2009
2012
2009
2012
2009
2012
30
30
30
30
30
30
30
30
30
30
15
16
19
26
15
19
21
22
21
22
0,48
0,51
0,62
0,86
0,48
0,64
0,69
0,72
0,69
0,72
Density
(1m²)
173
150
470
667
373
473
183
270
138
400
Ntot
Gtot
103800
90000
282000
400200
223800
283800
109800
162000
82800
240000
49824
45900
174840
344172
107424
181632
75762
116640
57132
172800
%Gsampled Gshared
0,03
0,03
0,01
0,01
0,01
0,01
0,03
0,02
0,04
0,01
6
1
5
1
2
Supplementary Table S2 Spring densities for 0.1m²-quadrat. Three counts were performed in each
sampling quadrat.
Arradon Q1
2004
27.0
2005
14.7
2006
15.3
2007
11.7
2008
14.5
2009
10.8
2010
16.5
2011
23.8
2012
6.7
Arradon Q2
45.0
14.3
11.7
17.7
35.5
13.8
16.5
29.5
40.0
Roscanvel Q1
43.0
30.3
22.0
22.7
-
20.7
20.0
29.0
-
Roscanvel Q2
39.3
21.7
21.0
24.3
28.0
18.3
22.3
27.0
-
-
12.0
19.7
32.3
0.0
17.3
21.0
24.0
15.0
Sainte-Marguerite Q1
46.7
14.5
18.5
15.8
29.0
19.3
23.3
33.0
-
Sainte-Marguerite Q2
-
-
-
-
0.0
21.0
23.3
26.0
-
Callot Q1
25.0
18.7
30.3
37.3
72.0
22.7
14.3
21.0
27.5
Molène
Callot Q2
25.7
26.0
26.3
34.3
66.0
32.3
13.7
19.0
20.5
Arcouest Q1
48.0
44.0
30.3
33.3
38.0
24.7
19.3
55.8
11.0
Arcouest Q2
47.3
29.3
27.7
35.0
33.0
26.0
28.3
37.3
30.5
Saint-Malo Q1
-
27.0
36.0
33.3
54.7
47.0
56.8
85.5
66.7
Saint-Malo Q2
-
27.3
38.0
27.7
32.7
37.3
37.5
72.8
47.3
Supplementary Table S3 Matrix of genetic distance (Fst). Fst-values were calculated following Weir &
Cockerham (1984), for each pair of samples. Significance of values: * for P < 0.05; ** for P < 0.01 and
*** for P < 0.001.
Arcouest 1
Arcouest 2
Arradon 1
Arradon 2
Callot 1
Callot 2
Molène
Roscanvel 1
Roscanvel 2
Sainte-Marguerite 1
Sainte-Marguerite 2
Saint-Malo 1
Saint-Malo 2
Arc 2
0.02*
-
Arr 1
0.09***
0.17***
-
Arr 2
0.09***
0.15***
0.00
-
Cal 1
0.03***
0.06***
0.08***
0.08***
-
Cal 2
0.01*
0.06**
0.08***
0.08***
0.01
-
Mol
0.31***
0.35***
0.30***
0.28***
0.30***
0.31***
-
Ros 1
0.14***
0.14***
0.16***
0.13***
0.07***
0.11***
0.29***
-
Ros 2
0.13***
0.14***
0.15***
0.12***
0.09***
0.12***
0.25***
0.01*
-
Smar 1
0.19***
0.24***
0.20***
0.20***
0.21***
0.19***
0.18***
0.24***
0.21***
-
Smar 2
0.28***
0.32***
0.26***
0.27***
0.29***
0.26***
0.17***
0.28***
0.24***
0.04***
-
Smal 1
0.09***
0.08***
0.19***
0.16***
0.08***
0.10***
0.26***
0.12***
0.11***
0.24***
0.30***
-
Smal 2
0.19***
0.20***
0.26***
0.22***
0.19***
0.19***
0.23***
0.18***
0.15***
0.27***
0.30***
0.05***
-
Supplementary Table S4 Network values of Clustering Coefficient (CC). Each value corresponds to
the average value of CC inside local networks for one pair of quadrats at a location.
Arradon
Roscanvel
Molène
Sainte-Marguerite
Callot
Arcouest
Saint-Malo
Average
2009
0.42
0.17
0.33
0.16
0.33
0.31
0.52
0.32
2012
0.43
0.19
0.50
0.34
0.50
0.58
0.43
0.42
Supplementary Figure S1 Locations of the seven intertidal meadows of Zostera marina. For each
location, both quadrats sampled in 2009 were also sampled in 2011, except in Molène, where a
single quadrat was studied. Approximately 35 sampling units collected from each quadrat, according
to randomly-generated coordinates.
Supplementary Figure S2 Isolation-by-distance for Z. marina. The full line corresponds to the
significant regression combining the two distance-scales, indicating that pairs within a location are
less distinct than pairs among locations. The left-hand dashed line corresponds to the regression with
pairs of quadrats within a location (local scale), and the right-hand dashed line to the regression with
pairs of quadrats among locations (regional scale). The observed pattern is very similar to the ones
observed in 2009. The pair of quadrats of Sainte-Marguerite (the diamond-shaped point in the topleft corner) is a noticeable exception indicating that a strong divergence occurred during the threeyear period between samplings.