doi:10.1111/j.1420-9101.2010.02198.x
Two reproductively isolated cytotypes and a swarm of highly
inbred, disconnected populations: a glimpse into Salicornia’s
evolutionary history and challenging taxonomy
A. VANDERPOORTEN*, O. J. HARDY , J. LAMBINON* & O. RASPÉà
*Institute of Botany, University of Liège, Liège, Belgium
Faculté des Sciences, Université Libre de Bruxelles, Bruxelles, Belgium
àNational Botanic Garden of Belgium, Meise, Belgium
Keywords:
Abstract
hybridization;
microsatellite;
phylogeography;
polyploidy;
reproductive isolation;
Salicornia;
selfing;
species concept.
The main factor of differentiation at six nuclear microsatellite and seven
cpDNA loci in Salicornia from the Mediterranean and Atlantic coasts of France
is cytotypic identity, suggesting the presence of a strong reproductive barrier
among sympatric cytotypes. Within cytotypes, a substantial proportion of the
differentiation between species is due to confounded phylogeographic signal.
Conspecific individuals tend to be significantly more related than individuals
from different species at the population scale, but mean kinship coefficients
among pairs of conspecific and nonconspecific individuals from different
populations are not significantly different, suggesting the absence of reproductive isolation among species of the same cytotype. The observed association
between morphology and genetic variation within populations would thus
result from the selfing mating system (Fis = 0.70) generating substantial
linkage within the genome, linkage that would quickly disappear among
unrelated individuals from different populations. Salicornia species thus
function as a network of inbred populations, strongly challenging taxonomic
concepts.
Introduction
Polyploidization is an important evolutionary force.
Estimates suggest that 70% of all angiosperms have
experienced one or more episodes of polyploidization
(Masterson, 1994), and polyploids represent the bulk of
the species diversity in many major lineages of land
plants including pteridophytes (95%, Soltis & Soltis,
1999) and mosses (79%, Wyatt et al., 1988). As opposed
to the traditional view of polyploids as genetically
depauperate species because of the sudden reproductive
isolation after a single polyploidization event, an increasing body of literature suggests that individual polyploid
species evolved multiple times (see Albach, 2007, for
review), often after the fusion of nonreduced gametes
Correspondence: A. Vanderpoorten, Institute of Botany,
University of Liège, B22 Sart Tilman, B-4000 Liège, Belgium.
Tel.: +32 4366 3842; fax: +32 4366 2925;
e-mail: [email protected]
630
from different species. The greater diversity resulting
from increased heterozygosity found within individual
genomes theoretically provides selective advantages
in changing environments, explaining for example the
striking increase in polyploids with latitude, associated
intensity of climatic fluctuations, and frequent recolonization events (Abbott & Brochmann, 2003; Brochmann
et al., 2004). At the population level, by contrast, recently
formed polyploids might, owing to founding effects,
harbour less genetic diversity if polyploid formation is
a rare and ⁄ or recent event (see Soltis & Soltis, 1999, for
review). The recurrent series of range contractions and
expansions during the Pleistocene glacials and interglacials would further reinforce this effect (Hewitt, 2004).
An ancient, possibly polyphyletic origin of polyploids, as
well as subsequent introgression, may, however, account
for the high genetic diversity that has increasingly been
reported among polyploid populations (Ramsey &
Schemske, 2002; Luttikhuizen et al., 2007).
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Reproductive isolation in Salicornia
Salicornia (Amaranthaceae), a sub-cosmopolitan genus
of annual species, is a model of choice for exploring the
origin and evolution of polyploid formation and intercytotypic barriers. Two chromosome series, one diploid with 2n = 18 and the other tetraploid with 2n = 36,
are present in the genus (Dalby, 1962; Shepherd & Yan,
2003), and the distinction between cytotypes is fully
supported by amplified fragment length polymorphism
(AFLP) and DNA sequences (Le Goff, 1999; Kadereit
et al., 2007; Murakeözy et al., 2007). Tetraploids, whose
seedling radicles grow more rapidly than those of
diploids, typically occur in areas exposed to tidal
disturbance, whereas diploids colonize more stable,
upshore habitats (Davy et al., 2001). Thus, perhaps
more than in other groups of plants that are often
characterized by cytotypic vicariance along major ecological gradients (see Duckert-Henriod & Favarger,
1987; and Vamosi et al., 2007; for review), diploid and
tetraploid Salicornia species are commonly found in
sympatry along tidal gradients (Davy et al., 2001), if not
completely intermixed in complex vegetation mosaics
(Lorenzoni et al., 1993; van Hulzen et al., 2006), making
the genus a keystone for the phytosociological classification of European salt marshes (Géhu, 1992; Géhu &
Bioret, 1992).
Owing to their strict occurrence in extremely constraining salty environments, Salicornia species display a
strikingly reduced, convergent morphology offering a
limited number of characters for taxonomy. The flowers
631
are uniformously tepaloid and the leaves reduced to
scarious rims. The level of variation of ITS, ETS and
trnL sequence data is extremely low within cytotypes
(Kadereit et al., 2007; Murakeözy et al., 2007), which
results in poorly resolved relationships among species.
None of the traditionally defined species, even those
whose distinction is straightwforward, such as S. pusilla,
which is the only species among diploids with solitary
flowers, were, however, resolved as monophyletic
(Kadereit et al., 2007; Murakeözy et al., 2007). The genus
has, consequently, long puzzled taxonomists, and systematic treatments range from recognition of an array of
species and their putative hybrids (e.g. Lahondère, 2004;
Stace, 2010) to severe reductions to synonymy, keeping
one or a few species within each cytotype (e.g. Valdés &
Castroviejo, 1990; Piirainen, 2001).
In the present study, we use a combination of PCRRFLP markers across a range of cpDNA loci as well as
specific nuclear microsatellites to define the hierarchy
of factors accounting for Salicornia’s genetic variation
patterns along the Mediterranean and Atlantic coasts of
France and address the following questions at different
nested taxonomic and spatial scales: (i) What is the origin
of the tetraploids and what are the genetic relationships
between diploid and tetraploid lineages? (ii) Are species
reproductively isolated? (iii) What are the dispersal
ranges of seeds and pollen and does dispersal rates
exceed mutation rates, or is a strong phylogeographic
signal present in the data?
Fig. 1 Localization of the 43 populations
of Salicornia sampled along the Mediterranean and Atlantic coasts of France.
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A. VANDERPOORTEN ET AL.
Materials and methods
Taxon sampling
In total, 566 specimens were collected from 13 French
Mediterranean (including Corsica) and 30 localities along
the Atlantic coast (sensu lato, including the English
Channel and the North sea) (see Table S1, Fig. 1).
Diploids and tetraploids differ in a suite of nonequivocal
morphological features (e.g. Dalby, 1962; Lahondère,
2004), and a recent study combining flow cytometry and
thorough morphometric analyses confirmed that morphological variation conforms to cytotypic differentiation
(Kaligaric et al., 2008). The degree of size difference
between the median and the lateral flowers proved, in
particular, to be most useful to distinguish individuals
attributed to either the diploid or tetraploid lineage based
upon their morphology. The reliability of the morphological criterion was confirmed by a trial on a subset of 30
freshly collected specimens whose ploidy level was
determined by flow cytometry using the protocol
described in Kaligaric et al. (2008). Based on the morphological criterion, the 566 specimens included 194
tetraploids (comprising collectively S. fragilis, S. emericii
and S. dolichostachya) and 372 diploids (comprising
collectively S. ramosissima, S. obscura, S. patula and
S. pusilla). The application of the morphological criterion
was, however, complicated in the case of S. obscura, a
species that differs from all other diploids by flowers of
sub-equal size. Therefore, the cytotype of 98 such
specimens was scored as ambiguous, and those specimens were not included in the analyses explicitly
partitioning the genetic variation among cytotypes.
The taxonomic system of Lahondère (2004), which
closely matches the most recent treatment of Stace
(2010), was used here for species identification. We
followed, however, the species names accepted by
Kadereit et al. (2007), and hence, considered S. brachystachya to be synonym to S. ramosissima. Our sampling
thus included specimens of S. pusilla, S. ramosissima,
S. patula and S. obscura, which are diploid, and S. emericii,
S. dolichostachya and S. fragilis, which are tetraploid. Four
species are restricted to the Atlantic coasts of Europe:
S. pusilla and S. obscura have been reported from France
to The Netherlands and the British Isles; and S. dolichostachya and S. fragilis from the Iberian Peninsula to
Denmark and the British Isles. One species, S. patula, is
restricted to the Mediterranean, where it has been
reported from the Iberian Peninsula to the Adriatic.
Finally, S. emericii and S. ramosissima occur along both the
Atlantic and Mediterranean coasts of Europe. Each
specimen was placed in individual Eppendorfs and kept
at )80 C pending DNA extraction.
Molecular protocols
Samples were cooled in liquid nitrogen and immediately
ground using a Retsch MM 301 mill, followed by DNA
isolation using the CTAB protocol of Doyle & Doyle
(1987) without any RNase treatment. Each specimen was
genotyped at six nuclear microsatellite loci as described
in Vanderpoorten et al. (2010a), and at seven cpDNA loci
using PCR-RFLP (Table 1).
Amplification of cpDNA regions was performed in 20 lL
containing 1· enzyme buffer, 3.5 mM MgCl2, 200 lg mL)1
BSA, 200 lM dNTPs, 0.2 lM of each primer and 0.5 U Taq
polymerase (Roche, Penzberg, Germany) or DreamTaq
polymerase (Fermentas, St Leon-Rot, Germany). PCR
cycling conditions were as follows, with annealing temperature and elongation time depending on the amplified
region (Table 1): 94 C for 3 min, 35 cycles of 30 s at 93 C,
30 s at 52–62 C, 2–4 min at 72 C and a final extension of
7 min at 72 C. Amplicons were then digested with one, or
a cocktail of two restriction endonucleases (Table 1).
Restriction products were separated on 9% polyacrylamide gels (29 : 1 acrylamide : bisacrylamide) for trnK1trnK2, trnK2-trnQ, trnC-trnD, trnT-psbC, trnS-trnfM, and
trnV-rbcL, and 1.8% agarose gels for trnH-trnK. Gels were
stained with ethidium bromide and photographed under
UV light. For each polymorphic band in the restriction
profile, size variants were numbered with decreasing size,
whereas restriction-site polymorphisms were coded as 0
for the highest molecular weight band when it contained
the restriction site or, if one of the two bands observed
Table 1 Chloroplast DNA amplicons analysed in a PCR-RFLP analysis of diploid and tetraploid Salicornia, with primer information, PCR
conditions and restriction enzymes.
Amplicon
Reference for primers
Ta (C)
Extension
time (min)
Restriction
enzyme(s)
Number of polymorphic
bands (alleles)
trnH-trnK
trnK1-trnK2
trnK2-trnQ
trnC-trnD
trnT-psbC
trnS-trnfM
trnV-rbcL
Demesure et al. (1995)
Demesure et al. (1995)
Dumolin-Lapègue et al. (1997)
Demesure et al. (1995)
Dumolin-Lapègue et al. (1997)
Demesure et al. (1995)
Dumolin-Lapègue et al. (1997)
60
58
54
58
52
56
56
2
3
3
3
4
2
3
Hsp92II
HinfI + MboI
HinfI + RsaI
HinfI + RsaI
HinfI + MboI
HinfI + MboI
HinfI + MboI
1
3
4
4
5
2
4
(9)
(2,
(5,
(2,
(2,
(3,
(4,
2,
2,
6,
3,
2)
2,
5)
2, 2)
3, 2)
3, 3, 2)
2, 6)
Ta, annealing temperature.
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Reproductive isolation in Salicornia
when the restriction site was present showed size variants,
this band was coded as 0 in specimens in which the
restriction site was absent.
For simple sequence repeats (SSRs), the genotypes at
each locus were directly scored for diploid species from
the electropherograms using GE N E MA P P E R 4.1 (Applied
Biosystems, Foster, CA, USA). For tetraploids, the dosage
of the different alleles within a genome was unknown.
Microsatellite DNA Allele Counting using Peak Ratios
was introduced to quantify allelic configuration in
polyploids (Esselink et al., 2004). This technique relies
on the assumption that during PCRs, abundant alleles
within a locus should amplify more often than less
abundant ones. The relative peak areas found in peak
diagrams are therefore thought to be correlated with the
relative number of copies of that allele within the
genome. However, PCR selection caused by differential
primer affinity, allele size and PCR drift resulting from
events during early cycles of PCR causes a bias in
simultaneously amplified products (Wagner et al.,
1994). Peak area ratios in heterozygous diploid individuals thus nearly always differ from 1, requiring a
correction for differences in amplification success of
alleles. In hybrids, however, unequal amplification efficiency at microsatellite loci between species may occur,
so that individuals that are hybrids between these two
groups may not possess the same allele dosage (Vergilino
et al., 2009). In allotetraploids such as Salicornia (see
below), wherein differences in amplification patterns
between primer pairs are most likely due to incomplete
complementarity of the designed primers to the target
regions in the heterologous chromosomes (Catalán et al.,
2006; Palop-Esteban et al., 2007), the definition of clear
correction factors proves most difficult (Scheepens et al.,
2007; Gonzalez-Perez et al., 2009; Helsen et al., 2009). As
a consequence, each microsatellite allele was scored for
each specimen using a binary coding similar to that
employed with dominant markers, i.e. each allele was
scored as present ⁄ absent. This way of coding genotypes
can cause some redundancy (one locus can occur several
times in the data matrix), but it solves the problem of
assessing allele dosage and reduces the risk of mixing
genotypes from homeologous loci in tetraploids.
Data analysis
Global patterns of genetic variation
We first explored the SSR data matrix without making
any a priori hypothesis of structure in the data. The
mating system of Salicornia is characterized by high levels
of selfing (Dalby, 1962; Ferguson, 1964; Jefferies &
Gottlieb, 1982), hampering the use of techniques
such as those implemented by ST R U C T U R E (Pritchard
et al., 2000) that rely on Hardy–Weinberg equilibrium to
perform groupings. IN S T R U C T , an extension of ST R U C T U R E
that performs groupings from expected genotype frequencies based on inbreeding or selfing rates (Gao et al.,
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2007), and which is thus potentially well suited to strong
selfers like Salicornia, does not allow for both diploids and
tetraploids to be analysed simultaneously. We therefore
submitted the matrix of presence–absence of alleles to a
principal components analysis (PCA) performed on the
correlation matrix and implemented the Markov chains
Monte Carlo (MCMC) of IN S T R U C T within each of the
diploid and tetraploid cytotypes. The admixture model of
IN S T R U C T was run for K = 2–20 groups, and the deviance
information criterion was employed to determine the
optimal value of K. For each K, three MCMCs were run
for 500 000 iterations each and 100 000 steps of burnin.
Convergence of the three runs was assessed by means of
the Gelman–Rubin statistics.
For the chloroplast data, the minimum spanning haplotype network was computed using AR L E Q U I N 3.1. Support for clades was assessed by a cladistic analysis of the
matrix of presence ⁄ absence of bands through an adaptation of Jukes-Cantor’s substitution model for binary
characters (Lewis, 2001). Restriction-site data typically
suffer from a severe sampling bias because constant
characters are never recorded, leading to an overestimation of the transition rates and hence, the necessity to
apply a correction to the model (Felsenstein, 1992). This
correction was implemented by using the ‘variable’ coding
option of MrBayes 3.1 (Ronquist & Huelsenbeck, 2003).
The model was implemented in a Bayesian framework.
Four Metropolis-coupled Markov chain Monte Carlo of
four chains each were run for 10 000 000 generations with
MrBayes 3.1. Trees and model parameters were sampled
every 10 000 generations. The number of generations
needed to reach stationarity and chain convergence was
estimated by visual inspection of the plot of the loglikelihood score at each sampling point. The trees of the
‘burn-in’ for each run were excluded from the tree set,
and the remaining trees from each run were combined
to form the full sample of trees assumed to be representative of the posterior probability distribution.
Cytotypic partitioning of genetic diversity and variation
The MrBayes analysis was used to explicitly test whether
the cpDNA patterns were compatible with the expectations of a monophyletic origin of the tetraploids. The
analysis described above was re-run under the constraint
that only trees, which are compatible with a monophyletic origin of the diploid and tetraploid cytotypes, were
sampled. Bayes factors, measured by twice the difference
of the log marginal likelihoods of the two competing
models, were used to assess the significance of the
difference of the log-likelihoods returned by the constrained and unconstrained analyses. Threshold values of
2, 5 and 10 were taken as positive, strong and very strong
evidence for selecting a model over another, respectively
(Raftery, 1996).
We then measured the level of genetic diversity and
differentiation within and among cytotypes. Allelic richness (expected number of alleles when resampling a
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A. VANDERPOORTEN ET AL.
constant number of individuals, here three individuals)
was computed using the method of El Mousadik & Petit
(1996) for samples of unequal size. A t-test was then used
to compare the average allelic richness per population in
diploids vs. tetraploids. Fst and Nst were employed as a
measure of genetic differentiation for the SSR and cpDNA
data matrix, respectively. Nst is a measure of genetic
differentiation among populations analogous to Fst but
taking into account the phylogenetic relationships
between alleles (Pons & Petit, 1996). The latter was derived
from a matrix of mean character differences among
haplotypes as implemented by P A U P 4.0b10 (Swofford,
2003). Significance of Fst and Nst was tested by constructing
the distribution of the null hypothesis by means of 999
random permutations of individuals among populations as
implemented by SP A G E D I 1.3 (Hardy & Vekemans, 2002).
Finally, a discriminant analysis was employed to find
the allelic combinations that best allow for the determination of the cytotypes. Discriminant functions were
constructed for each of the SSR and cpDNA data sets. To
avoid that the frequencies of diploids and tetraploids in
the data directly influence the analysis, equal a priori
probabilities to belong to one of the two cytotypes were
given to each specimen. The discriminant functions were
then used to determine the probability that the specimens characterized by sub-equal flowers and attributed
to S. obscura, belong to the diploid or tetraploid cytotype.
The robustness of the discriminant functions was tested
by a cross-validation procedure, wherein the data were
divided into a training set and a test set. For that purpose,
the 5th, 10th, 15th, etc. lines of the matrix were removed
from the training set and included in the test set. The
analyses were re-performed on the training set, and the
probability to belong to the diploid or tetraploid cytotype
was determined for each specimen of the test set based
upon the discriminant functions independently derived
from the training set.
Geographic and taxonomic partitioning of genetic
variation
Genetic variation was partitioned among species and
geographic regions (Mediterranean and Atlantic) using
Fst and Nst for the SSRs and cpDNA data, respectively.
The existence of a phylogeographic signal in the chloroplast data was tested by assessing the significance of the
observed difference between Nst and Fst values by means
of 999 random permutations of the mean character
difference matrix among haplotypes. Indeed, when Nst
is significantly larger than Fst, it means that mutations
creating new haplotypes occur at a higher rate than the
gene flow of haplotypes among subdivisions, generating
a phylogeographic pattern. Fis, whose significance was
determined by means of 999 permutations of alleles
among individuals from the same population, was computed using SP A G E D I 1.3.
Finally, we investigated patterns of genetic differentiation at the scale of individuals within geographic
regions, both within and among species, along gradients
of geographic distance. For that purpose, we computed
pairwise similarity coefficients between conspecific individuals on the one hand, and between individuals
belonging to two different species on the other. For
SSR data, we estimated pairwise kinship coefficients
between individuals, Fij, using J. Nason’s estimator
(Loiselle et al., 1995). For the cpDNA data, Fij was also
computed as well as a Fij analogue for ordered alleles,
called Nij, taking the phylogenetic relationship among
haplotypes into account. More specifically, the estimated
and
parameters Fij and Nij are defined as Fij 1 hij =h
Nij 1 mij =m, where hij is the probability that two gene
copies from individuals i and j carry different alleles (or
haplotypes), mij is the phylogenetic distance between the
haplotypes carried by individuals i and j (mean character
and m are the
differences among haplotypes), while h
averages over all pairs of individuals in the sample of hij
and mij, respectively. Both Fij and Nij were computed from
global allele frequencies within each geographic region.
To test for isolation by distance, the significance of the
slope of the regression of Fij or Nij on the logarithm of
spatial distance between individuals, ln(dij), was tested by
means of 999 random permutations of population locations (Mantel test). The mean Fij or Nij values were also
computed over i, j pairs separated by predefined geographic distance intervals, d, giving F(d) and N(d).
Threshold distance separating intervals were 0, 10, 50,
100, 250, 500 and 1000 km, the first interval corresponding to pairs of individuals from the same population. For cpDNA data, the difference between N(d) and
F(d) was tested by means of 999 random permutations
of the genetic distance matrix to test the presence of a
phylogeographic signal at different spatial scales. All
computations were performed using SP A G E D I 1.3.
Results
Nuclear SSR and cpDNA data
The number of nuclear microsatellite alleles at each locus
is given in Table 2. Diploids and tetraploids substantially
Table 2 Number of alleles and proportion (%) of homozygous
and heterozygous genotypes at six nuclear microsatellite loci
in diploid and tetraploid Salicornia sampled along the Atlantic
and Mediterranean coasts of France.
2n
S2
S5
S7
S8
S10
S19
4n
Nb of
alleles
Homozygous ⁄
heterozygous
Nb of
alleles
Homozygous ⁄
heterozygous
3
4
5
4
5
5
98 ⁄ 2
98 ⁄ 2
82 ⁄ 18
91 ⁄ 9
88 ⁄ 12
93 ⁄ 7
4
3
5
5
5
6
10 ⁄ 90
35 ⁄ 65
46 ⁄ 54
51 ⁄ 49
88 ⁄ 12
7 ⁄ 93
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Reproductive isolation in Salicornia
Fig. 2 Principal component analysis of diploid and tetraploid
Salicornia sampled along the Atlantic and Mediterranean coasts
of France and genotyped at six nuclear microsatellite loci.
differ by their degree of heterozygosity at all loci but S10,
at which the vast majority of specimens were homozygous for both cytotypes (Table 2).
The PCR-RFLP analysis of the cpDNA revealed a total
of 23 polymorphic bands, allowing for the distinction of
37 haplotypes. Fifteen haplotypes were restricted to
diploids, which exhibited a strong biogeographic differentiation because 10 were sampled from the Atlantic
(haplotypes F, G, H, I, J, K, L, Q, R, AH) and four from
the Mediterranean (D, T, U, V). Sixteen other haplotypes
were restricted to tetraploids, with 11 sampled from the
Atlantic (E, W, Z, AB, AD, AE, AF, AG, AI, AJ, AL) and
four from the Mediterreanean (A, B, C, AC). The six
remaining haplotypes were shared by diploids and
tetraploids, with five restricted to the Atlantic (M, N, O,
P, AK) and one to the Mediterranean (X).
Global genetic structure
The PCA performed on the presence ⁄ absence of alleles
at the six nuclear microsatellite loci shows that the main
differentiation among specimens is among cytotypes
(Fig. 2). Along PCA1, which accounts for 18.1% of the
total variance, the correlation coefficient between the
specimen scores and their cytotypic identity is 0.87
(P < 0.001). PCA2, which accounts for another 7.1% of
the total variance, discriminates diploid Mediterranean
and Atlantic specimens. PCA1 is positively correlated
with allele S5-106 (0.72), which is opposed to alleles
S2-124 ()0.85), S5-102 ()0.77), S5-104 ()0.76), S8-128
()0.71), S19-183 ()0.83). Several alleles are thus
diagnostic for cytotypic identity, but none are strictly
635
restricted to a cytotype. For example, allele S2-124 and
S19-183 have a frequency of 2 and 1% in diploids,
whereas they reach a frequency of 90 and 88% in
tetraploids, respectively. Along PCA2, S8-124 (0.52) and
S19-175 (0.49) are opposed to S19-177 ()0.46), S10-172
()0.58) and S8-126 ()0.66).
For the IN S T R U C T analyses, the Gelman–Rubin statistics
was systematically < 1.10 for each value of K, indicating
good chain convergence. In both the diploid and tetraploid datasets, the log-likelihoods increased with the
value of K, and the model with the highest value of K set
here, i.e. K = 20, was systematically identified as the
best-fit model based upon the deviance information
criterion.
The network representation of the relationships among
the cpDNA haplotypes of the sampled Salicornia is
presented in Fig. 3. Five groups of haplotypes, hereafter
referred to as groups I–V, can be distinguished. Groups I,
IV and V, which have a posterior probability (p.p.) of
0.83, 1.00 and 1.00 in the Bayesian analysis, respectively,
include nearly exclusively tetraploids. Group V almost
only contains Atlantic tetraploid haplotypes, but AK
includes 6% of diploid specimens. Group I mostly groups
both Mediterranean and Atlantic tetraploid haplotypes,
although some Mediterranean diploids (9% of haplotype
X and 58% of haplotype AA) are also included. Groups II
and III, which have a p.p. of 0.97, only contain diploids.
None of the species were defined by monophyletic
chloroplast lineages, and most haplotypes are shared
among several species. By contrast, most haplotypes tend
to be unique to either the Mediterranean or Atlantic
regions, and a strong geographic structure is evident
within each of the diploid and tetraploid lineages. In
diploids, all the Mediterranean haplotypes are clustered
within group II, whereas all the Atlantic haplotypes
cluster together within group III.
Genetic diversity and differentiation between diploid
and tetraploid cytotypes
The 50% majority-rule consensus from the trees sampled
from the posterior probability distribution generated by
the MrBayes analysis of the cpDNA dataset included a
large polytomy at the basis of the tree. However,
constraining the diploid and tetraploid cytotypes to
monophyly led to a significant difference in marginal
log-likelihood ()1482 and )1542 in the unconstrained
and constrained analyses, respectively).
Fst values among cytotypes are all significant at the
0.001 level and are only marginally higher than intracytotypic comparisons among biogeographic regions for
both the nuclear and cpDNA markers (Table 3).
The cytotypic identity of all the specimens was
correctly recovered by the discriminant analyses employing the cpDNA data at 96.4% (97% for the diploids and
95.5% for the tetraploids). After cross-validation, the
correct classification rate remained at 96%. With the SSR
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A. VANDERPOORTEN ET AL.
Fig. 3 cpDNA haplotypic network from the
analysis of PCR-RFLP markers in diploid and
tetraploid Salicornia sampled along the
Atlantic and Mediterranean coasts of France.
Bars along the branches represent single
mutational steps. Roman numbers identify
clades of haplotypes discussed in the text.
Each circle represents a haplotype. Circle size
is proportional to the number of specimens
included in the haplotype, and colour patterns indicate the species identification and
geographic origin of those specimens. Numbers within clades are the posterior probabilities, as assessed by a Bayesian inference
implementing a transition model among
states for the PCR-RFLP profiles (see text
for details).
Table 3 Genetic differentiation between geographic regions
(Atlantic and Mediterranean) and cytotypes (diploid and tetraploid)
in Salicornia: pairwise Fst values for the SSRs and Nst for the cpDNA
markers are indicated above and below the diagonal, respectively.
All Fst and Nst values are significant at the 0.001 level.
2n
2n
4n
4n
Mediterranean
Atlantic
Mediterranean
Atlantic
2n
Mediterranean
2n
Atlantic
4n
Mediterranean
4n
Atlantic
–
0.89
0.63
0.52
0.26
–
0.70
0.63
0.43
0.31
–
0.38
0.49
0.41
0.19
–
data matrix, the correct classification rate was 99.4%
(99.7% for the diploids and 99% for the tetraploids), and
these values only slightly dropped after cross-validation
(overall correct classification rate of 96.5%, with 98.9%
for the diploids and 92% for the tetraploids). The
Table 4 Pairwise comparisons of Fst (SSR, above the diagonal)
and Nst (cpDNA, below the diagonal) among Salicornia patula,
S. ramosissima, S. obscura and S. pusilla sampled along the Atlantic
and Mediterranean coasts of France. All of the presented Fst and Nst
values are significantly different from 0 at the 0.001 probability level.
S.
S.
S.
S.
patula
ramosissima
obscura
pusilla
S. patula
S. ramosissima
S. obscura
S. pusilla
–
0.67
0.88
0.94
0.17
–
0.17
0.18
0.25
0.03
–
0.25
0.30
0.05
0.02
–
discriminant functions derived from the SSR and cpDNA
data were consistent in identifying the same 35% of the
specimens attributed to S. obscura as being tetraploids.
Allelic richness for diploid and tetraploid populations
is documented in Table S1. Average allelic richness did
not significantly differ between diploids and tetraploids,
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Reproductive isolation in Salicornia
637
Table 5 Geographic (Atlantic vs. Mediterranean) and taxonomic (species assignation) partitioning of genetic variation in diploid
Salicornia from the Mediterranean and Atlantic coasts of France. Values above and below the diagonal are Fst derived from the analysis
of nuclear SSR data and Nst derived from the analysis of PCR-RFLP patterns at seven cpDNA loci, respectively. All Fst and Nst are significant
at the 0.001 level except for the Fst between S. patula and S. ramosissima from the Mediterranean (P < 0.05).
S. patula, Mediterranean
S. ramosissima, Atlantic
S. obscura, Atlantic
S. pusilla, Atlantic
S. ramosissima,
Mediterranean
S. patula,
Mediterranean
S. ramosissima,
Atlantic
S. obscura,
Atlantic
S. pusilla,
Atlantic
S. ramosissima,
Mediterranean
–
0.91
0.88
0.94
< 0.01
0.28
–
0.11
0.05
0.88
0.25
0.03
–
0.25
0.88
0.30
0.05
0.02
–
0.92
0.03
0.30
0.28
0.33
–
neither in the chloroplast (1.38 vs. 1.34) nor in the
nucleus (1.89 vs. 1.78).
Taxonomic and geographic partitioning
of genetic variation
Taxonomic partitioning (Table 4) accounted substantially
less than geographic partitioning (Table 5) for the
observed patterns of genetic variation. The global Fst
and Nst resulting from taxonomic partitioning (i.e. among
species) of the SSR and cpDNA data, respectively, were
0.010 (P < 0.001) and 0.69 (P < 0.001). By comparison,
the Fst and Nst resulting from the geographic partitioning
of the SSR and cpDNA data between the Atlantic and
Mediterranean reached 0.27 (P < 0.001) and 0.89
(P < 0.001), respectively. The latter was significantly
higher than the cpDNA Fst (0.41), indicating the presence
of a significant phylogeographic signal.
Pairwise species differentiation levels substantially
droped when performed within the same geographic
region (Table 5). For example, while SSR Fst and cpDNA
Nst between S. ramosissima and S. patula were 0.17 and
0.67, respectively (Table 4), these values both droped to
0.03 within the Mediterranean. By comparison, SSR Fst
and cpDNA Nst within S. ramosissima reached respectively
Table 6 Geographic (Atlantic vs. Mediterranean) and taxonomic
(species assignation) partitioning of genetic variation for tetraploid
Salicornia from the Mediterranean and Atlantic coasts of France.
Values above and below the diagonal are Fst derived from the
analysis of nuclear SSR data and Nst derived from the analysis of
PCR-RFLP patterns at seven cpDNA loci, respectively. All Fst and
Nst are significant at the 0.001 level.
S.
fragilis
S. fragilis
S. dolichostachya
S. emericii
(Mediterranean)
S. emericii
(Atlantic)
S.
dolichostachya
S. emericii
(Mediterranean)
S. emericii
(Atlantic)
–
0.09
0.56
0.05
–
0.60
0.21
0.24
–
0.05
0.04
0.11
< 0.01
< 0.01
0.56
–
0.30 and 0.52 between Mediterranean and Atlantic
populations.
Similar results were obtained with the tetraploids.
Geographic partitioning of the genetic variation included
in the SSR and cpDNA data resulted in Fst and Nst values
between the Mediterranean and Atlantic coasts of
0.18 (P < 0.001) and 0.37 (P < 0.001), respectively. By
comparison, taxonomic partitioning among the three
tetraploid species resulted in a global Fst for SSR and Nst
for the cpDNA data set of 0.08 (P < 0.001) and 0.20
(P < 0.001). Pairwise comparisons among species and
geographic regions (Table 6) similarly showed that the
highest values of genetic differentiation were reached
at the inter-regional level.
F and N statistics and Mantel tests among
individuals within geographic regions
The variation of the average kinship coefficients within
and among species along a gradient of geographic
distance was very similar in diploids (Figs 4 and 5) and
tetraploids (see Figs S1 and S2). Globally, mean SSR Fij
among conspecific individuals were only significant at
the local scale. Average Fij among pairs of individuals
from different species were lower, but significant at that
scale. Beyond the population scale, none of the Fij
comparisons were significant. For the cpDNA data, all
mean Nij values within and among species reached 1 at
the local scale and were significantly different from 0, but
not significantly higher than the respective mean Fij
values. There was a progressive decrease of Nij values
with geographic distance; hence, the slope of regression
between the Nij and geographic distance was significantly
different from 0.
Discussion
Differentiation and reproductive isolation
between diploids and tetraploids
The clear and almost complete segregation of diploids
and tetraploids based on the PCA of the nuclear SSRs
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A. VANDERPOORTEN ET AL.
(a)
P > 0.05
P > 0.05
P < 0.01
(b)
P < 0.01
P < 0.001
P < 0.001
Fig. 4 Mean Fij resulting from the comparisons of individual SSR genotypes (a) and
mean Nij resulting from the comparisons of
individual cpDNA haplotypes (b) within and
among the diploid Salicornia patula and
S. ramosissima from the Mediterranean coasts
of France depending on the geographic
distance separating them. The P-value of the
slope of the regression between the Fij and
Nij values and geographic distance are
given in the upper right corners.
and the cpDNA network suggests the presence of a
strong reproductive barrier among cytotypes in Salicornia, in agreement with several observations of complete
reproductive barriers among sympatric cytotypes (Hardy
et al., 2000; Husband & Sabara, 2003; Kloda et al.,
2008). SSR and cpDNA alleles typical for tetraploids
were, however, occasionally found in diploids Allele
sharing between diploids and tetraploids at SSRs loci
may be because of homoplastic mutations or null
alleles. The sharing of SSR alleles or cpDNA haplotypes
by diploid and tetraploid lineages may also be explained
by the retention of ancestral polymorphisms from their
diploid ancestors within the tetraploid lineages. Such an
interpretation is fully compatible with the very recent
origin of the genus, dated to 1.8–1.4 myrs (Kadereit
et al., 2006). However, the sharing of alleles among
sympatric individuals from different cytotypes, along
with the occurrence of specimens with a typical diploid
morphology and nuclear SSR patterns but with a
cpDNA haplotype characteristic for tetraploids, is rather
indicative of instances of inter-cytotypic gene flow. This
hypothesis is favoured by Kaligaric et al. (2008) to
explain the sharing of cpDNA haplotypes between
Mediterranean diploids and tetraploids and in fact,
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639
(a)
(b)
Fig. 5 Mean Fij resulting from the comparisons of individual SSR genotypes (a) and
mean Nij resulting from the comparisons of
individual cpDNA haplotypes (b) within and
among the diploid Salicornia ramosissima,
S. obscura and S. pusilla from the Atlantic
coasts of France depending on the
geographic distance separating them. The
P-value of the slope of the regression
between the Fij and Nij values and
geographic distance are given in the
upper right corners.
although there are few examples of the process in the
wild, the existence of gene flow among cytotypes has
long been recognized (see Chapman & Abbott, 2010, for
review).
The high correlation between cytotypes and genotypes
observed here confirms previous assessments on the
reliability of flower morphology to distinguish among
diploid and tetraploid Salicornia (e.g. Lahondère, 2004;
Kaligaric et al., 2008). Those cpDNA and SSR markers
may therefore prove useful for the determination of the
ploidy level of herbarium material, juvenile specimens or
specimens with an ambiguous flower morphology. This is
especially the case of S. obscura, whose lateral and
median flowers are subequal in size, rendering the
confusion with both diploid and tetraploid species possible (see Lambinon & Vanderpoorten, 2009, for review).
In fact, the molecular analysis of the specimens attrib-
uted to S. obscura in the present study revealed that about
one-third of them were tetraploids. This suggests that
S. obscura has served as a convenient taxonomic repository for morphologically ill-characterized diploid or
tetraploid specimens.
Origin and evolution of the tetraploids
The tetraploids exhibit high levels of heterozygosity.
Heterozygosity is fixed in the vast majority of populations
at loci S2 and S19, with > 95% of heterozygous profiles.
More than 50% of specimens are heterozygous at loci S5,
S7 and S8. These patterns are consistent with previous
isozyme analyses, wherein diploids were strictly homozygous and tetraploids showed either a homozygous or a
fixed heterozygous profile (Wolff & Jefferies, 1987).
Although the chances of producing an autopolyploid
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A. VANDERPOORTEN ET AL.
from two unreduced gametes fusing are greatly increased
in strong selfers like Salicornia (Shepherd & Yan, 2003),
the most parsimonious explanation is that the tetraploids
are of allopolyploid origin. Indeed, although the lack of
fixed heterozygosity may strengthen the evidence for
autopolyploidy (Soltis & Rieseberg, 1986), fixed heterozygosity at many loci in a polyploid is commonly used as
evidence for allopolyploidy (Arft & Ranker, 1998; Såstad
et al., 2001; Nyberg Berglund et al., 2006). In allopolyploids, indeed, parental genomes may be different enough
for chromosome pairing to occur only between chromosomes that originate from the same parental genome.
Alleles of each parental genome segregate as if they were
from a diploid with disomic inheritance. If the parental
genomes are homozygous for different alleles, all gametes
will be heteroallelic and all offspring will be heterozygous, i.e. heterozygosity will be fixed. The increased
heterozygosity resulting from hybridization in tetraploid
Salicornia may, ultimately, result in the formation of new
gene combinations and generation of new forms of
enzymes, and be critical for the successful establishment
in unstable environments with recurrent flooding periods where they typically occur.
At the population level, given the strong reproductive
barrier among cytotypes, and provided that the diploids
did not suffer more substantially than the tetraploids in
the course of the last glaciations, the comparable levels of
diversity observed in tetraploids and diploids either
suggest an ancient and ⁄ or multiple origin of polyploids.
Indeed, polyploids are expected to harbour less genetic
diversity if polyploid formation is a rare and ⁄ or recent
event. This is because, although allotetraploids potentially accumulate genetic variation at a faster rate than
diploids, newly formed polyploids start out with limited
genetic diversity because of founding effects. It therefore
takes a considerable amount of time to reach equilibrium
between mutation and drift, and ultimately higher levels
of genetic diversity (Luttikhuizen et al., 2007). An
ancient origin of allotetraploid Salicornia would be consistent with the idea that fast mutation rates at the
microsatellite loci would have regenerated the loss of
diversity following speciation. The only marginally higher differentiation among than within cytotypes with the
SSRs, and the actually higher differentiation within
diploids than between diploids and tetraploids in the
chloroplast, contrast with the ancient origin hypothesis.
The latter is further weakened by the recent origin of the
genus (Kadereit et al., 2006) and the lack of phylogenetic
resolution within cytotypes, which has been interpreted
in terms of a recent and rapid expansion (Kadereit et al.,
2007; Murakeözy et al., 2007).
Thus, an alternative explanation for the observed
patterns of diversity in tetraploids is that the latter
evolved recurrently from diploids. Although a monophyletic origin of European tetraploids was resolved from
the phylogenetic analysis of nrDNA ETS sequences
(Kadereit et al., 2007), which is consistent with the
clear differentiation among cytotypes observed here
using nuclear SSRs, a monophyletic origin of the cpDNA
lineages observed among tetraploids was statistically
rejected. This suggests that allopolyploidization has happened several times from a common gene pool, adding
to the mounting evidence for a recurrent origin of
polyploids (see Albach, 2007, for review).
Geographic and taxonomic partitioning of genetic
variation within cytotypes
Although none of the species, as circumscribed by the
most complex taxonomic treatments of, e.g. Lahondère
(2004) and Stace (2010), are defined by monophyletic
chloroplast lineage, partitioning of genetic variation
among species is weak, but significant. The strong
underlying phylogeographic structure, however, largely
contributes to this differentiation. Levels of differentiation between populations of different species within the
same region are indeed very low when compared to
those among conspecific populations from different
regions. A significant phylogeographic signal is in fact
present in the cpDNA data between the Mediterranean
and the Atlantic, suggesting that these two regions were
colonized anciently and accumulated mutations at a
faster rate than migration events. These findings are
consistent with the eastern ⁄ western pattern of differentiation found by Kadereit et al. (2007).
At the local scale, the results further indicate that
taxonomy cannot be retrieved from analyses of genetic
relationships. In fact, mean cpDNA Nij kinship coefficients are equal to 1 within and among species. Similarly,
although conspecific individuals tend to be significantly
more related than individuals from different species at
the population scale (see below), kinship coefficients
derived from SSR variation among individuals from
different species are not significantly lower than those
within species as soon as individuals from different
populations are considered. The poor relation between
taxonomy and genetic variation documented here is
consistent with previous analyses using AFLPs (Le Goff,
1999) and DNA sequences (Murakeözy et al., 2007;
Kadereit et al., 2007), which failed to resolve monophyletic species groups. This is, however, counter-intuitive
given the apparently clear circumscription of at least
some species, like S. pusilla, which is readily recognized
by its solitary flowers.
In Salicornia, phenotypic plasticity has been suggested
as the main factor accounting for the incongruence
observed between traditional species concepts and patterns of genetic differentiation (Murakeözy et al., 2007).
Owing to their extremely reduced morphology, Salicornia
species are defined based on global branching architecture and shape; colour; and size of the lateral vs. central
flowers, i.e. a suite of quantitative characters that are
indeed arguably more prone to plasticity than complex
flower characters found in other groups. The hypothesis
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Reproductive isolation in Salicornia
that plasticity accounts for the incongruence between
species concepts and patterns of genetic differentiation is,
however, at odds with the fact that mean Fij among pairs
of conspecific individuals in the SSR data tend to be
significantly higher than those between pairs of individuals from different species at the population scale,
thereby suggesting that the morphological differentiation
has a genetical basis. Transplantation experiments indeed
revealed that individuals tend to retain their specific
morphology (Kadereit et al., 2007).
Another interpretation of the incongruence between
taxonomy and genetic variation in Salicornia is the lack of
inter-specific reproductive barriers. Salicornia species
have, however, traditionally been considered as strong
if not complete selfers (Dalby, 1962; Ferguson, 1964). A
100% inbreeding rate was, for instance, reported based
upon the genetic identity between 38 maternal plants
and 2112 F1 progenies (Noble et al., 1992). In diploid
species indeed, the anthers usually dehisce before they
are exserted and ripe dehiscing anthers may be seen in
contact with presumably receptive stigmas, their pollen
spilling onto the stigmatic papillae. Cleistogamy has, in
addition, been reported in many instances (see Kadereit
et al., 2007, for review). Pistillate flowers can, however,
be observed in S. ramosisima (Kadereit et al., 2007),
suggesting that outbreeding by wind pollination cannot
be completely ruled out. In addition, heterozygosity was
recurrently observed in diploids, and the mean level of
inbreeding observed within diploid populations
(Fis = 0.70) indicates an outcrossing rate of 18% if only
selfing accounts for the heterozygote deficit. Such an
interpretation is consistent with the existence of fertile
inter-specific hybrids such as S. · marshallii between
S. pusilla and S. ramosissima, which is readily distinguished by a combination of inflorescences with a single
flower, as in S. pusilla, two flowers, and three flowers, as
in S. ramosissima. The hybrid nature of such specimens
with an intermediate morphology was confirmed by use
of the present SSR markers because they display heterozygous genotypes at loci with different homozygous
genotypes within sympatric S. pusilla and S. ramosissima.
The absence of interspecifric reproductive barriers is also
suggested by the partial incongruence reported among
nuclear and cpDNA sequences (Murakeözy et al., 2007;
Kaligaric et al., 2008). Salicornia is comparable, in this
respect, to Quercus, wherein species are not well differentiated genetically owing to the sharing of common
cpDNA haplotypes among sympatric species, suggesting
appreciable localized cytoplasmic gene flow and high
levels of cpDNA fixation within populations (Whittemore
& Schaal, 1991), whilst haplotypes sampled among
allopatric conspecific specimens are highly divergent
(Petit et al., 1993; Manos et al., 1999).
A third and not mutually exclusive interpretation is
that the observed morphological differences are not
the result of divergent genomes, but are based on single
or few point mutations, or even to changes in the
641
mechanisms of gene regulation affecting when and
where a gene is expressed. In the beach mouse, for
example, a single amino acid mutation contributes to
adaptive colour pattern (Hoekstra et al., 2006). In other
taxa with reduced morphologies like mosses, Hedenäs &
Eldenäs (2008) and Sotiaux et al. (2009) similarly evoked
the possibility that a single or a few genes may be
responsible for dramatic morphological modifications,
whereas the remaining of the genome had no time to sort
out. This interpretation is in line with Kadereit’s et al.
(2007) hypothesis of recurrent evolution of a species
such as S. pusilla from S. ramosissima. The observed
association between morphology and genetic variation
within populations would thus result from the selfing
mating system generating substantial linkage within the
genome, linkage that would quickly disappear among
unrelated individuals from different populations.
Kinship coefficients derived from both the nuclear and
cpDNA markers significantly decreased as soon as individuals from different populations were considered,
indicating an extremely low mobility of both seeds and
pollen. In fact, Salicornia seeds lack specialized devices for
dispersal. Fifty per cent of the seeds can be found within
a distance of 10 cm from the mother plant, and most are
trapped in sediments, algae or marsh vegetation (see
Kadereit et al., 2007, for review). Whilst rare events of
long-distance dispersal, probably aided by zoochory,
might explain some trans-oceanic phylogenetic patterns
(Kadereit et al., 2007), this suggests that routine dispersal
at the regional scale is extremely limited in the genus.
The existence of strongly inbred, disconnected populations might explain why, although a significant geographic partitioning of genetic variation is evidenced by
the F statistics described above, the log-likelihood values
of the models implemented by IN S T R U C T steadily
increased with the value of K. Under this hypothesis,
distinctive traits among species of a same cytotype would
represent genetic variation within a single gene pool
displaying discontinuous variation because of the coexistence of inbred lineages.
Whatever the evolutionary mechanisms behind it, our
results thus strongly suggest that the observed range of
morphological variation in Salicornia is unparalleled by
genetic differentiation. The results presented here do not
support the most complex taxonomic treatments of
Lahondère (2004) and Stace (2010) but rather fit with
the broad species aggregate concept of, e.g. Valdés &
Castroviejo (1990) and Piirainen (2001). Given the
strong geographic signal in the data, one possibility
would be to recognize, within the study area, one
Mediterranean and one Atlantic species within each of
the diploid and tetraploid lineages. Such species would be
‘cryptic’ in the sense that they would not be characterized morphologically. ‘Cryptic’ species have increasingly
been recognized in other organisms with reduced morphologies like bryophytes (see Vanderpoorten et al.,
2010b, for review). Recognition of such lineages solely
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A. VANDERPOORTEN ET AL.
characterized by their geographic range would, however,
dramatically increase the number of ‘species’ in taxa with
a substantial geographic structure across their distribution range. Definite taxonomic conclusions will, therefore, be presented elsewhere based upon a comparative
analysis of all source of information available, including
morphology, cytology and various genetic markers
collected for a representative numbers of all Salicornia
species across their entire distribution range.
Acknowledgments
This study was made possible thanks to grant 2. 4504.05
of the Belgian Funds for Scientific Research (F.R.S.
FNRS). The authors are very thankful to C. Lahondère,
G. Paradis and B. Toussaint, who contributed to the
fieldwork and checked our identifications, and to two
reviewers for their constructive comments on the manuscript. We also acknowledge the Cornell Computational
Biology Service Unit (CBSU).
References
Abbott, R.J. & Brochmann, C. 2003. History and evolution of the
arctic flora: in the footsteps of Eric Hultén. Mol. Ecol. 12: 299–
313.
Albach, D.C. 2007. Amplified fragment length polymorphisms
and sequence data in the phylogenetic analysis of polyploids:
multiple origins of Veronica cymbalaria (Plantaginaceae). New
Phytol. 176: 481–498.
Arft, A.M. & Ranker, T.A. 1998. Allopolyploid origin and
population genetics of the rare orchid Spiranthes diluvialis.
Am. J. Bot. 85: 110–122.
Brochmann, C., Brysting, A.K., Alsos, I.G., Borgen, L., Grundt,
H.H., Scheen, A.C. et al. 2004. Polyploidy in arctic plants. Biol.
J. Linn. Soc. 82: 521–536.
Catalán, P., Segarra-Moragues, J.G., Palop-Esteban, M., Moreno,
C. & González-Candelas, F. 2006. A Bayesian approach for
discriminating among alternative inheritance hypotheses in
plant polyploids: the allotetraploid origin of genus Borderea
(Dioscoreaceae). Genetics 172: 1939–1953.
Chapman, R.A. & Abbott, R. 2010. Introgression of fitness genes
across a ploidy barrier. New Phytol. 186: 63–71.
Dalby, D.H. 1962. Chromosome number, morphology and
breeding behaviour in the British Salicorniae. Watsonia 5:
150–162.
Davy, A.J., Bishop, F. & Costa, C.S.B. 2001. Salicornia L.
(Salicornia pusilla J. Woods, S. ramosissima J. Woods, S. europaea
L., S. obscura P.W. Ball & Tutin, S. nitens P.W. Ball & Tutin,
S. fragilis P.W. Ball & Tutin and S. dolichostachya Moss). J. Ecol.
89: 684–707.
Demesure, B., Sodzi, N. & Petit, R.J. 1995. A set of universal
primers for amplification of polymorphic non-coding regions
of mitochondrial and chloroplast DNA in plants. Mol. Ecol. 4:
129–131.
Doyle, J.J. & Doyle, J.L. 1987. Preservation of plant samples for
DNA restriction endonuclease analysis. Taxon 36: 715–722.
Duckert-Henriod, M.M. & Favarger, C. 1987. Contribution à la
cytotaxonomie et à la cytogéographie des Poa de la Suisse. Birkhäuser, Basel.
Dumolin-Lapègue, S., Pemonge, M.H. & Petit, R.J. 1997. An
enlarged set of consensus primers for the study of organelle
DNA in plants. Mol. Ecol. 6: 393–397.
El Mousadik, A. & Petit, R.J. 1996. High level of genetic
differentiation for allelic richness among populations of the
argan tree (Argania spinosa (L.) Skeels) endemic to Morocco.
Theor. Appl. Genet. 92: 832–839.
Esselink, G.D., Nybom, H. & Vosman, B. 2004. Assignment of
allelic configuration in polyploids using the MAC-PR (microsatellite DNA allele counting-peak ratios) method. Theor. Appl.
Genet. 109: 402–408.
Felsenstein, J. 1992. Phylogenies from restriction sites: a maximum-likelihood approach. Evolution 46: 159–173.
Ferguson, I.K. 1964. Notes on the stigma morphology and
flowering behaviour in British Salicorniae. Watsonia 6: 25–27.
Gao, H., Williamson, S. & Bustamante, C.D. 2007. A Markov
chain Monte Carlo approach for joint inference of population
structure and inbreeding rates from multilocus genotype data.
Genetics 176: 1635–1651.
Géhu, J.M. (ed.) 1992. Phytosociologie littorale et Taxonomie.
J. Cramer, Berlin.
Géhu, J.M. & Bioret, F. 1992. Etude synécologique et phytocoenotique des communautés à salicornes des vases salées du
littoral breton. Bull. Soc. Bot. Centre-Ouest 23: 347–419.
Gonzalez-Perez, M.A., Lledo, M.D., Lexer, C., Fay, M., Marrero,
M., Banares-Baudet, A. et al. 2009. Genetic diversity and
differentiation in natural and reintroduced populations of
Bencomia exstipulata and comparisons with B. caudata (Rosaceae) in the Canary Islands: an analysis using microsatellites.
Bot. J. Linn. Soc. 160: 429–441.
Hardy, O.J. & Vekemans, X. 2002. SPAGeDi: a versatile
computer program to analyse spatial genetic structure at the
individual or population levels. Mol. Ecol. Notes 2: 618–620.
Hardy, O.J., Vanderhoeven, S., De Loose, M. & Meerts, P. 2000.
Ecological, morphological and allozymic differentiation
between diploid and tetraploid knapweeds (Centaurea jacea)
from a contact zone in the Belgian Ardennes. New Phytol. 146:
281–290.
Hedenäs, L. & Eldenäs, P. 2008. Relationships in Scorpidium
(Calliergonaceae, Bryophyta), especially between S. cossonii
and S. scorpioides. Taxon 57: 121–130.
Helsen, P., Verdyck, P., Tye, A. & Van Dongen, S. 2009. Low
levels of genetic differentiation between Opuntia echios
varieties on Santa Cruz (Galapagos). Plant Syst. Evol. 279: 1–10.
Hewitt, G.M. 2004. Genetic consequences of climatic oscillations in the Quaternary. Phil. Trans. Roy. Soc. London 359: 183–
195.
Hoekstra, H.E., Hirschmann, R.J., Bundey, R.A., Insel, P.A. &
Crossland, J.P. 2006. A single amino acid mutation contributes to adaptive beach mouse color pattern. Science 313: 101–
104.
van Hulzen, J.B., van Soelen, J., Herman, P.M.J. & Bouma, T.J.
2006. The significance of spatial and temporal patterns of algal
mat deposition in structuring salt marsh vegetation. J. Veg. Sci.
17: 291–298.
Husband, B.C. & Sabara, H.A. 2003. Reproductive isolation
between autotetraploids and their diploid progenitors in
fireweed, Chamerion angustifolium (Onagraceae). New Phytol.
161: 703–713.
Jefferies, R.L. & Gottlieb, L.D. 1982. Genetic differentiation of
the microspecies Salicornia europaea L. (sensu stricto) and
S. ramosissima J. Woods. New Phytol. 92: 123–129.
ª 2010 THE AUTHORS. J. EVOL. BIOL. 24 (2011) 630–644
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Reproductive isolation in Salicornia
Kadereit, G., Mucina, L. & Freitag, H. 2006. Phylogeny of
Salicornioideae (Chenopodiaceae): diversification, biogeography, and evolutionary trends in leaf and flower morphology.
Taxon 55: 617–642.
Kadereit, G., Ball, P., Beer, S., Mucina, L., Sokoloff, D., Teege, P.
et al. 2007. A taxonomic nightmare comes true: phylogeny
and biogeography of glassworts (Salicornia L., Chenopodiaceae). Taxon 56: 1143–1170.
Kaligaric, M., Bohanec, B., Simonovik, B. & Sajna, N. 2008.
Genetic and morphologic variability of annual glassworts
(Salicornia L.) from the Gulf of Trieste (Northern Adriatic).
Aquat. Bot. 89: 275–282.
Kloda, J.M., Dean, P.D.G., Maddren, C., McDonald, D.W. &
Mayes, S. 2008. Using principle component analysis acorss
polyploidy levels within plant complexes: an example from
british restharrows (Ononis spinosa and Ononis repens). Heredity
100: 253–260.
Lahondère, C. 2004. Les salicornes s.l. (Salicornia L., Sarcocornia
A. J. Scott et Arthrocnemum Moq.) sur les côtes françaises. Bull.
Soc. Bot. Centre-Ouest Numéro Spécial 24: 1–122.
Lambinon, J. & Vanderpoorten, A. 2009. Les salicornes (Salicornia s.l.), groupe taxonomique emblème de la flore des sols
salés et de sa complexité. Bull. Soc. Linn. Nord-Picardie 2007:
11–17.
Le Goff, F. 1999. Analyse des paramètres biotiques et abiotiques pour
une exploitation maı̂trisée des salicornes. PhD thesis, Rennes.
Lewis, P.O. 2001. A likelihood approach to estimating phylogeny
from discrete morphological character data. Syst. Biol. 50: 913–
925.
Loiselle, B.A., Sork, V.L., Nason, J. & Graham, C. 1995. Spatial
genetic structure of a tropical understory shrub, Psychotria
officinalis (Rubiaceae). Am. J. Bot. 82: 1420–1425.
Lorenzoni, C., Géhu, J.M., Lahondère, C. & Paradis, G. 1993.
Description phytosociologique et cartographique de la végétation de l’étang de Santa Giulia (Corse-du-Sud). Bull. Soc. Bot.
Centre-Ouest 24: 121–150.
Luttikhuizen, P.C., Stift, M., Kuperus, P. & van Tienderen, P.H.
2007. Genetic diversity in diploid vs. tetraploid Rorippa
amphibia (Brassicaceae). Mol. Ecol. 16: 3544–3553.
Manos, P.S., Doyle, J.J. & Nixon, K.C. 1999. Phylogeny,
biogeography, and processes of molecular differentiation in
Quercus subgenus Quercus (Fagaceae). Mol. Phylogenet. Evol. 12:
333–349.
Masterson, J. 1994. Stomatal size in fossil plants: evidence
for polyploidy in majority of angiosperms. Science 264: 421–
423.
Murakeözy, E.P., Ainouche, A., Meudec, A., Deslandes, E. &
Poupart, N. 2007. Phylogenetic relationships and genetic
diversity of the Salicornieae (Chenopodiaceae) native to the
Atlantic coasts of France. Plant Syst. Evol. 264: 217–237.
Noble, S.M., Davy, A.J. & Oliver, R.M. 1992. Ribosomal DNA
variation and population differentiation in Salicornia L. New
Phytol. 122: 553–565.
Nyberg Berglund, A.B., Saura, A. & Westerbergh, A. 2006.
Electrophoretic evidence for disomic inheritance and allopolyploid origin of the octoploid Cerastium alpinum (Caryophyllaceae). J. Hered. 97: 296–302.
Palop-Esteban, M., Segarra-Moragues, J.G. & Gonzalez-Candelas, F. 2007. Historical and biological determinants of
genetic diversity in the highly endemic triploid sea lavender
Limonium dufourii (Plumbaginaceae). Mol. Ecol. 16: 3814–
3827.
643
Petit, R.J., Kremer, A. & Wagner, D.B. 1993. Geographic
structure of chloroplast DNA polymorphisms in European
oaks. Theor. Appl. Genet. 87: 122–128.
Piirainen, M. 2001. Salicornia. In: Flora Nordica 2 (B. Jonsell, ed.),
pp. 50–54. Royal Swedish Academy of Sciences, Stockholm.
Pons, O. & Petit, R.J. 1996. Measuring and testing genetic
differentiation with ordered versus unordered alleles. Genetics
144: 1237–1245.
Pritchard, J.K., Stephens, M. & Donnelly, P. 2000. Inference of
population structure using multilocus genotype data. Genetics
155: 945–959.
Raftery, A.E. 1996. Hypothesis testing and model selection. In:
Markov Chain Monte Carlo in Practice (W.R. Gilks, S. Richardson
& D.J. Spiegelhalte, eds), pp. 163–187. Chapman & Hall,
London.
Ramsey, J. & Schemske, D.W. 2002. Neopolyploidy in flowering
plants. Annu. Rev. Ecol. Syst. 33: 589–639.
Ronquist, F. & Huelsenbeck, J.P. 2003. MrBayes 3: Bayesian
phylogenetic inference under mixed models. Bioinformatics 19:
1572–1574.
Såstad, S.M., Stenoien, H.K., Flatberg, K.I. & Bakken, S. 2001.
The narrow endemic Sphagnum troendelagicum is an allopolyploid derivative of the widespread S. balticum and S. tenellum.
Syst. Bot. 26: 66–74.
Scheepens, J.F., Veeneklaas, R.M., Van de Zande, L. & Bakker,
J.P. 2007. Clonal structure of Elytrigia atherica along different
successional stages of a salt marsh. Mol. Ecol. 16: 1115–1124.
Shepherd, K.A. & Yan, G. 2003. Chromosome number and size
variation in the Australian Salicornioideae (Chenopodiaceae)
– evidence of polyploidisation. Aust. J. Bot. 51: 441–452.
Soltis, D.E. & Rieseberg, L.H. 1986. Autopolyploidy in Tolmiea
menziesii (Saxifragaceae): genetic insights from enzyme electrophoresis. Am. J. Bot. 73: 310–318.
Soltis, D.E. & Soltis, P.S. 1999. Polyploidy: recurrent formation
and genome evolution. Trends Ecol. Evol. 14: 348–352.
Sotiaux, A., Enroth, J., Quandt, D., Olsson, S. & Vanderpoorten,
A. 2009. When morphology and molecules tell us different
stories: a case-in-point with Leptodon corsicus, a new and unique
endemic moss species from Corsica. J. Bryol. 31: 186–196.
Stace, C.A. 2010. New Flora of the British Isles, 3rd edn. Cambridge
University Press, Cambridge.
Swofford, D.L. 2003. P A U P *. Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4. Sinauer Associates,
Sunderland, MA.
Valdés, R. & Castroviejo, S. 1990. Salicornia. In: Flora Iberica, Vol.
II (S. Castroviejo, M. Laı́nz, G. López González, P. Montserrat,
F. Muñoz Garmendia, J. Paiva & L. Villar, eds), pp. 531–534.
Real Jardı́n Botánico, Madrid.
Vamosi, J.C., Goring, S.J., Kennedy, B.F., Mayberry, R.J.,
Moray, C.M., Neame, L.A. et al. 2007. Pollination, floral
display, and the ecological correlates of polyploidy. Funct.
Ecosys. Comm. 1: 1–9.
Vanderpoorten, A., Raspé, O., Risterrucci, A.M., Gohy, L. &
Hardy, O.J. 2010a. Identification and characterization of eight
nuclear microsatellite loci in the glasswort genus Salicornia
(Chenopodiaceae). Belg. J. Bot. 142: 204–208.
Vanderpoorten, A., Gradstsein, S.R., Carine, M.A. & Devos, N.
2010b. The ghosts of Gondwana and Laurasia in modern
liverwort distributions. Biol. Rev. 85: 471–487.
Vergilino, R., Belzile, C. & Dufresne, F. 2009. Genome size
evolution and polyploidy in the Daphnia pulex complex
(Cladocera: Daphniidae). Biol. J. Linn. Soc. 97: 68–79.
ª 2010 THE AUTHORS. J. EVOL. BIOL. 24 (2011) 630–644
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
644
A. VANDERPOORTEN ET AL.
Wagner, A., Blackstone, N., Cartwright, P., Dick, M., Misof, B.,
Snow, P. et al. 1994. Surveys of gene families using polymerase chain reaction: PCR selection and PCR drift. Syst. Biol. 43:
250–261.
Whittemore, A.T. & Schaal, B.A. 1991. Interspecific gene flow
in sympatric oaks. Proc. Natl Acad. Sci. USA 88: 2540–2544.
Wolff, S.L. & Jefferies, R.L. 1987. Morphological and isozyme
variation in Salicornia europaea (s.l.) (Chenopodiaceae) in
northeastern America. Can. J. Bot. 65: 1410–1419.
Wyatt, R., Rdrzykoski, I.J., Stoneburner, A., Bass, H.W. & Galau,
G.A. 1988. Allopolyploidy in bryophytes: multiple origins of
Plagiomnium medium. Proc. Natl Acad. Sci. USA 85: 5601–5604.
Supporting information
Additional Supporting Information may be found in the
online version of this article:
Table S1 Number of individuals, geographic origin
(locality, latitude, longitude) and allelic richness in
diploid [Salicornia patula (p), S. ramosissima (r), S. pusilla
(u), S. obscura (o)] and tetraploid [S. fragilis (f), S. emericii
(e), and S. dolichostachya (d)] populations of Salicornia
from the Mediterranean and Atlantic coasts of France
collected for this study (see Fig. 1).
Figure S1 Mean Fij resulting from the comparisons of
individual SSR genotypes (a) and mean Nij resulting from
the comparisons of individual cpDNA haplotypes (b)
between pairs of individuals within each of the tetraploid
species Salicornia emericii, S. fragilis, and S. dolichostachya
from the Mediterranean coasts of France depending on
the geographic distance separating them. The P-value of
the slope of the regression between the Fij and Nij values
and geographic distance are given in the upper right
corners.
Figure S2 Mean Fij resulting from the pairwise comparisons of individual SSR genotypes (a) and mean Nij
resulting from the pairwise comparisons of individual
cpDNA haplotypes (b) among the tetraploid species
Salicornia emericii, S. fragilis, and S. dolichostachya from
the Mediterranean coasts of France depending on the
geographic distance separating them. The P-value of the
slope of the regression between the Fij and Nij values and
geographic distance are given in the upper right corners.
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addressed to the authors.
Received 28 April 2010; revised 27 October 2010; accepted 12 November
2010
ª 2010 THE AUTHORS. J. EVOL. BIOL. 24 (2011) 630–644
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY