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Molecular Ecology (2002) 11, 329 – 345
The contribution of haploids, diploids and clones to
fine-scale population structure in the seaweed
Cladophoropsis membranacea (Chlorophyta)
Blackwell Science Ltd
H . J . V A N D E R S T R A T E , * † L . V A N D E Z A N D E , † W . T . S T A M and J . L . O L S E N *
*Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, PO Box 14, 9750 AA Haren,
The Netherlands, †Department of Population Genetics, Centre for Ecological and Evolutionary Studies, University of Groningen, PO
Box 14, 9750 AA Haren, The Netherlands
Abstract
Local populations of Cladophoropsis membranacea exist as mats of coalesced thalli composed
of free-living haploid and diploid plants including clonally reproduced plants of either
phase. None of the phases are morphologically distinguishable. We used eight microsatellite
loci to explore clonality and fine-scale patch structure in C. membranacea at six sites on the
Canary Islands. Mats were always composites of many individuals; not single, large clones.
Haploids outnumbered diploids at all sites (from 2:1 to 10:1). In both haploid and diploid
plants, genetic diversity was high and there was no significant difference in allele frequencies.
Significant heterozygote deficiencies were found in the diploid plants at five out of six sites
and linkage disequilibrium was associated with the haploid phase at all sites. Short dispersal
distances of gametes/spores and small effective population sizes associated with clonality
probably contribute to inbreeding. Spatial autocorrelation analysis revealed that most clones
were found within a radius of ≈ 60 cm and rarely further than 5 m. Dominance of the haploid
phase may reflect seasonal shifts in the relative frequencies of haploids and diploids, but
may alternatively reflect superiority of locally adapted and competitively dominant, haploid
clones; a strategy that is theoretically favoured in disturbed environments. Although sexual
reproduction may be infrequent in C. membranacea, it is sufficient to maintain both life history
phases and supports theoretical modelling studies that show that haploid–diploid life histories
are an evolutionarily stable strategy.
Keywords: alga, clonality, dispersal, isomorphic diplohaplontic life history, microsatellites, population
genetic structure
Received 2 August 2001; revision received 15 November 2001; accepted 15 November 2001
Introduction
Population structure is the complex result of many interacting
genetic and demographic factors set against a dynamic
environmental background. A description of spatial genetic
structure at different scales provides a way to examine the
processes that have affected the evolution and ecology of
species. One of these processes is dispersal, the amount of
which is directly related to the reproductive system (Loveless
& Hamrick 1984). Studies in terrestrial plants (mainly
Correspondence: Han van der Strate. Present address: †Department
of Marine Biology, Centre for Ecological and Evolutionary Studies,
University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
Fax: + 31– 50 – 3632261; E-mail: [email protected]
© 2002 Blackwell Science Ltd
angiosperms) show that mating systems and dispersal
capabilities are excellent predictors of genetic diversity,
structure and gene flow over a range of spatial scales
(Hamrick et al. 1979; Loveless & Hamrick 1984; Schoen &
Brown 1991). Marine plants, including seaweeds, exhibit
an extremely diverse range of life histories (Van den Hoek
et al. 1995) which offer a unique opportunity to investigate
the relationship between life history strategies and
dispersal capacity in the aquatic realm.
The seaweed Cladophoropsis membranacea (Hofman Bang
ex. C. Agardh) Børgesen grows on rocky benches and reefs
in wave-swept or high-surge, intertidal zones in the tropics
and subtropics. In the Canary Islands it grows on basalt
benches (50–500 m wide). Thalli consist of intertwined,
loosely branched, filaments. The multinucleate construction
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330 H . J . V A N D E R S T R A T E E T A L .
Fig. 1 Haplodiplo-isomorphic life history of Cladophoropsis membranacea. It is not known whether gametophytes are dioecious or
monoecious. Asexual reproduction via gametes and/or mitospores has been reported for both generational phases.
and paucity of cross-walls gives rise to the distinctive siphonous
morphology. Thalli form dense cushions and mats 1–2 cm
thick that may extend > 50 cm. C. membranacea is most
abundant from the autumn to late spring. In some habitats
it can become transiently dominant.
The life history of Cladophoropsis (SiphonocladalesCladophorales Complex, Ulvophyceae) is characterized
by a diplohaplontic–isomorphic alternation of generations
(Van den Hoek et al. 1995). As illustrated in Fig. 1, populations
consist of independent, free-living haploid and diploid
phases that are morphologically indistinguishable. In
addition, asexual reproduction (Bodenbender et al. 1988;
Bodenbender & Schnetter 1990; Kapraun & Nguyen 1994)
via parthenogenesis of gametes and/or the production of
mitospores in one or both phases, may add clonal structure
to the population. It is not known whether the haploid or
diploid phases of the life history sequentially dominate or
coexist throughout the year, nor is it known to what extent
asexual reproduction augments one or both phases.
Over the past 20 years many efforts have been made to
gain a better understanding of direct dispersal (reviewed
by Norton 1992b) and population genetic structure in
algae using isozyme electrophoresis (reviewed by Sosa &
Lindstrom 1999) as an indirect measurement of gene flow.
Low levels of allozyme variation have often limited the power
of these studies and led to the questionable conclusion of
extremely broad-scale panmixis. However, a handful of
studies have shown population differentiation at scales
of tens to hundreds of kilometres, that roughly matched
predictions related to dispersal capability and the mating
system. In seaweeds, dioecy and outcrossing have been
investigated by Lu & Williams (1994) in Halidrys, by Benzie
et al. (1997) in Caulerpa, and by Hwang et al. (1998) in Porphyra.
Monoecy and selfing have been investigated by Williams
& Di Fiori (1996) in Pelvetia; and asexuality by Innes (1988)
in Enteromorpha and Ulva, by Intasuwan et al. (1993) in
Gracilaria, by Pearson & Murray (1997) in Lithothrix, by
Sosa et al. (1996) in Gracilaria and by Sosa et al. (1998) in
Gelidium.
Evidence for short-distance dispersal at a scale of metres
has come mainly from direct measurements of settling or
spore filtration studies [reviewed in Destombe et al. 1990,
1992; Norton 1992a,b; (Gracilaria); Berndt & Brawley 2000
(Fucus)]. These studies report distances ranging from 1 to
20 m for most species. Indirect measurements using highresolution, genetic markers (such as fingerprinting and
microsatellite loci), at very small scales, are still in the early
stages. Coyer et al. (1997) investigated dispersal within
clusters of Postelsia to be limited to < 5 m; Engel et al. (1999)
examined paternity relationships in Gracilaria and found
80% of the mate pairs within 85 cm; and Wright et al. (2000)
examined patch structure in Delisea and found differences
at scales between 50 and 300 cm.
The contribution of clonal reproduction has been investigated in only a few cases. Cheney & Babbel (1978) found
new recruits of Eucheuma near the parent plants; Innes &
Yarish (1984) observed only 13 out of a possible 5400
multilocus phenotypes in more than 1000 individuals of
Enteromorpha linza; Sosa et al. (1996) found almost no
variation in Gracilaria cervicornis populations; and Sosa
et al. (1998) found significant differences in allele frequencies between the haploid and diploid phases in Gelidium
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P O P U L A T I O N S T R U C T U R E O F C L A D O P H O R O P S I S M E M B R A N A C E A 331
arbuscula with strong differentiation in the haploid, asexual
phase.
In this study we used recently developed microsatellite
loci (Van der Strate et al. 2000) to explore fine-scale patch
structure and dispersal in C. membranacea. The aims of
the present study were to: (i) determine the relative contributions of haploid and diploid individuals including their
respective genetic diversities; (ii) assess the contribution
of clonal reproduction; (iii) determine the approximate
size of genetic individuals within mats; and (iv) estimate
small-scale dispersal processes in relation to population
differentiation.
Materials and methods
Study area and sampling design
In January 1998, 580 samples of Cladophoropsis membranacea
were collected from three different locations on Tenerife
and Gran Canaria (Fig. 2). A nested hierarchical sampling
design was used in order to assess the spatial scale of
differentiation. Cushions were mapped within each 1-m2
quadrat. From each quadrat, 20 samples were taken at
random (20 × 4 × 6 = 480 samples). In order to estimate
the size of a genetic individual in relation to cushion size,
an additional 100 samples (5–18 samples per cushion) were
collected at ≈ 1-cm intervals from a range of cushion sizes
(≈ 4 – 50 cm). In order to minimize the chance of inadvertently
sampling more than one genetic individual from a single
cushion, one sample consisted of ≈ 20 filaments plucked from
an area of < 0.5 cm2. Filaments were rinsed, blotted dry and
placed directly in silica gel for later DNA extraction.
DNA extraction
DNA was isolated according to the cethyltrimethylammonium
bromide (CTAB) protocol of De Jong et al. (1998). Silicadried, algal material (0.02 g) was incubated at 60 °C for 30 min
in 500 µL of preheated extraction buffer [2% CTAB (w/v),
1.4 m NaCl, 20 mm ethylenediaminetetraacetic acid, 100 mm
Tris–HCl pH 8.0, and 0.2% β-mercaptoethanol (v/v)]
followed by a 15-min, slow rotation at room temperature.
The crude extract was centrifuged at 2000 g for 15 min and
the supernatant was decanted to a fresh tube. The remainder
of the extraction followed standard procedures involving
two extractions with an equal volume of chloroform–
isoamylalcohol (24 : 1, v/v). This was followed by DNA
precipitation using two-thirds volume of cold isopropanol
(4 °C, 1 h), centrifugation (4 °C, 15 min, 10 000 g), and
washing of the pellet with 80% ethanol. The DNA was
dissolved in 100 µL of 0.1× TE (10 mm Tris/HCl and 1 mm
EDTA pH8). Further purification steps were not required.
The typical yield was ≈ 1 µg.
Microsatellite amplification and locus visualization
We have recently developed microsatellite loci for C.
membranacea (Van der Strate et al. 2000). The eight loci
Fig. 2 Six study sites, and as an example, the
hierarchical sampling design for the location
Tenerife south for Cladophoropsis membranacea.
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332 H . J . V A N D E R S T R A T E E T A L .
used in this study are summarized in Table 1. This study
represents the first application of these loci. Loci were
amplified individually using the polymerase chain reaction
(PCR). Each 10-µL reaction contained the following: 2 µL
template DNA (≈ 10 ng/µL), 1 µL each of unlabelled,
microsatellite primer (5 µm), 1 µL 10 d× NTP mix (2 mm
of dATP, dGTP, dTTP and 0.2 mm dCTP; Pharmacia
Biotech), 1 µL 10× Taq polymerase buffer (Pharmacia Biotech),
0.08 µL Taq polymerase (5 units/µL, Pharmacia Biotech),
1 µL bovine serum albumin (0.1% w/v) and 0.14 µCi
[α32P]dCTP (3000 Ci/mmol, Amersham) in 3 µL H2O.
Amplifications were performed in a PTC-100™ machine
(MJ Research, Inc.). The profile consisted of an initial
denaturation step of 3 min at 94 °C followed by 30 cycles of:
1 min at 94 °C, 2 min at the optimal annealing temperature
(Tm) (Table 1), 1.5 min at 72 °C and 10 min at 72 °C. Each
PCR run included a positive (2 ng of the plasmid clone
DNA containing the microsatellite) and a negative (no
template DNA) control. The PCR products were separated
by electrophoresis on 5% denaturing polyacrylamide
gels (Biozym, Sequagel XR). Loci were visualized using
standard autoradiography (medical X-ray film, Fuji Film)
and scored manually against a size standard (sequence
ladder of the plasmid vector pBluescript SK+) and the
amplified clone from the locus of interest (i.e. positive
control above).
Identification of haploid and diploid individuals
Haploid and diploid individuals of C. membranacea cannot
be distinguished morphologically. Individuals were considered
diploid if any of the eight loci were heterozygous. However,
individuals that are homozygous at all eight loci may, in
principle, be haploid or diploid. In order to correct for
this potential bias, we calculated the expected number of
diploid homozygous individuals for all eight loci at each
site. This was done by calculating the expected homozygosity
for each locus under Hardy–Weinberg equilibrium using
the observed allele frequencies and then taking the product
of the individual loci times the number of individuals
(N = 80). The expected number of eight-locus homozygotes
per site was always smaller or, equal to one, indicating
that our eight-locus, homozygote assignment to haploids
was not biased.
Data analysis
Genetic diversity was compared separately in the haploid
and diploid phases. Basic diversity measures, tests for
Hardy–Weinberg equilibrium and F-statistics (Wright 1965)
were calculated in the program package genetix ver. 4.0
(Belkhir et al. 1998) or arlequin version. 1.1 (Schneider
et al. 1997). Clonal reproduction must be accounted for
in population genetic analyses in order to prevent biases in
estimates of FIS, FST and linkage disequilibrium. Duplicate
haplotypes/diplotypes were therefore used only once.
If this is not done, estimates of FIS can be upwardly or
downwardly biased depending on whether an excess
of homozygotes or heterozygotes is present. Estimates of
linkage disequilibrium will also be affected. Withinpopulation differentiation will tend to be downwardly
biased, whereas between-population differentiation will
tend to be overestimated.
Significance testing was carried out using permutation
(1000 permutations) followed by sequential Bonferroni
correction (Rice 1988). The frequency of expected null
alleles was calculated using the method of Brookfield (1996).
Clonal diversity (Pd) for each phase was calculated as
the proportion of distinguishable haplotypes or diplotypes
divided by the total number of samples for the specific
phase at the spatial scale of interest (Ellstrand & Roose
1987). A value of 1 indicates that each individual has a unique
haplo- or diplotype and therefore, the area has maximal
clonal diversity. A value approaching 0 indicates that
all individuals have the same haplotype or diplotype
(i.e. are monoclonal). Reliable detection of clonality is
dependent upon the resolving power of the loci. If a
multilocus haplotype or diplotype has many, relatively
low-frequency alleles at several loci, the likelihood that
it occurs more than once by chance is extremely low. In
contrast, if a multilocus haplotype or diplotype is composed of only a few, high-frequency alleles at several loci,
the likelihood of multiple occurrences by chance alone may
be fairly high. Since we were specifically interested in
assessing the clonal contribution to population structure
and dispersal, we assessed the resolving power of our eight
loci in three ways.
The number of unique diplotypes Nd possible is:
Nd =
Terminology related to haploid and diploid individuals
For clarity, we use the term haplotype to refer to the
multilocus genotypes of haploid individuals (i.e. free-living
gametophytes); and the term diplotype to refer to the
multilocus genotypes of diploid individuals (i.e. free-living
sporophytes). The term clone is used to refer to identical
haplotypes or identical diplotypes that are the result of
asexual reproduction of the respective phases.
L
∏ [a ( a + 1)] /2
i =1
where L is the number of loci (eight in this study), and ai is
the number of alleles at locus i.
For haplotypes there is only one allele possible per locus,
which means that the number of unique haplotypes Nh
present in a given area is the product of all of the possible
alleles at the eight different loci.
For each eight-locus diplotype (Pdip) the expected
probability is:
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P O P U L A T I O N S T R U C T U R E O F C L A D O P H O R O P S I S M E M B R A N A C E A 333
Pdip = (Π pi)2h
where pi is the frequency in the population of each allele
(two per locus) represented in the diplotype and h is the
number of loci that are heterozygous (Parks & Werth 1993).
For the expected probability of each eight-locus haplotype
(Phap) h is set at zero. The above equation also describes the
likelihood of occurrence of a given haplotype or diplotype
at a given location. If identical haplotypes or diplotypes
had P values of < 0.05 and were found in close proximity
(e.g. within or between quadrats), they were considered
clones. Finally, the likelihood of encountering the same
haplotype or diplotype at least twice (Pse) in a more
distantly spaced sample was calculated according to:
Pse = 1 – (1 – Phap)H, or Pse = 1 – (1 – Pdip)D
where H and D are the number of distinct haplotypes and
diplotypes identified (Parks & Werth 1993).
Linkage disequilibrium was tested for haploid and
diploid phases separately. This was done across all 28
[i.e. L(L – 1)/2] possible pairings of loci across all populations
and per population (i.e. site) using genepop version. 3.2d
(Raymond & Rousset 1995). Linkage disequlibrium was
further analysed using the D-statistics of Black & Krafsur
(1985) as implemented in genetix version. 4.0 (Belkhir
et al. 1998). Significance testing was done using permutation
followed by sequential Bonferroni correction (Rice 1988).
In order to avoid linkage due to the clonal contribution,
duplicate haplotypes and diplotypes were removed prior
to analysis.
Spatial-autocorrelation was used to estimate clonal
dispersal and patch structure from mapped quadrats. Spatial
auto-correlation analysis was performed using Moran’s I
coefficient in autocorrg version. 2.0 (Hardy & Vekemans
1999). Significance testing per distance class was done using
permutation. In order to increase statistical power per class,
alternative distance classes were tried, as was combining
the four quadrats within a site (including necessary distance
corrections). Due to the paucity of diploid individuals,
spatial autocorrelation analyses were performed on haploid
individuals only. The analysis was first run on the full data
set including all duplicated haplotypes and then with the
duplicates removed.
Population differentiation was investigated at spatial
scales of 1, 5 and 20 m and at 5 km (between sites) using a
two-level and three-level amova (Excoffier et al. 1992) as
implemented the program package arlequin version. 1.1
(Schnieder et al. 1997). Due to the low number of diploids
this was done for haploids only. For the two-level amova
differentiation was estimated with FST (Weir & Cockerham
1984) and for the three-level analysis FCT and FSC (amova
analogues). Duplicate haplotypes/diplotypes were used
only once to avoid overestimation of FST.
Results
Eight microsatellite loci provided a total of 65 alleles (N = 478)
(Table 1) of which 35 had a frequency ≥ 0.05 (Fig. 3). Six
of the eight loci were moderately to highly polymorphic
with expected heterozygosities ranging from 0.59 to 0.89.
Consistency of the eight loci and their relative contribution
in subsequent calculations was evaluated by computing
the jack-knifed mean and standard deviation of FST over
all 478 samples (Table 2). Six of the eight loci were found
to behave similarly. Loci Cmb5 and Cmb6 were nearly
monomorphic and thus contributed little information.
In order to assess whether the sampling intensity was
sufficient to capture the allelic diversity present, we plotted
individuals sampled against the mean number of alleles/
locus (Fig. 4). Even with 20 samples, the major diversity
was recovered.
Null alleles are a commonly reported problem with
microsatellite loci (Hare et al. 1996). This can be the result of
Table 1 Microsatellite loci for Cladophoropsis membranacea and their properties based on a sample of N = 478 individuals
Locus
Repeat
Tm (°C)
Size range (bp)
No. of alleles
HE
Cmb 1
Cmb 2
Cmb 3
Cmb 4
Cmb 5
Cmb 6
Cmb 7
Cmb 8
Total
(GT)6GC(GT)11
(GT)7 GCGGCTACT(GT)18
(GT)12 N26*(GT)14
(GT)4AT (GT)15
(GT)13
(GT)13
(GT)13
(GT)19
60
60
60
60
60
60
55
60
126–136, clone 130
168–186, clone 172
171–185, clone 175
118–136, clone 126
208–212, clone 208
116–120, clone 116
202–228, clone 202
135–161, clone 139
6
10
7
8
3
3
14
14
65
0.70
0.79
0.65
0.59
0.12
0.14
0.83
0.89
0.59
Tm is optimal annealing temperature and HE is unbiased average expected heterozygosity (Nei 1978). See van der Strate et al. (2000) for details.
* = AT(CA) (TA) (TG) T(CA) T(AC) .
N 26
2
2
3
2
2
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334 H . J . V A N D E R S T R A T E E T A L .
0.35
allele frequency
0.4
Locus Cmb 1
Locus Cmb 2
0.30
0.25
0.3
0.20
0.2
Fig. 3 Global allele frequencies for eight
microsatellite loci in Cladophoropsis membranacea.
Haploid thalli (black, N = 401); diploid thalli
(grey, N = 77). Note that y-axes are not the
same.
0.15
0.10
0.1
0.05
0.0
0.00
126
128
130
132
136
Locus Cmb 3
0.5
allele frequency
134
168
170
172
174
176
178
0.6
184
186
Locus Cmb 4
0.4
0.3
0.3
0.2
0.2
0.1
0.0
0.0
171
173
175
177
179
181
183
185
118
120
122
124
126
128
130
132
134
136
1.0
1.0
Locus Cmb 6
Locus Cmb 5
allele frequency
182
0.5
0.4
0.1
0.5
0.5
0.0
0.0
208
210
116
212
118
120
0.25
0.30
Locus Cmb 8
Locus Cmb 7
0.25
allele frequency
180
0.20
0.20
0.15
0.15
0.10
0.10
0.05
0.05
0.00
0.00
202 204 206 208 210 212 214 216 218 220 222 224 226 228
135 137 139 141 143 145 147 149 151 153 155 157 159 161
allele size (bp)
allele size (bp)
Table 2 Consistency of microsatellite loci in Cladophoropsis membranacea. Jack-knifed mean FST values for all haploid samples (without
duplicates) over the six sites
Mean FST
SD
FST
Cmb 1
Cmb 2
Cmb 3
Cmb 4
Cmb 5
Cmb 6
Cmb 7
Cmb 8
All loci
0.183
0.069
> 0*
0.210
0.079
> 0*
0.279
0.087
> 0**
0.215
0.071
> 0*
0.009
0.021
=0
0.375
0.197
=0
0.166
0.062
> 0*
0.244
0.047
> 0**
0.205
0.015
> 0***
SD, standard deviations.
*P < 0.05; **P < 0.01; ***P < 0.001.
mutations in flanking sequences that result in failed amplification of one or both alleles. Because we were dependent
on molecular identification of haploids vs. diploids, it was
critical to assess whether or not nulls could be a significant
problem. In our study, all eight loci amplified without
difficulty and amplification failure rates were < 0.006. We
tested further for null alleles in two ways; first, a set of
homozygous individuals was reamplified for all of the
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Mean number of alleles/locus
P O P U L A T I O N S T R U C T U R E O F C L A D O P H O R O P S I S M E M B R A N A C E A 335
6
5
4
3
Playa del Hombre
Penascos
Palm-Mar
Las Galletas
La Barranquera
Punta del Hidalgo
all
2
1
0
0
10
20
30
40
50
60
70
80
Number of individuals
Fig. 4 Relationship between sampling effort and mean number of
alleles per locus for the six sites, plotted separately.
different loci under relaxed annealing temperatures. No
additional alleles were amplified. Second, we calculated
the expected frequency of null alleles for diploid individuals according to equation (4) of Brookfield (1996)
(Table 3). These values were all less than one. Although
neither of these tests is foolproof, we could find no evidence
that homozygous individuals were the result of null alleles.
The number of alleles per locus ranged from 3 to 14
with an overall mean of 7.6 alleles per locus for the haploid
individuals and 6.8 alleles per locus in the diploid individuals
(Table 3). This difference is not significant (two-sample t-test).
Likewise, allele frequencies between haploids and diploids
were not significantly different within sites (exact test for
population differentiation). There were no haploid/diploid-
specific alleles detected that might be suggestive of different
gene pools.
Diploid populations were not in Hardy–Weinberg
equilibrium as assessed by significantly positive FIS values
(Table 3) at five of the six sites (diplotypes used once;
duplicates removed). Although diploid sample sizes were
small in most cases, those at Las Galletas (N = 16) and La
Barranquera (N = 25) still maintained high values of
heterozygote deficiency across loci.
Thirteen out of 28 pairwise comparisons among the
eight loci revealed significant linkage disequilibrium (LD)
(Table 4). Further testing revealed that LD in the haploid
phase was due to drift (DIS < DST and D’IS < D’ST) and not
systematic selection (notation of Black & Krafsur 1985).
LD was found at all sites in the haploid phase and at La
Barranquera in the diploid phase (Table 4). The apparent
absence of LD at the other sites probably reflects insufficient
sample sizes.
Haploids, diploids and clones
Among the 478 samples taken, 401 were found to be haploid
and 77 diploid (Table 3). The chance that plants assigned as
haploids might actually be fully homozygous diploids was
extremely low (Table 3). Therefore we are confident that
the number of haploids observed is a good estimate of the
true condition, in which haploid plants greatly outnumbered
diploid plants at all six sites. At Peñascos this was nearly
10 : 1 whereas at La Barranquera it was 2 : 1. Even when
identical haplotypes and diplotypes were subsequently
Table 3 Comparison of the clonal diversity (Pd), per cent polymorphic loci (P; 99% criterion), mean number of alleles per locus (NA) and
genetic diversity (HE, expected heterozygosity) between haploid (1n) and diploid (2n) thalli of Cladophoropsis membranacea at six sites
Location
Site
Gran Canaria
Playa del Hombre
Peñascos
Tenerife south
Palm-Mar
Las Galletas
Tenerife north
La Barranquera
Punta del Hidalgo
Overall
Life
history
phase
No. of
indiv.
Diploid
homoE
n
2n
n
2n
n
2n
n
2n
n
2n
n
2n
n
2n
71
9
76
4
70
10
62
16
55
25
67
13
401
77
0.03
1.18
0.92
0.82
0.18
0.32
No. of indiv.
minus
clonality
Pd
P(%)
NA
HE
40
9
37
4
41
10
25
16
41
24
42
13
219
76
0.56
1
0.49
1
0.59
1
0.40
1
0.75
0.96
0.63
1
0.55
0.98
100
100
100
75
87.5
75
75
87.5
100
87.5
100
87.5
100
100
5.6
3.9
5.4
2.9
4.0
2.8
3.3
3.1
3.6
3.9
4.3
3.8
7.6
6.8
0.64
0.56
0.51
0.49
0.46
0.38
0.42
0.38
0.50
0.48
0.49
0.44
0.62
0.59
HO
NullsE
FIS
0.31
<1
0.53
<1
0.25
<1
0.36***
0.21
<1
0.44***
0.26
1
0.45***
0.23
<1
0.49***
0.26
<7
0.47***
– 0.10
The number of expected diploid homozygotes (HomoE) per site is 1.2 (see Materials and methods section). Observed heterozygosity (HO),
NullsE (Brookfield 1996) and the inbreeding coefficient (FIS) are calculated for diploids.
***P < 0.001.
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 329–345
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336 H . J . V A N D E R S T R A T E E T A L .
Table 4 Results of Fisher’s exact test for linkage disequilibrium as implemented in genepop version 3.2 (Raymond & Rousset 1995)
Pairwise comparison of loci
Locus
Cmb 1
Cmb 1
Cmb 2
Cmb 3
Cmb 4
Cmb 5
Cmb 6
Cmb 7
Cmb 8
—
***
—
—
—
—
—
Linkage disequilibrium per site
Cmb 2
Cmb 3
Cmb 4
Cmb 5
Cmb 6
Cmb7
Cmb 8
Site
Haploids
Diploids
***
***
***
***
***
—
—
—
—
—
—
***
—
—
—
***
***
—
***
—
***
***
***
***
—
—
—
—
Playa del Hombre
Peñascos
Palm-Mar
Las Galletas
La Barranquera
Punta del Hidalgo
12/28 (40)
11/28 (37)
1/21 (41)
5/15 (25)
5/28 (41)
2/28 (42)
0/28 (9)
0/6 (4)
0/15 (10)
0/21 (16)
4/21 (24)
0/21 (13)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Pairwise comparison of loci (Cmb 1 to Cmb 8) across all the six sites for Cladophoropsis membranacea. Haploids (upper right) and diploids
(lower left) were treated separately. Duplicated clones were removed. Right: number of pairwise comparisons across loci showing linkage
disequlibrium per site. Calculations for diploids are questionable due to small sample sizes at most sites. Duplicate clones were removed.
Sample sizes in parentheses
***P < 0.001 after Bonferroni correction.
removed, the ratio still ranged from 4 : 1 to 2 : 1 in favour
of haploids. Within-site clonal diversity varied between
25 and 60% for haploids. Cladophoropsis membranacea can
reproduce either the diploid or haploid phases through
asexual production of spores. This contributes yet another
element to the population structure, which had to be
identified. Using the observed allele frequencies for each
locus, the possible number of unique eight-locus haplotypes
(Nh) was 3.3 × 106 and the number of possible unique
diplotypes (Nd) was 1.79 × 1010. We identified 219 unique
haplotypes and 76 unique diplotypes. An additional 65
haplotypes and only one diplotype (La Barranquera)
occurred two or more times. These were considered clones
(or duplicates). Clonal diversity (Pd) was high, ranging
from 0.40 to 0.75 (x̄ = 0.55) for the haploid phase and from
0.96 to 1.0 (x̄ = 0.99) for the diploid phase over the six sites.
The haploid phase contained a significantly higher number
of clones per site then the diploid phase (P = 0.0003, t-test).
As illustrated in Table 5, between nine and 15 clonal haplotype
types were detected per site and most were unique for a
given site.
Although the calculated values of Nh and Nd were
large, the presence of a few alleles in high frequencies at
each locus might significantly reduce these numbers and
lead to the occurrence of identical multilocus haplotypes/
diplotypes by chance alone. In order to assess this factor we
calculated the probabilities of occurrence Pgen and Pdip for
all haplotypes and diplotypes that were found more than
once (Table 5). In 100% of the cases the probabilities were
< 0.05 and in > 80% of the cases they were < 0.001. This
meant that the likelihood of any eight-locus haplotype or
diplotype occurring by chance was extremely low. Thus
we attributed identical eight-locus, microsatellite haplotypes
or diplotypes to asexual reproduction, especially if the
individuals were in close proximity. However, some
identical haplotypes were found at scales of 5 – 26 m, as
illustrated in Fig. 5. These individuals may or may not be
dispersed clones based on probabilities of second encounter
(Pse). Although this calculation is somewhat circular (because
higher haplotype frequencies will increase the chance of
encounter), it does provide some indication of where
dispersal boundaries might be expected, especially when
used in conjunction with spatial auto-correlation and
spatial-hierarchical analyses of population differentiation.
As shown in Table 5, more than half of the Pse values are
much larger than 0.05 and most of these occurred at
distances of > 5 m. At Playa del Hombre and Peñascos, for
example, only two identical haplotypes (Fig. 5, dark blue
and light blue cushions) were found between quadrats
(> 5 m) and in three cases at distances > 20 m (Fig. 5 blue,
red and purple cushions). Over the entire study, identical
haplotypes were found at distances > 5 km (between sites)
in only seven cases.
Mats and individuals
Individual thalli of C. membranacea coalesce with one another
to form small cushions or more extensive mats. In order
to investigate the clonal diversity of a mat, we sampled
several cushions of different sizes as exemplified in Fig. 6.
Eight-locus haplotypes showed that cushions of ≈ 1– 5 cm
in diameter were typically one genetic individual (N = 3
cushions, 15 samples). Cushions 7 –10 cm in diameter
contained two haplotypes (N = 2 cushions, 10 samples).
In mats (> 50 cm), up to six haplotypes and eight genotypes
were detected (N = 3 mats, 54 samples). Mats thus represent
a mixture of different genetic individuals and are not single,
fast-growing clones.
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 329 – 345
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P O P U L A T I O N S T R U C T U R E O F C L A D O P H O R O P S I S M E M B R A N A C E A 337
Table 5 Clones in Cladophoropsis membranacea
Haplotype
Frequency
Phap
Table 5 Continued
Pse
Gran Canaria
Playa del Hombre
1
13
2
4
3
4
4
4
5
3
6
2
7
2
8
2
9
2
10
2
11
2
12
2
13
2
14
1
15
1
0.0003
0.0005
7.6E-05
0.0003
1.0E-05
5.1E-05
4.4E-05
4.6E-05
0.0001
2.0E-08
2.8- 05
9.1E-07
0.0001
0.0003
0.0009
0.0133
0.0216
0.0031
0.0109
0.0004
0.0020
0.0017
0.0018
0.0046
8.1E-07
0.0011
3.6E-05
0.0054
0.0115
0.0349
Peñascos
1
2
10
14
15
16
17
18
19
20
0.0147
0.0250
4.2E-07
0.0029
0.0125
0.0078
8.4E-05
0.0046
0.0033
1.6E-06
0.4225
0.6087
1.5E-05
0.1003
0.3726
0.2522
0.0031
0.1569
0.1140
5.8E-05
17
8
2
2
6
4
3
3
2
2
Tenerife south
Palm-Mar
21
22
23
24
25
26
27
28
29
30
31
32
33
10
5
4
4
4
3
2
2
2
2
2
1
1
0.0207
0.0055
0.0097
0.0103
0.0087
0.0031
0.0098
0.0058
0.0003
2.5E-07
0.0017
0.0010
0.0015
0.5763
0.2009
0.3294
0.3450
0.3018
0.1179
0.3327
0.2123
0.0138
1.0E-05
0.0666
0.0385
0.0609
Las Galletas
32
33
34
35
36
37
38
39
40
41
42
5
4
10
10
4
3
3
3
2
2
2
0.0030
0.0151
0.0302
0.0013
0.0181
0.0085
0.0002
0.0038
0.0004
0.0027
0.0151
0.0729
0.3167
0.5357
0.0315
0.3671
0.1928
0.0053
0.0904
0.0106
0.0658
0.3166
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 329–345
Haplotype
Frequency
Phap
Pse
Tenerife north
La Barranquera
43
44
45
46
47
48
49
50
51
D
4
3
3
3
2
2
2
2
2
2
0.0034
0.0004
0.0002
0.0011
0.0051
0.0059
0.0102
6.4E-05
0.0008
1.4E-05
0.1298
0.0182
0.0073
0.0441
0.1885
0.2163
0.3421
0.0026
0.0327
0.0003
Punta del Hidalgo
52
53
54
55
56
57
58
59
60
61
62
63
64
65
5
4
3
3
3
3
3
3
2
2
2
2
2
2
0.0150
0.0115
0.0099
0.0081
0.0129
0.0007
7.7E-06
0.0002
0.0001
7.8E-05
0.0002
0.0029
0.0008
0.0062
0.4704
0.3844
0.3417
0.2891
0.4198
0.0292
0.0003
0.0097
0.0043
0.0032
0.0073
0.1161
0.0334
0.2294
Sixty-five distinct multilocus haplotypes that occurred more then
once and their estimated probability of encounter (Phap) and reencounter (Pse). Haplotypes in bold were found between the sites.
The only diplotype (D) that occurred more then once was found at
La Barranquera.
Dispersal
At each site individuals were mapped within quadrats
from which a spatial autocorrelation analysis was performed
(Fig. 7). Individuals in closer proximity to one another, i.e.
in smaller distance classes, were expected to share a higher
correlation of alleles. In the first analysis, all haplotypes
were included. A comparison of all sampled haplotypes
(Fig. 7, solid lines) reveals a significant correlation at up to
80 cm at four of the six sites. The intercept of the x-axis is
taken as the approximate radius of the patch size of dispersal.
At Playa del Hombre, for example, this suggests clonal
dispersal of ≈ 1.2 m. When duplicate clones were removed
so that each haplotype was counted only once in the analysis
(Fig. 7, dotted lines), there was no correlation. This suggests
that dispersal has two components: the initial random
recruitment at the site followed by asexual amplification of
the resident thalli to ‘fill-in’ the space.
Fig. 5 Mapped quadrats of Cladophoropsis membranacea cushions at Playa del Hombre (top) and Peñascos (bottom). Uncoloured and numbered cushions contain unique eight-locus
haplotypes or diplotypes. Within Playa del Hombre or within Peñascos, cushions with the same colour contain identical eight-locus haplotypes.
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338 H . J . V A N D E R S T R A T E E T A L .
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P O P U L A T I O N S T R U C T U R E O F C L A D O P H O R O P S I S M E M B R A N A C E A 339
Table 6 Overview of diploid individuals of Cladophoropsis membranacea assignable to parents present within the same quadrat
Parents assignable
Fig. 6 Relationships between thallus size and individuality in
Cladophoropsis membranacea. (A) A 50-cm long, continuous mat was
sampled at the points shown. (B) Schematic representation of the
mat in (A) and of three other cushions each sampled five times.
Colours correspond to haplotypes in haploid plants (d) and
diplotypes in diploid plants (j).
Where diploid plants were identified, we examined whether
or not they could be assigned to nearby haploid parents from
the same quadrat (1 m2). About 50% could be assigned to
one or both parents (Table 6).
Population differentiation
At each site we tested for population differentiation at three
scales; 1, 5 and 20 m. With the exception of La Barranquera,
there was no significant population differentiation detected
at these scales (Table 7). Using a two level amova we also
tested for differences between sites. In all three cases, significant
FST values were found at 6 –10 km. Approximately 90% of
the variance was accounted for within sites and only about
10% between sites.
Discussion
Haploid and diploid plants were found at all sites but haploid
plants were always dominant and the two phases were not
in Hardy–Weinberg equilibrium as shown by heterozygote
deficiencies and significant linkage disequilibrium.
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 329–345
Site
No.of
diploids
both
one
none
Playa del Hombre
Peñascos
Palm-Mar
Las Galletas
La Barranquera
Punta del Hidalgo
9
4
10
16
24
13
3
0
0
1
1
1
1
2
6
8
11
4
5
2
4
7
12
8
Total
76
6
32
38
The chance for inbreeding is probably high in Cladophoropsis
membranacea. This is because dispersal distances for gametes/
spores are short (≈ 1 m), the local dominance of a few clones
would reduce the effective population sizes considerably
(Table 3) and because the haploid phase is the gametophyte.
Playa del Hombre (Fig. 5) typifies such a setting in which
71 of 80 individuals were haploid. Of these, 31 haplotypes
occurred just once, 15 occurred more than once and one
haplotype occurred 13 times. If there are no barriers to selfing,
the opportunities for outcrossing would be further reduced
as suggested by parental assignment of diploids in Table 6.
While inbreeding is most likely, heterozygote deficiencies
can also be the result of a Wahlund effect, null alleles or
sampling artefacts. A Wahlund effect is unlikely given the
small spatial scale of the study and the lack of population
differentiation at scales < 5 km. Null alleles are unlikely
based on tests presented in the Results section. Small
diploid sample sizes might have led to spurious results
in cases where N was very small, but results from La
Barranquera (N = 24) suggest that heterozygote deficiencies
are real.
Linkage disequilibrium was attributed to genetic drift.
However, the nature of small-scale population dynamics
in C. membranacea may be promoting the disequilibrium
via small effective population sizes, i.e. founder events, few
diploids and low clonal diversity of the haploids and
nonrandom mating. Populations may also be in a permanent
state of nonequilibrium. Since drift is expected to be stronger
in small populations, we looked for correlations between
linkage disequilibrium and clonal diversity, i.e. stronger
disequilibrium expected at those sites having the lowest
clonal diversity (Tables 3 and 4). No correlations were found.
The only other report of linkage disequilibrium in seaweeds
is in Gelidium arbuscula (Sosa et al. 1998). There it was
attributed to significant differences in allele frequencies found
between sporophytes and gametophytes combined with
dominant asexual reproduction. The high genetic diversity
found in both phases of C. membranacea and the lack of
differences in allele frequencies indicates that the two
MEC_1448.fm Page 340 Wednesday, February 20, 2002 9:07 AM
340 H . J . V A N D E R S T R A T E E T A L .
0.5
0.6
*
Playa del Hombre
*
0.4
Penascos
0.4
Moran’s I
Moran’s I
0.3
* *
0.2
0.1
*
0.2
*
0.0
0.0
–0.2
–0.1
–0.2
–0.4
0
20
40
60
80
100
120
140
0
20
Central radius of distance class (in cm)
60
80
100
120
140
0.3
0.4
*
La Barranquera
0.3
* *
*
0.1
*
0.2
Moran’s I
*
0.2
Moran’s I
40
Central radius of distance class (in cm)
Punta del Hidalgo
**
0.1
*
0.0
0.0
–0.1
–0.1
*
–0.2
–0.2
0
20
40
60
80
100
120
0
140
20
40
60
80
100
120
140
Central radius of distance class (in cm)
Central radius of distance class (in cm)
0.4
0.5
Palm-Mar
*
0.2
Las Galletas
0.4
Moran’s I
Moran’s I
0.3
0.0
–0.2
0.2
0.1
0.0
–0.1
–0.4
–0.2
–0.6
–0.3
0
20
40
60
80
100
120
140
Central radius of distance class (in cm)
0
20
40
60
80
100
120
140
Central radius of distance class (in cm)
Fig. 7 Spatial auto-correlation plots for all haploid thalli of Cladophoropsis membranacea at each site. Solid lines (all samples); dotted lines
(duplicate clones removed); * significant spatial auto-correlation at P < 0.05 for specified distance class.
phases are sufficiently refreshed by sexual reproduction
to prevent loss of one or both stages. An interesting and,
as yet unresolved, question is whether C. membranacea is
actually a perennial alga that dies back each year but
regenerates from microscopic, over-wintering thalli. In
that case, the balance of diploids and haploids may depend
more on chance events of initial recruitment and subsequent
density-dependent factors. Future studies will test for
temporal stability or lack thereof.
Dominance of the haploid phase
Only a few studies have investigated the proportions of
isomorphic haploid and diploid plants under field conditions.
As shown in Fig. 6, cushions and mats are composed of
multiple haploid, diploid, or clonal individuals with haploids
dominating.
Studies in two rhodophytic seaweeds from the Canary
Islands, Gelidium canariensis (Sosa & Garcia-Reina 1993)
and in Gelidium arbuscula (Sosa & Garcia-Reina 1992)
showed a permanent dominance of the diploid phase over
a 2-year period in both species and significant differences
in allele frequencies between the two phases in some
populations of G. arbuscula (Sosa et al. 1998). Equal distributions of haploids and diploids have also been reported
in Gracilaria gracilis (Engel et al. 1999) and in Gracilaria
verrucosa (now: gracilis) (Destombe et al. 1989). In both species
an excess of haploids was observed only transiently over a
1-month period. All of these species, however, are relatively
long-lived plants that persist for several years. They also
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 329 – 345
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P O P U L A T I O N S T R U C T U R E O F C L A D O P H O R O P S I S M E M B R A N A C E A 341
Table 7 Hierarchical amova analysis of spatial scales of differentiation in Cladophoropsis membranacea
Source of variation
Scale
d.f.
Variance
components
Per cent
variation
Fixation indices
(FCT, FSC, FST)
P value
between plots within a site
between quadrats within a plot
within quadrats
20 m
4.5–5 m
1 m2
1
2
41
0.05335
–0.06774
2.56379
2.09
–2.66
100.56
0.02093
–0.02714
–0.00564
0.33920
0.84066
0.67644
Peñascos
between plots within a site
between quadrats within a plot
within quadrats
16 m
4–6 m
1 m2
1
2
42
–0.06018
0.06098
1.83996
–3.27
3.31
99.96
–0.03269
0.03208
0.00044
0.66471
0.10948
0.26686
PdH and Peñ
between sites
within sites
6.8 km
26–29.5 m
1
89
0.17541
2.19363
7.40
92.60
between plots within a site
between quadrats within a plot
within quadrats
20 m
2.5–5 m
1 m2
1
2
45
0.21418
–0.00033
1.89437
10.16
–0.02
89.86
0.10159
–0.00017
0.10144*
0.33724
0.41349
0.01271
Punta del Hidalgo
between plots within a site
between quadrats within a plot
within quadrats
21 m
2.6–4 m
1 m2
1
2
46
–0.04415
0.07245
1.91459
–2.27
3.73
98.54
–0.02272
0.03646
0.01457
1.00000
0.07527
0.11926
LB and PH
between sites
within sites
10 km
27.5–27.6 m
1
94
0.37990
1.97417
16.14
83.86
between plots within a site
between quadrats within a plot
within quadrats
22 m
4.5–6 m
1 m2
1
2
49
0.00612
–0.00889
1.83131
0.33
–0.49
100.15
0.00335
–0.00488
–0.00151
0.33333
0.52297
0.48778
Las Galletas
between plots within a site
between quadrats within a plot
within quadrats
21 m
6.4–7 m
1 m2
1
2
41
–0.01060
–0.00742
1.61212
–0.66
–0.47
101.13
–0.00665
–0.00462
–0.01130
0.30401
0.45748
0.65200
PM and LG
between sites
within sites
8.1 km
32.5–34.4 m
1
93
0.29093
1.72775
14.41
85.59
Site
Gran Canaria
Playa del Hombre
Tenerife North
La Barranquera
Tenerife South
Palm-Mar
0.07404***
0.16138***
0.14412***
0.00000
0.00000
0.00000
Only haploid thalli were used and duplicate haplotypes were counted only once. Small negative values can occur by chance because
individuals being drawn at random from one group/subgroup have a higher probability of being identical to an individual from another
group/subgroup of origin (Excoffier et al. 1992).
*P < 0.05; ***P < 0.001 after Bonferroni correction.
possess an additional life history stage involving a diploid
carposporophyte stage, which mitotically amplifies the
diploid stage in a manner analogous to the haploid stage
in C. membranacea.
The finding that haploid thalli of C. membranacea accounted
for 84% of the samples and outnumbered diploid thalli
by at least 2 : 1 (and sometimes as much as 10 : 1) was not
expected at the outset of the study. Although plant biomass
waxes and wanes throughout the year, the balance between
life history stages had been expected to be relatively even.
At least three explanations may account for why this was
not so.
First, differences in habitat requirement between gametophytes and sporophytes might result in a biased sampling.
This is known to be important for heteromorphic seaweed
species such as the rhodophyte Asparagopsis/Falkenbergia
where gametophytes are subtidal and the sporophytes
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 329–345
intertidal. In C. membranacea, however, plants do not occur
in the subtidal or in the very high intertidal zones. This leaves
only the possibility of nonapparent microhabitat heterogeneity
within the relatively narrow zone in which it occurs. In
Enteromorpha, an alga with a similar isomorphic life history
and habitat, Joint et al. (2000) performed laboratory experiments in which they found that diploid spores preferentially
settled on bacterial films. This type of phase-specific resource
partitioning may play a substantial role in determining
which part of the life history can recruit. Unfortunately, it
will be very difficult to assess in field populations without
prior knowledge of the resources being competed for.
Second, seasonality might also provide an explanation
for the different proportions of haploid and diploid plants.
All sampling was conducted in January and thus represents
a single time point. During the summer months, shifts in
temperature and general exposure of reefs result in senescence
MEC_1448.fm Page 342 Wednesday, February 20, 2002 9:07 AM
342 H . J . V A N D E R S T R A T E E T A L .
and die-back of C. membranacea that may lead to its
apparent disappearance at some sites. Although reproduction
appears to be continuous, the specific type of reproduction
(i.e. sexual or asexual) is actually not known. There are no
specialized gametangia or sporangia, and biflagellate
gametes and quadriflagellate spores can only be distinguished
under the microscope. Thus, field identification of reproductive plants is, by itself, insufficient. Repeat sampling of
the study sites at other times throughout the year could,
in principle, reveal a temporal dominance of diploids,
especially if this could be related to nutrient availability.
Third, the dominance of haploids might also reflect
competitive advantage with or without seasonality. Locally
adapted haplotypes that propagate asexually may rapidly
minimize the availability of unoccupied space and thus
limit or even prevent opportunities for recruitment of new
diplotypes via sexual reproduction. This phenomenon
has been documented in (semi)aquatic plants (Grace 1993;
Neuhaus et al. 1993), and in corals (Hunter 1993). In algae,
there is at least some evidence for competitive dominance
in Gracilaria verrucosa (Destombe et al. 1993). They compared
competitive abilities between isomorphic haploid and
diploid thalli and found that haploids grew better than
diploids in low-nutrient conditions. They concluded that
haploid-competitive advantage would allow them to
dominate under a broad range of environmental conditions.
Cladophoropsis membranacea may follow a similar pattern in
which more stressful conditions in the late summer and
autumn favour the haploid phase. Modelling studies (Hughes
& Otto 1999) show that asexual reproduction of the haploid
phase is low cost to the plant and may even provide a selective
advantage to the persistence of the species. For example,
advantageous alleles would be exposed to direct selection
in haploids while any deleterious alleles carried during the
diploid phase could be rapidly purged during the haploid
phase. Even a small amount of sexual reproduction is
sufficient to maintain genetic diversity, therefore making
the numerical dominance of haploid plants at any given
time unimportant.
Initial dominance
Spatial auto-correlation analysis showed that haploid and
diploid individuals (i.e. duplicate haplotypes or diplotypes
removed) were randomly distributed. This implies that
the initial recruitment at a site is simply dependent on the
mix of gametes and spores in the propagule pool. An initial
skew one way or the other would simply change the rate
at which a mature patch would come to be dominated by
haploids or diploids. Since clonal reproduction of the haploids
accounted for between 50 and 75% of the individuals,
density-dependent effects on subsequent recruitment are
likely to play a significant role under our competitive
superiority hypothesis as long as the local area remains
environmentally stable. This would be especially true if
microscopic stages of C. membranacea are able to over-winter
in the porous substrata.
Many algae on (sub)tropical reefs are viewed as
ephemeral components of the intertidal turf community
and assumed to be annuals. Whether or not the same clonal
haplotypes persist year after year, or are replaced by a new
set of haplotypes from near by or far away is not known.
Perennial microscopic forms and propagule banks have
been postulated many times for seaweeds but their actual
presence is very difficult to prove (Norton 1992a; Santelices
et al. 1995). Such microscopic reserves in the substratum
could differentially respond to environmental selection
pressures and would go a long way towards testing the
fundamental question of population persistence since
natural environmental fluctuations in the intertidal are
expected to select for more than a few haplotypes/
diplotypes on a long-term basis.
Dispersal
Short- and long-distance dispersal affect population structure
differently. Most studies of direct dispersal of algal gametes
and spores, including the present study, have shown that
dispersal is limited to a few metres. In Gracilaria verrucosa,
the viability of male gametes is restricted to 6 h and dispersal
within 2 m (Destombe et al. 1990, 1992). In Gracilaria gracilis
80% of the actual mate pairs were separated by < 1 m
(Engel et al. 1999). Nevertheless, 11% of the cystocarps were
attributed to foreign males, probably from a population
25 m away. Fingerprinting analyses in the sea palm kelp
Postelsia palmaeformis indicated spore dispersal of 1– 5 m
(Coyer et al. 1997) and a microsatellite study in the fucoid
Ascophyllum nodosum indicated dispersal of 70 cm (Olsen
unpublished).
Wave-swept environments may actually retard dispersal
by maintaining a kind of retention zone in which the local
spore/propagule pool is trapped in tidepools and pockets
(Coyer et al. 1997; Engel et al. 1999; Wright et al. 2000).
An exception to the short-distance generalization is found
in the green alga Enteromorpha linza. Innes (1987) was able
to track spores for at least 200 m and Zechmann & Mathieson
(1985) showed that green algal gametes, spores and zygotes
were more often found in surface water samples along the
coast of New Hampshire (USA) than were gametes, spores
and zygotes from rhodophytic or phaeophytic seaweeds.
In C. membranacea, a few identical haplotypes were found
at a distance of 20 m and, in seven cases, between sites
5–10 km apart. While the 20-m scale fits with the observations of Zechmann & Mathieson (1985), the larger scale remains
difficult to interpret. We know that gene flow occurs at these
scales as shown by significant population differentiation
(FST 0.07–0.16; P < 0.001) in Table 7. However, it is not
possible to distinguish whether our observations represent
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 329 – 345
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P O P U L A T I O N S T R U C T U R E O F C L A D O P H O R O P S I S M E M B R A N A C E A 343
true long-distance dispersal of gametes/spores or a chance
occurrence of the same multilocus haplotype. At these
distances the role of plant fragments, either rafted or floating probably plays the key role. Although fragments of C.
membranacea are unlikely to reattach, spores can be released
at any time and floating debris containing C. membranacea
is relatively common. Whole-plant dispersal may therefore
play a role at intermediate distances, thus augmenting the
limits of spore dispersal. It may also account for the relatively
weak isolation by distance observed around and between
islands (Van der Strate unpublished).
Biphasic life histories as an evolutionary stable strategy
The persistence of species with free-living haploid and
diploid stages has generally been viewed as a transitory,
evolutionary state (Maynard Smith 1978; Stebbins & Hill
1980) in the evolution towards diploidy. However, recent
theoretical modelling suggests that biphasic life histories
represent an evolutionary stable strategy (ESS) (Kondrashov
& Crow 1991; Bengtsson 1992; Richerd et al. 1993, 1994;
Otto & Marks 1996) and that seaweeds are able to maintain
it indefinitely across many lineages (Valero et al. 1992;
Klinger 1993; Hughes & Otto 1999). Diploids are thought to
have an automatic fitness advantage over haploids because
they have two copies of every gene and are better able to
survive the effects of deleterious mutations. In contrast,
deleterious mutations in haploid individuals will be more
rapidly eliminated by selection and advantageous mutations
may confer greater local fitness. Species able to switch
between these two modes clearly have an advantage.
Otto & Marks (1996) were able to show in their modelling
simulations that assortative mating, selfing and apomixis
acted to maintain genetic associations that favoured the
haploid phase; whereas conditions that promoted random
mating and outcrossing favoured diploidy. More recently,
Hughes & Otto (1999) developed a combined model allowing
for life-cycle differences and competition which allowed
them to explore the effects of ecological factors on the
maintenance of biphasic life histories in organisms with
isomorphic haploid and diploid stages. They found that
linked reproductive phases had few confounding effects
on competition, and if an environment could be exploited
more efficiently by haploids and diploids, then these two
entities could coexist in an evolutionarily stable strategy.
They also explored the conditions that favoured shifts between
haploidy and diploidy. Using demographic data from
the isomorphic alga Gracilaria (Destombe et al. 1993) they
found that competitive abilities of haploids could overcome
the advantages of being diploid when niche differentiation
and haploid resistance to competition were sufficient to
compensate for higher diploid survival and reproduction,
but not so great that diploids were lost altogether. To test
the Hughes & Otto (1999) model with C. membranacea
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 329–345
would require detailed information about the demography
and competitive interactions between the two isomorphic
phases which is currently unavailable. Using a combination
of indirect genetic data with demographic data from the
field it should be possible to test the life-history part of the
model. Designing experiments to test competition, however,
will probably be limited to laboratory experiments.
In conclusion, high-resolution genetic markers have
allowed us to explore population structure in a common
tropical marine alga characterized by an isomorphic, biphasic
life history. The proportions of haploids, diploids and
clones suggests dominance of the haploid phase and its
probable competitive superiority. Future studies must add
a temporal component. The importance of seasonality (or
lack thereof ), persistence vs. new recruitment (possibly from
propagule banks) and competition between phases needs to
be assessed. There is also a critical need for demographic data.
With microsatellites it is now possible to follow fertility and
distinguish haplotypes and diplotypes in the field.
Acknowledgements
We thank Ricardo Haroun for arranging and assisting with the field
work on Gran Canaria and Tenerife. We also thank Pedro Sosa,
Mark Vermeij and Rike Walda for assistance with the fieldwork,
and Stella Boele-Bos for assistance with the laboratory analyses.
This research was supported by NWO-SLW Project Number
805–42048.
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Han van der Strate is a PhD student in population genetics and
marine biology interested in the relationships between population
structure, dispersal and adaptation in marine algae. He is
supervised by Louis van de Zande, Wytze Stam and Jeanine
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the descriptive and functional importance of genetic variation of
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