Download Fine-scale spatial genetic structure and clonal distribution of the cold

Coral Reefs (2012) 31:1135–1148
DOI 10.1007/s00338-012-0937-5
REPORT
Fine-scale spatial genetic structure and clonal distribution
of the cold-water coral Lophelia pertusa
M. P. Dahl • R. T. Pereyra • T. Lundälv
C. André
•
Received: 3 November 2011 / Accepted: 16 July 2012 / Published online: 2 August 2012
Ó Springer-Verlag 2012
Abstract Determining the spatial genetic structure within
and among cold-water coral populations is crucial to
understanding population dynamics, assessing the resilience of cold-water coral communities and estimating
genetic effects of habitat fragmentation for conservation.
The spatial distribution of genetic diversity in natural
populations depends on the species’ mode of reproduction,
and coral species often have a mixed strategy of sexual and
asexual reproduction. We describe the clonal architecture
of a cold-water coral reef and the fine-scale population
genetic structure (\35 km) of five reef localities in the NE
Skagerrak. This study represents the first of this type of
analysis from deep waters. We used thirteen microsatellite
loci to estimate gene flow and genotypic diversity and to
describe the fine-scale spatial distribution of clonal individuals of Lophelia pertusa. Within-population genetic
diversity was high in four of the five reef localities. These
four reefs constitute a genetic cluster with asymmetric gene
flow that indicates metapopulation dynamics. One locality,
the Säcken reef, was genetically isolated and depauperate.
Asexual reproduction was found to be a highly important
mode of reproduction for L. pertusa: 35 genetic individuals
Communicated by Biology Editor Dr. Ruth Gates
Electronic supplementary material The online version of this
article (doi:10.1007/s00338-012-0937-5) contains supplementary
material, which is available to authorized users.
M. P. Dahl (&) R. T. Pereyra C. André
Department of Marine Ecology-Tjärnö, University
of Gothenburg, 452 96 Strömstad, Sweden
e-mail: [email protected]
T. Lundälv
Sven Lovén Centre of Marine Sciences-Tjärnö,
University of Gothenburg, Strömstad, Sweden
were found on the largest reef, with the largest clone
covering an area of nearly 300 m2.
Keywords Cold-water coral Clonality Spatial genetic structure Genotypic diversity Connectivity Conservation genetics
Introduction
Genetic diversity within and among populations is influenced
by the species’ mode of reproduction. Sexual reproduction
resulting in recombination increases genetic diversity within
populations, while dispersal of larvae connects populations. In
contrast, clonal reproduction (asexual), which lacks sexual
recombination, may decrease diversity, potentially hampering
adaptation to environmental change (Lasker and Coffroth
1999). On the other hand, clonal propagation allows organisms to produce progeny without sexual reproduction and thus
enables species to persist when unable to complete the sexual
reproductive life cycle (Honnay and Bossuyt 2005). Clonal
propagation also allows genetic individuals to spread out
by clonal growth and to monopolize resources locally (Pan
and Price 2002). Nevertheless, all species that are considered
clonal have some level of genetic recombination (Bengtsson
2003; de Meeûs et al. 2007; Gladychev et al. 2008). The
degree of clonality has been shown to influence the genetic
structure within and among populations by reducing effective
population size and altering gene flow (Loveless and Hamrick
1984; Baums et al. 2006; Whitaker 2006). However, the
importance of clonal reproduction for the spatial distribution
of genetic diversity in natural populations of cold-water corals
is largely unknown.
Clonal establishment benefits species with a mixed
strategy of sexual and asexual reproduction particularly in
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unfavourable local environments where occasional sexual
recombination keeps genetic diversity higher and decreases
likelihood of extinction (Bengtsson 2003). In some species,
sexual reproduction and asexual reproduction contribute
equally to population growth, while in others, one reproductive mode dominates. The relative contribution of
sexual and asexual reproduction can vary among populations of a single species in response to biotic and abiotic
factors, for example latitude (Hoffman 1986; Dorken and
Eckert 2001) and population density (van Kleunen et al.
2001). The extent of clonality tends to increase at the limits
of geographical distribution of the species compared to
those populations located in the centre of the range and also
tends to increase with population age (Lesica and Allendorf
1995; Eckert 2002; Silvertown 2008).
Population connectivity occurs through the larval stage,
which is the only long-distance dispersal phase. Marine
larvae are, however, notoriously difficult to track in the
natural environment, and genetic markers are therefore often
used to infer population connectivity. In general, marine
populations are believed to be highly connected, resulting in
weak genetic structures. But this view has been recently
challenged (Cowen et al. 2000; Hauser and Carvalho 2008).
Indeed, corals show a wide range of genetic structures, from
panmixia over 7,000 km (Takabayashi et al. 2003) to locally
subdivided populations (see van Oppen and Gates (2006) for
a review).
Within coral reefs, genotypic richness depends on the
frequency of larval replenishment, recruitment dynamics
and longevity of genets (Eriksson 1993). The term ‘ramet’
is used for a physiologically distinct colony, and the term
‘genet’ or clone describes all ramets that are genetically
identical (sensu Harper 1977). Genotypic richness is
expected to decrease over time due to elimination of genets
by intraspecific competition, selection and stochastic
effects (Hartnett and Bazzaz 1985; Eriksson 1989). However, genotypic richness can be maintained at high levels if
the life span of genets is long and sexual reproduction
occurs occasionally (Eriksson 1993; Bengtsson 2003). The
spatial arrangement of ramets in a genet (i.e. the clonal
architecture) is known to affect mating opportunities and
prospective intraspecific competition.
Lophelia pertusa (henceforward referred to as Lophelia)
is the main reef-building cold-water coral species in the NE
Atlantic Ocean (Rogers 1999). It is gonochoristic; asexual
reproduction occurs by fragmentation, and sexual reproduction follows an annual gametogenic cycle. The fecundity is very high: a 30-cm2 colony is estimated to have
nearly 100,000 oocytes (Waller and Tyler 2005). After
successful fertilization, larvae capable of spending up to
7 weeks in the water column develop (M. Dahl, pers. obs.).
Reproduction by fragmentation allows clonal growth to act
as a means of small-scale spatial dispersal by horizontal
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Coral Reefs (2012) 31:1135–1148
spreading of clonal individuals (Gliddon et al. 1987).
Hence, persistent clonal reproduction may enable genets
(all genetically identical members of a clone) to grow
laterally, and the age of individual Lophelia clones can be
estimated by recording annual size increments (Witte and
Stöcklin 2010). Some corals are capable of producing
asexual propagules with long-distance dispersal abilities,
which may help them to recover from environmental disturbances (van Oppen et al. 2008). To date, however, this
reproductive strategy has not been reported in Lophelia.
Lophelia occurs frequently along the European continental
margin on ridges, seamounts and mound structures and in
fjords (Rogers 1999) and shows broad-scale (1,000 km)
genetic structuring (Le Goff-Vitry et al. 2004). The relative
contribution of each mode of reproduction has been
reported to vary among Lophelia populations along the
continental margin (Le Goff-Vitry et al. 2004). However,
these reported estimates of clonality might not be accurate
since sampling was conducted with dredge and trawl where
coral polyps may break and mix, making it hard to determine the precise sampling location of individual specimens. In contrast, recent advancements in sampling using
camera-assisted ROVs now allow precise sampling,
thereby allowing accurate assessment of clonal reproduction and mapping of the clonal architecture over entire
reefs.
In the North East Skagerrak, there are currently five
known Lophelia reef localities. These reefs provide an
excellent opportunity to map the distribution of individual
clones and also assess the genetic structure among local
reefs. In this study, we (1) assess the relative contribution
of sexual and asexual reproduction, (2) assess the dispersal
through clonal growth and larval transport and (3) discuss
the implications for reef conservation.
Materials and methods
Study sites and sampling
The Norwegian trench supplies the Skagerrak with Atlantic
deep water, providing suitable habitats for cold-water
corals. Through complex seabed topography, the Norwegian trench is further connected to the Oslofjord and the
deep troughs running along the Swedish west coast. Polyps
from a total of 142 Lophelia coral colonies were sampled
from five spatially distinct coral reef complex localities
in the NE Skagerrak (Fig. 1, Table 1). The locations
span from the outer Oslofjord to the northern part of the
Kosterfjord; the maximum distance between any reef pair
is less than 35 km (Fig. 1). Coral samples were collected
during numerous cruises over 6 years (2003–2009), preserved in ethanol (96 %) and maintained at -20 °C prior to
Coral Reefs (2012) 31:1135–1148
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Table 1 Lophelia pertusa
Ns
G
R
Prop clones
A
Rare alleles
Hexp
Hobs
FIS
Fjellknausene
12
7
0.55
0.417
5.1
0.05
0.81
0.71
0.12
West Søstrene
4
4
1
0.000
5.8
0.10
0.88
0.75
0.15
East Søstrene
26
13
0.48
0.500
5.6
0.08
0.86
0.75
0.12*
Tisler
87
35
0.40
0.598
5.6
0.17
0.84
0.72
0.15*
Säcken
13
5
0.33
0.615
2.4
0.05
0.48
0.69
-0.44*
Number of ramets (Ns), number of genets (G), genotypic richness (R), proportion of clones, allelic richness (A), rare alleles (expressed as mean
number of alleles per locus normalized by genotypic richness), expected heterozygosity, observed heterozygosity and inbreeding coefficient (FIS)
* significantly different from zero at p \ 0.05
genetic analyses. Samples were collected with a remotely
operated vehicle (ROV) to minimize damage and to allow a
precise geographical position of each sample. A detailed
analysis of the spatial distribution of clones was performed
for the Tisler reef (n = 87, Table 1), the largest known reef
in the NE Skagerrak. Here, live coral extends over ca
250 ha (*20–30 % coverage, Fig. 2), and damage from
extensive trawling activity has been documented (Lundälv
2003). Positions of the samples were obtained with a USBL
underwater positioning system of type Simrad HPR 410 P
in combination with a Furuno GPS gyro type SC 110 and a
DGPS instrument type GBX Pro. Data were visualized and
logged in the navigational software Olex. Accuracy of
obtained positions was approximately ±2 m. All ramets
belonging to a putative genet were sampled at a distance
greater than the precision level of the positioning system.
Genotyping
DNA was extracted from coral polyp tissue with the Viogene Blood & Tissue Genomic DNA Extraction Miniprep
System, following the manufacturer’s protocol. All samples were genotyped using thirteen microsatellite loci
developed for Lophelia: three dinucleotide (Lp loci, LeGoff
and Rogers 2002) and ten tri- or tetranucleotide loci (Lpe,
Morrison et al. 2008). The Lp loci were amplified following LeGoff and Rogers (2002), whereas the Lpe loci were
amplified in 10 lL PCR containing 2-60 ng/lL of template
DNA, 0.05 U recombinant Taq DNA polymerase (TaKaRa
TaqTM), 0.125 lM of forward and reverse primer, 1X
buffer, 1.5 mM MgCl2 and 0.2 mM of each dNTP. PCR
amplifications for Lpe loci were performed under the following conditions: initial denaturation at 94 °C (2 min),
followed by 30 cycles at 94 °C (30 s), 58 °C (40 s) and
72 °C (30 s), with a final extension at 72 °C (10 min). Sets
of three labelled primer pairs were poolplexed and sized on
a CEQ 8000 Genetic Analysis System.
Control for the presence of scoring errors due to stuttering during PCR amplification, null alleles and large
allele dropout were performed with MICROCHECKER
version 2.2.3 (van Oosterhout et al. 2004). Estimated frequencies of putative null alleles were subsequently calculated with FREENA (Chapuis and Estoup 2007) using the
EM algorithm (Dempster et al. 1977) and the ENA method
to calculate unbiased FST estimates adjusted for the presence of null alleles.
Genotypic richness and diversity
Estimation of components of clonal diversity (clonal richness, clonal heterogeneity and clonal evenness) was performed in GENCLONE version 2.0 (Arnaud-Haond and
Belkhir 2007). Genotypic richness was determined as
R = (G–1)/(N–1), where N is the number of genotyped
samples and G the number of genotypes. GENCLONE was
used to calculate the unique genotype probability (psex) to
assess the presence of putative clonal genotypes as a result
of sexual reproduction (Parks and Werth 1993). GENCLONE was also used to analyse intra-reef spatial genetic
structure using spatial autocorrelation of genotypes (see
Sokal and Oden 1978), represented by the kinship coefficient Fij (Loiselle et al. 1995). The spatial autocorrelation
analysis was performed on two reef localities (Tisler and
East Søstrene) where sample sizes allowed analysis of
intra-reef spatial genetic structure. Spatial autocorrelation
was also used to assess the impact of bottom trawling at
Tisler reef by performing the analysis before and after
removing all ramets sampled inside areas known to be
affected by bottom trawling (Lundälv 2003). The spatial
distance where the ramet and genet level correlograms
intersect defines the clonal subrange (Harada et al. 1997;
Alberto et al. 2005), a spatial measure that describes the
scale at which clonality affects the genetic structure in a
population. The clonal architecture of Tisler and East
Søstrene was described with the spatial aggregation index
(Ac) in GENCLONE. Finally, a permutation test was performed to obtain an accurate estimate of the minimum,
average and maximum number of discriminated genetic
individuals for the given number of samples from Tisler
reef.
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West Søstrene
(110 m)
East Søstrene
(95 - 120 m)
Fjellknausene
(95 - 115 m)
Säcken
(85 m)
Tisler
(70-145 m)
Fig. 1 Sampling locations and depth for L. pertusa in north-east Skagerrak
EstimateS (Colwell 2000) was used to calculate the
expected total number of distinct genetic individuals at
Tisler reef, by fitting an asymptotic function to the samplebased rarefaction curve from GENCLONE. The nonparametric richness estimators ICE and Chao2 were used (50
randomizations for each sample).
Age of genets
The minimum age of three different genets from Tisler reef
was estimated by calculating the time required to grow
linearly to cover the measured area. Linear growth rates
for corals in this area have previously been measured to
5–7 mm year-1 (HERMES community report 2008). These
three genets (referred to as the orange, blue and red genet)
contained sufficient numbers of ramets to assume that the
clone continuously covered the area. We used a conservative
approach where only genets that appeared continuously
distributed were included (see marked areas at the insert in
Fig. 3). Individuals of the same clone scattered further afield
and not within the continuous distribution were excluded
123
because ramets with a different genetic identity may confound the interpretation. During ROV sampling, no trawling
damage was observed in the vicinity of these clones, something that would have caused an overestimation of the age
estimate. The IVS FLEDERMAUS software was used to
calculate the area covered by the clones. The calculated area
was based on horizontal distances, thus representing horizontal coral growth. Since corals predominately grow vertically, age was calculated assuming an average coral colony
height of 60 cm and a circular shape of the clone. By measuring a large number of coral colonies, we estimated an
average 19-degree angle vertical growth. Repeated genotypes (ramets that belong to one genet) were included in the
age and area coverage analysis described above. In subsequent genetic analysis, only unique multilocus genotypes
were kept.
Summary statistics
Allele frequencies, observed heterozygosity (Ho), expected
heterozygosity (He), inbreeding coefficients (f), Hardy–
Coral Reefs (2012) 31:1135–1148
10° 56
59° 0.75
10° 56.5
1139
10° 57
10° 57.5
10° 58
10° 58.5
10° 59
10° 59.5
11° 0
59° 0.75
-240 -220 -200 -180 -160 -140 -120 -100 -80 -60 -40 -20
Water depth m
59° 0.5
59° 0.5
59° 0.25
59° 0.25
59° 0
59° 0
58° 59.75
58° 59.75
58° 59.5
58° 59.5
10° 56
10° 56.5
10° 57
10° 57.5
10° 58.5
10° 58
10° 59
10° 59.5
11° 0
Fig. 2 Bathymetric map of Tisler reef location generated from
multibeam data. Reef is the dominant structure at the sill between the
two basins. Photograph insert of Tisler reef illustrates the difficulty in
distinguishing genetic individuals without molecular methods. The
within-population demography is hidden by the identical appearance
of different genetic individuals. The sponge Mycale lingua is
commonly observed growing next to and competing with L. pertusa.
Photograph by Tomas Lundälv
Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LE) were calculated using GENEPOP 4.0.6
(Rousset 2008). Allelic richness using the rarefaction
method was calculated using FSTAT version 2.9.3.2
(Goudet 2001). The significance levels were adjusted by
Bonferroni correction when multiple tests were applied.
statistic DK (Evanno et al. 2005) in the program STRUCTURE HARVESTER version 0.6.8 (Earl and vonHoldt
2011) to estimate the most likely number of K clusters.
To corroborate the results from STRUCTURE and to
assess recruitment and migration, we used genetic assignment performed with GENECLASS 2 (Piry et al. 2004),
which is an individual-based classification method. Groups
are defined a priori and individuals are assigned to known
sources using the Bayesian allele frequency estimation
method (Rannala and Mountain 1997) with the leave-oneout procedure. A simulation logarithm is implemented,
which makes it possible to detect first-generation migrants
(Paetkau et al. 2004). We performed the simulations with
10 000 individuals and a threshold p value of 0.01.
Genetic differentiation
Differentiation between sites was described by the FST
estimator h (Weir and Cockerham 1984), and the null
hypothesis of no differentiation was tested using Fisher’s
exact test. We used factorial correspondence analysis
implemented in GENETIX version 4.05.2 (Belkhir et al.
1996–2004) for visualizing the spatial variation in genetic
composition among sampling localities.
We used STRUCTURE 2.2 (Pritchard et al. 2000) to
identify the number of different K genetic clusters. Five
replicate runs were performed under the admixture model
with correlated allele frequencies (K = 1–6; burn-in =
20,000 and 105 iterations). The program DISTRUCT
(Rosenberg 2004) was used to plot individual membership
assignments to each cluster. We calculated the ad hoc
Results
Genetic diversity
All 13 microsatellite loci were highly polymorphic, averaging 22 alleles per locus and ranging from 7 (LpeC120)
to 42 (Lp462), respectively (Electronic Supplemental
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Fig. 3 Lophelia pertusa.
Bathymetric map of Tisler reef,
facing westward, generated
from multibeam data. Coloured
dots represent spatial
distribution of genets. Each
colour is one genet; white dots
indicate multilocus genotypes
found only once. Insert is an
enlargement of the central part
of the reef. See Fig. 2 for scale
Material, ESM Table S1); the mean number of alleles per
population was 9.1. Locus-specific allele frequency distributions are shown in ESM Fig. S1, and locus-by-locus
statistics on the number of alleles, Ho, He and FIS are
presented in ESM Table S1. Overall loci values of He
ranged between 0.48 and 0.88, Ho from 0.69 to 0.75 and
FIS from -0.44 to 0.15 (Table 1). West Søstrene exhibited
the highest allelic richness (5.8) in contrast to Säcken reef
(2.4) that had values significantly lower than the average
richness over all populations (4.88; p \ 0.001) (Table 1).
Tisler had the highest mean number of private alleles
(0.17), while Säcken reef and Fjellknausene had the lowest
(0.05). Fisher’s exact test revealed that 16 of 65 locus/
population specific comparisons (24.6 %) deviated from
Hardy–Weinberg expectations; six remained significant
after Bonferroni correction (a = 0.05/65 = 0.0008). All
deviations were due to heterozygote deficits, five were
from Tisler reef and one from East Søstrene. Significant
genotypic linkage disequilibrium was found in eight of 390
comparisons (2.1 %), but none remained significant after
Bonferroni correction. Linkage between loci was found in
three of 78 comparisons; only one of these comparisons
(LpeD3 and Lp355) remained significant after Bonferroni
correction. MICROCHECKER revealed no evidence of
scoring errors, large allele dropouts or null alleles except
in one case where the potential occurrence of null alleles in
LpeC126 was indicated. The estimated frequency of the
null allele varied between 0.27 and 0.39 in all populations,
123
and the unbiased FST values obtained using the ENA
method did not vary significantly from the uncorrected
ones. Overall uncorrected and corrected FST values across
loci and populations were 0.038 (95 % CI: 0.029–0.047)
and 0.039 (95 % CI: 0.030–0.047), respectively.
Genotypic richness, indicating the relative importance
of sexual versus asexual reproduction, ranged from 0.3 in
the Säcken population to 1 in the West Søstrene population
(Table 1). The probability that any of the different genets
would have the same genotype by chance was significantly
low (pgen \ 1.00E-15), and the probability that the repeated genotypes would have originated from distinct sexual
reproductive events was also significantly low (psex \
1.8E-24). Hence, all ramets with identical multilocus
genotype were assumed to be clones.
We found a total of 64 multilocus genotypes, and none
of these were found at more than one sampling locality.
This suggests that Lophelia does not develop asexual larvae and that coral fragments are not transported over long
distances.
Within-reef genetic structure
The longest distance between two ramets of the same genet
was 253 m (Fig. 3; black dots). In total, the 87 sampled
ramets from Tisler reef were distributed among 35 genetic
individuals (Fig. 4). The distribution of clonal ramets
conformed to a Pareto distribution with a shallow slope
Number of distinct MLG
Coral Reefs (2012) 31:1135–1148
1141
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88
Number of individuals
Fig. 4 Lophelia pertusa. Boxplot showing the genotypic richness of Tisler reef samples. Central line depicts average number of genotypes.
Edges of the box indicate minimum and maximum number of genotypes
(b = 1.61) indicating low evenness. Clonal heterogeneity
estimate and the equitability index (i.e. Simpson’s complement (D) = 0.94 and evenness (V) = 0.89, respectively) also show low evenness. Among the 35 genets
detected at Tisler reef, fourteen appeared more than once
(clones) and 21 were singletons. However, a few large
clones dominated the reef; the three most frequently
occurring genets made up 37 % of all sampled colonies.
The most frequently sampled genet accounted for 16 %
(Fig. 3; blue), while the second and third most frequently
observed genets accounted for 13 and 8 %, respectively
(Fig. 3; red and orange). Clonal distribution, heterogeneity
and evenness estimates for East Søstrene were similar
to those of Tisler (b = 1.95, D = 0.93, V = 0.83). East
Søstrene had a higher evenness than Tisler, where six
genets had ramets appearing more than once and seven
were singlets. Indices of spatial aggregation were significant for both sites tested (p \ 0.001; Ac = 0.46 and 0.63,
for Tisler and East Søstrene, respectively).
The expected total number of genets at Tisler reef varied
from a point estimate of 76 (ICE) to 90 (Chao2; 95 %
confidence interval from 52 to 219). The estimated minimum age of genets at Tisler reef indicates that clones are of
substantial age. The orange clone covered an area
of 299 m2, which translates to an estimated age range of
4,408–6,172 years. Corresponding values for the blue and
red clones are 164 and 130 m2, with estimated ages ranging
from 3,251 to 4,569 and 2,906–4,068 years, respectively.
Spatial autocorrelations revealed that the clonal subrange at Tisler reef extends up to 120 m. Ramets less than
28 m from each other have a 25 % probability of clonal
identity. Probability of clonal identity decreased with
increasing distances between ramets. When ramets sampled in trawled areas were excluded from the analysis, the
clonal subrange decreased to 66 m (Fig. 5); the probability
of clonal identity was higher at shorter distances (27 % at
25 m) and exhibited a steeper decrease with increasing
distance (Fig. 5). The clonal subrange at East Søstrene
extended 55 m. Additionally, the probability of clonal
identity was high at short distances (57 % at 14 m) and
decreased rapidly to 9 % at a distance of 60 m (data not
shown).
Population genetic structure
The factorial correspondence analysis revealed that the
four reef complexes located in the outer Oslofjord and
outer Hvaler (West Søstrene, East Søstrene, Fjellknausene
and Tisler) clustered on the right side of the first component axis, which explains most of the variation observed
(41.3 %, Fig. 6). All genets from Säcken were distinct and
formed a separate cluster in the multivariate space.
The Bayesian clustering analysis in STRUCTURE
(Fig. 7) and DK estimation (ESM Fig. S2 A, B) also suggested two distinct genetic clusters with Säcken reef as one
cluster and the other four reefs as another. Likewise,
individual assignment tests using GENECLASS showed
that all individuals sampled at Säcken were assigned back
to the Säcken location (Fig. 8), indicative of their distinct
genetic composition. Fifty-five per cent of the individuals
sampled at East Søstrene and 46 % of the individuals
sampled at Tisler were assigned back to their sample
location, whereas 36 % of individuals from East Søstrene
were assigned to Tisler and 38 % of the individuals from
Tisler were assigned to East Søstrene (Fig. 8). None of the
individuals at Fjellknausene were assigned to its sampling
location; all individuals were suggested to originate from
East Søstrene (57 %) or Tisler (43 %). Similarly, at West
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Coral Reefs (2012) 31:1135–1148
Fig. 5 Spatial autocorrelation
analysis of kinship coefficients
for L. pertusa colonies at Tisler
reef. Three different analyses
are shown for both nontrawled
(dashed lines) and trawled
(filled lines) data sets in the
diagram: (i) at ramet level
(triangles); (ii) at genet level
(circles); and (iii) the
probability of clonal identity
(squares)
0,3
Probability of clonal identity F(r) (87)
Probability of clonal identity F(r) (82)
Coancestry (Fij) All pairs of ramets included (87)
Coancestry (Fij) All pairs of ramets included (82)
Coancestry (Fij) Pairs of genets included (87)
Coancestry (Fij) Pairs of genets included (82)
0,25
0,2
0,15
0,1
0,05
0
0
100
200
300
400
500
600
700
-0,05
Spatial distance (m)
Axis 2 (22,49 %)
110
100
90
80
70
60
50
40
30
20
10
0
-10
-20
-30
-40
-50
-60
-70
-20 000
-18 000
-16 000
-14 000
-12 000
-10 000
8 000
4 000
-8 000
Axis 1 (41,31% )
-6 000
-4 000
-2 000
0
2 000
4 000
0
-4 000
-8 000
Axis
-12 000
-16 000
3 (18,69 %)
Fig. 6 Factorial correspondence analysis based on allele frequencies
from 13 microsatellite loci genotyped in five L. pertusa populations
from NE Skagerrak. Colours represent sample locations, orange =
West Søstrene, green = East Søstrene, blue = Fjellknausene, yellow = Tisler, red = Säcken
Søstrene, none were assigned back to that sampling location. Fifty per cent were assigned to Fjellknausene and
25 % to both Tisler and East Søstrene. Ten individuals
across all populations were detected to be first-generation
migrants. Of six migrants detected at Tisler reef, five
originated from East Søstrene and one from West Søstrene.
At East Søstrene, one first-generation migrant came from
Tisler and a second from Fjellknausene. The two remaining
first-generation migrants were found at Fjellknausene and
West Søstrene; both originated from Tisler.
Discussion
123
We present here the first empirical characterization of
the fine-scale genetic structure of the cold-water coral
L. pertusa. We show that reefs in Skagerrak consist of
large and old clones, with an overall incidence of
clonality of ca. 50 %. We found genetic differentiation
and no sharing of clones among reefs, indicating that
larval transport is the predominant mode of inter-reef
dispersal.
Coral Reefs (2012) 31:1135–1148
1143
Within-reef genetic structure and clonal spatial
distribution
K= 2
K= 3
K= 4
K= 5
en
ck
Sa
ler
Tis
Fje
l
We lkna
st use
So
n
str e
Ea
en
st
e
So
str
en
e
K= 6
Fig. 7 Bayesian STRUCTURE analysis of L. pertusa populations
using combined data from 13 microsatellite loci. Results are shown
for five levels of K (2–6). True K = 2 (DK = 65.3). Each individual
is represented by a vertical line partitioned into K coloured segments
that represent the individual’s estimated membership fractions. Black
lines separate individuals from different sampling sites, which are
labelled below the figure
Genetic diversity was high in all Lophelia populations
except at Säcken reef (Table 1). Säcken reef is characterized by low heterozygosity and allelic diversity, and such
levels of diversity have been previously reported from East
Africa coral populations (Souter and Grahn 2007; Ridgway
et al. 2008). The other four reef populations exhibited
levels of allelic richness similar to those described for
many tropical, shallow-water coral species (e.g. Baums
2008; Shearer et al. 2009; Underwood et al. 2009). The
total and mean number of alleles per locus for all reefs
except Säcken reef were similar to average levels found in
more than 70 populations of scleractinian corals (Shearer
et al. 2009). Interestingly, the level of heterozygosity in
Lophelia seems to be higher than those reported for many
tropical coral species (cf. Table 3 in Baums 2008). This
may be attributed to the gonochoristic breeding system that
enforces outcrossing compared to the hermaphroditism
common in tropical corals. However, these high levels of
heterozygosity may be also expected in populations with
high rates of clonal reproduction by retention of more
alleles (Balloux et al. 2003). In addition, asexual reproduction and long generation times slow down the loss of
genetic diversity through genetic drift (Orive 1993; Young
et al. 1996).
Despite the high values of expected heterozygosity in
Lophelia, our results also show a considerable number
of heterozygote deficiencies. Heterozygote deficiencies in
adult sessile marine invertebrates with planktonic larvae
Fig. 8 Individual-based selfassignment test using the leaveone-out procedure on five L.
pertusa populations in NE
Skagerrak. Vertical bars
represent per cent individuals
assigned back to sample
location. Colours represent
assigned locations:
orange = West Søstrene,
green = East Søstrene,
blue = Fjellknausene,
yellow = Tisler, red = Säcken
100%
75%
50%
25%
Fjellknausene
0%
West Søstrene
East Søstrene
Tisler reef
Säcken reef
123
1144
have been reported in numerous studies (e.g. Johnson and
Black 1982; Andrade and Solferini 2007; reviewed in
Brownlow et al. 2008) and are also found in many studies
of scleractinian corals (e.g. Ayre and Hughes 2000; van
Oppen et al. 2008; Underwood et al. 2009). Sampling
artefacts and attributes of molecular markers such as null
alleles may contribute to these heterozygote deficiencies.
However, clonal reproduction can largely account for this
deficiency. Facultative clonal reproduction is common
among sessile marine invertebrates (Hughes and Cancino
1985; Hughes 1989) and is also found in all hermatypic
corals (Veron 2000). This life-history trait causes heterozygote deficiencies at the population level due to deviation
from random mating (van Oppen et al. 2008).
Clonal reproduction in Lophelia has been previously
addressed in studies of genetic structure at larger spatial
scales (LeGoff-Vitry and Rogers 2004; Morrison et al.
2011), where minimum and maximum distances between
reefs ranged from 50 to 9,000 km. The proportions of
clones were low (average 0.09 and 0.13, respectively),
indicating that sexual reproduction is the predominant
mode of reproduction. LeGoff-Vitry et al. (2004) reported
evidence of clonality in only three out of ten populations
investigated. La Galicia and Porcupine Seabight reefs had
relatively low levels of asexual reproduction (0.15 and
0.20, respectively) compared to Darwin Mounds (0.49).
The high proportion of clones at Darwin Mounds was
attributed to low rates of sexual reproduction, patchy distribution of available habitat and bottom trawling. Similarly, Morrison et al. (2011) reported no clonality at 5 of
16 populations examined from mainly western Atlantic
localities. The proportion of clones (0.42–0.62) and range
of genotypic richness estimated in the present study
(0.33–0.55, excluding the West Søstrene locality where
only four specimens were collected) suggest that asexual
reproduction in Lophelia is more important for reef
development than previously shown. Thus, clonality may
play a key role in cold-water coral reef establishment and
maintenance. Our results suggest that asexual reproduction
is the natural state found in cold-water coral reef development in the Skagerrak although trawling may also cause
fragmentation. The dominance of few clones (as expressed
by low evenness) has been observed in several clonal
organisms (reviewed in Arnaud et al. 2007). Similarly,
Lophelia reefs and shallow-water tropical corals appear
dominated by few clones (Coffroth and Lasker 1998;
Whitaker 2006; Baums et al. 2006). Intraspecific competition influences evenness, and this influence will have
larger effects as population age increases and levels of
sexual reproduction decrease. However, low evenness can
also be explained by differences in time of establishment of
genets (early established genets will have more time to
increase their size and acquire resources). While a balance
123
Coral Reefs (2012) 31:1135–1148
between larval replenishment and genet longevity largely
determines genotypic richness, evenness is more dependent
on genet longevity and genet size and hence directly linked
to clonal growth (Coffroth and Lasker 1998). Therefore,
the evenness values calculated for Lophelia suggest that
clonal growth contributes more to reef development than
would be expected if only seedling recruitment were
operating. Natural mechanisms of spatial extension of
clones are governed by a relationship between growth and
bioeroders such as clionid sponges that cause weakening
and breakage of the skeleton. A growth pattern or patch
development of Lophelia, often referred to as Wilson rings
(Wilson 1979), arises from the initial settling of a larva and
subsequent growth until the colony is so large and weakened that fragments fall off. These fragments will grow
until they meet the same fate. The mechanism for the
development of these rings supports the idea that clone size
is directly related to genet age. Our estimates of genet ages
suggest that the large clones are of considerable age, possibly being the same individuals that first settled after the
glacial ice retreated several thousand years ago. Thus,
genet longevity is an evolutionary consequence of clonal
propagation and constitutes a key life-history trait of
Lophelia. Consequently, the longevity and low number
of genets observed in the present study suggest that the
turnover rate is extremely slow. Unique genotypes (singletons) that appear within areas occupied by larger clones
are indicative of subsequent immigration and settlement.
Hence, development of populations is continuous with low
rates of replenishment.
Clonal reproduction also affects the reproductive
dynamics within a population by influencing the clonal
architecture and thus the opportunities for mating. Generally, there are two types of clonal architecture: (a) the
‘guerrilla’, characterized by a high level of intermingling
of genotypes and (b) the ‘phalanx’, where high aggregations of clones result in a mosaic of clumped ramets of the
same genotype. The phalanx architecture may decrease
mate availability in gonochoristic species with external
fertilization since the probability of successful fertilization
decreases with distance. However, once this architecture is
established, it can be advantageous for local persistence by
optimizing resource capture and space occupation (Herben
and Hara 1997). All genets at Tisler with one exception
seem to conform to the phalanx strategy. The ‘blue’ clone
exhibits a complex distribution of ramets that might have
either a biological (fragmentation) or an anthropogenic
(spreading by anchors) origin.
Population structure
The five Skagerrak reef complexes constitute two distinct
genetic clusters: one comprised of the four reefs located in
Coral Reefs (2012) 31:1135–1148
the outer Oslofjord and Hvaler area (West Søstrene, East
Søstrene, Fjellknausene and Tisler) and a second consisting
of only the isolated Säcken reef located further inside the
coastal system of deep troughs. This pattern was supported
by FST estimates (ESM Table S2), the high posterior
probabilities in STRUCTURE (Fig. 7), as well as the
individual assignment tests (Fig. 8). The gene flow/connectivity pattern observed within the larger cluster indicates asymmetric connectivity among subpopulations in a
larger metapopulation. Further, the results suggest that
Tisler and East Søstrene reefs are important sources of
larvae. Such connectivity patterns are likely the result of
the complex local hydrographic circulation conditions
(Lavaleye et al. 2009), which in turn is attributed to the
topographic complexity of the seabed in the area. In
comparison to the restricted gene flow at a local scale
within the NE Skagerrak, Lophelia populations on both
sides of the Atlantic have been found to be both restricted
and moderately connected genetically over larger spatial
scales. LeGoff-Vitry et al. (2004) reported moderate levels
of gene flow among populations on the European continental margin and isolated fjord populations. Recently,
Morrison et al. (2011) showed significant population subdivision among Gulf of Mexico and West and East Atlantic
Ocean, but high connectivity within regions. This geographical variation emphasizes that connectivity patterns
are not solely a species-specific biological trait but also a
reflection of local environmental conditions and stochastic
oceanographic processes.
Clonal reproduction influences population genetic
structure in at least two ways: (1) reproductive output
increases as a function of total genet size (Highsmith 1982;
Hämmerli and Reusch 2003) and (2) the probability
of genet death decreases as a function of number of ramets
or size of genets/ramets (Highsmith 1982). In the coral
Goniastrea aspera, for example, the largest colonies produce 25 % of the annual egg production even though
numerically these colonies only comprise 3 % of the
population (Babcock 1984). Hence, large genets, as was
observed at Tisler reef, may contribute more to the gametic
gene pool. Once genets become dominant, the probability
of genet death approaches zero and their genes will persist
in the population until the ancient genotype dies. This
‘swamping’ of the local gene pool means that most larvae
are descendants of the dominant genotype at a reef.
Implication for conservation and future directions
A striking feature of cold-water coral reefs is that they are
generally composed of single coral species. In tropical
coral reef ecosystems, community composition (species,
species diversity and abundance) likely affects the ability
to respond to environmental changes (Connell et al. 2004).
1145
In contrast, cold-water coral reefs such as Lophelia appear
to be more dependent on the genotypic diversity within
populations to respond to environmental changes.
Recently, it has been recognized that genetic diversity may
influence ecological processes at all possible levels of
organization (Hughes et al. 2008). Genetic diversity has
been shown to have similar effects on, for example, fitness,
responses to disturbance and ecosystem function as
species diversity has for a wide range of organisms
including plants, invertebrates and vertebrates (Gamfeldt
et al. 2005; Pearman and Garner 2005; Johnson et al. 2006;
Crutsinger et al. 2006; Mattila and Seeley 2007; Hughes
and Stachowicz 2009). Clonal species with high genotypic
diversity are described as having higher resistance to parasites and pathogens (Booth and Grime 2003; Altermatt
and Ebert 2008). Additionally, experimental studies have
shown positive effects of genotypic diversity on survival
and faster recovery after extreme climatic events (Hughes
and Stachowicz 2004; Reusch et al. 2005). Here, we show
that the genotypic diversity was typically less than 50 %
for the reefs. We detected 35 genetically distinct individuals representing at best 90 individuals in total at the
largest reef, Tisler. Shearer et al. (2009) estimated from
tropical corals that 35 randomly sampled colonies are
required to maintain [90 % of the genetic diversity. Taking these estimates as reference, for highly clonal species
as Lophelia, nearly 100 colonies would be needed to
maintain similar levels of diversity.
Rare species have been prioritized in conservation
efforts due to the high risk of extinction, but it has become
increasingly clear that more common habitat-forming
species such as Lophelia may be equally important to
conserve (Gaston and Fuller 2008). Conservation efforts
in Lophelia could be improved with information of key
life-history traits such as levels of clonality and genetic
diversity within populations. For example, results from this
study on clumped spatial distribution of clones indicate that
trawling activities can eliminate unique genets from the
population. At Tisler, there are large areas of dead coral
structure at both ends of the reef surrounded by numerous
trawl scars (Lundälv 2003). Our results show an increase in
the clonal subrange at Tisler reef where high trawling
activity has been documented (Fig. 5). The large distances
between ramets suggest that bottom trawling has altered
the genetic structure. Prior to these damages, the reef had a
length of about 2 km (Lavaleye et al. 2009); at present, the
length of living coral reef is approximately 1,200 m.
Trawling on cold-water coral habitats disrupts the threedimensional structure of reefs and subsequently alters the
hydrodynamic and sedimentary conditions around it
(Rogers 1999). Genetic changes in the form of increased
clonal subranges have also been observed in seagrass
habitats in the Mediterranean Sea (Diaz-Almela et al.
123
1146
2007). However, to our knowledge, changes in the genetic
structure of scleractinian corals specifically produced by
trawling have not been previously reported. Thus,
description of genetic and genotypic diversity distribution
is increasingly urgent for cold-water coral ecosystems. Due
to longevity of individuals in cold-water corals, even small
genetic differences between Lophelia populations should
be considered in conservation decisions. Sampling strategy,
accuracy and spatial resolution will all affect the measured
level of genotypic richness and should therefore be stated
explicitly. Description of the genetic diversity distribution
is an important step, but the factors and processes
responsible for the observed patterns remain untested.
In 2009, Sweden and Norway established two protected
areas, which together form a transboundary marine national
park covering 751 km2. The park encompasses all the reefs
examined in the present study; thus, all reefs are protected.
The Säcken reef is in a severely degraded condition; the
low genetic variation in combination with the apparent lack
of gene flow to Säcken reef from other reefs suggests that
the potential for future adaptation to environmental change
may be low. The distances between the living colonies also
suggest that successful fertilization may be limited. Consequently, active restoration of Säcken reef population
is recommended. The present study demonstrates the usefulness of investigating the small-scale spatial genetic
structure to improve our understanding of reef development, maintenance and the conservation of these habitats.
Acknowledgments We thank Lisbeth Jonsson and Armin Form for
assistance in collecting samples and Sophie Arnaud-Haond for
valuable advice and comments on an earlier version. Funding was
provided by grants from Oscar and Lili Lamm, Helge Ax:son Johnsons stiftelse, Wilhelm och Martina Lundgrens Vetenskaps- och
Understödsfond and Colliander. We would also like to acknowledge
the help of three anonymous reviewers whose suggestions helped
to improve the manuscript considerably. Funding for sampling was
supported by the two European projects: HERMES EC contract no
GOCE-CT-2005-511234, funded by the European Commission’s
Sixth Framework Programme under the priority ‘Sustainable Development, Global Change and Ecosystems’ and the HERMIONE project, EC contract no 226354, funded by the European Commission’s
7th Framework Programme under the theme Environment (including
climate change).
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