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Genetic diversity of the imperilled bath sponge Spongia
officinalis Linnaeus, 1759 across the Mediterranean Sea:
patterns of population differentiation and implications
for taxonomy and conservation
T. DAILIANIS,*† C. S. TSIGENOPOULOS,† C. DOUNAS† and E. VOULTSIADOU*
*Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece, †Institute of Marine
Biology and Genetics, Hellenic Centre for Marine Research, Thalassocosmos, PO Box 2214, 71003 Heraklion, Crete, Greece
Abstract
The Mediterranean bath sponge Spongia officinalis is an iconic species with high socio-
economic value and precarious future owing to unregulated harvesting, mortality
incidents and lack of established knowledge regarding its ecology. This study aims to
assess genetic diversity and population structure of the species at different geographical
scales throughout its distribution. For this purpose, 11 locations in the Eastern
Mediterranean (Aegean Sea), Western Mediterranean (Provence coast) and the Strait of
Gibraltar were sampled; specimens were analysed using partial mitochondrial cyto-
chrome oxidase subunit I (COI) sequences, along with a set of eight microsatellite loci.
According to our results (i) no genetic differentiation exists among the acknowledged
Mediterranean morphotypes, and hence, S. officinalis can be viewed as a single,
morphologically variable species; (ii) a notable divergence was recorded in the Gibraltar
region, indicating the possible existence of a cryptic species; (iii) restriction to gene flow
was evidenced between the Aegean Sea and Provence giving two well-defined regional
clusters, thus suggesting the existence of a phylogeographic break between the two
systems; (iv) low levels of genetic structure, not correlated to geographical distance, were
observed inside geographical sectors, implying mechanisms (natural or anthropogenic)
that enhance dispersal and gene flow have promoted population connectivity; (v) the
genetic diversity of S. officinalis is maintained high in most studied locations despite
pressure from harvesting and the influence of devastating epidemics. These findings
provide a basis towards the effective conservation and management of the species.
Keywords: microsatellites, mitochondrial DNA, morphological variability, population structure,
Porifera
Received 31 December 2010; revision received 21 June 2011; accepted 5 July 2011
Introduction
Mediterranean sponges of the family Spongiidae
(Porifera: Demospongiae: Dictyoceratida) have been
exploited since ancient times for their cleansing and
therapeutic qualities (Voultsiadou 2007). Spongia offici-
nalis, the main harvested species, is distributed on
hard sublittoral substrata throughout the Mediterra-
nean Sea and along the adjacent coasts of the Eastern
Atlantic up to Galicia and down to Cape Verde
Islands (Vacelet 1959; Carballo et al. 1994). Its popula-
tions are very abundant in the Eastern Mediterranean,
forming the so-called ‘sponge grounds’ or ‘sponge
beds’, whereas in the Western Mediterranean, they are
not commercially exploitable (Pronzato & Manconi
2008); the Eastern Mediterranean is more favourable
for dictyoceratid sponges, which show here increased
Correspondence: Thanos Dailianis, Fax: +30 2810 337870;
E-mail: thanosd@her.hcmr.gr
2011 Blackwell Publishing Ltd
Molecular Ecology (2011) 20, 3757–3772 doi: 10.1111/j.1365-294X.2011.05222.x
diversity in comparison to other poriferan orders (Vo-
ultsiadou 2005). Moreover, it appears that the east to
west decrease in bath sponge abundance reflects tem-
perature and salinity gradients, as generally shown for
the Mediterranean poriferan distribution (Voultsiadou
2009). However, our knowledge regarding mechanisms
of dispersal for S. officinalis is very limited and has
mainly been inferred from data on other Porifera
(Mariani et al. 2005; Maldonado 2006).
Currently, Mediterranean bath sponge abundance
appears considerably reduced in comparison with past
time periods (Pronzato & Manconi 2008; Voultsiadou
et al. 2011). Besides intensive harvesting over more than
two centuries, a series of disease outbreaks during the
last two decades (Webster 2007; Garrabou et al. 2009)
severely affected their populations. Accordingly, S. offi-
cinalis is included in the international conventions of
Bern and Barcelona for protection and regulation of its
exploitation, yet harvesting is still a profitable activity
because the smaller quotas have resulted in increased
prices for natural sponges.
Despite their constant socio-economic interest, the
classification of bath sponges has been complicated and
controversial ever since the first poriferan species, S. of-
ficinalis, was proposed by Linnaeus in 1759. Their taxon-
omy at the species level has undergone numerous
changes through time, because of the presence of sub-
stantial intraspecific morphological variation (Cook &
Bergquist 2001). Thus, different morphotypes of S. offi-
cinalis have been consecutively considered as varieties
(Schulze 1879), subspecies (Vacelet 1959) or distinct spe-
cies (Pronzato & Manconi 2008), exclusively based on
their external morphology (see next section). Notably,
during a recent research (Voultsiadou et al. 2011) on
the status of bath sponges in the Aegean Sea, the arche-
typal sponge harvesting area, an impressive phenotypic
overlap between the ‘mollissima’ and ‘adriatica’ mor-
photypes of S. officinalis was recorded, and the re-eval-
uation of their taxonomic status was suggested. Such
taxonomic uncertainties are not uncommon in the porif-
eran phylum, which is characterized by high levels of
phenotypic plasticity that can result in controversial
species-level systematics (Knowlton 2000).
The absence of sound classification and the limited
knowledge on the current abundance and distribution
of bath sponge populations in the Mediterranean (see
Voultsiadou et al. 2011 and references therein) prevent
any comprehensive attempt towards their conservation
and effective management. This need becomes more
urgent under ongoing climate change, to which recent
epidemics of sessile invertebrates in the Mediterranean
(Garrabou et al. 2009) and other present and predicted
threats for the Mediterranean biodiversity (Coll et al.
2010; Lejeusne et al. 2010) have been linked.
To address such concerns, molecular approaches are
proving increasingly useful, as they can reveal under-
lying intra- and interspecific diversity and assist iden-
tification of phylogeographic patterns. Several
molecular markers have been utilized to aid taxon-
omy (Ca
´rdenas et al. 2010; Po
¨ppe et al. 2010) and
investigate phylogeography (Duran et al. 2004a,b;
DeBiasse et al. 2010) in Porifera. The standard barcod-
ing COI fragment exhibits adequate diversity to effec-
tively distinguish between taxa of the same genus
and ⁄or family described utilizing morphological traits
(Heim et al. 2007; Ca
´rdenas et al. 2010) or implied by
ecological traits (Duran & Ru
¨tzler 2006; Wulff 2006),
and trace cryptic speciation (see the study by Xavier
et al. 2010). During the last decade, microsatellite
DNA loci have been used extensively as markers for
population studies in a variety of marine inverte-
brates, including commercial ones (Addison & Hart
2004; Gutie
´rrez-Rodrı
´guez & Lasker 2004; Baums et al.
2005; Pe
´rez-Portela & Turon 2008; Ledoux et al. 2010).
Polymorphic microsatellites have been isolated for six
sponge species up to date, among which are the
Mediterranean bath sponges Spongia lamella (Noyer
et al. 2009, as Spongia agaricina) and S. officinalis
(Dailianis & Tsigenopoulos 2010). Nevertheless, only
three studies on noncommercial sponge natural popu-
lations utilizing some of these sets have been pub-
lished (Duran et al. 2004c; Blanquer et al. 2009;
Blanquer & Uriz 2010).
Genetic approaches are increasingly relevant to the
conservation and management of wild populations,
through the resolution of taxonomic uncertainties
(Frankham 2010), identification of management units
(Palsbøll et al. 2007) and evaluation of the impact of
anthropogenic changes to natural ecosystems (Schwartz
et al. 2007). The main objectives of this study are (i) to
provide evidence regarding the taxonomic status of the
principal Mediterranean bath sponge S. officinalis and
(ii) to estimate the levels of population connectivity
and gene flow among individuals and populations in
the main distribution areas of the species. The first
objective was addressed with analysis of partial mito-
chondrial cytochrome oxidase subunit I (COI)
sequences, while for the second, a set of eight micro-
satellite markers was used. This is the first attempt
towards the investigation of geographical and phyloge-
netic variation of bath sponges using molecular
approaches. Such data will be valuable in identifying
actual stocks of these harvested organisms and reveal-
ing patterns regarding their geographical structure and
population connectivity; these are critical requirements
to establish comprehensive management strategies and
actions towards their protection and sustainable exploi-
tation.
3758 T. DAILIANIS ET AL.
2011 Blackwell Publishing Ltd
Materials and methods
Sampling, assignment of specimens to morphotypes
and DNA extraction
Specimens of Spongia officinalis (522 individuals) were
collected from 11 locations, distributed in three broader
geographical sectors (Fig. 1; Table 1): Eastern Mediter-
ranean (Aegean Sea), Western Mediterranean (Provence
coast) and the Strait of Gibraltar (Ceuta). Sampling was
performed between 2005 and 2008 by diving at depths
from 4 to 42 m. Variation in geographical distance
between sampled locations (Table S1, Supporting infor-
mation) allowed assessment of interpopulation diversity
at three distance levels: low, between adjacent locations
in Provence (7–93 km apart); intermediate, between
Aegean islands (103–539 km); and high, between the
above two regions, or between each of them and Gibral-
tar (minimum pairwise distance >1900 km).
All specimens were analysed collectively under the
generally accepted species name S. officinalis based on
the following considerations. Two morphotypes of
S. officinalis are encountered in the Eastern Mediterra-
nean (Fig. S1, Supporting information) discriminated
exclusively according to their external morphology:
irregularly flattened to massive body shape with scat-
tered oscula in the ‘adriatica’ morphotype, cup-shaped
with oscula inside the concave upper surface in the
‘mollissima’ morphotype (Vacelet 1987; Pronzato &
Manconi 2008: as Spongia mollissima). Furthermore, pop-
ulations of ‘adriatica’ exhibit a difference in macro-mor-
phological traits as observed by Pronzato et al. (2003),
being massive with several large oscula and few small
ones in the Western Mediterranean, while flattened
with many small and few large oscula in the Eastern
Mediterranean. The examination of external morphol-
ogy and skeletal features in our extensive specimen col-
lection revealed no apparent discontinuities between
the Aegean ‘mollissima’ and ‘adriatica’. Actually, a vari-
able number of ‘typical’ specimens along with a series
of intermediate types were observed in each of the sam-
pling stations (Table 2), making it almost impossible to
draw the borders between them. The specimens from
Provence and Gibraltar were closer to the western ‘adri-
atica’ morphotype.
Sponge fragments were preserved in 95%ethanol
replaced with fresh ethanol twice after intervals of sev-
eral hours; they were then stored at )20 C prior to
DNA extraction. A segment from the choanosome of
each sample, approximating a cube with a side of 2–
3 mm, was cut up and inspected under a stereomicro-
scope to avoid contamination by foreign tissues. Geno-
mic DNA was extracted with DNeasy Blood & Tissue
kit (Qiagen) following manufacturer’s protocol.
W06°06' W05°42' W05°18' W04°54' W04°30'
N35°42'
N36°06'
N36°30'
N42°42'
N43°06'
N43°30'
E05°06' E05°30' E05°54' E06°18' E06°42'
E20°00' E22°00' E24°00' E26°00' E28°00'
N36°00'
N38°00'
N40°00'
N30°00'
N35°00'
N40°00'
N45°00'
W05°00' 00°00' E05°00' E10°00' E15°00' E20°00' E25°00' E30°00' E35°00'
A
B
C
(a)
(b)
C
100 km
30 km
20 km
CEU
SVE MAR
LAC
PCP
LRO
SPO
CYC
KAR
ECR
WCR
Fig. 1 Maps of sampling locations in the Aegean Sea (A),
Provence coast (B) and Gibraltar (C). For labelling, see Table 1.
GENETIC DIVERSITY OF SPONGIA OFFICINALIS 3759
2011 Blackwell Publishing Ltd
Extracted DNA was stored at )20 C prior to amplifica-
tion.
Amplification and sequencing of cytochrome oxidase
subunit I (COI)
The 5¢partition of the cytochrome coxidase subunit I
(COI) gene was amplified using the degenerate barcod-
ing primers dgLCO1490 and dgHCO2198 (Meyer et al.
2005). Reactions were performed in a total volume of
50 lL containing 0.1 lMof each primer, 0.05 mMdNTPs,
0.02 U Taq polymerase, 2.5 mMMgCl
2
and 1 lL
(approximately 20 ng) of template DNA. An initial step
of 3 min at 94 C was followed by six cycles using a
low annealing temperature (94 C for 30 s, 43 C for
90 s, 72 C for 60 s) and 36 cycles using a higher
annealing temperature (94 C for 30 s, 51 C for 90 s,
72 C for 60 s). The final extension step was performed
at 72 C for 5 min. The PCR product was run and
screened in 1%agarose and subsequently purified fol-
lowing standard sodium acetate precipitation.
A total of 33 specimens, three from each of the 11
sampled geographical locations, were sequenced with
the BigDyeTerminator v3.1 Cycle Sequencing kit
(ABI) following manufacturer’s recommendations and
using the same primers as in the amplification step, on
an automated ABI PRISM3700 Genetic Analyzer.
Sequences were manually edited and aligned in BIOEDIT
v7.0.5.1 (Hall 1999) using the implemented CLUSTALW
utility. Consensus sequences were checked against
GenBank with BLASTN (Altschul et al. 1990) to confirm
poriferan origin and exclude any case of potential con-
tamination. For the translation of nucleotide sequences
into amino acid sequences, NCBI ORF Finder was used
with the mould, protozoan and coelenterate mitochon-
drial genetic code table. The nucleotide sequence data
reported in this study have been deposited in the NCBI
GenBank nucleotide sequence database with accession
numbers HQ830362–HQ830364.
Phylogenetic inference
Additional sequences from keratose species were
acquired from GenBank for phylogenetic analysis: Hip-
pospongia lachne EU237484, Ircinia strobilina GQ337013
and Vaceletia sp. EU237489. Plakinastrella sp. (EU237487)
(Porifera: Homosclerophorida) was used as outgroup.
Phylogenetic reconstructions for the COI sequence were
performed with the maximum likelihood (ML) method
as implemented in PAUP* v4.0b10 (Swofford 2002). The
best-fit model of evolution was determined by JMODEL-
TEST v0.1.1 (Posada 2008) using the Akaike information
criterion. The suggested model of nucleotide substitu-
tion was the general time-reversible model with a pro-
portion of invariable sites (GTR + I, I = 0.52). Trees
were calculated using heuristic searches and a tree-
bisection–reconnection branch swapping algorithm.
Nodal support was estimated by bootstrap re-sampling
(10 000 replicates). An additional phylogenetic recon-
struction was performed under Bayesian inference (BI)
criterion with MRBAYES v3.1.2 (Ronquist & Huelsenbeck
2003) using the same evolutionary model (GTR + I) and
the default priors. Four Markov chains were run for
100 000 generations with sampling every 100 genera-
tions. The average standard deviation in split frequen-
cies was <0.01 at the end of the run. The trees of the
first 250 generations were discarded until the probabili-
ties reached a stable plateau (burn-in), and the remain-
ing trees were used to generate a 50%majority-rule
consensus tree. Corrected p-distances between haplo-
types were calculated in PAUP* using the GTR + I
model.
Microsatellite screening and analysis
Samples from all studied geographical locations were
screened for variation at eight microsatellite loci
(Spof054, Spof057, Spof069, Spof102, Spof130, Spof136,
Spof148 and Spof240) previously described for S. offici-
Table 2 Percent contribution of specimens assigned to Spongia
officinalis morphotypes in the stations of the Aegean Sea (for
labelling, see Table 1)
Assignment to morphotypes SPO CYC KAR WCR ECR
‘adriatica’ morphotype 83.3 2.9 6.9 80.4 0
‘mollissima’ morphotype 0 11.6 31.9 0 75.6
Intermediate type 16.7 85.5 61.1 19.6 24.4
Table 1 Sampling stations and number of collected Spongia of-
ficinalis specimens (N)
Geographical
region Location Label Depth (m) N
Aegean Sea Sporades SPO 10–25 90
Aegean Sea Cyclades CYC 6–7 68
Aegean Sea Karpathos KAR 7–25 51
Aegean Sea Western Crete WCR 4–10 47
Aegean Sea Eastern Crete ECR 20–42 35
Provence coast La Ciotat LAC 6–14 14
Provence coast Le Rove LRO 6–15 45
Provence coast Marseille MAR 6–32 80
Provence coast Sec du Veyron SVE 22 15
Provence coast Porquerolles ⁄
Port-Cros
PCP 6–15 64
Gibraltar Ceuta CEU 17 13
3760 T. DAILIANIS ET AL.
2011 Blackwell Publishing Ltd
nalis by Dailianis & Tsigenopoulos (2010), with amplify-
ing conditions as described therein. The sizes of the flu-
orescently labelled PCR products were estimated
according to an internal size marker (GeneScan500
LIZ) on an ABI Prism3700 sequencer (Applied Bio-
systems) and analysed using STRAND v.2.3.48 (UC Davis
Veterinary Genetics Laboratory, http://www.vgl.ucdavis.
edu/informatics/strand.php).
Allelic frequencies, the expected and observed hetero-
zygosities (H
E
and H
O
, respectively), along with the
inbreeding coefficient F
IS
values as in Weir & Cocker-
ham (1984) were estimated in GENETIX v4.05.2 (Belkhir
et al. 2000). A rarefaction method (Petit et al. 1998) was
used to assess allelic richness [Ar(g)] and private allelic
richness [Ap(g)] independently of the sample size, with
grepresenting the minimum number of genes observed
at one locus in one of the samples. Computations were
performed with ADZE v1.0 (Szpiech et al. 2008) for
g= 22 (the number of genes scored for locus Spof148 in
the CEU population). Significance of departure from
Hardy–Weinberg equilibrium (HWE) was estimated
with GENEPOP v4.0.10 (Rousset 2008) for each locus and
location, with the null hypothesis of random mating
and an alternative hypothesis of heterozygote defi-
ciency. The data were also analysed for evidence of
recent bottleneck events using the software BOTTLENECK
v1.2.02 (Piry et al. 1999). Deviations from expected het-
erozygosity were estimated according to a two-phased
model of mutation with the proportion of stepwise
mutation model set at 95%and variance among multi-
ple steps at 12. Significance of deviations was tested
using the Wilcoxon sign-rank test with 1000 iterations.
Finally, data were inspected with MICRO-CHECKER v2.2.3
(Van Oosterhout et al. 2004) for presence of null alleles.
Estimation of variation among populations
Differentiation among samples was estimated using
Jost’s (2008) actual measure of differentiation D
est
calcu-
lated with the software SMOGD v1.2.5 (Crawford 2010).
The package DEMEtics (Gerlach et al. 2010) within the
statistical package Rv2.12.1 (R Development Core Team
2009) was also used to estimate global single- and mul-
tilocus D
est
values and 95%confidence interval with
1000 bootstraps for the latter. Additionally, Weir &
Cockerham’s (1984) F
ST
(h), which assumes an infinite
allele model, and Slatkin’s (1995) R
ST
(q), which assumes
a stepwise mutation model, were used. Values of F
ST
and R
ST
were estimated using FSTAT v2.9.3 (Goudet 2001)
and RST CALC v2.2 (Goodman 1997), respectively. Signifi-
cance levels (P= 0.05) were determined using the differ-
entiation tests implemented in the above packages, with
1100 permutations for FSTAT and 1000 permutations for
RST CALC, and adjusted for multiple comparisons based
on a false discovery rate control following Benjamini &
Yekutieli’s (2001) procedure (B-Y, Narum 2006). Isolation
by distance was analysed by correlation between pair-
wise F
ST
⁄(1)F
ST
) values (Rousset 1997) and the loga-
rithm of the geographical distances between locations.
The significance of the relationship between geographi-
cal and genetic distance was evaluated with a Mantel
test in IBD v1.52 (Bohonak 2002) with 10 000 randomiza-
tions. An analysis of molecular variance (AMOVA, Excof-
fier et al. 1992) was performed based on the number of
different alleles (F
ST
) and on the sum of squared size dif-
ferences (R
ST
) as implemented in ARLEQUIN v3.5 (Excoffier
et al. 2005). Assessment of structure within studied
genotypes without a priori assumptions regarding popu-
lations was performed using a Bayesian algorithm as
implemented in STRUCTURE v2.3 (Pritchard et al. 2000).
The number of genetically homologous groups (K) was
determined using the ad hoc statistic DJ(Evanno et al.
2005). The software was run first with the total data set
and K= 1–22 to determine potential strong trends for
population subdivision, followed by two secondary runs
(with K= 1–12) using only the genotypes corresponding
to the Eastern and Western Mediterranean sector,
respectively, to detect substructuring. For each run, 10
6
Markov chain Monte Carlo (MCMC) repetitions were
performed following 20 000 burn-in iterations, in ten
replicate tests. For graphical display of the results, DI-
STRUCT v1.1 (Rosenberg 2004) was used. In addition, a
discriminant analysis of principal components (DAPC,
Jombart et al. 2010) was performed to infer population
subdivision. DAPC is a multivariate analysis that inte-
grates principal component analysis (PCA) with discri-
minant analysis to summarize genetic differentiation
between groups. It was implemented with the adegenet
package (Jombart 2008) within the statistical package R
v2.12.1; 170 principal components of the PCA were
retained, which accounted for approximately 90%of the
total genetic variability.
Results
COI sequence variation and phylogenetic inference
Thirty-three nucleotide sequences 630 bp in length were
obtained. All screened specimens from the Mediterra-
nean basin (Aegean and Provence) yielded identical
haplotypes (MEDIT). The specimens from Gibraltar
yielded two distinct haplotypes (GIBR 1 and 2). Porifer-
an origin of the haplotypes was confirmed by the BLASTN
search; the best match was a closely related dictyocera-
tid (Hippospongia lachne), followed by other sponge spe-
cies.
A relatively high number of substitutions were
recorded between the Mediterranean haplotype and
GENETIC DIVERSITY OF SPONGIA OFFICINALIS 3761
2011 Blackwell Publishing Ltd
those from Gibraltar, reaching a maximum of 20 nucleo-
tide differences; these mutation events were mostly
silent, and only two resulted in amino acid differences
(Table S2, Supporting information). Between GIBR 1
and 2, only two nucleotide substitutions were recorded,
one of them leading to amino acid substitution. Nucleo-
tide substitutions between Spongia officinalis (MEDIT,
GIBR 1 and 2) and H. lachne,Ircinia strobilina and Vacel-
etia sp. varied between 19 and 58. Pairwise genetic
divergence between studied haplotypes, as expressed
by corrected p-distance, showed comparable values
throughout the data set, ranging from 0.031 to 0.062,
with the exception of Vaceletia sp., which exhibited
higher divergence (0.087–0.119) from all other studied
taxa. Differentiation between the two Gibraltar haplo-
types was low, with a p-distance of 0.003.
Phylogenetic reconstructions using the ML algorithm
and BI for the COI region of the studied specimens
along with dictyoceratid sequences acquired from Gen-
Bank resulted in identical topologies (Fig. 2). Two dis-
tinct clusters were formed: one containing the Gibraltar
haplotypes and the second grouping the Mediterranean
S. officinalis with the Western Atlantic H. lachne and
I. strobilina. Bootstrap values for all clusters were below
90%while BI posterior probabilities ranged from 0.88
to 1.
Microsatellite variation within populations
Population parameters inferred from microsatellite data
exhibited substantial variation across most populations
(Table 3). Mean values for rarefied allelic richness
Ar(22) per locus ranged from 7.4 for Spof148 to 14 for
Spof136; the corresponding range per geographical loca-
tion was from 8.5 for CEU to 12.1 for WCR. Global rare-
fied private allelic richness [Ap(22)] values ranged from
0.9 to 2.4 across different loci. Regarding different geo-
graphical locations, SPO showed the lowest value of
Ap(22) (0.8) and CEU the highest (2.2). Expected hetero-
zygosity (H
E
) was overall high across all populations,
its mean values ranging from 0.81 to 0.90; low to mod-
erate values were exhibited only by locus Spof148 and
mostly for the Aegean locations, ranging from 0.43 to
0.81. Heterozygote deficiency was indicated with a sig-
nificant (P< 0.001) deviation from the HWE observed
over all populations and loci, while observed heterozy-
gosity (H
O
) was consistently lower than H
E
with few
exceptions (mean values ranging from 0.54 to 0.73).
Inbreeding coefficient F
IS
exhibited mostly positive val-
ues, often >0.1, with multilocus values ranging from
0.192 to 0.352 over different populations.
Analysis with MICRO-CHECKER revealed no evi-
dence of scoring errors because of stuttering or large
allele dropout, while presence of null alleles was sug-
gested for almost all loci (Table S3A, Supporting infor-
mation). Locus Spof136 was the least susceptible,
showing presence of null alleles in only one population,
while all others showed evidence for six of the studied
populations at a minimum. Failed amplifications also
occurred in our samples (Table S3B, Supporting infor-
mation), and their percentages respective to the total
number of genotypes averaged from 3.6%(spof148) to
12.4%(spof057).
The bottleneck analysis revealed no significant evi-
dence for a recent event, indicating that all studied pop-
ulations were under mutation–drift equilibrium.
Probability values for H
E
excess were >0.98 for all pop-
ulations except CEU (P= 0.769), and plots of allelic fre-
quency distribution were L-shaped.
Differentiation between populations
Diversity between S. officinalis populations at low
(within the Provence region) and intermediate (within
Plakinastrella sp.
Vaceletia sp.
SPOF Medit.
Ircinia strobilina
Hippospongia lachne
SPOF Gibr. 1
SPOF Gibr. 2
0.01 substitutions/site
EU237487
EU237489
GQ337013
EU237484
65.2
0.88
66.1
0.92
87.9
1.00
Fig. 2 Maximum likelihood (ML) and Bayesian inference (BI)
phylogenetic reconstruction for the COI partition of studied
Spongia officinalis specimens (SPOF Medit.: haplotype from
Aegean and Provence; SPOF Gibr. 1-2: haplotypes from Gibral-
tar) alongside with other keratose sequences (GenBank acces-
sion numbers given). Numbers above branches indicate
bootstrap values under ML criterion. Below branches are BI
posterior probabilities. Branch length corresponds to the BI.
3762 T. DAILIANIS ET AL.
2011 Blackwell Publishing Ltd
Table 3 Summary of genetic variation for Spongia officinalis at the eight studied microsatellite loci and 11 Mediterranean sampled
locations (for labelling, see Table 1)
WCR ECR KAR CYC SPO LAC LRO MAR PCP SVE CEU HWE
N47 35 51 68 90 14 45 80 64 15 13
spof054
H
O
0.84 0.70 0.46 0.48 0.52 0.50 0.93 0.76 0.80 0.36 0.77
H
E
0.92 0.92 0.91 0.90 0.92 0.82 0.72 0.82 0.81 0.75 0.83
F
IS
0.094 0.258 0.508 0.480 0.443 0.422 )0.292 0.079 0.012 0.549 0.111
HWE * *** *** *** *** ** ns *** *** *** * ***
Ar(22) 12.4 12.9 12.6 11.3 12.1 9.5 7.2 9.4 9.6 7.9 10.7
Ap(22) 0.7 1.4 2.4 0.4 0.4 2.0 0.1 2.5 2.6 1.2 3.0
spof057
H
O
0.60 0.35 0.37 0.39 0.33 0.43 0.60 0.66 0.53 0.45 0.00
H
E
0.97 0.91 0.89 0.91 0.91 0.82 0.91 0.95 0.94 0.88 0.69
F
IS
0.388 0.621 0.599 0.579 0.642 0.506 0.358 0.312 0.441 0.522 1.000
HWE *** *** *** *** *** *** *** *** *** *** *** ***
Ar(22) 17.3 13.4 12.5 13.6 12.6 8.3 12.2 14.9 14.2 12.0 4.0
Ap(22) 4.3 2.6 3.7 2.5 1.4 1.2 2.3 1.6 2.0 2.5 1.8
spof069
H
O
0.93 0.79 0.56 0.85 0.74 0.93 0.55 0.71 0.76 0.77 0.62
H
E
0.95 0.94 0.94 0.95 0.95 0.91 0.93 0.96 0.92 0.85 0.92
F
IS
0.028 0.180 0.419 0.117 0.219 0.017 0.421 0.264 0.179 0.134 0.368
HWE ns *** *** *** *** ns *** *** *** ns *** ***
Ar(22) 15.0 15.0 14.8 14.8 14.4 13.2 13.3 15.6 12.3 9.5 13.9
Ap(22) 0.9 0.6 1.6 0.7 0.4 0.9 2.3 2.9 0.3 1.7 3.8
spof102
H
O
0.43 0.69 0.33 0.51 0.60 0.31 0.67 0.59 0.56 0.54 1.00
H
E
0.84 0.77 0.79 0.86 0.78 0.72 0.90 0.81 0.87 0.73 0.74
F
IS
0.490 0.122 0.587 0.405 0.240 0.597 0.274 0.276 0.367 0.297 )0.313
HWE *** *** *** *** *** *** *** *** *** * ns ***
Ar(22) 8.5 8.7 7.1 8.7 7.2 8.4 11.2 8.4 9.7 7.2 6.7
Ap(22) 1.3 0.9 0.7 1.4 0.3 2.1 1.6 0.4 0.9 0.1 0.6
spof130
H
O
0.57 0.86 0.51 0.77 0.66 0.86 0.68 0.68 0.78 0.87 0.45
H
E
0.91 0.86 0.83 0.91 0.76 0.84 0.81 0.82 0.87 0.88 0.89
F
IS
0.387 0.014 0.393 0.155 0.139 0.013 0.174 0.182 0.115 0.055 0.524
HWE *** ns *** *** *** ns * *** *** ns *** ***
Ar(22) 11.7 9.1 9.1 10.8 6.9 11.0 8.9 9.1 9.8 10.7 12.0
Ap(22) 2.1 0.9 1.4 1.2 0.4 3.0 0.8 0.7 2.3 0.5 6.1
spof136
H
O
0.87 0.89 0.90 0.71 0.90 1.00 0.96 0.89 0.89 1.00 0.77
H
E
0.93 0.93 0.93 0.92 0.94 0.93 0.94 0.95 0.96 0.87 0.89
F
IS
0.078 0.062 0.043 0.231 0.053 )0.034 )0.001 0.069 0.081 )0.114 0.178
HWE *** * ns ** ns ns ns *** * ns * ***
Ar(22) 14.5 13.9 12.8 12.3 13.9 16.7 15.0 15.3 15.5 11.2 12.8
Ap(22) 1.7 0.6 0.4 0.6 1.0 3.1 1.4 1.6 2.1 1.0 0.9
spof148
H
O
0.49 0.53 0.40 0.65 0.67 0.50 0.66 0.78 0.90 0.80 0.45
H
E
0.48 0.69 0.43 0.81 0.79 0.88 0.85 0.88 0.91 0.80 0.64
F
IS
)0.017 0.241 0.088 0.205 0.153 0.460 0.239 0.116 0.013 0.034 0.333
HWE ns ** ns *** ** *** *** ** * ns ns ***
Ar(22) 4.3 5.7 2.2 6.6 6.2 10.8 11.7 10.4 11.9 7.1 4.0
Ap(22) 0.9 0.1 0.1 0.5 0.2 2.7 4.1 1.2 2.4 0.7 0.8
spof240
H
O
0.80 0.91 0.82 0.74 0.84 0.71 0.56 0.61 0.59 0.33 0.17
H
E
0.93 0.92 0.88 0.90 0.91 0.85 0.79 0.88 0.88 0.72 0.23
F
IS
0.149 0.023 0.070 0.188 0.088 0.193 0.309 0.319 0.336 0.561 0.313
HWE ** ns * ** ns * *** *** *** *** ns ***
GENETIC DIVERSITY OF SPONGIA OFFICINALIS 3763
2011 Blackwell Publishing Ltd
the Aegean region) geographical distance, as expressed
by pairwise multilocus values of D
est
between sampled
populations, was moderate with averages of
0.232 ± 0.074 SD and 0.285 ± 0.067 SD, respectively
(Table S4, Supporting information). Advancing to the
higher geographical distance scale (between Provenc¸al
and Aegean populations), D
est
values were substan-
tially higher, with an average of 0.596 ± 0.088. While
average global D
est
values between Gibraltar and Med-
iterranean populations were comparable to those
between the Provence and the Aegean, a substantial
variation of D
est
was observed among loci, with differ-
entiation values estimated for loci Spof057 and Spof240
approximating a value of 1 (Table S5, Supporting
information). Estimates F
ST
and R
ST
were substantially
lower than D
est
regarding their global values per locus
(Table 4) as well as their pairwise values between
populations at all geographical distance scales
(Table S6A, Supporting information). Most values,
however, were significant, indicating significant genetic
differentiation within and between the studied regions.
Nonsignificant values of genetic differentiation were
observed only with R
ST
estimate within the Provenc¸al
cluster (Table S6B, Supporting information). The high-
est values of F
ST
and R
ST
were recorded between
Gibraltar (CEU) and the Mediterranean, with averages
of 0.126 ± 0.016 and 0.510 ± 0.029, respectively. A sig-
nificant positive correlation (R
2
= 0.565; P£0.002)
between genetic and geographical distance was
observed when all Mediterranean geographical loca-
tions were included in the analysis (Fig. 3). However,
this resulted entirely from inter-basin genetic distances,
as was shown by the lack of a significant pattern of
isolation by distance when locations of the Aegean or
Provence cluster were treated independently (P£0.40
and P£0.28, respectively).
Investigation of genetic structure
Analysis of molecular variance attributed the majority
of variation to intrapopulation differences (between
individuals in each geographical location) (Table 5).
However, it also detected a significant amount of varia-
tion between the two Mediterranean regions (Aegean
and Provence); when utilizing the R
ST
estimate, the per-
centage of variation between the two groups was sub-
stantially higher than that calculated with F
ST
(24.11%
vs. 3.61%). Moreover, percentages of variation assigned
Table 3 (Continued)
WCR ECR KAR CYC SPO LAC LRO MAR PCP SVE CEU HWE
Ar(22) 13.1 13.2 9.9 11.1 12.3 8.3 7.7 9.9 9.4 5.6 3.8
Ap(22) 1.4 2.4 2.8 0.7 2.1 0.5 1.0 1.2 0.7 0.0 1.0
Multilocus
Mean H
O
0.69 0.71 0.54 0.64 0.66 0.65 0.70 0.71 0.73 0.64 0.53
Mean H
E
0.86 0.87 0.83 0.90 0.87 0.85 0.86 0.88 0.89 0.81 0.73
F
IS
0.211 0.192 0.352 0.295 0.251 0.261 0.197 0.203 0.195 0.247 0.315
HWE *** *** *** *** *** *** *** *** *** *** *** ***
Mean Ar(22) 12.1 11.5 10.1 11.1 10.7 10.8 10.9 11.6 11.6 8.9 8.5
Mean Ap(22) 1.7 1.2 1.6 1.0 0.8 1.9 1.7 1.5 1.7 1.0 2.2
Total pA 17 6 12 6 7 8 16 27 23 2 13
N, number of individuals; H
O
, observed heterozygosity; H
E
, expected heterozygosity; F
IS
, inbreeding coefficient; HWE, departure
from Hardy–Weinberg equilibrium; Ar(22), rarefied allelic richness; Ap(22), rarefied private allelic richness; pA, number of private
alleles.
*P< 0.05, **P< 0.01, ***P< 0.001.
Table 4 Global single- and multilocus F
ST
,R
ST
and D
est
values for studied Spongia officinalis specimens
Locus
Multilocus 95%CI
Spof054 Spof057 Spof069 Spof102 Spof130 Spof136 Spof148 Spof240
F
ST
0.086 0.043 0.025 0.051 0.077 0.024 0.135 0.052 0.061 0.040–0.087
R
ST
0.265 0.036 0.042 0.103 0.099 0.215 0.281 0.116 0.149 0.142–0.183
D
est
0.637 0.803 0.575 0.376 0.430 0.458 0.550 0.700 0.566 0.543–0.584
CI, confidence interval.
3764 T. DAILIANIS ET AL.
2011 Blackwell Publishing Ltd
between the 10 Mediterranean locations were low—-
though significant—with both approaches.
Following analysis with STRUCTURE, the model with
two genetically homogenous groups of specimens (K)
was suggested by the method of Evanno as the one
with the uppermost hierarchical level of structure. This
was indicated by a high value for the ad hoc statistic DK
(2515.1) for K= 2; the value of DKrapidly decreased
after that, ranging from 0.2 to 12.2 for successive values
of Kfrom 3 to 22. Results for K= 2 revealed the parti-
tion into two well-differentiated clusters corresponding
to the Aegean and Provenc¸al populations (Fig. 4a), the
latter including specimens from Gibraltar (CEU). The
second run of STRUCTURE, using genotypes parti-
tioned according to the first analysis, indicated three
groups (DK= 12.3) in the Aegean and five groups
(DK= 16.9) in Provence-Gibraltar (Fig. 4b, c). Karpathos
was clearly distinguished from other locations within
the first cluster and Gibraltar within the second; apart
from those, no clear pattern for assignment of individu-
als to geographical origin was observed.
The DAPC method including all samples, revealed a
clear separation of the Gibraltar cluster in the second
PCA axis, while the Provenc¸al and Aegean clusters
were also separated from each other on the first axis
(Fig. 5a). When the contribution of different alleles to
the second principal component of the DAPC was
investigated, it became evident that loci Spof057 and
Spof240 were responsible for the segregation of Gibral-
tar individuals (Fig. S2, Supporting information). Anal-
ysis excluding individuals from Gibraltar allowed for a
better resolution regarding the structure of Mediterra-
nean populations (Fig. 5b). Within the Aegean cluster,
Karpathos was again clearly separated from the other
populations, while a less explicit pattern of segregation
was observed between Cretan populations (WCR and
ECR) on the one hand and the Sporades–Cyclades pla-
teaus (SPO and CYC) on the other. No obvious struc-
ture was evident within the Provenc¸al cluster. In both
analyses, the genetic structure of studied populations
was captured by the first two principal components,
which retained 62%and 66%of the total variation,
respectively, for each analysis, as evidenced by the cor-
responding eigenvalues.
Discussion
Genetic variation and taxonomic status of Spongia
officinalis
The single haplotype of the COI fragment characteriz-
ing all Mediterranean specimens of Spongia officinalis
indicates that the ‘adriatica’ and ‘mollissima’ morpho-
types as well as the morphologically divergent eastern
and western ‘adriatica’ do not correspond to taxonomic
FST/(1-FST)
0
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Log(D)
123
Among provence populations
Among aegean populations
Between aegean and provence populations
Fig. 3 Genetic isolation by distance for Mediterranean sam-
pled locations of Spongia officinalis inferred from multilocus
estimates of genetic differentiation F
ST
⁄(1)F
ST
) and the loga-
rithm of the geographical distance with a Mantel test. The uni-
form regression line corresponds to all pairs of values
(r= 0.751; P< 0.01), while the dashed (r= 0.230; NS) and the
dotted line (r= 0.027; NS) correspond to regression among
populations of the Provence coast and the Aegean Sea, respec-
tively.
Table 5 Analysis of molecular variance (AMOVA) among Mediterranean geographical regions (Aegean and Provence) and sampled
locations for Spongia officinalis, based on the number of different alleles (F
ST
) and on the sum of squared size differences (R
ST
)
Source of variation
Degrees of
freedom
F
ST
R
ST
Variation
(%)
Significance
(P-value)
Variation
(%)
Significance
(P-value)
Among geographical regions 1 3.61 0.000 ± 0.000 24.11 0.000 ± 0.000
Among sampled locations 8 4.06 0.000 ± 0.000 3.42 0.000 ± 0.000
Within sampled locations 1008 92.33 0.006 ± 0.002 72.47 0.007 ± 0.002
GENETIC DIVERSITY OF SPONGIA OFFICINALIS 3765
2011 Blackwell Publishing Ltd
units at any level, and presumably S. officinalis can be
viewed as a single, morphologically variable species.
This finding was also supported by microsatellite analy-
sis. Genetic differentiation, as expressed by both F
ST
and R
ST
, was uniformly low among Aegean locations
independently of the percent contribution of the ‘adriat-
ica’ or ‘mollissima’ morphotypes, while D
est
values
observed within the Aegean cluster had a similar range
to those recorded within the Provenc¸al one, where no
distinction concerning varieties has been evidenced.
The above observations allow us to assume that the
morphological variability characterizing S. officinalis is
not linked to underlying genetic differentiation, at least
associated to the examined markers, and does not corre-
spond to discrimination of subspecies. Phenotypic plas-
ticity has been acknowledged as an inherent trait for
several sponges and evidently connected with abiotic
and biotic factors, such as flow regime (Mendola et al.
2008), substratum (Mercurio et al. 2006) or symbiotic
relationships (Carballo et al. 2006). Very few studies,
however, demonstrate lack of genetic differentiation
between acknowledged morphotypes or distinct sponge
species (Lo
´pez-Legentil et al. 2010). A detailed mor-
phological description of S. officinalis morphotypes
(Dailianis & Voultsiadou, in preparation) would consid-
erably improve our knowledge of this highly variable
commercial species.
The lack of intraspecific variation in the Mediterra-
nean data set detected with COI sequences is in accor-
dance with recent reports in Porifera (Duran et al.
2004b; Whalan et al. 2008; Lo
´pez-Legentil & Pawlik
2009—but see the study by DeBiasse et al. 2010) and is
attributed to the low rate of mitochondrial DNA evolu-
tion acknowledged for sponges and other nonbilaterian
metazoans (Hebert et al. 2003; Wo
¨rheide 2006; Shearer
& Coffroth 2007). On the other hand, the high genetic
divergence in COI haplotypes between Gibraltar and
Mediterranean S. officinalis specimens, accompanied by
clear separation under all phylogenetic reconstructions,
is directly comparable to that between the latter and
other Western Atlantic Dictyoceratida. Similar levels of
interspecific genetic variation between Pacific dictyo-
ceratid species were evidenced by Po
¨ppe et al. (2010).
Interspecific values of COI divergence in sponges rarely
reach the thresholds suggested for distinction of bilate-
rian metazoan species (Hebert et al. 2003); nevertheless,
these are not low per se, as interspecific values ranging
from approximately 1%to 22%have been reported
across different poriferan taxa (Wulff 2006; Blanquer &
Uriz 2007; Ca
´rdenas et al. 2010; Reveillaud et al. 2010;
Xavier et al. 2010). The genetic divergence of the Gibral-
tar specimens is further supported by microsatellites, as
evidenced in the DAPC, in the complete differentiation
between Gibraltar and the Mediterranean indicated by
D
est
for two microsatellite loci, as well as in the high
number of private alleles observed in CEU despite its
low allelic richness. These observations suggest the exis-
tence of a cryptic species distributed at least in the
vicinity of Gibraltar; this segregation could possibly be
related to the circulation patterns prevailing in the Alb-
oran Sea, which have been shown to pose a phylogeo-
graphic barrier for marine organisms in the Almeria-
Oran border (see the study by Patarnello et al. 2007).
However, the low support for this clade by individual-
based Bayesian analysis and the cross-amplification of
microsatellite markers developed for the Mediterranean
S. officinalis stress the need for a targeted sampling
survey, supplemented by thorough morphological
Aegean Sea Provence
WCR
ECR
KAR
CYC
SPO
LAC
LRO
MAR
PCP
SVE
CEU
WCR
ECR
KAR
CYC
SPO
LAC
LRO
MAR
PCP
SVE
CEU
(a)
(b) C
K = 2
K = 3 K = 5
Gibraltar
Fig. 4 Assignment of Spongia officinalis individual genotypes to genetically homologous groups (K) as inferred by Bayesian analysis
using STRUCTURE for all studied locations (A) and for each one of the two groups defined after the first run (B, C). Each individual
is represented by a vertical bar partitioned into K-coloured segments that represent its estimated membership fraction in each of the
inferred groups. For labelling, see Table 1.
3766 T. DAILIANIS ET AL.
2011 Blackwell Publishing Ltd
examination and sequencing of nuclear markers, to
assess adequately the taxonomic status, affinities and
actual distribution range of this newly discovered clade.
Patterns of diversity between populations
Weak, yet significant levels of differentiation at both
low and intermediate geographical scale and strong dif-
ferentiation at a long-distance scale (i.e. between the
two Mediterranean basins) were detected by microsatel-
lite-based analyses. However, the employed estimates
of genetic distance differed as to the absolute level of
detected variation. The use of F
ST
to assess differentia-
tion with hypervariable markers has recently been criti-
cized (Jost 2008) as F-statistics depend on within-
population heterozygosity and tend to underestimate
differentiation between populations as variation
increases. On the other hand, while R
ST
has been put
forward for markers that follow a stepwise mutation
model (as it is sometimes the case for microsatellites)
and is not supposed to be affected by within-population
variation, its effectiveness is reduced when mutations
do not occur in a strictly stepwise pattern, as is usually
the case in practice (Meirmans & Hedrick 2011). Thus,
for our analyses characterized by high levels of within-
population variation (as evidenced by high values of
heterozygosity and allelic richness), D
est
is expected to
more accurately illustrate the actual magnitude of dif-
ferentiation between populations. Nevertheless, as
Whitlock (2011) recently argues, estimations of F
ST
and
R
ST
should still be reported, as they better convey the
evolutionary and demographic processes that lead to in-
terpopulation differentiation.
The explicit restriction to gene flow detected with mi-
crosatellites between the Aegean and Provence popula-
tions might suggest the existence of two discrete groups
of S. officinalis corresponding to each of the two major
Mediterranean basins. The Strait of Sicily separating
them has been indicated as a boundary to gene flow
affecting the population connectivity of several marine
species (e.g. Bahri-Sfar et al. 2000; Arnaud-Haond et al.
2007; Tarnowska et al. 2010). However, further research
is needed to check whether the observed genetic differ-
entiation of the eastern S. officinalis reflects a differentia-
tion between the two Mediterranean basins or is limited
to the Aegean Sea as a result of its hydrographic isola-
tion from the rest of the Mediterranean (Borrero-Pe
´rez
et al. 2011). In any case, past events in the geological
history of the Mediterranean such as the Pleistocene
glacial episodes, together with the current oceano-
graphic processes, have been considered as the most
important evolutionary drives of spatial genetic differ-
entiation in the Mediterranean subareas (Arnaud-Ha-
ond et al. 2007).
The significant estimates of genetic distance observed
for S. officinalis within the two Mediterranean basins
imply the existence of structured populations through
reduced gene flow. Although the lecithotrophic larvae
of Dictyoceratida have shown higher swimming effi-
ciency than those of other sponges (Mariani et al. 2006),
d = 5
1
2
3
4
5
6
7
8
9
10
11 Gibraltar
Provence
Aegean Sea
(a)
d = 5
1
2
3
4
5
6
7
8
9
10
Aegean Sea
Provence
(b)
Fig. 5 Subdivision of the studied Spongia officinalis populations
according to the DAPC method: (a) for all areas and (b) for the
Aegean and Provence clusters. Sampled geographical locations
are indicated with different colours; dots represent individuals;
95%inertia ellipses are included for each cluster. (1: WCR; 2:
ECR; 3: KAR; 4: CYC; 5: SPO; 6: LAC; 7: LRO; 8: MAR; 9: PCP;
10: SVE; 11: CEU).
GENETIC DIVERSITY OF SPONGIA OFFICINALIS 3767
2011 Blackwell Publishing Ltd
their dispersal capabilities are expected to be quite lim-
ited as often observed in Porifera (Maldonado 2006;
Shanks 2009). However, the observed within-basin
levels of differentiation were modest and not equally
distributed between population pairs. For instance,
Karpathos appears differentiated from other Aegean
locations, while population connectivity can be assumed
between the Sporades and Cyclades plateaus. A more
explicit and strong structure pattern, similar to that
already revealed for sponges with microsatellites
(Duran et al. 2004c; Blanquer & Uriz 2010), could be
expected for the Aegean Sea, because of the fragmented
coastline and an observed sporadic occurrence of S. offi-
cinalis populations (see the study by Voultsiadou et al.
2011). We argue that hydrographic patterns can influ-
ence the dispersion potential of propagation vectors
and, consequently, either pose barriers to gene flow, or
potentially override limitations posed by distance and
seascape features, and promote genetic admixture
(Whalan et al. 2008). Thus, the population connectivity
observed between Sporades and Cyclades despite sub-
stantial intermediate distance and discontinuous habitat
could be attributed to the effect of the cold, low salinity
current flowing from north to south in the Aegean
archipelago (Zervakis et al. 2005). On the other hand,
the segregation observed for the populations of Karpa-
thos can rather be attributed to the influence of the
strong saline and warm current, flowing from the Lev-
antine northwards, on the latter.
In the case of the Aegean Sea, in which S. officinalis is
systematically exploited for centuries, harvesting itself
might also enhance dispersal; fishermen travel long dis-
tances and process harvested specimens on-board, this
involving squeezing of the sponge body to free the skel-
eton from living tissue. As S. officinalis is a gonochoris-
tic species reproductively active throughout the year
and bears developed embryos and larvae from Novem-
ber to July (Baldacconi et al. 2007) overlapping with the
harvesting period, release of sperm and larvae between
harvested locations could potentially enhance propaga-
tion. A similar assistance to dispersal can rely in
embryo-bearing transportable propagules (Maldonado
& Uriz 1999); fragments of S. officinalis indeed occur on
the sea bottom (Dailianis, personal observation), proba-
bly resulting from fission events or detachment of indi-
viduals. However, both aforementioned hypotheses
should be confirmed experimentally.
Diversity at the intrapopulation level
Homozygote excess was observed in most occasions,
accompanied by high positive F
IS
values and significant
departure from HWE; however, this should primarily
be associated to the widespread presence of null alleles
detected in our analysis (Selkoe & Toonen 2006). Null
alleles can be frequent in invertebrates (Launey &
Hedgecock 2001) and are usually attributed to muta-
tions in the flanking regions of the microsatellites (Cal-
len et al. 1993). A positive role of inbreeding cannot be
ruled out, as this has been rather commonly reported in
marine invertebrates, especially sessile ones with free-
swimming larvae such as corals and ascidians (e.g. Le
Goff-Vitry et al. 2004; Pe
´rez-Portela & Turon 2008);
however, its relative role compared to the artefact
caused by null alleles cannot be determined.
Remarkably, genetic variation was consistently high
at the intrapopulation level, as indicated by the AMOVA,
as well as the heterozygosity values and allelic richness.
This is an interesting observation, because at least shal-
low Aegean populations of S. officinalis have been sub-
jected to centuries-long harvesting pressure and
undergone several mass mortality incidents, which
severely affected its populations in broad distance
scales (see the study by Voultsiadou et al. 2011).
Although the first documented such incident influenc-
ing the studied areas is quite recent, dating to 1986,
mortality events of bath sponges have been reported
from other regions since the late 19th century (Webster
2007), and consequently, a persistent periodical occur-
rence of these phenomena in the Mediterranean can be
assumed. Under these forces, genetic diversity would
be expectedly reduced through population bottlenecks
and genetic drift. The maintenance of high intrapopula-
tion genetic variability throughout our data set could be
explained by (i) adequate levels of population connec-
tivity, as indicated to an extent by our results, (ii) the
potential effect of regeneration of partially harvested
individuals that would help the species maintenance
and (iii) the existence of robust populations, scarcely
influenced by fisheries and epidemics, that could pro-
mote re-colonization of affected areas. The latter
hypothesis can be supported by the reported occurrence
of shallow populations that tolerated recent mortality
events, probably due to a beneficial current flow regime
(Voultsiadou et al. 2011). An additional source of regen-
eration could be the populations occupying the species’
deepest bathymetric range (>40 m) which, growing
below the Mediterranean thermocline, are presumably
less susceptible to both temperature-induced stress and
harvesting pressure.
Implications for management and research priorities
Our results indicate that shallow S. officinalis popula-
tions in the Mediterranean Sea, despite exhibiting
reduced abundance and fragmented distribution owing
to harvesting and large-scale mortality incidents (Voul-
tsiadou et al. 2011), remain fairly vigorous regarding
3768 T. DAILIANIS ET AL.
2011 Blackwell Publishing Ltd
genetic variability, hence presenting a potential for
regeneration if effectively managed. A regulatory proto-
col, aimed at reducing harvesting pressure and promot-
ing conservation of natural populations in selected
areas, could possibly assist re-colonization at a broader
scale, as suggested by the trends favouring population
connectivity indicated between some locations within
the two main Mediterranean basins. Furthermore, artifi-
cial restocking of areas (Baldacconi et al. 2010) from
adjacent—or even remote—populations is not expected
to induce alterations in indigenous populations at host
habitats, as the genetic identity of S. officinalis appears
to be uniform within Mediterranean boundaries.
Research towards the linking of hydrographic parame-
ters to stress-inducing factors emerges as an urgent
need to rank existing S. officinalis populations on a scale
from less to more susceptible to damage and to recog-
nize protection priorities and possible re-colonization
sources; this becomes more crucial for shallow water
populations, the status of which might be irreversibly
affected in the long term by climate change-induced
phenomena (Lejeusne et al. 2010).
The present study is the first providing sound clues
regarding patterns of variation for a poorly studied spe-
cies with high socio-economical importance; however, a
wide range of questions concerning the Mediterranean
bath sponges remain to be answered, regarding both
their systematic status and population characteristics.
Three more species await evaluation (Spongia lamella,
Spongia zimocca and Hippospongia communis); research at
a pan-Mediterranean scale and a wider bathymetric
range should be implemented to identify stepping
stones regarding population connectivity throughout
their distribution. The potential for concealed biodiver-
sity at the vicinity of the Gibraltar area suggested by
our results should be further investigated with studies
focusing on the spongiids distributed along the Atlantic
coasts adjacent to the strait, to resolve the taxonomic
status of specimens formerly identified as S. officinalis.
Finally, affinities between Mediterranean Spongiidae
and their congeners of the Western Atlantic should also
be examined to elucidate phylogeographic patterns and
gain insight into the evolutionary history of bath
sponges.
Acknowledgements
We are grateful to Thierry Pe
´rez for providing samples from
Provence and Gibraltar. Thanks are due to Zacharias Skouras
for critically commenting on the manuscript and Theodore
Abatzopoulos for valuable advice on phylogeographic issues.
Helpful laboratory assistance with microsatellite genotyping
was provided by Muriel Berge
´. Field research was supported
by funding from the Greek Ministry of Rural Development
and Food (EPAL 2006–2008) and laboratory work was partially
funded by the programme ECIMAR (Agence Nationale de la
Recherche, France). We finally thank three anonymous review-
ers for their constructive comments and suggestions that led to
a greatly improved manuscript.
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The present work is part of the doctoral research of T.D. on
the taxonomy and population genetics of Mediterranean bath
sponges, supervised by E.V. C.S.T. is a research associate
working on molecular systematics and population genetics of
marine organisms. C.D. is a research director involved in the
study of marine biodiversity at the species and community
level and the management of marine resources. E.V. is an asso-
ciate professor, whose research focuses on taxonomy, zoogeog-
raphy and biology of Porifera.
Data accessibility
DNA sequences: GenBank accession numbers HQ830362–
HQ830364.
Phylogenetic data: TreeBASE Study accession number. S11667.
Microsatellite data: DRYAD entry doi:10.5061/dryad.hm304.
Sample location information can be obtained from the authors
upon request.
Supporting information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Photographs of representative Aegean Spongia officinalis
specimens assigned to the morphotypes ‘adriatica’ (A), ‘mol-
lissima’ (B), or the intermediate type (C ), and Provence speci-
mens assigned to the ‘western adriatica’ form (D).
Fig. S2 Contributions of alleles to the second principal compo-
nent of the DAPC analysis with all individuals included.
Table S1 Pairwise geographic distance (km) between sampled
locations.
Table S2 Pairwise amino acid differences (above diagonal),
base pair differences (below diagonal) and genetic divergence
as corrected p-distance (inside parentheses) between cyto-
chrome oxidase subunit I haplotypes of Spongia officinalis from
sampled locations along with other keratose species.
Table S3 (A) Indicated existence and frequencies of null alleles
across eight microsatellite loci and 11 geographic locations for
Spongia officinalis, as suggested by the program MICRO-CHECKER.
(B) Percentage of failed amplifications across loci and popula-
tions.
Table S4 Pairwise values of Jost’s D
est
across all eight micro-
satellite loci (based on harmonic mean) between sampled geo-
graphic locations for Spongia officinalis (for labeling see
Table 1).
Table S5 Average values of Jost’s (2008) D
est
per microsatellite
locus within the studied Mediterranean regions (Aegean and
Provence), between them, and between the studied Gibraltar
population and Mediterranean ones.
Table S6 (A) Pairwise F
ST
(above diagonal) and R
ST
values
(below diagonal) between sampled geographic locations for
Spongia officinalis estimated from analysis of all studied micro-
satellite loci. (B) Probability values for significant deviation
from zero for each corresponding F
ST
and R
ST
value.
Please note: Wiley-Blackwell are not responsible for the content
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authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
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2011 Blackwell Publishing Ltd
