Molecular phylogeography of western Mediterranean dusky grouper Epinephelus marginatus
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C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
© 2000 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés
S0764446900001153/FLA
Animal biology / Biologie animale
Molecular phylogeography of western
Mediterranean dusky grouper Epinephelus
marginatus
André Gillesa, Anne Miquelisa, Jean-Pierre Quignardb,c, Éric Faurea,c*
a
Upres 2202, Biodiversité, hydrobiologie, ICB, case 31, place Victor-Hugo, université de Provence, 13331
Marseille cedex 3, France
b
Laboratoire d’ichtyologie, université Montpellier II, place E.-Bataillon, case 102, 34095 Montpellier cedex,
France
c
Groupe d’étude du mérou (GEM), BP 230, Six-Fours-les-Plages, France
Received 14 June 1999; accepted 3 October 1999
Communicated by André Adoutte
Abstract – Intraspecific sequence variation in a portion of the gene coding for cyto-
chrome b in the dusky grouper (Epinephelus marginatus Lowe 1834), an endangered fish
species in various regions of the Mediterranean sea, was examined in 29 individuals from
the western Mediterranean sea. Sixty-four phylogenetically informative nucleotide posi-
tions were present in a 353-base pair cytochrome b sequence, amplified using the
polymerase chain reaction. Statistical analysis of the sequence data using a variety of
tree-building algorithms separated the taxa into one group of dusky groupers corre-
sponding to some of the Algerian individuals and another regrouped set of fishes
originating in France, Tunisia and the remaining Algerian specimens. Although, on the
basis of their morphology, E. marginatus are now considered as a single species, our
results suggest that a subgroup of the Algerian dusky grouper constitutes a cryptic
(undescribed) species. These results suggest that morphological and genetic evolution
may be uncoupled in dusky grouper, resulting in morphological similarity between
species despite extensive genetic divergence. In addition, we cannot rule out the
possibility of gene introgression with other species of grouper. A more in depth phylo-
genetic analysis (i.e. between and within the different Epinephelus species) would likely
affect many conservation management decisions about this assemblage of groupers.
© 2000 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS
cytochrome b / Teleostei / Serranidae / Epinephelus marginatus / grouper /
Mediterranean sea
Résumé – Phylogéographie moléculaire du mérou brun de Méditerranée occi-
dentale Epinephelus marginatus. Les relations phylogéniques intraspécifiques entre
29 individus d’Epinephelus marginatus provenant de la Méditerranée occidentale
(Algérie, France, Tunisie) ont été étudiées. Une portion du gène codant le cytochrome
b a été séquencé (353 paires de bases) et 64 bases se sont révélées être phylogénéti-
quement informatives. Les arbres phylogénétiques obtenus (méthodes de parcimonie et
de distance) montrent que les séquences de mérou se séparent en deux groupes. L’un,
contient une partie des mérous algériens et l’autre, comprend tous les mérous français
* Correspondence and reprints: e_faure@hotmail.com
195A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
et tunisiens ainsi que des mérous algériens. Pour expliquer ces résultats, plusieurs
hypothèses peuvent être formulées : présence en Algérie d’une espèce cryptique ou
d’hybrides, ou d’individus issus d’une « population » atlantique. © 2000 Académie des
sciences/Éditions scientifiques et médicales Elsevier SAS
cytochrome b / Teleostei / Serranidae / Epinephelus marginatus / mérou / mer
Méditerranée
Version abrégée Les deux analyses mettent en évidence une bifurca-
tion au sein des séquences de mérou brun de Méditer-
Le mérou brun (Epinephelus marginatus) est l’une ranée occidentale. Les valeurs de bootstrap supportent
des sept espèces de mérous dénombrées en Méditer- fortement cette séparation en un groupe nommé A,
ranée. Les plus fortes densités de mérou brun se situent contenant des mérous algériens (100% NJ et 100% MP)
sur les côtes nord et nord-ouest de l’Afrique, de la et en un autre appelé B, contenant les mérous français,
Tunisie au Sénégal. Réputé sédentaire et territorial, E. tunisiens et des mérous algériens (95% NJ et 99% MP).
marginatus affectionne les fonds rocheux littoraux Au sein du groupe B, les multifurcations dans les
riches en abris. analyses en NJ ou MP mettent en évidence les difficul-
Jusqu’à ces dernières années, le mérou brun ne tés de positionnement des mérous français et tunisiens.
semblait pas se reproduire sur les côtes nord de la De plus, dans l’arbre en NJ « bootstrappé », à l’inverse
Méditerranée occidentale mais plus au sud (en dessous de celui en MP, les populations française et tunisienne
du 41°5 de latitude nord), en particulier sur les côtes ne sont pas différenciées. Dans l’arbre en MP, une
d’Afrique du Nord. Les côtes nord-africaines sont séquence de mérous français se positionne avec les
d’ailleurs considérées comme les « pépinières » proba- mérous tunisiens. Lors des analyses, les mérous algé-
bles de cette espèce pour la Méditerranée. Toutefois, riens du groupe B constituent toujours un groupe
depuis quelques années la présence sur le littoral monophylétique supporté par de fortes valeurs de
méditerranéen français de juvéniles pesant environ 10 bootstrap (87% NJ et 97% MP). De plus, les longueurs
g semble indiquer que l’aire de reproduction de l’espèce de branches sont similaires pour les deux groupes A et
se serait étendue vers le nord. Ces observations récen- B (24 à 41/26 à 45) suggérant des taux d’évolution
tes sont encourageantes car a contrario divers facteurs équivalents.
participent fortement à la diminution des effectifs. Ces Les analyses phylogénétiques suggèrent une très
derniers éléments, associés au fait que le développe- grande diversité génétique au sein des mérous bruns
ment sexuel est de type hermaphrodite successif pro- de Méditerranée occidentale. Plusieurs hypothèses
térogyne et que la maturité sexuelle est tardive contri- pourraient expliquer ces résultats. Premièrement, nous
buent à faire d’Epinephelus marginatus une espèce à pourrions être en présence d’une espèce cryptique en
risque sur le plan des effectifs. Cette espèce est d’ailleurs Algérie, Sur les côtes algériennes, deux espèces puta-
classée comme vulnérable dans l’inventaire de la faune tives vivraient en sympatrie et ne présenteraient pas de
menacée en France. Aujourd’hui, le mérou bénéficie différences morphologiques apparentes. D’autre part,
officiellement d’un statut légal de protection dans cer- en Algérie des représentants des groupes A et B sont
tains pays riverains de la Méditerranée. trouvés dans les eaux d’Annaba et d’Alger. Le groupe B
Ce travail s’inscrit dans le cadre des recherches pourrait faire parti d’un pool de mérous strictement
entreprises afin de connaître l’origine des mérous pré- méditerranéens, tandis que les mérous du groupe A
sents sur les côtes de la Méditerranée nord occidentale seraient issus d’une population atlantique. L’analyse de
et d’essayer de détecter d’éventuels cheminements séquences de mérous de l’Atlantique, qui est en cours,
migratoires. devrait apporter des éléments de réponse.
Lors des analyses phylogéographiques, 29 séquen- L’absence de barrière géographique entre les deux
ces de cyt b de mérou brun ont été alignées avec la groupes, laisse supposer que d’autres hypothèses sont
séquence homologue d’un Carangidae Trachurus tra- envisageables. Par exemple, il est impossible d’exclure
churus. Ce dernier a été utilisé comme groupe exté- la présence d’hybrides puisqu’expérimentalement il a
rieur car des essais préliminaires ont montré qu’il été montré que l’hybridation était possible. Des phé-
s’agissait d’une des séquences publiées dans GenBank nomènes d’introgression ne sont peut-être pas impos-
parmi les plus proches de celles des mérous bruns. sibles dans le milieu naturel. Parmi les espèces de
L’alignement de séquence comprend 353 nucléoti- mérous présentes en Méditerranée, trois coexistent sur
des alignés, dont 115 sont variables et 64 informatifs. les côtes algériennes avec le mérou brun (E. costae,
Deux méthodes d’analyse phylogénétique ont été uti- badèche, E. aeneus, mérou blanc, et E. caninus, mérou
lisées (méthode de distance – neighbour joining (NJ)) gris). Toutefois les périodes de frai du mérou brun et de
et méthode de parcimonie (MP)). la badèche ne sont pas exactement identiques bien que
196A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
ces deux espèces soient sympatriques. De plus, sur les juvéniles dans notre échantillonnage et que jusqu’à ces
côtes algériennes, le mérou blanc et le mérou gris, qui dernières années il n’y avait pas de reproduction sur les
sont très rares, n’ont pas les mêmes biotopes que le côtes françaises, on aurait pu s’attendre à trouver les
mérou brun. En Algérie, le mérou brun coexisterait mérous français (Provence) en position apicale. Ce
avec le mérou d’Haïfa (E. haïfensis) mais leurs biotopes résultat suggère qu’une population existe depuis long-
sont relativement distincts. temps sur les côtes françaises, ce qui reste à confirmer.
L’analyse du groupe B suggère que les mérous Afin d’essayer de répondre aux questions soulevées
tunisiens et français sont plus proches entre eux que par cette étude, nous allons d’une part, analyser des
des mérous algériens du même groupe. De plus, une mérou bruns de l’Atlantique pour vérifier l’hypothèse
séquence de mérous français présente de fortes homo- de l’origine atlantique du groupe A. D’autre part, afin
logies avec celles des mérous tunisiens. Ces résultats de confirmer ou d’infirmer les hypothèses d’une colo-
sont en accord avec les travaux portant sur l’étude de la nisation des régions septentrionales par des mérous
parasitofaune branchiale des mérous tunisiens pré- originaires du sud, des analyses d’individus de Corse,
sente des affinités avec celle des individus des côtes Sardaigne, Sicile, Baléares et des côtes continentales
françaises. D’autre part, l’arbre phylogénétique en MP italiennes et espagnoles seront réalisées. De plus, il est
montre que les mérous français sont à la partie basale nécessaire d’étudier d’autres espèces d’Epinephelus
du groupe B. Étant donné que nous n’avions pas de afin de détecter d’éventuels hybrides.
1. Introduction It is now well known that sequence variations of genes
or portions of genes amplified by polymerase chain reac-
tion (PCR) can be used to assess relationships between and
within species, genera and higher taxa. The ultimate goal
In the Mediterranean sea, the dusky grouper Epinephelus is to reconstruct phylogenetic trees and to decipher the
marginatus is one of the seven species belonging to the underlying evolutionary and biogeographical history. A
genus Epinephelus. This species is absent from the Black useful marker is the mitochondrial cytochrome b gene (cyt
sea. In the Atlantic, it is reported as far as the British Isles in b) which displays enough sequence variation to assess the
the north, South Africa in the south and the Brazilian coast phylogenetic relationships in fishes and other vertebrates
in the west [1, 2]. The highest densities of the dusky at the intraspecific, generic and familial levels [9, 10]. In
grouper occur on the north and north western coasts of an attempt to establish the origin of the population(s) of
Africa, from Tunisia to Senegal [3]. Reputed to be seden- dusky grouper present on the French coasts, we have
tary and territorial, E. marginatus favours rocky coastal determined and compared partial sequences of the cyt b
bottoms that offer plenty of shelter [4]. The dusky grouper gene to analyse the phylogenetic relationships between
was considered to be fairly common in the Mediterranean, the dusky grouper of the western Mediterranean sea. The
but because of underwater fishing, poaching, overfishing taxonomic distinctions of grouper subspecies and popula-
and to a lesser extent pollution, it has become scarce on tions are relevant not only to systematic issues but also to
the north western coasts of the Mediterranean. Other still species conservation, because species, subspecies and
unidentified factors may also contribute to the decline in population are the units of protection. In addition, the
numbers. Unexplained mortality phenomena have been existence of a population-specific genetic subdivision is
reported from Malta, Port-Cros (France) and Skikda (Alge- important in deciding whether reintroduction or reloca-
ria) [5]. Even in a region that is considered as richly tion programmes need to take account of the genetic
stocked, such as eastern Algeria, the stock is in demo- distinctiveness of isolated populations.
graphic instability [5]. In addition, until recent years, the
dusky grouper appeared to breed only south of a line
running from Barcelona to Napoli, in particular along the 2. Materials and methods
coast of North Africa. The North African coast is some-
times considered as the region from which the individuals 2.1. Sample collection
populating the north western Mediterranean originated [6].
All these factors mean that E. marginatus is an endangered Figure 1 shows sampling locations in the western Medi-
species, which is now classified as a ‘vulnerable’ species terranean sea. Twenty-nine dusky groupers have been
in the inventory of endangered fauna in France [7]. Today, collected from the Fench coast (near Marseilles: Fr1, Fr2,
the grouper enjoys legally protected status in certain Medi- Fr3, Fr4, Fr5, Fr6, Fr7, Fr8), from Algerian coasts (near
terranean countries. Since 1986 an organization known as Algiers, Al1, Al2, Al3, Al4, Al5, Al6, Al7, Al8; and near
the GEM (Groupe d’Étude du Mérou – Grouper Study Annaba, Al8, Al9, Al10, Al11, Al12, Al13, Al14, Al15,
Group) has been setting up and running research pro- Al16, Al17, Al18) and from Tunisian coasts (near Zembra,
grammes on the ecology, ethology and biology of this Tu1, Tu2; and near Tabarka, Tu3). The fishes were identi-
member of the Serranidae [8]. fied by the collectors. A piece of caudal fin of approxi-
197A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
Figure 1. Collection locations for dusky groupers of the western Mediterranean sea.
mately 1 cm2 was cut off from each dead or live grouper. 2.3. Sequence analysis
The pieces of fin were stored in 70 % ethyl alcohol at
ambient temperature or at –20 °C as soon as possible.
In order to facilitate outgroup comparisons, the cyt b
sequences of nine other Percomorphs species were
2.2. DNA extraction and PCR reaction
extracted from GenBank: one Labroidei (Cichlidae, Tila-
Total DNA was extracted from approximately 0.25 cm2 pia mossambica, accession number X81565); three Per-
of caudal fin by using modifications of the method of coidei (Sparidae, Boops boops, X81567), (Moronidae,
Taberlet and Bouvet [11]. An approximately 350-bp sec- Dicentrachus labrax, X81566), (Carangidae, Trachurus tra-
tion of the mitochondrial (mt) DNA genome from the cyt b churus, X81568), three Scombroidei (Scombridae;
gene was amplified using published universal primers Scomber scombrus, X81564; Sarda sarda, X81562; Thun-
28-For 5’-CGAACGTTGATATGAAAAACCATCGTTG- nus thynnus, X81563) and two Gobioidei (Gobiidae, Pro-
3’ [12] and 34-Rev 5’-AAACTGCAGCCCCTCAGAATGA- terorhinus marmoratus, U53678 and Neogobius melanos-
TATTTGTCCTCA-3’ [13]. Polymerase chain reaction (PCR) tomus, U53677). DNA sequences were aligned by using
components per 50 mL reaction were as follows: 50 ng the Clustal W program [14]. Neighbour joining (NJ) analy-
template DNA, 0.2 mM of each primer, 2.0 U. HiTaq Taq sis [15] was performed using MUST package [16]. A cla-
polymerase, dNTPs 0.2 mM, 5 mL of the reaction buffer distic approach following the maximum parsimony (MP)
provided by the Taq manufacturer (Bioprobe, France). The criterion has also been used [17]. Bootstrap analyses
cycling parameters were as follows 92 °C for 2 min, five (1 000) were performed to obtain confidence estimates for
times (92 °C for 15 s, 48 °C for 45 s, and 72 °C for 1.5 each furcation. Base composition was studied using
min), 30 times (92 °C for 15 s, 52 °C for 45 s, and 72 °C for MUST [16]. Absolute saturation plots were performed
1.5 min), and 72 °C for 8 min. Using the single-stranded using the NET, AF_PAUP3, and COMP_MAT programs of
DNA as a template, the nucleotide sequence was deter- MUST and PAUP [17]. In addition, the absolute saturation
mined with an automated DNA sequencer (Genome was performed on transitions and transversions separately
Express, Grenoble, France). for each codon position [18].
198A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
Figure 2. Aligned sequences for partial cytochrome b gene in dusky grouper.
The sequence data include the cyt b sequence of the Carangidae Trachurus trachurus. See figure 1 for sample name abbreviations and collection
site information (base same as that of reference sequence; (–) alignment; (N) base could not be determined for that sequence).
3. Results b gene sequences were analysed using two phylogenetic
methods (unweighted maximum parsimony (MP) and
neighbour joining (NJ)). A heuristic search of the most
3.1. DNA phylogeographic study of the dusky grouper
parsimonious tree [19] showed more than 1 000 equipar-
In various preliminary assays, all the comparative simonious trees of 211 steps with a consistency index (CI)
sequence data for the percomorph species of Perciforms from 0.699 to 0.706 and a retention index (RI) from 0.834
taken from GenBank (except the cyt b sequences of Scom- to 0.840 (figure 3.A). The MP bootstrapped tree is shown
bridae which are too distant from those of dusky grouper) in figure 3.B.
were chosen as an outgroup. In all the trees obtained, the
topology of the trees and the approximate value of the The results of each of these analyses show that a highly
nodes are similar (data not shown). Also, for the phylogeo- significant bifurcation separated the western Mediterra-
graphic analysis of E. marginatus, 29 partial cyt b nean dusky grouper cyt b sequences. Bootstrap resam-
sequences of dusky grouper were aligned with those from pling support for the separation was strong for a lineage
the Carangidae Trachurus trachurus which was one of the (named group A) containing some of the Algerian dusky
closest to the cyt b sequences of dusky grouper and this grouper (100 % NJ and 100 % MP) compared to the other
sequence was chosen as an outgroup. The sequence align- lineage (named group B) containing the rest of Algerian
ment included 353 aligned nucleotide positions, of which grouper and all the French and Tunisian fishes (99 % NJ
115 are variable and 64 are phylogenetically informative and 95 % MP). In group B, the multifurcation in the NJ and
(that is, parsimony sites) (figure 2). Within this alignment, MP analyses shows the difficulty of positioning the French
we observed 56 unique substitutions among 29 individu- and Tunisian groupers. In the MP bootstrapped tree, in
als of dusky grouper analysed for the cyt b sequence. contrast to the NJ bootstrapped tree, the Tunisian and
Thirty four of these 56 mutations are transitions (10 G-to-A, French populations are not differentiated. As expected,
24 C-to-T), while the other 22 are transversions (5 G-to-T, one of the groupers collected on the French coast (Fr1)
2 G-to-C, 12 A-to-T and 3 A-to-C). which has a sequence identical to two of the Tunisian
When the absolute saturation [18] was performed on fishes constitutes a group with Tunisian groupers (figure
transitions and transversions separately for each codon 3.A). The Algerian grouper belonging to group B, forms a
position, no saturation was observed (data not shown), monophyletic subgroup. Bootstrapped distance analysis
and all the positions were used for the construction of clearly supports this monophyly (97 % NJ and 100 % MP).
phylogenetic trees. Using the alignment of figure 2, the cyt The branches in the two different groups A and B have
199A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
Figure 3. A. Neighbour joining on a matrix of the Jukes and Cantor model.
Bootstrap analyses carried out with 1 000 iterations among 29 taxa of dusky groupers of western Mediterranean sea, with T. trachurus as an
outgroup (bootstrap proportion on the top, in bold).
B. Strict consensus of 1 000 equiparsimonious trees of 211 steps with a consistency index (CI) from 0.699 to 0.706 and a retention index (RI) from
0.834 to 0.840 for 29 taxa of dusky groupers with T. trachurus as an outgroup, using an unweighted maximum parsimony (number of steps on the
top of the branches). Bootstrap analyses carried out with 1 000 iterations (bootstrap proportion at the bottom in bold).
similar length ranges (24–41/26–45), indicating a similar absolute saturation [18] was performed on transitions and
rate of evolution. transversions separately for each codon position (figure 5)
and showed that transitions and transversions at third
3.2. Phylogenetic position of the dusky grouper positions (TS3 and TV3, respectively) exhibit a clear pla-
teau showing mutational saturation, while the second
For the study of phylogenetic relationships within the positions (TS2 and TV2) showed rather dispersed plots. In
Perciformes order, nine partial cyt b sequences belonging addition, transitions and transversions at first positions
to four different suborders (Labroidei, Percoidei, Scom- showed a bias of composition as already reported by
broidei, Gobiidei) were extracted from GenBank and were Meyer [20]. Accordingly, only the transversions of the first
added to six cyt b partial sequences of E. marginatus. Each and second positions will be used for the reconstruction
suborder was represented by one family except Percoidei; methods. We have therefore worked on 48 variable sites of
in the latter suborder, the cyt b sequences of four families which 28 are phylogenetically informative (data not
(Carangidae, Moronidae, Serranidae, Sparidae) were used shown).
for the construction of phylogenetic trees. This sequence
alignment included 344 aligned nucleotide positions (data The tree constructed from these data provided some
not shown). The base composition of the third position of interesting information on phylogenetic relationships in
codons shows a strong heterogeneity between bases and Perciforms (figure 6). In spite of the non-utilization of
species. For the first positions, a slight fluctuation between various informative (but saturated) sites, they present a
the species is apparent, and for the second, a strong topology similar to those obtained by Cantatore et al. [13].
homogeneity for each taxonomic level (figure 4). The In the MP analysis of the same data, the Scombridae are
200A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
Figure 4. Base composition of the cytochrome b sequence for each species (classified according to their subfamilies) and for each codon position
taken separately.
The first, second and third codons are on the top, middle and bottom of the figure, respectively.
shown to be a monophyletic suborder (bootstrap value and 2, respectively. In addition, the most parsimonious
85 %), and as already shown by Block et al. [21] and tree by ‘branch and bound’ is of 33 steps with a CI of 0.56
Cantatore et al. [13], our data support the placing of the and a RI of 0.52 (data not shown).
more closely related S. Sarda and T. Thynnus in the same The NJ tree presents a similar topology, that is a separa-
subfamily (Sardinae) and of S. scombrus in the related tion of the Scombrodei from the other suborders (bootstrap
subfamily Scombrinae (figure 6). A contrario, within the value 80 %). Surprisingly, we noted a differentiation of the
Perciforms, the multifurcation of the tree shows that other Moronidae-Sparidae group from the other families (boot-
interfamily relationships remain undefined. Surprisingly, strap value 95 %) and the paraphyly of the Percoidei
this tree does not correspond to the traditional classifica- (figure 6). The Goboiidei remain monophyletic but with a
tion of the Perciforms [22]. The paraphyly of the Percoidei low bootstrap value (44 %). The dusky groupers constitute
was already demonstrated by Cantatore et al. [13]. Within the terminal part of the Percoidei, showing here too a very
the Gobiidae, the intrafamily relationships were shown to strong differentiation of the species into two quite distinct
be non-significant, having bootstrap support values below taxonomic units (bootstrap values in groups A and B were
54 %. In addition, all the dusky groupers constitute a 99 and 89 %, respectively).
monophyletic taxon (bootstrap value 66 %) separated into
two groups (bootstrap value 90 % and 91 %). The phylo-
genetic divergence nodes between the two groups of 4. Discussion
dusky grouper are very deep, comparable to, or greater
than, those seen between two species belonging to the 4.1. Phylogenetic analysis of the dusky grouper
same family but not to the same genus, for example, the
distance which separates T. thynnus from S. sarda (branch Phylogenetic analysis of partial mitochondrial cyt b
lengths 1/1) – the branch lengths are the number of steps sequences of dusky grouper from the western Mediterra-
separating the first species (or taxa) to the second com- nean sea led to two major findings. First, the phylogenetic
pared to the exclusive node – or P. marmoratus from N. differentiation between the two groups of dusky groupers
melanostomus (branch lengths 3/0), whereas the branches is wide, greater than that usually observed between two
separating the grouper of groups A and B have lengths of 3 species of fishes belonging to the same genus; this suggests
201A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
Figure 5. Absolute saturation plots for the cytochrome b sequences
for transitions (TS, left) and transversions (TV, right) separately and for Figure 6. Bootstrap analyses carried out with 1 000 iterations among
each codon position taken separately (first, second and third codon 13 taxa of Perciforms, with S. scombrus as an outgroup: using a
positions were in A, B, C and D, E, F for TS and TV, respectively). weighted maximum parsimony (branch length value on the top and
The Y axis is the pairwise number of observed differences (in percent), bootstrap proportion at the bottom right), neighbour joining on a
the X axis is the pairwise number of inferred substitutions as recon- matrix of the jukes and cantor model (bootstrap proportion at the
structed in the MP tree. bottom left).
The likelihood ratio test and Templeton’s test indicate that the two
trees are not significantly different in topology so only the maximum
the existence of a cryptic species. Second, there is a parsimony tree is shown.
genetic differentiation within one of the groups of western
dusky groupers. This lineage referred to as group B, con-
tains French, Tunisian and the rest of the Algerian popula- from N. melanostomus. Because these fishes are widely
tions of dusky groupers, suggesting recent gene flow. In recognized as distinct species, with clear morphological,
addition, the monophyly of each of the two groups of ecological and behavioural differences (references in [23,
dusky groupers was supported by high bootstrap values 24]), they could provide a measure of genus-level distance
using NJ or MP methods, and the analysis of the trees among genera of Percidae. Could there perhaps be, within
suggests that the first separation between the western the Percoidei, a differential evolution rate between the
Mediterranean dusky groupers might have probably families?
occurred in Algeria. Surprisingly, the present study suggests the presence of
cryptic species (morphologically indistinguishable spe-
4.1.1. Presence of putative cryptic species cies) within E. marginatus. A more complete morphologi-
Using the cyt b sequence, the estimated genetic dis- cal study and an allozymic analysis of these species were
tances between the two groups of dusky groupers of the clearly called for based on these cyt b sequences. These
western Mediterranean sea approach or exceed the level future studies could permit us to establish the breaking of
of genetic distance measured between other species and the genetic flow. Further re-analysis might reveal subtle
even other genera of Percomorphs. This genetic distance morphological differences which could be used to distin-
exceeds that which separated fishes belonging to the same guish between sympatric, genetically distinct populations
family but not to the same genus, for example, the distance in both of the two groups. Relatively recently, Ben
which separated T. thynnus from S. sarda or P. marmoratus Tuvia [25] identified in the Mediterranean a new species
202A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
for science E. haïfensis, morphologically very close to E. morphological change is thought to occur at a much
marginatus. Differentiation has even been proposed slower rate than genetic evolution, resulting in morpho-
between the juveniles of E. haïfensis and E. caninus [26]. logically indistinguishable cryptic species. In the case of
Despite the existence of maps showing the geographical scincid lizards, the presence of cryptic species was due to
distribution of E. marginatus and E. haïfensis [27], the the probable lack of gene flow between geographically
sympatry of these two species on the coast of North Africa isolated Pacific island groups [38]. Finally, species might
is still uncertain Heemstra and Randall [2] did not observe remain morphologically similar despite extensive genetic
specimens of E. haïfensis in this area. Further complicating divergence due to stabilizing selection on morphology,
classification is the presence of various colour patterns and convergence due to similar evolutionary pressures in
observed during the reproductive period [28]. different locations (see [38] and references there-in).
The dusky groupers from Algeria were collected in two
regions more than 400 km apart (from Algiers to Annaba), 4.1.2. Relationships within the dusky grouper of group B
and surprisingly in each region dusky groupers belonging In group B, there is an important genetic diversity within
to groups A and B were found. An important criterion for the populations from three different regions (Algeria,
recognizing species is the achievement of reproductive France and Tunisia), but no apparent genetic divergence
isolation in nature [29, 30]. Because the two Algerian between two of them (France–Tunisia).
grouper populations are not isolated and morphological The MP bootstrapped phylogenetic reconstruction sug-
distinctions did not delineate the marked molecular phy- gests that in group B, the Tunisian and French dusky
logenetic differences reported among these two popula- groupers are closely related. In addition, one of the partial
tion mitochondrial lineages, we cannot conclude defini- cyt b of a French dusky grouper (Fr1) shares 100 % of the
tively that the two groups belong to two different species. sequence with two Tunisian dusky grouper. In spite of the
Because there are no geographical barriers (no obvious small numbers of sequences belonging to this last sub-
barriers), only putative behavioural or physiological repro- group, this result does not exclude that some of the Tuni-
ductive barriers that have evolved between the two Alge- sian groupers could have colonized the French coast. This
rian population mitochondrial lineages (but, we cannot be hypothesis is in accordance with the study of Oliver [39]
certain of this explanation). The consistent evidence of which showed that on the basis of the study of parasites
substantial genetic differentiation implies that effective or among the dusky groupers, relationships might be envis-
persistent hybridization has been rare or absent in nature aged between the grouper from the Tunisian coast and
for a very long time span, but a clonally inherited marker those from the French coast.
such as mtDNA does not permit any conclusions about the The analysis of the trees shown in figure 3.A suggests
occurrence of hybridization and nuclear genes must be that all the French grouper except one (Fr1) displayed a
sequenced. As already shown experimentally [31], we basal position and the Tunisian and Algerian groupers of
cannot rule out the possibility of gene introgression with group B displayed an apical position. This is not in agree-
other species of grouper. Seven species of grouper belong- ment with the opinion that the French grouper immigrated
ing to the genus Epinephelus occur in the Mediterra- from the southern Mediterranean sea. Various authors
nean [2, 27, 32, 33] and on the Algerian coast. E. margina- have hypothesized that the dusky grouper could not breed
tus coexists with three of these species E. costae, E. aeneus, north of the 42nd parallel [6], but for some years this
E. haïfensis and E. caninus. E. costae (golden grouper) does hypothesis has been contradicted by various observations.
not have exactly the same spawning period as E. margina- Nuptial displays have been observed on the French
tus [1, 34], which does not share the same biotopes as E. coast [28] and groupers have been seen spawning in the
aeneus (white grouper), which is particularly rare [35], or Medes island marine reserve on the Costa Brava
E. caninus (dogtooth grouper). In Algeria, E. marginatus (Spain) [40]. In addition, a rejuvenation and an increase in
may coexist with E. haïfensis [27], but this requires confir- the grouper population has been observed in the Parc
mation. In any case, their biotope is relatively distinct [2, national de Port-Cros, France (GEM, 1996). We do not
34]. know whether the slight warming of the Mediterranean
While morphological stasis in the dusky grouper is (0.12–0.60 °C over 30 years) could alone explain this
suggested in the present work for the first time, genetic phenomenon, with the temperature increase facilitating
divergence and morphological similarity have been well the northward migration of young females and then offer-
documented in Lake Tanganyika’s Tropheus cichlid ing conditions that are more suitable for the reproduction
fish [36]. In that case, common morphology is not due to a of the species [41]. Nevertheless, this hypothesis would
slower rate of evolution, because in a fraction of the time appear to be the only plausible one to date.
in which lake Tanganyika’s cichlids remained unchanged,
a species flock of over 200 species flourished in Lake 4.2. Phylogenetic relationships
Victoria [12]. The common morphology in these cichilds within the Perciformes order
is thought to be maintained by stabilizing selection due to
ecological niche packing [36]. An alternate mechanism Our results, like those of other studies [13], do not
has been proposed for morphological similarity in pleth- provide an adequate basis for clarifying the phylogenetic
odondid salamanders [37] and scincid lizards [38]. Where relationships within the Perciformes order. Except for the
203A. Gilles et al. / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 323 (2000) 195–205
Scombroidae family, other interfamily relationships have phological similarity leaves open the question of ecologi-
been shown to be non-significant in the latter study and cal difference between these species when found in
have not been resolved in our work. If the monophyly of sympatry. Ongoing studies are investigating the selective
the Percomorpha is generally accepted, there is no con- pressures responsible for the high degree of morphological
sensus and insufficient data regarding the number and the similarity and genetic differentiation.
monophyly of suborders and subfamilies (Lecointre G. The lack of substantial molecular genetic differentiation
pers. comm.). The families of the Cichlidae and the Gobi- among French and North African populations (only Tuni-
idae are little differentiated from the Percoidei and only sian) does not indicate that there is no genetic imperative
seem in fact to constitute families of Percoidei and not to protect the French grouper. However, these negative
suborders in their own right. It could be too that it is the results should be interpreted cautiously, because they do
suborder of the Percoidei that includes families solely by not assess the reduction of potential fitness after putative
morphological convergence, and in this case it is the hybridization, which might reflect ecological or ethologi-
suborder of Percoidei that should disappear. The data of cal adaptedness. In addition, in spite of the fact that in the
Cantatore et al. [13] suggest that the Acipenseriformes- phylogenetic trees, cyt b sequences of French, Tunisian
Perciformes ancestor is about 200 million years (MY) old, and some of the Algerian dusky grouper are in the same
in the low range of the known fossil records (205–355 group (B), we cannot affirm that the French and North
MY). According to this last hypothesis, the ancient origin African groupers are the same population. In addition, in
of Perciforms may justify some of the uncertainties in our our study, the majority of the groupers analysed were fairly
phylogenetic analysis. old (size > 30 cm). A deeper understanding of various
factors, including a specific study of juveniles in France
4.3. Conclusion and the confirmation or invalidation of the putative errat-
ism of the juveniles is important for developing effective
Future ecological and allozymic studies of Mediterra- management programmes for the protection of endan-
nean grouper fauna should consider the possibility that gered species.
morphologically similar populations can represent geneti-
cally and historically distinct cryptic species. Despite Acknowledgements: This project was funded partially
nearly identical morphologies, the high level of mtDNA by the GEM (Six-Fours-les-Plages, France) and Nausi-
sequence divergence between groups A and B of dusky caa (Boulogne, France). We thank all the people who
grouper clades suggest more ancient evolutionary diver- provided groupers for this study (F. Bachet, K. Boubezari,
gence. With regard to the Algerian population, this mor- A. El Hili, H. Kara, A. Marc).
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