Journal of Molluscan Studies Advance Access published 21 January 2011
THE UNIONIDS OF TUNISIA: TAXONOMY AND PHYLOGENETIC
RELATIONSHIPS, WITH REDESCRIPTION OF UNIO RAVOISIERI
DESHAYES, 1847 AND U. DURIEUI DESHAYES, 1847
NOUREDDINE KHALLOUFI 1 , CARLOS TOLEDO 2, ANNIE MACHORDOM 2,
MONCEF BOUMAÏZA 1 AND RAFAEL ARAUJO 2
1
Unite´ D’Hydrobiologie, Laboratoire de Biosurveillance de l’Environment, Faculty of Sciences of Bizerte, 7021 Jarzouna, Tunisia; and
2
Museo Nacional de Ciencias Naturales (CSIC), Jose´ Gutie´rrez Abascal 2, 28006 Madrid, Spain
Correspondence: R. Araujo; e-mail: [email protected]
ABSTRACT
Using an approach based on molecular, reproductive and morphological characters, four independent evolutionary lineages represented by the following species were identified in Tunisia: Unio ravoisieri Deshayes, U. gibbus Spengler, U. durieui Deshayes and Potomida littoralis (Cuvier). The species
U. ravoisieri and U. durieui are redescribed, including details of their distribution, biology and glochidium morphology. The puzzling distribution of the former (Algeria, Tunisia and two localities in
Northeast Spain) could reflect introduction or natural dispersal. In Tunisia, the release of the glochidia of U. ravoisieri starts in March, but in Spain this is delayed until August or September. The entire
interlamellar space of the outer demibranchs acts as a marsupium (ectobranchy). Glochidia are
rounded-triangular, globose, and either hooked or hookless. Unio durieui is distributed across Algeria
and Tunisia, where it releases glochidia in December and in March. Glochidia are always hooked,
but the elaboration of the hook is variable. The molecular phylogenetic analysis supports two clusters
within Unio, each including both European and North African species. Strong bootstrap support was
obtained for the two clades within U. ravoisieri, of which one includes specimens from the Iberian
Peninsula as well as Tunisia. Unio durieui appeared as the sister species of U. gibbus. The Tunisian
populations of P. littoralis were not segregated from those in Europe, and U. fellmanni should be considered a synonym.
INTRODUCTION
Naiads or freshwater mussels (Unionoida) comprise some 850
species that are widespread in the rivers of all continents
except Antarctica (Graf & Cummings, 2006; Bogan, 2008).
According to Haas (1969), an authority on unionoids, many
names introduced by early authors should be relegated to synonymy. Recently, new conchological, anatomical, reproductive
and molecular data have begun to resolve taxonomic problems
in this group (Nagel, Badino & Alessandria, 1996; Huff et al.,
2004; Araujo, Gómez & Machordom, 2005; Graf &
Cummings, 2007; Zanatta, Ngo & Lindel, 2007; Campbell
et al., 2008; Pin Chong et al., 2008; Araujo et al., 2009a, c;
Araujo, Toledo & Machordom, 2009b; Reis & Araujo, 2009).
A molecular approach has been particularly helpful
(Machordom et al., 2003; Graf & Cummings, 2006; Araujo
et al., 2009b, c).
The taxonomy of the Palaearctic unionids is especially
poorly resolved in countries such as Algeria and Tunisia, for
which the most relevant malacological works were often published long ago (Deshayes, 1847; Bourguignat, 1864, 1866;
Issel, 1880; Kobelt, 1884; Letourneux & Bourguignat, 1887;
Pallary, 1923, 1926, 1927, 1936). Since then, with the exception
of the book by Haas (1969), only the reviews by Van Damme
(1984), Mandahl-Barth (1988) and Daget (1998) have
included information on the species inhabiting these areas.
However, these latter authors did not critically review the taxonomy, but only described the shells and anatomy of available
material. Two recent papers have addressed the Tunisian
Anodonta species (Khalloufi & Boumı̈za, 2005) and Unio gibbus
(Khalloufi & Boumı̈za, 2009).
Hence for North Africa, and particularly Tunisia, the
nomenclature of this variable group is still ambiguous. Indeed,
several available names of species and subspecies (e.g.
U. elongatulus durieui Deshayes, 1847 and U. pictorum ravoisieri
Deshayes, 1847) have not yet been validated, and it is
unknown how many species really exist (Haas, 1969; Van
Damme, 1984; Mandahl-Barth, 1988; Daget, 1998).
In the present study, we aim to clarify the taxonomy and
phylogenetic relationships of the Tunisian unionids, based on
molecular and morphological characters.
MATERIAL AND METHODS
Living specimens were collected from the rivers of the main
Tunisian basins: Medjerda, Sejenane, El Maâden, and El
Kébir (Fig. 1). Some Spanish specimens were also included in
this study (Fig. 1).
To gather recent and historical data we reviewed the following literature: Deshayes (1847), Bourguignat (1864, 1866),
Kobelt (1884), Rossmässler (1884), Letourneux & Bourguignat
(1887), Drouet (1893), Germain (1908), Pallary (1923, 1926,
1927, 1936), Azpeitia (1933), Pérès (1944), Haas (1969), Van
Damme (1984), Mandahl-Barth (1988), Daget (1998) and
Khalloufi & Boumı̈za (2005, 2009). We also examined the
North African naiads of the collections of the Museo Nacional
de Ciencias Naturales (MNCN, Madrid, Spain), Muséum
National d’Histoire Naturelle (MNHN, Paris, France),
Forschungsinstitut Senckenberg (SMF, Frankfurt, Germany)
and Museum d’Histoire Naturelle (MHN, Geneva,
Switzerland).
Journal of Molluscan Studies (2011) 0: 1 –13
# The Author 2011. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved
doi:10.1093/mollus/eyq046
Downloaded from mollus.oxfordjournals.org at Centro de Información y Documentación Científica on January 23, 2011
(Received 1 July 2010; accepted 14 October 2010)
N. KHALLOUFI ET AL.
For the species redescribed, U. ravoisieri and U. durieui,
descriptions were made of shell shape and outline, colour,
umbonal sculpture and hinge characteristics. Maximum length
(L), height (H) and width (W) were measured, and the
indices L/H and L/W calculated for 164 specimens of U. ravoisieri (6 from Algeria, 96 from Tunisia and 62 from Spain) and
47 of U. durieui (all from Tunisia). These data were subjected
to statistical tests (descriptive statistics: K-W ANOVA,
Spearman rank R) using the STATISTICA 6.0 package.
Glochidia were collected from the mussels by removing them
from gravid gills. They were cleaned with KOH and goldcoated for scanning electron microscopy.
Seventy-one specimens were used in the molecular study
(Table 1), including Tunisian specimens, sequences from
GenBank and the newly sequenced Spanish specimens. Two
pieces of foot were removed from specimens and frozen at
2758C or preserved in absolute ethanol. DNA was extracted
from these samples using the Qiagen DNeasy Blood Kit, following the corresponding protocol. A partial sequence of the
cytochrome oxidase subunit I (COI) gene (658 bp) was amplified by PCR using the primers of Machordom et al. (2003). A
partial 16S rRNA (487 bp) gene sequence was amplified using
two pairs of primers: 16sar-L-myt 50 -CGACTGTTTAACAAA
AACAT-30 , 16sbr-H-myt 50 -CCGTTCTGAACTCAGCTC
ATGT-30 (Lydeard, Mulvey & Davis, 1996), 16Sar-L 50 -CGC
CTGTTTATCAAAAACAT-30 and 16Sbr-H 50 -CCGGTCT
GAACTCAGATCACGT-30 (Palumbi, 1996). The PCR mix
contained 2 ml of DNA, 5 ml of the corresponding buffer (10
with 2 mM MgCl2), 1 ml of dNTP mix (10 mM), 0.8 ml of
each primer (10 mM), 0.3 ml Taq DNA polymerase (5 U/ml)
2
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Figure 1. Sampling sites in Tunisia and Spain.
UNIONIDS OF TUNISIA
Table 1. List of specimen samples examined, genes sequenced and GenBank accession numbers.
Species
Locality
COI
16S rRNA
Reference
Unio delphinus
Tua River, Portugal
EF571413
EF571345
Reis, Machordom & Araujo (submitted for publication)
Aravil River, Portugal
EF571417
EF571351
Reis et al. (submitted for publication)
Coa River, Portugal
EF571419
EF571353
Reis et al. (submitted for publication)
Austria
AF156499
DQ060163
Graf and Foighil (2000)
Poland
AF468684
EU518634
Soroka et al. (unpubl.)
Ebro River, Spain
AY522849
EF635011
Araujo et al. (2005)
AY522850
EF635012
Araujo et al. (2005)
EF571405
EF571338
Reis et al. (submitted for publication)
Unio pictorum
Unio mancus
Unio tumidiformis
Odeleite River, Portugal
EF571408
EF571340
Reis et al. (submitted for publication)
Sao Pedro River, Portugal
EF571409
EF571341
Reis et al. (submitted for publication)
Unio crassus
Poland
DQ060174
DQ060162
Kallersjo et al. (2005)
Unio gibbus
Barbate River, Spain
EU735755
EU735770
Araujo et al. (2009b)
EU735756
EU735771
Araujo et al. (2009b)
EU735758
EU735773
Araujo et al. (2009b)
Sejenane River, Tunisia
GU070980
GU071028
This study
GU070981
GU071029
This study
Medjerda River, Tunisia
GU070982
GU071030
This study
GU070984
GU071032
This study
DQ060176
DQ060161
Kallersjo et al. (2005)
Unio tumidus
Sweden
Potomida littoralis
Imperial channel, Spain
AF303348
AF303307
Machordom et al. (2003)
AF303349
AF303308
Machordom et al. (2003)
GU070946
GU070994
This study
GU070949
GU070997
This study
El-Maâden River, Tunisia
GU070950
GU070998
This study
GU070952
GU071000
This study
Sejenane River, Tunisia
GU070953
GU071001
This study
GU070954
GU071002
This study
Psilunio littoralis*
Banyolas Lake, Spain
AF120652
–
Giribet and Wheeler (2002)
Unio ravoisieri
Ser River, Spain
GU070955
GU071003
This study
GU070961
GU071009
This study
Banyolas Lake, Spain
GU070962
GU071010
This study
GU070963
GU071011
This study
El-Kébir River, Tunisia
GU070964
GU071012
This study
GU070969
GU071017
This study
Medjerda River, Tunisia
GU070970
GU071018
This study
GU070977
GU071025
This study
Sejenane River, Tunisia
GU070971
GU071019
This study
GU070973
GU071021
This study
GU070978
GU071026
This study
GU070979
GU071027
This study
El-Maâden River, Tunisia
GU070975
GU071023
This study
GU070976
GU071024
This study
Ziatine River, Tunisia
GU070985
GU071033
This study
GU070987
GU071035
This study
El-Kébir River, Tunisia
GU070988
GU071036
This study
GU070989
GU071037
This study
El-Maâden River, Tunisia
GU070990
GU071038
This study
GU070993
GU071041
This study
Ziatine River, Tunisia
Unio durieui
*Specimen cited as Psilunio littoralis but ascribed here to Unio ravoisieri.
(Biotools) and ddH2O was added to give a final volume of
50 ml. Amplification reactions were performed as an initial
denaturation cycle at 948C for 4 min; 40 cycles of 948C for
45 s, 458C (for COI amplification) or 508C (for 16S amplification) for 1 min, 728C for 1 min and a final extension at 728C
for 10 min. The PCR products were separated on 1% agarose
gels stained with SYBR Safe (Invitrogen) to check the size of
the fragments and cleaned by ethanol precipitation. The
amplified DNA was sequenced in an ABI 3730 genetic analyser
(Applied Biosystems) and the sequences obtained were
assembled and edited using Sequencher v. 4.6 (Gene Codes
Corporation) after removing primer regions. Alignments were
adjusted by eye taking into account previously published
GenBank sequences for Potomida and several Unio species
(Table 2). For the 16S sequences, we tried to include the
lowest possible number of gaps, whereas COI required no
3
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Sado River, Portugal
1.44
3.16
15.71
16.40
16.65
15.88
15.98
16.06
15.61
15.90
14.71
16.73
17.03
16.98
16.51
16.61
16.93
16.52
16.64
15.42
13.35
12.41
13.25
13.35
6.49
10.16
4.85
10.43
10.23
5.38
5.63
10.17
13.07
10.90
11.17
11.92
13.19
13.52
13.92
12.77
13.52
13.46
13.48
13.53
6.53
6.18
1.92
2.91
1.24
1.97
0.56
1.92
11.33
11.84
12.89
12.32
12.74
13.30
14.16
12.63
14.26
14.04
13.47
12.10
10.91
9.84
12.79
14.41
12.98
8.57
11.80
10.81
13.04
13.46
13.48
6.18
1.92
1.24
0.00
14.08
7.42
9.56
9.41
9.08
9.08
13.58
7.95
9.10
9.51
9.20
9.20
3.83
15.35
14.06
6.56
7.70
9.10
7.37
8.32
9.56
9.39
7.69
7.69
9.39
5.15
4.29
5.44
2.75
4.54
4.88
2.45
5.79
12.21
12.66
13.68
6.40
7.53
8.48
7.85
7.85
13.03
0.36
4.11
13.23
13.64
6.79
5.97
7.52
7.71
8.25
8.05
7.83
8.04
8.04
7.83
3.90
4.52
1.81
1.41
5.07
2.91
2.44
4.86
(Ib. P.)
(Tun.)
P. littoralis
U.
tumidus
durieui
U.
U. gibbus
(Tun.)
(M.)
U. gibbus
U. gibbus
(Ib. P.)
crassus
U.
pictorum
(Tun.)
U.
U.
U. ravoisieri
The alignment included 1,150 positions, of which 276 were
parsimony-informative and 816 were constant. All methods of
tree construction were generally consistent and showed a
similar topology, although with some differences in the
relationships of U. delphinus, U. mancus, U. pictorum and U. ravoisieri. Analyses with and without codon partitions did not differ.
Three independent Bayesian runs using different numbers of
generations converged on the same tree for the combined
dataset, but posterior probabilities increased slightly with the
number of generations. A ML tree based on the combined
dataset for the COI and 16S sequences was selected as representative (Fig. 2).
Regarding the genus Unio, the trees revealed the following
lineages: U. ravoisieri, U. durieui, U. delphinus Spengler, 1793,
U. mancus Lamarck, 1819, U. pictorum Linnaeus, 1757, U. gibbus
Spengler, 1793, U. tumidus Philipsson, 1788, U. crassus Philipsson,
1788 and U. tumidiformis Castro, 1885, all supported by high NJ,
MP and ML bootstrap values, as well as posterior probabilities
(PP ¼ 100%). The analysed populations of U. ravoisieri formed a
Differences expressed as percentages.
16.08
17.05
16.08
P. littoralis (Ib)
17.32
16.76
P. littoralis (Tun.)
16.36
13.77
11.45
10.77
10.57
U. tumidus
12.75
13.21
U. durieui
13.70
12.42
13.59
12.08
12.84
U. gibbus (M.)
U. gibbus (Tun.)
12.45
12.02
12.63
U. gibbus (Ib. P.)
13.80
10.04
11.03
U. crassus
11.03
11.08
12.02
11.36
U. tumidiformis
2.48
4.41
U. pictorum
5.89
5.23
4.44
U. ravoisieri (Tun.)
4.84
4.69
U. ravoisieri (Ib. P.)
4.06
2.06
1.03
1.64
4.36
U. mancus
U. delphinus
mancus
(Ib. P.)
U.
delphinus
U. ravoisieri
RESULTS
U.
Table 2. Pairwise genetic distance matrix constructed according to 487 bp of the 16S gene (above the diagonal) and 658 bp of the COI gene (below).
gaps. Our objective was not to establish phylogenetic relationships between Potomida and Unio, but to resolve relationships
among Unio species. We used Amblema plicata (Say, 1817) as
outgroup, because it is closely related to the Unionini (Graf,
2002; Graf & Cummings, 2006).
Phylogenetic analyses of the COI and 16S rRNA datasets
were conducted both separately and for the combined dataset.
Congruence among data was tested using a partition homogeneity test implemented in PAUP*.
Maximum-parsimony (MP) and neighbour-joining (NJ)
analyses were performed using PAUP* v. 4.0b10 (Swofford,
2001). Parsimony analyses were conducted through equally
weighted heuristic searches using the TBR (tree-bisectionreconnection) branch-swapping algorithm after limiting the
number of rearrangements to 2,000,000. Gaps were treated as
missing data. Bootstrap support for tree branches was assessed
through 1,000 replicates. The PHYML program was used for
maximum-likelihood (ML) analyses, using the optimal basepair substitution model. ML bootstrap values were calculated
using 500 replicates. Majority rule consensus trees were created
using PAUP.
Modeltest v. 3.7 (Posada & Crandall, 1998) was used to
select the most appropriate model and starting parameters for
the ML and NJ analyses, according to the Akaike test
implemented in this program. This indicated the transversion
model (TVM), with four categories of substitution rates following a gamma distribution and allowing for invariant sites, as
the best substitution model to fit the combined dataset
(COI þ 16S). However, we used a general-time reversible
model, with gamma distributed rate variation among sites
approximated with four categories and allowing invariant sites
(GTR þ I þ G), because of the program’s limitation to ML
searches. Separate analyses for each gene gave different models:
TVM þ G for COI and GTR þ G þ I for 16S.
Bayesian analyses were performed using Mr Bayes v. 3.1.2
(Huelsenbeck & Ronquist, 2001). For COI, both independently and in the combined dataset, we compared analyses
with and without partitioning by codon position. For these
analyses, three simultaneous independent runs were performed
starting from different random trees. The program estimated
the best-fit model and, for each run, the tree search was performed through four chains of a Markov Chain Monte Carlo
(MCMC) algorithm for 5 million generations, sampling every
100 generations. Finally, we constructed a consensus tree,
having removed the first 5,000 trees (10%) from the analysis as
burn-in.
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tumidiformis
P. littoralis
N. KHALLOUFI ET AL.
UNIONIDS OF TUNISIA
monophyletic group composed of two clades: one of specimens
from the Tunisian rivers Sejenane, Medjerda and El-Maâden
(100% support in NJ, MP and ML bootstrap and PP values),
the other of specimens inhabiting the rivers of the Iberian
Peninsula and the Tunisian Kébir and El-Maâden rivers (moderate to high NJ, MP and ML bootstrap and PP values; Fig. 2).
One sequence from GenBank (AF120652), identified as Psilunio
littoralis (¼ Potomida littoralis), also appeared in the second U.
ravoisieri group according to its COI sequence.
Unio delphinus, U. mancus and U. pictorum formed a wellsupported clade with U. ravoisieri, but relationships among
these four were not clearly resolved (Fig. 2). Despite this poor
resolution, high divergences among U. delphinus, U. mancus and
U. pictorum were observed (4.06–4.44% for COI; Table 2) and
their evolutionary independence was well supported. Unio tumidiformis (Iberian Peninsula) and U. crassus (represented by one
sequence from Poland) formed a well-supported clade with the
above-mentioned species.
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Figure 2. Consensus tree showing phylogenetic relationships (ML analysis) among different unionid species based on partial 16S and COI gene
sequences. Numbers above branches are bootstrap values for NJ and MP models; those below branches indicate ML bootstrap values and Bayesian
inference posterior probabilities.
N. KHALLOUFI ET AL.
A second large clade within Unio contained U. gibbus and
U. durieui, with U. tumidus in a basal position. The Tunisian
specimens of U. durieui formed a homogeneous group with no
differences among rivers.
The group of the Potomida littoralis specimens was well supported by all the phylogenetic analyses, although considerable
population differentiation was observed between specimens
from the Tunisian rivers and the Iberian Peninsula (up to
3.3% for COI and 1.65% for 16S; Table 2).
At the level of populations inhabiting different Tunisian
river courses, we found no genetic structure in U. gibbus or
P. littoralis, in contrast to U. ravoisieri. Specimens of U. gibbus
from Spain, Morocco and Tunisia clustered together as a consistent group, within which those from the first two areas were
closest (Fig. 2).
Life cycle: Mature glochidia found in specimens from the El
Kébir (March 2008) and Sejenane rivers (April and May
2010). Specimens from El Kébir had empty gills in December,
and those obtained in March from the Sejenane River had no
glochidia, although some carried embryos in the external gills.
In Spanish specimens from Banyoles Lake and the Ser River,
the external gills were replete with eggs from June to July.
Glochidia rounded-triangular, globose, hooked or hookless
even inside same water tube (Fig. 4). Some glochidia with one
hooked and one hookless valve. Mean glochidium dimensions
(measured by SEM): length 208.83 mm (n ¼ 5), height
176.95 mm (n ¼ 4), width 171.66 mm (n ¼ 10).
SYSTEMATIC DESCRIPTIONS
Unio ravoisieri Deshayes, 1847
(Figs 3, 4)
Unio ravoisieri Deshayes, 1847: no description, pl. 108, figs 4– 7
(Oubeira Lake, La Calle, Algeria; type lost).
Unio moreleti Deshayes, 1847: no description, pl. 109, figs 1 –4
(Oubeira Lake, La Calle, Algeria; type lost).
Unio penchinatianus Bourguignat, 1865: 342 –343, pl. 21, figs 1 –7
(Banyoles Lake, Girona, Spain; types MHN BGT1269).
Unio medjerdae Kobelt, 1886: 23, pl. 42, figs 257, 259
(Medjerda River, Beja, Tunisia; holotype SMF 4397).
Unio micelii Kobelt, 1886: 23, pl. 43, figs 260, 261 (Medjerda
River, Beja, Tunisia; holotype SMF 4400).
Description: Specimens look very different depending on
habitat, and Tunisian specimens differ profoundly from
Spanish ones. From El Kébir River (Fig. 3B): green,
elongated, with prominent umbo (resembling U. mancus). From
Sejenane River (Fig. 3C): brown, elongated, with flat umbo
(resembling type specimens of U. ravoisieri, Fig. 3A and U. moreleti). Specimens from Medjerda River are similar to those
from the El Kébir. Umbonal sculpture and pseudocardinal
teeth of the valve are similar in all populations (see below).
From Ser River, Spain (Fig. 3D): shell small, thin,
elongated, compressed. Colour brown, sometimes green or
yellow, with closely spaced external growth rings. Length
60 mm, rarely to 95 mm. Dorsal and ventral edges parallel, the
former slightly ascendant towards posterior region. Rounded
shape of antero-dorsal edge of shell is pronounced and characteristic of the population. Umbos flat, not projecting, with
tubercles arranged in two rows, sometimes sharp (see Araujo
et al., 2009a); umbonal sculpture can be lacking even in specimens with well-preserved umbo. Hinge and ligament thin.
Pseudocardinal teeth small, laminar, the two on left valve
fused.
From Banyoles Lake (Fig. 3E, F): large, to 105 mm, thick,
swollen. Colour yellow-brown, never green; posterior region
with chalky deposit. Outline quadrangular, umbo in anterior
position so posterior region is large. Hinge and ligament well
developed. Pseudocardinal teeth strong, crenulated, separated
in left valve. Umbonal sculpture as described above.
The populations showed significant morphometric differences (P , 0.05), mostly in their L/W and L/H indices,
forming the following four groups: specimens from Algeria,
from Banyoles, from Ser River and all specimens from Tunisia.
For all populations, the median L/W index was 2.94 (+0.26;
range 1.93 –4.27) and the median L/H index was 1.97 (+0.17;
range 1.25 –2.91).
Remarks: The presence of a chalky deposit in the posterior
region of the shell is a typical character of the specimens from
Banyoles Lake (Haas, 1916; Araujo et al., 2009a).
Unio durieui Deshayes, 1847
(Figs 5A–D, 6A–E)
Unio durieui Deshayes, 1847: no description, pl. 109, figs 5– 8
(Algeria; type lost).
Unio tafnanus Kobelt, 1884: 66, pl. 28, fig. 216 (Tafna River,
Oran Province, Algeria; holotype SMF 4404).
Description: Less variable than U. ravoisieri and distinguished
from it by near rectangular outline and rounded (not pointed)
posterior margin. Posterior region elevated, resembling shape
of U. gibbus, hence highest point of shell is behind umbo. Shells
from Ziatinne River are brown, and from El Kébir green.
Specimens from both rivers are more elongated than type
specimen (Fig. 5A– C). A constant character is the gap
between the two pseudocardinal teeth of the left valve, which
sometimes overlap but never fuse, being always separated.
Anterior pseudocardinal tooth external to posterior one and
nearer shell edge. Sculpture of the umbos, when present, of
rows of strong, wavy W-shaped rugae, resembling sculpture of
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Distribution and conservation status: The species shows a disjunct
distribution, including rivers in Algeria (Senam, St Dona, La
Calle and Zenati rivers, and Oubeira Lake: MNHN uncatalogued lots), Tunisia (Abid and Meliane rivers at Cap Bon, NE
Tunisia; Sejenane, Habbes and Joumine rivers in N Tunisia;
Béja, El Kébir, Ghrib, Medjerda and Mellague rivers in NW
Tunisia) and two sites in northeastern Spain (Banyoles Lake
and Ser River, both in Girona province, Catalonia). At
Banyoles Lake it lives in sympatry with U. mancus, Potomida littoralis and Anodonta anatina, and in Tunisia with Potomida littoralis, U. gibbus and U. durieui.
Although in Tunisia U. ravoisieri was formerly probably the
most frequent Unio species, current observations indicate a
reduction. We have confirmed its total disappearance from
certain irrigation canals where it was frequent (N.K., personal
observation). There are anecdotal reports of large quantities of
valves mixed with the sand extracted from the Medjerda River
for building work during the 1980s (N.K., personal
observation).
In Spain, the species is scarce and difficult to find, inhabiting only two localities in two separate Mediterranean river
basins. In the Ser River (Fluviá basin) specimens live hidden
in banks and slopes, generally in the shade of trees and among
submerged roots. At the Banyoles Lake (Ter basin) they are
buried in the substratum from the shore to a depth of 10 m.
The restricted distribution in Spain and low population
density of U. ravoisieri suggest that it is vulnerable and we
therefore recommend its inclusion under the European
Habitats Directive and the Spanish Catalogue of Endangered
Species (Araujo et al., 2009a).
UNIONIDS OF TUNISIA
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Figure 3. Unio ravoisieri. A. Figure of the holotype of Unio ravoisieri Deshayes, 1847 (by permission of the Museo Nacional de Ciencias Naturales,
Madrid), Oubeira Lake, La Calle, Algeria. B. El Kébir River, Tunisia. C. Sejenane River, Tunisia. D. Ser River, Spain. E. Banyoles Lake, Spain.
F. Paratype of Unio penchinatianus Bourguignat, 1865 (Museum d’Histoire Naturelle, Geneva bgt1269-4), Banyoles Lake, Spain. Scale bar ¼ 1 cm.
P. littoralis. Median L/W index was 1.80 (+0.09; range 1.62 –
2.06); median L/H index 2.76 (+0.11; range 2.48 –3.11).
in U. ravoisieri (Fig. 6). Glochidia always hooked, but hook
varies from small spiculated ventral structure (Fig. 6C) to a
normal hook (Fig. 6D). Mean glochidial dimensions (measured
by SEM): length 208.61 mm (n ¼ 11), height 232.09 mm
(n ¼ 10), width 158 mm (n ¼ 1).
Life cycle: Mature glochidia were found in several specimens
collected from the El Kébir and El Maâden in December
2008. Three of the six specimens collected in March 2009
from El Maâden also contained glochidia. None of the five
specimens obtained from the Ziatinne River in December 2008
were gravid. Glochidia rounded-triangular; less globose than
Distribution: In Tunisia it has been recorded from the Ziatinne
River (in sympatry with P. littoralis), El Kébir River (with
U. ravoisieri) and El Maâden river (with P. littoralis and
7
N. KHALLOUFI ET AL.
U. ravoisieri). It is less common than U. ravoisieri, and does not
occur in the Medjerda and Sejenane rivers. Distribution in
Algeria: Tafna River (Kobelt, 1884), Senam and Rhumel
rivers (MNHN uncatalogued lots).
accompanying text. Subsequently, Kobelt (1884) and
Letourneux & Bourguignat (1887) described six new species
from Tunisia (see below). Haas (1969) compiled synonymies
and recognized only three Mediterranean North African taxa:
U. pictorum ravoisieri, U. elongatulus durieui and Potomida littoralis
fellmanni. No recent material from Algeria is available and the
Deshayes collection is missing (P. Bouchet, personal communication). Accordingly, our information for Algeria was limited
to the original colour figures of Deshayes’ types, data from
Haas (1969) and available museum specimens.
Our discovery of U. ravoisieri in Banyoles Lake and comparison
of the specimens with the types of U. penchinatianus from the same
locality (Fig. 3F) indicate that the Bourguignat species is a
DISCUSSION
The taxonomic designations of the Tunisian species examined
are based on our previous (Araujo et al., 2009b) and current
molecular interpretations as well as on comparisons among
field and museum specimens and the available figured types.
Three of these species were described by Deshayes (1847) from
Algeria (Figs 3A, 5A), based only on figures and with no
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Figure 4. Glochidia of Unio ravoisieri. A, B. External view (El Kébir River). C. Hooked glochidium (Sejenane River). D. Hookless glochidium
(Sejenane River). E. Lateral view of a closed glochidium (El Kébir River). F. Spicules (El Kébir River). G, H. Hook (Sejenane River).
UNIONIDS OF TUNISIA
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Figure 5. A–D. Unio durieui. A. Figure of the holotype of Unio durieui Deshayes, 1847 (by permission of the Museo Nacional de Ciencias Naturales,
Madrid), Algeria. B. Ziatinne River, Tunisia. C. El Kébir River, Tunisia. D. El Maâden River, Tunisia. E. Unio gibbus, Sejenane River, Tunisia.
F. Potomida littoralis, Tunisia. Scale bar ¼ 1 cm.
synonym. Other synonyms of U. ravoisieri are U. moreleti, U. micelii
and U. medjerdae, although Haas (1969) considered the last two to
be synonyms of U. durieui. Besides U. durieui, Letourneux &
Bourguignat (1887) listed four new species from Tunisia:
U. delevieleusae Hagenmüller, 1887, U. doumeti Bourguignat, 1887,
U. zenaticus Letourneux, 1887 and U. rouieri Bourguignat, 1887.
9
N. KHALLOUFI ET AL.
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Figure 6. A–E. Glochidia of Unio durieui. A. El Kébir River. B. El Maâden River. C. Detail of a small hook (El Maâden River). D. Detail of
normal hook (El Maâden River). E. Lateral view of closed glochidium (El Kébir River). F – H. Glochidia of U. gibbus (Medjerda River).
F. Internal view. G. Detail of hook. H. Lateral view of opened glochidium.
10
UNIONIDS OF TUNISIA
specimen from GenBank (AF120652) was evidently a
misidentified U. ravoisieri.
In summary, Tunisia is home to at least four species of freshwater mussels: Unio ravoisieri, U. gibbus, U. durieui and Potomida
littoralis. We were unable to find living populations of Anodonta,
although one species of this genus has been recently cited for
this country (Khalloufi & Boumı̈za, 2005). Our data begin to
unravel the complex taxonomy of Unio first reviewed by Haas
(1969) and indicate that: (1) U. elongatulus penchinatianus is not
a distinct subspecies; (2) despite U. durieui being the only
North African (Algeria, Morocco and Tunisia) species assigned
by Haas (1969) to the elongatulus group, we here demonstrate
that this interpretation was wrong.
The phylogenetic relationships presented here, although preliminary, suggest a complex biogeographical history, the two
large clusters within the genus Unio each containing both
European and North African species. The disjunct distribution
of U. ravoisieri in North Africa and Spain is intriguing.
Although we have no data related to the time needed for divergence of freshwater mussel taxa, the identity of the haplotypes
of the Spanish and Tunisian populations of U. ravoisieri indicates recent gene flow between them. Extinction of intermediate populations in the Mediterranean rivers of Spain is a
reasonable hypothesis, although there are no old records or
fossils to support the idea. As we did not find this species in
Morocco, we suggest that the western boundary of the distribution of U. ravoisieri could be the Molouya river (between
Morocco and Algeria), which is a known biogeographical frontier for freshwater fish (Machordom & Doadrio, 2001), lizards
(Harris et al., 2002) and plants (Molero & Montserrat, 2006).
Bearing in mind that the dispersal method of these molluscs
relies upon the fishes that act as hosts for their glochidia, we
propose two possible alternatives to the extinction theory.
Some historical human transport perhaps by the Phoenicians,
Romans or Almohads (deliberate or not) from the Kebir or
El-Maâden rivers, as has been claimed for other animal groups
(Recuero et al., 2007). Another, more dubious, possibility is a
connection among freshwater courses between these areas after
the Messinian crisis which dried up the Mediterranean
(5.5 Ma), followed by subsequent extinction of connecting
populations.
The contrasting pattern found in U. gibbus, with Iberian and
Moroccan specimens forming a cluster separated from those
from Tunisia, is worthy of note. A similar pattern is shown, for
example, by cyprinid fish (Machordom & Doadrio, 2001),
which act as a host for the glochidia of Unio, as well as by
other taxa (Perdices, Machordom & Doadrio, 1995;
Garcı́a-Parı́s, Alcobendas & Alberch, 1998; Tremetsberger
et al., 2004; Carretero, 2008). This closer relationship between
the southern Iberian and northwestern Maghreb species rather
than among north African populations provides further evidence of the presence during the Messinian of the Betic-Rifian
Massif which disappeared some 5.5 million years ago
(Krijgsman et al., 1999).
Unio durieui is phylogenetically closer to U. gibbus than the
other Unio species included in the analysis. This does not
support the relationship proposed by Haas (1969) between
U. durieui and the Mediterranean U. elongatulus group, since the
similarity of U. elongatulus to U. mancus (Araujo et al., 2005)
implies its position in the clade of U. ravoisieri, U. delphinus,
U. mancus and U. pictorum (Fig. 2). The Tunisian specimens of
U. gibbus clustered with the ones from the Iberian Peninsula
and Morocco (Fig. 2; Table 2). This is probably the most
common and widespread North African Unio species, inhabiting rivers from Morocco to Tunisia (Araujo et al., 2009b;
Khalloufi & Boumı̈za, 2009).
In P. littoralis, population differentiation between specimens
from different Tunisian rivers and specimens from the Iberian
Peninsula showed a mean divergence of 3.16% for COI. This
value is less than that between other pairs of species, and in
the absence of diagnostic morphological characters we propose
that, for now, these populations can be considered as belonging
to a single species. Unio fellmanni Deshayes, 1847 was based on
an Algerian specimen of Potomida littoralis. The P. littoralis
ACKNOWLEDGEMENTS
We thank Virginie Héros and Philippe Bouchet (Muséum
National d’Histoire Naturelle, Paris) and Yves Finet (Museum
d’Histoire Naturelle, Geneva) for the loan of specimens.
Ronald Janssen (Forschungsinstitut Senckenberg, Frankfurt)
provided us with photographs and the loan of material. We
also thank Jordi Ruiz Olmo (Servei de Protecció de la Fauna,
Departament de Medi Ambient i Habitatge, Generalitat
de Catalunya), Miquel Campos (Consorci de l’Estany de
Banyoles), Jordi Nebot and Francesc Uribe (Museu de
Ciencies Naturals de Barcelona) and J. Reis for their help with
collecting the specimens from Catalonia. The SEM
11
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Haas (1969) listed three other synonyms of U. durieui: U. ravoisieri
var. issericus Kobelt, 1884, U. tetuanensis Kobelt, 1884 and U. tafnanus Kobelt, 1884. Based on the figures of Rossmässler (1884) we
consider the first a synonym of U. ravoisieri and confirm the synonymy of the latter, but not of U. tetuanensis owing to its appearance and the absence of U. durieui in Morocco (personal
observation). It is likely that U. tetuanensis belongs to the Atlantic
species U. delphinus. Other names listed by Haas (1969) and
Daget (1998) as synonyms of U. durieui that cannot be assigned to
this taxon with certainty are U. sitifensis Morelet, 1852, U. delevieleusae Hagenmüller, 1887 and U. doumeti Bourguignat, 1887.
Finally, the names U. zenaticus and U. rouieri were synonymized
by Haas (1969) and ascribed to the genus Potomida.
The shell shape of U. ravoisieri may be similar to that of
U. pictorum and U. mancus, but its species status is quite clear.
The two U. ravoisieri groups observed here are considered to
represent divergent populations; divergence values between
them were no higher than 2.48% for the COI gene, considerably lower than those between U. ravoisieri and U. pictorum
(4.54 and 4.41%) and U. ravoisieri and U. mancus (4.84 and
5.89%) (Table 2).
Morphometric comparisons (data not shown) among
U. ravoisieri populations revealed large differences, even
between populations from the same area (the Ser and Banyoles
populations in Spain, or between Algeria and Tunisia in North
Africa). Given that these differences were not confirmed by the
molecular tests, we assume that they are related to morphological adaptation. For example, the distinctive shape of the
specimens from Banyoles Lake (Fig. 3E, F) may be related to
their lacustrine habitat, as in some other unionids (e.g.
U. tumidiformis from Ruidera Lakes; Reis & Araujo, 2009).
The L/W index was the macroscopic character that was best
able to separate U. ravoisieri from U. durieui, being mostly
greater than 2 in the former and lower than 2 in the latter.
The glochidia of U. ravoisieri can be distinguished from those of
U. durieui by the consistent presence of a hook, and by the
higher and less globose shape of the latter. Although a recent
report on the glochidium of U. gibbus by Araujo et al. (2009b)
described it as hookless, other specimens of this species may
have the normal Unio-type hooked glochidia (Fig. 6F, G, H)
(see also Khalloufi & Boumı̈za, 2009). The variability of this
character observed in some North African Unio species has not
been recorded before and warrants further study.
The period of glochidia release for U. ravoisieri in Tunisia
starts in March, but in Spain this seems to occur later (August,
September), while U. durieui appears to show two different
reproductive seasons (December and March).
N. KHALLOUFI ET AL.
photographs were prepared by Laura Tormo and Alberto
Jorge from the Museo Nacional de Ciencias Naturales
(Madrid). Other photographs were provided by Fernando
Señor and the plates were compiled by Jesús Muñoz, both
from the photography facility of the MNCN. Ana Burton
revised the English version of the manuscript. The comments
by three anonymous referees, D. Graf and D.G. Reid were also
very helpful.
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