Taxonomic research on Squalus megalops ( Macleay , 1881 ) and Squalus blainvillei ( Risso , 1827 ) ( Chondrichthyes : Squalidae ) in Tunisian waters ( central Mediterranean Sea )

1 Institut National des Sciences et Technologies de la Mer (centre de Sfax), B.P1035 Sfax 3018, Tunisia. E-mail: sondesmarouani@yahoo.fr 2 Faculté des Sciences de Sfax, B.P802 Sfax 3018, Tunisia. 3 Università di Pisa Dipartimento di Biologia Unità di Biologia Marina e Ecologia Via Derna 1, 56126 Pisa, Italy. 4 CSIRO Marine and Atmospheric Research, GPO Box 1538, Hobart, TAS, 7001, Australia. 5 Muséum national d’Histoire naturelle, Département Systématique et Evolution CP5155, rue Buffon, 75231 Paris cedex 05, France.


INTRODUCTION
The genus Squalus includes 25 species, 11 of which have been described recently from the Indo-West Pa-cific (Last et al. 2007a).In the Mediterranean Sea, two species occur commonly (Bradai et al. 2004, Serena et al. 2009): Squalus acanthias (Linnaeus, 1758) and Squalus blainvillei (Risso, 1827).However, a third species has been recorded as Squalus megalops by Muñoz-Chápuli et al. (1984) and Muñoz-Chápuli and Ramos (1989).Squalus megalops (Macleay, 1881) has been recorded from many localities of the eastern Atlantic and Indo-West Pacific (Compagno 2005), but Duffy and Last (2007) and Last and Stevens (1994) suggested that it may belong to a species complex, and stated that records of Squalus megalops outside Australia need to be confirmed.
Outside its main distributional area (Mediterranean Sea and eastern Atlantic), Squalus blainvillei has also been recorded erroneously off Australia and New Zealand (Garrick 1960).It was thought to be widespread in the Atlantic, Indian and Pacific Oceans (Bigelow andSchroeder 1948, 1957), as well as off Japan (Chen et al. 1979).Compagno et al. (2005) restricted the distribution of Squalus blainvillei to the Mediterranean Sea and eastern Atlantic, and questioned the Pacific records.The confusion is due largely to the poor original description given by Risso (1827) and the lack of type material.
The doubtful taxonomic status of the Tunisian dogfish led us to make a taxonomic study of these sharks, including both morphological and genetic analyses.The detailed descriptions provided here should allow the species to be clearly differentiated and contribute to a better understanding of the geographical distribution of Squalus megalops.

Morphological studies
The studied material consisted in 32 specimens of Squalus blainvillei (18 females ranging from 27 to 96 cm TL, and 14 males from 34 to 72 cm TL) and 26 specimens of Squalus megalops (14 females ranging from 33 to 70 cm TL, and 12 males from 35 to 53.5 cm TL).They were caught by commercial trawlers in the Gulf of Gabès (Fig. 1) between January 2007 and May 2009.
A subsample of 9 specimens of each species (S. blainvillei: 5 females ranging from 63.0 to 82.0 cm TL and 2 males from 66.5 to 68.5 cm TL as well as a 96.0 cm TL female (INSTM /SQUAL 02) and a 69.6 cm TL (INSTM /SQUAL 09) male; S. megalops: 6 females (INSTM /SQUAL 03-08) ranging from 33.0 to 69.5 cm TL and 3 males (INSTM /SQUAL 10-12) from 46.8 to 51.1 cm TL) were used for the morphometry study.All encoded samples were preserved whole in 10% formalin at the National Institute of Marine Sciences and Technology (Center of Sfax, Tunisia).The morphometric methodology followed Compagno (1984) and Last et al. (2007a), and measurements were expressed in a percentage of the total length (TL).Some measurements were provided as proportions to make comparison with previous studies possible.
The skeletal anatomy (neurocranium, clasper components) was studied using the rest of the sam-ples (23 specimens of S. blainvillei ranging from 27 to 71 cm TL, and 17 specimens of S. megalops ranging from 35 to 70 cm TL).They were dissected to reveal the structure of the chondrocrania and claspers and to count meristic characters.Chondrocranial measurements follow Muñoz-Chápuli and Ramos (1989) and were expressed as a percentage of the total length of the chondrocranium (TLC).Meristic characters were obtained separately for the trunk (monospondylous centra), precaudal (monospondylous + diplospondylous centra to origin of the caudal-fin upper lobe) and caudal (centra of the caudal fin) vertebrae.Tooth rows were counted directly on specimens by making incisions at the jaw angles to expose the teeth, and the tooth shape was noted.The terminal cartilages of claspers, called spurs and claws by Leigh-Sharpe (1920), were described and used to discriminate between the two species.Skin samples, observed by optic microscopy for characterising the dermal denticles, were obtained from the laterodorsal area, anterior to the first dorsal spine.

ISSR technique
The ISSR genetic analysis was performed on tissue samples of 7 specimens of S. blainvillei (4 females ranging from 36 to 64 cm TL and 3 males from 28.5 to 34.5 cm TL) and 7 specimens of S. megalops (4 females ranging from 50.1 to 68 cm TL and 3 males from 35 to 49.1 cm TL) from the dissected samples.
DNA inter-simple sequence repeat (ISSR) markers were introduced by Gupta et al. (1994) andZietkiewics et al. (1994).The ISSR technique is based on amplifying anonymous nuclear DNA sequences delimited by two inverted microsatellites.Using a single primer, composed of a short microsatellite sequence with one to four degenerate nucleotides anchored at the 5' or 3' end, allows several DNA regions to be amplified, which are treated as loci.In the present work, ISSRs were used to verify the presence of two species of the genus Squalus in the Gulf of Gabès.
Portions of the muscle were removed from each individual and stored in 95% ethanol at 4°C.For the DNA extraction, highly pure genomic DNA was isolated from approximately 25 mg of muscle tissue using the Speedtools tissue DNA extraction kit (Biotools B and M Lab.S.A. Spain) according to the manufacturer's instructions.Once the DNA had been extracted it was stored at 4°C until amplification through polymerase chain reaction (PCR).
We used eight primers provided by Operon Molecules for Life (Table 1).The PCR reaction mixture contained up to 30 mg of genomic DNA, 2.5 mM Mg Cl2, 0.2 µM primer, 200 µM of each dNTP, 0.5 U of Taq DNA polymerase and 10X reaction buffer in a final volume of 25 µl.The amplification program was 3 min at 94°C, with the following cycle repeated 45 times: 40 s at 94°C, 45 s at 55°C and 1 min 40 s at 72°C; finally 5 min at 72°C.In order to exclude PCR artefacts and verify the repeatability of the results, a negative control and replicates were included.After separation by electrophoresis on a 2% agarose gel, the PCR products were stained with ethidium bromide.One hundred base pair ladders (100 bp DNA ladder, Promega) were used for reference with each primer.
For the ISSR analysis, based on the total band presence/absence, we obtained a triangular matrix of Nei (1978) inter-individual genetic distance.UPGMA cluster analysis was carried out using the program TFPGA (Miller, 1997).The nodes of the dendrogram were tested by bootstrapping with 10000 replicates.

Molecular barcoding methods
For this study we used a specimen of each species (a 96 cm TL female S. blainvillei and a 69.5 cm TL female S. megalops) from the conserved individuals used for morphometric analysis.White tissue samples were preserved in 95% ethanol.DNA was extracted using a method outlined by Ward et al. (2007) for barcoding Australian chondrichthyans.The Barcoding analysis was mainly used to compare the Tunisian specimen of S. megalops with specimens from Australia.The analysis was performed with CSIRO and the sequences of the two Tunisian specimens were inserted in the results already obtained by Ward et al. (2007) for discriminating spurdogs of the genus Squalus.

Description of the Tunisian specimens of Squalus blainvillei
Table 2 shows the proportional measurements of the Tunisian specimens of S. blainvillei and Table 3 the ratios between some of these measurements.Table 4 provides the chondrocranial measurements of these specimens.
Coloration: When fresh, uniform grey brown dorsally, white below; dorsal and caudal fins grey, first dorsal-fin anterior base and free rear tip paler than rest of fin, second dorsal-fin anterior base not darker than rest of fin, dorsal-fin tips and upper posterior margin with narrow black margin; pectoral, pelvic and caudal fins grey with white posterior margins and tips; naked axils of fins and pectoral origin dusky; eyes bright green in life.

Description of the Tunisian specimens of Squalus megalops
The proportional measurements of the Tunisian specimens of S. megalops and the ratios between some of these measurements are given in Tables 5 and 3 respectively.Chondrocranial measurements are shown in Table 4.
S. megalops is a moderate-sized species of Squalus of the "megalops-cubensis" group: the maximum TLs of the specimens observed in Tunisia were 74.2 and 72.0 cm for females and males respectively.
Coloration: When fresh, uniform grey brown dorsally, white below; dorsal and caudal fins grey, first dorsal-fin anterior base and free rear tip paler than rest of fin; dorsal fins apex dusky; pectoral, pelvic and caudal fins grey with white posterior margins and tips; naked axils of fins and pectoral origin dusky; eyes bright green in life.
Table 3 summarises the main differences between the two Squalus species occurring in Tunisian waters.The external characters listed in this table are sufficient for field identification of these commercial species.

ISSR method
The ISSR molecular analysis separated the Tunisian specimens into two clusters (species).The ISSR profiles were characterised by 71 polymorphic loci.The amplified DNA fragment lengths (for all primers) were approximately from 247 to 1922 pb.Maltagliati et al. (2005) defined a locus as ''fully diagnostic'' when it produced bands in all individuals of one species and not in the other, and ''nearly diagnostic'' when it pro- duced species-specific bands in some individuals (but not all) of one species and not in the other species.We detected 2 fully diagnostic and 9 nearly diagnostic loci in the presumed species S. blainvillei and only 6 nearly diagnostic loci in the second species S. megalops.The UPGMA dendrogram of Rogers and Tanimoto's (1960) dissimilarities was characterised by high bootstrap support and clearly separated the studied individuals into two clusters (Fig. 9).

Molecular barcoding method
Figures 10a and 10b provide the topology of the neighbour-joining K2P tree for the sequences analysed by Ward et al. (2007), among which we inserted the sequences of the Tunisian samples.The sequence of the Tunisian specimen of S. blainvillei appears as a separate cluster set apart from the Australasian Squalus species (Fig. 10a), whereas the Tunisian specimen of S. megalops is included in the S. megalops cluster (Fig. 10b), which indicates that it has similarities with the Australian S. megalops.

DISCUSSION
Morphological and biological similarities among squalids have led to considerable confusion over their taxonomy (Myakov and Kondyurin, 1986).The taxonomic status of S. blainvillei is problematical as there are no extant types and the description and figures of Risso (1827) do not correspond to any known species of Squalus (Chen et al. 1979, Muñoz-Chápuli andRamos 1989).This led Garrick (1960), in a review of the Australian species of Squalus, to incorrectly synonymise S. griffini and S. fernandinus (Molina, 1782) with S. blainvillei.However, in a review of Japanese Squalus, Chen et al. (1979) defined S. blainvillei as a species with high dorsal fins and long dorsal-fin spines based on their examination of Japanese material and descriptions of S. blainvillei from its type locality, the northern Mediterranean.They observed that Squalus, referring to S. fernandinus and S. blainvillei by Bigelow and Schroeder (1948) and Garrick (1960), had short dorsal-fin spines and were more similar to S. mitsukurii from Japan, and suggested that nominal S. blainvillei from New Zealand could be identical to S. mitsukuri.Compagno (1984) also noted that dogfish resembling S. mitsukurii occurred off Australia and New Zealand, and he did not recognise S. blainvillei from the Southern Hemisphere.
Actually, both S. blainvillei and S. megalops were identified from the Mediterranean Sea based on morphometry in a previous study (Muñoz-Chápuli and Ramos 1989), but the occurrence of S. megalops in the region has been questioned by other authors (Last and Stevens 1994).Thus, we expanded this study by including other morphometric characters and a molecular study to confirm that S. megalops occurs as a valid species in the Mediterranean Sea.
The Tunisian Squalus megalops species are consistent for characters typifying the "megalops-cubensis" group and fit the description of S. megalops from Australian waters (Last et al. 2007b), as well as the eastern Atlantic-Mediterranean (Muñoz-Chápuli and Ramos 1989) and Mediterranean waters (Muñoz-Chápuli et al. 1984).Its presence in the Mediterranean Sea has been considered doubtful by many authors.In addition, Last and Stevens (1994) suggested that the southern Fig. 10.-A, simplified tree of Squalus spp.analysis (on this simplified tree, the branches are not proportional to the distance between groups).B, Squalus megalops grouping extract from the general analysis of Squalus spp.(on this simplified tree, the branches are not proportional to the distance between groups).
Australian S. megalops could be distinct from megalops like spurdogs in other parts of the world, and that S. megalops might be endemic to Australia.However, the story may be even more complicated.Recent morphological studies have shown that more than a single form of this species exists in Australian seas (Last, unpublished data).Specimens described from other areas clearly agree with our Tunisian samples of S. megalops for most of the morphometric characters (Table 5).However, there are some morphometric differences: the Australian S. megalops studied by Last et al. (2007b) has a larger internarial space 4.5 (4.3-4.7)versus 3.9 (3.7-4.1)%TL, a larger eye 5.0 (4.9-5.0)versus 4.1 (3.5-4.8)%TL, a longer second dorsal fin 12.0 (11.0-12.7)versus 10.2 (9.1-11.6)%TL; a longer anterior margin and base second dorsal fin 10.1 (9.4-10.6)versus 8.2 (7.4-9.3)%TL and 7.1 (6.4-5.2) versus 5.2 (4.6-6.1)%TLrespectively, and a higher pelvic fin 4.8 (4.3-5.2) versus 2.9 Eastern Atlantic-Mediterranean specimens have a higher first dorsal fin 8.5±0.8 versus 6.0 (±0.2 / 5.6-6.5)%TL and a longer pectoral-fin inner margin 9.3±0.7 versus 7.4 (6.6-8.2 / ±0.2)%TL.The Mediterranean S. megalops studied by Muñoz-Chápuli et al. (1984) also had a longer pectoral-fin inner margin 9.2±0.6 versus 7.4 (6.6-8.2 / ±0.2)%TL.Tunisian specimens of Squalus megalops had similar vertebral counts to those studied by Springer and Garrick (1964) (Indo-Pacific), Bass et al. (1976) (South Africa), Muñoz-Chápuli et al. (1984) (Mediterranean coasts of Spain), Muñoz-Chápuli and Ramos (1989) (east Atlantic) and Last et al. (2007b) (south Western Australia, Queensland).They had a low number of monospondylous centra (37), typical of the Squalus megalops-cubensis group (Muñoz-Chápuli and Ramos, 1989), and a relatively high number of precaudal centra (81), which is somewhat fewer than in S. blainvillei (Table 6).Ledoux (1970) and Merret (1973) pointed out a close similarity between S. blainvillei and S. megalops, when compared with S. acanthias and S. asper.Moreover, the relationships between the snout tip and nostril distance and the distance from the nostril to the preoral clefs, which were proposed by Bass et al. (1976) as the best features for discriminating between species of the genus Squalus, proved to be of little use for discriminating our material.However, the differences in morphometrics observed in our study allow these two species to be differentiated.In addition, the presence of two lateral processes on each side of the basal plate in S. megalops (only one in S. blainvillei) also allows the two species to be discriminated.The chondrocranium of S. megalops has a narrower interorbital distance and smaller olfactory capsules than that of S. blainvillei.
Molecular analysis (ISSR and barcoding methods) evidenced the non-conspecificity of these species and the similarity between the Tunisian Squalus megalops and the "true" Australian S. megalops.

Fig. 6 .
Fig. 6. -Dissected chondrocranium of (a) S. blainvillei, adult male 780 mm TL, showing the single lateral process of the basal plate and (b) S. megalops, adult female 714 mm TL, showing the two lateral processes of the basal plate.

Fig. 9 .
Fig. 9. -UPGMA consensus dendrogram of Rogers and Tanimoto's (1960) dissimilarity among individuals analysed.Bootstrap values on branching points represent the number of times (in percentage) a particular cluster group was formed out of 10000 iterations.

Table 1 .
-Primer sequences used in the ISSR analysis, number of polymorphic loci per primer and range of band molecular weights in base pairs (bp) amplified by PCR-ISSR for the 14 individuals of Squalus analysed.

Table 2 (
Cont.).-Proportional dimensions as percentages (±SD) of total length for specimens of Squalus blainvillei from southern Tunisia, eastern Atlantic-Mediterranean, New Zealand, equatorial, western Indian Ocean and Mediterranean coasts of Spain.

Table 4 .
-Chondrocranial measurements of Squalus blainvillei and Squalus megalops from the Gulf of Gabès, and comparison through a t-Student test (p<0.05;bold values: statistically different).

Table 5
Mean Min.Max.Mean Min.Max.Mean Min.Max.Mean Min.Max.
. -Proportional dimensions as percentages of total length for specimens of Squalus megalops from southern Tunisia, southern Australia, Qeensland, Western Australia, eastern Atlantic-Mediterranean and Spanish waters.Reference / area Present study Last et al. (2007) Last et al. (2007) Last et al. (2007) Chápuli and Chápuli et al.

Table 5 (
Cont.).-Proportional dimensions as percentages of total length for specimens of Squalus megalops from southern Tunisia, southern Australia, Qeensland, Western Australia, eastern Atlantic-Mediterranean and Spanish waters.