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Molecular phylogeny and biometrics of lessepsian puffer fish Lagocephalus sceleratus (Gmelin, 1789) from Mediterranean and Red Seas, Egypt
Egyptian Journal of Aquatic Research (2015) 41, 323–335
H O S T E D BY
National Institute of Oceanography and Fisheries
Egyptian Journal of Aquatic Research http://ees.elsevier.com/ejar www.sciencedirect.com
FULL LENGTH ARTICLE
Molecular phylogeny and biometrics of lessepsian puffer fish Lagocephalus sceleratus (Gmelin, 1789) from Mediterranean and Red Seas, Egypt Mahmoud M.S. Farrag a,*, Taha B.H. Soliman c, El-Sayed Kh.A. Akel c, Alaa A.K. Elhaweet b, Mohsen A. Moustafa a a
Faculty of Science, Al-Azhar University (Assiut Branch), Egypt College of Fisheries Technology and Aquaculture, Arab Academy for Science, Technology and Maritime Transportation, Alexandria, Egypt c National Institute of Oceanography & Fisheries (Alexandria), Egypt b
Received 16 May 2015; revised 26 August 2015; accepted 26 August 2015 Available online 26 September 2015
KEYWORDS Lagocephalus sceleratus; Biometric; Phylogeny; Mediterranean and Red Seas; Egypt
Abstract Phylogenetic comparison for lessepsian puffer fish Lagocephalus sceleratus (Gmelin, 1789) from the Egyptian waters (Mediterranean and Red Seas) was performed using morphological and genetic characteristics to confirm its identification and to recognize any variations due to differences in both habitats. A similar identity in body description from both habitats was observed. Slight phenotyping variations in some morphological measurements were observed with no significant difference. The correlation between the reference length and most of morphological indices was similar (0.99) for both habitats. Molecular phylogeny, using two primers (16S rRNA and Cytochrome b) showed similar size bands at 600 bp and 400 bp ladders for both primers respectively in both habitats. The obtained sequences were aligned showing an identity (more than 99%) to the published sequences of L. sceleratus indicating high genetic resemblance. So, the present populations from both habitats don’t appear to be separated species, indicating no obvious genetic variations after its migration into new habitat, expecting that, genetic differentiation may occur very slowly. This supported the idea that both morphological and genetic characteristics are recommended to be used together in identification and phylogenetic relations of the population from two habitats. The specific primers 16S rRNA and Cyt b showed confirmable tools to support the same target where the environmental variations may alter phenotypic variations. Ó 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
* Corresponding author at: Department of Zoology, Faculty of Science, Al-Azhar University (Assiut Branch), 71524 Assiut, Egypt. Tel.: +20 1007253531. E-mail address: [email protected] (M.M.S. Farrag). Peer review under responsibility of National Institute of Oceanography and Fisheries. http://dx.doi.org/10.1016/j.ejar.2015.08.001 1687-4285 Ó 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Introduction Pufferfish Lagocephalus sceleratus (Gmelin, 1789) has received considerable public attention after its migration from Red Sea via Suez Canal, since its rapid distribution in different countries of Mediterranean Basin, with social impacts (Farrag, 2014). It originally has expanded its distribution widely from Indo-Pacific regions (Smith and Heemstra, 1986). This species is the common and largest distributed one among puffer fish species in the Egyptian Mediterranean waters (Farrag, 2014). The invasion of species to Mediterranean Basin has become an ecological and economic issue which must be addressed, studied and monitored (Bariche et al., 2004; IUCN, 2008; Oral, 2010). Since its moving from a certain habitat to a different one, the environmental differences in the habitats may lead to the detection of morphological differentiation by phenotypic markers, which may be a practical level of partitioning among self-recruiting stocks (Turan, 1999). So, stock identification is very important, hence it is a multidisciplinary field of fisheries science, biometrics, genetics and life history studies (ICES, 1996; Pawson and Jennings, 1996; Begg et al., 1999). Hence, it is necessary to identify specimens correctly to investigate biological traits as growth, mortality, fecundity, trophic relation, parasite relationship, historical and paleontological events (Ibanez et al., 2007). Moreover, measures of gene flow and genetic differentiation are typically used to distinguish one population from another allowing a better analysis of fish species (Omer and Abukashwa, 2012). So, this work aims to confirm the identification and compare between the L. sceleratus population from two water bodies (The Mediterranean Sea as a new habitat and The Red Sea as the origin one) using the combination of biometric and genetic characteristics. Materials and methods This study was constructed on phylogenetic comparison using both biometric and genetic characteristics of puffer fish L. sceleratus from two water bodies (The Mediterranean Sea, Egypt as new habitat) and The Red Sea (Hurghada) as the origin of its migration (Fig. 1). Biometric characteristics
Figure 1 The study area along the Egyptian Mediterranean coast and fishing ports {(1) Port Said; (2) Ezbet-ElBorg (Damietta); (3) El-Burullus; (4) El-Maadia; (5) Abu-Qir; (6) Alexandria (Anfoushy)} and (7) Hurghada, Red Sea.
of these indices and their standard deviation. The meristic characteristics were counted as: Dorsal fin rays, anal rays, pectoral fin rays, caudal fin rays and number of vertebrae (Templeman, 1970). For genetic studies DNA was isolated from the muscle tissues that preserved in 99% ethanol using modified protocol of phenol- chloroform (Sambrook and Russell, 2001). The isolated DNA was diluted and stored at 20 °C until used. PCR amplification and genotyping
The biometric characteristics (Morphometric and meristic) were carried out at the laboratory on 44 and 35 fresh specimens from Red Sea and Mediterranean Sea, respectively having the same length range to get more accuracy of results and to avoid the errors due to variations in length; the length measurements were taken to the nearest 0.1 cm of each fish; then pooled monthly and analyzed keeping the size interval as 1 cm. The morphometric measurements were taken for each species (Fig. 2). The head length (H.L) was used as a reference for pre orbital length (Pre O. L), eye width (E.W) and inner orbital width (I. Orbit), while the total body length (T.L) was used as a reference for Standard length (S.L.), Forked length (F.L.), Pre dorsal length (Pre D. L.), Post dorsal length (Post D. L.), Pre pectoral length (Pre P.L.), Pre anal length (Pre A.L.), Max. body depth (Max. B. D), Head depth (H. D). Then the ratio index for each characteristic (Morphometric measurements / relevant reference 100) was calculated, the different length groups were pooled to calculate mean values
Partial region of the universal primers 16S rRNA with region 600 bp (16Sar, 50 -CGCCTGTTTATCAAAAACAT-30 and 16Sbr, 50 CCGGTCGAAACTCAGATCACGT-30 ) (Palumbi et al., 1991), and Cyt b with region 400 bp (F: 50 -CTACG CAAAACGCACCCAC-30 and R: 50 GGGGAGGACGTAG CCCACAAAG-30 ) were amplified according to Meyer (1994). PCR amplification for 21 individuals from Mediterranean Sea and 18 individuals from Red Sea using 16S rRNA primer was done at the lab of Graduate School of Science and Engineering for Research, University of Toyama, Japan. Due to the poor preservation of specimens from Red Sea, only specimens from Mediterranean Sea were sequenced. So, the other six specimens of puffer fish L. sceleratus (3 from Mediterranean Sea and 3 from Red Sea, Egypt) have used the second primer (Cytochrome b) at the lab of biotechnology, Faculty of veterinary Medicine, Kafr El-Sheikh University, Egypt in order to give more of results about the present comparison. PCR amplification for both primers was performed in
Molecular phylogeny and biometrics of lessepsian puffer fish
Figure 2
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Lagocephalus sceleratus showing morphometric measurements.
15-ll reaction volumes, 100 ng/ll of DNA template, 1.2 ll dNTP mixture 2.5 mM each, 1.5 ll 10 Ex Taq buffer (Takara), 1.5 ll of each primer (5 mM), 1 U of Taq polymerase, and purified water to complete the final volume. Thermal cycler conditions consisted of an initial denaturation step at 94 °C for 1 min, 35 cycles of 15 s at 94 °C for annealing, 15 s at 50 °C, and 30 s at 72 °C, with a final extension step of 7 min at 72 °C, and cooling to 4 °C until the PCR products were removed from the thermocycler. A 4-ll sample of each PCR product was run on 1.5% agarose gel. A 11 ll volume of each amplified product was purified using ExoSAP-IT (Amersham Pharmacia Biotech) and, labeled using Big Dye Terminator V.3.1 Cycle sequencing Kit. The purified samples were sent to Macogen Inc., (South Korea) to sequence in both directions by ABI 3730XL DNA sequence (Applied Biosystem, USA). Data analysis Geneious 4.8.4 software was used for sequencing annotation and assembly. The obtained sequences were aligned by Clustal W program software version 2.1 provided by DNA Data Bank of Japan (DDBJ) (http://clustalw.ddbj.nig.ac.jp/index.php) (Larkin et al., 2007) and submitted to Gen Bank database. The neighbor-joining phylogenetic trees of Cyt b and 16S rRNA genes were conducted by MEGA software version 6 (Tamura et al., 2013). Results Morphological comparison The morphological description of L. sceleratus (Gmelin, 1789) from two water bodies (Mediterranean and Red Seas) were similar whereas the body is elongated, somewhat compressed laterally and inflatable; color is gray to greenish with dorsal regular black spots; the small spinules on the dorsal surface
extending to origin of the dorsal fin, while the small spinules ventrally do not reach the cloacae opening. A wide silver band is located on the lower parts of the flanks from the mouth to the caudal fin with silver blotch in front and below the eye. The pectoral fin base is black, it has wide based, and has a round posterior edge and the caudal fin is lunate (Fig. 2). The biometric indices (Table 1) for the same population from two habitats were slightly similar at the same length range (19.9–55.5 cm) for indices of S.L/T.L (%); F.L/T.L (%); Pre D. L./T.L (%); Post D. L/T.L (%); Pre P. L./T.L (%); Pre A. L./T.L (%); E.W./H.L (%), these indices were relevant to total length as reference. However, slightly variations were observed for some indices (Max. B. D./T.L (%); Min. B. D./T.L (%); HL/T.L (%)) where these indices for Red Sea were 13.28, 3.59, 12.24, 31.42 higher than those of Mediterranean Sea species. While the indices of Pre nostril/H.L (%), Pre orbit/H.L (%), I. O/H.L (%) showed an inverse trend of variations giving values of Mediterranean Sea higher than those of Red Sea and most of these indices are proportional to the head length. The correlation coefficients (Fig. 3) of S.L, F.L, Pre D. L, Post D.L. and Pre A.L. gave a similar value (0.990) for both Mediterranean Sea and Red Sea species. The Max. B.D., Min. B.D., Pre P.L., H.D, Pre O., Post O. and Inner O. for L. sceleratus from Mediterranean Sea were 0.97, 0.98, 0.99, 0.97, 0.99, 0.97 and 0.96 respectively; these values were higher than those of Red Sea which were 0.94, 0.78, 0.91, 0.96, 0.94, 0.96 and 0.94 for the same parameters respectively. The prenostril value 0.97 in Mediterranean was less than that of Red Sea 0.99. Eye width parameters gave the same correlation value (0.88) for both habitats. Generally, insignificant difference between biometric indices of L. sceleratus from two habitats was observed at p P 0.95. Moreover, both of the two populations of L. sceleratus from two habitats showed nearly the same range of meristic characteristic as given in Table 1. For Mediterranean waters, the dorsal fin rays was X + (10–12), the anal fin rays was
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Table 1
Comparison in indices of biometric characteristics of L. sceleratus from the Mediterranean and Red Seas, Egypt.
Species
L. sceleratus (Mediterranean Sea)
L. sceleratus (Red Sea)
Length range
20–55 (cm) (T.L)
20–55 (cm) (T.L)
No. specimens
35
Indices/values
Min.
Max.
Av.
±SD
Min.
Max.
Av.
±SD
T.L (cm) S.L/T.L (%) F.L/T.L (%) Pre D.L/T.L (%) Post D.L/T.L (%) Pre P.L/T.L (%) Pre A.L./T.L (%) H.D/H.L (%) Max. B. D/T.L (%) Min. B. D/T.L (%) Pre nostril/H.L (%) Pre Orbit/H.L (%) Inner O./H.L (%) E. W/H.L (%) H.L/T.L (%) No. D.F. rays No. A.F. rays No. P.F. rays No. C.F. rays No. V
19.9 83.23 91.67 50.18 60.12 25.05 53.85 35.71 10.42 2.51 25.33 40.00 29.41 18.06 25.35 10–12 10–11 16–18 16–19 16–17
55 85.45 94.52 57.54 64.51 27.96 57.93 52.42 15.44 3.33 35.54 56.61 46.81 31.43 31.99
39.58 84.32 93.17 55.54 62.53 26.12 56.46 46.19 12.89 2.95 32.32 50.51 38.69 22.84 27.84
13.25 0.75 0.79 1.74 1.42 0.72 1.10 4.89 1.11 0.22 3.09 6.26 6.41 3.35 2.12
20.00 81.98 90.74 53.33 58.89 19.44 53.70 38.73 11.51 2.59 24.62 39.35 28.46 17.26 28.70 11–12 10–12 16–18 17–19 16–17
55.50 86.63 97.16 59.56 65.44 28.25 60.41 47.41 15.41 4.89 31.61 49.08 39.88 29.85 33.48
39.30 84.20 93.49 57.22 63.03 25.62 57.87 42.64 13.28 3.59 28.63 44.27 34.23 22.53 31.42
13.29 1.01 1.55 1.71 1.55 2.50 1.87 2.61 1.04 0.67 2.10 3.04 2.95 3.45 1.20
Figure 3
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Biometric characteristics indices of L. sceleratus from the Egyptian Mediterranean Sea and Red Seas, Egypt.
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Figure 3 (continued)
X + (10–11), the pectoral fin rays was X + (16–18) and the caudal fin rays was X + (16–19). For the Red Sea species, the dorsal fin rays was X + (11–12), the anal fin rays was X + (10–12), the pectoral fin rays was X + (16–18) and the caudal fin rays was X + (17–19). Number of vertebrae of two populations from two habitats ranged from 16 to 17+ urostyle and post cranial vertebra in all fish examined showing no appreciable variation between the populations. Molecular identification and phylogenetic analyses The task of molecular identification and phylogenetic comparison was conducted using different two primers (16S rRNA and cytochrome b) through the following processes. Amplification of 16S rRNA (600 bp) and cytochrome b (400 bp) The obtained results of the present molecular profiles of L. sceleratus from both habitats (Mediterranean and Red Seas) showed similarity to the fragments of size bands at 600 bp and 400 bp of ladders giving the same band size in the right expected molecular weight of both 16S rRNA and cytochrome b genes respectively (Fig. 4A, B). The PCR products were sequenced only for Mediterranean Sea specimens and the sequences were similar, a single representative sequence of all
obtained sequences was uploaded to Genbank under the accession number KF938589. This sequence was aligned and conferred with previously published sequences that used the same primer for the same population of L. sceleratus from different areas and showed high level of identity (Fig. 5); the identity was 100% with accession numbers of AB194240 and AB742031 from Japan by Ishizaki et al. (2006) and Igarashi et al. (unpublished) respectively, while it was 98% with KC862306 from Turkey that constituted by Tuney (2016). Likewise, the PCR products obtained from the amplification of the second primer Cytochrome b gave also the same band size and expected molecular weight for all specimens from two locations (Fig. 6). The PCR products from both habitats were sequenced and they were similar; one representative sequence from each habitat was uploaded on the Genebank under accession numbers of KF031124 (Red Sea), and KF031125 (Mediterranean Sea). These sequences were aligned with those whose amplified partial mitochondrial Cyt b for the same population from different localities (Fig. 7); the alignment of present Red Sea sequence has showed a high level of identity (99%) with JQ681894 from USA; was 99% for AY267356 from Taiwan. The identity of KF031125 (Mediterranean Sea, Egypt, present work) was 99% with the sequence (KF031124) from Red Sea, Egypt, present work), while it was 100% with EF362414 from Greece water, Mediterranean Sea.
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Figure 3 (continued)
The phylogenetic relations with the same population and others The phylogenetic tree based on the distance analysis of sequences generated using Neighbor-Joining (NJ) tree (MEGA software with 500 bootstrap replicates) illustrated that, the phylogenetic relation with the same population from different locations used 16S rRNA sequence has produced two clusters or clades (Fig. 8A). One such clade contains KC862306 from Turkey with the identity of bootstrap 98%, while the second clade has two subclades of present sequence (KF938589) from Mediterranean Sea, Egypt) in one, while the second subclade represented by sister group contains two sequences (AB194240 and AB742031) from Japan with bootstrap 100%. Moreover, the phylogenetic tree of L. sceleratus with other seven different pufferfish species under the same genus Lagocephalus using the same primer (16S rRNA) has produced two primary clades (Fig. 8B); one of them consists of two groups having L. sceleratus in one group while the other group contained L. inermis as a sister group to L. laevigatus, L. wheeleri, L. lagocephalus and L. gloveri. The second clade was represented only by L. lunaris. For the second primer (Cyt b), the phylogenetic relation with the same population from different locations has produced two clusters (Fig. 9A). The first one contains JQ681894 from USA with bootstrap of an identity (99%),
and AY267356 99% from Taiwan, while the second cluster has two subclusters forming sister groups. One of these subclusters contains KF031124 from Egypt (Red Sea), and second subcluster contains both the present sequence KF031125 from Egypt (Mediterranean Sea) and EF362414 from Greece (Mediterranean Sea). The identity of bootstrap of KF031125 (Mediterranean Sea, Egypt) with that of KF031124 (Red Sea, Egypt) was 99% and 100% for EF362414 from Greece (Mediterranean Sea). Moreover, its phylogenetic tree with other different nine pufferfish species using Cyt b gene has produced two main clusters (Fig. 9B); one of them contains L. sceleratus and L. suezensis as two subclusters, while the second cluster in phylogenetic tree has contained L. gloveri, L. guentheri, L. lagocephalus, L. laevigatus, L. lunaris, L. inermis and L. wheeleri together as a sister groups. Discussion The objective of a racial study was to establish, with some degree of confidence, the taxonomic identity of the common puffer fish species L. sceleratus from different water bodies (Mediterranean and Red Seas). Interactive effects of environment produce morphometric differences within a species, the variability in growth, development, and maturation creates a
Molecular phylogeny and biometrics of lessepsian puffer fish
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Figure 3 (continued)
variety of body shapes within a species (ICES, 1996; Pawson and Jennings, 1996; Begg et al., 1999). Since the morphometric characteristics are sensitive to any environmental changes, the genetic differentiation was used to confirm the identification and identity of such species from two Water bodies. The present description and morphology of L. sceleratus from Mediterranean and Red Seas showed an agreement and similarity to its congeneric tropical species Mozambique and southern African shores, as well as the Red Sea (Smith and Heemstra, 1986; Ni and Kwon, 1999; Froese and Pauly, 2003). Moreover, they were similar to those studied by Akyol et al. (2005); Bilecenoglu et al. (2006); Kasapidis et al. (2007); Golani et al. (2011); Jribi and Bradai (2012) and Milazzo et al. (2012) and this confirms its identification.
The comparison between the immigrant L. sceleratus from Mediterranean Sea and the same population from Red Sea is very important, since this species represented the common and largest one of puffer fish species in the Egyptian Mediterranean waters and needs to be studied more (Farrag, 2014). The biometric comparison has used the same length range for specimens from both locations to avoid the size effects (even with the presence of gap in length range of one population, the author removed the same gap in the second population to get more accuracy of results). The insignificance for the mean of different biometric indices of two populations was detected. Moreover most indices (morphometric and meristic counts) showed a high similarity between different locations reflecting no change in its identification and taxo-
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Figure 4 Gel electrophoresis of 16S rRNA PCR product (600 bp) for: (A) 21 specimens of L. sceleratus from Mediterranean Sea (1–21) and (B) eighteen specimens of L. sceleratus from Red Sea (Lanes 1–18); M-200 bp DNA ladder.
nomic classification. These findings may be attributed to many reasons as the same length range used from both habitats. Moreover, the ecological conditions such as salinity and temperature in eastern Mediterranean became similar to those of the Red Sea (Avsar, 1999), the mean salinity of Eastern Mediterranean is 38.5%, and it may rise to 39.5% in some areas such as Iskenderun Bay while the salinity in the Suez Bay ranged between 33.5% and 40.3%. On the other hand, TUDAV (2006) claimed that global warming has increased
M.M.S. Farrag et al. the temperature of the Mediterranean facilitating the establishment of lessepsian species in the Eastern Mediterranean. So, the similar conditions may refer to slowly/not changing morphological measurements, in addition to that the similarity may be taken in further consideration to a change in the genetic characteristics. However, slight variations in some indices as Max. B.D/T.L (%); Min. B.D/T.L (%) and Pre. O/H.L (%) were observed, this may be attributed to variations in ecological and geographical variations of different areas. Huet (1949) found variations in fin rays of the salt water and fresh water races of sticklebacks in relation to both temperature and salinity. The width index of the eye of L. sceleratus from the Mediterranean Sea was somewhat larger than that of Red Sea and this could be related to the higher turbidity of the waters of the Mediterranean Sea. Similar results were found in the rainbow trout Oncorhynchus mykiss (Pankhurst and Montgomery, 1994) and Mugil curema from the Atlantic (Ibanez-Aguirre et al., 2006). In some cases, the body width can be strongly influenced by sexual maturation and the preservation quality of fish specimen (Ibanez-Aguirre et al., 2006). The observed differences in Max. B.D/T.L (%); Min. B.D/T.L (%); Pre. O/H.L (%) in the present work may be attributed to sexual maturation of the examined specimens and fullness of stomach as well as the inflating of the body especially for puffer fish that can inflate itself. On the other hand, the differences in body shape and measurements could be due to the used methods as reported by Corti and Crosetti (1996) and Mamuris et al. (1998). These findings can be seen in comparison between the present results and others where the scientists measured the head length from the beginning of the snout to the end eye (Jribi and Bradai, 2012); the present measure of head length was from the beginning of the snout to the end of real skull that located behind the pectoral fine. Even, while using the same method to measure head length, the indices of head length varied between two habitats causing slight changes in different characteristics which used head length as a reference. These variations may be attributed to the previously mentioned reasons as well as food contents which can appear as variations in the ventral part of the head of puffer fish. So, the slight variations between two populations from Red Sea
Figure 5 Gel electrophoresis of Cyt b PCR product (400 bp), each specimen of L. sceleratus was run in two lanes: A, B and C from Mediterranean Sea (Left side), while D, E and F were from Red Sea (Right side); M-100 bp DNA ladder.
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Figure 6 Aligned DNA sequences using CLUSTALW 2.1 of the amplified 16S rRNA region of mitochondrial DNA from puffer fish from different locations: KF938589, Egypt; AB194240-AB742031, Japan; KC862306, Turkey. Asterisk (*) indicates identity with Lagocephalus sceleratus sequences. A gap introduced into the sequences to optimize the alignment is represented by a dash (-).
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Figure 7 Aligned DNA sequences using CLUSTALW 2.1 of the amplified Cyt b gene of mitochondrial DNA for puffer fish from different locations: EF362414.1, Greece; KF031124.1-KF031125.1, Egypt; JQ681894.1, USA; AY267356.1, Taiwan. Asterisk (*) indicates identity with L. sceleratus sequences. A gap introduced into the sequences to optimize the alignment is represented by a dash (-).
and Mediterranean Sea in the present work are not enough to discriminate groups to be a separate species. The gene frequency was utilized in fish molecular studies in a wide range to detect the taxonomy and systematic status of the species due to its conservative properties (Gilles et al., 2001 and Li et al., 2008). Genetic differentiation and phylogenetic analysis of L. sceleratus in Egypt did not gain sufficient attention. So, genetic differentiation was used in combination with biometric characteristics in the present work to confirm the identification of L. sceleratus and its identity with the same population from other locations and its phylogenetic trees. The two genes/primers (16S rRNA and Cyt b) are famous in the field of taxonomy, diversity and phylogenetic relationship at
various taxonomic levels were used by various authors (Gilles et al., 1998; Nguyen et al., 2006; Li et al., 2008 and Faddagh et al., 2012). The present gel electrophoresis profiles for both genes (16S rRNA and Cyt b) for L. sceleratus from both habitats showed similar fragments of molecular size bands at 600 bp and 400 bp ladders in both 16S rRNA and cytochrome b genes respectively. This may reflects the genetic similarity of two populations from Mediterranean Sea and Red Sea which is the origin place of migration. The present sequence of 16S rRNA (KF938589) from Mediterranean Sea was aligned with other sequences of L. sceleratus from different locations; the identity was 100% with those sequences (AB194240 and AB742031) from Japan by
Molecular phylogeny and biometrics of lessepsian puffer fish
Figure 8a Neighbor-Joining tree inferred from Tamura-Nei distances between sequences of 16S rRNA gene in L. sceleratus and the same species from different locations.
Figure 8b Neighbor-Joining distance tree between sequences of 16S rRNA gene of L. sceleratus and other different pufferfish (Genus: Lagocephalus) obtained from NCBI GenBank (http:// www.ncbi.nlm.nih.gov).
Figure 9a Neighbor-Joining tree inferred from Tamura-Nei distances between sequences of Cyt b gene in L. sceleratus and the same species from five different locations.
Figure 9b Neighbor-Joining distance tree for the Cyt b gene sequence data of L. sceleratus and other different pufferfish (Genus: Lagocephalus) obtained from NCBI GenBank (http:// www.ncbi.nlm.nih.gov).
Ishizaki et al. (2006) and Igarashi, et al. (unpublished) respectively. This finding has reflected the confirmation of its identification and systematic levels where the Japanese waters are considered among the origin places of present population. While, the identity of the present sequence (KF938589) from Mediterranean Sea, Egypt was 98% with KC862306 from
333 Turkey (Tuney, 2016) and was 99% with AP011932 mitochondrial DNA complete genome (Yamanoue et al., 2011), these identities were lower than those from Japan although both the present one and those from Turkey were from the same Mediterranean Basin, creating somewhat doubt in the presence of difference. This variation may be attributed to a few nucleotide differences from the analysis after sequencing; however this variation did not live up to the occurrence of difference in its identification. Regarding the other primer Cyt b gene, the identity of KF031125 (Mediterranean Sea, Egypt) was 99% with the KF031124 (present work, Red Sea, Egypt); both present sequences used Cyt b showed an identity of 99% with JQ681894 and AY267356 from USA and Taiwan respectively. Even for mitochondrial DNA complete genome, the identity was 99% with AP011932. Although, the identity of two present species was more than 99%, the identity of that from Mediterranean Sea (KF031125) was 100% with EF362414 from Greece, indicating the difference in habitat may induce slight differences. From the alignment and comparison, the present two sequences from Mediterranean and Red Seas reflected a high level of identity which was more than 99% indicating a high level of genetic resemblance. In general the identity ranged from 99% to 100% confirming the taxonomic level of L. sceleratus. Moreover, the percentage of identity has affected on the phylogenetic analysis of such species, the phylogenetic relations illustrated that, the presence of present sequence (KF938589) from Mediterranean Sea, Egypt as sister group to AB194240 and AB742031 from Japan in one cluster for the same species reflects a high level of genetic distance and strong Neighbor-Joining (NJ) relationship which confirms its identification, taxonomic level and its origin place. This relation was stronger than that with KC862306 from Turkey as it is found in a separate second cluster. The NeighborJoining (NJ) relationship expected that the present sequence from Mediterranean Sea will be closer to that from Turkish water than other countries as they located on the same Mediterranean Basin, this finding may be attributed to the variation in identity between various sequences, however the similarity between the present work and that from Turkish water was 98% indicating the same taxonomic level of two populations. The phylogenetic analysis with other seven pufferfish species of genus Lagocephalus used (16S rRNA) showed two primary clads. One of them contained L. sceleratus and L. inermis as a sister group to L. laevigatus, L. wheeleri, L. lagocephalus and L. gloveri, while the second clad was L. lunaris as a separate cluster. The presence of those different species together in two clads or clusters reflected that all species belong to the same genus. Moreover, the presence of L. lunaris in a separate clad reflected the low genetic distance to present species. For the second primer, the phylogenetic tree of L. sceleratus from different locations has produced two clusters, the first one contained JQ681894 from USA and AY267356 from Taiwan, while the second cluster has two sub clusters, one subcluster for KF031124 from Egypt (Red Sea, present work), and second subcluster included KF031125 from Egypt (Mediterranean Sea, present work) and EF362414 from Greece together as sister group. These findings of Neighbor-Joining (NJ) relationships indicated the more of identity between present sequences of Mediterranean and Red Seas where they
334 were in one cluster, also the presence of present sequence from Mediterranean Sea in one subcluster with that from Greece, Mediterranean Sea reflected the accuracy of its identification and strongly Neighbor-Joining (NJ) relationship that was closer to the species of Greece. Moreover, the presence of the present species and other nine species in two clusters reflected the Neighbor-Joining (NJ) relationship between these species; while the nearest species was L. suezensis reflecting the strong Neighbor-Joining (NJ) relationship between two species and this is supported by Farrag (2014) who illustrated that the morphological characteristics of L. suezensis has a higher similarity to L. sceleratus than other puffer fishes. In conclusion, the environmental variations between habitats induced slight phenotypic variation in some morphological characteristics of present populations of L. sceleratus; however, there was insignificant difference in the mean of biometric indices between two populations from Mediterranean and Red Seas, Egypt combined with high similarity and high correlation. The most suitable characteristics can be used are standard length, fork length, pre-dorsal and post-dorsal, pre-anal, pre-pectoral, head length, and other meristic characteristics. The pair wise comparison of genetic distance revealed that L. sceleratus from Mediterranean and Red Seas were more similar and the identity was more than 99% indicating no obvious genetic variations observed for this species after its migration into new habitat, and can be expected that, genetic differentiation may occur very slowly in the typically large population of commercial marine fishes as supported by Ward et al. (1994). The results of this work recommended use of the combined morphological and molecular analysis to support the identification, taxonomy and phylogenetic relationships of the population. The specific genes, 16S rRNA and cytochrome b, were highly useful as they showed confirmable tools in identification, phylogenetic relationships, and identity between populations especially during the comparison from different habitats. Acknowledgments The authors would like to thank Prof. Yuji Yamazaki, University of Toyama, Japan and Dr. Mohamed El-Ghanam, Kafr El-Sheikh University, Egypt for their permission to analyze the specimens at their labs. Moreover, many thanks are due Dr. Moustafa Sarhan, Al-Azhar University, Assiut, Egypt and Dr. Jessica Gordon, The University of the Ryukyus, Okinawa, Japan for their revisions and comments on this work. References Akyol, O., Unal, V., Ceyhan, T., Bilecenoglu, M., 2005. First confirmed record of Lagocephalus sceleratus (Gmelin, 1789) in the Mediterranean. J. Fish. Biol. 66, 1183–1186. Avsar, D., 1999. Physico-chemical characteristics of the Eastern Mediterranean in relation to distribution of the new Scyphomedusae (Rhopilema nomadica). Turk. J Zool. 23 (2), 605–616. Bariche, M., Letourneur, Y., Harmelin-Vivien, M., 2004. Temporal Fluctuations and settlement patterns of native and Lessepsian herbivorous fishes on the Lebanese coast (Eastern Mediterranean). Environ. Biol. Fishes 70, 81–90. Begg, G.A., Friedland, K.D., Pearce, J.B., 1999. Stock identification and its role in stock assessment and fisheries management: an overview. J. Fish. Res. 43, 1–8.
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335 Sambrook, J., Russell, D.W., 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York. Smith, M.M., Heemstra, P.C., 1986. Tetraodontidae. In: Smith, M.M., Heemstra, P.C. (Eds.), Smiths’ Sea Fishes. Springer-Verlag, Berlin, pp. 894–903. Tamura, T., Sone, M., Nakamura, Y., Shimamura, T., Imoto, S., Miyano, S., Okazawa, H., 2013. A restricted level of PQBP1 is needed for the best longevity of Drosophila. Neurobiol. Aging 34 (1), 356.e11–356.e20 (Export to RIS). Templeman, W., 1970. Vertebral and other meristic characteristic halibut, Reinharditus hippologossoides, from the north-west Atlantic. J. Fish. Res. Bd. Can. 27 (9), 1459–1562. TUDAV, 2006. Kuresel Isinma ve Turkiye Denizleri Raporu. Turk Deniz Arastirmalari Vakfi. Available from:. Cited 25.04.2006 (in Turkish). Tuney, I., 2016. Molecular identification of puffer fish Lagocephalus sceleratus (Gmelin, 1789) and Lagocephalus spadiceus (Richardson, 1845) from Eastern Mediterranean, Turkey. Fresen. Environ. Bull., in press. Turan, C., 1999. A note on the examination of morphometric differentiation among fish populations: the truss system. Turk. J. Zool. 23, 259–263. Ward, J.M., Fox, J.G., Anver, M.R., Haines, D.C., George, C.V., Collins, M.J., Gorelick, P.L., Nagashima, K., Gonda, M.A., Gilden, R.V., Tully, J.G., Russell, R.J., Benveniste, R.E., Paster, B.J., Dewhirst, F.E., Donovan, J.C., Anderson, L.M., Rice, J.M., 1994. Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J. National Cancer Inst. 86, 1222–1227. Yamanoue, Y., Miya, M., Doi, H., Sakai, H., Nishida, M., 2011. Multiple invasions into freshwater by pufferfishes (Teleostei: Tetraodontidae): a mitogenomic perspective. PLoS One 6 (2), 1–13.
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National Institute of Oceanography and Fisheries
Egyptian Journal of Aquatic Research http://ees.elsevier.com/ejar www.sciencedirect.com
FULL LENGTH ARTICLE
Molecular phylogeny and biometrics of lessepsian puffer fish Lagocephalus sceleratus (Gmelin, 1789) from Mediterranean and Red Seas, Egypt Mahmoud M.S. Farrag a,*, Taha B.H. Soliman c, El-Sayed Kh.A. Akel c, Alaa A.K. Elhaweet b, Mohsen A. Moustafa a a
Faculty of Science, Al-Azhar University (Assiut Branch), Egypt College of Fisheries Technology and Aquaculture, Arab Academy for Science, Technology and Maritime Transportation, Alexandria, Egypt c National Institute of Oceanography & Fisheries (Alexandria), Egypt b
Received 16 May 2015; revised 26 August 2015; accepted 26 August 2015 Available online 26 September 2015
KEYWORDS Lagocephalus sceleratus; Biometric; Phylogeny; Mediterranean and Red Seas; Egypt
Abstract Phylogenetic comparison for lessepsian puffer fish Lagocephalus sceleratus (Gmelin, 1789) from the Egyptian waters (Mediterranean and Red Seas) was performed using morphological and genetic characteristics to confirm its identification and to recognize any variations due to differences in both habitats. A similar identity in body description from both habitats was observed. Slight phenotyping variations in some morphological measurements were observed with no significant difference. The correlation between the reference length and most of morphological indices was similar (0.99) for both habitats. Molecular phylogeny, using two primers (16S rRNA and Cytochrome b) showed similar size bands at 600 bp and 400 bp ladders for both primers respectively in both habitats. The obtained sequences were aligned showing an identity (more than 99%) to the published sequences of L. sceleratus indicating high genetic resemblance. So, the present populations from both habitats don’t appear to be separated species, indicating no obvious genetic variations after its migration into new habitat, expecting that, genetic differentiation may occur very slowly. This supported the idea that both morphological and genetic characteristics are recommended to be used together in identification and phylogenetic relations of the population from two habitats. The specific primers 16S rRNA and Cyt b showed confirmable tools to support the same target where the environmental variations may alter phenotypic variations. Ó 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
* Corresponding author at: Department of Zoology, Faculty of Science, Al-Azhar University (Assiut Branch), 71524 Assiut, Egypt. Tel.: +20 1007253531. E-mail address: [email protected] (M.M.S. Farrag). Peer review under responsibility of National Institute of Oceanography and Fisheries. http://dx.doi.org/10.1016/j.ejar.2015.08.001 1687-4285 Ó 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Introduction Pufferfish Lagocephalus sceleratus (Gmelin, 1789) has received considerable public attention after its migration from Red Sea via Suez Canal, since its rapid distribution in different countries of Mediterranean Basin, with social impacts (Farrag, 2014). It originally has expanded its distribution widely from Indo-Pacific regions (Smith and Heemstra, 1986). This species is the common and largest distributed one among puffer fish species in the Egyptian Mediterranean waters (Farrag, 2014). The invasion of species to Mediterranean Basin has become an ecological and economic issue which must be addressed, studied and monitored (Bariche et al., 2004; IUCN, 2008; Oral, 2010). Since its moving from a certain habitat to a different one, the environmental differences in the habitats may lead to the detection of morphological differentiation by phenotypic markers, which may be a practical level of partitioning among self-recruiting stocks (Turan, 1999). So, stock identification is very important, hence it is a multidisciplinary field of fisheries science, biometrics, genetics and life history studies (ICES, 1996; Pawson and Jennings, 1996; Begg et al., 1999). Hence, it is necessary to identify specimens correctly to investigate biological traits as growth, mortality, fecundity, trophic relation, parasite relationship, historical and paleontological events (Ibanez et al., 2007). Moreover, measures of gene flow and genetic differentiation are typically used to distinguish one population from another allowing a better analysis of fish species (Omer and Abukashwa, 2012). So, this work aims to confirm the identification and compare between the L. sceleratus population from two water bodies (The Mediterranean Sea as a new habitat and The Red Sea as the origin one) using the combination of biometric and genetic characteristics. Materials and methods This study was constructed on phylogenetic comparison using both biometric and genetic characteristics of puffer fish L. sceleratus from two water bodies (The Mediterranean Sea, Egypt as new habitat) and The Red Sea (Hurghada) as the origin of its migration (Fig. 1). Biometric characteristics
Figure 1 The study area along the Egyptian Mediterranean coast and fishing ports {(1) Port Said; (2) Ezbet-ElBorg (Damietta); (3) El-Burullus; (4) El-Maadia; (5) Abu-Qir; (6) Alexandria (Anfoushy)} and (7) Hurghada, Red Sea.
of these indices and their standard deviation. The meristic characteristics were counted as: Dorsal fin rays, anal rays, pectoral fin rays, caudal fin rays and number of vertebrae (Templeman, 1970). For genetic studies DNA was isolated from the muscle tissues that preserved in 99% ethanol using modified protocol of phenol- chloroform (Sambrook and Russell, 2001). The isolated DNA was diluted and stored at 20 °C until used. PCR amplification and genotyping
The biometric characteristics (Morphometric and meristic) were carried out at the laboratory on 44 and 35 fresh specimens from Red Sea and Mediterranean Sea, respectively having the same length range to get more accuracy of results and to avoid the errors due to variations in length; the length measurements were taken to the nearest 0.1 cm of each fish; then pooled monthly and analyzed keeping the size interval as 1 cm. The morphometric measurements were taken for each species (Fig. 2). The head length (H.L) was used as a reference for pre orbital length (Pre O. L), eye width (E.W) and inner orbital width (I. Orbit), while the total body length (T.L) was used as a reference for Standard length (S.L.), Forked length (F.L.), Pre dorsal length (Pre D. L.), Post dorsal length (Post D. L.), Pre pectoral length (Pre P.L.), Pre anal length (Pre A.L.), Max. body depth (Max. B. D), Head depth (H. D). Then the ratio index for each characteristic (Morphometric measurements / relevant reference 100) was calculated, the different length groups were pooled to calculate mean values
Partial region of the universal primers 16S rRNA with region 600 bp (16Sar, 50 -CGCCTGTTTATCAAAAACAT-30 and 16Sbr, 50 CCGGTCGAAACTCAGATCACGT-30 ) (Palumbi et al., 1991), and Cyt b with region 400 bp (F: 50 -CTACG CAAAACGCACCCAC-30 and R: 50 GGGGAGGACGTAG CCCACAAAG-30 ) were amplified according to Meyer (1994). PCR amplification for 21 individuals from Mediterranean Sea and 18 individuals from Red Sea using 16S rRNA primer was done at the lab of Graduate School of Science and Engineering for Research, University of Toyama, Japan. Due to the poor preservation of specimens from Red Sea, only specimens from Mediterranean Sea were sequenced. So, the other six specimens of puffer fish L. sceleratus (3 from Mediterranean Sea and 3 from Red Sea, Egypt) have used the second primer (Cytochrome b) at the lab of biotechnology, Faculty of veterinary Medicine, Kafr El-Sheikh University, Egypt in order to give more of results about the present comparison. PCR amplification for both primers was performed in
Molecular phylogeny and biometrics of lessepsian puffer fish
Figure 2
325
Lagocephalus sceleratus showing morphometric measurements.
15-ll reaction volumes, 100 ng/ll of DNA template, 1.2 ll dNTP mixture 2.5 mM each, 1.5 ll 10 Ex Taq buffer (Takara), 1.5 ll of each primer (5 mM), 1 U of Taq polymerase, and purified water to complete the final volume. Thermal cycler conditions consisted of an initial denaturation step at 94 °C for 1 min, 35 cycles of 15 s at 94 °C for annealing, 15 s at 50 °C, and 30 s at 72 °C, with a final extension step of 7 min at 72 °C, and cooling to 4 °C until the PCR products were removed from the thermocycler. A 4-ll sample of each PCR product was run on 1.5% agarose gel. A 11 ll volume of each amplified product was purified using ExoSAP-IT (Amersham Pharmacia Biotech) and, labeled using Big Dye Terminator V.3.1 Cycle sequencing Kit. The purified samples were sent to Macogen Inc., (South Korea) to sequence in both directions by ABI 3730XL DNA sequence (Applied Biosystem, USA). Data analysis Geneious 4.8.4 software was used for sequencing annotation and assembly. The obtained sequences were aligned by Clustal W program software version 2.1 provided by DNA Data Bank of Japan (DDBJ) (http://clustalw.ddbj.nig.ac.jp/index.php) (Larkin et al., 2007) and submitted to Gen Bank database. The neighbor-joining phylogenetic trees of Cyt b and 16S rRNA genes were conducted by MEGA software version 6 (Tamura et al., 2013). Results Morphological comparison The morphological description of L. sceleratus (Gmelin, 1789) from two water bodies (Mediterranean and Red Seas) were similar whereas the body is elongated, somewhat compressed laterally and inflatable; color is gray to greenish with dorsal regular black spots; the small spinules on the dorsal surface
extending to origin of the dorsal fin, while the small spinules ventrally do not reach the cloacae opening. A wide silver band is located on the lower parts of the flanks from the mouth to the caudal fin with silver blotch in front and below the eye. The pectoral fin base is black, it has wide based, and has a round posterior edge and the caudal fin is lunate (Fig. 2). The biometric indices (Table 1) for the same population from two habitats were slightly similar at the same length range (19.9–55.5 cm) for indices of S.L/T.L (%); F.L/T.L (%); Pre D. L./T.L (%); Post D. L/T.L (%); Pre P. L./T.L (%); Pre A. L./T.L (%); E.W./H.L (%), these indices were relevant to total length as reference. However, slightly variations were observed for some indices (Max. B. D./T.L (%); Min. B. D./T.L (%); HL/T.L (%)) where these indices for Red Sea were 13.28, 3.59, 12.24, 31.42 higher than those of Mediterranean Sea species. While the indices of Pre nostril/H.L (%), Pre orbit/H.L (%), I. O/H.L (%) showed an inverse trend of variations giving values of Mediterranean Sea higher than those of Red Sea and most of these indices are proportional to the head length. The correlation coefficients (Fig. 3) of S.L, F.L, Pre D. L, Post D.L. and Pre A.L. gave a similar value (0.990) for both Mediterranean Sea and Red Sea species. The Max. B.D., Min. B.D., Pre P.L., H.D, Pre O., Post O. and Inner O. for L. sceleratus from Mediterranean Sea were 0.97, 0.98, 0.99, 0.97, 0.99, 0.97 and 0.96 respectively; these values were higher than those of Red Sea which were 0.94, 0.78, 0.91, 0.96, 0.94, 0.96 and 0.94 for the same parameters respectively. The prenostril value 0.97 in Mediterranean was less than that of Red Sea 0.99. Eye width parameters gave the same correlation value (0.88) for both habitats. Generally, insignificant difference between biometric indices of L. sceleratus from two habitats was observed at p P 0.95. Moreover, both of the two populations of L. sceleratus from two habitats showed nearly the same range of meristic characteristic as given in Table 1. For Mediterranean waters, the dorsal fin rays was X + (10–12), the anal fin rays was
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Table 1
Comparison in indices of biometric characteristics of L. sceleratus from the Mediterranean and Red Seas, Egypt.
Species
L. sceleratus (Mediterranean Sea)
L. sceleratus (Red Sea)
Length range
20–55 (cm) (T.L)
20–55 (cm) (T.L)
No. specimens
35
Indices/values
Min.
Max.
Av.
±SD
Min.
Max.
Av.
±SD
T.L (cm) S.L/T.L (%) F.L/T.L (%) Pre D.L/T.L (%) Post D.L/T.L (%) Pre P.L/T.L (%) Pre A.L./T.L (%) H.D/H.L (%) Max. B. D/T.L (%) Min. B. D/T.L (%) Pre nostril/H.L (%) Pre Orbit/H.L (%) Inner O./H.L (%) E. W/H.L (%) H.L/T.L (%) No. D.F. rays No. A.F. rays No. P.F. rays No. C.F. rays No. V
19.9 83.23 91.67 50.18 60.12 25.05 53.85 35.71 10.42 2.51 25.33 40.00 29.41 18.06 25.35 10–12 10–11 16–18 16–19 16–17
55 85.45 94.52 57.54 64.51 27.96 57.93 52.42 15.44 3.33 35.54 56.61 46.81 31.43 31.99
39.58 84.32 93.17 55.54 62.53 26.12 56.46 46.19 12.89 2.95 32.32 50.51 38.69 22.84 27.84
13.25 0.75 0.79 1.74 1.42 0.72 1.10 4.89 1.11 0.22 3.09 6.26 6.41 3.35 2.12
20.00 81.98 90.74 53.33 58.89 19.44 53.70 38.73 11.51 2.59 24.62 39.35 28.46 17.26 28.70 11–12 10–12 16–18 17–19 16–17
55.50 86.63 97.16 59.56 65.44 28.25 60.41 47.41 15.41 4.89 31.61 49.08 39.88 29.85 33.48
39.30 84.20 93.49 57.22 63.03 25.62 57.87 42.64 13.28 3.59 28.63 44.27 34.23 22.53 31.42
13.29 1.01 1.55 1.71 1.55 2.50 1.87 2.61 1.04 0.67 2.10 3.04 2.95 3.45 1.20
Figure 3
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Biometric characteristics indices of L. sceleratus from the Egyptian Mediterranean Sea and Red Seas, Egypt.
Molecular phylogeny and biometrics of lessepsian puffer fish
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Figure 3 (continued)
X + (10–11), the pectoral fin rays was X + (16–18) and the caudal fin rays was X + (16–19). For the Red Sea species, the dorsal fin rays was X + (11–12), the anal fin rays was X + (10–12), the pectoral fin rays was X + (16–18) and the caudal fin rays was X + (17–19). Number of vertebrae of two populations from two habitats ranged from 16 to 17+ urostyle and post cranial vertebra in all fish examined showing no appreciable variation between the populations. Molecular identification and phylogenetic analyses The task of molecular identification and phylogenetic comparison was conducted using different two primers (16S rRNA and cytochrome b) through the following processes. Amplification of 16S rRNA (600 bp) and cytochrome b (400 bp) The obtained results of the present molecular profiles of L. sceleratus from both habitats (Mediterranean and Red Seas) showed similarity to the fragments of size bands at 600 bp and 400 bp of ladders giving the same band size in the right expected molecular weight of both 16S rRNA and cytochrome b genes respectively (Fig. 4A, B). The PCR products were sequenced only for Mediterranean Sea specimens and the sequences were similar, a single representative sequence of all
obtained sequences was uploaded to Genbank under the accession number KF938589. This sequence was aligned and conferred with previously published sequences that used the same primer for the same population of L. sceleratus from different areas and showed high level of identity (Fig. 5); the identity was 100% with accession numbers of AB194240 and AB742031 from Japan by Ishizaki et al. (2006) and Igarashi et al. (unpublished) respectively, while it was 98% with KC862306 from Turkey that constituted by Tuney (2016). Likewise, the PCR products obtained from the amplification of the second primer Cytochrome b gave also the same band size and expected molecular weight for all specimens from two locations (Fig. 6). The PCR products from both habitats were sequenced and they were similar; one representative sequence from each habitat was uploaded on the Genebank under accession numbers of KF031124 (Red Sea), and KF031125 (Mediterranean Sea). These sequences were aligned with those whose amplified partial mitochondrial Cyt b for the same population from different localities (Fig. 7); the alignment of present Red Sea sequence has showed a high level of identity (99%) with JQ681894 from USA; was 99% for AY267356 from Taiwan. The identity of KF031125 (Mediterranean Sea, Egypt, present work) was 99% with the sequence (KF031124) from Red Sea, Egypt, present work), while it was 100% with EF362414 from Greece water, Mediterranean Sea.
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Figure 3 (continued)
The phylogenetic relations with the same population and others The phylogenetic tree based on the distance analysis of sequences generated using Neighbor-Joining (NJ) tree (MEGA software with 500 bootstrap replicates) illustrated that, the phylogenetic relation with the same population from different locations used 16S rRNA sequence has produced two clusters or clades (Fig. 8A). One such clade contains KC862306 from Turkey with the identity of bootstrap 98%, while the second clade has two subclades of present sequence (KF938589) from Mediterranean Sea, Egypt) in one, while the second subclade represented by sister group contains two sequences (AB194240 and AB742031) from Japan with bootstrap 100%. Moreover, the phylogenetic tree of L. sceleratus with other seven different pufferfish species under the same genus Lagocephalus using the same primer (16S rRNA) has produced two primary clades (Fig. 8B); one of them consists of two groups having L. sceleratus in one group while the other group contained L. inermis as a sister group to L. laevigatus, L. wheeleri, L. lagocephalus and L. gloveri. The second clade was represented only by L. lunaris. For the second primer (Cyt b), the phylogenetic relation with the same population from different locations has produced two clusters (Fig. 9A). The first one contains JQ681894 from USA with bootstrap of an identity (99%),
and AY267356 99% from Taiwan, while the second cluster has two subclusters forming sister groups. One of these subclusters contains KF031124 from Egypt (Red Sea), and second subcluster contains both the present sequence KF031125 from Egypt (Mediterranean Sea) and EF362414 from Greece (Mediterranean Sea). The identity of bootstrap of KF031125 (Mediterranean Sea, Egypt) with that of KF031124 (Red Sea, Egypt) was 99% and 100% for EF362414 from Greece (Mediterranean Sea). Moreover, its phylogenetic tree with other different nine pufferfish species using Cyt b gene has produced two main clusters (Fig. 9B); one of them contains L. sceleratus and L. suezensis as two subclusters, while the second cluster in phylogenetic tree has contained L. gloveri, L. guentheri, L. lagocephalus, L. laevigatus, L. lunaris, L. inermis and L. wheeleri together as a sister groups. Discussion The objective of a racial study was to establish, with some degree of confidence, the taxonomic identity of the common puffer fish species L. sceleratus from different water bodies (Mediterranean and Red Seas). Interactive effects of environment produce morphometric differences within a species, the variability in growth, development, and maturation creates a
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Figure 3 (continued)
variety of body shapes within a species (ICES, 1996; Pawson and Jennings, 1996; Begg et al., 1999). Since the morphometric characteristics are sensitive to any environmental changes, the genetic differentiation was used to confirm the identification and identity of such species from two Water bodies. The present description and morphology of L. sceleratus from Mediterranean and Red Seas showed an agreement and similarity to its congeneric tropical species Mozambique and southern African shores, as well as the Red Sea (Smith and Heemstra, 1986; Ni and Kwon, 1999; Froese and Pauly, 2003). Moreover, they were similar to those studied by Akyol et al. (2005); Bilecenoglu et al. (2006); Kasapidis et al. (2007); Golani et al. (2011); Jribi and Bradai (2012) and Milazzo et al. (2012) and this confirms its identification.
The comparison between the immigrant L. sceleratus from Mediterranean Sea and the same population from Red Sea is very important, since this species represented the common and largest one of puffer fish species in the Egyptian Mediterranean waters and needs to be studied more (Farrag, 2014). The biometric comparison has used the same length range for specimens from both locations to avoid the size effects (even with the presence of gap in length range of one population, the author removed the same gap in the second population to get more accuracy of results). The insignificance for the mean of different biometric indices of two populations was detected. Moreover most indices (morphometric and meristic counts) showed a high similarity between different locations reflecting no change in its identification and taxo-
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Figure 4 Gel electrophoresis of 16S rRNA PCR product (600 bp) for: (A) 21 specimens of L. sceleratus from Mediterranean Sea (1–21) and (B) eighteen specimens of L. sceleratus from Red Sea (Lanes 1–18); M-200 bp DNA ladder.
nomic classification. These findings may be attributed to many reasons as the same length range used from both habitats. Moreover, the ecological conditions such as salinity and temperature in eastern Mediterranean became similar to those of the Red Sea (Avsar, 1999), the mean salinity of Eastern Mediterranean is 38.5%, and it may rise to 39.5% in some areas such as Iskenderun Bay while the salinity in the Suez Bay ranged between 33.5% and 40.3%. On the other hand, TUDAV (2006) claimed that global warming has increased
M.M.S. Farrag et al. the temperature of the Mediterranean facilitating the establishment of lessepsian species in the Eastern Mediterranean. So, the similar conditions may refer to slowly/not changing morphological measurements, in addition to that the similarity may be taken in further consideration to a change in the genetic characteristics. However, slight variations in some indices as Max. B.D/T.L (%); Min. B.D/T.L (%) and Pre. O/H.L (%) were observed, this may be attributed to variations in ecological and geographical variations of different areas. Huet (1949) found variations in fin rays of the salt water and fresh water races of sticklebacks in relation to both temperature and salinity. The width index of the eye of L. sceleratus from the Mediterranean Sea was somewhat larger than that of Red Sea and this could be related to the higher turbidity of the waters of the Mediterranean Sea. Similar results were found in the rainbow trout Oncorhynchus mykiss (Pankhurst and Montgomery, 1994) and Mugil curema from the Atlantic (Ibanez-Aguirre et al., 2006). In some cases, the body width can be strongly influenced by sexual maturation and the preservation quality of fish specimen (Ibanez-Aguirre et al., 2006). The observed differences in Max. B.D/T.L (%); Min. B.D/T.L (%); Pre. O/H.L (%) in the present work may be attributed to sexual maturation of the examined specimens and fullness of stomach as well as the inflating of the body especially for puffer fish that can inflate itself. On the other hand, the differences in body shape and measurements could be due to the used methods as reported by Corti and Crosetti (1996) and Mamuris et al. (1998). These findings can be seen in comparison between the present results and others where the scientists measured the head length from the beginning of the snout to the end eye (Jribi and Bradai, 2012); the present measure of head length was from the beginning of the snout to the end of real skull that located behind the pectoral fine. Even, while using the same method to measure head length, the indices of head length varied between two habitats causing slight changes in different characteristics which used head length as a reference. These variations may be attributed to the previously mentioned reasons as well as food contents which can appear as variations in the ventral part of the head of puffer fish. So, the slight variations between two populations from Red Sea
Figure 5 Gel electrophoresis of Cyt b PCR product (400 bp), each specimen of L. sceleratus was run in two lanes: A, B and C from Mediterranean Sea (Left side), while D, E and F were from Red Sea (Right side); M-100 bp DNA ladder.
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Figure 6 Aligned DNA sequences using CLUSTALW 2.1 of the amplified 16S rRNA region of mitochondrial DNA from puffer fish from different locations: KF938589, Egypt; AB194240-AB742031, Japan; KC862306, Turkey. Asterisk (*) indicates identity with Lagocephalus sceleratus sequences. A gap introduced into the sequences to optimize the alignment is represented by a dash (-).
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Figure 7 Aligned DNA sequences using CLUSTALW 2.1 of the amplified Cyt b gene of mitochondrial DNA for puffer fish from different locations: EF362414.1, Greece; KF031124.1-KF031125.1, Egypt; JQ681894.1, USA; AY267356.1, Taiwan. Asterisk (*) indicates identity with L. sceleratus sequences. A gap introduced into the sequences to optimize the alignment is represented by a dash (-).
and Mediterranean Sea in the present work are not enough to discriminate groups to be a separate species. The gene frequency was utilized in fish molecular studies in a wide range to detect the taxonomy and systematic status of the species due to its conservative properties (Gilles et al., 2001 and Li et al., 2008). Genetic differentiation and phylogenetic analysis of L. sceleratus in Egypt did not gain sufficient attention. So, genetic differentiation was used in combination with biometric characteristics in the present work to confirm the identification of L. sceleratus and its identity with the same population from other locations and its phylogenetic trees. The two genes/primers (16S rRNA and Cyt b) are famous in the field of taxonomy, diversity and phylogenetic relationship at
various taxonomic levels were used by various authors (Gilles et al., 1998; Nguyen et al., 2006; Li et al., 2008 and Faddagh et al., 2012). The present gel electrophoresis profiles for both genes (16S rRNA and Cyt b) for L. sceleratus from both habitats showed similar fragments of molecular size bands at 600 bp and 400 bp ladders in both 16S rRNA and cytochrome b genes respectively. This may reflects the genetic similarity of two populations from Mediterranean Sea and Red Sea which is the origin place of migration. The present sequence of 16S rRNA (KF938589) from Mediterranean Sea was aligned with other sequences of L. sceleratus from different locations; the identity was 100% with those sequences (AB194240 and AB742031) from Japan by
Molecular phylogeny and biometrics of lessepsian puffer fish
Figure 8a Neighbor-Joining tree inferred from Tamura-Nei distances between sequences of 16S rRNA gene in L. sceleratus and the same species from different locations.
Figure 8b Neighbor-Joining distance tree between sequences of 16S rRNA gene of L. sceleratus and other different pufferfish (Genus: Lagocephalus) obtained from NCBI GenBank (http:// www.ncbi.nlm.nih.gov).
Figure 9a Neighbor-Joining tree inferred from Tamura-Nei distances between sequences of Cyt b gene in L. sceleratus and the same species from five different locations.
Figure 9b Neighbor-Joining distance tree for the Cyt b gene sequence data of L. sceleratus and other different pufferfish (Genus: Lagocephalus) obtained from NCBI GenBank (http:// www.ncbi.nlm.nih.gov).
Ishizaki et al. (2006) and Igarashi, et al. (unpublished) respectively. This finding has reflected the confirmation of its identification and systematic levels where the Japanese waters are considered among the origin places of present population. While, the identity of the present sequence (KF938589) from Mediterranean Sea, Egypt was 98% with KC862306 from
333 Turkey (Tuney, 2016) and was 99% with AP011932 mitochondrial DNA complete genome (Yamanoue et al., 2011), these identities were lower than those from Japan although both the present one and those from Turkey were from the same Mediterranean Basin, creating somewhat doubt in the presence of difference. This variation may be attributed to a few nucleotide differences from the analysis after sequencing; however this variation did not live up to the occurrence of difference in its identification. Regarding the other primer Cyt b gene, the identity of KF031125 (Mediterranean Sea, Egypt) was 99% with the KF031124 (present work, Red Sea, Egypt); both present sequences used Cyt b showed an identity of 99% with JQ681894 and AY267356 from USA and Taiwan respectively. Even for mitochondrial DNA complete genome, the identity was 99% with AP011932. Although, the identity of two present species was more than 99%, the identity of that from Mediterranean Sea (KF031125) was 100% with EF362414 from Greece, indicating the difference in habitat may induce slight differences. From the alignment and comparison, the present two sequences from Mediterranean and Red Seas reflected a high level of identity which was more than 99% indicating a high level of genetic resemblance. In general the identity ranged from 99% to 100% confirming the taxonomic level of L. sceleratus. Moreover, the percentage of identity has affected on the phylogenetic analysis of such species, the phylogenetic relations illustrated that, the presence of present sequence (KF938589) from Mediterranean Sea, Egypt as sister group to AB194240 and AB742031 from Japan in one cluster for the same species reflects a high level of genetic distance and strong Neighbor-Joining (NJ) relationship which confirms its identification, taxonomic level and its origin place. This relation was stronger than that with KC862306 from Turkey as it is found in a separate second cluster. The NeighborJoining (NJ) relationship expected that the present sequence from Mediterranean Sea will be closer to that from Turkish water than other countries as they located on the same Mediterranean Basin, this finding may be attributed to the variation in identity between various sequences, however the similarity between the present work and that from Turkish water was 98% indicating the same taxonomic level of two populations. The phylogenetic analysis with other seven pufferfish species of genus Lagocephalus used (16S rRNA) showed two primary clads. One of them contained L. sceleratus and L. inermis as a sister group to L. laevigatus, L. wheeleri, L. lagocephalus and L. gloveri, while the second clad was L. lunaris as a separate cluster. The presence of those different species together in two clads or clusters reflected that all species belong to the same genus. Moreover, the presence of L. lunaris in a separate clad reflected the low genetic distance to present species. For the second primer, the phylogenetic tree of L. sceleratus from different locations has produced two clusters, the first one contained JQ681894 from USA and AY267356 from Taiwan, while the second cluster has two sub clusters, one subcluster for KF031124 from Egypt (Red Sea, present work), and second subcluster included KF031125 from Egypt (Mediterranean Sea, present work) and EF362414 from Greece together as sister group. These findings of Neighbor-Joining (NJ) relationships indicated the more of identity between present sequences of Mediterranean and Red Seas where they
334 were in one cluster, also the presence of present sequence from Mediterranean Sea in one subcluster with that from Greece, Mediterranean Sea reflected the accuracy of its identification and strongly Neighbor-Joining (NJ) relationship that was closer to the species of Greece. Moreover, the presence of the present species and other nine species in two clusters reflected the Neighbor-Joining (NJ) relationship between these species; while the nearest species was L. suezensis reflecting the strong Neighbor-Joining (NJ) relationship between two species and this is supported by Farrag (2014) who illustrated that the morphological characteristics of L. suezensis has a higher similarity to L. sceleratus than other puffer fishes. In conclusion, the environmental variations between habitats induced slight phenotypic variation in some morphological characteristics of present populations of L. sceleratus; however, there was insignificant difference in the mean of biometric indices between two populations from Mediterranean and Red Seas, Egypt combined with high similarity and high correlation. The most suitable characteristics can be used are standard length, fork length, pre-dorsal and post-dorsal, pre-anal, pre-pectoral, head length, and other meristic characteristics. The pair wise comparison of genetic distance revealed that L. sceleratus from Mediterranean and Red Seas were more similar and the identity was more than 99% indicating no obvious genetic variations observed for this species after its migration into new habitat, and can be expected that, genetic differentiation may occur very slowly in the typically large population of commercial marine fishes as supported by Ward et al. (1994). The results of this work recommended use of the combined morphological and molecular analysis to support the identification, taxonomy and phylogenetic relationships of the population. The specific genes, 16S rRNA and cytochrome b, were highly useful as they showed confirmable tools in identification, phylogenetic relationships, and identity between populations especially during the comparison from different habitats. Acknowledgments The authors would like to thank Prof. Yuji Yamazaki, University of Toyama, Japan and Dr. Mohamed El-Ghanam, Kafr El-Sheikh University, Egypt for their permission to analyze the specimens at their labs. Moreover, many thanks are due Dr. Moustafa Sarhan, Al-Azhar University, Assiut, Egypt and Dr. Jessica Gordon, The University of the Ryukyus, Okinawa, Japan for their revisions and comments on this work. References Akyol, O., Unal, V., Ceyhan, T., Bilecenoglu, M., 2005. First confirmed record of Lagocephalus sceleratus (Gmelin, 1789) in the Mediterranean. J. Fish. Biol. 66, 1183–1186. Avsar, D., 1999. Physico-chemical characteristics of the Eastern Mediterranean in relation to distribution of the new Scyphomedusae (Rhopilema nomadica). Turk. J Zool. 23 (2), 605–616. Bariche, M., Letourneur, Y., Harmelin-Vivien, M., 2004. Temporal Fluctuations and settlement patterns of native and Lessepsian herbivorous fishes on the Lebanese coast (Eastern Mediterranean). Environ. Biol. Fishes 70, 81–90. Begg, G.A., Friedland, K.D., Pearce, J.B., 1999. Stock identification and its role in stock assessment and fisheries management: an overview. J. Fish. Res. 43, 1–8.
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