Journal of Fish Biology (2008) 73, 1228–1248 doi:10.1111/j.1095-8649.2008.01996.x, available online at http://www.blackwell-synergy.com
Molecular phylogeny of Turkish Trachurus species (Perciformes: Carangidae) inferred from mitochondrial DNA analyses Y. B EKTAS *
AND
A. O. B ELDUZ †‡
*Faculty of Fisheries and Aquatic Sciences, Rize University, 53100 Rize, Turkey and ‡Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey (Received 9 January 2008, Accepted 19 June 2008) Genetic variation among three species of Trachurus (T. trachurus, T. mediterraneus and T. picturatus) from Turkey was investigated by phylogenetic analysis of the entire mtDNA control region (CR) (862 bp, n ¼ 182) and partial cytochrome (cyt) b (239 bp, n ¼ 174) sequences. Individuals were collected at nine stations in four geographic locations: North-eastern Mediterranean Sea, Aegean Sea, Sea of Marmara and Black Sea. Polymerase chain reactiondirect sequencing of the CR and the partial cyt b genes produced 28 and 131 distinct haplotypes, respectively. Maximum likelihood, neighbour-joining and maximum parsimony methods produced similar tree topologies. The results of both CR and cyt b sequence analyses revealed the existence of several species-specific nucleotide sites that can be used to discriminate between the three species. Genetic distances indicated that T. mediterraneus and T. picturatus are more closely related to each other than either is to T. trachurus. Inter-nucleotide and intra-nucleotide diversities of T. picturatus were larger than those of T. mediterraneus and T. trachurus. There was no evidence of a geographical difference in haplotype frequencies of these two mtDNA regions # 2008 The Authors to be clustered. Journal compilation # 2008 The Fisheries Society of the British Isles
Key words: control region; cytochrome b; genetic variation; mackerel; marine fish; mtDNA.
INTRODUCTION Species of Trachurus are widely distributed along coastal and oceanic waters of temperate, tropical and subtropical seas (Eschmeyer, 2003). The genus Trachurus is included in the family Carangidae (Perciformes), which consists of 33 genera and 140 species (Froese & Pauly, 2001). Species of Trachurus are pelagic and many are of economic importance. Although numerous studies have focused on morphological and ecological variability among species of Trachurus, the taxonomy and phylogenetic relationships among species remain controversial (Shaboneyev, 1981; Kijima et al., 1988; Ben Salem, 1995). The taxonomy
†Author to whom correspondence should be addressed. Tel.: þ90 462 377 2522; fax: þ90 462 325 3195; email:
[email protected]
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of the genus Trachurus was first described by Nichols (1920), who recognized 12 species in three geographically isolated groups (T. trachurus, T. mediterraneus and T. picturatus). Cardenas et al. (2005) characterized the phylogenetic relationships among 11 recognized species of Trachurus with mtDNA cytochrome (cyt) b and control region (CR) sequences. They suggested that the diversification of the ancestral stock of Trachurus occurred between 16 and 20 million years ago (MYA) and was likely associated with the closure of the Tethys Sea. In that study, CR and cyt b sequences showed similar low levels of divergence between species. The genus Trachurus is represented by three species in Turkish waters, including horse mackerel, Trachurus trachurus (L.), Mediterranean horse mackerel, Trachurus mediterraneus (Steindachner), and blue jack mackerel, Trachurus picturatus (Bowdich) (Whitehead et al., 1986; Fischer et al., 1987). Trachurus trachurus has a global distribution, extending from the eastern Atlantic Ocean, including the Mediterranean Sea, the Black Sea and the Sea of Marmara, to the western Atlantic, and also to the western Pacific and Indian Oceans. Trachurus mediterraneus and T. picturatus have more restricted distributions. The distribution of T. picturatus extends from the Bay of Biscay (France) in the north to Morocco in the south and to the Mediterranean Sea in the east. Trachurus mediterraneus occurs only in the Sea of Marmara and Black Sea. The mtDNA sequences, particularly cyt b and the CR, are frequently used in population genetic and phylogenetic studies of fishes (Liu & Chen, 2003; Peng et al., 2004; Perdices et al., 2004). The CR is a rapidly evolving locus in most species and has been used for both phylogenetic analysis and intraspecific phylogeographic studies of fishes (Avise, 2000). The cyt b gene encodes a protein and hence evolves relatively more slowly (Sbisa` et al., 1997) and has proved to be a robust genetic marker for reconstructing phylogenies of fishes at various levels of evolutionary divergence (Meyer, 1993). Recently, Karaiskou et al. (2003) used partial sequences of cyt b and the 16S rDNA gene to estimate phylogenetic relationships among T. trachurus, T. mediterraneus and T. picturatus. The results indicated that T. picturatus and T. mediterraneus were most closely related and suggested that the origins of the three species reflected isolations across the Straits of Gibraltar 2–5 MYA in the late Miocene (Messinian) and Pliocene. Karaiskou et al. (2003) used restriction enzyme analysis of the CR to investigate the genetic population structures of these species in Atlantic and Mediterranean waters and did not find genetic differences between the Atlantic and Mediterranean populations in these three species. Recently, Comesan´a et al. (2008) studied CR sequence differentiation between Atlantic and Mediterranean samples of T. trachurus from both coasts of the Iberian Peninsula and found high levels of genetic homogeneity among Atlantic and Mediterranean T. trachurus populations, most likely because of the high dispersion capability of fish in this species. The objective of this study is to use cyt b gene and CR variability to investigate the phylogenetic relationships, geographic distributions and genetic divergence of the three Turkish species of Trachurus and to determine if genetic differences can be detected among populations in the North-eastern Mediterranean, Aegean Sea, Sea of Marmara and Black Sea. # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 73, 1228–1248
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MATERIALS AND METHODS SAMPLING AND DNA EXTRACTIONS Individuals of three Trachurus species, T. trachurus, T. mediterraneus and T. picturatus, were collected from five geographical regions in Turkey between November 2005 and January 2006 (Table I and Fig. 1). Species identifications of fish were performed using morphometric and meristic characters according to Nichols (1920), Fischer et al. (1987) and Smith-Vaniz (1986). Trachurus picturatus were sampled at two localities in the Aegean Sea and Northeastern Mediterranean Sea; T. trachurus were sampled at five localities in the western and eastern Marmara Sea, Aegean Sea and at two localities in the Mediterranean Sea and T. mediterraneus were collected from all these localities. Fish were immediately placed on wet ice in the field and transported to the laboratory for identification and dissection. Each specimen was labelled with a tag inserted into the operculum and kept at 80° C until tissue samples could be taken. Approximately 10 mg of white muscle tissue was removed using sterile scalpel blades and forceps. Total genomic DNA was extracted from frozen white muscle tissue by the WizardÒ Genomic DNA Purification kit (Promega Corporation, Madison, WI, U.S.A.). Proteinase K was used during extractions to promote cell lysis and protein digestion. The resuspended DNA was treated with RNase A (1 mg per 100 ml total volume) to remove RNA and incubated at 37° C for 30 min. Extracted DNA was resuspended in DNA rehydration solution. The concentration of extracted DNA was determined using Agilent 8453E UV-visible Spectroscopy System (Agilent Technologies, Santa Clara, CA, U.S.A.), and extractions were stored at 20° C.
P C R A M P L I F I C A T I O N A N D DN A S E Q U EN C I N G Amplifications by polymerase chain reaction (PCR) were performed using the universal primers H15149 and L14841 (Kocher et al., 1989) to amplify sequences of cyt b TABLE I. Origin and number of individuals from three Trachurus species sequenced for mtDNA cytochrome b and control region Genus species
Location
Trachurus mediterraneus
Trachurus trachurus
Trachurus picturatus Total
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Black Sea1 Black Sea2 Black Sea3 Black Sea4 Marmara Sea1 Marmara Sea2 Aegean Sea North-eastern Mediterranean North-eastern Mediterranean Marmara Sea1 Marmara Sea2 Aegean Sea North-eastern Mediterranean North-eastern Mediterranean Aegean Sea North-eastern Mediterranean
Sea1 Sea2
Sea1 Sea2 Sea1
Cytochrome b
Control region
20 6 6 6 10 6 7 6 10 11 10 10 10 12 20 24 174
20 6 6 6 10 10 10 6 10 12 12 12 11 13 18 20 182
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FIG. 1. Map showing sampling locations of Turkish Trachurus species.
(Table II). The entire CR was amplified using primers (Table II) designed from Trachurus CR sequences (GenBank accession numbers AY533482, AY533479 and AY533477). Primers were designed using the web-based programme Primer3 (http://www.frodo.wi. mit.edu/). PCR amplifications were performed with 04–05 mg of template DNA in a reaction mixture of 50 ml containing 5 ml of 10 reaction buffer, 25 mM MgCl2, 1 unit of Taq DNA polymerase (Go Taq, Promega), 02 mM of each dNTP and 1 mM of each primer. Thermal cycling amplification were performed in a BioRad Thermal Cycler (MJ Research, Inc., Walthan, MA, U.S.A.), with an initial denaturation at 95° C for 3 min, followed by 35 cycles of strand denaturation at 94° C for 45 s, annealing at 50° C for 45 s, primer extension at 72° C for 1 min and a final 7 min elongation at 72° C for the cyt b. For the CR, the PCR cycles consisted of an initial denaturation at 95° C for 3 min, followed by 35 cycles of strand denaturation at 94° C for 30 s, annealing at 40° C for 30 s and primer extension at 72° C for 45 s and a final 5 min elongation at 72° C. Sizes of polymerase chain reaction (PCR) products were estimated with a 100-bp DNA ladder (Gibco BRL, Madison, WI, U.S.A.) in 12% agarose gel, run in 1 TBE buffer, stained with 05 mg ml1 ethidium bromide and exposed
TABLE II. List of amplification and sequencing primers used in this study Gene
Name
Cytochrome b H15149*
Primer sequences
59-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-39 59-AAAAAGCTTCCATCCAACL14841*† ATCTCAGCATGATGAAA-39 Control region TracCR_F* 59-CCTTTGCGCAGCGCATATATA-39 TracCR_R*† 59-ATGTGAAATATTATAATAATT-39
Reference Kocher et al. (1989) Kocher et al. (1989) Present study Present study
*Primers used for the amplification. †Primers used for the sequencing.
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under UV lights. The amplified products of partial cyt b and whole CR were 239 and 862 bp, respectively, in length. PCR amplifications were sequenced using BigDye (Applied Biosystems Inc., Foster City, CA, U.S.A.) terminator cycling protocols. PCR products were purified using ethanol precipitation and run on an Automatic Sequencer (ABI 3730x1; Applied Biosystems) by a contract laboratory. Sequencing of the 59 end of cyt b was carried out using the primer L14841 (Kocher et al., 1989). Sequences with ambiguous sites were resequenced from the 39 end (Table II). The CR was sequenced with degenerate TracCR_R designed by the authors (Table II). PCR product purification and DNA sequence analysis were performed by Macrogen Inc. (Seoul, Korea).
PHYLOGENETIC ANALYSES Sequences of cyt b and CR were aligned with homologous sequences in related species from the GenBank database with BIOEDIT (Thompson et al., 1997), and alignments were checked by eye and repeated using different parameter values. New sequences of cyt b were deposited in GenBank under accession numbers EU246350–EU246377 (Table III) and sequences of CR under accession numbers EU246378–EU246508 (Table IV). Nucleotide composition and basic diversity statistics were calculated with DNASP 3.5.3 (Rozas & Rozas, 1999) and MEGA 3.1 (Kumar et al., 2004). Phylogenetic analyses were performed with MEGA using neighbour-joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) methods of tree construction and using 1000 bootstrap iterations (Felsenstein, 1985). For cyt b analyses, sequences of Seriola dumerili (Risso) (GenBank EU036499) and Caranx crysos (Mitchill) (GenBank EF392575) were selected as closely related outgroups. Phylogenetic trees of CR haplotypes were constructed without an outgroup. Sequence divergences were calculated using the Kimura’s two-parameter (K2P) distance model (Kimura, 1980). NJ trees of K2P distances provided a graphical representation of divergence between species (Saitou & Nei, 1987). Global and pair-wise differentiation between taxa was assessed using the exact test of Raymond & Rousset (1995), as implemented in ARLEQUIN (Schneider et al., 2000).
RESULTS SEQUENCE VARIATIONS OF
CYT B
The 59 end of cyt b (239 bp) was amplified from 174 individuals (Table I). A total of 30 (13%) nucleotide sites were variable of which 17 (7%) positions were parsimony informative (Table III). Variable sites included 24 transitions and five transversions with reference to haplotype TmCytb01. Position 220 had both transitions (ti) and transversions (tv). Within species, T. mediterraneus had 10 ti only, T. trachurus had seven ti and one tv and T. picturatus eight ti and two tv. Mean ti/tv ¼ 416 over species. A major codon base compositional bias existed at the third codon position, with 23 of 26 substitutions occurring in this position. One mutation (143 G-A transitions) occurred at the second position of codon, producing a glycine to glutamic acid replacement in haplotype TmCytb10 (amino acid position 48), and two mutations (the 70 A-C and 181 G-A) were in first codon position, producing an asparagine to histidine replacement in haplotype TpCytb07 (amino acid position 24) and valine to isoleucine replacement in haplotype TtCytb08 (amino
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11111111111111122222 127777900124445678889902233 147020136436274584382473920628
GCCGACATGCCTTCGGACGAGCGATGACTA .........................A.... ............C................. ..........T................... ...A.......................... .............................G ..T........................... ..............A............... ........................C..... ................G............. .......C......................
..T.G.C..T...T.C..A..TT...G... ..T......T.....C..A..TT...G... ..T......T...T.C..A..TT...G..G ..T......T...T.C..A..TT...G... ..T......T...T.C..A..TT...G... ..T......T...T.C..A..TT...G.C. ..T......T...T.C..A..TTC..GT.. .T.......T...T.C..A..TT...G...
Haplotype numbers
TmCytb 01 02 03 04 05 06 07 08 09 10 11
TpCytb 01 02 03 04 05 06 07 08
Haplotype variable site
EU246361 EU246362 EU246363 EU246364 EU246365 EU246366 EU246367 EU246368 Total
EU246350 EU246351 EU246352 EU246353 EU246354 EU246355 EU246356 EU246357 EU246358 EU246359 EU246360 Total
GenBank accession numbers 6
6
2 1 1 20
BS2
16
BS1
6
10
1
1 1
6
2
1
2
7
MS1
3
BS4
4
BS3
Black Sea
6
1
5
MS2
Sea of Marmara
Locality
4 12 1 1 1 1 20
7
3 1 1
2
AS
Aegean Sea
1 24
1 3 2 13 4
6
4 1 1
NMS1
10
1
1
8
NMS2
North-eastern Mediterranean Sea
TABLE III. Nucleotide variation and frequencies of the cytochrome b haplotypes in samples of three species of Trachurus from Turkish waters. Position numbers indicate variable sites and a full stop indicate identical bases to the reference sequence
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11111111111111122222 127777900124445678889902233 147020136436274584382473920628
GCCGACATGCCTTCGGACGAGCGATGACTA ...A.T.C.....T.CG.AG..T..AT... ...A.T.C.....T.C..AG..T..AT... ...A.T.CA....T.C..AG..T..AT... ...A.T.C.....T.C.AAG..T..AT... ...A.T.C.....T.C..AGA.T..AT... ...A.T.C.....T.C..AG..T.CAT... ...A.T.C...C.T.C..AG..T..AT... .T.A.T.C.....T.C..AG..T..AT... A..A.T.C.....T.C..AG..T..AT...
Haplotype numbers
TtCytb 01 02 03 04 05 06 07 08 09
Haplotype variable site
EU246369 EU246370 EU246371 EU246372 EU246373 EU246374 EU246375 EU246376 EU246377 Total
GenBank accession numbers BS1
BS2
BS3
Black Sea
TABLE III. Continued
BS4
11
1
1 9
MS1
1 10
2
7
MS2
Sea of Marmara
Locality
10
10
AS
Aegean Sea
10
6 1 3
NMS1
12
1 1
2
8
NMS2
North-eastern Mediterranean Sea
1234 Y. BEKTAS AND A. O. BELDUZ
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TABLE IV. Variable positions of 131 mtDNA control region haplotypes among three species of Trachurus from Turkey. Full stops represent matches with nucleotides in haplotype TmCR1. Shaded regions show species-specific nucleotide positions
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TABLE IV. Continued
Y. BEKTAS AND A. O. BELDUZ
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acid position 61), respectively. A G-A transition at site 75 occurred in the third codon position, resulting in a methionine to isoleucine replacement in haplotype TtCytb05 (amino acid position 25). Finally, an isoleucine occurred at position 67 in T. trachurus but methionine in T. mediterraneus and T. picturatus. A total of 28 cyt b haplotypes appeared among the three species of Trachurus (Table III). Eleven haplotypes appear in 77 individuals of T. mediterraneus, with haplotype TmCytb01 appearing at a high frequency (55 fish) in all samples. Haplotype TmCytb03 was found in 10 individuals from six locations, and haplotype TmCytb07 was found in three fish from three locations. Eight singleton haplotypes, one substitution from haplotype TmCytb01, were found in T. mediterraneus. A total of nine haplotypes appeared in 53 individuals of T. trachurus. Haplotype TtCytb02 (37 individuals) appeared in all samples; haplotype TtCytb04 occurred in five individuals from two locations; seven haplotypes were singletons. A total of eight haplotypes appeared in 44 individuals of T. picturatus, with each of these haplotypes TpCytb03, TpCytb04, TpCytb05 and TpCytb08 appearing in two populations. The remaining haplotypes were singletons. Several single nucleotide polymorphisms (SNP) in cyt b sequences showed fixed differences between taxa. Eight differences were found between T. mediterraneus, T. trachurus and T. picturatus, which can be used to identify individuals of each species. Trachurus mediterraneus had G at positions 145, 163 and 187, whereas T. trachurus and T. picturatus had C, A and T, respectively, in these position (Table III). At positions 70 and 178, T. trachurus has T and G, whereas T. mediterraneus had C and A, and T. picturatus had C and A, respectively. At positions 94 and 184, T. picturatus had T, whereas T. mediterraneus and T. trachurus had C. Finally, at position 220, T. trachurus had T, whereas T. mediterraneus had A and T. picturatus had G (Table III).
CR VARIATION
The overall length of the CR (862 bp, n ¼ 182) was the same in the three species (Table I). A total of 110 (13%) positions were variable of which 77 (9%) were parsimony informative and defined 131 haplotypes (Table IV). Sixty-four haplotypes appeared in 84 individuals of T. mediterraneus, with haplotypes TmCR01 and TmCR06 occurring in seven and three individuals, respectively, from three regions. Another six haplotypes were found in two regions. The remaining 56 haplotypes occurred in only one fish (singletons) and were each one substitution removed from TmCR1. In T. trachurus, 40 haplotypes appeared in 60 individuals. Haplotypes TtCR01 and TtCR 08 were shared by eight and four individuals, respectively, from four regions, and haplotypes TtCR02 and TtCR09 were each shared by three individuals from three regions. Haplotypes TtCR06 and TtCR 11 were shared by two and three individuals, respectively, from two regions, and 34 haplotype were singletons. In T. picturatus, 27 haplotypes appeared in 38 individuals, with haplotypes TpCR03, TpCR 12, TpCR 15 and TpCR21 shared by two populations. Twenty-three haplotypes occurred as singletons (Table IV).
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Several CR SNPs distinguished the three species. Nucleotide positions 153(C), 183 (T), 184 (T), 217 (T), 252 (A), 290 (A), 471 (T), 509 (A), 625 (C), 546 (A) and 701 (C) distinguished T. mediterraneus from the other species. For T. trachurus, diagnostic nucleotide positions included 133 (T), 184 (A), 246 (A), 268 (A), 271 (C), 540 (T), 558 (C) and 701 (C). Finally, T. picturatus had an eight species-specific SNPs at positions 40 (T), 63 (C), 75 (A), 113 (A), 184 (G), 590 (G), 98 (A) and 142 (A) (Table IV). POPULATION ANALYSIS
Cytochrome b haplotypic diversities were larger for T. picturatus (h ¼ 0652) than for T. trachurus (0426) and T. mediterraneus (0476) (Table V). Nucleotide diversities within and among populations for the three species were larger in T. picturatus (average, 00041 and 00048, respectively) than in T. trachurus (average, 00020 and 00024) and T. mediterraneus (average, 00023 and 00026) (Table V). Global tests of population differentiation were not significant among the nine populations of T. mediterraneus (P ¼ 0126), the two populations of T. picturatus (P ¼ 0299) and five populations of T. trachurus (P ¼ 0052). CR haplotypic diversities were h ¼ 0979 in T. picturatus, 0969 in T. trachurus and 0989 in T. mediterraneus (Table V). Nucleotide diversities estimated within and among populations for the three species were larger in T. picturatus (average, 00067 and 00072, respectively) than in T. trachurus (average, 00038 and 00042) and T. mediterraneus (average, 00086 and 00087) (Table V). Global tests of population differentiation were not significant among the nine populations of T. mediterraneus (P ¼ 0387), two populations of T. picturatus (P ¼ 0408) or five populations of T. trachurus (P ¼ 0341). P H Y L O G E N E T I C A N A L Y SI S
Pair-wise sequence divergences between cyt b haplotypes ranged between 042 and 084% in T. mediterraneus, 042 and 170% in T. picturatus and 042 and 084% in T. trachurus. Sequence divergences between species ranged from 256 to 478% between T. picturatus and T. mediterraneus, 390 to 525% between T. picturatus and T. trachurus and 388 to 523% between T. trachurus and T. mediterraneus. Sequence divergences between CR haplotypes ranged between 012 and 201% in samples of T. mediterraneus, 028 and 115% in T. picturatus and 028 and 114% in T. trachurus. Divergences between species ranged from 284 to 418% between T. picturatus and T. mediterraneus, 308 to 444% between T. picturatus and T. trachurus and 332 to 556% between T. trachurus and T. mediterraneus. Neighbour-joining trees of cyt b and CR haplotypes appear in Figs 2 and 3. Three major lineages corresponding to the three species were identified within Trachurus. These lineages clustered into two groups. One group included the haplotypes of T. picturatus and T. mediterraneus and was supported by high bootstrap values, whereas the second group included the lineage of T. trachurus. The topologies of the MP and ML trees were similar to that of the neighbourjoining tree. # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 73, 1228–1248
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TABLE V. Locations of samples of Trachurus, sample size, number of haplotypes, haplotype diversity (h) and nucleotide diversity (p) in the control region and cytochrome b partial sequences (S.D. in parentheses) Species Cytochrome b T. mediterraneus
T. trachurus
T. picturatus
Control region T. mediterraneus
T. trachurus
T. picturatus
Sample size
Number of haplotypes
BS1 BS2 BS3 BS4 MS1 MS2 AS NMS1 NMS2 Total MS1 MS2 AS NMS1 NMS2 Total AS NMS1 Total
20 6 6 6 10 6 7 6 10 77 11 10 10 10 12 53 20 24 44
4 1 2 4 3 2 4 3 3 11 3 3 1 3 4 9 6 6 8
0363 0000 0533 0800 0511 0333 0810 0600 0378 0476 0346 0511 0000 0600 0561 0426 0621 0681 0652
( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (
0046) 0000) 0076) 0078) 0090) 0067) 0045) 0084) 0078) 0062) 0049) 0056) 0000) 0086) 0071) 0052) 0069) 0066) 0067)
000163 000000 000223 000418 000233 000139 000480 000279 000168 000225 000152 000232 000000 000279 000266 000199 000375 000423 000406
( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (
000039) 000000) 000060) 000084) 000036) 000056) 000082) 000028) 000047) 000048) 000058) 000037) 000000) 000062) 000043) 000040) 000055) 000082) 000071)
BS1 BS2 BS3 BS4 MS1 MS2 AS NMS1 NMS2 Total MS1 MS2 AS NMS1 NMS2 Total AS NMS1 Total
20 6 6 6 10 10 10 6 10 84 12 12 12 11 13 60 18 20 38
14 5 6 6 9 10 9 6 9 64 10 10 9 11 12 40 15 16 27
0953 0933 1000 1000 0978 1000 0978 1000 0978 0990 0955 0970 0909 1000 0987 0969 0980 0974 0979
( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (
0088) 0094) 0000) 0000) 0067) 0000) 0082) 0000) 0050) 0043) 0055) 0061) 0038) 0092) 0054) 0060) 0071) 0063) 0066)
000904 000766 000858 000758 000786 000838 000985 000700 000866 000859 000339 000476 000327 000388 000345 000384 000673 000652 000669
( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (
000086) 000077) 000028) 000079) 000057) 000047) 000059) 000084) 000063) 000062) 000069) 000058) 000087) 000090) 000075) 000076) 000046) 000077) 000063)
Location
h ( S.D.)
p ( S.D.)
DISCUSSION G E O GR A P H I C D I S T R IB U T I O N
Although Demir (1961) proposed that only T. mediterraneus was present along the Turkish Black Sea coast, Smith-Vaniz (1986) reported the existence of T. trachurus in the Black Sea. In this study, no T. trachurus were found in
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FIG. 2. Neighbour-joining tree of 28 haplotypes of Trachurus species recovered from cytochrome b sequences. Seriola dumerili and Caranx rhoncus (Carangidae) were used to root the tree. Only bootstrap values based on 1000 replications larger than 50% are displayed. Maximum likelihood and the maximum parsimony trees had similar topologies.
samples from the Turkish Black Sea coast, supporting the findings of Demir (1961). Biometric indices were insufficient to distinguish two horse mackerel subpopulations in the Black Sea (Yankova & Raykov, 2006). According to Prodanov et al. (1997), Black Sea horse mackerel consist of a single population as the environmental conditions are almost the same throughout the Black Sea. The present mtDNA analysis also indicated that there were no subpopulations of T. mediterraneus along the Turkish Black Sea coast. The samples from this region were also genetically similar to samples from the other regions (Aegean Sea, Sea of Marmara and North-eastern Mediterranean Sea). This genetic similarity among geographically contiguous populations can be attributed to the gene flow. The widespread genetic homogeneity also discredits sub-species designations for populations of T. mediterraneus in Turkish Black Sea waters. CYT B
AND CR SEQUENCE VARIATION
CR sequences showed a greater level of divergence between haplotypes, on average, than did sequences of cyt b, and hence larger values of intra-nucleotide and inter-nucleotide diversities. This may have been because of the longer CR # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 73, 1228–1248
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sequences that were examined in this study, which uncovered a greater number of variable SNPs than in cyt b. Also, the CR may also have a greater rate of mutation than in cyt b, which produces longer phylogenetic branches in the same population than for cyt b. The present results show that the cyt b and the CR sequences provide suitable markers for the discrimination of the three species of Trachurus in Turkish waters and provide insights into their phylogenetic relationships. Based on the cyt b gene sequence comparisons among species, eight diagnostic SNPs allow the identification of individuals of these species. Only one difference resulted in an amino acid change (amino acid position 67), which distinguished T. trachurus from T. mediterraneus and T. picturatus. In a previous study, a phylogenetic analysis of cyt b was made on the European Trachurus species (Karaiskou et al., 2003). Haplotype Tm1 (AF489409) in the earlier study is identical with the haplotype TmCytb01 (EU246350) in the present study and occurs at a high frequency in T. mediterraneus. Haplotype Tp7 (AF489420) is identical with haplotype TpCytb04 (EU246364) and is characteristic of T. picturatus. Haplotype Tt3 (AF489405) is the same as haplotype TtCytb02 (EU246370) and is characteristic of T. trachurus. The CR sequences also provide species diagnostic SNPs. The first 300 bp of this region are highly conserved and can be used as a molecular marker for molecular identification of the three Trachurus species. In an earlier study, Cardenas et al. (2005) determined the origin, diversification and historical biogeography of some of Trachurus species with the CR sequences of T. mediterraneus, T. trachurus and T. picturatus. The identification of fish from the three species in the present study was further supported by their geographic distributions and by published sequences from Karaiskou et al. (2003) and Cardenas et al. (2005). POPULATION STRUCTURE
Based on cyt b sequence analysis, haplotype diversities in T. trachurus (h ¼ 0476) and T. mediterraneus (0426) were similar but less than diversities in T. picturatus (0652) (Table V). Inter-nucleotide and intra-nucleotide diversities in T. picturatus were also larger (0406 and 0477%, respectively) than those in the other two species. These differences may indicate a difference in the effective population sizes of these species. For the CR, the high values of haplotype diversity (Table V) found in the present study were similar to those found by Karaiskou et al. (2004) and Comesan´a et al. (2008). Few genetic differences have been found among populations of Trachurus. This lack of geographical variation, even on large geographical scales, is likely because of the migratory ability of pelagic fishes and high levels of gene flow between populations (Grewe & Hampton, 1998). Although Karaiskou et al. (2003) found significant differences among T. picturatus populations, the lack of differences in the present study may be because of the inclusion of samples FIG. 3. Neighbour-joining tree of the 131 control region haplotypes of Trachurus species without an outgroup. Only bootstrap values based on 1000 replications >50% are displayed. Maximum likelihood and maximum parsimony trees had similar topologies.
# 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 73, 1228–1248
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from only two contiguous T. picturatus populations, which were not separated by a physical barrier to migration. Hence, mitochondrial CR and partial cyt b gene sequences did not provide clear evidence for the existence of population differentiation within species in the Turkish waters. PHYLOGENETIC RELATIONSHIPS
Sequence divergences based on cyt b between Turkish Trachurus species (mean 256–525%; present study) represent some of the lowest values reported between marine fish species (mean 37–13%; Johns & Avise, 1998). Similar low cyt b sequence divergences between species of Trachurus were reported by Karaiskou et al. (2003; 213–525%) and Cardenas et al. (2005; 343 1%). Cardenas et al. (2005) also reported low levels of divergence between Trachurus species (mean 35 1%) for the CR. The low level of CR divergence between species (mean 284–556%) in the present study also supports the hypothesis of Cardenas et al. (2005) that low levels of divergence between species might be a common characteristic for the mtDNA in Trachurus. The phylogenetic analyses, based on three haplotype trees (NJ, ML and MP), were in accordance with the taxonomies of T. trachurus, T. picturatus and T. mediterraneus as no haplotypes were shared between species. The phylogenetic reconstruction showed that T. mediterraneus and T. picturatus were most closely related (Figs 2 and 3) and that T. trachurus was in the most basal position of these three Trachurus species. This basal position suggests that T. trachurus differentiated earlier from a common ancestor than the other two species.
DI V E R G E NC E T IM E A ND Z O O GE O GR A P HY
Both cyt b and CR sequences indicated shallow divergences among the three Trachurus species were low (39 13 and 42 14%, respectively). These sequence divergences indicated times of separation among the three Trachurus species of 26–65 MYA during the late Miocene or early Pliocene, using a temporal calibration of 1–12% per million years for cyt b (Bermingham et al., 1997). At this time, the Mediterranean Sea became isolated from the Atlantic Ocean, and the Paratethys Sea became fragmented into the present-day Black, Caspian and Aral Seas (Bianco, 1990). The Black Sea was severely affected by the Mediterranean Messinian Salinity Crisis and suffered a drastic lowering of water level at the end of the Messinian (Gillet et al., 2007). Most of the present-day Mediterranean species entered the region during the early Pliocene when the connection with the Atlantic was re-established slightly
