Phylogeny of the genus Merluccius based on mitochondrial and nuclear genes

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Gene 406 (2007) 171 – 179 www.elsevier.com/locate/gene

Phylogeny of the genus Merluccius based on mitochondrial and nuclear genes Daniel Campo, Gonzalo Machado-Schiaffino, Juliana Perez, Eva Garcia-Vazquez ⁎ Departamento de Biologia Funcional, Universidad de Oviedo. C/ Julian Claveria s/n. 33006-Oviedo, Spain Received 15 January 2007; received in revised form 24 August 2007; accepted 6 September 2007 Available online 19 September 2007

Abstract Genus Merluccius is considered one of the most important groups within the Teleostei, and comprises 12 extant species distributed along the coasts of America, Europe and Africa, being its fisheries very important in these continents. Despite their noticeable economical importance for humans, to date the phylogeny of hakes has not been clearly established. In this study we used mitochondrial sequences (the ribosomal genes 12S rDNA and 16S rDNA, the coding gene cytochrome b and the control region) and the nuclear 5S rDNA conserved region in order to determine the phylogenetic and biogeographical relationships within the genus Merluccius. This is the first time that all the species of this genus recognized by the FAO are included in a phylogeny. Maximum Parsimony, Maximum Likelihood and Bayesian analyses of the mitochondrial sequences suggest that the geographical origin of the genus was the North Atlantic Ocean, and indicate that two main clades, early separated in the evolution, exist within the genus: one American (7 species) and one Euro-African (5 species). Among the American species, M. bilinearis seems to be the most ancient one, and the rise of the Panama Isthmus could act as a physical barrier leading to further processes of speciation. Within the Euro-African clade, successive events of geographical differentiation could explain the observed pattern of species distribution. Therefore, we propose both vicariant speciation and geographical dispersion as main mechanisms to explain the evolutionary history of the genus Merluccius. © 2007 Elsevier B.V. All rights reserved. Keywords: 12S rDNA; 16S rDNA; Cytochrome b; Control region; 5S rDNA; Merluccius; Phylogeny; Biogeography; Speciation mechanisms

1. Introduction One of the most important groups within the Teleostei is the genus Merluccius (Rafinesque, 1810), which comprises 12 morphologically well characterized species (Inada, 1981). They are distributed along the coasts of Europe (Merluccius merluccius) and West Africa (from north to south M. senegalensis, M. polli, M. capensis, M. paradoxus), northeast America (M. bilinearis, M. albidus), south east America (M. hubbsi, M. australis), northwest America (M. productus, M. angustimanus), southwest America (M. gayi, M. australis), and the east of New Zealand (M. australis). Their distribution is shown in Fig. 1. Another species (M. hernandezi) was described from the west American coast based Abbreviations: rDNA, ribosomal DNA; PCR, Polymerase Chain Reaction; dNTP, Deoxyribonucleotide triphosphate; bp, base pairs; ML, Maximum Likelihood; MP, Maximum Parsimony; TBR, Tree-Bisection-Reconnection; AIC, Akaike Information Criterion; mtDNA, mitochondrial DNA; CR, Control Region; Cytb, Cytochrome b. ⁎ Corresponding author. Tel.: +34 985102726; fax: +34 985103534. E-mail address: [email protected] (E. Garcia-Vazquez). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.09.008

on morphological traits but its taxonomic position is not clear, being considered by the FAO a variant of M. angustimanus (Cohen et al., 1990). Hake fisheries are very important for many regions (Pitcher and Alheit, 1995), both in America (Argentina, Uruguay, Peru, and the west coast of the USA from Washington to California) and in the Old Continents (Namibia, west Europe, South Africa). Despite their noticeable economical importance for humans, phylogenetic relationships among the species of this genus have not been widely studied. A few studies have been published about the evolution of the genus Merluccius based on genetic data. Roldán et al. (1999) traced genetic relationships among nine Merluccius species on the basis of variation at 21 allozyme loci; Quinteiro et al. (2000) analyzed sequence variation of the left domain of the mitochondrial DNA control region (450 base pairs) for 26 individuals of 11 species; Grant and Leslie (2001) made a review of previous published genetic data bearing on the phylogeny of hakes in the genus Merluccius, and combined them with new 20 allozyme loci data. According to these and other morphological and biogeographical studies, hakes may have

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Fig. 1. Map of distribution of the twelve Merluccius species (Pitcher and Alheit 1995; modified by Castillo, 2002).

emerged in the North Atlantic during the middle Oligocene (Kabata and Ho, 1981; Inada, 1981; Fedotov and Bannikov, 1989). The ancestral lineage was probably divided between two geographical regions, east (Europe and Africa) and west (America) (Roldán et al., 1999; Quinteiro et al., 2000; Grant and Leslie, 2001). The genus Merluccius probably reached the Pacific through the then-open Isthmus of Panama (i.e. Inada, 1981; Ho, 1990). The origin of Argentine hake, M. hubbsi, was not clear. Szidat (1955) and Inada (1981) postulated a south Pacific origin, whereas Ho (1990) and Kabata and Ho (1981) suggested that the species was derived from the North Atlantic stock. Genetic data existing to date (Stepien and Rosenblatt, 1996; Roldán et al., 1999; Quinteiro et al., 2000; Grant and Leslie, 2001) supports Inada's hypothesis (Grant and Leslie, 2001). In this study we use partial mitochondrial sequences (a total of 1593 base pairs long comprising 12S and 16S rDNA, cytochrome b gene and control region) to reconstruct the phylogeny of the genus Merluccius, in an attempt to trace the evolutionary history of hakes. The conserved region of the nuclear 5S rDNA gene (120 base pairs long) was also sequenced in all species. 2. Materials and methods 2.1. Sampling Eight to ten adult individuals per species were collected for all species except for M. angustimanus, for which only two samples were obtained. The individuals were sampled from different regions (two to three individuals per region) to cover

the geographical distribution range of each species. Tissue samples were muscle or gill biopsies preserved in ethanol and stored at 4 °C until analysis. A total number of 107 Merluccius individuals were collected for this study (Table 1). 2.2. DNA extraction and PCR amplification Total DNA was extracted using a Chelex Resin protocol (Estoup et al., 1996). PCR amplification of 12S and 16S rDNA fragments were done using the universal primers described by Kocher et al. (1989) and Palumbi et al. (1991) respectively. PCR amplification of the cytochrome b was performed using the primers H15149 and L14841 described by Kocher et al. (1989). The primers M.mer HK01 and M.mer HK02 (Lundy et al., 2000) were employed to amplify the control region, as in Quinteiro et al. (2000). PCR reactions were carried out separately in a total volume of 40 μl containing Dynazyme Buffer 1×, 250 μM of each dNTP, 40 pmol of each primer, 0.6 μl of Dynazyme II DNA polymerase (Finnzymes), and 4 μl of total DNA. PCR was performed using the GeneAmp PCR system 2400 by Perkin Elmer Cetus with the following conditions: an initial denaturing step at 95 °C for 5 min, followed by 30 cycles of denaturing at 95 °C for 30 s, annealing (for 30 s) at 60 °C for 12S rDNA, 53 °C for the control region and 50 °C for 16S rDNA and cytochrome b; and an extension at 72 °C for 30 s, ending with a final extension at 72 °C for 10–15 min. PCR amplification of 5S rDNA genes followed the protocol described by Pérez and García Vázquez (2004), employing the primers 5S C: 5′-AAGCTTACAGCACCTGGTATT-3′ and 5S MD: 5′-TTCAACATGGGCTCCGACGGA-3′ described therein.

D. Campo et al. / Gene 406 (2007) 171–179 Table 1 Species analyzed, number of individuals sequenced per species and number of haplotypes found with the program Collapse1.2 (Posada, 2004) Species

Individuals sequenced

Haplotypes for CR + Cytb

Haplotypes for 12S + 16S + CR + Cytb

M. M. M. M. M. M. M. M. M. M. M. M.

10 2 10 9 9 8 9 10 10 10 10 10

3 2 3 5 6 6 4 4 2 4 4 2

4 2 2 3 3 4 2 5 2 5 4 2

productus angustimanus gayi australis bilinearis albidus hubbsi merluccius senegalensis polli capensis paradoxus

CR + Cytb: combined dataset of control region and cytochrome b sequences. 12S + 16S + CR + Cytb: combined dataset of four partial mitochondrial genes (12S, 16S, control region and cytochrome b).

2.3. DNA purification and sequencing PCR products were visualized in 50 ml 1.5% agarose gels with 3 μl of 10 mg/ml ethidium bromide. Stained bands were excised from the gel and DNA was purified with an Eppendorf PerfectPrep Gel CleanUp Kit prior to sequencing. Automated fluorescence sequencing was performed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) with BigDye 3.1 Terminator system, in the Unit of Genetic Analysis of the University of Oviedo (Spain). 2.4. Phylogenetic analyses Sequences were edited using the BioEdit Sequence Alignment Editor software (Hall, 1999) and aligned with the ClustalW application (Thompson et al., 1994) included in BioEdit with a penalty of 6 for gap opening and 4 for gap extension. Some gaps were introduced in order to solve the alignments for the 16S and control region sequences; however, as all positions could be clearly aligned they were included in the analyses. The different haplotypes found for each species were obtained with the program Collapse 1.2 (Posada, 2004). Two datasets were tested for congruence employing the partition homogeneity test using 1000 replicates as implemented in PAUP (ver. 4.0b10; Swofford, 2003), combined control region and cytochrome b sequences with a total length of 744 base pairs (bp) on the one hand, and a combination of four partial mitochondrial gene sequences (12S, 16S, control region, cytochrome b) with a total length of 1593 bp on the other hand. Maximum Likelihood (ML) trees were obtained with PHYML (Guindon and Gascuel, 2003). Maximum Parsimony (MP) analyses were done with program PAUP using a heuristic search with 10 random-addition-sequence replicates and the TBR algorithm for branch-swapping; gaps were treated as a fifth state. The statistical robustness of tree nodes was tested with 100 bootstrap replicates (Felsenstein, 1985) in both cases. Bayesian analyses were performed in MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001) with default settings to establish the initial

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heating values for four Markov chains, which ran simultaneously and were sampled every 100 cycles. The ModelTest (ver. 3.06) software (Posada and Crandall, 1998) was employed to determine the model of sequence evolution that best fitted our datasets, and to calculate the proportion of invariable sites and the value of the gamma distribution shape parameter. MrModelTest ver. 2.2 (Nylander, 2004) was employed to obtain the same information for each gene fragment to be included in MrBayes 3.1.2, since this program permits to implement an independent model of molecular evolution and model parameters for each gene partition of the dataset. In both cases the Akaike Information Criterion (AIC) was followed. Gadus morhua (Gadiformes: Gadidae) was included in all analyses as outgroup. The sequences of this species for the genes here analyzed were obtained from GenBank, with Accession numbers AY842450 for the 12S rDNA, AY850363 for 16S rDNA U12063 for the control region and DQ174046 for the cytochrome b. The program MEGA3 (Kumar et al., 2004) was employed to calculate the Kimura 2-parameters (Kimura, 1980) genetic distances between all the cytochrome b sequences of Merluccius species here analyzed and to draw the phylogenetic trees. 3. Results Mitochondrial and nuclear sequences were obtained, edited and aligned. Inconsistent 5′ and 3′ fragments containing undetermined nucleotides were removed manually. For the 107 individuals of Merluccius collected both 12S and 16S were sequenced; 45 of them were also sequenced for the control region and the cytochrome b. Sequences are available in the GenBank database (http://www.ncbi.nlm.nih.gov/). 12S rDNA haplotypes have the Accession numbers DQ274000 to DQ274015 and 16S rDNA haplotypes correspond to Accession numbers DQ274016 to DQ274041. Cytochrome b haplotypes are EF362882 to EF362915. Control region haplotypes correspond to EF362839 to EF362874. 5S rDNA sequences are EF362916 to EF362948. 3.1. Combined control region–cytochrome b Partition homogeneity test revealed no significant differences between the control region and the cytochrome b datasets (P = 0.32), and therefore phylogenetic analysis was conducted with combined sequences. Forty five different haplotypes were obtained from the 107 combined control region–cytochrome b sequences of Merluccius (Table 1). Amongst the 744 sites examined, 288 were variable, of which 142 were parsimony informative. The G + C content was 0.438. The model of evolution obtained from ModelTest (Posada and Crandall, 1998) was the Hasegawa, Kishino, Yano 85 (HKY) (Hasegawa et al., 1985), with a proportion of invariable sites of 0.3308, a gamma distribution shape parameter of 0.5732 and a transition/ transversion ratio of 3.193. All these parameters were used to perform ML analysis. The models of evolution obtained with MrModelTest (Nylander, 2004) were the HKY for the control region and the General Time Reversible (GTR) (Tavaré, 1986) for

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the cytochrome b. Likelihood values of Bayesian trees reached stationarity after 1,000,000 of generations. The final tree obtained from this dataset is shown in Fig. 2. 3.2. Combined 12S–16S-control region–cytochrome b For this dataset, the partition homogeneity test revealed a statistically significant difference between the four gene fragments (P = 0.02). However, subsequent analyses were done with this combination of four mitochondrial genes in an attempt to solve the deeper branches of the phylogenetic trees, since combining both

slow evolving (12S, 16S) and fast evolving genes (control region, cytochrome b) in a single phylogenetic analysis can help to solve ambiguous nodes. Thirty eight different haplotypes were obtained from the 46 combined 12S–16S-control region–cytochrome b sequences of Merluccius (Table 1). Amongst the 1593 sites examined, 426 were variable, from which 200 were parsimony informative. The G + C content was 0.468. The model of evolution obtained from ModelTest (Posada and Crandall, 1998) was the Tamura–Nei (Tamura and Nei, 1993), with a proportion of invariable sites of 0.5459 and a gamma distribution shape parameter of 0.5807;

Fig. 2. ML tree topology for Merluccius species based on combined control region and cytochrome b sequences (744 bp). MP and Bayesian analyses gave identical estimates of relationships. Bootstrap support values above 50% after 100 replicates are indicated above the branches for ML and MP analyses respectively, and Bayesian posterior probabilities are given below the branches. Outgroup: Gadus morhua.

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these parameters were used to perform ML analysis. The model of evolution revealed by MrModelTest (Nylander, 2004) was the Symmetrical model (Zharkikh, 1994) for both the 12S and the 16S sets of sequences (see above for control region and cytochrome b); these parameters were used for the construction of the Bayesian phylogeny, which reached stationarity after 600,000 generations. The final tree obtained from this dataset is shown in Fig. 3.

species employing the cytochrome b sequences. Divergence values ranged from 0.5% between M. productus and M. angustimanus and 13.5% between M. productus and M. merluccius. Among all the American species of Merluccius, the mean divergence (±SE) was 4.7 ± 0.007%, whereas the mean divergence between the European and African species was 5.3 ± 0.009%. The mean value of divergence between hakes from different continents was 11.5%.

3.3. Genetic distances

3.4. Phylogenetic relationships

Table 2 presents the matrix of genetic distances estimated with the Kimura 2-parameters method for all the Merluccius

Tree topologies obtained from two different datasets (combined control region–cytochrome b and combined 12S–16S-control

Fig. 3. ML tree topology for Merluccius species based on the combination of four mitochondrial partial gene sequences (12S, 16S, control region and cytochrome b) (1593 bp). MP and Bayesian analyses gave identical estimates of relationships. Bootstrap support values above 50% after 100 replicates are indicated above the branches for ML and MP analyses respectively, and Bayesian posterior probabilities are given below the branches. Outgroup: Gadus morhua.

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Table 2 Matrix of Kimura 2-parameters genetic distances (Kimura, 1980) between cytochrome b sequences of 12 Merluccius species Species

1

2

3

4

5

6

7

8

9

10

11

1: M. productus 2: M. angustimanus 3: M. gayi 4: M. australis 5: M. bilinearis 6: M. albidus 7: M. hubbsi 8: M. merluccius 9: M. senegalensis 10: M. polli 11: M. capensis 12: M. paradoxus

0.005 0.017 0.040 0.074 0.049 0.045 0.135 0.133 0.113 0.129 0.110

0.015 0.041 0.072 0.047 0.048 0.132 0.131 0.111 0.126 0.109

0.046 0.070 0.060 0.061 0.122 0.121 0.105 0.116 0.107

0.067 0.035 0.042 0.123 0.121 0.105 0.116 0.099

0.077 0.067 0.113 0.110 0.101 0.117 0.103

0.038 0.126 0.132 0.108 0.106 0.106

0.117 0.115 0.100 0.119 0.104

0.015 0.075 0.050 0.079

0.074 0.053 0.084

0.076 0.040

0.074

region–cytochrome b sequences) were almost identical (Figs. 2 and 3). In both cases, the three methods employed for phylogenetic reconstruction (ML, MP and Bayesian) yielded identical results for each dataset, with high statistical support for most nodes. The main result of these trees is the early separation of two major lineages within the genus Merluccius, one American clade including both east Pacific and west Atlantic species, and one Euro-African clade comprising all the east Atlantic hakes. Within the American clade, the northern Atlantic hake M. bilinearis was clearly separated from the rest of species in both trees. The next level of clustering grouped Pacific species (M. productus, M. angustimanus and M. gayi) together, apart from the other three species (the North Atlantic M. albidus in one branch as a sister taxon of the clade comprising the two South American M. hubbsi and M. australis). The only difference between the two tree topologies was the relative position of the three North Pacific hakes. In the tree inferred from combined control region– cytochrome b (Fig. 2) M. productus is placed as a sister taxon of the clade formed by the other two species (M. angustimanus and M. gayi); however, the node which ranked these two latter species as sister taxa obtained a very low bootstrap support in ML and MP analyses. On the other hand, the tree obtained from the combination of four mitochondrial genes (Fig. 3) yielded M. gayi as the most basal taxon of this North Pacific clade, whereas M. productus was placed as the most derived species; in this case, the internal nodes got a higher statistical support with the three methods (ML, MP and Bayesian). With respect to the Euro-African Merluccius species relationships, the two trees yielded identical branching pattern (Figs. 2 and 3). This clade was bifurcated into two well differentiated

groups. The first included the South African M. capensis and its sister group M. merluccius/M. senegalensis. The second comprised the sister species M. paradoxus and M. polli. All nodes were highly supported in both cases. 3.5. 5S rDNA coding region All the 107 individuals sampled were sequenced for the nuclear 5S rDNA gene, and only two different sequences were obtained (Fig. 4), with two nucleotide substitutions (transitions) at the positions 3 and 25. One sequence (Sequence A) was obtained for M. merluccius, M. senegalensis and M. capensis. The other (Sequence B) was obtained for the rest of hake species. Within-species variation was not found. Heterozygotes were not found. 4. Discussion 4.1. Phylogenetic relationships and genetic divergence The two tree topologies obtained in the present study were almost identical, and were statistically well supported by high bootstrap and posterior probability values (although the bootstrap values for MP analyses were low in some nodes, in clear discordance with the other two methods). They confirm the existence of two major lineages of hakes already suggested by other authors (Soliman, 1973; Inada, 1981; Kabata and Ho, 1981; Ho, 1990; Lombarte and Castellón, 1991; Roldán et al., 1999; Quinteiro et al., 2000; Grant and Leslie, 2001), one American and one Euro-African. The mean value of genetic

Fig. 4. Alignment of the two haplotypes of the 5S rDNA coding region found in all the individuals sequenced from the Merluccius species. In bold letters (positions 3 and 25) are shown the only two nucleotide substitutions found.

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divergence for the cytochrome b between the American and the Euro-African species of Merluccius is 11.5%, which is slightly lower than the value estimated by Quinteiro et al. (2000) from a more variable mitochondrial marker (14 ± 0.021%). 4.1.1. American clade According to our results, M. bilinearis is the most divergent taxon among the American species, and shows the highest values of divergence within this clade, which agrees with results obtained by Roldán et al. (1999), Quinteiro et al. (2000) and Grant and Leslie (2001). In this study we present for the first time genetic data in a phylogenetic analysis for the tropical eastern Pacific species M. angustimanus. On the basis of morphological similarity, this species is considered to be a recently derived taxon of M. gayi (Mathews, 1975; Ho, 1990). Analysis of otolith morphology shows that of all the species of hakes M. angustimanus is most similar to M. productus and M. gayi, but does not indicate which is the sister taxon of M. angustimanus (Grant and Leslie, 2001). The two tree topologies obtained in this work (Figs. 2 and 3) confirmed that M. angustimanus is more related to M. productus and M. gayi than to other species of Merluccius, clustering these three species in a well separated group within the American clade, in concordance with their geographical distribution. However, the different branching patterns for this group between the two trees make it difficult to solve the sister-taxon relationship of M. angustimanus. On the other hand, the four mitochondrial genes combination tree (Fig. 3) showed higher bootstrap and posterior probability values for the nodes within this group, and the value of genetic divergence between M. angustimanus and M. productus (0.5%) is lower than between the former and M. gayi (1.5%). For these two reasons, we are inclined to suggest that M. angustimanus and M. productus are sister species. Kabata and Ho (1981) suggested that M. hubbsi and M. australis were closely related species in the basis of similarity of copepod parasites, and proposed a North Atlantic origin of M. hubbsi. Nevertheless, allozyme data refused this possibility clustering M. australis as a sister taxon to M. productus–M. gayi (Stepien and Rosenblatt, 1996; Roldán et al., 1999; Grant and Leslie, 2001). Our results support Kabata and Ho's scenario ranking M. hubbsi and M. australis as sister species, and clustering them together with M. albidus in a well differentiated group within the American clade. 4.1.2. Euro-African clade All previously available genetic data (Stepien and Rosenblatt, 1996; Roldán et al., 1999; Quinteiro et al., 2000; Grant and Leslie, 2001) agree in a deep separation of two EuroAfrican lineages of hakes, one comprising M. paradoxus and M. polli and another one comprising M. merluccius, M. senegalensis and M. capensis. Our results confirm these two well separated groups, with a mean value of divergence of 7.7%. In addition, this separation is also supported by the prevalence of a new derived polymorphism in the highly conserved coding region of the nuclear 5S rDNA gene, only found within the M. merluccius–M. senegalensis–M. capensis

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group, which could also indicate that this group is the most derived one in the genus Merluccius. However, there is no agreement in the branching order within the M. merluccius–M. senegalensis–M. capensis group. Allozyme data (Roldán et al., 1999; Grant and Leslie, 2001) indicate that M. senegalensis and M. capensis are sister taxa, whereas mtDNA data (Quinteiro et al., 2000) suggest that M. merluccius and M. senegalensis are sister species. In this work, the analysis of combined slow (12S and 16S) and fast evolving genes (control region and cytochrome b) allowed us to avoid the potential incidence of homoplasies in rapidly evolving sequences suggested by Grant and Leslie (2001), yielding tree topologies which agree with the latter hypothesis with high statistical support. 4.2. Biogeography of hakes and modes of speciation The results obtained in the present study seems to be consistent with a North Atlantic origin of the genus Merluccius (Kabata and Ho, 1981; Inada, 1981; Fedotov and Bannikov, 1989). An early separation of American and Euro-African hakes is also consistent with our results, being the mean divergence between these two clades of 11.5%. Taking a rate of evolution of 0.9%–2.5% for coding regions of mitochondrial DNA calibrated by McCune (1997), the time of divergence estimated from cytochrome b sequences between M. bilinearis and the M. productus–M. angustimanus–M. gayi group ranks between 4.1 and 1.5 millions of years (my), which is consistent with the rise of the Panama Isthmus (4–3 my ago) acting as a physical barrier to gene flow, allowing further processes of speciation following geographical patterns of dispersion. By that time, a southern population of the ancient North Atlantic original stock of Merluccius could have migrated to the south along the South America coastal waters, following speciation processes to originate M. albidus, M. hubbsi and M. australis. The co-occurrence of M. bilinearis and M. albidus would be attributable to a northerly expansion of M. albidus along North America. This scenario does not agree the combined allozyme data (Grant and Leslie, 2001) and mtDNA (Quinteiro et al., 2000), but is recognized by Grant and Leslie (2001) as an alternative explanation. Tree topological patterns and genetic distances estimated in the present study support this hypothesis. The early separation of the M. merluccius–M. senegalensis– M. capensis group from the M. paradoxus–M. polli suggested by previously available genetic data (Stepien and Rosenblatt, 1996; Roldán et al., 1999; Quinteiro et al., 2000; Grant and Leslie, 2001) is in concordance with our data. The mean value of divergence between these two groups is 7.7%, which indicates a time of divergence of 4.2–3 my. Grant et al. (1988) proposed two biogeographical scenarios which could explain the sympatric distribution of the southern Africa hake species (M. capensis and M. paradoxus): (1) past episodes of oceanic cooling displaced northward and isolated ancestral populations of southern African Merluccius, and (2) the two species represent different biogeographical dispersals of North Atlantic taxa along the west coast of Africa. Our data clearly support the latter hypothesis, since both species are clustered in well separated groups. The occurrence of a sympatric overlap

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between them is due to secondary contact after a considerable time of divergence. Our results suggest that the evolution of the genus Merluccius has probably involved two different types of speciation events. Separation between American and Euro-African hakes and differentiation events associated to the rise of the Panama Isthmus are examples of vicariant speciation. On the other hand, in both continents, the clear pattern of phylogeographic speciation is surprising for hakes since they are demersal species with high dispersal capacity. Geographic differentiation is supported for many species; in the Euro-African clade M. merluccius and M. senegalensis are closer to each other than to the South African M. capensis; in the American lineage, the Pacific M. angustimanus appears here closely related to M. productus, in concordance with their geographical distribution, being M. gayi in another branch. The same can be applied to the southern species M. hubbsi and M. australis, separated from the northern M. albidus. Speciation associated to geographical distribution, without apparent physical barriers in the oceanic environment suggests a sort of spawning homing for this genus. This is supported by within-species genetic differentiation associated to geographic distance, that has been described for M. merluccius (Castillo et al., 2004) and M. paradoxus (von der Heyden et al., 2007). Acknowledgments We are grateful to Ivan G. Pola (Universidad de Oviedo, Spain) for collaboration in laboratory tasks. We are indebted to Dr. David Posada (Universidad de Vigo, Spain) and Dr. Salvador Carranza (Universidad de Barcelona, Spain) for precious help with data analysis. Hake samples were kindly provided by Prof. Francis Juanes (University of Massachusetts, USA), Dr. Ignacio Sobrino (IEO Cadiz, Spain), Dr. Luis O. Bala (CONICET, Argentina), Dr. Mauricio Ponte (University of Santiago, Chile), Dr. Francisco Sanchez (IEO Santander, Spain), Dr. Robin Tilney (Department of Environmental Affairs, Cape Town, South Africa), and Dr. Eduardo Vallarino (University of Mar del Plata, Argentina). We also want to thank anonymous referees for highly valuable comments on the original manuscript. This work has been supported by the EU Project FISH & CHIPS (STREP GOCE CT2003-505491). References Castillo, A.G.F., 2002. Desarrollo y aplicación de marcadores microsatélites a la filogenia del género Merluccius. Tesis de Licenciatura. Universidad de Oviedo. Castillo, A.G.F., Martinez, J.L., Garcia-Vazquez, E., 2004. Fine spatial structure of Atlantic hake (Merluccius merluccius) stocks revealed by variation at microsatellite loci. Mar. Biotechnol. 6 (4), 299–306. Cohen, D.M., Inada, T., Iwamoto, T., Scialabba, N., 1990. FAO Species Catalogue, vol. 10, Gadiform fishes of the world (Order Gadiformes). An annotated and illustrated catalogue of cods, hakes, grenadiers and other gadiform fishes known to date. FAO Fisheries Synopsis n° 125, vol. 10. FAO, Rome. Estoup, A., Largiadèr, C.R., Perrot, E., Chourrout, D., 1996. Rapid one-tube DNA extraction for reliable PCR detection of fish polymorphic marker and transgenes. Mol. Mar. Biol. Biotech. 5 (4), 295–298.

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