J. Quinteiro á R. Vidal á M. Rey-Me ndez

Phylogeny and biogeographic history of hake (genus Merluccius),inferred from mitochondrial DNA control-region sequences

Received: 12 December 1998 /Accepted: 15 October 1999

Abstract Phylogenetic analyses of the left domain of themitochondrial DNA control-region sequence have beenused to examine the relationships among species of thegenus Merluccius (Ra®nesque, 1810), and to comparethese with hypotheses based on morphological, meristicand allozyme characters. Analysis of aligned sequencesrevealed that transition bias was much lower than inmammalian mtDNA, and that nucleotide compositionof control-region sequences was biased toward A and T.We have roughly calibrated a molecular clock for thegenus, based on the rise of the Isthmus of Panama ,which is believed to have created a barrier to dispersalbetween marine species of the Atlantic and Paci®cOceans. Our mtDNA-based phylogeny was highly con-gruent with allozyme-based phylogenies, but poorly sowith a previously described phylogeny based on mor-phology. Speci®cally, our phylogeny resolved two well-supported principal clades, one of American (westAtlantic and east Paci®c) species and the other of Euro±African (east Atlantic) species. This suggests an evolu-tionary history during which the ancestral lineage ofMerluccius was divided between two geographic regions,with subsequent dispersal and vicariant events resultingin the evolution and distribution of extant taxa. How-ever, the relationships between some taxa within theAmerican clade could not be resolved. We suggest thatthis is consistent with an hypothesis of a rapid origin andradiation of these taxa.

Introduction

Species of the genus Merluccius (hake) are widely dis-tributed throughout cool waters (4 to 14 °C) of the maincurrent system of the Atlantic, the Southern Ocean andthe margin of the east Paci®c (Soliman 1973). Althoughthe taxonomy of this genus is complex, it is currentlybelieved to comprise 12 species with highly conservativeexternal morphology (Inada 1981; Cohen et al. 1990).Hake can perform long migrations, and physiologicaladaptations of the swimbladder and blood system fa-cilitate vertical mobility, enabling them to migrate fromnear the bottom during the day to mid-water or near-surface waters to feed at night (Olivar et al. 1988). Theireggs are pelagic, and the larvae remain in the watercolumn for �2 mo. before descending to the bottom(Fahay 1974). In most areas, two hake species overlapfor a considerable part of their geographical ranges. Forexample, the two species of Cape hake, M. capensis andM. paradoxus, overlap o� southern Africa, the silverhake M. bilinearis and the o�shore hake M. albidus o�the eastern North American seaboard, the austral hakeM. australis and the Argentine hake M. hubbsi o� Pat-agonia, the Senegalese hake M. senegalensis and theBenguela hake M. polli o� west Africa, and the australhake M. australis and the Chilean hake M. gayi o�southern Chile (Fig. 1) (Cohen et al. 1990; Howes 1991;Lo pez Abella n and Ariz Tellerõ a 1993).

Our knowledge of the systematics and biogeographyof Merluccius spp. relies mainly on the works of Inada(1981, 1989), Kabata and Ho (1981), Ferna ndez (1985),Howes (1989), Cohen et al. (1990), and Ho (1990).Nevertheless, their interrelationships are poorly under-stood, and there is little agreement on the position ofthis group in relation to kindred taxa. Some investi-gators considered that Merluccius spp. should be in-cluded in the family Gadidae (see Inada 1989 fordetails), while others view the Merluccidae as a sepa-rate family occupying a relatively primitive positionwithin the Gadidae (Marshall 1966; Cohen 1984;

Marine Biology (2000) 136: 163±174 Ó Springer-Verlag 2000

Communicated by S.A. Poulet, Rosco�

J. Quinteiro á R. Vidal á M. Rey-Me ndez (&)Universidade de Santiago de Compostela,Facultade de Bioloxõ a,Departamento de BioquõÂmica e Bioloxõ a Molecular,E-15706 Santiago de Compostela,Galicia, Spain

Fax: 0034 (0)981 596904e-mail: bnreymen@usc.es

Okamura 1989). Markle (1989) ranked Merluccidae asa sister group to the Lotidae plus Gadidae families,while Howes recognized the derived character of thefamily Merluccidae (Howes 1990, 1991), and restrictedit to include only the genus Merluccius, ranking it as asister group of the Gadidae.

Paleontological information suggests that the genusarose in the middle Oligocene (Fedotov and Bannikov1989). There are two hypotheses concerning its originand dispersal: both agree that hake originated in theNorth Atlantic and entered the Paci®c through thethen-open Panamanian seaway, but di�er as to whetherM. hubbsi derived from an eastern South Paci®c stock(Szidat 1955, 1961; Inada 1981) or from a westernNorth Atlantic stock (Kabata and Ho 1981; Ho 1990).Likewise, morphological and biogeographic studies(Inada 1981; Ho 1990) suggest that hake from the Oldand the New World represent distinct clades. However,details of hake evolution in the eastern and westernAtlantic and the eastern Paci®c are still unclear, andtheories based on larval drift are unconvincing (Inada1981).

Despite their importance in the pelagic ecosystem(Pitcher and Alheit 1995), relatively little attention hasbeen devoted to the evolution of these organisms, anddispersal distances and the extent of consequent gene¯ow are currently unknown. With the exception of a fewstudies on allozyme polymorphism and restrictionfragment-length polymorphism (RFLP) of mitochon-

drial DNA (mtDNA) (Becker et al. 1988; Grant et al.1988; Rolda n 1995; Stepien and Rosenblatt 1996), thephylogenetic relationships among Merluccius spp. havenot been investigated at the DNA-sequence level.

An understanding of phylogenetic relationshipsamong species is necessary to a rigorous comparativeapproach in studies of adaptation (Felsenstein 1988),and can also illuminate biogeographic patterns (Avise1994). Studies on interspeci®c variation based on theanalysis of mtDNA sequences provide a powerful toolfor investigating the evolutionary history of ®shes(Moritz et al. 1987; Avise 1989). The usefulness ofmtDNA in evolutionary studies derives from its pre-dominantly maternal inheritance, high substitution rate,and apparent lack of recombination (reviewed by Wil-son et al. 1985). In addition, di�erent regions of themolecule and di�erent lineages of ®shes can evolve atdi�erent rates. Evolution of the control region can bethree to ®ve times faster than the remainder of the mi-tochondrial genome in some groups (Brown et al. 1986).However, several recent reports have challenged thegenerality of this evolutionary rate, especially in ®shes(Shedlock et al. 1992; Bernatchez and Danzmann 1993;Zhu et al. 1994).

In this study, we determined the sequence of the leftdomain of the control region of mtDNA in samplesfrom 11 of 12 currently recognized species of Merluccius.We then used tree-reconstruction techniques to investi-gate the evolutionary history of this genus on the basisof these sequences, and compared our conclusions withthose obtained previously by other techniques.

Fig. 1 Merluccius spp. Approximate distributions of major species

164

Materials and methods

Sampling

Nucleotide sequences of the left domain of the mtDNA controlregion of 26 individuals of 11 of the currently recognized species ofthe genus Merluccius (Ra®nesque, 1810) (Table 1) and two tenta-tive outgroup species, Macruronus magellanicus and M. novae-zelandiae (Quinteiro et al. unpublished data), were determined.Tissue from Merluccius angustimanus and the species recently de-scribed by Mathews (1985), M. hernandezi, was not available foranalysis. Tissue samples (muscle or heart) were collected and storedin 70% ethanol. The sampling strategy included an initial screeningto investigate intraspeci®c variation (Table 1), so that only non-identical haplotypes were included in the analysis.

Laboratory protocols

Total DNA was isolated by standard phenol/chloroform/isoamylalcohol procedure, and resuspended in 50 to 100 ll of sterile water(Kocher et al. 1989; Sambrook et al. 1989). Ampli®cations of con-trol-region sequences were carried out in 25 ll reaction volumescontaining Promega Bu�er A 1x, 1.5 mM MgCl2, 200 lM of each

dNTP, 0.1 lM of each primer and 0.5 or 1 ll of a 1/10 dilution ofstock total DNA. Two primers designed speci®cally for ®shes wereused: the light-strand primer L15998 (5¢-TACCCCAAACTCC-CAAAGCTA-3¢) complementary to tRNApro (Alvarado-Bremer1994); and a heavy-strand primer, B (5¢-ACGCTGGAAA-GAACGCCCGGCATGG-3¢), complementary to the central regionof the D-loop in gadoids (Lee et al. 1995). The ampli®cation proce-dure, performed in a Gene Amp PCR System 2400 (Perkin Elmer),was as follows: an initial denaturing step of 3 min at 94 °C, followedby 30 cycles, with each cycle comprised denaturing at 94 °C for 30 s,annealing at 50 to 54 °C for 40 s, followed by extension at 72 °C for1 min and a ®nal extension at 72 °C for 5 min. Standard precau-tions, including the use of negative controls, were taken to detectcontamination and related problems. The polymerase chain-reac-tion (PCR) mixture was digested for 30 min at 37 °C, with 1 ll ofshrimp alkaline phosphatase (2.0 U ll)1) and 1 ll of exonuclease I(10.0 U ll)1) contained in the PCR product pre-sequencing kit(USB, Cleveland, Ohio, USA). After enzyme inactivation, an ali-quot of 6 ll of the PCR mixture was used for cycle-sequencing withABI PRISMÒ DNA polymerase, FS (Perkin-Elmer Corporation,USA). Labelled extension products were gel-separated and detectedwith an automated DNA-sequencer (Model ABI PRISM 377, Per-kin-Elmer Corporation, USA). All samples were sequenced in theforward direction; problematic samples were subsequently se-quenced in the reverse direction to con®rm nucleotide assignments.

Table 1 Merluccius spp. Initialscreening of intraspeci®c varia-tion and sampling locations anddates of collection (MED Med-iterranean; NWA NW Atlantic;NEA NE Atlantic; NEP NEPaci®c; SWA SW Atlantic; SEASE Atlantic; SEP SE Paci®c)

Species abbreviations Common name Location Haplotypes

M. bilinearis Silver hake NWA, USA 3M. bil-1M. bil-2M. bil-3M. bil-4

M. australis Austral hake SEP, Chile 2M. aus-1M. aus-2M. aus-3

M. productus Paci®c hake NEP, USA 2M. pro-1M. pro-2

M. albidus O�shore hake NWA, USA 1M. alb-1M. alb-2

M. gayi Chilean hake SEP, Chile 1M. gay-1M. gay-2

M. hubbsi Argentine hake SWA, Argentina 1M. hub-1M. hub-2

M. capensis Shallow-water Cape hake SEA, South Africa 2M. cap-1M. cap-2

M. senegalensis Senegalese hake SEA, Senegal 2M. sen-1M. sen-2

M. merluccius European hake MED, NEA, Spain 4M. mer-1M. mer-2M. mer-3M. mer-4

M. polli Benguela hake SEA, Mauritania 2M. poll1-1M. polli-2

M. paradoxus Deep-water Cape hake SEA, South Africa 1M. pardx-1

165

Sequence analyses

Sequence alignments were performed using Clustal W (Thompsonet al. 1994), with subsequent checking and adjustment by eye. Thealigned sequences were then compared with published sequencesfor Gadiformes (Lee et al. 1995) to verify the boundaries of thetRNApro gene. Statistics on nucleotide composition were compiledusing the MEGA program (Kumar et al. 1993). Previous phylo-genetic analyses based on mtDNA have demonstrated that, as di-vergence time increases, many sites become saturated by multipletransitional substitutions, thus obscuring more ancient divergences(Brown et al. 1982). Transversions, however, appear to accumulatelinearly over time (Miyamoto and Boyle 1989; Irwin et al. 1991).Two approaches were used to investigate whether transition satu-ration occurred. (1) The number of transitions and transversionswere plotted against total sequence divergence (%) for all pairwisecomparisons. At saturation, the rate of increase in transitionsreaches a plateau, and transversions outnumber transitions. Se-quence divergences were estimated with the Tamura±Nei model(Tamura and Nei 1993), which corrects for nucleotide compositionbias and substitution bias. (2) We calculated the observed meanratio of transitions to transversions (TS:TV), and determinedwhether this ratio di�ered from that expected at phylogenetic sat-uration. The transition-to-transversion ratio expected at saturation(Rs:v) can be estimated from empirical-base frequencies (Holmquist1983): Rs:v � �PA � PG � PC � PT � : �PA � PG��PC � PT �where PA, PC,PT and PG are the base frequencies.

Phylogenetic analyses

Phylogenetic analyses were performed using two standard tree-re-construction methods, because no single approach has been shownto be consistently superior for ®nding the correct tree (Hillis andHuelsenbeck 1992; Hillis 1995).

First, a neighbor-joining (NJ) analysis without the assumptionof equal rates of evolution (Saitou and Nei 1987) was performedusing MEGA Version 1.01 (Kumar et al. 1993), considering bothtransitions and transversions, and the Tamura±Nei model of nu-cleotide substitution (Tamura and Nei 1993). In addition, we alsoperformed analyses considering transversions only to counteractthe potential e�ect of homoplasy. Missing data together with in-sertions and indels were handled with the complete-deletion option.The reliability of the NJ tree was evaluated by two 2000 bootstrapreplicates (Hedges 1992).

Second, the software package PHYLIP Version 3.5 (Felsenstein1993) was used to execute a maximum-likelihood (ML) analysis(Felsenstein 1981). Parameters speci®ed in the analysis includedone category of substitution, empirical base frequencies, and a 2.0:1value for the TS:TV ratio. This ratio, obtained from the sequencedata, constituted the mean TS:TV ratio for all pairwise compari-sons between ingroup taxa, estimated with the Tamura±Nei modelin the MEGA program, which corrects for transition bias (Wakeley1996) to give corrected ratios, R. Because the order of taxa in thedata set can a�ect the results (Ferris et al. 1995), we analyzed eachdata set ten times, using a randomized input order. Insertion/de-letion events are di�cult to take into account in the maximum-likelihood procedure, since sites with gaps in any of the sequencesunder consideration are excluded from the analysis (Goldman1997). The robustness of the ML tree was inferred from P valuescalculated for branch lengths.

In order to test whether the two tree topologies di�ered sig-ni®cantly, the Kishino and Hasegawa (1989) likelihood-ratio test,implemented in PHYLIP, was used: this test compares tree top-ologies on the basis of di�erences between their log-likelihoodvalues. We tested for the heterogeneity of rates of evolutionarychange along the di�erent lineages using the maximum-likelihoodmethod of Felsenstein (1993). The null hypothesis for this test isthat the rate of nucleotide substitutions is constant for all branchesof the tree.

Despite the fact that monophyly of the genus Merluccius hasbeen veri®ed by studies of both morphological and allozyme

characters (Ho 1990; Rolda n 1995), our choice of outgroup wasconstrained by the lack of available sequences and by the complextaxonomy of the genus (see ``Introduction''). Reported mtDNAsequences in GenBank for several members of the family Gadidae(Gadus morhua X99772; Melanogramus aegle®nus U59475; Micro-gadus tomcod U12058 and Pollachius virens U12069) are too di-vergent to provide informative comparisons. We therefore usedsequence data from the Patagonian grenadier and blue grenadier(Macruronus magellanicus and M. novaezelandiae: Quinteiro et al.unpublished data) to polarize the characters in some of our ana-lyses. Although our results are based on midpoint rooting, thetopologies of the trees remain unaltered when the Patagoniangrenadier and blue grenadier are included as an outgroup.

Results

After ampli®cations, a fragment of about 450 base pairs(bp) was obtained from each sampled individual ofMerluccius spp. Alignment gave a 412 bp sequence, withthe ®rst nucleotide located in Position 22 of the controlregion, since nucleotide assignments were uncertain forthe 5¢ end of some sequences.

Intraspeci®c sequence-divergence (range 0.3 to 0.8%)was negligible in comparison with observed interspeci®cdivergence, although pairwise comparisons using Me-rluccius merluccius did reveal somewhat higher values(range 0.5 to 1.8%: Table 2).

Patterns of nucleotide substitutionsand genetic divergence

A total of 121 variable sites and 107 phylogenetically in-formative sites were detected (Table 3). Base compositionwas strongly biased: adenine and thymine were repre-sented roughly equally, occurring at mean (�SE) per-centages of 31.8 � 0.28% and 31.7 � 0.16%,respectively, while cytosine occurred at 25.4 � 0.29% ofsites, and guanine at only 11.1 � 0.18% of sites. Thesebiases are similar to those found in the mtDNA control-region of other ®shes, including the left domain of thecontrol-region of other Gadiformes (Meyer et al. 1990;Bernatchez and Danzmann 1993; Brown et al. 1993; Zhuet al. 1994; Lee et al. 1995;McMillan and Palumbi 1997).

The ratio of transitions to transversions (TS:TV)(after correction for composition and transition bias) forpairwise interspeci®c comparisons ranged from 0.959 to7.146, with a mean of 2.0 � 0.76. The highest TS:TVratios were between species from the Paci®c Ocean,namely Merluccius productus and M. gayi (mean 7.1),the lowest between M. capensis and M. polli (mean 1.0).Among hake from the American and Euro-Africancoasts, the mean ratios were 3.0 � 1.39 and 2.0 � 1.04,respectively. TSs outnumbered TVs when closely relatedsequences were compared, but TVs were as common asTSs when some highly divergent sequences were com-pared, indicating possible saturation. However, a plot ofthe observed number of TSs and TVs against sequencedivergences revealed a fairly linear relationship, sug-gesting that saturation had not been reached (Fig. 2).

166

Furthermore, in no case did the TS:TV ratio (minimum0.959) approach the value expected at saturation (0.47;estimated following Holmquist 1983), and the meanTS:TV ratio (2.0) was signi®cantly higher than that ex-pected at saturation (Student t-test; t = 5.67, P <0.001).

Table 2 presents estimates of genetic distance be-tween all the individuals examined. Divergence rangedfrom as little as 2% between Merluccius gayi andM.productus to asmuch as 20%betweenM.bilinearis andM. senegalensis. Among the hake species of the westernAtlantic and the eastern Paci®c, the mean divergence(�SE) was 8.8 � 0.015%, while the mean divergenceamong species of the eastern Atlantic coast was slightlylower (6.0 � 0.012%). The mean divergence betweenspecies from di�erent continents was 14.0 � 0.021%.

Phylogenetic relationship

Gene tree-topologies obtained by the two methods (NJand ML) are shown in Fig. 3. The two trees are broadlysimilar, both showing two well-de®ned principal clades.The branching patterns obtained by the two methodswere largely congruent, although some branches exhib-ited low bootstrap support and con¯icting topology. Ingeneral, deeper nodes were well de®ned, whereas inter-mediate nodes had lower bootstrap values.

The early evolutionary history of hake is character-ized by the divergence into two major basic lineages: oneAmerican clade comprising west Atlantic and east Pa-ci®c species, and the other Euro±African clade includingeast Atlantic species. The Euro±African clade comprisesspecies that are relatively homogeneous in size andecology. This clade was well-resolved, and bifurcatedinto two groups. The ®rst comprised Merluccius polli, ablackish species distributed along the west coast ofequatorial Africa, and M. paradoxus, which is distrib-uted along the continental shelf o� southern and south-western Africa. The second group comprisedM. capensisand its sister group M. senegalensis plus M. merluccius.The distribution of M. polli overlaps with that of M.senegalensis o� the coasts of Mauritania and Senegal,although the former inhabits deeper waters. Likewise, afurther two species with similar external morphology,M. capensis and M. paradoxus, overlap o� the south-western coast of Africa, but with the latter occurring indeeper water (Inada 1981). Despite such microallopatricdistributions (deep and shallow water), M. senegalensisand M. polli were separated by at least 37 nucleotidesubstitutions (sequence divergence: 10.2%), and M.capensis and M. paradoxus by at least 30 nucleotidesubstitutions (sequence divergence = 7.6%).

The American clade, containing all species occurringin the western Atlantic and eastern Paci®c, is more dif-®cult to interpret in view of the alternative topologiesobtained and the low bootstrap support for some nodesin the NJ tree. In the ML analysis, Merluccius hubbsiwas ranked as the sister taxon of the clade comprisingT

able

2Merlucciusspp.Genetic-distance

matrix

(Tamura±Nei

model)ofhakemtD

NA

control-regionsequences(Speciescodes

asin

Table

1)

Species

12

34

56

78

910

11

12

13

14

15

16

17

18

19

20

1:Mbil-1

2:Mbil-2

0.005

3:Mbil-3

0.008

0.003

4:Maus-1

0.171

0.164

0.167

5:Maus-2

0.164

0.158

0.161

0.005

6:Mpro-1

0.120

0.113

0.113

0.073

0.073

7:Mpro-2

0.122

0.116

0.116

0.075

0.075

0.003

8:Malb-1

0.144

0.139

0.138

0.090

0.091

0.053

0.055

9:Mgay-1

0.107

0.101

0.101

0.082

0.082

0.021

0.023

0.047

10:Mhub-1

0.123

0.116

0.116

0.078

0.078

0.034

0.036

0.053

0.034

11:Mcap-1

0.182

0.176

0.179

0.153

0.147

0.131

0.134

0.134

0.121

0.128

12:Mcap-2

0.185

0.178

0.182

0.156

0.150

0.134

0.137

0.137

0.124

0.131

0.008

13:Msen-1

0.198

0.191

0.195

0.168

0.162

0.139

0.142

0.136

0.139

0.140

0.067

0.069

14:Msen-2

0.201

0.195

0.198

0.171

0.165

0.142

0.139

0.139

0.142

0.143

0.069

0.072

0.003

15:Mmer-1

0.178

0.171

0.174

0.146

0.140

0.124

0.127

0.127

0.121

0.121

0.053

0.055

0.034

0.036

16:Mmer-2

0.178

0.172

0.175

0.143

0.136

0.118

0.121

0.25

0.121

0.115

0.050

0.053

0.031

0.034

0.013

17:Mmer-3

0.178

0.172

0.175

0.149

0.143

0.124

0.127

0.124

0.121

0.121

0.050

0.053

0.026

0.029

0.008

0.005

18:Mmer-4

0.191

0.185

0.188

0.156

0.156

0.137

0.141

0.138

0.134

0.134

0.058

0.061

0.045

0.048

0.016

0.018

0.018

19:Mpolli-1

0.172

0.166

0.169

0.146

0.140

0.130

0.133

0.140

0.127

0.133

0.075

0.072

0.100

0.103

0.086

0.077

0.083

0.098

20:Mpolli-2

0.169

0.163

0.166

0.149

0.143

0.127

0.130

0.137

0.124

0.130

0.075

0.072

0.100

0.103

0.086

0.077

0.083

0.098

0.003

21:Mpardx-1

0.169

0.162

0.165

0.153

0.146

0.115

0.118

0.125

0.112

0.124

0.078

0.075

0.109

0.112

0.100

0.092

0.098

0.107

0.034

0.031

167

M. australis, M. productus, M. albidus and M. gayi. Inthe NJ analysis, in contrast, M. australis and M. albidusdid not cluster as sister-species, and 55% of the pseu-doreplicates placed M. hubbsi as the sister taxon ofM. productus plus M. gayi. An analysis considering onlytranversions revealed basically the same topology,although with di�erent patterns for M. australis,M. albidus and M. hubbsi, for which bootstrap valueswere not signi®cantly improved. Despite this, the twoanalyses agree in at least two respects: (1) The primaryspeciation event gave rise toM. bilinearis, a silvery-whitespecies distributed along the North American Atlanticcoast (Inada 1981); (2) both analyses clustered M. prod-uctus (from the North Paci®c) and M. gayi (from thewest coast of South America) as sister-species, with arelatively high bootstrap support for this node (72%).Comparison of the NJ and ML trees by the log-likeli-hood test indicated that both are equally likely (NJln = )1689.8274, ML ln = )1678.5834; SD = 5.8281).

Phenograms based on genetic distance of allozymesin nine hake species (Rolda n 1995) were more similar tophylogenies found with mtDNA control-region se-quences than to phylogenies based on morphologicalcharacters (Ho 1990) (Fig. 4). There was a signi®cantcorrelation of allozymic genetic distances and sequence-divergence (Fig. 5; r = 0.8, Mantel's test: P < 0.001).

Felsenstein's maximum-likelihood test indicated thatthe rate of evolutionary change did not di�er signi®-cantly among the di�erent lineages (P < 0.05), sug-gesting that hake mtDNA control-region is evolving in aclock-like fashion. The optimal log-likelihood valueunder the constraint of a molecular clock was

)1668.1753 (vs )1656.1553 when this constraint wasrelaxed).

Discussion

Like many other ®sh groups, Merluccius spp. have verysimilar body forms and must be diagnosed by an arrayof subtle and statistically-determined meristic characters(Inada 1981, 1989). Nucleotide-sequence data from mi-tochondrial DNA, on the other hand, provide a largesource of potentially phylogenetic characters that areindependent of morphology, including problematicmeristic characters.

Rate of variation

As has been reported for other ®sh mtDNA control-regions (Meyer et al. 1990; Sturmbauer and Meyer 1992;Zhu et al. 1994), we found that transitions accumulatemore rapidly than transversions in hake mtDNA, whichhas a high A + T content (>60%). However, our re-sults indicate a low rate of nucleotide substitution and alow TS:TV ratio. There are indications from the litera-ture that the mitochondrial control-region may not be asvariable in some ®sh species as it is in mammals (Meyeret al. 1990; Bernatchez et al. 1992; Bernatchez andDanzmann 1993; Zhu et al. 1994; Turner 1997). Fur-thermore, a low rate of evolution has been reported forthe control region of other gadiforms (Theragra chal-

Table 3 Merluccius spp. Aligned sequences for the 11 species ofhake examined; only variable nucleotide positions are shown(? Ambiguities; dots indicate identity with ®rst sequence for

M. bilinearis, Mbil-1; species codes as in Table 1). Representativesequences of each species reported here have been submitted toGenBank: Accession Nos.: AF112245-AF112255

SpeciesCode No.

Variable nucleotide positions

1111 1111111111 11111111111122 2233344555 5566677778 8888890000 0001111111 2223333333

3456785746 8901534235 6702336890 2456751236 7890234789 1590134568

Mbil-1 AGCACAATCT TAAAATGATA TTTGGGGCCA ATTGAAAGAC AATAAAATCC ACTTTTTAGAMbil-2 .......... ......A... .......... .......... .......... ..........Mbil-3 .......... ..C...A... .......... .......... .......... ..........Maus-1 .......AGC .T..G.AG.T ...AATATTT ...TGG.... ......T..T TTCCCCCCATMaus-2 .......AGC ....G.AG.T ...AA.ATTT ...TGG.... ......T..T TTCCCCCCATMpro-1 ...G...AGC .GG.G.A..T ......AGTT ...TGG.... .........T T.C....TACMpro-2 ...G...AGC .GG.GAA..T ......AGTT ...TGG.... .........T T.C....TACMalb-1 .....G.AGC ..G.G.A..T C.....A..T ...TGG...T ........TT TTC....TACMgay-1 .......AGC .GG.G.A..T ......A.TT ...TGG...T .........T T.C....TACMhub-1 .....G..GC C.G.G.A... ......A.TT ...TGG.... .........T TTC....TACMcap-1 G....T..GC .G....AC.T C.AA..ATTT .ACTGGCAGT ..A.TG...T .TCG.A.TACMcap-2 G....T..GC .G....AC.T C.AA..ATTT .ACTGGCAGT ..A.TG...T .TCG.A.TACMsen-1 GCACTT..GC .G....ATCC C.ATA.ATTT .A.TGGT.GT G.A.T..CTT CTCG...TACMsen-2 GCACTT..GC .G...AATCC C.ATA.ATTT .A.TGGT.GT G.A.T..CTT CTCG...TACMmer-1 G....TG.GC ??.C..ATCC ..ATA.ATTT .A.TGGTA.T ..A.T..CTT CTCG...TACMmer-2 G....TG.GC G.....AT.C C.ATA.ATTT .A.TGGTA.T ..A.T..CTT CTCG...TACMmer-3 G....TG.GC G.....ATCC C.ATA.ATTT .A.TGGTA.T ..A.T..CTT CTCG...TACMmer-4 G....TG.GC ??....AC.? CAATATATTT GA.TGGTA.T .GAGT..CTT CTCG...TACMpolli-1 .....T.AGC .G....AT.C C.AA..ATT. .CCTGGTAGT ..A.T..A.T C.CC.C.TACMpolli-2 .....T.AGC .G....AT.C C.AA..ATT. .CCTGGTAGT ..A.T..A.T C.C..C.TACMpardx-1 ....TT.AGC .G....AC.. C.A...ATTT ..CTGGTAGT ..A.T..A.T C.C..A.TAC

168

cogramma: Shields and Gust 1995; Gadus morhua:S. Carr personal communication).

Although no mtDNA sequences have been previouslypublished for Merluccius spp., a nucleotide-sequencevariation of 11.6% has been reported between thesympatric species M. capensis and M. paradoxus on thebasis of restriction-enzyme analyses of mtDNA (Beckeret al. 1988). This value of genetic divergence is markedlyhigher than that obtained between these species in thepresent study (mean 7.7%).

There have been several studies of ®sh species in-volving direct comparisons of levels of genetic variation

as inferred from mtDNA sequence and RFLP data (Birdet al. 1986; Carr and Marshall 1991; McVeigh et al.1991; Bernatchez and Danzmann 1993; Brown et al.1993; Taylor and Dodson 1994). In several studies, directsequencing of the control region has revealed consider-ably higher levels of mtDNA variation; but this was notthe case in the present study, probably because of a slowevolution rate and a transition rate approaching satura-tion in highly divergent pairwise comparisons. Despitethis, both techniques agree that Merluccius capensis andM. paradoxus are well-di�erentiated congeneric species.

Phylogenetic relationships and rate of evolution

The two tree topologies obtained in the present studywere very similar, indicating two clearly de®ned lineages,the American and Euro±African clades. This agrees withthe results of other morphological and biogeographicstudies (Soliman 1973; Inada 1981; Ho 1990). Studies oncopepod ectoparasites, allozyme polymorphisms, andotoliths also indicate the existence of these two evolu-tionary linages (Kabata and Ho 1981; Lombarte andCastello n 1991; Rolda n 1995). However, the phyloge-netic relationships inferred from our mtDNA-sequencedata were more in agreement with those suggested byRolda n on the basis of allozyme polymorphism thanwith those suggested by Ho (1990) on the basis ofmorphological characters. The basic agreement ofmtDNA topologies with allozyme phylogenies suggeststhat su�cient internodal times have elapsed for the twophylogenies to reach a state of monophyly. Agreement

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TTATATTTTA ATCGTCACTC CAGTTAGGCG GAACGTTCGA ATCAAAGACA TACGTATTAC T.......... ...T...... .......... .......... .......... .......... ........... ...T...... .......... .......... .......... .......... .C......CC. ..TTCTTTC. .T.CCC.ATC A...AG..A. ..TG.CCGG. ..TAA..C.G CC......CC. ..TTCTTTC. .T.CCC.ATC A...AG..A. ..TG.CC.G. ..TAA..C.G CC.G....CC. ..TTC.T.C. T..CCC..T. ....AG.... ...G.CCGG. .......C.G .C.G....CC. ..TTC.T.C. T..CCC..T. ....AG.... ...G.CCGG. .......C.G .CAG..C.CC. ..TTC.T.C. ..ACCC...C .G.GAG.... .....CCGG. ..TAC..C.G .C.G...CCC. ...TC.T... ...CCC..T. ....AG.... .....CCGG. .......C.G .C.G....CC. ...TC.T.CT ...CCC..T. ....AG...G ..TG.CCGG. ..G.C..C.. .C.G.T.CCC. ..TTC.TTC. ..TCCCA.TC .......T.. CC...CC..G A...CTC.C. .C.G.T.CCCT .ATTC.TTC. ..TCCCA.TC .......... CC...CC..G A...CTC.C. .C.G.T..CC. T.TTC.TTC. ..TCCC..TT .G....C... CC...CC..G A...CT..C. .C.G.T..CC. T.TTC.TTC. ..TCCC..TT .G....C... CC...CC..G A...CT..C. .C.G.T..CC. T.TTC.TTC. ..TCCC..TC .......... CC...CC..G A...CT..C. .C.G.T..CC. T.TTC.TTC. ..TCCC..TT .......... CC.G.CC..G A...CT..C. .C.G.T..CC. T.TTC.TTC. ..TCCC..TT .......... CC...CC..G A...CT..C. .C.G.T..CC. T.TTC.TTC. ..TCCC..TT .......... CC...CC..G A...CT..C. .CAGCT..CCT ...TC.TTC. ..TCCC..TC ..G....... .C.GC..... ....CTC.C. CCAGCT..CCT ...TC.TTC. ..TCCC..TC ..G....... .C.GC..... ....CTC.C. CCAGC..CCCT ..TTC.T.C. ..TCCC.?TC ?.G....... ...GC..... .G..CTC.C. C

Fig. 2 Merluccius spp. Plot of observed number of transitions (TS )and transversions (TV) as a function of percentage sequencedivergence (estimated by method of Tamura and Nei 1993)

169

between allozyme- and mtDNA-based phylogenies isprobably less close for species separated by short inter-nodal times (notably Merluccius australis, M. albidusand M. hubbsi, species not considered by Rolda n), as aresult of the stochastic process of lineage-sorting andextinction during speciation (Niegel and Avise 1986). Inhis cladistic analysis of the genus, Ho (1990) found alarger number of most parsimonious trees and a highlevel of homoplasy (73.9%). This may be attributable toconsideration of insu�cient characters (seven, of whichonly six were informative) by Ho, and/or to genuineincongruence between molecular and morphologicalphylogenies.

Numerical taxonomy based on myogen electropho-resis (Lleonart and Agell 1980) and morphology-based

cladistic analyses (Ho 1990) have indicated a close re-lationship between the species of the eastern Atlantic,Merluccius senegalensis and M. polli. Our analyses donot support this ®nding, and are in keeping with theallozyme±polymorphism analysis of Rolda n (1995), inwhich these two species were not closely grouped,M. polli being placed as sister-species of M. paradoxus.

Our results for Merluccius productus and M. gayiappear to agree with the phylogenetic analysis of Ho(1990), based on osteological and meristic data, in whichM. gayi and M. angustimanus were considered as sister-species, together forming the sister group of M. prod-uctus. Likewise, Stepien and Rosenblatt (1996) suggesteda close relationship between these species on the basis ofallozyme polymorphism. These ®ndings are supportedby the close morphological and meristic similarityamong these species (Inada 1981).

Ho (1990) suggested that Merluccius australis andM. hubbsi are closely related species, sharing a morerecent common ancestry with M. bilinearis, an Atlanticspecies that putatively crossed the Panamanian seawayand speciated into M. productus to the north and laterM. angustimanus and M. gayi to the south. In our study,

Fig. 3 Merluccius spp. Molecular phylogeny for 11 species of hakeanalyzed. Both trees were midpoint-rooted. A Neighbor-joining treeconsidering both transitions and transversions (w indicate alternativepatterns obtained using transversions only; values above branchesbootstrap percentages). B Maximum-likelihood tree (log-likelihoodscore = )1656.15532); all branch lengths (except within species) weresigni®cantly di�erent from zero (P < 0.01); branch lengths are drawnproportional to amount of sequence divergence

170

the phylogenetic relationships among M. australis, M.albidus and M. hubbsi are uncertain, because of the al-ternative tree topologies, weak bootstrap support, andthe very short internode lengths compared with the longterminal branches. We suggest that the inability to re-solve clearly between these species may arise from theirrapid origin and radiation over a short time-span.

Pamilo and Nei (1988) argued that a tree constructedfrom DNA sequences of a single gene may di�er con-siderably from the species tree, if the time of divergencebetween species is short. This makes it di�cult to reachthe resolution required to clarify the phylogenetic rela-tionships between groups in fast cladogenetic lineages.As evolution proceeds along the terminal branches, it ismore likely to result in changes that are homoplasticwith the character states of the internal branches (Fel-

senstein 1978, 1988). Our control-region sequences ap-pear to resolve both basal and terminal but notintermediate relationships among hake. Thus, the lackof resolution at the intermediate level of divergence mayre¯ect rapid phyletic radiation.

The fossil record for hake is notoriously poor and in-complete, and it is di�cult to calibrate a rate of evolutionspeci®c to the genus Merluccius on the basis of our se-quence data. However, of particular interest is the puta-tive role played by the Isthmus of Panama in the evolutionof hake, since its closure created a barrier to dispersal. Weconsider that divergence between allopatric western At-lantic and eastern Paci®c species may provide a usefulbench mark by which to assess the time of divergence ofsplits in the genus Merluccius. Taking the earliestestimated time of separation of the tropical and Paci®c

Fig. 4 Merluccius spp. Summa-ry of current status of phyloge-netic relationships in hake.A Cladistic analysis of mor-phological and meristic data(adapted from Ho 1990);B UPGMA tree based on Nei'sgenetic distance (adapted fromRolda n 1995)

171

Oceans [3.2 to 2.8 million years ago (mya); Coates et al.1992] as a conservative estimate of the time of divergenceofM. productus andM. albidus (the closest north Atlanticand Paci®c species of veri®ed certain geographic origin),the rate of evolution expected is 1.7 to 2.0% per millionyears. This calibration assumes that rates of mtDNA se-quence-divergence are similar among Merluccius species,and thatM. albidus andM. productus diverged at the timeof the rise of the Isthmus of Panama . While this estimatemust be interpreted with caution (see Avise 1994), twofactors support it. (1) Using RFLP data and an assumedrate of evolution of 2% per million years (Brown et al.1979), Becker et al. (1988) estimated the time of diver-gence betweenM. capensis andM. paradoxus as 5.8 mya.If our estimate of the rate of mitochondrial DNA evolu-tion is correct, the age estimate for the split betweenM. capensis and M. paradoxus is similar (3.8 to 4.5 mya)to that of Becker et al., and falls within the Pliocene. (2)The relatively short genetic distance between M. prod-uctus and M. gayi indicates that speciation events mayhave been associated with dispersal and founder-events inthe Pleistocene, coinciding with glaciation±deglaciationcycles (Webb and Bartlein 1992). Our estimate of the dateof separation of M. productus and M. gayi (1.3 to1.1 mya), together with fossil evidence (Zinsmeister1970), is also consistent with the origin of these species inthe Pleistocene, and corroborates its recent divergence.

Speciation mode and evolutionary history

Hake have a number of behavioural characteristicswhich would tend to reduce their speciation rate. Theyform large populations and can perform extensivemigrations, altering their geographical distribution inresponse to ¯uctuations in abundance or environmentalperturbations. They also have extended spawning

seasons with multiple spawning; some spawning is ob-served year-round (Inada 1981; Cohen et al. 1990;Pitcher and Alheit 1995). The morphological characterssegregating the di�erent species (see Inada 1981) do notindicate ecological separation.

The evolution of this genus may thus have been de-termined by a series of factors that combined elements ofdispersal and vicariance. For example, it seems likelythat the Benguela system, on the west coast of southernAfrica, may to some degree act as a barrier to migrationbetween Namibian hake and hake of the Cape region.The area of maximum upwelling, which lies betweenthese two regions, may reinforce the reproductive iso-lation of the populations of these species, since temper-ature is below the minimum required for normal larvaldevelopment of hake.

The opening of the southern Atlantic Ocean basinbetween South America and Africa began during theCretaceous (Cox and Moore 1993). However, thenorthern margins of these continents remained relativelyclose to each other until the early Oligocene or lateEocene (�30 to 40 mya; Van Syoc 1995), i.e. the periodin which the genus Merluccius arose (Fedotov andBannikov 1989). By this time, the Atlantic may havebecome an e�ective barrier to gene ¯ow via planktoniclarvae. Van Andel (1979) suggested that a slowing ofequatorial currents at the boundary of the Eocene/Oli-gocene epochs resulted in a concomitant drop in pro-ductivity throughout the equatorial tropical convergencezones. This slowing of equatorial currents across thewidening Atlantic Ocean may have been the cause of theprimary taxon bifurcation in the ancestral stock of hake,giving rise to the South American and Euro±Africanclades.

Acknowledgements The authors thank the following people forcooperation in collection of samples: J. Gonzalez, UniversidadCato lica del Norte, Chile; C. Gonzalez Sotelo and C. PinÄ eiro, In-stituto de Investigaciones Marinas, Vigo, Spain; R. Method,Alaska Fisheries Science Center, USA; J. Gilbraith, USA; J. Pereiroand B. PatinÄ o, Instituto EspanÄ ol de Oceanografõ a, Vigo, Spain; andI. Sobrino, Instituto EspanÄ ol de Oceanografõ a, Cadiz, Spain. Wealso thank C. Stepien for valuable comments. This research wassupported partially by a grant from CICYT ALI 95±0053. R.V.was receiving a predoctoral fellowship from the Government ofChile (MIDEPLAN), and this paper represents part of his PhDdissertation.

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