Molecular Organization and Evolution of 5S rDNA in the Genus

Molecular Organization and Evolution of 5S rDNA in the Genus Merluccius and Their Phylogenetic ImplicationsDaniel Campo ÆGonzalo Machado-Schiaffino ÆJose Luis Horreo ÆEva Garcia-VazquezReceived:25November 2008/Accepted:29January 2009/Published online:27February 2009ÓSpringer Science+Business Media,LLC 2009Abstract The molecular organization of the 5S rRNA gene family has been studied in a wide variety of animal taxa,including many bony fish species.It is arranged in tandemly repeated units consisting of a highly conserved 120base pair–long region,which encodes for the 5S rRNA,and a nontranscribed spacer (NTS)of variable length,which contains regulatory elements for the transcription of the coding sequence.In this work,a comparative analysis of 5S ribosomal DNA (rDNA)organization and evolution in the 12species of the genus Merluccius ,which are dis-tributed in the Atlantic and Pacific oceans,was carried out.Two main types of 5S rDNA (types A and M)were iden-tified,as differentiated by the absence or presence of a simple sequence repeat within the NTS.Four species exhibited the 2types of 5S rDNA,whereas the rest showed only 1type.In addition,the species M.albidus and M.bilinearis showed 2variants (S and L)of type-M 5S rDNA,which differentiated by length.The results obtained here support the hypothesis of a 5S rRNA dual system as an ancient condition of the Piscine genome.In contrast,some inconsistencies were found between the phylogeny of the genus Merluccius based on mitochondrial genes and that obtained from nuclear markers (5S rDNA,microsatellite loci,and allozyme data).Hybrid origin of the American species M.australis is suggested based on these results.Keywords Merluccius Á5S rDNA ÁCoding sequence ÁNTS A ÁNTS M ÁMolecular evolution ÁPhylogeny ÁIncongruence ÁSpeciation ÁHybridizationIntroductionThe minor class of ribosomal DNA (rDNA)comprises the 5S rRNA gene family,which is arranged in higher eukaryotes in several thousands of copies of tandemly repeated units.Each unit consists of a highly conserved coding sequence of 120base pairs (bp)encoding for the 5S rRNA,and a flanking region of variable length that is not transcribed (nontranscribed spacer [NTS])and contains some regulatory elements for the transcription of the cod-ing sequence (Sajdak et al.1998;Wasko et al.2001).Because it is nontranscribed,the NTS is neutral and it is expected to freely mutate.However,the 5S rDNA fits in aconcerted evolution model (Drouin and Moniz de Sa`1995),which allows for the homogenization of the repeated sequences,thus decreasing intraindividual and intrapopu-lation heterogeneity (Dover 1982).For this reason,the NTS has been widely employed as a molecular marker for species identification and phylogenetic studies,although its application for this last purpose in closely related species is being currently discussed (Pasolini et al.2006).Many studies have been published dealing with the structure,chromosomal location,and sequence variation of the 5S rRNA genes in fungi (Kramer et al.1978;Cihlar and Sypherd 1980;Tabata 1980;Cassidy and Pukkila 1987;Duchesne and Anderson 1990;Amici and Rollo 1991),plants (Ganal et al.1988;Nedi et al.2002),and animals (Brown et al.1977;Bogenhagen et al.1980;Bogenhagen and Brown 1981;Komiya et al.1986),including freshwaterand marine fishes (Penda´s et al.1994;Mora ´n et al.1996;Electronic supplementary material The online version of thisarticle (doi:10.1007/s00239-009-9207-8)contains supplementary material,which is available to authorized users.D.Campo (&)ÁG.Machado-Schiaffino ÁJ.L.Horreo ÁE.Garcia-VazquezDepartamento de Biologia Funcional,Universidad de Oviedo,C.Julian Claveria s/n,33006Oviedo,Spain e-mail:campodaniel@uniovi.esJ Mol Evol (2009)68:208–216DOI 10.1007/s00239-009-9207-8Sajdak et al.1998;Martins and Galetti2001;Wasko et al. 2001;Sola et al.2003;Robles et al.2005;Pasolini et al. 2006;Gornung et al.2007).Two different types of5S rDNA have been found in Xenopus laevis,one expressed in somatic cells and the other in oocytes,derived from the somatic type by gene duplication(Komiya et al.1986).This dual system of paralogous5S rRNA genes has been documented in other animal taxa,including manyfish species(Sajdak et al. 1998;Martins and Galetti2001;Wasko et al.2001;Sola et al.2003;Robles et al.2005;Pasolini et al.2006).The main difference between these two types of5S rDNA relies on the length of the NTS,although in some cases nucleo-tide substitutions in the120bp-long coding sequence of the two5S rDNA types have been reported(Penda´s et al. 1994;Martins and Galetti2001;Wasko et al.2001;Paso-lini et al.2006;Gornung et al.2007).The genus Merluccius is included in the family Mer-lucciidae,which is considered the most basal group within the Gadoidei(Teletchea et al.2006).The12species of the genus Merluccius are distributed in the Atlantic(Euro-pean–African coasts:M.merluccius,M.senegalensis,M. polli,M.capensis,and M.paradoxus;American coasts:M. albidus,M.bilinearis,and M.hubbsi)and the Pacific(M. productus,M.angustimanus,M.gayi,and M.australis) oceans.Hakefisheries are a priority for many regions (Pitcher and Alheit1995);thus,many works published about hake genetics are mainly focused on the population structure of Merluccius species(Lundy et al.1999;Castillo et al.2004;Cimmaruta et al.2005;von der Heyden et al. 2007)for application infisheries management.The phy-logeny of the genus has been scarcely studied,based only on allozyme variation(Rolda´n et al.1999;Grant and Leslie 2001)and mitochondrial loci(Quinteiro et al.2000;Campo et al.2007).Nuclear ribosomal RNA genes have been studied in the genus Merluccius for application as species-specific markers to identify commercial seafood based on the length of polymerase chain reaction(PCR)products(Pe´rez and Garcı´a-Va´zquez2004;Pe´rez et al.2005).Simulta-neous occurrence of C2amplification products of different lengths for the5S rDNA locus in species,such as Mer-luccius paradoxus,M.gayi,and M.bilinearis(Pe´rez and Garcı´a-Va´zquez2004),suggests the existence of[1locus at this gene in the genus.However,the type or origin of these duplicated loci has not been investigated until now.Two main objectives were achieved in this study.First,a comparative analysis of the5S rDNA organization among the12species of Merluccius was carried out to contribute to deciphering the pattern of evolution of this multigene family.Second,phylogenetic relations were inferred for all the species in the genus based on the5S rDNA sequences here obtained and for some American hake species based on genetic distances estimated with5microsatellite loci. They were compared with the molecular phylogeny and speciation patterns previously proposed for Merluccius (Stepien and Rosenblatt1996;Rolda´n et al.1999; Quinteiro et al.2000;Grant and Leslie2001;Campo et al. 2007).Material and MethodsSampling,DNA Extraction,and PCR AmplificationThe Merluccius samples analyzed in this work(2to10for each of the12Merluccius species yielding a total of51 individuals)belong to the collection already analyzed by Campo et al.(2007).Total DNA was extracted from muscle tissue according to Chelex resin protocol(Estoup et al.1996).Amplification of the NTS and partial-coding sequence of the5S rDNA was done using the universal primers5SA and5SBR(Penda´s et al.1994).The total coding sequences were amplified according to the protocol described by Pe´rez and Garcı´a-Va´zquez(2004)by employing the primers5S C(50-AAGCTTACAGCACCTGGTATT-30) and5S MD(50-TTCAACATGGGCTCCGACGGA-30) described therein.PCR reactions were carried out in a total volume of40l l containing Promega buffer19(Promega, Madison,WI), 2.5mM MgCl2,250l M each dNTP, 40pmol each primer,0.2l l Promega GoTaq polymerase (Promega,Madison,WI),and2-l l sample of DNA.PCR was performed in a GeneAmp PCR System9700(Applied Biosystems)with the following conditions:initial dena-turing step at95°C for5minutes,followed by30cycles of denaturing at95°C for30seconds,annealing(for30sec-onds)at65°C for both pair of primers,and extension at 72°C for30seconds,ending with afinal extension at72°C for15to minutes.In addition,for M.bilinearis,M.albidus, M.hubbsi,M.australis and M.gayi,5dinucleotide microsatellite loci were analyzed(in24,24,25,50,and25 individuals,respectively):Maus7,Maus30,and Maus32 (Machado-Schiaffino and Garcia-Vazquez2009);Mmer-UEAW01(Rico et al.1997),and Mmer-Hk20(Mora´n et al. 1999).PCR amplifications were performed on reaction mixtures containing approximately50ng extracted hake DNA template,10mM Tris-HCl(pH8.8),2.5mM MgCl2, 50mM KCl,0.1%Triton x-100,0.35l Mfluorescently labelled primers,0.5U Promega Taq polymerase,and 250l M each dNTP in afinal volume of20l L.DNA Purification and SequencingPCR products were loaded in50-ml2.5%agarose gels and stained with2l l10mg/ml ethidium bromide.Bandscorresponding to the5S rDNA fragments amplified were removed from the gel,and DNA was purified using the Wizard SV Gel and PCR Clean-Up System and then sequenced.Automatedfluorescence sequencing was per-formed with both primers in every case on an ABI PRISM 3100Genetic Analyzer(Applied Biosystems)with BigDye 3.1Terminator system,in the Unit of Genetic Analysis of the University of Oviedo(Spain).For the microsatellite markers,size of the labelled PCR products were deter-mined employing the same genetic analyzer,and the results were visualized employing GENESCAN V.3.7software (Applied Biosystems).Phylogenetic AnalysesSequences were edited with BioEdit(Hall1999)and aligned with ClustalW(Thompson et al.1994)with a penalty of6for gap opening and4for gap extension. However,alignments had to be edited manually a posteri-ori because due to the enormous differences in length, some regions were not properly aligned by the program.To perform phylogenetic analyses,gaps were coded according to the methods proposed by Simmons and Ochoterna (2000)(i.e.,simple and complex indel coding methods)as implemented in SeqState(Mu¨ller2005).We constructed two phylogenetic trees:(1)one with a mixed data set composed of the sequence alignment(without gaps)plus the codification of the gaps according to the simple indel coding method and(2)another one using only the gaps coded with the complex indel coding procedure.The phylogenetic analysis of thefirst data set was done in MrBayes 3.1.2(Huelsenbeck and Ronquist2001)with default settings to establish the initial heating values for four Markov chains,which ran simultaneously and were sampled every100cycles.MrModelTest software version 2.2(Nylander2004)was employed to determine the model of sequence evolution that bestfitted the DNA data (according to Akaike criterion),and this information was implemented in the Bayesian analysis.In contrast,for the data set containing only gap information,maximum par-simony(MP)analysis was done with the program PAUP (ver.4.0b10;Swofford2003)using an heuristic search with 10random-addition sequence replicates and the Tree-Bisection-Reconnection(TBR)algorithm for branch-swapping.The statistical robustness of MP tree nodes was tested with100bootstrap replicates(Felsenstein1985).We also constructed a neighbor-joining tree with a distance matrix calculated from frequency data for thefive microsatellite loci previously mentioned in computer package PHYLIP(Felsenstein1989).Statistical support of nodes was calculated in this case with1000bootstrap replicates.Finally,the program FigTree1.1.2(Rambaut 2008)was employed to visualize the trees.ResultsMolecular Organization of5S rDNA in Merluccius speciesThe electrophoretic banding pattern of the fragments amplified with the primers5SA and5SBR(Penda´s et al. 1994)was very heterogeneous among the12species of Merluccius(Table1).Some species exhibited only1band (M.angustimanus,M.australis,M.capensis,M.gayi,M. merluccius,M.productus,and M.senegalensis),whereas others yielded2(M.albidus,M.hubbsi,M.paradoxus,and M.polli)or3bands(M.bilinearis).After sequencing the fragments obtained with the2pairs of primers A-BR and C-MD for the51individuals analyzed,a consensus sequence of the5S rDNA repeat unit(coding sequence plus NTS)was obtained for each band.These sequences were deposited in GenBank under the accession numbers FJ196623to FJ196640(Table1).As reported by Campo et al.(2007),only two types of coding sequences were found(Fig.1),differentiated by two nucleotide substitutions at positions3and25.One of the sequences(sequence A)was obtained for M.merluc-cius,M.senegalensis,and M.capensis.The other (sequence B)was obtained for all the bands of the rest of hake species.Neither heterozygotes nor intraspecific vari-ation were found.All of the internal control regions(ICRs) were identified in the coding sequence of all species(box Table1Size(bp),type,and GenBank accesion number for each of the5S rDNA repeat units(coding sequence plus nontranscribed spacer)found for all Merluccius speciesSpecies Size and type Accession no.M.albidus652,type-ML FJ196623416,type-MS FJ196624 M.angustimanus401,type-M FJ196625 M.australis400,type-M FJ196626 M.bilinearis223,type-A FJ196627759,type-ML FJ196628661,type-MS FJ196629 M.capensis371,type-A FJ196630 M.gayi386,type-M FJ196631 M.hubbsi241,type-A FJ196632660,type-M FJ196633 M.merluccius371,type-A FJ196634 M.paradoxus371,type-A FJ196635492,type-M FJ196636 M.polli371,type-A FJ196637501,type-M FJ196638 M.productus404,type-M FJ196639 M.senegalensis365,type-A FJ196640A,internal element,and box C in Fig.1).No nucleotide variation was found within these ICRs.All5S rDNA sequences contained the TATA box con-trol element within the NTS at position-30bp(base pairs) upstream from the next array(Sajdak et al.1998;Wasko et al.2001).In M.merluccius,M.senegalensis,and M. capensis it has been modified to AATA.All of the sequences analyzed exhibited an additional TATA-like region at exactly16residues upstream from the TATA box. They also presented the5thymidine residues required for transcription termination(Bogenhagen and Brown1981)at positions119to123and a second T-cluster2bases downstream from the primary one.Comparative Analyses of the NTS SequencesIn M.hubbsi,M.polli,and M.paradoxus,the NTS of the longer band contained of a simple sequence repeat(SSR), which also appeared in the single band of M.productus,M. angustimanus,M.gayi,and M.australis;in the two bands of M.albidus;and in the two longer bands of M.bilinearis. The SSR consisted of a variable number of repeats of the CA motif(between4and14)preceded by a variable number of Cs(between1and13).Its location was always similar,starting at position118,119,or121of the NTS.Based on the absence or presence of this microsatellite sequence within the NTS,we classified the5S rDNAs of hakes in type-A(absent)and type-M(microsatellite pres-ent).In addition,two variants of different length because of insertions and/or deletions were found within type-M sequences of M.albidus and M.bilinearis.These two variants were called‘‘S’’and‘‘L’’(short and long, respectively).In contrast,some intraspecific variation at the size of the NTS type-M caused by the number of Cs and CA motif repeats was found for M.productus,M.biline-aris,M.albidus,and M.hubbsi but not for M.polli and M. paradoxus.However,intraindividual variation cannot be ruled out for the two latter species because the methodol-ogy employed in this study(direct sequencing of PCR products without cloning them)does not allow unambig-uously identification of minor differences in the chromatogram.Because there is some variation,only the most frequent size found for each type-M band is listed in Table1.After introducing long gaps for solving the alignment, four groups of sequences were inferred based on nucleotide similarity and position of gaps.Group A comprised all type-A sequences(M.merluccius-A,M.senegalensis-A, M.capensis-A,M.paradoxus-A,and M.polli-A),except the sequences of M.bilinearis-A and M.hubbsi-A,which were223bp–and241bp–long,respectively.These two sequences could be considered a group apart based on their short length.Group M-I was composed of type M sequences of M.productus,M.angustimanus,M.gayi,M. australis,M.bilinearis-M-L,and M.albidus-M-S.Group M-II comprised the type-M sequences of M.polli and M. paradoxus.Finally,group M-III included the sequences M. bilinearis-M-S,M.albidus-M-L,and M.hubbs i-M.Two conserved‘‘blocks’’were identified in the NTS alignment as evidenced by high similarity between all sequences(Fig.2).Thefirst block(block-1)corresponded to the1to66nucleotides within the NTS,and the second block(block-2)comprised the last105residues of each sequence,with the exceptions being the short M.hubbsi and M.bilinearis type-A sequences,which only matched partially to these blocks because of long deletions.In addition,alignment regions with nucleotide homology between C2sequences(i.e.,with no gaps)showed little variation.The three groups of sequences carrying micro-satellites(M-I,M-II,and M-III)were clearly different from each other,although group M-II exhibited more fragments of the alignment in common with group M-III than with group M-I.Phylogenetic Relations Among NTS SequencesThe model of evolution obtained from MrModelTest (Nylander2004)was the Hasegawa–Kishino–Yano85Fig.1Alignment of the two haplotypes of the5S rDNA codingregion found in all of the individuals sequenced from the Merlucciusspecies.In bold letters(positions3and25)are shown the twonucleotide substitutions found.The internal control regions(boxA=position50to64;internal element=positions67to72;boxC=nucleotides80to97)areshadedFig.2Schematic representation of the alignment of the eighteentype-A and type-M sequences found for all the species within thegenus Merluccius.Dark grey represents fragments with nucleotidealignment,whereas light grey indicate gaps.The numbers below thediagram point:1=first conserved block(positions1to66);2=zone with the SSR;3=zone of nucleotide homology betweentype-A and type-M sequences;and4=second conserved block(positions570to675)(Hasegawa et al.1985),with a proportion of invariable sites of0.6076and equal rate of substitution for all sites. We did an analysis with all NTS sequences but later decided to remove M.hubbsi and M.bilinearis type-A sequences in thefinal phylogenetic reconstruction because due to their much shorter length they introduced noise into the phylogenetic inference.Thefinal phylogenetic tree is shown in Fig.3a.Almost identical topologies were recovered in Bayesian(DNA?’’simple indel’’coded gaps)and MP(‘‘complex indel’’coded gaps)analyses,with only two minor differences between them(see later text). Three main clades can be depicted from the tree,and the sequences belonging to the same group inferred from the alignment(A,M-I,M-II,and M-III)clustered together in all cases.From up to down,thefirst clade consisted of all the M-I sequences divided into two subgroups:(1)M. productus-M?M.angustimanus-M?M.gayi-M?M. australis-M]and(2)M.bilinearis-M-L?M.albidus-M-S. For this clade,the Bayesian tree exhibited a multifurcated pattern involving thefirst subgroup taxa,whereas in the MP tree they were clustered in a separate branch with61% of bootstrap support(tree not shown).The next branch clustered M-II and M-III sequences.The Bayesian tree placed M.bilinearis-M-S as a sister taxon of groupM-IIFig.3a Tree topology obtained in the bayesian phylogenetic analyses of the type-A and type-M sequences of the12Merluccius species plus the alignment gaps coded according to the simple indel coding method of Simmons and Ochoterna(2000).Values above the branches indicate posterior probability support,whereas numbers below the branches indicate bootstrap support after100replicates for MP analyses of only the alignment gaps coded according to the complex indel coding method of Simmons and Ochoterna(2000)as implemented in SeqState(Mu¨ller2005).Type-A sequences of M. bilinearis and M.hubbsi were excluded in both analyses(see reasons given in the text).b Neighbor-joining tree estimated from frequency data of5microsatellite loci for5American hake species.c ML tree topology for Merluccius species based on the combination of4r mitochondrial partial gene sequences(12S,16S,control region,and cytochrome b)as adapted from Campo et al.(2007).Outgroup= Gadus morhua(M.paradoxus-M?M.polli-M),whereas the MP tree split both groups in separate branches.Finally,all type-A sequences(group A)clustered in a well-separated branch with two subclades:one comprising M.paradoxus-A?M. polli-A and the other comprising M.merluccius-A?M. senegalensis-A?M.capensis-A.The four groups of sequences seem to constitute well-differentiated evolutionary clades.This was supported by high values of bootstrap and posterior probability for almost all nodes supporting these groups.Figure3b shows the midpoint-rooted neighbor-joining tree obtained from frequency data offive microsatellite loci(Maus7,Maus30, Maus32,Mmer-UEAW01,and Mmer-Hk20)forfive American hake species.M.bilinearis was separated as the most divergent taxon,whereas M.australis and M.gayi were clustered as sister species in the most derived branch. DiscussionMolecular Organization and Evolution of5S rDNAin Merluccius speciesAll sequences analyzed here likely correspond to func-tional genes because they exhibit all the necessary features for the correct gene expression:the three ICRs(box A, internal element,and box C in Fig.1),the TATA box,and the poly T region.The second T cluster that was found two bases downstream from the primary one could be a ‘‘backup’’cluster,a feature already described for Xenopus 5S RNA genes(Bogenhagen and Brown1981).This has also been reported in otherfishes(Gornung et al.2007). Similarly,the second TATA-like region,found16residues upstream from the TATA box,could be a‘‘backup’’TATA box.For the genus Merluccius,we found at least2types of 5S rDNA,of different length,in5species(M.bilinearis, M.albidus,M.hubbsi,M.paradoxus,and M.polli)of12. The existence of2classes of5S rDNA differing mainly in the size of the NTS,and sometimes also in the nucleotide sequence of the coding region,has been described for many animal species,includingfish(Komiya et al.1986;Penda´s et al.1994;Martins and Galetti2001;Wasko et al.2001; Sola et al.2003;Pasolini et al.2006;Gornung et al.2007). In addition,the existence of conserved blocks within NTS sequences and the low number of nucleotide substitutions found in the homologous sequence alignment regions indicate that the differences between the NTS sequences found in Merluccius species are mainly caused by inser-tions and deletions(more than nucleotide substitutions), such as in Characiformes(Wasko et al.2001)and other taxa as separate as sturgeons(Robles et al.2005).How-ever,the organization of Merluccius5S rRNA genes may be somewhat different from that of otherfish taxa.First,4 types of NTS sequences(groups A,M-I,M-II,and M-III), instead of2,could be considered for this genus,clustering the type-M sequences into3well-separated groups in the reconstructed phylogenetic tree.Second,differences in the coding sequence between the2types(long type-II and short type-I NTS)of5S rDNA,which have been reported for otherfish(Komiya et al.1986;Penda´s et al.1994; Martins and Galetti2001;Wasko et al.2001;Sola et al. 2003;Pasolini et al.2006;Gornung et al.2007),did not occur in Merluccius,where nucleotide substitutions in the coding region were found only for the clades M.merluc-cius,M.senegalensis,and M.capensis,the3most recently diverged species within the genus(Campo et al.2007).Distinct families of5S rRNA genes,often characterized by variants of spacers,have been described associated with differential expression in somatic and oocyte cells(Komiya et al.1986;Martins and Galetti2001;Wasko et al.2001; Pasolini et al.2006).Such kinds of tissue specialization can not be generalized for the genus Merluccius because only one type of NTS(type-A or type-M)exists for seven spe-cies,such as in European M.merluccius(type-A),Pacific M.australis(type-M),and others.Pasolini et al.(2006)suggested that the dual5S rRNA gene system corresponds to the ancestral condition of the Piscine genome and that the loss of a5S rRNA gene cluster might have occurred secondarily infish taxa that bear only one type of5S rDNA.M.bilinearis,supposed to be the most ancient species of the genus(e.g.,Quinteiro et al. 2000;Campo et al.2007)exhibited the two types of5S rDNA.In the most recent M.merluccius–M.senegalensis–M.capensis lineage,the loss of type-M5S rDNA in the ancestral species could have led to the current presence of only type-A5S rDNA.Deletion or loss of the type-A locus in all species within the Pacific Ocean lineage(M.pro-ductus,M.angustimanus,and M.gayi)explains their5S rDNA organization.However,the evolution of this gene family in the remaining clade can not be explained by simple loss of one type of5S rDNA.The north Atlantic American M.albidus and M.bilinearis exhibit two dif-ferent types of NTS containing microsatellites;M. bilinearis possesses one additional NTS without SSR, which absent in M.albidus.This could be explained by a duplication of type-M locus in the M.albidus–M.bilinearis lineage plus a loss of type-A in M.albidus.Additional deletions in M.bilinearis type-A could explain its short feature.A complex combination of duplications,insertions, and deletions,in general genome rearrangements,has likely been involved in the evolution of this gene family in the genus Merluccius.With respect to M.hubbsi-A and M.bilinearis-A,in addition to not having a microsatellite,they do not present any of the other features shared by the rest of type-Asequences,being just short sequences that match only the common blocks of the general alignment.They may have been originated through deletions from a longer sequence, but whether this ancestral state was type-A or type-M cannot be determined using the present data.Phylogenetic InferenceAccording to the phylogenetic trees constructed from NTS sequences and gaps alignment(Fig.3),the species of the genus Merluccius generallyfit a vicariant model of distri-bution(sequences of Pacific species cluster in the same branches as do Atlantic American and Atlantic Euro-Afri-can species),with a couple of exceptions.Type-M sequences of two African species(M.paradoxus and M. polli)cluster within the clade formed by three North American Atlantic hakes(M.bilinearis,M.albidus,and M. hubbsi).In contrast,one of the two type-M sequences of the Northwestern Atlantic species,M.albidus and M.biline-aris,MS and ML,respectively,cluster together as a sister clade of the group formed by the four Pacific hakes(M. productus,M.angustimanus,M.gayi,and M.australis).When comparing the phylogenetic relations here obtained from the analysis of NTS sequences of the12 Merluccius species with the phylogeny constructed from mitochondrial genes(Campo et al.2007),the main scheme is maintained(Fig.3c).Species cluster together by geo-graphic proximity,with M.bilinearis being likely the most ancient species.However,there is an important difference between the two trees.The South American hake M.aus-tralis,clustered as a sister species of the Argentine M. hubbsi from mitochondrial genes(in geographic concor-dance;their distributions overlap in Southwest Atlantic waters),was grouped with the Pacific hakes M.productus, M.angustimanus,and M.gayi in NTS sequences analysis. In addition,the phylogenetic tree constructed fromfive microsatellite frequency data infive American hakes (Fig.3b)also supports this pattern,placing M.australis as the sister species of the Pacific M.gayi.Moreover,phy-logenetic relations among Merluccius species inferred from other nuclear markers,such as allozyme loci(Stepien and Rosenblatt1996;Rolda´n et al.1999;Grant and Leslie 2001)also place M.australis more related to the Pacific lineage than to the Atlantic one.Although not uncommon,it is not easy to explain large incongruence of nuclear and mitochondrial phylogenies.In other cases—fromfish(e.g.,Egger et al.2007;Koblmu¨ller et al.2007)to lizards(e.g.,Leache´and McGuire2006)to mammals(e.g.,Ting et al.2008)—it has been interpreted as a signal of repeated hybridization and introgression, leading to the hybrid origin of some species and/or retic-ulate phylogeny.In the present case,phylogenetic incongruence between nuclear and mitochondrial markers for only one species could be explained by ancient asym-metric hybridization leading to the formation of a species of ancestral hybrid origin,M.australis.This process would have involved two ancestral populations,one from the M. productus–M.angustimanus–M.gayi branch(Pacific Ocean)and other from M.albidus–M.hubbsi lineage (Atlantic Ocean).If male individuals from the former population successfully reproduced atfirst with female individuals from the latter,and then also with the new hybrid female individuals with higherfitness than male individuals coming from the latter population and from the new hybrid pool,after a considerable number of genera-tions the nuclear genome of the M.albidus–M.hubbsi lineage could have been lost in the new hybrid population pool,being replaced by that of the M.productus–M.an-gustimanus–M.gayi ancestor,whereas they would have kept the mitochondrial lineage of the latter.Then these two ancestral populations could have split and migrated to the North Pacific and the South Atlantic oceans,respectively (Campo et al.2007).Therefore,the analysis of the nuclear sequences would place M.australis together with M.pro-ductus–M.angustimanus–M.gayi lineage,whereas mitochondrial DNA phylogeny would cluster it closely related to the Atlantic species M.albidus and M.hubbsi.Thus,we propose hybridization as a third mechanism of speciation(together with vicariance and geographic dis-persion;Campo et al.2007)to explain the evolutionary history of the genus Merluccius.Further work,such as extensive genome and karyotype analysis,should be done to confirm this hypothesis because hybridization can result in genomic changes,including alterations of gene expres-sion,chromosomal structure,and genome size(Baack and Rieseberg2007).Acknowledgments Hake samples were kindly provided by Francis Juanes(University of Massachusetts),Ignacio Sobrino(Instituto Es-pan˜ol de Oceanografia Cadiz,Spain),Luis O.Bala(Consejo Nacional de Investigaciones Cientificas y Tecnicas,Argentina),Mauricio Ponte (University of Santiago,Chile),Francisco Sanchez(Instituto Espan˜ol de Oceanografia Santander,Spain),Robin Tilney(Department of Environmental Affairs,Cape Town,South Africa),and Eduardo Vallarino(University of Mar del Plata,Argentina). 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